Note: Descriptions are shown in the official language in which they were submitted.
~0 94/20~23 2 1 S ~ 9 ~ 2 PCr/US94102610
TUMOR TAR~ r~G WITH L_ENANTIO~OERIC
Ol.IGONU~;1.EOTIDE CON~GATES OF
IMhU~C)k~:~GF.r~T.~ OF t~t~F~T~T~n R~ oN~
F~ el ~1 of the Invont~ or~
This invention relates to sequential targeting and
delivery of ~r-~noreagent compositions which find
S particular utility in the therapy and diagnostic imaging
of c~ncer by means of a tumor targeted sequential
delivery system comprising a primary non-radioactive
targeting immunoreagent and a secon~Ary radioactive
delivery agent. This invention al80 relates to novel
0 methods for the attachment of L~ nt~ omeric
oligonucleotides, complementary L-enantiomeric
oligonucleotides, and chelates, to proteins, and to
bifurcated tumor targeting and delivery vectors for the
treatment and ~ nostic imaging of tumors.
R~ckcrro~nr~ of the Tnvent~ ~n
The various, currently aVA~lAhle~ radiolabeled
~mm~noreactive proteins and methods which are employed
in diagnostic imaging and targeted the ~euLic
applications suffer from certain disadvantages. For
example, rA~o~mm~notherapy and diagnostic imaging with
the various currently available radioph~r~-ceuticals
which include radionuclide-con~ n ~ n~ immunoreactive
proteins can be less than optimal because these
radioph~rm~ceuticals may bind to non-target normal
tissue. This binding can result in undesirable toxicity
to normal tissue during therapeutic applications as well
as in high background signals during diagnostic imaging
applications. Inefficient covalent hondtng of the
. 30 radioactive component with protein in conjugate
preparation can be another problem due to release of the
radioactive component which may then deposit in healthy
tissue. Also, long plasma half-lives of currently
available radionuclide-cont~;n~ng ~mm~lnoreactive
~472Q5~ ~~ ~ PCT~S94/02610
proteins and slow clearance of radionuclide from the
body can result in prolonged exposure of normal tissue
to damaging effects of radiation and can produce
unacceptable toxic effects in otherwise normal and
disease free tissues in the body, especially in those
tissues and cells most sensitive to radiation damage,
e.g., the stem cells of the bone marrow and
gastrointestinal tract. While the number of ionic
radionuclides that can be associated with an
0 immunoreactive protein is restricted by the number of
sites of chelation available, an increase in that number
which can be achieved by increasing the number of
chelating agents conjugated to the protein can produce a
decrease in the immunoreactivity of the protein. This
can limit the number of such chelating agents that can
be attached to the protein. The number of chelating
agents that can be attached to an immunoreactive protein
is also limited by the number of available groups such
as, for example, amino groups suitable for use in
attachment of the chelating agents and by the potential
immunogenicity of the thus modified protein which, being
highly derivatized, could be recognized by a host immune
system as being haptenated.
It is an object of the present invention to
overcome some of the aforementioned disadvantages of the
currently available radiolabeled immunoreactive
proteins.
Nucleic acids in the form of deoxyribonucleic acid
(DNA) and ribonucleic acid (RNA) encode and transfer
genetic information for the cellular synthesis of
proteins and enzymes. Naturally occuring nucleic acids
are composed of nucleosides such as 2'-deoxyadenosine
(dA), 2'-deoxyguanosine (dG), 2'-deoxycytidine (dC), and
thymidine (T) in DNA and of adenosine (A), guanocine
(G), cytidine (C), and uridine (U) in RNA. Naturally
occuring modified nucleosides such as those containing
~ W094/205~ 215 7 ~ 0 2 PCT~594102610
2'-O-methylribosyl groups and those containing bases
such as N4,N4-dimethyladenine and N7-methylguanidine are
found in messenger, transfer, and ribosomal RNA.
Internucleosidyl phosphodiester bonds link nucleosides
at the oxygen of the 3'-hydroxyl group of a D-ribose or
of a D-deoxyribose sugar moiety in one nucleoside to the
oxygen of a 5'-hydroxyl group in a D-ribose or D-
deoxyribose sugar moiety in another nucleoside in RNA
and in DNA, respectively. Separate ch~; ns of nucleic
0 acids interact with one another via hydrogen bonds
formed between complementary pairs of nucleosidyl purine
and pyrimidine bases: adenine with thymine and guanine
with cytosine in DNA, and adenine with uracil and
guanine with cytosine in RNA. When the sequences of
bases in two separate oligonucleotide strands or in two
regions of a single strand are complementary to each
other, the complementary sequences can hybridize with
each other via hydrogen bonds between complementary base
pairs to form a right-handed double helix with a B-type
conformation, the two phosphate-ribose ester backbones
of which are antiparallel.
Structure I
o~p~o
~ O' ~O 5 Base
~cO~,
~,~z
O R1
D-Enantiomers
D Dl`J~: R1, H
~RNA: R1 - OH
.
21579~2
W094/205~ PCT~S94/02610
In naturally occurring DNA each nucleoside unit is
a D-enantiomer whose s~ucture is defined by the
chirality of the D-deoxyribose ring which has
substituents at the l'-(b), 3'-(a), and 4'-(b) positions
as represented schematically in Structure I wherein R
is H and "Base" represents an adenine, guanine, thymine
or cytosine moiety. In naturally occurring RNA each
nucleoside unit is also a D-enantiomer whose structure
is defined by the chirality of the D-ribose ring which
has substituents at the l'-(b), 2'-(a), 3'-(a), and 4'-
(b) positions as represented schematically in Structure
I wherein Rl is OH and ~IBase~ represents an adenine,
guanine, uracil or cytosine moiety. In both naturally
occurring DNA and RNA, the phosphate diesters and the
bases do not comprise chiral centers.
As noted by P. S. Miller in Biocon~ugate Chem.
l990, l, 187-l9l, synthetically prepared
oligonucleotides composed of naturally configured D-
enantiomers have been used as primers for nucleic acid
polymerizing enzymes, as synthons in the construction of
artificial genes for the preparation of proteins by
recombinant DNA techniques, and as diagnostic probes to
detect and characterize cellular nucleic acid sequences.
In addition, synthetically prepared oligonucleotides
have been investigated for use in the control of gene
expression in living cells, and as therapeutic agents in
the inhibition of viral replication and in the treatment
of cancer. These applications rely on the sequence
specific complementary binding properties of the
synthetic oligonucleotides with natural D-enantiomeric
oligonucleotide target sequences.
Synthetically prepared oligonucleotides composed of
naturally configured D-enantiomers can be generated by a
variety of methods, the currently most useful of which
include solid phase synthesis via phosphoramidite
interm~ tes and solid phase synthesis via H-
~0 g4/205~ 2 1 5 ~ 9 0 2 PCT~S94/02610
phosphonate intermediates as described by E. Uhl ~nn and
A. Peyman in Chemical Reviews, l990, ~Q, 544. In the
phosphoramidite method, a 5'-hydroxyl group of a growing
DNA oligomer chain of D-enantiomers having amide
protecting groups on the exocylic amine groups of the
bases therein and which is attached to a solid phase
support reacts with an activated D-nucleoside 5'-O-
dimethoxytrityl-3'-(2-cyanoethyl N,N-diisopropyl)-
phosphoramidite in the presence of lH-tetrazole as a
0 catalyst. Commonly used protecting groups include the
benzoyl group for the protection of the exocyclic amino
groups of A~n~ne and cytosine and the isobutyryl group
for the protection of the exocyclic amino group of
guanine. Any unreacted 5'-hydroxyl groups are capped
lS with acetate groups by reaction with acetic anhydride in
the presence of 4-N,N-dimethyl~m~ nopyridine. The
resulting phosphite is then oxidized with iodine to form
a phosphotriester. The 5'-O-dimethoxytrityl group is
removed under acid conditions using dichloroacetic acid,
and the reaction sequence is repeated using another
activated D-nucleoside 5'-O-dimethoxytrityl-3'-(2-
cyanoethyl N,N-diisopropyl) phosphoramidite. At the end
of the synthetic sequence, the oligonucleotide is freed
from amide protecting groups and cleaved from the
support by treatment with ammonium hydroxide. The
oligonucleotide is then purified or isolated by methods
such as precipitation, electrophoresis, or
chromatography.
Many in vivo applications of synthetic D-
enantiomeric oligonucleotides are limited by the rapid
destruction of the oligomers by nucleases which are
encountered both extracellularly and intracellularly.
As reviewed by J. Goodchild in Bioconjugate Chem. l990,
l, l65, strategies to introduce resistance to nuclease
activity included modification of the nucleoside bases,
and of the phosphate and ribose components of the
2157~2
W094/205~ PCT~S94/02610
polymer backbone. All of these were done with the need
to maintain the ability of the'modified oligonucleotide
to hybridize with a na~'uràlly occuring D-enantiomeric
DNA or RNA target sequence or to bind with an enzyme
that uniquely recognizes the D-enantiomer.
The effects on the properties of D-oligonucleotide~
of a single modification of oligonucleoside chirality
such as can be achieved through inversion at the l'-
ribose site to form an a-anomer, represented in
0 Structure II, have been studied by Bloch et al in Gene,
1988, 72, 349. These workers have found that alpha-
anomeric DNA hybridizes with complementary beta-anomeric
RNA, the naturally occurring enantiomeric oligomer, to
form an a:b DNA:RNA hybrid, and that the hybrid is
nuclease resistant.
Structure II
o~p~o
--o' ~O s
~Base
O R1
a-Anomers
a~N~ Rl.H
a~N~ R~.CH
Thuong and Chassignol in Tetrahedron Letters, 1988,
2~, 5905 reported the solid phase synthesis employing 2-
cyanoethyl phosphoramidite synthons of oligo-a-
deoxynucleotides containing a-d-T, a-d-C, and a-d-A
covalently linked at the 5'-phosphate to 2-methoxy-6-
chloro-9-(5-hydroxypentylamino)acridine. Analogous
oligo-a-deoxynucleotides without the acridine moiety
~ W094/205~ 2 1 5 7 ~ 0 2 PCT~S94/02610
were reported to be resistant to nucleases and to form
hybrids with natural D-RNA that were more stable than
those with b-deoxynucleotides. Morvan et al in Nucleic
Acid Research, 1988, 16, 833, reported the preparation
of oligo-a-deoxynucleotides containing a-d-T, a-d-C, a-
d-G, and a-d-A which formed double helix hybrids or
duplexes with complementary a and b strands with
parallel and antiparallel polarity, respectively. These
duplexes retained specific Watson-Crick base pairing
specificity, and the heteroduplexes were shown to belong
to the B-DNA family. They also showed higher thermal
stability when compared to the corresponding naturally
occurring bb-DNA duplexes. Again, however, the target
oligomer was a naturally occurring D-oligomer.
Complete inversion of all chiral sites of the D-
enantiomer in Structure I provides the mirror image,
non-naturally occurring L-enantiomer which is
represented in Structure III. In the absence of a
chiral environment or a chiral reagent such as an enzyme
which can distinguish between D- and L-enantiomers, the
L-enantiomer is identical in chemical reactivity to the
D-enantiomer. Urata et al in J. Am. Chem. Soc. 1991,
11~, 8174 reported the synthesis (via the 2-cyanoethyl
phosphoramidite method) and properties of the self-
complementary L-DNA oligomer duplex, L-d(CGCGCG) and
compared them to those of the mirror image, natural D-
d(CGCGCG) oligomer. The unnatural all-L-duplex was
resistant to digestion by nuclease P-1 while the natural
all-D-duplex was rapidly degraded to the component D-
nucleotides and 5'-end deoxycytidine. As expected, both
the DD- and the LL-duplexes exhibited identical HPLC
retention times and their circular dichroism (CD)
spectra were mirror images of one another. In 0.1 M
NaCl, the D-enantiomer showed the CD profile of a
standard B form while the L-enantiomer exhibited the
same magnitude but opposite sign at all wavelengths.
2 ~ 5 ~
W094/205~ PCT~S94/02610
This implied the existance of a mirror image B form
helix with left-handed double-helical conformation. In
4 M NaCl, the D-enantiomer,s~owed the pro~ile of a left-
handed Z form while the L-ënantiomer exhibited the ~ame
S magnitude but opposite sign at all wavelengths
indicating the existance of a mirror image Z form with a
right-handed double helical conformation. The CD
spectra of both the all D-duplex and all L-duplex forms
exhibited the same B to Z conformational transition
0 (with opposite signs) as a function of salt
concentration as well as the same temperature dependence
under both low and high salt concentrations. It was
concluded that the D- and L-DNA have the same type and
strength of hydrogen bonding and base stacking
interactions, and that the structure of the L-DNA is the
exact mirror image of that of the D-DNA. This all L-
enantiomeric oligomer, being self-complementary, could
not be used to target either a separate natural D- or
unnatural L-enantiomer.
Structure III
0~,
Base S O' `O--
1~C~/ ~
z/ ~ 5
Rl O
L-Enantiomers
L~ R, . H
L-RNA: R, ~ OH
Fujimori et al in Nucleic Acid Research, l990, 22,
97 reported the synthesis of 9-(2-deoxy-b-L-erythro-
~ W094/205~ 215 7 9 ~ 2 PCT~S94/02610
pentafuranosyl)-9H-purin-6-amine, L-dA, and the
hexameric L-DNA, L-d(AAAAAA). They observed L-d ~ R)
to have resistance to bovine spleen phosphodiesterase
while D-d(AAAAAA) was completely degraded. They also
noted that the L-d~AAAAAA) formed a complex with a
natural D-RNA enantiomer, D-poly(U), which was weaker
relative to that formed between the enantiomeric natural
D-d(AAAAAA) and D-poly(U). No complex was formed
between the unnatural L-d (A~ ) and the natural DNA
0 enantiomer D-poly(T). These results suggested that
while L-enantiomeric DNA has the ability to distinguish
complementary RNA from DNA, the unnatural L-DNA:D-RNA
complexes are not nearly as strong as the natural
corresponding D-DNA:D-RNA complexes.
Although L-DNA has been shown to hybridize in vitro
with natural enantiomeric D-RNA and to be stable to
nuclease resistance, in practice such a duplex formation
with targeted D-RNA is not readily achievable in vivo
since it is necessary for the L-DNA to cross at least
one cell membrane to find the intact D-RNA.
Furthermore, natural D-RNA has a relatively short
lifetime in the presence of naturally occurring nuclease
enzymes.
.~llmm~ry of the Inv~nt;on
The present invention is directed to a non-
radioactive targeting ~ lnoreagent that comprises a
tumor antigen recognizing moiety, one or more L-
enantiomeric oligonucleotides comprising non-self-
associating L-enantiomeric oligonucleotide sequences,
and one or more l;nk;ng groups.
The present invention is also directed to a
radioactive targeting immunoreagent that comprises an L-
enantiomeric oligonucleotide comprising an L-
enantiomeric oligonucleotide sequence that is
complementary in sequence to and capable of
2~7~2
W094/205~ PCT~S94/02610
hybridization with one ~or more fragments of a non-self-
associating L-ena~iomeric oligonucleotide sequence, one
or more chelating agents, one or more linking groups and
one or more radionuclides.
The present invention is also directed to
pharmaceutical compositions comprising one or more of
the above-described immllnoreagents and a
pharmaceutically acceptable carrier.
The present invention is further directed to
0 methods for treating and imaging disease sites such as
tumor sites in a patient. Said methods comprise
a~m; ni stration to the patient of an effective amount of
the above-described non-radioactive targeting
;mmllnoreagent followed at an effective time interval by
an effective amount of the above-described radioactive
targeting immunoreagent.
The present invention provides many advantages
co~r~red to conventional targeting immunoreagents. For
example, the non-radioactive targeting immunoreagent can
accumulate at a tumor site in vivo while it is not
accumulated at normal tissue sites.
The in vivo residence half life of the non-
radioactive targeting immunoreagent is long enough to
permit its accumulation at a tumor site.
The in vivo residence half life of the radioactive
targeting reagent is shorter than the residence half
life of the non-radioactive targeting immunoreagent.
The portion of the radioactive targeting reagent
that does not hybridize to tumor associated non-
radioactive targeting reagent is rapidly cleared from
the patient.
With respect to the same degree of modification of
a targeting immunoreagent by a directly labeled
radionuclide or a chelate cont~;ning a radionuclide, an
amplification of the number of radionuclides per site of
~O
~ W094/205~ 215 7 9 0 2 PCT~S94/02610
modification per targeting immunoreagent can be
obtained.
A segment of the complementary sequenced L-
enantiomeric oligonucleotide of the non-radioactive
targeting immunoreagent and a segment of the L-
enantiomeric oligonucleotide of the radioactive
targeting reagent can hybridize in vitro and in vivo.
The complementary sequenced L-enantiomeric
oligonucleotides of the non-radioactive targeting
0 immunoreagent and the radioactive targeting reagent will
not hybridize with isomeric, complementary sequenced,
naturally occurring D-enantiomeric oligonucleotides.
The complementary sequenced L-enantiomeric
oligonucleotides of the non-radioactive targeting
;mmllnoreagent and the radioactive targeting reagent are
stable to nuclease activity.
The complementary sequenced L-enantiomeric
oligonucleotides of the non-radioactive targeting
~llnoreagent and the radioactive targeting reagent and
the hybrid formed between the complementary L-
enantiomeric oligonucleotides will not bind to enzymes
which are specific for binding to isomeric D-
enantiomeric oligonucleotides.
The hybrid formed between complementary sequenced
L-enantiomeric oligonucleotides of the non-radioactive
targeting immunoreagent and the radioactive targeting
reagent s stable to nuclease activity.
The complementary sequenced L-enantiomeric
oligonucleotides of the non-radioactive targeting
immunoreagent and the radioactive targeting reagent do
not self hybridize.
The non-radioactive targeting immunoreagent and the
radioactive targeting reagent can comprise a wide
variety of spacing, linking, and chelating groups, L-
enantiomeric oligonucleotide sequences and
radionuclides.
W094/205~ 21~ 7 ~ ~ 2 PCT~S94/02610 ~
The complementary sequenced L-enantiomeric
oligonucleotides of the non-radioactive targeting
immunoreagent can comprise ~-enantiomeric
oligonucleotide seque~ces which can be tandemly linked
by spacing groups, wherein a segment of each L-
enantiomeric oligonucleotide sequence can hybridize with
a segment of the radioactive targeting reagent.
The complementary sequenced L-enantiomeric
oligonucleotides of the non-radioactive targeting
immunoreagent can be linked to an antibody by means of
either a 5'- or a 3'-substituent such as a 5'-amine or
3'-amine.
Reagents are provided that have a specificity for
tumors and a wide variety of compositions can be
prepared in accordance with the present invention.
A particular advantage of the present invention is
that L-enantiomeric oligonucleotide sequence lengths and
spacing groups can be selected such that on
hybridization of two complementary radioactive targeting
reagent L-enantiomeric oligonucleotide sequences to a
single L-enantiomeric oligonucleotide-cont~;n;ng strand
comprising adjacent, tandemly linked L-enantiomeric
oligonucleotide sequences of a non-radioactive targeting
~mm~lnoreagent, the proximal end groups of the sequences
of two such radioactive targeting moieties are
orthogonal to each other because of their relative
spacial configuration on the double stranded helix so
formed.
Other advantageous features of this invention will
become readily apparent upon reference to the following
description of the preferred e-mbodiments.
nescr;pt;on of the P~eferre~ ~mho~;m~nts
This invention describes various novel
bioconjugates which possess utility in therapeutic and
diagnostic imaging compositions and methods. This
~ W094/205~ 21~ 7 9 0 2 PCT~S94/02610
invention further describes novel methods of preparing
bioconjugates by the attachment of various L-
enantiomeric oligonucleotide sequences to chelating
agents, preferably terpyridine containing chelating
agents, and to immunoreagents such as proteins,
antibodies, and receptors.
In particular, this invention describes novel
bioconjugates useful for sequential targeting and
amplified delivery of novel radioactive ;mm~lnoreagent
compositions which find particular utility in
therapeutic and diagnostic imaging compositions and
methods.
More particularly, this invention describes the
preparation and use of targeting immunoreagents that
comprise a tumor antigen recognizing moiety, one or more
L-enantiomeric oligonucleotides comprising non-self-
associating L-enantiomeric oligonucleotide sequences,
and one or more link~ng groups. These targeting
immunoreagents react with a radioactive sequential
targeting reagent that comprises an L-enantiomeric
oligonucleotide sequence that is complementary in
sequence to and capable of hybridization with one or
more fragments of the said non-self-associating L-
enantiomeric oligonucleotide sequences, one or more
chelating agents, one or more linking groups, and having
one or more radionuclides associated therewith. Most
preferably, the L-enantiomeric nucleotides are L-
enantiomers of natural D-deoxyribonucleotides.
In a preferred embodiment, the above-described
targeting immunoreagents form a co~pound that comprises
moieties represented by the structure IV:
W094/205~ 2 1 5 7 ~ ~ 2
PCT~S94/02610
Structure IV
Z Lz- I . Ql Ii LQ E
a
P
wherein:
Z is the residue of an immunoreactive protein;
Lz and LQ are independently a chemical bond or a
;nking group;
I is an L-enantiomeric oligonucleotide comprising a
0 contiguous sequence of from 12 to about 50 L-
enantiomeric nucleotide units wherein said contiguous
sequence contains one or more members of a family of
homologous contiguous sequences, the individual homologs
of said family comprising from 12 to about 30 L-
enantiomeric nucleotide units, and provided that
contiguous sequences of six or more L-enantiomeric
nucleotide units of said L-enantiomeric oligonucleotide
do not hybridize with any other contiguous sequences of
six or more contiguous L-enantiomeric nucleotide units
anywhere in structure IV;
QI is a spacing group;
a is 0 or an integer from 1 to about 6;
Ii is an L-enantiomeric oligonucleotide comprising
a contiguous sequence of from 12 to about 50 L-
enantiomeric nucleotide units, a contiguous sequence
therein comprising a portion of I;
E is an end capping group; and
p is an integer from 1 to about 10.
More preferably, a is an integer from 1 to about 6.
3 0 In another preferred embodiment, the above-
described targeting reagent comprises moieties
represented by the structure V:
21~79Q2
W094l205~ PCT~S94/02610
described targeting reagent comprises moieties
represented by the structure V:
Structure V
W,- Ll_ cI Q cI L2 W 2
b
~Mllx -L~ - [M2] z
W 3
[M31y
S -- -- w
wherein:
cI is a contiguous sequence of from 12 to about 50
L-enantiomeric nucleotide units wherein said contiguous
sequence contains one or more members of a family of
0 homologous contiguous sequences, the individual homologs
of said family comprising from 12 to about 30 L-
enantiomeric nucleotide units, where the nucleotide
sequences of said homologs are complementary to the
nucleotide sequences of members of the set of L-
enantiomeric oligonucleotides in a co-a~m; n~ sterable
targeting immunoreagent, and where contiguous sequences
of six or more L-enantiomeric nucleotide units of said
complementary L-enantiomeric oligonucleotide do not
hybridize with any other contiguous sequences of six or
more contiguous L-enantiomeric nucleotide units anywhere
in structure ~,
QCI is a spacing group;
L1, L2, and L3 are independently a chemical bond or
a linking group;
W1, W2, and W3 are each a residue of a chelating
group;
l~
W094/205~ 2 ~5 ~ ~ ~ 2 PCT~S94/02610
M1, M2, and M3 comprise elements with oxidation
states equal to or greater than +1, and at least one of
M1, M2 and M3 is a radionuclide;
x, y, and z are independently zero or one provided
that at least one of x, y, or z is one; and
w and b are independently zero or an integer from 1
to about 4.
As used herein, an L-enantiomeric nucleotide unit
is defined as the mirror image of the naturally
0 occurring, isomeric D-enantiomer nucleotide unit; an L-
enantiomeric nucleoside is defined as the mirror image
of the naturally occurring, isomeric D-enantiomeric
nucleoside; and an L-enantiomeric oligonucleotide
sequence is defined as the mirror image of the naturally
occurring, isomeric D-enantiomeric oligonucleotide
sequence.
Non-limiting examples of L-enantiomeric nucleosides
are set forth below.
NH2
t ~ ~ H2N~[N o --OH
- ~3H 2 :~H
2-deoxy-L-adenosil,e, L dA 2-deoxy-Lgual)os;ne, L-dG
o O NH2
ol~O ~OH ~O~ OH ,,
~ 3 , 3 .
3 ~H 4 :~H 5 ~H
2-deoxy-L-ufidine, L-dU 2-deoxy-L-thymidine, LdT 2-deoxy-Lcytidine, L-dC
1~ -
~WO 94/205~ 21~ 7 9 0 ~ PCT~S94/02610
Methods of synthesis of L-enantiomeric
oligonucleotides from L-enantiomeric nucleotide
intermediates are identical to those used in the
synthesis of mirror image D-enantiomeric
oligonucleotides from isomeric D-enantiomeric nucleotide
intermediates. Examples of such methods include those
discussed in "Chemistry of Nucleosides and Nucleotides",
edited by Leroy B. Townsend, Plenum Press, N.Y., 1988.
The synthesis of L-enantiomeric oligonucleotides by such
methods requires the use of L-enantiomeric reagents such
as L-enantiomeric nucleoside and nucleotide derivatives.
The preparations of various L-enantiomeric nucleosides
are well documented by Robins, M. et al (1970), J. Org.
Chem., 35, p636-639; Holy, A. (1972), Coll. Czechoslov.
Chem. Commun., 37, p4072-4087; Visser, G. et al (1986),
Rec. Travaux Chim. Pay-Bas, 105/12 p528-537; Anderson,
D. et al (1984), Nucleosides and Nucleotides, 3/5,
p499-512; Uhlm~nn~ E. et al (1990), Chem. Review,
(1990), 90/4 p543-584; Smejkal, I. et al (1964) Coll.
Czecholsov.Chem. Commun., 29, p2809-2813, and others.
Of the methods known in the art for the synthesis
of oligonucleotides, a preferred method of synthesis of
L-enantiomeric oligonucleotides of this invention
comprises solid phase synthesis utilizing L-enantiomeric
nucleotide phosphoramidite intermediates which contain
blocking groups on the hydroxyl groups therein. The
blocking of a 5'-hydroxyl group in a ribonucleoside
moiety and in a deoxyribonucleoside moiety is
preferrably done with an acid labile trityl group such
as, for example, a monomethoxytrityl group (sometimes
hereafter referred to as an MMT group) or a
dimethoxytrityl group (sometimes hereafter referred to
as a DMT group) employing the respective trityl
chlorides as reagents. The deblocking of such a trityl-
blocked 5'-hydroxyl group is preferably done with an
W094/205~ 21~ 7 ~ a 2 PCT~S94/02610
acid such as, for example, acetic acid in water, a
chloroacetic acid in a solvent such as dichloromethane,
or benzenesulfonic in a solvent such as chloroform or
methanol as a reagent. The blocking of a 2'-hydroxyl
group in a ribonucleic acid m~oièty is preferably done
with a silyl chloride reagent such as, for example, t-
butyldimethylchlorosilane which reacts with a ribosyl
2'-hydroxyl group to form a t-butyldimethylsilyl ether.
The deblocking of such a t-butyldimethylsilyl ether can
be achieved by treatment with, for example, sodium
hydroxide dissolved in a solvent such as, for example,
methanol.
Preferred L-enantiomeric nucleotides are L-2-
deoxyribonucleotides, and preferred L-enantiomeric
oligonucleotides are oligo-L-2-deoxyribonucleotides.
Derivatives of L-enantiomeric deoxyribonucleotides that
are useful in the synthesis of the L-enantiomeric oligo-
L-deoxyribonucleotides of this invention are L-
enantiomeric deoxyribonucleosides such as, for example,
derivatives of compounds 1 to 5 which have the 5'-OH in
each blocked, for example, with a dimethoxytrityl (DMT)
group, each of which is activated at the 3'-OH for
phosphate bond formation in the o igonucleotide, for
example, by treatment with 2-cyanoethyl-N,N-
diisopropylphosphoryl chloride to produce the respective
3'-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite
intermediates. These are utilized for the synthesis of
oligo-L-deoxyribonucleotide moieties, for example, on a
solid phase, automated DNA synthesizer.
