CN105377304B - Amanita hemolysin derivative - Google Patents
Amanita hemolysin derivative Download PDFInfo
- Publication number
- CN105377304B CN105377304B CN201480012523.6A CN201480012523A CN105377304B CN 105377304 B CN105377304 B CN 105377304B CN 201480012523 A CN201480012523 A CN 201480012523A CN 105377304 B CN105377304 B CN 105377304B
- Authority
- CN
- China
- Prior art keywords
- group
- amanita hemolysin
- amanita
- antibody
- iii
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 0 CC(C)CC[C@]1C(*)C#CCC1 Chemical compound CC(C)CC[C@]1C(*)C#CCC1 0.000 description 5
Landscapes
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The present invention relates to the treatment of tumour.On the one hand, the present invention relates to the conjugate that amanita hemolysin is connected with target conjugated group such as antibody by given joint, it can be used for treating cancer and other illnesss and disease.On the other hand, the present invention relates to the pharmaceutical composition for including the conjugate.
Description
Technical field
The present invention relates to oncotherapy.On the one hand, the present invention relates to amanita hemolysin and target conjugated group such as antibody to pass through
The conjugate of given joint connection, for treating cancer and other illnesss and disease.On the other hand, the present invention relates to including described
The pharmaceutical composition of conjugate.
Background technology
Amanita hemolysin is the cyclic peptide of 8 amino acid compositions.For example, they can separate or synthesize system from goose cream gill fungus mushroom
It is standby.Amanita hemolysin specifically suppresses the DNA dependent rna polymerase II of mammalian cell, so as to suppress impacted thin
The transcription of born of the same parents and the biosynthesis of protein.The stopping that the Transcription inhibition of cell causes to grow and breeds.Although it is not covalently to connect
Connect, amanita hemolysin and rna plymerase ii can closely connect (KD=3nM).Because the dissociation from enzyme amanitin is one
Slowly process, so the possibility that impacted cell recovers is little.When the suppression overlong time of transcription, cell will be through
Course programmed cell death (apoptosis).
In 1981, by using the connector (amino acid 4 for the indole ring for being connected to tryptophan；Referring to Fig. 1), through diazonium
Change, connect anti-Thy1.2 antibody to α-goose cream gill fungus, inquired into and controlled by the use of amanita hemolysin as cytotoxic group for tumour
Treat (Davis ＆ Preston, Science 1981,213,1385-1388).Davis and Preston determine connection site 7 '.
Morris and Venton prove replacements in site 7 ' cause holding cytotoxicity derivative (Morris ＆ Venton,
Int.J.Peptide Protein Res 1983,21 419-430)。
Conjugate described in patent application EP1859811 A1 (announcement on November 28th, 2007), wherein, β amanitins
Amanita hemolysin amino acid/11 γ C- atoms it is direct, i.e., albumin or monoclonal antibody are not connected to by joint design
HEA125, OKT3, or PA-1.Further there is illustrated these conjugates to breast cancer cell (MCF-7), Burkitt lymphoma cells
The inhibition of (Raji cells) and T- lymphoma cells (Jurkat cell) propagation.It has prompted the use of connector, including this
The connector of sample, the connector includes the elements such as such as acid amides, ester, ether, thioether, disulphide, urea, thiocarbamide, alkyl, but it does not have
Such construct is actually shown, also without more details are provided, such as the attachment site of amanita hemolysin.
Patent application WO 2010/115629 and WO 2010/115630 (equal 14 days October in 2010 announces) description are total to
Yoke thing, wherein antibody, such as anti-EpCAM antibody such as humanization huHEA125, pass through the γ of amanita hemolysin amino acid/11 (i) described
6 ' C- atoms of C- atoms (ii) the amanita hemolysin amino acid 4, or (iii) pass through the δ C- of δ C atom amanita hemolysins amino acid 3
Atom is connected to amanita hemolysin, can be connected at each occurrence directly or by the connector between antibody and amanita hemolysin.
The connector of prompting includes the elements such as such as ester, ether, carbamate, peptide bond.It is thin to breast cancer further there is illustrated these conjugates
Born of the same parents' (cell line mcf-7), pancreas cancer (cell line CAPAN-1), colon cancer (Colo205 of cell line) and cholangiocarcinoma (cell line OZ)
The inhibitory action of propagation.
Wieland has examined structure-activity relationship (T.Wieland, Peptides of Poisonous of amanita hemolysin
Amanita Mushrooms,Springer series in molecular biology,Springer Verlag New
York,1986).It is assumed that described in the hydroxyl group (hydroxy-proline) and amino acid 3 of amino acid 2-hydroxyl group (dihydroxy-different
Leucine) it is required for activity, and in amino acid/11 (aspartic acid or asparagine), the work(of amino acid 4 (6- hydroxytryptophans)
Can group and amino acid 3-hydroxyl group is more tolerant to chemical modification.This shows that the latter is the preferred site of connector attachment, and answers
Avoid the former modification.
It is known that when being connected to the carrier of big biomolecule, amanita hemolysin is relative nontoxic, such as antibody molecule,
And they just play their cytotoxicity only after biomolecule carrier is cut away.In view of the toxicity of amanita hemolysin, especially
Be to liver cell, most importantly, after applying plasma for the amanita hemolysin conjugate of targeting therapy for tumor, it
Keep highly stable, and the release of amanita hemolysin occurs after target cell internalization.In this case, it is conjugated stable small
Improvement can produce serious influence the security of therapeutic window and amanita hemolysin conjugate treatment method.
Goal of the invention
The target conjugated group amanita hemolysin conjugate that the present invention seeks to further provide for stablizing in blood plasma, to minimize
To the harmful side effect of non-target cell.
The content of the invention
The present invention is found surprisingly that based on following:Two oxygen atoms are connected with γ the and δ C atoms of amanita hemolysin amino acid 3
The ring of formation can improve target conjugated group amanita hemolysin conjugate tolerance, without disturbing amanita hemolysin and their target
The DNA dependent rna polymerase II interaction of mammalian cell.So far hydroxyl group and amanita hemolysin amino acid 3 are thought
The connections of γ and δ C atoms be vital to the effect of amanita hemolysin and amanita hemolysin conjugate.Wieland
(T.Wieland,Peptides of Poisonous Amanita Mushrooms,Springer Series in
Molecular Biology,Springer Verlag New York,1986；Wieland T,Rempel D,Gebert U,
Buku A,Boehringer H.über die Inhaltsstoffe des grünen
XXXII.Chromatographische Auftrennung der Gesamtgifte und Isolierung der neuen
Nebentoxine Amanin und Phallisin sowie des ungiftigen Amanullins.Liebigs Ann
Chem..1967；704:226-236) think that the naturally-produced amanita hemolysin toxicity for lacking γ-hydroxyl significantly reduces, such as a hydroxyl
Amatoxin acid amides.
On the one hand, the present invention relates to the amanita hemolysin of Formulas I
Wherein：
R1Selected from C=O, C=S, C=NR6And CR7R8；
R2Selected from S=O, SO2And S；
R3Selected from NHR5And OR5；
R4Selected from H, OR5, and OC1-6- alkyl；
R6Selected from C1-6- alkylidene-R5, cycloalkylidene-R5, miscellaneous cycloalkylidene-R5, arlydene-R5, and heteroarylidene-R5；
R7And R8Independently selected from H, C1-6- alkylidene-R5, cycloalkylidene-R5, miscellaneous cycloalkylidene-R5, arlydene-R5, and
Heteroarylidene-R5；
Wherein：
(i) each R5It is H；
(ii)R5In one be-Ln- X, wherein L are connectors, and n is selected from 0 and 1, X are the chemistry that can be connected with targeting group
Group, and remaining R5For H；Or
(iii)R5In one be-Ln-X*-Y, wherein L is connector, n be selected from 0 and 1, Y be targeting group, X* is X and Y
The chemical part that produces of functional group's connection, and remaining R5It is H.
On the other hand, the present invention relates to the amanita hemolysin as medicine.
Another aspect, the present invention relates to the amanita hemolysin for treating the cancer in patient, wherein the cancer is selected from breast
Gland cancer, colorectal cancer in gastrointestinal cancer, such as the group of composition, cancer of pancreas, cholangiocarcinoma, hepatocellular carcinoma, osteosarcoma, lung cancer are preceding
Row gland cancer, squamous cell carcinoma, oophoroma, carcinoma of testis, carcinoma of urinary bladder, stomach cancer, head and neck cancer, cervical carcinoma, kidney, glioma, skin
Skin cancer, such as：Malignant mela noma, thyroid cancer, leukaemia, malignant lymphoma.
Another aspect, the present invention relates to drug conjugate, according to the present invention, it includes amanita hemolysin further include it is a kind of or
A variety of pharmaceutically available diluents, carrier, excipient, filler, adhesive, lubricant, glidant, disintegrant, adsorbent；
And/or preservative.
Another aspect, the present invention relates to a kind of method, and for synthesizing the amanita hemolysin of the present invention, it comprises the following steps：
By the amanita hemolysin of Formula II
Wherein：
R2Selected from S=O, SO2And S；
R3Selected from NHR5And OR5；
R4Selected from H, OR5, and OC1-6- alkyl；
R6Selected from C1-6- alkylidene-R5, cycloalkylidene-R5, miscellaneous cycloalkylidene-R5, arlydene-R5, and heteroarylidene-R5；
R7And R8Independently selected from H, C1-6- alkylidene-R5, cycloalkylidene-R5, miscellaneous cycloalkylidene-R5, arlydene-R5, and
Heteroarylidene-R5；
Wherein R5One of be H, R5In one be-Ln- X, wherein L are connectors, and n is selected from 0 and 1, X can be with targeting group
The chemical group of connection, and remaining R5For H；Or R5In one be-Ln-X*-Y, wherein L is connector, and n is selected from 0 and 1,
Y is targeting group, and X* is that the functional group of X and Y connects the chemical group produced, and remaining R5It is H；
With following reaction：(i) bis- succinimdyl carbonate of N, N'- (DSC), (ii) thion reagent, particularly sulphur, 1,
1'- thiocarbonyldiimidazoles or 1,1'- thiocarbonyls -2 (1H)-pyridone；(iii) dichloro of amino carbonyl reagent, particularly isocyanides
Compound or isothiocyanic acid benzene；Or (iv) aldehyde, ketone or acyclic acetals.
