Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/545—Heterocyclic compounds
• • A—HUMAN NECESSITIES
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  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/68033—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a maytansine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
  • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
  • A61K47/6855—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D257/00—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms
  • C07D257/02—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms not condensed with other rings
  • C07D257/08—Six-membered rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
  • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
  • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
  • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
  • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
  • C07D498/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains three hetero rings
  • C07D498/14—Ortho-condensed systems
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  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
  • C07F9/6524—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having four or more nitrogen atoms as the only ring hetero atoms
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  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
  • C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
  • C07F9/65583—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system each of the hetero rings containing nitrogen as ring hetero atom
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  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
  • C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
  • C07F9/65586—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system at least one of the hetero rings does not contain nitrogen as ring hetero atom
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  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
  • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings

Definitions

• the invention relates to the field of bioconjugation of functional entities (payloads) to targeting agents, in particular biological targeting agents, such as antibody drug conjugates (ADCs), where one or more payload molecules are conjugated to a targeting agent, as for example monoclonal antibody.
• ADCs antibody drug conjugates
• the present invention relates to novel hydrophilic tetrazine molecules and their preparation, which tetrazines allow a more efficient conjugation of payload molecules to targeting agents, like monoclonal antibodies.
• the present invention also relates to particular tetrazine intermediates useful for the preparation of correspondingly functionalized payload molecules.
• the present invention also relates to respective conjugates, in particular bio-conjugates and methods of their preparation.
• the invention also relates to the use of such conjugates of the present invention for use in medicine, to corresponding pharmaceutical compositions as well as to corresponding diagnostic and analytical kits.
• ADCs are a fast growing class of oncology therapeutics that perceive major attention, which is reflected in the growing number of approved ADC drugs and increasing numbers of clinical trials.
• mAbs monoclonal antibodies
• DARs drug-to-antibody ratios
• a solution to overcome these limitations is the production of homogenous ADCs/radioimmunoconjugates (RICs) via site-specific conjugation methods.
• Site-specific ligation methods offer the possibility for tight control of the DAR, pharmacokinetic properties and production of uniform batches of administered drugs. This also leads to an improvement of the therapeutic index, because side effects of unwanted species of ADCs are erased.
• Another substantial component of every ADC is the linker used for attachment of the payload.
• SUBSTITUTE SHEET (RULE 26)
• linker chemistry also influences the properties of the actual released active metabolite. Increasing hydrophilicity for example leads to decreased rates diffusion across membranes and higher retention inside the cell, as well as less sensitivity to multi drug resistance mechanism of cancer cells.
• US2019247513A1 discloses tetrazine compounds and dienophile capable of undergoing inverse electron demand Diels Alder reaction with the said tetrazines and their use in bio-orthogonal drug activation.
• the Compounds 333 and 14.5 to 14.7 relate to tetrazine derivatives presenting a fluorescent payload, spaced apart from the tetrazine core by a spacer of various chemical nature, like aryl or peptidyl or alkyl-based spacers as well as polyoxyalkly-based hydrophylic groups.
• WO2020256544A1 relates to substituted tetrazines characterized by high click conjugation yield.
• the tartrazine core is attached to pyridyl-based spacers, themselves linked to a payload R87 (such as a cytotoxic drug) and/or polyoxyalkyl hydrophilic groups through glutarly groups.
• Mao et al. (Angew. Chem Int Ed (2021), 602393-2397) as well as W02020239039A1 disclose tetrazine compounds in which an hydrophilic or chemical moieties capable of forming a chemical bond (such as carboxyl, hydroxyl or phosphate-based groups) are spaced apart from the tetrazine core by alkyl-based spacers.
• an hydrophilic or chemical moieties capable of forming a chemical bond such as carboxyl, hydroxyl or phosphate-based groups
• Mao et al describes also compounds in which said hydrophilic or chemical moieties capable of forming a chemical bond are replaced by fluorescent groups.
• Shainyan.B. A et. al (CHEMICAL ABSTRACTS SERVICE, COLUMBUS, OHIO, US; Data base accession no. 1983: 453711) relates to tetrazines presenting alkylsulphone groups as para-substituents.
• W02014081301A1 discloses tetrazines whereby the tetrazine core is linked to various hydrophilic or chemical moieties capable of forming a chemical bond via aryl-based spacers.
• Oiler-Salvia et. al (Angew. Chem Int Ed (2018), 57 2831-2834) relates to the field of antibody drug conjugates and discloses a construct in which trastuzumab was site-selectively conjugated to tetrazine-modified monomethyl auristatin E (MMAE) via inverse-electron demand Diels-Alder cycloaddition.
• MMAE monomethyl auristatin E
• the tetrazine handle used for said site-specific conjugation consists of an aryl moiety attached to the tetrazine core.
• Handula et al ( Molecules 2021, 26, 4640) relates to the use of bio-orthogonal reactions such as tee I EDDA reactions in pre-targeting strategies.
• Various tetrazine surrogates are disclosed as suitable dienes, which dienes are characterized by the presence of a p-substituted tetrazine core presenting alkyl and/or aryl spacers.
• Fig. 2 of the disclosure relates to a classification of said tetrazines surrogates based on the respective reactivity.
• the above-mentioned problem was surprisingly solved by the provision of 1 ,2,4,5- tetrazines functionalized payload molecules carrying as tetrazine C-substituent a small hydrophilic group, as for example a phosphonate residue.
• the invention enables easy use of payloads for bioorthogonal bioconjugation via SPIEDAC in aqueous buffers without the need for possibly disruptive added organic solvents and therefore use of sensitive biological agents. It also aids in prevention of aggregation and better solubility of formed bioconjugated agents. Due to its relatively small size it should not exhibit much steric hindrance compared to bigger solubilizing units, like PEGs, glycosides, etc. and allow for attachment of minimal size payloads.
• the above-mentioned problem is solved by the provision of derivatives of phosphonate group carrying tetrazines further carrying as chemical moieties allowing for coupling with a payload molecule, for example via amide coupling or formation of carbamates.
• Corresponding tetrazine-functionalized payload molecules are represented by the general formula I as referred to herein below; the corresponding tetrazine-functionalized intermediates are represented by the general formula II as referred to herein below.
• FIG. 1 IEDDA of a TCO derivative, incorporated site-specifically in a mAb, with a 1 ,2,4,5-tetrazine.
• FIG. 1 Purification of TrastuzumabA132TCO*-5; A: Superdex S200 run, B: Coomassie stained SDS-PAGE analyzing fractions 10-20 of the S200 run
• ADC antibody drug conjugate
• BCN 2-amino-6-(9-biocyclo[6.1.0]non-4-ynylmethoxycarbonylamino)hexanoid acid
• DI PEA /V,/V-diisopropylethylamine
• HATU 1-[Bis(dimethylamino)methylene]-1 H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate, a coupling agent
• HOBt Hydroxybenzotriazole
• MMAE Monomethyl auristatin E ((S)-/V-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1 S,2F?)-1- hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3- methoxy-5-methyl-1-oxoheptan-4-yl)-/ ⁇ /,3-dimethyl-2-((S)-3-methyl-2- (methylamino)butanamido)butanamide), a anti-neoplastic agent
• NLS nuclear localization signal
• O-tRNA orthogonal tRNA
• O-RS orthogonal RS
• PBS phosphate buffered saline
• PNP Chloroformate 4-Nitrophenyl chloroformate
• POI polypeptide of interest
• pRS prokaryotic RS
• ptRNA prokaryotic tRNA
• PylRS AF mutant M. mazei pyrrolysyl tRNA synthetase comprising amino acid substitutions
• SDS-PAGE sodium sodecyl sulfate polyacrylamide gel electrophoresis
• SPIEDAC Strain-promoted Inverse Electron-Demand Diels-Alder cycloaddition
• 5-TAMRA-OSu 5-Carboxytetramethylrhodamine N-succinimidyl ester, a fluorophore
• TCO Trans-cyclooctene
• TCO-Lys N-£-((trans-Cyclooct-4-en-1-yloxy)carbonyl)-L-lysine
• TCO*-Lys N-£-((trans-Cyclooct-2-en-1-yloxy)carbonyl)-L-lysine
• TCO # -Lys N-£-((trans-Cyclooct-3-en-1-yloxy)carbonyl)-L-lysine
• TCO- E- Lys N6-(( ((R, E)-cyclooct-4-en-1-yl) oxy)carbonyl)-L-lysine
• UNAA unnatural amino acid, synonym to ncAA
• U6 promoter promoter that normally controls expression of the U6 RNA (a small nuclear RNA) in mammalian cells
• purified refers to the state of being free of other, dissimilar compounds with which a compound of the invention is normally associated in its natural state, so that the “purified”, “substantially purified,” and “isolated” subject comprises at least 0.5%, 1%, 5%, 10%, or 20%, or at least 50% or 75% of
• SUBSTITUTE SHEET (RULE 26) the mass, by weight, of a given sample.
• these terms refer to the compound of the invention comprising at least 95, 96, 97, 98, 99 or 100%, of the mass, by weight, of a given sample.
• the terms “purified”, “substantially purified,” and “isolated” when referring to a nucleic acid or protein also refers to a state of purification or concentration different than that which occurs naturally, for example in a prokaryotic or eukaryotic environment, like, for example in a bacterial or fungal cell, or in the mammalian organism, especially human body.
• nucleic acid or protein or classes of nucleic acids or proteins, described herein may be isolated, or otherwise associated with structures or compounds to which they are not normally associated in nature, according to a variety of methods and processes known to those of skill in the art.
• one or more or the similar term “at least one” refers to e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.
• any numerical value and any inclusive range falling within that range is specifically disclosed, including its upper and lower end value.
• every range of values disclosed herein should be understood to mean every value and narrower range that falls within the broader range.
• substantially describes a range of values of from about 80 to 100%, such as, for example, 85-99.9%, in particular 90 to 99.9%, more particularly 95 to 99.9%, or 98 to 99.9% and especially 99 to 99.9%.
• “Predominantly” refers to a proportion in the range of above 50%, as for example in the range of 51 to 100%, particularly in the range of 75 to 99,9%; more particularly 85 to 98,5%, like 95 to 99%.
• halogen denotes in each case a fluorine, bromine, chlorine or iodine radical, in particular a fluorine radical.
• Alkyl relates to a straight-chain or branched alkyl group having from 1 to 6, in particular 1 to 4 or 1 , 2 or 3 carbon atoms. Examples include methyl, C 1 -C 4 -alkyl residues, such as methyl, ethyl, n-propyl, /so-propyl, n-butyl, 2-butyl, /so-butyl or tert-butyl; n-pentyl, 1- methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 1 -ethylpropyl, 1,1- dimethylpropyl, 1,2-dimethylpropyl; n-hexyl, 1 -methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1 -di methyl butyl, 1 ,2-dimethylbutyl, 1,3-di methyl butyl, 4-
• “Lower alkyl” relates to a straight-chain or branched alkyl group having 1 , 2, 3 or 4, in particular 1 or 2 carbon atoms. Examples include methyl, ethyl, n-propyl, /so-propyl, n-butyl, 2-butyl, /so-butyl or tert-butyl.
• Alkenyl relates to a mono-unsaturated hydrocarbon radical comprising a single chemical carbon-carbon double bond, having 2, 3, 4, 5 or 6 carbon atoms, e.g. vinyl, allyl (2- propen-1-yl), 1 -propen-1 -yl, 2-propen-2-yl, methallyl (2-methylprop-2-en-1-yl) and the like.
• “Lower alkenyl” relates to a mono-unsaturated hydrocarbon radical comprising a single chemical carbon-carbon double bond, having 2, 3 or 4 carbon atoms, e.g. vinyl, allyl (2- propen-1-yl), 1 -propen-1 -yl, 2-propen-2-yl, methallyl (2-methylprop-2-en-1-yl) and the like.
• Alkinyl and “lower alkinyl” relate to the analogs of the abovementioned alkenyl or lower alkenyl groups and represent to a mono-unsaturated hydrocarbon radical comprising a single chemical carbon-carbon triple bond.
• Alkylene relates to a straight-chain or branched alkylene group having froml to 6, in particular 1 to 4 carbon atoms. Examples include methylene, ethylene, 1 ,2-ethylene, 1,3- propylene, isopropylene; 1-4-butylene, 1-5-pentylene 1-6- hexylene and the respective branched analogues thereof.
• “Lower alkylene” relates to a straight-chain or branched alkylene group having from 1 to 4 carbon atoms. Examples include methylene, ethylene, 1,2-ethylene, 1,3-propylene, isopropylene, 1-4-butylene and the respective branched analogues thereof.
• Alkoxy relates to a radical of the formula R-O-, wherein R is a straight-chain or branched alkyl group as defined above having from 1 to 6, in particular 1 to 4 or 1 to 3 carbon atoms as defined herein.
• R is a straight-chain or branched alkyl group as defined above having from 1 to 6, in particular 1 to 4 or 1 to 3 carbon atoms as defined herein.
• Non limiting examples are methoxy, ethoxy, n-propoxy, /so- propoxy, n-butoxy, 2-butoxy, /so-butoxy or tert-butoxy.
• Alkyleneoxy relates to a radical of the formula -R-O-, wherein R is a straight-chain or branched alkylene group having from 1 to 6, in particular 1 to 4 or 1 to 3 carbon atoms as defined herein.
• “Lower alkyleneoxy” relates to a radical of the formula -R-O-, wherein R is a straightchain or branched lower alkylene group having from 1 to 4 or 1 to 3 carbon atoms as defined herein. Examples include methyleneoxy, ethyleneoxy, 1 ,2-ethyleneoxy, 1,3-propyleneoxy, isopropyleneoxy and 1-4-butyleneoxy.
