METHOD FOR MODIFYING MICROCYSTINS AND NODULARINS
20200377922 ยท 2020-12-03
Assignee
Inventors
- Heike Enke (Berlin, DE)
- Wolfram Lorenzen (Berlin, DE)
- Stefan Jahns (Berlin, DE)
- Dan Enke (Berlin, DE)
- Timo Niedermeyer (Halle Saale, DE)
- Julia Moschny (Sulzbach-Rosenberg, DE)
Cpc classification
C12N2500/33
CHEMISTRY; METALLURGY
C12P13/00
CHEMISTRY; METALLURGY
A61K47/64
HUMAN NECESSITIES
C07K7/56
CHEMISTRY; METALLURGY
A61K47/6829
HUMAN NECESSITIES
International classification
C12P21/02
CHEMISTRY; METALLURGY
A61K47/68
HUMAN NECESSITIES
Abstract
A method is used for producing a modified non-ribosomal peptide, e.g. a modified microcystin and/or modified nodularin (together CA), including the steps of a) growing a modified non-ribosomal peptide producing cyanobacteria strain in a culture media, b) adding one or more modified substrates, preferably modified amino acids to said culture, and c) inoculating the non-ribosomal peptide, producing strain the presence of said modified substrates. The thus modified non-ribosomal peptide may be used for the therapy of various diseases.
Claims
1-19. (canceled)
20: A method of producing a modified non-ribosomal peptide from cyanobacteria, comprising: a) growing a non-ribosomal peptide producing cyanobacteria strain in a culture medium, b) adding one or more modified substrates to said culture medium, and c) growing the cyanobacteria strain in the presence of said one or more modified substrates, wherein the one or more modified substrates are either i) a modified amino acid, which comprises an anchor group directly accessible or transformable for use in conjugation chemistry for the attachment of a targeting moiety or a label, or for additional structural modifications, or ii) a modified substrate, which is not directly derived from a naturally incorporated substrate.
21: The method according to claim 20, wherein the cyanobacteria strain is selected such that the incorporation of the one or more modified substrates into the non-ribosomal peptide occurs at a defined position.
22: The method according to claim 20, wherein the non-ribosomal peptide is a microcystin of the following general structure: D-Ala.sub.1-X.sub.2-D-MeAsp.sub.3-Z.sub.4-Adda.sub.5-DGlu.sub.6-Mdha.sub.7 and wherein the one or more modified substrates are incorporated in at least one position other than Adda.sub.5 and DGlu.sub.6.
23: The method according to claim 22, wherein the incorporation of said one or more modified substrates occurs in position X2 and/or Z4.
24: The method according to claim 20, wherein the non-ribosomal peptide is a nodularin of the following general structure:
D-MeAsp.sub.1-Arg.sub.2-Adda.sub.3-DGlu.sub.4-Mdhb.sub.5 and wherein the one or more modified substrates are incorporated at any position other than Adda.sub.3 and DGlu.sub.4.
25: The method according to claim 24, wherein the incorporation of said one or more modified substrates occurs in position Arg2.
26: The method according to claim 22, wherein the modified position is X.sub.2 and the one or more modified substrates are a modified amino acid.
27: The method according to claim 22, wherein the modified position is Z.sub.4 and the one or more modified substrates are a modified amino acid.
28: The method according to claim 24, wherein the modified position is Arg2 and the one or more modified substrates are a modified amino acid.
29: The method according to claim 20, wherein the one or more modified substrate is at least one substrate selected from the group consisting of (2S)-2-amino-3-azidopropanoic acid, (2S)-2-amino-6-azidohexanoic acid, (S)-2-Amino-5-azidopentanoic acid, (2S)-2-amino-3-(4-prop-2-ynyloxyphenyl)propanoic acid, (2S)-2-amino-5-(N-nitrocarbamimidamido)pentanoic acid, (2S)-2-amino-3-(furan-2-yl)propanoic acid, (S)-Amino-6-((prop-2-ynyloxy)carbonylamino)hexanoic acid, N-Propargyl-Lysine, (2S)-2-Amino-3-(4-azidophenyl)propanoic acid, and L--Amino--guanidinohexanoic acid.
30: The method according to claim 22, wherein, independently of one another, D-Ala.sub.1 is selected from the group consisting of D-Ala, D-Ser and D-Leu, D-MeAsp.sub.3 is selected from the group consisting of D-MeAsp and D-Asp, Adda.sub.5 is selected from the group consisting of Adda, DM-Adda, (6Z)Adda and ADM-Adda, DGlu.sub.6 is selected from the group consisting of D-Glu and D-Glu(OCH3), Mdha.sub.7 is selected from the group consisting of Mdha, Dha, L-Ser, L-MeSer, Dhb, and MeLan, and X.sub.2 and/or Z.sub.4 comprise the at least one modified substrate.
31: The method according to claim 24, wherein, independently of one another, MeAsp.sub.1 is selected from the group consisting of D-MeAsp and D-Asp, Arg.sub.2 is selected from the group consisting of Arg and Homo-Arg, Adda.sub.3 is selected from the group consisting of Adda, DM-Adda, (6Z)Adda and Me-Adda, DGlu.sub.4 is selected from the group consisting of D-Glu and D-Glu(OCH3), Mdhb.sub.5 is selected from the group consisting of Mdhb and Dhb, and wherein the position for MeAsp.sub.1, Arg.sub.2 and Mdhb.sub.5 comprises the at least one modified substrate.
32: The method according to claim 20, wherein the concentration of the one or more modified substrates in the culture medium is between 5 M and 500 M and/or DMSO is added as an additional ingredient.
33: The method according to claim 20, wherein the conjugation chemistry is at least one selected from the group consisting of copper(I)-catalyzed azide-alkyne cycloaddition, strain promoted azide-alkyne cycloaddition, alkyne-azide cycloaddition, alkene-tetrazine inverse-demand Diels-Alder reaction, and reactions exploiting the specific reactivities of primary amines, thiols, aldehydes, carboxyls, and oximes.
34: The method according to claim 20, wherein the cyanobacteria strain is at least one selected from the group consisting of Microcystis, Planktothrix, Oscillatoria, Nostoc, Anabaena, Aphanizomenon, Hapalosiphon, Nodularia, Lyngbya, Phormidium, Spirulina, Halospirulina, Arthrospira, Trichodesmium, Leptolyngbya, Plectonema, Myxosarcina, Pleurocapsa, Pseudanabaena, Geitlerinema, Euhalothece, Calothrix, Tolypothrix, Scytonema, Fischerella, Mastigocladus, Westiellopsis, Stigonema, Chlorogloeopsis, Cyanospira, Cylindrospermopsis, Cylindrospermum, Microchaete, Rivularia, Autosira, Trichonema, Trichodesmium, Symploca, Starria, Prochlorothrix, Microcoleus, Limnothrix, Crinalium, Borzia, Chroococcidiopsis, Cyanocystis, Dermocarpella, Staniera, Xenococcus, Chamaesiphon, Chroococcus, Cyanobacterium, Cyanobium, Cyanothece, Dactylococcopsis, Gloeobacter, Gloeocapsa, and Gloeothece.
35: A method of producing a compound for targeted therapy comprising a non-ribosomal peptide linked to a targeting moiety, the method comprising: A) providing a targeting moiety and a non-ribosomal peptide comprising at least one modified amino acid, wherein the at least one modified amino acid comprises an anchor group directly accessible or transformable for use in conjugation chemistry by performing a method according to claim 20, and B) attaching said targeting moiety to said non-ribosomal peptide via chemical conjugation to said anchor group.
36: The method according to claim 35, wherein the targeting moiety is attached via a linker arranged between the modified amino acid and the targeting moiety.
37: The method according to claim 35, wherein the targeting moiety is an antibody.
38: The method according to claim 20, wherein the one or more modified substrates are the modified amino acid which comprises an anchor group directly accessible or transformable for use in click chemistry.
Description
BRIEF DESCRIPTION OF DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0143] The invention relates to a method of producing a modified non-ribosomal peptide, especially cytotoxic non-ribosomal peptides such as modified microcystin and/or modified nodularin (both CA), comprising the steps of: [0144] a) growing a microcystin and/or nodularin producing cyanobacteria strain (CA-STRAIN) in a culture media, [0145] b) adding one or more modified substrates preferably modified amino acids to said culture, and [0146] c) cultivation the strain in the presence of said modified substrates.
[0147] The invention relates to a method of producing a modified non-ribosomal peptide from cyanobacteria, comprising the steps of: [0148] a) growing a non-ribosomal peptide producing cyanobacteria strain in culture media, [0149] b) adding one or more modified substrates to said culture, and [0150] c) growing the strain in the presence of said modified substrates, [0151] d) wherein the modified substrate, is either [0152] i) a modified amino acid, which comprises an anchor group directly accessible or transformable for use in conjugation chemistry incl. click chemistry, for the attachment of a targeting moiety or a label, or a linker or for any other structural modification [0153] ii) or, the modified substrate in the non-ribosomal peptide is a modified substrate which is not directly derived from the naturally incorporated substrate, such as preferably an amino acid or a modified amino acid which is, in nature, not incorporated at the specific position in said non-ribosomal peptide and which is also not a substitution of the naturally incorporated substrate with functional groups which are not directly accessible or transformable for use in conjugation chemistry incl. click chemistry, for the attachment of a targeting moiety or a label.
[0154] In the first option the modified substrate carries an anchor group directly accessible or transformable for use in conjugation chemistry incl. click chemistry, for the attachment of a targeting moiety or a label or a linker or for any other structural modification of the non-ribosomal peptide. This will allow for example connecting antibodies to the CA.
[0155] In the second option the modified substrate allows for the generation of new CAs wherein the CA carries an amino acid in the CA at a position where, in nature such an amino acid does not exist and which is also not a substitution of the naturally incorporated substrate with functional groups which are not directly accessible or transformable for use in conjugation chemistry. The amino acid may be modified. This allows for the creation of great compound libraries with CAs with novel structures.