3 0 A preferred method of preparing a blocked 2'-deoxy-
L-adenosine 3'-O-phosphoramidite derivative, A10, that
is suitable for use in the synthesis of the L-
enantiomeric oligo-L-deoxyribonucleotide materials of
this invention is outlined in Scheme 1. 1-O-Methyl-3,5-
di-O-p-toluyl-2-deoxy-L-erythro-pentofuranose (A2 in
Scheme 1) is prepared from L-arabinal (A1) by treatment
l&
W094/205~ 21~ 7 9 0 ~ PCT~S94/02610
with p-toluoyl chloride and methanol according to the
method described by Smejkal, I. et al (1964), Coll.
Czechoslov. Chem. Commun., 29, p2809-2813. Fusion of A2
and 2,6-dichloropurine (A3) provides 2,6-dichloro-9-
(3,5-di-O-p-toluyl-2-deoxy-alpha-L-erythro-
pentofuranosyl)purine (A4). Treatment of A4 with
methanol and AmmO~; ~ selectively aminates the purine at
position 6 and concurrently deblocks the acylated
hydroxyl groups at the 3'- and 5'-positions. The
0 re~;n;ng chloride is removed by hydrogenolysis to
afford the desired L-nucleoside, L-dA, (A6). The
exocyclic amine of A6 is then blocked by diacylation
with benzoyl chloride to form A7 which contains two
benzoate esters in addition to the blocked amine. The
benzoate esters are removed by saponification with
sodium hydroxide in water to form A8. This diol is then
treated with dimethoxytrityl chloride (available from
Aldrich Chemical Cs~rAny) to form A9 which has a DMT
ether at the 5'-position. A9 is then treated with 2-
cyanoethyl N,N-diisopropylchlorophosphoramidite
(available from Aldrich Chemical Comr~ny) to provide
A10, the desired diprotected 5'-O-dimethoxytrityl-2'-
deoxy-L-adenosine 3'-0-2-cyanoethylphosphoramidite. A10
is used as an interm~Ate in the synthesis of L-
oligonucleotide sequences of the compositions of
Structure IV and Structure V of this invention.
Production of such sequences can be done, for example,
using an automated oligonucleotide synthesizer using
procedures described by the manufacturer for the
synthesis and purification of isomeric D-
oligonucleotides.
>
~9
W094/20523 215 7 9 ~ 2 PCT~S94/02610
Scheme 1 r~pa,~ ion of a Blocked L~eoxyad~nos;l,e phosphor~"i-Jile
cl
CHO CH30 rOCOPh-p-CH3 1 N
< 1. CH30H/H~ -,~~/ , N I ~
HO~ 2. ClCOPh-p-CI~ ~ ~,,N ~~r
OH p-CH3-Phoc'~ C~N~;N
L-arabinal A2 A3 p-CH3-PhOC" A4
A1
NH3~CH30H N~ N~ o ~H N;~N~
~ ~ H2O ~N N O ~OH
at 40C/48hr , ~ ~ Ph-COCI
~HNH~OH OH
AS A6
N~)Nph)2 N~)Nph)2 N~h~2
pyridi e
oCOPh ~H I ~H
A7 A8 A~
N(COPh)2
,N(iPr)2 NJ~rN D~ ;loA~b~it~
Cl' o~CN l~N1N O ~ODMT
-
A10 ~p~--CN
N(iPr)2
A preferred method of preparing a blocked 2'-deoxy-
L-guanosine 3'-O-phosphoramidite derivative, G8, that is
suitable for use in the synthesis of the L-enantiomeric
oligo-L-deoxyribonucleotide materials of this invention
is outlined in Scheme 2. 1-O-Methyl-3,5-di-O-p-toluyl-
2-deoxy-L-erythro-pentofuranose ~A2 in Scheme 1) is
prepared from L-arabinal (Al) by treatment with p-
2.0
~ W094/20~ 21 S 7 9 ~ 2 PCT~S94/02610
toluoyl chloride and methanol according to the method
described by Smejkal, I. et al (1964), Coll. Czechoslov.
Chem. Commun., 29, p2809-2813. The methyl acetal (A2)
is hydrolyzed in dilute hydrochloric acid to provide the
hemiacetal (G2) which is acylated with acetic anhydride
to form 1-acetyl 3,5-di-0-p-toluyl-beta-L-erythro-
pentofuranose (G3). Fusion of G3 and 2-fluoro-6-
benzyloxypurine (G4) provides G5 which is then treated
with alcoholic ammonia to provide the desired product
0 (G6) which is purified by chromatography. The 6-benzyl
protecting group of G6 is removed by hydrogenolysis with
palladium on carbon in ammonium hydroxide to yield 2-
amino-9-(2-deoxy-L-erythro-pentofuranosyl)purin-6-one
(G7; L-dG). The exocyclic 2-amino group of G7 is then
protected by acylation with isobutyryl chloride, the 5'-
hydroxyl group is protected as a DMT ether using
dimethoxytrityl chloride, and the 3'-hydroxyl is treated
with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite
to provide G8, the desired diprotected 5'-0-
dimethoxytrityl-2'-deoxy-L-guanosine 3'-0-2-
cyanoethylphosphoramidite. G8 is used as an
intermediate in the synthesis of L-oligonucleotide
sequences of the compositions of Structure IV and
Structure V of this invention. Production of such
sequences can be done, for example, using an automated
oligonucleotide synthesizer using procedures described
by the manufacturer for the synthesis and purification
of isomeric D-oligonucleotides.
W094/20523 21~ 7 9 ~ ~ - PCT~S94/02610
Scheme ~ Preparation of a blocl<ed L-deoxyguanosine pl~os~,hord", li~e
A2 , HO o rOCOPh p CH3 ACO o rOCOPh-p CH3
dilute HCUH 20 A~zO
or HOAc/H20 PhCH20
p-CH3-PhOCC~ p-CH3-PhOC'~ F~XN
G2 G3 G4
PhCH20 PhCH20
Fl~XN o rOCOPh p-CH3 H2N~XN o~/--OH r
NH3/CH30 H NH~OH
d,.~,,.~tuy.~ll~
p-CH3-PhOC' ~ sep~ n of nH
G5 G6
o o
HN~[N~ (C~2CH-COCI o N~N~
~o~r DMT-CUP"i ' e H C>~NH N N O rODMT
N(~Pr)2
a~P~O~CN
0 DMT ~ u~ l N~)2
A preferred method of preparing a blocked 2'-deoxy-
L-uridine 3'-O-phosphoramidite derivative, U7, that is
suitable for use in the synthesis of the L-enantiomeric
oligo-L-deoxyribonucleotide materials of this invention
is outlined in Scheme 3. The reaction of L-arabinose
(U1) with cyanamide yields 2'-amino-1,2-oxazoline (U2)
which upon treatment with methyl propiolate (U3) affords
0 anhydro-L-uridine (U4). The anhydro derivative U4 is
opened with HBr to give the 2'-bromo-nucleoside (U5),
which upon catalytic hydrogenation affords 2-deoxy-L-
uridine (U6). The 5'-hydroxyl group of U6 is protected
as a DMT ether using dimethoxytrityl chloride, and the
3'-hydroxyl is treated with 2-cyanoethyl N,N-
~ W094/205~ 215 7 ~ 0 2 PCT~S94/02610
diisopropylchlorophosphoramidite to provide U7, the
desired protected 5'-O-dimethoxytrityl-2'-deoxy-L-
uridine 3'-0-2-cyanoethylphosphoramidite. U7 is used as
an intermediate in the synthesis of L-oligonucleotide
sequences of the compositions o~ Structure IV and
Structure V of this invention. Production of such
sequences can be done, for example, using an automated
oligonucleotide synthesizer using procedures described
by the manufacturer for the synthesis and purification
of isomeric D-oligonucleotides.
Scheme 3: r~,oar~lion of a Blocked L-deoxyuridine pl,ospl)or~."idil~
OH H2N~ H ~ 3
L-arabinose U2 U3
U1
HNJ`3
,~\N ~r OlN OOH
N~ HBr/anhydrous ~, 1 0%PdUC/~
jH Ether/dioxane Br OH
U4 U5
O O
HN~ HN~
OlN~ o ~OH ,OlN O rODMT
y ~ DMT-CUpyridine \~
N~iPr)2 ~_
~H Cl O~ O~P~O--CN
U6 DMT-dirr~Gthoxyb;~rl N(iPr)2
U7
2157~0~
W~94/205~ PCT~S94/02610 O
A preferred me~hod of preparing a blocked L-
thymidine 3'-O-phosphoramidite derivative, T7, that is
suitable for use in the synthesis of the L-enantiomeric
oligo-L-deoxyribonucleotide materials of this invention
is outlined in Scheme 4. The synthetic method is
analogous to the preparation of the 2'-deoxy-L-uridine
3'-O-phosphoramidite derivative, U7, described in Scheme
3 using L-arabinose as a chiral starting material.
0 Accordingly, L-arabinose is treated with cyanamide to
provide 2'-amino-l,2-oxazoline (U2) which is then
reacted with methyl methacrylate with heating to yield
the anhydro-nucleoside (T4). T4 is opened with
anhydrous HBr to give 2-bromo-L-thymidine (T5). Upon
catalytic hydrogenation T5 affords L-thymidine (T6).
The 5'-hydroxyl group of T6 is protected as a DMT ether
using dimethoxytrityl chloride, and the 3'-hydroxyl is
treated with 2-cyanoethyl N,N-
diisopropylchlorophosphoramidite to provide T7, the
desired protected 5'-O-dimethoxytrityl-L-thymidine 3'-O-
2-cyanoethylphosphoramidite. T7 is used as an
intermediate in the synthesis of L-oligonucleotide
sequences of the compositions of Structure IV and
Structure V of this invention. Production of such
sequences can be done, for example, using an automated
oligonucleotide synthesizer using procedures described
by the manufacturer for the synthesis and purification
of isomeric D-oligonucleotides.
~ Wo 94/205~ 215 7 9 0 2 PCT~S94/02610
~cheme 4: r~par~lion of a Blocked L-thymidine ~I.ospl~or~,.idite
U 1 N ~r H3C~N ~ r
H2N-CN ~ ,OCH3N ~~';;5--OH
U2 1`3 T4
O O
HN J~'CH3 HN ~CH3
. O N O~ DMF, MgO \~
HBr/anhydrous ~ 1 0%Pd/C/~
Ether/dioxane
Br ~H OH
O
HN~CH3
T6 ~OlN~ O --ODMT
DMT-CUpyridine ~ ~
,N(iPr)2 ~_
Cl' `o~CN ~,p, --CN
DMT~dil . atl loxyb ;Iyl N(iPr)2
17
A preferred method of preparing a blocked 2'-deoxy-
L-cytidine 3'-O-phosphoramidite derivative, C7, that is
suitable for use in the synthesis of the L-enantiomeric
oligo-L-deoxyribonucleotide materials of this invention
is outlined in Scheme 5. Thus, L-arabinal, A1, is
treated with HCl and toluyl chloride to provide the 3,5-
di-O-p-toluyl-2-deoxy-L-ribofuranosyl chloride (C2).
0 Fusion of (C2) with 4-chloro-2-trimethylsiloxypyrimidine
(C3) affords the 1-(3,5-di-O-p-toluyl-2-deoxy-L-
ribofuranosyl)-4-chloropyrimidin-2-one (C4), which upon
W~94/20523 2 1 5 ~ ~ ~ 2
PCT~S94/02610
treatment with ammonia in methanol yields 2-deoxy-L-
cytidine (C5). The exocyclic amine of C5 is protected
by treatment with benzoyl chloride, and the esters at
the 3'- and 5'-positions are saponified to regenerate C6
with free 3'- and 5'-hydroxyl groups. The 5'-hydroxyl
group of C~ is protected as a D~ ether using
dimethoxytrityl chloride, and the 3'-hydroxyl is treated
with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite
to provide C7, the desired protected 5'-O-
0 dimethoxytrityl-2'-deoxy-L-cytidine 3'-0-2-
cyanoethylphosphoramidite. C7 is used as an
interme~;~te in the synthesis of L-oligonucleotide
sequences of the compositions of Structure IV and
Structure V of this invention. Production of such
sequences can be done, for example, using an automated
oligonucleotide synthesizer using procedures described
by the manufacturer for the synthesis and purification
of isomeric D-oligonucleotides.
a~
~WO 94/205~ 21 S 7 9 0 2 PCT~S94/02610
~cheme 5: Preparation of a Blocked L-deoxycytidine phosphoramidite
.~
HO ~ r ~OCOPh-~CH3
- --'< HCI ~N
--~ p-Clt.-PhCOCI ~ 1
;)H ' oCOPh-p-CH3 N OSi(CH3)3
C1 C2 C3
Cl NH2
\~ NH3/CI-130 H \~
NaOH
OCOPh-p-CH3 ~H
C4 C5
NHCOPh NHCOPh
HN~ . HNJ~
OlN~ ~OH DMT-CUpyridine OlN O ~ODMT
~~N~iPr)2 \~
Cl 'P`O~CN ~_~
:)H DMT~dimethox~fityl )~p~--CN
C7 N(iPr)2
The 2'-deoxy-L-adenosine (A6) is sometimes
hereinafter referred to as L-dA; the 2'-deoxy-L-
guanosine (G7) is sometimes hereinafter referred to as
L-dG; the 2'-deoxy-L-uridine (U6) is sometimes
hereinafter referred to as L-dU; the L-thymidine (T6)
is sometimes hereinafter referred to as L-T; and the 2'-
deoxy-L-cytidine (C5) is sometimes hereinafter referred
0 to as L-dC. Accordingly, as a definition of abbreviated
nomenclature for the L-enantiomeric oligonucleotides of
this invention, an irrelevant L-enantiomeric
W094/205~ 215 7 ~ ~ 2 PCT~S94/02610 ~
ollgonucleotide having a sequence containing, for
example, a 2'-deoxy-L-adenosine, L-dA, which is linked
by a phosphate diester group to a 2'-deoxy-L-guanosine,
L-dG, which in turn is linked by a phosphate diester
group to a 2'-deoxy-L-uridine, L-dU, which in turn is
linked by a phosphate diester group to an L-thymidine,
L-T, which in turn is linked by a phosphate diester
group to a 2'-deoxy-L-cytidine, L-dC, would be defined
and sometimes referred to as L-d(AGUTC). If the 5'-end
0 of this oligomer is attached to a group Rl and the 3'-
end of this oligomer is attached to a group R2, then the
oligomer would sometimes be referred to as Rl-5'-L-
d(AGUTC)-3'-R2 or sometimes as 5'-Rl-L-d(AGUTC)-R2-3'.
Conversely, if the 3'-end of this oligomer is attached
to a group Rl and the 5'-end of this oligomer is
attached to a group R2, then the oligomer would
sometimes be referred to as Rl-3'-L-d(AGUTC)-5'-R2 or
sometimes as 3'-Rl-L-d(AGUTC)-R2-5'. Reference is made
to the L-oligonucleotides of this invention and
particularly to the presently preferred oligonucleotides
which have the sequences as described hereinbelow using
this nomenclature.
The term n residue" is used herein in context with a
chemical entity. Said chemical entity comprises, for
example, a chelating group, or a l;nk;ng group, or a
protein reactive group, or an imm-unoreactive group, or
an ; ~oreactive protein, or an antibody, or an
antibody fragment, or a cross-l;nk;ng agent such as a
heterobifunctional cross-l;nk;ng agent, or an L-
3 0 enantiomeric oligonucleotide, or a spacing group, or an
end capping group. The term "residue" is defined as
that portion of the chemical entity which exclusively
r~m~ins when one or more chemical bonds therein when
considered as an independent chemical entity, is
altered, modified, or replaced to comprise one or more
covalent bonds to one or more other chemical entities.
~$
~ W094/205~ 215 7 9 ~ 2 PCT~S94/02610
Thus, for example, in one aspect, a "residue of an L-
enantiomeric oligonucleotide" in the context of, for
example, I and Ii in Structure IV or of cI in Structure
V comprises an ~-enantiomeric oligonucleotide modified
at least for divalent attachment to the residue of
another chemical entity, i.e., the residue of said L-
enantiomeric oligonucleotide comprises at least a
divalent L-enantiomeric oligonucleotidyl sequence. In
another aspect, for example, "the residue of a chelating
group" in the context of Wl, W2 or W3 of Structure V
comprises a chelating group which is at least
monovalently modified through attachment to the residue
of another chemical entity such as, for example, to the
residue of a l;nk;ng group.
lS In Structure IV above, Z preferably is an antibody
or antibody fragment which recognizes and is specific
for a tumor associated antigen. In some em.bodiments,
the above-described protein can contain an
;mmllnoreactive group covalently bonded thereto through a
chemical bond or a link;ng group derived from the
residue of a protein reactive group and the residue of a
reactive group on the protein. As used herein, the term
'l;mmllnoreactive protein" which can be abbreviated by
"IRP" also includes an organic compound which is capable
of covalently bonding to the protein and which is found
in a living organism or is useful in the diagnosis,
treatment or genetic engineering of cellular material or
living org~n;sm~, and which has a capacity for
interaction with another component which may be found in
biological fluids or associated with cells to be treated
such as tumor cells.
The immunoreactive group can be selected from a
wide variety of naturally occurring or synthetically
prepared materials, including, but not limited to
enzymes, amino acids, peptides, polypeptides, proteins,
lipoproteins, glycoproteins, lipids, phospholipids,
~q
W094/205~ 2 ~5 ~ 9 0 2 PCT~S94/02610
hormo~es, growth factors, steroids, vit~m;ns,
polysaccharides, viruses, protozoa, fungi, parasites,
rickettsia, molds, and components thereof, blood
components, tissue and organ components,
pharmaceuticals, haptens, lectins, toxins, nucleic acids
(including oligonucleotides), antibodies (monoclonal and
polyclonal), anti-antibodies, anti~ody fragments,
antigenic materials (including proteins and
carbohydrates), avidin and derivatives thereof, biotin
and derivatives thereof, and others known to one skilled
in the art. In addition, an immunoreactive group can be
any substance which when presented to an ~mm~lnocompetent
host will result in the production of a specific
antibody capable of binding with that substance, or the
antibody so produced, which participates in an antigen-
antibody reaction.
Preferred i~mllnoreactive groups are antibodies and
various immunoreactive fragments thereof, as long as
they contain at least one reactive site for reaction
with a protein reactive group as described herein on the
residue of the L-enantiomeric oligonucleotide or with
1 ;nk;ng groups as described herein. That site can be
inherent to the ;m~llnoreactive species or it can be
introduced through appropriate chemical modification of
the immunoreactive species. In addition to antibodies
produced by the techniques outlined above, other
antibodies and proteins produced by the techniques of
molecular biology are specifically included.
Preferably, the ;m~llnoreactive group does not bind
to the residue of an L-enantiomeric oligonucleotide in
structure IV so as to inhibit the binding of the L-
enantiomeric oligonucleotide to a complementary
sequenced L-enantiomeric oligonucleotide of structure V.
As used herein, the term "antibody fragment-l refers
to an ;mmllnoreactive material which comprises a residue
of an antibody, which an~ibody characteristically
3C
W094/205~ 21~ 7 ~ 0~ PCT~S94/02610
exhibits an affinity for binding to an antigen. The term
affinity for binding to an antigen, as used herein,
refers to the thermodynamic expression of the strength
of interaction or binding between an antibody combining
site and an antigenic determinant and, thus, of the
stereochemical compatibility between them. As such, it
is the expression of the equilibrium or association
constant for the antibody-antigen interaction. The term
"affinity" as used herein also refers to the
0 thermodynamic expression of the strength of interaction
or binding between a ligand and a receptor and, thus, of
the stereochemical compatibility between them. As such,
it is the expression of the equilibrium or association
constant for the ligand-receptor interaction.
With respect to the affinity of binding of an
antibody to an antigen, antibody fragments exhibit a
percentage of said affinity for binding to said antigen,
that percentage being in the range of 0.001 per cent to
1,000 per cent, preferably 0.01 per cent to 1,O00 per
cent, more preferably 0.1 per cent to 1,000 per cent,
and most preferably 1.0 per cent to 100 per cent, of the
relative affinity of said antibody for binding to said
antigen.
An antibody fragment can be produced from an
antibody by a chemical reaction comprising one or more
chemical bond cleaving reactions; by a chemical reaction
comprising of one or more chemical bond forming
reactions employing as reactants one or more chemical
components selected from a group comprising amino acids,
peptides, carbohydrates, linking groups as defined
herein, spacing groups as defined herein, protein
reactive groups as defined herein, and antibody
fragments such as are produced as described herein and
by a molecular biological process, a bacterial process,
or by a process comprising or resulting from the genetic
engineering of antibody genes.
31
W094/205~ 21~ 7 ~ ~ 2 PCT~S94/02610
An antibody fragment can be derived from an
antibody by a chemical reaction comprising one or more
of the following reactions:
(a) cleavage of one or more chemical bonds of which
an antibody is comprised, said~onds being selected
from, for example, carbon-nitrogen bonds, sulfur-sulfur
bonds, carbon-carbon bonds, carbon-sulfur bonds, and
carbon-oxygen bonds, and wherein the method of said
cleavage is selected from:
0 (i) a catalysed chemical reaction comprising the
action of a biochemical catalyst such as an enzyme such
as papain or pepsin which enzymes to those skilled in
the art are known to produce antibody fragments commonly
referred to as Fab and Fab'2, respectively;
(ii) a catalysed chemical reaction comprising the
action of an electrophilic chemical catalyst such as a
hydronium ion which, for example, favorably occurs at a
pH equal to or leQs than 7;
(iii) a catalysed chemical reaction comprising the
action of a nucleophilic catalyst such as a hydroxide
ion which, for example, favorably occurs at a pH equal
to or greater than 7;
(iv) a chemical reaction comprising a substitution
reaction employing a reagent such which is consumed in a
stoichiometric manner such as, for example, a
substitution reaction at a sulfur atom of a disulfide
bond by a reagent comprising a sulfhydryl group
(comprising a -SH group) or an anionic sulfide group
(comprising an -S~ group in the form of a salt such as a
-S~ Na+ group);
(v) a chemical reaction comprising a reduction
reaction such as, for example, the reduction of a
disulfide bond; and
(vi) a chemical reaction comprising an oxidation
reaction such as the oxidation of a carbon-oxygen bond
of a hydroxyl group or the oxidation of a carbon-carbon
3~
~ W094/205~ 215 7 9 0 2 PCT~S94/02610
bond of a vicinal diol group such as occurs in a
carbohydrate moiety; or
(b) formation of one or more chemical bonds between
one or more reactants, such as formation of one or more
covalent bonds selected from, for example, carbon-
nitrogen bonds (such as, for example, amide bonds, amine
bonds, hydrazone bonds, imine bonds, and thiourea
bonds), sulfur-sulfur bonds such as disulfide bonds,
carbon-carbon bonds, carbon-sulfur bonds, and carbon-
0 oxygen bonds, and employing as reactants in said
chemical bond formation one or more reagents comprising
amino acids, peptides, carbohydrates, linking groups as
defined herein, spacing groups as defined herein,
protein reactive groups as defined herein, and antibody
fragments such as are produced as described in (a),
above; or
(c) an antibody fragment can be derived by
formation of one or more non-covalent bonds between one
or more reactants. Such non-covalent bonds comprise
hydrophobic interactions such as occur in an aqueous
medium between chemical species that independently
- comprise mutually acce~sible regions of low polarity
such as regions comprising aliphatic and carbocyclic
groups, and of hydrogen bond interactions such as occur
in the binding of an oligonucleotide with a
complementary oligonucleotide; or
(d) an antibody fragment can be produced as a
result of the methods of molecular biology or by genetic
engineering of antibody genes, for example, in the
genetic engineering of a single chain immunoreactive
group or a Fv fragment.
An antibody fragment can be produced as a result a
combination of one or more of the above methods.
~n certain embodiments, the immunoreactive group
can be an enzyme which has a reactive group for
attachment to the residue of an L-enantiomeric
W094l205~ 215 7 9 ~ 2 PCT~S94/02610
oligonucleotide, I, by means of a linking group Lz.
Representative enzymes include, but are not limited to,
aspartate aminotransaminase, alanine aminotransaminase,
lactate dehydrogenase, creatine phosphokinase, gamma
glutamyl transferase, alkaline acid phosphatase,
prostatic acid phosphatase, horseradish peroxidase and
various esterases.
If desired, the immunoreactive group can be
modified or chemically altered to provide a reactive
0 group for use in the attachment to the residue of the L-
enantiomeric oligonucleotide, I, through a linking group
as described below by techniques known to those skilled
in the art. Such techniques include the use of linking
moieties and chemical ~odification such as described in
WO-A-89/02931 and Wo-A-89/2932, which are directed to
modification of oligonucleotides, and U.S. Patent No.
4,719,182.
Two highly preferred uses for the compositions of
this invention are for the diagnostic imaging of tumors
and the radiological treatment of tumors. Preferred
immunological groups therefore include antibodies to
tumor-associated antigens. An antibody is sometimes
hereinafter referred to as Ab. Specific non-limiting
examples of antibodies include B72.3 and related
antibodies (described in U.S. Patent Nos. 4,522,918 and
4,612,282) which recognize colorectal tumors; 9.2.27 and
related anti-melanoma antibodies; D612 and related
antibodies which recognize colorectal tumors; UJ13A and
related antibodies which recognize small cell lung
carcinomas; NRLU-10, NRCO-02 and related antibodies
which recognize small cell lung carcinomas and
colorectal tumors (Pan-carcinoma); 7EllC5 and related
antibodies which recognize prostate tumors; CC49 and
related antibodies which recognize colorectal tumors;
TNT and related antibodies which recognize necrotic
tissue; PRlA3 and related antibodies which recognize
34
WOg4/205~ 21~ 7 9 0 2 PCT~S94/02610
colon carcinoma; ING-l and related antibodies, which are
described in International Patent Publication WO-A-
90/02569; B174, C174 and related antibodies which
recognize squamous cell carcinomas; B43 and related
S antibodies which are reactive with certain lymphomas and
leukemias; and anti-HLB and related monoclonal
antibodies. An especially preferred antibody is ING-l.
Referring to structure IV again, Lz and LQ are
independently a chemical bond or the residue of a
0 linking group. The phrase "residue of a linking group"
as used herein refers to a moiety that rem~;nS, results,
or is derived from the reaction of a protein reactive
group with a reactive site on the protein. The phrase
"protein reactive group" as used herein refers to any
lS group which can react with a functional group typically
found on a protein. However, it is specifically
contemplated that a protein reactive groups can also
react with a functional group typically found on a non-
protein biomolecule. Thus a linking group useful in the
practice of this invention derives from a group which
can react with any biological molecule contA;n;ng an
immunoreactive group, whether or not the biological
molecule is a protein, to form a l~nk;ng group between
the immunoreactive group and the L-enantiomeric
oligonucleotide containing species as described below.