Brief description of the drawings
Fig. 1 shows the structural formula of different amanita hemolysins.Bold numerals (1 to 8) represent to form eight kinds of amanita hemolysin
The standard number of amino acid.Also show amino acid/11, in 3 and 4 atom title (be respectively from alpha to
γ, alpha is to δ, and from 1' to 7').
After Fig. 2 is shown when cell culture 72 is small, different amanita hemolysin-trastuzumab (trade name He Sai in BrdU detections
Spit of fland) conjugate is to the toxicity of SK-OV-3 (oophoroma) cell.
After Fig. 3 is shown when cell culture 72 is small, different amanita hemolysin-trastuzumab conjugates are to SK- in BrdU detections
The cytotoxic activity of OV-3 (oophoroma) cell.
After Fig. 4 is shown when cell culture 72 is small, different amanita hemolysin-trastuzumab conjugates pair in BrdU detections
The cytotoxic activity of SKBR-3 (breast cancer) cell.
After Fig. 5 is shown when cell culture 72 is small, different amanita hemolysin-trastuzumab conjugates pair in BrdU detections
The cytotoxic activity of JIMT-1 (breast cancer) cell.
Fig. 6 shows the body of two kinds of different amanita hemolysins-Herceptin conjugates in Breast Cancer Xenograft Model
Interior effect.
Fig. 7 shows the body of two kinds of different amanita hemolysins-Herceptin conjugates in female NMRI nu/nu mouse
Interior tolerance.
Embodiment
Embodiment and the embodiment being included therein by reference to the present invention, can be more easily understood this hair
It is bright.
On the one hand, the present invention relates to the amanita hemolysin of Formulas I.
Wherein：
R1Selected from C=O, C=S, C=NR6And CR7R8；
R2Selected from S=O, SO2And S；
R3Selected from NHR5And OR5；
R4Selected from H, OR5, and OC1-6- alkyl；
R6Selected from C1-6- alkylidene-R5, cycloalkylidene-R5, miscellaneous cycloalkylidene-R5, arlydene-R5, and heteroarylidene-R5；
R7And R8Independently selected from H, C1-6- alkylidene-R5, cycloalkylidene-R5, miscellaneous cycloalkylidene-R5, arlydene-R5, and
Heteroarylidene-R5；
Wherein：
(i) each R5It is H；
(ii)R5In one be-Ln- X, wherein L are connectors, and n is selected from 0 and 1, X are the chemistry that can be connected with targeting group
Group, and remaining R5For H；Or
(iii)R5In one be-Ln-X*-Y, wherein L is connector, n be selected from 0 and 1, Y be targeting group, X* is X and Y
The chemical part that produces of functional group's connection, and remaining R5It is H.
Term " target conjugated group ", as used herein, refers to be specifically bound to any of target molecule or target epitope
Molecule or molecule group.Preferred target conjugated group is (i) antibody or antigen-binding fragment in the context of the application；(II) antibody
Sample albumen；(iii) aptamer." the target conjugated group " used suitable for the present invention typically has 40000Da (40kDa's)
Or the molecular weight of higher.
As used herein, if its dissociation constant KD reaches described 100 μM or less, preferably 50 μM or less, preferably
30 μM or less, preferably 20 μM or less, preferably 10 μM or less, are preferably 5 μM or less, more preferably 1 μM or less, more excellent
Elect 900nM or less, more preferably 800nM or less, more preferably 700nM or less, more preferably 600nM or less as,
More preferably 500nM or less, more preferably 400nM or less, more preferably more preferably 300nM or less, 200nM or few,
Even more preferably 100nM or less, even more preferably 90nM or less, even more preferably 80nM or less, even more preferably
For 70nM or less, 60nM or less, even more preferably below 50nM, even more preferably 40nM or less are even more preferably,
Even more preferably 30nM or less, even more preferably 20nM or less, even more preferably 10nM or the second less chemical combination
Thing, then it is assumed that the first compound (such as antibody) is " specifically combining " to second compound (such as antigen, such as target protein).
In the context of the application, term " target molecule " and " target epitope " are referred respectively to respectively by particular target conjugated group
With reference to antigen and epitope.Preferable target molecule is tumor associated antigen, is especially in the presence of thin in one or more tumours
Antigen or epitope in born of the same parents' type or Tumor-associated cell surface, compared to non-tumor cell surface, it have higher concentration and/
Or different three-dimensional structure.Preferably, the antigen or epitope are present in one or more tumours or mesenchyma stroma of tumors cell type
Surface on, but be not present on the surface of non-tumor cell.In a particular embodiment, target conjugated group is specifically bound
HER-2/neu's or epithelial cell adhesion molecule (EpCAM's) epitope.In other embodiments, the antigen or epitope
Preferentially expressed in the cell for participating in autoimmune disease.In other embodiments, the antigen or epitope are preferentially being joined
With being expressed in the cell of inflammatory disease.
Term as used herein " antibody or its antigen-binding fragment ", refers to immunoglobulin molecules and immune globulin
The immunoactive portions of white molecule, that is, contain antigen binding site, the molecule of energy immunologic opsonin combination antigen.Also include passing through
Technology selects immunoglobulin-like albumen, such as, phage specificities binding target molecule, such as to target protein HER-2/
Neu or EpCAM.Immunoglobulin molecules in the present invention can be any types (for example, IgG, IgE, IgM, IgD, IgA and
IgY), the immunoglobulin molecules of class (for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.Suitable for this hair
Bright " antibody and antigen-binding fragment " includes, but not limited to polyclonal, monoclonal, and unit price, bispecific is heterologous, more specifically
Property, the mankind, humanization (particularly CDR transplanting), desensitization, or chimeric antibody, single-chain antibody (such as scFv), Fab fragments, F
(ab')2Fragment, the fragment that Fab expression libraries produce, double antibody or four antibody (Holliger P. et al., 1993), nanometer resists
Body, antiidiotype (anti-Id) antibody (including for example, anti-Id antibody) in the present invention, and the epitope of any of above antibody
Binding fragment.
In some embodiments, antigen-binding fragment is human antigen binding antibody's fragment of the present invention, including but unlimited
In Fab, Fab' and F (ab') 2, Fd, scFv s (scFv), single-chain antibody the Fvs (dsFv) of disulfide bond connection and includes V
The fragment in L or V H structures domain.Antigen binding antibody fragment, including single-chain antibody, can individually include Variable Area or with it is following complete
Portion or part are combined：Hinge area, CL, CH1, CH2, and CH3 domains.The present invention antigen-binding fragment also include variable domain with
One hinge area, CL, CH1, CH2, and any combinations of CH3 domains.
The antibody that the present invention uses can be any animal origin, including birds and mammal.Preferably, antibody is derived from
People, rodent (such as mouse, rat, cavy, or rabbit) chicken, pig, sheep, goat, camel, ox, horse, donkey, cat, or dog.Especially
Preferable antibody is people or mouse source.As used herein, " human antibody " is to include resisting for human immunoglobulin(HIg) amino acid sequence
Body, and one or more human immunoglobulin antibody of human immunoglobulin(HIg) library or transgenic animals are isolated from, and not
Express endogenous immunoglobulin, such as the U.S. Patent number 5939598 that Kucherlapati＆Jakobovits is obtained.
Term " antibody-like protein " refer to screen (for example, by circulating mutagenesis) go out can with target molecule specifically bind
Protein.In general, such antibody-like protein connects both ends to albumen stent including at least one variable peptide loop.This dual knot
Structure constraint drastically increases antibody-like protein binding affinity.The length of classical variable peptide loop is 10 to 20 amino acid.
Scaffolding protein can be any albumen for having good solubility.Preferably, scaffolding protein is small globular protein.Antibody sample
Albumen includes but not limited to the ankyrin repeat protein matter of affine body, analog antibody, and design (referring to summary：Binz etc.,
2005).Antibody-like protein can be derived from the large-scale library of mutant, such as from big phage display library screening and can be with
Separated similar to conventional antibody.In addition, antibody sample associated proteins can be lured by the combination of surface exposed residue in globular preteins
Become to obtain.
Term " aptamer " refer to by the external selection of more wheels repeatedly or SELEX (system of ligand index concentration into
Change) filter out can be combined with target molecule nucleic acid molecules (see summarize：Brody and Gold,2000).Aptamer can be with
It is DNA or RNA molecule.Aptamer can contain modification, such as modified nucleotide, the pyrimidine substituted such as 2'- fluorine.
As used herein, " aptamer conjugate " refers to target conjugated group toxin conjugate, wherein the target conjugated group is
The aptamer of above-mentioned replacement (III).
" connector " in invention context refers to the molecule for connecting two components, and each component is connected to one end of connector,
This can with the distance between two components of increase and alleviating the space between these components and disturbing, such as this paper targets conjugated group and
Between amanita hemolysin.There is no connector, amanita hemolysin polymerize with the amanita hemolysin that is directly connected to reduce of target conjugated group with RNA
The ability of enzyme II interactions.In specific embodiments, the main chain of connector have 1 to 30 atom (i.e. 1,2,3,4,5,6,
7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29, or 30 atoms).
