Modified microcystins and nodularins
11512114 · 2022-11-29
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
A61K47/6855
HUMAN NECESSITIES
A61K47/6829
HUMAN NECESSITIES
C07K2319/30
CHEMISTRY; METALLURGY
C07K7/64
CHEMISTRY; METALLURGY
A61K47/6851
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
A compound includes cytotoxic agents such as microcystin and nodularin. More specifically, a modified microcystin and/or nodularin compound contains one or more modified substrates, wherein one or more modified substrates include an anchor group directly accessible or transformable for use in conjugation chemistry (incl. click chemistry), for the attachment of a targeting moiety. This compound is useful for cancer treatment.
Claims
1. An antibody-drug conjugate, comprising: an antibody or a fragment thereof containing an antigen binding site that immuno-specifically binds to a tumor associated antigen, a modified microcystin comprising an amino acid with an anchor group attached to said antibody or said fragment thereof, wherein the modified microcystin has the following general structure
cyclo(-D-Ala.sub.1-X.sub.2-D-MeAsp.sub.3-Z.sub.4-Adda.sub.5-D-Glu.sub.6-Mdha.sub.7), wherein, independently from each other, D-Ala.sub.1 and D-MeAsp.sub.3 each represent a variable D-amino acid position, Adda.sub.5 is selected from the group consisting of Adda, DM-Adda, (6Z) Adda, and ADM-Adda, D-Glu.sub.6 is selected from the group consisting of D-Glu and D-Glu(OCH.sub.3), Mdha.sub.7 is selected from the group consisting of Mdha, Dha, L-Ser, L-MeSer, Dhb, and MeLan, and X.sub.2 and Z.sub.4 are, independently from each other, an L-amino acid, wherein said amino acid with said anchor group is incorporated in at least one position other than Adda.sub.5 and DGlu.sub.6 and wherein said anchor group is derived from chemically conjugating a group selected from the group consisting of an azido group, an alkyne group, a tetrazine, a primary amine, a thiol, an aldehyde, a carboxyl, and an oxime of said amino acid to said antibody or said fragment thereof, thereby attaching said modified microcystin to said antibody or said fragment thereof via conjugation chemistry, and wherein said antibody-drug conjugate is cytotoxic.
2. The antibody-drug conjugate according to claim 1, wherein said amino acid with said anchor group is incorporated in position X.sub.2 and/or Z.sub.4.
3. The antibody-drug conjugate according to claim 1, wherein said at least one position is X.sub.2.
4. The antibody-drug conjugate according to claim 1, wherein said at least one position is Z.sub.4.
5. The antibody-drug conjugate according to claim 1, wherein said amino acid with said anchor group is derived 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, and (2S)-2-amino-3-(4-azidophenyl)propanoic acid.
6. The antibody-drug conjugate according to claim 1, wherein, 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, and/or X.sub.2 and/or Z.sub.4 comprises said amino acid with said anchor group.
7. The antibody-drug conjugate according to claim 1, 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 a reaction exploiting the specific reactivity of the primary amine, the thiol, the aldehyde, the carboxyl, and the oxime.
8. The antibody-drug conjugate according to claim 1, wherein said amino acid with said anchor group comprises: a modified amino acid which is, in nature, not incorporated at the specific position in said modified microcystin and which is also not a substitution of the naturally incorporated amino acid with functional groups which are not directly accessible or transformable for use in conjugation chemistry, for the attachment of the antibody or the fragment thereof or a label.
9. The antibody-drug conjugate according to claim 1, wherein the modified microcystin comprises: a chemical structure, wherein a natural receptor or transporter in a target cell which, in-vivo, is capable of intracellular uptake of members of the same microcystin group into the target cell, is capable of uptake of the modified microcystin comprising the chemical structure only at a very low rate which is at least 50 times lower than the rate for microcystin-LR transported by organic anion transporting polypeptide 1B1 (OATP1B1) to restrict uptake of the compound into human cells that express OATP1B1.
10. The antibody-drug conjugate according to claim 1, wherein the modified microcystin comprises: a chemical structure having drug-like properties that are changed to its advantage for their application as payload for an antibody-drug conjugate, in comparison to microcystin-LR, by at least 10%.
11. The antibody-drug conjugate according to claim 10, wherein the drug-like properties comprise solubility in water, stability under low pH, pharmacokinetics, and pharmacodynamics.
12. The antibody-drug conjugate according to claim 1, wherein D-MeAsp.sub.3 is selected from the group consisting of D-MeAsp and D-Asp, and wherein said amino acid with said anchor group is incorporated in at least one position selected from the group consisting of D-Ala.sub.1, X.sub.2, Z.sub.4 and Mdha.sub.7.
13. The antibody-drug conjugate according to claim 1, wherein D-MeAsp.sub.3 is selected from the group consisting of D-MeAsp and D-Asp, and D-Ala.sub.1 is selected from the group consisting of D-Ala, D-Ser and D-Leu, and wherein said amino acid with said anchor group is incorporated in at least one position selected from the group consisting of X.sub.2 and Z.sub.4.
14. The antibody-drug conjugate according to claim 12, wherein said anchor group is attached to said antibody or said fragment thereof via a linker.
15. The antibody-drug conjugate according to claim 14, wherein said anchor group is included in a click chemistry attachment that conjugates said modified microcystin via said linker with said antibody.
