Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~2~5~33~
METAL CHELATE CONJUGATED MONOCLONAL ANTIBODIES
This invention relates generally to m etal chelate conjugated
monoclonal Antibodies. This invention also relates to a method for
treating cellular disorders, particularly cancer, which employs a
radiometal chelate conjugated monoclonal antibody.
Effective therapeutic methods for the treatment of cellular
disorders such as cancer have been the object of intensive research.
Conventional therapy employs surgery, radiation and chemotherapy. Each
of these methods suffers a serious drawback in that it is not highly
selective between healthy and cancerous cells. In order to be effective,
these methods kill or remove large amounts of healthy tissue.
Furthermore, chemotherapy adversely affects the immune system so that
death or serious illness often arises from fungFl, bacterial or viral
inf ections.
The development of monoclonal antibodies has opened the
possibility of selectively delivering therapeutic agents or diagnostic agents
to specific target cells. Monoclonal antibodies are immunoglobulins of
well-defined chemical structure. A characteristic feature of monoclonal
antibodies is reproducability of function and high specificity.
Radioiodine bour.d directly to monoclonal antibodies has been used
for diagnosis and therapy. Iodin~131 has hsd some therapeutic success
for large tumors, but radioiodine labled antibodies have been ineffective
in the treatment of small tumor foci or metastases. In addition,
specifically bonded antibodies are relatively rapidly catabolized by the
2 5 target cell. Catabolism, therefore, leads to the incorporation of
metabolized iodine in the excretory organs, i.e., kidney, bladder and
stomach. In addition, attempts to transport toxins via monoclonal
antibodies to tumor cells have not resulted in a successful therapeutic
method.
It has been suggested in the literature that
diethylenetriaminepentaacetic acid (DTPA) can form stable met~l
complexes when attached to protein. Krejcarek et al., Biochem. ~
Biophys. Res. Commun. 77:581 (1977) Imaging of target sites in vivo
with radiometal-DTPA conjugated polyclonal antibodies prepared according
to the method of Krejcarek have been reported by Khaw et al., Science
209:295 ~980). Despite separation, by gel chromatography and dialysis,
of free and chelated metal from metal chelate conjugated polyclonal
antibodies the gamma images included in the article show that a high
proportion of the radiometal localized in the liver.
2~33~
-- 2
In one of its aspects, the present invention provides a method of
treating cellular disorders comprising contacting a target cell with
radiometal chelate conjugated monoclonal antibodies wherein said
radiometal is ~n alph~ particle emitting met~l nuclide. In another
embodiment, the present invention contemplates a method comprising
introducing into body fluid metal chelate conjugated monoclons~ antibodies
wherein said conjugated chelate is a derivative of
diethylenetriaminepentsacetic acid, said conjugate being substanti~lly free
of adventitiously bound ions of said metal and retaining substanti~lly all
of the activity and selectivity of the antibody. Such a technique is
suitable for both diagnostic and therapeutic purposes. The present
invention also provides 8 method for producing 8 metal chelate conjugated
monoclonal antibody of an alpha emitting radiometal wherein the metal
chelate conjugated antibody is psssed through a chromotography column
having one or more layers selected from the group of ion retardPtion
resins, anion exchange resins, cation exchange resins and a chelating ion
exchange resin, and a final layer comprising a sizing matrix.
The present invention employes metal chelate conjugated
monoclonal ~antibodies for therapeutic techniques, particularly in vivo.
This invention also provides metal chelated conjugated antibodies
which retain their biological activity and specificity, and which are
substantially free of adventitiously bonded metals. Adventitiously bonded
metals are not stable and result in free metal entering the blood.
Metals which are released in the blood can be bound by transferrin or
25 other metal binding proteins (e.g., ferritin) which are present in blood.
Such bound metals are retained in the circulatory system for considerable
periods of time and are cleared by the reticuloendothelial system (RES).
Such clearance results in a concentration of the metal in the liver and
spleen. It is apparent that random, long term circ~dation of radioactive
3 metals in the body or concentration of radioactive metals in the liver
and spleen are hig}dy undesirable. The practice of this invention can
alleviate these serious problems.
MonocIonal antibodies are immunoglobulins of well-defined chemical
structure, in contrast to polyclonal antibodies which are heterogeneous
35 mixtures of immunoglobulins. A characteristic feature of monoclonal
antibodies is reproducibility of function and specificity, and such
antibodies can be and have been developed for a wide variety of target
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3 ~2255~3~
antigens, including tumor cells. Methods for obtaining monoclonal
antibodies have been extensively discussed and are well-known in the
art. A useful text is Monoclonal Antibodies (R.H. Kennett, T.J. McKearn
~ K.B Bec~tol e~. 1980). 9ee ~ls~ Koprowski et al. Il.S. Patent
:
.
