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 antibody-linker conjugates by microbial transglutaminase. The invention further provides antibody-linker conjugates, antibody-drug conjugates, linker constructs and pharmaceutical compositions comprising the antibody-linker conjugates or antibody-drug conjugates of the invention and uses thereof. [Background technology]

[0002] Antibody-drug conjugates (ADCs) are typically composed of an antibody and a small molecule drug conjugated to the antibody by a chemical linker. After decades of preclinical and clinical research, brentuximab vedotin (Adcetris®) for relapsed Hodgkin's lymphoma and systemic anaplastic large cell lymphoma, and gemtuzumab ozogamicin (for acute myeloid leukemia) Mylotarg(R)), ado-trastuzumab emtansine (Kadcyla(R)) for HER2-positive metastatic breast cancer, inotuzumab ozogamicin (Besponsa(R)) for B-cell malignancies and more recently Poratuzumab A series of ADCs, such as zumabvedotin-piiq (Polivy®), have been approved to treat specific tumor types. More recently, enfortumab vedotin (Padcev®), trastuzumab deruxtecan (Enhertu®), sacituzumab govitecan (Trodelvy®) and belantamab mafodotin (Blenrep®) (registered trademark)) has received marketing approval. For a review on ADCs, see for example (Zhao P. et al., 2020, Acta Pharmaceutica Sinica B, 10, 1589-1600). Although many ADCs have shown excellent anticancer activity, many patients do not respond to these treatments or experience significant side effects before efficacy or relapse after a period of time. Therefore, there is a significant medical need for new ADC formats that have favorable drug-like properties, can be produced in sufficient quantities and quality at reasonable costs to support drug development, and are suitable as therapeutic agents. It still exists.

[0003] A key step in the preparation of ADCs is the covalent conjugation step of the payload to the antibody. Most ADCs currently in clinical development are based on the antibody's endogenous lysine or cysteine ​​residues, while carefully controlling the average degree of modification so that the average drug-to-antibody ratio (DAR) is in the range of 3.5 to 4.0. created by conjugation to groups. Historically, this ratio has been problematic in manufacturing and formulation due to (a) minimizing the amount of unconjugated antibody and (b) higher hydrophobicity and lower solubility. (Lambert JM and Berkenbilt A., 2018, Annu. Rev. Med. 69, 191-207) and typically have poor pharmacokinetic properties (Lyon RP, et al., 2015, Nat Biotechnol, 33, 733-735) were selected on the basis of avoiding species in the mixture with very high DAR. In recent years, various genetic, chemical, and enzymatic methods have been developed for site-specific conjugation and may make it possible to achieve a DAR of 2 (or 4) while avoiding over- or under-modification of antibodies. . An overview of these methodologies is provided in Yamada et al. (reviewed in Kei Yamada and Yuji Ito, 2019, ChemBioChem, 20, 2729-2739).

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

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

[0006] Another approach to generate ADCs by 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, substituting N297 with another amino acid affects the overall stability of the entire Fc domain (Subedi GP and Barb AW., 2015, Structure, 23, 1573-1583) and the overall efficacy of the conjugate. may have undesirable effects. As a result, this can lead to increased antibody aggregation and decreased solubility, which is particularly important for hydrophobic payloads. Furthermore, the glycans present on N297 have important immunomodulatory effects as they induce effector functions such as antibody-dependent cellular cytotoxicity (ADCC). These immunomodulatory effects will be lost upon deglycosylation or any of the other approaches discussed above to obtain aglycosylated antibodies. Additionally, modifying the sequence of any established antibody can also pose regulatory issues, which is often the case when an approved and clinically validated antibody is used as an ADC conjugate. This is problematic because it is used as a starting point for Recently, Spycher et al. disclosed a transglutaminase-based conjugation approach that does not require prior deglycosylation of antibodies for payload conjugation (Spycher et al., WO2019 / 057772). The ability to conjugate natively glycosylated antibodies offers considerable advantages in manufacturing embodiments: the enzymatic deglycosylation step not only removes the cleaved glycans but also the deglycosylating enzyme (e.g., PNGase F). It is not desirable in good manufacturing practice (GMP) practice as it must be ensured that it is removed from the reaction mixture. Furthermore, it is not necessary to genetically engineer the antibody for attachment of the payload, thereby avoiding the insertion of sequences that could increase immunogenicity and reduce the overall stability of the antibody. obtain. [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 to solve the problem]

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

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

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

[0011] 1. A method for producing antibody-linker conjugates by microbial transglutaminase (MTG), the structure (shown in the N→C direction) (Sp 1 )-RK-(Sp 2 )-B-(Sp 3 ) or (Sp 1 )-B-(Sp 2 )-RK-(Sp 3 ) conjugating a linker containing Gln to a Gln residue contained in the antibody, wherein - (Sp 1 ) is a chemical spacer or is absent; - (Sp 2 ) is a chemical spacer or is absent; - (Sp 3 ) is a chemical spacer or is absent; - R is arginine or an arginine derivative or an arginine mimetic; - K is lysine or a ricin derivative or a ricin mimetic; - B is the concatenation part or payload; and the linker is conjugated to a Gln residue included in the antibody via a primary amine included in the side chain of the lysine residue, ricin derivative, or ricin mimetic.

[0012] 2. Chemical spacer (Sp 1 ), (Sp 2 ) and (Sp 3 ) each independently comprises 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] Four. 4. The method of any one of embodiments 1-3, wherein the net charge of the linker is neutral or positive.

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

[0016] 6. 6. The method according to 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 according to 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 according to 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 according to any one of embodiments 1-8, wherein B is a linking moiety.

[0020] Ten. The connecting part B is - bioorthogonal marker group, or - Non-bioorthogonal entities for cross-linking The method of embodiment 9, comprising:

[0021] 11. The bioorthogonal marker group or the non-bioorthogonal entity for cross-linking is - -N-N≡N, or -N 3 ; - Lys(N 3 ); - Tetrazine; - alkynes; - Distorted cyclooctyne; -BCN; - distorted alkenes; - photoreactive groups; - aldehydes; - Acyl trifluoroborate; - Proteolytic agents (“PROTACs”); - cyclopentadiene / spirolocyclopentadiene; - thioselective electrophile; - -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 as in any one of embodiments 9-11, comprising the further step of conjugating one or more payloads to linking moiety B.

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

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

[0025] 15. The payload is - toxin; - Cytokines; - growth factors; - radionuclides; - Hormones; - antiviral; - antibacterial agent; - Fluorescent dye: - Immunomodulators / immunostimulants; - half-life increasing part; - solubility increasing part; - Polymer-toxin conjugates; - Nucleic acids; - biotin or streptavidin moiety; - vitamins; - Proteolytic agents (“PROTACs”); - target binding moiety; and / or - Anti-inflammatory agent 15. The method of any one of embodiments 12-14, comprising at least one of:

[0026] 16. The toxin is - pyrrolobenzodiazepines (e.g. PBD); - Auristatin (e.g. MMAE, MMAF); - maytansinoids (e.g. maytansine, DM1, DM4, DM21); - duocarmycin; - Nicotinamide phosphoribosyltransferase (NAMPT) inhibitor; - Tubulisin; - Engine (e.g. Kalicare Sewing Machine); - anthracycline derivatives (PNU) (e.g. doxorubicin); - Kinesin spindle protein (KSP) inhibitors based on pyrrole; - cryptophycin; - drug efflux pump inhibitor; - Sandramycin; - amanitin (e.g. α-amanitin); and - camptothecin (e.g. exatecan, deruxtecan) 16. The method of embodiment 15, wherein the method is at least one selected from the group consisting of.

[0027] 17. Chemical spacer (Sp 2 ) comprises a self-destructive moiety.

[0028] 18. 18. The method of embodiment 17, wherein the self-destructive portion 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 according to any one of embodiments 1 to 19, wherein the antibody is an IgG antibody, in particular 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 contained in the Fc domain of the IgG antibody. 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. A Gln residue introduced into the heavy or light chain of an antibody by molecular engineering can be H 23. The method of embodiment 22, wherein the method is 2-domain N297Q (EU numbering).

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

[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. IgG antibodies are glycosylated IgG antibodies, especially IgG antibodies are C H The method according to any one of embodiments 20 to 22 or 24 to 25, wherein the 2 domain is glycosylated at residue N297 (EU numbering).

[0037] 27. 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 enfortuzumab. 27. The method as in any one of embodiments 1-26, wherein:

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

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

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

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

[0042] 32. 32. A method according to any one of embodiments 1 to 31, wherein the microbial transglutaminase is derived from a Streptomyces species, in particular Streptomyces mobaraensis.

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

[0044] 34. a) antibodies; and b) Structure: (Sp 1 )-RK-(Sp 2 )-B-(Sp 3 )or (Sp 1 )-B-(Sp 2 )-RK-(Sp 3 ) (In the formula, - (Sp 1 ) is a chemical spacer or is absent; - (Sp 2 ) is a chemical spacer or is absent; - (Sp 3 ) is a chemical spacer or is absent; - R is arginine or an arginine derivative or an arginine mimetic; - K is lysine or a ricin derivative or a ricin mimetic; - B is the concatenation part or payload) linker containing An antibody-linker conjugate comprising: A linker is formed between the γ-carboxamide group of the glutamine residue in the antibody and the primary amine contained in the side chain of the lysine residue, lysine derivative, or lysine mimetic contained in the RK motif contained in the linker. An antibody-linker conjugate that is conjugated to an antibody through an isopeptide bond.

[0045] 35. Chemical spacer (Sp 1 ), (Sp 2 ) and (Sp 3 ) each independently comprises 0 to 12 amino acid residues.

[0046] 36. The antibody-linker 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. Conjugate.

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

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

[0049] 39. Any of embodiments 34-38, 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). The antibody-linker conjugate described in one.

[0050] 40. The antibody according to 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). Linker conjugate.

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

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

[0053] 43. The connecting part B is - bioorthogonal marker group, or - Non-bioorthogonal entities for cross-linking 43. The antibody-linker conjugate of embodiment 42, comprising:

[0054] 44. The bioorthogonal marker group or the non-bioorthogonal entity for cross-linking is - -N-N≡N, or -N 3 ; - Lys(N 3 ); - Tetrazine; - alkynes; - Distorted cyclooctyne; -BCN; - distorted alkenes; - photoreactive groups; - aldehydes; - Acyl trifluoroborate; - Proteolytic agents (“PROTACs”); - cyclopentadiene / spirolocyclopentadiene; - thioselective electrophile; - -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 according to any one of embodiments 42-44, wherein one or more payloads are conjugated to linking moiety B.

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

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

[0058] 48. The payload is - toxin; - Cytokines; - growth factors; - radionuclides; - Hormones; - antiviral; - antibacterial agent; - Fluorescent dye: - Immune regulators / immunostimulants; - half-life increasing part; - solubility increasing part; - Polymer-toxin conjugates; - Nucleic acids; - biotin or streptavidin moiety; - vitamins; - Proteolytic agents (“PROTACs”); - target binding moiety; and / or - Anti-inflammatory agent 48. The antibody-linker conjugate of any one of embodiments 45-47, comprising at least one of:

[0059] 49. The toxin is - pyrrolobenzodiazepines (e.g. PBD); - Auristatin (e.g. MMAE, MMAF); - maytansinoids (e.g. maytansine, DM1, DM4, DM21); - duocarmycin; - Nicotinamide phosphoribosyltransferase (NAMPT) inhibitor; - Tubulisin; - Engine (e.g. Kalicare Sewing Machine); - anthracycline derivatives (PNU) (e.g. doxorubicin); - Kinesin spindle protein (KSP) inhibitors based on pyrrole; - cryptophycin; - drug efflux pump inhibitor; - Sandramycin; - amanitin (e.g. α-amanitin); and - camptothecin (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. Chemical spacer (Sp 2 ) 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. 53. An antibody-linker conjugate according to 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 contained in the Fc domain of the IgG antibody. H 54. The antibody-linker conjugate of embodiment 53, wherein Gln residue Q295 (EU numbering) of the 2 domain.

[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. A Gln residue introduced into the heavy or light chain of an antibody by molecular engineering can be H The antibody-linker conjugate according to embodiment 55, which is two domain N297Q (EU numbering).

[0067] 57. A Gln residue introduced into the heavy or light chain of an antibody by molecular engineering is either (a) incorporated into the heavy or light chain of the antibody, or (b) at the N- or C-terminus of the heavy or light chain of the antibody. An antibody-linker conjugate according to embodiment 55 comprised in a peptide fused to.

[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. IgG antibodies are glycosylated IgG antibodies, especially IgG antibodies are C H The antibody-linker conjugate according to any one of embodiments 53 to 55 or 57 to 58, which is glycosylated at residue N297 (EU numbering) of the 2 domain.

[0070] 60. 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 enfortuzumab. The antibody-linker conjugate according to any one of embodiments 34-59, wherein

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

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

[0073] 63. a) IgG antibodies; and b) a linker comprising a drug moiety B, wherein the drug moiety B is 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); a linker that is covalently linked to an amino acid sequence that An antibody-drug conjugate comprising: The linker is C of the antibody. H It is conjugated to an IgG antibody by an isopeptide bond formed between the γ-carboxamide group of the glutamine residue Q295 (EU numbering) in the 2 domain and the primary amine contained in the side chain of the lysine residue contained in the linker. Gated antibody-drug conjugate.

[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 included 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. IgG antibodies are glycosylated IgG antibodies, especially IgG antibodies are C H 66. The antibody-drug conjugate according to 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 according to any one of embodiments 63-66, wherein the IgG antibody is an IgG1 antibody.

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

[0079] 69. 68. The antibody-drug conjugate according to any one of embodiments 63-67, wherein the IgG antibody is trastuzumab or an antibody comprising a heavy chain as set forth in SEQ ID NO: 7 and a light chain as set forth in SEQ ID NO: 8.

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

[0081] 71. The drug is - pyrrolobenzodiazepines (e.g. PBD); - Auristatin (e.g. MMAE, MMAF); - maytansinoids (e.g. maytansine, DM1, DM4, DM21); - duocarmycin; - Nicotinamide phosphoribosyltransferase (NAMPT) inhibitor; - Tubulisin; - Engine (e.g. Karikea Sewing Machine); - anthracycline derivatives (PNU) (e.g. doxorubicin); - Kinesin spindle protein (KSP) inhibitors based on pyrrole; - cryptophycin; - drug efflux pump inhibitor; - Sandramycin; - amanitin (e.g. α-amanitin); and - camptothecin (e.g. exatecan, deruxtecan) The antibody-drug conjugate according to any one of embodiments 63-70, wherein the antibody-drug conjugate is a toxin selected from the group consisting of.

[0082] 72. Embodiments in which the linker has the structure RKAA-PABC-B, in particular where B is auristatin or maytansinoid, in particular auristatin is MMAE and the maytansinoid is DM1 or maytansine. An antibody-drug conjugate according to any one of 63 to 71.

[0083] 73. Embodiments in which the linker has the structure RKA-PABC-B, in particular where B is auristatin or a maytansinoid, in particular auristatin is MMAE and the maytansinoid is DM1 or maytansine. An antibody-drug conjugate according to any one of 63 to 71.

[0084] 74. Embodiments in which the linker has the structure ARK-PABC-B, in particular where B is auristatin or a maytansinoid, in particular the auristatin is MMAE and the maytansinoid is DM1 or maytansine. An antibody-drug conjugate according to any one of 63 to 71.

[0085] 75. Embodiments in which the linker has the structure RKR-PABC-B, in particular where B is auristatin or a maytansinoid, in particular auristatin is MMAE and the maytansinoid is DM1 or maytansine. An antibody-drug conjugate according to any one of 63 to 71.

[0086] 76. structure: (Sp 1 )-RK-(Sp 2 )-B-(Sp 3 )or (Sp 1 )-B-(Sp 2 )-RK-(Sp 3 ) (In the formula, - (Sp 1 ) is a chemical spacer or is absent; - (Sp 2 ) is a chemical spacer or is absent; - (Sp 3 ) is a chemical spacer or is absent; - R is arginine or an arginine derivative or an arginine mimetic; - K is lysine or a ricin derivative or a ricin mimetic; - B is the concatenation part or payload) A linker construct containing.

[0087] 77. Chemical spacer (Sp 1 ), (Sp 2 ) and (Sp 3 ) each independently comprises 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. The linker construct according to any one of embodiments 76-78, wherein the net charge of the linker is neutral or positive.

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

[0091] 81. The 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. A linker construct according to any one of embodiments 76-81, wherein B is a linking moiety.

[0093] 83. The connecting part B is - bioorthogonal marker group, or - Non-bioorthogonal entities for cross-linking 83. The linker construct of embodiment 82, comprising:

[0094] 84. The bioorthogonal marker group or the non-bioorthogonal entity for cross-linking is - -N-N≡N, or -N 3 ; - Lys(N 3 ); - Tetrazine; - alkynes; - distorted cyclooctyne; -BCN; - distorted alkenes; - photoreactive groups; - aldehydes; - Acyl trifluoroborate; - Proteolytic agents (“PROTACs”); - cyclopentadiene / spirolocyclopentadiene; - thioselective electrophile; - -SH; and - Cysteine 84. A linker construct according to embodiment 83, consisting of or comprising at least one molecule or moiety selected from the group consisting of:

[0095] 85. Structure RKAA-B (in particular, where B is Lys(N 3 ) or cysteine) according to any one of embodiments 76-84.

[0096] 86. Structure RKA-B (in particular, where B is Lys(N 3 ) or cysteine) according to any one of embodiments 76-84.

[0097] 87. Structure ARK-B (in particular, B is Lys(N 3 ) or cysteine) according to any one of embodiments 76-84.

[0098] 88. Structure B-RKR (in particular, where B is Lys(N 3 ) or cysteine) according to any one of embodiments 76-84.

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

[0100] 90. The payload is - toxin; - Cytokines; - growth factors; - radionuclides; - Hormones; - antiviral; - antibacterial agent; - Fluorescent dye: - Immune regulators / immunostimulants; - half-life increasing part; - solubility increasing part; - Polymer-toxin conjugates; - Nucleic acids; - biotin or streptavidin moiety; - vitamins; - Proteolytic agents (“PROTACs”); - target binding moiety; and / or - Anti-inflammatory agent 90. A linker construct according to embodiment 89, comprising at least one of:

[0101] 91. The toxin is - pyrrolobenzodiazepines (e.g. PBD); - Auristatin (e.g. MMAE, MMAF); - maytansinoids (e.g. maytansine, DM1, DM4, DM21); - duocarmycin; - Nicotinamide phosphoribosyltransferase (NAMPT) inhibitor; - Tubulisin; - Engine (e.g. Karikea Sewing Machine); - anthracycline derivatives (PNU) (e.g. doxorubicin); - Kinesin spindle protein (KSP) inhibitors based on pyrrole; - cryptophycin; - drug efflux pump inhibitor; - Sandramycin; - amanitin (e.g. α-amanitin); and - camptothecin (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. Chemical spacer (Sp 2 92. The linker construct according to any one of embodiments 89-91, wherein ) comprises a self-immolative moiety.

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

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

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

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

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

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

[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 a 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 according to embodiment 65 or 66, wherein the antibody is polatuzumab or trastuzumab or enfortuzumab.

[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) Antibody drug-conjugate according to any one of embodiments 63 to 75. A pharmaceutical composition comprising at least one pharmaceutically acceptable ingredient.

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

[0114] 104. In particular, 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, for use in therapy and / or diagnosis. 63. The antibody-linker conjugate of any one of embodiments 33-62, comprising:

[0115] 105. In particular, neoplastic, neurological, autoimmune, inflammatory or infectious diseases. - Are you suffering from the disease? - are at risk of developing it and / or - have been diagnosed with it Embodiments 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, for use in the treatment of a patient. The antibody-linker conjugate according to any one of 33 to 62.

[0116] 106. Antibody-linker conjugate or antibody-drug conjugate 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 B-cell associated cancer. gate, antibody-drug conjugate or pharmaceutical composition.

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

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

[0119] 109. Embodiments wherein the antibody-linker conjugate or antibody-drug conjugate comprised in the pharmaceutical composition comprises trastuzumab and the neoplastic disease is HER2-positive cancer, particularly HER2-positive breast cancer, gastric cancer, ovarian cancer or lung cancer. Antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use as described in 105.

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

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

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

[0123] 113. In particular, the antibody-linker conjugate Neoplastic, neurological, autoimmune, inflammatory or infectious diseases - Are you suffering from the disease? - are at risk of developing it and / or - have been diagnosed with it 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, for the manufacture of a medicament for the treatment of a patient. , use of an antibody-linker conjugate according to any one of embodiments 33-62.

[0124] 114. A method of 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 an antibody-linker conjugate according to any one of embodiments 33 to 62. The method, wherein the 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] Accordingly, in one embodiment, the present invention provides a method for producing antibody-linker conjugates by microbial transglutaminase (MTG), the structure (shown in the N→C direction) (Sp 1 )-RK-(Sp 2 )-B-(Sp 3 ) or (Sp 1 )-B-(Sp 2 )-RK-(Sp 3 ) conjugating a linker containing Gln to a Gln residue contained in the antibody, wherein - (Sp 1 ) is a chemical spacer or is absent; - (Sp 2 ) is a chemical spacer or is absent; - (Sp 3 ) is a chemical spacer or is absent; - R is arginine or an arginine derivative or an arginine mimetic; - K is lysine or a ricin derivative or a ricin mimetic; - B is the concatenation part or payload; and wherein the linker is conjugated to a Gln residue included in the antibody via a primary amine included in the side chain of the lysine residue, ricin derivative, or ricin mimetic.

[0126] Thus, 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. In patent application WO2019 / 057772, it was demonstrated that peptide-based linkers can be efficiently conjugated to glutamine residues of glycosylated antibodies via lysine residues in the linker. However, here it was surprisingly shown that the extended motif RK led to further improved conjugation efficiency.

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

[0128] Within the scope of the present invention, the linker is defined by the structure (Sp 1 )-RK-(Sp 2 )-B-(Sp 3 ) or (Sp 1 )-B-(Sp 2 )-RK-(Sp 3 ), and the linker is preferably conjugated to a glutamine residue in the antibody by a primary amine contained in residue K contained 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 lysine derivative as 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 "ricin mimetic," as used herein, has a structure different from ricin, but has similar characteristics to ricin, thus significantly altering the function and / or structure of a peptide or protein. Refers to a compound that can be used to replace lysine in the peptide or protein without causing damage. In certain embodiments, the lysine mimetic may differ from lysine in the length or composition of the primary amine and the fatty chain attached to 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-homolysine.

[0130] In certain embodiments, residue K may be a lysine derivative. The term "ricin derivative" as used herein refers to a ricin or ricin mimetic in which one or more functional groups contained in the ricin or ricin mimetic have been modified or substituted. Within the scope of the present invention, it is preferred that the amino groups of the side chains of the lysine derivatives are not modified in such a way that they are available for conjugation to glutamine residues in proteins. In embodiments where the residue K is located at the C-terminal position of the linker, K may be a lysine derivative in which the α-carboxy group is 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. The term "arginine mimetic," as used herein, has a structure different from arginine, but has similar characteristics to arginine, thus significantly altering the function and / or structure of a peptide or protein. Refers to a compound that can be used to replace arginine in the peptide or protein without causing damage. Arginine mimetics may differ from arginine in the length or composition of the guanidino group and the fatty chain connected to the alpha carbon atom. Alternatively, or in addition, the arginine mimetic may be different from the arginine of the guanidino group itself. That is, the arginine mimetic may include a functional group with similar physiochemical properties to a guanidino group. In certain embodiments, the 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 an arginine mimetic in which the guanidino group is substituted or modified. In certain embodiments, the arginine derivative may be omega-methylarginine. In embodiments where the residue R is located at the N-terminal position of the linker, R may be an arginine derivative in which the α-amino group is modified or substituted. In certain embodiments, the alpha-amino group of the arginine mimetic may be acetylated.