Description
BRIEF DESCRIPTION OF DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
(99) The inventors for the first time have incorporated modified amino acids into microcystins which carry so called clickable anchor groups which allow the fast and easy binding of the entire molecule to e.g. peptide linker or other functional units like e.g. antibodies (see
(100) It is shown that feeding of any combination of a clickable substrate with e.g. amino acids naturally occurring in microcystins and/or nodularins, modified versions of these amino acids or any other substrate might potentially lead to an incorporation of the fed substrate combinations into the non-ribosomal peptide (see
(101) 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 microcystin or nodularin (e.g. the respective non-modified amino acid) (see
(102) That means in the past successful feedings were only regarded to structural variants directly derived from the naturally (native) incorporated amino acid (e.g. o-methyl-tyrosine or chloro-tyrosine instead of tyrosine or homo-arginine instead of arginine). Considering the new results of the inventors it is obvious that the structural and functional diversity of non-ribosomal peptides generated by feeding significantly increases if also substrates can be used that are structurally not directly derived from the substrate which is naturally incorporated into the respective 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.
(103) The invention relates to a modified microcystin and/or nodularin compound comprising at least one modified substrate, wherein the at least one modified substrate comprises an anchor group directly accessible or transformable for use in click chemistry, for the attachment of a targeting moiety and/or label or for additional structural modifications. This will allow for example connecting antibodies to the microcystins and nodularins and the creation of great compound libraries of microcystins and nodularins with novel structures.
(104) In one embodiment of the invention, the one or more modified substrates are incorporated at a defined position of the microcystin and/or nodularin.
(105) The CAs can carry an amino acid in the CA at a position where, in nature such an amino acid does not exist. The amino acid may be modified.
(106) The inventors can astonishingly modify multiple positions other than the so-called variable positions 2 and 4 of microcystins or the respective Arg2 position in nodularins.
(107) The invention further relates to a compound, wherein the one or more modified substrates are incorporated at any position of the microcystin other than Adda5 and DGlu6, which has the following general structure:
D-Alar-X.sub.2-D-MeAsp.sub.3-Z.sub.4-Adda.sub.5-DGlu.sub.6-Mdha.sub.7, SEQ ID NO: 1
(108) and wherein X.sub.2 and/or Z.sub.4 are positions of preferred incorporation of said modified substrate.
(109) In 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:
D-MeAsp.sub.1-Arg.sub.2-Adda.sub.3-DGlu.sub.4-Mdhb.sub.5, SEQ ID NO: 2.
(110) and wherein Arg.sub.2 is the position of preferred incorporation of said modified substrate.
(111) Preferably, if the CA is microcystin, the modified position is X.sub.2 or Z.sub.4 and the modified substrate is a modified amino acid (see
(112) Also, preferably if the CA is microcystin, the modified position is X.sub.2 and Z.sub.4 and the modified substrate is a modified amino acid (see
(113) In nodularin, preferably, the modified position is Arg.sub.2 and the modified substrate is a modified amino acid.
(114) 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
(115) 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: 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. Alkyne (e.g. propargyl 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. 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. 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. Thiols that can subsequently be modified e.g. by reaction with maleimides, haloacetyls, pyridyldisulfides, thiosulfonares 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. 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. 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. 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.
(116) 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
(117) 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 (inc. click chemistry) amenable functional groups as described above.
(118) Ideally, modified amino acids which are directly accessible or transformable for use in conjugation chemistry (inc. click chemistry), is selected from the group of the following table (see
(119) TABLE-US-00001 Systematic name CAS Number Short name Supplier Order 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 (2S)-2-Amino-3-(4- 33173-53-4 Azido-L- Iris Biotech HAA1850 azidophenyl)propanoic acid Phe GmbH L-α-Amino-ε-guanidinohexanoic acid 156-86-5 H-homo- Bachem 4016423 Arg-OH
(120) The invention relates to a compound, wherein the microcystin has one of the following formula:
D-Ala-X.sub.2-D-MeAsp.sub.3-Z.sub.4-Adda.sub.5-DGlu.sub.6-Mdha.sub.7,
(121) TABLE-US-00002 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 D-Ala variable D-MeAsp variable Adda D-Glu Mdha acids D-Ser A-Asp DM-Adda D-Glu(OCH.sub.3) Dha D-Leu (6Z)Adda L-Ser ADM-Adda L-MeSer Dhb MeLan
(122) wherein
(123) TABLE-US-00003 Ala.sub.1 X.sub.2 D-MeAsp.sub.3 Z.sub.4 Mdha.sub.7
(124) comprise the position of the incorporation of one or more modified substrates, 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:
(125) TABLE-US-00004 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 (2S)-2-Amino-3-(4- 33173-53-4 Azido-L- Iris Biotech HAA1850 azidophenyl)propanoic acid Phe GmbH L-α-Amino-ε-guanidinohexanoic acid 156-86-5 H-homo- Bachem 4016423 Arg-OH
(126) The invention also relates to a compound, wherein the nodularin has one of the following formula:
(127) TABLE-US-00005 Position 1 2 3 4 5 Possible MeAsp.sub.1 Arg.sub.2 Adda.sub.3 DGlu.sub.4 Mdhb.sub.5 amino D-MeAsp Homo-Arg Adda D-Glu Mdhb acid D-Asp DM-Adda D-Glu(OCH.sub.3) Dhb (6Z)Adda Me-Adda
(128) wherein
(129) TABLE-US-00006 MeAsp.sub.1 Arg.sub.2 Mdhb.sub.5
(130) comprise the one or more modified substrates, wherein preferably the modified substrate is an amino acid selected from the group of:
(131) TABLE-US-00007 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-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 L-α-Amino-ε-guanidinohexanoic acid 156-86-5 H-homo- Bachem 4016423 Arg-OH
(132) Ideally, the nodularin is modified at the Arg.sub.2 position.