:
F
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4,196,265. The selection of a monoclon&l antibody for the practice of
this invention will depend upon the end use for which the met~l chelate
conjugated monoclonal antibody will be employed. Such selection is
within the skill of the art.
The antibodies are generally maintained in an aqueous solution
that contains an ionic compound. A physiologic normal saline solution
is very often employed and is widely available. Other ionic solutions,
such as those containing sodium or potassium phosphate, sodium carbonate
and the like, are known in the art and may also be employed.
A wide variety of organic chelating agents or ligands can be
conjugated to monoc3onal antibodies. Organic ligands to be conjugated
to monoclonal antibodies may be chosen from among either the natural
or synthetic amines, porphyrins, aminocarboxylic acids, iminocarboxylic
acids, ethers, thiols, phenols, ~Iycols and alcohols or the polyamines,
polyaminocarboxylic acids, polyiminocarboxylic acids, aminopolycarboxylic
acids, iminopolycarboxylic acids, nitrilocarboxylic acids,
dinitrilopolycarboxylic acid, polynitrilopolycarboxylic acids,
ethylenediaminetetracetates, diethylenetriaminepenta or tetracetates,
polyethers, polythiols, cryptands, polyetherphenolates, polyetherthiols,
ethers of thioglycols or alcohols, polyaminephenols, all either acyclic,
macrocyclic, ~cyclic, macrobicylclic or polycyclic9 or other similar ligands
which produce highly stable metal chelates or cryptates. Obviously, the
choice of the iigand depends upon the metal to be chelated and is
within the skill of the art.
The ligand used in certain embodiments of this invention possesses
a nonmetal bonded organic functional group suitable for bonding to the
monoclonal antibody. Functional groups may be chosen from among the
carboxylic acid groups, diazotiazable amine groups, succinimide esters,
anhydrides, mixed anhydrides, benzimidates, nitrenes, isothiocyanates9
azides, sulfonamides, bromoacetamides, iodoacetamides, carbodiimides,
sulfonylchlorides, hydrazides, thioglycols, or any reactive functional group
known in the art as a biomolecular conjugating or coupling agent.
The present inven$ion preferabIy employs a derivative of
diethylenetriaminepentaacetic acid (DTPA). It has been found that DTPA
ligands tightly bind metal ions and that the DTPA derivative (hereinafter
~ 5 ~ iL2Z5930
referred to as chelate~ forms a chelate conjugated monoclonal antibody
that is highly stable7 both with respect to the metal chelate binding
and with respect to chelate-antibody conjugate. These properties are
of great importance, particularly for in vivo applications. Por example,
if the chel&te releases the metal ion after introduction into the blood,
these ions will tend to be bound by transferrin, or the like, and be
distributed generslly in the circulatory system of the body. Moreover,
the ions will ultimately tend to collect and remain in organs such as
the liver and spleen. These effects can have serious consequences
depending on the toxicity of the met~l and its radioactivity.
Furthermore, if the chelate does not form a highly stable conjugate
with the antibody, there is a significant reduc$ion in the amount of
metal delivered to the target site and a corresponding decrease in
efficscy.
In the preparation of the metal chelate conjugated monoclonal
antibodies of the present invention, it is important to avoid m etal
contamination from outside sources. Labware should be plastic or glass
cleansed of exogenous metal. All stock solutions should be metal depleted
by, for example, column chromotography with a suitable resin.
For ease of presentation, the present invention will be described
with respect to the DTPA chelate. The preferred chelate is prepsred
from an amine salt of DTPA. Amine is used broa~y and includes
primary, secondary and tertiary amines that will completely deprotonate
the DTPA. Selection of an appropriate amine is within the skill of the
art and the effica~y of any amine (including ammonia) can readily be
determined. A particldarly preferred amine is triethylamine. At least
about 5 equivalents of the amine is added to an aqueous solution of
DTPA and warmed to complete the reaction. The reaction produces a
pentakis(smine)DTPA salt according to the following equstion wherein
triethylsmine is the amine:
(CH3CH2)3N ~ DTP~ --7 [(CH3CH2)3N] 5DTPA
Solid DTPA-amine s~lt csn be recovered by evsporating or freeze-drying
the solution to rem ove the wster and excess amine.
.
- 6 - 1;~2593~
The actual chelate is a DTPA derivative. A functioP~al group is
added to the DTPA and the DTPA is bonded through it to amine groups
on the monoclonal antibody. Esters of a haloformic acid ~re reacted
with the DTPA-amine salt to make the chelate employed by the present
invention. By ester of a h~loformic acid is meant an ester of the
general formula XC(O)-C~R wherein X is a hslogen, preferably a chloride,
and R is any suitable functional group, preferably containing not more
than about 6 carbon atoms. The selection of R and X is within the
skill of the ~art taking into consideration the stability of the ehelate
and steric hindeance when the chelate is reacted with the monoc~onal
antibody. A preferred ester is isobutylchloroformate.
In an exemplary preparation, approximately equimolar amounts of
haloformic acid ester and DTPA-amine salt are dissolved in a polar
organic solvent such as pure, dry acetonitrile. Excess of the halomformic
acid ester should be avoided because it ~ will block a metal chelation
site on the modified DTPA ligand. The temperature of the reaction is
generally not critical and can be chosen to provide a salt that is either
partially or substantially precipitated. The reaction preferrsbly is carried
out at a temperature low enough to precipitate substantially all of the
haloamine ~salt by-product ~of the reaction. When the amine employed
is triethylamine ~and the ester is an ester of chloroformic acid, the
temperature sho~d be in the range of ~from about -20 C to about -
70C. ~Maintaining the temperature in this range drives the equilibrium
reaction to the~ right, producing a high yield of a mixed carboxycarbonic
anhydridè of DPTA according to the following equation:
X-C~O)-O-R + ~(CH3CH~)3N] 5DTPA ~'
O O
[(CH3CH2)3N] 4DTPA-C~ ,C-O-R ~ [(CH3CH2)3
By carrying out the above reaction in the temperature range
specified, a high concentration of the chelate can be produced
substantially free of the haloamine salt by-product. For example,
approximately 0.25 mM of the pentakis(triethylamine)DTPA salt can be
- 7 - lZ25~3~)
.
dissolved in 0.5 ml acetoni ~ile and reacted with 35 microliters of
isobutylchloroformate. After approximately 45 minutes ~t -70 C, the
solution can be ~entrifuged to remove the precipitate leaving a
supernatant liquid containing the desired chelate at a concentration of
about 0.5 M. The chelate is desirably introduced into the chelat~
antibody conjugation reaction at a concentration of at least about D.25
M in the organic solvent. Such concentrations of chelate permit the
use of relatively small amounts of organic solvents in the conjugation
reaction mixture. ~xcessive amounts of organic solvent in the reaction
mixture sho~d be avoided because the solvent can produce adverse
effects with respect to the biologicAl activity and speci~ city of the
antibody.
The chelate conjugated mono~on01 antibody is formed by adding
the chelate in the organic solvent to an aqueous saline antibody solution.
It is important to carry out the reaction of the modified DTPA and
antibody at a pH not higher than about 7.2. The chelate-antibody
reaction competes with the decomposition of the chelate caused by its
reaction with water. lf the pH is too low, however, the chelate
undergoes acid catalysed decomposition and the biological activity and
specificity of the antibody is diminished. The pH is desirably in the
range of from about 6.0 to about 7.2, preferably as close to 7.0 as
practicable. In this range, the reaction of the DTPA chelate with water
is less detrimental to the chelate-antibody reaction.
While the above discussion has focused on DTPA, it is within the
skill of the art to form conjugates employing other ligands. See, e.g.,
73 Proc. Natl. Acad. Sci. USA 3803 (Nov. 1976)~
To preserve the maximum biological activity of the antibody, the
use of strong acids or bases to adjust pH should be avoided for any
chelate-anti~ody preparation. Use of a strong acid or base can cause
localized denaturation in the solution~ The pH can be controlled in the
aqueous solution of monoclonal antibody by inc3uding a suitable buffer.
For example, NaHCO3 at a concentration of approximately 0.1 M can
be used. Other buffers such as MES (2-(N-morpholino)ethane sulfonic
acid) are known in the art and may also be employed. The choice of
an appropriate buffer is within the skill of the art.
`' .'
- 8 - 122~930
When the chelate solution is added to the aqueous antibody
solution, both should be at about 0C. The temperature of the solution
generally should not be ~llowed to rise above abou~ 4 C during the
course of the reaction. Use of temperatures in the range of about 0
to about 4C tends to ~void decomposing the antibody and also reduce
chelate decomposition. Duration of the reac~ion is not critical so long
as the reaction is permitted to go to completion and the solution may
be lef t in the cold overnight.