Preferred linking groups are derived from protein
reactive groups selected from but not limited to:
(1) A group that will react directly with amine,
alcohol, or sulfhydryl groups on the protein or
biological molecule contA;n;ng the immunoreactive group,
for example, active halogen containing groups including,
for example, chloromethylphenyl groups and chloroacetyl
[ClCH2C(=O)-] groups, activated 2-(leaving group
substituted)-ethylsulfonyl and ethylcarbonyl groups such
as 2-chloroethylsulfonyl and 2-chloroethylcarbonyl;
vinylsulfonyl; vinylcarbonyl; epoxy; isocyanato;
W094/20~ 2 1 ~ 7 ~ 0 2 PCT~S94/02610
isothiocyanato; aldehyde; aziridine;
succinimidoxycarbonyl; activated acyl groups such as
carboxylic acid halides; mixed anhydrides and the like;
and other groups known to bç~useful in conventional
photographic gelatin hardening agents;
(2) A group that can react readily with modified
proteins or biological molecules containing the
immunoreactive group, i.e., proteins or biological
molecules containing the immunoreactive group modified
to contain reactive groups such as those mentioned in
(1) above, for example, by oxidation of a group such as
a hydroxyl group of a protein to an aldehyde or to a
carboxylic acid, in which case the "l;nk;ng group" can
be derived from protein reactive groups selected from
amino, alkylamino, arylamino, hydrazino, alkylhydrazino,
arylhydrazino, carbazido, semicarbazido, thiocarbazido,
thiosemic~rh~7ido, sulfhydryl, sulfhydrylalkyl,
sulfhydrylaryl, hydroxy, carboxy, carboxyalkyl and
carboxyaryl. The alkyl portions of said l;nk;ng groups
can contain from 1 to about 20 carbon atoms. The aryl
portions of said l;nk;ng groups can contain from about 6
to about 20 carbon atoms;
(3) A group that can be linked to the protein or
biological molecule cont~;n;ng the immllnoreactive group,
or to the modified protein as noted in (1) and (2) above
by use of a crossl;nk;ng agent. The residues of certain
useful crosslinking agents, such as, for example,
homobifunctional and heterobifunctional gelatin
hardeners, bisepoxides, and bisisocyanates can become a
part of, i.e., a linking group in, the protein~
enantiomeric oligonucleotide-cont~;n;ng species)
conjugate during the crosslink;ng reaction. Other
useful crosslinking agents, however, can facilitate the
crosslinking, for example, as consumable catalysts, and
are not present in the final conjugate. Examples of
such crossl;nk;ng agents are carbodiimide and
3G
~ W094/205~ 215 7 9 0 2 PCT~S94/02610
carbamoylonium crosslinking agents as disclosed in U.S.
Patent No. 4,421,847 and the ethers of U.S. Patent No.
4,877,724. ~ith these crosslinking agents, one of the
reactants, such as the immunoreactive group, must have a
carboxyl group and the other, such as the L-enantiomeric
oligonucleotide-containing species, must have a reactive
amine, alcohol, or sulfhydryl group. In amide bond
formation, the crosslinking agent first reacts
selectively with the carboxyl group, then is split out
0 during reaction of the thus "activated" carboxyl group
with an amine to form an amide linkage between the
protein and L-enantiomeric oligonucleotide containing
species, thus covalently bonding the two moieties. An
advantage of this approach is that crosslinking of like
molecules, e.g., proteins with proteins or L-
enantiomeric oligonucleotide cont~ining species with
themselves is avoided, whereas the reaction of, for
example, homo-bifunctional crosslink~ng agents is
nonselective and unwanted crosslinked molecules are
obtained.
Preferred useful l;nking groups are derived from
various heterobifunctional cross-link;ng reagents such
as those listed in the Pierce Chemical Comr~ny
Immunotechnology Catalog - Protein Modification Section,
(1991 and 1992).
Useful non-limiting examples of such reagents
include:
Sulfo-SMCC Sulfosuccinimidyl 4-(N-
maleimidomethyl)cyclohexane-1-
carboxylate;
.
Sulfo-SIAB Sulfosuccinimidyl (4-
iodoacetyl)aminobenzoate;
~ = :~
W094/205~ 2 ~ 5 7 ~ ~ 2 PCT~S94/02610
Sulfo-SMPB Sulfosuccinimidyl 4-(p-
maleimidophenyl)butyrate;
2-IT 2-Iminothiolane; and
SATA N-Succ;n; m; dyl S-acetylthioacetate.
In addition to the foregoing description, the
linking groups, in whole or in part, can also comprise
and be derived from nucleotides and residues of
nucleotides, both naturally occurring and modified.
Particularly useful, non-limiting reagents for
incorporation of modified nucleotide moieties containing
reactive functional groups, such as amine and sulfhydryl
groups, into an L-enantiomeric oligonucleotide sequence
of this invention are commercially available from, for
example, Clonetech Laboratories Inc. (Palo Alto
California) and include Uni-Link AminoModifier (Catalog
# 5190), Biotin-ON phosphoramidite (Catalog ~ 5191), N-
MNT-C6-AminoModifier (Catalog # 5202), AminoModifier II
(Catalog # 5203), DMT-C6-3'Amine-ON (Catalog # 5222),
C6-ThiolModifier (Catalog # 5211), and the like. In one
aspect, l; nk; ng groups of this invention are derived
from the reaction of a reactive functional group such as
an amine or sulfhydryl group as are available in the
above Clonetech reagents, one or more of which has been
incorporated into an L-enantiomeric oligonucleotide
sequence of this invention, with, for example, one or
more of the previously described protein reactive groups
such as heterobifunctional protein reactive groups, one
or more of which has been incorporated into an
immunoreagent of this invention.
Referring to Structure IV again, I and Ii each
independently comprise an L-enantiomeric oligonucleotide
of a contiguous sequence of from 12 to about 50 L-
enantiomeric nucleotide units wherein said contiguous
3B
W094/205~ 21~ 7 9 0 ~ PCT~S94/02610
sequence contains one or more members of a family of
homologous contiguous sequences; wherein the individual
homologs of said family comprise from 12 to about 30 L-
enantiomeric nucleotide units; wherein the homologs of
said family of sequences, both individually or as a set
of homologous sequences being hereinafter sometimes
referred to as "the Sequence"; and wherein any
contiguous sequence of six or more L-enantiomeric
nucleotide units does not hybridize with any other
0 contiguous sequence of six or more contiguous L-
enantiomeric nucleotide units anywhere in structure IV.
Members of the set of homologous contiguous sequences
which comprise "the Sequence" can be found in both the
sequence I and the sequence Ii, and at least one such
sequence is co~o~ to both I and Ii.
The L-enantiomeric oligonucleotide sequence of I
and Ii in Structure IV can comprise L-DNA, L-RNA, purine
and pyrimidine base-modified L-DNA or L-RNA, backbone-
modified L-DNA or L-RNA such as methyl phosphonate or
thiophosphonate or carbohydrate modified L-DNA or L-RNA
analogs, whole or partially modified, or combinations
thereof as long as a complementary L-enantiomeric
oligonucleotide sequence incorporated into the
radioactive targeting moiety of Structure V described
below can hybridize to said L-enantiomeric
oligonucleotide sequence to form a hybrid which exhibits
a Tm (melting temperature) greater than about 37 C.
Preferred L-enantiomeric oligonucleotides are non-base-
modified and non-backbone-modified L-DNA and L-RNA, more
preferred are L-DNA comprising L-dA, L-T, L-dG, L-dU and
L-dC L-enantiomeric nucleotide units. Currently
especially preferred L-enantiomeric oligonucleotides are
L-DNA comprising L-dA, L-T, L-dG, and L-dC L-
enantiomeric nucleotide units.
In a preferred embodiment, the L-enantiomeric
oligonucleotide sequence I and Ii can comprise double
39
.,
21~7~02
W094120~ PCT~S94/02610
stranded L-DNA or L-RNA. That is, the L-enantiomeric
oligonucleotide sequence may comprises complementary L-
DNA or L-RNA which forms a double helix molecule. The
complementary L-enantiomeric oligonucleotide sequence
incorporated into the radioactive targeting moiety,
composed of L-DNA or L-RNA, then~hybridizes to one or
the other of the strands of the double stranded L-DNA or
L-RNA comprising I and Ii. In this way, the
complementary L-enantiomeric oligonucleotide sequence
0 incorporated into the radioactive targeting moiety
interacts with the duplex L-DNA or L-RNA of I and Ii in
such a way as to form triplex (triple helix) L-DNA,
triplex L-RNA, or a triplex L-DNA:L-RNA hybrid.
Preferred non-limiting examples of L-enantiomeric
oligonucleotide sequences comprising the "Sequence" are
shown below. The following sequences comprise a set of
homologous L-enantiomeric oligonucleotide sequences
which when considered individually or in any combination
comprise a set herein defined as the ~Sequence":
(i) L-d(TTATGGACGGAG) (SEQ ID NO:l);
(ii) L-d(TTATGGACGGAGA) (SEQ ID NO:2);
(iii) L-d(TTATGGACGGAGAA) (SEQ ID NO:3);
(iv) L-d(TTATGGACG~AAG) (SEQ ID NO:4);
(v) L-d(TTATGGACGÇA~AAGC) (SEQ ID NO:5);
(vi) L-d(TTATGGACG~AGAAGCT) (SEQ ID NO:6);
(vii) L-d(TTATGGACG~A~AAGCTA) (SEQ ID NO:7); and
(viii) L-d(TTATGGACGGA~AAGCTAA) (SEQ ID NO:8).
Of course, sequence (viii) contains sequence (vii)
which contains sequence (vi) which contains sequence
(v), and so on. Thus, in structure IV, if I contains,
for example sequence (iii), and an Ii contains, for
example, sequence (v), then both I and Ii contain at
least sequence (iii). Another Ii in structure IV can
contain (viii), in which case it would also contain (i)
through (vii) as well as (viii). In this case, all
three sequences would contain at least (iii) [as well as
4~
~ W094/205~ 215 ~ ~ 0 2 PCT~S94/02610
(i) and (ii)], and the two Ii's would contain at least
(v) tas well as (i) through (iv)]. In this regard, an
L-enantiomeric oligonucleotide that comprises a
contiguous sequence of L-enantiomeric nucleotides, which
sequence being complementary to at least sequence (i),
would hybridize to all sequences (i) through (viii) as
would any member of a set of contiguous complementary
sequences, the individual members of which comprise the
sequence complementary to any of (i) through (viii).
Such a set of contiguous complementary sequences can
comprise cI as will be described below.
Another set of preferred homologous L-enantiomeric
oligonucleotide sequences comprising the "Sequence" is:
(ix) L-d(CG~A~GCTAA) (SEQ ID NO:9);
(x) L-d(ACGGAÇAAGCTAA) ~SEQ ID NO:lO);
(xi) L-d(GACG~A~AAGCTAA) (SEQ ID NO:ll);
(xii) L-d(GGACG~A~.AAGCTAA) (SEQ ID NO:12);
(xiii) L-d(TGGACGGA~AGCTAA) (SEQ ID NO:13);
(xiv) L-d(ATGGACGGAGA~GCTAA) (SEQ ID NO:14);
(xv) L-d(TATGGACGGA~AAGCTAA) (SEQ ID NO:15); and
(xvi) L-d(TTATGGAcG~r~AAGcTAA) (SEQ ID NO:8).
An especially preferred sequence is: L-
d(TTATGGACG~A~A~GCTAA) (SEQ ID NO-8).
Two or more of the L-enantiomeric oligonucleotide
sequences of this invention can be t~n~emly linked by
means of chemical bonds, by llnking groups such as
described above, or by spacing groups as described
below. The sequential order of L-enantiomeric
nucleotides in the L-enantiomeric oligonucleotide
sequences of this invention can be from the 5' to the 3'
end or from the 3' to the 5' end. Attachment of the L-
enantiomeric oligonucleotide sequences of this invention
via linking groups as described above to the immune
reactive group as described above can be accomplished
via 3' or via 5' sites or via derivatives attached to 3'
or 5' sites of the L-enantiomeric oligonucleotide.
41
W094/205~ 215 7 ~ ~ 2 PCT~S94/02610
As discussed above, the "Sequence~ may also be
composed of a double stranded L-DNA or L-RNA. That is,
the "Sequence" may consist of complementary L-
enantiomeric oligonucleotides which non-covalently
interact to form double stranded L-D~or L-RNA.
Attachment of this double stranded nucleic acid to the
immune reactive group as described above can be
accomplished via 3' or via 5' sites or via derivatives
attached to 3' or 5' sites of the L-enantiomeric
oligonucleotide.
Referring to structure IV again, QI is a spacing
group which separates and links two or more L-
enantiomeric oligonucleotide sequences of this
invention. QI can comprise a linking group as defined
above, alone, or in combination with an L-enantiomeric
nucleotide or an L-enantiomeric oligonucleotide
comprising 2 to about 20 L-enantiomeric nucleotide
units, the sequence of which is not self-associating or
such that contiguous sequences of six or more L-
enantiomeric nucleotide units therein do not hybridize
with any other contiguous sequences of six or more
contiguous L-enantiomeric nucleotide units anywhere in
structure IV. QI can also comprise a residue of an
amino acid group, a peptide group, or a poly(alkylene
oxide) group such as a poly(ethylene glycol) group. It
is contemplated that each spacing group can be linked to
from two to about six L-enantiomeric oligonucleotide
sequences, at least two of which contain the Sequence of
this invention. Preferably, the spacing group is linked
to two L-enantiomeric oligonucleotide sequences each of
which contains the Sequence of this invention.
Preferably, the spacing group is an L-enantiomeric
oligonucleotide sequence.
Non limiting examples of preferred spacing groups
are L-enantiomeric oligonucleotides comprising the
following sequences:
4~
~ W094/20~ 215 7 ~ 0 2 PCT~S94/02610
L-d(ACT);
L-d(ACTC);
L-d(ACTCT);
~-d(CTC);
L-d(TCTC); and
L-d(CTCTC).
An especially preferred spacing group is an L-
enantiomeric oligonucleotide: L-d(ACTCTC).
Of course, the condition described above, i.e.,
0 that the L-enantiomeric oligonucleotide sequence of this
invention comprises a non-self associating sequence,
still applies when considering the selection of L-
enantiomeric oligonucleotide spacing groups linked in
combination with the L-enantiomeric oligonucleotide
Sequence groups.
In structure IV, a is from 0 to about 6, preferably
an integer from l to about 6, more preferably one to
about 4, and most preferably one or two.
In structure IV, p is an integer from l to lO,
preferably l to about 6, and more preferably l to 3. It
is also contemplated that mixtures of immunoreactive
proteins comprising mixtures of Z modified as defined in
structure IV together with Z not so modified will also
be useful in this invention. In this case, the bulk
mixture properties of -p" of such mixtures would
comprise fractional values from about zero to about lO.
Preferably, the bulk p values would be from about O.l to
about lO.0, more preferably from about 0.2 to about 5.0,
and most preferably from about 0.4 to about 3.
In structure IV, E is an end capping group. E is
preferably an L-enantiomeric nucleotide group or a group
of one or more D-oligonucleotides that is modified so as
to reduce or prevent the action of exonuclease enzymatic
activity on the D-enantiomeric oligonucleotide sequence
therein. E can be a 3'- or 5'-phosphate-linked ribose
group contalning one or more sub~tituents such as an
43
W094/205~ 21~ 7 ~ ~ 2 ~ PCT~S94/02610 ~
alkyl group of 1 to ab~'u~ 1~ carbon atoms. E can be a
5'- or 3'-ether group such as an alkyl ether, an aryl
ether, an aralkyl ether, a substituted aryl ether or an
aralkyl ether wherein the alkyl groups contain ~rom 1 to
about 10 carbon atoms and the aryl groups contain from 6
to 10 carbon atoms, and wherein the alkyl or aryl groups
may contain oxygen, nitrogen or sulfur atoms or be
substituted by alkyl or aryl groups containing oxygen,
nitrogen or sulfur atoms. E can be a 5'-O- or 3'-O-
0 phosphate ester group such as an alkyl ester, an aryl
ester, an aralkyl ester, a substituted aryl ester or an
aralkyl ester wherein the alkyl groups contain from 1 to
about 10 carbon atoms and the aryl groups contain from 6
to 10 carbon atoms, and wherein the alkyl or aryl groups
may contain oxygen, nitrogen or sulfur atoms or be
substituted by alkyl or aryl groups cont~;ning oxygen,
nitrogen or sulfur atoms. E can be a poly(alkylene
oxidyl) group on the ribosyl moiety, preferably at the
5'- or 3'- position, either as an ether group or linked
by a phosphate ester to the 5'- or 3'-oxygen of the L-
ribosyl group. The poly(alkylene oxidyl) group can be,
for example, a poly(ethylene oxidyl) or poly(propylene
oxidyl) or a poly(propylene oxidyl-co-ethylene oxidyl)
group, each polymer cont~; n; ng from 2 to about 100
repeating units. A phosphate ester comprising such
entities is also useful, as well as a phosphate ester or
modified ribose comprising elements of a suitable
linking group as defined above. E can also comprise a
Z or it can be attached to QI by elements of Lz as
defined above to form a cyclic structure. E can also
comprise compounds with a two carbon-one nitrogen atom
internucleoside linkage. Preferably, E comprises a
poly(alkylene glycol) phosphate diester. With respect
to E, the poly(alkylene glycol) moiety can have from 2
to about 100 repeating units. Preferably, the
poly(alkylene glycol) is a poly(ethylene glycol). A
~ W094/205~ 2 1 5 7 9 0 2 PCT~S94/02610
currently preferred poly(alkylene glycol) phosphate
diester is a tetra(ethylene glycol) phosphate diester,
hereinafter sometimes referred to as a "Teg" or "Teg
unit". Such poly(alkylene glycol) phosphate diesters
can be linked in tandem to each other to form a dimer
phosphate ester sequence, a trimer sequence, a tetramer
sequence, and so forth. One or two such units is
preferred. Such units can also be attached to residues
of QI, Lz, LQ, I, Ii or Z described above. A preferred
end group E comprises a Teg unit linked by a phosphate
ester bond to an L-enantiomeric nucleotide such as T.
Other preferred end capping units for an L-DNA ~equence
comprise a residue of L-dA, L-dG, L-T, L-dC, and L-dU or
an oligonucleotide sequence comprised therefrom.
In the context of this invention, the term
"modified nucleotide moiety" is intended to mean a
chemical entity which comprises one or more chemical
groups that are analogous to one or more portions of a
naturally occurring D-enantiomeric nucleotide or of a
residue of a naturally occurring D-enantiomeric
nucleotide. A "modified D-enantiomeric nucleotide
moiety" comprises that chemical entity which exclusively
remains when one or more chemical bonds, of which said
naturally occurring D-enantiomeric nucleotide is
otherwise comprised when considered as an independent
chemical entity, is altered, modified, or replaced to
comprise one or more covalent bonds to one or more other
chemical entities, or comprises that chemical entity
which exclusively remains after removal or deletion of a
portion, such as, for example, a purine or pyrimidine
base portion, a hydroxyl portion, a ribose portion, and
the like or combinations thereof, of the naturally
occurring D-enantiomeric nucleotide in one location
simultaneously with said replacement of another portion
3~ of said L-enantiomeric nucleotide. Particularly useful,
non-limiting examples of modified D-enantiomeric
4~
W094/205~ 21~ 7 9 ~ 2 PCT~S94/02610
nucleotide moieties comprise reactive functional groups,
such as amine and sulfhydryl groups. They can be
commercially available such as, for example, those
modified D-enantiomeric nucleotide moieties and
precursors thereto which are available from Clonetech
Laboratories Inc. ~Palo Alto, ~aiifornia). Said
modified D-enantiomeric nucleotide moieties and
precursors thereto include Uni-Link ~m; noModifier
(Catalog ~5190), Biotin-ON phosphoramidite (Catalog
#5191), N-MNT-C6-AminoModifier (Catalog $5202),
AminoModifier II (Catalog #5203), DMT-C6-3'Amine-ON
(Catalog ~5222), C6-ThiolModifier (Catalog ~5211), and
the like. One or more of said moieties can be
incorporated into an L-enantiomeric oligonucleotide
sequence comprising this invention. In one aspect,
l;nk~ng groups of this invention are derived from the
reaction of a reactive functional group such as an amine
or sulfhydryl group as are available in the above
Clonetech reagents, one or more of which has been
incorporated into an L-enantiomeric oligonucleotide
sequence of this invention, with, for example, one or
more of the previously described protein reactive groups
such as heterobifunctional protein reactive groups, one
or more of which has been incorporated into an
lmmllnoreagent of this invention.
A "modified D-enantiomeric nucleotide moiety" can
comprise a D-enantiomeric nucleotide moiety that is
modified so as to reduce or prevent the action of
exonuclease enzymatic activity on the D-enantiomeric
oligonucleotide sequence. It can be a 3'- or 5'-
phosphate linked ribose or a 3'- or 5'-phosphate linked
2'-deoxyribose group containing one or more substituents
such as an alkyl group of 1 to about 10 carbon atoms, or
an ether group such as alkyl or aryl or aralkyl or
substituted aryl or aralkyl ether wherein the alkyl
groups contain from 1 to about 10 carbon atoms and such
~6
~ wo 94,20~ 2 1 5 7 9 0 2 PCT~S94/02610
alkyl or aryl groups may contain or be substituted by
substituents containing oxygen, nitrogen or sulfur
atoms, or a poly(alkylene oxidyl) group, preferably at
the 5'- or 3'-ribose position, respectively, or
elsewhere on the ribose group, which substituent will
reduce or prevent the action of exonuclease enzymatic
activity. A "modified D-enantiomeric nucleotide moiety"
comprising a phosphate ester comprising said
substituents is also useful, as well as a phosphate
ester or modified ribose comprising elements of a
suitable linking group as defined above. A "modified D-
enantiomeric nucleotide moiety" can also comprise a
residue of Z or it can be attached to QI by elements of
Lz as defined above to form a cyclic structure. A
"modified D-enantiomeric nucleotide moiety" can also
comprise compounds with a two carbon-one nitrogen atom
internucleoside linkage.
Preferably, a "modified D-enantiomeric nucleotide
moiety" comprises a poly(alkylene glycol) phosphate
diester. With respect to said "modified D-enantiomeric
nucleotide moietyn, the poly(alkylene glycol) moiety can
have from 2 to about lO0 repeating units. Preferably,
the poly(alkylene glycol) is a poly(ethylene glycol). A
currently preferred poly(alkylene glycol) phosphate
diester is a tetra(ethylene glycol) phosphate diester,
hereinafter sometimes referred to as a "Teg" or "Teg
unit~. Such poly(alkylene glycol) phosphate diesters
can be linked in tandem to each other to form a dimer
phosphate ester sequence, a trimer sequence, a tetramer
sequence, and so forth. One or two such units is
preferred. Such units can also be attached to residues
f QI~ LZ, LQ, I, Ii or Z described herein. A preferred
"modified D-enantiomeric nucleotide moiety" comprises a
Teg unit linked by a phosphate ester bond to a D-
enantiomeric nucleotide such as ~-T.
47
W0941205~ 215 7 ~ ~ 2 PCT~S94/02610
Referring to the radioactive targeting
immunoreagent described in~Structure V, preferred non-
limiting examples of a set of L-enantiomeric
oligonucleotides, CI, which are complementary to the
5members of the set of L-enantiomeri~c oligonucleotides
comprising the "Sequence" of.I;in structure IV include
the L-DNA L-enantiomeric oligonucleotides:
(xvii) L-d(TTAGCTTCTCCG) (SEQ ID NO:l6);
(xviii) L-d(TTAGCTTCTCCGT) (SEQ ID NO:l7);
0(xix) L-d(TTAGC~ CCGTC) (SEQ ID NO:18);
(xx) L-d(TTAGCTTCTCCGTCC) (SEQ ID NO:19);
(xxi) L-d(TTAGCTTCTCCGTCCA) (SEQ ID NO:20);
(xxii) L-d(TTAGCll~lCCGTCCAT) (SEQ ID NO:21);
(xxiii) L-d(TTAG~lL~lCCGTCCATA) (SEQ ID NO:22);
15 and
(xxiv) L-d(TTAGCTTCTCCGTCC~TA~) (SEQ ID NO:23)
Another set of preferred homologous L-enantiomeric
oligonucleotide sequences which can comprise a set
complementary to the nSequence" of structure IV is:
20(xxv) L-d(CTCCGTCCATAA) (SEQ ID NO:24);
(xxvi) L-d(TCTCCGTCCATPA) (SEQ ID NO:25);
(xxvii) L-d(TTCTCCGTCCATA~) (SEQ ID NO:26);
(xxviii) L-d(CTTCTCCGTCCAT~) (SEQ ID NO:27);
(xxix) L-d(GCll~lCCGTCCATAA) (SEQ ID NO:28);
25(xxx) L-d(AGCTTCTCCGTCC~TA~) (SEQ ID NO:29);
(xxxi) L-d(TAGCTTCTCCGTCC~TA~) (SEQ ID NO:30);
(xxxii) L-d(TTAGCTTCTCCGTCCATAA) (SEQ ID NO:23)
An especially preferred complementary sequence
comprises:
30L-d(TTAGCTTCTCCGTCC~TA~) (SEQ ID NO: 23)
The complementarity of the above cI L-enantiomeric
oligonucleotide sequences of structure V with respect to
the previously listed "I" L-enantiomeric oligonucleotide
sequences of structure IV depends on the relative
35orientation of each, i.e., whether the sequences of I
and of cI are constructed from 5' to 3' or 3' to 5', or
4a
~ W094/205~ 2 I S 7 9 ~ 2 PCT~S94102610
vice versa, respectively. Preferably, the Tm of a
hybridized complex formed between the respective I and
cI sequences is greater than 37C.
In a preferred embodiment, the L-enantiomeric
oligonucleotide sequence cI can comprise double stranded
L-DNA or L-RNA. That is, the L-enantiomeric
oligonucleotide sequence may comprise complementary L-
DNA or L-RNA which forms a double helix molecule. The
complementary L-enantiomeric oligonucleotide sequence
0 incorporated into the non-radioactive targeting
immunoreagent composed of L-DNA or L-RNA then hybridizes
to one or the other of the strands of the double
stranded L-DNA or L-RNA comprising cI. In this way, the
complementary L-enantiomeric oligonucleotide sequence
incorporated into the non-radioactive targeting
~mm~noreagent interacts with the duplex L-DNA or L-RNA
of cI in such a way as to form triplex (triple helix) L-
DNA, triplex L-RNA, or a triplex L-DNA:L-RNA hybrid.
QCI in structure V is a spacing group; QCI can be
selected from QI as described for structure IV.
Preferably, QCI comprises an L-enantiomeric
oligonucleotide contiguous sequence of from 2 to about
30 L-enantiomeric nucleotides wherein the sequence of
which is not self-associating and wherein a contiguous
sequence of six or more L-enantiomeric nucleotide units
does not hybridize with any other contiguous sequence of
six or more contiguous L-enantiomeric nucleotide units
anywhere in structure V. QCI can also comprise an L-
enantiomeric oligonucleotide, preferably a sequence such
as ~xxvii) to (xxxii) above which ~s complementary to
the "Sequence" in structure IV. Preferably, QCI
comprises one or two such sequences.
Ll, L2, and L3 in structure V are independently a
chemical bond, preferably a phosphate ester bond, or a
linking group which are defined as Lz and LQ in the
~9
. ,
WO94/205~ 2 ~ ~ 7 9 0 2
PCT~S94/02610
above structure V. ~ ~2, and L3 can also
independently comprise components of QCI.
W1, W2, and W3 in structure V are residues of
chelating groups. The chelating groups of this
invention can comprise the residue of one or more of a
wide variety of chelating agents that can have a
radionuclide associated therewith. As is well known, a
chelating agent is a compound containing donor atoms
that can combine by coordinate bonding with a metal atom
to form a cyclic structure called a chelation complex or
chelate. This class of compounds is described in the
Kirk-Othmer Encyclopedia of Chemical Technology, Vol. 5,
339-368.
