U.S. patent application number 17/415759 was filed with the patent office on 2022-07-14 for drug conjugate.
This patent application is currently assigned to Sapreme Technologies B.V.. The applicant listed for this patent is Charite - Universitatsmedizin Berlin, Sapreme Technologies B.V.. Invention is credited to Hendrik Fuchs, Ruben Postel.
Application Number | 20220218837 17/415759 |
Document ID | / |
Family ID | 1000006304673 |
Filed Date | 2022-07-14 |
United States Patent
Application |
20220218837 |
Kind Code |
A1 |
Postel; Ruben ; et
al. |
July 14, 2022 |
DRUG CONJUGATE
Abstract
The invention relates to antibody-drug conjugates (ADC) that are
potentiated by co-administration of the ADC with a moiety
comprising covalently linked saponin. The invention also relates to
antibody-oligonucleotide conjugates (AOC) that are potentiated by
co-administration of the AOC with a moiety comprising covalently
linked saponin. The invention also relates to ADCs and AOCs which
are conjugated with a saponin via a covalent linker. The invention
further relates to an effector moiety such as a toxin or an
antisense oligonucleotide such as for example a BNA, conjugated
with a saponin via a covalent linkage. The invention also relates
to a BNA covalently conjugated with a targeting moiety such as an
antibody. The invention also relates to therapeutic combinations
comprising a first pharmaceutical composition comprising a
conjugate of a cell-targeting moiety such as an antibody and an
antisense oligonucleotide such as a BNA covalently bound thereto,
and comprising a second pharmaceutical composition comprising
either a free saponin, or a conjugate of a cell-targeting moiety
such as an antibody with a saponin covalently linked thereto.
Furthermore, the invention relates to any of these conjugates or
therapeutic compositions or therapeutic combinations, for use as a
medicament. The invention also relates to any of these conjugates
or therapeutic compositions or therapeutic combinations, for use in
a method for the treatment or the prophylaxis of a cancer. Finally,
the invention relates to methods for producing any of these
conjugates of the invention.
Inventors: |
Postel; Ruben; (Utrecht,
NL) ; Fuchs; Hendrik; (Berlin, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sapreme Technologies B.V.
Charite - Universitatsmedizin Berlin |
Bilthoven
Berlin |
|
NL
DE |
|
|
Assignee: |
Sapreme Technologies B.V.
Bilthoven
NL
Charite - Universitatsmedizin Berlin
Berlin
DE
|
Family ID: |
1000006304673 |
Appl. No.: |
17/415759 |
Filed: |
December 9, 2019 |
PCT Filed: |
December 9, 2019 |
PCT NO: |
PCT/EP2019/084210 |
371 Date: |
June 18, 2021 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/6415 20170801;
A61K 47/6885 20170801; A61P 35/00 20180101; A61K 47/6825 20170801;
A61K 47/6849 20170801; A61K 47/6857 20170801; A61K 47/6855
20170801; A61K 47/6807 20170801; A61K 47/549 20170801; A61K 47/60
20170801; A61K 47/6889 20170801; A61K 47/642 20170801; A61K 47/6845
20170801 |
International
Class: |
A61K 47/68 20060101
A61K047/68; A61K 47/64 20060101 A61K047/64; A61K 47/60 20060101
A61K047/60; A61K 47/54 20060101 A61K047/54; A61P 35/00 20060101
A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 21, 2018 |
NL |
2022283 |
Jul 10, 2019 |
NL |
2023468 |
Jul 25, 2019 |
NL |
2023568 |
Claims
1. Therapeutic molecule with chemical structure of COMPOUND I:
A1.sub.m((-L9.sub.w)((-L1.sub.q-B1.sub.n).sub.u((-L2.sub.r-L3.sub.s)(-L4.-
sub.v-C).sub.p).sub.t)).sub.x (compound I), wherein A1 is a first
ligand if B1 is a first effector moiety, or A1 is the first
effector moiety if B1 is the first ligand; C is a saponin; m=0 or 1
if A1 is the first ligand and B1 is the first effector moiety;
m=0-32 if A1 is the first effector moiety and B1 is the first
ligand; n=0 or 1 if B1 is the first ligand and A1 is the first
effector moiety, or if A1 is the first ligand and B1 is the first
effector moiety; p=any of 1-128; L1 is at least one linker for
covalently coupling two chemical groups; L2 is at least one linker
for covalently coupling two chemical groups; L3 is at least one
oligomeric or polymeric scaffold for covalently coupling two
chemical groups; L4 is at least one linker for covalently coupling
two chemical groups; L9 is a tri-functional linker for covalently
coupling three chemical groups; q=0 or 1; r=0 or 1; s=0 or 1; t=0,
1 or 2 if s=0, and t=any of 0-16 if s=1; u=any of 0-32 if A1 is the
first ligand and B1 is the first effector moiety, or u=1 if A1 is
the first effector moiety and B1 is the first ligand; v=0 or 1; w=1
or 0; and x=1-16.
2.-3. (canceled)
4. Therapeutic molecule of claim 1, wherein the first ligand A1 or
B1 comprises or consists of an immunoglobulin, a binding domain of
an immunoglobulin or a binding fragment of an immunoglobulin, such
as an antibody, an IgG, a molecule comprising or consisting of a
Vhh domain or Vh domain, a Fab, an scFv, an Fv, a dAb, an
F(ab).sub.2, Fcab fragment, or comprise(s) or consist(s) of at
least one non-proteinaceous ligand and/or at least one
proteinaceous ligand, the ligand for binding to a cell-surface
molecule such as EGF or a cytokine, with the proviso that the first
ligand and the second ligand are the same or are different.
5. Therapeutic molecule of claim 1, wherein the first ligand A1 or
B1 binds to a tumor-cell epitope, preferably a tumor-cell specific
epitope, of a tumor-cell receptor, preferably a tumor-cell specific
receptor, preferably selected from CD71, CA125, EpCAM(17-1A), CD52,
CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin
alpha-V beta-3, HER2, EGFR, CD20, CD22, Folate receptor 1, CD146,
CD56, CD19, CD138, CD27L receptor, PSMA, CanAg, integrin-alphaV,
CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239, CD70, CD123,
CD352, DLL3, CD25, ephrinA4, MUC1, Trop2, CEACAM5, CEACAM6, HER3,
CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1,
CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2, more preferably selected from
CD71, EGFR and HER2, with the proviso that the first ligand and the
second ligand bind to the same or to a different tumor-cell
epitope, preferably a tumor-cell specific epitope, and/or wherein
the tumor-cell receptor, preferably the tumor-cell specific
receptor, to which the first ligand can bind is the same as, or is
different from the tumor-cell receptor, preferably the tumor-cell
specific receptor, to which the second ligand can bind.
6. Therapeutic molecule of claim 1, wherein the first ligand A1 or
B1 comprises or consists of cetuximab, daratumumab, gemtuzumab,
trastuzumab, panitumumab, brentuximab, inotuzumab, moxetumomab,
polatuzumab, obinutuzumab, OKT-9 anti-CD71 monoclonal antibody of
the IgG type, pertuzumab, rituximab, ofatumumab, Herceptin,
alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal antibody, an
antibody of Table A2 or Table A3 or Table A4, preferably cetuximab
or trastuzumab or OKT-9, or at least one tumor-cell receptor
binding-domain thereof and/or at least one tumor-cell receptor
binding-fragment thereof which are preferably (a) tumor-cell
specific receptor binding-domain(s) and/or (a) tumor-cell specific
receptor binding-fragment(s), with the proviso that the first
ligand is the same or different from the second ligand.
7.-8. (canceled)
9. Therapeutic molecule of claim 1, wherein the first effector
moiety A1 or B1 comprises or consist of at least one of any one or
more of an oligonucleotide, a nucleic acid and a xeno nucleic acid,
preferably selected from any one or more of a vector, a gene, a
cell suicide inducing transgene, deoxyribonucleic acid (DNA),
ribonucleic acid (RNA), anti-sense oligonucleotide (ASO, AON),
short interfering RNA (siRNA), microRNA (miRNA), DNA aptamer, RNA
aptamer, mRNA, mini-circle DNA, peptide nucleic acid (PNA),
phosphoramidate morpholino oligomer (PMO), locked nucleic acid
(LNA), bridged nucleic acid (BNA), 2'-deoxy-2'-fluoroarabino
nucleic acid (FANA), 2'-O-methoxyethyl-RNA (MOE),
2'-0,4'-aminoethylene bridged nucleic acid, 3'-fluoro hexitol
nucleic acid (FHNA), a plasmid, glycol nucleic acid (GNA) and
threose nucleic acid (TNA), or a derivative thereof, more
preferably a BNA, for example a BNA for silencing HSP27 protein
expression, with the proviso that the first effector moiety and the
second effector moiety are the same or are different.
10. Therapeutic molecule of claim 1, wherein the first effector
moiety A1 or B1 comprises or consists of at least one proteinaceous
molecule, preferably selected from any one or more of a peptide, a
protein, an enzyme such as urease and Cre-recombinase, a
proteinaceous toxin, a ribosome-inactivating protein, at least one
protein toxin selected from Table A5 and/or a bacterial toxin, a
plant toxin, more preferably selected from any one or more of a
viral toxin such as apoptin; a bacterial toxin such as Shiga toxin,
Shiga-like toxin, Pseudomonas aeruginosa exotoxin (PE) or exotoxin
A of PE, full-length or truncated diphtheria toxin (DT), cholera
toxin; a fungal toxin such as alpha-sarcin; a plant toxin including
ribosome-inactivating proteins and the A chain of type 2
ribosome-inactivating proteins such as dianthin e.g. dianthin-30 or
dianthin-32, saporin e.g. saporin-S3 or saporin-S6, bouganin or
de-immunized derivative debouganin of bouganin, shiga-like toxin A,
pokeweed antiviral protein, ricin, ricin A chain, modeccin,
modeccin A chain, abrin, abrin A chain, volkensin, volkensin A
chain, viscumin, viscumin A chain; or an animal or human toxin such
as frog RNase, or granzyme B or angiogenin from humans, or any
fragment or derivative thereof; preferably the protein toxin is
dianthin and/or saporin, with the proviso that the first effector
moiety/moieties and the second effector moiety/moieties are the
same or are different.
11. (canceled)
12. Therapeutic molecule of claim 1, wherein the therapeutic
molecule comprises or consists of any one of Gemtuzumab ozogamicin,
Brentuximab vedotin, Trastuzumab emtansine, Inotuzumab ozogamicin,
Moxetumomab pasudotox and Polatuzumab vedotin and an antibody-drug
conjugate of Table A2 and Table A3, or at least one tumor-cell
specific receptor binding-domain thereof and/or at least one
tumor-cell specific receptor binding-fragment thereof which are
preferably (a) tumor-cell specific receptor binding-domain(s)
and/or (a) tumor-cell specific receptor binding-fragment(s), with
the proviso that the therapeutic molecule and the second
therapeutic molecule are the same or are different.
13. Therapeutic molecule of claim 1, wherein the saponin C is a
triterpenoid saponin or a bisdesmosidic triterpene saponin,
belonging to the type of a 12,13-dehydrooleanane with an aldehyde
function in position C-23 and optionally comprising a glucuronic
acid function in a carbohydrate substituent at the C-3beta-OH group
of the saponin, and/or a saponin isolated from a Gypsophila species
and/or a Saponaria species and/or an Agrostemma species and/or a
Quillaja species such as Quillaja saponaria- or, wherein the
saponin C is a single specific saponin or is a mixture of two or
more different saponins, such as one or more of the saponins in
Table A1 or Scheme I, SO1861, SA1657, GE1741, SA1641, QS-21,
QS-21A, QS-21 A-api, QS-21 A-xyl, QS-21B, QS-21 B-api, QS-21 B-xyl,
QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS1861, QS1862,
Quillajasaponin, Saponinum album, QS-18, Quil-A, Gyp1, gypsoside A,
AG1, AG2, 501542, 501584, 501658, 501674, 501832, or any of their
stereomers and/or any combinations thereof, preferably the saponin
is SO1861 and/or GE1741 and/or SA1641 and/or QS-21 and/or saponin
with a quillaic acid aglycon core, a
Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA carbohydrate substituent
at the C-3beta-OH group and a
Glc-(1.fwdarw.3)-Xyl-(1.fwdarw.4)-Rha-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)-4-OA-
c-Qui-(1.fwdarw.4)]-Fuc carbohydrate substituent at the C-28-OH
group, and/or is
3-O-beta-D-galactopyranosyl-(1.fwdarw.2)-[beta-D-xylopyranosyl--
(1.fwdarw.3)]-beta-D-glucuronopyranosyl quillaic acid
28-O-beta-D-glucopyranosyl-(1.fwdarw.3)-beta-D-xylopyranosyl-(1.fwdarw.4)-
-alpha-L-rhamnopyranosyl-(1.fwdarw.2)-[beta-D-xylopyranosyl-(1.fwdarw.3)-4-
-OAc-beta-D-quinovopyranosyl-(1.fwdarw.4)]-beta-D-fucopyranoside,
more preferably the saponin is SO1861 and/or QS-21 wherein the
saponin C is a bisdesmosidic saponin having a molecular mass of at
least 1.500 Dalton and comprising an oleanan-type triterpene
containing an aldehyde group at the C-23 position and optionally a
hydroxyl group at the C-16 position, with a first branched
carbohydrate side chain at the C-3 position which first branched
carbohydrate side chain optionally contains glucuronic acid,
wherein the saponin contains an ester group with a second branched
carbohydrate side chain at the C-28 position which second branched
carbohydrate chain preferably comprises at least four carbohydrate
units, optionally containing at least one acetyl residue such as
two acetyl residues and/or at least one deoxy carbohydrates and/or
a quinovose and/or a glucose and/or 4-methoxycinnamic acid and/or
optionally comprising
5-O-[5-O-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl--
octanoic acid and/or optionally comprising
5-O[5-O-Rha-(1.fwdarw.2)-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dih-
ydroxy-6-methyl-octanoic acid bound to a carbohydrate via an ester
bond, or wherein the at least one saponin is QS-21 or any one or
more of QS-21A, QS-21 A-api, QS-21 A-xyl, QS-21B, QS-21 B-api,
QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS-18,
QS1861, protonated QS1861 (QS1862), Quil-A, or wherein the saponin
C is a bisdesmosidic triterpene saponin belonging to the type of a
12,13-dehydrooleanane with an aldehyde function in position C-23,
wherein the saponin C is covalently coupled to an amino-acid
residue of the first ligand A1 or B1 and/or the first effector
moiety B1 or A1 and/or the second ligand A2 or B2 and/or the second
effector moiety B2 or A2 via the aldehyde function in the saponin
C, preferably said aldehyde function in position C-23, preferably
via a linker L2, L4, L6, L8, and/or L9 and/or L10, more preferably
via a cleavable linker L2, L4, L6, L8, and/or L9 and/or L10,
wherein the amino-acid residue preferably is selected from cysteine
and lysine.
14.-19. (canceled)
20. Therapeutic molecule of claim 1, wherein the first ligand A1 or
B1 and/or the first effector moiety B1 or A1 comprise(s) one or
more than one covalently bound saponin C, preferably 2, 3, 4, 5, 6,
8, 10, 16, 32, 64, 128 or 1-100 saponins, or any number of saponins
therein between, such as 7, 9, 12 saponins.
21. Therapeutic molecule of claim 1, wherein the first ligand A1 or
B1 and/or the first effector moiety B1 or A1 comprise(s) one or
more than one covalently bound saponin C, wherein the saponin(s) C
is/are covalently bound directly to an amino-acid residue of the
first ligand A1 or B1 and/or the first effector moiety B1 or A1
when r, s and v are 0, preferably to a cysteine and/or to a lysine,
and/or is/are covalently bound via at least one linker L2, L4
and/or L9, or via at least one cleavable linker L2, L4 and/or L9
and/or via at least one oligomeric or polymeric scaffold L3,
preferably 1-8 of such scaffolds or 2-4 of such scaffolds, wherein
the at least one scaffold is optionally based on a dendron, wherein
1-32 saponins, preferably 2, 3, 4, 5, 6, 8, 10, 16, 32 saponins, or
any number of saponins therein between, such as 7, 9, 12 saponins,
are covalently bound to the at least one scaffold.
22. Therapeutic molecule of claim 1, wherein the saponin C is a
bisdesmosidic triterpene saponin belonging to the type of a
12,13-dehydrooleanane with an aldehyde function in position C-23,
wherein the saponin C is covalently coupled to an amino-acid
residue of the first ligand A1 or B1 and/or the first effector
moiety B1 or A1 via the aldehyde function in the saponin C,
preferably said aldehyde function in position C-23, via a cleavable
linker L2, L4 and/or L9, wherein the amino-acid residue preferably
is selected from cysteine and lysine, and wherein the cleavable
linker L2, L4 and/or L9 is subject to cleavage under acidic
conditions, reductive conditions, enzymatic conditions or
light-induced conditions, and preferably the cleavable linker
comprises a hydrazone bond or a hydrazide bond subject to cleavage
under acidic conditions when bound to saponin, and/or comprises a
bond susceptible to proteolysis, for example proteolysis by
Cathepsin B, when bound to saponin, and/or the cleavable linker
comprises a disulphide bond susceptible to cleavage under reductive
conditions.
23. Therapeutic molecule of claim 1, wherein the saponin C is a
bisdesmosidic triterpene saponin belonging to the type of a
12,13-dehydrooleanane with an aldehyde function in position C-23,
wherein the saponin C is covalently coupled to an amino-acid
residue of the first ligand A1 or B1 and/or the first effector
moiety B1 or A1 via the aldehyde function in the saponin C,
preferably said aldehyde function in position C-23, via a cleavable
linker L2, L4 and/or L9, wherein the amino-acid residue preferably
is selected from cysteine and lysine, and wherein the cleavable
linker L2, L4 and/or L9 is subject to cleavage in vivo under acidic
conditions as present in endosomes and/or lysosomes of mammalian
cells, preferably human cells, preferably at pH 4.0-6.5, and more
preferably at pH.ltoreq.5.5.
24. Therapeutic molecule of claim 1, wherein the polymeric or
oligomeric scaffold L3 comprises a polymeric or oligomeric
structure and comprises a chemical group, the chemical group for
covalently coupling of the polymeric or oligomeric scaffold L3 to
the amino-acid residue of the first ligand and/or the first
effector moiety and/or the second ligand and/or the second effector
moiety.
25.-28. (canceled)
29. Therapeutic molecule of claim 1, wherein the at least one
saponin is covalently bound to the first ligand and/or to the first
effector moiety via at least one linker comprising a tri-functional
linker L9 when j=1, to which tri-functional linker both the first
ligand and the at least one first effector moiety are bound,
preferably the tri-functional linker is the trifunctional linker of
Scheme II.
30. Therapeutic molecule of claim 1, wherein the first ligand A1 or
B1 and/or the first effector moiety B1 or A1 comprise(s) one or
more than one covalently bound saponin C, wherein the saponin(s) C
is/are covalently bound via at least one oligomeric or polymeric
scaffold L3, preferably 1-8 of such scaffolds or 2-4 of such
scaffolds, wherein 1-32 saponins, preferably 2, 3, 4, 5, 6, 8, 10,
16, 32 saponins, or any number of saponins therein between, such as
7, 9, 12 saponins, are covalently bound to the at least one
scaffold, and wherein the polymeric or oligomeric structure of the
scaffold L3 comprises a linear, branched and/or cyclic polymer,
oligomer, dendrimer, dendron, dendronized polymer, dendronized
oligomer, a DNA, a polypeptide, poly-lysine, a poly-ethylene
glycol, or an assembly of these polymeric or oligomeric structures
which assembly is preferably built up by covalent
cross-linking.
31. Therapeutic molecule of claim 1, wherein the first ligand A1 or
B1 and/or the first effector moiety B1 or A1 comprise(s) one or
more than one covalently bound saponin C, wherein the saponin(s) C
is/are covalently bound directly to an amino-acid residue of the
first ligand A1 or B1 and/or the first effector moiety B1 or A1
when r, s and v are 0, preferably to a cysteine and/or to a lysine,
and/or is/are covalently bound via at least one linker L2, L4
and/or L9, or via at least one cleavable linker L2, L4 and/or L9
and/or via at least one oligomeric or polymeric scaffold L3,
preferably 1-8 of such scaffolds or 2-4 of such scaffolds, wherein
the at least one scaffold is optionally based on a dendron, wherein
1-32 saponins, preferably 2, 3, 4, 5, 6, 8, 10, 16, 32 saponins, or
any number of saponins therein between, such as 7, 9, 12 saponins,
are covalently bound to the at least one scaffold, and wherein the
first ligand A1 or B1 is covalently bound to the first effector
moiety B1 or A1, respectively, via at least one linker L1.
32. (canceled)
33. Therapeutic molecule of claim 1, wherein r=0, s=0, v=0, s=0,
v=0, p=0, t=0, ligand A1 is a monoclonal antibody or at least one
binding fragment or -domain thereof selected from an
immunoglobulin, a binding domain of an immunoglobulin or a binding
fragment of an immunoglobulin, such as an antibody, an IgG, a
molecule comprising or consisting of a Vhh domain or Vh domain, a
Fab, an scFv, an Fv, a dAb, an F(ab).sub.2, Fcab fragment, m=1,
effector moiety B1 is at least one of an oligonucleotide, a nucleic
acid and a xeno nucleic acid, preferably selected from any one or
more of a vector, a gene, a cell suicide inducing transgene,
deoxyribonucleic acid (DNA), ribonucleic acid (RNA), anti-sense
oligonucleotide (ASO, AON), short interfering RNA (siRNA), microRNA
(miRNA), DNA aptamer, RNA aptamer, mRNA, mini-circle DNA, peptide
nucleic acid (PNA), phosphoramidate morpholino oligomer (PMO),
locked nucleic acid (LNA), bridged nucleic acid (BNA),
2'-deoxy-2'-fluoroarabino nucleic acid (FANA),
2'-O-methoxyethyl-RNA (MOE), 2'-0,4'-aminoethylene bridged nucleic
acid, 3'-fluoro hexitol nucleic acid (FHNA), a plasmid, glycol
nucleic acid (GNA) and threose nucleic acid (TNA), or a derivative
thereof, preferably a BNA, either q=0, n=1 and u=2-4, or q=1,
n=2-4, u=2-4 and linker L1 is at least one oligomeric or polymeric
scaffold L3, preferably 1-8 of such scaffolds or 2-4 of such
scaffolds, wherein the at least one scaffold is optionally based on
a dendron, wherein 1-32 saponins, preferably 2, 3, 4, 5, 6, 8, 10,
16, 32 saponins, or any number of saponins therein between, such as
7, 9, 12 saponins, are covalently bound to the at least one
scaffold.
34. (canceled)
35. Pharmaceutical composition comprising the therapeutic molecule
with chemical structure of COMPOUND I according to claim 1, and
optionally further comprising a pharmaceutically acceptable
excipient.
36.-37. (canceled)
38. Method for the treatment or prophylaxis of cancer in a patient
in need thereof comprising administering to the patient the
pharmaceutical composition of claim 35, wherein the ligand A1 or B1
can bind to a tumor-cell epitope, preferably a tumor-cell specific
epitope, on a tumor-cell surface molecule, preferably a tumor
cell-specific surface molecule.
39.-42. (canceled)
Description
TECHNICAL FIELD
[0001] The invention relates to antibody-drug conjugates (ADC) that
are potentiated by co-administration of the ADC with a moiety
comprising covalently linked saponin. The invention also relates to
antibody-oligonucleotide conjugates (AOC) that are potentiated by
co-administration of the AOC with a moiety comprising covalently
linked saponin. The invention also relates to ADCs and AOCs which
are conjugated with a saponin via a covalent linker. The invention
further relates to an effector moiety such as a toxin or an
antisense oligonucleotide such as for example a BNA, conjugated
with a saponin via a covalent linkage. The invention also relates
to a BNA covalently conjugated with a targeting moiety such as an
antibody. The invention also relates to therapeutic combinations
comprising a first pharmaceutical composition comprising a
conjugate of a cell-targeting moiety such as an antibody and an
antisense oligonucleotide such as a BNA covalently bound thereto,
and comprising a second pharmaceutical composition comprising
either a free saponin, or a conjugate of a cell-targeting moiety
such as an antibody with a saponin covalently linked thereto.
Furthermore, the invention relates to any of these conjugates or
therapeutic compositions or therapeutic combinations, for use as a
medicament. The invention also relates to any of these conjugates
or therapeutic compositions or therapeutic combinations, for use in
a method for the treatment or the prophylaxis of a cancer. Finally,
the invention relates to methods for producing any of these
conjugates of the invention.
BACKGROUND
[0002] Molecules with a therapeutic biological activity are in many
occasions in theory suitable for application as an effective
therapeutic drug for the treatment of a disease such as a cancer in
human patients in need thereof. A typical example are
small-molecule biologically active moieties. However, many if not
all potential drug-like molecules and therapeutics currently used
in the clinic suffer from at least one of a plethora of
shortcomings and drawbacks. When administered to a human body,
therapeutically active molecules may exert off-target effects, in
addition to the biologically activity directed to an aspect
underlying a to-be-treated disease or health problem. Such
off-target effects are undesired and bear a risk for induction of
health- or even life-threatening side effects of the administered
molecule. It is the occurrence of such adverse events that cause
many drug-like compounds and therapeutic moieties to fail phase III
clinical trials or even phase IV clinical trials (post-market entry
follow-up). Therefore, there is a strong desire to provide drug
molecules such as small-molecule therapeutics, wherein the
therapeutic effect of the drug molecule should, e.g., (1) be highly
specific for a biological factor or biological process driving the
disease, (2) be sufficiently safe, (3) be sufficiently efficacious,
(4) be sufficiently directed to the diseased cell with little to no
off-target activity on non-diseased cells, (5) have a sufficiently
timely mode of action (e.g. the administered drug molecule should
reach the targeted site in the human patient within a certain time
frame and should remain at the targeted site for a certain time
frame), and/or (6) have sufficiently long lasting therapeutic
activity in the patient's body, amongst others. Unfortunately, to
date, `ideal` therapeutics with many or even all of the beneficial
characteristics here above outlined, are not available to the
patients, despite already long-lasting and intensive research and
despite the impressive progress made in several areas of the
individually addressed encountered difficulties and drawbacks.
[0003] Chemotherapy is one of the most important therapeutic
options for cancer treatment. However, it is often associated with
a low therapeutic window because it has no specificity towards
cancer cells over dividing cells in healthy tissue. The invention
of monoclonal antibodies offered the possibility of exploiting
their specific binding properties as a mechanism for the targeted
delivery of cytotoxic agents to cancer cells, while sparing normal
cells. This can be achieved by chemical conjugation of cytotoxic
effectors (also known as payloads or warheads) to antibodies, to
create antibody-drug conjugates (ADCs). Typically, very potent
payloads such as emtansine (DM1) are used which have a limited
therapeutic index (a ratio that compares toxic dose to efficacious
dose) in their unconjugated forms. The conjugation of DM1 to
trastuzumab (ado-trastuzumab emtansine), also known as Kadcycla,
enhances the tolerable dose of DM1 at least two-fold in monkeys. In
the past few decades tremendous efforts and investments have been
made to develop therapeutic ADCs. However, it remains challenging
to bring ADCs into the clinic, despite promising preclinical data.
The first ADC approved for clinical use was gemtuzumab ozogamicin
(Mylotarg, CD33 targeted, Pfizer/Wyeth) for relapsed acute
myelogenous leukemia (AML) in 2000. Mylotarg was however, withdrawn
from the market at the request of the Federal Drug Administration
(FDA) due to a number of concerns including its safety profile.
Patients treated with Mylotarg were more often found to die than
patients treated with conventional chemotherapy. Mylotarg was
admitted to the market again in 2017 with a lower recommended dose,
a different schedule in combination with chemotherapy or on its
own, and a new patient population. To date, only five ADCs have
been approved for clinical use, and meanwhile clinical development
of approximately fifty-five ADCs has been halted. However, interest
remains high and approximately eighty ADCs are still in clinical
development in nearly six-hundred clinical trials at present.
[0004] Despite the potential to use toxic payloads that are
normally not tolerated by patients, a low therapeutic index (a
ratio that compares toxic dose to efficacious dose) is a major
problem accounting for the discontinuance of many ADCs in clinical
development, which can be caused by several mechanisms such as
off-target toxicity on normal cells, development of resistance
against the cytotoxic agents and premature release of drugs in the
circulation. A systematic review by the FDA of ADCs found that the
toxicity profiles of most ADCs could be categorized according to
the payload used, but not the antibody used, suggesting that
toxicity is mostly determined by premature release of the payload.
Of the approximately fifty-five ADCs that were discontinued, it is
estimated that at least twenty-three were due to a poor therapeutic
index. For example, development of a trastuzumab tesirine conjugate
(ADCT-502, HER-2 targeted, ADC therapeutics) was recently
discontinued due to a narrow therapeutic index, possibly due to an
on-target, off-tissue effect in pulmonary tissue which expresses
considerable levels of HER2. In addition, several ADCs in phase 3
trials have been discontinued due to missing primary endpoint. For
example, phase 3 trials of a depatuxizumab mafodotin conjugate
(ABT-414, EGFR targeted, AbbVie) tested in patients with newly
diagnosed glioblastoma, and a mirvetuximab soravtansine conjugate
(IMGN853, folate receptor alpha (FR.alpha.) targeted, ImmunoGen)
tested in patients with platinum-resistant ovarian cancer, were
recently stopped, showing no survival benefit. It is important to
note that the clinically used dose of some ADCs may not be
sufficient for its full anticancer activity. For example,
ado-trastuzumab emtansine has an MTD of 3.6 mg/kg in humans. In
preclinical models of breast cancer, ado-trastuzumab emtansine
induced tumor regression at dose levels at or above 3 mg/kg, but
more potent efficacy was observed at 15 mg/kg. This suggests that
at the clinically administered dose, ado-trastuzumab emtansine may
not exert its maximal potential anti-tumor effect.
[0005] ADCs are mainly composed of an antibody, a cytotoxic moiety
such as a payload, and a linker. Several novel strategies have been
proposed and carried out in the design and development of new ADCs
to overcome the existing problems, targeting each of the components
of ADCs. For example, by identification and validation of adequate
antigenic targets for the antibody component, by selecting antigens
which have high expression levels in tumor and little or no
expression in normal tissues, antigens which are present on the
cell surface to be accessible to the circulating ADCs, and antigens
which allows internalizing of ADCs into the cell after binding; and
alternative mechanisms of activity; design and optimize linkers
which enhance the solubility and the drug-to-antibody ratio (DAR)
of ADCs and overcome resistance induced by proteins that can
transport the chemotherapeutic agent out of the cells; enhance the
DAR ratio by inclusion of more payloads, select and optimize
antibodies to improve antibody homogeneity and developability. In
addition to the technological development of ADCs, new clinical and
translational strategies are also being deployed to maximize the
therapeutic index, such as, change dosing schedules through
fractionated dosing; perform biodistribution studies; include
biomarkers to optimize patient selection, to capture response
signals early and monitor the duration and depth of response, and
to inform combination studies.
[0006] An example of ADCs with clinical potential are those ADCs
such as brentuximab vedotin, inotuzumab ozogamicin, moxetumomab
pasudotox, and polatuzumab vedotin, which are evaluated as a
treatment option for lymphoid malignancies and multiple myeloma.
Polatuzumab vedotin, binding to CD79b on (malignant) B-cells, and
pinatuzumab vedotin, binding to CD22, are tested in clinical trials
wherein the ADCs each were combined with co-administered rituximab,
a monoclonal antibody binding to CD20 and not provided with a
payload [B. Yu and D. Liu, Antibody-drug conjugates in clinical
trials for lymphoid malignancies and multiple myeloma; Journal of
Hematology & Oncology (2019) 12:94]. Combinations of monoclonal
antibodies such as these examples are yet a further approach and
attempt to arrive at the `magic bullet` which combines many or even
all of the aforementioned desired characteristics of ADCs.
[0007] Meanwhile in the past few decades, nucleic acid-based
therapeutics are under development. Therapeutic nucleic acids can
be based on deoxyribonucleic acid (DNA) or ribonucleic acid (RNA),
Anti-sense oligonucleotides (ASOs, AONs), and short interfering
RNAs (siRNAs), MicroRNAs, and DNA and RNA aptamers, for approaches
such as gene therapy, RNA interference (RNAi). Many of them share
the same fundamental basis of action by inhibition of either DNA or
RNA expression, thereby preventing expression of disease-related
abnormal proteins. The largest number of clinical trials is being
carried out in the field of gene therapy, with almost 2600 ongoing
or completed clinical trials worldwide but with only about 4%
entering phase 3. This is followed by clinical trials with ASOs.
Similarly to ADCs, despite the large number of techniques being
explored, therapeutic nucleic acids share two major issues during
clinical development: delivery into cells and off-target effects.
For instance, ASOs such as peptide nucleic acid (PNA),
phosphoramidate morpholino oligomer (PMO), locked nucleic acid
(LNA) and bridged nucleic acid (BNA), are being investigated as an
attractive strategy to inhibit specifically target genes and
especially those genes that are difficult to target with small
molecules inhibitors or neutralizing antibodies. Currently, the
efficacy of different ASOs is being studied in many
neurodegenerative diseases such as Huntington's disease,
Parkinson's disease, Alzheimer's disease, and amyotrophic lateral
sclerosis and also in several cancer stages. The application of
ASOs as potential therapeutic agents requires safe and effective
methods for their delivery to the cytoplasm and/or nucleus of the
target cells and tissues. Although the clinical relevance of ASOs
has been demonstrated, inefficient cellular uptake, both in vitro
and in vivo, limit the efficacy of ASOs and has been a barrier to
therapeutic development. Cellular uptake can be <2% of the dose
resulting in too low ASO concentration at the active site for an
effective and sustained outcome. This consequently requires an
increase of the administered dose which induces off-target effects.
Most common side-effects are activation of the complement cascade,
the inhibition of the clotting cascade and toll-like receptor
mediated stimulation of the immune system.
[0008] Chemotherapeutics are most commonly small molecules,
however, their efficacy is hampered by the severe off-target side
toxicity, as well as their poor solubility, rapid clearance and
limited tumor exposure. Scaffold-small-molecule drug conjugates
such as polymer-drug conjugates (PDCs) are macromolecular
constructs with pharmacologically activity, which comprises one or
more molecules of a small-molecule drug bound to a carrier scaffold
(e.g. polyethylene glycol (PEG)).
[0009] Such conjugate principle has attracted much attention and
has been under investigation for several decades. The majority of
conjugates of small-molecule drugs under pre-clinical or clinical
development are for oncological indications. However, up-to-date
only one drug not related to cancer has been approved (Movantik, a
PEG oligomer conjugate of opioid antagonist naloxone, AstraZeneca)
for opioid-induced constipation in patients with chronic pain in
2014, which is a non-oncology indication. Translating application
of drug-scaffold conjugates into treatment of human subjects
provides little clinical success so far. For example, PK1
(N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer doxorubicin;
development by Pharmacia, Pfizer) showed great anti-cancer activity
in both solid tumors and leukemia in murine models, and was under
clinical investigation for oncological indications. Despite that it
demonstrated significant reduction of nonspecific toxicity and
improved pharmacokinetics in man, improvements in anticancer
efficacy turned out to be marginal in patients, and as a
consequence further development of PK1 was discontinued.
[0010] The failure of scaffold-small-molecule drug conjugates is at
least partially attributed to its poor accumulation at the tumor
site. For example, while in murine models PK1 showed 45-250 times
higher accumulation in the tumor than in healthy tissues (liver,
kidney, lung, spleen, and heart), accumulation in tumor was only
observed in a small subset of patients in the clinical trial.
[0011] A potential solution to the aforementioned problems is
application of nanoparticle systems for drug delivery such as
liposomes. Liposomes are sphere-shaped vesicles consisting of one
or more phospholipid bilayers, which are spontaneously formed when
phospholipids are dispersed in water. The amphiphilicity
characteristics of the phospholipids provide it with the properties
of self-assembly, emulsifying and wetting characteristics, and
these properties can be employed in the design of new drugs and new
drug delivery systems. Drug encapsulated in a liposomal delivery
system may convey several advantages over a direct administration
of the drug, such as an improvement and control over
pharmacokinetics and pharmacodynamics, tissue targeting property,
decreased toxicity and enhanced drug activity. An example of such
success is liposome-encapsulated form of a small molecule
chemotherapy agent doxorubicin (Doxil: a pegylated
liposome-encapsulated form of doxorubicin; Myocet: a non-pegylated
liposomal doxorubicin), which have been approved for clinical
use.
[0012] Therefore, a solution still needs to be found that allows
for drug therapies such as anti-tumor therapies, applicable for
non-systemic use when desired, wherein the drug has for example an
acceptable safety profile, little off-target activity, sufficient
efficacy, sufficiently low clearance rate from the patient's body,
etc.
SUMMARY
[0013] For an embodiment of the present invention, it is a first
goal to provide an improved biologically active compound or
composition comprising such improved biologically active
compound.
[0014] It is one of several objectives of embodiments of the
current invention to provide a solution to the problem of
non-specificity, encountered when administering small-molecule
therapeutically active compounds to a human patient in need
thereof. It is one of several objectives of embodiments of the
current invention to provide a solution to the problem of drugs
with non-optimal specificity for a biological factor or biological
process driving a disease. It is one of several objectives of
embodiments of the current invention to provide a solution to the
problem of insufficient safety characteristics of current drugs,
when administered to human patients in need thereof. It is one of
several objectives of embodiments of the current invention to
provide a solution to the problem of current drugs being less
efficacious than desired, when administered to human patients in
need thereof. It is one of several objectives of embodiments of the
current invention to provide a solution to the problem of current
drugs being not sufficiently directed to the diseased cell with
little to no off-target activity on non-diseased cells, when
administered to human patients in need thereof. It is one of
several objectives of embodiments of the current invention to
provide a solution to the problem that current drugs do not have a
sufficiently timely mode of action (e.g. the administered drug
molecule should reach the targeted site in the human patient within
a certain time frame and should remain at the targeted site for a
certain time frame), when administered to human patients in need
thereof. It is one of several objectives of embodiments of the
current invention to provide a solution to the problem that current
drugs have not sufficiently long lasting therapeutic activity in
the patient's body, when administered to human patients in need
thereof.
[0015] At least one of the above objectives of embodiments of the
invention is achieved by providing a conjugate of the invention,
preferably comprising a cell-targeting moiety and at least one
saponin, conjugate also being suitable for use as a medicament or
suitable for implication in a pharmaceutical combination according
to the invention, and suitable for use as a semi-finished product
in the manufacture of an ADC or an antibody-oligonucleotide
conjugate (AOC) of the invention, according to the invention. The
therapeutic combination comprises for example a conjugate of the
invention comprising covalently bound saponin and for example
comprises a second conjugate comprising an effector molecule, also
referred to as an effector moiety or payload, wherein the
conjugates comprise a different binding site for a different
epitope exposed on a different cell-surface molecule of a targeted
cell, wherein the different cell-surface molecules are expressed by
the same target cell and exposed on the surface of the same target
cell, or wherein the conjugates comprise the same binding site for
the same epitope on the same cell-surface molecule of said targeted
cell.
[0016] The present invention will be described with respect to
particular embodiments but the invention is not limited thereto but
only by the claims. The embodiments of the invention described
herein can operate in combination and cooperation, unless specified
otherwise.
[0017] An aspect of the invention relates to a conjugate comprising
or consisting of an antibody and an antisense oligonucleotide such
as an antisense BNA, covalently linked together. In FIG. 1-5, the
gene-silencing activity of such a conjugate is depicted (in vivo
test in an animal tumor model). Reference is also made to the
Examples section.
[0018] An aspect of the invention relates to a combination of a
first composition comprising a conjugate comprising or consisting
of an antibody and an antisense oligonucleotide such as an
antisense BNA, covalently linked together, and a second composition
comprising free saponin of the invention (see Table A1, Scheme I).
In FIG. 1-7A and in FIG. 1-7C, the gene-silencing activity of such
a conjugate is depicted (in vitro cell-based bioassay with human
tumor cells). Reference is also made to the Examples section.
[0019] An aspect of the invention relates to a pharmaceutical
combination comprising or consisting of a first composition
comprising a first conjugate comprising or consisting of an
antibody and an antisense oligonucleotide such as an antisense BNA,
and a second composition comprising a first conjugate comprising or
consisting of the same antibody and at least one saponin of the
invention. In FIG. 1-5, the gene-silencing activity of such a
conjugate is depicted (in vivo test in an animal tumor model). In
FIG. 8-5, the gene-silencing activity of such a conjugate is
depicted (in vitro cell-based bioassay with human tumor cells).
Reference is also made to the Examples section.
[0020] An aspect of the invention relates to a pharmaceutical
combination comprising or consisting of a fourth composition
comprising a fourth conjugate comprising or consisting of an
antibody and an antisense oligonucleotide such as an antisense BNA,
and a fifth composition comprising a first conjugate comprising or
consisting of a different antibody and at least one saponin of the
invention. In FIG. 10-6A and in FIG. 10-6C, the gene-silencing
activity of such a conjugate is depicted (in vitro cell-based
bioassay with human tumor cells). Reference is also made to the
Examples section.
[0021] An aspect of the invention relates to a conjugate comprising
or consisting of an antisense oligonucleotide such as an antisense
BNA, covalently linked to at least one saponin of the invention. In
FIG. 1-3, the gene-silencing activity of such a conjugate is
depicted (in vitro cell-based bioassay with human tumor cells).
Reference is also made to the Examples section.
[0022] An aspect of the invention relates to a conjugate comprising
or consisting of an antisense oligonucleotide such as an antisense
BNA, covalently coupled to a polymeric scaffold such as a dendron
such as a G4-dendron, wherein the polymeric scaffold is covalently
conjugated with one or more saponin molecules of the invention,
such as four saponin molecules. In FIG. 1-3, the gene-silencing
activity of such a conjugate is depicted (in vitro cell-based
bioassay with human tumor cells). Reference is also made to the
Examples section.
[0023] An aspect of the invention relates to a conjugate comprising
or consisting of an antibody such as a monoclonal antibody with
specificity for a tumor marker or tumor-cell receptor, covalently
linked to at least one antisense oligonucleotide molecule such as
antisense BNA, and covalently linked to at least one saponin
molecule of the invention. In FIG. 2-4, the gene-silencing activity
of such a conjugate is depicted (in vivo test in an animal tumor
model). Reference is also made to the Examples section.
[0024] An aspect of the invention relates to a conjugate comprising
or consisting of an antibody such as a monoclonal antibody with
specificity for a tumor marker or tumor-cell receptor, covalently
linked to at least one antisense oligonucleotide molecule such as
antisense BNA via a tri-functional linker such as the linker of
Scheme II, and covalently linked to at least one saponin molecule
of the invention via the same tri-functional linker. In FIG. 1-1,
the gene-silencing activity of such a conjugate is depicted (in
vivo test in an animal tumor model). Reference is also made to the
Examples section.
[0025] An aspect of the invention relates to a therapeutic
combination consisting or comprising of a eighth composition
comprising a conjugate comprising or consisting of an antibody,
preferably a monoclonal antibody with specificity for a tumor
marker or tumor-cell receptor, covalently linked to at least one
saponin molecule of the invention, preferably via at least one
linker, preferably at least one cleavable linker, cleavable under
physiological acidic conditions, and further comprising a ninth
composition comprising an antisense oligonucleotide such as an
antisense BNA molecule. In FIG. 6-2, the gene-silencing activity of
such a conjugate is depicted (in vivo test in an animal tumor
model). In FIG. 5-2A and FIG. 5-2C, the gene-silencing activity of
such a conjugate is depicted (in vitro cell-based bioassay with
human tumor cells). Reference is also made to the Examples
section.
[0026] An aspect of the invention relates to any of the
aforementioned conjugates or compositions or therapeutical
combinations, for use as a medicament.
[0027] An aspect of the invention relates to any of the
aforementioned conjugates or compositions or therapeutical
combinations, for use in the treatment or prophylaxis of a
cancer.
[0028] An aspect of the invention relates to a therapeutic molecule
with chemical structure of COMPOUND I:
A1.sub.m((-L9.sub.w)((-L1.sub.q-B1.sub.n).sub.u((-L2.sub.r-L3.sub.s)(-L4-
.sub.v-C).sub.p).sub.t)).sub.x (compound I),
wherein A1 is a first ligand if B1 is a first effector moiety, or
A1 is the first effector moiety if B1 is the first ligand; C is a
saponin; m=0 or 1 if A1 is the first ligand and B1 is the first
effector moiety; m=0-32 if A1 is the first effector moiety and B1
is the first ligand; n=0 or 1 if B1 is the first ligand and A1 is
the first effector moiety, or if A1 is the first ligand and B1 is
the first effector moiety; p=any of 1-128; L1 is at least one
linker for covalently coupling two chemical groups; L2 is at least
one linker for covalently coupling two chemical groups; L3 is at
least one oligomeric or polymeric scaffold for covalently coupling
two chemical groups; L4 is at least one linker for covalently
coupling two chemical groups; L9 is a tri-functional linker for
covalently coupling three chemical groups; q=0 or 1; r=0 or 1; s=0
or 1; t=0, 1 or 2 if s=0, and t=any of 0-16 if s=1; u=any of 0-32
if A1 is the first ligand and B1 is the first effector moiety, or
u=1 if A1 is the first effector moiety and B1 is the first ligand;
v=0 or 1; w=1 or 0; and x=1-16.
[0029] An embodiment relates to the therapeutic combination
comprising the therapeutic molecule according to the invention and
a second therapeutic molecule with chemical structure of COMPOUND
II:
A2.sub.a((-L10.sub.i)((-L5.sub.d-B2.sub.b).sub.h((-L6.sub.e-L7.sub.f)(-L-
8.sub.i-C).sub.c).sub.q)).sub.k (compound II),
wherein A2 is a second ligand if B2 is a second effector moiety, or
A2 is the second effector moiety if B2 is the second ligand; C is a
saponin; a=0 or 1 if A2 is the second ligand and B2 is the second
effector moiety, or a=0-32 if A2 is the second effector moiety and
B2 is the second ligand; b=0 or 1 if B2 is the second ligand and A2
is the second effector moiety, or if A2 is the second ligand and B2
is the second effector moiety; c=any of 1-128; L5 is at least one
linker for covalently coupling two chemical groups; L6 is at least
one linker for covalently coupling two chemical groups; L7 is at
least one oligomeric or polymeric scaffold for covalently coupling
two chemical groups; L8 is at least one linker for covalently
coupling two chemical groups; L10 is a tri-functional linker for
covalently coupling three chemical groups; d=0 or 1; e=0 or 1; f=0
or 1; g=0, 1 or 2 if f=0, and g=any of 0-16 if f=1; h=any of 0-32
if A2 is the second ligand and B2 is the second effector moiety, or
h=1 if A2 is the second effector moiety and B2 is the second
ligand; i=0 or 1; j=1 or 0 and; k=1-16.
[0030] An embodiment relates to the therapeutic molecule of the
invention, wherein r=0, s=0, v=0, s=0, v=0, p=0, t=0, ligand A1 is
a monoclonal antibody or at least one binding fragment or -domain
thereof according to any one of the claims 3-7, m=1, effector
moiety B1 is an effector moiety according to claim 8, preferably a
BNA, either q=0, n=1 and u=2-4, or q=1, n=2-4, u=2-4 and linker L1
is the oligomeric or polymeric scaffold L3 according to any one of
the claims 21-30.
[0031] An embodiment is the therapeutic combination of the
invention, wherein the therapeutic molecule is the therapeutic
molecule of the previous embodiment, and wherein the second ligand
A2 is a monoclonal antibody or at least one binding fragment or
-domain thereof according to the invention, a=1, d=0, b=0, h=0,
e=0, L7 is the scaffold according to the invention and f=1, or L7
is absent and f=0, L8 is a linker or a cleavable linker according
to the invention, i=1, c=2-4 and g=2-4 if f=1 and g=0 if f=0, and
saponin C is a saponin according to the invention, preferably the
saponin C is SO1861 and/or QS-21, with the proviso that the ligand
A1 and the ligand A2 are the same or are different.
[0032] An aspect of the invention relates to a therapeutic
combination, wherein the therapeutic combination comprises: (a) a
first pharmaceutical composition comprising the therapeutic
molecule with chemical structure of COMPOUND I according to the
invention, the first pharmaceutical composition optionally further
comprising a pharmaceutically acceptable excipient; and (b) a
second pharmaceutical composition comprising the second therapeutic
molecule with chemical structure of COMPOUND II according to the
invention, the second pharmaceutical composition optionally further
comprising a pharmaceutically acceptable excipient.
[0033] An aspect of the invention relates to the first
pharmaceutical composition of the invention for use as a
medicament.
[0034] An aspect of the invention relates to a therapeutic
combination for use in the treatment or prevention of cancer in a
human subject, wherein the therapeutic combination comprises: (a)
the first pharmaceutical composition of the invention; and (b) the
second pharmaceutical composition of the invention, wherein the
ligand A1 or B1 and the ligand A2 or B2 can bind to a tumor-cell
epitope, preferably to a tumor-cell specific epitope, on a
tumor-cell surface molecule, preferably on a tumor cell-specific
surface molecule, with the proviso that the tumor-cell epitope or
tumor-cell specific epitope to which the ligand A1 or B1 can bind
is the same as, or is different from the tumor-cell epitope or the
tumor-cell specific epitope to which the ligand A2 or B2 can
bind.
[0035] An aspect of the invention relates to the first
pharmaceutical composition of the invention further comprising the
second therapeutic molecule of the invention.
[0036] An aspect of the invention relates to the first
pharmaceutical composition of the invention further comprising the
second therapeutic molecule of the invention, for use as a
medicament.
[0037] An aspect of the invention relates to the first
pharmaceutical composition of the invention further comprising the
second therapeutic molecule of the invention, for use in the
treatment or prevention of a cancer in a human subject.
[0038] An aspect of the invention relates to any of the following
ADCs and AOCs, and their semi-finished conjugates, comprising a
conjugate comprising covalently linked saponin of the invention
and/or comprising a conjugate comprising a payload or effector
moiety linked to e.g. an antibody of the invention and either
optionally further comprising at least one effector molecule of the
invention or optionally further comprising at least one saponin of
the invention, respectively, or both:
Anti-EGFR antibody--saponin; Anti-EGFR antibody--triterpenoid
saponin and/or a bisdesmosidic triterpene saponin belonging to the
type of a 12,13-dehydrooleanane with an aldehyde function in
position C-23 and optionally comprising a glucuronic acid function
in a carbohydrate substituent at the C-3beta-OH group of the
saponin; Anti-EGFR antibody--SO1861; Anti-EGFR antibody--GE1741;
Anti-EGFR antibody--SA1641; Anti-EGFR antibody--Quil-A; Anti-EGFR
antibody--QS-21; Anti-EGFR antibody--saponins in water soluble
saponin fraction of Quillaja saponaria; Cetuximab--saponin;
Cetuximab--triterpenoid saponin and/or a bisdesmosidic triterpene
saponin belonging to the type of a 12,13-dehydrooleanane with an
aldehyde function in position C-23 and optionally comprising a
glucuronic acid function in a carbohydrate substituent at the
C-3beta-OH group of the saponin;
Cetuximab--SO1861;
Cetuximab--GE1741;
Cetuximab--SA1641;
Cetuximab--Quil-A;
Cetuximab--QS-21;
[0039] Cetuximab--saponins in water soluble saponin fraction of
Quillaja saponaria; Anti-HER2 antibody--saponin; Anti-HER2
antibody--triterpenoid saponin and/or a bisdesmosidic triterpene
saponin belonging to the type of a 12,13-dehydrooleanane with an
aldehyde function in position C-23 and optionally comprising a
glucuronic acid function in a carbohydrate substituent at the
C-3beta-OH group of the saponin; Anti-HER2 antibody--SO1861;
Anti-HER2 antibody--GE1741; Anti-HER2 antibody--SA1641; Anti-HER2
antibody--Quil-A; Anti-HER2 antibody--QS-21; Anti-HER2
antibody--saponins in water soluble saponin fraction of Quillaja
saponaria; Trastuzumab--saponin; Trastuzumab--triterpenoid saponin
and/or a bisdesmosidic triterpene saponin belonging to the type of
a 12,13-dehydrooleanane with an aldehyde function in position C-23
and optionally comprising a glucuronic acid function in a
carbohydrate substituent at the C-3beta-OH group of the
saponin;
Trastuzumab--SO1861;
Trastuzumab--GE1741;
Trastuzumab--SA1641;
Trastuzumab--Quil-A;
Trastuzumab--QS-21;
[0040] Trastuzumab--saponins in water soluble saponin fraction of
Quillaja saponaria; Anti-CD71 antibody--saponin; Anti-CD71
antibody--triterpenoid saponin and/or a bisdesmosidic triterpene
saponin belonging to the type of a 12,13-dehydrooleanane with an
aldehyde function in position C-23 and optionally comprising a
glucuronic acid function in a carbohydrate substituent at the
C-3beta-OH group of the saponin; Anti-CD71 antibody--SO1861;
Anti-CD71 antibody--GE1741; Anti-CD71 antibody--SA1641; Anti-CD71
antibody--Quil-A; Anti-CD71 antibody--QS-21; Anti-CD71
antibody--saponins in water soluble saponin fraction of Quillaja
saponaria; OKT-9--saponin; OKT-9--triterpenoid saponin and/or a
bisdesmosidic triterpene saponin belonging to the type of a
12,13-dehydrooleanane with an aldehyde function in position C-23
and optionally comprising a glucuronic acid function in a
carbohydrate substituent at the C-3beta-OH group of the
saponin;
OKT-9--SO1861;
OKT-9--GE1741;
OKT-9--SA1641;
OKT-9--Quil-A;
OKT-9--QS-21;
[0041] OKT-9--saponins in water soluble saponin fraction of
Quillaja saponaria; Anti-EGFR antibody--oligonucleotide; Anti-EGFR
antibody--antisense oligonucleotide; Anti-EGFR antibody--siRNA;
Anti-EGFR antibody--antisense BNA; Anti-EGFR antibody--antisense
BNA(HSP27); Anti-EGFR antibody--proteinaceous toxin; Anti-EGFR
antibody--ribosome inactivating protein; Anti-EGFR
antibody--dianthin; Anti-EGFR antibody--saporin;
Cetuximab--oligonucleotide; Cetuximab--antisense oligonucleotide;
Cetuximab--siRNA; Cetuximab--antisense BNA; Cetuximab--antisense
BNA(HSP27); Cetuximab--proteinaceous toxin; Cetuximab--ribosome
inactivating protein; Cetuximab--dianthin; Cetuximab--saporin;
Anti-HER2 antibody--oligonucleotide; Anti-HER2 antibody--antisense
oligonucleotide; Anti-HER2 antibody--siRNA; Anti-HER2
antibody--antisense BNA; Anti-HER2 antibody--antisense BNA(HSP27);
Anti-HER2 antibody--proteinaceous toxin; Anti-HER2
antibody--ribosome inactivating protein; Anti-HER2
antibody--dianthin; Anti-HER2 antibody--saporin;
Trastuzumab--oligonucleotide; Trastuzumab--antisense
oligonucleotide; Trastuzumab--siRNA; Trastuzumab--antisense BNA;
Trastuzumab--antisense BNA(HSP27); Trastuzumab--proteinaceous
toxin; Trastuzumab--ribosome inactivating protein;
Trastuzumab--dianthin; Trastuzumab--saporin; Anti-CD71
antibody--oligonucleotide; Anti-CD71 antibody--antisense
oligonucleotide; Anti-CD71 antibody--siRNA; Anti-CD71
antibody--antisense BNA; Anti-CD71 antibody--antisense BNA(HSP27);
Anti-CD71 antibody--proteinaceous toxin; Anti-CD71
antibody--ribosome inactivating protein; Anti-CD71
antibody--dianthin; Anti-CD71 antibody--saporin;
OKT-9--oligonucleotide; OKT-9--antisense oligonucleotide;
OKT-9--siRNA; OKT-9--antisense BNA; OKT-9--antisense BNA(HSP27);
OKT-9--proteinaceous toxin; OKT-9--ribosome inactivating protein;
OKT-9--dianthin; OKT-9--saporin; Anti-EGFR antibody
(-oligonucleotide)(-saponin), wherein the oligonucleotide is any
one or more of antisense oligonucleotide, siRNA, antisense BNA, and
antisense BNA(HSP27), and wherein the saponin is any one or more of
a triterpenoid saponin and/or a bisdesmosidic triterpene saponin
belonging to the type of a 12,13-dehydrooleanane with an aldehyde
function in position C-23 and optionally comprising a glucuronic
acid function in a carbohydrate substituent at the C-3beta-OH group
of the saponin, SO1861, GE1741, SA1641, Quil-A, QS-21, and saponins
in water soluble saponin fraction of Quillaja saponaria, wherein
the anti-EGFR antibody preferably is cetuximab; Anti-EGFR antibody
(-proteinaceous toxin)(-saponin), wherein the proteinaceous toxin
is any one or more of a ribosome inactivating protein, dianthin and
saporin, and wherein the saponin is any one or more of a
triterpenoid saponin and/or a bisdesmosidic triterpene saponin
belonging to the type of a 12,13-dehydrooleanane with an aldehyde
function in position C-23 and optionally comprising a glucuronic
acid function in a carbohydrate substituent at the C-3beta-OH group
of the saponin, SO1861, GE1741, SA1641, Quil-A, QS-21, and saponins
in water soluble saponin fraction of Quillaja saponaria, wherein
the anti-EGFR antibody preferably is cetuximab; Anti-HER2 antibody
(-oligonucleotide)(-saponin), wherein the oligonucleotide is any
one or more of antisense oligonucleotide, siRNA, antisense BNA, and
antisense BNA(HSP27), and wherein the saponin is any one or more of
a triterpenoid saponin and/or a bisdesmosidic triterpene saponin
belonging to the type of a 12,13-dehydrooleanane with an aldehyde
function in position C-23 and optionally comprising a glucuronic
acid function in a carbohydrate substituent at the C-3beta-OH group
of the saponin, SO1861, GE1741, SA1641, Quil-A, QS-21, and saponins
in water soluble saponin fraction of Quillaja saponaria, wherein
the anti-HER2 antibody preferably is trastuzumab; Anti-HER2
antibody (-proteinaceous toxin)(-saponin), wherein the
proteinaceous toxin is any one or more of a ribosome inactivating
protein, dianthin and saporin, and wherein the saponin is any one
or more of a triterpenoid saponin and/or a bisdesmosidic triterpene
saponin belonging to the type of a 12,13-dehydrooleanane with an
aldehyde function in position C-23 and optionally comprising a
glucuronic acid function in a carbohydrate substituent at the
C-3beta-OH group of the saponin, SO1861, GE1741, SA1641, Quil-A,
QS-21, and saponins in water soluble saponin fraction of Quillaja
saponaria, wherein the anti-HER2 antibody preferably is
trastuzumab; Anti-CD71 antibody (-oligonucleotide)(-saponin),
wherein the oligonucleotide is any one or more of antisense
oligonucleotide, siRNA, antisense BNA, and antisense BNA(HSP27),
and wherein the saponin is any one or more of a triterpenoid
saponin and/or a bisdesmosidic triterpene saponin belonging to the
type of a 12,13-dehydrooleanane with an aldehyde function in
position C-23 and optionally comprising a glucuronic acid function
in a carbohydrate substituent at the C-3beta-OH group of the
saponin, SO1861, GE1741, SA1641, Quil-A, QS-21, and saponins in
water soluble saponin fraction of Quillaja saponaria, wherein the
anti-CD71 antibody preferably is OKT-9; and Anti-CD71 antibody
(-proteinaceous toxin)(-saponin), wherein the proteinaceous toxin
is any one or more of a ribosome inactivating protein, dianthin and
saporin, and wherein the saponin is any one or more of a
triterpenoid saponin and/or a bisdesmosidic triterpene saponin
belonging to the type of a 12,13-dehydrooleanane with an aldehyde
function in position C-23 and optionally comprising a glucuronic
acid function in a carbohydrate substituent at the C-3beta-OH group
of the saponin, SO1861, GE1741, SA1641, Quil-A, QS-21, and saponins
in water soluble saponin fraction of Quillaja saponaria, wherein
the anti-CD71 antibody preferably is OKT-9.
[0042] An embodiment is the first proteinaceous molecule of the
invention, the semi-finished conjugate of the invention or the
conjugate of the invention, wherein the first binding site is
selected from cetuximab, trastuzumab, OKT-9, and/or wherein the
effector molecule is selected from dianthin, saporin and antisense
BNA(HSP27), and/or wherein the saponin is selected from SO1861,
GE1741, SA1641, Quil-A, QS-21, and saponins in water soluble
saponin fraction of Quillaja saponaria.
[0043] An embodiment is the conjugate according to the invention,
wherein the first proteinaceous molecule is selected from
cetuximab, trastuzumab, OKT-9, and/or wherein the effector molecule
is selected from dianthin, saporin and antisense BNA(HSP27), and/or
wherein the saponin is selected from SO1861, GE1741, SA1641,
Quil-A, QS-21, and saponins in water soluble saponin fraction of
Quillaja saponaria.
[0044] An aspect of the invention relates to an ADC or an AOCs or a
semi-finished ADC conjugate or a semi-finished AOC conjugate
comprising an antibody-saponin conjugate of the invention and
comprising at least one effector molecule of the invention and/or
comprising at least one saponin of the invention, of Structure
C:
A(-S)b(-E)c Structure C,
wherein A is the first binding site; S is the saponin; E is the
effector molecule; b=0-64, preferably 0, 1, 2, 3, 4, 8, 16, 32, 64
or any whole number or fraction therein between; c=0-8, preferably
0, 1, 2, 3, 4, 6, 8 or any whole number or fraction therein
between, wherein S is coupled to A and/or E, E is coupled to A
and/or S, preferably S is coupled to A and E is coupled to A.
[0045] An embodiment is the Structure C of the invention, wherein A
is an anti-EGFR antibody such as cetuximab, an anti-HER2 antibody
such as trastuzumab, an anti-CD71 antibody such as OKT-9, and/or
wherein S is any one or more of a saponin, a triterpenoid saponin
and/or a bisdesmosidic triterpene saponin belonging to the type of
a 12,13-dehydrooleanane with an aldehyde function in position C-23
and optionally comprising a glucuronic acid function in a
carbohydrate substituent at the C-3beta-OH group of the saponin,
SO1861, GE1741, SA1641, Quil-A, QS-21, and saponins in water
soluble saponin fraction of Quillaja saponaria, and/or wherein E is
any one or more of an oligonucleotide, an antisense
oligonucleotide, an siRNA, an antisense BNA, and an antisense
BNA(HSP27), and/or any one or more of a proteinaceous toxin, a
ribosome inactivating protein, dianthin and saporin.
[0046] An embodiment is the Structure C of the invention, the
conjugate of the invention or the semi-finished conjugate of the
invention or the first proteinaceous molecule of the invention,
wherein the saponin, if present, and/or the effector molecule, if
present, is covalently coupled via at least one linker, such as a
cleavable linker, and/or via at least one oligomeric or polymeric
scaffold, such as a linker based on N-.epsilon.-maleimidocaproic
acid hydrazide (EMCH) succinimidyl 3-(2-pyridyldithio)propionate or
3-(2-Pyridyldithio)propionic acid N-hydroxysuccinimide ester
(SPDP), and
1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid hexafluorophosphate (HATU), and such as a scaffold based on
a Dendron such as a G4-Dendron or a tri-functional linker such as
the tri-functional linker of Scheme II, and/or wherein at least a
lysine side chain and/or a cysteine side chain of the first binding
site of the antibody, preferably a monoclonal antibody or fragments
or domains thereof, is involved in the covalent bond with the
saponin and/or the effector molecule and/or the linker and/or the
cleavable linker and/or the scaffold, wherein preferably the
saponin and/or the effector molecule is covalently linked to the
first binding site of the antibody, preferably a monoclonal
antibody, wherein the covalent link comprises or consists of an
amide bond, a hydrazone bond, a disulphide bond.
[0047] An aspect of the invention relates to the use of any of the
aforementioned conjugates of the invention or any of the
semi-finished conjugates of the invention or the antibody-saponin
conjugate of the invention, as a medicament.
[0048] An aspect of the invention relates to the use of any of the
conjugates of the invention or the semi-finished conjugates of the
invention or the antibody-saponin conjugates of the invention, for
use in the treatment or prophylaxis of a cancer or an auto-immune
disease.
[0049] FIG. 25 and FIG. 26 show examples of ADCs of the invention
with covalently coupled saponin(s) and OACs of the invention with
covalently coupled saponin(s).
[0050] Of course, any and all of a, b, c, d, e, f, g, h, I, j, k,
m, n, p, q, r, s, t, u, v, w and/or x have the value in accordance
with each individual embodiment and aspect of the invention for any
and all of the aforementioned aspects and embodiments according to
the invention. In addition, (tri-functional) linkers L1, L2, L4,
L5, L6, L8, L9 and/or L10, if present in a molecule or conjugate or
moiety of the invention, are the (tri-functional) linkers as
indicated for each and any of the aforementioned aspects and
embodiments of the invention, as is readily appreciated by the
skilled person. The oligomeric or polymeric scaffolds L3 and/or L7,
if present in a molecule or conjugate or moiety of the invention,
are the oligomeric or polymeric scaffolds as indicated for each and
any of the aforementioned aspects and embodiments of the invention,
as is also readily appreciated by the skilled person. Furthermore,
the first ligand A1 and the first effector moiety B1, if present,
and the second ligand A2 and the second effector moiety B2, if
present, and the first effector moiety A1 and the first ligand B1,
if present, and the second effector moiety A2 and the second ligand
B2, if present, are the selected and indicated ligands and effector
moieties, as disclosed for the first, second, third, fourth, fifth,
and sixth series of embodiment and aspects of the invention, and
all further embodiments and aspects of the invention, outlined here
above. Saponin C is any one or more of the saponins referred to and
listed in any of the aforementioned aspects and embodiments of the
invention, in particular one or more saponins selected from Scheme
I and/or Table A1.
Definitions
[0051] The term "linker" has its regular scientific meaning, and
here refers to a chemical moiety or a linear stretch of amino-acid
residues complexed through peptide bonds, which attaches a molecule
or an atom to another molecule, e.g. to a ligand or to an effector
molecule or to a scaffold. Typically, the linker comprises a chain
of atoms linked by chemical bonds. Any linker molecule or linker
technology known in the art can be used in the present disclosure.
Where indicated, the linker is a linker for covalently binding of
molecules through a chemical group on such a molecule suitable for
forming a covalent linkage or bond with the linker. The linker may
be a non-cleavable linker, e.g., the linker is stable in
physiological conditions. The linker may be a cleavable linker,
e.g. a linker that is cleavable, in the presence of an enzyme or at
a particular pH range or value, or under physiological conditions
such as intracellular conditions in the endosomes such as the late
endosomes and the lysosomes of mammalian cells such as human cells.
Exemplary linkers that can be used in the context of the present
disclosure includes, but is not limited to,
N-.epsilon.-maleimidocaproic acid hydrazide (EMCH), succinimidyl
3-(2-pyridyldithio)propionate or 3-(2-Pyridyldithio)propionic acid
N-hydroxysuccinimide ester (SPDP), and
1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid hexafluorophosphate (HATU).
[0052] The term "tri-functional linker" has its regular scientific
meaning, and here refers to a linker which attaches three molecules
via a chemical group on each of the three molecules. The skilled
person is able to design such tri-functional linkers, based on the
present disclosure and the common general knowledge. Such
tri-functional linker can exhibit, for instance, a maleimido group
that can be used for conjugation to targeting ligands that exhibit
thiol groups to perform a thiol-ene reaction. In addition, the
tri-functional linker could exhibit a dibenzocyclooctyne (DBCO)
group to perform the so-called strain-promoted alkyne-azide
cycloaddition (SPAAC, click chemistry) with an azido bearing
saponin. Finally, the tri-functional linker could obtain a third
functional group such as a trans-cyclooctene (TCO) group to perform
the so-called inverse electron demand Diels-Alder (IEDDA) reaction
with a tetrazine (Tz) bearing effector molecule. The skilled person
will appreciate that the chemical groups of the tri-functional
linker can be all three the same, or different, or the linker may
comprise two of the same chemical groups for linking a molecule to
the tri-functional linker. The formed bonds between the
tri-functional linker can be covalent or non-covalent, and covalent
bonds are preferred. The formed bonds between the tri-functional
linker and the one or two or three bound molecules via respective
chemical groups, can be cleavable (labile) bonds, such as cleavable
under acidic conditions inside cells such as endosomes and
lysosomes of mammalian cells such as human cells, or can be
non-cleavable bonds. Of course, the tri-functional linker may
encompass one or two chemical groups for forming covalent bonds
while the further two or one chemical group(s), respectively,
are/is for forming a non-covalent bond. Of course, the
tri-functional linker may encompass one or two chemical groups for
forming cleavable bonds while the further two or one chemical
group(s), respectively, are/is for forming a non-cleavable
bond.
[0053] The term "cleavable", such as used in the term "cleavable
linker" or "cleavable bond" has its regular scientific meaning, and
here refers to being subject to cleavage under acidic conditions,
reductive conditions, enzymatic conditions or light-induced
conditions. For example, a cleavable linker may be subject to
cleavage under acidic conditions, preferably said cleavable linker
is subject to cleavage in vivo under acidic conditions as present
in endosomes and/or lysosomes of mammalian cells, preferably human
cells, preferably at pH 4.0-6.5, and more preferably at pH 5.5. As
another example, a cleavable linker may be subject to cleavage by
an enzyme, e.g. by cathepsin. Furthermore, an example of a covalent
bond cleavable under reductive conditions is a disulphide bond.
[0054] The terms "oligomer" and "polymer" in the context of an
oligomeric or polymeric scaffold has its regular scientific
meaning. A polymer here refers to a substance which has a molecular
structure built up chiefly or completely from a large number of
equal or similar units bonded together; an oligomer here refers to
a polymer whose molecules consist of relatively few repeating
units. For example, a structure comprising 5-10 or less equal or
similar units, may be called an oligomeric structure, whereas a
structure comprising 10-50 monomeric units or more may be called a
polymeric structure, whereas a structure of 10 monomeric units may
be called either oligomeric or polymeric.
[0055] The term "binding site" has its regular scientific meaning,
and here refers to a region or an epitope on a molecule, e.g. a
protein, DNA or RNA, to which another molecule can bind.
[0056] The term "scaffold" has its regular scientific meaning, and
here refers to an oligomeric or polymeric template or a carrier or
a base (base molecule or base structure), to which one or more
molecules, e.g. ligand molecule, effector molecule, can be
covalently bound, either directly, or via a linker, such as a
cleavable linker. A scaffold may have a structurally ordered
formation such as a polymer, oligomer, dendrimer, dendronized
polymer, or dendronized oligomer or have an assembled polymeric
structure such as a hydrogel, microgel, nanogel, stabilized
polymeric micelle or liposome, but excludes structures that are
composed of non-covalent assemblies of monomers such as
cholesterol/phospholipid mixtures. A scaffold may comprise a
polymeric or oligomeric structure, such as poly- or oligo(amines),
e.g., polyethylenimine and poly(amidoamine); or structures such as
polyethylene glycol, poly- or oligo(esters), such as poly(lactids),
poly(lactams), polylactide-co-glycolide copolymers; or
poly(dextrin), poly- or oligosaccharides, such as cyclodextrin or
polydextrose; or structures such as natural and/or artificial poly-
or oligoamino acids such as poly-lysine or a peptide or a protein,
DNA oligo- or polymers, stabilized RNA polymers or PNA (peptide
nucleic acid) polymers. Preferably, the polymeric or oligomeric
structures are biocompatible, wherein biocompatible means that the
polymeric or oligomeric structure does not show substantial acute
or chronic toxicity in organisms and can be either excreted as it
is or fully degraded to excretable and/or physiological compounds
by the body's metabolism.