That is it is the number of the atom of most short connection needs or key between amanita hemolysin group and target conjugated group that the length of connector, which defines, its
Described in connector main chain side and amanita hemolysin reaction, opposite side and target conjugated group react.It is excellent in the context of the present invention
The connector of choosing is C1-20- alkylidene, C1-20- miscellaneous alkylidene, C2-20- alkenylene, C2-20- miscellaneous alkenylene, C2-20- alkynylene, C2-20-
Miscellaneous alkynylene, cycloalkylidene, miscellaneous cycloalkylidene, arlydene, heteroarylidene, sub- aralkyl, or miscellaneous sub- aralkyl, its optionally by
Substitution.Connector can include one or more structural details, such as carboxylic acid amides, ester, ether, thioether, disulfide, urea, thiocarbamide, hydrocarbon portion
Grade.Connector can also include the combination of two or more structural details.Each structural detail can reside in connector once
More than, such as twice, three times, four times, five times, six times.In some embodiments, connector can include a disulfide bond.Can be with
Understand, the connector must be connected by single step or two or more subsequent steps with amanita hemolysin and target conjugated group
Connect.For this reason, the connector will connect two groups, preferably proximally and distally, its can (I) and one of the component to be connected base
Group, the preferably activated group of amanita hemolysin or target binding peptide, form covalent bond；Or (ii) its (can) be activated with formed connection
The covalent bond of group on amanita hemolysin.It is therefore preferred that chemical group in connector proximally and distally, this is the knot of coupling reaction
Fruit, such as ester, ether, carbamate, peptide bond etc..
In the context of the present invention, term " amanita hemolysin " is included from the separated all 8 amino acid compositions of Amanita
Cyclic peptide, and by Wieland, T. and Faulstich H descriptions (Wieland T, Faulstich H., CRC Crit Rev
Biochem.1978Dec；5(3):185-260), and then including all chemical derivatives；In addition it is all semi-synthetic similar
Thing；Further comprise synthesis congener (cricoid, 8 amino acid) of all main structures using native compound as template, also
Including all synthesis or semi-synthetic analogs containing non-hydroxylated amino acid, rather than hydroxylated amino acids, additionally include
All synthesis or semi-synthetic analog, wherein sulfide, sulfone, or the different atom substituted thioethers sulfuryl parts with sulphur, example
If amanitin carbon is for the carbon atom of analog, in any case, suppress to feed wherein this all analog derivative or the like passes through
Newborn animal rna plymerase ii plays its functional activity.
As used herein, compound " chemical derivative " (or referred to as：" derivative ") refer to the species that there are chemical constitution,
It is analogous to compound, but contains the chemical group and/or at least one chemical combination of missing being not present at least one compound
Chemical group present in thing.Compared with the derivative, compound is referred to as " mother " compound.In general, one or moreization
Learning the parent compound of reactions steps can produce " derivative ".
As used herein, " analog " of compound is related on compound structure but differs, and shows at least one
The activity of kind compound.Compared with the analog, compound is referred to as " mother " compound.Foregoing active includes but not limited to：
And the combination activity of another compound；Inhibitory activity, such as enzyme inhibition activity；Toxic action；Activation Activity, such as enzyme activition
Activity.Analog and parent compound is not required to have identical activity.In the context of the application, if the phase that compound is shown
Close activity be at least up to parent compound activity 1% (more preferably at least 5%, more preferably at least 10%, more preferably at least
20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%), then this compound is analog.Therefore,
" analog of amanita hemolysin ", as used herein, refer in structure with the α amanitins shown in Fig. 1, β amanitins, γ geese
Cream gill fungus alkali, ε amanitins, three hydroxyl amanita hemolysins, three hydroxyl amanita hemolysin acid amides, carboxyl goose green grass or young crops phallotoxins acid amides and a hydroxyl amanita hemolysin
Any relevant compound of compound in carboxylic acid, and with α amanitins, β amanitins, γ amanitins, ε amanitins,
Three hydroxyl amanita hemolysins, three hydroxyl amanita hemolysin acid amides are at least one in carboxyl goose green grass or young crops phallotoxins acid amides and a hydroxyl amanita hemolysin carboxylic acid
Compare, its inhibitory activity to mammalian RNA polymerase II show as at least 1% (more preferably at least 5%, more preferably at least
10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%).With α goose cream gill fungus
Alkali, β amanitins, γ amanitins, ε amanitins, three hydroxyl amanita hemolysins, three hydroxyl amanita hemolysin acid amides, carboxyl goose green grass or young crops phallotoxins
Any in acid amides and a hydroxyl amanita hemolysin carboxylic acid, one is compared, and " analog of amanita hemolysin " in the present invention moves lactation
Thing rna plymerase ii inhibitory activity possibility or even stronger.The concentration of inhibitor when can react suppressed half by measuring
(IC 50 is worth), measures inhibitory activity.The inhibitory activity for crossing measurement cell proliferation carrys out indirect determination it gathers mammalian rna
The inhibitory activity of synthase II.The appropriate method of measure cell inhibitory effect is described in embodiment.
" semi-synthetic analog " refer to using natural origin (such as vegetable material, bacterial cultures, fungal cultures or
Cell culture) compound as starting material the analog as obtained from chemical synthesis.In general, the present invention's is " hemizygous
Into analog " make to synthesize by the compound for being isolated from family of Amanitaceae mushroom.In contrast, " synthetic analogues " refer to by
The analog that small (typical petrochemical industry) module passes through so-called fully synthetic synthesis.Generally, it is this fully synthetic biological assistant to be not required.
Functionally, amanita hemolysin is defined as suppressing the peptide or depsipeptide of mammalian RNA polymerase II.Preferable goose
Cream phallotoxins has the functional group that can be reacted with linkers or target conjugated group, and (such as carboxyl, amino, hydroxyl, mercaptan or mercaptan are caught
Obtain group).The amanita hemolysin for being particularly suitable for conjugate in the present invention is the α amanitins shown in Fig. 1, β amanitins, γ goose cream
Gill fungus alkali, ε amanitins, three hydroxyl amanita hemolysins, three hydroxyl amanita hemolysin acid amides, carboxyl goose green grass or young crops phallotoxins acid amides and a hydroxyl amanita hemolysin carboxylic
Acid and salt, chemical derivative, semi-synthetic analog, and the analog of synthesis.Particularly preferred amanita hemolysin in the present invention
It is α amanitins, β amanitins and three hydroxyl amanita hemolysin acid amides.
In specific embodiments of the present invention, the amanita hemolysin of (iii), wherein the Y functional groups of the amanita hemolysin of (iii)
It is amino.In specific such embodiment, X* is urea groups.
In specific embodiment of the present invention, described is-LnThe residue R of-X*-Y5:
(i) it is present in R1；
(ii) it is present in R3；
(iii) it is present in R4；Or
(iv) it is present in R5.
In specific embodiment of the present invention, the amanita hemolysin is selected from α amanitins, β amanitins, three hydroxyl goose cream poison
Peptide, three hydroxyl amanita hemolysin acid amides, or selected from its salt or the like.
In specific embodiment, the connector L of (ii) or (iii) are that have 1 to 20 atom for being independently selected from C, O, N and S
Straight chain, particularly 2 to 16 atoms, more particularly 5 to 14 atoms, or even more particularly 6 to 12 atoms.Specifically
In embodiment, at least 60% atom is C atoms in straight chain.In specific embodiment, the atom in straight chain is by singly-bound
Connection.
In specific embodiment, the length of (ii) or (iii) connector is 12 atoms, particularly from 2 to 10, particularly
It is and most particularly 6 to 8 atoms from 4 to 9.
In specific embodiment, connector L is alkylidene, alkenylene, miscellaneous alkenylene, alkynylene, miscellaneous alkynylene, sub- cycloalkanes
Base, miscellaneous cycloalkylidene, arlydene, heteroarylidene, sub- aralkyl, or miscellaneous sub- aralkyl, including 1 to 4 miscellaneous selected from N, O, and S
Atom, wherein the connector is optionally substituted.
Term " alkylidene " refers to the divalent straight saturated hydrocarbyl for having 1 to 20 carbon atom, includes 1 to 10 carbon atom
Group.In some embodiments, alkylidene can be low-grade alkylidene.Term " low-grade alkylidene " refers to there is 1 to 6 carbon original
The alkylidene of son, and in certain embodiments, there is 1 to 5 or 1 to 4 carbon atom.The example of alkylidene includes, but unlimited
In methylene (- CH2-), ethylidene (- CH2-CH2-), positive propylidene, positive butylidene, positive pentylidene, and n-hexyl.
Term " alkenylene " refers to the divalent straight group for having 2 to 20 carbon atoms, wherein the carbon-carbon bond at least one
A is double bond, and other keys can be singly-bound or other double bond.Term " alkynylene " herein refers to there are 2 to 20 carbon originals
The group of son, wherein the carbon-carbon bond at least one be three keys, and other keys can be singly-bound, double bond or three other keys.
The example of alkenylene includes ethenylidene (- CH=CH-), 1- allylidenes, 2- allylidenes, 1- butenylidenes, 2- Aden alkene
Base, 3- butenylidenes, and analog.The example of alkynylene includes ethynylene, 1- Asias propinyl, 2- Asias propinyl, etc..
As used herein, " cycloalkylidene " means divalence ring, this divalence ring is the monocyclic or multi-loop system of any stabilization
A part, wherein the ring has 3 to 12 carbon atoms, but does not have hetero atom, and the wherein described ring is fully saturated, and
And term " sub- cycloalkenyl group " means divalence ring, this divalence ring is monocyclic or multi-loop system a part for any stabilization, wherein institute
Stating ring has 3 to 12 carbon atoms, but does not have hetero atom, and wherein described ring be at least part it is undersaturated (but include appoint
What aromatic ring).The example of cycloalkylidene includes, but not limited to cyclopropylidene, sub- cyclobutyl, cyclopentylene, cyclohexylidene, and Asia
Suberyl.The example of sub- cycloalkenyl group includes, but not limited to cyclopentenylidene and cyclohexadienylidene.