The residues of suitable chelating agents can be
independently selected from polyphosphates, such as
sodium tripolyphosphate and hex~metaphosphoric acid;
aminocarboxylic acids, such as
ethyl~ne~ Am; netetraacetic acid, N-(2-
hydroxyethyl)ethylene-~i~;netriacetic acid,
nitrilotriacetic acid, N,N-di(2-hydroxyethyl)glycine,
ethylenebis(hydroxyphenylglycine) and diethylenetriamine
pentacetic acid; 1,3-diketones, such as acetylacetone,
trifluoroacetylacetone, and thenoyltrifluoroacetone;
hydroxycarboxylic acids, such as tartaric acid, citric
acid, gluconic acid, and 5-sulfosalicylic acid;
polyamines, such as ethylene~m;ne, diethylenetriamine,
triethylenetetramine, and triaminotriethylamine;
aminoalcohols, such as triethanolamine and N-(2-
hydroxyethyl)ethylene~ ne; aromatic heterocyclic
bases, such as 2,2'-dipyridyl, 2,2'-diimidazole,
dipicoline amine and 1,10-phenanthroline; phenols, such
as 3~1l~ylaldehyde, disulfopyrocatechol, and
chromotropic acid; amin~ph~nols, such as 8-
hydroxyquinoline and oximesulfonic acid; oximes, such as
dimethylglyoxime and salicylaldoxime; peptides
containing proximal chelating functionality such as
5G
~ W094/205~ 215 7 ~ 0 2 PCT~S94/02610
polycysteine, polyhistidine, polyaspartic acid,
polyglutamic acid, or combinations of such amino acids;
Schiff bases, such as disalicylaldehyde 1,2-
propylenediimine; tetrapyrroles, such as
S tetraphenylporphin and phthalocyanine; sulfur
compounds, such as toluenedithiol, meso-2,3-
dimercaptosuccinic acid, dimercaptopropanol,
thioglycolic acid, potassium ethyl xanthate, sodium
diethyldithiocarbamate, dithizone, diethyl
dithiophosphoric acid, and thiourea; synthetic
macrocylic compounds, such as dibenzo[18]crown-6,
(CH3)6-[14]-4,11-diene-N4, and (2.2.2-cryptate); and
phosphonic acids, such as nitrilotrimethylene-phosphonic
acid, ethylenediaminetetra(methylenephosphonic acid),
and hydroxyethylidenediphosphonic acid, or combinations
of two or more of the above agents.
Preferred residues of chelating agents contain
polycarboxylic acid groups and include: ethylene~1 ~m~ ne-
N, N, N',N'-tetraacetic acid (EDTA); N,N,N',N",N"-
diethylene-triaminepentaacetic acid (DTPA); 1,4,7,10-
tetraazacyclododecane-N,N',N",Nn'-tetraacetic acid
(DOTA); 1,4,7,10-tetraazacyclododecane-N~N'~Nn-triacetic
acid (DO3A); l-oxa-4,7,10-triazacyclododecane-N,N',N"-
triacetic acid (OTTA); trans(1,2)-
cycl~he~nodiethylenetriamine pentaacetic acid (CDTPA);
Preferred residues of chelating agents contain
polycarboxylic acid groups and include the following:
W094/20S23 215 7 ~ 0 2 PCT~S94/02610
COOH
~ ~ N GOOH ~ N ~
r ~COOH ~ COOH r ~ H HOOC r ~ ~- COOH
B4A p4~ s DCDTPA
,
OCH3OCH3
HOOC COOH ~G~OH HOOC ~ G`OOH
TMTMacroTMT
~ N ~ ~ GOC~ r ~ ~ COOH
HOOC COOH COOHHOOC N
COOH
PheMTM~urllC~AT
In one aspect, other suitable residues of chelating
agents comprise proteins modified for the chelation of
metals such as technetium and rhenium as described in
U.S. Patent No. 5,078,985, the disclosure of which is
hereby incorporated by reference.
In another aspect, suitable residues of chelating
agents are derived from N3S and N2S2 cont~;n;ng
compounds, as for example, those disclosed in U.S.
Patent Nos. 4,444,690; 4,670,545; 4,673,562; 4,897,255;
4,965,392; 4,980,147; 4,988,496; 5,021,556 and
5,075,099.
Other suitable residues of chelating agents are
described in PCT/US91/08253, the disclosure of which is
~WO 94/205~ 215 7 9 0 2 PCT~S94/02610
hereby incorporated by reference. In structure V above,
if Wl, W2, and W3 comprise the residue of multiple
chelating agents, such agents can be linked together by
a linking group such as described above in structure IV.
A residue of each of the chelating agents Wl, W2,
and W3 in structure V is independently linked to the
complementary L-enantiomeric oligonucleotide moiety cI
or spacing group QCI through a chemical bond or a
l;nk;ng group, i.e., Ll, L2 and L3 in structure V,
0 above. Preferred linking groups include nitrogen atoms
in groups such as amino, imido, nitrilo and imino
groups; alkylene, preferably cont~;n;ng from l to 18
carbon atoms such as methylene, ethylene, propylene,
butylene and hexylene, such alkylene optionally being
interrupted by l or more heteroatoms such as oxygen,
nitrogen and sulfur or heteroatom-cont~;n;ng groups;
carbonyl;
sulfonyl;
sulfinyl;
ether;
thioether;
ester, i.e., carbonyloxy and oxycarbonyl;
thioester, i.e., carbonylthio, thiocarbonyl,
thiocarbonyloxy, and oxythiocarboxy;
amide, i.e., iminocarbonyl and carbonylimino;
thioamide, i.e., iminothiocarbonyl and
thiocarbonyl ;m; no;
thio;
dithio;
phosphate; phosphonate;
urelene;
thiourelene;
urethane, i.e., iminocarbonyloxy, and oxycarbonylimino;
thiourethane, i.e., iminothiocarbonyloxy and
oxythiocarbonylimino;
an amino acid linkage, i.e., a
W094/205~ 21~ 7 ~ 0 2 PCT~S94/02610 ~
N ~ k or ~ N ~ k
X1 X~
,.
group wherein k=1 and X1, X2, X~ independently are H,
alkyl, cont~;n;ng from 1 to i8, preferably 1 to 6 carbon
atoms, such as methyl, ethyl and propyl, such alkyl
optionally being interrupted by 1 or more heteroatoms
such as oxygen, nitrogen and sulfur, substituted or
unsubstituted aryl, contA;n;ng from 6 to 18, preferably
6 to 10 carbon atoms such as phenyl, hydroxyiodophenyl,
hydroxyphenyl, fluorophenyl and naphthyl, aralkyl,
preferably cont~in;ng from 7 to 12 carbon atoms, such as
benzyl, heterocyclyl, preferably contA;n;ng from 5 to 7
nuclear carbon and one or more heteroatoms such as S, N,
P or O, examples of preferred heterocyclyl groups being
pyridyl, quinolyl, imidazolyl and thienyl;
heterocyclylalkyl, the heterocyclyl and alkyl portions
of which preferably are described above;
or a peptide link~ge~ i.e., a
~ X
N ~ k or ~ N ~ k
X1 X1
group wherein k>1 and each of X1, X2, and X3 is
independently represented by a group as described for
X1, X2, and X3 above. Two or more l;nk;ng groups can be
used, such as, for example, alkyleneimino and
iminoalkylene. It is contemplated that other linking
groups may be suitable for use herein, such as linking
groups commonly used in protein heterobifunctional and
homobifunctional conjugation and crossl~nk;ng chemistry
as described for Lz or LQ in structure IV.
~4
~ W094/205~ 215 7 ~ 0 2 PCT~S94/02610
Especially preferred linking groups include
unsubstituted or substituted phosphate ester groups
containing amino groups which when linked to the residue
of a chelating agent via an isothiocyanate group on the
chelating agent form a thiourea group.
The linking groups can contain various substituents
which do not interfere with the coupling reaction
between chelate W1, W2, or W3 and L-enantiomeric
oligonucleotide cI or the spacing group. The linking
groups can also contain substituents which can otherwise
interfere with such reaction, but which during the
coupling reaction, are prevented from so doing with
suitable protecting groups commonly known in the art and
which substituents are regenerated after the coupling
reaction by suitable deprotection. The linking groups
can also contain substituents that are introduced after
the coupling reaction. For example, the l;nk;ng group
can be substituted with a group such as a halogen, such
as F, Cl, Br or I; an ester group; an amide group;
alkyl, preferably containing from 1 to about 18, more
preferably, 1 to 4 carbon atoms such as methyl, ethyl,
propyl, i-propyl, butyl, and the like; substituted or
unsubstituted aryl, preferably containing from 6 to
about 20, more preferably 6 to 10 carbon atoms such as
phenyl, naphthyl, hydroxyphenyl, iodophenyl,
hydroxyiodophenyl, fluorophenyl and methoxyphenyl;
substituted or unsubstituted aralkyl, preferably
containing from 7 to about 12 carbon atoms, such as
benzyl and phenylethyl; alkoxy, the alkyl portion of
3 0 which preferably contains from 1 to about 18 carbon
atoms as described for alkyl above; alkoxyaralkyl, such
as ethoxybenzyl; substituted or unsubstituted
heterocyclyl, preferably containing from 5 to 7 nuclear
carbon and heteroatoms such as S, N, P or O, examples of
preferred heterocyclyl groups being pyridyl, quinolyl,
imidazolyl and thienyl; a carboxyl group; a carboxyalkyl
~5
W094/20~ 215 7 9 ~ 2 PCT~S94/02610
group, the alkyl portion of which preferably contains
from l to 8 carbon atoms; or the residue of a chelating
group.
In structure V, Ml, M2 and M3 each comprise
S elements with oxidation states equal to or greater than
+l, and at least one of wh~ch is a radionuclide.
Preferably each of Ml, M2 and M3 comprise a metal
isotope, preferably a radioactive metal isotope,
sometimes herein referred to as a metal radioisotope,
0 which radioisotope is useful in a therapeutic or in a
diagnostic imaging application. Preferred metal
radioisotopes are selected from, for example, Sc, Fe,
Pb, Ga, Y, Bi, Mn, Cu, Cr, Zn, Ge, Mo, Tc, Ru, In, Sn,
Re, Sr, Sm, Lu, Eu, Ru, Dy, Sb, W, Re, Po, Ta and Tl.
Useful emissions from such radioisotopes include
spontaneous alpha emissions, beta emissions, gamma
emissions, X-ray emissions, positron emissions, and such
emissions as are induced by the processes of electron
capture and internal conversion. Said emissions can be
purely of one kind such as pure alpha, pure beta, pure
gamma and the like, or of combinations of nuclear
emissions such as beta and gamma emissions and the like.
Radioisotopes with emissions comprising, for
example, alpha radiation or beta radiation are useful in
therapeutic applications, especially in the therapy of
cancer. Useful isotopes in therapeutic applications
include, for example, alpha radiation emitting isotopes
such as, for example, 207pb, 2llpb, 208pb, 2l2pb, 2l2Bi
207Ti, and 223Ra; beta radiation emitting isotopes such
as, for example 47Sc, 66Ga, 67CU, 77As, 30y, lO5Rh
109Pd lllAg 121Sn, 127Te, 143pr~ 149pm~ 153Sm, 161Tb,
l66Ho, l69Er, l77LU, l88Re, l86Re, l9los and l99Au; and
isotopes which emit radiation as a result of the
processes of electron capture and internal conversion
3~ such as, for example, 97Ru, 177msn~ l99Sb, l28Ba and
l97Hg. Radioisotopes especially preferred in
~ W094/205~ 215 7 ~ 0 2 PCT~S94/02610
therapeutic applications include 2l2Pb, 2l2Bi, 90Y,
l;7Lu, l86Re, and l88Re. Currently the most preferred
radioisotope is 90Y.
Radioisotopes with emissions comprising, for
example, gamma radiation or positron radiation are
useful in diagnostic imaging applications, especially in
diagnostic imaging of cancer. Useful isotopes in
diagnostic imaging applications include, for example,
gamma radiation emitting isotopes such as 47Sc, 5lCr,
0 67Cu, 67Ga, 97Ru, 99mTC, lllIn, ll7msn, l4lCe, l67Tm,
l99Au, 87y and 203Pb; and positron radiation emitting
isotopes such as 44Sc, 48v, 64CU, 66Ga, 69Ge, 72AS, 86y
and 89Zr. Radioisotopes especially preferred in
diagnostic imaging applications include 64Cu, 99mTc,
lllIn and 87y. Currently, the most preferred are 99mTc
and lllIn.
In another aspect, other suitable radionuclides can
be incorporated, for example, by covalent bonding into
QCI and include radioacti~e isotopes of halogens such as
radioactive isotopes of iodine, for example, l23I, l24I,
l25I and l3lI as well as radioactive isotopes of
astatine such as 2llAt.
Methods of generating an image useful in the
diagnostic imaging of, for example, cancer in a mammal
comprise detecting emissions imagewise from
radioisotopes as employed in the compositions and
methods of this invention. Said image generating
methods comprise the use of, for example, a collimated
camera detector such as a gamma camera co~only employed
3 0 in radioimmunoscintigraphy (RIS), and the use of linked
X-ray detectors commonly employed in positron emission
tomography (PET) and in single photon emission
- tomography (SPET).
In structure V, x, y, and z are independently zero
or l provided that at least one of x, y, or z is one;
and
W094/205~ 2 i ~ 7 ~ 0 2 PCT~S94/02610
w and b are zero or an integer from l to about 4.
Preferred compositions can be prepared as outlined
in the schemes that follow.
~ h~m~ 6
Derivatization of antibody~amine groups with
heterobifunctional linking reagents SMCC, 2-IT, or SATA.
V SMCC V
~ NH2 ' ~Ar~ll~ N~
amine¦ 7 ¦Ab-M ¦
\ / NH
V 2-l~ otl ~iolane `f ~ SH
~NH2 a
¦Ab-SH¦
\ SATAINH20H \ / o
¦Ab-amine ~ SH
6 8b
In scheme 6, the protein (antibody such as ING-l,
antibody fragment, enzyme, receptor) is chemically
modified for later covalent coupling to a thiolated L-
enantiomeric oligonucleotide (to Ab-M) or to a
maleimido-group-cont~; n; ng L-enantiomeric
oligonucleotide (to Ab-SH). Chemical modification is
effected using a bifunctional cross link;ng agent,
preferably a heterobifunctional cross l;nk;ng agent
having both a group capable of reacting with protein
functional groups (e.g. amine in Ab-amine) and also
having a further group capable of reacting with thiol
groups. The latter is selected from haloacetyl, halo-
~ W094/205~ 215 7 9 0 2 PCT~S94/02610
acetamidyl, maleimido, and activated disulfide
functions.
Maleimido and thioalkyl groups are introduced to an
antibody by utilizing the heterobifunctional linkers,
sulfosuccinimido-4-(M-maleimidomethyl)-cyclohexane-l-
carboxylate (Sulfo-SMCC), 2-iminothiolane (2-IT), or
succinimidyl-S-acetylthioacetate(SATA). The reaction of
a sample containing antibody with a linking agent is for
a time sufficient to introduce an average of about 0.5-3
0 l;nk;ng agent molecules per antibody molecule in the
sample. The derivatized antibody is purified using a
gel filtration column, and more preferably a Sephadex G-
25 column.
Non-limiting examples of preferred protein
conjugates are listed below:
ING-l-NH-CO-cyclohexAne-CH2-Maleimide;
ING-l-NH-C(=NH)-(CH2)3-SH; and
ING-l-NH-CO-CH2-SH.
ING-l is a preferred protein contA;n;ng amine groups
such as lysine amines as represented by Ab-amine.
s5
W094/20523 2 ~ 5 7 ~ 0 2 PCT~S94/02610
S~h~me 7
Derivatization of L-enantiomeric oligonucleotide "~-d(I-
QI-I)" via amine groups with heterobifunctional linking
reagents SATA and SMCC.
S~TEG-Wfl-QI-1)-3'{P]-O~N
5-TEG-L-d(l~;lrl)-3'-[P~ O~ NHOH H
N~ s OH H
¦ L~Oligo~3~H2 ¦ SA~AH 5~ TEG L d(l - Ql - I) ~'--N~
wh~ ~ i~ ~, ~ gmup o
¦ L d(Oii~o) 3'~H ¦
~J
o o
5'-TEG-L-d(l-Q,-I)-3'-[p] -o~ N~N~
OH O
TEG L~(l - Q~ N~
¦ L~(Oli90) 3'-M
GO
~ W094/205~ 215 7 9 0 2 PCT~S94/02610
Sch~me 8
Derivatization of L-enantiomeric oligonucleotide "L-d(I-
QI-I)" with amine groups and with sulfhydryl groups for
use with heterobifunctional linking reagents.
S
~O-DMT
TrS~\~O N(iPrk NC~ N(iPr)z Nl I r.. ,Oc
[for 5' attachment] lfor 3' end blocked]
+ ~MT=d;,~. L~AY I I
rrr ~ ~1 Ag
~OH
HS~ O[P~-5'-L-d(l-QI-1)-3'-[P]-O~NH2
¦L-d(Oligo)-5'-SH ¦
5'HS-L-d(l-QI-l)-NH2 3'
S~h~me 9
Derivatization of L-enantiomeric oligonucleotide "5'-
0 TrS-L-d(I-QI-I)-3'-NH2" with biotin for use with
heterobifunctional linking reagents.
~1 ,
WO 94/20523 2 15 7 ~ 0 2 PCT/US94/02610
TrS-5'-L-d(Oligo)-3'-N H2
" n
~H
_1`N O O
~O-N~ ~
O ~ H H
O ~ N
TrS-5'-L-d(01igo)-3'-NH/~
TrS-5'-L-d(l-Q 1-1)-3'-Biotin
TrS-5'-L-d(01igo)-3'-Biotin
Ag+
H
~__H~
HS-5'-L~(01igo)-3'-NI~ ~ H
HS-5'-L-d(l-QI-1)-3'-Biotin
HS-5'-L-d(01igo)-3'-Biotin
6~
~WO 94/20S23 2 1 5 7 ~ O ~ PCT/US94/02610
h eme 10
Preparation of "5'-HS-L-d(I-QI-I)-3'-biotin" with
biotinylated phosphoramidite reagent for use with
heterobifunctional linking reagents.
s
rO-DMT
NC~O~P~o CH2)sNH-Biotin
N(iPr)2
[for 3' end blocked]
Solid Phase Sy"ll,esi~ of
L-d(l~rl)-3'-Biotin
H0-5'-L-d(l-QI-1)-3'-Biotin
TrS~\/~O N(iPr)2
for 5' ~ Cl lllle~q
OH
Trs~~`o[p]-5~-L~(l - Ql-1)-3'-[P]-O~
~(CH~)5NH-Biotin
¦ Ag+
HS-5'-L-d(1-Q~-1)-3'-Biotin
HS-5'-L-d(01igo)-3'-Biotin
L-enantiomeric oligonucleotides and modified L-
enantiomeric oligonucleotides are synthesized according
0 to standard methods such as solid phase synthesis that
are well known in the art for the synthesis of D-
enantiomeric oligonucleotides. Derivatizations of L-
enantiomeric oligonucleotides L-d(I-QI-I) are achieved
using the reaction of 5'-TEG-L-enantiomeric oligomer-
NH2-3' with SATA or SMCC to afford L-enantiomeric
oligomer-3'-SH or L-enantiomeric oligomer-3'-M (see
Scheme 7~.
C~3
W094/205~ 2 ~ 2 PCT~S94/02610
The followin.g; L, çnantiomeric oligonucleotides are
preferred:
(a) L-d(TTATGGACGGAGAAGCTAA) (SEQ ID NO: 8), and
(b) {5'-Teg-L-d(TCTTATGGACGGAGAAGCTAA-[ACTCTC]-
S TTATGGACGÇA~GCTAATCT)-3'-amine-L-T},
(SEQ ID No:31)
wherein the square bracketed portion of SEQ ID No:31
denotes the preferred spacer, -L-d[ACTCTC]-, which
separates two sequences each containing the preferred
sequence -L-d(TTATGGACGGAGAAGCTAA)- (SEQ ID No: 8).
These L-enantiomeric oligomers are.also derivatized
to afford a bifunctio~l;7.ed L-enantiomeric oligomer:
5'-HS-L-d(I-QI-I)-NH2-3' via introduction of
bifunctional reagents at the 5'- and 3'-positions, as
shown in Scheme 8. The introduction of biotin at the
3'-position is also achieved by the reaction of
5'-HS-L-d(I-QI-I)-NH2-3'with biotin-imidocarboxylate as
shown in Scheme 9.
There are useful spacers with a protected amine,
thiol, or carboxyl group on one end and a
phosphoramidite at the other (see Scheme lO).
The applications of these spacers are shown in
reactions below.
I~wo 94/20523 2 1 5 7 ~ O ~
PCT/US94/02610
~S ch ~me 11
Assembly of Ab-M + L-d(Oligo)-5~-SH
~/ , o
~NH~N~ Ab-M ¦
5' HS-L-d(l -Ql -I)-NH2 3'
\ / o
~ ~ o
~NH ~NJI~
~-6'-L-d(l -Ql-l )-3'-NH2
¦Ab-M-5'-S-L-d(Oligo)-3'-NH2 ¦
.c: ~.h ~?me 1 ?
Ass~r,ll~lJ of Ab SH I L-d(01i~o)-3'-M
y~
!j`TEG-L~(I ~- 1)-3'-N~
y~.,"l~
~N 3'-L-d(l ~-1 ~TEG ~i'
¦ Ab-S-M-3'-L-d(Oli~o) ¦
The modified maleimido antibody (Ab-M, Scheme 6)
and the thiolated L-enantiomeric oligonucleotide (5'-HS-
1 0 L-d (I-QI-I)-NH2-3') can be assembled to yield the
modified antibody-L-enantiomeric oligonucleotide
W094/20S~ 21~ 7 ~ ~ 2 PCT~S94/02610
conjugate (Ab-M-5'-S-succinimido-L-d(I-QI-I)-NH2-3') as
shown in Scheme 11. Similarly, Ab-S-M-3'-L-d(I-QI-I)-
TEG-5' is prepared from Ab-SH and 5'-TEG-L-d(I-QI-I)-
maleimide-3' as shown in Scheme 13.
~ c~h ~m~
Complementary L-Oligonuc~otide ~6'-TMT-L~(cl)-lMT-3'~
N(iPr)2
F~ NH~~ 0~ P~o~CN
To form 5' amine
J 2 TMT-NCS
5'H2N-L~(Cl)-NH2 3' r 5~TMT-L-d(cl)-TMT 3
NC~O~p~~ NHFmoc
(iP~ ODMT
To form 3~ arnine
0 In these s~hem~S~ L-d(oligo) represents an L-
enantiomeric oligodeoxyribonucleotide such as an L-
deoxyribonucleotide, L-d(I); d(cI) represents the
complementary L-enantiomeric oligodeoxyribonucleotide
sequence; L-d(I-QI-I) is an L-enantiomeric
oligodeoxyribonucleotide; and TMT represents a member of
a class of terpyridine chelates, preferably as described
above. A preferred TMT is 4'-(3-isothiocyanato-4-
methoxyphenyl)-6,6"-bistN,N-di(carboxymethyl)-
aminomethyl~-2,2':6',2"-terpyridine, TMT-NCS.
The reaction of 5l-H2N-L-d(cI)-NH2-3~ with 2 moles
of TMT-NCS affords the desired 5'-TMT-L-d(cI)-TMT-3' as
shown in Scheme 13.
G~
~ W094/205~ 215 7 9 ~ ~ PCT~S94/02610
There are other useful linking agents with a
protected amine, thiol, or carboxyl group on one end and
a phosphoramidite at the other. The applications of
these agents are shown in the Examples.
In a preferred embodiment, an effective dose of a
radioactive targeting reagent as described above in a
pharmaceutically acceptable medium is prepared by
exposing a composition comprising a complementary L-
enantiomeric oligonucleotide sequence contA;n;ng one or
more chelating groups such as the
oligodeoxyribonucleotide sequence as described above to
a composition cont~;n;ng a radioactive metal isotope
such that the molar amount of the radionuclide metal
isotope is less than the molar amount of the chelating
groups. The exposure lasts an effective time during
which uptake of of the radionuclide metal isotope into
the chelating agents occurs.
In a preferred embodiment, an effective dose of a
non-radioactive targeting immunoreagent as described
above in a pharmaceutically acceptable medium is
~m; n;stered to a patient and the non-radioactive
targeting immunoreagent is allowed to accumulate at the
target site such as at a tumor site in the patient.
Subsequently, an effective dose of a radioactive
targeting reagent as described above in a
pharmaceutically acceptable medium is administered to
the patient, and the radioactive targeting reagent is
allowed to accumulate at the target site, said target
site being the non-radioactive targeting immunoreagent
which has accumulated at the tumor site in the patient.
The present invention also comprises one or more of
the immunoreagents of this invention formulated into
compositions together with one or more non-toxic
physiologically acceptable carriers, adjuvants or
vehicles which are collectively referred to herein as
carriers, for parenteral injection for oral
(~7
WO 94l20523 2 ~ 5 7 ~ ~ 2 PCT/US94/02610 ~Ij
a~m; n; stration in solid or liquid form, for rectal or
topical administration, or the like.
The compositions can be administered to humans and
An;m~ls either orally, rectally, parenterally
~intravenously, intramuscularly or subcutaneously),
intracisternally, intravaginaliy, intraperitoneally,
intravesically, intra-articularly, locally (in the form
of powders, ointments or drops), or as a buccal or nasal
spray.
Compositions suitable for parenteral injection may
comprise physiologically acceptable sterile aqueous or
nonaqueous solutions, dispersions, suspensions or
emulsions and sterile powders for reconstitution into
sterile injectable solutions or dispersions. Examples
of suitable aqueous and nonaqueous carriers, diluents,
solvents or vehicles include water, ethanol, polyols
(propylene glycol, polyethylene glycol, glycerol, and
the like), suitable mixtures thereof, vegetable oils
(such as olive oil) and injectable organic esters such
as ethyl oleate. Proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by
the maintenance of the required particle size in the
case of dispersions and by the use of surfactants.
These compositions may also contain adjuvants such
as preserving, wetting, emulsifying, and dispensing
agents. Prevention of the action of microorg~n;s~ can
be ensured by various antibacterial and antifungal
agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, and the like. It may also be desirable to
include isotonic agents, for example sugars, sodium
chloride and the like. Prolonged absorption of the
injectable pharmaceutical form can be brought about by
the use of agents delaying absorption, for example,
aluminum monostearate and gelatin.
Solid dosage forms for oral a~m;n;stration include
capsules, tablets, pill8, powders and granules. In such
~8
W094l205~ 215 7 9 0~ PCT~S94/02610
solid dosage forms, the active compound is admixed with
at least one inert customary excipient (or carrier) such
as sodium citrate or dicalcium phosphate or (a) fillers
or extenders, as for example, starches, lactose,
sucrose, glucose, mannitol and silicic acid, (b)
binders, as for example, carboxymethylcellulose,
alignates, gelatin, polyvinylpyrrolidone, sucrose and
acacia, (c) humectants, as for example, glycerol, (d)
disintegrating agents, as for example, agar-agar,
0 calcium carbonate, potato or tapioca starch, alginic
acid, certain complex silicates and sodium carbonate,
(e) solution retarders, as for example paraffin, (f)
absorption accelerators, as for example, quaternary
ammonium compounds, (g) wetting agents, as for example,
cetyl alcohol and glycerol monostearate, (h) adsorbents,
as for example, kaolin and bentonite, and (i)
lubricants, as for example, talc, calcium stearate,
magnesium stearate, solid polyethylene glycols, sodium
lauryl sulfate or mixtures thereof. In the case of
capsules, tablets and pills, the dosage forms may also
comprise buffering agents.
Solid compositions of a similar type may also be
employed as fillers in soft and hard-filled gelatin
capsules using such excipients as lactose or milk sugar
as well as high molecular weight polyethylene glycols,
and the like.
Solid dosage forms such as tablets, dragees,
capsules, pills and granules can be prepared with
coatings and shells, such as enteric coatings and others
well known in the art. They may contain opacifying
agents, and can also be of such composition that they
release the active compound or compounds in a certain
part of the intestinal tract in a delayed manner.
Examples of embedding compositions which can be used are
polymeric substances and waxes.
~9
W0941205~ 2 1 5 7 ~ ~ 2 PCT~S94102610 ~
The active compounds can also be in micro-
encapsulated form, if appropriate, with one or more of
the above-mentioned excipients.
Liquid dosage forms for oral administration include
pharmaceutically acceptable emulsions, solutions,
suspensions, syrups and elixirs. In addition to the
active compounds, the liquid dosage forms may contain
inert diluents commonly used in the art, such as water
or other solvents, solubilizing agents and emulsifiers,
as for example, ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl
benzoate, propylene glycol, l,3-butylene glycol,
dimethylformamide, oils, in particular, cottonseed oil,
groundnut oil, corn germ oil, olive oil, castor oil and
sesame oil, glycerol, tetrahydrofurfuryl alcohol,
polyethylene glycols and fatty acid esters of sorbitan
or mixtures of these substances, and the like.
