1
50229-435
AN ORGANIC CATION TRANSPORTER
PREFERENTIALLY EXPRESSED IN
HEMATOPOIETIC CELLS AND LEUKEMIAS
AND USES THEREOF
Cross Reference to Related Applications
[01] This application claims the benefit of U.S. Provisional Application No. 60/471,709,
filed May 20, 2003.
Field of the Invention
[02] The invention relates to a gene encoding an organic cation transporter, 0CT6, and its
use as a target for the treatment of hematological malignancies, and in particular, leukemia.
The invention fiirther relates to screening methods for identifying agonists and antagonists/
binding partners of 0CT6 transport activity.
Background of the Invetion
[03] The lipid bilayer of the cellular membrane insulates the intracellular milieu fi-om
exposure to hydrophilic compounds. Unlike lipophilic compounds that can diffuse through
cellular membranes, water-soluble compounds usually require specific transport mechanisms
to gain access to the intracellular space. The regulation of the traffic of polar compounds in
both directions across the cellular membrane is a complex process involving several large
families of transport proteins.
2
[04] Most often in cancer research, drug transport is thought of as a mechanism of cellular
drug resistance, as drug efflux pumps such as the products of the MDRl and MRP genes have
been shown to be mechanisms of resistance to iipid-soiuble anticancer drugs. However, drug
transport is a two-way street, and mechanisms also exist for pmnping drugs into cells. For
polar, water-soluble anticancer agents, drug uptake, and not drug efflux, is the critical
determinant of cellular drug accumulation.
[05] Most cancer chemotherapy employs drugs that are lipid-soluble that can easily
penetrate the cell membrane of cancer cells. One advantage of using lipid-soluble drugs is
that they easily gain intracellular access to different types of cancer cells, so many cancer
cells appear to be initially sensitive to these drugs. The disadvantage is that cancer cells
leam to increase the activity of drug efflux pumps in the cell membrane to pump lipid-
soluble drugs out of the cell, resulting in drug resistance.
[06] In contrast, potential water-soluble anticancer drugs may not survive the preclinical
screening process since there is a great deal of variability in the expression of drug transport
genes in different types of cancer cells. Variability in transport gene expression may result in
variability in accumulation of polar, water-soluble drugs. One approach to more effectively
utilize water-soluble anticancer drugs is to identify which of the dozens of transport genes are
actually expressed in tumors.
[07] The importance of carrier-mediated anticancer drug uptake is exempHfied in reduced
folate carrier (RFC) mediated uptake of methotrexate (MTX). Methotrexate (MTX), a
reduced folate analogue, is scavenged and retained in cells by mechanisms designed to secure
folates from the environment. The major mechanism of MTX uptake at pharmacologic
concentrations is the reduced folate carrier (RFC), an OAT transporter with a Km for MTX
between approximately 0,8-26 \xM. Decreased RFC activity has been observed in several in
vitro models of transport-mediated MTX resistance (Biochem. Pharmacol. 11: 1233-1234,
1960). Once rodent and human genes encoding proteins with RFC activity were isolated, the
molecular explanations for decreased RFC activity emerged. RFCl transfection into the
transport-deficient MTX^ ZR75 cell line resulted in a 20-fold increase in 6-hour MTX uptake
and a concomitant 250-fold increase in sensitivity to MTX relative to control cell clones.
3
showing that the RFCl gene reconstitutes RFC activity and has a significant impact on MTX
cytotoxicity (Moscow, et al., Cancer Res. 55: 3790-3794, 1995).
[08] In different ceil iines, MTX transport deficiency has been ascribed either to mutations
in the RFC gene or in decreased expression of the RFC gene product. Several studies haye
demonstrated that RFCl gene expression is an important determinant of sensitivity to MTX.
In in vitro studies, we have found that RFCl RNA levels correlate with MTX sensitivity in a
panel of non-selected cell lines, including breast cancer cell lines (Moscow et al., Int J
Cancer. 72: 184-190, 1997).
[09] A plethora of genes with the ability to transport MTX out of the cell have been
reported, including MRPl, MRP2, MRP3, MRP4, the organic anion transporters hOAT2 and
hOAT3, and the mitoxantrone-resistance protein (BCRP/MXR). However, despite the
multitude of MTX export genes, clinical studies have shown a relationship between the
expression of RFCl, the mechanism of MTX uptake, and prognosis in Acute Lymphoid
Leukemia (ALL) and osteosarcoma. As a result, RFCl expression and MTX uptake are now
imphcated as determinants of clinical sensitivity in several types of tumors. Thus, the role of
RFCl in mediating sensitivity of its cytotoxic drug substrates has become a prototype that
illustrates the potential role of transporters, like OAT and OCT genes, in determination of
anticancer drug selectivity and toxicity.
[10] However, there is a need to identify additional channels, or transporters, that are
found in specific cancers, to enable the targeting of different cancers with anticancer agents
that are substrates for those transporters.
Summary of the Invention
[11] The present invention is directed towards a membrane protein that functions to
transport hydrophilic substances across cellular membranes. The protein, 0CT6, is a new
member of the organic cation transporter (OCT) family (SLC22 gene family). Tissue
distribution of this protein is distinct from other OCT protein family members; being
detected in leukemia, leukemia blast cells and CD34+ cells.
[12] In one aspect, the present invention provides a novel target for hematological
malignancies such as leukemia, an 0CT6 transporter.
4
[13] In another aspect of the present invention there is a method for screening potential
substrates that selectively bind the 0CT6 transporter. The method involves contacting a cell
which overexpresses an 0CT6 transporter gene with a test compound and determining
whether the test compoxmd is a substrate for the 0CT6 transporter.
[14] In another aspect, there is a method for screening potential anti-cancer agents in a cell
overexpressing an 0CT6 transporter gene. The method comprises determining viability of a
cell which expresses 0CT6 transporter gene incubated in the presence and absence of a test
compound and identifying the test compound as a potential anti-cancer agent if there is
cellular influx of the test compound and cell death.
[15] In another aspect of the invention, a test kit is provided for screening candidate drugs
for hematologic malignancies comprising a mammalian cell line or cells which overexpress
0CT6, a control substrate and a detectable substance.
[16] In still another aspect of the invention, there are immunogenic compositions for
treating hematological malignancies. In a preferred embodiment, immunogenic
compositions for treating leukemia comprise a substrate that binds selectively to a leukemia
cell expressing the 0CT6 transporter gene. In another preferred embodiment of the
invention, the substrate comprises an antibody that selectively binds to the 0CT6 transporter
protein. Preferably, the 0CT6 transporter protein allows cellular uptake of the substrate
which then causes cell death. In one embodiment the substrate is cytotoxic and in another
preferred embodiment the substrate is coupled with a cytotoxic agent.
[17] In still another aspect, the present invention provides a method for impairing a
leukemia cell comprising contacting the cell with a cytotoxic 0CT6 transporter protein. In
one embodiment the substrate is a cytotoxin and in another embodiment the substrate is
coupled to a cytotoxic agent.