• Polyalkyleneoxy relates to a moiety comprising at least two, as for example 2 to 20, 2 to 15, 2 to 10 or 2 to 5 repetitive units of covalently linked, identical or different, in particular identical, lower alkyleneoxy groups having at least two carbon atoms, as defined above, in particular polyethyleneoxy and polypropyleneoxy groups, having 2 to 20, 2 to 15, 2 to 10 or 2 to 5 identical repetitive units.
• Alkenoxy relates to a radical of the formula R-O-, wherein R is a straight-chain or branched alkenyl group as having from 1 to 6, in particular 1 to 4 or 1 to 3 carbon atoms as defined herein.
• Alkanoyloxy relates to a radical of the formula R-(CO)-O-, wherein R is a straightchain or branched alkyl group having from 1 to 6, in particular 1 to 4 or 1 to 3 carbon atoms as defined herein.
• Alkylaminocarbonyloxy relates to a radical of the formula R-NH-(CO)-O-, wherein R is a straight-chain or branched alkyl group having from 1 to 6, in particular 1 to 4 or 1 to 3 carbon atoms as defined herein.
• Alkylthio relates to a radical of the formula R-S-, wherein R is an alkyl radical having from 1 to 4, preferably from 1 to 3 carbon atoms as defined herein.
• Alkylamino relates to a radical of the formula R-NH- wherein R is an alkyl radical having from 1 to 6, in particular from 1 to 4 carbon atoms as defined herein. Examples include methylamino, ethylamino, n-propylamino, iso-propylamino, n-butylamino, 2- butylamino, iso-butylamino, tert-butylamino and the like.
• Dialkylamino relates to a radical of the formula RR’N- wherein R and R’ are independently of each other an alkyl radical having from 1 to 6, in particular from 1 to 4 carbon atoms as defined herein. Examples include dimethylamino, diethylamino, N-methyl- N-ethylamino and the like.
• Alkenylamino relates to a radical of the formula R-NH- wherein R is an alkenyl radical having from 2 to 6, in particular from 2 to 4 carbon atoms as defined herein. Examples include vinylamino, allylamino (2-propen-1-yl-amino), 1 -propen-1 -yl-amino, 2-propen-2-yl- amino, methallylamino (2-methylprop-2-en-1 -yl-amino) and the like.
• N-Alkyl-N-alkenylamino relates to a radical of the formula RR’N- wherein R is an alkyl radical having from 1 to 6, in particular from 1 to 4 carbon atoms as defined herein and R’ an alkenyl radical having from 2 to 6, in particular from 2 to 4 carbon atoms as defined herein.
• Examples include N-methyl-N-vinylamino, N-methyl-N-allylamino (N-methyl-N-2-propen-1 -yl- amino), N-methyl-N-1 -propen-1 -yl-amino, N-methyl-N-2-propen-2-yl-amino, N-methyl-N- methallylamino (N-methyl-N-2-methylprop-2-en-1 -yl-amino) and the like.
• Dialkenylamino relates to a radical of the formula RR’N- wherein R and R’ are independently of each other an alkyl radical having from 2 to 6, in particular from 2 to 4 carbon atoms as defined herein. Examples include divinylamino, diallylamino (di-(2-propen- 1-yl)-amino), N-vinyl-N-allyl-amino and the like.
• Aryl relates to monovalent mono- or polycyclic aromatic moieties, in particular having 6 to 14 ring carbon atoms, in particular, phenyl, fluorenyl, naphthenyl and phenantrenyl
• “Arylene” relates to the bivalent analog of the above-mentioned aryl groups, in particular 1,2-, 1,3- and 1,4- phenylene,
• Halogen relates to F, Cl, Br or I
• substituted are selected from halogen, C1-C 4 - alkyl, CN, CF3, hydroxyl, -O-CF3, C 1 -C 4 -alkoxy, C2-C 4 -alkanoyloxy, C1-C 4 - alkylaminocarbonyloxy and C 1 -C 4 -alkylthio; carboxy and carboxy-C 1 -C 4 -alkyl.
• substituted means that a radical is substituted with 1 , 2 or 3, especially 1 or 2, substituent(s).
• a “linkage” is formed between two neighbored structural motifs of a compound of the invention and is, unless otherwise indicated, either a chemical bond, or is selected from a ether, thioether, ester, amide, carbamate, dicarbamate, carbonate, hydrazine, urea, alkylene oxide or linear or branched polyalkylene oxide linkage in any possible orientation.
• An “ether” linkage contains at least one group of the type: (-O-).
• a “thioether” linkage contains at least one group of the type: (-S-).
• a “dicarbamate” linkage contains at least one group of the type:
• a “hydrazine” linkage contains at least one group of the type: -NH-NH-
• R independently of each other may represent H or lower alkyl, lower alkenyl or lower alkenyl, in particular methyl , or ethyl; R’ represents a lower alkylene group or lower alkenylene group; in particular methylene or ethylene.
• a “cleavable group” encompasses any group, which may be cleaved enzymatically or chemically, in particular under in vivo or ex vivo conditions; an enzymatic cleavage may be effected, for example, through the action of a protease; a chemical cleavage, may be effected for example through hydrolytic cleavage or reductive cleavage of S-S bonds.
• a “tetrazine” group represents, unless otherwise defined, a residue that consists of a six-membered aromatic ring containing four nitrogen atoms with the molecular formula -C2N4-, in particular derived from the 1 ,2,4,5-tetrazine or s- tetrazine isomer, and liked to neighboring groups via ring carbon positions 3 and 6.
• a “tetrazine-reactive group” is a chemical moiety that has the ability to chemically react, in particular, via a so-called “bioorthogonal” or “click reaction”, with a tetrazine group as defined herein.
• tetrazine reactive group is selected from dienophiles. More particularly, it is selected from dienophiles having the ability to react in a biological environment with the tetrazine group.
• isonitrile groups norbornene groups, bicyclononynyl groups, cyclooctenyl groups,
• SUBSTITUTE SHEET (RULE 26) cyclooctinyl groups, cyclopropenyl groups, cyclobutynyl groups, and spirohexenyl groups and their stereoisomers, alkene or allyl groups or dihydro azete groups.
• Tetrazine ligation refers to the reaction of a trans-cyclooctene and an s-tetrazine in an inverse-demand Diels Alder reaction followed by retro Diels Alder reaction to eliminate nitrogen (N2). A reaction of this type proceeds with high velocity, allowing bio molecule modification at extremely low concentrations.
• Compounds as herein described may contain one or more asymmetric elements such as stereogenic centers, stereogenic axes and the like, e.g. asymmetric carbon atoms, so that the compounds can exist in different stereoisomeric forms. These compounds can be, for example, racemates or optically active forms. All stereoisomers, diastereomers, Z- and E- forms, in purified and mixture forms are included. Accordingly, when a compound is recited by specific name or a class of compounds is recited, all these forms are intended to be included.
• Compounds as herein described may also exist in more than one form of structural isomers also designated as constitutional isomers or regioisomers. These are molecules that differ only in the different sequence of their atoms or atomic groups while having the same gross formula.
• any such potential stereo- or regiosomeric form or mixture of more than one stereo- and /or regiosomeric form is within the scope of the present invention.
• An “inverse electron-demand Diels-Alder (IEDDA) cycloaddition” is a reaction between an electron-poor diene and an electron-rich dienophile and represents only one example of different types of “bioorthogonal reactions”.
• the diene used may be a 1 ,2,4,5-tetrazine or a 1 ,2,4-triazine.
• the dienophiles encompass a variety of molecules including strained cyclic alkenes, such as trans-cyclooctenes (TCO, norbornenes, cyclopropenes or azetines). Of these, the reaction between a tetrazine and TCO is the fastest reported to date and suitable for in vivo applications (Smeek et al, Current Opinion in Chemical Biology Volume 60, February 2021, Pages 79-88).
• bioorthogonal refers to any chemical reaction that can occur inside of living systems, i.e. in aqueous environment, without interfering with native biochemical processes.
• Tetrazine ligation may for example be mentioned as one type of bioorthogonal reaction.
• Bioorthogonal chemistry typically proceeds in two steps. First, a cellular substrate is modified with a bioorthogonal functional group (also designated chemical reporter) as for example one of the above-identified “tetrazine reactive groups”.
• a cellular substrate include for example immunoglobulins, like natural or recombinant antibodies, etc.
• the chemical reporter must not
• SUBSTITUTE SHEET (RULE 26) alter the structure of the substrate dramatically to avoid affecting its bioactivity.
• a probe containing the complementary functional group as for example a tetrazine group as described herein, is introduced to react and label the substrate. .
• “Acid or base addition salts” of compounds of the invention are especially addition salts with physiologically tolerated acids or bases.
• Physiologically tolerated acid addition salts can be formed by treatment of the base form of a compound of the invention with appropriate organic or inorganic acids.
• Compounds of the invention containing an acidic proton may be converted into their non-toxic metal or amine addition salt forms by treatment with appropriate organic and inorganic bases.
• the compounds and salts of the invention also comprise the hydrates and solvent addition forms thereof, e.g. hydrates, alcoholates and the like.
• “Physiologically tolerated” acids or bases are in particular those which are tolerated by the system used for the incorporation of the first and second dienophiles (e.g. a biological system such as a translation system used for preparation of polypeptides with trans- cyclooctenyl or cyclooctynyl groups), e.g. which are substantially non-toxic to living cells.
• a biological system such as a translation system used for preparation of polypeptides with trans- cyclooctenyl or cyclooctynyl groups
• a “pharmaceutical composition” comprises in addition to an ADC of the invention one or more substances such as selected from the group consisting of pharmaceutically acceptable preservatives, pharmaceutically acceptable colorants, pharmaceutically acceptable protective colloids, pharmaceutically acceptable pH regulators and pharmaceutically acceptable osmotic pressure regulators. Such substances are described in the art. A more detailed description of pharmaceutical compositions of the invention is provided below.
• the term "effective amount” refers to the amount of a therapy which is sufficient to reduce or ameliorate the severity and/or duration of a disorder or one or more symptoms thereof, prevent the advancement of a disorder, cause regression of a disorder, prevent the recurrence, development, onset or progression of one or more symptoms associated with a disorder, detect a disorder, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy (e.g., prophylactic or therapeutic agent).
• a “polypeptide” is any oligomer of amino acid residues (natural or unnatural, or a combination thereof), of any length, typically but not exclusively joined by covalent peptide bonds.
• a polypeptide can be from any source, e.g., a naturally occurring polypeptide, a polypeptide produced by recombinant molecular genetic techniques, a polypeptide from a cell or translation system, or a polypeptide produced by cell-free synthetic means.
• SUBSTITUTE SHEET (RULE 26) polypeptide is characterized by its amino acid sequence, e.g., the primary structure of its component amino acid residues.
• amino acid sequence of a polypeptide is not limited to full-length sequences, but can be partial or complete sequences. Furthermore, it is not intended that a polypeptide be limited by possessing or not possessing any particular biological activity.
• protein is synonymous with polypeptide.
• peptide refers to a small polypeptide, for example but not limited to, from 2-25 amino acids in length.
• a protein having incorporated into its amino acid sequence at least one ncAA in a particular embodiment is utilized to form a “targeting agent”.
• the primary object of such targeting agent is the formation of a covalent or noncovalent linkage with a particular “target”.
• a secondary object of the targeting agent is the targeted transport of a “payload molecule” to said target.
• said targeting agent has to be combined (reversibly or irreversibly) with at least one “payload molecule”.
• said targeting agent is functionalised by said at least one ncAA.
• the functionalized targeting agent carrying said at least one ncAA may then be linked to said at least one payload molecule through bioconjugation via said ncAA residue.
• Said ncAA is reactive with the payload molecule which in turn carries a corresponding moiety, in the present case a particular tetrazine moiety, reactive with said ncAA residue of the targeting agent.
• the thus obtained bioconjugate allows the transfer of the payload molecule to the intended target.
• the term "to incorporate an unnatural amino acid", e.g., into a targeting polypeptide, refers to the direct addition of an unnatural amino acid to a growing polypeptide chain during primary construction of the target polypeptide, e.g., via translation or chemical synthesis.
• an unnatural amino acid (“UNAA”) can be directly incorporated into targeting polypeptides using any of a number of methods known in the art. While many embodiments utilize orthogonal translation systems as the route of direct incorporation of unnatural amino acids, other direct incorporation methods (e.g., in vitro translation systems, solid-phase synthesis, etc.) can be used alternatively. It will be appreciated that in typical embodiments herein, an unnatural amino acid is preferably incorporated into target polypeptide, i.e. , during construction of the polypeptide, and is not added via post-translational chemical derivatization.
• the unnatural amino acids can be site- specifically incorporated into a targeting polypeptide with high efficiency and high fidelity using “orthogonal tRNA/aminoacyl-tRNA synthetase pairs”.
• translation system refers to the components necessary to incorporate an amino acid in a growing polypeptide chain (protein).
• Components of a translation system can include, e.g., ribosomes, tRNAs, synthetases, mRNA and the like.
• the translation system may be an in vivo or an in vitro translation system.
• An “in vitro translation system” may be a cell-free translation system.
• a cell-free translation system is a system for synthesizing a desired protein by obtaining protein factors required for mRNA translation, e.g., in form of a cell extract, followed by reconstituting this reaction in vitro.
• Such cell-free systems and their use for protein synthesis are known in the art. Examples include extracts of E. coli, wheat germ extract, or rabbit reticulocyte lysate (Spirin and Swartz, Cell-free Protein Synthesis, Wiley VCH Verlag, Weinheim, Germany, 2008).
• An aminoacyl tRNA synthetase is an enzyme capable of acylating a tRNA with an amino acid or amino acid analog.
• the RS used in the methods of the invention is capable of acylating a tRNA with an unnatural amino acid.
• the methods of the invention expediently utilize a “tRNA I aminoacyl tRNA synthetase (tRNA/RS) pair”.
