Note: Descriptions are shown in the official language in which they were submitted.
wo 93/09816 2 ~ ~ 3 5 ~ 8 Pcr/uss2/09874
DescriptiQn
METHOD FOR DIAGNOSING AND TREATING CANCER
5 Statement of Government Interest
This invention was made with government support under contract
DE-A606-76RLO 1830, awarded by the U.S. Department of Energy. The
government has certain rights in the invention.
10 Technical Field
The present invention relates generally to methods for diagnosing
a~d treating cancer.
BackFround of the Invention
Cancer accounts for one-fifth of the total mortality in the United
States~ and is the second leading cause of death af~er cardiovascul;lr diseases and
stroke. The three leading types of tumors found in man are lung, prostate, and
colorectal cancer, and the three leading types of tumors found in women are
breast, lung, and colorectal cancer. Common therapeutic approaches for the
20 treatment of cancer generally involve the surgical removal of solid tumors,
followed by chemotherapy and/or radiotherapy. One ~isadv~ntage of this general
approach, however, is that most chemotherapeutic or radiotherapeutic a8ents are
not tumor-cell specific, thu~ damaging norm~l tissue during the course of
treatment.
Various method~ have been utilized in order to more effectively
direct or target therapeutic agent~ to tumor cells. For ex~mple, many tumor cells
have an increased number of certain cell surface antigens as compared to normal
cells. This dif~erence benveen tumor and normal cells may be exploited in order
to more effectively target therapeutic agents to tumor cells~ More specifically,30 targeting agents such as monoclonal slntibodies may be used tn specifically target
and bind to the tumor cells, resulting in the localization and internalization of the
therapeutic agents. For example, monoclonal antibodies such as the anti-~pl6()
antibody for human lung cancer (see Sugiyama et al~, "Selective Growth Inhibition
of Human Lung Cancer Ccll Lines Bearing a Surface Glycoprotein gpl60 by ~
35 Labeled Anti-gpl60 Monoclonal Antibody," Cancer Re~. 4-~:2768-2773, lg88), a
~NT-1" monoclonal antibody for hum;m cervical carcinoma (see Chen etal.,
"Tumor Necrosis Treatment of ME-180 Human Cervical Carcinoma Model with
SUBSTI~E ~
W O 93/09816 PC~r/US92/09X74
2~23~
l3l1-Labeled TNT-l Monoclonal Antibo(ly~" Dep~rtment of Pathology, University
of Southern California School of Me~icine, Los Angeles, Californi;l), and
antibodies against the epidermal growth f~ctor receptor for KB c~rcinnm~ (.see
Aboud-Pirak et al., "Efficacy of Antibodies to Epidermal Growth F~ctor Receptor
S Against KB Carcinoma In ~tro and in Nude Mice," J. National Cancer In. titute
80(20):1605-1611, 198~) have been use~ to specifically localize tumor cells.
Monoclonal antibodies, however, are disadvantageous because they are typically
developed in mouse systems, and injection of such antibodies into hum~ns resultsin the generation of an extensive immune response against the antibody itself, thus
10 lirniting its effectiveness in killing tumor ce~ls.
In order to kill tumor cells, t~rgeting agents have been coupled to
various chemotherapeutic agents including~ among others, ricin, abrin, diptheriatoxin, cholera toxin, gelonin, Pseudomonas toxin, Slli~ella toxin, and Pokeweed
antiviral toxin (see U.S. Patent ~o. 4,545,985; see al~-> Jansen et al.,
15 "Immunotoxins: Hybrid Molecules Combining Hi~h Specificity and Potent
~rtotoxicity," mmunological Review 62:18S-216, 1982; see also Thorpe and Ross,
"The Preparation and Cytotoxic Properties of Antibody-Toxin Conjugates,"
ImmunologicalReview 62:119-158). Similarly, various radiotherapeutic agents
have also been utilized to kill tumor cells including, for example, the bet~ emitter~
20 13lI, 67Cu, 186Re, and 90Y. Beta emitters, howe~/er, ~re disadvantageous bec~use
of their low specific activity, low linear energy transfer, low dose rates (allowing
for cell repair of radiation damage), dam~ge to surrounding normal tissues, and in
some cases the lack of an associated imageable photon (e.g., yttrium-9(1).
The present inven~ion overcomes the disadvantages discussed
25 above, and further provides other related advantages.
Summar~of the Invention
The present invention provi~les reagents and meth~ds for detecting
and treating cancer. Within one aspect of the present invention, a conjugate of a
30 growth factor and an alpha-emitting radionuclide is provided, the growth factor
being capable of specifically binding to a defined popul~tion of cancer cells.
Within various embodiments, the growth factor is coupled to the alph~-emitting
radionuclide by a linker, such as a short polycarbon compound, to sep~rate the
alpha-emitting radionuclide from the growth factor. Preferred linkers may be
35 selccted from the group consisting of disulfides, dic3rboxylic acids, and multi-
carbon chain linkers (polycarbons). A particularly preferred linker is
hexamethylene diamine. This linker may be coupled to 3 portion of the growth
lTE ~
wo 93/09816 Pcr/us92/os874
2~23S88
factor selected from the group consistin~ of the N-terminus ~nd the C-terminus.
In addition, within other embodiments of the invention, the alpha-emitting
radionuclide is bound to a sequestering agent, such as, for example, a macrocyclic
complexing agent. Preferred macrocyclic complexing agents include crown ethers
5 such as a 21-crown-7 or an 18-crown-6 ether. Within another aspect of the present
invention, a pharmaceutical composition is provided comprising a conjugate of a
growth factor and an alpha-emitting radionuclide, and ~ pharmaceutically
acceptable carrier or diluent, the growth factor being capable of specifically
binding to a defined population of cancer cells.
Within various embodiments of the present invention, the alpha-
emitting radionuclide is selected from the group consisting of lead-?12/bismuth-212, bismuth-213/polonium-213, bismuth-212m, bismuth-~ 12, polonium-2(K,
polonium-210, astatine-211, radium-223, radium-224, and actinium-~25.
Within another aspect of the present inven~ion, a conjugate of a
15 growth factor and non-radioactive iodine is provided, the growth factor beingcapable of specifically binding to a defined population of cancer cells.
Pharmaceutical compositions are also provided, comprising conjugate of a
growth factor and non-radioactive iodine, and a pharmaceutically acceptable
carrier or diluent, the growth factor being capable of specifically binding to
20 defined population of cancer cells~
Within other aspects of the invention, a method for treating cancer
in warm-blooded animals is provided, comprising administering to a warm-
blooded animal an effective amount of a conjugate of a growth factor and an
alpha-emitting radionuclide, a conjugate of a growth factor and non-radioactive
25 iodine, a conjugate of a growth factor and yttrium-90, or a conjugate of a growth
factor and an oxyanion of a metal, the growth factor being c~pable of specifically
binding to a defined population of cancer cells. Within particularly preferred
embodiments of the invention, the above-described method further comprises,
prior to the step of administering an effective amount of a conjugate,
30 administering an unlabeled growth factor capable of specifically binding to the
defirled population of cancer cells, in ~n amount sufficient to masl; growth factor
receptors in healthy tissues of the animal.