[18] In yet another aspect, the present invention provides a method for treating
hematological malignancies comprising administering to a subject in need thereof an
immunogenic composition comprising a substrate that binds selectively to a cell expressing
the 0CT6 transporter gene. In a preferred embodiment the 0CT6 transporter protein allows
cellular uptake of the substrate which then causes cell death. In another preferred
5
embodiment the substrate is cytotoxic. In another preferred embodiment, the substrate is
coupled with a cytotoxic agent.
Brief Description of the Drawings
[ 1 9] Figure 1 . A. shows the predicted hydropathy profile of 0CT6.
[20] Figure !• is a dendrogram showing phylogenic relationship between 0CT6 (SEQ
ID N0:3) and other OCT and OAT proteins, including, OCTNl (SEQ ID N0:4), 0CT3
(SEQ ID N0:5), 0CTN2 (SEQ ID N0:6), 0CT2 (SEQ ID N0:7), OCT! (SEQ ID N0:8),
OATS (SEQ ID N0:9), 0AT4 (SEQ ID NO:10), OATS (SEQ ID N0:11), and OATl (SEQ
IDN0:12).
[21] Figure 2. is the CLUSTALW aUgnment of 0CT6 and other OCT and OAT proteins.
The bottom row represents areas of consensus.
[22] Figure 3, shows the normal tissue distribution of 0CT6 RNA determined by RT-PCR
using a cDNA panel. Only lOOOX (highest) cDNA concentration is shown. Panel A. 1,
salivary gland; 2, thyroid; 3, adrenal; 4, pancreas; 5, ovary; 6, uterus; 7, prostate; 8, skins; 9,
peripheral blood leukocytes; 10, bone marrow; 11, fetal brain; 12, fetal liver. Panel B. 1,
brain; 2, heart; 3, kidney; 4, spleen; 5, liver; 6, colon; 7, lung; 8, small intestine; 9, muscle;
10, stomach, 11, testis; 12, placenta.
[23] Figure 4. shows quantitative RT-PCR for the transporter gene 0CT6 performed with
RNA extracted from peripheral blood leukocytes, CD34+ cells and additional hematopoietic
cell lines. Fresh discarded buffy coats that were twice sorted by FACS using CD 14
(monocytes), CD15 (granulocytes), CD3 (T-cells) and CD20 (B-cells). Purities of 99% or
better were obtained. For peripheral WBC and sorted subsets, the average ± SD represent
pooled results from samples from 2 individuals performed in triplicate or quadruplicate. For
CD34-selected mobilized peripheral blood (MPB), the results from each of 3 individuals are
shown. For CD34-selected bone marrow (CD34+-BM), the results are from one individual.
0CT6 levels were normalized to the expression of actin RNA, as a control for equivalence of
mRNA template. The units, in log scale, are arbitrary and based on a standard curve of
0CT6 RT-PCR in serially diluted HL60 RNA. Unity is defined as the level of 0CT6 RNA
found in M0LT4 cells.
6
[24] Figure 5. shows quantitative RT-PCR for the gene 0CT6 using RNA extracted from
leukemic blasts obtained from patients at the time of initial diagnosis. 0CT6 levels were
normalized to the expression of actin RNA, as a control for equivalence of mRNA lemplate.
The 0CT6 RNA levels in placenta, liver, kidney and MOLT-4 cell line were determined
concurrently and shown for comparison. The units, in log scale, are arbitrary and based on a
standard curve of 0CT6 RT-PCR in serially diluted HL60 RNA. Unity is defined as the
level of 0CT6 RNA found in M0LT4 cells.
Detailed Description of the Invention
[25] The present invention is based on the discovery and isolation of a new member of the
SLC22 gene family (the OCT family of proteins) that is unusual for its distinct pattern of
tissue distribution. Rather than the typical high levels of expression in liver, kidney or
placenta, high levels of RNA for this transporter were found in some leukemia cell lines, in
CD34+ cells, and in circulating leukemia blast cells.
[26] All patents, patent applications and literature cited in this description are incorporated
herein by reference in their entirety. In the case of inconsistencies, the present disclosure,
including definitions, will prevail.
OCT Familv
[27] Two families of proteins involved in maintaining homeostasis of charged organic
compounds are the organic anion transporters (OATs) which carry the SLC21 designation
and the organic cation transporters (OCTs), which carry the SLC22 designation {See Table
1). OATs and OCTs each have characteristic patterns of tissue expression, with predominant
expression in a tissue involved in the transport of xenobiotics, i.e., liver, kidney or placenta.
7
[28] Table 1. Organic anion and cation transported genes
SLC21
SLC22
Gene Name
Locus Link
Alternative Names
SLC21A1
6577
SLC21A2
6578
PGT
SLC21A3
6579
OATP, 0ATP1. OATPIb, OATP-A
SLC21A4
28237
0AT-K1,0AT-K2
SLC21A5
28236
0ATP2, OATP-2
SLC21A6
10599
LST-1,0ATP-C
SLC21A7
28235
0ATP3. OATP-3
SLC21A8
28234
LST2, OATP8. SLC21A8, OATP-8
SLC21A9
11309
OATP-B
SLC21A10
28233
0ATP4
SLC21A11
28232
OATP-D
SLC21A12
28231
LOC51737. OATP-E. POAT
SLC21A13
28230
0ATP5. OATP-5
SLC21A14
53919
OATP-F
SLC22A1
6580
0CT1
SLC22A2
6582
0CT2
SLC22A3
6581
0CT3
SLC22A4
6583
0CTN1
SLC22A5
6584
0CTN2, CDSP, SCO
SLC22A6
9356
NKT, 0AT1.0AT-1
SLC22A7
10864
NLT. 0AT2. OAT-2
SLC22A8
9376
0AT3. OAT-3
SLC22A9
0AT4, OAT-4
[29] The OAT and OCT carriers result in increased cellular accumulation of their
respective substrates, despite the fact that they are carriers that mediate facilitative diffusion.
For carriers, the degree of intracellular accumulation may not exceed the extracellular
concentration. However, the presence of the carrier allows uptake in comparison to no
uptake in the absence of the carrier, and drugs that bind an intracellular target or which are
chemically modified in the cells, e.g., by phosphorylation or polyglutamylation, may be
eliminated from the substrate pool and not available for transport back across the cellular
membrane.
[30] The first five members of the SLC22 family of transporters, OCTl, 0CT2, 0CT3,
OCTNl, and 0CTN2, have been characterized as organic cation transporters. The uptake of
many cations, such as tetraethylammonium (TEA), N-l-methylnicotineamide (NMN),
choline, procainamide, amantadine and morphine are mediated by these polyspecific
transporters. In general, these transporters are potential-dependent, but independent of
sodium and proton gradients. These genes are all characterized by the presence of 1 1 or 12
8
transmembrane domains, as predicted by hydrophobicity analysis, and all have a large
hydrophilic loop between transmembrane domain (TMD)l and TMD2.