[0156]
[0157] The two anchor groups (the azido group and the propargyl group, also known as alkyne group) of the two modified microcystins described above can be directly used for conjugation chemistry, more specific for click chemistry. Hereby the respective click reaction is based on the reaction between these two groups with each other. That means an azido group reacts with a propargyl group (alkyne group) forming a triazole conjugate as shown in
[0158] The selection of a suitable strain for the feeding of modified substrates needs to be identified by screening (feeding experiments with a high number of diverse modified substrates incl. the use and variation of strain-specific cultivation conditions for a high number of strains). Such screening is preferably done in small scale cultures (e.g. in 1.6 ml to 10 ml scale) in order to assure throughput and efficiency. In addition the detection of modified non-ribosomal peptides is preferably done by mass spectrometry (MS) whereas the MS method is preferably suited for analyses of small scale cultures without the need of extensive extractions and sample preparations, e.g. MALDI-ToF-MS (see
[0159] In the context of the establishment of a screening for strains that can be fed with modified substrates for the generation of novel non-ribosomal peptides the inventors found that feeding of O-methyl-tyrosine and homo-arginine at the same time to a strain producing MC-YR and MC-LR (Y-for tyrosine; R-for arginine; L for leucine) resulted in the incorporation of O-methyl-tyrosine instead of tyrosine and the incorporation of homo-arginine instead of arginine. Consequently, by feeding of these two modified amino acids the fed strain additionally produced MC-Y-homo-R, MC-O-methyl-YR, MC-O-methyl-Y-homo-R, and MC-L-homo-R (see
[0160] Furthermore the selection of suitable substrates for feedings is ideally done based on non-ribosomal peptides naturally produced by a specific strain, e.g. naturally produced microcystins and nodularins. Hereby not only substrates which are directly derived from the native substrates can be selected.
[0161] The fact that strains might produce several structural variants with different amino acids at a specific position of the non-ribosomal peptide significantly increases the number of suitable substrates. This counts even more if a strain naturally produces variants with structurally distant amino acids at a specific position, e.g. the Microcystis strain CBT 480 primarily produces MC-LR and MC-YR as major microcystins in comparable amounts (besides further structural variants produced in minor amounts), although the hydrophobic aliphatic amino acid leucine (L) is structurally rather distant from the aromatic amino acid tyrosine (Y).
[0162] Interestingly, by genome sequencing of said strain Microcystis CBT 480 the inventors found that despite the fact that the two major microcystin variants MC-LR and MC-YR are synthesized by this strain, there is only one microcystin synthetase gene cluster encoded in the genome of said Microcystis strain CBT480. Subsequent DNA sequence comparison with another microcystin synthetase gene cluster from Microcystis strain CBT265 (also PCC7806) which primarily produces MC-LR did not reveal significant sequence differences which might explain the differences in the abundance of microcystin variants produced by both strains (see
[0163] Therefore the inventors conclude, that preferably, the strain is selected by cultivation/feeding/chemical analysis screening and the chemical structures of the produced non-ribosomal peptides are known such that the suited modified substrates can be selected and the incorporation of the modified substrate into the non-ribosomal peptide during cultivation occurs at a defined position.
[0164] Preferable cyanobacterial strains can be of a variety of suitable genera, including but not limited to genera of the group comprising Microcystis, Planktothrix, Oscillatoria, Nostoc, Anabaena, Aphanizomenon, Hapalosiphon, Nodularia, Lyngbya, Phormidium, Spirulina, Halospirulina, Arthrospira, Trichodesmium, Leptolyngbya, Plectonema, Myxosarcina, Pleurocapsa, Pseudanabaena, Geitlerinema, Euhalothece, Calothrix, Tolypothrix, Scytonema, Fischerella, Mastigocladus, Westiellopsis, Stigonema, Chlorogloeopsis, Cyanospira, Cylindrospermopsis, Cylindrospermum, Microchaete, Rivularia, Autosira, Trichonema, Trichodesmium, Symploca, Starria, Prochlorothrix, Microcoleus, Limnothrix, Crinalium, Borzia, Chroococcidiopsis, Cyanocystis, Dermocarpella, Staniera, Xenococcus, Chamaesiphon, Chroococcus, Cyanobacterium, Cyanobium, Cyanothece, Dactylococcopsis, Gloeobacter, Gloeocapsa, Gloeothece. The only pre-requisite for strains to be used by the here described method is the synthesis of a least one non-ribosomal peptide by the respective strain.
[0165] Preferably the non-ribosomal peptide(s) produced by the cyanobacterial strain can be a variety of CA, including but not limited to cytotoxic non-ribosomal peptides of the group comprising microcystins, nodularins, cryptophycins, largarzoles, apratoxins, hectochlorines, aurilides, bisebromoamides, grassypeptolides, carmaphycins, symplocamides, lagunamides, coibamides, desmethoxy-majusculamides, curacins or it can be a non-ribosomal peptide with another bioactivity including but not limited to bioactive non-ribosomal peptides of the group comprising aeruginosins (synonyms: microcin, spumigin), microginins (synonyms: cyanostatin, oscillaginin, nostoginin), anabaenopeptins (oscillamide, ferintoic acid, nodulapeptin, plectamide, schizopeptin), cyanopaptolins (synonyms: aeruginopeptin, anabaenopeptilide, dolostatin, hofmannoline, microcystillide, micropeptin, nostocyclin, nostopeptin, oscillapeptilide, oscillapeptin, planctopeptin, scyptolin, somamide, symplostatin, tasipeptin), cyclamides (synonyms: aanyascyclamide, dendroamide, microcyclamide, nostocyclamide, raocyclamide, tenuecycyclamide, ulongamide, westiellamide. The term in this list refers to names in original publications.
[0166] Preferably, the modified substrate is a modified amino acid. Preferably, the modified substrate allows for conjugation chemistry (incl. click chemistry).
[0167] The modified substrate allows for a broad diversity of functional groups being placed into the non-ribosomal peptide, e.g. the CA for coupling to the label or targeting moiety, etc.
[0168] Preferably, it allows for conjugation chemistry including click chemistry whereas conjugation chemistry is characterized as all chemical reactions that are able to join together two molecules with functional groups that can react with each other and whereas click chemistry is characterized as chemical reactions with a high thermodynamic driving force that drives it quickly and irreversibly to high yield of a single reaction product, with high reaction specificity (in some cases, with both regio- and stereo-specificity) generating minimal and inoffensive byproducts. Click reactions are not disturbed by water. These qualities make click reactions (beside other, e.g. diagnostic applications) particularly suitable to the problem of an efficient and site-specific coupling of drug-like molecules at different positions of the drug and with different click chemistry to different positions of monoclonal antibodies via suited linker peptides or without linkers in order to create the ADC for the targeted treatment of diseases such as cancer, infection diseases, thrombosis and other diseases and disorders.
[0169] The main difference of the here described method of introduction of conjugation chemistry incl. click chemistry into the drug-like molecules compared to conventional methods is based on the way of introduction (via feeding of pre-selected cyanobacteria strains with pre-selected substrates and their site-specific introduction into the non-ribosomal peptides), the access to a broad diversity of suited modified substrates with structural features and different kind of conjugation and click chemistry, resp., and the resulting structural diversity of clickable drug-like molecules (see
[0170] The cyanobacteria strain may be selected from the group comprising Microcystis, Planktothrix, Oscillatoria, Nostoc, Anabaena, Aphanizomenon, Hapalosiphon, Nodularia, Lyngbya, Phormidium, Spirulina, Halospirulina, Arthrospira, Trichodesmium, Leptolyngbya, Plectonema, Myxosarcina, Pleurocapsa, Pseudanabaena, Geitlerinema, Euhalothece, Calothrix, Tolypothrix, Scytonema, Fischerella, Mastigocladus, Westiellopsis, Stigonema, Chlorogloeopsis, Cyanospira, Cylindrospermopsis, Cylindospermum, Microchaete, Rivularia, Autosira, Trichonema, Trichodesmium, Symploca, Starria, Prochlorothrix, Microcoleus, Limnothrix, Crinalium, Borzia, Chroococcidiopsis, Cyanocystis, Dermocarpella, Staniera, Xenococcus, Chamaesiphon, Chroococcus, Cyanobacterium, Cyanobium, Cyanothece, Dactylococcopsis, Gloeobacter, Gloeocapsa, Gloeothece.
[0171] The inventors for the first time have incorporated modified amino acids into non-ribosomal peptides from cyanobacteria which carry so called clickable anchor groups which allow for the fast and easy binding of the entire molecule to e.g. linkers or other functional units like e.g. antibodies (see
[0172] It is shown that feeding of any combination of a clickable substrate with e.g. amino acids naturally occurring in the respective non-ribosomal peptide, modified versions of these amino acids or any other modified substrates might potentially lead to an incorporation of the fed substrate combinations into the non-ribosomal peptide (see
[0173] The inventors show for the first time that a successfully fed substrate (e.g. the modified amino acid) is structurally not necessarily directly related to the substrate that is naturally incorporated into the respective non-ribosomal peptide (e.g. the respective non-modified amino acid) (see
[0174] The invention relates to a method of producing a modified non-ribosomal peptide, preferably a modified CA, wherein the strain and the modified substrates are selected and the chemical structure(s) of the produced non-ribosomal peptide(s) is/are known such that the incorporation of the modified substrates during cultivation into the non-ribosomal peptide occurs at a defined position.
[0175] Preferably, if the non-ribosomal peptide is a CA which is microcystin and the one or more modified substrates are incorporated at any position other than Adda.sub.5 and DGlu.sub.6, which has the following general structure: [0176] D-Ala.sub.1-X.sub.2-D-MeAsp.sub.3-Z.sub.4-Adda.sub.5-DGlu.sub.6-Mdha.sub.7,
and wherein X.sub.2 and Z.sub.4 are positions of preferred incorporation of said modified amino acid. If the CA is nodularin the one or more modified substrates are incorporated at any position other than Adda.sub.3 and DGlu.sub.4, which has the following general structure: [0177] D-MeAsp-Arg.sub.2-Adda.sub.3-DGlu.sub.4-Mdhb.sub.5.