(133) 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.
(134) 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.
(135) 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.
(136) In one embodiment of the invention the targeting moiety (TM) is selected form the group of antibody, antibody fragment, fab fragment, peptide, modified antibody, fluorophores, and chromatographic columns. Concerning the targeting moiety, 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
(137) According to the invention, the compound can be used as a medicament. Furthermore, it can be used as a medicament for the diagnosis or treatment of cancer and/or other diseases and disorders.
(138) 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.
(139) 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.
(140) BMPR1B (bone morphogenetic protein receptor-type IB, Genbank accession no. NM-001203);
(141) E16 (LAT1, SLC7A5, Genbank accession no. NM-003486);
(142) STEAP1 (six transmembrane epithelial antigen of prostate, Genbank accession no. NM-012449);
(143) 0772P (CA125, MUC16, Genbank accession no. AF361486);
(144) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank accession no. NM-005823);
(145) 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);
(146) 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);
(147) PSCA hIg (2700050C12Rik, C530008016Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession no. AY358628);
(148) ETBR (Endothelin type B receptor, Genbank accession no. AY275463);
(149) MSG783 (RNF124, hypothetical protein F1120315, Genbank accession no. NM-017763);
(150) STEAP2 (HGNC-8639, IPCA-1, PCANAP1, STAMP1, 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);
(151) TrpM4 (BR22450, F1120041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession no. NM-017636);
(152) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor, Genbank accession no. NP-003203 or NM-003212);
(153) CD21 (CR2 (Complement receptor 2) or C3DR(C3d/Epstein Barr virus receptor) or Hs.73792 Genbank accession no. M26004);
(154) CD79b (CD79B, CD79β, IGb (immunoglobulin-associated beta), B29, Genbank accession no. NM-000626 or 11038674);
(155) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a), SPAP1B, SPAP1C, Genbank accession no. NM-030764, AY358130);
(156) HER2 (ErbB2, Genbank accession no. M11730); Coussens L., et al Science (1985) 230(4730):1132-1139);
(157) NCA (CEACAM6, Genbank accession no. M18728); Barnett T., et al Genomics 3, 59-66, 1988;
(158) MDP (DPEP1, Genbank accession no. BC017023);
(159) IL20Rα (IL20Ra, ZCYTOR7, Genbank accession no. AF184971);
(160) Brevican (BCAN, BEHAB, Genbank accession no. AF229053);
(161) EphB2R (DRT, ERK, HekS, EPHT3, Tyro5, Genbank accession no. NM-004442);
(162) ASLG659 (B7h, Genbank accession no. AX092328); US20040101899 (Claim 2);
(163) PSCA (Prostate stem cell antigen precursor, Genbank accession no. AJ297436);
(164) GEDA (Genbank accession No. AY260763); AAP14954 lipoma HMGIC fusion-partner-like protein/pid=AAP14954.1 Homo sapiens (human);
(165) (26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3, BR3, Genbank accession No. AF116456); BAFF receptor/pid=NP-443177.1-Homo sapiens; Thompson, J. S., et al Science 293 (5537), 2108-2111 (2001); WO2004058309; WO2004011611; WO2003045422 (Example; Page 32-33); WO2003014294 (Claim 35;
(166) CD22 (B-cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814, Genbank accession No. AK026467);
(167) 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);
(168) 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);
(169) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen) that binds peptides and presents them to CD4+ T lymphocytes);
(170) 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);
(171) CD72 (B-cell differentiation antigen CD72, Lyb-2); 359 aa), pl: 8.66, MW: 40225 TM: 1 [P] Gene
(172) Chromosome: 9p13.3, Genbank accession No. NP-001773.1); 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);
(173) 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);
(174) IRTA2 (FcRHS, Immunoglobulin superfamily receptor translocation associated 2, a putative immunoreceptor with possible roles in B cell development and lymphomagenesis;
(175) TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, putative transmembrane proteoglycan, related to the EGF/heregulin family of growth factors and follistatin);
(176) MUC1 (Tumor-associated MUC1 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.
(177) 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 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.
(178) 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
(179) 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.
(180) A further embodiment of the invention is a microcystin and/or nodularin compound, comprising one or more modified substrates, wherein the one or more modified substrate 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.
Examples
(181) Successful feedings of modified substrates were performed in different cultivation systems and scales allowing for screening (small scales of up to 10 ml; see
(182) 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.
(183) 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 μmol/s*m.sup.2. The temperature was again strain-specific varied between 20° C. and 30° C.
(184) 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 μmol/s*m.sup.2.
(185) 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 μmol/s*m.sup.2.
(186) 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 μmol/s*m.sup.2.
(187) 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.
(188) 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.
(189) Exemplary feeding scheme for the 10 ml scale:
(190) All strains were cultivated in BG11 medium (see below), according to strain-specific cultivation conditions determined before.
(191) Cells were pre-cultivated in Erlenmeyer flasks under low light conditions (30 μmol/s*m2) for 4 days at 25° C. and on a shaker at 70 rpm.
