Note: Descriptions are shown in the official language in which they were submitted.
HYDROPHILIC LINKERS FOR CONJUGATION OF A CYTOTOXIC AGENT OR
CHROMOPHORE MOLECULE TO A CELL-BINDING MOLECULE
FIELD OF THE INVENTION
The present invention relates to the preparation of hydrophilic linkers used
for the
conjugation of a drug, in particular, a cytotoxic agent or a chromophore
molecule to a cell binding
molecule. The present invention also relates to methods of making cell-binding
agent-drug (e.g.
cytotoxic agent) conjugates comprising either modification of drugs with these
hydrophilic linkers
first, followed by reaction with cell-binding agents; or modification of cell-
binding agents with
these hydrophilic linkers first, followed by reaction with drugs.
BACKGROUND OF THE INVENTION
Targeted therapies have been the much focused in the pharmaceutical research
and
development for many years. They are a cornerstone of "precision medicine"
(President Barack
Obama, State of the Union Address, Jan. 20, 2015,
www.whitehouse.gov/precisionmedicine) that
uses specific information about a person's tumor to help diagnose, plan
treatment, find out how well
treatment is working, or make a prognosis (F. S. Collins, New Engl. J. Med.
2015; 372:793-795).
So far many different targeted therapies have been approved for use in cancer
treatment by US
FDA, European EMA and Chinese CFDA. These therapies include hormone therapies,
signal
transduction inhibitors, gene expression modulator, apoptosis inducer,
angiogenesis inhibitor,
immunotherapies, and toxin delivery molecules. The hormone therapies act by
preventing the body
from producing the hormones or by interfering with the action of the hormones,
which in turn can
slow or stop the growth of hormone-sensitive tumors. Signal transduction
inhibitors block a cell
responds to signals from its environment, in particular, prevent the ability
of cancer cells to
multiply quickly and invade other tissues. Gene expression modulator can
modify the function of
proteins that play a role in controlling gene expression. Apoptosis inducers
cause cancer cells to
undergo a process of controlled cell death. Angiogenesis inhibitors block the
growth of new blood
vessels to tumors (a process called tumor angiogenesis). Immunotherapies
trigger the immune
system to destroy cancer cells. Most immunotherapies are monoclonal antibodies
that recognize
specific molecules on the surface of cancer cells. Toxin delivery molecules
use a transport
vehicle/method to deliver a toxin drug specifically to the cancer cells.
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The much promising approach that has been studied extensively during the past
three
decades is the toxin delivery molecules. There are several systemic deliveries
of chemo-
therapeutical drugs for targeted treatment of tumor: Heat-activated targeted
drug delivery;
Tissue-selective drug delivery for cancer using carrier-mediated transport
systems; Tumor-
activated prodrug therapy for targeted delivery of chemotherapy; Pressure-
induced filtra-
tion of drug across vessels to tumor; Promoting selective permeation of the
anticancer
agent into the tumor; Two-step targeting using a bispecific antibody; Site-
specific delivery
by an antibody conjugate; And light-activation of macromolecules. Many
carriers have
been studied in different forms or in special formulations for the target
delivery of anti-
cancer drugs, such as Albumin-based drug carriers; Carbohydrate-enhanced
chemotherapy;
Proteins and peptides based drug carriers; Fatty acids as targeting vectors
linked to active
drugs; Microsphere carriers; Monoclonal antibodies as carriers; Vitamins, e.g.
folates as
carriers; Nanoparticle carriers,; Liposome carriers, e. g. pegylated liposomes
(enclosed in a
polyethylene glycol bilayer); Polyethylene glycol (PEG) carriers; Single-chain
antigen-
binding molecule carriers; Polymeric micelle carriers; Lipoprotein-based drug
carriers;
Dendrimers; etc. Ideally the delivery vehicle has to be site-specific, non-
toxic, biocompati-
ble, non-immunogenic, and biodegradable (Scott, R; et al (2008) Expert Opin.
Drug Deli.
5, 459) and avoid recognition by the host's defense mechanisms (Saltzman, W.;
(2008).
"Drug delivery systems" Access Science. McGraw-Hill Co.). For this criterion,
using a
monoclonal antibody (mAb) as a delivery vehicle for chemotherapeutical drugs
has been
much successful. By combining the unique targeting capabilities of inonoclonal
antibodies
with the cancer-killing ability of cytotoxic drugs, the antibody-drug
conjugates (ADCs)
allow sensitive discrimination between healthy and diseased tissue (ADC
Review, J.
Antibody-drug Conjugates, - Jun 1, 2013). Beside the successful market
approval of ado-
trasuaturiab emtansine (T-DM) in 2013 and Brentuximab vedotin in 2011 by US
FDA,
there are over 40 different ADC drugs currently in clinical trial.s in USA
(ww-w.clinicaltrials.gov). But there are still many challenges among ADC
development,
such as the linker selection for improvement of the therapeutic index, InAbs
linked to a
careful selection of the targets, a better understanding of the mechanism of
action, and the
management and understanding of ADC off-target toxicities. It has been known
that the
linker between the delivery vehicles, in particular, an antibodies and the
cell-killing toxins
plays a critical role in the development of targeted drug delivery systems, as
the nature of
the linker significantly affects the potency, selectivity and the
pharmacokinetics of the
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resulting conjugates (Zhao, R. Y. et al (2011) J. Med. Chem. 54, 3606;
Acchionea, M. et al
(2012) mAbs, 4, 362; Doronina, S. eta], (2006) Bioconjug Chem, 17, 114;
Hamann, P. et
al. (2005) Bioconjug Chem. 16, 346). So far. four types of linkers had often
been used for
preparation of cell binding agent-drug conjugates that have entered the
clinic: (a) acid-
labile linkers, exploiting the acidic endosomal and lysosomal intracellular
microenviron-
ment; (b) linkers cleavable by lysosomal proteases; (c) chemically stable
thioether linkers
that release a lysyl adduct after proteolytic degradation of the antibody
inside the cell; and
(d) disulfide-containing linkers, which are cleaved upon exposure to an
intracellular thiol
(Zhao, R. Y. eta!, 2011 J. Med. Chem. 36, 5404).
Conjugates of cell-binding agents with drugs or modified chemical compounds
via
different types of linkers have been described (U.S. Patent Nos. 4,680.338,
5,122,368,
5,141,648, 5,208,020, 5,416,064; 5,475,092, 5,543,390, 5,563,250 5,585,499,
5,880,270,
6,214,345, 6.436,931, 6,372,738, 6,340,701, 6,989,452, 7,129,261, 7,375,078,
7,498,302,
7,507,420, 7,691,962, 7,910,594, 7,968,586, 7,989,434, 7,994,135, 7,999,083,
8,153,768,
8,236.319, W02014080251. Zhao, R.; et al, (2011) J. Med. Chem. 36, 5404;
Doronina, S.;
et al, (2006) Bioconjug Chem, 17, 114; Hamann, P.; et al. (2005) Bioconjug
Chem. 16,
346). Typically, in these conjugates, the cell-binding agents are first
modified with a
bifunctional agent such as SPDP (N-succinimidyl 3-(2-pyridyldithio)
propionate), SMPDP
(N-succinimidyl 4-methyl-4-(2-pyridyldithio) pentanoate), SPDB (N-succinimidyl
4-(2-
pyridyldithio) butanoate), or SMCC (succinimidy1-4-(N-maleimidomethyl)
cyclohexane-l-
carboxylate), to introduce an active disulfide or a maleimido moiety. Reaction
with a thiol-
containing cytotoxic drug provides a conjugate in which the cell-binding
agent, such as a
monoclonal antibody, and drug are linked via disulfide bonds or thioether
bonds.
However, the use of the cell binding molecule-drug conjugates, such as
antibody
drug conjugates (ADCs), in developing therapies for a wide variety of cancers
has been
limited both by the availability of specific targeting agents (carriers) as
well as the conjuga-
tion methodologies which result in the formation of protein aggregates when
the amount of
the drugs that are conjugated to the carrier (i.e., the drug loading) is
increased. Normally
the tendency for cytotoxic drug conjugates to aggregate is especially
problematic when the
conjugation reactions are performed with the hydrophobic linkers. Since higher
drug
loading increases the inherent potency of the conjugate, it is desirable to
have as much drug
loaded on the carrier as is consistent with retaining the affinity of the
carrier protein. The
presence of aggregated protein, which may be nonspecifically toxic and
immunogenic, and
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therefore must he removed for therapeutic applications, makes the scale-up
process for the
production of these conjugates more difficult and decreases the yield of the
products.
We have invented a series of hydrophilic linkers containing phosphinate,
sulfonyl,
and/or sulfoxide groups which can improve methods for conjugating cytotoxic
drugs to a
carrier (cell binding molecules) in high a drug loading without aggregation
(PCT/M2012/056700 and PCT/CN2014/072769). Here we describe the extending
innova-
tion of the hydrophilic linkers for better conjugation, much conditional drug
release,
loading with two different kinds of drugs or molecules per a linker, and/or
linking with a
pair of sites of a cell binding molecule per a linker for specific or better
conjugation.
SUMMARY OF THE INVENTION
The present invention provides hydrophilic linkers containing phosphamide,
phosphinate, sulfonamide, sulfonyl, sulfonimide, and/or sulfoxide groups to
link drugs to a
cell-binding agent (e.g., an antibody). The preferred formula of the cell
binding molecule ¨
hydrophilic linker- drug conjugates can be represented as: Cb-(-L-Drug)õ.
wherein Cb is a
cell-binding agent, L is a hydrophilic linker, Drug is a drug molecule, and n
is an integer
from 1 to 20. The advantages in applying the hydrophilic linker in the cell
molecule-drug
conjugate are: a). Reducing the aggregation of the conjugates in water based
media; h).
enabling higher drug-per-cell binding molecule-ratio conjugate, resulting in
higher poten-
cy; c). Being retained inside the target cell after the drug-linker released
from the conju-
gates, which can combat permeability-glycoprotein (Pgp)-expressing multidrug
resistant
(MDR) cells; d). Enabling loading two different kinds of drugs per a linker;
f). Conjugating
a pair of sites of a cell binding molecule per a linker.
In one aspect of the present invention, the hydrophilic linker is represented
by For-
mula (I) wherein Y can react with a cell-binding agent and Z can react with a
cytotoxic
drug:
Y¨RifQ¨R2HT¨R3-1---R4¨Z
n
R5 R6 (I)
Wherein:
Y represents a functional group that enables reaction with a cell-binding
agent;
Q and T are either ¨X1¨P(=0)(0M)-. or X1-S(02)-, or ¨Xi¨S(0)-; or ¨Xi-
P(=0)(0M)-X2-, or ¨Xi¨P(=0)[X2-R4-4-X3-,or ¨X1¨P(=0)[X2-R1-Y]-X3-, or
X2-, or ¨Xi¨S(0)-X2-;
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Xi, X2 and X3 are independently selected from N(R7), 0, or S; In addition,
when Xi
is either N(R7), or 0, or S, then either X/ or X3, or another X1 connects to -
P(=0), -5(0),
or -S(02) can be CH2.
in and n are integer from 0 to 5, but not 0 at the same time;
Z represents a functional group that enables linkage of a cytotoxic drug via a
disul-
fide, thioether, thioester, peptide, hydrazone, ether, ester, carbamate,
carbonate, amine
(secondary, tertiary, or quartary), imine, cycloheteroalkyane, heteroaromatic,
alkoxime or
amide bond;
R1. R2, R3, R4, R5, R6, and R7 are the same or different and are H, linear
alkyl having
from 1-6 carbon atoms, branched or cyclic alkyl having from 3 to 6 carbon
atoms, linear,
branched or cyclic alkenyl or alkynyl, or 1-6 carbon atoms of esters, ether,
amide, or
polyethyleneoxy unit of formula (0CH1CH2)p, wherein p is an integer from 0 to
about
1000, or combination thereof.
Additionally R1, R2, R3 and R4 are respectively a chain of atoms selected from
C, N,
0,5, Si, and P that covalently connects the cell-surface binding ligand, the
phosphinate or
sulfonyl group, the conjugated drug and among themselves (R4, R2 , R3 and R4).
The atoms
used in forming the hydrophilic linker may be combined in all chemically
relevant ways,
such as forming alkylane, alkylene, alkenylene, and alkynylene, ethers,
polyoxyalkylene,
esters, amines, imines, polyamines, hydrazines, hydrazones, amides, ureas,
semicarbazides,
carbazides, alkoxyamincs, alkoxylamines, urethanes, amino acids, peptides,
acyloxylamines, hydroxamic acids, or combination thereof.
M is H, or Na, or K, or N+R1R1R3 or a pharmaceutical salt. R1, R2 and R3 are
de-
scribed above.
In another embodiment, when the hydrophilic linkers of the Formula (I) has two
or
more Y groups, in particular the two same Y groups, at such case, either Q,
or/and T is -
X1-P(=0)[X2-R1-Y]-X3-, then the hydrophilic linkers of the Formula (I) can be
linked to
two or more sites, particularly to a pair of sites of cell binding molecules.
In yet another embodiment, when the hydrophilic linkers of the Formula (I) has
two
Z or more groups, at such case, either Q, or/and T is -X1-13(=0)[X2-R4-Z]-X3-,
then the
hydrophilic linkers of the Formula (I) can be linked to two or more drugs, in
particular, two
different drugs.
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In another aspect, this invention provides a cell-binding agent-drug conjugate
of
Formula (II), in which the cell-binding agent, Cb, and the drug, Drug, have
reacted at the
two ends of the hydrophilic linker:
Cb-(RifQ-R2-1-[T R4 -Dru)
n R5 R6 q (II)
wherein:
Cb represents a cell-binding agent;
Drug represents the drug linked to the cell-binding agent via the hydrophilic
linker
by a disulfide, thioether, thioester, peptide, hydrazone, ether, ester,
carbamate, carbonate,
cycloheteroalkyane, heteroaromatic, alkoxime or amide bond;
q is 1 - 20; m, n, Ri, 129, R3, RI, R5, R6 and M are described the same
previously in
Formula (I).
Q and T are either -X1-P(=0)(0M)-, or -X1-S(02)-, or -X1-S(0)-; or -X1-
P(=0)(0M)-X2-, or -Xi-P(=0)[X2-R4-Drugl-X3-, or -X1-P(=0)[X2-R1-Cbl-X3-, or -
X1-
S(0/)-X7-, or -X1-S(0)-X2-;
Xi X2 and X3 are independently selected from N(R7), 0, or S; In addition, when
Xi
is either N(R7), or 0, or S. then either X,, or X3, or another Xi that
connects to -P(=0), -
S(0), or -S(02) can be CH2.
In a further aspect, the present invention provides a modified cell-binding
agent of
Formula (III), in which the cell-binding agent, Cb, has reacted with the
hydrophilic linker,
which still has Z, a group capable of reacting with a drug:
Cb-(Ri-FQ T -R3-1---R4-Z
n R5 R6 (III)
Wherein Cb, Z, m, n, q, Ri, R/. R3, R4, R5 and R6 are defined the same as in
Formula
(I) and (II).
Q and T are either -X1-P(=0)(0M)-, or -X1-S(02)-. or -X1-S(0)-; or -X1-
P(=0)(0M)-X2-, or -Xi-P(=0)P(2-R4-4-X3-, or -Xi-P(=0)P(2-R1-C14-X3-, or -X1-
S(02)-X2-, or
Xi X2 and X3 are independently selected from N(R7), 0, or S; In addition, when
X1 is
either N(R7), or 0, or S. then either X2, or X3, or another X1 that connects
to -P(=0), -S(0),
or -S(02) can be Cth.
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In an even further aspect, the present invention provides a modified drug of
Formula
(IV), in which the drug, Drug, has reacted with the hydrophilic linker, which
still has Y, a
group capable of reacting with the cell-binding agent:
Y¨RifQ-R2F-ET-R31¨R4¨Drug
n
R5 R6 (IV)
Wherein Y, Drug, m, n, q, R1, R2, R3, R4, R5 and R6 are defined the same as in
For-
mula (I) and ( II).
Q and T are either -X1-P(=0)(0M)-, or -Xi-S(02)-. or -Xi-S(0)-; or -Xi-
P(=0)(0M)-X2-, or -Xi-P(=0)[X2-R4-Drug]-X3-, or -X1-P(=0)[X2-R1-Y]-X3-. or -X1-
S(02)-X,-. or -X1-S(0)-X2-;
Xi, X2 and X3 are independently selected from N(R7), 0, or S; In addition,
when X1 is
either N(R7), or 0, or S, then either X,), or X3, or another Xi that connects
to -P(=0), -S(0),
or -S(02) can be Cfb.
The present invention further relates to a method of making a cell-binding
molecule-
drug conjugate of Formula (II), wherein the drug is linked to a cell-binding
agent via the
hydrophilic linker.
The present invention also relates to a method of making a modified cell-
binding
molecule of Formula wherein the cell-binding molecule is reacted with
the hydrophilic
linker.
The present invention also relates to a method of making a modified drug of
Formula
(IV), wherein the drug is reacted with the hydrophilic linker.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows synthesis of phosphamide linkers containing rnaleimide groups
and
the application of these linkers in the conjugation of an antibody with drugs.
Figure 2 shows the synthesis of phosphamide linkers containing disulfide bonds
and
the application of these linkers in the conjugation of an antibody with drugs.
Figure 3 shows the synthesis of phosphamide linkers containing malcimide
groups
and the application of these linkers in the conjugation of an antibody with
drugs.
Figure 4 shows the synthesis of phosphamide linkers containing maleimide,
hydra-
zone, or thioether groups and the application of these linkers in the
conjugation of an
antibody with drugs.
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Figure 5 shows the synthesis of hinder phosphamide linkers containing
maleimide
groups and the application of these linkers in the conjugation of an antibody
with drugs.
Figure 6 shows the synthesis of phosphamide linkers containing disulfide or
oxime
groups and the application of these linkers in the conjugation of an antibody
with drugs.
Figure 7 shows the synthesis of phosphamide linkers containing maleimide and
pol-
yethylene glycol groups and the application of these linkers in the
conjugation of an
antibody with drugs.
Figure 8 shows the synthesis of phosphamide linkers containing maleimide,
polyeth-
ylene glycol and azido groups and the application of these linkers in the
conjugation of an
antibody with two different compounds. Drugi and Drug2 here can be a cytotoxic
agent for
therapeutic application or a chromophore compound for monitoring the
interaction of the
conjugates with targeted cells as well.
Figure 9 shows the synthesis of phosphamide linkers containing disulfide,
polyeth-
ylene glycol or ketone groups, and these linkers are used for conjugation of a
protein with
two different compounds. Drug' and Drug,' here can be a cytotoxic agent for
therapeutic
application or a chromophore compound for monitoring the interaction of the
conjugates
with targeted cells.
Figure 10 shows the synthesis of the sulfonamide and the sulfinamide linkers
con-
taining a maleimide group and the application of these linkers in the
conjugation of an
antibody with a cytotoxic drug.
Figure 11 shows the synthesis of a sulfonamide linker containing a disulfide,
poly-
ethylene glycol or ketone group, and the linker is used for conjugation of a
protein with two
different compounds. Drug' and Drug2 here can be a cytotoxic agent for
therapeutic
application or a chromophore compound for monitoring the interaction of the
conjugate
with targeted cells.
Figure 12 shows the synthesis of phosphamide linkers containing thioether or
disul-
fide groups, and these linkers are used to link two drug/compounds per linker.
Drug here
can be a cytotoxic agent for therapeutic application or a chromophore compound
for
monitoring the interaction of the conjugates with targeted cells.
Figure 13 shows the synthesis of an antibody conjugate via the phosphamide
linkers
containing two function groups, wherein one group is linked to a cytotoxic
agent, MMAF
for targeted killing, and the other one is linked to a fluorochrome group for
monitoring the
interaction of the conjugate with a targeted cell.
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Figure 14 shows the synthesis of the phosphamide linkers containing two
function
groups.
Figure 15 shows the synthesis of an antibody conjugate via a phosphamide
linker
containing both a tubulysin analog and a MMAF analog on the linker.
Figure 16 shows the synthesis of the phosphamide linkers containing two
different
cytotoxic drugs (PBD dimer & MMAF analogs).
Figure 17 shows the synthesis of antibody conjugates via the phosphamide
linkers of
the present patent. The linkers can be conjugated two different drugs (e.g. a
PBD analog
and a MMAF analog) per linker, or can be linked to a pair of cysteine sites of
an antibody.
Figure 18 shows the synthesis of a sulfonamide linker containing a disulfide,
poly-
ethylene glycol, azido, or a triazole group, and the linker is for conjugation
with two
different compounds.
DETAILED DESCRIPTION OF THE INVENTION
DEFINITIONS
"Alkyl" refers to an aliphatic hydrocarbon group which may be straight or
branched
having 1 to 8 carbon atoms in the chain. "Branched" means that one or more
lower C
numbers of alkyl groups such as methyl, ethyl or propyl are attached to a
linear alkyl chain.
Exemplary alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, t-
butyl, n-pentyl,
3-pentyl, octyl, nonyl, decyl, cyclopentyl, cyclohexyl, 2,2-dimethylbutyl, 2,3-
dimethylbutyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 3,3-dimethylpentyl,
2,3,4-
trimethylpentyl, 3-methyl-hexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-
dimethylhexyl,
3,5-dimethylhexyl, 2,4-dimethylpentyl, 2-methylheptyl. 3-methylheptyl, n-
heptyl,
isoheptyl, n-octyl, and isooctyl. A C1-C8 alkyl group can be unsubstituted or
substituted
with one or more groups including, but not limited to, -C1-C8 alkyl,-0-(CI-C8
alkyl), -aryl,
-C(0)R', -0C(0)R', -C(0)OR', -C(0)NH2, -C(0)NHR', -C(0)N(R)2, -NHC(0)R', -SR',
-
S(0)2R', -S(0)R', -OH, -halogen, -N3, -NH2, -NH(R.), -N(R) 2 and -CN; where
each R' is
independently selected from -C I-C8 alkyl and aryl."Halogen" refers to
fluorine, chlorine,
bromine or iodine atom; preferably fluorine and chlorine atom.
"Heteroalkyl" refers to C2-.C8 alkyl in which one to four carbon atoms are
inde-
pendently replaced with a heteroatom from the group consisting of 0, S and N.
-Carbocycle" refers to a saturated or unsaturated ring having 3 to 8 carbon
atoms as a
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monocycle or 7 to 13 carbon atoms as a bicycle. Monocyclic carbocycles have 3
to 6 ring
atoms, more typically 5 or 6 ring atoms. Bicyclic carbocycles have 7 to 12
ring atoms,
arranged as a bicycle [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring
atoms arranged as a
bicycle [5,6] or [6,6] system. Representative C3-C8 carbocycles include, but
are not limited
to, -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclopentadienyl, -cyclohexyl, -
cyclohexenyl,
-1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl, -1,3-
cycloheptadienyl, -1,3,5-
cycloheptatrienyl, -cyclooctyl, and -cyclooctadienyl.
A "C3-C8 carbocycle" refers to a 3-, 4-, 5-, 6-, 7- or 8-membered saturated or
unsatu-
rated nonaromatic carbocyclic ring. A C3-C8 carbocycle group can be
unsubstituted or
substituted with one or more groups including, but not limited to, -C1-C8
alkyl), -aryl, -C(0)W, -0C(0)R', -C(0)OR', -C(0)NH2, -C(0)NHR', -C(0)N(W)2, -
NHC(0)R', -SW, -S(0)W,-S(0)2R'. -OH, -halogen, -N3, -NH2, -NH(R'), -N(R) 2 and
-CN;
where each R' is independently selected from -C1-C8 alkyl and aryl.
"Alkenyl" refers to an aliphatic hydrocarbon group containing a carbon-carbon
double
bond which may be straight or branched having 2 to 8 carbon atoms in the
chain. Exempla-
ry alkenyl groups include ethenyl, propenyl, n-butenyl, i-butenyl. 3-meth
ylbut-2-en yl, n-
pentenyl, hexylenyl, heptenyl, octenyl.
