CN116801910A - Means and methods for producing antibody-linker conjugates - Google Patents

Means and methods for producing antibody-linker conjugates Download PDF

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CN116801910A
CN116801910A CN202180073108.1A CN202180073108A CN116801910A CN 116801910 A CN116801910 A CN 116801910A CN 202180073108 A CN202180073108 A CN 202180073108A CN 116801910 A CN116801910 A CN 116801910A
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antibody
linker
conjugate
moiety
amino acid
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P·施皮歇尔
P·普罗布斯特
I·阿廷格-托勒
R·伯特兰
R·斯塔克
D·格拉布罗夫斯基
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Alaris Biotechnology Co ltd
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Alaris Biotechnology Co ltd
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Priority claimed from PCT/EP2021/079560 external-priority patent/WO2022084560A1/en
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Abstract

The present invention relates to a method for producing an antibody-payload conjugate by Microbial Transglutaminase (MTG). The method comprises the steps of incorporating the structure (shown in the N.fwdarw.C direction) (Sp 1 )‑RK‑(Sp 2 )‑B‑(Sp 3 ) Or (Sp) 1 )‑B‑(Sp 2 )‑RK‑(Sp 3 ) A step of coupling 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 arginine jigA mimetic; k is lysine or a lysine derivative or lysine mimetic; b is a linking moiety or payload; and wherein the linker is coupled to the Gln residue comprised in the antibody via a primary amine comprised in a side chain of the lysine residue, lysine derivative or lysine mimetic. Further, the invention relates to antibody-linker conjugates, antibody-drug conjugates and linker constructs comprising an RK motif.

Description

Means and methods for producing antibody-linker conjugates
The present invention relates to a method for producing an antibody-linker conjugate 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.
Antibody-drug conjugates (ADCs) generally consist of an antibody and a small molecule drug conjugated to the antibody via a chemical linker. After decades of preclinical and clinical studies, a range of ADCs have been approved for the treatment of specific tumor types, such as velutinab for recurrent hodgkin's lymphoma and systemic anaplastic large cell lymphoma (brentuximab vedotin,) Gituzumab for acute myelogenous leukemia (gemtuzumab ozogamicin,/v)>) Enmetrastuzumab for HER2 positive metastatic breast cancerAnti (ado-trastuzumab emtansine,)>) Ottotuzumab for B-cell malignancies (inotuzumab ozogamicin,/I)>) And the most recent poloxamers of poloxamers Shan Kangwei (polatuzumab vedotin-piiq,). Recently, enrolment Shan Kangwei statins (enfortumab vedotin,/j>) Detrastuzumab (trastuzumab deruxtecan,/->) The binding agent of the gorgon Sha Tuozhu mab (sacituzumab govitecan,) And Bei Lan Tamab Mo Futing (belantamab mafadotin,>) Has been approved for marketing. For reviews of ADCs see, for example (Zhao P et al 2020,Acta Pharmaceutica Sinica B,10, 1589-1600). While many ADCs have shown impressive anticancer activity, many patients do not respond to these treatments, experience serious side effects before signs of efficacy or relapse after a certain period of time, and thus there remains a need in medicine for new forms of ADCs with favorable drug-like properties that can be produced at reasonable cost in sufficient quantity and quality to support drug development and are suitable as therapeutic agents.
The key step in the preparation of ADCs is the covalent coupling step of the payload to the antibody. Most ADCs in current clinical development are performed by coupling to endogenous lysine or cysteine residues of the antibody, carefully controlling the average degree of modification to produce an average drug-to-antibody ratio (DAR) in the range of 3.5-4.0. Historically, this ratio was chosen based on (a) minimizing the amount of unconjugated antibody and (b) avoiding species in the mixture using very high DARs, which can be problematic in manufacturing and formulation due to higher hydrophobicity and lower solubility (Lambert JM and Berkenbilt a.,2018, annu. Rev. Med.69, 191-207), and generally results in poor pharmacokinetic properties (Lyon RP et al 2015,Nat Biotechnol,33, 733-735). Recently, a variety of genetic, chemical, and enzymatic methods have been developed for site-specific conjugation, which can achieve DAR of 2 (or 4) while avoiding under-or over-modification of antibodies. These methods are outlined by Yamada et al (reviewed in Kei Yamada and Yuji Ito,2019, chemBiochem, 2729-2739).
Enzymatic coupling has shown great interest because these coupling reactions are generally rapid, site-specific, and can be performed under physiological conditions. Among the enzymes available, microbial Transglutaminase (MTG) from the species streptomyces mobaraensis (Streptomyces mobaraensis) is of increasing interest as an attractive alternative to conventional chemical protein coupling of functional moieties, including antibodies. MTG catalyzes the transamidation reaction between the "reactive" glutamine of a protein or peptide and the "reactive" lysine residue of the protein or peptide under physiological conditions, the latter also being a simple low molecular weight primary amine such as 5-aminopentyl (Jeger S et al, 2010, angel. Chem. Int. Ed.,49, 9995-9997).
Jeger et al describe that conjugation of antibodies using transglutaminase as the enzyme occurs at residue Q295, however, conjugation is only possible when the glycan moiety at asparagine residue 297 (N297) is removed with PNGase F, whereas glycosylated antibodies are not efficiently conjugated (coupling efficiency less than 20%) (Jeger S et al, 2010, angelw. Chem. Int, ed.,49, 9995-9997; mindt T et al, 2008, bioconj Chem,9, 271-278).
Other methods of producing ADCs by MTG are based on the use of non-glycosylated (aglycosylated) antibodies, in which residue N297 is replaced with an amino acid residue that is not amenable to glycosylation. However, substitution of N297 for another amino acid may lead to unwanted effects, as it may affect the overall stability of the whole Fc domain (Subedi GP and Barb AW.,2015, structure,23, 1573-1583) and the efficacy of the whole conjugate. As a result, increased aggregation and reduced solubility of antibodies may result, which becomes particularly important for hydrophobic payloads. Further, the glycan present at N297 has important immunomodulatory effects because it triggers effector functions such as Antibody Dependent Cellular Cytotoxicity (ADCC), etc. These immunomodulatory effects will be lost in deglycosylation or any other method described above to obtain non-glycosylated antibodies. Furthermore, any sequence modification of the established antibodies may also lead to regulatory problems, which is problematic, since accepted and clinically validated antibodies are typically used as the origin of ADC coupling.
More recently, spycher et al disclose a transglutaminase-based coupling method that does not require prior deglycosylation of the antibody for payload coupling (Spycher et al, WO 2019/057772). The ability to couple naturally glycosylated antibodies provides significant advantages in terms of production: in terms of Good Manufacturing Process (GMP), an enzymatic deglycosylation step is undesirable, as it must be ensured that both the deglycosylating enzyme (e.g. PNGase F) and the cleaved glycans are removed from the reaction mixture. Furthermore, genetic engineering of the antibody for payload attachment is not required, so that sequence insertions that may increase immunogenicity and reduce the overall stability of the antibody can be avoided.
In view of the foregoing, there remains a need in the art for improved methods for generating ADCs with high coupling efficiency.
Further, there is a need in the art for novel ADCs with improved therapeutic and/or pharmacokinetic properties and highly defined (defined) drug-to-antibody ratios.
Disclosure of Invention
The invention is characterized by the embodiments and claims provided herein. In particular, the invention relates in particular to the following embodiments:
1. production of antibody-linker conjugates by Microbial Transglutaminase (MTG) The method comprising the steps of (a) introducing a compound comprising (as shown in the N.fwdarw.C direction) (Sp 1 )-RK-(Sp 2 )-B-(Sp 3 ) Or (Sp) 1 )-B-(Sp 2 )-RK-(Sp 3 ) A step of coupling a linker of the structure 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 arginine mimetic;
k is lysine or a lysine derivative or a lysine mimetic;
b is a linking part or payload;
and wherein the linker is coupled to the Gln residue comprised in the antibody via a primary amine comprised in a side chain of the lysine residue, lysine derivative or lysine mimetic.
2. The method according to embodiment 1, wherein the chemical spacer (Sp 1 )、(Sp 2 ) Sum (Sp) 3 ) Each independently comprises 0 to 12 amino acid residues.
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.
4. The method according to any one of embodiments 1 to 3, wherein the net charge of the linker is neutral or positive.
5. The method according to any one of embodiments 1 to 4, wherein the linker does not comprise negatively charged amino acid residues.
6. The method according to any 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).
7. The method according to any 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).
8. The method according to any of embodiments 1 to 7, wherein the linker comprises the amino acid sequence RKAA (SEQ ID NO: 1).
9. The method according to any one of embodiments 1 to 8, wherein B is a linking moiety.
10. The method according to embodiment 9, wherein the connecting portion B comprises
Bio-orthogonal labelling group, or
Non-bioorthogonal entities for cross-linking.
11. The method according to embodiment 10, wherein the bio-orthogonal labeling group or the non-bio-orthogonal entity for cross-linking consists of or comprises at least one molecule or moiety selected from the group consisting of:
-N-N≡N or-N 3 ；
·Lys(N 3 )；
Tetrazine;
alkynes;
strained cyclooctyne (strained cyclooctyne);
·BCN；
strained olefins;
photoreactive groups;
aldehyde;
acyl trifluoroborates;
Protein degrading agent ('PROTAC');
cyclopentadiene/spirocyclopentadiene (spironolycyclopentadiene);
a thio-selective electrophile;
-SH; and
cysteine.
12. The method according to any one of embodiments 9 to 11, comprising the further step of coupling one or more payloads to the linking moiety B.
13. The method according to embodiment 12, wherein the one or more payloads are coupled to the linking moiety B via a click reaction.
14. The method according to any one of embodiments 1 to 8, wherein B is a payload.
15. The method according to any one of embodiments 12 to 14, wherein the payload comprises at least one of:
toxins;
cytokines;
growth factors;
radionuclides;
hormones;
antiviral agents;
an antimicrobial agent;
fluorescent dye;
immunomodulators/immunostimulants;
half-life increasing moiety;
a solubility-increasing moiety;
polymer-toxin conjugate;
nucleic acid;
biotin or streptavidin moiety;
vitamins;
protein degrading agent ('PROTAC');
a target binding moiety; and/or
Anti-inflammatory agents.
16. The method of embodiment 15, wherein the toxin is at least one selected from the group consisting of:
Pyrrolobenzodiazepines (e.g., PBDs);
auristatin (e.g. MMAE, MMAF);
maytansinoids (e.g., maytansinoids, DM1, DM4, DM 21);
duocarmycin (duocarmycin);
nicotinamide phosphoribosyl transferase (NAMPT) inhibitors;
tubulysin (tubulysin);
enediyne (e.g., calicheamicin);
anthracycline derivatives (PNUs) (e.g., doxorubicin);
inhibitors of the pyrrolyl Kinesin Spindle Protein (KSP);
candidiasis (cryptophycin);
drug efflux pump inhibitors;
mountain Zhuo Meisu;
amanitine (e.g., α -amanitine); and
camptothecins (e.g., exenatide, deluxetans).
17. The method according to any one of embodiments 14 to 16, wherein the chemical spacer (Sp 2 ) Including self-cleaving (self-immolative) moieties.
18. The method of embodiment 17, wherein the self-cleaving portion is directly attached to the payload B.
19. The method according to embodiment 17 or 18, wherein the self-cleaving moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety.
20. The method according to any one of embodiments 1 to 19, wherein the antibody is an IgG antibody, in particular an IgG1 antibody.
21. The method according to embodiment 20, wherein the linker-conjugated Gln residue is comprised in the Fc domain of an antibody, in particular wherein the linker-conjugated Gln residue is the C of an IgG antibody H Gln residue Q295 of the 2 domain (EU numbering).
22. The method according to embodiment 20, wherein the linker-coupled Gln residues are introduced into the heavy or light chain of the antibody by molecular engineering.
23. The method according to embodiment 22, wherein the Gln residue introduced into the heavy or light chain of the antibody by molecular engineering is the C of an aglycosylated IgG antibody H 2 domain N297Q (EU numbering).
24. The method according to embodiment 22, wherein the Gln residue introduced into the heavy or light chain of the antibody by molecular engineering is contained in a peptide that has been (a) integrated into the heavy or light chain of the antibody or (b) fused to the N-terminal or C-terminal end of the heavy or light chain of the antibody.
25. The method according to embodiment 24, wherein the peptide comprising a Gln residue has been fused to the C-terminus of the heavy chain of the antibody.
26. The method according to any one of embodiments 20 to 22 or 24 to 25, wherein the IgG antibody is a glycosylated IgG antibody, particularly wherein the IgG antibody is at C H Glycosylation at residue N297 (EU numbering) of the 2 domain.
27. The method according to any one of embodiments 1 to 26, wherein the antibody is selected from the group consisting of: vitamin b (Brentuximab), trastuzumab (Trastuzumab), gemtuzumab (Gemtuzumab), oxtuzumab (Inotuzumab), avizumab (Avelumab), cetuximab (Cetuximab), rituximab (Rituximab), up Lei Tuoyou mab (Daratumumab), pertuzumab (Pertuzumab), vedolizumab (Vedolizumab), oxuzumab (occtuzumab), touzumab (tocuzumab), wu Sinu mab (usetuzumab), golimumab (Golimumab), oxuzumab (obuzumab), sha Xituo mab (sacitumab), bei Lantuo mab (belantalumab), loxuzumab (pouzumab), and enrouzumab (entuzumab).
28. The method according to any one of embodiments 1 to 27, wherein the antibody is selected from the group consisting of: velutinab, gemtuzumab, trastuzumab, oxtuzumab, poltuzumab, enrolment mab, sha Xituo bead mab, and Bei Lantuo mab.
29. The method of any one of embodiments 1 to 28, wherein the antibody is either pertuzumab or trastuzumab or enrolment mab.
30. The method according to any one of embodiments 1 to 29, wherein the linker is coupled to the γ -carboxamide group of the Gln residue comprised in the antibody.
31. The method according to any one of embodiments 1 to 30, wherein the linker is adapted to couple to the glycosylated antibody with a coupling efficiency of at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95%.
32. The method according to any one of embodiments 1 to 31, wherein the microbial transglutaminase is derived from a Streptomyces (Streptomyces) species, in particular Streptomyces mobaraensis (Streptomyces mobaraensis).
33. An antibody-linker conjugate prepared by the method according to any one of embodiments 1 to 32.
34. An antibody-linker conjugate comprising:
a) An antibody; and
b) A joint comprising the structure:
(Sp 1 )-RK-(Sp 2 )-B-(Sp 3 ) Or (b)
(Sp 1 )-B-(Sp 2 )-RK-(Sp 3 ) The method comprises the steps of carrying out a first treatment on the surface of the Wherein the method comprises the steps of
·(Sp 1 ) Is a chemical spacer or is absent;
·(Sp 2 ) Is a chemical spacer or is absent;
·(Sp 3 ) Is a chemical spacer or is absent;
r is arginine or an arginine derivative or arginine mimetic;
k is lysine or a lysine derivative or a lysine mimetic;
b is a linking part or payload;
wherein the linker is coupled to the antibody via an isopeptide bond formed between a γ -carboxamide group of a glutamine residue contained in the antibody and a primary amine contained in a side chain of a lysine residue, a lysine derivative or a lysine mimetic contained in an RK motif contained in the linker.
35. The antibody-linker conjugate according to embodiment 34, wherein the chemical spacer (Sp 1 )、(Sp 2 ) Sum (Sp) 3 ) Each independently comprises 0 to 12 amino acid residues.
36. The antibody-linker conjugate according to 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.
37. The antibody-linker conjugate according to any one of embodiments 34 to 36, wherein the net charge of the linker is neutral or positive.
38. The antibody-linker conjugate according to any one of embodiments 34 to 37, wherein the linker does not comprise a negatively charged amino acid residue.
39. The antibody-linker conjugate according to any one of embodiments 34 to 38, wherein the linker comprises an amino acid sequence selected from the group consisting of: RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), and RKR (SEQ ID NO: 4).
40. The antibody-linker conjugate 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).
41. The antibody-linker conjugate according to any one of embodiments 34 to 40, wherein the linker comprises the amino acid sequence RKAA (SEQ ID NO: 1).
42. The antibody-linker conjugate according to any one of embodiments 34 to 41, wherein B is a linking moiety.
43. The antibody-linker conjugate according to embodiment 42, wherein linking moiety B comprises
Bio-orthogonal labelling group, or
Non-bioorthogonal entities for cross-linking.
44. The antibody-linker conjugate according to embodiment 43, wherein the bio-orthogonal labeling group or the non-bio-orthogonal entity for cross-linking consists of or comprises at least one molecule or moiety selected from the group consisting of:
-N-N≡N or-N 3 ；
·Lys(N 3 )；
Tetrazine;
alkynes;
strained cyclooctyne;
·BCN；
strained olefins;
photoreactive groups;
aldehyde;
acyl trifluoroborates;
protein degrading agent ('PROTAC');
cyclopentadiene/spirocyclopentadiene;
a thio-selective electrophile;
-SH; and
cysteine.
45. The antibody-linker conjugate according to any one of embodiments 42 to 44, wherein one or more payloads are conjugated to linking moiety B.
46. The antibody-linker conjugate according to embodiment 45, wherein one or more payloads are coupled to linking moiety B via a click reaction.
47. The antibody-linker conjugate according to any one of embodiments 34 to 41, wherein B is a payload.
48. The antibody-linker conjugate according to any one of embodiments 45 to 47, wherein the payload comprises at least one of:
toxins;
cytokines;
growth factors;
radionuclides;
hormones;
antiviral agents;
an antimicrobial agent;
fluorescent dye;
immunomodulators/immunostimulants;
half-life increasing moiety;
a solubility-increasing moiety;
polymer-toxin conjugate;
nucleic acid;
biotin or streptavidin moiety;
vitamins;
protein degrading agent ('PROTAC');
a target binding moiety; and/or
Anti-inflammatory agents.
49. The antibody-linker conjugate according to embodiment 48, wherein the toxin is at least one selected from the group consisting of:
pyrrolobenzodiazepines (e.g., PBDs);
auristatin (e.g., MMAE, MMAF);
maytansinoids (e.g., maytansine, DM1, DM4, DM 21);
sesquicomycin;
nicotinamide phosphoribosyl transferase (NAMPT) inhibitors;
microtubule lysin;
enediyne (e.g., calicheamicin);
anthracycline derivatives (PNUs) (e.g., doxorubicin);
Inhibitors of the pyrrolyl Kinesin Spindle Protein (KSP);
candidiasis;
drug efflux pump inhibitors;
mountain Zhuo Meisu;
amanitine (e.g., α -amanitine); and
camptothecins (e.g., irinotecan, delutinacon).
50. The antibody-linker conjugate according to any one of embodiments 47 to 49, wherein the chemical spacer (Sp 2 ) Including self-cleaving moieties.
51. The antibody-linker conjugate according to embodiment 50, wherein the self-cleaving moiety is directly attached to payload B.
52. The antibody-linker conjugate according to embodiment 50 or 51, wherein the self-cleaving moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety.
53. The antibody-linker conjugate according to any of embodiments 34 to 52, wherein the antibody is an IgG antibody, in particular an IgG1 antibody.
54. The antibody-linker conjugate according to embodiment 53, wherein the linker-conjugated Gln residue is comprised in the Fc domain of an antibody, in particular wherein the linker-conjugated Gln residue is the C of an IgG antibody H Gln residue Q295 of the 2 domain (EU numbering).
55. The antibody-linker conjugate according to embodiment 53, wherein the linker-conjugated Gln residues are introduced into the heavy or light chain of the antibody by molecular engineering.
56. The antibody-linker conjugate according to embodiment 55, whereinIntroduction of Gln residues in the heavy or light chain of antibodies by molecular engineering into the C of non-glycosylated IgG antibodies H 2 domain N297Q (EU numbering).
57. The antibody-linker conjugate according to embodiment 55, wherein the Gln residue of the heavy or light chain of the antibody introduced by molecular engineering is comprised in a peptide that has been (a) integrated into the heavy or light chain of the antibody or (b) fused to the N-terminus or C-terminus of the heavy or light chain of the antibody.
58. The antibody-linker conjugate according to embodiment 57, wherein the peptide comprising a Gln residue has been fused to the C-terminus of the heavy chain of the antibody.
59. The antibody-linker conjugate according to any of embodiments 53 to 55 or 57 to 58, wherein the IgG antibody is a glycosylated IgG antibody, particularly wherein the IgG antibody is at C H Glycosylation at residue N297 (EU numbering) of the 2 domain.
60. The antibody-linker conjugate according to any one of embodiments 34 to 59, wherein the antibody is selected from the group consisting of: vibutuzumab, trastuzumab, gemtuzumab, oxtuzumab, avermectin, cetuximab, rituximab, up Lei Tuoyou mab, pertuzumab, vedolizumab, oreuzumab, tolizumab, wu Sinu mab, golimumab, otouzumab, sha Xituo mab, bei Lantuo mab, polotouzumab, and enrolment mab.
61. The antibody-linker conjugate according to any one of embodiments 34 to 60, wherein the antibody is selected from the group consisting of: velutinab, gemtuzumab, trastuzumab, oxtuzumab, poltuzumab, enrolment mab, sha Xituo bead mab, and Bei Lantuo mab.
62. The antibody-linker conjugate according to any one of embodiments 34 to 61, wherein the antibody is polotophyllizumab or trastuzumab or enrolment monoclonal antibody.
63. An antibody-drug conjugate comprising:
a) IgG antibodies; and
b) A linker comprising a drug moiety B, wherein the drug moiety B is covalently linked to an amino acid sequence selected from the group consisting of: RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3) or RKR (SEQ ID NO: 4);
wherein the linker is via C in the antibody H An isopeptide bond formed between the gamma-carboxamide group of glutamine residue Q295 (EU numbering) of the 2 domain and the primary amine contained in the side chain of the lysine residue contained in the linker is coupled to an IgG antibody.
64. The antibody-drug conjugate according to embodiment 63, wherein drug moiety B is linked to the N-terminus or C-terminus of the amino acid sequence contained in the linker via a self-cleaving moiety.
65. The antibody-drug conjugate according to embodiment 64, wherein the self-cleaving moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety.
66. The antibody-drug conjugate according to any of embodiments 63-65, wherein the IgG antibody is a glycosylated IgG antibody, particularly wherein the IgG antibody is at C H Glycosylation at residue N297 (EU numbering) of the 2 domain.
67. The antibody-drug conjugate according to any one of embodiments 63-66, wherein the IgG antibody is an IgG1 antibody.
68. The antibody-drug conjugate according to any one of embodiments 63-67, wherein the IgG antibody is a poloxamer 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.
69. 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.
70. The antibody-drug conjugate according to any one of embodiments 63-67, wherein the IgG antibody is enrolment mab 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.
71. The antibody-drug conjugate according to any one of embodiments 63-70, wherein the drug is a toxin selected from the group consisting of:
pyrrolobenzodiazepines (e.g., PBDs);
auristatin (e.g., MMAE, MMAF);
maytansinoids (e.g., maytansine, DM1, DM4, DM 21);
sesquicomycin;
nicotinamide phosphoribosyl transferase (NAMPT) inhibitors;
microtubule lysin;
enediyne (e.g., calicheamicin);
anthracycline derivatives (PNUs) (e.g., doxorubicin);
inhibitors of the pyrrolyl Kinesin Spindle Protein (KSP);
candidiasis;
drug efflux pump inhibitors;
mountain Zhuo Meisu;
amanitine (e.g., α -amanitine); and
camptothecins (e.g., irinotecan, delutinacon).
72. The antibody-drug conjugate according to any one of embodiments 63-71, wherein the linker has the structure RKAA-PABC-B, in particular wherein B is auristatin or maytansinoid, in particular wherein auristatin is MMAE, and wherein maytansinoid is DM1 or maytansinoid.
73. The antibody-drug conjugate according to any one of embodiments 63-71, wherein the linker has the structure RKA-PABC-B, in particular wherein B is auristatin or maytansinoid, in particular wherein auristatin is MMAE, and wherein maytansinoid is DM1 or maytansinoid.
74. The antibody-drug conjugate according to any one of embodiments 63-71, wherein the linker has the structure ARK-PABC-B, in particular wherein B is auristatin or maytansinoid, in particular wherein auristatin is MMAE, and wherein maytansinoid is DM1 or maytansinoid.
75. The antibody-drug conjugate according to any one of embodiments 63-71, wherein the linker has the structure RKR-PABC-B, in particular wherein B is auristatin or maytansinoid, in particular wherein auristatin is MMAE, and wherein maytansinoid is DM1 or maytansinoid.
76. A linker construct comprising the structure:
(Sp 1 )-RK-(Sp 2 )-B-(Sp 3 ) Or (b)
(Sp 1 )-B-(Sp 2 )-RK-(Sp 3 ) The method comprises the steps of carrying out a first treatment on the surface of the Wherein the method comprises the steps of
·(Sp 1 ) Is a chemical spacer or is absent;
·(Sp 2 ) Is a chemical spacer or is absent;
·(Sp 3 ) Is a chemical spacer or is absent;
r is arginine or an arginine derivative or arginine mimetic;
k is lysine or a lysine derivative or a lysine mimetic;
b is the connection portion or payload.
77. The linker construct according to embodiment 76, wherein the chemical spacer (Sp 1 )、(Sp 2 ) Sum (Sp) 3 ) Each independently comprises 0 to 12 amino acid residues.
78. The linker construct according to 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.
79. The linker construct according to any one of embodiments 76 to 78, wherein the net charge of the linker is neutral or positive.
80. The linker construct according to any one of embodiments 76 to 79, wherein the linker does not comprise negatively charged amino acid residues.
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).
82. The linker construct according to any one of embodiments 76 to 81, wherein B is a linking moiety.
83. The linker construct according to embodiment 82, wherein linker moiety B comprises
Bio-orthogonal labelling group, or
Non-bioorthogonal entities for cross-linking.
84. The linker construct according to embodiment 83, wherein the bio-orthogonal labeling group or the non-bio-orthogonal entity for crosslinking consists of or comprises at least one molecule or moiety selected from the group consisting of:
-N-N≡N or-N 3 ；
·Lys(N 3 )；
Tetrazine;
alkynes;
strained cyclooctyne;
·BCN；
strained olefins;
photoreactive groups;
aldehyde;
acyl trifluoroborates;
Protein degrading agent ('PROTAC');
cyclopentadiene/spirocyclopentadiene;
a thio-selective electrophile;
-SH; and
cysteine.
85. The linker construct according to any one of embodiments 76 to 84, wherein the linker construct consists of or comprises the structure RKAA-B, in particular wherein B is Lys (N 3 ) Or cysteine.
86. The linker construct according to any one of embodiments 76 to 84, wherein the linker construct consists of or comprises the structure RKA-B, in particular wherein B is Lys (N 3 ) Or cysteine.
87. The linker construct according to any one of embodiments 76 to 84, wherein the linker construct consists of or comprises structure ARK-B, in particular wherein B is Lys (N 3 ) Or cysteine.
88. The linker construct according to any one of embodiments 76 to 84, wherein the linker construct is composed of a structureB-RKR composition or comprising the structure B-RKR, in particular wherein B is Lys (N 3 ) Or cysteine.
89. The linker construct according to any one of embodiments 76 to 81, wherein B is a payload.
90. The linker construct according to embodiment 89, wherein the payload comprises at least one of:
Toxins;
cytokines;
growth factors;
radionuclides;
hormones;
antiviral agents;
an antimicrobial agent;
fluorescent dye;
immunomodulators/immunostimulants;
half-life increasing moiety;
a solubility-increasing moiety;
polymer-toxin conjugate;
nucleic acid;
biotin or streptavidin moiety;
vitamins;
protein degrading agent ('PROTAC');
a target binding moiety; and/or
Anti-inflammatory agents.
91. The linker construct according to embodiment 90, wherein the toxin is at least one selected from the group consisting of:
pyrrolobenzodiazepines (e.g., PBDs);
auristatin (e.g., MMAE, MMAF);
maytansinoids (e.g., maytansine, DM1, DM4, DM 21);
sesquicomycin;
nicotinamide phosphoribosyl transferase (NAMPT) inhibitors;
microtubule lysin;
enediyne (e.g., calicheamicin);
anthracycline derivatives (PNUs) (e.g., doxorubicin);
inhibitors of the pyrrolyl Kinesin Spindle Protein (KSP);
candidiasis;
drug efflux pump inhibitors;
mountain Zhuo Meisu;
amanitine (e.g., α -amanitine); and
camptothecins (e.g., irinotecan, delutinacon).
92. The linker construct according to any one of embodiments 89 to 91, wherein the chemical spacer (Sp 2 ) Including self-cleaving moieties.
93. The linker construct according to embodiment 92, wherein the self-cleaving moiety is directly attached to payload B.
94. The linker construct according to embodiment 92 or 93, wherein the self-cleaving moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety.
95. The linker construct according to any one of embodiments 89 to 94, wherein the linker construct consists of or comprises the structure RKAA-PABC-B, in particular wherein B is auristatin or maytansinoid, in particular wherein auristatin is MMAE, and wherein maytansinoid is DM1 or maytansinoid.
96. The linker construct according to any one of embodiments 89 to 94, wherein the linker construct consists of or comprises the structure RKA-PABC-B, in particular wherein B is auristatin or maytansinoid, in particular wherein auristatin is MMAE, and wherein maytansinoid is DM1 or maytansinoid.
97. The linker construct according to any one of embodiments 89 to 94, wherein the linker construct consists of or comprises the structure ARK-PABC-B, in particular wherein B is auristatin or maytansinoid, in particular wherein auristatin is MMAE, and wherein maytansinoid is DM1 or maytansinoid.
98. The linker construct according to any one of embodiments 89 to 94, wherein the linker construct consists of or comprises the structure B-PABC-RKR, in particular wherein B is auristatin or maytansinoid, in particular wherein auristatin is MMAE, and wherein maytansinoid is DM1 or maytansinoid.
99. Use of the linker construct according to any one of embodiments 76 to 98 in the production of an antibody-linker conjugate by microbial transglutaminase.
100. The use according to embodiment 99, wherein the antibody is an IgG antibody, in particular an IgG1 antibody.
101. The method of embodiment 65 or 66, wherein the antibody is either poloxamer or trastuzumab or enrolment mab.
102. A pharmaceutical composition comprising:
a) The antibody-linker conjugate according to any one of embodiments 33 to 62, in particular wherein the antibody-linker conjugate comprises at least one payload;
or (b)
b) The antibody-drug conjugate according to any one of embodiments 63-75; and is also provided with
The pharmaceutical composition comprises at least one pharmaceutically acceptable ingredient.
103. The pharmaceutical composition according to embodiment 102, comprising at least one additional therapeutically active agent.
104. The antibody-linker conjugate according to any one of embodiments 33 to 62, in particular wherein the antibody-linker conjugate comprises at least one payload, the antibody-drug conjugate according to any one of embodiments 63 to 75, or the pharmaceutical composition according to embodiments 102 or 103, for use in therapy and/or diagnosis.
105. The antibody-linker conjugate according to any one of embodiments 33 to 62, in particular wherein the antibody-linker conjugate comprises at least one payload, the antibody-drug conjugate according to any one of embodiments 63 to 75, or the pharmaceutical composition according to embodiments 102 or 103, for use in therapy
● Patients suffering from neoplastic diseases, neurological diseases, autoimmune diseases, inflammatory diseases or infectious diseases,
● Patients at risk of developing neoplastic, neurological, autoimmune, inflammatory or infectious diseases, and/or
● Patients diagnosed with neoplastic disease, neurological disease, autoimmune disease, inflammatory disease or infectious disease.
106. The antibody-linker conjugate, antibody-drug conjugate, or pharmaceutical composition for use according to embodiment 105, wherein the antibody-linker conjugate or antibody-drug conjugate comprised in the pharmaceutical composition comprises poloxamer, and wherein the neoplastic disease is a B cell-related cancer.
107. The antibody-linker conjugate, antibody-drug conjugate, or pharmaceutical composition for use according to embodiment 106, wherein the B cell-related cancer is non-hodgkin's lymphoma, particularly wherein the B cell-related cancer is diffuse large B cell lymphoma.
108. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use according to embodiment 106 or 107, wherein the antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition is administered in combination with bendamustine (bendamustine) and/or rituximab.
109. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use according to embodiment 105, wherein the antibody-linker conjugate or antibody-drug conjugate comprised in the pharmaceutical composition comprises trastuzumab, and wherein the neoplastic disease is a HER2 positive cancer, in particular HER2 positive breast cancer, gastric cancer, ovarian cancer or lung cancer.
110. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use according to embodiment 109, wherein the antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition is administered in combination with lapatinib (lapatinib), capecitabine (capecitabine), and/or a taxane.
111. The antibody-linker conjugate, antibody-drug conjugate, or pharmaceutical composition for use according to embodiment 105, wherein the antibody-linker conjugate or antibody-drug conjugate comprised in the pharmaceutical composition comprises enrolment mab or an enrolment mab variant, and wherein the neoplastic disease is a binding element (Nectin) -4 positive cancer, in particular a binding element-4 positive pancreatic cancer, lung cancer, bladder cancer, or breast cancer.
112. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use according to embodiment 111, wherein the antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition is administered in combination with a cisplatin-based chemotherapeutic agent and/or pambrizumab (Pembrolizumab).
113. Use of an antibody-linker conjugate according to any one of embodiments 33 to 62, in particular wherein the antibody-linker conjugate comprises at least one payload, an antibody-drug conjugate according to any one of embodiments 63 to 75 or a pharmaceutical composition according to embodiments 102 or 103 for the manufacture of a medicament for the treatment of
● Patients suffering from neoplastic diseases, neurological diseases, autoimmune diseases, inflammatory diseases or infectious diseases,
● Patients at risk of developing neoplastic, neurological, autoimmune, inflammatory or infectious diseases, and/or
● Patients diagnosed with neoplastic disease, neurological disease, autoimmune disease, inflammatory disease or infectious disease.
114. A method of treating or preventing a neoplastic disease, the method comprising administering the antibody-linker conjugate according to any one of embodiments 33 to 62 to a patient in need thereof, in particular wherein the antibody-linker conjugate comprises at least one payload, the antibody-drug conjugate according to any one of embodiments 63 to 75, or the pharmaceutical composition according to embodiments 102 or 103.
Thus, in one embodiment, the invention relates to a method for producing an antibody-linker conjugate by Microbial Transglutaminase (MTG), comprising administering a polypeptide comprising (as shown in the N.fwdarw.C direction) (Sp 1 )-RK-(Sp 2 )-B-(Sp 3 ) Or (Sp) 1 )-B-(Sp 2 )-RK-(Sp 3 ) A step of coupling a linker of the structure 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 arginine mimetic;
K is lysine or a lysine derivative or a lysine mimetic;
b is a linking part or payload;
and wherein the linker is coupled to the Gln residue comprised in the antibody via a primary amine comprised in a side chain of the lysine residue, lysine derivative or lysine mimetic.
That is, the present invention is based, at least in part, on the surprising discovery that: a linker comprising the peptide motif RK (arginyl-lysyl) can be efficiently coupled to glycosylated antibodies. In patent application WO 2019/057772, it was demonstrated that peptide-based linkers can be efficiently coupled to glutamine residues of glycosylated antibodies via lysine residues in the linker. However, it has now surprisingly been shown that the extended motif RK provides a further improved coupling efficiency.
The inventors have shown that lysine-containing linkers that do not contain an RK motif result in a coupling efficiency of 27% to 77% when directly attached to a drug molecule (see table 4). As provided herein, linkers comprising RK motifs are coupled to glycosylated antibodies with an efficiency of at least 82%, and in some cases up to 100% (see table 3 and table 5). Thus, for MTG-based coupling to glycosylated antibodies, particularly when the payload is directly coupled to the glycosylated antibody in a one-step reaction, linkers comprising the RK motif are particularly preferred over other lysine-based linkers.
In the present invention, it is preferable that the linker includes a structure (Sp 1 )-RK-(Sp 2 )-B-(Sp 3 ) Or (Sp) 1 )-B-(Sp 2 )-RK-(Sp 3 ) Wherein the linker is via R at the linkerPrimary amines contained in residue K contained in the K motif are coupled to glutamine residues in the antibody. In certain embodiments, residue K is a lysine residue. However, in certain embodiments, residue K may also be a lysine mimetic or lysine derivative, provided that the lysine mimetic or lysine derivative includes a primary amine in its amino acid side chain.
Thus, in certain embodiments, residue K may be a lysine mimetic. As used herein, the term "lysine mimetic" refers to a compound having a structure other than lysine, but having similar characteristics to lysine, and thus can be used to replace lysine in a peptide or protein without significantly altering the function and/or structure of the peptide or protein. In certain embodiments, the lysine mimetic may differ from lysine in the length or composition of the aliphatic chain linking the primary amine and the α -carbon atom. Thus, in certain embodiments, the lysine mimetic may be ornithine or 2, 7-diaminoheptanoic acid. In certain embodiments, the lysine mimetic may be a β -amino acid, such as β -homolysine.
In certain embodiments, residue K may be a lysine derivative. As used herein, the term "lysine derivative" refers to lysine or a lysine mimetic in which one or more functional groups contained in the lysine or lysine mimetic are modified or substituted. In the present invention, it is preferable that the amino group in the side chain of the lysine derivative is unmodified so as to be available for coupling with the glutamine residue in the protein. In embodiments where 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 -carboxyl group of the lysine mimetic may be amidated.
The linker also includes 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 also be an arginine mimetic or an arginine derivative.
Thus, in certain embodiments, residue R may be an arginine mimetic. As used herein, the term "arginine mimetic" refers to a compound that has a structure other than arginine but has similar characteristics to arginine, and thus can be used to replace arginine in a peptide or protein without significantly altering the function and/or structure of the peptide or protein. Arginine mimics may differ from arginine in the length or composition of the aliphatic chain connecting the guanidine group to the alpha-carbon atom. Alternatively or in addition, the arginine mimetic may differ from arginine in the guanidino group itself. That is, the arginine mimetic may comprise a functional group having similar physicochemical properties as the guanidine group. In certain embodiments, the arginine mimetic may be homoarginine, 2-amino-3-guanidine-propionic acid, β -ureido alanine, or citrulline.
In certain embodiments, residue R may be an arginine derivative. As used herein, the term "arginine derivative" refers to arginine or an arginine mimetic in which one or more functional groups contained in the arginine or arginine mimetic are modified or substituted. The arginine derivative may be arginine or an arginine mimetic in which a guanidino group is substituted or modified. In certain embodiments, the arginine derivative may be omega-methyl arginine. In embodiments where residue R is located at the N-terminal position of the linker, R may be an arginine derivative in which the α -amino group is modified or substituted. In certain embodiments, the α -amino group of the arginine mimetic may be acetylated.
It will be appreciated that the RK motif preferably consists of the amino acids arginine and lysine. However, arginine or lysine residues, or both, may be replaced with mimics or derivatives as disclosed above. In certain embodiments, the RK motif may consist of the amino acids arginine and ornithine. In certain embodiments, the RK motif may consist of the amino acids arginine and 2, 7-diaminoheptanoic acid. In certain embodiments, the RK motif may consist of the amino acids homoarginine and lysine. In certain embodiments, the RK motif may consist of the amino acids 2-amino-3-guanidino-propionic acid and lysine. In certain embodiments, the RK motif may consist of the amino acids homoarginine and ornithine. In certain embodiments, the RK motif may consist of the amino acids homoarginine and 2, 7-diaminoheptanoic acid. In certain embodiments, the RK motif may consist of the amino acids 2-amino-3-guanidino-propionic acid and ornithine. In certain embodiments, the RK motif may consist of the amino acids 2-amino-3-guanidino-propionic acid and 2, 7-diaminoheptanoic acid.
In the present invention, RK motif-embedding structure (Sp 1 )-RK-(Sp 2 )-B-(Sp 3 ) Or (Sp) 1 )-B-(Sp 2 )-RK-(Sp 3 ) Is a kind of medium. That is, the linker may include one or more chemical spacers (Sp). The term "chemical spacer" as used herein describes a chemical residue covalently attached to a linker and/or a chemical moiety between two chemical residues of a linker.
In a particular embodiment, the invention relates to a method according to the invention, wherein the chemical spacer (Sp 1 )、(Sp 2 ) Sum (Sp) 3 ) Each independently comprises 0 to 12 amino acid residues.
That is, in certain embodiments, the chemical spacer (Sp 1 )、(Sp 2 ) And/or (Sp) 3 ) May be present or absent. In the presence (Sp 1 )、(Sp 2 ) And/or (Sp) 3 ) In an embodiment (Sp) 1 )、(Sp 2 ) And/or (Sp) 3 ) May comprise one or more amino acid residues. In such embodiments, (Sp) 1 )、(Sp 2 ) And/or (Sp) 3 ) May comprise from 0 to 12 amino acid residues. It has to be noted that the chemical spacer (Sp 1 )、(Sp 2 ) And/or (Sp) 3 ) Non-amino acid residues may also be included, as will be disclosed in more detail below.
Chemical spacer (Sp) 1 )、(Sp 2 ) And/or (Sp) 3 ) The "amino acid residue" contained in (a) may be an amino acid, an amino acid mimetic or an amino acid derivative. It is understood that the term amino acid includes not only alpha-amino acids but also other amino acids such as beta-amino acids, gamma-amino acids or delta-amino acids. The alpha-amino acid residues may be present in the chemical spacer (Sp) in either their L-or D-form 1 )、(Sp 2 ) And/or (Sp) 3 ) Is a kind of medium. At (Sp 1 )、(Sp 2 ) And/or (Sp) 3 ) In embodiments comprising chiral β -amino acids, γ -amino acids, or δ -amino acids, the chiral β -amino acids, γ -amino acids, or δ -amino acids may exist in their S-or R-forms. Thus, in the broadest sense, the term "amino acid residue" as used herein may refer to a residue that contains an amino group (-NH) 2 ) And any organic compound of carboxyl (-COOH). Thus, whenever reference is made in this disclosure to "amino acid" or "amino acid residue", it is to be understood that the term amino acid residue may also include amino acid mimics or derivatives.
Further, it is understood that the term amino acid residue is not limited to the known group of proteinogenic amino acids, i.e. alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, but also encompasses non-classical amino acids and non-natural amino acids. As used herein, a "non-classical amino acid (non-canonical amino acid)" may be any amino acid that is not part of the group of proteinogenic amino acids, but may be obtained from a natural source. However, it must be noted that some non-classical amino acids may also be found in naturally occurring peptides and/or proteins.
As used herein, an "unnatural amino acid" or "synthetic amino acid" can be any molecule that belongs to the general definition of amino acids (i.e., includes amino and carboxyl groups) but is not found in nature. Thus, the unnatural amino acid is preferably obtained by chemical synthesis. It will be appreciated that in some cases the distinction between non-classical amino acids and non-natural amino acids may be ambiguous. For example, amino acids defined as unnatural amino acids can be identified in nature at a later point in time, and thus reclassified as nonclassical amino acids.
Examples of non-classical amino acids or non-natural amino acids may be, but are not limited to, D-amino acids (such as D-alanine, D-arginine, D-methionine), homoamino acids (such as homoserine, homoarginine, homocysteine, a-aminoadipic acid), N-methylated amino acids (such as sarcosine, N-Me-leucine), a-methyl amino acids (such as a-methyl-histidine, a-aminoisobutyric acid), β -amino acids (such as β -alanine, D-3-aminoisobutyric acid, L- β -homoalanine), γ -amino acids (such as γ -aminobutyric acid), alanine mimetics or derivatives (such as β -cyclopropylalanine, phenylglycine, dehydroalanine, β -cyanoalanine, β - (3-pyridyl) -alanine, β - (1, 2, 4-triazol-1-yl) -alanine, β - (1-piperazinyl) -alanine), phenylalanine mimetics or derivatives (such as 4-iodophenylalanine, pentafluoro-phenylalanine, naphtyl-alanine, 4-aminophenylalanine), mimetics or derivatives (such as ω -methyl alanine, ω -alanine derivatives (such as ω -methyl alanine), (3- (3-methyl-3H-diazacyclopropan-3-yl) propylamino) carbonyl-L-lysine, N epsilon-trimethyllysine), histidine mimetics or derivatives (such as 2, 5-diiodohistidine, 1-methylhistidine), tyrosine mimetics or derivatives (such as 3-aminotyrosine, thyronine, 3, 5-dinitrotyrosine, 3-hydroxy-methyl-tyrosine, O-phospho-L-tyrosine), tryptophan mimetics or derivatives (such as 5-hydroxy-tryptophan, 1-methyltryptophan), serine mimetics or derivatives (such as beta- (2-thienyl) -serine, beta- (3, 4-dihydroxyphenyl) -serine, O-phosphoserine), threonine mimetics or derivatives (such as isoleucine, O-phosphothreonine), proline mimetics or derivatives (such as hydroxyproline, 3, 4-dehydro-proline, pyroglutamic acid, sulfane, cis-octahydroindole-2-carboxylic acid), leucine and isoleucine mimetics or derivatives (such as isoleucine, norleucine, leucine, 4, 5-dehydroleucine, (4-hydroxy-tryptophan), norvaline, valine, or derivatives (such as norvaline) Citrulline mimetics or derivatives (such as, for example, thiocitrulline, homocysteine), cysteine mimetics or derivatives (such as, for example, penicillamine, selenocysteine, butylsulfanilic acid-sulfoximine), methionine mimetics or derivatives (such as, for example, S-methyl methionine, L-methionine sulfone, L-methionine sulfoxide, L-methionine sulfoxime, selenomethionine), aspartic acid mimetics or derivatives (such as, for example, DL-threo-beta-hydroxy aspartic acid, L-aspartic acid beta-methyl ester), glutamic acid mimetics or derivatives (such as, gamma-methylglutamate, gamma-carboxyglutamate, gamma-hydroxyglutamate, L-glutamate 5-methyl ester, L-2-aminopimelic acid), asparagine mimetics or derivatives (such as L-threo-3-hydroxyasparagine, N-dimethyl-L-asparagine, L-2-amino-2-carboxyethane sulfonamide, 5-diazo-4-oxo-L-norvaline), glutamine mimetics or derivatives (such as 4-F- (2S, 4 r) -fluoroglutamine, gamma-glutamyl formamide, theanine, L-glutamate gamma-mono oxalate), amino acids containing cyclic moieties (such as 4-aminopiperidine-4-carboxylic acid, azetidine-2-carboxylic acid, piperidine acid, 1-aminocyclopentanecarboxylic acid, spinine), or amino acids comprising bioorthogonal moieties (such as propargylglycine, α -allylglycine, L-azido-homoalanine, p-benzoyl-1-phenylalanine, p-2-fluoroacetyl-1-phenylalanine, (S) -2-amino-3- (4- (6-methyl-1, 2,4, 5-tetrazin-3-yl) phenyl) propionic acid).
In addition to the above alpha-amino acids, chemical spacers (Sp 1 )、(Sp 2 ) And/or (Sp) 3 ) May comprise one or more beta-amino acids, gamma-amino acids, delta-amino acids or delta 1-amino acids. Thus, in certain embodiments, the linker may be a peptidomimetic. The peptidomimetic may not exclusively comprise a classical peptide bond formed between two delta 2-amino acids, but may additionally or alternatively comprise one or more amide bonds formed between an alpha amino acid and a delta 3-amino acid, a gamma amino acid, a delta 0-amino acid, or a delta 5-amino acid, or between two delta 6-amino acids, gamma amino acids, delta 4-amino acids, delta 7-amino acids, or epsilon-amino acids, respectively. Thus, in any instance of the invention in which the linker is described as a peptide, it is to be understood that the linker may also be a peptidomimetic and thus consist of, not exclusively, alpha-amino acids, but may comprise one or more beta-amino acids, gamma-amino acids, delta-amino acids or epsilon-amino acids or molecules not classified as amino acids. Beta-amino acids, gamma-amino acids, delta which may be included in the linkers of the inventionExamples of the-amino acid or epsilon-amino acid include, but are not limited to, beta-alanine, gamma-aminobutyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, 6-aminocaproic acid and statins.
Furthermore, chemical spacer (Sp 1 )、(Sp 2 ) And/or (Sp) 3 ) Amino acid derivatives and/or amino acid mimics may be included. At (Sp 1 )、(Sp 2 ) And/or (Sp) 3 ) In embodiments comprising one or more amino acid derivatives, it is preferred that the amino acid derivatives have free amino and carboxyl groups such that they may form peptide or isopeptide bonds. At (Sp 1 )、(Sp 2 ) And/or (Sp) 3 ) In embodiments comprising one or more amino acid derivatives, the amino acid mimics may have free amino and carboxyl groups such that they may form peptide or isopeptide bonds. However, in certain embodiments, the amino acid mimetic or derivative may have a substituted amino group that does not prevent peptide bond formation. Examples of such amino acid mimics or derivatives may be N-methylated amino acids such as sarcosine or N-methylleucine (N-Me-leucone).
In a range included in (Sp 1 ) Or (Sp) 3 ) In embodiments in which the amino acid residue is a terminal amino acid residue, the terminal amino acid residue may comprise a modified, protected or substituted N-terminal amino group or C-terminal carboxyl group.
Further, the amino acid mimetic or derivative may be a mimetic or derivative of an amino acid comprising a derivatized amino group, such as proline or other cyclic amino acids (such as azetidine-2-carboxylic acid, piperidine acid, or spinine). Further, the amino acid mimetic may also include other functional groups that replace the amino and/or carboxyl groups of a standard amino acid, which allows the amino acid mimetic to form alternative bonds with adjacent amino acids, amino acid derivatives, and/or amino acid mimetics, and form peptide mimetics.