[0178] Preferably, if the non-ribosomal peptide is a CA which is microcystin, the modified position is X.sub.2 or Z.sub.4 and the modified substrate is a modified amino acid (see
[0179] Also, preferably if the non-ribosomal peptide is a CA which is microcystin, the modified position is X.sub.2 and Z.sub.4 and the modified substrate is a modified amino acid (see
[0180] The modified substrate, preferably modified amino acid, preferably contains an anchor group directly accessible or transformable for use in conjugation chemistry (incl. click chemistry), for the attachment of a targeting moiety or a label or for additional structural modifications (see
[0181] In the method according to the invention, the conjugation chemistry reaction (incl. click chemistry reaction) of the clickable substrate is selected from reactions comprising copper(I)-catalyzed azide-alkyne cycloaddition, strain promoted azide-alkyne cycloaddition, alkyne-azide cycloaddition, or alkyne-tetrazine inverse-demand Diels-Alder reaction. Additional conjugation chemistry can be selected from reactions exploiting the specific reactivities of primary amines, thiols, aldehydes, carboxyls, and oximes. Therefore, the anchor group of at least one modified substrate which is directly accessible for use in conjugation chemistry (incl. click chemistry), for the attachment of a targeting moiety can be selected from the group of: [0182] Azido groups that can subsequently be modified e.g. by reaction with alkynes, activated alkenes, or phosphines, whereas the azido group of the cytotoxin reacts with the respective functional group of a linker, antibody, or other functional molecule such as a fluorescent dye or polymer matrix. [0183] Alkyne (e.g. propargy or diaryl-strained cyclooctyne) groups that can subsequently be modified e.g. by reaction with azides, whereas the alkyne group of the cytotoxin reacts with the respective functional group of a linker, antibody, or other functional molecule such as a fluorescent dye or polymer matrix. [0184] Tetrazines that can subsequently be modified e.g. by reaction with alkynes or alkenes, whereas the tetrazine group of the cytotoxin reacts with the respective functional group of a linker, antibody, or other functional molecule such as a fluorescent dye or polymer matrix. [0185] Primary amines that can subsequently be modified e.g. by reaction with isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, phosphines, or fluorophenyl esters, whereas the amino group of the cytotoxin reacts with the respective functional group of a linker, antibody, or other functional molecule such as a fluorescent dye or polymer matrix. [0186] Thiols that can subsequently be modified e.g. by reaction with maleimides, haloacetyls, pyridyldisulfides, thiosulfonares, cyanobenzothiazoles, or vinylsulfones, whereas the thiol group of the cytotoxin reacts with the respective functional group of a linker, antibody, or other functional molecule such as a fluorescent dye or polymer matrix. [0187] Aldehydes that can subsequently be modified e.g. by reaction with amines, aminothiols, Ellman's Reagent, alkoxyamines, hydrazides or thiols, whereas the aldehyde group of the cytotoxin reacts with the respective functional group of a linker, antibody, or other functional molecule such as a fluorescent dye or polymer matrix. [0188] Carboxyls that can subsequently be modified e.g. by reaction with carbodiimides, whereas the cyrboxy group of the cytotoxin reacts with the respective functional group of a linker, antibody, or other functional molecule such as a fluorescent dye or polymer matrix. [0189] Oximes that can subsequently be modified e.g. by reaction with acetophenones such as p-acetylphenylalanine, whereas the oxime group of the cytotoxin reacts with the respective functional group of a linker, antibody, or other functional molecule such as a fluorescent dye or polymer matrix.
[0190] Also claimed is the introduction of at least one modified substrate with a functional group that is directly transformable for use in conjugation chemistry (incl. click chemistry) for the attachment of a targeting moiety. One examples for this is the introduction of a substrate containing a nitro group that can be reduced to yield a primary amino group, which, as described above, can be used for conjugation chemistry (incl. click chemistry). Another example is the introduction of a substrate containing a furanyl that can subsequently be modified e.g. by photoreaction with nucleophiles such as hydrazines, whereas the furanyl group reacts after activation to an unsaturated dicarbonyl residue with the respective nucleophilic functional group of a targeting moiety like a linker, antibody, or other functional molecule such as a fluorescent dye or a polymer matrix (see
[0191] Tyrosine containing microcystins can also be functionalized using 4-phenyl-3H-1,2,4-triazoline-3,5(4H)-diones (PTADs) to introduce additional conjugation chemistry (incl. click chemistry) amenable functional groups as described above.
[0192] Ideally, modified amino acids which are directly accessible or transformable for use in conjugation chemistry (incl. click chemistry), are selected from the group of the following table (see
TABLE-US-00005 Short Order Systematic name CAS Number name Supplier number (2S)-2-amino-3-azidopropanoic acid 105661-40-3 Azido-L- Iris Biotech HAA1880 hydrochloride Ala GmbH (2S)-2-amino-6-azidohexanoic acid 159610-92-1 Azido-Lys Iris Biotech HAA1625 hydrochloride GmbH (S)-2-Amino-5-azidopentanoic acid 156463-09-1 Azido- Iris Biotech HAA1620 hydrochloride Norval GmbH (2S)-2-amino-3-(4-prop-2- 610794-20-2 Prg-Tyr Iris Biotech HAA1971 ynoxyphenyl)propanoic acid GmbH hydrochloride (2S)-2-amino-5-(N- 2149-70-4 Nitro-Arg Sigma-Aldrich 2149-70-4 nitrocarbamimidamido)pentanoic acid Chemie GmbH (2S)-2-amino-3-(furan-2-yl)propanoic 127682-08-0 Furyl-Ala Iris Biotech HAA2930 acid GmbH (S)-Amino-6-((prop-2- 1428330-91-9 Lys(Poc) Iris Biotech HAA2090 ynyloxy)carbonylamino)hexanoic acid GmbH hydrochloride N-Propargyl-Lysine 1428330-91-9 Prg-Lys SiChem SC-8002 (2S)-2-Amino-3-(4- 33173-53-4 Azido-L- Iris Biotech HAA1850 azidophenyl)propanoic acid Phe GmbH L--Amino--guanidinohexanoic acid 156-86-5 H-homo- Bachem 4016423 Arg-OH
[0193] The invention relates to a method, wherein one of the following microcystins is produced, D-Ala.sub.1-X.sub.2-D-MeAsp.sub.3-Z.sub.4-Adda.sub.5-DGlu.sub.6-Mdha.sub.7,
TABLE-US-00006 Position 1 2 3 4 5 6 7 Possible Ala.sub.1 X.sub.2 D-MeAsp.sub.3 Z.sub.4 Adda.sub.5 DGlu.sub.6 Mdha.sub.7 amino acids D-Ala variable D-MeAsp variable Adda D-Glu Mdha D-Ser D-Asp DM-Adda D-Glu(OCH.sub.3) Dha D-Leu (6Z)Adda L-Ser ADM-Adda L-MeSer Dhb MeLan
wherein
TABLE-US-00007 Ala.sub.1 X.sub.2 D-MeAsp.sub.3 Z.sub.4 Mdha.sub.7
comprise the position of the incorporation of at least one modified substrate wherein preferably the modified substrate which are directly accessible or transformable for use in conjugation (click) chemistry is an amino acid selected from the group of:
TABLE-US-00008 Short Order Systematic name CAS Number name Supplier number (2S)-2-amino-3-azidopropanoic acid 105661-40-3 Azido-L- Iris Biotech HAA1880 hydrochloride Ala GmbH (2S)-2-amino-6-azidohexanoic acid 159610-92-1 Azido-Lys Iris Biotech HAA1625 hydrochloride GmbH (S)-2-Amino-5-azidopentanoic acid 156463-09-1 Azido- Iris Biotech HAA1620 hydrochloride Norval GmbH (2S)-2-amino-3-(4-prop-2- 610794-20-2 Prg-Tyr Iris Biotech HAA1971 ynoxyphenyl)propanoic acid GmbH hydrochloride (2S)-2-amino-5-(N- 2149-70-4 Nitro-Arg Sigma-Aldrich 2149-70-4 nitrocarbamimidamido)pentanoic acid Chemie GmbH (2S)-2-amino-3-(furan-2-yl)propanoic 127682-08-0 Furyl-Ala Iris Biotech HAA2930 acid GmbH (S)-Amino-6-((prop-2- 1428330-91-9 Lys(Poc) Iris Biotech HAA2090 ynyloxy)carbonylamino)hexanoic acid GmbH hydrochloride N-Propargyl-Lysine 1428330-91-9 Prg-Lys SiChem SC-8002 (2S)-2-Amino-3-(4- 33173-53-4 Azido-L- Iris Biotech HAA1850 azidophenyl)propanoic acid Phe GmbH L--Amino--guanidinohexanoic acid 156-86-5 H-homo- Bachem 4016423 Arg-OH
[0194] The invention also relates to a method, wherein one of the following nodularins is produced,
TABLE-US-00009 Position 1 2 3 4 5 Possible MeAsp.sub.1 Arg.sub.2 Adda.sub.3 DGlu.sub.4 Mdhb.sub.5 amino acid D-MeAsp Homo- Adda D-Glu Mdhb Arg D-Asp DM-Adda D-Glu(OCH.sub.3) Dhb (6Z)Adda Me-Adda
Wherein
[0195]
TABLE-US-00010 MeAsp.sub.1 Arg.sub.2 Mdhb.sub.5
comprise the position for the at least one modified substrate, wherein preferably the modified substrate is an amino acid selected from the group of:
TABLE-US-00011 CAS Short Order Systematic name Number name Supplier number (2S)-2-amino-3-azidopropanoic acid 105661-40-3 Azido-L-Ala Iris Biotech HAA1880 hydrochloride GmbH (2S)-2-amino-6-azidohexanoic acid 159610-92-1 Azido-Lys Iris Biotech HAA1625 hydrochloride GmbH (S)-2-Amino-5-azidopentanoic acid 156463-09-1 Azido- Iris Biotech HAA1620 hydrochloride Norval GmbH (2S)-2-amino-3-(4-prop-2- 610794-20-2 Prg-Tyr Iris Biotech HAA1971 ynoxyphenyl)propanoic acid GmbH hydrochloride (2S)-2-amino-5-(N- 2149-70-4 Nitro-Arg Sigma-Aldrich 2149-70-4 nitrocarbamimidamido)pentanoic acid Chemie 3mbH (2S)-2-amino-3-(furan-2-yl)propanoic 127682-08-0 Furyl-Ala Iris Biotech HAA2930 acid GmbH (S)-Amino-6-((prop-2- 1428330-91-9 Lys(Poc) Iris Biotech HAA2090 ynyloxy)carbonylamino)hexanoic acid GmbH hydrochloride N-Propargyl-Lysine 1428330-91-9 Prg-Lys SiChem SC-8002 (2S)-2-Amino-3-(4- 33173-53-4 Azido-L- Iris Biotech HAA1850 azidophenyl)propanoic acid Phe GmbH L--Amino--guanidinohexanoic acid 156-86-5 H-homo- Bachem 4016423 Arg-OH
[0196] Ideally, the nodularin is modified at the Arg.sub.2 position.