[0134] It should be understood that it is preferred that the RK motif consists of the amino acids arginine and lysine. However, the arginine or lysine residues, or both, may be replaced by the mimetics or derivatives 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 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 defined by the structure (Sp 1 )-RK-(Sp2 )-B-(Sp 3 ) or (Sp 1 )-B-(Sp 2 )-RK-(Sp 3 ). That is, the linker may include one or more chemical spacers (Sp). The term "chemical spacer" as used herein refers to a chemical that is covalently attached to a chemical residue of a linker and / or inserted between two chemical residues of a linker. Describe the relevant parts.

[0136] In a particular embodiment, the invention provides a method according to the invention, comprising a chemical spacer (Sp 1 ), (Sp 2 ) and (Sp 3 ) relate to methods, each independently comprising from 0 to 12 amino acid residues.

[0137] That is, in certain embodiments, a chemical spacer (Sp 1 ), (Sp 2 ) and / or (Sp 3 ) may be present or absent. (Sp 1 ), (Sp 2 ) and / or (Sp 3 ) is present, in embodiments where (Sp 1 ), (Sp 2 ) and / or (Sp 3 ) may contain one or more amino acid residues. In such embodiments, (Sp 1 ), (Sp 2 ) and / or (Sp 3 ) may contain 0 to 12 amino acid residues. Chemical spacer (Sp 1 ), (Sp 2 ) and / or (Sp 3 It must be noted that ) may also include non-amino acid residues, which are disclosed in more detail below.

[0138] Chemical spacer (Sp 1 ), (Sp 2 ) and / or (Sp 3 ) 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. The α-amino acid residue can be used in its L- or D-form with a chemical spacer (Sp 1 ), (Sp 2 ) and / or (Sp 3 ). (Sp 1 ), (Sp 2 ) and / or (Sp 3 ) comprises a chiral β-, γ- or δ-amino acid, the chiral β-, γ- or δ-amino acid may be present in its S- or R-form. Thus, in its broadest sense, the term "amino acid residue" as used herein refers to the amino group (-NH 2 ) and any organic compound containing a carboxy group (-COOH). Thus, whenever "amino acid" or "amino acid residue" is referred to throughout this disclosure, it is to be understood that the term amino acid residue may also encompass amino acid mimetics or derivatives.

[0139] Furthermore, the term amino acid residue refers to the known set of amino acids that make up proteins, namely alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, It should be understood that non-canonical and unnatural amino acids are also included, but are not limited to proline, serine, threonine, tryptophan, tyrosine and valine. A "non-canonical amino acid" as used herein can be any amino acid that is not part of the set of amino acids that make up proteins, but that can be obtained from natural sources. However, it must be noted that some non-canonical amino acids may also be found in naturally occurring peptides and / or proteins.

[0140] "Unnatural amino acid" or "synthetic amino acid" as used herein means any molecule under the general definition of amino acid, i.e., any molecule that contains an amino group and a carboxy group but is not found in nature. It may be a molecule of Therefore, unnatural amino acids are preferably obtained by chemical synthesis. It should be understood that the distinction between non-canonical and unnatural amino acids may be unclear in some instances. For example, an amino acid defined as an unnatural amino acid may at a later point 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-methylamino 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 mimetics or derivatives (e.g. β-cyclopropylalanine, phenylglycine, dehydro-alanine, β- cyanoalanine, β-(3-pyridyl)-alanine, β-(1,2,4-triazol-1-yl)-alanine, β-(1-piperazinyl)-alanine), phenylalanine mimetics or derivatives (e.g. 4-iodophenylalanine, pentafluoro-phenylalanine, naphthyl-alanine, 4-aminophenylalanine), arginine mimetics or derivatives (e.g. β-ureidoalanine, ω-methylarginine), lysine mimetics or derivatives (e.g. (3- (3-methyl-3H-diazilin-3-yl)propamino)carbonyl-1-lysine, Nε,Nε,Nε-trimethyllysine), histidine mimetics or derivatives (e.g., 2,5-di-iodohistidine, 1- methylhistidine), tyrosine mimetics or derivatives (e.g. 3-aminotyrosine, thyronine, 3,5-dinitrotyrosine, 3-hydroxy-methyl-tyrosine, O-phospho-L-tyrosine), tryptophan mimetics or derivatives (e.g. , 5-hydroxy-tryptophan, 1-methyltryptophan), serine mimetics or derivatives (e.g. β-(2-thienyl)-serine, β-(3,4-dihydroxyphenyl)-serine, O-phosphoserine), threonine mimetics or derivatives (e.g. allo-threonine, O-phosphothreonine), proline mimetics or derivatives (e.g. hydroxyproline, 3,4-dehydro-proline, pyroglutamic acid, thiaproline, cis-octahydroindole-2-carvone) acid), leucine and isoleucine mimetics or derivatives (e.g. allo-isoleucine, norleucine, 4,5-dehydroleucine, (4S)-4-hydroxy-L-isoleucine), valine mimetics or derivatives (e.g. norvaline, γ -hydroxyvaline), citrulline mimetics or derivatives (e.g. thiocitrulline, homocitrulline), cysteine ​​mimetics or derivatives (e.g. penicillamine, selenocysteine, buthionine-sulfoximine), methionine mimetics or derivatives (e.g. S-methylmethionine) , L-methionine sulfone, L-methionine sulfoxide, L-methionine sulfo-ximine, selenomethionine), aspartic acid mimetics or derivatives (e.g. DL-threo-β-hydroxyaspartic acid, L-aspartic acid β-methyl ester) , glutamic acid mimetics or derivatives (e.g. γ-methyleneglutamic acid, γ-carboxyglutamic acid, γ-hydroxyglutamic acid, L-glutamic acid 5-methyl ester, L-2-aminoheptanedioic acid), asparagine mimetics or derivatives (e.g. L-threo-3-hydroxyasparagine, N,N-dimethyl-L-asparagine, L-2-amino-2-carboxyethanesulfonamide, 5-diazo-4-oxo-L-norvaline), 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 -carboxylic acid, azetidine-2-carboxylic acid, pipecolic acid, 1-aminocyclopentanecarboxylic acid, spinacin), or amino acids containing bioorthogonal moieties (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-tetrazine-3) -yl)phenyl)propanoic acid).

[0142] In addition to the above alpha-amino acids, chemical spacers (Sp 1 ), (Sp 2 ) and / or (Sp 3) may contain one or more β-, γ-, δ- or ε-amino acids. Thus, in certain embodiments, the linker may be a peptidomimetic. A peptidomimetic may not exclusively contain a classical peptide bond formed between two alpha-amino acids, but may additionally or alternatively contain an alpha-amino acid and a beta-, gamma-, delta- or epsilon-amino acid. or between two β-, γ-, δ- or ε-amino acids, respectively. Thus, in any instance of the invention where a linker is described as a peptide, the linker may be a peptidomimetic and thus may not consist exclusively of α-amino acids and instead be not classified as an amino acid. It is to be understood that it may contain one or more β-, γ-, δ- or ε-amino acids or molecules. 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- Includes 5-phenylpentanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, 6-aminohexanoic acid and statins.

[0143] In addition, chemical spacers (Sp 1 ), (Sp 2 ) and / or (Sp 3 ) may include amino acid derivatives and / or amino acid mimetics. (Sp 1 ), (Sp 2 ) and / or (Sp 3 In embodiments where ) comprises one or more amino acid derivatives, it is preferred that the amino acid derivatives have a free amino group and a carboxy group so that they can undergo the formation of peptide or isopeptide bonds. (Sp 1 ), (Sp 2 ) and / or (Sp 3 In embodiments where ) comprises one or more amino acid mimetics, the amino acid mimetics may have free amino groups and carboxy groups such that they can undergo peptide or isopeptide bond formation. However, in certain embodiments, amino acid mimetics or derivatives may have substituted amino groups that do not interfere with peptide bond formation. An example of such an amino acid mimetic or derivative may be an N-methylated amino acid, such as sarcosine or N-Me-leucine.

[0144] (Sp 1 ) or (Sp 3 In embodiments where the amino acid residue included in ) is a terminal amino acid residue, the terminal amino acid residue may include a modified, protected or substituted N-terminal amino group or C-terminal carboxy group.

[0145] Furthermore, 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. good. Additionally, amino acid mimetics replace the amino and / or carboxy groups of standard amino acids, in which the amino acid mimetic undergoes the formation of alternative bonds with adjacent amino acids, amino acid derivatives and / or amino acid mimetics, and peptidomimetics. It may also contain other functional groups that allow it to form a body.

[0146] The term "amino acid mimetic," as used herein, refers to the purpose of having a structure different from a particular amino acid, but functioning similarly to said particular amino acid, and thus replacing said particular amino acid. Refers to compounds that can be used for An amino acid mimetic is said to function similarly to a particular amino acid if it fulfills, at least to some extent, similar structural and / or functional characteristics as 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 includes one or more terminal amino acid residues, the terminal amino acid residues may be protected. For example, (Sp 1 ) comprises an N-terminal amino acid residue, the N-terminal amino group may be protected. For example, in certain embodiments, a spacer (Sp 1 ) may be acetylated. In other embodiments, the R residue included 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, eg, by acetylation. In certain embodiments, linking moiety B or payload B may be or based on amino acids. In such embodiments, the amino acid-based payload or N-terminal amino group of linking moiety B may be protected, for example by acetylation.

[0148] Similarly, (Sp 3 ) comprises a C-terminal amino acid residue, the C-terminal carboxy group may be protected. For example, in certain embodiments, a spacer (Sp 3 ) may be amidated. In other embodiments, the K residue included 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, for example by amidation. In certain embodiments, linking moiety B or payload B may be or based on amino acids. In such embodiments, the amino acid-based payload or the C-terminal carboxy group of linking moiety B may be protected, for example by amidation.

[0149] In certain embodiments, a chemical spacer (Sp 1 ), (Sp 2 ) and / or (Sp 3 ) may contain 0 to 12 amino acid residues, including amino acid derivatives and amino acid mimetics. That is, in certain embodiments, (Sp 1 ) may contain 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues, (Sp 2 ) may contain 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues, (Sp 3 ) 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 provides a method according to the invention, wherein the linkers are 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or less. comprising the amino acid residue of.

[0151] That is, in certain embodiments, the linkers include amino acid mimetics and amino acid derivatives. It may contain 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid residues. Amino acid residues included in linkers, including amino acid mimetics and amino acid derivatives, can be linked to RK motifs, chemical spacers (Sp 1 ), (Sp 2 ) and / or (Sp 3 ), as well as, in certain embodiments, preferred amino acid residues included in B when B is an amino acid-based linking moiety or payload. In embodiments where the linker includes only two amino acid residues, the two amino acid residues are included in the RK motif. In such embodiments, (Sp 1 ), (Sp 2 ) and / or (Sp 3 ) is absent or does not include any amino acids, amino acid mimetics or amino acid derivatives.

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

[0153] In a particular embodiment, the invention relates to a method according to the 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). i.e. chemical spacer (Sp 1 ), (Sp 2 ) and / or (Sp 3), if present, is exclusive from amino acids, amino acid mimetics or amino acid derivatives. The net charge of a peptide is usually calculated at neutral pH (7.0). In the simplest approach, the net charge is calculated 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); Determined by calculating the difference between two groups. If the linker comprises a non-canonical amino acid or amino acid derivative in which a charged functional group has been modified or substituted, one skilled in the art would know how to determine the charge of the non-canonical amino acid or amino acid derivative at neutral pH. .

[0155] In certain embodiments, (Sp 1 ), (Sp 2 ) and / or (Sp 3 ) 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 a linker is calculated solely based on the amino acid residues included in the linker, including amino acid mimetics and amino acid derivatives. Thus, in a particular embodiment, the invention relates to a method according to the invention, wherein the net charge of the amino acid residues comprised in the linker is neutral or positive.

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

[0158] That is, the linker may be free of negatively charged amino acid residues, including amino acid mimetics and amino acid derivatives. A negatively charged amino acid residue is an amino acid, amino acid mimetic, or amino acid derivative that has a negative charge at neutral pH (7.0). The 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 a particular embodiment, the invention relates to a method according to the invention, wherein the linker comprises at least one positively charged amino acid residue outside the RK motif. That is, (Sp 1 ), (Sp 2 ) and / or (Sp 3 ) contains at least one positively charged amino acid. In certain embodiments, (Sp 1 ), (Sp 2 ) and / or (Sp 3 ) contains at least one histidine residue.

[0160] In addition to or in place of amino acid residues, including amino acid mimetics and derivatives, chemical spacers (Sp 1 ), (Sp 2 ) and / or (Sp 3 ) may contain or consist of non-amino acid moieties.

[0161] That is, in certain embodiments, a chemical spacer (Sp 1 ), (Sp 2 ) and / or (Sp 3 ) may not consist exclusively of amino acids, amino acid mimetics or amino acid derivatives. i.e. chemical spacer (Sp 1 ), (Sp 2 ) and / or (Sp 3 ) may include or be exclusive of non-amino acid components. In certain embodiments, a chemical spacer (Sp 1 ), (Sp 2 ) and / or (Sp 3 ) may include amino acids and non-amino acid components.

[0162] For example, but not limited to, chemical spacers (Sp 1 ), (Sp 2 ) and / or (Sp 3 ), each of which contains a framework containing 1 to 200 carbon atoms, optionally at least 10 atoms, such as 10 to 100 atoms or 20 to 100 atoms, substituted with one or more atoms Optionally, the carbon-containing framework may be a linear hydrocarbon or a cyclic group, a symmetrically or asymmetrically branched hydrocarbon, a monosaccharide, a disaccharide. , linear or branched oligosaccharides (asymmetrically branched or symmetrically branched), other natural linear or branched oligomers (asymmetrically branched or symmetrically branched), or more generally , any dimer, trimer, or higher order oligomer (linear, asymmetrically branched or symmetrically branched) resulting from any chain growth or step growth polymerization process.

[0163] (Sp 1 ), (Sp 2 ) and / or (Sp 3 ) is any linear, branched and / or cyclic C 2~30 Alkyl, C 2~30 alkenyl, C 2~30 Alkynyl, C 2~30 heteroalkyl, C 2~30 heteroalkenyl, C 2~30 heteroalkynyl; especially any linear or branched C in which x1 and x2 are independently integers selected from the range 0 to 20 2~5 Alkyl, C 5~10 Alkyl, C 11~20 Alkyl, -O-C 1~5 Alkyl, -O-C 5~10 Alkyl, -O-C 11~20 Alkyl, or (CH 2 -CH 2 -O-) 1~24 or (CH 2 ) x1 -(CH 2 -O-CH 2 ) 1~24 -(CH 2 ) x2 It may be a group, an amino acid, an oligopeptide, a glycan, a sulfate, a phosphate, or a carboxylate. In some embodiments, (Sp 1 ), (Sp 2 ) and / or (Sp 3 ) is C 2~6 It may also contain an alkyl group.

[0164] In certain embodiments, a chemical spacer (Sp 1 ), (Sp 2 ) and / or (Sp 3 ) is 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) May include. Polyethylene glycol (PEG) is a polyether compound that has many applications from industrial production to medicine. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE) depending on its molecular weight. The structure of PEG is usually H-(O-CH 2 -CH 2 ) n Expressed as -OH. Those skilled in the art know how to functionalize condensation polymers so that they can be linked to amino acid residues or payloads.

[0165] We 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, linker ARK-PEG 2 -PABC-MMAE (Figure 14) and ARK-PEG 2 -(NH)-(CH 3 )-S-C4-Maytansine (Figure 15) was conjugated to glycosylated polatuzumab with 92 and 90% efficiency (compared to 94% for ARK-PABC-MMAE), respectively. Another example is the linker ARK-PEG 2 -PABC-MMAE (Figure 14) and ARK-PEG 2 -(NH)-(CH 3 )-S-C4-Maytansine (Figure 15) was conjugated to glycosylated trastuzumab with 99% efficiency (compared to 100% for ARK-PABC-MMAE).

[0166] Thus, in a particular embodiment, the invention relates to a method according to the invention, wherein the linker comprises one or more PEG moieties. In certain embodiments, the PEG moiety is surrounded by a chemical spacer (Sp 1 ), (Sp 2 ) and / or (Sp 3 ) may be included. In certain embodiments, each PEG moiety included in the linker comprises 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. It may also contain monomers. In certain embodiments, the PEG moiety is such that it connects to the RK motif or directly to the payload (Sp 2 )include. In certain embodiments, the PEG moiety is (Sp2 ) to the linking moiety or payload to the amino acid residues contained in (Sp 2 )include. In certain embodiments, the PEG moiety connects the RK motif to the self-destructive moiety, which in turn connects the payload (Sp 2 )include. In certain embodiments, the PEG moiety is (Sp 2 ) is connected to the self-destructive moiety, which in turn is connected to the payload (Sp 2 )include.

[0167] In certain embodiments, a chemical spacer (Sp 1 ), (Sp 2 ) and / or (Sp 3 ) may contain dextran. The term "dextran" as used herein refers to a complex branched glucan composed of chains of varying lengths that can have weights ranging from 3 to 2000 kDa. Straight chains typically consist of alpha-1,6 glycosidic linkages between glucose molecules, whereas branches begin with alpha-1,3 linkages. Dextran can be synthesized from sucrose by, for example, lactic acid bacteria. In the context of the present invention, the dextrans used as carriers may preferably have a molecular weight of about 15 to 1500 kDa.

[0168] In certain embodiments, a chemical spacer (Sp 1 ), (Sp 2 ) and / or (Sp 3 ) may include oligonucleotides. The term "oligonucleotide" as used herein refers to non-naturally occurring oligonucleotides as well as oligomers or polymers of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). Preferably, the oligonucleotide is a polymer of DNA because of its greater stability.

[0169] In certain embodiments, a chemical spacer (Sp 1 ), (Sp 2 ) and / or (Sp 3 ), when present, is exclusive from amino acid residues, including amino acid mimetics and derivatives, and PEG moieties. In certain embodiments, a chemical spacer (Sp 1 ), (Sp 2 ) and / or (Sp 3 ), when present, is exclusive of amino acid residues, including amino acid mimetics and derivatives. In certain embodiments, a chemical spacer (Sp 1 ), (Sp 2 ) and / or (Sp 3 ) All amino acid residues contained in ) are α-L-amino acids. That is, in certain embodiments, the payload or linker, excluding linking moiety B, consists exclusively of amino acid residues. In certain embodiments, the payload or linker excluding linking moiety B consists exclusively of α-L-amino acid residues. Such peptide-based linkers may include protecting groups at the N- and / or C-terminus. That is, the N-terminal amino group may be acetylated and / or the C-terminal carboxy group may be amidated.

[0170] Chemical spacer (Sp 1 ), (Sp 2 ) and / or (Sp 3 ) may have the same structure. However, chemical spacers (Sp 1 ), (Sp 2 ) and / or (Sp 3 ) have different structures and / or chemical spacers (Sp 1 ), (Sp 2 ) and / or (Sp 3 ) are preferably not all present at the same time. That is, in certain embodiments, a chemical spacer (Sp 1 ), (Sp 2 ) and / or (Sp 3 ) 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] Thus, in a particular embodiment, the invention provides a method according to the invention, wherein the linker comprises the amino acid sequences RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 3), 4) or RK-Val-Cit (SEQ ID NO: 54). It should be understood that the RK motif used to conjugate the linker to the glutamine residue of the antibody may be comprised of the amino acid sequence RKAA, RKA, ARK, RKR or RK-Val-Cit.

[0173] In a particular embodiment, the 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 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 invention relates to a method according to the invention, wherein the linker comprises the amino acid sequence RK-Val-Cit (SEQ ID NO: 54).

[0176] Within the scope of the invention, it is preferred that the linker is conjugated to the antibody via a primary amine contained in the side chain of residue K contained in the RK motif. Therefore, the chemical spacer (Sp 1 ), (Sp 2 ) and / or (Sp 3 ) preferably does not contain additional lysine residues, lysine mimetics or lysine derivatives that may serve as further amine donors in transglutaminase-based conjugation reactions. In other embodiments, any free N-terminal amino group included in the linker may be substituted, eg, acetylated, such that it cannot serve as a substrate for a microbial transglutaminase.

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

table 1

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

[0179] "Linking moiety" as used herein generally refers to a molecule that is at least bifunctional. Within the scope of the invention, the linking moiety may be used to attach a first functional group and a further molecule to the linker, before or after the linker is conjugated to the antibody. Contains a second functional group that can. In certain embodiments, the linking moiety of the invention is an amino acid, an amino acid mimetic, or an amino acid derivative. In such embodiments, the linking moiety is preferably connected to the linker via its amino group, while the functional group contained in the amino acid side chain is used to attach further molecules to the linker. be able to. Alternatively, the linking moiety may be connected to the linker via its carboxy group, while the functional group contained in the amino acid side chain can be used to attach further molecules to the linker.

[0180] In a particular embodiment, the invention provides a method according to the invention, wherein the connecting moiety B is - bioorthogonal marker group, or - Non-bioorthogonal entities for cross-linking Relating to a method, including.

[0181] The term "bioorthogonal marker group" is used to refer to a reactive group that allows a chemical reaction to occur inside a biological system without interfering with native biochemical processes.Sletten and Bertozzi (A Bioorthogonal Quadricyclane Ligation) J Am Chem Soc 2011, 133 (44), 17570-17573). A "non-bioorthogonal entity for cross-linking" may be any molecule comprising or consisting of a first functional group, where the first functional group is a compatible second functional group. Payloads containing functional groups can be chemically or enzymatically crosslinked. Even if the cross-linking reaction is a non-bioorthogonal reaction, it is preferred that the reaction does not introduce any further modifications to the antibody other than cross-linking the payload to the linker. In view of the above, the linking moiety B may consist of a "bioorthogonal marker group" or a "non-bioorthogonal entity" or a "bioorthogonal marker group" or a "non-bioorthogonal entity". It may also include "substance". For example, the connected part Lys(N 3 ), then Lys(N 3 ) and the azide group alone may be considered bioorthogonal marker groups within the scope of the present invention. Lys(N 3 ) is K(N 3 ) refers to 6-azido-L-lysine, which may be abbreviated as 6-azido-L-lysine.

[0182] In a particular embodiment, the invention provides a method according to the invention, wherein the bioorthogonal marker group or the non-bioorthogonal entity for crosslinking comprises: - -N-N≡N, or -N 3 ; - Lys(N 3 ); - Tetrazine; - alkynes; - Distorted cyclooctyne; -BCN; - distorted alkenes; - photoreactive groups; - aldehydes; - Acyl trifluoroborate; - Proteolytic agents (“PROTACs”); - cyclopentadiene / spirolocyclopentadiene; - thioselective electrophile; - -SH; and - Cysteine comprising or comprising at least one molecule or moiety selected from the group consisting of:

[0183] A bioorthogonal marker group included in the linker or a non-bioorthogonal entity for crosslinking can intervene in any of the binding 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 cysteine, a cysteine ​​mimetic, or a cysteine ​​derivative with a free sulfhydryl group.

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

[0187] It should be noted that other thio-selective electrophiles such as 3-arylpropionitrile (APN) or phosphonamidates can also be used in place of maleimide in the method of the invention.

[0188] Providing a Cys residue in the linker according to the invention therefore allows ready-made toxin-maleimide constructs to be used to create antibody-payload conjugates, more generally Cys-maleimide It has the advantage that the advantages of bonding chemistry can be fully utilized. At the same time, ready-made antibodies that do not need to be deglycosylated can be used. In certain embodiments, the Cys residue can be at the C-terminus or intrachain of an amino acid-based linker.