(192) 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.
(193) The feeding of cultures started at inoculation by adding the respective modified substrate(s) to a concentration of 10 mM in the medium. Daily additions of modified substrates remained constant over 4 days by feeding of additional 10 μM 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 μM 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.
(194) 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 μM media concentration were used (if strain tolerated such concentrations) in order to increase the yield of modified non-ribosomal peptides.
(195) TABLE-US-00008 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 μg/L μM H.sub.3BO.sub.3 2.86 46.3 MnCl.sub.2•4H.sub.2O 1.8 9.15 ZnSO.sub.4•7H.sub.2O 0.22 0.77 Na.sub.2MoO.sub.4•2H.sub.2O 0.390 1.61 CuSO.sub.4•5H.sub.2O 0.079 0.32 Co(NO.sub.3).sub.2•6H.sub.2O 0.0494 0.17
(196) Table: Recipe for BG11 medium which has been used for feeding experiments
(197) For the following strains feeding of at least one modified and clickable substrate were demonstrated.
(198) TABLE-US-00009 Cyano Biotech Strain ID Main microcystin variants NO. Genera produced 1 Microcystis MC-YR 275 Microcystis MC-LR MC-LW MC-LF 329 Planktothrix (D-Asp3, Dhb7)MC-RR 480 Microcystis MC-LR MC-YR 633 Microcystis MC-RR 959 Microcystis MC-LR MC-YR
(199) 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 is phenylalanine. D-MAsp3 is D-erythro-β-methylaspartic acid at position 3 and Dhb7 is dehydrobutyrate at position 7.
(200)
(201) 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.
(202) TABLE-US-00010 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. Natu- rally Posi- Naturally incor- tion produced porated of NRP amino natu- NRP variate Monoiso- acid Monoiso- rally variants which is topic which is Sum topic incor- naturally effected Sum formula mass of replaced formula mass porated CBT produced by fed of naturally naturally by (zwitterion) (Zwitterion) amino strain Genera/ by the modified produced produced modified of natural of natural acid in no. Species strain substrate NRP variant NRP variant substrate substrate substrate NRP 1 Microcystis MC-LR MC-YR C52H72N10O13 1044.52803 Arg C6H14N4O2 174.111679 2 sp. MC-YR 1 Microcystis MC-LR MC-YR C52H72N10O13 1044.52803 Tyr C9H11NO3 181.073898 4 sp. MC-YR 1 Microcystis MC-LR MC-YR C52H72N10O13 1044.52803 Arg C6H14N4O2 174.111679 2 sp. MC-YR 1 Microcystis MC-LR MC-YR C52H72N10O13 1044.52803 Tyr C9H11NO3 181.073898 4 sp. MC-YR 265 Microcystis MC-LR, MC-LR C49H74N10O12 994.548767 Arg C6H14N4O2 174.111679 4 aeruginosa Cyano- peptolin A, B, C, D und 963A; Micro- cyclamide, Aeruginosin, Aeru- cyclamide A, B, C, D 265 Microcystis MC-LR, MC-LR C49H74N10O12 994.548767 Leu C6H13NO2 131.094635 2 aeruginosa Cyano- peptolin A, B, C, D und 963A; Micro- cyclamide, Aeruginosin, Aeru- cyclamide A, B, C, D 275 Microcystis MC-LR MC-LR C49H74N10O12 994.548767 Arg C6H14N4O2 174.111679 4 aeruginosa MC-LW MC-LF 275 Microcystis MC-LR MC-LR C49H74N10O12 994.548767 Arg C6H14N4O2 174.111679 4 aeruginosa MC-LW MC-LF 275 Microcystis MC-LR MC-LR C49H74N10O12 994.548767 Arg C6H14N4O2 174.111679 4 aeruginosa MC-LW MC-LF 275 Microcystis MC-LR MC-LF C52H71N7O12 985.516071 Phe C9H11NO2 165.078979 4 aeruginosa MC-LW MC-LF 275 Microcystis MC-LR MC-LF C52H71N7O12 985.516071 Phe C9H11NO2 165.078979 4 aeruginosa MC-LW MC-LF 275 Microcystis MC-LR MC-LR C49H74N10O12 994.548767 Arg C6H14N4O2 174.111679 4 aeruginosa MC-LW MC-LF 275 Microcystis MC-LR MC-LR C49H74N10O12 994.548767 Arg C6H14N4O2 174.111679 4 aeruginosa MC-LW MC-LF 275 Microcystis MC-LR MC-LW C54H72N8O12 1024.52697 Trp C11H12N2O2 204.089878 4 aeruginosa MC-LW MC-LF 275 Microcystis MC-LR MC-LW C54H72N8012 1024.52697 Trp C11H12N2O2 204.089878 4 aeruginosa MC-LW MC-LF Calculated Mass monoiso- Measured Monoiso- difference topic monoiso- topic between mass of topic MS Sum mass natural mutasyn- mass of Peak Short formula (zwitter und thesis mutasyn + EIC UV CBT name of (zwitterion) ion) of modified product thesis (Mass Peak strain Genera/ modified of modified modified substrate (novel product specto- PDA no. Species substrate substrate substrate (Da) microcystin) [M + H].sup.