[0197] The invention also relates to a method, wherein one of the following anabaenopeptins is produced,
TABLE-US-00012 Position 1 3 4 5 6 Possible Tyr Val HTyr MeAla Phe amino acid Arg Ile MeHTyr MeLeu Tyr Lys HPhe MeHTyr Ile Phe MeTyr Leu Ile HArg
Wherein
[0198]
TABLE-US-00013 Tyr Phe
comprise the position for the at least one modified substrate, wherein preferably the modified substrate is an amino acid selected from the group of:
TABLE-US-00014 CAS Short Order Systematic name Number name Supplier number (2S)-2-amino-3-azidopropanoic acid 105661-40-3 Azido-L- Iris Biotech HAA1880 hydrochloride Ala GmbH (2S)-2-amino-6-azidohexanoic acid 159610-92-1 Azido-Lys Iris Biotech HAA1625 hydrochloride GmbH (S)-2-Amino-5-azidopentanoic acid 156463-09-1 Azido- Iris Biotech HAA1620 hydrochloride Norval GmbH (2S)-2-amino-3-(4-prop-2- 610794-20-2 Prg-Tyr Iris Biotech HAA1971 ynoxyphenyl)propanoic acid GmbH hydrochloride (2S)-2-amino-5-(N- 2149-70-4 Nitro-Arg Sigma-Aldrich 2149-70-4 nitrocarbamimidamido)pentanoic acid Chemie GmbH (2S)-2-amino-3-(furan-2-yl)propanoic 127682-08-0 Furyl-Ala Iris Biotech HAA2930 acid GmbH (S)-Amino-6-((prop-2- 1428330-91-9 Lys(Poc) Iris Biotech HAA2090 ynyloxy)carbonylamino)hexanoic acid GmbH hydrochloride N-Propargyl-Lysine 1428330-91-9 Prg-Lys SiChem SC-8002 (2S)-2-Amino-3-(4- 33173-53-4 Azido-L- Iris Biotech HAA1850 azidophenyl)propanoic acid Phe GmbH L--Amino--guanidinohexanoic acid 156-86-5 H-homo- Bachem 4016423 Arg-OH
[0199] The invention also relates to a method, wherein one of the following oscillamides is produced,
TABLE-US-00015 Position 1 3 4 5 6 Possible Tyr Met HTyr MeAla Phe amino acid Arg Ile MeHTyr
Wherein
[0200]
TABLE-US-00016 Tyr HTyr
comprise the position for the at least one modified substrate, wherein preferably the modified substrate is an amino acid selected from the group of:
TABLE-US-00017 CAS Short Order Systematic name Number name Supplier number (2S)-2-amino-3-azidopropanoic acid 105661-40-3 Azido-L- Iris Biotech HAA1880 hydrochloride Ala GmbH (2S)-2-amino-6-azidohexanoic acid 159610-92-1 Azido-Lys Iris Biotech HAA1625 hydrochloride GmbH (S)-2-Amino-5-azidopentanoic acid 156463-09-1 Azido- Iris Biotech HAA1620 hydrochloride Norval GmbH (2S)-2-amino-3-(4-prop-2- 610794-20-2 Prg-Tyr Iris Biotech HAA1971 ynoxyphenyl)propanoic acid GmbH hydrochloride (2S)-2-amino-5-(N- 2149-70-4 Nitro-Arg Sigma-Aldrich 2149-70-4 nitrocarbamimidamido)pentanoic acid Chemie GmbH (2S)-2-amino-3-(furan-2-yl)propanoic 127682-08-0 Furyl-Ala Iris Biotech HAA2930 acid GmbH (S)-Amino-6-((prop-2- 1428330-91-9 Lys(Poc) Iris Biotech HAA2090 ynyloxy)carbonylamino)hexanoic acid GmbH hydrochloride N-Propargyl-Lysine 1428330-91-9 Prg-Lys SiChem SC-8002 (2S)-2-Amino-3-(4- 33173-53-4 Azido-L- Iris Biotech HAA1850 azidophenyl)propanoic acid Phe GmbH L--Amino--guanidinohexanoic acid 156-86-5 H-homo- Bachem 4016423 Arg-OH
[0201] The invention also relates to a method, wherein modified cryptophycins are produced, wherein the O-methyl-chloro-Tyrosine in cryptophycin 1 comprise the position for the at least one modified substrate, wherein preferably the modified substrate is an amino acid selected from the group of:
TABLE-US-00018 CAS Short Order Systematic name Number name Supplier number (2S)-2-amino-3-azidopropanoic acid 105661-40-3 Azido-L- Iris Biotech HAA1880 hydrochloride Ala GmbH (2S)-2-amino-6-azidohexanoic acid 159610-92-1 Azido-Lys Iris Biotech HAA1625 hydrochloride GmbH (S)-2-Amino-5-azidopentanoic acid 156463-09-1 Azido- Iris Biotech HAA1620 hydrochloride Norval GmbH (2S)-2-amino-3-(4-prop-2- 610794-20-2 Prg-Tyr Iris Biotech HAA1971 ynoxyphenyl)propanoic acid GmbH hydrochloride (2S)-2-amino-5-(N- 2149-70-4 Nitro-Arg Sigma-Aldrich 2149-70-4 nitrocarbamimidamido)pentanoic acid Chemie GmbH (2S)-2-amino-3-(furan-2-yl)propanoic 127682-08-0 Furyl-Ala Iris Biotech HAA2930 acid GmbH (S)-Amino-6-((prop-2- 1428330-91-9 Lys(Poc) Iris Biotech HAA2090 ynyloxy)carbonylamino)hexanoic acid GmbH hydrochloride N-Propargyl-Lysine 1428330-91-9 Prg-Lys SiChem SC-8002 (2S)-2-Amino-3-(4- 33173-53-4 Azido-L- Iris Biotech HAA1850 azidophenyl)propanoic acid Phe GmbH L--Amino--guanidinohexanoic acid 156-86-5 H-homo- Bachem 4016423 Arg-OH
[0202] In the method according to the invention, the at least one modified amino acid comprises an anchor group directly accessible or transformable for use in conjugation chemistry (incl. click chemistry), for the attachment of a targeting moiety and/or a label via a linker or w/o a linker between the modified amino acid and the targeting moiety and/or a label. Such anchor groups are described above for the modified substrates.
[0203] In the method according to the invention, the conjugation chemistry reaction (incl. click chemistry reaction) of the clickable substrate is selected from the group comprising copper(I)-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, alkyne-azide cycloaddition, or alkyne-tetrazine inverse-demand Diels-Alder reaction. Additional conjugation chemistry can be selected from reactions exploiting the specific reactivities of primary amines, thiols, aldehydes, carboxyls, and oximes.
[0204] However, regarding the modification of the CA of microcystins and nodularins by the introduction of modified substrates most preferred are the genera Microcystis, Planktothrix, Oscillatoria, Nostoc, Anabaena, Aphanizomenon, Hapalosiphon, Nodularia.
[0205] The invention relates to a modified non-ribosomal peptide, including a modified CA compound comprising at least one modified amino acid, wherein the at least one modified amino acid comprises an anchor group directly accessible or transformable for use in conjugation chemistry (incl. click chemistry), for the attachment of a targeting moiety and/or a label via a linker or w/o a linker between the modified amino acid and the targeting moiety and/or a label.
[0206] Ideally, the at least one modified CA compound is a microcystin, a nodularin or a cryptophycin or one of the CA listed in the above table with cyanobacterial CA.
[0207] Preferably, the modified amino acid in the CA is linked to a targeting moiety or a label. Concerning the targeting moiety (TM), in one embodiment, the ADC specifically binds to a receptor encoded by an ErbB gene. The TM may bind specifically to an ErbB receptor selected from EGFR, HER2, HER3 and HER4. The ADC may specifically bind to the extracellular domain (ECD) of the HER2 receptor and inhibit the growth of tumor cells which overexpress HER2 receptor (see
[0208] The ADC of the invention may be useful in the treatment of cancer including, but are not limited to, antibodies against cell surface receptors and tumor-associated antigens (TAA). Such tumor-associated antigens are known in the art, and can prepared for use in generating antibodies using methods and information which are well known in the art. In attempts to discover effective cellular targets for cancer diagnosis and therapy, researchers have sought to identify transmembrane or otherwise tumor-associated polypeptides that are specifically expressed on the surface of one or more particular type(s) of cancer cell as compared to on one or more normal non-cancerous cell(s). Often, such tumor-associated polypeptides are more abundantly expressed on the surface of the cancer cells as compared to on the surface of the non-cancerous cells. The identification of such tumor-associated cell surface antigen polypeptides has given rise to the ability to specifically target cancer cells for destruction via targeted antibody-based therapies.
[0209] Examples of TAA include, but are not limited to, Tumor-Associated Antigens listed below. Tumor-associated antigens targeted by antibodies include all amino acid sequence variants and isoforms possessing at least about 70%, 80%, 85%, 90%, or 95% sequence identity relative to the sequences identified in the cited references, or which exhibit substantially the same biological properties or characteristics as a TAA having a sequence found in the cited references. For example, a TAA having a variant sequence generally is able to bind specifically to an antibody that binds specifically to the TAA with the corresponding sequence listed. The sequences and disclosure in the reference specifically recited herein are expressly incorporated by reference.