[0057] The term "ligand" has its regular scientific meaning, and
here refers to any molecule or molecules which may selectively bind
to a target cell-surface molecule or target cell-surface receptor
expressed at target cells, e.g. target cancer cells or target
auto-immune cells. The ligand may bind to an epitope comprised by
receptors or other antigens on the target cells. Preferably, the
cell-binding ligands are antibodies.
[0058] The term "antibody" as used herein is used in the broadest
sense, which may refer to an immunoglobulin (Ig) defined as a
protein belonging to the class IgG, IgM, IgE, IgA, or IgD (or any
subclass thereof), or a functional binding fragment or binding
domain of an immunoglobulin. In the context of the present
invention, a "binding fragment" or a "binding domain" of an
immunoglobulin is defined as antigen-binding fragment or -domain or
other derivative of a parental immunoglobulin that essentially
maintains the antigen binding activity of such parental
immunoglobulin. Functional fragments and functional domains are
antibodies in the sense of the present invention even if their
affinity to the antigen is lower than that of the parental
immunoglobulin. "Functional fragments and -domains" in accordance
with the invention include, but are not limited to, F(ab')2
fragments, Fab' fragments, Fab fragments, scFv, dsFv, single-domain
antibody (sdAb), monovalent IgG, scFv-Fc, reduced IgG (rIgG),
minibody, diabodies, triabodies, tetrabodies, Fc fusion proteins,
nanobodies, variable V domains such as VHH, Vh, and other types of
antigen recognizing immunoglobulin fragments and domains. The
fragments and domains may be engineered to minimize or completely
remove the intermolecular disulphide interactions that occur
between the CH1 and CL domains. Functional fragment and -domains
offer the advantage of greater tumor penetration because of their
smaller size. In addition, the functional fragment or -domain can
be more evenly distributed throughout the tumor mass as compared to
whole immunoglobulin.
[0059] The antibodies (immunoglobulins) of the present invention
may be bi- or multifunctional. For example, a bifunctional antibody
has one arm having a specificity for one receptor or antigen, while
the other arm recognizes a different receptor or antigen.
Alternatively, each arm of the bifunctional antibody may have
specificity for a different epitope of the same receptor or antigen
of the target cell.
[0060] The antibodies (immunoglobulins) of the present invention
may be, but are not limited to, polyclonal antibodies, monoclonal
antibodies, human antibodies, humanized antibodies, chimeric
antibodies, resurfaced antibodies, anti-idiotypic antibodies, mouse
antibodies, rat antibodies, rat/mouse hybrid antibodies, llama
antibodies, llama heavy-chain only antibodies, heavy-chain only
antibodies, and veterinary antibodies. Preferably, the antibody
(immunoglobulin) of the present invention is a monoclonal antibody.
The resurfaced, chimeric, humanized and fully human antibodies are
also more preferred because they are less likely to cause
immunogenicity in humans. The antibodies of the ADC of the present
invention preferably specifically binds to an antigen expressed on
the surface of a cancer cell, an autoimmune cell, a diseased cell,
an aberrant cell, while leaving any healthy cell essentially
unaltered (e.g. by not binding to such normal cell, or by binding
to a lesser extent in number and/or affinity to such healthy
cell).
[0061] Specific antibodies that can be used for the ADCs of the
present invention include, but are not limited to, anti-HER2
monoclonal antibody such as trastuzumab and pertuzumab, anti-CD20
monoclonal antibody such as rituximab, ofatumumab, tositumomab and
ibritumomab, anti-CA125 monoclonal antibody such as oregovomab,
anti-EpCAM (17-1A) monoclonal antibody such as edrecolomab,
anti-EGFR monoclonal antibody such as cetuximab, panitumumab and
nimotuzumab, anti-CD30 monoclonal antibody such brentuximab,
anti-CD33 monoclonal antibody such as gemtuzumab and huMy9-6,
anti-vascular integrin alpha-v beta-3 monoclonal antibody such as
etaracizumab, anti-CD52 monoclonal antibody such as alemtuzumab,
anti-CD22 monoclonal antibody such as epratuzumab, anti-CEA
monoclonal antibody such as labetuzumab, anti-CD44v6 monoclonal
antibody such as bivatuzumab, anti-FAP monoclonal antibody such as
sibrotuzumab, anti-CD19 monoclonal antibody such as huB4,
anti-CanAg monoclonal antibody such as huC242, anti-CD56 monoclonal
antibody such huN901, anti-CD38 monoclonal antibody such as
daratumumab, anti-CA6 monoclonal antibody such as DS6, anti-IGF-IR
monoclonal antibody such as cixutumumab and 3B7, anti-integrin
monoclonal antibody such as CNTO 95, and anti-syndecan-1 monoclonal
antibody such as B-134.
[0062] Any other molecules than antibodies that bind to a cell
receptor or antigen of a target cell can also be used as the
cell-binding ligand for the ligand-drug conjugates of the present
invention and the ligands provided with covalently bound saponin
according to the invention. These ligands include, but are not
limited to, proteins, polypeptides, peptides, small molecules.
Examples of these non-antibody ligands are interferons (e.g.
IFN-.alpha., IFN-.beta., and IFN-.gamma.), transferrins, lectins,
epidermal growth factors (EGF) and EGF-like domains,
gastrin-releasing peptides (GRP), platelet-derived growth factors
(PDGF), transforming growth factors (TGF), vaccinia growth factor
(VGF), insulin and insulin-like growth factors (IGF, e.g. IGF-1 and
IGF-2), other suitable hormones such as thyrotropin releasing
hormones (TRH), melanocyte-stimulating hormones (MSH), steroid
hormones (e.g. estrogen and androgen), somatostatin, lymphokines
(e.g. IL-2, IL-3, IL-4, and IL-6), colony-stimulating factors (CSF,
e.g. G-CSF, M-CSF and GM-CSF), bombesin, gastrin, Arg-Gly-Asp or
RGD, aptamers (e.g. AS-1411, GBI-10, RNA aptamers against HIV
glycoprotein), small molecules (e.g. folate, anisamide
phenylboronic acid), vitamins (e.g., vitamin D), carbohydrates
(e.g. hyaluronic acid, galactose).
[0063] An "effector molecule" or "effector moiety" or "payload" has
its regular scientific meaning and in the context of this invention
is any substance that affects the metabolism of a cell by
interaction with an intracellular effector molecule target, wherein
this effector molecule target is any molecule or structure inside
cells excluding the lumen of compartments and vesicles of the
endocytic and recycling pathway but including the membranes of
these compartments and vesicles. Said structures inside cells thus
include the nucleus, mitochondria, chloroplasts, endoplasmic
reticulum, Golgi apparatus, other transport vesicles, the inner
part of the plasma membrane and the cytosol.
[0064] The effector molecule or -moiety is a pharmaceutically
active substance, such as a toxin such as a proteinaceous toxin, a
drug, a polypeptide or a polynucleotide. A pharmaceutically active
substance in this invention is an effector molecule or -moiety that
is used to achieve a beneficial outcome in an organism, preferably
a vertebrate, more preferably a mammal such as non-human subjects
or a human being/subject. Benefits include diagnosis, prognosis,
treatment, cure and prevention (prophylaxis) of diseases and/or
symptoms and/or health problems. The pharmaceutically active
substance may also lead to undesired and sometimes even harmful
side effects (adverse events such as observed during clinical
trials). In this case, pros and cons must be weighed to decide
whether the pharmaceutically active substance is suitable in the
particular case. If the effect of the pharmaceutically active
substance inside a cell is predominantly beneficial for the
organism as a whole, the cell is called a target cell. If the
effect inside a cell is predominantly harmful for the organism as a
whole, the cell is called an off-target cell. In artificial systems
such as cell cultures and bioreactors, target cells and off-target
cells depend on the purpose and are defined by the user. Examples
of effector molecules and -moieties are a drug, a toxin, a
polypeptide (such as an enzyme), a polynucleotide (including
polypeptides and polynucleotides that comprise non-natural amino
acids or nucleic acids), and any combination thereof.
[0065] An effector molecule or effector moiety that is a drug may
include, but not limited to, anti-cancer agents, anti-inflammatory
agents, and anti-infective (e.g., anti-fungal, antibacterial,
anti-parasitic, anti-viral) agents. Preferably, the drug molecule
of the present invention is an anti-cancer agent or an
anti-auto-immune agent. Suitable anti-cancer agents include, but
are not limited to, alkylating agents, antimetabolites, spindle
poison plant alkaloids, cytotoxic/antitumor antibiotics,
topoisomerase inhibitors, photosensitizers, and kinase inhibitors.
Also included in the definition of "anti-cancer agent" are: e.g.
(i) anti-hormonal agents that act to regulate or inhibit hormone
action on tumors such as anti-estrogens and selective estrogen
receptor modulators; (ii) aromatase inhibitors that inhibit the
enzyme aromatase, which regulates estrogen production in the
adrenal glands; (iii) anti-androgens; (iv) protein kinase
inhibitors; (v) lipid kinase inhibitors; (vi) antisense
oligonucleotides, particularly those which inhibit expression of
genes in signaling pathways implicated in aberrant cell
proliferation; (vii) ribozymes such as VEGF expression inhibitors
and HER2 expression inhibitors; (viii) vaccines such as gene
therapy vaccines; topoisomerase 1 inhibitors; (ix) anti-angiogenic
agents; and pharmaceutically acceptable salts, acids, solvates and
derivatives of any of the above.
[0066] An effector molecule or -moiety that is a toxin may include,
but is not limited to, proteinaceous toxins (e.g. bacterial-derived
toxins, and plant-derived toxins), toxins targeting tubulin
filaments, toxins targeting DNA, toxins targeting RNA. Examples of
proteinaceous toxins are saporin, dianthin, ricin, modeccin, abrin,
volkensin, viscumin, shiga toxin, shiga-like toxin, pseudomonas
exotoxin (PE, also known as exotoxin A), diphtheria toxin (DT), and
cholera toxin. Examples of tubulin filaments-targeting toxins are
maytansinoids (e.g. DM1 and DM4), auristatins (e.g. Monomethyl
auristatin E (MMAE) and Monomethyl auristatin F (MMAF)), toxoids,
tubulysins, cryptophycins, rhizoxin. Examples of DNA-targeting
toxins are calicheamicins: N-Acetyl-.gamma.-calicheamicin, CC-1065
analogs, duocarmycins, doxorubicin, methotrexate, benzodiazepines,
camptothecin analogues, and anthracyclines. Examples of
DNA-targeting toxins are amanitins, spliceostatins, and
thailanstatins. A toxin, as used in this invention, is defined as a
pharmaceutically active substance that is able to kill or
inactivate a cell. Preferably, a targeted toxin is a toxin that is
only, or at least predominantly, toxic for target cells but not for
off-target cells. The net effect of the targeted toxin is
preferably beneficial for the organism as a whole.
[0067] An effector molecule or -moiety that is a polypeptide may
be, e.g., a polypeptide that recover a lost function, such as for
instance enzyme replacement, gene regulating functions, or a toxin.
Examples of polypeptides as effector molecules are, e.g., Cas9;
toxins (e.g. saporin, dianthin, gelonin, (de)bouganin, agrostin,
ricin (toxin A chain); pokeweed antiviral protein, apoptin,
diphtheria toxin, pseudomonas exotoxin) metabolic enzymes (e.g.
argininosuccinate lyase, argininosuccinate synthetase), enzymes of
the coagulation cascade, repairing enzymes; enzymes for cell
signaling; cell cycle regulation factors; gene regulating factors
(transcription factors such as NF-.kappa.B or gene repressors such
as methionine repressor).
[0068] An effector molecule or an effector moiety that is a
polynucleotide may, e.g., be a polynucleotide that comprises coding
information, such as a gene or an open reading frame encoding a
protein. It may also comprise regulatory information, e.g. promotor
or regulatory element binding regions, or sequences coding for
micro RNAs. Such polynucleotide may comprise natural and artificial
nucleic acids. Artificial nucleic acids include, e.g. peptide
nucleic acid (PNA), Morpholino and locked nucleic acid (LNA), as
well as glycol nucleic acid (GNA) and threose nucleic acid (TNA).
Each of these is distinguished from naturally occurring DNA or RNA
by changes to the backbone of the molecule. Examples of nucleotides
as effector molecules are, but not limited to, e.g., DNA: single
stranded DNA (e.g. DNA for adenine phosphoribosyltransferase);
linear double stranded DNA (e.g. clotting factor IX gene); circular
double stranded DNA (e.g. plasmids); RNA: mRNA (e.g. TAL effector
molecule nucleases), tRNA, rRNA, siRNA, miRNA, antisense RNA;
anti-sense oligonucleotides (ASOs, AONs e.g. PNA, PMO, LNA and
BNA).
[0069] The term "proteinaceous", used in e.g. "proteinaceous
molecule" and "proteinaceous toxin", are molecules and toxins
comprising at least a string of amino acid residues that can be
obtained as an expression product from a single mRNA. Such a
molecule or toxin may further comprise any post-translational
modifications, a carbohydrate such as an N- or O-linked
carbohydrate, disulphide bonds, phosphorylations, sulphatations,
etc., as a result of any post-translational modification, and/or
may further comprise any other modification such as those resulting
from chemical modifications (e.g., linking of effector moieties,
saponin, scaffolds, ligands, etc., either directly to e.g. an
amino-acid side chain, or via at least one linker (covalently)
bound to the molecule for chemically modifying the proteinaceous
molecule, and chemically bound (covalently) to the proteinaceous
molecule). The term "proteinaceous" also encompasses and includes
assemblies of such molecules, e.g. homodimers, heterotrimers,
heterohexamers or complex assemblies such as ribosomes.
[0070] The terms "specific" and "specifically", in the context of
for example "specific binding" and "receptor or molecular target
specifically present or expressed at the surface of a tumor cell"
and the like, have their normal scientific meaning known in the
art, and here refer to e.g. a binding interaction of a first
molecule with a second molecule which occurs with a higher affinity
relative to any putative binding of the first molecule to a further
molecule different from the second molecule, or e.g. to the
expression or expression to a higher extent when e.g. the number of
receptors or molecular targets is considered, of a cell-surface
receptor or molecular target on the surface of a first type of cell
such as a tumor cell, autoimmune cell, diseased cell, aberrant
cell, relative to the extent of expression of the same receptor or
molecular target at a second type of cell such as a healthy cell,
etc., wherein expression at the second type of cell can be fully
absent or very low, relative to any extent of expression on the
tumor cell, etc. Furthermore, the term "specific", for example in
"specific binding", has its normal scientific meaning known in the
art, and here has the meaning of indicating a molecule that can
have an interaction with another molecule with higher binding
affinity than background interactions between molecules. Similarly,
the term "specificity" refers to an interaction, for example,
between two molecules or between a cell and a molecule, which has
higher binding affinity than background interactions between
molecules. Binding molecules such as immunoglobulins bind via their
binding site such as immunoglobulin variable regions of the
immunoglobulin, to binding sites on molecules, such as epitopes,
cell-surface receptors, etc., with a higher binding affinity than
background interactions between molecules. In the context of the
invention, background interactions are typically interactions with
an affinity lower than a K.sub.D of 10E-4 M. Similarly, "specific
binding domains" are domains that preferentially bind to binding
sites on molecules, such as epitopes, cell-surface receptors, etc.,
with a higher binding affinity than background interactions between
molecules. In the context of the invention, "background
interactions" are typically interactions with an affinity lower
than a K.sub.D of 10E-4 M. Preferably, specific binding domains
bind with an affinity higher than a K.sub.D of about 10E-5 M.
[0071] The term "binding" is defined as interactions between
molecules that can be distinguished from background
interactions.
[0072] Throughout the specification, the term "fragment" refers to
an amino acid sequence which is part of a protein domain or which
builds up an intact protein domain. Binding fragments according to
the invention must have binding specificity for the respective
target such as a cell-surface receptor, e.g. on the surface of a
diseased cell such as a tumor cell.
[0073] The term "ADC" or "antibody-drug conjugate" has its regular
scientific meaning known to the skilled person, and here refers to
a class of biopharmaceutical drugs designed as a targeted therapy
for treating e.g. cancer. Unlike chemotherapy, ADCs are intended to
target and kill tumor cells while sparing healthy cells. ADCs are
composed of an antibody linked to a biologically active cytotoxic
(anticancer) payload or drug. ADCs combine the targeting
capabilities of monoclonal antibodies with the cancer-killing
ability of cytotoxic drugs. They are designed with the intention to
discriminate between healthy cells and diseased tissue such as
tumor cells in a tumor.
[0074] The term "Saponinum album" has its normal meaning and here
refers to a mixture of saponins produced by Merck KGaA (Darmstadt,
Germany) containing saponins from Gypsophila paniculata and
Gypsophila arostii, containing SA1657 and mainly SA1641.
[0075] The term "Quillajasaponin" has its normal meaning and here
refers to the saponin fraction of Quillaja saponaria and thus the
source for all other QS saponins, mainly containing QS-18 and
QS-21.
[0076] "QS-21" or "QS21" has its regular scientific meaning and
here refers to a mixture of QS-21 A-apio (.about.63%), QS-21 A-xylo
(.about.32%), QS-21 B-apio (.about.3.3%), and QS-21 B-xylo
(.about.1.7%).
[0077] Similarly, "QS-21A" has its regular scientific meaning and
here refers to a mixture of QS-21 A-apio (.about.65%) and QS-21
A-xylo (.about.35%).
[0078] Similarly, "QS-21B" has its regular scientific meaning and
here refers to a mixture of QS-21 B-apio (.about.65%) and QS-21
B-xylo (.about.35%).
[0079] The term "Quil-A" refers to a commercially available
semi-purified extract from Quillaja saponaria and contains variable
quantities of more than 50 distinct saponins, many of which
incorporate the triterpene-trisaccharide substructure
Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA- at the C-3beta-OH group
found in QS-7, QS-17, QS18, and QS-21. The saponins found in Quil-A
are listed in van Setten (1995), Table 2 [Dirk C. van Setten,
Gerrit van de Werken, Gijsbert Zomer and Gideon F. A. Kersten,
Glycosyl Compositions and Structural Characteristics of the
Potential Immuno-adjuvant Active Saponins in the Quillaja saponaria
Molina Extract Quil A, RAPID COMMUNICATIONS IN MASS SPECTROMETRY,
VOL. 9, 660-666 (1995)]. Quil-A and also Quillajasaponin are
fractions of saponins from Quillaja saponaria and both contain a
large variety of different saponins with largely overlapping
content. The two fractions differ in their specific composition as
the two fractions are gained by different purification
procedures.
[0080] The term "QS1861" and the term "QS1862" refer to QS-7 and
QS-7 api. QS1861 has a molecular mass of 1861 Dalton, QS1862 has a
molecular mass of 1862 Dalton. QS1862 is described in Fleck et al.
(2019) in Table 1, row no. 28 [Juliane Deise Fleck, Andresa Heemann
Betti, Francini Pereira da Silva, Eduardo Artur Troian, Cristina
Olivaro, Fernando Ferreira and Simone Gasparin Verza, Saponins from
Quillaja saponaria and Quillaja brasiliensis: Particular Chemical
Characteristics and Biological Activities, Molecules 2019, 24, 171;
doi:10.3390/molecules24010171]. The described structure is the
api-variant QS1862 of QS-7. The molecular mass is 1862 Dalton as
this mass is the formal mass including proton at the glucuronic
acid. At neutral pH, the molecule is deprotonated. When measuring
in mass spectrometry in negative ion mode, the measured mass is
1861 Dalton.
[0081] The terms first, second, third and the like in the
description and in the claims, are used for distinguishing between
similar elements and not necessarily for describing a sequential or
chronological order. The terms are interchangeable under
appropriate circumstances. The embodiments of the invention can
operate in other sequences than described or illustrated
herein.
[0082] Furthermore, the various embodiments, although referred to
as "preferred" or "e.g." or "for example" or "in particular" are to
be construed as exemplary manners in which the invention may be
implemented rather than as limiting the scope of the invention.
[0083] The term "comprising", used in the claims, should not be
interpreted as being restricted to the elements or steps listed
thereafter; it does not exclude other elements or steps. It needs
to be interpreted as specifying the presence of the stated
features, integers, steps or components as referred to, but does
not preclude the presence or addition of one or more other
features, integers, steps or components, or groups thereof. Thus,
the scope of the expression "a pharmaceutical composition
comprising A and B" should not be limited to a pharmaceutical
composition consisting only of components A and B, rather with
respect to the present invention, the only enumerated components of
the pharmaceutical composition are A and B, and further the claim
should be interpreted as including equivalents of those components.
Similarly, the scope of the expression "a method comprising step A
and step B" should not be limited to a method consisting only of
steps A and B, rather with respect to the present invention, the
only enumerated steps of the method are A and B, and further the
claim should be interpreted as including equivalents of those
steps.
[0084] In addition, reference to a feature by the indefinite
article "a" or "an" does not exclude the possibility that more than
one of the features such as for example a component, excipient,
saponin, etc. are present, unless the context clearly requires that
there is one and only one of the features. The indefinite article
"a" or "an" thus usually means "at least one".
BRIEF DESCRIPTION OF THE DRAWINGS
[0085] FIG. 1-1: in vivo HSP27 expression in A431 xenograph `nude`
mouse tumor model treated with 30 mg/kg
cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA), 25 mg/kg
Cetuximab-(Lys-L-HSP27BNA).sup.4 or 25 mg/kg
Cetuximab-(Cys-L-SO1861).
[0086] FIG. 2-1: in vitro enhanced HSP27 gene silencing in EGFR
expressing A431 cells by treatment with
cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA),
Cetuximab-(Lys-L-HSP27BNA).sup.4 or Cetuximab-(Cys-L-SO1861).
[0087] FIG. 3-1: The legends and axes for Figures A, B, C and D are
the same. A. cell killing activity in EGFR expressing cells
(MDA-MB-468) by cetuximab, cetuxamib+10 pM cetuximab-saporin,
cetuximab-Cys-(dendron(-L-SO1861).sup.4).sup.3,9 and
cetuximab-Cys-(dendron(-L-SO1861).sup.4).sup.3,9+10 pM
cetuximab-saporin. B. cell killing activity in HER2 expressing
cells (SK-BR-3) by trastuzumab, trastuzumab+50 pM
trastuzumab-saporin,
Trastuzumab-Cys-(dendron(-L-SO1861).sup.4).sup.4 and
Trastuzumab-Cys-(dendron(-L-SO1861).sup.4).sup.4+50 pM
trastuzumab-saporin. C. cell killing activity in EGFR expressing
cells (HeLa) by cetuximab, cetuxamib+10 pM cetuximab-saporin,
cetuximab-Cys-(dendron(-L-SO1861).sup.4).sup.3,9 and
cetuximab-Cys-(dendron(-L-SO1861).sup.4).sup.3,9+10 pM
cetuximab-saporin. D. cell killing activity in HER2 expressing
cells (JIMT-1) by trastuzumab, trastuzumab+50 pM
trastuzumab-saporin,
Trastuzumab-Cys-(dendron(-L-SO1861).sup.4).sup.4 and
Trastuzumab-Cys-(dendron(-L-SO1861).sup.4).sup.4+50 pM trastuzu
mab-saporin.
[0088] FIG. 4-1: The legends and axes for Figures A, B, C and D are
the same. A. cell killing activity in EGFR.sup.++/CD71.sup.+ cells
(MDA-MB-468) of cetuximab, cetuximab+10 pM CD71mab-saporin,
Cetuximab-Cys-(dendron(-L-SO1861).sup.4).sup.3,9,
Cetuximab-Cys-(dendron(-L-SO1861).sup.4).sup.3,9+10 pM
CD71mab-saporin, Cetuximab-Lys-(dendron(-L-SO1861).sup.4).sup.4,4
or Cetuximab-Lys-(dendron(-L-SO1861).sup.4).sup.4,4+10 pM
CD71mab-saporin. B. cell killing activity in HER2.sup.++/CD71.sup.+
(SK-BR-3) cell lines of trastuzumab, trastuzumab+10 pM
CD71mab-saporin, trastuzumab-Cys-(dendron(-L-SO1861).sup.4).sup.4,
trastuzumab-Cys-(dendron(-L-SO1861).sup.4).sup.4+10 pM
CD71mab-saporin, trastuzumab-Lys-(dendron(-L-SO1861).sup.4).sup.4,7
or trastuzumab-Lys-(dendron(-L-SO1861).sup.4).sup.4,7+10 pM
CD71mab-saporin. C. cell killing activity in EGFR.sup.+/CD71.sup.+
cells (CaSki) of cetuximab, cetuximab+10 pM CD71 mab-saporin,
Cetuximab-Cys-(dendron(-L-SO1861).sup.4).sup.3,9,
Cetuximab-Cys-(dendron(-L-SO1861).sup.4).sup.3,9+10 pM
CD71mab-saporin, Cetuximab-Lys-(dendron(-L-SO1861).sup.4).sup.4,4
or Cetuximab-Lys-(dendron(-L-SO1861).sup.4).sup.4,4+10 pM
CD71mab-saporin. D. cell killing activity in
HER2.sup.+/-/CD71.sup.+ cells (JIMT-1) of trastuzumab,
trastuzumab+10 pM CD71 mab-saporin,
trastuzumab-Cys-(dendron(-L-SO1861).sup.4).sup.4,
trastuzumab-Cys-(dendron(-L-SO1861).sup.4).sup.4+10 pM
CD71mab-saporin, trastuzumab-Lys-(dendron(-L-SO1861).sup.4).sup.4,7
or trastuzumab-Lys-(dendron(-L-SO1861).sup.4).sup.4,7+10 pM
CD71mab-saporin.
[0089] FIG. 5-1: cell killing activity in HER2 expressing cells
(SK-BR-3) of T-DM1, T-DM1+25.6 nM trastuzumab or T-DM1+5.3 nM
trastuzumab-Cys-(dendron(-L-SO1861).sup.4).sup.4.
[0090] FIG. 6-1: HSP27 gene silencing activity of
HSP27BNA-dendron(-L-SO1861).sup.4 compared to the HSP27BNA
alone.
[0091] FIG. 7-1: Schematic representation of release of SO1861 from
dendron(-L-SO1861).sup.4 under acidic conditions.
[0092] FIG. 8-1: The legends and axes for Figures A and B are the
same. A. cell killing activity in EGFR expressing A431 cells by the
`naked` dendron (Dendron(NEM).sup.4), Dendron(NEM).sup.4+10 pM
EGFdianthin, dendron(-L-SO1861).sup.4 or dendron(L-SO1861).sup.4+10
pM EGFdianthin. B. cell killing activity in EGFR expressing HeLa
cells by the `naked` dendron (Dendron(NEM).sup.4),
Dendron(NEM).sup.4+10 pM EGFdianthin, dendron(-L-SO1861).sup.4 or
dendron(L-SO1861).sup.4+10 pM EGFdianthin.
[0093] FIG. 9-1: The legends and axes for Figures A, B and C are
the same. A. Effect of trastuzumab on the cell viability of a range
of cancer cells. B. Effect of cetuximab on the cell viability of a
range of cancer cells. C. Effect of T-DM1 on the cell viability of
a range of cancer cells.
[0094] FIG. 10-1: Schematic representation of the monoclonal
antibody-(SO1861-scaffold-antisense BNA oligo) conjugate.
[0095] FIG. 11-1: Schematic representation of the 1-target
2-component system using a monoclonal antibody bound to a toxin and
the same monoclonal antibody bound to a scaffold comprising
saponin.
[0096] FIG. 12-1: Schematic representation of the 2-target
2-component system using a monoclonal antibody bound to a toxin and
a different monoclonal antibody with a different target bound to a
scaffold comprising a saponin.
[0097] FIG. 13-1: Schematic representation of
Dendron(-L-SO1861).sup.4.
[0098] FIG. 14-1: Schematic representation of
Dendron(-L-SO1861).sup.8.
[0099] FIG. 15-1: Schematic representation of
SO1861-L-trifunctional linker-L-HSP27BNA.
[0100] FIG. 16-1: Reaction scheme of the synthesis of the
Dendron(SO1861).sup.4-HSP27BNA oligo conjugate.
[0101] FIG. 17-1: Model scaffold consisting of four molecular arms
for saponin binding via a Schiff base (imine) and one arm for click
chemistry. The polymeric structure is a pentavalent polyethylene
glycol-based dendrimer of the first generation.
[0102] FIG. 18-1: Cell viability of HER14 cells after treatment
with a pentameric dendrimer (pentrimer), the pentrimer in the
presence of SA1641, dianthin-EGF, dianthin-EFG in the presence of
SA1641, the pentrimer in presence of dianthin-EGF, and the
pentrimer in presence of dianthin-EGF as well as SA1641.
[0103] FIG. 19-1: SO1861 structure with highlighted chemical groups
for conjugation of endosomal escape enhancing saponins to a
polymeric structure. Highlighted groups are aldehyde (black
circle), carboxylic acid (dashed circle), alkene (dashed pentagon),
and alcohol (dashed box).
[0104] FIG. 20-1: A. Standard molecular structure of SO-1861-EMCH
conjugate. Maleimide group is marked with a circle. B. 3D model of
SO1861-EMCH conjugate. Maleimide group is marked with a circle.
[0105] FIG. 21-1: Reaction scheme for the generation of
poly(SO1861) using SO1861-EMCH as monomer, the APS/TMEDA system as
polymerization initiator, and aminopropanethiol as radical
quencher.
[0106] FIG. 22-1: Schematic representation of the DNA approach.
Usage of the principle of DNA-origami to generate a DNA based
scaffold that is able to conjugate and release glycoside molecules.
In addition, one of the DNA strands obtains a click chemistry
moiety that can be used for conjugation to a targeted toxin to form
a functionalized scaffold. bp: base pair.
[0107] FIG. 23-1: Schematic representation of the
poly(peptide-SO1861) approach. Usage of a peptide sequence that can
conjugate and release glycoside molecules and which can react with
itself to form a poly(peptide-SO1861) construct. The poly(peptide)
chain endings can be further modified with click chemistry moieties
(e.g., BCN--NHS linker) that can be used for conjugation to a
toxin.
[0108] FIG. 24-1: Molecular structure of G4-dendron with protected
amino groups.
[0109] FIG. 25-1: Schematic representation of a basic scaffold with
click chemistry function to link any desired effector molecule.
[0110] FIG. 26-1: Schematic representation of a functionalized
scaffold with pre-bound effector molecule and click chemistry
function to link any desired ligand. Optionally, a pH-sensitive
linkage can be provided to release the effector molecule from the
scaffold after reaching the endosomes.
[0111] FIG. 1-2. Antibody-protein toxin+unconjugated SO1861 vivo
study. BT474 tumor bearing mice treated with various concentrations
of Trastuzumab-saporin (i.v.)+1.5 mg/kg unconjugated SO1861 (subQ
injection 1 hour before trastuzumab-saporin treatment).
[0112] FIG. 2-2. unconjugated saponin-mediated endosomal escape and
target cell killing enhancement. A) Cell viability analyses of HeLa
cells (EGFR.sup.+) treated with SO1861, SO1832, SO1862 (isomer of
SO1861) or SO1904 with or without 1.5 pM EGFdianthin B) Cell
viability analyses of HeLa cells (EGFR.sup.+) treated with
EGFdianthin and fixed concentrations of SO1861, SO1832, SO1862
(isomer of SO1861) or SO1904. C) Cell viability analyses of HeLa
cells (EGFR.sup.+) treated with SO1861 or GE1741 with or without
1.5 pM EGFdianthin. D) Cell viability analyses of HeLa cells
(EGFR.sup.+) treated with various QSmix (saponin mixture from
Quillaia Saponaria) with or without 1.5 pM EGFdianthin.
[0113] FIG. 3-2. unconjugated SO1861 versus SO1861-EMCH activity.
EGFR targeted antisense BNA oligo delivery and gene silencing in
cancer cells, according to the invention. A, B, C) Cell viability
analyses of A431 (EGFR.sup.++), HeLa (EGFR.sup.+) or A2058
(EGFR.sup.-) cells treated with SO1861 or SO1861-EMCH with or
without 1.5 pM EG dianthin. D, E) Cell viability analyses of A431
(EGFR.sup.++) or HeLa (EGFR.sup.+) cells treated with SO1861 or
SO1861-N3 with or without 1.5 pM EGFdianthin.
[0114] FIG. 4-2. unconjugated SO1861 versus SO1861-EMCH (labile)
versus SO1861-S(stable). Cell viability analyses of HeLa cells
(EGFR.sup.+) treated with SO1861, SO1861-S(S=HATU, stable linker)
and SO1861-EMCH (labile linker) with or without EGFdiantin.
[0115] FIG. 5-2. EGFR targeted antisense BNA oligonucleotide
delivery and gene silencing. HSP27 mRNA expression analyses of A431
(EGFR.sup.++) and A2058 (EGFR.sup.-) cells treated with
cetuximab-(Cys-L-SO1861).sup.39 or cetuximab-(Cys-L-SO1861).sup.39
100 nM HSP27BNA.
[0116] FIG. 6-2. Tumor targeted antisense BNA oligo nucleotide
delivery and gene silencing in tumor bearing mice. Mice treated
with HSP27BNA+cetuximab-(Cys-L-SO1861).sup.3,9 in A431 tumor
bearing mice reveals efficient tumor targeted gene silencing,
compared to the controls.
[0117] FIG. 1-3: HSP27BNA gene silencing activity of HSP27BNA,
HSP27BNA-SO1861 and HSP27BNA-dendron-(SO1861).sup.4 in A431 cancer
cell lines.
[0118] FIG. 1-4: Tumor targeted protein toxin delivery results in
tumor volume reduction and tumor growth inhibition, in tumor
bearing mice. A. Dose escalation (intraperitoneal, i.p.) of
cetuximab-(Cys-L-SO1861).sup.3,9(Lys-S-dianthin).sup.2 in A431
tumor bearing mice reveals tumor volume reduction, compared to the
control. B. Dose escalation intraperitoneal, i.p, of
cetuximab-(Cys-L-SO1861).sup.3,9(Lys-L-dianthin).sup.2 in A431
tumor bearing mice reveals tumor growth reduction, compared to the
controls. C. Dose escalation intravenous, i.v., of
cetuximab-(Cys-L-SO1861).sup.3,9(Lys-L-dianthin).sup.2 in A431
tumor bearing mice reveals tumor growth reduction, compared to the
controls.
[0119] FIG. 2-4: Tumor targeted antisense BNA oligo nucleotide
delivery and gene silencing in tumor bearing mice. 30 mg/kg
cetuximab-(Cys-L-SO1861).sup.3,9(Lys-L-HSP27BNA).sup.1,8 in A431
tumor bearing mice reveals induced efficient tumor targeted gene
silencing, compared to the controls.
[0120] FIG. 3-4: Tumor targeted antisense BNA oligo nucleotide
delivery and gene silencing in tumor bearing mice. 30 mg/kg
cetuximab-Cys-(SO1861-L-trifunctional
linker-L-HSP27BNA).sup.3'.sup.7 in A431 tumor bearing mice reveals
induced efficient tumor targeted gene silencing, compared to the
controls.
[0121] FIG. 4-4: HER2 or EGFR targeted protein toxin delivery and
cell killing in cancer cells, according to the invention. A.
Trastuzumab-(Cys-L-SO1861).sup.3,8(Lys-L-dianthin).sup.1,7 or
Trastuzumab-(Cys-L-SO1861).sup.3,8(Lys-S-dianthin).sup.1,7
treatment and controls on SK-BR-3 cells (HER2.sup.++) B.
Trastuzumab-(Cys-L-SO1861).sup.3,8(Lys-L-dianthin).sup.1,7 or
Trastuzumab-(Cys-L-SO1861).sup.3,8(Lys-S-dianthin).sup.1,7
treatment and controls on MDA-MB-468 cells (HER2.sup.-). C.
Cetuximab-(Cys-L-SO1861).sup.3,8(Lys-L-dianthin).sup.1,7 or
Cetuximab-(Cys-L-SO1861).sup.3,8(Lys-S-dianthin).sup.1,7 treatment
and controls on A431 cells (EGFR.sup.++) D.
Cetuximab-(Cys-L-SO1861).sup.3,8(Lys-L-dianthin).sup.1,7 or
Cetuximab-(Cys-L-SO1861).sup.3,8(Lys-S-dianthin).sup.1,7 treatment
and controls onA2058 cells (EGFR).
[0122] FIG. 5-4: EGFR targeted antisense BNA oligo delivery and
gene silencing in cancer cells, according to the invention. A.
Cetuximab-(Cys-L-SO1861).sup.3,8(Lys-L-HSP27BNA).sup.1,7 treatment
and controls on A431 cells (EGFR.sup.++) B.
Cetuximab-(Cys-L-SO1861).sup.3,8(Lys-L-HSP27BNA).sup.1,7 treatment
and controls on A2058 cells (EGFR).
[0123] FIG. 6-4: HER2 targeted antisense BNA oligo delivery and
gene silencing in cancer cells, according to the invention.
Trastuzumab-(Cys-L-SO1861).sup.3,8(Lys-L-HSP27BNA).sup.3,5
treatment and controls on SK-BR-3 cells (HER2.sup.++).
[0124] FIG. 7-4: EGFR targeted antisense BNA oligo delivery and
gene silencing in cancer cells, according to the invention. A.
Cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA).sup.3,7
treatment and controls on A431 cells (EGFR.sup.++). B.
Cetuximab-Cys-(SO1861-L-trifunctional
linker-L-HSP27BNA).sup.3'.sup.7 treatment and controls on A2058
cells (EGFR.sup.-).
[0125] FIG. 8-4: Control treatments on all cell lines. Cell
viability when trastuzumab (A), cetuximab (B), T-DM1, (C) free
toxins: saporin and dianthin (D) or saporin coupled to a non-cell
binding IgG (D) are used as treatment on the indicated cell lines
SK-BR-3, JIMT-1, MDA-MB-468, A431, CaSki, HeLa, A2058, BT-474.
[0126] FIG. 9-4: (S)n-(L)(E) concept: mAb-(SO1861).sup.n(protein
toxin).sup.n. Both, SO1861 at the cysteine residues (Cys) and
protein toxin (ribosomal inactivating protein) at the lysine
residues are conjugated to the same antibody (mAb) for delivery and
internalization into the target cells. 1)
mAb-(Cys-L-SO1861).sup.4(Lys-protein toxin).sup.2 bind to its
corresponding cell surface receptor, 2) receptor-mediated
endocytosis the conjugate occurs, 3) at low endolysosomal pH and
appropriate concentration, SO1861 becomes active to enable
endolysosomal escape, 4) release of toxin into cytoplasm occurs and
5) toxin induces cell death.
[0127] FIG. 10-4: (S)n-(L)(E) concept: mAb-(SO1861).sup.n(antisense
BNA oligo).sup.n. Both, SO1861, at the cysteine residues (Cys) and
the antisense BNA oligo nucleotide, at the lysine residues are
conjugated to the same antibody (mAb) for delivery and
internalization into the target cells. 1)
mAb-(Cys-SO1861).sup.4(Lys-BNAoligo).sup.2 bind to its
corresponding cell surface receptor, 2) receptor-mediated
endocytosis of both conjugates occurs, 3) at low endolysosomal pH
and appropriate concentration, SO1861 becomes active to enable
endolysosomal escape, 4) release of BNA oligo into cytoplasm occurs
and 5) target gene silencing is induced.
[0128] FIG. 11-4: (S)n--(L)(E) concept:
mAb-(SO1861-scaffold-antisense BNA oligo)n. the
(SO1861-trifunctional linker-BNAoligo).sup.n is conjugated to an
antibody (mAb) for delivery and internalization into the target
cells. 1) mAb-(SO1861-trifunctional linker-BNAoligo).sup.4 binds to
its corresponding cell surface receptor, 2) receptor-mediated
endocytosis of both conjugates occurs, 3) at low endolysosomal pH
and appropriate concentration, SO1861 becomes active to enable
endolysosomal escape, 4) release of BNA oligo into cytoplasm occurs
and 5) target gene silencing is induced.
[0129] FIG. 12-4: Antibody-SO1861 conjugation procedure. Shown is
the coupling reaction of the linking of four moieties of a
plant-derived saponin SO1861 to the four cysteines in the light
chain of an antibody. First, the disulphide bonds in the IgG are
disrupted under influence of exposure to TCEP
(Tris(2-carboxyethyl)phosphine); second, the saponin SO1861
comprising a chemical linker bound to it, is added together with
trifluoro acetic acid, and four saponin moieties are linked to the
IgG. For producing cleavable `ready to conjugate` saponins the
aldehyde group of SO1861 was reacted with an EMCH
(.epsilon.-maleimidocaproic acid hydrazide) linker. The hydrazide
group of EMCH forms an acid cleavable hydrazone bond with the
aldehyde of SO1861. At the same time the EMCH linker presents a
maleimide group that is thiol (sulfhydryl group) reactive and thus
can be conjugated to thiols of the IgG, i.e. the ligand moiety.
Herewith, an endosomal escape enhancing conjugate of the invention
is provided, and/or a first binding molecule of the invention is
provided.
[0130] FIG. 1-5: 1T2C in vivo activity. The 1T2C combination of 50
mg/kg cetuximab-(Cys-L-SO1861).sup.4+25 mg/kg
cetuximab-(-L-HSP27BNA).sup.4 in A431 tumor bearing mice reveals
strong tumor targeted gene silencing, compared to the controls.
[0131] FIG. 2-5: 1T2C in vivo activity. The 1T2C combination of 40
mg/kg trastuzumab-(Cys-L-SO1861).sup.4+0.02/0.03 mg/kg
trastuzumab-saporin in a PDX tumor mouse model (high HER2
expression) shows effective tumor growth inhibition.
[0132] FIG. 3-5: 1-target 2-component. EGFR targeted cell killing
in A431 cells (EGFR.sup.++) (A, C) and CaSKi cells (EGFR.sup.+) (B,
D) by a therapeutic combination according to the invention. A, B)
Cetuximab-(Cys-L-SO1861).sup.3,7 titration+fixed concentration 10
pM cetuximab-saporin and controls on A431 (A) and CaSKi (B) cells.
C, D) Cetuximab-saporin titration+fixed concentration of 75 nM
cetuximab-(Cys-L-SO1861).sup.3,7 and controls on A431 (C) and CaSKi
(D) cells. Remark: For target receptor expression data of each cell
line (determined by FACS analysis) see table 19.
[0133] FIG. 4-5: 1-target 2-component. EGFR targeted cell killing
in HeLa cells (EGFR.sup.+/-) (A, C) and A2058 cells (EGFR.sup.-)
(B, D) by a therapeutic combination according to the invention. A,
B) Cetuximab-(Cys-L-SO1861).sup.3,7 titration+fixed concentration
10 pM cetuximab-saporin and controls on HeLa (A) and CaSKi (B)
cells. C, D) Cetuximab-saporin titration+fixed concentration of 75
nM cetuximab-(Cys-L-SO1861).sup.3,7 and controls on Hela (C) and
A2058 (D) cells. Remark: For target receptor expression data of
each cell line (determined by FACS analysis) see table 19.
[0134] FIG. 5-5: 1-target 2-component. HER2 targeted cell killing
in SKBR3 cells (HER2.sup.++) (A, B) by a therapeutic combination
according to the invention. A) Trastuzumab-(Cys-L-SO1861).sup.4
titration+fixed concentration 50 pM trastuzumab-saporin and
controls on SKBR3 cells. B) Trastuzumab-saporin titration+fixed
concentration of 2.5 nM trastuzumab-(Cys-L-SO1861).sup.4 and
controls on SKBR3 cells. Remark: For target receptor expression
data of each cell line (determined by FACS analysis) see table
19.
[0135] FIG. 6-5: 1-target 2-component. HER2 targeted cell killing
in JIMT-1 cells (HER2.sup.+/-) (A, C) and MDA-MB-468 cells
(HER2.sup.-) (B, D) by a therapeutic combination according to the
invention. A, B) Trastuzumab-(Cys-L-SO1861).sup.4 titration+fixed
concentration of 50 pM trastuzumab-saporin and controls on JIMT-1
(A) and MDA-MB-468 (B) cells. C, D) Trastuzumab-saporin
titration+fixed concentration of 2.5 nM
trastuzumab-(Cys-L-SO1861).sup.4 and controls on JIMT-1 (C) and
MDA-MB-468 (D) cells. Remark: For target receptor expression data
of each cell line (determined by FACS analysis) see table 19.
[0136] FIG. 7-5: Chloroquine inhibits the 1-target 2-component.
HER2 and EGFR targeted cell killing in SK-BR-3 (HER2.sup.++) and
A431 cells (EGFR.sup.++), by a therapeutic combination according to
the invention+chloroquine. A) Trastuzumab-saporin titration+fixed
concentration of 5 nM trastuzumab-(Cys-L-SO1861).sup.4+0.5 pM
chloroquine and control on SK-BR-3 cells. B) Cetuximab-saporin
titration+fixed concentration of 5 nM
cetuximab-(Cys-L-SO1861).sup.3,8+0.5 pM chloroquine and control on
A431 cells. Remark: For target receptor expression data of each
cell line (determined by FACS analysis) see table 19.
[0137] FIG. 8-5: 1-target 2-component. EGFR targeted gene silencing
in A431 cells (EGFR.sup.++) and A2058 cells (EGFR.sup.-) by a
therapeutic combination according to the invention. A,B)
Cetuximab-(Cys-L-SO1861).sup.3,8 titration+fixed concentration of
100 nM Cetuximab-(Lys-L-HSP27BNA).sup.4 and control on A431 cells
(A) and A2058 cells (B). C, D) Cetuximab-(Lys-L-HSP27BNA).sup.4
titration+fixed concentration of 77 nM
Cetuximab-(Cys-L-SO1861).sup.3,8 and control on A431 cells (C) and
A2058 cells (D). Remark: For target receptor expression data of
each cell line (determined by FACS analysis) see table 19.
[0138] FIG. 9-5: 2-target 2-component. A) EGFR and HER2 targeted
cell killing in MDA-MB-468 cells (EGFR.sup.++) and HeLa cells
(EGFR.sup.+/-) and HER2 targeted cell killing in SK-BR-3 cells
(HER2.sup.++) and JIMT-1 cells (HER2.sup.+/-) by a therapeutic
combination according to the invention. A)
Cetuximab-Cys-(dendron(-L-SO1861).sup.4).sup.3'.sup.9
titration+fixed concentration 10 pM cetuximab-saporin and controls
on MDA-MB-468 cells (A) and HeLa cells (B). C,D)
Trastuzumab-Cys-(dendron(-L-SO1861).sup.4).sup.4 titration+fixed
concentration 50 pM trastuzumab-saporin and controls on SK-BR-3
cells (C) and JIMT-1 cells (D). Remark: For target receptor
expression data of each cell line (determined by FACS analysis) see
table 19.
[0139] FIG. 10-5: 1-target 2-component. SK-BR-3 cells
(HER2.sup.+/-) can efficiently be killed with the therapeutic
combination according to the invention, Tratuzumab-saporin+2.5 nM
trastuzumab-(Cys-L-SO1861).sup.4, however titration of T-DM1+2.5 nM
trastuzumab-(Cys-L-SO1861).sup.4 is not effective at such low toxin
concentrations. T-DM1 is Trastuzumab-emtansine (Kadcyla.RTM.),
carrying .about.3.5 emtansine (DM1) toxin molecules per antibody
(DAR3.5). Remark: For target receptor expression data of each cell
line (determined by FACS analysis) see table 19.
[0140] FIG. 11-5: Control treatments on all cell lines. A-D) Cell
viability when trastuzumab (A), cetuximab (B), T-DM1, (C) free
toxins: saporin and dianthin (D) or saporin coupled to a non-cell
binding IgG (D) are treated with the indicated cell lines SK-BR-3,
JIMT-1, MDA-MB-468, A431, CaSki, HeLa, A2058, BT-474. Remark: For
target receptor expression data of each cell line (determined by
FACS analysis) see table 19.
[0141] FIG. 12-5: 1-target 2-component. EGFR targeted cell killing
in A431 cells (EGFR.sup.++) (A) and CaSKi cells (EGFR.sup.+) (B)
and A2058 cells (EGFR.sup.-) by a therapeutic combination according
to the invention. A, B, C) Cetuximab-(Cys-L-QSmix).sup.4'.sup.1
titration+fixed concentration 10 pM cetuximab-saporin or 10 pM
cetuximab-dianthin and controls in A431 cells (A), CaSKi cells (B)
and A2058 cells (C). QSmix is a mixture of saponins from an extract
Quillaja saponaria. Remark: For target receptor expression data of
each cell line (determined by FACS analysis) see table 19.
[0142] FIG. 13-5: 1-target 2-component concept:
mAb1-SO1861+mAb1-protein toxin. SO1861 and toxin (ribosomal
inactivating protein) are each, independently, conjugated to an
antibody (mAb1) for delivery and internalization into target cells.
1) mAb1-SO1861 and mAb1-protein toxin bind to the cell surface
receptor, 2) receptor-mediated endocytosis of both conjugates
occurs, 3) at low endolysosomal pH and appropriate concentration,
SO1861 becomes active to enable endolysosomal escape, 4) release of
toxin into cytoplasm occurs and 5) toxin induces cell death
[0143] FIG. 14-5: 1-target 2-component concept:
mAb1-SO1861+mAb2-BNA oligo. SO1861 and antisense BNA oligo
nucleotide are each, independently, conjugated to an antibody
(mAb1) for delivery and internalization into target cells. 1)
mAb1-SO1861 and mAb1-BNAoligo bind to the cell surface receptor, 2)
receptor-mediated endocytosis of both conjugates occurs, 3) at low
endolysosomal pH and appropriate concentration, SO1861 becomes
active to enable endolysosomal escape, 4) release of BNA oligo into
cytoplasm occurs and 5) target gene silencing.
[0144] FIG. 15-5: 1-target 2-component concept:
mAb1-(scaffold(-SO1861)n)n+mAb1-protein toxin.
Dendron(.about.SO1861).sup.n and protein toxin (ribosomal
inactivating protein) are each, independently, conjugated to an
antibody (mAb1) for delivery and internalization into target cells.
1) mAb1-dendron(-SO1861).sup.4 and mAb1-protein toxin bind to the
cell surface receptor, 2) receptor-mediated endocytosis of both
conjugates occurs, 3) at low endolysosomal pH and appropriate
concentration, SO1861 becomes active to enable endolysosomal
escape, 4) release of toxin into cytoplasm occurs and 5) toxin
induces cell death.
[0145] FIG. 1-6: The 2T2 component system tested in A431 tumor
bearing mice model reveals tumor regression.
[0146] FIG. 2-6: The 2T2 component system tested in A431 tumor
bearing mice model reveals tumor regression and eradication.
[0147] FIG. 3-6: 2-target 2-component. EGFR/HER2 targeted cell
killing in A431 cells (EGFR.sup.++/HER2.sup.+/-) (A, C) and CaSKi
cells (EGFR.sup.++/HER2.sup.+/-) (B, D) by a therapeutic
combination according to the invention. A, B)
Cetuximab-(Cys-L-SO1861).sup.3,7 titration+fixed concentration 50
pM trastuzumab-saporin and controls on A431 cells. C, D)
Trastuzumab-saporin titration+fixed concentration of 75 nM
cetuximab-(Cys-L-SO1861).sup.3,7 and controls on Caski cells. The
legends and/or axes are the same for all the A,B, C or D.
[0148] FIG. 4-6: 2-target 2-component. EGFR/HER2 targeted cell
killing in HeLa cells (EGFR.sup.+/-/HER2.sup.+/-) (A, C) and A2058
cells (EGFR/HER2.sup.+/-) (B, D) by a therapeutic combination
according to the invention. A, B) Cetuximab-(Cys-L-SO1861).sup.3,7
titration+fixed concentration 50 pM trastuzumab-saporin and
controls on HeLa cells. C, D) Trastuzumab-saporin titration+fixed
concentration of 75 nM cetuximab-(Cys-L-SO1861).sup.3,7 and
controls on A2058 cells. The legends and/or axes are the same for
all the A,B, C or D.
[0149] FIG. 5-6: 2-target 2-component. HER2/EGFR targeted cell
killing in SKBR3 cells (HER2.sup.++/EGFR.sup.+/-) (A, B) by a
therapeutic combination according to the invention. A
Trastuzumab-(Cys-L-SO1861).sup.4 titration+fixed concentration 1.5
pM EGFdianthin and controls on SKBR3 cells. B) EGFdianthin
titration+fixed concentration of 2.5 nM
trastuzumab-(Cys-L-SO1861).sup.4 and controls on SKBR3 cells.
[0150] FIG. 6-6: 2-target 2-component. HER2/EGFR targeted cell
killing in JIMT-1 cells (HER2.sup.+/-EGFR.sup.+/-) (A, C) and
MDA-MB-468 cells (HER2.sup.-/EGFR.sup.++) (B, D) by a therapeutic
combination according to the invention. A, B)
Trastuzumab-(Cys-L-SO1861).sup.4 titration+fixed concentration 1.5
pM EGFdianthin and controls on JIMT-1 cells. C, D) EGFdianthin
titration+fixed concentration of 2.5 nM
trastuzumab-(Cys-L-SO1861).sup.4 and controls on MDA-MB-468 cells.
The legends and/or axes are the same for all the A,B, C or D.
[0151] FIG. 7-6: 2-target 2-component. HER2/EGFR targeted cell
killing in SKBR3 cells (HER2.sup.++/EGFR.sup.+/-) (A, B) by a
therapeutic combination according to the invention. A)
Trastuzumab-(Cys-L-SO1861).sup.4 titration+fixed concentration 10
pM cetuximab-saporin and controls on SKBR3 cells. B)
Cetuximab-saporin titration+fixed concentration of 2.5 nM
trastuzumab-(Cys-L-SO1861).sup.4 and controls on SKBR3 cells.
[0152] FIG. 8-6: 2-target 2-component. HER2/EGFR targeted cell
killing in JIMT-1 cells (HER2.sup.+/-EGFR.sup.+/-) (A, C) and
MDA-MB-468 cells (HER2.sup.-/EGFR.sup.++) (B, D) by a therapeutic
combination according to the invention. A, B)
Trastuzumab-(Cys-L-SO1861).sup.4 titration+fixed concentration 10
pM cetuximab-saporin and controls on JIMT-1 cells. C, D)
Cetuximab-saporin titration+fixed concentration of 2.5 nM
trastuzumab-(Cys-L-SO1861).sup.4 and controls on MDA-MB-468 cells.
The legends and/or axes are the same for all the A,B, C or D.
[0153] FIG. 9-6: Chloroquine inhibits the 2-target 2-component.
EGFR/HER2, EGFR/CD71 or HER2/CD71 targeted cell killing in A431
cells (EGFR.sup.++/HER2.sup.+/-/CD71.sup.+) (A, B), MDA-MB-468
cells (EGFR.sup.++/HER2.sup.-/CD71.sup.+) (C) or SK-BR-3
(HER2.sup.++/EGFR.sup.+/-/CD71.sup.+) (D) by a therapeutic
combination according to the invention+chloroquine. A)
Trastuzumab-dianthin or trastuzumab-saporin titration+fixed
concentration of 75 nM cetuximab-(Cys-L-SO1861).sup.3,9+800 nM
chloroquine and controls on A431 cells. B) CD71mab-saporin
titration+fixed concentration of 10.5 nM
cetuximab-(Cys-L-SO1861).sup.3,9+500 nM chloroquine and control on
A431 cells. C) CD71mab-saporin titration+fixed concentration of
10.5 nM cetuximab-(Cys-L-SO1861).sup.3,9+500 nM chloroquine and
control on MDA-MB-468 cells. D) CD71mab-saporin titration+fixed
concentration of 5 nM trastuzumab-(Cys-L-SO1861).sup.3'.sup.9+500
nM chloroquine and control on SK-BR-3 cells.
[0154] FIG. 10-6: 2-target 2-component. EGFR/HER2 targeted gene
silencing in A431 cells (EGFR.sup.++/HER2.sup.+/-) (A) and A2058
cells (EGFR/HER2.sup.+/-) (B) by a therapeutic combination
according to the invention. A) Cetuximab-(Cys-L-SO1861).sup.3,9
titration+fixed concentration of 100 nM
trastuzumab-(Lys-L-HSP27BNA).sup.4,4 and control on A431 cells (A)
and A2058 cells (B). C, D) Trastuzumab-(Lys-L-HSP27BNA).sup.4,4
titration+fixed concentration of 77 nM
cetuximab-(Cys-L-SO1861).sup.3,9 and controls on A431 cells (A) and
A2058 cells (B). The legends and/or axes are the same for all the
A,B, C or D.
[0155] FIG. 11-6: 2-target 2-component. A) EGFR/CD71 or HER2/CD71
targeted cell killing in MDA-MB-468 cells (EGFR.sup.++/CD71.sup.+)
(A) HeLa cells (EGFR.sup.+/-/CD71.sup.+), SK-BR-3 cells
(HER2.sup.++/CD71.sup.+) (B) and JIMT-1 cells
(HER2.sup.+/-/CD71.sup.+) by a therapeutic combination according to
the invention. A) Cetuximab-Cys-(dendron(-L-SO1861).sup.4).sup.3,9
titration+fixed concentration 10 pM CD71 mab-saporin and controls
on MDA-MB-468 cells. B) A)
Cetuximab-Cys-(dendron(-L-SO1861).sup.4).sup.3,9 titration+fixed
concentration 10 pM CD71mab-saporin and controls on HeLa cells. C)
Trastuzumab-Cys-(dendron(-L-SO1861).sup.4).sup.4 titration+fixed
concentration 10 pM CD71mab-saporin and controls on SK-BR-3 cells.
D) Trastuzumab-Cys-(dendron(-L-SO1861).sup.4).sup.4 titration+fixed
concentration 10 pM CD71mab-saporin and controls on JIMT-1
cells.
[0156] FIG. 12-6: 2-target 2-component versus T-DM1. A431 cells
(EGFR.sup.++/HER2.sup.+/-) can efficiently be killed with the
therapeutic combination according to the invention,
Tratuzumab-saporin+75 nM cetuximab-(Cys-L-SO1861).sup.3,9, however
titration of T-DM1+75 nM cetuximab-(Cys-L-SO1861).sup.3,9 is not
effective at such low toxin concentrations. T-DM1 is
Trastuzumab-emtansine (Kadcyla.RTM.), carrying=3.5 emtansine (DM1)
toxin molecules per antibody.
[0157] FIG. 13-6: 2-target 2-component. EGFR/CD71 and EGFR/HER2
targeted cell killing in A431 cells (EGFR+.sup.++/HER2.sup.+/-) (A)
and CaSKi cells (EGFR.sup.++/HER2.sup.+/-) (B) and A2058 cells
(EGFR/HER2.sup.+/-) by a therapeutic combination according to the
invention. A, B,C) Cetuximab-(Cys-L-QSmix).sup.4'.sup.1
titration+fixed concentration 10 pM trastuzumab-saporin or 10 pM
CD71mab-saporin and controls on A431 cells (A). CaSKi cells (B) and
A2058 cells (C). QSmix is a mixture of saponins from an extract
Quillaja saponaria.
[0158] FIG. 14-6: Control treatments on all cell lines. A-D) Cell
viability when trastuzumab (A), cetuximab (B), T-DM1, (C) free
toxins: saporin and dianthin (D) or saporin coupled to a non-cell
binding IgG (D) are treated with the indicated cell lines SK-BR-3,
JIMT-1, MDA-MB-468, A431, CaSki, HeLa, A2058, BT-474. The legends
and/or axes are the same for all the A,B, C or D.
[0159] FIG. 15-6: 2-target 2-component concept:
mAb1-SO1861+mAb2-protein toxin. SO1861 and toxin (ribosomal
inactivating protein) are each, separately, conjugated to an
antibody (mAb) for delivery and internalization into target cells.
1) mAb1-SO1861 and mAb2-protein toxin bind to their corresponding
cell surface receptor, 2) receptor-mediated endocytosis of both
conjugates occurs, 3) at low endolysosomal pH and appropriate
concentration, SO1861 becomes active to enable endolysosomal
escape, 4) release of toxin into cytoplasm occurs and 5) toxin
induces cell death.
[0160] FIG. 16-6: 2-target 2-component concept:
mAb1-SO1861+mAb2-BNA oligo. SO1861 and antisense BNA oligo
nucleotide are each, separately, conjugated to an antibody (mAb)
for delivery and internalization into target cells. 1) mAb1-SO1861
and mAb2-BNAoligo bind to their corresponding cell surface
receptor, 2) receptor-mediated endocytosis of both conjugates
occurs, 3) at low endolysosomal pH and appropriate concentration,
SO1861 becomes active to enable endolysosomal escape, 4) release of
BNA oligo into cytoplasm occurs and 5) target gene silencing.
[0161] FIG. 1-7: EGFR targeted gene silencing in A431 cells
(EGFR.sup.++) (A) and A2058 cells (EGFR.sup.-) (B) by a combination
with unconjugated SO1861-EMCH and HSP27BNA, and SO1861-EMCH and
Cetuximab-(Lys-L-HSP27BNA) titration+fixed concentration of 4000 nM
SO1861-EMCH and controls on A431 cells (A) and A2058 cells (B). (A)
and (B) are HSP27BNA or Cetuximab-HSP27BNA conjugate (nM) based
graphs. EGFR targeted gene silencing in A431 cells (EGFR.sup.++)
(A) and A2058 cells (EGFR.sup.-) (B) by a combination with
unconjugated SO1861-EMCH with free HSP27BNA, and SO1861-EMCH and
conjugate Cetuximab-(Lys-L-HSP27BNA) titration+fixed concentration
of 4000 nM SO1861-EMCH and controls on A431 cells (C) and A2058
cells (D). (C) and (D) are HSP27BNA (nM) based graphs. The legend
with FIGS. 1-7B and D also applies for FIGS. 1-7A and C.
DETAILED DESCRIPTION
[0162] In order for a bioactive molecule to work, the molecule must
be able to engage with its target, e.g. in the blood serum, on the
outside of the cell surface or inside a cell or an organelle. The
active moiety of almost all protein-based targeted toxins, e.g.,
must enter the cytosol of the target cell to mediate its target
modulatory effect. In many constellations the toxin remains
ineffective since (1) the targeting moiety is poorly internalized
and remains bound to the outside of the cells, (2) is recycled back
to the cell surface after internalization or (3) transported to the
endolysosomes where it is degraded. Although these fundamental
issues are known for decades and more than 500 targeted toxins have
been investigated in the past decades, the problems have not been
solved yet and only one antibody-targeted protein toxin,
moxetumomab pasudotox-tdfk (LUMOXITI.RTM., AstraZeneca
Pharmaceuticals LP), has been approved for relapsed or refractory
hairy cell leukemia by the FDA to date.
[0163] To overcome these problems, many strategies have been
described including approaches to redirect the toxins to endogenous
cellular membrane transport complexes of the biosynthetic pathway
in the endoplasmic reticulum and techniques to disrupt or weaken
the membrane integrity of endosomes, i.e. the compartments of the
endocytic pathway in a cell, and thus facilitating the endosomal
escape. This comprises the use of lysosomotropic amines, carboxylic
ionophores, calcium channel antagonists, various cell-penetrating
peptides of viral, bacterial, plant, animal, human and synthetic
origin, other organic molecules and light-induced techniques.
Although the efficacy of the targeted toxins was typically
augmented in cell culture hundred- or thousand-fold, in exceptional
cases more than million-fold, the requirement to co-administer
endosomal escape enhancers with other substances harbors new
problems including additional side effects, loss of target
specificity, difficulties to determine the therapeutic window and
cell type-dependent variations.
[0164] All strategies, including physicochemical techniques,
require enhancer molecules that interact more or less directly with
membranes and comprise essentially small chemical molecules,
secondary metabolites, peptides and proteins. A common feature of
all these substances is that they are per se not target
cell-specific and distribute with other kinetics than the targeted
toxins. This is one major drawback of the current approaches.
[0165] The present invention will be described with respect to
particular embodiments but the invention is not limited thereto but
only by the claims. The embodiments of the invention described
herein can operate in combination and cooperation, unless specified
otherwise.
[0166] While the invention has been described in terms of several
embodiments, it is contemplated that alternatives, modifications,
permutations and equivalents thereof will become apparent to one
having ordinary skill in the art upon reading the specification and
upon study of the drawings and graphs. The invention is not limited
in any way to the illustrated embodiments. Changes can be made
without departing from the scope which is defined by the appended
claims.
[0167] An aspect of the invention relates to a therapeutic molecule
with chemical structure of COMPOUND I:
A1.sub.m((-L9.sub.w)((-L1.sub.q-B1.sub.n).sub.u((-L2.sub.r-L3.sub.s)(-L4-
.sub.v-C).sub.p).sub.t)).sub.x (compound I),
wherein A1 is a first ligand if B1 is a first effector moiety, or
A1 is the first effector moiety if B1 is the first ligand; C is a
saponin; m=0 or 1 if A1 is the first ligand and B1 is the first
effector moiety; m=0-32 if A1 is the first effector moiety and B1
is the first ligand; n=0 or 1 if B1 is the first ligand and A1 is
the first effector moiety, or if A1 is the first ligand and B1 is
the first effector moiety; p=any of 1-128; L1 is at least one
linker for covalently coupling two chemical groups; L2 is at least
one linker for covalently coupling two chemical groups; L3 is at
least one oligomeric or polymeric scaffold for covalently coupling
two chemical groups; L4 is at least one linker for covalently
coupling two chemical groups; L9 is a tri-functional linker for
covalently coupling three chemical groups; q=0 or 1; r=0 or 1; s=0
or 1; t=0, 1 or 2 if s=0, and t=any of 0-16 if s=1; u=any of 0-32
if A1 is the first ligand and B1 is the first effector moiety, or
u=1 if A1 is the first effector moiety and B1 is the first ligand;
v=0 or 1; w=1 or 0; and x=1-16.
[0168] An aspect of the invention relates to a therapeutic
combination comprising the therapeutic molecule according to the
invention and a second therapeutic molecule with chemical structure
of COMPOUND II:
A2.sub.a((-L10.sub.i)((-L5.sub.d-B2.sub.b).sub.h((-L6.sub.e-L7.sub.f)(-L-
8.sub.i-C).sub.c).sub.q)).sub.k (compound II),
wherein A2 is a second ligand if B2 is a second effector moiety, or
A2 is the second effector moiety if B2 is the second ligand; C is a
saponin; a=0 or 1 if A2 is the second ligand and B2 is the second
effector moiety, or a=0-32 if A2 is the second effector moiety and
B2 is the second ligand; b=0 or 1 if B2 is the second ligand and A2
is the second effector moiety, or if A2 is the second ligand and B2
is the second effector moiety; c=any of 1-128; L5 is at least one
linker for covalently coupling two chemical groups; L6 is at least
one linker for covalently coupling two chemical groups; L7 is at
least one oligomeric or polymeric scaffold for covalently coupling
two chemical groups; L8 is at least one linker for covalently
coupling two chemical groups; L10 is a tri-functional linker for
covalently coupling three chemical groups; d=0 or 1; e=0 or 1; f=0
or 1; g=0, 1 or 2 if f=0, and g=any of 0-16 if f=1; h=any of 0-32
if A2 is the second ligand and B2 is the second effector moiety, or
h=1 if A2 is the second effector moiety and B2 is the second
ligand; i=0 or 1; j=1 or 0 and; k=1-16.
[0169] An embodiment is the second therapeutic molecule of the
invention, wherein g=0, 1 or 2 if f=0 and t>0, g=1 or 2 if f=0
and t=0, g=any of 0-16 if f=1 and t>0, and g=any of 1-16 if f=1
and t=0.
An embodiment is the therapeutic molecule of the invention or the
second therapeutic molecule of the invention, wherein the first
ligand A1 or B1 and/or the second ligand A2 or B2 comprise(s) or
consist(s) of an immunoglobulin, a binding domain of an
immunoglobulin or a binding fragment of an immunoglobulin, such as
an antibody, an IgG, a molecule comprising or consisting of a Vhh
domain or Vh domain, a Fab, an scFv, an Fv, a dAb, an F(ab).sub.2,
Fcab fragment, or comprise(s) or consist(s) of at least one
non-proteinaceous ligand and/or at least one proteinaceous ligand,
the ligand for binding to a cell-surface molecule such as EGF or a
cytokine, with the proviso that the first ligand and the second
ligand are the same or are different.
[0170] An embodiment is the therapeutic molecule of the invention
or the second therapeutic molecule of the invention, wherein the
first ligand A1 or B1 and/or the second ligand A2 or B2 bind(s) to
a tumor-cell epitope, preferably a tumor-cell specific epitope, of
a tumor-cell receptor, preferably a tumor-cell specific receptor,
preferably selected from CD71, CA125, EpCAM(17-1A), CD52, CEA,
CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin
alpha-V beta-3, HER2, EGFR, CD20, CD22, Folate receptor 1, CD146,
CD56, CD19, CD138, CD27L receptor, PSMA, CanAg, integrin-alphaV,
CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239, CD70, CD123,
CD352, DLL3, CD25, ephrinA4, MUC1, Trop2, CEACAM5, CEACAM6, HER3,
CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1,
CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2, more preferably selected from
CD71, EGFR and HER2, with the proviso that the first ligand and the
second ligand bind to the same or to a different tumor-cell
epitope, preferably a tumor-cell specific epitope, and/or wherein
the tumor-cell receptor, preferably the tumor-cell specific
receptor, to which the first ligand can bind is the same as, or is
different from the tumor-cell receptor, preferably the tumor-cell
specific receptor, to which the second ligand can bind.
[0171] An embodiment is the therapeutic molecule of the invention
or the second therapeutic molecule of the invention, wherein the
first ligand A1 or B1 and/or the second ligand A2 or B2 comprise(s)
or consist(s) of cetuximab, daratumumab, gemtuzumab, trastuzumab,
panitumumab, brentuximab, inotuzumab, moxetumomab, polatuzumab,
obinutuzumab, OKT-9 anti-CD71 monoclonal antibody of the IgG type,
pertuzumab, rituximab, ofatumumab, Herceptin, alemtuzumab,
pinatuzumab, OKT-10 anti-CD38 monoclonal antibody, an antibody of
Table A2 or Table A3 or Table A4, preferably cetuximab or
trastuzumab or OKT-9, or at least one tumor-cell receptor
binding-domain thereof and/or at least one tumor-cell receptor
binding-fragment thereof which are preferably (a) tumor-cell
specific receptor binding-domain(s) and/or (a) tumor-cell specific
receptor binding-fragment(s), with the proviso that the first
ligand is the same or different from the second ligand.
[0172] An embodiment is the therapeutic molecule of the invention
or the second therapeutic molecule of the invention, wherein the
first ligand A1 or B1 is internalized by a tumor cell after binding
of the first ligand to its binding partner on the tumor cell, and
wherein preferably binding of the first ligand to the tumor cell is
followed by tumor-cell receptor-mediated internalization, e.g. via
endocytosis, of a complex of the first ligand and the binding
partner of the first ligand on the tumor cell.
