Note: Descriptions are shown in the official language in which they were submitted.
CA 03127903 2021-07-27
Description
BI-LIGAND DRUG CONJUGATE AND USE THEREOF
Technical Field
The present application relates to the field of biomedical chemistry. More
particularly, the present application relates to a bi-ligand drug conjugate, a
pharmaceutical composition comprising the bi-ligand drug conjugate, a method
for
delivering a payload to a subject in need thereof by using the bi-ligand drug
3.13 conjugate, and a method for treating a disease with the bi-ligand drug
conjugate.
Background Art
In general, diseased cells and nottnal cells are significantly different in
both
pathological and physiological characteristics, and one of the differences
lies in that
diseased cells have specific or overexpressed materials (such as antigens,
chemical
signals and receptors) on their surfaces, whereas these materials are absent
or lowly
expressed in nottnal cells. On this basis, an antibody-drug conjugate (ADC)
and a
polypeptide-drug conjugate (PDC) are developed for the treatment of diseases.
At
present, some ADC- and PDC-based drugs have been marketed or have entered
clinical researches; however, due to their design rationale, ADC- and PDC-
based
drugs have a great limitation in clinical application.
Due to the complexity and relatively large molecular weight of ADCs, the
development thereof faces many difficulties, including lack of suitable
targets,
difficulty in production and poor drug stability. Currently, ADCs are mainly
used in
the field of tumor treatment. In some instances, the affinity of a targeting
antibody
to a cancer cell surface antigen can reach 10-9-10-12 (Kd, mole/liter).
Therefore,
when having a high specificity to target cells, ADCs also have a high
specificity to
normal cells with the same targeting receptor as the target cells. Moreover,
it may
take a long time (1 to 3 weeks) to metabolize ADCs in vivo, during which ADCs
continuously kill nottnal cells, leading to significantly increased toxic and
side
effects. Therefore, ADCs are more applicable to diseases characterized by a
very
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large difference in the amount of cell surface antigens between tumor cells
and
normal cells. However, very few diseases known in the art can meet such strict
requirement.
PDCs have been used to treat a variety of diseases in clinical or pre-clinical
researches, but in such case chemotherapeutics are simply connected to
polypeptides, or polypeptides are added to nanoparticles or polymer materials
embedded with chemotherapeutic drugs, which will make most of the polypeptides
unable to enter cells due to their large molecular weights and bearing
charges.
Therefore, most of the PDCs are currently only suitable for an extracellular
treatment and the application scopes and efficacy of PDCs are severely
limited.
A drug conjugate compound can also be a ligand-drug conjugate (LDC),
wherein the ligand may be a peptide or a small molecule. However, in terms of
bioavailability, stability, efficacy, toxicity, etc., there are various
problems in the
application of LDCs. For example, due to large molecular weights,
lipophilicity or
other properties, many ligands are incapable of entering cells, which limits
their
therapeutic applications. In addition, ligands usually have low efficacy when
conjugated with conventional chemotherapeutics (such as doxorubicin and
paclitaxel), and when conjugated with highly effective drug molecules (such as
MMAE and DM1), ligands may produce a high toxicity, thereby poisoning and
even killing animals before a therapeutically effective amount for treating a
tumor is
achieved.
Therefore, there is also an urgent need in the art to obtain an improved LDC,
which can act on highly-expressed receptors widely existing on the surface of
diseased cells, broaden a targeting range and a treatment window, enhance drug
efficacy and avoid drug side effects.
Summary of the Invention
In one aspect, the present application discloses a conjugate compound or a
pharmaceutically acceptable salt thereof, comprising a payload and two
targeting
molecules, wherein the two targeting molecules are a synergistic molecule
moiety
and a prostate-specific membrane antigen ligand moiety, respectively.
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In another aspect, the present application discloses a conjugate compound or a
pharmaceutically acceptable salt thereof, comprising a payload and two
targeting
molecules, wherein the two targeting molecules are a synergistic molecule
moiety
and a ligand moiety represented by formula (I), respectively:
0
(D-Phe)-Cys-Tyr-(D-Trp)-Lys-Thr-Cys-Thr A-
/
(I).
In another aspect, the present application discloses a conjugate compound or a
pharmaceutically acceptable salt thereof, comprising a payload and two
targeting
molecules, wherein the two targeting molecules are a synergistic molecule
moiety
and P10, respectively, and the payload is camptothecin or any derivative
thereof
In some embodiments, the prostate-specific membrane antigen ligand
comprised in the conjugate compound or the pharmaceutically acceptable salt
thereof has the following structure:
HO 00 0 OH
H 0 (s) N AN
H H H
0 .
In some embodiments, the prostate-specific membrane antigen ligand
comprised in the conjugate compound or the pharmaceutically acceptable salt
thereof has the following structure:
HO 0 0 OH
0 0
HO (s) NANNAN
H H H H
0
In some embodiments, the prostate-specific membrane antigen ligand
comprised in the conjugate compound or the pharmaceutically acceptable salt
thereof has the following structure:
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HO 0 0õOH
y j 0 --- 0 HO
HO ANAN 0
(s) N N (s) 0
H H H H
0 j(i\j,&ti
N (s)
H
0 \
0
HO .
In some embodiments, the prostate-specific membrane antigen ligand
comprised in the conjugate compound or the pharmaceutically acceptable salt
thereof has the following structure:
H2N
HO 0 0 OH
0 0 HO
_
0
HO (s) NANNAN 0 1.4 0
H H H H
0 j(N,(sA
H H
0 \
0 0
In some embodiments, the two targeting molecules comprised in the conjugate
compound or the pharmaceutically acceptable salt thereof are different.
In some embodiments, the synergistic molecule comprised in the conjugate
compound or the pharmaceutically acceptable salt thereof is a cell-interacting
molecule.
In some embodiments, the two targeting molecules comprised in the conjugate
compound or the pharmaceutically acceptable salt thereof are cell-interacting
molecules that interact with different cell molecules.
In some embodiments, the synergistic molecule comprised in the conjugate
compound or the pharmaceutically acceptable salt thereof is an endocytosis
molecule capable of mediating endocytosis.
In some embodiments, the synergistic molecule comprised in the conjugate
compound or the pharmaceutically acceptable salt thereof binds to a molecule
selected from the group consisting of FOLR1, TRPV6, FOLH1 (PMSA), GNRHR,
Her2, Trop2, Her3, NECTIN4, LRP1, GLUT1, EGFR1, AXL, CA9, CD44,
Claudin18.2, APN, DLL3, CEACAM5, FZD10, TFRC, MET, IGFR1, SSTR2,
CCKBR, LFA1, ICAM, GPR87, GM-CSF, GM-CSFR, TIM3, TLR family, CD40,
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CD4OL, 0X40, OX4OL, GITRL, GITR, 4-BBL, 4-1BB, CD70, CD27, ICOSL,
ICOS, HHLA2, CD28, CD86/80, CD28, MHCII antigen, TCR, CTLA-4, CD155,
CD122, CD113, IGIT, PD-L1, PD1, Galectin-9, TIM-3, HVEM, BTLA, CD160,
VISTA, B7-H4, B7-H3, phosphatidylserine, HHLA2, LAG3, Galectin-3, LILRB4,
SIGLEC15, NKG2A, NKG2D, SLAMF7, KIR2DL1, KIR2DL2, KIR2DL3, FGFR1,
FGFR2, FGFR4, NeuGcGM3 and CXCR4.
In some embodiments, the conjugate compound or the pharmaceutically
acceptable salt thereof comprises a prostate-specific membrane antigen ligand
moiety and a synergistic molecule moiety, wherein the synergistic molecule
moiety
binds to a molecule selected from the group consisting of FOLR1, TRPV6, FOLH1
(PMSA), SSTR2 and GNRHR.
In some embodiments, the conjugate compound or the pharmaceutically
acceptable salt thereof comprises a ligand moiety represented by formula (I)
and a
synergistic molecule moiety, wherein the synergistic molecule moiety binds to
a
molecule selected from the group consisting of FOLR1, TRPV6, SSTR2 and
GNRHR.
In yet some embodiments, the conjugate compound or the pharmaceutically
acceptable salt thereof comprises P10 and a synergistic molecule moiety,
wherein
the synergistic molecule binds to a molecule selected from the group
consisting of
FOLR1, TRPV6, FOLH1 (PMSA) and GNRHR.
In some embodiments, the synergistic molecule comprised in the conjugate
compound or the pharmaceutically acceptable salt thereof is a folate or an
analog
thereof In some embodiments, the analog of folate is selected from the group
consisting of 5-methyltetrahydrofolate, 5-foitnyltetrahydrofolate,
methotrexate, and
5,10-methylenetetrahydrofolate.
In some embodiments, the conjugate compound or the pharmaceutically
acceptable salt thereof comprises one, two, three, four or more payloads. In
some
embodiments, the payload is selected from the group consisting of a small
molecule
compound, a nucleotide, a peptide and a protein. In some embodiments, the
payload
is a small molecule compound. In some embodiments, the small molecule
compound is selected from the group consisting of camptothecin and any
derivative
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thereof, auristatin and any derivative thereof, maytansine and any derivative
thereof,
a radionuclide complex, a cyclooxygenase-2 inhibitor, paclitaxel and any
derivative
thereof, epothilone and any derivative thereof, bleomycin and any derivative
thereof,
dactinomycin and any derivative thereof, plicamycin and any derivative
thereof, and
mitomycin C. In some embodiments, the small molecule compound is camptothecin
or any derivative thereof, auristatin or any derivative thereof, maytansine or
any
derivative thereof, a radionuclide complex or a cyclooxygenase-2 inhibitor.
In some embodiments, the payload comprised in the conjugate compound or
the pharmaceutically acceptable salt thereof of the present application is
linked to at
least one targeting molecule via a linker.
In some embodiments, the linker comprised in the conjugate compound or the
pharmaceutically acceptable salt thereof of the present application is a
peptide
linker, a disulfide linker, a pH-dependent linker or a combination of the
above-mentioned linkers.
In some embodiments, the peptide linker is cleavable under a certain
physiological environment by protease cleavage or reduction. In some
embodiments,
the peptide linker is selected from the group consisting of cysteine, lysine,
lysine-lysine, valine-citrulline, phenylalanine-lysine, valine-lysine,
cysteine-lysine,
cysteine-glutamic acid, aspartic acid-aspartic acid, and aspartic acid-
aspartic
acid-lysine, and optionally, the carboxylic acid in the above-mentioned amino
acids
is amidated.
In some embodiments, the disulfide linker is selected from the group
consisting of DMDS, MDS, DSDM, and NDMDS.
In some embodiments, the pH-dependent linker is a cis-aconitic anhydride.
In some embodiments, the linker of the conjugate compound or the
pharmaceutically acceptable salt thereof of the present application has the
following
structure:
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N H2
NH
0 0
,(SA
4N
0 HN (S) 0 *
0 0 IN
0 ,
N H2
0
NH
0 0
N.(sA
N N (S)
0 H 0
0
0 0 ,
N H2
0
NH
0 Ho
[N1
N N
0 0 y\
HN0
0 0
s'
N H2
9
N H2
0
NH
0 0
N N (s)
N
= H
(R) 0 0 *
H N0 0 0 1\
0
H 0
0
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N H2
0
NH
0 H 0
N
N (S)
(R) 0
HNO 0
61-=!),r0
0 H N
0
NH2
0
NH
0 H 0
N rE1
N N (s)
H
(R) 1/4.= 0
HN0 0
? 0
OY\J NH2
6L
HO
H
0 HN N
= H H
0
00H
0
Nizz.N
0
0
0
Iir0 0 0
0
o
NIrorN 00j
)y31 $ 0 0
0
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/1\1_-:N
0
3&) 0 0
N 1\11rOf N
0 0
HN
H2N
/NH
ArCNO
0
0
\jLO 110 0 0)
N NlrorN
0 0
HN
H2N
/NH N.õ..N
(s)
0 H2N0 0
\)L0 0O 0)
N (s) N IrOThrN0(:))
0 0
HN
H2N
ON H2 0
H (s)
HN
or the linker is a combination of the above-mentioned structures and a peptide
linker.
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In some embodiments, the two targeting molecules comprised in the conjugate
compound or the phannaceutically acceptable salt thereof of the present
application
are linked to each other via a spacer. In some embodiments, the spacer
described in
the present application comprises an amino acid sequence selected from the
group
consisting of SEQ ID NOs: 1-14, Arg-Arg, Ala-Ser-Asn, Ala-Ala-Ala, Ser-Ser-
Arg,
Pro-Arg and Pro-Leu-Gly.
In some embodiments, the conjugate compound of the present application is
CB-20B which has the following structural foimula:
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OH
OH
H2N HN 0
)=I21
HN Fl(s)
N N OH
HN
HN
HNO
HN 0
(s) u<OH
0
NH HN
(=-======,(OH
0
NH 0
0 (S) HOy..
0
H2N HN NH
0 s
0-1,1 0
)NH..= (s)
0
HN 0
0(S) N'ILNH 2
NH
0
0,
HmN)
0
.0
0
0 (R)
(R)
0
NH
(R)
(8) OH
In some embodiments, the conjugate compound of the present application is
CB-20BK which has the following structural foimula:
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OH
0
OH
(s)
HN 0
HN 0
H2N (s)
)=N OH
HNO
N N
HN
HN HN
HN 0
(s)
OH HN
(s) OH
Hoy
0 0
NH NH
0 HN
(s) 0
H2N
(R
N 0
).. NEclo
HN 0
NH
P
0
(D
0
HI)_<
(s)
0
(s) NET
ER
(sN)
0 (R)
(R)
0
NH
(R)
(S) OH
In some embodiments, the conjugate compound of the present application is
CB-60S which has the following structural foimula:
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OH
0
(s)
H2N HN 0
)=N
HN 0 HN 0
N)¨ N (s)
OH
HN
HN
HN
0
HN 0
6s) OH
0
NH HN
(s) OH
0
NH
HOy.. (s)
(s) 0
0
H2N HN NH
(s)
0
ro,N_NõN
0
NH
01
0 NH
(s)
0
HN
OH
0 Nz
0 ¨
\ N
0 (s)
0 0
In some embodiments, the conjugate compound of the present application is
CB-60SK which has the following structural foimula:
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OH
0
(s)
HN 0
HN 0
H2N (s) =
)=N OH
HN tO
N
HN
HN HN
0
HN 0
(s) OH HN
(s) __ yOH
0 0
NH NH
HO((s)
0
= HN
0 (s) NH2
(s) 0
H2N HN NH
(s)
0
ro,N,NoN
0
'o
NH
0
0
C)
0 NH
(s)
H2N N
0
HN
OH
O N z
O ¨
\ N
0 (s)
0
In some embodiments, the conjugate compound of the present application is
CB-20C which has the following structural foimula:
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OH
0
OH
(s)
HN 0
HN 0
H2N (s)
)=N OH
HN
N N
HN
HN HN
=
HN 0
(s)
OH HN
(s) OH
0 0
NH NH
HOy, (s)
0
0 HN
0 (s) NH2
(S) 0
H2N HN NH
(R)
0
N
Oi
NH
)1..
0
HN 0
of\I)1N1H2
NH
P
0
(D
NH
:S
0'
N¨N
\
3
In some embodiments, the conjugate compound of the present application is
CB-1020 which has the following structural foimula:
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OH
0 H
OH p ...,,))õ,,N,ri,NI-12
0 HN NH
0
1/....(OH (s)
(S) N
HN 0 o(1=..,---1,
Ht.( 0 0 HN
(s) ===.<
OH HO (s) o
0 NH
(R) ....(
HN HN
0 0
HN H2N (s)
NH
o (s) ...,,OH
HN
(s) o
HN N HN--""
01..õ)/N
0
NH 0 HN
HOy.,..= 0
0 (s)
0 HN NH
,N H2 01 0
0 ...,
NH HN
HO,ir,- 0-
0 NH
01
0
NI HN
tO
HN-..õ....õ..,,....
\O
S 0)
0 0-)
.L0
0 N
j
) ii\IH
0
HN 0
o2
H
NH
P
0
0
>... si.s7
0
FII\..<
(s)
0
\etl¨
i .H0
\
0
(isµl)
(R)
(R)
0
NH
(R)
60H
In some embodiments, the conjugate compound of the present application is
CB-1320 which has the following structural foi tnula:
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0 11
F1 ....
OH
(s)
HN 0
H6s) ....80
I,
\OH
HN
0
HN
0
HNa
HN HN
0H N¨'
(s) 0
NH 0 I NH (s)
HOy,. HN N
0 01 ¶ 01-1 1 NH
2
0 HN HN
0 HN NH
0 (,$)0
NH
L H* (S) NH HoN¨c.,_<
O
H HN
H N
N-... js ..,..õ.,õ.,..- 0
0 (0
is
0
0 C-0 ¨) \_ JO
N
Oi
>. /NH
0
HN 0
4Ths..,""N)I'NH2
0 H
NH
P
0
o
)... ,(Ns¨)
o
HN
o
s) N-
- 0 .0
\
o
N\ (s)
Q(R)
(R-
0
NH
(R)
(S) OH
In some embodiments, the conjugate compound of the present application is
CB-1820 which has the following structural foimula:
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OH
0
1....OH
HN 0
0
H
HN 0 N OH
HN . /
FINC))NHO ,
HN (s) NH 0
0 0 0
HN HN (RiN H ,
H041,õ3),
(R
0
O
(c... ;
HN Hd 0
4-Thr(S OH HN 0
0 M
NH NH2
HO(0
HN
HN
0 NH OH HO,r,.... 04
0 NH
01
0
0
HN--_ _______
3
0.--1-:\I
Oj
/ 0
HN I
0NH2
NH
0
oP \ ....sisT
/ o
Ni...<
o
x_to
\
\ (is)
o (R)
(R)
0
NH
((R)
8) OH
In some embodiments, the conjugate compound of the present application is
CR19428 which has the following structural foimula:
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OH
0
OH
(s)
HN 0
HN 0
H2N (s) =
)=N OH
HN
N N
HN
HN HN
0
HN 0
(s)
OH HN
(s) OH
0 0
NH NH
HO( = (s)
0
HN
(s) NH2
= (S) 0
H2N HN NH
(R)
0
N
NH
HN
NH
(s)
0
HN
OR
NH
(o
0 HN
s V \
0 ==
'OH
In some embodiments, the conjugate compound of the present application is
20R-SMO9 which has the following structural foimula:
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H2N
HO 00 0 OH 0 HO
HO (s) N AN isN )-LN 0 0
H H 0 H H 0 0 .$) H 0 (s) ?
N (S) 'LN (s) NH2
H 0 = H 0
0
FIC::
00, _OH HN
0 rCOOH
H
(s) 0 7----
0 NfrN
N -Th
0 N N¨\
N)'NN
1 H \N) COON
H2N N N
H
HOOCH
In some embodiments, the conjugate compound of the present application is
CB-20R which has the following structural foimula:
H2N
HO 0 0, _OH
0 0 HO
HO (s) N)-LN N AN 0 (1::i 0 j.rH 0
H H H H
0 N ka,[1, H j-1,
N (s) N (s) NH2
H , H
0 0
0
HO
0 õOH HN
0 rCOOH
N f-ENI (s) o0 /---_
0
HN- ( "Th
N H )cNN 0
1 H c___.._) COON
H2N N N
H
HOOCH
9
wherein M is a radionuclide.
In some embodiments, the conjugate compound of the present application is
CB-18G which has the following structural foimula:
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OH
H2N 0 H 0
_________________________________________ HN
(s)
(W(s) *
0 NH 0
HN (1'NH
HO
0 (/µµ
0 HN (R)
O
NH
H2N = (S)
)=N HO 0
HN)_t0 HN 0
N N NH2
HN
HO _,_0
N(S) 0
0 0 NH
HN 0 OH
(s) = 4DH
0
NO) NH 0
R)
oi
0
0 N
NH
0
HN 0
0
NH
0
C)
N¨
I(S)
HN
(s)
0
Xs) tFT
0
(SN
0 (R)
(R)
0
NH
(R)
(S) OH
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In some embodiments, the conjugate compound of the present application is
CR19426 which has the following structural foimula:
OH
H 2 N 0 = H 0
(s)
___________________________________________ HN
(s)
Ws) NH
HN (17-NH
HO Vs)
(11'
0
0
NH
H2N
)=N HO 0
HN 0 HN 0
N N NH2
HN
HO HN
(s) 0
0 0 NH
HN 0 OH 0
O
(s) OH
0
HN õIR) 2\IH 0
\ 0
0 N
co
HN
OR
NH
ioHN
NH
(
0
0 HN
0 \ N
s V \
o
In some embodiments, the conjugate compound of the present application is
CB-10S which has the following structural foimula:
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OH
0
(s) H2
HN NH
0
(s)
0
(s)
0 HN
0
HO (s)
NH
0
(R) (
HN
0
H2 N (s)
NH
0
(s)
HN
HN 0
OH
)N (s)
HNON
0 (s)
N N
HN
0
HN (s)
0 NH
(s) =.,,
HN
HN 0
(s)
OH 0 NH
0
H N NH
0
0 N
NH
ro
NH
01
0
NH
0
0
(s 0
0 ,
/ \
N z
OH
0
In some embodiments, the conjugate compound of the present application is
CR19425 which has the following structural foimula:
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OH
o (s)
HN NH
0
o
(s)
0
(s)
HN
0
HO (s)
NH
0
(R) (
HN
0
H2N (s)
NH
0
(s)
HN
HN 0
)=N (s)
HNI)_t0
ON (s)
N N
HN
0
HN (s)
0 NH 0
(s)
0 HN
HN 0
(s)
OH 0 NH
0 NH
HN NH
\O
0 N
NH
HN
NH
(s)
0
HN
NH
(o
0 HN
0 \ N
s V \
0
'OH
In some embodiments, the conjugate compound of the present application is
CB-50S which has the following structural foimula:
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OH
0 (s) FN1 y NH2
HN NH
0
O
(s)
0
(s)
HN
HO (s)
N H
0
(R) .. (
HN
0
H2 N (s)
N H
0
(s)
HN
H2 N 0
)=N (s)
H N 0 N H
)¨ N N 0(s)
HN
0
H N (s)
= 0 N H
(s)
0
HN 0 0
(s)
" OH .. HO
0 N H
0
0 N H2
H N N H
N -14/N
0
NH
0
0
NH
=
0
0 0
0
0
/
N z
OH
0
In another aspect, the present application discloses a pharmaceutical
composition comprising the conjugate compound or the pharmaceutically
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CA 03127903 2021-07-27
acceptable salt thereof of the present application, and a pharmaceutically
acceptable
carrier.
In some embodiments, the composition is administered intravenously,
subcutaneously, orally, intramuscularly or intraventricularly.
In another aspect, the present application discloses a method for delivering a
payload to a subject in need thereof, comprising administering to the subject
a
therapeutically effective amount of the conjugate compound or the
pharmaceutically
acceptable salt thereof of the present application, or the pharmaceutical
composition
of the present application.
In another aspect, the present application discloses a method for treating a
disease in a subject, comprising administering to the subject a
therapeutically
effective amount of the conjugate compound or the pharmaceutically acceptable
salt
thereof of the present application, or the pharmaceutical composition of the
present
application.
In some embodiments, the method for treating a disease in a subject of the
present application further comprises administering one or more therapeutic
agents
in combination with the conjugate compound or the pharmaceutically acceptable
salt thereof, or the pharmaceutical composition.
In yet another aspect, the present application discloses use of the conjugate
compound or the pharmaceutically acceptable salt thereof of the present
application,
or the pharmaceutical composition of the present application in the
preparation of a
drug for treating a disease in a subject.
In yet another aspect, the present application discloses the conjugate
compound
or the pharmaceutically acceptable salt thereof of the present application, or
the
pharmaceutical composition of the present application for treating a disease
in a
subject.
In some embodiments, the disease is selected from the group consisting of a
cancer, an immunological disease, a cardiovascular disease, a metabolic
disease,
and a neurological disease.
In some embodiments, the cancer is selected from the group consisting of
prostatic cancer, breast cancer, lung cancer, renal cancer, leukemia, ovarian
cancer,
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gastric cancer, uterine cancer, endometrial carcinoma, liver cancer, colon
cancer,
thyroid cancer, pancreatic cancer, colorectal cancer, esophageal cancer, skin
cancer,
lymphoma, and multiple myeloma.
In some embodiments, the immunological disease is an autoimmune disease.
In some embodiments, the autoimmune disease is selected from the group
consisting of connective tissue disease, systemic sclerosis, rheumatoid
arthritis, and
systemic lupus erythematosus.
In some embodiments, the cardiovascular disease is selected from the group
consisting of angina, myocardial infarction, stroke, heart attack,
hypertensive heart
disease, rheumatic heart disease, cardiomyopathy, arrhythmia, and congenital
heart
disease.
In some embodiments, the metabolic disease is selected from the group
consisting of diabetes, gout, obesity, hypoglycemia, hyperglycemia, and
dyslipidemia.
In some embodiments, the neurological disease is selected from the group
consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease,
head
injury, multiple sclerosis, vertigo, coma, and epilepsy.
Brief Description of the Drawings
Fig. 1 shows chemical structural foiniulas of conjugate compounds CB-20B,
CB-20BK, CB-60S, CB-60SK, CB-20C, CB-1020, CB-1320, CB-1820, CR19428,
20R-SM09, CB-20R, CB-18G, CR19426, CB-10S, CR19425 and CB-50S.
Fig. 2A shows a curve of Cy5-pep-20BK binding and endocytosis by different
cells (from top to bottom: LNCaP cells, SKOV3 cells, DU145 cells and NCI-H460
cells) over time. Fig. 2B shows a curve of Cy5-pep-20AK binding and
endocytosis
by different cells (from top to bottom: LNCaP cells, SKOV3 cells, DU145 cells
and
NCI-H460 cells) over time.
Fig. 3 shows fluorescence images of Cy5-FA binding and endocytosis by
different cells over time, wherein the fluorescence shown as a complete circle
is the
fluorescence of cell nuclei, and the fluorescence distributed in a spotty
pattern is the
fluorescence of Cy5-FA.
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Fig. 4A shows an inhibitory activity of conjugate compound CB-20BK on the
amplification of tumor cells as shown. Fig. 4B shows an inhibitory activity of
conjugate compound CB-20B on the amplification of tumor cells as shown. Fig.
4C
shows an inhibitory activity of conjugate compound CB-10S on the amplification
of
tumor cells as shown. Fig. 4D shows an inhibitory activity of conjugate
compound
CB-605 on the amplification of tumor cells as shown. Fig. 4E shows an
inhibitory
activity of conjugate compound CB-605K on the amplification of tumor cells as
shown. Fig. 4F shows an inhibitory activity of conjugate compound CB-18G on
the
amplification of tumor cells as shown. Fig. 4G shows an inhibitory activity of
conjugate compound CB-505 on the amplification of tumor cells as shown.
Figs. 5A-5E show tumor suppressive effects of conjugate compound CB-20BK
in mice.
Figs. 6A-6C show tumor suppressive effects of conjugate compound CB-20B
in mice.
Figs. 7A-7E show tumor suppressive effects of conjugate compound CB-18G
in mice.
Figs. 8A-8B show effects of CBP-1018 for injection on tumor volumes of
LU2505 lung cancer model and LU1206 lung cancer model.