[0210] BMPR1B (bone morphogenetic protein receptor-type IB, Genbank accession no. NM-001203);
[0211] E16 (LAT1, SLC7A5, Genbank accession no. NM-003486);
[0212] STEAP1 (six transmembrane epithelial antigen of prostate, Genbank accession no. NM-012449);
[0213] 0772P (CA125, MUC16, Genbank accession no. AF361486);
[0214] MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank accession no. NM-005823);
[0215] Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession no. NM-006424);
[0216] Sema 5b (F1110372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B, Genbank accession no. AB040878);
[0217] PSCA hlg (2700050C12Rik, C530008016Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession no. AY358628);
[0218] ETBR (Endothelin type B receptor, Genbank accession no. AY275463);
[0219] MSG783 (RNF124, hypothetical protein F1120315, Genbank accession no. NM-017763);
[0220] STEAP2 (HGNC-8639, IPCA-1, PCANAP1, STAMP, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein, Genbank accession no. AF455138);
[0221] TrpM4 (BR22450, F1120041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession no. NM-017636);
[0222] CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor, Genbank accession no. NP-003203 or NM-003212);
[0223] CD21 (CR2 (Complement receptor 2) or C3DR(C3d/Epstein Barr virus receptor) or Hs.73792 Genbank accession no. M26004);
[0224] CD79b (CD79B, CD79, IGb (immunoglobulin-associated beta), 29, Genbank accession no. NM-000626 or 11038674);
[0225] FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a), SPAPiB, SPAPIC, Genbank accession no. NM-030764, AY358130);
[0226] HER2 (ErbB2, Genbank accession no. M11730); Coussens L., et al Science (1985) 230(4730):1132-1139);
[0227] NCA (CEACAM6, Genbank accession no. M18728); Barnett T., et al Genomics 3, 59-66, 1988;
[0228] MDP (DPEP1, Genbank accession no. BC017023);
[0229] IL20R (IL20Ra, ZCYTOR7, Genbank accession no. AF184971);
[0230] Brevican (BCAN, BEHAB, Genbank accession no. AF229053);
[0231] EphB2R (DRT, ERK, HekS, EPHT3, Tyro5, Genbank accession no. NM-004442);
[0232] ASLG659 (B7h, Genbank accession no. AX092328); US20040101899 (Claim 2);
[0233] PSCA (Prostate stem cell antigen precursor, Genbank accession no. AJ297436);
[0234] GEDA (Genbank accession No. AY260763); AAP14954 lipoma HMGIC fusion-partner-like protein/pid=AAP14954.1 Homo sapiens (human);
[0235] (26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3, BR3, Genbank accession No. AF116456); BAFF receptor/pid=NP-443177.-Homo sapiens; Thompson, J. S., et al Science 293 (5537), 2108-2111 (2001); WO2004058309; WO2004011611; WO2003045422 (Example; Page 32-33); WO2003014294 (Claim 35;
[0236] CD22 (B-cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FU22814, Genbank accession No. AK026467);
[0237] CD79a (CD79A, CD79a, immunoglobulin-associated alpha, a B cell-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex on the surface with Ig M molecules, transduces a signal involved in B-cell differentiation);
[0238] CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptor that is activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and perhaps development of AIDS, lymphoma, myeloma, and leukemia);
[0239] HLA-DOB (Beta subunit of MHC class II molecule (la antigen) that binds peptides and presents them to CD4+T lymphocytes);
[0240] P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, deficiency may contribute to the pathophysiology of idiopathic detrusor instability);
[0241] CD72 (B-cell differentiation antigen CD72, Lyb-2); 359 aa), pl: 8.66, MW: 40225 TM: 1 [P] Gene Chromosome: 9p13.3, Genbank accession No. NP-001773.1);
[0242] LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of the leucine rich repeat (LRR) family, regulates B-cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosis);
[0243] FcRH1(Fc receptor-like protein 1, a putative receptor for the immunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains, may have a role in B-lymphocyte differentiation);
[0244] IRTA2 (FcRH5, Immunoglobulin superfamily receptor translocation associated 2, a putative immunoreceptor with possible roles in B cell development and lymphomagenesis;
[0245] TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, putative transmembrane proteoglycan, related to the EGF/heregulin family of growth factors and follistatin);
[0246] MUC1 (Tumor-associated MUC glycopeptide epitopes); Human adenocarcinomas overexpress a hypoglycosylated, tumor-associated form of the mucin-like glycoprotein MUC1 containing abnormal mono- and disaccharide antigens, such as Tn, sialyl-Tn, and TF, as well as stretches of unglycosylated protein backbone in the variable number of tandem repeats (VNTR) region.
[0247] The ADC which can be produced based on the present invention may be used to treat various diseases or disorders in a patient, such as cancer and autoimmune conditions including those characterized by the overexpression of a disease-associated antigen, including but not limited to tumor-associated antigen. Exemplary conditions or disorders include infection diseases, thrombosis and others and specifically benign or malignant tumors; leukemia and lymphoid malignancies; other disorders such as neuronal, glial, astrocytal, hypothalamic, glandular, macrophagal, epithelial, stromal, blastocoelic, inflammatory, angiogenic and immunologic disorders. Cancer types susceptible to ADC treatment include those which are characterized by the overexpression of certain tumor associated antigens or cell surface receptors, e.g. HER2.
[0248] One method is for the treatment of cancer in a mammal, wherein the cancer is characterized by the overexpression of an ErbB receptor. The mammal optionally does not respond, or responds poorly, to treatment with an unconjugated anti-ErbB antibody. The method comprises administering to the mammal a therapeutically effective amount of an antibody-drug conjugate compound. The growth of tumor cells that overexpress a growth factor receptor such as HER2 receptor or EGF receptor may be inhibited by administering to a patient an ADC according to the invention which binds specifically to said growth factor receptor and a chemotherapeutic agent wherein said antibody-drug conjugate and said chemotherapeutic agent are each administered in amounts effective to inhibit growth of tumor cells in the patient (see
[0249] Examples of cancer to be treated herein include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, gastrointestinal stromal tumor (GIST), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer.
EXAMPLES
[0250] Successful feedings of modified substrates were performed in different cultivation systems and scales allowing for screening (small scales of up to 10 ml; see
[0251] 1.6 ml cultures cultivated in ca. 2.2 ml deep-well microtiter plates (dw-MTP) whereas CO.sub.2 supply was assured by intense shaking of 600 rpm and a constant CO.sub.2 concentration of 5% in the head space above the dw-MTP. Illumination occurred via LED panel or vial fluorescence bulbs for 24 hours a day. Light intensity was adjusted in dependence of the strain and its growth phase between 35-250 mol/s*m.sup.2. The temperature was strain-specific varied between 20 C. and 30 C.
[0252] A cultivation according to the method is thus preferred wherein the shaking is between 400-800 rpm and a constant CO.sub.2 concentration of 1 to 10% in the head space, preferably 3 to 8% in the head space.
[0253] 10 ml cultures cultivated in 40 ml polystyrene tubes whereas CO.sub.2 supply was assured by intense shaking of 250-350 rpm and a constant CO.sub.2 concentration of 5% below the culture vessel. Hereby CO.sub.2 got introduced into the culture via a CO.sub.2 permeable polypropylene membrane on the bottom of the culture vessels. Illumination occurred via fluorescence bulbs for 24 hours a day and light intensity was again adjusted in dependence of the strain and its growth phase between 35-250 mol/s*m.sup.2. The temperature was again strain-specific varied between 20 C. and 30 C.
[0254] A cultivation according to the method is thus preferred wherein the illumination occurred via fluorescence bulbs for 24 hours a day and is between 20-450 mol/s*m2.
[0255] 50 ml cultures cultivated in glass flasks whereas CO.sub.2 supply was assured by bubbling with constant CO.sub.2 concentration of 5%. The cultures were mixed via stirring with a magnetic stir bar at 100 rpm. Illumination occurred via fluorescence bulbs for 24 hours a day and intensity was adjusted in dependence of strain and growth phase between 35-250 mol/s*m2.
[0256] In addition, feeding experiments were also performed in a production scale between 2 L and 20 L whereas CO.sub.2 supply and mixing was assured by bubbling with constant CO.sub.2 concentration of 0.5-5.0%. Illumination occurred via fluorescence bulbs and intensity was adjusted in dependence of strain and growth phase between 35-250 mol/s*m2
[0257] Optionally, the cultivations were performed under day-night-cycles of 16 hours light/8 hours at the same light intensities during the day period as described above.
[0258] Optionally, the cultivations were performed with different light sources (e.g. LED lights or sulfur-plasma lamps) and using strain-specific variations of light intensity, CO.sub.2 concentration, shaking/stirring intensity and media composition.
[0259] Exemplary feeding scheme for the 10 ml scale:
[0260] All strains were cultivated in BG11 medium (see below), according to strain-specific cultivation conditions determined before.
[0261] Cells were pre-cultivated in Erlenmeyer flasks under low light conditions (30 mol/s*m2) for 4 days at 25 C. and on a shaker at 70 rpm.
[0262] For the feeding experiment in the 10 ml scale, the cells were inoculated at optical density at 750 nm (OD750 nm) of 0.5 in ca. 40 ml polystyrene tubes. The medium was buffered by addition of TES to a concentration of 10 mM in the medium. Optionally DMSO was added to a concentration of 1% in the medium.
[0263] The feeding of cultures started at inoculation by adding the respective modified substrate(s) to a concentration of 10 M in the medium. Daily additions of modified substrates remained constant over 4 days by feeding of additional 10 M per day (day 1-4). Alternatively, additions of modified substrate(s) were done on day one and day three after inoculation by feeding of the modified substrate(s) to a concentration of 30 M in the medium at each of the days. Growth of cultures was monitored daily by measurements of optical density at 750 nm (OD750 nm). Cultivation was finished by adding methanol to the culture to an end concentration of 20%. Subsequently extraction was done via a standard solid phase extraction procedure using C18-modified silica cartridges.
[0264] For other scales mentioned above the protocols were similar and only slightly varied. For example, at 2 and 20 L scale the medium was not always buffered and due to the slower growth rate the duration of cultivation was prolonged for another week. Furthermore, in some cases increased amounts of added modified substrates up to 300 M media concentration were used (if strain tolerated such concentrations) in order to increase the yield of modified non-ribosomal peptides.
TABLE-US-00019 TABLE Recipe for BG11 medium which has been used for feeding experiments Component mg/L mM NaNO.sub.3 1500 17.6 K.sub.2HPO.sub.4*3H.sub.2O 40 0.23 MgSO.sub.4*7H.sub.2O 75 0.3 CaCl.sub.2*2H.sub.2O 36 0.24 Na.sub.2CO.sub.3 20 0.19 Ferric ammon. citrate 6 0.021 Citric acid 6 0.031 Na.sub.2EDTA*2H.sub.2O 1 0.0027 Trace elements g/L M H.sub.3BO.sub.3 2.86 46.3 MnCl.sub.24H.sub.2O 1.8 9.15 ZnSO.sub.47H.sub.2O 0.22 0.77 Na.sub.2MoO.sub.42H.sub.2O 0.390 1.61 CuSO.sub.45H.sub.2O 0.079 0.32 Co(NO.sub.3).sub.26H.sub.2O 0.0494 0.17
[0265] For the following strains feeding of at least one modified and clickable substrate were demonstrated.
TABLE-US-00020 Cyano Biotech Main non-ribosomal Strain ID No. Genera peptide variants produced 1 Microcystis MC-YR 265 Microcystis MC-LR MC-LR, Cyanopeptolin A, B, C, D und 963A; Microcyclamide, Aeruginosin, Aerucyclamide A, B, C, D 275 Microcystis MC-LR MC-LW MC-LF 280 Planktothrix MC-LR 329 Planktothrix (D-Asp3, Dhb7)MC-RR 332 Planktothrix (D-Asp3, Dhb7)MC-RR, Anabaenopeptin A, B, E/F, NZ867 480 Microcystis MC-LR MC-YR 633 Microcystis MC-RR 786 Nodularia NOD 861 Microcystis MC-RY MC-LY 959 Microcystis MC-LR MC-YR 1161 Planktothrix (D-Asp3, E-Dhb7)MC-RR Anabaenopeptin A, E/F, B Oscillamide Y
[0266] MC is microcystin, the two letters behind MC define the amino acids at the variable positions 2 and 4 whereas R is arginine, Y is tyrosine, L is leucine, W is tryptophan, and F ist phenylalanine. D-MAsp3 is D-erythro-f-methylaspartic acid at position 3 and Dhb7 is dehydrobutyrate at position 7. NOD is Nodularin.