The chelate-to-antibody mole ratio may vary widely depending
upon the use for which the conjugate is intended. The mole ratio of
chelate-to-antibody can broadly range from about 0.1 to about 10 or
higher and preferably from about 0.25 to about 5. In many instances
the mole ratio of chelate to antibody will range from about 0.5 to
about 3.
In general an excess of chelate is employed in the reaction because
the chelate will decompose to ssme extent in the aqueous solution. The
number of chelates bound per molecule of antibody will be a function
of both the concentration of the chelate and the concentration of the
antibody in the reaction mixture, with high concentrations tending to
provide more chelates per antibody. If the amount of antibody employed
is relatively small and a relatively dilute solution is employed, a
substantial ~ excess of chelate may be required. For example, a molar
excess of approximately 600:1 of chelate may be required to react with
an antibody solution having a protein concentration of about 5 to 10
mg per ml in order to provide approximately 1.5 chelates bonded per
molecule of antibody. Molar excesses as low as 100:1 can be employed,
however, and still produce an average of about O.S chelates bonded per
moleclde of antibody. Adding too many chelate molecules per antibody
molecule can reduce the biological activity and specificity of the
antibody.
When the addition of the chelate to the anffbody has gone to
completion, substantial amounts of decomposed chelate may be present
in the soiution. This can occur for any chelate-antibody conjugate.
The decomposed chelate should be removed while retaining the biological
activity and specificity of the antibody. Dialysis or chromatography
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~-- 9 --
1225930
can, for example, be employed. If desired, a first dialysis against dilute
ascorbic acid and EDTA solution to remove any residual iron which may
be present in the chelate or the protein. A purified chelate conjugated
antibody can be produced by dialysis of the reaction mixture over a 48
hour period against three l liter changes of an aqueous solution at about
4C and a pH of about 6 containing 50 mM ci~ate and 200 mM sodium
chloride with 1 ml Chelex lOO*resin (13io-Rad)~n the dialysis vessel. A
final dialysis into l liter of solution containing lO mM MES and 200 mM
sodium chloride at 4C and pH 6 completes purification of the protein.
10 Variations of dialysis procedures are known and are within the skill of
the art.
Metal chelation is carried out in an aqueous solution and, once
again, desirably av~ids the use of strong acids or bases. Metal chelation
for any chelat~antibody conjugate is carried out at a pH which does
15 not significan'dy reduce the biological activity or specificity of the
antibody. ~enerally, the acceptable range is from about pH 3.2 to
about pH 9, however, parffcular anffbodies may have to be restricted
to a narrower range. At a pH below about 3.5, advenfftious binding
of metal ions to anffbodies is substantially impaired for many metals.
20 A preferred range, therefore, is often from about pH 3.2 to about pH
3.5. Factors peculiar to solutions of the metal employed, however, may
permit a pH above 3.5. The selection of the appropriate pH within
the range is within the skill of the art.
In the present invention, a weakly chelating acid or base is
25 desirably employed as a buffer. Citric acid or glycine are useful buffers.
Still other buffers are, of course, known in the art. The present
invention contemplates a solution of chelate conjugated antibodies
adjusted to the desired pH with a wea~dy chelating acid or base buffer
and without the addition of a strong acid or base. To this solution is
30 added a metal salt. If the metal salt is in solution, that solution also
has its pH adjusted with a chelating buffer. The pH of the metal
solution, however, can be adjusted with strong acids or bases prior to
its addition to the chelate conjugated antibody solution7
Any acceptable metal salt can be employed to make the met~l
3 5 chelate conjugated monoclonal antibodies. Typical salts may include
* Tr adema rks
- 10 - 1225g3~
h~lides (e.g, cMolqdes), nitr~tes, perc~orates, or the like. The metal
s~lt is employed in as high a concentration ~s is practicable; Radiation
exposure of individuals h~ndling the prep~ration will generally set a limit
below one equivalent metal per chelate binding site.