Detailed Description of Embodiments
While various aspects and embodiments will be disclosed in the present
application, it is apparent that a person skilled in the art may make various
equivalent changes and modifications to the aspects and embodiments without
deviating from the subject spirit and scope of the present application. The
various
aspects and embodiments disclosed in the present application are only for the
purposes of illustration and are not intended to be limiting, with the true
scope being
indicated by the appended claims. All publications, patents or patent
applications
cited in the present application are incorporated by reference in their
entirety.
Unless defined otherwise, the technical and scientific teinis as used herein
have the
same meanings as commonly understood by a person skilled in the art to which
the
present application belongs.
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As used herein and in the appended claims, the singular foinis "a", "an", and
"the" include plural reference unless the context clearly dictates otherwise.
The
Wails "a" (or "an"), "one or more" and "at least one" can be used
interchangeably
herein. It is also to be noted that the teinis "comprise/comprising",
"include/including", and "have/having" can be used interchangeably.
As used herein and in the appended claims, the teini "analog" includes a
structural analog and a functional analog. The structural analog refers to a
class of
compounds with similar chemical structures, which may comprise one or more
different atoms or one or more different functional groups. The functional
analog
refers to a class of compounds that have the same or similar chemical,
biological or
pharmacological effects. For example, the analogs of folate include
5-methyltetrahydrofolate, 5-formyltetrahydrofolate, methotrexate,
and
5,10-methyl enetetrahydro folate .
As used herein and in the appended claims, the teini "derivative" refers to a
relatively complex compound derived from a parent compound molecule in which
one or more atoms or atomic groups are substituted with other atoms or atomic
groups. For example, camptothecin derivatives include irinotecan, SN-38, Dxd,
topotecan, GI-147211C, 9-aminocamptothecin, 7-hydroxymethyl camptothecin,
7-aminomethyl camptothecin, 10-hydroxyc amptothe c in, (20 S)-c amptothec in,
9-nitrocamptothecin, gimatecan, karenitecin, silatecan, exatecan,
diflomotecan,
belotecan, lurtotecan and S39625
In one aspect, the present application discloses a conjugate compound or a
pharmaceutically acceptable salt thereof, comprising a payload and two
targeting
molecules, wherein the two targeting molecules are a synergistic molecule
moiety
and a prostate-specific membrane antigen ligand moiety, respectively.
In another aspect, the present application discloses a conjugate compound or a
pharmaceutically acceptable salt thereof, comprising a payload and two
targeting
molecules, wherein the two targeting molecules are a synergistic molecule
moiety
and a ligand moiety represented by foimula (I), respectively:
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0
(D-Phe)-Cys-Tyr-(D-Trp)-Lys-Thr-Cys-Thr A¨
/
(I).
In another aspect, the present application discloses a conjugate compound or a
pharmaceutically acceptable salt thereof, comprising a payload and two
targeting
molecules, wherein the two targeting molecules are a synergistic molecule
moiety
and P10, respectively, and the payload is camptothecin or any derivative
thereof
The tem" "payload" as used herein refers to a molecule or material to be
delivered to a target cell or tissue. Without limitation, the payload may be
any
molecule or material that is intended for use in the diagnosis, treatment, or
prevention of a disease in a subject. In some embodiments, the payload has a
molecular weight of less than or equal to about 5 kDa. In some embodiments,
the
payload has a molecular weight of less than or equal to about 1.5 kDa. In some
embodiments, the payload is a drug or a diagnostic reagent that has been
deemed
safe and effective for use by appropriate drug approval and registration
agencies
(such as FDA, EMEA, or NMPA).
In some embodiments, the payload of the present application is a small
molecule compound, a nucleotide (such as a DNA, a plasmid DNA, an RNA, an
siRNA, an antisense oligonucleotide and an aptamer), a peptide, or a protein
(for
example, an enzyme). In some embodiments, the payload is a small molecule
compound.
In some embodiments, the payloads of the present application include, but are
not limited to: an anticancer drug, a radioactive substance, a vitamin, an
anti-AIDS
drug, an antibiotic, an immunosuppressant, an antiviral drug, an enzyme
inhibitor, a
neurotoxin, an opioid, a regulator of cell-extracellular matrix interaction, a
vasodilator, an antihypertensive drug, a hypnotic, an antihistamine, an
anticonvulsant, a muscle relaxant, an anti-Parkinson substance, an
anticonvulsant
and a drug for inhibiting muscle contraction, an antiparasitic drug and/or an
anti-antiprotozoal drug, an analgesic drug, an antipyretic, a steroidal or
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non-steroidal anti-inflammatory drug, an anti-angiogenic factor, an
antisecretory
factor, an anticoagulant and/or an antithrombotic agent, a local anesthetic, a
prostaglandin, an antidepressant, an antipsychotic, an antiemetic, or an
imaging
agent.
In some embodiments, the payload of the present application has a free amino
or carboxyl group before being conjugated with the conjugate compound of the
present application, and the payload is conjugated with the conjugate compound
through an acylation reaction between the above-mentioned amino or carboxyl
group and the corresponding part (for example, a linker) of the conjugate
compound.
In some embodiments, a modification on the above-mentioned free amino or
carboxyl group (for example, by conjugating with the conjugate compound of the
present application) may significantly reduce the activity of the payload (for
example, by at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%).
The "small molecule compound" as used herein refers to a compound having a
molecular weight of less than or equal to about 2 kDa. In some embodiments,
the
small molecule compound has a molecular weight of less than or equal to about
1.5
kDa. In some preferred embodiments, the small molecule compound has a
molecular weight of less than or equal to about 1 kDa, 800 Da, 700 Da, 600 Da,
or
500 Da. In some embodiments, the small molecule compound of the present
application is selected from the group consisting of camptothecin and any
derivative
thereof (for example, SN38 or Dxd), auristatin and any derivative thereof (for
example, MMAE and MMAF), maytansine and any derivative thereof, a
cyclooxygenase-2 inhibitor (for example, celecoxib), a radionuclide complex,
paclitaxel and any derivative thereof, epothilone and any derivative thereof,
bleomycin and any derivative thereof, dactinomycin and any derivative thereof,
plicamycin and any derivative thereof, and mitomycin C. In some embodiments,
the
small molecule compound is camptothecin or any derivative thereof, auristatin
or
any derivative thereof, a radionuclide complex or a cyclooxygenase-2
inhibitor. In
some embodiments, the small molecule compound described in the present
application is a drug for relieving or treating a cancer. In some embodiments,
the
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small molecule compound described in the present application is a drug for
relieving or treating an autoimmune disease.
The ten," "camptothecin" as used herein refers to a cytotoxic alkaloid, mainly
derived from Camptotheca acuminata (Nyssaceae) and showing a strong anti-tumor
activity. The camptothecin and derivative thereof of the present application
include
a camptothecin and a derivative thereof that are already in existence or will
be
produced later. The camptothecin and derivative thereof of the present
application
include, but are not limited to: camptothecin, irinotecan, SN-38, Dxd,
topotecan,
GI-147211C, 9-aminocamptothecin, 7-hydroxymethyl
camptothecin,
7-aminomethyl camptothecin, 10-hydroxyc amptothe c in, (20 S)-c amptothec in,
9-nitrocamptothecin, gimatecan, karenitecin, silatecan, exatecan,
diflomotecan,
belotecan, lurtotecan and S39625.
The tettn "auristatin and any derivative thereof/auristatin or any derivative
thereof' as used herein refers to a natural anti-tumor product, aplysiatoxin
10, and a
series of derivatives thereof, wherein such compounds interfere with
microscopic
self-assembly to arrest cells in a mitotic phase, which has a strong lethality
to the
cells. The auristatin and any derivative thereof of the present application
include
auristatin and any derivative thereof that are already in existence or will be
produced later. The auristatin and derivative thereof of the present
application
include, but are not limited to auristatin, monomethyl auristatin E (MMAE),
monomethyl auristatin F (MMAF), monomethyl auristatin D (MMAD), AFP and
AFHPA.
The tettn "cyclooxygenase-2 inhibitor" as used herein is a class of specific
cyclooxygenase-2 inhibitors. Cyclooxygenase-2 participates in the development
and
infiltration of malignant tumors through a variety of mechanisms.
Cyclooxygenase-2 inhibitors can inhibit tumor cell migration and adhesion and
intravascular infiltration, thereby inhibiting the occurrence and development
of
malignant tumors. The cyclooxygenase-2 inhibitors of the present application
include cyclooxygenase-2 inhibitors that are already in existence or will be
produced later. Cyclooxygenase-2 inhibitors include, but are not limited to
celecoxib, rofecoxib, parecoxib, valdecoxib and etoricoxib.
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The tem" "radionuclide complex" as used herein refers to a special class of
complexes containing radionuclides, wherein the chelating agent in the complex
can
be chelated with the radionuclide and provide a linking part that more stably
binds
to a target substance. The tem" "radionuclide" as used herein refers to an
element
that can spontaneously emit radiation (such as a-rays, I3-rays, or y-rays).
The
radionuclides of the present application include all radionuclides for
treatment and
diagnosis that are already in existence or will be produced later. The
radionuclides
of the present application include, but are not limited to 67cu, 64cu., 90y,
109pd, 111Ag,
149pm, 153sm, 165H0, 166H0, 177Lu, 186-e
K,
188Re, 99mTC, 67Ga, 68Ga, "In, 90y, 177Lu,
186Re, 188Re, 197AU, 198AU, 'Au, io5Rh, 161Tb, 149pm, 44se, 47se, 70As, 71As,
72As,
73AS, 74AS, 76AS, 77As, 212pb, 212Bi, 213Bi, 225Ac, 117msb, 67Ga, 201T1, 1231,
1311, 160Gd,
148Nd, 89Sr and 211At. In some embodiments, the chelating agent is a
macrocyclic
chelating agent. The chelating agents of the present application include, but
are not
limited to H2dedpa, H4octapa, H2azapa, DTPA, CHX-A"-DTPA, DTPA-bis
anhydride, Maleimide-DTPA, DTPA(tBu)4, DiamSar CB-TE2A, Cyclam, DO2A,
DOTA, OTA-GA(tBu)4, Maleimide-DOTA-GA, p-NCS-Bz-DOTA-GA,
NH2-DOTA-GA, DOTA-GA anhydride, DOTA-tris(tBu)
ester,
Propargyl-DOTA-tris(tBu) ester, DO3AM-acetic
acid,
DO3AM-N-(2-aminoethyl)ethanamide,
DO3AtBu-N-(2-aminoethyl)ethanamide,
DOTA-di(tBu)ester, DOTA-tris(tBu)ester NHS ester, DOTA-NHS ester,
Propargyl-DOTA-tris(tBu) ester, DOTADOTA-GA anhydride, DOTA-GA(tBu)4,
p-NCS-Bz-DOTA-GA, NH2-DOTA-GA, Maleimide-DOTA-GA, AGuIX, Gado-H,
CYCLEN, DO2AtBu, DO3AtBu, DO3AEt, DO3AM, DOTAEt, DOTPrEt,
cis-Glyoxal-Cyclen, Mono-N-Benzyl-Cyclen,
trans-N-Dibenyl-Cyclen,
TriB0C-Cyclen, Mono-N-Benzyl-TACN, DiB0C-TACN, Cross-bridge-Cyclam
(CB-Cyclam), (1 3)aneN4, TACN, TACN=3HC1, TACD, Mono-N-benzyl-TACD,
DiB0C-TACD, 1,7-Dioxa-4,10-diazacyclododecane, C-Methyl-Ester-Cyclam,
C-Carboxylic-Acid-Cyclam, trans-N-Dimethyl-Cyclam, TETRAM, TETAEt,
TETAMEt2, TETAMMe2, TETAM, CPTA, CB-Cyclam derivatives, CB-TE2A,
Methylamino-(13)aneN4, Bis-(13)aneN4, Oxo-(1
3)aneN4,
Mono-N-Benzyl-(13)aneN4, TriB0C-(1 3)aneN4, TRITRAM, TRI3AEt, TRI3AtBu,
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TRITAM, TRITA, Mono-N-Benzyl-Cyclam, Foi _______________
inaldehyde-Cyc lam,
c is -Glyoxal-Cyc lam, Dioxocyclam, Oxocyc lam, trans-N-D ibenzyl-Cyc lam,
TriB0C-Cyclam, DOTP, DOTMA, TETA, DOTAM, DiAmSar, CB-Cyclam,
CB-TE2A, NOTA, NOTAM, NH2-NODA-GA, Iodo-NODA-GA, NCS-MP-NODA,
NH2-MPAA-NODA, NODA-GA(tBu)3, NODA-GA-NHS ester,
Maleimide-NODA-GA, NO TA -NH S ester,
Maleimide-NOTA,
Propargyl-NOTA(tBu)2, p-NCS-benzyl-NODA-GA, NOTA(tBu)2, NCS-MP-NODA,
NH2-MPAA-NODA, NH2-NODA-GA, Iodo-NODA-GA and TACN.
In some embodiments, the conjugate compound or the pharmaceutically
acceptable salt thereof of the present application comprises one payload. In
some
embodiments, the conjugate compound or the pharmaceutically acceptable salt
thereof of the present application comprises two or more payloads. For
example, the
conjugate compound or the pharmaceutically acceptable salt thereof of the
present
application comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19,
20 or more payloads. In a conjugate compound containing multiple payloads,
each
of the payloads may be identical to or different from each other. In some
embodiments, at least two of the payloads are different from each other.
The term "targeting molecule" as used herein refers to any molecule or moiety
capable of targeting the conjugate compound of the present application to a
target
site, a target tissue, a target organ, a target cell, or a target
intracellular region. In
some embodiments, the targeting molecule allows the conjugate compound of the
present application to be distributed more in a target site, a target tissue,
a target
organ, a target cell or a target intracellular region compared with a non-
target site, a
non-target tissue, a non-target organ, a non-target cell or a non-target
intracellular
region, for example, at least 10% more, 20% more, 50% more, 80% more, 100%
more, 150% more, 200% more, 300% more, 400% more, 500% more, etc. In some
embodiments, the targeting molecule allows a conjugate compound containing a
targeting molecule, compared with a conjugate compound containing no targeting
molecule, to be distributed more in a target site, a target tissue, a target
organ, a
target cell or a target intracellular region, for example, at least 10% more,
20% more,
50% more, 80% more, 100% more, 150% more, 200% more, 300% more, 400%
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more, 500% more, etc. In some embodiments, the targeting molecule can trigger
or
promote a conjugate compound containing such targeting molecules to
specifically
bind to a target molecule, trigger or promote endocytosis of the conjugate
compound by a target cell, and trigger or promote the conjugate compound to be
enriched around a target cell and/or enter a target cell.
In some embodiments, the conjugate compound of the present application
comprises at least two targeting molecules. In some embodiments, the two or
more
targeting molecules comprised in the conjugate compound of the present
application
are identical or different. In some embodiments, at least two targeting
molecules of
the two or more targeting molecules comprised in the conjugate compound of the
present application are different. In some embodiments, the two or more
targeting
molecules comprised in the conjugate compound of the present application are
all
different from each other. In some embodiments, at least two targeting
molecules of
the two or more targeting molecules comprised in the conjugate compound of the
present application can specifically bind to different cell surface proteins
or markers.
In some embodiments, the two or more targeting molecules comprised in the
conjugate compound of the present application can specifically bind to
different cell
surface proteins or markers.
In some embodiments, the conjugate compound of the present application
comprises at least two targeting molecules, at least one of which is a
synergistic
molecule.
The teini "synergistic molecule" as used herein refers to any molecule or
moiety that is capable of working synergistically with other targeting
molecules
comprised in the conjugate compound of the present application to better
trigger or
promote the conjugate compound to specifically bind to a target molecule,
trigger or
promote the endocytosis of the conjugate compound by a target cell, trigger or
promote the conjugate compound to be enriched around a target cell and/or
enter a
target cell, and/or cause the conjugate compound, in a different manner, to
specifically bind to a target cell and be maintained. In some embodiments, the
synergistic molecule allows the conjugate compound of the present application
to be
distributed more in a target site, a target tissue, a target organ, a target
cell or a
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target intracellular region compared with a non-target site, a non-target
tissue, a
non-target organ, a non-target cell or a non-target intracellular region, for
example,
at least 10% more, 20% more, 50% more, 80% more, 100% more, 150% more,
200% more, 300% more, 400% more, 500% more, etc. In some embodiments, the
synergistic molecule allows a conjugate compound containing a synergistic
molecule, compared with a conjugate compound containing no synergistic
molecule,
to be distributed more in a target site, a target tissue, a target organ, a
target cell or a
target intracellular region, for example, at least 10% more, 20% more, 50%
more,
80% more, 100% more, 150% more, 200% more, 300% more, 400% more, 500%
more, etc. In some embodiments, the synergistic molecule allows a conjugate
compound containing a synergistic molecule, compared with a conjugate compound
containing no synergistic molecule, to have a higher activity on a target
cell, for
example, at least 10% higher, 20% higher, 50% higher, 80% higher, 100% higher,
150% higher, 200% higher, 300% higher, 400% higher, 500% higher, etc.
In some embodiments, the synergistic molecule of the present application is a
cell-interacting molecule.
The teini "cell-interacting molecule" as used herein refers to a molecule that
is
capable of interacting with a cell surface material of a target cell to
trigger or
promote a conjugate compound containing such cell-interacting molecules to
specifically bind to a cell, to trigger or promote endocytosis of the
conjugate
compound by a target cell, and/or to trigger or promote the conjugate compound
to
be enriched around a target cell and/or enter a target cell.
The cell-interacting molecule may be a small chemical molecule or a large
biomolecule. In some embodiments, the cell-interacting molecule is a small
molecule compound or a polypeptide. In some embodiments, the cell-interacting
molecule is a small molecule compound, or a polypeptide comprising 2-50, 2-40,
2-30, 2-25, 2-22, 2-20, 2-18, 2-15, 2-12, 2-10, 2-8, 4-50, 5-50, 5-40, 5-30, 5-
25,
5-22, 5-20, 5-18, 5-15, 5-12, 5-10, 6, 7, 8, or 9 amino acids.
In some embodiments, the targeting molecule is a ligand capable of binding to
a cell surface receptor or other molecules. In some embodiments, at least one
of the
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targeting molecules is a ligand capable of binding to a cell surface receptor
or other
molecules.
The ligands of the present application can include a variety of chemical or
biological molecules, which can have a specific binding affinity to a selected
target,
wherein the selected target can be, for example, a cell surface receptor, a
cell
surface antigen, a cell, a tissue, an organ, etc. In some embodiments, the
ligand can
specifically bind to a protein or a marker expressed on the surface of a
target cell. In
some embodiments, the ligand of the present application binds to a cell
surface
protein or marker with an affinity of 1 0-6-10-11M (Ka value). In some
embodiments,
113 the ligand of the present application binds to a cell surface protein
or marker with an
affinity of at least 10-7, at least 10-8 and at least 10-9 M (Ka value). In
some
embodiments, the ligand of the present application binds to a cell surface
protein or
marker with an affinity of less than 10-6, less than 10-7 and less than 10-8 M
(Ka
value). In some embodiments, the ligand of the present application binds to a
cell
surface protein or marker with a certain affinity, wherein the certain
affinity refers
to the affinity of the ligand to a target cell surface protein or marker which
is at least
two, three, four, five, six, eight, ten, twenty, fifty, one hundred or more
times higher
than that to a non-target cell surface protein or marker. In some embodiments,
the
expression of the cell surface protein or marker of the present application in
target
cells (e.g. cancer cells) is significantly higher than that in nottnal cells.
The tettn
"significantly" as used herein refers to statistically significant
differences, or
significant differences that can be recognized by a person skilled in the art.
In some embodiments, the expression level of the cell surface protein or
marker of the present application in target cells (e.g. cancer cells) are 2 to
1,000,000
times higher than that in normal cells; for example, the expression level in
target
cells (e.g. cancer cells) are 2 to 10, 2 to 100, 2 to 1,000, 2 to 10,000, 2 to
100,000 or
2 to 1,000,000 (which can be equal to any value within the above numerical
range,
and the end values of this range included) times higher than that in nottnal
cells. In
some embodiments, the expression level of the cell surface receptor in target
cells
(e.g. cancer cells) is at least 10 times higher, or 100 times higher, or 1,000
times
higher, or 10,000 times higher, or 100,000 times higher than that in nottnal
cells. In
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some embodiments, compared with the level of the cell surface protein or
marker on
target cells (e.g. cancer cells), the level of the cell surface receptor on
nonnal cells is
reduced by at least 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some
embodiments, the cell surface protein or marker described in the present
application
is undetectable in nonnal cells.
In some embodiments, the cell surface protein or marker of the present
application is a cell surface receptor.
In some embodiments, the cell surface receptor of the present application is
selected from the group consisting of a transferrin receptor (TFR), a low-
density
lipoprotein receptor (LDLR), a folate receptor (FR), a growth hoinione-
inhibiting
hormone receptor, a uric acid kinase receptor, a tumor necrosis factor
receptor
(TNFR), an integrin receptor (LFA-1), an SST-14 receptor (SSTR2), a GNRH
receptor (GNRHR), a TRPV6 and an integrin a receptor.
In some embodiments, the cell surface protein or marker of the present
application is a cell surface antigen.
In some embodiments, the cell surface antigen of the present application is
selected from the group consisting of a prostate-specific membrane antigen, a
MUC1 mucin, an acute lymphoblast common antigen, a Thy-1 cell surface antigen,
a Melan-A protein, a squamous cell carcinoma antigen, a galectin 3 and a human
leukocyte antigen.
In some embodiments, the cell-interacting molecule of the present application
can bind to a molecule selected from the group consisting of FOLR1, TRPV6,
FOLH1 (PMSA), GNRHR, Her2, Trop2, Her3, NECTIN4, LRP1, GLUT1, EGFR1,
AXL, CA9, CD44, Claudin18.2, APN, DLL3, CEACAM5, FZD10, TFRC, MET,
IGFR1, SSTR2, CCKBR, LFA1, ICAM, GPR87, GM-CSF, GM-CSFR, TIM3, TLR
family, CD40, CD4OL, 0X40, OX4OL, GITRL, GITR, 4-BBL, 4-1BB, CD70,
CD27, ICOSL, ICOS, HHLA2, CD28, CD86/80, CD28, MHCII antigen, TCR,
CTLA-4, CD155, CD122, CD113, IGIT, PD-L1, PD1, Galectin-9, TIM-3, HVEM,
BTLA, CD160, VISTA, B7-H4, B7-H3, phosphatidylserine, HHLA2, LAG3,
Galectin-3, LILRB4, 5IGLEC15, NKG2A, NKG2D, SLAMF7, KIR2DL1,
KIR2DL2, KIR2DL3, FGFR1, FGFR2, FGFR4, NeuGcGM3 and CXCR4.
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In some embodiments, the conjugate compound or the pharmaceutically
acceptable salt thereof of the present application comprises a prostate-
specific
membrane antigen ligand moiety and a synergistic molecule moiety, wherein the
synergistic molecule moiety binds to a molecule selected from the group
consisting
of FOLR1, TRPV6, FOLH1 (PMSA), SSTR2 and GNRHR.
In some embodiments, the conjugate compound or the pharmaceutically
acceptable salt thereof of the present application comprises a ligand moiety
represented by formula (I) and a synergistic molecule moiety, wherein the
synergistic molecule moiety binds to a molecule selected from the group
consisting
of FOLR1, TRPV6, SSTR2 and GNRHR.
In yet some embodiments, the conjugate compound or the pharmaceutically
acceptable salt thereof of the present application comprises P10 and a
synergistic
molecule moiety, wherein the synergistic molecule binds to a molecule selected
from the group consisting of FOLR1, TRPV6, FOLH1 (PMSA) and GNRHR.
In some embodiments, one of the synergistic molecules in the conjugate
compound or the pharmaceutically acceptable salt thereof of the present
application
is an endocytosis molecule moiety capable of mediating endocytosis. The term
"endocytosis" as used herein means that the conjugate compound or the
pharmaceutically acceptable salt thereof interacts with a target cell and then
is
capable of mediating its own endocytosis, internalization or uptake by the
target cell.
The teini "endocytosis molecule" as used herein refers to a molecule that
interacts
with a target cell and then is capable of mediating the endocytosis,
internalization,
or uptake of the conjugate compound or the pharmaceutically acceptable salt
thereof
of the present application by the target cell.
In some embodiments, the endocytosis molecule is selected from the group
consisting of a folate and an analog thereof, a peptide capable of mediating
endocytosis, and a cell-penetrating peptide.
In some embodiments, the endocytosis molecule of the present application is a
folate or an analog thereof
Folate is beneficial for forming a chemical bond with other groups due to its
small molecule weight, non-immunogenicity, and good stability. Folate can be
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associated with a folate receptor expressed on a cell surface with a high
affinity to
mediate a cellular uptake of the folate. Although expressed at a very low
level in
most normal cells, a folate receptor is expressed at a high level in numerous
cancer
cells to meet the high folate demand of rapidly dividing cells under a low
folate
condition (see Kelemen LE, Int J Cancer, 2006; 119:243-50; Kane MA, et al., J
Clin Invest. 1988; 81: 1398-406; Matsue H, et al., Proc Natl Acad Sci USA.
1992;
89: 6006-9; Zhao R, et al., Annu Rev Nutr. 2011; 31: 177-201). Folate is
capable of
specifically binding to a folate receptor on a cell surface, and is also
capable of
mediating endocytosis of a conjugate compound or a pharmaceutically acceptable
salt thereof into target cells.
In some embodiments, the analog of folate is selected from the group
consisting of 5-methyltetrahydrofolate, 5-formyltetrahydrofolate,
methotrexate, and
5,10-methyl enetetrahydro folate .
In some embodiments, the endocytosis molecule is a peptide capable of
mediating endocytosis.
In some embodiments, the peptide capable of mediating endocytosis comprises
an amino acid sequence selected from the group consisting of SEQ ID NO: 16,
SEQ
ID NO: 17, SEQ ID NO: 18 and Arg-Gly-Asp (named as RGD), and homologous
peptides having at least 70%, at least 80%, at least 85%, at least 90%, at
least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at
least 98%, at least 99% amino acid sequence homology to any of SEQ ID NOs:
16-18, wherein the homologous peptides are functional equivalents of the
peptides
as shown in SEQ ID NOs: 16-18, respectively.
In some embodiments, the peptide capable of mediating endocytosis as
described in the present application has a conservative substitution of an
amino acid
at only one amino acid site compared to the sequences of SEQ ID NOs: 16-20 and
RGD. In some embodiments, the peptide capable of mediating endocytosis as
described in the present application has a conservative substitution of an
amino acid
at 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid sites compared to the sequences of
SEQ ID
NOs: 16-20.
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Under the premise of not affecting its biological activity, the peptide
capable of
mediating endocytosis as described in the present application may also contain
non-naturally occurring amino acids, including, for example, I3-fluoroalanine,
1-methyl-histidine, y-methylene-glutamic acid, a-
methyl-leucine,
4,5 -dehydro-lysine, hydroxypro line, 3 -fluoro-phenylalanine, 3-amino-
tyrosine,
4-methyl-tryptophan, and the like.
The percentage of homology can be determined by various well-known
methods in the art. For example, the comparison of sequences can be achieved
by
the following publically available tools: BLASTp software (available from the
website of National Center for Biotechnology Infoitnation (NCBI):
http://blast.ncbi.nlm.nih.gov/Blast.cgi; also see: Altschul S.F. et al., J.