"Alkynyl" refers to an aliphatic hydrocarbon group containing a carbon-carbon
triple
bond which may be straight or branched having 2 to 8 carbon atoms in the
chain. Exempla-
ry alkynyl groups include ethynyl, propynyl, n-butynyl, 2-butynyl, 3-
methylbutynyl, 5-
pentynyl, n-pentynyl, hexylynyl, heptynyl, and octynyl.
"Alkylene" refers to a saturated, branched or straight chain or cyclic
hydrocarbon rad-
ical of 1-18 carbon atoms, and having two monovalent radical centers derived
by the
removal of two hydrogen atoms from the same or two different carbon atoms of a
parent
alkane. Typical alkylene radicals include, but are not limited to: methylene (-
CH2-), 1,2-
ethyl (-CH2C1-11-), 1,3-propyl (-CH2CH2CH2-), 1,4-butyl (-CH2CH,CH2CH2-), and
the like.
-Alkenylene" refers to an unsaturated, branched or straight chain or cyclic
hydrocar-
bon radical of 2-18 carbon atoms, and having two monovalent radical centers
derived by
the removal of two hydrogen atoms from the same or two different carbon atoms
of a
parent alkene. Typical alkenylene radicals include, but are not limited to:
1,2-ethylene (-
CH=CH-).
"Alkynylene" refers to an unsaturated, branched or straight chain or cyclic
hydrocar-
bon radical of 2-18 carbon atoms, and having two monovalent radical centers
derived by
the removal of two hydrogen atoms from the same or two different carbon atoms
of a parent
alkyne. Typical alkynylene radicals include, but are not limited to:
acetylene, propargyl and
4-pentynyl.
"Aryl" or Ar refers to an aromatic or hetero aromatic group, composed of one
or several
rings, comprising three to fourteen carbon atoms, preferentially six to ten
carbon atoms. The term
of "hetero aromatic group' refers one or several carbon on aromatic group,
preferentially one, two,
three or four carbon atoms are replaced by 0, N, Si, Se, P or S,
preferentially by 0, S, and N. The
term aryl or Ar also refers to an aromatic group, wherein one or several H
atoms are replaced
independently by -R', -halogen, -OR', or -SR', -NR'R", -N=NR', -N=R', -NR'R", -
NO2, -S(0)R',
-S(0)2R', -S(0)2OR', -0S(0)20R', -PR'R", -P(0)R'R", -P(OR')(OR"), -
P(0)(OR')(OR") or
-0P(0)(OR')(OR") wherein R', R" are independently H, alkyl, alkenyl, alkynyl,
heteroalkyl, aryl,
arylalkyl, carbonyl, or pharmaceutical salts.
"Heterocycle" refers to a ring system in which one to four of the ring carbon
atoms are
independently replaced with a heteroatom from the group of 0, N, S. Se, B, Si
and P. Preferable
heteroatoms are 0, N and S. Heterocycles are also described in The Handbook of
Chemistry and
Physics, 78th Edition, CRC Press, Inc., 1997-1998, p. 225 to 226. Preferred
nonaromatic heterocy-
clic include, but are not limited to epoxy, aziridinyl, thiiranyl,
pyrrolidinyl, pyrazolidinyl,
imidazolidinyl, oxiranyl, tetrahydrofuranyl, dioxolanyl, tetrahydropyranyl,
dioxanyl, dioxolanyl,
piperidyl, piperazinyl, morpholinyl, pyranyl, imidazolinyl, pyrrolinyl,
pyrazolinyl, thiazolidinyl,
tetrahydrothiopyranyl, dithianyl, thiomorpholinyl, dihydropyranyl,
tetrahydropyranyl,
dihydropyranyl, tetrahydropyridyl, dihydropyridyl, tetrahydropyrinidinyl,
dihydrothiopyranyl,
azepanyl, as well as the fused systems resulting from the condensation with a
phenyl group.
The term "heteroaryl" or aromatic heterocycles refers to a 5 to 14, preferably
5 to 10
membered aromatic hetero, mono-, bi- or multicyclic ring. Examples include
pyrrolyl, pyridyl,
pyrazolyl, thienyl, pyrimidinyl, pyrazinyl, tetrazolyl, indolyl, quinolinyl,
purinyl, imidazolyl,
thienyl, thiazolyl, benzothiazolyl, furanyl, benzofuranyl, 1,2,4-thiadiazolyl,
isothiazolyl, triazoyl,
tetrazolyl, isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl, carbazolyl,
benzimidazolyl,
isoxazolyl, pyridyl-N-oxide, as well as the fused systems resulting from the
condensation with a
phenyl group.
"Alkyl", "cycloalkyl", "alkenyl", "alkynyl", "aryl", "heteroaryl",
"heterocyclic" and
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the like refer also to the corresponding "alkylene, "cycloalkylene".
"alkenylene,
"alkynylene", "arylene", "heteroarylene", "heterocyclene and the likes which
are formed
by the removal of two hydrogen atoms.
"Arylalkyl" refers to an acyclic alkyl radical in which one of the hydrogen
atoms
bonded to a carbon atom, typically a terminal or 5p3 carbon atom, is replaced
with an aryl
radical. Typical arylalkyl groups include, but are not limited to, benzyl, 2-
phenylethan-1-yl,
2-phenylethen-l-yl, naphthylmethyl, 2-naphthylethan-l-yl, 2-naphthylethen-l-
yl,
naphthobenzyl, 2-naphthophenylethan-l-y1 and the like.
-Heteroarylalkyl" refers to an acyclic alkyl radical in which one of the
hydrogen at-
oms bonded to a carbon atom, typically a terminal or sp3 carbon atom, is
replaced with a
heteroaryl radical. Typical heteroarylalkyl groups include, but are not
limited to, 2-
benzimidazolylmethyl, 2-furylethyl and the like.
Examples of a "hydroxyl protecting group" include, but arc not limited to,
methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranyl ether,
benzyl ether,
p-methoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether,
triisopropylsilyl ether, t-
butyklimethylsilyi ether, triphenylmethylsilyl ether, acetate ester,
substituted acetate esters,
pivaloate, benzoate, methanesulfonate and p-toluenesulfonate.
"Leaving group" refers to a functional group that can be substituted by
another func-
tional group. Such leaving groups are well known in the art, and examples
include, but are
not limited to, a halide (e.g., chloride, bromide, and iodide),
methanesulfonyl (mesyl), p-
toluenesulfonyl (tosyl), trifluoromethylsulfonyl (triflate), and
trifluoromethylsulfonate.
The following abbreviations may be used herein and have the indicated
definitions:
Boc, tert-butoxy carbonyl; BroP, bromotrispyrrolidinophosphonium
hexafluorophosphate;
CDI, 1, l'-carbonyldiimidazole; DCC, dicyclohexylcarbodiimide; DCM,
dichloromethane;
DIAD, diisopropylazodicarboxylate; DIBAL-H, diisobutylaluminium hydride;
DlPEA,
diisopropylethylamine; DEPC, diethyl phosphorocyanidate; DMA, N,N-dimethyl
acetamide; DMAP, 4-(N, N-dimethylamino)pyridine; DMF, NV-dimethylformamide;
DMSO, dimethylsulfoxide; DTT, dithiotheritol; EDC, 1-(3-dimethylaminopropy1)-3-
ethylcarbodiimide hydrochloride; ESI-MS, electospray mass spectrometry; HATU,
047-
azabenzotriazol-1-y1)-N, N, N' N'-tetramethyluronium hexafluorophosphate;
HOBt, 1-
hydroxybenzotriazole; HPLC, high pressure liquid chromatography; NHS, N-
Hydroxysuccinimide; MMP, 4-methylmorpholine; PAB. p-aminobeznyl; PBS,
phosphate-
buffered saline (pH 7.0-7.5); PEG, polyethylene glycol; SEC, size-exclusion
chromatog-
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raphy; TCEP, tris(2-carboxyethyl)phosphine; TFA, trifluoroacetic acid; THF,
tetrahydrofuran; Val, valine.
"Pharmaceutically" or "pharmaceutically acceptable" refer to molecular
entities and
compositions that do not produce an adverse, allergic or other untoward
reaction when
administered to an animal, or a human, as appropriate.
"Pharmaceutically acceptable solvate" or "solvate" refer to an association of
one or
more solvent molecules and a disclosed compound. Examples of solvents that
form phar-
maceutically acceptable solvates include, but are not limited to, water,
isopropanol, etha-
nol, methanol, DMSO, ethyl acetate, acetic acid and ethanolamine.
"Pharmaceutically acceptable excipient" includes any carriers, diluents,
adjuvants, or
vehicles, such as preserving or antioxidant agents, fillers, disintegrating
agents, wetting
agents, emulsifying agents, suspending agents, solvents, dispersion media,
coatings,
antibacterial and antifungal agents, isotonic and absorption delaying agents
and the like.
The use of such media and agents for pharmaceutical active substances is well
known in
the art. Except insofar as any conventional media or agent is incompatible
with the active
ingredient, its use in the therapeutic compositions is contemplated.
Supplementary active
ingredients can also be incorporated into the compositions as suitable
therapeutic combina-
tions.
As used herein, "pharmaceutical salts" refer to derivatives of the disclosed
corn-
pounds wherein the parent compound is modified by making acid or base salts
thereof. The
pharmaceutically acceptable salts include the conventional non-toxic salts or
the quaternary
ammonium salts of the parent compound formed, for example, from non-toxic
inorganic or
organic acids. For example, such conventional non-toxic salts include those
derived from
inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic,
phosphoric. nitric
and the like; and the salts prepared from organic acids such as acetic,
propionic, succinic,
tartaric, citric, methanesulfonic, benzenesulfonic, glucoronic, glutamic,
benzoic, salicylic,
toluenesulfonic, oxalic, fumaric, maleic, lactic and the like. Further
addition salts include
ammonium salts such as tromethamine, meglumine, epolamine, etc., metal salts
such as
sodium, potassium, calcium, zinc or magnesium.
The pharmaceutical salts of the present invention can be synthesized from the
parent
compound which contains a basic or acidic moiety by conventional chemical
methods.
Generally, such salts can be prepared via reaction the free acidic or basic
forms of these
compounds with a stoichiometric amount of the appropriate base or acid in
water or in an
13
organic solvent, or in a mixture of the two. Generally, non-aqueous media like
ether, ethyl acetate,
ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts
are found in Remington's
Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, 1985,
p. 1418.
The novel conjugates disclosed herein use the hydrophilic linkers. Examples of
some
suitable linkers and their synthesis are shown in Figures 1 to 17.
THE HYDROPHILIC LINKERS
The synthetic routes to produce hydrophilic linkers as well as the preparation
of the
conjugates of drugs to a cell binding molecules of the present invention are
shown in Figures 1-18.
The hydrophilic linkers possess three elements: a) Substituents that are
either phosphamide, or
phosphinate, or sulfonamide, or sulfonyl, or sulfonamide, and/or sulfoxide, or
mixed of these
groups, b) A group, such as but not limited to, a N-hydroxysuccinimide ester
group, maleimido
group, disulfide group, haloacetyl group, alkoxyamino group, and/or hydrazide
group, capable of
reaction with a cell- binding agent, and c) A group, such as but not limited
to, a disulfide,
maleimide, haloacetyl, aldehyde, ketone, azide, amine, alkoxyamine and
hydrazide, capable of
reaction with a drug. The hydrophilic substituents can be introduced by
methods described herein.
For example of the phosphamide substituents, they can be formed through
directly condensation of
phosphorus (V) oxychloride with amino molecules which are described in the
figures 1, 2, 3, 4 and
5. For the mixed the phosphinate/ phosphamide substituents, they can be
introduced by first
treating a commercially available ammonium phosphinate with an acrylate via
Michael addition,
then substitution of excess amount of dibromo alkane to a phosphinate group,
and followed by
condensation with an amino compound, which are exampled in the figures 6, 7, 8
and 9. For
example of the sulfonamide and the sulfinamide substituents, they can be
through directly
condensation of sulfuryl chloride and thionyl chloride with amino compounds,
which are exampled
in the figure 10. The sulfonyl/ sulfonamide substituent can be formed through
condensation of
chlorosulfonic acid with an amine which is exampled in the figures 11 and 18.
The detail synthesis
of the hydrophilic linkers and their uses for the preparation of cell binding
ligand-drug conjugates
of this invention are disclosed in the figures 1-18.
Preferably, the hydrophilic linkers are compounds of the Formula (I) below:
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Y¨RifQ-R2HT-R3-1--R4¨Z
n /
R5 R6 (I)
wherein:
Y represents a functional group that enables reaction with a cell-binding
agent;
Q and T are either -X1-P(=NOM)-. or -X1-S(02)-, or -Xi-S(0)-; or -Xi-
P(=0)(0M)-X2-, or -X1-P(=0)[X2-R4-4-X3-, or -X1-P(=0)[X2-R1-Y1-X3-, or
X2-, or -X1-S(0)-X2-;
X1, X2 and X3 are independently selected from N(R7), 0, or S; In addition,
when Xi
is either N(R7), or 0, or S, then either X/ or X3, or another Xi connects to -
P(=0), -S(0),
or -S(02) can be CH2.
m and n are integer from 0 to 5, but not 0 at the same time;
Z represents a functional group that enables linkage of a cytotoxic drug via
an alkyl,
alkylene, alkenylene, alkynylene, aromatic, heteroalkyl, disulfide, thioether,
thioester,
peptide, hydrazone, ether, ester, carbamate, carbonate, amine (secondary,
tertiary, or
quartary), imine, cycloheteroalkyane, heteroaromatic, alkoxime or amide bond;
Ri, R2, R3, R4, R5, R6, R7 and R8 are the same or different and are H, linear
alkyl hav-
ing from 1-6 carbon atoms, branched or cyclic alkyl having from 3 to 6 carbon
atoms,
linear, branched or cyclic alkenyl or alkynyl, or 1-6 carbon atoms of esters,
ether, amide,
or polyethyleneoxy unit of formula (0CH2CH2)p, wherein p is an integer from 0
to about
1000, or combination thereof.
M is H, or Na, or K, or N+R1R2R3 or a pharmaceutical salt. R1, R2 and R3 are
de-
scribed above.
In another embodiment, Ri, R,, R3, and R4 can be respectively a chain of atoms
se-
lected from C, N. 0, S. Si, and P that covalently connects the cell-surface
binding ligand,
the phosphinate or sulfonyl or sulfoxide group, the conjugated drug and
themselves (Ri,
R3 and R4). The atoms used in forming the hydrophilic linker may be combined
in all
chemically relevant ways, such as forming alkylanc, alkylenc, alkenylene, and
alkynylcne,
ethers, polyoxyalkylene, esters, amines, imines, polyamines, hydrazines,
hydrazones,
amides, ureas, semicarbazides, carbazides, alkoxyamines, alkoxyl amines,
urethanes, amino
acids, acyloxylamines, hydroxamic acids, and many others. In addition, it is
to be under-
stood that the atoms forming the linker (L) may be either saturated or
unsaturated, or may
be radicals, or may be cyclized upon each other to form divalent cyclic
structures, includ-
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ing cyclo alkanes, cyclic ethers, cyclic amines, arylenes, heteroarylenes, and
the like in the
linker.
Examples of the functional group, Y, that enables reaction with a cell-binding
agent
include amine reacting agents such as but not limited to N-hydroxysuccinimide
esters, p-
nitrophenyl esters, dinitrophenyl esters, pentafluorophenyl esters; thiol
reactive agents such
as but not limited to pyridyldisulfides, nitropyridyldisulfides, maleimides,
haloacetates and
carboxylic acid chlorides.
Examples of the functional group, Z, which enables linkage of a cytotoxic
drug, in-
clude groups that enable linkage via a disulfide, thioether, thioester,
peptide, hydrazone,
ester, carbamate, carbonate, alkoxime or an amide bond. Such functional groups
include,
but are not limited to, thiol, disulfide, amino, carboxy. aldehydes,
maleimido, haloacetyl,
hydrazines, alkoxylamino, and/or hydroxy.
In preferred embodiments, RI, R3, R3, and R4, are linear alkyl having from 1-6
car-
bon atoms, or polyethyleneoxy unit of formula (OCH,CH))p, p = 1-100,
In another embodiments, when the hydrophilic linkers of the Formula (I) has
two or
more Y groups, in particular the two same Y groups, at such case, either Q,
or/and T is -
X1-P(=0)[X2-R1-Y]-X3-, then the hydrophilic linkers of the Formula (I) can be
for linking
to two or more sites, particularly to a pair of sites of cell binding
molecules. Wherein X1, X2
and X3 are independently selected from N(R7), 0, CH2 or S; R1 is defined
above.
In yet another embodiments, when the hydrophilic linkers of the Formula (I)
has two
Z or more groups, at such case, either Q, or/and T is - X1-13(=0)[X7-R4-Zi-X3-
, then the
hydrophilic linkers of the Formula (I) can be for linking for two or more
drugs, in particular,
two different drugs. Wherein X1, X? and X3 are independently selected from
N(R7), 0, CH2
or S; R4 is defined above.
The synthesis of 2-dithio-pyridyl containing cross-linkers of Formulae (I) is
shown,
for example, in figures 2, 6, 9. 11 and 12. The synthesis of maleimido-
containing cross
linkers of the Formula (I) is shown, for example, in figures 1, 3, 4, 5, 7, 8,
10, 13, and 17.
The synthesis of thioether-containing cross linkers of the Formula (I) is
shown, for exam-
ple, in figures 1, 3, 4, 5, 7, 8, 10, 12 and 17. The synthesis of polyethylene
glycol-
containing hydrophilic cross linkers of Formula (I) is shown, for example, in
figures 7, 8, 9,
11, 12, 13, 14, 15, 16, 17 and 18. The synthesis of azide-containing
hydrophilic cross
linkers of Fon-nula (I) for Huisgen 1,3-dipolar cycloaddition of azides to
alkynes (also
called click chemistry) is shown, for example, in figures 8, 9, 13 and 18. The
synthesis of
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hydrophilic cross linkers of Formula (I) bearing hydrazide, or ketone, or
alkoxime moieties
enabling linkage via acid-labile bonds is shown, for example, in figures 6, 9,
11, 14, 15 and
16. The synthesis of hydrophilic linkers of Formula (I) which can be linked
two drugs per a
linker, or linked two sites per a linker is shown, for example, in figure 1,
2, 3, 4, 5, 6. 7, 17
and 18. The synthesis of hydrophilic linkers of Formula (I), which can be
linked two
different compounds /drugs per a linker, is shown, for example, in figures 8,
9, 11, 14, 15,
16 and 17. The synthesis of hydrophilic linkers of Formula (I), which can be
linked one
cytotoxic compound and one chromophore molecule per a linker, is shown, for
example, in
figure 13.
CELL-BINDING AGENT-DRUG CONJUGATES
The conjugates of the present invention can be represented by the following
formula,
Cb-(-L-Drug),õ wherein Cb is a cell-binding agent, L is a hydrophilic linker,
Drug is a drug
molecule, and n is an integer from 1 to 20.
The hydrophilic linker L may be composed of one or more linker components. Ex-
emplary linker components include 6-maleimidocaproyl ("MC"),
maleimidopropanoyl
("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-
phe" or "af"), p-
aminnbenzyloxycarbonyl ("PAR"), 4-thiopentanoate ("SPP"), 4-(N-
maleimidomethyl)cyclohexane-1 carboxylate ("MCC"), (4-acetyl)aminobenzoate
("SIAB"), 4-thio-butyrate (SPDB), 4-thio-2-hydroxysulfonyl-butyrate (2-Sulfo-
SPDB),
ethyleneoxy ¨CH2CH20-- as one or more repeating units ("EO" or "PEO").
Additional
linker components are known in the art and some are described herein.
Example structures of these components containing linkers are:
0 0 0
0 0
II H A 0
0 0
jiµl\NMA/11?\s %1NT ='"13 ¨NN)V\/11'.1?\s
H 0 H H II 0
(MC, 6-maleimidocaproyl containing)
0 0 0
,NH
455\4 V\ 1Nlj-N3¨S
0
0 (MP, maleimidopropanoyl containing)
(rHN H 0
I (a, HN H0
I I
0 N,/ ? N_P
H
0 0 0 H on
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(PAB, p-aminobenzyloxycarbonyl containing)
0 0 s 0 0
kliN4''41.-P\sA, N/kr=Ilp-'4,/--N A
0 0 OH 0
0
0
ki_r-T1?\s,`?2,
0
i 0 0 IN/\ II 1114?\s A
1%9\ 0-1N1
I A 0
S HNTõ,-.....
0 , ,
0
0
k..I.,. NVH Q ' A H...f.N y
I \ P
0 I 0
HN,../.-----
(ME, maleimidoethyl containing).
0 H 0 0 H 0
H2N
y14NH, N/\.,N....11,_ ill ,=\.,.N,t4
t N
r''N/VH ii VI
1-1 HN ki OH H2N
H HNNS
V Or rr
(valine-citrulline containing)
0 0 1 0
0 0 W 0
t-NH * c. '0-4(N/\eNI-SS
H OH
o I 1 H NjeZZI
H
0
(MCC, 4-(N-maleimidomethyl)cyclohexane-1 carboxylate)
0 c) is NH
Nje221 'CT V\N * NA;42"
OH H H 0 H H
((4-acetyl)aminobenzoate containing)
0 0 H
S S si,lk ^ 11N1 Pi ki
(' H N HO3S
II
HO3S H OH 0
(4-thio-2-hydroxysulfonyl-butyrate, 2-sulfo-SPDB)
Preferably, the conjugates have the following Formula (II):
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Cb-(Ri-FQ-R2]-fT-R3-1---R4-Dru)
n R5 R6
wherein:
Cb represents a cell-binding agent;
Drug represents the drug linked to the cell-binding agent via the hydrophilic
linkers
of this invention by an alkyl, alkylene, alkenylene, alkynylene, ether,
polyoxyalkylene,
ester, amine, imine, polyamine, hydrazine, hydrazone, amide, urea,
semicarbazide,
carbazide, alkoxyamine, urethanes, amino acid, peptide, acyloxylamine,
hydroxamic acid,
disulfide, thioether, thioester, carbamate, carbonate, heterocyclic ring,
heteroalkyl,
heteroaromatic, or alkoxime bond, or combination thereof.
q is 1 ¨ 30; m, n, R1, R2, R3, RI, R5, R6 and M are described the same
previously in
Formula (I).
Q and T are either ¨X1¨P(=0)(0M)-. or ¨X1-S(02)-, or ¨X1¨S(0)-; or ¨X1¨
P(=0)(0M)-X2-, or ¨X1¨P(=O)[X2-R4-Drugl-X3-, or ¨X1-13(=0)PC2-R1-Cbl-X3-, or
¨X1-
S(02)-X/-, or ¨Xi¨S(0)-X2-; Wherein X1, X/ and X3 are the same described in
Formula (I).
In another embodiment, when the conjugate of the Formula (II) has two or more
Cb
groups, in particular the two same Cb groups, at such case, either Q, or/and T
is ¨ X1¨
P(=0)[X2-R1-CbJ-X3-. then the conjugate of the Formula (11) is linked to two
or more sites,
particularly a pair of sites of cell binding molecules.
In yet another embodiments, when the conjugate of the Formula (II) has two or
more
Drug groups, at such case, either Q, or/and T is ¨Xi¨P(=0)11X2-R4-Drug]-X3-,
then the
conjugate of the Formula (II) is linked to two or more drugs, in particular,
two or more
different drugs.
As described in more detail below, the drug can be any of many small molecule
drugs, including, but not limited to, tubulysins, calicheamicins, auristatins,
maytansinoids,
CC-1065 analogs, morpholinos doxorubicins, taxanes, cryptophycins,
epothilones, and
benzodiazepine dimers (e.g., dimmers of pyrrolobenzodiazepine (PBD) or
tomaymycin),
indolinobenzodiazepines, imidazobenzothiadiazepines, or
oxazolidinobenzodiazepines).