[0189] In another embodiment, linking moiety B may include an azido group. A person skilled in the art will appreciate that molecules containing an azido group can be incorporated into the linker according to the invention, for example 6-azido-lysine (Lys(N 3 )) or 4-azido-homoalanine (Xaa(N 3 ))know. Linking moieties containing azide groups can be used as substrates in various bioorthogonal reactions, such as strain-promoted azide-alkyne cycloaddition (SPAAC), copper-catalyzed azide-alkyne cycloaddition (CuAAC) or Staudinger ligation. may be done. For example, in certain embodiments, a payload comprising a cyclooctyne derivative such as DBCO, DIBO, BCN or BARAC may be attached to a linker comprising an azide group by SPAAC.

[0190] In yet another embodiment, linking moiety B may include a tetrazine group. Those skilled in the art are aware of tetrazine-containing molecules, preferably amino acid derivatives containing a tetrazine group, which can be incorporated into linkers according to the invention. Linking moieties containing tetrazine can be used as substrates in bioorthogonal tetrazine ligations. For example, in certain embodiments, a payload that includes a cyclopropene, norbolene, norbolene derivative, or cyclooctyne group, such as bicyclo[6.1.0]nonine (BCN), can be attached to a linker that includes a tetrazine group.

[0191] In certain embodiments, linking moiety B may include 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 Amant et al., A Reactive Antibody Platform for One-Step Production of Antibody--Drug Conjugates through a Diels--Alder Reaction with Maleimide; Bioconjugate Chem. 2019, 30, 9, 2340-2348.

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

[0193] In a particular embodiment, the invention relates to a method according to the invention, comprising a further step of conjugating one or more payloads to a linking moiety B.

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

[0195] The term "payload" as used herein refers to a low molecular weight molecule or chemical entity that can be chemically synthesized, and that must be produced by fermentation of a host cell or that is chemically synthesized. and represents any naturally occurring or synthetically produced molecule, including larger molecules or biological entities that confer novel functions to antibodies. If the payload does not contain a linking moiety or other portion of the linker contained in the payload's linker, such as a chemical spacer (Sp 1 ) and / or (Sp 3 ) or may contain additional structures or functional groups that allow for attachment to the RK motif.

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

[0197] A number of bioorthogonal reactions that can be used to link the payload to the bioorthogonal marker group included in linking moiety B are known in the art. For example, between azide and cyclooctyne (also known as 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)), 1,3-dipole between nitrone and cyclooctyne (Ning et al ("Protein Modification by Strain-Promoted Alkyne-Nitrone Cycloaddition". Angewandte Chemie International Edition. 49 (17): 3065)) Cycloaddition, oxime / hydrazone formation from aldehydes and ketones (Yarema, et al ("Metabolic Delivery of Ketone Groups to Sialic Acid Residues. Application To Cell Surface Glycoform Engineering". Journal of Biological Chemistry. 273 (47): 31168- 79)), tetrazine ligation (Blackman et al ("The Tetrazine Ligation: Fast Bioconjugation based on Inverse-electron-demand Diels-Alder Reactivity". Journal of the American Chemical Society. 130 (41): 13518-9)), Isonitrile-based click reactions (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 ACS. Chem. Biol. 1: 644-648)) or strain-promoted alkyne-nitrone cycloadditions (SPANC), MacKenzie et al ("Strain-promoted cycloadditions involving nitrones and alkyne--rapid tunable reactions for bioorthogonal labeling". Curr Opin Several chemical ligation strategies have been developed that meet bioorthogonality requirements, including Chem Biol. 21: 81-8)). All of these documents are incorporated herein by reference to provide a full enabling disclosure and to avoid redundant repetition.

[0198] After the linker has been conjugated to the Gln residue of the antibody by a microbial transglutaminase, the payload is attached to a bioorthogonal marker group or a non-bioorthogonal entity for cross-linking included in the linker according to the invention. It should be understood that preferred. However, the present invention provides for coupling one or more payloads to a linker containing at least one linking moiety B in a first step, and converting the resulting linker-payload construct in a second step to an antibody by microbial transglutaminase. Conjugated antibody-linker conjugates are also included.

[0199] In a particular embodiment, the invention relates to a method according to the invention, wherein one or more payloads are conjugated to linking moiety B by a click reaction.

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

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

[0202] In another particularly preferred embodiment, at least one payload may be conjugated to the linking moiety B comprised in the linker by strain-promoted azide-alkyne cycloaddition (SPAAC). That is, in certain embodiments, the payload may include an alkyne group, such as, but not limited to, a cycloocytne group, and the linking moiety B may include a molecule containing an azide group, such as, but not limited to, a cycloocytne group. Lysine derivative Lys(N 3 ) may be used. However, B may also be a non-amino acid molecule containing a free azide group. In another embodiment, the payload may include an alkyne group, such as a cyclooctyne group, and linking moiety B may include an azido group.

[0203] In addition to a click reaction between the linking moiety of the linker and the payload functional group, 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 covalently linked to the linking moiety. However, in certain embodiments, the payload may be linked to the linking moiety by a strong non-covalent bond. That is, in certain embodiments, linking moiety B may include a biotin moiety, such as, but not limited to, the lysine derivative biocytin. In such embodiments, a payload containing a streptavidin moiety can be linked to a linker containing a biotin moiety.

[0205] In a particular embodiment, the 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. Preferably, the payload is attached to a chemical spacer included in a linker or directly to the RK motif. In embodiments where 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 the 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 an amino acid residue. Those skilled in the art will know how to functionalize the payload so that it can be attached to carboxy groups, amino groups or amino acid side chains.

[0207] Additionally, those skilled in the art know how to attach payloads to amino acid-based linkers by chemical synthesis. For example, an amine-containing payload, or a thiol-containing payload (e.g., for maytansine analogs), or a hydroxy-containing payload (e.g., for SN-38 analogs) is attached to the C-terminus of an amino acid-based linker by chemical synthesis. Good too. However, those skilled in the art are aware of further reactions and reactive groups that can be utilized to attach payloads to the N-terminus, C-terminus or side chains of amino acids or amino acid derivatives by chemical synthesis. Typical reactions that can be used to attach payloads to amino acid-based linkers by chemical synthesis include, but are not limited to, peptide bonds, activated ester bonds (NHS ester, PFP ester), click reactions (CuAAC, SPAAC) and Michael addition (thiolmaleimide conjugation). Conjugation of the payload to the peptide is performed according to the prior art, e.g. Costoplus et al. 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 TM antibody-drug conjugate (TDC). RSC Adv., 2017,7, 24828-24832), Doronina et al. (Enhanced activity of monomethylauristatin F through monoclonal antibody delivery: effects of linker technology on efficacy and toxicity. Bioconjug Chem. 2006 Jan -Feb;17(1):114-24), Nakada et al. (Novel antibody drug conjugates containing exatecan derivative-based cytotoxic payloads. Bioorg Med Chem Lett. 2016 Mar 15;26(6):1542-1545) and Dickgiesser (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 the C-terminus of a linker based on or comprising a peptide according to the invention. In certain embodiments, the payload can be attached directly to the N-terminal amino or C-terminal carboxy group of a peptide or amino acid residue (see, eg, Figure 22).

[0209] Those skilled in the art are aware of reactive groups that are suitable for attaching payloads to amino acid residues. For example, an amine-containing payload can be attached to the C-terminal carboxy group of an amino acid residue by an amide bond (Figure 22). Alternatively, a payload containing a thiol or hydroxy group can be attached to the C-terminal carboxy group of an amino acid by a thioester or ester bond, respectively. A payload containing a carboxylic acid group can be attached to the N-terminal amino group of an amino acid residue by an amide bond.

[0210] In certain embodiments, the payload may be indirectly attached to the N- or C-terminus of a peptide or amino acid residue comprised in a linker according to the invention. Those skilled in the art are aware of linker molecules that can be used to attach the payload to the N-terminal amino group or the C-terminal carboxy group of the amino acid residues comprised in the linker according to the 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 that includes a thiol group can be attached to the N-terminus of an amino acid residue by a linker molecule. For example, a payload containing a thiol group can be attached to the N-terminal amino group by a thiocarbamate linker molecule (Figure 28). Alternatively, a payload containing a thiol group can be attached to the N-terminal amino group by an alkyl linker molecule containing a carboxy group and a thiol group. In certain embodiments, the alkyl linker molecule may be a 3-mercaptopropionic acid linker molecule, and the payload forms a di-sulfur bond with a thiol group contained in the 3-mercaptopropionic acid linker molecule (Figure 29 ).

[0213] In certain embodiments, a payload containing an amide group can be attached to the N-terminus of an amino acid residue by a linker molecule. For example, a payload containing an amine group can be attached to an N-terminal amino group by a dicarboxylic acid linker molecule, where the dicarboxylic acid linker forms an amide bond with the payload and the amino group of the N-terminal amino acid residue. Examples of dicarboxylic acids that can be used as linker molecules in the present invention are, but are not limited to, succinic acid or pimelic acid (see Figures 9 and 30).

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

[0215] In a particular embodiment, the invention provides a method according to the invention, wherein the payload comprises: - toxin; - Cytokines; - growth factors; - radionuclides; - Hormones; - antiviral; - antibacterial agent; - Fluorescent dye: - Immunomodulators / immunostimulants; - half-life increasing part; - solubility increasing part; - Polymer-toxin conjugates; - Nucleic acids; - biotin or streptavidin moiety; - vitamins; - Proteolytic agents (“PROTACs”); - target binding moiety; and / or - Anti-inflammatory agent Relating to a method comprising at least one of:

[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 the two-step conjugation process disclosed herein. It can be attached to a linking moiety included in the antibody-linker conjugate produced as part of the process.

[0217] In certain embodiments, the payload may be a cytokine. The term "cytokine" as used herein refers to any secreted polypeptide that affects the function of other cells and modulates interactions between cells in an immune or inflammatory response. Cytokines include, but are not limited to, monokines, lymphokines, and chemokines, regardless of whether the cell produces them. For example, monokines are commonly said to be produced and secreted by monocytes, but are also produced by many other cells, such as natural killer cells, fibroblasts, basophils, neutrophils, Endothelial cells, brain astrocytes, bone marrow stromal cells, epidermal keratinocytes, and B lymphocytes 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). It will be done.

[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 to another therapeutic class that has a useful anti-inflammatory effect. It also means any other drug derived from it. Such anti-inflammatory agents include, but are not limited to, non-steroidal anti-inflammatory drugs (NSAIDs), disease-modifying anti-rheumatic drugs (DMARDs), macrolide antibiotics, and statins. Preferably, the NSAID includes, but is not limited to, salicylic acid (e.g. aspirin), arylpropionic acid (e.g. ibuprofen), anthranilic acid (e.g. mefenamic acid), pyrazole (e.g. phenylbutazone), cyclic acetic acid (e.g. indomethicin) and oxicams (eg, piroxicam). Preferably, anti-inflammatory agents for use in the method of the invention include sulindac, diclofenac, tenoxicam, ketorolac, naproxen, nabumetone, diflunasal, ketoprofen, arypropionic acids, tenidap, hydroxychloroquine, sulfasalazine. , Celecoxib, Rofecoxib, Meloxicam, Etoricoxib, Valdecoxib, Methotrexate, Etanercept, Infliximab, Adalimumab, Atorvastatin, Fluvastatin, Lovastatin, Pravastatin, Simvastatin, Clarithromycin, Azithromycin, Roxithromycin, Erythromycin, Ibuprofen, Dexibuprofen, Flur Includes biprofen, 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 attenuate 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 naturally occurring substances 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 in 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 include basic fibroblast growth factor, adrenomedullin, angiopoietin, autocrine cell motility stimulating factor, bone morphogenetic protein, brain-derived neurotrophic factor, epidermal growth factor, Endothelial growth factor, fibroblast growth factor, glial cell lineage-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 substance released by cells or glands in one part of the body that sends out messages that affect 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 norepinephrine. (NRE), dopamine (DPM or DA), anti-Müllerian hormone or Müllerian-inhibiting hormone (AMH), adiponectin (Acrp30), adrenocorticotropic hormone or corticotropin (ACTH), angiotensinogen and angiotensin (AGT), antidiuresis. hormones or vasopressin (ADH), 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, insulin (INS), insulin-like growth factor or somatomedin (IGF), leptin (LEP), progestin (LH), melanocyte-stimulating hormone (MSH or α-MSH), orexin, oxytocin (OXT) , parathyroid hormone (PTH), prolactin (PRL), relaxin (RLN), secretin (SCT), somatostatin (SRIF), thrombopoietin (TPO), thyroid-stimulating hormone or thyrotropin (TSH), thyrotropin-releasing hormone (TRH), cortisol , aldosterone, testosterone, dehydroepiandrosterone (DHEA), androstenedione, dihydrotestosterone (DHT), estrone, estriol (E3), progesterone, calcitriol, calcidiol, prostaglandin (PG), leukotriene (LT) , prostacyclin (PGI2), thromboxane (TXA2), prolactin-releasing hormone (PRH), lipotropin (PRH), brain natriuretic peptide (BNP), neuropeptide Y (NPY), histamine, endothelin, pancreatic polypeptide, renin, and It is 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) that is effective in inhibiting the formation and / or replication of viruses in mammals. This includes agents that interfere with host or viral mechanisms necessary for virus formation and / or replication in mammals. Examples of antiviral agents include ribavirin, amantadine, VX-497 (merimepodib, Vertex Pharmaceuticals), VX-498 (Vertex Pharmaceuticals), Levovirin, Viramidine, Ceplene (Maxamine), XTL-001 and XTL-002 (XTL Biopharmaceuticals) can be mentioned.

[0223] In certain embodiments, the payload may be an antibacterial agent. The term "antibacterial agent" as used herein: (i) inhibits, reduces or prevents the growth of bacteria; (ii) inhibits or reduces the ability of bacteria to produce an infection in a subject; or (iii) refers to any substance, compound, combination of substances, or combination of compounds capable of inhibiting or reducing the ability of bacteria to increase or persist in the environment. The term "antibacterial agent" also refers to compounds capable of reducing the infectivity or virulence of bacteria.

[0224] In certain embodiments, the payload may be an immunomodulatory agent. The term "immunomodulatory agent," as used herein for 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 (eg, antisense nucleic acid molecules, iRNA and triple helices), small molecules, organic compounds, and inorganic compounds. In particular, immunomodulators include, but are not limited to, methotrexate, leflunomide, cyclophosphamide, cytoxan, Immuran, cyclosporine A, minocycline, azathioprine, antibiotics (e.g., FK506 (tacrolimus)), methylprednisolone (MP). , corticosteroids, steroids, mycophenolate mofetil, rapamycin (sirolimus), mizoribine, deoxyspergualine, brequinal, malononitriloamindes (e.g., leflunamide), T cell receptor modulators , and cytokine receptor modulators.

[0225] In certain embodiments, an immunomodulatory agent may be an immunostimulatory agent. The term "immunostimulant" as used herein preferably refers to any substance or substance capable of eliciting an immune response (eg, an immune response to a particular pathogen). Compounds that activate immune cells include Toll-like receptor (TLR) agonists. Such agonists include pathogen-associated molecular patterns (PAMPs), e.g. compositions that mimic infection, such as bacterially derived immunomodulators (also known as danger signals) and damage-associated molecular patterns (DAMPs), e.g. , including compositions that mimic stressed or damaged cells. TLR agonists include nucleic acids or lipid compositions (eg, monophosphoryl lipid A (MPLA)). In one example, the TLR agonist is a TLR9 agonist, e.g., a cytosine-guanosine oligonucleotide (CpG-ODN), a poly(ethyleneimine) (PEI)-fused oligonucleotide (ODN), e.g., a PEI-CpG-ODN, or a double Contains chain deoxyribonucleic acid (DNA). In another example, the TLR agonist is a TLR3 agonist, e.g., polyinosine-polycytidic acid (poly(I:C)), PEI-poly(I:C), polyadenylic-polyuridylic acid (poly(A:U)), PEI -Contains poly(A:U), or double-stranded ribonucleic acid (RNA). Other exemplary vaccine immunostimulatory compounds include lipopolysaccharide (LPS), chemokines / cytokines, fungal beta-glucans (eg, 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. Half-life increasing moieties are, for example, PEG moieties (polyethylene glycol moieties; PEGylated), other polymeric moieties, PAS moieties (oligopeptides containing proline, alanine and serine; PASylated), or serum albumin binding agents. 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. Polymer-toxin conjugates are polymers that can have many payload molecules. Such conjugates are also called fleximers, which are marketed, for example, by Mersana therapeutics. The polymer-toxin conjugate may include any of the toxins disclosed herein.

[0228] In certain embodiments, the payload may be a nucleotide. An example of a nucleic acid payload is MCT-485, a very small non-coding double-stranded RNA with oncolytic and immune activating properties developed by MultiCell Technologies, Inc.

[0229] In certain embodiments, the payload may be a fluorescent dye. The term "fluorescent dye" as used herein refers to a dye that absorbs light at a first wavelength and emits at a second wavelength that is longer than the first wavelength. In certain embodiments, the fluorescent dye is a near-infrared fluorescent dye that emits light at a wavelength between 650 and 900 nm. In this region, there is less tissue autofluorescence and low fluorescence quenching enhances deep tissue penetration while minimizing background interference. Therefore, near-infrared fluorescence imaging can be used to intraoperatively visualize tissue to which the antibody-payload conjugates of the invention are bound. "Near-infrared fluorescent dyes" are known in the art and are 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 include a radionuclide. The term "radionuclide" as used herein refers to positively charged ions of radioactive metals such as, for example, Y, In, Tb, Ac, Cu, Lu, Tc, Re, Co, Fe, etc. medically useful radionuclides, including, e.g. 90 Y, 111 In, 67 Cu, 77 Lu, 99 Tc, 161 Tb, 225 Regarding Ac etc. The radionuclide may be included in a chelating agent such as DOTA or NODA-GA. Additionally, the radionuclide may be a therapeutic radionuclide or a radionuclide that can be used as a contrast agent in 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. The vitamin may be selected from the group consisting of folate, including folic acid, holacin, and vitamin B9.

[0232] In a particular embodiment, the invention provides a method according to the invention, wherein the toxin is - pyrrolobenzodiazepines (e.g. PBD); - Auristatin (e.g. MMAE, MMAF); - maytansinoids (e.g. maytansine, DM1, DM4, DM21); - duocarmycin; - Nicotinamide phosphoribosyltransferase (NAMPT) inhibitor; - Tubulisin; - Engine (e.g. Kalicare Sewing Machine); - anthracycline derivatives (PNU) (e.g. doxorubicin); - Kinesin spindle protein (KSP) inhibitors based on pyrrole; - cryptophycin; - drug efflux pump inhibitor; - Sandramycin; - amanitin (e.g. α-amanitin); and - camptothecin (e.g. exatecan, deruxtecan) at least one selected from the group consisting of.

[0233] That is, the antibody-linker conjugate produced using the methods of the invention preferably includes a toxin payload. The term "toxin" as used herein relates 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, necrotoxins, hematotoxins and cytotoxins. In certain embodiments, the toxin is a toxin used in the treatment of neoplastic diseases. That is, toxins can be conjugated to antibodies using the methods of the invention and delivered to or into malignant cells depending on the targeting specificity of the antibody.

[0234] In certain embodiments, the toxin may be 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 dolastatin.

[0235] In certain embodiments, the toxin may be a maytansinoid. In the context of the present invention, the term "maytansinoid" originally refers to a class of highly cytotoxic drugs isolated from the African shrub Maytenus ovatus and also to maytansinol and the natural maytansinol. C-3 ester of synthetic 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. Patent No. 5,416,064); 4,265,814; 4,308,268; 4,308,269; 4,309,428; 4,317,821; 4,322,348; and 4,331,598); Refers to esters (U.S. Pat. No. 4,137,230; U.S. Pat. No. 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 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 duocarmycin. Suitable duocarmycins are, for example, duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA, duocarmycin MA, and duocarmycin It may be CC-1065. The term "duocarmycin" should also be understood to refer to synthetic analogs of duocarmycin, such as adzelesin, vizeresin, calzelesin, 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 an inhibitor that reduces the activity of NAMPT. The term "NAMPT inhibitor" may include prodrugs of NAMPT inhibitors. Examples of NAMPT inhibitors include, but are not limited to, FK866 (also referred to as APO866), GPP 78 hydrochloride, ST 118804, STF31, pyridylcyanoguanidine (also referred to as CH-828), GMX-1778, and P7C3. can be mentioned. Additional NAMPT inhibitors are known in the art and may be suitable for use in the compositions and methods described herein. See, eg, PCT Publication WO2015 / 054060, US Pat. No. 8,211,912, and US Pat. No. 9,676,721, which are incorporated herein by reference in their entirety. In some embodiments, the NAMPT inhibitor is FK866. In some embodiments, the NAMPT inhibitor is GMX-1778.

[0238] In certain embodiments, the toxin may be tubulicin. Tubulysin is a cytotoxic peptide containing nine members (AI). Tubulysin A has potential use as an anticancer agent. Tubulysin A arrests cells in G2 / M phase. Tubrisin A inhibits polymerization more efficiently than vinblastine and induces depolymerization of isolated microtubules. Tubulysin A has a potent cytostatic effect on various tumor cell lines with an IC50 in the picomolar range. Another tubulycin that can be used in the methods of the invention may be tubulycin 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 9- and 10-membered rings containing two triple bonds separated by a double bond (e.g. See K. C. Nicolaou; A. L. Smith; E. W. Yue (1993). "Chemistry and biology of natural and designed enediynes". PNAS 90 (13): 5881-5888, the entire contents of which are incorporated herein by reference. ). Some enediynes can undergo Bergmann cyclization, and the resulting diradical, a 1,4-dehydrobenzene derivative, is capable of extracting hydrogen atoms from the sugar backbone of DNA, which leads to the formation of DNA chains. (e.g., S. Walker; R. Landovitz; W. D. Ding; G. A. Ellestad; D. Kahne (1992), the entire contents of which are incorporated herein by reference. T". Proc Natl Acad Sci U.S.A. 89 (10): 4608-12). Their reactivity with DNA gives many enediynes antibiotic characteristics, and some enediynes are being investigated clinically as anticancer antibiotics. Non-limiting examples of enediynes are dynemicin, neocarzinostatin, calicheamicin, esperamicin (e.g., Adrian L. Smith and K. C. Bicolaou, "The Enediyne Antibiotics", the entire contents of which are incorporated herein by reference) 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) ). In certain embodiments, the toxin may be 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 genus Streptomyces 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 compounds that inhibit kinesin spindle protein, which is involved in bipolar spindle assembly during cell division. Kinesin spindle protein inhibitors are being investigated for the treatment of cancer. An example of a kinesin spindle protein inhibitor includes ispinesib. Additionally, the term "kinesin spindle protein inhibitor" includes SB715992 or SB743921 from GlaxoSmithKline and pentamidine / chlorpromaline from CombinatoRx.

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

[0243] In certain embodiments, the toxin may be sandramycin. Sandramycin is a depsipeptide that was originally isolated from Nocardioides sp. (ATCC 39419) and has been shown to have cytotoxic and antitumor activity.

[0244] In certain embodiments, the toxin may be an amatoxin. Amatoxins (including alpha-amanitin, beta-amanitin and amanitin) are cyclic peptides composed of eight amino acids. These are isolated from the Amanita mushroom and can be prepared synthetically from binding blocks. Amatoxin specifically inhibits DNA-dependent RNA polymerase II in mammalian cells, thereby inhibiting transcription and protein biosynthesis in affected cells. Inhibition of transcription in cells arrests growth and proliferation. Although not covalently bound, the complex between amanitin and RNA-polymerase II is very tight (KD=3nM). Dissociating amanitin from the enzyme is a very slow process that reduces the chance of recovery for affected cells. If transcription is inhibited for too long within a cell, the cell undergoes programmed cell death (apoptosis). In a preferred embodiment, the term "amatoxin" as used herein refers to alpha -refers to amanitin or its variants.