[31] OCT substrates are shown below in Table 2. Tetraethyi ammoniimi (TEA) is the
classic substrate for OCT transporters. In addition, OCTl, 0CT2 and 0CT3 transport 1-
methyl-4-phenylpyridinium (MPP). Compared to 0CT2, OCTl has a higher affinity for
some cations (for example mepiperphenidol and procainamide), a similar affinity for others
(for example, decynium 22 and quinidine), and a lower affinity for corticosterone (See
Koepsell et al, Ann. Rev. Physiol. 60: 243-266, 1998.). 0CT3 is an electrogenic transporter
for TEA and guanidine. Other physiologic substrates for OCT transporters include
dopamine, histamine, epinephrine and norepinephrine, acetylcholine and 5-
hydroxytryptamine ( Burckhardt, et al.. Am J Physiol Renal Physiol. 278: F853-66., 2000.),
suggesting an important role for these transporters in the central nervous system, in addition
to their role in hepatic and renal clearance. Interestingly, despite its cationic nature, recent
studies have identified cimetidine as a selective inhibitor, but not a substrate for several
organic cation transporters, including rOCTl, rOCT2, rOCT3, hOCTNl, and hOCTN2.
9
[32] Table 2. OCT Substrates
Common Name Gene Name
Cell Tvoe
Substrate
KT(uM)
OCTl
SLC22A1
HeLa
TEA
229
OCTl
SLC22A1
Xenopus
MPP
14.6
0CT2
SLC22A2
Xenopus
Norepinephrine
1900
0CT2
SLC22A2
Xenopus
Histamine
1300
0CT2
SLC22A2
Xenopus
Dopamine
390
0CT2
SLC22A2
Xenopus
Serotonin
80
0CT2
SLC22A2
HEK293
MPP
16
0CT2
SLC22A2
HEK293
Dopamine
330
0CT2
SLC22A2
Xenopus
Amantadine
27
0CT2
SLC22A2
Xenopus
Memantine
34
0CT3
SLC22A3
HeLa
TEA .
2500
0CT3
SLC22A3
HRPE
MPP
47
OCTNl
SLC22A4
Fibroblasts
L-Camitine
6.6
0CTN2
SLC22A5
HEK293
L-Camitine
4.34
0CTN2
SLC22A5
HEK293
L-Camitine
4.3
0CTN2
SLC22A5
HEK293
D-Camitine
10.9
0CTN2
SLC22A5
HEK293
Acetyl-L-camitine
8.5
0CTN2
SLC22A5
Xenopus
L-Camitine
4.8
0CTN2
SLC22A5
Xenopus
D-Camitine
98
0CTN2
SLC22A5
JAR
L-Camitine
3.5
[33] OCTl and 0CT2 are predominantly expressed in the kidney and liver. These
transporters are located on the basolateral surface of renal tubules and, therefore, play a role
in the removal of organic cations from the blood. 0CT3 is most abundantly expressed in
placenta. In addition, other tissue-specific roles have been implicated for these transporters.
As noted above, OCTs may play a role in transport of endogenous neuroleptic substrates, and
0CT3 has been implicated in the disposition of cationic neurotoxins and neurotransmitters in
the brain( Wu, et al., J Biol Chem. 273: 32776-86, 1998). Dhillon et al (Clin Pharmacol
Ther. 65: 205, 19996) used RT-PCR followed by functional transport studies (TEA) to
identify OCTl expression in a human mammary epithelial cell line (MCF12A). Further, the
OCTl gene has been shown to be up regulated in lactating mammary epithelial cells.
[34] The OCTNl gene, cloned from a cDNA, shows sequence similarity to organic cation
transporter genes, which is highly expressed in kidney as well as trachea, bone marrow and
10
fetal liver. Recombinant OCTNl expressed in mammalian cells exhibited saturable uptake
of TEA that was pH sensitive. Several others suggest that OCTNl is a renal proton/organic
cation antiporter functioning at the epithelial apical membrane. The uptake of pyriiamine,
quinidine, verapamil and L-camitine were increased by expression of OCTNl in Xenopus
oocytes.
[35] Another OCT protein family member, 0CTN2, cloned from a human placental
trophoblast cell line, is expressed widely in human tissues including kidney, placenta and
heart. 0CTN2 is more closely related to OCTNl than to OCTl, 0CT2 and 0CT3 (Biochem
Biophys Res Commun. 246: 589-95, 1998). Transfection of 0CTN2 has demonstrated its
role in the transport of TEA and carnitine. 0CTN2-mediated transport of TEA is sodium
independent, whereas transport of carnitine is sodium-dependent. The role of sodium in
0CTN2-mediated carnitine transport not only involves the electrogenic gradient, but the
presence of sodium also alters the affinity of 0CTN2 for camitine. Germline mutations of
0CTN2 result in primary camitine deficiency, a syndrome of progressive cardiomyopathy
and skeletal myopathy. The symptoms associated with this syndrome are thought to result
not only from generalized camitine deficiency from decreased renal camitine reabsorbtion,
but also from inability of cardiac and skeletal myocytes, which ordinarily express 0CTN2, to
accumulate camitine. This syndrome demonstrates that tissue-specific OCT-mediated
transport is essential for accumulation of required cations in specific tissues.
[36] The present invention identifies a new transport protein in the OCT family, 0CT6,
preferentially expressed in leukemia cell lines, leukemia blast cells and CD34+ cells. The
cell surface localization and the transporter function of the 0CT6 gene product suggest its
usefulness as a target in the diagnosis and treatment of hematologic malignancies.
[37] As used herein, the term "antibody" refers to an immunoglobulin molecule with a
specific amino acid sequence evoked in by an antigen, and characterized by reacting
specifically with the antigen in some demonstrable way.
[38] As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle
with which the compositions of the present invention are administered.
11
[39] As used herein, "compound" refers to any agent, chemical, substance, or substrate,
whether organic or inorganic, or any protein including antibodies, peptides, polypeptides,
peptoids, and the like.
[40] As used herein, the term cytotoxin" or cytoxic agent includes any specific substance,
which may or may not be antibody, that inhibits or prevents the functions of cells, causes
destruction of cells, or both.
[41] As used herein, the term "derivative" refers to something produced by modification of
something pre-existing; for example, a substance or chemical compound that may be
produced from another substance or compound of similar structure in one or more steps.
[42] As used herein, the term "fragment" refers to a part of a larger entity, said larger
entity comprising by non-limiting example, an antibody, compound or substance.
[43] As used herein, the term "leukemia blast" or "leukemic blast" refers to lymphoblasts,
the abnormal immature white blood cells associated with leukemia.
[44] As used herein, the term "monoclonal antibody" is not limited to antibodies produced
through hybridoma technology. The term "monoclonal antibody" refers to an antibody that is
derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not
the method by which it is produced.
[45] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier that
may be administered to a subject, together with one or more liver protecting agents and one
or more mushroom powder or extract of the present invention, and which does not destroy
the pharmacological activity thereof and is nontoxic when administered in doses sufficient to
deliver a therapeutic amount of the compound.
[46] As used herein, the term "substrate" refers to a substance, compound, agent, antibody
or derivatives and/or fragment thereof, acted upon by the OCT6 transporter protein (e.g., a
substance that is taken across the cellular membrane by action of the 0CT6 transporter
protein).
[47] 0CT6 (SEQ ID N0:1) was first identified as a potential OCT gene by assembling and
sequencing ESTs as described in Example 1 (amino acid sequence of 0CT6 is SEQ ID
N0:2). The gene sequence proved to be identical to the recently submitted cDNA 0KB 1
(GenBank AF268892) submitted by M. Okabe and T, Abe, incorporated herein in its entirety.