Within yet another aspect of the present invention, a method for
detecting cancer is provided, comprising the steps of (a) administering to a warm-
3S blooded a~nal an effect;ve amount of a conjugate of a growth factor and analpha-cmitting radionuclide, the growth factor being capable of specifically
binding to a de~med population of cancer cells; and (b) detecting the presence and
SUBS~llTUrE SHEEl
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'2 1'' 3 ~ ~, S
location of the conju~te within th~ w;lrm-t)loo~le(l ~nim;d ~n~l therefrom
determining the presence of cancer.
Within another aspect of the present invention, a method for
detecting the presence of cancer in warm-blooded anim~ls is provided, comprising5 the steps of (a) administering to the animal an unlabeled growth factor capable of
specifically binding to a defined population of cancer cells, in an amount sufficient
to mask growth factor receptor sites in healthy tissues of the animal, (b)
administering to the animal an effective amount of a conjugate of the growth
factor and a radioactive isotope which emits gamma radi~tion, an-~ (c) detecting10 the presence and location of the conjugate with;n the w~rm-blooded animal andtherefrom detennining the presence of cancer. Within one embodiment, the
radioactive isotope is selected from the group consisting of rhenium-186,
technetium-99m, iodine-131, selenium-75, iodine~ iodine-12~ iodine-124,
indium-111, copper-67, radium-223, gold-19K, yttrium-9()~ chromium-SI, iron-52~
15 copper-64, gallium-67, gallium-66, ~allium-72~ gallium-68, zirconium-89,
ruthenium-97, lead-203, rhodium-1()5, rhenium-18~, gold-l99, astatine-211,
bromine-76, bromine-77, fluorine-18, bismuth-~()6, mercury-197, and mercury-203.Within yet another aspect of the present invention, a method for
diagnosing and treating cancer in warm-blooded animals is provided, comprising
20 the steps of (a) administering to the animal an unlabeled growth factor capable of
specifically binding to a defined population of cancer cells, in ~n amount sufficient
to mask growth factor receptor sites in healthy tissues of the animal~ (b)
administering to the animal an effective amount of conjugate of the ~rowth factor
and a radioactive isotope which emits gamma radiation, (c) detectin~ the presence
25 and location of the conjugate within the warm^~looded animal and therefrom
determining the presence of the cancer, and (d) administering an effective amount
of a second conjugate of a growth factor and a radioactive isotope or non-
radioactive iodine, such that the cancer is treated.
Within another aspect of the present invention~ a method for
30 diagnosing and treating cancer in warm-blooded animals is provided, cnmprising
the steps of (a) administering to the animal an unlabeled &rowth factor capable of
specifically binding to a defined population of cancer cells, in an amount sufficient
to mask growth factor receptor sites in healthy tissues of the animal, (b)
administering to the animal an effective amount of a first conjugate of a growth3S factor and a radioactive isotope which emits gamma radiation, (c) detecting the
presence and location of the conjugate within the warm-blooded animal and
therefrom determining the presence of the cancer~ and (d) administering an
wo 93~09816 Pcr/us92/os8~4
2123~
effective amount of a second conju~ate of a growth factor ~n~l ~ cytotoxic metail
ion, such that the cancer is tre~ited. Within one embodiment, the cytotoxic agent
is an oxyanion of a metal selected from the group consisting of manganese,
technetium, rhenium, chromium, molybdenum, tungsten, vanadium, and tellurium.
S Within another embodiment, the ~totoxic agent is an alpha particle emitting
radioactive isotope selected from the group consisting of lead-212/bismuth-212,
bismuth-213/polonium-213, bismuth-212m, bismuth-212, polonium-206, radium-
224, and actinium-22S.
Within yet other embodiments of the above invention~ the growth
10 factor is selected from the group consisting of epidermal growth f~ictor,
transforming growth factor - alpha, fibroblast growth factors, insulin-like growth
factor I and II, and nerve growth factor.
These and other aspects of the present invention will become
evident upon reference to the following drawings and detailed description.
Brief Description of the Drawines
FIGURE 1 is a table listing various radioactive nuclides which emit `alpha-particle radiation.
FIGURE 2 schematically illustrates a decay series starting with
Cm-243. Radium-223 is a member of this decay series.
FIGURE 3 is a graph which illustrates the iodination profile of 1311 to
epidermal growth factor.
FIGURE 4 ;S a graph which compares cells treated with 13~ 311-
epidermal growth factor, and epidermal growth factor alone.
2S FIGU~E; 5 jS a graph which illustrates the effects of various
concentrations of 1311-epidermal growth factor on A4~s1 cells. ` -
FIGURE 6 ;S a graph which illustrates the effects of various
concentrations of 131I-epidermal growth factor on L cells.
FIGU~E 7 is a graph which illustrates the effects of various
30 concentrations of non-radioactive iodine-epidermal growth factor on A431 cells~
FIGURE 8 is a graph which illustrates the effects of various
concentrations of non-radioactive iodine-epidcrmDI growth factor on L cells~
.
Detailed Descri~don of the Invention
A~ noted above, the present invention provides reagents for
detecting and treating cancer~ These reagents generally comprise a conjugate of a
growth factor and an alpha-emitting radionuclide~ ~ conjugate of a growth factor
~mU~E SHEEr
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2 ~ 2 ~
and non-radioactive io~line, or any of a numher of growth factor conju~te~ whichare described in more detail below, the ~rowth factor being chosen such th~t it is
capable of specifically binding to a define~l population of cancer cells.
Many growth factors known to one of ordinary skill in the art may
S be utilized within the present invention. Representative examples include
platelet derived growth &ctors, transforming growth factor-beta, interleukins (i.e.,
IL-I, IL-2, IL-3, IL4, IL-S, IL-6, IL-7, IL-8, or IL-9), granulocyte-m~crophage
colony stimulating factor (GMCSF), erythropoietin, tumor necrosis &ctor,
endothelial cell growth factor, platelet basic proteins, capillary endothelial cell
10 growth factor, cartilage-derived growth factor, chondrosarcoma-derived growthfactor, retina-derived growth factor, hepatoma derived growth factor, bombesin~
~nd parathyroid hormone. Particularly preferred growth factors include
epidermal growth &ctor, transforming growth factor - alph~, fibrobl~st growth
factors, insulin like growth factor I and 11, and nerve growth f~ctor.
The growth factor should be selected such that it is capable of
specifically binding to a defined population of cancer cells which include, for
cxample, preneoplastic cells, premetastatic cells, and tumor cells (both benign and
malignant). As will be understood by one of ordinary skill in the art, a definedpopulation of cancer cells may generally be differentiated from normal cells bascd
20 upon the greater number of growth factor receptors on the cell surface.
Consequently, within the context of the present invention a growth factor may bedefined to be "specifically binding" to a (3efined population of cancer cells if this
population of cells has greater than approximately two times the number of
growth factor receptors on its surface as compare~ to norm~l cells, and preferably
25 greater than five to ten times the number of growth factor receptors~ In addition,
this difference in the number of growth factor receptors on cancer cells, as
compared to normal cells, may be exploited in order to more specifically target
growth factor conjugates. In particular, the number of growth factor receptors on
cells in healthy tissue may be determined, and compared to the number of growth
30 factor receptors on cancer cells. A~s ~escribed in more detail below~ unlabeled
growth factor capable of speci~lcally binding to a defined population of cancer
cells may then be administered in an amount sufflcient to masl; growth fac~or
receptor sitcs on the norm31 cells of he~lthy tissucs, in order to mask the lessabundant growth faaor receptors on normal cells (if present) prior to the addition
35 of a conjugated growth factor.