• tRNA/RS tRNA I aminoacyl tRNA synthetase
• the tRNA/RS pair used in the processes of the invention is orthogonal to the translation system.
• orthogonal refers to a molecule (e.g., an orthogonal tRNA (O-tRNA) and/or an orthogonal aminoacyl tRNA synthetase (O-RS)) that is used with reduced efficiency by a translation system of interest (e.g., a cell).
• O-tRNA orthogonal tRNA
• O-RS orthogonal aminoacyl tRNA synthetase
• Orthogonal refers to the inability or reduced efficiency, e.g., less than 20% efficient, less than 10% efficient, less than 5% efficient, or e.g., less than 1% efficient, of an orthogonal tRNA or an orthogonal aminoacyl tRNA synthetase to function with the endogenous aminoacyl tRNA synthetases or endogenous tRNAs of the translation system of interest.
• an orthogonal tRNA in a translation system of interest is acylated by any endogenous aminoacyl tRNA synthetase of a translation system of interest with reduced or even zero efficiency, when compared to acylation of an endogenous tRNA by the endogenous aminoacyl tRNA synthetase.
• an orthogonal aminoacyl tRNA synthetase acylates any endogenous tRNA in the translation system of interest with reduced or even zero efficiency, as compared to acylation of the endogenous tRNA by an endogenous aminoacyl tRNA synthetase.
• Orthogonal tRNA/RS pairs used in processes of the invention preferably have following properties: the O-tRNA is preferentially acylated with the unnatural amino acid of the invention by the O-RS.
• the orthogonal pair functions in the translation system of interest, e.g., the translation system uses the unnatural amino acid acylated O-tRNA to
• SUBSTITUTE SHEET incorporate the unnatural amino acid of the invention in a polypeptide chain. Incorporation occurs in a site specific manner, e.g., the O-tRNA recognizes a selector codon, e.g., an amber stop codon, in the mRNA coding for the polypeptide.
• preferentially acylates refers to an efficiency of, e.g., about 50% efficient, about 70% efficient, about 75% efficient, about 85% efficient, about 90% efficient, about 95% efficient, or about 99% or more efficient, at which an O-RS acylates an O-tRNA with an unnatural amino acid compared to an endogenous tRNA or amino acid of a translation system of interest.
• the unnatural amino acid is then incorporated in a growing polypeptide chain with high fidelity, e.g., at greater than about 75% efficiency for a given selector codon, at greater than about 80% efficiency for a given selector codon, at greater than about 90% efficiency for a given selector codon, at greater than about 95% efficiency for a given selector codon, or at greater than about 99% or more efficiency for a given selector codon.
• the term “selector codon” refers to codons recognized by the O-tRNA in the translation process and not recognized by an endogenous tRNA.
• the O-tRNA anticodon loop recognizes the selector codon on the mRNA and incorporates its amino acid, e.g., an unnatural amino acid, at this site in the polypeptide.
• Selector codons can include, e.g., nonsense codons, such as stop codons, e.g., amber, ochre, and opal codons; four or more base codons; codons derived from natural or unnatural base pairs and the like.
• a selector codon can also include one of the natural three base codons (i.e. natural triplets), wherein the endogenous system does not use said natural triplet, e.g., a system that is lacking a tRNA that recognizes the natural triplet or a system wherein the natural triplet is a rare codon.
• An “anticodon” has the reverse complement sequence of the corresponding codon.
• An O-tRNA/O-RS pair is composed of an O-tRNA, e.g., a suppressor tRNA, or the like, and an O-RS.
• a “suppressor tRNA” is a tRNA that alters the reading of a messenger RNA (mRNA) in a given translation system.
• a suppressor tRNA can read through, e.g., a stop codon, a four base codon, or a rare codon.
• the O-tRNA is not acylated by endogenous synthetases and is capable of decoding a selector codon, as described herein.
• the O-RS recognizes the O-tRNA, e.g., with an extended anticodon loop, and preferentially acylates the O-tRNA with an unnatural amino acid.
• tRNA and the RS used in the processes of the invention can be naturally occurring or can be derived by mutation of a naturally occurring tRNA and/or RS from a variety of
• the tRNA and RS are derived from at least one organism.
• the tRNA is derived from a naturally occurring or mutated naturally occurring tRNA from a first organism and the RS is derived from naturally occurring or mutated naturally occurring RS from a second organism.
• a suitable tRNA/RS pair may be selected from libraries of mutant tRNA and RS, e.g. based on the results of a library screening.
• a suitable tRNA/RS pair may be a heterologous tRNA/synthetase pair that is imported from a source species into the translation system.
• the cell used as translation system is different from said source species.
• a suitable orthogonal O-tRNA can be derived from an archaebacterium, such as Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium such as Haloferax volcanii and Halobacterium species NRC-I, Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, Methanococcus maripaludis, Methanopyrus kandleri, Methanosarcina mazei (Mm), Pyrobaculum aerophilum, Pyrococcus abyssi, Sulfolobus solfataricus (Ss), Sulfolobus tokodaii, Thermoplasma acidophilum, Thermoplasma volcanium, or the like, or a eubacterium, such as Escherichia coli, Thermus thermophilus, Bacillus subtilis, Bacillus stearother
• eukaryotic sources e.g., plants, algae, protists, fungi, yeasts, animals, e.g., mammals, insects, arthropods, or the like can also be used as sources of O-tRNAs and O-RSs
• the RS is a pyrrolysyl tRNA synthetase (pylRS) capable of acylating a tRNA with the unnatural amino acid of the invention.
• the pyrrolysyl tRNA synthetase used in methods of the invention may be a wildtype or a genetically engineered pylRS.
• wildtype pylRS include, but are not limited to pylRS from archaebacteria and eubacteria such as Methanosarcina mazei, Methanosarcina barkeri, Methanococcoides burtonii,
• SUBSTITUTE SHEET (RULE 26) Methanosarcina acetivorans, Methanosarcina thermophila, and Desulfitobacterium hafniense.
• Pyrrolysyl tRNA synthetase is an aminoacyl tRNA synthetase (RS).
• RSs are enzymes capable of acylating a tRNA with an amino acid or amino acid analog.
• the PylRS of the invention is enzymatically active, i.e. is capable of acylating a tRNA (tRNA Pyl ) with a certain amino acid or amino acid analog, preferably with an UNAA or salt thereof
• archaeal pyrrolysyl tRNA synthetase refers to a PylRS, wherein at least a segment of the PylRS amino acid sequence, or the entire PylRS amino acid sequence, has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at last 99%, or 100% sequence identity to the amino acid sequence of a naturally occurring PylRS from an archaeon, or to the amino acid sequence of an enzymatically active fragment of such naturally occurring PylRS.
• the PylRS of the present invention may comprise a mutant archaeal PylRS, or an enzymatically active fragment thereof.
• mutant archaeal PylRSs or “mutated archaeal PylRSs” differ from the corresponding wildtype PylRSs in comprising additions, substitutions and/or deletions of one or more than one amino acid residue. Preferably, these are modifications which improve PylRS stability, alter PylRS substrate specificity and/or enhance PylRS enzymatic activity. Particularly preferred “mutant archaeal PylRSs” or “mutated archaeal PylRSs” are described in more detail herein below.
• nuclear export signal refers to an amino acid sequence which can direct a polypeptide containing it (such as a NES-containing PylRS of the invention) to be exported from the nucleus of a eukaryotic cell. Said export is believed to be mostly mediated by Crm1 (chromosomal region maintenance 1 , also known as karyopherin exportin 1).
• NESs are known in the art. For example, the database ValidNESs (http://validness.ym.edu.tw/) provides sequence information of experimentally validated NES- containing proteins. Further, NES databases like, e.g., NESbase 1.0 (www.cbs.dtu.
• these NESs comprise the conserved amino acid sequence pattern LxxLxL (SEQ ID NOU 11) or LxxxLxL (SEQ ID NOU 12), wherein each L is independently selected from leucine, isoleucine, valine, phenylalanine and methionine amino acid residues, and each x is independently selected from any amino acid (see La Cour et al., Protein Eng Des Sei 17(6):527-536, 2004).
• nuclear localization signal refers to an amino acid sequence which can direct a polypeptide containing it (e.g., a wild-type archaeal PylRS) to be imported into the nucleus of a eukaryotic cell. Said export is believed to be mediated by binding of the NLS-containing polypeptide to importin (also known as karyopherin) so as to form a complex that moves through a nuclear pore. NLSs are known in the art.
• NLSdb see Nair et al., Nucl Acids Res 31(1), 2003
• cNLS Mapper www.nls-mapper.aib.keio.ac.jp; see Kosugi et al., Proc Natl Acad Sci U S A.
• Mutant archaeal PylRSs of the invention as defined above can be further modified by removing the NLS optionally present in said naturally occurring PylRS where the mutant is derived from and/or by introducing at least one NES.
• the NLS in the naturally occurring PylRS can be identified using known NLS detection tools such as, e.g., cNLS Mapper.
• the removal of a NLS from and/or the introduction of a NES into an archaeal PylRS or mutant thereof can change the localization of the thus modified polypeptide when expressed in a eukaryotic cell, and in particular can avoid or reduce accumulation of the polypeptide in the nucleus of the eukaryotic cell.
• the localization of a PylRS mutant of the invention expressed in a eukaryotic cell can be changed compared to a PylRS or PylRS mutant, which differs from the PylRS mutant of the invention in that it (still) comprises the NLS and lacks the NES.
• the archaeal PylRS of the invention comprises a NES but (still) comprises an NLS
• the NES is preferably chosen such that the strength of the NES overrides the NLS preventing an accumulation of the PylRS in the nucleus of a eukaryotic cell.
• PylRS enzymatic activity is maintained at basically the same level, i.e. the PylRS of the invention has at least 50%, at least 60%, at least 70%, at
• SUBSTITUTE SHEET (RULE 26) least 80%, at least 90%, or at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% of the enzymatic activity of the corresponding wild-type or mutant PylRS.
• the NES is expediently located within the PylRS or mutant PylRS of the invention such that the NES is functional.
• a NES can be attached to the C-terminus (e.g., C-terminal of the last amino acid residue) or the N-terminus (e.g., in between amino acid residue 1 , the N-terminal methionine, and amino acid residue 2) of a wild-type or mutant archaeal PylRS.
• tetrazine compounds of the invention described herein below having the general formula (I) are used for preparation of conjugates, in particular bioconjugates.
• a compound of formula (I) is reacted via its functional tetrazine moiety with a suitable tetrazine-reactive second functional group carried by a conjugation partner, as for example a biomolecule, in particular a targeting molecule, like for example an antibody molecule.
• Said tetrazine-reactive functional group may for example be a cyclooctynyl or transcyclooctenyl group.
• the invention provides processes for preparing such polypeptides, in vivo or in vitro.
• said tetrazine-reactive functional group can be translationally incorporated in a polypeptide that is encoded by a polynucleotide comprising one or more than one selector codon(s).
• the present invention relates to the following main aspects and particular embodiments thereof.
• a first aspect of the present invention relates to payload molecules functionalized by means of a particular tetrazine group, which are adapted to conjugation with a second molecule, in particular a bio-molecule, carrying a functional counterpart group reactive with said tetrazine group of the functionalized payload molecule.
• SUBSTITUTE SHEET (RULE 26) wherein m is 0 or 1 n represents an integer selected from 1 and 2 o is 0 or represents an integer selected from 1 or 2
• A represents a cleavable moiety
• Sp 1 and Sp 2 independently of each other represent a spacer moiety
• X represents a self-immolative moiety
• Y represents a payload residue (or cargo)
• Z represents a hydrophilic group
• m is 0.
• m as 1.
• n 1
• n is 2.
• o 0.
• o is 1.
• o is 2.
• Sp 1 and Sp 2 are different, according to another particular embodiment Sp 1 and Sp 2 are identical.
• residue Z in said compound general formula I of the first embodiment is selected from one of the following hydrophilic groups: a) phosphor and/or sulfur containing hydrophilic moieties Z, in particular (R 1 O) 2 P(O)-, (R 1a O) 2 P(O)-O-, (R 2 O) 3 P-O-, R 3 S(O) 2 -, (R 4 O)S(O) 2 O-, or (R 4a O)S(O) 2 -, wherein residues R 1 to R 4 , R 1a and R 4a are same or different and independently of each other represent H or lower alkyl, in particular methyl or ethyl;
• SUBSTITUTE SHEET (RULE 26) or a salt form, as for example a physiologically tolerated salt, of said phosphor and/or sulfur containing hydrophilic moieties; b) linear or branched mono- or poly-alkylene oxide moieties Z, in particular selected from linear the moieties -((CH2)x-O)y-R 5 , -(O-(CH2)x)y-H and -(O-(CH2) x ) y -OR 6 , and the branched analogues thereof; wherein residues R 5 and R 6 independently of each other represent H or lower alkyl, in particular H, methyl or ethyl; x independently of each other represent an integer selected from 1 , 2, 3 or 4; in particular 1 or 2; and y independently of each other represent an integer from 1 to 20, in particular 1 to 15,
• Z is (R 1 O)2P(O)-, (R 1a O)2P(O)-O- or (R 2 O)sP-O- and more particularly (R 1 O)2P(O)-; wherein residues R 1 ; R 2 and R 1a are same or different independently of each other represent H or methyl or ethyl;
• Z is a linear mono- or poly-alkylene oxide moietiy.
• Z is selected from linear the moieties - ((CH 2 )x-O) y -R 5 , -(O-(CH2)x) y -H and -(O-(CH2)x) y -OR 6 , wherein residues R 5 and R 6 independently of each other represent H, methyl or ethyl;
• x independently of each other represent therein an integer selected from 1 or 2.