To synthesize the conjugate, the cell-binding agent can be first modified with
the
hydrophilic linkers of the present invention to introduce reactive groups of
disulfide,
maleimido, haloacetyl, azide, 1-yne, ketone, aldehyde, alkoxyamino, or
hydrazide. Synthe-
sis of the cell-binding agent-drug conjugates linked via disulfide bonds is
achieved by a
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disulfide exchange between the disulfide bond in the modified cell-binding
agent and a
drug containing a free thiol group. Synthesis of the cell-binding agent-drug
conjugates
linked via thioether is achieved by reaction of the maleimido or haloacetyl or
ethylsulfonyl
modified cell-binding agent and a drug containing a free thiol group.
Synthesis of conju-
gates bearing an acid labile hydrazone link can be achieved by reaction of a
carbonyl group
with the hydrazide moiety in the linker, by methods known in the art (see, for
example, P.
Hamann et al., Hinman, L. M., et al. Cancer Res. 53, 3336-334, 1993; B.
Laguzza et al., J.
Med. Chem., 32; 548-555, 1959; P. Trail et al., Cancer Res., 57; 100-105,
1997).
Alternatively, the drug can be modified with the hydrophilic linkers of the
present
invention to give a modified drug of Formula (IV) bearing functionality
capable of reacting
with a cell binding agent. For example a thiol-containing drug can be reacted
with the
hydrophilic linker of Formula (I) bearing a maleimdo, or a haloacetyl, or an
ethylsulfonyl
substituent at neutral pH in aqueous buffer to give a drug connected to the
hydrophilic
linker via a thioether link. A thiol-containing drug can undergo disulfide
exchange with a
hydrophilic linker bearing a pyrdiyldithio moiety to give a modified drug
attached via a
disulfide bond to the hydrophilic cross linker. A drug bearing a hydroxyl
group or a thiol
group can be reacted with a hydrophilic linker bearing a halogen of this
invention, in the
presence of a mild base, to give a modified drug bearing an ether or thiol
ether link. A
hydroxyl group containing drug can be condensed with a hydrophilic cross
linker of
Formula (I) bearing a carboxyl group, in the presence of a dehydrating agent,
such as EDC
or DCC, to give an ester link. An amino group containing drug can similarly
undergo
condensation with a carboxyl group on the hydrophilic linker of Formula (I) to
give an
amide bond.
The conjugate may be purified by standard biochemical means, such as gel
filtration
on a Sephadex G25 or Sephacryl S300 column, adsorption chromatography, and ion
exchange or by dialysis. In some cases, a small molecule as a cell-binding
agent (e.g. folic
acid, melanocyte stimulating hormone, EGF etc) conjugated with a small
molecular drugs
can be purified by chromatography such as by HPLC, medium pressure column
chromatog-
raphy or ion exchange chromatography.
MODIFIED CELL-BINDING AGENTS
The cell-binding agent modified by reaction with linkers of the present
invention are
preferably represented by the Formula (III)
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C4Ri-FQ-R21-1-T
/ =
n R5 R6 (III)
Wherein Cb, q, m, n, R1, R2, R3. R4, R5, R6 and Z, as well as the substituents
inside
are described the same previously in Formula (I) and (II).
Q and T are either ¨X1¨P(=0)(0M)-. or ¨X1-S(02)-, or ¨X1¨S(0)-; or ¨X1-
P(=0)(0M)-X2-, or ¨X1-1p(=O)[X2-R4-4-X3-, or ¨Xi¨P(=O)IIX2-Ri-Cb]-X3-, or ¨X1-
S(02)-X/-, or ¨Xi¨S(0)-X2-; Wherein X1, X2 and X3 are the same described in
Formula (I).
In another embodiment, when the compound of the Formula (III) has two or more
Cb groups, in particular the two same Cb groups, at such case, either Q,
or/and T is ¨ X1¨
P(=0)[X2-Ri-Cbl-X3-, then the compound of the Formula (III) is linked to two
or more
sites, particularly a pair of sites of cell binding molecules.
In yet another embodiment, when the compound of the Formula (III) has two or
more Z groups, at such case, either Q, or/and T is ¨X1¨P(=0)P(7-R4-4-X3-, then
the
compound of the Formula (III) can be used to link to two or more drugs, in
particular, two
different drugs.
In preferred embodiments, Z is a disulfide substituent, maleimido, haloacetyl,
alkoxyamine, hydrazine group, or an N-hydroxysuccinimide ester, and Cb linked
with R1 is
through thioether, hydrazone, amide, alkoxime, carbamate, or disulfide bond.
The modi-
fied cell-binding agent can be prepared via a reaction of the cell-binding
agent with the
hydrophilic linkers by methods known in the art for other cross-linkers (U.S.
Patent Nos.
5,846,545, 5,585,499, 5,475,092, 5,414,064, 5,208,020, and 4,563,304;
Carlsson, J. et al.
Biochern. J. (1978) 173, 723-737(1978); Goff, D. A., Bioconj. Chem. (1990), 1.
381-386;
L. Delprino et al. J. Phartn. Sci. (1993), 82, 506-512; S. Arpicco et al.,
Bioconjugate
Chern(1997), 8, 327-337).
Advantageously, because the phosphamide, phosphinate, sulfonamide, sulfonyl,
sulfonimide, and/or sulfoxide groups on the hydrophilic linkers are soluble in
water or
require only a small percentage of organic solvent to maintain solubility in
aqueous solu-
tion, the reaction between the cell-binding agent and the cross-linker can be
conducted in
aqueous solution. The cross-linking reagent is dissolved in aqueous buffer,
optionally
containing a small amount (typically <10% by volume) of a polar organic
solvent that is
miscible with water, for example different alcohols, such as methanol,
ethanol, and propa-
nol, acetone, acetonitrile, tetrahydrofuran (THF), 1,4-dioxane, dimethyl
formamide (DMF),
21
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dimethyl acetamide (DMA), or dimethylsulfoxide (DMSO) at a high concentration,
for
example 1-100 mM, and then an appropriate aliquot is added to the buffered
aqueous
solution of the cell-binding agent. An appropriate aliquot is an amount of
solution that
introduces 1-10 cross-linking groups per cell-binding agent, preferably 1-6
groups, and the
volume to be added should not exceed 10 %. preferably 5 %, and most preferably
0-3 % of
the volume of the cell-binding agent solution. The aqueous solutions for the
cell-binding
agents are buffered between pH 6 and 9, preferably between 6.5 and 7.5 and can
contain
any non-nucleophilic buffer salts useful for these pH ranges. Typical buffers
include
phosphate, triethanolamine HC1, HEPES, and MOPS buffers, which can contain
additional
components, such as cyclodextrins, sucrose and salts, for examples, NaCl and
KC1. After
the addition the reaction is incubated at a temperature of from 4 C to 45 C,
preferably at
ambient temperature. The progress of the reaction can be monitored by
measuring the
increase in the absorption at 280, or 320 nm, or another appropriate
wavelength. After the
reaction is complete, isolation of the modified cell-binding agent can be
performed in a
routine way, using for example gel filtration chromatography, or adsorptive
chromatog-
raphy.
The extent of modification can be assessed by measuring the absorbance of the
nitropridine thione, dinitropyridine dithione, pyridine thione,
carboxamidopyridine
dithione and dicarboxamidopyridine dithione group released. The hydrophilic
cross-linkers
described herein have diverse functional groups that can react with any cell-
binding agent
that possesses a suitable substituent. For example cell-binding agents bearing
an amino or
hydroxyl substituent can react with cross linkers bearing an N-
hydroxysuccinimide (NHS)
ester, cell-binding agents bearing a thiol substituent can react with cross
linkers bearing a
maleimido or haloacetyl group. Additionally, cell-binding agents bearing a
carbonyl
substituent can react with the linkers bearing a hydrazide or an alkoxyamine.
One skilled in
the art can readily determine which linker to use based on the known
reactivity of the
available functional group on the cell-binding agent.
MODIFIED CYTOTOXIC DRUGS
The cytotoxic drugs modified by reaction with cross-linkers of the present
invention
are preferably represented by the Formula (IV):
Y¨RifQH-
-R2T¨R31¨R4¨Drug
- R5 R6 (IV)
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PCT/IB2015/054521
wherein Y, m. n. R1, R2, R3, R4, R5, R6 and Drug, as well as the substituents
inside
the Formula (IV) are described the same previously in Formula (I) and (II).
Q and T are either ¨X1¨P(=0)(01\4)-. or ¨X1-S(02)-, or ¨Xi¨S(0)-; or ¨Xi¨
P(=0)(0M)-X2-, or ¨X1¨P(=O)[X2-R4-Drug]-X3-, or ¨X1¨P(=0)[X2-R1-Y] -X3-, or
¨Xi-
S(0/)-X/-, or ¨X1¨S(0)-X2-; Wherein X1, X2 and X3 are the same described in
Formula (I).
In another embodiment, when the compound of the Formula (IV) has two or more Y
groups, in particular the two same Y groups, at such case, either Q. or/and T
is ¨ X1¨
P(=0)[X2-R1-Y]-X3-, then compound of the Formula (IV) can be linked to two or
more
sites, particularly a pair of sites of cell binding molecules.
In yet another embodiment, when the compound of the Formula (IV) has two or
more Drug groups, at such case, either Q, or/and T is ¨ Xi¨P(=0)[X2-R4-Drug]-
X3-, then
the compound of the Formula (II) is linked to two or more drugs, in
particular, two or more
different drugs.
In preferred embodiments, Y is a disulfide substituent, a malcimido, a
haloacetyl, an
alkoxylamino group, carboxylic acid, or an N-hydroxysuccinimide ester.
The modified drugs can be prepared by reacting the drug with the linkers of
the pre-
sent invention to give a modified drug of Formula (IV) bearing functionality
capable of
reacting with a cell binding agent. For example a thiol-containing drug can be
reacted with
the linker of Formula (I) bearing a maleimdo substituent at neutral pH in
aqueous buffer to
give a drug connected to the hydrophilic linker via thioether linkage. A thiol-
containing
drug can undergo disulfide exchange with a hydrophilic linker bearing a
pyrdiyldithio
moiety to give a modified drug attached via a disulfide bond to the
hydrophilic linker. A
drug bearing a hydroxyl group can be reacted with a linker bearing a halogen,
in the
presence of a mild base, to give a modified drug bearing ether linkage. A
hydroxyl group
containing drug can be condensed with a linker of Formula (I) bearing a
carboxyl group, in
the presence of a dehydrating agent, such as EDC or dicyclohexylcarbodiimide
(DCC), to
give ester linkage. A drug bearing a thiol group can be reacted with a linker
bearing a
maleimido or a vinylsulfonyl, or a haloacetyl group, to give a modified drug
bearing
thiocther linkage. An amino group containing drug can similarly undergo
condensation
with a carboxyl group on the hydrophilic linker of Formula (I) to give an
amide bond. The
modified drug can be purified by standard methods such as column
chromatography over
silica gel or alumina, crystallization, preparatory thin layer chromatography,
ion exchange
chromatography, or HPLC.
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CELL-BINDING AGENTS
The cell-binding molecule that comprises the conjugates and the modified cell-
binding agents of the present invention may be of any kind presently known, or
that
become known, molecule that binds to, complexes with, or reacts with a moiety
of a cell
population sought to be therapeutically or otherwise biologically modified.
The cell binding agents include, but are not limited to, large molecular
weight pro-
teins such as, for example, full-length antibodies (polyclonal antibodies,
monoclonal
antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific
antibodies); single
chain antibodies; fragments of antibodies such as Fab, Fab', F(a17)2, F
Parham, J.
Immunol. 131, 2895-2902 (1983)1, fragments produced by a Fab expression
library, anti-
idiotypic (anti-Id) antibodies. CDR's, and epitope-binding fragments of any of
the above
which immuno-specifically bind to cancer cell antigens, viral antigens,
microbial antigens
or a protein generated by the immune system that is capable of recognizing,
binding to a
specific antigen or exhibiting the desired biological activity (Miller et al
(2003) J. of
Immunology 170:4854-4861); interferons (such as type I, II, III); peptides;
lymphokines
such as IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, GM-CSF, interferon-gamma (IFN-y);
hormones
such as insulin, TRH (thyrotropin releasing hormones), MSH (melanocyte-
stimulating
hormone), steroid hormones, such as androgens and estrogens, melanocyte-
stimulating
hormone (MSH); growth factors and colony-stimulating factors such as epidermal
growth
factors (EGF), granulocyte-macrophage colony-stimulating factor (GM-CSF),
transforming
growth factors (TGF), such as TGFa, TGF[3, insulin and insulin like growth
factors (IGF-I,
IGF-II) G-CSF, M-CSF and GM-CSF [Burgess, Immunology Today, 5, 155-158
(1984)];
vaccinia growth factors (VGF); fibroblast growth factors (FGFs); smaller
molecular weight
proteins, poly-peptide, peptides and peptide hormones, such as bombesin,
gastrin, gastrin-
releasing peptide; platelet-derived growth factors; interleukin and cytokines,
such as
interleukin-2 (IL-2), interleukin-6 (IL-6), leukemia inhibitory factors,
granulocyte-
macrophage colony-stimulating factor (GM-CSF); vitamins, such as folate;
apoproteins and
glycoproteins, such as transferrin [O'Keefe et al, 260 J. Biol. Chem. 932-937
(1985)1;
sugar-binding proteins or lipoproteins, such as lectins; cell nutrient-
transport molecules;
and small molecular inhibitors, such as prostate-specific membrane antigen
(PSMA)
inhibitors and small molecular tyrosine kinase inhibitors (TKI), non-peptides
or any other
cell binding molecule or substance, such as bioactive polymers (Dhar, et al.
Proc. Natl.
Acad. Sci. 2008, 105, 17356-61); bioactive dendrimers (Lee, et al, Nat.
Biotechnol. 2005,
24
23, 1517-26; Almutairi, eta!; Proc. Natl. Acad. Sci. 2009, 106, 685-90);
nanoparticles (Liong, eta!,
ACS Nano, 2008, 19, 1309-12; Medarova, et al, Nat. Med. 2007, 13, 372-7;
Javier, eta!,
Bioconjugate Chem. 2008, 19, 1309-12); liposomes (Medinai, et al, Curr. Phar.
Des. 2004, 10,
2981-9); viral capsides (Flenniken, et al, Viruses Nanotechnol. 2009, 327,71-
93).
In general, a monoclonal antibody is preferred as a cell-surface binding agent
if an
appropriate one is available. And the antibody may be murine, human,
humanized, chimeric, or
derived from other species.
Production of antibodies used in the present invention involves in vivo or in
vitro
procedures or combinations thereof. Methods for producing polyclonal anti-
receptor peptide
antibodies are well-known in the art, such as in U.S. Pat. No. 4,493,795 (to
Nestor et al). A
monoclonal antibody is typically made by fusing myeloma cells with the spleen
cells from a mouse
that has been immunized with the desired antigen (Kohler, G.; Milstein, C.
(1975). Nature 256:
495-497). The detailed procedures are described in "Antibodies--A Laboratory
Manual", Harlow
and Lane, eds., Cold Spring Harbor Laboratory Press, New York (1988).
Particularly monoclonal
antibodies are produced by immunizing mice, rats, hamsters or any other mammal
with the antigen
of interest such as the intact target cell, antigens isolated from the target
cell, whole virus,
attenuated whole virus, and viral proteins. Splenocytes are typically fused
with myeloma cells
using polyethylene glycol (PEG) 6000. Fused hybrids are selected by their
sensitivity to HAT
(hypoxanthine-aminopterin-thymine). Hybridomas producing a monoclonal antibody
useful in
practicing this invention are identified by their ability to immunoreact
specified receptors or inhibit
receptor activity on target cells.
A monoclonal antibody used in the present invention can be produced by
initiating a
monoclonal hybridoma culture comprising a nutrient medium containing a
hybridoma that secretes
antibody molecules of the appropriate antigen specificity. The culture is
maintained under
conditions and for a time period sufficient for the hybridoma to secrete the
antibody molecules into
the medium. The antibody-containing medium is then collected. The antibody
molecules can then
be further isolated by well-known techniques, such as using protein-A affinity
chromatography;
anion, cation, hydrophobic, or size exclusive chromatographies (particularly
by affinity for the
specific antigen after protein A, and sizing column chromatography);
centrifugation, differential
solubility, or by any other standard technique for the purification of
proteins.
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Media useful for the preparation of these compositions are both well-known in
the
art and commercially available and include synthetic culture media. An
exemplary synthet-
ic medium is Dulbecco's minimal essential medium (DMEM; Dulbecco et al.,
Virol. 8, 396
(1959)) supplemented with 4.5 gm/1 glucose, 0-20 mM glutamine, 0-20% fetal
calf serum,
several ppm amount of heavy metals, such as Cu, Mn, Fe, or Zn, etc, or/and the
heavy
metals added in their salt forms, and with an anti-foaming agent, such as
polyoxyethylene-
polyoxypropylene block copolymer.
In addition, antibody-producing cell lines can also be created by techniques
other
than fusion, such as direct transformation of B lymphocytes with oncogenic
DNA. or
transfection with an oncovirus, such as Epstein-Barr virus (EBV, also called
human
herpesvirus 4 (HHV-4)) or Kaposi's sarcoma-associated herpesvirus (KSHV). See,
U.S.
Pat. Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917;
4,472,500;
4,491,632; 4,493,890. A monoclonal antibody may also be produced via an anti-
receptor
peptide or peptides containing the carboxyl terminal as described well-known
in the art.
See Niman et al., Proc. Natl. Acad. Sci. USA, 80: 4949-4953 (1983); Geysen et
al., Proc.
Natl. Acad. Sci. USA, 82: 178-182 (1985); Lei et al. Biochemistry 34(20): 6675-
6688,
(1995). Typically, the anti-receptor peptide or a peptide analog is used
either alone or
conjugated to an immunogenic carrier, as the immunogen for producing anti-
receptor
peptide monoclonal antibodies.
There are also a number of other well-known techniques for making monoclonal
an-
tibodies as binding molecules in this invention. Particularly useful are
methods of making
fully human antibodies. One method is phage display technology which can be
used to
select a range of human antibodies binding specifically to the antigen using
methods of
affinity enrichment. Phage display has been thoroughly described in the
literature and the
construction and screening of phage display libraries are well known in the
art, see, e.g.,
Dente et al, Gene. 148(1):7-13 (1994); Little et al, Biotechnol Adv. 12(3):539-
55 (1994);
Clackson et al., Nature 352:264-628 (1991); Huse et al., Science 246:1275-1281
(1989).
Monoclonal antibodies derived by hybridoma technique from another species than
human, such as mouse, can be humanized to avoid human anti-mouse antibodies
when
infused into humans. Among the more common methods of humanization of
antibodies are
complementarity-determining region grafting and resurfacing. These methods
have been
extensively described, see e.g. U.S. Pat. Nos. 5,859,205 and 6,797,492; Liu et
al, Immunol
Rev. 222:9-27 (2008); Almagro et al, Front Biosci. 13: 1619-33 (2008); Lazar
et al, Mol
26
Immunol. 44(8):1986-98 (2007); Li et al, Proc. Natl. Acad. Sci. U S A.
103(10):3557-62 (2006).
Fully human antibodies can also be prepared by immunizing transgenic mice,
rabbits, monkeys, or
other mammals, carrying large portions of the human immunoglobulin heavy and
light chains, with
an immunogen. Examples of such mice are: the Xenomouse. (Abgenix/Amgen), the
HuMAb-
Mouse (Medarex/BMS), the VelociMouse (Regeneron), see also U.S. Pat. No.
6,596,541,
6,207,418, No. 6,150,584, No. 6,111,166, No. 6,075,181, No. 5,922,545, Nos.
5,661,016,
5,545,806, 5,436,149 and 5,569,825. In human therapy, murine variable regions
and human
constant regions can also be fused to construct called "chimeric antibodies"
that are considerably
less immunogenic in man than murine mAbs (Kipriyanov et at, Mol Biotechnol.
26:39-60 (2004);
Houdebine, Curr Opin Biotechnol. 13:625-9 (2002)). In addition, site-directed
mutagenesis in the
variable region of an antibody can result in an antibody with higher affinity
and specificity for its
antigen (Brannigan eta!, Nat Rev Mot Cell Biol. 3:964-70, (2002)); Adams et
al, J Immunol
Methods. 231:249-60 (1999)) and exchanging constant regions of a mAb can
improve its ability to
mediate effector functions of binding and cytotoxicity.
Antibodies immunospecific for a malignant cell antigen can also be obtained
commercially
or produced by any method known to one of skill in the art such as, e.g.,
chemical synthesis or
recombinant expression techniques. The nucleotide sequence encoding antibodies
immunospecific
for a malignant cell antigen can be obtained commercially, e.g., from the
GenBank database or a
database like it, the literature publications, or by routine cloning and
sequencing.
Apart from an antibody, a peptide or protein that bind/block/target or in some
other way
interact with the epitopes or corresponding receptors on a targeted cell can
be used as a binding
molecule. These peptides or proteins could be any random peptide or proteins
that have an affinity
for the epitopes or corresponding receptors and they don't necessarily have to
be of the
immunoglobulin family. These peptides can be isolated by similar techniques as
for phage display
antibodies (Szardenings, J Recept Signal Transduct Res. 2003; 23(4):307-49).
The use of peptides
from such random peptide libraries can be similar to antibodies and antibody
fragments. The
binding molecules of peptides or proteins may be conjugated on or linked to a
large molecules or
materials, such as, but is not limited, an albumin, a polymer, a liposome, a
nano particle, a
dendrimer, as long as such attachment permits the peptide or protein to retain
its antigen binding specificity.
27
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Examples of antibodies used for conjugation of drugs via the hydrophilic
linkers of
this prevention for treating cancer, autoimmune disease, and/or infectious
disease include,
but are not limited to, 3F8 (anti-GD2), Abagovomab (anti CA-125), Abciximab
(anti CD41
(integrin alpha-IIb), Acialimumab (anti-TNF-a), Adecatumumab (anti-EpCAM,
CD326),
Afelimomab (anti-TNF-a); Afutuzumab (anti-CD20), Alacizumab pegol (anti-
VEGFR2),
ALD518 (anti-IL-6). Alemtuzumab (Campath, MabCampath, anti- CD52), Altumomab
(anti-CEA), Anatumomab (anti-TAG-72), Anrukinzumab (IMA-638, anti-IL-13).