Besides such inert diluents, the composition can
also include adjuvants, such as wetting agents,
emulsifying and suspending agents, sweetening, flavoring
and perfuming agents. Suspensions, in addition to the
active compounds, may contain suspending agents, as for
example, ethoxylated isostearyl alcohols,
polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide,
bentonite, agar-agar and tragacanth, or mixtures of
these substances, and the like.
Compositions for rectal A~m; n; strations are
preferably suppositories which can be prepared by mixing
3 0 the compounds of the present invention with suitable
non-irritating excipients or carriers such as cocoa
butter, polyethylene glycol or a suppository wax, which
are solid at ordinary temperatures but liquid at body
temperature and, therefore, melt in the rectum or
vaginal cavity and release the active component.
~0
W094/205~ 21 S ~ 9 0 ~ PCT~S94/02610
Dosage forms for topical administration of a
compound of this invention include ointments, powders,
sprays and inhalants. The active component is admixed
under sterile conditions with a physiologically
acceptable carrier and any preservatives, buffers or
propellants as may be required. Ophth~lm;c
formulations, eye ointments, powders and solutions are
also contemplated as being within the scope of this
invention.
Actual dosage levels of active ingredient in the
compositions of the present invention may be varied so
as to obtain an amount of active ingredient that is
effective to obtain a desired therapeutic response for a
particular composition and method of A~m; n; stration.
The selected dosage level therefore depends upon the
desired therapeutic effect, on the route of
a~mi n; stration, on the desired duration of treatment and
other factors.
The total daily therapeutic dose of the compounds
of this invention a~m; n; stered to a host in a single or
divided dose may be in amounts, for example, of from
about l00 picomol to about 5 micromols per kilogram of
body weight. Dosage unit compositions may contain such
amounts of such submultiples thereof as may be used to
make up the daily dose. It will be understood, however,
that the specific dose level for any particular patient
will depend upon a variety of factors including the body
weight, general health, sex, diet, time and route of
a~m; n; stration, rates of absorption and excretion,
combination with other drugs and the severity of the
particular disease being treated.
In another embodiment, the present invention is
directed to a method of diagnosis comprising the
a~m; n; stration of a diagnostic imaging effective amount
of the compositions of the present invention to a m~m~-l
in need of such diagnosis. A method for diagnostic
2157~02
W094/205~ PCT~S94/02610
imaging for use in medical procedures in accordance with
this invention comprises administering to the body of a
test subject in need of a diagnostic image an effective
diagnostic image producing amount of the above-described
compositions. In this method, an efective diagnostic
image producing amount of a non-radioactive targeting
immunoreagent as described above~-in a pharmaceutically
acceptable medium is administered to a patient and said
non-radioactive targeting immunoreagent is allowed to
accumulate at the target site such as at a tumor site in
said patient. Subsequently, a diagnostic imaging
effective dose of a radioactive targeting reagent as
described above in a pharmaceutically acceptable medium
is a~ministered to said patient, and said radioactive
targeting reagent is allowed to accumulate at the target
site, said target site being the said non-radioactive
targeting ir-llnoreagent accumulated at said tumor site
in said patient. The image pattern can then be
visualized.
The total ~;~gnostic imaging effective dose of the
compounds of this invention a~m; n; stered to a host in a
single or divided dose may be in amounts, for example,
of from about l picomol to about 0.5 micromols per
kilogram of body weight. Dosage unit compositions may
contain such amounts of such submultiples thereof as may
be used to make up the effective diagnosting imaging
dose. It will be understood, however, that the specific
dose level for any particular patient will depend upon a
variety of factors including the body weight, general
health, sex, diet, time and route of A~m; n; stration,
rates of absorption and excretion, combination with
other drugs and the severity of the particular disease
being treated.
In addition to human patients, the test subjects
can include m~mm~l ian species such as rabbits, dogs,
.
W094/205~ 215 7 9 0 ~ PCT~S94/02610
cats, monkeys, sheep, pigs, horses, bovine ~n;m~l S and
the like.
After A~m; n; stration of the compositions of the
present invention, the subject m~ 1 iS maintained for
an effective time which is a time period sufficient for
the administered compositions to be distributed
throughout the subject and to enter the targeted tissues
of the mA~l. A sufficient time period for the non-
radioactive targeting immunoreagent is generally from
0 about l hour to about 2 weeks or more and, preferably
from about 2 hours to about l week. A sufficient time
period for the radioactive targeting reagent such as the
preferred 90Y is generally measured in terms of half-
life of the radionuclide and as such is in the range of
from about l to about lO half-lives or more and,
preferably from about 2 hours to about 6 half-lives.
The following examples further illustrate the
invention and are not to be construed as limiting of the
specification and claims in any way. Specific
embodiments of the invention are illustrated in the
following examples.
EXAMPLES
Example l
L-Enantiomeric Oligonucleotide Design
A targeting immunoreagent of this invention as
described in Structure IV comprising an antibody 2,
l;nk;ng groups Lz and LQ, an L-enantiomeric
oligonucleotide sequence I, a spacing group QI~ a second
sequence Ii, and an end capping group E is designed as
follows utilizing anticipated complementary binding
properties between complementary pairs of mirror image
D-enantiomeric oligonucleotides as a model for analogous
physical properties imputed to the desired L-
enantiomeric oligonucleotide.
W094/205~ 2 ~ ~ 7 ~ ~ 2 PCT~S94/02610
A sample mirror image, naturally occurring D-
enantiomeric oligodeoxyribonucleotide with the following
sequence (herein referred to as SEQ ID NO: 32) is
analyzed for conformity to the criteria described
previously for Structure IV wherein groups Lz, I, QI,
Ii, and LQ and E are as represented below as D-
enantiomers: ~:
Lz Iz QI Ii LQ E
5'D-
0 tTCITTATGGACGGATcCGcTAAlTclTTATGGAcGGATccGcTAAl TCT ] 3 '
(mirror image D-enantiomer SEQ ID NO: 32)
Analysis of this D-enantiomeric oligonucleotide
sequence using "oligo" computer software (National
Biosciences) designed for D-enantiomers reveals it to
contain 5 regions of self-complementarity. All such
regions are hairpin loops with differing degrees of
overlap. The sum of the negative free energy changes
(dG's) for these self-complementary regions is -28.3
kcal/mole which predicts a melting temperature of 86C
in l.0 molar salt for this D-enantiomeric
oligonucleotide. The most stable region of self-
complementarity (dG ~ -15 kcal/mol) contains 8 base
pairs most of which correspond to a BAM-l restriction
site palindrome. In the presence of these regions of
self-complementarity a D-enantiomeric oligonucleotide
sequence complementary to this sequence cannot hybridize
successfully to its full length. By inference, a mirror
image L-enantiomeric oligonucleotide of the same
sequence and an L-enantiomeric oligonucleotide sequence
complementary to the L-enantiomeric oligonucleotide
sequence can also not hybridize successfully to its full
length in the presence of mirror image regions of ~elf-
complementarity. Modifications to the D-enantiomeric
oligonucleotide sequence (as well as respective
modifications to the mirror image L-enantiomeric
oligonucleotide) are then made in accordance with the
7~t '
~ W094/205~ 215 7 ~ 0 2 PCT~S94/02610
criteria outlined for structure IV to replace selected
bases in regions of self-complementarity such that
excessive base pairing is removed in iterative analysis.
Additional D-enantiomeric and additional L-enantiomeric
nucleotides are also inserted into the respective spacer
sequences to ensure that, in helical conformations, the
respective t~rm;n~l groups of two respective
complementary sequences, D-cI and L-cI, when hybridized
to the two D-enantiomer sequences I and II and to the
0 two L-enantiomer sequences, respectively are orthogonal
to each other.
In this manner, a new D-enantiomeric
oligonucleotide (SEQ ID NO: 33) with the following
sequence is designed. The mirror image L-enantiomer
(SEQ ID NO: 34) is synthesized and the L-enantiomer is
conjugated to an immunoreactive molecule (i.e., to an
antibody such as ING-l):
~ - ~n~nt ~ ~m~ r 01 igonll~leot~e
2 O LZ I QI I
LQ E
5'X-
DtTCITTATGGACGGAGAAGCTAAlAcTcTclTTATGGAcGGA~AAGcTAAlTcTy
T]-3'
2 5 ( SEQ ID NO: 33)
T. - ~n~nt~ ~m~r 01 ~gonll~l~ot~e
L-d(LzI QI I
LQ E)
5'X-L-
d(TCITTATGGACGGAGAAGCTAAlAcTcTclTTATGGAcG~A~A~GcTA-A-lTcTyT)
3'
( SEQ ID NO: 34)
This L-enantiomeric oligonucleotide (SEQ ID NO: 34)
comprises two copies of the same L-enantiomeric
W094l205~ 215 7 9 0 2 PCT~S94/02610
nucleotide sequence (i.e., I = Ii = L-
d(TTATGGACGGAGAAGCTAA); SEQ ID NO : 8) linked through 6
L-enantiomeric nucleotide spacer [QI = L-d(ACTCTC)].
The groups Lz and LQE at the 5' or the 3' end comprise
groups X and Y. X and Y can each independently be an
amine-containing group (such as, ~or example, those
available from Clontech Industries, each of which amine-
containing groups being sometimes hereinafter
cryptically referred to as "NH2" or as "amine") or one
of X and Y is selected from an amine-containing group
and the other is selected from one or more TEG groups as
described above. Additionally, one of X and Y can also
be a term; n~l hydroxyl group ~in which case the 3'-YT in
the above sequence becomes a deoxyribosyl-OH group on
removal from the solid phase support, and more
preferably X is a 5'OH group available by lc...o-v~l of a
DMT group with acid at the end of the synthetic sequence
before deprotecting the amine groups with ammonium
hydroxide), or one of X and Y can also be a ter~;nAl
phosphate ester group (in which case the 3'-YT or the
5'-X becomes a -OPO3H2 group or an ionized salt such as
a sodium salt thereof), as desired. Such phosphate
term;nAl groups can be made by treatment of the 5'-OH
term;n~l group with 2-cyanoethyl N,N-diisopropyl
chlorophosphoramidite at the end of the synthesis.
Subsequent reaction with ammonium hydroxide and
oxidation provides the term; n~ 1 phosphate group at X. A
3'-phosphate group can be introduced after synthesis of
the oligomer and its r el--oval from the solid support by
treatment of the 3-hydroxyl group with phosphoric acid
anhydide followed by hydrolysis in water.
Thus, in one embodiment, this L-enantiomeric
oligonucleotide, cryptically referred to as 5'-Teg-L-
d(I-QI-I)-3'-amine, is:
5'Teg-L-
d(TCTTATGGACG~G~GCTAAACTCTCTTATGGACGGAGAAGCTAATCT)-3'-
~6
W094/205~ 21~ 7 ~ 0 ~ ~ PCT~S94102610
NH2-T, i.e., (SEQ ID NO: 31), or 5'-Teg-L-d(I-QI-I)-
3'amine.
In another embodiment, this L-enantiomeric
oligonucleotide, cryptically referred to as 5'-amine-L-
d(I-QI-I)-3lTeg, is:
5'H2N-L-
d(TCTTATGGACGGAGAAGCTAAACTCTCTTATGGACG~ GCTAATCT-3'-
Teg-T) or 5'-amine-L-d(I-QI-I)-3'-Teg or (SEQ ID NO: 35)
In another embodiment, this L-enantiomeric
oligonucleotide, cryptically referred to as 5'-amine-L-
d(I-QI-I)-3'0H, is:
5'H2N-L-
d(TCTTATGGACG~,~G~AGCTAAACTCTCTTATGGACG~A~A~GCTAATCT-3'-
OH) or 5'-amine-L-d(I-QI-I)-3'-OH of (SEQ ID NO: 36)
In another embodiment, this L-enantiomeric
oligonucleotide, cryptically referred to as 5'-amine-L-
d(I-QI-I)-3'0PO3H2, is:
5'H2N-L-
d(TCTTATGGACG~AGA~GCTAAACTCTCTTATGGACGG~A~GCTAATCT-3'-
OPO3H2) or 5'-amine-L-d(I-QI-I)-3'-OPO3H2 or (SEQ ID NO:
37)-
Example 2
(2a) Synthesis of L-d(I-QI-I); 5'-Teg-L-d(I-QI-I)-3'-NH2
(SEQ ID NO: 31)
This L-enantiomeric oligonucleotide is prepared on
an Applied Biosystems oligonucleotide synthesizer
originally designed by the manufacturer to be used for
the synthesis of naturally occurring D-enantiomeric
oligonucleotides. The method applicable to the trityl-
on protocol for the synthesis of naturally occurring D-
enantiomeric oligonucleotides is used as directed by the
equipment manufacturer but is modified to instead use L-
2-deoxynucleotide phosphoramidite reagent precursors 5'-
dimethoxytrityl L-cytidine-3'-O-phosphoramidite, 5'-
dimethoxytrityl L-adenosine-3'-O-phosphoramidite, S'-
W094/20S~ 21~ 2 PCT~S94/02610
dimethoxytrityl ~-guanosine-3'-O-phosphoramidite, and
5'-dimethoxytrityl L-thymidine-3'-O-phosphoramidite
prepared as described above rather than the D-
enantiomers. Clonetech's Uni-link Amino Modifier is
used as the precursor to the 3'-amine group. The TEG
group, a tetra(ethylene glycol) pho~sphate diester linked
in this invention by a phosphate estér bond to 5'-
dimethoxytrityl L-thymidine-3'-O-phosphoramidite twhich
is the L-enantiomer (mirror image) of the reagent
0 disclosed in WO/92/02534 which refers to a
tetra(ethylene glycol) phosphate diester linked by a
phosphate ester bond to 5'-dimethoxytrityl D-thymidine-
3'-O-phosphoramidite] is used at the 5'-end of the
strand. Following synthesis of the entire L-
enantiomeric oligonucleotide, the base protecting groups
and solid support are removed with ammonium hydroxide
and the resulting 5'-protecting group is ell-o~ed with 3%
trichloroacetic acid. The L-enantiomeric
oligonucleotide is desalted and further purified by
elution down an OPEC Cartridge (Clonetech) with
deionized water. Electrophoresis on a 12%
polyacryiamide gel is used to further purify the L-
enantiomeric oligonucleotide. The L-DNA band is
visualized by ultraviolet light shadowing. It is cut
out, minced, and extracted with a buffer comprising l0mM
Tris HCl and lmM EDTA at pH 7.5 at 4C for 24 hours.
The gel pieces are then ~ ed by centrifugation, and
the L-DNA is purified through a spun column of Sephadex
G-25. The concentration of L-enantiomeric
oligonucleotide is estimated using absorbance at 260 nm.
(2b) Synthesis of 5'-H2N-L-d(I-QI-I)-3'-Teg (SEQ ID NO:
35)
This L-enantiomeric oligonucleotide is prepared on
an Applied Biosystems oligonucleotide synthesizer by the
trityl-off protocol otherwise used for D-enantiomers as
094/205~ 215 7 9 0 ~ PCT~S94/02610
directed by the equipment manufacturer but modified to
use L-2-deoxynucleotide phosphoramidite reagent
precursors (i.e., 5'-dimethoxytrityl L-cytidine-3'-O-
phosphoramidite, 5'-dimethoxytrityl L-adenosine-3'-O-
phosphoramidite, 5'-dimethoxytrityl L-guanosine-3'-O-
phosphoramidite, and 5'-dimethoxytrityl L-thymidine-3'-
O-phosphoramidite as described above). Clonetech's 6
carbon monomethoxytrityl AminoModifier (N-MMT-C6-
AminoModifier) is used as the precursor of the 5'-amine
group. The TEG group is used as the ter~; n~ l group at
the 3'-end. The base protecting group and solid support
are le...o~ed with ammonium hydroxide and the L-
enantiomeric oligonucleotide is further purified by
polyacrylamide gel electrophoresis.
(2c) Synthesis of 5'-Trityl-S-L-d(I-QI-I)-3'-NH2
The L-enantiomeric oligonucleotide sequence of
Example 2a minus the Teg group is prepared on an Applied
Biosystems oligonucleotide synthesizer by the trityl-on
protocol as directed by the e~lipme~t manufacturer using
L-2-deoxynucleotide phosphoramidite reagent precursors
(5'-dimethoxytrityl L-cytidine-3'-O-phosphoramidite, 5'-
dimethoxytrityl L-adenosine-3'-O-phosphoramidite, 5'-
dimethoxytrityl L-guanosine-3'-O-phosphoramidite, and
5'-dimethoxytrityl L-thymidine-3'-O-phosphoramidite as
described above). Clonetech's Uni-link Amino Modifier
is used as the precursor to the 3'-amine group.
Clonetech's C6-ThioModifier is used as the precursor to
the 5'-thiol group (which is herein referred to
cryptically as 5-S~ added to the sequence in place of
TEG in Example 2a. Following synthesis of the whole L-
enantiomeric oligonucleotide, the base protecting groups
and solid support are ~el-,o~ed with ~m~o~ ium hydroxide.
The L-enantiomeric oligonucleotide is desalted and
further purified by elution down an OPEC Cartridge
(Clonetech) with deionized water. The concentration of
~9
W094/205~ 2 ~ ~ ~ 9 ~ ~ PCT~S94/02610
L-enantiomeric oligonucleotide is estimated using
absorbance at 260 nm.
(2d) Preparation of fluorescent 5'Teg-L-d(I-QI-I)-3'-NH-
Cy5.18
A sample (30 nmoles) of 5'-Te,g-L-d(I-QI-I)-3'-NH2,
prepared according to Example 2a,~ s evaporated to
dryness and redissolved in 500 mL 0.l M bicarbonate
buffer at pH 9 using a vortex mixer. This sample is
then added to a vial contA; n ; ng the dried succinimidyl
ester of the dye Cy5.18 (Biological Detection Systems;
Pittsburg PA). After thorough mixing the reaction is
allowed to proceed at room temperature for one hour with
frequent mixing. The product, 5'-Teg-L-d(I-QI-I)-3'-NH-
Cy5.18, is purified by elution from a Sepahadex G-25
column with distilled water.
Example 3
(3a) Synthesis of cI ~; ~m; ne, 5'-NH2-L-d(cI)-3'-NH2
An L-enantiomeric oligonucleotide, L-d(cI),
cont~;n;ng the L-enantiomeric nucleotide ~equence 5'X-L-
d~TTAGClL~lCCGTCC~T~AYT)-3'(SEQ ID NO: 38) complementary
to the L-enantiomeric oligonucleotide I is prepared on
an Applied Biosystems oligonucleotide synthesizer as
outlined in Example 2a. The 3'- (Y) and 5'- (X) amine-
cont~;n;ng groups are incorporated as directed by the
equipment manufacturer using Uni-link Amino Modifier
(Clonetech) for the precursor to the 3'-amine group, and
Clonetech's 6 carbon monomethoxytrityl ~m; noModifier (N-
3 0 MMT-C6-AminoModifier: Catalog # 5202) as precursor to
the 5'-amine group. After final deblocking and removal
from the solid supportr the protecting groups are
removed with ~mmon;um hydroxide, and the amine-
function~l;7ed L-enantiomeric oligonucleotide is
purified by elution down an OPEC Cartridge (Clonetech)
with deionized water. The L-enantiomeric
W094/205~ 215 7 ~ 0 ~ PCT~S94/02610
oligonucleotide is further purified by polyacrylamide
gel electrophoresis or reverse-phase HPLC. The
concentration of L-enantiomeric oligonucleotide is
estimated using absorbance at 260 nm.
(3b) Synthesis of I ~iAm;ne, 5'-NH2-L-d(I)-3'-NH2
An L-enantiomeric oligonucleotide, I, containing
the L-enantiomeric nucleotide sequence 5'X-L-
d(TTATGGACGGAGAAGCTAAYT)-3'(SEQ ID NO: 39) is prepared
on an Applied Biosystems oligonucleotide synthesizer as
outlined in Example 3a. The 3'- (Y) and 5'- (X) amine-
containing groups are incorporated as directed by the
equipment manufacturer using Uni-link Amino Modifier
(Clonetech) for the precursor to the 3'-amine group, and
Clonetech's 6 carbon monomethoxytrityl AminoModifier (N-
MMT-C6-AminoModifier: Catalog # 5202) as the precursor
to the 5'-amine group. After final deblocking and
cleavage from the solid support, the protecting groups
are lc--,oved with A~O~ ium hydroxide, and the amine-
functionalized L-enantiomeric oligonucleotide is
purified by elution down an OPEC Cartridge (Clonetech)
with deionized water. The L-enantiomeric
oligonucleotide is further purified by polyacrylamide
gel electrophoresis or reverse-phase HPLC. The
concentration of L-enantiomeric oligonucleotide is
estimated using absorbance at 260 nm.
(3c) Synthesis of L-d(cI)
An L-enantiomeric oligonucleotide, cI, contAining
the L-enantiomeric nucleotide sequence 5'-L-
d(TTAGC~ CCGTCCATAA)-3' (SEQ ID NO: 23) complementary
to (I) is prepared on an Applied Biosystems
oligonucleotide synthesizer as outlined in Example 2a.
After final deblocking and cleavage from the solid
support, the protecting groups are removed with ammonium
hydroxide. The L-enantiomeric oligonucleotide is
81
W094/20~ 2 ~ 2 PCT~S94/02610
purified by elution down an OPEC Cartridge (Clonetech)
with deionized water. The L-enantiomeric
oligonucleotide is further purified by polyacrylamide
gel electrophoresis. The concentration of L-
enantiomeric oligonucleotide~is estimated using
absorbance at 260 nm.
(3dJ Synthesis of L-d(I)
An L-enantiomeric oligonucleotide, I, contAin;ng
the L-enantiomeric nucleotide sequence 5'-L-
d(TTATGGACGÇ~.A~GCTAA)-3' (SEQ ID NO: 8t is prepared on
an Applied Biosystems oligonucleotide synthesizer as
outlined in Example 3c. After final deblocking and
cleavage from the solid support, the protecting groups
are removed with ammonium hydroxide, and the L-
enantiomeric oligonucleotide is purified by elution down
an OPEC Cartridge (Clonetech) with deionized water. The
L-enantiomeric oligonucleotide is further purified by
polyacrylamide gel electrophoresis. The concentration
of L-enantiomeric oligonucleotide is estimated using
absorbance at 260 nm.
(3e) Preparation of fluorescent cI ~; ~m; ne, 5 ' -NH2-L-
d(cI)-3'-NH2, and fluorescent I ~; ~m; ne, L-d(I)
A sample (30 nmoles) of 5'-NH2-L-d(cI)-3'-NH2,
prepared according to Example 3a, is evaporated to
dryness and redissolved in 500 mL 0.1 M bicarbonate
buffer at pH 9 by vortexing. This sample is then added
to a vial cont~;n;ng the dried N-hydroxysucc;n;~;dyl
ester of the dye Cy5.18 (Biological Detection Systems;
Pittsburg PA). After thorough vortexing the reaction is
allowed to proceed at room temperature for one hour with
frequent mixing. CY5.18 labeled 5'-NH2-L-d(cI)-3'-NH2
i~ purified by elution from a Sep~h~x G-25 column with
distilled water.
~ 094/205~ 2 1 ~ 7 ~ ~ ~ PCT~S94/02610
A sample of Cy5.18 labeled I ~- ~mi ne, 5'-NH2-L-
d(I)-3'-NH2 of Example 3b is prepared in the same
manner.
Example 4
(4a) Annealing of L-d(cI) to 5'-Teg-L-d(I-QI-I)-3'-NH2
A sample of 5'-Teg-L-d(I-QI-I)-3'-NH2 (2 mmoles),
prepared according to Example 2a, is mixed with aliquots
containing increasing amounts of L-d(cI) prepared by the
0 method of Example 3c, [from 100:5 to 100:200 of L-d(I-
QI-I):L-d(cI)], in 6SSC buffer at room temperature. The
mixtures are loaded into a sample cuvette and analysed
by UV light t260 nm) in a Cary 13 instrument while the
cuvette temperature is ramped up from 30C to 90C and
then back down to 30C. The presence of binary
complexes (L-d(cI):5'-Teg-L-d(I-QI-I)-3'-NH2 and/or 5'-
Teg-L-d(I-QI-I)-3'-NH2:L-d(cI)) are indicated at
concentrations of L-d(cI) below equimolar with respect
to 5'-Teg-L-d(I-QI-I)-3'-NH2. At higher concentrations
of L-d(cI), a ternary complex (L-d(cI):5'-Teg-L-d(I-QI-
I)-3'-NH2:L-d(cI)) can be observed.
(4b) Hybridization of TMT-cI-TMT to L-d(I-QI-I)
Similar experiments using L-d(cI)-TMT (see Example
6a below) ~^mo~strate the annealing of TMT-L-d(cI)-TMT
to 5'-Teg-L-d(I-QI-I)-3'-NH2. A ternary complex is
formed.
Example 5
3 0 Preparation of 35S-labeled TMT-NCS
A suspension of about 40 mmoles of TMT amine (PCT
US91/08253) in 650 mL methanol is stirred at room
temperature and deionized water is added dropwise (about
70 mL) until a clear pale yellow solution develops. The
solution is cooled in an ice bath to 10C and about 60
mmoles 35S-thiophosgene is added dropwise over about 3
83
W094/205~ 2 ~ 5 7 ~ ~ ~ PCT~S94/02610
minutes. A precipate of TMT isothiocyanate forms and
the solution is stirred continuously for a further 2.5
hours. The solution and precipitate are concentrated to
near dryness on a rotovap under reduced pressure ( -
15mm Hg) at room temperature. The near-dry solid is
diluted further with 750 mL of m~t;~nol and is stirred
until the solids appear homogeneous. The solid TMT
isothiocyanate ~TMT-NCS) is then collected by filtration
and is rinsed repeatedly with methanol. The product is
0 dried overnight in a vacuum chamber.
Example 6
(6a) Preparation 5'-H2N-L-d(cI)-3'-NH2 conjugated to a
chelating agent, TMT-NCS, to form 5'-TMT-L-d(cI)-
3'-TMT and preparation 5~-H2N-L-d(I)-3~-NH2
conjugated to a chelating agent, TMT-NCS, to form
5'-TMT-L-d(I)-3'-TMT.
To 300 nmoles of one of the L-enantiomeric
ol~gonucleotides, either 5'-H2N-L-d(cI)-3'-NH2 solution
of Example 3a or 5'-H2N-L-d(I)-3'-NH2 solution of
Example 3b, in S00 microL of 1.0 M carbonate/bicarbonate
buffer at pH 9.0 is added 12 mg of TMT isothiocyanate
(PCT US91/08253). The reaction mixture is vortex mixed
and kept at 37C for 2 hours and at room temperature for
overnight. The resulting reaction mixture is quenched
with ethanolamine (15 microM) and the product is
purified by Sephadex G-25 column chromatography using
deionized water as the eluting solvent.
The number of TMT's per molecule of L-enantiomeric
oligonucleotide ~;Am;ne is quantified by an asssay using
the time resolved fluorescence of Europium metal
chelated to the TMT.
8~
W094/205~ 215 7 ~ ~ ~ PCT~S94/02610
(6b) General procedure for labelling of Ing-1-TMT, 5'-
TMT-L-d(cI)-3'-TMT, and S'-TMT-L-d(I)-3'-TMT
conjugates with fluorescent metals.
Binding of lanthanides such as europium (3+) to
chelating agents that contain an aromatic moiety held
close to the co-ordination sphere can lead to
"sensitized'l fluorescence wherein light is absorbed
through the aromatic system and the energy is
transferred to the metal. The metal then produces
0 emissions characterized by a very large Stokes shift and
fluorescence lifetimes of up to several seconds. The
fluorescence at 615 nm is measured at a time-delay of
400 microseconds after an excitation pulse at 340 nm.
This time delay is useful for high sensitivity
measurements since short lived background fluorescence
is el~m~n~ted and up to a 1,500 fold enhancement in
sensitivity over normal Eu-fluorescence is achievable.