The term "amino acid mimetic" as used herein refers to a compound that has a structure that is different from a particular amino acid, but that functions in a manner similar to a particular amino acid, and thus can be used to replace a particular amino acid. Amino acid mimetics are said to function in a manner similar to a particular amino acid if they at least to some extent satisfy the structural and/or functional characteristics similar to the amino acid they mimic. The term "amino acid derivative" refers to an amino acid as defined herein wherein one or more functional groups contained in the amino acid are modified or substituted. The amino acid derivative may preferably be a derivative of a proteinogenic amino acid or a non-classical amino acid. Any functional group of the amino acid derivative may be substituted or modified.
In embodiments in which the linker comprises one or more terminal amino acid residues, the terminal amino acid residues may be protected. For example, in (Sp 1 ) In embodiments comprising an N-terminal amino acid residue, the N-terminal amino group may be protected. For example, in certain embodiments, a spacer (Sp 1 ) The N-terminal amino acid residue in (a) may be acetylated. In other embodiments, the R residue contained in the RK motif may be the N-terminal amino acid of the linker. In such embodiments, the N-terminal amino group of arginine, arginine mimetic, or arginine derivative may be protected, for example, by acetylation. In certain embodiments, the linking moiety B or payload B may be amino acids or amino acid based. In such embodiments, the amino acid-based payload or the N-terminal amino group of the linking moiety B may be protected, for example, by acetylation.
Likewise, in (Sp 3 ) In embodiments comprising a C-terminal amino acid residue, the C-terminal carboxyl group may be protected. For example, in certain embodiments, the spacer (Sp 3 ) The C-terminal amino acid residue of (C) may be amidated. In other embodiments, the K residue contained in the RK motif may be the C-terminal amino acid of the linker. In such embodiments, the C-terminal carboxyl group of the lysine, lysine mimetic, or lysine derivative may be protected, for example, by amidation. In certain embodiments, the linking moiety B or payload B may be amino acids or amino acid based. In such embodiments, the amino acid-based payload or the C-terminal carboxyl group of the linking moiety B may be protected, for example, by amidation.
In certain embodiments, the chemical spacer (Sp 1 )、(Sp 2 ) And/or (Sp) 3 ) May comprise from 0 to 12 amino acid residues, including amino acid derivatives and amino acid mimics. That is, in certain embodiments, (Sp) 1 ) May comprise 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues, (Sp) 2 ) May comprise 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues, and (Sp 3 ) May comprise 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino residues.
In a particular embodiment, the invention relates to a method according to the invention, wherein the linker comprises no more than 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 amino acid residues.
That is, in certain embodiments, a linker may comprise 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid residues, including amino acid mimics and amino acid derivatives. It will be appreciated that when B is an amino acid based linker or payload, the amino acid residues (including amino acid mimics and amino acid derivatives) contained in the linker are preferably in the RK motif, chemical spacer (Sp 1 )、(Sp 2 ) And/or (Sp) 3 ) Amino acid residues that are also comprised in B, as well as in certain embodiments. In embodiments where the linker comprises only two amino acid residues, the two amino acid residues are comprised in the RK motif. In such embodiments, (Sp) 1 )、(Sp 2 ) And/or (Sp) 3 ) Absent, or not comprising any amino acid, amino acid mimetic, or amino acid derivative.
In certain embodiments, the linker may comprise 2 to 25 amino acid residues, including amino acid mimics and amino acid derivatives. In other embodiments, the linker may comprise 2 to 20 amino acid residues, including amino acid mimics and amino acid derivatives. In other embodiments, the linker may comprise 2 to 15 amino acid residues, including amino acid mimics and amino acid derivatives. In other embodiments, the linker may comprise 2 to 10 amino acid residues, including amino acid mimics and amino acid derivatives. In other embodiments, the linker may comprise 3 to 10 amino acid residues, including amino acid mimics and amino acid derivatives. In other embodiments, the linker may comprise 3 to 8 amino acid residues, including amino acid mimics and amino acid derivatives. In other embodiments, the linker may comprise 3 to 6 amino acid residues, including amino acid mimics and amino acid derivatives.
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.
In certain embodiments, the linker is a peptide linker (or a peptide mimetic as disclosed herein). That is, the chemical spacer (Sp 1 )、(Sp 2 ) And/or (Sp) 3 ) Consisting of amino acids, amino acid mimics or amino acid derivatives, if present. The net charge of a peptide is typically calculated at neutral pH (7.0). In the simplest method, the net charge is determined by adding the number of positively charged amino acid residues (Arg and Lys and optionally His) and the number of negatively charged amino acid residues (Asp and Glu) and calculating the difference between the two groups. In the case where the linker comprises a non-classical amino acid or an amino acid derivative in which the charged functional group is modified or substituted, the person skilled in the art knows the methods of determining the charge of the non-classical amino acid or amino acid derivative at neutral pH.
In certain embodiments, the composition is described in (Sp 1 )、(Sp 2 ) And/or (Sp) 3 ) The payload, linker moiety B or any non-amino acid moiety contained in (c) may also contribute to the net charge of the linker. However, those skilled in the art know methods to calculate the net charge of the entire linker (including any non-amino acid moieties) preferably at neutral pH (7.0).
In certain embodiments, the net charge of the linker is calculated based solely on the amino acid residues (including amino acid mimics and amino acid derivatives) contained in the linker. 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.
In a particular embodiment, the invention relates to a method according to the invention, wherein the linker does not comprise negatively charged amino acid residues.
That is, the linker may be free of negatively charged amino acid residues (including amino acid mimics and amino acid derivatives). Negatively charged amino acid residues are amino acids, amino acid mimics or amino acid derivatives that carry a negative charge at neutral pH (7.0). Typical negatively charged amino acids are glutamic acid and aspartic acid. However, negatively charged non-classical amino acids, amino acid mimics and amino acid derivatives are known in the art.
In a specific 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 ) Comprising at least one positively charged amino acid. In certain embodiments, (Sp) 1 )、(Sp 2 ) And/or (Sp) 3 ) Comprising at least one histidine residue.
In addition to or in place of amino acid residues (including amino acid mimics and derivatives), chemical spacers (Sp 1 )、(Sp 2 ) And/or (Sp) 3 ) May include or consist of non-amino acid moieties.
That is, in certain embodiments, the chemical spacer (Sp 1 )、(Sp 2 ) And/or (Sp) 3 ) May not consist entirely of amino acids, amino acid mimics or amino acid derivatives. That is, the chemical spacer (Sp 1 )、(Sp 2 ) And/or (Sp) 3 ) May include or may consist of only non-amino acid components. In certain embodiments, the chemical spacer (Sp 1 )、(Sp 2 ) And/or (Sp) 3 ) Amino acid and non-amino acid components may be included.
Such as, but not limited to, chemical spacers (Sp 1 )、(Sp 2 ) And/or (Sp) 3 ) Each of which may includeA carbon-containing backbone of 1 to 200 atoms, optionally at least 10 atoms (e.g., 10 to 100 atoms or 20 to 100 atoms) substituted at one or more atoms, optionally wherein the carbon-containing backbone is a linear hydrocarbon or an oligosaccharide comprising cyclic groups, symmetrical or asymmetrical branched hydrocarbons, monosaccharides, disaccharides, linear or branched (asymmetrically branched or symmetrically branched), other natural linear or branched (asymmetrically branched or symmetrically branched) oligomers, or more generally any dimer, trimer or higher oligomer (linear, asymmetrically branched or symmetrically branched) resulting from any chain-growth or step-growth polymerization process.
(Sp 1 )、(Sp 2 ) And/or (Sp) 3 ) Can be 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 groups, optionally wherein one or more homocyclic aromatic or heterocyclic compound groups may be inserted; in particular C, any straight or branched chain 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 -a group (wherein x1 and x2 are independently integers selected from 0 to 20), 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 ) May include C 2-6 An alkyl group.
In certain embodiments, the chemical spacer (Sp 1 )、(Sp 2 ) And/or (Sp) 3 ) One or more polyethylene glycol (PEG) moieties or similar polycondensates, such as poly (carboxybetaine methacrylate) (pcmma), polyoxazoline, polyglycerol, polyvinylpyrrolidone, or poly (hydroxyethyl methacrylate) (pHEMA), may be included. Polyethylene glycol (PEG) is a polyether compound that has many applications from industrial manufacturing to medicine. PEG is also known as polyethylene oxide(PEO) or Polyoxyethylene (POE), depending on its molecular weight. The structure of PEG is generally denoted as H- (O-CH) 2 -CH 2 ) n -OH. The person skilled in the art is aware of methods of functionalizing polycondensates so that they can be coupled to amino acid residues or payloads.
The inventors have shown that a linker comprising a PEG moiety can be coupled to a glycosylated antibody as efficiently as a similar linker without the PEG moiety. For example, the linker ARK-PEG 2 -PABC-MMAE (FIG. 14) and ARK-PEG 2 -(NH)-(CH 3 ) S-C4-maytansinoids (FIG. 15) were coupled to glycosylated Polotuzumab at an efficiency of 92% and 90% (compared to 94% of ARK-PABC-MMAE), respectively. In another example, the linker ARK-PEG 2 -PABC-MMAE (FIG. 14) and ARK-PEG 2 -(NH)-(CH 3 ) S-C4-maytansinoid (FIG. 15) was coupled to glycosylated trastuzumab with an efficiency of 99% (compared to 100% for ARK-PABC-MMAE).
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 may be contained within a chemical spacer (Sp 1 )、(Sp 2 ) And/or (Sp) 3 ) Is a kind of medium. In certain embodiments, each PEG moiety included in the linker may include 2 to 20 ethylene glycol monomers, 2 to 15 ethylene glycol monomers, 2 to 10 ethylene glycol monomers, or 2 to 5 ethylene glycol monomers. In certain embodiments, the PEG moiety is contained in (Sp 2 ) To directly link the linking moiety or payload to the RK motif. In certain embodiments, the PEG moiety is contained in (Sp 2 ) To connect a connecting portion or payload to a connecting portion or payload in (Sp 2 ) Amino acid residues comprised in the amino acid sequence. In certain embodiments, the PEG moiety is contained in (Sp 2 ) To link the RK motif to a self-cleaving moiety, which in turn is linked to the payload. In certain embodiments, the PEG moiety is contained in (Sp 2 ) Is to be arranged in (Sp) 2 ) The amino acid residues comprised in (a) are linked to a self-cleaving moiety, which in turn is linked to a payload.
In certain embodiments, the chemical spacer (Sp 1 )、(Sp 2 ) And/or (Sp) 3 ) Dextran may be included. The term "dextran" as used herein refers to complex branched dextran consisting of chains of different lengths, which may have a weight ranging from 3kDa to 2000 kDa. The straight chain typically consists of alpha-1, 6 glycosidic linkages between glucose molecules, while the branched chain starts with alpha-1, 3 linkages. Dextran can be synthesized from sucrose, for example from lactic acid bacteria. In the context of the present invention, the dextran used as a carrier may preferably have a molecular weight of about 15kDa to 1500 kDa.
In certain embodiments, the chemical spacer (Sp 1 )、(Sp 2 ) And/or (Sp) 3 ) Oligonucleotides may be included. The term "oligonucleotide" as used herein refers to oligomers or polymers of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), as well as non-naturally occurring oligonucleotides. The oligonucleotide is preferably a polymer of DNA due to higher stability.
In certain embodiments, the chemical spacer (Sp 1 )、(Sp 2 ) And/or (Sp) 3 ) Consists only of amino acid residues (including amino acid mimics and derivatives) and PEG moieties. In certain embodiments, the chemical spacer (Sp 1 )、(Sp 2 ) And/or (Sp) 3 ) Consists only of amino acid residues (including amino acid mimics and derivatives). In certain embodiments, the compound is contained within a chemical spacer (Sp 1 )、(Sp 2 ) And/or (Sp) 3 ) All amino acid residues in (a) are alpha-L-amino acids. That is, in certain embodiments, the linker that does not include a payload or linking moiety B consists of only amino acid residues. In certain embodiments, the linker that does not include a payload or linking moiety B consists only of α -L-amino acid residues. Such peptide-based linkers may comprise protecting groups at the N-terminal and/or C-terminal end. That is, the N-terminal amino group may be acetylated and/or the C-terminal carboxyl group may be amidated.
It must be noted that chemical spacers (Sp 1 )、(Sp 2 ) And/or (Sp) 3 ) May have the same structure. However, it is preferred that the chemical spacer(Sp 1 )、(Sp 2 ) And/or (Sp) 3 ) Each of which has a different structure, and/or not all chemical spacers (Sp 1 )、(Sp 2 ) And/or (Sp) 3 ) Are all present at the same time. That is, in certain embodiments, the chemical spacer (Sp 1 )、(Sp 2 ) And/or (Sp) 3 ) Only one or two of which may be present in the joint.
In certain embodiments, the RK motif may be directly linked to one or more small hydrophobic amino acid residues. For example, in certain embodiments, the RK motif can be directly linked to one or more alanine residues.
That is, in a specific 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), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4) or RK-Val-Cit (SEQ ID NO: 54). It will be appreciated that the RK motif used to couple the linker to the glutamine residues of the antibody may be contained in the amino acid sequence RKAA, RKA, ARK, RKR or RK-Val-Cit.
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).
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).
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).
In the present invention, it is preferred that the linker is coupled to the antibody by a primary amine in the side chain of residue K contained in the RK motif. Therefore, it is preferable that the chemical spacer (Sp 1 )、(Sp 2 ) And/or (Sp) 3 ) No additional lysine residues, lysine mimetics or lysine derivatives are included, which can be used as additional amine donors in transglutaminase-based coupling reactions. In other embodiments, any free N-terminal amino group contained in the linker may be takenGeneration (e.g., acetylation) such that it cannot serve as a substrate for microbial transglutaminase.
The joint of the present invention further comprises at least one connection portion or payload B. The linker of the invention can be used to couple the payload directly to an antibody in a one-step coupling process. In other embodiments, a linker comprising one or more linking moieties may be coupled to the antibody in a first step, and then one or more payloads may be attached to the antibody-linker conjugate in a second step. Table 1 below sets forth two terms as used herein:
TABLE 1 one-step coupling and two-step coupling
In some embodiments, the joint may include one or more connecting portions B. Thus, in a particular embodiment, the invention relates to a method according to the invention, wherein B is a linking moiety.
As used herein, "linking moiety" generally refers to an at least bifunctional molecule. In the present invention, the linking moiety comprises a first functional group that allows the linking moiety to be coupled to the linker of the present invention and a second functional group that can be used to couple additional molecules to the linker either before or after the linker is coupled to the antibody. 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 linked to the linker via its amino group, while the functional group contained in the amino acid side chain may be used to couple additional molecules to the linker. Alternatively, the linking moiety may be linked to the linker through its carboxyl group, while the functional group contained in the amino acid side chain may be used to couple additional molecules to the linker.
In a particular embodiment, the invention relates to a method according to the invention, wherein the connecting portion B comprises
Bio-orthogonal labelling group, or
Non-bioorthogonal entities for cross-linking.
The term "bioorthogonal labeling group" has been established by Sletten and Bertozzi (bioorthogonal bicyclic linkage (ABioorthogonal Quadricyclane Ligation), J Am Chem Soc 2011, 133 (44), 17570-17573) to designate reactive groups that can cause chemical reactions to occur within living systems without interfering with natural biochemical processes. The "non-bioorthogonal entity for cross-linking" may be any molecule comprising or consisting of a first functional group, wherein the first functional group may be chemically or enzymatically cross-linked to a payload comprising a compatible second functional group. Even in the case where the cross-linking reaction is a non-bioorthogonal reaction, it is preferred that the reaction does not introduce additional modifications to the antibody other than cross-linking of the payload to the linker. In view of the above, the linking moiety B may be composed of a "bio-orthogonal labeling group" or a "non-bio-orthogonal entity", or may include a "bio-orthogonal labeling group" and a "non-bio-orthogonal entity". For example, at the linker moiety Lys (N 3 ) In the present invention, the whole Lys (N 3 ) And azido groups can both be considered bio-orthogonal labeling groups. Lys (N) 3 ) Refers to 6-azido-L-lysine, which may also be abbreviated as K (N) 3 )。
In a specific embodiment, the invention relates to a method according to the invention, wherein the bio-orthogonal labeling group or the non-bio-orthogonal entity for cross-linking consists of or comprises at least one molecule or moiety selected from the group consisting of:
-N-N≡N or-N 3 ；
·Lys(N 3 )；
Tetrazine;
alkynes;
strained cyclooctyne;
·BCN；
strained olefins;
photoreactive groups;
aldehyde;
acyl trifluoroborates;
protein degrading agent ('PROTAC');
cyclopentadiene/spirocyclopentadiene;
a thio-selective electrophile;
-SH; and
cysteine.
The bio-orthogonal labeling groups or non-bio-orthogonal entities contained in the linker for cross-linking may for example participate in any of the binding reactions shown in table 2:
TABLE 2
The linking moiety B may be or comprise the so-called "binding partner 1" or "binding partner 2" in table 2.
In certain embodiments, the linking moiety B may be a cysteine, a cysteine mimic, or a cysteine derivative having a free thiol group.
The free thiol group of such Cys residues (or mimetics or derivatives) can be coupled to a payload construct comprising a thio-selective electrophile, such as maleimide. Toxin constructs comprising maleimide moieties have been frequently used and are also approved by medical authorities, such as Adcetris. Thus, toxin constructs comprising MMAE toxins can be coupled to the free thiol group of a Cys residue in the linker of the invention.
It must be noted that other thio-selective electrophiles, such as 3-Aryl Propionitrile (APN) or phosphoamidate (phosphoramidate), may also be used instead of maleimide in the process of the present invention.
Thus, providing Cys residues in the linker according to the invention has the advantage of allowing the use of off-the-shelf toxin-maleimide constructs to generate antibody-payload conjugates, or more generally, the advantage of being able to fully exploit Cys-maleimide binding chemistry. At the same time, ready-made antibodies can be used, which do not have to be deglycosylated. In particular embodiments, the Cys residue may be C-terminal or intra-chain in an amino acid based linker.
In another embodiment, the linking moiety B may comprise an azide group. Those skilled in the art are aware of molecules comprising an azide group that may incorporate a linker according to the invention, such as 6-azido-lysine (Lys (N) 3 ) Or 4-azido-homoalanine (Xaa (N) 3 )). The linking moiety comprising an azide group may be used as a substrate in different bio-orthogonal reactions, such as strain-promoted azide-alkyne cycloaddition (sparc), copper-catalyzed azide-alkyne cycloaddition (CuAAC) or Staudinger ligation. For example, in certain embodiments, a payload comprising a cyclooctyne derivative (such as DBCO, DIBO, BCN or BARAC) can be coupled to a linker comprising an azide group via sparc.
In another embodiment, the linking moiety B may comprise a tetrazine group. Those skilled in the art are aware of tetrazine containing molecules, preferably amino acid derivatives comprising tetrazine groups, which may be incorporated into linkers according to the invention. The linker moiety comprising a tetrazine may be used as a substrate in a bio-orthogonal tetrazine linker. For example, in certain embodiments, a payload comprising a cyclopropene, norbornene derivative, or cyclooctyne group, such as bicyclo [6.1.0] nonyne (BCN), may be coupled to a linker comprising a tetrazine group.
In certain embodiments, the linking moiety B may comprise a cyclic diene, such as a cyclopentadiene derivative. Amant et al, tuning the Diels-Alder Reaction for Bioconjugation to Maleimide Drug-linker (modulating Diels-Alder reaction of bioconjugate to maleimide drug linker) 2018, 29,7, 22406-2414; and Amant et al A Reactive Antibody Platform for One-Step Production of Antibody-Drug Conjugates through a Diels-Alder Reaction with Maleimide (reactive antibody platform for one-step production of antibody-drug conjugates by Diels-Alder reaction with maleimide), bioconjugate Chem;2019 30,9, 2340-2348 have described potential cyclopentadiene derivatives that can be linked to maleimide-containing payload molecules.
In certain embodiments, the linking moiety B may comprise a photoreactive group. The term "photoreactive group" as used herein refers to a chemical group that reacts to an applied external energy source to produce an active species, thereby covalently bonding to an adjacent chemical structure (e.g., extractable hydrogen). Examples of photoreactive groups are, 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, biazidine, psoralen, or benzophenone.
In a particular embodiment, the invention relates to a method according to the invention, comprising the further step of coupling one or more payloads to the linking moiety B.
Instead of coupling the linker comprising one or more payloads directly to the antibody in a one-step process, in certain embodiments the invention relates to a two-step process, wherein the linker comprising at least one linking moiety B is coupled to the antibody in a first step and one or more payloads may be subsequently coupled to the linking moiety B in a second step.
The term "payload" as used herein refers to any naturally occurring or synthetically produced molecule, including small molecular weight molecules or chemically synthesized chemical entities as well as macromolecules or biological entities that are required to be produced by fermentation of host cells or that can also be chemically synthesized and confer new functions on antibodies. It will be appreciated that the payload may include further structures or functional groups that allow the payload to be attached to a linking moiety contained in the linker or other moiety of the linker (such as a chemical spacer (Sp 1 ) And/or (Sp) 3 ) Or RK motif).
In a two-step coupling method, the payload may be attached to the linking moiety by any suitable method known in the art. Preferably, the payload may be attached to any of the bio-orthogonal labeling groups disclosed herein or non-bio-orthogonal entities for cross-linking. That is, the payload preferably comprises a functional group compatible with the bio-orthogonal labeling group contained in at least one of the linking moieties B or the non-bio-orthogonal entity used for crosslinking.
Several bio-orthogonal reactions that can be used to attach the payload to the bio-orthogonal label groups included in the linking moiety B are known in the art. For example, many chemical ligation strategies have been developed to meet the requirement of bio-orthogonality, including 1, 3-dipolar cycloaddition between azide and cyclooctyne (also known as Copper-free click chemistry, baskin et al ("Copper-free click chemistry for dynamic in vivo imaging (Copper-free click chemistry for dynamic in vivo imaging)", proceedings of the National Academy of sciences.104 (43): 16793-7)), between nitrone and cyclooctyne (Ning et al ("strain-Promoted Alkyne-nitrone cycloaddition modified protein (Protein Modification by Strain-protein-Nitrone Cycloaddition)", angewandte Chemie International edition.49 (17): 3065) "); aldehydes and ketones form oxime/hydrazones (Yarema et al ("metabolic transfer of ketone groups to sialic acid residues, applied to cell surface glycoprotein engineering (Metabolic Delivery of Ketone Groups to Sialic Acid residues. Application To Cell Surface Glycoform Engineering)", journal of biochemistry 273 (47): 31168-79)); tetrazine linkages (Blackman et al ("tetrazine linkages: quick bioconjugate based on anti-electron demand Diels-Alder Reactivity (The Tetrazine Ligation: fast Bioconjugation based on Inverse-electron-demand Diels-Alder reactivities)", journal of the American Chemical,130 (41): 13518-9)); click reaction based on isonitrile Et al ("exploration of isonitrile-based click chemistry and biomolecular ligation (Exploring isonitrile-based click chemistry for ligation with biomolecules)", organic)&Biomolecular Chemistry,9 (21): 7303)); and the recent tetracycloalkane linkages (Sletten and Bertozzi (JACS, "bioorthogonal tetracycloalkane linkages (A Bioorthogonal Quadricyclane Ligation)", J Am Chem Soc,2011, 133 (44), 17570-17573 A) is set forth; copper (I) catalyzed azide-alkyne cycloaddition (CuAAC, kolb&Sharpless ("increasing impact of click chemistry on Drug discovery (The growing impact of click chemistry on Drug discovery)", drug discovery today.8 (24): 1128-1137)), strain-promoted azide-alkyne cycloaddition (SPAAC, agard et al ("comparative study of bioorthogonal reactions with azides (A Comparative Study of Bioorthogonal Reactions with Azides)", ACS Chem. Biol. 1:644-648)), or Strain-promoted alkyne-nitroketone cycloaddition (SPANC, macKenzie et al ("Strain-promoted cycloaddition involves nitroketone and alkyne rapid tunable reactions for bioorthogonal labeling (Strain-promoted cycloadditions involving nitrones and alkynes-rapid tunable reactions for bioorthogonal labeling)", curr Opin Chem biol. 21:81-8)). All of these documents are incorporated by reference herein to provide a full disclosure and avoid lengthy repetition.
It will be appreciated that after the linker has been coupled to the Gln residues of the antibody by microbial transglutaminase, the payload is preferably coupled to a bio-orthogonal labeling group comprised in the linker of the invention or to a non-bio-orthogonal entity for cross-linking. However, the invention also includes antibody-linker conjugates, wherein in a first step one or more payloads are coupled to a linker comprising at least one linking moiety B, and wherein in a second step the resulting linker-payload construct is coupled to an antibody by a microbial transglutaminase.
In a particular embodiment, the invention relates to a method according to the invention, wherein one or more payloads are coupled to the linking moiety B via a click reaction.
That is, one or more payloads may be attached to linking moiety B in a click reaction, particularly any of the click reactions disclosed herein.
In a particularly preferred embodiment, at least one payload may be coupled to the linking moiety B comprised in the linker via thiol-maleimide coupling. That is, in certain embodiments, the payload may include a maleimide group, and the linking moiety B may be a molecule including 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 comprising 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.
In another particularly preferred embodiment, at least one payload may be coupled to the linking moiety B comprised in the linker via a strain-promoted azide-alkyne cycloaddition (sparc). That is, in certain embodiments, the payload may comprise an alkynyl group, such as but not limited to cyclooctynyl (cycloocytne group), and the linking moiety B may be an azide group-containing molecule, such as but not limited to lysine derivative Lys (N) as disclosed herein 3 ). However, B may also be a non-amino acid molecule comprising a free azide group. In another embodiment, the payload may include an alkynyl group (such as a cyclooctynyl group), and the linking moiety B may include an azide group.
In addition to a click reaction between the linking moiety in the linker and the functional group in the payload, the payload may be covalently bound to the linking moiety by any enzymatic or non-enzymatic reaction known in the art.
Preferably the payload is attached to the linking moiety via a covalent bond. However, in some embodiments, the payload may be attached to the linking moiety via a strong non-covalent bond. That is, in certain embodiments, the linking moiety B may include a biotin moiety, such as, but not limited to, a lysine derivative biocytin. In such embodiments, the payload comprising the streptavidin moiety may be linked to a linker comprising the biotin moiety.
In a particular embodiment, the invention relates to a method according to the invention, wherein B is a payload.
In certain embodiments, the payload may already be part of the linker, such that the payload may be coupled to the antibody in a one-step process. In such embodiments, the linker is preferably coupled to the linker by chemical synthesis. The payload is preferably coupled to a chemical spacer comprised in the linker or directly to the RK motif. In embodiments where the payload is coupled to an amino acid residue (including amino acid mimics and derivatives), the payload may be coupled to the C-terminal carboxyl or N-terminal amino group of the amino acid residue. Alternatively, the payload may be coupled to a functional group contained in a side chain of the amino acid residue. The skilled person is aware of methods of functionalizing payloads so that they can be coupled to carboxyl, amino or amino acid side chains.
Further, the person skilled in the art is aware of methods for coupling payloads to amino acid based linkers by chemical synthesis. For example, an amine-containing payload, or a thiol-containing payload (e.g., maytansinoid), or a hydroxyl-containing payload (e.g., SN-38 analog) may be attached to the C-terminus of an amino acid-based linker by chemical synthesis. However, those skilled in the art are aware of further reactive and reactive groups that can be used to couple payloads to the N-terminus, C-terminus, or side chains of an amino acid or amino acid derivative by chemical synthesis. Typical reactions that may be used to couple payloads to amino acid based linkers by chemical synthesis include, but are not limited to: peptide coupling, activated ester coupling (NHS ester, PFP ester), click reaction (CuAAC, sparc), michael addition (thiol-maleimide coupling). Conjugation of payloads to peptides has been widely described in the prior art, for example by Costonlus et al (Peptide-Cleavable Self-cleaving maytansinoid antibody-drug conjugates designed to provide improved Bystander killing (Peptide-Cleavable Self-cleaving Self-immolative Maytansinoid Antibody-Drug Conjugates Designed To Provide Improved Bystander Killing), ACS Med Chem Lett.2019Sep 27;10 (10): 1393-1399); sonzini et al (use of host-Guest Chemistry to improve the physical stability of antibody-drug conjugates (Improved Physical Stability of an Antibody-Drug Conjugate Using Host-Guest Chemistry), bioconjugate chem.2020Jan 15;31 (1): 123-129); bodero et al (RGD and isoDGR peptide mimetic for tumor targeting-synthesis and biological evaluation of alpha-amanitine conjugates (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 (next generation maleimides and thioMAB) TM The combined use of antibody technology provides a highly stable, efficient and nearly homogeneous THIOMAB TM Antibody-drug conjugate (TDC) (Use of a next generation maleimide in combination with THIOMAB) TM antibody technology delivers a highly stable,potent and near homogeneous THIOMAB TM anti-drug connect (TDC), RSC adv, 2017,7, 24828-24832; doronina et al (enhanced activity of monomethyl auristatin F delivered by monoclonal antibodies: effect of linker technology on efficacy and toxicity (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 cytotoxic payloads based on irinotecan derivatives (Novel antibody drug conjugates containing exatecan derivative-based cytotoxic payloads), bioorg Med Chem Lett.2016Mar 15;26 (6): 1542-1545); and Dickgierter et al (Site-specific conjugation of natural antibodies using engineered microbial transglutaminase (Site-Specific Conjugation of Native Antibodies Using Engineered Microbial Transglutaminases), bioconjug chem.2020, mar 12, doi:10.1021/acs. Bioconjchem.0c00061).
It will be appreciated that the payload may be coupled to the N-terminus or the C-terminus of a peptide-based or peptide-containing linker according to the invention. In certain embodiments, the payload may be directly coupled to the N-terminal amino or C-terminal carboxyl group of the peptide or amino acid residue (see, e.g., fig. 22).
The skilled person is aware of reactive groups suitable for coupling payloads to amino acid residues. For example, an amine-containing payload may be coupled to the C-terminal carboxyl group of an amino acid residue via an amide bond (fig. 22). Alternatively, payloads containing thiol or hydroxyl groups may be coupled to the C-terminal carboxyl group of an amino acid via a thioester or ester bond, respectively. The payload comprising the carboxylic acid group may be coupled to the N-terminal amino group of the amino acid residue via an amide bond.
In certain embodiments, the payload may be indirectly coupled to the N-terminus or the C-terminus of a peptide or amino acid residue contained in a linker according to the invention. Those skilled in the art are aware of linker molecules that can be used to couple a payload to the N-terminal amino or C-terminal carboxyl group of an amino acid residue comprised in a linker according to the invention.
In certain embodiments, the payload comprising the hydroxyl group may be coupled to the N-terminus of the amino acid residue via a linker molecule. For example, a payload comprising a hydroxyl group may be coupled to an N-terminal amino group via a urethane linker molecule (fig. 24).
In certain embodiments, payloads comprising thiol groups may be coupled to the N-terminus of an amino acid residue via a linker molecule. For example, payloads containing thiol groups can be coupled to the N-terminal amino group via a thiocarbamate linker molecule (fig. 28). Alternatively, payloads containing thiol groups may be coupled to the N-terminal amino group via an alkyl linker molecule containing a carboxyl group and a thiol group. In certain embodiments, the alkyl linker molecule may be a 3-mercaptopropionic acid linker molecule, wherein the payload forms a disulfide bond with a thiol group contained in the 3-mercaptopropionic acid linker molecule (fig. 29).
In certain embodiments, the payload comprising the amide group may be coupled to the N-terminus of the amino acid residue via a linker molecule. For example, a payload comprising an amine group may be coupled to an N-terminal amino group via a dicarboxylic acid linker molecule, wherein 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 may be used as linker molecules in the present invention are, but are not limited to succinic acid or pimelic acid (see fig. 9 and 30).
Alternative linker molecules for indirectly coupling a payload to the N-terminus of an amino acid residue comprised in a linker according to the invention have been described in the art or linker molecules suitable for directly coupling a payload to the C-terminus of an amino acid residue comprised in a linker according to the invention are comprised in the invention.
In a particular embodiment, the invention relates to a method according to the invention, wherein the payload comprises at least one of the following:
toxins;
cytokines;
growth factors;
radionuclides;
hormones;
antiviral agents;
an antimicrobial agent;
fluorescent dye;
immunomodulators/immunostimulants;
half-life increasing moiety;
a solubility-increasing moiety;
polymer-toxin conjugate;
nucleic acid;
biotin or streptavidin moiety;
vitamins;
protein degrading agent ('PROTAC');
a target binding moiety; and/or
Anti-inflammatory agents.
Any of the payloads disclosed herein may be coupled directly to a linker for use in the one-step coupling process disclosed herein, or may be attached to a linking moiety contained in an antibody-linker conjugate that is produced as part of the two-step process disclosed herein.
In certain embodiments, the payload may be a cytokine. The term "cytokine" as used herein refers to any secreted polypeptide that affects other cellular functions and modulates interactions between cells in an immune or inflammatory response. Cytokines include, but are not limited to, monokines, lymphokines, and chemokines, regardless of which cell produces them. For example, monokines are commonly referred to as being produced and secreted by monocytes, however, many other cells produce monokines (such as natural killer cells, fibroblasts, basophils, neutrophils, endothelial cells, brain astrocytes, bone marrow stromal cells, epidermal keratinocytes, and B lymphocytes). Lymphokines are generally thought 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 (TNF alpha), and tumor necrosis factor beta (TNF beta).
In certain embodiments, the payload may be an anti-inflammatory agent. As used herein, the term "anti-inflammatory agent" refers to those agents whose primary mode of action and use is in the field of treating inflammation, as well as any other agents from another therapeutic class that have useful anti-inflammatory effects. Such anti-inflammatory agents include, but are not limited to, non-steroidal anti-inflammatory drugs (NSAIDs), disease modifying antirheumatic drugs (DMARDs), macrolide antibiotics, and statins. Preferably, NSAIDs include, but are not limited to, salicylates (e.g., aspirin), aryl propionic acids (e.g., ibuprofen), anthranilic acids (e.g., mefenamic acid), pyrazoles (e.g., phenylbutazone), cyclic acetic acids (indomethacin), and oxicams (oxicam) (e.g., piroxicam). Preferably, anti-inflammatory agents useful in the methods of the invention include sulindac, diclofenac, tenoxicam, ketorolac, naproxen, nabumetone, diferusal (diferusal), ketoprofen, aliskiren (arlypropionic acid), tenidap (tenidap), hydroxychloroquine, sulfasalazine, celecoxib (celecoxib), rofecoxib (rofecoxib), meloxicam (meloxicam), etoricoxib (etoricoxib), valdecoxib (valdecoxib), methotrexate, etanercept, infliximab, adalimumab, atorvastatin, fluvastatin, lovastatin, simvastatin, clarithromycin, azithromycin, roxithromycin, erythromycin, ibuprofen, dexibuprofen (debufen), flurbiprofen, fenoprofen, benoxicam, profen, and profen.
In certain embodiments, the anti-inflammatory agent may be an anti-inflammatory cytokine that, when bound to a target-specific antibody, may ameliorate inflammation caused, for example, by an autoimmune disease. Cytokines having anti-inflammatory activity may be, but are not limited to, IL-1RA, IL-4, IL-6, IL-10, IL-11, IL-13, or TGF-beta.
In certain embodiments, the payload may be a growth factor. The term "growth factor" as used herein refers to a naturally occurring substance capable of stimulating cell growth, proliferation, cell differentiation and/or cell maturation. The growth factors are present in the form of proteins or steroid hormones. Growth factors are important for regulating a variety of cellular processes. Growth factors generally act as signal molecules between cells. However, their ability to promote cell growth, proliferation, cell differentiation and cell maturation varies with growth factors. A non-limiting list of examples of growth factors includes: basic fibroblast growth factor, adrenomedullin, angiogenin, autotaxin, bone morphogenic protein, brain-derived neurotrophic factor, epidermal growth factor, epithelial growth factor, fibroblast growth factor, glial cell line-derived neurotrophic factor, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, growth differentiation factor-9, hepatocyte growth factor, hepatoma-derived growth factor, insulin-like growth factor, migration-stimulating factor, myostatin, nerve growth factor and other neurotrophic factors, 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 growth factor, and cytokines (e.g., IL-1-cofactors of IL-3 and IL-6, IL-2-t-cell growth factor, IL-3, IL-4, IL-5, IL-6 and IL-7).
In certain embodiments, the payload may be a hormone. The term "hormone" as used herein refers to a chemical substance released by a cell or gland in a part of the body that gives information about cells affecting other parts of the organism. Examples of hormones useful in the present invention are, but are not limited to, melatonin (MT), serotonin (5-HT), thyroxine (T4), triiodothyronine (T3), epinephrine (epiephrine) or epinephrine (adrenaline) (EPI), norepinephrine (norepinephrine) or norepinephrine (norradrenine) (NRE), dopamine (DPM or DA), antimalexin (anti-mullein) or Mu Leshi (mullein) inhibitory hormone (AMH), adiponectin (Acrp 30), corticotropin or corticotropin (ACTH), angiotensinogen and Angiotensin (AGT), anti-diuretic or vasopressin (ADH), atrial natriuretic peptide or atrial propeptide (ANP), calcitonin (CT), cholecystokinin (CCK), corticotropin-releasing hormone (CRH), erythropoietin (EPO), follicle stimulating hormone (GRP), growth hormone (GhGRP), anti-mullein (anti-Muller (anti-Ing), human hormone (GhGH), human hormone (GL), human hormone (GLG), human hormone (GhG), hormone (GLG), hormone (GL), hormone (GH), human hormone (hG), or hormone (hG), hG) or hG (RH), hG (hG) or hG (hG) are useful Melanocyte stimulating hormone (MSH or alpha-MSH), orexin (orexin), oxytocin (OXT), parathyroid hormone (PTH), prolactin (PRL), relaxin (RLN), secretin (SCT), somatostatin (SRIF), thrombopoietin (TPO), thyroid stimulating hormone or Thyroid Stimulating Hormone (TSH), thyroid stimulating hormone releasing hormone (TRH), cortisol, aldosterone, testosterone, dehydroepiandrosterone (DHEA), androstenedione, dihydrotestosterone (DHT), estrone, estriol (E3), progesterone, calcitriol, calcitonin, prostaglandins (PG), leukotrienes (LT), prostacyclin (PGI 2), thromboxane (TXA 2), prolactin Releasing Hormone (PRH), lipotropin (PRH), brain Natriuretic Peptide (BNP), neuropeptides Y, histamine, endothelin, polypeptides, renin and enkephalins.
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 viral formation and/or replication in a mammal. This includes agents that interfere with the host or viral machinery required for viral formation and/or replication in a mammal. Antiviral agents include, for example, ribavirin, amantadine, VX-497 (merimeprob), vertex Pharmaceuticals, VX-498 (Vertex Pharmaceuticals), levovirin (Levovirin), talivirine (Viramidine), histamine dihydrochloride (histamine dihydrochloride), XTL-001 and XTL-002 (XTL Biopharmaceuticals).
In certain embodiments, the payload may be an antimicrobial agent. As used herein, the term "antimicrobial" refers to a compound capable of: (i) inhibiting, reducing or preventing bacterial growth, (ii) inhibiting or reducing the ability of bacteria to produce an infection in a subject, or (iii) inhibiting or reducing the ability of bacteria to reproduce or maintain an infection in the environment, a compound, a combination of substances, or a combination of compounds. The term "antibacterial agent" also refers to a compound capable of reducing bacterial infectivity or virulence.
In certain embodiments, the payload may be an immunomodulatory agent. The term "immunomodulator" as used herein in combination therapy refers to a substance that acts to inhibit, mask or enhance the immune system of the host. Examples of immunomodulators include, but are not limited to, protein agents such as cytokines, peptidomimetics and antibodies (e.g., human, humanized, chimeric, monoclonal, polyclonal, fv, scFv, fab, or F (ab) 2 fragments or epitope binding fragments), nucleic acid molecules (e.g., antisense nucleic acid molecules, iRNA, and triple helix nucleic acid molecules), small molecules, organic compounds, and inorganic compounds. In particular, immunomodulators include, but are not limited to: methotrexate (methotrexate), leflunomide (leflunomide), cyclophosphamide (cyclophosphamide), cyclophosphamide (Cytoxan), pyrazole anhydride thiourea (Imuran), cyclosporin A (cyclosporine A), minocycline (minocycline), azathioprine (azathioprine), antibiotics (e.g., FK506 (tacrolimus)), methylprednisolone (MP), corticosteroids, steroids, mycofenozide (mycophenolate mofetil), rapamycin (sirolimus), bromophenol (mizoribine), deoxyspergualin (deoxyspergualin), brequinar (malononitrilobalamide) (e.g., leflunomide)), T cell receptor modulators and cytokine receptor modulators.
In certain embodiments, the immunomodulator may be an immunostimulant. The term "immunostimulant" as used herein preferably refers to any substance or substances capable of triggering an immune response (e.g., an immune response against a specific pathogen). Immune cell activating compounds include Toll-like receptor (TLR) agonists. Such agonists include pathogen-associated molecular patterns (PAMPs), for example compositions mimicking infection, such as bacterial-derived immunomodulators (also known as danger signals), and damage-associated molecular patterns, for example compositions mimicking stress or damage cells. TLR agonists include nucleic acids or lipid compositions (e.g., monophosphoryl lipid a (MPLA)). In one example, the TLR agonist comprises a TLR9 agonist, such as a cytosine guanosine oligonucleotide (CpG-ODN), a poly (ethyleneimine) (PEI) -condensed Oligonucleotide (ODN) (such as PEI-CpG-ODN-or double-stranded deoxyribonucleic acid (DNA)). In another example, TLR agonists include TLR3 agonists such as poly inosine-polycytidylic acid (poly (I: C)), PEI-poly (I: C), poly adenylate-poly uridylic acid (poly (a: U)), PEI-poly (a: U), or double stranded ribonucleic acid (RNA). Other exemplary vaccine immunostimulatory compounds include Lipopolysaccharide (LPS), chemokines/cytokines, fungal beta-glucans (such as lentinan), imiquimod, CRX-527, and OM-174.
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 polymer moieties, PAS moieties (oligopeptides constituting proline, alanine and serine; PAS acylated) or serum albumin binders. The solubility-increasing moiety is, for example, a PEG-moiety (PEGylation) or a PAS moiety (PAS acylation).
In certain embodiments, the payload may be a polymer-toxin conjugate. A polymer-toxin conjugate is a polymer capable of carrying a number of payload molecules. Such conjugates are sometimes also referred to as flexible bodies (flexmers), for example sold by Mersana treatment company. The polymer-toxin conjugate may comprise any of the toxins disclosed herein.
In certain embodiments, the payload may be a nucleotide. An example of a nucleic acid payload is MCT-485, which is a very small non-coding double stranded RNA with oncolytic and immune activating properties, developed by MultiCell Technologies, inc.
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 650nm and 900 nm. In this region, tissue autofluorescence is lower and less fluorescence extinction enhances deep tissue penetration with minimal background interference. Thus, near infrared fluorescence imaging can be used to visualize tissue to which the antibody payload conjugates of the invention bind during surgery. "near infrared fluorescent dyes" are known in the art and are commercially available. In certain embodiments, the near infrared fluorescent dye may be IRDye 800CW, cy7, cy7.5, NIR CF750/770/790, dylight 800 or Alexa Fluor 750.
In certain embodiments, the payload may comprise a radionuclide. The term "radionuclide" as used herein relates to medically useful radionuclides including, for example, positively charged ions of a radiometal such as Y, in, tb, ac, cu, lu, tc, re, co, fe and the like such as 90 Y、 111 In、 67 Cu、 77 Lu、 99 Tc、 161 Tb、 225 Ac, etc. The radionuclide may be contained in a chelator such as DOTA or NODA-GA. Further, the radionuclide may be a therapeutic radionuclide or a radionuclide that can be used as a contrast agent in imaging techniques, as described below. Radionuclides or molecules comprising radionuclides are known in the art and are commercially available.
In certain embodiments, the payload may be a vitamin. The vitamins may be selected from the group consisting of folate (including folic acid, and vitamin B9).
In a particular embodiment, the invention relates to a method according to the invention, wherein the toxin is at least one selected from the group consisting of:
pyrrolobenzodiazepines (e.g., PBDs);
auristatin (e.g., MMAE, MMAF);
maytansinoids (e.g., maytansine, DM1, DM4, DM 21);
sesquicomycin;
Nicotinamide phosphoribosyl transferase (NAMPT) inhibitors;
microtubule lysin;
enediyne (e.g., calicheamicin);
anthracycline derivatives (PNUs) (e.g., doxorubicin);
inhibitors of the pyrrolyl Kinesin Spindle Protein (KSP);
candidiasis;
drug efflux pump inhibitors;
mountain Zhuo Meisu;
amanitine (e.g., α -amanitine); and
camptothecins (e.g., irinotecan, delutinacon).
That is, the antibody-linker conjugates produced by the methods of the invention preferably comprise a toxin payload. The term "toxin" as used herein relates to any compound that is produced by and toxic to a living cell or organism. Thus, the toxin may be, for example, a small molecule, a peptide or a protein. Specific examples are neurotoxin, necrotic toxin, blood toxin and cytotoxin. In certain embodiments, the toxin is a toxin for use in treating neoplastic disease. That is, the toxin may be conjugated to an antibody using the methods of the invention and delivered to or into malignant cells due to the target specificity of the antibody.
In certain embodiments, the toxin may be auristatin. As used herein, the term "auristatin" refers to a family of antimitotics. The term "auristatin" is 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 (dolastatin).
In certain embodiments, the toxin may be a maytansinoid. In the context of the present invention, the term "maytansinoid" refers to a class of highly cytotoxic drugs originally isolated from the african shrubs maytansinoid (Maytenus ovatus) as well as additional maytansinols (Maytansinol) and C-3 esters of natural Maytansinol (U.S. Pat. No. 4,151,042); c-3 ester analogues of maytansinol were synthesized (Kupchan et al, J.Med. Chem.21:31-37, 1978; higashide et al, nature 270:721-722, 1977; kawai et al, chem. Farm. Bull.32:3441-3451; and U.S. Pat. No. 5,416,064); c-3 esters of simple carboxylic acids (U.S. Pat. Nos. 4,248,870;4,265,814;4,308,268;4,308,269;4,309,428;4,317,821;4,322,348; and 4,331,598); and C-3 esters and derivatives of N-methyl-L-alanine (U.S. Pat. No. 4,137,230;4,260,608; and Kawai et al chem.pharm Bull.12:3441,1984). Exemplary maytansinoids that may be used in the methods of the invention or that may be included in the antibody-payload conjugates of the invention are maytansinoids, DM1, DM3, DM4 and/or DM21.
In certain embodiments, the toxin may be a duocarmycin (duocarmycin). Suitable macbecins can be, for example, macbecins A, bl, B2, CI, C2, D, SA, MA and CC-1065. The term "duocarmycin" is understood to also refer to synthetic analogues of duocarmycin, such as adozelesin, bizelesin, carbozelesin, KW-2189 and CBI-TMI.
In certain embodiments, the toxin may be a NAMPT inhibitor. As used herein, the terms "NAMPT inhibitor" and "nicotinamide phosphoribosyl transferase inhibitor" refer to inhibitors that reduce NAMPT activity. The term "NAMPT inhibitor" may also include prodrugs of NAMPT inhibitors. Examples of NAMPT inhibitors include, but are not limited to, FK866 (also known as APO 866), GPP 78 hydrochloride, ST 118804, STF31, pyridylcyanguanide (also known as CH-828), GMX-1778, and P7C3. Additional NAMPT inhibitors are known in the art and may be suitable for use in the compositions and methods described herein. See, for example, PCT publication WO 2015/054060, U.S. patent nos. 8,211,912 and 9,676,721, the entire contents of which are incorporated herein by reference. In some embodiments, the NAMPT inhibitor is FK866. In some embodiments, the NAMPT inhibitor is GMX-1778.
In certain embodiments, the toxin may be tubulysin. Tubulysin is a cytotoxic peptide comprising 9 members (a-I). Tubulysin a has potential application prospects as an anticancer agent. It blocks cells during the G2/M phase. Tubulysin a inhibits polymerization more effectively than vinca alkaloid (vinblastine) and induces depolymerization of isolated microtubules. Tubulysin a has a powerful cytostatic effect against a variety of tumor cell lines with IC50 in the picomolar range. Other tubulysins that may be used in the methods of the invention may be tubulysin E.
In certain embodiments, the toxin may be enediyne. The term "enediyne" as used herein refers to a class of bacterial natural products characterized by nine-membered and ten-membered rings containing two triple bonds separated by a double bond (see, e.g., K.C.Nicolaou; A.L.Smith; E.W.Yue (1993), "chemistry and biology of natural and engineered enediynes (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 are capable of Bergman cyclization and the resulting diradicals (1, 4-dehydrobenzene derivatives) are capable of abstracting a hydrogen atom from the sugar backbone of DNA, which results in DNA strand cleavage (see e.g., s.walker; r.landovitz; w.d. ding; g.a. Elestad; d.kahne (1992) "cleavage behavior of calicheamicin γ1and calicheamicin T (Cleavage behavior of calicheamicin gamma 1and calicheamicin T)", proc Nat1 Acad Sci u.s.a.89 (10): 4608-12; the entire contents of which are incorporated herein by reference). Their reactivity with DNA confers many enediyne antibiotic properties, and some enediyne are being clinically investigated as anticancer antibiotics. Non-limiting examples of enediynes are dactinomycin (dymicin), neocarcinostatin (neocarcinostatin), calicheamicin (calicheamicin), epothilone (esperamicin) (see, e.g., adrian L.Smith and K.C. Bicolau, "enediyn antibiotics (The Enedizyne Antibiotics)", J.Med. Chem.,1996, 39 (11), pp 2103-2117; and Donald Borders, "enediyn antibiotics as antitumor agents (Enedizyne antibiotics as antitumor agents)", informa Healthcare; version 1 (Nov. 23, 1994, ISBN-10:082489385; the entire contents of which are incorporated herein by reference).