+ metry) (HPLC) 1 Microcystis Azido-lys C6H12N4O2 172.0960 2.0157 1042.5124 1043.5197 yes yes sp. 1 Microcystis Prg-Tyr C12H13NO3 219.0895 −38.0156 1082.5437 1083.5510 yes yes sp. 1 Microcystis Nitro-Arg C6H13N5O4 219.0968 −44.9851 1089.5131 1090.5204 yes yes sp. 1 Microcystis Azido-L- C9H10N4O2 206.0804 −25.0065 1069.5345 1070.5418 yes yes sp. Phe 265 Microcystis Prg-Tyr C12H13NO3 219.0895 −44.9779 1039.5266 1040.5339 yes no aeruginosa 265 Microcystis Prg-Tyr C12H13NO3 219.0895 −87.9949 1082.5437 1083.5510 yes no aeruginosa 275 Microcystis Nitro-Arg C6H13N5O4 219.0968 −44.9851 1039.5338 1040.5411 yes undeter- aeruginosa mined 275 Microcystis Furyl-Ala C7H9NO3 155.0582 19.0534 975.4953 976.5026 yes undeter- aeruginosa mined 275 Microcystis Lys(Poc) C10H16N2O4 228.1110 −53.9993 1048.5481 1049.5554 yes undeter- aeruginosa mined 275 Microcystis Prg-Tyr C12H13NO3 219.0895 −54.0106 1039.5266 1040.5339 yes yes aeruginosa 275 Microcystis Azido-L- C9H10N4O2 206.0804 −41.0014 1026.5175 1027.5247 yes yes aeruginosa Phe 275 Microcystis Azido-Lys C6H12N4O2 172.0960 2.0157 992.5331 993.5404 yes yes aeruginosa 275 Microcystis Azido- C5H10N4O2 158.0804 16.0313 978.5175 979.5247 yes yes aeruginosa Norval 275 Microcystis Prg-Tyr C12H13NO3 219.0895 −14.9997 1039.5266 1040.5339 yes yes aeruginosa 275 Microcystis Azido-L- C9H10N4O2 206.0804 −1.9905 1026.5175 1027.5247 yes yes aeruginosa Phe
(203) TABLE-US-00011 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 Naturally incor- produced porated Position NRP amino of NRP variate Monoiso- acid Monoiso- naturally variants which is topic which is topic incor- naturally effected Sum formula mass of replaced Sum formula mass porated CBT produced by fed of naturally naturally by (zwitterion) (Zwitterion) amino strain Genera/ by the modified produced produced modified of natural of natural acid in no. Species strain substrate NRP variant NRP variant substrate substrate substrate NRP 329 Planktothrix (D-Asp3, (D-Asp3, C48H73N13O12 1023.5502 Arg C6H14N4O2 174.11168 4 agardhii E-Dhb7) E-Dhb7) MC-RR MC-RR 332 Planktothrix Anabaeno- (D-Asp3, C48H73N13O12 1023.5502 Arg C6H14N4O2 174.11168 2 rubescens peptin E-Dhb7) A, B, MC-RR E/Fund NZ857, (D-Asp3, E-Dhb7) MC-RR 480 Microcystis MC-LR MC-LR C49H74N10O12 994.548767 Leu C6H13NO2 131.09464 2 aeruginosa (D-Asp3) MC-YR 480 Microcystis MC-LR MC-LR C49H74N10O12 994.548767 Arg C6H14N4O2 174.11168 4 aeruginosa (D-Asp3) MC-YR 480 Microcystis MC-LR MC-LR C49H74N10O12 994.548767 Arg C6H14N4O2 174.11168 4 aeruginosa (D-Asp3) MC-YR 480 Microcystis MC-LR MC-YR C52H72N10O13 1044.52803 Arg C6H14N4O2 174.11168 4 aeruginosa (D-Asp3) MC-YR 480 Microcystis MC-LR MC-YR C52H72N10O13 1044.52803 Tyr C9H11NO3 181.0739 2 aeruginosa (D-Asp3) MC-YR 480 Microcystis MC-LR MC-YR C52H72N10O13 1044.52803 Arg C6H14N4O2 174.11168 4 aeruginosa (D-Asp3) MC-YR 480 Microcystis MC-LR MC-YR C52H72N10O13 1044.52803 Tyr C9H11NO3 181.0739 2 aeruginosa (D-Asp3) MC-YR 480 Microcystis MC-LR MC-YR C52H72N10O13 1044.52803 Tyr C9H11NO3 181.0739 2 aeruginosa (D-Asp3) MC-YR 480 Microcystis MC-LR MC-LR C49H74N10O12 994.548767 Leu C6H13NO2 131.09464 2 aeruginosa (D-Asp3) MC-YR 480 Microcystis MC-LR MC-YR C52H72N10O13 1044.52803 Tyr C9H11NO3 181.0739 2 aeruginosa (D-Asp3) MC-YR Calculated Mass monoiso- Measured Monoiso- difference topic monoiso- topic between mass of topic MS Sum mass natural mutasyn- mass of Peak Short formula (zwitter und thesis mutasyn + EIC UV CBT name of (zwitterion) ion) of modified product thesis (Mass Peak strain Genera/ modified of modified modified substrate (novel product specto- PDA no. Species substrate substrate substrate (Da) microcystin) [M + H].sup.