The number of growth factor receptors on a cell may be readily
detem~ined based upon the ability of the cell to bind to the growth factor
wo 93/09816 Pcr/us92/09874
2 L 2 '~J ~
receptor's substr~e. For example, ~ y~ such ~ r;l~lioreceptor l in~in~ y~
which determine the quantity of receptor suhstr~te that binds to ~ cell over thecourse of time may be readily utilized to determine the number ~nLI type of cellsurface receptors (see, for example, Ladd~ et al., Anal. Biocl~em. 93:2~6-294, 1979).
5 Briefly, utilizing radiolabeled growth factor and membrane prep~rations isolated
from both normal and tumor cells, one can readily determine both growth factor
receptor number and affinity by a standard competitive binding assay followed bya Scatchard plot analysis (see Scatchard, Anal. N.Y. Acad. Sci. 51:66()-672, 1949). -: `
In order to determine which growth factor conjugate would be the
10 most effective therapeutically or diagnostically, within one embodiment the cells
of interest (e.g., tumor cells) are removed from the patient. The removal of cells
~pay typically be accomplished through surgical procedures, ~lthough m~ny other
methods may also be utilized, dependent of course on the type of tumor and its -
location. Once the tumor cells have been removed, they may he maintained in an
15 in vitro culture using conventional media (see, f~r ex~mplc, "Media F(>rmulations,"
ATCC Cell Lines & Hybridomas, 1988). The number and type of receptors may
thcn be readily determined using methods described above; and ~ growth factor
conjugate selected on the basis of its ability to specifically bind to the tumor cells.
Additionally, the therapeutic (or dia~nostic) effectiveness of the growth factor20 conjugate upon tumor cells may be readily determined by in vitro ass~ys. A
representative assay is described below in Examples l and 2.
Alternatively, within another embodiment growth factor conju~ates
may be utilized for therapeutic or diagnostic purposes based only upon the knowncharacteristics of certain tumors. For example, certain types t)F tumors such as25 human epidermal carcinomas are already well defined, and h;lve been shown to
possess abnormally high numbers of epiderm~l growth f~ctor receptors (see
Berger et al., "Epiderm;ll Growth Factor Receptors in Lung Tumors,`' J. Patl20l0gy
152:297-307, 1987; Dotzlaw et al., "Epidermal Growth Factor Gene Expression in
Human Breast Biopsy Samples: Relationship to Estrogen and Progesterone
30 Rcceptor Gcne Express;on," Cancer Re~: s~:4~n4-42n8~ 199(~; Maddy et al.,
nEpidermal Growth Factor Receptors in Human Prostate Cancer: Correlation
with Histological Differentiation of the Tumor," Br. J. Cancer h():41-44, l989;
Liberman et al., "Expression of Epidermal Growth Factor Receptors in Hum~n
Brain Tumors," Cancer Res. 44:573~ ), 1984; Neal et al., "Epidermal Growth
35 Factor Receptors in Human Bladder Cancer: Comparisons of Invasive and
Superficial Tumors," Lancet 1:366-368, 1985; and Moorghen et al., "Epiderrnal
Growth Factor Receptors in Colorectal Carcinoma," Anticancer Re~. 10:6n~-612,
wo g3/098I6 PCr/usg2/os874
2 ~
1990). Thus, an epidermal growth f~ctor conjug~te m;ly he re~ pplied to an
epidermal carcinoma without the neeù to first determine which gr(lwth factor to
use.
Similarly, lnterleukin-? receptors are expressed by abnL)rm;31 T cells
S in patients with certain Iymphoid malignancies or autoimmune disorders, but not
by resting cells. For example, HTLV-I associated adult T-cell leukemia cells
constitutively produce large numbers of IL-2 Tac receptors (se~ Waldmann,
Cancer Surv~ys 8(4):891-903, 1989, see also Waldmann, J. Na~L Canc. /nSL
81(12):914-923, 1989). Once a m~lignancy has heen classified a~ an HTLV-I
10 associated adult T-cell leukemi~, an IL-2 growth factor conju~;lte may he utilized
therapeutically without the need to further cl~ssify the m;~lignancy as discussed
?bove.
Similarly, a combination of growth factor conjugates may be utilized
based upon the known distribution of tumor types in a given disease. For
example, if 80% of human lung tumors express growth factor receptor type A,
15% of human lung tumors express growth factor receptor type B, and the
remaining S% of human lung tumors express growth factor type C; a conjugate
may be prepared for the treatment of lun~ cancer comprising a ct)mbination of
growth factors conjugates A, B, and C.
Within one aspect of the present invention the ~rowth factor is
conjugated to an alpha-emitting radionuclide. Alpha-emitting radionuclides are
particularly preferred because they have short range (35-7() l~m through solid
tissue and 3~-700 ILm through lung tissue), and are extremely efficient in killing
cells. On tbe average, only about 1 to 3 alpha particle emissions must penetratethrough the nucleus of a cell to kill the cell. lf the three-dimensional geometry of
cells is considered, about 2S alpha-particle emissions are needed per single cell to
achieve complete cell killing in a tumor mas.s with uniform labeling of the cellsurface by a radiolabeled protein (see 4th Int. Radiopharmaceutical Dosimetry
Symposium, CONF-851113, pp 26-36, 19~5). Many alph~-emitting radionuclides
30 are well known in the art, and may be utilized within the present invention. A
representative list is presented in Figure 1. Preferred alpha-emitting
radionuclides include lead-212/bismuth-21~, bismuth-213/polonium-21~, bismuth-
212m, bismuth-212, polonium-2()6, polonium-21(), ;IstZltine-211, radium-223,
radium-224, and actinium-225.
Particularly preferred alpha-emitters are radium-~ (half-
life = 11.4 days) and actinium-~ (h;llf-life = 1().() days). Radium-~3 is a
member of the natural uranium-~5 decay series (sce Figure 2)~ It exists naturally
.
`SlJBSTm~rE S~EFr
WOY3/0g816 2i"~3`)~ Pcr/US92/~g874
in all soils containing ur~nium an~ d;~uvhter pro~lucts, but m;lv h~ foun(l ~I hi~her
concentrations in uranium mill tailin~ piles. It may be removeL~ by chemical
separation from tailin~ sands by recoverine, its predecessor ~ctinium-~7.
Briefly, actinium-227 decays n~turally to Th-227~ which decays
5 naturally to Ra-223 (see Figure 2). Radium-223 may be separa~ed chemically from
both Ac-227 and Th-227 by, for example, passing a saline solution over an ion-
exchange resin containing the parent radionuclides. The purified salt radium-223may thus be eluted from the column (see Pilger, UCRL-3877, 1957, University of
California Radiation Laboratory, Berl;eley, California; Muller, "Praparative
10 Arbeiten uber Ac-~27 und seine Folgeprodukte, S~nderdmcl; a~ Rc~ chiJnica
Acta 9:181-186, 1968; and Atcher etal., "A Radionuclide Generator for the
Production of Pb-211 and ies Daughters," J. R~lioanl~L Nucl. C/~CI71. (Letters)
135(3):215-Zl, 1989). Radium-223 may also similarly be sep~r;~ted from enriched
U-235 stockpiles in which natural r~dioactive decay has allOwed the build-up of
15 Ac-227.
An alternative method of producing radium-223 for medical
applications is to stare with natural radium-æfi. Within this method, radium-226 is
first ir~adiated in a nuclear reactor to produce radium-227. The radium-227 thenbeta decays to actinium-227. For example, 1.() curies of radium-226 is irradiated
20 for about 120 days in a hydride asscmbly (such as the Fast Flux Test Facility,
Ricbland, Washington). This assembly produces neutrons of epithermal enetgy,
optimum for conversion of Ra-226 to Ra-227, which then beta-decays to Ac-227.