Apolizumab (anti-HLA-DR), Arcitumomab (anti-CEA), Aselizumab (anti-L-selectin
(CD62L). Atlizumab (tocilizumab, Actemra, RoActemra, anti-IL-6 receptor),
Atorolimumab (anti-Rhesus factor), Bapineuzumab (anti-beta amyloid),
Basiliximab
(Simulect, antiCD25 (a chain of IL-2 receptor), Bavituximab (anti-
phosphatidylserine),
Bectumomab (LymphoS can, anti-CD22), Belimumab (Benlysta. LymphoStat-B, anti-
BAFF), Bcnralizumab (anti-CD125), Bertilimumab (anti-CCL11 (cotaxin-1)),
Besilesomab
(Scintimun, anti-CEA-related antigen), Bevacizumab (Avastin, anti-VEGF-A),
Biciromab
(FibriScint. anti-fibrin IT beta chain), Bivatuzumab (anti-CD44 v6),
Blinatumomab (BiTE,
anti-CD19), Brentuximab (cAC10, anti-CD30 TNERSF8), Briakinumab (anti-IL-12,
IL-
23) Canakinumab (Ilaris, anti-IL-1), Cantuzumab (C242, anti-CanAg), Capromab,
Catumaxomab (Removab, anti-EpCAM, anti-CD3), CC49 (anti-TAG-72), Cedelizumab
(anti-CD4), Certolizumab pegol (Cianzia anti-TNF-a), Cetuximab (Erbitux, EVIC-
C225,
anti-EGFR), Citatuzumab bogatox (anti-EpCAM), Cixutumumab (anti-IGF-1),
Clenoliximab (anti-CD4), Clivatuzumab (anti-MUC1), Conatumumab (anti-TRAIL-
R2),
CR6261 (anti-Influenza A hemagglutinin), Dacetuzumab (anti-CD40), Daclizumab
(Zenapax, anti-CD25 (a chain of IL-2 receptor)), Daratumumab (anti-CD38
(cyclic ADP
ribose hydrolase), Denosumab (Prolia, anti-RANKL), Detumomab (anti-B-lymphoma
cell),
Dorlimomab, Dorlixizumab. Ecromeximab (anti-GD3 ganglioside), Eculizumab
(Soliris,
anti-CS), Edobacomab (anti-endotoxin), Edrecolomab (Panorex, MAb17-1A, anti-
EpCAM), Efalizumab tRaptiva, anti-LFA-1 (CD I la), Efungumab (Mycograb, anti-
Hsp90),
Elotuzumab (anti-SLAMF7), Elsilimomab (anti-IL-6), Enlimomab pegol (anti-ICAM-
1
(CD54)), Epitumomab (anti-cpisialin), Epratuzumab (anti-CD22), Erlizumab (anti-
ITGB2
(CD18)), Ertumaxomab (Rexomun, anti-HER2/neu, CD3), Etaracizumab (Abegrin,
anti-
integrin a,r33), Exbivirumab ( anti-hepatitis B surface antigen), Fanolesomab
(NeutroSpec,
anti-CD15), Faralimomab (anti-interferon receptor), Farletuzumab (anti-folate
receptor 1),
Felvizumab (anti-respiratory syncytial virus), Fezakinumab (anti-IL-22),
Figitumumab
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(anti-IGF-1 receptor), Fontolizumab (anti-TN-7), Foravirumab (anti-rabies
virus glycopro-
tein), Fresolimumab (anti-TGF-13), Galiximab (anti-CD80), Gantenerumab (anti-
beta
amyloid), Gavilimomab (anti-CD147 (basigin)), Gemtuzumab (anti-CD33),
Girentuximab
(anti-carbonic anhydrase 9), Glembatumumab (CR011, anti-GPNMB), Golimumab
(Simponi, anti-TNF-a). Gomiliximab (anti-CD23 (IgE receptor)), Ibalizumab
(anti-CD4),
Ibritumomab (anti-CD20), Igovomab (Indimacis-125, anti-CA-125), Imciromab
(Myoscint,
anti-cardiac myosin), Infliximab (Remicade, anti-TNF-a), Intetumumab (anti-
CD51),
Inolimomab (anti-CD25 (a chain of IL-2 receptor)), Inotuzumab (anti-CD22),
Ipilimumab
(anti-CD152), Iratumumab (anti- CD30 (TNFRSF8)), Keliximab (anti-CD4),
Labetuzumab
(CEA-Cide, anti-CEA), Lebrikizumab (anti- IL-13), Lemalesomab (anti-NCA-90
(granulo-
cyte antigen)), Lerdelimumab (anti-TGF beta 2), Lexatumumab (anti-TRAIL-R2),
Libivirumab (anti-hepatitis B surface antigen), Lintuzumab (anti-CD33),
Lucatumumab
(anti-CD40), Lumiliximab (anti- CD23 (IgE receptor), Mapatumumab (anti-TRAIL-
R1),
Maslimomab (anti- T-cell receptor), Matuzumab (anti-EGFR), Mepolizumab
(Bosatria,
anti-IL-5), Metelimumab (anti-TGF beta 1), Milatuzumab (anti-CD74),
Minretumomab
(anti-TAG-72), Mitumomab (BEC-2, anti-GD3 ganglioside), Morolimumab (anti-
Rhesus
factor), Motavizumab (Numax, anti-respiratory syncytial virus), Muromonab-CD3
(Orthoclone OKT3, anti-CD3). Nacolomab (anti-C242), Naptumomab (anti-5T4),
Natalizumab (Tysabri, anti-integrin a4),Nebacumab (anti-endotoxin),
Necitumumab (anti-
EGFR), Nerelimomab (anti-TNF-a), Nimotuzumab (Theracim, Theraloc, anti-EGFR),
Nofetumomab, Ocrelizumab (anti-CD20), Odulimomab (Afolimomab, anti-LFA-1
(CD11 a)), Ofatumumab (Arzerra, anti-CD20), Olaratumab (anti-PDGF-R a),
Omalizumab
(Xolair, anti-IgE Fe region), Oportuzumab (anti-EpCAM), Oregovomab (OvaRex,
anti-
CA-125), Otelixizumab (anti-CD3), Pagibaximab (anti-lipoteichoic acid),
Palivizumab
(Synagis, Abbosynagis, anti-respiratory syncytial virus), Panitumumab
(Vectibix, ABX-
EGF, anti-EGFR), Panobacumab (anti- Pseudomonas aeruginosa), Pascolizumab
(anti-IL-
4), Pcmtumomab (Thcragyn, anti-MUC1), Pertuzumab (Omnitarg, 2C4, anti-
HER2/neu),
Pexclizumab (anti-05). Pintumomab (anti-adcnocarcinoma antigen), Priliximab
(anti-
CD4), Pritumumab (anti-vimcntin), PRO 140 (anti-CCR5), Racotumomab (1E10, anti-
(N-
glycolylneuraminic acid (NeuGc, NGNA)-gangliosides GM3)), Rafivirumab (anti-
rabies
virus glycoprotein), Ramucirumab (anti-VEGFR2), Ranibizumab (Lucentis, anti-
VEGF-
A), Raxibacumab (anti-anthrax toxin, protective antigen). Regavirumab (anti-
cytomegalovirus glycoprotein B). Reslizumab (anti-IL-5), Rilotumumab (anti-
HGF),
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Rituximab (Mab'Thera, Rituxanmab, anti-CD20), Robatumumab (anti-IGF-1
receptor),
Rontalizumab (anti-IFN-a). Rovelizumab (LeukArrest. anti-CD11, CD18),
Ruplizumab
(Antova, anti-CD154 (CD4OL)), Satumomab (anti-TAG-72). Sevirumab (anti-
cytomegalovirus), Sibrotuzumab (anti-FAP), Sifalimumab (anti-IFN-a),
Siltuximab (anti-
IL-6), Siplizumab (anti-CD2), (Smart) MI95 (anti-CD33), Solanezumab (anti-beta
amy-
loid), Sonepcizumab (anti-sphingosine-l-phosphate), Sontuzumab (anti-
episialin),
Stamulumab (anti-myostatin), Sulesomab (LeukoScan, (anti-NCA-90 (granulocyte
anti-
gen), Tacatuzumab (anti-alpha-fetoprotein), Tadocizumab (anti-integrin
ct1ff,133), Talizumab
(anti-IgE), Tanezumab (anti-NGF), Taplitumomab (anti-CD19), Tefibazumab
(Aurexis,
(anti-clumping factor A), Telimomab. Tenatumomab (anti-tenascin C),
Teneliximab (anti-
CD40), Teplizumab (anti-CD3), TGN1412 (anti-CD28), Ticilimumab (Tremelimumab,
(anti-CTLA-4), Tigatuzumab (anti-TRAIL-R2), TNX-650 (anti-IL-13), Tocilizumab
(Atlizumab, Actemra, RoActemra, (anti-IL-6 receptor), Toralizumab (anti-CD154
(CD4OL)), Tositumomab (anti-CD20), Trastuzumab (Herceptin. (anti-HER2/neu),
Tremelimumab (anti-CTLA-4), Tucotuzumab celmoleukin (anti-EpCAM), Tuvirumab
(anti-hepatitis B virus), Urtoxazumab (anti- Eseheriehia call), Ustekinumab
(Stelara, anti-
IL-12. IL-23), Vapaliximab (anti-A0C3 (VAP-1)), Vedolizumab, (anti-
integrina4P7),
Veltuzumab (anti-CD20), Vepalimomab (anti-A0C3 (VAP-1), Visilizumab (Nuvion,
anti-
CD3), Vitaxin (anti-vascular integrin avb3), Volociximab (anti-integrin
a5131), Votumumab
(HumaSPECT, anti-tumor antigen CTAA16.88), Zalutumumab (HuMax-EGFr, (anti-
EGFR), Zanolimumab (HuMax-CD4, anti-CD4), Ziralimumab (anti-CD147 (basigin)),
Zolimomab (anti-CD5), Etanercept (Enbre110), Alefacept (Amevive ), Abatacept
(Orencia ), Rilonacept (Arcalyst), 14F7 [anti-IRP-2 (Iron Regulatory Protein
2)], 14G2a
(anti-GD2 ganglioside, from Nat. Cancer Inst. for melanoma and solid tumors),
J591 (anti-
PSMA, Weill Cornell Medical School for prostate cancers), 225.28S [anti-HMW-
MAA
(High molecular weight-melanoma-associated antigen), Sorin Radiofarmaci S.R.L.
(Milan,
Italy) for melanoma], COL-1 (anti-CEACAM3, CGM1, from Nat. Cancer Inst. USA
for
colorectal and gastric cancers), CYT-356 (Oncoltada for prostate cancers),
HNK20
(OraVax Inc. for respiratory syncytial virus), ImmuRAIT (from Immunomcdics for
NHL),
Lym-1 (anti-HLA-DR10, Peregrine Pharm. for Cancers), MAK-195F [anti-TNF (tumor
necrosis factor; TNFA, TNF-alpha; TNFSF2), from Abbott / Knoll for Sepsis
toxic shock],
MEDI-500 [T10B9, anti-CD3, TRaP (T cell receptor alpha/beta), complex, from
MedImmune Inc for Graft-versus-host disease], RING SCAN [ anti-TAG 72 (tumour
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associated glycoprotein 72), from Neoprobe Corp. for Breast, Colon and Rectal
cancers],
Avicidin (anti-EPCAM (epithelial cell adhesion molecule), anti-TACSTD1 (Tumor-
associated calcium signal transducer 1), anti-GA733-2 (gastrointestinal tumor-
associated
protein 2), anti-EGP-2 (epithelial glycoprotein 2); anti-KSA; KS1/4 antigen;
M4S; tumor
antigen 17-1A; CD326, from NeoRx Corp. for Colon, Ovarian, Prostate cancers
and NHL];
LymphoCide (Immunomedics, NJ), Smart liD10 (Protein Design Labs), Oncolym
(Techniclone Inc, CA), Allomune (BioTransplant, CA), anti-VEGF (Genentech,
CA);
CEAcide (Immunomedics. NJ), IMC-1C11 (ImClone Systems, NJ) and Cetuximab
(ImClone, NJ) .
Other antibodies as cell binding molecules/ligands include, but are not
limited to, are
antibodies against the following antigens: Aminopeptidase N (CD13), Annexin
Al, B7-H3
(CD276, various cancers), CA125 (ovarian), CA15-3 (carcinomas), CA19-9
(carcinomas),
L6 (carcinomas), Lewis Y (carcinomas). Lewis X (carcinomas), alpha fetoprotein
(carci-
nomas), CA242 (colorectal), placental alkaline phosphatase (carcinomas),
prostate specific
antigen (prostate), prostatic acid phosphatase (prostate), epidermal growth
factor (carcino-
mas), CD2 (Hodgkin's disease, NHL lymphoma, multiple myeloma), CD3 epsilon (T
cell
lymphoma, lung, breast, gastric, ovarian cancers, autoimrnune diseases,
malignant ascites),
CD19 (B cell malignancies), CD20 (non-Hodgkin's lymphoma), CD22 (leukemia, lym-
phoma, multiple myeloma, SLE), CD30 (Hodgkin's lymphoma), CD33 (leukemia, auto-
immune diseases), CD38 (multiple myeloma), CD40 (lymphoma, multiple myeloma,
leukemia (CLL)), CD51 (Metastatic melanoma, sarcoma), CD52 (leukemia), CD56
(small
cell lung cancers, ovarian cancer, Merkel cell carcinoma, and the liquid
tumor, multiple
myeloma), CD66e (cancers), CD70 (metastatic renal cell carcinoma and non-
Hodgkin
lymphoma), CD74 (multiple myeloma), CD80 (lymphoma), CD98 (cancers), mucin
(carcinomas), CD221 (solid tumors), CD227 (breast, ovarian cancers). CD262
(NSCLC
and other cancers), CD309 (ovarian cancers), CD326 (solid tumors), CEACAM3
(colorec-
tal, gastric cancers), CEACAM5 (carcinoembryonic antigen; CEA, CD66e) (breast,
colo-
rectal and lung cancers), DLL4 (delta-like-4), EGFR (Epidermal Growth Factor
Receptor,
various cancers), CTLA4 (melanoma), CXCR4 (CD184, Heme-oncology, solid
tumors),
Endoglin (CD105, solid tumors), EPCAM (epithelial cell adhesion molecule,
bladder, head,
neck, colon, NI-IL prostate, and ovarian cancers), ERBB2 (Epidermal Growth
Factor
Receptor 2; lung, breast, prostate cancers), FCGR1 (autoimmune diseases), FOLR
(folate
receptor, ovarian cancers), GD2 ganglioside (cancers), G-28 (a cell surface
antigen
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glyvolipid, melanoma), GD3 idiotype (cancers), Heat shock proteins (cancers).
HER1
(lung, stomach cancers), HER2 (breast, lung and ovarian cancers), HLA-DR10
(NHL),
HLA-DRB (NHL, B cell leukemia), human chorionic gonadotropin (carcinoma),
IGF1R
(insulin-like growth factor 1 receptor, solid tumors, blood cancers), IL-2
receptor (interleu-
kin 2 receptor, T-cell leukemia and lymphomas), IL-6R (interleukin 6 receptor,
multiple
myeloma, RA, Castleman's disease, IL6 dependent tumors), Integrins (avf33,
0[31, a6f34,
a11133, 0135, av135, for various cancers), MAGE-1 (carcinomas), MAGE-2
(carcinomas),
MAGE-3 (carcinomas), MAGE 4 (carcinomas), anti-transferrin receptor
(carcinomas), p97
(melanoma), MS4A1 (membrane-spanning 4-domains subfamily A member 1, Non-
Hodgkin's B cell lymphoma, leukemia), MUC1 or MUC1-KLH (breast, ovarian,
cervix,
bronchus and gastrointestinal cancer), MUC16 (CA125) (Ovarian cancers), CEA
(colorec-
tal), gp100 (melanoma), MARTI (melanoma). MPG (melanoma), MS4A1 (membrane-
spanning 4-domains subfamily A, small cell lung cancers, NHL), Nucleolin, Neu
oncogene
product (carcinomas), P21 (carcinomas), Paratope of anti-(N-glycolylneuraminic
acid,
Breast, Melanoma cancers), PLAP-like testicular alkaline phosphatase (ovarian,
testicular
cancers), PSMA (prostate tumors), PSA (prostate), ROB04, TAG 72 (tumour
associated
glycoprotein 72, AML, gastric, colorectal, ovarian cancers), T cell
transmembrane protein
(cancers). Tie (CD202b), TNFRSF1OB (tumor necrosis factor receptor superfamily
mem-
ber 10B, cancers), TNFRSF13B (tumor necrosis factor receptor superfamily
member 13B,
multiple myeloma, NHL, other cancers, RA and SLE), TPBG (trophoblast
glycoprotein,
Renal cell carcinoma), TRAIL-R1 (Tumor necrosis apoprosis Inducing ligand
Receptor
1,1ymphoma, NHL, colorectal, lung cancers), VCAM-1 (CD106, Melanoma), VEGF,
VEGF-A. VEGF-2 (CD309) (various cancers). Some other tumor associated antigens
recognized by antibodies have been reviewed (Gerber, et al, mAbs 1:3, 247-253
(2009);
Novellino et al, Cancer Immunol Immunother. 54(3), 187-207 (2005). Franke, et
al, Cancer
Biother Radiopharm. 2000, 15. 459-76).
The cell binding agents, more preferred antibodies, can be any agents that are
able to
against tumor cells, virus infected cells, microorganism infected cells,
parasite infected
cells, autoimmune cells, activated cells, myeloid cells, activated T-cells, B
cells, or mela-
nocytes. More specifically the cell binding agents can be any agent/molecule
that is able to
against any one of the following antigens or receptors: CD3, CD4, CD5, CD6,
CD7, CD8,
CD9, CD10, CD11a, CD11b. CD11c, CD12w, CD14, CD15, CD16, CDw17, CD18,
CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30,
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CD31, CD32, CD33, CD34, CD35, CD36, CD37. CD38, CD39, CD40, CD41, CD42,
CD43, CD44, CD45, CD46, CD47, CD48, CD49b, CD49c, CD51, CD52, CD53, CD54.
CD55, CD56, CD58, CD59, CD61, CD62E, CD62L, CD62P, CD63, CD66, CD68, CD69,
CD70, CD72, CD74, CD79, CD79a, CD79b, CD80, CD81, CD82, CD83, CD86, CD87,
CD88, CD89, CD90, CD91, CD95, CD96, CD98, CD100, CD103, CD105, CD106,
CD109, CD117, CD120, CD125, CD126, CD127, CD133, CD134, CD135, CD138.
CD141, CD142, CD143, CD144, CD147, CD151, CD147, CD152, CD154, CD156.
CD158, CD163, CD166, .CD168, CD174, CD180, CD184. CDw186, CD194, CD195,
CD200, CD200a. CD200b, CD209, CD221, CD227, CD235a, CD240, CD262, CD271,
CD274, CD276 (B7-H3), CD303, CD304, CD309, CD326, 4-1BB, 5AC, 5T4 (Trophoblast
glycoprotein, TPBG, 5T4, Wnt-Activated Inhibitory Factor 1 or WAIF1),
Adenocarcinoma
antigen, AGS-5, AGS-22M6, Activin receptor-like kinase 1, AFP, AKAP-4, ALK,
Alpha
intergrin, Alpha v beta6, Amino-peptidase N, Amyloid beta, Androgen receptor,
Angiopoictin 2, Angiopoietin 3, Anncxin Al, Anthrax toxin protective antigen,
Anti-
transferrin receptor, A0C3 (VAP-1), B7-H3, Bacillus anthracis anthrax, BAFF (B-
cell
activating factor), B-lymphoma cell, bcr-abl, Bombesin, BORIS, C5, C242
antigen, CA125
(carbohydrate antigen 125, MUC16), CA-IX (or CAIX, carbonic anhydrase 9),
CALLA,
CanAg, Canis lupus familiaris IL31, Carbonic anhydrase IX, Cardiac myosin,
CCL11(C-C
motif chemokine 11), CCR4 (C-C chemokine receptor type 4, CD194), CCR5, CD3E
(epsilon), CEA (Carcinoembryonic antigen), CEACAM3, CEACAM5 (carcinoembryonic
antigen), CFD (Factor D), Ch4D5. Cholecystokinin 2 (CCK2R), CLDN18 (Claudin-
18),
Clumping factor A, CRIPTO, FCSF1R (Colony stimulating factor 1 receptor,
CD115),
CSF2 (colony stimulating factor 2, Granulocyte-macrophage colony-stimulating
factor
(GM-CSF)), CTLA4 (cytotoxic T-lymphocyte-associated protein 4), CTAA16.88
tumor
antigen, CXCR4 (CD184), C-X-C chemokine receptor type 4, cyclic ADP ribose
hydrolase,
Cyclin Bl, CYP1B1, Cytomegalovirus, Cytomegalovirus glycoprotein B,
Dabigatran,
DLL4 (delta-like-ligand 4), DPP4 (Dipeptidyl-peptidase 4), DR5 (Death receptor
5), E.
coli shiga toxin type-1, E. coli shiga toxin type-2, ED-B, EGFL7 (EGF-like
domain-
containing protein 7), EGFR, EGFR1I, EGFRvIII, Endoglin (CD105), Endothclin B
receptor, Endotoxin, EpCAM (epithelial cell adhesion molecule), EphA2,
Episialin,
ERBB2 (Epidermal Growth Factor Receptor 2), ERBB3, ERG (TMPRSS2 ETS fusion
gene), Escherichia coli. ETV6-AML, FAP (Fibroblast activation protein alpha),
FCGR1,
alpha-Fetoprotein, Fibrin II, beta chain, Fibronectin extra domain-B, FOLR
(folate recep-
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tor), Folate receptor alpha, Folate hydrolase, Fos-related antigen 1.F protein
of respiratory
syncytial virus, Frizzled receptor, Fucosyl GM l , GD2 ganglioside, G-28 (a
cell surface
antigen glyvolipid), GD3 idiotype, GloboH. Glypican 3, N-glycolylneuraminic
acid, GM3,
GMCSF receptor a-chain, Growth differentiation factor 8, GP100, GPNMB
(Transmembrane glycoprotein NMB), GUCY2C (Guanylate cyclase 2C, guanylyl
cyclase
C(GC-C), intestinal Guanylate cyclase, Guanylate cyclase-C receptor, Heat-
stable entero-
toxin receptor (hSTAR)), Heat shock proteins, Hemagglutinin, Hepatitis B
surface antigen,
Hepatitis B virus, HER1 (human epidermal growth factor receptor 1), HER2,
HER2/neu,
HER3 (ERBB-3), IgG4, HGF/SF (Hepatocyte growth factor/scatter factor), HHGFR,
HIV-
1, Histone complex, HLA-DR (human leukocyte antigen), HLA-DR10, HLA-DRB ,
HMWMAA, Human chorionic gonadotropin, HNGF, Human scatter factor receptor
kinase,
HPV E6/E7, Hsp90, hTERT, ICAM-1 (Intercellular Adhesion Molecule 1), Idiotype,
IGF1R (IGF-1, insulin-like growth factor 1 receptor), IGHE, IFN-T. Influen-
za hemagglutinin, IgE, IgE Fc region, IGHE, IL-1, IL-2 receptor (interleukin 2
receptor),
IL-4, IL-5, IL-6, IL-6R (interleukin 6 receptor), IL-9. IL-10, IL-12, IL-13,
IL-17, IL-17A,
IL-20, IL-22, IL-23, IL31RA, ILGF2 (Insulin-like growth factor 2), Integrins
(a4, a1b133,
avf33, a437, a5131, a6134, a7f37, al1[33, a5135, av[35), Interferon gamma-
induced protein,
ITGA2, ITGB2, KIR2D, LCK, Le, Legumain, Lewis-Y antigen, LFA-1(Lymphocyte
function-associated antigen 1, CD11 a), LHRH, LINGO-1, Lipoteichoic acid,
LIVIA,
LMP2, LTA, MAD-CT-1, MAD-CT-2, MAGE-1, MAGE-2, MAGE-3, MAGE Al, MAGE
A3, MAGE 4, MART I, MCP-1, MW (Macrophage migration inhibitory factor, or
glyco-
sylation-inhibiting factor (GIF)), MS4A1 (membrane-spanning 4-domains
subfamily A
member 1), MSLN (mcsothelin), MUC1(Mucin 1, cell surface associated (MUC1) or
polymorphic epithelial mucin (PEM)), MUC1-KLH, MUC16 (CA125), MCP1(monocyte
chemotactic protein 1), MelanA/MART1, ML-IAP, MPG, MS4A1 (membrane-spanning 4-
domains subfamily A), MYCN, Myelin-associated glycoprotein, Myostatin, NA17,
NARP-
1, NCA-90 (granulocyte antigen), Nectin-4 (ASG-22ME), NGF, Neural apoptosis-
regulated proteinase 1, NOGO-A, Notch receptor, Nucleolin, Neu oncogene
product, NY-
BR-1, NY-ESO-1, OX-40, OxLDL (Oxidized low-density lipoprotein). 0Y-TES 1,
P21,
p53 nonmutant. P97. Page4, PAP, Paratope of anti-(N-glycolylneuraminic acid),
PAX3,
PAX5, PCSK9, PDCD1 (PD-1, Programmed cell death protein 1,CD279), PDGF-Ra
(Alpha-type platelet-derived growth factor receptor), PDGFR-13, PDL-1, PLAC1,
PLAP-
like testicular alkaline phosphatase, Platelet-derived growth factor receptor
beta, Phos-
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phate-sodium co-transporter, PMEL 17, Polysialic acid, Proteinase3 (PRI),
Prostatic
carcinoma, PS (Phosphatidylserine), Prostatic carcinoma cells, Pseudomonas
aeruginosa,
PSMA, PSA, PSCA, Rabies virus glycoprotein, RHD (Rh polypeptide 1 (RhPI),
CD240),
Rhesus factor, RANKL, RhoC, Ras mutant, RGS5, ROB04, Respiratory syncytial
virus,
RON, Sarcoma translocation breakpoints, SART3, Sclerostin. SLAMF7 (SLAM family
member 7), Selectin P, SDC1 (Syndecan 1), sLe(a), Somatomedin C, SIP
(Sphingosine-l-
phosphate), Somatostatin, Sperm protein 17, SSX2. STEAP1 (six-transmembrane
epithelial
antigen of the prostate 1), STEAP2, STn, TAG-72 (tumor associated glycoprotein
72),
Survivin, T-cell receptor, T cell transmembrane protein, TEM1 (Tumor
endothelial marker
1), TENB2, Tenascin C (TN-C), TGF-a, TGF-I3 (Transforming growth factor beta),
TGF-
131, TGF-132 (Transforming growth factor-beta 2), Tie (CD202b), Tie2, TIM-1
(CDX-014),
Tn, TNF, TNF-a, TNFRSF8, TNFRSFIOB (tumor necrosis factor receptor superfamily
member 10B), TNERSF13B (tumor necrosis factor receptor superfamily member
13B).