• R’, R” and R’ independently of each other are selected from H and C 1 -t o C 4 -alkyl;
• SUBSTITUTE SHEET (RULE 26) d) combinations of at least two identical or, more particularly, different moieties, selected from M1, M2 and M3.
• Moiety M1 is a monocyclic non-substituted aromatic moiety having 6 carbon atoms, in particular 1,4-phenylene, or
• Moiety M2 represents heterocyclic residues of the general formula X, wherein
• One or two of the ring moieties Xi to X4 represents N and the other represent >CH;
• Moiety M3 represents a linear lower-alkylene, in particular -(CH2)ni-, wherein n1 is an integer from 1 or 2; more particularly methylene;
• R’, R” and R’ independently of each other are selected from H and C 1 -to C 4 -alkyl; (Moiety M1); b) heterocyclic residues of the general formula X
• SUBSTITUTE SHEET (RULE 26) wherein one, two or three of the ring moieties Xi to X4 represents N and the other represent >CH; (Moiety M2); c) linear or branched lower-alkylene, in particular -(CH2)ni-, wherein n1 is an integer from 1 to 4; more particularly methylene; (Moiety M3); d) linear or branched polyalkylene oxide moieties, in particular selected from linear the moieties -((CH2)xi-O) y i- or -(O-(CH2)xi) y i- and the branched analogues thereof; wherein x1 independently of each other represent an integer selected from 1 , 2, 3 or 4; in particular 1 or 2; and y1 independently of each other represent an integer from 1 to 20, in particular 1 to 4; (Moiety M4); e) a heteroatom containing moiety selected from
• R are independently of each other selected from H and C 1 - C 4 -alkyl x2 represents an integer selected from 1, 2, 3 or 4; in particular 1 or 2; x3 represents an integer selected from 1, 2, 3 or 4; in particular 1 or 2; and x4 represents an integer selected from 1, 2, 3 or 4; in particular 1 or 2;
• SUBSTITUTE SHEET (RULE 26) (Moiety M5); or f) combinations of at least two identical or, more particularly, different moieties selected from M1, M2, M3 and M4; or combinations of at least two identical or, more particularly, different moieties selected from M1 , M2, M3, M4 and M5.
• Moiety M1 is a monocyclic non-substituted aromatic moiety having 6 carbon atoms, particularly 1 ,2-phenylene 1 ,3-phenylene or 1 ,4 phenylene, more particularly 1,4-phenylene, or
• Moiety M2 represents heterocyclic residues of the general formula X, wherein
• One or two of the ring moieties Xi to X4 represents N and the other represent >CH;
• Moiety M3 represents a linear lower-alkylene, in particular -(CH2)ni-, wherein n1 is an integer from 1 or 2; more particularly methylene;
• Moiety 4 represents a linear polyalkylene oxide moiety, selected from linear the moieties -((CH2)xi-O) y i- or -(O-(CH2)xi) y i-; wherein x1 independently of each other represent an integer selected from 1 or 2; and y1 independently of each other represent an integer from 2 to 20, in particular 2 to 4;
• Moiety 5 represents -N(R””)-, -N(R””)-(CH2)x3-C(O)- or -(CH 2 )X2-N(R””)- wherein
• SUBSTITUTE SHEET (RULE 26) R” are independently of each other selected from H and C 1 - C 4 -alkyl, more particularly H; and x2 represents an integer selected from 1 , 2, 3 or 4; in particular 1 or 2.
• said group A of the compound (I) of anyone of the preceding embodiments is an enzymatically or chemically cleavable linker group, selected from a) a peptidyl group, in particular di-, tri- or tetra-peptidyl group; b) a disulfide group of the formula -(CR 7 R 8 ) n2 -S-S-(C R 7 R 8 ) n2 -X 5 - or X 5 -(CR 7 R 8 ) n2 -S-S-(C R 7 R 8 ) n2 -X 5 - wherein n2 represents an integer from 1 to 4 residues R 7 and R 8 independently of each other are selected from H or lower alkyl, in particular methyl; or two residues R 7 and R 8 together with the carbon atom which they are attached to form a cyclic C 4 -to Cs-alkyl group; and moiety X5 is selected from -C(O
• R 9 is H or lower alkyl; and d) beta-glucuronidase-sensitive cleavable linker groups (glucuronide-linker groups), in particular carrying a beta-glucuronic acid derived trigger residue;
• the cleavable linker is a peptidyl group according to feature a).
• the cleavable linker is a glucuronide-linker group according to feature d).
• said self-immolative group X of a compound (I) of anyone of the preceding embodiments is selected from a) p-amino-benzyl alcohol (PAB) derived groups of the formula
• R 20 independently of each other, represent H or a lower alkyl group
• R 10 and R 11 independently of each other, represent H or lower alkyl group
• R 12 and R 13 independently of each other, represent H, methyl or ethyl, in particular H or methyl, especially H;
• X 1 and X 2 independently of each other represent O, S or NR 10 d) methylene alkoxy carbamates (MAC) type linkages of the formula -OC(O)-NR 13 -C(R 14 R 15 )-(O)-
• R 13 , R 14 , R 15 , and R 16 independently of each other represent H or lower alkyl, in particular, C 1 to C 4 -alkyl.
• the said self-immolative group X is a PAB derived group according to feature a).
• said payload residue Y of a compound of anyone of the preceding embodiments is selected from bioactive compounds; labeling agents, such as in particular dyes, radiolabels, protein degraders, photosensitizers; and chelators.
• said compound of anyone of the preceding embodiments corresponds to acompound of general formula la wherein linkages a, p, y and 5 are independently from each other selected from a chemical bond, or an ether, thioether, ester, amide, carbamate, carbonyl (in particular keto),
• SUBSTITUTE SHEET (RULE 26) dicarbamate, carbonate, hydrazine, urea, alkylene oxide or linear or branched polyalkylene oxide linkage.
• Said polyalkylene oxide linkage selected from linear the moieties -((CH2)xi-O) y i- or - (O-(CH 2 )xi) y i-; wherein x1 independently of each other represent an integer selected from 1 or 2; and y1 independently of each other represent an integer from 2 to 20, in particular 2 to 4;
• linkages a, p, y and 5 each are a chemical bond.
• linkages a, p, y and 5 is a chemical bond.
• linkages p is a chemical bond.
• linkages y is a chemical bond.
• linkages a and p each are a chemical bond.
• linkages a, p and y each are a chemical bond.
• linkages a, p, y each are a chemical bond
• 5 is an ether, thioether, ester, amide, carbamate, carbonyl (in particular keto), dicarbamate, carbonate, hydrazine, urea, alkylene oxide or linear or branched polyalkylene oxide linkage.
• linkages a, p, y each are a chemical bond, and 5 is an, ester, amide, carbamate, dicarbamate, carbonate, alkylene oxide or linear or branched polyalkylene oxide linkage.
• Said polyalkylene oxide linkage selected from linear the moieties -((CH2)xi-O) y i- or - (O-(CH 2 )xi) y i-; wherein x1 independently of each other represent an integer selected from 1 or 2; and y1 independently of each other represent an integer from 2 to 4;
• said spacer Sp 1 of a compound of anyone of the preceding embodiments is selected from one of the following combinations of moieties:
• linkages between said moieties M1, M2, M3 are independently selected from a chemical bond, an ether, thioether, ester, amide, carbamate, dicarbamate, carbonate, hydrazine or urea, and alkylene oxide or a linear or branched polyalkylene oxide linkage.
• said spacer Sp 2 of a compound of anyone of the preceding embodiments is selected from one of the following combinations of moieties
• linkages between said moieties M1 , M2, M3, M4 and M5 are independently selected from a chemical bond, an ether, thioether, ester, amide, carbamate, dicarbamate, carbonate, hydrazine or urea, and alkylene oxide or a linear or branched polyalkylene oxide linkage.
• linkages between said moieties M1 , M2, M3 and M4 are each are a chemical bond.
• the second aspect of the present invention relates to conjugates, and more particularly bio-conjugates. They are formed by the reaction of at least one functionalized payload molecule of the general formula (I) of the above first aspect of the invention, functionalized by means of a particular tetrazine group as defined above. Said payload molecule is adapted to conjugation with a functionalized targeting agent, in particular functionalized bio-molecule, carrying a functional counterpart group, which is reactive with said tetrazine group of the functionalized payload molecule in a biorthogonal chemical reaction.
• a conjugate, and more particularly bio-conjugate is provided, which is obtainable by reacting a functionalized targeting agent, with a tetrazine compound of formula I, of anyone of the preceding embodiments in order to form a covalent linkage between said functionalized targeting agent, and said tetrazine compound of formula I.
• said functionalized targeting agent of the eleventh embodiment is selected from correspondingly functionalized forms of the following entities: viruses, whole cells, phages, liposomes, biomolecules and low- or- high-molecular weight chemical compounds, antibodies, antibody derivatives,
• SUBSTITUTE SHEET (RULE 26) antibody fragments, antibody (fragment) fusions, enzymes, proteins, peptides, peptide mimetics, carbohydrates, monosaccharides, polysaccharides, oligo- or polynucleotides, in particular DNA, RNA, PNA and LNA molecules, aptamers, drugs, glycoproteins, glycans, lipids, polymers, chemotherapeutic agents, receptor agonists and antagonists, cytokines, hormones, steroids, toxins and derivatives thereof.
• the targeting agent is selected from antibodies, antibody derivatives, antibody fragments, and antibody (fragment) fusions.
• a conjugate of anyone of the embodiments eleven and twelve wherein said functionalized targeting agent comprises as a functional group at least one dienophilic moiety reactive with said tetrazin moiety of said compound of formula I.
• said functionalized targeting agent comprises at least one polypeptide sequence, having at least one non-natural amino acid residue within its amino acid sequence, which non-natural amino acid residue comprises at least one dienophile moiety reactive with said tetrazin moiety of said compound of formula I.
• a conjugate of anyone of the embodiments eleven to fourteen is provided, wherein said functionalized biomolecule is a polyclonal or monoclonal immunoglobulin molecule, in particular a monoclonal antibody or fragment thereof.
• a conjugate of anyone of the embodiments eleven to fifteen is provided, which is formed by biorthogonal bioconjugation of a tetrazine-compound of formula I and a functionalized biomolecule carrying a functional group capable of reaction via a Diels-Alder-type cycloaddition reaction, as for example cyclooctinyl-dienophiles, trans-cyclooctenyl-dienophiles, norbornenyl dienophiles, cyclopropenyl dienophiles, cyclobutenyl dienophiles, spirohexenyl dienophiles, BCN dienophiles, azetine dienophiles, or alkenes.
• cyclooctinyl-dienophiles trans-cyclooctenyl-dienophiles
• norbornenyl dienophiles norbornenyl dienophiles
• a conjugate of embodiment sixteen is provided, wherein said functional group capable of reaction via a Diels-Alder-type cycloaddition reaction is selected from
• R 1 is hydrogen, halogen, C 1 -C 4 -alkyl, (R a O) 2 P(O)O-C 1 -C 4 -alkyl, (R b O) 2 P(O)-C 1 - C 4 -alkyl, CF3, CN, hydroxyl, C 1 -C 4 -alkoxy, -O-CF3, C2-Cs-alkenoxy, C2-C5- alkanoyloxy, C 1 -C 4 -alkylaminocarbonyloxy or C 1 -C 4 -alkylthio, C1-C 4 - alkylamino, Di-(C 1 -C 4 -alkyl)amino, C2-C5-alkenylamino, C2-Cs-alkenyl-C 1 -C 4 - alkyl-amino or Di-(C2-C5-alkenyl)amino; and
• R a , R b independently are hydrogen or C2-C5-alkanoyloxymethyl
• R 2 is hydrogen, halogen, C 1 -C 4 -alkyl, (R c O)2P(O)O-C 1 -C 4 -alkyl, (R d O)2P(O)-C 1 -C 4 - alkyl, CF3, CN, hydroxyl, C 1 -C 4 -alkoxy, -O-CF3, C2-Cs-alkenoxy, C2-C5- alkanoyloxy, C 1 -C 4 -alkylaminocarbonyloxy or C 1 -C 4 -alkylthio, C1-C 4 - alkylamino, Di-(C 1 -C 4 -alkyl)amino, C2-C5-alkenylamino, C2-Cs-alkenyl-C 1 -C 4 - alkyl-amino or Di-(C2-C5-alkenyl)amino; and
• R c , R d independently are hydrogen or C2-C5-alkanoyloxymethyl.
• a third aspect of the present invention relates to methods of preparing bio-conjugates.
• a method of preparing a bio-conjugate of anyone of the embodiments eleven to seventeen comprises reaction in an aqueous, optionally buffered reaction medium a tetrazin compound as defined in anyone of the embodiments one to ten with a functionalized biomolecule
• SUBSTITUTE SHEET (RULE 26) carrying a functional dienophile group and performing a Diels-Alder-type cycloaddition reaction between said molecules.
• a fourth aspect of the present invention relates to certain tetrazine intermediates, for example useful for preparing the tetrazine compounds of formula I.
• a tetrazine intermediate of the general formula II is provided, wherein n3 represent an integer selected from 1 or 2
• Sp 1 and Sp 2 are as defined above, linkages a, p, and y are independently from each other selected from a chemical bond, or an ether, thioether, ester, amide, carbonyl (in particular keto), carbamate, dicarbamate, carbonate, hydrazine, urea, alkylene oxide or linear or branched polyalkylene oxide linkage;
• Z represents a phosphor containing hydrophilic group, in particular (R 1 O) 2 P(O)-, (R 1a O) 2 P(O)-O-, and (R 2 O) 3 P-O-;
• R 1 , R 1a and R 2 are same or different and independently of each other represent H or lower alkyl, in particular methyl or ethyl; and even more particularly H;
• R represents H or a chemical group capable of forming a chemical bond, or capable of forming an ether, thioether, ester, such as active esters like succinimidyl- or pentafluorophenyl- ester, amide, carbamate, dicarbamate, carbonate, hydrazine, urea, alkylene oxide or linear or branched polyalkylene oxide linkage; and optionally with the proviso that R does not represent a
• SUBSTITUTE SHEET (RULE 26) chemical protecting group, in particular does not represents a cleavable protecting group, and more particularly not a N-, O-, or S- protecting group.