Other nuclear reaceors may, however~ also be used to activ~te Ra-~fi to Ra-227.
This procedure produces about 9.5 curies of ~ctinium-2~7. Radium-~s may then
2S be chemically separated from actinium-2~7 and thorium-~?7 utilizing methods
described above.
The growth factor may be c!~njugated t(~ the alph;l-emitting
radionuclide by various methods, although it is p3rticularly preferred to bind the
alpha-emitting radionuclide to a sequestering agent, for example by positioning
30 the alpha-emilting radionuclide within the sequestering ~gent, which is in turn
coupled by a linker to the growth factor. A variety of diverse organic macrocyclic
complexing agents may be used to se4uester the alpha-emitting radionuclide
includin& among others, the followin~ groups: ( I ) spherands, (2) crypt~Lsphcrands,
(3) cryptands, (4) hemisphcrands, (5) corr3nds (modified crown ethers), ~md
35 (6) podands (acyclic host~s) (sce Cram, Sciencc 240:76() 67, I~X~). In general, these
macrocyclic ring compounds are large~ somewhat spheric;ll org~n;c compounds
which resemble cage structures, and have the ability to hold a heavy radionuclide
~ urrT
wo 93/09816 Pcr/US92/09874
2 1 2 '3 ` ~
as a ligand hold~ a met~l ion. The se~uesterin~ ~ent shoul~ he selecte~ such th~t
it has both a high affinity and specificity for the alph~-emittin~ radionuclide as
well as a low intrinsic mammalian toxicity. High specificity is es.~enti;ll to avoid
displacement by other divalent cations (Mg+2 and C~+2) that are prevalent in
5 physiological fluids. Additionally, the compound should either contain a
functional group, or have chemistry which is compatible with the introduction ofan appropriate functional group, to allow ~ttachment to the linker.
The affinity of the sequestering agent for the alpha-emitting
radionuclide is defined by the system energetics ~s de~cribed ~ y Cr~m (supra).
10 More specifically, as inferred by X-r~y crystallographic ~;lt~ of complexed and
non-complexed crown ethers, it is believe~ th~t the solution conformations of non-
complexed ethers lack well-defined cavities with the associated convergently
aligned binding sites. During the process of complexation, the crown ether
undergoes desolvation and reordering of structure, a process which requires
15 cnergy~ If the sequestering agent presents a rigid prestructured and desolvated
cavity to the ion (as is the case for spherands), the energy normally consumed by
desolvation and reorganization is reflected in a larger binding constant for the ion.
Based on this fundamental principle of reorganization, Cram lists the affinity of
hosts for their most complimentary guests as: spherands > crypt~spherands >
20 c~yptands > hemispherands ~ corrands ~ podands. The difference in binding
affmity between spherands and podan~s is dr;lmatic~ for ex~mple, the binding
constant of a lithium sequestering spheran~ was found to be 1012 higher than itscorresponding open-ch~in podand (see Cram, suprn). Thus, although many
different sequestering agents may be utilized within the context of the present
25 invention, spherands which are designed and synthesized specifically to sequester
radium-223 are particularly preferred.
Particularly preferred sequestering agents inclu~e lX-crown-6 or 21-
crown-7 ethers, including for example mo~lified crown ethers such as
dicyclohexano-21-crown-7 (Case ~nd McDowell~ Rcldioact. ~a~i~cl~m. 1:58, 1990;
30 McDowell et al., Solvent E;xtr. Ion Excl~. 7:377, 1989; for other crown`ethers or
macrocyclic polyethers, see Pedersen, Science 241:536-540, 19~8~ U.S. Patent No.4,943,375, Eia et al., ~eteroycles 32(4):711-722~ 1991; Wai and Du,Anal. C)lem
62(21):2412-14, 1990; Tang and Wai, Anabst(L~ndon) 114(4):451-4S3, 1989).
Briefly, Ra2+ is bound by thc ether~te oxygen network comprising the interior
35 cavity of the spherical crown-ether molecule. This binding ;s bclieved to be pH
dependent: Ra2+ complexes with a combination of ~ proton and sm;lller Group
IA ions for the binding site within the crown C:lvity. These crown ethers may
wo 93/09816 2 1 2 3 .~ ~ ~ Pc~r/US92/o9874
addition~lly be mo~ified with polarizal-le function;ll group~ (simil~r to changes
made with closo- and nido-carboarnyl species used in boron-neutron c~pture
therapy), resulting in compounds with gre~ter solubility in ~queou~ media (see
generally, Mizusawa et al., Inorg. Cllen~. 24:1911, 198S). Such ch;ln~es improveS retention of biological specificity after conjugation, and improve the conjugate
loading capability of the biological ;Igent. These modific~tion~ may be
accomplished in tandem with the synthesis of the above-noted crown ethers under
appropriate conditions for mild conjugation to the biological delivery system.
Additional crown ethers suitable for use within the prescnt
10 invention may be synthesized~ or purchased fr-)m various sources including, among
others, Aldrich Chemical Co. (Milwaukee, ~is.)~ Fluk:l Chemical Corp.
(Ronkonkoma, N.Y.), and Nisso Research Chemicals, (Iw;li Co. Ltd., Tokyo,
Japan). Sequestration of the alpha-emitting r~dionuclide may be achieved by
mLxing the sequestering agent with a salt of the alpha-emittin~ radionuclide whicb
15 has bcen dissolved in solvent. The particular solvent chosen depends of course on
the solubility of the sequestering agent and alpha-emitting radionuclide. For
example, Cram and co-workers prepared the sodium complex of a spherand
simply by adding excess salt dissolved in acetonitrile to a methylene chloride
solution of the spherand (see Cram anà Lein, J. Am. C~2~m. soc. 1()7:36~7-3668,
20 198S).
The ability of the crown ether to sequester or complex with the
alpha-emitting radionuclide may be readily determined (see Cox et ~1., "Rates and
Equilibria of Alkaline-Earth-Metal Complexes with Diaz;l Crown Ethers in
Methanol," Inorg. Chem., 27:4018~021, 198~; see also Mohite and Khopkar,
25 HSeparation of Barium From Alkaline Earths and ~ssociated Elements by
Extraction with Dibenzo-1~-crown-6 From a Picrate Medium~" Analytica Cllimica
Acta, 206:363-367, 1988). Briefly, separation of the complexed and free
radionuclide can be accomplished by partitioning between an organic solvent
(such as chloroforrn) and water. The complexed radionucli~ie will p~rtition into30 the organic phase, whereas the free r;ldionuclide will reside exclusively in the
- aqueous phase. Alternatively, a variety of chromatographic techniques such as
High Performance Liquid Chromatography (HPLC) or Reverse-Phase High
Performance Liquid Chromatography (RP-HPLC) may be utilized to separate
sequestered radionuclide from the free cation. Once isolated, veri~lc~tion of the
35 molecular architecture may be accomplished. Briefly, the mode of cation binding
can take two forms: (1) through external association (i~, anion/cati()n pairing
without bond formation), or (2) via coordination of the cation to the cro~n-ether
SUBSmUTF ~
wo 93/09816 Pcr/us92/og874
21235~8
oxygen network. Specificity and stron~ hindin~, which ~re preferred for the
present applications, are dependent on the latter type of associ~tion. Single
crystal X-ray diffraction techniques may he u~ed to unambiguously ~ssign the type
of interaction for the solid materials, and I7O, 13C and lH-NMR m;ly be used to
5 determine the structures of target materials in solution.