TPBG (trophoblast glycoprotein), TRAIL-R1 (Tumor necrosis apoprosis Inducing
ligand
Receptor 1), TRAILR2 (Death receptor 5 (DRS)), tumor-associated calcium signal
trans-
ducer 2, tumor specific glycosylation of MUC1, TWEAK receptor,
TYRP1(glycoprotein
75), TRP-2, Tyrosinase, VCAM-1 (CD106), VEGF, VEGF-A, VEGF-2 (CD309),
VEGFR-1, VEGFR2, or vimentin, WT1, XAGE 1, or cells expressing any insulin
growth
factor receptors, or any epidermal growth factor receptors.
In another specific embodiment, the cell-binding ligand -drug conjugates via
the hy-
drophilic linkers of this invention are used for the targeted treatment of
cancers. The
targeted cancers include, but are not limited, Adrenocortical Carcinoma, Anal
Cancer,
Bladder Cancer, Brain Tumor (Adult, Brain Stem Glioma, Childhood, Cerebellar
Astrocy-
toma, Cerebral Astrocytoma, Ependymoma, Medulloblastoma, Supratentorial
Primitive
Neuroectodermal and Pineal Tumors, Visual Pathway and Hypothalamic Glioma),
Breast
Cancer, Carcinoid Tumor, Gastrointestinal, Carcinoma of Unknown Primary,
Cervical
Cancer, Colon Cancer, Endometrial Cancer, Esophageal Cancer, Extrahepatic Bile
Duct
Cancer, Ewings Family of Tumors (PNET), Extracranial Germ Cell Tumor, Eye
Cancer,
Intraocular Melanoma, Gallbladder Cancer, Gastric Cancer (Stomach), Germ Cell
Tumor,
Extragonadal, Gestational Trophoblastic Tumor, Head and Neck Cancer,
Hypopharyngeal
Cancer, Islet Cell Carcinoma, Kidney Cancer (renal cell cancer), Laryngeal
Cancer, Leu-
kemia (Acute Lymphoblastic, Acute Myeloid, Chronic Lymphocytic, Chronic
Myelogenous, Hairy Cell), Lip and Oral Cavity Cancer, Liver Cancer, Lung
Cancer (Non-
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Small Cell, Small Cell, Lymphoma (AIDS-Related. Central Nervous System,
Cutaneous T-
Cell, Hodgkin's Disease, Non-Hodgkin's Disease, Malignant Mesothelioma,
Melanoma,
Merkel Cell Carcinoma, Metasatic Squamous Neck Cancer with Occult Primary,
Multiple
Myeloma, and Other Plasma Cell Neoplasms, Mycosis Fungoides, Myelodysplastic
Syn-
drome, Myeloproliferative Disorders, Nasopharyngeal Cancer, Neuroblastoma,
Oral
Cancer, Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer (Epithelial, Germ
Cell
Tumor, Low Malignant Potential Tumor), Pancreatic Cancer (Exocrine, Islet Cell
Carci-
noma), Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer, Penile
Cancer,
Pheochromocytoma Cancer, Pituitary Cancer, Plasma Cell Neoplasm, Prostate
Cancer
Rhabdomyosarcoma, Rectal Cancer, Renal Cell Cancer (kidney cancer), Renal
Pelvis and
Ureter (Transitional Cell), Salivary Gland Cancer, Sezary Syndrome, Skin
Cancer, Skin
Cancer (Cutaneous T-Cell Lymphoma, Kaposi's Sarcoma, Melanoma), Small
Intestine
Cancer, Soft Tissue Sarcoma, Stomach Cancer, Testicular Cancer, Thymoma
(Malignant),
Thyroid Cancer, Urethral Cancer, Uterine Cancer (Sarcoma), Unusual Cancer of
Child-
hood, Vaginal Cancer, Vulvar Cancer, Wilms' Tumor.
In another specific embodiment, the cell-binding-drug conjugates via the
hydrophilic
likers of this invention are used in accordance with the compositions and
methods for the
treatment or prevention of an autoirnmune disease. The autoimmune diseases
include, but
are not limited, Achlorhydra Autoimmune Active Chronic Hepatitis, Acute
Disseminated
Encephalomyelitis, Acute hemorrhagic leukoencephalitis, Addison's Disease,
Agammaglobulinemia, Alopecia areata, Amyotrophic Lateral Sclerosis, Ankylosing
Spondylitis, Anti-GBM/TBM Nephritis, Antiphospholipid syndrome, Antisynthetase
syndrome, Arthritis, Atopic allergy, Atopic Dermatitis. Autoimmune Aplastic
Anemia,
Autoimmune cardiomyopathy, Autoimmune hemolytic anemia. Autoimmune hepatitis,
Autoimmune inner ear disease, Autoimmune lymphoproliferative syndrome,
Autoimmune
peripheral neuropathy, Autoimmune pancreatitis, Autoimmune polyendocrine
syndrome
Types I, II, & III, Autoimmune progesterone dermatitis, Autoimmune
thrombocytopenic
purpura, Autoimmune uvcitis, Balo discase/Balo concentric sclerosis. Bechcts
Syndrome,
Berger's disease, Bickerstaffs encephalitis, Blau syndrome, Bullous
Pcmphigoid,
Castleman's disease, Chagas disease, Chronic Fatigue Immune Dysfunction
Syndrome,
Chronic inflammatory demyelinating polyneuropathy, Chronic recurrent
multifocal
ostomyelitis, Chronic lyme disease, Chronic obstructive pulmonary disease,
Churg-Strauss
syndrome, Cicatricial Pemphigoid, Coeliac Disease, Cogan syndrome, Cold
agglutinin
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disease, Complement component 2 deficiency, Cranial arteritis, CREST syndrome,
Crohns
Disease (a type of idiopathic inflammatory bowel diseases), Cushing's
Syndrome, Cutane-
ous leukocytoclastic angiitis, Dego's disease, Dercum's disease, Dermatitis
herpetiformis,
Dermatomyositis, Diabetes mellitus type 1, Diffuse cutaneous systemic
sclerosis, Dressler's
syndrome, Discoid lupus erythematosus, Eczema, Endometriosis, Enthesitis-
related arthri-
tis, Eosinophilic fasciitis, Epidermolysis bullosa acquisita, Erythema
nodosum, Essential
mixed cryoglobulinemia, Evan's syndrome, Fibrodysplasia ossificans
progressiva, Fibrom-
yalgia, Fibromyositis, Fibrosing aveolitis, Gastritis, Gastrointestinal
pemphigoid, Giant cell
arteritis, Glomerulonephritis, Goodpasture's syndrome, Graves' disease,
Guillain-Barre
syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, Haemolytic
anaemia,
Henoch-Schonlein purpura. Herpes gestationis, Hidradenitis suppurativa, Hughes
syn-
drome (See Antiphospholipid syndrome), Hypogammaglobulinemia, Idiopathic
Inflamma-
tory Demyclinating Diseases, Idiopathic pulmonary fibrosis, Idiopathic
thrombocytopenic
purpura (See Autoimmune thrombocytopenic purpura), lgA nephropathy (Also
Berger's
disease), Inclusion body myositis, Inflammatory demyelinating polyneuopathy,
Interstitial
cystitis, Irritable Bowel Syndrome , Juvenile idiopathic arthritis, Juvenile
rheumatoid
arthritis, Kawasaki's Disease. Lambert-Eaton myasthenic syndrome,
Leukocytoclastic
vasculitis, Lichen planus, Lichen sclerosus, Linear IgA disease (LAD), Lou
Gehrig's
Disease (Also Amyotrophic lateral sclerosis), Lupoid hepatitis, Lupus
erythematosus,
Majeed syndrome, Meniere's disease, Microscopic polyangiitis, Miller-Fisher
syndrome,
Mixed Connective Tissue Disease, Morphea, Mucha-Habermann disease,
Muckle¨Wells
syndrome, Multiple Myeloma, Multiple Sclerosis, Myasthenia gravis, Myositis,
Narcolep-
sy, Neuromyelitis optica (Devic's Disease), Neuromyotonia. Occular cicatricial
pemphigoid, Opsoclonus myoclonus syndrome, Ord thyroiditis, Palindromic
rheumatism,
PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with
Streptococ-
cus), Paraneoplastic cerebellar degeneration, Paroxysmal nocturnal
hemoglobinuria, Parry
Romberg syndrome, Parsonnage-Turner syndrome, Pars planitis, Pemphigus,
Pcmphigus
vulaaris, Pernicious anaemia, Perivenous encephalomyelitis, POEMS syndrome,
Polyarteritis nodosa, Polymyalgia rhcumatica, Polymyositis, Primary biliary
cirrhosis,
Primary sclerosing cholangitis, Progressive inflammatory neuropathy,
Psoriasis, Psoriatic
Arthritis, Pyoderma gangrenosum, Pure red cell aplasia, Rasmussen's
encephalitis, Ray-
naud phenomenon, Relapsing polychondritis, Reiter's syndrome, Restless leg
syndrome,
Retroperitoneal fibrosis, Rheumatoid arthritis, Rheumatoid fever, Sarcoidosis,
Schizophre-
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nia. Schmidt syndrome, Schnitzler syndrome, Scleritis, Scleroderma, Sjogrents
syndrome,
Spondyloarthropathy, Sticky blood syndrome, Still's Disease, Stiff person
syndrome,
Subacute bacterial endocarditis, Susac's syndrome, Sweet syndrome, Sydenham
Chorea,
Sympathetic ophthalmia, Takayasu's arteritis, Temporal arteritis (giant cell
arteritis),
Tolosa-Hunt syndrome, Transverse Myelitis, Ulcerative Colitis (a type of
idiopathic
inflammatory bowel diseases), Undifferentiated connective tissue disease,
Undifferentiated
spondyloarthropathy, Vasculitis, Vitiligo, Wegener's granulomatosis, Wilson's
syndrome,
Wiskott-Aldrich syndrome
In another specific embodiment, a binding molecule used for the conjugate via
the
hydrophilic linkers of this invention for the treatment or prevention of an
autoimmune
disease can be, but are not limited to, anti-elastin antibody; Abys against
epithelial cells
antibody; Anti-Basement Membrane Collagen Type IV Protein antibody; Anti-
Nuclear
Antibody; Anti ds DNA; Anti ss DNA, Anti Cardiolipin Antibody IgM, IgG; anti-
celiac
antibody; Anti Phospholipid Antibody IgK, IgG; Anti SM Antibody; Anti
Mitochondrial
Antibody; Thyroid Antibody; Microsomal Antibody, T-cells antibody;
Thyroglobulin
Antibody, Anti SCL-70; Anti-Jo; Anti-U1RNP; Anti-La/SSB; Anti SSA; Anti
SSB;
Anti Perital Cells Antibody; Anti Histones; Anti RNP; C-ANCA; P-ANCA; Anti
centro-
mere; Anti-Fibrillarin, and Anti GBM Antibody, Anti-ganglioside antibody; Anti-
Desmogein 3 antibody; Anti-p62 antibody; Anti-sp100 antibody; Anti-
Mitochondrial(M2)
antibody; Rheumatoid factor antibody; Anti-MCV antibody; Anti-topoisomerase
antibody;
Anti-neutrophil cytoplasmic(cANCA) antibody;
In certain preferred embodiments, the binding molecule for the conjugate in
the
present invention, can bind to both a receptor or a receptor complex expressed
on an
activated lymphocyte which is associated with an autoimmune disease. The
receptor or
receptor complex can comprise an immunoglobulin gene superfamily member (e.g.
CD2,
CD3. CD4, CD8, CD19, CD20, CD22, CD28, CD30, CD33, CD37. CD38, CD56, CD70,
CD79, CD79b, CD90, CD125, CD152/CTLA-4, PD-1. or ICOS), a TNF receptor super-
family member (e.g. CD27, CD40, CD95/Fas, CD134/0X40, CD137/4-1BB, INF-R1,
TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2/TRAIL-R1, TRAIL-R2, TRAIL-
R3, TRAIL-R4, and APO-3), an integrin, a cytokine receptor, a chemokine
receptor, a
major histocompatibility protein, a lectin (C-type, S-type, or I-type), or a
complement
control protein.
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In another specific embodiment, useful cell binding ligands that are
immunospecific
for a viral or a microbial antigen are humanized or human monoclonal
antibodies. As used
herein, the term "viral antigen" includes, but is not limited to, any viral
peptide, polypep-
tide protein (e.g. HIV gp120, HIV nef, RSV F glycoprotein, influenza virus
neuramimidase, influenza virus hemagglutinin, HTLV tax, herpes simplex virus
glycopro-
tein (e.g. gB, gC, gD, and gE) and hepatitis B surface antigen) that is
capable of eliciting an
immune response. As used herein, the term "microbial antigen" includes, but is
not limited
to, any microbial peptide, polypeptide, protein, saccharide, polysaccharide,
or lipid mole-
cule (e.g., a bacterial, fungi, pathogenic protozoa, or yeast polypeptides
including, e.g.,
LPS and capsular polysaccharide 5/8) that is capable of eliciting an immune
response.
Examples of antibodies available 1 for the viral or microbial infection
include, but are not
limited to, Palivizumab which is a humanized anti-respiratory syncytial virus
monoclonal
antibody for the treatment of RSV infection; PR0542 which is a CD4 fusion
antibody for
the treatment of HIV infection; Ostavir which is a human antibody for the
treatment of
hepatitis B virus; PROTVIR which is a humanized IgG1 antibody for the
treatment of
cytomegalovirus; and anti-LPS antibodies.
The cell binding molecules¨drug conjugates via the hydrophilic linkers of this
inven-
tion can be used in the treatment of infectious diseases. These infectious
diseases include,
but are not limited to, Acinetobacter infections. Actinomycosis. African
sleeping sickness
(African trypanosomiasis). AIDS (Acquired immune deficiency syndrome),
Amebiasis,
Anaplasmosis, Anthrax, Arcanobacterium haemolyticum infection, Argentine
hemorrhagic
fever, Ascariasis, Aspergillosis, Astrovirus infection, Babesiosis, Bacillus
cereus infection.
Bacterial pneumonia, Bacterial vaginosis, Bacteroides infection,
Balantidiasis,
Baylisascaris infection. BK virus infection, Black piedra, Blastocystis
hominis infection,
Blastomycosis, Bolivian hemorrhagic fever, Borrelia infection, Botulism (and
Infant
botulism), Brazilian hemorrhagic fever, Brucellosis, Burkholderia infection,
Buruli ulcer,
Calicivirus infection (Norovirus and Sapovirus), Campylobacteriosis,
Candidiasis
(Moniliasis; Thrush), Cat-scratch disease, Cellulitis, Chagas Disease
(American
trypanosomiasis), Chancroid, Chickenpox, Chlamydia, Chlamydophila pncumoniac
infection, Cholera, Chromoblastomycosis, Clonorchiasis, Clostridium difficile
infection,
Coccidioidomycosis, Colorado tick fever, Common cold (Acute viral
rhinopharyngitis;
Acute coryza), Creutzfeldt-Jakob disease, Crimean-Congo hemorrhagic fever.
Cryptococcosis, Cryptosporidiosis, Cutaneous larva migrans, Cyclosporiasis,
Cysticercosis,
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Cytomegalovirus infection, Dengue fever, Dientamoebiasis, Diphtheria,
Diphyllobothriasis,
Dracunculiasis, Ebola hemorrhagic fever, Echinococcosis, Ehrlichiosis,
Enterobiasis
(Pinworm infection), Enterococcus infection, Enterovirus infection, Epidemic
typhus,
Erythema infectiosum (Fifth disease), Exanthem subitum, Fasciolopsiasis,
Fasciolosis,
Fatal familial insomnia, Filariasis, Food poisoning by Clostridium
perfringens, Free-living
amebic infection, Fusobacterium infection, Gas gangrene (Clostridial
myonecrosis),
Geotrichosis, Gerstmann-Straussler-Scheinker syndrome, Giardiasis, Glanders,
Gnathostomiasis, Gonorrhea, Granuloma inguinale (Donovanosis), Group A
streptococcal
infection, Group B streptococcal infection, Haemophilus influenzae infection,
Hand, foot
and mouth disease (HFMD), Hantavirus Pulmonary Syndrome, Helicobacter pylori
infec-
tion, Hemolytic-uremic syndrome, Hemorrhagic fever with renal syndrome,
Hepatitis A,
Hepatitis B, Hepatitis C, Hepatitis D, Hepatitis E, Herpes simplex,
Histoplasmosis, Hook-
worm infection, Human bocavirus infection, Human ewingii ehrlichiosis, Human
granulo-
cytic anaplasmosis, Human metapneumovirus infection, Human monocytic
ehrlichiosis,
Human papillomavirus infection, Human parainfluenza virus infection,
Hymenolepiasis.
Epstein-Barr Virus Infectious Mononucleosis (Mono), Influenza, Isosporiasis,
Kawasaki
disease, Keratitis, Kingella kingae infection, Kuru, Lassa fever,
Legionellosis (Legion-
naires' disease), Legionellosis (Pontiac fever), Leishmaniasis, Leprosy,
Leptospirosis,
Listeriosis, Lyme disease (Lyme borreliosis), Lymphatic filariasis
(Elephantiasis), Lym-
phocytic choriomeningitis. Malaria, Marburg hemorrhagic fever, Measles,
Melioidosis
(Whitmore's disease), Meningitis, Meningococcal disease, Metagonimiasis,
Microsporidiosis, Molluscum contagiosum, Mumps, Murine typhus (Endemic
typhus),
Mycoplasma pneumonia, Mycetoma, Myiasis, Neonatal conjunctivitis (Ophthalmia
neonatorum), (New) Variant Creutzfeldt-Jakob disease (vCJD, nvCJD),
Nocardiosis,
Onchocerciasis (River blindness), Paracoccidioidomycosis (South American
blastomycosis), Paragonimiasis, Pasteurellosis, Pediculosis capitis (Head
lice), Pediculosis
corporis (Body lice), Pediculosis pubis (Pubic lice, Crab lice), Pelvic
inflammatory disease,
Pertussis (Whooping cough), Plague. Pneumococcal infection, Pneumocystis
pneumonia,
Pneumonia, Poliomyelitis, Prevotella infection, Primary amoebic
meningoencephalitis,
Progressive multifocal leukoencephalopathy, Psittacosis, Q fever, Rabies, Rat-
bite fever,
Respiratory syncytial virus infection. Rhinosporidiosis, Rhinovirus infection,
Rickettsial
infection, Rickettsialpox, Rift Valley fever, Rocky mountain spotted fever,
Rotavirus
infection, Rubella, Salmonellosis, SARS (Severe Acute Respiratory Syndrome),
Scabies,
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Schistosomiasis, Sepsis, Shigellosis (Bacillary dysentery), Shingles (Herpes
zoster).
Smallpox (Variola), Sporotrichosis, Staphylococcal food poisoning,
Staphylococcal
infection, Strongyloidiasis, Syphilis, Taeniasis, Tetanus (Lockjaw), Tinea
barbae (Barber's
itch), Tinea capitis (Ringworm of the Scalp), Tinea corporis (Ringworm of the
Body),
Tinea cruris (Jock itch), Tinea manuum (Ringworm of the Hand), Tinea nigra,
Tinea pedis
(Athlete's foot), Tinea unguium (Onychomycosis), Tinea versicolor (Pityriasis
versicolor),
Toxocariasis (Ocular Larva Migrans), Toxocariasis (Visceral Larva Migrans),
Toxoplas-
mosis, Trichinellosis, Trichomoniasis, Trichuriasis (Whipworm infection),
Tuberculosis,
Tularemia, Ureaplasma urealyticum infection, Venezuelan equine encephalitis,
Venezuelan
hemorrhagic fever, Viral pneumonia, West Nile Fever, White piedra (Tinea
blanca),
Yersinia pseudotuberculosis infection, Yersiniosis, Yellow fever, Zygomycosis.
The cell binding molecule, which is more preferred to be an antibody described
in
this patent that are against pathogenic strains include, but are not limit,
Acinetobacter
baumannii, Actinomyces israclii, Actinomyccs gerencseriae and
Propionibacterium
propionicus, Trypanosoma brucei, HIV (Human immunodeficiency virus), Entamoeba
histolytica, Anaplasma genus, Bacillus anthracis. Arcanobacterium
haemolyticum, Junin
virus, Ascaris lumbricoides, Aspergillus genus, Astroviridae family, Babesia
genus,
Bacillus cereus, multiple bacteria, Bacteroides genus, Balantidium coli,
Baylisascaris
genus, BK virus, Piedraia hortae, Blastocystis hominis, Blastomyces
dermatitides,
Machupo virus, Borrelia genus, Clostridium botulinum, Sabia, Brucella genus,
usually
Burkholderia cepacia and other Burkholderia species, Mycobacterium ulcerans,
Caliciviridae family, Campylobacter genus, usually Candida albicans and other
Candida
species, Bartonella henselae, Group A Streptococcus and Staphylococcus,
Trypanosoma
cruzi, Haemophilus ducreyi, Varicella zoster virus (VZV), Chlamydia
trachomatis,
Chlamydophila pneumoniae, Vibrio cholerae, Fonsecaea pedrosoi, Clonorchis
sinensis,
Clostridium difficile, Coccidioides immitis and Coccidioides posadasii,
Colorado tick fever
virus, rhinoviruses. coronaviruses, CJD prion, Crimean-Congo hemorrhagic fever
virus,
Cryptococcus ncoformans, Cryptosporidium genus, Ancylostoma braziliense;
multiple
parasites, Cyclospora cayetanensis, Taenia solium, Cytomegalovirus, Dengue
viruses
(DEN-1, DEN-2, DEN-3 and DEN-4) ¨ Flaviviruses, Dientamoeba fragilis,
Corynebacterium diphtheriae, Diphyllobothrium, Dracunculus medinensis,
Ebolavirus,
Echinococcus genus, Ehrlichia genus, Enterobius vermicularis, Enterococcus
genus,
Enterovirus genus, Rickettsia prowazekii, Parvovirus B19, Human herpesvirus 6
and
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Human herpesvirus 7, Fasciolopsis buski, Fasciola hepatica and Fasciola
gigantica, FFT
prion, Filarioidea superfamily, Clostridium perfringens, Fusobacterium genus,
Clostridium
perfringens; other Clostridium species, Geotrichum candidum, GSS prion,
Giardia
intestinalis, Burkholderia mallei. Gnathostoma spinigerum and Gnathostoma
hispidum,
Neisseria gonorrhoeae, Klebsiella granulomatis, Streptococcus pyogenes,
Streptococcus
agalactiae, Haemophilus influenzae. Enteroviruses, mainly Coxsackie A virus
and
Enterovirus 71, Sin Nombre virus, Helicobacter pylori, Escherichia coli
0157:H7,
Bunyaviridae family, Hepatitis A Virus, Hepatitis B Virus, Hepatitis C Virus,
Hepatitis D
Virus, Hepatitis E Virus, Herpes simplex virus 1, Herpes simplex virus 2,
Histoplasma
capsulatum, Ancylostoma duodenale and Necator americanus, Hemophilus
influenzae.
Human bocavirus, Ehrlichia ewingii, Anaplasma phagocytophilum, Human
metapneumovirus, Ehrlichia chaffeensis, Human papillomavirus, Human
parainfluenza
viruses, Hymenolepis nana and Hymenolepis diminuta, Epstein-Barr Virus.