• R represents an amino or carboxyl group
• linkages a, p, and y each are a chemical bond.
• linkages a is a chemical bond.
• linkages p is a chemical bond.
• linkages y is a chemical bond.
• linkages a and p each are a chemical bond.
• linkages a, p and y each are a chemical bond.
• Z is (R 1 O)2P(O)-, (R 1a O)2P(O)-O-, and (R 2
• R 1 , R 1a and R 2 are same or different and independently of each other represent H or lower alkyl, in particular methyl or ethyl; and even more particularly H;
• a fifth aspect of the present invention relates to methods of preparing certain tetrazine intermediates.
• a method of preparing a tetrazine intermediate of general formula II comprising the steps of: i. reacting a first cyano compound of the general formula III
• R and Sp 2 and n3 are as defined above in the presence of a hydrazine hydrate; ii. subsequent oxidation, in particular of an1 ,4-dihydro-s-tetrazine compound formed in step 1 , as for example with oxidants selected from NaNC>2, Phl(OAc)2, DDQ, or air oxidation iii. optionally isolating the obtained tetrazine compound; and iv. optionally deprotecting the hydroxyl groups of residue Z.
• step i) is performed in the absence of a catalyst.
• step i) is performed in the presence of a catalyst.
• the catalyst is a metal-containing catalyst, as for example Zn(OTf)2.
• the catalyst is a metal-free catalyst.
• the metal-free catalyst is an organic catalyst.
• the organic catalyst is a sulfur containing catalyst.
• the sulfur containing catalyst is selected from 3- mercaptopropionic acid, L-cysteine, glutathione, 2-aminoethanethiol, 1,3-propanedithiol, thioglycolic acid and N-acetyl-L-cysteine, and in particular 3-mercaptopropionic acid.
• reaction step i) is carried out in a molar excess, as for example 1 to 20-fold molar excess, of the hydrazine compound over the compounds of formula (III) and (IV).
• reaction step i) is carried out in an alcoholic solvent, in particular ethanol.
• reaction step ii) is carried out in a molar excess, as for example 1 to 20-fold molar excess, of the oxidant over the compounds of formula (III) and (IV).
• the oxidant of step ii. is NaNC>2.
• the oxidant of step ii. is Phl(OAc)2
• the oxidant of step ii. is air.
• a conjugate as defined in anyone of the embodiments eleven to seventeen, for use in medicine, in particular for use in diagnosis and/or therapy is provided.
• a pharmaceutical composition comprising in a pharmaceutically acceptable carrier at least one conjugate as defined in anyone of the embodiments eleven to seventeen
• a diagnostic or analytical kit comprising at least one tetrazin compound as defined in anyone of the embodiments one to ten.
• tetrazine intermediates of general formula II can be prepared in analogy to methods, which are well known in the art. Suitable methods are found in the various publications cited herein, all of which are incorporated herein by reference in their entireties. Some methods are outlined herein.
• the metal-catalysed one-pot synthesis of tetrazine intermediates of formula II may be performed in line with the disclosure of Yang et al, Angew. Chem Int Ed (2012), 51 , 5222.
• a method of preparing a tetrazine intermediate of general formula II is provided.
• step i) is performed in a one-pot reaction.
• a solution of the cyano educts of the general formulae (III) and (IV) is provided, which is supplemented with a hydrazine compound, in particular hydrazine hydrate, and optionally in the presence of a suitable catalyst.
• catalysts are acidic metal-free organo-catalysts mercapto compounds as for example 3-mercaptopropionic acid, L-cysteine, glutathione, 2-aminoethanethiol, 1 ,3- propanedithiol, thioglycolic acid and N-acetyl-L-cysteine, and in particular 3- mercaptopropionic acid, like 3-mercato propionic acid.
• Alternative catalysts are metalcontaining catalyst, as for example Zn(OTf)2.
• step i) The reaction of step i) is performed under temperature control until completion. Subsequently any resulting dihydro tetrazine intermediate may be oxidized.
• conventional oxidants in particular sodium nitrite/HCI or nitric acid may be applied.
• the mixture of said cyano educts is provided in a suitable solvent.
• suitable solvents that may be used are selected from polar organic solvents, like THF and organic alcohols, in particular ethanol.
• the compounds of formulae (III) and (IV) are provided in a molar ratio in the range of 1 : 10 to 10 : 1 , as for example 1 : 5 to 5: 1. More particularly the compound if formula III is provided in 1 to 10 or 1 to 5-fold molar excess
• the hydrazine compound is added in molar excess over the cyano educts, as for example in a 1 to 20 or 4 to 15 fold excess over compound (III) or (IV)
• the acid catalyst used in catalytic or, more particular, it we molar amounts relative to compound (III) or (IV).
• the accident is applied in at least it we molar, more preferably in a molar excess over the hydrazine compound.
• the reaction temperature is controlled in a range of - 10 to +10 °C.
• step ii. converts the dihydrotetrazine intermediate as formed by step i) to the respective tetrazine.
• a suitable oxidant as for example NaNC>2, , p-benzoquinone, DDQ, or Phl(OAc)2.
• Phl(OAc)2 as oxidant is describe for example by Selvaraj, R. et al in Tetrahedron Lett. 2014; 55(34): 4795-4797
• step iii. for isolating the product of step ii. may be performed by means of conventional purification methods. Chromatographic methods, like flash chromatography or HPLC shall be particularly mentioned.
• the process of the present invention may also comprise step iv., provided that an educt of formula III was applied, wherein residue Z comprises protected, for example esterified hydroxyl groups, and it is intended to make use of the produced tetrazine in deprotected form.
• residue Z comprises protected, for example esterified hydroxyl groups, and it is intended to make use of the produced tetrazine in deprotected form.
• Methods for the protecting esterified hydroxyl groups are well known. Deprotection by means of treatment with trimethylsilylbromide in organic solvent may be mentioned as non-limiting example.
• methylphosphonate tetrazines may be synthesized. These compounds contain functional groups allowing reactive with further chemical moieties, for example, through amide coupling or formation of carbamate or ester linkages. Such chemical moieties are selected from the above identified groups
• SUBSTITUTE SHEET (RULE 26) Y payload residue (cargo) or combinations thereof, as for example -A-X-Y, -A-Y, -X-Y, -A-X-X-Y, -X-X-Y -Sp 2 -A-X-Y, -Sp 2 -A-Y,-Sp 2 -X-Y, -Sp 2 -A-X-X-Y
• Payload molecules Y typically used as constituent of a tetrazine compound of the general formula I may be selected from Bioactive compounds, labeling agents, and chelators. Non-limiting examples thereof are given in the following sections.
• Bioactive compounds include, but are not limited to, the following:
• Bioactive compounds applicable according to the present invention include but are not limited to: small organic molecule drugs, steroids, lipids, proteins, aptamers, oligopeptides, oligonucleotides, oligosaccharides, as well as peptides, peptoids, amino acids, nucleotides, oligo- or polynucleotides, nucleosides, DNA, RNA, toxins, glycans and immunoglobulins.
• the bioactive compound is a low to medium molecular weight compound (e.g. about 200 to 5000 Da, about 200 to about 1500 Da, preferably about 300 to about 1000 Da).
• Exemplary cytotoxic drugs are particularly those which are used for cancer therapy.
• Such drugs include, in general, DNA damaging agents, anti-metabolites, natural products and their analogs, enzyme inhibitors such as dihydro folate reductase inhibitors and thymidylate synthase inhibitors, DNA binders, DNA alkylators, radiation sensitizers, DNA intercalators, DNA cleavers, microtubule stabilizing and destabilizing agents, topoisomerases inhibitors.
• Examples include but are not limited to platinum-based drugs, the anthracycline
• SUBSTITUTE SHEET family of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, taxanes, lexitropsins, the pteridine family of drugs, diynenes, the podophyllotoxins, dolastatins, maytansinoids, differentiation inducers, and taxols.
• Particularly useful members of those classes include, for example, auristatins, maytansines, maytansinoids, calicheamicins, dactinomycines, duocarmycins, CC1065 and its analogs, camptothecin and its analogs, SN-38 and its analogs; DXd, tubulysin M, cryptophycins, pyrrolobenzodiazepines and pyrrolobenzodiazepine dimers (PBDs), pyridinobenzodiazepines (PDDs) and indolinobenzodiazepines (IBDs) (cf.
• auristatins maytansines, maytansinoids, calicheamicins, dactinomycines, duocarmycins, CC1065 and its analogs, camptothecin and its analogs, SN-38 and its analogs
• DXd tubulysin M, cryptophycins, pyrrolobenzodiazepines and
• exemplary drug classes are angiogenesis inhibitors, cell cycle progression inhibitors, P13K/m-TOR/AKT pathway inhibitors, MAPK signaling pathway inhibitors, kinase inhibitors, protein chaperones inhibitors, HDAC inhibitors, PARP inhibitors, Wnt/Hedgehog signaling pathway inhibitors, RNA polymerase inhibitors, and protein degraders (cf. https://pubs.acs.org/doi/10.1021/acschembio.0c00285).
• auristatins examples include dolastatin 10, monomethyl auristatin E (MMAE), auristatin F, monomethyl auristatin F (MMAF), auristatin F hydroxypropylamide (AF HPA), auristatin F phenylene diamine (AFP), monomethyl auristatin D (MMAD), auristatin PE, auristatin EB, auristatin EFP, auristatin TP and auristatin AQ.
• Suitable auristatins are also described in U.S. ;Publication Nos. 2003/0083263, 2011/0020343, and 2011/0070248; PCT Application ; Publication Nos. WO09/117531 , W02005/081711 , W004/010957;
• Exemplary drugs include the dolastatins and analogues thereof including: dolastatin A ( U.S. Pat No. 4,486,414), dolastatin B (U.S. Pat No. 4,486,414), dolastatin 10 (U.S. Pat No.
• SUBSTITUTE SHEET (RULE 26) 4,486,444, 5,410,024, 5,504,191, 5,521 ,284, 5,530,097, 5,599,902, 5,635,483, 5,663,149, 5,665,860, 5,780,588, 6,034,065, 6,323,315), dolastatin 13 (U.S. Pat No. 4,986,988), dolastatin 14 (U.S. Pat No. 5,138,036), dolastatin 15 (U.S. Pat No. 4,879,278), dolastatin 16 (U.S. Pat No. 6,239,104), dolastatin 17 (U.S. Pat No. . 6,239,104), and dolastatin 18 (U.S. Pat No. . 6,239,104), each patent incorporated herein by reference in their entirety.
• maytansines maytansinoids, such as DM-1 and DM-4, or maytansinoid analogs, including maytansinol and maytansinol analogs, are described in U.S. Patent Nos. 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371 ,533; 5,208,020; 5,416,064; 5,475,092; 5,585,499; 5,846,545; 6,333,410; 6,441,163; 6,716,821 and 7,276,497.
• PBDs PBDs
• dimers and analogs include but are not limited to those described in [Denny, Exp. Opin. Ther. Patents, 10(4):459-474 (2000)], [Hartley et al., Expert Opin Investig Drugs. 2011, 20(6):733-44], Antonow et al., Chem Rev. 2011, 111(4), 2815- 64],
• Calicheamicins include, e.g. enediynes, esperamicin, and those described in U.S. Patent Nos. 5,714,586 and 5,739,116.
• duocarmycins and analogs examples include CC1065, duocarmycin SA, duocarmycin A, duocarmycin B I, duocarmycin B2, duocarmycin Cl, duocarmycin C2, duocarmycin D, DU- 86, KW-2189, adozelesin, bizelesin, carzelesin, seco- adozelesin.
• Other examples include those described in, for example, US Patent No.
• Exemplary vinca alkaloids include vincristine, vinblastine, vindesine, and navelbine, and those disclosed in U.S. Publication Nos. 2002/0103136 and 2010/0305149, and in U.S. Patent No. 7,303,749, the disclosures of which are incorporated herein by reference in their entirety.
• Exemplary epothilone compounds include epothilone A, B, C, D, E, and F, and derivatives thereof. Suitable epothilone compounds and derivatives thereof are described, for example, in U.S. Patent Nos. 6,956,036; 6,989,450; 6,121 ,029; 6,117,659; 6,096,757; 6,043,372; ; 5,969,145; and 5,886,026; and WO97/19086; WO98/08849; W098/22461; W098/25929; W098/38192; WO99/01124; WO99/02514; WO99/03848; WO99/07692;
• Exemplary platinum compounds include cisplatin, carboplatin, oxaliplatin, iproplatin, ormaplatin, tetraplatin.
• Exemplary DNA binding or alkylating drugs include CC-1065 and its analogs, anthracyclines, calicheamicins, dactinomycines, mitromycines, pyrrolobenzodiazepines, and the like.
• microtubule stabilizing and destabilizing agents include taxane compounds, such as paclitaxel, docetaxel, tesetaxel, and carbazitaxel; maytansinoids, auristatins and analogs thereof, vinca alkaloid derivatives, epothilones and cryptophycins.
• topoisomerase inhibitors include camptothecin and camptothecin derivatives, camptothecin analogs and non-natural camptothecins, such as, for example, CPT-11 , SN-38, topotecan, 9-aminocamptothecin, rubitecan, gimatecan, karenitecin, silatecan, lurtotecan, exatecan, DXd, diflometotecan, belotecan, lurtotecan and S39625.