[0173] An embodiment is the therapeutic molecule of the invention
or the second therapeutic molecule of the invention, wherein the
second ligand A2 or B2 is internalized by a tumor cell after
binding of the second ligand to its binding partner on the tumor
cell, and wherein preferably binding of the second ligand to the
tumor cell is followed by tumor-cell receptor-mediated
internalization, e.g. via endocytosis, of a complex of the second
ligand and the binding partner of the second ligand on the tumor
cell.
[0174] An embodiment is the therapeutic molecule of the invention
or the second therapeutic molecule of the invention, wherein the
first effector moiety A1 or B1 and/or the second effector moiety A2
or B2 comprise(s) or consist(s) of at least one of any one or more
of an oligonucleotide, a nucleic acid and a xeno nucleic acid,
preferably selected from any one or more of a vector, a gene, a
cell suicide inducing transgene, deoxyribonucleic acid (DNA),
ribonucleic acid (RNA), anti-sense oligonucleotide (ASO, AON),
short interfering RNA (siRNA), microRNA (miRNA), DNA aptamer, RNA
aptamer, mRNA, mini-circle DNA, peptide nucleic acid (PNA),
phosphoramidate morpholino oligomer (PMO), locked nucleic acid
(LNA), bridged nucleic acid (BNA), 2'-deoxy-2'-fluoroarabino
nucleic acid (FANA), 2'-O-methoxyethyl-RNA (MOE),
2'-0,4'-aminoethylene bridged nucleic acid, 3'-fluoro hexitol
nucleic acid (FHNA), a plasmid, glycol nucleic acid (GNA) and
threose nucleic acid (TNA), or a derivative thereof, more
preferably a BNA, for example a BNA for silencing HSP27 protein
expression, with the proviso that the first effector moiety and the
second effector moiety are the same or are different.
[0175] An embodiment is the therapeutic molecule of the invention
or the second therapeutic molecule of the invention, wherein the
first effector moiety A1 or B1 and/or the second effector moiety A2
or B2 comprise(s) or consist(s) of at least one proteinaceous
molecule, preferably selected from any one or more of a peptide, a
protein, an enzyme such as urease and Cre-recombinase, a
proteinaceous toxin, a ribosome-inactivating protein, at least one
protein toxin selected from Table A5 and/or a bacterial toxin, a
plant toxin, more preferably selected from any one or more of a
viral toxin such as apoptin; a bacterial toxin such as Shiga toxin,
Shiga-like toxin, Pseudomonas aeruginosa exotoxin (PE) or exotoxin
A of PE, full-length or truncated diphtheria toxin (DT), cholera
toxin; a fungal toxin such as alpha-sarcin; a plant toxin including
ribosome-inactivating proteins and the A chain of type 2
ribosome-inactivating proteins such as dianthin e.g. dianthin-30 or
dianthin-32, saporin e.g. saporin-S3 or saporin-S6, bouganin or
de-immunized derivative debouganin of bouganin, shiga-like toxin A,
pokeweed antiviral protein, ricin, ricin A chain, modeccin,
modeccin A chain, abrin, abrin A chain, volkensin, volkensin A
chain, viscumin, viscumin A chain; or an animal or human toxin such
as frog RNase, or granzyme B or angiogenin from humans, or any
fragment or derivative thereof; preferably the protein toxin is
dianthin and/or saporin, with the proviso that the first effector
moiety/moieties and the second effector moiety/moieties are the
same or are different.
[0176] An embodiment is the therapeutic molecule of the invention
or the second therapeutic molecule of the invention, wherein the
first effector moiety A1 or B1 and/or the second effector moiety A2
or B2 comprise(s) or consist(s) of at least one payload, preferably
selected from any one or more of a toxin targeting ribosomes, a
toxin targeting elongation factors, a toxin targeting tubulin, a
toxin targeting DNA and a toxin targeting RNA, more preferably any
one or more of emtansine, pasudotox, maytansinoid derivative DM1,
maytansinoid derivative DM4, monomethyl auristatin E (MMAE,
vedotin), monomethyl auristatin F (MMAF, mafodotin), a
Calicheamicin, N-Acetyl-.gamma.-calicheamicin, a
pyrrolobenzodiazepine (PBD) dimer, a benzodiazepine, a CC-1065
analogue, a duocarmycin, Doxorubicin, paclitaxel, docetaxel,
cisplatin, cyclophosphamide, etoposide, docetaxel, 5-fluorouracyl
(5-FU), mitoxantrone, a tubulysin, an indolinobenzodiazepine,
AZ13599185, a cryptophycin, rhizoxin, methotrexate, an
anthracycline, a camptothecin analogue, SN-38, DX-8951f, exatecan
mesylate, truncated form of Pseudomonas aeruginosa exotoxin (PE38),
a Duocarmycin derivative, an amanitin, a-amanitin, a spliceostatin,
a thailanstatin, ozogamicin, tesirine, Amberstatin269 and
soravtansine, or a derivative thereof.
[0177] An embodiment is the therapeutic molecule of the invention
or the second therapeutic molecule of the invention, wherein the
therapeutic molecule and/or the second therapeutic molecule
comprise(s) or consist(s) of any one of Gemtuzumab ozogamicin,
Brentuximab vedotin, Trastuzumab emtansine, Inotuzumab ozogamicin,
Moxetumomab pasudotox and Polatuzumab vedotin and an antibody-drug
conjugate of Table A2 and Table A3, or at least one tumor-cell
specific receptor binding-domain thereof and/or at least one
tumor-cell specific receptor binding-fragment thereof which are
preferably (a) tumor-cell specific receptor binding-domain(s)
and/or (a) tumor-cell specific receptor binding-fragment(s), with
the proviso that the therapeutic molecule and the second
therapeutic molecule are the same or are different.
[0178] An embodiment is the therapeutic molecule of the invention
or the second therapeutic molecule of the invention, wherein the
saponin C is a triterpenoid saponin or a bisdesmosidic triterpene
saponin, belonging to the type of a 12,13-dehydrooleanane with an
aldehyde function in position C-23 and optionally comprising a
glucuronic acid function in a carbohydrate substituent at the
C-3beta-OH group of the saponin, and/or a saponin isolated from a
Gypsophila species and/or a Saponaria species and/or an Agrostemma
species and/or a Quillaja species such as Quillaja saponaria.
[0179] An embodiment is the therapeutic molecule of the invention
or the second therapeutic molecule of the invention, wherein the
saponin C is a single specific saponin or is a mixture of two or
more different saponins, such as one or more of the saponins in
Table A1 or Scheme I, SO1861, SA1657, GE1741, SA1641, QS-21,
QS-21A, QS-21 A-api, QS-21 A-xyl, QS-21B, QS-21 B-api, QS-21 B-xyl,
QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS1861, QS1862,
Quillajasaponin, Saponinum album, QS-18, Quil-A, Gyp1, gypsoside A,
AG1, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, or any of their
stereomers and/or any combinations thereof, preferably the saponin
is SO1861 and/or GE1741 and/or SA1641 and/or QS-21 and/or saponin
with a quillaic acid aglycon core, a
Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA carbohydrate substituent
at the C-3beta-OH group and a
Glc-(1.fwdarw.3)-Xyl-(1.fwdarw.4)-Rha-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)-4-OA-
c-Qui-(1.fwdarw.4)]-Fuc carbohydrate substituent at the C-28-OH
group, and/or is
3-O-beta-D-galactopyranosyl-(1.fwdarw.2)-[beta-D-xylopyranosyl--
(1.fwdarw.3)]-beta-D-glucuronopyranosyl quillaic acid 28-O-beta-D-g
lucopyranosyl-(1.fwdarw.3)-beta-D-xylopyranosyl-(1.fwdarw.4)-alpha-L-rham-
nopyranosyl-(1.fwdarw.2)-[beta-D-xylopyranosyl-(1.fwdarw.3)-4-OAc-beta-D-q-
uinovopyranosyl-(1.fwdarw.4)]-beta-D-fucopyranoside, more
preferably the saponin is SO1861 and/or QS-21.
[0180] An embodiment is the therapeutic molecule of the invention
or the second therapeutic molecule of the invention, wherein the
saponin C is a bisdesmosidic saponin having a molecular mass of at
least 1.500 Dalton and comprising an oleanan-type triterpene
containing an aldehyde group at the C-23 position and optionally a
hydroxyl group at the C-16 position, with a first branched
carbohydrate side chain at the C-3 position which first branched
carbohydrate side chain optionally contains glucuronic acid,
wherein the saponin contains an ester group with a second branched
carbohydrate side chain at the C-28 position which second branched
carbohydrate chain preferably comprises at least four carbohydrate
units, optionally containing at least one acetyl residue such as
two acetyl residues and/or at least one deoxy carbohydrates and/or
a quinovose and/or a glucose and/or 4-methoxycinnamic acid and/or
optionally comprising
5-O-[5-O-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl--
octanoic acid and/or optionally comprising
5-O-[5-O-Rha-(1.fwdarw.2)-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-di-
hydroxy-6-methyl-octanoic acid bound to a carbohydrate via an ester
bond, or wherein the at least one saponin is QS-21 or any one or
more of QS-21A, QS-21 A-api, QS-21 A-xyl, QS-21B, QS-21 B-api,
QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS-18,
QS1861, protonated QS1861 (QS1862), Quil-A.
[0181] An embodiment is the therapeutic molecule of the invention
or the second therapeutic molecule of the invention, wherein the
saponin C is a bisdesmosidic triterpene saponin belonging to the
type of a 12,13-dehydrooleanane with an aldehyde function in
position C-23, wherein the saponin C is covalently coupled to an
amino-acid residue of the first ligand A1 or B1 and/or the first
effector moiety B1 or A1 and/or the second ligand A2 or B2 and/or
the second effector moiety B2 or A2 via the aldehyde function in
the saponin C, preferably said aldehyde function in position C-23,
preferably via a linker L2, L4, L6, L8, L9 and/or L10, more
preferably via a cleavable linker L2, L4, L6, L8, L9 and/or L10,
wherein the amino-acid residue preferably is selected from cysteine
and lysine.
[0182] An embodiment is the therapeutic molecule of the invention
or the second therapeutic molecule of the invention, wherein the
saponin C is a bisdesmosidic triterpene saponin belonging to the
type of a 12,13-dehydrooleanane with an aldehyde function in
position C-23, wherein the aldehyde function in position C-23 of
the at least one saponin is covalently coupled to linker
N-.epsilon.-maleimidocaproic acid hydrazide, which linker is
covalently coupled via a thio-ether bond to a sulfhydryl group in
the first ligand A1 or B1 and/or in the first effector moiety B1 or
A1 and/or in the second ligand A2 or B2 and/or in the second
effector moiety B2 or A2, such as a sulfhydryl group of a
cysteine.
[0183] An embodiment is the therapeutic molecule of the invention
or the second therapeutic molecule of the invention, wherein the
saponin C is a bisdesmosidic triterpene saponin belonging to the
type of a 12,13-dehydrooleanane with an aldehyde function in
position C-23 and comprising a glucuronic acid function in a
carbohydrate substituent at the C-3beta-OH group of the saponin,
wherein the saponin C is covalently coupled to the amino-acid
residue of the first ligand A1 or B1 and/or the first effector
moiety B1 or A1 and/or the second ligand A2 or B2 and/or the second
effector moiety B2 or A2 via the glucuronic acid function in the
saponin C, if present, preferably via a linker L2, L4, L6, L8, L9
and/or L10, wherein the amino-acid residue preferably is selected
from cysteine and lysine, more preferably the amino-acid residue is
lysine.
[0184] An embodiment is the therapeutic molecule of the invention
or the second therapeutic molecule of the invention, wherein the
saponin C is a bisdesmosidic triterpene saponin belonging to the
type of a 12,13-dehydrooleanane with an aldehyde function in
position C-23 and comprising a glucuronic acid function in a
carbohydrate substituent at the C-3beta-OH group of the saponin,
wherein the glucuronic acid function in the carbohydrate
substituent at the C-3beta-OH group of the at least one saponin is
covalently coupled to linker
1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid hexafluorophosphate, which linker is covalently coupled via
an amide bond to an amine group in the first ligand A1 or B1 and/or
in the first effector moiety B1 or A1 and/or in the second ligand
A2 or B2 and/or in the second effector moiety B2 or A2, such as an
amine group of a lysine or an N-terminus of the first ligand A1 or
B1 and/or the first effector moiety B1 or A1 and/or the second
ligand A2 or B2 and/or the second effector moiety B2 or A2.
[0185] An embodiment is the therapeutic molecule of the invention
or the second therapeutic molecule of the invention, wherein the
first ligand A1 or B1 and/or the first effector moiety B1 or A1
and/or the second ligand A2 or B2 and/or the second effector moiety
B2 or A2 comprise(s) one or more than one covalently bound saponin
C, preferably 2, 3, 4, 5, 6, 8, 10, 16, 32, 64, 128 or 1-100
saponins, or any number of saponins therein between, such as 7, 9,
12 saponins.
[0186] An embodiment is the therapeutic molecule of the invention
or the second therapeutic molecule of the invention, wherein the
first ligand A1 or B1 and/or the first effector moiety B1 or A1
and/or the second ligand A2 or B2 and/or the second effector moiety
B2 or A2 comprise(s) one or more than one covalently bound saponin
C, wherein the saponin(s) C is/are covalently bound directly to an
amino-acid residue of the first ligand A1 or B1 and/or the first
effector moiety B1 or A1 and/or the second ligand A2 or B2 and/or
the second effector moiety B2 or A2 when r, s, v, e, f and i are 0,
preferably to a cysteine and/or to a lysine, and/or is/are
covalently bound via at least one linker L2, L4, L6, L8, L9 and/or
L10, or via at least one cleavable linker L2, L4, L6, L8, L9 and/or
L10 and/or via at least one oligomeric or polymeric scaffold L3
and/or L7, preferably 1-8 of such scaffolds or 2-4 of such
scaffolds, wherein the at least one scaffold is optionally based on
a dendron, wherein 1-32 saponins, preferably 2, 3, 4, 5, 6, 8, 10,
16, 32 saponins, or any number of saponins therein between, such as
7, 9, 12 saponins, are covalently bound to the at least one
scaffold.
[0187] An embodiment is the therapeutic molecule of the invention
or the second therapeutic molecule of the invention, wherein the
saponin C is a bisdesmosidic triterpene saponin belonging to the
type of a 12,13-dehydrooleanane with an aldehyde function in
position C-23, wherein the saponin C is covalently coupled to an
amino-acid residue of the first ligand A1 or B1 and/or the first
effector moiety B1 or A1 and/or the second ligand A2 or B2 and/or
the second effector moiety B2 or A2 via the aldehyde function in
the saponin C, preferably said aldehyde function in position C-23,
preferably via a linker L2, L4, L6, L8, L9 and/or L10, more
preferably via a cleavable linker L2, L4, L6, L8, L9 and/or L10,
wherein the amino-acid residue preferably is selected from cysteine
and lysine, and wherein the cleavable linker L2, L4, L6, L8, L9
and/or L10 is subject to cleavage under acidic conditions,
reductive conditions, enzymatic conditions or light-induced
conditions, and preferably the cleavable linker comprises a
hydrazone bond or a hydrazide bond subject to cleavage under acidic
conditions when bound to saponin, and/or comprises a bond
susceptible to proteolysis, for example proteolysis by Cathepsin B,
when bound to saponin, and/or the cleavable linker comprises a
disulphide bond susceptible to cleavage under reductive
conditions.
[0188] An embodiment is the therapeutic molecule of the invention
or the second therapeutic molecule of the invention, wherein the
saponin C is a bisdesmosidic triterpene saponin belonging to the
type of a 12,13-dehydrooleanane with an aldehyde function in
position C-23, wherein the saponin C is covalently coupled to an
amino-acid residue of the first ligand A1 or B1 and/or the first
effector moiety B1 or A1 and/or the second ligand A2 or B2 and/or
the second effector moiety B2 or A2 via the aldehyde function in
the saponin C, preferably said aldehyde function in position C-23,
preferably via a linker L2, L4, L6, L8, L9 and/or L10, more
preferably via a cleavable linker L2, L4, L6, L8, L9 and/or L10,
wherein the amino-acid residue preferably is selected from cysteine
and lysine, and wherein the cleavable linker L2, L4, L6, L8, L9
and/or L10 is subject to cleavage in vivo under acidic conditions
as present in endosomes and/or lysosomes of mammalian cells,
preferably human cells, preferably at pH 4.0-6.5, and more
preferably at pH.ltoreq.5.5.
[0189] An embodiment is the therapeutic molecule of the invention
or the second therapeutic molecule of the invention, wherein the
polymeric or oligomeric scaffold L3 and/or L7 comprises a polymeric
or oligomeric structure and comprises a chemical group, the
chemical group for covalently coupling of the polymeric or
oligomeric scaffold L3 and/or L7 to the amino-acid residue of the
first ligand and/or the first effector moiety and/or the second
ligand and/or the second effector moiety.
[0190] An embodiment is the therapeutic molecule of the invention
or the second therapeutic molecule of the invention, wherein the
saponin C is a bisdesmosidic triterpene saponin belonging to the
type of a 12,13-dehydrooleanane with an aldehyde function in
position C-23 and comprising a glucuronic acid function in a
carbohydrate substituent at the C-3beta-OH group of the saponin,
wherein the glucuronic acid function in the carbohydrate
substituent at the C-3beta-OH group of the at least one saponin is
covalently coupled to linker
1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid hexafluorophosphate, which linker is covalently coupled via
an amide bond to an amine group in the first ligand A1 or B1 and/or
in the first effector moiety B1 or A1 and/or in the second ligand
A2 or B2 and/or in the second effector moiety B2 or A2, such as an
amine group of a lysine or an N-terminus of the first ligand A1 or
B1 and/or the first effector moiety B1 or A1 and/or the second
ligand A2 or B2 and/or the second effector moiety B2 or A2, and
wherein the at least one saponin is covalently bound to the
polymeric or oligomeric structure of the scaffold L3 and/or L7 via
a cleavable linker L4 and/or L8 according to the invention.
[0191] An embodiment is the therapeutic molecule of the invention
or the second therapeutic molecule of the invention, wherein the
chemical group of the polymeric or oligomeric scaffold L3 and/or
L7, for covalently coupling of the scaffold to the amino-acid
residue of the first ligand and/or the first effector moiety and/or
the second ligand and/or the second effector moiety, is a click
chemistry group, preferably selected from a tetrazine, an azide, an
alkene or an alkyne, or a cyclic derivative of these groups, more
preferably the click chemistry group is an azide.
[0192] An embodiment is the therapeutic molecule of the invention
or the second therapeutic molecule of the invention, wherein the
first ligand A1 or B1 and/or the first effector moiety B1 or A1
and/or the second ligand A2 or B2 and/or the second effector moiety
B2 or A2 comprise(s) one or more than one covalently bound saponin
C, preferably 2, 3, 4, 5, 6, 8, 10, 16, 32, 64, 128 or 1-100
saponins, or any number of saponins therein between, such as 7, 9,
12 saponins, and wherein the at least one saponin is covalently
bound to the first ligand and/or to the first effector moiety
and/or to the second ligand and/or to the second effector moiety,
either directly or via at least one linker such as a bi-functional
linker, for example based on N-.epsilon.-maleimidocaproic acid
hydrazide and/or based on
1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid hexafluorophosphate, or a tri-functional linker L9 when w=1
and/or a tri-functional linker L10 when j=1, such as the
tri-functional linker of Scheme II.
[0193] An embodiment is the therapeutic molecule of the invention
and encompassing the previous embodiment, or the second therapeutic
molecule of the invention encompassing the previous embodiment,
wherein the tri-functional linker L9 when w=1 and/or the
tri-functional linker L10 when j=1, comprises a second chemical
group with at least one saponin covalently bound thereto, a third
chemical group for covalent binding to the first and/or second
ligand and a first chemical group for covalent binding to at least
one first and/or second effector moiety, preferably the
tri-functional linker is the trifunctional linker of Scheme II.
[0194] An embodiment is the therapeutic molecule of the invention
or the second therapeutic molecule of the invention, wherein the at
least one saponin is covalently bound to the first ligand and/or to
the first effector moiety and/or to the second ligand and/or to the
second effector moiety via at least one linker comprising a
tri-functional linker L9 when j=1 and/or a tri-functional linker
L10 when w=1, to which tri-functional linker both the first ligand
and the at least one first effector moiety are bound and/or to
which tri-functional linker both the second ligand and the at least
one second effector moiety are bound, preferably the tri-functional
linker is the trifunctional linker of Scheme II.
[0195] An embodiment is the therapeutic molecule of the invention
or the second therapeutic molecule of the invention, wherein the
polymeric or oligomeric structure of the scaffold L3 and/or L7
comprises a linear, branched and/or cyclic polymer, oligomer,
dendrimer, dendron, dendronized polymer, dendronized oligomer, a
DNA, a polypeptide, poly-lysine, a poly-ethylene glycol, or an
assembly of these polymeric or oligomeric structures which assembly
is preferably built up by covalent cross-linking.
[0196] An embodiment is the therapeutic molecule of the invention
or the second therapeutic molecule of the invention, wherein the
first ligand A1 or B1 and/or the first effector moiety B1 or A1
and/or the second ligand A2 or B2 and/or the second effector moiety
B2 or A2 comprise(s) one or more than one covalently bound saponin
C, preferably 2, 3, 4, 5, 6, 8, 10, 16, 32, 64, 128 or 1-100
saponins, or any number of saponins therein between, such as 7, 9,
12 saponins, and wherein the first ligand A1 or B1 is covalently
bound to the first effector moiety B1 or A1, respectively, via at
least one linker L1, and/or wherein the second ligand A2 or B2 is
covalently bound to the second effector moiety B2 or A2,
respectively, via at least one linker L5.
[0197] An embodiment is the therapeutic combination of the
invention, wherein the first ligand A1 is a monoclonal antibody or
at least one binding fragment or -domain thereof according to any
one of the claims 3-7, m=1, q=0, n=0, u=0, r=0, L3 is the scaffold
according to the invention and s=1, or L3 is absent and s=0, L4 is
a linker or a cleavable linker according to the invention, v=1,
p=2-4 and t=2-4 if s=1 and t=0 if s=0, and saponin C is a saponin
according to the invention, preferably the saponin C is SO1861
and/or QS-21, and effector moiety A2 is an effector moiety selected
from any one or more of an oligonucleotide, a nucleic acid and a
xeno nucleic acid, preferably selected from any one or more of a
vector, a gene, a cell suicide inducing transgene, deoxyribonucleic
acid (DNA), ribonucleic acid (RNA), anti-sense oligonucleotide
(ASO, AON), short interfering RNA (siRNA), microRNA (miRNA), DNA
aptamer, RNA aptamer, mRNA, mini-circle DNA, peptide nucleic acid
(PNA), phosphoramidate morpholino oligomer (PMO), locked nucleic
acid (LNA), bridged nucleic acid (BNA), 2'-deoxy-2'-fluoroarabino
nucleic acid (FANA), 2'-O-methoxyethyl-RNA (MOE),
2'-0,4'-aminoethylene bridged nucleic acid, 3'-fluoro hexitol
nucleic acid (FHNA), a plasmid, glycol nucleic acid (GNA) and
threose nucleic acid (TNA), or a derivative thereof, more
preferably a BNA, for example a BNA for silencing HSP27 protein
expression, a=1, d=0, b=0, h=0, e=0, f=0, i=0, c=0 and g=0.
[0198] An embodiment is the therapeutic molecule of the invention,
wherein r=0, s=0, v=0, s=0, v=0, p=0, t=0, ligand A1 is a
monoclonal antibody or at least one binding fragment or -domain
thereof according to any one of the invention, m=1, effector moiety
B1 is an effector moiety selected from any one or more of an
oligonucleotide, a nucleic acid and a xeno nucleic acid, preferably
selected from any one or more of a vector, a gene, a cell suicide
inducing transgene, deoxyribonucleic acid (DNA), ribonucleic acid
(RNA), anti-sense oligonucleotide (ASO, AON), short interfering RNA
(siRNA), microRNA (miRNA), DNA aptamer, RNA aptamer, mRNA,
mini-circle DNA, peptide nucleic acid (PNA), phosphoramidate
morpholino oligomer (PMO), locked nucleic acid (LNA), bridged
nucleic acid (BNA), 2'-deoxy-2'-fluoroarabino nucleic acid (FANA),
2'-O-methoxyethyl-RNA (MOE), 2'-0,4'-aminoethylene bridged nucleic
acid, 3'-fluoro hexitol nucleic acid (FHNA), a plasmid, glycol
nucleic acid (GNA) and threose nucleic acid (TNA), or a derivative
thereof, more preferably a BNA, for example a BNA for silencing
HSP27 protein expression, either q=0, n=1 and u=2-4, or q=1, n=2-4,
u=2-4 and linker L1 is the oligomeric or polymeric scaffold L3
according to the invention.
[0199] An embodiment is the therapeutic combination of the
invention, wherein the therapeutic molecule is the therapeutic
molecule of the previous embodiment, and wherein the second ligand
A2 is a monoclonal antibody or at least one binding fragment or
-domain thereof according to the invention, a=1, d=0, b=0, h=0,
e=0, L7 is the scaffold according to the invention and f=1, or L7
is absent and f=0, L8 is a linker or a cleavable linker according
to the invention, i=1, c=2-4 and g=2-4 if f=1 and g=0 if f=0, and
saponin C is a saponin according to the invention, preferably the
saponin C is SO1861 and/or QS-21, with the proviso that the ligand
A1 and the ligand A2 are the same or are different.
[0200] An aspect of the invention relates to a therapeutic
combination, wherein the therapeutic combination comprises: (a) a
first pharmaceutical composition comprising the therapeutic
molecule with chemical structure of COMPOUND I according to the
invention, the first pharmaceutical composition optionally further
comprising a pharmaceutically acceptable excipient; and (b) a
second pharmaceutical composition comprising the second therapeutic
molecule with chemical structure of COMPOUND II according to the
invention, the second pharmaceutical composition optionally further
comprising a pharmaceutically acceptable excipient.
[0201] An aspect of the invention relates to the first
pharmaceutical composition of the invention for use as a
medicament.
[0202] An aspect of the invention relates to a therapeutic
combination for use in the treatment or prevention of cancer in a
human subject, wherein the therapeutic combination comprises: (a)
the first pharmaceutical composition of the invention; and (b) the
second pharmaceutical composition of the invention, wherein the
ligand A1 or B1 and the ligand A2 or B2 can bind to a tumor-cell
epitope, preferably to a tumor-cell specific epitope, on a
tumor-cell surface molecule, preferably on a tumor cell-specific
surface molecule, with the proviso that the tumor-cell epitope or
tumor-cell specific epitope to which the ligand A1 or B1 can bind
is the same as, or is different from the tumor-cell epitope or the
tumor-cell specific epitope to which the ligand A2 or B2 can
bind.
[0203] An aspect of the invention relates to the first
pharmaceutical composition of the invention, for use in the
treatment or prophylaxis of cancer in a patient in need thereof,
wherein the ligand A1 or B1 can bind to a tumor-cell epitope,
preferably a tumor-cell specific epitope, on a tumor-cell surface
molecule, preferably a tumor cell-specific surface molecule.
[0204] An embodiment is the first pharmaceutical composition for
use according to the invention or the therapeutic combination for
use according to the invention, wherein the second pharmaceutical
composition of the invention and the first pharmaceutical
composition of the invention are administered to the patient in
need thereof.
[0205] An aspect of the invention relates to the first
pharmaceutical composition of the invention further comprising the
second therapeutic molecule of the invention.
[0206] An aspect of the invention relates to the first
pharmaceutical composition of the invention further comprising the
second therapeutic molecule of the invention, for use as a
medicament.
[0207] An aspect of the invention relates to the first
pharmaceutical composition of the invention further comprising the
second therapeutic molecule of the invention, for use in the
treatment or prevention of a cancer in a human subject.
[0208] An aspect of the invention within a first series of aspects
and embodiments of the invention relates to a scaffold suitable for
covalently binding at least one biologically active molecule to a
carrier molecule, the scaffold comprising a polymeric or oligomeric
structure and at least one of said biologically active molecules
covalently bound to said polymeric or oligomeric structure, wherein
the scaffold further comprises a first chemical group for
covalently coupling of the scaffold to the carrier molecule.
[0209] An embodiment within the first series of aspects and
embodiments of the invention is the scaffold according to the
invention, wherein the at least one biologically active molecule
has a molecular mass of 3.000 Dalton or less, preferably 2.500
Dalton or less, more preferably 2.300 Dalton or less, most
preferably, 2.000 Dalton or less, such as 1.700 Dalton-1.950
Dalton.
[0210] An embodiment within the first series of aspects and
embodiments of the invention is the scaffold according to the
invention, wherein the at least one biologically active molecule is
an amphiphilic molecule.
[0211] An embodiment within the first series of aspects and
embodiments of the invention is the scaffold according to the
invention, wherein the at least one biologically active molecule is
a single specific molecule or is a mixture of different molecules,
when more than one biologically active molecules are covalently
bound to the polymeric or oligomeric structure comprised by the
scaffold.
[0212] An embodiment within the first series of aspects and
embodiments of the invention is the scaffold according to the
invention, wherein the at least one biologically active molecule is
a glycoside, preferably a bisdesmosidic triterpene or triterpenoid
saponin, more preferably a bisdesmosidic triterpene saponin, most
preferably a bisdesmosidic triterpene saponin belonging to the type
of a 12,13-dehydrooleanane with an aldehyde function in position
C-23 and optionally comprising a glucuronic acid function in a
carbohydrate substituent at the C-3beta-OH group of the
saponin.
[0213] An embodiment within the first series of aspects and
embodiments of the invention is the scaffold according to the
invention, wherein the at least one biologically active molecule is
a saponin that can be isolated from a Gypsophila species and/or a
Saponaria species and/or an Agrostemma species and/or a Quillaja
species such as Quillaja saponaria or is a single specific saponin
or is a mixture of two or more different saponins, such as one or
more of the saponins in Table A1 or Scheme I, SO1861, SA1657,
GE1741, SA1641, QS-21, QS-21A, QS-21 A-api, QS-21 A-xyl, QS-21B,
QS-21 B-api, QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl,
QS1861, QS1862, Quillajasaponin, Saponinum album, QS-18, Quil-A,
Gyp1, gypsoside A, AG1, AG2, SO1542, SO1584, SO1658, SO1674,
SO1832, or any of their stereomers and/or any combinations thereof,
preferably the saponin is SO1861 and/or GE1741 and/or SA1641 and/or
QS-21 and/or saponin with a quillaic acid aglycon core, a
Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA carbohydrate substituent
at the C-3beta-OH group and a
Glc-(1.fwdarw.3)-Xyl-(1.fwdarw.4)-Rha-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)-4-OA-
c-Qui-(1.fwdarw.4)]-Fuc carbohydrate substituent at the C-28-OH
group, and/or is
3-O-beta-D-galactopyranosyl-(1.fwdarw.2)-[beta-D-xylopyranosyl--
(1.fwdarw.3)]-beta-D-glucuronopyranosyl quillaic acid
28-O-beta-D-glucopyranosyl-(1.fwdarw.3)-beta-D-xylopyranosyl-(1.fwdarw.4)-
-alpha-L-rhamnopyranosyl-(1.fwdarw.2)-[beta-D-xylopyranosyl-(1.fwdarw.3)-4-
-OAc-beta-D-quinovopyranosyl-(1.fwdarw.4)]-beta-D-fucopyranoside,
more preferably the saponin is SO1861 and/or QS-21.
[0214] An embodiment within the first series of aspects and
embodiments of the invention is the scaffold according to the
invention, wherein the at least one biologically active molecule is
a bisdesmosidic saponin having a molecular mass of at least 1.500
Dalton and comprising an oleanan-type triterpene containing an
aldehyde group at the C-23 position and optionally a hydroxyl group
at the C-16 position, with a first branched carbohydrate side chain
at the C-3 position which first branched carbohydrate side chain
optionally contains glucuronic acid, wherein the saponin contains
an ester group with a second branched carbohydrate side chain at
the C-28 position which second branched carbohydrate chain
preferably comprises at least four carbohydrate units, optionally
containing at least one acetyl residue such as two acetyl residues
and/or optionally comprising deoxy carbohydrates and/or optionally
comprising quinovose and/or optionally comprising glucose and/or
optionally comprising 4-methoxycinnamic acid and/or optionally
comprising
5-O-[5-O-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl--
octanoic acid and/or optionally comprising
5-O-[5-O-Rha-(1.fwdarw.2)-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-di-
hydroxy-6-methyl-octanoic acid bound to a carbohydrate via an ester
bond, or wherein the at least one saponin is QS-21 or any one or
more of QS-21A, QS-21 A-api, QS-21 A-xyl, QS-21B, QS-21 B-api,
QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS-18,
QS1861, protonated QS1861 (QS1862), Quil-A.
[0215] An embodiment within the first series of aspects and
embodiments of the invention is the scaffold according to the
invention, wherein the at least one biologically active molecule is
covalently bound to the polymeric or oligomeric structure via a
non-cleavable bond or via a cleavable bond, wherein preferably said
cleavable bond is subject to cleavage under acidic conditions,
reductive conditions, enzymatic conditions or light-induced
conditions, more preferably the cleavable bond is a hydrazone bond
or a hydrazide bond subject to cleavage under acidic conditions,
and/or is a bond susceptible to proteolysis, for example
proteolysis by Cathepsin B, and/or is a bond susceptible for
cleavage under reductive conditions such as a disulphide bond.
[0216] An embodiment within the first series of aspects and
embodiments of the invention is the scaffold according to the
invention, wherein the at least one biologically active molecule is
covalently bound to the polymeric or oligomeric structure via a
cleavable bond, wherein said cleavable bond is subject to cleavage
in vivo under acidic conditions as present in endosomes and/or
lysosomes of mammalian cells, preferably human cells, preferably at
pH 4.0-6.5, and more preferably at pH.ltoreq.5.5.
[0217] An embodiment within the first series of aspects and
embodiments of the invention is the scaffold according to the
invention, wherein the at least one biologically active molecule is
covalently bound to the polymeric or oligomeric structure of the
scaffold via an imine bond, a hydrazone bond, a hydrazide bond, an
oxime bond, a 1,3-dioxolane bond, a disulphide bond, a thio-ether
bond, an amide bond, a peptide bond or an ester bond, preferably
via at least one linker.
[0218] An embodiment within the first series of aspects and
embodiments of the invention is the scaffold according to the
invention, wherein the aldehyde function in position C-23 of the at
least one saponin is involved in the covalent bonding to the
polymeric or oligomeric structure of the scaffold, and/or, if
present, the glucuronic acid function in the carbohydrate
substituent at the C-3beta-OH group of the at least one saponin, is
involved in the covalent bonding to the polymeric or oligomeric
structure of the scaffold, either via direct binding or via at
least one linker.
[0219] An embodiment within the first series of aspects and
embodiments of the invention is the scaffold according to the
invention, wherein the aldehyde function in position C-23 of the at
least one saponin is covalently coupled to linker
N-.epsilon.-maleimidocaproic acid hydrazide, which linker is
covalently coupled via a thio-ether bond to a sulfhydryl group in
the polymeric or oligomeric structure of the scaffold, such as a
sulfhydryl group of a cysteine.
[0220] An embodiment within the first series of aspects and
embodiments of the invention is the scaffold according to the
invention, wherein the glucuronic acid function in the carbohydrate
substituent at the C-3beta-OH group of the at least one saponin is
covalently coupled to linker
1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid hexafluorophosphate, which linker is covalently coupled via
an amide bond to an amine group in the polymeric or oligomeric
structure of the scaffold, such as an amine group of a lysine or an
N-terminus of a proteinaceous molecule.
[0221] An embodiment within the first series of aspects and
embodiments of the invention is the scaffold according to the
invention, wherein the chemical group for covalently coupling of
the scaffold to the carrier molecule is a click chemistry
group.
[0222] An embodiment within the first series of aspects and
embodiments of the invention is the scaffold according to the
invention, wherein the click chemistry group is a tetrazine, an
azide, an alkene or an alkyne, or a cyclic derivative of any of
these groups, preferably an azide.
[0223] An embodiment within the first series of aspects and
embodiments of the invention is the scaffold according to the
invention, wherein the scaffold is a tri-functional linker
comprising a second chemical group with at least one biologically
active molecule covalently bound thereto, comprising a third
chemical group for covalent binding to a molecule and comprising
the first chemical group for covalent binding to the carrier,
preferably the tri-functional linker is the tri-functional linker
of Scheme II and Structure B.
[0224] An embodiment within the first series of aspects and
embodiments of the invention is the scaffold according to the
invention, wherein the at least one biologically active molecule is
a defined number of glycoside molecules or a defined range of
glycoside molecules, preferably 1-128 or at least 2, 3, 4, 5, 6, 8,
10, 16, 32, 64 or 128 glycoside molecules, or any number of
glycoside molecules therein between, such as 7, 9, 12 glycoside
molecules.
[0225] An embodiment within the first series of aspects and
embodiments of the invention is the scaffold according to the
invention, wherein the polymeric or oligomeric structure comprises
a linear, branched and/or cyclic polymer, oligomer, dendrimer,
dendron, dendronized polymer, dendronized oligomer, a DNA, a
polypeptide, a poly-lysine, a poly-ethylene glycol, or an assembly
of these polymeric or oligomeric structures which assembly is
preferably built up by covalent cross-linking.
[0226] An embodiment within the first series of aspects and
embodiments of the invention is the scaffold according to the
invention, wherein the carrier molecule comprises or consists of
any of a proteinaceous molecule, a protein, a peptide, a nucleic
acid, an oligonucleotide, a lipid, a fat, a fatty acid, a
nanoparticle, a carbohydrate, or any covalently bound conjugate or
covalently bound complex of combinations thereof.
[0227] An embodiment within the first series of aspects and
embodiments of the invention is the scaffold according to the
invention, wherein the carrier molecule comprises or consists of an
immunoglobulin, at least one binding domain of an immunoglobulin
and/or at least one binding fragment of an immunoglobulin, such as
an antibody, an IgG, a molecule comprising or consisting of a Vhh
domain or Vh domain, a Fab, an scFv, an Fv, a dAb, an F(ab).sub.2,
Fcab fragment, or comprises or consists of at least one
non-proteinaceous ligand and/or at least one proteinaceous ligand
for binding to a cell-surface molecule such as EGF or a
cytokine.
[0228] An embodiment within the first series of aspects and
embodiments of the invention is the scaffold according to the
invention, wherein the carrier molecule comprises or consists of at
least one binding domain and/or at least one binding fragment for
binding to a cell-surface receptor such as a tumor-cell specific
cell-surface receptor selected from CD71, CA125, EpCAM(17-1A),
CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular
integrin alpha-V beta-3, HER2, EGFR, CD20, CD22, Folate receptor 1,
CD146, CD56, CD19, CD138, CD27L receptor, PSMA, CanAg,
integrin-alphaV, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239,
CD70, CD123, CD352, DLL3, CD25, ephrinA4, MUC1, Trop2, CEACAM5,
CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3,
CD7, PD-L1, CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2, preferably
selected from CD71, EGFR, HER2.
[0229] An embodiment within the first series of aspects and
embodiments of the invention is the scaffold according to the
invention, wherein the carrier molecule comprises or consists of
any one of cetuximab, daratumumab, gemtuzumab, trastuzumab,
panitumumab, brentuximab, inotuzumab, moxetumomab, polatuzumab,
obinutuzumab, OKT-9 anti-CD71 monoclonal antibody of the IgG type,
pertuzumab, rituximab, ofatumumab, Herceptin, alemtuzumab,
pinatuzumab, OKT-10 anti-CD38 monoclonal antibody, an antibody of
Table A2 or Table A3 or Table A4, preferably cetuximab or
trastuzumab or OKT-9, or at least one tumor-cell receptor
binding-fragment thereof and/or at least one tumor-cell receptor
binding-domain thereof, such as at least one tumor-cell specific
receptor binding-fragment thereof and/or at least one tumor-cell
specific receptor binding-domain thereof.
[0230] An embodiment within the first series of aspects and
embodiments of the invention is the scaffold according to the
invention, wherein the scaffold is suitable for forming a covalent
bond with the carrier molecule, said covalent bond preferably
involving a cysteine side-chain of the carrier molecule and/or a
lysine side-chain of the carrier molecule when the carrier molecule
comprises at least a cysteine and/or a lysine.
[0231] An embodiment within the first series of aspects and
embodiments of the invention is the scaffold according to the
invention, wherein the carrier molecule comprises or consists of at
least one effector molecule, or wherein the carrier further
comprises at least one effector molecule, wherein the effector
molecule is at least one of an active pharmaceutical substance,
such as any one or more of a payload, a toxin, a drug, a
polypeptide, an oligonucleotide, a nucleic acid, a xeno nucleic
acid, an enzyme such as urease and Cre-recombinase, a protein
toxin, a ribosome-inactivating protein.
[0232] An embodiment within the first series of aspects and
embodiments of the invention is the scaffold according to the
invention, wherein the protein toxin comprises or consists of any
one or more of a protein toxin selected from Table A5 and/or a
viral toxin such as apoptin; a bacterial toxin such as Shiga toxin,
Shiga-like toxin, Pseudomonas aeruginosa exotoxin (PE) or exotoxin
A of PE, full-length or truncated diphtheria toxin (DT), cholera
toxin; a fungal toxin such as alpha-sarcin; a plant toxin including
ribosome-inactivating proteins and the A chain of type 2
ribosome-inactivating proteins such as dianthin e.g. dianthin-30 or
dianthin-32, saporin e.g. saporin-S3 or saporin-S6, bouganin or
de-immunized derivative debouganin of bouganin, shiga-like toxin A,
pokeweed antiviral protein, ricin, ricin A chain, modeccin,
modeccin A chain, abrin, abrin A chain, volkensin, volkensin A
chain, viscumin, viscumin A chain; or an animal or human toxin such
as frog RNase, or granzyme B or angiogenin from humans, or any
fragment or derivative thereof; preferably the protein toxin is
dianthin and/or saporin.
[0233] An embodiment within the first series of aspects and
embodiments of the invention is the scaffold according to the
invention, wherein the oligonucleotide, the xeno nucleic acid or
the nucleic acid comprises or consists of any one or more of a
vector, a gene, a cell suicide inducing transgene, deoxyribonucleic
acid (DNA), ribonucleic acid (RNA), anti-sense oligonucleotide
(ASO, AON), short interfering RNA (siRNA), microRNA (miRNA), DNA
aptamer, RNA aptamer, mRNA, mini-circle DNA, peptide nucleic acid
(PNA), phosphoramidate morpholino oligomer (PMO), locked nucleic
acid (LNA), bridged nucleic acid (BNA), 2'-deoxy-2'-fluoroarabino
nucleic acid (FANA), 2'-O-methoxyethyl-RNA (MOE),
2'-0,4'-aminoethylene bridged nucleic acid, 3'-fluoro hexitol
nucleic acid (FHNA), a plasmid, glycol nucleic acid (GNA) and
threose nucleic acid (TNA), or a derivative thereof, preferably a
BNA, for example a BNA for silencing HSP27 protein expression.
[0234] An embodiment within the first series of aspects and
embodiments of the invention is the scaffold according to the
invention, wherein the effector molecule comprises or consists of
at least one payload, preferably selected from any one or more of a
toxin targeting ribosomes, a toxin targeting elongation factors, a
toxin targeting tubulin, a toxin targeting DNA and a toxin
targeting RNA, more preferably any one or more of emtansine,
pasudotox, maytansinoid derivative DM1, maytansinoid derivative
DM4, monomethyl auristatin E (MMAE, vedotin), monomethyl auristatin
F (MMAF, mafodotin), a Calicheamicin,
N-Acetyl-.gamma.-calicheamicin, a pyrrolobenzodiazepine (PBD)
dimer, a benzodiazepine, a CC-1065 analogue, a duocarmycin,
Doxorubicin, paclitaxel, cisplatin, cyclophosphamide, etoposide,
docetaxel, 5-fluorouracyl (5-FU), mitoxantrone, a tubulysin, an
indolinobenzodiazepine, AZ13599185, a cryptophycin, rhizoxin,
methotrexate, an anthracycline, a camptothecin analogue, SN-38,
DX-8951f, exatecan mesylate, truncated form of Pseudomonas
aeruginosa exotoxin (PE38), a Duocarmycin derivative, an amanitin,
a-amanitin, a spliceostatin, a thailanstatin, ozogamicin, tesirine,
Amberstatin269 and soravtansine, or a derivative thereof.
[0235] An embodiment within the first series of aspects and
embodiments of the invention is the scaffold according to the
invention, wherein the carrier molecule comprises or consists of a
covalently linked combination of an effector molecule and a
monoclonal antibody, preferably selected from Gemtuzumab
ozogamicin, Brentuximab vedotin, Trastuzumab emtansine, Inotuzumab
ozogamicin, Moxetumomab pasudotox and Polatuzumab vedotin and an
antibody-drug conjugate of Table A2 and Table A3.
[0236] An aspect of the invention within the first series of
aspects and embodiments of the invention relates to a method for
producing a scaffold suitable for covalently binding at least one
biologically active molecule to a carrier molecule, the method
comprising: a) providing a polymeric or oligomeric structure
comprising a first chemical group for covalently coupling of the
polymeric structure or the oligomeric structure to the carrier
molecule and comprising at least one of a second chemical group
different from the first chemical group, wherein each second
chemical group is for covalently coupling one of the at least one
biologically active molecules to the oligomeric or polymeric
structure; and b) covalently coupling at least one biologically
active molecule to said polymeric or oligomeric structure via the
second chemical group(s), wherein preferably the biologically
active molecule(s) is/are any one of the biologically active
molecules of the invention, more preferably SO1861 and/or GE1741
and/or SA1641 and/or QS-21, therewith providing the scaffold.
[0237] An embodiment within the first series of aspects and
embodiments of the invention is the method according to the
invention, the scaffold comprising at least one covalently bound
biologically active molecule, the method comprising: a) providing a
scaffold comprising at least one biologically active molecule
covalently bound to a polymeric or oligomeric structure in said
scaffold, preferably providing a scaffold according to the
invention or the scaffold obtainable by the method according to the
invention or the scaffold obtained with the method according to the
invention; and b) covalently coupling the scaffold of a) to a
carrier molecule according to the invention, therewith providing
the scaffold covalently bound to a carrier molecule, the scaffold
comprising at least one covalently bound biologically active
molecule.
[0238] An embodiment within the first series of aspects and
embodiments of the invention is the method according to the
invention, wherein the scaffold is able to augment endosomal escape
and/or lysosomal escape of the effector molecule according to the
invention when either said effector molecule is covalently bound to
the scaffold and contacted with a mammalian cell, or when said
effector molecule is contacted with a mammalian cell in the
presence of the scaffold.
[0239] An aspect of the invention within a second series of aspects
and embodiments of the invention relates to a first proteinaceous
molecule comprising a first binding site for binding to a first
epitope of a first cell-surface molecule, the first proteinaceous
molecule provided with at least one saponin covalently bound via at
least one linker and/or via an oligomeric or polymeric scaffold to
an amino-acid residue of said first proteinaceous molecule, or
covalently bound directly to an amino-acid residue of said first
proteinaceous molecule.
[0240] An embodiment within the second series of aspects and
embodiments of the invention is the first proteinaceous molecule
according to the invention, wherein the first binding site
comprises or consists of an immunoglobulin, or at least one binding
domain of an immunoglobulin and/or at least one binding fragment of
an immunoglobulin, such as an antibody, an IgG, a molecule
comprising or consisting of a Vhh domain or Vh domain, a Fab, an
scFv, an Fv, a dAb, an F(ab).sub.2, Fcab fragment, and/or comprises
or consists of at least one ligand for binding to a cell-surface
molecule such as EGF or a cytokine.
[0241] An embodiment within the second series of aspects and
embodiments of the invention is the first proteinaceous molecule
according to the invention, wherein the first epitope of the first
cell-surface molecule is a tumor-cell specific first epitope of a
first tumor-cell surface molecule, more preferably a tumor-cell
specific first epitope of a first tumor-cell surface receptor
specifically present on a tumor cell.
[0242] An embodiment within the second series of aspects and
embodiments of the invention is the first proteinaceous molecule
according to the invention, wherein the at least one saponin is a
triterpenoid saponin and/or a bisdesmosidic triterpene saponin
belonging to the type of a 12,13-dehydrooleanane with an aldehyde
function in position C-23 and optionally comprising a glucuronic
acid function in a carbohydrate substituent at the C-3beta-OH group
of the saponin, and/or a saponin isolated from a Gypsophila species
and/or a Saponaria species and/or an Agrostemma species and/or a
Quillaja species such as Quillaja saponaria.
[0243] An embodiment within the second series of aspects and
embodiments of the invention is the first proteinaceous molecule
according to the invention, wherein the at least one saponin is a
single specific saponin or is a mixture of two or more different
saponins, such as one or more of the saponins in Table A1 or Scheme
I, SO1861, SA1657, GE1741, SA1641, QS-21, QS-21A, QS-21 A-api,
QS-21 A-xyl, QS-21B, QS-21 B-api, QS-21 B-xyl, QS-7-xyl, QS-7-api,
QS-17-api, QS-17-xyl, QS1861, QS1862, Quillajasaponin, Saponinum
album, QS-18, Quil-A, Gyp1, gypsoside A, AG1, AG2, SO1542, SO1584,
SO1658, SO1674, SO1832, or any of their stereomers and/or any
combinations thereof, preferably the saponin is SO1861 and/or
GE1741 and/or SA1641 and/or QS-21 and/or saponin with a quillaic
acid aglycon core, a Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA
carbohydrate substituent at the C-3beta-OH group and a
Glc-(1.fwdarw.3)-Xyl-(1.fwdarw.4)-Rha-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)-4-OA-
c-Qui-(1.fwdarw.4)]-Fuc carbohydrate substituent at the C-28-OH
group, and/or is
3-O-beta-D-galactopyranosyl-(1.fwdarw.2)-[beta-D-xylopyranosyl--
(1.fwdarw.3)]-beta-D-glucuronopyranosyl quillaic acid
28-O-beta-D-glucopyranosyl-(1.fwdarw.3)-beta-D-xylopyranosyl-(1.fwdarw.4)-
-alpha-L-rhamnopyranosyl-(1.fwdarw.2)-[beta-D-xylopyranosyl-(1.fwdarw.3)-4-
OAc-beta-D-quinovopyranosyl-(1.fwdarw.4)]-beta-D-fucopyranoside,
more preferably the saponin is SO1861 and/or QS-21.
[0244] An embodiment within the second series of aspects and
embodiments of the invention is the first proteinaceous molecule
according to the invention, wherein the at least one saponin is a
bisdesmosidic saponin having a molecular mass of at least 1.500
Dalton and comprising an oleanan-type triterpene containing an
aldehyde group at the C-23 position and optionally a hydroxyl group
at the C-16 position, with a first branched carbohydrate side chain
at the C-3 position which first branched carbohydrate side chain
optionally contains glucuronic acid, wherein the saponin contains
an ester group with a second branched carbohydrate side chain at
the C-28 position which second branched carbohydrate chain
preferably comprises at least four carbohydrate units, optionally
containing at least one acetyl residue such as two acetyl residues
and/or optionally comprising deoxy carbohydrates and/or optionally
comprising quinovose and/or optionally comprising glucose and/or
optionally comprising 4-methoxycinnamic acid and/or optionally
comprising
5-O-[5-O-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl--
octanoic acid and/or optionally comprising
5-O-[5-O-Rha-(1.fwdarw.2)-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-di-
hydroxy-6-methyl-octanoic acid bound to a carbohydrate via an ester
bond, or wherein the at least one saponin is QS-21 or any one or
more of QS-21A, QS-21 A-api, QS-21 A-xyl, QS-21B, QS-21 B-api,
QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS-18,
QS1861, protonated QS1861 (QS1862), Quil-A.
[0245] An embodiment within the second series of aspects and
embodiments of the invention is the first proteinaceous molecule
according to the invention, wherein the at least one saponin is a
bisdesmosidic triterpene saponin belonging to the type of a
12,13-dehydrooleanane with an aldehyde function in position C-23,
wherein the at least one saponin is covalently coupled to the
amino-acid residue of the first proteinaceous molecule via an
aldehyde function in the saponin, preferably said aldehyde function
in position C-23, preferably via at least one linker, more
preferably via at least one cleavable linker, wherein the
amino-acid residue preferably is selected from cysteine and
lysine.
[0246] An embodiment is the first proteinaceous molecule according
to the invention, wherein the at least one saponin is a
bisdesmosidic triterpene saponin belonging to the type of a
12,13-dehydrooleanane with an aldehyde function in position C-23
and comprising a glucuronic acid function in a carbohydrate
substituent at the C-3beta-OH group of the saponin, wherein the at
least one saponin is covalently coupled to the amino-acid residue
of the first proteinaceous molecule via the glucuronic acid
function in the carbohydrate substituent at the C-3beta-OH group of
the saponin, preferably via at least one linker, wherein the
amino-acid residue preferably is selected from cysteine and
lysine.
[0247] An embodiment within the second series of aspects and
embodiments of the invention is the first proteinaceous molecule
according to the invention, wherein the aldehyde function in
position C-23 of the at least one saponin is covalently coupled to
linker N-.epsilon.-maleimidocaproic acid hydrazide, which linker is
covalently coupled via a thio-ether bond to a sulfhydryl group in
the first proteinaceous molecule, such as a sulfhydryl group of a
cysteine.
[0248] An embodiment within the second series of aspects and
embodiments of the invention is the first proteinaceous molecule
according to the invention, wherein the glucuronic acid function in
the carbohydrate substituent at the C-3beta-OH group of the at
least one saponin is covalently coupled to linker
1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid hexafluorophosphate, which linker is covalently coupled via
an amide bond to an amine group in the first proteinaceous
molecule, such as an amine group of a lysine or an N-terminus of
the first proteinaceous molecule.
[0249] An embodiment within the second series of aspects and
embodiments of the invention is the first proteinaceous molecule
according to the invention, wherein the first epitope of the first
cell-surface molecule to which the first binding site of the first
proteinaceous molecule binds is a tumor-cell specific first epitope
of the tumor-cell specific receptor preferably selected from CD71,
CA125, EpCAM(17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin,
syndecan-1, vascular integrin alpha-V beta-3, HER2, EGFR, CD20,
CD22, Folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor,
PSMA, CanAg, integrin-alphaV, CA6, CD33, mesothelin, Cripto, CD3,
CD30, CD239, CD70, CD123, CD352, DLL3, CD25, ephrinA4, MUC1, Trop2,
CEACAM5, CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3,
CD38, FGFR3, CD7, PD-L1, CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2, more
preferably selected from CD71, EGFR, HER2.
[0250] An embodiment within the second series of aspects and
embodiments of the invention is the first proteinaceous molecule
according to the invention, wherein the tumor cell-specific first
epitope, first tumor-cell surface molecule or first tumor-cell
specific receptor, are a first epitope or a first molecule or a
first receptor that are internalized by the tumor cell after
binding of the first proteinaceous molecule of the invention to the
first epitope or first molecule or first receptor, and wherein
preferably the first proteinaceous molecule is subjected to
tumor-cell receptor-mediated internalization, e.g. via endocytosis,
or tumor-cell surface molecule mediated internalization, e.g. via
endocytosis, when bound to the cell-surface molecule comprising the
first epitope, the tumor-cell surface molecule or the tumor-cell
specific receptor.
[0251] An embodiment within the second series of aspects and
embodiments of the invention is the first proteinaceous molecule
according to the invention, wherein the first binding site of the
first proteinaceous molecule comprises or consists of any one of
cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab,
brentuximab, inotuzumab, moxetumomab, polatuzumab, obinutuzumab,
OKT-9 anti-CD71 monoclonal antibody of the IgG type, pertuzumab,
rituximab, ofatumumab, Herceptin, alemtuzumab, pinatuzumab, OKT-10
anti-CD38 monoclonal antibody, an antibody of Table A2 or Table A3
or Table A4, preferably cetuximab or trastuzumab or OKT-9, or at
least one tumor-cell receptor binding-fragment thereof and/or at
least one tumor-cell receptor binding-domain thereof, preferably at
least one tumor-cell specific receptor binding-fragment thereof
and/or at least one tumor-cell specific receptor binding-domain
thereof.
[0252] An aspect of the invention within the second series of
aspects and embodiments of the invention relates to a therapeutic
combination, wherein the therapeutic combination comprises: (a) a
first pharmaceutical composition comprising the first proteinaceous
molecule according to the invention and optionally a
pharmaceutically acceptable excipient; and (b) a second
pharmaceutical composition comprising a second proteinaceous
molecule different from the first proteinaceous molecule, the
second proteinaceous molecule comprising a second binding site for
binding to a second epitope of a second cell-surface molecule
different from the first cell-surface molecule, and comprising an
effector moiety, the second pharmaceutical composition optionally
further comprising a pharmaceutically acceptable excipient, wherein
the second epitope is different from the first epitope.
[0253] An embodiment within the second series of aspects and
embodiments of the invention is the therapeutic combination
according to the invention, wherein the therapeutic combination
comprises: (a) the first pharmaceutical composition according to
the invention comprising the first proteinaceous molecule according
to the invention, wherein the first epitope on the first
cell-surface molecule is a tumor-cell specific first epitope on a
first tumor cell-specific surface molecule, preferably a tumor-cell
specific first epitope on a first cell-surface receptor
specifically present at a tumor cell; and (b) the second
pharmaceutical composition according to the invention, wherein the
second cell-surface molecule is a second tumor cell-specific
surface molecule different from the first tumor cell-specific
surface molecule, preferably a second cell-surface receptor
specifically present at a tumor cell different from the first
cell-surface receptor specifically present at said tumor cell, and
wherein the second epitope is a tumor-cell specific second
epitope.
[0254] An embodiment within the second series of aspects and
embodiments of the invention is the therapeutic combination
according to the invention, wherein the therapeutic combination
comprises: (a) the first pharmaceutical composition according to
the invention comprising the first proteinaceous molecule according
to the invention and comprising the first binding site for binding
to the first epitope on the first cell-surface molecule, the first
pharmaceutical composition optionally further comprising a
pharmaceutically acceptable excipient; and (b) a third
pharmaceutical composition comprising a third proteinaceous
molecule, the third proteinaceous molecule comprising the first
binding site for binding to the first epitope on the cell-surface
molecule of (a) and an effector moiety, the third pharmaceutical
composition optionally further comprising a pharmaceutically
acceptable excipient, wherein the first binding site of the first
proteinaceous molecule and the first binding site of the third
proteinaceous molecule are the same, and wherein the first
cell-surface molecule and the first epitope on the first
cell-surface molecule, to which the first proteinaceous molecule
can bind, and the first cell-surface molecule and the first epitope
on the first cell-surface molecule, to which the third
proteinaceous molecule can bind, are the same.
[0255] An embodiment within the second series of aspects and
embodiments of the invention is the therapeutic combination
according to the invention, wherein the therapeutic combination
comprises: (a) the first pharmaceutical composition according to
the invention; and (b) the third pharmaceutical composition
according to the invention, wherein the first cell-surface molecule
is expressed on a tumor cell surface, and preferably the first
cell-surface molecule is a tumor cell-specific surface molecule,
and wherein preferably the first epitope is a first tumor-cell
specific epitope.
[0256] An embodiment within the second series of aspects and
embodiments of the invention is the first proteinaceous molecule
according to the invention or the therapeutic combination according
to the invention, wherein the first binding site for binding to the
first epitope on the first cell surface molecule is a binding site
for a tumor-cell specific first epitope on a first cell-surface
receptor specifically present at a tumor cell.
[0257] An embodiment within the second series of aspects and
embodiments of the invention is the therapeutic combination
according to the invention, wherein the second binding site of the
second proteinaceous molecule and/or the first binding site of the
third proteinaceous molecule comprises or consists of an
immunoglobulin, at least one binding domain of an immunoglobulin
and/or at least one binding fragment of an immunoglobulin, such as
an antibody, an IgG, a molecule comprising or consisting of a Vhh
domain or Vh domain, a Fab, an scFv, an Fv, a dAb, an F(ab)2, Fcab
fragment, and/or comprises or consists of at least one ligand for
binding to a cell-surface molecule such as EGF or a cytokine.
[0258] An embodiment within the second series of aspects and
embodiments of the invention is the therapeutic combination
according to the invention, wherein the second binding site of the
second proteinaceous molecule for binding to the second epitope is
a second binding site for a tumor-cell specific second epitope on a
second cell-surface receptor specifically present at the tumor
cell, wherein the second binding site is different from the first
binding site.
[0259] An embodiment within the second series of aspects and
embodiments of the invention is the first proteinaceous molecule
according to the invention or the therapeutic combination according
to the invention, wherein said first and second proteinaceous
molecules comprise the first and second binding site respectively
for binding to a first and a second tumor-cell specific epitope on
a first and a second tumor-cell specific receptor respectively, the
receptors being different and being present at the same tumor cell,
wherein the first and second binding site are different and the
first and second tumor cell specific epitope are different.
[0260] An embodiment within the second series of aspects and
embodiments of the invention is the first proteinaceous molecule
according to the invention or the therapeutic combination according
to the invention, wherein said first and third proteinaceous
molecules comprise the same first binding site for binding to a
first tumor-cell specific epitope on a first tumor-cell specific
receptor.
[0261] An embodiment within the second series of aspects and
embodiments of the invention is the first proteinaceous molecule
according to the invention or the therapeutic combination according
to the invention wherein the first receptor and/or the second
receptor are selected from CD71, CA125, EpCAM(17-1A), CD52, CEA,
CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin
alpha-V beta-3, HER2, EGFR, CD20, CD22, Folate receptor 1, CD146,
CD56, CD19, CD138, CD27L receptor, PSMA, CanAg, integrin-alphaV,
CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239, CD70, CD123,
CD352, DLL3, CD25, ephrinA4, MUC1, Trop2, CEACAM5, CEACAM6, HER3,
CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1,
CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2, preferably selected from CD71,
EGFR and HER2.
[0262] An embodiment within the second series of aspects and
embodiments of the invention is the first proteinaceous molecule
according to the invention and/or the therapeutic combination
according to the invention, wherein the first and second tumor-cell
specific receptors are internalized by the tumor cell after binding
to the first proteinaceous molecule according to the invention
and/or the second proteinaceous molecule according to the
invention, and wherein preferably binding of the first
proteinaceous molecule and/or the second proteinaceous molecule to
the first and second tumor-cell specific receptors respectively,
results in tumor-cell receptor-mediated internalization, e.g. via
endocytosis, of a complex of the first proteinaceous molecule and
the first tumor-cell specific receptor and of a complex of the
second proteinaceous molecule and the second tumor-cell specific
receptor.
[0263] An embodiment within the second series of aspects and
embodiments of the invention is the therapeutic combination or the
first pharmaceutical composition according the invention, wherein
the first tumor-cell receptor, preferably the first tumor-cell
specific receptor, is internalized by the tumor cell after binding
to the first proteinaceous molecule according to the invention
and/or after binding to the third proteinaceous molecule according
to the invention, and wherein preferably binding of the first
proteinaceous molecule and/or the third proteinaceous molecule to
the first tumor-cell receptor, such as the first tumor-cell
specific receptor, is followed by tumor-cell receptor-mediated
internalization, e.g. via endocytosis, of a complex of the first
proteinaceous molecule and the first tumor-cell receptor and of a
complex of the third proteinaceous molecule and the first
tumor-cell receptor.
[0264] An embodiment within the second series of aspects and
embodiments of the invention is the first proteinaceous molecule
according to the invention, and/or therapeutic combination
according to the invention, wherein the first binding site and/or
the second binding site is/are or comprise(s) a monoclonal antibody
or at least one cell-surface molecule binding fragment and/or
-domain thereof, and preferably comprise or consist of any one of
cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab,
brentuximab, inotuzumab, moxetumomab, polatuzumab, obinutuzumab,
OKT-9 anti-CD71 monoclonal antibody of the IgG type, pertuzumab,
rituximab, ofatumumab, Herceptin, alemtuzumab, pinatuzumab, OKT-10
anti-CD38 monoclonal antibody, and an antibody of Table A4,
preferably cetuximab or trastuzumab or OKT-9, or at least one
cell-surface molecule binding fragment or -domain thereof, with the
proviso that the first binding site of the first proteinaceous
molecule is different from the second binding site of the second
proteinaceous molecule.
[0265] An embodiment within the second series of aspects and
embodiments of the invention is the therapeutic combination
according to the invention or the first pharmaceutical composition
according to the invention, wherein the first binding site of the
first proteinaceous molecule and the third proteinaceous molecule
comprises a monoclonal antibody or at least one of a cell-surface
molecule binding domain and/or -fragment thereof, and preferably
comprise or consist of any one of cetuximab, daratumumab,
gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab,
moxetumomab, polatuzumab, obinutuzumab, OKT-9 anti-CD71 monoclonal
antibody of the IgG type, pertuzumab, rituximab, ofatumumab,
Herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal
antibody, an antibody of Table A2 or Table A3 or Table A4,
preferably cetuximab or trastuzumab or OKT-9, or at least one
cell-surface molecule binding fragment and/or -domain thereof, with
the proviso that the first binding site of the first proteinaceous
molecule is the same as the first binding site of the third
proteinaceous molecule.
[0266] An embodiment within the second series of aspects and
embodiments of the invention is the therapeutic combination
according to the invention, wherein the second binding site of the
second proteinaceous molecule and/or the first binding site of the
third proteinaceous molecule is or comprises a monoclonal antibody
or at least one cell-surface molecule binding fragment or -domain
thereof, and preferably comprises or consists of any one of
Gemtuzumab ozogamicin, Brentuximab vedotin, Trastuzumab emtansine,
Inotuzumab ozogamicin, Moxetumomab pasudotox and Polatuzumab
vedotin and an antibody-drug conjugate of Table A2 and Table
A3.
[0267] An embodiment within the second series of aspects and
embodiments of the invention is the therapeutic combination
according to the invention, wherein the effector moiety that is
comprised by the second proteinaceous molecule and/or by the third
proteinaceous molecule comprises or consists of any one or more of
an oligonucleotide, a nucleic acid, a xeno nucleic acid, preferably
selected from any one or more of a vector, a gene, a cell suicide
inducing transgene, deoxyribonucleic acid (DNA), ribonucleic acid
(RNA), anti-sense oligonucleotide (ASO, AON), short interfering RNA
(siRNA), microRNA (miRNA), DNA aptamer, RNA aptamer, mRNA,
mini-circle DNA, peptide nucleic acid (PNA), phosphoramidate
morpholino oligomer (PMO), locked nucleic acid (LNA), bridged
nucleic acid (BNA), 2'-deoxy-2'-fluoroarabino nucleic acid (FANA),
2'-O-methoxyethyl-RNA (MOE), 2'-0,4'-aminoethylene bridged nucleic
acid, 3'-fluoro hexitol nucleic acid (FHNA), a plasmid, glycol
nucleic acid (GNA) and threose nucleic acid (TNA), or a derivative
thereof, more preferably a BNA, for example a BNA for silencing
HSP27 protein expression.
[0268] An embodiment within the second series of aspects and
embodiments of the invention is the therapeutic combination
according to the invention, wherein the effector moiety that is
comprised by the second proteinaceous molecule and/or by the third
proteinaceous molecule comprises or consists of at least one
proteinaceous molecule, preferably selected from any one or more of
a peptide, a protein, an enzyme such as urease and Cre-recombinase,
a ribosome-inactivating protein, a proteinaceous toxin, more
preferably selected from any one or more of a protein toxin
selected from Table A5 and/or a viral toxin such as apoptin; a
bacterial toxin such as Shiga toxin, Shiga-like toxin, Pseudomonas
aeruginosa exotoxin (PE) or exotoxin A of PE, full-length or
truncated diphtheria toxin (DT), cholera toxin; a fungal toxin such
as alpha-sarcin; a plant toxin including ribosome-inactivating
proteins and the A chain of type 2 ribosome-inactivating proteins
such as dianthin e.g. dianthin-30 or dianthin-32, saporin e.g.
saporin-S3 or saporin-S6, bouganin or de-immunized derivative
debouganin of bouganin, shiga-like toxin A, pokeweed antiviral
protein, ricin, ricin A chain, modeccin, modeccin A chain, abrin,
abrin A chain, volkensin, volkensin A chain, viscumin, viscumin A
chain; or an animal or human toxin such as frog RNase, or granzyme
B or angiogenin from humans, or any fragment or derivative thereof;
preferably the protein toxin is dianthin and/or saporin.
[0269] An embodiment within the second series of aspects and
embodiments of the invention is the therapeutic combination
according to the invention, wherein the effector moiety comprised
by the second proteinaceous molecule and/or by the third
proteinaceous molecule comprises or consists of at least one
payload, preferably selected from any one or more of a toxin
targeting ribosomes, a toxin targeting elongation factors, a toxin
targeting tubulin, a toxin targeting DNA and a toxin targeting RNA,
more preferably any one or more of emtansine, pasudotox,
maytansinoid derivative DM1, maytansinoid derivative DM4,
monomethyl auristatin E (MMAE, vedotin), monomethyl auristatin F
(MMAF, mafodotin), a Calicheamicin, N-Acetyl-.gamma.-calicheamicin,
a pyrrolobenzodiazepine (PBD) dimer, a benzodiazepine, a CC-1065
analogue, a duocarmycin, Doxorubicin, paclitaxel, docetaxel,
cisplatin, cyclophosphamide, etoposide, docetaxel, 5-fluorouracyl
(5-FU), mitoxantrone, a tubulysin, an indolinobenzodiazepine,
AZ13599185, a cryptophycin, rhizoxin, methotrexate, an
anthracycline, a camptothecin analogue, SN-38, DX-8951f, exatecan
mesylate, truncated form of Pseudomonas aeruginosa exotoxin (PE38),
a Duocarmycin derivative, an amanitin, a-amanitin, a spliceostatin,
a thailanstatin, ozogamicin, tesirine, Amberstatin269 and
soravtansine, or a derivative thereof.
[0270] An embodiment within the second series of aspects and
embodiments of the invention is the first proteinaceous molecule
according to the invention, wherein the first proteinaceous
molecule comprises more than one saponin, preferably 2, 3, 4, 5, 6,
8, 10, 16, 32, 64 or 1-100 saponins, or any number of saponins
therein between, such as 7, 9, 12 saponins, covalently bound
directly to an amino-acid residue of the first proteinaceous
molecule, preferably to a cysteine and/or to a lysine, and/or
covalently bound via at least one linker and/or via at least one
cleavable linker and/or via at least one polymeric or oligomeric
scaffold, preferably 1-8 of such scaffolds or 2-4 of such
scaffolds, wherein the at least one scaffold is optionally based on
a dendron, wherein 1-32 saponins such as 2, 3, 4, 5, 6, 8, 10, 16,
32 saponins, or any number of saponins therein between, such as 7,
9, 12 saponins, are covalently bound to the at least one
scaffold.