[0267]
[0268] The following tables summarize results of feeding experiments of different cyanobacterial genera and strains, resp. with one or two modified substrates each comprising an anchor group directly accessible or transformable for use in conjugation chemistry (incl. click chemistry), for the attachment of a targeting moiety and/or a label via a linker or w/o a linker between the modified amino acid and the targeting moiety and/or a label.
TABLE-US-00021 TABLE 1 Part 1 of summary of results of feeding one modified substrate to different cyanobacterial strains of the genera Microcystis and Planktothrix. MC - microcystin with letters behind MC indicating the amino acids at the variable position 2 and 4 in the one-letter-code. Cyano Biotech GmbH CH Kilger Anwaltspartnerschaft mbB Germany Fasanernstrae 29 Our Ref.: B111-0003WO1 29 10719 Berlin Naturally NKP Naturally visible produced amino acid
NKP variant
of which is CBT produced which is naturally replaced strain Genera/ by the effected produced by modified no. Species strain by
NRP
1 Microcystis
Arg
sp. 1 Microcystis
Tyr
sp. 1 Microcystis
Arg
sp. 1 Microcystis
Tyr
sp.
Arg
275
Arg
275
Arg
Arg
275
275
Arg
Arg
Trp
Trp
CBT strain Position Mass MS Peak UV Peak no. of
PDA
1 2
yes yes 1 4
yes
1
yes yes 1 4
yes yes
6
yes no
2
yes no 275
yes
275 4
yes
6
yes
4
yes yes 275 2
yes yes 275 4
yes yes
4
yes yes
4
yes yes
4
yes yes
indicates data missing or illegible when filed
TABLE-US-00022 TABLE 2 Part 2 of summary of results of feeding one modified substrate to different cyanobacterial strains of the genera Microcystis and Planktothrix. MC - microcystin with letters behind MC indicating the amino acids at the variable position 2 and 4 in the one-letter-code. Naturally produced
amino acid
variant which is CBT produced which is replaced strain Genera/ by the effected by modified Position no. Species strain by
substrate
of
329
Arg
4 322
Arg
2 4
2 4
Arg
4
Arg
4 4
Arg
4
2 4
Arg
4
4
Tyr
2 4
4
Tyr
2 Mass CBT difference UV Peak strain between Calculated Measured MS Peak PDA no.
329 Nitro-Arg
yes yes 322
yes yes 4
yes
4
Nitro-Arg
yes yes
yes yes 4
yes yes
yes yes 4
Nitro-Arg
yes yes 4
yes yes 4
Arg-Tyr
yes yes 4
yes yes 4
yes yes
indicates data missing or illegible when filed
TABLE-US-00023 TABLE 3 Part 3 of summary of results of feeding one modified substrate to different cyanobacterial strains of the genera Microcystis and Planktothrix. MC - microcystin with letters behind MC indicating the amino acids at the variable position 2 and 4 in the one-letter-code Naturally Naturally NKP produced incorporated variant NKP variant amino acid naturally which is which is CBT produced effected by replaced by strain Genera/ by the fed modified modified no. species strain substrate
substrate
Microcystis
Arg
sp.
Microcystis
Arg
sp. 635 Microcystis
Arg
sp.
Microcystis
Arg
sp.
Arg
Microcystis
Arg
sp.
Microcystis
Arg
sp.
Microcystis
Arg
sp.
Micro
Arg
sp.
Microcystis
Arg
sp.
Microcystis
Arg
sp.
Microcystis
T
sp.
Microcystis
T
sp.
Arg
Arg
T
Position of naturally Short CBT incorporated
UV Peak strain amino acid of modified MS Peak PDA no. in
substrate
2
yes yes
2
yes yes 635
yes yes
yes yes
yes yes
4 Nitro-Arg
yes
4 Nitro-Arg
yes
yes yes
4
yes yes
yes yes
yes yes
yes yes
2
yes yes
yes yes
7
yes yes
7
yes yes
4
yes yes
indicates data missing or illegible when filed
TABLE-US-00024 TABLE 4 Summary of results of feeding two modified substrates to different cyanobacterial culture of the genera Microcystis. MC - microcystin with letters behind MC indicating the amino acids at the variable position 2 and 4 in the one-letter-code. Monoisotopic mass of Naturally Naturally naturally incorporated; incorporated; produced amino acid amino acid microcystin which is which is Microcystin variant replaced by replaced by Monoisotopic variants which is modified modified Short mass Short CBT naturally effected substrate 1 substrate 2 names of (zwitterion) names of strain Genera/ produced by by the (position (position modified of modified modified no. Species the strain substrates in MC) in MC) substrate 1 substrate 1 substrate 2 1 Microcystis MC-YR 1044.528032 Tyr Arg Prg-Tyr 219.0895433 Nitro-Arg sp. (pos. 4) (pos. 2) 1 Microcystis MC-YR 1044.528032 Arg Tyr Nitro-Arg 219.0967539 Azido- sp. (pos. 2) (pos. 4) L-Phe 480 Microcystis MC-LR 1044.528032 Arg Tyr Nitro-Arg 219.0967539 Prg-Tyr aeruginosa (D-Asp3)MC- (pos. 4) (pos. 2) YR 480 Microcystis MC-LR 1044.528032 Arg Tyr Nitro-Arg 219.0967539 Azido- aeruginosa (D-Asp3)MC- (pos. 4) (pos. 2) L-Phe YR Mass Mass difference difference Calculated Measured Monoisotopic between between monoisotopic monoisotopic mass natural und natural und mass of mass of CBT (zwitterion) modified modifeid mutasynthesis mutasynthesis MS Peak UV Peak strain of modified substrate 1 substrate 2 product (novel product EIC (Mass PDA no. substrate 2 (Da) (Da) microcystin) [M + H]+ spectrometry) (HPLC) 1 219.0967538 38.01564528 44.885 1127.5288 1128.3360 yes yes 1 206.0803756 44.08507492 25.00647758 1114.5196 1115.5269 yes yes 480 219.0899433 44.98507492 38.03564528 1127.5288 1128.5360 yes yes 480 206.0803756 44.98507492 25.00547758 1134.5396 3115.5269 yes yes
FIGURE CAPTIONS
[0269]
[0270] Upper left: General structure of Microcystins. X.sub.2 and Z.sub.4 indicate variable L-amino acids. D-Ala=D-Alanine, D-Me-Asp=D-methyl aspartic acid, Arg=Arginine, Adda=3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid, D-Glu=D-glutamic acid, Mdha=N-methyldehydroaanine. Upper right: General structure of Nodularins. Arg.sub.2 indicates the variable L-amino acid corresponding to Z.sub.4 in the microcystin molecule. D-Me-Asp=D-methyl aspartic acid, Arg=Arginine, Adda=3-amino-9-methoxy-2,6,8-trimethyl-10-phenydeca-4,6-dienoic acid, D-Glu=D-glutamic acid, Mdhb=N-methyldehydrobutyrate.
[0271] Down right: General structure of anabaenopeptin A and schematic general structure of anabaenopeptin type peptides (incl. oscillamides): Anabaenopeptins (and oscillamides) are cyclic peptides that are characterized by a lysine in position 5 and the formation of the ring by an N-6-peptide bond between Lys and the carboxy group of the amino acid in position 6 A side chain of one amino acid unit is attached to the ring by an ureido bond formed between the a-N of Lys and the a-N of the side chain amino acid. All other positions in the ring and side chain are variable.
[0272] Down right: Chemical structure of cryptophycin-1. Cryptophycins are a class of macrocyclic depsipeptides produced as secondary metabolites by cyanobacteria of the genus Nostoc. Isolation of the first representative, cryptophycin-1, from cultivated Nostoc species ATCC 53789 was published in 1990 by researchers at Merck.
[0273]
[0274] Comparison between different cultivation systems and scales and different mass spectrometry detections in the context of suitable screening approaches towards strains that are suited for feeding of modified substrates for modifying non-ribosomal peptides including CA (
[0275]
[0276] Detection of modified microcystins by two different mass spectrometry method after feeding of modified substrates to a Microcystis aeruginosa strain CBT 480 in a 50 ml scale (above of each of the four figures A, B, C, D detection with ESI-IT-ToF-MS; below of each of the four figures A, B, C, D detection with MALDI-ToF-MS).
TABLE-US-00025 A: Control (no feeding with O- B: Control (no feeding with methyltyrosine) homoarginine) C: Feeding with O-methyltyrosine D: Feeding with homoarginine
[0277] Molecule masses of naturally produced microcystins:
[0278] 995 Da=MC-LR, 1045 Da=MC-YR
[0279] Molecule masses of modified microcystins generated by feeding with O-methyltyrosine (OMetY) and homoarginine (hR)
[0280] 1059 Da=MC-OMetYR or MC-YhR; 1009 Da=MC-LhR
[0281]
[0282] Detection of modified microcystins by two different mass spectrometry method after feeding of modified substrates to a Microcystis aeruginosa strain CBT 480 in a 6 ml scale (above of each of the two figures A/A and B/B detection with ESI-IT-ToF-MS; below of each of the two figures A/A and B/B detection with MALDI-ToF-MS).
TABLE-US-00026 A, A: CBT 480 culture fed with B, B: CBT 480 culture fed with O-methyltyrosine homoarginine
[0283] Molecule masses of naturally produced microcystins:
[0284] 995 Da=MC-LR, 1045 Da=MC-YR
[0285] Molecule masses of modified microcystins generated by feeding with O-methyltyrosine (OMetY) and homoarginine (hR)
[0286] 1059 Da=MC-OMetYR or MC-YhR; 1009 Da=MC-LhR;
[0287]
[0288] Detection of modified microcystins by two different mass spectrometry method after feeding of modified substrates to a Microcystis aeruginosa strain CBT 480 with O-methyltyrosine in a 1.6 ml (dw-MTP) scale (ESI-IT-ToF-MS on the left; MALDI-ToF-MS on the right)
[0289] A, A: feeding of 300 M O-methyltyrosine (OMetY), w/o DMSO
[0290] B, B: feeding of 30 M O-methyltyrosine (O-MetY), w/o DMSO
[0291] C, C: feeding of 300 M O-methyltyrosine (OMetY), w/ 1% DMSO
[0292] D, D: feeding of 30 M O-methyltyrosine (OMetY), w/ 1% DMSO
[0293] E, E: control (no feeding)
[0294] Molecule masses of naturally produced microcystins:
[0295] 995 Da=MC-LR, 1045 Da=MC-YR
[0296] Molecule masses of modified microcystin generated by feeding with O-methyltyrosine
[0297] 1059 Da=MC-OMetYR
[0298]
[0299] Detection of modified microcystins by two different mass spectrometry method after feeding of modified substrates to a Microcystis aeruginosa strain CBT 480 with homoarginine in a 1.6 ml (dw-MTP) scale (ESI-IT-ToF-MS detection on the left; MALDI-ToF-MS detection on the right)
[0300] A, A: feeding of 300 M homoarginine (hR), w/o DMSO
[0301] B, B: feeding of 30 M homoarginine (hR), w/o DMSO
[0302] C, C: feeding of 300 M homoarginine (hR), w/ 1% DMSO
[0303] D, D: feeding of 30 M homoarginine (hR), w/ 1% DMSO
[0304] E, E: control (no feeding)
[0305] Molecule masses of naturally produced microcystins:
[0306] 995 Da=MC-LR, 1045 Da=MC-YR
[0307] Molecule masses of modified microcystins generated by feeding with homoarginine
[0308] 1059 Da=MC-YhR; 1009 Da=MC-LhR
[0309] All modified microcystins could be detected with both MS methods. However, most samples resulting from feeding without the addition of DMSO of 1% in the culture medium could not be detected with MALDI-ToF-MS but with ESI-IT-ToF-MS. Therefore, it is recommended to use DMSO for feeding experiments in screenings of small scale cultures (between 1 and 10 ml culture volumes) especially if the MS detection of modified non-ribosomal peptides is based on MALDI-ToF-MS.