• camptothecin compounds that can be used in the present invention include those described in, for example, J. Med. Chem., 29:2358-2363 (1986); J. Med. Chem., 23:554 (1980); J. Med Chem., 30: 1774 (1987).
• Angiogenesis inhibitors include, but are not limited to, MetAP2 inhibitors, VEGF inhibitors, PIGF inhibitors, VGFR inhibitors, PDGFR inhibitors, MetAP2 inhibitors.
• Exemplary VGFR and PDGFR inhibitors include sorafenib, sunitinib and vatalanib.
• Exemplary MetAP2 inhibitors include fumagillol analogs, meaning compounds that include the fumagillin core structure.
• Exemplary cell cycle progression inhibitors include CDK inhibitors such as, for example, BMS-387032 and PD0332991; Rho-kinase inhibitors such as, for example, AZD7762; aurora kinase inhibitors such as, for example, AZD1152, MLN8054 and MLN8237; PLK inhibitors such as, for example, Bl 2536, BI6727, GSK461364, GN-01910; and KSP inhibitors such as, for example, SB 743921, SB 715992, MK-0731 , AZD8477, AZ3146 and ARRY-520.
• CDK inhibitors such as, for example, BMS-387032 and PD0332991
• Rho-kinase inhibitors such as, for example, AZD7762
• aurora kinase inhibitors such as, for example, AZD1152, MLN8054 and MLN8237
• PLK inhibitors such as, for example, B
• Exemplary P13K/m-TOR/AKT signalling pathway inhibitors include phosphoinositide 3- kinase (P13K) inhibitors, GSK-3 inhibitors, ATM inhibitors, DNA-PK inhibitors and PDK-1 inhibitors.
• P13K phosphoinositide 3- kinase
• Exemplary P13 kinases are disclosed in U.S. Patent No. 6,608,053, and include BEZ235, BGT226, BKM120, CAL263, demethoxyviridin, GDC-0941, GSK615, IC87114, LY294002, Palomid 529, perifosine, PF-04691502, PX-866, SAR245408, SAR245409, SF1126, Wortmannin, XL147 and XL765.
• Exemplary AKT inhibitors include, but are not limited to AT7867.
• Exemplary MAPK signaling pathway inhibitors include MEK, Ras, JNK, B-Raf and p38 MAPK inhibitors.
• MEK inhibitors are disclosed in U.S. Patent No. 7,517,944 and include GDC- ;0973, GSKI 120212, MSC1936369B, AS703026, R05126766 and R04987655, PD0325901, AZD6244, AZD8330 and GDC-0973.
• Exemplary B-raf inhibitors include CDC-0879, PLX-4032, and SB590885.
• Exemplary B p38 MAPK inhibitors include BIRB 796, LY2228820 and SB 202190.
• Exemplary receptor tyrosine kinases inhibitors include but are not limited to AEE788 (NVP- AEE 788), BIBW2992 (Afatinib), Lapatinib, Erlotinib (Tarceva), Gefitinib (Iressa), AP24534 (Ponatinib), ABT-869 (linifanib), AZD2171 , CHR-258 (Dovitinib), Sunitinib (Sutent), Sorafenib (Nexavar), and Vatalinib.
• Exemplary protein chaperon inhibitors include HSP90 inhibitors.
• Exemplary inhibitors include 17AAG derivatives, BIIB021, BIIB028, SNX-5422, NVP-AUY-922 and KW-2478.
• HDAC inhibitors include Belinostat (PXD101), CUDC-101, Droxinostat, ITF2357 (Givinostat, Gavinostat), JNJ-26481585, LAQ824 (NVP-LAQ824, Dacinostat), LBH- 589 (Panobinostat), MC1568, MGCD0103 (Mocetinostat), MS-275 (Entinostat), PCI- 24781, Pyroxamide (NSC 696085), SB939, Trichostatin A and Vorinostat (SAHA).
• Exemplary PARP inhibitors include iniparib (BSI 201), olaparib (AZD-2281), ABT-888 (Veliparib), AG014699, CEP9722, MK 4827, KU-0059436 (AZD2281), LT-673, 3- aminobenzamide, A-966492, and AZD2461.
• Exemplary Wnt/Hedgehog signalling pathway inhibitors include vismodegib, cyclopamine and XAV-939.
• Exemplary RNA polymerase inhibitors include amatoxins.
• Exemplary amatoxins include alpha-amanitins, beta amanitins, gamma amanitins, eta amanitins, amanullin, amanullic acid, amanisamide, amanon, and proamanullin.
• Exemplary cytokines include IL-2, IL-7, IL-10, IL-12, IL-15, IL-21 , TNF.
• Auristatins As non-limiting examples of particular drugs there may be mentioned Auristatins, Maytansinoids, PBDs, topoisomerase inhibitors, anthracyclines
• the bioactive compound may be selected from any synthetic or naturally occurring compounds comprising one or more natural and/or nonnatural, proteinogenic and/or non-proteinogenic amino acid residues, such as in particular oligo- or polypeptides or proteins.
• a particular group of such compounds comprises immunoglobulin molecules as for example antibodies, antibody derivatives, antibody fragments, antibody (fragment) fusions (e.g. bi-specific and tri-specific mAb fragments or derivatives), polyclonal or monoclonal antibodies, such as human, humanized, mouse or chimeric antibodies.
• immunoglobulin molecules as for example antibodies, antibody derivatives, antibody fragments, antibody (fragment) fusions (e.g. bi-specific and tri-specific mAb fragments or derivatives), polyclonal or monoclonal antibodies, such as human, humanized, mouse or chimeric antibodies.
• Typical non-limiting examples of antibodies for use in the present invention are selected form biologically, in particular pharmacologically active antibody molecules.
• Nonlimiting examples are selected form the following group: trastuzumab, bevacizumab, cetuximab, panitumumab, ipilimumab, rituximab, alemtuzumab, ofatumumab, gemtuzumab, brentuximab, ibritumomab, tositumomab, pertuzumab, adecatumumab, IGN101 , INA01 labetuzumab, hua33, pemtumomab, oregovomab, minretumomab (CC 4 9), cG250, J591 , MOv-18, farletuzumab (MGRAb-003), 3F8, ch14, 18, KW-2871 , hu3S193, l
• SUBSTITUTE SHEET (RULE 26) naptumomab, narnatumab, necitumumab, nesvacumab, nimotuzumab, nivolumab, nofetumomab, obinutuzumab, ocaratuzumab, ofatumumab, olaratumab, onartuzumab, ontuxizumab, oportuzumab, oregovomab, otlertuzumab, pankomab, parsatuzumab, pasotuxizumab, patritumab, pembrolizumab, pemtumomab, pidilizumab, pintumomab, polatuzumab, pritumumab, quilizumab, racotumomab, ramucirumab, rilotumumab, robatumumab
• Labeling agents which may be used according to the invention can comprise any type of label known in the art which does not inhibitor negatively affect reactivity of the tetrazine moiety.
• Labels of the invention include, but are not limited to, dyes (e.g. fluorescent, luminescent, or phosphorescent dyes, such as dansyl, coumarin, fluorescein, acridine, rhodamine, silicon-rhodamine, BODIPY, or cyanine dyes), chromophores (e.g., phytochrome, phycobilin, bilirubin, etc.), radiolabels (e.g.
• dyes e.g. fluorescent, luminescent, or phosphorescent dyes, such as dansyl, coumarin, fluorescein, acridine, rhodamine, silicon-rhodamine, BODIPY, or cyanine dyes
• chromophores e.g., phytochrome, phycobilin, bilirubin, etc.
• radiolabels e.g.
• radioactive forms of hydrogen, fluorine, carbon, phosphorous, sulphur, or iodine such as tritium, fluorine-18, carbon-11, carbon-14, phosphorous-32, phosphorous-33, sulphur-33, sulphur-35, iodine-123, or iodine-125), MRI- sensitive spin labels, affinity tags (e.g.
• exemplary dyes can include an NIR contrast agent that fluoresces in the near infrared region of the spectrum.
• exemplary near-infrared fluorophores can include dyes and other fluorophores with emission wavelengths (e.g., peak emission wavelengths) between about 630 and 1000 nm, e.g., between about 630 and 800 nm, between about 800 and 900 nm, between about 900 and 1000 nm, between about 680 and 750 nm, between about 750 and 800 nm, between about 800 and 850 nm, between about 850 and 900 nm, between about 900 and 950 nm, or between about 950 and 1000 nm.
• Fluorophores with emission wavelengths (e.g., peak emission wavelengths) greater than 1000 nm can also be used in the methods described herein.
• exemplary fluorophores include 7-amino-4-methylcoumarin-3 - acetic acid (AMCA), TEXAS REDTM (Molecular Probes, Inc., Eugene, Oreg.), 5-(and -6)- carboxy-X-rhodamine, lissamine rhodamine B, 5-(and -6)-carboxyfluorescein, fluorescein-5- isothiocyanate (FITC), 7-diethylaminocoumarin-3-carboxylic acid, tetramethylrhodamine-5- (and -6)-isothiocyanate, 5 -(and -6)-carboxytetramethylrhodamine, 7-hydroxycoumarin-3- carboxylic acid, 6-[fluorescein 5-(and -6)-carboxamido]hexanoic acid, N-(4,4-difluoro-5,7- dimethyl-4-bora-3a,4a diaza-3-indacen
• fluorophores are for example described in EP3572468A1.
• Further labelling agents are 177-Lutetium, 89-Zirkonium, 131-lod, 68-Gallium, 99m-Technecium, 225-Actinium, 213-Bismut, 90-Ytrium, 212-Plumbum, 111-lndium, 64-Copper, 67-Copper, 124-lodine, 227-Thorium and 188-Rhenium.
• Acetyl acetone (ACAC), ethylene diamine (EN), 2-(2-aminoethylamino)ethanol (AEEA), diethylene triamine (DIEN), iminodiacetate (IDA), triethylene tetramine (TRIEN), triaminotriethylamine, nitrilotriacetate (NTA) and its saltslike NasNTA or FeNTA, ethylenediaminotriacetate (TED), ethylenediamine tetraacetate (EDTA) and its salts like Na2EDTA and CaNa2EDTA, diethylene triaminpentaacetate (DTPA), 1,4,7,10- ztetraazacyclododecane-1 ,4,7, 10-tetraacetate (DOTA), 1 ,4,7-triazacyclononane-1 ,4,7- triacetic acid (NOTA), Oxalate (OX), tartrate (TART), citrate (CIT), dimethylglyoxime (DMG), 8-hydroxyquinoline
• PROTACs in general, but there is a plethora of different E3 ligase binding molecules in combination with specific targeted proteins to degrade (vgl WO2017201449A1). (Maneiro, M. et al ACS Chem. Biol. 2020, 15, 6, 1306-1312
• Suitable cleavable moieties A are well known from the prior art.
• p-glucuronide linkers carrying a betaglucuronic acid derived trigger residue.
• Non-limiting examples thereof are:
• Suitable self-immolative moieties X are well known from the prior art.
• a bio-conjugate of the present invention is provided by bioconjugation, wherein a suitably functionalized biomolecule is reacted with a tetrazine compound of formula I in order to form a covalent linkage between said functionalized biomolecule and said tetrazine.
• Said biomolecule acts as “targeting agent” which targets the payload moiety Y, which is part of the tetrazine compound of formula I, to a particular place of interest (“target”).
• target a respective suitable “targeting agent” can be selected.
• the bio-conjugate comprising the payload moiety may then further act on the target or the biological structure comprising said target....
• the primary object of such targeting agent is the formation of a covalent or noncovalent linkage with a particular “target”.
• a secondary object of the targeting agent is the targeted transport of a “payload molecule” to said target.
• the POI has to be combined (reversibly or irreversibly) with at least one payload molecule.
• said POI has to be functionalised by introducing said at least one ncAA.
• the functionalized POI carrying said at least one ncAA may then be linked to said at least one payload molecule through bioconjugation via said ncAA residue.
• Said ncAA is reactive with a payload molecule which in turn carries a corresponding moiety reactive with said at least one ncAA residue of the POI.
• the thus obtained bioconjugate i.e. the targeting agent, allows the transfer of the payload molecule to the intended target.
• a “target” can be any molecule, which is present in and/or on an organism, tissue or cell. Such targets may be nonspecific or specific for a particular organism, tissue or cell.
• Targets include cell surface targets, e.g. receptors, glycoproteins, glycans, carbohydrates; structural proteins, e.g. amyloid plaques; abundant extracellular targets such as in stroma, extracellular matrix targets such as growth factors, and proteases; intracellular targets, e.g. surfaces of Golgi bodies, surfaces of mitochondria, RNA, DNA, enzymes, components of cell signaling pathways; and/or foreign bodies, e.g. pathogens such as viruses, bacteria, fungi, yeast or parts thereof.
• targets include compounds such as proteins of which the presence or expression level is correlated with a certain tissue or cell type or of which the expression level is up- regulated or down-regulated in a certain disorder.
• such target is a protein such as a (internalizing or non- internalizing) receptor.
• Targets can be selected from any suitable targets within the human or animal body or on a pathogen or parasite.