Mol. Biol.,
215:403-410 (1990); Stephen F. et al, Nucleic Acids Res., 25:3389-3402
(1997)),
ClustalW2 (available from the website of European Bioinfounatics Institute:
http://www.ebi.ac.uk/Tools/msa/clustalw2/; also see: Higgins D. G. et al.,
Methods
in Enzymology, 266:383-402 (1996); Larkin M.A. et al., Bioinformatics (Oxford,
England), 23(21): 2947-8 (2007)), and Tcoffee (available from the website of
Sweden Bioinformatics Institute; also see: Poirot 0. et al., Nucleic Acids
Res.,
31(13): 3503-6 (2003); Notredame C. et al., J. Mol. Boil., 302(1): 205-17
(2000)). If
the alignment of sequences is performed using a software, the default
parameters
available in the software may be used, or otherwise the parameters may be
customized to suit the alignment purpose. All of these are within the scope of
knowledge of a person skilled in the art.
The term "functional equivalent" as used herein refers to a derivative peptide
that retains a biological activity that is substantially similar to that of
the original
peptide sequence that the derivative peptide derives from. The functional
equivalent
may be a natural derivative or is prepared synthetically. Exemplary functional
equivalents include amino acid sequences having substitutions, deletions, or
additions of one or more amino acids, provided that the biological activity of
a
peptide is maintained. The amino acid used for substitution desirably has
chemico-physical properties similar to the amino acid to be substituted.
Desirable
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similar chemico-physical properties include, similarities in charges,
bulkiness,
hydrophobicity, hydrophilicity, and the like.
In some embodiments, the functional equivalents include a conservative
substitution of an amino acid residue. The conservative substitution of an
amino
acid residue refers to a substitution between amino acids with similar
properties, for
example, a substitution between polar amino acids (such as a substitution
between
glutamine and asparagine), a substitution between hydrophobic amino acids
(such
as a substitution among leucine, isoleucine, methionine and valine), a
substitution
between amino acids with identical charges (such as a substitution among
arginine,
lysine and histidine, or a substitution between glutamic acid and aspartic
acid), etc.
In some embodiments, the endocytosis molecule is a cell-penetrating peptide.
Cell-penetrating peptides (CPPs), also known as protein transduction domains
(PTDs), are short peptides (generally less than 40 amino acids), with the
ability to
gain access to the interior of cells in a receptor-independent manner. The
cell-penetrating peptides, when conjugated with payloads, are capable of
mediating
the transmembrane transport of the payloads and have a protein transduction
activity.
In some embodiments, the cell-penetrating peptide described in the present
application is selected from the group consisting of a tumor-homing peptide, a
mitochondrial penetrating peptide, an activatable cell-penetrating peptide,
and an
antibacterial peptide. In some embodiments, the cell-penetrating peptide
comprises
an amino acid sequence selected from the group consisting of SEQ ID NO: 19
(RRRRRRRRR, named as R9) and SEQ ID NO: 20 (GRKKRRQRRRPPQ, which
is a Tat peptide, i.e. a cell-penetrating peptide of the HIV transactivator of
transcription protein).
In some embodiments, one targeting molecule in the conjugate compound or
the pharmaceutically acceptable salt thereof of the present application is a
prostate-specific membrane antigen ligand moiety.
The term "prostate-specific membrane antigen" as used herein refers to a type
II transmembrane glycoprotein that exists in the membrane of prostate
epithelial
cells and consists of 750 amino acids, comprising 19 amino acids in the
intracellular
region, 24 amino acids in the transmembrane region and 707 amino acids in the
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extracellular region. The prostate-specific membrane antigen is expressed in
normal
prostate epithelial cells, but is expressed at a much higher level in prostate
cancer
cells. Compared with the traditional prostate-specific antigen used for
clinical
detection, the prostate-specific membrane antigen is a more sensitive and
specific
prostate cancer tumor marker, and especially, it is highly expressed in both
hormone-refractory prostate cancer and prostate cancer metastatic lesions, and
has a
high sensitivity and specificity in distinguishing prostate cancer from other
types of
malignant tumors. Moreover, in a variety of nonprostate solid tumors (such as
lung
cancer, bladder cancer, gastric cancer, pancreatic cancer, kidney cancer and
colorectal cancer), the prostate-specific membrane antigen is also highly and
specifically expressed on tumor vascular endothelial cells.
The tem" "prostate-specific membrane antigen ligand" as used herein refers to
an antibody, an aptamer and a small molecule that is capable of specifically
recognizing and binding to a prostate-specific membrane antigen. The
prostate-specific membrane antigen ligands of the present application include
the
prostate-specific membrane antigen ligands that are already in existence or
will be
produced later, as well as fragments of the aforementioned ligands, as long as
these
fragments still retain the ability to bind to a prostate-specific membrane
antigen.
Antibody ligands are the most common prostate-specific membrane antigen
ligands,
which include, but are not limited to monoclonal antibodies J591, J533, J415
and
E99 (for example, see Liu H, Rajasekaran AK, Moy P et al. Constitutive and
antibody-induced internalization of prostate-specific memberane antigen [J].
Cancer Res, 1998, 58 (18): 4055-4060). The aptamer is a single-stranded DNA or
RNA that is obtained through technical screening by an exponential enrichment
ligand system and can bind to prostate-specific membrane antigens with a high
affinity and a high specificity. Such prostate-specific membrane antigen
ligands
include, but are not limited to an xPSM-A10 aptamer and a derivative thereof
and
an xPSM-A9 aptamer and a derivative thereof (for example, see Lupoid SE et
al.,
Identification and Characterization of nuclease-stabilized RNA molecules that
bind
human prostate cancer cells via the prostate-specific membrane antigen, Cncer
Res,
2002, 62(14):4029-4033). Compared with antibody ligands and aptamer ligands,
the
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prostate-specific membrane antigen small molecule ligands have the advantages
of
small molecular weight, high pettneability, low immunogenicity, and ease of
synthesis, and include but are not limited to glutamine urea small molecule
ligands
and phosphoramidate small molecule ligands.
In some embodiments, the prostate-specific membrane antigen small mole
cule ligands of the present application can be selected from the group consist
ing of 2-[[methylhydroxyphosphinyl]methyl]glutaric acid; 2-[[ethylhydroxyphos
phinyl]methyl]glutaric acid; 2-[[propylhydroxyphosphinyl]methyl]glutaric acid;
2-[[butylhydroxyphosphinyl]methyl]glutaric acid; 2-[[cyclohexylhydroxyphosphin
yl]methyl] glutaric acid; 2- [[phenylhydroxyphosphinyl]methyl] glutaric acid;
2- [ [2
-(tetrahydrofuranyl)hydroxyphosphinyl]methyl]glutaric acid; 2-[[(2-
tetrahydropyra
nyl)hydroxyphosphinyl]methyl] glutaric acid; 2 - [ [((4-
pyridyl)methyl)hydroxyphos
phinyl]methyl] glutaric acid; 2 - [ [((2-
pyridyl)methyl)hydroxyphosphinyl]methyl] gl
utaric acid; 2-[[(phenylmethyl)hydroxyphosphinyl]methyl]glutaric acid; 2-[[((2-
p
henylethyl)methyl)hydroxyphosphinyl]methyl] glutaric acid; 2- [ [((3 -
phenylpropyl)
methyl)hydroxyphosphinyl]methyl] glutaric acid; 2- [ [((3 -
phenylbutyl)methyl)hydr
oxyphosphinyl]methyl]glutaric acid; 2-[[((2-phenylbutyl)methyl)hydroxyphosphin
yl]methyl] glutaric acid; 2- [ [(4-phenylbutyl)hydroxypho sphinyl] methyl]
glutaric ac
id; and 2-[[(aminomethyl)hydroxyphosphinyl]methyl]glutaric acid; 2-[[methyl h
ydroxyphosphinyl] oxy] glutaric acid; 2- [ [ethyl hydroxyphosphinyl] oxy]
glutaric a
cid; 2-[[propyl hydroxyphosphinyl]oxy]glutaric acid; 2-[[butyl hydroxyphosphin
yl] oxy] glutaric acid; 2- [ [phenyl hydroxypho sphinyl] oxy] glutaric acid; 2-
[ [((4-py
ridyl)methyl)hydroxyphosphinyl] oxy] glutaric acid; 2- [ [((2-
pyridyl)methyl)hydrox
yphosphinyl] oxy] glutaric acid; 2- [ [(phenylmethyl)hydroxyphosphinyl] oxy]
glutaric
acid; and 2[[((2-phenylethyl)methyl)hydroxyphosphinyl]oxy]glutaric acid; 2-
[[(n-hydroxyl)carbamoyl]methyl]glutaric acid; 2-[[(n-hydroxyl-n-methyl)carbamo
yl]methyl]glutaric acid; 2-[[(n-butyl-n-hydroxyl)carbamoyl]methyl]glutaric
acid;
2-[[(n-benzyl-n-hydroxyl)carbamoyl]methyl]glutaric acid; 2-[[(n-hydroxyl-n-
phen
yl)carbamoyl]methyl]glutaric acid; 2-[[(n-hydroxyl-n-2-phenylethyl)carbamoyl]m
ethyl]glutaric acid; 2-[[(n-ethyl-n-hydroxyl)carbamoyl]methyl]glutaric acid; 2-
[ [(n-hydroxyl-n-propyl)carbamoyl]methyl] glutaric acid; 2- [ [(n-hydroxyl-n-3
-phen
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ylpropyl)carbamoyl]methyl]glutaric acid; 2-[[(n-hydroxyl-n-4-
pyridyl)carbamoyl]
methyl]glutaric acid; 2-[[(n-hydroxyl)amide]methyl]glutaric acid; 2-[[n-
hydroxyl
(methyl)amide]methyl]glutaric acid; 2-[[n-
hydroxyl(benzyl)amide]methyl]glutaric
acid; 2-[[n-hydroxyl(phenyl)amide]methyl]glutaric acid; 2-[[n-hydroxyl(2-pheny
lethyl)amide]methyl]glutaric acid; 2-[[n-hydroxyl(ethyl)amide]methyl]glutaric
aci
d; 2-[[n-hydroxyl(propyl)amide]methyl]glutaric acid; 2-[[n-hydroxyl(3-
phenylpro
pyl)amide]methyl]glutaric acid; and 2-[[n-hydroxyl(4-
pyridyl)amide]methyl]gluta
ric acid; 2-[(thionyl)methyl]glutaric acid; 2-[(methylthionyl)methyl]glutaric
acid;
2-[(ethylthionyl)methyl]glutaric acid; 2-[(propylthionyl)methyl]glutaric acid;
2-
[(butylthionyl)methyl]glutaric acid; 2-[(phenylthionyl]methyl]glutaric acid; 2-
[[(2-phenylethyl)thionyl]methyl]glutaric acid; 2-[[(3-
phenylpropyl)thionyl]methyl]
glutaric acid; 2-[[(4-pyridyl)thionyl]methyl]glutaric acid; 2-
[(benzylthionyl)meth
yl]glutaric acid; 2-[(sulfonyl)methyl]glutaric acid; 2-
[(methylsulfonyl)methyl]glu
taric acid; 2-[(ethylsulfonyl)methyl]glutaric acid; 2-
[(propylsulfonyl)methyl]gluta
ric acid; 2-[(butylsulfonyl)methyl]glutaric acid; 2-
[(phenylsulfonyl]methyl]glutar
ic acid; 2-[[(2-phenylethyl)sulfonyl]methyl]glutaric acid; 2-[[(3-
phenylpropyl)sul
fonyl]methyl]glutaric acid; 2-[[(4-pyridyl)sulfonyl]methyl]glutaric acid; 2-
[(benz
ylsulfonyl)methyl]glutaric acid; 2-[(sulfoximinyl)methyl]glutaric acid; 2-
[(methy
lsulfoximinyl)methyl]glutaric acid; 2-[(ethylsulfoximinyl)methyl]glutaric
acid; 2-
[(propylsulfoximinyl)methyl]glutaric acid; 2-
[(butylsulfoximinyl)methyl]glutaric
acid; 2-[(phenylsulfoximinyl]methyl]glutaric acid; 2-[[(2-
phenylethyl)sulfoximiny
l]methyl]glutaric acid; 2-[[(3-phenylpropyl)sulfoximinyl]methyl]glutaric acid;
2-
[[(4-pyridyl)sulfoximinyl]methyl]glutaric acid; and 2-
[(benzylsulfoximinyl)methy
l]glutaric acid; n- [methyl hydroxyphosphinyl]glutamic acid; n- [ethyl
hydroxyph
osphinyl]glutamic acid; n-[propyl hydroxyphosphinyl]glutamic acid; n4butyl hy
droxyphosphinyl]glutamic acid; n- [phenyl hydroxyphosphinyl]glutamic acid; n-
[(phenylmethyl)hydroxyphosphinyl]glutamic acid; n-[((2-phenylethyl)methyl)hydr
oxyphosphinyl]glutamic acid; and N-methyl-N4phenylhydroxyphosphinyl]gluta
mic acid. The prostate-specific membrane antigen ligands of the present appli
cation also include all prostate-specific membrane antigen small molecule liga
nds disclosed in PCT applications WO 2010/108125 and WO 2006/093991, t
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CA 03127903 2021-07-27
he above-mentioned two patent applications are incorporated herein in their e
ntirety.
In some embodiments, the prostate-specific membrane antigen small molecule
ligand of the present application is a glutaric acid derivative. In some
embodiments,
the prostate-specific membrane antigen small molecule ligand of the present
application is an aminocarbonyl derivative of glutaric acid.
In some embodiments, the prostate-specific membrane antigen small molecule
ligand of the present application has the following structure:
HO 0
0
H0(
(s) NAN1/4
H H
0
In some embodiments, the prostate-specific membrane antigen ligand
comprised in the conjugate compound or the pharmaceutically acceptable salt
thereof has the following structure:
HO 00 0 OH
HO
(s) NANiNC.,
H H H
0 .
In some embodiments, the prostate-specific membrane antigen ligand
comprised in the conjugate compound or the pharmaceutically acceptable salt
thereof has the following structure:
HO 00 0, _OH
0
HO (S) NANNAN 0
H H H H
0
In some embodiments, the prostate-specific membrane antigen ligand
comprised in the conjugate compound or the pharmaceutically acceptable salt
thereof has the following structure:
HO 0 0 , -OH
0 0 HO
H 0 N AN : NA N 0
(s) (S) 0
H H H H
N (S)
H
0 \
0
H 0 .
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In some embodiments, the prostate-specific membrane antigen ligand
comprised in the conjugate compound or the pharmaceutically acceptable salt
thereof has the following structure:
H2N
HO 0 0 OH
0 0 HO
_
0
HO
(S) NANNAN 0 i_i 0
H H H H
0 j(isj,(sA
H i H
0 \
0 0
HO
In some embodiments, one targeting molecule in the conjugate compound or
the pharmaceutically acceptable salt thereof of the present application is a
ligand
moiety represented by formula (I):
0
> _____________________ (D-Phe)-Cys-Tyr-(D-Trp)-Lys-Thr-Cys-Thr A¨
/
(I),
or a ligand moiety having at least 70%, at least 80%, at least 85% or at least
90% amino acid sequence homology thereto or having at most 3, 2 or 1 amino
acid
substitutions (for example, conservative substitutions) therewith.
In some embodiments, the one targeting molecule in the conjugate compound
or the pharmaceutically acceptable salt thereof of the present application is
P10 or a
ligand moiety having at least 70%, at least 80%, at least 85%, at least 90%,
at least
91%, at least 92% or at least 93% amino acid sequence homology thereto or
having
at most 3, 2 or 1 amino acid substitutions (for example, conservative
substitutions)
therewith.
The term "P10" as used herein refers to a peptide having an amino acid
sequence Cys-Lys-Glu-Phe-Leu-His-Pro-Ser-Lys-Val-Asp-Leu-Pro-Arg.
In some embodiments, the conjugate compound of the present application has
targeting molecules selected from the group consisting of (1) a folate ligand
and a
prostate-specific membrane antigen ligand; (2) a TRPV6 ligand and a
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prostate-specific membrane antigen ligand; (3) a GNRHR ligand and a
prostate-specific membrane antigen ligand; (4) an SSTR2 ligand and a
prostate-specific membrane antigen ligand; (5) a folate ligand and an SSTR2
ligand;
or (6) a TRPV6 ligand and a folate ligand.
In some embodiments, the two targeting molecules in the conjugate compound
or the pharmaceutically acceptable salt thereof provided in the present
application
are a synergistic molecule moiety and a prostate-specific membrane antigen
ligand
moiety, respectively. In some embodiments, the synergistic molecule is capable
of
mediating endocytosis. In some embodiments, the two targeting molecules in the
conjugate compound or the pharmaceutically acceptable salt thereof provided in
the
present application are a folate or an analog thereof and a prostate-specific
membrane antigen ligand moiety, respectively. Without wishing to be limited by
the
theory, a specific folate or an analog thereof and a prostate-specific
membrane
antigen ligand moiety that have a better stability than the ligand
combinations in the
prior art are selected.
In some embodiments, the two targeting molecules in the conjugate compound
or the pharmaceutically acceptable salt thereof provided in the present
application
are a synergistic molecule moiety and a ligand moiety represented by formula
(I),
respectively. In some embodiments, the synergistic molecule is capable of
mediating endocytosis. In some embodiments, the two targeting molecules in the
conjugate compound or the pharmaceutically acceptable salt thereof provided in
the
present application are a folate or an analog thereof and a ligand moiety
represented
by formula (I), respectively.
In some embodiments, the two targeting molecules of the conjugate compound
or the pharmaceutically acceptable salt thereof provided in the present
application
are a synergistic molecule moiety and P10, respectively. In some embodiments,
the
synergistic molecule is capable of mediating endocytosis. In some embodiments,
the two targeting molecules of the conjugate compound or the pharmaceutically
acceptable salt thereof provided in the present application are a folate or an
analog
thereof and P10, respectively.
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In some embodiments, the conjugate compound provided in the present
application only comprises a single payload conjugated with the two targeting
molecules. In some embodiments, the conjugate compound provided in the present
application comprises multiple payloads conjugated with the two targeting
molecules.
The teini "conjugated" as used herein refer to the linking through a covalent
bond of two chemical groups, either directly founing a covalent bond between
the
two chemical groups, or indirectly linking the two chemical groups via a
linker.
In some embodiments, the conjugate compound or the phannaceutically
acceptable salt thereof comprises a payload(s) (for example, 1 payload) and
two
targeting molecules, wherein the payload is directly covalently linked to at
least one
of the targeting molecules. In some embodiments, the payload is directly
covalently
linked to the two targeting molecules.
In some embodiments, the conjugate compound or the phannaceutically
acceptable salt thereof comprises a payload(s) (for example, 1 payload) and
two
targeting molecules, wherein the payload is covalently linked to at least one
of the
targeting molecules via a linker. In some embodiments, the payload is
covalently
linked to the two targeting molecules via a linker.
The teini "linker" as used herein refers to a molecule or moiety that
covalently
links a payload to a targeting molecule. The linkers include a functional
group for
linking a payload to at least one targeting molecule. In some embodiments, the
functional group may comprise two reactive moieties, one for linking to a
payload
and the other for linking to a targeting molecule. In some embodiments, the
functional groups are different from each other. In some embodiments, the
functional groups include a group containing a thiol-reacting moiety and an
amine-reacting moiety. In some embodiments, the functional groups are
identical to
each other. In some embodiments, the functional groups are maleimide groups.
In
some embodiments, the linker contains an amino acid. In some embodiments, the
carboxylic acid in the amino acid contained in the linker is amidated. In some
embodiments, the linker contains a short chain polyethylene glycol (for
example,
comprising 2-10, 2-8, 3-8, 4-8, 4-7, 4-6, or 5 repeating units).
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In some embodiments, the linker of the present application is a multivalent
linker capable of binding to at least one (for example, 1, 2, 3, 4, 5, 6, 7,
8, 9, 10 or
more) payload and at least one targeting molecule. The payloads bound to the
multivalent linker may be identical or different, and the targeting molecules
bound
to the multivalent linker may be identical or different.
In one aspect, the linker shall be sufficiently stable to avoid from
unintended
release of payloads during a blood circulation to increase an effective amount
of
payloads delivered to target cells or tissues and avoid toxicity. In another
aspect, the
linker shall be capable of releasing the payloads around or within target
cells to
efficiently kill target cells or block functions of target cells. In some
embodiments,
the linker comprises at least one cleavable functional group. Preferably, the
cleavable functional group is sufficiently stable outside a target cell, but
upon entry
into the target cell, is cleaved to release a payload(s). In some embodiments,
the
cleavable functional group is cleaved at least 10, 20, 30, 50, 100 times or
more
efficiently in target cells than in the blood or serum.
The cleavable linker may be cleaved by a hydrolysis, an enzymatic reaction, or
a reduction reaction, or by a pH change. In some embodiments, the linker is
cleavable under a certain physiological environment (for example, under an
appropriate pH environment). In some embodiments, the linker is cleavable in
an
acidic environment with a pH of about 6.5 or lower, or by reagents such as
enzymes.
In some embodiments, the linker is susceptible to cleavage agents, for
example, pH,
redox potential or the presence of degradative molecules.
In some embodiments, the linker is non-cleavable. Non-cleavable linkers as
used herein refer to linkers which remain basically intact during
intracellular
metabolism.
In some embodiments, the linker is a peptide linker consisting of a straight
or
branched chain amino acids linked by peptide bonds. In some embodiments, the
peptide linker is cleavable by a protease that is highly or specifically
expressed
around or in target cells, for example, cathepsin B in the lysosome or
endosome.
The peptide linker as used herein can be of varying lengths. Typically, the
peptide
linker of the present application is from 1 to 50 amino acids in length. In
some
Date Recue/Date Received 2021-07-27
CA 03127903 2021-07-27
embodiments, the peptide liker is from 1 to 45, from 1 to 40, from 1 to 35,
from 1 to
30, from 1 to 25, from 1 to 20, from 1 to 15, from 1 to 10, from 1 to 9, from
1 to 8,
from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, from 1 to 2
or 1
amino acids in length. In some embodiments, the peptide liker is from 2 to 45,
from
2 to 40, from 2 to 35, from 2 to 30, from 2 to 25, from 2 to 20, from 2 to 15,
from 2
to 10, from 2 to 9, from 2 to 8, from 2 to 7, from 2 to 6, from 2 to 5, from 2
to 4,
from 2 to 3 or 2 amino acids in length. The number of amino acids of the
peptide
linker as described in the present application can be equal to any integer
value
within the above numerical range, including the end values of this range. In
some
embodiments, the peptide linker is preferred to be 1, 2, 3, 4 or 5 amino acids
in
length. In some embodiments, the peptide linker is cysteine, lysine, lysine-
lysine,
valine-citrulline, phenylalanine-lysine, valine-lysine,
cysteine-lysine,
cysteine-glutamic acid, aspartic acid-aspartic acid, and aspartic acid-
aspartic
acid-lysine, and optionally, the carboxylic acid in the above-mentioned amino
acids
is amidated.
In some embodiments, the linker is a disulfide linker containing a disulfide
bond. The disulfide bond may be cleaved under an intracellular reductive
environment, while remains stable in a circular system. The disulfide linker
of the
present application may be DSDM, DMDS, MDS, or NDMDS. The structures of
these disulfide linkers are shown in Table 1 below.
Table 1: Structures of DSDM, DMDS, MDS and NDMDS
Name Structure
1 0
DSDM NcS,s0.1\j
0
0
0
0
I
DMDS N S -S7).L ,11-
0
0
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0
0
MDS I
,)-L _II?
N SS 0
0
I H
NS'70-11\i'l\r el 0
NDMDS 0
0.---.................---).(0,
0
0
In some embodiments, the linker is a pH-dependent linker. The pH-dependent
linker as described in the present application is cleavable under a certain pH
environment. In some embodiments, the pH-dependent linker may be stable under
an alkaline condition, while cleavable under an acidic condition, for example,
under
a pH value of 6.5 or lower. In some embodiments, the pH-dependent linker is a
cis-aconitic anhydride.
In some embodiments, the linker of the conjugate compound or the
pharmaceutically acceptable salt thereof is
NH2
0
NH
0 0
H H
N,(sA N 4 40
H
0 0 Oy\
0
0 ,
or a combination of the above-mentioned structure and a peptide linker (for
example, binding to a targeting molecule through a peptide linker containing 1-
3
amino acids).
In some embodiments, the linker of the conjugate compound or the
pharmaceutically acceptable salt thereof has the following structure:
52
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NH2
NH
0 H 0
\N(R) s
0 NtLz HN (S) 0 N 110 Oy\
0
0 0 ,
or a combination of the above-mentioned structure and a peptide linker (for
example, binding to a targeting molecule through a peptide linker containing 1-
3
amino acids).
In some embodiments, the linker of the conjugate compound or the
pharmaceutically acceptable salt thereof has the following structure:
NH2
NH
0 0
(S) N
- (R) 0 H 0 =
01r\
HN0 0
0
(S)
NH2 9
or a combination of the above-mentioned structure and a peptide linker (for
example, binding to a targeting molecule through a peptide linker containing 1-
3
amino acids).
In some embodiments, the linker of the conjugate compound or the
pharmaceutically acceptable salt thereof has the following structure:
NH2
NH
0 0
J,r1\1
N (S)
= H
0 0 Oy\
0
HNO 0
HOs,
0
53
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or a combination of the above-mentioned structure and a peptide linker (for
example, binding to a targeting molecule through a peptide linker containing 1-
3
amino acids).
In some embodiments, the linker of the conjugate compound or the
pharmaceutically acceptable salt thereof has the following structure:
NH2
NH
0 0
N-11\j'LSAN (S) N
= H
110 Olf\
HN0 0
HO
0
(s) 0
0
0
or a combination of the above-mentioned structure and a peptide linker (for
example, binding to a targeting molecule through a peptide linker containing 1-
3
amino acids).
In some embodiments, the linker of the conjugate compound or the
pharmaceutically acceptable salt thereof has the following structure:
NH2
NH
0 0
N N (s) N
- H
HN0 0
0
(S) 0 a 0 N H2
0 (S)
HN N,CSAN
= H H
0
00H
or a combination of the above-mentioned structure and a peptide linker (for
example, binding to a targeting molecule through a peptide linker containing 1-
3
amino acids).
54
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In some embodiments, the linker of the conjugate compound or the
pharmaceutically acceptable salt thereof has the following structure:
0
4N -rN 1,c!
0 (:)
0
)
0
H H
NI-r-rNi 0())
le 10 0 0
0 ,
or a combination of the above-mentioned structure and a peptide linker (for
example, binding to a targeting molecule through a peptide linker containing 1-
3
amino acids).
In some embodiments, the linker of the conjugate compound or the
pharmaceutically acceptable salt thereof has the following structure:
--2r0
(30
)
0
H H
N 10r N 023)
0 101 0 0
0 ,
or a combination of the above-mentioned structure and a peptide linker (for
example, binding to a targeting molecule through a peptide linker containing 1-
3
amino acids).
In some embodiments, the linker of the conjugate compound or the
pharmaceutically acceptable salt thereof has the following structure:
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CA 03127903 2021-07-27
s I
0
0
)
3.L0 110 0 0
H H
H 0 0
HN
0
H2N 9
or a combination of the above-mentioned structure and a peptide linker (for
example, binding to a targeting molecule through a peptide linker containing 1-
3
amino acids).