12
It is also contained within the submitted BAG clone CTA-331P3 (SEQ ID NO: 3) (GenBank
AG002464) located at chromosome 6q21, incorporated herein in its entirety. The gene has a
predicted protein structure typical of transport proteins with two groups of six
transmembrane domains separated by a hydrophilic region (Figure lA). CLUSTALW
alignment produced a dendrogram showing the phylogenic relationship between 0CT6 and
other OAT and OCT proteins (Figure IB). This dendrogram suggests that the distinction
between OAT and OCT genes, based on functional studies, obscures the conamon origin of
both families of transporters. The actual CLUSTALW alignment of these genes is shown in
Figure 2 and demonstrates multiple regions of conservation among all of these genes.
[48] Next, according to the methods described in Example 3, quantitative RT PCR
analysis of the expression of 0CT6 was performed, along with the expression of other OCT
genes, in 50 cell lines. The results are shown in Table 3. The two highest expressing cell
lines for 0CT6 in this panel were two leukemia cell lines, HL60, a human promyelocytic
leukemia cell line, and M0LT4, a human acute lymphoblastic leukemia (T-cell) cell line.
There was only a low level of expression detected in most of the other cell lines.
13
Table 3. OCT expression in 50 cell lines of the NCI Drug Screen
■NO.
oeii Line
source
UU 1 1
1 ^
1 o
1 IN £.
LIU 1 0
1
Leukemia
D.f
U.r
n 9
n 1
K 7
O.f
ML-DU
Leukemia
U.D
1 1
1 .o
0 n
n 4
#10
LeuKemia
1.4
0 9
1.4
^^ 9
O.Z
4
IVIUL I -4
Leukemia
A t
\J.l
1 .1
n
0 ft
U.O
AA Q
40.0
5
Leukemia
^.U
n 1
7
o.U^
A
D
CD
Leukemia
i.y
1 .1
0.0
0.3
9 ft
^.0
7
ARiiQ/ATr^r'
A04y/A 1 LrO
Lung cancer
4 7
1 .f
161
1 9
o
o
nUr-OZ
LunQ cancer
n Q
U.o
A ft
4.0
n
2 4
46.. *t
A i
A
9
Lung cancer
4.0
U.O
0 1
21.1
A A
1U
Lung cancer
U.O
U.f
0 0
0
9
•f I
11
iNUI-n4DU
Lung cancer
U.r
n .u
0.0
1.7
1 A
1 .0
oolon ua.
4.y
o.o
30.9
2.2
ft
O.O
lo
oolon oa.
1.0
i n
1 .u
0 0
2.6
o.*f
14
UOT "lift
ooion oa.
l.f
n 1
9 ft
Q 7
y./
15
no 1 -lo
oolon ua.
u.y
1 7
l.f
n 1
A 9
1o
LIT OO
n 1 -zy
uoion ua.
I.y
1.^
ifi 1
1
1 .o
1 .0
17
Ifftk AO
Wvi-I^
oolon ua.
u.o
1.U
15 7
7
9 1
Z. 1
1o
Q\A/ Ron
ouion oa.
1 r)
9 ft
40.4
1.9
0. 1
19
Or-ZD<5
UNo 1 umor
n A
U.4
U.O
0 n
oc one
ONo 1 umor
U.D
1 1
9 R
Z.O
21
SF-539
CNS Tumor
0.5
A £5
0.0
n 9
U.Z
O.o
22
SNB-75
CNS Tumor
U.O
1.0
n n
n ft
U.O
Z.O
23
U251
CNS Tumor
0.8
A A
0.9
0.0
0.6
(A
24
LOCIMVI
Melanoma
2.9
2.1
U.l
n A
U.H
o.o
25
MALME-SM
Melanoma
1.5
1.5
u.u
9 '5
o
0
26
M14
Melanoma
1.9
1.4
n n
u.u
1 Q
1 .y
4. A
27
SK-MEL-z
Melanoma
2.1
4 A
1 .9
0 n
u.u
9 9
o.y
28
SK-MEL-5
Melanoma
2.6
1 .5
u.u
1 .y
ZJ
29
UACC-257
Melanoma
3.2
3.6
0.0
1 .1
C /I
0.4
30
IGROV 1
Ovarian Ca.
4.9
5015
17 Q
1 A
1 .0
^.0
31
OVCAR-3
Ovarian Ca.
1.4
0.1
u.u
9 9
14
32
UVt/AK-4
Ovarian Ca.
^.0
i A
1.4
A
UVOAK-0
ijvarian oa.
0.0
Z. r
105
10.0
A A
'S>l
o4
VJVOAK-O
L^varian oa.
1 . 1
1 n
1 .u
0.0
0.8
1 ft
uvarian oa.
Q
1 QQ^
9.2
8.5
Q ft
36
A498
Renal Ca.
1o.4
1 An
1 ou
1 .o
37
ACHN
Renal Ca.
1.1
1.1
n 7
U, f
1 9
1 .1
38
CAKI_1
Renal Ca.
3.5
2.5
A O
4.0
4 Q
1 .O
2.8
39
RXF-393
Renal Ca.
1.7
1.2
3.0
0.6
1.2
40
TK-10
Renal Ca.
3.6
5.0
16.8
2.5
8
41
UO-31
Renal Ca.
4.4
1.6
31.2
1.2
2.3
42
PC-3
Prostate Ca.
2.1
0.8
9.6
3.3
4.7
43
DU-145
Prostate Ca.
1.1
1.1
3.4
1.6
3
44
MCF-7
Breast Ca.
0,8
1.8
0.0
10.4
3.5
45
NCI/ADR-RES
Breast Ca.
1.4
1.3
1.1
2.0
2.1
46
MDA-MB-231
Breast Ca.
1.2
0.4
3.9
4.8
1.8
47
HS578T
Breast Ca.
1.0
1.5
0.0
1.2
8.3
48
MDA-MB-435
Breast Ca.
1.9
0.6
0.1
0.7
2.7
49
BT-549
Breast Ca.
1.2
0.8
0.1
0.3
2.6
50
T-47D
Breast Ca.
0.7
1.1
0.1
4.2
8.7
14
[50] 0CT6 is unique among the known members of OCT and OAT genes because of its
pattem of tissue distribution. The pattern of expression of the 0CT6 gene in the 50 cell Unes
suggested that its expression miglit be restricted to hematopoietic tissues. Tlie restricted
pattem of expression observed for 0CT6 also suggests that therapies using 0CT6-specific
substrates are unlikely to have widespread toxicity to normal tissues. Therefore, we
examined 0CT6 expression in a cDNA panel representing a wide cross-section of normal
tissues according to the methods of Example 4 (Figure 3). This study revealed that 0CT6
RNA levels are highest in testis and fetal liver, with lower but detectable levels in peripheral
blood leukocytes and bone marrow. Since fetal hematopoiesis occurs in the liver, it is
possible that the fetal liver sample may have included both hepatocytes and hematopoietic
cells. 0CT6 RNA levels were also barely detectable in pancreatic and adrenal tissue. Unlike
other OCT genes, expression was not detectable in liver, kidney or placenta.