As noted above, within one embodiment of the precent invention
the alpha-emitting radionuclide is positioned within a sequestering agent which is
in turn coupled by a linker to preferably either the amino ("1~") or carboxy ("C')
terminus of the growth factor. The linker serves to place an inert "spacer"
10 between the biologically active growth factnr and the alph~a-emitting radionuclide
containing complex. This space minimizes steric inter~ctinns th;lt may interferewith the growth factor's affinity toward~ its target. The optimum length of the
spacer ann is primarily dependent on the ~ffinity of the gr~-wth factor for its t~rget
receptor. The higher this affinity, the smaller the rel~tive import;lnce of stearic
15 repulsion between the sequestering agent and the target receptors. A virtual~y
linutless number of linkers may be selected which are suit;~ble for use within the
present invention, although presently preferred linkers include disulfides,
dicarboxylic acids, polycarbon chains, and modi~led polycarbon chains. Preferredlinkers include hydrocarbon chains which range in length from 4 to 18 carbon
20 atoms. Particularly preferred linkers have at least six methylene units such as
hexamethylene diamine.
The linker may be attachetl to any of a numher of extraanular
functionalities on the sequestering agent, although c3rboxy and amino
functionalities are particularly preferred. Within one aspect of the invention, if
25 the extraanular functionalization is a carboxy group, then a first synthetic step
could involve reaction of the sequestering agent with hex~methyiene diamine.
Subsequent reaction with the C-term;nus of the growth factnr wnuld complete
sy~thesis of the conjugate~ Alternatively, as noted ahove~ the linker may be
coupled to other aspects of the growth factor such as the N-terminus~ Within this
30 cmbodiment, after reaction with hexame~hylene di~mine the se~luesterin~ agentmay be reacted with succinic anhydri~e. Subsequent coupling of the linker to thegrowth factor m~y then be accomplishel3 through the ~-terminus of the growth
factor.
Alternatively, within another aspect of the present invention, the
35 sequestcring agent may contain an amino functionality~ In these cases, a
dicarboxylic acid linker (for example~ octanedinic acid) may t)e utilized to couple
the sequestering agent to the I~J-terminus nf the growth factor~ On the other hand~
wo 93/09816 P~/US92/09874
2~2t~ ~3~J;~ ~
1 . .
if the sequestering agent is reacted with ethylene diamine after condensation with
the dicarboxylic acid, linkage to the growth factor may be accomplished through
the C-terminus.
Within one embodiment of the present invention, in order to allow
5 the covalent attachment of the sequestering agent to the linker an appropriatefunctionality is inserted into the sequestering agent. For example, a bromine atom
may be incorporated into the appropriate position of an aromatic constituent
during synthesis of the macrogrclic compound (see Skowronska-Ptasinska et al., J.
O~p. C~lem 53:5484-91, 1988). Sequential treatment of thi~ compound with n-
10 butyllithium and CO2 yields the carboxy analog:
B~ 1) n-bulyUithium HOOC~
~`O? Ra+2, `"`"~O ` Ra~
Macrocyclic
' Sequetste~ing
Agent ..
It should be noted, however, thslt synthetic reactions leading to these
15 types of sequestering agents may pro~uce very low yields.
- Since the growth factor is likely to be the limiting reactant, the next
step within this embodiment of the invention is the reaction bet~veen the
functionalized sequestering agent and the linker:
COOH CONH (CH2) 6NH2
<O> ~NH2(CH2) 6NH2 ~ ` O)
.s~ ~"
If the scquestering agcnt is not immobilized on a rigid support the
follotwing by-product may also be produced: `
: `
wo 93/09816 P~/US92/09874
2 1 2 3 . j X b l~
CONH (CH2) 6 NHOC
\"'/'/" ~/"'''0"\>
.. . ..
Thus, chromatographic purification of the reaction mixture to
isolate the desired product may be necess~ry before proceedin~. Briefly, standard
semi-preparative chromatographic separations base~l upon, for example, RP-
HPLC or HPLC purification, may be utilized to purify the target compounds from
10 the synthetic mixtures. Products may be detected either by refractive index or by
the more sensitive technique of ultraviolet adsorption detection. Within one
embodiment, a chromophoric benzene moiety is incorporated into ~he
sequestering agent to facilitate detection during chromatographic purification.
The final reaction within this embodiment invnlves a similar
15 reaction bet~veen the sequestering agent-linker (organic soluble) and the c~rboxy
terminus of the growth factor (water soluhle) as summ:lrized below:
CONH (~H2) 6 NH 2 ~ . CONH (CH2) 6 NHOC~rowth F~ctor
(O ~ ~, FaOOCr-t3rowth ~ O ~
Solubility incompatibilities may be overcome by use of a 50:50
dimethylformamide:water solvent system (see generally Cooper, T~2e Tools of
Biochemist y, Wiley, New York, pp. 234-255, 1977; C:uatrecas~s, "Protein
Purification by Affinity Chromatography on Polyacrylamide Beads," J~ BioL Cllem.25 245:3059, 1970; and Cuatrecasas, HAffinity Chrnmatography of Macromolecules,HinAdvances in Eny nology, A~ Meister (ed~), Wiley~ l~ew Yor~;, p. 2~ 197~)~
W0 93/09816 2 l 2 ~ PCl'/US92/098~4
Within ;lnother aspect of the pre~ent invention, the ~rowth f;lctor is
conjugated to non-radioactive iodine. Briefly, non-radioactive iodine m~y be
obtained from many commercial sources, including, for example, Si~m~ Chemic;ll
Co. (St. Louis, Mo.). Various me~hods which are typically used to label proteinsS with radioactive iodine may also be utilized to conjugate non-r~dio;lctive iodine to
the growth factor. For example, iodide (normally supplie(l ~s Nal) may be
oxidized to form 12, which then att;lcks tyrosyl and histidyl side chains.
Representative methods utilizing this technique include the Chloramine T method
(Hunter and Greenwood, Nanlre 194:495496, 1962), the lodogen method (see
10 ~raker and Speck, Biocltem. Biopllys. Re~. C~>mmun. 8():~s49-~7, 197~), and the
lactoperoxidase method (see Hubbard 3nd Cohn, J. Cell Bi~ 55:'7~ )5, 1972).
~Iternatively, an iodinated reagent cont;lining ~ reactive couplin~ group may bebound to the protein (sec Bolton and Hunter, Bi~cll~m. J. ]~ 5?~_5~9, 197~).
Within other aspects of the invention, numerou~ additional growth
15 factor conjugates are provided. Within one embodiment, these ~rowth f;lctor
conjugates comprisc a growth factor, and a radioactive isotope which emits
gamma radiation. Representative examples of such radioactive isotopes includc
rhenium-186, technetium-99m, iodine-131, selenium-75, iodine-l?~, iodine-125,
iodine-124, indium-lll, copper-67, radium-?~3, gold-19X, yttrium-9~), chromium-
20 51, iron-52, copper-64, gallium-67, g~llium-66, gallium-72, g:~llium-~, zirconium-
89, ruthenium-97, lead-2()3, rhodium-l()~, rhenium-18~, gol~l-l9~, ast3tine-?ll,bromine-76, bromine-77, fluorine-18, bismuth-2()6, mercury-1')7, and mercury-203.