In some embodiments, the linker of the conjugate compound or the
pharmaceutically acceptable salt thereof has the following structure:
/NH N,N
1
Ari\Aõ..N 0
0 0
0
)
H
N ) Ny,,cy...---)i,N 0C).õõ=-i
H
0 0
HN
0
H2N ,
or a combination of the above-mentioned structure and a peptide linker (for
example, binding to a targeting molecule through a peptide linker containing 1-
3
amino acids).
In some embodiments, the linker of the conjugate compound or the
pharmaceutically acceptable salt thereof has the following structure:
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/NH N,N
0 H2N 00 0)
5,?,,H 0
(S) N
0000
0
HN
H2N
or a combination of the above-mentioned structure and a peptide linker (for
example, binding to a targeting molecule through a peptide linker containing 1-
3
amino acids).
In some embodiments, the linker of the conjugate compound or the
pharmaceutically acceptable salt thereof has the following structure:
H2
0
N(S = " \/\ Nss
(s)
H N
or a combination of the above-mentioned structure and a peptide linker (for
example, binding to a targeting molecule through a peptide linker containing 1-
3
amino acids).
In some embodiments, the linker of the present application may comprise any
one of or a combination of the linkers as described above.
In some embodiments, the payload is conjugated with a first targeting
molecule directly or indirectly, and the first targeting molecule is
conjugated with a
second targeting molecule directly or indirectly. In some embodiments, the
payload
is conjugated with each of the first and the second targeting molecule
directly. In
some embodiments, the payload is conjugated with each of the first and the
second
targeting molecule indirectly. In some embodiments, the payload is conjugated
with
the first targeting molecule indirectly, e.g. via a linker, and the first
targeting
molecule is conjugated with the second targeting molecule directly or
indirectly. In
some embodiments, the payload is conjugated with the first targeting molecule
via a
57
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first linker, and the payload is conjugated with the second targeting molecule
via a
second linker. In some embodiments, the linker is a multivalent linker that
binds to
at least one (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) payload and
two
targeting molecules.
In some embodiments, the two targeting molecules are linked to each other via
a spacer. In some embodiments, the spacer is cleavable by a protease that is
specifically expressed in target cells or to be expressed in target cells.
Such
proteases include, for example, the proteases as listed in Table 2 below. In
some
embodiments, the spacer comprises the amino acid sequence selected from any
one
of the amino acid sequences as listed in Table 2 below.
Table 2: List of Enzymatically Cleavable Sequences
Amino acid sequence of
Protease SEQ ID NO.
recognition site
Cathepsin B RR -
Legumain ASN -
Matripase KSRAEDE SEQ ID NO: 1
MMP-2 PLGLAG SEQ ID NO: 2
Prostate-specific antigen SSLY SEQ ID NO: 3
Stromelysin-3 AAA -
TMPRSS2 LLRSLIG SEQ ID NO: 4
Urokinase-type
SSR -
plasminogen activator
Activated protein C LVKR SEQ ID NO: 5
Factor Ixa LVVR SEQ ID NO: 6
Factor VIIa QLTR SEQ ID NO: 7
Factor Xa LEGR SEQ ID NO: 8
Thrombin PR -
Calpain-a PLFAEP SEQ ID NO: 9
Calpain-2 GLGSEP SEQ ID NO: 10
Enteropeptidase DDDDK SEQ ID NO: 11
MMP-8 GPSG SEQ ID NO: 12
Cathepsin L PLG -
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Proprotein convertase 5 RSKR SEQ ID
NO: 13
Calpain-3 VGVF SEQ ID
NO: 14
The Wails "cleavable" or "cleaved" as used herein refer to a metabolic process
or reaction process on the conjugate compound provided in the present
application,
whereby a linker between a payload and a targeting molecule, or a spacer
between
targeting molecules are broken to release free payload or targeting molecule.
The
linker and spacer is either cleaved by a protease or cleaved under a certain
physiological environment, e.g. a pH environment.
In some embodiments, the conjugate compound has a structure of foimula I, II,
III, or IV shown as follows, wherein n, m, p and q are independently 0 or 1,
which
represent that the linker and spacer are present or absent independently. The
"molecule" in the following formula is an abbreviation for "targeting
molecule".
Payload Linker )0 __ Molecule 14.J ( Spacer
)03 ¨.Molecule 2
( Formula i)
,N10 ec ule 1
4.0v
Payload
(Li
nicer
" Molecule
Formula II)
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Molecule 1 4-
Payload I,
Multivalent
linker
Pa-y-1-o- ad 2 Molecule 2
( Formula 0)
Molecule I +I
.09**
Multivalent
Payload
linker
Molecule 2
( Formula M
In some embodiments, the conjugate compound or the pharmaceutically
acceptable salt thereof provided in the present application comprises at least
one
(for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) payload as provided in
the present
application, two targeting molecules as provided in the present application
and
optionally a linker or spacer as provided in the present application. In some
embodiments, the conjugate compound or the pharmaceutically acceptable salt
thereof provided in the present application comprises one payload as provided
in the
present application, one ligand that specifically binds to a cell surface
protein or
marker as provided in the present application, one synergistic molecule as
provided
in the present application, and a linker or spacer as provided in the present
application.
In some embodiments, the conjugate compound has a structure of formula V,
VI, VII, or VIII shown as follows, wherein n, m, p, q and s are independently
0 or 1,
which represent that the linker, multivalent linker and spacer are present or
absent
independently.
Date Recue/Date Received 2021-07-27
CA 03127903 2021-07-27
Payload _______________ ( Linker )n Ligand __ ( Spacer )m Synergistic
molecule
( Formula V)
Synergistic
Payload ( Linker ) ¨ _________________ molecule ( Spacer )mn Ligand
( Formula VI)
_________________________________________________ Ligand
Payload
.ver 2.4 _______________________________________
Synergistic molecule
( Formula VII)
Ligand
C
Payload
__________________________________ Multivalent
linker
¨^ Synergistic molecule
Formula VIII)
In some embodiments, the conjugate compound or the pharmaceutically
acceptable salt thereof provided in the present application comprises a
payload and
two targeting molecules, wherein the two targeting molecules are a synergistic
molecule moiety and a prostate-specific membrane antigen ligand moiety,
respectively, for example, CB-20B, CB-20BK, CB-60S, CB-60SK, CB-20C,
CB-1020, CB-1320, CB-1820, CR19428, 20R-SMO9 and CB-20R.
In some embodiments, the conjugate compound or the pharmaceutically
acceptable salt thereof provided in the present application comprises one or
more
payloads and two targeting molecules, wherein the two targeting molecules are
a
synergistic molecule moiety and a ligand moiety represented by formula (I),
respectively, for example, CB-18G, CB-1820 and CR19426.
In some embodiments, the conjugate compound or the pharmaceutically
acceptable salt thereof provided in the present application comprises one
payload
61
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and two targeting molecules, wherein the two targeting molecules are a
synergistic
molecule moiety and P10, respectively, and the payload is camptothecin or any
derivative thereof, such as CB-10S, CR19425 and CB-50S.
In some embodiments, the conjugate compound of the present application is
selected from the group consisting of the following compounds: CB-20B, CB-
20BK,
CB-60S, CB-60SK, CB-20C, CB-1020, CB-1320, CB-1820, CR19428, 20R-SM09,
CB-20R, CB-18G, CR19426, CB-10S, CR19425 and CB-50S (the specific structure
of each conjugate compound is shown in Fig. 1). In some embodiments, the
conjugate compound of the present application is fottned by linking a linker-
drug
moiety to a ligand moiety via a covalent bond. The linker-drug moiety of the
present application comprises a payload and a linker, and the ligand moiety of
the
present application comprises two targeting molecules and an optional spacer
or
linker, wherein the two moieties foal" the conjugate compound of the present
application by reacting and fottning a covalent bond, and the covalent bond
can be
fottned between the linker in the linker-drug moiety and the ligand molecule
in the
ligand moiety, or can be formed between the linker in the linker-drug moiety
and
the spacer or linker in the ligand moiety.
The conjugate compound of the present application, CB-20B, is fottned by
linking a linker-drug moiety LT1002 to a ligand moiety 20B-SMO9 via a covalent
bond. The conjugate compound of the present application, CB-20BK, is fottned
by
linking a linker-drug moiety LT1002 to a ligand moiety 20BK-SMO9 via a
covalent
bond. The conjugate compound of the present application, CB-60S, is fottned by
linking a linker-drug moiety LT2000C to a ligand moiety 60S-SM09 via a
covalent
bond. The conjugate compound of the present application, CB-60SK, is fottned
by
linking a linker-drug moiety LT2000C to a ligand moiety 60SK-SM09 via a
covalent bond. The conjugate compound of the present application, CB-20C, is
fottned by linking a linker-drug moiety LD1001 to a ligand moiety 20BK-SM09
via
a covalent bond. The conjugate compound of the present application, CB-1020,
is
fottned by linking a linker-drug moiety LT1002 to a ligand moiety 1020BK-SMO9
via a covalent bond. The conjugate compound of the present application, CB-
1320,
is fottned by linking a linker-drug moiety LT1002 to a ligand moiety
62
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1320BK-SMO9 via a covalent bond. The conjugate compound of the present
application, CB-1820, is fonned by linking a linker-drug moiety LT1002 to a
ligand
moiety 1820BK-SMO9 via a covalent bond. The conjugate compound of the present
application, CR19428, is fonned by linking a linker-drug moiety CR19423 to a
ligand moiety 20BK-SMO9 via a covalent bond. The conjugate compound of the
present application. CB-20R. is fonned by complexing 20R-SMO9 with a
radionuclide ion M. The conjugate compound of the present application, CB-18G,
is
fonned by linking a linker-drug moiety LT1002 to a ligand moiety 18G-SMO9 via
a
covalent bond. The conjugate compound of the present application, CR19426, is
fonned by linking a linker-drug moiety CR19423 to a ligand moiety 18G-SMO9 via
a covalent bond. The conjugate compound of the present application, CB-10S, is
fonned by linking a linker-drug moiety LT1000 to a ligand moiety CBSMO9 via a
covalent bond. The conjugate compound of the present application, CR19425, is
fonned by linking a linker-drug moiety CR19423 to a ligand moiety CBSMO9 via a
covalent bond. The conjugate compound of the present application, CB-50S, is
fonned by linking a linker-drug moiety LT1000N3 to a ligand moiety 50S-SMO9
via a covalent bond. Each structure is shown in Table 3 below.
Table 3: Structures of Linker-drug Moiety and Ligand Moiety
Abbreviation Structure
NH2
0
NH
CF3
0 0
LD1001 N¨
H
N
I N
- H H 140
0 0 N
0 Y
OH
Hµ!`i
\
0 0
LT1000 N/
0
0
0,0
N
0
(s) 0
0 0
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Abbreviation Structure
OH
H H
cN ,ro.iN
NI \
LT 1000N3 N3
() 0
0
0 0
y N
0 0
(s) 0
¨) 0 0)
\ / 0 0
NH2
o
NH
0 0 H
LT1002 H ii
N Ali
? I V ? H
' H
0 0 WI 0--- R N
R OH
0 y (e) N (S) (R)
(s) 010
0 õA., 0 ,0 0
1
HO
\ /
N N 0
I
/
0
A (s)
0 0 00 0
LT2000C H H ii
N, 0
iNlrOr - N
H
0 NH
0
(K--) 0 0¨? NH2
\ /
CBSMO9 H2N
NH NH2
HN
HO Hr_N 9
H \- 0 = H H
s) Ni,,,,,, s)N
. s) N .
(s) N lifiRN (s) N yrsl'N IfISI'N
Nlr'NH
H H H H H H H
0000000
0
OH 0
HO 0
NH2
(R)
0
0 OH /
0 ao
N NH
N)?r N 'N 0
_, I
H2N N N
H
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Abbreviation Structure
18G-SMO9 o
Ho it 0 FIN 0
J\
HN (Rs
0,$)
'S
0, ,NH2
HN H 0 0 H r-o-
,,..$) (R) ki (si s) ki 0 0)
NH -
HN60 0 õ H H
OH 0
---7" 0
HN (s)
H)
;--- 0 0 N
,..) .(R)
HO HO (S)
/
H2N HO4
0
0
(R) õ SH
OOH
/ /
0
0
NH
N"..----"Thr
H
N)-,NN 0
1 H
H2N Nle
H
20B-SMO9 HO 0 (:),OH
0 0 HO
0
Vli N 0 H 0
0 N,@-L
N (s) NH
H
0
0
HO
(R) SH
0
0
OOH
H
N NH
0 N(-r
H
NJ,NN 0
0=,N H2
1 H
H2N NN
H
Date Recue/Date Received 2021-07-27
CA 03127903 2021-07-27
Abbreviation Structure
20BK-SMO9 H2N
HO 0 0, _OH
0 0 HO
0
HO (s) N)N(N)-1\1
r I
0
H i H
0
0 0
HO
(R) SH
0 0, _OH
0
r
/H
NiNNH
0
H 0
0NH2
1\1)NN
1 H
H2N NIµl
H
20R-SMO9 H2N
D 0 0 0,0H 0 HO
HO A A 0
(s) N N N N
H H H H 0 ,$) CI?
0
(s) 'LNH2
H : H
0 - 0 -)
\O
HO
00 OH HN
COOH
,--4 0 /----..---\
0 la [\ilfil
HN-Q - )
1\1)?rN 0 N N-\
I H c-NJ b 0H
H2N N N
H
Hooc)
50S-SMO9 H2N
NH HO X
NH2
HN N0 0 ¨( 0 , H \ 0 ---
q
ro o
HrH,
HolomAvli,i,,N)sN-1.0N S) NINA(c.)s)NN s)N Ir
r.,
(s) N N'NH
0 H 0 H 0 H 0 H 0 H 0 0 \,
0
OH 0
HO 0
NH2
0, ,OH /
0
NH
0 el 11 ic)
r-N
N N
1 __
H2N N N
H
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Abbreviation Structure
60S-SMO9 HO 0 0, _OH
0 0 HO
0
HO (S) N -LINI(.N)-NI 0 H
0
H H H H
0
N (s) . NH
H 0 \c)
HO
(s)
0 1
0
0,0H
H
Nriµl-,QSNIH
0
H
N, NN 0
0N H2
1 H
H2N NIµl
H
60SK-SMO9 H2N
yEiof 0 (:),OH HO
0
HO .
(s)NA NIN1j-LN 0 9
H H H H
0 N.(sP kil
N (s)
H = H
0 -\ 0
0
HO
(s) \
0 /0
0 OH
H
N _iN,a NH
0
H
NJ-,NN 0
0NH2
H
H2N N N
H
1020BK-SMO9 H2N
H NI-12
HN N
Q 9
HO H
0 ---( 0 H 0 H ,0 0H2OH,0
HONNN NN.k....--..,o,
011 H 011 H 011 H OH H 01 1 H
01 1 H HH (:)
0
OH 0 OHO
NH2 HNC)
0
H2N
(S) HO 0 0 0 ,OH HO
HO A
(s)N N NA N 0 LSHH 0
H H H H
0
N (s) N (s)
H
0 -\ 0
0
HO
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Abbreviation Structure
1320BK-SMO9 142N
H
HN
0 0 y
H2NN s) N11.,4 .õ
HN 0
,--,IH
0
OH H
, 0 i 0 HN N 0
O(R), s) Q H
11 li-rsiN ysiN (2) (:)
o " o "
o)
¨
NH A HN
OH
0
H2N
(s) SH
;JO 0 0OH HO
p
0
0
H H H H
0 N p NkSAN kl1)-LN
H i H H
0 0 0
HO
1820BK-SMO9 o
41 ',, NH
,t)
HO do 0 HN 0
,, HN-3\
os) ss 0 NH2
HN 0 H r-o-
õ....s: (,õi-I, ? (-
S) N 0 (21
HN r
NH i H H
3- 0 0 (157 0
HN (s) (5/4 - ' 0
'OH , j )
0 ki
) I-K/(-17 HO _c _.....1s)
1
NH
HO
H2N) 0
H2N \O
(R)
HO 0 0 OH
0 0 HO
HO (s) r\l)N1N)N 0 .,,,(0 0 0
H H H H H , H
/
0 NkS),.)-L.
....i.kl.õ....¨..õ--,}___
N (s) NH
N (s) 0
0
0
HO
CR19423
0 0 0 0 F
H H H
1
N )-L N ,(s-L N
H H H 1
0 0 b 0 , N
0
N
\
0
¨ OH
:
p -
o-
0
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In some embodiments, the conjugate compound or the pharmaceutically
acceptable salt thereof provided in the present application enters the blood
circulation and the outside of cells (in an intercellular substance). Since
the linker is
very stable in an extracellular environment and drug molecules cannot be
released,
the toxicity of the drug molecules is blocked. The conjugate is a drug with no
cytotoxicity or with a low toxicity and will not have a toxic effect on normal
cells.
In some embodiments, the conjugate compound or the pharmaceutically
acceptable salt thereof provided in the present application binds to multiple
receptors or antigens and other acting molecules that are simultaneously
highly
expressed on diseased cells, and the synergistic effect thereof greatly
increases the
affinity of the conjugate compound to target cells, reducing the possibility
of
binding to normal cells. Therefore, a highly effective toxin drug such as
MMAE/Dxd/5N38/a radionuclide complex can be carried to enhance the efficacy of
drugs, broaden a therapeutic window and avoid drug side effects.
In some embodiments, the conjugate compound or the pharmaceutically
acceptable salt thereof provided in the present application enters the
interior of
targeted cells, and then the linker can be cleaved through changes of the
internal
environment of the cells (by a specific enzyme digestion, a pH change, a
disulfide
bond reduction, etc.) to release drug molecules (equivalent to removing
modifying
groups of drug molecules), which has a therapeutic effect on tumor cells.
In some embodiments, the conjugate compound or the pharmaceutically
acceptable salt thereof of the present application can be used to specifically
deliver
a payload to target cells in a target tissue environment. Generally, the two
targeting
molecules of the conjugate compound or the pharmaceutically acceptable salt
thereof have three advantages. Firstly, the two targeting molecules can act in
multiple modes (often synergistically), resulting in improved therapeutic
effects
while reducing side effects. Secondly, the binding of the two targeting
molecules
increases the affinity and avidity of the conjugate compound or the
pharmaceutically acceptable salt thereof to target receptors or target cells,
thereby
enhancing its specificity and avoiding off-target toxicity. Finally, when
properly
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designed, a combination of the two targeting molecules can fulfill multi-
functional
properties required for a drug conjugate.
The conjugate compound or the pharmaceutically acceptable salt thereof of the
present application achieves unexpected technical effects, including but not
limited
to: (1) a combination of a ligand capable of binding to cell surface receptors
and a
endocytosis molecule capable of mediating endocytosis enable the conjugate
compound to specifically enter target cells; (2) the conjugate compound or the
pharmaceutically acceptable salt thereof enhances an affinity and targeting
specificity of drug compounds, thereby delivering highly effective
chemotherapeutic agents such as MMAE to a patient, broadening the therapeutic
window of such agents and avoiding side effects; (3) a linker can prevent the
release
of a payload outside of target cells (for example, in a blood circulation
system,
intercellular substance, etc.), which ensures the stability of the conjugate
compound
during the blood circulation, and reduces the toxicity of the drug; after
entering
target cells, the linker is cleaved to release the payload and exert the
effect of the
drug, and meanwhile multiple drug resistance (MDR) can be avoided; and (4) a
wide variety of drugs may be delivered in a form of the conjugate compound of
the
present application, and therefore, the application scopes of relevant drugs
are
widened. Therefore, the conjugate compound or the pharmaceutically acceptable
salt thereof of the present application not only broadens the targeting scope
and
therapeutic window of LDC-based drugs, but also reduces toxicity and side
effects
of some drugs.
The terms "polypeptide", "protein" and "peptide" as used herein can be a
single amino acid or a polymer of amino acids. The polypeptide, protein or
peptide
as described in the present application may contain naturally-occurring amino
acids
and non-naturally-occurring amino acids, or analogs and mimetics thereof The
polypeptide, protein or peptide can be obtained by any method well known in
the art,
for example, but not limited to, by an isolation and a purification from
natural
materials, a recombinant expression, a chemical synthesis, etc.
In another aspect, the present application discloses a pharmaceutical
composition comprising the conjugate compound or the pharmaceutically
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acceptable salt thereof provided in the present application, and a
pharmaceutically
acceptable carrier.
The term "pharmaceutically acceptable" as used herein means, within the
scope of sound medical judgment, being suitable for contact with cells of
human
beings and other animals without undue toxicity, irritation, allergic
response, etc.,
and being commensurate with a reasonable benefit/risk ratio.
The term "pharmaceutically acceptable salt" as used herein refers to a
relatively non-toxic, inorganic and organic acid addition salt and base
addition salt,
of the conjugate compound of the present application. Representative acid
addition
salts include hydrobromides, hydrochlorides, sulfates, bisulfates, phosphates,
nitrates, acetates, oxalates, valerates, oleates, palmitates, stearates,
laurates, borates,
benzo ate s, lactates, phosphates, to sylate s, citrates, male ate s,
fumarates, succinates,
tartrates, naphthylates, me sylate s, glucoheptonates, lactiobionates,
sulphamates,
malonates, salicylates, propionates, methylene-b is -b-hydroxynaphthoate s,
genti s ate s,
is ethionate s, di-p-toluoyltartrates, methane
sulphonate s, ethane sulphonate s,
benzene sulphonate s, p-toluenesulphonates,
cyclohexylsulphamates,
quinateslaurylsulphonate salts, and the like. Base addition salts include
pharmaceutically acceptable metal and amine salts. Suitable metal salts
include
sodium, potassium, calcium, barium, zinc, magnesium, and aluminum salts. In
some
embodiments, sodium and potassium salts are preferred. Suitable inorganic base
addition salts are prepared from metal bases which include, for example,
sodium
hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum
hydroxide, lithium hydroxide, magnesium hydroxide, and zinc hydroxide.
Suitable
amine base addition salts are prepared from amines which have sufficient
basicity to
form a stable salt, and preferably include the following amines which are
frequently
used in medicinal chemistry because of their low toxicity and acceptability
for
medical use: ammonia, ethylenediamine, N-methyl-glucamine, lysine, arginine,
ornithine, choline, N,N' -dibenzylethylenediamine, chloroprocaine,
diethanolamine,
procaine, N-benzylphenethylamine, diethylamine,
piperazine,
tris(hydroxymethyl)-aminomethane,
tetramethylammonium hydroxide,
triethylamine, dibenzylamine, ephenamine, dehydroabietylamine, N-
ethylpiperidine,
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benzylamine, tetramethylammonium, tetraethylammonium, methylamine,
dimethylamine, trimethylamine, ethylamine, basic amino acids, e.g., lysine and
arginine, dicyclohexylamine, and the like.
The term "pharmaceutically acceptable carrier" as used herein refers to a
pharmaceutically acceptable solvent, suspension or any other pharmacologically
inert vehicle for delivering the conjugate compound provided in the present
application to a subject, without interfering with the structures and
properties of the
conjugate compound. Such carriers enable the conjugate compound to be
formulated as, for example, tablets, pills, capsules, liquids, gels, syrups,
slurries,
suspensions and pastilles, for oral ingestion by a subject. Such carriers
enable the
conjugate compound to be formulated as injections, infusions or preparations
for
local administration.
The pharmaceutically acceptable carriers for use in the pharmaceutical
composition provided in the present application may include, but are not
limited to,
for example, pharmaceutically acceptable liquids, gels, or solid carriers,
aqueous
vehicles (such as sodium chloride injection, Ringer's injection, isotonic
dextrose
injection, sterile water injection, or dextrose and lactated Ringer's
injection),
nonaqueous vehicles (such as fixed oils derived from vegetables, cottonseed
oil,
corn oil, sesame oil, or peanut oil), antimicrobial agents, isotonic agents
(such as
sodium chloride or dextrose), buffers (such as phosphate or citrate buffers),
antioxidants (such as sodium bisulfate), anesthetics (such as procaine
hydrochloride), suspensions/dispersions (such as sodium
carboxymethylcellulose,
hydroxypropyl methylcellulose, or polyvinylpyrrolidone), chelating agents
(such as
EDTA (ethylenediamine tetraacetic acid) or EGTA (ethylene glycol tetraacetic
acid)), emulsifying agents (such as polysorbate 80 (Tween-80)), diluents,
adjuvants,
excipients or non-toxic auxiliary substances, other components well known in
the
art, or various combinations thereof Suitable components may include, for
example,
fillers, binders, buffers, preservatives, lubricants, flavoring agents,
thickening
agents, coloring agents, or emulsifying agents.
In some embodiments, the pharmaceutical composition is an injection
preparation. The injection preparations include sterile water solutions or
dispersions,
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suspensions or emulsions. In all cases, the injection preparations should be
sterile
and be a fluid for easy injection. It should be stable under the conditions of
manufacture and storage and must be preserved against the contaminating action
of
microorganisms such as bacteria and fungi. The carriers can be solvents or
dispersion mediums containing, for example, water, ethanol, polyol (for
example,
glycerol, propylene glycol, liquid polyethylene glycol, and the like), and
suitable
mixtures thereof and/or vegetable oils. The injection preparations should
maintain
appropriate fluidity. The appropriate fluidity can be maintained, for example,
by the
use of coatings such as lecithin, by the use of surfactants, and the like.
Prevention of
the action of microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid,
thimerosal, and the like.
In some embodiments, the pharmaceutical composition is an oral preparation.
The oral preparations include, but are not limited to, capsules, cachets,
pills, tablets,
lozenges (using a flavored basis, usually sucrose and acacia or tragacanth),
powders,
granules, or as solutions or suspensions in aqueous or non-aqueous liquids, or
as
oil-in-water or water-in-oil liquid emulsions, or as elixirs or syrups, or as
pastilles
(using an insert base, such as gelatin and glycerin, or sucrose and acacia)
and/or as
mouth washes.
In solid dosage forms for oral administration (e.g., capsules, tablets, pills,
dragees, pulvis, granules and the like), the conjugate compound is mixed with
one
or more pharmaceutically acceptable carriers, such as sodium citrate or
dicalcium
phosphate, and/or any of the followings: (1) fillers or extenders, such as
starches,
lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as
carboxymethylcellulose, alginates, gelatins, polyvinyl pyrrolidone, sucrose
and/or
acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as
agar-agar,
calcium carbonate, potato or tapioca starch, alginic acid, certain silicates,
and
sodium carbonate; (5) solution retarding agents, such as paraffin; (6)
absorption
accelerators, such as quaternary ammonium compounds; (7) wetting agents, such
as
acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and
bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium
stearate,
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solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; and
(10)
coloring agents.
In liquid dosage forms for oral administration, the conjugate compound is
mixed with any of the followings: pharmaceutically acceptable emulsions,
microemulsions, solutions, suspensions, syrups and elixirs. In addition to the
conjugate compound, the liquid dosage forms may contain inert diluents
commonly
used in the art, such as, water or other solvents, solubilizing agents and
emulsifying
agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl
acetate,
benzyl alcohol, benzyl benzoate, isopropanol, 1,3-butylene glycol, oils (in
particular,
cottonseed oil, peanut oil, corn oil, olive oil, castor oil and sesame oil),
glycerol,
tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of
sorbitan, and
mixtures thereof Besides inert diluents, an oral composition can also include
adjuvants such as wetting agents, emulsifying agents and suspensions,
sweetening
agents, flavoring agents, coloring agents, perfuming agents and preservatives.