[0245] In certain embodiments, the toxin may be camptothecin. The term "camptothecin" as used herein is intended to mean camptothecin or camptothecin derivatives that function as topoisomerase I inhibitors. Exemplary camptothecins include, for example, topotecan, exatecan, deruxtecan, irinotecan, DX-8951f, SN38, BN 80915, raltothecan, 9-nitrocamptothecin, and aminocamptothecin. A variety of camptothecins have been described, including camptothecins used to treat human cancer patients. Some camptothecins, for example, Kehrer et al., Anticancer Drugs, 12 (2) : 89-105, (2001) or Li et al., ACS Med. Chem. Lett. 2019, 10, 10, 1386-1392 )It is described in.

[0246] Toxins may also be inhibitors of drug efflux transporters within the meaning of the invention. An antibody-payload conjugate containing a toxin and an inhibitor of a drug efflux transporter has the advantage that, when translocated into a cell, an inhibitor of a drug efflux transporter prevents the toxin from exiting the cell. obtain. Within the scope of the 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, They include paroxetine, reserpine, saquinavir, sertraline, quinidine, tamoxifen, verapamil, and duloxetine. Elacridar and CP 100356 are other common P-gp inhibitors. Zosquidar and Tarikidar were also developed with this in mind. Finally, valspodar and reversan are other examples of such drugs.

[0247] It should be understood that the payload B as defined herein should not be understood exclusively as the actual payload itself, but rather as a payload molecule. The payload molecule, as used herein, may include additional structures that facilitate attachment of the payload to a 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 included in the payload molecule that is linked to the linker of the invention. The payload molecule has the structure: X-(spacer)-payload, (where payload represents the actual payload, e.g., one of the compounds disclosed herein, and Represents a reactive group that is suitable for attachment to a compatible functional group of the RK motif (one-step process), where (spacer) is a chemical that spatially separates the actual payload from the reactive group X. (representing a specific spacer). 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 include a peptide or an amino acid residue, and the reactive group X may be the amino group of the N-terminal amino acid residue included in the spacer. In other embodiments, no spacer may be present. In embodiments where no spacer is present, the functional group may be included in the actual payload. In certain embodiments, spacers can be used to attach functional groups of interest to the actual payload, ie, functional groups that are compatible with the functional groups included in the linking moiety. 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 a particular embodiment, the invention provides a method according to the invention, comprising a chemical spacer (Sp 2 ) relates to methods, including self-destructive parts.

[0250] That is, the linker may include a self-immolative moiety that facilitates release of the payload of the target cell or tissue. A self-immolative moiety can be included in any part of the linker. However, the self-immolative moiety is separated by a chemical spacer (Sp 2 ) is preferably included. Alternatively, the self-immolative moiety may be included (spacer) in the payload molecule as defined above.

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

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

[0253] More preferably, the self-destructive part is attached directly to the payload B, such that the payload is released upon disassembly of the self-destructive part. In certain embodiments, the self-immolative moiety is located between the payload and the RK motif included in the linker. That is, the self-destructive moiety may be attached to the N-terminus of residue R or the C-terminus of residue K. Alternatively, the self-destructive part can be combined with the payload and a chemical spacer (Sp 2 ), preferably at the N- or C-terminus of said amino acid residues. Additionally, the self-immolative portion can be separated from the payload by any method known in the art. 2 ) may be located between non-amino acid residues included in

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

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

[0256] That is, in certain embodiments, the linker may include the self-immolative moiety p-aminobenzylcarbamoyl (PABC). PABC contains a free amine group, which is suitable for attachment to an amino acid residue or the C-terminus of a peptide, and a carbamoyl group, by means of which a payload, particularly a payload containing an amine, can be attached. However, those skilled in the art know how to functionalize the payload so that it contains amine groups. Preferably, the self-immolative moiety PABC is located between the payload and the amino acid residues included in the linker. Amino acid residues are either residue K included in the RK motif or chemical spacers (Sp 2 ) is preferable. In certain embodiments, the self-immolative moiety PABC comprises a payload and a chemical spacer (Sp 2 ) is located between the alanine residues contained in In certain embodiments, the self-immolative moiety may be located between the payload and the peptidase cleavage site. In certain embodiments, the self-immolative moiety may be located between the payload and the cathepsin cleavage site. That is, the self-immolative moiety may be located between the payload and a motif known to be cleavable by cathepsins.

[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. include. 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. Additional motifs that can be specifically hydrolyzed by peptidases are described in Salomon et al., Optimizing Lysosomal Activation of Antibody-Drug Conjugates (ADCs) by Incorporation of Novel Cleavable Dipeptide Linkers, Mol Pharm. 2019, 16(12), p.4817 -4825.

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

[0259] Thus, in certain embodiments, the linker has the structure (Sp 1 )-RK-(Sp 2 )-Val-Cit-(self-destructive part)-may contain a payload. In certain embodiments, the linker has the structure (Sp 1 )-RK-(Sp 2 )-Val-Cit-payload. In certain embodiments, the linker has the structure (Sp 1 )-RK-(Sp 2 )-Val-Cit-PABC-payload.

[0260] In certain embodiments, the linker may include the structure RK-Val-Cit (SEQ ID NO: 54). In certain embodiments, the linker may include or consist of the structure RK-Val-Cit-(self-destructive moiety)-payload. In certain embodiments, the linker may include or consist of the structure RK-Val-Cit-PABC-payload. In certain embodiments, the linker may include 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] The peptide cleavage site is cleavable by other peptidases, such as caspase 3, legumain or neutrophil elastase, or Dal Corso et al., Innovative Linker Strategies for Tumor-Targeted Drug Conjugates; Chemistry;25(65) ; It should be noted that it may be the motif described on 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 has the structure (Sp 1 )-RK-(Sp 2 )-PABC-payload (wherein, (Sp 2 ) may be absent or consist of amino acid residues).

[0263] In certain embodiments, the linker has the structure (Sp 1 )-RK-(Sp 2 )-PABC-payload (wherein, (Sp 2 ) is the PABC part and (Sp 2 ) or a PEG moiety between the most C-terminal amino acid residues included in the RK motif.

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

[0265] It should be noted that the payload can be attached to the self-immolative PABC moiety by an additional linker molecule. For example, an amine-containing payload may be attached to the PABC moiety by a p-nitrophenol (PNP) group. Additional linker molecules that allow attachment of payloads containing reactive groups other than amines to the PABC moiety are described 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 may be attached to the PABC by an ethylenediamine (EDA) linker (see Figures 18 and 19).

[0266] In a particular embodiment, the invention relates to a method according to the invention, wherein the self-immolative moiety comprises a methylamine group. It has been previously 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, the self-destructive moiety containing a methylamine group may be attached to the C-terminus of the amino acid residue by an amide bond formed between the α-carboxy group of the amino acid residue and the amine contained in the methylamine group. good. Amino acid residues are (Sp 2 ) or the residue K included in the RK motif. The methyl group contained in the methylamine group may be bonded to the payload by an ether or thioether bond. Thus, methylamine groups may be preferably used as self-immolative groups when the payload contains hydroxy or thiol groups. In certain embodiments, the hydroxy-containing payload may be a camptothecin, eg, an anthracycline such as the exatecan derivative Dxd or PNU-159682. In certain embodiments, the thiol-containing payload may be a maytansinoid, such as DM1, DM4 or DM21.

[0268] Linkers containing methylamine groups have the molecular structure C-(NH)-(CH 3 )-O-C or C-(NH)-(CH 3 )-S-C. Exemplary linkers containing methylamine groups are shown in FIGS. 15, 17 and 21.

[0269] It should be appreciated that self-immolative moieties comprising PABC and methylamine groups are preferably used to attach the payload to the C-terminal carboxy group of the 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 p-aminobenzylethanol (PABE) for attaching a phenol-containing payload to the C-terminal carboxy group of an amino acid residue. ) linker (Zhang et al., Bioconjugate Chem. 2018, 29, 6, 1852-1858) or para-methyl to attach a payload containing a tertiary amine or heteroaryl moiety to the C-terminal carboxy group of an amino acid residue. Aniline (PMA) linker (Staben et al., Nature Chemistry volume 8, pages 1112-1119 (2016)) (see Figures 20 and 23, respectively). Non-limiting examples of payloads containing phenolic groups are duocarmycin GA or pyrrolobenzodiazepine PBD. A non-limiting example of a payload containing a tertiary amine is duocarmycin GA.

[0271] However, the payload may be attached to the N-terminal amino group by a self-immolative moiety. For example, the payload may be attached to the N-terminal amino group of an amino acid residue by 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 phenolic payload such as PBD to the N-terminal amino group of an amino acid residue (see Figure 27). Preferably, the OHPAS moiety contains 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 can be a functionalized PEG linker, such as, but not limited to, a functionalized (PEG) linker. 2 It may be attached to the N-terminal amino group of the amino acid residue by a linker. Preferably, the PEG linker is functionalized at one end with an amino group to allow attachment to the carboxy group contained in the OHPAS moiety, and functionalized at the other end with a carboxy group to allow attachment of the amino acid residue. Coupling to the N-terminal amino group is possible (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 OHPAS and the payload to allow attachment of a non-phenolic payload to OHPAS. For example, para-hydroxybenzyl (PHB) linker molecules may be used to enable attachment of payloads containing primary or secondary amines to OHPAS via carbamate formation (see Figures 31 and 32). I want to be). Payloads containing tertiary amines may be attached to OHPAS containing linkers via quaternary ammonium formation (see Figures 33 and 34). Additionally, para-hydroxybenzylethylenediamine (PHB-EDA) linker molecules can be used to attach hydroxy-containing payloads to OHPAS moieties via carbamate formation (Park et al., Bioconjugate Chem. 2019, 30, 7, 1957-1968) (see Figure 25).

[0273] In certain embodiments, the payload can be attached to amino acid residues included in the linker by a cleavable moiety. A "cleavable moiety" as used herein is a chemical unit that can be separated from the actual payload by enzymatic or non-enzymatic hydrolysis. In certain embodiments, the cleavable moiety may be an amino acid motif that is hydrolyzable by a peptidase or protease.

[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, a 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, a cleavable moiety may be a moiety that is cleavable by beta-lysosomal acid pyrophosphatase or acid phosphatase.

[0276] An example of further cleavable moieties that can be used to release payload from a linker molecule is Bargh et al., Cleavable linkers in antibody-drug conjugates; Chem Soc Rev. 2019 Aug 12;48(16):4361- 4374. In certain embodiments, the linker may include a structure (cleavable portion)-(self-destructive portion)-payload. In such embodiments, the self-destructive portion can disintegrate and release the payload upon cutting of the severable portion.

[0277] In a particular embodiment, the invention provides a method according to the invention, wherein the linker is , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34. Relating to any one method.

[0278] In certain embodiments, a linker may include two or more linking moieties and / or payload B. That is, in certain embodiments, the linker a)(Sp 1 )-RK-(Sp 2 )-B 1 -(Sp 3 )-B 2 -(Sp 4 ), b)(Sp 4 )-B 2 -(Sp 1 )-RK-(Sp 2 )-B 1 -(Sp 3 ), c)(Sp 1 )-B 1 -(Sp 2 )-RK-(Sp 3 )-B 2 -(Sp 4 ),or d)(Sp 4 )-B 2 -(Sp 1 )-B 1 -(Sp 2 )-RK-(Sp 3 ) may include.

[0279] In such embodiments, a chemical spacer (Sp 1 ), (Sp 2 ), (Sp 3 ) and RK motifs may have the same characteristics as defined above. Furthermore, part B 1 and B 2 may be any one of the linking moieties and / or payloads defined above. In addition, chemical spacers (Sp 4 ) is a chemical spacer (Sp 1 ), (Sp 2 ) or (Sp 3 ) or may be absent.

[0280] Thus, in a particular embodiment, the invention provides a method according to the invention, in which the linker comprises a second linking moiety or a payload B. 2 including, especially B 2 However, a chemical spacer (Sp 1 ) or (Sp 3 ) connected to the linker.

[0281] i.e. payload or concatenated part B 2 is a chemical spacer (Sp 1 ) or (Sp 3 ) or payload or connecting part B 1 may be directly connected to. Payload or connecting part B 2 is B 2 (Sp 1 ), (Sp 3 ) or B 1 may include any functional group that is suitable for bonding to a functional group contained in.

[0282] In certain embodiments, the payload or concatenated portion B 2 is B 2 is(Sp 3 ) or B 1 It may also contain an amino group connected to. That is, B 2 is due to the amino group (Sp 3 ) or B 1 may be connected to a carboxy group contained in In certain embodiments, (Sp 3 ) contains a chemical spacer (Sp 3 ) may be a carboxy group contained in the C-terminal amino acid residue. In certain embodiments, B 1 The carboxy group contained in may be an α-carboxy group of an amino acid-based payload or linking moiety. In certain embodiments, B 2 is formed by the linker molecule (Sp 3 ) or B 1 may be bonded to a carboxy group contained in In certain embodiments, the linker molecule may include a self-immolative moiety.

[0283] In certain embodiments, the payload or concatenated portion B 2 is B 2 is(Sp 1 ) or B 1 may contain a carboxy group connected to. That is, B 2 is due to the carboxy group (Sp 1 ) or B 1 may be connected to an amine group contained in In certain embodiments, (Sp 1 ) contains a chemical spacer (Sp 1 ) may be an amine group contained in the N-terminal amino acid residue. In certain embodiments, B 1 The amine group included in may be an α-amino group of an amino acid-based payload or linking moiety. In certain embodiments, B 2 is formed by the linker molecule (Sp 1 ) or B 1 may be bonded to an amine group contained in In certain embodiments, the linker molecule may include a self-immolative moiety.

[0284] However, B 2 It should be noted that may contain functional groups other than amine or carboxy groups. In such embodiments, B 2 (Sp 1 ), (Sp 3 ) or B 1 may be combined with

[0285] In certain embodiments, the payload or concatenated portion B 2 is (Sp 1 ) or (Sp 3 ) may be bonded to an amino acid side chain contained in That is, B 2 (Sp 1 ) or (Sp 3) may be connected to the functional group of the amino acid side chain contained in

[0286] In certain embodiments, (Sp 1 ), (Sp 2 ), (Sp 3 ) and RK motifs consist exclusively of amino acids, amino acid mimetics and / or amino acid derivatives. In certain embodiments, B 1 and / or B 2 also contains an amino acid skeleton. In such embodiments, the linker may be a linear peptide or peptidomimetic. B 1 In embodiments where is an amino acid, an amino acid mimetic or an amino acid derivative, the linker has the structure (Sp 1 )-RK-(Sp 2 )-B 1 (In the formula, (Sp 1 )-RK-(Sp 2 )-B 1 may have linear peptides or peptidomimetics). B 1 In embodiments where is an amino acid, an amino acid mimetic or an amino acid derivative, the linker has the structure (Sp 1 )-RK-(Sp 2 )-B 1 -(Sp 3 )(wherein, (Sp 1 )-RK-(Sp 2 )-B 1 -(Sp 3 ) may be a linear peptide or a peptidomimetic. B 1 In embodiments where is an amino acid, amino acid mimetic or amino acid derivative, the linker has the structure RK-(Sp 2 )-B 1 -(Sp 3 )(wherein, RK-(Sp 2 )-B 1 -(Sp 3 ) may be a linear peptide or a peptidomimetic. B 1 In embodiments where is an amino acid, amino acid mimetic or amino acid derivative, the linker has the structure RK-(Sp 2 )-B 1 (In the formula, RK-(Sp 2 )-B 1 may have linear peptides or peptidomimetics). B 1 In embodiments where is an amino acid, an amino acid mimetic or an amino acid derivative, the linker has the structure RK-B 1 -(Sp 3 )(In the formula, RK-B 1 -(Sp 3 ) may be a linear peptide or a peptidomimetic. B 1 In embodiments where is an amino acid, an amino acid mimetic or an amino acid derivative, the linker has the structure RK-B 1 (In the formula, RK-B 1 may have linear peptides or peptidomimetics).

[0287] B 1 and B 2 In embodiments where is an amino acid, an amino acid mimetic or an amino acid derivative, the linker has the structure (Sp 1 )-RK-(Sp 2 )-B 1 -(Sp 3 )-B 2 -(Sp 4 )(wherein, (Sp 1 )-RK-(Sp 2 )-B 1 -(Sp 3 )-B 2 -(Sp 4 ) may be a linear peptide or a peptidomimetic. B 1 and B 2 In other embodiments where is an amino acid, amino acid mimetic, or amino acid derivative, the linker has the structure (Sp 4 )-B 2 -(Sp 1 )-RK-(Sp 2 )-B 1 -(Sp 3 )(wherein, (Sp 4 )-B 2 -(Sp 1 )-RK-(Sp 2 )-B 1 -(Sp 3 ) may be a linear peptide or a peptidomimetic. B 1 and B 2 In other embodiments where is an amino acid, amino acid mimetic, or amino acid derivative, the linker has the structure (Sp 4 )-B 2 -(Sp 1 )-B 1 -(Sp 2 )-RK-(Sp 3 )(wherein, (Sp 4 )-B 2 -(Sp 1 )-B 1 -(Sp 2 )-RK-(Sp 3 ) may be a linear peptide or a peptidomimetic.

[0288] B 1 In embodiments where is not an amino acid, an amino acid mimetic, or an amino acid derivative, the linker has the structure (Sp 1 )-RK-(Sp 2 )-B 1 -(Sp 3 )(wherein, (Sp 1 )-RK-(Sp 2 ) is a linear peptide or peptidomimetic, and B 1 は(Sp 2 ) connected to the C-terminal carboxy group contained in ). B 1 In embodiments where is not an amino acid, an amino acid mimetic, or an amino acid derivative, the linker has the structure (Sp 1 )-B 1 -(Sp 2 )-RK-(Sp 3 )(wherein, (Sp 2 )-RK-(Sp 3 ) is a linear peptide or peptidomimetic, and B 1 は(Sp 2 ) connected to the N-terminal amino group contained in ). However, B 1 It should be noted that the peptide or peptidomimetic does not necessarily have to be directly linked to the peptide or peptidomimetic. Instead, B 1 may be attached to the peptide or peptidomimetic by a linker molecule and / or a self-immolative moiety.

[0289] B 1 is an amino acid, amino acid mimetic or amino acid derivative, and B 2 In embodiments where is not an amino acid, an amino acid mimetic, or an amino acid derivative, the linker has the structure (Sp 1 )-RK-(Sp 2 )-B 1 -(Sp 3 )-B 2 -(Sp 4 ), (Sp 4 )-B 2 -(Sp 1 )-RK-(Sp 2 )-B 1 -(Sp 3 ), (Sp 1 )-B 1 -(Sp 2 )-RK-(Sp 3 )-B 2 -(Sp 4 ) or (Sp 4 )-B 2 -(Sp 1 )-B 1 -(Sp 2 )-RK-(Sp 3 )(wherein, (Sp 1 )-RK-(Sp 2 )-B 1 -(Sp 3 ) or (Sp 1 )-B 1 -(Sp 2 )-RK-(Sp 3 ) is a linear peptide or peptidomimetic, and B 2 は(Sp 3 ), B 1 or the C-terminal carboxy group contained in RK or (Sp 1 ), B 1 or attached to the N-terminal amino group of RK).

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

[0291] In a particular embodiment, the invention provides a method according to the invention, comprising: 1 and B 2 are the same or different from each other.

[0292] i.e. payload or concatenated part B 1 and B 2 may be identical, ie, have the same chemical structure, or may be structurally different. In certain embodiments, B 1 and B 2 are both payloads or are concatenated parts. B 1 and B 2 are both payloads, payload B 1 and B 2 may be the same payload or different payloads. B 1 and B 2 In embodiments in which both are connecting portions, the connecting portion B1 and B 2 may be the same connecting portion or different connecting portions. In certain embodiments, B 1 may be a connected part, B 2 may be the payload and vice versa.

[0293] All payloads or concatenations are B 1 For example, this means that not all payloads or linking moieties can function as intrachain payloads or linking moieties on the one hand (Sp 2 ) or RK and on the other hand (Sp 3 ), (Sp 1 ) or B 2 It should be understood that this is because it does not necessarily have a functional group that forms a covalent bond with. Therefore, B 1 In embodiments where B is an intrachain payload or linking moiety, B 1 is preferably a divalent or polyvalent molecule. For example, B 1 may be an amino acid, an amino acid mimetic or an amino acid derivative. In such embodiments, B 1 is (Sp 2 ) or to the C-terminal carboxy group of RK and by its carboxy group (Sp 3 ) or B 2 can be bonded to the N-terminal amino group of Or B 1 is (Sp 2 ) or to the N-terminal amino group of RK and by its amino group (Sp 1 ) or B 2 can be bonded to the C-terminal carboxy group of

[0294] In certain embodiments, the linker connects two connecting moieties B 1 and B 2 May include.

[0295] Thus, in certain embodiments, the invention encompasses linkers that include two bioorthogonal marker groups and / or non-bioorthogonal entities. For example, a linker according to the invention may include an azide-containing linking moiety, such as Lys(N 3 ) or Xaa(N 3 ), and linking moieties containing sulfhydryls, such as cysteine. In certain embodiments, a linker according to the invention comprises an azide-containing linking moiety, for example Lys(N 3 ) or Xaa(N 3 ), and a linking moiety that includes a tetrazine, such as a tetrazine-modified amino acid. In certain embodiments, a linker according to the invention may include a linking moiety that includes a sulfhydryl, eg, cysteine, and a linking moiety that includes a tetrazine, eg, a tetrazine-modified amino acid. Linkers that include two different bioorthogonal marker groups and / or non-bioorthogonal entities mean that they accept two separate payloads, thus resulting in an antibody-payload conjugate containing more than one payload. has advantages.

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

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

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

[0299] Additionally, dual-labeled probes can be used for non-invasive imaging and therapy or intra-operative / post-operative imaging / surgery. In such embodiments, tumor patients may be selected by non-invasive imaging. The tumor can then be surgically removed using other imaging agents (e.g., fluorescent dyes) to help the surgeon or robot identify any cancerous tissue during surgery.

[0300] In certain embodiments, B 1 and B 2 One of B may be a linking moiety containing a thiol group, for example cysteine, and B 1 and B 2 The other is a linking moiety containing an azide moiety, e.g. Lys(N 3 ) may be used. 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 include two payloads. A linker containing only the payload but no linking moiety may be conjugated to the antibody in a one-step process.

[0302] B 1 and B 2 In embodiments where both are payloads, B 1 and B 2 It is to be understood that the two may be the same or different in structure. In certain embodiments, a linker containing one or more payloads may be chemically synthesized. Alternatively, one or more payloads may be attached to a linking moiety contained in a linker by any of the methods disclosed herein before the linker is conjugated to the antibody.

[0303] In certain embodiments, linkers of the invention link two different payloads to the antibody's C H 2 domain can be conjugated to residue Q295. The use of a second payload allows the development of an entirely new class of antibody-payload conjugates that exceed current therapeutic approaches in terms of efficacy and efficacy. Also, new fields of application are envisaged, e.g. dual-type imaging for imaging and therapy or intraoperative / postoperative surgery (Azhdarinia A. et al., Dual-Labeling Strategies for Nuclear and Fluorescence Molecular Imaging: A Review and Analysis. Mol Imaging Biol. 2012 Jun; 14(3): 261-276). For example, dual-labeled antibodies, including molecular imaging agents for preoperative positron emission tomography (PET) and near-infrared fluorescence (NIRF) dyes to guide the delineation of surgical margins, can be used to diagnose and stage cancer. , and can greatly enhance resection (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, allowing non-invasive whole-body imaging to localize disease and intraoperative tumor margin identification, respectively. However, to date the generation of such dual-labeled probes has been difficult due to the lack of suitable site-specific methods, and when attaching two different probes by chemical means, random conjugation of the probes has made it difficult to analyze and Reproducibility becomes almost impossible.