Within other embodiments of the invention, the growth f;lctor conjug3te
comprises a growth factor and a cytotoxic agent. Represent~tive examples of
25 ~ytotoxic agents include (in addition to the v~rious alpha and gamma emittersdiscussed above) oxyanions of a metal selected from the ~roup consisting of
manganese, technetium, rhenium, chromium, molyhdenum, tungsten, v~nadium,
and tellurium.
Conjugated growth factors of the present invention may additinn~lly
30 be purified utilizing a v3riety of techni4ues, including ~mong others~ column- chromatography, HPLC, and RP-HPLC.
Conjugates of the prcsent invention m~y be utilized in v~rious w~ys~
For example, they may be employed in in vitro a~ssays as described below in ordcr
to kill spccific cells.
Additionally, ~s noted ~hove, the conjug;ltes of the present
invention may be utilized for the treatment and detection of cancer in warm-
blooded animals. Many warm-bloodetl ;mim~ls m3y he tre;lted ~nd di;lYno~ed for
SUBSTITUTE S~EET
WO g3/09816 Pcr/US92/09874
21~358~
cancer, including for ex~mple, mice, r;lt~, sheep, COW!i, pi~, mon~ey~, ;m-l hum~ns.
Briefly, as noted above, within one aspect of the present invention a method fortreating cancer in warm-blooded anim~ls is provided, comprising the step of
administering to the animal an effective ~mount of a conjug~te of ~ growth f~ctor
5 and an alpha-emitting radionuclide, the growth factor conjugate being cap;lble of
specifically binding to a defined population of cancer cells. Within one
embodiment, the alpha-emitting radionuclide is selected from the group consisting
of lead 212/bismuth-212, bismuth-213/polonium-213, bismuth-212m, bismuth-212,
polonium-206, polonium-223, radium-224, and actinium-225.
Within another aspect of the present invention, a method for
trcating cancer in warm-b!ooded animals is provided, comprising administering totbe animal an effective amount of a conjugate of a growth f~ctor and yttrium-90,the growth factor conjugate being cap~ble of specifically bin~lin~ to a defined
population of cancer cells.
Within yet another aspect of the present invention, a method for
treating cancer in warm-bloolled animals is provided, comprisin~ the step of
adrninistering to the animal an effective amount of a conjugate of a growth factor
and an o~yanion of a metal selected from the group consisting of manganese,
technetium, rhenium, chromium, molybdenum, tungsten, vanadium, and tellurium,
20 the growth factor conjugate being capable of specific~lly binding to a defined
population of cancer cells.
Within another aspect of the present invention, sl metho~3 for
t~rcating cancer in warm-blooded anim;~ls is provide~l, comprising the step of
administering to the animal an effective amount of a conjugate of ~ growth factor
25 and non-radioactive iodine, the growth factor conjugate heing capable of
speci~lcally binding to a defined population of cancer cells.
Within particularly preferred emhod;ments of the invention, the
present invention provides, prior to the step of administerin~ an effective amount
of a conjugate as described above, administering an unlaheleù growth factor
30 capable of specifically binding to the defined population of cancer cells~ in an
amount sufflcient to mask growth factor receptors in healthy tissues of the animal.
Briefly, in order to concentrate the radioisotope preferentially in cancer cells and
avoid excessive damage to nortnal cells, ~dministr~tion of the conjug~ted growthfactor is preccdcd by the step of sdministering a "cold" or unlabcled growth factor
35 capable of binding to growth &ctor receptors in both normal and cancer cells~thereby reducing the number of receptnr sites on nnrmal cells avail;lble for
binding and thus minimizing radiation damage to nnrmal cells~ M~king of growth
~)BS1~TUTE SHEEr
wo 93/09816 ~ 3 ~ ~ ~ Pcr/uss2/os874
factor receptors m;ly he ~ccompli~he(l in metho~ for hoth tre;nin~ anul ~ unosin~
cancer, as described herein.
Within one aspect of the present invention, a method for detecting
cancer is provided, comprising the steps of (a) administering to a warm-blooded
5 animal an effective amount of a conjugate of a growth factor and an alpha-
ernitting radionuclide, the growth factor being capable of specifically binding to a
defined population of cancer cells, and (b) detecting the presence of the conjugate
within the warm-blooded animal, and therefrom determining the presence of
cancer. Briefly, conjugates or pharmaceutical composition~ a~ described above
10 may be adrninistered in an effective amount as determine~ by experimental trials.
The presence of the conjugate may be detected by any suitable nuclear medicine
radiation ca~mera which detects the requisite particle emissiOns (c.g., alph;l or
gamma). In the case of alpha emitters, a Nuclear Medicine An~er camera fitted
with a collimator for the Tc99m energy window is particularly preferred.
lS Within another aspect of the present invention, a method for
detecting the presence of cancer in w;lrm-blooded animals is provided comprisingthe steps of (a) administering to the warm-blooded animal an effective amount ofa conjugate of a growth factor and an alpha-emitting radionuclide, the growth
factor conjugate being capable of specifically binding to a defined population of
20 cancer cellsl and (b) detecting the presence and location of the conjugate within
the warrn-blooded animal and therefrom determining the pre~ence of cancer.
Within yet another aspect of the present invention a method for
detecting the presence of cancer in w~rrn-blooded animal~ prnvided~ comprising
the steps of (a) administering to the animal an unlabeled growth factor cap~ble of
25 specifically binding to a defmed population of cancer cells, in an amount suf~lcient
to mask growth factor receptor sites in healthy tissues of the animal, (b)
administering to the animal an effective amount of a conjugate of the growth
factor and a radioactive isotope which emits gamma radiation, and (c) detecting
the presence and location of the conjugate within the warm-blooded ;lnimal and
30 therefrom determining the presence of cancer. Within variou~ emhodiments of
the present invention, ~he radio~ctive isotope is selected from the group consisting
of rhenium-186, technctium-99m, iodine-131, selenium-75, iodine-12~, iodine-125,iodine-124, indium-111, copper-67, radium-223, gold-19X, yttrium-9(), cbromium-
S1, i~on-52, copper-64, gallium-67, gallium-66, gallium-72, gallium-68, zirconium-
3S 89, ruthcnium-97, lead-203, rhodium-105, rhenium-1~s, gold-l99, astatine-211,;~ bromine-76, bromine-77, fluorine-18~ bismuth-2()6, mercurv-197, and mercurv-2()3~
SU~SllTUTE SHEEr
wo 93/09816 PCr/us92/09874
21~3~jS ~ 1~
Within ~nother aspect of the pre~ent invenlion, ;I metho~l for
diagnosing and treating cancer in warm-blooded animals is provided, comprising
(a) administering to the animal an unla~eled growth factor capable of specifically
binding to a defined population of cancer cells, in an amount sufficient to maskS growth factor receptor sites in healthy tissues of the animal, (b) administering to
the animal an effective amount of conjug~te of the growth fslctor and a radioactive
isotope which emits gamma radiation, (c) detecting the presence and location of
the conjugate within the warm-bloodeu~ animal and therefrom determining the
presence of the cancer, and (d) administering an effective amount of a second
10 conjugate of a growth factor and a radioactive isotope or non-radioactive iodine,
such that the cancer is treated.
Within yet another aspect of the present invention, a method for
diagnosing and treating cancer in warm-blooded animals is provide~, comprising
the steps of (a) administering to the animal ~n unlabeled growth factor capable of
15 specifically binding to a defined population of cancer cells, in an amount sufficient
to mask growth factor receptor sites in healthy tissues of the animal, (b)
administenng to the animal an effective amount of a first conjugate of a growth
factor and a radioactive isotope which emits gamma radi~tion, (c) detecting the
presence and location of the conjugate within the warm-blooded animal and
20 therefrom determining the presence of the cancer, and (d) administering an
effective amount of a second conjugate of a growth factor and a cytotoxic metal
ion, such that the cancer is treated.
Within various embodiments of the invention, the cvtotoxic agent is
an oxyanion of a metal selected from the group ct)nsisting of manganese,
2S technetium, rhenium, chromium, molybdenum, tun~sten, vanadium, and tellurium~
Within yet other embodiments of the invention, the cytotoxic agent is an alpha
partide emitting radioactive isotope selected from the group consisting of lead-212/bismuth-212, bismuth-213/polonium-~13, bismuth-212m, bi~muth-212,
polonium-206, radium-224, and actinium-2'~5~
3Q Within a further emhodiment of the invention, pharmaceutical
compositions are provided. Briefly, represent~tive extlmple~ of pharmaceutical
compositions include a conjugate of a ~rowth factor and an ;llpha-emitting
radionuclide, or a conjugate of a growth factor and non-radi~active io~Jine, or ~my
of the other growth factor conjugates discussed above, along with a
35 pharmaceutically acceptable carrier or diluent. Suitable pharmaceutically
acceptable carriers or diluents include neutr~l buffered saline or saline~
Additionally, the pharmaceutical composition may contain other constituents,
~UBSrlTUTE SHEET
wo 93/09816 2 l 2 ~ PCl iUS92/09874
I')
including for example buffers, c~rbohy~r;ne~ such a~ ~luco.~e. !iucro~se, or dextrose,
preservatives, as well as other stabilizer~ or excipients. Although appropriate
dosages may be determined by experimental trials, about Sx101O to 5xlOl I
conjugate complexes/70kg of adult weight may be administered assuming a l:I
5 ratio of growth factor to the alpha-emitter or non-radioactive iodine.
Nevertheless, the amount and frequency of administration will depend of course
on many factors such as the condition of the patient, the nature and severity of the
disease, as well as the type of cancer being treated. In addition, as discussed
above, it is generally preferable to first mask growth factor receptors with
- 10 unlabeled growth factor, in order to minimize ~lamage to normal healthy tissues.
The following examples are offered by way of illustration, and not
by way of limitation.
SUBS~llUrE SHEE~
WO 93/09816 PCI`/US92/09874
2 1 2 3 ;~
EXAM PLES
EXAMPLE 1
E~FE(~S O~ 131I RADIOLA~ELED EGF ON A43 1 CELL~
A. Preparation of ~ell~
The human cervic~l epidermoid carcinoma cell line A431 (av~ilable
from the American Type Culture Collection or "ATCC," Rockville, Maryland,
under accession number CRL 1555) wa~ grown in Dulhecco'.s Modified E~gle's
10 Medium with 10% fet~l bovine serum. The cells were h;lrvested hy trypsinization
with 0.0S5'o trypsin and counted with trypan blue to obtain the numl er of live cells.
A431 cells have approximately 1-2 x lo6 EGF receptors per cell.
B. Radioiodination of Epidermal Growth Factnr
One hundred micro~r~ms of murine EGF (GlBCO
Laboratories/Life Technologies, Inc., Grand Island, N.Y~) w;ls r:ldiol~beled by
iodination with 13lI (Dupont, Wilmington, Del.) using ~ modifie~l method of
lactoperoxidase procedure ~s descrihed by Leung et ~l. (Pr)c S.J-:. &p. Bi~L M~,196(4):385-9, 1991) which modifies the procedure of Thorell and Joh~nsson
(Bioc~lim. BiopJtys. Acta 251:363 1971). Briefly, H22 w~s added in four or fivealiquots at l-min intervals to a reaction mixture of ln mCi of l?511, l()() ~g of EGF,
and 100 ~g of lactoperoxid~se. The l~beled EGF was then separate-3 from the free[131]NaI and lactoperoxidase by gel filtr~tion on a Seph~cryl S-2()() column ( I x 3()
cm) that was previously equilibr~ted with ().ns M pho~ph;~te-t)uffere~ saline
containing 0.1~o bovine serum albumin pH 7.~). Figure 3 shows the iodination
profile of the 1311 to EGF, demonstr~tin~ that ~9()% of the l~ll W;IS l~heled into
the EGF molecule.
C. totoxi~ Assav
A431 cells were grown in 6 well cultured pl~tes. Two of the wells
were exposed to approximately 5()1) ~Ci of radiolabeled EGF ( l~ II-EGF), two ofthe wdls were cxposed to free 1311 similar tn the quantity of 1311-EGF, and the
other two wclls of A431 cells were exposed to unlabeled EGF s;milar to the
quanti~ of 13lI-EGF. The A431 cells were exposed to the three different
35 treatments for one hour. Cells were washed twice w;th PBS buffer, and fed with
DMEM and cultured for S days. The experiment was repeated in four replicate of
SIJBSTI~E SHEE~ "
wo 93/0981 6 2 ~ ~ 3 !J ~ ~ PCl / IJS92/09874
6 well plates. At the en~l of ~;ly 5, cell~ were h;lrve~te~ n~ counte~l. Fi~ure ~.
shows that the wells of A431 cells exp(l~e~ to 1311-EGF have signific~ntly fewerviable cells as comp~red to cells expo~ed tO free 1311 or cells exposed to unl~beled
EGF. Thus, radiolabeled growth factor c~n be used as a specific cytotoxic ~gent
S for human tumor cells which possess high numbers of the growth f~ctor receptor.
EXAMPLE 2
EFFEcrs OF ~ON-RADIOACr VE IODI;~.E LABELED EGF ON A43 1 AI~D L CELL~.
10 A. Preparation of Cell~
A431 cells and L cell~ were prepared ~s descrihed ;lhove in Ex~lmpie
1. L cells are a murine ~Ibroblast cell line which is av~ ble from the ATCC
under accession number CRL 6362. Unlike A43 1 cells, L cells h;lve le~s than 1()00
EGF reccptors per cell. Cells were grown and h~rvested as described above in
15 Example lA.
B. lodination of Epidermal Growth Factor
Epidermal Growth Factor wa~ iodinated utilizin" a procedure
identical to that described in Ex~mple lB above, except th;lt non-radio3ctive
20 iodine (Sigma Chemical Co., St~ Louis, Mo.) was utilizea in pl~ce of radioactive
od~ne.
C. Cytotoxicitv Assav
Cells were prepared and an~lyzed essentially a~ described in
2S Example lC. As illustrated in Figure~ 4 through 7~ EGF ha~ a cytotoxic
effect on A431 cells (see Figure 4), but not on cells with low numl ers of receptors
such as L cells (see Figure 6). When experiment~ were perft)rme(l with EGF
labeled with non-r~dioactive iodine, there was a surprising Cyt~ xic effect similar
to that of EGF labeled 13 lI. Furthermore, the cytotoxic effect did not extend to L
30 cells, indicating that the cytoxic effect w3s mediated by EGF binding to the cell.
EXAMPLE 3
MASIU~G OFEGF-GROWrH FACrt)R RE~:En~)R~ PRIORT ) ADMI~ ~ ~n1~ 231-EGF
In order to determine the biodistribution of 1~31 EGF, the following
experiment was undertaken. Briefly, approximately 1 x 1()6 A431 cells were
SUBSml)TE SHEFr
Pcr/US92/o9874
wo 93/09816
~ 23~ ~8 ~.
injected subcutaneously into nude mice. The cell~ were ~ )w~ t" grow in the
mice for one to two weeks, after which the mice were injecte~ either with or
without unlabeled EGF, followed by the injection of 1231-E(iF. The mice were
then sacrificed and the percent of injecte~l dose per gram determined in the blood,
5 tumor, muscle, lung, kidney, spleen, liver, intestine, thyroid, urine and stomach.
The results of this experiment are set forth below in Tables I and 11.
TABLE I
A-431 With Lsbeled EGF (1-123) Biodistribution
Summarv of Percent Injected Dose Per Gr~m
TL~I, E
.~. . ._ .
Tum~)r Lune ~çen Intes~ine Urine
~QQII ¦ Mu~cle ¦ ~iS~Y ¦ L;ver ¦ Thvroid ¦ ~itomach
~IOUSE
Dormal 3.2 --- 0.6 2.3 14.6 1.7 3S.X 0.~ 0.0 3~.4 2.1
1-1 5.8 3.S 0.6 2.~ 2S.9 2.1 33.1 0.7 ~.5 2.7 1.4
1-2 5.1 2.6 0.6 2.S 24.~ 2.4 43.9 0.3 3.' --- 1
1-3 2.g 13 0.3 1.~; 1().( 1.4 6.0 1.9 2.0 --- 0.6
14 4~1 2.7 0.7 2.~ 22.() 1.~ 14.9 0.~ 3.0 2.( 0.
average4.5 2.5 2.2 2.5 22.3 1.'3 24.5 0.9 2.') 2.7 1. I
5-1 2.~i 4.7 0.7 2.~ 16.~ 2.2 24.1 0.~ 3.9 75.1 8.0
5-2 2.S 2.~i 0.6 2.~ 14.~ 1.9 2~.~ 0.~ ) 24.~ 7.3
S-3 4.7 3.9 1.2 4.6 17.~ 3~ 1 22.() 1 .~ 7.4 39.2 7.0
5-4 4.7 3.9 1.2 4.~ 17.~ 3.1 22.0 1.3 7.4 ~').2 1i.2
average3.2 3.6 O.t~ 3.2 15.6 2.3 2S.5 0.~ S. 1 46.n 9.4
50-3-1 18.6 4.0 1.2 ~.4 30.. ~ 5.4 6.7 3.~~.... --- 3.2
S0-3-2 20.9 1~7 13 9.~ 23.1 6.6 6.3 4.~; 7.() --- 3.5
a~erage19.8 2.9 13 9.1 2~.X 6.0 fi.5 4.2 6.3 --- 3.4
25-10 110.1 2.1 1.3 6.~; 4X.fi 3.' 7.4 2.~ 3.~ 3.6
25-14-1 0.0 0.4 0.0 l () I () ~ ~ 1.5 0-4
- -
5014~ 0.0 03 0.0 0.1 0.1 O.U 0.0 u.n o.~ 13 0.6
SUBSTI~UTE SI~EET -
Wo 93/09816 ~ 1 2 ~ Pcrfus92/09874
TABLE 11
A-431 With Labeled EGF (1-123) Biodistribution
Summarv of Tissue to Blood R~tios
TI~S-~E
Tumor ~ ~Snleen Inte~line yrine
Muscle Killnev Liver Thvroid ~;tomach
MOUSE I I _ I I
normal --- 0.2 0.74.(- O.. S 11.2 I).~ 0.() 11.4 0.7
1-1 O.fi0.1 ~ 4.5 0.~ ~.7 l).l n~ 0.'
1-2 O.S 0.1 O.~S 4.~; 0.~ ().I U.(~ 0.
1.3 O.S 0.1 0.(, ~ .. 2.1 t).7 ().7 --- 0.'
1-4 0.71).2 O.l) j.4 U. 1 3.(~ (). I().7 ~).() 0.'
average 0.6 0.1 0.65.1 0.: 4.0 O~s 0.~ O.fi 0.~ -
~1 1.6 0.2 1.0~.9 0.~ 8.5 0.2 1.4 26.6 2.
S-2 1.1 0.2 1.04.7 0.7 9.S 0.3 1.4 15.6 2.~
5-3 1.0 0.2 1.04.7 0.7 95 0.3 1.4 15.6 2.4
5-4 0.8 0.3 1.03.~Y 0.7 4.7 0.?~ .4 33
a~rerage 1.10.~ 1.0S.() 0.~; ~.S U t 1.(- lS.I 2.9
50-3-1 0.2 0.1 O.S1.~- 0.~ 0.~ U.~ -- O.t
2 0.1l).l 0.~I.l ().~s0.3 0.2 l).:~ --- 0.2
avera~e 0.20.1 O.S1.~ 0.~ 0.~ 0.' ().~ --- 0.'25-10-1 0.20.1 0.74.~ 0.? 0.7 ().~ O.~s -- 0
25-1~1 14.70.~ 2.73.7 1.' 1.7 1.~ I().`~SX.'~ 14.:
50-14-1 9,t 0,'~ 3.(- 0.~ 7.. ; 3X.
. . ,. . , ~
Notes: 1. Norm~l mouse s~crificed at 3 minutes.
2. Group 1 mice sacriffced ~t 3 minutes.
3. Group 5 mice sacr;ficed at 48 minutes.
4. Mice 50-3-1 and 2 were blocked with 50 ug native EGF 3 minutes
prior to labeled EGF; sacrificed at 3 minutes.
5. Mouse 25-1~1 was blocked with 25 Ug n~tive EGF ~ minutes prior
to labeled EGF; sacr;ficed at 1() minutes
6. Mice 25-14-1 and 50-14-1 were injected with 25 u~ ~nd S() u~ n~tive
EGF respectively, ~ minutes prior to labeled EGF; s~criffced ~t 14
hours.
SU~SrllU~E SHEET
wo g3/09816 Pcr/uss2/o9874
2~
21~3~8
As shown in Table II, administration of unlabeled growth factor
sufficient to mask growth factor receptors in normal healthy tissue, results in the
more specific targeting of ~ EGF.
From the foregoing it will be appreciated that, although specific
embodiments of the invention have been described herein for the purposes of
illustration, various modifications may be made without deviating from the spirit
and scope of the invention. Furthermore, various references have been cited
herein which provide additional detail and experimental insight, and are therefore
10 hereby incorporated by reference. Accordingly, the invention is not limited except
as by the appended claims.
While the applicant has disclosed the invention in the context of
diagnosis and treatment of cancer, it is believed that the invention in its broadest
aspect is applicable to the delivery of therapeutic agents to treat other disease
15 conditions; for example, use of growth factor conjugated to a therapeutic agent
sucb as an antibiotic would enhance the efficacy of the therapeutic agent for
trcating diseases such as rheumatoid arthritis.
~ ~ SUBSrlTUrE SHEET