In some embodiments, the pharmaceutical composition is a mouth spray
preparation or a nasal spray preparation. The spray preparations include, but
are not
limited to, aqueous aerosols, nonaqueous suspensions, lipidosome preparations
or
solid granular preparations. Aqueous aerosols are prepared by mixing aqueous
solutions or suspensions of agents with conventional pharmaceutically
acceptable
carriers and stabilizers. The carriers and stabilizers vary according to the
requirements of specific compounds, but in general, they include nonionic
surfactants (Tweens or polyethylene glycol), oleic acid, lecithin, amino acids
such
as glycine, buffer solution, salts, sugar or sugar alcohol. Aerosols are
generally
prepared from isotonic solutions, and can be delivered by sprayers.
In some embodiments, the pharmaceutical composition can be used by mixing
with one or more other drugs. In some embodiments, the pharmaceutical
composition comprises at least one other drug. In some embodiments, the other
drugs are antineoplastic drugs, cardiovascular drugs, anti-inflammatory drugs,
antiviral drugs, digestive system drugs, nervous system drugs, respiratory
system
drugs, immune system drugs, dermatologic drugs, metabolic drugs, and the like.
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In some embodiments, the pharmaceutical compositions can be administered to
a subject in need thereof by appropriate routes, including without limitation,
oral,
injection (such as intravenous, intramuscular, subcutaneous, intracutaneous,
intracardiac, intrathecal, intrapleural and intraperitoneal injection),
mucosal (such as
nasal and intraoral administration), sublingual, rectal, percutaneous,
intraocular, and
pulmonary administration. In some embodiments, the pharmaceutical composition
can be administered intravenously, subcutaneously, orally, intramuscularly or
intraventricularly.
Due to the properties of some payloads, such as high toxicity and high
hydrophilicity, it is desired to deliver the payloads more specifically and
more
efficiently to a subject in need thereof For example, in cancer treatment, it
is
desired to specifically deliver chemotherapeutic agents to cancer cells
without
toxicity to normal cells. Therefore, in another aspect, the present
application
discloses a method for delivering a payload to a subject in need thereof,
comprising
administering to the subject a therapeutically effective amount of the
conjugate
compound or the pharmaceutically acceptable salt thereof provided in the
present
application, or the pharmaceutical composition provided in the present
application.
The payloads described in the present application may be any pharmaceutical
agent
that elicits biological or medicinal responses in a tissue, system, animal,
individual
or human that is being sought by a researcher, veterinarian, medical doctor or
other
clinicians in preventing, inhibiting, ameliorating or treating a disease.
The term "object" as used herein refers to human and non-human animals.
Non-human animals include all vertebrates, for example, mammals and
non-mammals. The subject may also be a livestock animal such as cattle, swine,
sheep, poultry and horse, or a domestic animal such as dog and cat. The
subject may
be male (e.g. man) or female (e.g. woman), may be elderly, and may be an
adult,
adolescent, child, or infant. A human subject may be Caucasian, African,
Asian,
Semitic, or human with other racial backgrounds or a mixture of such racial
backgrounds.
The term "therapeutically effective amount" as used herein refers to an amount
of the conjugate compound or the pharmaceutically acceptable salt thereof, or
the
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pharmaceutical composition which relieves to some extent one or more symptoms
of a disease or disorder in a subject, returns to normal either partially or
completely
one or more physiological or biochemical parameters associated with or
causative
of the disease or disorder, and/or reduces the likelihood of the onset of the
disease
or disorder. Such amounts generally vary according to a number of factors
which
can be detettnined and explained, according to the scope of the specification
provided in the present application, by those of ordinary skill in the art.
These
factors include, without limitation: the particular subject and the age,
weight, height,
general physical condition, and medical history thereof, the particular
compound
used, the carrier of the preparation and the administration route selected,
and the
nature and severity of the condition being treated.
In some embodiments, the amount of the conjugate compound, or the
pharmaceutically acceptable salt thereof, or the pharmaceutical composition is
sufficient to inhibit a disease or disorder in a subject, or prophylactically
inhibit or
prevent the onset of a disease or disorder in a subject. Although the
therapeutically
effective amount may vary in different subjects, it is generally ranged from
0.01 to
100 mg/kg, for example, 0.01 to 90 mg/kg, 0.01 to 80 mg/kg, 0.01 to 70 mg/kg,
0.01 to 60 mg/kg, 0.01 to 50 mg/kg, 0.01 to 40 mg/kg, 0.01 to 30 mg/kg, 0.01
to 20
mg/kg, 0.01 to 10 mg/kg, 0.01 to 5 mg/kg, 0.01 to 4 mg/kg, 0.01 to 3 mg/kg,
0.01 to
2 mg/kg, 0.01 to 1 mg/kg, and 0.01 to 0.1 mg/kg. The therapeutically effective
amount as described in the present application can be equal to any value
within the
above numerical range, including the end values of this range.
In another aspect, the present application discloses a method for delivering a
payload to a subject in need thereof, comprising administering to the subject
a
therapeutically effective amount of the conjugate compound or the
pharmaceutically
acceptable salt thereof provided in the present application, or the
pharmaceutical
composition provided in the present application.
In another aspect, the present application discloses a method for treating a
disease in a subject, comprising administering to the subject a
therapeutically
effective amount of the conjugate compound or the pharmaceutically acceptable
salt
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thereof provided in the present application, or the pharmaceutical composition
provided in the present application.
In some embodiments, the disease is a cancer, including but not limited to,
prostatic cancer, breast cancer, lung cancer, renal cancer, leukemia, ovarian
cancer,
gastric cancer, uterine cancer, endometrial carcinoma, liver cancer, thyroid
cancer,
pancreatic cancer, colon cancer, colorectal cancer, esophageal cancer, skin
cancer,
lymphoma, and multiple myeloma.
In some embodiments, cancer cells of the cancers have an expression of the
cell surface receptors or antigens mentioned in the present application. In
some
embodiments, cancer cells of the cancers have a high expression (for example,
according to data from Depmap (see: https://depmap.org/portal/), the
corresponding
gene expression is at least 0, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8,
9, 10 or more
than 10) of cell surface receptors or antigens mentioned in the present
application.
In some embodiments, cancer cells of the cancers have a high expression of
FOLR1
and FOLH1, TRPV6 and FOLH1, GNRHR and FOLH1, SSTR2 and FOLH1,
FOLR1 and SSTR2, or TRPV6 and FOLR1. In some embodiments, the disease is
an immunological disease, for example, an autoimmune disease, including but
not
limited to, connective tissue disease, systemic sclerosis, rheumatoid
arthritis, and
systemic lupus erythematosus.
In some embodiments, the disease is a cardiovascular disease, including but
not limited to, angina, myocardial infarction, stroke, heart attack,
hypertensive heart
disease, rheumatic heart disease, cardiomyopathy, arrhythmia, and congenital
heart
disease.
In some embodiments, the disease is a metabolic disease, including but not
limited to, diabetes, gout, obesity, hypoglycemia, hyperglycemia, and
dyslipidemia.
In some embodiments, the disease is a neurological disease, including but not
limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease,
head
injury, multiple sclerosis, vertigo, coma, and epilepsy.
In some embodiments, the method provided in the present application further
comprises administering one or more therapeutic agents in combination with the
conjugate compound or the pharmaceutically acceptable salt thereof, or the
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pharmaceutical composition. In some embodiments, the therapeutic agents target
an
anti-cancer therapeutic target, induce or boost an immune response against
cancer,
or are chemotherapeutic agents.
The present application will be described in greater detail by way of specific
examples. The following examples are offered for illustrative purposes only,
and are
not intended to limit the invention in any manner. A person skilled in the art
will
readily recognize a variety of noncritical parameters which can be changed or
modified to yield essentially the same results.
EXAMPLES
The following examples are intended to further illustrate the present
application. The advantages and features of the present application will
become
clear with the descriptions. However, these illustrations are merely
exemplary, and
should not be constructed as limitations to the scope of the present
application.
Example 1: Preparation of Conjugate Compounds
Synthesis of conjugate compounds CB-20BK, CB-18G, CB-20B, CB-10S,
CB-20C, FA-MMAE, CB-20AK, CB-1020, CB-1320 and CB-1820
1. 10 g of Rink amide-am resin (hereafter referred to as "Rink
Resin", from
Sunresin New Materials Co. Ltd., Cat#: 183599-10-2) with a degree of
substitution
of 0.45 mmol/g was weighed and loaded onto a solid phase reaction column; DCM
was added, and nitrogen gas was bubbled through the solvent to swell the resin
for
minutes; the solvent was pumped off; and Fmoc protecting groups on the resin
were removed by DBLK, and then the resin was washed 5 times with DMF. 4.79 g
(9 mmol) of Fmoc-Lys(Dde)-OH and 1.47 g (10.8 mmol) of HOBt were weighed
25 and dissolved in DMF. To the above-mentioned solution at 0 C in an ice-
water bath,
1.67 ml (10.8 mmol) of DIC was added and mixed to activate for 5 minutes. The
solution was added to the above-mentioned reaction column and reacted for 3
hours,
and then the solvent was pumped off; and the resin in the reaction column was
washed 3 times. Then Fmoc protecting groups were removed by DBLK.
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2. The above-mentioned operations were repeated, and Fmoc-Cys(Trt)-0H,
Fmoc-Lys(Boc)-0H, Fmoc-Asp(OtBu)-0H, Fmoc-Asp(OtBu)-OH and inteitnediate
108 were successively conjugated according to the structures.
tBuO 0 0 ,OtB u
0 0
tBuO (s) N N N 0
H H H H
0
OH
(Intel _______ me diate 108)
3. Dde protecting groups were removed twice with 2% hydrazine
hydrate/DMF, for 10 minutes each time, and then the resin was washed 5 times
with
DMF. Fmoc-Glu-OtBu and pteroic acid were conjugated successively. Finally, the
resin was condensed twice with methanol, and the solvent was pumped off to
obtain
17.4 g of a protected peptide resin.
4. 17.4 g of the peptide resin obtained in the previous step was added to a
250 ml single-necked flask; 139 ml of lysis buffer (TFA : H20 : TIS = 95 : 3 :
2
(volume ratio)) was previously prepared, and 2.1 g of DTT was weighed and
added
to the lysis buffer. The lysis buffer was added to the above-mentioned flask,
and the
mixture was reacted at room temperature for 2.5 hours and filtered; the resin
was
continued to be washed with 30 ml of TFA; the above-mentioned filtrate was
combined, and the mixture was added to 834 ml of absolute ether, with a yellow
solid being precipitated, and centrifuged to obtain a solid, which was washed
with
absolute ether and dried in vacuo to obtain 6.4 g of a crude product as a
yellow solid
and with a yield of 93.4% and an HPLC purity of 82.3%. The product was
separated
by prep-HPLC (condition: C18 column, mobile phase A: 0.1% trifluoroacetic acid
in water, B: acetonitrile, elution gradient: (15-25)%B, elution time: 60
minutes;
fractions were collected); and the fraction containing the qualified product
was
lyophilized to obtain 4.73 g of 20BK-SM09, with a purity of 98.8%.
5. 4.09 g (3.11 mmol) of Mc-Val-Cit-PAB-MMAE (LT1002) was weighed
and added to a 1000 ml single-necked flask, and 500 ml of phosphate buffer and
100 ml of acetonitrile were added; the solution was stirred and maintained at
pH =
7.2 until a clear solution was obtained; and 4.73 g (3.11 mmol) of
inteimediate
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20BK-SM09 was added, and the mixture was reacted at room temperature for 2
hours, during which the reaction was monitored by HPLC. After the reaction was
completed, the mixture was filtered, and the filtrate was separated by prep-
HPLC
(condition: C18 column, mobile phase A: ammonium bicarbonate solution (pH =
7.2), B: acetonitrile, elution gradient: (25-35)%B, elution time: 60 minutes;
fractions were collected); the fraction containing the qualified product was
lyophilized to obtain 6.96 g of CB-20BK product, with a purity of 98.8% and a
yield of 78.8%.
In the same way, conjugate compounds CB-18G, CB-20B, CB-10S, CB-20C,
FA-MMAE (with a structure shown as follows), CB-20AK (with a structure shown
as follows), CB-1020, CB-1320 and CB-1820 can be obtained through similar
steps
in the above-mentioned methods.
H2NyJN
,
0
NH
0
0 OH 0 oH 0 0
4N NH N LC)JL1
N
(R) S 0 H 8 H 0 H I
N
0
0
0
,OH
(s)
0
0
0 NH2
(FA-MMAE)
1-4N
HO
0 C)
HO H Y N Cri4õ))))),,1
-101 (s) J(rA N (s)
0
NF-
o o r_f(.ti 0 H
HO
0 OyN/S,LLN N r
0 H 0 (s) }D
1-12M
(CB-20AK)
Synthesis of conjugate compounds CB-50S, CB-60S and CB-60SK
1. 10 g of Wang Resin (from Sunresin New Materials Co. Ltd., Cat#:
1365700-43-1) with a degree of substitution of 1.1 mmolig was weighed and
loaded
onto a solid phase reaction column; DMF was added, and nitrogen gas was
bubbled
through the solvent to swell the resin for 30 minutes; 14.3 g (22 mmol) of
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Fmoc-Arg(pbf)-0H, 3.56 g (26.4 mmol) of HOBt, and 0.27 g (2.2 mmol) of DMAP
were weighed and dissolved in DMF; at 0 C in an ice-water bath, 4.1 ml (26.4
mmol) of DIC was added and mixed to activate for 5 minutes; the solution was
added to the reaction column and reacted for 3 hours, and then the solvent was
pumped off; and the resin was washed 3 times.
2. 10.4 ml of acetic oxide and 8.9 ml of pyridine were dissolved in 50 ml
of
DMF and mixed, and the resin after washing in the above steps was added; the
mixture was blocked at room temperature for 5 hours and washed three times
with
DMF; and the resin was condensed with methanol, and then the solvent was
pumped off to obtain Fmoc-Arg(pbf)-Wang Resin, which has a degree of
substitution detet __ mined to be 0.53 mmol/g.
3. 3.8 g (2 mmol) of Fmoc-Arg(pbf)-Wang Resin (Sub = 0.53 mmol/g) was
weighed and loaded onto a reaction column; the resin was washed 3 times with
DMF and then was swelled for 30 minutes by means of adding DMF. Then Fmoc
protecting groups were removed by DBLK, and the resin was washed 6 times with
DMF. 2.0 g (6 mmol) of Fmoc-Pro-OH and 0.97 g (7.2 mmol) of HOBt were
weighed and dissolved in DMF; at 0 C in an ice-water bath, 1.1 ml (7.2 mmol)
of
DIC was added and mixed to activate for 5 minutes; the mixture was added to
the
reaction column and reacted for 2 hours; and then Fmoc protecting groups were
removed by DBLK.
4. The above-mentioned operations were repeated, and Fmoc-Leu-OH,
Fmoc-Asp(OtBu)-0H, Fmoc-Val-OH, Fmoc-Lys(Boc)-0H, Fmoc-Ser(tBu)-0H,
Fmoc-Pro-OH, Fmoc-His(Trt)-0H, Fmoc-Leu-OH,
Fmoc-Phe-OH,
Fmoc-Glu(OtBu)-0H, Fmoc-Lys(Boc)-0H,
Fmoc-propargyl-Gly-OH,
Fmoc-Glu-OtBu and pteroic acid were conjugated successively according to the
structures. The resin was condensed twice with methanol, and the solvent was
pumped off to obtain 8.4 g of a peptide resin.
5. 8.4 g of the peptide resin obtained in the previous step was added to a
250
ml single-necked flask; 67 ml of lysis buffer (TFA : H20: TIS = 95 : 3 : 2
(volume
ratio)) was previously prepared, and 0.92 g of DTT was weighed and added to
the
lysis buffer. The lysis buffer was added to the flask, and the mixture was
reacted at
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room temperature for 2.5 hours; the resin was filtered and washed with 20 ml
of
TFA; the filtrate was combined, and the mixture was added to 402 ml of
absolute
ether, with a yellow solid being precipitated, and centrifuged to obtain a
solid,
which was washed with absolute ether and dried in vacuo to obtain 4.06 g of a
crude
product as a yellow solid and with a yield of 97.3% and an HPLC purity of
84.6%.
The product was separated by prep-HPLC (condition: C18 column, mobile phase A:
0.1% trifluoroacetic acid in water, B: acetonitrile, elution gradient: (20-
29)%B,
elution time: 60 minutes; fractions were collected); and the fraction
containing the
qualified product was lyophilized to obtain 2.86 g of 50S-SM09, with a purity
of
97.6%.
6. 1.29 g (1.37
mmol) of LT1000N3 was weighed and added to a 500 ml
single-necked flask, and 270 ml of a mixed solvent (CAN : H20 = 1 : 4) and 393
mg (2.74 mmol) of CuBr were added and stirred. 2.86 g (1.37 mmol) of
intermediate 50S-SMO9 was added, and the mixture was reacted at room
temperature for 2-3 hours, during which the reaction was monitored by HPLC.
After
the reaction was completed, the mixture was filtered, and the filtrate was
separated
by prep-HPLC (condition: C18 column, mobile phase A: 0.1% trifluoroacetic acid
in water, B: acetonitrile, elution gradient: (22-40)%B, elution time: 60
minutes;
fractions were collected); and the fraction containing the qualified product
was
lyophilized to obtain 3.17 g of CB-50S, with a purity of 98.6% and a yield of
76.4%.
In the same way, conjugate compounds CB-60S and CB-60SK can be obtained
through similar steps in the above-mentioned methods.
Synthesis of CB-20R
1. 5 g of Rink Resin with a degree of substitution of 0.45 mmol/g was
weighed and loaded onto a solid phase reaction column; DCM was added, and
nitrogen gas was bubbled through the solvent to swell the resin for 30
minutes; the
solvent was pumped off; Fmoc protecting groups on the resin were removed by
DBLK; and then the resin was washed 5 times with DMF. 2.4 g (4.5 mmol) of
Fmoc-Lys(Dde)-OH and 0.74 g (5.4 mmol) of HOBt were weighed and dissolved in
DMF; at 0 C in an ice-water bath, 0.84 ml (5.4 mmol) of DIC was added and
mixed
82
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to activate for 5 minutes; the mixture was added to the reaction column and
reacted
for 3 hours; and the solvent was pumped off, and the resin was washed 3 times.
Then Fmoc protecting groups were removed by DBLK.
2. The above-mentioned operations were repeated, and Fmoc-Cys(Trt)-0H,
Fmoc-Lys(Boc)-0H, Fmoc-Asp(OtBu)-0H, Fmoc-Asp(OtBu)-OH and inteitnediate
108 were successively conjugated according to the structures.
3. Dde protecting groups were removed twice with 2% hydrazine
hydrate/DMF, for 10 minutes each time, and then the resin was washed 5 times
with
DMF. DOTA-tris(tBu) ester was conjugated. Then Dde protecting groups were
removed twice with 2% hydrazine hydrate/DMF, for 10 minutes each time, and
then
the resin was washed 5 times with DMF. Fmoc-Glu-OtBu and pteroic acid were
conjugated successively, and finally, the resin was condensed twice with
methanol
and the solvent was pumped off to obtain 9.2 g of a protected peptide resin.
4. 9.2 g of the peptide resin obtained in the previous step was added to a
250
ml single-necked flask; 74 ml of lysis buffer (TFA : H20: TIS = 95 : 3 : 2
(volume
ratio)) was previously prepared, and 1.05 g of DTT was weighed and added to
the
lysis buffer. The lysis buffer was added to the flask, and the mixture was
reacted at
room temperature for 2.5 hours; the resin was filtered and washed with 20 ml
of
TFA; the filtrate was combined, and the mixture was added to 560 ml of
absolute
ether, with a yellow solid being precipitated, and centrifuged to obtain a
solid,
which was washed with absolute ether and dried in vacuo to obtain 3.8 g of a
crude
product as a yellow solid and with a yield of 87.4% and an HPLC purity of
81.2%.
The product was separated by prep-HPLC (condition: C18 column, mobile phase A:
0.1% trifluoroacetic acid in water, B: acetonitrile, elution gradient: (15-
25)%B,
elution time: 60 minutes; fractions were collected); and the fraction
containing a
synthetic product was lyophilized to obtain 2.9 g of 20R-SM09, with a purity
of
97.8%.
5. 20R-SMO9 was complexed with radionuclide ion M to obtain CB-20R.
Specifically, the radioactive label 177Lu (about 50 MBq) was mixed with 100
[t.1 of
0.5 M sodium acetate buffer (pH = 5). 40 [t.1 of aqueous solution of 1 mM CB-
20R
dissolved in 10% DMSO, 2 1 of saturated ascorbic acid solution and 100 [t.1
of
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solution containing 177Lu were mixed, and the mixture was heated to 95 C for
10
minutes. The label was detected by radio-HPLC (within 5 minutes; 0%-100% ACN
in water; C18 column).
Synthesis of compound CR19425
--- `-..'11112 44080311 11 9 g
*õ..õ.=-- ..1 i N...,
...)..,.....),. ,,,,, 1 Molecular weight : 324.396 (7i's 'NH F N
tis ,---,
Si... ` CR19420 . , . 4:t , Piped(MK
....., b simi HATUDIPEA Y X 114_,(P
.,
HO-,:'-"-µ,N ".=, = -.'", -,, ,
,)---
sil.õ,i,J
Molecular weight : 435.46
0N19418 FICrts,
\ b
Molecular weight : 801,8.284
CR19421
11''r"'il
i 1
rt, 0 ? H Y
Q
0
0 - soH
Molecular weight : 472.60
---l',..%, ..-, HATU,DIPEA
F '' 'N
A.µ,..,..
P
\ b
Molecular weight : 57'9.5854
eft18422
.,
ri n ri i
N ,,..,..i.õ . , . N. ,.." = i- , ,,..F
-4
'0' 0 N µ!,;coshio9
0
H " , -. . Ni __ o= CR19425
,
. ...
Molecular we i dm :1 034,01 N ..:=f ., Molecular
weight: 3126.43/4
Cil1-113423,
.0-
0:
1. Under N2 protection, to a reaction flask, 441.4 mg of CR19420
(with a
structure as shown in the above reaction step) and 8.0 mL of DMF were added,
10 stirred, dissolved and cooled in an ice bath; then 459.2 mg of HATU and
380 [IL of
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DIPEA were added and stirred for half an hour; and then 500.0 mg of CR19419
(with a structure as shown in the above reaction step) and 190 [IL of DIPEA
were
added, and the mixture was reacted at room temperature until the reaction was
completed. After the reaction was completed, the reaction solution was poured
into
an acetic acid aqueous solution; a solid was precipitated and filtered, and
the filter
cake was washed with an acetic acid aqueous solution and water, and dried in
vacuo
to obtain 835.7 mg of CR19421 (with a structure as shown in the above reaction
step) as a brown powder and with an HPLC purity of 90.0% and a yield of 90.6%.
2. Under N2 protection, to a reaction flask, 835.7 mg of CR19421 and 16 mL
of 10% piperidine DMF solution were added and reacted at room temperature for
half an hour. After the reaction was completed, the reaction solution was
poured into
TFA/MTBE; a solid was precipitated and filtered, and the filter cake was
washed
with MTBE and dried in vacuo to obtain 599.7 mg of CR19422 (with a structure
as
shown in the above reaction step) as a field gray powder and with an HPLC
purity
of 84.7% and a yield of 83.0%.
3. Under N2 protection, to a reaction flask, 599.7 mg of CR19422, 586.7 mg
of CR19424 (with a structure as shown in the above reaction step) and 15 mL of
DMF were added, stirred, dissolved and cooled in an ice bath; then 495.7 mg of
HATU and 410 [IL of DIPEA were added and reacted at room temperature. After
the
reaction was completed, the reaction solution was subjected to a preparative
purification, and the acetonitrile was removed under reduced pressure from the
pure
product solution; the residue was extracted with a mixed solvent of
dichloromethane
and methanol, concentrated and dried to obtain 674.9 mg of CR19423 (with a
structure as shown in the above reaction step) as a yellow powder and with an
HPLC purity of 88.4% and a yield of 63.1%.
4. Under N2 protection, to a reaction flask, 14.8 mg of CR19423, 3.0 mL of
PBS buffer (pH = 6.6) and 3.0 mL of acetonitrile were added, stirred and
dissolved;
then 32.9 mg of CBSMO9 was added, and the mixture was adjusted to pH 6.6-6.8
with Na2HPO4 and reacted for half an hour. After the reaction was completed,
the
reaction solution was subjected to a preparative purification, and the pure
product
Date Recue/Date Received 2021-07-27
CA 03127903 2021-07-27
was lyophilized to obtain 21.8 mg of CR19425 as a yellow powder and with an
HPLC purity of 95.6% and a yield of 48.8%.
Synthesis of compound CR19426
9
H ' 1 ,
e`ir = 'N y'"*. 0 e' =:' :
H
t4: N = =L,
18G-SIV109
H H CR19426
Molecular weight :10t)7 N
Molecular weight : 3312.63
CRI.S4.0 ,OH
1
0
1. Under N2 protection, to a reaction flask, 25.2 mg of CR19423, 3.0 mL of
PBS buffer (pH = 6.6) and 3.0 mL of acetonitrile were added, stirred and
dissolved;
then 55.5 mg of 18G-5M09 was added, and the mixture was adjusted to pH 6.6-6.8
with Na2HPO4 and reacted for half an hour. After the reaction was completed,
the
reaction solution was subjected to a preparative purification, and the pure
product
113 was
lyophilized to obtain 44.6 mg of CR19426 as a yellow powder and with an
HPLC purity of 95.4% and a yield of 55.2%.
Synthesis of compound CR19428
.0 t
rt
- z
0Z BK-SIV109
H. " CR19428
0
4 ?
MO leCUlar t:tglit. )3.4 07
Molecular weight : 25b,6384
0114043 .0H
0
.(;)
1. Under N2
protection, to a reaction flask, 486 mg of CR19423, 3.0 mL of
PBS buffer (pH = 6.6) and 3.0 mL of acetonitrile were added, stirred and
dissolved;
then 712 mg of 20BK-SMO9 was added, and the mixture was adjusted to pH 6.6-6.8
with Na2HPO4 and reacted for half an hour. After the reaction was completed,
the
reaction solution was subjected to a preparative purification, and the pure
product
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was lyophilized to obtain 508 mg of CR19428 as a yellow powder and with an
HPLC purity of 96.7% and a yield of 42.3%.
Example 2: Determination of Affinity of Conitmate Compounds to Tamet
Proteins
1. Determination of binding affinity of CB-20BK to protein FOLR1
Experimental instruments, materials and reagents:
BIAcore T200 (GE)
CMS chip (GE, Cat#: 29104988)
Buffer: HBS-EP+ buffer 10X (GE, Cat#: BR100669), diluted 10 folds with
deionized water before use.
Amino coupling kit (GE, Cat#: BR100050)
Regeneration reagents: 10 mM Glycine 2.0 (GE, Cat#: BR100355)
10 mM Glycine 3.0 (GE, Cat#: BR100357)
Experimental steps
The experiment was carried out according to BIAcore T200 (GE) instruction
manual to determine the affinity of analytes CB-20BK, CB-20AK, folate (FA) and
FA-MMAE to FOLR1. In the experiment, CMS chip was conjugated with ligand
FOLR1 (R&D System, Cat#: 5646-FR). The experimental results are as shown in
Table 4.