+ metry) (HPLC) 329 Planktothrix Nitro- C6H13N5O4 219.0968 44.9851 1068.5353 1069.5426 yes yes agardhii Arg 332 Planktothrix Prg-Tyr C12H13NO3 219.0895 −44.9779 1068.5281 1069.5353 yes yes rubescens 480 Microcystis Azido- C5H10N4O2 158.0804 −26.9857 1021.5345 1022.5418 yes undeter- aeruginosa Norval mined 480 Microcystis Nitro- C6H13N5O4 219.0968 −44.9851 1039.5338 1040.5411 yes yes aeruginosa Arg 480 Microcystis Azido- C6H12N4O2 172.0960 2.0157 992.5331 993.5404 yes yes aeruginosa Lys 480 Microcystis Azido- C6H12N4O2 172.0960 2.0157 1042.5124 1043.5197 yes yes aeruginosa Lys 480 Microcystis Prg-Tyr C12H13NO3 219.0895 −38.0156 1082.5437 1083.5510 yes yes aeruginosa 480 Microcystis Nitro- C6H13N5O4 219.0968 −44.9851 1089.5131 1090.5204 yes yes aeruginosa Arg 480 Microcystis Azido-L- C9H10N4O2 206.0804 −25.0065 1069.5345 1070.5418 yes yes aeruginosa Phe 480 Microcystis Prg-Tyr C12H13NO3 219.0895 −38.0156 1082.5437 1083.5510 yes yes aeruginosa 480 Microcystis Azido- C6H12N4O2 172.0960 −41.0014 1035.5502 1036.5574 yes yes aeruginosa Lys 480 Microcystis Azido- C6H12N4O2 172.0960 8.9779 1035.5502 1036.5574 yes yes aeruginosa Lys
(204) TABLE-US-00012 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 incor- produced porated NRP Monoiso- amino Position NRP variate topic acid of variants which is mass of which is Sum Monoiso- naturally naturally effected Sum formula naturally replaced formula mass porated CBT produced by fed of naturally produced by (zwitterion) (Zwitterion) amino strain Genera/ by the modified produced NRP modified of natural of natural acid in no. Species strain substrate NRP variant variant substrate substrate substrate NRP 633 Microcystis MC-RR MC-RR C49H75N13O12 1037.5658 Arg C6H14N4O2 174.11168 2 sp. 633 Microcystis MC-RR MC-RR C49H7SN13O12 1037.5658 Atr C6H14N4O2 174.11168 2 sp. 633 Microcystis MC-RR MC-RR C49H75N13O12 1037.5658 Arg C6H14N4O2 174.11168 2 sp. 633 Microcystis MC-RR MC-RR C49H75N13O12 1037.5658 Arg C6H14N4O2 174.11168 2 sp. 786 Microcystis NOD NOD C41H60N8O10 824.44324 Arg C7H16N4O2 174.11168 2 sp. (MeAsp1?) (MeAsp1?) 959 Microcystis MC-LR MC-LR C49H74N10O12 994.548767 Arg C6H14N4O2 174.11168 4 sp. MC-YR 959 Microcystis MC-LR MC-YR C52H72N10O13 1044.52803 Arg C6H14N4O2 174.11168 4 sp. MC-YR 959 Microcystis MC-LR MC-LR C49H74N10O12 994.548767 Arg C6H14N4O2 174.11168 4 sp. MC-YR 959 Microcystis MC-LR MC-YR C52H72N10O13 1044.52803 Arg C6H14N4O2 174.11168 4 sp. MC-YR 959 Microcystis MC-LR MC-LR C49H74N10O12 994.548767 Arg C6H14N4O2 174.11168 4 sp. MC-YR 959 Microcystis MC-LR MC-YR C52H72N10O13 1044.52803 Arg C6H14N4O2 174.11168 4 sp. MC-YR 959 Microcystis MC-LR MC-YR C52H72N10O13 1044.52803 Tyr C9H11NO3 181.0739 2 sp. MC-YR 959 Microcystis MC-LR MC-YR C52H72N10O13 1044.52803 Tyr C9H11NO3 181.0739 2 sp. MC-YR 1161 Planktothrix (D-Asp3, (D-Asp3, C48H73N13O12 1023.5502 Arg C6H14N4O2 174.11168 2 rubescens E-Dhb7) E-Dhb7) MC-RR, MC-RR Anabaeno- peptin A, B, E/F, Oscilla- mide Y 861 Microcystis MC-LY MC-LY C52H71N7O13 1001.51099 Leu C6H13NO2 131.09464 2 R aeruginosa MC-RY 861 Microcystis MC-LY MC-RY C52H72N10O13 1044.52803 Arg C6H14N4O2 174.11168 2 R aeruginosa MC-RY 861 Microcystis MC-LY MC-RY C52H72N10O13 1044.52803 Tyr C9H11NO3 181.0739 4 R aeruginosa MC-RY Calculated Mass monoiso- Measured Monoiso- difference topic monoiso- topic between mass of topic MS Sum mass natural mutasyn- mass of Peak Short formula (zwitter und thesis mutasyn + EIC UV CBT name of (zwitterion) ion) of modified product thesis (Mass Peak strain Genera/ modified of modified modified substrate (novel product specto- PDA no. Species substrate substrate substrate (Da) microcystin) [M + H].sup.+ metry) (HPLC) 633 Microcystis Azido-L- C3H6N4O2 130.0491 44.0626 993.5032 994.5105 yes yes sp. Ala 633 Microcystis Azido- C5H10N4O2 158.0804 16.0313 1021.5345 1022.5418 yes yes sp. Norval 633 Microcystis Nitro- C6H13N5O4 219.0968 −44.9851 1082.5509 1083.5582 yes yes sp. Arg 633 Microcystis Azido- C6H12N4O2 172.0960 2.0157 1035.5501 1036.5574 yes yes sp. Lys 786 Microcystis H-homo- C7H16N4O2 188.1273 −14.0156 838.4589 839.4661 yes yes sp. Arg-OH 959 Microcystis Nitro- C6H13N5O4 219.0968 −44.9851 1039.5338 1040.5411 yes undeter- sp. Arg mined 959 Microcystis Nitro- C6H13N5O4 219.0968 −44.9851 1089.5131 1090.5204 yes undeter- sp. Arg mined 959 Microcystis Azido-L- C3H6N4O2 