[0310] On the other side MALDI-ToF-MS detection of modified non-ribosomal peptides after feeding of modified substrates to small scale cultures of 1.6 ml cultivated in deep-well-microtiter plates (dw-MTW) allows for high throughput screening (HTS). Both cultivation (with and without feeding of modified substrates) and sample preparation for MALDI-ToF-MS can be done using a pipetting robot allowing for the parallel test of diverse strains and substrates as described in Tillich et al. BMC Microbiology 2014, 14:239.
[0311]
[0312] McyBI represent the first of two enzyme modules of McyB and is responsible for the incorporation of the amino acid at the position 2 of the microcystin molecule. This is the amino acid leucine in case of the Microcystin aeruginosa strain PCC7806 whereas it is leucine OR tyrosine in the Microcystis aeruginosa strain CBT 480. The so called core motifs A2-A6 of the adenylation (A) domain of McyBI are highlighted in black (A2-A6) and the amino acids responsible for substrate (amino acid) recognition and activation during the biosynthesis of the respective microcystin are indicated by big and bold white letters. These amino acids form the active pocket of the A domains and the sequence in their one-letter amino acid code represent the so called specificity-conferring code of A domains which shall allow for the prediction of substrate specificity of A domains. The box and the arrow indicate the only difference in the amino acid sequence of McyBI of both strains. Only one of nine pocket-forming amino acids of the A domains of both strains is different between the strains and also the remaining parts of the A domain as well as of the whole biosynthetic gene clusters are almost identical between the strains leading to the conclusion that the incorporation of leucine and tyrosine at position 2 of the microcystin in the strain CBT 480 is a strain-specific feature but cannot be explained by differences in the DNA sequence of the biosynthetic gene clusters and amino acid sequence of the microcystin synthetases, resp.
[0313]
[0314] Exemplary embodiment No. 1: Incorporation of the modified substrate Azido-L-Phe (Phe=phenylalanine) into Microcystin-YR in position 2 produced by strain CBT959. HPLC-PDA Chromatogram at 238 nm for sample of control cultivation (a) for sample of cultivation with added modified substrate (b). Extracted ion chromatogram from HPLC-MS data of mass value of protonated molecular ion of novel Microcystin variant for sample of control cultivation (c) and for sample of cultivation with added modified substrate (d) in the positive ionization mode. Finally, (e) shows the averaged mass spectrum of the peak visible in chromatogram d). Detector signal intensities (y-Axis) are measured in milli-absorption units (mAU) und counts (dimensionless quantity) for PDA and mass spectrometry data, respectively.
[0315] The growth of strain CBT 959 could not be followed by measurement of optical density at 750 nm (OD.sub.750 nm) as the cell formed aggregates making it impossible to measure reliable OD.sub.750 nm values.
[0316]
[0317] Exemplary embodiment No. 2: Incorporation of the modified substrate Prg-Tyr (Tyr=Tyrosine) into Microcystin YR in position 2 produced by strain CBT 480.
[0318] HPLC-PDA Chromatogram at 238 nm for sample of control cultivation (a) for sample of cultivation with added modified substrate (b). Extracted ion chromatogram from HPLC-MS data of mass value of protonated molecular ion of novel Microcystin variant for sample of control cultivation (c) and sample of cultivation with added modified substrate (d) in the positive ionization mode. Finally, e) shows the averaged mass spectrum of the peak visible in chromatogram d). Detector signal intensities (y-Axis) are measured in milli-absorption units (mAU) und counts (dimensionless quantity) for PDA and mass spectrometry data respectively.
[0319]
[0320] Exemplary embodiment No. 2: Growths curve of CBT 480 cultures with and without Prg-Tyr (Tyr=Tyrosine) added.
[0321]
[0322] Exemplary embodiment No. 3: Incorporation of the modified substrate Azido-Lys (Lys=Lysine) into Microcystin LR in position 4 produced by strain CBT 275.
[0323] HPLC-PDA Chromatogram at 238 nm for sample of control cultivation (a) for sample of cultivation with added modified substrate (b). Extracted ion chromatogram from HPLC-MS data of mass value of protonated molecular ion of novel Microcystin variant for sample of control cultivation (c) and sample of cultivation with added modified substrate (d) in the positive ionization mode. Finally, e) shows the averaged mass spectrum of the peak visible in chromatogram d). Detector signal intensities (y-Axis) are measured in milli-absorption units (mAU) und counts (dimensionless quantity) for PDA and mass spectrometry data respectively.
[0324]
[0325] Exemplary embodiment No. 3: Growths curve of CBT 275 cultures with and without Azido-Lys (Lys=Lysine) added.
[0326]
[0327] Exemplary embodiment No. 4: Incorporation of the modified substrate Prg-Tyr (Tyr=Tyrosine) into Microcystin LW in position 4 produced by strain CBT 275.
[0328] HPLC-PDA Chromatogram at 238 nm for sample of control cultivation (a) for sample of cultivation with added modified substrate (b). Extracted ion chromatogram from HPLC-MS data of mass value of protonated molecular ion of novel Microcystin variant for sample of control cultivation (c) and sample of cultivation with added modified substrate (d) in the positive ionization mode. Finally, e) shows the averaged mass spectrum of the peak visible in chromatogram d). Detector signal intensities (y-Axis) are measured in milli-absorption units (mAU) und counts (dimensionless quantity) for PDA and mass spectrometry data respectively.
[0329]
[0330] Exemplary embodiment No. 4: Growths curve of CBT 275 cultures with and without Prg-Tyr (Tyr=Tyrosine) added.
[0331]
[0332] Exemplary embodiment No. 5: Incorporation of the modified substrate Nitro-Arg (Arg=Arginine) into Microcystin YR in position 4 produced by strain CBT 1.
[0333] HPLC-PDA Chromatogram at 238 nm for sample of control cultivation (a) for sample of cultivation with added modified substrate (b). Extracted ion chromatogram from HPLC-MS data of mass value of protonated molecular ion of novel Microcystin variant for sample of control cultivation (c) and sample of cultivation with added modified substrate (d) in the positive ionization mode. Finally, e) shows the averaged mass spectrum of the peak visible in chromatogram d). Detector signal intensities (y-Axis) are measured in milli-absorption units (mAU) und counts (dimensionless quantity) for PDA and mass spectrometry data respectively.
[0334]
[0335] Growths curve of CBT 1 cultures with and without Nitro-Arg (Arg=Arginine) added.
[0336]
[0337] Exemplary embodiment No. 6: Incorporation of the modified substrate Furyl-L-Ala (Ala=Alanine) into Microcystin LR in position 4 produced by strain CBT 275.
[0338] HPLC-PDA Chromatogram at 238 nm for sample of control cultivation (a) for sample of cultivation with added modified substrate (b). Extracted ion chromatogram from HPLC-MS data of mass value of protonated molecular ion of novel Microcystin variant for sample of control cultivation (c) and sample of cultivation with added modified substrate (d) in the positive ionization mode. Finally, e) shows the averaged mass spectrum of the peak visible in chromatogram d). Detector signal intensities (y-Axis) are measured in milli-absorption units (mAU) und counts (dimensionless quantity) for PDA and mass spectrometry data respectively. The PDA-Signal of the novel Furyl-Ala variant of Microcystin LR is not visible due to the low concentration.
[0339]
[0340] Exemplary embodiment No. 6: Growths curve of CBT 275 cultures with and without Fury-Ala (Ala=Alanine) added.
[0341]
[0342] Exemplary embodiment No. 7: Incorporation of the modified substrate Nitro-Arg (Arg=Arginine) and Prg-Tyr (Tyr=Tyrosine) into Microcystin YR in position 2 and 4 respectively produced by strain CBT 480.
[0343] HPLC-PDA Chromatogram at 238 nm for sample of control cultivation (a) for sample of cultivation with added modified substrate (b). Extracted ion chromatogram from HPLC-MS data of mass value of protonated molecular ion of novel Microcystin variant for sample of control cultivation (c) and sample of cultivation with added modified substrate (d) in the positive ionization mode. Finally, e) shows the averaged mass spectrum of the peak visible in chromatogram d). Detector signal intensities (y-Axis) are measured in milli-absorption units (mAU) und counts (dimensionless quantity) for PDA and mass spectrometry data respectively.
[0344]
[0345] Exemplary embodiment No. 7: Growths curve of CBT 480 cultures with and without Nitro-Arg (Arg=Arginine) and Prg-Tyr (Tyr=Tyrosine) added.
[0346]
[0347] Exemplary embodiment No. 8: Incorporation of the modified substrate Nitro-Arg (Arg=Arginine) into Microcystin (D-Asp3, E-Dhb7)-RR in position 2/4 produced by strain CBT 329.
[0348] HPLC-PDA Chromatogram at 238 nm for sample of control cultivation (a) for sample of cultivation with added modified substrate (b). Extracted ion chromatogram from HPLC-MS data of mass value of double protonated molecular ion of novel Microcystin variant for sample of control cultivation (c) and sample of cultivation with added modified substrate (d) in the positive ionization mode. Finally, e) shows the averaged mass spectrum of the peak visible in chromatogram d). Detector signal intensities (y-Axis) are measured in milli-absorption units (mAU) und counts (dimensionless quantity) for PDA and mass spectrometry data respectively.
[0349]
[0350] Exemplary embodiment No. 8: Growths curve of CBT 329 cultures with and without Nitro-Arg (Arg=Arginine) added.