Table 4. Binding Affinity of CB-20BK and Related Compounds to FOLR1
ka kd KD
FA (folate) 3.34 x 106 M's' 2.26 x 10-4 s-1 6.77 x
10-11M
FA-MMAE 1.79 x 10 M's' 1.15 x 10-4 s-1 6.43 x
10-11M
CB-20AK N/D N/D N/D
CB-20BK 1.03 x 10 M's' 1.31 x 10-4 s-1 1.27 x
10-1 M
"N/D" means that no specific binding was detected.
Table 4 shows that CB-20BK specifically binds to FOLR1 with a good affinity.
The binding affinity of CB-20BK to FOLR1 is slightly weaker than the binding
affinity of FA or FA-MMAE to FOLR1. CB-20AK does not comprise the folate
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moiety of CB-20BK and has not been detected to specifically bind to FOLR1 in
the
experiment.
2. Determination of bindin2 affinity of CB-20BK to protein FOLH1
Experimental instruments, materials and reagents:
GatorTM (Probe Life)
SA probe (Probe Life, Cat#: 1906018)
Buffer: Q buffer (Probe Life), 10 mM, pH = 7.4
Experimental steps
(1) Synthesis steps of analyte Biotin-CB-20BK
1) 5.1 g of Rink Resin with a degree of substitution of 0.45 mmol/g was
weighed and loaded onto a solid phase reaction column; DCM was added, and
nitrogen gas was bubbled to swell the resin for 30 minutes; the solvent was
pumped
off; Fmoc protecting groups were removed by DBLK; and then the resin was
washed 5 times with DMF. 2.45 g of Fmoc-Lys(Dde)-OH and 0.75 g of HOBt were
weighed and dissolved in DMF; at 0 C in an ice-water bath, 0.83 ml of DIC was
added to activate for 5 minutes; the mixture was added to the reaction column
and
reacted for 3 hours; and the solvent was pumped off, and the resin was washed
3
times. Then Fmoc protecting groups were removed by DBLK.
2) The above-mentioned operations were repeated, and Fmoc-Cys(Trt)-0H,
Fmoc-Lys(Boc)-0H, Fmoc-Asp(OtBu)-0H, Fmoc-Asp(OtBu)-OH and inteitnediate
108 were successively conjugated according to the structures.
3) Dde protecting groups were removed twice with 2% hydrazine hydrate/DMF,
for 10 minutes each time, and then the resin was washed 5 times with DMF.
Fmoc-Lys(Biotin)-0H, Fmoc-Glu-OtBu and pteroic acid were conjugated
successively, and after pteroic acid was conjugate, the resin was washed twice
with
DMF; and finally, the resin was condensed with methanol, and the solvent was
pumped off to obtain 9.7 g of a protected peptide resin.
4) 9.7 g of the peptide resin obtained in the previous step was added to a 250
ml single-necked flask; 77.6 ml of lysis buffer (TFA : H20 : TIS = 95 : 3 : 2
(volume ratio)) was previously prepared, and 1.1 g of DTT was weighed and
added
to the lysis buffer. The lysis buffer was added to the flask, and the mixture
was
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reacted at room temperature for 2.5 hours; the resin was filtered and washed
with 20
ml of TFA; the filtrate was combined, and the mixture was added to 466 ml of
methyl tert-butyl ether, with a yellow solid being precipitated, and
centrifuged to
obtain a solid, which was washed with methyl tert-butyl ether and dried in
vacuo to
obtain 4.6 g of a yellow solid with an HPLC purity of 83.2%. The product was
separated by prep-HPLC and lyophilized to obtain 2.1 g of Biotin-20BK-SM09
with
a purity of no less than 95%.
5) 500 mg of Mc-Val-Cit-PAB-MMAE (LT1002) was weighed and added to a
250 ml single-necked flask, and 65 ml of phosphate buffer and 20 ml of
acetonitrile
were added; the solution was stirred and maintained at pH = 7.2 until a clear
solution was obtained; and 785 mg of intermediate Biotin-20BK-SMO9 was added,
and the mixture was reacted at room temperature for 2 hours, during which the
reaction was monitored by HPLC. After the reaction was completed, the mixture
was filtered, separated by prep-HPLC, and lyophilized to obtain 723 mg of
Biotin-CB-20BK, with a purity of 96.8% and a yield of 59.4%.
(2) The experiment was carried out according to GatorTM (Probe Life)
instruction manual to determine the binding affinity of Biotin-CB-20BK to
FOLH1.
In the experiment, SA probe binds to ligand Biotin-CB-20BK, and the analyte is
FOLH1 (Sino Biologicals, Cat#: 15877-H07H). The experimental results are as
shown in Table 5.
Table 5. Binding Affinity of CB-20BK to FOLH1
ka kd KD
FOLH1 1.19 x 104 A4-1s-1 1.70 x 10-4 s-1 6.98 x 10-9 M
Table 5 shows that CB-20BK binds to FOLH1 with a good affinity.
3. FOLH1 binds to CB-20BK-FOLR1 complex
Experimental materials and reagents:
BIAcore T200 (GE)
CMS chip (GE, Cat#: 29104988)
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Buffer: HBS-EP+ buffer 10X (GE, Cat#: BR100669), diluted 10 folds with
deionized water before use.
Amino coupling kit (GE, Cat#: BR100050)
Regeneration reagents: 10 mM Glycine 2.0 (GE, Cat#: BR100355)
10 mM Glycine 3.0 (GE, Cat#: BR100357)
Experimental steps
CM5 chip was conjugated with a ligand: FOLR1 (R&D System, Cat#:
5646-FR)
Analyte: CB-20BK, FA-MMAE and FOLH1 (Sino Biologicals, Cat#:
15877-H07H)
According to BIAcore T200 (GE) operating manual, FOLR1 was conjugated to
the CMS chip; CB-20BK or FA-MMAE was loaded first, and then FOLH1 was
loaded; and the binding of FOLH1 to the CB-20BK-FOLR1 complex was detected.
The experimental results are as shown in Table 6.
Table 6. Binding of CB-20BK-FOLR1 Complex to FOLH1 Solution at Different
Concentrations
Concentration (04) FA-MMAE (RU) CB-20BK (RU)
0.5 9.8 23.9
0.25 8.4 14.1
0.125 4.2 4.6
0.0625 -1 -0.9
Table 6 shows that with regard to an FOLH1 solution at a high concentration,
the amount of FOLH1 bound to a CB-20BK-FOLR1 complex is significantly higher
than that bound to an FA-MMAE-FOLR1 complex, which shows a continuous
rising trend. The binding of FOLH1 to an FA-MMAE-FOLR1 complex tends to
saturate at a high concentration. This experiment has proved that CB-20BK can
bind to both FOLR1 and FOLH1 receptors with a good affinity.
Example 3: Study on Binding and Endocytosis of Ligand Conjugates to Target
Cells
Date Recue/Date Received 2021-07-27
CA 03127903 2021-07-27
1. Cell binding and endocytosis experiment of conjugate compound
CB-20BK
Synthesis of labeled samples Cy5-pep-20BK, Cy5-FA and Cy5-pep-20AK
(1) 2 g of Rink Resin with a degree of substitution of 0.45 mmol/g was
weighed and loaded onto a solid phase reaction column; DCM was added, and
nitrogen gas was bubbled through the solvent to swell the resin for 30
minutes; the
solvent was pumped off; Fmoc protecting groups were removed by DBLK; and then
the resin was washed 5 times with DMF. 0.96 g (1.8 mmol) of Fmoc-Lys(Dde)-OH
and 0.3 g (2.2 mmol) of HOBt were weighed and dissolved in DMF; at 0 C in an
ice-water bath, 0.33 ml (2.2 mmol) of DIC was added and mixed to activate for
5
minutes; the mixture was added to the reaction column and reacted for 3 hours;
and
the solvent was pumped off, and the resin was washed 3 times. Then Fmoc
protecting groups were removed by DBLK.
(2) The above-mentioned operations were repeated, and Fmoc-Lys(Boc)-0H,
Fmoc-Asp(OtBu)-0H, Fmoc-Asp(OtBu)-OH and intermediate 108 were conjugated
successively according to the structures:
(3) Dde protecting groups were removed twice with 2% hydrazine
hydrate/DMF, for 10 minutes each time, and then the resin was washed 5 times
with
DMF. Fmoc-Lys(Dde)-0H, Fmoc-Glu-OtBu and pteroic acid were conjugated
successively.
(4) Dde protecting groups were removed twice with 2% hydrazine
hydrate/DMF, for 10 minutes each time, and then the resin was washed 5 times
with
DMF; the resin was conjugated with Cy5-COOH and then washed twice with DMF;
and finally, the resin was condensed twice with methanol and the solvent was
pumped off to obtain 3.6 g of a protected peptide resin.
(5) 3.6 g of the peptide resin obtained in the previous step was added to a 50
ml single-necked flask; 29 ml of lysis buffer (TFA : H20: TIS = 95 : 3 : 2
(volume
ratio)) was previously prepared, and 0.4 g of DTT was weighed and added to the
lysis buffer. The lysis buffer was added to the flask, and the mixture was
reacted at
room temperature for 2.5 hours; the resin was filtered and washed with 8 ml of
TFA;
the filtrate was combined, and the mixture was added to 173 ml of absolute
ether,
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with a yellow solid being precipitated, and centrifuged to obtain a solid,
which was
washed with absolute ether and dried in vacuo to obtain 1.9 g of a crude
product as
a blue solid and with a yield of 89.6% and an HPLC purity of 76.3%. The
product
was separated by prep-HPLC (condition: C18 column, mobile phase A: 0.1%
trifluoroacetic acid in water, B: acetonitrile, elution gradient: (20-28)%B,
elution
time: 50 minutes; fractions were collected); and the fraction containing the
qualified
product was lyophilized to obtain 736 mg of Cy5-pep-20BK, with a purity of
93.4%.
In the same way, compounds Cy5-FA and Cy5-pep-20AK can be obtained
through similar steps in the above-mentioned methods.
H2N
HO 0 0, _OH
0 0 HO
0 '03S HO (s) NAN 0 0 ,.rH 0
H H H H
0
N (s) (s) N NH2
H H
0 0
N
HO
0, _OH HN -03S
0 0
0
0
N N 0
I H
H2N
SOS-
(Cy5-pep-20BK)
-03s
H2N
HO 0 0,0H
0 0 HO
HO (s)NANNAN 0 (rH 0 0
H H H H
0 NksA N,)-LN H2 -03s
N (S) N (S)
H H
0 \(:) 0 \
\_H
HO
0
SOB-
(Cy5-pep-20AK)
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-03s
N-
J-1
-03S
0 H2N
0
frN
0 N
0 0
I
H2N N
SO3-
(Cy5 -FA)
Experiment for detecting cell binding and endocytosis of samples
Cy5-pep-20AK/Cy5-pep-20BK by flow cytometry method
Sample infoimation: Cy5-pep-20AK and Cy5-pep-20BK
Cell lines: LNCaP human prostate cancer cells, Du145 human prostate cancer
cells, SKOV3 human ovarian cancer cells and NCI-H460 human lung cancer cells
Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin
solution, L-glutamine and PBS
Experimental operations:
(1) Cell culture: the cells were digested with trypsin, collected and counted,
and then the cells were added to several culture flasks containing a complete
medium; the culture flasks were placed in an incubator at 37 C, 5% CO2 for
culturing the cells, and finally about 5 x 106 cells were collected into a
centrifuge
tube.
(2) Sample incubation: Cy5-pep-20AK/Cy5-pep-20BK samples were diluted to
8 nmol/L with PBS; the cell suspension was centrifuged at 1000 rpm for 5
minutes;
the supernatant was removed, and the residue was washed once with PBS; the
cells
were suspended uniformly and divided equally into different centrifuge tubes
according to the experimental scheme; PBS was removed by centrifugation; 200
[t.1
of a sample working solution was added to each tube and incubated at 37 C for
15,
30, 60, and 90 minutes, respectively, and 1 tube to which only PBS was added
was
used as a blank control; all operations were performed avoiding light
exposure.
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(3) Washing and loading: the working solution was removed by centrifugation
at 1000 rpm for 5 minutes, and the cells were washed 3 times with PBS, and an
appropriate amount of PBS was added to suspend the cells to 1 x 106 cells/ml;
Beckman CytoFLEX flow cytometer was turned on in advance to complete the
startup cleaning process; and the cell samples were loaded successively, and
the
fluorescence of 10,000 living cells was read in the APC channel; all
operations were
performed avoiding light exposure.
(4) Data analysis: the absolute MFI (mean fluorescence intensity) of the APC
fluorescence of each cell sample and the relative MFI of the relative blank
control
were obtained, and a data line graph was completed according to the relative
MFI.
Results and analysis
Table 7. Expression Levels of FOLR1 and FOLH1 in Different Cell Lines
(with reference to data from Depmap, https://depmap.org/portal/)
RNA-Seq LNCaP SKOV3 Du145 NCI-H460
FOLR1 +/- 6+ +/- +1-
FOLH1 10+ + +/- +/-
According to data from Depmap, the data being 0 or negative is expressed as
"-", the data being 0.001-0.499 is expressed as "+/-", the data being 0.500-
1.499 is
expressed as "+", the data being 1.500-2.499 is expressed as "2+", and so on.
The fluorescence intensity of cells was respectively detected at 15 minutes,
30
minutes, 60 minutes, and 90 minutes of incubation, and the results were
plotted and
shown in Figs. 2A and 2B. The binding and endocytosis of the sample
Cy5-pep-20BK cells can be seen from the figures. Compared with a DU145 cell
line with a relatively low expression of FOLR1 and a NCI-H460 cell line with a
relatively low expression of FOLH1, an LNCaP cell line with a relatively high
expression of FOLR1 and an SKOV3 cell line with a relatively high expression
of
FOLH1 have a significantly higher fluorescence intensity of CB-20BK that was
bond and endocytosed. Since Cy5-pep-20AK only comprises an FOLH1 ligand and
does not comprise the FOLR1 ligand, FA, Cy5-pep-20AK shows a high level of
binding and endocytosis in LNCaP cells with a high expression of FOLH1, and
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shows a moderate level of binding and endocytosis in SKOV3 cells with a
moderate
expression of FOLH1; in the two cell lines, the binding and endocytosis level
of
Cy5-pep-20AK was significantly lower than that of Cy5-pep-CB-20BK. The degree
of binding and internalization of a ligand conjugate to cells is positively
correlated
with expression levels of cell-related receptors.
2. Binding and endocytosis experiment of single-ligand conjugate
Cy5-FA to cells
Sample infoimation: Cy5-FA
Cell lines: Hela cervical cancer cells, SKOV3 human ovarian cancer cells and
A549 human lung cancer cells
Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin
solution, L-glutamine, PBS, and anti-fluorescence quenching mounting medium
containing DAPI
Experimental operations:
(1) Cells growing on coverslips: coverslips were placed in a 24-well plate;
the
cells were digested with trypsin, collected and counted, and then the cells
were
diluted with a complete cell culture medium to about 2 x 105 cells/ml; 500 IA
of the
diluted cell solution was added to each well of the 24-well plate; and the
plate was
placed in an incubator at 37 C, 5% CO2 for culturing 48 hours.
(2) Sample incubation: the Cy5-FA sample was diluted in an IMDM medium
to 73 nmol/L; the culture medium in the 24-well plate was discarded; 200 IA of
the
sample working solution was added to the cell coverslip in each well and
incubated
at 37 C for 15, 30, and 60 minutes, respectively, and 1 well to which only an
IMDM
medium was added was used as a blank control; all operations were perfoinied
avoiding light exposure.
(3) Washing and mounting: the working solution in the 24-well plate was
discarded, and the plate was washed 3 times with preheated PBS at 37 C; the
cell
coverslips were taken out, and 5 [t.1 of anti-fluorescence quenching mounting
medium containing DAPI was added dropwise onto the slides and then covered
with the cell coverslips to complete the mounting; all operations were
perfoinied
avoiding light exposure.
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(4) Image interpretation for samples: Leica fluorescence microscope DM2500
was turned on to preheat the fluorescence exciter for 15 minutes; at the same
position, cell nucleus and Cy5 fluorescein were photographed at the
corresponding
fluorescence channel, and the image fusion was completed in the software.
The binding and endocytosis of the sample Cy5-FA to cells at 15 minutes and
30 minutes can be seen from Fig. 3, and the fluorescence intensity in cell
lines Hela
and SKOV-3 with a high expression of FOLR1 is significantly higher than that
in a
cell line A549 with a relatively low expression of FOLR1. The degree of
binding
and internalization of a ligand conjugate to cells is positively correlated
with
expression levels of cell-related receptors.
Example 4: Experiment of Inhibitin2 Cell Expansion in Conjugate Compounds
1. Experiment of inhibiting tumor cell expansion in CB-20BK
Sample infoimation: CB-20BK
Cell lines: KB human oral epidermoid carcinoma cells, T-47D human breast
cancer cells, NCI-H460 human lung cancer cells, CALU-3 human lung
adenocarcinoma cells, HuH-7 human liver cancer cells and LNCaP human prostate
cancer cells
Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin
solution, L-glutamine and CCK8
Experimental operations:
1) Cell plating
The cells were prepared in advance, digested with trypsin, collected and
counted; with a complete cell culture medium, KB cells were diluted to 2.5 x
104
cells/ml, T-47D cells were diluted to 1.3 x 104 cells/ml, NCI-H460 cells were
diluted to 3.5 x 104 cells/ml, CALU-3 cells were diluted to 4.0 x 104
cells/ml,
HuH-7 cells were diluted to 3.0 x 104 cells/ml, and LNCaP cells were diluted
to 8.0
x 104 cells/ml; 100 IA of the diluted cell solution was added to each well of
96-well
plates; and negative control and blank control wells were set on each plate.
The
96-well plates added with the cells were placed in an incubator at 37 C, 5%
CO2 for
culturing overnight.
2) Diluting and adding samples
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The samples were diluted with a culture medium to desired concentrations (see
Table 8). To each well of the 96-well plates that were cultured overnight, 50
1.11 of
the diluted samples were added, with 3 replicate wells; negative controls and
blank
controls were set; and the plates were placed in an incubator at 37 C, 5% CO2
for
culturing 72 hours.
Table 8: Concentration of Sample (CB-20BK) After Dilution
Concentration after
Tube number
dilution (mon)
1 201
2 50
3 13
4 3.1
5 0.8
6 0.2
7 0.05
8 0.01
9 0.003
0.0008
3) Color development and reading plate
To each well, 15 p1(10% of the liquid volume in a well) of a color developing
10 solution from CCK-8 was added; the plates were incubated at 37 C for an
appropriate time (try to keep the OD value in the range of 1.0-2.0); the
covers of the
96-well plates were removed, and the plates were read at 450 nm on a
microplate
reader (Molecular Devices Spectra MAX Plus).
4) Data processing
The data was edited using SoftMax Pro and a four-parameter fitting curve was
plotted.
5) Experimental results and analysis
According to the four-parameter fitting curve graph (Fig. 4A, wherein C value
corresponds to IC5o), CB-20BK has an inhibitory effect on the growth and
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expansion of KB, T-47D, NCI-H460, CALU-3, HuH-7, and LNCaP tumor cell lines.
Since expression levels of the corresponding receptors of conjugate compounds
in
the cells are different, the inhibition levels are different. The ICso for
cell lines
T-47D, KB, LNCaP, HuH-7 and CALU-3 with a relatively high expression of the
receptors is significantly lower than that for cell line NCI-H460 with a
relatively
low expression of the receptors. CB-20BK shows a correlation between tumor
growth inhibition effects and expression levels of cell-related receptors.
2. Experiment of conjugate compound CB-20BK and related
compounds inhibiting expansion of tumor cell lines LNCaP (human prostate
cancer cells) and 22RV1 (human prostate cancer cells)
Since different cells have different sensitivity to a cell proliferation
inhibitory
toxicity of the payload in a ligand conjugate, the quantitative analysis may
be
interfered occasionally. A series of cell lines with known expression levels
of
receptors of different ligands were selected and tested for the response to
inhibitory
effects of the cell proliferation of a ligand conjugate and a single payload.
The ICso
ratio of a single payload to that of a ligand conjugate in same cell line were
taken to
remove such interference.
Related samples: MMAE, CB-20BK, CB-20AK and FA-MMAE
Experimental operations: LNCaP and 22RV1 cells were prepared in advance,
counted, and plated in 96-well cell culture plates at a cell density of 4 x
104 cells/ml
and 2.0 x 104 cells/ml, respectively (100 [a/well); negative control and blank
control wells were set on each plate. After adherent cell culture overnight,
the
diluted samples to be tested were added at 50 [d/well. The plates were placed
in an
incubator at 37 C, 5% CO2 and cultured for 68-72 hours. To each well, 15
p1(10%
of the liquid volume in a well) of a color developing solution from CCK8 was
added; and the plates were incubated at 37 C for 45-70 minutes and each plate
was
read at 450 nm on a microplate reader. The data was edited using SoftMax Pro
and a
4-P fitting curve was plotted. The calculated data is shown in Table 9.
Table 9. Inhibitory Effects of MMAE, FA-MMAE, CB-20BK and CB-20AK on
Proliferation of Cell Lines and Receptor Gene Expression Levels in Cell Lines
(with reference to data from Depmap)
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FOL FOL
ICso
RI HI IC50
IC50
ICso ratio
gene gene IC50 IC50 ratio
ratio
Cell IC50 (FA- (MM
expr expr (CB-2 (CB-20
(MMA (MMA
line (MMAE) MMA AE/F
essio essio OAK) BK)
E/CB-2 E/CB-2
E) A-M
n n OAK)
OBK)
MAE)
level level
LNCaP +/- 10+ 0.00255 0.264 0.102 0.0958 0.010 0.025 0.0267
22RV1 +/- 6+ 0.00648 1.01 1.05 0.502 0.006 0.006 0.013
It can be seen from the above table that there is not much difference in the
expression level of FOLR1 between LNCaP and 22RV1, and the expression level of
FOLH1 in LNCaP is significantly higher than the expression level of FOLH1 in
22RV1. CB-20BK (with FOLH1 ligand and FOLR1 ligand) and CB-20AK (only
with FOLH1 ligand) have inhibitory effects on the expansion of both LNCaP and
22RV1 tumor cell lines. The inhibitory effects of CB-20BK and CB-20AK on the
cells with different expression levels of receptor FOLH1 are different. The
ICso ratio
in cell line LNCaP with a relatively high expression of receptor FOLH1 is 2-4
times
higher than the ICso ratio in cell line 22RV1 with a relatively low expression
of the
receptor. FA-MMAE also has an inhibitory effect on the expansion of LNCaP and
22RV1 tumor cell lines. Since the expression level of FOLR1 in LNCaP and 22RV1
is very low, the expression in LNCaP cells (FOLR1 gene expression level is
0.3219,
with reference to data from Depmap) is slightly higher than that in 22RV1
(FOLR1
gene expression level is 0.0704, with reference to data from Depmap), and the
ICso
ratio of FA-MMAE between LNCaP and 22RV1 only differs by a factor of 1.5. The
above data indicates that inhibitory effects on cell proliferation is
positively
correlated with expression levels of cell expression receptors FOLH1 and
FOLR1.
3. Experiment of conjugate compound CB-20BK and related
compounds inhibiting expansion of tumor cell lines PANC-1 (human
pancreatic cancer cells) and CFPAC-1 (human pancreatic cancer cells)
Related samples: MMAE and CB-20BK
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Experimental operations: PANC-1 and CFPAC-1 cells were prepared in
advance, counted, and plated in 96-well cell culture plates at a cell density
of 4 x
104 cells/ml (100 [d/well); negative control and blank control wells were set
on each
plate. After adherent cell culture overnight, the diluted samples to be tested
were
added at 50 [d/well. The plates were placed in an incubator at 37 C, 5% CO2
and
cultured for 68-72 hours. To each well, 15 p1(10% of the liquid volume in a
well)
of a color developing solution from CCK8 was added; and the plates were
incubated
at 37 C for 45-70 minutes and each plate was read at 450 nm on a microplate
reader.
The data was edited using SoftMax Pro and a 4-P fitting curve was plotted. The
calculated data is shown in the following table:
Table 10. Inhibitory Effects of CB-20BK on Proliferation of Cell Lines and
Receptor Gene Expression Levels in Cell Lines
(with reference to data from Depmap)
FOLR1 gene FOLH1 gene
ICso ICso ICso (MMAE)/
Cell line expression expression
(MMAE) (CB-20BK) ICso (CB-20BK)
level level
CFPAC-1 4+ +1- 0.015 0.557 0.027
PANC-1 +1- +1- 0.002 0.126 0.020
It can be seen from Table 10 that the expression level of FOLH1 in CFPAC-1
is very low, and the expression level of FOLR1 in CFPAC-1 is significantly
higher
than that in PANC1 cell lines. CB-20BK has an inhibitory effect on the
expansion
of PANC-1 and CFPAC-1 tumor cell lines. The inhibitory effects on the cells
with
different expression levels of receptor FOLR1 are different. The ICso ratio in
cell
line CFPAC-1 with a relatively high expression of receptor FOLR1 is
significantly
higher than the ICso ratio in the PANC-1 with a relatively low expression of
receptor
FOLR1, and the inhibitory effects on cell proliferation is positively
correlated with
expression levels of cell-related receptor FOLR1.
Experiments 2 and 3 prove that the two ligands in CB-20BK and the receptors
thereof (FOLR1 and FOLH1) expressed on the cell surface play an important role
in
the compound inhibiting tumor cell proliferation.
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4. Experiment of inhibiting tumor cell expansion in CB-20B
Sample infoimation: CB-20B
Cell lines: A549 human lung cancer cells, HuH-7 human liver cancer cells, KB
human oral epideituoid carcinoma cells, LNCaP human prostate cancer cells,
DU145 human prostate cancer cells and T-47D human breast cancer cells
Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin
solution, L-glutamine and CCK8
Experimental operations:
1) Cell plating
The cells were prepared in advance, digested with trypsin, collected and
counted; diluted with a complete cell culture medium, A549 cells, HuH-7 cells,
KB
cells and DU145 cells were plated at a density of 2 x 104 cells/ml, T-47D
cells were
plated at a density of 1 x 105 cells/ml, and LNCaP cells were plated at a
density of 6
x 104 cells/ml in 96-well plates; 100 1.11 of the diluted cell solution was
added to
each well; and negative control and blank control wells were set on each
plate. The
96-well plates added with the cells were placed in an incubator at 37 C, 5%
CO2 for
culturing overnight.
2) Diluting and adding samples
The samples were diluted with a culture medium to desired concentrations (see
Table 11). To each well of the 96-well plates that were cultured overnight, 50
1.11 of
the diluted samples were added, with 3 replicate wells; negative controls and
blank
controls were set; and the plates were placed in an incubator at 37 C, 5% CO2
for
culturing 72 hours.
Table 11: Concentration of Sample (CB-20B) After Dilution
Concentration after
Tube number
dilution (mon)
1 200
2 66.7
3 22.2
4 7.4
5 2.5
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6 0.8
7 0.3
8 0.09
9 0.03
0.01
3) Color development and reading plate
To each well, 15 p1(10% of the liquid volume in a well) of a color developing
solution from CCK-8 was added; the plates were incubated at 37 C for an
5 appropriate time (try to keep the OD value in the range of 1.0-2.0); the
covers of the
96-well plates were removed, and the plates were read at 450 nm on a
microplate
reader (Molecular Devices Spectra MAX Plus).