[51] To determine whether 0CT6 RNA expression in hematopoietic cells was lineage-
specific, leukocytes were sorted from discarded buffy coat specimens by flow cytometry, and
purified subpopulations were examined for 0CT6 RNA expression according to the methods
described in Example 5. 0CT6 expression was also examined in a population of CD34+
cells. As can be seen in Figure 4, the expression of 0CT6 was highly enriched in CD34+
cells in comparison to the other cell populations. Also,significant levels of 0CT6 expression
(relative to M0LT4) were found in other hematopoietic cell lines: U937, a human histiocytic
lymphoma cell line; THP-1, a human acute monocytic leukemia cell line; KG-1, a human
erythroleukemia cell line; and MV-4-1 1, a hxmian biphenotypic (B-cell and myelomonocytic)
leukemia cell line.
[52] The high levels of 0CT6 RNA in some leukemia cell lines and CD34+ cells also
raised the question as to whether this gene was highly expressed in actual leukemias. To
address this issue, the RNA levels of 0CT6 in 25 samples of peripheral leukemic cells were
measured according to the methods set out in Example 6. The FAB classification of these
samples are shown in Table 4. These results are shown in Figure 5, and demonstrate that the
majority of specimens contained RNA levels for 0CT6 that exceeded the level found in
M0LT4 cell line, the second highest expressing cell line among those examined, and exceed
by orders of magnitude the levels found in placenta, kidney and liver.
15
[53] Table 4. Phenotypes of leukemia specimens
Sample Number Description
1
CML, blast crisis
2
CML, blast crisis
3
CML, stable phase
4
CML, probably stable phase
5
CML, accelerated phase
6
ALL
7
ALL
8
AML
9
ALL
10
ALL
11
ALL
12
AML
13
AML
14
AML
15
AML
16
ALL, biphenotypic
17
ALL, biphenotypic
18
AML
19
AML, M2
20
AML, M2
21
AML, M4
22
AML, M4
23
AML, M1
24
AML
25
AML, M4
[54] Due to the 0CT6 protein's location on the cellular membrane and its function as an
intracellular transporter, the 0CT6 transporter protein has been identified as a therapeutic
target. Basic principles of cellular pharmacology suggest that increase in intracellular
accumulation will lead to increased intracellular effect. For anticancer drugs, this principle
has been studied extensively in the context of lipophilic drugs, which require no specific
mechanism for cellular uptake, and export pumps such as the product of the multidrug
resistance gene, MDRl, whose overexpression of MDRl leads to increased cellular
resistance by decreasing intracellular concentrations of drug ( Moscow, J. A., Schneider, E.
S., Ivy, S. P., and Cowan, K. H. Multidrug resistance. In: H. M. Pinedo, D. L. Longo, and B.
A. Chabner (eds.), Cancer chemotherapy and biological response modifiers. Annual 17. New
York: Elsevier, 1997). The same principle applies to charged, hydrophilic drugs of the
16
present invention, except that the determinants of sensitivity depend on uptake as opposed to
efflux. As such, cells overexpressing an 0CT6 transporter are likely to be highly sensitive to
cytotoxic 0CT6 substrates.
Drug Screening
[55] Accordingly, the present invention provides methods for screening potential
substrates of, and potential therapeutic agents against hematological malignancies like
leukemia that overexpress, the 0CT6 transporter. In particular, potential therapeutic agents
are screened for the ability to be a substrate recognized by an 0CT6 transporter protein.
Preferably, potential substrates are screened for the ability to confer cytotoxic effects on a
cell overexpressing 0CT6 transporter protein. More preferably, agents are screened for the
abihty to preferentially cause cellular uptake into, and cell death of, cells overexpressing the
0CT6 transporter. Most preferably, the agents are screened for the ability to cause cell death
of cancer cells such as leukemia overexpressing the 0CT6 transporter as compared to normal
cells.
[56] A method for screening potential substrates of the 0CT6 transporter protein
comprises providing a cell or cell line which expresses 0CT6 and a test compound,
incubating the test compound and cell line and analyzing the cell or cell line to determine if
there was a cellular influx of the test compound. Analysis of the cell line to determine
whether cellular uptake of the test compound occurred can be accomplished by any means
known in the art. For example, a test compound can be tagged with a detectable label prior
to contact with a cell and then observed under microscopy or by other means for its location.
Non-limiting examples of labels include green fluorescent protein, alkaline phosphatase,
horseradish peroxidase, rease, f3-galactosidase, CAT, luciferase, an immunogenic tag peptide
sequence, an extrinsically activatable enzyme, an extrinsically activatable toxin, an
extrinsically activatable fluor, an extrinsically activatable quenching agent, a radioactive
element or an antibody.
[57] A method for screening candidate anti-cancer agents comprises determining the
viability of a mammalian cell which expresses 0CT6 incubated in the presence and absence
of a test compound and identifying the test compound as a potential anti-leukemia agent if
17
there is a cellular uptake of the test compound and cell death. Analysis of cell viability can
be accomplished by any means known in the art.
[58] It is well known in the art that viability of a cell caii be determined by contacting the
cell with a dye and viewing it under a microscope. Viable cells can be observed to have an
intact membrane and do not stain, whereas dying or dead cells having "leaky" membranes do
stain. Incorporation of the dye by the cell indicates the death of the cell. The most common
dye used in the art for determining viability is trypan blue. Viability of cells can also be
determined by detecting DNA synthesis. Cells can be cultured in cell medium with labeled
nucleotides (e.g., thymidine). The uptake or incorporation of the labeled nucleotides
indicates DNA synthesis and cell viability. In addition, colonies formed by cells cultured in
medium indicate cell growth and is another means to test viability of the cells.
[59] Identification and/or observation of cells undergoing apoptosis can be another method
of determining cell viability. Apoptosis is a specific mode of cell death recognized by a
characteristic pattern of morphological, biochemical, and molecular changes. Cells going
through apoptosis appear shrunken, and roimded; they also can be observed to become
detached from culture dish. Thermophological changes involve a characteristic pattern of
condensation of chromatin and cytoplasm which can be readily identified by microscopy.
When stained with a DNA-binding dye, such as H33258, apoptotic cells display classic
condensed and punctate nuclei instead of homogeneous and round nuclei.
[60] The hallmark of apoptosis is the endonucleolysis, a molecular change in which
nuclear DNA is initially degraded at the linker sections of nucleosomes to give rise to
fragments equivalent to single and multiple nucleosomes. When these DNA fragments are
subjected to gel electrophoresis, they reveal a- series of DNA bands which are positioned
approximately equally distant from each other on the gel. The size difference between the
two bands next to each other is about the length of one nucleosome (i.e., 20 base pairs). This
characteristic display of the DNA bands is called a DNA ladder and it indicates apoptosis of
the cell. Apoptotic cells can be identified by flow cytometric methods based on
measurement of cellular DNA content, increased sensitivity of DNA to denaturation, or
altered light scattering properties. These methods are well known in the art and are within
the contemplation of the invention.
18
[61] Abnormal DNA breaks are also characteristic of apoptosis and can be detected by any
means known in the art. In one embodiment, DNA breaks are labeled with biotinylated
dUTP (b-dUTP). Ceils are fixed and incubated in the presence of biotinylated dUTP with
either exogenous terminal transferase (terminal DNA transferase assay; tdT assay) or DNA
polymerase (nick translation assay; NT assay). The biotinylated dUTP is incorporated into
the chromosome at the places where abnormal DNA breaks are repaired, and are detected
with fluorescein conjugated to avidin under fluorescence microscopy.