[0304] Furthermore, in the study of Levengood M. et al., (Orthogonal Cysteine ​​Protection Enables Homogeneous Multi-Drug Antibody-Drug Conjugates. Angewandte Chemie, Volume56, Issue3, January 16, 2017) Cell lines and xenograft models in which drug-labeled antibodies are resistant to ADCs composed of individual auristatin components (with different physiochemical properties and exerting complementary anticancer activities) imparted activity. This suggests that dual-labeled ADCs can address cancer heterogeneity and resistance more effectively than a single conventional ADC alone. Because one resistance mechanism to ADCs involves actively pumping the cytotoxic moiety out of cancer cells, another dual-drug application would be to use drugs that specifically block the efflux mechanisms of cytotoxic drugs. may include the addition and simultaneous delivery of. Thus, such dual-labeled ADCs can help overcome cancer resistance to ADCs more effectively than conventional ADCs.

[0305] The term "antibody" is used herein in its broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies), and antibody fragments of length that exhibit the desired biological activity. The term "antibody(s)" broadly encompasses naturally occurring forms of antibodies (eg, IgG, IgA, IgM, IgE).

[0306] Preferably, the antibody is a monoclonal antibody. Antibodies may be of human origin, but may equally be derived from mice, rats, goats, donkeys, hamsters, or rabbits. If the conjugate is for therapeutic use, the mouse or rabbit antibody may optionally be chimerized or humanized.

[0307] C H Fragments or recombinant variants of antibodies containing two domains can be, for example, - Just a heavy chain domain (shark antibody / IgNAR(V H -C H 1-C H 2-C H 3-C H 4-C H Five) 2 or camelized antibody / hcIgG(V H -C H 2-C H 3) 2 ) including antibody formats - scFv-Fc(VH-VL-CH2-CH3)2 - Fc fusion peptides containing an Fc domain and one or more receptor domains It may be.

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

[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 a recognized immunoglobulin gamma gene. In humans, IgG includes the subdivisions 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 two immunoglobulin chains, each pair having one light chain and one heavy chain, each light chain containing the immunoglobulin domains VL and CL, each Heavy chains include the immunoglobulin domains VH, Cγ1 (also called CH1), Cγ2 (also called CH2), and Oγ3 (also called CH3). In the context of human IgG1, according to Kabat's EU index, "CH1" refers to positions 118-215, CH2 domain refers to positions 231-340, and CH3 domain refers to positions 341-447. IgG1 also contains a hinge domain, which in the case of IgG1 points to positions 216-230.

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

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

[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, enfortuzumab, 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 enfortuzumab.

[0315] Thus, in certain embodiments, the invention relates to antibody-linker conjugates, where 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, where 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, where 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] Thus, in certain embodiments, the antibody may be an IgG antibody, preferably glycosylated at residue N297. Thus, in a particular embodiment, the invention provides a method according to the invention, wherein the IgG antibody is a glycosylated IgG antibody, in particular the IgG antibody is a C-glycosylated IgG antibody. H 2 domain is glycosylated at residue N297 (EU numbering).

[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 the glycan at residue N297 has been cleaved with the enzyme PNGase F. Furthermore, the antibody may be an aglycosylated antibody, preferably residue N297 is 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 are conjugated to endogenous Gln residues of the Fc domain of the antibody, or to Gln residues introduced into the antibody by molecular engineering. Good too.

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

[0324] A linker of the invention may be conjugated to any Gln residue of 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 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 invention H 2,C H 3, and if applicable, C H It may be conjugated to 4 domains.

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

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

[0327] Additionally, engineered conjugates using Q295 for payload attachment showed good pharmacokinetics and efficacy (Lhospice et al., Site-Specific Conjugation of Monomethyl Auristatin E to Anti-Cd30 Antibodies Improves Their Pharmacokinetics and Therapeutic Index in Rodent Models, Mol Pharm; 2015; 12(6), p.1863-1871), even unstable toxins that are easily degraded (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). Thus, similar effects are expected to be seen for this site-specific method since the same residues are modified, except for those of the glycosylated antibody. Glycosylation can further contribute to overall ADC stability, and removal of glycan moieties, similar to the mentioned approaches, has been shown to result in less stable antibodies (Zheng et al.; The impact of glycosylation on monoclonal antibody conformation and stability. Mabs-Austin; 2011, 3(6), p.568-576).

[0328] C of linker by transglutaminase H 2 The literature discussing conjugation to Gln residues focuses on small, low molecular weight substrates. However, in the prior art literature, a deglycosylation step at the N297 position, or the use of an aglycosylated antibody, as appropriate, is always described to accomplish such conjugation (WO2015 / 015448; WO2017 / 025179;WO2013 / 092998).

[0329] However, very surprisingly and against all expectations, site-specific conjugation of glycosylated antibodies to Q295 is possible quite efficiently by using the linker structure discussed above. be. In particular, attachment of linkers containing toxin molecules was achieved with conjugation efficiency 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 a specific linker. enable.

[0331] As shown, the method according to the invention does not require prior enzymatic deglycosylation of N297, use of an aglycosylation antibody, substitution of N297 with another amino acid, or T299A to prevent glycosylation. There is also no need to introduce mutations.

[0332] These two points provide considerable advantages in manufacturing aspects. Enzymatic deglycosylation steps are not desirable in GMP embodiments, as one must ensure that not only the cleaved glycans but also the deglycosylating enzyme (eg, PNGase F) are removed from the medium.

[0333] Furthermore, it is necessary not to genetically manipulate the antibody for attachment of the payload, thereby avoiding insertion of sequences that could increase immunogenicity and reduce the overall stability of the antibody. can be done.

[0334] Substitution of N297 with another amino acid also has undesirable effects, which may affect the overall stability of the entire Fc domain (Subedi et al, The Structural Role of Antibody N-Glycosylation in Receptor Interactions. Structure 2015, 23 ( 9), 1573-1583) and overall conjugate efficacy, which can result in increased antibody aggregation and decreased solubility, which is particularly important for hydrophobic payloads such as PBD (Zheng et al.; The impact of glycosylation on monoclonal antibody conformation and stability. Mabs-Austin 2011, 3 (6), 568-576). Furthermore, the glycans present on N297 have important immunomodulatory effects, as they induce antibody-dependent cellular cytotoxicity (ADCC). These immunomodulatory effects will be lost upon deglycosylation or any of the other approaches discussed above to obtain aglycosylated antibodies. Additionally, modifying the sequence of any established antibody can also pose regulatory issues, which is often the case when an approved and clinically validated antibody is used as an ADC conjugate. This is problematic because it is used as a starting point for

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

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

[0337] Thus, in a particular embodiment, the invention relates to a method according to the 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.

[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 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. Initially, a single residue in the heavy or light chain of an antibody can be replaced with a Gln residue. A Gln-containing peptide tag consisting of two or more amino acid residues can then be incorporated into the heavy or light chain of the antibody. To that end, the peptide tag may be incorporated at an internal position in the heavy or light chain, i.e. between two existing amino acid residues of the heavy or light chain, or by replacing them. , or the peptide tag may be fused (attached) to the N- or C-terminus of the heavy or light chain of the antibody.

[0339] For example, an amino residue in the heavy or light chain of an antibody may be replaced with a Gln residue so long as the resulting antibody can be conjugated to the linker of the invention by a microbial transglutaminase. In certain embodiments, the antibody is an IgG antibody. H 2 domain in which amino acid residue N297 (EU numbering) is substituted, in particular, the substitution is an N297Q substitution. Antibodies containing the N297Q mutation may be conjugated to more than one linker per heavy chain of the antibody. For example, an antibody containing the N297Q mutation may be conjugated to four linkers, one linker being conjugated to residue Q295 of the first heavy chain of the antibody; one linker is conjugated to residue N297Q of the first heavy chain of the antibody, one linker is conjugated to residue Q295 of the second heavy chain of the antibody, and one linker is conjugated to residue N297Q of the second heavy chain of the antibody. is conjugated to residue N297Q of the heavy chain of A person skilled in the art knows to replace residue N297 of an IgG antibody with a Gln residue to obtain an aglycosylated antibody.

[0340] Thus, in a particular embodiment, the invention provides a method according to the invention, in which a Gln residue introduced into the heavy or light chain of an antibody by molecular engineering is a H2 domain N297Q (EU numbering), regarding the method.

[0341] In certain embodiments, the invention provides a method according to the invention, comprising: (a) a Gln residue introduced into the heavy or light chain of an antibody by molecular engineering; or (b) contained in a peptide fused to the N- or C-terminus of an antibody heavy or light chain.

[0342] Instead of replacing a single amino acid residue in an antibody, a peptide tag containing a Gln residue that is accessible to transglutaminase may be introduced into the heavy or light chain of the antibody. Such peptide tags 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, a peptide tag containing a transglutaminase accessible Gln residue is fused to the C-terminus of the heavy chain of the antibody. Even more preferably, a peptide tag containing a transglutaminase accessible Gln residue is fused to the C-terminus of the heavy chain of an IgG antibody. Some peptide tags that can be fused to the C-terminus of the heavy chain of an antibody and serve as substrates 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 an antibody.

[0344] Exemplary peptide tags that can be introduced into the heavy or light chain of an antibody, in particular fused to the C-terminus of the heavy chain of an antibody, are LLQGG (SEQ ID NO: 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), 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 will be familiar with methods of substituting amino acid residues in antibodies, or with methods of molecular cloning as described, for example, in Sambrook, Joseph. (2001). Molecular cloning: a laboratory manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press. We know how to introduce peptide tags into antibodies by the method of

[0346] In general, those skilled in the art will know how to determine the location of the antibody to which the linker is conjugated. For example, conjugation sites can be determined by proteolytic digestion of the antibody-payload conjugate and LC-MS analysis of the resulting fragments. For example, samples can be deglycosylated with GlyciNATOR (Genovis) and then digested with Trypsin Gold (mass spectrometry grade, Promega), respectively, according to the instructions manual. Therefore, 1 μg of protein can be incubated with 50 ng of trypsin overnight at 37°C. LC-MS analysis can be performed using a nanoAcquity HPLC system coupled to a Synapt-G2 mass spectrometer (Waters). To that end, 100 ng of peptide solution was loaded onto an Acquity UPLC Symmetry C18 capture column (Waters, part number 186006527) and mixed with 1% buffer A (Water, 0.1% formic acid) and 99% buffer B (acetonitrile, 0.1 % formic acid) for 3 min at a flow rate of 5 µL / min. Peptides can then be eluted with a linear gradient of buffer B from 3% to 65% for 25 minutes. Data can be obtained in positive polarity resolution mode and mass range from 50 to 2000 m / z. Other equipment settings may be as follows: capillary voltage 3,2 kV, sampling cone 40 V, extraction cone 4.0 V, source temperature 130 °C, cone gas 35 L / h, nanoflow gas 0.1 bar, and purge gas 150 L / h. The mass spectrometer can be calibrated with [Glu1]-fibrinopeptide.

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

[0348] For hydrophobic interaction chromatography (HIC), samples were adjusted to 0.5 M ammonium sulfate and purified by MAB PAK HIC Butyl column (5 μm, 4.6 × 100 mm, Thermo Scientific). A full gradient from pH 7.5) to B (20% isopropanol, 25mM Tris HCl, pH 7.5) at 1 mL / min over 20 min at 30°C can be used for evaluation. Typically, 40 μg of sample can be used and the signal can be recorded at 280 nm. The relative HIC retention time (HIC-RRT) can be calculated by dividing the absolute retention time of the two ADC DARs by the retention time of each unconjugated mAb.

[0349] To determine the LC-MS DAR, the ADC is 4 HCO 3 to a final concentration of 0.025 mg / mL. Next, 40 μL of this solution was reduced with 1 μL TCEP (500 mM) for 5 min at room temperature, then alkylated by adding 10 μL chloroacetamide (200 mM), and then allowed to stand at 37 °C in the dark. Can be incubated overnight. For reversed-phase chromatography, the Dionex U3000 system can be used in combination with the software Chromeleon. The system can be equipped with an RP-1000 column (1000 Å, 5 μm, 1.0×100 mm, Sepax) heated to 70° C., and a UV detector set at a wavelength of 214 nm. Solvent A may consist of water with 0.1% formic acid and solvent B may consist of 85% acetonitrile with 0.1% formic acid. The reduced and alkylated sample can be loaded onto the column and separated by a 30-55% gradient of solvent B over 14 minutes. A liquid chromatography system can be coupled to a Synapt-G2 mass spectrometer for identification of DAR species. The capillary voltage of the mass spectrometer can be set to 3 kV, the sampling cone can be set to 30 V, and the extraction cone can be applied with a voltage of up to 5 V. The source temperature can be set to 150°C, the desolvation temperature to 500°C, the cone gas to 20l / h, and the desolvation gas to 600l / h. It can be done in mode. The instrument can be calibrated with sodium iodide. Spectral deconvolution can be performed by MassLynx's MaxEnt1 algorithm until convergence. After assigning DAR species to chromatographic peaks, they can be calculated based on the integrals of the peak areas of the reversed phase chromatogram.

[0350] In a particular embodiment, the invention relates to a method according to the 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 invention links the side chain of the Gln residue contained in the antibody, preferably any one of the Gln residues disclosed herein, more preferably Gln residue Q295 (EU numbering). Preferably, it is conjugated to an amide group.

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

[0353] That is, in certain embodiments, the linker is glycosylated with an efficiency of at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95%. It can be a linker that can be conjugated to an antibody. 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 will know how to determine the efficiency of conjugation of an antibody with a particular linker. For example, conjugation efficiency can be determined as described herein. That is, antibodies, particularly IgG1 antibodies, are incubated with 5-20 eq molar equivalents of linker and 3-6 U of microbial transglutaminase per mg of antibody in a suitable buffer at 37°C for 20-48 hours or as described in Example 1. As such, it can be incubated at a concentration of 1-5 mg / mL. After an 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 will vary and can be highly dependent 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). can be 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 can be conjugated as described in Example 1. Briefly, 5 mg / ml of native glycosylated monoclonal antibody was prepared in a rotary thermomixer at 50 mM containing microbial transglutaminase (MTG, Zedira) at a concentration of 5 U per mg of antibody and 5 molar equivalents of the designated linker-payload. Can be incubated for 24 hours at 37°C in Tris pH 7.6.

[0356] In a particular embodiment, the invention relates to a method according to the invention, wherein the microbial transglutaminase is derived from a Streptomyces species, in particular Streptomyces mobaraensis.

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

[0358] One such microbial transglutaminase is commercially available from Zedira (Germany). It is produced recombinantly 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 NO: 13 and 14).

[0359] In another embodiment, microbial transglutaminase from Streptomyces ladakanum (previously known as Streptoverticillium ladakanum) may be used. Transglutaminase of Streptomyces ladakanum (US 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 higher percentage 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 the four N-terminal amino acids but has similar activity.

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

[0363] In certain embodiments, mutant variants of microbial transglutaminase can be used for conjugation of linkers to antibodies. That is, the microbial transglutaminase used in the method of the present invention may be a variant of S. mobaraensis transglutaminase shown in SEQ ID NO: 12 or 13. In certain embodiments, the recombinant S. morabaensis transglutaminase set forth in SEQ ID NO: 12 may include the mutation G254D. In certain embodiments, the recombinant S. morabaensis transglutaminase shown in SEQ ID NO: 12 may include mutations G254D and E304D. In certain embodiments, the recombinant S. morabaensis transglutaminase set forth in SEQ ID NO: 12 may include mutations D8E and G254D. In certain embodiments, the recombinant S. morabaensis transglutaminase shown in SEQ ID NO: 12 may include mutations E124A and G254D. In certain embodiments, the recombinant S. morabaensis transglutaminase shown in SEQ ID NO: 12 may include mutations A216D and G254D. In certain embodiments, the recombinant S. morabaensis transglutaminase shown in SEQ ID NO: 12 may include 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 a conjugation reaction can depend on the amount of antibody used in the same reaction. For example, microbial transglutaminase contains 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, and 30 U per mg of antibody. It may be added to the conjugation reaction at a concentration of less than 20 U, 10 U per mg of antibody, or 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, the microbial transglutaminase is present in an amount of 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, and even more preferably 2 to 20 U per mg of antibody. It may be added to the conjugation reaction at a concentration range of 6 U, 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 invention involves the use of microbial transglutaminase. However, it should be noted that equivalent reactions may be performed with enzymes containing transglutaminase activity that are not of microbial origin. Therefore, antibody-linker conjugates according to the invention may also be produced with enzymes containing transglutaminase activity that are not of microbial origin.

[0367] Antibodies may be added to the conjugation reaction at any concentration. However, it is preferred that the antibody is added to the conjugation reaction at a concentration in the range of 0.1-20 mg / ml. Thus, in a particular embodiment, the invention provides a method according to the invention, wherein the antibody is 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 is added to the conjugation reaction at a concentration of 1 to 10 mg / mL, even more preferably 2 to 7.5 mg / mL, 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, most preferably from 5 to 17 mg / ml. .

[0369] In order to obtain efficient conjugation, the linker is preferably added to the antibody in molar excess. That is, in certain embodiments, the antibodies are mixed in at least 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 molar equivalents of linker.

[0370] Thus, in certain embodiments, the invention provides a method according to the invention, wherein the antibody contains 2 to 100 molar equivalents of linker, preferably 2 to 80 molar equivalents of linker, more preferably 2 to 70 molar equivalents of linker. even more preferably 2 to 60 molar equivalents of linker, even more preferably 2 to 50 molar equivalents of linker, even more preferably 2 to 40 molar equivalents of linker, even more preferably 2 to 30 molar equivalents of 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, most preferably 2 to 10 molar equivalents of the linker, Regarding the method.

[0371] Alternatively, the antibody contains 2.5 to 100 molar equivalents of linker, preferably 2.5 to 80 molar equivalents of linker, more preferably 2.5 to 70 molar equivalents of linker, even more preferably 2.5 to 60 molar equivalents of 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 linker, even more preferably 2.5 to 10 molar equivalents of linker, most preferably 2.5 to 8 molar equivalents of linker may be contacted.

[0372] Alternatively, the antibody contains 5 to 100 molar equivalents of linker, preferably 5 to 80 molar equivalents of linker, more preferably 5 to 70 molar equivalents of linker, even more preferably 5 to 60 molar equivalents of 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, most preferably 5 to 10 molar equivalents of linker may be contacted.

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

[0374] The methods of the invention may be performed in any buffer suitable for conjugation of payload to a linker. Buffers suitable for the methods of the invention include, but are not limited to, Tris, MOPS, HEPES, PBS or BisTris buffers. The concentration of the buffer depends, inter alia, on the concentration of the antibody and / or linker and may range from 10-1000mM, 10-500mM, 10-400mM, 10-250mM, 10-150mM or 10-100mM. Additionally, the buffer may contain any salt concentration that is suitable for carrying out the methods of the invention. For example, the buffer used in the method of the invention may be 150mM or less, 140mM or less, 130mM or less, 120mM or less, 110mM or less, 100mM or less, 90mM or less, 80mM or less, 70mM or less, 60mM or less, 50mM or less, 40mM or less, It may have a salt concentration of 30mM or less, 20mM or less or 10mM or less, or it may have no salt. In a particular embodiment, the methods of the invention are performed in 50 mM Tris (pH 7.6), preferably without salt.

[0375] It must be noted that optimal reaction conditions (eg, pH, buffer, salt concentration) vary between payloads and may depend to some extent on the physicochemical properties of the linker and / or payload. However, one skilled in the art will not require undue experimentation to identify reaction conditions that are suitable for carrying out the method of the invention.

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

[0377] In a preferred embodiment, the invention provides a method for producing an antibody-linker conjugate by a microbial transglutaminase (MTG), comprising: a structure (shown in the N→C direction); (Sp 1 )-RK-(Sp 2 )-B-(Sp 3 ) or (Sp 1 )-B-(Sp 2 )-RK-(Sp 3 ) to a Gln residue contained in the antibody, wherein the linker comprises: - (Sp 1 ) is a chemical spacer or is absent; - (Sp 2 ) is a chemical spacer or is absent; - (Sp 3 ) is a chemical spacer or is absent; - R is arginine or an arginine derivative or an arginine mimetic; - K is lysine or a ricin derivative or a ricin mimetic; - B is the concatenation part or payload; the linker is conjugated to a Gln residue contained in the antibody by a primary amine contained in the side chain of the lysine residue, lysine derivative or lysine mimetic; and the antibody is contacted with 2 to 80 molar equivalents of linker; and / or Relating to a method, wherein microbial transglutaminase is added to the conjugation reaction at a concentration ranging from 1 to 20 U per 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 invention provides a method for producing an antibody-linker conjugate by a microbial transglutaminase (MTG), comprising: a structure (shown in the N→C direction); (Sp 1 )-RK-(Sp 2 )-B-(Sp 3 ) or (Sp 1 )-B-(Sp 2 )-RK-(Sp 3 ) to a Gln residue contained in the antibody, wherein the linker comprises: - (Sp 1 ) is a chemical spacer or is absent; - (Sp 2 ) is a chemical spacer or is absent; - (Sp 3 ) is a chemical spacer or is absent; - R is arginine or an arginine derivative or an arginine mimetic; - K is lysine or a ricin derivative or a ricin mimetic; - B is the concatenation part or payload; the linker is conjugated to a Gln residue contained in the antibody by a primary amine contained in the side chain of the lysine residue, lysine derivative or lysine mimetic; and the antibody is contacted with 2-50 molar equivalents of linker; and / or Relating to a method, wherein a microbial transglutaminase is added to the conjugation reaction at a concentration ranging from 1 to 10 U per 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 invention provides a method for producing an antibody-linker conjugate by a microbial transglutaminase (MTG), comprising: a structure (shown in the N→C direction); (Sp 1 )-RK-(Sp 2 )-B-(Sp 3 ) or (Sp 1 )-B-(Sp 2 )-RK-(Sp 3 ) to a Gln residue contained in the antibody, wherein the linker comprises: - (Sp 1 ) is a chemical spacer or is absent; - (Sp 2 ) is a chemical spacer or is absent; - (Sp 3 ) is a chemical spacer or is absent; - R is arginine or an arginine derivative or an arginine mimetic; - K is lysine or a ricin derivative or a ricin mimetic; - B is the concatenation part or payload; the linker is conjugated to a Gln residue contained in the antibody by a primary amine contained in the side chain of the lysine residue, lysine derivative or lysine mimetic; and the antibody is contacted with 2-30 molar equivalents of linker; and / or Relating to a method, wherein microbial transglutaminase is added to the conjugation reaction at a concentration ranging from 2 to 10 U per 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 invention provides a method for producing an antibody-linker conjugate by a microbial transglutaminase (MTG), comprising: a structure (shown in the N→C direction); (Sp 1 )-RK-(Sp 2 )-B-(Sp 3 ) or (Sp 1 )-B-(Sp 2 )-RK-(Sp 3 ) to a Gln residue contained in the antibody, wherein the linker comprises: - (Sp 1 ) is a chemical spacer or is absent; - (Sp 2 ) is a chemical spacer or is absent; - (Sp 3 ) is a chemical spacer or is absent; - R is arginine or an arginine derivative or an arginine mimetic; - K is lysine or a ricin derivative or a ricin mimetic; - B is the concatenation part or payload; the linker is conjugated to a Gln residue contained in the antibody by a primary amine contained in the side chain of the lysine residue, lysine derivative or lysine mimetic; and The antibody is contacted with about 2 to 20 molar equivalents of linker; and / or microbial transglutaminase is added to the conjugation reaction at a concentration ranging from 2 to 10 U per mg of antibody; 20 mg / mL is added to the conjugation reaction at concentrations ranging from ~20 mg / mL.