[0351]
[0352] Exemplary embodiment No. 9: Incorporation of the modified substrate Azido-Lys (Lys=Lysine) into Microcystin YR in position 4 produced by strain CBT 1.
[0353] HPLC-PDA Chromatogram at 238 nm for sample of control cultivation (a) for sample of cultivation with added modified substrate (b). Extracted ion chromatogram from HPLC-MS data of mass value of protonated molecular ion of novel Microcystin variant for sample of control cultivation (c) and sample of cultivation with added modified substrate (d) in the positive ionization mode. Finally, e) shows the averaged mass spectrum of the peak visible in chromatogram d). Detector signal intensities (y-Axis) are measured in milli-absorption units (mAU) und counts (dimensionless quantity) for PDA and mass spectrometry data respectively. The PDA-Signal of the novel Azido-Lys (Lys=Lysine) variant of Microcystin YR is not visible due to overlapping peaks in the sample.
[0354]
[0355] Exemplary embodiment No. 9: Growths curve of CBT 1 cultures with and without Azido-Lys (Lys=Lysine) added.
[0356]
[0357] Exemplary embodiment No. 10: Incorporation of the modified substrate Azido-Norval (Norval=Norvaline) into Microcystin RR in position 2 produced by strain CBT 633.
[0358] HPLC-PDA Chromatogram at 238 nm for sample of control cultivation (a) for sample of cultivation with added modified substrate (b). Extracted ion chromatogram from HPLC-MS data of mass value of protonated molecular ion of novel Microcystin variant for sample of control cultivation (c) and sample of cultivation with added modified substrate (d) in the positive ionization mode. Finally, e) shows the averaged mass spectrum of the peak visible in chromatogram d). Detector signal intensities (y-Axis) are measured in milli-absorption units (mAU) und counts (dimensionless quantity) for PDA and mass spectrometry data respectively.
[0359]
[0360] Growths curve of CBT 633 cultures with and without Azido-Norval (Norval=Norvaline) added.
[0361]
[0362] Exemplary embodiment No. 11: Incorporation of the modified substrate H-homoarg-OH (homoarg=homoarginine) into Nodularin in position 2 produced by strain CBT 786.
[0363] HPLC-PDA Chromatogram at 238 nm for sample of control cultivation (a) for sample of cultivation with added modified substrate (b). Extracted ion chromatogram from HPLC-MS data of mass value of protonated molecular ion of novel Nodularin variant for sample of control cultivation (c) and sample of cultivation with added modified substrate (d) in the positive ionization mode. Finally, e) shows the averaged mass spectrum of the peak visible in chromatogram d). Detector signal intensities (y-Axis) are measured in milli-absorption units (mAU) und counts (dimensionless quantity) for PDA and mass spectrometry data respectively.
[0364]
[0365] Exemplary embodiment No. 12: Incorporation of the modified substrate Azido-L-Phe (Phe=phenylalanine) into Microcystin YR in position 2 produced by strain CBT 480 in a large scale (2 l) cultivation system.
[0366] HPLC-PDA Chromatogram at 238 nm for sample of control cultivation (a) for sample of cultivation with added modified substrate (b). Extracted ion chromatogram from HPLC-MS data of mass value of protonated molecular ion of novel Microcystin variant for sample of control cultivation (c) and sample of cultivation with added modified substrate (d) in the positive ionization mode. Finally, e) shows the averaged mass spectrum of the peak visible in chromatogram d). Detector signal intensities (y-Axis) are measured in milli-absorption units (mAU) und counts (dimensionless quantity) for PDA and mass spectrometry data respectively.
[0367]
[0368] Exemplary embodiment No. 13: Feeding of Microcystis aeruginosa strain CBT 480 with different amounts of modified substrate 4-azido-L-phenylalanine (0 M, 10 M, 30 M) results an increasing amount of produced modified microcystin with increasing amount of fed modified substrate 4-azido-L-phenylalanine. This result allows for optimization of feeding protocols for respective productions of modified non-ribosomal peptides (here modified microcystins).
[0369] The upper part of the figure shoes overlaid HPLC-PDA Chromatograms at 238 nm for sample of control cultivation, sample of cultivation with added substrate 4-azido-L-phenylalanine of 10 M in culture medium and sample of cultivation with added substrate 4-azido-L-phenylalanine of 30 M in culture medium. The lower figure shows the averaged mass spectrum of the newly formed peak visible at about 10 min in the HPLC chromatogram. Detector signal intensities (y-Axis) are measured in milli-absorption units (mAU) and counts (dimensionless quantity) for PDA and mass spectrometry data, respectively.
[0370]
[0371] Exemplary embodiment No. 14: Incorporation of the modified substrate Prg-Tyr (Tyr=Tyrosine) into (D-Asp.sup.3, E-Dhb.sup.7) Microcystin-RR in position 2 produced by strain CBT 280.
[0372] HPLC-PDA Chromatogram at 238 nm for sample of control cultivation (a) for sample of cultivation with added modified substrate (b). Extracted ion chromatogram from HPLC-MS data of mass value of protonated molecular ion of novel Microcystin variant for sample of control cultivation (c) and sample of cultivation with added modified substrate (d) in the positive ionization mode. Finally, e) shows the averaged mass spectrum of the peak visible in chromatogram d). Detector signal intensities (y-Axis) are measured in milli-absorption units (mAU) und counts (dimensionless quantity) for PDA and mass spectrometry data respectively.
[0373]
[0374] Exemplary embodiment No. 15: Incorporation of the modified substrate Prg-Tyr (Tyr=Tyrosine) into Anabaenopeptin A in position 2 produced by strain CBT 280.
[0375] HPLC-PDA Chromatogram at 210 nm for sample of control cultivation (a) for sample of cultivation with added modified substrate (b). Extracted ion chromatogram from HPLC-MS data of mass value of protonated molecular ion of novel Anabaenopeptin variant for sample of control cultivation (c) and sample of cultivation with added modified substrate (d) in the positive ionization mode. Finally, e) shows the averaged mass spectrum of the peak visible in chromatogram d). Detector signal intensities (y-Axis) are measured in milli-absorption units (mAU) und counts (dimensionless quantity) for PDA and mass spectrometry data respectively.
[0376]
[0377] Exemplary embodiment No. 16: Incorporation of the modified substrate Azido-Phe (Phe=Phenylalanine) into Anabaenopeptin NZ857 produced by strain CBT 332.
[0378] HPLC-PDA Chromatogram at 210 nm for sample of control cultivation (a) for sample of cultivation with added modified substrate (b). Extracted ion chromatogram from HPLC-MS data of mass value of protonated molecular ion of novel Anabaenopeptin variant for sample of control cultivation (c) and sample of cultivation with added modified substrate (d) in the positive ionization mode. Finally, e) shows the averaged mass spectrum of the peak visible in chromatogram d). Detector signal intensities (y-Axis) are measured in milli-absorption units (mAU) und counts (dimensionless quantity) for PDA and mass spectrometry data respectively.
[0379]
[0380] Exemplary embodiment No. 17: Incorporation of the modified substrate Azido-Phe (Phe=Phenylalanine) into Oscillamide Y produced by strain CBT 1161.
[0381] HPLC-PDA Chromatogram at 210 nm for sample of control cultivation (a) for sample of cultivation with added modified substrate (b). Extracted ion chromatogram from HPLC-MS data of mass value of protonated molecular ion of novel Oscillamide variant for sample of control cultivation (c) and sample of cultivation with added modified substrate (d) in the positive ionization mode. Finally, e) shows the averaged mass spectrum of the peak visible in chromatogram d). Detector signal intensities (y-Axis) are measured in milli-absorption units (mAU) und counts (dimensionless quantity) for PDA and mass spectrometry data respectively.
[0382]
[0383] Exemplary embodiment No. 18: Incorporation of the modified substrate Prg-Tyr (Tyr=Tyrosine) into Oscillamide Y produced by strain CBT 1161.
[0384] HPLC-PDA Chromatogram at 210 nm for sample of control cultivation (a) for sample of cultivation with added modified substrate (b). Extracted ion chromatogram from HPLC-MS data of mass value of protonated molecular ion of novel Oscillamide variant for sample of control cultivation (c) and sample of cultivation with added modified substrate (d) in the positive ionization mode. Finally, e) shows the averaged mass spectrum of the peak visible in chromatogram d). Detector signal intensities (y-Axis) are measured in milli-absorption units (mAU) und counts (dimensionless quantity) for PDA and mass spectrometry data respectively.
[0385]
[0386] Exemplary embodiment No. 19: Incorporation of the modified substrate Prg-Tyr (Tyr=Tyrosine) into Cryptophycin 1 produced by strain CBT 567.
[0387] Extracted ion chromatogram from HPLC-MS data of mass value of protonated molecular ion of novel Cryptophycin variant for sample of control cultivation (a) and sample of cultivation with added modified substrate (b) in the positive ionization mode. Finally, c) shows the averaged mass spectrum of the additional peak in chromatogram b). Detector signal intensities (y-Axis) are measured in counts (dimensionless quantity).
[0388]
[0389] Exemplary embodiment No. 20: Produced ADCs and results of analytical SEC-HPLC. In analytical SEC-HPLC the conjugates Microcystin-ADC1 and Microcystin-ADC2 showed a high level of purity with 98.9% and 99.0% monomers. In both cases, aggregates and small fragments were detected with rates of 0.8% and 0.2%.
[0390]
[0391] Exemplary embodiment No. 21: Coomassie stained Gelelectrophoresis gels demonstrating the binding of Microcystin variants 1 and 2 as payloads on monoclonal antibodies. In Coomassie staining under reducing conditions all samples showed a signal for the heavy chain at app. 50 kDa and the light chain at app. 25 kDa. All conjugates showed an up-shift of the protein signal of the heavy and the light chain compared to the naked MAB indicating toxin conjugation to both antibody chains. For all ADCs a double-signal was detected for the light chain indicating both, conjugated and unconjugated species. In Coomassie staining under non-reducing conditions the naked antibody showed a double signal at app. 150 kDa for the intact antibody. The ADCs showed a variety of signals between 25 kDa and 150 kDa, since in both cases the toxin was conjugated to reduced interchain disulfides leading to instability of the antibody during incubation at 37 C.
[0392]
[0393] Exemplary embodiment No. 22: Successful in vitro proof of concept of Microcystin-based ADCs. The cell viability is monitored in an in-vitro-assay with a cancer cell line for the different concentrations of the Microcystin ADC for two Microcystin variants as payloads. The ADC carries a non-cleavable linker. For Microcystin-ADC-2 an EC.sub.50 values of 220 pM was determined. Differences between structural payload variants underline huge potential of further structural optimizations.