• Non-limiting examples of suitable targets include but are not limited to a group comprising cellular components such as cell membranes and cell walls, receptors such as cell membrane receptors, intracellular structures such as Golgi bodies or mitochondria, enzymes, receptors, DNA, RNA, viruses or viral particles, macrophages, tumor-associated macrophages, antibodies, proteins, carbohydrates, monosaccharides, polysaccharides, cytokines, hormones, steroids, somatostatin receptor, monoamine oxidase, muscarinic receptors, myocardial sympatic nerve system, leukotriene receptors, e.g. on leukocytes, urokinase plasminogen activator receptor (uPAR), folate receptor, apoptosis marker, (anti-)
• cellular components such as cell membranes and cell walls, receptors such as cell membrane receptors, intracellular structures such as Golgi bodies or mitochondria, enzymes, receptors, DNA, RNA, viruses or viral particles, macrophages, tumor-
• SUBSTITUTE SHEET (RULE 26) angiogenesis marker, gastrin receptor, dopaminergic system, serotonergic system, GABAergic system, adrenergic system, cholinergic system, opioid receptors, GPIIb/llla receptor and other thrombus related receptors, fibrin, calcitonin receptor, tuftsin receptor, P- glycoprotein, neurotensin receptors, neuropeptide receptors, substance P receptors, NK receptor, CCK receptors, sigma receptors, interleukin receptors, herpes simplex virus tyrosine kinase, human tyrosine kinase, integrin receptor, fibronectin targets, AOC3, ALK, AXL, C242, CA-125, CCL11 , CCR5, CD2, CD3, CD4, CD5, CD15, CA15-3, CD18, CD19, CA19-9, CD20, CD21, CD22, CD23, CD25, CD28, CD30, CD31 , CD33
• fibronectin targets are the alternatively spliced extra-domain-A (ED-A) and extra-domain-B (ED-B) of fibronectin.
• ED-A extra-domain-A
• ED-B extra-domain-B
• Nonlimiting examples of targets in stroma can be found in V. Hofmeister, D. Schrama, J. C. Becker, Cancer Immun. Jmmunother. 2008, 57, 1, the contents of which are hereby incorporated by reference.
• the targeting agent can comprise compounds comprising an ncAA-functionalized peptide sequence.
• Such compounds include but are not limited to antibodies, antibody derivatives, antibody fragments, antibody (fragment) fusions (e.g. bi-specific and tri-specific mAb fragments or derivatives), proteins, peptides, e.g. octreotide and derivatives, VIP, MSH, LHRH, chemotactic peptides, bombesin, elastin, peptide mimetics, receptor agonists and antagonists, cytokines, hormones, steroids, toxins.
• the target is a receptor and a targeting agent is employed, which is capable of specific binding to the target.
• Suitable targeting agents include but are not limited to, the ligand of such a receptor or a part thereof, which still binds to the receptor, e.g. a receptor binding peptide in the case of receptor binding protein ligands.
• targeting agents of protein nature include insulin, transferrin, fibrinogen-gamma fragment, thrombospondin, claudin, apolipoprotein E, Affibody molecules such as for example ABY-025, Ankyrin repeat proteins, ankyrin-like repeat proteins, interferons, e.g. alpha, beta, and gamma interferon, interleukins, lymphokines, colony stimulating factors and protein growth factor, such as tumor growth factor, e.g. alpha, beta tumor growth factor, platelet-derived growth factor (PDGF), uPAR targeting protein, apolipoprotein, LDL, annexin V, endostatin, and angiostatin.
• PDGF platelet-derived growth factor
• peptides molecules like antibody, as used in targeting agents include LHRH receptor targeting peptides, EC-1 peptide, RGD peptides, HER2-targeting peptides, PSMA targeting peptides, somatostatin-targeting peptides, bombesin.
• targeting agents include lipocalins, such as anticalins.
• One particular embodiment uses AffibodiesTM and multimers and derivatives.
• antibodies are used to form a targeting agent. While antibodies or immunoglobulins derived from IgG antibodies are particularly well-suited for use in this invention, immunoglobulins from any of the classes or subclasses may be selected, e.g. IgG, IgA, IgM, IgD and IgE. Suitably, the immunoglobulin is of the class IgG including but not limited to IgG subclasses (lgG1, 2, 3 and 4) or the class IgM which is able to specifically bind to a specific epitope on an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins.
• Antibodies may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, camelized single domain antibodies, recombinant antibodies, anti-idiotype antibodies, multispecific antibodies, antibody fragments, such as, Fv, VHH, Fab, F(ab)2, Fab', Fab'-SH, F(ab')2, single chain variable fragment antibodies (scFv), tandem/bis-scFv, Fc, pFc', scFv-Fc, disulfide Fv (dsFv), bispecific antibodies (bc-scFv) such as BiTE antibodies, trispecific antibody derivatives such as tribodies, camelid antibodies, minibodies, nanobodies, resurfaced antibodies, humanized antibodies, fully human antibodies, single domain antibodies (sdAb, also known as NanobodyTM), chimeric antibodies, chimeric antibodies comprising at least one human constant region, dual-affinity antibodies such as dual-affinity retargeting proteins (DARTTM), and multimers and
• Antibody fragment refers to at least a portion of the variable region of the immunoglobulin that binds to its target, i.e. the antigen-binding region.
• antibody mimetics as targeting agents, such as but not limited to Affimers, Anticalins, Avimers, Alphabodies, Affibodies, DARPins, and multimers and derivatives thereof; reference is made to [Trends in Biotechnology 2015, 33, 2, 65], the contents of which is hereby incorporated by reference.
• antibody is meant to encompass all of the antibody variations, fragments, derivatives, fusions, analogs and mimetics outlined in this paragraph, unless specified otherwise.
• the targeting agent is selected from agents derived from antibodies and antibody derivatives such as antibody fragments, fragment fusions, proteins, peptides, peptide mimetics.
• the targeting agent is selected from agents derived from antibody fragments, fragment fusions, and other antibody derivatives that do not contain a Fc domain.
• Typical non-limiting examples of antibody molecules to be further modified to form ncAA modified targeting agents of the present invention are selected form biologically, in particular pharmacologically active antibody molecules.
• Non-limiting examples are selected form the following group: trastuzumab, bevacizumab, cetuximab, panitumumab, ipilimumab, rituximab, alemtuzumab, ofatumumab, gemtuzumab, brentuximab, ibritumomab, tositumomab, pertuzumab, adecatumumab, IGN101 , INA01 labetuzumab, hua33, pemtumomab, oregovomab, minretumomab (CC 4 9), cG250, J591 , MOv-18, farletuzumab (MGRAb-003), 3F8, ch14,18, KW
• SUBSTITUTE SHEET (RULE 26) demcizumab, denintuzumab, depatuxizumab, derlotuximab, detumomab, dinutuximab, drozitumab, duligotumab, durvalumab, dusigitumab, ecromeximab, edrecolomab, elgemtumab, emactuzumab, enavatuzumab emibetuzumab, enfortumab, enoblituzumab, ensituximab, epratuzumab, ertumaxomab, etaracizumab, farletuzumab, ficlatuzumab, figitumumab, flanvotumab, futuximab, galiximab, ganitumab, icrucumab, i
• the target and targeting agent are selected so as to result in the specific or increased targeting of a tissue or disease, such as cancer, an inflammation, an infection, a cardiovascular disease, e.g. thrombus, atherosclerotic lesion, hypoxic site, e.g. stroke, tumor, cardiovascular disorder, brain disorder, apoptosis, angiogenesis, an organ, and reporter gene/enzyme.
• a tissue or disease such as cancer, an inflammation, an infection, a cardiovascular disease, e.g. thrombus, atherosclerotic lesion, hypoxic site, e.g. stroke, tumor, cardiovascular disorder, brain disorder, apoptosis, angiogenesis, an organ, and reporter gene/enzyme.
• a tissue or disease such as cancer, an inflammation, an infection, a cardiovascular disease, e.g. thrombus, atherosclerotic lesion, hypoxic site, e.g. stroke, tumor, cardiovascular disorder, brain disorder, apoptosis, angiogenesis
• the targeting agent specifically binds or complexes with a cell surface molecule, such as a cell surface receptor or antigen, for a given cell population. Following specific binding or complexing of the targeting agent with the receptor, the drug will enter the cell.
• a cell surface molecule such as a cell surface receptor or antigen
• a targeting agent that "specifically binds or complexes with” or “targets” a cell surface molecule, an extracellular matrix target, or another target, preferentially associates with the target via intermolecular forces.
• the ligand can preferentially associate with the target with a dissociation constant (Kd or KD) of less than about 50 nM, less than about 5 nM, or less than about 500 pM.
• the targeting agents in particular biomolecules, normally have to be functionalised in order to enable the covalent binding of a tetrazine compound of the general formula I.
• dienophiles suitable for reacting with a tetrazine moiety are well known in the art. In general, more- or poly-cyclic, in particular mono- and bi-cyclic unsaturated dienophile are applicable.
• Such dienophiles are capable of reaction via a Diels-Alder-type cycloaddition reaction, as for example cyclooctynyl-dienophiles, trans-cyclooctenyl-dienophiles, norbornenyl dienophiles, cyclopropenyl dienophiles, cyclobutenyl dienophiles, spirohexenyl dienophiles, BCN dienophiles, or azetine dienophiles.
• a targeting agent in particular a targeting agent comprising a polypeptide portion, comprising one or more than one UNAA residue can be prepared according to the present invention using a suitable translation system, in particular in vivo translation system.
• An in vivo translation system can be a cell, e.g. a prokaryotic or eukaryotic cell.
• the cell can be a bacterial cell, e.g. E. coir, a fungal cell such as a yeast cell, e.g. S. cerevisiae or a methylotrophic yeast; a plant cell, or an animal cell such as an insect cell or a mammalian
• SUBSTITUTE SHEET (RULE 26) cell e.g. a HEK cell or a HeLa cell.
• Eukaryotic cells used for polypeptide expression may be single cells or parts of a multicellular organism.
• the applied cellular system comprises (e.g., is fed with) at least one unnatural amino acid or a salt thereof corresponding to the UNAA residue(s) of the targeting agent to be prepared.
• the cellular system further comprises:
• a PylRS of the invention and a tRNA Pyl , wherein the PylRS is capable of (preferably selectively) acylating the tRNA Pyl with the UNAA or salt thereof;
• a polynucleotide encoding the targeting agent wherein any position of the targeting agent, occupied by an UNAA residue is encoded by a codon (e.g. selector codon) that is the reverse complement of the anticodon of the tRNA Pyl .
• a codon e.g. selector codon
• the cellular system is cultured so as to allow translation of the targeting agent, - encoding polynucleotide (ii), thereby producing the targeting agent.
• the translation in step (b) can be achieved by culturing the cellular system under suitable conditions, preferably in the presence of (e.g., in a culture medium containing) the UNAA or salt thereof, for a time suitable to allow translation at a ribosome of the cell.
• suitable conditions preferably in the presence of (e.g., in a culture medium containing) the UNAA or salt thereof, for a time suitable to allow translation at a ribosome of the cell.
• a compound inducing transcription such as, e.g., arabinose, isopropyl /3-D-thiogalactoside (IPTG) or tetracycline.
• mRNA that encodes the targeting agent (and comprises one or more than codon that is the reverse complement of the anticodon comprised by the tRNA Pyl ) is bound by the ribosome.
• the polypeptide is formed by stepwise attachment of amino acids and UNAAs at positions encoded by codons which are recognized (bound) by respective aminoacyl tRNAs.
• the UNAA(s) is/are incorporated in the targeting agent, at the position(s) encoded by the codon(s) that is/are the reverse complement of the anticodon comprised by the tRNA Pyl .
• the cellular system may comprise a polynucleotide sequence encoding the PylRS of the invention which allows for expression of the PylRS by the cell.
• the tRNA Pyl may be produced by the cellular system based on a tRNA Pyl -encoding polynucleotide sequence comprised by the cell.
• the PylRS-encoding polynucleotide sequence and the tRNA Pyl - encoding polynucleotide sequence can be located either on the same polynucleotide or on separate polynucleotides.
• the present invention provides a method for producing a targeting agent, comprising one or more than one UNAA residue, wherein the method comprises the steps of:
• SUBSTITUTE SHEET (RULE 26) (a) providing a cellular system comprising polynucleotide sequences encoding: at least one PylRS of the invention, at least one tRNA (tRNA Pyl ) that can be acylated by the PylRS, and at least one targeting agent, wherein any position of the targeting agent, occupied by an UNAA residue is encoded by a codon that is the reverse complement of the anticodon of the tRNA Pyl ; and
• the cellular system used for preparing a targeting agent, comprising one or more than one unnatural amino acid residue as described herein can be prepared by introducing polynucleotide sequences encoding the PylRS, the tRNA Pyl and the targeting agent, into a (host) cell.
• Said polynucleotide sequences can be located on the same polynucleotide or on separate polynucleotides, and can be introduced into the cell by methods known in the art (such as, e.g., using virus-mediated gene delivery, electroporation, microinjection, lipofection, or others).
• the targeting agent prepared according to the present invention may optionally be recovered and purified, either partially or substantially to homogeneity, according to procedures generally known in the art. Unless the targeting agent, is secreted into the culture medium, recovery usually requires cell disruption. Methods of cell disruption are well known in the art and include physical disruption, e.g., by (ultrasound) sonication, liquid-sheer disruption (e.g., via French press), mechanical methods (such as those utilizing blenders or grinders) or freeze-thaw cycling, as well as chemical lysis using agents which disrupt lipid-lipid, protein-protein and/or protein-lipid interactions (such as detergents), and combinations of physical disruption techniques and chemical lysis.
• physical disruption e.g., by (ultrasound) sonication, liquid-sheer disruption (e.g., via French press), mechanical methods (such as those utilizing blenders or grinders) or freeze-thaw cycling, as well as chemical lysis using agents which disrupt lipid-lipid, protein-protein and/or protein-lipid
• Standard procedures for purifying polypeptides from cell lysates or culture media are also well known in the art and include, e.g., ammonium sulfate or ethanol precipitation, acid or base extraction, column chromatography, affinity column chromatography, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, lectin chromatography, gel electrophoresis and the like. Protein refolding steps can be used, as desired, in making correctly folded mature proteins. High performance liquid chromatography (HPLC), affinity chromatography or other suitable methods can be employed in final purification steps where high purity is desired. Antibodies made against the polypeptides of the invention can be used as purification reagents, i.e. for affinity-based
• SUBSTITUTE SHEET (RULE 26) purification of the polypeptides.