As used herein, term " miscellaneous cycloalkylidene " and " miscellaneous Asia cycloalkenyl group " mean divalence ring, this divalence ring is any steady
Fixed monocyclic or multi-loop system a part, wherein the ring has 3 to 12 carbon atoms, and wherein described ring by carbon atom and
At least one hetero atom composition, in particular at least one hetero atom are independently selected from the group being made of N, O and S, and miscellaneous cycloalkylidene refers to
Be fully saturated ring, miscellaneous Asia cycloalkenyl group refers to that at least partly undersaturated ring (but does not include any arlydene or miscellaneous
Arlydene ring).
Term " arlydene " means divalence ring or loop system, this divalence ring or ring system are the monocyclic or polycyclic systems of any stabilization
A part for system, wherein the ring or loop system have 3 to 20 carbon atoms, but does not have hetero atom, is advised by " 4n+2 " a pi-electron
Then determine that its ring or ring system are made of aromatic portion, including phenylene.
As used herein, term " heteroarylidene " refers to divalence ring or loop system, this divalence ring or ring system are any stabilizations
Monocyclic or multi-loop system a part, wherein the ring or loop system have 3 to 20 carbon atoms, is advised by " 4n+2 " a pi-electron
Then determine that its ring or ring system are made of aromatic portion, and contain carbon atom and one or more nitrogen, sulphur and/or oxygen heteroatom.
In the context of the present invention, term " substitution " is intended to suggest that is designated group with one or more hydrogen of connector main chain
One of replaced, if specified atom has a normal chemical valence, or substituted group atom properly without excessive, and should
Substitution produces stable compound.As defined herein, term " being optionally substituted " means that one or more substituents do not take
Generation or the substitution connector.When substituent is ketone (or oxygen, i.e. ,=O) base, sulphur or imino group etc., then replacement adaptor backbone atoms
On two hydrogen.Typical substituent includes, for example, alkyl, alkenyl, alkynyl, cycloalkyl, Heterocyclylalkyl, aryl, aralkyl,
Heteroaryl, acyl group, aroyl, heteroaryl, carboxyl, alkoxy, aryloxy group, acyloxy, aryl acyloxy, miscellaneous base, alkoxy, halogen
Element, (thio) ester, cyano group, phosphoryl, amino, imino group, (thio) acid amides, sulfydryl, alkylthio group, acyl sulfenyl, sulfonyl, sulfuric acid
Ester, sulphonic acid ester, sulfonamides, sulphonyl, nitro, azido, haloalkyl, including perfluoroalkyl (such as trifluoromethyl), haloalkoxy
Base, alkyl sulfenyl, alkyl sulphinyl, alkyl sulphonyl, alkyl sulfonyl-amino, arylalkyl sulfonyl amino, phosphoryl, phosphorus
Acid esters, phosphonate ester, phosphinate, alkyl carboxyl, acid amides, oxo, hydroxyl, sulfydryl, amino (optional one or two substitutions, for example, logical
Cross alkyl, aryl or heteroaryl), imino group, carboxylic acid amides, carbamoyl (optional one or two substitutions, such as by alkyl, aryl,
Or heteroaryl), amidino groups, amino-sulfonyl, acyl amino, aroylamino, (thio) urea groups, arylthio) urea groups, alkyl (sulphur
Generation) urea groups, cycloalkyl (thio) urea groups, aryloxy group, aralkoxy or-O (CH2)n-OH,-O(CH2)n-NH2,-O(CH2)nCOOH,-(CH2)nCOOH,-C(O)O(CH2)nR,-(CH2)nN(H)C(O)OR,or-N(R)S(O)2R, wherein n are 1-4 and R
Hydrogen is independently chosen from ,-alkyl ,-alkenyl, alkynyl, cycloalkyl ,-cycloalkenyl group ,-(C connections-Heterocyclylalkyl) ,-(C connections-heterocycle
Alkenyl) ,-aryl, and heteroaryl, it is permissible to have multiple substitutions.It will be appreciated by those skilled in the art that if condition is closed
It is suitable, substituent, such as Heterocyclylalkyl, aryl, heteroaryl, alkyl etc., or functional group, such as-OH ,-NHR etc., itself can be taken
Generation.Those skilled in the art is also, it should be understood that when the conditions are suitable, substitution part can also be substituted itself.
In specific embodiment, the group that connector L includes is selected from：Disulfide (- SS-), ether (- O-), thioether (- S-), amine
(- NH-), ester (- OC (=O)-or-C (=O)-O-), carboxylic acid amides (- NH-C (=O)-or-C (=O)-NH-), carbamate
(- NH-C (=O)-O- or-OC (=O)-NH-), and urea groups (- NH-C (=O)-NH-).
In specific embodiments of the present invention, the m groups of the connector L of (ii) or (III) are selected from:Alkylidene, alkenylene are sub-
Alkynyl, cycloalkylidene, miscellaneous alkyl, miscellaneous alkenylene, miscellaneous alkynylene, heterocyclic radical, arlydene, heteroaryl, sub- aralkyl, and miscellaneous sub- virtue
Alkyl, wherein each group can individually substitute at random, following groups are independently chosen from the n groups of the connector：Disulfide (- SS-),
Ether (- O-), thioether (- S-), amine (- NH-), ester (- OC (=O)-or-C (=O)-O-), carboxylic acid amides (- NH-C (=O)-or-C
(=O)-NH-), carbamate (NH-C (=O)-O- or-OC (=O)-NH-), and urea groups (- NH-C (=O)-NH-), wherein
M=n+1.In specific embodiment, m is that 2 and n is 1, or m is that 3 and n is 2.In specific embodiment, connector includes respectively
2 or 3 unsubstituted alkylidenes, and the disulfide of 1 or 2 connection unsubstituted alkylidene, ether, thioether, amine, ester, carboxylic
Acid amides, carbamate or urea groups.
In specific embodiment, the carbon atom in straight chain be independently part optionally substituted methylene (-
CH2-).Especially, in this embodiment, the optional substituent is independently selected from halogen and C1-6- alkyl, particularly first
Base.
In specific embodiment, connector L, particularly the connector L shown in [0043] section, selected from following linking group：
Amanita hemolysin end:-(CH2)2- target conjugated group end；
Amanita hemolysin end:-(CH2)3- target conjugated group end；
Amanita hemolysin end:-(CH2)4- target conjugated group end；
Amanita hemolysin end:-(CH2)5- target conjugated group end；
Amanita hemolysin end:-(CH2)6- target conjugated group end；
Amanita hemolysin end:-(CH2)7- target conjugated group end；
Amanita hemolysin end:-(CH2)8- target conjugated group end；
Amanita hemolysin end:-(CH2)9- target conjugated group end；
Amanita hemolysin end:-(CH2)10- target conjugated group end side；
Amanita hemolysin end:-(CH2)11- target conjugated group end；
Amanita hemolysin end:-(CH2)12- target conjugated group end；
Amanita hemolysin end:-(CH2)16- target conjugated group end；
Amanita hemolysin end:-(CH2)2-S-S-(CH2)2- target conjugated group end；
Amanita hemolysin end:-(CH2)3-S-S-(CH2)2- target conjugated group end；
Amanita hemolysin end:-(CH2)2-S-S-(CH2)3- target conjugated group end；
Amanita hemolysin end:-(CH2)3-S-S-(CH2)3- target conjugated group end；
Amanita hemolysin end:-(CH2)4-S-S-(CH2)4- target conjugated group end；
Amanita hemolysin end:-(CH2)2-CMe2-S-S-(CH2)2- target conjugated group end；
Amanita hemolysin end:-(CH2)2-S-S-CMe2-(CH2)2- target conjugated group end；
Amanita hemolysin end:-(CH2)2-O-(CH2)2- target conjugated group end；
Amanita hemolysin end:-(CH2)2-O-(CH2)2-O-(CH2)2- target conjugated group end；
Amanita hemolysin end:-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2- target-conjugated group end；
Amanita hemolysin end:-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2- target conjugated group end；
Amanita hemolysin end:-CH2-C6H4-NH-Cit-Val-(CH2)6- target conjugated group end；
Amanita hemolysin end:-CH2-C6H4-NH-Lys-Phe-(CH2)6- target conjugated group end；
Amanita hemolysin end:-CH2-C6H4-NH-Val-Val-(CH2)6- target conjugated group end；
Amanita hemolysin end:-CH2-C6H4-NH-Ile-Val-(CH2)6- target conjugated group end；
Amanita hemolysin end:-CH2-C6H4-NH-His-Val-(CH2)6- target conjugated group end；
Amanita hemolysin end:-CH2-C6H4-NH-Met-Val-(CH2)6- target conjugated group end；
With
Amanita hemolysin end:-CH2-C6H4-NH-Asn-Lys-(CH2)6- target conjugated group end
In specific embodiment, target conjugated group is specific to be combined with the epitope being present on tumour cell, especially
It is that the wherein described specific epitope with human epidermal growth factor receptor 2 (HER2) of target conjugated group is combined.
In specific embodiment, the antibody is trastuzumab or HEA125 or antibody fragment, and it includes trastuzumab
Or the antigen-binding fragment of HEA125.
In specific embodiment, at least one amanita hemolysin molecule is connected with target conjugated group.Often it is conjugated amanita hemolysin
The increase of number can also increase its toxicity.Therefore, in specific embodiment, the ratio of target conjugated group and amanita hemolysin is
1 target conjugated group and 1 to 15 amanita hemolysin molecule, particularly 1,2,3,4,5,6,7,8,9,10,11,12,13,14, or
15.It is counted as a target conjugated group than antibody the dimer such as ratio in the case of IgGs, the dimer to calculate.
In specific embodiments, target conjugated group is selected from:
(i) antibody or its antigen-binding fragment；
(ii) antibody-like protein；With
(iii) aptamer.
In specific embodiments, the antibody or antigen-binding fragment are selected from double antibody, four antibody, nano antibody,
Chimeric antibody, goes immune antiboidy, humanized antibody or human antibody.
In specific embodiments, the antigen-binding fragment is selected from Fab, F (ab ')2, Fd, Fv, scFv, two sulphur
The Fvs (dsFv) of key connection.