Kits
[62] The present invention provides kits that can be used in the above screening methods.
In one embodiment, a kit comprises a substantially isolated polypeptide comprising an 0CT6
epitope which is specifically immunoreactive with only test compound(s) that are substrates
of the OCT6 transporter protein. Binding of a test compound to the 0CT6 epitope is
indicative that the test compound is a 0CT6 substrate. In another embodiment, a kit
comprises a cell line that overexpresses an 0CT6 transporter protein. Binding and/or cellular
uptake of a test compound via the 0CT6 protein is indicative that the test compound is a
0CT6 substrate. Preferably, the kits of the present invention further comprise a control
compound or antibody which does not react with the 0CT6 transporter protein. In another
specific embodiment, the kits of the present invention contain a means for detecting the
binding of a test compound to an 0CT6 epitope and/or cellular uptake of a test compound.
For example, the test compound may be conjugated to a detectable substrate such as a
fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent
compound, or a second antibody which recognizes the first antibody may be conjugated to a
detectable substrate,
[63] The detectable substance may be coupled or conjugated either directly to the test
compound (or fi-agment thereof) or indirectly, through an intermediate (such as, for example,
a linker known in the art) using techniques known in the art. See, for example, U.S. Pat. No.
4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics
according to the present invention. Further non-limiting examples of detectable substances
include various enzymes, prosthetic groups, fluorescent materials, luminescent materials,
19
bioluminescent materials, radioactive materials, positron emitting metals using various
positron emission tomographies, nonradioactive paramagnetic metal ions, immunogenic tag
peptide sequences, extrinsically activatable toxins, extfinsicaiiy activatabie quenching agents,
or antibodies. Non-limiting examples of suitable enzymes include horseradish peroxidase,
alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable
prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of
suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate,
rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example
of a luminescent material includes luminol; examples of bioluminescent materials include
luciferase, luciferin, and aequorin; and examples of suitable radioactive material include
'^'l,^"lnor^^Tc.
Inununo genie Compositions
[64] The present invention also provides immunogenic compositions for the treatment of
hematological malignancies. Non-limiting exemplary hematological malignancies include,
but are not limited to, Hodgkin's disease, leukemia such as, acute lymphoid (lymphocytic or
lymphoblastic) leukemia (ALL), acute myeloid (myelogenous or myeloblastic) leukemia
(AML), acute lymphoid leukemia, biphenotypic (ALL, biphentoypic), acute undifferentiated
leukemia (AUL), chronic myeloid (myelogenous or granulocytic) leukemia (CML),
erythroleukemia, granuloxytic leukemia, lymphoma, monocytic leukemia, myleoma,
myelomonocytic leukemia, myelodysplastic syndromes, non-Hodgkin lymphoma,
progranulocytic leukemia.
[65] According to the invention immunogenic compositions for the treatment of
hematological malignancies comprise a substrate recognized by an 0CT6 transporter protein.
Preferably, the substrate is a compound that binds selectively or specifically to a 0CT6
transporter protein. In a preferred embodiment, the compound binds selectively to the 0CT6
transporter protein encoded by a nucleotide sequence of SEQ ID N0:1. The compound may
be a cytotoxin or coupled or conjugated with a cytoxic agent. Preferably the cytoxin or
cytotoxic agent is a chemotherapeutic agent.
20
[66] The present invention also provides pharmaceutical compositions. Such compositions
comprise a therapeutically effective amount of a compound, and a pharmaceutically
acceptable carrier.
[67] Cell surface proteins like the 0CT6 transporter can be utilized in antibody-based
targeting strategies. In still another aspect of the invention, antibodies can be developed by
known methods in the art against the external epitope of 0CT6 transporter protein. In a
preferred embodiment, antibodies are substrates of the 0CT6 protein. The antibodies may be
polyclonal antibodies or monoclonal antibodies.
[68] Polyclonal antibodies to an antigen-of-interest can be produced by various procedures
well known in the art. For example, a polypeptide of the invention can be administered to
various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the
production of sera containing polyclonal antibodies specific for the antigen. Various
adjuvants may be used to increase the immunological response, depending on the host
species, and include but are not limited to, Freund's (complete and incomplete), mineral gels
such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole Umpet hemocyanins, dinitrophenol, and
potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and
corynebacterium parvum. Such adjuvants are also well known in the art.
[69] Monoclonal antibodies can be prepared using a wide variety of techniques known in
the art including the use of hybridoma, recombinant, and phage display technologies, or a
combination thereof For example, monoclonal antibodies can be produced using hybridoma
techniques including those known in the art and taught, for example, in Harlow et al.,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);
Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier,
N.Y., 1981) (said references incorporated by reference in their entireties).
[70] The present invention further encompasses antibodies or fragments thereof
conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically to,
for example, monitor the development or progression of a tumor as part of a clinical testing
procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be
facilitated by coupling the antibody to a detectable substance. Examples of detectable
21
substances include various enzymes, prosthetic groups, fluorescent materials, luminescent
materials, bioluminescent materials, radioactive materials, positron emitting metals using
various positron emission tomographies, and noruadioactive paiwiagnetic metal ions. The
detectable substance may be coupled or conjugated either directly to the antibody (or
fragment thereof) or indirectly, through an intermediate, such as, for example, a linker known
in the art, using techniques known in the art. (See, for example, U.S. Pat. No. 4,741,900 for
metal ions which can be conjugated to antibodies for use as diagnostics according to the
present invention.) Examples of suitable enzymes include horseradish peroxidase, alkaline
phosphatase, beta-galactosidase, or acetylchoUnesterase; examples of suitable prosthetic
group complexes include streptavidin/biotin and avidin/biotin; examples of suitable
fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate,
rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example
of a luminescent material includes luminol; examples of bioluminescent materials include
luciferase, luciferin, and aequorin; and examples of suitable radioactive material include ^^^I,
'^'l.'%or^'Tc.
[71] Further, an antibody or fragment thereof may be conjugated to a therapeutic moiety
such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive
metal ion, e.g., alpha-emitters such as, for example, Bi. Non-limiting examples include
paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,
tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy
anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,
glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or
homologs thereof Therapeutic agents include, but are not limited to, antimetabolites (e.g.,
methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine),
alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine
(BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,
streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),
anthracycUnes (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g.,
dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)),
and anti-mitotic agents (e.g., vincristine and vinblastine).
22
[72] The conjugates of the invention can be used for modifying a given biological
response such as inducing cell death for the treatment and prevention of hematological
malignancies like leukemia. The therapeutic agent or drag moiety is not lo be construed as
limited to classical chemical therapeutic agents. For example, the drug moiety may be a
protein or polypeptide possessing a desired biological activity for inducing cell death. Such
proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria toxin; a protein such as tumor necrosis factor, a-interferon, .beta.-interferon, nerve
growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic
agent, e.g., TNF-alpha, TNF-beta, AIM I (See, International Publication No. WO 97/33899),
AIM II (See, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al., Int.