In certain embodiments, the toxin may be doxorubicin. As used herein, "doxorubicin" refers to a member of the anthracycline family derived from the Streptomyces (Streptomyces) bacterium Streptomyces Boseki, apparent variety (Streptomyces peucetius var. Caesius), and includes doxorubicin, daunorubicin, epirubicin, and idarubicin.
In certain embodiments, the toxin may be a kinesin spindle protein inhibitor. The term "kinesin spindle protein inhibitor" refers to a compound that inhibits kinesin spindle proteins that are involved in bipolar spindle assembly during cell division. Kinesin spindle protein inhibitors are being investigated for the treatment of cancer. Examples of kinesin spindle protein inhibitors include iss Ping Si (ispinestib). Further, the term "kinesin spindle protein inhibitor" includes GlaxoSmithKline SB715992 or SB743921 and combinatosx's pentanamidine/chlorpromazine.
In certain embodiments, the toxin may be nostoc as described in US 20180078656 A1, which is incorporated by reference.
In certain embodiments, the toxin may be altretamycin. Mountain Zhuo Meisu is an ester peptide (depsipeptide), which was first isolated from nocardiops (Nocardioides sp.) (ATCC 39419), and has been demonstrated to have cytotoxic and antitumor activity.
In certain embodiments, the toxin may be amatoxins. Amatoxins (including alpha-amanitine, beta-amanitine, and amanitine) are cyclic peptides consisting of 8 amino acids. They may be isolated from amanita (Amanita phalloides) mushrooms or may be prepared synthetically from building blocks. Amatoxins specifically inhibit DNA-dependent RNA polymerase II in mammalian cells and thereby affect transcription and protein biosynthesis of the cells. Inhibition of transcription in cells results in cessation of growth and proliferation. Although not covalently bound, the complex between amanitine and RNA polymerase II is very tight (kd=3 nM). Dissociation of amanitine from enzymes is a very slow process that makes recovery of the affected cells less likely. When transcription inhibition in a cell is prolonged for too long, the cell undergoes programmed cell death (apoptosis). In a preferred embodiment, the term "Amatoxin" as used herein refers to a-amanitin or variants thereof as described in, for example, WO 2010/115630, WO 2010/115629, WO 2012/119787, WO 2012/04504 and WO 2014/135282.
In certain embodiments, the toxin may be camptothecin (camptothecin). The term "camptothecin" as used herein refers to camptothecin or camptothecin derivatives having the function of a topoisomerase I inhibitor. Exemplary camptothecins include, for example, topotecan (topotecan), exenatide (exatecan), deluxe (deruxecan), irinotecan (irinotecan), DX-8951f, SN38, BN 80915, lurtotecan (lurotecan), 9-nitrocamptothecine, and aminocamptothecin. Various camptothecins have been described, including camptothecins for use in the treatment of human cancer patients. Several camptothecins are described, for example, in Kehrer et al, anticancer Drugs,12 (2): 89-105, (2001) or Li et al, ACS Med. Chem. Lett.2019,10,10,1386-1392).
Toxins may also be inhibitors of drug efflux transporters in the sense of the present invention. An antibody-payload conjugate comprising a toxin and an inhibitor of a drug efflux transporter may have the advantage that the inhibitor of the drug efflux transporter prevents the toxin from flowing out of the cell when internalized into the cell. In the present invention, the drug efflux transporter may be a P-glycoprotein. Some common P-glycoprotein drug inhibitors include: amiodarone, clarithromycin, cyclosporine, colchicine, diltiazem, erythromycin, felodipine, ketoconazole, lansoprazole, omeprazole and other proton pump inhibitors, nifedipine, paroxetine, reserpine, saquinavir, sertraline, quinidine, tamoxifen, verapamil, and duloxetine. Icleidar (eladridar) and CP 100356 are other common P-gp inhibitors. The development of zoquidazole (zosuquidar) and taroquidazole (tariqidar) also takes this into account. Finally, valspoda (valspodar) and reverse are other examples of such agents.
It should be understood that the payload B as defined herein is not to be understood as the actual payload itself, but as the payload molecule. Payload molecules as used herein may include additional structures, for example to facilitate coupling of the payload to the linking moiety B or RK motif or chemical spacer via chemical synthesis.
That is, in certain embodiments, the actual payload may be contained in a payload molecule attached to a linker of the invention. The payload molecule may have the following structure:
x- (spacer) -payload,
wherein the payload represents an actual payload, e.g. one of the compounds disclosed herein, X represents a reactive group suitable for attaching the payload molecule to a compatible functional group in a chemical spacer or RK motif of a linking moiety (two-step process) or linker (one-step process), and wherein (spacer) represents a chemical spacer spatially separating the actual payload from the reactive group X. However, it should be understood that in certain embodiments, the reactive group X may be part of a spacer or actual payload. For example, the spacer may comprise a peptide or amino acid residue, wherein the reactive group X may be the amino group of the N-terminal amino acid residue comprised in the spacer. In certain embodiments, the spacer may not be present. In embodiments where no spacer is present, the functional group may be included in the actual payload. In certain embodiments, the spacer may be used to attach a functional group of interest (i.e., a functional group compatible with the functional groups contained in the linking moiety) to the actual payload. In certain embodiments, the reactive group X may be a maleimide group or a cyclooctyne group, such as, but not limited to, a DBCO or BCN group.
In a particular embodiment, the invention relates to a method according to the invention, wherein the chemical spacer (Sp 2 ) Including self-cleaving moieties.
That is, the linker may comprise a self-cleaving moiety to facilitate release of the payload in the target cell or tissue. The self-cleaving moiety may be contained in any portion of the linker. However, the self-cleaving moietyPreferably contained in a chemical spacer (Sp) separating the payload from the RK motif 2 ) Is a kind of medium. Alternatively, the self-cleaving moiety may be comprised in a (spacer) comprised in a payload molecule as defined above.
As used herein, the term "self-cleaving moiety (self-immolative moiety)" refers to at least a bifunctional molecule that may be contained in a linker and spontaneously degrade after an initial reaction occurs, thereby releasing the payload. The initial reaction may be hydrolysis of the covalent bond between the self-cleaving moiety and the amino acid residue. In certain embodiments, the covalent bond between the self-cleaving moiety and the amino acid residue may be an amide bond formed between an a-carboxy group of the amino acid and an amine group contained in the self-cleaving moiety, and the initial reaction may be catalysed by a peptidase or protease. However, the present invention includes other chemicals.
In a particular embodiment, the invention relates to a method according to the invention, wherein the self-cleaving part is directly attached to the payload B.
More preferably, the self-cleaving portion is directly attached to the payload B such that the payload is released upon degradation of the self-cleaving portion. In certain embodiments, the self-cleaving moiety is located between the payload and an RK motif contained in the linker. That is, the self-cleaving moiety may be coupled to the N-terminus of residue R or the C-terminus of residue K. Alternatively, the self-cleaving moiety may be located in a payload and chemical spacer (Sp 2 ) Preferably at the N-terminus or C-terminus of the amino acid residue. Further, the self-cleaving moiety may be located on the payload with a moiety contained in a chemical spacer (Sp 2 ) Is a non-amino acid residue.
It will be appreciated that the choice of self-cleaving moiety will depend inter alia on the functional groups available in the payload molecule.
In a particular embodiment, the invention relates to a method according to the invention, wherein the self-cleaving moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety.
That is, in certain embodiments, the linker may be To contain a self-cleaving moiety, a p-aminobenzylcarbamoyl (PABC) moiety. PABC includes free amine groups and carbamoyl groups suitable for coupling to the C-terminus of an amino acid residue or peptide, and PABC may be coupled to payloads, particularly payloads containing amines, through the carbamoyl group. However, the skilled artisan is aware of methods of functionalizing the payload so that it contains amine groups. The self-cleaving moiety PABC is preferably located between the payload and the amino acid residues comprised in the linker. The amino acid residue is preferably residue K contained in the RK motif or in a chemical spacer (Sp 2 ) Comprising an amino acid. In certain embodiments, the self-cleaving moiety PABC is located between the payload and the chemical spacer (Sp 2 ) Between the alanine residues in the inclusion. In certain embodiments, the self-cleaving moiety may be located between the payload and the peptidase cleavage site. In certain embodiments, the self-cleaving moiety may be located between the payload and the cathepsin cleavage site. That is, the self-cleaving moiety may be located between the payload and a motif known to be cleavable by a cathepsin.
The term "cathepsin" as used herein refers to a family of proteases. The term cathepsin includes cathepsin a, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin F, cathepsin G, cathepsin H, cathepsin K, cathepsin L1, cathepsin L2, cathepsin O, cathepsin S, cathepsin W and cathepsin Z. In particular embodiments, the cleavable moiety may be a motif specifically hydrolyzed by cathepsin B, such as valine-alanine, valine-citrulline, or alanine-alanine. Salomon et al, "lysosomal activation by incorporation of novel cleavable dipeptide linkers to optimize antibody-Drug Conjugates (ADC) (Optimizing Lysosomal Activation of Antibody-Drug Conjugates (ADCs) by Incorporation of Novel Cleavable Dipeptide Linkers)", mol Pharm,2019, 16 (12), disclose additional motifs that can be specifically hydrolyzed by peptidases.
A typical dipeptide structure used in ADC linkers is the valine-citrulline motif, provided for example in Brentuximab Vedotin; and a cathepsin B labile dipeptide linker at Dubowchik and Firestone "for lysosomal release of doxorubicin from internalized immunoconjugates: model studies of enzymatic drug release and antigen-specific in vitro anti-cancer activity (Cathepsin B-labile dipeptide linkers for lysosomal release of doxorubicin from internalizing immunoconjugates: model studies of enzymatic drug release and antigen-specific in vitro anticancer activity) "; bioconjug Chem;2002;13 (4); pages 855-69. Such linkers may be cleaved by cathepsin B to release the actual payload at the disease site. The same applies to valine-alanine motifs, which are provided, for example, in SGN-CD 33A.
Thus, in certain embodiments, the linker may comprise a structure (Sp 1 )-RK-(Sp 2 ) Val-Cit- (self-cleaving moiety) -payload. In certain embodiments, the linker may comprise a structure (Sp 1 )-RK-(Sp 2 ) Val-Cit-payload. In certain embodiments, the linker may comprise a structure (Sp 1 )-RK-(Sp 2 ) Val-Cit-PABC-payload.
In some embodiments, the linker may include the structure RK-Val-Cit (Seq ID NO: 54). In certain embodiments, the linker may comprise or consist of the structure RK-Val-Cit- (self-cleaving moiety) -payload. In certain embodiments, the linker may comprise or consist of the structure RK-Val-Cit-PABC-payload. In certain embodiments, the linker may comprise or consist of the structure RK-Val-Cit-PABC-MMAE. In certain embodiments, the linker may comprise or consist of the structure RK-Val-Cit-PABC-maytansine.
It must be noted that the peptide cleavage site may also be a motif cleavable by other peptidases, such as caspase 3, endoasparaginase (Legumain) or neutrophil elastase, or innovative ligation strategies for tumor targeted drug conjugates as described in Dal Corso et al (Innovative Linker Strategies for Tumor-Targeted Drug Conjugates), chemistry 25 (65), pages 14740-14757.
However, it must be noted that cells comprise a wide range of cellsPeptidases, and other less conserved amino acid motifs can also be efficiently cleaved by peptidases. Thus, in certain embodiments, the linker may comprise a structure (Sp 1 )-RK-(Sp 2 ) -a PABC-payload, wherein (Sp) 2 ) Absent or composed of amino acid residues.
In certain embodiments, the linker may comprise a structure (Sp 1 )-RK-(Sp 2 ) -a PABC-payload, wherein (Sp) 2 ) Comprises a PEG moiety which is conjugated to (Sp) at a PABC moiety 2 ) Or between the most C-terminal amino acid residues contained in the RK motif.
In certain embodiments, the linker comprising the self-cleaving moiety PABC is coupled to a payload comprising an amine (particularly a payload comprising a primary or secondary amine). In certain embodiments, the amine-containing payload is an oseltatin (ausristine), such as MMAE. In certain embodiments, the amine-containing payload is a maytansinoid, such as maytansinoid.
It must be noted that the payload may be coupled to the self-cleaving PABC moiety via an additional linker molecule. For example, an amine-containing payload may be coupled to a PABC moiety via a p-nitrophenol (PNP) group. Su et al Bioconjugate chem.2018, 29,4, 1155-1167 and Dokter et al Mol Cancer ter.2014nov; 13 (11): 2618-29 have disclosed other linker molecules that allow the coupling of payloads containing reactive groups other than amines to PABC moieties. For example, payloads containing alcohol or phenol groups may be coupled to PABCs via Ethylenediamine (EDA) linkers (see fig. 18 and 19).
In a particular embodiment, the invention relates to a method according to the invention, wherein the self-cleaving moiety comprises a methylamino group. It has been previously demonstrated that methylamino groups can be used as self-cleaving moieties in peptide-based linkers of ADCs (costaplus et al, ACS med. Chem. Lett.,2019, 10, 10, 1393-1399 and Li et al, ACS med. Chem. Lott.2019, 10, 10, 1386-1392).
In particular, the self-cleaving moiety comprising a methylamino group may be coupled by an amide bond formed between the α -carboxyl group of the amino acid residue and the amine comprised in the methylamino groupTo the C-terminus of the amino acid residue. The amino acid residue may be a residue contained in (Sp 2 ) Or residue K contained in the RK motif. The methyl groups contained in the methylamino groups may be coupled to the payload through ether or thioether linkages. Thus, when the payload includes a hydroxyl group or a thiol group, it may be preferable to use a methylamino group as the self-cleaving group. In certain embodiments, the hydroxyl-containing payload may be a camptothecin (such as the irinotecan derivative Dxd) or an anthracycline (such as PNU-159582). In certain embodiments, the payload comprising a thiol may be a maytansinoid (such as DM1, DM4, or DM 21).
The linker comprising a methylamino group may comprise the molecular structure C- (NH) - (CH) 3 ) -O-C or C- (NH) - (CH) 3 ) -S-C. Exemplary linkers including methylamino groups are shown in fig. 15, 17, and 21.
It will be appreciated that the payload is preferably coupled to the C-terminal carboxyl group of the amino acid residue using PABC and a self-cleaving moiety comprising a methylamino group.
Other self-cleaving moieties that may be used to couple a payload to the C-terminal carboxyl group of an amino acid residue include a p-aminobenzyl alcohol (PABE) linker (Zhang et al, bioconjugate chem.2018,29,6,1852-1858) for coupling a phenol-containing payload to the C-terminal carboxyl group of an amino acid residue or a p-methylaniline (PMA) linker (Staben et al, nature Chemistry volume 8, pages 1112-1119 (2016)) for coupling a payload comprising a tertiary amine or heteroaryl moiety to the C-terminal carboxyl group of an amino acid residue (see fig. 20 and 23, respectively). Non-limiting examples of payloads containing phenolic groups are the sesquialter mycin GA or the pyrrolobenzodiazepine PBD. A non-limiting example of a payload comprising a tertiary amine is the sesquialter mycin GA.
However, the payload may also be coupled to the N-terminal amino group via a self-cleaving moiety. For example, the payload may be coupled to the N-terminal amino group of an amino acid residue via a self-cleaving moiety comprising an ortho-hydroxy protected aryl sulfate. For example, ortho-hydroxy protected aryl sulfate (OHPAS) may be used to couple a phenolic payload, such as PBD, to the N-terminal amino group of an amino acid residue (see fig. 27). The OHPS part is preferably Comprising a carboxyl group, the OHPAS moiety may be directly coupled to the N-terminal amino group of the amino acid residue via the carboxyl group. Alternatively, the OHPAS moiety may be attached via a functionalized PEG (e.g., without limitation, functionalized (PEG) 2 Linker) is coupled to the N-terminal amino group of the amino acid residue. Preferably, one end of the PEG linker is functionalized with an amino group to allow coupling to the carboxyl group contained in the OHPS moiety and the other end is functionalized with a carboxyl group to allow coupling to the N-terminal amino group of an amino acid residue (Park et al Bioconjugate chem.2019, 30,7, 1957-1968) (see FIG. 26).
Alternatively or in addition, a linker molecule may be located between the sulfate group of the OHPAS and the payload to allow coupling of the non-phenolic payload to the OHPAS. For example, a p-hydroxybenzyl (PHB) linker molecule may be used to allow a payload comprising a primary or secondary amine to be coupled to an OHPS moiety by carbamate formation (see FIGS. 31 and 32). The tertiary amine-containing payload may be coupled to the OHPAS-containing linker by forming a quaternary ammonium (see fig. 33 and 34). Further, p-hydroxybenzyl ethylenediamine (PHB-EDA) linker molecules can be used to couple hydroxyl-containing payloads to OHPS moieties by carbamate formation (Park et al Bioconjugate chem.2019, 30,7, 1957-1968) (see FIG. 25).
In certain embodiments, the payload may be coupled to an amino acid residue contained in the linker via a cleavable moiety. As used herein, a "cleavable moiety" is a chemical unit that can be separated from the actual payload by enzymatic or non-enzymatic hydrolysis. In certain embodiments, the cleavable moiety may be an amino acid motif that is hydrolyzable by a peptidase or protease.
In other embodiments, the cleavable moiety contained in the linker may be a carbohydrate moiety. In such embodiments, the cleavable moiety may be a moiety that is cleavable by a glucosidase. Thus, in certain embodiments, the cleavable moiety may be a moiety that is cleavable by β -glucuronidase or β -galactosidase.
In other embodiments, the cleavable moiety contained in the linker may be a phosphate moiety. In such embodiments, the cleavable moiety may be a moiety that is cleavable by a phosphatase. Thus, in certain embodiments, the cleavable moiety may be a moiety that is cleavable by a beta-lysosomal acid pyrophosphatase or acid phosphatase.
Examples of other cleavable moieties that can be used to release a payload from a linker molecule have been described by Bargh et al, cleavable linkers in antibody-drug conjugates (Cleavable linkers in antibody-drug conjugates), chem Soc rev.2019, 8 months 12; 48 (16) 4361-4374. In some embodiments, the linker may include a structure (cleavable moiety) - (self-cleaving moiety) -payload. In such embodiments, the self-cleaving portion may degrade and release the payload upon cleavage of the cleavable portion.
In a particular embodiment, the invention relates to a method according to the invention, wherein the joint is any one of the joints shown in fig. 1, fig. 2, fig. 3, fig. 8, fig. 9, fig. 14, fig. 15, fig. 17, fig. 18, fig. 19, fig. 20, fig. 21, fig. 22, fig. 23, fig. 24, fig. 25, fig. 26, fig. 27, fig. 28, fig. 29, fig. 30, fig. 31, fig. 32, fig. 33 or fig. 34.
In some embodiments, the joint may include two or more connection portions and/or payload B. That is, in some embodiments, the joint may include a structure
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 (b)
d)(Sp 4 )-B 2 -(Sp 1 )-B 1 -(Sp 2 )-RK-(Sp 3 )。
In such embodiments, the chemical spacer (Sp 1 )、(Sp 2 )、(Sp 3 ) And the RK motif may have the same properties as defined above. In addition, part B 1 And B 2 May be any of the connection portions and/or payloads defined above. Further, chemical spacer (Sp 4 ) May have a chemical spacer (Sp 1 )、(Sp 2 ) Or (Sp) 3 ) The same characteristics may or may not be present.
Thus, in a particular embodiment, the invention relates to a method according to the invention, wherein the joint comprises a second connection part or payload B 2 In particular wherein B 2 Via chemical spacers (Sp 1 ) Or (Sp) 3 ) Is connected to the joint.
That is, payload or connection portion B 2 May be attached to a chemical spacer (Sp 1 ) Or (Sp) 3 ) Or directly to the payload or to the connection part B 1 . Payload or connection part B 2 May include a catalyst suitable for mixing B 2 Is coupled to a metal complex in (Sp 1 )、(Sp 3 ) Or B is a 1 Any of the functional groups contained in the (c) polymer.
In certain embodiments, payload or attachment portion B 2 May include amino, B 2 Is linked to (Sp) 3 ) Or B is a 1 . That is, B 2 Can be linked to (Sp) through the amino group 3 ) Or B is a 1 Carboxyl groups contained in the (b) amino acid. In certain embodiments, the compositions are contained in (Sp 3 ) The carboxyl group in (c) may be a carboxyl group in a chemical spacer (Sp 3 ) Carboxyl groups contained in the C-terminal amino acid residues of (C). In certain embodiments, B 1 The carboxyl group comprised in (a) may be an alpha-carboxyl group based on the payload or linking moiety of an amino acid. In certain embodiments, B 2 Can be linked to the amino acid sequence in (Sp 3 ) Or B is a 1 The carboxyl groups contained in (a) are coupled. In certain embodiments, the linker molecule may include a self-cleaving moiety.
In certain embodiments, payload or attachment portion B 2 May include carboxyl groups, B 2 Is linked to (Sp) 1 ) Or B is a 1 . That is, B 2 Can be linked to (Sp) 1 ) Or B is a 1 An amine group contained in the (b). In some casesIn an embodiment, (Sp) 1 ) The amine groups contained therein may be chemical spacers (Sp 1 ) An amine group contained in the N-terminal amino acid residue of (C). In certain embodiments, B 1 The amine groups contained in (a) may be alpha-amino groups based on the payload or linking moiety of the amino acid. In certain embodiments, B 2 Can be linked to the amino acid sequence in (Sp 1 ) Or B is a 1 The amine groups contained in (a) are coupled. In certain embodiments, the linker molecule may include a self-cleaving moiety.
However, care must be taken that B 2 Other functional groups besides amine or carboxyl groups may also be included. In such an embodiment, B 2 The cleavage group may be linked to (Sp) directly or via a linker or self-cleavage group by any method known in the art 1 )、(Sp 3 ) Or B is a 1 And (3) coupling.
In certain embodiments, payload or attachment portion B 2 Can be combined with the components in the (Sp 1 ) Or (Sp) 3 ) The amino acid side chains comprised in (a) are coupled. That is, B 2 Can be linked to the amino acid sequence in (Sp 1 ) Or (Sp) 3 ) Functional groups of amino acid side chains comprised in the amino acid.
In certain embodiments, (Sp) 1 )、(Sp 2 )、(Sp 3 ) And the RK motif consists only of amino acids, amino acid mimics and/or amino acid derivatives. In certain embodiments, B 1 And/or B 2 Also comprises an amino acid backbone. In such embodiments, the linker may be a linear peptide or a peptidomimetic. At B 1 In embodiments that are amino acids, amino acid mimics, or amino acid derivatives, the linker may have the structure (Sp 1 )-RK-(Sp 2 )-B 1 Wherein, (Sp) 1 )-RK-(Sp 2 )-B 1 Is a linear peptide or peptidomimetic. At B 1 In embodiments that are amino acids, amino acid mimics, or amino acid derivatives, the linker may have the structure (Sp 1 )-RK-(Sp 2 )-B 1 -(Sp 3 ) Wherein, (Sp) 1 )-RK-(Sp 2 )-B 1 -(Sp 3 ) Is a linear peptide or peptidomimetic. At B 1 Is amino acid,In embodiments of the amino acid mimetic or amino acid derivative, the linker may have the structure RK- (Sp) 2 )-B 1 -(Sp 3 ) Wherein RK- (Sp) 2 )-B 1 -(Sp 3 ) Is a linear peptide or peptidomimetic. At B 1 In embodiments that are amino acids, amino acid mimics, or amino acid derivatives, the linker may have the structure RK- (Sp) 2 )-B 1 Wherein RK- (Sp) 2 )-B 1 Is a linear peptide or peptidomimetic. At B 1 In embodiments that are amino acids, amino acid mimics or amino acid derivatives, the linker may have the structure RK-B 1 -(Sp 3 ) Wherein RK-B 1 -(Sp 3 ) Is a linear peptide or peptidomimetic. At B 1 In embodiments that are amino acids, amino acid mimics or amino acid derivatives, the linker may have the structure RK-B 1 Wherein RK-B 1 Is a linear peptide or peptidomimetic.
At B 1 And B 2 In embodiments that are amino acids, amino acid mimics, or amino acid derivatives, the linker may have 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 ) Is a linear peptide or peptidomimetic. At B 1 And B 2 In other embodiments, which are amino acids, amino acid mimics or amino acid derivatives, the linker may have 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 ) Is a linear peptide or peptidomimetic. At B 1 And B 2 In other embodiments, which are amino acids, amino acid mimics or amino acid derivatives, the linker may have 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 ) Is a linear peptide or peptidomimetic.
At B 1 In embodiments other than amino acids, amino acid mimics or amino acid derivatives, the linker may have 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 Is connected to at (Sp 2 ) The C-terminal carboxyl group contained in (C) is provided. At B 1 In embodiments other than amino acids, amino acid mimics or amino acid derivatives, the linker may have 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 Is connected to at (Sp 2 ) Comprising an N-terminal amine group. However, care must be taken that B 1 It is not necessarily necessary to couple directly to the peptide or peptide mimetic. In contrast, B 1 The coupling to the peptide or peptidomimetic can be via a linker molecule and/or a self-cleaving moiety.
At B 1 Is an amino acid, an amino acid mimetic or an amino acid derivative and B 2 In embodiments other than amino acids, amino acid mimics or amino acid derivatives, the linker may have 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 Coupled to (Sp 3 )、B 1 Or the C-terminal carboxyl group contained in RK or coupled to (Sp 1 )、B 1 Or the N-terminal amino group of RK.
In such embodiments, an antibody-payload conjugate may be generated wherein, for example, the ratio of antibody to payload is 2 or 4, for example, one or two payloads are coupled to each Q295 residue.
In a particular embodiment, the invention relates to a method according to the invention, wherein B 1 And B 2 The same as or different from each other.
That is, payload or connection portion B 1 And B 2 May be identical (i.e., have the same chemical structure) or may be structurally different. In certain embodiments, B 1 And B 2 Are both payloads or are both connection parts. At B 1 And B 2 In embodiments where both are payloads, payload B 1 And B 2 May be the same or different payloads. At B 1 And B 2 In the embodiment in which both are the connecting portions, the connecting portion B 1 And B 2 May be the same or different connecting portions. In certain embodiments, B 1 May be a connecting part, B 2 May be a payload and vice versa.
It should be appreciated that not all payloads or linking portions may be present at position B 1 Acting as in-chain payloads or linking moieties, e.g. because they do not have a chain length (Sp 2 ) Or RK and the other side (Sp 3 )、(Sp 1 ) Or B is a 2 A covalent bond forming functional group. Thus, preferably at B 1 In embodiments that are in-chain payloads or linking portions, B 1 Is a divalent or multivalent molecule. For example, B 1 May be an amino acid, an amino acid mimetic or an amino acid derivative. In such an embodiment, B 1 Can be linked to (Sp) via its amino group 2 ) Or the C-terminal carboxyl group of RK, and is coupled via its carboxyl group to (Sp 3 ) Or B is a 2 Is coupled to the N-terminal amino group of (C). Alternatively, B 1 Can be bound to (Sp) via its carboxyl group 2 ) Or the N-terminal amino group of RK, and is coupled to (Sp) via its amino group 1 ) Or B is a 2 C-terminal carboxyl coupling of (C).
In some embodiments, the joint may include two connecting portions B 1 And B 2 。
That is, in certain embodiments, the present invention encompasses compositions comprising two organisms A linker of an orthogonal labeling group and/or a non-bioorthogonal entity. For example, a linker according to the invention may comprise an azide-containing linking moiety (such as Lys (N) 3 ) Or Xaa (N) 3 ) And thiol-containing linking moieties (such as cysteine). In certain embodiments, a linker according to the invention may comprise an azide-containing linking moiety (such as Lys (N) 3 ) Or Xaa (N) 3 ) And a tetrazine-containing linking moiety (such as a tetrazine modified amino acid). In certain embodiments, a linker according to the invention may comprise a thiol-containing linking moiety (such as a cysteine) and a tetrazine-containing linking moiety (such as a tetrazine modified amino acid). Linkers comprising two different bio-orthogonal label groups and/or non-bio-orthogonal entities have the advantage that they can accept two different payloads, thereby producing an antibody-payload conjugate comprising more than one payload.
In this way, an antibody payload ratio of 2+2 can be obtained. The use of a second payload may allow the development of a completely new class of antibody-payload conjugates that surpass current methods of treatment in terms of efficacy and potency.
Such embodiments may specifically allow targeting two different structures (e.g., DNA and microtubules) in a cell. Since some cancers are resistant to a drug, such as microtubule (microshutter) toxins, DNA toxins can still kill cancer cells.
According to another embodiment, two drugs may be used, which are only fully effective when released at the same time and in the same tissue. In the event of partial degradation of the antibody in healthy tissue or premature loss of one of the drugs, this may lead to reduced off-target toxicity.
Moreover, the dual-labeled probes can be used for non-invasive imaging and therapy or intra-operative/post-operative imaging/surgery. In such embodiments, the tumor patient may be selected by non-invasive imaging. The tumor may then be surgically removed using other imaging agents (e.g., fluorochromes) that assist the surgeon or robot in identifying all cancerous tissue during the surgery.
In certain embodiments, B 1 And B 2 One of (a) may be a thiol-containing linking moiety (such as cysteine), and B 1 And B 2 May be another of an azide moiety-containing linking moiety (such as Lys (N) 3 )). In such embodiments, two different payloads may be coupled to the linker, one via a sulfhydryl maleimide coupling and the other via a sparc reaction.
In some embodiments, the joint may contain two payloads. A linker comprising only a payload but no linking moiety may be coupled to the antibody in a one-step process.
It will be appreciated that at B 1 And B 2 In embodiments where both are payloads, B 1 And B 2 May be identical or different in structure. In certain embodiments, a linker comprising one or more payloads may be chemically synthesized. Alternatively, one or more payloads may be coupled to the linking moiety contained in the linker by any of the methods disclosed herein prior to coupling the linker to the antibody.
In certain embodiments, the linker of the invention may allow coupling of two different payloads to the C of an antibody H Residue Q295 of the 2 domain. The use of a second payload allows the development of a completely new class of antibody-payload conjugates that surpass current methods of treatment in terms of efficacy and efficacy. New fields of application are also envisaged, for example Dual Imaging for Imaging and therapy or intra-and post-operative surgery (see Azhdarinia A et al, dual labeling strategy for nuclear and fluorescent molecular Imaging: review and analysis (Dual-Label Strategies for Nuclear and Fluorescence Molecular Imaging: A Review and Analysis), mol Imaging biol.2012Jun;14 (3): 261-276). For example, dual-labeled antibodies comprising a molecular imaging agent for preoperative Positron Emission Tomography (PET) and a near infrared fluorescence (NIRF) dye for directing demarcation of the operative margin can greatly enhance diagnosis, staging and excision of cancer (see Houghton JL. et al, PET, NIRF and multimodal PET/NIRF imaging for pancreatic cancer) Site-directed labeling CA19.9 targeting immunoconjugate (Site-specifically labeled CA 19.9.19.9-targeted immunoconjugates for the PET, NIRF, and multimodal PET/NIRF imaging of pancreatic cancer), proc Natl Acad Sci U S A.2015Dec29; 112 (52):15850-5). PET and NIRF optical imaging provide complementary clinical applications enabling non-invasive whole body imaging to locate disease and identify tumor margins during surgery, respectively. However, the generation of such dual-labeled probes is currently difficult due to the lack of suitable site-specific methods; the chemical ligation of two different probes results in almost impossible analysis and reproducibility due to random coupling of the probes.
Furthermore, in the studies of Levengood M et al (orthorhombic cysteine protection made possible homogeneous multi-Drug Antibody-Drug Conjugates) (Orthogonal Cysteine Protection Enables Homogeneous Multi-Drug antibodies-Drug Conjugates), angewandte Chemie, volume 56, stage 3, month 1, 16 of 2017), the dual Drug labeled antibodies had the attachment of two different auristatin toxins (with different physiochemical properties and complementary anticancer activity) conferring activity in cell lines and xenograft models that were refractory to ADCs comprising the auristatin component alone. This suggests that dual-labeled ADCs are more effective in addressing cancer heterogeneity and drug resistance than single conventional ADCs alone. Since one mechanism of drug resistance to ADCs involves active pumping of the cytotoxic moiety from cancer cells, another dual drug application may involve additional and simultaneous delivery of drugs that specifically block the efflux mechanism of the cytotoxic drug. Thus, such dual-labeled ADCs may be more effective in helping to overcome cancer resistance to ADCs than traditional ADCs.
The term "antibody" as used herein is the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. The terms "an antibody (anti)" and "antibodies (anti) broadly include naturally occurring forms of antibodies (e.g., igG, igA, igM, igE).
The antibody is preferably a monoclonal antibody. Antibodies may be of human origin but are also of mouse, rat, goat, donkey, hamster or rabbit origin. In the case of conjugates for use in therapy, the murine or rabbit antibodies may optionally be chimeric or humanized.
Comprising C H Fragments or recombinant variants of antibodies to the 2 domain may be, for example,
antibody forms comprising only heavy chain domains (shark antibody/IgNAR (VH-C) H 1-C H 2-C H 3-C H 4-C H 5) 2 Or camelid antibody/hcIgG (VH-C) H 2-C H 3) 2 )
Single chain antibody (scFV) -Fc (VH-VL-C) H 2-C H 3) 2
An Fc fusion peptide comprising an Fc domain and one or more receptor domains.
Antibodies may also be bispecific (e.g., DVD-IgG, cross mab, additional IgG-HC fusion) or bispecific. See Brinkmann and Kontermann; bispecific antibodies (Bispecific antibodies); drug Discov Today;2015;20 (7); pages 838-47, for review.
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.
"IgG" as used herein refers to a polypeptide belonging to the class of antibodies substantially encoded by the putative immunoglobulin gamma gene. In humans, igG includes subclasses or isotypes IgG1, igG2, igG3 and IgG4. In mice, igG includes IgG1, igG2a, igG2b, igG3. Full length IgG consists of two identical pairs of two immunoglobulin chains, each pair having one light chain and one heavy chain, each light chain comprising immunoglobulin domains VL and CL, and each heavy chain comprising immunoglobulin domains VH, cγ1 (also known as CH 1), cγ2 (also known as CH 2), and oγ3 (also known as CH 3). In the context of human IgG1, the "CH1" refers to positions 118-215, the CH2 domain refers to positions 231-340, and the CH3 domain refers to positions 341-447 according to the EU index in Kabat. IgG1 also includes a hinge domain, which in the case of IgG1 refers to positions 216-230.
The antibody used in the method of the invention or the antibody-payload conjugate of the invention may be or comprise any antibody, preferably any antibody of the IgG type. For example, the antibody may be, but is not limited to, vitamin b (Brentuximab), trastuzumab (Trastuzumab), gemtuzumab (Gemtuzumab), oxuzumab (Inotuzumab), avizumab (Avelumab), cetuximab (Cetuximab), rituximab (Rituximab), up Lei Tuoyou mab (Daratumumab), pertuzumab (Pertuzumab), vedolizumab (Vedolizumab), oxuzumab (oxelizumab), tolizumab (Tocilizumab), wu Sinu mab (useumab), golimumab (Golimumab), oxuzumab (obituzumab), sha Xituo mab (Sacituzumab), bei Lantuo mab (betuzumab), poisuzumab (pouzumab) and entuzumab (entuzumab).
Thus, in a specific embodiment, the invention relates to a method according to the invention, wherein the antibody is selected from the group consisting of: vibutuzumab, trastuzumab, gemtuzumab, oxtuzumab, avermectin, cetuximab, rituximab, up Lei Tuoyou mab, pertuzumab, vedolizumab, oreuzumab, tolizumab, wu Sinu mab, golimumab, otouzumab, sha Xituo mab, bei Lantuo mab, polotouzumab and enrolment mab.
In a specific embodiment, the invention relates to a method according to the invention, wherein the antibody is selected from the group consisting of: velutinab, gemtuzumab, trastuzumab, oxtuzumab, poltuzumab, enrolment mab, sha Xituo bead mab, bei Lantuo mab.
In a more preferred embodiment, the invention relates to a method according to the invention, wherein the antibody is either polotophyllizumab or trastuzumab or enrolment mab.
That is, in a particular embodiment, the invention relates to an antibody-linker conjugate, wherein the antibody is a poloxamer, and wherein the linker is any of the linkers disclosed herein.
In another embodiment, the invention relates to an antibody-linker conjugate, wherein the antibody is trastuzumab, and wherein the linker is any one of the linkers disclosed herein.
In another embodiment, the invention relates to an antibody-linker conjugate, wherein the antibody is enrolment mab, and wherein the linker is any one of the linkers disclosed herein.
Antibodies for use in the methods according to the invention may be glycosylated, deglycosylated or non-glycosylated (aglycosylated) antibodies.
That is, in certain embodiments, the antibody may be an IgG antibody that is preferably glycosylated at residue N297. Thus, in a particular embodiment, the invention relates to a method according to the invention, wherein the IgG antibody is glycosylated, in particular wherein the IgG antibody is at C H Glycosylation at residue N297 (EU numbering) of the 2 domain.
As described herein, an IgG antibody glycosylated at residue N297 has several advantages over an unglycosylated antibody.
However, the antibody may also be a deglycosylated antibody, preferably wherein the glycan at residue N297 has been cleaved off by the enzyme PNGase F. Further, the antibody may be a non-glycosylated antibody, preferably wherein residue N297 has been replaced with a non-asparagine residue. Methods for deglycosylating antibodies and generating non-glycosylated antibodies are known in the art.
In certain embodiments, the linker of the invention may be coupled to endogenous Gln residues in the Fc domain of an antibody, or to Gln residues that have been introduced into an antibody by molecular engineering.
Thus, in a specific embodiment, the invention relates to a method according to the invention, wherein the linker-coupled Gln residue is comprised in the Fc domain of an antibody, in particular wherein the linker-coupled Gln residue is the C of an IgG antibody H Gln residue Q295 of the 2 domain (EU numbering).
The linker of the invention can be coupled to any Gln residue in the Fc domain of an antibody that can be used as a substrate for microbial transglutaminase. Generally, the term Fc structure as used hereinThe domains refer to the last two constant region immunoglobulin domains of IgA, igD and IgG (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 may be attached to the C of an antibody H 2、C H 3 and C H 4 (where applicable) domain coupling.
In certain embodiments, the endogenous gin residue may be gin residue Q295 (EU numbering) of the CH2 domain of an IgG antibody. Thus, in a specific embodiment, the invention relates to a method according to the invention, wherein the Gln residue in the Fc domain of an antibody is Gln residue Q295 (EU numbering) of the CH2 domain of an IgG antibody.
It is important to understand that Q295 is an extremely conserved amino acid residue in IgG-type antibodies. It is conserved among human IgG1, 2, 3, 4, and rabbit and rat antibodies. Thus, the ability to use Q295 is a considerable advantage for preparing therapeutic antibody-payload conjugates or diagnostic conjugates, where the antibody is typically of non-human origin. The method according to the invention thus does provide 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 have this residue. Thus, it will be appreciated that the antibodies used in the methods of the invention preferably comprise C H An IgG type antibody of residue Q295 of the 2 domain (EU numbering).
Further, it has been shown that engineering conjugates with Q295 for payload attachment exhibit good pharmacokinetics and efficacy (Lhosice et al, site-specific conjugation of monomethyl auristatin E to anti-Cd30 antibodies improves their pharmacokinetic and therapeutic index in rodent models (Site-Specific Conjugation of Monomethyl Auristatin E to Anti-Cd30 Antibodies Improves Their Pharmacokinetics and Therapeutic Index in Rodent Models), mol Pharm;2015;12 (6), p.1863-1871), and are capable of carrying even labile readily degradable toxins (Dorywalska et al; site-dependent degradation of unclassifiable auristatin-Based Linker-payloads in rodent plasma and their effect on ADC efficacy (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): E0132822). It is expected that a similar effect will be seen with this site-specific approach, as the same residues are modified, but the antibody is glycosylated. Glycosylation can further contribute to the overall stability of the ADC, and removal of glycan moieties as described above has been shown to result in reduced antibody stability (Zheng et al; influence of glycosylation on monoclonal antibody conformation and stability (The impact of glycosylation on monoclonal antibody conformation and stability), mabs Austin;2011,3 (6), pages 568-576).
Coupling of the linker to C by transglutaminase is discussed H In the literature for 2-Gln residues, the emphasis is on small, low molecular weight substrates. However, in the prior art documents, the use of deglycosylated or non-glycosylated antibodies at position N297 is always described as necessary in order to achieve such coupling (WO 2015/015448; WO 2017/025179; WO 2013/092998).
However, quite surprisingly, contrary to all expectations, site-specific coupling to Q295 of glycosylated antibodies was indeed effective by using the linker structure described above. In particular, coupling of the linker comprising the toxin molecule is achieved with a coupling efficiency of greater than 80%.
Even though Q295 is very close to N297, N297 is glycosylated in its native state, the use of a designated linker according to the methods of the present invention allows the linker or payload to be coupled thereto.
As shown, the method according to the invention does not require prior enzymatic deglycosylation of N297 nor the use of non-glycosylated antibodies nor the substitution of N297 for another amino acid nor the introduction of a T299A mutation to prevent glycosylation.
These two points provide significant advantages in terms of manufacturing. In terms of GMP, an enzymatic deglycosylation step is undesirable, as it must ensure that both the deglycosylating enzyme (e.g. PNGase F) and the cleaved glycans must be removed from the culture medium.
Furthermore, genetic engineering of the antibody for payload attachment is not required, so that sequence insertions that may increase immunogenicity and reduce the overall stability of the antibody can be avoided.
N297 substitution against another amino acid also has unwanted effects, as it may affect overall stability of the entire Fc domain (Subedi et al, structural role of antibody N-glycosylation in receptor interactions (The Structural Role of Antibody N-Glycosylation in Receptor Interactions), structure2015, 23 (9), 1573-1583), and efficacy of the entire conjugate, which may result in increased antibody aggregation and decreased solubility (Zheng et al, influence of glycosylation on monoclonal antibody conformation and stability (The impact of glycosylation on monoclonal antibody conformation and stability), mabs Austin 2011,3 (6), 568-576), which is particularly important for hydrophobic payloads such as PBD. Further, the glycan present in N297 has an important immunomodulatory effect because it triggers antibody-dependent cellular cytotoxicity (ADCC) and the like. These immunomodulatory effects will be lost in deglycosylation or any of the other methods described above to obtain non-glycosylated antibodies. Further, any sequence modification of the established antibodies may also lead to regulatory problems, which is problematic, since acceptable and clinically validated antibodies are typically used as the origin of ADC coupling.
Thus, the method according to the invention allows for easy and defect-free manufacturing of stoichiometric well-defined ADCs with site-specific payload binding.
In view of the above, it is pointed out that the method of the invention is preferably used for IgG antibodies at antibody C H Conjugation at residue Q295 of the 2 domain (EU numbering), wherein the antibody is at C H The 2 domain is glycosylated at residue N297 (EU numbering). However, it is explicitly noted that the methods of the invention also encompass the coupling of deglycosylated or non-glycosylated antibodies at residue Q295 or any other suitable Gln residue of the antibody, wherein the Gln residue may be an endogenous Gln residue or a Gln residue introduced by molecular engineering.
Thus, in a specific embodiment, the invention relates to a method according to the invention, wherein the Gln residues coupled to the linker have been introduced into the heavy or light chain of the antibody by molecular engineering.
The term "molecular engineering" as used herein refers to the manipulation of nucleic acid sequences using molecular biological methods. In the present invention, molecular engineering can be used to introduce Gln residues into the heavy or light chain of an antibody. Generally, two different strategies for introducing Gln residues into the heavy or light chain of an antibody are contemplated in the present invention. First, a single residue of the heavy or light chain of an antibody may be substituted with a Gln residue. Second, a Gln-containing peptide tag consisting of two or more amino acid residues may be integrated into the heavy or light chain of an antibody. To this end, the peptide tag may be integrated into the internal position of 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 (appended) to the N-terminal or C-terminal end of the heavy or light chain of the antibody.
For example, the amino residues of the heavy or light chain of an antibody may be substituted with Gln residues, provided that the resulting antibody may be coupled to a linker of the invention by microbial transglutaminase. In certain embodiments, the antibody is C wherein the IgG antibody H An antibody in which amino acid residue N297 (EU numbering) of the 2 domain is substituted, particularly wherein the substitution is an N297Q substitution. Antibodies comprising the N297Q mutation may be coupled to more than one linker on each heavy chain of the antibody. For example, an antibody comprising an N297Q mutation may be coupled to four linkers, wherein one linker is coupled to residue Q295 of the first heavy chain of the antibody, one linker is coupled to residue Q297Q of the first heavy chain of the antibody, one linker is coupled to residue Q295 of the second heavy chain of the antibody, and one linker is coupled to residue N297Q of the second heavy chain of the antibody. Those skilled in the art know that substitution of residue N297 of an IgG antibody with a Gln residue results in a non-glycosylated antibody.
Thus, in a particular embodiment, the invention relates to a method according to the invention, wherein the Gln residues introduced into the heavy or light chain of the antibody by molecular engineering are the C of a non-glycosylated IgG antibody H 2 domain N297Q (EU numbering).
In a particular embodiment, the invention relates to a method according to the invention, wherein the Gln residues introduced into the heavy or light chain of the antibody by molecular engineering are comprised in a peptide which has been (a) integrated into the heavy or light chain of the antibody or (b) fused to the N-or C-terminus of the heavy or light chain of the antibody.
Instead of replacing a single amino acid residue of an antibody, a peptide tag comprising a transglutaminase accessible Gln residue may be introduced into the heavy or light chain of the antibody. Such peptide tags may be fused to the N-terminus or C-terminus of the heavy or light chain of an 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 comprising a transglutaminase accessible Gln residue is fused to the C-terminus of the heavy chain of the antibody. Even more preferably, a peptide tag comprising a transglutaminase accessible Gln residue is fused to the C-terminus of the heavy chain of an IgG antibody. Several peptide tags that can be fused to the C-terminus of the heavy chain of an antibody and used as substrates for microbial transglutaminases are described in WO 2012/059882 and WO 2016/144608.
Thus, in a specific embodiment, the invention relates to a method according to the invention, wherein a peptide comprising a Gln residue has been fused to the C-terminus of the heavy chain of an antibody.
Exemplary peptide tags that may be incorporated into the heavy or light chain of an antibody, particularly fused to the C-terminus of the heavy chain of an antibody, are LLQGG (SEQ ID NO: 16), LLQG (SEQ ID NO: 17), LSLSQG (SEQ ID NO: 18), GGGLLQGG (SEQ ID NO: 19), GLLQG (SEQ ID NO: 20), LLQ (SEQ ID NO: 21), GSPLAQSHGG (SEQ ID NO: 22), GLLQGGG (SEQ ID NO: 23), GLLQGG (SEQ ID NO: 24), GLLQ (SEQ ID NO: 25), LLQGA (SEQ ID NO: 26), LLQGA (SEQ ID NO: 27), LLQYQGA (SEQ ID NO: 28), LLQGSG (SEQ ID NO: 29), LLQQQG (SEQ ID NO: 30), LLQQG (SEQ ID NO: 31), SLLQG (SEQ ID NO: 32), LLQQ (SEQ ID NO: 22), GLLQGGG (SEQ ID NO: 35), LLQGYQ (SEQ ID NO: 37), EEQYYQ (SEQ ID NO: 37), LLQYTY (SEQ ID NO: 35), LLQYYQ (SEQ ID NO: 37), LLQYTY (QYY (SEQ ID NO: 40), LLQYYY (QYID NO: 35), LLQYQ (SEQ ID NO: 37), LLQQQQYQ (SEQ ID NO: 35) LQR (SEQ ID NO: 48) and YQR (SEQ ID NO: 49).
Those skilled in the art are aware of molecular cloning, for example, by Sambrook, joseph (2001): the method of molecular cloning described in the laboratory Manual (Molecular cloning: a laboratory manual) Cold Spring Harbor, N.Y.: cold Spring Harbor Laboratory Press replaces amino acid residues of antibodies or introduces peptide tags into antibodies.
In general, the skilled person knows the method of determining at which position of the antibody the linker is coupled. For example, the conjugation site may be determined by proteolytic digestion of the antibody-payload conjugate and LC-MS analysis of the resulting fragment. For example, the sample may be deglycosylated with GlyciNATOR (Genovis) according to the instructions and then digested with trypsin gold (mass spectrometry grade, promega), respectively. Thus, 1 μg of protein can be incubated with 50ng trypsin overnight at 37 ℃. LC-MS analysis can be performed using a nanoAcquisy HPLC system coupled to a Synta-G2 mass spectrometer (Waters). To this end, 100ng of peptide solution can be loaded onto Acquity UPLC Symmetry C capture column (Waters, part No. 186006527) and captured for 3min with a flow rate of 5 μl/min in 1% buffer a (water, 0.1% formic acid) and 99% buffer B (acetonitrile, 0.1% formic acid). The peptide may then be eluted in a linear gradient from 3% to 65% buffer B over 25 min. The data may be acquired in a resolution mode with positive polarity and in a mass range from 50 to 2000 m/z. Other instrument settings may be as follows: capillary voltage 3.2kV, sampling cone 40V, extraction cone 4.0V, source temperature 130 ℃, cone gas 35L/h, nano-flow gas 0.1 bar, and purge gas 150L/h. The mass spectrometer can be calibrated with [ Glu1] -fibrinopeptides.