On the one hand, the present invention relates to the amanita hemolysin as medicine.
Another aspect, the present invention relates to the amanita hemolysin for treating cancer patient, particularly wherein described cancer is selected from
Breast cancer, cancer of pancreas, cholangiocarcinoma, colorectal cancer, lung cancer, prostate cancer, oophoroma, prostate cancer, stomach cancer, kidney are pernicious
Melanoma, leukaemia and malignant lymphoma.
As used herein, " patient " refers to from target conjugated group toxin conjugate described herein treatment to benefit
Any mammal or birds.Preferably, " patient " is selected from:Experimental animal (such as mouse or rat), domestic animal (including
Such as cavy, rabbit, chicken, pig, sheep, goat, camel, ox, horse, donkey, selects in the group of cat, or dog), or primate, bag
Include the mankind.Particularly preferred " patient " is people.
As used herein, " treatment " of disease or illness, " processing " or " therapy " refer to complete following one or more:(a)
Reduce the seriousness of disease；(b) control or prevent progression of the disease；(c) prevent that sb.'s illness took a turn for the worse；(d) control or prevent patient disease
Recurrence；Control or prevent to suffer from symptom recurrence (e).
As used herein, according to the present invention, treatment includes applying conjugate or pharmaceutical composition to patient, wherein, " apply
With " include applying in vivo, and be administered to outside organizer, such as vein transplantation.
In a particular embodiment, using the conjugate of the present invention of effective therapeutic dose.
" effective therapeutic dose " is the therapeutic dose that can be accomplished the end in view.The effective dose of given therapeutic agent will with factor,
Such as the property of reagent, method of administration, is changed using the species and size and administration purpose of therapeutic agent animal.According in this area
Some schemes, technical staff can empirically determine the effective dose of each case.
On the other hand, the present invention relates to pharmaceutical composition, including amanita hemolysin according to the present invention, further include it is a kind of or
A variety of pharmaceutically acceptable diluents, carrier, excipient, filler, adhesive, lubricant, glidant, disintegrant, absorption
Agent；And/or preservative.
" pharmaceutically acceptable " refers to through Federal Regulatory Agencies or state government or American Pharmacopeia or other generally accepted medicines
Allusion quotation is ratified, available for animal and people.
In a particular embodiment, pharmaceutical composition is used in the form of Formulations for systemic administration.Including non-bowel, including note
Penetrate and infuse.Injection is prepared in the form of ampoule or directly to use injection, such as the syringe or disposable for preparing to use
Using syringe and pierce through in bottle be discontinued more.The form of medication of injection:Subcutaneously (sc), intramuscular (IM), intravenous (iv) or skin
Interior (IC) is injected.Particularly, the preparation such as crystal for being adapted to injection, solution, the suspension of nano particle or scattered can be produced
System, such as colloid, such as hydrosol.
The concentrate of ejection preparation can be further produced, is dissolved with the isotonic diluent agent of water or scattered.Preserved material can be made
It is standby into infusion isotonic solution, fat emulsion, Liposomal formulation and microemulsion.Similar to injection, infusion preparation can be prepared into
Dilutable concentrate.In hospital and therapy of walking about can be using the ejection preparation of permanent infusion format, such as the side for passing through micropump
Formula.
Gastrointestinal drug preparation can be added, for example, albumin, blood plasma, expansion, surface reactive material, organic diluent,
PH influences thing, complex compound or polymer, and the target-conjugated group toxin conjugate that can particularly influence the present invention is adsorbed onto protein
Or the material of polymer, or reduce the material such as syringe that target-conjugated group toxin conjugate of the invention is adsorbed onto material
Tool or packaging material, for example, plastics or glass.
The amanita hemolysin that the present invention includes target conjugated group can be connected with non-bowel microcarrier or nano particle, for example,
The particle of fine dispersion is based on poly- (methyl) acrylate, poly-lactic acid ester, polyglycolic acid salt, polyaminoacid or Polymer Technology Group
Ethyl ester.Parenteral administration can also be modified to depot formulation, such as based on " multiple unit principles ", if respectively with fine point
Scattered, scattered and of the invention suspended form target-conjugated group toxin conjugate, or as the Crystal suspensions in medicine, or base
In " individual unit principle is " if target-conjugated group toxin conjugate of the closing present invention is such as subsequently implanted tablet into preparation
It is or bar-shaped.These single units and multiple-unit preparation implant or depot's medicine generally include such as so-called biodegradable poly-
Compound, such as lactic acid and hydroxyacetic acid, poly(ether-urethane), polyaminoacid, the polyester of poly- (methyl) acrylate or polysaccharide.
Auxiliary agent and carrier, preferably sterile water (aqua sterilisa) are added in the non-bowel pharmaceutical composition of the present invention, pH value influences
Material etc., such as organic or inorganic acid or alkali, and their salt, buffer solution, for adjusting the buffer solution of pH value, isotonic solution
Such as sodium chloride, sodium acid carbonate, glucose and fructose, surfactant and surfactant, take off with emulsifying agent, such as polyoxyethylene
The fatty acid partial ester (for example, tween) of water sorbierite, alternatively, as aliphatic acid polyethenoxy acid esters (for example,
), as fat oil such as peanut oil, soybean oil or castor oil, as fatty acid synthetic ester such as ethyl oleate, isopropyl myristate
With neutral oil (for example,) and as reagent and additive in polymerization such as gelatin, glucan, polyvinylpyrrolidone, additive, it
Can increase the dissolubility of organic solvent, such as propane diols, ethanol, n,N-dimethylacetamide, propane diols or as complex compound example
Such as citrate and urea, as preservative such as benzoic acid hydroxypropyl ester and methyl ester, benzylalcohol, as antioxidant such as sulfurous acid
Sodium and as stabilizer such as EDTA.
In preferred embodiment, pharmaceutical composition of the invention is configured to supensoid agent, thickener prevent the target of the present invention-
The precipitation of conjugated group toxin conjugate, or surfactant and polyelectrolyte, to ensure the suspension of deposit and/or complexing
Thing forming agent, for example, adding EDTA.The compound of various polymer active ingredients can also be prepared in fact.The example of this polymer
Son is polyethylene glycol, polystyrene, carboxymethyl cellulose,Or polyethylene glycol sorbitan fatty acid ester.Invention
Target-conjugated group toxin conjugate can also mix the liquid preparation such as cyclodextrin of inclusion compound form.Specifically implementing
In scheme, dispersant can be used as other auxiliary agents to add.Available for the lyophilized scaffold agent of production as mannitol, glucan, sucrose,
Human albumin, lactose, PVP or kind gelatin.
In specific embodiments of the present invention, X be carbamic acid derivative-NH-C (O)-Z, wherein Z be can be by target
The primary amine of the nucleophilic group of conjugated group, particularly target conjugated group, the leaving group replaced.
In a particular embodiment, Z is selected from：Tertbutyloxycarbonyl, succinimide epoxide, 1-O- succinimides epoxide-
3- sulfonic acid (sulfo group-NHS), O- (4-nitrophenoxy), O- (3- nitro-phenoxies), O- (2,4-nitrophenoxy), O- (2,4-
Two chloro- 6- nitro-phenoxies), phenyl-pentafluoride, pentachlorobenzene, O- (2,4,5- phenoxy benzoic acid), O- (3,4- dihydro-3-hydroxy 4-
Oxo -1,2,3- phentriazine -3- bases), O- (interior -1- hydroxyls -5- norbornene -2,3- dicarboximide -1- bases), 1- neighbour's benzene
Diformyloxy, 1- benzotriazole epoxides, 1- (7- azabenzotriazols) epoxide), and TMSIM N imidazole base.
On the other hand, the present invention relates to the method for synthesizing amanita hemolysin of the present invention, including following reactions steps：By Formula II
Amanita hemolysin
Wherein：
R2Selected from S=O, SO2And S；
R3Selected from NHR5And OR5；
R4Selected from H, OR5, and OC1-6- alkyl；
R6Selected from C1-6- alkylidene-R5, cycloalkylidene-R5, miscellaneous cycloalkylidene-R5, arlydene-R5, and heteroarylidene-R5；
R7And R8Independently selected from H, C1-6- alkylidene-R5, cycloalkylidene-R5, miscellaneous cycloalkylidene-R5, arlydene-R5, and
Heteroarylidene-R5；
Wherein R5One of be H, R5In one be-Ln- X, wherein L are connectors, and n is selected from 0 and 1, X can be with targeting group
The chemical group of connection, and remaining R5For H；Or R5In one be-Ln-X*-Y, wherein L is connector, and n is selected from 0 and 1,
Y is targeting group, and X* is that the functional group of X and Y connects the chemical group produced, and remaining R5It is H；
With following reaction：(i) bis- succinimdyl carbonate of N, N'- (DSC), (ii) thion reagent, particularly sulphur, 1,
1'- thiocarbonyldiimidazoles or 1,1'- thiocarbonyls -2 (1H)-pyridone；(iii) dichloro of amino carbonyl reagent, particularly isocyanides
Compound or isothiocyanic acid benzene；Or (iv) aldehyde, ketone or acyclic acetals.