[0271] An embodiment within the second series of aspects and
embodiments of the invention is the first proteinaceous molecule
according to the invention, wherein the at least one linker is a
non-cleavable linker or a cleavable linker, wherein the cleavable
linker is for example subject to cleavage under acidic conditions,
reductive conditions, enzymatic conditions or light-induced
conditions, and preferably the cleavable linker comprises a
hydrazone bond or a hydrazide bond subject to cleavage under acidic
conditions when bound to saponin, and/or comprises a bond
susceptible to proteolysis, for example proteolysis by Cathepsin B,
and/or is a bond susceptible for cleavage under reductive
conditions such as a disulphide bond, when bound to saponin.
[0272] An embodiment within the second series of aspects and
embodiments of the invention is the first proteinaceous molecule
according to the invention, wherein the cleavable linker is subject
to cleavage in vivo under acidic conditions as present in endosomes
and/or lysosomes of mammalian cells, preferably human cells,
preferably at pH 4.0-6.5, and more preferably at pH 5.5, when the
cleavable linker is bound to a saponin.
[0273] An embodiment within the second series of aspects and
embodiments of the invention is the first proteinaceous molecule
according to the invention, wherein the oligomeric or polymeric
scaffold comprises a polymeric or oligomeric structure and
comprises a chemical group, the chemical group for covalently
coupling of the scaffold to the amino-acid residue of said first
proteinaceous molecule.
[0274] An embodiment within the second series of aspects and
embodiments of the invention is the first proteinaceous molecule
according to the invention, wherein the at least one saponin is
covalently bound to the polymeric or oligomeric structure of the
oligomeric or polymeric scaffold via at least one cleavable linker
according to the invention.
[0275] An embodiment within the second series of aspects and
embodiments of the invention is the first proteinaceous molecule
according to the invention, wherein the chemical group of the
oligomeric or polymeric scaffold, for covalently coupling of the
oligomeric or polymeric scaffold to the amino-acid residue of said
first proteinaceous molecule, is a click chemistry group,
preferably selected from a tetrazine, an azide, an alkene or an
alkyne, or a cyclic derivative of these groups, more preferably
said chemical group is an azide.
[0276] An embodiment within the second series of aspects and
embodiments of the invention is the first proteinaceous molecule
according to the invention, wherein the polymeric or oligomeric
structure of the oligomeric or polymeric scaffold comprises a
linear, branched and/or cyclic polymer, oligomer, dendrimer,
dendron, dendronized polymer, dendronized oligomer, a DNA, a
polypeptide, poly-lysine, a poly-ethylene glycol, or an assembly of
these polymeric or oligomeric structures which assembly is
preferably built up by covalent cross-linking.
[0277] An aspect of the invention within the second series of
aspects and embodiments of the invention relates to a composition
comprising the first proteinaceous molecule according to the
invention and the second proteinaceous molecule according to the
invention.
[0278] An aspect of the invention within the second series of
aspects and embodiments of the invention relates to a composition
comprising the first proteinaceous molecule according to the
invention and the third proteinaceous molecule according to the
invention.
[0279] An embodiment within the second series of aspects and
embodiments of the invention is the composition according to the
invention, wherein the effector moiety that is comprised by the
second proteinaceous molecule or by the third proteinaceous
molecule is any one of the effector moieties according to the
invention, preferably a BNA.
[0280] An aspect of the invention within the second series of
aspects and embodiments of the invention relates to a composition
comprising the first proteinaceous molecule according to the
invention and any one or more of an oligonucleotide, a nucleic acid
and a xeno nucleic acid, preferably selected from at least one of a
vector, a gene, a cell suicide inducing transgene, deoxyribonucleic
acid (DNA), ribonucleic acid (RNA), anti-sense oligonucleotide
(ASO, AON), short interfering RNA (siRNA), microRNA (miRNA), DNA
aptamer, RNA aptamer, mRNA, mini-circle DNA, peptide nucleic acid
(PNA), phosphoramidate morpholino oligomer (PMO), locked nucleic
acid (LNA), bridged nucleic acid (BNA), 2'-deoxy-2'-fluoroarabino
nucleic acid (FANA), 2'-O-methoxyethyl-RNA (MOE),
2'-0,4'-aminoethylene bridged nucleic acid, 3'-fluoro hexitol
nucleic acid (FHNA), a plasmid, glycol nucleic acid (GNA) and
threose nucleic acid (TNA), or a derivative thereof, more
preferably a BNA, for example a BNA for silencing HSP27 protein
expression.
[0281] An aspect of the invention within the second series of
aspects and embodiments of the invention relates to an
antibody-drug conjugate or a ligand-drug conjugate comprising the
first proteinaceous molecule according to the invention and an
effector moiety.
[0282] An embodiment within the second series of aspects and
embodiments of the invention is the antibody-drug conjugate or the
ligand-drug conjugate according to the invention, wherein the
antibody can bind to any one of CD71, CA125, EpCAM(17-1A), CD52,
CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin
alpha-V beta-3, HER2, EGFR, CD20, CD22, Folate receptor 1, CD146,
CD56, CD19, CD138, CD27L receptor, PSMA, CanAg, integrin-alphaV,
CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239, CD70, CD123,
CD352, DLL3, CD25, ephrinA4, MUC1, Trop2, CEACAM5, CEACAM6, HER3,
CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1,
CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2, preferably CD71, HER2, EGFR,
and/or is or comprises any one of cetuximab, daratumumab,
gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab,
moxetumomab, polatuzumab, obinutuzumab, OKT-9 anti-CD71 monoclonal
antibody of the IgG type, pertuzumab, rituximab, ofatumumab,
Herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal
antibody, an antibody of Table A2 or Table A3 or Table A4,
preferably cetuximab or trastuzumab or OKT-9, or at least one
tumor-cell receptor binding-fragment thereof and/or at least one
tumor-cell receptor binding-domain thereof, and/or wherein the
antibody-drug conjugate comprises any one of Gemtuzumab ozogamicin,
Brentuximab vedotin, Trastuzumab emtansine, Inotuzumab ozogamicin,
Moxetumomab pasudotox and Polatuzumab vedotin and an antibody-drug
conjugate of Table A2 and Table A3, or wherein the ligand-drug
conjugate comprises at least one ligand for binding to a
cell-surface molecule such as EGF or a cytokine.
[0283] An embodiment within the second series of aspects and
embodiments of the invention is the antibody-drug conjugate or the
ligand-drug conjugate according to the invention, wherein the
effector moiety is any one or more of the effector moieties
according to the invention.
[0284] An aspect of the invention within the second series of
aspects and embodiments of the invention relates to a
pharmaceutical composition comprising the composition according to
the invention or the antibody-drug conjugate according to the
invention or the ligand-drug conjugate according to the invention,
and optionally further comprising a pharmaceutically acceptable
excipient.
[0285] An aspect of the invention within the second series of
aspects and embodiments of the invention relates to the therapeutic
combination or the composition or the antibody-drug conjugate or
the ligand-drug conjugate or the pharmaceutical composition
according to the invention, for use as a medicament.
[0286] An aspect of the invention within the second series of
aspects and embodiments of the invention relates to the therapeutic
combination or the composition or the antibody-drug conjugate or
the ligand-drug conjugate or the pharmaceutical composition
according to the invention, for use in the treatment or prevention
of a cancer or an autoimmune disease.
[0287] An aspect of the invention within a third series of aspects
and embodiments of the invention relates to an effector moiety
capable of inducing an intracellular effect when present inside a
mammalian cell, the effector moiety conjugated with at least one
saponin, wherein the at least one saponin is covalently bound to
the effector moiety via at least one linker, or is covalently bound
directly to said effector moiety.
[0288] An embodiment within the third series of aspects and
embodiments of the invention is the effector moiety according to
the invention, comprising or consisting of at least one
oligonucleotide, a nucleic acid, a xeno nucleic acid, preferably
selected from any one or more of a vector, a gene, a cell suicide
inducing transgene, deoxyribonucleic acid (DNA), ribonucleic acid
(RNA), anti-sense oligonucleotide (ASO, AON), short interfering RNA
(siRNA), microRNA (miRNA), DNA aptamer, RNA aptamer, mRNA,
mini-circle DNA, peptide nucleic acid (PNA), phosphoramidate
morpholino oligomer (PMO), locked nucleic acid (LNA), bridged
nucleic acid (BNA), 2'-deoxy-2'-fluoroarabino nucleic acid (FANA),
2'-O-methoxyethyl-RNA (MOE), 2'-0,4'-aminoethylene bridged nucleic
acid, 3'-fluoro hexitol nucleic acid (FHNA), a plasmid, glycol
nucleic acid (GNA) and threose nucleic acid (TNA), or a derivative
thereof, more preferably a BNA, for example a BNA for silencing
HSP27 protein expression.
[0289] An embodiment within the third series of aspects and
embodiments of the invention is the effector moiety according to
the invention, comprising at least one proteinaceous molecule, the
proteinaceous molecule preferably selected from any one or more of
a peptide, a protein, an enzyme such as urease and Cre-recombinase,
a ribosome-inactivating protein, a proteinaceous toxin such as any
one or more of a protein toxin selected from Table A5 and/or a
bacterial toxin or plant toxin, more preferably selected from any
one or more of a viral toxin such as apoptin; a bacterial toxin
such as Shiga toxin, Shiga-like toxin, Pseudomonas aeruginosa
exotoxin (PE) or exotoxin A of PE, full-length or truncated
diphtheria toxin (DT), cholera toxin; a fungal toxin such as
alpha-sarcin; a plant toxin including ribosome-inactivating
proteins and the A chain of type 2 ribosome-inactivating proteins
such as dianthin e.g. dianthin-30 or dianthin-32, saporin e.g.
saporin-S3 or saporin-S6, bouganin or de-immunized derivative
debouganin of bouganin, shiga-like toxin A, pokeweed antiviral
protein, ricin, ricin A chain, modeccin, modeccin A chain, abrin,
abrin A chain, volkensin, volkensin A chain, viscumin, viscumin A
chain; or an animal or human toxin such as frog RNase, or granzyme
B or angiogenin from humans, or any fragment or derivative thereof;
preferably the protein toxin is dianthin and/or saporin.
[0290] An embodiment within the third series of aspects and
embodiments of the invention is the effector moiety according to
the invention, comprising at least one payload, the payload
preferably selected from any one or more of a toxin targeting
ribosomes, a toxin targeting elongation factors, a toxin targeting
tubulin, a toxin targeting DNA and a toxin targeting RNA, more
preferably any one or more of emtansine, pasudotox, maytansinoid
derivative DM1, maytansinoid derivative DM4, monomethyl auristatin
E (MMAE, vedotin), monomethyl auristatin F (MMAF, mafodotin), a
Calicheamicin, N-Acetyl-.gamma.-calicheamicin, a
pyrrolobenzodiazepine (PBD) dimer, a benzodiazepine, a CC-1065
analogue, a duocarmycin, Doxorubicin, paclitaxel, docetaxel,
cisplatin, cyclophosphamide, etoposide, docetaxel, 5-fluorouracyl
(5-FU), mitoxantrone, a tubulysin, an indolinobenzodiazepine,
AZ13599185, a cryptophycin, rhizoxin, methotrexate, an
anthracycline, a camptothecin analogue, SN-38, DX-8951f, exatecan
mesylate, truncated form of Pseudomonas aeruginosa exotoxin (PE38),
a Duocarmycin derivative, an amanitin, a-amanitin, a spliceostatin,
a thailanstatin, ozogamicin, tesirine, Amberstatin269 and
soravtansine, or a derivative thereof.
[0291] An embodiment within the third series of aspects and
embodiments of the invention is the effector moiety according to
the invention, wherein the at least one saponin is a triterpenoid
saponin and/or a bisdesmosidic triterpene saponin belonging to the
type of a 12,13-dehydrooleanane with an aldehyde function in
position C-23 and optionally comprising a glucuronic acid function
in a carbohydrate substituent at the C-3beta-OH group of the
saponin, and/or a saponin isolated from a Gypsophila species and/or
a Saponaria species and/or an Agrostemma species and/or a Quillaja
species such as Quillaja saponaria.
[0292] An embodiment within the third series of aspects and
embodiments of the invention is the effector moiety according to
the invention, wherein the at least one saponin is a single
specific saponin or is a mixture of two or more different
saponins.
[0293] An embodiment within the third series of aspects and
embodiments of the invention is the effector moiety according to
the invention, wherein the saponin is one or more of the saponins
in Table A1 or Scheme I, SO1861, SA1657, GE1741, SA1641, QS-21,
QS-21A, QS-21 A-api, QS-21 A-xyl, QS-21B, QS-21 B-api, QS-21 B-xyl,
QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS1861, QS1862,
Quillajasaponin, Saponinum album, QS-18, Quil-A, Gyp1, gypsoside A,
AG1, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, or any of their
stereomers and/or any combinations thereof, preferably the saponin
is SO1861 and/or GE1741 and/or SA1641 and/or QS-21 and/or saponin
with a quillaic acid aglycon core, a
Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA carbohydrate substituent
at the C-3beta-OH group and a
Glc-(1.fwdarw.3)-Xyl-(1.fwdarw.4)-Rha-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)-4-OA-
c-Qui-(1.fwdarw.4)]-Fuc carbohydrate substituent at the C-28-OH
group, and/or is
3-O-beta-D-galactopyranosyl-(1.fwdarw.2)-[beta-D-xylopyranosyl--
(1.fwdarw.3)]-beta-D-glucuronopyranosyl quillaic acid
28-O-beta-D-glucopyranosyl-(1.fwdarw.3)-beta-D-xylopyranosyl-(1.fwdarw.4)-
-alpha-L-rhamnopyranosyl-(1.fwdarw.2)-[beta-D-xylopyranosyl-(1.fwdarw.3)-4-
-OAc-beta-D-quinovopyranosyl-(1.fwdarw.4)]-beta-D-fucopyranoside,
more preferably the saponin is SO1861 and/or QS-21.
[0294] An embodiment within the third series of aspects and
embodiments of the invention is the effector moiety according to
the invention, wherein the saponin is a bisdesmosidic saponin
having a molecular mass of at least 1.500 Dalton and comprising an
oleanan-type triterpene containing an aldehyde group at the C-23
position and optionally a hydroxyl group at the C-16 position, with
a first branched carbohydrate side chain at the C-3 position which
first branched carbohydrate side chain optionally contains
glucuronic acid, wherein the saponin contains an ester group with a
second branched carbohydrate side chain at the C-28 position which
second branched carbohydrate chain preferably comprises at least
four carbohydrate units and optionally contains at least one acetyl
residue such as two acetyl residues and/or optionally comprises one
or more deoxy carbohydrates and/or quinovose and/or glucose and/or
4-methoxycinnamic acid and/or optionally comprising
5-O-[5-O-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl--
octanoic acid and/or optionally comprising
5-O-[5-O-Rha-(1.fwdarw.2)-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-di-
hydroxy-6-methyl-octanoic acid bound to a carbohydrate via an ester
bond, or wherein the at least one saponin is QS-21 or any one or
more of QS-21A, QS-21 A-api, QS-21 A-xyl, QS-21B, QS-21 B-api,
QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS-18,
QS1861, protonated QS1861 (QS1862), Quil-A.
[0295] An embodiment within the third series of aspects and
embodiments of the invention is the effector moiety according to
the invention, wherein the at least one saponin is a bisdesmosidic
triterpene saponin belonging to the type of a 12,13-dehydrooleanane
with an aldehyde function in position C-23, wherein the at least
one saponin is covalently coupled to an amino-acid residue, when
present, of the effector moiety via an aldehyde function in the
saponin, preferably said aldehyde function in position C-23,
preferably via at least one linker, more preferably via at least
one cleavable linker, wherein the amino-acid residue preferably is
selected from cysteine and lysine.
[0296] An embodiment within the third series of aspects and
embodiments of the invention is the effector moiety according to
the invention, wherein the at least one saponin is a bisdesmosidic
triterpene saponin belonging to the type of a 12,13-dehydrooleanane
with an aldehyde function in position C-23 and comprising a
glucuronic acid function in a carbohydrate substituent at the
C-3beta-OH group of the saponin, wherein the at least one saponin
is covalently coupled to an amino-acid residue, when present, of
the effector moiety via the glucuronic acid function in the
carbohydrate substituent at the C-3beta-OH group of the saponin,
preferably via at least one linker, wherein the amino-acid residue
preferably is selected from cysteine and lysine.
[0297] An embodiment within the third series of aspects and
embodiments of the invention is the effector moiety according to
the invention, wherein the at least one linker comprises at least
one non-cleavable linker and/or at least one cleavable linker,
wherein optionally said cleavable linker is subject to cleavage
under acidic, reductive, enzymatic or light-induced conditions, and
preferably the cleavable linker comprises a cleavable bond selected
from a hydrazone bond and a hydrazide bond subject to cleavage
under acidic conditions, and/or a bond susceptible to proteolysis,
for example proteolysis by Cathepsin B, and/or a bond susceptible
for cleavage under reductive conditions such as a disulphide
bond.
[0298] An embodiment within the third series of aspects and
embodiments of the invention is the effector moiety according to
the invention, wherein the at least one linker comprises at least
one cleavable linker which is subject to cleavage in vivo under
acidic conditions as present in endosomes and/or in lysosomes of
mammalian cells, preferably of human cells, preferably at pH
4.0-6.5, and more preferably at pH.ltoreq.5.5.
[0299] An embodiment within the third series of aspects and
embodiments of the invention is the effector moiety according to
the invention, wherein the at least one saponin is covalently bound
to a lysine side chain, forming an amide bond, and/or to a cysteine
side chain, forming a thio-ether linkage, or a disulphide bond,
wherein the lysine and/or cysteine is/are comprised by the effector
moiety, and wherein the saponin is bound either directly to the
effector moiety, or bound via at least one linker.
[0300] An embodiment within the third series of aspects and
embodiments of the invention is the effector moiety according to
the invention, wherein the at least one saponin is covalently bound
to the effector moiety via at least one linker, wherein the linker
is or comprises a scaffold comprising a polymeric or oligomeric
structure and a chemical group for covalently coupling of the
scaffold to the effector moiety.
[0301] An embodiment within the third series of aspects and
embodiments of the invention is the effector moiety according to
the invention, wherein the at least one saponin is covalently bound
to the polymeric or oligomeric structure of the scaffold via a
cleavable bond.
[0302] An embodiment within the third series of aspects and
embodiments of the invention is the effector moiety according to
the invention, wherein the cleavable bond is subject to cleavage
under any of acidic conditions, reductive conditions, enzymatic
conditions and light-induced conditions, more preferably the
cleavable bond is a hydrazone bond or a hydrazide bond subject to
cleavage under acidic conditions, and/or is a bond susceptible to
proteolysis, for example proteolysis by Cathepsin B, and/or is a
bond susceptible for cleavage under reductive conditions such as a
disulphide bond.
[0303] An embodiment within the third series of aspects and
embodiments of the invention is the effector moiety according to
the invention, wherein the cleavable bond is subject to cleavage in
vivo under acidic conditions as present in endosomes and/or in
lysosomes of mammalian cells, preferably of human cells, preferably
at pH 4.0-6.5, and more preferably at pH.ltoreq.5.5.
[0304] An embodiment within the third series of aspects and
embodiments of the invention is the effector moiety according to
the invention, wherein the at least one saponin is covalently bound
to the polymeric or oligomeric structure of the scaffold via an
imine bond, a hydrazone bond, a hydrazide bond, an oxime bond, a
1,3-dioxolane bond, a disulphide bond, a thio-ether bond, an amide
bond, a peptide bond and/or an ester bond, preferably via at least
one linker, preferably an amide bond, a hydrazide bond, a
thio-ether bond and/or a hydrazone bond.
[0305] An embodiment within the third series of aspects and
embodiments of the invention is the effector moiety according to
the invention, wherein the at least one saponin is covalently bound
to the polymeric or oligomeric structure of the scaffold, involving
in the covalent bond the aldehyde function in position C-23 of the
at least one saponin, when present, the covalent bond being
preferably an imine bond or a hydrazone bond or an amide bond or a
thio-ether bond or a disulphide bond, and/or involving in the
covalent bond the glucuronic acid function in the carbohydrate
substituent at the C-3beta-OH group of the at least one saponin,
when present, wherein preferably the covalent bond is an amide bond
or a disulphide bond or a thio-ether bond.
[0306] An embodiment within the third series of aspects and
embodiments of the invention is the effector moiety according to
the invention, wherein the aldehyde function in position C-23 of
the at least one saponin is covalently coupled to linker
N-.epsilon.-maleimidocaproic acid hydrazide, which linker is
covalently coupled via a thio-ether bond to a sulfhydryl group in
the polymeric or oligomeric structure of the scaffold, such as a
sulfhydryl group of a cysteine.
[0307] An embodiment within the third series of aspects and
embodiments of the invention is the effector moiety according to
the invention, wherein the glucuronic acid function in the
carbohydrate substituent at the C-3beta-OH group of the at least
one saponin is covalently coupled to linker
1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid hexafluorophosphate, which linker is covalently coupled via
an amide bond to an amine group in the polymeric or oligomeric
structure of the scaffold, such as an amine group of a lysine or an
N-terminus of the polymeric or oligomeric structure of the
scaffold.
[0308] An embodiment within the third series of aspects and
embodiments of the invention is the effector moiety according to
the invention, wherein the chemical group of the scaffold, for
covalently coupling of the scaffold to the effector moiety, is a
click chemistry group, preferably selected from a tetrazine, an
azide, an alkene or an alkyne, or a cyclic derivative of these
groups, more preferably the click chemistry group is an azide.
[0309] An embodiment within the third series of aspects and
embodiments of the invention is the effector moiety according to
the invention, wherein the polymeric or oligomeric structure of the
scaffold comprises a linear, branched and/or cyclic polymer,
oligomer, dendrimer, dendron, dendronized polymer, dendronized
oligomer, a DNA, a polypeptide, poly-lysine, a poly-ethylene
glycol, or an assembly of these polymeric or oligomeric structures
which assembly is preferably built up by covalent
cross-linking.
[0310] An embodiment within the third series of aspects and
embodiments of the invention is the effector moiety according to
the invention, wherein the at least one saponin is a defined number
of saponins or a defined range of saponins, preferably 1-128
saponins or at least 2, 3, 4, 5, 6, 8, 10, 16, 32, 64 or 128
saponins, or any number of saponins therein between, such as 7, 9,
12 saponins.
[0311] An embodiment within the third series of aspects and
embodiments of the invention is the effector moiety according to
the invention, wherein the effector moiety comprises more than one
saponin, preferably 2, 3, 4, 5, 6, 8, 10, 16, 32, 64 or 1-100
saponins, or any number of saponins therein between, such as 7, 9,
12 saponins, covalently bound directly to an amino-acid residue of
the effector moiety, preferably to a cysteine and/or to a lysine,
and/or covalently bound via at least one linker and/or via at least
one cleavable linker and/or via at least one polymeric or
oligomeric scaffold of any one of the claims 14-23, preferably 1-8
of such scaffolds or 2-4 of such scaffolds, wherein 1-32 saponins,
preferably 2, 3, 4, 5, 6, 8, 10, 16 or 32 saponins, are covalently
bound to the at least one scaffold.
[0312] An aspect of the invention within the third series of
aspects and embodiments of the invention relates to an
antibody-drug conjugate comprising the effector moiety according to
the invention, or a ligand-drug conjugate comprising the effector
moiety according to the invention.
[0313] An embodiment within the third series of aspects and
embodiments of the invention is the antibody-drug conjugate or the
ligand-drug conjugate according to the invention, wherein the
antibody can bind to any one of CD71, CA125, EpCAM(17-1A), CD52,
CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin
alpha-V beta-3, HER2, EGFR, CD20, CD22, Folate receptor 1, CD146,
CD56, CD19, CD138, CD27L receptor, PSMA, CanAg, integrin-alphaV,
CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239, CD70, CD123,
CD352, DLL3, CD25, ephrinA4, MUC1, Trop2, CEACAM5, CEACAM6, HER3,
CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1,
CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2, preferably CD71, HER2, EGFR,
and/or wherein the antibody of the antibody-drug conjugate is or
comprises any one of cetuximab, daratumumab, gemtuzumab,
trastuzumab, panitumumab, brentuximab, inotuzumab, moxetumomab,
polatuzumab, obinutuzumab, OKT-9 anti-CD71 monoclonal antibody of
the IgG type, pertuzumab, rituximab, ofatumumab, Herceptin,
alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal antibody, an
antibody of Table A2 or Table A3 or Table A4, preferably cetuximab
or trastuzumab or OKT-9, or at least one tumor-cell specific
receptor binding-fragment thereof and/or at least one tumor-cell
specific receptor binding-domain thereof, and/or wherein the
antibody-drug conjugate comprises any one of Gemtuzumab ozogamicin,
Brentuximab vedotin, Trastuzumab emtansine, Inotuzumab ozogamicin,
Moxetumomab pasudotox and Polatuzumab vedotin and an antibody-drug
conjugate of Table A2 and Table A3, and/or wherein the ligand-drug
conjugate comprises or consists of at least one non-proteinaceous
ligand and/or at least one proteinaceous ligand for binding to a
cell-surface molecule such as EGF or a cytokine.
[0314] An aspect of the invention within the third series of
aspects and embodiments of the invention relates to a therapeutic
combination comprising: (a) the effector moiety according to the
invention and optionally a pharmaceutically acceptable excipient;
and (b) an antibody-drug conjugate or a ligand-drug conjugate, and
optionally a pharmaceutically acceptable excipient.
[0315] An embodiment within the third series of aspects and
embodiments of the invention is the therapeutic combination
according to the invention, wherein the antibody-drug conjugate can
bind to any one of tumor-cell receptors CD71, CA125, EpCAM(17-1A),
CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular
integrin alpha-V beta-3, HER2, EGFR, CD20, CD22, Folate receptor 1,
CD146, CD56, CD19, CD138, CD27L receptor, PSMA, CanAg,
integrin-alphaV, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239,
CD70, CD123, CD352, DLL3, CD25, ephrinA4, MUC1, Trop2, CEACAM5,
CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3,
CD7, PD-L1, CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2, preferably CD71,
HER2, EGFR, and/or wherein the antibody of the antibody-drug
conjugate is or comprises any one of cetuximab, daratumumab,
gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab,
moxetumomab, polatuzumab, obinutuzumab, OKT-9 anti-CD71 monoclonal
antibody of the IgG type, pertuzumab, rituximab, ofatumumab,
Herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal
antibody, an antibody of Table A2 or Table A3 or Table A4,
preferably cetuximab or trastuzumab or OKT-9, or at least one
tumor-cell specific receptor binding-fragment thereof and/or at
least one tumor-cell specific receptor binding-domain thereof,
and/or wherein the antibody-drug conjugate comprises any one of
Gemtuzumab ozogamicin, Brentuximab vedotin, Trastuzumab emtansine,
Inotuzumab ozogamicin, Moxetumomab pasudotox and Polatuzumab
vedotin and an antibody-drug conjugate of Table A2 and Table A3,
and/or wherein the ligand-drug conjugate comprises or consists of
at least one non-proteinaceous ligand and/or at least one
proteinaceous ligand for binding to a cell-surface molecule such as
EGF or a cytokine.
[0316] An aspect of the invention within the third series of
aspects and embodiments of the invention relates to a
pharmaceutical composition comprising the effector moiety according
to the invention or the antibody-drug conjugate according to the
invention or the ligand-drug conjugate according to the invention,
and optionally a pharmaceutically acceptable excipient.
[0317] An aspect of the invention within the third series of
aspects and embodiments of the invention relates to the effector
moiety according to the invention or the antibody-drug conjugate
according to the invention or the therapeutic combination according
to the invention or the ligand-drug conjugate according to the
invention or the pharmaceutical composition according to the
invention, for use as a medicament.
[0318] An aspect of the invention within the third series of
aspects and embodiments of the invention relates to the effector
moiety according to the invention or the antibody-drug conjugate
according to the invention or the ligand-drug conjugate according
to the invention or the therapeutic combination according to the
invention or the pharmaceutical composition according to the
invention, for use in the treatment or prevention of a cancer or an
autoimmune disease.
[0319] An aspect of the invention within a fourth series of aspects
and embodiments of the invention relates to a conjugate comprising
a cell-surface molecule targeting molecule and at least one
effector moiety and further comprising at least one covalently
bound saponin.
[0320] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the at least one saponin is a triterpenoid saponin and/or a
bisdesmosidic triterpene saponin belonging to the type of a
12,13-dehydrooleanane with an aldehyde function in position C-23
and optionally comprising a glucuronic acid function in a
carbohydrate substituent at the C-3beta-OH group of the saponin,
and/or a saponin isolated from any one or more of a Gypsophila
species and/or a Saponaria species and/or an Agrostemma species
and/or a Quillaja species such as Quillaja saponaria.
[0321] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the at least one saponin is a single specific saponin or is
a mixture of two or more different saponins.
[0322] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the at least one saponin has a molecular mass of 3.000
Dalton or less, preferably 2.500 Dalton or less, more preferably
2.300 Dalton or less, most preferably, 2.000 Dalton or less, such
as 1.500 Dalton-1.900 Dalton.
[0323] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the at least one saponin is one or more of the saponins in
Table A1 or Scheme I, SO1861, SA1657, GE1741, SA1641, QS-21,
QS-21A, QS-21 A-api, QS-21 A-xyl, QS-21B, QS-21 B-api, QS-21 B-xyl,
QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS1861, QS1862,
Quillajasaponin, Saponinum album, QS-18, Quil-A, Gyp1, gypsoside A,
AG1, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, or any of their
stereomers and/or any combinations thereof, preferably the saponin
is SO1861 and/or GE1741 and/or SA1641 and/or QS-21 and/or saponin
with a quillaic acid aglycon core, a
Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA carbohydrate substituent
at the C-3beta-OH group and a
Glc-(1.fwdarw.3)-Xyl-(1.fwdarw.4)-Rha-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)-4-OA-
c-Qui-(1.fwdarw.4)]-Fuc carbohydrate substituent at the C-28-OH
group, and/or is
3-O-beta-D-galactopyranosyl-(1.fwdarw.2)-[beta-D-xylopyranosyl--
(1.fwdarw.3)]-beta-D-glucuronopyranosyl quillaic acid
28-O-beta-D-glucopyranosyl-(1.fwdarw.3)-beta-D-xylopyranosyl-(1.fwdarw.4)-
-alpha-L-rhamnopyranosyl-(1.fwdarw.2)-[beta-D-xylopyranosyl-(1.fwdarw.3)-4-
-OAc-beta-D-quinovopyranosyl-(1.fwdarw.4)]-beta-D-fucopyranoside,
more preferably the at least one saponin is SO1861 and/or
QS-21.
[0324] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the at least one saponin is a bisdesmosidic saponin having
a molecular mass of at least 1.500 Dalton and comprising an
oleanan-type triterpene containing an aldehyde group at the C-23
position and optionally a hydroxyl group at the C-16 position, with
a first branched carbohydrate side chain at the C-3 position which
first branched carbohydrate side chain optionally contains
glucuronic acid, wherein the saponin contains an ester group with a
second branched carbohydrate side chain at the C-28 position which
second branched carbohydrate chain preferably comprises at least
four carbohydrate units, optionally containing at least one acetyl
residue such as two acetyl residues and/or optionally comprising
deoxy carbohydrates and/or optionally comprising quinovose and/or
optionally comprising glucose and/or optionally comprising
4-methoxycinnamic acid and/or optionally comprising
5-O-[5-O-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl--
octanoic acid and/or optionally comprising
5-O-[5-O-Rha-(1.fwdarw.2)-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-di-
hydroxy-6-methyl-octanoic acid bound to a carbohydrate via an ester
bond, or wherein the at least one saponin is QS-21 or any one or
more of QS-21A, QS-21 A-api, QS-21 A-xyl, QS-21B, QS-21 B-api,
QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS-18,
QS1861, protonated QS1861 (QS1862), Quil-A.
[0325] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the cell-surface molecule targeting molecule comprises or
consists of a ligand or a proteinaceous ligand or a proteinaceous
binding molecule for binding to the cell-surface molecule.
[0326] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the cell-surface molecule targeting molecule comprises or
consists of a non-proteinaceous ligand and/or a proteinaceous
ligand for binding to a cell-surface molecule such as EGF or a
cytokine, and/or comprises or consists of an immunoglobulin, at
least one binding domain of an immunoglobulin and/or at least one
binding fragment of an immunoglobulin, such as an antibody, an IgG,
a molecule comprising or consisting of a Vhh domain or Vh domain, a
Fab, an scFv, an Fv, a dAb, an F(ab).sub.2, Fcab fragment, which
can bind to the cell-surface molecule.
[0327] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the cell-surface molecule targeting molecule can bind to a
tumor-cell surface molecule, preferably a tumor-cell receptor such
as a tumor-cell specific receptor, more preferably a receptor
selected from CD71, CA125, EpCAM(17-1A), CD52, CEA, CD44v6, FAP,
EGF-IR, integrin, syndecan-1, vascular integrin alpha-V beta-3,
HER2, EGFR, CD20, CD22, Folate receptor 1, CD146, CD56, CD19,
CD138, CD27L receptor, PSMA, CanAg, integrin-alphaV, CA6, CD33,
mesothelin, Cripto, CD3, CD30, CD239, CD70, CD123, CD352, DLL3,
CD25, ephrinA4, MUC1, Trop2, CEACAM5, CEACAM6, HER3, CD74, PTK7,
Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA4, CD52,
PDGFRA, VEGFR1, VEGFR2, preferably selected from CD71, HER2 and
EGFR.
[0328] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the tumor-cell receptor is internalized by the tumor cell
after binding to the cell-surface molecule targeting molecule of
the invention and the conjugate of the invention, and wherein
preferably binding of the conjugate to the tumor-cell receptor is
followed by tumor-cell receptor-mediated internalization, e.g. via
endocytosis, of a complex of the conjugate and the tumor-cell
receptor.
[0329] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the cell-surface molecule targeting molecule is or
comprises a monoclonal antibody or at least one cell-surface
molecule binding fragment or -domain thereof, and preferably
comprises or consists of any one of cetuximab, daratumumab,
gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab,
moxetumomab, polatuzumab, obinutuzumab, OKT-9 anti-CD71 monoclonal
antibody of the IgG type, pertuzumab, rituximab, ofatumumab,
Herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal
antibody, an antibody of Table A2 or Table A3 or Table A4,
preferably cetuximab or trastuzumab or OKT-9, or at least one
cell-surface molecule binding fragment or -domain thereof.
[0330] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the at least one effector moiety comprises or consists of
any one or more of an oligonucleotide, a nucleic acid and a xeno
nucleic acid, preferably selected from any one or more of a vector,
a gene, a cell suicide inducing transgene, deoxyribonucleic acid
(DNA), ribonucleic acid (RNA), anti-sense oligonucleotide (ASO,
AON), short interfering RNA (siRNA), microRNA (miRNA), DNA aptamer,
RNA aptamer, mRNA, mini-circle DNA, peptide nucleic acid (PNA),
phosphoramidate morpholino oligomer (PMO), locked nucleic acid
(LNA), bridged nucleic acid (BNA), 2'-deoxy-2'-fluoroarabino
nucleic acid (FANA), 2'-O-methoxyethyl-RNA (MOE),
2'-0,4'-aminoethylene bridged nucleic acid, 3'-fluoro hexitol
nucleic acid (FHNA), a plasmid, glycol nucleic acid (GNA) and
threose nucleic acid (TNA), or a derivative thereof, more
preferably a BNA, for example a BNA for silencing HSP27 protein
expression.
[0331] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the at least one effector moiety comprises or consists of
at least one proteinaceous molecule, preferably selected from any
one or more of a peptide, a protein, an enzyme such as urease and
Cre-recombinase, a ribosome-inactivating protein, a proteinaceous
toxin selected from Table A5 and more preferably selected from any
one or more of a viral toxin such as apoptin; a bacterial toxin
such as Shiga toxin, Shiga-like toxin, Pseudomonas aeruginosa
exotoxin (PE) or exotoxin A of PE, full-length or truncated
diphtheria toxin (DT), cholera toxin; a fungal toxin such as
alpha-sarcin; a plant toxin including ribosome-inactivating
proteins and the A chain of type 2 ribosome-inactivating proteins
such as dianthin e.g. dianthin-30 or dianthin-32, saporin e.g.
saporin-S3 or saporin-S6, bouganin or de-immunized derivative
debouganin of bouganin, shiga-like toxin A, pokeweed antiviral
protein, ricin, ricin A chain, modeccin, modeccin A chain, abrin,
abrin A chain, volkensin, volkensin A chain, viscumin, viscumin A
chain; or an animal or human toxin such as frog RNase, or granzyme
B or angiogenin from humans, or any fragment or derivative thereof;
preferably the protein toxin is dianthin and/or saporin.
[0332] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the at least one effector moiety comprises or consists of
at least one payload, preferably selected from any one or more of a
toxin targeting ribosomes, a toxin targeting elongation factors, a
toxin targeting tubulin, a toxin targeting DNA and a toxin
targeting RNA, more preferably any one or more of emtansine,
pasudotox, maytansinoid derivative DM1, maytansinoid derivative
DM4, monomethyl auristatin E (MMAE, vedotin), monomethyl auristatin
F (MMAF, mafodotin), a Calicheamicin,
N-Acetyl-.gamma.-calicheamicin, a pyrrolobenzodiazepine (PBD)
dimer, a benzodiazepine, a CC-1065 analogue, a duocarmycin,
Doxorubicin, paclitaxel, docetaxel, cisplatin, cyclophosphamide,
etoposide, docetaxel, 5-fluorouracyl (5-FU), mitoxantrone, a
tubulysin, an indolinobenzodiazepine, AZ13599185, a cryptophycin,
rhizoxin, methotrexate, an anthracycline, a camptothecin analogue,
SN-38, DX-8951f, exatecan mesylate, truncated form of Pseudomonas
aeruginosa exotoxin (PE38), a Duocarmycin derivative, an amanitin,
a-amanitin, a spliceostatin, a thailanstatin, ozogamicin, tesirine,
Amberstatin269 and soravtansine, or a derivative thereof.
[0333] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the at least one effector moiety is covalently bound to the
cell-surface molecule targeting molecule, either via at least one
linker or bound directly to the cell-surface molecule targeting
molecule.
[0334] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the at least one effector moiety is covalently bound to the
cell-surface molecule targeting molecule, thereby forming any one
of antibody-drug conjugates Gemtuzumab ozogamicin, Brentuximab
vedotin, Trastuzumab emtansine, Inotuzumab ozogamicin, Moxetumomab
pasudotox and Polatuzumab vedotin and an antibody-drug conjugate of
Table A2 and Table A3.
[0335] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the at least one saponin is covalently bound to the
cell-surface molecule targeting molecule preferably an amino-acid
residue of the cell-surface molecule targeting molecule, via an
aldehyde function in the saponin, and/or to the at least one
effector moiety preferably via an amino-acid residue in the at
least one effector moiety, via an aldehyde function in the saponin,
preferably an aldehyde function in position C-23 in a bisdesmosidic
triterpene saponin belonging to the type of a
12,13-dehydrooleanane.
[0336] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the aldehyde function in the at least one saponin,
preferably the aldehyde function in position C-23 of the at least
one saponin, is covalently coupled to linker
N-.epsilon.-maleimidocaproic acid hydrazide, which linker is
covalently coupled via a thio-ether bond to a sulfhydryl group in
the cell-surface molecule targeting molecule and/or in the at least
one effector moiety, such as a sulfhydryl group of a cysteine.
[0337] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the at least one saponin is a bisdesmosidic triterpene
saponin belonging to the type of a 12,13-dehydrooleanane, with an
aldehyde function in position C-23 and comprising a glucuronic acid
function in a carbohydrate substituent at the C-3beta-OH group of
the saponin, wherein the saponin is covalently bound to an
amino-acid residue of the cell-surface molecule targeting molecule
and/or to the at least one effector moiety via said glucuronic acid
function and preferably via an amino-acid residue in the at least
one effector moiety.
[0338] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the glucuronic acid function in the carbohydrate
substituent at the C-3beta-OH group of the at least one saponin is
covalently coupled to linker
1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid hexafluorophosphate, which linker is covalently coupled via
an amide bond to an amine group in the cell-surface molecule
targeting molecule and/or in the at least one effector moiety, such
as an amine group of a lysine or an N-terminus of the cell-surface
molecule targeting molecule and/or of the at least one effector
moiety.
[0339] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the at least one saponin is covalently bound to the
cell-surface molecule targeting molecule and/or to the at least one
effector moiety either directly or via at least one linker such as
a bi-functional linker, for example based on
N-.epsilon.-maleimidocaproic acid hydrazide and/or based on
1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid hexafluorophosphate, or a tri-functional linker, such as the
tri-functional linker of Scheme II and Structure B.
[0340] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the tri-functional linker comprises a second chemical group
with at least one saponin covalently bound thereto, a third
chemical group for covalent binding to the cell-surface molecule
targeting molecule and a first chemical group for covalent binding
to the at least one effector moiety, preferably the tri-functional
linker is the trifunctional linker of Scheme II and Structure
B.
[0341] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the at least one saponin is covalently bound to the
cell-surface molecule targeting molecule and to the at least one
effector moiety via at least one linker comprising a tri-functional
linker to which tri-functional linker both the cell-surface
molecule targeting molecule and the at least one effector moiety
are bound, preferably the tri-functional linker is the
trifunctional linker of Scheme II and Structure B.
[0342] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the at least one linker comprises at least one cleavable
linker, wherein optionally said cleavable linker is subject to
cleavage under acidic, reductive, enzymatic or light-induced
conditions, and preferably the cleavable linker comprises a
cleavable bond selected from a hydrazone bond or a hydrazide bond
subject to cleavage under acidic conditions, and/or a bond
susceptible to proteolysis, for example proteolysis by Cathepsin B,
and/or a bond susceptible for cleavage under reductive conditions
such as a disulphide bond.
[0343] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the at least one linker comprises at least one cleavable
linker, wherein said cleavable linker is subject to cleavage in
vivo under acidic conditions as present in endosomes and/or in
lysosomes of mammalian cells, preferably of human cells, preferably
at pH 4.0-6.5, and more preferably at pH.ltoreq.5.5.
[0344] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the at least one saponin is covalently bound to a lysine
side chain, forming an amide bond, and/or to a cysteine side chain,
forming a thio-ether linkage or a disulphide bond, wherein the
lysine and/or cysteine is/are comprised by the cell-surface
molecule targeting molecule and/or is/are comprised by the at least
one effector moiety, and wherein the at least one saponin is either
directly bound to the lysine and/or cysteine, or is bound via at
least one linker optionally comprising a cleavable linker and/or a
tri-functional linker such as the tri-functional linker of Scheme
II and Structure B.
[0345] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the linker is based on N-.epsilon.-maleimidocaproic acid
hydrazide and/or based on
1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid hexafluorophosphate, a tri-functional linker such as the
tri-functional linker of Scheme II and Structure B, a cleavable
linker, and/or involves any one or more of a disulphide bond, a
thio-ether bond, an amide bond, a hydrazide bond.
[0346] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the at least one saponin is covalently bound to the
cell-surface molecule targeting molecule and/or to the at least one
effector moiety via at least one linker, wherein the linker is or
comprises a scaffold comprising a polymeric or oligomeric structure
and further comprising at least one fourth chemical group for
covalently coupling of the scaffold to the cell-surface molecule
targeting molecule and/or to the at least one effector moiety.
[0347] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the at least one saponin is covalently bound to the
polymeric or oligomeric structure of the scaffold via a cleavable
bond and/or via a non-cleavable bond.
[0348] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the cleavable bond is subject to cleavage under any of
acidic conditions, reductive conditions, enzymatic conditions and
light-induced conditions, and preferably the cleavable bond
comprises a hydrazone bond or a hydrazide bond subject to cleavage
under acidic conditions, and/or a bond susceptible to proteolysis,
for example proteolysis by Cathepsin B, and/or a bond susceptible
for cleavage under reductive conditions such as a disulphide
bond.
[0349] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the cleavable bond is subject to cleavage in vivo under
acidic conditions as present in endosomes and/or in lysosomes of
mammalian cells, preferably of human cells, preferably at pH
4.0-6.5, and more preferably at pH.ltoreq.5.5.
[0350] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the at least one saponin is covalently bound to the
polymeric or oligomeric structure of the scaffold via any one or
more of an imine bond, a hydrazone bond, a hydrazide bond, an oxime
bond, a 1,3-dioxolane bond, a disulphide bond, a thio-ether bond,
an amide bond, a peptide bond or an ester bond, preferably via at
least one linker.
[0351] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the at least one saponin is covalently bound to the
polymeric or oligomeric structure of the scaffold via any one or
more of an imine bond, a hydrazone bond and a hydrazide bond, which
bond is preferably cleavable according to the invention, wherein
preferably the at least one saponin is covalently bound to the
polymeric or oligomeric structure of the scaffold via the aldehyde
function in position C-23 of the at least one saponin.
[0352] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the aldehyde function in position C-23 of the at least one
saponin is covalently coupled to linker
N-.epsilon.-maleimidocaproic acid hydrazide, which linker is
covalently coupled via a thio-ether bond to a sulfhydryl group in
the polymeric or oligomeric structure of the scaffold, such as a
sulfhydryl group of a cysteine.
[0353] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the at least one saponin is covalently bound to the
polymeric or oligomeric structure of the scaffold via an amide
bond, wherein preferably the at least one saponin is covalently
bound to the polymeric or oligomeric structure of the scaffold via
the glucuronic acid function in the carbohydrate substituent at the
C-3beta-OH group of the at least one saponin, when present.
[0354] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the glucuronic acid function in the carbohydrate
substituent at the C-3beta-OH group of the at least one saponin is
covalently coupled to linker
1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid hexafluorophosphate, which linker is covalently coupled via
an amide bond to an amine group in the polymeric or oligomeric
structure of the scaffold, such as an amine group of a lysine or an
N-terminus of the polymeric or oligomeric structure of the
scaffold.
[0355] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the at least one saponin is covalently bound to the
polymeric or oligomeric structure of the scaffold, involving in the
covalent bond the aldehyde function in position C-23 of the at
least one saponin, when present, and/or involving in the covalent
bond the glucuronic acid function in the carbohydrate substituent
at the C-3beta-OH group of the at least one saponin, when
present.
[0356] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the at least one fourth chemical group of the scaffold, for
covalently coupling of the scaffold to the cell-surface molecule
targeting molecule and/or to the at least one effector moiety, is a
click chemistry group, preferably selected from any one or more of
a tetrazine, an azide, an alkene or an alkyne, or a cyclic
derivative of these groups, preferably an azide group.
[0357] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the polymeric or oligomeric structure of the scaffold
comprises a linear, branched and/or cyclic polymer, oligomer,
dendrimer, dendron, dendronized polymer, dendronized oligomer, a
DNA, a polypeptide, poly-lysine, a poly-ethylene glycol, or an
assembly of these polymeric or oligomeric structures which assembly
is preferably built up by covalent cross-linking.
[0358] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the at least one saponin is a defined number of saponins or
a defined range of saponins, preferably 1-128 saponins or at least
2, 3, 4, 5, 6, 8, 10, 16, 32, 64 or 128 saponins, or any number of
saponins therein between, such as 7, 9, 12 saponins.
[0359] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the conjugate comprises more than one saponin, preferably
2, 3, 4, 5, 6, 8, 10, 16, 32, 64 or 1-100 saponins, or any number
of saponins therein between, such as 7, 9, 12 saponins, covalently
bound directly to an amino-acid residue of the cell-surface
molecule targeting molecule and/or to the at least one effector
moiety and preferably via an amino-acid residue in the at least one
effector moiety, preferably to a cysteine and/or to a lysine,
and/or covalently bound via at least one linker and/or via at least
one cleavable linker and/or via at least one polymeric or
oligomeric scaffold of the invention, preferably 1-8 of such
scaffolds or 2-4 of such scaffolds, wherein 1-32 saponins,
preferably 2, 3, 4, 5, 6, 8, 10, 16 or 32 saponins, or any number
of saponins therein between, such as 7, 9, 12 saponins, are
covalently bound to the at least one scaffold.
[0360] An embodiment within the fourth series of aspects and
embodiments of the invention is the conjugate of the invention,
wherein the at least one saponin is covalently bound to the
cell-surface molecule targeting molecule and to the at least one
effector moiety via a tri-functional linker, the tri-functional
linker comprising a second chemical group with at least one saponin
covalently bound thereto either directly or via a linker such as a
cleavable linker and/or via the scaffold comprising a polymeric or
oligomeric structure and a fourth chemical group according to the
invention for covalently coupling of the scaffold to the
tri-functional linker, the tri-functional linker further comprising
a third chemical group for covalent binding to the cell-surface
molecule targeting molecule and comprising a first chemical group
for covalent binding to the at least one effector moiety, wherein
the cell-surface molecule targeting molecule is bound to the third
chemical group and/or the at least one effector moiety is bound to
the first chemical group, preferably the trifunctional linker is
the trifunctional linker of Scheme II and Structure B.
[0361] An aspect of the invention within the fourth series of
aspects and embodiments of the invention relates to a
pharmaceutical composition comprising the conjugate of the
invention and optionally a pharmaceutically acceptable excipient
and/or a pharmaceutically acceptable diluent.
[0362] An aspect of the invention within the fourth series of
aspects and embodiments of the invention relates to the conjugate
of the invention or the pharmaceutical composition of the
invention, for use as a medicament.
[0363] An aspect of the invention within the fourth series of
aspects and embodiments of the invention relates to the conjugate
of the invention or the pharmaceutical composition of the
invention, for use in the treatment or prevention of a cancer or an
autoimmune disease.
[0364] An aspect of the invention within a fifth series of aspects
and embodiments of the invention relates to a therapeutic
combination for use as a medicament, wherein the therapeutic
combination comprises: (a) a fourth pharmaceutical composition
comprising a fourth proteinaceous molecule comprising a binding
site for binding to an epitope on a cell-surface molecule and at
least one saponin covalently bound to said fourth proteinaceous
molecule preferably to an amino-acid residue of said fourth
proteinaceous molecule, the fourth pharmaceutical composition
optionally further comprising a pharmaceutically acceptable
excipient; and (b) a fifth pharmaceutical composition comprising a
fifth proteinaceous molecule, the fifth proteinaceous molecule
comprising a binding site for binding to the epitope on the
cell-surface molecule of (a) and an effector moiety, the fifth
pharmaceutical composition optionally further comprising a
pharmaceutically acceptable excipient, wherein the binding site of
the fourth proteinaceous molecule and the binding site of the fifth
proteinaceous molecule are the same, and wherein the cell-surface
molecule and the epitope on the cell-surface molecule, to which the
fourth proteinaceous molecule can bind, and the cell-surface
molecule and the epitope on the cell-surface molecule, to which the
fifth proteinaceous molecule can bind, are the same.
[0365] An aspect of the invention within the fifth series of
aspects and embodiments of the invention relates to the fourth
pharmaceutical composition of the invention for use as a
medicament.
[0366] An aspect of the invention within the fifth series of
aspects and embodiments of the invention relates to a therapeutic
combination for use in the treatment or prevention of a cancer in a
human subject, wherein the therapeutic combination comprises: (a)
the fourth pharmaceutical composition of the invention; and (b) the
fifth pharmaceutical composition of the invention, wherein the
cell-surface molecule is expressed on a tumor cell surface, and
preferably the cell-surface molecule is a tumor cell-specific
surface molecule, and wherein preferably the epitope is a
tumor-cell specific epitope.
[0367] An aspect of the invention within the fifth series of
aspects and embodiments of the invention relates to the fourth
pharmaceutical composition of the invention, for use in the
treatment or prophylaxis of a cancer in a patient in need thereof,
wherein the cell-surface molecule is expressed on a tumor cell
surface, and preferably the cell-surface molecule is a tumor
cell-specific surface molecule, and wherein preferably the epitope
is a tumor-cell specific epitope.
[0368] An embodiment within the fifth series of aspects and
embodiments of the invention is the fourth pharmaceutical
composition for use according to the invention or the therapeutic
combination for use of the invention, wherein the fifth
pharmaceutical composition of the invention and the fourth
pharmaceutical composition are administered to the patient in need
thereof.
[0369] An embodiment within the fifth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, or the fourth pharmaceutical composition for use
according to the invention, wherein the binding site of the fourth
proteinaceous molecule and the fifth proteinaceous molecule
comprises or consists of an immunoglobulin or a binding fragment or
binding domain of said immunoglobulin, such as any one or more of
an antibody, an IgG, a molecule comprising or consisting of a Vhh
domain or Vh domain, a Fab, an scFv, an Fv, a dAb, an F(ab)2, Fcab
fragment, and/or comprises or consists of at least one ligand, the
ligand for binding to a cell-surface molecule such as EGF or a
cytokine.
[0370] An embodiment within the fifth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, or the fourth pharmaceutical composition for use
according to the invention, wherein the tumor-cell surface molecule
is a cell-surface receptor specifically present at a tumor cell,
and wherein preferably the epitope is a tumor-cell specific
epitope.
[0371] An embodiment within the fifth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, or the fourth pharmaceutical composition for use
according to the invention, wherein the at least one saponin is a
triterpenoid saponin and/or a bisdesmosidic triterpene saponin
belonging to the type of a 12,13-dehydrooleanane with an aldehyde
function in position C-23 and optionally comprising a glucuronic
acid function in a carbohydrate substituent at the C-3beta-OH group
of the saponin, and/or a saponin isolated from a Gypsophila species
and/or a Saponaria species and/or an Agrostemma species and/or a
Quillaja species such as Quillaja saponaria.
[0372] An embodiment within the fifth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, or the fourth pharmaceutical composition for use
according to the invention, wherein the at least one saponin is a
single specific saponin or is a mixture of two or more different
saponins, such as one or more of the saponins in Table A1 or Scheme
I, SO1861, SA1657, GE1741, SA1641, QS-21, QS-21A, QS-21 A-api,
QS-21 A-xyl, QS-21B, QS-21 B-api, QS-21 B-xyl, QS-7-xyl, QS-7-api,
QS-17-api, QS-17-xyl, QS1861, QS1862, Quillajasaponin, Saponinum
album, QS-18, Quil-A, Gyp1, gypsoside A, AG1, AG2, SO1542, SO1584,
SO1658, SO1674, SO1832, or any of their stereomers and/or any
combinations thereof, preferably the saponin is SO1861 and/or
GE1741 and/or SA1641 and/or QS-21 and/or saponin with a quillaic
acid aglycon core, a Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA
carbohydrate substituent at the C-3beta-OH group and a
Glc-(1.fwdarw.3)-Xyl-(1.fwdarw.4)-Rha-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)-4-OA-
c-Qui-(1.fwdarw.4)]-Fuc carbohydrate substituent at the C-28-OH
group, and/or is
3-O-beta-D-galactopyranosyl-(1.fwdarw.2)-[beta-D-xylopyranosyl--
(1.fwdarw.3)]-beta-D-glucuronopyranosyl quillaic acid
28-O-beta-D-glucopyranosyl-(1.fwdarw.3)-beta-D-xylopyranosyl-(1.fwdarw.4)-
-alpha-L-rhamnopyranosyl-(1.fwdarw.2)-[beta-D-xylopyranosyl-(1.fwdarw.3)-4-
-OAc-beta-D-quinovopyranosyl-(1.fwdarw.4)]-beta-D-fucopyranoside,
more preferably the saponin is SO1861 and/or QS-21.
[0373] An embodiment within the fifth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, or the fourth pharmaceutical composition for use
according to the invention, wherein the at least one saponin is a
bisdesmosidic saponin having a molecular mass of at least 1.500
Dalton and comprising an oleanan-type triterpene containing an
aldehyde group at the C-23 position and optionally a hydroxyl group
at the C-16 position, with a first branched carbohydrate side chain
at the C-3 position which first branched carbohydrate side chain
optionally contains glucuronic acid, wherein the saponin contains
an ester group with a second branched carbohydrate side chain at
the C-28 position which second branched carbohydrate chain
preferably comprises at least four carbohydrate units, optionally
containing at least one acetyl residue such as two acetyl residues
and/or optionally at least one deoxy carbohydrate and/or a
quinovose and/or a glucose and/or 4-methoxycinnamic acid and/or
optionally comprising
5-O-[5-O-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl--
octanoic acid and/or optionally comprising
5-O-[5-O-Rha-(1.fwdarw.2)-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-di-
hydroxy-6-methyl-octanoic acid bound to a carbohydrate via an ester
bond, or wherein the at least one saponin is QS-21 or any one or
more of QS-21A, QS-21 A-api, QS-21 A-xyl, QS-21B, QS-21 B-api,
QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS-18,
QS1861, protonated QS-1861 (QS1862), Quil-A.
[0374] An embodiment within the fifth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, or the fourth pharmaceutical composition for use
according to the invention, wherein the at least one saponin is a
bisdesmosidic triterpene saponin belonging to the type of a
12,13-dehydrooleanane with an aldehyde function in position C-23,
wherein the saponin is covalently coupled to the fourth
proteinaceous molecule, preferably covalently coupled to an
amino-acid residue of the fourth proteinaceous molecule, via an
aldehyde function in the saponin, preferably said aldehyde function
in position C-23, preferably via at least one linker, and/or via at
least one cleavable linker, wherein the amino-acid residue
preferably is selected from cysteine and lysine.
[0375] An embodiment within the fifth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, or the fourth pharmaceutical composition for use
according to the invention, wherein the aldehyde function in
position C-23 of the at least one saponin is covalently coupled to
linker N-.epsilon.-maleimidocaproic acid hydrazide, which linker is
covalently coupled via a thio-ether bond to a sulfhydryl group in
the fourth proteinaceous molecule, such as a sulfhydryl group of a
cysteine.
[0376] An embodiment within the fifth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, or the fourth pharmaceutical composition for use
according to the invention, wherein the at least one saponin is a
bisdesmosidic triterpene saponin belonging to the type of a
12,13-dehydrooleanane with an aldehyde function in position C-23
and comprising a glucuronic acid function in a carbohydrate
substituent at the C-3beta-OH group of the saponin, wherein the
saponin is covalently coupled to an amino-acid residue of the
fourth proteinaceous molecule via the glucuronic acid function in
the carbohydrate substituent at the C-3beta-OH group of the
saponin, preferably via at least one linker, wherein the amino-acid
residue preferably is selected from cysteine and lysine.
[0377] An embodiment within the fifth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, or the fourth pharmaceutical composition for use
according to the invention, wherein the glucuronic acid function in
the carbohydrate substituent at the C-3beta-OH group of the at
least one saponin is covalently coupled to linker
1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid hexafluorophosphate, which linker is covalently coupled via
an amide bond to an amine group in the fourth proteinaceous
molecule, such as an amine group of a lysine or an N-terminus of
the fourth proteinaceous molecule.
[0378] An embodiment within the fifth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, or the fourth pharmaceutical composition for use
according to the invention, wherein the epitope is an epitope on a
tumor-cell receptor, preferably a tumor-cell specific epitope, and
wherein the receptor is preferably a tumor-cell specific receptor,
more preferably a receptor selected from CD71, CA125, EpCAM(17-1A),
CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular
integrin alpha-V beta-3, HER2, EGFR, CD20, CD22, Folate receptor 1,
CD146, CD56, CD19, CD138, CD27L receptor, PSMA, CanAg,
integrin-alphaV, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239,
CD70, CD123, CD352, DLL3, CD25, ephrinA4, MUC1, Trop2, CEACAM5,
CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3,
CD7, PD-L1, CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2, most preferably
selected from CD71, HER2 and EGFR.
[0379] An embodiment within the fifth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, or the fourth pharmaceutical composition for use
according to the invention, wherein the tumor-cell receptor,
preferably the tumor-cell specific receptor, is internalized by the
tumor cell after binding to the fourth proteinaceous molecule of
the invention and/or after binding to the fifth proteinaceous
molecule of the invention, and wherein preferably binding of the
fourth proteinaceous molecule and/or the fifth proteinaceous
molecule to the tumor-cell receptor, such as the tumor-cell
specific receptor, is followed by tumor-cell receptor-mediated
internalization, e.g. via endocytosis, of a complex of the fourth
proteinaceous molecule and the tumor-cell receptor and of a complex
of the fifth proteinaceous molecule and the tumor-cell
receptor.
[0380] An embodiment within the fifth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, or the fourth pharmaceutical composition for use
according to the invention, wherein the binding site of the fourth
proteinaceous molecule and the fifth proteinaceous molecule
comprises a monoclonal antibody or at least one of a cell-surface
molecule binding domain and/or -fragment thereof, and preferably
comprise or consist of any one of cetuximab, daratumumab,
gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab,
moxetumomab, polatuzumab, obinutuzumab, OKT-9 anti-CD71 monoclonal
antibody of the IgG type, pertuzumab, rituximab, ofatumumab,
Herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal
antibody, an antibody of Table A2 or Table A3 or Table A4,
preferably cetuximab or trastuzumab or OKT-9, or at least one
cell-surface molecule binding fragment and/or -domain thereof, with
the proviso that the fourth binding site is the same as the fifth
binding site.
[0381] An embodiment within the fifth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, wherein the effector moiety comprised by the
fifth proteinaceous molecule comprises or consists of any one or
more of an oligonucleotide, a nucleic acid and a xeno nucleic acid,
preferably selected from any one or more of a vector, a gene, a
cell suicide inducing transgene, deoxyribonucleic acid (DNA),
ribonucleic acid (RNA), anti-sense oligonucleotide (ASO, AON),
short interfering RNA (siRNA), microRNA (miRNA), DNA aptamer, RNA
aptamer, mRNA, mini-circle DNA, peptide nucleic acid (PNA),
phosphoramidate morpholino oligomer (PMO), locked nucleic acid
(LNA), bridged nucleic acid (BNA), 2'-deoxy-2'-fluoroarabino
nucleic acid (FANA), 2'-O-methoxyethyl-RNA (MOE),
2'-0,4'-aminoethylene bridged nucleic acid, 3'-fluoro hexitol
nucleic acid (FHNA), a plasmid, glycol nucleic acid (GNA) and
threose nucleic acid (TNA), or a derivative thereof, more
preferably a BNA, for example a BNA for silencing HSP27 protein
expression.
[0382] An embodiment within the fifth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, wherein the effector moiety comprised by the
fifth proteinaceous molecule comprises or consists of at least one
proteinaceous molecule, preferably selected from any one or more of
a peptide, a protein, an enzyme such as urease and Cre-recombinase,
a proteinaceous toxin, a ribosome-inactivating protein, a protein
toxin selected from Table A5 and/or a bacterial toxin, a plant
toxin, more preferably selected from any one or more of a viral
toxin such as apoptin; a bacterial toxin such as Shiga toxin,
Shiga-like toxin, Pseudomonas aeruginosa exotoxin (PE) or exotoxin
A of PE, full-length or truncated diphtheria toxin (DT), cholera
toxin; a fungal toxin such as alpha-sarcin; a plant toxin including
ribosome-inactivating proteins and the A chain of type 2
ribosome-inactivating proteins such as dianthin e.g. dianthin-30 or
dianthin-32, saporin e.g. saporin-S3 or saporin-S6, bouganin or
de-immunized derivative debouganin of bouganin, shiga-like toxin A,
pokeweed antiviral protein, ricin, ricin A chain, modeccin,
modeccin A chain, abrin, abrin A chain, volkensin, volkensin A
chain, viscumin, viscumin A chain; or an animal or human toxin such
as frog RNase, or granzyme B or angiogenin from humans, or any
fragment or derivative thereof; preferably the protein toxin is
dianthin and/or saporin.
[0383] An embodiment within the fifth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, wherein the effector moiety comprised by the
fifth proteinaceous molecule comprises or consists of at least one
payload, preferably selected from any one or more of a toxin
targeting ribosomes, a toxin targeting elongation factors, a toxin
targeting tubulin, a toxin targeting DNA and a toxin targeting RNA,
more preferably any one or more of emtansine, pasudotox,
maytansinoid derivative DM1, maytansinoid derivative DM4,
monomethyl auristatin E (MMAE, vedotin), monomethyl auristatin F
(MMAF, mafodotin), a Calicheamicin, N-Acetyl-y-calicheamicin, a
pyrrolobenzodiazepine (PBD) dimer, a benzodiazepine, a CC-1065
analogue, a duocarmycin, Doxorubicin, paclitaxel, docetaxel,
cisplatin, cyclophosphamide, etoposide, docetaxel, 5-fluorouracyl
(5-FU), mitoxantrone, a tubulysin, an indolinobenzodiazepine,
AZ13599185, a cryptophycin, rhizoxin, methotrexate, an
anthracycline, a camptothecin analogue, SN-38, DX-8951f, exatecan
mesylate, truncated form of Pseudomonas aeruginosa exotoxin (PE38),
a Duocarmycin derivative, an amanitin, a-amanitin, a spliceostatin,
a thailanstatin, ozogamicin, tesirine, Amberstatin269 and
soravtansine, or a derivative thereof.
[0384] An embodiment within the fifth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, wherein the fifth proteinaceous molecule
comprises or consists of any one of Gemtuzumab ozogamicin,
Brentuximab vedotin, Trastuzumab emtansine, Inotuzumab ozogamicin,
Moxetumomab pasudotox and Polatuzumab vedotin and an antibody-drug
conjugate of Table A2 and Table A3, or at least one tumor-cell
receptor binding-domain thereof and/or at least one tumor-cell
receptor binding-fragment thereof, wherein said domain(s) or
fragment(s) comprise(s) the effector moiety and are preferably (a)
tumor-cell specific receptor binding-domain(s) and/or (a)
tumor-cell specific receptor binding-fragment(s).
[0385] An embodiment within the fifth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, wherein the fourth proteinaceous molecule
comprises more than one covalently bound saponin, preferably 2, 3,
4, 5, 6, 8, 10, 16, 32, 64, 128 or 1-100 saponins, or any number of
saponins therein between, such as 7, 9, 12 saponins.
[0386] An embodiment within the fifth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, wherein the at least one saponin is covalently
bound directly to an amino-acid residue of the fourth proteinaceous
molecule, preferably to a cysteine and/or to a lysine, and/or is
covalently bound via at least one linker and/or via at least one
cleavable linker and/or via at least one oligomeric or polymeric
scaffold, preferably 1-8 of such scaffolds or 2-4 of such
scaffolds, wherein the at least one scaffold is optionally based on
a dendron, wherein 1-32 saponins, such as 2, 3, 4, 5, 6, 8, 10, 16,
32 saponins, or any number of saponins therein between, such as 7,
9, 12 saponins, are covalently bound to the at least one
scaffold.
[0387] An embodiment within the fifth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, wherein the cleavable linker is subject to
cleavage under acidic conditions, reductive conditions, enzymatic
conditions or light-induced conditions, and preferably the
cleavable linker comprises a cleavable bond selected from a
hydrazone bond and a hydrazide bond subject to cleavage under
acidic conditions, and/or a bond susceptible to proteolysis, for
example proteolysis by Cathepsin B, and/or a bond susceptible for
cleavage under reductive conditions such as a disulphide bond.
[0388] An embodiment within the fifth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, wherein the cleavable linker is subject to
cleavage in vivo under acidic conditions as present in endosomes
and/or lysosomes of mammalian cells, preferably human cells,
preferably at pH 4.0-6.5, and more preferably at pH.ltoreq.5.5.
[0389] An embodiment within the fifth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, wherein the oligomeric or polymeric scaffold
comprises a polymeric or oligomeric structure and comprises at
least one chemical group, the at least one chemical group for
covalently coupling of the scaffold to the amino-acid residue of
said fourth proteinaceous molecule.
[0390] An embodiment within the fifth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, wherein the at least one saponin is covalently
bound to the polymeric or oligomeric structure of the scaffold via
a cleavable linker according to the invention.
[0391] An embodiment within the fifth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, wherein the at least one saponin is covalently
bound to the polymeric or oligomeric structure of the scaffold via
any one or more of an imine bond, a hydrazone bond, a hydrazide
bond, an oxime bond, a 1,3-dioxolane bond, a disulphide bond, a
thio-ether bond, an amide bond, a peptide bond or an ester bond,
preferably via at least one linker.
[0392] An embodiment within the fifth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, wherein the at least one saponin comprises an
aldehyde function in position C-23 and optionally a glucuronic acid
function in the carbohydrate substituent at the C-3beta-OH group of
the at least one saponin, which aldehyde function is involved in
the covalent bonding to the polymeric or oligomeric structure of
the scaffold, and/or, if present, the glucuronic acid function is
involved in the covalent bonding to the polymeric or oligomeric
structure of the scaffold, either via direct binding or via at
least one linker.
[0393] An embodiment within the fifth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, wherein the aldehyde function in position C-23 of
the at least one saponin is covalently coupled to linker
N-.epsilon.-maleimidocaproic acid hydrazide, which linker is
covalently coupled via a thio-ether bond to a sulfhydryl group in
the polymeric or oligomeric structure of the scaffold, such as a
sulfhydryl group of a cysteine.
[0394] An embodiment within the fifth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, wherein the glucuronic acid function in the
carbohydrate substituent at the C-3beta-OH group of the at least
one saponin is covalently coupled to linker
1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid hexafluorophosphate, which linker is covalently coupled via
an amide bond to an amine group in the polymeric or oligomeric
structure of the scaffold, such as an amine group of a lysine or an
N-terminus of a proteinaceous molecule.
[0395] An embodiment within the fifth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, wherein the at least one chemical group of the
scaffold, for covalently coupling of the oligomeric or polymeric
scaffold to the amino-acid residue of said fourth proteinaceous
molecule, is a click chemistry group, preferably selected from a
tetrazine, an azide, an alkene or an alkyne, or a cyclic derivative
of these groups, more preferably the click chemistry group is an
azide.
[0396] An embodiment within the fifth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, wherein the polymeric or oligomeric structure of
the scaffold comprises a linear, branched and/or cyclic polymer,
oligomer, dendrimer, dendron, dendronized polymer, dendronized
oligomer, a DNA, a polypeptide, poly-lysine, a poly-ethylene
glycol, or an assembly of these polymeric or oligomeric structures
which assembly is preferably built up by covalent
cross-linking.
[0397] An embodiment within the fifth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, wherein the fourth pharmaceutical composition and
the fifth pharmaceutical composition are administered to the
patient in need thereof.
[0398] An aspect of the invention within the fifth series of
aspects and embodiments of the invention relates to the fourth
pharmaceutical composition of the invention, further comprising the
fifth proteinaceous molecule of the invention.
[0399] An aspect of the invention within the fifth series of
aspects and embodiments of the invention relates to the fourth
pharmaceutical composition of the invention, further comprising the
fifth proteinaceous molecule of the invention, for use as a
medicament.
[0400] An aspect of the invention within the fifth series of
aspects and embodiments of the invention relates to the fourth
pharmaceutical composition of the invention, further comprising the
fifth proteinaceous molecule of the invention, for use in the
treatment or prophylaxis of cancer in a patient in need
thereof.
[0401] An aspect of the invention within a sixth series of aspects
and embodiments of the invention relates to a therapeutic
combination for use as a medicament, wherein the therapeutic
combination comprises: (a) a sixth pharmaceutical composition
comprising a sixth proteinaceous molecule comprising a sixth
binding site for binding to a sixth cell-surface molecule and at
least one saponin covalently bound to said sixth proteinaceous
molecule preferably covalently bound to an amino-acid residue of
said sixth proteinaceous molecule, the sixth pharmaceutical
composition optionally further comprising a pharmaceutically
acceptable excipient; and (b) a seventh pharmaceutical composition
comprising a seventh proteinaceous molecule preferably different
from the sixth proteinaceous molecule, the seventh proteinaceous
molecule comprising a seventh binding site for binding to a seventh
cell-surface molecule different from the sixth cell-surface
molecule and an effector moiety, the seventh pharmaceutical
composition optionally further comprising a pharmaceutically
acceptable excipient.