130.0491 44.0626 950.4862 951.4934 yes yes sp. AJa 959 Microcystis Azido-L- C3H6N4O2 130.0491 44.0626 1000.4654 1001.4727 yes yes sp. AJa 959 Microcystis Azido- C5H10N4O2 158.0804 16.0313 978.5175 979.5247 yes yes sp. Norval 959 Microcystis Azido- C5H10N4O2 158.0804 16.0313 1028.4967 1029.5040 yes yes sp. Norval 959 Microcystis Prg-Tyr C12H13NO3 219.0895 −38.0156 1082.5437 1083.5510 yes yes sp. 959 Microcystis Azido-L- C9H10N4O2 206.0804 −25.0065 1069.5345 1070.5418 yes yes sp. Phe 1161 Planktothrix Prg-Tyr C12H13NO3 219.0895 −44.9779 1068.5281 1069.5353 yes yes rubescens 861 Microcystis Azido- C6H12N4O2 172.0960 −41.0014 1042.5124 1043.5197 yes yes R aeruginosa Lys 861 Microcystis Azido- C6H12N4O2 172.0960 2.0157 1042.5124 1043.5197 yes yes R aeruginosa Lys 861 Microcystis Azido- C6H12N4O2 172.0960 8.9779 1035.5502 1036.5574 yes yes R aeruginosa Lys
FIGURE CAPTIONS
(205)
(206) General structure of Microcystins. X2 and Z4 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-methyldehydroalanine.
(207)
(208)
(209) 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 like microcystins and nodularins (
(210)
(211) 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).
(212) A: Control (no feeding with O-methyltyrosine) B: Control (no feeding with homoarginine)
(213) C: Feeding with O-methyltyrosine D: Feeding with homoarginine
(214) Molecule masses of naturally produced microcystins:
(215) 995 Da=MC-LR, 1045 Da=MC-YR
(216) Molecule masses of modified microcystins generated by feeding with O-methyltyrosine (OMetY) and homoarginine (hR)
(217) 1059 Da=MC-OMetYR or MC-YhR; 1009 Da=MC-LhR
(218)
(219) 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).
(220) A, A′: CBT 480 culture fed with O-methyltyrosine B, B′: CBT 480 culture fed with homoarginine
(221) Molecule masses of naturally produced microcystins: 995 Da=MC-LR, 1045 Da=MC-YR
(222) Molecule masses of modified microcystins generated by feeding with O-methyltyrosine (OMetY) and homoarginine (hR)
(223) 1059 Da=MC-OMetYR or MC-YhR; 1009 Da=MC-LhR;
(224)
(225) 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)
(226) A, A′: feeding of 300 μM O-methyltyrosine (OMetY), w/o DMSO
(227) B, B′: feeding of 30 μM O-methyltyrosine (OMetY), w/o DMSO
(228) C, C′: feeding of 300 μM O-methyltyrosine (OMetY), w/1% DMSO
(229) D, D′: feeding of 30 μM O-methyltyrosine (OMetY), w/1% DMSO
(230) E, E′: control (no feeding)
(231) Molecule masses of naturally produced microcystins:
(232) 995 Da=MC-LR, 1045 Da=MC-YR
(233) Molecule masses of modified microcystin generated by feeding with O-methyltyrosine
(234) 1059 Da=MC-OMetYR
(235)
(236) 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) sclale (ESI-IT-ToF-MS detection on the left; MALDI-ToF-MS detection on the right)
(237) A, A′: feeding of 300 μM homoarginine (hR), w/o DMSO
(238) B, B′: feeding of 30 μM homoarginine (hR), w/o DMSO
(239) C, C′: feeding of 300 μM homoarginine (hR), w/1% DMSO
(240) D, D′: feeding of 30 μM homoarginine (hR), w/1% DMSO
(241) E, E′: control (no feeding)
(242) Molecule masses of naturally produced microcystins:
(243) 995 Da=MC-LR, 1045 Da=MC-YR
(244) Molecule masses of modified microcystins generated by feeding with homoarginine
(245) 1059 Da=MC-YhR; 1009 Da=MC-LhR
(246) 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.
(247) 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.
(248)
(249) 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 only difference in the amino acid sequence of McyBI of both strains is in amino acid position 672. 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. The consensus sequence as between the “Query” and “Sbjct” sequences is indicated by “Consensus” and corresponds to SEQ ID NO: 6. Each letter in the consensus sequence is an identical match. The blank spaces in the consensus sequence are where the match indicated a zero or negative score. The “+” symbol in the consensus sequence represents a conservative substitution. The “Query” sequence shown corresponds to SEQ ID NO: 4 and the “Sbjct” sequence shown corresponds to SEQ ID NO: 5.