Embodiment
The synthesis of 1 amanita hemolysin conjugated molecule of embodiment
The synthesis of 1.1 6 '-N-Boc- (6- Aminohexyls)-α-amanitin HDP 30.0132
α-amanitin HDP 30.0132
In argon gas and at room temperature, 180mg (196 μm of ol) vacuum drying α amanitins are dissolved in 5000 μ l anhydrous dimethyls
Sulfoxide (DMSO).Add Aminohexyl bromine (439mg, 8 equivalents) and 1M sodium hydroxides (215.5 μ l, 1.1 equivalents).At room temperature 2
After hour, with acetic acid solution acidified reaction mixture in 100 μ l 1M DMSO to pH=5.Volatile matter is evaporated in vacuo, will be residual
Excess is dissolved in 500 μ l methanol, and is added in the ice-cold methyl tertiary butyl ether(MTBE)s (MTBE) of 40ml.Centrifugal sediment, and lead to
Cross to be resuspended in 40ml MTBE and wash.Sediment is placed in 4000 μ l methanol, and passes through C18 column preparatives with 500 μ l every time
HPLC filtered and purified (RP-18,10 μm of 250x21.2mm, Luna,).Solvent orange 2 A：Water；Solvent B：Methanol；Ladder
Degree：0min 5%B；5min 5%B, 20min 100%B；25min 100%B；27min 5%B, 35min, 5%B；Flow velocity
30ml/min.Collect the fraction of 18.4-19.1min retention times and by evaporation of the solvent to 126mg (57%) HDP30.0132, be
Colorless solid.
MS(ESI+)1118.5[M+H]+,1140.5[M+Na]+
Pass through evaporation, fraction 35mg (19%) the α amanitins 1.2HDP of collection 12.8-13.4min retention times
The synthesis of 30.0134 6'- (6- Aminohexyls)-α-amanitin HDP 30.0134
6'- (6- Aminohexyls)-α-amanitin (HDP 30.0132,81.82mg, 73.17 μm of ol) is dissolved in 300 μ l tri-
Fluoroacetic acid (TFA).At room temperature reaction mixture is stirred in argon gas.The acid is removed in vacuum after 2min at 20 DEG C, and passes through
Remaining TFA is removed with the coevaporation of 2 × 3ml dry toluenes.Residue is dissolved in 95 of 3000 μ l containing 0.05%TFA：5 water/
In methanol, and purified with 500 μ l every time by the preparation HPLC on C18 columns.Solvent orange 2 A：Water (contains 0.05%TFA), solvent
B：Methanol (contains 0.05%TFA), gradient：0min 5%B；5min 5%B, 20min 100%B；25min 100%B；27min
5%B, 35min 5%B；Flow velocity 30ml/min.Collect 13.4-13.9min retention times fraction and by evaporation of the solvent extremely
175.52mg (89%) HDP 30.0134, is colorless solid.
HR-MS(ESI+)1018.46749calc.1018.46679for C45H68N11O14S[M+H]+
1.3HDP 30.0643 6 '-(6- (succinimidyloxycarbonyl)-Aminohexyl-α-amanitin HDP30.0643
Synthesis
HDP 30.0134 (160.52mg, 141.78 μm of ol) is dissolved in 1000 μ l anhydrous dimethyl formamides (DMF),
Bis- succinimdyl carbonates (DSC) of 63.19mg (10eq.) N, N'- are once added in 4000 μ anhydrous DMFs, then add 39.3
μ l (2 equivalent) triethylamine, stirs mixture at room temperature.After 30 minutes, by reaction mixture equal portions be added dropwise to two added with
The centrifuge tube of the ice-cold MTBE of 40ml.Centrifugal sediment, and by being resuspended to 40ml MTBE washings.Precipitation is dried in vacuo, it is molten
Solve and merge in 95% methanol of the 2400 μ l containing 0.05%TFA, it is pure by the progress of C18 columns preparation HPLC with 400 μ l every time
Change (RP-18,10 μm of 250x21.2mm, Luna,) solvent orange 2 As：Water (contains 0.05%TFA), solvent B：Methanol (contains
0.05%TFA), gradient：0min 5%B；5min 5%B, 20min 100%B；25min 100%B；27min 5%B,
35min 5%B；Flow velocity 30ml/min collects the fraction of 15.4-16.5 retention times and by evaporation of the solvent to 151.46mg
(92%) HDP 30.0643, are white powder.
MS(ESI+)1159.1[M+H]+,1181.0[M+Na]+
The synthesis of 1.4 cyclic carbonate derivative HDP 30.1165
HDP 30.0134 is dissolved in 500 μ l anhydrous dimethyl formamides (DMF), and 452 μ l are once added in anhydrous DMF
Bis- succinimdyl carbonate solution (DSC) of the N of (10 equivalent) 0.2M, N'-.Add triethylamine (2.51 μ l, 2 equivalents), room temperature
Lower stirring mixture 90min.Then, volatile matter is removed in vacuum under 40 DEG C of bath temperatures.Residue is dissolved in 500 μ l containing 0.05%
95% methanol of TFA, and purified by preparation HPLC on a cl 8 column (RP-18,10 μm of 250x21.2mm, Luna,) solvent orange 2 As：Water (contains 0.05%TFA), solvent B：Methanol (contains 0.05%TFA), gradient：0min 5%B；5min 5%
B, 20min 100%B；25min 100%B；27min 5%B, 35min 5%B；Flow velocity 30ml/min.Collect 15.4-16.5
The fraction of retention time, evaporates solvent, and residue is freezed as 9.06 milligrams of (85%) HDP 30.1065 from the tert-butyl alcohol of 2ml, is
White powder.
MS(ESI+)1185.10[M+H]+,1207.07[M+Na]+
The synthesis of 1.5 6'-N-BOC- (6- Aminohexyls)-S- deoxidation α amanitins HDP 30.0741
Added into solution of the HDP 30.0132 (18.64mg, 16.67 μm of ol) in 2ml ethanol molybdenum six carbonyl (51mg,
11.6 equivalents), in seal pipe by 75 DEG C of mixture heating 25 it is small when.Then, volatile matter is removed in a vacuum, and by residue
It is dissolved in 2ml methanol.Insoluble substance is removed by centrifugation and by supernatant concentration to 500 μ l, passes through the preparation HPLC on C18 columns
Purified (RP-18,10 μm of 250x21.2mm, Luna,).Solvent orange 2 A：Water, solvent B：Methanol (contains 0.05%TFA),
Gradient：0min 5%B；5min5%B, 20min 100%B；25min 100%B；27min 5%B, 35min 5%B；Flow velocity
30ml/min.Collect the fraction of 17.9-18.6 retention times and by evaporation of the solvent to 10.95mg (89%) HDP 30.0134, be
Colorless solid.
MS(ESI+)1102.3[M+H]+,1124.5[M+Na]+
The synthesis of 1.6 6'- (6- Aminohexyls)-S- deoxidation α amanitins HDP 30.0743
HDP 30.0741 (10.95mg, 9.93 μm of ol) is dissolved in 500 μ l TFA and in incubation at room temperature 2min.Then in vacuum
Middle evaporating volatile substances, residual TFA is removed by being co-evaporated with 2x 1ml dry toluenes.12.38mg crude products are without further pure
Change is used for next step.
MS(ESI+)1002.4[M+H]+,1024.3[M+Na]+
1.7 6'- (the synthesis of 6- (succinimidyloxycarbonyl)-Aminohexyl-S deoxidation α amanitins HDP 30.1033
HDP 30.0743 (12.38mg, 10.51 μm of ol) is dissolved in 500 μ l anhydrous dimethyl formamides (DMF), and once adds
Enter solution of bis- succinimdyl carbonate (DSC) of 525 μ l (10 equivalent) 0.2N N, N'- in anhydrous DMF, then add
2.91 μ l (2 equivalent) triethylamine, stirs mixture at room temperature.After 30 minutes, reaction mixture equal portions are added dropwise to two and are added
There is the centrifuge tube of the ice-cold MTBE of 10ml.Centrifugal sediment, and by being resuspended to 10ml MTBE washings.Precipitation is dried in vacuo,
It is dissolved in 95% methanol of the 500 μ l containing 0.05%TFA, purified by the preparation HPLC on C18 columns (250x21.2mm,
Luna RP-18,10μm,).Solvent orange 2 A：Water (contains 0.05%TFA), solvent B：Methanol (contains 0.05%TFA), gradient：
0min 5%B；5min 5%B, 20min 100%B；25min 100%B；27min 5%B, 35min5%B；Flow velocity 30ml/
min.The fraction of 16.0-17.2min retention times is collected, evaporates solvent, and residue is freezed from the tert-butyl alcohol of 3ml and is
10.93mg (91%) HDP 30.1033, is white powder.
MS(ESI+)1143.3[M+H]+
The synthesis of 1.8 cyclic carbonate derivative HDP 30.1036
Thick HDP30.0743 (12.38mg, 9.93 μm of ol) is dissolved in 500 μ l anhydrous dimethyl formamides (DMF), and once adds
Enter in solution of the bis- succinimdyl carbonate solution (DSC) of 0.2N N, N'- of 497 μ l (10 equivalent) in anhydrous DMF.With
Triethylamine (2.75 μ l, 2 equivalents) is added afterwards, stirs mixture 90min at room temperature.Then, removed under 40 DEG C of bath temperatures in vacuum
Volatile matter.Residue is dissolved in 95% methanol of the 500 μ l containing 0.05%TFA, and is carried out by preparation HPLC on a cl 8 column
Purifying (RP-18,10 μm of 250x21.2mm, Luna,).Solvent orange 2 A：Water (contains 0.05%TFA), solvent B：Methanol (contains
0.05%TFA), gradient：0min 5%B；5min 5%B, 20min 100%B；25min 100%B；27min 5%B,
35min 5%B；Flow velocity 30ml/min.The fraction of 15.9-17.2 retention times is collected, evaporates solvent, and by residue from 3ml
The tert-butyl alcohol freeze as 10.06mg (87%) HDP 30.1036, be white powder.