[0402] An aspect of the invention within the sixth series of
aspects and embodiments of the invention relates to the sixth
pharmaceutical composition of the invention for use as a
medicament.
[0403] An aspect of the invention within the sixth series of
aspects and embodiments of the invention relates to a therapeutic
combination for use in the treatment or prevention of cancer in a
human subject, wherein the therapeutic combination comprises: (a)
the sixth pharmaceutical composition of the invention, wherein the
sixth cell-surface molecule is a sixth tumor-cell surface molecule,
preferably a sixth tumor cell-specific surface molecule; and (b)
the seventh pharmaceutical composition of the invention, wherein
the seventh cell-surface molecule is a seventh tumor-cell surface
molecule different from the sixth tumor-cell surface molecule,
preferably the seventh cell-surface molecule is a seventh tumor
cell-specific surface molecule different from the sixth tumor
cell-specific surface molecule.
[0404] An aspect of the invention within the sixth series of
aspects and embodiments of the invention relates to the sixth
pharmaceutical composition of the invention, for use in the
treatment or prophylaxis of cancer in a patient in need thereof,
wherein the sixth cell-surface molecule is a sixth tumor-cell
surface molecule, preferably a sixth tumor cell-specific surface
molecule.
[0405] An embodiment within the sixth series of aspects and
embodiments of the invention is the sixth pharmaceutical
composition for use according to the invention or the therapeutic
combination of the invention, wherein the seventh pharmaceutical
composition of the invention and the sixth pharmaceutical
composition are administered to the patient in need thereof, and
wherein the seventh tumor-cell surface molecule is different from
the sixth tumor-cell surface molecule, preferably the seventh tumor
cell-specific surface molecule is different from the sixth tumor
cell-specific surface molecule.
[0406] An embodiment within the sixth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention or the sixth pharmaceutical composition for use
according to the invention, wherein the sixth binding site of the
sixth proteinaceous molecule comprises or consists of an
immunoglobulin or at least one binding fragment or -domain of said
immunoglobulin for binding to the sixth cell-surface molecule, such
as any one or more of an antibody, an IgG, a molecule comprising or
consisting of a Vhh domain or Vh domain, a Fab, an scFv, an Fv, a
dAb, an F(ab)2, Fcab fragment, and/or comprises or consists of at
least one ligand, preferably at least one ligand for binding to the
sixth cell-surface molecule such as EGF or a cytokine.
[0407] An embodiment within the sixth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention or the sixth pharmaceutical composition for use
according to the invention, wherein the sixth binding site for
binding to the sixth tumor-cell surface molecule, preferably a
tumor cell-specific surface molecule, is a sixth binding site for a
sixth cell-surface receptor present at a tumor cell, preferably
specifically present at a tumor cell.
[0408] An embodiment within the sixth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention or the sixth pharmaceutical composition for use
according to the invention, wherein the at least one saponin is a
triterpenoid saponin or a bisdesmosidic triterpene saponin,
belonging to the type of a 12,13-dehydrooleanane with an aldehyde
function in position C-23 and optionally comprising a glucuronic
acid function in a carbohydrate substituent at the C-3beta-OH group
of the saponin, and/or a saponin isolated from a Gypsophila species
and/or a Saponaria species and/or an Agrostemma species and/or a
Quillaja species such as Quillaja saponaria.
[0409] An embodiment within the sixth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention or the sixth pharmaceutical composition for use
according to the invention, wherein the at least one saponin is a
single specific saponin or is a mixture of two or more different
saponins, such as one or more of the saponins in Table A1 or Scheme
I, SO1861, SA1657, GE1741, SA1641, QS-21, QS-21A, QS-21 A-api,
QS-21 A-xyl, QS-21B, QS-21 B-api, QS-21 B-xyl, QS-7-xyl, QS-7-api,
QS-17-api, QS-17-xyl, QS1861, QS1862, Quillajasaponin, Saponinum
album, QS-18, Quil-A, Gyp1, gypsoside A, AG1, AG2, SO1542, SO1584,
SO1658, SO1674, SO1832, or any of their stereomers and/or any
combinations thereof, preferably the saponin is SO1861 and/or
GE1741 and/or SA1641 and/or QS-21 and/or saponin with a quillaic
acid aglycon core, a Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA
carbohydrate substituent at the C-3beta-OH group and a
Glc-(1.fwdarw.3)-Xyl-(1.fwdarw.4)-Rha-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)-4-OA-
c-Qui-(1.fwdarw.4)]-Fuc carbohydrate substituent at the C-28-OH
group, and/or is
3-O-beta-D-galactopyranosyl-(1.fwdarw.2)-[beta-D-xylopyranosyl--
(1.fwdarw.3)]-beta-D-glucuronopyranosyl quillaic acid
28-O-beta-D-glucopyranosyl-(1.fwdarw.3)-beta-D-xylopyranosyl-(1.fwdarw.4)-
-alpha-L-rhamnopyranosyl-(1.fwdarw.2)-[beta-D-xylopyranosyl-(1.fwdarw.3)-4-
-OAc-beta-D-quinovopyranosyl-(1.fwdarw.4)]-beta-D-fucopyranoside,
more preferably the saponin is SO1861 and/or QS-21.
[0410] An embodiment within the sixth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention or the sixth pharmaceutical composition for use
according to the invention, wherein the at least one saponin is a
bisdesmosidic saponin having a molecular mass of at least 1.500
Dalton and comprising an oleanan-type triterpene containing an
aldehyde group at the C-23 position and optionally a hydroxyl group
at the C-16 position, with a first branched carbohydrate side chain
at the C-3 position which first branched carbohydrate side chain
optionally contains glucuronic acid, wherein the saponin contains
an ester group with a second branched carbohydrate side chain at
the C-28 position which second branched carbohydrate chain
preferably comprises at least four carbohydrate units, optionally
containing at least one acetyl residue such as two acetyl residues
and/or at least one deoxy carbohydrates and/or a quinovose and/or a
glucose and/or 4-methoxycinnamic acid and/or optionally comprising
5-O-[5-O-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl--
octanoic acid and/or optionally comprising
5-O-[5-O-Rha-(1.fwdarw.2)-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-di-
hydroxy-6-methyl-octanoic acid bound to a carbohydrate via an ester
bond, or wherein the at least one saponin is QS-21 or any one or
more of QS-21A, QS-21 A-api, QS-21 A-xyl, QS-21B, QS-21 B-api,
QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS-18,
QS1861, protonated QS1861 (QS1862), Quil-A.
[0411] An embodiment within the sixth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention or the sixth pharmaceutical composition for use
according to the invention, wherein the at least one saponin is a
bisdesmosidic triterpene saponin belonging to the type of a
12,13-dehydrooleanane with an aldehyde function in position C-23,
wherein the saponin is covalently coupled the sixth proteinaceous
molecule, preferably covalently coupled to an amino-acid residue of
the sixth proteinaceous molecule, via an aldehyde function in the
saponin, preferably said aldehyde function in position C-23,
preferably via at least one linker, and/or via at least one
cleavable linker, wherein the amino-acid residue preferably is
selected from cysteine and lysine.
[0412] An embodiment within the sixth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention or the sixth pharmaceutical composition for use
according to the invention, wherein the aldehyde function in
position C-23 of the at least one saponin is covalently coupled to
linker N-.epsilon.-maleimidocaproic acid hydrazide, which linker is
covalently coupled via a thio-ether bond to a sulfhydryl group in
the sixth proteinaceous molecule, such as a sulfhydryl group of a
cysteine.
[0413] An embodiment within the sixth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention or the sixth pharmaceutical composition for use
according to the invention, wherein the at least one saponin is a
bisdesmosidic triterpene saponin belonging to the type of a
12,13-dehydrooleanane with an aldehyde function in position C-23
and comprising a glucuronic acid function in a carbohydrate
substituent at the C-3beta-OH group of the saponin, wherein the
saponin is covalently coupled to an amino-acid residue of the sixth
proteinaceous molecule via the glucuronic acid function in the
carbohydrate substituent at the C-3beta-OH group of the saponin,
preferably via at least one linker, wherein the amino-acid residue
preferably is selected from cysteine and lysine.
[0414] An embodiment within the sixth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention or the sixth pharmaceutical composition for use
according to the invention, wherein the glucuronic acid function in
the carbohydrate substituent at the C-3beta-OH group of the at
least one saponin is covalently coupled to linker
1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid hexafluorophosphate, which linker is covalently coupled via
an amide bond to an amine group in the sixth proteinaceous
molecule, such as an amine group of a lysine or an N-terminus of
the sixth proteinaceous molecule.
[0415] An embodiment within the sixth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention or the sixth pharmaceutical composition for use
according to the invention, wherein the seventh binding site of the
seventh proteinaceous molecule comprises or consists of an
immunoglobulin, at least one binding domain of said immunoglobulin
and/or at least one binding fragment of said immunoglobulin for
binding to the seventh cell-surface molecule, such as an antibody,
an IgG, a molecule comprising or consisting of a Vhh domain or Vh
domain, a Fab, an scFv, an Fv, a dAb, an F(ab)2, Fcab fragment,
and/or comprises or consists of at least one ligand, preferably a
ligand for binding to the seventh cell-surface molecule such as EGF
or a cytokine.
[0416] An embodiment within the sixth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention or the sixth pharmaceutical composition for use
according to the invention, wherein the seventh binding site for
binding to the seventh tumor-cell surface molecule, preferably the
seventh tumor cell-specific surface molecule, is a seventh binding
site for a seventh cell-surface receptor present at a tumor cell,
preferably specifically present at a tumor cell.
[0417] An embodiment within the sixth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention or the sixth pharmaceutical composition for use
according to the invention, wherein the sixth binding site and the
seventh binding site are binding sites for binding to a sixth and
seventh tumor-cell receptor respectively, preferably for binding to
a sixth and seventh tumor-cell specific receptor respectively,
preferably present at the same tumor cell, and wherein the sixth
and seventh tumor-cell receptor are preferably tumor-cell specific
receptors, and/or are selected from CD71, CA125, EpCAM(17-1A),
CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular
integrin alpha-V beta-3, HER2, EGFR, CD20, CD22, Folate receptor 1,
CD146, CD56, CD19, CD138, CD27L receptor, PSMA, CanAg,
integrin-alphaV, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239,
CD70, CD123, CD352, DLL3, CD25, ephrinA4, MUC1, Trop2, CEACAM5,
CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3,
CD7, PD-L1, CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2, more preferably
selected from HER2, CD71 and EGFR, with the proviso that the sixth
binding site is different from the seventh binding site and with
the proviso that the sixth and seventh tumor-cell specific
receptor, preferably the sixth and seventh tumor-cell specific
receptor, are different receptors.
[0418] An embodiment within the sixth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention or the sixth pharmaceutical composition for use
according to the invention, wherein the sixth binding site and the
seventh binding site comprise or consist of cetuximab, daratumumab,
gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab,
moxetumomab, polatuzumab, obinutuzumab, OKT-9 anti-CD71 monoclonal
antibody of the IgG type, pertuzumab, rituximab, ofatumumab,
Herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal
antibody, an antibody of Table A2 or Table A3 or Table A4,
preferably cetuximab or trastuzumab or OKT-9, or at least one
tumor-cell receptor binding-domain thereof and/or at least one
tumor-cell receptor binding-fragment thereof, with the proviso that
the sixth binding site is different from the seventh binding
site.
[0419] An embodiment within the sixth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention or the sixth pharmaceutical composition for use
according to the invention, wherein the sixth tumor-cell receptor
is internalized by the tumor cell after binding to the sixth
proteinaceous molecule of the invention, and wherein preferably
binding of the sixth proteinaceous molecule to the sixth tumor-cell
receptor is followed by tumor-cell receptor-mediated
internalization, e.g. via endocytosis, of a complex of the sixth
proteinaceous molecule and the sixth tumor-cell receptor, wherein
the sixth tumor-cell receptor is preferably a sixth tumor-cell
specific receptor.
[0420] An embodiment within the sixth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, wherein the seventh tumor-cell receptor is
internalized by the tumor cell after binding to the seventh
proteinaceous molecule of the invention, and wherein preferably
binding of the seventh proteinaceous molecule to the seventh
tumor-cell receptor is followed by tumor-cell receptor-mediated
internalization, e.g. via endocytosis, of a complex of the seventh
proteinaceous molecule and the seventh tumor-cell receptor, wherein
the seventh tumor-cell receptor is preferably a seventh tumor-cell
specific receptor.
[0421] An embodiment within the sixth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention or the sixth pharmaceutical composition for use
according to the invention, wherein the effector moiety comprised
by the seventh proteinaceous molecule comprises or consists of at
least one of an oligonucleotide, a nucleic acid and a xeno nucleic
acid, preferably selected from any one or more of a vector, a gene,
a cell suicide inducing transgene, deoxyribonucleic acid (DNA),
ribonucleic acid (RNA), anti-sense oligonucleotide (ASO, AON),
short interfering RNA (siRNA), microRNA (miRNA), DNA aptamer, RNA
aptamer, mRNA, mini-circle DNA, peptide nucleic acid (PNA),
phosphoramidate morpholino oligomer (PMO), locked nucleic acid
(LNA), bridged nucleic acid (BNA), 2'-deoxy-2'-fluoroarabino
nucleic acid (FANA), 2'-O-methoxyethyl-RNA (MOE),
2'-0,4'-aminoethylene bridged nucleic acid, 3'-fluoro hexitol
nucleic acid (FHNA), a plasmid, glycol nucleic acid (GNA) and
threose nucleic acid (TNA), or a derivative thereof, more
preferably a BNA, for example a BNA for silencing HSP27 protein
expression.
[0422] An embodiment within the sixth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention or the sixth pharmaceutical composition for use
according to the invention, wherein the effector moiety comprised
by the seventh proteinaceous molecule comprises or consists of at
least one proteinaceous molecule, preferably selected from any one
or more of a peptide, a protein, an enzyme such as urease and
Cre-recombinase, a proteinaceous toxin, a ribosome-inactivating
protein, a protein toxin selected from Table A5 and/or a bacterial
toxin, a plant toxin, more preferably selected from any one or more
of a viral toxin such as apoptin; a bacterial toxin such as Shiga
toxin, Shiga-like toxin, Pseudomonas aeruginosa exotoxin (PE) or
exotoxin A of PE, full-length or truncated diphtheria toxin (DT),
cholera toxin; a fungal toxin such as alpha-sarcin; a plant toxin
including ribosome-inactivating proteins and the A chain of type 2
ribosome-inactivating proteins such as dianthin e.g. dianthin-30 or
dianthin-32, saporin e.g. saporin-S3 or saporin-S6, bouganin or
de-immunized derivative debouganin of bouganin, shiga-like toxin A,
pokeweed antiviral protein, ricin, ricin A chain, modeccin,
modeccin A chain, abrin, abrin A chain, volkensin, volkensin A
chain, viscumin, viscumin A chain; or an animal or human toxin such
as frog RNase, or granzyme B or angiogenin from humans, or any
fragment or derivative thereof; preferably the protein toxin is
dianthin and/or saporin.
[0423] An embodiment within the sixth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention or the sixth pharmaceutical composition for use
according to the invention, wherein the effector moiety comprised
by the seventh proteinaceous molecule comprises or consists of at
least one payload, preferably selected from any one or more of a
toxin targeting ribosomes, a toxin targeting elongation factors, a
toxin targeting tubulin, a toxin targeting DNA and a toxin
targeting RNA, more preferably any one or more of emtansine,
pasudotox, maytansinoid derivative DM1, maytansinoid derivative
DM4, monomethyl auristatin E (MMAE, vedotin), monomethyl auristatin
F (MMAF, mafodotin), a Calicheamicin,
N-Acetyl-.gamma.-calicheamicin, a pyrrolobenzodiazepine (PBD)
dimer, a benzodiazepine, a CC-1065 analogue, a duocarmycin,
Doxorubicin, paclitaxel, docetaxel, cisplatin, cyclophosphamide,
etoposide, docetaxel, 5-fluorouracyl (5-FU), mitoxantrone, a
tubulysin, an indolinobenzodiazepine, AZ13599185, a cryptophycin,
rhizoxin, methotrexate, an anthracycline, a camptothecin analogue,
SN-38, DX-8951f, exatecan mesylate, truncated form of Pseudomonas
aeruginosa exotoxin (PE38), a Duocarmycin derivative, an amanitin,
a-amanitin, a spliceostatin, a thailanstatin, ozogamicin, tesirine,
Amberstatin269 and soravtansine, or a derivative thereof.
[0424] An embodiment within the sixth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, wherein the seventh proteinaceous molecule
comprises or consists of any one of Gemtuzumab ozogamicin,
Brentuximab vedotin, Trastuzumab emtansine, Inotuzumab ozogamicin,
Moxetumomab pasudotox and Polatuzumab vedotin and an antibody-drug
conjugate of Table A2 and Table A3, or at least one tumor-cell
receptor binding-domain thereof and/or at least one tumor-cell
receptor binding-fragment thereof, wherein said domain(s) or
fragment(s) comprise(s) the effector moiety and are preferably (a)
tumor-cell specific receptor binding-domain(s) and/or (a)
tumor-cell specific receptor binding-fragment(s).
[0425] An embodiment within the sixth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention or the sixth proteinaceous molecule for use
according to the invention, wherein the sixth proteinaceous
molecule comprises more than one covalently bound saponin,
preferably 2, 3, 4, 5, 6, 8, 10, 16, 32, 64, 128 or 1-100 saponins,
or any number of saponins therein between, such as 7, 9, 12
saponins.
[0426] An embodiment within the sixth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention or the sixth proteinaceous molecule for use
according to the invention, wherein the more than one covalently
bound saponin are covalently bound directly to an amino-acid
residue of the sixth proteinaceous molecule, preferably to a
cysteine and/or to a lysine, and/or are covalently bound via at
least one linker and/or via at least one cleavable linker and/or
via at least one oligomeric or polymeric scaffold, preferably 1-8
of such scaffolds or 2-4 of such scaffolds, wherein the at least
one scaffold is optionally based on a dendron, wherein 1-32
saponins, preferably 2, 3, 4, 5, 6, 8, 10, 16, 32 saponins, or any
number of saponins therein between, such as 7, 9, 12 saponins, are
covalently bound to the at least one scaffold.
[0427] An embodiment within the sixth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, wherein the cleavable linker is subject to
cleavage under acidic conditions, reductive conditions, enzymatic
conditions or light-induced conditions, and preferably the
cleavable linker comprises a cleavable bond selected from a
hydrazone bond and a hydrazide bond subject to cleavage under
acidic conditions, and/or a bond susceptible to proteolysis, for
example proteolysis by Cathepsin B, and/or a bond susceptible for
cleavage under reductive conditions such as a disulphide bond.
[0428] An embodiment within the sixth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, wherein the cleavable linker is subject to
cleavage in vivo under acidic conditions as present in endosomes
and/or lysosomes of mammalian cells, preferably human cells,
preferably at pH 4.0-6.5, and more preferably at pH 5.5.
[0429] An embodiment within the sixth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, wherein the oligomeric or polymeric scaffold
comprises a polymeric or oligomeric structure and comprises at
least one chemical group, the at least one chemical group for
covalently coupling of the scaffold to the amino-acid residue of
said sixth proteinaceous molecule.
[0430] An embodiment within the sixth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, wherein the at least one saponin is covalently
bound to the polymeric or oligomeric structure of the scaffold via
a cleavable linker according to the invention.
[0431] An embodiment within the sixth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, wherein the at least one saponin is covalently
bound to the polymeric or oligomeric structure of the scaffold via
any one or more of an imine bond, a hydrazone bond, a hydrazide
bond, an oxime bond, a 1,3-dioxolane bond, a disulphide bond, a
thio-ether bond, an amide bond, a peptide bond or an ester bond,
preferably via at least one linker.
[0432] An embodiment within the sixth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, wherein the at least one saponin comprises an
aldehyde function in position C-23 and optionally a glucuronic acid
function in the carbohydrate substituent at the C-3beta-OH group of
the at least one saponin, which aldehyde function is involved in
the covalent bonding to the polymeric or oligomeric structure of
the scaffold, and/or, if present, the glucuronic acid function is
involved in the covalent bonding to the polymeric or oligomeric
structure of the scaffold, either via direct binding or via at
least one linker.
[0433] An embodiment within the sixth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, wherein the aldehyde function in position C-23 of
the at least one saponin is covalently coupled to linker
N-.epsilon.-maleimidocaproic acid hydrazide, which linker is
covalently coupled via a thio-ether bond to a sulfhydryl group in
the polymeric or oligomeric structure of the scaffold, such as a
sulfhydryl group of a cysteine.
[0434] An embodiment within the sixth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, wherein the glucuronic acid function in the
carbohydrate substituent at the C-3beta-OH group of the at least
one saponin is covalently coupled to linker
1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid hexafluorophosphate, which linker is covalently coupled via
an amide bond to an amine group in the polymeric or oligomeric
structure of the scaffold, such as an amine group of a lysine or an
N-terminus of a proteinaceous molecule.
[0435] An embodiment within the sixth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, wherein the chemical group of the polymeric or
oligomeric scaffold, for covalently coupling of the scaffold to the
amino-acid residue of the sixth proteinaceous molecule, is a click
chemistry group, preferably selected from a tetrazine, an azide, an
alkene or an alkyne, or a cyclic derivative of these groups, more
preferably the click chemistry group is an azide.
[0436] An embodiment within the sixth series of aspects and
embodiments of the invention is the therapeutic combination for use
of the invention, wherein the polymeric or oligomeric structure of
the scaffold comprises a linear, branched and/or cyclic polymer,
oligomer, dendrimer, dendron, dendronized polymer, dendronized
oligomer, a DNA, a polypeptide, poly-lysine, a poly-ethylene
glycol, or an assembly of these polymeric or oligomeric structures
which assembly is preferably built up by covalent
cross-linking.
[0437] An aspect of the invention within the sixth series of
aspects and embodiments of the invention relates to the therapeutic
combination for use of the invention, wherein the sixth
pharmaceutical composition and the seventh pharmaceutical
composition are administered to the patient in need thereof.
[0438] An aspect of the invention within the sixth series of
aspects and embodiments of the invention relates to the sixth
pharmaceutical composition of the invention, further comprising the
seventh proteinaceous molecule of the invention.
[0439] An aspect of the invention within the sixth series of
aspects and embodiments of the invention relates to the sixth
pharmaceutical composition of the invention, further comprising the
seventh proteinaceous molecule of the invention, for use as a
medicament.
[0440] An aspect of the invention within the sixth series of
aspects and embodiments of the invention relates to the sixth
pharmaceutical composition of the invention, further comprising the
seventh proteinaceous molecule of the invention, for use in the
treatment or prophylaxis of cancer in a patient in need
thereof.
[0441] Of course, any and all of a, b, c, d, e, f, g, h, I, j, k,
m, n, p, q, r, s, t, u, v, w and/or x have the value in accordance
with each individual embodiment and aspect of the invention for any
and all of the aforementioned aspects and embodiments according to
the invention. In addition, (tri-functional) linkers L1, L2, L4,
L5, L6, L8, L9 and/or L10, if present in a molecule or conjugate or
moiety of the invention, are the (tri-functional) linkers as
indicated for each and any of the aforementioned aspects and
embodiments of the invention, as is readily appreciated by the
skilled person. The oligomeric or polymeric scaffolds L3 and/or L7,
if present in a molecule or conjugate or moiety of the invention,
are the oligomeric or polymeric scaffolds as indicated for each and
any of the aforementioned aspects and embodiments of the invention,
as is also readily appreciated by the skilled person. Furthermore,
the first ligand A1 and the first effector moiety B1, if present,
and the second ligand A2 and the second effector moiety B2, if
present, and the first effector moiety A1 and the first ligand B1,
if present, and the second effector moiety A2 and the second ligand
B2, if present, are the selected and indicated ligands and effector
moieties, as disclosed for the first, second, third, fourth, fifth,
and sixth series of embodiment and aspects of the invention, and
all further embodiments and aspects of the invention, outlined here
above. Saponin C is any one or more of the saponins referred to and
listed in any of the aforementioned aspects and embodiments of the
invention, in particular one or more saponins selected from Scheme
I and/or Table A1.
[0442] An embodiment is the first, fourth or sixth proteinaceous
molecule of the invention comprising a saponin comprising one or
several or all of the indicated structural features of the saponin
of Structure A in Scheme I, the saponin of structure A referred to
as a saponin with an `ideal` structure when endosomal escape
enhancing activity towards an effector moiety present in the
endosome of a cell contacted with first proteinaceous molecule,
and/or a saponin selected from any one or more of the further
saponins in Scheme I:
##STR00001## ##STR00002## ##STR00003## ##STR00004##
##STR00005##
According to the invention, a glycoside, such as a saponin C
according to the invention, bound to the first, fourth and/or sixth
proteinaceous molecule of the invention, which has the `ideal`
structure for the purpose of enhancing endosomal escape of an
effector molecule bound to the second or third or fifth or seventh
proteinaceous molecule of the invention is a bisdesmosidic saponin
according to Structure A of Scheme I, having a molecular mass of at
least 1.500 Dalton and comprising an oleanan-type triterpene
containing an aldehyde group at the C-23 position and optionally a
hydroxyl group at the C-16 position, with a first branched
carbohydrate side chain at the C-3 position which first branched
carbohydrate side chain optionally contains glucuronic acid,
wherein the saponin contains an ester group with a second branched
carbohydrate side chain at the C-28 position which second branched
carbohydrate chain preferably comprises at least four carbohydrate
units, optionally containing at least one acetyl residue such as
two acetyl residues and/or optionally comprising deoxy
carbohydrates and/or optionally comprising quinovose and/or
optionally comprising glucose and/or optionally comprising
4-methoxycinnamic acid and/or optionally comprising
5-O-[5-O-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl--
octanoic acid and/or optionally comprising
5-O-[5-O-Rha-(1-2)-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-
-6-methyl-octanoic acid bound to a carbohydrate via an ester
bond.
[0443] SO1861 is different from the "ideal structure" displayed in
Scheme I, Structure A, only in having only one acetyl residue at
the quinovose and having an additional xylose. The "ideal
structure" of a saponin for enhancing endosomal escape of an
effector molecule or effector moiety or a payload, is a saponin
which preferably has the Structure A of Scheme I, and saponins
which display the endosomal escape enhancing activity have one or
more of the structural features displayed in Structure A of Scheme
I. Without wishing to be bound by any theory, the inventors belief
that the Structure A of Scheme I represents an "ideal saponin" (and
not a minimum requirement saponin) for endosomal escape enhancing
activity, which means that not all of the structures (chemical
groups) can or must be present in each saponin with at least
sufficient endosomal escape enhancing activity to promote
accumulation of the effector moiety in the cytosol, and which means
that some saponins might have other structure elements such as acyl
chains, and/or for yet other saponins that display endosomal escape
enhancing activity, the sugars can be different than the sugars
displayed in Scheme I. For example, the QS-21 saponin and some of
the saponins in the water soluble fraction of Quillaja saponaria
(Quillaja saponins; Quil-A) differ in the carbohydrate modification
at C-28 when the ideal structure of Structure A in Scheme I is
considered: presence of an acyl chain in QS-21 for example. In the
water soluble fraction of Quillaja saponaria, saponins such as
QS-7, QS1862, are similar to the ideal Structure A, and are similar
to SO1861.
[0444] An embodiment is the first, fourth or sixth proteinaceous
molecule of the invention, comprising the oligomeric tri-functional
linker as the scaffold core structure, according to Scheme II:
##STR00006##
wherein the saponins are covalently bound to the tri-functional
linker scaffold L9 and/or L10 via labile, cleavable hydrazone
linkers (acid sensitive) and/or via a maleimide comprising bond,
whereas the binding of the scaffold to the binding site such as an
antibody is established via labile, cleavable hydrazone linkers
(acid sensitive) and/or via a maleimide comprising bond with
cysteines in the binding site such as 1, 2, 3 or 4 cysteines,
therewith forming Structure B:
##STR00007##
such that 1-4 scaffolds are covalently bound to a single e.g.
antibody such as a monoclonal antibody. According to the invention,
one of the two displayed saponins in Structure B can also be
replaced by a covalently coupled effector moiety, effector
molecule, payload, or both saponins are absence with two covalently
coupled effector moieties, effector molecules, payloads coupled to
the tri-functional linker. The binding site is for example an
antibody or a binding fragment or binding domain thereof.
[0445] The inventors established that the therapeutic window of an
antibody drug conjugate, such as the second and third and fifth and
seventh proteinaceous molecules in the second or third or fifth or
seventh pharmaceutical composition of the invention, respectively,
increases when administered to a tumor-bearing mammal (mouse) to
whom also the first, fourth or sixth pharmaceutical composition is
administered. The first, fourth or sixth proteinaceous protein has
at least one glycoside such as a saponin C bound thereto,
preferably covalently, more preferably via a cleavable linker. The
saponin C augments the therapeutic efficacy of the effector moiety
A1, B1, A2 or B2, bound to the second and third and fifth and
seventh proteinaceous molecule, likely by enhancing the endosomal
escape of the effector moiety into the cytosol where the activity
of the effector moiety is desired. This way, already at a lower
dose than the conventional dose of the ADC, i.e. the second or
third or fifth or seventh proteinaceous molecule, therapeutic
effect is established under influence of the presence of the first,
fourth or sixth proteinaceous molecule comprising the saponin C
near, at and/or inside the targeted cell. The targeted cell is for
example a diseased cell such as a tumor cell or an auto-immune cell
or a B-cell disease related B-cell, etc. The effector moiety A1,
B1, A2 or B2 is for example a toxin as part of an ADC or an
oligonucleotide such as a BNA as part of an AOC according to the
invention.
[0446] By targeting (two) different cell-surface molecules with the
first and second, or fourth and fifth, or sixth and seventh
proteinaceous molecule, the delivery of the saponin C and the
effector molecule A1, B1, A2 or B2 at and inside the cytosol of the
very same targeted cell, exposing (both different) cell-surface
molecules on the cell surface, is improved and more specific,
compared to exposure of such cells to only the second, third, fifth
or seventh proteinaceous molecule such as an ADC or an AOC, without
the presence of the cell-targeted saponin C (first, fourth or sixth
proteinaceous molecule). An aberrant cell selected for separate
targeting by the binding site of the first, fourth or sixth
proteinaceous molecule and by the binding site of the second,
third, fifth or seventh proteinaceous molecule, wherein the binding
sites are different and wherein the epitope to which the first and
second proteinacous molecules bind are different and are located
in/on a different kind and type of cell-surface molecule such as
two different receptors, ideally bears the first epitope and the
second epitope on the first cell-surface molecule and the second
cell-surface molecule respectively, to a high extent (i.e.
relatively higher expression of the two distinct and different
cell-surface molecules on the targeted cell such as for example a
tumor cell or an auto-immune cell, than the expression on a
non-targeted cell such as for example a healthy cell) and/or expose
the first and second cell-surface molecules specifically, when
(neighboring) healthy cells in a patient are considered.
Preferably, both cell-surface molecules targeted by the first and
second binding sites are relatively highly and/or specifically
expressed on the targeted (diseased, tumor) cell compared to
healthy cells, which are not intended to be targeted with the
molecules and conjugates of the invention. An embodiment is the
pharmaceutical combination, wherein at least one of the first
(fourth, sixth) and second (fifth, seventh) binding site and thus
at least one of the first (fourth, sixth) and second (fifth,
seventh) cell-surface molecule such as a first (fourth, sixth) and
second (fifth, seventh) tumor-cell receptor, is expressed
specifically or to a relatively higher extent when compared to
expression of the first (fourth, sixth) cell-surface molecule
and/or the second (fifth, seventh) cell-surface molecule on the
surface of a healthy (neighbouring) cell. Thus, the first (fourth,
sixth) epitope or the second (fifth, seventh) epitope, preferably
the first (fourth, sixth) epitope and the second (fifth, seventh)
epitope, on the targeted cell-surface molecule is/are ideally
unique to the targeted diseased cells, and is/are at least
specifically present and exposed at the surface of the targeted
cells. Binding of the first (fourth, sixth) and second (fifth,
seventh) proteinaceous molecules to their respective first (fourth,
sixth) and second (fifth, seventh) epitope on a targeted cell is
followed by endocytosis of the complexes of the first (fourth,
sixth) proteinaceous molecule and the first (fourth, sixth) target
cell-surface molecule and the second (fifth, seventh) proteinaceous
molecule and the second (fifth, seventh) target cell-surface
molecule. Since the first and second proteinaceous molecules have
to enter the same target cell through binding interaction with two
different cell-surface molecules both expressed to a sufficient
extent or uniquely on the targeted cell when compared to healthy
cells that should not be targeted, accumulation of a
therapeutically active amount of first and second proteinaceous
molecules inside the target cells is only possible and occurring if
expression levels of the two distinct targeted cell-surface
molecules is both above a certain minimal expression threshold. At
the same time, the fact that the effector moiety bound to the
second (fifth, seventh) proteinaceous molecule is only capable of
exerting its intracellular (e.g. cytotoxic or gene silencing)
activity in the presence of the first (fourth, sixth) proteinaceous
molecule bearing the covalently bound saponin, when both the first
and second proteinaceous molecules were capable to enter the target
cell in sufficient amounts by binding to sufficiently exposed and
expressed first and second cell-surface molecules, also provides a
safeguard against negative and undesired side effects of the
effector moiety towards e.g. healthy cells and healthy tissue not
meant to be targeted and affected by the effector moiety, when
expression of at least on of the first and second cell-surface
molecules is sufficiently low at the healthy cells and preferably
when expression of both the first and second targeted cell-surface
molecules is sufficiently low at the healthy cells. That is to say,
sufficiently low expression or even absence of exposed first and
second cell-surface molecules with regard to the first and second
cell-surface molecules, and at least either the first cell-surface
molecule or the second cell-surface molecule, bound by the first
and second binding site of the first and second proteinaceous
molecules respectively, does ideally not allow entrance into
(non-targeted) healthy cells of both the first and second
proteinaceous molecules to amounts that would in concert result in
endosomal escape of the effector moiety under influence of the
saponin bound to the first proteinaceous molecule. Since the ADC or
the AOC can be used at lower dose compared to when the first
proteinaceous molecule was not added to the therapeutic regimen,
ADC or AOC entrance in healthy cells to low extent already bears a
lower risk for occurrence of unwanted side effects when for example
the targeting and killing of target diseased cells such as tumor
cells and auto-immune cells is considered.
[0447] Synchronization is the missing link between a successful
delivery strategy for mice and its application in humans. Indeed,
the inventors established in a series of in vivo mouse tumor models
that separately administering to the mice a dose of free saponin
and a dose of ADC (second or third or fifth or seventh
proteinaceous molecule according to the invention) did not result
in any desired anti-tumor activity such as delayed tumor growth,
tumor regression, diminished and slower tumor growth, compared to
control animals not treated with the ADC and free saponin. The free
saponin was administered using various routes of administration and
using various time points of administering the free saponin
compared to the moment of administering the ADC (administering free
saponin before, during and after administering the ADC). The ADC
tested in in vivo tumor models was cetuximab-dianthin (with free
SO1861), or trastuzumab-saporin (with free SO1861). Varying the
dose of free saponin did not provide for an efficacious anti-tumor
activity. The ADCs referred to were administered at a dose that in
itself did not inflict any beneficial anti-tumor effect on the
tumor-bearing animals. Surprisingly, the inventors now established
that beneficial anti-tumor activity in various in vitro mammalian
cell-based bioassays and/or in various in vivo animal tumor models
can be achieved by treating the animals with conjugates according
to the invention, optionally comprising a scaffold according to the
invention, i.e. combinations of first and second or first and third
or fourth and fifth or sixth and seventh proteinaceous molecules of
the invention. The scaffold for example being a tri-functional
linker with a covalently bound saponin (e.g. SO1861, QS-21) via a
cleavable or non-cleavable linkage, and/or with a covalently bound
effector moiety (e.g. dianthin, silencing BNA (HSP27) via a
non-cleavable bond or a cleavable bond, and/or with a covalently
bound monoclonal antibody such as cetuximab, trastuzumab, OKT-9, or
the scaffold being a dendron, such as a dendron to which for
example four moieties can bind such as four saponin molecules, or a
dendron for binding for example two saponins and two effector
molecules, the dendron comprising a chemical group for (covalent)
coupling to a ligand or an antibody or fragment or domain thereof.
Reference is made to the Examples section, exemplifying various of
these scaffolds according to the invention, showing in vivo and/or
in vitro anti-tumor cell activity when cell toxicity exerted by
e.g. a proteinaceous toxin is considered or when gene silencing in
the tumor cell is considered.
[0448] Without wishing to be bound by any theory, in view of the
failures observed when treatment of tumor-bearing animals with an
ADC together with free saponin is considered, it is preferred to
synchronize the presence of both, the at least one saponin, and the
effector moiety, preferably a toxin or an (antisense)
oligonucleotide, in compartments or vesicles of the endocytic
pathway of the target cell, e.g. a tumor cell or an auto-immune
cell. With ADC (and/or AOC) and free saponin, synchronizing the
presence of the molecules in the late endosomes, in order to obtain
the synergistic effects in vivo was not beneficially obtainable
according to attempts of the inventors. In one aspect, the
invention preferably solves at least the following problem with
respect to combining the effector moiety comprised by the second,
third, fifth or seventh proteinaceous molecule and the saponins
comprised by the first, fourth or sixth proteinaceous molecule:
without wishing to be bound by any theory the only reasonable
chemical group within, e.g., the saponins that can be used for
(covalent), in particular single and cleavable, retainable coupling
is required for the endosomal escape activity. Known restrictions
are most likely the reason why saponins have not been used in
combination with pharmaceutically active substances in clinical
investigations other than the application of saponins in
vaccination regimes wherein the use of an immune-potentiating
adjuvant substance was implied, although the striking endosomal
escape enhancer effect of, e.g., saponins listed in Table A1 and
Scheme I is known for more than 10 years. For example providing a
first, fourth or sixth proteinaceous molecule of the invention with
a covalently conjugated scaffold solves these difficulties, at
least in part. Surprisingly, the saponins previously applied for
their immune-potentiating activity in the vaccination context
involving saponins as adjuvant component, are now also suitably for
(covalent) coupling to the first, fourth or sixth proteinaceous
molecule of the invention, for anti-tumor activity in vitro and in
vivo.
[0449] An effector moiety useful in the present invention
preferably relies on late endosomal escape for exerting its effect.
Some effectors, such as, e.g., a pseudomonas exotoxin, are rerouted
to other organelles prior to the "late endosomal stage" and, thus,
would normally not benefit from coupling to the second
proteinaceous molecule according to the present invention. However,
such toxin may be adapted for use with the present invention, e.g.,
by deleting the signal peptide responsible rerouting. In particular
toxins that are highly toxic and would require only one molecule to
escape the endosomes to kill a cell maybe modified to be less
potent. It is preferred to use a toxin that kills a cell if at
least 2, more preferably at least 5, more preferably at least 10,
more preferably at least 20, more preferably at least 50, most
preferably at least 100 toxin molecules escape the endosome. It is
further preferred that a second proteinaceous molecule of the
invention comprises a covalently conjugated functionalized
scaffold, i.e. a scaffold comprising covalently bound effector
moietie(s) for targeting the scaffold comprising the bound effector
moietie(s) at a target cell such as a tumor cell or an auto-immune
cellFurther, in order to reduce off-target toxicity, cell membrane
non-permeable small molecule toxins are preferred effector
molecules over cell membrane permeable toxins.
[0450] The term "ligand" as used in this invention has its ordinary
meaning and preferably means a molecule or structure that is able
to bind another molecule or structure on the cell surface of a
target cell, wherein said molecule or structure on the cell surface
can be endocytosed and is preferably absent or less prominent on
off-target cells. Preferably, said molecule or structure on the
cell surface is constitutively endocytosed. More preferably a
ligand in this invention induces endocytosis of said molecule or
structure on the cell surface of target cells after binding to said
molecule or structure. This is for instance the case for Epidermal
Growth Factor Receptor (EGFR), present on the surface of a variety
of cancer cells. Examples of molecules or structures on the cell
surface of target cells that are constitutively endocytosed, are
for instance Claudin-1 or major histocompatibility complex class II
glycoproteins. A ligand can, e.g., be an antibody, a growth factor
or a cytokine. Combining in a carrier molecule a toxin with a
ligand is one possibility to create a targeted toxin. A toxin that
is only toxic in a target cell because it interferes with processes
that occur in target cells only can also be seen as a targeted
toxin (as in off-target cells it cannot exert its toxic action,
e.g. apoptin). Preferably, a targeted toxin is a toxin that is
combined with a ligand or e.g. a monoclonal antibody in order to be
active in target cells and not in off-target cells (as it is only
bound to and endocytosed by target cells). In a functionalized
scaffold comprising a carrier molecule comprising a ligand and an
effector moiety (i.e. a second or third proteinaceous molecule),
the ligand or the monoclonal antibody guides the effector moiety
and scaffold to the target cells. After internalization, the at
least one glycoside, preferably a saponin comprised by the
conjugate of the first proteinaceous molecule and the saponin,
mediates the endosomal escape of the effector moiety. The saponin
is typically a saponin listed in Table A1 and Scheme I, and
preferably the saponin is SO1861 and/or QS-21, and/or SA1641 and/or
GE1741.
[0451] The inventors established that immunoglobulins, domains
thereof, ligands, etc., are particularly suitable for application
as the (first) binding site of the first, fourth or sixth
proteinaceous molecule (and the same binding site of the third
proteinaceous molecule) comprising the (first) binding site.
Similarly, the inventors established that immunoglobulins, domains
thereof, ligands, etc., are particularly suitable for application
as the second, fifth or seventh binding site of the second, fifth
or seventh proteinaceous molecule comprising the second, fifth or
seventh binding site. For example, antibodies and binding domains
of antibodies are suitable for targeting an epitope on the exposed
surface of a selected cell-surface molecule, resulting in targeting
the first and third and fourth and sixth (and separately the
second, fifth and seventh) proteinaceous molecule to target cells
expressing the cell-surface molecule targeted by the first and
third and fourth and sixth proteinaceous molecule and/or target
also cells expressing the second or fifth or seventh cell-surface
molecule targeted by the second or fifth or seventh proteinaceous
molecule, these cells also expressing the first and third and
fourth and sixth cell-surface molecule (which is the same
cell-surface molecule), and having said cell-surface molecules on
their cell surface. Similarly, ligands such as EGF, targeting the
EGFR on target cells, are suitable for application as the binding
site in the first and third and fourth and sixth proteinaceous
molecules, or as the second or fifth or seventh binding site in the
second or fifth or seventh proteinaceous molecule with the proviso
that the second, fifth and seventh binding site is different from
the first and third and fourth and sixth binding site which first
and third binding site are the same. Preferred are binding sites
for the first and third, fourth, sixth epitope or for the second,
fifth, seventh epitope, which are specific for the binding of the
first and third, fourth, sixth proteinaceous molecules to the
first, fourth, sixth cell-surface molecule and/or for the binding
of the second, fifth, seventh proteinaceous molecule to the second,
fifth, seventh cell-surface molecule, the first (third, fourth,
sixth) and second (fifth, seventh) cell-surface molecules exposed
on the very same target cell. Binding sites based on antibodies or
domains or binding fragments thereof for example provide for such
desired specificity for a selected first, second, third, fourth,
fifth, sixth, seventh epitope on a selected first or second or
third (same as the first), fourth, fifth, sixth, seventh
cell-surface molecule of a selected cell for targeting such as a
diseased cell, a tumor cell, an auto-immune cell, etc. Therefore,
first, second and third and fourth, fifth, sixth and seventh
binding sites based on antibodies or binding molecules (fragments,
domains) are preferred for the first and second and third and
fourth and fifth and sixth ad seventh proteinaceous molecules.
[0452] By targeting the same cell-surface molecule with the first
and third, and fourth and fifth proteinaceous molecule, the
delivery of the saponin C and the effector moiety A1, B1, A2 or B2
at and inside the cytosol of the very same targeted cell is
improved and more specific. An aberrant cell selected for targeting
by the binding site of the first and third, or the fourth and fifth
proteinaceous molecule ideally bears the cell-surface molecule to a
high extent and/or specifically, when (neighboring) healthy cells
in a patient are considered. Thus, the epitope on the targeted
cell-surface molecule is ideally unique to the targeted diseases
cells, and is at least specifically present and exposed at the
surface of the targeted cells. Binding of the first and third or
fourth and fifth proteinaceous molecules is followed by endocytosis
of the complexes of the first proteinaceous molecule and the target
cell-surface molecule and the third proteinaceous molecule and the
target cell-surface molecule, or of the fourth proteinaceous
molecule and the target cell-surface molecule and the fifth
proteinaceous molecule and the target cell-surface molecule. Since
the first and third, or fourth and fifth, proteinaceous molecules
have to enter the same target cell through binding interaction with
the very same cell-surface molecules, accumulation of a
therapeutically active amount of first and third, or fourth and
fifth, proteinaceous molecules inside the target cells is only
possible and occurring if expression levels of the targeted
cell-surface molecule is above a certain minimal expression
threshold. At the same time, the fact that the effector moiety
bound to the third and fifth proteinaceous molecule is only capable
of exerting its intracellular (e.g. cytotoxic or gene silencing)
activity in the presence of the first or fourth proteinaceous
molecule bearing the covalently bound saponin, when both the first
and third, or both the fourth and fifth, proteinaceous molecules
were capable to enter the target cell in sufficient amounts by
binding to sufficiently exposed and expressed cell-surface
molecule, also provides a safeguard against negative and undesired
side effects of the effector moiety towards e.g. healthy cells and
healthy tissue not meant to be targeted and affected by the
effector moiety, when expression of the targeted cell-surface
molecule is sufficiently low at the healthy cells. That is to say,
low expression of the cell-surface molecule bound by the binding
site of the first and third, or fourth and fifth, proteinaceous
molecules, does not allow entrance of both the first and third, or
both the fourth and fifth, proteinaceous molecules to amounts that
would in concert result in endosomal escape of the effector moiety
under influence of the saponin bound to the first and fourth
proteinaceous molecule. Since the ADC or AOC can be used at lower
dose compared to when the first or fourth proteinaceous molecule
was not added to the therapeutic regimen, ADC or AOC entrance in
healthy cells to low extent already bears a lower risk for
occurrence of unwanted side effects when for example the targeting
and killing of target diseased cells such as tumor cells and
auto-immune cells is considered.
[0453] Throughout the description and claims (the whole
application), the terms `first` and `third` have the same meaning
when the first and third epitope, the first and third binding site,
the first and third cell-surface molecule are considered. That is
to say, for the first and third proteinaceous molecules, the
targeted epitope is the same, the binding site is the same, the
targeted cell-surface molecule such as a tumor-cell (specific)
receptor is the same. The same for the terms `fourth` and
`fifth`.
[0454] Tables A2, A3 and A4 list preferred examples of the first,
third, fourth and sixth cell-surface molecule comprising the first
(third, fourth and sixth) epitope for the first (third, fourth and
sixth) binding site of the first (third, fourth and sixth)
proteinaceous molecule. In addition, Tables A2, A3 and A4 also list
preferred examples of the second, fifth and seventh cell-surface
molecule comprising the second, fifth and seventh epitope for the
second, fifth and seventh binding site of the second, fifth,
seventh proteinaceous molecule. When the first, third, fourth,
sixth and/or second, fifth, seventh cell-surface molecule is
specifically expressed on the target cell, preferably both the
first, third, fourth and sixth, and the second, fifth, seventh
cell-surface molecules, and when the first and second epitopes on
the first and second cell-surface molecules respectively, to which
the first binding site and/or the second binding site can bind
respectively, is specifically present in the first and/or second
cell-surface molecule, specific targeting of the first, third
and/or second proteinaceous molecule to the same desired target
cell such as a tumor cell exposing the first and second tumor-cell
surface molecules, is facilitated, whereas other cells such as
healthy cells, which do not express the first and/or second
cell-surface molecule or do express the first and/or second
cell-surface molecule to a lower extent, preferably which do not
express the first and second cell-surface molecule or do express
the first and second cell-surface molecule to a lower extent
compared to expression of the cell-surface molecule(s) on the
targeted (aberrant) cell, are not targeted by the first, third and
second proteinaceous molecule or are targeted to a lower
extent.
[0455] A pharmaceutically active substance in this invention is an
effector moiety that is used to achieve a beneficial outcome in an
organism, preferably a vertebrate, more preferably a human being
such as a cancer patient or an auto-immune patient. Benefit
includes diagnosis, prognosis, treatment, cure and/or prevention of
diseases and/or symptoms. The pharmaceutically active substance may
also lead to undesired harmful side effects. In this case, pros and
cons must be weighed to decide whether the pharmaceutically active
substance is suitable in the particular case. If the effect of the
pharmaceutically active substance inside a cell is predominantly
beneficial for the whole organism, the cell is called a target
cell. If the effect inside a cell is predominantly harmful for the
whole organism, the cell is called an off-target cell. In
artificial systems such as cell cultures and bioreactors, target
cells and off-target cells depend on the purpose and are defined by
the user.
[0456] An effector moiety that is a polypeptide may be, e.g., a
polypeptide that recover a lost function, such as for instance
enzyme replacement, gene regulating functions, or a toxin.
[0457] An embodiment is the first, fourth or sixth proteinaceous
molecule of the invention, wherein the first, fourth or sixth
proteinaceous molecule comprises more than one saponin C,
preferably 2, 3, 4, 5, 6, 8, 10, 16, 32, 64 or 1-100 saponins, or
any number of saponins therein between, such as 7, 9, 12 saponins,
covalently bound directly to an amino-acid residue of the first,
fourth or sixth proteinaceous molecule, preferably to a cysteine
and/or to a lysine, and/or covalently bound via at least one linker
and/or via at least one cleavable linker and/or via at least one
polymeric or oligomeric scaffold L3, L7, preferably 1-8 of such
scaffolds or 2-4 of such scaffolds, wherein the at least one
scaffold is optionally based on a dendron, wherein 1-32 saponins
such as 2, 3, 4, 5, 6, 8, 10, 16, 32 saponins, or any number of
saponins therein between, such as 7, 9, 12 saponins, are covalently
bound to the at least one scaffold.
[0458] Table A1 and Scheme I and the above embodiments summarize a
series of saponins C that have been identified for their endosomal
escape enhancing activity when contacted to mammalian cells, in
particular human tumor cells, in free form together with a second
molecule (e.g. an effector moiety or effector molecule, payload,
such as a toxin, an oligonucleotide). Indeed, in cell-based
bioassays using human tumor cells it was established for the
saponins tabulated in Table A1 and those in Scheme I and in the
various embodiments of the invention described herein, that under
influence of these saponins, when bound to the first, fourth or
sixth proteinaceous molecule, a second molecule (effector moiety)
such as a nucleic acid and/or a toxin such as a protein toxin (e.g.
one or more of the protein toxins listed in Table A5), bound to the
second or third or fifth or seventh proteinaceous molecule, is
delivered into the cytosol with increased efficiency and/or
efficacy, presumably through intracellular release from the (late)
endosomes and lysosomes. That is to say, endosomal and/or lysosomal
escape of such second molecules (effector moieties bound to a
second or to a third or to a fifth or to a seventh proteinaceous
molecule of the invention), e.g. nucleic acids and/or toxins, is
less efficient in the absence of the saponin.
[0459] Surprisingly, the inventors now demonstrate that a
water-soluble saponin fraction from Quillaja saponaria, comprising
QS-21 and its family members QS-21A, QS-21 A-api, QS-21 A-xyl,
QS-21B, QS-21 B-api, QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api,
QS-17-xyl, QS1861, QS1862, QS-18 and Quil-A, also exhibits the
ability to potentiate a biological effect in vitro of e.g. a
nucleic acid bound to a monoclonal antibody or a protein toxin
bound to a monoclonal antibody (examples of a second and/or third
and/or fifth and/or seventh proteinaceous molecule of the invention
comprising covalently bound oligonucleotide or payload such as a
(protein) toxin), when administered to tumor cells of a mammalian
species (human) in the form of a covalent conjugate comprising a
monoclonal antibody (first, fourth, sixth proteinaceous molecule of
the invention), together with the second and/or third and/or fifth
and/or seventh proteinaceous molecule comprising the effector
moiety (the aforementioned second and/or third and/or fifth and/or
seventh proteinaceous molecule) and the at least one glycoside such
as the QS-21 and its family member saponins encompassed by such
QS-21 preparation (e.g. water soluble fraction of Quillaja
saponaria), comprised by the first, fourth or sixth proteinaceous
molecule as a covalent conjugate, wherein the effector molecule and
the glycoside, e.g. saponin fraction of Quillaja saponaria, QS-21,
SO1861, SA1641, GE1741, are covalently bound to for example the
proteinaceous molecules directly or via a linker or via a polymeric
or oligomeric scaffold, either directly or via at least one linker.
Without wishing to be bound by any theory, the observed stimulation
or potentiation of for example antisense BNA mediated reduction of
tumor-cell HSP27 expression (HSP27 gene silencing) in the presence
of saponins derived from Quillaja saponaria in vitro may (also)
relate to activation of the inflammasome in the tumor cell by the
saponins, for example resulting in tumor cell pyroptosis. The
inventors established that second and third and fifth and seventh
proteinaceous molecules conjugated to for example antisense BNA or
dianthin or saporin, exerted any anti-tumor cell activity in vitro
at all or improved anti-tumor cell activity when contacted with
cells in bio-based cell assays, when in the presence of the first
or fourth or sixth proteinaceous molecule of the invention,
comprising the saponin, and targeted to the same (tumor) cells as
the cell surface molecule targeted by the second and/or third
and/or fifth and/or seventh proteinaceous molecule, whereas in the
absence of the first, fourth, sixth proteinaceous molecule and thus
in the absence of saponin, no such activity towards the tumor cell
was observed.
[0460] QS-21, and also the water-soluble saponins fraction
comprising QS-21 from Quillaja saponaria is already for a long time
known and previously intensively applied for its
immune-potentiating abilities, e.g. as an adjuvant in e.g. sub-unit
vaccines. For example, QS-21 is applied in two phase III clinical
trials with human patients, who were vaccinated with a sub-unit
vaccine mixed with an adjuvant comprising QS-21 (Glaxo-Smith-Kline,
MAGRIT trial, DERMA study), wherein the sub-unit was MAGE-A3
protein, which is specifically expressed and presented by tumor
cells. The anti-tumor vaccinations, potentiated with QS-21, aimed
for extension of disease-free survival of the cancer patients
(melanoma; non-small cell lung cancer). In addition, QS-21 has been
tested as an adjuvant in clinical trials for developing anti-cancer
vaccine treatment, for vaccines for HIV-1 infection, in development
of a vaccine against hepatitis B, and for anti-malaria vaccine
development using QS-21 comprising adjuvants AS01 and AS02 of
Glaxo-Smith-Kline. Previous studies revealed an immune response
elicited against MAGE-A3 peptides presented at the cancer cell
surface, under influence of the QS-21 saponin comprising adjuvant
(AS15; GSK). To the surprise of the inventors, the saponin fraction
of Quillaja saponaria, and thus likely QS-21 (as part of the water
soluble saponin fraction of Quillaja saponaria) potentiates the
anti-tumor cell activity of e.g. a payload such as a protein toxin
(dianthin), bound to the second, fifth, seventh proteinaceous
molecule (e.g. the ligand EGF).
[0461] The inventors show that a tumor-cell targeting monoclonal
antibody provided with covalently coupled antisense BNA such as
BNA(HSP27), and contacted with the tumor cells together with a
first or fourth proteinaceous molecule of the invention with
covalently coupled saponin (e.g. SO1861, QS-21), both the BNA and
the saponin coupled to the respective antibody (e.g. cetuximab) of
the first and third or fourth and fifth proteinaceous molecule via
a cleavable bond is capable of silencing HSP27 in vivo in tumors,
compared to control and compared to AOC (third and fifth
proteinaceous molecule) only, without presence of first or fourth
proteinaceous molecule with coupled saponin. Co-administering an
ADC or an antibody-oligonucleotide conjugate (AOC), such as an
antibody-BNA conjugate, with a first or fourth proteinaceous
molecule with a saponin thus endows the ADC or AOC with anti-tumor
cell activity not seen with only the ADC or only the AOC at the
same dose. Noteworthy, the AOC (the second or third or fifth
proteinaceous molecule) and the monoclonal antibody with covalently
coupled saponin (first or fourth proteinaceous molecule) increase
HSP27 expression in tumor cells, when administered to tumor-bearing
mice separately in separate groups of mice, compared to a control
group (vehicle administered, only). Only co-administration of the
AOC comprising the effector moiety of the invention (second or
third or fifth proteinaceous molecule) and the first or fourth
proteinaceous molecule with covalently coupled saponin, displays
reduced HSP27 expression when compared to controls. The antisense
BNA (HSP27) was BNA with oligo nucleic acid sequence
5'-GGCacagccagtgGCG-3' according to Zhang et al. (2011) [Y Zhang, Z
Qu, S Kim, V Shi, B Liao1, P Kraft, R Bandaru, Y Wu, LM Greenberger
and ID Horak, Down-modulation of cancer targets using locked
nucleic acid (LNA)-based antisense oligonucleotides without
transfection, Gene Therapy (2011) 18, 326-333]. Noteworthy, to the
best of the knowledge of the inventors, BNA is designed for
application as a free nucleic acid. The inventors are now the first
to demonstrate that the antisense BNA can be covalently coupled
through a (non-)cleavable linker with a ligand or an antibody, in a
way that gene-silencing activity is retained in vitro and more
importantly in vivo in the tumor cells of a tumor-bearing animal.
This approach of providing BNA-based AOCs opens new ways to
administer targeted BNA to human (cancer) patients in need
thereof.
[0462] The inventors disclose here that covalently coupling
saponins such as saponins in the water-soluble fraction of Quillaja
saponaria, QS-21, SA1641, SO1861, Table A1, Scheme I, to a first or
fourth or sixth proteinaceous molecule, such as via a
tri-functional linker, e.g. the tri-functional linker of Scheme II
and Structure B, or via an oligomeric or polymeric structure of a
scaffold comprising covalently bound saponins, results in improved
cell toxicity exerted by the effector moiety such as a toxin,
comprised by the second and/or third and/or fifth and/or seventh
proteinaceous molecule, under influence of the covalently coupled
saponin in the first, fourth or sixth proteinaceous molecule.
[0463] According to the invention, typically the saponin is a
saponin listed in Table A1, Scheme I. It has been proven beneficial
for the activity of the saponin, e.g. the endosomal escape
enhancing activity inside cells when the entry into the cell and
the accumulation inside the cytosol of an effector moiety
covalently coupled to the second or third or fifth or seventh
proteinaceous molecule, is considered, when the saponin is
covalently coupled to the first or fourth or sixth proteinaceous
molecule involving a hydrazone bond, and/or a hydrazide bond,
and/or a disulphide bond. Such bond types readily cleave under the
acidic conditions inside (late) endosomes and lysosomes of
mammalian cells, e.g. human cells, and/or under the reductive
conditions. Alternatively, the inventors also demonstrate that
covalent coupling of saponin to the first, fourth or sixth
proteinaceous molecule via a bond that is not readily cleavable
under the physiological conditions inside cells, e.g. (late)
endosomes, lysosomes, cytosol, is also beneficial to the
potentiating activity of the saponin on the biological effect of
e.g. an effector moiety such as a nucleic acid (e.g. BNA silencing
HSP27) and a proteinaceous toxin such as saporin. Throughout the
application, including the claims, the term `cleavable linker`,
`cleavable bond`, etc., is also referred to as `labile linker`
(`L`) and `labile bond`, for example in the context of cleavage of
such a bond or linker in the (late) endosome and/or lysosome when a
conjugate of the invention, e.g. a first, fourth or sixth
proteinaceous molecule optionally comprising a scaffold with
saponins coupled to the first, fourth or sixth proteinaceous
molecule through a linker and/or via the scaffold via hydrazone
bonds or disulphide bonds, is referred to. For example, FIG. 1-1,
FIG. 6-2, FIG. 2-4 and FIG. 1-5 show the in vivo HSP27 gene
silencing in human tumors in mice. The tumor-bearing mice were for
example treated with a first proteinaceous molecule consisting of
monoclonal antibody with saponin bound thereto via a labile linker
(hydrazone bond) according to the invention, whereas the third
proteinaceous molecule comprised bound antisense BNA for silencing
the HSP27 gene in the tumor cells, covalently coupled to the
monoclonal antibody (same type as the first monoclonal antibody)
via a disulphide bond. That is to say, without wishing to be bound
by any theory, the hydrazone bond and the disulphide bond are
cleaved in the (late) endosomes and/or lysosomes of the targeted
tumor cells that express the epitope on the targeted cell-surface
molecule, here the EGFR, at the cell surface, once the therapeutic
combination of the invention is internalized by e.g. endocytosis.
Cleavage of the bonds likely contributes to the endosomal escape
enhancing activity of the saponin when the entry of the BNA from
the endosome and/or lysosome into the cytosol is considered,
although such cleavage is not a necessity for observing the gene
silencing effect of the combination of the cetuximab-SO1861
conjugate and the cetuximab-BNA conjugate of the invention.
[0464] The skilled person will appreciate that a tri-functional
linker is a scaffold of the invention suitable for covalently
coupling one, two or three saponin moieties. For the tri-functional
linker covalent coupling of one or two saponin moieties is
preferred. The second and/or third binding site is for example
suitable for covalent coupling a proteinaceous ligand such as the
first, fourth or sixth proteinaceous molecule. Typical
proteinaceous ligands are EGF for targeting (tumor) cells
expressing EGFR at the cell surface, and cytokines for targeting
tumor cells or autoimmune cells. Moreover, the second or third
binding site of the tri-functional linker is suitable for covalent
coupling of an immunoglobulin such as a monoclonal antibody, i.e.
the first, fourth or sixth proteinaceous molecule for binding to a
cell surface molecule such as a tumor cell surface molecule,
preferably a tumor-cell specific molecule, more preferably a tumor
cell receptor that is specifically (over-)expressed at the surface
of the tumor cell. Similarly, the immunoglobulin, or any
fragment(s) and/or domain(s) thereof which encompass the binding
specificity of the immunoglobulin, is suitable for binding to a
cell surface molecule such as a receptor, expressed at the surface
of an autoimmune cell. Thus, in an embodiment, the first, fourth or
sixth proteinaceous molecule comprises the tri-functional linker,
said linker comprises or consists of a covalently bound saponin,
e.g. QS-21, SO1861, and the covalently bound binding site such as a
cell targeting moiety such as a ligand or an antibody for
(specific) binding to a tumor cell, an auto-immune cell, a diseased
cell, an aberrant cell, a non-healthy cell, a B-cell disease.
[0465] A first, fourth or sixth proteinaceous molecule according to
the invention thus comprises at least one saponin. With "at least
one" in this context is meant that the first, fourth or sixth
proteinaceous molecule comprises one saponin molecule but may also
comprise a couple (e.g. two, three or four) of saponins or a
multitude (e.g. 10, 20 or 100) of saponins. Depending on the
application, the first, fourth or sixth proteinaceous molecule may
comprise a covalently bound scaffold with covalently bound
saponins, wherein the scaffold may be designed such that it
comprises a defined number of saponins. Preferably, a first, fourth
or sixth proteinaceous molecule according to the invention
comprises a defined number or range of saponins, rather than a
random number. This is especially advantageous for drug development
in relation to marketing authorization. A defined number in this
respect means that a first, fourth or sixth proteinaceous molecule
preferably comprises a previously defined number of saponins. This
is, e.g., achieved by designing a scaffold comprising a polymeric
structure with a certain number of possible moieties for the
saponin(s) to attach. Under ideal circumstances, all of these
moieties are coupled to a saponin and the scaffold than comprises
the prior defined number of saponins. It is envisaged to offer a
standard set of scaffolds, comprising, e.g., two, four, eight,
sixteen, thirty-two, sixty-four, etc., saponins so that the optimal
number can be easily tested by the user according to his needs. An
embodiment is the first, fourth or sixth proteinaceous molecule of
the invention comprising the scaffold of the invention, wherein the
saponin is present in a defined range as, e.g., under non-ideal
circumstances, not all moieties present in a polymeric structure
bind a saponin. Such ranges may for instance be 2-4 saponin
molecules per scaffold, 3-6 saponin molecules per scaffold, 4-8
saponin molecules per scaffold, 6-8 saponin molecules per scaffold,
6-12 saponin molecules per scaffold and so on. In such case, a
first proteinaceous molecule comprising a scaffold according to the
invention thus comprises 2, 3 or 4 saponins if the range is defined
as 2-4.
[0466] The scaffold is fundamentally independent of the type of
saponin covalently bound to the scaffold, the scaffold subsequently
(in sequential order) covalently coupled to the first, fourth or
sixth proteinaceous molecule. Thus, first, fourth or sixth
proteinaceous molecule comprising the scaffold is the basis product
for a new platform technology. Since the at least one covalently
bound saponin mediates intracellular delivery of the effector
moiety bound to the second, third, fifth or seventh proteinaceous
molecule, the scaffold technology according to the invention is the
first system known that mediates controlled intracellular effector
moiety delivery by saponins. The scaffold provides an optimized and
functionally active unit that can be linked to the saponin(s) and
to the binding site comprised by the first, fourth or sixth
proteinaceous molecule, e.g. a ligand, an antibody, etc., at a
single and defined position.
[0467] An embodiment is the first, fourth or sixth proteinaceous
molecule comprising a scaffold according to the invention, wherein
the number of monomers of the polymeric or oligomeric structure is
an exactly defined number or range. Preferably, the polymeric or
oligomeric structure comprises structures such as poly(amines),
e.g., polyethylenimine and poly(amidoamine), or structures such as
polyethylene glycol, poly(esters), such as poly(lactides),
poly(lactams), polylactide-co-glycolide copolymers, poly(dextrin),
or a peptide or a protein, or structures such as natural and/or
artificial polyamino acids, e.g. poly-lysine, DNA polymers,
stabilized RNA polymers or PNA (peptide nucleic acid) polymers,
either appearing as linear, branched or cyclic polymer, oligomer,
dendrimer, dendron, dendronized polymer, dendronized oligomer or
assemblies of these structures, either sheer or mixed. Preferably,
the polymeric or oligomeric structures are biocompatible, wherein
biocompatible means that the polymeric or oligomeric structure does
not show substantial acute or chronic toxicity in organisms and can
be either excreted as it is or fully degraded to excretable and/or
physiological compounds by the body's metabolism. Assemblies can be
built up by covalent cross-linking or non-covalent bonds and/or
attraction. They can therefore also form nanogels, microgels, or
hydrogels, or they can be attached to carriers such as inorganic
nanoparticles, colloids, liposomes, micelles or particle-like
structures comprising cholesterol and/or phospholipids. Said
polymeric or oligomeric structures preferably bear an exactly
defined number or range of coupling moieties for the coupling of
glycoside molecules (and/or effector molecules and/or carrier
molecules such as a ligand, monoclonal antibody or a fragment
thereof). Preferably at least 50%, more preferably at least 75%,
more preferably at least 85%, more preferably at least 90%, more
preferably at least 95%, more preferably at least 98%, more
preferably at least 99%, most preferably 100% of the exactly
defined number or range of coupling moieties in the polymeric or
oligomeric structure is occupied by a glycoside molecule in a
scaffold according to the invention.
[0468] Preferably, a dendron is a branched, clearly defined
tree-like polymer with a single chemically addressable group at the
origin of the tree, called the focal point. A dendrimer is a
connection of two or more dendrons at their focal point. A
dendronized polymer is a connection of the focal point of one or
more dendrons to a polymer. In a preferred embodiment, a scaffold
according to the invention is provided, wherein the polymeric or
oligomeric structure comprises a linear, branched or cyclic
polymer, oligomer, dendrimer, dendron, dendronized polymer,
dendronized oligomer or assemblies of these structures, either
sheer or mixed, wherein assemblies can be built up by covalent
cross-linking or non-covalent attraction and can form nanogels,
microgels, or hydrogels, and wherein, preferably, the polymer is a
derivative of a poly(amine), e.g., polyethylenimine and
poly(amidoamine), and structures such as polyethylene glycol,
poly(esters), such as poly(lactids), poly(lactams),
polylactide-co-glycolide copolymers, and poly(dextrin), and
structures such as natural and/or artificial polyamino acids such
as poly-lysine, or a peptide or a protein or DNA polymers,
stabilized RNA polymers or PNA (peptide nucleic acid) polymers.
Preferably, the polymeric or oligomeric structures are
biocompatible.
[0469] An embodiment is the therapeutic combination of the
invention or the therapeutic combination for use according to the
invention, wherein the first, fourth or sixth proteinaceous
molecule comprises more than one covalently bound saponin,
preferably 2, 3, 4, 5, 6, 8, 10, 16, 32, 64, 128 or 1-100 saponins,
or any number of saponins therein between, such as 7, 9, 12
saponins.
[0470] An embodiment is the first, fourth or sixth proteinaceous
molecule of the invention, wherein the at least one saponin is
covalently bound to the polymeric or oligomeric structure of the
oligomeric or polymeric scaffold via at least one cleavable linker
according to the invention.
[0471] An embodiment is the first, fourth or sixth proteinaceous
molecule of the invention, wherein the chemical group of the
oligomeric or polymeric scaffold, for covalently coupling of the
oligomeric or polymeric scaffold to the amino-acid residue of said
first, fourth or sixth proteinaceous molecule, is a click chemistry
group, preferably selected from a tetrazine, an azide, an alkene or
an alkyne, or a cyclic derivative of these groups, more preferably
said chemical group is an azide.
[0472] An embodiment is the first, fourth or sixth proteinaceous
molecule of the invention, wherein the polymeric or oligomeric
structure of the oligomeric or polymeric scaffold comprises a
linear, branched and/or cyclic polymer, oligomer, dendrimer,
dendron, dendronized polymer, dendronized oligomer, a DNA, a
polypeptide, poly-lysine, a poly-ethylene glycol, or an assembly of
these polymeric or oligomeric structures which assembly is
preferably built up by covalent cross-linking.
[0473] The inventors established that covalent coupling, preferably
via cleavable bonds or linkers, of the saponin to the first, fourth
or sixth proteinaceous molecule, according to any of the
embodiments here above, provides efficient and cell-targeted
potentiation of the activity of an effector moiety bound to the
second and to the third and to the fifth and to the seventh
proteinaceous molecule, wherein the first and third and fourth and
sixth proteinaceous molecules comprise the same first, fourth or
sixth binding site and wherein the first (fourth, sixth) and second
(fifth, seventh) proteinaceous molecules comprise a first (third,
fourth, sixth) and second (fifth, seventh) binding site which are
different. Coupling saponin to a cysteine side chain or a lysine
side chain of the first, fourth or sixth proteinaceous molecule
such as a monoclonal antibody, directly or via a linker, proved to
be a beneficial way of specific and efficient delivery of
effector-moiety potentiating activity inside the target cell, when
also the effector moiety is delivered in the same target cell by
using the second and/or third and/or fifth and/or seventh
proteinaceous molecule comprising the same first binding site as
the first proteinaceous molecule when the third or fifth
proteinaceous molecule is considered and comprising different first
and second, seventh binding sites respectively when the first and
second, seventh proteinaceous molecules are considered.