Orthomy-
xoviridac family, Isospora belli, Kingella kingac, Klebsiella pncumoniac,
Klebsiella
ozaenas, Klebsiella rhinoscleromotis, Kuru prion, Lassa virus, Legionella
pneumophila,
Legionella pneumophila, Leishmania genus, Mycobacterium leprae and
Mycobacterium
lepromatosis, Leptospira genus, Listeria monocytogenes, Borrelia burgdorferi
and other
Borrelia species, Wuchereria bancrofti and Brugia malayi, Lymphocytic
choriomeningitis
virus (LCMV), Plasmodium genus. Marburg virus, Measles virus, Burkholderia
pseudomallei, Neisseria meningitides, Metagonimus yokagawai, Microsporidia
phylum,
Molluscum contagiosum virus (MCV), Mumps virus, Rickettsia typhi, Mycoplasma
pneumoniae, numerous species of bacteria (Actinomycetoma) and fungi
(Eumycetoma),
parasitic dipterous fly larvae, Chlamydia trachomatis and Neisseria
gonorrhoeae, vCJD
prion, Nocardia asteroides and other Nocardia species, Onchocerca volvulus,
Paracoccidioides brasiliensis, Paragonimus westermani and other Paragonimus
species,
Pasteurella genus, Pediculus humanus capitis, Pediculus humanus corporis,
Phthirus pubis,
Bordetella pertussis, Yersinia pcstis, Streptococcus pneumoniac, Pneumocystis
jirovccii,
Poliovirus, Prevotella genus, Nacgleria fowleri, JC virus, Chlamydophila
psittaci, Coxiella
bumetii, Rabies virus, Streptobacillus moniliformis and Spirillum minus,
Respiratory
syncytial virus, Rhinosporidium seeberi, Rhinovirus, Rickettsia genus,
Rickettsia akari,
Rift Valley fever virus, Rickettsia rickettsii, Rotavirus, Rubella virus,
Salmonella genus,
SARS coronavirus, Sarcoptes scabiei, Schistosoma genus, Shigella genus,
Varicella zoster
virus, Variola major or Variola minor, Sporothrix schenckii, Staphylococcus
genus, Staph-
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ylococcus genus, Staphylococcus aureus, Streptococcus pyogenes, Strongyloides
stercoralis, Treponema pallidum, Taenia genus, Clostridium tetani,
Trichophyton genus,
Trichophyton tonsurans, Trichophyton genus, Epidermophyton flocco sum,
Trichophyton
rubrum, and Trichophyton mentagrophytes, Trichophyton rubrum, Hortaea
werneckii,
Trichophyton genus, Malassezia genus, Toxocara canis or Toxocara cati,
Toxoplasma
gondii, Trichinella spiralis, Trichomonas vaginalis, Trichuris trichiura,
Mycobacterium
tuberculosis, Francisella tularensis, Ureaplasma urealyticum, Venezuelan
equine encephali-
tis virus, Vibrio colerae, Guanarito virus, West Nile virus, Trichosporon
beigelii, Yersinia
pseudotuberculosis, Yersinia enterocolitica, Yellow fever virus, Mucorales
order
(Mucormycosis) and Entomophthorales order (Entomophthoramycosis), Pseudomonas
aeruginosa. Campylobacter (Vibrio) fetus. Aeromonas hydrophila, Edwardsiella
tarda,
Yersinia pestis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei,
Salmonella
typhimurium, Treponema pertenue, Treponema carateneum, Borrelia vincentii,
Borrelia
burgdorfcri, Leptospira icterohemorrhagiae, Pneumocystis carinii, BruceIla
abortus,
Brucella suis, BruceIla melitensis, Mycoplasma spp., Rickettsia prowazeki,
Rickettsia
tsutsugumushi, Clamydia spp.; pathogenic fungi (Aspergillus fumigatus, Candida
albicans,
Histoplasma capsulatum); protozoa (Entomoeba histolytica, Trichomonas tenas,
Trichomonas hominis, Tryoanosoma gambiense, Trypanosoma rhodesiense,
Leishmania
donovani, Leishmania tropica, Leishmania braziliensis, Pneumocystis pneumonia,
Plasmo-
dium vivax, Plasmodium falciparum, Plasmodium malaria); or Helminiths
(Schistosoma
japonicum, Schistosoma mansoni, Schistosoma haematobium, and hookworms).
Other antibodies as cell binding ligands used in this invention for treatment
of viral
disease include, but are not limited to, antibodies against antigens of
pathogenic viruses,
including as examples and not by limitation: Poxyiridae, Herpesviridae,
Adenoviridae,
Papovaviridae, Enteroviridae, Picornaviridae, Parvoviridae, Reoviridae,
Retroviridae,
influenza viruses, parainfluenza viruses, mumps, measles, respiratory
syncytial virus,
rubella, Arboviridae, Rhabdoviridae, Arenaviridae, Non-A/Non-B Hepatitis
virus,
Rhinoviridae, Coronaviridac, Rotoviridae. Oncovirus [such as, HBV
(Hepatocellular
carcinoma), HPV (Cervical cancer, Anal cancer), Kaposi's sarcoma-associated
herpesvirus
(Kaposi's sarcoma), Epstein-Barr virus (Nasopharyngeal carcinoma, Burkitt's
lymphoma,
Primary central nervous system lymphoma), MCPyV (Merkel cell cancer), SV40
(Simian
virus 40), HCV (Hepatocellular carcinoma), HTLV-I (Adult T-cell
leukemia/lymphoma)],
Immune disorders caused virus: [such as Human Immunodeficiency Virus (AIDS)];
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Central nervous system virus: [such as, ICV (Progressive multifocal
leukoencephalopathy),
MeV (Subacute sclerosing panencephalitis), LCV (Lymphocytic choriomeningitis),
Arbovirus encephalitis. Orthomyxoviridae (probable) (Encephalitis lethargica),
RV (Ra-
bies), Chandipura virus, Herpesviral meningitis, Ramsay Hunt syndrome type II;
Poliovirus
(Poliomyelitis, Post-polio syndrome), HTLV-I (Tropical spastic paraparesis)];
Cytomegal-
ovirus (Cytomegalovirus retinitis, HSV (Herpetic keratitis)); Cardiovascular
virus [such as
CBV (Pericarditis, Myocarditis)]; Respiratory system/acute viral
nasopharyngitis/viral
pneumonia: [Epstein-Barr virus (EB V infection/Infectious mononucleosis),
Cytomegalovi-
rus; SARS coronavirus (Severe acute respiratory syndrome) Orthomyxoviridae:
Influenzavirus A/B/C (Influenza/Avian influenza), Paramyxovirus: Human
parainfluenza
viruses (Parainfluenza), RSV (Human respiratory syncytial virus), hMPV];
Digestive
system virus [MuV (Mumps), Cytomegalovirus (Cytomegalovirus esophagitis);
Adenovi-
rus (Adenovirus infection); Rotavirus, Norovirus, Astrovirus, Coronavirus; HBV
(Hepatitis
B virus), CB V, HAV (Hepatitis A virus), HCV (Hepatitis C virus), HDV
(Hepatitis D
virus), HEY (Hepatitis E virus), HGV (Hepatitis G virus)]; Urogenital virus
[such as, BK
virus, MuV (Mumps)].
According to a further object, the present invention also concerns
pharmaceutical
compositions comprising the conjugate via the hydrophilic linkers of the
invention together
with a pharmaceutically acceptable carrier, diluent, or excipient for
treatment of cancers,
infections or autoimmune disorders. The method for treatment of cancers,
infections and
autoimmune disorders can be practiced in vitro, in vivo, or ex vivo. Examples
of in vitro
uses include treatments of cell cultures in order to kill all cells except for
desired variants
that do not express the target antigen; or to kill variants that express
undesired antigen.
Examples of ex vivo uses include treatments of hematopoietic stem cells (HSC)
prior to the
performance of the transplantation (HSCT) into the same patient in order to
kill diseased or
malignant cells. For instance, clinical ex vivo treatment to remove tumour
cells or lymphoid
cells from bone marrow prior to autologous transplantation in cancer treatment
or in
treatment of autoimmune disease, or to remove T cells and other lymphoid cells
from
allogencic bone marrow or tissue prior to transplant in order to prevent graft-
versus-host
disease, can be carried out as follows. Bone marrow is harvested from the
patient or other
individual and then incubated in medium containing serum to which is added the
conjugate
of the invention, concentrations range from about 1 pM to 0.1 mM, for about 30
minutes to
about 48 hours at about 37 'C. The exact conditions of concentration and time
of incubation
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(=dose) are readily determined by the skilled clinicians. After incubation the
bone marrow
cells are washed with medium containing serum and returned to the patient by
i.v. infusion
according to known methods. In circumstances where the patient receives other
treatment
such as a course of ablative chemotherapy or total-body irradiation between
the time of
harvest of the marrow and reinfusion of the treated cells, the treated marrow
cells are stored
frozen in liquid nitrogen using standard medical equipment.
For clinical in vivo use, the conjugate via the linkers of the invention will
be supplied
as solutions or as a lyophilized solid that can be redissolved in sterile
water for injection.
Examples of suitable protocols of conjugate administration are as follows.
Conjugates are
given weekly for 8 weeks as an i.v. bolus. Bolus doses are given in 50 to 500
ml of normal
saline to which human serum albumin (e.g. 0.5 to 1 mL of a concentrated
solution of
human serum albumin, 100 mg/mL) can be added. Dosages will be about 50 i.t.g
to 20
mg/kg of body weight per week, i.v. (range of 10 g to 200 mg/kg per
injection). 8 weeks
after treatment, the patient may receive a second course of treatment.
Specific clinical
protocols with regard to route of administration, excipients, diluents,
dosages, times, etc.,
can be determined by the skilled clinicians.
Examples of medical conditions that can be treated according to the in vivo or
ex
vivo methods of killing selected cell populations include malignancy of any
types of
cancer, autoimmune diseases, graft rejections, and infections (viral,
bacterial or parasite).
The amount of a conjugate which is required to achieve the desired biological
effect,
will vary depending upon a number of factors, including the chemical
characteristics, the
potency, and the bioavailability of the conjugates, the type of disease, the
species to which
the patient belongs, the diseased state of the patient, the route of
administration, all factors
which dictate the required dose amounts, delivery and regimen to be
administered.
In general terms. the conjugates via the linkers of this invention may be
provided in
an aqueous physiological buffer solution containing 0.1 to 10% w/v conjugates
for paren-
teral administration. Typical dose ranges are from 1 pg/kg to 0.1 g/kg of body
weight per
day; a preferred dose range is from 0.01 mg/kg to 20 mg/kg of body weight per
day or an
equivalent dose in a human child. The preferred dosage of drug to be
administered is likely
to depend on such variables as the type and extent of progression of the
disease or disorder,
the overall health status of the particular patient, the relative biological
efficacy of the
compound selected, the formulation of the compound, the route of
administration (intrave-
nous, intramuscular, or other), the pharmacokinetic properties of the compound
by the
chosen delivery route, and the speed (bolus or continuous infusion) and
schedule of administrations
(number of repetitions in a given period of time).
The conjugates via the linkers of the present invention are also capable of
being
administered in unit dose forms, wherein the term "unit dose" means a single
dose which is capable
of being administered to a patient, and which can be readily handled and
packaged, remaining as a
physically and chemically stable unit dose comprising either the active
conjugate itself, or as a
pharmaceutically acceptable composition, as described hereinafter. As such,
typical total
daily/weekly/biweekly/monthly dose ranges are from 0.01 to 100 mg/kg of body
weight. By way of
general guidance, unit doses for humans range from 1 mg to 3000 mg per day, or
per week, per two
week or per month. Preferably the unit dose range is from 1 to 500 mg
administered one to four
times a day, and even more preferably from 10 mg to 500 mg, once a day.
Conjugates provided
herein can be formulated into pharmaceutical compositions by admixture with
one or more
pharmaceutically acceptable excipients. Such unit dose compositions may be
prepared for use by
oral administration, particularly in the form of tablets, simple capsules or
soft gel capsules; or
.. intranasal, particularly in the form of powders, nasal drops, or aerosols;
or dermally, for example,
topically in ointments, creams, lotions, gels or sprays, or via trans-dermal
patches.
DRUGS/CYTOTOXIC AGENTS
Drugs that can be conjugated to a cell-binding molecule in the present
invention are small
molecule drugs including cytotoxic agents, which can be linked to or after
they are modified for
linkage to the cell-binding agent. A "small molecule drug" is broadly used
herein to refer to an
organic, inorganic, or organometallic compound that may have a molecular
weight of for example
100 to 1800, more suitably from 120 to 1400. Small molecule drugs are well
characterized in the
art, such as in W005058367A2, and in U.S. Patent No. 4,956,303. The drugs
include known drugs
and those that may become known drugs.
Drugs that are known include, but not limited to,
1). Chemotherapeutic agents: a). Alkylating agents: such as Nitrogen mustards:
chlorambucil, chlornaphazine, cyclophosphamide, dacarbazine, estramustine,
ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, mannomustine,
mitobronitol,
melphalan, mitolactol, pipobroman, novembichin, phenesterine, prednimustine,
thiotepa,
trofosfamide, uracil mustard; CC-1065 (including its adozelesin, carzelesin
and bizelesin
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synthetic analogues); Duocarmycin (including the synthetic analogues, KW-2189
and CBI-
TMI); Benzodiazepine dimers (e.g., dimmers of pyrrolobenzodiazepine (PBD) or
tomaymycin, indolinobenzodiazepines, imidazobenzothiadiazepines, or
oxazolidino-
benzodiazepines); Nitrosoureas: (carmustine, lomustine, chlorozotocin,
fotemustine,
nimustine, ranimustine); Alkylsulphonates: (busulfan, treosulfan, improsulfan
and
piposulfan); Triazenes: (dacarbazine); Platinum containing compounds:
(carboplatin,
cisplatin, oxaliplatin); aziridines, such as benzodopa, carboquone,
meturedopa, and
uredopa; ethylenimines and methylamelamines including altretamine,
triethylenemel-
amine, trietylenephosphoramide. triethylenethiophosphaoramide and
trimethylolomelamine]; b). Plant Alkaloids: such as Vinca alkaloids:
(vincristine, vinblas-
tine, vindesine, vinorelbine, navelbin); Taxoids: (paclitaxel, docetaxol) and
their analogs.
Maytansinoids (DM1, DM2, DM3, DM4, maytansine and ansamitocins) and their
analogs,
cryptophycins (particularly cryptophycin 1 and cryptophycin 8); epothilones,
eleutherobin,
discodermolide, bryostatins, dolostatins, auristatins, tubulysins,
cephalostatins;
pancratistatin; a sarcodictyin; spongistatin; c). DNA Topoisomerase
Inhibitors: such as
[Epipodophyllins: (9-aminocamptothecin, camptothecin, crisnatol, daunomycin,
etoposide,
etopo side phosphate, irinotecan, mitoxantrone, novantrone, retinoic acids
(retinols),
teniposide, topotecan, 9-nitrocamptothecin (RFS 2000)); mitomycins: (mitomycin
C)]; d).
Anti-metabolites: such as {[Anti-folate: DHFR inhibitors: (methotrexate,
trimetrexate,
denopterin, pteropterin, aminopterin (4-aminopteroic acid) or the other folic
acid ana-
logues); IMP dehydrogenase Inhibitors: (mycophenolic acid, tiazofurin,
ribavirin, EICAR);
Ribonucleotide reductase Inhibitors: (hydroxyurea, deferoxamine)]; [Pyrimidine
analogs:
Uracil analogs: (ancitabine, azacitidine. 6-azauridine, capecitabine (Xeloda),
carmofur,
cytarabine, dideoxyuridine, doxifluridine, enocitabine, 5-Fluorouracil,
floxuridine,
ratitrexed (Tomudex)); Cytosine analogs: (cytarabine, cytosine arabinoside,
fludarabine);
Purine analogs: (azathioprine, fludarabine, mercaptopurine, thiamiprine,
thioguanine)];
folic acid replenisher, such as frolinic acid I; c). Hormonal therapies: such
as {Receptor
antagonists: {Anti-estrogen: (mcgestrol, raloxifene, tamoxifen); LHRH
agonists: (aoscrclin,
leuprolide acetate); Anti-androgens: (bicalutamidc, flutamide, calusterone,
dromostanolonc
propionate, epitiostanol, goserelin, leuprolide, mepitiostane, nilutamide,
testolactone,
trilostane and other androgens inhibitors)]; Retinoids/Deltoids: [Vitamin D3
analogs: (CB
1093, EB 1089 KH 1060, cholecalciferol, ergocalciferol); Photodynamic
therapies:
(verteporfin, phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A);
Cytokines:
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(Interferon-alpha, Interferon-gamma, tumor necrosis factor (TNFs), human
proteins con-
taining a TNF domain)]}; f). Kinase inhibitors, such as BIBW 2992 (anti-
EGFR/Erb2),
imatinib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib,
nilotinib, lapatinib,
axitinib, pazopanib. vandetanib. E7080 (anti-VEGFR2), mubritinib, ponatinib
(AP24534),
bafetinib (INNO-406). bosutinib (SKI-606), cabozantinib, vismodegib, iniparib,
ruxolitinib,
CYT387, axitinib. tivozanib, sorafenib, bevacizumab, cetuximab, Trastuzumab,
Ranibizumab. Panitumumab, ispinesib; g). antibiotics, such as the enediyne
antibiotics (e.g.
calicheamicins, especially calicheamicin y1,61, al and 131, see, e.g., J. Med.
Chem., 39
(11), 2103-2117 (1996), Angew Chem Intl. Ed. Engl. 33:183-186 (1994);
dynemicin,
including dynemicin A and deoxydynemicin; esperamicin, kedarcidin, C-1027,
maduropeptin, as well as neocarzinostatin chromophore and related
chromoprotein
enediyne antiobiotic chromomophores), aclacinomysins, actinomycin,
authramycin,
azaserine, blcomycins, cactinomycin, carabicin, carminomycin, carzinophilin;
chromomycins, dactinomycin, daunorubicin, dctorubicin, 6-diazo-5-oxo-L-
norleucine,
doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-
doxorubicin and deoxydoxorubicin, epirubicin, esorubicin, idarubicin,
marcellomycin,
nitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin,
potfiromycin,
puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex,
zinostatin, zorubicin; f). Others: such as Polyketides (acetogenins),
especially bullatacin
and bullatacinone; gemcitabine, epoxomicins (e. g. carfilzomib), bortezomib,
thalidomide,
lenalidomide, pomalidomide, tosedostat, zybrestat, PLX4032, STA-9090,
Stimuvax,
allovectin-7, Xegeva, Provenge, Yervoy, Isoprenylation inhibitors (such as
Lovastatin).
Dopaminergic neurotoxins (such as 1-methyl-4-phenylpyridinium ion), Cell cycle
inhibi-
tors (such as staurosporine), Actinomycins (such as Actinomycin D,
dactinomycin),
Bleomycins (such as bleomycin A2, bleomycin B2, peplomycin), Anthracyclines
(such as
daunorubicin, doxorubicin (adriamycin), idarubicin, epirubicin, pirarubicin,
zorubicin,
mtoxantronc, MDR inhibitors (such as verapamil). Ca2+ATPase inhibitors (such
as
thapsigargin), Histonc deacetylase inhibitors (Vorinostat, Romidcpsin,
Panobinostat,
Valproic acid, Mocctinostat (MGCD0103), Belinostat, PCI-24781, Entinostat,
SB939,
Resminostat, Givinostat, AR-42, CUDC-101, sulforaphane, Trichostatin A) ;
Thapsigargin,
Celecoxib, glitazones, epigallocatechin gallate, Disulfiram, Salinosporamide
A.; Anti-
adrenals, such as aminoglutethimide, mitotane, trilostane; aceglatone;
aldophosphamide
glycoside; aminolevulinic acid; amsacrine; arabinoside, bestrabucil;
bisantrene; edatraxate;
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defofamine; demecolcine; di aziquone; eflornithine (DFMO), elfomithine;
elliptinium
acetate, etoglucid; gallium nitrate; gacytosine, hydroxyurea; ibandronate,
lentinan;
lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin;
phenamet;
pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK ;
razoxane; rhizoxin;
sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2',2"-
trichlorotriethylamine;
trichothecenes (especially T-2 toxin, verrucarin A, roridin A and anguidine);
urethane,
siRNA, antisense drugs, and a nucleolytic enzyme.
2). An anti-autoimmune disease agent includes, but is not limited to,
cyclosporine,
cyclosporine A, aminocaproic acid, azathioprine, bromocriptine. chlorambucil,
chloroquine, cyclophosphamide, corticosteroids (e.g. amcinonide,
betamethasone,
budesonide, hydrocortisone, flunisolide, fluticasone propionate, fluocortolone
danazol,
dexamethasone, Triamcinolone acetonide, beclometasone dipropionate), DHEA,
enancrcept, hydroxychloroquinc, infliximab, meloxicam, methotrexate, mofctil,
mycophenylatc, prednisonc, sirolimus, tacrolimus.
3). An anti-infectious disease agent includes, but is not limited to, a).
Aminoglyco-
sides: amikacin, astromicin, gentamicin (netilmicin, sisomicin, isepamicin),
hygromycin B,
kanamycin (amikacin, arbekacin, bekanamycin, dibekacin, tobramycin). neomycin
(framycetin, paromomycin, ribostamycin), netilmicin, spectinomycin,
streptomycin,
tobramycin, verdamicin; b). Amphenicols: azidamfenicol, chloramphenicol,
florfenicol,
thiamphenicol; c). Ansamycins: geldanamycin, herbimycin; d). Carbapenems:
biapenem,
doripenem, ertapenem, imipenern/cilastatin, meropenem, panipenem; e). Cephems:
carbacephem (loracarbef), cefacetrile, cefaclor, cefradine, cefadroxil,
cefalonium,
cefaloridine, cefalotin or cefalothin, cefalexin, cefaloglycin, cefamandole.
cefapirin,
cefatrizine, cefazaflur, cefazedone, cefazolin, cefbuperazone, cefcapene,
cefdaloxime,
cefepime. cefminox, cefoxitin, cefprozil, cefroxadine, ceftezole, cefuroxime,
cefixime,
cefdinir, cefditoren, cefepime, cefetamet, cefmenoxime, cefodizime, cefonicid,
cefoperazone, ceforanidc, cefotaxime, ccfotiam, cefozopran, ccphalexin,
cefpimizole,
cdpiramide, ccfpirome, cefpodoxime, ccfprozil, cefquinome, cefsulodin,
ceftazidime,
cefteram, ceftibuten, ccftiolene, ceftizoxime, ceftobiprolc, ceftriaxonc,
cefuroxime,
cefuzonam, cephamycin (cefoxitin, cefotetan, cefmetazole), oxacephem
(flomoxef,
latamoxef); Glycopeptides: bleomycin, vancomycin (oritavancin,
telavancin),
teicoplanin (dalbavancin), ramoplanin; g). Glycylcyclines: e. g. tigecycline;
g).P-
Lactamase inhibitors: penam (sulbactam, tazobactam), clavam (clavulanic acid);
i).
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Lincosamides: clindamycin, lincomycin; j). Lipopeptides: daptomycin, A54145,
calcium-
dependent antibiotics (CDA); k). Macrolides: azithromycin, cethromycin,
clarithromycin,
dirithromycin, erythromycin, flurithromycin, josamycin, ketolide
(telithromycin,
cethromycin), midecamycin, miocamycin, oleandomycin, rifamycins (rifampicin,
rifampin,
rifabutin, rifapentine), rokitamycin, roxithromycin, spectinomycin,
spiramycin, tacrolimus
(FK506), troleandomycin, telithromycin; 1). Monobactams: aztreonam, tigemonam;
m).