MS(ESI+)1191.08[M+Na]+
Synthesis-Herceptin of 2 amanita hemolysin antibody conjugates of embodiment and preactivated amanita hemolysin-NHS- amino
Formic acid esters it is conjugated
1.1 Herceptins 30.0643
1.15mg pre-activate amanita hemolysin-linker derivative HDP 30.0643 is dissolved in 230 μ l anhydrous dimethyl sulphoxides
(DMSO), and phosphate buffer (PBS, pH value=7.4) solution of the antibody of 3.33ml 6mg/ml is added to.It is molten by what is obtained
Liquid separates (PD-10 columns in 4 DEG C of shaken over night, and by the gel filtration of cross-link dextran G-25；GE Healthcare Life
Sciences).PD-10 columns are pre-washed with the PBS solution of 6 × 5ml pH 7.4.Conjugate level is detected by Bradford solution
Point, and conjugate fraction is merged.Then, the PBS Slide-A-Lyzer cassette by the solution to 1L pH 7.4
(Thermo Scientific；0.5-3ml；20.000 molecular cut offs) 4 DEG C of dialysed overnights of dialysis cassette.Pass through RotiQuant-
Assay(Carl Roth；Germany protein concentration) is measured.With the Amicon ultracentrifugation filter (micropores of 50'000MWCO；
4000rpm is centrifuged) concentration ADC to 3mg/ml.Toltrazuril is determined by the UV absorption for measuring A=280nm and A=310 nanometers
The payload of the amanita hemolysin of monoclonal antibody.
1.2 Herceptins -30.1033
2.00mg pre-activate amanita hemolysin-linker derivative HDP 30.1033 is dissolved in 400 μ l anhydrous dimethyl sulphoxides
(DMSO), and phosphate buffer (PBS, pH value=7.4) solution of the antibody of 2.62ml 10mg/ml is added to.It is molten by what is obtained
Liquid separates (PD-10 columns in 4 DEG C of shaken over night, and by the gel filtration of cross-link dextran G-25；GE Healthcare Life
Sciences).PD-10 columns are pre-washed with the PBS solution of 6 × 5ml pH 7.4.Conjugate level is detected by Bradford solution
Point, and conjugate fraction is merged.Then the PBS Slide-A-Lyzer cassette by the solution to 1L pH 7.4
(Thermo Scientific；0.5-3ml；20.000 molecular cut offs) 4 DEG C of dialysed overnights of dialysis cassette.Pass through RotiQuant-
Assay(Carl Roth；Germany protein concentration) is measured.With the Amicon ultracentrifugation filter (micropores of 50'000MWCO；
4000rpm is centrifuged) concentration ADC to 3mg/ml.Toltrazuril is determined by the UV absorption for measuring A=280nm and A=310 nanometers
The payload of the amanita hemolysin of monoclonal antibody.
1.3 Herceptins -30.1036
Pre-activate amanita hemolysin-linker derivative HDP 30.1036 is dissolved in 400 μ l anhydrous dimethyl sulphoxides (DMSO), and
Add to phosphate buffer (PBS, pH value=7.4) solution of the antibody of 2.57ml 10mg/ml.Obtained solution is shaken at 4 DEG C
It is dynamic to stay overnight, and the gel filtration for passing through cross-link dextran G-25 separates (PD-10 columns；GE Healthcare Life
Sciences).PD-10 columns are pre-washed with the PBS solution of 6 × 5ml pH 7.4.Conjugate level is detected by Bradford solution
Point, and conjugate fraction is merged.Then the PBS Slide-A-Lyzer cassette by the solution to 1L pH 7.4
(Thermo Scientific；0.5-3ml；20.000 molecular cut offs) 4 DEG C of dialysed overnights of dialysis cassette.Pass through RotiQuant-
Assay(Carl Roth；Germany protein concentration) is measured.With the Amicon ultracentrifugation filter (micropores of 50'000MWCO；
4000rpm is centrifuged) concentration ADC to 3mg/ml.Toltrazuril is determined by the UV absorption for measuring A=280nm and A=310 nanometers
The payload of the amanita hemolysin of monoclonal antibody.
1.4 Herceptins -30.1165
Pre-activate 0.90mg amanita hemolysins-linker derivative HDP 30.1165 is dissolved in 180 μ l anhydrous dimethyl sulphoxides
(DMSO), and add to the antibody of 2.00ml 5mg/ml in phosphate buffer (PBS, pH value=7.4) solution.By what is obtained
Solution separates (PD-10 columns in 4 DEG C of shaken over night, and by the gel filtration of cross-link dextran G-25；GE Healthcare
Life Sciences).PD-10 columns are pre-washed with the PBS solution of 6 × 5ml pH 7.4.Detected and be conjugated by Bradford solution
Thing fraction, and conjugate fraction is merged.Then the PBS Slide-A-Lyzer by the solution to 1L pH 7.4
cassette(Thermo Scientific；0.5-3ml；20.000 molecular cut offs) 4 DEG C of dialysed overnights of dialysis cassette.Pass through
RotiQuant-Assay(Carl Roth；Germany protein concentration) is measured.With the Amicon ultracentrifugation filters of 50'000MWCO
(micropore；4000rpm is centrifuged) concentration ADC to 3mg/ml.Determined by the UV absorption for measuring A=280nm and A=310 nanometers
The payload of the amanita hemolysin of Herceptin.
Embodiment 3Her-30.0643 [4.4], Her-30.1033 [3.9], Her-30.1036 [4.0] and
Her-
Cytotoxicity of 30.1165 [5.0] to external HER2 positive tumor cells system
With chemiluminescence BrdU immunofluorescence techniques (Roche Diagnostics), HER2 positive tumor cells in vitro
It is SK-OV-3 (oophoroma), Herceptin amanita hemolysin conjugation is assessed in SKBR-3 (breast cancer) and JIMT-1 (breast cancer)
The cytotoxicity of thing.In 37 DEG C and 5%CO2, after being incubated the conjugate 72h of various concentrations, in BMG Labtech Optima enzymes
Mark in instrument, the cell being fixed and permeated with anti-BrdU-HRP antibody tests determines cytoactive.With Graphpad Prism
4.0 softwares calculate the EC of dose-response curve50Value.
The EC of Herceptin-amanita hemolysin conjugate in different Her2 positive cell lines50Value is (referring also to Fig. 2 to 5)：
| Conjugate | SKOV-3 | SKBR-3 | JIMT-1 |
| Her-30.1036[4.0] | 3.8x10-11M | 3.3x10-11M | 2.1x10-9M |
| Her-30.0643[4.4] | 2.9x10-11M | 2.0x10-11M | 1.0x10-9M |
| Her-30.1165[5.0] | 1.1x10-10M | - | - |
| Her-30.1033[3.9] | 2.7x10-11M | 1.3x10-11M | 7.9x10-9M |
Her-30.0643 [4.4] and Her- in the JIMT-1 heteroplastic transplantation models of 4 Herceptin of embodiment tolerance
The internal effect of 30.1036 [4.0]
Buy six week old completely female NMRI NU/NU nude mices (Janvier), and be randomly divided into three groups, every group 8.
5x 10 is subcutaneously injected in the side of each mouse6JIMT-1 cells.After tumor inoculation 14 days, according to dosage 30 μ g/kg and 150 μ g/kg are quiet
Arteries and veins injection Herceptin amanita hemolysin conjugate Her-30.0643 [4.4] and Her-30.1036 [4.0], and give negative control
Injection carrier (PBS buffer).Record gross tumor volume (referring to Fig. 6).Two kinds of conjugates show high antitumor activity, cause to swell
Knurl is completely reduced into 30 μ g and 150 μ g/kg.
The tolerance of Her-30.0643 [4.4] and Her-30.1036 [4.0] in 5 mouse of embodiment
Seven week old completely female NMRI NU/NU nude mices (Janvier) are bought, are randomly divided into three groups, every group 3.Once
It is injected intravenously the Herceptin amanita hemolysin conjugate Her-30.0643 [4.4] and Her-30.1036 that dosage is 300 μ g/kg
[4.0], negative control injection carrier (PBS buffer) is given.Record the weight of mouse (referring to Fig. 7).Use Her-30.0643
[4.4] all animals for the treatment of are dead in 9 days, and are shown afterwards within 14 days with all animals of Her-30.1036 [4.0] treatments
Go out and the weight similar with negative control group.
Claims (25)
1. the amanita hemolysin of Formulas I
Wherein：
R1Selected from C=O, C=S, C=NR6And CR7R8；
R2Selected from S=O, SO2And S；
R3Selected from NHR5And OR5；
R4Selected from H, OR5, and OC1-6- alkyl；
R6Selected from C1-6- alkylidene-R5, cycloalkylidene-R5, miscellaneous cycloalkylidene-R5, arlydene-R5, and heteroarylidene-R5；
R7And R8Independently selected from H, C1-6- alkylidene-R5, cycloalkylidene-R5, miscellaneous cycloalkylidene-R5, arlydene-R5, and miscellaneous Asia
Aryl-R5；
Wherein：
(i) each R5It is H；
(ii)R5In one be-Ln- X, wherein L are connectors, and n is selected from 0 and 1, X are the chemical groups that can be connected with targeting group,
And remaining R5For H；Or
(iii)R5In one be-Ln-X*-Y, wherein L is connector, n be selected from 0 and 1, Y be targeting group, X* is the function of X and Y
The chemical part that group's connection produces, and remaining R5It is H.
2. the amanita hemolysin described in claim 1 (iii), wherein the functional group of the Y is amino.
3. the amanita hemolysin described in claim 2, wherein X* are urea groups.
4. claim 1 (iii), the amanita hemolysin described in any one of 2 and 3, wherein the residue R5For-Ln- X*-Y,
Its:
(v) it is present in R1；
(vi) it is present in R3；
(vii) it is present in R4；Or
(viii) it is connected to the nitrogen-atoms of tryptophan.
5. claim 1 (ii), 1 (iii) and 2-4 any one of them amanita hemolysins, wherein n are 1, and wherein described connector
Up to 12 atoms.
6. claim 1 (ii), 1 (iii) neutralizes 2-5 any one of them amanita hemolysins, wherein the connector L is alkylidene,
Miscellaneous alkylidene, alkenylene, miscellaneous alkenylene, alkynylene, miscellaneous alkynylene, cycloalkylidene, miscellaneous cycloalkylidene, arlydene, heteroarylidene,
Sub- aralkyl, or miscellaneous sub- aralkyl, including 1 to 4 hetero atom for being selected from N, O and S, wherein the connector is optionally substituted.