[0474] To explain the invention in more detail, the process of
cellular uptake of substances (although the inventors do not wish
to be bound by any theory) and the used terminology in this
invention is described. The uptake of extracellular substances into
a cell by vesicle budding is called endocytosis. Said vesicle
budding can be characterized by (1) receptor-dependent ligand
uptake mediated by the cytosolic protein clathrin, (2) lipid-raft
uptake mediated by the cholesterol-binding protein caveolin, (3)
unspecific fluid uptake (pinocytosis), or (4) unspecific particle
uptake (phagocytosis). All types of endocytosis run into the
following cellular processes of vesicle transport and substance
sorting called the endocytic pathways. The endocytic pathways are
complex and not fully understood. Without wishing to be bound by
any theory, organelles may be formed de novo and mature into the
next organelle along the endocytic pathway. It is however, now
hypothesized that the endocytic pathways involve stable
compartments that are connected by vesicular traffic. A compartment
is a complex, multifunctional membrane organelle that is
specialized for a particular set of essential functions for the
cell. Vesicles are considered to be transient organelles, simpler
in composition, and are defined as membrane-enclosed containers
that form de novo by budding from a preexisting compartment. In
contrast to compartments, vesicles can undergo maturation, which is
a physiologically irreversible series of biochemical changes. Early
endosomes and late endosomes represent stable compartments in the
endocytic pathway while primary endocytic vesicles, phagosomes,
multivesicular bodies (also called endosome carrier vesicles),
secretory granules, and even lysosomes represent vesicles. The
endocytic vesicle, which arises at the plasma membrane, most
prominently from clathrin-coated pits, first fuses with the early
endosome, which is a major sorting compartment of approximately pH
6.5. A large part of the cargo and membranes internalized are
recycled back to the plasma membrane through recycling vesicles
(recycling pathway). Components that should be degraded are
transported to the acidic late endosome (pH lower than 6) via
multivesicular bodies. Lysosomes are vesicles that can store mature
lysosomal enzymes and deliver them to a late endosomal compartment
when needed. The resulting organelle is called the hybrid organelle
or endolysosome. Lysosomes bud off the hybrid organelle in a
process referred to as lysosome reformation. Late endosomes,
lysosomes, and hybrid organelles are extremely dynamic organelles,
and distinction between them is often difficult. Degradation of an
endocytosed molecule occurs inside an endolysosome or lysosome.
Endosomal escape is the active or passive release of a substance
from the inner lumen of any kind of compartment or vesicle from the
endocytic pathway, preferably from clathrin-mediated endocytosis,
or recycling pathway into the cytosol. Endosomal escape thus
includes but is not limited to release from endosomes,
endolysosomes or lysosomes, including their intermediate and hybrid
organelles.
[0475] Unless specifically indicated otherwise and in particular
when relating to the endosomal escape mechanism of the glycoside
molecule such as the saponin of the invention, whenever the word
"endosome" or "endosomal escape" is used herein, it also includes
the endolysosome and lysosome, and escape from the endolysosome and
lysosome, respectively. After entering the cytosol, said substance
might move to other cell units such as the nucleus.
[0476] In formal terms, a glycoside is any molecule in which a
sugar group is bound through its anomeric carbon to another group
via a glycosidic bond. Glycoside molecules, such as saponins, in
the context of the invention are such molecules that are further
able to enhance the effect of an effector moiety, without wishing
to be bound by any theory, in particular by facilitating the
endosomal escape of the effector moiety. Without wishing to be
bound by any theory, the glycoside molecules (saponins, such as
those listed in Table A1) interact with the membranes of
compartments and vesicles of the endocytic and recycling pathway
and make them leaky for said effector moieties resulting in
augmented endosomal escape. With the term "the scaffold is able to
augment endosomal escape of the effector moiety" is meant that the
at least one saponin (glycoside molecule), which is coupled to the
polymeric or oligomeric structure of the scaffold, is able to
enhance endosomal escape of an effector moiety when both molecules
are within an endosome, e.g. a late endosome, optionally and
preferably after the at least one glycoside such as a saponin is
released from the first, fourth, sixth proteinaceous molecule such
as from a linker or polymeric or oligomeric structure comprised by
said first, fourth, sixth proteinaceous molecule, e.g., by cleavage
of a cleavable bond between the at least one glycoside (saponin)
and the first, fourth, sixth proteinaceous molecule (for example
via a polymeric or oligomeric structure of a scaffold and/or via a
linker). Even though a bond between the at least one glycoside such
as a saponin according to the invention and the first, fourth,
sixth proteinaceous molecule, optionally via a linker or a
scaffold, may be a "stable bond", that does not mean that such bond
cannot be cleaved in the endosomes by, e.g., enzymes. For instance,
the glycoside or saponin, optionally together with a linker or a
part of the oligomeric or polymeric structure of a scaffold, may be
cleaved off from the remaining linker fragment or oligomeric or
polymeric structure. It could, for instance be that a protease cuts
a (proteinaceous) linker or proteinaceous polymeric structure,
e.g., albumin, thereby releasing the at least one glycoside,
saponin. It is, however, preferred that the glycoside molecule
(preferably saponin) is released in an active form, preferably in
the original form that it had before it was (prepared to be)
coupled to the first, fourth, sixth proteinaceous molecule
optionally via a linker and/or an oligomeric or polymeric scaffold;
thus the glycoside (saponin) has its natural structure after such
cleavage or the glycoside (saponin) has (part of) a chemical group
or linker bound thereto, after such cleavage, while glycoside
biological activity (saponin biological activity), e.g.
endosomal/lysosomal escape enhancing activity towards an effector
moiety present in the same endosome or lysosome, is maintained or
restored upon said cleavage of the bond between the glycoside
(saponin) and the carrier molecule, i.e. the first, fourth, sixth
proteinaceous molecule optionally comprising a linker and/or a
scaffold of the invention. With regard to the present invention the
term "stable" with respect to bonds between e.g. saponins and
amino-acid residues of the first, fourth, sixth proteinaceous
molecule, a linker, a polymeric or oligomeric structures (of the
scaffold), ligands, (monoclonal) immunoglobulins or binding domains
or -fragments thereof, and/or effectors (effector moieties,
effector molecules), is meant that the bond is not readily broken
or at least not designed to be readily broken by, e.g., pH
differences, salt concentrations, or UV-light, reductive
conditions. With regard to the present invention the term
"cleavable" with respect to bonds between e.g. saponins and the
first, fourth, sixth proteinaceous molecule, linkers, amino-acid
residues, polymeric or oligomeric structures of the scaffold,
ligands, antibodies and/or effectors, is meant that the bond is
designed to be readily broken by, e.g., pH differences, salt
concentrations, under reductive conditions, and the like. The
skilled person is well aware of such cleavable bonds and how to
prepare them.
[0477] Before the present invention one of the major hurdles of
introducing ADCs and AOCs on the market was the small therapeutic
window: a therapeutically effective dose of an ADC or an AOC is
accompanied with (unacceptable) side effects, hampering development
and implication in treatment of patients with the ADCs. By the
application of the first, fourth or sixth proteinaceous molecule of
the invention it has now become possible to guide one or multiple
glycoside molecules (saponin) to a (target) cell, together with the
ADC carrying a payload or together with a (monoclonal) antibody
conjugated with an oligonucleotide such as a BNA according to the
invention (i.e. a particular second or third or fifth or seventh
proteinaceous molecule of the invention). In particular, it was
previously not possible to specifically guide an effector moiety of
a second or third or fifth or seventh proteinaceous molecule and a
(predefined, controllable) particular number or range of glycoside
molecules (saponins) per effector moiety at the same time to the
cytosol of cells, such as via the endocytic pathway of a cell.
[0478] A solution provided for by the invention comprises the
covalent binding of at least one saponin to the first or fourth or
sixth proteinaceous molecule. A further solution provided for by
the invention comprises (first) polymerizing the glycoside
molecules (saponins) using an oligomeric or polymeric scaffold, and
providing the first or fourth or sixth proteinaceous molecule with
a cluster of covalently bound saponins, enabling re-monomerization
of the one or more saponins at the intracellular site where the
mode of action of the saponin is desired, e.g. after endocytosis.
"Polymerizes" in this context means the reversible and/or
irreversible multiple conjugation of saponin molecules to the
first, fourth, sixth proteinaceous molecule, either via linker, or
directly or via a polymeric or oligomeric structure to form a
scaffold or the reversible and/or irreversible multiple conjugation
of (modified) saponins thereby forming a polymeric or oligomeric
structure to form a scaffold. "Re-monomerization" in this context
means the cleavage of the saponins from the first, fourth, sixth
proteinaceous molecule, from the linker linking the saponin(s) to
the first, fourth, sixth proteinaceous molecule or from the
scaffold, for example after endocytosis, and regaining the (native)
chemical state of the unbound saponins, which unbound saponins may
or may not comprise additional chemical groups such as a chemical
group for linking the saponin to a linker, an amino-acid residue of
the first proteinaceous molecule or to the scaffold, and/or a
(chemical) linker bound to a chemical group of the saponin such as
an aldehyde group or carboxylic acid group. Due to the complex
chemistry of the saponins for example the `polymerization` of
saponins at a scaffold or other linking linker and their
"re-monomerization" at a desired location such as intracellularly
e.g. after endocytosis, was a challenging task. In particular, the
chemical reactions used for providing the linkers and the scaffold
comprising covalently linked glycosides for covalent binding to the
first, fourth, sixth proteinaceous molecule, e.g. triterpenoid
saponins (polymerization of the glycosides), normally occur in
water-free organic solvents, but saponins and for example
biocompatible polymers applied as a scaffold for bearing bound
saponins, are water-soluble molecules. The chemical properties of
the unmodified saponin further prohibited polymerization by itself
and, one other possible solution, to bind multiple saponins
(directly) to the effector molecule was estimated not to be very
promising, as an effector molecule (drug, toxin, polypeptide or
polynucleotide) does typically not provide sufficient binding sites
and because the coupling product would become quite heterogeneous
and/or coupling biologically active molecules such as a saponin and
e.g. a peptide, a toxin, a nucleic acid together bears the risk for
influencing and hampering the activity of one or even both
molecules bound together in such saponin-comprising conjugate.
Further, there was a considerable risk that the effector moiety
comprised by the second or third or fifth or seventh proteinaceous
molecule loses its function after coupling of a saponin to the e.g.
ADC or antibody-oligonucleotide conjugate (AOC). Embodiments of the
present invention solves at least one of these drawbacks.
[0479] An aspect of the invention relates to a composition
comprising the first or fourth or sixth proteinaceous molecule of
the invention and the second, third, fifth or seventh proteinaceous
molecule of the invention.
[0480] An aspect of the invention relates to a composition
comprising the first, fourth or sixth proteinaceous molecule of the
invention and the third or fifth proteinaceous molecule of the
invention.
[0481] An embodiment is the composition comprising the first,
fourth or sixth proteinaceous molecule of the invention and the
second, or seventh proteinaceous molecule of the invention, or is
the composition comprising the first, fourth or sixth proteinaceous
molecule of the invention and the third or fifth proteinaceous
molecule of the invention, wherein the effector moiety that is
comprised by the second, fifth or seventh proteinaceous molecule or
by the third proteinaceous molecule is any one of the effector
moieties according to the invention, preferably a BNA.
[0482] An aspect of the invention relates to a composition
comprising the first, fourth or sixth proteinaceous molecule of the
invention and any one or more of an oligonucleotide, a nucleic acid
and a xeno nucleic acid, preferably selected from at least one of a
vector, a gene, a cell suicide inducing transgene, deoxyribonucleic
acid (DNA), ribonucleic acid (RNA), anti-sense oligonucleotide
(ASO, AON), short interfering RNA (siRNA), microRNA (miRNA), DNA
aptamer, RNA aptamer, mRNA, mini-circle DNA, peptide nucleic acid
(PNA), phosphoramidate morpholino oligomer (PMO), locked nucleic
acid (LNA), bridged nucleic acid (BNA), 2'-deoxy-2'-fluoroarabino
nucleic acid (FANA), 2'-O-methoxyethyl-RNA (MOE),
2'-O,4'-aminoethylene bridged nucleic acid, 3'-fluoro hexitol
nucleic acid (FHNA), a plasmid, glycol nucleic acid (GNA) and
threose nucleic acid (TNA), or a derivative thereof, more
preferably a BNA, for example a BNA for silencing HSP27 protein
expression (antisense BNA(HSP27)).
[0483] An effector molecule, or effector moiety, in the context of
this invention is any substance that affects the metabolism of a
cell by interaction with an intracellular effector molecule target,
wherein this effector molecule target is any molecule or structure
inside cells excluding the lumen of compartments and vesicles of
the endocytic and recycling pathway but including the membranes of
these compartments and vesicles. Said structures inside cells thus
include the nucleus, mitochondria, chloroplasts, endoplasmic
reticulum, Golgi apparatus, other transport vesicles, the inner
part of the plasma membrane and the cytosol. Cytosolic delivery of
an effector moiety in the context of the invention preferably means
that the effector moiety is able to escape the endosome (and/or
lysosome), which, as defined previously, also includes escaping the
endolysosome and the lysosome, and is preferably able to reach the
effector moiety target as described herein. The invention also
encompasses a new type of molecule, referred to as scaffold that
serves to bring both an effector moiety and at least one glycoside
molecule such as a saponin of the invention in an endosome at the
same time in a pre-defined ratio, when the effector moiety is
comprised by the second or third or fifth or seventh proteinaceous
molecule of the invention and the saponin is comprised by the
first, fourth or sixth proteinaceous molecule. Within the context
of the present invention, the polymeric or oligomeric structure of
the scaffold is a structurally ordered formation such as a polymer,
oligomer, dendrimer, dendronized polymer, or dendronized oligomer
or it is an assembled polymeric structure such as a hydrogel,
microgel, nanogel, stabilized polymeric micelle or liposome, but
excludes structures that are composed of non-covalent assemblies of
monomers such as cholesterol/phospholipid mixtures. The terms
"polymer, oligomer, dendrimer, dendronized polymer, or dendronized
oligomer" have their ordinary meaning. In particular a polymer is a
substance which has a molecular structure built up chiefly or
completely from a large number of equal or similar units bonded
together and an oligomer is a polymer whose molecules consist of
relatively few repeating units. There is no consensus about one
specific cut-off for "many" and "a few" as used in the above
definition of polymer and oligomer, respectively. However, as the
scaffold may comprise a polymeric or an oligomeric structure, or
both, the full range of numbers of similar units bonded together
applies to such structure, i.e. from 2 monomeric units to 100
monomeric units, 1000 monomeric units, and more. A structure
comprising 5 or less, for instance maybe called an oligomeric
structure, whereas a structure comprising 50 monomeric units maybe
called a polymeric structure. A structure of 10 monomeric units
maybe called either oligomeric or polymeric. A scaffold as defined
herein, further comprises at least one glycoside molecule such as a
saponin of the invention. A scaffold preferably includes a
polymeric or oligomeric structure such as poly- or oligo(amines),
e.g., polyethylenimine and poly(amidoamine), and biocompatible
structures such as polyethylene glycol, poly- or oligo(esters),
such as poly(lactids), poly(lactams), polylactide-co-glycolide
copolymers, and poly(dextrin), poly- or oligosaccharides, such as
cyclodextrin or polydextrose, and poly- or oligoamino acids, such
as poly-lysine or a peptide or a protein, or DNA oligo- or
polymers. An assembled polymeric structure as defined herein
comprises at least one scaffold and, optionally, other individual
polymeric or oligomeric structures. Other individual polymeric or
oligomeric structures of said assembly may be (a) scaffolds (thus
comprising at least one glycoside molecule such as a saponin of the
invention), (b) functionalized scaffolds (thus comprising at least
one glycoside molecule such as a saponin, and a ligand, antibody,
etc. as the first proteinaceous molecule, (c) polymeric or
oligomeric structures without a glycoside molecule such as a
saponin of the invention (See Table A1 for example), without a
ligand, antibody, etc., as the first proteinaceous molecule. A
functionalized assembled polymeric structure is an assembled
polymeric structure that contains (a) at least one functionalized
scaffold or (b) at least one scaffold and at least one polymeric
structure comprising at least one ligand, antibody, etc. as the
first proteinaceous molecule. Polymeric or oligomeric structures
within an assembled polymeric structure that do not comprise any of
the above mentioned molecules (i.e. no glycosides such as saponins,
no first proteinaceous molecule such as ligands, antibodies) are in
particular added as structural components of the assembled
structures, which help to build up or to stabilize the assembled
structure ("glue-like").Without wishing to be bound by any theory,
the acidic environment seems to be a prerequisite for the
synergistic action between glycoside (saponin) and effector
moiety.
[0484] Whether or not a first, fourth or sixth proteinaceous
molecule comprising saponins, either or not further comprising one
or more (cleavable) linkers and/or optionally a scaffold, is able
to disturb the acidic environment and inhibit the endosomal escape
function of the at least one glycoside (saponin) can be easily
determined with an assay known in the art. The inhibition is
described as "fold amount increases of glycoside necessary to
induced 50% cell killing". It is preferred that the scaffold does
not lead to an increase that is at least the increase in glycoside
molecules (saponins) necessary to obtain 50% cell killing observed
when using Chloroquine as a positive control. Alternatively, and
preferably, the first, fourth, sixth proteinaceous molecule
comprising saponins, either or not further comprising one or more
(cleavable) linkers and/or optionally a scaffold does not lead to
an at least 4-fold increase of glycoside molecules to induce 50%
cell killing, more preferably does not lead to an at least 2-fold
increase. The fold increase is to be measured in assay, wherein
Chloroquine, as a positive control, induces a 2-fold increase in
glycoside amount, preferably saponin amount wherein the saponin is
any one or more of the saponins of the invention (see Table A1,
Scheme I, previous embodiments) to observe 50% cell killing.
[0485] With the term "improving or enhancing an effect of an
effector moiety" is meant that the glycoside molecule, preferably a
saponin of the invention, increases the functional efficacy of that
effector moiety (e.g. the therapeutic index of a toxin or a drug or
an oligonucleotide such as a BNA; the metabolic efficacy of a
modifier in biotechnological processes; the transfection efficacy
of genes in cell culture research experiments), preferably by
enabling or improving its target engagement. Acceleration,
prolongation, or enhancement of antigen-specific immune responses
are preferably not included. Therapeutic efficacy includes but is
not limited to a stronger therapeutic effect, preferably with lower
dosing and/or with less side effects. "Improving an effect of an
effector moiety" can also mean that an effector moiety, which could
not be used because of lack of effect (and was e.g. not known as
being an effector moiety), becomes effective when used in
combination with the present invention. Any other effect, which is
beneficial or desired and can be attributed to the combination of
effector moiety and the second or third proteinaceous molecule, as
provided by the invention is considered to be "an improved effect".
In an embodiment, the scaffold comprising bound saponin(s) and
comprised by the first, fourth or sixth proteinaceous molecule
enhances an effect of the effector moiety comprised by the second,
third, fifth or seventh proteinaceous molecule which effect is
intended and/or desired. In case of a first, fourth or sixth
proteinaceous molecule comprising saponin bound to a proteinaceous
scaffold, the proteinaceous polymeric structure of the scaffold as
such may have, for instance, an effect on colloid osmotic pressure
in the blood stream. If such effect is not the intended or desired
effect of such a functionalized scaffold comprised by the first,
fourth or sixth proteinaceous molecule, the proteinaceous structure
of the scaffold is not an effector moiety as defined in the
invention. Or, for instance in case of a DNA- or RNA-based scaffold
carrying bound saponins and comprised by the first proteinaceous
molecule, parts of that DNA or RNA may have an (unintended)
function, e.g., by interfering with expression. If such
interference is not the intended or desired effect of the ultimate
functionalized scaffold, the DNA- or RNA polymeric structure of the
scaffold is not the effector moiety as defined in the
invention.
[0486] A number of preferred features can be formulated for
endosomal escape enhancers comprised by the first, fourth or sixth
proteinaceous molecule, i.e. a glycoside or saponin, preferably a
saponin according to the invention: (1) they are preferably not
toxic and do not invoke an immune response, (2) they preferably do
not mediate the cytosolic uptake of the effector moiety into
off-target cells, (3) their presence at the site of action is
preferably synchronized with the presence of the effector moiety,
(4) they are preferably biodegradable or excretable, and (5) they
preferably do not substantially interfere with biological processes
of the organism unrelated to the biological activity of the
effector molecule with which the endosomal escape enhancer is
combined with, e.g. interact with hormones. Examples of glycoside
molecules such as saponins of the invention that fulfill the before
mentioned criteria, at least to some extent, are bisdesmosidic
triterpenes, preferably bisdesmosidic triterpene saponins, such as
SO1861, SA1641, QS-21, GE1741, and the saponins in Table A1, Scheme
I.
[0487] An aspect of the invention relates to an antibody-drug
conjugate or an antibody-oligonucleotide conjugate or a ligand-drug
conjugate comprising the first, fourth or sixth proteinaceous
molecule of the invention and an effector moiety.
[0488] As said before, the at least one saponin that is comprised
by the first, fourth or sixth proteinaceous molecule according to
the invention increases the efficacy of at least current and new
effector moieties as defined in this invention. Potential
side-effects will be decreased due to lowering of dosing of the
effector moiety comprised by the second or third or fifth or
seventh proteinaceous molecule, without lowering the efficacy.
Therefore, the invention provides a first, fourth or sixth
proteinaceous molecule according to the invention for use in
medicine or for use as a medicament. Thus, an aspect of the
invention relates to a first, fourth or sixth proteinaceous
molecule according to the invention, the first, fourth or sixth
proteinaceous molecule comprising at least a saponin, for use as a
medicament. Also provided is the use of a first, fourth or sixth
proteinaceous molecule according to the invention for manufacturing
a medicament. Especially cancer medicines, and in particular the
classical chemotherapy medicaments, are notorious for their side
effects. Because of targeting and synchronization in time and place
of both the pharmaceutically active substance comprised by the
second or third or fifth or seventh proteinaceous molecule and the
saponin comprised by the first, fourth or sixth proteinaceous
molecule, since the first and third, and/or the fourth and fifth,
proteinaceous molecule bear the same binding site for the same
epitope on the same cell-surface molecule, or since the first and
second, or sixth and seventh, proteinaceous molecule bear different
binding sites for different first and second, or sixth and seventh,
epitopes on the first and second, or on the sixth and seventh,
cell-surface molecules respectively, a therapeutic combination
according to the invention is especially valuable for use as a
medicament, in particular for use in a method of treating cancer.
The invention thus provides a therapeutic combination according to
the invention or a first, fourth or sixth proteinaceous molecule of
the invention for use in a method of treating cancer. The invention
also provides a therapeutic combination according to the invention
or a first, fourth or sixth proteinaceous molecule of the invention
for use in a method of treating acquired or hereditary disorders,
in particular monogenic deficiency disorders. The therapeutic
combination thus comprises the first and second proteinaceous
molecule and/or comprises the first and third proteinaceous
molecule, and/or the fourth and fifth proteinaceous molecule,
and/or the sixth and seventh proteinaceous molecule. Thus, an
aspect of the invention relates to a therapeutic combination
according to the invention, wherein the second or third or fifth or
seventh proteinaceous molecule comprises a covalently bound
effector moiety, for use in a method for the treatment of a cancer
or an auto-immune disease.
[0489] A further application of the first, second and third,
fourth, fifth, sixth, seventh proteinaceous molecules of the
invention in medicine is the substitution of intracellular enzymes
in target cells that produce these enzymes in insufficient amount
or insufficient functionality. The resulting disease might be
hereditary or acquired. In most cases, only symptomatic treatment
is possible and for a number of rare diseases, insufficient
treatment options lead to a shortened life span of concerned
patients. An example for such a disease is phenylketonuria, which
is an inborn error of metabolism that results in decreased
metabolism of the amino acid phenylalanine. The disease is
characterized by mutations in the gene for the hepatic enzyme
phenylalanine hydroxylase. Phenylketonuria is not curable to date.
The incidence is approximately 1:10,000 with the highest known
incidence in Turkey with 1:2,600. A second or third or fifth or
seventh proteinaceous molecule, preferably an antibody, with bound
phenylalanine hydroxylase or with a bound polynucleotide that
encodes phenylalanine hydroxylase can be used to target liver cells
by use of a suitable specific antibody, and to substitute the
defect enzyme in hepatocytes. This is one example of use of the
therapeutic combination of the invention comprising a first, fourth
or sixth proteinaceous molecule with a saponin bound thereto and a
second or third or fifth or seventh proteinaceous molecule with the
enzyme or the oligonucleotide bound thereto according to the
invention for substitution or gene therapy. In a preferred
embodiment, a therapeutic combination according to the invention
for use in a method of gene therapy or substitution therapy is
provided.
[0490] The present invention also provides a method of treating
cancer, the method comprising administering a medicament comprising
a therapeutic combination according to the invention to a patient
in need thereof, preferably administering an effective dose of said
medicament to a patient in need thereof, preferably a human cancer
patient.
[0491] Considerations concerning forms suitable for administration
are known in the art and include toxic effects, solubility, route
of administration, and maintaining activity. For example,
pharmacological compositions injected into the bloodstream should
be soluble.
[0492] Suitable dosage forms, in part depend upon the use or the
route of entry, for example transdermal or by injection. Such
dosage forms should allow the compound to reach a target cell
whether the target cell is present in a multicellular host. Other
factors are known in the art, and include considerations such as
toxicity and dosage form which retard the compound or composition
from exerting its effect.
[0493] An embodiment is the combination of an endosomal escape
enhancing conjugate according to the invention, comprising the
first, fourth or sixth proteinaceous molecule comprising at least
one covalently bound saponin, and a binding moiety, wherein the
binding moiety comprises at least one effector moiety, the binding
moiety being the second or third or fifth or seventh proteinaceous
molecule comprising the bound effector moiety, wherein the
endosomal escape enhancing conjugate and the binding moiety are,
independently from one another, able to specifically bind to a
target cell-specific surface molecule or structure, thereby
inducing receptor-mediated endocytosis of a complex of the
endosomal escape enhancing conjugate and the target cell-specific
surface molecule, and of the complex of the binding moiety and the
target cell-specific surface molecule, wherein the endosomal escape
enhancing conjugate and the binding moiety can bind to the same
target cell-specific surface molecule via their same binding site,
or wherein the endosomal escape enhancing conjugate and the binding
moiety can bind to the different target cell-specific surface
molecules via their different binding sites. An embodiment is the
combination according to the invention, wherein the endosomal
escape enhancing conjugate is able to compete with the binding
moiety for binding to the target cell-specific surface molecule or
structure. An embodiment is the combination according to the
invention, wherein the endosomal escape enhancing conjugate and the
binding moiety are, independently from one another, able to
specifically bind to the same epitope, or to a different epitope.
An embodiment is the combination for use in a method for the
treatment of an aberrancy such as a cancer according to the
invention, wherein said endosomal escape enhancing conjugate and
said binding moiety are to be administered concomitant or
sequentially, preferably concomitant.
[0494] An aspect of the invention relates to a kit comprising a
first container containing an endosomal escape enhancing conjugate
according to the invention (i.e. the first, fourth or sixth
proteinaceous molecule) and a second container containing a binding
moiety according to the invention (i.e. the second and/or third
and/or fifth and/or seventh proteinaceous molecule), the kit
further comprising instructions for using the binding molecules
(i.e. the therapeutic combination comprising the first and second
or the first and third or the fourth and fifth or the sixth and
seventh pharmaceutical compositions).
[0495] It is part of the invention that the therapeutic
combination, the first pharmaceutical composition, the first
proteinaceous molecule, the second or third pharmaceutical
composition or the second or third proteinaceous molecule of the
invention is further combined with a covalent conjugate (complex)
of a binding molecule or a binding moiety and a saponin, or is
further combined with a pharmaceutical compound, an antibody, etc.,
therewith providing a composition comprising three or more
enhancers, pharmaceutically active ingredients, etc., e.g. a
conjugate of the invention (e.g. a first proteinaceous molecule
and/or a second or third proteinaceous molecule) combined with a
binding moiety complexed with an effector molecule, further
combined with a pharmaceutical, which is either or not linked to a
saponin, and which is either or not coupled to a ligand such as a
targeting immunoglobulin, a domain or a fragment thereof.
Furthermore, an embodiment is the therapeutic combination, the
first pharmaceutical composition, the first proteinaceous molecule,
the second or third pharmaceutical composition or the second or
third proteinaceous molecule of the invention, wherein the second
or third proteinaceous molecule is provided with two or more
effector moieties such as a toxin or immunotoxin, wherein the two
or more effector moieties are the same or different.
TABLE-US-00001 TABLE A1 Saponins displaying (late)
endosomal/lysosomal escape enhancing activity, and saponins
comprising a structure reminiscent to such saponins displaying
(late) endosomal/lysosomal escape enhancing activity Carbohydrate
substituent Saponin Name Aglycon core at the C-3beta-OH group
Carbohydrate substituent at the C-28-OH group NP-005236 2alpha-
GlcA- Glc/Gal- Hydroxyoleanolic acid AMA-1 16alpha- Glc-
Rha-(1.fwdarw.2)-[Xyl-(1.fwdarw.4)]-Rha- Hydroxyoleanolic acid AMR
16alpha- Glc-
Rha-(1.fwdarw.2)-[Ara-(1.fwdarw.3)-Xyl-(1.fwdarw.4)]-Rha-
Hydroxyoleanolic acid alpha-Hederin Hederagenin (23-
Rha-(1.fwdarw.2)-Ara- -- Hydroxyoleanolic acid) NP-012672
16alpha,23- Ara/Xyl-(1.fwdarw.4)-Rha/Fuc- Ara/Xyl-
Dihydroxyoleanolic (1.fwdarw.2)-Glc/Gal-(1.fwdarw.2)- acid
Rha/Fuc-(1.fwdarw.2)-GlcA- NP-017777 Gypsogenin
Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Xyl-(1.fwdarw.4)-Rha-(1.fwdarw.2)-[R-(.fwdarw.4)]-Fuc- (R = 4E-
Methoxycinnamic acid) NP-017778 Gypsogenin
Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Xyl-(1.fwdarw.4)-Rha-(1.fwdarw.2)-[R-(.fwdarw.4)]-Fuc- (R = 4Z-
Methoxycinnamic acid) NP-017774 Gypsogenin
Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Xyl-(1.fwdarw.4)-[Gal-(1.fwdarw.3)]-Rha-(1.fwdarw.2)-4-OAc- Fuc-
NP-018110.sup.c, Gypsogenin
Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Xyl-(1.fwdarw.4)-[Glc-(1.fwdarw.3)]-Rha-(1.fwdarw.2)-3,4-di-
NP-017772.sup.d OAc-Fuc- NP-018109 Gypsogenin
Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Xyl-(1.fwdarw.4)-[Glc-(1.fwdarw.3)]-Rha-(1.fwdarw.2)-[R-(.fwdarw.4)]-
3-OAc-Fuc- (R = 4E-Methoxycinnamic acid) NP-017888 Gypsogenin
Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Glc-(1.fwdarw.3)-Xyl-(1.fwdarw.4)-[Glc-(1.fwdarw.3)]-Rha-
(1.fwdarw.2)-4-OAc-Fuc- NP-017889 Gypsogenin
Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Glc-(1.fwdarw.3)-Xyl-(1.fwdarw.4)-Rha-(1.fwdarw.2)-4-OAc-Fuc-
NP-018108 Gypsogenin Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Ara/Xyl-(1.fwdarw.3)-Ara/Xyl-(1.fwdarw.4)-Rha/Fuc-
(1.fwdarw.2)-[4-OAc-Rha/Fuc-(1.fwdarw.4)]-Rha/Fuc- SA1641.sup.a,
Gypsogenin Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Xyl-(1.fwdarw.3)-Xyl-(1.fwdarw.4)-Rha-(1.fwdarw.2)-[Qui- AE
X55.sup.b (1.fwdarw.4)]-Fuc- NP-017674 Quillaic acid
Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Api-(1.fwdarw.3)-Xyl-(1.fwdarw.4)-[Glc-(1.fwdarw.3)]-Rha-
(1.fwdarw.2)-Fuc- NP-017810 Quillaic acid
Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Xyl-(1.fwdarw.4)-[Gal-(1.fwdarw.3)]-Rha-(1.fwdarw.2)-Fuc- AG1
Quillaic acid Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Xyl-(1.fwdarw.4)-[Glc-(1.fwdarw.3)]-Rha-(1.fwdarw.2)-Fuc- NP-003881
Quillaic acid Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Ara/Xyl-(1.fwdarw.4)-Rha/Fuc-(1.fwdarw.4)-[Glc/Gal-
(1.fwdarw.2)]-Fuc- NP-017676 Quillaic acid
Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Api-(1.fwdarw.3)-Xyl-(1.fwdarw.4)-[Glc-(1.fwdarw.3)]-Rha-
(1.fwdarw.2)-[R-(.fwdarw.4)]-Fuc- (R =
5-O-[5-O-Ara/Api-3,5-dihydroxy-6-
methyl-octanoyl]-3,5-dihydroxy-6-methyl- octanoic acid) NP-017677
Quillaic acid Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Api-(1.fwdarw.3)-Xyl-(1.fwdarw.4)-Rha-(1.fwdarw.2)-[R-(.fwdarw.4)]-
Fuc- (R = 5-O-[5-O-Ara/Api-3,5-dihydroxy-6-
methyl-octanoyl]-3,5-dihydroxy-6-methyl- octanoic acid) NP-017706
Quillaic acid Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Api-(1.fwdarw.3)-Xyl-(1.fwdarw.4)-Rha-(1.fwdarw.2)-[Rha-
(1.fwdarw.3)]-4-OAc-Fuc- NP-017705 Quillaic acid
Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Api-(1.fwdarw.3)-Xyl-(1.fwdarw.4)-[Glc-(1.fwdarw.3)]-Rha-
(1.fwdarw.2)-[Rha-(1.fwdarw.3)]-4-OAc-Fuc- NP-017773 Quillaic acid
Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
6-OAc-Glc-(1.fwdarw.3)-Xyl-(1.fwdarw.4)-Rha-(1.fwdarw.2)-[3-
OAc-Rha-(1.fwdarw.3)]-Fuc- NP-017775 Quillaic acid
Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Glc-(1.fwdarw.3)-Xyl-(1.fwdarw.4)-Rha-(1.fwdarw.2)-[3-OAc--
Rha-(1.fwdarw.3)]-Fuc- SA1657 Quillaic acid
Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Xyl-(1.fwdarw.3)-Xyl-(1.fwdarw.4)-Rha-(1.fwdarw.2)-[Qui-
(1.fwdarw.4)]-Fuc- AG2 Quillaic acid
Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Glc-(1.fwdarw.3)-[Xyl-(1.fwdarw.4)]-Rha-(1.fwdarw.2)-[Qui-
(1.fwdarw.4)]-Fuc- SO1861 Quillaic acid
Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Glc-(1.fwdarw.3)-Xyl-(1.fwdarw.4)-Rha-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)-
4-OAc-Qui-(1.fwdarw.4)]-Fuc- GE1741 Quillaic acid
Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Xyl-(1.fwdarw.3)-Xyl-(1.fwdarw.4)-Rha-(1.fwdarw.2)-[3,4-di-OAc-
Qui-(1.fwdarw.4)]-Fuc- SO1542 Quillaic acid
Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Glc-(1.fwdarw.3)-[Xyl-(1.fwdarw.4)]-Rha-(1.fwdarw.2)-Fuc- SO1584
Quillaic acid Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
6-OAc-Glc-(1.fwdarw.3)-[Xyl-(1.fwdarw.4)]-Rha-(1.fwdarw.2)- Fuc-
SO1658 Gypsogenin Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Glc-(1.fwdarw.3)-[Xyl-(1.fwdarw.3)-Xyl-(1.fwdarw.4)]-Rha-
(1.fwdarw.2)-Fuc- SO1674 Quillaic acid
Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Glc-(1.fwdarw.3)-[Xyl-(1.fwdarw.3)-Xyl-(1.fwdarw.4)]-Rha-
(1.fwdarw.2)-Fuc- SO1832 Quillaic acid
Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Xyl-(1.fwdarw.3)-Xyl-(1.fwdarw.4)-Rha-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)-
4-OAc-Qui-(1.fwdarw.4)]-Fuc- QS-7 (also referred Quillaic acid
Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Api/Xyl-(1.fwdarw.3)-Xyl-(1.fwdarw.4)-[Glc-(1.fwdarw.3)]-Rha- to as
QS1861) (1.fwdarw.2)-[Rha-(1.fwdarw.3)]-4OAc-Fuc- QS-7 api (also
Quillaic acid Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Api-(1.fwdarw.3)-Xyl-(1.fwdarw.4)-[Glc-(1.fwdarw.3)]-Rha- referred
to as (1.fwdarw.2)-[Rha-(1.fwdarw.3)]-4OAc-Fuc- QS1862) QS-17
Quillaic acid Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Api/Xyl-(1.fwdarw.3)-Xyl-(1-4)-[Glc-(1.fwdarw.3)]-Rha-
(1.fwdarw.2)-[R-(.fwdarw.4)]-Fuc- (R =
5-O-[5-O-Rha-(1.fwdarw.2)-Ara/Api-3,5-
dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy- 6-methyl-octanoic acid)
QS-18 Quillaic acid Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Api/Xyl-(1.fwdarw.3)-Xyl-(1.fwdarw.4)-[Glc-(1.fwdarw.3)]-Rha-
(1.fwdarw.2)-[R-(.fwdarw.4)]-Fuc- (R =
5-O-[5-O-Ara/Api-3,5-dihydroxy-6-
methyl-octanoyl]-3,5-dihydroxy-6-methyl- octanoic acid) QS-21
A-apio Quillaic acid Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Api-(1.fwdarw.3)-Xyl-(1.fwdarw.4)-Rha-(1.fwdarw.2)-[R-(.fwdarw.4)]-
Fuc- (R = 5-O-[5-O-Ara/Api-3,5-dihydroxy-6-
methyl-octanoyl]-3,5-dihydroxy-6-methyl- octanoic acid) QS-21
A-xylo Quillaic acid Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Xyl-(1.fwdarw.3)-Xyl-(1.fwdarw.4)-Rha-(1.fwdarw.2)-[R-(.fwdarw.4)]-
Fuc- (R = 5-O-[5-O-Ara/Api-3,5-dihydroxy-6-
methyl-octanoyl]-3,5-dihydroxy-6-methyl- octanoic acid) QS-21
B-apio Quillaic acid Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Api-(1.fwdarw.3)-Xyl-(1.fwdarw.4)-Rha-(1.fwdarw.2)-[R-(.fwdarw.3)]-
Fuc- (R = 5-O-[5-O-Ara/Api-3,5-dihydroxy-6-
methyl-octanoyl]-3,5-dihydroxy-6-methyl- octanoic acid) QS-21
B-xylo Qui!laic acid Gal-(1.fwdarw.2)-[Xyl-(1.fwdarw.3)]-GlcA-
Xyl-(1.fwdarw.3)-Xyl-(1.fwdarw.4)-Rha-(1.fwdarw.2)-[R-(.fwdarw.3)]-
Fuc- (R = 5-O-[5-O-Ara/Api-3,5-dihydroxy-6-
methyl-octanoyl]-3,5-dihydroxy-6-methyl- octanoic acid) beta-Aescin
Protoaescigenin-21(2- Glc-(1.fwdarw.2)-[Glc-(1.fwdarw.4)]-GlcA- --
(described: methylbut-2-enoate)-22- Aescin la) acetat Teaseed
saponin I 23-Oxo-barringtogenol C -
Glc-(1.fwdarw.2)-Ara-(1.fwdarw.3)-[Gal- -- 21,22-bis(2-methylbut-2-
(1.fwdarw.2)]-GlcA- enoate) Teaseedsaponin J 23-Oxo-barringtogenol
C - Xyl-(1.fwdarw.2)-Ara-(1.fwdarw.3)-[Gal- --
21,22-bis(2-methylbut-2- (1.fwdarw.2)]-GlcA- enoate) Assamsaponin F
23-Oxo-barringtogenol C - Glc-(1.fwdarw.2)-Ara-(1.fwdarw.3)-[Gal-
-- 21(2-methylbut-2-enoate)- (1.fwdarw.2)]-GlcA- 16,22-diacetat
Digitonin Digitogenin Glc-(1.fwdarw.3)-Gal-(1.fwdarw.2)-[Xyl- --
(1.fwdarw.3)]-Glc-(1.fwdarw.4)-Gal- Primula acid 1 3,16,28-
Rha-(1.fwdarw.2)-Gal-(1.fwdarw.3)-[Glc- -- Trihydroxyoleanan-12-en
(1.fwdarw.2)]-GlcA- AS64R Gypsogenic acid --
Glc-(1.fwdarw.3)-[Glc-(1.fwdarw.6)]-Gal- Carbohydrate substituent
at the C-23-OH group AS6.2 Gypsogenic acid Gal-
Glc-(1.fwdarw.3)-[Glc-(1.fwdarw.6)]-Gal- .sup.a, .sup.bDifferent
names refer to different isolates of the same structure .sup.c,
.sup.dDifferent names refer to different isolates of the same
structure
TABLE-US-00002 TABLE A2 ADCs which were previously investigated in
the human clinical setting, and subsequently retracted from further
clinical investigation Last Development Drug Name Indication Target
Stage Monoclonal Oncology Cells Expressing Epidermal Growth Factor
Discovery Antibody Receptor (Proto Oncogene c ErbB 1 or Conjugate
to Receptor Tyrosine Protein Kinase erbB 1 Target EGFR for or HER1
or ERBB1 or EGFR or EC Oncology 2.7.10.1) Affilutin Multiple
Myeloma (Kahler Disease) Discovery IMGN-779 Myelodysplastic
Syndrome Cells Expressing Myeloid Cell Surface IND/CTA Filed
Antigen CD33 (Sialic Acid Binding Ig Like Lectin 3 or gp67 or CD33)
Neuradiab Non-Hodgkin Lymphoma Cells Expressing Tenascin
(Cytotactin or Phase I GMEM or GP 150-225 or Glioma Associated
Extracellular Matrix Antigen or Hexabrachion or JI or Myotendinous
Antigen or Neuronectin or Tenascin C or TNC) IMGN-779 Refractory
Acute Myeloid Leukemia; Cells Expressing Myeloid Cell Surface Phase
I Relapsed Acute Myeloid Leukemia Antigen CD33 (Sialic Acid Binding
Ig Like Lectin 3 or gp67 or CD33) AGS-67E Acute Myelocytic Leukemia
(AML, Cells Expressing Leukocyte Antigen CD37 Phase I Acute
Myeloblastic Leukemia) (Tetraspanin 26 or CD37) AGS-67E Hairy Cell
Leukemia; Non-Hodgkin Cells Expressing Leukocyte Antigen CD37 Phase
I Lymphoma; Refractory Chronic (Tetraspanin 26 or CD37) Lymphocytic
Leukemia (CLL); Relapsed Chronic Lymphocytic Leukemia (CLL); T-Cell
Leukemia ASG-15ME Metastatic Transitional (Urothelial) Cells
Expressing SLIT And NTRK Like Phase I Tract Cancer Protein 6
(SLITRK6) vandortuzumab Metastatic Hormone Refractory Cells
Expressing Metalloreductase Phase I vedotin (Castration Resistant,
Androgen- STEAP1 (Six Transmembrane Epithelial Independent)
Prostate Cancer Antigen Of The Prostate 1 or STEAP1 or EC 1.16.1.)
CDX-014 Ovarian Cancer Cells Expressing Hepatitis A Virus Cellular
Phase I Receptor 1 (Kidney Injury Molecule 1 or T Cell
Immunoglobulin And Mucin Domain Containing Protein 1 or T-Cell
Immunoglobulin Mucin Receptor 1 or T Cell Membrane Protein 1 or
CD365 or HAVCR1) AGS-16M18 Liver Cancer; Renal Cell Carcinoma Phase
I vorsetuzumab Non-Hodgkin Lymphoma; Renal Cell Cells Expressing
CD70 Antigen (CD27 Phase I mafodotin Carcinoma Ligand or Tumor
Necrosis Factor Ligand Superfamily Member 7 or CD70) denintuzumab
Acute Lymphocytic Leukemia (ALL, Cells Expressing B Lymphocyte
Antigen Phase I mafodotin Acute Lymphoblastic Leukemia); B- CD19 (B
Lymphocyte Surface Antigen B4 Cell Non-Hodgkin Lymphoma; Burkitt or
Differentiation Antigen CD19 or T Cell Lymphoma; Lymphoblastic
Surface Antigen Leu 12 or CD19) Lymphoma; Mantle Cell Lymphoma
SGN-CD70A Diffuse Large B-Cell Lymphoma; Cells Expressing CD70
Antigen (CD27 Phase I Follicular Lymphoma; Mantle Cell Ligand or
Tumor Necrosis Factor Ligand Lymphoma; Metastatic Renal Cell
Superfamily Member 7 or CD70) Carcinoma; Non-Hodgkin Lymphoma
RG-7636 Metastatic Melanoma Endothelin B Receptor (Endothelin Phase
I Receptor Non Selective Type or EDNRB) SC-006 Metastatic
Colorectal Cancer Phase I MM-310 Breast Cancer; Endometrial Cancer;
Ephrin Type A Receptor 2 (Epithelial Cell Phase I Esophageal
Cancer; Gastric Cancer; Kinase or Tyrosine Protein Kinase
Gastroesophageal (GE) Junction Receptor ECK or EPHA2 or EC
2.7.10.1) Carcinomas; Head And Neck Cancer Squamous Cell Carcinoma;
Non-Small Cell Lung Cancer; Ovarian Cancer; Pancreatic Ductal
Adenocarcinoma; Prostate Cancer; Small-Cell Lung Cancer; Soft
Tissue Sarcoma; Solid Tumor; Transitional Cell Carcinoma
(Urothelial Cell Carcinoma) PF-06647263 Metastatic Breast Cancer;
Ovarian Cells Expressing Ephrin A4 (EPH Related Phase I Cancer
Receptor Tyrosine Kinase Ligand 4 or EFNA4) PF-06263507 Solid Tumor
Cells Expressing Trophoblast Glycoprotein Phase I (M6P1 or 5T4
Oncofetal Antigen or 5T4 Oncofetal Trophoblast Glycoprotein or Wnt
Activated Inhibitory Factor 1 or TPBG) PF-06650808 Metastatic
Breast Cancer; Non-Small Cells Expressing Neurogenic Locus Notch
Phase I Cell Lung Cancer; Ovarian Cancer Homolog Protein 3 (NOTCH3)
XMT-1522 Breast Cancer; Gastric Cancer; Non- Receptor Tyrosine
Protein Kinase ERBB 2 Phase I Small Cell Lung Cancer (Metastatic
Lymph Node Gene 19 Protein or Proto Oncogene Neu or Proto Oncogene
C ErbB 2 or Tyrosine Kinase Type Cell Surface Receptor HER2 or
p185erbB2 or HER2 or CD340 or ERBB2 or EC 2.7.10.1); Tubulin
AMG-595 Anaplastic Astrocytoma; Recurrent Cells Expressing
Epidermal Growth Factor Phase I Glioblastoma Multiforme (GBM)
Receptor (Proto Oncogene c ErbB 1 or Receptor Tyrosine Protein
Kinase erbB 1 or HER1 or ERBB1 or EGFR or EC 2.7.10.1) pinatuzumab
Chronic Lymphocytic Leukemia (CLL) Cells Expressing B Cell Receptor
CD22 (B Phase I vedotin Lymphocyte Cell Adhesion Molecule or Sialic
Acid Binding Ig Like Lectin 2 or T Cell Surface Antigen Leu 14 or
CD22) cantuzumab Colorectal Cancer; Non-Small Cell Phase I
ravtansine Lung Cancer; Pancreatic Cancer; Solid Tumor AVE-9633
Acute Myelocytic Leukemia (AML, Cells Expressing Myeloid Cell
Surface Phase I Acute Myeloblastic Leukemia) Antigen CD33 (Sialic
Acid Binding Ig Like Lectin 3 or gp67 or CD33) BIWI-1.sup.(1)
Breast Cancer; Carcinomas; Cells Expressing CD44 Antigen (CDw44 or
Phase I Esophageal Cancer; Head And Neck Epican or Extracellular
Matrix Receptor III Cancer Squamous Cell Carcinoma or GP90
Lymphocyte Homing/Adhesion Receptor or HUTCH I or Heparan Sulfate
Proteoglycan or Hermes Antigen or Hyaluronate Receptor or
Phagocytic Glycoprotein 1 or CD44) RG-7882 Epithelial Ovarian
Cancer; Fallopian Cells Expressing Mucin 16 (Ovarian Phase I Tube
Cancer; Pancreatic Cancer; Cancer Related Tumor Marker CA125 or
Peritoneal Cancer Ovarian Carcinoma Antigen CA125 or MUC16) ASG-5ME
Adenocarcinoma; Hormone Cells Expressing Choline Transporter Like
Phase I Refractory (Castration Resistant, Protein 4 (Solute Carrier
Family 44 Androgen-Independent) Prostate Member 4 or SLC44A4)
Cancer; Metastatic Adenocarcinoma of The Pancreas DCDS-0780A B-Cell
Non-Hodgkin Lymphoma Phase I SC-004 Endometrial Cancer; Epithelial
Phase I Ovarian Cancer; Fallopian Tube Cancer; Peritoneal Cancer
RG-7600 Ovarian Cancer; Pancreatic Ductal Phase I Adenocarcinoma
sofituzumab Epithelial Ovarian Cancer; Fallopian Cells Expressing
Mucin 16 (Ovarian Phase I vedotin Tube Cancer; Ovarian Cancer;
Cancer Related Tumor Marker CA125 or Pancreatic Cancer; Peritoneal
Cancer Ovarian Carcinoma Antigen CA125 or MUC16) IMGN-289 Breast
Cancer; Esophageal Cancer; Cells Expressing Epidermal Growth Factor
Phase I Gastric Cancer; Head And Neck Receptor (Proto Oncogene c
ErbB 1 or Cancer Squamous Cell Carcinoma; Receptor Tyrosine Protein
Kinase erbB 1 Non-Small Cell Lung Cancer; Solid or HER1 or ERBB1 or
EGFR or EC Tumor 2.7.10.1) SAR-428926 Breast Cancer; Colorectal
Cancer; Cells Expressing Lysosome Associated Phase I Gastric
Cancer; Non-Small Cell Lung Membrane Glycoprotein 1 (CD107 Antigen
Cancer; Ovarian Cancer; Prostate Like Family Member A or CD107a or
Cancer; Solid Tumor LAMP1) SGNCD-19B B-Cell Non-Hodgkin Lymphoma;
Cells Expressing B Lymphocyte Antigen Phase I Diffuse Large B-Cell
Lymphoma; CD19 (B Lymphocyte Surface Antigen B4 Follicular Lymphoma
or Differentiation Antigen CD19 or T Cell Surface Antigen Leu 12 or
CD19) SGNCD-123A Refractory Acute Myeloid Leukemia; Cells
Expressing Interleukin 3 Receptor Phase I Relapsed Acute Myeloid
Leukemia Subunit Alpha (CD123 or IL3RA) SGNCD-352A Refractory
Multiple Myeloma; Cells Expressing SLAM Family Member 6 Phase I
Relapsed Multiple Myeloma (Activating NK Receptor or NK T B Antigen
or CD352 or SLAMF6) RG-7841 Breast Cancer; Non-Small Cell Lung
Cells Expressing Lymphocyte Antigen 6E Phase I Cancer; Solid Tumor
(Retinoic Acid Induced Gene E Protein or Stem Cell Antigen 2 or
Thymic Shared Antigen 1 or LY6E) IMGN-388 Solid Tumor Cells
Expressing Integrin Alpha V Phase I (Vitronectin Receptor Subunit
Alpha or CD51 or ITGAV) lorvotuzumab Refractory Multiple Myeloma;
Cells Expressing Neural Cell Adhesion Phase I mertansine Relapsed
Multiple Myeloma Molecule 1 (Antigen Recognized By Monoclonal
Antibody 5.1H11 or CD56 or NCAM1) lorvotuzumab Neuroendocrine
Carcinoma; Cells Expressing Neural Cell Adhesion Phase I mertansine
Neuroendocrine Tumors; Non-Small Molecule 1 (Antigen Recognized By
Cell Lung Cancer; Ovarian Cancer; Monoclonal Antibody 5.1H11 or
CD56 or Skin Cancer NCAM1) BAY-794620 Lung Cancer; Solid Tumor
Cells Expressing Carbonic Anhydrase 9 Phase I (Carbonate
Dehydratase IX or pMW1 or Membrane Antigen MN or P54/58N or Renal
Cell Carcinoma Associated Antigen G250 or CA9 or EC 4.2.1.1)
RG-7598 Refractory Multiple Myeloma; Phase I Relapsed Multiple
Myeloma Oncolysin B B-Cell Leukemia; Lymphoma Cells Expressing B
Lymphocyte Antigen Phase I CD19 (B Lymphocyte Surface Antigen B4 or
Differentiation Antigen CD19 or T Cell Surface Antigen Leu 12 or
CD19) ADCT-502.sup.(1) Bladder Cancer; Breast Cancer; Cells
Expressing Receptor Tyrosine Phase I Esophageal Cancer; Gastric
Cancer; Protein Kinase ERBB 2 (Metastatic Lymph Non-Small Cell Lung
Cancer Node Gene 19 Protein or Proto Oncogene Neu or Proto Oncogene
C ErbB 2 or Tyrosine Kinase Type Cell Surface Receptor HER2 or
p185erbB2 or HER2 or CD340 or ERBB2 or EC 2.7.10.1) AMG-172 Renal
Cell Carcinoma Cells Expressing CD70 Antigen (CD27 Phase I Ligand
or Tumor Necrosis Factor Ligand Superfamily Member 7 or CD70)
ImmuRAIT-LL2 B-Cell Non-Hodgkin Lymphoma Cells Expressing B Cell
Receptor CD22 (B Phase I/II Lymphocyte Cell Adhesion Molecule or
Sialic Acid Binding Ig Like Lectin 2 or T Cell Surface Antigen Leu
14 or CD22) indusatumab Adenocarcinoma Of The Gastro- Cells
Expressing Heat Stable Enterotoxin Phase I/II vedotin esophageal
Junction; Gastric Cancer Receptor (Guanylyl Cyclase C or or
Intestinal Guanylate Cyclase or GUCY2C or EC 4.6.1.2) clivatuzumab
Pancreatic Cancer Cells Expressing Mucin 1 (Breast Phase I/II
tetraxetan Carcinoma Associated Antigen DF3 or Episialin or H23AG
or Krebs Von Den Lungen 6 or PEMT or Peanut Reactive Urinary Mucin
or Polymorphic Epithelial Mucin or Tumor Associated Epithelial
Membrane Antigen or Tumor Associated Mucin or CD227 or MUC1)
depatuxizumab Recurrent Malignant Glioma Epidermal Growth Factor
Receptor (Proto Phase I/II mafodotin.sup.(2) Oncogene c ErbB 1 or
Receptor Tyrosine Protein Kinase erbB 1 or HER1 or ERBB1 or EGFR or
EC 2.7.10.1)
CDX-014 Metastatic Renal Cell Carcinoma; Cells Expressing Hepatitis
A Virus Cellular Phase I/II Papillary Renal Cell Carcinoma Receptor
1 (Kidney Injury Molecule 1 or T Cell Immunoglobulin And Mucin
Domain Containing Protein 1 or T-Cell Immunoglobulin Mucin Receptor
1 or T Cell Membrane Protein 1 or CD365 or HAVCR1) vadastuximab
Refractory Acute Myeloid Leukemia; Cells Expressing Myeloid Cell
Surface Phase I/II talirine.sup.(1) Relapsed Acute Myeloid Leukemia
Antigen CD33 (Sialic Acid Binding Ig Like Lectin 3 or gp67 or CD33)
vadastuximab Myelodysplastic Syndrome Cells Expressing Myeloid Cell
Surface Phase I/II talirine Antigen CD33 (Sialic Acid Binding Ig
Like Lectin 3 or gp67 or CD33) MLN-2704 Metastatic Hormone
Refractory Cells Expressing Glutamate Phase I/II (Castration
Resistant, Androgen- Carboxypeptidase 2 (Folate Hydrolase 1 or
Independent) Prostate Cancer Prostate Specific Membrane Antigen or
PSMA or Pteroylpoly Gamma Glutamate Carboxypeptidase or Cell Growth
Inhibiting Gene 27 Protein or FOLH1 or EC 3.4.17.21) Oncolysin B
AIDS - Related Lymphoma Cells Expressing B Lymphocyte Antigen Phase
I/II CD19 (B Lymphocyte Surface Antigen B4 or Differentiation
Antigen CD19 or T Cell Surface Antigen Leu 12 or CD19) coltuximab
Diffuse Large B-Cell Lymphoma Cells Expressing B Lymphocyte Antigen
Phase II ravtansine CD19 (B Lymphocyte Surface Antigen B4 or
Differentiation Antigen CD19 or T Cell Surface Antigen Leu 12 or
CD19) coltuximab Acute Lymphocytic Leukemia (ALL, Cells Expressing
B Lymphocyte Antigen Phase II ravtansine Acute Lymphoblastic
Leukemia) CD19 (B Lymphocyte Surface Antigen B4 or Differentiation
Antigen CD19 or T Cell Surface Antigen Leu 12 or CD19) coltuximab
Diffuse Large B-Cell Lymphoma Cells Expressing B Lymphocyte Antigen
Phase II ravtansine CD19 (B Lymphocyte Surface Antigen B4 or
Differentiation Antigen CD19 or T Cell Surface Antigen Leu 12 or
CD19) indusatumab Adenocarcinoma Of The Gastro- Cells Expressing
Heat Stable Enterotoxin Phase II vedotin.sup.(2) esophageal
Junction; Gastric Cancer; Receptor (Guanylyl Cyclase C or or
Metastatic Adenocarcinoma of The Intestinal Guanylate Cyclase or
GUCY2C Pancreas or EC 4.6.1.2) depatuxizumab Squamous Non-Small
Cell Lung Epidermal Growth Factor Receptor (Proto Phase II
mafodotin Cancer Oncogene c ErbB 1 or Receptor Tyrosine Protein
Kinase erbB 1 or HER1 or ERBB1 or EGFR or EC 2.7.10.1)
depatuxizumab Anaplastic Astrocytoma; Anaplastic Epidermal Growth
Factor Receptor (Proto Phase II mafodotin.sup.(2) Oligoastrocytoma;
Gliosarcoma; Oncogene c ErbB 1 or Receptor Tyrosine High-Grade
Glioma; Oligoden- Protein Kinase erbB 1 or HER1 or ERBB1 droglioma;
Pediatric Diffuse Intrinsic or EGFR or EC 2.7.10.1) Pontine Glioma;
Recurrent Glioblastoma Multiforme (GBM) lifastuzumab Non-Small Cell
Lung Cancer Sodium Dependent Phosphate Transport Phase II vedotin
Protein 2B (Sodium Phosphate Transport Protein 2B or NaPi3b or
Sodium/Phosphate Cotransporter 2B or NaPi 2b or Solute Carrier
Family 34 Member 2 or SLC34A2) lifastuzumab Ovarian Cancer Sodium
Dependent Phosphate Transport Phase II vedotin Protein 2B (Sodium
Phosphate Transport Protein 2B or NaPi3b or Sodium/Phosphate
Cotransporter 2B or NaPi 2b or Solute Carrier Family 34 Member 2 or
SLC34A2) Bismab-A Acute Myelocytic Leukemia (AML, Cells Expressing
Myeloid Cell Surface Phase II Acute Myeloblastic Leukemia) Antigen
CD33 (Sialic Acid Binding Ig Like Lectin 3 or gp67 or CD33)
denintuzumab Diffuse Large B-Cell Lymphoma; Cells Expressing B
Lymphocyte Antigen Phase II mafodotin Follicular Lymphoma CD19 (B
Lymphocyte Surface Antigen B4 or Differentiation Antigen CD19 or T
Cell Surface Antigen Leu 12 or CD19) Avicidin.sup.(1) Colorectal
Cancer; Prostate Cancer Cells Expressing Epithelial Cell Adhesion
Phase II Molecule (Adenocarcinoma Associated Antigen or Cell
Surface Glycoprotein Trop 1 or Epithelial Cell Surface Antigen or
Epithelial Glycoprotein 314 or KS 1/4 Antigen or KSA or Tumor
Associated Calcium Signal Transducer 1 or CD326 or EPCAM)
pinatuzumab Diffuse Large B-Cell Lymphoma; Cells Expressing B Cell
Receptor CD22 (B Phase II vedotin Follicular Lymphoma Lymphocyte
Cell Adhesion Molecule or Sialic Acid Binding Ig Like Lectin 2 or T
Cell Surface Antigen Leu 14 or CD22) SGN-15 Metastatic Breast
Cancer; Non-Small Cells Expressing Lewis Y Antigen (CD174) Phase II
Cell Lung Cancer; Ovarian Cancer; Prostate Cancer cantuzumab
Gastric Cancer; Gastroesophageal Phase II ravtansine (GE) Junction
Carcinomas ASP-6183 Ovarian Cancer Phase II SAR-566658 Metastatic
Breast Cancer Cells Expressing Sialoglycotope CA6 Phase II Antigen
Oncolysin S Small-Cell Lung Cancer Cells Expressing Neural Cell
Adhesion Phase II Molecule 1 (Antigen Recognized By Monoclonal
Antibody 5.1H11 or CD56 or NCAM1) lorvotuzumab Small-Cell Lung
Cancer Cells Expressing Neural Cell Adhesion Phase II mertansine
Molecule 1 (Antigen Recognized By Monoclonal Antibody 5.1H11 or
CD56 or NCAM1) glembatumumab Metastatic Melanoma; Metastatic Cells
Expressing Transmembrane Phase II vedotin Uveal Melanoma;
Osteosarcoma; Glycoprotein NMB (Transmembrane Squamous Non-Small
Cell Lung Glycoprotein HGFIN or GPNMB) Cancer MM-302 Metastatic
Breast Cancer Cells Expressing Receptor Tyrosine Phase II/III
Protein Kinase ERBB 2 (Metastatic Lymph Node Gene 19 Protein or
Proto Oncogene Neu or Proto Oncogene C ErbB 2 or Tyrosine Kinase
Type Cell Surface Receptor HER2 or p185erbB2 or HER2 or CD340 or
ERBB2 or EC 2.7.10.1) Neuradiab Brain Cancer; Glioblastoma Cells
Expressing Tenascin (Cytotactin or Phase III Multiforme (GBM) GMEM
or GP 150-225 or Glioma Associated Extracellular Matrix Antigen or
Hexabrachion or JI or Myotendinous Antigen or Neuronectin or
Tenascin C or TNC) clivatuzumab Metastatic Adenocarcinoma of The
Cells Expressing Mucin 1 (Breast Phase III tetraxetan Pancreas
Carcinoma Associated Antigen DF3 or Episialin or H23AG or Krebs Von
Den Lungen 6 or PEMT or Peanut Reactive Urinary Mucin or
Polymorphic Epithelial Mucin or Tumor Associated Epithelial
Membrane Antigen or Tumor Associated Mucin or CD227 or MUC1)
depatuxizumab Glioblastoma Multiforme (GBM) Epidermal Growth Factor
Receptor (Proto Phase III mafodotin.sup.(2) Oncogene c ErbB 1 or
Receptor Tyrosine Protein Kinase erbB 1 or HER1 or ERBB1 or EGFR or
EC 2.7.10.1) vadastuximab Acute Myelocytic Leukemia (AML, Cells
Expressing Myeloid Cell Surface Phase III talirine.sup.(1) Acute
Myeloblastic Leukemia) Antigen CD33 (Sialic Acid Binding Ig Like
Lectin 3 or gp67 or CD33) glembatumumab Metastatic Breast Cancer
Cells Expressing Transmembrane Phase III vedotin.sup.(2)
Glycoprotein NMB (Transmembrane Glycoprotein HGFIN or GPNMB)
Oncolysin B B-Cell Leukemia; Lymphoma Cells Expressing B Lymphocyte
Antigen Phase III CD19 (B Lymphocyte Surface Antigen B4 or
Differentiation Antigen CD19 or T Cell Surface Antigen Leu 12 or
CD19) ImmuRAIT-LL2 B-Cell Leukemia Cells Expressing B Cell Receptor
CD22 (B Preclinical Lymphocyte Cell Adhesion Molecule or Sialic
Acid Binding Ig Like Lectin 2 or T Cell Surface Antigen Leu 14 or
CD22) indusatumab Metastatic Colorectal Cancer Cells Expressing
Heat Stable Enterotoxin Preclinical vedotin Receptor (Guanylyl
Cyclase C or or Intestinal Guanylate Cyclase or GUCY2C or EC
4.6.1.2) ASG-15ME Lung Cancer Cells Expressing SLIT And NTRK Like
Preclinical Protein 6 (SLITRK6) HTI-1511 Bile Duct Cancer Cells
Expressing Epidermal Growth Factor Preclinical
(Cholangiocarcinoma); Breast Cancer; Receptor (Proto Oncogene c
ErbB 1 or Colorectal Cancer; Non-Small Cell Receptor Tyrosine
Protein Kinase erbB 1 Lung Cancer or HER1 or ERBB1 or EGFR or EC
2.7.10.1) ZW-33 Gastric Cancer; Metastatic Breast Cells Expressing
Receptor Tyrosine Preclinical Cancer Protein Kinase ERBB 2
(Metastatic Lymph Node Gene 19 Protein or Proto Oncogene Neu or
Proto Oncogene C ErbB 2 or Tyrosine Kinase Type Cell Surface
Receptor HER2 or p185erbB2 or HER2 or CD340 or ERBB2 or EC
2.7.10.1) ZW-33 Ovarian Cancer Cells Expressing Receptor Tyrosine
Preclinical Protein Kinase ERBB 2 (Metastatic Lymph Node Gene 19
Protein or Proto Oncogene Neu or Proto Oncogene C ErbB 2 or
Tyrosine Kinase Type Cell Surface Receptor HER2 or p185erbB2 or
HER2 or CD340 or ERBB2 or EC 2.7.10.1) SGNCD-352A Non-Hodgkin
Lymphoma Cells Expressing SLAM Family Member 6 Preclinical
(Activating NK Receptor or NK T B Antigen or CD352 or SLAMF6)
HuMax-CD74-ADC Oncology Cells Expressing HLA Class II Preclinical
Histocompatibility Antigen Gamma Chain (HLA DR Antigens Associated
Invariant Chain or Ia Antigen Associated Invariant Chain or p33 or
CD74) sacituzumab Pancreatic Ductal Adenocarcinoma Cells Expressing
Tumor Associated govitecan Calcium Signal Transducer 2 (Cell
Surface Glycoprotein Trop 2 or Membrane Component Chromosome 1
Surface Marker 1 or Pancreatic Carcinoma Marker Protein GA733-1 or
TACSTD2) sacituzumab Adenocarcinoma; Cervical Cancer; Cells
Expressing Tumor Associated govitecan Colorectal Cancer;
Endometrial Calcium Signal Transducer 2 (Cell Surface Cancer;
Epithelial Ovarian Cancer; Glycoprotein Trop 2 or Membrane
Esophageal Cancer; Follicular Thyroid Component Chromosome 1
Surface Cancer; Gastric Cancer; Glioblastoma Marker 1 or Pancreatic
Carcinoma Marker Multiforme (GBM); Head And Neck Protein GA733-1 or
TACSTD2) Cancer Squamous Cell Carcinoma; Hepatocellular Carcinoma;
Kidney Cancer (Renal Cell Cancer); Metastatic Hormone Refractory
(Castration Resistant, Androgen- Independent) Prostate Cancer;
Metastatic Transitional (Urothelial) Tract Cancer; Transitional
Cell Cancer (Urothelial Cell Cancer) sacituzumab Hepatocellular
Carcinoma Cells Expressing Tumor Associated govitecan Calcium
Signal Transducer 2 (Cell Surface Glycoprotein Trop 2 or Membrane
Component Chromosome 1 Surface Marker 1 or Pancreatic Carcinoma
Marker Protein GA733-1 or TACSTD2) sacituzumab Metastatic Breast
Cancer; Transitional Cells Expressing Tumor Associated govitecan
Cell Cancer (Urothelial Cell Cancer) Calcium Signal Transducer 2
(Cell Surface Glycoprotein Trop 2 or Membrane Component Chromosome
1 Surface
Marker 1 or Pancreatic Carcinoma Marker Protein GA733-1 or TACSTD2)
sacituzumab Non-Small Cell Lung Cancer; Small- Cells Expressing
Tumor Associated govitecan Cell Lung Cancer Calcium Signal
Transducer 2 (Cell Surface Glycoprotein Trop 2 or Membrane
Component Chromosome 1 Surface Marker 1 or Pancreatic Carcinoma
Marker Protein GA733-1 or TACSTD2) sacituzumab Metastatic Breast
Cancer Cells Expressing Tumor Associated govitecan Calcium Signal
Transducer 2 (Cell Surface Glycoprotein Trop 2 or Membrane
Component Chromosome 1 Surface Marker 1 or Pancreatic Carcinoma
Marker Protein GA733-1 or TACSTD2) .sup.(1)Discontinued due to
adverse events .sup.(2)Discontinued due to lack of efficacy
TABLE-US-00003 TABLE A3 ADCs that reached phase III clinical
development Last Development Development Drug Name Indication Stage
Stage Reason for Discontinuation trastuzumab emtansine Gastric
Cancer Marketed Phase II/III Unspecified MM-302 Metastatic Breast
Discontinued Phase II/III Business/Strategic Decision Cancer
trastuzumab emtansine Metastatic Breast Marketed Phase III
Unspecified Cancer trastuzumab emtansine Gastric Cancer Marketed
Phase III Unspecified ibritumomab tiuxetan Diffuse Large B-
Marketed Phase III Cell Lymphoma inotuzumab ozogamicin Follicular
Lymphoma Marketed Phase III inotuzumab ozogamicin Diffuse Large B-
Marketed Phase III Lack of Efficacy Cell Lymphoma; Non-Hodgkin
Lymphoma rovalpituzumab tesirine Small-Cell Lung Phase III Phase
III Cancer rovalpituzumab tesirine Small-Cell Lung Phase III Phase
III Cancer Neuradiab Brain Cancer; Inactive Phase III Unspecified
Glioblastoma Multiforme (GBM) clivatuzumab tetraxetan Metastatic
Inactive Phase III Unspecified Adenocarcinoma of The Pancreas
depatuxizumab mafodotin Glioblastoma Inactive Phase III Lack of
Efficacy Multiforme (GBM) vadastuximab talirine Acute Myelocytic
Discontinued Phase III Adverse Events Leukemia (AML, Acute
Myeloblastic Leukemia) glembatumumab vedotin Metastatic Breast
Discontinued Phase III Lack of Efficacy Cancer Oncolysin B B-Cell
Leukemia; Discontinued Phase III Business/Strategic Decision
Lymphoma
TABLE-US-00004 TABLE A4 Tumor-specific cell-surface receptor
targets which can be targeted by immunoglobulins according to the
invention, and antibodies that can be used for the ADCs and the
antibodies provided with a saponin, and the ADCs provided with a
saponin, of the present invention (not presented as a limitation;
further immunoglobulins are equally suitable for the invention)
Target cell- surface receptor Example monoclonal antibodies HER2
anti-HER2 monoclonal antibody such as trastuzumab and pertuzumab
CD20 anti-CD20 monoclonal antibody such as rituximab, ofatumumab,
tositumomab and ibritumomab CA125 anti-CA125 monoclonal antibody
such as oregovomab EpCAM (17-1A) anti-EpCAM (17-1A) monoclonal
antibody such as edrecolomab EGFR anti-EGFR monoclonal antibody
such as cetuximab, panitumumab and nimotuzumab CD30 anti-CD30
monoclonal antibody such brentuximab CD33 anti-CD33 monoclonal
antibody such as gemtuzumab and huMy9-6 vascular integrin
anti-vascular integrin alpha-v beta-3 monoclonal antibody such as
alpha-v beta-3 etaracizumab CD52 anti-CD52 monoclonal antibody such
as alemtuzumab CD22 anti-CD22 monoclonal antibody such as
epratuzumab CEA anti-CEA monoclonal antibody such as labetuzumab
CD44v6 anti-CD44v6 monoclonal antibody such as bivatuzumab FAP
anti-FAP monoclonal antibody such as sibrotuzumab CD19 anti-CD19
monoclonal antibody such as huB4 CanAg anti-CanAg monoclonal
antibody such as huC242 CD56 anti-CD56 monoclonal antibody such
huN901 CD38 anti-CD38 monoclonal antibody such as daratumumab CA6
anti-CA6 monoclonal antibody such as DS6 IGF-IR anti-IGF-IR
monoclonal antibody such as cixutumumab and 3B7 integrin
anti-integrin monoclonal antibody such as CNTO 95 syndecan-1
anti-syndecan-1 monoclonal antibody such as B-B4
TABLE-US-00005 TABLE A5 RIPs from plants* Plant Family Plant
Species Proteins Classification Adoxaceae Sambucus ebulus L.