Immunol., 6:1567-1574 (1994)), VEGI (See, Intemational Publication No. WO 99/23105), a
thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, biological
response modifiers such as, for example, lymphokines, interleukin-1 ("IL-T'), interleukin-2
C'IL-2"), interleukin-6 ('TL-6"), granulocyte macrophage colony stimulating factor ("GM-
CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.
Therapeutic Treatment
[73] The present invention is further directed to methods for preventing and treating
hematological malignancies such as leukemia. According to the invention, hematological
malignancies comprise without limitation, Hodgkin's disease, leukemia such as, acute
lymphoid (lymphocytic or lymphoblastic) leukemia (ALL), acute myeloid (myelogenous or
myeloblastic) leukemia (AML), acute lymphoid leukemia, biphenotypic (ALL,
biphentoypic), acute undifferentiated leukemia (AUL), chronic myeloid (myelogenous or
granulocytic) leukemia (CML), erythroleukemia, granuloxytic leukemia, lymphoma,
monocytic leukemia, myleoma, myelomonocytic leukemia, myelodysplastic syndromes, non-
Hodgkin lymphoma, progranulocytic leukemia.
[74] Methods of treatment of the present invention comprise administering to a subject in
need thereof an immunogenic composition of the present invention. The compositions may
be administered with a pharmaceutically acceptable carrier.
23
[75] Such pharmaceutical carriers can be sterile liquids, such as water and oils, including
those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical
composition is administered intravenously. Saline solutions and aqueous dextrose and
glycerol solutions can also be employed as liquid carriers, particularly for injectable
solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose,
gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc,
sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
The composition, if desired, can also contain minor amounts of wetting or emulsifying
agents, or pH buffering agents. These compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and
the like. The composition can be formulated as a suppository, with traditional binders and
carriers such as triglycerides. Oral formulation can include standard carriers such as
pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine,
cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are
described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions
will contain a therapeutically effective amoxmt of the compound, preferably in purified form,
together with a suitable amount of carrier so as to provide the form for proper administration
to the patient. The formulation should suit the mode of administration.
[76] In a preferred embodiment, the composition is formulated in accordance with routine
procedures as a pharmaceutical composition adapted for intravenous administration to human
beings. Typically, compositions for intravenous administration are solutions in sterile
isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing
agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
Generally, the ingredients are supplied either separately or mixed together in unit dosage
form, for example, as a dry lyophilized powder or water free concentrate in a hermetically
sealed container such as an ampoule or sachette indicating the quantity of active agent.
Where the composition is to be administered by infusion, it can be dispensed with an infusion
bottle containing sterile pharmaceutical grade water or saline. Where the composition is
24
administered by injection, an ampoule of sterile water for injection or saline can be provided
so that the ingredients may be mixed prior to administration.
[77] The compGunds of the invention can be fonnulated as neutral or salt forms.
Pharmaceutically acceptable salts include those formed with anions such as those derived
from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with
cations such as those derived from sodium, potassium, ammonium, calcium, ferric
hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[78] The amount of the compound of the invention which will be effective in the
treatment, inhibition and prevention of hematological malignancies can be determined by
standard clinical techniques. In addition, in vitro assays may optionally be employed to help
identify optimal dosage ranges. The precise dose to be employed in the formulation will also
depend on the route of administration, and the seriousness of the disease or disorder, and
should be decided according to the judgment of the practitioner and each patient*s
circumstances. Effective doses may be extrapolated from dose-response curves derived from
in vitro or animal model test systems.
[79] Various other deHvery systems are known and can be used to administer a
composition of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules,
recombinant cells capable of expressing the compound, receptor-mediated endocytosis (See,
e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid as
part of a retroviral or other vector, etc. Methods of introduction include but are not limited to
intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural,
and oral routes. The compounds or compositions may be administered by any convenient
route, for example by infiision or bolus injection, by absorption through epithelial or
mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be
administered together with other biologically active agents. Administration can be systemic
or local. In addition, it may be desirable to introduce the pharmaceutical compounds or
compositions of the invention into the central nervous system by any suitable route, including
intraventricular and intrathecal injection; intraventricular injection may be facilitated by an
intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.
25
Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and
formulation with an aerosolizing agent.
[80] in a specific embodiment, it may be desirable to administer the pharmaceutical
compounds or compositions of the invention locally to the area in need of treatment; this may
be achieved by, for example, and not by way of limitation, local infusion during surgery,
topical application, e.g., in conjunction with a wound dressing afler surgery, by injection, by
means of a catheter, by means of a suppository, or by means of an implant, said implant
being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic
membranes, or fibers. Preferably, when administering a protein, including an antibody, of
the invention, care must be taken to use materials to which the protein does not absorb.
[81] In another embodiment, the compound or composition can be delivered in a vesicle,
in particular a liposome (See Langer, . Science 249:1527-1533 (1990); Treat et al., in
Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler
(eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see
generally ibid.)
[82] In yet another embodiment, the compovmd or composition can be delivered in a
controlled release system. In one embodiment, a pump may be used (see Langer, supra;
Seflon, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980);
Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials
can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC
Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and
Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J.,
Macromol. Sci. Rev, Macromol. Chem. 23:61 (1983); see also Levy et al.. Science 228:190
(1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105
(1989)). In yet another embodiment, a controlled release system can be placed in proximity
of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose
(see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-
138(1984)).
[83] In a specific embodiment where the compound of the invention is a nucleic acid
encoding a protein, the nucleic acid can be administered in vivo to promote expression of its
26
encoded protein, by constructing it as part of an appropriate nucleic acid expression vector
and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see
U.S. Pat. No. 4,980,286), or by direct injection, or by use of microparticle bombardment
(e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or
transfecting agents, or by administering it in linkage to a homeobox-like peptide which is
known to enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci. USA 88:1864-1868
(1991)), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated
within host cell DNA for expression, by homologous recombination.
Examples
[84] The following examples are presented for the illustrative purposes and it is to be
understood that the present invention is not limited to those precise embodiments, and that
various changes and modifications can be effected therein by one skilled in the art without
departing from the scope and spirit of the invention as defined by the appended claims.
[85] Example 1. 0CT6 Nucleotide Sequence Identification and Analysis
[86] 0CT6 was first identified as a potential OCT gene by assembling and sequencing
ESTs. BLAST searches of human ESTs in GenBank data base identified AI040384 (654 bp),
AA033971 (714 bp) and H70190 (474 bp) sequences from three fetal liver IMAGE clones,
1656502, 429904 and 212935 respectively. IMAGE clone 1656502 (3', insert 1337 bp)
ended the predicted 3' stop codon, whereas IMAGE clone 429904 (5', insert 996 bp) and
IMAGE clone 212935 (5', insert 966 bp) aUgned with the 5 -coding region. All clones were
obtained from the IMAGE Consortium through the American Type Culture Collection
(Manassas, VA). Each clone was sequenced in both directions. The sequences were
determined using ABI Prism TM 377 DNA sequencer (Perkin-Ehner). Our assemblage
proved to be identical to the recently submitted cDNA 0KB 1 (AF268892) submitted by M.
Okabe and T. Abe. We have dubbed this gene 0CT6 as OCTNl and 0CTN2 may be
considered as 0CT4 and 0CT5 respectively.