7. claim 1 ((ii), 1 (iii) and any amanita hemolysins of 2-6, wherein the connector L is included with lower part
One of:Disulfide, ether, thioether, amine, ester, carboxylic acid amides, carbamate and urea part.
8. -7 any one of them amanita hemolysin of claim 1 (iii), wherein target conjugated group specific binding is present in
Epitope on tumour cell.
9. any amanita hemolysin of claim 1 (iii) -8, wherein the target conjugated group is selected from:
(i) antibody or its antigen-binding fragment；
(ii) antibody-like protein；With
(iii) aptamer
10. the amanita hemolysin described in claim 9, wherein the antibody or antigen-binding fragment are selected from double antibody, four antibody, receive
Meter Kang Ti, chimeric antibody, goes immune antiboidy, humanized antibody or human antibody.
11. the amanita hemolysin described in claim 9 or 10, wherein the antigen-binding fragment is selected from Fab, F (ab') 2, Fd, Fv,
ScFv, the Fvs (dsFv) of disulfide bond connection.
12. -11 any one of them amanita hemolysin of claim 1 (iii), as medicine.
13. -11 any one of them amanita hemolysin of claim 1 (iii), for treating the cancer of patient.
14. a kind of pharmaceutical composition, including -11 any one of them amanita hemolysin of claim 1 (iii), and further include one
Kind or a variety of pharmaceutically acceptable diluents, carrier, excipient, filler, adhesive, lubricant, glidant, disintegrant,
Adsorbent；And/or preservative.
15. the amanita hemolysin described in claim 1 (ii), wherein X be carbamic acid derivative-NH-C (O)-Z, wherein Z be from
Group is removed, the leaving group can be replaced by the nucleophilic group of target conjugated group.
16. the amanita hemolysin described in claim 15, wherein Z are selected from-tertbutyloxycarbonyl,-succinimide epoxide, -1-O- ambers
Amber imide epoxide -3- sulfonic acid (sulfo group-NHS),-O- (4-nitrophenoxy),-O- (3- nitro-phenoxies),-O- (2,4-
2,4-dinitrophenoxy) ,-O- (2,4- bis- chloro- 6- nitro-phenoxies) ,-phenyl-pentafluoride ,-pentachlorobenzene oxygen ,-O- (2,4,5- chlorobenzene) ,-O-
(3,4- dihydro-3-hydroxy -4- oxos -1,2,3- phentriazine -3- bases),-O- (interior -1- hydroxyls -5- norbornene -2,3- bis-
Carboximide -1- bases), -1- O-phthalic acyloxy, -1- benzotriazole epoxides, -1- (7- azabenzotriazols) epoxide),
With-TMSIM N imidazole base.
17. the amanita hemolysin described in claim 5, wherein the connector grows 2 to 10 atoms.
18. the amanita hemolysin described in claim 5, wherein the connector grows 4 to 9 atoms.
19. the amanita hemolysin described in claim 5, wherein the connector grows 6 to 8 atoms.
20. the amanita hemolysin described in claim 8, wherein the target conjugated group specifically binds human epidermal growth factor acceptor
The epitope of 2 (HER2).
21. the amanita hemolysin described in claim 13, wherein the cancer is selected from breast cancer, cancer of pancreas, cholangiocarcinoma, colorectum
Cancer, lung cancer, prostate cancer, oophoroma, stomach cancer, kidney, malignant mela noma, leukaemia and malignant lymphoma.
22. the amanita hemolysin described in claim 15, wherein Z are leaving groups, the leaving group can be by target conjugated group
Primary amine replaces.
23. the synthetic method of the amanita hemolysin described in claim 15,16 or 22, comprises the following steps：By the goose cream poison of formula II
Peptide
Wherein：
R2Selected from S=O, SO2And S；
R3Selected from NHR5And OR5；
R4Selected from H, OR5, and OC1-6- alkyl；
R6Selected from C1-6- alkylidene-R5, cycloalkylidene-R5, miscellaneous cycloalkylidene-R5, arlydene-R5, and heteroarylidene-R5；
R7And R8Independently selected from H, C1-6- alkylidene-R5, cycloalkylidene-R5, miscellaneous cycloalkylidene-R5, arlydene-R5, and miscellaneous Asia
Aryl-R5；
Wherein R5One of be H, R5In one be-Ln- X, wherein L are connectors, and n is that can be connected with targeting group selected from 0 and 1, X
Chemical group, and remaining R5For H；Or R5In one be-Ln-X*-Y, wherein L is connector, n be selected from 0 and 1, Y be
Group is targeted, X* is that the functional group of X and Y connects the chemical group produced, and remaining R5It is H；
With following reaction：(i) bis- succinimdyl carbonate of N, N'- (DSC), (ii) thion reagent；(iii) amino carbonyl tries
Agent；Or (iv) aldehyde, ketone or acyclic acetals.
24. the synthetic method of the amanita hemolysin described in claim 23, wherein the thion reagent is sulphur, 1,1'- thiocarbonyl groups
Diimidazole or 1,1'- thiocarbonyls -2 (1H)-pyridone.
25. the synthetic method of the amanita hemolysin described in claim 23, wherein the amino carbonyl reagent is the dichloride of isocyanides
Or isothiocyanic acid benzene.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/EP2014/000614 WO2014135282A1 (en) | 2013-03-04 | 2014-03-10 | Amatoxin derivatives |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105377304A CN105377304A (en) | 2016-03-02 |
| CN105377304B true CN105377304B (en) | 2018-05-15 |
Family
ID=55400962
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201480012523.6A Active CN105377304B (en) | 2014-03-10 | 2014-03-10 | Amanita hemolysin derivative |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JP6321687B2 (en) |
| CN (1) | CN105377304B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3222292A1 (en) * | 2016-03-03 | 2017-09-27 | Heidelberg Pharma GmbH | Amanitin conjugates |
| JP7062647B2 (en) * | 2016-06-17 | 2022-05-06 | マジェンタ セラピューティクス インコーポレイテッド | Compositions and Methods for Depleting CD117 + Cells |
| WO2017219025A1 (en) * | 2016-06-17 | 2017-12-21 | Magenta Therapeutics, Inc. | Compositions and methods for the depletion of cells |
| US11446388B2 (en) * | 2016-12-23 | 2022-09-20 | Heidelberg Pharma Research Gmbh | Amanitin antibody conjugates |
| CN109125736B (en) * | 2017-08-18 | 2022-01-04 | 四川百利药业有限责任公司 | Non-natural amatoxin antibody conjugate |
| CA3082166A1 (en) * | 2017-11-29 | 2019-06-06 | Magenta Therapeutics, Inc. | Compositions and methods for the depletion of cd2+ cells |
| JP2021517133A (en) * | 2018-03-08 | 2021-07-15 | マジェンタ セラピューティクス インコーポレイテッドＭａｇｅｎｔａ Ｔｈｅｒａｐｅｕｔｉｃｓ， Ｉｎｃ． | Anti-CD252 antibody, conjugate, and usage |
| CN109796537A (en) * | 2019-02-28 | 2019-05-24 | 中国农业大学 | A kind of β-amanita hemolysin artificial antigen and the preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010115629A2 (en) * | 2009-04-08 | 2010-10-14 | Deutsches Krebsforschungszentrum | Amatoxin-armed therapeutic cell surface binding components designed for tumour therapy |
| WO2012041504A1 (en) * | 2010-09-30 | 2012-04-05 | Heidelberg Pharma Gmbh | Amatoxin-conjugates with improved linkers |
| WO2012119787A1 (en) * | 2011-03-10 | 2012-09-13 | Heidelberg Pharma Gmbh | Amatoxin-conjugates with improved linkages |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2649114A1 (en) * | 2006-04-12 | 2007-10-25 | Crosslink Genetics Corporation | Compositions and methods for modulating gene expression |
| EP2416805B1 (en) * | 2009-04-08 | 2013-07-24 | Heinz Dr. Faulstich | Amatoxin antibody conjugates for the treatment of cancer |
-
2014
- 2014-03-10 JP JP2015560586A patent/JP6321687B2/en active Active
- 2014-03-10 CN CN201480012523.6A patent/CN105377304B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010115629A2 (en) * | 2009-04-08 | 2010-10-14 | Deutsches Krebsforschungszentrum | Amatoxin-armed therapeutic cell surface binding components designed for tumour therapy |
| WO2012041504A1 (en) * | 2010-09-30 | 2012-04-05 | Heidelberg Pharma Gmbh | Amatoxin-conjugates with improved linkers |
| WO2012119787A1 (en) * | 2011-03-10 | 2012-09-13 | Heidelberg Pharma Gmbh | Amatoxin-conjugates with improved linkages |
Also Published As
| Publication number | Publication date |
|---|---|
| JP6321687B2 (en) | 2018-05-09 |
| JP2016516010A (en) | 2016-06-02 |
| CN105377304A (en) | 2016-03-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105377304B (en) | Amanita hemolysin derivative | |
| EP2964264B1 (en) | Amatoxin derivatives | |
| EP2416805B1 (en) | Amatoxin antibody conjugates for the treatment of cancer | |
| US20180185509A1 (en) | Amatoxin-conjugates with improved linkages | |
| CN107636016A (en) | Amatoxin anti-body conjugates | |
| CN103153345B (en) | There is the amatoxin conjugates of the connector of improvement | |
| CN110099698A (en) | Amanitin antibody conjugates | |
| CN106029083A (en) | Hydrophilic antibody-drug conjugates | |
| CN102596922A (en) | Potent conjugates and hydrophilic linkers | |
| CN107198778A (en) | With the cell binding agent maytansinoid conjugate connected through not cleavable joint | |
| RU2575854C2 (en) | Amatoxin conjugates with improved bonds | |
| NZ614548B2 (en) | Amatoxin-conjugates with improved linkages |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |