U.S. patent application number 09/316452 was filed with the patent office on 2002-02-07 for three-step pretargeting methods and compounds.
Invention is credited to GUSTAVSON, LINDA M., RENO, JOHN M., THEODORE, LOUIS J..
Application Number | 20020015705 09/316452 |
Document ID | / |
Family ID | 27496256 |
Filed Date | 2002-02-07 |
United States Patent
Application |
20020015705 |
Kind Code |
A1 |
THEODORE, LOUIS J. ; et
al. |
February 7, 2002 |
THREE-STEP PRETARGETING METHODS AND COMPOUNDS
Abstract
Methods, compounds, compositions and kits that relate to
pretargeted delivery of diagnostic and therapeutic agents are
disclosed. In particular, three-step pretargeting methods are
described.
Inventors: |
THEODORE, LOUIS J.;
(LYNNWOOD, WA) ; RENO, JOHN M.; (BRIER, WA)
; GUSTAVSON, LINDA M.; (SEATTLE, WA) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE
SUITE 6300
SEATTLE
WA
98104-7092
US
|
Family ID: |
27496256 |
Appl. No.: |
09/316452 |
Filed: |
May 21, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09316452 |
May 21, 1999 |
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08753445 |
Nov 25, 1996 |
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09316452 |
May 21, 1999 |
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08156614 |
Nov 23, 1993 |
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5578287 |
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09316452 |
May 21, 1999 |
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PCT/US93/05406 |
Jun 7, 1993 |
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09316452 |
May 21, 1999 |
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07995383 |
Dec 23, 1992 |
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07995383 |
Dec 23, 1992 |
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07895588 |
Jun 9, 1992 |
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5283342 |
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Current U.S.
Class: |
424/178.1 |
Current CPC
Class: |
A61K 51/1268 20130101;
A61K 47/557 20170801; A61K 47/68 20170801; A61K 47/6887 20170801;
A61K 51/0497 20130101; A61K 47/6897 20170801; A61K 2123/00
20130101; A61K 47/6898 20170801; A61K 47/6893 20170801; A61K
2121/00 20130101; B82Y 5/00 20130101; A61K 47/665 20170801 |
Class at
Publication: |
424/178.1 |
International
Class: |
A61K 039/395 |
Claims
What is claimed is:
1. A method of enhancing active agent localization at a target site
in a mammalian recipient, which method comprises: administering to
the recipient a first conjugate comprising a targeting moiety and a
biotin, whereupon the first conjugate localizes to the target site;
administering to the recipient avidin or streptavidin; and
thereafter administering to the recipient a second conjugate
comprising biotin, a linker resistant to biotinidase cleavage and
an active agent, wherein second conjugate localization at the
target site is enhanced as a result of prior localization of the
first conjugate.
2. A method of claim 1 wherein the targeting moiety is
proteinaceous.
3. A method of claim 1 wherein the targeting moiety is an
oligonucleotide, a peptide, a polypeptide, a cytokine, a monoclonal
antibody, a monovalent fragment thereof.
4. A method of claim 3 wherein the monoclonal antibody is; a human,
a humanized or a chimeric monoclonal antibody.
5. A method of claim 3 wherein the monoclonal antibody or fragment
thereof is reactive with an antigen recognized by the antibody
NR-LU-10.
6. A method of claim 1 wherein the active agent is selected from
the group consisting of radionuclides, chemotherapeutic drugs,
anti-tumor agents and toxins.
7. A method of claim 6 wherein the active agent is a radionuclide
selected from the group consisting of Re-186, Re-188, Tc-99m, Y-90,
At-211, Pb-212, Bi-212, Sm-153, Eu-169, Lu-177, Cu-67, Rh-105,
In-111, Au-198, I-123 and I-131.
8. A method of claim 6 wherein the active agent is a cytokine or a
lectin inflammatory response promoter.
9. A method of claim 1 wherein the step of administering the second
conjugate is conducted by intralesional or intraarterial
injection.
10. A method of claim 9 wherein the second conjugate is
administered via an artery supplying target site tissue.
11. A method of claim 9 wherein the second conjugate is
administered via an artery selected from the group consisting of
hepatic artery, carotid artery, bronchial artery and renal
artery.
12. A method of claim 1 wherein the second conjugate is
administered intravenously.
13. A method of claim 1 wherein the second conjugate comprises a
biotin-DOTA compound of the following formula: 47wherein a linker L
is selected from the group comprising: 1) a D-amino acid-containing
linker of the formula 482) a linker of the formula 493) a linker of
the formula 504) a Linker of the formula 51wherein L' is selected
from the group comprising: a) --NH--CO--(CH.sub.2).sub.n--O--; b)
--NH--; c) --NH--CO--CH.sub.2--N--R"--; R'd) --NH--CS--NH--; and e)
--NH--CO--(CH.sub.2).sub.n--NH--, wherein R.sup.1 is hydrogen,
lower alkyl; lower alkyl substituted with one or more hydrophilic
groups including (CH.sub.2).sub.m--OH, (CH.sub.2).sub.m--OSO.sub.3,
(CH.sub.2).sub.m--SO.sub.3, and 52where m is 1 or 2;
glucuronide-substituted amino acids; or other glucuronide
derivatives; R.sup.2 is hydrogen; lower alkyl; substituted lower
alkyl having one or more substituents selected from the group
comprising hydroxy, sulfate, and phosphonate; or a hydrophilic
moiety; R.sup.3 is hydrogen; an amine; a lower alkyl; a hydroxy-,
sulfate- or phosphonate-substituted lower alkyl; a glucuronide; or
a glucuronide-derivatized amino acid; R.sup.4 is hydrogen, lower
alkyl or 53R.sup.1 is hydrogen; --(CH.sub.2).sub.2--OH or a sulfate
or phosphonate derivative thereof; or 54R" is a bond or
--(CH.sub.2).sub.n--CO--NH--; and n ranges from 0-5.
14. A method of claim 13 wherein L is a D-amino acid-incorporating
linker of the formula 55
15. A method of claim 14 wherein R.sup.1 is CH.sub.3 and R.sup.2 is
H.
16. A method of claim 13 wherein L is a linker of the formula
56
17. A method of claim 16 wherein R.sup.3 is hydrogen; R.sup.4 is
CH.sub.3; and n is 4.
18. A method of claim 16 wherein R.sup.3 is hydrogen; R.sup.4 is
CH.sub.3; and n is 0.
19. A method of claim 16 wherein R.sup.3 is hydrogen; R.sup.4 is
57and n is 4.
20. A method of claim 13 wherein L is a linker of the formula
58wherein L' is selected from the group comprising: a)
--NH--CO--(CH.sub.2).sub.n--- O--; b) --NH--; c)
--NH--C(--CH.sub.2--N--R"--; R'd) --NH--CS--NH--; and e)
--NH--CO--(CH.sub.2).sub.n--NH-- or a bis-DOTA derivative thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of pending PCT
Patent Application No. PCT/US93/05406, filed Jun. 7, 1993 and
designating the United States, which, in turn, is a
continuation-in-part of pending U.S. patent application Ser. No.
07/995,383, filed Dec. 23, 1992, which is, in turn, a
continuation-in-part of pending U.S. patent application Ser. No.
07/895,588, filed Jun. 9, 1992.
TECHNICAL FIELD
[0002] The present invention relates to methods, compounds,
compositions and kits useful for delivering to a target site a
targeting moiety that is conjugated to one member of a
ligand/anti-ligand pair. After localization and clearance of the
targeting moiety conjugate, direct or indirect binding of a
diagnostic or therapeutic agent conjugate at the target site
occurs. Methods for radiometal labeling of biotin and for improved
radiohalogenation of biotin, as well as the related compounds, are
also disclosed.
SUMMARY OF THE INVENTION
[0003] The present invention describes three-step pretargeting
diagnostic and therapeutic methods. Three-step pretargeting
protocols feature administration of a targeting moiety-ligand
conjugate, which is allowed to localize at a target site and to
dilute in the circulation. Subsequently administered anti-ligand
binds to the targeting moiety-ligand conjugate in both blood and at
a target site and clears unbound antibody-ligand conjugate from the
blood. A diagnostic or therapeutic agent-ligand conjugate that
exhibits rapid whole body clearance is then administered and binds
to the targeting moiety-ligand-anti-ligand localized at a target
site, thereby constituting the third target site-localized
component in the protocol.
[0004] Preferred three-step pretargeting methods of the present
invention employ biotin/avidin as the ligand/anti-ligand binding
pair. These preferred three-step pretargeting methods involve the
administration of biotin conjugated to therapeutic or diagnostic
radionuclides or other active agents such as chemotherapeutic
drugs, anti-tumor agents such as cytokines and the like.
Y-90-DOTA-biotin conjugates are particularly preferred in the
practice of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0005] FIG. 1 illustrates blood clearance of biotinylated antibody
following intravenous administration of avidin.
[0006] FIG. 2 depicts radiorhenium tumor uptake in a three-step
pretargeting protocol, as compared to administration of
radiolabeled antibody (conventional means involving antibody that
is covalently linked to chelated radiorhenium).
DETAILED DESCRIPTION OF THE INVENTION
[0007] Prior to setting forth the invention, it may be helpful to
set forth definitions of certain terms to be used within the
disclosure.
[0008] Targeting moiety: A molecule that binds to a defined
population of cells. The targeting moiety may bind a receptor, an
enzymatic substrate, an antigenic determinant, or other binding
site present on the target cell population. Antibody is used
throughout the specification as a prototypical example of a
targeting moiety.
[0009] Ligand/anti-ligand pair: A complementary/anti-complementary
set of molecules that demonstrate specific binding, generally of
relatively high affinity. Exemplary ligand/anti-ligand pairs
include zinc finger protein/dsDNA fragment, enzyme/inhibitor,
hapten/antibody, ligand/receptor, and biotin/avidin. Biotin/avidin
is used throughout the specification as a prototypical example of a
ligand/anti-ligand pair.
[0010] Anti-ligand: As defined herein, an "anti-ligand"
demonstrates high affinity, and preferably, multivalent binding of
the complementary ligand. Preferably, the anti-ligand is large
enough to avoid rapid renal clearance, and contains sufficient
multivalency to accomplish crosslinking and aggregation of
targeting moiety-ligand conjugates. Univalent anti-ligands also
find utility in the practice of the present invention.
[0011] Avidin: As defined herein, "avidin" includes avidin,
streptavidin and derivatives and analogs thereof that are capable
of high affinity, multivalent or univalent binding of biotin.
[0012] Ligand: As defined herein, a "ligand" is a relatively small,
soluble molecule that exhibits rapid serum, blood and/or whole body
clearance when administered intravenously in an animal or
human.
[0013] Active Agent: A diagnostic or therapeutic agent ("the
payload"), including radionuclides, drugs, anti-tumor agents,
toxins and the like.
[0014] N.sub.xS.sub.y Chelates: As defined herein, the term
"N.sub.xS.sub.y chelates" includes bifunctional chelators that are
capable of (i) coordinately binding a metal or radiometal and (ii)
covalently attaching to a targeting moiety. Particularly preferred
N.sub.xS.sub.y chelates have N.sub.2S.sub.2 andc N.sub.3S cores.
Exemplary N.sub.xS.sub.y chelates are described in Fritzberg et
al., Proc. Natl. Acad. Sci. USA 85:4024-29, 1988; in Weber et al.,
Bioconj. Chem. 1:431-37, 1990; and in the references cited therein,
for instance.
[0015] Pretarqeting: As defined herein, pretargeting involves
target site localization of a targeting moiety that: is conjugated
with one member of a ligand/anti-ligand pair; after a time period
sufficient for optimal target-to-non-target accumulation of this
targeting moiety conjugate, active agent conjugated to the opposite
member of the ligand/anti-ligand pair is administered and is bound
(directly or indirectly) to the targeting moiety conjugate at the
target site (two-step pretargeting). Three-step pretargeting
protocols are also provided by the present invention, involving,
for example, administration of targeting moiety-ligand,
administration of anti-ligand to clear circulating targeting
moiety-ligand and to localize to previously target-localized
targeting moiety ligand, and administration of active
agent-ligand.
[0016] Conjugate: A conjugate encompasses chemical conjugates
(covalently or non-covalently bound), fusion proteins and the
like.
[0017] A recognized disadvantage associated with in vivo
administration of targeting moiety-radioisotopic conjugates for
imaging or therapy is localization of the attached radioactive
agent at both non-target and target sites. Until the administered
radiolabeled conjugate clears from the circulation, normal organs
and tissues are transitorily exposed to the attached radioactive
agent. For instance, radiolabeled whole antibodies that are
administered in vivo exhibit relatively slow blood clearance;
maximum target site localization generally occurs 1-3 days
post-administration. Generally, the longer the clearance time of
the conjugate from the circulation, the greater the radioexposure
of non-target organs.
[0018] These characteristics are particularly problematic with
human radioimmunotherapy. In human clinical trials, the long
circulating half-life of radioisotope bound to whole antibody
causes relatively large doses of radiation to be delivered to the
whole body. In particular, the bone marrow, which is very
radiosensitive, is the dose-limiting organ of non-specific
toxicity.
[0019] In order to decrease radioisotope exposure of non-target
tissue, potential targeting moieties generally have been screened
to identify those that display minimal non-target reactivity, while
retaining target specificity and reactivity. By reducing non-target
exposure (and adverse non-target localization and/or toxicity),
increased doses of a radiotherapeutic conjugate may be
administered; moreover, decreased non-target accumulation of a
radiodiagnostic conjugate leads to improved contrast between
background and target.
[0020] Therapeutic drugs, administered alone or as targeted
conjugates, are accompanied by similar disadvantages. Again, the
goal is administration of the highest possible concentration of
drug (to maximize exposure of target tissue), while remaining below
the threshold of unacceptable normal organ toxicity (due to
non-target tissue exposure). Unlike radioisotopes, however,
therapeutic drugs need to be taken into a target cell to exert a
cytotoxic effect. In the case of targeting moiety-therapeutic drug
conjugates, it would be advantageous to combine the relative target
specificity of a targeting moiety with a means for enhanced target
cell internalization of the targeting moiety-drug conjugate.
[0021] In contrast, enhanced target cell internalization is
disadvantageous if one administers diagnostic agent-targeting
moiety conjugates. Internalization of diagnostic conjugates results
in cellular catabolism and degradation of the conjugate. Upon
degradation, small adducts of the diagnostic agent or the
diagnostic agent per se may be released from the cell, thus
eliminating the ability to detect the conjugate in a
target-specific manner.
[0022] One method for reducing non-target tissue exposure to a
diagnostic or therapeutic agent involves "pretargeting" the
targeting moiety at a target site, and then subsequently
administering a rapidly clearing diagnostic or therapeutic agent
conjugate that is capable of binding to the "pretargeted" targeting
moiety at the target site. A description of some embodiments of the
pretargeting technique may be found in U.S. Pat. No. 4,863,713
(Goodwin et al.).
[0023] A typical pretargeting approach ("three-step") is
schematically depicted below. 1
[0024] Briefly, this three-step pretargeting protocol features
administration of an antibody-ligand conjugate, which is allowed to
localize at a target site and to dilute in the circulation.
Subsequently administered anti-ligand binds to the antibody-ligand
conjugate and clears unbound antibody-ligand conjugate from the
blood. Preferred anti-ligands are large and contain sufficient
multivalency to accomplish crosslinking and aggregation of
circulating antibody-ligand conjugates. The clearing by anti-ligand
is probably attributable to anti-ligand crosslinking and/or
aggregation of antibody-ligand conjugates that are circulating in
the blood, which leads to complex/aggregate clearance by the
recipient's RES. It is preferred that the ligand-anti-ligand pair
displays relatively high affinity binding.
[0025] A diagnostic or therapeutic agent-ligand conjugate that
exhibits rapid whole body clearance is then administered. When the
circulation brings the active agent-ligand conjugate in proximity
to the target cell-bound antibody-ligand-anti-ligand complex,
anti-ligand binds the circulating active agent-ligand conjugate and
produces an antibody-ligand:anti-ligand:ligand-active agent
"sandwich" at the target site. Because the diagnostic or
therapeutic agent is attached to a rapidly clearing ligand (rather
than antibody, antibody fragment or other slowly clearing targeting
moiety), this technique promises decreased non-target exposure to
the active agent.
[0026] Alternate pretargeting methods eliminate the step of
parenterally administering an anti-ligand clearing agent. These
"two-step" procedures feature targeting moiety-ligand or targeting
moiety-anti-ligand administration, followed by administration of
active agent conjugated to the opposite member of the
ligand-anti-ligand pair.
[0027] The present invention provides methods for radiolabeling
biotin with technetium-99m, rhenium-186 and rhenium-188 are
disclosed. Previously, biotin derivatives were radiolabeled with
indium-ill for use in pretargeted immunoscintigraphy (for instance,
Virzi et al., Nucl. Med. Biol. 18:719-26, 1991; Kalofonos et al.,
J. Nucl. Med. 31: 1791-96, 1990; Paganelli et al., Canc. Res.
51:5960-66, 1991). However, .sup.99mTc is a particularly preferred
radionuclide for immunoscintigraphy due to (i) low cost, (ii)
convenient supply and (iii) favorable nuclear properties.
Rhenium-186 displays chelating chemistry very similar to
.sup.99mTc, and is considered to be an excellent therapeutic
radionuclide (i.e., a 3.7 day half-life and 1.07 MeV maximum
particle that is similar to .sup.131I). Therefore, the claimed
methods for technetium and rhenium radiolabeling of biotin provide
numerous advantages.
[0028] The present invention is also directed to radiolabeling with
yttrium-90, lutetium-177, sumarium-153, and other appropriate +3
metals. Y-90 is a particularly preferred beta particle emitting
radionuclide for therapy, because it exhibits favorable nuclear
properties including high specific activity, long path length with
respect to deposition of radiation in tissue, high equilibrium dose
constant and favorable half-life properties. More specifically, the
beta emission of Y-90 (Beta.sub.av=0.937 MeV) is one of the most
energetic of all beta emitters. The X.sub.90 value of Y-90 is 5.34
mm (i.e., 90% of the energy emitted from a point source is absorbed
in a sphere of 5.34 mm radius). Y-90 has a high equilibrium dose
constant or mean energy/nuclear transition, Delta=1.99
Rad-gram/microcurie-hour, and a 64 hour half-life suitable for
targeted therapy. Y-90 can be manufactured at high specific
activity and is available as a generator product. Specific
advantages of Y-90 are (1) that it has the capability to kill
neighboring target cells not directly targeted by the pretargeted
targeting moiety-ligand or targeting moiety-anti-ligand conjugate
and (2) that more radiation is deposited per microcurie localized
than for other beta emitters of lower mean particle energy
(provided that a sufficiently large target volume is
available).
[0029] Lu-177 is a particularly preferred radionuclide for targeted
nuclide therapy, since it has a moderately energetic beta emission
(Beta.sub.av=0.140 MeV); it is available in high specific activity;
its radiochemicaL production is efficient; it emits two gammas of
ideal energy and abundance for imaging (208 keV, 11% and 113 keV,
7%); and it has a relatively long half-life (161 hours). The
X.sub.90 for Lu-177 is 0.31 mm, i.e., 90% of the energy emitted
form a point source is absorbed in a sphere of radius 0.31 mm.
Lu-177 has an equilibrium dose constant or mean energy/nuclear
transition of 0.31 Rad-gram/microcuries-hour and an adequate
half-life to serve as a targeted therapeutic radionuclide. Specific
advantages of Lu-177 are (1) that its emitted energy is efficiently
absorbed in smaller targeted tumor volumes such as metastatic tumor
foci or involved lymph nodes and (2) that its long physical
half-life makes optimal use of the tumor retention property of the
pretargeting delivery method. Lu-177 has the additional advantage
of being imagable by commonly available nuclear medicine
cameras.
[0030] The "targeting moiety" of the present invention binds to a
defined target cell population, such as tumor cells. Preferred
targeting moieties useful in this regard include antibody and
antibody fragments, peptides, and hormones. Proteins corresponding
to known cell surface receptors (including low density
lipoproteins, transferrin and insulin), fibrinolytic enzymes,
anti-HER2, platelet binding proteins such as annexins, and
biological response modifiers (including interleukin, interferon,
erythropoietin and colony-stimulating factor) are also preferred
targeting moieties. Also, anti-EGF receptor antibodies, which
internalize following binding to the EGF receptor and which traffic
to the nucleus, are preferred targeting moieties for use in the
present invention to facilitate delivery of Auger emitters and
nucleus binding drugs to target cell nuclei. Oligonucleotides,
e.g., antisense oligonucleotides that are complementary to portions
of target cell nucleic acids (DNA or RNA), are also useful as
targeting moieties in the practice of the present invention.
Oligonucleotides binding to cell surfaces are also useful. Analogs
of the above-listed targeting moieties that retain the capacity to
bind to a defined target cell population may also be used within
the claimed invention. In addition, synthetic targeting moieties
may be designed.
[0031] Functional equivalents of the aforementioned molecules are
also useful as targeting moieties of the present invention. One
targeting moiety functional equivalent is a "mimetic" compound, an
organic chemical construct designed to mimic the proper
configuration and/or orientation for targeting moiety-target cell
binding. Another targeting moiety functional equivalent is a short
polypeptide designated as a "minimal" polypeptide, constructed
using computer-assisted molecular modeling and mutants having
altered binding affinity, which minimal polypeptides exhibit the
binding affinity of the targeting moiety.
[0032] Preferred targeting moieties of the present invention are
antibodies (polyclonal or monoclonal), peptides, oligonucleotides
or the like. Polyclonal antibodies useful in the practice of the
present invention are polyclonal (Vial and Callahan, Univ. Mich.
Med. Bull., 20: 284-6, 1956), affinity-purified polyclonal or
fragments thereof (Chao et al., Res. Comm. in Cher. Path. &
Pharm., 9: 749-61, 1974).
[0033] Monoclonal antibodies useful in the practice of the present
invention include whole antibody and fragments thereof. Such
monoclonal antibodies and fragments are producible in accordance
with conventional techniques, such as hybridoma synthesis,
recombinant DNA techniques and protein synthesis. Useful monoclonal
antibodies and fragments may be derived from any species (including
humans) or may be formed as chimeric proteins which employ
sequences from more than one species. See, generally, Kohler and
Milstein, Nature, 256: 495-97, 1975; Eur. J. Immunol., 6: 511-19,
1976.
[0034] Human monoclonal antibodies and "humanized" murine
antibodies are also useful as targeting moieties in accordance with
the present invention. Human monoclonal antibodies may be obtained
from human serum, from hybrid mice or other mammals having a
functional human immune system, using hybridoma technology, or the
like. Also, a murine monoclonal antibody, for example, may be
"humanized" by genetically recombining the nucleotide sequence
encoding the murine Fv region (i.e., containing the antigen binding
site which antibodies are also known as chimeric antibodies) or the
complementarity determining regions thereof with the nucleotide
sequence encoding a human constant domain region and an Fc region,
e.g., in a manner similar to that disclosed in European Patent
Application No. 0,411,893 A2. Some additional murine residues may
also be retained within the human variable region framework domains
to ensure proper target site binding characteristics. Humanized
targeting moieties are recognized to decrease the immunoreactivity
of the antibody or polypeptide in the host recipient, permitting an
increase in the half-life and a reduction in the possibility of
adverse immune reactions.
[0035] Types of active agents (diagnostic or therapeutic) useful
herein include toxins, drugs, anti-tumor agents, and radionuclides.
Several of the potent toxins useful within the present invention
consist of an A and a B chain. The A chain is the cytotoxic portion
and the B chain is the receptor-binding portion of the intact toxin
molecule (holotoxin). Because toxin B chain may mediate non-target
cell binding, it is often advantageous to conjugate only the toxin
A chain to a targeting protein. However, while elimination of the
toxin B chain decreases non-specific cytotoxicity, it also
generally leads to decreased potency of the toxin A chain-targeting
protein conjugate, as compared to the corresponding
holotoxin-targeting protein conjugate.
[0036] Preferred toxins in this regard include holotoxins, such as
abrin, ricin, modeccin, Pseudomonas exotoxin A, Diphtheria toxin,
pertussis toxin and Shiga toxin; and A chain or "A
chain-like"molecules, such as ricin A chain, abrin A chain,
modeccin A chain, the enzymatic portion of Pseudomonas exotoxin A,
Diphtheria toxin A chain, the enzymatic portion of pertussis toxin,
the enzymatic portion of Shiga toxin, gelonin, pokeweed antiviral
protein, saporin, tritin, barley toxin and snake venom peptides.
Ribosomal inactivating proteins (RIPs), naturally occurring protein
synthesis inhibitors that lack translocating and cell-binding
ability, are also suitable for use herein. Extremely highly toxic
toxins, such as palytoxin and the like, are also contemplated for
use in the practice of the present invention.
[0037] Charge modification of proteinaceous targeting moieties and
conjugates containing such targeting moieties and diagnostically or
therapeutically active agents is discussed in published European
Patent Application No. EP 329,184. Preferred charge modification in
accordance with the present invention involves treatment of a
proteinaceous active agent with a anion-forming reagent to provide
a charge-modified moiety or conjugate exhibiting an acidic shift in
isoelectric point. Preferably, the shift in isoelectric point is
one-tenth of a pH unit or greater. Generally, charge-modified
proteins exhibit a serum half-life that is at least 10% greater
than the half-life of native proteins. A 50% or greater increase in
half-life is not uncommon following charge modification to a
protein.
[0038] Anion-forming agents useful in the practice of the present
invention are structured to react with functional groups of the
protein to be charge-modified and incorporate a negatively charged
group to impart an acidic shift in the pI of the protein to be
charge-modified. Preferred anion-forming agents useful in the
practice of the present invention are structured to react with
primary amines on lysine residues of the protein to be charge
modified. Such anion-forming agents include active esters
(carboxylic and imide), maleimides and anhydrides. Preferred active
esters include N-hydroxysuccinimidyl, thiophenyl,
2,3,5,6-tetrafluorophenyl, and 2,3,5,6,-tetrafluorothiophenyl
esters. Derivatization of other protein residues may also be
employed in the practice of the present invention (e.g.,
derivatization of arginine residues with glyoxal, phenyl glyoxal or
cyclohexanedione). Negatively charged groups which may be used to
impart an acidic shift to proteinaceous active agents include
phosphates, phosphonates, sulfates, nitrates, borates, silicates,
carbonates, and carboxyl groups such as native carboxyl groups or
carboxyl groups generated from an anhydride during the reaction of
the anion-forming agent with the protein.
[0039] Useful anion-forming agents include compounds incorporating
an anhydride and/or at least one COOH group, such as succinic
anhydride, other cyclic acid anhydrides, phthalic anhydride, maleic
anhydride, N-ethyl maleimide substituted with carboxyl groups,
aliphatic anhydrides (e.g., acetic anhydride), aromatic anhydrides,
pH-reversible anhydrides (e.g., citraconic anhydride and dimethyl
maleic anhydride), alpha halo acids such as bromoacetate and
iodoacetate, and diacids or triacids substituted with a functional
group that reacts with an amino acid on a protein to be
charge-modified.
[0040] For example, succinic anhydride is dissolved in DMSO or
another dry organic solvent at a concentration of 40 mg per 200
microliters. This succinic anhydride solution (or a dilution
thereof up to 2.5 ml in anhydrous DMSC), 1.73.times.10.sup.-2M) is
added to a protein (e.g., holotoxin or toxin domain or conjugate
containing one or more of these components) solution (e.g., 3-5
mg/ml in carbonate/bicarbonate buffer, pH 8.5-9.0) at molar ratios
of succinic anhydride to protein of 1:5, 1:10 and 1:25 (with higher
molar ratios preferred). The reaction is carried out at room
temperature for 15-30 minutes. After reaction completion, succinic
acid is removed by ultrafiltration or by gel filtration. The degree
of isoelectric shift is determined by isoelectric focusing. The
toxicity of charge-modified active agents is tested in accordance
with known procedures for toxicity testing.
[0041] Preferred drugs suitable for use herein include conventional
chemotherapeutics, such as vinblastine, doxorubicin, bleomycin,
methotrexate, 5-fluorouracil, 6-thioguanine, cytarabine,
cyclophosphamide and cis-platinum, as well as other conventional
chemotherapeutics as described in Cancer: Principles and Practice
of Oncology, 2d ed., V. T. DeVita, Jr., S. Hellman, S. A.
Rosenberg, J. B. Lippincott Co., Philadelphia,, Pa., 1985, Chapter
14. A particularly preferred drug within the present invention is a
trichothecene.
[0042] Trichothecenes are drugs produced by soil fungi of the class
Fungi imperfecti or isolated from Baccharus megapotamica (Bamburg,
J. R. Proc. Molec. Subcell. Biol. 8:41-110, 1983; Jarvis &
Mazzola, Acc. Chem. Res. 15:338-395, 1982). They appear to be the
most toxic molecules that contain only carbon, hydrogen and oxygen
(Tamm, C. Fortschr. Chem. Org. Naturst. 31:61-117, 1974). They are
all reported to act at the level of the ribosome as inhibitors of
protein synthesis at the initiation, elongation, or termination
phases.
[0043] There are two broad classes of trichothecenes: those that
have only a central sesquiterpenoid structure and those that have
an additional macrocyclic ring (simple and macrocyclic
trichothecenes, respectively). The simple trichothecenes may be
subdivided into three groups (i.e., Group A, B, and C) as described
in U.S. Pat. Nos. 4,744,981 and 4,906,452 (incorporated herein by
reference). Representative examples of Group A simple
trichothecenes include: Scirpene, Roridin C, dihydrotrichothecene,
Scirpen-4, 8-diol, Verrucarol, Scirpentriol, T-2 tetraol,
pentahydroxyscirpene, 4-deacetylneosolaniol, trichodermin,
deacetylcalonectrin, calonectrin, diacetylverrucarol,
4-monoacetoxyscirpenol, 4,15-diacetoxyscirpenol,
7-hydroxydiacetoxyscirpe- nol, 8-hydroxydiacetoxy-scirpenol
(Neosolaniol), 7,8-dihydroxydiacetoxysci- rpenol,
7-hydroxy-8-acetyldiacetoxyscirpenol, 8-acetylneosolaniol, NT-1,
NT-2, HT-2, T-2, and acetyl T-2 toxin. Representative examples of
Group B simple trichothecenes include: Trichothecolone,
Trichothecin, deoxynivalenol, 3-acetyldeoxynivalenol,
5-acetyldeoxynivalenol, 3,15-diacetyldeoxynivalenol, Nivalenol,
4-acetylnivalenol (Fusarenon-X), 4,15-idacetylnivalenol,
4,7,15-triacetylnivalenol, and tetra-acetylnivalenol.
Representative examples of Group C simple trichothecenes include:
Crotocol and Crotocin. Representative macrocyclic trichothecenes
include Verrucarin A, Verrucarin B, Verrucarin J (Satratoxin C),
Roridin A, Roridin D, Roridin E (Satratoxin D), Roridin H,
Satratoxin F, Satratoxin G, Satratoxin H, Vertisporin, Mytoxin A,
Mytoxin C, Mytoxin B, Myrotoxin A, Myrotoxin B, Myrotoxin C,
Myrotoxin D, Roritoxin A, Roritoxin B, and Roritoxin D. In
addition, the general "trichothecene" sesquiterpenoid ring
structure is also present in compounds termed "baccharins" isolated
from the higher plant Bacchazris megapotamica, and these are
described in the literature, for instance as disclosed by Jarvis et
al. (Chemistry of Alleopathy, ACS Symposium Series No. 268: ed. A.
C. Thompson, 1984, pp. 149-159).
[0044] Experimental drugs, such as mercaptopurine,
N-methylformamide, 2-amino-1,3,4-thiadiazole, melphalan,
hexamethylmelamine, gallium nitrate, 3% thymidine,
dichloromethotrexate, mitoguazone, suramin, bromodeoxyuridine,
iododeoxyuridine, semustine, 1-(2-chloroethyl)-3-(2,6--
dioxo-3-piperidyl)-1-nitrosourea, N,N'-hexamethylene-bis-acetamide,
azacitidine, dibromodulcitol, Erwinia asparaginase, ifosfamide,
2-mercaptoethane sulfonate, teniposide, taxol, 3-deazauridine,
soluble Baker's antifol, homoharringtonine, cyclocytidine,
acivicin, ICRF-187, spiromustine, levamisole, chlorozotocin,
aziridinyl benzoquinone, spirogermanium, aclarubicin, pentostatin,
PALA, carboplatin, amsacrine, caracemide, iproplatin, misonidazole,
dihydro-5-azacytidine, 4'-deoxy-doxorubicin, menogaril, triciribine
phosphate, fazarabine, tiazofurin, teroxirone, ethiofos,
N-(2-hydroxyethyl)-2-nitro-1H-imidazole- -1-acetamide,
mitoxantrone, acodazole, amonafide, fludarabine phosphate,
pibenzimol, didemnin B, merbarone, dihydrolenperone,
flavone-8-acetic acid, oxantrazole, ipomeanol, trimetrexate,
deoxyspergualin, echinomycin, and dideoxycytidine (see NCI
Investigational Drugs, Pharmaceutical Data 1987, NIH Publication
No. 88-2141, Revised November 1987) are also preferred.
[0045] Radionuclides useful within the present invention include
gamma--emitters, positron-emitters, Auger electron-emitters, X-ray
emitters and fluorescence-emitters, with beta- or alpha-emitters
preferred for therapeutic use. Radionuclides are well-known in the
art and include .sup.123I, .sup.125I, .sup.130I, .sup.131I,
.sup.133I, .sup.135I, .sup.47Sc, .sup.72As, .sup.72Se, .sup.90Y,
.sup.88Y, .sup.97Ru, .sup.100Pd, .sup.101mRh, .sup.119Sb,
.sup.128Ba, .sup.197Hg, .sup.211At, .sup.212Bi, .sup.153Sm,
.sup.169Eu, .sup.212Pb, .sup.109Pd, .sup.111, In, .sup.67Ga,
.sup.68Ga, .sup.67Cu, .sup.75Br, .sup.76Br, .sup.77Br, .sup.99mTc,
.sup.11C, .sup.13N, .sup.15O and .sup.18F. Preferred therapeutic
radionuclides include .sup.188Re, .sup.186Re, .sup.203Pb,
.sup.212Pb, .sup.212Bi, .sup.109Pd, .sup.64Cu, .sup.67Cu, .sup.90Y,
.sup.125I, .sup.131I, .sup.77Br, .sup.211At, .sup.97Ru, .sup.105Rh,
.sup.198Au, .sup.177Lu and .sup.199Ag.
[0046] Other anti-tumor agents, e.g., agents active against
proliferating cells, are administrable in accordance with the
present invention. Exemplary anti-tumor agents include cytokines,
such as IL-2, tumor necrosis factor or the like, lectin
inflammatory response promoters (selecting), such as L-selectin,
E-selectin, P-selectin or the like, and like molecules.
[0047] Ligands; suitable for use within the present invention
include biotin, haptens, lectins, epitopes, dsDNA fraguents, enzyme
inhibitors and analogs and derivatives thereof. Useful
complementary anti-ligands include avidin (for biotin),
carbohydrates (for lectins), antibody, fragments or analogs
thereof, including mimetics (for haptens and epitopes), zinc finger
proteins (for dsDNA fragments) and enzymes (for enzyme inhibitors.
Preferred ligands and anti-ligands bind to each other with an
affinity of at least about k.sub.D.gtoreq.10.sup.-9 M.
[0048] The 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetra acetic
acid (DOTA)-biotin conjugate (DOTA-LC-biotin) depicted below has
been reported to have desirable in vivo biodistribution and is
cleared primarily by renal excretion. 2
[0049] DOTA may also be conjugated to other ligands or to
anti-ligands in the practice of the present invention.
[0050] Because DOTA strongly binds Y-90 and other radionuclides, it
has been proposed for use in radioimmunotherapy. For therapy, it is
very important that the radionuclide be stably bound within the
DOTA chelate and that the DOTA chelate be stably attached to a
ligand or anti-ligand. For illustrative purposes, DOTA-biotin
conjugates are described. Only radiolabeled DOTA-biotin conjugates
exhibiting those two characteristics are useful to deliver
radionuclides to the targets. Release of the radionuclide from the
DOTA chelate or cleavage of the biotin and DOTA conjugate
components in serum or at non-target sites renders the conjugate
unsuitable for use in therapy.
[0051] Serum stability of DOTA-LC-biotin (where LC refers to the
"long chain" linker, including an aminocaproyl spacer between the
biotin and the DOTA conjugate components) shown above, while
reported in the literature to be good, has proven to be
problematic. Experimentation has revealed that DOTA-LC-biotin is
rapidly cleared from the blood and excreted into the urine as
fragments, wherein the biotinamide bond rather than the DOTA-amide
bond has been cleaved, as shown below. 3
[0052] Additional experimentation employing PIP-biocytin conjugates
produced parallel results as shown below. 4
[0053] Cleavage of the benzamide was not observed as evidenced by
the absence of detectable quantities of iodobenzoic acid in the
serum.
[0054] It appears that the cleavage results from the action of
serum biotinidase. Biotinidase is a hydrolytic enzyme that
catalyzes the cleavage of biotin from biotinyl peptides. See, for
example, Evangelatos, et al., "Biotinidase Radioassay Using an
I-125-Biotin Derivative, Avidin, and Polyethylene Glycol Reagents,"
Analytical Biochemistry, 196: 385-89, 1991.
[0055] Drug-biotin conjugates which structurally resemble biotinyl
peptides are potential substrates for cleavage by plasma
biotinidase. Poor in vivo stability therefore limits the use of
drug-biotin conjugates in therapeutic applications. The use of
peptide surrogates to overcome poor stability of peptide
therapeutic agents has been an area of intense research effort.
See, for example, Spatola, Peptide Backbone Modification: A
Structure-Activity Analysis of Peptide Containing Amide Bond
Surrogates, "Chemistry and Biochemistry of Amino Acids, Peptides
and Proteins," vol. 7, Weinstein, ed., Marcel Dekker, New York,
1983; and Kim et al., "A New Peptide Bond Surrogate: 2-Isoxazoline
in Pseudodipeptide Chemistry," Tetrahedron Letters, 45: 6811-14,
1991.
[0056] Elimination of the aminocaproyl spacer of DOTA-LC-biotin
gives DOTA-SC-biotin (where the SC indicates the "short chain"
linker between the DOTA and biotin conjugate components), which
molecule is shown below: 5
[0057] DOTA-SC-biotin exhibits significantly improved serum
stability in comparison to DOTA-LC-biotin. This result does not
appear to be explainable on the basis of biotinidase activity
alone. The experimentation leading to this conclusion is summarized
in the Table set forth below.
[0058] Time Dependent Cleavage of DOTA-Biotin Conjugates
1 % Avidin Binding Time at PIP- Y-90-LC Y-90-SC 37.degree. C.
Biocytin DOTA-Biotin DOTA-Biotin 5 Minutes 75% 50% -- 15 Minutes
57% 14% -- 30 Minutes 31% 12% -- 60 Minutes -- 0% 98% 20 Hours --
0% 60% where "--" indicates that the value was not measured.
[0059] The difference in serum stability between DOTA-LC-biotin and
DOTA-SC-biotin might be explained by the fact that the SC
derivative contains an aromatic amide linkage in contrast to the
aliphatic amide linkage of the LC derivative, with the aliphatic
amide linkage being more readily recognized by enzymes as a
substrate therefor. This argument cannot apply to biotinidase,
however, because biotinidase very efficiently cleaves aromatic
amides. In fact, it is recognized that the simplest and most
commonly employed biotinidase activity measuring method uses
N-(d-biotinyl)-4-aminobenzoate (BPABA) as a substrate, with the
hydrolysis of BPABA resulting in the liberation of biotin and
4-aminobenzoate (PABA). See, for example, B. Wolf, et al., "Methods
in Enzymology," pp. 103-111, Academic Press Inc., 1990.
Consequently, one would predict that DOTA-SC-biotin, like its LC
counterpart, would be a biotinidase substrate. Since DOTA-SC-biotin
exhibits serum stability, biotinidase activity alone does not
adequately explain why some conjugates are serum stable while
others are not. A series of DOTA-biotin conjugates was therefore
synthesized by the present inventors to determine which structural
features conferred serum stability to the conjugates.
[0060] Some general strategies for improving serum stability of
peptides with respect to enzymatic action are the following:
incorporation of D-amino acids, N-methyl amino acids and
alpha-substituted amino acids.
[0061] In vivo stable biotin-DOTA conjugates are useful within the
practice of the present invention. In vivo stability imparts the
following advantages:
[0062] 1) increased tumor uptake in that more of the radioisotope
will be targeted to the previously localized targeting
moiety-streptavidin; and
[0063] 2) increased tumor retention, if biotin is more stably bound
to the radioisotope.
[0064] In addition, the linkage between DOTA and biotin may also
have a significant impact on biodistribution (including normal
organ uptake, target uptake and the like) and pharmacokinetics.
[0065] The strategy for design of the DOTA-containing molecules and
conjugates of the present invention involved three primary
considerations:
[0066] 1) in vivo stability (including biotinidase and general
peptidase activity resistance), with an initial acceptance
criterion of 100% stability for 1 hour;
[0067] 2) renal excretion; and
[0068] 3) ease of synthesis.
[0069] The DOTA-bziotin conjugates of the present invention reflect
the implementation of one or more of the following strategies:
[0070] 1) substitution of the carbon adjacent to the cleavage
susceptible amide nitrogen;
[0071] 2) alkylation of the cleavage susceptible amide
nitrogen;
[0072] 3) substitution of the amide carbonyl with an alkyl amino
group;
[0073] 4) incorporation of D-amino acids as well as analogs or
derivatives thereof; or
[0074] 5) incorporation of thiourea linkages.
[0075] DOTA-biotin conjugates in accordance with the present
invention may be generally characterized as follows: conjugates
that retain the biotin carboxy group in the structure thereof and
those that do not (i.e., the terminal carboxy group of biotin has
been reduced or otherwise chemically modified. Structures of such
conjugates represented by the following general formula have been
devised: 6
[0076] wherein L may alternatively be substituted in one of the
following ways on one of the --CH.sub.2--COOH branches of the DOTA
structure: --CH(L)--COOH or --CH.sub.2COOL or --CH.sub.2COL). When
these alternative structures are employed, the portion of the
linker bearing the functional group for binding with the DOTA
conjugate component is selected for the capability to interact with
either the carbon or the carboxy in the branch portions of the DOTA
structure, with the serum stability conferring portion of the
linker structure being selected as described below.
[0077] In the case where the linkage is formed on the core of the
DOTA structure as shown above, L is selected according to the
following principles, with the portion of the linker designed to
bind to the DOTA conjugate component selected for the capability to
bind to an amine.
[0078] A. One embodiment of the present invention includes linkers
incorporating a D-amino acid spacer between a DOTA aniline amine
and the biotin carboxy group shown above. Substituted amino acids
are preferred for these embodiments of the present invention,
because alpha-substitution also confers enzymatic cleavage
resistance. Exemplary L moieties of this embodiment of the present
invention may be represented as follows: 7
[0079] where R.sup.1 is selected from lower alkyl, lower alkyl
substituted with hydrophilic groups (preferably,
(CH.sub.2).sub.n--OH, (CH.sub.2).sub.n--OSO.sub.3,
(CH.sub.2).sub.n--SO.sub.3, 8
[0080] where n is 1 or 2), glucoronide-substituted amino acids or
other glucuronide derivaties; and
[0081] R.sup.2 is selected from hydrogen, lower alkyl, substituted
lower alkyl (e.g., hydroxy, sulfate, phosphonate or a hydrophilic
moiety (preferably OH).
[0082] For the purposes of the present disclosure, the term "lower
alkyl" indicates an alkyl group with from one to five carbon atoms.
Also, the term "substituted" includes one or several substituent
groups, with a single substituent group preferred.
[0083] Preferred L groups of this embodiment of the present
invention include the following:
[0084] R.sup.1=CH.sub.3 and R.sup.2=H (a D-alanine derivative, with
a synthetic scheme therefor shown in Example XI);
[0085] R.sup.1=CH.sub.3 and R.sup.2=CH.sub.3 (an N-methyl-D-alanine
derivative);
[0086] R.sup.1=CH.sub.2--OH and R.sup.2=H (a D-serine
derivative);
[0087] R.sup.1=CH.sub.2OSO.sub.3 and R.sup.2=H (a
D-serine-O-sulfate-deriv- ative); and 9
[0088] R.sup.2=H (a D-serine-O-phosphonate-derivative);
[0089] Other preferred moieties of this embodiment of the present
invention include molecules wherein R.sup.1 is hydrogen and
R.sup.2=--(CH.sub.2).sub.nOH or a sulfate or phosphonate derivative
thereof and n is 1 or 2 as well as molecules wherein R.sup.1 is
10
[0090] Preferred moieties incorporating the glucuronide of D-lysine
and the glucuronide of amino pimelate are shown below as I and II
respectively. 11
[0091] A particularly preferred linker of this embodiment of the
present invention is the D-alanine derivative set forth above.
[0092] B. Linkers incorporating alkyl substitution on one or more
amide nitrogen atoms are also encompassed by the present invention,
with some embodiments of such linkers preparable from L-amino
acids. Amide bonds having a substituted amine moiety are less
susceptible to enzymatic cleavage. Such linkers exhibit the
following general formula: 12
[0093] where R.sup.4 is selected from hydrogen, lower alkyl, lower
alkyl substituted with hydroxy, sulfate, phosphonate or the like
and 13
[0094] R.sub.3 is; selected from hydrogen; an amine; lower alkyl;
an amino- or a hydroxy-, sulfate- or phosphonate-substituted lower
alkyl; a glucuronide or a glucuronide-derivatized amino groups;
and
[0095] n ranges from 0-4.
[0096] Preferred linkers of this embodiment of the present
invention include:
[0097] R.sup.3=H and R.sup.4=CH.sub.3 when n=4, synthesizable as
discussed in Example XI;
[0098] R.sup.3=H and R.sup.4=CH.sub.3 when n=0, synthesizable from
N-methyl-glycine (having a trivial name of sarcosine) as described
in Example XI;
[0099] R.sup.3=NH.sub.2 and R.sup.4=CH.sub.3, n=0;
[0100] R.sup.3=H and R.sup.4= 14
[0101] when
[0102] n=4 (Bis-DOTA-LC-biotin), synthesizable from bromohexanoic
acid as discussed in Example XI; and
[0103] R.sup.3=H and R.sup.4= 15
[0104] when n=0 (bis-DOTA-SC-biotin), synthesizable from
iminodiacetic acid.
[0105] The synthesis of a conjugate including a linker wherein
R.sup.3 is H and R.sup.4 is --CH.sub.2CH.sub.2OH and n is 0 is also
described in Example XI. Schematically, the synthesis of a
conjugate of this embodiment of the present invention wherein n is
0, R.sup.3 is H and R.sup.4 is --CH.sub.2--COOH is shown below.
16
[0106] Bis-HOTA-LC-biotin, for example, offers the following
advantages:
[0107] 1) incorporation of two DOTA molecules on one biotin moiety
increases the overall hydrophilicity of the biotin conjugate and
thereby directs in vivo distribution to urinary excretion; and
[0108] 2) substitution of the amide nitrogen adjacent to the biotin
carboxyl group blocks peptide and/or biotinidase cleavage at that
site.
[0109] Bis-DOTA-LC-biotin, the glycine-based linker and the
N-methylated linker where R.sup.3=H, R.sup.4=CH.sub.3, n=4 are
particularly preferred linkers of this embodiment of the present
invention.
[0110] C. Another linker embodiment incorporates a thiourea moiety
therein. Exemplary thiourea adducts of the present invention
exhibit the following general formula: 17
[0111] where R.sup.5 is selected from hydrogen or lower alkyl;
[0112] R.sup.6 is selected from H and a hydrophilic moiety; and
[0113] n ranges from 0-4.
[0114] Preferred linkers of this embodiment of the present
invention are as follows:
[0115] R.sup.5=H and R.sup.6=H when n=5;
[0116] R.sup.5=H and R.sup.6COOH when n=5; and
[0117] R.sup.5=CH.sub.3 and R.sup.6=COOH when n=5.
[0118] The second preferred linker recited above can be prepared
using either L-lysine or D-lysine. Similarly, the third preferred
linker can be prepared using either N-methyl-D-lysine or
N-methyl-L-lysine.
[0119] Another thiourea adduct of minimized lipophilicity is 18
[0120] which may be formed via the addition of biotinhyclrazide
(commercially available from Sigma Chemical Co., St. Louis, Mo.)
and DOTA-benzyl-isothiocyanate (a known compound synthesized in one
step from DOTA-aniline), with the thiourea-containing compound
formed as shown below. 19
[0121] D. Amino acid-derived linkers of the present invention with
substitution of the carbon adjacent to the cleavage susceptible
amide have the general formula set forth below: 20
[0122] wherein Z is --(CH.sub.2).sub.2--, conveniently synthesized
form glutamic acid; or
[0123] Z=--CH.sub.2--S--CH.sub.2--, synthesizable from cysteine and
iodo-acetic acid; or
[0124] Z=--CH.sub.2--, conveniently synthesized form aspartic acid;
or
[0125] Z=--(CH.sub.2).sub.n--CO--O--CH.sub.2--, where n ranges from
1-4 and which is synthesizable from serine.
[0126] E. Another exemplary linker embodiment of the present
invention has the general formula set forth below: 21
[0127] and n ranges from 1-5.
[0128] F. Another embodiment involves disulfide-containing linkers,
which provide a metabolically cleavable moiety (--S--S--) to reduce
non-target retention of the biotin-DOTA conjugate. Exemplary
linkers of this type exhibit the following formula: 22
[0129] where n and n' preferably range between 0 and 5.
[0130] The advantage of using conditionally cleavable linkers is an
improvement in target/non-target localization of the active agent.
Conditionally cleavable linkers include enzymatically cleavable
linkers, linkers that are cleaved under acidic conditions, linkers
that are cleaved under basic conditions and the like. More
specifically, use of linkers that are cleaved by enzymes, which are
present in non-target tissues but reduced in amount or absent in
target tissue, can increase target cell retention of active agent
relative to non-target cell retention. Such conditionally cleavable
linkers are useful, for example, in delivering therapeutic
radionuclides to target cells, because such active agents do not
require internalization for efficacy, provided that the linker is
stable at the target cell surface or protected from target cell
degradation.
[0131] Cleavable linkers are also useful to effect target site
selective release of active agent at target sites. Active agents
that are preferred for cleavable linker embodiments of the present
invention are those that are substantially non-cytotoxic when
conjugated to ligand or anti-ligand. Such active agents therefore
require release from the ligand- or anti-ligand-containing
conjugate to gain full potency. For example, such active agents,
while conjugated, may be unable to bind to a cell surface receptor;
unable to internalize either actively or passively; or unable to
serve as a binding substrate for a soluble (intra- or
inter-cellular) binding protein or enzyme. Exemplary of an active
agent-containing conjugate of this type is chemotherapeutic
drug-cis-aconityl-biotin. The cis-aconityl linker is acid
sensitive. Other acid sensitive linkers useful in cleavable linker
embodiments of the present invention include esters, thioesters and
the like. Use of conjugates wherein an active acent and a ligand or
an anti-ligand are joined by a cleavable linker will result in the
selective release of the active agent at tumor cell target sites,
for example, because the inter-cellular millieu of tumor tissue is
generally of a lower pH (more highly acidic) than the
inter-cellular milieu of normal tissue.
[0132] G. Ether, thioether, ester and thioester linkers are also
useful in the practice of the present invention. Ether and
thioether linkers are stable to acid and basic conditions and are
therefore useful to deliver active agents that are potent in
conjugated form, such as radionuclides and the like. Ester and
thioesters are hydrolytically cleaved under acidic or basic
conditions or are cleavable by enzymes including esterases, and
therefore facilitate improved target:non-target retention.
Exemplary linkers of this type have the following general formula:
23
[0133] where X is O or S; and
[0134] Q is a bond, a methylene group, a --CO-- group or
--CO--(CH.sub.2).sub.n--NH--; and
[0135] n ranges from 1-5.
[0136] Other such linkers have the general formula:
[0137] --CH.sub.2--X--Q, where Q and X are defined as set forth
above.
[0138] H. Another amino-containing linker of the present invention
is structured as follows: 24
[0139] preferably methyl.
[0140] In this case, resistance to enzymatic cleavage is conferred
by the alkyl substitution on the amine.
[0141] I. Polymeric linkers are also contemplated by the present
invention. Dextran and cyclodextran are preferred polymers useful
in this embodiment of the present invention as a result of the
hydrophilicity of the polymer, which leads to favorable excretion
of conjugates containing the same. Other advantages of using
dextran polymers are that such polymers are substantially non-toxic
and non-immunogenic, that they are commercially available in a
variety of sizes and that they are easy to conjugate to other
relevant molecules. Also, dextran-linked conjugates exhibit
advantages when non-target sites are accessible to dextranase, an
enzyme capable of cleaving dextran polymers into smaller units
while non-target sites are not so accessible.
[0142] Other linkers of the present invention are produced prior to
conjugation to DOTA and following the reduction of the biotin
carboxy moiety. These linkers of the present invention have the
following general formula: 25
[0143] Embodiments of linkers of this aspect of the present
invention include the following:
[0144] J. An ether linkage as shown below may be formed in a
DOTA-biotin conjugate in accordance with the procedure indicated
below.
L'=--NH--CO--(CH.sub.2).sub.n--O--
[0145] where n ranges from 1 to 5, with 1 preferred. 26
[0146] This linker has only one amide moiety which is bound
directly to the DOTA aniline (as in the structure of
DOTA-SC-biotin). In addition, the ether linkage imparts
hydrophilicity, an important factor in facilitating renal
excretion.
[0147] K. An amine linker formed from reduced biotin (hydroxybiotin
or aminobiotin) is shown below, with conjugates containing such a
linker formed, for example, in accordance with the procedure
described in Example XI.
L'=--NH--
[0148] This linker contains no amide moieties and the unalkylated
amine may impart favorable biodistribution properties since
unalkylated DOTA-aniline displays excellent renal clearance.
[0149] L. Substituted amine linkers, which can form conjugates via
amino-biotin intermediates, are shown below. 27
[0150] where R.sup.8 is H; --(CH.sub.2).sub.2--OH or a sulfate or
phosphonate derivative thereof; 28
[0151] or the like;
[0152] and R.sup.9 is a bond or --(CH.sub.2).sub.n--CO--NH--, where
n ranges from 0-5 and is preferably 1 and where q is 0 or 1. These
moieties exhibit the advantages of an amide only directly attached
to DOTA-aniline and either a non-amide amine imparting a positive
charge to the linker in vivo or a N-alkylated glucuronide
hydrophilic group, each alternative favoring renal excretion.
[0153] M. Amino biotin may also be used as an intermediate in the
production of conjugates linked by linkers having favorable
properties, such as a thiourea-containing linker of the
formula:
L'=--NH--CS--NH--
[0154] Conjugates containing this thiourea linker have the
following advantages: no cleavable amide and a short, fairly polar
linker which favors renal excretion.
[0155] A bis-DOTA derivative of the following formula can also be
formed from amino-biotin. 29
[0156] where n ranges from 1 to 5, with 1 and 5 preferred. This
molecule offers the advantages of the previously discussed bis-DOTA
derivatives with the added advantage of no cleavable amides.
[0157] Additional linkers of the present invention which are
employed in the production of conjugates characterized by a reduced
biotin carboxy moiety are the following:
[0158] L=--(CH.sub.2).sub.4--NH--, wherein the amine group is
attached to the methylene group corresponding to the reduced biotin
carboxy moiety and the methylene chain is attached to a core carbon
in the DOTA ring. Such a linker is conveniently synthesizable from
lysine.
[0159] L=--(CH.sub.2).sub.q--CO--NH--, wherein q is 1 or 2, and
wherein the amine group is attached to the methylene group
corresponding to the reduced biotin carboxy moiety and the
methylene group(s) are attached to a core carbon in the DOTA ring.
This moiety is synthesizable from amino-biotin.
[0160] The linkers set forth above are useful to produce conjugates
having one or more of the following advantages:
[0161] bind avidin or streptavidin with the same or substantially
similar affinity as free biotin;
[0162] bind metal M.sup.+3 ions efficiently and with high kinetic
stability;
[0163] are excreted primarily through the kidneys into urine;
[0164] are stable to bodily fluid amidases;
[0165] penetrate tissue rapidly and bind to pretargeted avidin or
streptavidin; and
[0166] are excreted rapidly with a whole body residence half-life
of less than about 5 hours.
[0167] Synthetic routes to an intermediate of the DOTA-biotin
conjugates depicted above, nitrobenzyl-DOTA, have been proposed.
These proposed synthetic routes produce the intermediate compound
in suboptimal yield, however. For example, Renn and Meares, "Large
Scale Synthesis of Bifunctional Chelating Agent
Q-(p-nitrobenzyl)-1,4,7,10-tetraazacyclodode-
cane-N,N',N",N'"-tetra acetic acid, and the Determination of its
Enantiomeric Purity by Chiral Chromatography", Bioconj. Chem., 3:
563-9, 1992, describe a nine-step synthesis of nitrobenzyl-DOTA,
including reaction steps that either proceed in low yield or
involve cumbersome transformations or purifications. More
specifically, the sixth step proceeds in only 26% yield, and the
product must be purified by preparative HPLC. Additionally, step
eight proceeds in good yield, but the process involves copious
volumes of the coreactants.
[0168] These difficulties in steps 6-8 of the prior art synthesis;
are overcome in the practice of the present invention through the
use of the following synthetic alternative therefor. 30
[0169] The poor yield in step six of the prior art synthesis
procedure, in which a tetra amine alcohol is converted to a
tetra-toluenesulfonamide toluenesulfonate as shown below, is the
likely result of premature formation of the O-toluenesulfonate
functionality (before all of the amine groups have been converted
to their corresponding sulfonamides. 31
[0170] Such a sequence of events would potentially result in
unwanted intra- or inter-molecular displacement of the reactive
O-toluenesulfonate by unprotected amine groups, thereby generating
numerous undesirable side-products.
[0171] This problem is overcome in the aforementioned alternative
synthesis scheme of the present invention by reacting the
tetra-amine alcohol with trifluoroacetic anhydride.
Trifluoroacetates, being much poorer leaving groups than
toluenesulfonates, are not vulnerable to analogous side reactions.
In fact, the easy hydrolysis of trifluoroacetate groups, as
reported in Greene and Wuts, "Protecting Groups in Organic
Synthesis," John Wiley and Sons, Inc., New York, p. 94, 1991.,
suggests that addition of methanol to the reaction mixture
following consumption of all amines should afford the
tetra-fluoroacetamide alcohol as a substantially exclusive product.
Conversion of the tetra-fluoroacetamide alcohol to the
corresponding toluenesulfonate provides a material which is
expected to cyclize analogously to the tetra-toluenesulfonamide
toluenesulfonate of the prior art. The cyclic tetra-amide product
of the cyclization of the toluenesulfonate of tetra-fluoroacetamide
alcohol, in methanolic sodium hydroxide at 15-25.degree. C. for 1
hour, should afford nitro-benzyl-DOTA as a substantially exclusive
product. As a result, the use of trifluoracetamidie protecting
groups circumvents the difficulties associated with cleavage of the
very stable toluenesulfonamide protecting group, which involves
heating with a large excess of sulfuric acid followed by
neutralization with copious volumes of barium hydroxide.
[0172] Another alternative route to nitro-benzyl-DOTA is shown
below. 32
[0173] This alternative procedure involves the cyclizaton of
p-nitrophenylalanyltriglycine using a coupling agent, such as
diethylycyanophosphate, to give the cyclic tetraamide. Subsequent
borane reduction provides
2-(p-nitrobenzyl)-1,4,7,10-tetraazacyclododecane, a common
precursor used in published routes to DOTA including the Renn and
Meares article referenced above. This alternative procedure of the
present invention offers a synthetic pathway that is considerably
shorter than the prior art Renn and Meares route, requiring two
rather than four steps between p-nitrophenylalanyltriglycine to the
tetraamine. The procedure of the present invention also avoids the
use of tosyl amino protecting groups, which were prepared in low
yield and required stringent conditions for removal. Also, the
procedure of the present invention poses advantages over the route
published by Gansow et al., U.S. Pat. No. 4,923,985, because the
crucial cyclization step is intramolecular rather than
intermolecular. Intramolecular reactions typically proceed in
higher yield and do not require high dilution techniques necessary
for successful intermolecular reactions.
[0174] An additional aspect of the present invention is directed to
the use of targeting moieties that are monoclonal antibodies or
fragments thereof that localize to an antigen that is recognized by
the antibody NR-LU-10. Such monoclonal antibodies or fragments may
be murine or of other non-human mammalian origin, chimeric,
humanized or human.
[0175] NR-LU-10 is a 150 kilodalton molecular weight IgG2b
monoclonal antibody that recognizes an approximately 40 kilodalton
glycoprotein antigen expressed on most carcinomas. In vivo studies
in mice using an antibody specific for the NR-LU-10 antigen
revealed that such antibody was not rapidly internalized, which
would have prevented localization of the subsequently administered
active-agent- containing conjugate to the target site.
[0176] NR-LU-10 is a well characterized pancarcinoma antibody that
has been safely administered to over 565 patients in human clinical
trials. The hybridoma secreting NR-LU-10 was developed by fusing
mouse splenocytes immunized with intact cells of a human small cell
lung carcinoma with P3.times.63/Ag8UI murine myeloma cells. After
establishing a seed lot, the hybridoma was grown via in vitro cell
culture methods, purified and verified for purity and
sterility.
[0177] Radioimmunoassays, immunoprecipitation and
Fluorescence-Activated Cell Sorter (FACS) analysis were used to
obtain reactivity profiles of NR-LU-10. The NR-LU-10 target antigen
was present on either fixed cultured cells or in detergent extracts
of various types of cancer cells. For example, the NR-LU-10 antigen
is found in small cell lung, non-small cell lung, colon, breast,
renal, ovarian, pancreatic, and other carcinoma tissues. Tumor
reactivity of the NR-LU-10 antibody is set forth in Table A, while
NR-LU-10 reactivity with normal tissues is set forth in Table B.
The values in Table B are obtained as described below. Positive
NR-LU-10 tissue reactivity indicates NR-LU-10 antigen expression by
such tissues. The NR-LU-10 antigen has been further described by
Varki et al., "Antigens Associated with a Human Lung Adenocarcinoma
Defined by Monoclonal Antibodies," Cancer Research, 44: 681-687,
1984, and Okabe et al., "Monoclonal Antibodies to Surface Antigens
of Small Cell Carcinoma of the Lung," Cancer Research, 44:
5273-5278, 1984.
[0178] The tissue specimens were scored in accordance with three
reactivity parameters: (1) the intensity of the reaction; (2) the
uniformity of the reaction within the cell type; and (3) the
percentage of cells reactive with the antibody. These three values
are combined into a single weighted comparative value between 0 and
500, with 500 being the most intense reactivity. This comparative
value facilitates comparison of different tissues. Table B includes
a summary reactivity value, the number of tissue samples examined
and the number of samples that reacted positively with
NR-LU-10.
[0179] Methods for preparing antibodies that bind to epitopes of
the NR-LU-10 antigen are described in U.S. Pat. No. 5,084,396.
Briefly, such antibodies may be prepared by the following
procedure:
[0180] absorbing a first monoclonal antibody directed against a
first epitope of a polyvalent antigen onto an inert, insoluble
matrix capable of binding immunoglobulin, thereby forming an
immunosorbent;
[0181] combining the immunosorbent with an extract containing
polyvalent NR-LU-10 antigen, forming an insolubilized immune
complex wherein the first epitope is masked by the first monoclonal
antibody;
2TABLE A TUMOR REACTIVITY OF ANTIBODY Organ/Cell #Pos/
Intensity.sup.a Percent.sup.b Uniformity.sup.c Type Tumor Exam Avg.
Range Avg. Range Avg. Range Pancreas 6/9 3 3 100 100 2.3 2-3
Carcinoma Prosate 9/9 2.8 2-3 95 80-100 2 1-3 Carcinoma Lung 8/8 3
3 100 100 2.2 1--3 Adeno- carcinoma Lung Small 2/2 3 3 100 100 2 2
Cell Carcinoma Lung 8/8 2.3 2-3 73 5-100 1.8 1-3 Squamous Cell
Carcinoma Renal 8/9 2.2 2-3 83 75-100 1 1 Carcinoma Breast 23/23
2.9 2-3 97 75-100 2.8 1-3 Adeno- carcinoma Colon 12/12 2.9 2-3 98
98-100 2.9 2-3 Carcinoma Malignant 0/2 0 0 0 0 0 0 Melanoma Ocular
Malignant 0/11 0 0 0 0 0 0 Melanoma Ovarian 35/25 2.9 2-3 200 100
2.2 1-3 Carcinoma Undifferent- 1/1 2 2 90 90 2 2 iated Carcinoma
Osteosarcoma 1/1 2 2 20 20 1 1 Synovial 0/1 0 0 0 0 0 0 Sarcoma
Lymphoma 0/2 0 0 0 0 0 0 Liposacroma 0/1 0 0 0 0 0 0 Utenne 0/1 0 0
0 0 0 0 Leiomyo- sacroma .sup.aRated from 0-3, with 3 representIng
highest intensity .sup.bPercentage of cells stained within the
examined tissue section. .sup.cRates from 0-3, with 3 representing
highest uniformity
[0182]
3TABLE B Organ/Cell Type # Pos/Exam Summary Reactivity Adencid
Epithelium 3/3 433 Lymphoid Foilicie-Central 0/3 1 Lymphoid
Foilicie-Peripheral 0/3 0 Mucus Gland 2/2 400 Adipose Tissue Fat
Cells 0/3 0 Adrenal Zona Fasciculata Cortex 0/3 0 Zona Glomerulosa
Cortex 0/3 0 Zona Reticulans Cortex 0/3 0 Medulla 0/3 0 Aorta
Endotheilum 0/3 0 Elastic Interna 0/3 0 Tunica Adventitia 0/3 0
Tunica Media 0/3 0 Brain-Cerebellum Axons. Myelinated 0/3 0
Microglia 0/3 0 Neurons 0/3 0 Purkenje's Cells 0/3 0 Brain-Cerebrum
Axons. Myelinated 0/3 0 Microglia 0/3 0 Neurons 0/3 0
Brain-Midbrain Axons, Myelinated 0/3 0 Microglia 0/3 0 Neurons 0/3
0 Colon Mucosal Epithelium 3/3 500 Musculans Externa 0/3 0
Musculans Mucosa 0/3 0 Nerve Ganglia 0/3 0 Serosa 0/1 0 Duodenum
Mucosal Epithelium 3/3 500 Musculans Mucosa 0/3 0 Epididymis
Epithelium 3/3 419 Smooth Muscle 0/3 0 Spermatozoa 0/1 0 Esophagus
Epithelium 3/3 86 Mucosal Gland 2/2 450 Smooth Muscle 0/3 0 Gall
Bladder Mucosal Epithelium 0/3 467 Smooth Muscle 0/3 0 Heart
Myocardium 0/3 0 Serosa 0/1 0 Iluem Lymph Node 0/2 0 Muccsal
Epithelium 0/2 0 Musculans Externa 0/1 0 Musculans Mucosa 0/2 0
Nerve Ganglia 0/1 0 Serosa 0/1 0 Jejunum Lymph Node 0/1 0 Muccsal
Epithelium 2/2 400 Musculans Externa 0/2 0 Musculans Mucosa 0/2 0
Nerve Ganglia 0/2 0 Serosa 0/1 0 Kidney Collecting Tubules 2/3 160
Distal Convoluted Tubules 3/3 500 Glomerular Epithelium 0/3 0
Mesangial 0/3 0 Proximal Convoluteo Tubules 3/3 500 Liver Bile Duct
3/3 500 Central Lobular Hepatccyte 1/3 1 Periportal Hepatocyte 1/3
40 Kupffer Cells 0/3 0 Lung Alveolar Macrcphage 0/3 500 Bronchial
Epithtelium 0/2 0 Bronchial Smooth Muscle 0/2 0 Pneumocyte Type 3/3
354 Pneumocyte Type II 3/3 387 Lymph Node Lymohoid Follicle-Central
0/3 0 Lymphoid Follicle-Peripheral 0/3 0 Mammary Gland Aveolar
Epithelium 3/3 500 Duct Epithelium 3/3 500 Myceoithelium 0/3 0
Muscle Skeletat Muscle Fiber 0/3 0 Nerve Axon. Myelinated 0/2 0
Endoneurium 0/2 0 Neurclemma 0/2 0 Neuron 0/2 0 Perineurium 0/2 0
Ovary Corpus Luteum 0/3 0 Epithelium 1/1 270 Granulosa 1/3 400
Serosa 0/3 0 Theca 0/3 0 Oviduct Eprthelium 1/1 500 Smooth Muscle
0/3 0 Pancreas Acinar Cell 3/3 500 Duct Epithelium 3/3 500 Islet
Call 3/3 500 Peritoneum Mesothelium 0/1 0 Pituitary Adenohypophysis
2/2 500 Neurchypophysis 0/2 0 Placenta Trophoblasts 0/3 0 Prostate
Concerions 0/3 0 Glandular Epitheilum 3/3 400 Smooth Muscle 0/3 0
Rectum Lymph Node 0/2 0 Mucosal Epithelium 0/2 0 Muscularis Externa
0/1 0 Muscularis Mucosa 0/3 0 Nerve Ganglia 0/3 0 Salivary Gland
Acinar Epithelium 3/3 500 Duct Epithelium 3/3 500 Skin Apocrine
Glands 3/3 280 Basal Layer 3/3 33 Epithelium 1/3 10 Follicle 1/1
190 Stratum Corneum 0/3 0 Spinal Cord Axons. Myelinated 0/2 0
Microglial 0/2 0 Neurons 0/2 0 Spleen Lymphoid Follicle-Central 0/3
0 Lymphoid Follicle-Peripheral 0/3 0 Trabecular Smooth Muscle 0/3 0
Stomach Chief Cells 3/3 290 Mucosal Epithelium 3/3 367 Muscularis
Mucosa/Externa 0/3 0 Parietal Cells 3/3 290 Smooth Muscle 0/3 0
Stromal Tissue Adipose 0/63 0 Artericlar Smooth Muscle 0/120 0
Endothelium 0/120 0 Fibrous Connective Tissue 0/120 0 Macrophages
0/117 0 Mast Cells/Eosinphilis 0/86 0 Testis Interstitial Cells 0/3
0 Sertoli Cells 3/3 93 Thymus Hassal's Epithelium 3/3 147 Hassal's
Keratin 3/3 333 Lymphoid Cortex 0/3 0 Lymphoid Medulla 3/3 167
Thyroid C-cells 0/3 0 Colloid 0/3 0 Foilicularr Epithelium 3/3 500
Tonsil Epithelium 1/3 500 Lymphoid Follicle-Central 0/3 0 Lymphoid
Follicle-Peripheral 0/3 0 Mucus Gland 1/1 300 Striated Muscle 0/3 0
Umbilical cord Epithelium 0/3 0 Urinary Bladder Mucosal Epithelium
3/3 433 Serosa 0/1 0 Smooth Muscle 0/3 0 Uterus Endometnal
Epithelium 3/3 500 Endometnal Glands 3/3 500 Smooth Muscle 0/3 0
Vagina/Cervix Epitherial Glands 1/1 500 Smooth Muscle 0/2 0
Squamous Epithelium 1/1 200
[0183] immunizing an animal with the insolubilized immune
complex;
[0184] fusing spleen cells from the immunized animal to myeloma
cells to form a hybridoma capable of producing a second monoclonal
antibody directed against a second epitope of the polyvalent
antigen;
[0185] culturing the hybridoma to produce the second monoclonal
antibody; and
[0186] collecting the second monoclonal antibody as a product of
the hybridoma.
[0187] Consequently, monoclonal antibodies NR-LU-01, NR-LU-02 and
NR-LU-03, prepared in accordance with the procedures described in
the aforementioned patent, are exemplary targeting moieties useful
in this aspect of the present invention.
[0188] Additional antibodies reactive with the NR-LU-10 antigen may
also be prepared by standard hybridoma production and screening
techniques. Any hybridoma clones so produced and identified may be
further screened as described above to verify antigen and tissue
reactivity.
[0189] The invention is further described through presentation of
the following examples. These examples are offered by way of
illustration, and not by way of limitation.
EXAMPLE I
Synthesis of a Chelate-Biotin conjugate
[0190] A chelating compound that contains an N.sub.3S chelating
core was attached via an amide linkage to biotin. Radiometal
labeling of an exemplary chelate-biotin conjugate is illustrated
below. 33
[0191] The spacer group "X" permits the biotin portion of the
conjugate to be sterically available for avidin binding. When
"R.sup.1" is a carboxylic acid substituent (for instance,
CH.sub.2COOH), the conjugate exhibits improved water solubility,
and further directs in vivo excretion of the radiolabeled biotin
conjugate toward renal rather than hepatobiliary clearance.
[0192] BriefLy, N-.alpha.-Cbz-N-.SIGMA.-t-BOC protected lysine was
converted to the succinimidyl ester with NHS and DCC, and then
condensed with aspartic acid .beta.-t-butyl ester. The resultant
dipeptide was activated with NHS and DCC, and then condensed with
glycine t-butyl ester. The Cbz group was removed by hydrogenolysis,
and the amine was acylated using tetrahydropyranyl mercaptoacetic
acid succinimidyl ester, yielding
S-(tetrahydropyranyl)-mercaptoacetyl-lysine. Trifluorcacetic acid
cleavage of the N-t-BOC group and t-butyl esters, followed by
condensation with LC-biotin-NHS ester provided
(.SIGMA.-caproylamide biotin)-aspartyl glycine. This synthetic
method is illustrated below. 34
EXAMPLE II
Preparation of a Technetium or Rhenium Radiolabeled Chelate-Biotin
Conjugate
[0193] The chelate-biotin conjugate of Example I was radiolabeled
with either .sup.99mTc pertechnetate or .sup.186Re perrhenate.
Briefly, .sup.99mTc pertechnetate was reduced with stannous
chloride in the presence of sodium gluconate to form an
intermediate Tc-gluconate complex. The chelate-biotin conjugate of
Example I was added and heated to 100.degree. C. for 10 min at a pH
of about 1.8 to about 3.3. The solution was neutralized to a pH of
about 6 to about 8, and yielded an N.sub.3S-coordinated
.sup.99mTc-chelate-biotin conjugate. C-18 HPLC gradient elution
using 5-60% acetonitrile in 1% acetic acid demonstrated two anomers
at 97% or greater radiochemical yield using .delta. detection.
[0194] Alternatively, .sup.186Re perrhenate was spiked with cold
ammonium perrhenate, reduced with stannous chloride, and complexed
with citrate. The chelate-biotin conjugate of Example I was added
and heated to 90.degree. C. for 30 min at a pH of about 2 to 3. The
solution was neutralized to a pH of about 6 to about 8, and yielded
an N.sub.3S-coordinated .sup.186Re-chelate-biotin conjugate. C-18
HPLC gradient elution using 5-60% acetonitrile in 1% acetic acid
resulted in radiochemical yields of 85-90%. Subsequent purification
over a C-18 reverse phase hydrophobic column yielded material of
99% purity.
EXAMPLE III
In Vitro Analysis of Radiolabeled Chelate-Biotin Conjugates
[0195] Both the .sup.99mTc- and .sup.186Re-chelate-biotin
conjugates were evaluated in vitro. When combined with excess
avidin (about 100-fold molar excess), 100% of both radiolabeled
biotin conjugates complexed with avidin.
[0196] A .sup.99mTc-biotin conjugate was subjected to various
chemical challenge conditions. Briefly, .sup.99mTc-chelate-biotin
conjugates were combined with avidin and passed over a 5 cm size
exclusion gel filtration column. The radiolabeled biotin-avidin
complexes were subjected to various chemical challenges (see Table
1), and the incubation mixtures were centrifuged through a size
exclusion filter. The percent of radioactivity retained (indicating
avidin-biotin-associated radiolabel) is presented in Table 1. Thus,
upon chemical challenge, the radiometal remained associated with
the macromolecular complex.
4TABLE 1 Chemical Challenge of .sup.99mTc-Chelate- Biotin-Avidin
Complexes Challenge % Radioactivity Retained Medium pH 1 h,
37.degree. C. 18 h, RT PBS 7.2 99 99 Phosphate 8.0 97 97 10 mM
cysteine 8.0 92 95 10 mM DTPA 8.0 99 98 0.2 M carbonate 10.0 97
94
[0197] In addition, each radiolabeled biotin conjugate was
incubated at about 50 .mu.g/ml with serum; upon completion of the
incubation, the samples were subjected to instant thin layer
chromatography (ITLC) in 80% methanol. Only 2-4% of the
radioactivity remained at the origin (i.e., associated with
protein); this percentage was unaffected by the addition of
exogenous biotin. When the samples were analyzed using size
exclusion H-12 FPLC with 0.2 M phosphate as mobile phase, no
association of radioactivity with serum macromolecules was
observed.
[0198] Each radiolabeled biotin conjugate was further examined
using a competitive biotin binding assay. Briefly, solutions
containing varying ratios of D-biotin to radiolabeled biotin
conjugate were combined with limiting avidin at a constant total
biotin:avidin radio. Avidin binding of each radiolabeled biotin
conjugate was determined by ITLC, and was compared to the
theoretical maximum stoichiometric binding (as determined by the
HABA spectrophotometric assay of Green, Biochem. J. 94:23c-24c,
1965). No significant differences in avidin binding was observed
between each radiolabeled biotin conjugate and D-biotin.
EXAMPLE IV
In vivo Analysis of Radiolabeled Chelate-Biotin Conjugates
Administered After Antibody Pretargeting
[0199] The .sup.186Re-chelate-biotin conjugate of Example I was
studied in an animal model of a three-step antibody pretargeting
protocol. Generally, this protocol involved: (i) prelocalization of
biotinylated monoclonal antibody; (ii) administration of avidin for
formation of a "sandwich" at the target site and for clearance of
residual circulating biotinylated antibody; and (iii)
administration of the 186Re-biotin conjugate for target site
localization and rapid blood clearance.
[0200] A. Preparation and Characterization of Idiotinylated
Antibody
[0201] Biotinylated NR-LU-10 was prepared according to either of
the following procedures. The first procedure involved
derivatization of antibody via lysine .epsilon.-amino groups.
NR-LU-10 was radioiodinated at tyrosines using chloramine T and
either .sup.1251 or .sup.131I sodium iodide. The radioiodinated
antibody (5-10 mg/ml) was then biotinylated using biotinamido
caproate NHS ester in carbonate buffer, pH 8.5, containing 5% DMSO,
according to the scheme below. 35
[0202] The impact of lysine biotinylation on antibody
immunoreactivity was examined. As the molar offering of
biotin:antibody increased from 5:1 to 40:1, biotin incorporation
increased as expected (measured using the HABA assay and
pronase-digested product) (Table 2, below). Percent of biotinylated
antibody immunoreactivity as compared to native antibody was
assessed in a limiting antigen ELISA assay. The immunoreactivity
percentage dropped below 70% at a measured derivitization of
11.1:1; however, at this level of derivitization, no decrease in
antigen-positive cell binding (performed with LS-180 tumor cells at
antigen excess). Subsequent experiments used antibody derivitized
at a biotin:antibody ratio of 10:1.
5TABLE 2 Effect of Lysine Biotinylation on Immunoreactivity Molar
Measured Offering Derivitization Immunoassessment (%) (Biotins/Ab)
(Biotins/Ab) ELISA Cell Binding 5:1 3.4 86 10:1 8.5 73 100 13:1
11.1 69 102 20:1 13.4 36 106 40:1 23.1 27
[0203] Alternatively, NR-LU-10 was biotinylated using thiol groups
generated by reduction of cystines. Derivitization of thiol groups
was hypothesized to be less compromising to antibody
immunoreactivity. NR-LU-10 was radioiodinated using p-aryltin
phenylate NHS ester (PIP-NHS) and either .sup.125I or .sup.131I
sodium iodide. Radioiodinated NR-LU-10 was incubated with 25 mM
dithiothreitol and purified using size exclusion chromatography.
The reduced antibody (containing free thiol groups) was then
reacted with a 10- to 100-fold molar excess of
N-iodoacetyl-n'-biotinyl hexylene diamine in phosphate-buffered
saline (PBS), pH 7.5, containing 5% DMSO (v/v).
6TABLE 3 Effect of Thiol Biotinylation on Immunoreactivity Molar
Measured Offering Derivitization Immunoassessment (%) (Biotins/Ab)
(Biotins/Ab) ELISA Cell Binding 10:1 4.7 114 50:1 6.5 102 100 100:1
6.1 95 100
[0204] As shown in Table 3, at a 50:1 or greater biotin:antibody
molar offering, only 6 biotins per antibody were incorporated. No
significant impact on immunoreactivity was observed.
[0205] The lysine- and thiol-derivitized biotinylated antibodies
("antibody (lysine)" and "antibody (thiol)", respectively) were
compared. Molecular sizing on size exclusion FPLC demonstrated that
both biotinylation protocols yielded monomolecular IgGs.
Biotinylated antibody (lysine) had an apparent molecular weight of
160 kD, while biotinylated antibody (thiol) had an apparent
molecular weight of 180 kD. Reduction of endogenous sulfhydryls
(i.e., disulfides) to thiol groups, followed by conjugation with
biotin, may produce a somewhat unfolded macromolecule. If so, the
antibody (thiol) may display a larger hydrodynamic radius and
exhibit an apparent increase in molecular weight by chromatographic
analysis. Both biotinylated antibody species exhibited 98% specific
binding to immobilized avidin-agarose.
[0206] Further comparison of the biotinylated antibody species was
performed using non-reducing SDS-PAGE, using a 4% stacking gel and
a 5% resolving gel. Biotinylated samples were either radiolabeled
or unlabeled and were combined with either radiolabeled or
unlabeled avidin or streptavidin. Samples were not boiled prior to
SDS-PAGE analysis. The native antibody and biotinylated antibody
(lysine) showed similar migrations; the biotinylated antibody
(thiol) produced two species in the 50-75 kD range. These species
may represent two thiol-capped species. Under these SDS-PAGE
conditions, radiolabeled streptavidin migrates as a 60 kD tetramer.
When 400 .mu.g/ml radiolabeled streptavidin was combined with 50
.mu.g/ml biotinylated antibody (analogous to "sandwiching"
conditions in vivo), both antibody species formed large molecular
weight complexes. However, only the biotinylated antibody
(thiol)-streptavidin complex moved from the stacking gel into the
resolving gel, indicating a decreased molecular weight as compared
to the biotinylated antibody (lysine)-streptavidin complex.
[0207] B. Blood Clearance of Biotinylated Antibody Species
[0208] Radioiodinated biotinylated NR-LU-10 (lysine or thiol) was
intravenously administered to non-tumored nude mice at a dose of
100 .mu.g. At 24 h post-administration of radioiodinated
biotinylated NR-LU-10, mice were intravenously injected with either
saline or 400 .mu.g of avidin. With saline administration, blood
clearances for both biotinylated antibody species were biphasic and
similar to the clearance of native NR-LU-10 antibody.
[0209] In the animals that received avidin intravenously at 24 h,
the biotinylated antibody (lysine) was cleared (to a level of 5% of
injected dose) within 15 min of avidin administration
(avidin:biotin=10:1). With the biotinylated antibody (thiol),
avidin administration (10:1 or 25:1) reduced the circulating
antibody level to about 35% of injected dose after two hours.
Residual radiolabeled antibody activity in the circulation after
avidin administration was examined in vitro using immobilized
biotin. This analysis revealed that 85% of the biotinylated
antibody was complexed with avidin. These data suggest that the
biotinylated antibody (thiol)-avidin complexes that were formed
were insufficiently crosslinked to be cleared by the RES.
[0210] Blood clearance and biodistribution studies of biotinylated
antibody (lysine) 2 h post-avidin or post-saline administration
were performed. Avidin administration significantly reduced the
level of biotinylated antibody in the blood (see FIG. 1), and
increased the level of biotinylated antibody in the liver and
spleen. Kidney levels of biotinylated antibody were similar.
EXAMPLE V
In Vivo Characterization of .sup.186Re-Chelate-Biotin Conjugates In
a Three-Step Pretargeting Protocol
[0211] A .sup.186Re-chelate-biotin conjugate of Example I
(MW.apprxeq.1000; specific activity=1-2 mCi/mg) was examined in a
three-step pretargeting protocol in an animal model. More
specifically, 18-22 g female nude mice were implanted
subcutaneously with LS-180 human colon tumor xenografts, yielding
100-200 mg tumors within 10 days of implantation.
[0212] NR-LU-10 antibody (MW.apprxeq.150 kD) was radiolabeled with
.sup.125I/Chloramine T and biotinylated via lysine residues (as
described in Example VI.A, above). Avidin (MW.apprxeq.66 kD) was
radiolabeled with .sup.131I/PIP-NHS (as described for
radioiodination of NR-LU-10 in Example IV.A., above). The
experimental protocol was as follows:
7 Group 1 Time 0, inject 100 .mu.g .sup.125I-labeled, biotinylated
NR-LU-10 Time 24 h, inject 400 .mu.g hu 131I-labeled avidin Time 26
h, inject 60 .mu.g .sup.186Re-chelate- biotin conjugate Group 2
Time 0, inject 400 .mu.g .sup.131I-labeled avidin (control) Time 2
h, inject 60 .mu.g .sup.186Re-chelate biotin conjugate Group 3 Time
0, inject 60 .mu.g .sup.186Re-chelate- (control) biotin
conjugate
[0213] The three radiolabels employed in this protocol are capable
of detection in the presence of each other. It is also noteworthy
that the sizes of the three elements involved are logarithmically
different--antibody.about.150,000; avidin.about.66,000; and
biotin.about.1,000. Bicodistribution analyses were performed at 2,
6, 24, 72 and 120 h after administration of the
.sup.186Re-chelate-biotin conjugate.
[0214] Certain preliminary studies were performed in the animal
model prior to analyzing the .sup.186Re-chelate-biotin conjugate in
a three-step pretargeting protocol. First, the effect of
biotinylated antibody on blood clearance of avidin was examined.
These experiments showed that the rate and extent of avidin
clearance was similar in the presence or absence of biotinylated
antibody. Second, the effect of biotinylated antibody and avidin on
blood clearance of the .sup.186Re-chelate-biotin conjugate was
examined; blood clearance was similar in the presence or absence of
biotinylated antibody and avidin.
[0215] Third, tumor uptake of biotinylated antibody administered at
time 0 or of avidin administered at time 24 h was examined. At 25
h, about 350 pmol/g biotinylated antibody was present at the tumor;
at 32 h the level was about 300 pmol/g; at 48 h, about 200 pmol/g;
and at 120 h, about 100 pmol/g. Avidin uptake at the same time
points was about 250, 150, 50 and 0 pmol/g, respectively. From the
same experiment, tumor to blood ratios were determined for
biotinylated antibody and for avidin. From 32 h to 120 h, the
ratios of tumor to blood were very similar.
[0216] The three-step pretargeting protocol (described for Group 1,
above) was then examined. More specifically, tumor uptake of the
.sup.186Re-chelate-biotin conjugate in the presence or absence of
biotinylated antibody and avidin was determined. In the absence of
biotinylated antibody and avidin, the .sup.186Re-chelate-biotin
conjugate displayed a slight peak 2 h post-injection, which was
substantially cleared from the tumor by about 5 h. In contrast, at
2 h post-injection in the presence of biotinylated antibody and
avidin (specific), the .sup.186Re-chelate-biotin conjugate reached
a peak in tumor approximately 7 times greater than that observed in
the absence of biotinylated antibody and avidin. Further, the
specifically bound .sup.186Re-chelate-biotin conjugate was retained
at the tumor at significant levels for more than 50 h. Tumor to
blood ratios determined in the same experiment increased
significantly over time (i.e., T:B=.apprxeq.8 at 30 h;.apprxeq.15
at 100 h;.apprxeq.35 at 140 h).
[0217] Tumor uptake of the .sup.186Re-chelate-biotin conjugate has
further been shown to be dependent on the dose of biotinylated
antibody administered. At 0 .mu.g of biotinylated antibody, about
200 pmol/g of .sup.186Re-chelate-biotin conjugate was present at
the tumor at 2 h after administration; at 50 .mu.g antibody, about
500 pmol/g of .sup.186Re-chelate-biotin conjugate; and at 100 .mu.g
antibody, about 1,300 pmol/g of .sup.186Re-chelate-biotin
conjugate.
[0218] Rhenium tumor uptake via the three-step pretargeting
protocol was compared to tumor uptake of the same antibody
radiolabeled through chelate covalently attached to the antibody
(conventional procedure) The results of this comparison are
depicted in FIG. 2. Blood clearance and tumor uptake were compared
for the chelate directly labeled rhenium antibody conjugate and for
the three-step pretargeted sandwich. Areas under the curves (AUC)
and the ratio of AUC.sub.tumor/AUC.sub.blood were determined. For
the chelate directly labeled rhenium antibody conjugate, the ratio
of AUC.sub.tumor/AUC.sub.blood=24055/10235; for the three-step
pretargeted sandwich, the ratio of
AUC.sub.tumor/AUC.sub.blood=46764/6555- .
EXAMPLE VI
Preparation of Chelate-Biotin Conjugates Having Improved
Biodistribution Properties
[0219] The biodistribution of .sup.111In-labeled-biotin derivatives
varies greatly with structural changes in the chelate and the
conjugating group. Similar structural changes may affect the
biodistribution of technetium- and rhenium-biotin conjugates.
Accordingly, methods for preparing technetium- and rhenium-biotin
conjugates having optimal clearance from normal tissue are
advantageous.
[0220] A. Neutral MAMA Chelate/Conjugate
[0221] A neutral MAMA chelate-biotin conjugate is prepared
according to the following scheme. 36
[0222] The resultant chelate-biotin conjugate shows superior kidney
excretion. Although the net overall charge of the conjugate is
neutral, the polycarboxylate nature of the molecule generates
regions of hydrophilicity and hydrophobicity. By altering the
number and nature of the carboxylate groups within the conjugate,
excretion may be shifted from kidney to gastrointestinal routes.
For instance, neutral compounds are cleared by the kidneys; anionic
compounds are cleared through the GI system.
[0223] B. Polvlysine Derivitization
[0224] Conjugates containing polylysine may also exhibit beneficial
biodistribution properties. With whole antibodies, derivitization
with polylysine may skew the biodistribution of conjugate toward
liver uptake. In contrast, derivitization of Fab fragments with
polylysine results in low levels of both liver and kidney uptake;
blood clearance of these conjugates is similar to that of Fab
covalently linked to chelate. An exemplary polylysine derivitized
chelate-biotin conjugate is illustrated below. 37
[0225] Inclusion of polylysine in radiometal-chelate-biotin
conjugates is therefore useful for minimizing or eliminating RES
sequestration while maintaining good liver and kidney clearance of
the conjugate. Polylysine derivatives offer the further advantages
of: (1) increasing the specific activity of the
radiometal-chelate-biotin conjugate; (2) permitting control of rate
and route of blood clearance by varying the molecular weight of the
polylysine polymer; and (3) increasing the circulation half-life of
the conjugate for optimal tumor interaction.
[0226] Polylysine derivitization is accomplished by standard
methodologies. Briefly, poly-L-lysine is acylated according to
standard amino group acylation procedures (aqueous bicarbonate
buffer, pH 8, added biotin-NHS ester, followed by chelate NHS
ester). Alternative methodology involves anhydrous conditions using
nitrophenyl esters in DMSO and triethyl amine. The resultant
conjugates are characterized by UV and NMR specta.
[0227] The nmmber of biotins attached to polylysine is determined
by the HABA assay. Spectrophotometric titration is used to assess
the extent of amino group derivitization. The
radiometal-chelate-biotin conjugate is characterized by size
exclusion.
[0228] C. Cleavable Linkaae
[0229] Through insertion of a cleavable linker between the chelate
and biotin portion of a radiometal-chelate-biotin conjugate,
retention of the conjugate at the tumor relative to normal tissue
may be enhanced. More specifically, linkers that are cleaved by
enzymes present in normal tissue but deficient or absent in tumor
tissue can increase tumor retention. As an example, the kidney has
high levels of .gamma.-glutamyl transferase; other normal tissues
exhibit in vivo cleavage of .gamma.-glutamyl prodrugs. In contrast,
tumors are generally deficient in enzyme peptidases. The
glutamyl-linked biotin conjugate depicted below is cleaved in
normal tissue and retained in the tumor. 38
[0230] D. Serine Linker With O-Polar Substituent Sugar substitution
of N.sub.3S chelates renders such chelates water soluble.
Sulfonates, which are fully ionized at physiological pH, improve
water solubility of the chelate-biotin conjugate depicted below.
39
[0231] This compound is synthesized according to the standard
reaction procedures. Briefly, biocytin is condensed with
N-t-BOC-(O-sulfonate or O-glucose) serine NHS ester to give
N-t-BOC-(O-sulfonate or O-glucose) serine biocytinamide. Subsequent
cleavage of the N-t-BOC group with TFA and condensation with ligand
NHS ester in DMF with triethylamine provides
ligand-amidoserine(O-sulfonate or O-glucose)biocytinamide.
EXAMPLE VII
Preparation and Characterization of PIP-Radioiodinated Biotin
[0232] Radioiodinated biotin derivatives prepared by exposure of
poly-L-lysine to excess NHS-LC-biotin and then to Bolton-Hunter
N-hydroxysuccinimide esters in DMSO has been reported. After
purification, this product was radiolabeled by the iodogen method
(see, for instance, Del Rosario et al., J. Nucl. Med. 32:5, 1991,
993 (abstr.)). Because of the high molecular weight of the
resultant radioiodinated biotin derivative, only limited
characterization of product (i.e., radi.o-HPLC and binding to
immobilized streptavidin) was possible.
[0233] Preparation of radioiodonated biotin according to the
present: invention provides certain advantages. First, the
radioiodobiotin derivative is a low molecular weight compound that
is amenable to complete chemical characterization. Second, the
disclosed methods for preparation involve a single step and
eliminate the need for a purification step.
[0234] Briefly, iodobenzamide derivatives corresponding to biocytin
(R.dbd.COOH) and biotinamidopentylamine (R.dbd.H) were prepared
according to the following scheme. In this scheme, "X" may be any
radiohalogen, including .sup.125I, .sup.131I, .sup.123I, .sup.211At
and the like. 40
[0235] Preparation of 1 was generally according to Wilbur et al.,
J. Nucl. Med. 30:216-26, 1989, using a tributyltin intermediate.
Water soluble carbodiimide was used in the above-depicted reaction,
since the NHS ester 1 formed intractable mixtures with DCU. The NHS
ester was not compatible with chromatography; it was insoluble in
organic and aqueous solvents and did not react with biocytin in DMF
or in buffered aqueous acetonitrile. The reaction between 1 and
biocytin or 5-(biotinamido) pentylamine was sensitive to base. When
the reaction of 1 and biocytin or the pentylamine was performed in
the presence of triethylamine in hot DMSO, formation of more than
one biotinylated product resulted. In contrast, the reaction was
extremely clean and complete when a suspension of 1 and biocytin (4
mg/ml) or the pentylamine (4 mg/ml) was heated in DMSO at
117.degree. C. for about 5 to about 10 min. The resultant
.sup.125I-biotin derivatives were obtained in 94% radiochemical
yield. Optionally, the radioiodinated products may be purified
using C-18 HPLC and a reverse phase hydrophobic column.
Hereinafter, the resultant radioiodinated products 2 are referred
to as PIP-biocytin (R.dbd.COOH) and PIP-pentylamine (R.dbd.H).
[0236] Both iodobiotin derivatives 2 exhibited .gtoreq.95% binding
to immobilized avidin. Incubation of the products 2 with mouse
serum resulted in no loss of the ability of 2 to bind to
immobilized avidin. Biodistribution studies of 2 in male BALB/c
mice showed rapid clearance from the blood (similar to
.sup.186Re-chelate-biotin conjugates described above). The
radioiodbiotin 2 had decreased hepatobiliary excretion as compared
to the .sup.186Re-chelate-biotin conjugate; urinary excretion was
increased as compared to the .sup.186Re-chelate-biotin conjugate.
Analysis of urinary metabolites of 2 indicated deiodination and
cleavage of the biotin amide bond; the metabolites showed no
binding to immobilized avidin. In contrast, metabolites of the
.sup.186Re-chelate-biotin conjugate appear to be excreted in urine
as intact biotin conjugates. Intestinal uptake of 2 is <50% that
of the .sup.186Re-chelate-biotin conjugate. These biodistribution
properties of 2 provided enhanced whole body clearance of
radioisotope and indicate the advantageous use of 2 within
pretargeting protocols.
[0237] .sup.131I-PIP-biocytin was evaluated in a two-step
pretargeting procedure in tumor-bearing mice. Briefly, female nude
mice were injected subcutaneously with LS-180 tumor cells; after 7
d, the mice displayed 50-100 mg tumor xenografts. At t=0, the mice
were injected with 200 .mu.g of NR-LU-10-avidin conjugate labeled
with .sup.125I using PIP-NHS (see Example IV.A.). At t=36 h, the
mice received 42 .mu.g of .sup.131I-PIP-biocytin. The data showed
immediate, specific tumor localization, corresponding
to.apprxeq.1.5 .sup.131I-PIP-biocytin molecules per avidin
molecule.
[0238] The described radiohalogenated biotin compounds are amenable
to the same types of modifications described in Example VI above
for .sup.186Re-chelate-biotin conjugates In particular, the
following PIP-polylysine-biotin molecule is made by trace labeling
polylysine with .sup.125I-PIP, followed by extensive biotinylation
of the polylysine. 41
[0239] Assessment: of .sup.125I binding to immobilized avidin
ensures that all radioiodinated species also contain at least an
equivalent of biotin.
EXAMPLE VIII
Preparation of Biotinylated Antibody (Thiol) Through Endogenous
Antibody Sulfhydryl Groups or Sulfhydryl-Generating Compounds
[0240] Certain antibodies have available for reaction endogenous
sulfhydryl groups. If the antibody to be biotinylated contains
endogenous sulfhydryl groups, such antibody is reacted with
N-iodoacetyl-n'-biotinyl hexylene diamine (as described in Example
IV.A., above). The availability of one or more endogenous
sulfhydryl groups obviates the need to expose the antibody to a
reducing agent, such as DTT, which can have other detrimental
effects on the biotinylated antibody.
[0241] Alternatively, one or more sulfhydryl groups are attached to
a targeting moiety through the use of chemical compounds or linkers
that contain a terminal sulfhydryl group. An exemplary compound for
this purpose is iminothiolane. As with endogenous sulfhydryl groups
(discussed above), the detrimental effects of reducing agents on
antibody are thereby avoided.
EXAMPLE IX
Two-Step Pretargeting Methodology That Does Not Induce
Internalization
[0242] A NR-LU-13-avidin conjugate is prepared as follows.
Initially, avidin is derivatized with N-succinimidyl
4-(N-maleimidomethyl)cyclohexan- e-1-carboxylate (SMCC).
SMCC-derived avidin is then incubated with NR-LU-13 in a 1:1 molar
ratio at pH 8.5 for 16 h. Unreacted NR-LU-13 and SMCC-derived
avidin are removed from the mixture using preparative size
exclusion HPLC. Two conjugates are obtained as products--the
desired 1:1 NR-LU-13-avidin conjugate as the major product; and an
incompletely characterized component as the minor product.
[0243] A .sup.99mTc-chelate-biotin conjugate is prepared as in
Example I:, above. The NR-LU-13-avidin conjugate is administered to
a recipient and allowed to clear from the circulation. One of
ordinary skill in the art of radioimmunoscintigraphy is readily
able to determine the optimal time for NR-LU-13-avidin conjugate
tumor localization and clearance from the circulation. At such
time, the .sup.99mTc-chelate-biotin conjugate is administered to
the recipient. Because the .sup.99mTc-chelate-biotin conjugate has
a molecular weight of 1,000, crosslinking of NR-LU-13-avidin
molecules on the surface of the tumor cells is dramatically reduced
or eliminated. As a result, the .sup.99mTc diagnostic agent is
retained at the tumor cell surface for an extended period of time.
Accordingly, detection of the diagnostic agent by imaging
techniques is optimized; further, a lower dose of radioisotope
provides an image comparable to that resulting from the typical
three-step pretargeting protocol.
[0244] Optionally, clearance of NR-LU-13-avidin from the
circulation may be accelerated by plasmapheresis in combination
with a biotin affinity column. Through use of such column,
circulating NR-LU-13-avidin will be retained extracaporeally, and
the recipient's immune system exposure to a large, proteinaceous
immunogen (i.e., avidin) is minimized.
[0245] An alternative procedure for clearing NR-LU-13-avidin from
the circulation without induction of internalization involves
administration of biotinylated, high molecular weight molecules,
such as liposomes, IgM and other molecules that are size excluded
from ready permeability to tumor sites. When such biotinylated,
high molecular weight molecules aggregate with NR-LU-13-avidin, the
aggregated complexes are readily cleared from the circulation via
the RES.
EXAMPLE X
Enhancement of Therapeutic Agent Internalization Through Avidin
Crosslinking
[0246] The ability of multivalent avidin to crosslink two or more
biotin molecules (or chelate-biotin conjugates) is advantageously
used to improve delivery of therapeutic agents. More specifically,
avidin crosslinking induces internalization of crosslinked
complexes eat the target cell surface.
[0247] Biotinylated NR-CO-04 (lysine) is prepared according to the
methods described in Example IV.A., above. Doxorubicin-avidin
conjugates are prepared by standard conjugation chemistry. The
biotinylated NR-CO-04 is administered to a recipient and allowed to
clear from the circulation. One of ordinary skill in the art of
radioimmunotherapy is readily able to determine the optimal time
for biotinylated NR-CO-04 tumor localization and clearance from the
circulation. At such time, the doxorubicin-avidin conjugate is
administered to the recipient. The avidin portion of the
doxorubicin-avidin conjugate crosslinks the biotinylatead NR-CO-04
on the cell surface, inducing internalization of the complex. Thus,
doxorubicin is more efficiently delivered to the target cell.
[0248] In a first alternative protocol, a standard three-step
pretargeting methodology is used to enhance intracellular delivery
of a drug to a tumor target cell. By analogy to the description
above, biotinylated NR-LU-05 is administered, followed by avidin
(for blood clearance and to form the middle layer of the sandwich
at the target cell-bound biotinylated antibody). Shortly
thereafter, and prior to internalization of the biotinylated
NR-LU-05-avidin complex, a methotrexate-biotin conjugate is
administered.
[0249] In a second alternative protocol, biotinylated NR-LU-05 is
further covalently linked to methotrexate. Subsequent
administration of avidin induces internalization of the complex and
enhances intracellular delivery of drug to the tumor target
cell.
[0250] In a third alternative protocol, NR-CO-04-avidin is
administered to a recipient and allowed to clear from the
circulation and localize at the target site. Thereafter, a
polybiotinylated species (such as biotinylat(Bd poly-L-lysine, as
in Example IV.B., above) is administered. In this protocol, the
drug to be delivered may be covalently attached to either the
antibody-avidin component or to the polybiotinylated species. The
polybiotinylated species induces internalization of the (drug)
-antibody-avidin-polybiotin-(drug) complex.
EXAMPLE XI
Synthesis of DOTA-Biotin Conjugates
[0251] A. Synthesis of Nitro-Benzyl-DOTA.
[0252] The synthesis of aminobenzyl-DOTA was conducted
substantially in accordance with the procedure of McMurry et al.,
Bioconjugate Chem., 3: 108-117, 1992. The critical step in the
prior art synthesis is the intermolecular cyclization between
disuccinimidyl N-(tert-butoxycarbonyl) iminodiacetate and
N-(2-aminoethyl)-4-nitrophenyl alaninamide to prepare
1-(tert-butoxycarbonyl) -5-(4-nitrobenzyl)
-3,6,11-trioxo-1,4,7,10-tetraa- zacyclododecane. In other words,
the critical step is the intermolecular cyclization between the
bis-NHS ester and the diamine to give the cyclized dodecane.
McMurry et al. conducted the cyclization step on a 140 mmol scale,
dissolving each of the reagents in 100 ml DMF and adding via a
syringe pump over 48 hours to a reaction pot containing 4 liters
dioxane.
[0253] A 5x scale-up of the McMurry et al. procedure was not
practical in terms of reaction volume, addition rate and reaction
time. Process chemistry studies revealed that the reaction addition
rate could be substantially increased and that the solvent volume
could be greatly reduced, while still obtaining a similar yield of
the desired cyclization product. Consequently on a 30 mmol scale,
each of the reagents was dissolved in 500 ml DMF and added via
addition funnel over 27 hours to a reaction pot containing 3 liters
dioxane. The addition rate of the method employed involved a 5.18
mmol/hour addition rate and a 0.047 M reaction concentration.
[0254] B. Synthesis of a D-alanine-linked conjugate with a
preserved biotin carboxy moiety. A reaction scheme to form a
compound of the following formula is discussed below. 42
[0255] The D-alanine-linked conjugate was prepared by first
coupling D-alanine (Sigma Chemical Co.) to biotin-NHS ester. The
resultant biotinyl-D-alanine was then activated with
1-(3-dimethylaminopropyl)-3-et- hyl-carbodiimide hydrochloride
(EDCI) and N-hydroxysuccinimide (NHS). This NHS ester was reacted
in situ with DOTA-aniline to give the desired product which was
purified by preparative HPLC.
[0256] More specifically, a mixture of D-alanine (78 mg, 0.88 mmol,
1.2 equivalents), biotin-NHS ester (250 mg, 0.73 mmol, 1.0
equivalent), triethylamine (0.30 ml, 2.19 mmol, 3.0 equivalents) in
DMF (4 ml) was heated at 110.degree. C. for 30 minutes. The
solution was cooled to 23.degree. C. and evaporated. The product
solid was acidified with glacial acetic acid and evaporated again.
The product biotinyl-D-alanine, a white solid, was suspended in 40
ml of water to remove excess unreacted D-alanine, and collected by
filtration. Biotinyl-D-alanine was obtained as a white solid (130
mg, 0.41 mmol) in 47% yield.
[0257] NHS (10 mg, 0.08 mmol) and EDCI (15 mg, 0.07 mmol) were
added to a solution of biotinyl-D-alanine (27 mg, 0.08 mmol) in DMF
(1 ml). The solution was stirred at 23.degree. C. for 63 hours, at
which time TLC analysis indicated conversion of the carboxyl group
to the N- hydroxy succinimidyl ester. Pyridine (0.8 ml) was added
followed by DOTA-aniline (20 mg, 0.04 mmol). The mixture was heated
momentarily at approximately 100.degree. C., then cooled to
23.degree. C. and evaporated. The product,
DOTA-aniline-D-alanyl-biotinamide was purified by preparative
HPLC.
[0258] C. Synthesis of N-hydroxyethyl-linked conjugate.
[0259] Iminodiacetic acid dimethyl ester is condensed with
biotin-NHS-ester to give biotinyl dimethyl iminodiacetate.
Hydrolysis with one equivalent of sodium hydroxide provides the
monomethyl ester after purification from under and over hydrolysis
products. Reduction of the carboxyl group with borane provides the
hydroxyethyl amide. The hydroxyl group is protected with
t-butyl-dimethyl-silylchloride. The methyl ester is hydrolysed,
activated with EDCI and condensed with DOTA-aniline to form the
final product conjugate.
[0260] D. Synthesis of N-Me-LC-DOTA-biotin. A reaction scheme is
shown below. 43
[0261] Esterification of 6-Aminocaproic acid (Sigma Chemical Co.)
was carried out with methanolic HCl. Trifluoroacetylation of the
amino group using trifluoroacetic anhydride gave
N-6-(methylcaproyl)-trifluoroacetami- de. The amide nitrogen was
methylated using sodium hydride and iodomethane in tetrahydrofuran.
The trifluoroacetyl protecting group was cleaved in acidic methanol
to give methyl 6-methylamino-caproate hydrochloride. The amine was
condensed with biotin-NHS ester to give methyl
N-methyl-caproylamido-biotin. Saponification afforded the
corresponding acid which was activated with EDCI and NHS and, in
situ, condensed with DOTA-aniline to give
DOTA-benzylamido-N-methyl-caproylamido-biotin.
[0262] 1. Preparation of methyl 6-aminocaproate hydrochloride.
Hydrogen chloride (gas) was added to a solution of 20.0 g (152
mmol) of 6-aminocaproic acid in 250 ml of methanol via rapid
bubbling for 2-3 minutes. The mixture was stirred at 15-25.degree.
C. for 3 hours and then concentrated to afford 27.5 g of the
product as a white solid (99%):
[0263] H-NMR (DMSO) 9.35 (1 H, broad t), 3.57 (3H, s),
[0264] 3.14 (:2H, quartet), 2.28 (2H, t), 1.48 (4H,
[0265] multiplet), and 1.23 ppm (2H, multiplet).
[0266] 2. Preparation of N-6-(methylcaproyl)-trifluoroacetamide. To
a solution of 20.0 g (110 mmol) of methyl 6-aminocaproate
hydrochloride in 250 ml of dichloromethane was added 31.0 ml (22.2
mmol) of triethylamine. The mixture was cooled in an ice bath and
trifluoroacetic anhydride (18.0 ml, 127 mmol) was added over a
period of 15-20 minutes. The mixture was stirred at 0-10.degree. C.
for 1 hour and concentrated. The residue was diluted with 300 ml of
ethyl acetate and saturated aqueous sodium bicarbonate (3.times.100
ml). The organic phase was dried over anhydrous magnesium sulfate,
filtered and concentrated to afford 26.5 g of the product as a pale
yellow oil (100%):
[0267] H-NMR (DMSO) 3.57 (3H, s), 3.37 (2H, t), 3.08 (1.9H,
quartet, N-CH.sub.3), 2.93 (1.1H, s, N-CH.sub.3),
[0268] 2.30 (2H, t), 1.52 (4H, multiplet), and 1.23 ppm (2H,
multiplet).
[0269] 3. Preparation of methyl 6-N-methylamino-caproate
hydrochloride. To a solution of 7.01 g (29.2 mmol) of
N-6-(methylcaproyl)-trifluoroacetamid- e in 125 ml of anhydrous
tetrahydrofuran was slowly added 1.75 g of 60% sodium hydride (43.8
mmol) in mineral oil. The mixture was stirred at 15-25.degree. C.
for 30 minutes and then 6.2 g (43.7 mmol) of iodomethane was added.
The mixture was stirred at 15-25.degree. C. for 17 hours and then
filtered through celite. The solids were rinsed with 50 ml of
tetrahydrofuran. The filtrates were combined and concentrated. The
residue was diluted with 150 ml of ethyl acetate and washed first
with 5% aqueous sodium sulfite (2.times.100 ml) and then with 100
ml of 1 N aqueous hydrochloric acid. The organic phase was dried
over anhydrous magnesium sulfate, filtered and concentrated to
afford a yellow oily residue. The residue was diluted with 250 ml
of methanol and then hydrogen chloride (gas) was rapidly bubbled
into the mixture for 2-3 minutes. The resultant mixture was
refluxed for 18 hours, cooled and concentrated. The residue was
diluted with 150 ml of methanol and washed with hexane (3.times.150
ml) to remove mineral oil previously introduced with NaH. The
methanol phase was concentrated to afford 4.91 g of the product as
a yellow oil (86%):
[0270] H-NMR (DMSO) 8.80 (2H, broad s), 3.58 (3H, s),
[0271] 2.81 (2H, multiplet), 2.48 (3H, s), 2.30 (2H,
[0272] t), 1.52 (4H, multiplet), and 1.29 ppm (2H, multiplet).
[0273] 4. Preparation of methyl 6-(N-methylcaproylamido-biotin.
N-hydroxysuccinimidyl biotin (398 mg, 1.16 mmol) was added to a
solution of methyl 6-(N-methyl) aminocaproate hydrochloride (250
mg, 1.28 mmol) in DMF (4.0 ml) and triethylamine (0.18 ml, 1.28
mmol). The mixture was; heated in an oil bath at 100.degree. C. for
10 minutes. The solution was evaporated, acidified with glacial
acetic acid and evaporated again. The residue was chromatographed
on a 25 mm flash chromatography column manufactured by Ace Glass
packed with 50 g silica (EM Science, Gibbstown, N.J., particle size
0.40-0.63 mm) eluting with 15% MeOH/EtOAc. The product was obtained
as a yellow oil (390 mg) in 79% yield.
[0274] 5. Preparation of 6-(N-methyl-N-biotinyl) amino caproic
acid. To a solution of methyl 6-(N-methyl-caproylamido-biotin (391
mg, 1.10 mmol) in methanol (2.5 ml) was added a 0.95 N NaOH
solution (1.5 ml). This solution was stirred at 23.degree. C. for 3
hours. The solution was neutralized by the addition of 1.0 M HCl
(1.6 ml) and evaporated. The residue was dissolved in water,
further acidified with 1.0 M HCl (0.4 ml) and evaporated. The gummy
solid residue was suspended in water and agitated with a spatula
until it changed into a white powder. The powder was collected by
filtration with a yield of 340 mg.
[0275] 6. Preparation of
DOTA-benzylamido-N-methyl-caproylamido-biotin. A suspension of
6-(N-methyl-N-biotinyl)amino caproic acid (29 mg, 0.08 mmol) and
N-hydroxysuccinimide (10 mg, 0.09 mmol) in DMF (0.8 ml) was heated
over a heat gun for the short time necessary for the solids to
dissolve. To this heated solution was added EDCI (15 mg, 0.08
mmol). The resultant solution was stirred at 23.degree. C. for 20
hours. To this stirred solution were added aminobenzyl-DOTA (20 mg,
0.04 mmol) and pyridine (0.8 ml). The mixture was heated, over a
heat gun for 1 minute. The product was isolated by preparative
HPLC, yielding 3 mg.
[0276] E. Synthesis of a bis-DOTA conjugate with a preserved biotin
carboxy group. A reaction scheme is shown below. 44
[0277] 1. Preparation of methyl 6-bromocaproate (methyl
6-bromohe:Kanoate). Hydrogen chloride (gas) was added to a solution
of 5.01 g (25.7 mmol) of 6-bromocaproic acid in 250 ml of methanol
via vigorous bubbling for 2-3 minutes. The mixture was stirred at
15-25.degree. C. for 3 hours and then concentrated to afford 4.84 g
of the product as a yellow oil (90%):
[0278] H-NMR (DMSO) 3.58 (3H, s) , 3.51 (2H, t) , 2.29
[0279] (2H, t) , 1.78 (2H, pentet) , and 1.62-1.27 ppm (4H, m).
[0280] 2. Preparation of N,N-bis-(methyl 6-hexanoyl)-amine
hydrochloride. To a solution of 4.01 g (16.7 mmol) of N-(methyl
6-hexanoyl)-trifluoroace- tamide (prepared in accordance with
section D.2. herein) in 125 ml of anhydrous tetrahydrofuran was
added 1.0 g (25 mmol) of 60% sodium hydride in mineral oil. The
mixture was stirred at 15-25.degree. C. for 1 hour and then 3.50 g
(1.6.7 mmol) of methyl 6-bromocaproate was added and the mixture
heated to reflux. The mixture was stirred at reflux for 22 hours.
NMR assay of an aliquot indicated the reaction to be incomplete.
Consequently, an additional 1.00 g (4.8 mmol) of methyl
6-bromocaproate was added and the mixture stirred at reflux for 26
hours. NMR assay of an aliquot indicated the reaction to be
incomplete. An additional 1.0 g of methyl 6-bromocaproate was added
and the mixture stirred at reflux for 24 hours. NMR assay of an
aliquot indicated the reaction to be near complete. The mixture was
cooled and then directly filtered through celite. The solids were
rinsed with 100 ml of tetrahydrofuran. The filtrates were combined
and concentrated. The residue was diluted with 100 ml of methanol
and washed with hexane (3.times.100 ml) to remove the mineral oil
introduced with the sodium hydride. The methanol phase was treated
with 6 ml of 10 N aqueous sodium hydroxide and stirred at
15-25.degree. C. for 3 hours. The mixture was concentrated. The
residue was diluted with 100 ml of deionized water and acidified to
pH 2 with concentrated HCl. The mixture was washed with ether
(3.times.100 ml). The aqueous phase was concentrated, diluted with
200 ml of dry methanol and then hydrogen chloride gas was bubbled
through the mixture for 2-3 minutes. The mixture was stirred at
15-25.degree. C. for 3 hours and then concentrated. The residue was
diluted with 50 ml of dry methanol and filtered to remove inorganic
salts. The filtrate was concentrated to afford 1.98 g of the
product as a white solid (38%):
[0281] H-NMR (DMSO) 8.62 (2H, m) 3.58 (6H, s), 2.82
[0282] (4H, m) 2.30 (4H, t), 1.67-1.45 (8H, m) and
[0283] 1.38-1.22 ppm (4H, m).
[0284] 3. Preparation of N,N'-bis-(methyl 6-hexanoyl)-biotinamide.
To a solution of 500 mg (1.46 mmol) of N-hydroxysuccinimidyl biotin
in 15 ml of dry dimethyl-formamide was added 600 mg (1.94 mmol) of
N,N-bis-(methyl 6-hexanoyl)amine hydrochloride followed by 1.0 ml
of triethylamine. The mixture was stirred at 80-85.degree. C. for 3
hours and then cooled and concentrated.
[0285] The residue was chromatographed on silica gel, eluting with
20% methanol/ethyl acetate, to afford 620 mg of the product as a
near colorless oil (85%):
[0286] H-NMR (CDCl.sub.3) 5.71 (1H, s), 5.22 (1H, s), 4.52
[0287] (1H, m), 4.33 (1H, m), 3.60 (3H, s), 3.58 (3H,
[0288] s), 3.34-3.13 (5H, m), 2.92 (1H, dd), 2.75 (1H,
[0289] d), 2.33 (6H, m) and 1.82-1.22 ppm (18H, m);
[0290] TLC-R.sub.f 0.39 (20:80 methanol/ethyl acetate).
[0291] 4. Preparation of N,N-bis-(6-hexanoyl)-biotinamide. To a
solution of 610 mg (0.819 mmol) of N,N-bis-(methyl
6-hexanoyl)-biotinamide in 35 ml of methanol was added 5.0 ml of 1N
aqueous sodium hydroxide. The mixture was stirred at 15-25.degree.
C. for 4.5 hours and then concentrated. The residue was diluted
with 50 ml of deionized water acidified to pH 2 with 1N aqueous;
hydrochloric acid at 4.degree. C. The product, which precipitated
out as a white solid, was isolated by vacuum filtration and dried
under vacuum to afford 482 mg (84%):
[0292] H-NMR (DMSO) 6.42 (1H, s), 6.33 (1H, s), 4.29
[0293] (1H, m), 4.12 (1H, m), 3.29-3.04 (5H, m), 2.82
[0294] (1H, dd), 2.57 (1H, d), 2.21 (6H, m) and 1.70-1.10 ppm (18H,
m).
[0295] 5. Preparation of N',N'-bis-(N-hydroxy-succinimiclyl
6-hexanoyl)-biotinamide. To a solution of 220 mg (0.467 mmol) of
N,N-bis-(6-hexanoyl)-biotinamicle in 3 ml of dry dimethylformamide
was added 160 mg (1.39 mmol) of N-hydroxysuccinimide followed by
210 mg (1.02 mmol) of dicyclohexyl-carbodiimide. The mixture was
stirred at 15-25.degree. C. for 17 hours and then concentrated. The
residue was chromatographed on silica gel, eluting with 0.1:20:80
acetic acid/methanol/ethyl acetate, to afford 148 mg of the product
as a foamy off-white solid (48%):
[0296] H-NMR (DMSO) 6.39 (1H, s), 6.32 (1H, s), 4,29
[0297] (1H, m), 4,12 (1H, m), 3.30-3.03 (5H, m), 2.81 (9H, dd and
s), 2.67 (4H, m), 2.57 (1H, d),
[0298] 2.25 (2H, t), 1.75-1.20 (18H, m); TLC-RF 0.37 (0.1:20:80
acetic acid/methanol/ethyl acetate).
[0299] 6. Preparation of
N,N-bis-(6-hexanoylamidobenzyl-DOTA)-biotinamide. To a mixture of
15 mg of DOTA-benzylamine and 6.0 mg of
N',N'-bis-(N-hydroxy-succinimidyl 6-hexanoyl)-biotinamide in 1.0 ml
of dry dimethylformamide was added 0.5 ml of dry pyridine. The
mixture was stirred at 45-50.degree. C. for 4.5 hours and at
15-25.degree. C. for 12 hours. The mixture was concentrated and the
residue chromatographed on a 2.1.times.2.5 cm octadecylsilyl (ODS)
reverse-phase preparative HPLC column eluting with a--20 minute
gradient profile of 0.1:95:5 to 0.1:40:60 trifluoroacetic
acid:water:acetonitrile at 13 ml/minute to afford the desired
product. The retention time was 15.97 minutes using the
aforementioned gradient at a flow rate of 1.0 ml/minute on a 4.6
mm.times.25 cm ODS analytical HPLC column.
[0300] F. Synthesis of an N-methyl-glycine linked conjugate. A
reaction scheme for this synthesis is shown below. 45
[0301] The N-methyl glycine-linked DOTA-biotin conjugate was
prepared by an analogous method to that used to prepare
D-alanine-linked DOTA-biotin conjugates. N-methyl-glycine (trivial
name sarcosine, available from Sigma Chemical Co.) was condensed
with biotin-NHS ester in DMF and triethylamine to obtain N-methyl
glycyl-biotin. N-methyl-glycyl biotin was then activated with EDCI
and NHS. The resultant NHS ester was not isolated and was condensed
in situ with DOTA-aniline and excess pyridine. The reaction
solution was heated at 60.degree. C. for 10 minutes and then
evaporated. The residue was purified by preparative HPLC to give
[(N-methyl-N-biotinyl)-N-glycyl]-aminobenzyl-DOTA.
[0302] 1. Preparation of (N-methyl)glycyl biotin. DMF (8.0 ml) and
triethylamine (0.61 ml, 4.35 mmol) were added to solids N-methyl
glycine (182 mg, 2.05 mmol) and N-hydroxy-succinimidyl biotin (500
mg, 1.46 mmol). The mixture was heated for 1 hour in an oil bath at
85.degree. C. during which time the solids dissolved producing a
clear and colorless solution. The solvents were then evaporated.
The yellow oil residue was acidified with glacial acetic acid,
evaporated and chromatographed on a 27 mm column packed with 50 g
silica, eluting with 30% MeOH/EtOAc 1% HOAc to give the product as
a white solid (383 mg) in 66% yield.
[0303] H-NMR (DMSO): 1.18-1.25 (m, 6H, (CH.sub.2).sub.3), 2.15,
[0304] 2.35 (2 t's, 2H, CH.sub.2CO), 2.75 (m, 2H, SCH.sub.2),
[0305] 2.80, 3.00 (2 s's, 3H, NCH.sub.3), 3.05-3.15 (m, 1H,
[0306] SCH), 3.95, 4.05 (2 s's, 2H, CH.sub.2N), 4.15, 4.32
[0307] (2 m's, 2H, 2CHN's), 6.35 (s, NH), 6.45 (s, NH).
[0308] 2. Preparation of [(N-methyl-N-biotinyl)glycyl]
aminobenzyl-DOTA. N-hydroxysuccinimide (10 mg, 0.08 mmol) and EDCI
(15 mg, 6.08 mmol) were added to a solution of (N-methylglycyl
biotin (24 mg, 0.08 mmol) in DMF (1.0 ml). The solution was stirred
at 23 .degree. C. for 64 hours. Pyridine (0.8 ml) and
aminobenzyl-DOTA (20 mg, 0.04 mmol) were added. The mixture was
heated in an oil bath at 63.degree. C. for 10 minutes, then stirred
at 23.degree. C. for 4 hours. The solution was evaporated. The
residue was purified by preparative HPLC to give the product as an
off white solid (8 mg, 0.01 mmol) in 27% yield.
[0309] H-NMR (D.sub.2O): 1.30-1.80 (m, 6H), 2.40, 2.55 (2 t's, 2H,
CH.sub.2CO), 2.70-4.2 (complex multiplet), 4.35 (m, CHN), 4.55 (m,
CHN), 7.30 (m, 2H, benzene hydrogens), 7.40 (m, 2H, benzene
hydrogens).
[0310] G. Synthesis of a short chain amine-linked conjugate with a
reduced biotin carboxy group. A two-part reaction scheme is shown
below. 46
[0311] The biotin carboxyl group is reduced with diborane in THF to
give a primary alcohol. Tosylation of the alcohol with tosyl
chloride in pyridine affords the primary tosylate. Aminobenzyl DOTA
is acylated with trifluoroactetic anhydride in pyridine to give
(N-trifluoroacetyl)aminobe- nzyl-DOTA. Deprotonation with 5.0
equivalents of sodium hydride followed by displacement of the
biotin tosylate provides the
(N-trifluoracetamido-N-descarboxylbiotinyl)aminobenzyl-DOTA. Acidic
cleavage of the N-trifluoroacetamide group with HCl(g) in methanol
provides the amine-linked DOTA-biotin conjugate.
EXAMPLE XII
Human Clinical Trial: Three-Step Pretargeting
[0312] Patients were selected on the basis of a variety of
criteria. The three-step pretargeting protocol to which such
patients were subjected proceeded as follows:
[0313] Step 1--Patients received 10 mg whole NR-LU-10-LC-biotin
conjugate prepared in accordance with the procedure described in
Example IV. In some cases, the conjugate was radiolabeled with
Tc-99m in accordance with the procedure referenced above for
radiolabeling NR-LU-10 Fab to facilitate monitoring of the
conjugate in vivo. The NR-LU-10-LC-biotin conjugate, either
radiolabeled or non-radiolabeled, was diluted in 30 ML of normal
saline and administered by intravenous injection over 3-5 minutes.
The conjugate was administered within 4 hours after completion of
the radiolabeling procedure and exhibited 20-25 mCi activity when
administered.
[0314] Step 2--Avidin was administered intravenously 24-36 hours
after administration of the NR-LU-10-LC-biotin conjugate. The
avidin administration was conducted in two stages: 3-5 mg in 5 mL
of physiological solution as a rapid bolus dose and 40-80 mg in 100
mL of physiological solution 30 minutes later. Some patients
received radiolabeled avidin (10-15 mCi activity at the time of
administration) to facilitate in vivo monitoring of this component
of the three-step, pretargeting system. Avidin was also
radiolabeled substantially in accordance with the procedure
referenced above for NR-LU-10 Fab radiolabeling.
[0315] Step 3--Diethylenetriaminepentacetic acid-alpha,
W-bis(biocytinamide) (DTPA-bis-biotin, available from Sigma
Chemical Company, St. Louis, Missouri) was radiolabeled with In-111
as set forth below. DTPA-biotin was diluted in PBS, pH 7.4, to a
concentration of 2 micrograms/microliter. The solution was
sterilized by 0.22 mm millipore filtration. .sup.111InCl.sub.3 was
diluted in citrate buffer (0.02M; pH 6.5) to 740 kBq/pl. The two
reagents were mixed and allowed to react at room temperature for 10
minutes. Generally, a 98% chelation of In-111 to DTPA-biotin was
achieved, as verified by paper chromatography. Administration of
2-5 mg of In-111-DTPA-biotin (5-10 mCi activity) diluted in 5 mL of
saline was conducted intravenously, over 1 minute, 24 hours after
avidin administration. The patients were evaluated for 24 hours
following administration of In-111N-methyl-glycine (having a
trivial name of sarcosine) as described in Example XI;
DTPA-biotin.
[0316] A 66 year old male presented with a large primary lesion in
the ascending colon and a small lesion in the transverse colon
(polyp). This patient was subjected to a three-step pretargeting
protocol as follows:
[0317] t=0; 10 mg monoclonal antibody-biotin
[0318] t=25 hour; 10 mg avidin;
[0319] t=25.3 hour; 90 mg avidin; and
[0320] t=24 hour; 6 mCi In-111-DTPA-biotin.
[0321] Images were taken and analyzed by the attending physician.
The large lesion was visualized in a 2 hour SPECT image.
EXAMPLE XIII
Three-Step Pretargeting using Y-90
[0322] A patient presents with ovarian cancer. A monoclonal
antibody (MAb) directed to an ovarian cancer cell antigen, e.g.,
NR-LU-10, is conjugated to biotin to form a MAb-biotin conjugate.
The MAb-biotin conjugate is administered to the patient in an
amount sufficient to substantially saturate the available antigenic
sites at the target (which amount is at least sufficient to allow
the capture of a therapeutically effective radiation dose at the
target and which amount may be in excess of the maximum tolerated
dose of conjugate administrable in a targeted, chelate-labeled
molecule protocol, such as administration of monoclonal
antibody-chelate-radionuclide conjugate). The MAb-biotin so
administered is permitted to localize to target cancer cells for
24-48 hours. Next, an amount of avidin sufficient to clear
non-targeted MAb-biotin conjugate and to bind to the targeted
biotin is administered.
[0323] A biotin-radionuclide chelate conjugate of the type
discussed in Example XI(F) above is radiolabeled with Y-90 as set
forth below. Carrier free .sup.90YCl.sub.3 (available from
NEN-DuPont, Wilmington, Del.) at 20-200 .mu.l in 0.05 N HCl was
diluted with ammonium acetate buffer (0.5M, pH 5) to a total volume
of 0.4 ml. 50 .mu.l (500 mg/ml) of ascorbic acid and 50-100 .mu.l
(10 mg/ml) of DOTA-biotin were added to the buffered
.sup.90YCl.sub.3 solution. The mixture was incubated for one hour
at 80.degree. C. Upon completion of the incubation, 55 .mu.l of 100
mM DTPA was added to the mixture to chelate any unbound .sup.90Y.
The final preparation was diluted to 10 ml with 0.9% NaCl.
[0324] The radiolabeled DOTA-biotin conjugate is administered to
the patient in a therapeutically effective dose. The
biotin-radionuclide chelate conjugate localizes to the targeted
MAb-biotin-avidin moiety or is substantially removed from the
patient via the renal pathway.
[0325] Kits containing one or more of the components described
above are also contemplated. For instance, radiohalogenated biotin
may be provided in a sterile container for use in pretargeting
procedures. A chelate-biotin conjugate provided in a sterile
container is suitable for radiometallation by the consumer; such
kits would be particularly amenable for use in pretargeting
protocols. Alternatively, radiohalogenated biotin and a
chelate-biotin conjugate may be vialed in a non-sterile condition
for use as a research reagent.
[0326] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
* * * * *