Further, the skilled artisan is aware of methods of determining the drug to antibody (DAR) ratio or payload to antibody ratio of an antibody-payload construct. For example, DAR can be determined by Hydrophobic Interaction Chromatography (HIC) or LC-MS.
For Hydrophobic Interaction Chromatography (HIC), the sample can be adjusted to 0.5M ammonium sulfate and evaluated over a MAB PAK HIC butyl column (5. Mu.M, 4.6x 100mm,Thermo Scientific) at 1mL/min and 30℃in 20 minutes using a full gradient from A (1.5M ammonium sulfate, 25mm Tris-HCl, pH 7.5) to B (20% isopropanol, 25mm Tris-HCl, pH 7.5). 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 ADC DAR2 species by the retention time of the corresponding unconjugated mAb.
For LC-MS DAR measurement, ADC can use NH 4 HCO 3 Diluted to a final concentration of 0.025 mg/mL. Subsequently, 40. Mu.L of the solution can be reduced with 1. Mu.L of TCEP (500 mM) at room temperature for 5min, then alkylated by adding 10. Mu.L of chloroacetamide (200 mM), then incubated overnight in the dark at 37 ℃. For reverse phase chromatography, the Dionex U3000 system can be used in combination with Chromeleon software. The system can be equipped with RP-1000 column heated to 70 DEG C 5 μm, 1.0X100 mm, sepax) and an ultraviolet detector set at a wavelength of 214 nm. Solvent a may consist of water containing 0.1% formic acid, while solvent B may comprise 85% acetonitrile containing 0.1% formic acid. The reduced and alkylated samples may be loaded onto a column and separated by a gradient from 30-55% solvent B in 14 minutes. The liquid chromatography system may be connected to a Synapt-G2 mass spectrometer for identifying DAR species. The capillary voltage of the mass spectrometer can be set to 3kV, the sampling cone is set to 30V, and the extraction cone can reach a value of 5V. The source temperature may be set to 150 ℃, desolvation temperature to 500 ℃, cone gas to 20l/h, desolvation gas to 600l/h, and acquisition may be performed in positive mode in the mass range of 600-5000Da, scanning time 1s. The instrument can be calibrated with sodium iodide. Deconvolution of the spectra can be performed using MaxEnt1 algorithm of MassLynx until convergence. After the assignment of the DAR species to chromatographic peaks, the DAR can be calculated based on the integrated peak area of the reversed phase chromatograph.
In a particular embodiment, the invention relates to a method according to the invention, wherein the linker is coupled to the gamma-carboxamide group of the Gln residue comprised in the antibody.
That is, the linker according to the present invention is preferably coupled to an amide group in the side chain of a Gln residue comprised in an antibody, preferably any one of the Gln residues disclosed herein, more preferably Gln residue Q295 (EU numbering).
In a particular embodiment, the invention relates to a method according to the invention, wherein the linker is suitable for coupling to the glycosylated antibody with a coupling efficiency of at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95%.
That is, in certain embodiments, the linker may be a linker capable of coupling to a glycosylated antibody with an efficiency of at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%. In a preferred embodiment, the linker may be one that is capable of coupling to the glycosylated antibody with an efficiency of at least 70%. In another preferred embodiment, the linker may be one that is capable of coupling to the glycosylated antibody with an efficiency of at least 75%. In another preferred embodiment, the linker may be one that is capable of coupling to the glycosylated antibody with an efficiency of at least 80%. In another preferred embodiment, the linker may be one that is capable of coupling to the glycosylated antibody with an efficiency of at least 85%. In another preferred embodiment, the linker may be one that is capable of coupling to the glycosylated antibody with an efficiency of at least 90%. In another preferred embodiment, the linker may be one that is capable of coupling to the glycosylated antibody with an efficiency of at least 95%. Preferably, the glycosylated antibody is a glycosylated IgG antibody, more preferably an IgG antibody glycosylated at residue N297 (EU numbering).
The skilled person is aware of methods for determining the coupling efficiency of antibodies to specific linkers. For example, coupling efficiency may be determined as described herein. That is, the antibody, particularly an IgG1 antibody, may be incubated with 5-20eq molar equivalents of linker and 3-6U of microbial transglutaminase per mg of antibody in a suitable buffer at 37℃or as described in example 1 for 20-48 hours at a concentration of 1-5 mg/mL. After the incubation period, the coupling efficiency can be determined by LC-MS analysis under reducing conditions. The microbial transglutaminase can be MTG from Streptomyces mobaraensis available from Zedira (Germany). Suitable buffers may be Tris, MOPS, HEPES, PBS or BisTris buffers. However, it should be understood that the choice of buffer system may vary and will depend to a large extent on the chemistry of the linker. However, one skilled in the art can identify optimal buffering conditions based on the disclosure of the present invention. Alternatively, the coupling efficiency can be determined as described in Spycher et al (Dual Site specific modification of antibodies by use of immobilized microbial transglutaminase in solid phase (Dual, site-Specific Modification of Antibodies by Using Solid-Phase Immobilized Microbial Transglutaminase), chemBioChem 2019 18 (19): 1923-1927), and analyzed as described in Benjamin et al (sulfuration of Q295: site specific coupling of hydrophobic payloads without genetic engineering (thio of Q295: site-Specific Conjugation of Hydrophobic Payloads without the Need for Genetic Engineering), mol. Pharmaceuticals 2019, 16:2795-2807).
In certain embodiments, the antibodies may be conjugated as described in example 1. That is, 5mg/ml of the naturally glycosylated monoclonal antibody may be incubated in 50mM Tris pH 7.6 at 37℃for 24 hours in a rotating thermomixer, the 50mM Tris pH 7.6 comprising microbial transglutaminase (MTG, zedira) at a concentration of 5U/mg antibody and 5 molar equivalents of the indicated linker-payload.
In a particular embodiment, the invention relates to a method according to the invention, wherein the microbial transglutaminase is derived from Streptomyces, in particular Streptomyces mobaraensis.
That is, the microbial transglutaminase used in the method of the present invention may be derived from Streptomyces (especially from Streptomyces mobaraensis), preferably having 80% sequence identity with the native enzyme. Thus, MTG may be a native enzyme, or may be an engineered variant of a native enzyme class.
Such microbial transglutaminases are commercially available from Zedira (germany). It is recombinantly produced in E.coli. Streptomyces mobaraensis transglutaminase has the amino acid sequence disclosed in SEQ ID NO. 12. Variants of Streptomyces mobaraensis MTG having other amino acid sequences have been reported and are also encompassed by the present invention (SEQ ID NOS: 13 and 14).
In another embodiment, a microbial transglutaminase from Streptomyces dactylus (Streptomyces ladakanum) (previously known as Streptomyces dactylus (Streptoverticillium ladakanum)) can be used. Streptomyces dacarbazine transglutaminase (U.S. Pat. No. 6,60510 B2) has the amino acid sequence disclosed in SEQ ID NO. 15.
Both transglutaminases can be sequence modified. In several embodiments, transglutaminases having 80%, 85%, 90% or 95% or more sequence identity to any of SEQ ID NOS.12-15 may be used.
Another suitable microbial transglutaminase is commercially available from Ajinomoto under the name ACTIVA TG. Compared to Zedira's transglutaminase, ACTIVA TG lacks 4N-terminal amino acids, but has similar activity.
Other microbial transglutaminases useful in the present invention are disclosed in Kieliszek and Misiewicz (Folia Microbiol (Praha). 2014;59 (3): 241-250), WO 2015/191883A1, WO 2008/102007 A1 and US 2010/0143970), the contents of which are incorporated herein by reference in their entirety.
In certain embodiments, mutant variants of microbial transglutaminase can be used for coupling of the linker to the antibody. That is, the microbial transglutaminase used in the method of the present invention may be a variant of Streptomyces mobaraensis transglutaminase as set forth in SEQ ID NO:12 or 13. In certain embodiments, the recombinant Streptomyces mobaraensis transglutaminase as set forth in SEQ ID NO. 12 may comprise the mutation G254D. In certain embodiments, the recombinant Streptomyces mobaraensis transglutaminase as set forth in SEQ ID NO. 12 may comprise mutations G254D and E304D. In certain embodiments, the recombinant Streptomyces mobaraensis transglutaminase as set forth in SEQ ID NO. 12 may comprise the mutations D8E and G254D. In certain embodiments, the recombinant Streptomyces mobaraensis transglutaminase as set forth in SEQ ID NO. 12 may comprise mutations E124A and G254D. In certain embodiments, the recombinant Streptomyces mobaraensis transglutaminase as set forth in SEQ ID NO. 12 may comprise mutations A216D and G254D. In certain embodiments, the recombinant Streptomyces mobaraensis transglutaminase as set forth in SEQ ID NO. 12 may comprise the mutations G254D and K331T.
The microbial transglutaminase can be added to the coupling reaction at any concentration that allows the antibody to bind efficiently to the linker. In certain embodiments, the concentration of microbial transglutaminase in the coupling reaction may depend on the amount of antibody used in the same reaction. For example, microbial transglutaminase can be added to the coupling reaction at a concentration of less than 100U/mg antibody, 90U/mg antibody, 80U/mg antibody, 70U/mg antibody, 60U/mg antibody, 50U/mg antibody, 40U/mg antibody, 30U/mg antibody, 20U/mg antibody, 10U/mg antibody, or 6U/mg antibody. In certain embodiments, microbial transglutaminase can be added to the coupling reaction at a concentration of 1, 3, 5 or 6U/mg antibody.
That is, in certain embodiments, the microbial transglutaminase may be added to the coupling reaction at a concentration of 1-20U/mg of antibody, preferably 1-10U/mg of antibody, more preferably 1-7.5U/mg of antibody, even more preferably 2-6U/mg of antibody, even more preferably 2-4U/mg of antibody, or most preferably 3U/mg of antibody.
The method according to the invention comprises the use of a microbial transglutaminase. However, it should be noted that the equivalent reaction may be carried out by enzymes of non-microbial origin comprising transglutaminase activity. Thus, the antibody-linker conjugates of the invention may also be produced with enzymes of non-microbial origin comprising transglutaminase activity.
Antibodies may be added to the coupling reaction at any concentration. However, it is preferable to add the antibody to the coupling reaction at a concentration of 0.1-20 mg/ml. That is, in a particular embodiment, the invention relates to a method according to the invention, wherein the antibody is added to the coupling reaction at a concentration of 0.1-20mg/mL, preferably 0.25-15mg/mL, more preferably 0.5-12.5mg/mL, even more preferably 1-10mg/mL, even more preferably 2-7.5mg/mL, most preferably about 5 mg/mL.
Alternatively, the antibody may be added to the coupling reaction at a concentration of 1-20mg/ml, preferably 2.5-20mg/ml, more preferably 5-20mg/ml, most preferably 5-17 mg/ml.
In order to obtain efficient coupling, the linker is preferably added to the antibody in molar excess. That is, in certain embodiments, the antibody is mixed with at least 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 molar equivalents of the linker.
That is, in a specific embodiment, the invention relates to a method according to the invention, wherein the antibody is contacted with 2-100 molar equivalents of linker, preferably 2-80 molar equivalents of linker, more preferably 2-70 molar equivalents of linker, even more preferably 2-60 molar equivalents of linker, even more preferably 2-50 molar equivalents of linker, even more preferably 2-40 molar equivalents of linker, even more preferably 2-30 molar equivalents of linker, even more preferably 2-25 molar equivalents of linker, even more preferably 2-20 molar equivalents of linker, even more preferably 2-15 molar equivalents of linker, most preferably 2-10 molar equivalents of linker.
Alternatively, the antibody may be contacted with 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 linker, even more preferably 2.5 to 40 molar equivalents of linker, even more preferably 2.5 to 30 molar equivalents of linker, even more preferably 2.5 to 20 molar equivalents of 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.
Alternatively, the antibody may be contacted with 5-100 molar equivalents of linker, preferably 5-80 molar equivalents of linker, more preferably 5-70 molar equivalents of linker, even more preferably 5-60 molar equivalents of linker, even more preferably 5-50 molar equivalents of linker, even more preferably 5-40 molar equivalents of linker, even more preferably 5-30 molar equivalents of linker, even more preferably 5-20 molar equivalents of linker, even more preferably 5-15 molar equivalents of linker, most preferably 5-10 molar equivalents of linker.
The process according to the invention is preferably carried out in a pH range of 6 to 9. Thus, in a preferred embodiment, the invention relates to a method according to the invention, wherein the coupling of the linker to the antibody is effected at a pH in the range of 6 to 8.5, more preferably at a pH in the range of 6.5 to 8, even more preferably at a pH in the range of 7 to 8. In a most preferred embodiment, the invention relates to a method according to the invention, wherein the coupling of the linker to the antibody is effected at pH 7.6.
The method of the invention may be carried out in any buffer suitable for coupling a payload to a linker. Buffers suitable for use in 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 the linker and may vary in the range 10-1000mM, 10-500mM, 10-400mM, 10-250mM, 10-150mM or 10-100 mM. Further, the buffer may comprise any salt concentration suitable for carrying out the method of the invention. For example, the buffers used in the methods of the invention may have a salt concentration of.ltoreq.150 mM,.ltoreq.140 mM,.ltoreq.130 mM,.ltoreq.120 mM,.ltoreq.110 mM,.ltoreq.100 mM,.ltoreq.90 mM,.ltoreq.80 mM,.ltoreq.70 mM,.ltoreq.60 mM,.ltoreq.50 mM,.ltoreq.40 mM,.ltoreq.30 mM,.ltoreq.20 mM, or.ltoreq.10 mM, or no salt. In a particular embodiment, the method of the invention is performed in 50mM Tris (pH 7.6), preferably salt-free.
It is noted that the optimal reaction conditions (e.g., pH, buffer, salt concentration) may vary from payload to payload and depend to some extent on the physicochemical properties of the linker and/or the payload. However, one skilled in the art does not require undue experimentation to determine the reaction conditions suitable for practicing the methods of the present invention.
It is to be understood that the application includes any combination of the above disclosed linker, antibody MTG and/or buffer concentrations.
In a preferred embodiment, the application relates to a method for producing an antibody-linker conjugate by Microbial Transglutaminase (MTG), comprising administering a polypeptide comprising (as indicated in the N.fwdarw.C direction) (Sp 1 )-RK-(Sp 2 )-B-(Sp 3 ) Or (Sp) 1 )-B-(Sp 2 )-RK-(Sp 3 ) A step of coupling a linker of the structure 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 arginine mimetic;
k is lysine or a lysine derivative or a lysine mimetic;
b is a linking part or payload;
wherein the linker is coupled to a Gln residue comprised in the antibody via a primary amine comprised in a side chain of a lysine residue, a lysine derivative or a lysine mimetic; and is also provided with
Wherein the antibody is contacted with 2-80 molar equivalents of the linker; and/or
Wherein the microbial transglutaminase is added to the coupling reaction at a concentration of 1-20U/mg of antibody, and optionally wherein the antibody is added to the coupling reaction at a concentration of 0.1-20 mg/mL.
In a more preferred embodiment, the application relates to a method for producing an antibody-linker conjugate by Microbial Transglutaminase (MTG), comprising administering a polypeptide comprising (as indicated in the N.fwdarw.C direction) (Sp 1 )-RK-(Sp 2 )-B-(Sp 3 ) Or (Sp) 1 )-B-(Sp 2 )-RK-(Sp 3 ) A step of coupling a linker of the structure 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 arginine mimetic;
k is lysine or a lysine derivative or a lysine mimetic;
b is a linking part or payload;
wherein the linker is coupled to a Gln residue comprised in the antibody via a primary amine comprised in a side chain of a lysine residue, a lysine derivative or a lysine mimetic; and is also provided with
Wherein the antibody is contacted with 2-50 molar equivalents of the linker; and/or
Wherein the microbial transglutaminase is added to the coupling reaction at a concentration of 1-10U/mg of antibody, and optionally wherein the antibody is added to the coupling reaction at a concentration of 1-20 mg/mL.
In an even more preferred embodiment, the invention relates to a method for producing an antibody-linker conjugate by Microbial Transglutaminase (MTG), the method comprising administering a polypeptide comprising (as indicated in the N.fwdarw.C direction) (Sp 1 )-RK-(Sp 2 )-B-(Sp 3 ) Or (Sp) 1 )-B-(Sp 2 )-RK-(Sp 3 ) A step of coupling a linker of the structure 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 arginine mimetic;
k is lysine or a lysine derivative or a lysine mimetic;
b is a linking part or payload;
wherein the linker is coupled to a Gln residue comprised in the antibody via a primary amine comprised in a side chain of a lysine residue, a lysine derivative or a lysine mimetic; and is also provided with
Wherein the antibody is contacted with 2-30 molar equivalents of the linker; and/or
Wherein the microbial transglutaminase is added to the coupling reaction at a concentration of 2-10U/mg antibody, and optionally wherein the antibody is added to the coupling reaction at a concentration of 5-20 mg/mL.
In an even more preferred embodiment, the invention relates to a method for producing an antibody-linker conjugate by Microbial Transglutaminase (MTG), the method comprising administering a polypeptide comprising (as indicated in the N.fwdarw.C direction) (Sp 1 )-RK-(Sp 2 )-B-(Sp 3 ) Or (Sp) 1 )-B-(Sp 2 )-RK-(Sp 3 ) A step of coupling a linker of the structure 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 arginine mimetic;
K is lysine or a lysine derivative or a lysine mimetic;
b is a linking part or payload;
wherein the linker is coupled to a Gln residue comprised in the antibody via a primary amine comprised in a side chain of a lysine residue, a lysine derivative or a lysine mimetic; and is also provided with
Wherein the antibody is contacted with about 2-20 molar equivalents of the linker; and/or wherein the microbial transglutaminase is added to the coupling reaction at a concentration of 2-10U/mg antibody, and optionally wherein the antibody is added to the coupling reaction at a concentration of 5-20 mg/mL.
In an even more preferred embodiment, the invention relates to a method for producing an antibody-linker conjugate by Microbial Transglutaminase (MTG), the method comprising administering a polypeptide comprising (as indicated in the N.fwdarw.C direction) (Sp 1 )-RK-(Sp 2 )-B-(Sp 3 ) Or (Sp) 1 )-B-(Sp 2 )-RK-(Sp 3 ) A step of coupling a linker of the structure 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 arginine mimetic;
k is lysine or a lysine derivative or a lysine mimetic;
b is a linking part or payload;
wherein the linker is coupled to a Gln residue comprised in the antibody via a primary amine comprised in a side chain of a lysine residue, a lysine derivative or a lysine mimetic; and is also provided with
Wherein the antibody is contacted with about 2.5 to 15 molar equivalents of the linker; and/or wherein the microbial transglutaminase is added to the coupling reaction at a concentration of 2-10U/mg antibody, and optionally wherein the antibody is added to the coupling reaction at a concentration of 5-20 mg/mL.
In a most preferred embodiment, the invention relates to a method for producing an antibody-linker conjugate by Microbial Transglutaminase (MTG), comprising administering a polypeptide comprising (as indicated in the N.fwdarw.C direction) (Sp 1 )-RK-(Sp 2 )-B-(Sp 3 ) Or (Sp) 1 )-B-(Sp 2 )-RK-(Sp 3 ) A step of coupling a linker of the structure 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 arginine mimetic;
k is lysine or a lysine derivative or a lysine mimetic;
b is a linking part or payload;
wherein the linker is coupled to a Gln residue comprised in the antibody via a primary amine comprised in a side chain of a lysine residue, a lysine derivative or a lysine mimetic; and is also provided with
Wherein the antibody is contacted with about 2.5 to about 10 molar equivalents of the linker; and/or wherein the microbial transglutaminase is added to the coupling reaction at a concentration of 2-10U/mg antibody, and optionally wherein the antibody is added to the coupling reaction at a concentration of 5-20 mg/mL.
In a particular embodiment, the invention relates to antibody-linker conjugates prepared by the methods of the invention.
That is, the present invention relates to an antibody-linker conjugate produced by any one of the above steps.
In a particular embodiment, the invention relates to an antibody-linker conjugate comprising:
a) An antibody; and
b) A joint comprising the structure:
(Sp 1 )-RK-(Sp 2 )-B-(Sp 3 ) Or (b)
(Sp 1 )-B-(Sp 2 )-RK-(Sp 3 ) The method comprises the steps of carrying out a first treatment on the surface of the Wherein the method comprises the steps of
·(Sp 1 ) Is a chemical spacer or is absent;
·(Sp 2 ) Is a chemical spacer or is absent;
·(Sp 3 ) Is a chemical spacer or is absent;
r is arginine or an arginine derivative or arginine mimetic;
k is lysine or a lysine derivative or a lysine mimetic;
b is a linking part or payload;
wherein the linker is coupled to the antibody via an isopeptide bond formed between the γ -carboxamide group of a glutamine residue comprised in the antibody and a primary amine comprised in a side chain of a lysine residue, a lysine derivative or a lysine mimetic comprised in an RK motif comprised in the linker.
That is, the invention further relates to antibody-linker conjugates that have been produced by the methods of the invention. In particular, the invention relates to antibodies that are conjugated to any of the linkers disclosed herein for use in the methods of the invention at glutamine residues contained in the heavy or light chain of the antibody. That is, all linkers that have been disclosed above for use in the methods of the invention may be included in the antibody-linker constructs of the invention. Preferably, the linker of the invention is coupled to a glutamine residue in an antibody via an amide bond formed between the amide side chain of the glutamine residue comprised in the antibody and a primary amine comprised in residue K comprised in the RK motif of the linker. In certain embodiments, as disclosed herein, the primary amine contained in residue K is an amine group contained in a side chain of a lysine residue, lysine mimetic, or lysine derivative. In certain embodiments, K is a lysine residue and the primary amine via which the linker is coupled to the antibody is an epsilon-amino group contained in the lysine residue.
The chemical spacer included in the antibody-linker constructs disclosed herein may be any of the linkers disclosed herein that include RK. That is, the joint may be a joint comprising a single connection portion or payload B, or may be a joint comprising two or more connection portions and/or payloads B 1 、B 2 Etc.
In a particular embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the chemical spacer (Sp 1 )、(Sp 2 ) Sum (Sp) 3 ) Each independently comprises 0 to 12 amino acid residues.
In a particular embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the linker comprises no more than 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 amino acid residues.
In a particular embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the net charge of the linker is neutral or positive.
In a particular embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the linker does not comprise negatively charged amino acid residues.
In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the linker comprises an amino acid sequence selected from the group consisting of: RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3) and RKR (SEQ ID NO: 4).
In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the linker comprises an amino acid sequence selected from the group consisting of: RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2) and ARK (SEQ ID NO: 3).
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).
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).
In a particular embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein B is a linking moiety.
In a particular embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the linking moiety B comprises
Bio-orthogonal labelling group, or
Non-bioorthogonal entities for cross-linking.
In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the bio-orthogonal labeling group or the non-bio-orthogonal entity for cross-linking consists of or comprises at least one molecule or moiety selected from the group consisting of:
-N-N≡N or-N 3 ；
·Lys(N 3 )；
Tetrazine;
alkynes;
strained cyclooctyne;
·BCN；
strained olefins;
photoreactive groups;
aldehyde;
acyl trifluoroborates;
protein degrading agent ('PROTAC');
cyclopentadiene/spirocyclopentadiene;
a thio-selective electrophile;
-SH; and
cysteine.
That is, an antibody-linker conjugate according to the invention may be an antibody conjugated to a linker comprising one or more linking moieties. Such antibody-linker conjugates may be tailored later with one or more payloads (particularly with payloads that can be adapted for coupling to one or more linking moieties).
In a particular embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein one or more payloads have been coupled to linking moiety B.
In a particular embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein one or more payloads have been coupled to linking moiety B via a click reaction.
That is, the antibody-linker conjugate according to the present invention may be an antibody-payload conjugate produced in the two-step method disclosed herein.
In a particular embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein B is a payload.
In a particular embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the payload comprises at least one of the following:
toxins;
cytokines;
growth factors;
radionuclides;
hormones;
antiviral agents;
an antimicrobial agent;
fluorescent dye;
immunomodulators/immunostimulants;
half-life increasing moiety;
a solubility-increasing moiety;
polymer-toxin conjugate;
nucleic acid;
biotin or streptavidin moiety;
vitamins;
protein degrading agent ('PROTAC');
a target binding moiety; and/or
Anti-inflammatory agents.
In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the toxin is selected from at least one of the group consisting of:
pyrrolobenzodiazepines (e.g., PBDs);
auristatin (e.g., MMAE, MMAF);
maytansinoids (e.g., maytansine, DM1, DM4, DM 21);
sesquicomycin;
nicotinamide phosphoribosyl transferase (NAMPT) inhibitors;
microtubule lysin;
Enediyne (e.g., calicheamicin);
anthracycline derivatives (PNUs) (e.g., doxorubicin);
inhibitors of the pyrrolyl Kinesin Spindle Protein (KSP);
candidiasis;
drug efflux pump inhibitors;
mountain Zhuo Meisu;
amanitine (e.g., α -amanitine); and
camptothecins (e.g., irinotecan, delutinacon).
In a particular embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the chemical spacer (Sp 2 ) Including self-cleaving moieties.
In a particular embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the self-cleaving moiety is directly attached to payload B.
In a particular embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the self-cleaving moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety.
That is, an antibody-linker conjugate according to the invention may be an antibody-payload conjugate produced in a one-step process disclosed herein.
The antibodies comprised in the antibody-linker conjugate according to the invention may be any of the antibodies disclosed herein for use in the method according to the invention, in particular any of the IgG-type antibodies. That is, the antibodies comprised in the antibody-linker conjugates according to the invention may comprise the same glycosylation pattern, mutation and/or modification as the antibodies disclosed herein for the methods according to the invention.
In a specific embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the linker is any one of the linkers shown in fig. 1, fig. 2, fig. 3, fig. 8, fig. 9, fig. 14, fig. 15, fig. 17, fig. 18, fig. 19, fig. 20, fig. 21, fig. 22, fig. 23, fig. 24, fig. 25, fig. 26, fig. 27, fig. 28, fig. 29, fig. 30, fig. 31, fig. 32, fig. 33 or fig. 34.
In a specific 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.
In a particular embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the linker-conjugated Gln residue is comprised in the Fc domain of the antibody, in particular wherein the linker-conjugated Gln residue is the C of an IgG antibody H Gln residue Q295 of the 2 domain (EU numbering).
In a particular embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the Gln residue coupled to the linker has been introduced into the heavy or light chain of the antibody by molecular engineering.
In a particular embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the Gln residue of the heavy or light chain introduced into the antibody by molecular engineering is the C of a non-glycosylated IgG antibody H 2 domain N297Q (EU numbering).
In a particular embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the Gln residue of the heavy or light chain introduced into the antibody by molecular engineering is comprised in a peptide which has been (a) integrated to the heavy or light chain of the antibody or (b) fused to the N-or C-terminus of the heavy or light chain of the antibody.
In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the peptide comprising a Gln residue has been fused to the C-terminus of the heavy chain of the antibody.
In a particular embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the IgG antibody is a glycosylated IgG antibody, in particular wherein the IgG antibody is at C H Glycosylation at residue N297 (EU numbering) of the 2 domain.
In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the antibody is selected from the group consisting of: vibutuzumab, trastuzumab, gemtuzumab, oxtuzumab, avermectin, cetuximab, rituximab, up Lei Tuoyou mab, pertuzumab, vedolizumab, oreuzumab, tolizumab, wu Sinu mab, golimumab, otouzumab, sha Xituo mab, bei Lantuo mab, polotouzumab and enrolment mab.
In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the antibody is selected from the group consisting of: velutinab, gemtuzumab, trastuzumab, oxtuzumab, poltuzumab, enrolment mab, sha Xituo bead mab, bei Lantuo mab.
In a more preferred embodiment, the present invention relates to an antibody-linker conjugate according to the invention, wherein the antibody is either pertuzumab or trastuzumab or enrolment mab.
In a particular embodiment, the invention relates to an antibody-drug conjugate. That is, antibodies may be conjugated to a linker according to the invention, wherein the linker comprises one or more toxins.
Thus, in a particular embodiment, the invention relates to an antibody-drug conjugate comprising:
a) IgG antibodies; and
b) A linker comprising a drug moiety B, wherein the drug moiety B is covalently linked to an amino acid sequence selected from the group consisting of: RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3) or RKR (SEQ ID NO: 4);
wherein the linker is coupled to the IgG antibody via an isopeptide bond which is at antibody C H The gamma-carboxamide group of glutamine residue Q295 (EU numbering) of the 2 domain forms with the primary amine contained in the side chain of the lysine residue contained in the linker.
In certain embodiments, the invention relates to an antibody-drug conjugate comprising:
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 sequence RK-Val-Cit (SEQ ID NO: 54);
wherein the linker is coupled to the IgG antibody via an isopeptide bond which is at antibody C H The gamma-carboxamide group of glutamine residue Q295 (EU numbering) of the 2 domain forms with the primary amine contained in the side chain of the lysine residue contained in the linker.
That is, in certain embodiments, the linker may comprise any of the sequences RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4) or RK-Val-Cit (SEQ ID NO: 54), wherein the linker is coupled to the glutamine residue in the antibody via a primary amine comprised in residue K. It will be appreciated that drug moiety B need not be directly 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). Alternatively, drug moiety B may be indirectly linked to the structure RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4) or RK-Val-Cit (SEQ ID NO: 54). For example, the linker may include other chemical structures between drug moiety B and structure RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4) or RK-Val-Cit (SEQ ID NO: 54). Such chemical structures may be those disclosed herein for chemical spacers (Sp 1 )、(Sp 2 ) Or (Sp) 3 ) Any structure of (2). In certain embodiments, the linker may comprise a linker located between drug moiety B and structure RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NONO: 3), RKR (SEQ ID NO: 4) or RK-Val-Cit (SEQ ID NO: 54). In certain embodiments, the linker may include one or more PEG moieties between drug moiety B and structure RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4) or RK-Val-Cit (SEQ ID NO: 54). In certain embodiments, the linker may include a cleavable and/or self-cleavable moiety between drug moiety B and 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).
That is, in a specific embodiment, the present invention relates to an antibody-drug conjugate according to the present invention, wherein drug moiety B is linked to the N-terminal or C-terminal end of the amino acid sequence contained in the linker via a self-cleaving moiety.
In a particular embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the self-cleaving moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety.
That is, the self-cleaving moiety contained in a linker according to the present invention may be any of the self-cleaving moieties disclosed herein. In certain embodiments, the self-cleaving moiety may be a PABC or methylamino as disclosed herein.
In a particular embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the IgG antibody is a glycosylated IgG antibody, in particular wherein the IgG antibody is at C H Glycosylation at residue N297 (EU numbering) of the 2 domain.
In a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the IgG antibody is an IgG1 antibody.
That is, the antibody is preferably an IgG antibody, particularly an IgG1 antibody, particularly wherein IgG or IgG1 antibody is glycosylated at residue N297 (EU numbering).
The antibody-drug conjugate may comprise one or more toxins disclosed herein. Thus, in a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the drug is a toxin selected from the group consisting of:
pyrrolobenzodiazepines (e.g., PBDs);
auristatin (e.g., MMAE, MMAF);
maytansinoids (e.g., maytansine, DM1, DM4, DM 21);
Sesquicomycin;
nicotinamide phosphoribosyl transferase (NAMPT) inhibitors;
microtubule lysin;
enediyne (e.g., calicheamicin);
anthracycline derivatives (PNUs) (e.g., doxorubicin);
inhibitors of the pyrrolyl Kinesin Spindle Protein (KSP);
candidiasis;
drug efflux pump inhibitors;
mountain Zhuo Meisu;
amanitine (e.g., α -amanitine); and
camptothecins (e.g., irinotecan, delutinacon).
It is understood that the toxin may be directly coupled to the linker by chemical synthesis. However, in other embodiments, the toxin may also be attached to the linker moiety contained in the linker in a two-step process.
In a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure RKAA-B or RKAA- (linker molecule) -B.
That is, payload B may be coupled directly to the C-terminus of an alanine residue, or may be coupled to the C-terminus of an alanine residue via a linker molecule. It will be appreciated that the choice of linker molecule will depend to a large extent on the functional groups available in payload B. Disclosed herein are linker molecules suitable for coupling payloads having different functional groups to peptides. The linker molecule may be a cleavable or non-cleavable linker molecule. In particular, the linker molecule may comprise a self-cleaving moiety, in particular any of the self-cleaving moieties disclosed herein. Thus, in a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure RKAA- (self-cleaving moiety) -B.
Alternatively, the payload may be coupled to the N-terminus of the arginine residue directly or via a linker molecule (e.g., via any of the linker molecules disclosed herein). That is, in a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure B-RKAA or B- (linker molecule) -RKAA. In a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of structure B- (self-cleaving moiety) -RKAA. In certain embodiments, the self-cleaving moiety included in structure B- (self-cleaving moiety) -RKAA can be a self-cleaving moiety including an ortho-hydroxy protected aryl sulfate (OHPAS) moiety as disclosed herein.
In a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker has the structure RKAA-PABC-B, in particular wherein B is auristatin or maytansinoid, in particular wherein auristatin is MMAE, and wherein maytansinoid is DM1 or maytansinoid.
In certain embodiments, the linker may have the structure RKAA-PABC-B. That is, the linker may comprise a linear peptide RKAA in which the carboxyl group of the C-terminal alanine residue is coupled via an amide bond to an amino group comprised in the PABC. Toxin B may be attached to PABC by carbamate formation. It should be understood that not all toxins contain functional groups that allow carbamate formation with PABCs. Thus, toxins may be attached to PABCs via linkers.
In certain embodiments, the toxin may be a toxin comprising a primary or secondary amine. In certain embodiments, the toxin may be MMAE or maytansine.
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 8.
In some implementationsIn a mode, the linker may have the structure RKAA-PABC-MMAE. In certain embodiments, the linker may have the structure RKAA- (PEG) n -PABC-MMAE, wherein n is an integer between 2 and 20. In certain embodiments, the linker may have the structure RKAA- (PEG) 2 -PABC-MMAE. In certain embodiments, the linker may have the structure RKAA-MMAE. In certain embodiments, the linker may have the structure RKAA-Val-Cit-PABC-MMAE. In certain embodiments, the linker may include an additional linker between the PABC moiety and MMAE. In certain embodiments, the additional linker may be a p-nitrophenol (PNP) group.
It must be noted that the linker may comprise other self-cleaving moieties than PABC. That is, the linker may have the structure RKAA- (self-cleaving moiety) -toxin. Those skilled in the art will recognize other self-cleaving moieties that may be used in the present invention. Further, the skilled artisan is aware of toxins that can be coupled to self-cleaving moieties, optionally via additional linkers.
In certain embodiments, the toxin may be a toxin comprising a hydroxyl group, and the linker may comprise a self-cleaving methylamino group. That is, the linker may have the structure RKAA- (NH) - (CH) 3 ) -O-toxin. In certain embodiments, the hydroxyl-containing toxin may be a camptothecin (such as, for example, irinotecan or an irinotecan derivative, particularly the irinotecan derivative Dxd) or an anthracycline (such as, for example, PNU-159582).
In certain embodiments, the toxin may be a toxin comprising a thiol group, and the linker may comprise a self-cleaving methylamino group. That is, the linker may have the structure RKAA- (NH) - (CH) 3 ) -S-toxin. In certain embodiments, the thiol-containing toxin may be a maytansinoid (such as DM 1) or a thiol-containing derivative thereof.
In a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure RKA-B or RKA- (linker molecule) -B.
That is, payload B may be coupled directly to the C-terminus of an alanine residue, or may be coupled to the C-terminus of an alanine residue via a linker molecule. It will be appreciated that the choice of linker molecule will depend to a large extent on the functional groups available in payload B. Disclosed herein are linker molecules suitable for coupling payloads having different functional groups to peptides. The linker molecule may be a cleavable or non-cleavable linker molecule. In particular, the linker molecule may comprise a self-cleaving moiety, in particular any of the self-cleaving moieties disclosed herein. Thus, in a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure RKA- (self-cleaving moiety) -B.
Alternatively, the payload may be coupled to the N-terminus of the arginine residue directly or via a linker molecule (e.g., via any of the linker molecules disclosed herein). That is, in a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure B-RKA or B- (linker molecule) -RKA. In a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of structure B- (self-cleaving moiety) -RKA. In certain embodiments, the self-cleaving moiety included in structure B- (self-cleaving moiety) -RKA may be a self-cleaving moiety including an ortho-hydroxy protected aryl sulfate (OHPAS) moiety as disclosed herein.
In a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker has the structure RKA-PABC-B, in particular wherein B is auristatin or maytansinoid, in particular wherein auristatin is MMAE, and wherein maytansinoid is DM1 or maytansinoid.
In certain embodiments, the linker may have the structure RKA-PABC-B. That is, the linker may comprise the linear peptide RKA, wherein the carboxyl group of the C-terminal alanine residue is coupled via an amide bond to the amino group comprised in the PABC. Toxin B may be attached to PABC by carbamate formation. It should be understood that not all toxins contain functional groups that allow carbamate formation with PABCs. Thus, toxins may be attached to PABCs via linkers.
In certain embodiments, the toxin may be a toxin comprising a primary or secondary amine. In certain embodiments, the toxin may be MMAE or maytansine.
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.
In certain embodiments, the linker may have the structure RKA-PABC-MMAE. In certain embodiments, the linker may have the structure RKA- (PEG) n -PABC-MMAE, wherein n is an integer between 2 and 20. In certain embodiments, the linker may have the structure RKA- (PEG) 2 -PABC-MMAE. In certain embodiments, the linker may have the structure RKA-MMAE. In certain embodiments, the linker may have the structure RKA-Val-Cit-PABC-MMAE. In certain embodiments, the linker may include an additional linker between the PABC moiety and MMAE. In certain embodiments, the additional linker may be a p-nitrophenol (PNP) group.
It must be noted that the linker may comprise other self-cleaving moieties than PABC. That is, the linker may have the structure RKA- (self-cleaving moiety) -toxin. Those skilled in the art will recognize other self-cleaving moieties that may be used in the present invention. Further, the skilled artisan is aware of toxins that can be coupled to self-cleaving moieties, optionally via additional linkers.
In certain embodiments, the toxin may be a toxin comprising a hydroxyl group, and the linker may comprise a self-cleaving methylamino group. That is, the linker may have the structure RKA- (NH) - (CH) 3 ) -O-toxin. In certain embodiments, the hydroxyl-containing toxin may be a camptothecin (such as, for example, irinotecan or an irinotecan derivative, particularly the irinotecan derivative Dxd) or an anthracycline (such as, for example, PNU-159582).
In certain embodiments, the toxin may be a toxin comprising a thiol group, and the linker may comprise a self-cleaving methylamino group. That is, the linker may have the structure RKA- (NH) - (CH) 3 ) -S-toxin. In certain embodiments, a toxin comprising a thiol mayIs a maytansinoid (such as DM 1) or a thiol-containing derivative.
In a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure ARK-B or ARK- (linker molecule) -B.
That is, payload B may be coupled directly to the C-terminus of a lysine residue, or may be coupled to the C-terminus of a lysine residue via a linker molecule. It will be appreciated that the choice of linker molecule will depend to a large extent on the functional groups available in payload B. Disclosed herein are linker molecules suitable for coupling payloads having different functional groups to peptides. The linker molecule may be a cleavable or non-cleavable linker molecule. In particular, the linker molecule may comprise a self-cleaving moiety, in particular any of the self-cleaving moieties disclosed herein. Thus, in a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure ARK- (self-cleaving moiety) -B.
Alternatively, the payload can be coupled to the N-terminus of the alanine residue directly or via a linker molecule (e.g., via any of the linker molecules disclosed herein). That is, in a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure B-ARK or B- (linker molecule) -ARK. In a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of structure B- (self-cleaving moiety) -ARK. In certain embodiments, the self-cleaving moiety included in structure B- (self-cleaving moiety) -ARK may be a self-cleaving moiety including an ortho-hydroxy protected aryl sulfate (OHPAS) moiety as disclosed herein.
In a particular embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker has the structure ARK-PABC-B, in particular wherein B is auristatin or maytansinoid, in particular wherein auristatin is MMAE, and wherein maytansinoid is DM1 or maytansinoid.
In certain embodiments, the linker may have the structure ARK-PABC-B. That is, the linker may comprise a linear peptide ARK in which the carboxyl group of the C-terminal lysine residue is coupled to the amino group comprised in the PABC via an amide bond. Toxin B may be attached to PABC by carbamate formation. It should be understood that not all toxins contain functional groups that allow carbamate formation with PABCs. Thus, toxins may be attached to PABCs via linkers.
In certain embodiments, the toxin may be a toxin comprising a primary or secondary amine. In certain embodiments, the toxin may be MMAE or maytansine.
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.
In certain embodiments, the linker may have the structure ARK-PABC-MMAE. In certain embodiments, the linker may have the structure ARK- (PEG) n -PABC-MMAE, wherein n is an integer between 2 and 20. In certain embodiments, the linker may have the structure ARK- (PEG) 2 PABC-MMAE (see FIG. 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.
It must be noted that the linker may comprise other self-cleaving moieties than PABC. That is, the linker may have the structure ARK- (self-cleaving moiety) -toxin. Those skilled in the art will recognize other self-cleaving moieties that may be used in the present invention. Further, the skilled artisan is aware of toxins that can be coupled to self-cleaving moieties, optionally via additional linkers.
In certain embodiments, the toxin may be a toxin comprising a hydroxyl group, and the linker may comprise a self-cleaving methylamino group. That is, the linker may have the structure ARK- (NH) - (CH) 3 ) -O-toxin. In certain embodiments, the hydroxyl-containing toxins may beEither camptothecins (such as, for example, irinotecan or derivatives of irinotecan, in particular, derivatives of irinotecan Dxd) or anthracyclines (such as, for example, PNU-159582).
In certain embodiments, the toxin may be a toxin comprising a thiol group, and the linker may comprise a self-cleaving methylamino group. That is, the linker may have the structure ARK- (NH) - (CH) 3 ) O-toxin (similar to fig. 15). In certain embodiments, the thiol-containing toxin may be a maytansinoid (such as DM 1) or a thiol-containing derivative thereof.
In certain embodiments, the linker is the linker shown in fig. 14 or 15.
In a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure RKR-B or RKR- (linker molecule) -B.
That is, payload B may be coupled directly to the C-terminus of an arginine residue, or may be coupled to the C-terminus of an arginine residue via a linker molecule. It will be appreciated that the choice of linker molecule will depend to a large extent on the functional groups available in payload B. Disclosed herein are linker molecules suitable for coupling payloads having different functional groups to peptides. The linker molecule may be a cleavable or non-cleavable linker molecule. In particular, the linker molecule may comprise a self-cleaving moiety, in particular any of the self-cleaving moieties disclosed herein. Thus, in a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure RKR- (self-cleaving moiety) -B.
Alternatively, the payload may be coupled to the N-terminus of the arginine residue directly or via a linker molecule (e.g., via any of the linker molecules disclosed herein). That is, in a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure B-RKR or B- (linker molecule) -RKR. In certain embodiments, the linker may be a dicarboxylic acid linker (see fig. 9). In a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of structure B- (self-cleaving moiety) -RKR. In certain embodiments, the self-cleaving moiety included in structure B- (self-cleaving moiety) -RKR may be a self-cleaving moiety including an ortho-hydroxy protected aryl sulfate (OHPAS) moiety as disclosed herein.
In a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker has the structure RKR-PABC-B, in particular wherein B is auristatin or maytansinoid, in particular wherein auristatin is MMAE, and wherein maytansinoid is DM1 or maytansinoid.
In certain embodiments, the linker may have the structure RKR-PABC-B. That is, the linker may comprise a linear peptide RKR in which the carboxyl group of the C-terminal arginine residue is coupled via an amide bond to an amino group comprised in the PABC. Toxin B may be attached to PABC by carbamate formation. It should be understood that not all toxins contain functional groups that allow carbamate formation with PABCs. Thus, toxins may be attached to PABCs via linkers.
In certain embodiments, the toxin may be a toxin comprising a primary or secondary amine. In certain embodiments, the toxin may be MMAE or maytansine.
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 may have the structure RKR-PABC-MMAE. In certain embodiments, the linker may have the structure RKR- (PEG) n -PABC-MMAE, wherein n is an integer between 2 and 20. In certain embodiments, the linker may have the structure RKR- (PEG) 2 -PABC-MMAE. In certain embodiments, the linker may have the structure RKR-MMAE. In certain embodiments, the linker may have the structure RKR-Val-Cit-PABC-MMAE. In certain embodiments, the linker may include an additional linker between the PABC moiety and MMAE. In certain embodiments, the additional linker may be a p-nitrophenol (PNP) group.
It must be noted that the linker may comprise other self-cleaving moieties than PABC. That is, the linker may have the structure RKR- (self-cleaving moiety) -toxin. Those skilled in the art will recognize other self-cleaving moieties that may be used in the present invention. Further, the skilled artisan is aware of toxins that can be coupled to self-cleaving moieties, optionally via additional linkers.
In certain embodiments, the toxin may be a toxin comprising a hydroxyl group, and the linker may comprise a self-cleaving methylamino group. That is, the linker may have the structure RKR- (NH) - (CH) 3 ) -O-toxin. In certain embodiments, the hydroxyl-containing toxin may be a camptothecin (such as, for example, irinotecan or an irinotecan derivative, particularly the irinotecan derivative Dxd) or an anthracycline (such as, for example, PNU-159582).
In certain embodiments, the toxin may be a toxin comprising a thiol group, and the linker may comprise a self-cleaving methylamino group. That is, the linker may have the structure RKR- (NH) - (CH) 3 ) O-toxin (similar to fig. 15). In certain embodiments, the thiol-containing toxin may be a maytansinoid (such as DM 1) or a thiol-containing derivative thereof.
In a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure RK-Val-Cit-B or RK-Val-Cit- (linker molecule) -B.
That is, payload B may be coupled directly to the C-terminus of the citrulline residue, or may be coupled to the C-terminus of the citrulline residue via a linker molecule. It will be appreciated that the choice of linker molecule will depend to a large extent on the functional groups available in payload B. Disclosed herein are linker molecules suitable for coupling payloads having different functional groups to peptides. The linker molecule may be a cleavable or non-cleavable linker molecule. In particular, the linker molecule may comprise a self-cleaving moiety, in particular any of the self-cleaving moieties disclosed herein. Thus, in a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure RK-Val-Cit- (self-cleaving moiety) -B.
In a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker has the structure RK-Val-Cit-PABC-B, in particular wherein B is auristatin or maytansinoid, in particular wherein auristatin is MMAE, and wherein maytansinoid is DM1 or maytansinoid.
In certain embodiments, the linker may have the structure RK-Val-Cit-PABC-B. That is, the linker may comprise the linear peptide RK-Val-Cit, wherein the carboxyl group of the C-terminal citrulline residue is coupled via an amide bond to an amino group comprised in PABC. Toxin B may be attached to PABC by carbamate formation. It should be understood that not all toxins contain functional groups that allow carbamate formation with PABCs. Thus, toxins may be attached to PABCs via linkers.
In certain embodiments, the toxin may be a toxin comprising a primary or secondary amine. In certain embodiments, the toxin may be MMAE or maytansine.
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 may have the structure RK-Val-Cit-PABC-MMAE. In certain embodiments, the linker may have the structure RK- (PEG) n Val-Cit-PABC-MMAE, where n is an integer between 2 and 20. In certain embodiments, the linker may have the structure RK- (PEG-Val-Cit-PABC-MMAE. In certain embodiments, the linker may have the structure RK-Val-Cit-MMAE. In certain embodiments, the linker may include an additional linker between the PABC moiety and the MMAE. In certain embodiments, the additional linker may be a p-nitrophenol (PNP) group.
It must be noted that the linker may comprise other self-cleaving moieties than PABC. That is, the linker may have the structure RK-Val-Cit- (self-cleaving moiety) -toxin. Those skilled in the art will recognize other self-cleaving moieties that may be used in the present invention. Further, the skilled artisan is aware of toxins that can be coupled to self-cleaving moieties, optionally via additional linkers.
In certain embodiments, the toxin may be a hydroxyl-containing toxinA member, and the linker may comprise a self-cleaving methylamino group. That is, the linker may have the structure RK-Val-Cit- (NH) - (CH) 3 ) -O-toxin. In certain embodiments, the hydroxyl-containing toxin may be a camptothecin (such as, for example, irinotecan or an irinotecan derivative, particularly the irinotecan derivative Dxd) or an anthracycline (such as, for example, PNU-159582).
In certain embodiments, the toxin may be a toxin comprising a thiol group, and the linker may comprise a self-cleaving methylamino group. That is, the linker may have the structure RK-Val-Cit- (NH) - (CH) 3 ) -S-toxin. In certain embodiments, the thiol-containing toxin may be a maytansinoid (such as DM 1) or a thiol-containing derivative thereof.
In a particular embodiment, the invention relates to an antibody-linker conjugate or an antibody-drug conjugate comprising the antibody poloxamer or alternatively an anti-CD 79b antibody.
That is, in a particular embodiment, the invention relates to an antibody-linker conjugate comprising:
a) A poloxamer or an anti-CD 79b antibody; and
b) A joint comprising the structure:
(Sp 1 )-RK-(Sp 2 )-B-(Sp 3 ) Or (b)
(Sp 1 )-B-(Sp 2 )-RK-(Sp 3 ) The method comprises the steps of carrying out a first treatment on the surface of the Wherein the method comprises the steps of
·(Sp 1 ) Is a chemical spacer or is absent;
·(Sp 2 ) Is a chemical spacer or is absent;
·(Sp 3 ) Is a chemical spacer or is absent;
r is arginine or an arginine derivative or arginine mimetic;
k is lysine or a lysine derivative or a lysine mimetic;
b is a linking part or payload;
wherein the linker is coupled to the polotostuzumab or anti-CD 79b antibody via an isopeptide bond formed between the γ -carboxamide group of a glutamine residue contained in the polotostuzumab or anti-CD 79b antibody and a primary amine contained in the side chain of a lysine residue, lysine derivative or lysine mimetic contained in the RK motif contained in the linker.