The duration of the reaction is not critical unless the pH is near
the outside limits of pH ~cceptable to the antibody. At or near such
pH limits, the reaction times generally should be under about 1 hour
and prefer~bly about 30 minutes or less. Indeed, from the standpoint
of economy of time, reaction times generally within these periods are
desired. It is also preferred to carry out the chelation in the presence
of Q water soluble, nonchelatable, biologically innocuous reducing agent,
such as ascorbic acid, to prevent trace iron from being chelated. The
reaction is usually completed by adding trisodium citrate in sufficient
quantity so that the solution pH is raised to a point that the metal
conjugate is no longer labile. It has been determined that most DTPA
complexes are at especially stable at a pH of about 6. Other weak
bases, or acids when the reaction is above pH 6, may be used so long
as they do not adversely affect the antibodies. Their selection is within
the skill of ~the art.
The reaction solution will generally require purification prior to
its use in v_, and may ~lso require purification prior to in vitro use.
Nonbonded metal and adventitiously bonded metal should be removed.
The discussiqn herein refers to adventitiously bound metsl ions. Some
of the metal, however, may be insecurely held by the chelates and acts
in the same manner as adventitiously bound metP~ ions.
Wh0n a radioactive metal is employed which has a short half life,
it is especially important that the purification step be as expeditious
as possible. The present invention contemplates a relatively fast
purification by use of chromatography and this facet of the invention
is applicable to chelate-antibody conjugates in general. By employing
one or more ion exchange, retardation or chelating resins in conjunction
with a sizing matrix (e.g., gelj the met~l chelate conjugated monoclonal
antibodies of the present invention can be quickly and thorouglily purified.
Different ion exchange resins can be employed singly, or any
combination of an ion retardation resin, a cation exchange resin, an
122593~
anion exchange resin or a chelating ion exchange resin eQn be employed.
The selection of an appropriate resin or resins, th~ir extent of class-
linkage, chemical form and mesh size is within the skill of the art.
Cation exchange resins employed in the present invention frequently
S are stron~y acidic polystyrene gel-type resis (e.g., Dowex *50WX8) or
other non-polystyrene stron~y acidic resins such as Zeocarb*215 ~Permutit
Co.). Additional stntable 8cidic resins can in~ude weakly acidic gel
polystyrene resins, macroporous gel polystyrene resins, or macroreticular
carboxylic acid cation exchange resins. Anion exchange resins can include
strongly bQsic polystyrene gel-type resins (e.g., Dowex lX8) or other less
basic resins such as pyridinium polymer-t~pe and phenolic polyamin~type
resins. Chelating resins may be Chelex 100, or any resin of the type
which is a styrene divinyl benzene copolymer containing paired
imminodiacetate ions (e.g, Dowex A-l). Useful retardation resins include
those containing paired anion and cation exchange sites te.g., Bio-Rad
AG l~A8). These resins are usually made by polymerizing acrylic acid
inside a strongly b~sic resin such ~s one having quaternary ammonium
groups in a styrene-divinyl benzene copolymer lattice. The above
discussion includes only representative examples of each resin; still other
2 0 resins are~ also known in the art. A compendium of commercially
availsble resins with brief ~descriptions of their properties and applications
is contained, inter alia, in ~Bio-Rad Laboratories, 1982 Price List lI. The
choice and combination of resins will depend upon the particular
separation problem encountered and is within the skill of the art in
~5 view of the disclosure herein. A useful reference is J. Khym, Analytical
Ion-Exchange Procedures in Chemistry and Biology (1974).
Sizing matri~es dre also well known in the art. These include
polyacril~mides, agraroses, polysaccharides or the like. A particularly
useful sizing matrix is a polysaccharide gel (e.g., SephadeX'~ G-50 gel).
Examples of poiyacrilamide gels are the Bio-Gel*P Series (Bio-Rad Labs).
The choice of the sizing matrix will depend upon the protein to be
purified and is within the skill of the art.
In the practice of this invenffon the various resins can be
established as layers within a cdumn and the solution to be purified
3 5 can be fed either downwardly through the column or upwardly through
* Trademarks
~,
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- 12 - l.Z2S930
the column. Downward feed is a preferred laboratory technique when
radioactive compounds are employed because gravity flow requires little
or no auxilary equipment or instrumentation. The choice of bed heights,
nOw rates, and the like are easily within the skill of the art.
It appears that highly charged metals are adventiffously bound, at
times, by the antibody at ionic sites along the surface of the protein.
At other times adventiffously bound metals appear to be included with
the folds of the antibody protein. These metals can be released into
solution but also can be reabsorbed from the solution in an equilibrium-
type process. The retardation or ion exchange resins employed in the
purification oi this invention are used in order to shift the equilibrium
and permit metals ~ to be removed from the antibody. For example, as
the antibody passes through an ion retardation resin, the passage OI
metal ions in the solution is slowed, but the protein is not. Adventitiously
bonded, higly charged (+3 or higher), metal ions are then released into
the solution to reestablish equilibrium. As those ions are released into
solution, however, they in turn are retarded by the resin to cause a
continuing metal ion release by the antibody.