Oxazolidinones: linezolid; n). Penicillins: amoxicillin, ampicillin
(pivampicillin, hetacillin,
bacampicillin, metampicillin. talampicillin), azidocillin, azlocillin,
benzylpenicillin,
benzathine benzylpenicillin, benzathine phenoxymethyl-penicillin,
clometocillin, procaine
benzylpenicillin, carbenicillin (carindacillin), cloxacillin, dicloxacillin,
epicillin,
flucloxacillin, mecillinam (pivmecillinam), mezlocillin, meticillin,
nafcillin, oxacillin,
penamecillin, penicillin, pheneticillin, phenoxymethylpenicillin,
piperacillin, propicillin,
sulbenicillin, tcmocillin, ticarcillin; o). Polypeptides: bacitracin,
colistin, polymyxin B; p).
Quinolones: alatrofloxacin, balofloxacin, ciprofloxacin, clinafloxacin,
danofloxacin,
difloxacin, enoxacin, enrofloxacin, floxin, garenoxacin, gatifloxacin,
gemifloxacin,
grepafloxacin, kano trovafloxacin, levofloxacin, lomefloxacin, marbofloxacin,
moxifloxacin, nadifloxacin. norfloxacin, orbifloxacin, ofloxacin, pefloxacin,
trovafloxacin.
grepafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin,
trovafloxacin; q).
Streptogramins: pristinamycin, quinupristin/dalfopristin); r). Sulfonamides:
mafenide,
prontosil, sulfacetamide, sulfamethizole, sulfanilimide. sulfasalazine,
sulfisoxazole, trime-
thoprim, trimethoprim-sulfamethoxazole (co-trimoxazole); s). Steroid
antibacterials: e.g.
fusidic acid; t). Tetracyclines: doxycycline, chlortetracycline, clomocycline,
demeclocycline, lymecycline, meclocycline, metacycline, minocycline,
oxytetracycline,
penimepicycline, rolitetracycline, tetracycline, glycylcyclines (e.g.
tigecycline); u). Other
types of antibiotics: annonacin, arsphenamine, bactoprenol inhibitors
(Bacitracin),
DADAL/AR inhibitors (cycloserine), dictyostatin, discodermolide. eleutherobin.
epothilone, ethambutol, etoposide, faropenem, fusidic acid, furazolidone,
isoniazid,
laulimalide, metronidazole, mupirocin, mycolactone, NAM synthesis inhibitors
(c. g.
fosfomycin), nitrofurantoin, paclitaxcl, platcnsimycin, pyrazinamidc,
quinupristin/dalfopristin, rifampicin (rifampin), tazobactam tinidazole,
uvaricin;
4). Anti-viral drugs: a). Entry/fusion inhibitors: aplaviroc, maraviroc,
vicriviroc,
gp41 (enfuvirtide), PRO 140, CD4 (ibalizumab); b). Integrase inhibitors:
raltegravir,
elvitegravir, globoidnan A; c). Maturation inhibitors: bevirimat, vivecon; d).
Neuramini-
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dase inhibitors: oseltamivir, zanamivir, peramivir; e). Nucleosides
&nucleotides: abacavir,
aciclovir, adefovir, amdoxovir, apricitabine, brivudine, cidofovir, clevudine,
dexelyncitabine, didanosine (ddI), elvucitabine, emtricitabine (FTC),
entecavir,
famciclovir, fluorouracil (5-FU), 3.-fluoro-substituted 2', 3'-
dideoxynucleoside analogues
(e.g. 3'-fluoro-2',3'-dideoxythymidine (FLT) and 3'-fluoro-2',3'-
dideoxyguanosine (FLG),
fomivirsen, ganciclovir, idoxuridine, lamivudine (3TC), 1-nucleosides (e.g. /8-
1-thymidine
and fl-1-2.-deoxycytidine), penciclovir, racivir, ribavirin, stampidine.
stavudine (d4T).
taribavirin (viramidine), telbivudine, tenofovir, trifluridine valaciclovir,
valganciclovir,
zalcitabine (ddC), zidovudine (AZT); f). Non-nucleosides: amantadine,
ateviridine.
capravirine, diarylpyrimidines (etravirine, rilpivirine), delavirdine,
docosanol, emivirine,
efavirenz, foscamet (phosphonoformic acid), imiquimod, interferon alfa,
loviride,
lodenosine, methisazone, nevirapine, NOV-205, peginterferon alfa,
podophyllotoxin,
rifampicin, rimantadine, resiquimod (R-848), tromantadine; g). Protease
inhibitors:
amprenavir, atazanavir, boceprevir, darunavir, fosamprenavir, indinavir,
lopinavir,
nelfinavir, pleconaril, ritonavir, saquinavir, telaprevir (VX-950),
tipranavir; h). Other types
of anti-virus drugs: abzyme, arbidol, calanolide a, ceragenin, cyanovirin-n,
diarylpyrimidines, epigallocatechin gallate (EGCG), foscamet, griffithsin,
taiibavirin
(viramidine), hydroxyurea, KP-1461, miltefosine, pleconaril, portmanteau
inhibitors,
ribavirin, seliciclib.
5). The drugs used for conjugates via a hydrophilic linker of the present
invention al-
so include radioisotopes. Examples of radioisotopes (radionuclides) are H,
11C. 14C, 18F,
32 35 64 68 86 99 111 123 174 125 131 133 177 211 213 =
P, S. Cu, Ga, Y, Tc, In, I, L Xe, Lu, At, or
Radioi-
sotope labeled antibodies are useful in receptor targeted imaging experiments
or can be for
targeted treatment such as with the antibody-drug conjugates of the invention
(Wu et al
(2005) Nature Biotechnology 23(9): 1137-1146). The cell binding molecules,
e.g. an
antibody can be labeled with ligand reagents through the hydrophilic linkers
of the present
patent that bind, chelate or otherwise complex a radioisotope metal, using the
techniques
described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al,
Ed. Wiley-
Interscience, New York, N.Y., Pubs. (1991). Chelating ligands which may
complex a metal
ion include DOTA, DOTP, DOTMA, DTPA and TETA (Macrocyclics, Dallas, Tex.).
6). The pharmaceutically acceptable salts, acids or derivatives of any of the
above
drugs.
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In another embodiment, the drug in the Formula (II) and (IV) can a chromophore
molecule, for which the conjugate can be used for detection, monitoring, or
study the
interaction of the cell binding molecule with a target cell. Chromophore
molecules are a
compound that have the ability to absorb a kind of light, such as UV light,
floresceui light,
IR light, near a light, visual light; A chromatophore molecule includes a
class or subclass
of xanthophores, erythrophores, iriclophores, leucophores, melanophores; and
cyanophores;
a class or subclass of fluorophore molecules which are fluorescent chemical
compounds re-
mitting- light upon light; a class or subclass of visual phototransduction
molecules; a class
or subclass of photophore molecules; a class or subclass of luminescence
molecules; and
a class or subclass of luciferin compounds.
The chromophore molecule can be selected from, but not limited, Non-protein
organ-
ic fluorophores, such as: Xanthene derivatives (fluorescein, rhodamine, Oregon
green,
eosin, and Texas red); Cyanine derivatives: (cyanine, indocarbocyanine,
oxacarbocyaninc,
thiacarbocyaninc, and merocyanine); Squaraine derivatives and ring-substituted
squaraines,
including Seta, SeTau, and Square dyes; Naphthalene derivatives (dansyl and
prodan
derivatives); Coumarin derivatives; Oxadiazole derivatives (pyridyloxazole,
nitrobenzoxadiazole and benzoxadiazole); Anthracene derivatives
(anthraquinones, includ-
ing DRAQ5, DRAQ7 and CyTRAK Orange); Pyrene derivatives (cascade blue, etc);
Oxazine derivatives (Nile red, Nile blue, cresyl violet, oxazine 170 etc).
Acridine deriva-
lives (proflavin, acridine orange, acridine yellow etc). Arylmethine
derivatives (auramine,
crystal violet, malachite green). Tetrapyrrole derivatives (porphin,
phthalocyanine, biliru-
bin).
Or a chromophore molecule can be selected from any analogs and derivatives of
the
following fluorophore compounds: CF dye (Biotium), DRAQ and CyTRAK probes
(BioStatus), BODIPY (Invitrogen), Alexa Fluor (Invitrogen), DyLight Fluor
(Thermo
Scientific, Pierce), Atto and Tracy (Sigma Aldrich), FluoProbes (Interchim),
Abberior Dyes
(Abberior), DY and MegaStokes Dyes (Dyomics), Sulfo Cy dyes (Cyandye), HiLyte
Fluor
(AnaSpec), Seta, SeTau and Square Dyes (SETA BioMedicals), Quasar and Cal
Fluor dyes
(Biosearch Technologies), SureLight Dyes (APC, RPEPerCP,
Phycobilisomes)(Columbia
Biosciences), APC, APCXL, RPE, BPE (Phyco-Biotech);
Examples of the widely used fluorophore compounds which are reactive or
conjugatable with the linkers of the invention are: Allophycocyanin (APC),
Aminocoumarin, APC-Cy7 conjugates, BODIPY-FL, Cascade Blue, Cy2, Cy3, Cy3.5,
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Cy3B, Cy5, Cy5.5. Cy7, Fluorescein, FluorX, Hydroxycoumarin, Lissamine
Rhodamine B,
Lucifer yellow, Methoxycoumarin, NBD, Pacific Blue, Pacific Orange. PE-Cy5
conjugates.
PE-Cy7 conjugates, PerCP, R-Phycoerythrin(PE), Red 613, Seta-555-Azide, Seta-
555-
DBCO, Seta-555-NHS, Seta-580-NHS, Seta-680-NHS, Seta-780-NHS, Seta-APC-780,
Seta-PerCP-680, Seta-R-PE-670, SeTau-380-NHS, SeTau-405-Maleimide, SeTau-405-
NHS, SeTau-425-NHS, SeTau-647-NHS, Texas Red, TRITC, TruRed, X-Rhodamine.
The fluorophore compounds that can be linked to the linkers of the invention
for
study of nucleic acids or proteins are selected from the following compounds
or their
derivatives: 7-AAD (7-aminoactinomycin D, CG-selective), Acridine Orange,
Chromomycin A3, CyTRAK Orange (Biostatus, red excitation dark), DAPI. DRAQ5,
DRAQ7, Ethidium Bromide, Hoechst33258, Hoechst33342, LDS 751, Mithramycin,
PropidiumIodide (PI), SYTOX Blue, SYTOX Green, SYTOX Orange, Thiazole Orange,
TO-PRO: Cyaninc Monomer, TOTO-1, TO-PRO-1, TOTO-3, TO-PRO-3, YOSeta-1,
YOYO-1. The fluorophore compounds that can be linked to the linkers of the
invention for
study cells are selected from the following compounds or their derivatives:
DCFH
(2'7'Dichorodihydro-fluorescein, oxidized form), DHR (Dihydrorhodamine 123,
oxidized
form, light catalyzes oxidation), Fluo-3 (AM ester. pH > 6), Fluo-4 (AM ester.
pH 7.2),
Indo-1 (AM ester, low/high calcium (Ca2+)), SNARF(pH 6/9). The preferred
fluorophore
compounds that can be linked to the linkers of the invention for study
proteins/antibodies
are selected from the following compounds or their derivatives:
Allophycocyanin(APC),
AmCyanl (tetramer, Clontech), AsRed2 (tetramer, Clontech), Azami Green
(monomer,
MBL), Azurite, B-phycoerythrin(BPE), Cerulean, CyPet, DsRed monomer
(Clontech),
DsRed2 ("RFP", Clontech), EBFP, EBFP2, ECFP, EGFP (weak dimer, Clontech),
Emerald
(weak dimer, Invitrogen), EYFP (weak dimer, Clontech), GFP (S65A mutation).
GFP
(S65C mutation), GFP (S65L mutation), GFP (S65T mutation), GFP (Y66F
mutation), GFP
(Y66H mutation), GFP (Y66W mutation), GFPuv. HcRedl, J-Red, Katusha, Kusabira
Orange (monomer, MBL), mCFP, mCherry, mCitrine, Midoriishi Cyan (dimer, MBL),
mKate (TagFP635, monomer, Evrogen). mKeima-Red (monomer, MBL), mKO, mOrange,
mPlum, mRaspberry, mRFP1 (monomer, Tsicn lab), mStrawberry, mTFP1,
mTurquoise2,
P3 (phycobilisome complex), Peridinin Chlorophyll (PerCP), R-
phycoerythrin(RPE), T-
Sapphire, TagCFP (dimer, Evrogen), TagGFP (dimer, Evrogen). TagRFP (dimer,
Evrogen),
TagYFP (dimer, Evrogen), tdTomato (tandem dimer), Topaz, TurboFP602 (dimer,
Evrogen), TurboFP635 (dimer. Evrogen), Turbo GFP (dimer, Evrogen), TurboRFP
(dimer,
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Evrogen), TurboYFP (dimer, Evrogen), Venus, Wild Type GFP, YPet, ZsGreenl
(tetramer,
Clontech), ZsYellowl (tetramer, Clontech).
In yet another embodiment, the preferred cytotoxic agents that conjugated to a
cell-
binding molecule via a hydrophilic linker of this patent are tubulysins,
maytansinoids,
taxanoids (taxanes), CC-1065 analogs, daunorubicin and doxorubicin compounds,
benzodi-
azepine dimers (e.g., dimers of pyrrolobenzodiazepine (PBD), tomaymycin,
anthramycin,
indolinobenzodiazepines, imidazobenzothiadiazepines, or oxazolidino-
benzodiazepines),
calicheamicins and the enediyne antibiotics, actinomycin, azaserines,
bleomycins,
epirubicin, tamoxifen, idarubicin, dolastatins, auristatins (e.g. monomethyl
auristatin E,
MMAE , MMAF, auristatin PYE, auristatin TP, Auristatins 2-AQ, 6-AQ, EB (AEB),
and
EFP (AEFP)), duocarmycins, thiotepa, vincristines, hemiasterlins, nazumamides,
microginins, radiosumins, alterobactins, microsclerodermins, theonellamides,
esperamicins,
PNU-159682, and their analogues and derivatives above thereof.
Tubulysins that are preferred for conjugation in the present invention are
well known
in the art and can be isolated from natural sources according to known methods
or prepared
synthetically according to known methods (e. g. Balasubramanian, R.; et al. J.
Med. Chem.,
2009, 52, 238-240. Wipf, P.; et al. Org. Lett., 2004, 6, 4057-4060. Pando, O.;
et al. J. Am.
Chem. Soc., 2011, 133, 7692-7695. Reddy, J. A.; et al. Mol. Pharmaceutics,
2009,6,
1518-1525. Raghavan, B.; et al. J. Med. Chem., 2008, 51, 1530-1533. Patterson,
A. W.; et
al. J. Org. Chem., 2008, 73, 4362-4369. Pando, 0.; et al. Org. Lett., 2009,
11(24), pp
5567-5569. Wipf. P.; et al. Org. Lett., 2007, 9 (8), 1605-1607. Friestad, G.
K.; Org. Lett.,
2004, 6, pp 3249-3252. Hillary M. Peltier, H. M.; et al. J. Am. Chem. Soc.,
2006, 128,
16018-16019. Chandrasekhar, S.; etal. J. Org. Chem., 2009, 74, 9531-9534. Liu,
Y.; et al.
Mol. Pharmaceutics, 2012, 9, 168 175. Friestad, G. K.; et al. Org. Lett.,
2009, 11, 1095
1098. Kubicek, K.; et al., Angew Chem Int Ed Engl, 2010. 49: p. 4809-12. Chai,
Y.; et al.,
Chem Biol, 2010, 17: 296-309. Ullrich, A.; et al., Angew Chem Int Ed Engl,
2009, 48,
4422-5. Sani, M.; et al. Angew Chem Int Ed Engl, 2007, 46, 3526-9. Domling,
A.; et al.,
Angew Chem Int Ed Engl, 2006. 45, 7235-9. Patent applications: Zanda, M. ; et
al, Can.
Pat. Appl. CA 2710693 (2011). Chai, Y.; et al. Eur. Pat. Appl. 2174947 (2010),
PCT WO
2010034724. Leamon, C.; et al, PCT WO 2010033733, WO 2009002993. Ellman, J.;
et al,
PCT WO 2009134279; PCT WO 2009012958, US appl. 20110263650,20110021568,
Matschiner, G.; et al, PCT WO 2009095447.Vlahov, I.; et al, PCT WO 2009055562,
WO
2008112873. Low, P.; et al, PCT WO 2009026177. Richter, W., PCT WO 2008138561.
54
CA 02989269 2017-12-12
WO 2015/151078 PCT/IB2015/054521
Kjems, J.; et al, PCT WO 2008125116. Davis, M.; et al, PCT WO 2008076333.
Diener, J.;
et al, U.S. Pat. Appl. 20070041901, WO 2006096754. Matschiner, G.; et al, PCT
WO
2006056464. Vaghefi, F.; et al, 5 PCT WO 2006033913. Doemling, A., Ger. Offen.
DE
102004030227; PCT WO 2004005327; WO 2004005326; W02004005269. Stanton. M.; et
al, U.S. Pat. Appl. Publ. 20040249130. Hoefle, G.; et al, Ger. Offen. DE
10254439 ; DE
10241152; DE 10008089. Leung, D.; eta!, WO 2002077036. Reichenbach, H.; eta!,
Ger.
Offen. DE 19638870; Wolfgang, R.; US 20120129779, Chen, H.,US app!.
20110027274.
The preferred structure of tubulysins for conjugation of cell binding
molecules are de-
scribed in the patent application of PCT/IB2012/053554
Calicheamicins and their related enediyne antibiotics that are preferred for
cell-
binding molecule-drug conjugates of this patent are described in: Nicolaou, K.
C. et al,
Science 1992. 256, 1172-1178; Proc. Natl. Acad. Sci USA. 1993, 90, 5881-5888),
U.S.
Patent Nos. 4,970,198; 5,053,394; 5,108,912; 5,264,586; 5.384,412; 5,606,040;
5,712,374;
5,714,586; 5,739,116; 5,770,701; 5,770,710; 5,773,001; 5,877,296; 6,015,562;
6,124,310;
8,153.768.
Maytansinoids that are preferred to be used in the present invention including
maytansinol and maytansinol analogues are described in U.S. Patent Nos.
4,256,746,
4,361,650, 4,307,016, 4,294,757, 4.294,757, 4,371,533, 4,424,219, 4,331,598,
4,450,254,
4,364,866, 4,313,946, 4,315,929 4,362,663, 4,322,348, 4,371,533, 4.424,219,
5,208,020,
5,416,064, 5.208,020; 5,416,064; 6,333.410; 6,441,163; 6.716,821, 7,276,497,
7,301,019,
7,303,749, 7,368,565, 7,411,063, 7,851,432, and 8,163,888.
Taxanes, which includes Paclitaxel (Taxol), a cytotoxic natural product, and
docetaxel (Taxotere), a semi-synthetic derivative, and their analogs which are
preferred for
conjugation via the hydrophilic linkers of the present patent are exampled
in:. K C.
Nicolaou et al., J. Am. Chem. Soc. 117, 2409-2420, (1995); Ojima et al. J.
Med. Chem.
39:3889-3896 (1996); 40:267-278 (1997); 45, 5620-5623 (2002); Ojima et al.,
Proc. Natl.
Acad. Sci., 96:4256-4261 (1999; Kim et al.. Bull. Korean Chem. Soc., 20, 1389-
1390
(1999); Miller, et al. J. Med. Chem., 47, 4802-4805(2004); U.S. Patent No.
5,475,011
5,728,849, 5,811,452; 6,340,701; 6,372,738; 6,391,913, 6.436,931; 6,589,979;
6,596,757;
6,706,708; 7,008,942; 7,186,851; 7,217,819; 7,276,499; 7,598,290; and
7,667,054.
CC-1065 analogues and doucarmycin analogs are also preferred to be used for a
con-
jugate with the hydrophilic linkers of the present patent. The examples of the
CC-1065
analogues and doucarmycin analogs as well as their synthesis are described in:
CA 02989269 2017-12-12
WO 2015/151078 PCT/IB2015/054521
e.g.Warpehoski et al, J. Med. Chem. 31:590-603 (1988), D. Boger et al., J.
Org. Chem; 66;
6654-6661. 2001; U.S. Patent Nos: 4169888, 4391904, 4671958, 4816567, 4912227,
4923990, 4952394, 4975278, 4978757, 4994578, 5037993, 5070092, 5084468,
5101038,
5117006, 5137877, 5138059, 5147786, 5187186, 5223409, 5225539, 5288514,
5324483,
5332740, 5332837, 5334528. 5403484, 5427908, 5475092, 5495009, 5530101,
5545806,
5547667, 5569825, 5571698, 5573922, 5580717, 5585089, 5585499, 5587161,
5595499,
5606017, 5622929, 5625126, 5629430, 5633425, 5641780, 5660829, 5661016,
5686237,
5693762, 5703080, 5712374, 5714586, 5739116, 5739350, 5770429, 5773001,
5773435,
5786377 5786486, 5789650, 5814318, 5846545, 5874299, 5877296, 5877397,
5885793,
5939598, 5962216, 5969108, 5985908, 6060608, 6066742, 6075181, 6103236,
6114598,
6130237, 6132722, 6143901. 6150584, 6162963, 6172197, 6180370, 6194612,
6214345,
6262271, 6281354, 6310209, 6329497, 6342480, 6486326, 6512101, 6521404,
6534660,
6544731, 6548530, 6555313. 6555693, 6566336, 6,586,618, 6593081, 6630579,
6,756,397, 6759509, 6762179, 6884869, 6897034, 6946455, 7,049,316, 7087600,
7091186,
7115573, 7129261,7214663, 7223837, 7304032, 7329507, 7.329,760, 7,388,026,
7,655,660, 7,655,661, 7,906,545, and 8,012,978.
Daunorubicin/Doxorubicin Analogues are also preferred for conjugation via the
hy-
drophilic linkers of the present patent. The preferred structures and their
synthesis are
exampled in: Hurwitz, E., et al.. Cancer Res. 35, 1175-1181 (1975). Yang, H.
M.. and
Reisfeld, R. A., Proc. Natl. Acad. Sci. 85, 1189-1193 (1988); Pietersz, C. A.,
E., etal., E.,
et al.," Cancer Res. 48, 926-9311 (1988); Trouet, et al., 79, 626-629 (1982);
Z. Brich et al.,
J. Controlled Release, 19, 245-258 (1992); Chen et al., Syn. Comm., 33, 2377-
2390, 2003;
King et al., Bioconj. Chem., 10, 279-288, 1999; King et al., J. Med. Chem.,
45, 4336-4343,
2002; Kratz et al., J Med Chem. 45, 5523-33. 2002; Kratz et al., Biol Pharm
Bull. Jan. 21,
56-61 , 1998; Lau et al., Bioorg. Med. Chem. 3, 1305-1312, 1995; Scott et al.,
Bioorg.
Med.1 Chem. Lett. 6, 1491-1496; 1996; Watanabe et al., Tokai J. Experimental
Clin. Med.
15, 327-334, 1990; Zhou et al., J. Am. Chem. Soc. 126, 15656-7, 2004: WO
01/38318;
U.S. Patent No.). 5,106,951; 5,122,368; 5.146.064; 5,177,016; 5,208,323;
5,824,805;
6,146,658; 6,214,345; 7569358; 7,803,903; 8,084,586; 8,053,205.
Auristatins and dolastatins are preferred in conjugation via the hydrophilic
linkers of
this patent. The auristatins (e. g. auristain E (AE) auristatin EB (AEB),
auristatin EFP
(AEFP), monomethyl auristatin E (MMAE), Monomethylauristatin (MMAF), Amistatin
F
phenylene diamine (AFP) and a phenylalanine variant of MMAE) which are
synthetic
56
analogs of dolastatins, are described in Int. J. Oncol. 15:367-72 (1999);
Molecular Cancer
Therapeutics, vol. 3, No. 8, pp. 921-932 (2004); U.S. Application Nos.