Ebulitin .alpha., Ebulitin .beta., Ebulitin .gamma. RIP 1 Ebulin f,
Ebulin I, Ebulin r1, Ebulin r2, SEA RIP 2 SEAII, SELfd, SELId,
SELIm lectin Sambucus nigra L. .alpha.-Nigritin, .beta.-Nigritin,
.gamma.-Nigritin, Nigritin f1, Nigritin f2 RIP 1 basic Nigrin b,
Nigrin b = SNA-V, Nigrin f = SNA-Vf, RIP 2 Nigrin I1, Nigrin I2,
Nigrin s, SNA-I, SNA-I', SNA-If, SNAflu-I, SNLRP1, SNLRP2 SNA-Id,
SNA-Im, SNA-II, SNA-III, SNA-IV = SNA-IVf, lectin SNA-IVl,
SNApol-I, SNApol-II, TrSNA-I, TrSNA-If Sambucus racemosa L. basic
racemosin b, SRA RIP 2 SRLbm = SRAbm lectin Sambucus sieboldiana
SSA = SSA-b-1, Sieboldin-b = SSA-b-2 RIP 2 (Miq.) Blume ex Graebn.
SSA-b-3, SSA-b-4 lectin Aizoaceae Mesembryanthemum RIP1 RIP 1
crystallinum L. Amaranthaceae Amaranthus caudatus L. Amaranthin =
ACA lectin Amaranthus cruentus L. ACL lectin Amaranthus A.
leucocarpus lectin lectin hypochondriacus L. [Syn.: Amaranthus
leucocarpus S. Watson] Amaranthus mangostanus L. Amaramangin RIP 1
Amaranthus tricolor L. AAP-27 RIP 1 Amaranthus viridis L.
Amaranthin RIP 1 Beta vulgaris L. Beetin-27 = BE27, Beetin-29 =
BE29, Betavulgin RIP 1 Celosia argentea L. [Syn.: CCP-25, CCP-27
RIP 1 Celosia cristata L.] Chenopodium album L. CAP30 RIP 1
Spinacia oleracea L. SoRIP1 = BP31 RIP 1 SoRIP2 RIP 1 candidate
Araliaceae Aralia elata (Miq.) Seem. Aralin RIP 2 Panax ginseng C.
A. Mey Panaxagin peculiar RIP 1 candidate/RNase Panax quinquefolius
L. Quinqueginsin peculiar RIP 1 candidate/RNase Asparagaceae
Asparagus officinalis L. Asparin 1, Asparin 2 RIP 1 Drimia maritima
(L.) Stearn Charybdin RIP 1 [Syn.: Charybdis maritima (L.) Speta]
Muscari armeniacum Musarmin 1, Musarmin 2, Musarmin 3, Musarmin 4
RIP 1 Leichtlin ex Baker Polygonatum multiflorum PMRIPm, PMRIPt RIP
2 (L.) All. Yucca gloriosa var. tristis Yucca leaf protein = YLP
RIP 1 Carriere [Syn.: Yucca recurvifolia Salisb.] Basellaceae
Basella rubra L. Basella RIP 2a, Basella RIP 2b, Basella RIP 3 RIP
1 Caryophyllaceae Agrostemma githago L. Agrostin 2, Agrostin 5,
Agrostin 6, Agrostin RIP 1 Dianthus barbatus L. Dianthin 29 RIP 1
Dianthus caryophyllus L. Dianthin 30, Dianthin 32 RIP 1 Dianthus
chinensis L. [Syn.: D. sinensis RIP RIP 1 Dianthus sinensis Link]
Gypsophila elegans M. Bieb. Gypsophilin RIP 1 Silene chalcedonica
(L.) Lychnin RIP 1 E. H. L. Krause [Syn.: Lychnis chalcedonica L.]
Silene glaucifolia Lag. [Syn.: Petroglaucin 1, Petroglaucin 2 RIP 1
Petrocoptis glaucifolia (Lag.) Boiss.] Silene laxipruinosa Mayol
& Petrograndin RIP 1 Rossello [Syn.: Petrocoptis grandiflora
Rothm.] Saponaria ocymoides L. Ocymoidin RIP 1 Saponaria
officinalis L. Saporin-L1 = SO-L1, Saporin-L2 = SO-L2, Saporin-L3 =
RIP 1 SO-L3, Saporin-I = SO-I = SO-4, Saporin-R1 = SO-R1,
Saporin-R2 = SO-R2, Saporin-R3 = SO-R3, SO3a, SO3b, Saporin-S5 =
Saporin 5 = SO-S5, Saporin-S6 = Saporin 6 = SO-6 = SO-S6,
Saporin-S8 = SO-S8, Saporin-S9 = Saporin 9 = SO-S9, SAP-C, SAP-S
Myosoton aquaticum (L.) Stellarin RIP 1 Moench [Syn.: Stellaria
aquatica (L.) Scop.] Stellaria media (L.) Vill. RIP Q3 RIP 1
Vaccaria hispanica (Mill.) Pyramidatin RIP 1 Rauschert [Syn.:
Vaccaria pyramidata Medik.] Cucurbitaceae Benincasa hispida
(Thunb.) Hispin RIP 1 Cogn. .alpha.-benincasin, .beta.-benincasin
sRIP 1 Bryonia cretica subsp. Bryodin 1 = BD1, Bryodin 2,
Bryodin-L, Bryodin-R RIP 1 dioica (Jacq.) Tutin. [Syn.: BDA
lectin/RIP 2 like Bryonia dioica L.] Citrullus colocynthis (L.)
Colocin 1, Colocin 2 RIP 1 Schrad. Cucurbita foetidissima
Foetidissimin peculiar RIP 2 Kunth Foetidissimin II RIP 2 Cucumis
ficifolius A. Rich. Cucumis figarei RIP = CF-RIP RIP 1 candidate
[Syn.: Cucumis figarei Delile ex Naudin] Cucurbita maxima
Cucurmoschin sRIP 1 candidate Duchesne Cucurbita moschata
Cucurmosin, Cucurmosin 2, C. moschata RIP, RIP 1 Duchesne [Syn.:
Cucurbita Moschatin, PRIP 1, PRIP 2 moschata (Duchesne ex
.alpha.-moschin, .beta.-moschin sRIP 1 candidate Lam.) Duchesne ex
Poir.] Cucurbita pepo L. Pepocin RIP 1 Cucurbita pepo var. texana
Texanin RIP 1 (Scheele) D. S. Decker [Syn.: Cucurbita texana
(Scheele) A. Gray] Gynostemma pentaphyllum Gynostemmin RIP 1
(Thunb.) Makino Lagenaria siceraria (Molina) Lagenin RIP 1
candidate Standl. Luffa acutangula (L.) Roxb. Luffaculin-1,
Luffaculin-2 RIP 1 Luffangulin sRIP 1 Luffa acutangula fruit lectin
lectin Luffa cylindrica (L.) M. Roem Luffin, Luffin-a, Luffin-b,
.alpha.-luffin, .beta.-luffin, LRIP RIP 1 [Syn.: Luffa aegyptiaca
Mill.] Luffacylin, Luffin P1 sRIP 1 Luffin-S, LuffinS(1),
LuffinS(2) = luffin S2, LuffinS(3) sRIP 1 candidate Marah oreganus
(Torr. & A. MOR-I, MOR-II RIP 1 Gray) Howell Momordica
balsamina L. Balsamin, MbRIP-1, Momordin II RIP 1 Momordica
charantia L. MAP 30, .alpha.-momorcharin = .alpha.-MC =
.alpha.-MMC, .beta.- RIP 1 momorcharin = .beta.-MC = .beta.-MMC,
.delta.-momorcharin = .delta.- MMC, Momordin, Momordin = Momordica
charantia inhibitor, Momordin II, Momordin-a, Momordin-b
.gamma.-momorcharin = .gamma.-MMC, Charantin sRIP 1 RIP 1 candidate
RIP 1 candidate MCL = M. charantia lectin, anti-H Lectin, Momordica
lectin agglutinin, Momordin, protein fraction 1, protein fraction 2
MCL = Momordica charantia seed lectin = Momordica RIP 2 charantia
lectin, MCL1 Momordica cochinchinensis Cochinin B, Momorcochin,
Momorcochin-S RIP 1 Spreng. Siraitia grosvenorii (Swingle)
Momorgrosvin RIP 1 C. Jeffrey ex A. M. Lu & Zhi Y. Zhang [Syn.:
Momordica grosvenorii Swingle] Sechium edule (Jacq.) Sw. Sechiumin
RIP 1 Sechium edule fruit lectin lectin Trichosanthes anguina L.
Trichoanguin RIP 1 SGSL lectin/RIP 2 like Trichosanthes cordata
TCA-I, TCA-II lectin Roxb. Trichosanthes cucumerina L. TCSL
lectin/RIP 2 candidate Trichosanthes .beta.-trichosanthin =
.beta.-TCS RIP 1 cucumeroides (Ser.) Maxim. Trichosanthes kirilowii
.alpha.-kirilowin, .beta.-kirilowin, TAP 29, TK-35, Trichobitacin,
RIP 1 Maxim. Trichokirin, Trichomislin = TCM, Trichosanthin =
Trichosanthes antiviral protein = TAP = TCS = .alpha.-
trichosanthin = .alpha.-TCS = GLQ223, Trichosanthin, .beta.-
trichosanthin = .beta.-TCS, .gamma.-trichosanthin = .gamma.-TCS
Trichokirin S1, S-Trichokirin, Trichosanthrip sRIP 1 TKL-1 =
Trichosanthes kirilowii lectin-1 lectin/RIP 2 candidate TK-I,
TK-II, TK-III, Trichosanthes kirilowii lectin lectin Trichosanthes
kirilowii Karasurin-A, Karasurin-B, Karasurin-C RIP 1 Maximovicz
var. japonica (Miquel) Kitamura Trichosanthes lepiniate
Trichomaglin RIP 1 Trichosanthes dioica Roxb. TDSL lectin/RIP 2
candidate Trichosanthes sp. Bac Kan Trichobakin RIP 1 8-98
Cupressaceae Thuja occidentalis L. Arborvitae RIP RIP candidate
Euphorbiaceae Croton tiglium L. Crotin I RIP 1 candidate Crotin 2
RIP 1 Euphorbia characias L. E. characias lectin lectin Suregada
multiflora Gelonin = GAP 31 RIP 1 (A. Juss.) Baill. [Syn.: Gelonium
multiflorum A. Juss.] Hura Crepitans L. Hura crepitans RIP, Hura
crepitans RIP-5 RIP 1 Hura crepitans latex lectin RIP 2 Crepitin,
Hurin, Hura crepitans seed lectin lectin Jatropha curcas L. Curcin,
Curcin 2, Curcin-L, Jc-SCRIP RIP 1 Manihot palmata Mull. Arg.
Mapalmin RIP 1 Manihot esculenta Crantz. Manutin 1, Manutin 2 RIP 1
[Syn.: Manihot utilissima Pohl] Ricinus communis L. Ricin =
crystalline Ricin = Ricin D, Ricin E, RCA = RIP 2 Ricinus communis
agglutinin = RCAI = RCA120 = R. communis hemagglutinin = RCB-PHA I,
RCAII = RCA60 = RCB-PHA II Ricinus communis, USA Ricin 1, Ricin 2,
Ricin 3 RIP 2 Ricinus communis, India Ricin I, Ricin II, Ricin III
RIP 2 Ricinus sanguienus, France Ricin.sub.11, Ricin.sub.12,
Ricin.sub.2 RIP 2 Fabaceae Abrus precatorius L. Abrin, Abrin-a =
Abrin C = Abrin-III, Abrin-b, Abrin-c = RIP 2 Abrin A = Abrin-I,
Abrin-d, Abrin-II, APA = Abrus precatorius agglutinin = Abrus
lectin = AAG, APA-I, APA-II Abrus pulchellus Thwaites Pulchellin,
Pulchellin PI, Pulchellin PII, Pulchellin PIII RIP 2 Pisum sativum
subsp. .alpha.-pisavin, .beta.-pisavin RIP 1 sativum L. [Syn.:
Pisum sativum var. arvense (L.) Poir.] Pisum sativum var. Sativin
RIP 1 candidate macrocarpon Iridaceae Iris hollandica var.
Professor IrisRIP = IRIP, IrisRIP.A1, IrisRIP.A2, IrisRIP.A3 RIP 1
Blaauw IRA, IRAb, IRAr RIP 2 Lamiaceae Clerodendrum aculeatum (L.)
CA-SRI RIP 1 candidate Schltdl. Clerodendrum inerme (L.) CIP-29 RIP
1 Gaertn. CIP-34 RIP 1 candidate Leonurus japonicus Houtt. Leonurin
RIP candidate Lauraceae Cinnamomum bodinieri H. Bodinierin RIP 2
Lev. Cinnamomum camphora (L.) Camphorin RIP 1 J. Presl Cinnamomin,
Cinnamomin 1, Cinnamomin 2, RIP 2 Cinnamomin 3 Cinphorin sRIP 2
Cinnamomum Porrectin RIP 2 parthenoxylon (Jack) Meisn. [Syn.:
Cinnamomum porrectum (Roxb.) Kosterm.] Malvaceae Abelmoschus
esculentus Abelesculin RIP 1 (L.) Moench Nyctaginaceae Boerhaavia
diffusa L. Boerhaavia inhibitor RIP 1 candidate Bougainvillea
spectabilis BAP I, Bouganin = Bougainvillea RIP I RIP 1 Willd.
Bougainvillea .times. buttiana cv. BBP-24, BBP-28 RIP 1 Enid
Lancester Bougainvillea .times. buttiana cv. BBAP1 RIP 1 Mahara
Mirabilis expansa (Ruiz & ME1, ME2 RIP 1 Pav.) Standl.
Mirabilis jalapa L. MAP, MAP-2, MAP-3, MAP-4, MAP-S RIP 1 Olacaceae
Malania oleifera Chun & Malanin lectin/RIP 2 S. K. Lee
candidate Ximenia americana L. Riproximin = Rpx, Rpx-I, Rpx-II RIP
2 Passifloraceae Adenia digitata (Harv.) Engl. Modeccin = Modeccin
4B, Modeccin 6B RIP 2 Adenia ellenbeckii Harms A. ellenbeckii
lectin RIP 2 candidate Adenia fruticosa Burtt Davy A. fruticosa
lectin lectin Adenia glauca Schinz A. glauca lectin RIP 2 candidate
Adenia goetzei Harms A. goetzei lectin RIP 2 (unresolved name)
Adenia keramanthus Harms A. keramanthus lectin RIP 2 candidate
Adenia lanceolata Engl. Lanceolin RIP 2
Adenia racemosa W. J. de A. racemosa lectin lectin Wilde Adenia
spinosa Burtt Davy A. spinosa lectin RIP 2 candidate Adenia
stenodactyla Harms Stenodactylin RIP 2 Adenia venenata Forssk. A.
venenata lectin RIP 2 candidate Adenia volkensii Harms Volkensin
RIP 2 Phytolaccaceae Phytolacca americana L. .alpha.-PAP, PAP =
Phytolacca americana protein = RIP 1 pokeweed antiviral protein,
PAP-I, PAP-II, PAP-III, PAP-C, PAP-H, PAP-R, PAP-S, PAP-S1, PAP-S2
Phytolacca dioica L. Diocin 1, Diocin 2, PD-L1, PD-L2, PD-L3,
PD-L4, PD- RIP 1 S1, PD-S2, PD-S3 Phytolacca dodecandra
Dodecandrin, Dodecandrin C RIP 1 L'Her. Phytolacca heterotepala
Heterotepalin 4, Heterotepalin 5b RIP 1 H. Walter Phytolacca
insularis Nakai Insularin = PIP = Phytolacca insularis antiviral
protein, RIP 1 PIP2 = P. insularis antiviral protein 2 Poaceae
Hordeum vulgare L. Barley toxin = Barley translation inhibitor =
Barley RIP 1 Protein Synthesis Inhibitor = BPSI = RIP 30, Barley
toxin I = Barley translation inhibitor I, Barley toxin II = Barley
translation inhibitor II = Barley Protein Synthesis Inhibitor II =
BPSI II, Barley toxin III = Barley translation inhibitor III, JIP60
Oryza sativa L. Oryza sativa RIP RIP 1 Secale cereale L. RPSI RIP 1
Triticum aestivum L. Tritin, Tritin 1, Tritin 2, Tritin 3,
Tritin-S, Tritin-L RIP 1 Zea mays L. b-32 = maize RIP = maize
proRIP1, Maize proRIP2 RIP 3/peculiar RIP 1 Ranunculaceae Eranthis
hyemalis (L.) EHL RIP 2 Salisb. Santalaceae Phoradendron
californicum PCL RIP 2 Nutt. Viscum album L. HmRip, HmRip 1, HmRip
2, HmRip 3, HmRip 4 RIP 2 (Himalayan mistletoe) Viscum album L.
ML-I = Mistletoe lectin I = Viscumin = Eu-ML = EML-1 = RIP 2
(European mistletoe) VAA-I, ML-II = Mistletoe lectin II = VAA-II,
ML-III = Mistletoe lectin III = VAA-III Viscum articulatum Burm. f.
Articulatin-D RIP 2 Viscum coloratum (Kom.) KML, KML-C, KML-IIL,
KML-IIU, VCA RIP 2 Nakai [Syn.: Viscum album subsp. coloratum Kom.]
Solanaceae Nicotiana tabacum L. CIP31 RIP-like protein TRIP RIP 1
candidate Thymelaeaceae Phaleria macrocarpa P. macrocarpa RIP RIP
candidate (Scheff.) Boerl. *Schrot J, Weng A, Melzig M F, et al.
Ribosome-inactivating and related proteins. Toxins (Basel). 2015
May 8; 7(5): 1556-615.
[0496] An aspect of the invention relates to a conjugate comprising
or consisting of an antibody and an antisense oligonucleotide such
as an antisense BNA, covalently linked together. In FIG. 1-5, the
gene-silencing activity of such a conjugate is depicted (in vivo
test in an animal tumor model). Reference is also made to the
Examples section.
[0497] An aspect of the invention relates to a combination of a
first composition comprising a conjugate comprising or consisting
of an antibody and an antisense oligonucleotide such as an
antisense BNA, covalently linked together, and a second composition
comprising free saponin of the invention (see Table A1, Scheme I).
In FIG. 1-7A and in FIG. 1-7C, the gene-silencing activity of such
a conjugate is depicted (in vitro cell-based bioassay with human
tumor cells). Reference is also made to the Examples section.
[0498] An aspect of the invention relates to a pharmaceutical
combination comprising or consisting of a first composition
comprising a first conjugate comprising or consisting of an
antibody and an antisense oligonucleotide such as an antisense BNA,
and a second composition comprising a first conjugate comprising or
consisting of the same antibody and at least one saponin of the
invention. In FIG. 1-5, the gene-silencing activity of such a
conjugate is depicted (in vivo test in an animal tumor model). In
FIG. 8-5, the gene-silencing activity of such a conjugate is
depicted (in vitro cell-based bioassay with human tumor cells).
Reference is also made to the Examples section.
[0499] An aspect of the invention relates to a pharmaceutical
combination comprising or consisting of a fourth composition
comprising a fourth conjugate comprising or consisting of an
antibody and an antisense oligonucleotide such as an antisense BNA,
and a fifth composition comprising a first conjugate comprising or
consisting of a different antibody and at least one saponin of the
invention. In FIG. 10-6A and in FIG. 10-6C, the gene-silencing
activity of such a conjugate is depicted (in vitro cell-based
bioassay with human tumor cells). Reference is also made to the
Examples section.
[0500] An aspect of the invention relates to a conjugate comprising
or consisting of an antisense oligonucleotide such as an antisense
BNA, covalently linked to at least one saponin of the invention. In
FIG. 1-3, the gene-silencing activity of such a conjugate is
depicted (in vitro cell-based bioassay with human tumor cells).
Reference is also made to the Examples section.
[0501] An aspect of the invention relates to a conjugate comprising
or consisting of an antisense oligonucleotide such as an antisense
BNA, covalently coupled to a polymeric scaffold such as a dendron
such as a G4-dendron, wherein the polymeric scaffold is covalently
conjugated with one or more saponin molecules of the invention,
such as four saponin molecules. In FIG. 1-3, the gene-silencing
activity of such a conjugate is depicted (in vitro cell-based
bioassay with human tumor cells). Reference is also made to the
Examples section.
[0502] An aspect of the invention relates to a conjugate comprising
or consisting of an antibody such as a monoclonal antibody with
specificity for a tumor marker or tumor-cell receptor, covalently
linked to at least one antisense oligonucleotide molecule such as
antisense BNA, and covalently linked to at least one saponin
molecule of the invention. In FIG. 2-4, the gene-silencing activity
of such a conjugate is depicted (in vivo test in an animal tumor
model). Reference is also made to the Examples section.
[0503] An aspect of the invention relates to a conjugate comprising
or consisting of an antibody such as a monoclonal antibody with
specificity for a tumor marker or tumor-cell receptor, covalently
linked to at least one antisense oligonucleotide molecule such as
antisense BNA via a tri-functional linker such as the linker of
Scheme II, and covalently linked to at least one saponin molecule
of the invention via the same tri-functional linker. In FIG. 1-1,
the gene-silencing activity of such a conjugate is depicted (in
vivo test in an animal tumor model). Reference is also made to the
Examples section.
[0504] An aspect of the invention relates to a therapeutic
combination consisting or comprising of a eighth composition
comprising a conjugate comprising or consisting of an antibody,
preferably a monoclonal antibody with specificity for a tumor
marker or tumor-cell receptor, covalently linked to at least one
saponin molecule of the invention, preferably via at least one
linker, preferably at least one cleavable linker, cleavable under
physiological acidic conditions, and further comprising a ninth
composition comprising an antisense oligonucleotide such as an
antisense BNA molecule. In FIG. 6-2, the gene-silencing activity of
such a conjugate is depicted (in vivo test in an animal tumor
model). In FIG. 5-2A and FIG. 5-2C, the gene-silencing activity of
such a conjugate is depicted (in vitro cell-based bioassay with
human tumor cells). Reference is also made to the Examples
section.
[0505] An aspect of the invention relates to any of the
aforementioned conjugates or compositions or therapeutical
combinations, for use as a medicament.
[0506] An aspect of the invention relates to any of the
aforementioned conjugates or compositions or therapeutical
combinations, for use in the treatment or prophylaxis of a
cancer.
Of course, as said before, any and all of a, b, c, d, e, f, g, h,
I, j, k, m, n, p, q, r, s, t, u, v, w and/or x have the value in
accordance with each individual embodiment and aspect of the
invention for any and all of the aforementioned aspects and
embodiments according to the invention. In addition,
(tri-functional) linkers L1, L2, L4, L5, L6, L8, L9 and/or L10, if
present in a molecule or conjugate or moiety of the invention, are
the (tri-functional) linkers as indicated for each and any of the
aforementioned aspects and embodiments of the invention, as is
readily appreciated by the skilled person. The oligomeric or
polymeric scaffolds L3 and/or L7, if present in a molecule or
conjugate or moiety of the invention, are the oligomeric or
polymeric scaffolds as indicated for each and any of the
aforementioned aspects and embodiments of the invention, as is also
readily appreciated by the skilled person. Furthermore, the first
ligand A1 and the first effector moiety B1, if present, and the
second ligand A2 and the second effector moiety B2, if present, and
the first effector moiety A1 and the first ligand B1, if present,
and the second effector moiety A2 and the second ligand B2, if
present, are the selected and indicated ligands and effector
moieties, as disclosed for the first, second, third, fourth, fifth,
and sixth series of embodiment and aspects of the invention, and
all further embodiments and aspects of the invention, outlined here
above. Saponin C is any one or more of the saponins referred to and
listed in any of the aforementioned aspects and embodiments of the
invention, in particular one or more saponins selected from Scheme
I and/or Table A1.
[0507] The invention is further illustrated by the following
examples, which should not be interpreted as limiting the present
invention in any way.
EXAMPLES
Example A--Treating a Mammalian Tumor-Bearing Animal with a
Conjugate of the Invention in Combination with an ADC Results in
Survival and Tumor Regression
[0508] Female Balb/c nude mice were injected subcutaneously with a
suspension of human A431 tumor cells. Under the skin of the mice, a
human epidermal carcinoma developed in the xenograft animal tumor
model. After injection of the tumor cells, the xenograft tumor was
allowed to develop to a size of approximately 170-180 mm.sup.3. The
A431 tumor cells have the following characteristics: high EGFR
expressors, medium CD71 expressors, low HER2 expressors.
[0509] In Table A, the results of the treatment of control mice and
tumor-bearing mice are presented. Tumor-bearing mice were treated
with the indicated antibodies directed to either human Her2/neu,
human EGFR, or human CD71, which are cell-surface receptors on the
xenograft tumor. Cetuximab was covalently conjugated with saponin
SO1861. The SO1861 was first provided with the linker EMCH
(N-.epsilon.-maleimidocaproic acid hydrazide), which EMCH is a
maleimide-and-hydrazide crosslinker for covalently conjugating
sulfhydryls (reduced cysteines of the antibody)) to carbonyls
(aldehyde or ketones; here the carbonyl of the aldehyde at position
C-23 of the saponin). The saponin-EMCH was covalently coupled to
reduced cysteines of the Cetuximab, forming a covalent thio-ether
bond between the EMCH and the cysteine side chain. The ADCs
trastuzumab-saporin (covalent conjugate) and anti-CD71 mAb (OKT-9,
IgG)--saporin (covalent conjugate) were tested for their
tumor-attacking efficacy in the mice, measured as tumor volume in
time after start of the treatment with the ADCs. The dose of the
ADCs was sub-optimal in the tumor model. That is to say, from
previous experiments, it was established at which sub-optimal dose
of the ADCs no tumor-regression or arrest of tumor growth would be
observable.
TABLE-US-00006 TABLE A RESULTS OF TREATING A MAMMALIAN
TUMOR-BEARING ANIMAL WITH A CONJUGATE OF THE INVENTION IN
COMBINATION WITH AN ADC RESULTS IN SURVIVAL AND TUMOR REGRESSION
tumor size (volume in mm.sup.3 or `+` for growth, `-` for
regression, Treatment Patient/healthy and `stable` for growth nor
group animal treatment regression) 1 xenograft vehicle 2000
mm.sup.3 (death/euthanasia) 2 xenograft Trastuzumab-saporin 2000
mm.sup.3 (death/euthanasia) 3 xenograft Anti-CD71 mAb OKT-9 - 2000
mm.sup.3 (death/euthanasia) saporin (covalent conjugate) 4
xenograft Cetuximab-SO1861 2000 mm.sup.3 (death/euthanasia)
(covalent conjugate) 5 xenograft Cetuximab >170 mm.sup.3, but
<2000 mm.sup.3 (death/euthanasia) 6 xenograft
Trastuzumab-saporin Tumor regression from 180 mm.sup.3 (covalent
conjugate) + at the start of treatment back to Cetuximab-SO1861 80
mm.sup.3 (survival) (covalent conjugate) 7 xenograft Anti-CD71 mAb
OKT-9 - Tumor regression from 180 mm.sup.3 saporin (covalent at the
start of treatment back to conjugate) + Cetuximab- 40 mm.sup.3
(survival) SO1861 (covalent conjugate)
[0510] These results demonstrate that the combination therapy of an
ADC at a dose which is ineffective when treatment of tumor-bearing
mice with the ADC alone is considered (tumor growths, death of the
mice is not prevented (euthanasia)), with a conjugate of the
invention consisting of a tumor-cell specific receptor targeting
antibody covalently bound to a saponin, i.e. SO1861, the covalent
conjugate administered to the mice suffering from cancer, at a
non-effective dose when administered alone (tumor growths, death of
the mice is not prevented (euthanasia)), provides an efficient and
efficacious treatment regimen, expressed as tumors in regression
and prolonged survival of the treated animals (beyond the duration
of the experiment). The sub-optimal dose of ADC combined with a
covalently bound saponin-comprising conjugate of the invention
which has no anti-tumor activity when administered alone, thus
provide for an effective treatment option for cancer patients,
wherein a relative low dose of the ADC is efficacious. A lower dose
of ADC bears the promise of less risk for adverse events, or even
no side effects at all. In addition, the stimulatory effect of the
saponin-bearing conjugate of the invention when the efficacy of the
ADC is considered, shows that ADCs which previously have proven to
lack efficacy when tumor patient treatment is concerned, may gain
renewed attention and value, since ADC efficacy is improved in
combination therapy setting, as the current example demonstrated.
Reference is made to Table A2 and Table A3, summarizing ADCs which
were previously investigated in the human clinical setting, but
then were for some ADCs retracted from further clinical
investigation. Especially the ADCs for which clinical development
was terminated due to observed lack of efficacy and/or due to
occurrence of unacceptable adverse event are ADCs which may gain
renewed value for cancer patients when combined with a covalently
bound saponin-comprising conjugate of the invention, such as the
cetuximab-saponin tested.
Example B--Saponins Mixture of Quillaja saponaria Comprising QS-21,
with Endosomal/Lysosomal Escape Enhancing Activity
[0511] Scheme I displays the common molecular structure of a series
of QS-21 saponins (in part adapted from: Conrado Pedebos, Laercio
Pol-Fachin, Ramon Pons, Cilaine V. Teixeira Hugo Verli, Atomic
Model and Micelle Dynamics of QS-21 Saponin, Molecules 2014, 19,
3744-3760). A mixture of water-soluble saponins obtained from
Quillaja saponaria (Sigma-Aldrich, product No. S4521; Roth, Item
No. 6857; InvivoGen, product `Quit-A`) may be applied in the
endosomal/lysosomal escape enhancing conjugate, composition,
combination of the invention, based on endosomal/lysosomal escape
enhancing properties of at least one individual saponin present in
the mixture, e.g. QS-21, or based on a combination of two or more
of the saponins comprised by the mixture, such as QS-21 and
QS-7.
[0512] The inventors demonstrated that the mixture of saponins from
Quillaja saponaria at 2.5 microgram/ml dose was capable of
enhancing endosomal escape of dianthin, as tested with mammalian
tumor cells in a cell-based bioassay. The effector moiety exposed
to the cells was dianthin covalently coupled to the ligand EGF:
EGF-dianthin. Cells tested were tumor cell lines HeLa for free
saponins, and A431, MDA-MB-468, CaSki and A2058 for testing the
saponins when covalently coupled to cetuximab.
Example 1
[0513] A trifunctional linker scaffold was designed and produced
with specific chemical end groups (DBCO, TCO) for conjugation
(labile, (L) conjugation) with on one arm an SO1861 molecule and on
the other arm an antisense HSP27BNA oligo nucleotide (targeting and
inducing degradation of the onco-target hsp27 mRNA in cancer cells)
to produce SO1861-L-trifunctional linker-L-HSP27BNA (FIG. 16-1).
SO1861-L-trifunctional linker-L-HSP27BNA was conjugated with its
the third arm (maleimide) to the cysteine residues (Cys) anti-EGFR
antibody, cetuximab (cetuximab-Cys-(SO1861-L-trifunctional
linker-L-HSP27BNA).sup.4).
[0514] This scaffold comprising conjugate was tested in a A431
xenograph `nude` mouse tumor model for EGFR-mediated tumor targeted
gene silencing activity. Dosings started at day 12 when tumors
reached .about.170 mm.sup.3 in size and tumor samples were
collected at 72h after the first dosing and analysed for HSP27 gene
expression compared to cellular control mRNA expression (reference
genes). This revealed that 1 dosing of 25 mg/kg
cetuximab-Cys-(SO1861-L-trifunctional
linker-L-HSP27BNA).sup.3'.sup.7 resulted in a 40% reduction in
HSP27 gene expression in the tumors compared to single dosing of
cetuximab-(Cys-L-SO1861).sup.3,8 or
cetuximab-(Lys-L-HSP27BNA).sup.4 mono therapies (FIG. 1-1).
Compared to the vehicle control tumors a reduction of 25% gene
silencing was observed. This shows and enables that conjugated
SO1861 efficiently can induce targeted delivery of therapeutic
oligo nucleotides in tumors, in vivo.
[0515] To further strengthen this,
cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA DAR4).sup.4
was tested for enhanced HSP27 gene silencing in EGFR expressing
(A431), in vitro as illustrated in FIG. 2-1.
Cetuximab-Cys-(SO1861-L-trifunctional
linker-L-HSP27BNA).sup.3'.sup.7 efficiently induces HSP27 gene
silencing in A431 cells (IC50= . . . ) compared to
Cetuximab-(Lys-L-HSP27BNA).sup.4 or
Cetuximab-(Cys-L-SO1861).sup.3,8 alone (FIG. 2-1).
Example 2
[0516] 1 target 2-components system is the combination treatment of
mAb1-(dendron(SO1861)n)n and mAb1-effector as illustrated in FIG.
11-1 and whereas the 2 target 2-component system is the combination
of mAb1-(dendron(SO1861).sup.n).sup.n mAb2-effector as illustrated
in FIG. 12-1.
[0517] Dendron(-L-SO1861).sup.4 was conjugated to the anti-EGFR
antibody, cetuximab via cysteine residues (Cys) conjugation with a
DAR3,9, cetuximab-Cys-(dendron(-L-SO1861).sup.4).sup.3,9 and tested
for enhanced cell killing activity in combination with an anti-EGFR
antibody-protein toxin conjugate (cetuximab-saporin) in EGFR
expressing cells (MDA-MB-468).
Cetuximab-Cys-(dendron(-L-SO1861).sup.4).sup.3,9+10 pM
cetuximab-saporin efficiently induces toxin-mediated cell killing
in high EGFR expressing cells (IC50= . . . ), whereas this was not
induced by Cetuximab-Cys-(dendron(-L-SO1861).sup.4).sup.3,9 or
cetuximab (equivalent)+10 pM cetuximab-saporin or cetuximab (FIG.
3-1A). Similar experiments in cells that express low levels of EGFR
(HeLa) revealed no activity of
Cetuximab-Cys-(dendron(-L-SO1861).sup.4).sup.3,9 (FIG. 3-1C)
indicating that in the absence of sufficient EGFR receptor
expression, effective intracellular SO1861 concentrations are not
reached (threshold) to induce endosomal protein toxin escape and
toxin-mediated cell killing.
[0518] Next, dendron(-L-SO1861).sup.4 was conjugated to the
anti-HER2 antibody, trastuzumab via cysteine conjugation (Cys) with
a DAR4, trastuzumab-Cys-(dendron(-L-SO1861).sup.4).sup.4 and tested
for enhanced cell killing activity in combination with an anti-HER2
antibody-protein toxin conjugate (trastuzumab-saporin) in HER2
expressing cells (SK-BR-3).
trastuzumab-Cys-(dendron(-L-SO1861).sup.4).sup.4+50 pM
trastuzumab-saporin efficiently induce toxin-mediated cell killing
(IC50= . . . ), whereas this was not induced by
trastuzumab-Cys-(dendron(-L-SO1861).sup.4).sup.4 or trastuzumab
(equivalent)+50 nM trastuzumab-saporin or trastuzumab (FIG. 3-1B).
Similar experiments in cells that express low levels of HER2
(JIMT-1) revealed no activity of
Trastuzumab-Cys-(dendron(-L-SO1861).sup.4).sup.4 (FIG. 3-1D)
indicating that in the absence of sufficient HER2 receptor
expression, effective intracellular SO1861 concentrations are not
reached (threshold) to induce endosomal protein toxin escape and
toxin-mediated cell killing.
[0519] Next, Cetuximab-Cys-(dendron(-L-SO1861).sup.4).sup.3,9 or
Cetuximab-Lys-(dendron(-L-SO1861).sup.4).sup.4,4
(Lys=dendron(-L-SO1861).sup.4 conjugated to lysines of antibody)
was tested in combination with 10 pM CD71mab-saporin in a 2 target
2 components system in EGFR.sup.++/CD71.sup.+ cells (MDA-MB-468).
This showed for both conjugates a strong enhancement of the cell
killing activity (IC50= . . . IC50= . . . resp.), whereas this was
not induced by Cetuximab-Cys-(dendron(-L-SO1861).sup.4).sup.3,9 or
Cetuximab-Lys-(dendron(-L-SO1861).sup.4).sup.4,4 or cetuximab
(equivalent)+10 pM CD71mab-saporin or cetuximab (FIG. 4-1A).
Similar experiments in cells that express lower levels of EGFR
(CaSKi, EGFR.sup.+/CD71.sup.+) revealed reduced activity for both
cetuximab-Cys-(dendron(-L-SO1861).sup.4).sup.3,9 or
cetuximab-Lys-(dendron(-L-SO1861).sup.4).sup.4,4 (FIG. 4-1C)
compared to the activity in high expressors (FIG. 4-1A) indicating
that in cells with lower EGFR receptor expression levels, the
effective intracellular SO1861 concentrations is lower resulting in
reduced toxin-mediated cell killing activity.
[0520] Same experiment was performed with
trastuzumab-Cys-(dendron(-L-SO1861).sup.4).sup.4 or
trastuzumab-Lys-(dendron(-L-SO1861).sup.4).sup.47 in combination
with CD71mab-saporin on HER2.sup.++/CD71+(SK-BR-3) cell lines
revealing strong cell killing activity compared to the controls
(FIG. 4-16). When trastuzumab-Cys-(dendron(-L-SO1861).sup.4).sup.4
or trastuzumab-Lys-(dendron(-L-SO1861).sup.4).sup.4,7 was tested on
HER2.sup.+/-/CD71.sup.+ (JIMT-1) in combination with 10 pM
CD71mab-saporin no cell killing activity could be observed
indicating that in the absence of sufficient HER2 receptor
expression, effective intracellular SO1861 concentrations are not
reached (threshold) to induce endosomal protein toxin escape and
toxin-mediated cell killing.
[0521] Next,
trastuzumab-Cys-(dendron(-L-SO1861).sup.4).sup.4+trastuzumab-emtansine
(T-DM1, antibody-small molecule toxin conjugate) was tested for
enhanced cell killing activity in HER2 expressing cells (SK-BR-3).
No enhanced cell killing was observed with this combination,
compared to T-DM1 alone or T-DM1+equivalent trastuzumab, since the
endosomal membrane forms no barrier for small molecules to reach
the cytoplasm. (FIG. 5-1).
Example 3
Materials and Methods
Dendron(SO1861).sub.4-BNA Oligo Synthesis (FIG. 17-1)
[0522] HSP27BNA oligo disulfide (1.1 mg, 0.187 .mu.mol) was
dissolved in 20 mM NH.sub.4HCO.sub.3 with 1.0 mM TCEP (500 .mu.L)
and the mixture was shaken for 1 min and left standing at room
temperature. After 1 hour the reaction mixture was filtered by
using a centrifugal filter with a molecular weight cut-off of 3000
Da (14000.times.g for 30 min). The residue solution was diluted
with 20 mM NH.sub.4HCO.sub.3 with 1.0 mM TCEP (500 .mu.L) and the
resulting mixture was filtered again under the same conditions
described above. The residue solution was diluted with 20 mM
NH.sub.4HCO.sub.3/acetonitrile (3:1, v/v, 1.0 mL) and the resulting
mixture was added to dendron(SO1861)4-maleimide1 (3.54 mg, 0.375
.mu.mol) (FIG. 17-1). The reaction mixture was shaken for 1 min and
left standing at room temperature. After 10 min the reaction
mixture was subjected to preparative LC-MS..sup.4A Fractions
corresponding to the product were immediately pooled together,
frozen and lyophilized overnight to give the title compound (1.25
mg, 85%) as a white fluffy solid. Purity based on LC-MS 94%
[0523] LRMS (m/z): 1896 [M-8].sup.8-, 2167 [M-7].sup.7-
[0524] LC-MS r.t. (min): 3.77.sup.68
Results
[0525] HSP27BNA oligo, (antisense BNA oligo targeting the mRNA
transcript of the cancer target, heat shock protein 27 (HSP27BNA))
was conjugated to a dendron(-L-SO1861).sup.4
(HSP27BNA-dendron(-L-SO1861).sup.4, FIG. 17-1) and co-administrated
to A431 cancer cells. As readout, gene silencing of HSP27 mRNA in
A431 cells was determined. This revealed that
HSP27BNA-dendron(-L-SO1861).sup.4 treatment resulted in an
improvement of HSP27 gene silencing activity compared to the
HSP27BNA alone (FIG. 6-1).
Example 4
Methods
SO1861 Releasing Assay
[0526] To dendron(SO1861).sub.4-Cbz (0.05 mg) (FIG. 7-1) was added
50 .mu.L of solution containing water/acetonitrile/TFA (1.00
mL/1.00 mL/4 drops). The reaction mixture was shaken for 1 min and
left standing at room temperature. The SO1861 release was followed
overtime by using UPLC-MS..sup.4
Results
[0527] The release efficiency of the SO1861 molecules from the
dendron(-L-SO1861).sup.4 under acid conditions has been determined
(FIG. 7-1).
[0528] Next, dendron(-L-SO1861).sup.4 was tested for enhanced
delivery of a targeted toxin, EGFdianthin on EGFR expressing cells
(A431 and HeLa). This shows that dendron(L-SO1861).sup.4+10 pM
EGFdianthin can induce enhanced toxin-mediated cell killing (IC50 .
. . nM), whereas the `naked` dendron (Dendron(NEM).sup.4) or
dendron(-L-SO1861).sup.4 or Dendron(NEM).sup.4+10 pM EGFdianthin is
not showing enhanced cell killing at these concentrations (FIG.
8-1A, 8-1B).
Example 5
Materials and Methods
[0529] In our current work, we investigated a model scaffold
consisting of four molecular arms for saponin binding via a Schiff
base (imine) and one arm for click chemistry. The polymeric
structure (FIG. 19-1) is a pentavalent polyethylene glycol-based
dendrimer of the first generation (i.e. number of repeated
branching cycles) that was purchased from Iris Biotech GmbH
(Marktredwitz, Germany). The saponin (in this example SA1641) was
purified from a saponin composite raw extract from Gypsophila
species called Saponinum album obtained from Merck (Darmstadt,
Germany). The powdered raw extract (2.5 g) was hydrolyzed in water
(100 mL) with sodium hydroxide (0.2 g). The solution was stirred
for 20 h at 40.degree. C. and then supplemented with glacial acetic
acid until pH 5.0 was reached. To remove tannins, the solution was
shaken in a separatory funnel with 30 mL butanol. The aqueous phase
was recaptured and butanol extraction repeated two times. The
butanol phases were supplemented with anhydrous sodium sulfate,
filtered and pooled. Butanol was evaporated and the remaining
saponin powder resolved in 20% methanol to a final concentration of
30 mg/mL. After short sonication, different saponins were separated
by high performance liquid chromatography (HPLC). Tubes (excluding
column) were rinsed with warm water (40.degree. C.) at a flow of
1.5 mL/min and then including Eurospher RP-C18-column (5 .mu.m,
250.times.8 mm) with isopropanol (100%). Saponins were applied to
the column and eluted with a methanol gradient (20% methanol to 70%
methanol within 30 min at 1.5 mL/min in water supplemented with
0.01% trifluoroacetic acid followed by 70% methanol for further 60
min) (Sama et al, 2018). Aliquots of the fractions were analyzed
for their SA1641 content by electrospray ionization mass
spectrometry (ESI-MS). Fractions containing pure SA1641 were pooled
and methanol evaporated. The aqueous solution was frozen as a thin
film in a rotating round-bottom flask by use of dry ice. After
storage for 16 h at -80.degree. C., the sample was lyophilized. To
produce the scaffold as defined in the invention, the polymeric
structure (0.2 mM) and SA1641 (3.2 mM) were solved in water
(approx. pH 8) and equal volumes mixed and shaken for 24 h at
26.degree. C. Then sodium cyanoborohydride (NaCNBH.sub.3; 0,1 M)
was added in 4-fold molar excess referred to SA1641 and the sample
incubated for further 24 h. The structure was then verified by
ultra performance liquid chromatography (UPLC)/ESI-MS. The samples
were applied to a RP-C4-column and eluted with a methanol gradient
(25% methanol to 80% methanol within 15 min in water supplemented
with 0.01% trifluoroacetic acid followed by 80% methanol for
further 10 min). The fractions were analyzed by use of
LockSpray.TM. that is an ion source designed specifically for exact
mass measurement with electrospray ionization using
LC-time-of-flight (LC-TOF) mass spectrometers from Waters
Corporation.
Example 6
Materials and Methods
[0530] As an example for a pharmaceutical active substance, we used
the targeted toxin dianthin-Epidermal Growth Factor (dianthin-EGF).
The plasmid His-dianthin-EGF-pET11d (Weng et al, 2009) (100 ng) was
added to 20 .mu.L Escherichia coli Rosetta.TM. 2 (DE3) pLysS
Competent Cells (Novagen, San Diego, Calif., USA). Cells were
transformed by a heat-shock (30 min on ice, 90 s at 42.degree. C.
and 1 min on ice). Thereafter, 300 .mu.L lysogeny broth (LB) was
added and the suspension incubated for 1 h at 37.degree. C. while
shaking at 200 rpm. A preheated lysogeny broth agar plate with 50
pg/mL ampicillin was inoculated with 100 .mu.l bacteria suspension
and the plate incubated overnight at 37.degree. C. Lysogeny broth
(3 mL) with 50 pg/mL ampicillin was inoculated with a colony from
the plate and the bacteria were incubated for 8 h at 37.degree. C.
and 200 rpm. The suspension (50 .mu.L) was added to 500 mL of
lysogeny broth with 50 pg/mL ampicillin and incubated overnight at
37.degree. C. and 200 rpm. Subsequently, the volume was scaled-up
to 2.0 L and bacteria grew under the same conditions until an
optical density at wavelength 600 nm of 0.9 was reached.
Thereafter, protein expression was induced by the addition of
isopropyl .beta.-D-1-thiogalactopyranoside (IPTG) at a final
concentration of 1 mM. Protein expression lasted for 3 h at
37.degree. C. and 200 rpm. Finally, the bacterial suspension was
centrifuged at 5,000.times.g and 4.degree. C. for 5 min,
resuspended in 20 mL PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM
Na.sub.2HPO.sub.4, 1.47 mM KH.sub.2PO.sub.4) and stored at
-20.degree. C. until use. For purification, bacterial suspensions
were thawed and lysed by sonication. Lysates were centrifuged
(15,800.times.g, 4.degree. C., 30 min) and imidazole added to a
final concentration of 20 mM. The supernatant was incubated with 2
mL of Ni-nitrilotriacetic acid agarose under continuous shaking for
30 min at 4.degree. C. in the presence of 20 mM imidazole.
Subsequently, the material was poured into a 20-mL-column and
washed three times with 10 mL wash buffer (50 mM NaH.sub.2PO.sub.4,
300 mM NaCl, 20 mM imidazole) and dianthin-EGF eluted by
10-mL-portions of increasing concentrations of imidazole (31, 65,
125 and 250 mM) in wash buffer. Eluate fractions (2 mL) were
dialyzed overnight at 4.degree. C. against 2.0 L PBS. Desalted
dianthin-EGF was concentrated by an Amicon.RTM. Ultra-15 (10 kDa)
and the protein concentration quantified.
[0531] To introduce a suitable click chemistry group into
dianthin-EGF, alkyne-PEG.sub.5-N-hydroxysuccinimidyl ester in
8-fold molar excess referred to dianthin-EGF was solved in dimethyl
sulfoxide and added to 9 volumes of dianthin-EGF (1 mg in 0.2 M
NaH.sub.2PO.sub.4/Na.sub.2HPO.sub.4, pH 8). After incubation at
room temperature for 4 h, non-bound alkyne was separated by use of
a PD10 column (GE-Healthcare, Freiburg, Germany). Click chemistry
with the polymeric structure was conducted by copper(I)-catalyzed
alkyne-azide cycloaddition. Alkyne-dianthin-EGF (0.02 mM),
dendrimer (0.05 mM), CuSO4 (0.1 mM),
tris(3-hydroxypropyltriazolylmethyl)amine (0.5 mM) and sodium
ascorbate (5 mM) were incubated under gentle agitation for 1 h at
room temperature in 0.1 M NaH.sub.2PO.sub.4/Na.sub.2HPO.sub.4, pH
8. Low molecular mass substances were then separated using a PD10
column.
[0532] To test the efficacy of the invention, we conducted a
viability assay with HER14 cells. These cells are fibroblasts
stably transfected with the human epidermal growth factor receptor
and therefore target cells for the targeted toxin dianthin-EGF.
HER14 cells (2,000 cells/100 .mu.L/well) were seeded into wells of
96-well-cell culture plates and incubated for 24 h in DMEM medium
supplemented with 10% fetal calf serum and 1%
penicillin/streptomycin at 37.degree. C., 5% CO.sub.2 and 98%
humidity. The different test substances (see results and FIG. 21-1)
were then added in triplicates in a volume of 25 .mu.L and
supplemented with further 25 .mu.L of medium. After an incubation
of 72 h, 30 .mu.L
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (0.5
mg/mL in water) was added per well and incubated for 2 h.
Thereafter, the medium was carefully removed and replaced by an
aqueous solution containing 10% (v/v) isopropanol, 5% (w/v) sodium
dodecyl sulfate and 400 mM HCl, and incubated for 5 min.
Solubilized formazan was photometrically quantitated at 570 nM in a
microplate reader (Spectra MAX 340 PC, Molecular Devices,
Sunnyvale, Calif., USA). Untreated cells were normalized to 1 and
all samples referred to the untreated control. Significance was
determined by unpaired two-sample t-tests.
Results
[0533] The polymeric structure, in the example a pentameric
dendrimer (pentrimer), does not have any cytotoxic effect on the
target cells, neither in absence nor in presence of SA1641 (FIG.
21-1, column 2 and 3). In the absence of the scaffold, the targeted
toxin (dianthin-EGF) shows half maximal toxicity at a concentration
of 0.1 nM (column 4). In the presence of SA1641 the same
concentration results in death of all cells indicating the general
ability of SA1641 to act as an enhancer of the endosomal escape
(column 5). The presence of the polymeric structure does not affect
the toxicity of dianthin-EGF neither in the presence nor in the
absence of SA1641 (columns 6 and 7), indicating that the scaffold
does not affect the toxicity of dianthin-EGF. To couple the model
polymeric structure via click chemistry to the example
pharmaceutically active substance of dianthin-EGF, the substance
had to be coupled with an alkyne group before. A manufacturer of a
pharmaceutically active substance can introduce the click position
during synthesis directly into the substance at a position of his
choice where the activity of the substance remains unaffected.
There was no additional loss of activity when clicking the
alkyne-modified pharmaceutically active substance to the polymeric
structure indicating that the polymeric structure itself was not
toxic.
Example 7
[0534] Considering available chemical groups for conjugation
reactions to the SO1861 molecule, four chemical groups have been
identified. The alcohols and diols of the sugar residues, the
aldehyde group on the triterpenoid backbone, the carboxylic acid on
one of the sugar residues (glucuronic acid), and the alkene group
on the triterpenoid backbone as highlighted in FIG. 19-1.
[0535] In view of the pros and cons of each identified chemical
group (Table 1), the aldehyde and alcohol groups are best suitable
for reversible conjugation reactions, while the alkene and the
carboxylic acid (glucuronic acid) are the groups best suitable for
irreversible/stable conjugation reactions. The aldehyde group
within the molecule structure of SO1861, however, is the most
suitable for reversible conjugation reactions over the alcohols. On
the one hand, because there is only one aldehyde present in the
structure that allows chemoselective reactions. On the other hand,
because the aldehyde can perform reversible conjugation reactions
with a variety of chemical groups such as amines, hydrazides, and
hydroxylamines forming acid-cleavable moieties like imines,
hydrazones, and oximes. This factor enables a freedom of choice
over the chemical group for the desired reversible conjugation
reaction. Contrary, the alcohols are good candidates for reversible
conjugation reaction via the formation of acetals and ketals as
well, but lack in chemoselectivity since they are present in a
large quantity on the glycosidic structure.
[0536] For the formation of an irreversible and stable bond the
carboxylic acid is the most suitable since it can form amides and
esters with the common tools used in peptide chemistry (e.g.
reaction with amines via carbodiimide mediated amide
formation).
TABLE-US-00007 TABLE 1 Functional groups that are available for
saponin conjugation reactions Functional Group Pros Cons Alcohol
Suitable for reversible acetal/ketal Acetal/ketal formation without
(Diols) formation chemoselectivity Suitable for ester formations
with Ester formation without activated carboxylic acids
chemoselectivity Aldehyde Suitable for chemoselective Not suitable
for acetal formation in reversible hydrazone formation with the
presence of unprotected hydrazides saponin sugar diols Suitable for
chemoselective reversible imine formation with amines Suitable for
chemoselective reversible oxime formation with hydroxylamines
Alkene Suitable for chemoselective Not suitable for reversible
irreversible radical reactions conjugation reactions Not suitable
for reactions involving a hydrogenation step Carboxylic Suitable
for chemoselective amide/ Not suitable for reversible acid ester
formation with amines and conjugation reactions under mild alcohols
after activation conditions
[0537] Regarding an ideal EMCH spacer length for conjugation to a
polymeric structure, computer simulation (PerkinElmer, ChemBio3D,
Ver. 13.0.0.3015) shows that the maleimide group on SO1861-EMCH is
located at the periphery of the molecule and thus should be
accessible for thiol bearing polymeric structures (FIG. 27-1).
[0538] As a polymeric structure, a G4-dendron (PFd-G4-Azide-NH-BOC,
Polymer Factory) with 16 functional amino end groups and an azido
group at the focal point was utilized for the conjugation to SO1861
(FIG. 24-1). The advantage of using a dendron over a dendrimer is
the focal point that the dendron structure is exhibiting.
[0539] Another approach for the development of a SO1861 scaffold
among the discussed polymer, and protein approach is the
poly(SO1861) approach. The idea of this approach is to generate a
polymer that consists of SO1861 molecules only, with pH sensitive
cleavable bonds that release the SO1861. In addition, the
poly(SO1861) should be able to perform conjugation reactions to
toxins and biopolymers. The main goal with this approach is to keep
it as simple and cost effective as possible. Since a protocol for
the generation of acid cleavable SO1861 has been developed already
(SO1861-EMCH approach) it would be interesting to see if it is
possible to polymerize the SO1861-EMCH through simple addition of a
polymerization initiator without further modifying the SO1861 or
identifying other conjugation sites on the SO1861 molecule. In the
past, several papers have discussed the polymerization of maleimide
groups by using radical initiators which attack the double bond of
the maleimide group and thus initiate a radical polymerization
along the double bonds of the maleimides. Since SO1861-EMCH reveals
a maleimide group in its structure this group could potentially be
explored for radical polymerization reactions to yield a
poly(SO1861) with acid cleavable function. If the polymerization
reaction has a reasonable reaction time the generated SO1861
polymers could be quenched with a radical quencher that not only
quenches the reaction but also generates a functional group for
toxin or biopolymer conjugation. Here, the system of ammonium
persulfate (APS) and tetramethylethylenediamine (TMEDA) is
indicated in an exemplary way as radical generator and
aminopropanethiol serves as a model radical quencher. Using
aminopropanethiol as a quencher exemplary, the generated amine
group could be specifically further modified to a click-able group
or being used to directly conjugate the poly(SO1861) to a
toxin.
[0540] Another approach for the development of a SO1861 scaffold is
the DNA approach. The idea of this approach is to utilize the
concept of the so-called DNA-origami (Kolb et al, 2004; Bird et al,
1988). DNA-origami as the polymeric or assembled polymeric
structure to conjugate saponins to it, can offer several inherent
advantages including stability, scalability, and precise control of
the final size and shape of the resulting DNA-saponin scaffold.
Since these DNA nanocarriers are comprised of natural DNA, they are
biocompatible and do not show toxicity to living cells, and can
ease the release of cargo from internal cellular compartments. The
multivalency of such a structure can further allow fine-tuning
targeting capabilities and high capacity for a variety of payloads
such as fluorophores and toxins. Thus, in this approach DNA strands
are identified that offer chemical functional groups on the 3' and
5' endings respectively, and that are able to hybridize only in
certain wanted areas of the sequence that allow a control over the
final shape of the construct. The chemical groups should be
utilized to couple saponins, for instance though a thiol-ene
reaction between the already developed SO1861-EMCH and a thiol
group on one of the 3' and 5' DNA strands. The complementary DNA
strand can offer a click function group that can be used for
coupling to a targeted toxin. The concept is illustrated in FIG.
23-1.
[0541] A similar approach is imaginable by using a specific peptide
sequence instead of DNA strands that is able to bind and release
saponins and that can be polymerized forming a large
poly(peptide)-like structure. In this approach, a peptide sequence
has been identified and purchased that has a length fitting the
calculated size of a SO1861-EMCH molecule, that offers a cysteine
residue in the middle of the sequence, and that obtains an amine
group at both the N-terminus and C-terminus. The cysteine residue
can be utilized to conjugate SO1861-EMCH via a thiol-ene reaction
of the maleimide group of SO1861-EMCH and the thiol group of the
cysteine residue. The two amine groups can be utilized to
polymerize the peptide-SO1861 conjugate with a suitable
crosslinker.
Example 8
SO1861-BNA Oligo Conjugation
[0542] HSP27 BNA oligo disulfide (1.10 mg, 0.187 .mu.mol) was
dissolved in 20 mM NH.sub.4HCO.sub.3 with 1.0 mM TCEP (500 .mu.L)
and the mixture was shaken for 1 min and left standing at room
temperature. After 1 hour the reaction mixture was filtered by
using a centrifugal filter with a molecular weight cut-off of 3000
Da (14000.times.g for 30 min). The residue solution was diluted
with 20 mM NH.sub.4HCO.sub.3 with 1.0 mM TCEP (500 .mu.L) and the
resulting mixture was filtered again under the same conditions
described above. The residue solution was diluted with 20 mM
NH.sub.4HCO.sub.3/acetonitrile (3:1, v/v, 1.00 mL) and the
resulting mixture was added to SO1861-EMCH (3.54 mg, 0.375
.mu.mol). The reaction mixture was shaken for 1 min and left
standing at room temperature. After 10 min the reaction mixture was
subjected to preparative LC-MS..sup.4A Fractions corresponding to
the product were immediately pooled together, frozen and
lyophilized overnight to give the title compound (1.25 mg, 85%) as
a white fluffy solid. Purity based on LC-MS 100%.
[0543] LRMS (m/z): 1561 [M-5].sup.5-, 1951 [M-4].sup.4-
[0544] LC-MS r.t. (min): 2.46.sup.6B
Dendron(SO1861).sup.4-BNA Oligo Conjugation
[0545] HSP27 BNA oligo disulfide (1.1 mg, 0.187 .mu.mol) was
dissolved in 20 mM NH.sub.4HCO.sub.3 with 1.0 mM TCEP (500 .mu.L)
and the mixture was shaken for 1 min and left standing at room
temperature. After 1 hour the reaction mixture was filtered by
using a centrifugal filter with a molecular weight cut-off of 3000
Da (14000.times.g for 30 min). The residue solution was diluted
with 20 mM NH.sub.4HCO.sub.3 with 1.0 mM TCEP (500 .mu.L) and the
resulting mixture was filtered again under the same conditions
described above. The residue solution was diluted with 20 mM
NH.sub.4HCO.sub.3/acetonitrile (3:1, v/v, 1.0 mL) and the resulting
mixture was added to dendron(SO1861)4-maleimide1 (3.54 mg, 0.375
.mu.mol). The reaction mixture was shaken for 1 min and left
standing at room temperature. After 10 min the reaction mixture was
subjected to preparative LC-MS..sup.4A Fractions corresponding to
the product were immediately pooled together, frozen and
lyophilized overnight to give the title compound (1.25 mg, 85%) as
a white fluffy solid. Purity based on LC-MS 94%
[0546] LRMS (m/z): 1896 [M-8].sup.8-, 2167 [M-7].sup.7-
[0547] LC-MS r.t. (min): 3.77.sup.6B
Cell Culture
[0548] Cells were seeded in DMEM (PAN-Biotech GmbH) supplemented
with 10% fetal bovine serum (PAN-Biotech GmbH) and 1%
penicillin/streptomycin (PAN-Biotech GmbH), in a 96 well plate at
5,000 c/w in 100 .mu.L/well and incubated overnight at 37.degree.
C. and 5% CO.sub.2. The next day samples were prepared in DMEM and
cells were treated.
Gene Silencing
[0549] RNA isolation and Qper analysis was performed according to
standard procedures and protocols.
HSP27 primers: F: R:
HSP27BNA Oligo
[0550] HSP27BNA(-thiol) oligos (sequence 5'-GGCacagccagtgGCG-3')
(Zhang et al., 2011) were ordered at Bio-synthesis Inc.
(Lewisville, Tex.)
Results
[0551] BNAoligo, antisense BNA oligo targeting the mRNA transcript
of the cancer target (upregulated in cancer cells), heat shock
protein 27 (HSP27BNA) was conjugated to SO1861-EMCH
(HSP27BNA-L-SO1861) or dendron(-L-SO1861).sup.4
(HSP27BNA-dendron(-L-SO1861).sup.4) and co-administrated to an A431
cancer cell line, according to the invention. As readout, gene
silencing of HSP27 mRNA in A431 cells was determined. This revealed
that HSP27BNA-L-SO1861 treatment resulted in an improvement of
HSP27 gene silencing activity compared to the HSP27BNA alone,
whereas the activity of HSP27BNA-dendron(-L-SO1861).sup.4 (4 SO1861
molecules/BNA) is even stronger (3-fold) compared to the gene
silencing activity of HSP27BNA alone (FIG. 1-3). This shows that
conjugation of 1 or more SO1861 molecules improves the gene
silencing activity of the therapeutic BNA oligo nucleotide due to
the enhancement of SO1861-mediated endosomal escape and cytoplasmic
delivery of the antisense BNA.
Example 9
[0552] SO1861 was conjugated (labile) via cysteine residues (Cys)
and dianthin (protein toxin) was conjugated (stable) via lysine
residues (Lys) to cetuximab (monoclonal antibody recognizing and
binding human EGFR), resulting in the production of:
Cetuximab-(Cys-L-SO1861).sup.3,9(Lys-S-dianthin).sup.2. The
conjugate was tested in a A431 (EGFR.sup.++) xenograph mouse tumor
model for EGFR tumor targeted cell killing as illustrated in FIG.
9-4. Dosings started at day 12 when tumors reached .about.150
mm.sup.3 in size and tumor volume was determined after every
dosing. Mice (n=3) were treated (intraperitoneal; i.p.; dose
escalation) at day 12: 0.5 mg/kg; dayl5: 1 mg/kg and day24: 1.5
mg/kg with cetuximab-(Cys-L-SO1861).sup.3,9(Lys-S-dianthin).sup.2
or cetuximab-(Lys-S-dianthin).sup.1'.sup.6. At day 26, compared to
the control group, tumor volume reduction could be observed in the
tumor bearing mice treated with
cetuximab-(Cys-L-SO1861).sup.3,9(Lys-S-dianthin).sup.2 (FIG. 1-4A).
This shows that labile conjugation of SO1861 to an antibody-protein
toxin (stable) conjugate can enhance the targeted therapeutic
efficacy of the tumor targeted antibody-protein toxin, thereby
inducing a more effective tumor targeted therapy.
[0553] Next, SO1861 was conjugated (labile) via cysteine residues
(Cys) and dianthin (protein toxin) was conjugated (labile) via
lysine residues (Lys) to cetuximab (monoclonal antibody recognizing
and binding human EGFR), resulting in the production of:
Cetuximab-(Cys-L-SO1861).sup.3,9(Lys-L-dianthin).sup.2. The
conjugate was tested in a A431 (EGFR.sup.++) xenograph mouse tumor
model for EGFR tumor targeted cell killing as illustrated in FIG.
9-4. Dosings started at day 12 when tumors reached .about.150
mm.sup.3 in size and tumor volume was determined after every
dosing. Mice (n=3) were treated (intraperitoneal; i.p.; dose
escalation) at day 12: 0.5 mg/kg; dayl5: 1 mg/kg, day24: 1.5 mg/kg
with cetuximab-(Cys-L-SO1861).sup.3,9(Lys-L-dianthin).sup.2 or
cetuximab-(Lys-L-dianthin).sup.16. This revealed that after 35 days
compared to the control, tumor bearing mice treated with
cetuximab-(Cys-L-SO1861).sup.3,9(Lys-L-dianthin).sup.2 showed tumor
growth inhibition (FIG. 1-4B). When mice (n=3; were treated
(intravenous, i.v.; dose escalation) day 12: 0.5 mg/kg; dayl5: 1
mg/kg, dayl8: 2 mg/kg, day24: 2.5 mg/kg with the
cetuximab-(Cys-L-SO1861).sup.3,9(Lys-L-dianthin).sup.2 according to
the invention also tumor growth inhibition could be observed
compared to the control (data represents 1 mice, since 2 mice died
during the treatments). This shows that labile conjugation of
SO1861 to an antibody-protein toxin (labile) conjugate can enhance
the targeted therapeutic efficacy of the tumor targeted
antibody-protein toxin, thereby inducing a more effective tumor
targeted therapy.
[0554] Next, SO1861-EMCH was conjugated via cysteine residues (Cys)
to cetuximab (monoclonal antibody recognizing and binding human
EGFR), with a DAR 3,9 and the antisense HSP27BNA oligo nucleotide
(targeting and inducing degradation of the onco-target hsp27 mRNA
(gene silencing) in cancer cells) via a labile (L) linker to the
lysine residues (Lys) of the antibody, with a DAR 1,8 resulting in
the production of
cetuximab-(Cys-L-SO1861).sup.3,9(Lys-L-HSP27BNA).sup.1,8.
Cetuximab-(Cys-L-SO1861).sup.3,9(Lys-L-HSP27BNA).sup.1,8was tested
in a A431 xenograph `nude` mouse tumor model for EGFR-mediated
tumor targeted HSP27 gene silencing, according to the invention as
illustrated in FIG. 10-4. Dosing started at day 12 when tumors
reached .about.150 mm.sup.3 in size and HSP27 mRNA expression was
determined. For this, tumor samples were collected at 72h after the
first dosing and analysed for HSP27 gene expression levels compared
to cellular control mRNA expression levels (reference genes). Tumor
bearing mice (n=3) treated (intraperitoneal; i.p.) with 30 mg/kg
cetuximab-(Cys-L-SO1861).sup.3,9(Lys-L-HSP27BNA).sup.1,8 showed
after 1 dosing 40% reduction in HSP27 mRNA expression in the tumors
compared to single dosing of cetuximab-(Cys-L-SO1861).sup.3,8 or
cetuximab-(Lys-L-HSP27BNA).sup.1'.sup.5 (FIG. 2-4). Compared to the
tumor of the vehicle control a reduction of 25% HSP27 gene
expression was observed. This shows and enables that conjugation of
SO1861 and HSP27BNA to the same targeting antibody, according to
the invention, efficiently induces SO1861-mediated enhanced
cytoplasmic delivery of a therapeutic antisense oligo nucleotide in
solid tumors of tumor bearing mice, inducing tumor targeted gene
silencing. In another example, a trifunctional linker scaffold was
designed and produced with 3 specific chemical end groups for
conjugation with SO1861 on one arm and the HSP27BNA on the other
arm to produce SO1861-L-trifunctional linker-L-HSP27BNA. Next,
SO1861-L-trifunctional linker-L-HSP27BNA was conjugated with its
third arm to cysteine residues (Cys) of the anti-EGFR antibody,
cetuximab (cetuximab-Cys-(SO1861-L-trifunctional
linker-L-HSP27BNA).sup.3'.sup.7) and tested in a A431 xenograph
`nude` mouse tumor model for EGFR-mediated tumor targeted gene
silencing activity, according to the invention as illustrated in
FIG. 11-4. Dosings started at day 12 when tumors reached .about.150
mm.sup.3 in size and HSP27 mRNA expression was determined. For
this, tumor samples were collected at 72h after the first dosing
and analysed for HSP27 gene expression levels compared to cellular
control mRNA expression levels (reference genes). This revealed
that 1 dosing of 30 mg/kg cetuximab-Cys-(SO1861-L-trifunctional
linker-L-HSP27BNA).sup.3'.sup.7 resulted in a 40% reduction in
HSP27 gene expression in the tumors compared to single dosing of 25
mg/kg cetuximab-(Cys-L-SO1861).sup.3,8 or 25 mg/kg
cetuximab-(Lys-L-HSP27BNA).sup.4 mono therapies (FIG. 3-4).
Compared to the vehicle control tumors, a reduction of 25% HSP27
gene expression was observed in tumor bearing mice treated with 1
dosing of cetuximab-Cys-(SO1861-L-trifunctional
linker-L-HSP27BNA).sup.37. This shows and enables that
cetuximab-Cys-(SO1861-L-trifunctional
linker-L-HSP27BNA).sup.3'.sup.7 efficiently induces SO1861-mediated
enhanced cytoplasmic delivery of a therapeutic antisense oligo
nucleotide in a solid tumor of tumor bearing mice, inducing
targeted gene silencing, in vivo.
Example 10
[0555] In another example according to the invention, SO1861
(labile) and the protein toxin, dianthin (labile or stable) were
conjugated to the HER2 targeting antibody, trastuzumab.