In this method, a known amount of Ing-1-TMT, 5'-
TMT-L-d(cI)-3'-TMT, or 5'-TMT-L-d(I)-3'-TMT conjugate
is titrated with increasing amounts of added EuCl3 in an
aqueous buffer. Thus, one microliter of a solution
containing 1-30 picomoles of the conjugate is added, in
duplicate, to wells in a Costar EIA/RIA 96-well plate
containing a precalculated amount of Tris.HCl buffer (pH
7.4). The buffer volume is derived by subtracting from
99 the volume in microliters of aqueous EuCl3 (typically
10-4 M to 10-6 M in Tris.HCl buffer). The total volume
in each well is thereby fixed at 100 microliters.
Aqueous EuC13 is then added to the buffered solution of
the conjugate. The plate is then covered and shaken at
low speed for one hour. The time resolved fluorescence
is then measured using a Delfia 1232 time-resolved
fluorometer (Wallac Inc.) and the data are analyzed. It
is found that each conjugated TMT molecule chelates one
Europium ion and that both 5'-TMT-~-d(cI)-3'-TMT and 5'-
W094/205~ 2 1~ 7 ~ ~ 2 PCT~S94/02610
TM~-L-d(I)-3'-TMT conjugates bind two Europium ions per
molecule of conjugate.
(6c) Preparation of 35S-labeled TMT-L-d(cI)-TMT from 5'-
S H2N-L-d(cI)-3'-NH2 and 35S-labeled TMT-NCS.
35S-labeled TMT-NCS (prepared~as in Example 5) is
substituted for the 12 mg of TMT isothiocyanate (PCT
US91/08253) in the method of Example 6a and the reaction
carried out as described above. The number of TMT's per
molecule of 5'-H2N-L-d(cI)-3'-NH2 is quantified by
counting the TMT-L-d(cI)-TMT product in a liquid
scintillation counter optimized to detect 35S. From a
knowledge of the concentration of derivatized 5'-H2N-L-
d(cI)-3'-NH2 and the specific activity of 35S-
thiophosgene, the number of TMT molecules per 5'-H2N-L-
d(cI)-3'-NH2 ~;~m~ne may be calculated.
Example 7
(7a) Preparation of a Yttrium (90Y) radiolabeled- L-
enantiomeric oligonucleotides, 90Y-TMT-L-d(cI)-TMT-
90Y and 90Y-TMT-L-d(I)-TMT-9OY
A solution of the L-enantiomeric oligonucleotide
TMT conjugates, either (TMT-L-d(cI)-TMT) from Example 6a
or TMT-L-d(I)-TMT from Example 6a, in deionized water at
room temperature is treated with a solution of 9YC13
(>500 Ci/mg; from Amersham Corp.) in 0.5 M ~odium
acetate buffer at pH 6.0 to a specific activity of 0.1
Ci/pmole for one hour at room temperature. The labeling
efficiency is determined by lel,Lo~lng 1.0 microliter of
the sample and spotting it on to a Gelman ITLC-SG
strip. The strip is developed in a glass beaker
containing 0.1 M sodium citrate at pH 6.0 for a few
minutes until the solvent front reached three-quarters
of the way to the top of the paper. The strip is then
inserted into a System 200 Imaging Scanner (Bioscan)
W094/205~ 215 7 ~ ~ ~ PCT~S94/02610
which had been optimized for 90Y and which is controlled
by a Compaq 386/20e computer. In this system unbound
90Y migrates at the solvent front. The TMT's of the L-
enantiomeric oligonucleotide TMT conjugates chelate in
excess of 98 % of the added radioactivity.
-
(7b) Hybridization of radiolabeled 90Y-TMT-L-d(cI)-TMT-
90Y or of 90Y-TMT-L-d(I)-TMT-90Y to 5'-Teg-L-d(I-
QI - I)-3'-NH2
0 A 5 pmole sample of 90Y-TMT-L-d(cI)-TMT-9OY from
Example 7(a) is mixed with increasing amounts (0.375 to
12 pmoles) of 5'-Teg-L-d(I-QI-I)-3'-NH2 from Example 2a
in PBS at 37C for one hour. A 5 mL aliquot from each
of these hybridizations is removed, mixed with SDS-
containing buffer, and run on a 12% PAGE gel.
Autoradiographs of the gels reveal that 5'-Teg-L-d(I-QI-
I)-3'-NH2 is capable of binding to 2 molecules of cI.
Similar procedures using non-complementary,90Y-TMT-
L-d(I)-TMT-9OY from Example 7(a) fail to show
hybridization to 5~-Teg-L-d(I-QI-I)-3~-NH2.
(7c) ~m;n~stration of 90Y-TMT-L-d(cI)-TMT-9OY to Nude
Mice
TMT-L-d(cI)-TMT labeled with 90Y to a specific
activity of 28 mCi/28 mg is injected into a 25 g nude
mouse bearing a subcutaneous tumor in i~s right flank.
At time intervals after injection, blood samples are
taken from the tail and counted for 90Y radioactivity in
a liquid scintillation counter. The results reveal that
more than 95% of the injected dose of 90Y is Le.. o~ed
from the blood stream in the first 30 minutes following
injection. After 2 hours following injection the
radioactivity in the blood levels off and a minute
fraction (< 0.01% of injected dose ) continues to
circulate during the next 22 hours. These data
demonstrate that in the absence of a hybridizing site
W094/20~ 2 ~ 2 PCT~S94/02610
recognizing the L-d(cI) sequence, radiolabeled TMT-L-
d(cI)-TMT is lost quickly from the circulation.
Example 8
(8a) Preparation of Antibody-Maleimide (Ab-M) using
Sulfo-SMCC and ING-1; (ING-1-Maleimide).
A Sulfo-SMCC solution (108 ~moles) in phosphate
buffered saline (PBS) is added to a sample of a chimeric
antibody (ING-1; 18 nmoles) solution in phosphate buffer
0 (pH 7). The resulting mixture is allowed to stand for 30
minutes with occasional mixing at room temperature. The
reaction is stopped with 60 nmoles basic tris buffer.
The reaction mixture is diluted with phosphate buffered
saline, added to a prewashed PD-10 column, and eluted
with PBS to afford ING-l-maleimide. This material is
stored on ice until use.
(8b) Preparation of mercaptoalkyl-Antibody (Ab-SH) from
ING-l and 2-iminothiolane; (ING-1-SH)
A sample of a chimeric antibody (ING-1; 5 nmoles)
solution in 0.1 M carbonate buffer (pH 8.8) is mixed
with 200 nmoles of an aqueous solution of 2-
iminothiolane. The resulting mixture is allowed to
stand for 30 min with occasional mixing at room
temperature. The reaction mixture is diluted with
phosphate buffed saline, added to a prewashed PD-10
column (Pharmacia), and eluted with PBS to afford
mercaptoalkyl-ING-l. This material is stored on ice
until use.
Example 9
(9a) Preparation of a mercaptoalkyl-L-d(I-QI-I) using 2-
iminothiolane; 5'-Teg-L-d(I-QI-I)-3'-SH
A sample of a solution of 5'-Teg-L-d(I-QI-I)-3'-NH2
(30 nmoles) in water is mixed with 1 M carbonate buffer
(pH 9) to give a final buffer concentration of 890 mM.
88 '
-
094/20S~ ~ 9 0 ~ PCT~S94/02610
Into the buffered L-DNA is added 12 mmoles of an aqueous
solution of 2-iminothiolane hydrochloride. These
reactants are vortex mixed and kept at 37C for 30
minutes. The reaction mixture is quenched by the
addition of 12 mmoles of ethanolamine, diluted with
phosphate buffed saline, added to a prewashed NAP-25
column (Pharmacia), and eluted with PBS to afford 5'Teg-
L-d(I-QI-I)-3'-NH-C(N=H2+)CH2CH2CH2SH (as the
hydrochloride Cl-). For use in con~ugation to a
maleimide-derivatized antibody (Ab-M), the product is
eluted from the column directly into the antibody
solution. Otherwise, the mercaptoalkyl-L-d(I-QI-I) is
stored on ice until use.
(9b) Preparation of a mercaptoalkyl-L-d(I-QI-I) using 2-
iminothiolane; 5'-HS-L-d(I-QI-I)-3'-Teg
A sample of 5'-H2N-L-d(I-QI-I)-3'-Teg (30 nmoles)
is treated as in Example 9a to afford 5'-HS-
CH2CH2CH2C( NH2+)-HN-L-d(I-QI-I)-Teg-3' (Cl-).
(9c) Preparation of an L-d(I-QI-I)-Maleimide using
Sulfo-SMCC; 5'-Teg-L-d(I-QI-I)-3'-Maleimide
An aqueous solution contA ~ n ~ ng 20 nmoles of 5'-Teg-
L-d(I-QI-I)-3'-NH2 (prepared as in Example 2a) is
diluted into phosphate buffed saline. Sulfo-SMCC (100
nmoles) in PBS is added and the resulting mixture is
allowed to stand for 30 min with occasional mixing at
room temperature. The reaction mixture is diluted with
phosphate buffed saline, added to a prewashed PD-10
column, and eluted with PBS to afford 5'-Teg-L-d(I-QI-
I)-3'-Maleimide. This material is stored on ice until
use.
W094/205~ 215 ~ 9 a 2 PCT~S94/02610
(9d) Preparation of an L-d(I-QI-I)-Maleimide; 5'-
Maleimide-L-d(I-QI-I)-3'-Teg.
5'-H2N-L-d(I-QI-I)-3'-Teg is reacted with sulfo-
SMCC in the same manner as 5'-Teg-L-d(I-QI-I)-3'-NH2 in
9c to afford 5'-Maleimide-L-d(I-QI-I)-3'Teg.
(9e) Preparation of 3'-mercapto L-d(I-QI-I) using SATA
An aqueous solution cont~; n; ng 50 nmoles of 5'-Teg-
L-d(I-QI-I)-3'-NH2 (prepared as in Example 2a) is
diluted into PBS and 500 nmoles of SATA (in DMSO) is
added. After mixing and standing at room temperature
for 60 min, the reaction mixture is diluted with PBS and
eluted from a NAP-lO column with PBS to afford 5'-Teg-L-
d(I-QI-I)-3'NH-CO-CH2-S-CO-CH3. The sulfhydryl group of
the acetylthioacetylated L-enantiomeric oligonucleotide
is deacylated by the addition of 30 mL of a pH 7.5
solution containing lOO mM sodium phosphate, 25 mM EDTA,
and 500 mM NH20H. The reaction is allowed to proceed
for two hours at room temperature after which time the
material is passed down a NAP-5 column using PBS for the
elution. The product, 5'-Teg-L-d(I-QI-I)-3'NH-CO-CH2-
SH, is used immediately to obviate oxidative
dimerization.
(9f) Preparation of 5'mercapto-L-d(I-QI-I) using SATA
H2N-5'-L-d(I-QI-I)-3'-Teg is reacted with SATA in
the same manner as 5'-Teg-L-d(I-QI-I)-3'-NH2 in Example
9e to afford 5'-HS-CH2-OC-NH-L-d(I-QI-I)-3'-Teg.
Example lO
(lOa) Conjugation of 5'Teg-L-d(I-QI-I)-3'-NH-
C(=NH2+)CH2CH2CH2SH to Ab-M; ING-l-Maleimido-3'-S-
L-d(I-QI-I)-5'-Teg
A sample (108 nmoles) of 5'-Teg-L-d(I-QI-I)-3'-NH-
C(=NH2+)CH2CH2CH2SH (prepared according to Example 9a)
is eluted with PBS from a NAP-25 column directly into a
qC)
W094/205~ 215 7 9 0 2 PCT~Sg4/02610
solution of Ab-M (maleimide-derivatized ING-1; 18
nmoles) from Example 8a. After mixing, the reaction is
allowed to proceed for 20 hours at 4C. The reactants
are then loaded into Centricon-100~ ultrafiltration
concentration devices ~Amicon) which are then
centrifuged at 1000 g for 25 minutes. The sample
containing the product is resuspended in fresh PBS, and
ultrafiltration, concentration by centrifugation and
resuspension are repeated a further 3 times until the
0 ratio of optical densities at 260 nm and 280 nm is
constant. The final product is ING-1-Maleimido-S-
(CH2)3-C(=NH2+)-NH-3'-L-d(I-QI-I)-5'-Teg.
(lOb) Conjugation of 5'-HSCH2CH2CH2(NH2+~)C-NH-L-d(I-QI-
I)-Teg-3' to Antibody-Maleimide; ING-1-Maleimide-
5'-S-L-d(I-QI-I)-Teg-3'
A sample (108 nmoles) of 5'-HSCH2CH2CH2(NH2+=)C-NH-
L-d(I-QI-I)-Teg-3' prepared according to Example 9b is
conjugated to 18 nmoles of maleimide-derivatized ING-1
as in Example 8a to give ING-1-Maleimide-5'-S-(CH2)3-
C(=NH2+)-NH-L-d(I-QI-I)-3'-Teg.
(lOc) Assays on the ING-1-Maleimide-S-L-d(I-QI-I)
conjugates
The optical density of each o the ING-1-Maleimide-
S-(CH2)3-C(=NH)NH-L-d(I-QI-I) samples of example lO(a)
and lO(b) is ex~m;ned in a spectrophotometer at 260 nm
and 280 nm. The ratio of optical densities at these two
wavelengths is calculated, and, by using known
extinction coefficients for the antibody and for the L-
enantiomeric oligonucleotide (approximate molecular
weight 16,500) at each of these wavelengths, the number
of L-enantiomeric oligonucleotide molecules, L-d(I-QI-
I), is estimated to be between 1 and 2 L-d(I-QI-I) per
antibody in the sample.
~l '
W094/20s~ 2 ~ 5 ~ ~ ~ 2 PCT~S94/02610
The concentration of ING-1 in a conjugate solution
is determined by the BioRad protein assay using bovine
immunoglobulin as the protein standard. These data
agreed well with the antibody concentrations determined
by ex~m;n~tion of the optical density of the conjugate
at 280 nm once it has been corrected for absorbance due
to the conjugated L-d(I-QI-I). Both these sets of data
are further confirmed by subjecting t~è antibody-L-d(I-
QI-I) conjugates to acid digestion and amino acid
0 analysis.
Antibody-L-d(I-QI-I) conjugates are exAm1ned for
their ability to bind to antigens on the surface of a
human tumor cell line to which the antibody is raised.
The immunoreactivity of the conjugates is compared by
flow cytometry with a standard preparation of the
antibody before being subjected to modification and
conjugation to L-d(I-QI-I). Target HT29 cells (a human
adenocarcinoma cell line: ATTC) are grown to confluency
in tissue culture flasks using McCoy's media
supplemented with 10% fetal calf serum. The cells are
harvested by scraping the flask walls with a cell
scraper. Cells from many separate flasks are pooled,
centrifuged to a pellet, resuspended at 5 x 105/mL in a
solution of ice-cold 50 mM sodium phosphate with 150 mM
sodium chloride buffer pH 7.4 (PBS) supplemented with
0.1% bovine serum albumin (Sigma) and 0.02% sodium azide
(Flow buffer). The cells are washed in this ~ame buffer
and then counted. An antibody standard curve is
constructed by diluting a stock solution of ING-1 with
an irrelevant (non binding), isotype-matched control
antibody (human IgGl) to give a number of samples
ranging in ING-1 content from 10% to 100~. A standard
curve is made in flow buffer so that each sample
contains 1.0 mg antibody protein per mL. Samples from
the standard curve and unknowns are then incubated with
5 x 105 HT29 cells at 4C for 1 hour. After extensive
~ W094/205~ 215 7 ~ ~ ~ PCT~S94/02610
washing to remove unbound antibody, the cells are
resuspended in lO0 mL flow buffer and incubated at 4C
for l hour with goat-anti-human antibody labeled with
fluorescene isothiocyanate (FITC). After further
washing in flow buffer the samples are analyzed by flow
cytometry on a Coulter EPICS 753 flow cytometer.
Fluorescene from Fluorescene isothiocyanite (FITC) and
propidium iodide (PI) is excited using the 488 nm
emission line of an argon laser. The output is set at
0 500 mW in light regulation mode. Single cells are
identified by 90 degree and forward angle light scatter.
Analysis windows are applied to these parameters to
separate single cells from aggregates and cell debris.
Fluorescence from FITC and propidium are separated with
a 550 nm long pass dichroic filter and collected through
a 530 nm band pass filter (for FITC), and a 635 nm band
pass filter (for PI). Light scatter parameters are
collected as integrated pulses and fluorescence is
collected as log integrated pulses. Dead cells are
excluded from the assay by placing an analysis window on
cells negative for PI uptake. The mean fluorescence per
sample (weighted average from 2500 cells) is calculated
for each histogram. FITC calibration beads are analysed
in each experiment to establish a fluorescence standard
2~ curve. The average fluorescence intensity for each
sample is then expressed as the average FITC equivalents
per cell. Immunoreactivity is calculated by comparing
the average fluorescence intensity of the unknown sample
with values from the standard curve. From the
immunoreactivity assay, ING-l-Maleimide-S-(CH2)3-
C(=NH2+)-NH-3'L-d(I-QI-I)-5~-Teg is approximately 2/3 as
immunoreactive as the ING-l standard and ING-l-
Maleimide-S-(CH2)3-C(=NH2+)-NH-5'-L-d(I-QI-I)-3'-Teg is
approximately 4/5 as immunoreactive. In a separate set
of experiments, immunoreactivity of ING-l-L-d(I-QI-I)
(3'-conjugate) and ING-l-L-d(I-QI-I) (5'-conjugate) are
~3
W094/205~ 21~ 7 ~ ~ 2 PCT~S94/02610
determined. Samples of these conjugates are also
subjected to electrophoresis on Novex 6% polyacrylamide
gels using SDS-containing buffers in order to estimate
their apparent molecular weight and the degree of
heterogeneity of the preparation. Using standards of
known molecular weight run on the same gel, a standard
curve is constructed of the distan~e travelled (Rf)
versus the log of the molecular weight. From this
standard curve the relative molecular weights of the
0 bands associated with each conjugate preparation are
determined. The SDS PAGE gels of ING-l-L-d~I-QI-I) and
ING-l antibody demonstrate that the molecular weight of
the ING-l-L-d(I-QI-I) conjugates are higher than that of
the antibody ING-l alone.
(lOd) Conjugation of Teg-5'-L-d(I-QI-I)-3'-Maleimide to
mercaptoalkyl-Antibody, Ab-SH; ING-l-NH-CO-CH2-S-
Maleimido-3'-L-d(I - QI - I)-5'Teg
20 nmoles of Teg-5'-L-d(I-QI-I)-3'-Maleimide (from
Example 9c) are reacted with 5 nmoles of mercaptoalkyl-
ING-l (Ab-SH from Example 8b) in PBS pH 7. After
mixing, the reaction is allowed to continue at 4C for
16 hours to afford ING-l-NH-CO-CH2-S-Maleimido-3'-L-d(I-
QI-I)-5'-Teg. The reaction mixture is loaded into a
Centricon-lOO~ ultrafiltration concentration device
which is then centrifuged at l,OOO g for 25 minutes.
The sample is resuspended in fresh PBS and this sequence
of concentration by centrifugation is repeated a further
3 times until the ratio of optical densities at 260 nm
and 280 nm is constant.
Maleimide-5'-L-d(I-QI-I)-3'-Teg is reacted with
mercaptoalkyl-ING-l (Ab-SH) in the same way to afford
ING-l-NH-CO-CH2-S-Maleimide-5'-L-d(I-QI-I)-3'Teg.
q~
WO 94/205~ 21 5 7 9 0 ~ PCT~S94/02610
(lOe) Conjugation of Teg-5'-L-d(I-QI-I)-3'-NH-C(=O)-CH2-
SH to Antibody-Maleimide, Ab-M.
A 6 nmole sample of ING-l-Maleimide (Ab-M from
Example 8a) in PBS is reacted with 40 nmoles of Teg-5'-
L-d(I-QI-I)-3'-NH-CO-CH2-SH (from Example 9e) at 4C for
l6 hours. The reactants are diluted with PBS and eluted
in PBS from a PD-lO column to afford ING-l-Maleimido-S-
CH2-C(=O)-NH-3'-L-d~I-QI-I)-5'Teg. The product is
concentrated in a Centricon-300~ device by
0 centrifugation at lOOOg for 25 minutes. The sample is
resuspended in fresh PBS and concentration by
centrifugation. This process is repeated a further 3
times until the ratio of optical densities at 260 nm and
280 nm is constant.
HS-CH2-(0=)C-NH-5'-L-d(I-QI-I)-3'-Teg is reacted
with ING-l-Maleimide in the same way to afford ING-l-
Maleimido-S-CH2-(0=)C-NH-5'-L-d(I-QI-I)-3'-Teg. The
optical density of these samples is ~Amined in a
spectrophotometer at 260 nm and 280 nm. The ratio of
optical densities at these two wavelengths is then
calculated. By using known extinction coefficients for
-the antibody and for the L-enantiomeric oligonucleotide
(approx molecular weight 16,500) at each of these
wavelengths, the number of L-enantiomeric
oligonucleotide molecules per antibody is estimated.
(lOf) Conjugation of 5'-Trityl-~S)-L-d(I-QI-I)-3'-NH2 to
Antibody-Maleimide, Ab-M.
A sample (lO nmoles) of 5'-Trityl(S)-L-d(I-QI-I)-
3 0 3'-NH2 from Exa~ple 2c is diluted into PBS and a
solution of silver nitrate in water is added to a final
concentration of 85 mM. After vortexing, the reaction
is allowed to proceed at room temperature for 30
minutes. A precipitate forms which iQ centifuged to the
bottom of the tube. The clear supernatant which
contains the L-enantiomeric oligonucleotide with a 5'
qa
W094/205~ ~15 ~ ~ 0 2 PCT~S94102610
terminal thiol group and without the 5' trityl group
(HS-5'-L-d(I-QI-I)-3'-NH2) is kept at 4C until use.
A 6 nmole sample of ING-1-Maleimide (from Example
8a) in PBS is reacted with 40 nmoles of HS-5'-L-d(I-QI-
I)-3'-Teg at 4C for 16 hours. The reactants are
diluted with PBS and eluted in 4 mL from a pre-washed
Econopac 106-DG column (BioRad) to afford ING-1-
Maleimide-S-5'-L-d(I-QI-I)-3'-Teg.i The product is
concentrated in a Centricon-300~ concentration device by
0 centrifugation at 1000 g for 25 minutes. The sample is
resuspended in fresh PBS and concentration by
centrifugation is repeated a further 3 times until the
ratio of optical densities at 260 nm and 280 nm is
constant.
The optical density of the~e samples are r~X~m; ned
in a spectrophotometer at 260 nm and 280 nm. The ratio
of optical densities at these two wavelengths is
calculated, and by using known extinction coefficients
for the antibod~ and for the L-enantiomeric
oligonucleotide (approximate molecular weight=16500) at
each of these wavelengths, the number of L-enantiomeric
oligonucleotide molecules per antibody is estimated.
Example 11
(lla) Annealing of L-d(cI) to ING-1-L-d(I-QI-I)
A sample of ING-l-Maleimide-S-(CH2)3-C(=NH2+)-NH-
3'-L-d(I-QI-I)-5'-Teg (24 pmoles) from Example lOa and a
sample of ING-1-Maleimide-S-(CH2)3-C(~NH2+)-NH-5'L-d(I-
QI-I)-3'-Teg (25 pmoles) from Example lOd are mixed in
separate cuvettes with 16-fold excess of L-d(cI3 from
Example 3c in 50 mM PBS containing 1.0 mM EDTA and 100
mM NaCl, pH 7.2, at room temperature. The cuvettes are
cooled to 20C, loaded into a Cary 13 instrument, and
the absorbance is analysed by UV light (260 nm) while
the cuvette temperature is ramped up from 20C to 80C
and then back down to 20C at a rate of 0.5C/min.
9~ .
~ W094/205~ PCT~S94/02610
2157~2
Analysis of the data reveals that cI is able to
hydridize to both the 3'- and the 5'- conjugates to form
a ternary complex with each. Similar experiments
using TMT-L-d(cI)-TMT (from Example 6) demonstrate the
S annealing of TMT-L-d(cI)-TMT to ING-l-Maleimido-S-
(CH2)3-C(=NH2+)-NH-3'-L-d(I -QI - I)-5'-Teg.
(llb) Hybridization of ING-1-L-d(I-QI-I) to either 90Y-
TMT-L-d(cI)-TMT-9OY or 90Y-TMT-L-d(I)-TMT-9OY in
phosphate buffer and human serum
A solution of ING-l-Maleimido-S-(CH2)3-C(=NH2+)-NH-
3'-L-d(I-QI-I)-5'-Teg (4 ul; 1 mg antibody/ml from
Example lOa) and 50 microL 90Y-TMT-L-d(cI)-TMT-90Y
solution from Example 9a are mixed with freshly prepared
human serum (200 mL) or PBS (200 mL; pH7.2) and
incubated at 37C for 2 hours. Aliquots are then
subjected to SDS PAGE electrophoresis on an 8 to 16%
gel. The gels are e~1ned by both autoradiography and
on a phosphoimager system to show that 90Y-TMT-L-d(cI)-
TMT-9OY is able to hybridize with ING-1-L-d(I-QI-I) in
human serum as well as phosphate buffer solution (pH
7.2). Samples are left at room temperature for 14 days
in PBS and in serum and run on 8 to 16% SDS-PAGE to give
similar patterns to each other and to gels incubated at
37C for 2 hours. This indicates that the conjugates
ar~ stable in serum for up to two weeks. 90Y-TMT-L-d(I)-
TMT-90Y from Example 9a fails to show hybridization to
ING-1-L-d(I-QI-I) at any time.
(llc) Hybridization of 9OY-T~T-L-d(cI)-TMT-9OY to ING-1-
L-d~I-QI-I)
A solution of ING-1-Maleimido-S-(CH2)3-C(=NH2+)-NH-
3'-L-d(I-QI-I)-5'-Teg (4ul; lmg/ml from Example lOa) and
50 uL 90Y-TMT-L-d(cI)-TMT-9OY (from Example 9a) are
mixed in PBS (200 ul; pH 7.2) and incubated at 37C for
60 minutes. Aliquots of these mixtures are then
9~ .
W094/205~ 215 ~ ~ ~ 2 PCT~S94102610
subjected to SDS PAGE electrophoresis on an 8 to 16%
gel. The gels are autoradiographed on a phosphoimager
system to show that 90Y-TMT-L-d(cI)-TMT-90Y is able to
hybridize with ING-l-L-d(I-QI-I).
(lld) Western blot to detect TMT on T~T-L-d(cI)-TMT
hybridized with ING-l-L-d(I-QI-I).
A sample of L-d(I-QI-I) from Example 2a or of ING-
l-Maleimido-3'-S-(CH2)3-C(=NH2+)-NH-L-d(I-QI-I)-5'-Teg
from Example l0a is separately mixed with the ~-
enantiomeric oligonucleotide TMT conjugate TMT-L-d(cI)-
TMT from Example 6. The reaction mixture of each is
kept on ice for l0 minutes. Aliquots of each reaction
mixture are mixed with SDS buffer and loaded onto two
duplicate 8 to 16% polyacrylamide gels. The gels are
subjected to electrophoresis at a constant voltage for 2
hours. One gel is electroblotted onto nitrocellulose
paper using CAPS buffer for 20 minutes according to the
manufacturer's protocol (Hoefer semi-dry transfer
method). After washing thrice with a solution of 0.05
Tween 20 in PBS, the gel is blocked with a solution of
3% BSA in PBS at room temperature for l hour. Following
further washing with Tween/PBS, the gel is overlaid with
a solution of a murine anti-TMT antibody (l0mg/ml in
PBS/Tween) and left overnight at room temperature. The
western blot is developed using a goat anti-mouse IgG
antibody conjugated to horseradish peroxidase (BioRad
Western blot kit) and peroxidase substrate. The blot
demonstrates that the T~T-L-d(cI)-TMT can be detected
via the TMT's as being hybridized to bands that
contained either L-d(I-QI-I) or ING-l-L-d(I-QI-I).