(250)
(251) Exemplary embodiment No. 1: Incorporation of the modified substrate Azido-L-Phe (Phe=phenylalanine) into Microcystin YR in position 2 produced by strain CBT 959. HPLC-PDA Chromatogram at 238 nm for sample of control cultivation (
(252) The growth of strain CBT 959 could not be followed by measurement of optical density at 750 nm (OD750 nm) as the cell form aggregates making it impossible to measure reliable OD750 nm values.
(253)
(254) Exemplary embodiment No. 2: Incorporation of the modified substrate Prg-Tyr (Tyr=Tyrosine) into Microcystin YR in position 2 produced by strain CBT 480.
(255) HPLC-PDA Chromatogram at 238 nm for sample of control cultivation (
(256)
(257) Exemplary embodiment No. 2: Growths curve of CBT 480 cultures with and without Prg-Tyr (Tyr=Tyrosine) added.
(258)
(259) Exemplary embodiment No. 3: Incorporation of the modified substrate Azido-Lys (Lys=Lysine) into Microcystin LR in position 4 produced by strain CBT 275.
(260) HPLC-PDA Chromatogram at 238 nm for sample of control cultivation (
(261)
(262) Exemplary embodiment No. 3: Growths curve of CBT 275 cultures with and without Azido-Lys (Lys=Lysine) added.
(263)
(264) Exemplary embodiment No. 4: Incorporation of the modified substrate Prg-Tyr (Tyr=Tyrosine) into Microcystin LW in position 4 produced by strain CBT 275.
(265) HPLC-PDA Chromatogram at 238 nm for sample of control cultivation (
(266)
(267) Exemplary embodiment No. 4: Growths curve of CBT 275 cultures with and without Prg-Tyr (Tyr=Tyrosine) added.
(268)
(269) Exemplary embodiment No. 5: Incorporation of the modified substrate Nitro-Arg (Arg=Arginine) into Microcystin YR in position 4 produced by strain CBT 1.
(270) HPLC-PDA Chromatogram at 238 nm for sample of control cultivation (
(271)
(272) Growths curve of CBT 1 cultures with and without Nitro-Arg (Arg=Arginine) added.
(273)
(274) 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.
(275) HPLC-PDA Chromatogram at 238 nm for sample of control cultivation (
(276)
(277) Exemplary embodiment No. 6: Growths curve of CBT 275 cultures with and without Furyl-Ala (Ala=Alanine) added.
(278)
(279) 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.
(280) HPLC-PDA Chromatogram at 238 nm for sample of control cultivation (
(281)
(282) Exemplary embodiment No. 7: Growths curve of CBT 480 cultures with and without Nitro-Arg (Arg=Arginine) and Prg-Tyr (Tyr=Tyrosine) added.
(283)
(284) 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.
(285) HPLC-PDA Chromatogram at 238 nm for sample of control cultivation (
(286)
(287) Exemplary embodiment No. 8: Growths curve of CBT 329 cultures with and without Nitro-Arg (Arg=Arginine) added.
(288)
(289) Exemplary embodiment No. 9: Incorporation of the modified substrate Azido-Lys (Lys=Lysine) into Microcystin YR in position 4 produced by strain CBT 1.
(290) HPLC-PDA Chromatogram at 238 nm for sample of control cultivation (
(291)
(292) Exemplary embodiment No. 9: Growths curve of CBT 1 cultures with and without Azido-Lys (Lys=Lysine) added.
(293)
(294) Exemplary embodiment No. 10: Incorporation of the modified substrate Azido-Norval (Norval=Norvaline) into Microcystin RR in position 2 produced by strain CBT 633.
(295) HPLC-PDA Chromatogram at 238 nm for sample of control cultivation (
(296)
(297) Growths curve of CBT 633 cultures with and without Azido-Norval (Norval=Norvaline) added.
(298)
(299) Exemplary embodiment No. 11: Incorporation of the modified substrate H-homoarg-OH (homoarg=homoarginine) into Nodularin in position 2 produced by strain CBT 786.
(300) HPLC-PDA Chromatogram at 238 nm for sample of control cultivation (
(301)
(302) 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.
(303) HPLC-PDA Chromatogram at 238 nm for sample of control cultivation (
(304)
(305) Exemplary embodiment No. 13: Feeding of Microcystis aeruginosa strain CBT 480 with different amounts of modified substrate 4-azido-L-phenylalanine (0 μM, 10 μM, 30 μM) 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).
(306) The upper part of the figure shows overlaid HPLC-PDA Chromatograms at 238 nm for sample of control cultivation, sample of cultivation with added substrate 4-azido-L-phenylalanine of 10 μM in culture medium and sample of cultivation with added substrate 4-azido-L-phenylalanine of 30 μM in culture medium. The lower part of the 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.
(307)
(308) Exemplary embodiment No. 14: Incorporation of the modified substrate Prg-Tyr (Tyr=Tyrosine) into (D-Asp3, E-Dhb7) Microcystin-RR in position 2 produced by strain CBT 280.
(309) HPLC-PDA Chromatogram at 238 nm for sample of control cultivation (
(310)
(311) 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%.
(312)
(313) 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.
(314)
(315) 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 μM was determined. Differences between structural payload variants underline huge potential of further structural optimizations.