Trastuzumab-(Cys-L-SO1861).sup.3,8(Lys-L-dianthin).sup.1,7 or
trastuzumab-(Cys-L-SO1861).sup.3,8(Lys-S-dianthin).sup.1,7, were
produced and tested for enhanced cell killing in SK-BR-3
(HER2.sup.++) and MDA-MB-468 (HER2.sup.-) cells as illustrated in
FIG. 9-4. Both,
trastuzumab-(Cys-L-SO1861).sup.3,8(Lys-L-dianthin).sup.1,7
(IC50=0.8 nM) and
trastuzumab-(Cys-L-SO1861).sup.3,8(Lys-S-dianthin).sup.1,7
(IC50=0.8 nM) efficiently induces cell killing of SK-BR-3 cells
(HER2.sup.++) (FIG. 4-4A). This was not observed in SK-BR-3 cells
treated with trastuzumab, trastuzumab-(Lys-L-dianthin).sup.17,
trastuzumab-(Lys-S-dianthin).sup.17 or
trastuzumab-(L-SO1861).sup.3,8 alone (FIG. 4-4A). In MDA-MB-468
cells (HER2.sup.-) no cell killing activity can be observed for any
of the conjugates, according to the invention (FIG. 4-4B). This
shows that conjugation of SO1861 to an HER targeting
antibody-protein toxin conjugate, efficiently induces
SO1861-mediated enhanced cytoplasmic delivery of the protein toxin
in the target cell resulting in target cell death.
[0556] In another example according to the invention, SO1861
(labile) and the protein toxin, dianthin (labile or stable) were
conjugated to the EGFR targeting antibody, cetuximab.
Cetuximab-(Cys-L-SO1861).sup.3,9(Lys-L-dianthin).sup.2 or
cetuximab-(Cys-L-SO1861).sup.3,9(Lys-S-dianthin).sup.2, was tested
for enhanced cell killing in A431 cells (EGFR.sup.++) and A2058
cells (EGFR.sup.-) as illustrated in FIG. 9-4. Both,
cetuximab-(Cys-L-SO1861).sup.3,9(Lys-L-dianthin).sup.2 (IC50=0.3
nM) and cetuximab-(Cys-L-SO1861).sup.3,8(Lys-S-dianthin).sup.17
(IC50=0.3 nM) showed enhanced cell killing in A431 cells
(EGFR.sup.++) compared to cetuximab-(Lys-L-dianthin).sup.1'.sup.6
(IC50=2 pM), cetuximab-(Lys-S-dianthin).sup.1'.sup.6 (IC5=2 pM)
alone (FIG. 4-4C). In A2058 cells (EGFR.sup.-) the combination
according to the invention did not show any cell killing activity
(IC50>200 nM; FIG. 4-4D). This shows that conjugation of SO1861
to an EGFR targeting antibody-protein toxin conjugate, efficiently
enhances SO1861-mediated cytoplasmic delivery of the protein toxin
in the target cell resulting in enhanced target cell death.
Example 11
[0557] In another example according to the invention, SO1861
(labile) and the HSP27BNA oligo (labile) were conjugated to the
EGFR targeting antibody, cetuximab.
Cetuximab-(Cys-L-SO1861).sup.3,8(Lys-L-HSP27BNA).sup.3,8 was tested
for enhanced HSP27 gene silencing in A431 cells (EGFR.sup.++) and
A2058 (EGFR.sup.-) cells, according to the invention as illustrated
in FIG. 10-4.
Cetuximab-(Cys-L-SO1861).sup.3,8(Lys-L-HSP27BNA).sup.3,8
efficiently induces HSP27 gene silencing in A431 cells (IC50=3 nM)
compared to cetuximab, cetuximab-(Lys-L-HSP27BNA).sup.3'.sup.9 or
cetuximab-(Cys-L-SO1861).sup.3,8 alone
[0558] (FIG. 5-4A). In A2058 cells (EGFR.sup.-) no gene silencing
activity can be observed with
cetuximab-(Cys-L-SO1861).sup.3,8(Lys-L-HSP27BNA).sup.3,8
(IC50>100 nM; FIG. 5-4B). This shows and enables that
conjugation of SO1861 and HSP27BNA to the same targeting antibody,
according to the invention, efficiently induces SO1861-mediated
enhanced cytoplasmic delivery of a therapeutic antisense oligo
nucleotide in the target cells, inducing targeted gene
silencing.
[0559] In another example according to the invention, SO1861
(labile) and the HSP27BNA oligo (labile) were conjugated to the
HER2 targeting antibody, trastuzumab.
Trastuzumab-(Cys-L-SO1861).sup.3,8(Lys-L-HSP27BNA).sup.35 was
tested for enhanced HSP27 gene silencing in SK-BR-3 cells
(HER2.sup.++) cells, according to the invention as illustrated in
FIG. 10-4.
Trastuzumab-(Cys-L-SO1861).sup.3,8(Lys-L-HSP27BNA).sup.3,5
efficiently induces HSP27 gene silencing in SK-BR-3 cells (IC50=9
nM) compared to trastuzumab-(Lys-L-HSP27BNA).sup.4,4 alone (FIG.
6-4). This shows and enables that conjugation of SO1861 and
HSP27BNA to an HER2 targeting antibody, according to the invention,
efficiently induces SO1861-mediated enhanced cytoplasmic delivery
of a therapeutic antisense oligo nucleotide in the target cells,
inducing targeted gene silencing.
[0560] In another example, cetuximab-Cys-(SO1861-L-trifunctional
linker-L-HSP27BNA).sup.3'.sup.7 was tested for enhanced HSP27 gene
silencing in A431 (EGFR.sup.++) and A2058 (EGFR.sup.-) cells
according to the invention as illustrated in FIG. 11-4.
Cetuximab-Cys-(SO1861-L-trifunctional
linker-L-HSP27BNA).sup.3'.sup.7 efficiently induces HSP27 gene
silencing in A431 cells (IC50=2 nM) compared to
Cetuximab-(Lys-L-HSP27BNA).sup.4 or
Cetuximab-(Cys-L-SO1861).sup.3,7 alone (FIG. 7-4A). In A2058 cells
(EGFR.sup.-) gene silencing activity was only observed at high
(>80 nM) concentrations of Cetuximab-Cys-(SO1861-L-trifunctional
linker-L-HSP27BNA).sup.3,7 (IC50=100 nM; FIG. 7-4B). This shows and
enables that in high EGFR expressing cells
cetuximab-Cys-(SO1861-L-trifunctional
linker-L-HSP27BNA).sup.3'.sup.7 efficiently induces SO1861-mediated
enhanced cytoplasmic delivery of a therapeutic antisense oligo
nucleotide in the target cells, inducing targeted gene
silencing.
Example 12
[0561] FIG. 8-4A-D displays the relative cell viability when
trastuzumab (FIG. 8-4A), cetuximab (FIG. 8-4B) or T-DM1 (FIG.
8-4C), unconjugated protein toxins, saporin, dianthin and saporin
conjugated to a (non-cell binding) IgG antibody (FIG. 8-4D) are
administered to various cancer cell lines SK-BR-3, JIMT-1,
MDA-MB-468, A431, CaSki, HeLa, A2058.
[0562] Trastuzumab and cetuximab do not or hardly influence cell
viability when exposed to most of the cell lines, with some effect
on cell growth inhibition via blocking the function of the HER2
growth factor receptor when trastuzumab is exposed to SK-BR-3 cells
at relatively high dose and with some effect on cell growth
inhibition via blocking the function of the EGFR growth factor
receptor when cetuximab is exposed to MDA-MB-468 cells at
relatively high dose.
[0563] TDM-1, or ado-trastuzumab emtansine, is a targeted therapy
approved by the U.S. Food and Drug Administration to treat:
HER2-positive metastatic breast cancer that has previously been
treated with Herceptin (chemical name: trastuzumab) and taxane
chemotherapy; early-stage HER2-positive breast cancer after surgery
if residual disease was found after neoadjuvant (before surgery)
treatment with Herceptin and taxane chemotherapy. The TDM-1 is a
combination of Herceptin (Trastuzumab) and the chemotherapy
medicine emtansine. FIG. 8-4C shows that the TDM-1 results in
decreased cell viability for all cell lines tested at >1000 pM
concentrations
[0564] The free toxins saporin and dianthin and the toxin saporin
coupled to a control IgG with no affinity for any of the cell
surface molecules on the cell lines tested, do not or hardly have
any influence on cell viability over a wide range of concentrations
toxin tested, up to 100.000 pM (FIG. 8-4D).
Example 13 (Example 1 Invention 5)
[0565] The 1 target 2-components system (1T2C) is the combination
treatment of mAb1-protein toxin and mAb1-SO1861, as illustrated in
FIG. 13-5. SO1861-EMCH was conjugated via cysteine residues (Cys)
and HSP27BNA oligo was conjugated via lysine residues to cetuximab
(monoclonal antibody recognizing and binding human EGFR), both with
a DAR 4 resulting in the production of 2 conjugates:
cetuximab-(Cys-L-SO1861).sup.4 and
cetuximab-(Lys-L-HSP27BNA).sup.4. The combination of
cetuximab-(Cys-L-SO1861).sup.4 (intraperitoneal administration,
(i.p.)) and cetuximab-(Lys-L-HSP27BNA).sup.4 (intravenous
administration, (i.v.)) was tested in a A431 xenograph `mouse tumor
model for EGFR tumor targeted gene silencing activity. Dosings
started at day 12 when tumors reached .about.150 mm.sup.3 in size
and tumor samples were collected at 72h after the first dosing and
analysed for HSP27 gene expression compared to control gene mRNA
expression levels (reference genes). This revealed that 1 dosing of
50 mg/kg cetuximab-(Cys-L-SO1861).sup.4+25 mg/kg
cetuximab-(Lys-L-HSP27BNA).sup.4 resulted in a 50% reduction in
HSP27 gene expression in the A431 tumors compared to single dosing
of cetuximab-(Cys-L-SO1861).sup.4 or
cetuximab-(Lys-L-HSP27BNA).sup.4 mono therapies (FIG. 1-5).
Compared to the vehicle control tumors, a reduction of 40% HSP27
gene silencing was observed. This shows and enables that the
combination of cetuximab-conjugated SO1861+cetuximab-conjugated
HSP27BNA oligo, according to the 1T2C invention, induces efficient
targeted delivery of a therapeutic antisense oligo nucleotide in
the cytoplasm of solid tumor cells, thereby inducing tumor targeted
gene silencing, in vivo.
[0566] Next, SO1861-EMCH was conjugated via cysteine residues (Cys)
to trastuzumab (monoclonal antibody recognizing and binding human
HER2), with a DAR 4 resulting in the production of
trastuzumab-(Cys-L-SO1861).sup.4. The combination of
trastuzumab-(Cys-L-SO1861).sup.4 and trastuzumab-saporin
(trastuzumab protein toxin conjugate) was tested in a mouse tumor
model (patient derived xenograph tumor model, PDX) with high HER2
expression levels and resistant for trastuzumab mono therapy. The
combination, according to the 1T2C invention of 40 mg/kg
trastuzumab-(Cys-L-SO1861).sup.4 (intraperitoneal administration,
(i.p.))+0.03 (Dayl, 8)/0.02 (Day 15, 22, 30, 36,43) mg/kg
trastuzumab-saporin (intravenous administration, (i.v.)) revealed
strong tumor growth inhibition compared to the vehicle control and
the 40 mg/kg trastuzumab-(Cys-L-SO1861).sup.4 or 0.03/0.02 mg/kg
trastuzumab-saporin mono therapies (FIG. 2-5). Besides, in tumor
bearing mice that were treated with a lower dosing combination (40
mg/kg trastuzumab-(Cys-L-SO1861).sup.4+0.01 mg/kg
trastuzumab-saporin) no tumor growth inhibiting activity was
observed (FIG. 2-5). This shows and enables that the 1T2C
combination of trastuzumab conjugated SO1861+trastuzumab conjugated
protein toxin induces efficient targeted delivery of a therapeutic
protein toxin in the cytoplasm of solid tumor cells, thereby
inducing tumor cell death and tumor growth inhibition, in vivo.
Example 14
[0567] The 1 Target 2-Components System (1T2C) is the Combination
Treatment of mAb1-SO1861 and mAb1-Protein Toxin (FIG. 13-5)
[0568] SO1861-EMCH was conjugated via cysteine residues (Cys) to
cetuximab (monoclonal antibody recognizing and binding human EGFR),
with a DAR 3,7 (cetuximab-(Cys-L-SO1861).sup.3,7).
Cetuximab-(Cys-L-SO1861).sup.3,7 was titrated on a fixed
concentration of 10 pM cetuximab-saporin (cetuximab, conjugated to
the protein toxin, saporin) and targeted protein toxin mediated
cell killing on EGFR expressing cells (A431, EGFR++; CaSKi,
EGFR.sup.+) was determined. This revealed strong cell killing at
low concentrations of cetuximab-(Cys-L-SO1861).sup.3,7 (A431:
IC50=0.6 nM and Caski IC50=1 nM; FIG. 5A, 3-5B) whereas cetuximab,
cetuximab-(Cys-L-SO1861).sup.3,7 or cetuximab+10 pM
cetuximab-saporin could not induce any cell killing activity in
EGFR expressing cells. This shows that cetuximab conjugated SO1861
efficiently enhances endosomal escape of the cetuximab conjugated
protein toxin (at non-effective concentrations), thereby inducing
cell killing of EGFR expressing cells. The cell killing activity in
A431 is more effective compared to CaSki correlating with EGFR
expression levels in these cell lines. EGFR receptor binding
competition between both conjugates within the 1T2C is also
observed when cetuximab-(Cys-L-SO1861).sup.3,7 concentrations
increase, cell killing activity declines due to outcompeting
receptor binding and internalization of cetuximab-saporin (FIG.
3-5A, 3-5B).
[0569] Next, cetuximab-saporin was titrated on a fixed
concentration of 75 nM cetuximab-(Cys-L-SO1861).sup.3,7 and
targeted protein toxin mediated cell killing on EGFR expressing
cells was determined. This revealed that 75 nM
cetuximab-(Cys-L-SO1861).sup.3,7 in combination with low
concentrations cetuximab-saporin induced already efficient cell
killing in EGFR expressing cells (A431: IC50=0.4 pM; and CaSKi:
(IC50=2 pM; FIGS. 3-5C and 3-5D), whereas cetuximab-saporin alone
or cetuximab-saporin+75 nM cetuximab showed cell killing only at
high concentrations cetuximab-saporin (IC50=40 pM, IC50=1000 pM,
resp.) in both cell lines (FIG. 3-5C, 3-5D). All this shows that
relatively low concentrations of cetuximab-saporin can be effective
and induce cell killing only in combination with low
cetuximab-SO1861 concentrations in high EGFR expressing cells. The
receptor competition between both conjugates within the 1T2C system
is also observed in the cetuximab-toxin titration treatments when
the cell killing activity of cetuximab-saporin with and without 75
nM cetuximab was compared (FIG. 3-5C, 3-5D).
[0570] Next, cetuximab-(Cys-L-SO1861).sup.3,7 was titrated on a
fixed concentration of 10 pM cetuximab-saporin and targeted protein
toxin mediated cell killing on low EGFR expressing cells or cells
without EGFR expression (HeLa, EGFR.sup.+/-; A2058, EGFR.sup.-) was
determined. Cells with low (HeLa) or no (A2058) EGFR expression
were not sensitive at all for any combination of
cetuximab-(Cys-L-SO1861).sup.3,7+10 pM cetuximab-saporin (HeLa:
IC50>1000 nM; A2058: IC50>1000 nM; FIG. 4-5A, 4-5B). This
shows that in the absence of sufficient EGFR receptor expression,
effective intracellular delivered SO1861 concentrations are not
optimal (threshold) to induce endosomal protein toxin escape and
toxin-mediated cell killing. Next, cetuximab-saporin was titrated
on a fixed concentration of 75 nM cetuximab-(Cys-L-SO1861).sup.3,7
and targeted protein toxin mediated cell killing on low (HeLa) or
no (A2058) EGFR expressing cells was determined. Low EGFR
expressing cells (HeLa) showed cell killing only at high
cetuximab-saporin concentrations in combination with 75 nM
cetuximab-(Cys-L-SO1861).sup.3,7 (HeLa: IC50=60 pM), FIG. 4-5C),
whereas A2058 cells (EGFR.sup.-) are not sensitive at any of the
tested concentrations (A2058: IC50>10.000 pM; FIG. 4-5D). All
this shows that cells with low or no EGFR receptor expression are
not susceptible for the combination of
cetuximab-(Cys-L-SO1861).sup.3,7+cetuximab-saporin, due to a lack
of sufficient EGFR receptor that facilitates the antibody-mediated
delivery of sufficient SO1861 within the endolysosomal
compartments, to facilitate the escape of the protein toxin.
[0571] Next, SO1861-EMCH was conjugated via cysteine residues (Cys)
to trastuzumab (monoclonal antibody recognizing and binding human
HER2, with a DAR 4, (trastuzumab-(Cys-L-SO1861).sup.4).
Trastuzumab-(Cys-L-SO1861).sup.4 was titrated on a fixed
concentration of 50 pM trastuzumab-saporin (trastuzumab, conjugated
to the protein toxin, saporin) and targeted protein toxin mediated
cell killing on HER2 expressing cells (SK-BR-3, HER2.sup.++) was
determined. This revealed strong cell killing at low concentrations
of trastuzumab-(Cys-L-SO1861).sup.4 (SK-BR-3: IC50=0.8 nM; FIG.
5-5A) whereas equivalent concentrations trastuzumab,
trastuzumab-(Cys-L-SO1861).sup.4 or trastuzumab+50 pM
trastuzumab-saporin could not induce any cell killing activity in
HER2 expressing cells. This shows that trastuzumab conjugated
SO1861 efficiently enhances endosomal escape of the trastuzumab
conjugated protein toxin (at non-effective concentrations), thereby
inducing cell killing of HER2 expressing cells. The receptor
competition between both conjugates within the 1T2C is also
observed when trastuzumab-(Cys-L-SO1861).sup.4 concentrations
increase, cell killing activity declines due to outcompeting
receptor binding and internalization of trastuzumab-saporin (FIG.
5-5A).
[0572] Next, trastuzumab-saporin was titrated on a fixed
concentration of 2.5 nM trastuzumab-(Cys-L-SO1861).sup.4, according
to the invention and targeted protein toxin mediated cell killing
on HER2 expressing cells was determined. This revealed that 2.5 nM
trastuzumab-(Cys-L-SO1861).sup.4 in combination with low
concentrations trastuzumab-saporin induced already efficient cell
killing in HER2 expressing cells (SK-BR-3: IC50=2 pM; FIG. 5-5B),
whereas trastuzumab-saporin alone or trastuzumab-saporin+2.5 nM
trastuzumab showed cell killing only at high concentrations
trastuzumab-saporin (FIG. 5-5B). All this shows that relatively low
concentrations of trastuzumab-saporin can be effective and induce
cell killing only in combination with low
trastuzumab-(Cys-L-SO1861).sup.4 concentrations in high HER2
expressing cells.
[0573] Next, trastuzumab-(Cys-L-SO1861).sup.4 was titrated on a
fixed concentration of 50 pM trastuzumab-saporin, according to the
invention and targeted protein toxin mediated cell killing on low
HER2 expressing cells (A431 HER2.sup.+/-) or cells without HER2
expression (JIMT-1: HER2.sup.+; MDA-MB-468: HER2.sup.-) was
determined. Cells with low or no HER2 expression were not sensitive
at all for any combination of trastuzumab-(Cys-L-SO1861).sup.4+50
pM trastuzumab-saporin (JIMT-1: IC50>1000 nM; MDA-MB-468:
IC50>1000 nM; FIG. 6-5A, 6-5B). This shows that in the absence
of sufficient HER2 receptor expression, effective intracellular
delivered SO1861 concentrations are not optimal (threshold) to
induce endosomal protein toxin escape and toxin-mediated cell
killing. Next, trastuzumab-saporin was titrated on a fixed
concentration of 2.5 nM trastuzumab-(Cys-L-SO1861).sup.4 and
targeted protein toxin mediated cell killing on low or no HER2
expressing cells was determined. Low HER2 expressing cells (JIMT-1)
showed cell killing only at high trastuzumab-saporin concentrations
in combination with 2.5 nM trastuzumab-(Cys-L-SO1861).sup.4
(JIMT-1: IC50>10.000 pM; FIG. 6-5C), whereas MDA-MB-468 cells
(HER2.sup.-) are not sensitive at any of the tested concentrations
(MDA-MB-468: IC50>10.000 pM; FIG. 6-5D).
[0574] All this shows that cells with low or no HER2 receptor
expression are not susceptible for the combination of
trastuzumab-(Cys-L-SO1861).sup.3,7+trastuzumab-saporin, due to a
lack of sufficient HER2 receptor that facilitates the
antibody-mediated delivery of sufficient SO1861 within the
endolysosomal compartments, to facilitate the escape of the protein
toxin.
Example 15
[0575] In order to show that the activity of the 1T2C system is
driven by the acidification of the endolysosomal compartments, the
1T2C system, according to the invention was tested in combination
with an endosomal acidification inhibitor, chloroquine.
Trastuzumab-saporin was titrated in combination with 5 nM
trastuzumab-(Cys-L-SO1861).sup.4 in combination with or without
chloroquine. Trastuzumab-saporin+5 nM
trastuzumab-(Cys-L-SO1861).sup.4 showed a strong cell killing
activity in high HER2 expressing cells (SK-BR-3, HER2.sup.++;
IC50=0.2 pM;), however, trastuzumab-saporin+5 nM
trastuzumab-(Cys-L-SO1861).sup.4+0.5 pM chloroquine resulted in
strong inhibition of the 1T2C cell killing activity in SK-BR-3
(HER2.sup.++) cells (IC50=40 pM). This shows that activity of the
antibody conjugated SO1861 is reduced/blocked when acidification of
endoslysomes is prohibited (FIG. 7-5A). Same results were derived
with the 1T2C combination, according to the invention of
cetuximab-saporin+5 nM cetuximab-(Cys-L-SO1861).sup.3,8 (IC50=1 pM)
compared with cetuximab-saporin+5 nM
cetuximab-(Cys-L-SO1861).sup.3,8+0.5 pM chloroquine (IC50=200 pM)
in EGFR expressing cells (A431, EGFR++; FIG. 7-5B).
Example 16
[0576] The 1 target 2-components system (1T2C) can also be the
combination treatment of mAb1-SO1861 and mAb1-antisense BNA oligo
nucleotide as illustrated in FIG. 14-5. For this we used an
antisense BNA oligonucleotide against the mRNA of a cancer specific
target gene (upregulated in cancer cells), heat shock protein 27
(HSP27). Upon release into the cytoplasm the antisense BNA
recognizes and binds the mRNA encoding for HSP27, targeting the
mRNA for destruction thereby depleting the HSP27 mRNA expression
within the cancer cell. HSP27BNA was conjugated to cetuximab with a
DAR4 (Cetuximab-(Lys-L-HSP27BNA).sup.4) and tested in combination
with cetuximab-(Cys-L-SO1861).sup.3,8 for enhanced HSP27 gene
silencing activity in EGFR expressing cells (A431, EGFR.sup.++) and
non-expressing cells (A2058, EGFR), according to the invention
(FIG. 14-5). Cetuximab-(Cys-L-SO1861).sup.3,8+100 nM
cetuximab-(Lys-L-HSP27BNA).sup.4 showed strong HSP27 gene silencing
in EGFR expressing cells (A431: IC50=nM, FIG. 8-5A), whereas
cetuximab-(Cys-L-SO1861).sup.3,8 alone did not show any gene
silencing activity. In A2058 cells (EGFR.sup.-) no gene silencing
activity was observed in the 1T2C combination (FIG. 8-5B). Next,
cetuximab-(Lys-L-HSP27BNA).sup.4+76.9 nM
Cetuximab-(Cys-L-SO1861).sup.3,8 show strong HSP27 gene silencing
activity in EGFR expressing cells (A431: IC50=4 nM, FIG. 8-5C),
whereas cetuximab-(Lys-L-HSP27BNA).sup.4 or
cetuximab-(Cys-L-SO1861).sup.3,8 or the combination of
Cetuximab-(Lys-L-HSP27BNA).sup.4+77 nM cetuximab did not reveal any
significant gene silencing activity (IC50>100 nM). When the
experiment was performed in EGFR non-expressing cells (A2058) no
gene silencing activity was observed in the 1T2C combination
(IC50>100 nM; FIG. 8-5D). All this shows that the 1T2C system
efficiently delivers an antisense BNA oligo to the cytoplasm of
high EGFR expressing cells, thereby inducing mRNA degradation of
the BNA target mRNA resulting in target gene silencing.
Example 17
[0577] The 1 target 2-components system (1T2C) can also be the
combination treatment of mAb1-(scaffold(-SO1861).sup.n).sup.n and
mAb1-protein toxin as illustrated in FIG. 15-5.
Dendron(-L-SO1861).sup.4 was conjugated to cetuximab via cysteine
residues (Cys) conjugation with a DAR3,9 and
cetuximab-Cys-(dendron(-L-SO1861).sup.4).sup.3,9 was tested for
enhanced cell killing activity in combination with an anti-EGFR
antibody-protein toxin conjugate (cetuximab-saporin) in EGFR
expressing cells (MDA-MB-468).
Cetuximab-Cys-(dendron(-L-SO1861).sup.4).sup.3,9+10 pM
cetuximab-saporin efficiently induces toxin-mediated cell killing
in high EGFR expressing cells (IC50=0.4 nM; FIG. 9-5A), whereas
this was not induced by
cetuximab-Cys-(dendron(-L-SO1861).sup.4).sup.3,9 or cetuximab+10 pM
cetuximab-saporin or cetuximab (FIG. 9-5A). This shows that
according to the 1T2C invention, cetuximab conjugated
dendron(-L-SO1861).sup.4 efficiently enhances endosomal escape of
the cetuximab conjugated protein toxin (at non-effective
concentrations), thereby inducing cell killing of high HER2
expressing cells. Similar 1T2C experiments were performed in cells
that express low levels of EGFR (HeLa, EGFR.sup.+/-) and this
revealed no cell killing activity when the 1T2C combination,
according of the invention was used (IC50>100 pM; FIG. 9-5B)
indicating that in the absence of sufficient EGFR receptor
expression, effective intracellular SO1861 concentrations are not
optimal (threshold) to induce cytoplasmic delivery of the protein
toxin that results in toxin-mediated cell killing.
[0578] Next, dendron(-L-SO1861).sup.4 was conjugated to the
anti-HER2 antibody, trastuzumab via cysteine conjugation (Cys) with
a DAR4, trastuzumab-Cys-(dendron(-L-SO1861).sup.4).sup.4 and tested
for enhanced cell killing activity in combination with an anti-HER2
antibody-protein toxin conjugate (trastuzumab-saporin) in HER2
expressing cells (SK-BR-3, HER2.sup.++).
Trastuzumab-Cys-(dendron(-L-SO1861).sup.4).sup.4+50 pM
trastuzumab-saporin efficiently induces toxin-mediated cell killing
(IC50=2 nM, FIG. 9-5C), whereas this was not induced by
trastuzumab-Cys-(dendron(-L-SO1861).sup.4).sup.4 or trastuzumab+50
nM trastuzumab-saporin or trastuzumab (FIG. 9-5C). This shows that
trastuzumab conjugated dendron(-L-SO1861).sup.4 efficiently
enhances endosomal escape of the trastuzumab conjugated protein
toxin (at non-effective concentrations), thereby inducing cell
killing of high HER2 expressing cells. Similar experiments in cells
that express low levels of HER2 (JIMT-1, HER2.sup.+/-) revealed no
activity of Trastuzumab-Cys-(dendron(-L-SO1861).sup.4).sup.4+50 pM
trastuzumab-saporin (IC50>200 nM; FIG. 9-5D) indicating that in
the absence of sufficient HER2 receptor expression, effective
intracellular SO1861 concentrations are not optimal (threshold) to
induce endosomal protein toxin escape and toxin-mediated cell
killing.
Example 18
[0579] The clinical approved ADC, trastuzuzmab-emtansine (T-DM1) is
a conjugate of the anti-Her2 antibody, trastuzumab and the small
molecule toxin emtansine (DAR3.5). T-DM1 was titrated in
combination with trastuzumab-(Cys-L-SO1861).sup.4 and compared with
the antibody protein toxin conjugate,
trastuzumab-saporin+Trastuzumab-(Cys-L-SO1861).sup.4, according to
the invention. Whereas trastuzumab-saporin+2.5 nM
trastuzumab-(Cys-L-SO1861).sup.4 showed enhanced activity compared
to Trastuzumab-saporin+2.5 nM trastuzumab or trastuzumab-saporin
alone (IC50=2 pM, FIG. 10-5), T-DM1+25.6 nM
trastuzumab-(Cys-L-SO1861).sup.4 showed no enhanced cell killing
activity (IC50>100 pM; FIG. 10-5). This shows that the 1T2C
system, according to the invention, cannot enhance the delivery of
an antibody small molecule conjugate, since small molecules can
already passively cross (endolysosomal) membranes.
Example 19
[0580] FIG. 11-5A-D displays the relative cell viability when
trastuzumab (FIG. 11-5A), cetuximab (FIG. 11-5B) or T-DM1 (FIG.
11-5C), unconjugated protein toxins, saporin, dianthin and saporin
conjugated to a (non-cell binding) IgG antibody (FIG. 11-5D) are
administrated to various cancer cell lines SK-BR-3, JIMT-1,
MDA-MB-468, A431, CaSki, HeLa, A2058.
[0581] Trastuzumab and cetuximab do not or hardly influence cell
viability when exposed to most of the cell lines, with some effect
on cell growth inhibition via blocking the function of the HER2
growth factor receptor when trastuzumab is exposed to SK-BR-3 cells
at relatively high dose and with some effect on cell growth
inhibition via blocking the function of the EGFR growth factor
receptor when cetuximab is exposed to MDA-MB-468 cells at
relatively high dose.
[0582] TDM-1, or ado-trastuzumab emtansine, is a targeted therapy
approved by the U.S. Food and Drug Administration to treat:
HER2-positive metastatic breast cancer that has previously been
treated with Herceptin (chemical name: trastuzumab) and taxane
chemotherapy; early-stage HER2-positive breast cancer after surgery
if residual disease was found after neoadjuvant (before surgery)
treatment with Herceptin and taxane chemotherapy. The TDM-1 is a
combination of Herceptin (Trastuzumab) and the chemotherapy
medicine emtansine. FIG. 11-5C shows that the TDM-1 results in
decreased cell viability for all cell lines tested at >1000 pM
concentrations
[0583] The free toxins saporin and dianthin and the toxin saporin
coupled to a control IgG with no affinity for any of the cell
surface molecules on the cell lines tested, do not or hardly have
any influence on cell viability over a wide range of concentrations
toxin tested, up to 100.000 pM (FIG. 11-5D).
Example 20
[0584] The 1 target 2-components system (1T2C) can also be the
combination treatment of mAb1-QSmix (mixture of saponins from
Quillaja saponaria) and mAb1-protein toxin.
[0585] QSmix-EMCH was conjugated via cysteine residues (Cys) to
cetuximab (monoclonal antibody recognizing and binding human EGFR),
with a DAR 4.1 (cetuximab-(Cys-L-QSmix).sup.4.1).
Cetuximab-(Cys-L-QSmix).sup.4.1 was titrated on a fixed
concentration of 10 pM cetuximab-saporin or 10 pM
cetuximab-dianthin and targeted protein toxin mediated cell killing
on A431 (EGFR.sup.++), CaSKi (EGFR.sup.+) and A2058 (EGFR.sup.-)
cells was determined. This revealed strong cell killing at low
concentrations of cetuximab-(Cys-L-QSmix).sup.4.1+10 pM
cetuximab-saporin or 10 pM cetuximab-dianthin in A431 (EGFR.sup.++)
and CaSKi (EGFR.sup.+) cells (A431: IC50=3 nM, FIG. 12-5A; CaSKi:
IC50=1 nM, FIG. 12-5B) whereas all control treatments could not
induce any cell killing in EGFR expressing cells. In cells that do
not express EGFR (A2058; EGFR.sup.-) no HSP27 gene silencing is
observed with the combination, according to the invention
(IC50>1000 nM; FIG. 12-5C). This shows that cetuximab conjugated
QS21mix efficiently enhances endosomal escape of the cetuximab
conjugated protein toxin (at non-effective concentrations), thereby
inducing cell killing only in EGFR expressing cells.
Example 21 2 Target 2-Component System (In Vivo)
[0586] The 2 target 2-components system (2T2C) is the combination
treatment of mAb1-SO1861 and mAb2-protein toxin, (FIG. 15-6; 16-6;
17-6). SO1861-EMCH was conjugated via cysteine residues (Cys) to
cetuximab (monoclonal antibody recognizing and binding human EGFR),
with a DAR 4 resulting in the production of:
cetuximab-(Cys-L-SO1861).sup.4. The combination of
cetuximab-(Cys-L-SO1861).sup.4 and trastuzumab-saporin or
CD71mab-saporin was tested in a A431
(EGFR.sup.++/HER2.sup.+/-/CD71.sup.+) xenograph `nude` mouse tumor
model for EGFR tumor targeted cell killing as illustrated in FIG.
1-6 and FIG. 2-6. Dose escalation was performed to determine the
therapeutic efficacy (Day 9: 0.3 mg/kg trastuzumab-saporin or 0.1
mg/kg CD71mab-saporin+5 mg/kg cetuximab-(Cys-L-SO1861).sup.4; Day
14, 18: 0.1 mg/kg trastuzumab-saporin or 0.05 mg/kg
CD71mab-saporin+5 mg/kg cetuximab-(Cys-L-SO1861).sup.4; Day 21:
0.05 mg/kg trastuzumab-saporin or 0.05 mg/kg CD71mab-saporin+15
mg/kg cetuximab-(Cys-L-SO1861).sup.4; Day 28: 0.02 mg/kg
trastuzumab-saporin or 0.02 mg/kg CD71mab-saporin+15 mg/kg
cetuximab-(Cys-L-SO1861).sup.4
trastuzumab-saporin/cetuximab-SO1861. Controls were on the same
dosing scheme respectively, only cetuximab (i. v.) was given 25
mg/kg every treatment day). At day 32 (dashed line), 35 and 39 we
started the combination, according to the 2T2C invention of 25
mg/kg cetuximab-(Cys-L-SO1861).sup.4 (intraperitoneal injection
(i.p.)+0.02 mg/kg trastuzumab-saporin or 0.02 CD71mab-saporin
(intravenous administration, (i.v.)) and this revealed strong tumor
regression for both 2T2C combination groups compared to the vehicle
control, 25 mg/kg cetuximab-(Cys-L-SO1861).sup.4 or 0.02 mg/kg
trastuzumab-saporin/CD71mab-saporin mono therapies (FIG. 1-6, 2-6).
The 2T2C system even outcompetes cetuximab, the clinically used
monoclonal antibody against EGFR. Next we performed the same
experiment but then we started with 25 mg/kg
cetuximab-(Cys-L-SO1861).sup.4 (intraperitoneal injection
(i.p.)+0.03 mg/kg trastuzumab-saporin or 0.03 CD71mab-saporin
(intravenous administration, (i.v.)) treatment with a dosing at day
9 and 14 and thereafter 1 dosing per week. The 2T2C system
according to the invention showed tumor regression in all mice and
even in 1 mice in both 2T2C groups, complete tumor eradication
(tumor volume=0 mm.sup.3) (FIG. 2-6). Also here the controls showed
a strong increased in tumor volume whereas the positive control for
this A431 mice model, cetuximab showed only tumor growth
inhibition, but no regression (FIG. 2-6). This shows and enables
the 2T2C system approach, according to the invention, of cetuximab
conjugated SO1861+trastuzumab conjugated protein toxin or CD71mab
conjugated protein toxin inducing highly efficient targeted
delivery of a therapeutic protein toxin in the cytoplasm of solid
tumors of tumor bearing mice, in vivo, thereby inducing even full
tumor eradication in some mice and strong tumor regression in
others even in large size tumors (2000 mm.sup.3).
Example 22 2 Target 2-Component System (In Vitro)
Results
[0587] The 2 target 2-components system (2T2C) is the combination
treatment of mAb1-SO1861 and mAb2-protein toxin, (see also FIG.
1-6, 2-6, 15-6, 16-6, 17-6). SO1861-EMCH was conjugated via
cysteine residues (Cys) to cetuximab (monoclonal antibody
recognizing and binding human EGFR), with a DAR 3,7
(cetuximab-(Cys-L-SO1861).sup.3,7).
Cetuximab-(Cys-L-SO1861).sup.3,7 was titrated on a fixed
concentration of 50 pM trastuzumab-saporin (trastuzumab, conjugated
to the protein toxin, saporin) and targeted protein toxin mediated
cell killing on EGFR/HER2 expressing cells (A431,
EGFR.sup.++/HER2.sup.+/-; CaSKi, EGFR.sup.+E/HER2.sup.+/-) was
determined as illustrated in FIG. 3-6. This revealed strong cell
killing at low concentrations of cetuximab-(Cys-L-SO1861).sup.3,7
(A431: IC50=3 nM and CaSKi IC50=10 nM; FIG. 3-6A, 3-6B) whereas
equivalent concentrations cetuximab,
cetuximab-(Cys-L-SO1861).sup.3,7 or cetuximab+50 pM
trastuzumab-saporin could not induce any cell killing activity in
EGFR/HER2 expressing cells. This shows that relatively low
concentrations of cetuximab-SO1861 conjugate efficiently enhances
endosomal escape of the trastuzumab conjugated protein toxin (at
non-effective concentrations), thereby inducing efficient cell
killing of high EGFR/low HER2 expressing cells.
[0588] Next, trastuzumab-saporin was titrated on a fixed
concentration of 75 nM cetuximab-(Cys-L-SO1861).sup.3,7 and
targeted protein toxin mediated cell killing on EGFR/HER2
expressing cells was determined. This revealed that 75 nM
cetuximab-(Cys-L-SO1861).sup.3,7 in combination with low
concentrations trastuzumab-saporin induced already efficient cell
killing in EGFR/HER2 expressing cells (A431: IC50=5 pM; and CaSKi:
IC50=1 pM; FIGS. 3-6C and 3-6D), whereas trastuzumab-saporin alone
or trastuzumab-saporin+75 nM cetuximab did not show significant
cell killing activity (IC50>10.000 pM) in both cell lines (FIG.
3-6C, 3-6D). All this shows that relatively low concentrations of
trastuzumab-saporin can be effective and induce cell killing in
combination with low cetuximab-SO1861 conjugate concentrations in
high EGFR/low HER2 expressing cells.
[0589] Next, cetuximab-(Cys-L-SO1861).sup.3,7 was titrated on a
fixed concentration of 50 pM trastuzumab-saporin and targeted
protein toxin-mediated cell killing on HeLa
(EGFR.sup.+/-/HER2.sup.+/-) or A2058 (EGFR/HER2.sup.+/-) was
determined as illustrated in FIG. 4-6, 15-6=17-6. Both HeLa
(EGFR.sup.+/-/HER2.sup.+/-) and A2058 (EGFR/HER2.sup.+/-) cells do
not show cell killing at low concentrations of
cetuximab-(Cys-L-SO1861).sup.3,7+50 pM trastuzumab-saporin (HeLa:
IC50=400 nM; A2058: IC50>400 nM; FIG. 4-6A, 4-6B). This shows
that in the absence of sufficient receptor expression, effective
intracellular delivered SO1861 concentrations are not reached
(threshold) to induce endosomal escape and cytoplasmic delivery of
the protein toxin. Next, trastuzumab-saporin was titrated on a
fixed concentration of 75 nM cetuximab-(Cys-L-SO1861).sup.37 and
targeted protein toxin mediated cell killing on HeLa
(EGFR.sup.+/-/HER2.sup.+/-) or A2058 (EGFR/HER2.sup.+/-) was
determined. Both HeLa (EGFR.sup.+/-/HER2.sup.+/-) and A2058
(EGFR/HER2.sup.+/-) cells showed no cell killing activity (HeLa:
IC50>10.000 pM; A2058: IC50>10.000 pM; FIG. 4-6C, 4-6D). All
this shows that cells with low or no EGFR receptor expression are
not susceptible for the combination of
cetuximab-(Cys-L-SO1861).sup.3,7+trastuzumab-saporin, due to a lack
of sufficient EGFR receptor that facilitates the antibody-mediated
delivery of sufficient SO1861 (threshold) to ensure endosomal
escape of the toxin within the cytoplasm of the cell.
[0590] Next, SO1861-EMCH was conjugated via cysteine residues (Cys)
to trastuzumab (monoclonal antibody recognizing and binding human
HER2), with a DAR 4 (trastuzumab-(Cys-L-SO1861).sup.4).
Trastuzumab-(Cys-L-SO1861).sup.4 was titrated on a fixed
concentration of 1.5 pM EGFdianthin (EGFR targeted ligand toxin
fusion protein) and targeted protein toxin mediated cell killing on
HER2/EGFR expressing cells (SK-BR-3: HER2.sup.++/EGFR.sup.+/-) was
determined. This revealed strong cell killing at low concentrations
of trastuzumab-(Cys-L-SO1861).sup.4+1.5 pM EGFdianthin (SK-BR-3:
IC50=1 nM; FIG. 5-6A) whereas equivalent concentrations
trastuzumab, trastuzumab-(Cys-L-SO1861).sup.4 or trastuzumab+1.5 pM
EGFdianthin could not induce any cell killing activity in
HER2.sup.++/EGFR.sup.+/- expressing cells. This shows that
trastuzumab conjugated SO1861 efficiently enhances endosomal escape
of the EGF fusion protein toxin (at non-effective concentrations),
thereby inducing cell killing of high HER2/low EGFR expressing
cells.
[0591] Next, EGFdianthin was titrated on a fixed concentration of
2.5 nM trastuzumab-(Cys-L-SO1861).sup.4 and targeted protein toxin
mediated cell killing on SK-BR-3 (HER2.sup.++/EGFR.sup.+/-)
expressing cells was determined. This revealed that 2.5 nM
trastuzumab-(Cys-L-SO1861).sup.4 in combination with low
concentrations EGFdianthin induced already efficient cell killing
in HER2/EGFR expressing cells (SK-BR-3: IC50=1 pM) (FIG. 5-6B),
whereas EGFdianthin alone or EGFdianthin+2.5 nM trastuzumab showed
no cell killing activity (IC50>10.000 pM) (FIG. 5-6B). All this
shows that relatively low concentrations of EGFdianthin can be
effective and induce cell killing only in combination with low
trastuzumab-(Cys-L-SO1861).sup.4 concentrations in high HER2/low
EGFR expressing cells.
[0592] Next, trastuzumab-(Cys-L-SO1861).sup.4 was titrated on a
fixed concentration of 1.5 pM EGFdianthin and targeted protein
toxin mediated cell killing on JIMT-1 (HER2.sup.+/-/EGFR.sup.+/-)
or MDA-MB-468: HER2.sup.-/EGFR.sup.++) was determined. Both cell
lines were not sensitive for any combination of
trastuzumab-(Cys-L-SO1861).sup.4+1.5 pM EGFdianthin (JIMT-1:
IC50>1000 nM; MDA-MB-468: IC50>1000 nM; FIG. 6-6A, 6-6B).
This shows that in the absence of sufficient HER2 receptor
expression, effective intracellular delivered SO1861 concentrations
are not reached (threshold) to induce endosomal escape and
cytoplasmic delivery of the protein toxin.
[0593] Next, EGFdianthin was titrated on a fixed concentration of
2.5 nM trastuzumab-(Cys-L-SO1861).sup.4 and targeted protein toxin
mediated cell killing on JIMT-1 (HER2.sup.+/-/EGFR.sup.+/-) or
MDA-MB-468 (HER2.sup.-/EGFR.sup.++) was determined. Both cell lines
showed cell killing at high EGFdianthin concentrations with or
without 2.5 nM trastuzumab-(Cys-L-SO1861).sup.4(JIMT-1: IC50=10.000
pM; MDA-MB-468: IC50=200 pM FIG. 6-6C, 6-6D).
[0594] All this shows that cells with low or no HER2 receptor
expression are not susceptible for the combination of
trastuzumab-(Cys-L-SO1861).sup.3,7+1.5 pM EGFdianthin, due to a
lack of sufficient HER2 receptor that facilitates the
antibody-mediated delivery of sufficient SO1861 (threshold) to
ensure endosomal escape of the toxin within the cytoplasm of the
cell.
[0595] Next, SO1861-EMCH was conjugated via cysteine residues (Cys)
to trastuzumab (monoclonal antibody recognizing and binding human
HER2), with a DAR 4, (trastuzumab-(Cys-L-SO1861).sup.4).
Trastuzumab-(Cys-L-SO1861).sup.4 was titrated on a fixed
concentration of 5 pM cetuximab-saporin (EGFR targeting
antibody-protein toxin conjugate) and targeted protein toxin
mediated cell killing on HER2/EGFR expressing cells (SK-BR-3:
HER2.sup.++/EGFR.sup.+/-) was determined as illustrated in FIG.
15-6-17-6. This revealed strong cell killing at low concentrations
of trastuzumab-(Cys-L-SO1861).sup.4+5 pM cetuximab-saporin
(SK-BR-3: IC50=1 nM; FIG. 7-6A) whereas equivalent concentrations
trastuzumab, trastuzumab-(Cys-L-SO1861).sup.4 or trastuzumab+5 pM
cetuximab-saporin could not induce any cell killing activity in
HER2.sup.++/EGFR.sup.+/- expressing cells. This shows that
trastuzumab conjugated SO1861 efficiently enhances endosomal escape
of the cetuximab conjugated protein toxin (at non-effective
concentrations), thereby inducing cell killing of
HER2.sup.++/EGFR.sup.+/- expressing cells.
[0596] Next, cetuximab-saporin was titrated on a fixed
concentration of 2.5 nM trastuzumab-(Cys-L-SO1861).sup.4 and 75 nM
trastuzumab-(Cys-L-SO1861).sup.4 and targeted protein toxin
mediated cell killing on HER2/EGFR expressing cells (SK-BR-3:
HER2.sup.++/EGFR.sup.+/-) was determined. This revealed that 2.5 nM
trastuzumab-(Cys-L-SO1861).sup.4 in combination with low
concentrations cetuximab-saporin induced already efficient cell
killing in SK-BR-3 cells (SK-BR-3: IC50=1 pM; FIG. 7-6B), whereas
cetuximab-saporin alone or cetuximab-saporin+2.5 nM trastuzumab
showed cell killing only at high concentrations trastuzumab-saporin
(SK-BR-3: IC50>4000 pM; FIG. 7-6B). All this shows that
relatively low concentrations of cetuximab-saporin can be effective
and induce cell killing only in combination with low
trastuzumab-(Cys-L-SO1861).sup.4 concentrations in
HER2.sup.++/EGFR.sup.+/- expressing cells.
[0597] Next, trastuzumab-(Cys-L-SO1861).sup.4 was titrated on a
fixed concentration of 5 pM cetuximab-saporin and targeted protein
toxin mediated cell killing on JIMT-1 (HER2.sup.+/-/EGFR.sup.+/-)
and MDA-MB-468 (HER2.sup.-/EGFR.sup.++) cells was determined. Both
cell lines were not sensitive for the combination of
trastuzumab-(Cys-L-SO1861).sup.4+5 pM cetuximab-saporin (JIMT-1:
IC50>1000 nM; MDA-MB-468: IC50>1000 nM; FIG. 8-6A, 8-6B).
This shows that in the absence of sufficient HER2 receptor
expression, effective intracellular delivered SO1861 concentrations
are not reached (threshold) to induce endosomal escape and
cytoplasmic delivery of the protein toxin.
[0598] Next, cetuximab-saporin was titrated on a fixed
concentration of 2.5 nM trastuzumab-(Cys-L-SO1861).sup.4 and
targeted protein toxin mediated cell killing on JIMT-1
(HER2.sup.+/-/EGFR.sup.+/-) and MDA-MB-468 (HER2.sup.-/EGFR.sup.++)
cells was determined. Both cell lines showed cell killing at
similar cetuximab-saporin concentrations with or without 2.5 nM
trastuzumab-(Cys-L-SO1861).sup.4(JIMT-1: IC50=80 pM; MDA-MB-468:
IC50=100 pM; FIG. 8-6C, 8-6D).
[0599] All this shows that cells with low or no HER2 receptor
expression are not susceptible for the combination of
trastuzumab-(Cys-L-SO1861).sup.4+cetuximab-saporin, due to a lack
of sufficient HER2 receptor that facilitates the antibody-mediated
delivery of sufficient SO1861 (threshold) to ensure endosomal
escape of the toxin within the cytoplasm of the cell.
Example 23
[0600] In order to show that the activity of the conjugated SO1861
is driven by the acidification of the endosomal compartments, the
2T2 components system, according to the invention was tested in
combination with an endosomal acidification inhibitor, chloroquine.
Trastuzumab-saporin+77 nM cetuximab-(Cys-L-SO1861).sup.3,9 or
trastuzumab-dianthin+77 nM cetuximab-(Cys-L-SO1861).sup.3,9 showed
strong cell killing activity in A431 (EGFR.sup.++/HER2.sup.+/-)
cells, whereas this 2T2C activity, according to the invention, was
inhibited when 800 nM chloroquine was co-administrated to both
combinations (FIG. 9-6A). Same results were observed when
CD71mab-saporin+10.5 nM cetuximab-(Cys-L-SO1861).sup.3,9+500 nM
chloroquine was tested in A431 (EGFR.sup.++/CD71.sup.+) and
MDA-MB-468 (EGFR.sup.++/CD71.sup.+) cells (FIG. 9-6B, 9C) or when
CD71mab-saporin+5 nM trastuzumab-(Cys-L-SO1861).sup.4+500 nM
chloroquine was tested in SK-BR-3 (HER2.sup.++/CD71.sup.+) cells
(FIG. 9-6D). This shows that the intracellular activity of
conjugated SO1861 within the 2T2C system can be inhibited when
acidification of endosomes is blocked.
Example 24
[0601] The 2 target 2-components system (2T2C) is also the
combination treatment of mAb1-SO1861 and mAb2-antisense BNA oligo
nucleotide, (FIG. 16-6). Therefore, the 2T2C system was also tested
in combination with an antisense BNA oligonucleotide against the
mRNA of a cancer specific target gene, heat shock protein 27
(HSP27). Upon release into the cytoplasm the antisense BNA
recognizes and binds the mRNA encoding for HSP27, targeting the
mRNA for destruction thereby depleting the HSP27 expression within
the cancer cell. HSP27BNA was conjugated to trastuzumab with a
DAR4.4 (trastuzumab-(Lys-L-HSP27BNA).sup.4'.sup.4) and tested in
combination with Cetuximab-(Cys-L-SO1861).sup.3,9 for enhanced
HSP27 gene silencing activity in A431 (EGFR.sup.++/HER2.sup.+/-)
cells and A2058 (EGFR/HER2.sup.+/-) cells as illustrated in FIG.
16-6. Cetuximab-(Cys-L-SO1861).sup.3'.sup.9 was titrated on a fixed
concentration of 100 nM Trastuzumab-(Lys-L-HSP27BNA).sup.4,4 and
targeted HSP27BNA-mediated gene silencing activity was determined.
Cetuximab-(Cys-L-SO1861).sup.3,9+100 nM
Trastuzumab-(Lys-L-HSP27BNA).sup.4,4 show strong gene silencing
activity in A431 cells (EGFR.sup.++/HER2.sup.+/-) (A431: IC50=1 nM;
FIG. 10-6A), compared to Cetuximab-(Cys-L-SO1861).sup.3,9 alone. In
A2058 cells (EGFR.sup.-/HER2.sup.+/-), the combination according to
the invention showed no HSP27 gene silencing (A2058: IC50>100
nM; FIG. 10-6B). This shows that cetuximab conjugated SO1861
efficiently enhances endosomal escape of the trastuzumab conjugated
BNA oligo nucleotide (at non-effective concentrations), thereby
inducing target gene silencing in EGFR.sup.++/HER2.sup.+/-
expressing cells.
[0602] Next, Trastuzumab-(Lys-L-HSP27BNA).sup.4,4 was titrated on a
fixed concentration of Cetuximab-(Cys-L-SO1861).sup.3,9 and
targeted HSP27BNA-mediated gene silencing activity was determined
in A431 (EGFR.sup.++/HER2.sup.+/-) cells and A2058
(EGFR/HER2.sup.+/-) cells as illustrated in FIG. 16-6.
Trastuzumab-(Lys-L-HSP27BNA).sup.4,4+77 nM
Cetuximab-(Cys-L-SO1861).sup.3,9 show strong gene silencing
activity in A431 cells (EGFR.sup.++/HER2.sup.+/-) (A431: IC50=1 nM;
FIG. 10-6C), whereas trastuzumab-(Lys-L-HSP27BNA).sup.4,4 alone or
Cetuximab-(Cys-L-SO1861).sup.3,9 alone or
trastuzumab-(Lys-L-HSP27BNA).sup.4,4+77 nM cetuximab did not reveal
any significant gene silencing activity (IC50>100 nM). A2058
(EGFR.sup.-/HER2.sup.+/-) cells did not show any gene silencing
activity in the combination according to the invention (A2058:
IC50>100 nM; FIG. 10-6D). All this shows that relatively low
concentrations of trastuzumab-HSP27BNA can be effective and induce
cell killing only in combination with low concentrations of
cetuximab-(-L-SO1861) concentrations in HER2.sup.++/EGFR.sup.+/-
expressing cells.
Example 25
[0603] The 2 target 2-components system (2T2C) can also be the
combination treatment of mAb1-(dendron(-SO1861).sup.n).sup.n and
mAb2-protein toxin. Dendron(-L-SO1861).sup.4 was conjugated to the
anti-EGFR antibody, cetuximab via cysteine residues (Cys) with a
DAR3,9, (cetuximab-Cys-(dendron(-L-SO1861).sup.4).sup.3,9) and
tested for enhanced cell killing activity in combination with an
anti-CD71 antibody protein toxin conjugate (CD71mab-saporin) in
MDA-MB-468 (EGFR.sup.++/CD71.sup.+) expressing cells as illustrated
in FIG. 17-6. Cetuximab-Cys-(dendron(-L-SO1861).sup.4).sup.3,9+10
pM CD71mab-saporin efficiently induces toxin-mediated cell killing
in MDA-MB-468 (EGFR.sup.++/CD71.sup.+) expressing cells (IC50=0.4
nM, FIG. 11-6A), whereas this could not be induced by
Cetuximab-Cys-(dendron(-L-SO1861).sup.4).sup.3,9) or cetuximab+10
pM CD71mab-saporin or cetuximab (FIG. 11-6A). This shows that
cetuximab conjugated dendron(-L-SO1861).sup.4 efficiently enhances
endosomal escape of the CD71mab-protein toxin (at non-effective
concentrations), thereby inducing cell killing of
EGFR.sup.++/CD71.sup.+ expressing cells. Similar experiments were
performed in HeLa cells (HER2.sup.+/-/CD71.sup.+) cells and this
revealed no activity of
cetuximab-Cys-(dendron(-L-SO1861).sup.4).sup.3,9)+10 pM
CD71mab-saporin (IC50>100 nM FIG. 11-6B) indicating that in the
absence of sufficient EGFR receptor expression, effective
intracellular SO1861 concentrations are not reached (threshold) to
induce endosomal escape and cytoplasmic delivery of the protein
toxin.
[0604] Next, dendron(-L-SO1861).sup.4 was conjugated to the
anti-HER2 antibody, trastuzumab via cysteine conjugation (Cys) with
a DAR4, trastuzumab-Cys-(dendron(-L-SO1861).sup.4).sup.4 and tested
for enhanced cell killing activity in combination with an anti-CD71
antibody protein toxin conjugate (CD71mab-saporin) in SK-BR-3 cells
(HER2.sup.++/CD71.sup.+) expressing cells.
Trastuzumab-Cys-(dendron(-L-SO1861).sup.4).sup.4+10 pM
CD71mab-saporin efficiently induces toxin-mediated cell killing in
SK-BR3 cells (IC50=3 nM, FIG. 11-6C), whereas this was not induced
by trastuzumab-Cys-(dendron(-L-SO1861).sup.4).sup.4 or trastuzumab
(equivalent)+10 pM CD71mab-saporin or trastuzumab (FIG. 11-6C).
This shows that trastuzumab conjugated dendron(-L-SO1861).sup.4,
according to the invention efficiently enhances endosomal escape of
the CD71mab-protein toxin (at non-effective concentrations),
thereby inducing cell killing of HER2.sup.++/CD71.sup.+ expressing
cells. Similar experiments were performed in JIMT-1 cells
(HER2.sup.+/-/CD71.sup.+) and this revealed no activity of
trastuzumab-Cys-(dendron(-L-SO1861).sup.4).sup.4+10 pM
CD71mab-saporin (IC50>100 nM FIG. 11-6C) indicating that in the
absence of sufficient HER2 receptor expression, effective
intracellular SO1861 concentrations are not reached (threshold) to
induce endosomal escape and cytoplasmic delivery of the protein
toxin.
Example 26
[0605] The clinical approved ADC, trastuzumab-emtansine (T-DM1) is
a conjugate of the anti-Her2 antibody, trastuzumab and the small
molecule toxin emtansine (DAR3-4). T-DM1 was tested within the 2T2C
system, according to the invention in combination with
cetuximab-(Cys-L-SO1861).sup.4. T-DM1+77 nM
cetuximab-(Cys-L-SO1861).sup.3,9 showed no enhanced cell killing
activity compared to T-DM1 alone or T-DM1+77 nM cetuximab
(IC50=80.000 pM, FIG. 12-6), whereas trastuzumab-saporin+75 nM
cetuximab-(Cys-L-SO1861).sup.3,7, according to the invention showed
enhanced cell killing activity compared to trastuzumab-saporin+75
nM cetuximab or trastuzumab-saporin alone (IC50=3 pM, FIG. 12-6).
All this shows that the 2T2C system does not enhance the delivery
of antibody conjugated small molecules, that are already able to
passively cross cellular (endosomal) membranes.
Example 1-2
[0606] Various concentrations of trastuzumab-saporin (HER2 targeted
protein-toxin conjugate; intravenous) were tested in combination
with 1.5 mg/kg SO1861 (1 hour before antibody-toxin injection;
subcutaneous) for enhanced efficacy in a BT474 (HER2++) xenograph
mouse model. Dosing started at day 13 when tumors reached
.about.150 mm.sup.3 in size and tumor volume was determined after
every treatment. Although tumor growth inhibition was observed in
the mice treated with 1 mg/kg and 0.3 mg/kg trastuzumab-saporin,
there was no enhanced tumor growth inhibition observed in the mice
treated with the combination of trastuzumab-saporin+SO1861. This
shows that unconjugated SO1861 is not able to enhance
antibody-protein toxins within the current settings and mouse
model.
Example 2-2
Materials:
[0607] QSmix (1): S4521 (Sigma Aldrich); QSmix (2): 6857.1 (Carl
Roth) QSmix (3): Quil-A.RTM. Adjuvant: vac-quil
(InvivoGen/Brenntag).
[0608] Previously, the efficacy of various saponins (SO1861,
SO1642) were co administrated as `free` unconjughated molecules to
cells in combination with a ligand toxin fusion (e.g. EGFdianthin)
or an antibody-protein toxin conjugate, resulting in enhanced cell
killing activity of target expressing cells. Here, three different
saponin molecules (SO1861, SO1862 (isomer of SO1861), SO1832 and
SO1904) isolated from a root extract of Saponaria officinalis were
titrated in the presence and absence of a non-effective fixed
concentration of 1.5 pM EGFdianthin on HeLa (EGFR.sup.+) cells.
This revealed a strong enhancement of cell killing activity for all
tested saponin variants (IC50=300 nM; FIG. 2-2A) compared to the
treatments without EGFdianthin. Next, EGFdianthin was titrated with
a fixed concentration of saponin (.about.1000 nM) and this revealed
strong targeted cell killing enhancement at low pM concentrations
of EGFdianthin (IC50=0.4 pM; FIG. 2-2B), observed for all used
saponins SO1861, SO1862 (isomer of SO1861), SO1832 and SO1904.
EGF-dianthin alone could only induce cell killing at very high
concnetrations (IC50=10.000 pM). This shows that these specific
types of saponins, all have the intrinsic capacity to efficiently
induce endosomal escape with only a very low amount of targeted
toxin available.
[0609] To extend this test, saponins from other sources were
analyzed. A saponin purified from a root extract of Gypsophila
elegans M. Bieb. (GE1741) was titrated on HeLa cells in the
presence and absence of 1.5 pM EGFdianthin and compared with
purified SO1861. GE1741 also enhances the EGFdianthin induced HeLa
cell killing, but shows slightly less efficacy compared to SO1861.
(GE1741 IC50=800 nM; FIG. 2-2C) and also displays a higher general
toxicity (IC50=5.000 nM in absence of EGFdianthin; FIG. 2-2C). A
similar test in which different partially purified mixtures of
Quillaja saponaria saponins (QSmix 1-3) were co-administrated with
1.5 pM EGFdianthin on HeLa cells and this revealed for 2 out of 3
(QSmix 1 and QSmix 3) similar activity as SO1861 (IC50
QSmix/QSmix3=300 nM; FIG. 2-2D). QSmix (2) is less efficient in
enhancing 1.5 pM EGFdianthin induced cell killing (IC50=2000 nM;
FIG. 2-2D), however, no general toxicity is observed. This shows
that also in QS extracts, specific type of saponins are available
that efficiently induce endosomal escape of the targeting ligand
toxin EGFdianthin.
Example 3-2
[0610] In order to conjugate SO1861 molecules to antibodies,
according to the invention, labile/acid sensitive linkers (-EMCH or
-N3), was conjugated to SO1861 via the aldehyde group, producing
SO1861-EMCH or SO1861-N3. To verify the activity of SO1861-EMCH the
molecule was titrated in the presence and absence of a fixed
non-effective (1.5 pM) EGFdianthin concentration on EGFR expressing
(A431, HeLa) and non-expressing cells (A2058). In all three cell
lines SO1861 alone showed a strong cell viability reduction,
whereas SO1861-EMCH as single compound showed no toxicity up to
25.000 nM (FIG. 3-2A-C). When SO1861-EMCH was combined with 1.5 pM
EGFdianthin a strong target specific cell viability reduction is
observed in the EGFR+A431 and HeLa cells (IC50=3.000 nM; FIG.
3-2A,B), while the EGFR.sup.- A2058 cells are not affected at all
(FIG. 3-2C). Similar results were obtained for SO1861-N3. 501861-N3
co-administrated with 1.5 pM EGFdianthin also shows efficient cell
killing on A431 and HeLa cells (IC50=3.000 nM), but without
EGFdianthin a general toxicity is observed at above 10.000 nM (FIG.
3-2D, 3-2E).
[0611] For the stable conjugation of SO1861 to antibodies,
according to the invention, a stable linker (HATU) was conjugated
to SO1861 via the carboxylic acid group of SO1861 producing,
SO1861-(S). To determine the activity different concentrations of
SO1861-(S) were co-administrated with 1.5 pM EGFdianthin and tested
for cell killing activity in EGFR expressing HeLa cells. SO1861-(S)
showed a similar activity as SO1861, indicating that conjugation to
the carboxylic acid does not affect the endosomal escape enhancing
potency of the molecule as is observed with SO1861-EMCH (FIG.
4-2).
Example 4-2
[0612] Labile SO1861 was conjugated via cysteine residues (Cys) to
the anti-EGFR antibody cetuximab (monoclonal antibody recognizing
and binding human EGFR), with DAR3.9
(cetuximab-(Cys-L-SO1861).sup.39) and tested for its enhanced
delivery of antisense BNA oligo nucleotides resulting in enhanced
target gene silencing. In this study we used an antisense BNA
oligonucleotide against the mRNA of a cancer specific target gene,
heat shock protein 27 (HSP27). Within the cytoplasm of the cell
HSP27BNA bind the mRNA encoding for HSP27, target the mRNA for
destruction, thereby reducing the HSP27 expression within the
cancer cell. Cetuximab-(Cys-L-SO1861).sup.39 was titrated on fixed
concentration of 100 nM HSP27BNA on EGFR.sup.++ (A431) and
EGFR.sup.- (A2058) cells. The combination according of the
invention showed efficient HSP27 silencing on A431 (IC50=2 nM; FIG.
5-2A), while no silencing was observed for
cetuximab-(Cys-L-SO1861).sup.39 alone.
Cetuximab-(Cys-L-SO1861).sup.39+100 nM HSP27BNA showed no gene
silencing activity in EGFR.sup.- cells (A2058) (FIG. 5-2B). This
shows that low concentrations of antibody-conjugated SO1861
efficiently can enhance cytoplasmic delivery and endolysosomal
escape of an antisense BNA oligo nucleotide, thereby inducing
efficient gene silencing in target expressing cells.
[0613] Next the HSP27BNA was titrated on EGFR.sup.++ (A431) and
EGFR.sup.- (A2058) cells combined with fixed concentration of
cetuximab-(Cys-L-SO1861).sup.39. This shows that HSP27BNA in
combination with 28.6 nM cetuximab-(Cys-L-SO1861).sup.3.9 or 77 nM
cetuximab-(Cys-L-SO1861).sup.3.9 very efficiently enhances HSP27
gene silencing in A431 cells (IC50=10 nM; FIG. 5-2C). HSP27BNA
alone or combined with a fixed equivalent of 77 nM cetuximab are
less efficient (IC50=1.000 nM; FIG. 5-2C). The combination
treatment of HSP27BNA+77 nM cetuximab-(Cys-L-SO1861).sup.39 was
also tested on EGFR- cells (A2058) and this revealed no HSP27 gene
silencing enhancement (IC50=1.000 nM; FIG. 5-2D). This shows that
cells with low or no EGFR receptor expression are not susceptible
for the combination of cetuximab-(Cys-L-SO1861).sup.3,9+HSP27BNA,
while cetuximab targeted SO1861 can enhance HSP27 gene silencing
efficiently at low concentrations of non-targeted HS27BNA in high
EGFR cells.
[0614] Next, SO1861-EMCH was conjugated via cysteine residues (Cys)
to cetuximab (monoclonal antibody recognizing and binding human
EGFR), with a DAR 3,8. The combination according to the invention,
cetuximab-(Cys-L-SO1861).sup.3,8+HSP27BNA (antisense HSP27BNA oligo
nucleotide targeting and inducing degradation of the onco-target
hsp27 mRNA (gene silencing) in cancer cells) was tested in a A431
xenograph `nude` mouse tumor model for EGFR-mediated tumor targeted
HSP27 gene silencing. Dosing started at day 12 when tumors reached
.about.150 mm.sup.3 in size and HSP27 mRNA expression was
determined. For this, tumor samples were collected at 72h after the
first dosing and analysed for HSP27 gene expression levels compared
to cellular control mRNA expression levels (reference genes). Tumor
bearing mice (n=3) were treated (intraperitoneal; i.p.) at day 12:
25 mg/kg cetuximab-(Cys-L-SO1861).sup.3,8+25 mg HSP27BNA and at day
15: 25 mg/kg cetuximab-(Cys-L-SO1861).sup.3,8+10 mg HSP27BNA and
this revealed a 25% reduction in HSP27 mRNA expression in the
tumors compared to vehicle control or single dosing of 25 mg/kg
HSP27BNA (FIG. 6-2). This shows and enables that conjugation of
SO1861 to a targeting antibody, according to the invention,
efficiently induces SO1861-mediated enhanced cytoplasmic delivery
of a therapeutic antisense oligo nucleotide in solid tumors of
tumor bearing mice, inducing tumor targeted gene silencing, in
vivo.
Example 1-7--Targeted Antisense Oligonucleotide Coupled to an
Antibody
Cetuximab-(Lys-L-HSP27BNA)+Saponin SO1861
[0615] An antisense BNA oligonucleotide against the mRNA of a
cancer specific target gene, heat shock protein 27 (HSP27), will
upon release into the cytoplasm recognize and bind the mRNA
encoding for HSP27, target the mRNA for destruction and thereby
lower the HSP27 expression within the cancer cell. Non-targeted
HSP27BNA was titrated on EGFR.sup.++ (A431) and EGFR.sup.- (A2058)
cells to test for HSP27 gene silencing in combination with
saponins. Data revealed efficient HS27 silencing on both A431 A2058
cells when HSP27BNA was combined with 4000 nM SO1861-ECMH (IC50=10
nM; FIG. 1-7A, 1-7B), while no silencing was observed for single
HSP27BNA treatment (IC50 1.000 nM; FIG. 1-7A, 1-7B).
[0616] Next, HSP27BNA was conjugated to the lysines of cetuximab
with DAR1.5 and DAR3.9, resulting in
cetuximab-(Lys-L-HSP27BNA).sup.15 and
cetuximab-(Lys-L-HSP27BNA).sup.39. The conjugated
cetuximab-HSP27BNA samples were again titrated on EGFR.sup.++
(A431) and EGFR.sup.- (A2058) cells to test for targeted HSP27 gene
silencing in combination with saponins. These conjugates show very
efficient HSP27 gene silencing in A431 (EGFR.sup.++) cells in the
presence of 4000 nM SO1861-EMCH (DAR1.5 IC50=0.05 nM and DAR3.9
IC50=0.3 nM; FIG. 1-7A), while the silencing of the targeted
HSP27BNA samples is comparable to the non-targeted HSP27BNA in the
absence of SO1861-EMCH. The silencing in A2058 (EGFR.sup.-) cells
is not improved compared to non-targeted HSP27BNA in general. Both
in the presence and absence of SO1861 similar HSP27BNA (conjugate)
amounts are required to induce silencing (FIG. 1-7B). This shows
that cells with high EGFR receptor expression very efficient
targeted HSP27 gene silencing can be achieved using targeted
HSP27BNA in combination with SO1861.
[0617] An antisense BNA oligonucleotide against the mRNA of a
cancer specific target gene, heat shock protein 27 (HSP27), will
upon release into the cytoplasm recognize and bind the mRNA
encoding for HSP27, target the mRNA for destruction and thereby
lower the HSP27 expression within the cancer cell. Non-targeted
HSP27BNA was titrated on EGFR.sup.++ (A431) and EGFR.sup.- (A2058)
cells to test for HSP27 gene silencing in combination with
saponins. Data revealed efficient HS27 silencing on both A431 A2058
cells when HSP27BNA was combined with 4000 nM SO1861-ECMH (IC50=10
nM; FIG. 1-7C, 1-7D), while no silencing was observed for single
HSP27BNA treatment (IC50 1.000 nM; FIG. 1-7C, 1-7D).
[0618] Next, HSP27BNA was conjugated to the lysines of cetuximab
with DAR1.5 and DAR3.9, resulting in
cetuximab-(Lys-L-HSP27BNA).sup.1.5 and
cetuximab-(Lys-L-HSP27BNA).sup.3.9. The conjugated
cetuximab-HSP27BNA samples were again titrated on EGFR.sup.++
(A431) and EGFR.sup.- (A2058) cells to test for targeted HSP27 gene
silencing in combination with saponins. These conjugates show very
efficient HSP27 gene silencing in A431 (EGFR.sup.++) cells in the
presence of 4000 nM SO1861-EMCH, requiring less HSP27BNA oligo
(IC50=0.04 nM; FIG. 1-7C), while the silencing of the targeted
HSP27BNA samples is comparable to the non-targeted HSP27BNA in the
absence of SO1861-EMCH. The silencing in A2058 (EGFR.sup.-) cells
is not improved compared to non-targeted HSP27BNA in general. It
even seems that about 10.times. more HSP27BNA oligo is required in
the presence of SO1861, while no significant silencing is observed
without SO1861 (FIG. 1-7D). This shows that cells with high EGFR
receptor expression very efficient targeted HSP27 gene silencing
can be achieved using targeted HSP27BNA in combination with
SO1861.
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