In a particular embodiment, the invention relates to an antibody-payload conjugate comprising a poloxamer or an anti-CD 79b antibody, wherein the linker is any one of the linkers shown in fig. 1, fig. 2, fig. 3, fig. 8, fig. 9, fig. 14, fig. 15, fig. 17, fig. 18, fig. 19, fig. 20, fig. 21, fig. 22, fig. 23, fig. 24, fig. 25, fig. 26, fig. 27, fig. 28, fig. 29, fig. 30, fig. 31, fig. 32, fig. 33, or fig. 34.
The poloxamer is commercially available as the antibody-drug conjugate, poloxamer Shan Kangwei statin (Polatuzumab vedotin), and sold under the name Polivy. The poloxamers of the poloxamer Shan Kangwei include the anti-CD 79b antibody, the poloxamer Shan Kangwei, and the linker maleimidocaproyl-L-valine-L-citrulline-PAC-MMAE (mc-vc-PABC-MMAE), commonly referred to as Wei Duoting. The mc-vc-PABC-MMAE linker of the poloxamer Shan Kangwei statin was coupled to the free cysteine residues contained in the antibody. The antibody polotophyllizumab is disclosed in WO 2009/012668, which is incorporated herein by reference in its entirety. Further, cysteine engineered variants of polotophyllizumab have been disclosed in WO 2009/099728, which is also incorporated herein by reference in its entirety.
The inventors have shown that the poloxamer conjugate comprising the linker according to the invention has a longer half-life in plasma than the commercially available conjugate of poloxamer Shan Kangwei statin. Thus, antibody-linker conjugates conjugated to the linkers of the invention by microbial transglutaminase are unexpectedly more stable than antibodies produced by other techniques (e.g., by coupling a maleimide-containing linker to cysteine residues of an antibody). Thus, an antibody-linker conjugate according to the invention is more likely to reach its target cell or tissue without prematurely losing its payload.
In certain embodiments, the antibody is a poloxamer comprising the heavy chain set forth in SEQ ID NO. 5 and the light chain set forth in SEQ ID NO. 6. However, variants of the pertuzumab are also encompassed by the invention, wherein the heavy and/or light chain has 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. In particular, the antibody may comprise any of the sequence variants disclosed in WO 2009/012668 or WO 2009/099728.
Preferably herein, the poloxamer or anti-CD 79b antibody is present in glycosylated form in the antibody-linker conjugate. That is, the poloxamer or anti-CD 79b antibody is preferably glycosylated at residue N297 (EU numbering). However, the poloxamer or anti-CD 79b antibody may also be deglycosylated as described herein.
The poloxamer or anti-CD 79b antibody may be conjugated to any of the linkers disclosed herein (particularly for use in the methods according to the invention). Preferably, the linker is coupled to glutamine residue Q295 (EU numbering) of the antibody in an MTG-catalyzed manner. However, the linker may also be coupled to an engineered glutamine residue, such as N297Q (EU numbering), and/or to any of the glutamine-containing tags disclosed herein.
The linker coupled to the poloxamer or anti-CD 79B antibody may comprise a single linker moiety or payload B, or may comprise a plurality of linker moieties and/or payloads B 1 、B 2 Etc.
That is, in certain embodiments, the linker included in the polotouzumab-linker conjugate may include one or more linking moieties B. Such a poloxamer-linker conjugate may then be functionalized with a suitable payload in a two-step process as disclosed herein.
In certain embodiments, the linker included in the polotouzumab-payload conjugate may include one or more payloads B. Such a poloxamer-payload conjugate may be obtained in a two-step process, wherein the linker comprising the linking moiety has been conjugated to the poloxamer in a first step and the payload has been linked to the linking moiety in a second step. Alternatively, the poloxamer-payload conjugate may be obtained in a one-step process, wherein the linker comprising the payload is directly coupled to the poloxamer.
In certain embodiments, the invention relates to an antibody-drug conjugate comprising a poloxamer or an anti-CD 79B antibody. That is, the linker included in the antibody-drug conjugate may include one or more toxins described herein.
In certain embodiments, an antibody-drug conjugate comprising a poloxamer or an anti-CD 79b antibody may comprise a linker comprising 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).
That is, in a particular embodiment, the invention relates to an antibody-drug conjugate comprising:
a) A poloxamer or an anti-CD 79b antibody; and
b) A linker comprising a drug moiety B, wherein the drug moiety B is covalently linked to an amino acid sequence selected from the group consisting of: RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4) or RK-Val-Cit (SEQ ID NO: 54);
wherein the linker is coupled to the poloxamer or anti-CD 79b antibody via an isopeptide bond which is at antibody C H The gamma-carboxamide group of glutamine residue Q295 (EU numbering) of the 2 domain forms with the primary amine contained in the side chain of the lysine residue contained in the linker.
Preferably, the antibody is a poloxamer comprising the heavy chain shown in SEQ ID NO. 5 and the light chain shown in SEQ ID NO. 6. Thus, in a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the IgG antibody is a poloxamer or an antibody comprising a heavy chain as shown in SEQ ID No. 5 and a light chain as shown in SEQ ID No. 6.
In certain embodiments, the linker coupled to the Pololizumab or anti-CD 79b antibody may comprise the structure RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4) or RK-Val-Cit (SEQ ID NO: 54). In certain embodiments, a linker comprising the structure RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4) or RK-Val-Cit (SEQ ID NO: 54) may be coupled to residue Q295 of the poloxamer or anti-CD 79b antibody via a primary amine contained in residue K.
In certain embodiments, a poloxamer or an anti-CD 79B antibody may be conjugated to the linker RKAA-B disclosed herein, wherein B is preferably a toxin. In certain embodiments, a poloxamer or an anti-CD 79B antibody may be conjugated to the linker RKAA- (self-cleaving moiety) -B disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the C-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a PABC or a methylamine-containing group.
In certain embodiments, the poloxamer or anti-CD 79B antibody may be conjugated to the linker RKAA-PABC-B. In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker RKAA-PABC-MMAE (see fig. 1). In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker RKAA-PABC-maytansine (see fig. 8). In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker RKAA-MMAE. In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker RKAA-PABC-PNP-MMAE. In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker RKAA- (NH) - (CH) 3 ) -O-B coupling, wherein O is an oxygen atom of an ether linkage and B is a hydroxyl-containing toxin. In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker RKAA- (NH) - (CH) 3 ) -O-camptothecin coupling. In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker RKAA- (NH) - (CH) 3 ) -S-B coupling, wherein S is a sulfur atom of a thioether bond and B is a thiol-containing toxin. In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker RKAA- (NH) - (CH) 3 ) -S-DM1 coupling.
In certain embodiments, a poloxamer or an anti-CD 79B antibody may be conjugated to the linker B-RKAA disclosed herein, wherein B is preferably a toxin. In certain embodiments, a poloxamer or an anti-CD 79B antibody may be conjugated to the linker B- (self-cleaving moiety) -RKAA disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the N-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a self-cleaving moiety that contains an ortho-hydroxy protected aryl sulfate (OHPAS) moiety. In certain embodiments, similar to fig. 9, the amine-containing payload B can be coupled to the N-terminal arginine moiety via a dicarboxylic acid linker.
In certain embodiments, a poloxamer or an anti-CD 79B antibody may be conjugated to the linker RKA-B disclosed herein, wherein B is preferably a toxin. In certain embodiments, a poloxamer or an anti-CD 79B antibody may be conjugated to the linker RKA- (self-cleaving moiety) -B disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the C-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a PABC or a methylamine-containing group.
In certain embodiments, the poloxamer or anti-CD 79B antibody may be conjugated to the linker RKA-PABC-B. In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker RKA-PABC-MMAE (see fig. 2). In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker RKA-PABC-maytansine. In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker RKA-MMAE. In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker RKA-PABC-PNP-MMAE. In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker RKA- (NH) - (CH) 3 ) -O-B coupling, wherein O is an oxygen atom of an ether linkage and B is a hydroxyl-containing toxin. In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker RKA- (NH) - (CH) 3 ) -O-camptothecin coupling. In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to linker RKA-(NH)-(CH 3 ) -S-B coupling, wherein S is a sulfur atom of a thioether bond and B is a thiol-containing toxin. In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker RKA- (NH) - (CH) 3 ) -S-DM1 coupling.
In certain embodiments, a poloxamer or an anti-CD 79B antibody may be conjugated to the linker B-RKA disclosed herein, wherein B is preferably a toxin. In certain embodiments, a poloxamer or an anti-CD 79B antibody may be conjugated to the linker B- (self-cleaving moiety) -RKA disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the N-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a self-cleaving moiety comprising an ortho-hydroxy protected aryl sulfate (OHPAS) moiety. In certain embodiments, similar to fig. 9, the amine-containing payload B can be coupled to the N-terminal arginine moiety via a dicarboxylic acid linker.
In certain embodiments, a poloxamer or an anti-CD 79B antibody may be conjugated to the linker ARK-B disclosed herein, wherein B is preferably a toxin. In certain embodiments, a poloxamer or an anti-CD 79B antibody may be conjugated to the linker ARK- (self-cleaving moiety) -B disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the C-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a PABC or a methylamine-containing group.
In certain embodiments, the poloxamer or anti-CD 79B antibody may be conjugated to the linker ARK-PABC-B. In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker ARK-PABC-MMAE (see fig. 3). In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker ARK-PABC-maytansine. In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker ARK-MMAE. In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker ARK-PABC-PNP-MMAE. In certain embodiments, the Polollipop beadsMonoclonal antibodies or anti-CD 79b antibodies may be conjugated to linkers ARK- (NH) - (CH 3 ) -O-B coupling, wherein O is an oxygen atom of an ether linkage and B is a hydroxyl-containing toxin. In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker ARK- (NH) - (CH) 3 ) -O-camptothecin coupling. In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker ARK- (NH) - (CH) 3 ) -S-B coupling, wherein S is a sulfur atom of a thioether bond and B is a thiol-containing toxin. In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker ARK- (NH) - (CH) 3 ) -S-DM1 coupling.
In certain embodiments, a poloxamer or an anti-CD 79B antibody may be conjugated to the linker B-ARK disclosed herein, wherein B is preferably a toxin. In certain embodiments, a poloxamer or an anti-CD 79B antibody may be conjugated to the linker B- (self-cleaving moiety) -ARK disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the N-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a self-cleaving moiety comprising an ortho-hydroxy protected aryl sulfate (OHPAS) moiety.
In certain embodiments, a poloxamer or an anti-CD 79B antibody may be conjugated to the linker RKR-B disclosed herein, wherein B is preferably a toxin. In certain embodiments, a poloxamer or an anti-CD 79B antibody may be conjugated to the linker RKR- (self-cleaving moiety) -B disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the C-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a PABC or a methylamine-containing group.
In certain embodiments, the poloxamer or anti-CD 79B antibody may be conjugated to the linker RKR-PABC-B. In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker RKR-PABC-MMAE. In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker RKR-PABC-maytansine. In certain embodiments, poiseThe lotuzumab or anti-CD 79b antibody may be conjugated to a linker RKR-MMAE. In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker RKR-PABC-PNP-MMAE. In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker RKR- (NH) - (CH) 3 ) -O-B coupling, wherein O is an oxygen atom of an ether linkage and B is a hydroxyl-containing toxin. In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker RKR- (NH) - (CH) 3 ) -O-camptothecin coupling. In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker RKR- (NH) - (CH) 3 ) -S-B coupling, wherein S is a sulfur atom of a thioether bond and B is a thiol-containing toxin. In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker RKR- (NH) - (CH) 3 ) -S-DM1 coupling.
In certain embodiments, a poloxamer or an anti-CD 79B antibody may be conjugated to the linker B-RKR disclosed herein, wherein B is preferably a toxin. In certain embodiments, a poloxamer or an anti-CD 79B antibody may be conjugated to the linker B- (self-cleaving moiety) -RKR disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the N-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a self-cleaving moiety comprising an ortho-hydroxy protected aryl sulfate (OHPAS) moiety. In certain embodiments, as illustrated in fig. 9, the amine-containing payload B can be coupled to the N-terminal arginine moiety via a dicarboxylic acid linker.
In certain embodiments, a poloxamer or an anti-CD 79B antibody may be conjugated to the linker RK-Val-Cit-B disclosed herein, wherein B is preferably a toxin. In certain embodiments, a poloxamer or an anti-CD 79B antibody may be conjugated to the linker RK-Val-Cit- (self-cleaving moiety) -B disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the C-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a PABC or a methylamine-containing group.
In certain embodiments, the poloxamer or anti-CD 79B antibody may be conjugated to the linker RK-Val-Cit-PABC-B. In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker RK-Val-Cit-PABC-MMAE. In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker RK-Val-Cit-PABC-maytansine. In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker RK-Val-Cit-MMAE. In certain embodiments, the poloxamer or anti-CD 79b antibody may be conjugated to the linker RK-Val-Cit-PABC-PNP-MMAE. In certain embodiments, the Polotuzumab or anti-CD 79b antibody may be conjugated to the linker RK-Val-Cit- (NH) - (CH) 3 ) -O-B coupling, wherein O is an oxygen atom of an ether linkage and B is a hydroxyl-containing toxin. In certain embodiments, the Polotuzumab or anti-CD 79b antibody may be conjugated to the linker RK-Val-Cit- (NH) - (CH) 3 ) -O-camptothecin coupling. In certain embodiments, the Polotuzumab or anti-CD 79b antibody may be conjugated to the linker RK-Val-Cit- (NH) - (CH) 3 ) -S-B coupling, wherein S is a sulfur atom of a thioether bond and B is a thiol-containing toxin. In certain embodiments, the Polotuzumab or anti-CD 79b antibody may be conjugated to the linker RK-Val-Cit- (NH) - (CH) 3 ) -S-DM1 coupling.
In a particular embodiment, the invention relates to an antibody-linker conjugate or an antibody-drug conjugate comprising the antibody trastuzumab or alternatively an anti-HER 2 antibody.
That is, in a particular embodiment, the invention relates to an antibody-linker conjugate comprising:
a) A poloxamer or an anti-HER 2/neu antibody; and
b) A joint comprising the structure:
(Sp 1 )-RK-(Sp 2 )-B-(Sp 3 ) Or (b)
(Sp 1 )-B-(Sp 2 )-RK-(Sp 3 ) The method comprises the steps of carrying out a first treatment on the surface of the Wherein the method comprises the steps of
·(Sp 1 ) Is a chemical spacer or is absent;
·(Sp 2 ) Is a chemical spacer or is absent;
·(Sp 3 ) Is a chemical spacer or is absent;
r is arginine or an arginine derivative or arginine mimetic;
k is lysine or a lysine derivative or a lysine mimetic;
b is a linking part or payload;
wherein the linker is coupled to the trastuzumab or the anti-HER 2/neu antibody via an isopeptide bond formed between the γ -carboxamide group of a glutamine residue contained in the trastuzumab or the anti-HER 2/neu antibody and a primary amine contained in a side chain of a lysine residue, a lysine derivative or a lysine mimetic contained in an RK motif contained in the linker.
In a specific embodiment, the invention relates to an antibody-payload conjugate comprising trastuzumab or an anti-HER 2/neu antibody, wherein the linker is any one of the linkers shown in fig. 1, fig. 2, fig. 3, fig. 8, fig. 9, fig. 14, fig. 15, fig. 17, fig. 18, fig. 19, fig. 20, fig. 21, fig. 22, fig. 23, fig. 24, fig. 25, fig. 26, fig. 27, fig. 28, fig. 29, fig. 30, fig. 31, fig. 32, fig. 33, or fig. 34.
Trastuzumab is commercially available as an antibody-drug conjugate, enmetrastuzumab (Trastuzumab emtansine), and sold under the name Kadcyla. Enmetrastuzumab comprises the anti-HER 2/neu antibody trastuzumab and toxin DM1, which is conjugated to trastuzumab through an N-succinimidyl-4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (SMCC) linker. The linker-DM 1 construct may be coupled to up to eight different lysine residues contained in the antibody, thereby producing antibodies with different 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, variants of trastuzumab are also encompassed by the invention, wherein the heavy and/or light chain has 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. In certain embodiments, the antibody may be an anti-HER 2/neu antibody, such as disclosed in, but not limited to, WO 1998/006692, WO 1999/905536, WO 2003/087131, which is incorporated herein by reference in its entirety.
Preferably herein, trastuzumab or an anti-HER 2/neu antibody is present in glycosylated form in the antibody-linker conjugate. That is, trastuzumab or anti-HER 2/neu antibody is preferably glycosylated at residue N297 (EU numbering). However, trastuzumab or anti-HER 2/neu antibodies can also be deglycosylated as described herein.
Trastuzumab or an anti-HER 2/neu antibody can be conjugated to any one of the linkers disclosed herein (particularly for use in a method according to the invention). Preferably, the linker is coupled to glutamine residue Q295 (EU numbering) of the antibody in an MTG-catalyzed manner. However, the linker may also be coupled to an engineered glutamine residue, such as N297Q (EU numbering), and/or to any of the glutamine-containing tags disclosed herein.
The linker coupled to trastuzumab or an anti-HER 2/neu antibody may comprise a single linking moiety or payload B, or may comprise multiple linking moieties and/or payloads B 1 、B 2 Etc.
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 can be subsequently functionalized with an appropriate payload in a two-step process as disclosed herein.
In certain embodiments, the linker included in the trastuzumab-payload conjugate may include one or more payloads B. Such trastuzumab-payload conjugates can be obtained in a two-step process, wherein the linker comprising the linking moiety has been coupled to trastuzumab in a first step and the payload has been linked to the linking moiety in a second step. Alternatively, the trastuzumab-payload conjugate may be obtained in a one-step process, wherein the linker comprising the payload is directly coupled to trastuzumab.
In certain embodiments, the invention relates to an antibody-drug conjugate comprising trastuzumab or an anti-HER 2/neu antibody. That is, the linker included in the antibody-drug conjugate may include one or more toxins described herein.
In certain embodiments, an antibody-drug conjugate comprising trastuzumab or an anti-HER 2/neu antibody may comprise a linker comprising 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).
That is, in a particular embodiment, the invention relates to an antibody-drug conjugate comprising:
a) Trastuzumab or an anti-HER 2/neu antibody; and
b) A linker comprising a drug moiety B, wherein the drug moiety B is covalently linked to an amino acid sequence selected from the group consisting of: RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4) or RK-Val-Cit (SEQ ID NO: 54);
wherein the linker is coupled to the trastuzumab or anti-HER 2/neu antibody via an isopeptide bond which is at antibody C H The gamma-carboxamide group of glutamine residue Q295 (EU numbering) of the 2 domain forms with the primary amine contained in the side chain of the lysine residue contained in the linker.
Preferably, the antibody is trastuzumab comprising the heavy chain shown in SEQ ID NO. 7 and the light chain shown in SEQ ID NO. 8. Thus, in a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the IgG antibody is trastuzumab or an antibody comprising a heavy chain as shown in SEQ ID No. 7 and a light chain as shown in SEQ ID No. 8.
In certain embodiments, the linker conjugated to trastuzumab or an anti-HER 2/neu antibody may comprise the structure RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4) or RK-Val-Cit (SEQ ID NO: 54). In certain embodiments, a linker comprising the structure RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4) or RK-Val-Cit (SEQ ID NO: 54) may be coupled to residue Q295 of trastuzumab or an anti-HER 2/neu antibody via a primary amine comprised in residue K.
In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to a linker RKAA-B as disclosed herein, wherein B is preferably a toxin. In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to linker RKAA- (self-cleaving moiety) -B as disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the C-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a PABC or a methylamine-containing group.
In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to linker RKAA-PABC-B. In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to the linker RKAA-PABC-MMAE (see fig. 1). In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to the linker RKAA-PABC-maytansine (see fig. 8). In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to linker RKAA-MMAE. In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to the linker RKAA-PABC-PNP-MMAE. In certain embodiments, the trastuzumab or anti-HER 2/neu antibody can be conjugated to linker RKAA- (NH) - (CH) 3 ) -O-B coupling, wherein O is an oxygen atom of an ether linkage and B is a hydroxyl-containing toxin. In certain embodiments, the trastuzumab or anti-HER 2/neu antibody can be conjugated to linker RKAA- (NH) - (CH) 3 ) -O-camptothecin coupling. In certain embodiments, the trastuzumab or anti-HER 2/neu antibody can be conjugated to linker RKAA- (NH) - (CH) 3 ) -S-B coupling, wherein S is a sulfur atom of a thioether bond and B is a thiol-containing toxin. In certain embodiments, the trastuzumab or anti-HER 2/neu antibody can be conjugated to linker RKAA- (NH) - (CH) 3 ) -S-DM1 coupling.
In certain embodiments, a tobrazumab or anti-HER 2/neu antibody can be conjugated to a linker B-RKAA disclosed herein, wherein B is preferably a toxin. In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to a linker B- (self-cleaving moiety) -RKAA as disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the N-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a self-cleaving moiety comprising an ortho-hydroxy protected aryl sulfate (OHPAS) moiety. In certain embodiments, similar to fig. 9, the amine-containing payload B can be coupled to the N-terminal arginine moiety via a dicarboxylic acid linker.
In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to a linker RKA-B as disclosed herein, wherein B is preferably a toxin. In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to linker RKA- (self-cleaving moiety) -B as disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the C-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a PABC or a methylamine-containing group.
In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to linker RKA-PABC-B. In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to the linker RKA-PABC-MMAE (see fig. 2). In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to the linker RKA-PABC-maytansine. In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to linker RKA-MMAE. In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to linker RKA-PABC-PNP-MMAE. In certain embodiments, the trastuzumab or anti-HER 2/neu antibody can be conjugated to linker RKA- (NH) - (CH 3 ) -O-B coupling, wherein O is an oxygen atom of an ether linkage and B is a hydroxyl-containing toxin. In certain embodiments, the trastuzumab or anti-HER 2/neu antibody can be conjugated to linker RKA- (NH) - (CH 3 ) -O-camptothecin coupling. In certain embodiments, the trastuzumab or anti-HER 2/neu antibody can be conjugated to linker RKA- (NH) - (CH 3 ) -S-B coupling, wherein S is a sulfur atom of a thioether bond and B is a thiol-containing toxin. In certain embodiments, the trastuzumab or anti-HER 2/neu antibody can be conjugated to linker RKA- (NH) - (CH 3 ) -S-DM1 coupling.
In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to a linker B-RKA as disclosed herein, wherein B is preferably a toxin. In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to a linker B- (self-cleaving moiety) -RKA as disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the N-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a self-cleaving moiety comprising an ortho-hydroxy protected aryl sulfate (OHPAS) moiety. In certain embodiments, similar to fig. 9, the amine-containing payload B can be coupled to the N-terminal arginine moiety via a dicarboxylic acid linker.
In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to a linker ARK-B as disclosed herein, wherein B is preferably a toxin. In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to a linker ARK- (self-cleaving moiety) -B as disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the C-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a PABC or a methylamine-containing group.
In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to linker ARK-PABC-B. In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to a linker ARK-PABC-MMAE (see fig. 3). In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to a linker ARK-PABC-maytansine. In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to a linker ARK-MMAE. In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to a linker ARK-PABC-PNP-MMAE. In certain embodiments, the trastuzumab or anti-HER 2/neu antibody can be conjugated to a linker ARK- (NH) - (CH 3 ) -O-B coupling, wherein O is an oxygen atom of an ether linkage and B is a hydroxyl-containing toxin. In certain embodiments, the trastuzumab or anti-HER 2/neu antibody can be conjugated to a linker ARK- (NH) - (CH 3 ) -O-camptothecin coupling. In certain embodiments, the Qu Tuo bead embodiment mab or anti-HER 2/neu antibody can be conjugated to a linker ARK- (NH) - (CH) 3 ) -S-B coupling, wherein S is a sulfur atom of a thioether bond and B is a hydroxyl-containing toxin. In certain embodiments, the trastuzumab or anti-HER 2/neu antibody can be conjugated to a linker ARK- (NH) - (CH 3 ) -S-DM1 coupling.
In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to a linker B-ARK as disclosed herein, wherein B is preferably a toxin. In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to a linker B- (self-cleaving moiety) -ARK as disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the N-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a self-cleaving moiety comprising an ortho-hydroxy protected aryl sulfate (OHPAS) moiety.
In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to a linker RKR-B as disclosed herein, wherein B is preferably a toxin. In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to linker RKR- (self-cleaving moiety) -B as disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the C-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a PABC or a methylamine-containing group.
In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to linker RKR-PABC-B. In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to the linker RKR-PABC-MMAE. In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to the linker RKR-PABC-maytansine. In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to linker RKR-MMAE. In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to linker RKR-PABC-PNP-MMAE. In certain embodiments, trastuzumab or anti-HEThe R2/neu antibody may be conjugated to linker RKR- (NH) - (CH) 3 ) -O-B coupling, wherein O is an oxygen atom of an ether linkage and B is a hydroxyl-containing toxin. In certain embodiments, the trastuzumab or anti-HER 2/neu antibody can be conjugated to linker RKR- (NH) - (CH 3 ) -O-camptothecin coupling. In certain embodiments, the trastuzumab or anti-HER 2/neu antibody can be conjugated to linker RKR- (NH) - (CH 3 ) -S-B coupling, wherein S is a sulfur atom of a thioether bond and B is a thiol-containing toxin. In certain embodiments, the trastuzumab or anti-HER 2/neu antibody can be conjugated to linker RKR- (NH) - (CH 3 ) -S-DM1 coupling.
In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to a linker B-RKR as disclosed herein, wherein B is preferably a toxin. In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to a linker B- (self-cleaving moiety) -RKR as disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the N-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a self-cleaving moiety comprising an ortho-hydroxy protected aryl sulfate (OHPAS) moiety. In certain embodiments, as illustrated in fig. 9, the amine-containing payload B can be coupled to the N-terminal arginine moiety via a dicarboxylic acid linker.
In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to a linker RK-Val-Cit-B as disclosed herein, wherein B is preferably a toxin. In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to a linker RK-Val-Cit- (self-cleaving moiety) -B as disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the C-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a PABC or a methylamine-containing group.
In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to linker RK-Val-Cit-PABC-B. In certain embodiments, trastuzumab or anti-HER 2/neu antibody can be conjugated to linker RK-ValCit-PABC-MMAE coupling. In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to linker RK-Val-Cit-PABC-maytansine. In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to linker RK-Val-Cit-MMAE. In certain embodiments, trastuzumab or an anti-HER 2/neu antibody can be conjugated to linker RK-Val-Cit-PABC-PNP-MMAE. In certain embodiments, the trastuzumab or anti-HER 2/neu antibody can be conjugated to a linker RK-Val-Cit- (NH) - (CH) 3 ) -O-B coupling, wherein O is an oxygen atom of an ether linkage and B is a hydroxyl-containing toxin. In certain embodiments, the trastuzumab or anti-HER 2/neu antibody can be conjugated to a linker RK-Val-Cit- (NH) - (CH) 3 ) -O-camptothecin coupling. In certain embodiments, the trastuzumab or anti-HER 2/neu antibody can be conjugated to a linker RK-Val-Cit- (NH) - (CH) 3 ) -S-B coupling, wherein S is a sulfur atom of a thioether bond and B is a thiol-containing toxin. In certain embodiments, the trastuzumab or anti-HER 2/neu antibody can be conjugated to a linker RK-Val-Cit- (NH) - (CH) 3 ) -S-DM1 coupling.
In a particular embodiment, the present invention relates to an antibody-linker conjugate or an antibody-drug conjugate comprising the antibody enrolment monoclonal antibody or alternatively an anti-conjugated element-4 antibody.
That is, in a particular embodiment, the invention relates to an antibody-linker conjugate comprising:
a) Enrolment monoclonal antibodies or anti-conjugated element-4 antibodies; and
b) A joint comprising the structure:
(Sp 1 )-RK-(Sp 2 )-B-(Sp 3 ) Or (b)
(Sp 1 )-B-(Sp 2 )-RK-(Sp 3 ) The method comprises the steps of carrying out a first treatment on the surface of the Wherein the method comprises the steps of
·(Sp 1 ) Is a chemical spacer or is absent;
·(Sp 2 ) Is a chemical spacer or is absent;
·(Sp 3 ) Is a chemical spacer or is absent;
r is arginine or an arginine derivative or arginine mimetic;
k is lysine or a lysine derivative or a lysine mimetic;
b is a linking part or payload;
wherein the linker is coupled to the enrolment mab or the anti-conjugated-4 antibody via an isopeptide bond formed between the gamma-carboxamide group of a glutamine residue comprised in the enrolment mab or the anti-conjugated-4 antibody and a primary amine comprised in a side chain of a lysine residue, a lysine derivative or a lysine mimetic comprised in an RK motif comprised in the linker.
In a particular embodiment, the present invention relates to an antibody-payload conjugate comprising an enrolment monoclonal antibody or an anti-conjugated agent-4 antibody, wherein the linker is any one of the linkers shown in fig. 1, 2, 3, 8, 9, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34.
Enrolment mab is commercially available as antibody-drug conjugate enrolment mab Wei Duoting (Enfortumab vedotin) and sold under the name Padcev. Enrolment Shan Kangwei spines include the anti-conjugated-4 antibody enrolment monoclonal antibody and linker maleimidocaproyl-L-valine-L-citrulline-PAC-MMAE (mc-vc-PABC-MMAE), commonly referred to as Wei Duoting. The mc-vc-PABC-MMAE linker of enrolment Shan Kangwei statin was coupled to free cysteine residues comprised in the antibody. The antibody enrolment mab is disclosed in WO 2012/047724, which is incorporated herein by reference in its entirety.
In certain embodiments, the antibody is enrolment mab comprising a heavy chain as set forth in SEQ ID NO. 9 and a light chain as set forth in SEQ ID NO. 10. However, variants of enrolment monoclonal antibodies are also encompassed by the present invention wherein the heavy and/or light chain has 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. In particular, the antibody may comprise any of the sequence variants disclosed in WO 2012/047724. In certain embodiments, the light chain of enrolment mab may comprise a mutation in residue Q55 of SEQ ID NO. 10. In particular, the mutation is Q55N (SEQ ID NO: 11).
Preferably, the enrolment monoclonal antibody or anti-binding agent-4 antibody is present in glycosylated form in an antibody-linker conjugate. That is, the enrolment mab or anti-conjugated to-4 antibody is preferably glycosylated at residue N297 (EU numbering). However, enrolment monoclonal antibodies or anti-conjugated-4 antibodies may also be deglycosylated as described herein.
The enrolment mab or anti-conjugated-4 antibody may be conjugated to any of the linkers disclosed herein (particularly for use in the methods according to the invention). Preferably, the linker is coupled to glutamine residue Q295 (EU numbering) of the antibody in an MTG-catalyzed manner. However, the linker may also be coupled to an engineered glutamine residue, such as N297Q (EU numbering), and/or to any of the glutamine-containing tags disclosed herein.
The linker coupled to the enrolment monoclonal antibody or the anti-conjugated element-4 antibody may comprise a single linking moiety or payload B, or may comprise a plurality of linking moieties and/or payloads B 1 、B 2 Etc.
That is, in certain embodiments, the linker included in the enrolment mab-linker conjugate may include one or more linking moieties B. Such enrolment mab-linker conjugates can be subsequently functionalized with a suitable payload in a two-step process as disclosed herein.
In certain embodiments, the linker contained in the enrolment mab-payload conjugate may contain one or more payloads B. Such enrolment mab-payload conjugates may be obtained in a two-step process, wherein the linker comprising the linking moiety has been conjugated to enrolment mab in a first step and the payload has been linked to the linking moiety in a second step. Alternatively, the enrolment mab-payload conjugate may be obtained in a one-step process, wherein the linker comprising the payload is directly coupled to the enrolment mab.
In certain embodiments, the invention relates to an antibody-drug conjugate comprising an enrolment monoclonal antibody or an anti-conjugated agent-4 antibody. That is, the linker included in the antibody-drug conjugate may include one or more toxins described herein.
In certain embodiments, an antibody-drug conjugate comprising an enrolment monoclonal antibody or an anti-conjugated protein-4 antibody may comprise a linker comprising 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).
That is, in a particular embodiment, the invention relates to an antibody-drug conjugate comprising:
a) Enrolment monoclonal antibodies or anti-conjugated element-4 antibodies; and
b) A linker comprising a drug moiety B, wherein the drug moiety B is covalently linked to an amino acid sequence selected from the group consisting of: RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4) or RK-Val-Cit (SEQ ID NO: 54);
wherein the linker is coupled to the enrolment monoclonal antibody or the anti-conjugated protein-4 antibody via an isopeptide bond which is found at antibody C H The gamma-carboxamide group of glutamine residue Q295 (EU numbering) of the 2 domain forms with the primary amine contained in the side chain of the lysine residue contained in the linker.
Preferably, the antibody is enrolment mab comprising a heavy chain as set forth in SEQ ID NO. 9 and a light chain as set forth in SEQ ID NO. 10. Thus, in a specific embodiment, the present invention relates to an antibody-drug conjugate according to the present invention, wherein the IgG antibody is enrolment mab or an antibody comprising a heavy chain as shown in SEQ ID No. 9 and a light chain as shown in SEQ ID No. 10.
In certain embodiments, the linker conjugated to the enrolment mab or anti-conjugated-4 antibody may comprise the structure RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4) or RK-Val-Cit (SEQ ID NO: 54). In certain embodiments, a linker comprising the structure RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4) or RK-Val-Cit (SEQ ID NO: 54) may be coupled to residue Q295 of the enrolment monoclonal antibody or anti-conjugated agent-4 antibody via a primary amine contained in residue K.
In certain embodiments, an enrolment monoclonal antibody or an anti-conjugated protein-4 antibody may be conjugated to a linker RKAA-B as disclosed herein, wherein B is preferably a toxin. In certain embodiments, an enrolment mab or an anti-conjugated-4 antibody may be conjugated to the linker RKAA- (self-cleaving moiety) -B disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the C-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a PABC or a methylamine-containing group.
In certain embodiments, the enrolment monoclonal antibody or anti-conjugated to-4 antibody may be conjugated to the linker RKAA-PABC-B. In certain embodiments, the enrolment mab or anti-conjugated-4 antibody may be conjugated to a linker RKAA-PABC-MMAE (see fig. 1). In certain embodiments, the enrolment mab or anti-conjugated to-4 antibody may be conjugated to a linker RKAA-PABC-maytansine (see fig. 8). In certain embodiments, the enrolment monoclonal antibody or anti-conjugated to-4 antibody may be conjugated to a linker RKAA-MMAE. In certain embodiments, the enrolment monoclonal antibody or anti-conjugated to-4 antibody may be conjugated to the linker RKAA-PABC-PNP-MMAE. In certain embodiments, the enrolment monoclonal antibody or anti-conjugated protein-4 antibody may be conjugated to a linker RKAA- (NH) - (CH) 3 ) -O-B coupling, wherein O is an oxygen atom of an ether linkage and B is a hydroxyl-containing toxin. In certain embodiments, the enrolment monoclonal antibody or anti-conjugated protein-4 antibody may be conjugated to a linker RKAA- (NH) - (CH) 3 ) -O-camptothecin coupling. In certain embodiments, the enrolment monoclonal antibody or anti-conjugated protein-4 antibody may be conjugated to a linker RKAA- (NH) - (CH) 3 ) -S-B coupling, wherein S is a sulfur atom of a thioether bond and B is a thiol-containing toxin. In certain embodiments, the enrolment monoclonal antibody or anti-conjugated protein-4 antibody may be conjugated to a linker RKAA- (NH) - (CH) 3 ) -S-DM1 coupling.
In certain embodiments, an enrolment monoclonal antibody or an anti-conjugated protein-4 antibody may be conjugated to a linker B-RKAA as disclosed herein, wherein B is preferably a toxin. In certain embodiments, an enrolment mab or an anti-conjugated-4 antibody may be conjugated to a linker B- (self-cleaving moiety) -RKAA as disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the N-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a self-cleaving moiety comprising an ortho-hydroxy protected aryl sulfate (OHPAS) moiety. In certain embodiments, similar to fig. 9, the amine-containing payload B can be coupled to the N-terminal arginine moiety via a dicarboxylic acid linker.
In certain embodiments, an enrolment monoclonal antibody or an anti-conjugated protein-4 antibody may be conjugated to a linker RKA-B as disclosed herein, wherein B is preferably a toxin. In certain embodiments, an enrolment mab or an anti-conjugated-4 antibody may be conjugated to the linker RKA- (self-cleaving moiety) -B disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the C-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a PABC or a methylamine-containing group.
In certain embodiments, the enrolment monoclonal antibody or anti-conjugated to-4 antibody may be conjugated to a linker RKA-PABC-B. In certain embodiments, the enrolment mab or anti-conjugated-4 antibody may be conjugated to a linker RKA-PABC-MMAE (see fig. 2). In certain embodiments, the enrolment monoclonal antibody or anti-conjugated to-4 antibody may be conjugated to a linker RKA-PABC-maytansine. In certain embodiments, the enrolment monoclonal antibody or anti-conjugated to-4 antibody may be conjugated to a linker RKA-MMAE. In certain embodiments, the enrolment monoclonal antibody or anti-conjugated to-4 antibody may be conjugated to the linker RKA-PABC-PNP-MMAE. In certain embodiments, the enrolment monoclonal antibody or anti-conjugated protein-4 antibody may be conjugated to a linker RKA- (NH) - (CH) 3 ) -O-B coupling, wherein O is an oxygen atom of an ether linkage and B is a hydroxyl-containing toxin. In certain embodiments, the enrolment monoclonal antibody or anti-conjugated protein-4 antibody may be conjugated to a linker RKA- (NH) - (CH) 3 ) -O-camptothecin coupling. In certain embodiments, the enrolment monoclonal antibody or anti-conjugated protein-4 antibody may be conjugated to a linker RKA- (NH) - (CH) 3 ) -S-B coupling, wherein S is a sulfur atom of a thioether bond and B is a thiol-containing toxin. In certain embodiments, the enrolment monoclonal antibody or anti-conjugated protein-4 antibody may be conjugated to a linker RKA- (NH) - (CH) 3 ) -S-DM1 coupling.
In certain embodiments, an enrolment monoclonal antibody or an anti-conjugated protein-4 antibody may be conjugated to a linker B-RKA as disclosed herein, wherein B is preferably a toxin. In certain embodiments, an enrolment mab or an anti-conjugated-4 antibody may be conjugated to a linker B- (self-cleaving moiety) -RKA as disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the N-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a self-cleaving moiety comprising an ortho-hydroxy protected aryl sulfate (OHPAS) moiety. In certain embodiments, similar to fig. 9, the amine-containing payload B can be coupled to the N-terminal arginine moiety via a dicarboxylic acid linker.
In certain embodiments, an enrolment monoclonal antibody or an anti-conjugated-4 antibody may be conjugated to a linker ARK-B as disclosed herein, wherein B is preferably a toxin. In certain embodiments, an enrolment mab or an anti-conjugated-4 antibody may be conjugated to a linker, ARK- (self-cleaving moiety) -B, as disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the C-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a PABC or a methylamine-containing group.
In certain embodiments, an enrolment monoclonal antibody or an anti-conjugated-4 antibody may be conjugated to a linker ARK-PABC-B. In certain embodiments, the enrolment mab or anti-conjugated-4 antibody may be conjugated to a linker ARK-PABC-MMAE (see FIG. 3). In certain embodiments, the enrolment monoclonal antibody or anti-conjugated to-4 antibody may be conjugated to a linker ARK-PABC-maytansine. In certain embodiments, the enrolment monoclonal antibody or anti-conjugated to-4 antibody may be conjugated to a linker ARK-MMAE. In certain embodiments, the enrolment monoclonal antibody or anti-conjugated to-4 antibody may be conjugated to a linker ARK-PABC-PNP-MMAE. In certain embodiments, the enrolment monoclonal antibody or anti-conjugated protein-4 antibody may be conjugated to a linker ARK- (NH) - (CH) 3 ) -O-B coupling, wherein O is an oxygen atom of an ether linkage and B is a hydroxyl-containing toxin. In certain embodiments, the enrolment monoclonal antibody or anti-conjugated protein-4 antibody may be conjugated to a linker ARK- (NH) - (CH) 3 ) -O-camptothecin coupling. In certain embodiments, the enrolment monoclonal antibody or anti-conjugated protein-4 antibody may be conjugated to a linker ARK- (NH) - (CH) 3 ) -S-B coupling, wherein S is a thioether bondAnd B is a thiol-containing toxin. In certain embodiments, the enrolment monoclonal antibody or anti-conjugated protein-4 antibody may be conjugated to a linker ARK- (NH) - (CH) 3 ) -S-DM1 coupling.
In certain embodiments, an enrolment monoclonal antibody or an anti-conjugated-4 antibody may be conjugated to a linker B-ARK as disclosed herein, wherein B is preferably a toxin. In certain embodiments, an enrolment mab or an anti-conjugated-4 antibody may be conjugated to a linker B- (self-cleaving moiety) -ARK as disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the N-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a self-cleaving moiety comprising an ortho-hydroxy protected aryl sulfate (OHPAS) moiety.
In certain embodiments, an enrolment monoclonal antibody or an anti-conjugated protein-4 antibody may be conjugated to a linker RKR-B as disclosed herein, wherein B is preferably a toxin. In certain embodiments, an enrolment mab or an anti-conjugated-4 antibody may be conjugated to a linker RKR- (self-cleaving moiety) -B as disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the C-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a PABC or a methylamine-containing group.
In certain embodiments, an enrolment monoclonal antibody or an anti-conjugated protein-4 antibody may be conjugated to the linker RKR-PABC-B. In certain embodiments, the enrolment monoclonal antibody or anti-conjugated to-4 antibody may be conjugated to a linker RKR-PABC-MMAE. In certain embodiments, the enrolment monoclonal antibody or anti-conjugated to-4 antibody may be conjugated to a linker RKR-PABC-maytansine. In certain embodiments, an enrolment monoclonal antibody or an anti-conjugated protein-4 antibody may be conjugated to the linker RKR-MMAE. In certain embodiments, the enrolment monoclonal antibody or anti-conjugated to-4 antibody may be conjugated to a linker RKR-PABC-PNP-MMAE. In certain embodiments, the enrolment monoclonal antibody or anti-conjugated protein-4 antibody may be conjugated to a linker RKR- (NH) - (CH) 3 ) -O-B coupling, wherein O is an oxygen atom of an ether linkage and B is a hydroxyl-containing toxin. In certain embodiments, the enrolment monoclonal antibody or anti-conjugated agentThe-4 antibody may be conjugated to a linker RKR- (NH) - (CH) 3 ) -O-camptothecin coupling. In certain embodiments, the enrolment monoclonal antibody or anti-conjugated protein-4 antibody may be conjugated to a linker RKR- (NH) - (CH) 3 ) -S-B coupling, wherein S is a sulfur atom of a thioether bond and B is a thiol-containing toxin. In certain embodiments, the enrolment monoclonal antibody or anti-conjugated protein-4 antibody may be conjugated to a linker RKR- (NH) - (CH) 3 ) -S-DM1 coupling.
In certain embodiments, an enrolment monoclonal antibody or an anti-conjugated protein-4 antibody may be conjugated to a linker B-RKR as disclosed herein, wherein B is preferably a toxin. In certain embodiments, an enrolment mab or an anti-conjugated-4 antibody may be conjugated to a linker B- (self-cleaving moiety) -RKR as disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the N-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a self-cleaving moiety comprising an ortho-hydroxy protected aryl sulfate (OHPAS) moiety. In certain embodiments, as illustrated in fig. 9, the amine-containing payload B can be coupled to the N-terminal arginine moiety via a dicarboxylic acid linker.
In certain embodiments, an enrolment mab or an anti-conjugated-4 antibody may be conjugated to a linker RK-Val-Cit-B as disclosed herein, wherein B is preferably a toxin. In certain embodiments, an enrolment mab or an anti-conjugated-4 antibody may be conjugated to a linker RK-Val-Cit- (self-cleaving moiety) -B as disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the C-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a PABC or a methylamine-containing group.
In certain embodiments, an enrolment monoclonal antibody or an anti-conjugated-4 antibody may be coupled to the linker RK-Val-Cit-PABC-B. In certain embodiments, the enrolment mab or anti-conjugated to-4 antibody may be conjugated to the linker RK-Val-Cit-PABC-MMAE. In certain embodiments, the enrolment mab or anti-binding agent-4 antibody may be conjugated to the linker RK-Val-Cit-PABC-maytansine. In certain embodiments, the enrolment monoclonal antibody or anti-conjugated agent-4 antibodyThe body may be coupled to linker RK-Val-Cit-MMAE. In certain embodiments, the enrolment mab or anti-binding agent-4 antibody may be conjugated to the linker RK-Val-Cit-PABC-PNP-MMAE. In certain embodiments, the enrolment monoclonal antibody or anti-conjugated protein-4 antibody may be conjugated to a linker RK-Val-Cit- (NH) - (CH) 3 ) -O-B coupling, wherein O is an oxygen atom of an ether linkage and B is a hydroxyl-containing toxin. In certain embodiments, the enrolment monoclonal antibody or anti-conjugated protein-4 antibody may be conjugated to a linker RK-Val-Cit- (NH) - (CH) 3 ) -O-camptothecin coupling. In certain embodiments, the enrolment monoclonal antibody or anti-conjugated protein-4 antibody may be conjugated to a linker RK-Val-Cit- (NH) - (CH) 3 ) -S-B coupling, wherein S is a sulfur atom of a thioether bond and B is a thiol-containing toxin. In certain embodiments, the enrolment monoclonal antibody or anti-conjugated protein-4 antibody may be conjugated to a linker RK-Val-Cit- (NH) - (CH) 3 ) -S-DM1 coupling.
Further, the present invention relates to a linker construct comprising the motif RK. The linker construct according to the invention can be used for the coupling of a variety of antibodies. Because of the highly conserved conjugation site Q295, the linker conjugates according to the invention can be used "off the shelf" for the production of antibody-payload conjugates of essentially any IgG type antibody. The RK linkers of the present invention can be used for efficient coupling of glycosylated antibodies compared to linkers known in the art and even result in high coupling efficiency when containing large payloads such as toxins.
Thus, in a particular embodiment, the invention relates to a linker construct comprising the following structure:
(Sp 1 )-RK-(Sp 2 )-B-(Sp 3 ) Or (b)
(Sp 1 )-B-(Sp 2 )-RK-(Sp 3 ) The method comprises the steps of carrying out a first treatment on the surface of the Wherein the method comprises the steps of
·(Sp 1 ) Is a chemical spacer or is absent;
·(Sp 2 ) Is a chemical spacer or is absent;
·(Sp 3 ) Is a chemical spacer or is absent;
r is arginine or an arginine derivative or arginine mimetic;
k is lysine or a lysine derivative or a lysine mimetic;
b is the connection portion or payload.
It will be appreciated that the linker construct may have the same structure and/or properties as disclosed above for the method according to the invention, for the antibody-linker conjugate according to the invention and/or for the linker of the antibody-drug conjugate according to the invention.
In a particular embodiment, the invention relates to a linker construct according to the invention, wherein the chemical spacer (Sp 1 )、(Sp 2 ) Sum (Sp) 3 ) Each independently comprises 0 to 12 amino acid residues.
In a particular embodiment, the invention relates to a linker construct according to the invention, wherein the linker comprises no more than 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 amino acid residues.
In a particular embodiment, the invention relates to a linker construct according to the invention, wherein the net charge of the linker is neutral or positive.
In a particular embodiment, the invention relates to a linker construct according to the invention, wherein the linker does not comprise negatively charged amino acid residues.
In a particular embodiment, the invention relates to a linker construct according to the invention, wherein the linker comprises the amino acid sequence RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4) or RK-Val-Cit (SEQ ID NO: 54).
In a particular embodiment, the invention relates to a linker construct according to the invention, wherein B is a linking moiety.
In a particular embodiment, the invention relates to a linker construct according to the invention, wherein the linking moiety B comprises
Bio-orthogonal labelling group, or
Non-bioorthogonal entities for cross-linking.
In a specific embodiment, the invention relates to a construct according to the invention, wherein the bio-orthogonal labeling group or the non-bio-orthogonal entity for cross-linking consists of or comprises at least one molecule or moiety selected from the group consisting of:
-N-N≡N or-N 3 ；
·Lys(N 3 )；
Tetrazine;
alkynes;
strained cyclooctyne;
·BCN；
strained olefins;
photoreactive groups;
aldehyde;
acyl trifluoroborates;
protein degrading agent ('PROTAC');
cyclopentadiene/spirocyclopentadiene;
a thio-selective electrophile;
-SH; and
cysteine.
In a particular embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of the structure RKAA-B or B-RKAA, in particular wherein B is Lys (N 3 ) Or cysteine.
That is, in a particular embodiment, the invention relates to a linker comprising the structure RKAA-B or B-RKAA, wherein 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 can be a thiol-containing linker moiety (such as cysteine), an azide-containing linker moiety (such as Lys (N) 3 ) Or a tetrazine containing linking moiety. It is understood that linkers comprising the structure RKAA-B or B-RKAA may comprise additional amino acid residues, linking moieties, payloads and/or other chemical groups such as, but not limited to, PEG moieties. In certain embodiments, the linker construct consists of the structure RKAA-B or B-RKAA.