[0381] In an even more preferred embodiment, the invention provides a method for producing an antibody-linker conjugate by a microbial transglutaminase (MTG), comprising: a structure (shown in the N→C direction); (Sp 1 )-RK-(Sp 2 )-B-(Sp 3 ) or (Sp 1 )-B-(Sp 2 )-RK-(Sp 3 ) to a Gln residue contained in the antibody, wherein the linker comprises: - (Sp 1 ) is a chemical spacer or is absent; - (Sp 2 ) is a chemical spacer or is absent; - (Sp 3 ) is a chemical spacer or is absent; - R is arginine or an arginine derivative or an arginine mimetic; - K is lysine or a ricin derivative or a ricin mimetic; - B is the concatenation part or payload; the linker is conjugated to a Gln residue contained in the antibody by a primary amine contained in the side chain of the lysine residue, lysine derivative or lysine mimetic; and The antibody is contacted with about 2.5 to 15 molar equivalents of linker; and / or microbial transglutaminase is added to the conjugation reaction at a concentration ranging from 2 to 10 U per mg of antibody; 20 mg / mL is added to the conjugation reaction at concentrations ranging from ~20 mg / mL.

[0382] In a most preferred embodiment, the invention provides a method for producing an antibody-linker conjugate by a microbial transglutaminase (MTG), comprising: a structure (shown in the N→C direction); (Sp 1 )-RK-(Sp 2 )-B-(Sp 3 ) or (Sp 1 )-B-(Sp 2 )-RK-(Sp 3 ) to a Gln residue contained in the antibody, wherein the linker comprises: - (Sp 1 ) is a chemical spacer or is absent; - (Sp 2 ) is a chemical spacer or is absent; - (Sp 3 ) is a chemical spacer or is absent; - R is arginine or an arginine derivative or an arginine mimetic; - K is lysine or a ricin derivative or a ricin mimetic; - B is the concatenation part or payload; the linker is conjugated to a Gln residue contained in the antibody by a primary amine contained in the side chain of the lysine residue, lysine derivative or lysine mimetic; and The antibody is contacted with about 2.5 to 10 molar equivalents of linker; and / or microbial transglutaminase is added to the conjugation reaction at a concentration ranging from 2 to 10 U per mg of antibody; 20 mg / mL is added to the conjugation reaction at concentrations ranging from ~20 mg / mL.

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

[0384] Thus, the present invention relates to antibody-linker conjugates produced using any of the foregoing steps.

[0385] In certain embodiments, the invention provides: a) antibodies; and b) Structure: (Sp 1 )-RK-(Sp 2 )-B-(Sp 3 )or (Sp 1 )-B-(Sp 2 )-RK-(Sp 3 ); (In the formula, - (Sp 1 ) is a chemical spacer or is absent; - (Sp 2 ) is a chemical spacer or is absent; - (Sp 3 ) is a chemical spacer or is absent; - R is arginine or an arginine derivative or an arginine mimetic; - K is lysine or a ricin derivative or a ricin mimetic; - B is the concatenation part or payload) An antibody-linker conjugate comprising a linker comprising: A linker is formed between the γ-carboxamide group of the glutamine residue in the antibody and the primary amine contained in the side chain of the lysine residue, lysine derivative, or lysine mimetic contained in the RK motif contained in the linker. The antibody-linker conjugate is conjugated to an antibody through a bonded isopeptide bond.

[0386] Thus, the invention further relates to antibody-linker conjugates produced using the methods of the invention. In particular, the invention refers to an antibody conjugated with any one of the linkers disclosed herein with a glutamine residue comprised in the heavy or light chain of the antibody in connection with the methods of the invention. That is, all linkers disclosed above with respect to the methods of the invention may be included in the antibody-linker constructs of the invention. Preferably, the linker of the present invention binds the antibody by 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 contained in the RK motif of the linker. Conjugated to a glutamine residue. In certain embodiments, the primary amine included in residue K is an amine group included 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 (with which the linker is conjugated to the antibody) is an ε-amino group included in the lysine residue.

[0387] The chemical spacer included in the antibody-linker constructs disclosed herein may be any one of the RK-containing linkers disclosed herein. That is, the linker may be a linker that includes a single linking moiety or payload B, or it may include two or more linking moieties and / or payload B. 1 , B 2 It may also be a linker that includes the following.

[0388] In a particular embodiment, the invention provides an antibody-linker conjugate according to the invention, comprising a chemical spacer (Sp 1 ), (Sp 2 ) and (Sp 3 ) relate to antibody-linker conjugates, each independently comprising from 0 to 12 amino acid residues.

[0389] In certain embodiments, the invention provides antibody-linker conjugates according to the invention, wherein the linker is 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 , to antibody-linker conjugates containing four or fewer amino acid residues.

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

[0391] In certain embodiments, the invention relates to antibody-linker conjugates according to the invention, wherein the linker does not contain negatively charged amino acid residues.

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

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

[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 a particular embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein B is a linking moiety.

[0397] In a particular embodiment, the invention provides an antibody-linker conjugate according to the invention, wherein linking moiety B is - bioorthogonal marker group, or - Non-bioorthogonal entities for cross-linking An antibody-linker conjugate comprising:

[0398] In certain embodiments, the invention provides an antibody-linker conjugate according to the invention, wherein the bioorthogonal marker group or the non-bioorthogonal entity for crosslinking is - -N-N≡N, or -N 3 ; - Lys(N 3 ); - Tetrazine; - alkynes; - Distorted cyclooctyne; -BCN; - distorted alkenes; - photoreactive groups; - aldehydes; - Acyl trifluoroborate; - Proteolytic agents (“PROTACs”); - cyclopentadiene / spirolocyclopentadiene; - thioselective electrophile; - -SH; and - Cysteine Antibody-linker conjugates 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 invention may be an antibody that is conjugated to a linker that includes one or more linking moieties. Such antibody-linker conjugates can subsequently be customized with one or more payloads, particularly payloads that may be suitable for binding to one or more linking moieties.

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

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

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

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

[0404] In certain embodiments, the invention provides an antibody-linker conjugate according to the invention, wherein the payload is - toxin; - Cytokines; - growth factors; - radionuclides; - Hormones; - antiviral; - antibacterial agent; - Fluorescent dye: - Immune regulators / immunostimulants; - half-life increasing part; - solubility increasing part; - Polymer-toxin conjugates; - Nucleic acids; - biotin or streptavidin moiety; - vitamins; - Proteolytic agents (“PROTACs”); - target binding moiety; and / or - Anti-inflammatory agent Antibody-linker conjugates comprising at least one of:

[0405] In certain embodiments, the invention provides an antibody-linker conjugate according to the invention, wherein the toxin is - pyrrolobenzodiazepines (e.g. PBD); - Auristatin (e.g. MMAE, MMAF); - maytansinoids (e.g. maytansine, DM1, DM4, DM21); - duocarmycin; - Nicotinamide phosphoribosyltransferase (NAMPT) inhibitor; - Tubulisin; - Engine (e.g. Kalicare Sewing Machine); - anthracycline derivatives (PNU) (e.g. doxorubicin); - Kinesin spindle protein (KSP) inhibitors based on pyrrole; - cryptophycin; - drug efflux pump inhibitor; - Sandramycin; - amanitin (e.g. α-amanitin); and - camptothecin (e.g. exatecan, deruxtecan) The antibody-linker conjugate is at least one selected from the group consisting of:

[0406] In a particular embodiment, the invention provides an antibody-linker conjugate according to the invention, comprising a chemical spacer (Sp 2 ) comprises a self-immolative moiety.

[0407] In a particular embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the self-immolative moiety is directly attached 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, an antibody-linker conjugate according to the 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 for the method according to the invention, in particular any one of the IgG type antibodies. That is, the antibodies comprised in the antibody-linker conjugates according to the invention may contain the same glycosylation patterns, mutations and / or modifications as the antibodies disclosed herein in connection with the methods according to the invention.

[0411] In certain embodiments, the invention provides an antibody-payload conjugate according to the invention, wherein the linker is 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 For antibody-payload conjugates that are any one of the linkers shown.

[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 certain embodiments, the invention provides antibody-linker conjugates according to the invention, wherein the Gln residue to which the linker is conjugated is included in the Fc domain of the antibody, in particular to which the linker is conjugated. The Gln residue is the C of IgG antibody. H 2 domain Gln residue Q295 (EU numbering) for antibody-linker conjugates.

[0414] In a particular embodiment, the invention provides an antibody-linker conjugate according to the 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. - Regarding linker conjugates.

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

[0416] In certain embodiments, the invention provides an antibody-linker conjugate according to the invention, wherein a Gln residue introduced into the heavy or light chain of an antibody by molecular engineering (a) relates to antibody-linker conjugates that are incorporated into a chain or (b) included in a peptide fused to the N- or C-terminus of the heavy or light chain of the antibody.

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

[0418] In certain embodiments, the invention provides an antibody-linker conjugate according to the invention, wherein the IgG antibody is a glycosylated IgG antibody, in particular the IgG antibody is a glycosylated IgG antibody. H For antibody-linker conjugates that are glycosylated at residue N297 (EU numbering) of the 2 domain.

[0419] In certain embodiments, the invention provides an antibody-linker conjugate according to the invention, wherein the antibody is brentuximab, trastuzumab, gemtuzumab, inotuzumab, avelumab, cetuximab, rituximab, daratumumab, pertuzumab, vedolizumab, ocrelizumab, tocilizumab , ustekinumab, golimumab, obinutuzumab, sacituzumab, belantamab, polatuzumab and enfortuzumab.

[0420] In certain embodiments, the invention provides 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. The present invention relates to an antibody-linker conjugate.

[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 invention relates to antibody-drug conjugates. That is, the antibody may be conjugated to a linker according to the invention, the linker comprising one or more toxins.

[0423] Thus, in certain embodiments, the invention provides: a) IgG antibodies; and b) a linker comprising a drug moiety B, wherein the drug moiety B is 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); a linker that is covalently linked to an amino acid sequence that An antibody-drug conjugate comprising: The linker is C of the antibody. H It is conjugated to an IgG antibody by an isopeptide bond formed between the γ-carboxamide group of the glutamine residue Q295 (EU numbering) in the 2 domain and the primary amine contained in the side chain of the lysine residue contained in the linker. Relating to antibody-drug conjugates that are gated.

[0424] In certain embodiments, the invention provides: a) IgG antibodies; and b) a linker comprising 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); An antibody-drug conjugate comprising: The linker is C of the antibody. H It is conjugated to an IgG antibody by an isopeptide bond formed between the γ-carboxamide group of the glutamine residue Q295 (EU numbering) in the 2 domain and the primary amine contained in the side chain of the lysine residue contained in the linker. Relating to antibody-drug conjugates that are gated.

[0425] That is, in certain embodiments, the linker comprises 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). ), the linker being conjugated to the glutamine residue of the antibody via the primary amine contained in residue K. Drug moiety B is 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) It should be understood that this is not necessary. Alternatively, drug moiety B may be indirectly linked to the structures 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 connected to. For example, a linker can be used 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). It may further include a chemical structure located at . Such a chemical structure is a chemical spacer (Sp 1 ), (Sp 2 ) or (Sp 3) may be any of the structures disclosed herein. In certain embodiments, the linker connects drug moiety B 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: 4). 54). In certain embodiments, the linker connects drug moiety B 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: 4). 54). In certain embodiments, the linker connects drug moiety B 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: 4). 54) may also include a cuttable and / or self-destructive portion located between 54).

[0426] Thus, in certain embodiments, the invention provides an antibody-drug conjugate according to the 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. Concerning antibody-drug conjugates.

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

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

[0429] In certain embodiments, the invention provides an antibody-drug conjugate according to the invention, wherein the IgG antibody is a glycosylated IgG antibody, in particular the IgG antibody is a glycosylated IgG antibody. H For antibody-drug conjugates that are 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 an IgG or IgG1 antibody is glycosylated at residue N297 (EU numbering).

[0432] The antibody-drug conjugate may include 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); - Auristatin (e.g. MMAE, MMAF); - maytansinoids (e.g. maytansine, DM1, DM4, DM21); - duocarmycin; - Nicotinamide phosphoribosyltransferase (NAMPT) inhibitor; - Tubulisin; - Engine (e.g. Karikea Sewing Machine); - anthracycline derivatives (PNU) (e.g. doxorubicin); - Kinesin spindle protein (KSP) inhibitors based on pyrrole; - cryptophycin; - drug efflux pump inhibitor; - Sandramycin; - amanitin (e.g. α-amanitin); and - camptothecin (e.g. exatecan, deruxtecan) The antibody-drug conjugate is a toxin 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 linked to a linking moiety included in a linker in a two-step process.

[0434] In a particular embodiment, the invention provides an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure RKAA-B or RKAA-(linker molecule)-B. Regarding.

[0435] That is, payload B may be attached directly to the C-terminus of the alanine residue, or may be attached to the C-terminus of the alanine residue by a linker molecule. It should be understood that the choice of linker molecule is highly dependent on the functional groups available in payload B. Linker molecules suitable for attaching payloads with 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 include a self-immolative moiety, particularly any one of the self-immolative moieties disclosed herein. Thus, 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 RKAA-(self-immolative moiety)-B. .

[0436] Alternatively, the payload may be attached to the N-terminus of the arginine residue directly or by a linker molecule, eg, by any one of the linker molecules disclosed herein. Thus, in certain embodiments, the invention provides an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure B-RKAA or B-(linker molecule)-RKAA. Concerning conjugates. 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 B-(self-immolative moiety)-RKAA. In certain embodiments, the self-immolative moiety included in structure B-(self-immolative moiety)-RKAA is a self-immolative moiety comprising an ortho-hydroxy-protected aryl sulfate (OHPAS) moiety as disclosed herein. There may be.

[0437] In a particular embodiment, the invention provides an antibody-drug conjugate according to the invention, wherein the linker has the structure RKAA-PABC-B, in particular where B is auristatin or maytansinoid, and in particular 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 include the linear peptide RKAA, and the carboxy group of the C-terminal alanine residue is bonded to the amino group contained in PABC through 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 carbamate formation with PABC. Thus, the toxin may be connected to the PABC by a linker.

[0439] In certain embodiments, the toxin may be a toxin that includes a primary or secondary amine. In 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 the linker shown in FIG. 1 or FIG. 8.

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

[0442] It should be noted that the linker may contain self-immolative moieties other than PABC. That is, the linker may have the structure RKAA-(self-destructive moiety)-toxin. Those skilled in the art will be aware of other self-destructive parts that can be used within the scope of the present invention. Additionally, those skilled in the art are aware of toxins that can be attached to the self-destructive moiety, optionally by additional linkers.

[0443] In certain embodiments, the toxin may include a hydroxy group and the linker may include a self-immolative methylamine group. That is, the linker has the structure RKAA-(NH)-(CH 3)-O-toxin. In certain embodiments, the hydroxy-containing toxin may be a camptothecin, eg, exatecan or an exatecan derivative, particularly the exatecan derivative Dxd, or an anthracycline such as PNU-159682.

[0444] In certain embodiments, the toxin may include a thiol group and the linker may include a self-destructive methylamine group. That is, the linker has the structure RKAA-(NH)-(CH 3 )-S-toxin. In certain embodiments, the thiol-containing toxin may be a maytansinoid, such as DM1 or a thiol-containing derivative thereof.

[0445] In a particular embodiment, the invention provides an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure RKA-B or RKA-(linker molecule)-B. Regarding.

[0446] That is, payload B may be attached directly to the C-terminus of the alanine residue, or may be attached to the C-terminus of the alanine residue by a linker molecule. It should be understood that the choice of linker molecule is highly dependent on the functional groups available in payload B. Linker molecules suitable for attaching payloads with 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 include a self-immolative moiety, particularly any one of the self-immolative moieties disclosed herein. Thus, 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 RKA-(self-immolative moiety)-B. .

[0447] Alternatively, the payload may be attached to the N-terminus of the arginine residue directly or by a linker molecule, eg, by any one of the linker molecules disclosed herein. Thus, in certain embodiments, the invention provides an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure B-RKA or B-(linker molecule)-RKA. Concerning conjugates. 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 B-(self-immolative moiety)-RKA. In certain embodiments, the self-immolative moiety included in structure B-(self-immolative moiety)-RKA is a self-immolative moiety comprising an ortho-hydroxy-protected aryl sulfate (OHPAS) moiety as disclosed herein. There may be.

[0448] In a particular embodiment, the invention provides an antibody-drug conjugate according to the invention, wherein the linker is of the structure RKA-PABC-B, in particular where B is auristatin or maytansinoid, and in particular 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, and the carboxy group of the C-terminal alanine residue is bonded 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 carbamate formation with PABC. Thus, the toxin may be connected to the PABC by a linker.

[0450] In certain embodiments, the toxin may be a toxin that includes a primary or secondary amine. In 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 FIG. 2.

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

[0453] It should be noted that the linker may contain self-immolative moieties other than PABC. That is, the linker may have the structure RKA-(self-immolative moiety)-toxin. Those skilled in the art will be aware of other self-destructive parts that can be used within the scope of the present invention. Additionally, those skilled in the art are aware of toxins that can be attached to the self-destructive moiety, optionally by additional linkers.

[0454] In certain embodiments, the toxin may include a hydroxy group and the linker may include a self-immolative methylamine group. That is, the linker has the structure RKA-(NH)-(CH 3 )-O-toxin. In certain embodiments, the hydroxy-containing toxin may be a camptothecin, eg, exatecan or an exatecan derivative, particularly the exatecan derivative Dxd, or an anthracycline such as PNU-159682.

[0455] In certain embodiments, the toxin may include a thiol group and the linker may include a self-destructive methylamine group. That is, the linker has the structure RKA-(NH)-(CH 3 )-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 provides an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure ARK-B or ARK-(linker molecule)-B. Regarding.

[0457] That is, payload B may be attached directly to the C-terminus of the lysine residue, or may be attached to the C-terminus of the lysine residue by a linker molecule. It should be understood that the choice of linker molecule is highly dependent on the functional groups available in payload B. Linker molecules suitable for attaching payloads with 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 include a self-immolative moiety, particularly any one of the self-immolative moieties disclosed herein. Thus, 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-(self-immolative moiety)-B. .

[0458] Alternatively, the payload may be attached to the N-terminus of the alanine residue directly or by a linker molecule, eg, by any one of the linker molecules disclosed herein. Thus, in certain embodiments, the invention provides an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure B-ARK or B-(linker molecule)-ARK. Concerning conjugates. 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 B-(self-immolative moiety)-ARK. In certain embodiments, the self-immolative moiety included in structure B-(self-immolative moiety)-ARK is a self-immolative moiety comprising an ortho-hydroxy-protected aryl sulfate (OHPAS) moiety as disclosed herein. There may be.

[0459] In certain embodiments, the invention provides an antibody-drug conjugate according to the invention, wherein the linker is of the structure ARK-PABC-B, in particular where B is auristatin or maytansinoid, and in particular 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 include a linear peptide ARK, with the carboxy group of the C-terminal lysine residue attached 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 carbamate formation with PABC. Thus, the toxin may be connected to the PABC by a linker.

[0461] In certain embodiments, the toxin may be a toxin that includes a primary or secondary amine. In 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 FIG. 3.

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

[0464] It should be noted that the linker may contain self-immolative moieties other than PABC. That is, the linker may have the structure ARK-(self-destructive moiety)-toxin. Those skilled in the art will be aware of other self-destructive parts that can be used within the scope of the present invention. Additionally, those skilled in the art are aware of toxins that can be attached to the self-destructive moiety, optionally by additional linkers.

[0465] In certain embodiments, the toxin may include a hydroxy group and the linker may include a self-immolative methylamine group. That is, the linker has the structure ARK-(NH)-(CH 3 )-O-toxin. In certain embodiments, the hydroxy-containing toxin may be a camptothecin, eg, exatecan or an exatecan derivative, particularly the exatecan derivative Dxd, or an anthracycline such as PNU-159682.

[0466] In certain embodiments, the toxin may include a thiol group and the linker may include a self-destructive methylamine group. That is, the linker has the structure ARK-(NH)-(CH 3 )-S-toxin (similar to Figure 15). In certain embodiments, the thiol-containing toxin may be a maytansinoid, such as DM1 or a thiol-containing derivative thereof. In certain embodiments, the linker is the linker shown in FIG. 14 or FIG. 15.

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

[0468] That is, payload B may be attached directly to the C-terminus of the arginine residue, or may be attached to the C-terminus of the arginine residue by a linker molecule. It should be understood that the choice of linker molecule is highly dependent on the functional groups available in payload B. Linker molecules suitable for attaching payloads with 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 include a self-immolative moiety, particularly any one of the self-immolative moieties disclosed herein. Thus, 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 RKR-(self-immolative moiety)-B. .

[0469] Alternatively, the payload may be attached to the N-terminus of the arginine residue directly or by a linker molecule, eg, by any one of the linker molecules disclosed herein. Thus, in certain embodiments, the invention provides an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure B-RKR or B-(linker molecule)-RKR. Concerning conjugates. In certain embodiments, the linker may be a dicarboxylic acid linker (see Figure 9). 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 B-(self-immolative moiety)-RKR. In certain embodiments, the self-immolative moiety included in structure B-(self-immolative moiety)-RKR is a self-immolative moiety comprising an ortho-hydroxy-protected aryl sulfate (OHPAS) moiety as disclosed herein. There may be.

[0470] In a particular embodiment, the invention provides an antibody-drug conjugate according to the invention, wherein the linker is of the structure RKR-PABC-B, in particular where B is auristatin or maytansinoid, and in particular 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 include the linear peptide RKR, and the carboxy group of the C-terminal arginine residue is bonded 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 carbamate formation with PABC. Thus, the toxin may be connected to the PABC by a linker.

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

[0473] In certain embodiments, the linker may have a protected N-terminus. In 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 has the structure RKR-(PEG) n -PABC-MMAE, where n is an integer between 2 and 20. In certain embodiments, the linker has 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 MMAE. In certain embodiments, the additional linker may be a p-nitrophenol (PNP) group.

[0475] It should be noted that the linker may contain self-immolative moieties other than PABC. That is, the linker may have the structure RKR-(self-destructive moiety)-toxin. Those skilled in the art will be aware of other self-destructive parts that can be used within the scope of the present invention. Additionally, those skilled in the art are aware of toxins that can be attached to the self-destructive moiety, optionally by additional linkers.

[0476] In certain embodiments, the toxin may include a hydroxy group and the linker may include a self-immolative methylamine group. That is, the linker has the structure RKR-(NH)-(CH 3 )-O-toxin. In certain embodiments, the hydroxy-containing toxin may be a camptothecin, eg, exatecan or an exatecan derivative, particularly the exatecan derivative Dxd, or an anthracycline such as PNU-159682.

[0477] In certain embodiments, the toxin may include a thiol group and the linker may include a self-destructive methylamine group. That is, the linker has the structure RKR-(NH)-(CH 3 )-S-toxin (similar to Figure 15). In certain embodiments, the thiol-containing toxin may be a maytansinoid, such as DM1 or a thiol-containing derivative thereof.

[0478] In certain embodiments, the invention provides an antibody-drug conjugate according to the invention, wherein the linker comprises the structure RK-Val-Cit-B or RK-Val-Cit-(linker molecule)-B or An antibody-drug conjugate comprising the same.

[0479] That is, payload B may be attached directly to the C-terminus of the citrulline residue, or may be attached to the C-terminus of the citrulline residue by a linker molecule. It should be understood that the choice of linker molecule is highly dependent on the functional groups available in payload B. Linker molecules suitable for attaching payloads with 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 include a self-immolative moiety, particularly any one of the self-immolative moieties disclosed herein. Thus, in a particular embodiment, the invention provides an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure RK-Val-Cit-(self-immolative moiety)-B. Regarding drug conjugates.