[87] The 0CT6 gene (SEQ ID N0:1) is also contained within BAG clone CTA-331P3
(SEQ ID N0:3) (GenBank AC002464) located at chromosome 6q21. It is divided into 6
27
exons that span 42 kb on the human genome, from nucleotide 79,570 to nucleotide 120490
onCTA-331P3.
[88] The gene has a predicted protein stracture typical of transport proteins with 2 groups
of 6 transmembrane domains separated by a hydrophilic region (Figure lA). The large
hydrophilic region between TMDl and TMD2 is typical of OCT and OAT genes and . is
presumed to be located on the outside surface of the cell membrane. The 0CT6 protein
contains potential sites for N-glycosylation and phosphorylation, which will be described
below in Methods. Of interest, the protein sequence also contains a 22 amino acid leucine
zipper motif, starting at amino acid 146, suggesting that there may be a physical interaction
between 0CT6 and ion channels or other membrane-associated proteins.
[89] CLUSTALW alignment produced a dendrogram showing the phylogenic relationship
between 0CT6 and other OAT and OCT proteins (Figure IB). This dendrogram suggests
that the distinction between OAT and OCT genes, based on functional studies, obscures the
common origin of both famiUes of transporters. The actual CLUSTALW alignment of these
genes is shown in Figure 2 and demonstrates multiple regions of conservation among all of
these genes.
[90] The hydropathy profile analysis, multiple sequence alignments of amino acid
sequences using CLUSTALW and the phylogenetic tree were all produced with Mac Vector
software.
[91] Example 2 . Molecular cloning of 0CT6
[92] BLAST searches of human ESTs in GenBank data base identified AI040384 (654
bp), AA033971 (714 bp) and H70190 (474 bp) sequences from three fetal liver IMAGE
clones, 1656502, 429904 and 212935 respectively. IMAGE clone 1656502 (3', insert 1337
bp) ended the predicted 3' stop codon, whereas IMAGE clone 429904 (5', insert 996 bp) and
IMAGE clone 212935 (5', insert 966 bp) aligned with the 5'-coding region. All clones were
obtained from the IMAGE Consortium through the American Type Culture Collection
(Manassas, VA). Each clone was sequenced in both directions. The sequences were
determined using ABI Prism TM 377 DNA sequencer (Perkin-Elmer).
28
[93] Example 3. Quantitative RT-PCR of 0CT6 RNA levels in cancer cell lines
[94] Total RNA isolated from 50 cell lines used in the NCI drug screen program was
provided by Ihe Developmental Therapeutics Program, NCI. Quantitative RT-PCR for
detecting OAT-X transporter gene expression was performed by using a Roche LightCycler,
which uses real time fluorescence detection for quantitative measurement of PCR products.
A gene-specific primer pair was designed with Oligo 4.0 software and purchased from
Integrated DNA Technologies, Inc. (Coralville, lA) (F: 5'-
GGCACATTTATTCACCAAGACCAG-3') (SEQ ID N0:13) and (F: 5'-
TGTGGACCTCAGCAGCATTTGGAT -3') (SEQ ID NO: 14). The specificity of the PCR
reaction was confirmed by directly determining the DNA sequence of the PCR product.
First, cDNA was synthesized from total RNA using Superscript First-Strand Synthesis
System (GIBCO/BRL) in a 20 ^il volume following the instructions suppUed by the
manufacturer. The cDNA treated with RNAse H for 20 minutes at 37°C and stored at -
20°C. Then, 2ul of cDNA reaction was amplified in a standard PCR reaction condition,
using 0.3 |xM primer concentration, with the addition of SYBR Green I Dye. After 30
seconds denature at 95°C, the amplification reaction proceeded through 45-50 cycles of 95°C
denature for 0 second, 62-65 °C annealing for 10 seconds and a 72°C extension for 40
seconds, with slopes of 20^C/s, 20°C/s and 2°C/s, respectively,
[95] Fluorescence was acquired during each cycle after heating to a temperature just below
the product melting temperature. Quantification was performed using the LightCycler
analysis software. The log-linear portion of the standard amplification curve was identified,
and the 'crossing point', a threshold of relative fluorescence, was determined as the best fit
through the log-linear region above the background fluorescence (noise) band. The
quantification of PCR product then was derived by plotting fluorescence data in the log linear
region of each sample to determine a calculated number of cycles needed to reach the
fluorescence crossing point. The calculated number of cycles required to reach the crossing
point is proportional to the amount of target RNA in the sample. The relative amount of
product was described in arbitrary units by interpolation of the data using a standard curve of
a series of dilutions of a standard cell line RNA. The quantitative measurement of each gene
in each cell Une was normalized to the relative amount of actin RNA in each cell line, as a
29
control for equivalent cDNA loading in each sample. The results represent the average of 3
independent determinations performed in duplicate.
[96] A melting curve analysis was peffomied with positive control RNA prior analysis of
the cell lines to enhance sensitivity and the specificity of the data. Amplified products
usually melt quickly at a temperature characteristic for the products. The fluorescence signal
was acquired at a temperature just below the Tm of the specific PCR product and above the
Tm of the primer dimers. All specific PCR products displayed a single, sharply melting
curve with a narrow peak. In addition, PCR products were confirmed for specificity and
correct size by visualization of the LightCycler products on a 1% agarose gel.
[97] Example 4. Tissue distribution
[98] First strand cDNAs derived fi"om 24 aduh and fetal tissues (RAPID-SCAN gene
expression panel, OriGene Technologies, Rockville, MD). The PCR primers used in this
study were the same as used in the quantitative RT-PCR studies. The PCR reaction samples
o o
were denatured at 94 C for 30 seconds, annealed and extended at 64 C for 30 sec for 35
cycles. The PCR products were then visualized on 1% agarose gels.
[99] Example 5. Cell sorting
[100] All human specimens were obtained in accordance with institutional IRB guidelines.
Leukocytes from fresh discarded buffy coats were isolated after RBC lysis with ammonium
chloride and labeled with lineage specific antibodies (CD 14, monocytes; CD 15,
granulocytes; CD3, T-cells; and CD20, B-cells), and isolated using a FACSVantage flow
cytometer. Each population was sorted twice to ensure purities of at least 99%. CD34 cells
were obtained fi-om discarded aliquots of G-CSF-mobilized peripheral blood stem cell
collections fi-om cancer patients. For each sample, the PCR results represent the pooled
average of cells fi-om 2 individuals performed in triplicate or quadruplicate.
[101] Example 6. 0CT6 RNA levels in leukemic blasts
[102] Total RNA was extracted fi:om leukemia specimens using QIAGEN RNeasy midi kit.
150ng of total RNA were used as a template for the first strand cDNA synthesis with the
30
Oligo(dT) primer using the super script system (GEBCO BRL) according to the
manufacturer's protocol. Quantitative real-time RT-PCR was performed using an iCycler
tliermal cycler wltli methods similar to those described above for the Roche LightCycier.
The results represent the average of 3 independent determination performed in duplicate.
[103] Although illustrative embodiments of the present invention have been described in
detail, it is to be imderstood that the present invention is not limited to those precise
embodiments, and that various changes and modifications can be effected therein by one
skilled in the art without departing from the scope and spirit of the invention as defined by
the appended claims.