The second gel is stained with ethidium bromide
(5mg/ml in distilled water). ~x~m; n~tion of the stained
gel under UV light again demonstrates hybrization.
q~ .
~ W094/205~ 2 1 ~ 7 ~ 0 2 PCT~S94/02610
Example 12
Preparation of Ab-TMT by direct conjugation (ING-l/TMT)
TMT-NCS (or a suitable derivative thereof) can be
conjugated to an antibody molecule to yield an antibody-
TMT conjugate molecule that displays the ability to bind
to a target antigen recognized by the antibody variable
region. Such a conjugate molecule can be used to
deliver metal ions that are chelated by the TMT moiety
in order to localize and/or treat the tumor that is
targeted by such an immunoconjugate. In one preferred
embodiment, the antibody is selected such that it has a
broad reactivity with an antigen molecule expressed on
tumor cells, thereby providing an antibody-TMT conjugate
that can deliver radionuclides to the tumors for
therapeutic or diagnostic purposes. The ch ; m~riC
antibody, ING-1, (International patent publication WO
90/02569) consists of a murine variable region and a
human immunoglobulin constant region. The antibody is
produced by culturing a mouse myeloma cell line
expressing the ch;meriC antibody essentially as
described in the above-referenced publication. After
purification, ING-1 is used at a concentration of 5.0
mg/mL in 50 mM sodium acetate and 150 mM sodium chloride
buffered at pH 5.6.
The conjugation of ING-l to TMT-NCS is achieved by
first adding 1.0 M carbonate plus 150 mM sodium chloride
buffer, pH 9.3, to ING-1 until the antibody solu~ion
reaches a pH of 9Ø A sample of that ING-l solution
containing 5 mg of protein is then pipetted into an acid
washed, conical, glass reaction vial. A solution of
TMT-NCS is prepared by dissolving lO0 mg in lO mL of
1.0 M carbonate plus 150 mM sodium chloride buffer, pH
9Ø The conjugation reaction is started by the
addition of 96.5 mL of the TMT-NCS solution to the
antibody to give a 4-fold molar excess of TMT-NCS over
ING-1. The solution is stirred briefly to mix the
qq
-
W094/205~ 21~ 2 PCT~S94/02610
reactants and then left in the dark at room temperature.
After 16 hours, the ING-1/TMT conjugat~ is separated
from unconjugated TMT by applying the reaction mixture
to a PD10 chromatography column which has been pre-
washed and equilibriated with 50 mM sodium acetate in
lS0 mM sodium chloride buffer, pH;5.6. The pure
conjugate is eluted from the column with 2.5 mL of that
sAme buffer.
Example 13
Analytical tests on the ING-1/TMT conjugates.
(13a) Analysis of Chelator to Antibody Ratio
The protein concentrations of ING-l in the
con~ugate solutions are determined by the BioRad protein
assay using bovine immunoglobulin as the protein
st~nd~rd.
In order to calculate the number of functional TMT
molecules per antibody, ING-l/TMT is reacted with a
solution of Europium chloride until saturation of the
metal-binding capacity of the TMT occurs. A 0.375 mg
aliquot of the ING-l/TMT in 2.5 ml in 0.05 M Tris HCl
buffer pH 7.5 is pipetted into a 5 ml quartz cuvette. A
20 mM Europium chloride (Europium chloride hexahydrate;
Aldrich) solution in 0.05 M Tris HCl buffer pH 7.5 is
prepared. An aliquot (50 mL) of this Europium chloride
solution is added to the cuvette cont~in;ng ING-1/TMT
and the resulting solution is slowly stirred on a
magnetic stirrer at room temperature for 10 min using a
small magnetic stir bar placed in the cuvette. The
fluorescence of the metal-ING-l/TMT complex is
determined in a Perkin Elmer LS 50 spectrofluorometer
using an excitation wavelength of 340 nm (10 nm slit
width). The fluorescent emission is monitored at 618 nm
using a 10 nm slit width and a 430 nm cutoff filter.
The above procedure is repeated and fluorescent readings
are made after each addition. Aliquots of Europium
iVC
215~
W094/205~ PCT~S94/02610
chloride are added until the increase in fluorescence
intensity is less than 5~ of the preceding reading. A
dilution correction is applied to the fluorescence
intensity measured at each mole ratio to compensate for
the change in volume of the test solution. Since each
chelating site on the ING-l/TMT conjugate binds one
Europium ion, and since Europium ion has to be in a
chelate site for fluorescence to occur, this method
allows the number of functional chelation sites to be
0 quantitated.
Using this method, the ratio of TMT molecules per
molecule of antibody is in the range from 0.3:l to 2:l.
113b) ING-l/TMT Immunoreactivity assay by ELISA
The antigen to which the antibody, ING-l, binds is
prepared from ~S174T or ~T 29 cells (available from
American Type Ti~sue Collection, ATTC) by scraping
confluent monolayers of cells from the walls of culture
flasks with a cell scraper. The cells from many flasks
are combined and a sample is taken and counted to
estimate the total number of cells harvested. At all
times the cells are kept on ice. Following
centrifugation of the cells at 1500 rpm for lO minutes
at 4C, the cells are washed once in 25 mL ice-cold 50
mM sodium phosphate buffer supplemented with 150 mM
sodium chloride, p~ 7.4 (PBS), pelleted under the same
conditions and transfered in lO mL PBS to an ice-cold
glass mortar. The cells are homogenized at 4C using a
motor-driven pestle and then centrifuged at 3000 x g for
5 minutes. The antigen-rich supernatant is removed from
the other cell debris and subjected to further
centrifugation at lO0,000 x g for one hour at 4C. The
pellet ~antigen fraction) from this final step is
suspended in lO0 mL of PBS for every million cells
harvested. Following an estimate of the protein
concentration (BioRad BCA protein assay using bovine
l~i
WO 94/20523 2 ~ 5 ~ ~ ~ 2 PCT/US94/02610
immunoglobulin as the protein standard) the antigen is
stored at -20C until use. Each well of a 96-well
Costar microtiter plates is coated with antigen by
adding 100 mL/well of cell lysate (10 mg/ml) prepared as
above. The microtiter plates are allowed to dry
overnight in a 37C incubator. After w~sh;ng the plates
five times with 0.05% Tween-20 (Sigmaj they are blotted
dry. The wells of each plate are blocked by adding 125
mL/well of a 1% BSA (bovine serum àlbumin, Sigma A-7906)
solution in PBS and incubated for 1 hour at room
temperature. The plates are washed five times with
0.05% Tween-20. Samples (50 mL/well in duplicate) of
ING-1/TMT conjugates and stAn~l~rd ING-1 antibody
solutions are prepared at a range of concentrations in
1% BSA in PBS. Biotinylated ING-1 (1.0 mg/mL in 0.1%
8SA) is added to each well (50mL/well) and the plates
are then incubated for 2 hours at room temperature.
Following five washes with 0.05% Tween-20, the plates
are blotted dry and incubated at room temperature for
one hour with dilute (1:2000 in 0.196 BSA) streptavidin-
~lk~line phosphatase (Tago; ~6567). After a further
five washes, color is developed in each well upon the
addition of 100 mL per well of phosphatase substrate
reagent (Sigma). After one hour at room temperature,
the color is read using a 405 nm filter in a Titertek
Multiscan microplate reader.
When tested by this procedure, the ;m~noconjugates
of ING-1 with TMT are found to have immunoreactivity
comparable to native ING-1.
(13c) ING-1/TMT Immunoreactivity Assay ~y Flow Cytometry
Target ~T29 cells are grown to confluency in tissue
culture flasks using McCoy's media supplemented with 10%
fetal calf ~erum. The cells are harvested by scraping
the f~ask walls with a cell scraper. Cells from many
separate f~aRks are pooled,--centrifuged to a pellet,
I C~
~ W094/20~ 215 ~ ~ ~ 2 PCT~S94/02610
resuspended at 5 x 105/mL in a solution of ice-cold 50
mM sodium phosphate with 150 mM sodium chloride buffer
pH 7.4 (PBS) supplemented with 0.1% bovine serum albumin
(Sigma) and 0.02% sodium azide (Flow buffer). The cells
are washed in this same buffer and then counted. An
antibody standard curve is constructed by diluting ING-1
with an irrelevant (non binding), isotype-matched
control antibody (human IgG1) to give a number of
samples ranging in ING-1 content from 10% to 100%. The
0 standard curve is made in flow buffer so that each
sample contains 1.0 mg protein per mL. Samples from the
standard curve and unknowns are then incubated with 5 x
105 HT29 cells at 4C for 1 hour. After extensive
washing to remove unbound antibody, the cells are
resuspended in 100 mL flow buffer and incubated at 4C
for 1 hour with goat-anti-human antibody labeled with
fluorescene isothiocyanate (FITC). After further
washing in flow buffer the samples are analyzed by flow
cytometry on a Coulter EPICS 753 flow cytometer.
Fluorescene FITC and propidium iodide (PI) are excited
using the 488 nm emission line of an argon laser. The
output is set at 500 mw in light regulation mode.
Single cells are identified by 90 degree and forward
angle light scatter. Analysis windows are applied to
these parameters to separate single cells from
aggregates and cell debris. Fluorescence from FITC and
propidium are separated with a 550 nm long pass dichroic
filter and collected through a 530 nm band pass filter
(for FITC), and a 635 nm band pass filter (for PI).
Light scatter parameters are collected as integrated
pulses and fluorescence is collected as log integrated
pulses. Dead cells are excluded from the assay by
placing an analysis window on cells negative for PI
uptake. The mean fluorescence per sample (weighted
average from 2500 cells) is calculated for each
histogram. FITC calibration beads are analysed in each
~3
W094/205~ 2 ~ 2 PCT~S94/02610
experiment to establish a standard curve. The average
fluorescence intensity for each sample is then expressed
as the average FITC equiva~ents per cell.
Immunoreactivity is ca~culated by comparing the average
S f uorescence intensity of the unknown sample with values
from the standard curve. Samples of ING-1/TMT have
immunoreactivity values comparable to the native ING-1
antibody by this method.
(13d) Determin~tion of aggregate formation by size-
exclusion HPLC.
A 30 cm x 7.5 mm TSK-G3000SW size-exclusion ~PLC
column tSupelco) fitted with a guard column of the same
material is equilibrated with 12 column volumes of 10 mM
sodium phosphate buffer pH 6.0 supplemented with 150 mM
sodium chloride using a Waters 600E HPLC system with a
flow rate of 1.0 mL per minute at 400-600 PSI. A sample
(25 mL) of BioRad gel f~ltration protein st~n~Ards is
injected on to the column. The retention time of each
standard is monitored by a Waters 490 W detector set at
280 nm. Following the recovery of the final standard
from the column, it is washed with a further 10 volumes
of 10 mM sodium phosphate buffer, pH 6.0, supplemented
with 150 mM sodium chloride. Samples (50mL) of either
native ING-1 antibody or ING-1/TMT at 200 mg/mL are
$njected onto the column and their retention times are
recorded. From the areas of the retained peaks and the
retention time, the amount of aggregated material in the
ING-1/TMT sample is calculated.
By this method the native ING-1 antibody has a
retention time of 9.1 minutes. ING-1/TMT has a major
peak also at 9.1 minutes but a minor peak, attributable
to aggregates, can sometimes be seen at 7.3 minutes. By
comparison of the peak areas, the aggregate peak is le~s
than ~% of the total.
1~
~ W094/205~ 215 7 ~ ~ 2 PCT~S94/02610
(13e) Radiolabeling of ING-1/TMT with 90Y.
A volume of radioactive Yttrium chloride (90Y in
0.04 M hydrochloric acid at a specific activity of >500
Ci/mg; Amersham-Mediphysics) is neutralized using two
volumes of 0.5 M sodium acetate pH 6Ø The neutralized
90Y (1.0 mCi) is added to 1.0 mL of ING-1/TMT (1 mg/mL)
in 50 mM sodium acetate buffer containing 150 mM sodium
chloride at pH 5.6. The labelling is allowed to proceed
for one hour and then the reaction mixture is loaded
onto a PD-10 chromatography column which has been pre-
washed and equilibrated in a buffer cont~;n;ng ~0 mM
sodium phosphate with 150 mM sodium chloride pH 7.4
(PBS). The sample is eluted from the column with 1.5 mL
of PBS. Fractions of radiolabeled ING-1/TMT (0.5 mL)
are collected, assayed for radioactivity, and pooled.
The labeling efficiency is determined by le...o~ing 1.0 mL
of the sample and spotting it on to a Gelman ITLC-SG
strip. The strip is developed in a glass beaker
containing 0.1 M sodium citrate, pH 6.0, for a few
minutes until the solvent front has reached three-
quaters of the way to the top of the paper. The strip
is inserted into a System 200 Imaging Scanner (Bioscan)
which has been optimized for 90Y and is controlled by a
Compaq 38~/20e computer. In this system free 90Y
migrates at the solvent front while the ING-1/TMT/9OY
r~ins at the origin.
Using this system more than 98% of the total 90Y
radioactivity is always found associated with ING-l/TMT
at the origin.
(13f) Labeling ING-1/TMT with fluorescent metals.
Binding of lanthanides such as europium (3+) to
chelating agents that contain an aromatic moiety held
close to the co-ordination sphere can lead to
"sensitized" fluorescence wherein light is absorbed
through the aromatic system -and the energy is transfered
~0~
W094/205~ 2 i ~ 2 PCT~S94/02610
to the metal. The metal then produces emissions
characterized by a very large Stokes shift and
fluorescence lifetimes of up to several seconds. A 0.5
mg aliquot of the ING-l/TMT in 2.5 mL in 0.05 M Tris HCl
buffer pH 7.5 is pipetted into a 4 mL conical reaction
vial contA;n;ng a small stirring bar. A 250 mM europium
chloride (europium chloride hexahydrate: Aldrich)
solution in 0.05 M Tris HCl bùffer pH 7.5 is prepared.
An aliquot (50 mL) of this europium chloride solution is
added to the reaction vial containing ING-1/TMT, and the
resulting solution is stirred very slowly on a magnetic
stirrer at room temperature. The labelling is allowed
to proceed for one hour and then the reaction mixture is
loaded on to a PD-10 chromatography column which had
been pre-washed and equilibrated in a buffer cont~n;~g
10 mM sodium phosphate and 150 mM sodium chloride at pH
6.0 (PBS). The sample is eluted from the column with
3.5 mL of PBS. The fluorescence of a 50 mL sample of
the metal-ING-1/TMT complex is determined in a Perkin
Elmer LS 50 spectrofluorometer using an excitation
wavelength of 340 nm (10 nm slit width). The
fluorescent emission is recorded at 618 nm using a 10 nm
slit width and a 430 nm cutoff filter. Each functional
chelating site on the ING-1/TMT conjugate binds one
europium ion. Using this method, an average of between
0.1 and 3 fluorescent europium ions are bound per
molecule of antibody in solution.
Example 14
(14a) Flow Cytometry of binding of fluorescent L-d(cI)
to cells treated with ING-1-L-d(I-QI-I).
ING-1-Maleimide-S-(CH2)3-C(=NH2+)-NH-3'-L-d(I-QI-
I)-5'Teg is prepared as in Example lOa. ING-1-Maleimide-
S-(CH2)3-C(~NH2+)-NH-5'-L-d(I-QI-I)-3'-Teg is prepared
as in Example lOb. Fluorescently labeled CY5.18-L-d(I-
QI-I) is prepared as in Example 2d. Fluorescently
~~ .
~ W094/205~ 21 5 ~ 0 2 PCT~S94/02610
labeled CY5.18-~-d(cI) and CY5.18-L-d(I) are prepared as
in Example 3e. Flow cytometry is carried out
essentially as described in Example 12c except that the
fluorescent dye CY5.18 is used in place of FITC. HT-29
cells ~0.5 x 106) are incubated on ice for 30 min with 1
mg each of the ING-1-L-d(I-QI-I) samples. The cells are
washed twice with flow buffer and pelleted at 1400 rpm
for 5 minutes between washes. Next the cells in each
sample are incubated with 5 mg CY5.18-L-d(cI) or CY5.18-
L-d(I) for 3 hours on ice. Some cells are incubated
with CY5.18-L-d(cI) and CY5.18-L-d(I) alone. After
extensive washing with flow buffer, the cells are
subjected to analysis on a fluorescence activated cell
sorter. CY5-18 calibration beads are analysed to
establish a standard curve of relative fluorescence
intensity versus CY5-18 concentration. The mean
fluorescence per sample (weighted average from 2500
cells) is calculated for each histogram. The average
fluorescence intensity for each sample is then expressed
as the average CY5-18 equivalents per cell. Identical
experiments are carried out in which the medium used for
incubation of the cells with the components is 100%
fetal calf serum in place of flow buffer.
The time taken to ~-~ m~ 7e hybridization of CY5-18-
L-d(cI) to ING-1-Maleimide-S-(CH2)3-C(~NH2+)-NH-3'-L-
d(I-QI-I)-5'-Teg on the surface of HT-29 cells is about
3 hours. There is no difference between flow buffer and
100% FCS in the degree of hybridization in each
preparation suggesting that the end-capped L-
enantiomeric oligonucleotide strands are stable to
nuclease digestion. Large amounts of CY5.18-L-d(cI) can
hybridize to both conjugates bound to the surface of
cells. There is relatively little non-specifc binding
to the cells either by the fluorescent L-enantiomeric
. 35 oligonucleotides themselves or by hybridization of CY5-
18-L-d(I-QI-I) to the conjugates.
10 1
21~ 2~ PCT~S94/02610
(14b) Binding of 90Y-TMT-L-d(cI)-TMT-9OY to HT-29 cells
treated with ING-1-~-d~I-QI-I).
TMT-L-d(cI)-TMT is radiolabeled to a specific
acti~ity of 0.1 mCi/pmole as described in Example 7.
ING-l-TMT-90Y is prepared as in Example 13e. ING-1-
Maleimide-S-(CH2)3-C(=NH2+)-NH-3'-L-d(I-QI-I)-5'-Teg is
prepared as in Example lOa. Three tubes each containing
1 x 105 HT-29 cells in DMEM medium supplemented with 10%
0 fetal calf serum are prepared and kept at 4C
throughout. Using matched protein concentrations in all
tubes to ensure that the amount of added antibody is
equal, the tubes are treated as shown in Table 1 below.
Table 1
l~e Im; n)S~le 1 Sa~le ~ SAm~le 3
0 ~ING~ -d~I-QI-I) 0 0
Wa~h X2 Wash X2 Wash X2
+L-d(cI)-~TMT9OY)2 ING-1-TNT9OYPrehybridized
ING-l-L-d-(I-QI-I)
with
L-d~cI)-~TMT9OY) 2
150 Wa~h Y2 Wa~h X2 Wa~h X2
3 0 lôO Centr$fuge cell-~ C~ntr~fuge cell~ Centrifuge cell-~
and count pellet and count pellet and count pellet
radioactivity r~ioactivity radioactivity
Both sample 1 and sample 3 show higher
radioactivity associated with the cell pellet than does
sample 2 (directly labeled ING-l-TMT-9OY) suggesting
that a delivery system consisting of 2 separate
W094/205~ 215 7 ~ 0 ~ PCT~S94/02610
(14c) Binding of Europium labeled ING-1/TMT and TMT-L-
d(cI)-TMT conjugates to HT-29 cells and HT-29
cells treated with ING-1-L-d(I-QI-I), respectively
S The TMT-L-d(cI)-TMT conjugate is labeled with
Europium ions as described in Example ~b and ING-1/TMT
is labeled with Europium ions as described in Example
13f. Standard curves are created for both conjugates in
concentration ranges of 100 picomoles/100 microL to 6
0 femtomoles/100 microL. HT-29 cells are grown to
confluence in McCoys media cont~n;ng 10% FCS and 50
microgram/ml of gentamyacin. The cells are washed with
phosphate buffered saline, and 5 ml of Trypsin Versene
is added. The HT-29 cells are then incubated at 37C in
5% C2 for 15 minutes, complete media (5 ml) is added,
and the cells are removed and washed in PBS. The HT-29
cells are then blocked with 10 micrograms of sheared
salmon sperm (natural D-enantiomer) DNA per 106 cells at
4C for 30 minutes, washed in PBS and used in the
hybridization assay as follows. 5 x 105 HT-29 cells are
added to a 100 microL working dilution of Ing-1/TMT or
Ing-1-L-d(I-QI-I) and the mixture is incubated for 30
minutes on ice. The cells are washed twice in 2 ml of a
wash buffer (PBS + O.1% BSA + O.01% NaN3) at 1400 ~PM
2~ for 5 minutes. A working dilution of TMT-L-dtcI)-TMT
(100 microL) is then added to the appropriate tubes and
the reaction mixture is incubated on ice for 3 hours
followed by washing twice in 2 mL of wash buffer ~PBS +
0.1% BSA + 0.01% NaN3) at 1400 RPM for 5 minutes.
Each binding study is done in triplicate. The cell
suspensions after binding and hybridization as above are
kept at 4C in test tubes until use (10 minutes to one
hour). The Europium fluorescence is measured in a
Delfia 1232 time-resolved fluorimeter by aliquoting four
100 microL portions into separate wells in a Costar
EIA/RIA 96-well plate from each tube after vortexing.
W094/20~ 215 ~ PCT~S94/02610
The results are processed as described in Example 15f.
The counts per seconds (cps) of the Eu-TMT-L-d(cI)-TM~-
Eu treated cells are considered as background and are
subtracted from the cps data from the hybridization
experiments (Eu-TMT-L-d(cI)-TMT-Eu/Ing-l-L-d(I-QI-I)).
This result and the cps data from the Ing-l/TMT-Eu
binding experiment are translated as picomoles of bound
TMT molecules per cell from the individual standard
curves created independently.
0 The results are described as fluorescence counts
per second (cps) or number (#) of picomoles per 105
cells. ING-l/TMT-Eu and ING-con~ugates: 0.25 microgram
or 1.65 picomoles; Eu-TMT-L-d(cI)-TMT-Eu: 100 ng or 15
picomoles per 5 X 105 cells. For the same degree of
1~ modification of ING-1 by ~MT and by L-d(I-QI-I) to form
ING-l-TMT and ING-l-L-d(I-QI-I), respectively, an
increase or amplification in fluorescence is seem in the
latter when Eu is added to form ING-l/TMT-Eu from ING-l-
TMT versus when Eu-TMT-L-d(cI)-TMT-Eu is added to ING-l-
L-d(I-QI-I) to form the hybrid, respectively.
.
The invention has been described in detail with
particular reference to certain preferred embodiments
thereof, but it will be understood that variations and
2~ modifications can be effected within the spirit and
scope of the invention.
11~
~ 0 94/20523 215 7 9 0 2 PCTrUS94/02610
Sr;yu~N~ LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Black, Christopher D.V.
Snow, Robert A.
($i) TITLE OF lhv~-~ION: TUMOR TA~lN~ WITH L-ENANTIOMERIC
OLIGONUCLEOTIDE CONJUGATES OF IMMuNo~Gr~NTs AND OF
~T.I~Tr!n RADIONUCLIDES
(iii) NUMLER OF ~:yur;N~s: 39
(iv) CORRESPONDENCE PnDP~-~S:
(A) ADDRESSEE: Dressler, Goldsmith, Shore, Sutker &
M i 1 n ~ Ltd.
Bl STREET: 180 North Stet~on, Ste. 4700
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Dl STATE: Illinois
E I COUh~: USA
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(A~ APPLI QTION NUMBER:
(B) FILING DATE:
(C) CLASSIFI Q TIONs
(vii$) A~ /AGENT lhrORMATION:
(A) NAME: Katz, Martin L.
(B) ~EGTSTRATION ~ KS 25,011
(ix) ~n~ CATION INFORMATION:
(A) TELEPHONE: (312)616-5400
(B) TELEFAX: (312)616-5460
(2) lhrO~I3ATION FOR SEQ ID NO:ls
(i) SEQUENCE r~A~T-~TICSs
IA LENGTH: 12 ba~e pair~
BI TYPE: ~lcleic acid
C S~ N~ SSS ~inslle
~D TOPOLOGY: linear
(ii) ~nrrc~rr~ TYPE: DNA (y~ - ic)
(ix) FEATURE:
(A) NAME/XEY: mi~c feature
(B) LOCATION: 1..1~
(D) OTHER lNrORMATION: /note= ~Each nucleotide i~ the
L--e~nAnt`i t ~ iC form "
(xi) SEQUENCE D~S~TPTION: SEQ ID NO:1:
I II
W O 94/20523 215 ~ 9 ~ 2 PCTrUS94/02610
TTATGGACGG AG 12
(2) INFORMATION FOR SEQ ID NO:2:
( i ) 5~N~ CHARACTERISTICS:
/A LENGTH: 13 ba~e pair~
B TYPE: nucleic ~cid
CI sTR~Nn~nNFcs: ~Lngle
~D, TOPOLOGY: lLnear
(ii) ~T~CUT~ TYPE: DNA (~ c)
(ix) FEATURE:
(A) NAME/REY: misc feature
(B) LOCATION: 1..13
(D) OTHER INrO~MATION: /note= "Each nucleotide iQ the
L-en~nti: - ic form.~
(xi) S~:Q~n~ D~SCBTPTION: SEQ ID NO:2:
TTATGGACGG AGA 13
(2) lN~ORMATION FOR SEQ ID NO:3:
(L) SEQUENCE rU~RA~TFRTSTICSs
~A~l ~ENGTH: 14 ba~e pair~
IBI TYPE: nucleic acid
I C I STP~pN~ NlI ~CS sins~le
~D, TOPOLOGY: linear
( ii ) ~OT~c~T~ TYPE: DNA
(ix) FEATURE:
(A) NA~E/ Æ Y: minc featur~
(B) LO QTION: 1..1~
(D) OTHER lNr~ATION: /note~ ~Each nucleotide iB the
L-enantiomeric fonm.~
(xi) SEQUENCE ~-Cr~TPTION SEQ ID NOs3:
TTATGGACGG AGAA 14
(2) lNrO~ATION FOR SEQ ID NOs4:
(i) SEQUENCE ~U~RprT~T~sTIcs
~A'l LENGTH: 15 ba~e pair~
B, TYPE: n~lclei~ ~cid
,C, STR~N~ N~ S: ingle
~D,, TOPOLOGY: linear
( ii ) ~nT-FCuT ~ TYPE: DNA (ge~ ~c)
~xi) S~Qu~ D~CC~TPTION: SEQ ID NOs4:
TTATGGACGG AGAAG 15
Il ~
~ 0 94/20523 ~15 7 9 ~ 2 PCTrUS94/02610
(2) INFORMATION FOR SEQ ID NO:5:
(i) S~Qu~ CHARACTERISTICS:
I A I LENGTH: 16 bane pairs
,8 TYPE: nucleic acid
C STRANDEDNESS: 3ingle
,D, TOPOLOGY: linear
( ii ) M~T-T~C~TT T~ TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/XEY: miRc feature
(8) LOCATION: 1..1~
(D) OTHER INFORMATION: /note= ~Each nucleotide i6 the
L-enantiomeric form."
(Xi) SEYUL.._~- DT~SC~TPTION: SEQ ID NO:5:
TTATGGACGG AGAAGC 16
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE ~UAT~A~-T~T~TSTICS:
~A LENGTH: 17 base pair~
BI TYPE: ~cl-~ ~c~d
~C s~T~a~ cs~ ~ngl~
D) TOPOLOGY: l$n-ar
( ii ) ~nT-T~ITT-T~ TYPE: DNA (~ ~ r )
(ix) FEATURE:
(A) NAME/~EY: mi~c feature
(B) LOCATION: 1..1~
(D) OTHER lN~ORhATION: /note= ~Each nucleot~de i~ the
L-enantiomer~c form.~
(xi) SEQUENCE DES~TPTIoN: SEQ ID NO:6:
TTATGGACGG an~Ar,CT 17
(2 ) lNrGfiMATION FOR SEQ ID NO:7:
(i) SEQU~NCE CHARACTERISTICS:
IA LENGTH: 18 ba3e pairA
IB TYPE: nucl-lc acid
,C STPA~ N~SS: in~le
~D,I TOPOLOGY: llnear
( ii ) MnT T`C~TT ~ TYPE: DNA (
(ix) FEAT~RE:
~A) NAME/~EY: misc feature
(B) LOCATION: 1..1~
(D) OTHER INFORMATION: /note- "Each nucleotide i~ the
L-enantiomeric form."