11134826, 20060074008,
2006022925. U.S. Patent Nos. 4414205, 4753894, 4764368, 4816444, 4879278,
4943628,
4978744, 5122368, 5165923, 5169774, 5286637, 5410024, 5521284, 5530097,
5554725, 5585089,
5599902, 5629197, 5635483, 5654399, 5663149, 5665860, 5708146, 5714586,
5741892, 5767236,
5767237, 5780588, 5821337, 5840699, 5965537, 6004934, 6033876, 6034065,
6048720, 6054297,
6054561, 6124431, 6143721, 6162930, 6214345, 6239104, 6323315, 6342219,
6342221, 6407213,
6569834, 6620911, 6639055, 6884869, 6913748, 7090843, 7091186, 7097840,
7098305, 7098308,
7498298, 7375078, 7462352, 7553816, 7659241, 7662387, 7745394, 7754681,
7829531, 7837980,
7837995, 7902338, 7964566, 7964567, 7851437, 7994135.
The benzodiazepine dimers (e.g. dimmers of pyrrolobenzodiazepine (PBD) or
(tomaymycin), indolinobenzodiazepines, imidazobenzothiadiazepines, or
oxazolidinobenzodiazepines) which are preferred cytotoxic agents according to
the present
invention are exampled in the art: US Patent Nos. 8,163,736; 8,153,627;
8,034,808; 7,834,005;
7,741,319; 7,704,924; 7,691,848; 7,678,787; 7,612,062; 7,608,615; 7,557,099;
7,528,128;
7,528,126; 7,511,032; 7,429,658; 7,407,951; 7,326,700; 7,312,210; 7,265,105;
7,202,239;
7,189,710; 7,173,026; 7,109,193; 7,067,511; 7,064,120; 7,056,913; 7,049,311;
7,022,699;
7,015,215; 6,979,684; 6,951,853; 6,884,799; 6,800,622; 6,747,144; 6,660,856;
6,608,192;
6,562,806; 6,977,254; 6,951,853; 6,909,006; 6,344,451; 5,880,122; 4,935,362;
4,764,616;
4,761,412; 4,723,007; 4,723,003; 4,683,230; 4,663,453; 4,508,647; 4,464,467;
4,427,587;
4,000,304; US patent appl. 20100203007, 20100316656, 20030195196.
The drugs/cytotoxic agents used for conjugation via a hydrophilic linker of
the present
patent can be any analogues and/or derivatives of drugs/molecules described in
the present patent.
One skilled in the art of drugs/cytotoxic agents will readily understand that
each of the
drugs/cytotoxic agents described herein can be modified in such a manner that
the resulting
compound still retains the specificity and/or activity of the starting
compound. The skilled artisan
will also understand that many of these compounds can be used in place of the
drugs/cytotoxic
agents described herein. Thus, the drugs/cytotoxic agents of the present
invention include
analogues and derivatives of the compounds described herein.
57
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EXAMPLES
The invention is further described in the following examples, which are not
intended
to limit the scope of the invention. Cell lines described in the following
examples were
maintained in culture according to the conditions specified by the American
Type Culture
Collection (ATCC) or Deutsche Sammlung von Mikroorganismen und Zellkulturen
GmbH,
Braunschweig. Germany (DMSZ), or The Shanghai Cell Culture Institute of
Chinese
Acadmy of Science, unless otherwise specified. Cell culture reagents were
obtained from
Invitrogen Corp., unless otherwise specified. All anhydrous solvents were
commercially
obtained and stored in Sure-seal bottles under nitrogen. All other reagents
and solvents
were purchased as the highest grade available and used without further
purification. The
preparative HPLC separations were performed with Varain PreStar HPLC. NMR
spectra
were recorded on Varian Mercury 400 MHz Instrument. Chemical shifts (.delta.)
are
reported in parts per million (ppm) referenced to tetramethylsilane at 0.00
and coupling
constants (J) arc reported in Hz. The mass spectral data were acquired on a
Waters Xevo
Qtof mass spec with Waters Acquity UPLC separations module and Acquity TUV
detector.
Example 1: 3-((((2-(2,5-Dioxo-2,5-dihydro- IH-pyrrol -1-
yDethyl)amino)(hydroxy)-
phosphoryHamino)propanoic acid (4)
0 0 0 0 0
QN.N.NH2 _300.0PC13
11'NN43¨C1 N1T2 Q
-78 C, THF
CI N-N,õN¨IFL-N's=)kOH
'; am, H
01 0 2 0 HO
4
N-2-ethyl-malimide hydrochloride salt (1.0 g. 5.66 mmol) in THF (50 ml) cooled
at
-78 C was added phosphoryl trichloride (0.86 g. 5.66 mmol). After stirred at -
78 C for 2 h
to form (2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)phosphoramidic
dichloride (2), 3-
aminopropanoic acid (0.51g, 5.70 mmol) in the mixture of THF/H20 (2:1, 30 ml)
and
triethylamine (1.0 g, 9.90 mmol) was added to the solution. The resulting
mixture was
stirred at RT for 3 h, concentrated under vacuum and purified on the SiO2
column eluted
with H20/CH3CN (1:20 ¨1:10) to afford the title compound 4 (1.28 g, 78%
yield). ESI MS
m/z- C9[113N306P (M-H), cacld. 290.06, found 290.10.
Example 2. N-Hydroxysuccinimidyl 3-((((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-
yeethyeamino)(hydroxy)phosphoryl)amino)propanoate (5).
NHS/Eloc,
OH 0-
'1%T;r3
H DMA I H
0 HO 4 0 HO 5 0
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3-((((2-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)(hydroxy)-
phosphoryeamino)propanoic acid (4) (0.50 g, 1.72 mmol) in DMA (30 ml) was
added
NHS (0.20 g, 1.74 mmol) and EDC (0.81 g, 4.22 mmol). The mixture was stirred
under Ar
overnight, evaporated and purified on SiO2 chromatography eluted with
acetone/CH2C12
(1:10 -1:3). The fractions containing the product were pooled, evaporated,
solidified in
C2H5OH/Dioxane/Hexane to afford the title compound (392 mg, 58% yield). ESI MS
na/z-
C13H16N408P (M-H), cacld. 387.08, found 387.20.
Example 3. 3-((Bis((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-
phosphory1)-amino)propanoic acid (8).
0 0
2 Clivio..03
N-"N,NH I N'NeN--P, "NNP-N I
-78 C, THF
0 0 0 7
0 0 0 0
H
HOANH2 I N -NeN ¨N N===)(.0H
, I H
0 HN'''N I 8
0
N-2-ethyl-malimide hydrochloride salt (2.0 g, 11.32 mmol) in THF (100 ml)
cooled
at -78 C was added phosphoryl trichloride (0.86 g, 5.66 mmol). After stirred
at -78 C for 1
h, the mixture was added triethylamine (1.0 g, 9.90 mmol) and the resulting
solution was
stirred at RT for 3 h to generate bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-
yl)ethyl)-
phosphoramidic chloride (7). Then 3-aminopropanoic acid (0.51g, 5.70 mmol) in
the
mixture of THF/H20 (2:1, 30 ml) and triethylamine (1.51 g, 14.90 mmol) was
added to the
solution. The resulting mixture was stirred at 35 C for 3 h, concentrated
under vacuum and
purified on the SiO2 column eluted with H20/CH3CN (1:20 -1:10) to afford the
title
compound 8 (1.47 g, 63% yield). ESI MS na/z- C151-119N507P (M-H), cacld.
412.11, found
412.20.
Example 4. N-Hydroxysuccinimidyl 3-((Bis((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-
ypethyl)amino)phosphory1)-amino)propanoate (9).
0 H 0 0 0 H 0 0 0
K(<1µ1"\e=N H ¨N NHS/El:1r36e N
I 0 in o N
HN DMA
0 0 HNN 0
8 9
0 0
59
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3-((Bi s((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-y1 )ethyl
)amino)phosphoryl)amino)-
propanoic acid (8) (0.55 g, 1.33 mmol) in DMA (30 ml) was added NHS (0.20 g,
1.74
mmol) and EDC (0.78 g, 4.06 mmol). The mixture was stirred under Ar overnight,
evapo-
rated and purified on short SiO2 chromatography eluted with Et0Ac/CH2C12 (1:3
¨1:1).
The fractions containing the product were pooled, evaporated in vacuum to
afford the title
compound (536 mg, 79% yield). EST MS m/z+ Ci9H23N6Na09P (M+Na), cacld. 533.13,
found 533.20.
Example 5. 3-((hydroxy((2-(pyridin-2-yldisulfanyl)ethyl)amino)phosphory1)-
amino)propanoic acid (15).
_300.0PC13 s 0
S's .0N, NH2 N H II
13 Cl 14
0 0
0
HO)cr'-..xi-LI
2 OH
/ HOH 15
2-(pyridin-2-yldisulfanyl)ethanamine hydrochloride salt (1.40 g, 7.52 mmol) in
THE
(60 ml) cooled at -78 C was added phosphoryl trichloride (1.15 g, 7.56 mmol).
After stirred
at -78 C for 2 h to form (2-(pyridin-2-yldisulfanyl)ethyl)phosphoramidic
dichloride (14), 3-
aminopropanoic acid (0.67g, 7.52 mmol) in the mixture of THF/H20 (2:1, 30 ml)
and
triethylamine (20 g, 19.80 mmol) was added to the solution. The resulting
mixture was
stirred at RT for 3 h, concentrated under vacuum and purified on the SiO2
column eluted
with H20/CH3CN (1:20 ¨1:10) to afford the title compound 15 (1.69 g, 66%
yield). ESI
MS m/z- C10H15N304PS (M-H). cacld. 336.03, found 336.20.
Example 6. N-Hydroxysuccinimidyl 3-((((2-(pyridin-2-yldi sulfanyl)ethyl)amino)-
(hydroxy)phosphoryl)amino)propanoate (16).
0 0 0 0 0
N(5.S/\A NHS/EDC N S.
HO 1-1 HO
15 16 0
3-((((2-( pyridin-2-yldisulfanyl)ethyl)amino)(hydroxy)-
phosphoryl)amino)propanoic
acid (15) (0.60 g, 1.78 mmol) in DMA (30 ml) was added NHS (0.22 g, 1.91 mmol)
and
EDC (0.81 g, 4.22 mmol). The mixture was stirred under Ar overnight,
evaporated and
purified on short C-18 chromatography eluted with water/dioxane at 4 C. The
fractions
containing the product were pooled, freezed at -78 C, lyophilized to afford
the title com-
CA 02989269 2017-12-12
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pound 16 (477 mg, 61% yield). ESI MS m/z- C14H19N406PS2 (M-H), cacld. 433.05,
found
433.20.
Example 7. 3-((Bis((2-(pyridin-2-yldisulfanyl)ethyl)amino)phosphoryl)amino)-
propanoic acid (18).
N S 0
,y \r,NH2 itt_p_pisis,e,1\111
0 C, THF cji S/\"
C11
13 17
0 0
0
HO t(TN S S II I I /NAOH
N s II
\s,\,,,N14
18
2-(pyridin-2-yldisulfanyHethanamine hydrochloride salt (2.10 g, 11.29 mmol) in
THE (100 ml) cooled at -78 C was added phosphoryl trichloride (0.85 g, 5.59
mmol). After
stirred at -78 C for 1 h, the mixture was added triethylamine (1.0 g, 9.90
mmol) and the
resulting solution was stirred at RT for 3 h to generate bis(2-(pyridin-2-
yldisulfanyHethyl)-
phosphoramidic chloride (17). Then 3-aminopropanoic acid (0.61g, 6.85 mmol) in
the
mixture of THF/H20 (2:1, 30 ml) and triethylamine (1.80 g, 17.82 mmol) was
added to the
solution. The resulting mixture was stirred at 35 C for 3 h, concentrated
under vacuum and
purified on the SiO2 column eluted with H20/CH3CN (1:20 ¨1:10) to afford the
title
compound 18 (1.47 g, 63% yield). ESI MS m/z- C17H23N503PS (M-H), cacld.
504.05,
found 504.20.
Example 8. 2,5-dioxopyrrolidin-1-y1 3-((bis((2-(pyridin-2-yldisulfanyl)ethyl)-
amino)phosphoryl)amino)propanoate (19).
0 0
N s /NAOH N s 0
NH /NA 0
(..); EDC/N HS
N \s, s I H Nix I H
N s \.,.NH 18 DMA
19 0
3-((Bis((2-(pyridin-2-yldisulfanyHethyl)amino)phosphoryl)amino)propanoic acid
(18) (0.52 g, 1.03 mmol) in DMA (30 ml) was added NHS (0.20 g, 1.74 mmol) and
EDC
(0.80 g, 4.16 mmol). The mixture was stirred under Ar overnight, evaporated
and purified
on short SiO2 chromatography eluted with Et0Ac/CH2C12 (1:4 ¨1:1). The
fractions con-
taining the product were pooled, evaporated in vacuum to afford the title
compound 19
61
CA 02989269 2017-12-12
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(536 mg, 79% yield). EST MS m/z+ C21H27N6N,05PS4 (M+Na), cacld. 625.06, found
625.20.
Example 9. 1-(2-(Methylamino)ethyl)-1H-pyrrole-2,5-dione (43)
0
0 H
N'N'"=-..NI12 a -\__r -
H -pow
42
0 43
N1-methylethane-1,2-diamine (10.23 g, 138.08 mmol) in DMA (150 ml) was added
succinic anhydride (13.82 g. 138.08 mmol) at 0 C. After stirring under Ar at 0
C for 1 hr
then RT for 4 h, the mixture was evaporated, redissolved in acetic acid (100
ml, 98%) and
Ac20 ( 0.5 mL), then heated at 80 C for 8 h. The mixture was concentrated,
purified on C-
18 flush chromatography eluted with water/CH3OH (100% water to 60% water
containing
0.3% HC1) to afford the title compound as a hydrochloric acid salt. (13.68 g,
52% yield).
ESI MS m/z+ 155.10 (M + H).
Example 10. 3-((bis((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)(methyl)-
amino)phosphory1)(methyl)amino)propanoic acid (50)
0
OPC13
i N0111µ ¨DPP. QI N-N/NI 2P--NI Ni=-=N 1 Q
0 C, THF 1 0
HO 11
)c.,"..,µ,/'
H 45
0 43 0 0 49 -PP'
0
0 1 iil 1 H
I o N0H
N
0
15 1-(2-(Methylamino)ethyl)-1H-pyrrole-2,5-dione, HC1 salt (43) (4.05 g,
21.00 mmol)
in THF (100 ml) cooled at -78 C was added phosphoryl trichloride (1.59 g,
10.50 mmol).
After stirred at -78 C for 1 h, the mixture was added triethylamine (1.2 g,
11.88 mmol) and
the resulting solution was stirred at RT for 3 h to generate bis(2-(2,5-dioxo-
2,5-dihydro-
1H-pyrrol-1-yl)ethyhmethyl))-phosphoramidic chloride (49). Then 3-
20 (methylamino)propanoic acid (1.21 g. 11.73 mmol) in the mixture of THF
(30 ml) and
triethylamine (1.20 g, 11.88 mmol) was added to the solution. The resulting
mixture was
stirred at 45 C for 3 h, concentrated under vacuum and purified on the SiO2
column eluted
with H20/CH3CN (1:20 ¨1:10) to afford the title compound 50 (2.44 g. 51%
yield). ESI
MS m/z- C18H25N507P (M-H), cacld. 454.16, found 454.20.
62
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PCT/IB2015/054521
Example 11. 2,5-diox opyrrolidin-l-yl 3-((hi s((2-(2,5-dioxo-2,5-dihydro-1H-
pyrrol-
1-yl)ethyl )(methyl)amino)phosphory1)(methyl)amino)propanoate (51)
0 0 0 0 0 0 0
0
50 0 0 51
Compound 50 (1.10 g, 2.41 Imo') in DMA (40 ml) was added NHS (0.40 g, 3.48
mmol) and EDC (1.80 g, 9.37 mmol). The mixture was stirred under Ar overnight,
evapo-
rated and purified on short SiO2 chromatography eluted with acetone/CH2C12
(1:6 ¨1:2).
The fractions containing the product were pooled, evaporated in vacuum to
afford the title
compound 51 (971 mg, 73% yield). ESI MS m/z+ C22H29N6N209P (M+Na), cacld.
575.17,
found 575.20.
Example 12. Conjugation of two DM1 per linker with an antibody for (52a).
0 11 0 0 0 I 0 0
DM1-SII Dm 4NPN,11. mAb
NI 0 N
0 I
0 ===/"'N I 0 mAb¨(NH2)1111P
q DMi
51 0 0 52a
The compound 51 (35 L, 20 mM in DMA), buffers (60 L, 100 mM NaH2PO4, pH
5.0-7.0) and DM1 (85 L, 20 mM in DMA) were incubated at 15-30 C for 20 min
¨2.5 h.
Then the mixture was added to a mixture of 2.0 mL of 10 mg/ml antiHer2
antibody in pH
6.5-8.0 PBS buffer. 1.0-2.0 mL of 100 mM NaH2PO4, pH 7.5 buffer. The subject
mixture
solution was incubated at RT for 2-24 h, purified on G-25 column eluted with
100 mM
NaH2PO4, 50 mM NaCl pH 5.5-7.5 buffers to afford 16.5-18.3 mg of the compound
52a
(-86% yield) in 11.6-14.2 ml buffers. The DM1/antibody ratio was 6.8-7.8,
which was
calculated according to the reference (Zhao, R. Y. et al, J. Med. Chem. 2011,
54, 3606). It
was 95-99% monomer analyzed by SEC HPLC (Tosoh Bioscience, Tskgel G3000SW, 7.8
mm ID x 30 cm, 0.5 ml/min, 100 min)
Alternatively, the compound 51 (35 L. 20 mM in DMA), was added to a mixture
of
2.0 mL of 10 mg/ml antiHer2 antibody in pH 6.5-8.0 PBS buffers, 0.5-1.7 mL of
100 mM
NaH2PO4, pH 6.5-8.0 buffer. After incubated at RT for 2 h, the mixture was
purified on G-
63
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WO 2015/151078 PCT/IB2015/054521
25 column eluted with 100 mM NaH1PO4, 50 mM NaCl pH 5.5-7.5 buffers. Then to
the
collected subject solution (4.5-6.5 ml) were added DM1 (70 L, 20 mM in DMA)
and
DMA (0.1-0.5 m1). The mixture was then incubated at RT for 2-16 h, purified on
G-25
column eluted with 100 mM NaH2PO4, 50 mM NaC1 pH 5.5-7.5 buffesr to afford
15.8-17.3 mg of the compound 52a (-81% yield) in 15.5-17.4 ml buffers. The
DM1/antibody ratio was 7.1-7.7, which was calculated according to the
reference (Zhao, R.
Y. et al, J. Med. Chem. 2011, 54, 3606). It was 95-99% monomer analyzed by SEC
HPLC
(Tosoh Bioscience, Tskgel G3000SW, 7.8 mm ID x 30 cm, 0.5 ml/min, 100 mM).
Example 13. Compound 147a bearing a hydrophilic linker of the present patent
con-
jugated with a tubulysin analog.
<
1 0 0 {(C) CI at OH
1 0 N rl ,,1, l
, 0 OAc Ne OH 0
41FI NH2
N
0 , %.`/'-'N-1 0 N
N -1104 2:41
I S H
51 0 146a 0
0 OH
0 113 1 0
H 0 OAc 0
_DI. ClirN4s,I.L N...yk LIN---
kINI-Pi ---NO\-N))
0
147a OV
The compound 51 (120 mg, 0.217 mmol) in THF (3.0 ml) was added compound
146a (Huang Y. et al, Med Chem. #44, 249th ACS National Meeting, Dever, CO,
Mar.
22-26, 2015; W02014009774) (151 mg, 0.199 mmol) in THF (3.0 ml) and buffer (5
ml,
100 mM Na2HPO4, pH 7.2). After stirred at RT for 4 h, the mixture was
concentrated and
purified with C-18 preparative HPLC (250 mm x ID 30 mm), eluted with
water/ethanol
(90% water to 50% water in 35 min, v = 65 ml/min ). The fractions containing
the product
were pooled, concentrated and crystallized with Et0H/Hexane to afford the
title com-
pound (159 mg, 67% yield). ESI MS m/z+ C56H82NiiN,014PS (M+Na), cacld.
1218.35,
found 1218.40.
Example 14. Conjugated compound 147a to an antibody for 148a.
0 OH
_
H I 0
14721--)i- 4 4 N OH ;,-
mAb
N 0 . I IIAN
I S H
S
0 q _
148a _
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To a mixture of 2.0 mL of 10 mg/ml antiHer2 antibody in pH 6.0-8.0, were added
of
0.70 -2.0 mL PBS buffer of 100 mM NaH2PO4, pH 6.5-7.5 buffers, TCEP (28 p L,
20 mM
in water) and the compound 147a (35 pL, 20 mM in DMA). The mixture was
incubated at
RT for 2-16 h, then DHAA (135 L, 50 mM) was added in. After continuous
incubation
at RT overnight, the mixture was purified on G-25 column eluted with 100 mM
NaH2PO4,
50 mM NaC1 pH 6.0-7.5 buffer to afford 16.8-17.9 mg of the conjugate compound
148a
(-87% yield) in 13.1-14.9 ml buffer. The drug/antibody ratio (DAR) was 2.8-
3.7, which
was determined UPLC-Qtof mass spectrum. It was 96-99% monomer analyzed by SEC
HPLC (Tosoh Bioscience, Tskgel G3000SW, 7.8 mm ID x 30 cm, 0.5 ml/min, 100
min)
and a single band measured by SDS-PAGE gel.
Alternatively, a mixture of 2.0 mL of 10 mg/ml antiHer2 antibody in pH 6.0-8.0
PBS buffer, 0.50-1.5 mL of 100 mM NaH2PO4, pH 6.0-8.0 buffers, and DTT (30 pL,
20
mM in water) was incubated at 15-37 C for 1-5 h and then purified on G-25
column eluted
with 100 mM NaH4304, 50 mM NaC1 pH 6.0-8.0 buffer. The pooled fractions (3.8-
5.8 ml)
was added 0.2-0.6 mL DMA and compound 147a (35 p L, 20 mM in DMA), and incubat-
ed at RT for 4-12 h. After addition of DHAA (135 p L, 50 mM) and continuous
incubation
at RT overnight, the mixture was purified on G-25 column eluted with 100 mM
NaH2PO4,
50 mM NaC1 pH 7.5 buffer to afford 15.5-16.8 mg of the conjugate compound 148a
(-80%
yield) in 13.8-15.9 ml buffers. The drug/antibody ratio (DAR) was 2.6-3.8,
which was
determined UPLC-QTOF mass spectrum. It was 96-98% monomer analyzed by SEC
HPLC (Tosoh Bioscience, Tskgel G3000SW, 7.8 mm ID x 30 cm, 0.5 ml/min, 100
min)
and a single band measured by SDS-PAGE gel.
Example 15. In vitro cytotoxicity evaluation of conjugates 52a and 148a:
The targeted cells (e.g. N-87, SKOV3 and HL60 cells, 6,000 cells) were
cultured in
the presence of various concentrations the antiHer2 antibody conjugate 52a and
148a for
96 hours after which cell viability was measured by propidium iodide exclusion
and
analyzed by flow cytometry using a Becton Dickinson FACSort (Becton Dickinson,
Franklin Lakes, NJ). Red fluorescent intensity (emission at 617 nm in the FL2
channel) of
the cells excited at 488 nm was measured. The regions for viable cells were
also set using
both the forward light scatter and right-angle light scatter properties of the
cells. The loss of
viability was determined by the loss of cells from within the gated region
defining viable
cells. The average number of viable cells per 6 replicate cultures was
calculated. The
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survival fraction was plotted versus conjugate concentration to determine the
IC50 value
(50% cell killing concentration) of the conjugates 52a and 148a.
The cytotoxicity results:
IC50 nM N87 cell (Ag+) SK-OV-3 cell (Ag+) HL60 cell (Ag-)
Conjugate 52a 0.027 nM 0.021 nM >50 nM
Conjugate 148a 0.038 nM 0.032 nM >50 nM
Specificity of conjugate 52a for N87 cell was over 1850 (IC50> 50/ IC50=
0.027), and
for SK-OV-3 cell was over 2380.
Specificity of conjugate 148a for N87 cell was over 1315 (IC50> 50/ IC50=
0.038),
and for SK-OV-3 cell was over 1560.
Both conjugate 52a and conjugate 148a were extremely potent and much
specifical-
ly targeting the antigen positive tumor cells.
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