[0480] In certain embodiments, the invention provides an antibody-drug conjugate according to the invention, wherein the linker has the structure RK-Val-Cit-PABC-B (particularly where B is an auristatin or a maytansinoid). and in particular, 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 include the linear peptide RK-Val-Cit, and the carboxy group of the C-terminal citrulline residue is bonded 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 carbamate formation with PABC. Thus, the toxin may be connected to the PABC by a linker.

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

[0483] In certain embodiments, the linker may have a protected N-terminus. In 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 has the structure RK-(PEG) n -Val-Cit-PABC-MMAE, where n is an integer between 2 and 20. In certain embodiments, the linker has 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 MMAE. In certain embodiments, the additional linker may be a p-nitrophenol (PNP) group.

[0485] It should be noted that the linker may contain self-immolative moieties other than PABC. That is, the linker may have the structure RK-Val-Cit-(self-destructive moiety)-toxin. Those skilled in the art will be aware of other self-destructive parts that can be used within the scope of the present invention. Additionally, those skilled in the art are aware of toxins that can be attached to the self-destructive moiety, optionally by additional linkers.

[0486] In certain embodiments, the toxin may include a hydroxy group and the linker may include a self-immolative methylamine group. That is, the linker has the structure RK-Val-Cit-(NH)-(CH 3 )-O-toxin. In certain embodiments, the hydroxy-containing toxin may be a camptothecin, eg, exatecan or an exatecan derivative, particularly the exatecan derivative Dxd, or an anthracycline such as PNU-159682.

[0487] In certain embodiments, the toxin may include a thiol group and the linker may include a self-destructive methylamine group. That is, the linker has the structure RK-Val-Cit-(NH)-(CH 3 )-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 polatuzumab or, alternatively, an antibody-linker conjugate or antibody drug conjugate comprising an anti-CD79b antibody.

[0489] That is, in certain embodiments, the invention provides: a) Polatuzumab or anti-CD79b antibody: and b) Structure: (Sp 1 )-RK-(Sp 2 )-B-(Sp 3 )or (Sp 1 )-B-(Sp 2 )-RK-(Sp 3 ); (In the formula, - (Sp 1 ) is a chemical spacer or is absent; - (Sp 2 ) is a chemical spacer or is absent; - (Sp 3 ) is a chemical spacer or is absent; - R is arginine or an arginine derivative or an arginine mimetic; - K is lysine or a ricin derivative or a ricin mimetic; - B is the concatenation part or payload) linker containing An antibody-linker conjugate comprising: The linker is a γ-carboxamide group of a glutamine residue contained in polatuzumab or an anti-CD79b antibody, and a lysine residue contained in the RK motif contained in the linker, or a primary amine contained in the side chain of a lysine derivative or lysine mimetic. The antibody-linker conjugate is conjugated to polatuzumab or an anti-CD79b antibody through an isopeptide bond formed during the antibody-linker conjugate.

[0490] In certain embodiments, the invention provides an antibody-payload conjugate comprising polatuzumab or an anti-CD79b antibody, wherein the linker is 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 for an antibody-payload conjugate that is any one of the linkers shown in FIG.

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

[0492] Polatuzumab conjugates comprising a linker according to the invention have been shown by the inventors to have a longer half-life in plasma compared to the commercially available conjugate polatuzumab vedotin. There is. Thus, an antibody-linker conjugate conjugated to a linker according to the invention by a microbial transglutaminase, for example by conjugating a maleimide-containing linker to cysteine ​​residues of the antibody, is more effective than antibodies produced by other techniques. is also surprisingly more stable. Antibody-linker conjugates according to the invention are thus more accessible to 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 present invention provides polatuzumab in which the heavy chain and / or light chain comprises at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO: 5 and / or SEQ ID NO: 6, respectively. Also includes variants of 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 the anti-CD79b antibody, is present in a glycosylated form of the antibody-linker conjugate. That is, polatuzumab, or the anti-CD79b antibody, is preferably glycosylated at residue N297 (EU numbering). However, polatuzumab, or anti-CD79b antibody, may also be deglycosylated as described herein.

[0495] Polatuzumab, or the 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 MTG-catalyzed conjugated to glutamine residue Q295 (EU numbering) of the antibody. However, the linker may also be conjugated to an engineered glutamine residue such as N297Q (EU numbering) and / or to any one of the glutamine-containing tags disclosed herein.

[0496] A linker that is conjugated to polatuzumab, or an anti-CD79b antibody, may include a single linking moiety or payload B, or multiple linking moieties and / or payload B. 1 , B 2 It may also include.

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

[0498] In certain embodiments, the linker included in the polatuzumab-payload conjugate may include one or more payloads B. Such polatuzumab-payload conjugates may have been obtained in a two-step process, where the linker containing the linking moiety is conjugated to polatuzumab in the first step, and the payload is conjugated to the linking moiety in the second step. connected. Alternatively, the polatuzumab-payload conjugate may be obtained in a one-step process, with the payload-containing linker conjugated directly to polatuzumab.

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

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

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

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

[0503] In certain embodiments, polatuzumab, or a linker conjugated to an anti-CD79b antibody, has the structure RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4) Or it may contain 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) is , may be conjugated to polatuzumab, or residue Q295 of the anti-CD79b antibody, via a primary amine contained in residue K.

[0504] In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to the linker RKAA-B disclosed herein, where B is preferably a toxin. In certain embodiments, polatuzumab, or an anti-CD79b antibody, is conjugated to a linker RKAA-(self-immolative moiety)-B, where B is preferably a toxin, as disclosed herein. You can leave it there. 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 PABC or methyl-amine-containing group.

[0505] In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to a 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 the anti-CD79b antibody, has a linker RKAA-(NH)-(CH 3 )-O-B, where O is an ether-linked oxygen atom and B is a hydroxy-containing toxin. In certain embodiments, polatuzumab, or the anti-CD79b antibody, has a linker RKAA-(NH)-(CH 3 )-O-camptothecin. In certain embodiments, polatuzumab, or the anti-CD79b antibody, has a linker RKAA-(NH)-(CH 3 )-S-B, where S is a sulfur atom in a thioether bond and B is a thiol-containing toxin. In certain embodiments, polatuzumab, or the anti-CD79b antibody, has a linker RKAA-(NH)-(CH 3 )-S-DM1.

[0506] In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to a linker B-RKAA disclosed herein, where B is preferably a toxin. In certain embodiments, polatuzumab, or an anti-CD79b antibody, is conjugated to a linker B-(self-immolative moiety)-RKAA disclosed herein, where B is preferably a toxin. You can leave it there. 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 that includes 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 FIG. 9.

[0507] In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to the linker RKA-B disclosed herein, where B is preferably a toxin. In certain embodiments, polatuzumab, or an anti-CD79b antibody, is conjugated to a linker RKA-(self-immolative moiety)-B, where B is preferably a toxin, as disclosed herein. You can leave it there. 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 PABC or methyl-amine-containing group.

[0508] In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to a 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, has a linker RKA-(NH)-(CH 3 )-O-B, where O is an ether-linked oxygen atom and B is a hydroxy-containing toxin. In certain embodiments, polatuzumab, or an anti-CD79b antibody, has a linker RKA-(NH)-(CH 3 )-O-camptothecin. In certain embodiments, polatuzumab, or an anti-CD79b antibody, has a linker RKA-(NH)-(CH 3 )-S-B, where S is a sulfur atom in a thioether bond and B is a thiol-containing toxin. In certain embodiments, polatuzumab, or an anti-CD79b antibody, has a linker RKA-(NH)-(CH 3 )-S-DM1.

[0509] In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to the linker B-RKA disclosed herein, where B is preferably a toxin. In certain embodiments, polatuzumab, or an anti-CD79b antibody, is conjugated to a linker B-(self-immolative moiety)-RKA disclosed herein, where B is preferably a toxin. You can leave it there. 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 that includes 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 FIG. 9.

[0510] In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to the linker ARK-B disclosed herein, where B is preferably a toxin. In certain embodiments, polatuzumab, or an anti-CD79b antibody, is conjugated to a linker disclosed herein, ARK-(self-immolative moiety)-B, where B is preferably a toxin. You can leave it there. 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 PABC or methyl-amine-containing group.

[0511] In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to a 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, has a linker ARK-(NH)-(CH 3 )-O-B, where O is an ether-linked oxygen atom and B is a hydroxy-containing toxin. In certain embodiments, polatuzumab, or an anti-CD79b antibody, has a linker ARK-(NH)-(CH 3 )-O-camptothecin. In certain embodiments, polatuzumab, or an anti-CD79b antibody, has a linker ARK-(NH)-(CH 3 )-S-B, where S is a sulfur atom in a thioether bond and S is a thiol-containing toxin. In certain embodiments, polatuzumab, or an anti-CD79b antibody, has a linker ARK-(NH)-(CH 3 )-S-DM1.

[0512] In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to the linker B-ARK disclosed herein, where B is preferably a toxin. In certain embodiments, polatuzumab, or an anti-CD79b antibody, is conjugated to a linker B-(self-immolative moiety)-ARK, where B is preferably a toxin, as disclosed herein. You can leave it there. 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 that includes 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 disclosed herein, where B is preferably a toxin. In certain embodiments, polatuzumab, or an anti-CD79b antibody, is conjugated to a linker RKR-(self-immolative moiety)-B, where B is preferably a toxin, as disclosed herein. You can leave it there. 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 PABC or methyl-amine-containing group.

[0514] In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to a 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, has a linker RKR-(NH)-(CH 3 )-O-B, where O is an ether-linked oxygen atom and B is a hydroxy-containing toxin. In certain embodiments, polatuzumab, or an anti-CD79b antibody, has a linker RKR-(NH)-(CH 3 )-O-camptothecin. In certain embodiments, polatuzumab, or an anti-CD79b antibody, has a linker RKR-(NH)-(CH 3 )-S-B, where S is a sulfur atom in a thioether bond and S is a thiol-containing toxin. In certain embodiments, polatuzumab, or an anti-CD79b antibody, has a linker RKR-(NH)-(CH 3 )-S-DM1.

[0515] In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to the linker B-RKR disclosed herein, where B is preferably a toxin. In certain embodiments, polatuzumab, or an anti-CD79b antibody, is conjugated to a linker B-(self-immolative moiety)-RKR disclosed herein, where B is preferably a toxin. You can leave it there. 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 that includes 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 FIG.

[0516] In certain embodiments, polatuzumab, or an anti-CD79b antibody, is conjugated to a linker RK-Val-Cit-B disclosed herein, where B is preferably a toxin. Good too. In certain embodiments, polatuzumab, or an anti-CD79b antibody, comprises a linker RK-Val-Cit-(self-immolative moiety)-B as disclosed herein, where B is preferably a toxin. It may be conjugated to. 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 PABC or methyl-amine-containing group.

[0517] In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to a 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, has a linker RK-Val-Cit-(NH)-(CH 3 )-O-B, where O is an ether-linked oxygen atom and B is a hydroxy-containing toxin. In certain embodiments, polatuzumab, or an anti-CD79b antibody, has a linker RK-Val-Cit-(NH)-(CH 3 )-O-camptothecin. In certain embodiments, polatuzumab, or an anti-CD79b antibody, has a linker RK-Val-Cit-(NH)-(CH 3 )-S-B, where S is a sulfur atom in a thioether bond and B is a thiol-containing toxin. In certain embodiments, polatuzumab, or an anti-CD79b antibody, has a linker RK-Val-Cit-(NH)-(CH 3 )-S-DM1.

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

[0519] That is, in certain embodiments, the invention provides: a) Trastuzumab or anti-HER2 / neu antibody: and b) Structure: (Sp 1 )-RK-(Sp 2 )-B-(Sp 3 )or (Sp 1 )-B-(Sp 2 )-RK-(Sp 3 ); (In the formula, - (Sp 1 ) is a chemical spacer or is absent; - (Sp 2 ) is a chemical spacer or is absent; - (Sp 3 ) is a chemical spacer or is absent; - R is arginine or an arginine derivative or an arginine mimetic; - K is lysine or a ricin derivative or a ricin mimetic; - B is the concatenation part or payload) linker containing An antibody-linker conjugate comprising: The linker connects the γ-carboxamide group of the glutamine residue contained in trastuzumab or the anti-HER2 / neu antibody and the first group contained in the side chain of the lysine residue, lysine derivative or lysine mimetic contained in the RK motif contained in the linker. The present invention relates to an antibody-linker conjugate that is conjugated to trastuzumab or an anti-HER2 / neu antibody through an isopeptide bond formed between secondary amines.

[0520] In certain embodiments, the invention provides an antibody-payload conjugate comprising trastuzumab or an anti-HER2 / neu antibody, wherein the linker is 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, For antibody-payload conjugates that are any one of the linkers shown in FIG. 33 or FIG. 34.

[0521] Trastuzumab is commercially available as trastuzumab emtacin, an antibody-drug conjugate, sold under the name Kadcyla. Trastuzumab emtacine comprises trastuzumab, an anti-HER2 / neu antibody, and the toxin DM1 linked to trastuzumab by 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 varying drug-to-antibody ratios. Preferably, trastuzumab comprises a heavy chain as shown in SEQ ID NO: 7 and a light chain as shown in SEQ ID NO: 8. However, the present invention provides trastuzumab wherein the heavy chain and / or light chain comprises at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO: 7 and / or SEQ ID NO: 8, respectively. Also includes variants of In certain embodiments, the antibody is an anti-HER2 / neu antibody, such as, but not limited to, as disclosed in WO1998 / 006692, WO1999 / 905536, WO2003 / 087131, which are incorporated herein by reference in their entirety. There may be.

[0522] It is preferred herein that the trastuzumab, or anti-HER2 / neu antibody, is present in a glycosylated form of the antibody-linker conjugate. That is, trastuzumab or the anti-HER2 / neu antibody is preferably glycosylated at residue N297 (EU numbering). However, trastuzumab, or 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 methods according to the invention. Preferably, the linker is MTG-catalyzed conjugated to glutamine residue Q295 (EU numbering) of the antibody. However, the linker may also be conjugated to an engineered glutamine residue such as N297Q (EU numbering) and / or to any one of the glutamine-containing tags disclosed herein.

[0524] A linker that is conjugated to trastuzumab, or an anti-HER2 / neu antibody, may include a single linking moiety or payload B, or multiple linking moieties and / or payload B. 1 , B 2 It may also include.

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

[0526] In certain embodiments, the linker included in the trastuzumab-payload conjugate may include one or more payloads B. Such trastuzumab-payload conjugates may have been obtained in a two-step process, where the linker containing the linking moiety is conjugated to trastuzumab in the first step, and the payload is conjugated to the linking moiety in the second step. connected. Alternatively, the trastuzumab-payload conjugate may be obtained in a one-step process, with the payload-containing linker conjugated directly to trastuzumab.

[0527] In certain embodiments, the invention relates to antibody-drug conjugates comprising trastuzumab or anti-HER2 / neu antibodies. That is, the linker included in the antibody-drug conjugate may include one or more toxins as described herein.

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

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

[0530] Preferably, the antibody is trastuzumab, comprising a heavy chain as shown in SEQ ID NO: 7 and a light chain as shown in SEQ ID NO: 8. Thus, in certain embodiments, the invention provides an antibody-drug conjugate according to the invention, wherein the IgG antibody comprises trastuzumab or an antibody comprising a heavy chain as set forth in SEQ ID NO: 7 and a light chain as set forth in SEQ ID NO: 8. The present invention relates to an antibody-drug conjugate.

[0531] In certain embodiments, the linker conjugated to trastuzumab or anti-HER2 / neu antibody has 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) is , may be conjugated to trastuzumab, or residue Q295 of an anti-HER2 / neu antibody via a 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 disclosed herein, where B is preferably a toxin. . In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, is conjugated to the linker RKAA-(self-immolative moiety)-B, where B is preferably a toxin, as disclosed herein. May be gated. 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 PABC or methyl-amine-containing group.

[0533] In certain embodiments, trastuzumab, or anti-HER2 / neu antibody, may be conjugated to linker RKAA-PABC-B. In certain embodiments, trastuzumab, or anti-HER2 / neu antibody, may be conjugated to the linker RKAA-PABC-MMAE (see Figure 1). In certain embodiments, trastuzumab, or 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 anti-HER2 / neu antibody, may be conjugated to the linker RKAA-PABC-PNP-MMAE. In certain embodiments, the trastuzumab, or anti-HER2 / neu antibody has a linker RKAA-(NH)-(CH 3 )-O-B, where O is an ether-linked oxygen atom and B is a hydroxy-containing toxin. In certain embodiments, the trastuzumab, or anti-HER2 / neu antibody has a linker RKAA-(NH)-(CH 3 )-O-camptothecin. In certain embodiments, the trastuzumab, or anti-HER2 / neu antibody has a linker RKAA-(NH)-(CH 3 )-S-B, where S is a sulfur atom in a thioether bond and B is a thiol-containing toxin. In certain embodiments, the trastuzumab, or anti-HER2 / neu antibody has a linker RKAA-(NH)-(CH 3 )-S-DM1.

[0534] In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, may be conjugated to a linker B-RKAA disclosed herein, where B is preferably a toxin. . In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, is conjugated to the linker B-(self-immolative moiety)-RKAA disclosed herein, where B is preferably a toxin. May be gated. 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 that includes 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 FIG. 9.

[0535] In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, may be conjugated to a linker RKA-B disclosed herein, where B is preferably a toxin. . In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, is conjugated to the linker RKA-(self-immolative moiety)-B, where B is preferably a toxin, as disclosed herein. May be gated. 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 PABC or methyl-amine-containing group.

[0536] In certain embodiments, trastuzumab, or anti-HER2 / neu antibody, may be conjugated to 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 anti-HER2 / neu antibody, may be conjugated to the linker RKA-PABC-maytansine. In certain embodiments, trastuzumab, or anti-HER2 / neu antibody, may be conjugated to the linker RKA-MMAE. In certain embodiments, trastuzumab, or anti-HER2 / neu antibody, may be conjugated to the linker RKA-PABC-PNP-MMAE. In certain embodiments, the trastuzumab, or anti-HER2 / neu antibody has a linker RKA-(NH)-(CH 3 )-O-B, where O is an ether-linked oxygen atom and B is a hydroxy-containing toxin. In certain embodiments, the trastuzumab, or anti-HER2 / neu antibody has a linker RKA-(NH)-(CH 3 )-O-camptothecin. In certain embodiments, the trastuzumab, or anti-HER2 / neu antibody has a linker RKA-(NH)-(CH 3 )-S-B, where S is a sulfur atom in a thioether bond and B is a thiol-containing toxin. In certain embodiments, the trastuzumab, or anti-HER2 / neu antibody has a linker RKA-(NH)-(CH 3 )-S-DM1.

[0537] In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, may be conjugated to a linker B-RKA disclosed herein, where B is preferably a toxin. . In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, is conjugated to the linker B-(self-immolative moiety)-RKA, where B is preferably a toxin, as disclosed herein. May be gated. 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 that includes 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 FIG. 9.

[0538] In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, may be conjugated to the linker ARK-B disclosed herein, where B is preferably a toxin. . In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, is conjugated to the linker ARK-(self-immolative moiety)-B, where B is preferably a toxin, as disclosed herein. May be gated. 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 PABC or methyl-amine-containing group.

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

[0540] In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, may be conjugated to the linker B-ARK disclosed herein, where B is preferably a toxin. . In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, is conjugated to the linker B-(self-immolative moiety)-ARK, where B is preferably a toxin, as disclosed herein. May be gated. 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 that includes an ortho-hydroxy-protected aryl sulfate (OHPAS) moiety.

[0541] In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, may be conjugated to a linker RKR-B disclosed herein, where B is preferably a toxin. . In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, is conjugated to the linker RKR-(self-immolative moiety)-B, where B is preferably a toxin, as disclosed herein. May be gated. 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 PABC or methyl-amine-containing group.

[0542] In certain embodiments, trastuzumab, or anti-HER2 / neu antibody, may be conjugated to linker RKR-PABC-B. In certain embodiments, trastuzumab, or 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 anti-HER2 / neu antibody, may be conjugated to the linker RKR-MMAE. In certain embodiments, trastuzumab, or anti-HER2 / neu antibody, may be conjugated to the linker RKR-PABC-PNP-MMAE. In certain embodiments, the trastuzumab, or anti-HER2 / neu antibody has a linker RKR-(NH)-(CH 3 )-O-B, where O is an ether-linked oxygen atom and B is a hydroxy-containing toxin. In certain embodiments, the trastuzumab, or anti-HER2 / neu antibody has a linker RKR-(NH)-(CH 3 )-O-camptothecin. In certain embodiments, the trastuzumab, or anti-HER2 / neu antibody has a linker RKR-(NH)-(CH 3 )-S-B, where S is a sulfur atom in a thioether bond and S is a thiol-containing toxin. In certain embodiments, the trastuzumab, or anti-HER2 / neu antibody has a linker RKR-(NH)-(CH 3 )-S-DM1.

[0543] In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, may be conjugated to the linker B-RKR disclosed herein, where B is preferably a toxin. . In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, is conjugated to the linker B-(self-immolative moiety)-RKR, where B is preferably a toxin, as disclosed herein. May be gated. 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 that includes 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 FIG.

[0544] In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, is conjugated to the linker RK-Val-Cit-B disclosed herein, where B is preferably a toxin. You can leave it there. In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, comprises a linker RK-Val-Cit-(self-immolative moiety)-B as disclosed herein, where B can be a toxin. preferred). 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 PABC or methyl-amine-containing group.

[0545] In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, may be conjugated to a linker RK-Val-Cit-PABC-B. In certain embodiments, trastuzumab, or anti-HER2 / neu antibody, may be conjugated to the linker RK-Val-Cit-PABC-MMAE. In certain embodiments, trastuzumab, or anti-HER2 / neu antibody, may be conjugated to the linker RK-Val-Cit-PABC-maytansine. In certain embodiments, trastuzumab, or anti-HER2 / neu antibody, may be conjugated to the linker RK-Val-Cit-MMAE. In certain embodiments, trastuzumab, or anti-HER2 / neu antibody, may be conjugated to the linker RK-Val-Cit-PABC-PNP-MMAE. In certain embodiments, trastuzumab, or the anti-HER2 / neu antibody, has a linker RK-Val-Cit-(NH)-(CH 3 )-O-B, where O is an ether-linked oxygen atom and B is a hydroxy-containing toxin. In certain embodiments, trastuzumab, or the anti-HER2 / neu antibody, has a linker RK-Val-Cit-(NH)-(CH 3 )-O-B camptothecin. In certain embodiments, trastuzumab, or the anti-HER2 / neu antibody, has a linker RK-Val-Cit-(NH)-(CH 3 )-S-B, where S is a sulfur atom in a thioether bond and B is a thiol-containing toxin. In certain embodiments, trastuzumab, or the anti-HER2 / neu antibody, has a linker RK-Val-Cit-(NH)-(CH 3 )-S-DM1.

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

[0547] That is, in certain embodiments, the invention provides: a) enfortumab or anti-Nectin-4 antibody; and b) Structure: (Sp 1 )-RK-(Sp 2 )-B-(Sp 3 )or (Sp 1 )-B-(Sp 2 )-RK-(Sp 3 ); (In the formula, - (Sp 1 ) is a chemical spacer or is absent; - (Sp 2 ) is a chemical spacer or is absent; - (Sp 3 ) is a chemical spacer or is absent; - R is arginine or an arginine derivative or an arginine mimetic; - K is lysine or a ricin derivative or a ricin mimetic; - B is the concatenation part or payload) linker containing An antibody-linker conjugate comprising: The linker is contained in the γ-carboxamide group of the glutamine residue contained in enfortumab or the anti-Nectin-4 antibody and in the side chain of a lysine residue, lysine derivative or lysine mimetic contained in the RK motif contained in the linker. The present invention relates to an antibody-linker conjugate that is conjugated to enfortumab or an anti-Nectin-4 antibody through an isopeptide bond formed between primary amines.