4) Data processing
The data was edited using SoftMax Pro and a four-parameter fitting curve was
10 plotted.
5) Experimental results and analysis
According to the four-parameter fitting curve graph (Fig. 4B, wherein C value
corresponds to IC5o), CB-20B has an inhibitory effect on the growth and
expansion
of A549, HuH-7, KB, LNcap, DU145 and T-47D tumor cell lines. Since expression
levels of the corresponding receptors of conjugate compounds in the cells are
different, the inhibition levels are different. The IC5o for cell lines T-47D,
LNcap,
KB, HuH-7 and DU145 with a relatively high expression of the receptors is
significantly lower than that for cell line A549 with a relatively low
expression of
the receptors. CB-20B shows a correlation between tumor growth inhibition
effects
and expression levels of cell-related receptors.
5. Experiment of inhibiting tumor cell expansion in CB-10S
Sample info' _______ "nation: CB-10S
Cell lines: KB human oral epideimoid carcinoma cells, NCI-H460 human lung
cancer cells, RT4 human bladder cancer cells, T-47D human breast cancer cells
and LNcap human prostate cancer cells
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Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin
solution, L-glutamine and CCK8
Experimental operations:
1) Cell plating
The cells were prepared in advance, digested with trypsin, collected and
counted; with a complete cell culture medium, KB cells were diluted to 3.5 x
104
cells/ml, LNCaP cells were diluted to 8.0 x 104 cells/ml, T-47D cells were
diluted to
1.2 x 104 cells/ml, NCI-H460 cells were diluted to 2.5 x 104 cells/ml, and RT4
cells
were diluted to 1.2 x 104 cells/ml; to each well of 96-well plates, 100 [11 of
the
diluted cell solution was added; and negative control and blank control wells
were
set on each plate. The 96-well plates added with the cells were placed in an
incubator at 37 C, 5% CO2 for culturing overnight.
2) Diluting and adding samples
The samples were diluted with a culture medium to desired concentrations (see
Table 12). To each well of the 96-well plates that were cultured overnight, 50
[El of
the diluted samples were added, with 3 replicate wells; negative controls and
blank
controls were set; and the plates were placed in an incubator at 37 C, 5% CO2
for
culturing 72 hours.
Table 12: Concentration of Sample (CB-10S) After Dilution
Concentration after
Tube number
dilution (junol/L)
1 302
2 43
3 6
4 0.9
5 0.1
6 0.02
7 0.003
8 0.0004
9 0.00005
10 0.000007
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3) Color development and reading plate
To each well, 15 p1(10% of the liquid volume in a well) of a color developing
solution from CCK-8 was added; the plates were incubated at 37 C for an
appropriate time (try to keep the OD value in the range of 1.0-2.0); the
covers of the
96-well plates were removed, and the plates were read at 450 nm on a
microplate
reader (Molecular Devices Spectra MAX Plus).
4) Data processing
The data was edited using SoftMax Pro and a four-parameter fitting curve was
plotted.
5) Experimental results and analysis
According to the four-parameter fitting curve graph (Fig. 4C, wherein C value
corresponds to ICso), CB-10S has an inhibitory effect on the growth and
expansion
of KB, LNCaP, T-47D, RT4 and NCI-H460 tumor cell lines. Since expression
levels
of the corresponding receptors of conjugate compounds in the cells are
different, the
inhibition levels are different. The ICso for cell lines KB, LNcap, T-47D and
RT4
with a relatively high expression of the receptors is significantly lower than
that for
cell line NCI-H460 with a relatively low expression of the receptors. CB-10S
shows
a correlation between tumor growth inhibition effects and expression levels of
cell-related receptors.
6. Experiment of inhibiting tumor cell expansion in CB-60S
Sample infoimation: CB-605
Cell lines: KB human oral epideimoid carcinoma cells, T-47D human breast
cancer cells, NCI-H460 human lung cancer cells, CALU-3 human lung
adenocarcinoma cells, HuH-7 human liver cancer cells and LNCaP human prostate
cancer cells
Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin
solution, L-glutamine and CCK8
Experimental operations:
1) Cell plating
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The cells were prepared in advance, digested with trypsin, collected and
counted; with a complete cell culture medium, KB cells were diluted to 2.0 x
104
cells/ml, T-47D cells were diluted to 1.5 x 104 cells/ml, NCI-H460 cells were
diluted to 2.0 x 104 cells/ml, CALU-3 cells were diluted to 3.0 x 104
cells/ml,
HuH-7 cells were diluted to 4.0 x 104 cells/ml, and LNCaP cells were
diluted to 8.0
x 104 cells/ml; 100 IA of the diluted cell solution was added to each well of
96-well
plates; and negative control and blank control wells were set on each plate.
The
96-well plates added with the cells were placed in an incubator at 37 C, 5%
CO2 for
culturing overnight.
2) Diluting and adding samples
The samples were diluted with a culture medium to desired concentrations (see
Table 13). To each well of the 96-well plates that were cultured overnight, 50
[El of
the diluted samples were added, with 3 replicate wells; negative controls and
blank
controls were set; and the plates were placed in an incubator at 37 C, 5% CO2
for
culturing 72 hours.
Table 13: Concentration of Sample (CB-60S) After Dilution
Concentration after
Tube number
dilution (mon)
1 320
2 80
3 20
4 5
5 1.3
6 0.3
7 0.08
8 0.02
9 0.005
10 0.001
3) Color development and reading plate
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To each well, 15 p1(10% of the liquid volume in a well) of a color developing
solution from CCK-8 was added; the plates were incubated at 37 C for an
appropriate time (try to keep the OD value in the range of 1.0-2.0); the
covers of the
96-well plates were removed, and the plates were read at 450 nm on a
microplate
reader (Molecular Devices Spectra MAX Plus).
4) Data processing
The data was edited using SoftMax Pro and a four-parameter fitting curve was
plotted.
5) Experimental results and analysis
According to the four-parameter fitting curve graph (Fig. 4D, wherein C value
corresponds to IC5o), CB-605 has an inhibitory effect on the growth and
expansion
of KB, T-47D, NCI-H460, CALU-3, HuH-7, and LNCaP tumor cell lines. Since
expression levels of the corresponding receptors of conjugate compounds in the
cells are different, the inhibition levels are different. The IC50 for cell
lines LNCaP
and HuH-7 with a relatively high expression of the receptors is significantly
lower
than that for cell lines NCI-H460, KB, T-47D and CALU-3 with a relatively low
expression of the receptors. CB-605 shows a correlation between tumor growth
inhibition effects and expression levels of cell-related receptors.
7. Experiment of inhibiting tumor cell expansion in CB-60SK
Sample infoimation: CB-605K
Cell lines: KB human oral epideimoid carcinoma cells, T-47D human breast
cancer cells, NCI-H460 human lung cancer cells, CALU-3 human lung
adenocarcinoma cells, HuH-7 human liver cancer cells and LNCaP human prostate
cancer cells
Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin
solution, L-glutamine and CCK8
Experimental operations:
1) Cell plating
The cells were prepared in advance, digested with trypsin, collected and
counted; with a complete cell culture medium, KB cells were diluted to 2.5 x
104
cells/ml, T-47D cells were diluted to 1.3 x 104 cells/ml, NCI-H460 cells were
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diluted to 3.5 x 104 cells/ml, CALU-3 cells were diluted to 4.0 x 104
cells/ml,
HuH-7 cells were diluted to 3.0 x 104 cells/ml, and LNCaP cells were diluted
to 8.0
x 104 cells/ml; 100 [t.1 of the diluted cell solution was added to each well;
and
negative control and blank control wells were set on each plate. The 96-well
plates
added with the cells were placed in an incubator at 37 C, 5% CO2 for culturing
overnight.
2) Diluting and adding samples
The samples were diluted with a culture medium to desired concentrations (see
Table 14). To each well of the 96-well plates that were cultured overnight, 50
[11 of
the diluted samples were added, with 3 replicate wells; negative controls and
blank
controls were set; and the plates were placed in an incubator at 37 C, 5% CO2
for
culturing 72 hours.
Table 14: Concentration of Sample (CB-60SK) After Dilution
Concentration after
Tube number
dilution (mon)
1 289
2 72
3 18
4 4.5
5 1.1
6 0.3
7 0.07
8 0.02
9 0.004
10 0.001
3) Color development and reading plate
To each well, 15 p1(10% of the liquid volume in a well) of a color developing
solution from CCK-8 was added; the plates were incubated at 37 C for an
appropriate time (try to keep the OD value in the range of 1.0-2.0); the
covers of the
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96-well plates were removed, and the plates were read at 450 nm on a
microplate
reader (Molecular Devices Spectra MAX Plus).
4) Data processing
The data was edited using SoftMax Pro and a four-parameter fitting curve was
plotted.
5) Experimental results and analysis
According to the four-parameter fitting curve graph (Fig. 4E, wherein C value
corresponds to IC5o), CB-605K has an inhibitory effect on the growth and
expansion of KB, T-47D, NCI-H460, CALU-3, HuH-7, and LNCaP tumor cell lines.
Since expression levels of the corresponding receptors of conjugate compounds
in
the cells are different, the inhibition levels are different. The IC50 for
cell lines
LNCaP and HuH-7 with a relatively high expression of the receptors is
significantly
lower than that for cell lines T-47D, NCI-H460, CALU-3 and KB with a
relatively
low expression of the receptors. CB-605K shows a correlation between tumor
growth inhibition effects and expression levels of cell-related receptors.
8. Experiment of inhibiting tumor cell expansion in CB-18G
Sample info' _______ "nation: CB-18G
Cell lines: A549 human lung cancer cells, Hela human cervical cancer cells,
SCLC-21H small cell lung cancer cells, U-2 OS human osteosarcoma cells, T-47D
human breast cancer cells and NCI-H460 human lung cancer cells
Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin
solution, L-glutamine and CCK8
Experimental operations:
1) Cell plating
The cells were prepared in advance, digested with trypsin, collected and
counted; diluted with a complete cell culture medium, A549 cells, Hela cells,
SCLC-21H cells and U-2 OS cells were plated at a density of 2 x 104 cells/ml,
and
T-47D cells and NCI-H460 cells were plated at a density of 3 x 104 cells/ml in
96-well plates; to each well of the 96-well plates, 100 [t.1 of the diluted
cell solution
was added; and negative control and blank control wells were set on each
plate. The
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96-well plates added with the cells were placed in an incubator at 37 C, 5%
CO2 for
culturing overnight.
2) Diluting and adding samples
The samples were diluted with a culture medium to desired concentrations (see
Table 15). To each well of the 96-well plates that were cultured overnight, 50
[El of
the diluted samples were added, with 3 replicate wells; negative controls and
blank
controls were set; and the plates were placed in an incubator at 37 C, 5% CO2
for
culturing 72 hours.
Table 15: Concentration of Sample (CB-18G) After Dilution
Concentration after
Tube number
dilution (mon)
1 100
2 25
3 6.25
4 1.56
5 0.39
6 0.10
7 0.02
8 0.006
9 0.002
0.0004
3) Color development and reading plate
To each well, 15 p1(10% of the liquid volume in a well) of a color developing
solution from CCK-8 was added; the plates were incubated at 37 C for an
appropriate time (try to keep the OD value in the range of 1.0-2.0); the
covers of the
96-well plates were removed, and the plates were read at 450 nm on a
microplate
reader (Molecular Devices Spectra MAX Plus).
4) Data processing
The data was edited using SoftMax Pro and a four-parameter fitting curve was
plotted.
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5) Experimental results and analysis
According to the four-parameter fitting curve graph (Fig. 4F, wherein C value
corresponds to IC50), CB-18G has an inhibitory effect on the growth and
expansion
of A549, Hela, SCLC-21H, U-2 OS, T-47D and NCI-H460 tumor cell lines. Since
expression levels of the corresponding receptors of conjugate compounds in the
cells are different, the inhibition levels are different. The ICso for cell
line Hela with
a relatively high expression of the receptors is significantly lower than that
for cell
line NCI-H460 with a relatively low expression of the receptors. CB-18G shows
a
correlation between tumor growth inhibition effects and expression levels of
cell-related receptors.
9. Experiment of inhibiting tumor cell expansion in CB-50S
Sample infoimation: CB-505
Cell lines: KB human oral epideimoid carcinoma cells, NCI-H460 human lung
cancer cells, RT4 human bladder cancer cells, T-47D human breast cancer cells
and LNCaP human prostate cancer cells
Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin
solution, L-glutamine and CCK8
Experimental operations:
1) Cell plating
The cells were prepared in advance, digested with trypsin, collected and
counted; with a complete cell culture medium, KB cells were diluted to 3.5 x
104
cells/ml, LNCaP cells were diluted to 8.0 x 104 cells/ml, T-47D cells were
diluted to
1.2 x 104 cells/ml, NCI-H460 cells were diluted to 2.5 x 104 cells/ml, and RT4
cells
were diluted to 1.2 x 105 cells/ml; to each well of 96-well plates, 100 [1.1
of the
diluted cell solution was added; and negative control and blank control wells
were
set on each plate. The 96-well plates added with the cells were placed in an
incubator at 37 C, 5% CO2 for culturing overnight.
2) Diluting and adding samples
The samples were diluted with a culture medium to desired concentrations (see
Table 16). To each well of the 96-well plates that were cultured overnight, 50
[El of
the diluted samples were added, with 3 replicate wells; negative controls and
blank
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controls were set; and the plates were placed in an incubator at 37 C, 5% CO2
for
culturing 72 hours.
Table 16: Concentration of Sample (CB-50S) After Dilution
Concentration after
Tube number
dilution (umol/L)
1 314
2 45
3 6
4 0.9
0.1
6 0.02
7 0.003
8 0.0004
9 0.00005
0.000008
5 3) Color development and reading plate
To each well, 15 p1(10% of the liquid volume in a well) of a color developing
solution from CCK-8 was added; the plates were incubated at 37 C for an
appropriate time (try to keep the OD value in the range of 1.0-2.0); the
covers of the
96-well plates were removed, and the plates were read at 450 nm on a
microplate
10 reader (Molecular Devices Spectra MAX Plus).
4) Data processing
The data was edited using SoftMax Pro and a four-parameter fitting curve was
plotted.
5) Experimental results and analysis
According to the four-parameter fitting curve graph (Fig. 4G, wherein C value
corresponds to IC5o), CB-505 has an inhibitory effect on the growth and
expansion
of KB, LNCaP, T-47D, RT4 and NCI-H460 tumor cell lines. Since expression
levels
of the corresponding receptors of conjugate compounds in the cells are
different, the
inhibition levels are different. The ICso for cell lines KB, LNcap, T-47D and
RT4
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with a relatively high expression of the receptors is significantly lower than
that for
cell line NCI-H460 with a relatively low expression of the receptors. CB-50S
shows
a correlation between tumor growth inhibition effects and expression levels of
cell-related receptors.
10. Experiment of inhibiting tumor cell expansion in conjugate
compound CB-1020
Experiment purpose: to test inhibitory effects of CB-1020 and related
compounds on the expansion of tumor cell lines LNCaP (human prostate cancer
cells), SK-BR-3 (human breast cancer cells), NCI-H226 (human lung cancer
cells),
CFPAC-1 (pancreatic cancer cells) and PANC -1 (pancreatic cancer cells).
Related samples: MMAE and CB-1020
Experimental operations: LNCaP, SK-BR-3, NCI-H226, CFPAC-1 and
PANC-1 cells were prepared in advance and counted; with a culture medium
(IMDM + 10% FBS + 1X L-Glutamine + 1X P/S), the cells LNCaP, SK-BR-3 and
CFPAC-1 were diluted to 4 x 104 cells/ml, NCI-H226 was diluted to 2.0 x 104
cells/ml and PANC-1 was diluted to 3.0 x 104 cells/ml; the diluted cell
solution was
plated in 96-well plates at 100 [11/well; and negative control and blank
control wells
were set on each plate. After adherent cell culture overnight, the diluted
samples to
be tested were added at 50 1/well. The plates were placed in an incubator at
37 C,
5% CO2 and cultured for 68-72 hours. To each well, 15 p1(10% of the liquid
volume in a well) of a color developing solution from CCK8 was added; and the
plates were incubated at 37 C for 45-70 minutes and each plate was read at 450
nm
on a microplate reader. The data was edited using SoftMax Pro and a 4-P
fitting
curve was plotted. The data is shown in the following table:
Table 17. Inhibitory Effects of CB-1020 on Proliferation of Cell Lines and
Receptor
Gene Expression Levels in Cell Lines
(with reference to data from Depmap)
TRPV6 gene FOLH1 gene ICso
ICso ICso
Cell line expression expression
(MMAE)/ICso
(MMAE) (CB-1020)
level level (CB-1020)
LNCap 5+ 10+ 0.003 0.168 0.0179
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SK-BR-3 4+ 2+ 0.002 0.287 0.0070
CFPAC -1 +/- 0.015 3.000 0.005
PANC-1 +/- +/- 0.002 0.458 0.0044
NCI-H226 +/- 0.006 1.570 0.00384
It can be seen from the above table that both LNCaP and SK-BR-3 express
TRPV6 and FOLRH1, wherein LNCap has a relatively higher expression level of
the two receptors. NCI-H226, CFPAC-1 and PANC-1 express the two receptors at a
low or weak level. CB-1020 has an inhibitory effect on the growth and
expansion of
LNCaP, SK-BR-3, NCI-H226, CFPAC-1 and PANC-1 tumor cell lines. The ICso
ratio of CB-1020 for cell line LNCaP with a relatively high expression of the
two
receptors is higher than the IC50 ratio for cell line SK-BR-3 with a medium
expression of the receptors; the ICso ratio for SK-BR-3 is higher than the
IC50 ratios
for CFPAC-1 with a relatively low expression of the receptors and cell lines
NCI-H226 and PANC-1 with a weak expression of the two receptors. The
inhibitory
effect on cell proliferation is positively correlated with the expression
level of the
two receptors in the cells.
11. Experiment of inhibiting tumor cell expansion in conjugate
compound CB-1320
Experiment purpose: to test inhibitory effects of CB-1320 and related
compounds on the expansion of tumor cell lines LNCaP (human prostate cancer
cells), SK-BR-3 (human breast cancer cells), MDA-MB-468 (human breast cancer
cells) and CFPAC-1 (pancreatic cancer cells).
Related samples: MMAE and CB-1320
Experimental operations: LNCaP, SK-BR-3, MDA-MB-468 and CFPAC-1
cells were prepared in advance and counted; with a culture medium (IMDM + 10%
FBS + lx L-Glutamine + 1X P/S), the cells LNCaP, SK-BR-3, MDA-MB-468 and
CFPAC-1 were diluted to 4 x 104 cells/ml; the diluted cell solution was plated
in
96-well plates at 100 ul/well; and negative control and blank control wells
were set
on each plate. After adherent cell culture overnight, the diluted samples to
be tested
were added at 50 1.11/well. The plates were placed in an incubator at 37 C, 5%
CO2
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and cultured for 68-72 hours. To each well, 15 p1(10% of the liquid volume in
a
well) of a color developing solution from CCK8 was added; and the plates were
incubated at 37 C for 45-70 minutes and each plate was read at 450 nm on a
microplate reader. The data was edited using SoftMax Pro and a 4-P fitting
curve
was plotted. The specific data is shown in the following table:
Table 18. Inhibitory Effects of CB-1320 on Proliferation of Cell Lines and
Receptor
Gene Expression Levels in Cell Lines
(with reference to data from Depmap)
FOLH1 gene ICso
GNRHR gene ICso ICso
Cell line expression
(MMAE)/1C50
expression level (MMAE) (CB-1320)
level (CB-1320)
LNCaP +/- 10+ 0.003 0.060 0.043
SK-BR-3 +/- 2+ 0.002 0.093 0.016
CFPAC-1 +/- +/- 0.015 1.090 0.014
MDA-MB-468 +/- +/- 0.002 0.212 0.011
It can be seen from the above table that the expression levels of GNRHR in the
4 cell lines are roughly equivalent, and the expression level of FOLH1 in
LNCaP is
significantly higher than that in other cell lines. CB-1320 has an inhibitory
effect on
the expansion of 4 tumor cell lines. The inhibitory effects of CB-1320 on the
cells
with different expression levels of receptor FOLH1 are different. The ICso
ratio in
cell line LNCap with a relatively high expression of receptor FOLH1 is
significantly higher than the ICso ratio in other cell lines with a relatively
low
expression of the receptor.
12. Experiment of inhibiting tumor cell expansion in conjugate
compound CB-1820
Experiment purpose: to test inhibitory effects of CB-1820 and related
compounds on the expansion of tumor cell lines LNCaP (human prostate cancer
cells), MDA-MB-468 (human breast cancer cells), CFPAC-1 (pancreatic cancer
cells) and PANC-1 (pancreatic cancer cells).
Related samples: MMAE and CB-1820
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Experimental operations: LNCaP, MDA-MB-468, CFPAC-1 and PANC-1 cells
were prepared in advance and counted; with a culture medium (IMDM + 10% FBS
+ lx L-Glutamine + lx P/S), the cells LNCaP, MDA-MB-468 and CFPAC-1 were
diluted to 4 x 104 cells/ml, and PANC-1 was diluted to 3.0 x 104 cells/ml; the
diluted cell solution was plated in 96-well plates at 100 [tl/well; and
negative
control and blank control wells were set on each plate. After adherent cell
culture
overnight, the diluted samples to be tested were added at 50 p1/well. The
plates were
placed in an incubator at 37 C, 5% CO2 and cultured for 68-72 hours. To each
well,
p1(10% of the liquid volume in a well) of a color developing solution from
10 CCK8 was added; and the plates were incubated at 37 C for 45-70 minutes
and
each plate was read at 450 nm on a microplate reader. The data was edited
using
SoftMax Pro and a 4-P fitting curve was plotted. The specific values are shown
in
the following table:
Table 19. Inhibitory Effects of CB-1820 on Proliferation of Cell Lines and
Receptor
15 Gene Expression Levels in Cell Lines
(with reference to data from Depmap)
SSTR2 gene FOLH1 gene
ICso ICso ICso (MMAE)/ICso
Cell line expression expression
(MMAE) (CB-1820) (CB-1820)
level level
LNCap +/- 10+ 0.003 0.021 0.1429
MDA-MB-468 +/- +/- 0.002 0.062 0.0323
CFPAC-1 +/- +/- 0.015 0.356 0.0421
PANC-1 +/- +/- 0.002 0.055 0.0364
It can be seen from the above table that the expression levels of SSTR2 in
LNCap, MDA-MB-468, CFPAC-1 and PANC-1 are roughly equivalent, and the
expression level of FOLH1 in LNCaP is significantly higher than the expression
level of FOLH1 in other cell lines. CB-1820 has an inhibitory effect on the
expansion of 4 tumor cell lines. The inhibitory effects of CB-1820 on the
cells with
different expression levels of receptor FOLH1 are different. The IC50 ratio in
cell
line LNCaP with a relatively high expression of the receptor is significantly
higher
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than the ICso ratio in other cell lines with a relatively low expression of
the receptor,
and the inhibitory effects on cell proliferation is positively correlated with
expression levels of cell-related receptor FOLH1.
13. Experiment of inhibiting tumor cell expansion in conjugate
compounds CR19425, CR19426 and CR19428
Experiment purpose: to test inhibitory effects of CR19428, CR19425,
CR19426 and related compounds on the expansion of tumor cell lines NCI-H226
(human lung cancer cells), CFPAC-1 (human pancreatic cancer cells) and
MDA-MB-468 (human breast cancer cells).
Related samples: SN-38, Dxd0017, CR19428, CR19425 and CR19426
Experimental operations: NCI-H226, CFPAC-1 and MDA-MB-468 cells were
prepared in advance and counted; MDA-MB-468 and CFPAC-1 cells were plated at
a density of 2 x 104 cells/ml (100 p1/well) and NCI-H226 cells were plated at
a
density of 1 x 104 cells/ml (100 1/well) in 96-well plates; and negative
control and
blank control wells were set on each plate. After adherent cell culture
overnight, the
diluted samples to be tested were added at 50 1/well. The plates were placed
in an
incubator at 37 C, 5% CO2 and cultured for 68-72 hours. To each well, 15
p1(10%
of the liquid volume in a well) of a color developing solution from CCK8 was
added; and the plates were incubated at 37 C for 45-70 minutes and each plate
was
read at 450 nm on a microplate reader. The data was edited using SoftMax Pro
and a
4-P fitting curve was plotted. The specific data is shown in the following
table:
Table 20. Inhibitory Effects of CR19428, CR19425 and CR19426 on Proliferation
of Cell Lines
ICso ICso ICso ICso
ICso
Cell line
(SN-38) (Dxd0017) (CR19428) (CR19425) (CR19426)
CFPAC-1 0.0055 0.0064 0.9060 0.6210
1.6600
MDA-MB-468 0.0136 0.0061 1.0700 0.9510
2.3000
NCI-H226 0.0173 0.0080 1.6200 1.0100
4.3900
Example 5: Pharmacodynamic Study of Conjugate Compounds in CDX
Models
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1. Pharmacodynamic study 1 of CB-20BK in CDX models
1) Sample preparation:
CB-20BK lyophilized powder was weighed, dissolved with PBS and prepared
into a sample mother solution, and the mother solution was diluted with a
physiological saline for injection to a sample solution with a working
concentration
for later use.
2) CDX model construction
Main cell lines: KB human oral epidermoid carcinoma cells, MIA paca-2
human pancreatic cancer cells, HCC1954 human breast cancer cells, CALU-3
human lung adenocarcinoma cells and DU145 human prostate cancer cells.
Model construction: The cells were resuscitated, cultured, collected, counted,
and subcutaneously injected into the right flank of BALB/c-nude mice. When the
tumor grew and expanded to 80-160 mm3, grouping and administration were
performed, or rapidly expanded tumor masses were transferred and injected to
mice
for scale up.
3) Grouping, administration and observation
Grouping: When the tumor grew to about 80-160 mm3 on average, a grouping
was performed, and a model control group and different doses of administration
groups were set.
Administration: The mice were administrated via tail vein injection.
Experimental observation and measurement: After tumor inoculation, the
effects of tumor growth and treatment on noinial behaviors of animals,
specifically
including the activity of experimental animals, food intake and drinking
status,
weight gain or loss (measuring weight twice a week), and other abnoinial
conditions
in eyes, hair, etc., were conventionally monitored.
The weight of mice and the long diameter (a) and short diameter (b) of tumor
mass were measured twice a week. The foimula for the calculation of tumor
volume
(TV) is: TV= 1/2 x ax b2.
4) Experimental results and analysis
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According to changes in tumor volumes of mice (Figs. 5A-5E), it can be seen
that CB-20BK has a good inhibitory effect in CDX tumor models of KB, MIA aca-
2,
HCC1954, CALU-3 and DU145 cell lines in mice.