In a particular embodiment, the invention relates to a linker construct according to the inventionWherein the linker construct comprises or consists of the structure RKA-B or B-RKA, in particular wherein B is Lys (N 3 ) Or cysteine.
That is, in a particular embodiment, the invention relates to a linker comprising the structure RKA-B or B-RKA, wherein 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 can be a thiol-containing linker moiety (such as cysteine), an azide-containing linker moiety (such as Lys (N) 3 ) Or a tetrazine containing linking moiety. It is understood that linkers comprising the structure RKA-B or B-RKA may comprise additional amino acid residues, linking moieties, payloads and/or other chemical groups such as, but not limited to, PEG moieties. In certain embodiments, the linker construct consists of the structure RKA-B or B-RKA.
In a particular embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of the structure ARK-B or B-ARK, in particular wherein B is Lys (N 3 ) Or cysteine.
That is, in a particular embodiment, the invention relates to a linker comprising the structure ARK-B or B-ARK, wherein 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 can be a thiol-containing linker moiety (such as cysteine), an azide-containing linker moiety (such as Lys (N) 3 ) Or a tetrazine containing linking moiety. It is understood that linkers comprising the structure ARK-B or B-ARK may comprise additional amino acid residues, linking moieties, payloads and/or other chemical groups such as, but not limited to, PEG moieties. In certain embodiments, the linker construct consists of the structure ARK-B or B-ARK.
In a particular embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of the structure RKR-B or B-RKR, in particular wherein B is Lys (N 3 ) Or cysteine.
That is, in certain embodiments, the present invention relates to And a linker comprising the structure RKR-B or B-RKR, wherein 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 can be a thiol-containing linker moiety (such as cysteine), an azide-containing linker moiety (such as Lys (N) 3 ) Or a tetrazine containing linking moiety. It is understood that linkers comprising the structure RKR-B or B-RKR may comprise additional amino acid residues, linking moieties, payloads, and/or other chemical groups such as, but not limited to, PEG moieties. In certain embodiments, the linker construct consists of the structure RKR-B or B-RKR.
In a particular embodiment, the invention relates to a linker construct according to the invention, wherein B is a payload.
In a particular embodiment, the invention relates to a joint construct according to the invention, wherein the payload comprises at least one of the following:
toxins;
cytokines;
growth factors;
radionuclides;
hormones;
antiviral agents;
an antimicrobial agent;
fluorescent dye;
immunomodulators/immunostimulants;
half-life increasing moiety;
a solubility-increasing moiety;
Polymer-toxin conjugate;
nucleic acid;
biotin or streptavidin moiety;
vitamins;
protein degrading agent ('PROTAC');
a target binding moiety; and/or
Anti-inflammatory agents.
In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the toxin is selected from at least one of the group consisting of:
pyrrolobenzodiazepines (e.g., PBDs);
auristatin (e.g., MMAE, MMAF);
maytansinoids (e.g., maytansine, DM1, DM4, DM 21);
sesquicomycin;
nicotinamide phosphoribosyl transferase (NAMPT) inhibitors;
microtubule lysin;
enediyne (e.g., calicheamicin);
anthracycline derivatives (PNUs) (e.g., doxorubicin);
inhibitors of the pyrrolyl Kinesin Spindle Protein (KSP);
candidiasis;
drug efflux pump inhibitors;
mountain Zhuo Meisu;
amanitine (e.g., α -amanitine); and
camptothecins (e.g., irinotecan, delutinacon).
In a particular embodiment, the invention relates to a linker construct according to the invention, wherein the chemical spacer (Sp 2 ) Including self-cleaving moieties.
In a particular embodiment, the invention relates to a linker construct according to the invention, wherein the self-cleaving moiety is directly attached to the payload B.
In a particular embodiment, the invention relates to a linker construct according to the invention, wherein the self-cleaving moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety.
In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of the structure RKAA- (self-cleaving moiety) -B. That is, the linker construct may include any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling payload B to the C-terminus of a peptide. In certain embodiments, payload B may be a toxin. Those skilled in the art are aware of toxins that can be coupled to the C-terminus of a peptide via a suitable self-cleaving moiety. Preferably, the self-cleaving moiety is any of the self-cleaving moieties disclosed herein, such as, but not limited to, a PABC-based self-cleaving linker, a PABE-based self-cleaving linker, or a methylamine-containing self-cleaving moiety.
In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of structure B- (self-cleaving moiety) -RKAA. That is, the linker construct may comprise any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling payload B to the N-terminus of a peptide. In certain embodiments, payload B may be a toxin. Those skilled in the art are aware of toxins that can be coupled to the N-terminus of a peptide via a suitable self-cleaving moiety. Preferably, the self-cleaving moiety is any of the self-cleaving moieties disclosed herein, such as but not limited to a self-cleaving moiety comprising an OHPAS moiety.
In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of the structure B-RKAA or RKAA-B. That is, payload B may be coupled directly to the N-terminus or C-terminus of the peptide. In the event that the functional groups contained in the payload are not compatible with the N-terminus and/or the C-terminus of the peptide, the payload may be coupled to the N-terminus and/or the C-terminus of the peptide, respectively, using a linker molecule. Disclosed herein are suitable linker molecules for coupling a payload to the N-terminus or C-terminus of a peptide.
In a particular embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of the structure RKAA-PABC-B, in particular wherein B is auristatin or a maytansinoid, in particular wherein auristatin is MMAE and wherein maytansinoid is maytansinoid.
That is, in a particular embodiment, the present invention relates to a linker comprising the structure RKAA-B, wherein B is a payload. It should be appreciated that B may be any payload known in the art and/or disclosed herein. In some embodiments of the present invention, in some embodiments,b may be a toxin. In certain embodiments, payload B may be separated from peptide RKAA by a self-cleaving moiety. Thus, the linker may comprise or consist of the structure RKAA- (self-cleaving moiety) -B, wherein B is a payload. In certain embodiments, the self-cleaving moiety may be a PABC or methylamino. Thus, in certain embodiments, the linker may comprise or consist of the structure RKAA-PABC-B. In certain embodiments, the linker may include the structures RKAA- (NH) - (CH) 3 ) -O-B or consisting of, wherein O is an oxygen atom contained in an ether linkage and B is a payload containing a hydroxyl group. In certain embodiments, the linker may comprise the structures RKAA- (NH) - (CH) 3 ) -S-B or consisting of, wherein S is a sulfur atom contained in a thioether bond and B is a payload comprising a thiol. In certain embodiments, the payload may be an auristatin or maytansinoid. In certain embodiments, the auristatin may be MMAE. In such embodiments, the linker may comprise or consist of the structures RKAA-PABC-MMAE or RKAA-MMAE. In certain embodiments, the maytansinoid may be maytansinoid. In such embodiments, the linker may comprise or consist of the structure RKAA-PABC-maytansine or RKAA-maytansine. In certain embodiments, the maytansinoid may be DM1 or a DM1 derivative. In such embodiments, the linker may include the structures RKAA- (NH) - (CH) 3 ) -S-DM1 or consists thereof. In certain embodiments, the payload may be a camptothecin. In such embodiments, the linker may include the structures RKAA- (NH) - (CH) 3 ) -O-camptothecine or consist thereof.
In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of the structure RKA- (self-cleaving moiety) -B. That is, the linker construct may include any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling payload B to the C-terminus of a peptide. In certain embodiments, payload B may be a toxin. Those skilled in the art are aware of toxins that can be coupled to the C-terminus of a peptide via a suitable self-cleaving moiety. Preferably, the self-cleaving moiety is any of the self-cleaving moieties disclosed herein, such as, but not limited to, a PABC-based self-cleaving linker, a PABE-based self-cleaving linker, or a methylamine-containing self-cleaving moiety.
In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of structure B- (self-cleaving moiety) -RKA. That is, the linker construct may comprise any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling payload B to the N-terminus of a peptide. In certain embodiments, payload B may be a toxin. Those skilled in the art are aware of toxins that can be coupled to the N-terminus of a peptide via a suitable self-cleaving moiety. Preferably, the self-cleaving moiety is any of the self-cleaving moieties disclosed herein, such as but not limited to a self-cleaving moiety comprising an OHPAS moiety.
In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of the structure B-RKA or RKA-B. That is, payload B may be coupled directly to the N-terminus or C-terminus of the peptide. In the event that the functional groups contained in the payload are not compatible with the N-terminus and/or the C-terminus of the peptide, the payload may be coupled to the N-terminus and/or the C-terminus of the peptide, respectively, using a linker molecule. Disclosed herein are suitable linker molecules for coupling a payload to the N-terminus or C-terminus of a peptide.
In a particular embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of the structure RKA-PABC-B, in particular wherein B is an auristatin or a maytansinoid, in particular wherein auristatin is MMAE, and wherein maytansinoid is maytansinoid.
That is, in a particular embodiment, the present invention relates to a linker comprising the structure RKA-B, wherein B is a payload. It should be appreciated that B may be any payload known in the art and/or disclosed herein. In certain embodiments, B can be a toxin. In certain embodiments, payload B may be separated from peptide RKA by a self-cleaving moiety. Thus, the linker may comprise the structure RKA- (self-cleaving)Solution part) -B or consist of it, wherein B is the payload. In certain embodiments, the self-cleaving moiety may be a PABC or methylamino. Thus, in certain embodiments, the linker may comprise or consist of the structure RKA-PABC-B. In certain embodiments, the linker may include the structures RKA- (NH) - (CH) 3 ) -O-B or consisting of, wherein O is an oxygen atom contained in an ether linkage and B is a payload containing a hydroxyl group. In certain embodiments, the linker may comprise the structures RKA- (NH) - (CH) 3 ) -S-B or consisting of, wherein S is a sulfur atom contained in a thioether bond and B is a payload comprising a thiol. In certain embodiments, the payload may be an auristatin or maytansinoid. In certain embodiments, the auristatin may be MMAE. In such embodiments, the linker may comprise or consist of the structures RKA-PABC-MMAE or RKA-MMAE. In certain embodiments, the maytansinoid may be maytansinoid. In such embodiments, the linker may comprise or consist of the structure RKA-PABC-maytansine or RKA-maytansine. In certain embodiments, the maytansinoid may be DM1 or a DM1 derivative. In such embodiments, the linker may comprise the structures RKA- (NH) - (CH) 3 ) -S-DM1 or consists thereof. In certain embodiments, the payload may be a camptothecin. In such embodiments, the linker may comprise the structures RKA- (NH) - (CH) 3 ) -O-camptothecine or consist thereof.
In a particular embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of the structure ARK- (self-cleaving moiety) -B. That is, the linker construct may include any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling payload B to the C-terminus of a peptide. In certain embodiments, payload B may be a toxin. Those skilled in the art are aware of toxins that can be coupled to the C-terminus of a peptide via a suitable self-cleaving moiety. Preferably, the self-cleaving moiety is any of the self-cleaving moieties disclosed herein, such as, but not limited to, a PABC-based self-cleaving linker, a PABE-based self-cleaving linker, or a methylamine-containing self-cleaving moiety.
In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of structure B- (self-cleaving moiety) -ARK. That is, the linker construct may comprise any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling payload B to the N-terminus of a peptide. In certain embodiments, payload B may be a toxin. Those skilled in the art are aware of toxins that can be coupled to the N-terminus of a peptide via a suitable self-cleaving moiety. Preferably, the self-cleaving moiety is any of the self-cleaving moieties disclosed herein, such as but not limited to a self-cleaving moiety comprising an OHPAS moiety.
In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of the structure B-ARK or ARK-B. That is, payload B may be coupled directly to the N-terminus or C-terminus of the peptide. In the event that the functional groups contained in the payload are not compatible with the N-terminus and/or the C-terminus of the peptide, the payload may be coupled to the N-terminus and/or the C-terminus of the peptide, respectively, using a linker molecule. Disclosed herein are suitable linker molecules for coupling a payload to the N-terminus or C-terminus of a peptide.
In a particular embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of the structure ARK-PABC-B, in particular wherein B is auristatin or a maytansinoid, in particular wherein auristatin is MMAE and wherein maytansinoid is maytansinoid.
That is, in certain embodiments, the invention relates to a linker comprising the structure ARK-B, wherein B is a payload. It should be appreciated that B may be any payload known in the art and/or disclosed herein. In certain embodiments, B can be a toxin. In certain embodiments, payload B may be separated from peptide ARK by a self-cleaving moiety. Thus, the linker may comprise or consist of the structure ARK- (self-cleaving moiety) -B, wherein B is a payload. In certain embodiments, the self-cleaving moiety may be a PABC or methylamino. Thus, in certain embodimentsThe linker may comprise or consist of the structure ARK-PABC-B. In certain embodiments, the linker may include the structures ARK- (NH) - (CH) 3 ) -O-B or consisting of, wherein O is an oxygen atom contained in an ether linkage and B is a payload containing a hydroxyl group. In certain embodiments, the linker may comprise the structures ARK- (NH) - (CH) 3 ) -S-B or consisting of, wherein S is a sulfur atom contained in a thioether bond and B is a payload comprising a thiol. In certain embodiments, the payload may be an auristatin or maytansinoid. In certain embodiments, the auristatin may be MMAE. In such embodiments, the linker may comprise or consist of the structure ARK-PABC-MMAE or ARK-MMAE. In certain embodiments, the maytansinoid may be maytansinoid. In such embodiments, the linker may comprise or consist of the structure ARK-PABC-maytansine or ARK-maytansine. In certain embodiments, the maytansinoid may be DM1 or a DM1 derivative. In such embodiments, the linker may comprise the structures ARK- (NH) - (CH) 3 ) -S-DM1 or consists thereof. In certain embodiments, the payload may be a camptothecin. In such embodiments, the linker may comprise the structures ARK- (NH) - (CH) 3 ) -O-camptothecine or consist thereof.
In a particular embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct consists of or comprises the structure RKR- (self-cleaving moiety) -B. That is, the linker construct may include any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling payload B to the C-terminus of a peptide. In certain embodiments, payload B may be a toxin. Those skilled in the art are aware of toxins that can be coupled to the C-terminus of a peptide via a suitable self-cleaving moiety. Preferably, the self-cleaving moiety is any of the self-cleaving moieties disclosed herein, such as, but not limited to, a PABC-based self-cleaving linker, a PABE-based self-cleaving linker, or a methylamine-containing self-cleaving moiety.
In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of structure B- (self-cleaving moiety) -RKR. That is, the linker construct may comprise any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling payload B to the N-terminus of a peptide. In certain embodiments, payload B may be a toxin. Those skilled in the art are aware of toxins that can be coupled to the N-terminus of a peptide via a suitable self-cleaving moiety. Preferably, the self-cleaving moiety is any of the self-cleaving moieties disclosed herein, such as but not limited to a self-cleaving moiety comprising an OHPAS moiety.
In a particular embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct consists of or comprises the structure B-RKR or RKR-B. That is, payload B may be coupled directly to the N-terminus or C-terminus of the peptide. In the event that the functional groups contained in the payload are not compatible with the N-terminus and/or the C-terminus of the peptide, the payload may be coupled to the N-terminus and/or the C-terminus of the peptide, respectively, using a linker molecule. Disclosed herein are suitable linker molecules for coupling a payload to the N-terminus or C-terminus of a peptide.
In a particular embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct consists of or comprises the structure RKR-PABC-B, in particular wherein B is auristatin or maytansinoid, in particular wherein auristatin is MMAE, and wherein maytansinoid is maytansinoid.
That is, in a particular embodiment, the present invention relates to a linker comprising the structure RKR-B, wherein B is a payload. It should be appreciated that B may be any payload known in the art and/or disclosed herein. In certain embodiments, B can be a toxin. In certain embodiments, payload B may be separated from peptide RKR by a self-cleaving moiety. Thus, the linker may comprise or consist of the structure RKR- (self-cleaving moiety) -B, wherein B is a payload. In certain embodiments, the self-cleaving moiety may be a PABC or methylamino. Thus, in certain embodiments, the linker may comprise or consist of the structure RKR-PABC-B. In some embodiments, the joint may include a structureRKR-(NH)-(CH 3 ) -O-B or consisting of, wherein O is an oxygen atom contained in an ether linkage and B is a payload containing a hydroxyl group. In certain embodiments, the linker may comprise the structures RKR- (NH) - (CH) 3 ) -S-B or consisting of, wherein S is a sulfur atom contained in a thioether bond and B is a payload comprising a thiol. In certain embodiments, the payload may be an auristatin or maytansinoid. In certain embodiments, the auristatin may be MMAE. In such embodiments, the linker may comprise or consist of the structures RKR-PABC-MMAE or RKR-MMAE. In certain embodiments, the maytansinoid may be maytansinoid. In such embodiments, the linker may comprise or consist of the structure RKR-PABC-maytansine or RKR-maytansine. In certain embodiments, the maytansinoid may be DM1 or a DM1 derivative. In such embodiments, the linker may include the structures RKR- (NH) - (CH) 3 ) -S-DM1 or consists thereof. In certain embodiments, the payload may be a camptothecin. In such embodiments, the linker may include the structures RKR- (NH) - (CH) 3 ) -O-camptothecine or consist thereof.
In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct consists of or comprises the structure RK-Val-Cit- (self-cleaving moiety) -B. That is, the linker construct may include any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling payload B to the C-terminus of a peptide. In certain embodiments, payload B may be a toxin. Those skilled in the art are aware of toxins that can be coupled to the C-terminus of a peptide via a suitable self-cleaving moiety. Preferably, the self-cleaving moiety is any of the self-cleaving moieties disclosed herein, such as, but not limited to, a PABC-based self-cleaving linker, a PABE-based self-cleaving linker, or a methylamine-containing self-cleaving moiety.
In a particular embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct consists of or comprises the structure RK-Val-Cit-B. That is, payload B may be coupled directly to the C-terminus of the peptide. In the event that the functional group contained in the payload is not compatible with the C-terminus of the peptide, the linker molecule may be used to couple the payload to the C-terminus of the peptide. Disclosed herein are suitable linker molecules for coupling a payload to the C-terminus of a peptide.
In a particular embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct consists of or comprises the structure RK-Val-Cit-PABC-B, in particular wherein B is auristatin or maytansinoid, in particular wherein auristatin is MMAE, and wherein maytansinoid is maytansinoid.
That is, in a particular embodiment, the present invention relates to a linker comprising the structure RK-Val-Cit-B, wherein B is a payload. It should be appreciated that B may be any payload known in the art and/or disclosed herein. In certain embodiments, B can be a toxin. In certain embodiments, payload B may be separated from peptide RK-Val-Cit by a self-cleaving moiety. Thus, the linker may comprise or consist of the structure RK-Val-Cit- (self-cleaving moiety) -B, where B is a payload. In certain embodiments, the self-cleaving moiety may be a PABC or methylamino. Thus, in certain embodiments, the linker may comprise or consist of the structure RK-Val-Cit-PABC-B. In certain embodiments, the linker may include the structure RK-Val-Cit- (NH) - (CH) 3 ) -O-B or consisting of, wherein O is an oxygen atom contained in an ether linkage and B is a payload containing a hydroxyl group. In certain embodiments, the linker may comprise the structure RK-Val-Cit- (NH) - (CH) 3 ) -S-B or consisting of, wherein S is a sulfur atom contained in a thioether bond and B is a payload comprising a thiol. In certain embodiments, the payload may be an auristatin or maytansinoid. In certain embodiments, the auristatin may be MMAE. In such embodiments, the linker may comprise or consist of the structure RK-Val-Cit-PABC-MMAE or RK-Val-Cit-MMAE. In certain embodiments, the maytansinoid may be maytansinoid. In such embodiments, the linker may comprise or consist of the structure RK-Val-Cit-PABC-maytansine or RK-Val-Cit-maytansine. In certain embodiments, the maytansinoid may be DM1 or a DM1 derivative. In such embodiments, the linker may include the structure RK-Val-Cit- (NH) - (CH) 3 ) -S-DM1 or consists thereof. In certain embodiments, the payload may be a camptothecin. In such embodiments, the linker may include the structure RK-Val-Cit- (NH) - (CH) 3 ) -O-camptothecine or consist thereof.
In a specific embodiment, the invention relates to an antibody-linker conjugate or an antibody-drug conjugate according to the invention, wherein the antibody-linker conjugate or the antibody-drug conjugate comprises at least one toxin.
That is, an antibody-linker conjugate or antibody-drug conjugate according to the invention comprises an antibody conjugated to at least one linker, wherein one linker comprises at least one toxin. In certain embodiments, the antibody-linker conjugate or antibody-drug conjugate comprises two linkers, wherein each heavy chain of the antibody is conjugated to one linker. In certain embodiments, the antibody-linker conjugate or antibody-drug conjugate comprises four linkers, wherein each heavy chain of the antibody is conjugated to two linkers. In this case, each linker may contain one or more payloads, such as toxins.
In certain embodiments, an antibody-linker conjugate or antibody-drug conjugate according to the invention comprises two linkers, wherein each linker comprises one payload, such as a toxin. In other embodiments, an antibody-linker conjugate or antibody-drug conjugate according to the invention comprises two linkers, wherein each linker comprises two payloads, e.g., one toxin and one other payload or two identical or different toxins. In embodiments where the antibody-linker conjugate or antibody-drug conjugate comprises two linkers, it is preferred that the linkers are conjugated to residues Q295 of both heavy chains of the IgG antibody. Even more preferably, the antibody is an IgG antibody glycosylated at residue N297.
In certain embodiments, an antibody-linker conjugate or antibody-drug conjugate according to the invention comprises four linkers, wherein each linker comprises one payload, such as a toxin. In other embodiments, an antibody-linker conjugate or antibody-drug conjugate according to the invention comprises four linkers, wherein each linker comprises two payloads, e.g., one toxin and one other payload or two identical or different toxins. In embodiments where the antibody-linker conjugate or antibody-drug conjugate comprises four linkers, it is preferred that the linkers are conjugated to residues Q295 and N297Q of the two heavy chains of the IgG antibody.
In a specific embodiment, the invention relates to an antibody-linker conjugate or an antibody-drug conjugate according to the invention, wherein the antibody-linker conjugate or the antibody-drug conjugate comprises two different toxins.
In certain embodiments, an antibody-linker conjugate or antibody-drug conjugate according to the invention may comprise two different toxins. That is, in certain embodiments, an antibody-linker conjugate or antibody-drug conjugate may comprise two linkers, wherein each linker comprises two different toxins. An advantage of antibody-linker conjugates or antibody-drug conjugates comprising two different toxins is that they can have increased cytotoxic activity. This increased cytotoxic activity can be achieved by binding two toxins that target two different cellular mechanisms. For example, an antibody-linker conjugate or antibody-drug conjugate according to the invention may comprise a first toxin that inhibits cell division, and the second toxin is a toxin that interferes with replication and/or transcription of DNA.
Thus, in a specific embodiment, the invention relates to an antibody-linker conjugate or an antibody-drug conjugate according to the invention, wherein the first toxin is a toxin that inhibits cell division and the second toxin is a toxin that interferes with replication and/or transcription of DNA.
Toxins that inhibit cell division (such as antimitotics or spindle toxins) are agents that potentially inhibit or prevent cell mitosis. Spindle toxins are toxins that disrupt cell division by affecting the protein lines (called spindles) that connect the chromosomal centromere regions. Spindle toxins effectively stop the production of new cells by interrupting the mitotic phase of cell division at the Spindle Assembly Checkpoint (SAC). The mitotic spindle consists of microtubules (polymeric tubulin) that together with regulatory proteins assist in replicating the activity of chromosomes appropriately separated from each other. Certain compounds affecting the mitotic spindle have proven to be very effective against solid tumors and hematological malignancies.
Two specific families of antimitotic agents, vinca alkaloids and taxanes, interrupt cell division by agitation of microtubule dynamics. Vinca alkaloids act by causing inhibition of tubulin polymerization into microtubules, leading to G2/M arrest during the cell cycle and ultimately cell death. In contrast, taxanes prevent the mitotic cell cycle by stabilizing microtubules against depolymerization. Although there are many other spindle proteins that may be targets for novel chemotherapeutics, tubulin binding agents are the only type used clinically. Agents that affect kinesin begin to enter clinical trials. Another type of paclitaxel works through tubulin attached within existing microtubules. Preferred toxins that inhibit cell division in the present invention are auristatins (such as MMAE and MMAF) and maytansinoids (such as DM1, DM3, DM4 and/or DM 21).
In a specific embodiment, the invention relates to an antibody-linker conjugate or an antibody-drug conjugate according to the invention, wherein the at least one toxin is auristatin or maytansinoid.
Several agents that prevent the correct replication and/or transcription of DNA molecules and that have been shown to be suitable for use in cancer therapy are known to those skilled in the art. For example, antimetabolites (such as nucleotides or nucleoside analogs) that are falsely incorporated into newly formed DNA and/or RNA molecules are known in the art and have been summarized by Tsesmetzis et al Cancers (Basel), 2018, 10 (7): 240. Other toxins known to interfere with DNA replication and/or transcription are doxycycline (duomycin).
Thus, in certain embodiments, an antibody-linker conjugate or antibody-drug conjugate according to the invention comprises two different toxins, wherein the first toxin is doxycycline and wherein the second payload is auristatin or maytansinoid.
In certain embodiments, the invention relates to an antibody-linker conjugate or an antibody-drug conjugate according to the invention, wherein the antibody-linker conjugate comprises two different auristatins.
One major advantage of an antibody-linker conjugate or antibody-drug conjugate comprising two different toxins is that the antibody-linker conjugate or antibody-drug conjugate may still act on target cells that have escaped the mechanism of action of one of the toxins and/or the antibody-payload conjugate may have a higher potency against a heterogeneous tumor.
In a specific embodiment, the invention relates to an antibody-linker conjugate or an antibody-drug conjugate according to the invention, wherein the antibody-linker conjugate comprises a toxin and an inhibitor of a drug efflux transporter.
In a particular embodiment, the invention relates to an antibody-linker conjugate or an antibody-drug conjugate according to the invention, wherein the antibody-linker conjugate or the antibody-drug conjugate comprises a toxin and a solubility-increasing moiety.
That is, the antibody-linker conjugate or antibody-drug conjugate may include two payloads, wherein the first payload is a toxin and the second payload is a solubility-enhancing moiety. Alternatively, the antibody-linker conjugate or antibody-drug conjugate may be obtained by clicking the toxin to the azide-containing linking moiety of the linker and by clicking the solubility-increasing moiety comprising maleimide to the cysteine side chain of the same linker. Alternatively, the toxin and/or solubility-increasing moiety may be attached to the linker by chemical synthesis.
In a specific embodiment, the invention relates to an antibody-linker conjugate or an antibody-drug conjugate according to the invention, wherein the antibody-linker conjugate comprises a toxin and an immunostimulant.
As used herein and depending on the context, the term "immunostimulant" includes compounds that increase the immune response of a subject to an antigen. Examples of immunostimulants include immunostimulants and immune cell activating compounds. The antibody-linker conjugates of the invention may comprise an immunostimulant that aids in programming immune cells to recognize the ligand and enhance antigen presentation. Immune cell activating compounds include Toll-like receptor (TLR) agonists. Such agonists include pathogen-associated molecular patterns (PAMPs), e.g., compositions that mimic infection, such as bacterial-derived immunomodulators (also known as danger signals), and damage-associated molecular patterns (DAMP), e.g., compositions that mimic stress or damage cells. TLR agonists include nucleic acids or lipid compositions (e.g., monophosphoryl lipid a (MPLA)). In one example, the TLR agonist comprises a TLR9 agonist, such as a cytosine-guanosine oligonucleotide (CpG-ODN), a poly (ethyleneimine) (PEI) -condensed Oligonucleotide (ODN) (such as PEI-CpG-ODN), or a double-stranded deoxyribonucleic acid (DNA). In another example, TLR agonists include TLR3 agonists such as poly inosine-polycytidylic acid (poly (I: C)), PEI-poly (I: C), poly adenylate-poly (a: U)), PEI-poly (a: U), or double stranded ribonucleic acid (RNA). Other exemplary vaccine immunostimulatory compounds include Lipopolysaccharide (LPS), chemokines/cytokines, fungal beta-glucans (such as lentinan), imiquimod, CRX-527, and OM-174.
In a specific embodiment, the invention relates to an antibody-linker conjugate or an antibody-drug conjugate according to the invention, wherein the antibody-linker conjugate or the antibody-drug conjugate comprises two different immunostimulants.
In a particular embodiment, the invention relates to an antibody-linker conjugate or an antibody-drug conjugate according to the invention, wherein the at least one immunostimulatory agent is a TLR agonist.
As used herein, the term "TLR agonist" refers to a molecule capable of eliciting a signaling response through a TLR signaling pathway (either as a direct ligand or indirectly through the production of endogenous or exogenous sources). Agonist ligands for TLR receptors are (i) natural ligands for the actual TLR receptor or functionally equivalent variants thereof that retain the ability to bind to TLR receptors and induce co-stimulatory signaling thereon; or (ii) an agonist antibody, or a functionally equivalent variant thereof, directed against a TLR receptor, which is capable of specifically binding to the TLR receptor, more specifically to the extracellular domain of said receptor, and inducing some immune signals controlled by the receptor and related proteins. The binding specificity may be for a human TLR receptor or for a TLR receptor homologous to a human TLR of a different species.
In certain embodiments, an antibody-linker conjugate according to the invention may comprise one or more imaging agents. Thus, in a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the antibody-linker conjugate comprises a radionuclide and a fluorescent dye.
In a particular embodiment, the invention relates to an antibody-conjugate according to the invention, wherein the radionuclide is a radionuclide suitable for tomography, in particular Single Photon Emission Computed Tomography (SPECT) or Positron Emission Tomography (PET), and wherein the fluorescent dye is a near infrared fluorescent dye.
The term "radionuclide" as used herein has the same meaning as the radionuclide (radioactive nuclide), radioisotope (radioisoppe) or radioisotope (radioactive isotope) of radioactivity.
Radionuclides are preferably detectable by nuclear medicine molecular imaging techniques such as Positron Emission Tomography (PET), single Photon Emission Computed Tomography (SPECT), a mixture of SPECT and/or PET, or combinations thereof. Single Photon Emission Computed Tomography (SPECT) herein includes Planar Scintigraphy (PS).
The mixture of SPECT and/or PET is, for example, SPECT/CT, PET/IRM or SPECT/IRM.
SPECT and PET acquire information about the concentration (or uptake) of the radionuclide introduced into the subject. PET generates images by detecting pairs of gamma rays emitted indirectly by positron emitting radionuclides. PET analysis produces a series of thin slice images of the body over a region of interest (e.g., brain, breast, liver, etc.). These thin slice images may be assembled into a three-dimensional representation of the inspected area. SPECT is similar to PET, but the radioactive materials used in SPECT have longer decay times than the materials used in PET and emit single gamma rays instead of double gamma rays. Although SPECT images exhibit lower sensitivity and less detail than PET images, SPECT techniques are much cheaper than PET and offer the advantage of not requiring access to a particle accelerator. Practical clinical PET exhibits higher sensitivity and better spatial resolution than SPECT, and exhibits the advantage of accurate attenuation correction due to the high energy of photons; PET thus provides more accurate quantitative data than SPECT. Planar Scintigraphy (PS) is similar to SPECT in that it uses the same radionuclide. However, PS generates only 2D information.
SPECT produces computer-generated images of local radiotracer uptake, while CT produces 3D anatomical images of the X-ray density of the human body. The combined SPECT/CT imaging in turn provides functional information from SPECT and anatomical information from CT, which are obtained during a single examination. CT data is also used for fast and optimal attenuation correction of single photon emission data. SPECT/CT improves sensitivity and specificity by precisely locating areas of abnormality and/or physiologic tracer uptake, but also helps to achieve accurate dosimetry estimates as well as guide interventional procedures or better define the target volume for external beam radiation therapy. Gamma camera imaging using single photon emission radioactive tracers represents most surgical procedures.
The radionuclide can be technetium-99 m% 99m Tc, gallium-67% 67 Ga), ga-68% 68 Ga), yttrium-90% 90 Y), indium-111% 111 In), rhenium-186% 186 Re, fluorine-18% 18 F) Copper-64% 64 Cu, terbium-149% 149 Tb) or thallium-201% 201 TI). The radionuclide may be contained in the molecule or bound to a chelator.
In a particular embodiment, the invention relates to the use of a linker construct according to the invention for the production of an antibody-linker conjugate by a microbial transglutaminase.
That is, the linker constructs described above may be used to produce the antibody-linker conjugates described herein. Preferably, the antibody is an IgG antibody comprising endogenous glutamine residue Q295 (EU numbering). In certain embodiments, the linker according to the invention is used to produce an antibody-linker conjugate by applying any of the reaction conditions disclosed herein.
Thus, in a specific embodiment, the invention relates to a use according to the invention, wherein the antibody is an IgG antibody, in particular an IgG1 antibody.
In a more preferred embodiment, the invention relates to a use according to the invention, wherein the antibody is either polotophyllizumab or trastuzumab or enrolment mab.
Further, the present invention relates to a pharmaceutical composition comprising an antibody-linker conjugate or an antibody-drug conjugate according to the invention.
Thus, in a particular embodiment, the present invention relates to a pharmaceutical composition comprising:
a) The antibody-linker conjugate according to the invention, in particular wherein the antibody-linker conjugate comprises at least one payload;
or (b)
b) Antibody drug conjugates according to the invention; and is also provided with
The pharmaceutical composition comprises at least one pharmaceutically acceptable ingredient.
It is understood that a pharmaceutical composition may comprise an antibody-payload conjugate that has been prepared using the one-step or two-step methods disclosed herein.
The type of payload contained in the antibody-payload construct contained in the pharmaceutical composition depends on the use of the pharmaceutical composition. In embodiments where the pharmaceutical composition is used to treat a disease, the payload is preferably a drug. If the disease is a neoplastic disease, the payload is preferably a toxin. In embodiments where the pharmaceutical composition is for diagnostic purposes, the payload is preferably an imaging agent.
Alternatively, the drug may comprise an antibody-drug conjugate as disclosed herein. Pharmaceutical compositions comprising antibody-drug conjugates are preferred for the treatment of diseases.
In a particular embodiment, the present invention relates to a pharmaceutical composition according to the present invention comprising at least one additional therapeutically active agent.
The pharmaceutical composition according to the present invention may comprise at least one pharmaceutically acceptable ingredient.
Pharmaceutically acceptable ingredients refer to ingredients of the pharmaceutical formulation that are non-toxic to the subject, in addition to the active ingredient. Pharmaceutically acceptable ingredients include, but are not limited to, buffers, excipients, stabilizers, or preservatives.
The pharmaceutical formulations of the antibody-linker conjugates described herein are prepared by mixing the conjugate with the desired purity with one or more optional pharmaceutically acceptable ingredients (Flemington's Pharmaceutical Sciences 16th edition,Oslo,A.Ed. (1980)) in the form of a lyophilized formulation or an aqueous solution. At the dosages and concentrations employed, the pharmaceutically acceptable ingredients are generally non-toxic to the recipient and include, but are not limited to: buffers (such as phosphates, citrates and other organic acids); antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethylammonium chloride, benzalkonium chloride, phenol, butanol or benzyl alcohol, alkyl p-hydroxybenzoates, such as p-hydroxybenzoate or p-hydroxyphenylpropyl ester, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); a low molecular weight (less than about 10 residues) polypeptide; proteins (such as serum albumin, gelatin, or immunoglobulins); hydrophilic polymers (such as polyvinylpyrrolidone); amino acids (such as glycine, glutamine, asparagine, histidine, arginine, or lysine); monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents (such as EDTA); sugar (such as sucrose, mannitol, trehalose, or sorbitol); salt-forming counterions (such as sodium); metal complexes (such as Zn protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable ingredients herein also include in vivo (installment) pharmaceutical dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronan glycoprotein, such as rHuPH20 @, for example Baxter International, inc.). Certain exemplary shasegps and methods of use, including rHuPH20, are described in U.S. patent publication nos. 2005/026086 and 2006/0104968. For example, sHASEGP can be combined with one or more additional glycosaminoglycanases (such as a chondroitinase).
In a particular embodiment, the invention relates to an antibody-linker conjugate according to the invention, in particular wherein the antibody-linker conjugate comprises at least one payload, an antibody-drug conjugate according to the invention or a pharmaceutical composition according to the invention for use in therapy and/or diagnosis.
That is, the antibody-linker conjugates, antibody-drug conjugates or pharmaceutical compositions according to the invention may be used to treat a subject or to diagnose a disease or condition in a subject. The individual or subject is preferably a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates (such as macaques)), rabbits, and rodents (such as mice and rats). In certain embodiments, the individual or subject is a human. When the antibody-linker conjugate or the pharmaceutical composition comprising the antibody-linker conjugate according to the invention is used in therapy, it is preferred that the linker comprises a drug. When the antibody-linker conjugate or the pharmaceutical composition comprising the antibody-linker conjugate according to the invention is used for diagnosis, it is preferred that the linker comprises at least one imaging agent.
In a particular embodiment, the invention relates to an antibody-linker conjugate according to the invention, in particular wherein the antibody-linker conjugate comprises at least one payload, an antibody-drug conjugate according to the invention, or a pharmaceutical composition according to the invention, for use in therapy
● Patients suffering from neoplastic diseases, neurological diseases, autoimmune diseases, inflammatory diseases or infectious diseases,
● Patients at risk of developing neoplastic, neurological, autoimmune, inflammatory or infectious diseases, and/or
● Patients diagnosed with neoplastic disease, neurological disease, autoimmune disease, inflammatory disease or infectious disease.
In a particular embodiment, the invention relates to an antibody-linker conjugate according to the invention, in particular wherein the antibody-linker conjugate comprises at least one payload, an antibody-drug conjugate according to the invention or a pharmaceutical composition according to the invention for use in the treatment of a patient suffering from a neoplastic disease.
The term "neoplastic disease" as used herein refers to a disease characterized by uncontrolled abnormal growth of cells. Neoplastic diseases include cancer. Examples of cancers include, but are not limited to, malignant epithelial tumors (carbioma), lymphomas, sarcomas, and leukemias. More specific examples of such cancers include breast cancer, prostate cancer, colon cancer, squamous cell carcinoma, small-cell lung cancer, non-small cell lung cancer, ovarian cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, liver cancer (liver cancer), bladder cancer, hepatoma (hepatoma), colorectal cancer, cervical cancer, endometrial cancer, salivary gland cancer, kidney cancer, vulval cancer, thyroid cancer, primary liver cancer (hepatic carcinoma), skin cancer, melanoma, brain cancer, ovarian cancer, neuroblastoma, myeloma, various head and neck cancers, acute lymphoblastic leukemia, acute myelogenous leukemia, ewing's sarcoma (Ewing's sarcoma), and peripheral nerve epithelial tumors. Preferred cancers include liver cancer, lymphoma, acute lymphoblastic leukemia, acute myelogenous leukemia, ewing's sarcoma, and peripheral nerve epithelial tumors.
That is, the antibody-linker conjugate or antibody-drug conjugate according to the present invention is preferably used for the treatment of cancer. Thus, in certain embodiments, an antibody-linker conjugate or antibody-drug conjugate according to the invention comprises an antibody that specifically binds to an antigen present on a tumor cell. In certain embodiments, the antigen may be an antigen on the surface of a tumor cell. In certain embodiments, when the antibody-linker conjugate binds to an antigen, the antigen on the surface of the tumor cell can internalize into the cell along with the antibody-linker conjugate.
If the antibody-linker conjugate or antibody-drug conjugate according to the invention is used for the treatment of cancer, it is preferred that the antibody-linker conjugate or antibody-drug conjugate comprises at least one payload having the potential to kill or inhibit proliferation of tumor cells to which the antibody-linker conjugate or antibody-drug conjugate is bound. In certain embodiments, at least one payload exhibits its cytotoxic activity after the antibody-linker conjugate or antibody-drug conjugate has been internalized into a tumor cell. In certain embodiments, at least one payload is a toxin.
The inflammatory disease may be an autoimmune disease. The infectious disease may be a bacterial infection or a viral infection.
In certain embodiments, the antibody-linker conjugates, antibody-drug conjugates, and/or pharmaceutical compositions according to the invention may be used to treat B cell-related cancers.
Thus, in certain embodiments, the invention relates to an antibody-linker conjugate, an antibody-drug conjugate or a pharmaceutical composition for use according to the invention, wherein the antibody-linker conjugate or the antibody-drug-linker conjugate comprised in the pharmaceutical composition comprises poloxamer, and wherein the neoplastic disease is a B cell associated cancer.
Thus, preferably the antibody-linker conjugate or antibody-drug conjugate comprises an anti-CD 79b antibody as disclosed herein, preferably wherein the anti-CD 79b antibody internalizes into the target cell upon binding to CD79 b. In certain embodiments, the anti-CD 79b antibody is a Polotuzumab having a heavy chain as set forth in SEQ ID NO:5 and a light chain as set forth in SEQ ID NO: 6. Further, it is preferred that the antibody-linker conjugate or antibody-drug conjugate comprises at least one toxin.
In certain embodiments, an anti-CD 79b antibody comprised in an antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition may be conjugated to any one of the linkers shown in fig. 1, 2, 3, 8, 9, 14 or 15 or any one of the linkers disclosed herein.
The B cell-related cancer may be any one selected from the group consisting of: high, medium, low grade lymphomas (including B-cell lymphomas such as, for example, mucosa-associated lymphocytic B-cell lymphomas and non-hodgkin lymphomas (NHL), mantle cell lymphomas, burkitt's lymphomas, small lymphomas, marginal zone lymphomas, diffuse large B-cell lymphomas, follicular lymphomas, and hodgkin lymphomas and T-cell lymphomas) and leukemias (including secondary leukemias, chronic Lymphocytic Leukemias (CLL) such as B-cell leukemias (cd5+ B-lymphocytes), myelogenous leukemia (such as acute myelogenous leukemia, chronic myelogenous leukemia), lymphoblastic leukemias such as Acute Lymphoblastic Leukemia (ALL) and myelodysplasia), and other hematologic and/or B-cell or T-cell associated cancers (including cancers of other hematopoietic types including polymorphonuclear leukocytes such as basophils, eosinophils, neutrophils and monocytes, dendritic cells, thrombocytes, erythrocytes and natural cells) and leukemias well as cancer B-cell proliferative disorders selected from the group consisting of aggressive lymphomas, lymphomas of non-hodgkin's (NHL), indolent lymphomas, lymphomas of indolent lymphomas (ALL), lymphomas of indolent lymphomas, lymphomas of leukemias (NHL).
In a particular embodiment, the invention relates to an antibody-linker conjugate, an antibody-drug conjugate or a pharmaceutical composition for use, wherein the B cell-related cancer is non-hodgkin lymphoma, in particular wherein the B cell-related cancer is diffuse large B cell lymphoma.
Further, the anti-CD 79B antibody-linker conjugate, anti-CD 79B antibody-drug conjugate, and/or pharmaceutical compositions comprising the anti-CD 79B antibody-linker conjugate or anti-CD 79B antibody-drug conjugate may be used in combination with other therapies suitable for treating B cell-related cancers.
Thus, in a specific embodiment, the present invention relates to an antibody-linker conjugate, an antibody-drug conjugate or a pharmaceutical composition for use according to the invention, wherein the antibody-linker conjugate, the antibody-drug conjugate or the pharmaceutical composition is administered in combination with bendamustine and/or rituximab.
It will be appreciated that the antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition need not necessarily be administered simultaneously with additional therapeutic agents, such as bendamustine and/or rituximab. In contrast, the antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition may be administered on a different dosing schedule, and thus on a different day, as another therapeutic agent for treating the same disease.
In certain embodiments, the antibody-linker conjugates, antibody-drug conjugates and/or pharmaceutical compositions according to the invention may be used to treat HER2 positive cancers.
Thus, in a specific embodiment, the invention relates to an antibody-linker conjugate, an antibody-drug conjugate or a pharmaceutical composition for use according to the invention, wherein the antibody-linker conjugate or the antibody-drug conjugate comprised in the pharmaceutical composition comprises trastuzumab, and wherein the neoplastic disease is a HER2 positive cancer, in particular HER2 positive breast cancer, gastric cancer, ovarian cancer or lung cancer.
Thus, preferably the antibody-linker conjugate or antibody-drug conjugate comprises an anti-HER 2/neu antibody as disclosed herein, preferably wherein the anti-HER 2/neu antibody internalizes into the target cell upon binding to HER 2/neu. In certain embodiments, the anti-HER 2/neu antibody is trastuzumab having a heavy chain as set forth in SEQ ID NO:7 and a light chain as set forth in SEQ ID NO: 8. Further, it is preferred that the antibody-linker conjugate or antibody-drug conjugate comprises at least one toxin.
In certain embodiments, an anti-HER 2/neu antibody comprised in an antibody-linker conjugate, antibody-drug conjugate, or pharmaceutical composition may be conjugated to any of the linkers shown in fig. 1, 2, 3, 8, 9, 14, or 15 or any of the linkers disclosed herein.
HER2 positive cancer as used herein may be, but is not limited to, HER2 positive breast, stomach, ovarian or lung cancer. The skilled person is able to determine whether the cancer is a HER2 positive cancer. For example, tumor cells may be isolated in a biopsy and the presence of HER2/neu may be determined by any method known in the art.
Further, the anti-HER 2/neu antibody-linker conjugate, the anti-HER 2/neu antibody-drug conjugate, and/or a pharmaceutical composition comprising the anti-HER 2/neu antibody-linker conjugate or the anti-HER 2/neu antibody-drug conjugate may be used in combination with other therapies suitable for treating HER2 positive cancer.
Thus, in a specific embodiment, the present invention relates to an antibody-linker conjugate, an antibody-drug conjugate or a pharmaceutical composition for use according to the present invention, wherein the antibody-linker conjugate, the antibody-drug conjugate or the pharmaceutical composition is administered in combination with lapatinib, capecitabine and/or a taxane.
It will be appreciated that the antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition need not necessarily be administered simultaneously with additional therapeutic agents (such as lapatinib, capecitabine, and/or a taxane). In contrast, the antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition may be administered on a different dosing schedule, and thus on a different day, as another therapeutic agent for treating the same disease.
In certain embodiments, the antibody-linker conjugates, antibody-drug conjugates, and/or pharmaceutical compositions according to the invention may be used to treat a lectin-4 positive cancer.
Thus, in a specific embodiment, the present invention relates to an antibody-linker conjugate, an antibody-drug conjugate or a pharmaceutical composition for use according to the present invention, wherein the antibody-linker conjugate or the antibody-drug conjugate comprised in the pharmaceutical composition comprises enrolment mab or an enrolment mab variant, and wherein the neoplastic disease is a binder-4 positive cancer, in particular a binder-4 positive pancreatic, lung, bladder or breast cancer.
Thus, preferably the antibody-linker conjugate or antibody-drug conjugate comprises an anti-conjugated-4 antibody as disclosed herein, preferably wherein the anti-conjugated-4 antibody internalizes into the target cell upon binding to conjugated-4. In certain embodiments, the anti-binding agent-4 antibody is enrolment mab having a heavy chain as set forth in SEQ ID NO. 9 and a light chain as set forth in SEQ ID NO. 10. Further, it is preferred that the antibody-linker conjugate or antibody-drug conjugate comprises at least one toxin.
In certain embodiments, an anti-binding element-4 antibody included in an antibody-linker conjugate, antibody-drug conjugate, or pharmaceutical composition may be conjugated to any of the linkers shown in fig. 1, 2, 3, 8, 9, 14, or 15 or any of the linkers disclosed herein.
The lectin-4-positive cancer used herein can be, but is not limited to, a pancreatic cancer, a bladder cancer, or a breast cancer that is positive for lectin-4. The skilled artisan is able to determine whether the cancer is a lectin-4-positive cancer. For example, tumor cells may be isolated in a biopsy and the presence of integrin-4 may be determined by any method known in the art.
Further, the anti-conjugated agent-4 antibody-linker conjugate, the anti-conjugated agent-4 antibody-drug conjugate, and/or a pharmaceutical composition comprising the anti-conjugated agent-4 antibody-linker conjugate or the anti-conjugated agent-4 antibody-drug conjugate may be used in combination with other therapies suitable for treating conjugated agent-4 positive cancers.
Thus, in a specific embodiment, the invention relates to an antibody-linker conjugate, an antibody-drug conjugate or a pharmaceutical composition for use according to the invention, wherein the antibody-linker conjugate, the antibody-drug conjugate or the pharmaceutical composition is administered in combination with a cisplatin-based chemotherapeutic agent and/or pamobolizumab (pembrolizumab).
It will be appreciated that the antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition need not necessarily be administered simultaneously with additional therapeutic agents, such as cisplatin-based chemotherapeutic agents and/or palbociclizumab. In contrast, the antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition may be administered on a different schedule, and thus on a different day, as other therapeutic agents for treating the same disease.
In a particular embodiment, the invention relates to the use of an antibody-linker conjugate according to the invention (in particular wherein the antibody-linker conjugate comprises at least one payload), an antibody-drug conjugate according to the invention, or a pharmaceutical composition according to the invention for the manufacture of a medicament for the following treatment,
● Patients suffering from neoplastic diseases, neurological diseases, autoimmune diseases, inflammatory diseases or infectious diseases,
● Patients at risk of developing neoplastic, neurological, autoimmune, inflammatory or infectious diseases, and/or
● Patients diagnosed with neoplastic disease, neurological disease, autoimmune disease, inflammatory disease or infectious disease.
In a particular embodiment, the present invention relates to a method of treating or preventing a neoplastic disease, the method comprising administering an antibody-linker conjugate according to the present invention to a patient in need thereof, in particular wherein the antibody-linker conjugate comprises at least one payload, an antibody-drug conjugate according to the present invention, or a pharmaceutical composition according to the present invention.