1 13
WO 94/20523 2 15 ~ ~ 0 2 PCTAUS94/02610
(xi) ~:yur,~_r. D~c~RTpTIoN: SEQ ID NO:7:
TTATGGACGG AGAAGCTA 18
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
IA' LENGTH: 19 ba~e pair~
B TYPE: nucleic acid
CI STRA~ CS: ~ingle
,DI TOPO~4GY: lLnear
(ii) MOTTrC~ TYPE: DNA (g~r ~)
(ix) FEATURE:
(A) NA'ME/XEY: mi~c feature
(B) LOCATION: 1..1~
(D) OTHER l~r~KdATION: /note= ~E~ch nucleotide is the
L-enanti r ~ ic form."
(x~) SEQUEN OE DFSCRTPTION: SEQ ID NO:8:
TTATGGACGG An~AGCT~A 19
(2) 1~} ~ TION FOR SEQ ID NOs9:
($) SEQUEN OE rU~RP~T~TICS:
,'A'I LENGTH: 12 bace pair~
~B TYPEs n~Cl~i~ ac$~
C 5~2~1J~ S5 ~ng1;-
~D, TOPOLOGY: 1 inear
(~i) ~OT~Fc~T~Tc TYPE: DNA (~ r i~)
($x) FEATURE:
(A) NANE/XEY: mi~c f~L~.
(B) LOCATION: 1..1~
~D) OTHER INFORMATION: /note- ~E~ch nucleotide i~ the
L-en-nt~ - ic form.~
(x$) SEQUEN OE D~ T~lON: SEQ ID NOs9:
C~ P-CT AA 12
(2) lN~O}I~ATION FOR SEQ ID NO:10:
(i) SEQUENCE r~ARA~TT~`~TsTIcs:
A', LENGTH: 13 bn~e pair~
IB TYPE: n-lcleic acid
,C STRAN~N~SS: ~ingle
,D, TOPOLO&Y: line~r
(ii) ~T~C~TT~ TYPE: DNA (y~
(ix) FEATURE:
(A) NANE/Æ Y: minc feature
(8) LOCATION: 1..1~
11~
~ O 94/20523 215 7 ~ O ~ PCTrUS94102610
(D) OTHER INFORMATION /note= ~Each nucleotide i8 the
L-enantiomeric form n
(xi) SEQUENCE DESCRIPTION SEQ ID NO lO
ACGGAGAAGC TAA 13
(2) INFORMATION FOR SEQ ID NO 11
(i) ~Q~ ~ CHARACTERSSTICS
tA LENGTH 14 base pairs
B TYPE nucleic ac~d
C STRAh~ S single
D~ TOPOLOGY linear
( i~ ) M~r ~C~lr ~ TYPE DNA (9~ - ic)
(ix) FEATVRE
(A) NAME/KEY mi~c feature
(8) LOCATION 1 1~
(D) OTHER INFORNATION /note= ~Each nucleotide i~ the
L-enantiomeric form ~
~x~) SEQ~ENCE DFCrpTpTIoN SEQ ID NO 11
~OC~ CTAA 14
(2) lNrOkMATION FOR SEQ ID NO 12
(~) SEQVENCE ÇuAR~crFR TSTICS
~A ~ENGTH 15 base pa'rs
Bl TYPE nucle$c acid
C ST~h~ -5S cin~le
~DJ 50PO~OGY: l~near
( ii ) M~T.~rTlT.F TYPE DNA (~
(ix) FEATVRE
(A) NANE/ Æ Y m~ 8C feature
(B) LOCATION 1 15
(D) OTHER lN~Ok~ATION /note~ ~Each nucleot~de i8 the
L-enantiomeric form ~
(xi) SEQVEN OE DF-CC~TPTION SEQ ID NO 12
Gr~CGr-~r-~ GCTAA 15
(2) lNrO~ATION FOR SEQ ID NO 13
(i) ~;gU~r._~: CHARACTT~RTSTICS:
~A LENGTH 16 bace pair~
B TYPE nucleic acid
C S~ CS single
~D~ TOPOLOGY linear
ii ) Mnr FC~Tr ~ TYPE DNA (Y ~c)
1 15
wo 94/20s23 215 7 9 0 2 PCT~US94/02610
(ix) FEATURE:
(A) NAME/~EY: mi~c feature
(B) LOCATION: 1..1~
(D) OTHER INFORMATION: /note= "Each nucleotide i~ the
L-enantiomeric form.~
(xi) SEQUENCE DFS~TPTION: SEQ ID NO:13:
TGr~CG~-~r-~ AGCTAA 16
(2) lN~uK~ATION FOR SEQ ID NO:14:
U~n_~ CH~RACTERISTICS:
,A'I LENGTH: 17 ba~e pair~
B TYPE: nucl~ic acid
C STRANv~vN~SS: ~inyle
~D TOPOLOGY: lLnear
~CuT~ TYPE: DNA ~ ic)
~ix) FEATURE:
(A) NAME/Æ Y: mioc feature
(B) LOCATION: 1..17
~D) OTHER IN.~OfiMATION: /note~ ~Each nucleotide i5 the
L--~n-ntir ic fo~m. "
(xi) SEQUEN OE D~SCRTPTIoNs SEQ ~D NOsl4s
ATGr-~rCG~G A~C~AA 17
(2) lNrOh~ATION FOR 8EQ ID NOsl5s
~i) SEQUENCE ~W~RprT~RT~sTIcss
IA~I LENGTHs 18 ba~e pa~ro
B, TYPEs n~cleic acid
,C, ST~A~ N~-CSs sLngle
~D, TOPO~OGY: linear
( ii ) Y~T~FC~T-~ TYPE: DNA (- ~)
~ix) FEATUREs
~A) NAME/~EYs mi~c f~t
~B) ~OCATIONs 1..1~
~D) OTHER lN~OR~ATION: /note- ~Each nucleotide i3 the
L--~l-nt ~ ~ ic fo~m. "
~xi) SEQUENCE D~S~TPTIONs SEQ ID NOsl5:
TATGr~cGr~ n~GCTA~ 18
~2) lN~OR~ATION FOR SEQ ID NOsl6:
~i) SEQUEN OE ~UAp~-T~;pTsTIcs:
~A LENGTH: 12 ba~e pair~
,8 TYPE: ml~l~c acid
C STRANv~vh~SS: single
D, TOPO~CGY: linear
I 1~
WO 94120SZ3 215 7 9 0 2 PCT/US94/02610
( ii ) MOT~CUT~ TYPE: DNA ~genomie)
(ix) FEATURE
(A) NAME/KEY: m$~c feature
(B) LOCATION 1..1~
(D) OTHER lNrOR~ATION: /note= ~Each nucleotide iQ the
L-enantiomeric form ~
(xi) ~Eyu~ _~ DESCRIPTION: SEQ ID NO:16:
TTAGCTTCTC CG 12
(2) INFORMATION FOR SEQ ID NO:17:
(i) ~u~r_~ CHA~TF~TSTICS:
'A' LENGTH: 13 ba~e pair~
B TYPE: nucleie aeid
C, ST~h~ h~SS: uingle
~D, TOPOLOGY: lin-ar
( ii ) ~nT~CuT~ TYPE: DNA
(ix) FEATURE:
(A) NAME/ Æ Y: mi~e f.aLu~_
(B) LOCATION: 1..1~
(D) OTHER INFORMATION: /not-e ~Eaeh nucleot~de ~u the
L-enanti~ ~.ie form
(xi) SEQUENCE ~erPTPTION: SEQ ID NO~
TTAG~.. ....CGT 13
~2) lrl~OR~ATION FOR SEQ ID NOsl8s
(i) SEQUENCE ~CTF~T8TICS:
'A' LENGTH: 14 baue pairs
B TYPE: n~le~eie aeid
~C ST~ SS: ~ingle
~D, TOPO~OGY: l~n-ar
(ii) MnT~CT~ TYPE: DNA (, ~)
(~x) FEAT~RE:
(A) NAME/~EYs mi~c featurQ
(B) LOCATION: 1 .1~
(D)'OTHER INFORMATION: /note= ~Eaeh nueleotide i~ the
L-enantiomerie form.~
(xi) SEQUENCE D~-e~TPTIoN: SEQ ID NO:18:
TTAG~.. ~.C CGTC 14
(2) INFORMATION FOR SEQ ID NO:19:
i ) ~QU~N~: CHARACTERISTICS:
(A) LENGTH: 15 baee pair~
117
2 1 ~ 7 ~ ~ ~
W O 94/20~23 PCTrUS94/02610
(B) TYPE nucleic acid
(c) STRANDTCnNESS 8 ingle
(D) TOPOLOGY linear
(ii) ~nr~UT~ TYPE DNA (~ ir)
(ix) FEATURE
(A) NAME/REY mi~c feature
(B) LOCATION 1 1~ ~
(D) OTHER INFORMATION /note= ~Each nucleotide i~ the
L-enantiomeric form ~
~xi) ~Çu~ DTc~cpJpTIoN SEQ ID NO l9
TTAG~ C CGTCC 15
(2) ln~R~ATION FOR SEQ ID NOs20
( i ) ~;yu~~ r: ~ATRI~c~TF~T~e:TIcs
A LENCTH 16 ba~e pair~
~B TYPE nncle~ acid
,C STRA~ ~SS single
~D, TOPOLOGY linear
~ ii ) ~T FC~T~TC TYPE DNA (~
(ix) FEATUREs
(A) NAME/~LYs mi-c feature
(B) LOCATIONs 1 1~
(D) OTHER ~N~k~TIONs /note~ ~Eacb nucleotide i~ the
L ~ nt~ ~ ic form ~
(xi) SEQUEN OE ~S rl~. ~ON- s 8EQ ID NOs20s
TTAG~ C CGTCCA 16
(2) ~rOkhATION FOR SEQ lD NOs21s
(~) SEQUENOE CHARACI~RISTICSs
~Al LENGTHs 17 ba~e pair~
B TYPEs ~cl-jc acid
C sTR~NnTcnNEsss ingle
~D TOPOLOGYs lin ar
(i~ ) ~T~TCc~Tc TYPE DNA (9 ~)
(ix) FEAT~RL
(A) NAME/RIY: mi~c feature
(B) LOCATION 1 17
(D) OTHER ~N~khATION /note~ ~Each nucleotide in the
L--~n~nti~ ~ ic form
(xi) SEQUENCE DTccrRlpTIoN SEQ ID NO:21
TTAG~C~C CGTCCAT 1
118
0 94/20523 215 7 9 ~ 2 PCTrUS94/02610
(2) INFORMATION FOR SEQ ID NO:22:
( i ) S~Q~r._~ C~ARACTERISTICS:
~AI LENGTH: 18 base pairs
B TYPE: nucleic acid
,C, sTRAN~n~ss: single
~D, TOPOLOGY: linear
( ~ i ) MOT~TCC~TT~T~ TYPE: DNA (~_ ~c)
(ix~ FEATURE:
(A) NAME/XEY: misc feature
(B) LOCATION: l..l~
(D) OTHER Ih~Ok~ATION: /note= ~Each nucleotide is the
L-en~ntiomeric form. n
(xi) SEQUENCE ~T~S~RTpTION: SEQ ID NO:22s
TTAGCTTCTC CGTCCATA 18
(2) lNr~RMATION FOR SEQ ID NO:23:
(i) SEQ~ENCE CHARACTERISTICS:
'Al LENGTH: l9 ba~e pair~
B TYPEs nucl-~c ~cid
C S~AN~ S: in~l-
~D~ TOPOLOGY: linear
( i i ) ~T-T~TT-T~ TYPE: DNA (91 i r )
(ix) FEATURE:
(A) NAME/~EYs micc feature
(B) LOCATION: l..l~
(D) OTHER Ih~u.~ATION: /not-- ~Each n~cleotLde i3 the
L-enAnti~ - ic form.~
(xi) SEQUENCE DFSrRTPTION: SEQ ID NO:23:
TTAGu.. ~.~ CGTCCATAA l9
~2) lN~O~ATION FOR SEQ ID NOs24:
(i) SEQUENCE ~TARArT~RTSTICS:
~A' LENGTH: 12 ~ace pair~
B TYPE: n--cl~ i o ~c$d
C STR~ Nl~ ..Cs ingle
~D TOPOLOGY: l$near
( ii ) ~nT~T~`C~' ~ T' TYPE: DNA (Y ~ r )
(ix) FEATURE:
(A) NAME/XEY: mi~c feature
(B) LOCATION: l..l~
(D) OTHER lr.~OkhATION: /note- ~Each nucleotide is the
L ~n n~i r ~ ic form. n
1~ 9
2 ~ 2
WO 94120523 PCT/US94/02610
~x$) ~yuc.._~ D~CrpTpTIoN: SEQ ID NO:24:
C~lC~AT ~A 12
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQ~ENCE ~RAcTFRTsTIcs:
lAI LENGTB: 13 base pairR
B TYPE: nucleic acid
,C, STR~ S: ~ingle
~D~ TOPOLOÇY: line~r
(Li) ~nT~RCu~ TYPE: DNA (g~
(ix) FEATURE:
(A) NAME/~EYs mi~c feature
(B) LOCATION: 1..1~
(D) OTBER lNrORMATION: /not~ ~Each nucleot~de i~ the
L-enant~omerLc form.~
(xi) ~yu~ r, D~ 'K T~ lON: 8EQ ID NO:25:
CC-~ ~A TA~ 13
(2) ~N~ SATION FOR SEQ ID NOs26:
(i) SEQU~N OE r~a~P~TæTICSs
'A ~ENGTB: 14 ba-e palrs
IB TYPE: ~cl-~c acid
,C Sq'~ mFrlNESS: ins~le
~D~ TOPOLOGY: l$near
(~) ~nr~C~r~ TYPE: DNA (~ ~)
(~x) FEATURE:
(A) NA~E¦~EYs mi~c feature
(B) LOCATION:1..14
(D) OTHER lNrvk~ATION: /note- ~Each nucleotide i5 the
L - ~n~nt i ~ Lc form.~
(xi) 8EQUEN OE D~SrPTPTION: SEQ ID NOs26:
w .CC ATAA 14
(2) INrohc~TIoN FOR SEQ ID NOs27s
(i) SEQUENCE ~R~r~F~TSTICS:
A'I LENGTH: 15 ba~e pairs
B TYPE: ntlclei~ ac~d
,C STRPN~ h~SS: single
~DJ TOPOLOGY: linear
( ~ i) M~T~Cr~r TYPE: DNA (~ - ic)
(ix) FEATURE:
(A) NAME/~EY: misc feature
(B) LOCATION: 1..I~
1~0
~ 0 94/20~23 215 7 9 0 2 PCTrUS94102610
- ~21 -
(D) OTHER INFORMATION /note= "Each nucleotide i~ the
L-~nantiomeric form "
(xi) SEQUENCE D~C~RTPTION SEQ ID NO 27
u~, CC~C CATAA 15
~2) INFORMATION FOR SEQ ID NO 28
J~r,~l~: rR1~ACT~TSTTCS:
IA' LENGTH 16 basQ pa~r~
B TYPE ~ucl~c ac~d
,C STPA~ cS ~$ngle
~D~ TOPOLOGY linear
( ii ) ~nT FU~ ~ ~ TYPE DNA (gF
(~x) FEATURE
(A) NAME/XEY mi~c feature
(8) LOCATION~
(D) OTHER lNr~R~ATION /note= ~Each nucleotide i~ the
L-enanti~ - ic form n
(xi) SEQUENCE D~C~DTPTIoN: SEQ ID NO:28
C~.,-~ C~. C QTAA 16
(2) l~ru~ATION FOR SEQ ID NOs29s
CHARACTERISTICSs
lA' LENGTH: 17 ba~e pair-
B TYPEs ~ucl-i~ acid
C S~A~ 5Ss ingle
ID, TOPOLOGY: lLn-ar
( ii ) MnT~C~T~ TYPE DNA (~ ~c)
(ix) FEATURE
(A) NAME/XEY: m~c feature
(B) LOCATIONs 1 1~
(D) O } R ~h~ _~TION /note~ ~Each nucleotide i~ the
L-enantiomQrLc form ~
(x~) SEQUEN OE DFC~TPTION SEQ ID NO:29:
AGu.~.CCG TCCATAA 17
(2) ~hrORMATION FOR SEQ ID NO 30
($) SEQVENCE CHARACTERISTICS
~A' LENGTH 18 ba~e pa~rs
8 TYPE nucl eic acid
CI ST~AN~ eS ingle
~D, TOPOLOGY linear
( ii ) MnT~C~T~ TYPE DNA (~ ~ ~c)
W O 94120523 2 ~ 5 ~
~ PCTrUS94/02610
(lx) FEATURE:
(A) NAME/REY: mi~c feature
(B) LOCATION: l..18
(D) OTHER INFORMATION: /note= "Each nucleotide i~ the
L-enantiomeric form. r
(Xi) ~EQ~N~ D~ccRlpTIoN: SEQ ID NO:30:
TAGC.~CC GTCCATAA 18
(2) INFORMATION FOR SEQ ID NO:3l:
( i ) S~J~ CH`ARACTERISTICS:
A LENGTH: 52 ba~e pair~
B TYPE: nnçlelc acid
C, STR~h~ CS: single
~D TOPOLOGY: linear
(ii) M~T.~C~IT.F TYPE: DNA (g.r~ ic)
(ix) FEATURE:
(A) NAME/REY: mi~c feature
(B) LOCATION: l
(D) OTRER lN~O~ATION: /note~ ~N equal~ Teg.
~ix) FEATUREs
(A) NAME/~EYs mlsc f-ature
(B) LOCATION: 2..5~
(D) OTn~R ~NrOR~ATION: /note- ~Each nucleotLde i~ the
L ~ -n~i~ ic form.
(ix) FEATURE:
(A) NAME/XEYs mi~c feature
(8) LOCATIONs 5l
(D) OTHER ~N~O.I~ATION: /note~ ~N equal~ NR2.
(ix) FEATURE:
(A) NAME/XEY: mi~c feature
(B) LOCATION: 52
(D) OTHER lNr~R~ATION: /note= ~Thi~ nucleotLde iu the
L--~n~n~ ~ ic form.~
(xi) SEQUEN OE DFCr~pTIoN: SEQ ID NO:31:
NTCTTATG&A CGGPr-~GCT AAA~-C.. ATGG~CGr~PG AAGCTAATCT NT 52
(2) INrol~aTIoN FOR SEQ ID NO:32:
(i) ~u~..~ CHARACTERISTICS:
~AI LENGTR: 45 base paLr~
,8 TYPE: nucleic acid
C, S~R~ h~SS: ~ingle
~D, TOPOLOGY: linear
(Li) M~T rCUT ~ TYPE: DNA (~ ~ç)
(xi) S~-yu~_~ D~S~TPTION: SEQ ID NO:32:
ILL
~O 94/20~;23 2 1 5 7 9 0 ~ PCTIUS94/02610
TCTTATGGAC GGA-~CC~u,A ATCTTATGGA CGGATCCGCT AATCT 45
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUEN OE ÇU~R~TFRTSTICs
rA~ LENGTH: 52 base pair~
B TYPE: nucl~;c ~cid
,C, STR~ CS: ~ingle
~D, TOPOLOGY: lin~Ar
(i~ ) M~T~ T-F TYPE: DNA (~ r
(~x) FEATUREs
(A) NAM /XEY: mi~c feature
(B) LOCATION: 1
(D) OTHER I~rohdATIoN: /note= "N equal~ an ,
~minc co~t~;nin7 group.
(;x) FEATURE:
(A) NANE/KEY: misc feature
(B) LOCATION: 51
(D) OTHER l~u~ATION: /note~ N equals an
~min~ ~oll~;n;n~ group."
(x;) ~Qu~ u~S~ ON: SEQ ID NO:33:
NTC~TATGGA CCG~r~GCT AAA~.~ ATCG~Gr~ GC~CT NT 52
(2) INFORdATION FOR SEQ ID NO:34:
(i) SEQUEN OE ~U~RA~T~TcTICSs
~A' LENGTH: 52 base pairs
B TYPE: nucl-;~ ~oid
,C STR~ -5.S: ~ingle
~DJ TOPO~OGY: linQar
(ii) MnTT~!C~TF TYPE: DNA (~ i r
(ix) FEATURE:
(A) NANE/XEY: misc fea~u.
(B) LOCATION: 1
(D) OT~R l~v~MATIoN: /note~ ~N egual~ an
amil.e ro~; n ~ ng group.
(ix) FEATURE:
(A) NAME/REY: mi~c feature
(B) LOCATION: 2..5~
(D) OTHER lNru~ATION: /note- ~Each nucleotide is the
L-enantiomeric form.
(ix) FEATURE:
(A) NAME/XEY: misc feature
~ (B) LOCATION: 51
(D) OTHER INFORMATION: /note~ nN equal~ an
amine con~; n; ng group.
(ix) FEATURE:
(A) NAME/~EY: misc feature
(B) LOGaTION: 52
t,~ ~
W O 94l20~23 ~ ~ 5 7 9 0 2 PCTrUS94/02610
(D) OTHER INFORMATION: /note= "Thi~ nucleotide i~ the
L-enantiomeric~ form.~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
NTCTTATGGA CGr-A~-AAr,CT AAA~ - ATGr-ACGr-AG AAGCTAATCT NT 52
(2) lN~OR~ TION FOR SEQ ID NO:35s
( i ) ~i l :yu l~ .' rRARAcTFRTsTIcs:
~A' LENGTH: 52 ba~e paLr~
B~ TYPE: nucleic ac~d
C STRAN~ r-~Ss sinslo
,D, TOPOLOGY: linear
( ii ) MnT ~C~T~ TYPE: DNA (~
(ix) FEAT~RE:
(A) NANE/KEY: mi~c feature
~B) LOCATION: 1
(D) OTHER ~N~O~MATION: /note- ~N equ_l~ H2N. n
(ix) FEATURE:
(A) NAME/Æ Y: misc f-~tur~
(B) LOCATION: 2..5~
(D) OTHER lN~Ok~A~ION: /note= ~Each nucleotidQ i~ thQ
L-~n-nt~l ~c form."
(ix) FEATUREi:
~A) NAME/ B Ys m~cc fe~ture
(B) LOCATIONs 51
(D) OTHER ~ JI-~A~I~s /not-- ~N T~5-"
(ix) FEATUREs
(A) NAME/XEYs mi~c fe-
~(B) LOCATION: 52
(D) OTHER INFORMATION: /note~ ~Thie nucleotlde i~ the
L-~nantit - ic form."
(xi) SEQUENCE ~-Cr~PTION: SEQ ID NO:35:
NTCTTATGGA CGr-Ar~ CT AAA~ ATCC~Gr-Ar, AAGCTAATCT NT 52
(2) lN~ORMATION FOR SEQ ID NO:36:
(1) SEQUENCE r~A~Ar,~R~STICS:
~A' LENGTH: 50 ba~e pair~
IB TYPE: nucleic acld
,C, STRAN~ S: ~inqle
~D,I TOPOLOGY: line_r
(ii) ~T~C~ TYPE: DNA (ger iC)
(ix) FEATnRE:
(A) NAME/~EY: m~c feature
(B) LOCATION: 1
(D) OTHER lN~uk~ATION: /note= "N equal~ H2N."
1 ~4'
~V0 94/20!;23 215 7 9 0 2 PCT/US94/02610
-- 125 --
(ix) FEATURE
(A) NAME/KEY mi~c feature
(B) LOCATION 2 50
(D) OT B R INFORMATION /note= "Each nucleotide i~ the
L-enantiomeric form ~
(xi) S~YU~N~ DTESCRTPTION SEQ ID NO 36
NTCTTATGGA CG4ar-~PGCT AhA~.~ ~ ATGr-~r~G~G AAGCTAATCT 50
(2) lNrORMATION FOR SEQ ID NO 37
(i) SEQUENCE rRaRACT~TSTICS
,'A' LENGTH 51 base pairs
B T,YPE nucleic acid
C STRA~N~ SS 8ingle
~D, TOPOLOGY linear
( ii ) ~T~TCcm~ TYPE DNA (~ r- ~ r )
(ix) FEASURE
(A) NAME/XEY misc f~ature
(B) LO Q TIONs 1
(D) OT~R lhr~l~TION /note- "N equal~ H2N "
(ix) FEASUREs
(A) NAME/XEYs mi~c fJ~~
(B) LO Q TIONs 2 5~
(D) OTHER l~OAhATION /note~ ~Each nucleotide i~ the
L F -nti I - Lc form
(ix) FEATUREs
(A) NAME/~EYsmi~c r.-- ~
(B) LO Q TION 51
(D) OT~ER ~ ATION /note~ ~N qual~ OP03~2 "
(xi) S~ur,~ur; DF-~C~TPTION SEQ ID NO 37
NTCTTATGGA cGr~-a~cT A~Au.~. AT~r'~Gr-~ AP~CTaaSCT N 51
~2) l~rO~MATION FOR SEQ ID NOs38
(i) S~Qu~ aRA~TrC~ssIcss
'A'l LENGTHs 22 ba~c pair~
B TYPE nuclei~ acid
C, STRAN~ N~8 single
,DJ TOPOLOGY lin-ar
(ii) ~T-T`CI~-T~ SYPE DNA (~
(ix) FEATURE
(A) NAME/XEY misc feature
(B) LO Q TION
(D) OTHER ~Nruk~ATION /note~ "N equal~ an
amine CQI~t~nin~ ~roup "
(ix) FEATURE
(A) NAME/XEY misc feature
1~
W O 94/20523 215 7 ~ 0 2 PCTrUS94/02610
- 126 -
(B) LO Q TION: 2..20
(D) OTHER lNrOh~ATION: /notes "Each nucleotide i~ the
L-enantiomeric form.
(ix) FEATURE:
(A) NAME/REY: minc feature
(B) LO Q TION: 21 io, ~'
(D) OTHER INFORMATION: /note= ~N équalQ an
am~ne ContA i ni ng group.
(Lx) FEATURE:
(A) NAME/REY: misc feature
(B) LO QTION: 22
(D) OTHER lNrO~.TION: /note= ~This nucleotLde is the
L-enantLomeric form.~
~xi) SEQUENCE DFCc~TpTIoN: SEQ ID NOs38:
NTTAGCTTCT CC~. ~ATAA NT 22
(2) IN~ATION FOR SEQ ID NO:39:
(i) SEQUENCE ~RA~T~TSTICS:
A' LENGTH: 22 base paLrs
B TYPE: nucleLe acld
C STR~L~ Ss s~ngl~
~D, TOPOLOGY: lin-ar
(L~) MnT~ClTF TYPE: DNA t~
(ix) FEATUREs
(A) NAME/~EYs mL~c f--L
(8) ~OCATIONs 1
(D) OTHER l~ATION: /note~ ~N equ_l~ an
mLn~ _o.~ n ng group.
(ix) FEATURE:
~A) NAME/~EY: mL-c feature
(B) LOCATION: 20
(D) OTHER I~O~ATION: /note~ ~ach nucleotide is the
L--en-nt~ ic form.
(ix) FEAT~RE:
(A) NAME/~EY: misc 1-~
(B) LOCATION: 21
(D) OTHER l~rOh~ATION: /note= ~N equ~ls an
Lnc co.~ L~ i n i ng group.
(ix) FEATURE:
(A) NAME/REY: mLsc feature
(B) LO Q TION: 22
(D) OTHER l~rOR~ATION: /note= ~This nucleotide is the
L-enantiomeric form. n
(xi) SEQUENCE DESCRIPTION: SEQ ID NOs3g:
NTTATG&ACG G~G~A~-CT~ NT 22
'2'
, ~