2. Pharmacodynamic study 2 of CB-20BK in CDX models
Experiment purpose: to perfornt a phannacodynamic study of conjugate
compound CB-20BK in a subcutaneous xenograft model (CDX) of humanized
LNCaP, DU145 and NCI-H460 cell lines
Main CDX models: LNCaP, DU145 and NCI-H460
Experimental scheme:
The cells or tumor tissue mass were prepared and subcutaneously inoculated
into the anterior right flank of BALB/c-nude mice. When the tumor volume
expanded to 80-160 mm3, a randomized grouping was performed, and a model
control group and administration groups were set. The mice were administrated
from the first day of grouping, wherein the administration dose was adjusted
according to the latest weight measurement. The mice were administrated via
tail
vein injection at an administration volume of 10 1.11/g. After grouping and
administration, the effects of tumor growth and treatment on nothial behaviors
of
animals, specifically including the activity of experimental animals, food
intake and
drinking status, weight gain or loss status, and other abnoinial conditions in
eyes,
hair, etc., were monitored. After grouping and administration, the weight of
mice
was measured twice a week to calculate the rate of weight change; at the same
time,
the long diameter and short diameter of the tumor were measured with a vernier
caliper, and the tumor volume, relative tumor proliferation rate, tumor volume
inhibition rate and other indicators were calculated. The formula of tumor
volume is
TV = 0.5 a x b2, wherein a is the long diameter of a tumor and b is the short
diameter of a tumor.
Table 21. Inhibitory Effects of CB-20BK on Growth in CDX models and Receptor
Gene Expression Levels in Each Model
(with reference to data from Crown Bioscience Inc.)
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Tumor growth
Model FOLR1 FOLH1 inhibition rate
Administration dosage/time
(TGI) (%)
LNCaP 5+ 10+ 95.68 3 mg/kg D1, 8, 15
DU145 - - 52 5 mg/kg DI, 4, 7
NCI-H460 - - 32 10 mg/kg D1, 8, 15
It can be seen from the above table that the test sample CB-20BK shows
different degrees of tumor growth inhibition effects with a regimen of
administration via tail vein. For CDX models of humanized LNCaP, DU145 and
NCI-H460 cell lines, the test sample has a certain anti-tumor growth effect
compared with a negative control group. In the LNCaP model with dual
expression
of FOLR1 and FOLH1, the test sample was administered on day 1, day 8 and day
(D1, D8 and D15) at a dose of 3 mg/kg, has a TGI value of 95.68%, shows an
excellent anti-tumor growth effect, which is significantly better than that in
DU145
10 and NCI-
H460 models with a relatively low expression of FOLR1 and FOLH1. The
tumor growth inhibition effect has a certain correlation with the expression
level of
cell-related receptors.
3. Tumor growth inhibition experiment 1 of conjugate compound
CB-20BK in HuPrime xenograft PDX models
15
Experiment purpose: Phattnacodynamic study of conjugate compound
CB-20BK in PDX models
Main models: LU1206, LU1380 and LU0367
Experimental scheme: The tumor tissue mass were prepared and
subcutaneously inoculated into the anterior right flank of BALB/c-nude mice.
When
the tumor volume expanded to 80-160 mm3, a randomized grouping was perfottned,
and a model control group and administration groups were set. The mice were
administrated from the first day of grouping, wherein the administration dose
was
adjusted according to the latest weight measurement. The mice were
administrated
via tail vein injection at an administration volume of 10 1.11/g and an
administration
dosage of 3 mg/kg. After grouping and administration, the effects of tumor
growth
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and treatment on noinial behaviors of animals, specifically including the
activity of
experimental animals, food intake and drinking status, weight gain or loss
status,
and other abnoinial conditions in eyes, hair, etc., were monitored. After
grouping
and administration, the weight of mice was measured twice a week to calculate
the
rate of weight change; at the same time, the long diameter and short diameter
of the
tumor were measured with a vernier caliper, and the tumor volume, relative
tumor
proliferation rate, tumor volume inhibition rate and other indicators were
calculated.
The foimula of tumor volume is TV = 0.5 a x b2, wherein a is the long diameter
of a
tumor and b is the short diameter of a tumor.
Table 22. Inhibitory Effects of CB-20BK on Growth in Lung Cancer PDX Models
and Receptor Gene Expression Levels in Each Model
(with reference to data from Crown Bioscience Inc.)
FOLR1 gene FOLH1 gene
TGI (%)
Model expression level expression level Dosage
LU1206 4+ 2+ 90.89 3
mg/kg
LU1380 - 4+ 30.02 3
mg/kg
LU0367 - 5+ 71.34 3
mg/kg
It can be seen from the data in Table 22 that the test sample CB-20BK (3
mg/kg) shows a certain anti-tumor effect on LU1206, LU1380 and LU0367
humanized lung cancer PDX models. The TGI value in the LU1206 model with
dual expression of FOLR1 and FOLH1 is 90.89%, and the TGI values in LU1380
and LU0367 models with a single expression are 30.02% and 71.34%,
respectively.
In the tumor growth inhibition experiment, the tumor growth inhibition effect
has a
certain correlation with the expression level of cell-related receptors.
4. Tumor growth inhibition experiment 2 of conjugate compound
CB-20BK in HuPrime xenograft PDX models
Experiment purpose: Phannacodynamic study of conjugate compound
CB-20BK in PDX models
Main models: BR1283 and BR0438
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Experimental scheme: The tumor tissue mass were prepared and
subcutaneously inoculated into the anterior right flank of BALB/c-nude mice.
When
the tumor volume expanded to 80-160 mm3, a randomized grouping was perfottned,
and a model control group and administration groups were set. The mice were
administrated from the first day of grouping, wherein the administration dose
was
adjusted according to the latest weight measurement. The mice were
administrated
via tail vein injection at an administration volume of 10 lig and an
administration
dosage of 3 mg/kg. After grouping and administration, the effects of tumor
growth
and treatment on nottnal behaviors of animals, specifically including the
activity of
experimental animals, food intake and drinking status, weight gain or loss
status,
and other abnottnal conditions in eyes, hair, etc., were monitored. After
grouping
and administration, the weight of mice was measured twice a week to calculate
the
rate of weight change; at the same time, the long diameter and short diameter
of the
tumor were measured with a vernier caliper, and the tumor volume, relative
tumor
proliferation rate, tumor volume inhibition rate and other indicators were
calculated.
The fottnula of tumor volume is TV = 0.5 a x b2, wherein a is the long
diameter of a
tumor and b is the short diameter of a tumor.
Table 23. Inhibitory Effects of CB-20BK on Growth in Breast Cancer PDX Models
and Receptor Gene Expression Levels in Each Model
(with reference to data from Crown Bioscience Inc.)
FOLR1 gene expression FOLH1 gene
TGI (%)
Model level expression level Dosage
BR1283 7+ 6+ 96.49 3 mg/kg
BR0438 5+ 4+ 70.74 3 mg/kg
It can be seen from the above table that the TGI values of the test sample
CB-20BK at a dose of 3 mg/kg in BR1283 and BR0438 models with dual
expression of FOLR1 and FOLH1 are 96.49% and 70.74%, respectively, showing
an excellent anti-tumor growth effect. Moreover, the TGI of CB-20BK is higher
in
BR1283 with a higher expression of FOLR1 and FOLH1.
5. Pharmacodynamic study of CB-20B in CDX models
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1) Sample preparation:
CB-20B lyophilized powder was weighed, dissolved with PBS and prepared
into a sample mother solution, and the mother solution was diluted with a
physiological saline for injection to a sample solution with a working
concentration
for later use.
2) CDX model construction
Main cell lines: KB human oral epidermoid carcinoma cells, PC-9 human lung
cancer cells and DU145 human prostate cancer cells.
Model construction: The cells were resuscitated, cultured, collected, counted,
and subcutaneously injected into the right flank of BALB/c-nude mice. When the
tumor grew and expanded to 80-160 mm3, grouping and administration were
performed, or rapidly expanded tumor masses were transferred and injected to
mice
for scale up.
3) Grouping, administration and observation
Grouping: When the tumor grew to about 80-160 mm3 on average, a grouping
was performed, and a model control group and different doses of administration
groups were set.
Administration: The mice were administrated via tail vein injection.
Experimental observation and measurement: After tumor inoculation, the
effects of tumor growth and treatment on normal behaviors of animals,
specifically
including the activity of experimental animals, food intake and drinking
status,
weight gain or loss (measuring weight twice a week), and other abnoinial
conditions
in eyes, hair, etc., were conventionally monitored.
The weight of mice and the long diameter (a) and short diameter (b) of tumor
mass were measured twice a week. The foimula for the calculation of tumor
volume
(TV) is: TV= 1/2 x ax b2.
4) Experimental results and analysis
According to changes in tumor volume of mice (Figs. 6A-6C), it can be seen
that CB-20B has a good inhibitory effect in CDX tumor models of KB, PC-9 and
DU145 cell lines in mice.
6. Pharmacodynamic study of CB-18G in CDX models
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1) Sample preparation:
CB-18G lyophilized powder was weighed, dissolved with PBS and prepared
into a sample mother solution, and the mother solution was diluted with a
physiological saline for injection to a sample solution with a working
concentration
for later use.
2) CDX model construction
Main cell lines: KB human oral epidermoid carcinoma cells, PC-9 human lung
cancer cells, SPC-Al human lung adenocarcinoma cells, CALU-3 human lung
adenocarcinoma cells and DU145 human prostate cancer cells
Model construction: The cells were resuscitated, cultured, collected and
counted, and the cell solution was subcutaneously injected into the right
flank of
BALB/c-nude mice. When the tumor grew and expanded to 80-160 mm3, grouping
and administration were performed, or rapidly expanded tumor masses were
transferred and injected to mice for scale up.
3) Grouping, administration and observation
Grouping: When the tumor grew to about 80-160 mm3 on average, a grouping
was performed, and a model control group and different doses of administration
groups were set.
Administration: The mice were administrated via tail vein injection.
Experimental observation and measurement: After tumor inoculation, the
effects of tumor growth and treatment on noinial behaviors of animals,
specifically
including the activity of experimental animals, food intake and drinking
status,
weight gain or loss (measuring weight twice a week), and other abnoinial
conditions
in eyes, hair, etc., were conventionally monitored.
The weight of mice and the long diameter (a) and short diameter (b) of tumor
mass were measured twice a week. The formula for the calculation of tumor
volume
(TV) is: TV= 1/2 x ax b2.
4) Experimental results and analysis
According to changes in tumor volumes of mice (Figs. 7A-7E), it can be seen
that CB-18G has a good inhibitory effect in CDX tumor models of KB, PC-9,
SPC-Al, CALU-3 and DU145 cell lines in mice.
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7. Tumor growth inhibition experiment of conjugate compounds
CB-1020, CB-1320 and CB-1820 in subcutaneous xenograft model (CDX) of
humanized HPAF-II, NCI-11226 and SCLC-211I cell lines
Experiment purpose: Phannacodynamic study of ligand conjugates CB-1020,
CB-1320 and CB-1820 in CDX models
Main CDX models: HPAF-II (human pancreatic cancer cells), NCI-H226
(human lung cancer cells) and SCLC-21H (small cell lung cancer cells)
Experimental scheme:
The cells or tumor tissue mass were prepared and subcutaneously inoculated
into the anterior right flank of BALB/c-nude mice. When the tumor volume
expanded to 80-160 mm3, a randomized grouping was performed, and a model
control group and administration groups were set. The mice were administrated
from the first day of grouping, wherein the administration dose was adjusted
according to the latest weight measurement. The mice were administrated via
tail
vein injection at an administration volume of 10 1.11/g. After grouping and
administration, the effects of tumor growth and treatment on nothial behaviors
of
animals, specifically including the activity of experimental animals, food
intake and
drinking status, weight gain or loss status, and other abnoinial conditions in
eyes,
hair, etc., were monitored. After grouping and administration, the weight of
mice
was measured twice a week to calculate the rate of weight change; at the same
time,
the long diameter and short diameter of the tumor were measured with a vernier
caliper, and the tumor volume, relative tumor proliferation rate, tumor volume
inhibition rate and other indicators were calculated. The formula of tumor
volume is
TV = 0.5 a x b2, wherein a is the long diameter of a tumor and b is the short
diameter of a tumor.
Table 24. Inhibitory Effects of CB-1020, CB-1320 and CB-1820 on Growth in CDX
Models and Receptor Gene Expression Levels in Each Model (with reference to
data from Crown Bioscience Inc.)
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TRPV6 GNRHR SSTR2 FOLH1
gene gene gene gene TGI (%) TGI (%) TGI (%) P
Model Dosage
expression expression expression expression (CB-1020) (CB-1320) (CB-1820)
value
level level level level
0.283 0.461 3 mg/kg
HPAF-II + - +/- +/- 0.445 0.153 5
mg/kg
0.899 0.023 10 mg/kg
0.823 0.011 3 mg/kg
NCIH226 +/- +/- +/- - 0.962 0.006 3
mg/kg
0.992 0.005 10 mg/kg
0.995 0.131 3 mg/kg
SCLC-21H 2+ +/- 3+ - 0.361 0.610 3
mg/kg
0.985 0.134 10 mg/kg
CB-1020 has an obvious anti-tumor growth effect in subcutaneous xenograft
models of humanized HPAF-II cell lines; CB-1320 has an obvious anti-tumor
growth effect in subcutaneous xenograft models of NCI-H226 and SCLC-21H cell
lines; and CB-1820 has an obvious anti-tumor growth effect in subcutaneous
xenograft models of SCLC-21H cell lines.
8. Tumor growth inhibition experiment of conjugate compound
CR19428 in subcutaneous xenograft model (CDX) of humanized CALU-3,
SCLC-2111 and SPC-Al cell lines
Experimental scheme: The tumor tissue mass were prepared and
subcutaneously inoculated into the anterior right flank of BALB/c-nude mice.
When
the tumor volume expanded to 80-160 mm3, a randomized grouping was perfottned,
and a model control group and administration groups were set. The mice were
administrated from the first day of grouping, wherein the administration dose
was
adjusted according to the latest weight measurement. The mice were
administrated
via tail vein injection at an administration volume of 10 1.11/g and twice a
week for
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consecutive 3 weeks. After grouping and administration, the effects of tumor
growth
and treatment on noinial behaviors of animals, specifically including the
activity of
experimental animals, food intake and drinking status, weight gain or loss
status,
and other abnoinial conditions in eyes, hair, etc., were monitored. After
grouping
and administration, the weight of mice was measured twice a week to calculate
the
rate of weight change; at the same time, the long diameter and short diameter
of the
tumor were measured with a vernier caliper, and the tumor volume, relative
tumor
proliferation rate, tumor volume inhibition rate and other indicators were
calculated.
The foimula of tumor volume is TV = 0.5 a x b2, wherein a is the long diameter
of a
tumor and b is the short diameter of a tumor.
Table 25. Inhibitory Effects of CR19428 in Lung Cancer PDX models
Model TGI (%) Administration dosage/time
CALU-3 51 50 mg/kg q2/w*3
SCLC-21H 66 50 mg/kg q2/w*3
SPC -A1 75 50 mg/kg q2/w*3
SPC -A1 91 100 mg/kg q2/w*3
CR-19428 is a drug conjugate of ligands FOLR1 and FOLH1 and a payload
Dxd. It can be seen from the above table that the drug conjugate has an
obvious
anti-tumor growth effect in subcutaneous xenograft models of humanized CALU-3,
SCLC-21H and SPC-Al cell lines.
9. Study on effectiveness of conjugate compound CBP-1018 in lung
cancer LU2505 model (PDX model)
The 3rd generation of lung cancer PDX model LU2505 (from Asian female
patients), which is a rapid-growing tumor model, was used in this experiment.
BALB/c nude mice bore tumors subcutaneously and, when the tumor volume
reached about 150 mm3, were grouped into: low-dose, medium-dose and high-dose
test sample groups, a small molecule MMAE control group, a targeting
polypeptide
20BK-SMO9 control group, and a blank control group (a total of 6 groups, and 8
mice per group); the mice were administered on day 1, day 8 and day 15 after
the
grouping; and the observation was continued for 14 days after the last
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administration. The active substance of test sample CBP-1018 is CB-20BK, which
is obtained by mixing CB-20BK with auxiliary materials and then lyophilizing
same.
Table 26. Phannacodynamic Results of CBP-1018 for Injection in Lung
Cancer LU2505 models (first experiment)
Tumor
Test sample Tumor growth
Dosage Weight
Tumor weight
/Control volume inhibition
(mg/kg) (g) (mg)
sample (mm3) rate
TGI%
Blank control
/ 24.0 2219.62 / 2031.4
(D22)
group
4.5 22.5 0.00 100.00 0.0 (D29)
CBP-1018 1.5 22.1 386.16 82.60 400.7 (D29)
0.5 23.6 1634.50 26.36 2084.7 (D26)
20BK-SMO9 10 23.8 2024.75 8.78 1811.5
(D22)
MMAE 0.375 23.5 1132.61 48.97 1597.1 (D29)
Notes: 1) Due to requirements of animal welfare, animals in a group must be
euthanized when the mean tumor volume in the group reaches 2000 mm3, leading
to
different execution time for each group. 2) The data of weight, tumor volume,
and
tumor growth inhibition rate is the data obtained on day 22 (D22).
As shown in Table 26 and Fig. 8A:
animals in all groups show no abnoinial clinical manifestations and no deaths
after administration; and the weight of the animals in each group increases
slowly.
The animals in the blank control group, 20BK-SMO9 group, and low-dose
CBP-1018 group were euthanized on day 22, day 22, and day 26 (D26) because of
the mean tumor volume exceeding 2000 mm3. Therefore, the data of weight, tumor
volume, and tumor growth inhibition rate in Table 26 is the data obtained on
D22.
The effectiveness of CBP-1018 for injection has an obvious dose correlation,
wherein CBP-1018 for injection was ineffective in the low-dose group, and
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effective in the medium-dose group and the high-dose group. The tumor in the
high-dose group was completely cured on day 19 (D19), and no signs of tumor
growth were seen on day 29 (D29).
The polypeptide 20BK-SM09 group showed no obvious tumor growth
inhibition effect, suggesting that a single targeting polypeptide is not
enough to
produce a clear anti-tumor effect.
The MMAE group showed a clear tumor growth inhibition effect. Equimolar
MMAE is contained in MMAE at 0.375 mg/kg and in CBP-1018 at 1.5 mg/kg. It
can be seen from the comparison that the tumor growth inhibition effect of
CBP-1018 at 1.5 mg/kg is significantly better than that in the MMAE group,
suggesting the advantage of ligand targeting (tumor growth inhibition rate:
82.60%
vs 48.97%).
10. Study on effectiveness of conjugate compound CBP-1018 in lung
cancer LU1206 model (PDX model)
The 5th generation of lung cancer PDX model LU1206 (from Asian female
patients), which is a rapid-growing tumor model, was used in this experiment.
BALB/c nude mice bore tumors subcutaneously and, when the tumor volume
reached about 150 mm3, were grouped into: low-dose, medium-dose and high-dose
test sample groups, a small molecule MMAE control group, a targeting
polypeptide
20BK-SMO9 control group, and a blank control group (a total of 6 groups, and 8
mice per group); the mice were administered on day 1, day 8 and day 15 after
the
grouping; and the observation was continued for 14 days after the last
administration.
Table 27. Phannacodynamic Results of CBP-1018 for Injection in Lung Cancer
LU1206 models (first experiment)
Tumor
Tumor
Tumor volume Tumor weight
Dosage Weight
Groups volume Tumor growth weight Inhibition
(mg/kg) (g)
(mm3) inhibition rate (mg) ratio
(%) (%)
Blank control / 22.7 1216.67 / 856.14 /
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group
4.5 23.4 31.44 97.42 13.55 98.42
CBP-1018 1.5 23.7 301.64 75.21 185.10 78.38
0.5 22.7 1118.30 8.08 852.19 0.46
20BK-SMO9 10 23.3 1158.80 4.76 914.36 -6.80
MMAE 0.375 23.6 778.32 36.03 437.73 54.53
As shown in Table 27 and Fig. 8B:
animals in all groups show no abnoinial clinical manifestations and no deaths
after administration, and all animals were euthanized on day 29. The weight of
the
animals in each group remained basically unchanged, and is slightly higher
than that
on the first day of administration.
The effectiveness of CBP-1018 for injection has an obvious dose correlation,
wherein CBP-1018 for injection was ineffective in the low-dose group (with a
tumor growth inhibition rate of 8.08%), and effective in the medium-dose group
and
113 the high-dose group, with a tumor growth inhibition rate of 75.21% and
97.42%,
respectively. The difference in effectiveness between the low-dose group and
the
medium-dose group is relatively large.
The polypeptide 20BK-SM09 group showed no obvious tumor growth
inhibition effect, suggesting that a single targeting polypeptide is not
enough to
produce a clear anti-tumor effect in this model.
The MMAE group showed a clear tumor growth inhibition effect. Equimolar
MMAE is contained in MMAE at 0.375 mg/kg and in CBP-1018 at 1.5 mg/kg. It
can be seen from the comparison that the tumor growth inhibition effect of
CBP-1018 at 1.5 mg/kg is significantly better than that in the MMAE group,
suggesting the advantage of ligand targeting (tumor volume inhibition rate:
75.21%
vs 36.03%; tumor weight inhibition rate: 78.38% vs 54.53%).
11. Tissue distribution of tumor-bearing mice (PDX model LU2505)
12 female tumor-bearing mice inoculated with tumor mass of an HuPrime
lung cancer LU2505 model and 12 healthy male BALB/c nude mice were given 1.5
mg/140 fiCi/kg of [3fl]CBP-1018 (isotope labeling on MMAE) via single tail
vein
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injection. At 0.5 hours, 2 hours, 6 hours and 24 hours respectively, 3 male
mice and
3 female mice were placed in an induction box and anesthetized by inhaling an
appropriate amount of carbon dioxide; and blood was collected by cardiac
puncture,
and then euthanasia was carried out immediately to collect samples.
Table 28. Total Radioactivity in Various Tissues at Different Time Points
after
Single Intravenous Administration of [3I-1]CBP-1018
Radioactivity concentration (ng Eq./g)
Tissue 0.5 hours 2 hours 6
hours 24 hours (% C.)
Tumor 763 / 643 / 566 / 413
Esophagus 915 784 628 508 521 172 286 91.7
Body fat 341 479 288 276 231 177 67.2
43.7
Skeletal muscle 321 311 207 137 188 95.5 90.8
61.5
Spleen 744 693 701 524 671 421 253 163
Stomach wall 658 623 414 370 460 259 211
123
Whole brain 66.3 67.9 53.8 39.5 82.0 48.2 76.4
68.3
Testicular epididymis 350 494 279 298 183 248 92.3 64.8
Heart 785 795 415 326 219 143 114 69.2
Lung 1493 1807 997 920 416 392 109 93.7
Kidney 5682 6118 3300 4197 1227 1391 241 238
Liver 1423 1306 1257 865 1138 599 712 518
Small intestine wall 726 778 641 816 490 334 210
90.9
Large intestine wall 550 520 672 823 1074 490 215
128
Whole blood 2087 3194 684 691 211 132 86.5 64.6
Plasma 7372 7610 1844 1760 495 306 214 140
It can be seen from the above table that the distribution in animal tissues
shows
no difference between male and female; after administration, the drug is
mainly
distributed in kidney, whole blood (mainly distributed in plasma), liver and
lung; in
all tissues, the highest concentration appears at 0.5 hours, and then the drug
is
eliminated rapidly, wherein the slowest elimination appears in liver and
tumor; and
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the slow elimination in tumor can explain the advantages of CBP-1018 in terms
of
effectiveness.
12. Excretion experiment in rats
Six noinial SD rats, half male and half female, were given 0.75 mg/70 fiCi/kg
of [31-1]CBP-1018 via single tail vein injection. At designated time
intervals, samples
such as urine, feces, cage flushing/cleaning solution and corpses of integral
rats
were collected before administration and 0-168 hours after administration, and
samples such as bile, urine, feces and cage flushing/cleaning solution of BDC
rats
were collected before administration and 0-72 hours after administration. The
above-mentioned samples were cryopreserved in a low-temperature refrigerator
(-10 C to -30 C).
To each sample, an appropriate amount of scintillation fluid was added and
uniformly mixed, and then the amount of radioactivity was measured using a
liquid
scintillation counter. The amount of radioactivity measured in samples such as
bile,
urine, feces, cage flushing/cleaning solution and corpses was used to
calculate the
percentage in the given dose. The amount of radioactivity in the plasma sample
was
used to calculate the total radioactivity in each gram of the sample. The main
pharmacokinetic parameters of the total plasma radioactivity were calculated
using
WinNonLin software (version 7.0, Pharsight) according to a non-compartmental
model.
Table 29. Results of Mass Balance Study on Integral Rats After Administration
Cage Total
Sex (number of Urine Feces Corpse
flushing/cleaning
recovery
rats) (%) (%) (%)
solution (%) (%)
50.26 23.93 4.51 84.40
Female (n = 3) 5.70 2.63
6.11 0.98 1.19 4.02
53.60 20.61 4.83 83.28
Male (n = 3) 4.24 2.16
3.68 1.74 0.42 0.71
Female and male
51.93 22.27 4.67 83.84
(n = 3 female and 4.97 2.30
4.87 2.21 0.82 2.65
3 male)
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It can be seen from the above table that CBP-1018 is mainly excreted in urine,
which accounts for about 57% of the total given dose, and the amount excreted
in
feces accounts for less than 25%. The result of mainly excretion in urine
through the
kidney is consistent with the finding of large distribution in the kidney in
the tissue
distribution of nude mice.
13. Plasma stability
CBP-1008 (the active substance thereof is LDC1OB described in WO
2017025047 Al, and CBP-1008 is obtained by mixing LDC1OB with auxiliary
materials and lyophilizing same; WO 2017025047A1 is incorporated by reference
in its entirety) and CBP-1018 at a concentration of 1 [tg/mL, 10 [tg/mL and
100
[tg/mL were incubated with plasma of different species at 37 C for 2 hours to
observe the stability of the two compounds. The results show (in Table 30)
that
CBP-1018 is stable in plasma of various species, and the remaining percentage
thereof after 2 hours of incubation is above 90% relative to the concentration
at 0
hours. However, CBP-1008 is only stable in plasma of rats (with 87.92%-91.82%
remaining), and is unstable in plasma of other species, with only 0.751%-
24.52%
remaining.
The experimental results suggest that the plasma stability of CBP-1018 is
better than that of CBP-1008.
Table 30. Summary of Plasma Stability Data of CBP-1008 and CBP-1018 at
Different Concentrations in Various Species
(% relative to 0 hours)
Cynomolgus
Compound Concentration Mouse Rat Human
monkey
1 [tg/mL 0.805 87.92 0.961 0.751
CBP-1008 10 [tg/mL 1.27 91.47 0.988 0.807
100 [tg/mL 24.52 91.82 1.10 0.953
1 [tg/mL 94.4 92.2 97.3 99.1
CBP-1018 10 [tg/mL 96.5 95.0 95.8 102
100 [tg/mL 98.1 94.8 101 103
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Notes:
1) The data in the table is the percentage of the compound concentration in
plasma after plasma incubation for 2 hours relative to 0 hours.
2) Since the data in the table is obtained from two experiments, the effective
digits after the decimal point are inconsistent. For the traceability and
authenticity,
the data in the report is directly referred without uniformity.
14. Half-life in PK
Consistent with the comparison of plasma stability, the pharmacokinetic
half-life of CBP-1018 (about 1 hour) is significantly longer than that of CBP-
1008
(about 20 minutes) in rats and cynomolgus monkeys, suggesting that CBP-1018 is
more stable than CBP-1008.
Table 31. Comparison of Half-life (in hour) of CBP-1008 and CBP-1018 in PK of
Rats
0.15 mg/kg 0.5 mg/kg 1.5 mg/kg
Male Female Male Female Male Female
CBP-1008 0.245 0.215 0.249 0.202 0.222 0.368
CBP-1018 0.928 0.833 1.37 0.952 1.49 1.51
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