In a particular embodiment, the invention relates to an antibody-linker conjugate according to the invention (in particular wherein the antibody-linker conjugate comprises at least one payload), an antibody-drug conjugate according to the invention or a pharmaceutical composition according to the invention for use in preoperative, intra-operative or post-operative imaging.
That is, the antibody-linker conjugate according to the present invention may be used in medical imaging. To this end, the antibody-linker conjugate may be visualized upon binding to a specific target molecule, cell or tissue. Different techniques are known in the art to visualize a particular payload. For example, if the payload is a radionuclide, the molecule, cell, or tissue to which the antibody-linker conjugate binds may be visualized by PET or SPECT. If the payload is a fluorescent dye, the molecule, cell or tissue to which the antibody-linker conjugate binds may be visualized by fluorescent imaging. In certain embodiments, an antibody-linker conjugate according to the invention comprises two different payloads, e.g., a radionuclide and a fluorescent dye. In this case, two different and/or complementary imaging techniques (e.g., PET/SPECT and fluorescence imaging) can be used to visualize the molecule, cell, or tissue to which the antibody-linker conjugate binds.
The antibody-linker conjugate may be used for preoperative, intra-operative and/or post-operative imaging.
Preoperative imaging includes all imaging techniques that can be performed preoperatively to visualize a particular target molecule, cell or tissue when diagnosing a disease or condition, and optionally to provide guidance for surgery. Preoperative imaging may include the step of visualizing the tumor by PET or SPECT prior to performing the procedure by using an antibody-linker conjugate that includes an antibody that specifically binds to an antigen on the tumor and is coupled to a payload that includes a radionuclide.
Intraoperative imaging includes all imaging techniques that can be performed during surgery to visualize specific target molecules, cells or tissues, thereby providing guidance for the surgery. In certain embodiments, antibody-linker conjugates comprising near infrared fluorescent dyes may be used to view tumors by near infrared fluorescent imaging during surgery. Intraoperative imaging allows a surgeon to identify specific tissue (e.g., tumor tissue) during surgery so that the tumor tissue can be completely removed.
Post-operative imaging includes all imaging techniques that can be performed post-operatively to visualize a particular target molecule, cell or tissue and evaluate the outcome of the operation. Post-operative imaging may be performed similarly to pre-operative surgery.
In certain embodiments, the invention relates to antibody-linker conjugates comprising two or more different payloads. For example, the antibody-linker conjugate may include a radionuclide and a near infrared fluorescent dye. Such antibody-payload conjugates are useful for imaging by PET/SPECT and near infrared fluorescence imaging. An advantage of this antibody is that it can be used to view target tissue (e.g., a tumor) before and after surgery by PET or SPECT. Meanwhile, tumors can be visualized through near-fluorescence infrared imaging during surgery.
In a particular embodiment, the invention relates to an antibody-linker conjugate according to the invention, in particular wherein the antibody-linker conjugate comprises at least one payload, an antibody-drug conjugate according to the invention or a pharmaceutical composition according to the invention for use in intraoperative imaging guided cancer surgery.
As described above, the antibody-linker conjugates of the invention can be used to view a target molecule, cell, or tissue and guide a surgeon or robot during surgery. That is, the antibody-linker conjugate may be used to visualize tumor tissue during surgery, for example, by near infrared imaging, and allow for complete removal of tumor tissue.
The antibody-linker conjugates, antibody-drug conjugates or pharmaceutical compositions according to the invention may be administered to a human or animal subject in an amount or dose effective to treat a disease or sufficient for diagnostic purposes.
The antibody-linker conjugates, antibody-drug conjugates or pharmaceutical compositions according to the invention may be administered by any suitable means, including parenteral, intrapulmonary and intranasal administration, and if desired for topical treatment, intralesional, intrauterine or intravesical administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, for example by injection (such as intravenous or subcutaneous injection), depending in part on whether the administration is brief or chronic. Various dosing regimens are contemplated herein, including, but not limited to, single or multiple dosing at various time points, bolus dosing, and pulse infusion.
The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition according to the invention may be formulated, administered and administered in a manner consistent with the invention, and will be formulated, dosed and administered in a manner consistent with good medical practice. Factors considered herein include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to the physician. The antibody-linker conjugates, antibody-drug conjugates or pharmaceutical compositions according to the invention are not required, but are optionally formulated with one or more agents currently used for the prevention or treatment of problematic conditions. The effective amount of such other agents depends on the amount of antibody-linker conjugate present in the formulation, the type of disorder or treatment, and other factors discussed above. These are typically used at the same dosages and routes of administration as described herein, or about 1% to 99% of the dosages described herein, or any dosages and any routes as empirically/clinically determined to be appropriate.
For the prevention or treatment of a disease, the appropriate dosage of the antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition according to the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody-payload conjugate, the severity and course of the disease, whether the antibody-linker conjugate is administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody-linker conjugate, and the discretion of the attending physician. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition according to the invention is suitable for administration to a patient in one or a series of treatments.
Drawings
FIG. 1 shows the chemical structure of RKAA-MMAE linker-payload complex according to the invention in which N-terminally protected Ac-RKAA peptide is covalently linked to PABC and MMAE.
FIG. 2 shows the chemical structure of RKA-MMAE linker-payload complex according to the invention in which N-terminally protected Ac-RKA peptide is covalently linked to PABC and MMAE.
Figure 3 shows the chemical structure of an ARK-MMAE linker-payload complex according to the invention, wherein the N-terminally protected Ac-ARK peptide is covalently linked to PABC and MMAE.
Figure 4 shows the chemical structure of a KRA-MMAE linker-payload complex (not according to the invention) in which the N-terminally protected Ac-KRA peptide is covalently linked to PABC and MMAE.
Figure 5 shows the chemical structure of AKR-MMAE linker-payload complex (not according to the invention), wherein the N-terminally protected Ac-AKR peptide is covalently linked to PABC and MMAE.
Figure 6 shows the chemical structure of a KAAR-MMAE linker-payload complex according to the invention (not according to the invention), wherein the N-terminally protected Ac-KAAR peptide is covalently linked to PABC and MMAE.
Figure 7 shows the chemical structure of a KARA-MMAE linker-payload complex (not according to the invention) in which the N-terminally protected Ac-KARA peptide is covalently linked to PABC and MMAE.
FIG. 8 shows the chemical structure of RKAA-maytansinoid linker-payload complexes according to the invention in which the N-terminally protected Ac-RKAA peptide is covalently linked to PABC and maytansinoid.
FIG. 9 shows the chemical structure of a maytansinoid-RKR linker-payload complex according to the present invention in which maytansinoid is covalently attached to a C4 alkylamide spacer which is linked to (has a C-terminal amide protecting group) RKR peptide.
FIG. 10 shows Size Exclusion Chromatography (SEC) of the antibody-drug conjugate ARA-01-RKAA-PABC-MMAE of the invention.
FIG. 11 depicts the results of dose-dependent in vitro cytotoxicity of the antibody-drug conjugate ARA-01-RKAA-PABC-MMAE of the invention against three different CD79b overexpressing cell lines (a-c) and CD79b non-expressing cell line (d).
FIG. 12 shows Pololium bead mab (SEQ ID NOS: 5 and 6), antibody-drug conjugate of the invention (ARA 01-RKAA-PABC-MMAE) and Pololium bead Shan Kangwei statin at various time points after a single intravenous injection of 5mg/kg into CD1 Swiss miceIs a plasma concentration of (2). The concentration in plasma was determined by ELISA. Mean plasma concentrations of 5 mice are plotted versus time, with error bars representing Standard Error (SEM) of the mean. It should be understood that the abbreviation ARA01-RKAA-MMAE in FIG. 12 refers to the antibody-drug conjugate ARA01-RKAAC-PABC-MMAE.
Fig. 13 schematically illustrates a one-step coupling process.
FIG. 14 shows the chemical structure of ARK-PEG2-PABC-MMAE linker-payload complex in accordance with the present invention wherein the N-terminally protected Ac-ARK peptide is covalently attached to the PEG2 spacer and the PABC-MMMAE payload.
FIG. 15 shows ARK-PEG2- (NH) - (CH) according to the present invention 3 ) Chemical structure of the-S-C4-maytansinoid linker-payload complex, wherein the N-terminally protected Ac-ARK peptide is covalently linked to PEG2- (NH) - (CH) 3 ) -S-C4 alkyl spacer and maytansine payload.
FIG. 16 depicts Granta 519 mouse tumor model. Human B-cell lymphoma tumor cells (Granta 519) were inoculated subcutaneously in CB17 SCID mice (n=8 per treatment group). When the tumor reaches about 200mm 3 At a size of 0.53mg/kg or 2.1mg/kg of poloxamer Shan Kangwei statinsAnd a single injection of ARA01-RKAA-PABC-MMAE at 0.53mg/kg, 1mg/kg or 2.1 mg/kg. ARA01-RKAA-PABC-MMAE provided the same tumor growth inhibition and survival relative to the poloxamer Shan Kangwei statin at approximately half the payload dose (0.53 mg/kg dose in FIG. 16A, 16B). At approximately equal payload doses relative to the poloxamer Shan Kangwei statin, ARA01-RKAA-PABC-MMAE treatment resulted in greater anti-tumor efficacy and considerable survival advantage than the 0/8 complete remission of the tumor with the poloxamer Shan Kangwei statin (fig. 16B compares the 0.53mg/kg dose of the poloxamer Shan Kangwei statin with the 1mg/kg dose of ARA 01-RKAA-PABC-MMAE). Mean tumor volume is shown as Standard Error of Mean (SEM).
FIG. 17 shows the chemical structure of RKAA-payload complex according to the invention in which an N-terminally protected Ac-RKAA peptide is covalently linked to an irinotecan derivative Dxd via a self-cleaving methylamine linker.
FIG. 18 shows the chemical structure of RKAA-payload complex according to the invention in which the N-terminally protected Ac-RKAA peptide is covalently linked to the payload PNU-159582 via a self-cleaving moiety PABC-EDA.
FIG. 19 shows the chemical structure of RKAA-payload complex according to the invention in which the N-terminally protected Ac-RKAA peptide is covalently linked to the payload carcinomatomycin GA via a self-cleaving moiety PABC-EDA.
FIG. 20 shows the chemical structure of RKAA-payload complex according to the invention in which N-terminally protected Ac-RKAA peptide is covalently linked to the payload PBD via a self-cleaving moiety PABE.
FIG. 21 shows the chemical structure of RKAA-payload complex according to the invention in which N-terminally protected Ac-RKAA peptide is covalently linked to payload maytansinoids via a self-cleaving methylamine linker and an additional alkyl linker molecule.
FIG. 22 shows the chemical structure of RKR-payload complexes according to the invention in which the N-terminally protected Ac-RKR peptide is directly coupled to the payload MMAE.
FIG. 23 shows the chemical structure of RKAA-payload complex according to the invention in which N-terminally protected Ac-RKAA peptide is covalently linked to payload carcinomatomycin GA via self-cleaving para-methylaniline (PMA) moiety.
FIG. 24 shows the chemical structure of RKR-payload complex in accordance with the invention in which a C-terminally protected RKR amide peptide is covalently linked to the payload PNU-159582 via a carbamate.
FIG. 25 shows the chemical structure of RKR-payload complex in accordance with the invention in which the C-terminally protected RKR amide peptide is cleaved via the self-cleaving moiety OHPS-PHB-EDA and additional (PEG) 2 The moiety is covalently linked to the payload PNU-159582.
FIG. 26 shows the chemical structure of RKR-payload complex in accordance with the invention in which the C-terminally protected RKR amide peptide is via a self cleaving moiety OHPS and additional (PEG) 2 The moiety is covalently linked to the payload PBD.
FIG. 27 shows the chemical structure of RKR-payload complexes in accordance with the invention in which a C-terminally protected RKR-amide peptide is covalently linked to the payload PBD via a self-cleaving moiety OHPS.
FIG. 28 shows the chemical structure of RKR-payload complex in accordance with the invention in which the C-terminally protected RKR-amide peptide is directly coupled to payload DM21.
FIG. 29 shows the chemical structure of RKR-payload complex in accordance with the invention in which a C-terminally protected RKR amide peptide is covalently linked to payload DM4 via an alkyl linker molecule comprising a carboxy group and a thiol group.
FIG. 30 shows the chemical structure of RKR-payload complex in accordance with the invention in which a C-terminally protected RKR-amide peptide is covalently linked to the payload MMAE via a dicarboxylic acid linker molecule.
FIG. 31 shows the chemical structure of RKR-payload complex in accordance with the invention in which the C-terminally protected RKR amide peptide is cleaved via a self cleaving moiety OHPS-PHB and additional (PEG) 2 The moiety is covalently linked to the payload MMAE.
FIG. 32 shows the chemical structure of RKR-payload complex according to the invention in which a C-terminally protected RKR-amide peptide is covalently linked to the payload MMAE via a self-cleaving moiety OHPAS-PHB.
FIG. 33 shows the chemical structure of RKR-payload complex in accordance with the invention in which the C-terminally protected RKR amide peptide is cleaved via a self cleaving moiety OHPS quaternary ammonium and additional (PEG) 2 The moiety is covalently linked to the payload carcinomycin GA.
FIG. 34 shows the chemical structure of RKR-payload complex in accordance with the invention in which a C-terminally protected RKR-amide peptide is covalently linked to a payload carcinomatomycin GA via a self-cleaving moiety OHPS quaternary ammonium.
FIG. 35 depicts the results of dose-dependent in vitro cytotoxicity of trastuzumab-RKAA-PABC-MMAE or trastuzumab-RKAA-PABC-maytansinoid on the HER2 positive cell line SKBR-3 as an antibody-drug conjugate of the invention.
FIG. 36 depicts the results of dose-dependent in vitro cytotoxicity of the anti-CD 79b antibody-drug conjugates ARA01-ARK-PABC-MMAE, ARA01-RKA-PABC-MMAE and ARA01-RKValCit-PABC-MMAE on CD79b positive cell line Granta-519.
FIG. 37 depicts the results of dose-dependent in vitro cytotoxic effects of the anti-conjugated agent-4 antibody-drug conjugates of the invention ARA04-RKAA-PABC-MMAE, ARA04-ARK-PABC-MMAE, ARA04-RKA-PABC-MMAE and ARA04-RKValCit-PABC-MMAE on the conjugated agent-4 positive cell line SUM190 PT.
FIG. 38 depicts the results of dose-dependent in vitro cytotoxicity of the anti-conjugated agent-4 antibody-drug conjugates ARA04-RKAA-PABC-MMAE, ARA04-ARK-PABC-MMAE, ARA04-RKA-PABC-MMAE and ARA04-RKValCit-PABC-MMAE of the invention against conjugated agent-4 negative cell line A549.
FIG. 39 shows the Pololizumab/ARA 01 (SEQ ID NOS: 5 and 6) antibody-drug conjugates of the present invention (ARA 01-ARK-PABC-MMAE, ARA01-RKA-PBC-MMAE, ARA 01-RKValCit-PABC-MAE) and Pololizumab Shan Kangwei statins at various time points after a single intravenous injection of 5mg/kg into CD1 Swiss mice Is a plasma concentration of (2). The concentration of ADC in plasma was determined by ELISA. Mean plasma concentrations of 5 mice are plotted versus time, with error bars representing Standard Error (SEM) of the mean. It should be understood that the abbreviations ARA01-ARK/RKA/RKValCit-PABC-MMAE in FIG. 39 refer to the antibody-drug conjugate ARA01-ARK/RKA/RKValCit-PABC-MMAE.
FIG. 40 shows enrolment mab/ARA 04 (SEQ ID NOS: 9 and 11) and enrolment Shan Kangwei statins at various time points after a single intravenous injection of 5mg/kg into CD1 Swiss miceIs a plasma concentration of (2). The concentration of ADC in plasma was determined by ELISA. Mean plasma concentrations of 5 mice are plotted versus time, with error bars representing Standard Error (SEM) of the mean. It should be understood that the abbreviations ARA04-ARK/RKA/RKValCit-PABC-MMAE in FIG. 39 refer to the antibody-drug conjugate ARA04-ARK/RKA/RKValCit-PABC-MMAE.
Fig. 41 depicts Ramos mouse tumor model. Human burkitt lymphoma tumor cells (Ramos) were inoculated subcutaneously in CB17SCID mice (n=6 per treatment group). When the tumor reaches about 200mm 3 At a size of 1.43mg/kg of Polol-tobe beads Shan Kangwei multiple-heaterOr a single injection of 1.25mg/kg (payload adjusted dose of poloxamer Shan Kangwei statins) ARA01-RKAA-PABC-MMAE or ARA 01-ARK-PABC-MMAE. The two ADCs of the invention provide equal tumor growth inhibition and survival and a durable anti-tumor response at about half the payload dose relative to the poloxamer Shan Kangwei statin. In contrast, the poloxamer Shan Kangwei statin administered at the same payload dose showed only transient tumor elimination. Mean tumor volume is shown as Standard Error of Mean (SEM).
FIG. 42 depicts SUM190PT mouse tumor model. Breast cancer-tumor cells (SUM 190 PT) were inoculated into mammary fat (fatpat) of CB17 SCID mice (n=6 per treatment group). When the tumor size reaches about 200mm 3 At the time, animals received 1.5mg/kg enrolment monoclonal antibodies Wei Duoting, respectivelyOr a single injection of 3mg/kg (payload adjusted dose of enrolment Shan Kangwei statin) ARA04-RKAA-PABC-MMAE or ARA 04-ARK-PABC-MMAE. Both ADCs of the present invention provide a complete and durable anti-tumor response lasting for more than 103 days at the same payload dose as enrolment Shan Kangwei statin. In contrast, enrolment Shan Kangwei statins administered at the same payload dose showed a transient anti-tumor response. When fig. 42 and 43 are observed, it is apparent that the ADC, ARA04-RKAA-PABC-MMAE or ARA04-ARK-PABC-MMAE of the present invention shows excellent therapeutic effects even when administered only at 1/4 (=4 times or less) of the payload dose. The non-binding mAb-RKAA-PABC-MMAE did not show any effect on tumor growth. Mean tumor volume is shown as Standard Error of Mean (SEM).
FIG. 43 depicts SUM190PT mouse tumor model. Breast cancer-tumor cells (SUM 190 PT) were inoculated into the mammary fat of CB17 SCID mice (n=6 per treatment group). When the tumor size reaches about 200mm 3 At the time, the animals received 0.5mg/kg enrolment monoclonal antibody Wei DuotingOr a single injection of 1mg/kg (payload adjusted dose of enrolment Shan Kangwei statin) ARA04-RKAA-PABC-MMAE or ARA 04-ARK-PABC-MMAE. Both ADCs of the present invention provide a complete and durable anti-tumor response lasting for more than 103 days at the same payload dose as enrolment Shan Kangwei statin. In contrast, enrolment Shan Kangwei statins administered at the same payload dose resulted in only slight tumor growth retardation. Mean tumor volume is shown as Standard Error of Mean (SEM).
Examples
Example 1: conjugation of peptide MMAE linker to two different antibodies
Method
Antibody trastuzumab is commercially availableRoche, purchased from pharmacies), and all linker-payload constructs (custom synthesized by Levena Biopharma). Polotuzumab having heavy and light chains consisting of the sequences of SEQ ID NOs 5 and 6 was transiently transfected into suspension-adapted CHO-K1 cells and expressed in serum-free/animal-component-free medium. The protein was purified from the supernatant by protein A affinity chromatography (Mab Select Sure column; GE Healthcare).
For 1-step coupling (see FIG. 13), 5mg/ml of native glycosylated monoclonal antibody in 50mM Tris pH 7.6, microbial transglutaminase (MTG, zedira) at a concentration of 5U/mg in 50mM Tris pH 7.6 or water, and 5 molar equivalents of the indicated linker-payload were used and incubated in a rotary heat mixer for 24 hours at 37 ℃. Coupling efficiency was assessed by LC-MS under DTT reduction conditions. Reduction of the sample was achieved by incubating the antibody-drug conjugate (ADC) in 50mM DTT/50mM Tris buffer for 10 minutes at 37 ℃. Probes were analyzed in Xex G2-XS QTOF (Waters) attached to the Acquity UPLC class H system (Waters) and the Acquity UPLC BEH C column. The conjugation efficiency was calculated from deconvoluted spectra and expressed as linker-payload conjugated antibody (=adc)%. The total coupling efficiency was calculated taking into account the intensities generated by the two glycoforms (G1F and G0F), i.e
Total conjugation efficiency (%) = total intensity-intensity of unconjugated antibody, the following formula was derived:
coupling efficiency (%) =100 x (1- (intensity (G1F) +intensity (G0F))/total intensity)
Results
The coupling efficiency varies depending on the linker structure and antibody used, however, it can be observed that the binding efficiency is highest when the peptide linker containing lysine contains an RK motif (tables 3 and 4).
TABLE 3 shows coupling efficiency of linker-payload complexes (according to the invention)
TABLE 4 shows coupling efficiency of linker-payload (not according to the invention) complexes
Example 2: conjugation of peptide-maytansine to two different antibodies
To demonstrate that high coupling efficiency can also be achieved with another payload (i.e. a payload other than MMAE), a linker-payload construct containing maytansine was used to couple to two different antibodies.
Method
Coupling was performed in exactly the same way as described in example 1. The corresponding maytansine-linker constructs were custom synthesized by Levena Biopharm.
Results
When a payload different from MMAE was used, maytansine in this example was also very efficient in coupling when the peptide linker containing lysine contained the RK motif (table 4). This example shows that when the peptide linker containing lysine contains the RK motif, the coupling efficiency is high regardless of the payload.
TABLE 5 shows coupling efficiency of linker-payload complexes (according to the invention)
Example 3: coupling of linker-payload (according to the invention) to third antibody
To further demonstrate the high coupling efficiency obtained with the linker-payload construct (according to the invention), the third antibody was selected and successfully coupled with high efficiency (for two different payloads, its general applicability was further demonstrated).
Method
Coupling was performed in exactly the same way as described in example 1. Antibody enrolment mab with heavy chain consisting of SEQ ID No. 9 and light chain variant consisting of SEQ ID No. 10 was transiently transfected into suspension adapted CHO-K1 cells and expressed in serum/animal free medium. The protein was purified from the supernatant by protein A affinity chromatography (Mab Select Sure column; GE Healthcare).
Results
Using two different linker-payload constructs according to the invention, high coupling efficiencies were obtained with the antibody enrolment mab.
TABLE 6 shows coupling efficiency of linker-payload complexes (according to the invention)
Example 4: coupling of linker-payloads (according to the invention) comprising non-amino acid spacers
To further demonstrate the high coupling efficiency obtained with the linker-payload construct (according to the invention), a linker with a polyethylene glycol (PEG) spacer was used and efficiently coupled to two different antibodies.
Method
Coupling was performed in exactly the same way as described in example 1. All linker payload constructs were custom synthesized by Levena Biopharma.
Results
High coupling efficiency to two different antibodies was obtained with linker-payloads comprising PEG spacers (according to the invention).
TABLE 7 shows coupling efficiency of linker-payload complexes (according to the invention)
Example 5: the ADC of the present invention is monomeric and does not aggregate
The linker-payload RKAA-PABC-MMAE (fig. 1) was coupled to the antibody polotouzumab (SEQ ID NOs: 5 and 6) as described in example 1 above. The resulting ADC, designated ARA-01-RKAA-PABC-MMAE, had a drug-to-antibody ratio (DAR) of 1.9 (essentially as described by Richard Y.C. Huang and Guodong Chen (2016), the antibody-drug conjugate was characterized by mass spectrometry: evolution and future trends (Characterization of antibody-drug conjugates by mass spectrometry: advances and future trends) Drug Discover Today, volume 21, as determined by standard mass spectrometry, described in stage 5) and analyzed by size exclusion chromatography.
Method
Using a catalyst having Superdex TM 200 Increate10/300 (Amersham Pharmacia Biotech) columnFPLC (Amersham-Pharmacia Biotech) was subjected to Size Exclusion Chromatography (SEC). Proteins were detected by UV/VIS at a wavelength of 280 nm. Samples were analyzed in running buffer at pH 7.4, 50mM phosphate, 100mM NaCl at a flow rate of 1 mL/min.
Results
Size Exclusion Chromatography (SEC) profile after purification showed that ARA-01-RKAA-MMAE eluted as a single monomer peak, indicating that ADC had excellent biophysical properties (fig. 10).
Example 6: the ADC of the invention shows strong anti-tumor effect in vitro
Method
The growth inhibition of ARA01-RKAA-PABC-MMAE on the following three CD79b overexpressing cell lines was studied in vitro: granta-519 (DSMZ, acc No. 342), BJAB (CLS) and WSU-DLCL2 (DSMZ, acc 575). As a negative control, CD79 negative cell line HT (ATCC, reference number: CRL-2260) was used. 4000 cells were seeded into 96-well plates and incubated with ARA-01-RKAA-PABC-MMAE in a humidification chamber at 37℃and 5% CO 2 Medium incubation for 72 hours.
Viability of the treated cultures was quantitatively determined by ATP in CellTiterGloLuminescence Assay as described by the supplier (Promega). The% viability relative to untreated cells was calculated according to the following formula:
the mean% viability was plotted against log10 (concentration) and the resulting dose-response curve was analyzed by nonlinear regression with software Prism8 using a four parameter dose-response curve equation.
Results
FIG. 11 shows that ARA01-RKAA-PABC-MMAE has very high cytotoxic activity against CD79b overexpressing cells, wherein EC 50 The value is comparable to a conventional ADC. Cytotoxic activity is highly selective for cells that overexpress CD79b, as there is essentially no reduction in cell viability as expected in HT cell lines. In conclusion, ARA01-RKAA-PABC-MMAE showed antigen-specific, significant antiproliferative activity in vitro.
Example 7: the ADC of the invention shows good pharmacokinetic parameters in vivo
In mice were studiedThe pharmacokinetic profile of the anti-CD 79b ADC ARA01-RKAA-MMAE according to the invention was studied and compared with commercially available anti-CD 79b-ADC Pololytol beads Shan Kangwei statinA comparison was made. The poloxamer of the poloxamer Shan Kangwei is an ADC consisting of the anti-CD 79b antibody, poloxamer, wherein MMAE is conjugated to the cysteines of the antibodies resulting in an average of 3.5 linked MMAE moieties per antibody (european medicines agency (European Medicines Agency), assessment Report on #>Program number: EMEA/H/C/004879/0000, see https:// www.ema.europa.eu/en/media/human/EPAR/polivy).
Method
ARA01-RKAA-PABC-MMAE (produced internally as described in example 5 above),(Roche, purchased from a pharmacy) and the naked anti-CD 79b antibody, polotuzumab (SEQ ID NOS: 5 and 6; expressed and purified as described above) were intravenously injected into 5 female mice (CD 1 Swiss, janvier) at doses of 5mg/kg ADC or antibody, respectively. After 10 minutes, 5.5 hours, 24 hours, 48 hours, 96 hours, 144 hours, 168 hours and 360 hours, about 20 μl of blood was withdrawn from the saphenous vein and placed into EDTA-coated microreactor CB 300 (Sarstedt). Blood samples were centrifuged at 9500 and x g for 10 minutes and plasma was stored at-80 ℃ until ELISA analysis was performed. The concentration in plasma was determined by ELISA using His-tagged human CD79b as capture agent using a dilution series of the corresponding samples with known concentrations: 125ng of HisCD79b (Sinobiological, reference number 29750-H08H) diluted in PBS was added to nickel plates (Ni-NTA HisSorb, qiagen) and after blocking with 200. Mu.l of PBS, 4% milk (Rapid, migros, switzerland), 50. Mu.l of diluted plasma sample (4% milk in PBS) was added. After incubation for 1 hour and washing with PBS, the preparation was carried out by adding donkey anti-human IgG-HRP (B) iolegend, poly 24109) or, for total ADC detection, rabbit anti-MMAE antibody was added again at room temperature (Levena, ref: LEV-PAE 1) for 1 hour, washed and detected via anti-rabbit IgG-HRP. Peroxidase activity was detected by addition of 3,3', 5' -tetramethylbenzidine (Sigma) and stopped by addition of acid. Readings were measured at 450nm after 1 to 5 minutes. The concentration of the sample in plasma and the slope k (plotted on a semilogarithmic scale) of the elimination phase (time points 24h-360 h) determined by ELISA at various time points after injection, are determined by the formula t 1/2 Calculation of half-life of samples by =ln2/-k (t 1/2 )。
Results
The plasma concentrations measured in samples taken at different time points after injection are shown in figure 12. ARA01-RKAA-PABC-MMAE andthe half-life of (2) is given in table 4 below. It can be seen that according to the ADC of the present invention ARA01-RKAA-PABC-MMAE, the half-life in vivo is an approved ADC +.>At least 2 times greater than the above. The increased plasma stability may lead to better safety features, as the payload does not seem to be released prematurely.
TABLE 8 plasma half-life
Example 8: the anti-CD 79b ADCs of the invention inhibit tumor growth in vivo more effectively than the approved anti-CD 89b ADC poloxamer Shan Kangwei statins
The tumor growth inhibition by anti-CD 79b ADC ARA01-RKAA-PABC-MMAE was studied in vivo and compared to commercially available Pololium beads Shan Kangwei statin.
Method
Will be 20x 10 6 Personal B thinThe tumor cells Granta 519 (DSMZ, acc No. 342) of the cytolymphoma were subcutaneously implanted in CB17 SCID mice (Janvier). Tumor size and body weight were recorded three times a week. Volume= (width) according to formula 2 x length x 0.5 to calculate tumor volume. When the average tumor size reached about 200mm 3 When mice were assigned to treatment groups, each group comprising 8 mice, using a non-random stratification scheme. On day 0 (day of random grouping), ARA01-RKAA-PABC-MMAE at doses of 0.53mg/kg, 1mg/kg and 2.1mg/kg (produced internally as described in example 5 above) and Polollipop beads Shan Kangwei statins at doses of 0.53mg/kg and 2.1mg/kg were administered in a single intravenous injection. Control mice were injected with PBS. All mouse experiments were performed according to the swiss guidelines and were approved by the veterinary agency of zurich, switzerland (Veterinarian Office of Z ulrich). According to these guidelines, PBS group and all 0.53mg/kg dose mice must be sacrificed on day 10, while 2 mice in 1mg/kg group must be sacrificed on day 6 and day 30 (tumor ulcers).
Results:
the in vivo efficacy of ARA01-RKAA-PABC-MMAE (DAR 1.9) compared to the poloxamer Shan Kangwei statin (DAR 3.5) was evaluated against the Granta 519 tumor model. Specifically, these animals received 0.53mg/kg or 2.1mg/kg of Polol bead Shan Kangwei statinAnd a single injection of ARA01-RKAA-PABC-MMAE at 0.53mg/kg, 1mg/kg or 2.1 mg/kg. Importantly, the ARA01-RKAA-PABC-MMAE provided the same tumor growth inhibition and survival as the poloxamer Shan Kangwei, relative to the poloxamer Shan Kangwei, at about half the payload dose (comparison of 2mg/kg dose, see fig. 16a, comparison of 0.53mg/kg dose, see fig. 16B). The ARA01-RKAA-PABC-MMAE treatment resulted in greater anti-tumor efficacy and considerable survival advantage relative to the poloxamer Shan Kangwei statin at approximately equal payload doses than the poloxamer Shan Kangwei statin with a complete tumor remission of 0/8 times (compared to the ARA01-RKAA-PABC-MMAE at a dose of 0.53mg/kg for the poloxamer Shan Kangwei statin and 1mg/kg in fig. 16B). Total (S)In addition, ARA01-RKAA-PABC-MMAE treatment produced greater anti-tumor efficacy than poloxamer Shan Kangwei statin at approximately equal payload doses and had considerable survival advantage.
Example 9: coupling of various RK-motif-peptides with the antibody trastuzumab
All RK containing peptides tested were coupled with high efficiency.
Method
The coupling reaction was carried out according to the conditions described in example 1. Briefly, 5mg/ml of naturally glycosylated trastuzumab antibody, 1.5U/mg concentration of MTG, and 20 molar equivalents of the designated peptide-linker comprising the RK-motif were mixed in Tris 50mM pH 7.6 at 37℃for 24 hours in a rotary heat mixer. Coupling efficiency was assessed by LCMS as follows: coupling Efficiency (CE) was calculated from deconvoluted spectra and expressed in%. The intensities generated by the two glycoforms (G1F and G0F) were taken into account in the calculation according to the following formula:
wherein cj = coupled, ncj = uncoupledResults
All of the RK-motif-linkers tested were coupled well with native fully glycosylated trastuzumab with an efficiency of >50%, as shown in Table 9.
TABLE 9 coupling efficiency of peptide linkers containing RK-motif with trastuzumab
RK-motif peptide linker Coupling efficiency (%)
HRKHA(SEQ ID NO:55) 98％
HRKAH(SEQ ID NO:56) 91％
RKAH(SEQ ID NO:57) 91％
RKH(SEQ ID NO:58) 87％
RKAA(SEQ ID NO:1) 86％
RKA(SEQ ID NO:2) 86％
RKHA(SEQ ID NO:59) 86％
RKHH(SEQ ID NO:60) 85％
ARKAH(SEQ ID NO:61) 82％
ARKHA(SEQ ID NO:62) 82％
HRK(SEQ ID NO:63) 81％
RKAAH(SEQ ID NO:64) 81％
ARKHH(SEQ ID NO:65) 80％
RKAAA(SEQ ID NO:66) 80％
Example 10: coupling of RK-motif-peptides
Method
Reaction conditions: the 5mg/ml of the naturally glycosylated trastuzumab antibody, 5U/mg concentration of MTG and 5 molar equivalents of the indicated peptide-linker were mixed in Tris 50mM pH 7.6 at 37℃for 24 hours in a rotary heat mixer. Coupling efficiency was assessed by LCMS as described in example 9.
Results
Peptides containing the RK-motif were coupled with significant coupling efficiency, as shown in Table 10.
TABLE 10 coupling efficiency of peptide linkers containing RK motif with trastuzumab
RK-motif peptide linker Coupling efficiency (%)
RKAAR(SEQ ID NO:67) 95％
RRKAY(SEQ ID NO:68) 100％
RRK(SEQ ID NO:69) 99％
ARKRA(SEQ ID NO:70) 98％
Example 11: coupling of RK-motif-linker-payload to MMAE
To demonstrate that the RK-motif-linker-payload is also suitable for one-step antibody coupling, MMAE was used as the payload, and additional linker-payloads containing RK-motif were used for coupling with trastuzumab.
Method
The coupling reaction was performed by mixing 5mg/ml of naturally glycosylated trastuzumab antibody, 5U/mg of MTG and 5 molar equivalents of the indicated linker-payload in Tris 50mM pH 7.6 for 24 hours in a 37℃rotary heat mixer. Coupling efficiency was assessed by LCMS as described in example 9.
Results
Surprisingly, as shown in table 11A, excellent coupling efficiencies (over 85%) were obtained using various RK-motif-linker-payloads containing MMAE coupled with naturally glycosylated trastuzumab antibodies. Surprisingly, as shown in tables 11A and 11B, a significantly lower coupling efficiency was observed when the linker-payload did not contain the RK-motif.
TABLE 11 coupling efficiency of RK-motif linker-payload with trastuzumab containing MMAE (according to the invention)
RK-linker-payload with MMAE Coupling efficiency (%)
RKAA-PABC-MMAE(SEQ ID NO:1) 100％
RKA-PABC-MMAE(SEQ ID NO:2) 100％
ARK-PABC-MMAE(SEQ ID NO:3) 100％
RKAAR-PABC-MMAE(SEQ ID NO:67) 99％
RRKAY-PABC-MMAE(SEQ ID NO:68) 100％
RRKAY-PABC-MMAE(SEQ ID NO:69) 96％
ARKRA-PABC-MMAE(SEQ ID NO:70) 89％
ARKRA-PABC-MMAE(SEQ ID NO:54) 91％
ARK-PEG2-PABC-MMAE(SEQ ID NO:3) 99％
TABLE 11B coupling efficiency of non-RK-motif-linker-payload to MMAE (not according to the invention)
non-RK-linker-payload with MMAE Coupling efficiency(％)
ARKRA-PABC-MMAE(SEQ ID NO:50) 43％
KRA-PABC-MMAE(SEQ ID NO:51) 68％
KR-PABC-MMAE(SEQ ID NO:71) 46％
KAAR-PABC-MMAE(SEQ ID NO:52) 64％
KARA-PABC-MMAE(SEQ ID NO:53) 77％
KAA-PABC-MMAE(SEQ ID NO:72) 57％
Example 12: coupling RK-motif linker-payloads Using alternative payload classes
To demonstrate the popularity of the linker technology of the present invention, various RK-motif linker-payloads were coupled with trastuzumab. The payload is selected from the following payload categories: cytotoxins, steroids (cortisol=cs) and immunomodulators (i.e. STING agonists) were evaluated.
Method
The coupling reaction was performed by mixing 5mg/ml of the naturally glycosylated trastuzumab antibody, 5-10U/mg of MTG and 5-10 molar equivalents of the indicated linker-payload in Tris 50mM pH 7.6 for 24 hours in a 37℃rotary hot mixer. Coupling efficiency was assessed by LCMS as described in example 9.
Results
Surprisingly, excellent coupling efficiencies (over 80%) were obtained using various RK-motif linker versions and payload classes, as shown in table 4. Also surprisingly, it was observed that the payload at the N-terminal position was well tolerated (as demonstrated by May-C5-RKR).
Table 12. Coupling efficiencies of linker-payloads containing RK-motif linkers with 3 different payload classes.
RK-linker-payload containing multiple toxins and drugs Coupling efficiency (%)
RKAA-PABC-May(SEQ ID NO:1) 92％
RKAA-PABC-Exa(SEQ ID NO:1) 98％
RKAA-PABC-EDA-PNU(SEQ ID NO:1) 99％
RKAA-PABE-amanitine (SEQ ID NO: 1) 90％
RKAA-EDA-cortisol (SEQ ID NO: 1) 88％
RKAA-PABC-EDA-STING(SEQ ID NO:1) 96％
RKAAR-PABC-Exa(SEQ ID NO:67) 99％
RKAAR-EDA-CS(SEQ ID NO:67) 98％
ARK-S-C5-May(SEQ ID NO:3) 98％
ARK-PABC-Exa(SEQ ID NO:3) 94％
ARK-PEG2-S-C5-May(SEQ ID NO:3) 99％
May-C5-RKR(SEQ ID NO:4) 96％
RRK-PABC-Exa(SEQ ID NO:69) 83％
May: maytansine; exa: an irinotecan derivative; STING (interferon gene stimulators; immunostimulatory factors); PNU (anthracycline analogues).
Example 13: coupling of RK-motif linker-payload to three different antibodies
To demonstrate the general applicability of this reaction, RK-motif-linker-payloads containing MMAE or maytansine (May) were selected to be coupled to three different antibodies: trastuzumab, poisotoizumab and enrolment antibody variants (heavy chain SEQ ID NO:9 and light chain SEQ ID NO: 11).
Method
The coupling reaction was performed by mixing 5mg/ml of the indicated native glycosylated antibody, 5-10U/mg of MTG and 5-10 molar equivalents of the indicated linker-payload in Tris 50mM pH 7.6 for 24 hours in a 37℃rotary hot mixer. Coupling efficiency was assessed by LCMS as described in example 9.
Results
Surprisingly, as shown in table 13, high coupling efficiencies were obtained for all three antibodies tested and all RK-motif-MMAE or May linker payloads tested.
TABLE 13 coupling efficiency of linker-payload to three different antibodies
NT: not tested
Example 14: coupling of RK-motif linker-payload under different reaction conditions
To demonstrate that coupling with RK-linker-payload tolerates a variety of reaction conditions, linker-payload coupling with polotophyllizumab was performed using a series of reaction conditions with different parameters.
Method
As standard conditions, the following parameters were used: 5mg/ml of naturally glycosylated Pololium-bead mAb, MTG at a concentration of 5U/mg and 5 molar equivalents of RKAA-PABC-MMAE in Tris 50mM, pH 7.6 at 37℃for 24 hours in a rotating heat mixer. Coupling efficiency was assessed by LCMS as described in example 9 above.
The variable parameters are shown in table 14.
Results
RKAA-PABC-MMAE linker-payload was coupled with very high coupling efficiency over a very wide range of reaction conditions: coupling efficiencies of >80% were achieved using antibody concentrations between 5 and 17mg/ml, MTG concentrations (U/mg) between 2 and 10U/mg relative to the antibody concentrations. Further, a high coupling efficiency (67% coupling efficiency from pH 6.0 and 86% pH 8) was also obtained with a molar concentration of linker relative to antibody (2 to 8 equivalents) and a very broad range of pH.
Surprisingly, using less linker-payload excess resulted in higher coupling efficiency compared to antibodies, i.e., 2-20 equivalents of linker-payload resulted in higher coupling efficiency than using 80 equivalents, contrary to expectations (table 14).
TABLE 14 coupling efficiency of RK-linker-payload to Polotuzumab under different reaction conditions
Example 15: ADC containing RK-motif PABC-payload linker payload was effective in vitro using three different antibodies
To demonstrate that ADCs according to the invention (i.e. using RK-motif-MMAE/maytansinoid linker payload generation) produce potent release and target-specific toxicity to cancer cell lines, trastuzumab-, polotouzumab- (ARA 01) and enrolmumab- (SEQ ID 9 and SEQ ID 11; ARA 04) based ADCs were tested on targeted expression cells using linkers RKAA-PABC-MMAE, RKAA-PABC-maytansine, ARK-PABC-MMAE, RKA-PABC-MMAE and RKValCit-MMAE.
Method
The growth inhibition of HER-2 positive SKBR-3 (ATCC HTB-30) cells by trastuzumab-RKAA-PABC-MMAE and trastuzumab-RKAA-PABC-maytansine was studied, the inhibition of CD79 b-positive Granta-519 lymphoma cells by ARA01-ARK-PABC-MAE, ARA01-RKA-PABC-MMAE and ARA01-RKValCit-PABC-MMAE was tested, and the cytotoxic effects of the binding agent-4 positive breast cancer cells SUM190PT (BIOVT, 28068A 16284) by ARA04-RKAA-PABC-MMAE, ARA04-ARK-PABC-MMAE, ARA04-RKA-PABC-MMAE and ARA 04-KVALPCit-PABC-MMAE were studied. Target dependency and specificity were tested on the lectin-4 negative lung cancer cell A549 (ATCC CCL-185). Under all conditions 4000 cells were seeded into 96 well plates and incubated with respective ADCs in a humidification chamber at 37 ℃ with 5% co 2 Middle incubation72 hours.
Results
FIG. 35 shows that trastuzumab-RKAA-PABC-MMAE and trastuzumab-RKAA-PABC-maytansine of the invention exert very high cytotoxic activity on HER-2 overexpressing cells, EC thereof 50 The value is comparable to a conventional ADC.
ARA01 and ARA04 ADCs containing different linkers of the invention showed very high target-specific cytotoxic activity against target-positive Granta-519 (FIG. 36) and SUM190PT (FIG. 37) cells, respectively, in CD79b and in the binding agent-4 targeted ADCs. EC (EC) 50 The values are in the range of a conventional ADC. In contrast, the same ADC did not affect target negative a549 cells (fig. 38), and showed an effect comparable to that of the conventional ADC.
In summary, all ADCs using RK-motif linker-payloads according to the present invention (conjugated to trastuzumab, polotouzumab or enrolment mab as parent antibody) show target-specific and remarkable antiproliferative activity in vitro.
Example 16: ADCs containing RK-motif-PABC-MMAE linker-payload show good pharmacokinetic properties in vivo
To assess the in vivo stability of the RK-motif MMAE ADC, mouse pharmacokinetic studies were performed using different RK-motif linker payloads conjugated to polotoxin and enrolment mab.
anti-CD 79b ADCs ARA01-ARK-PABC-MMAE, ARA01-RKA-PABC-MMAE and ARA01-RKValCit-PABC-MMAE generated with the linker-payload of the invention; and the pharmacokinetic profile of anti-binding-4 ADC generated with the different linkers ARA04-ARK-PABC-MMAE, ARA04-RKA-PABC-MMAE and ARA04-RKValCit-PABC-MMAE was studied in mice and compared with commercially available anti-CD 79b ADC Polottal beads Shan Kangwei statinAnd commercially available anti-conjugated agent-4 ADC enrolment mab-Wei Duoding +.>A comparison was made.
Method
Pharmacokinetic studies were performed as described in example 7 and the sampling time points were adjusted: blood samples were drawn from saphenous vein after 10 minutes, 4 hours, 48 hours, 96 hours, 168 hours, 264 hours, 336 hours, and 504 hours. For anti-CD 79b ADC detection, the method described in example 7 was used. The anti-integrin-4 ADC assay was performed simply as follows: the concentration of ADC in plasma was determined by ELISA using His-tagged human conjugated-4 as a capture agent: 125ng of His-binding-4 (Sinobiological, ref.: 19771-H08H) was diluted in PBS and added to nickel plates (Ni-NTA HisSorb, qiagen). After blocking with 200 μl PBS, 4% milk (Rapilait, migros, switzerland), 50 μl diluted plasma sample (4% milk in PBS) was added. After incubation for 1 hour and washing with PBS, the total ADC was detected using a rabbit anti-MMAE antibody (Levena, ref: LEV-PAE 1) which was added for an additional 1 hour at room temperature, washed and detected by anti-rabbit IgG HRP. Peroxidase activity was detected by addition of 3,3', 5' -tetramethylbenzidine (Sigma) and stopped by addition of acid. Readings were measured at 450nm after 1 to 5 minutes. Half-life was calculated using ADC concentration plotted (semilog scale) of samples in plasma over time. Half-life (t) was determined using the resulting slope k of the elimination phase at time points 48-504h using the following formula 1/2 )：t 1/2 ＝ln2/-k。
Results
Plasma concentrations of intact ADC measured in samples taken at different time points after injection are shown for anti-CD 79b ADC (fig. 39) and lectin-4 ADC (fig. 40).
Half-lives of ARA01-ARK-PABC-MMAE, ARA01-RKA-PABC-MMAE and ARA01-RKValCit-PABC-MMAE are shown in Table 15 below. Surprisingly, an about 2-fold increase in half-life was observed for all ADCs according to the invention compared to the calculated porto bead Shan Kangwei statin.
ARA04-RKAA-MMAE, ARA04-ARK-MMAE, ARA04-RKA-MMAE, ARA04-RKValCit-MMAE andthe half-lives of these ADCs are shown in table 16 below, showing a 2 to 2.5 fold increase in the average half-life of these ADCs compared to approved enrolment Shan Kangwei statins.
In summary, all ADCs generated according to the present invention showed 2-2.5 fold increased half-lives compared to the Wei Duoting standard using either poluzumab or enrolment mab as the parent antibody. This shows that ADCs generated with linker-payloads according to the invention lead to improved in vivo ADC stability, which may lead to generally better safety profiles (profiles) and Therapeutic Indices (TI) since the payload is not released prematurely.
TABLE 15 plasma half-life of anti-CD 79b ADCs
TABLE 16 plasma half-life of anti-conjugated protein-4 ADCs
Example 17: ADC containing RK-motif-PABC-MMAE linker payload showed more effective tumor growth inhibition in vivo compared to baseline in CD79b positive liquid tumor model and in binder-4 positive solid tumor model
Tumor growth inhibition of anti-CD 79b ADCs ARA01-RKAA-PABC-MMAE and ARA01-ARK-PABC-MMAE according to the invention was studied in vivo in a Ramos (CD 79b positive, liquid tumor) model. The anti-tumor properties of the anti-conjugated protein-4 ADC ARA04-RKAA-PABC-MMAE and AR 04-ARK-PABC-MMAE according to the invention were tested in SUM190PT (conjugated protein-4 positive, solid tumor) xenograft model. Non-binding mAb-RKAA-PABC-MMAE control ADC was included to exclude non-specific ADC activity.
Method
For SUM190PT xenografts, 2X 10 will be 6 Individual cells were injected into mammary fat; for Ramos, 20X 10 6 Subcutaneous injection of individual cells into CB17SCID mice (Janvier). Tumor size and body weight were recorded three times a week. Volume= (width) according to formula 2 x length x 0.5 to calculate tumor volume. When the average tumor size reached about 200mm 3 When mice were assigned to treatment groups, each group comprising 6 mice, using a non-random stratification scheme. The ADC was injected intravenously once on the day of random grouping.
As described in example 5, all ADCs are produced internally (in-house).
ARA01-RKAA-PABC-MMAE and ARA01-RKAA-PABC-MMAE (both DAR 1.9) were injected at a dose of 1.25mg/kg (equivalent to a payload of 25ug per kg body weight). The poloxamer Shan Kangwei statins (PV, DAR 3.6) were injected at a dose of 1.43mg/kg (equivalent to 50ug/kg payload or twice the ARA01-ADC payload dose).
ARA04-RKAA-PABC-MMAE and ARA04-RKAA-PABC-MMAE (DAR 1.9) were injected at ADC doses of 1mg/kg and 3mg/kg (corresponding to 10 μg and 30 μg payloads per kg body weight) and compared to enrolment Shan Kangwei statins (EV, DAR 3.8) at doses of 0.5mg/kg and 1.5 mg/kg. Non-binding mAb-RKAA-PABC-MMAE ADC (with the same linker-payload and DAR as ARA04 ADC) was injected at 3 mg/kg. Control mice were injected with PBS. All mouse experiments were performed according to swiss guidelines and were approved by the swiss zurich veterinary office.