[0548] In certain embodiments, the invention provides an antibody-payload conjugate comprising enfortumab or an anti-Nectin-4 antibody, wherein the linker is , 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, any one of the linkers shown in FIG. 33 or FIG. 34.

[0549] Enfortumab is commercially available as the antibody-drug conjugate enfortumab vedotin, sold under the name Padcev. Enfortumab vedotin is a combination of the anti-Nectin-4 antibody enfortumab and the linker maleimidocaproyl-L-valine-L-citrulline-PABC-MMAE (mc-vc-PABC- MMAE). 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, which is incorporated herein by reference in its entirety.

[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 provides that the heavy chain and / or the light chain contain at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO: 9 and / or SEQ ID NO: 10, respectively. Variants of fortumab are also included. In particular, the antibody may contain any of the sequence variations disclosed in WO2012 / 047724. In certain embodiments, the light chain of enfortumab may include 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 the enfortumab, or anti-Nectin-4 antibody, is present in a glycosylated form of the antibody-linker conjugate. That is, enfortumab or the anti-Nectin-4 antibody is preferably glycosylated at residue N297 (EU numbering). However, enfortumab, or anti-Nectin-4 antibodies, may also be deglycosylated as described herein.

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

[0553] A linker that is conjugated to enfortumab, or an anti-Nectin-4 antibody, may include a single linking moiety or payload B, or multiple linking moieties and / or payload B. 1 , B 2 It may also include.

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

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

[0556] In certain embodiments, the invention relates to antibody-drug conjugates comprising enfortumab or anti-Nectin-4 antibodies. That is, the linker included in the antibody-drug conjugate may include one or more toxins as described herein.

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

[0558] That is, in certain embodiments, the invention provides: a) Enfortumab or anti-Nectin-4 antibody: and b) a linker comprising drug moiety B, wherein drug moiety B is RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4) or RK-Val-Cit; An antibody-drug conjugate comprising a linker covalently linked to an amino acid sequence selected from the group consisting of (SEQ ID NO: 54), The linker is C of the antibody. H Enfortumab or An antibody-drug conjugate conjugated to an anti-Nectin-4 antibody.

[0559] Preferably, the antibody is enfortumab, comprising a heavy chain as shown in SEQ ID NO: 9 and a light chain as shown in SEQ ID NO: 10. Thus, in certain embodiments, the invention provides an antibody-drug conjugate according to the invention, wherein the IgG antibody comprises enfortumab or a heavy chain as set forth in SEQ ID NO: 9 and a light chain as set forth in SEQ ID NO: 10. The present invention relates to an antibody-drug conjugate, which is an antibody comprising an antibody.

[0560] In certain embodiments, the linker conjugated to enfortumab, or the anti-Nectin-4 antibody, has 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) is , may be conjugated to enfortumab, or residue Q295 of the anti-Nectin-4 antibody, via a primary amine contained in residue K.

[0561] In certain embodiments, enfortumab, or an anti-Nectin-4 antibody, is conjugated to a linker RKAA-B disclosed herein, where B is preferably a toxin. Good too. In certain embodiments, enfortumab, or an anti-Nectin-4 antibody, comprises a linker RKAA-(self-immolative moiety)-B as disclosed herein, where B is preferably a toxin. It may be conjugated to. 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 PABC or methyl-amine-containing group.

[0562] In certain embodiments, enfortumab, or an anti-Nectin-4 antibody, may be conjugated to linker RKAA-PABC-B. In certain embodiments, enfortumab, or an anti-Nectin-4 antibody, may be conjugated to the linker RKAA-PABC-MMAE (see Figure 1). In certain embodiments, enfortumab, or an anti-Nectin-4 antibody, may be conjugated to the linker RKAA-PABC-maytansine (see Figure 8). In certain embodiments, enfortumab, or an anti-Nectin-4 antibody, may be conjugated to the linker RKAA-MMAE. In certain embodiments, enfortumab, or an anti-Nectin-4 antibody, may be conjugated to the linker RKAA-PABC-PNP-MMAE. In certain embodiments, enfortumab, or the anti-Nectin-4 antibody, has a linker RKAA-(NH)-(CH 3 )-O-B, where O is an ether-linked oxygen atom and B is a hydroxy-containing toxin. In certain embodiments, enfortumab, or the anti-Nectin-4 antibody, has a linker RKAA-(NH)-(CH 3 )-O-camptothecin. In certain embodiments, enfortumab, or the anti-Nectin-4 antibody, has a linker RKAA-(NH)-(CH 3 )-S-B, where S is a sulfur atom in a thioether bond and B is a thiol-containing toxin. In certain embodiments, enfortumab, or the anti-Nectin-4 antibody, has a linker RKAA-(NH)-(CH 3 )-S-DM1.

[0563] In certain embodiments, enfortumab, or an anti-Nectin-4 antibody, is conjugated to a linker B-RKAA disclosed herein, where B is preferably a toxin. Good too. In certain embodiments, enfortumab, or an anti-Nectin-4 antibody, comprises a linker B-(self-immolative moiety)-RKAA disclosed herein, where B is preferably a toxin. It may be conjugated to. 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 that includes 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 FIG. 9.

[0564] In certain embodiments, enfortumab, or an anti-Nectin-4 antibody, is conjugated to a linker RKA-B disclosed herein, where B is preferably a toxin. Good too. In certain embodiments, enfortumab, or an anti-Nectin-4 antibody, comprises a linker RKA-(self-immolative moiety)-B as disclosed herein, where B is preferably a toxin. It may be conjugated to. 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 PABC or methyl-amine-containing group.

[0565] In certain embodiments, enfortumab, or an anti-Nectin-4 antibody, may be conjugated to a linker RKA-PABC-B. In certain embodiments, enfortumab, or an anti-Nectin-4 antibody, may be conjugated to the linker RKA-PABC-MMAE (see Figure 2). In certain embodiments, enfortumab, or an anti-Nectin-4 antibody, may be conjugated to the linker RKA-PABC-maytansine. In certain embodiments, enfortumab, or an anti-Nectin-4 antibody, may be conjugated to the linker RKA-MMAE. In certain embodiments, enfortumab, or an anti-Nectin-4 antibody, may be conjugated to the linker RKA-PABC-PNP-MMAE. In certain embodiments, enfortumab, or the anti-Nectin-4 antibody, has a linker RKA-(NH)-(CH 3 )-O-B, where O is an ether-linked oxygen atom and B is a hydroxy-containing toxin. In certain embodiments, enfortumab, or the anti-Nectin-4 antibody, has a linker RKA-(NH)-(CH 3 )-O-camptothecin. In certain embodiments, enfortumab, or the anti-Nectin-4 antibody, has a linker RKA-(NH)-(CH 3 )-S-B, where S is a sulfur atom in a thioether bond and B is a thiol-containing toxin. In certain embodiments, enfortumab, or the anti-Nectin-4 antibody, has a linker RKA-(NH)-(CH 3 )-S-DM1.

[0566] In certain embodiments, enfortumab, or an anti-Nectin-4 antibody, is conjugated to a linker B-RKA disclosed herein, where B is preferably a toxin. Good too. In certain embodiments, enfortumab, or an anti-Nectin-4 antibody, comprises a linker B-(self-immolative moiety)-RKA as disclosed herein, where B is preferably a toxin. It may be conjugated to. 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 that includes 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 FIG. 9.

[0567] In certain embodiments, enfortumab, or an anti-Nectin-4 antibody, is conjugated to a linker ARK-B disclosed herein, where B is preferably a toxin. Good too. In certain embodiments, enfortumab, or an anti-Nectin-4 antibody, is a linker disclosed herein, ARK-(self-immolative moiety)-B, where B is preferably a toxin. It may be conjugated to. 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 PABC or methyl-amine-containing group.

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

[0569] In certain embodiments, enfortumab, or an anti-Nectin-4 antibody, is conjugated to a linker B-ARK disclosed herein, where B is preferably a toxin. Good too. In certain embodiments, enfortumab, or the anti-Nectin-4 antibody, comprises a linker disclosed herein, B-(self-immolative moiety)-ARK, where B is preferably a toxin. It may be conjugated to. 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 that includes an ortho-hydroxy-protected aryl sulfate (OHPAS) moiety.

[0570] In certain embodiments, enfortumab, or an anti-Nectin-4 antibody, is conjugated to a linker RKR-B disclosed herein, where B is preferably a toxin. Good too. In certain embodiments, enfortumab, or an anti-Nectin-4 antibody, comprises a linker RKR-(self-immolative moiety)-B as disclosed herein, where B is preferably a toxin. It may be conjugated to. 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 PABC or methyl-amine-containing group.

[0571] In certain embodiments, enfortumab, or an anti-Nectin-4 antibody, may be conjugated to 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 the anti-Nectin-4 antibody, has a linker RKR-(NH)-(CH 3 )-O-B, where O is an ether-linked oxygen atom and B is a hydroxy-containing toxin. In certain embodiments, enfortumab, or the anti-Nectin-4 antibody, has a linker RKR-(NH)-(CH 3 )-O-camptothecin. In certain embodiments, enfortumab, or the anti-Nectin-4 antibody, has a linker RKR-(NH)-(CH 3 )-S-B, where S is a sulfur atom in a thioether bond and S is a thiol-containing toxin. In certain embodiments, enfortumab, or the anti-Nectin-4 antibody, has a linker RKR-(NH)-(CH 3 )-S-DM1.

[0572] In certain embodiments, enfortumab, or an anti-Nectin-4 antibody, is conjugated to a linker B-RKR disclosed herein, where B is preferably a toxin. Good too. In certain embodiments, enfortumab, or an anti-Nectin-4 antibody, comprises a linker B-(self-immolative moiety)-RKR as disclosed herein, where B is preferably a toxin. It may be conjugated to. 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 that includes 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 FIG.

[0573] In certain embodiments, enfortumab, or an anti-Nectin-4 antibody, is conjugated to the linker RK-Val-Cit-B disclosed herein, where B is preferably a toxin. May be gated. In certain embodiments, enfortumab, or the anti-Nectin-4 antibody, has a linker RK-Val-Cit-(self-immolative moiety)-B as disclosed herein, where B is a toxin. (preferably). 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 PABC or methyl-amine-containing group.

[0574] In certain embodiments, enfortumab, or an anti-Nectin-4 antibody, may be conjugated to a 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 the anti-Nectin-4 antibody, has a linker RK-Val-Cit-(NH)-(CH 3 )-O-B, where O is an ether-linked oxygen atom and B is a hydroxy-containing toxin. In certain embodiments, enfortumab, or the anti-Nectin-4 antibody, has a linker RK-Val-Cit-(NH)-(CH 3 )-O-B camptothecin. In certain embodiments, enfortumab, or the anti-Nectin-4 antibody, has a linker RK-Val-Cit-(NH)-(CH 3 )-S-B, where S is a sulfur atom in a thioether bond and B is a thiol-containing toxin. In certain embodiments, enfortumab, or the anti-Nectin-4 antibody, has a linker RK-Val-Cit-(NH)-(CH 3 )-S-DM1.

[0575] Furthermore, the invention relates to linker constructs containing the motif RK. Linker constructs according to the invention can be used for conjugation of a wide variety of antibodies. Because of the highly conserved conjugation site Q295, linker conjugates according to the 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 high-efficiency conjugation of glycosylated antibodies, resulting in high conjugation efficiency even when containing bulky payloads such as toxins. can be obtained.

[0576] Thus, in certain embodiments, the present invention provides the structure: (Sp 1 )-RK-(Sp 2 )-B-(Sp 3 )or (Sp 1 )-B-(Sp 2 )-RK-(Sp 3 ); (In the formula, - (Sp 1 ) is a chemical spacer or is absent; - (Sp 2 ) is a chemical spacer or is absent; - (Sp 3 ) is a chemical spacer or is absent; - R is arginine or an arginine derivative or an arginine mimetic; - K is lysine or a ricin derivative or a ricin mimetic; - B is the concatenation part or payload) Concerning linker constructs containing.

[0577] It is understood that the linker construct may have the same structure and / or characteristics as the linker disclosed above for the method according to the invention, the antibody-linker conjugate according to the invention and / or the antibody-drug conjugate according to the invention. should be understood.

[0578] In a particular embodiment, the invention provides a linker construct according to the invention, comprising a chemical spacer (Sp 1 ), (Sp 2 ) and (Sp 3 ) relate to linker constructs, each independently containing from 0 to 12 amino acid residues.

[0579] In certain embodiments, the invention provides linker constructs according to the invention, wherein the linkers are 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 Concerning linker constructs containing the following amino acid residues:

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

[0581] In certain embodiments, the invention relates to linker constructs according to the invention, wherein the linker does not contain negatively charged amino acid residues.

[0582] In a particular embodiment, the invention provides a linker construct according to the invention, wherein the linker comprises the amino acid 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).

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

[0584] In a particular embodiment, the invention provides a linker construct according to the invention, wherein the linking moiety B is - bioorthogonal marker group, or - Non-bioorthogonal entities for cross-linking and linker constructs, including linker constructs.

[0585] In a particular embodiment, the invention provides a linker construct according to the invention, wherein the bioorthogonal marker group or the non-bioorthogonal entity for crosslinking is - -N-N≡N, or -N 3 ; - Lys(N 3 ); - Tetrazine; - alkynes; - Distorted cyclooctyne; -BCN; - distorted alkenes; - photoreactive groups; - aldehydes; - Acyl trifluoroborate; - Proteolytic agents (“PROTACs”); - cyclopentadiene / spirolocyclopentadiene; - thioselective electrophile; - -SH; and - Cysteine Linker constructs consisting of or comprising at least one molecule or moiety selected from the group consisting of:

[0586] In a particular embodiment, the invention provides a linker construct according to the invention consisting of or comprising the structure RKAA-B or B-RKAA, in particular where B is Lys(N 3 ) or cysteine.

[0587] Thus, in certain embodiments, the invention relates to linkers comprising the structure RKAA-B or B-RKAA, where B is a linking moiety. It is to be understood that B may be any linking moiety known in the art and / or disclosed herein. In certain embodiments, B is a thiol-containing linking moiety, e.g. cysteine, an azide-containing linking moiety, e.g. Lys(N 3 ), or a linking moiety containing tetrazine. It should be understood that a linker comprising structure RKAA-B or B-RKAA may include additional amino acid residues, linking moieties, payloads and / or other chemical groups, such as, but not limited to, PEG moieties. In certain embodiments, the linker construct consists of the structure RKAA-B or B-RKAA.

[0588] In a particular embodiment, the invention provides a linker construct according to the invention consisting of or comprising the structure RKA-B or B-RKA, in particular where B is Lys(N 3 ) or cysteine.

[0589] Thus, in certain embodiments, the invention relates to linkers comprising the structure RKA-B or B-RKA, where B is a linking moiety. It is to be understood that B may be any linking moiety known in the art and / or disclosed herein. In certain embodiments, B is a thiol-containing linking moiety, e.g. cysteine, an azide-containing linking moiety, e.g. Lys(N 3 ), or a linking moiety containing tetrazine. It should be understood that a linker comprising structure RKA-B or B-RKA may contain additional amino acid residues, linking moieties, payloads and / or other chemical groups, such as, but not limited to, PEG moieties. In certain embodiments, the linker construct consists of the structure RKA-B or B-RKA.

[0590] In a particular embodiment, the invention provides a linker construct according to the invention consisting of or comprising the structure ARK-B or B-ARK, in particular B is Lys(N 3 ) or cysteine.

[0591] Thus, in certain embodiments, the present invention relates to linkers comprising the structure ARK-B or B-ARK, where B is a linking moiety. It is to be understood that B may be any linking moiety known in the art and / or disclosed herein. In certain embodiments, B is a thiol-containing linking moiety, e.g. cysteine, an azide-containing linking moiety, e.g. Lys(N 3 ), or a linking moiety containing tetrazine. It should be understood that structure ARK-B or a linker comprising B-ARK may include additional amino acid residues, linking moieties, payloads and / or other chemical groups, such as, but not limited to, PEG moieties. In certain embodiments, the linker construct consists of the structure ARK-B or B-ARK.

[0592] In a particular embodiment, the invention provides a linker construct according to the invention consisting of or comprising the structure RKR-B or B-RKR, in particular where B is Lys(N 3 ) or cysteine.

[0593] Thus, in certain embodiments, the invention relates to linkers comprising the structure RKR-B or B-RKR, where B is a linking moiety. It is to be understood that B may be any linking moiety known in the art and / or disclosed herein. In certain embodiments, B is a thiol-containing linking moiety, e.g. cysteine, an azide-containing linking moiety, e.g. Lys(N 3 ), or a linking moiety containing tetrazine. It should be understood that a linker comprising structure RKR-B or B-RKR may include additional amino acid residues, linking moieties, payloads and / or other chemical groups, such as, but not limited to, PEG moieties. In certain embodiments, the linker construct consists of the structure RKR-B or B-RKR.

[0594] In a particular embodiment, the invention relates to a linker construct according to the invention, wherein B is a payload.

[0595] In a particular embodiment, the invention provides a linker construct according to the invention, wherein the payload is - toxin; - Cytokines; - growth factors; - radionuclides; - Hormones; - antiviral; - antibacterial agent; - Fluorescent dye: - Immunomodulators / immunostimulants; - half-life increasing part; - solubility in...

Claims

1. a) an IgG antibody; and b) a linker comprising a drug moiety B, wherein said 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).

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. 2. The antibody-drug conjugate of claim 1, wherein the drug moiety B is linked to the N- or C-terminus of the amino acid sequence contained in the linker by a self-immolative moiety.

3. 3. The antibody-drug conjugate of claim 2, wherein the self-immolative moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety.

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

5. The antibody-drug conjugate of claim 4, wherein the IgG antibody is glycosylated at residue N297 (EU numbering) of the C H 2 domain.

6. The antibody-drug conjugate of any one of claims 1 to 5, wherein the IgG antibody is an IgG1 antibody.

7. The antibody-drug conjugate of any one of claims 1 to 6, 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.

8. The antibody-drug conjugate of any one of claims 1 to 6, 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.

9. The antibody-drug conjugate of any one of claims 1 to 6, 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.

10. 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 9, selected from the group consisting of:

11. - the 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 10, wherein

12. 12. The antibody-drug conjugate of any one of claims 1 to 11, wherein the linker has the structure RKAA-PABC-B, RKA-PABC-B, ARK-PABC-B, or RK-Val-Cit-PABC-B.

13. The antibody-drug conjugate of claim 12, wherein B is an auristatin or a maytansinoid.

14. The antibody-drug conjugate of claim 13, wherein the auristatin is MMAE and the maytansinoid is DM1 or maytansine.

15. An antibody-drug conjugate described in any one of claims 1 to 14, wherein the linker has the following structure: 【Chemical 1】 16. A method for treating a cancer cell line comprising administering to a patient a therapeutically effective amount of an IgG antibody ... b) a linker having the above structure 【Chemistry 2】 1. An antibody-drug conjugate comprising: An antibody-drug conjugate, wherein the linker is conjugated to the IgG antibody by an isopeptide bond formed between the γ-carboxamide group of glutamine residue Q295 (EU numbering) in the C H 2 domain of the antibody and a primary amine contained in the side chain of a lysine residue contained in the linker.

17. A method for treating a cancer cell line comprising administering to a patient a therapeutically effective amount of an IgG antibody, comprising administering to a patient a therapeutically effective amount of an IgG antibody ... b) a linker having the above structure 【Chemistry 3】 1. An antibody-drug conjugate comprising: An antibody-drug conjugate, wherein the linker is conjugated to the IgG antibody by an isopeptide bond formed between the γ-carboxamide group of glutamine residue Q295 (EU numbering) in the C H 2 domain of the antibody and a primary amine contained in the side chain of a lysine residue contained in the linker.

18. A method for treating a leukemia comprising administering to a patient a therapeutically effective amount of an IgG antibody, comprising administering to a patient a therapeutically effective amount of an IgG antibody, the method ... b) a linker having the above structure 【Chemistry 4】 1. An antibody-drug conjugate comprising: An antibody-drug conjugate, wherein the linker is conjugated to the IgG antibody by an isopeptide bond formed between the γ-carboxamide group of glutamine residue Q295 (EU numbering) in the C H 2 domain of the antibody and a primary amine contained in the side chain of a lysine residue contained in the linker.

19. 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 the drug moiety Linker constructs.

20. wherein the drug moiety 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 20. The linker construct of claim 19, wherein the linker construct is at least one selected from the group consisting of:

21. The pyrrolobenzodiazepine of claim 21, wherein the 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 21. The linker construct of claim 20, wherein:

22. A linker construct according to any one of claims 19 to 21, wherein the drug moiety B is an auristatin or a maytansinoid.

23. The linker construct described in claim 22, wherein the auristatin is MMAE and the maytansinoid is DM1 or maytansine.

24. The linker of claim 23, wherein the linker has the structure: 【Chemistry 5】 24. The linker construct of any one of claims 19 to 23, having:

25. Use of a linker construct according to any one of claims 19 to 24 in the production of antibody-drug conjugates by microbial transglutaminase.

26. 26. The use according to claim 25, wherein the antibody is an IgG antibody.

27. 27. The use according to claim 25 or 26, wherein the antibody is polatuzumab or trastuzumab or enfortumab.

28. A pharmaceutical composition comprising the antibody drug-conjugate of any one of claims 1 to 18, wherein the pharmaceutical composition comprises at least one pharmaceutically acceptable ingredient.

29. 29. The pharmaceutical composition of claim 28, comprising at least one additional therapeutically active agent.

30. A composition comprising an antibody-drug conjugate according to any one of claims 1 to 18, or a pharmaceutical composition according to claim 28 or 29, for use in therapy and / or diagnosis.

31. A method for treating a neoplastic disease, a neurological disease, an autoimmune disease, an inflammatory disease, or an infectious disease. - 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 18, or the pharmaceutical composition of claim 28 or 29, for use in treating a patient.

32. The composition for use of claim 31, 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.

33. 33. The composition for use of claim 32, wherein the cancer associated with B cells is non-Hodgkin's lymphoma or diffuse large B-cell lymphoma.

34. The composition for use according to claim 32 or 33, wherein the antibody-drug conjugate or the pharmaceutical composition is administered in combination with bendamustine and / or rituximab.

35. The composition for use of claim 31, 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.

36. The composition for use described in claim 35, wherein the HER2-positive cancer is HER2-positive breast cancer, gastric cancer, ovarian cancer or lung cancer.

37. The composition for use according to claim 35 or 36, wherein the antibody-drug conjugate or the pharmaceutical composition is administered in combination with lapatinib, capecitabine and / or a taxane.

38. The composition for use of claim 31, 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.

39. The composition for use described in claim 38, wherein the Nectin-4 positive cancer is Nectin-4 positive pancreatic cancer, lung cancer, bladder cancer or breast cancer.

40. The composition for use of claim 38 or 39, wherein the antibody-drug conjugate or pharmaceutical composition is administered in combination with a cisplatin-based chemotherapy agent and / or pembrolizumab.

41. A method for treating a neoplastic disease, a neurological disease, an autoimmune disease, an inflammatory disease, or an infectious disease. - Are you suffering from - be at risk of developing it, and / or - diagnosed with 20. Use of an antibody-drug conjugate according to any one of claims 1 to 18, or a pharmaceutical composition according to claim 18 or 19, for the manufacture of a medicament for the treatment of a patient.

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