• a variety of purification/protein folding methods are well known in the art, including, e.g., those set forth in Scopes, Protein Purification, Springer, Berlin (1993); and Deutscher, Methods in Enzymology Vol. 182: Guide to Protein Purification, Academic Press (1990); and the references cited therein.
• polypeptides can possess a conformation different from the desired conformations of the relevant polypeptides.
• polypeptides produced by prokaryotic systems often are optimized by exposure to chaotropic agents to achieve proper folding.
• the expressed polypeptide is optionally denatured and then renatured. This is accomplished, e.g., by solubilizing the proteins in a chaotropic agent such as guanidine HCI.
• a chaotropic agent such as guanidine HCI.
• guanidine, urea, DTT, DTE, and/or a chaperonin can be added to a translation product of interest.
• Methods of reducing, denaturing and renaturing proteins are well known to those of skill in the art.
• Polypeptides can be refolded in a redox buffer containing, e.g., oxidized glutathione and L-arginine.
• the targeting agent thus prepared may then be converted to a respective bioconjugate by reaction with a tetrazine compound of the above general formula I
• conjugates or bioconjugates of the present invention are generally given as “pharmaceutical compositions” comprised of a therapeutically and/or prophylactically effective amount or a diagnostically effective amount of at least one such active ingredient or its pharmaceutically acceptable salt and optionally at least one pharmaceutically acceptable excipient.
• Said pharmaceutical compositions may be delivered via suitable routes of administration such as via oral, rectal, transmucosal, topical, ophthalmic, otologic, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, as the case may be.
• suitable routes of administration such as via oral, rectal, transmucosal, topical, ophthalmic, otologic, or intestinal administration
• parenteral delivery including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, as the case may be.
• said at least one additional pharmaceutical excipient may be different.
• excipient is a substance formulated alongside the active ingredient and is included for different purpose, as for example for long-term stabilization, bulking up solid
• SUBSTITUTE SHEET (RULE 26) formulations that contain potent active ingredients in small amounts (thus often referred to as “bulking agents", “fillers”, or “diluents”), or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as for example facilitating drug absorption, reducing viscosity, or enhancing solubility.
• Excipients can also be useful in the manufacturing process of the pharmaceutical composition, to aid in the handling of the active substance concerns such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation or aggregation over the expected shelf life. The selection of appropriate excipients not only depends upon the route of administration and the dosage form, but also on the particular active ingredient and other factors.
• Excipients may be selected from the following classes: immunological adjuvants, antiadherents, binders, coatings, colours, disintegrant, flavours, glidants, lubricants, preservatives, sorbents, sweeteners, and vehicles.
• Non limiting examples of excipients comprise diluents, preserving agents, stabilizers, emulsifying agents, like emulsifying polymers, such as polysorbates or poloxamers, antioxidants; anti-irritants, chelating agents and stabililizing salts, such as chlorides, sulfates, phosphates, diphosphates, hydrobromides and nitrates, suspending agents, antibacterial agents or antifungal agents.
• buffering agents such as buffering systems of low molecular weight organic acids together with the respective salts, or inorganic buffering substances, such as phosphate buffers, can be used.
• suitable ingredients are also known from relevant pharmacological standard literature.
• a pharmaceutical composition as used herein may be presented in the form of a “dosage form” or “unit dose” and may comprise one or more APC, in particular ADCs as described herein.
• a pharmaceutical composition as used herein could, for example, provide two active agents admixed together in a unit dose or provide two active agents combined in a dosage form wherein the active agents are physically separated.
• compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, emulsifying,
• SUBSTITUTE SHEET (RULE 26) encapsulating, entrapping or or combinations thereof. Proper formulation is dependent upon the route of administration chosen.
• phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable risk/benefit ratio.
• the invention includes all “pharmaceutically acceptable salt forms” of the active ingredient.
• Pharmaceutically acceptable salts are those in which the counter ions do not contribute significantly to the physiological activity or toxicity of the compounds and as such function as pharmacological equivalents. These salts can be made according to common organic techniques employing commercially available reagents. Some anionic salt forms include acetate, acistrate, besylate, bromide, chloride, citrate, fumarate, glucouronate, hydrobromide, hydrochloride, hydroiodide, iodide, lactate, maleate, mesylate, nitrate, pamoate, phosphate, succinate, sulfate, tartrate, tosylate, and xinofoate.
• Some cationic salt forms include ammonium, aluminum, benzathine, bismuth, calcium, choline, diethylamine, diethanolamine, lithium, magnesium, meglumine, 4-phenylcyclohexylamine, piperazine, potassium, sodium, tromethamine, and zinc.
• a “therapeutically effective amount” and/or “prophylactically effective amount” means an amount effective, when administered to a human or non-human patient, to provide any therapeutic and/or prophylactic benefit. More particularly, a “therapeutically effective amount” is an amount of an active ingredient disclosed herein or a combination of two or more such active ingredients, which inhibits, totally or partially, the progression of the condition or alleviates, at least partially, one or more symptoms of the condition.
• a “diagnostically effective amount” means an amount effective to allow obtaining from the patient a diagnostically valuable information on status or progression of a disease state.
• a therapeutic benefit may be an amelioration of symptoms of a diseased patient, e.g., an amount effective to decrease the symptoms of a diseased patient.
• a patient may not present symptoms of a condition for which the patient is being treated.
• a prophylactically effective amount of a compound is also an amount sufficient to provide a significant positive effect on any indicia of a disease, disorder or condition e.g. an amount sufficient to significantly reduce the frequency and severity of disease symptoms to occur.
• a therapeutically effective amount can also be an amount, which is prophylactically effective.
• a “patient” as used herein means human or non-human, in particular human, animals.
• a "dosage form” is any unit of administration (“unit dose”) of one or more active agents as described herein.
• treating refers to: (i) preventing a disease, disorder or condition from occurring in a patient which may be predisposed to the disease, disorder and/or condition but has not yet been diagnosed as having it; (ii) inhibiting the disease, disorder or condition, i.e., arresting its development; and (iii) relieving the disease, disorder or condition, i.e., causing regression of the disease, disorder and/or condition.
• treating refers to: (i) preventing a disease, disorder or condition from occurring in a patient which may be predisposed to the disease, disorder and/or condition but has not yet been diagnosed as having it; (ii) inhibiting the disease, disorder or condition, i.e., arresting its development; and (iii) relieving the disease, disorder or condition, i.e., causing regression of the disease, disorder and/or condition.
• a prophylactic or therapeutic treatment or combinations thereof are examples thereof.
• “Frequency” of dosage may vary depending on the compound used and the particular type of infection treated. A dosage regimen of once per day is possible. Dosage regimens in which the active agent is administered for several times daily, as for example 2 to 10 times, like 2, 3, 4, 5, 6, 7, 8, 9 or 10 times may occasionally be more helpful.
• the specific dose level and frequency for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease in the patient undergoing therapy. Patients may generally be monitored for therapeutic or prophylactic effectiveness using assays suitable for the condition being treated or prevented, which will be familiar to those of ordinary skill in the art.
• compositions according to the present invention are liquid form preparations such as solutions, suspensions, and emulsions and comprise, a therapeutically effective amount ofat least one APC, in particular ADC component as defined above, optionally together with at least one further pharmaceutically acceptable excipient as defined above and may be administered through any suitable route.
• compositions according to the present invention are solid form preparations such as powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.
• Reagents were purchased from commercial suppliers and used without further purification. All solvents, including anhydrous solvents, were used as obtained from the commercial sources. Air and water-sensitive reagents and reactions were generally handled under argon atmosphere.
• reaction progress was monitored by TLC on Merck silica gel plates 60 F254 or via UHPLC-MS. TLC-detection was executed either via UV-light at 254 nm or with potassium permanganate staining.
• Flash chromatographic purification was performed on a Biotage Isolera One purification system using silica gel (0.060-0.200 mm), KP-Sil cartridges.
• Preparative HPLC purification was performed on Agilent Infinity 1260 series equipment consisting of Agilent 1260 preparative pumps, a 1260 preparative autosampler, a 1260 fraction collector and a 1260 multiple wavelength detector VL.
• the preparative column used was a Waters X-Bridge Prep C18 column: 5 pm; 19x150 mm operated with a linear gradient of H2O and acetonitrile, both containing 0.1% TFA as solvents.
• TSTU /V,/V,/V ⁇ /V'-Tetramethyl-O-(/V- succinimidyl)uroniumtetrafluorborat
• the isolated dihydrotetrazine compound (9.9 mg) was dissolved in 0.5 mL DMF/MeOH, 2.7 mg p-benzoquinone was added and the mixture stirred for 5 min. The mixture was directly subjected to purification via HPLC.
• the isolated dihydrotetrazine compound (320 mg) was dissolved in 10 mL DMF/MeOH, 108 mg p-benzoquinone was added and the mixture stirred for 5 min.
• N-Boc-glycine 486 mg, 2.78 mmol, 2.00 eq
• THF 5 mL
• N-Methylmorpholine 763 ⁇ L, 6.94 mmol, 5.00 eq
• isobutyl chloroformate 360 ⁇ L, 2.78 mmol, 2.00 eq
• 18 450 mg, 1.39 mmol, 1.00 eq
• SUBSTITUTE SHEET (RULE 26) added N-methylmorpholine (0.401 mL, 3.64 mmol, 5.00 eq) and isobutyl chloroformate (0.189 mL, 1.46 mmol, 2.00 eq) at 0 °C and the mixture was stirred for 5 min. Then 52 (237 mg, 0.729 mmol, 1.00 eq) was added and the mixture was stirred at RT overnight. Water and EtOAc were added, the pahses were separated and the aqueous phase was extracted with EtOAc (2 x). The combined organic phases were washed with a saturated aqueous solution of NaHCCh, dried over Na2SO4 and the solvents evaporated under reduced pressure.
• TMS-Br (11.0 ⁇ L, 83.2 ⁇ mol, 10.0 eq) at 0 °C and the mixture stirred at RT for 6 h. Afterwards 20 ⁇ L of TMS-Br were added and the mixture stirred overnight. Afterwards 40 ⁇ L of TMS-Br were added and the mixture stirred for 8 d. Water was added and the mixture was directly subjected to purification via HPLC.
• Trastuzumab A132TCO*A was expressed in insect cells (Spodoptera frugiperda cells, Sf21) utilizing the baculovirus based transduction system. Therefore, the gene of the heavy chain of Trastuzumab containing an amber stop codon at position A132 and a C-terminal 6-
• SUBSTITUTE SHEET (RULE 26) His tag, was cloned into pACEBac-DUAL plasmid (as for example described in WO 2017/093254) into the first multicloning site.
• the gene of the light chain of Trastuzumab was cloned without further modifications into the second multicloning site of the plasmid.
• the resulting plasmid, pACEBacDUAL-Trastuzumab heavy A132TAG-6His-light was transformed into DH10MultiBac-TAG cells harboring a Bacmid with the expression cassette for NES-PylRS AF (as for example described in WO2018/069481) as well U6(Sf21)-tRNA p v (as for example described in WO 2017/093254) in the backbone of the Bacmid.
• the transformation results in the integration of the plasmid into the Bacmid-DNA.
• insect cells Sf21 cells
• Vo-Virus was harvested and used to transduce a fresh batch of Sf21 cells, resulting in the production of Vi-Virus.
• This Virus was used to transduce a large expression culture (liter scale). After adding TCO*A-Lys the expression was carried out for 4 days.
• the cells were harvested at 500 ref using a Beckman rotor (J LA 8.1000) for 1 hour at 4 °C.
• the cells were resuspended in lysis buffer (4 x PBS, 0.2 mM TCEP, 1 mM PMSF, 5 mM Imidazole, pH 8) and sonicated three times for 30 seconds on ice. After a centrifugation step at 27143.1 RCF for 1 hour at 4 °C in a fixed angle rotor (JA 25.50, Beckman), the cleared lysate was incubated on nickel beads for 1 hour at 4 °C on a rocker. The nickel beads were collected in a polypropylene (PP)-column (Qiagen, Cat. No.: 34964) and washed with lysis buffer, containing 10 nM Imidazole.
• PP polypropylene
• Trastuzumab was eluted from the nickel beads using 500 mM Imidazole in the lysis buffer. The elution fraction was loaded on a MabSelect PrismA column equilibrated in Buffer (0.02 M Na2PO4, 0.15 M NaCI, pH 7.2). After a washing step with Buffer A, Trastuzumab was eluted from the column using a gradient up to 100% Buffer B (0.1 M sodium citrate, pH 3.2). Fractions were collected, which contained 1 M Tris pH 10 to neutralize the eluting sample.
• the fractions containing Trastuzumab were pooled and concentrated using an amicon filter device (30 kDa cutoff). The sample was further purified using a Superdex S200 (10/30) column equilibrated in 1 x PBS buffer. Fractions were collected and analyzed on SDS- PAGE. After concentrating the corresponding fractions, the sample was used for labeling with the cytotoxic payload.
• Trastuzumab A132TCO*A were incubated in 1xPBS with 40 nmol of phosphonate-tetrazine-MMAE (5) at 37 °C shaking at 600 rpm for 1 hour. After washing the reaction mix in a filter device (Amicon Filter device, 30 kDa cutoff) with 1x PBS to remove
• the plates were taken from the incubator, warmed up for 30 minutes at RT and 100 pl CellTiter-Glo® 2.0 Cell (Promega) was added to each well. The plates were shaken at 50 rpm on a rocker for 2 minutes and 10 minutes incubated at RT. Then the luminescence signal was read out using a plate reader. The luminescence signal was normalized to the measurement at time point 0 nM (negative control).
• the plot in Figure 4 shows the normalized luminescence signal of the two ADC and Trastuzumab WT at different concentrations (in M).

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