Results:
in a fast-growing Ramos xenograft model, the ADCs ARA01-RKAA-PABC-MMAE and ARA01-ARK-PABC-MMAE of the invention were compared to the poloxamer Shan Kangwei statin (PV). Figure 41 shows that one intravenous injection of 1.25mg/kg (or 25ug payload per kg mouse body weight) produced a highly potent anti-tumor response in all mice. In contrast, PV administered at 1.43mg/kg or 50ug payload/kg body weight showed only brief tumor elimination at 20 days post-treatment, without tumor-free individuals. In contrast, it is very surprising that ARA01-RKAA-PABC-MMAE and ARA01-ARK-PABC-MMAE show a durable complete anti-tumor response at half the payload dose.
ARA04-RKAA-PABC-MMAE and ARA04-ARK-PABC-MMAE were compared with enrobed Shan Kangwei statins (EV) in a SUM190PT breast cancer model. Importantly, as shown in FIGS. 42 and 43, the two ADCs of the present invention, ARA04-RKAA-PABC-MMAE and ARA04-ARK-PABC-MMAE, were extremely effective at both 1mg/kg and 3mg/kg and resulted in complete tumor eradication and a durable response throughout the study (103 days post-injection).
At approximately equal payload doses relative to enrolment monoclonal antibody Wei Duoting, ARA04-RKAA-PABC-MMAE and ARA04-ARK-PABC-MMAE (3 mg/kg) treatments resulted in greater, longer lasting anti-tumor efficacy and considerable survival advantage, with complete remission of the tumor 4/6 times, and complete remission of the EV tumor 0/6 times (comparison of EV at 1.5mg/kg dose and ARA04-RKAA-PABC-MMAE at 3mg/kg dose in fig. 42). The non-binding mAb-RKAA-PABC-MMAE did not have any effect on tumor growth and demonstrated high target specificity of the targeted ADC. Remarkably, the ARA04-RKAA-PABC-MMAE and ARA04-ARK-PABC-MMAE ADCs provided the same tumor growth inhibition and survival at approximately one third of the dose and 1mg/kg (=10 ug/kg payload dose) ARA04-RKAA/ARK-MMAE dose relative to enrolment monoclonal antibody Wei Duoting (comparison of 1.5 mg/kg=30 ug/kg payload dose), see fig. 42 and 43. Overall, ARA04-RKAA-PABC-MMAE and ARA04-ARK-PABC-MMAE treatments produced greater and sustained anti-tumor efficacy with respect to enrolment mab Wei Duoting at about one third of the payload dose, as well as considerable survival advantage.
In summary, we summarize that anti-CD 79b and anti-binding-4 ADCs (consisting of the same antibodies and payloads as their corresponding reference ADCs) generated with RK-motif linker-payloads according to the invention are highly active in vivo and show 2-3 fold superior efficacy, providing a considerable survival advantage over Wei Duoting-based ADCs, which is quite surprising.
Sequence listing
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<211> 446
<212> PRT
<213> artificial sequence
<220>
<223> Polatuzumab heavy chain
<400> 5
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Ser Ser Tyr
20 25 30
Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Leu Pro Gly Gly Gly Asp Thr Asn Tyr Asn Glu Ile Phe
50 55 60
Lys Gly Arg Ala Thr Phe Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Arg Val Pro Ile Arg Leu Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
<210> 6
<211> 218
<212> PRT
<213> artificial sequence
<220>
<223> Polotuzumab light chain
<400> 6
Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Asp Tyr Glu
20 25 30
Gly Asp Ser Phe Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
35 40 45
Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn
85 90 95
Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105 110
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 7
<211> 450
<212> PRT
<213> artificial sequence
<220>
<223> Trastuzumab heavy chain
<400> 7
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 8
<211> 214
<212> PRT
<213> artificial sequence
<220>
<223> trastuzumab light chain
<400> 8
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 9
<211> 447
<212> PRT
<213> artificial sequence
<220>
<223> Ennomab heavy chain
<400> 9
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Asn Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Ser
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ala Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 10
<211> 214
<212> PRT
<213> artificial sequence
<220>
<223> enrolment monoclonal antibody light chain
<400> 10
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Gly Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Pro
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 11
<211> 447
<212> PRT
<213> artificial sequence
<220>
<223> enrolment mono heavy chain
<400> 11
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Asn Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Ser
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ala Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 12
<211> 335
<212> PRT
<213> artificial sequence
<220>
<223> Streptomyces mobaraensis (Streptomyces mobaraensis) MTG Zedira
<400> 12
Phe Arg Ala Pro Asp Ser Asp Asp Arg Val Thr Pro Pro Ala Glu Pro
1 5 10 15
Leu Asp Arg Met Pro Asp Pro Tyr Arg Pro Ser Tyr Gly Arg Ala Glu
20 25 30
Thr Val Val Asn Asn Tyr Ile Arg Lys Trp Gln Gln Val Tyr Ser His
35 40 45
Arg Asp Gly Arg Lys Gln Gln Met Thr Glu Glu Gln Arg Glu Trp Leu
50 55 60
Ser Tyr Gly Cys Val Gly Val Thr Trp Val Asn Ser Gly Gln Tyr Pro
65 70 75 80
Thr Asn Arg Leu Ala Phe Ala Ser Phe Asp Glu Asp Arg Phe Lys Asn
85 90 95
Glu Leu Lys Asn Gly Arg Pro Arg Ser Gly Glu Thr Arg Ala Glu Phe
100 105 110
Glu Gly Arg Val Ala Lys Glu Ser Phe Asp Glu Glu Lys Gly Phe Gln
115 120 125
Arg Ala Arg Glu Val Ala Ser Val Met Asn Arg Ala Leu Glu Asn Ala
130 135 140
His Asp Glu Ser Ala Tyr Leu Asp Asn Leu Lys Lys Glu Leu Ala Asn
145 150 155 160
Gly Asn Asp Ala Leu Arg Asn Glu Asp Ala Arg Ser Pro Phe Tyr Ser
165 170 175
Ala Leu Arg Asn Thr Pro Ser Phe Lys Glu Arg Asn Gly Gly Asn His
180 185 190
Asp Pro Ser Arg Met Lys Ala Val Ile Tyr Ser Lys His Phe Trp Ser
195 200 205
Gly Gln Asp Arg Ser Ser Ser Ala Asp Lys Arg Lys Tyr Gly Asp Pro
210 215 220
Asp Ala Phe Arg Pro Ala Pro Gly Thr Gly Leu Val Asp Met Ser Arg
225 230 235 240
Asp Arg Asn Ile Pro Arg Ser Pro Thr Ser Pro Gly Glu Gly Phe Val
245 250 255
Asn Phe Asp Tyr Gly Trp Phe Gly Ala Gln Thr Glu Ala Asp Ala Asp
260 265 270
Lys Thr Val Trp Thr His Gly Asn His Tyr His Ala Pro Asn Gly Ser
275 280 285
Leu Gly Ala Met His Val Tyr Glu Ser Lys Phe Arg Asn Trp Ser Glu
290 295 300
Gly Tyr Ser Asp Phe Asp Arg Gly Ala Tyr Val Ile Thr Phe Ile Pro
305 310 315 320
Lys Ser Trp Asn Thr Ala Pro Asp Lys Val Lys Gln Gly Trp Pro
325 330 335
<210> 13
<211> 335
<212> PRT
<213> artificial sequence
<220>
<223> Streptomyces mobaraensis MTG
<400> 13
Phe Arg Ala Pro Asp Ser Asp Glu Arg Val Thr Pro Pro Ala Glu Pro
1 5 10 15
Leu Asp Arg Met Pro Asp Pro Tyr Arg Pro Ser Tyr Gly Arg Ala Glu
20 25 30
Thr Ile Val Asn Asn Tyr Ile Arg Lys Trp Gln Gln Val Tyr Ser His
35 40 45
Arg Asp Gly Arg Lys Gln Gln Met Thr Glu Glu Gln Arg Glu Trp Leu
50 55 60
Ser Tyr Gly Cys Val Gly Val Thr Trp Val Asn Ser Gly Gln Tyr Pro
65 70 75 80
Thr Asn Arg Leu Ala Phe Ala Phe Phe Asp Glu Asp Lys Tyr Lys Asn
85 90 95
Glu Leu Lys Asn Gly Arg Pro Arg Ser Gly Glu Thr Arg Ala Glu Phe
100 105 110
Glu Gly Arg Val Ala Lys Asp Ser Phe Asp Glu Ala Lys Gly Phe Gln
115 120 125
Arg Ala Arg Asp Val Ala Ser Val Met Asn Lys Ala Leu Glu Asn Ala
130 135 140
His Asp Glu Gly Ala Tyr Leu Asp Asn Leu Lys Lys Glu Leu Ala Asn
145 150 155 160
Gly Asn Asp Ala Leu Arg Asn Glu Asp Ala Arg Ser Pro Phe Tyr Ser
165 170 175
Ala Leu Arg Asn Thr Pro Ser Phe Lys Asp Arg Asn Gly Gly Asn His
180 185 190
Asp Pro Ser Lys Met Lys Ala Val Ile Tyr Ser Lys His Phe Trp Ser
195 200 205
Gly Gln Asp Arg Ser Gly Ser Ser Asp Lys Arg Lys Tyr Gly Asp Pro
210 215 220
Glu Ala Phe Arg Pro Asp Arg Gly Thr Gly Leu Val Asp Met Ser Arg
225 230 235 240
Asp Arg Asn Ile Pro Arg Ser Pro Thr Ser Pro Gly Glu Ser Phe Val
245 250 255
Asn Phe Asp Tyr Gly Trp Phe Gly Ala Gln Thr Glu Ala Asp Ala Asp
260 265 270
Lys Thr Val Trp Thr His Gly Asn His Tyr His Ala Pro Asn Gly Ser
275 280 285
Leu Gly Ala Met His Val Tyr Glu Ser Lys Phe Arg Asn Trp Ser Asp
290 295 300
Gly Tyr Ser Asp Phe Asp Arg Gly Ala Tyr Val Val Thr Phe Val Pro
305 310 315 320
Lys Ser Trp Asn Thr Ala Pro Asp Lys Val Thr Gln Gly Trp Pro
325 330 335
<210> 14
<211> 407
<212> PRT
<213> artificial sequence
<220>
<223> Streptomyces mobaraensis MTG P81453
<400> 14
Met Arg Ile Arg Arg Arg Ala Leu Val Phe Ala Thr Met Ser Ala Val
1 5 10 15
Leu Cys Thr Ala Gly Phe Met Pro Ser Ala Gly Glu Ala Ala Ala Asp
20 25 30
Asn Gly Ala Gly Glu Glu Thr Lys Ser Tyr Ala Glu Thr Tyr Arg Leu
35 40 45
Thr Ala Asp Asp Val Ala Asn Ile Asn Ala Leu Asn Glu Ser Ala Pro
50 55 60
Ala Ala Ser Ser Ala Gly Pro Ser Phe Arg Ala Pro Asp Ser Asp Asp
65 70 75 80
Arg Val Thr Pro Pro Ala Glu Pro Leu Asp Arg Met Pro Asp Pro Tyr
85 90 95
Arg Pro Ser Tyr Gly Arg Ala Glu Thr Val Val Asn Asn Tyr Ile Arg
100 105 110
Lys Trp Gln Gln Val Tyr Ser His Arg Asp Gly Arg Lys Gln Gln Met
115 120 125
Thr Glu Glu Gln Arg Glu Trp Leu Ser Tyr Gly Cys Val Gly Val Thr
130 135 140
Trp Val Asn Ser Gly Gln Tyr Pro Thr Asn Arg Leu Ala Phe Ala Ser
145 150 155 160
Phe Asp Glu Asp Arg Phe Lys Asn Glu Leu Lys Asn Gly Arg Pro Arg
165 170 175
Ser Gly Glu Thr Arg Ala Glu Phe Glu Gly Arg Val Ala Lys Glu Ser
180 185 190
Phe Asp Glu Glu Lys Gly Phe Gln Arg Ala Arg Glu Val Ala Ser Val
195 200 205
Met Asn Arg Ala Leu Glu Asn Ala His Asp Glu Ser Ala Tyr Leu Asp
210 215 220
Asn Leu Lys Lys Glu Leu Ala Asn Gly Asn Asp Ala Leu Arg Asn Glu
225 230 235 240
Asp Ala Arg Ser Pro Phe Tyr Ser Ala Leu Arg Asn Thr Pro Ser Phe
245 250 255
Lys Glu Arg Asn Gly Gly Asn His Asp Pro Ser Arg Met Lys Ala Val
260 265 270
Ile Tyr Ser Lys His Phe Trp Ser Gly Gln Asp Arg Ser Ser Ser Ala
275 280 285
Asp Lys Arg Lys Tyr Gly Asp Pro Asp Ala Phe Arg Pro Ala Pro Gly
290 295 300
Thr Gly Leu Val Asp Met Ser Arg Asp Arg Asn Ile Pro Arg Ser Pro
305 310 315 320
Thr Ser Pro Gly Glu Gly Phe Val Asn Phe Asp Tyr Gly Trp Phe Gly
325 330 335
Ala Gln Thr Glu Ala Asp Ala Asp Lys Thr Val Trp Thr His Gly Asn
340 345 350
His Tyr His Ala Pro Asn Gly Ser Leu Gly Ala Met His Val Tyr Glu
355 360 365
Ser Lys Phe Arg Asn Trp Ser Glu Gly Tyr Ser Asp Phe Asp Arg Gly
370 375 380
Ala Tyr Val Ile Thr Phe Ile Pro Lys Ser Trp Asn Thr Ala Pro Asp
385 390 395 400
Lys Val Lys Gln Gly Trp Pro
405
<210> 15
<211> 395
<212> PRT
<213> artificial sequence
<220>
<223> Streptomyces dactylotheca (Streptoverticillium ladakanum) MTG
<400> 15
Met His Arg Arg Ile His Ala Val Gly Gln Ala Arg Pro Pro Pro Thr
1 5 10 15
Met Ala Arg Gly Lys Glu Thr Lys Ser Tyr Ala Glu Thr Tyr Arg Leu
20 25 30
Thr Ala Asp Asp Val Ala Asn Ile Asn Ala Leu Asn Glu Ser Ala Pro
35 40 45
Ala Ala Ser Ser Ala Gly Pro Ser Phe Arg Ala Pro Asp Ser Asp Asp
50 55 60
Arg Val Thr Pro Pro Ala Glu Pro Leu Asp Arg Met Pro Asp Pro Tyr
65 70 75 80
Arg Pro Ser Tyr Gly Arg Ala Glu Thr Val Val Asn Asn Tyr Ile Arg
85 90 95
Lys Trp Gln Gln Val Tyr Ser His Arg Asp Gly Arg Lys Gln Gln Met
100 105 110
Thr Glu Glu Gln Arg Glu Trp Leu Ser Tyr Gly Cys Val Gly Val Thr
115 120 125
Trp Val Asn Ser Gly Gln Tyr Pro Thr Asn Arg Leu Ala Phe Ala Ser
130 135 140
Phe Asp Glu Asp Arg Phe Lys Asn Glu Leu Lys Asn Gly Arg Pro Arg
145 150 155 160
Ser Gly Glu Thr Arg Ala Glu Phe Glu Gly Arg Val Ala Lys Glu Ser
165 170 175
Phe Asp Glu Glu Lys Gly Phe Gln Arg Ala Arg Glu Val Ala Ser Val
180 185 190
Met Asn Arg Ala Leu Glu Asn Ala His Asp Glu Ser Ala Tyr Leu Asp
195 200 205
Asn Leu Lys Lys Glu Leu Ala Asn Gly Asn Asp Ala Leu Arg Asn Glu
210 215 220
Asp Ala Arg Ser Pro Phe Tyr Ser Ala Leu Arg Asn Thr Pro Ser Phe
225 230 235 240
Lys Glu Arg Asn Gly Gly Asn His Asp Pro Ser Arg Met Lys Ala Val
245 250 255
Ile Tyr Ser Lys His Phe Trp Ser Gly Gln Asp Arg Ser Ser Ser Ala
260 265 270
Asp Lys Arg Lys Tyr Gly Asp Pro Asp Ala Phe Arg Ser Ala Pro Gly
275 280 285
Thr Gly Leu Val Asp Met Ser Arg Asp Arg Asn Ile Pro Arg Ser Pro
290 295 300
Thr Ser Pro Gly Glu Gly Phe Val Asn Phe Asp Tyr Gly Trp Phe Gly
305 310 315 320
Ala Gln Thr Glu Ala Asp Ala Asp Lys Thr Val Trp Thr His Gly Asn
325 330 335
His Tyr His Ala Pro Asn Gly Ser Leu Gly Cys His Ala Cys Leu Thr
340 345 350
Arg Ala Ser Ser Ala Thr Gly Ser Glu Gly Tyr Ser Asp Phe Asp Arg
355 360 365
Gly Glu Pro Tyr Val Val Ser Pro Ser Pro Ser Pro Arg Met Leu Glu
370 375 380
His Arg Pro Arg Gln Gly Lys Ala Gly Leu Ala
385 390 395
<210> 16
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 1
<400> 16
Leu Leu Gln Gly Gly
1 5
<210> 17
<211> 4
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 2
<400> 17
Leu Leu Gln Gly
1
<210> 18
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 3
<400> 18
Leu Ser Leu Ser Gln Gly
1 5
<210> 19
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 4
<400> 19
Gly Gly Gly Leu Leu Gln Gly Gly
1 5
<210> 20
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 5
<400> 20
Gly Leu Leu Gln Gly
1 5
<210> 21
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 6
<400> 21
Leu Leu Gln
1
<210> 22
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 7
<400> 22
Gly Ser Pro Leu Ala Gln Ser His Gly Gly
1 5 10
<210> 23
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 8
<400> 23
Gly Leu Leu Gln Gly Gly Gly
1 5
<210> 24
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 9
<400> 24
Gly Leu Leu Gln Gly Gly
1 5
<210> 25
<211> 4
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 10
<400> 25
Gly Leu Leu Gln
1
<210> 26
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 11
<400> 26
Leu Leu Gln Leu Leu Gln Gly Ala
1 5
<210> 27
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 12
<400> 27
Leu Leu Gln Gly Ala
1 5
<210> 28
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 13
<400> 28
Leu Leu Gln Tyr Gln Gly Ala
1 5
<210> 29
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 14
<400> 29
Leu Leu Gln Gly Ser Gly
1 5
<210> 30
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 15
<400> 30
Leu Leu Gln Tyr Gln Gly
1 5
<210> 31
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 16
<400> 31
Leu Leu Gln Leu Leu Gln Gly
1 5
<210> 32
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 17
<400> 32
Ser Leu Leu Gln Gly
1 5
<210> 33
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 18
<400> 33
Leu Leu Gln Leu Gln
1 5
<210> 34
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 19
<400> 34
Leu Leu Gln Leu Leu Gln
1 5
<210> 35
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 20
<400> 35
Leu Leu Gln Gly Arg
1 5
<210> 36
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 21
<400> 36
Glu Glu Gln Tyr Ala Ser Thr Tyr
1 5
<210> 37
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 22
<400> 37
Glu Glu Gln Tyr Gln Ser Thr Tyr
1 5
<210> 38
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 23
<400> 38
Glu Glu Gln Tyr Asn Ser Thr Tyr
1 5
<210> 39
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 24
<400> 39
Glu Glu Gln Tyr Gln Ser
1 5
<210> 40
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 25
<400> 40
Glu Glu Gln Tyr Gln Ser Thr
1 5
<210> 41
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 26
<400> 41
Glu Gln Tyr Gln Ser Thr Tyr
1 5
<210> 42
<211> 4
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 27
<400> 42
Gln Tyr Gln Ser
1
<210> 43
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 28
<400> 43
Gln Tyr Gln Ser Thr Tyr
1 5
<210> 44
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 29
<400> 44
Tyr Arg Tyr Arg Gln
1 5
<210> 45
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 30
<400> 45
Asp Tyr Ala Leu Gln
1 5
<210> 46
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 31
<400> 46
Phe Gly Leu Gln Arg Pro Tyr
1 5
<210> 47
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 32
<400> 47
Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu
1 5 10
<210> 48
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 33
<400> 48
Leu Gln Arg
1
<210> 49
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> Q-Tag 34
<400> 49
Tyr Gln Arg
1
<210> 50
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> Joint 5
<400> 50
Lys Arg Ala
1
<210> 51
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> Joint 6
<400> 51
Ala Lys Arg
1
<210> 52
<211> 4
<212> PRT
<213> artificial sequence
<220>
<223> Joint 7
<400> 52
Lys Ala Ala Arg
1
<210> 53
<211> 4
<212> PRT
<213> artificial sequence
<220>
<223> Joint 8
<400> 53
Lys Ala Arg Ala
1
<210> 54
<211> 4
<212> PRT
<213> artificial sequence
<220>
<223> Joint 9
<220>
<221> MOD_RES
<222> 4
<223> Xaa is L-citrulline
<400> 54
Arg Lys Val Xaa
1
<210> 55
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Joint 10
<400> 55
His Arg Lys His Ala
1 5
<210> 56
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Joint 11
<400> 56
His Arg Lys Ala His
1 5
<210> 57
<211> 4
<212> PRT
<213> artificial sequence
<220>
<223> Joint 12
<400> 57
Arg Lys Ala His
1
<210> 58
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> Joint 13
<400> 58
Arg Lys His
1
<210> 59
<211> 4
<212> PRT
<213> artificial sequence
<220>
<223> Joint 14
<400> 59
Arg Lys His Ala
1
<210> 60
<211> 4
<212> PRT
<213> artificial sequence
<220>
<223> Joint 15
<400> 60
Arg Lys His His
1
<210> 61
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Joint 16
<400> 61
Ala Arg Lys Ala His
1 5
<210> 62
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Joint 17
<400> 62
Ala Arg Lys His Ala
1 5
<210> 63
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> Joint 18
<400> 63
His Arg Lys
1
<210> 64
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Joint 19
<400> 64
Arg Lys Ala Ala His
1 5
<210> 65
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Joint 20
<400> 65
Ala Arg Lys His His
1 5
<210> 66
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Joint 21
<400> 66
Arg Lys Ala Ala Ala
1 5
<210> 67
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Joint 22
<400> 67
Arg Lys Ala Ala Arg
1 5
<210> 68
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Joint 23
<400> 68
Arg Arg Lys Ala Tyr
1 5
<210> 69
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> Joint 24
<400> 69
Arg Arg Lys
1
<210> 70
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Joint 25
<400> 70
Ala Arg Lys Arg Ala
1 5
<210> 71
<211> 2
<212> PRT
<213> artificial sequence
<220>
<223> Joint 26
<400> 71
Lys Arg
1
<210> 72
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> Joint 27
<400> 72
Lys Ala Ala
1

Claims (116)

1. A method for producing an antibody-linker conjugate by Microbial Transglutaminase (MTG), comprising reacting a polypeptide comprising (as shown in the N.fwdarw.C direction) (Sp 1 )-RK-(Sp 2 )-B-(Sp 3 ) Or (Sp) 1 )-B-(Sp 2 )-RK-(Sp 3 ) A step of coupling a linker of the structure 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 arginyl mimetic arginine mimetic;
k is lysine or a lysine derivative or a lysine mimetic;
B is a linking part or payload;
and wherein the linker is coupled to a Gln residue comprised in the antibody via a primary amine comprised in a side chain of a lysine residue, the lysine derivative or the lysine mimetic.
2. The method according to claim 1, wherein the chemical spacer (Sp 1 )、(Sp 2 ) Sum (Sp) 3 ) Each independently comprises 0 to 12 amino acid residues.
3. The method of claim 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.
4. A method according to any one of claims 1 to 3, wherein the net charge of the linker is neutral or positive.
5. The method of any one of claims 1 to 4, wherein the linker does not comprise negatively charged amino acid residues.
6. The method according to any one of claims 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), RKR (SEQ ID NO: 4) or RK-Val-Cit (SEQ ID NO: 54).
7. The method according to any one of claims 1 to 6, wherein the linker comprises the amino acid sequence RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3) or RK-Val-Cit (SEQ ID NO: 54).
8. The method according to any one of claims 1 to 7, wherein the linker comprises the amino acid sequence RKAA (SEQ ID NO: 1).
9. The method of any one of claims 1 to 8, wherein B is a linking moiety.
10. The method of claim 9, wherein the connecting portion B comprises
Bio-orthogonal labelling group, or
Non-bioorthogonal entities for cross-linking.
11. The method of claim 10, wherein the bio-orthogonal labeling group or the non-bio-orthogonal entity for cross-linking consists of or comprises at least one molecule or moiety selected from the group consisting of:
-N-N≡N or-N 3 ；
·Lys(N 3 )；
Tetrazine;
alkynes;
strained cyclooctyne;
·BCN；
strained olefins;
photoreactive groups;
aldehyde;
acyl trifluoroborates;
protein degrading agent ('PROTAC');
cyclopentadiene/spirocyclopentadiene;
a thio-selective electrophile;
-SH; and
cysteine.
12. The method according to any one of claims 9 to 11, comprising the further step of coupling one or more payloads to the linking moiety B.
13. The method of claim 12, wherein the one or more payloads are coupled to the linking moiety B via a click reaction.
14. The method of any one of claims 1 to 8, wherein B is a payload.
15. The method of any of claims 12 to 14, wherein the payload comprises at least one of:
toxins;
cytokines;
growth factors;
radionuclides;
hormones;
antiviral agents;
an antimicrobial agent;
fluorescent dye;
immunomodulators/immunostimulants;
half-life increasing moiety;
a solubility-increasing moiety;
polymer-toxin conjugate;
nucleic acid;
biotin or streptavidin moiety;
vitamins;
protein degrading agent ('PROTAC');
a target binding moiety; and/or
Anti-inflammatory agents.
16. The method of claim 15, wherein the toxin is at least one selected from the group consisting of:
pyrrolobenzodiazepines (e.g., PBDs);
auristatin (e.g., MMAE, MMAF);
maytansinoids (e.g., maytansine, DM1, DM4, DM 21);
sesquicomycin;
nicotinamide phosphoribosyl transferase (NAMPT) inhibitors;
Microtubule lysin;
enediyne (e.g., calicheamicin);
anthracycline derivatives (PNUs) (e.g., doxorubicin);
inhibitors of the pyrrolyl Kinesin Spindle Protein (KSP);
candidiasis;
drug efflux pump inhibitors;
mountain Zhuo Meisu;
amanitine (e.g., α -amanitine); and
camptothecins (e.g., irinotecan, delutinacon).
17. The method according to any one of claims 14 to 16, wherein the chemical spacer (Sp 2 ) Including self-cleaving moieties.
18. The method of claim 17, wherein the self-cleaving portion is directly attached to the payload B.
19. The method of claim 17 or 18, wherein the self-cleaving moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety.
20. The method according to any one of claims 1 to 19, wherein the antibody is an IgG antibody, in particular an IgG1 antibody.
21. The method according to claim 20, wherein the linker-coupled Gln residue is comprised in the Fc domain of the antibody, in particular wherein the linker-coupled Gln residue is C of an IgG antibody H Gln residue Q295 of the 2 domain (EU numbering).
22. The method of claim 20, wherein the linker-coupled Gln residues are introduced into the heavy or light chain of the antibody by molecular engineering.
23. The method of claim 22, wherein the Gln residues introduced into the heavy or light chain of the antibody by molecular engineering are C of a non-glycosylated IgG antibody H 2 domain N297Q (EU numbering).
24. The method of claim 22, wherein the Gln residues introduced into the heavy or light chain of the antibody by molecular engineering are contained in a peptide that has been (a) integrated into the heavy or light chain of the antibody, or (b) fused to the N-terminus or C-terminus of the heavy or light chain of the antibody.
25. The method of claim 24, wherein the peptide comprising a Gln residue has been fused to the C-terminus of the heavy chain of the antibody.
26. The method according to any one of claims 20 to 22 or 24 to 25, wherein the IgG antibody is a glycosylated IgG antibody, in particular wherein the IgG antibody is at C H Glycosylation at residue N297 (EU numbering) of the 2 domain.
27. The method of any one of claims 1 to 26, wherein the antibody is selected from the group consisting of: vibutuzumab, trastuzumab, gemtuzumab, oxtuzumab, avermectin, cetuximab, rituximab, up Lei Tuoyou mab, pertuzumab, vedolizumab, oreuzumab, tolizumab, wu Sinu mab, golimumab, otouzumab, sha Xituo mab, bei Lantuo mab, polotouzumab and enrolment mab.
28. The method of any one of claims 1 to 27, wherein the antibody is selected from the group consisting of: velutinab, gemtuzumab, trastuzumab, oxtuzumab, poltuzumab, enrolment mab, sha Xituo bead mab, bei Lantuo mab.
29. The method of any one of claims 1 to 28, wherein the antibody is poloxamer or trastuzumab or enrolment mab.
30. The method of any one of claims 1 to 29, wherein the linker is coupled to a γ -carboxamide group of a Gln residue comprised in the antibody.
31. The method of any one of claims 1 to 30, wherein the linker is adapted to couple to a glycosylated antibody with a coupling efficiency of at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95%.
32. The method according to any one of claims 1 to 31, wherein the microbial transglutaminase is derived from a streptomyces species, in particular streptomyces mobaraensis.
33. An antibody-linker conjugate prepared by the method of any one of claims 1 to 32.
34. An antibody-linker conjugate comprising:
a) An antibody; and
b) A joint, the joint comprising the following structure:
(Sp 1 )-RK-(Sp 2 )-B-(Sp 3 ) Or (b)
(Sp 1 )-B-(Sp 2 )-RK-(Sp 3 ) The method comprises the steps of carrying out a first treatment on the surface of the Wherein the method comprises the steps of
·(Sp 1 ) Is a chemical spacer or is absent;
·(Sp 2 ) Is a chemical spacer or is absent;
·(Sp 3 ) Is a chemical spacer or is absent;
r is arginine or an arginine derivative or arginine mimetic;
k is lysine or a lysine derivative or a lysine mimetic;
b is a linking part or payload;
wherein the linker is coupled to the antibody via an isopeptide bond formed between a γ -carboxamide group of a glutamine residue contained in the antibody and a primary amine contained in a side chain of a lysine residue, a lysine derivative or a lysine mimetic contained in an RK motif contained in the linker.
35. The antibody-linker conjugate of claim 34 wherein the chemical spacer (Sp 1 )、(Sp 2 ) Sum (Sp) 3 ) Each independently comprises 0 to 12 amino acid residues.
36. The antibody-linker conjugate of claim 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.
37. The antibody-linker conjugate of any one of claims 34 to 36 wherein the net charge of the linker is neutral or positive.
38. The antibody-linker conjugate of any one of claims 34 to 37 wherein the linker does not comprise a negatively charged amino acid residue.
39. The antibody-linker conjugate of any one of claims 34 to 38 wherein said 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), RKR (SEQ ID NO: 4), RK-Val-Cit (SEQ ID NO: 54).
40. The antibody-linker conjugate of any one of claims 34 to 39, wherein said 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), RK-Val-Cit (SEQ ID NO: 54).
41. The antibody-linker conjugate according to any one of claims 34 to 40, wherein said linker comprises the amino acid sequence RKAA (SEQ ID NO: 1).
42. The antibody-linker conjugate of any one of claims 34 to 41 wherein B is a linking moiety.
43. The antibody-linker conjugate according to claim 42, wherein the linking moiety B comprises
Bio-orthogonal labelling group, or
Non-bioorthogonal entities for cross-linking.
44. The antibody-linker conjugate according to claim 43, wherein the bio-orthogonal labeling group or the non-bio-orthogonal entity for cross-linking consists of or comprises at least one molecule or moiety selected from the group consisting of:
-N-N≡N or-N 3 ；
·Lys(N 3 )；
Tetrazine;
alkynes;
strained cyclooctyne;
·BCN；
strained olefins;
photoreactive groups;
aldehyde;
acyl trifluoroborates;
protein degrading agent ('PROTAC');
cyclopentadiene/spirocyclopentadiene;
a thio-selective electrophile;
-SH; and
cysteine.
45. The antibody-linker conjugate of any one of claims 42 to 44 wherein one or more payloads are coupled to the linking moiety B.
46. The antibody-linker conjugate according to claim 45, wherein one or more payloads are coupled to the linking moiety B via a click reaction.
47. The antibody-linker conjugate of any one of claims 34 to 41 wherein B is a payload.
48. The antibody-linker conjugate of any one of claims 45 to 47, wherein the payload comprises at least one of:
Toxins;
cytokines;
growth factors;
radionuclides;
hormones;
antiviral agents;
an antimicrobial agent;
fluorescent dye;
immunomodulators/immunostimulants;
half-life increasing moiety;
a solubility-increasing moiety;
polymer-toxin conjugate;
nucleic acid;
biotin or streptavidin moiety;
vitamins;
protein degrading agent ('PROTAC');
a target binding moiety; and/or
Anti-inflammatory agents.
49. The antibody-linker conjugate according to claim 48, wherein the toxin is at least one selected from the group consisting of:
pyrrolobenzodiazepines (e.g., PBDs);
auristatin (e.g., MMAE, MMAF);
maytansinoids (e.g., maytansine, DM1, DM4, DM 21);
sesquicomycin;
nicotinamide phosphoribosyl transferase (NAMPT) inhibitors;
microtubule lysin;
enediyne (e.g., calicheamicin);
anthracycline derivatives (PNUs) (e.g., doxorubicin);
inhibitors of the pyrrolyl Kinesin Spindle Protein (KSP);
candidiasis;
drug efflux pump inhibitors;
mountain Zhuo Meisu;
amanitine (e.g., α -amanitine); and
camptothecins (e.g., irinotecan, delutinacon).
50. The antibody-linker conjugate of any one of claims 47 to 49 wherein the chemical spacer (Sp 2 ) Including self-cleaving moieties.
51. The antibody-linker conjugate according to claim 50, wherein the self-cleaving moiety is directly attached to the payload B.
52. The antibody-linker conjugate of claim 50 or 51 wherein the self-cleaving moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety.
53. The antibody-linker conjugate of any one of claims 34 to 52 wherein the antibody is an IgG antibody, particularly an IgG1 antibody.
54. The antibody-linker conjugate according to claim 53, wherein theThe linker-conjugated Gln residues are comprised in the Fc domain of said antibody, in particular wherein the linker-conjugated Gln residues are C of an IgG antibody H Gln residue Q295 of the 2 domain (EU numbering).
55. The antibody-linker conjugate according to claim 53, wherein the linker-conjugated Gln residue has been introduced into the heavy or light chain of the antibody by molecular engineering.
56. The antibody-linker conjugate of claim 55, wherein Gln residues introduced into the heavy or light chain of the antibody by molecular engineering are C of a non-glycosylated IgG antibody H 2 domain N297Q (EU numbering).
57. The antibody-linker conjugate according to claim 55, wherein the Gln residue introduced into the heavy or light chain of the antibody by molecular engineering is contained in a peptide that has been (a) integrated into the heavy or light chain of the antibody, or (b) fused to the N-terminus or C-terminus of the heavy or light chain of the antibody.
58. The antibody-linker conjugate according to claim 57, wherein the peptide comprising a Gln residue has been fused to the C-terminus of the heavy chain of the antibody.
59. The antibody-linker conjugate of any one of claims 53 to 55 or 57 to 58, wherein the IgG antibody is a glycosylated IgG antibody, particularly wherein the IgG antibody is at C H Glycosylation at residue N297 (EU numbering) of the 2 domain.
60. The antibody-linker conjugate of any one of claims 34 to 59 wherein said antibody is selected from the group consisting of: vibutuzumab, trastuzumab, gemtuzumab, oxtuzumab, avermectin, cetuximab, rituximab, up Lei Tuoyou mab, pertuzumab, vedolizumab, oreuzumab, tolizumab, wu Sinu mab, golimumab, otouzumab, sha Xituo mab, bei Lantuo mab, polotouzumab and enrolment mab.
61. The antibody-linker conjugate of any one of claims 34 to 60 wherein said antibody is selected from the group consisting of: velutinab, gemtuzumab, trastuzumab, oxtuzumab, poltuzumab, enrolment mab, sha Xituo bead mab, bei Lantuo mab.
62. The antibody-linker conjugate of any one of claims 34 to 61, wherein the antibody is either pertuzumab or trastuzumab or enrolment mab.
63. An antibody-drug conjugate comprising:
a) IgG antibodies; and
b) A linker comprising a drug moiety B, wherein the drug moiety B is covalently linked to an amino acid sequence selected from the group consisting of: RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4) or RK-Val-Cit (SEQ ID NO: 54);
wherein the linker is via a C-terminus of the antibody H An isopeptide bond formed between the gamma-carboxamide group of glutamine residue Q295 (EU numbering) of the 2 domain and the primary amine contained in the side chain of the lysine residue contained in the linker is coupled to the IgG antibody.
64. The antibody-drug conjugate of claim 63 wherein drug moiety B is linked to the N-terminus or C-terminus of the amino acid sequence contained in the linker via a self-cleaving moiety.
65. The antibody-drug conjugate of claim 64 wherein the self-cleaving moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety.
66. The antibody-drug conjugate of any of claims 63 to 65 wherein the IgG antibody is a glycosylated IgG antibody, particularly wherein the antibody is at C H Glycosylation at residue N297 (EU numbering) of the 2 domain.
67. The antibody-drug conjugate of any of claims 63-66 wherein the IgG antibody is an IgG1 antibody.
68. The antibody-drug conjugate of any of claims 63 to 67 wherein the IgG antibody is a poloxamer 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.
69. The antibody-drug conjugate of any of claims 63-67 wherein the IgG antibody is trastuzumab or enrolment mab 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.
70. The antibody-drug conjugate of any of claims 63-67 wherein the IgG antibody is enrolment mab 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.
71. The antibody-drug conjugate of any one of claims 63 to 70 wherein the drug is selected from the group consisting of toxins of:
pyrrolobenzodiazepines (e.g., PBDs);
auristatin (e.g., MMAE, MMAF);
maytansinoids (e.g., maytansine, DM1, DM4, DM 21);
sesquicomycin;
nicotinamide phosphoribosyl transferase (NAMPT) inhibitors;
microtubule lysin;
enediyne (e.g., calicheamicin);
anthracycline derivatives (PNUs) (e.g., doxorubicin);
inhibitors of the pyrrolyl Kinesin Spindle Protein (KSP);
candidiasis;
drug efflux pump inhibitors;
mountain Zhuo Meisu;
amanitine (e.g., α -amanitine); and
camptothecins (e.g., irinotecan, delutinacon).
72. Antibody-drug conjugate according to any one of claims 63 to 71, wherein the linker has the structure RKAA-PABC-B, in particular wherein B is an auristatin or a maytansinoid, in particular wherein the auristatin is MMAE, and wherein the maytansinoid is DM1 or maytansinoid.
73. Antibody-drug conjugate according to any one of claims 63 to 71, wherein the linker has the structure RKA-PABC-B, in particular wherein B is an auristatin or a maytansinoid, in particular wherein the auristatin is MMAE, and wherein the maytansinoid is DM1 or maytansinoid.
74. The antibody-drug conjugate of any one of claims 63 to 71, wherein the linker has the structure ARK-PABC-B, in particular wherein B is an auristatin or a maytansinoid, in particular wherein the auristatin is MMAE, and wherein the maytansinoid is DM1 or maytansinoid.
75. Antibody-drug conjugate according to any one of claims 63 to 71, wherein the linker has the structure RKR-PABC-B, in particular wherein B is an auristatin or a maytansinoid, in particular wherein the auristatin is MMAE, and wherein the maytansinoid is DM1 or maytansinoid.
76. Antibody-drug conjugate according to any one of claims 63 to 71, wherein the linker has the structure RK-Val-Cit-PABC-B, in particular wherein B is auristatin or maytansinoid, in particular wherein the auristatin is MMAE, and wherein the maytansinoid is DM1 or maytansinoid.
77. A linker construct comprising the structure:
(Sp 1 )-RK-(Sp 2 )-B-(Sp 3 ) Or (b)
(Sp 1 )-B-(Sp 2 )-RK-(Sp 3 ) The method comprises the steps of carrying out a first treatment on the surface of the Wherein the method comprises the steps of
·(Sp 1 ) Is a chemical spacer or is absent;
·(Sp 2 ) Is a chemical spacer or is absent;
·(Sp 3 ) Is a chemical spacer or is absent;
r is arginine or an arginine derivative or arginine mimetic;
K is lysine or a lysine derivative or a lysine mimetic;
b is the connection portion or payload.
78. The linker construct according to claim 77, wherein said chemical spacer (Sp 1 )、(Sp 2 ) Sum (Sp) 3 ) Each independently comprises 0 to 12 amino acid residues.
79. The linker construct of claim 77 or 78, wherein said linker comprises no more than 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 amino acid residues.
80. The linker construct of any one of claims 77 to 79, wherein the net charge of said linker is neutral or positive.
81. The linker construct of any one of claims 77 to 80, wherein said linker does not comprise a negatively charged amino acid residue.
82. The linker construct according to any one of claims 77 to 81, wherein said linker comprises the amino acid sequence RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4) or RK-Val-Cit (SEQ ID NO: 54).
83. The linker construct of any one of claims 77 to 82, wherein B is a linking moiety.
84. The linker construct of claim 83, wherein said linking moiety B comprises
Bio-orthogonal labelling group, or
Non-bioorthogonal entities for cross-linking.
85. The linker construct according to claim 84, wherein said bio-orthogonal labeling group or said non-bio-orthogonal entity for cross-linking consists of or comprises at least one molecule or moiety selected from the group consisting of:
-N-N≡N or-N 3 ；
·Lys(N 3 )；
Tetrazine;
alkynes;
strained cyclooctyne;
·BCN；
strained olefins;
photoreactive groups;
aldehyde;
acyl trifluoroborates;
protein degrading agent ('PROTAC');
cyclopentadiene/spirocyclopentadiene;
a thio-selective electrophile;
-SH; and
cysteine.
86. The linker construct according to any one of claims 77 to 85, wherein said linker construct consists of or comprises the structure RKAA-B, in particular wherein B is Lys (N 3 ) Or cysteine.
87. The linker construct according to any one of claims 77 to 85, wherein said linker construct consists of or comprises the structure RKA-B, in particular wherein B is Lys (N 3 ) Or cysteine.
88. The linker construct according to any one of claims 77 to 85, wherein said linker construct consists of or comprises structure ARK-B, in particular wherein B is Lys (N 3 ) Or cysteine.
89. The linker construct according to any one of claims 77 to 85, wherein said linker construct consists of or comprises structure B-RKR, in particular wherein B is Lys (N 3 ) Or cysteine.
90. The joint construct according to any one of claims 77 to 82, wherein B is a payload.
91. The joint construct of claim 90, wherein the payload comprises at least one of:
toxins;
cytokines;
growth factors;
radionuclides;
hormones;
antiviral agents;
an antimicrobial agent;
fluorescent dye;
immunomodulators/immunostimulants;
half-life increasing moiety;
a solubility-increasing moiety;
polymer-toxin conjugate;
nucleic acid;
biotin or streptavidin moiety;
vitamins;
protein degrading agent ('PROTAC');
a target binding moiety; and/or
Anti-inflammatory agents.
92. The linker construct of claim 91, wherein said toxin is at least one selected from the group consisting of:
pyrrolobenzodiazepines (e.g., PBDs);
auristatin (e.g., MMAE, MMAF);
maytansinoids (e.g., maytansine, DM1, DM4, DM 21);
Sesquicomycin;
nicotinamide phosphoribosyl transferase (NAMPT) inhibitors;
microtubule lysin;
enediyne (e.g., calicheamicin);
anthracycline derivatives (PNUs) (e.g., doxorubicin);
inhibitors of the pyrrolyl Kinesin Spindle Protein (KSP);
candidiasis;
drug efflux pump inhibitors;
mountain Zhuo Meisu;
amanitine (e.g., α -amanitine); and
camptothecins (e.g., irinotecan, delutinacon).
93. The linker construct according to any one of claims 90 to 92, wherein said chemical spacer (Sp 2 ) Including self-cleaving moieties.
94. The linker construct of claim 93, wherein said self-cleaving moiety is directly attached to said payload B.
95. The linker construct of claim 93 or 94, wherein said self-cleaving moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety.
96. The linker construct according to any one of claims 90 to 95, wherein the linker construct consists of or comprises the structure RKAA-PABC-B, in particular wherein B is auristatin or maytansinoid, in particular wherein the auristatin is MMAE, and wherein the maytansinoid is DM1 or maytansinoid.
97. The linker construct according to any one of claims 90 to 95, wherein the linker construct consists of or comprises the structure RKA-PABC-B, in particular wherein B is an auristatin or maytansinoid, in particular wherein the auristatin is MMAE, and wherein the maytansinoid is DM1 or maytansinoid.
98. The linker construct according to any one of claims 90 to 95, wherein the linker construct consists of or comprises the structure ARK-PABC-B, in particular wherein B is auristatin or maytansinoid, in particular wherein the auristatin is MMAE, and wherein the maytansinoid is DM1 or maytansinoid.
99. The linker construct according to any one of claims 90 to 95, wherein the linker construct consists of or comprises the structure B-PABC-RKR, in particular wherein B is auristatin or maytansinoid, in particular wherein the auristatin is MMAE, and wherein the maytansinoid is DM1 or maytansinoid.
100. The linker construct according to any one of claims 90 to 95, wherein said linker construct consists of or comprises the structure RK-Val-Cit-PABC-B, in particular wherein B is an auristatin or maytansinoid, in particular wherein said auristatin is MMAE, and wherein said maytansinoid is DM1 or maytansinoid.
101. Use of the linker construct according to any one of claims 77 to 100 in the production of an antibody-linker conjugate by microbial transglutaminase.
102. The use of claim 99, wherein the antibody is an IgG antibody, in particular an IgG1 antibody.
103. The use of claim 101 or 102, wherein the antibody is either pertuzumab or trastuzumab or enrolment mab.
104. A pharmaceutical composition comprising:
a) The antibody-linker conjugate of any one of claims 34 to 63, in particular wherein the antibody-linker conjugate comprises at least one payload;
or (b)
b) The antibody-drug conjugate of any one of claims 64 to 76; and is also provided with
The pharmaceutical composition comprises at least one pharmaceutically acceptable ingredient.
105. The pharmaceutical composition of claim 104, comprising at least one additional therapeutically active agent.
106. The antibody-linker conjugate of any one of claims 34 to 63, particularly wherein said antibody-linker conjugate comprises at least one payload, the antibody-drug conjugate of any one of claims 64 to 76, or the pharmaceutical composition of claim 104 or 105 for use in therapy and/or diagnosis.
107. The antibody-linker conjugate of any one of claims 34 to 63, particularly wherein said antibody-linker conjugate comprises at least one payload, the antibody-drug conjugate of any one of claims 64 to 76, or the pharmaceutical composition of claim 104 or 105, for use in therapy
● Patients suffering from neoplastic diseases, neurological diseases, autoimmune diseases, inflammatory diseases or infectious diseases,
● Patients at risk of developing neoplastic, neurological, autoimmune, inflammatory or infectious diseases, and/or
● Patients diagnosed with neoplastic disease, neurological disease, autoimmune disease, inflammatory disease or infectious disease.
108. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use according to claim 107, wherein the antibody-linker conjugate or antibody-drug-linker conjugate comprised in the pharmaceutical composition comprises poloxamer and wherein the neoplastic disease is B cell-related cancer.
109. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use according to claim 108, wherein the B cell-related cancer is non-hodgkin's lymphoma, in particular wherein the B cell-related cancer is diffuse large B cell lymphoma.
110. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use according to claim 108 or 109, wherein said antibody-linker conjugate, said antibody-drug conjugate or said pharmaceutical composition is administered in combination with bendamustine and/or rituximab.
111. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use according to claim 107, wherein the antibody-linker conjugate or antibody-drug conjugate comprised in the pharmaceutical composition comprises trastuzumab, and wherein the neoplastic disease is a HER2 positive cancer, in particular HER2 positive breast cancer, gastric cancer, ovarian cancer or lung cancer.
112. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use according to claim 111, wherein said antibody-linker conjugate, said antibody-drug conjugate or said pharmaceutical composition is administered in combination with lapatinib, capecitabine, and/or a taxane.
113. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use according to claim 107, wherein the antibody-linker conjugate or antibody-drug conjugate comprised in the pharmaceutical composition comprises enrolment mab or an enrolment mab variant, and wherein the neoplastic disease is a binding-4 positive cancer, in particular a binding-4 positive pancreatic cancer, lung cancer, bladder cancer or breast cancer.
114. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use according to claim 113, wherein said antibody-linker conjugate, said antibody-drug conjugate or said pharmaceutical composition is administered in combination with a cisplatin-based chemotherapeutic agent and/or palbociclib.
115. The antibody-linker conjugate of any one of claims 34 to 63, particularly wherein said antibody-linker conjugate comprises at least one payload, the antibody-drug conjugate of any one of claims 64 to 76 or the pharmaceutical composition of claim 104 or 105 for use in the manufacture of a medicament for use in the treatment of
● Patients suffering from neoplastic diseases, neurological diseases, autoimmune diseases, inflammatory diseases or infectious diseases,
● Patients at risk of developing neoplastic, neurological, autoimmune, inflammatory or infectious diseases, and/or
● Patients diagnosed with neoplastic disease, neurological disease, autoimmune disease, inflammatory disease or infectious disease.
116. A method of treating or preventing a neoplastic disease, the method comprising administering the antibody-linker conjugate of any one of claims 34 to 63 to a patient in need thereof, particularly wherein the antibody-linker conjugate comprises at least one payload, the antibody-drug conjugate of any one of claims 64 to 76, or the pharmaceutical composition of claim 104 or 105.
CN202180073108.1A 2020-10-25 2021-10-25 Means and methods for producing antibody-linker conjugates Pending CN116801910A (en)

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