A lev~el :of ion exchange resin may be employed below the ion
retardation resin to tigmly bind the separated, hig~y charged ions and
to continue the separation process. As the resin depletes the protein
solution of free, highly charged ions, equilibrium is again reestablished
between free and adventitious metal ions. However, throughout this
process, met~l ions inside the chelate are retained with the antibody.
It has been determined, however, that mere use of an ion
retardation or ion exchange resin is not satisfa~tory to provide an
effective removal of substantially all adventitiously bound metals. In
order to complete the purification a sizing matrix is employed. The
antibody solution which enters the matrix is areadly partially depleted
in free, highly charged metal ion content. In the sizing matrix, further
depletion occurs. As the solution moves through the matrix, the
ani tbodies are not retarded while the ions are. The resulting solution
taken off from the sizing matrix is substantially free of adventitiously
bound metals. Such loosely bound metals can be reduced to not more
than about six percent of the total rnetal content of conjugate so that
.~.~
- 13 - 92Z59:~0
at least about 94% of the met~l carried by the conjugate is bolold by
the chelate stably. Desirably, at least about 97% of the toW metals
bound by the chelate. It is possible to obtain metal levels in which
98% or more of the metal is bound by the chelate. Dialysis can be
employed to determine stably bo~d metal content.
A preferred method of purification is an ion retardation resin
(Bio-Rad AG l~A8) over a cation exchange resin (Bi~Rad AG 50WX8)
and a gel (Pharmacia Sephadex G-50). In the puriScation of technicium
chelate conjugated mono~on~ antibodies for the present invention, the
preferred column contains an ion ret~rda~ion resin (Bio-Rad AG l~A8)
over a cation exchange resin (Bio-Rad 50WX8) over an anion exchange
resin ~Bio-Rad AG lX8) and a si~ing matrix gel (Pharmacia Sephadex
G-50).
In the process of this invention, the antibody is retained in
nonaggregated form. Aggregation of antibodies, whether ~y clumping or
by cross-linking, res~ts in Q loss of antibody specificty which, of course,
is undesirable. Aggregation can be caused by excessively high
concentrations of antibodies in a carrier, or by contact with chemicals
that cause protein cross-linhng such as, for ex~lmple, carbodiimdes.
Standard sedimentation tests, size matrixing, or the like, can be employed
to determine if the antibodies have aggregated. Indeed, the antibody
specificity tests discussed herein will reflect aggregation as a loss of
specificity.
The activity and specificity of the conjugated antibody products
of this invention are maintained at a level of at least about 80%, and
preferably at least about 90% of the activity and specificity of the
antibody that was employed to produce the conjugate. Partic~arly
preferred solutions are characterized by antibody activity and specificity
of at least about 95% and, indeed, products have been produced which
retain the activity and specificity of the original antibody virtually
unchanged. The activity and specificity of antibodies are routinely
messured in the art by binding of antibodies, in vitr~ to an epitope.
The degree of activity and specificity of the final antibody product can
readily be determined simply by repeating the initial test with the final
conjugsted product.
- 14 - ~LZ25930
The invention contemplates an in vivo ther~peutic procedure in
which radiometal chelate conjugated monoclonal antibodies are introduced
into the body and ~llowed to concentrate in the target region. There
are a variety of radiometal isotopes which form stable DTPA complexes
and emit cytotoxic alpha particles. The therapeùtic effect occurs when
the conjugates are near or in contact with and bind to the targeted
cells. Cell death, it is believed, is a direct or indirect result of the
radiation event of the radiometal which is positioned in close proximity
to the cell.
The benefits of this aspect of the invention are several. ~irst,
the high specificity of the conjugated monoclonal antibody minimizes
the total radiation dosage. Only enough radiation for the target cells
need be employed. In addition, radiomet~l chelates generally are cleared
rapidly from the body sho~d $he conjugated antibody be disrupted. The
isotope can be short-lived and the affinity constant by which the isotope
is retained in the DTPA chelate is very high resulting in a stably bound
metal. Finally, since the the amount of radiometal employed is
minimized, the radiation hazard to persons preparing and administering
the radiometal chelate conjugated antibody is significantly reduced.
E~ecause of the properties of the DTPA radiometal chelate
conJugated monocIonal antibody employed by the present invention, tissue
damage ~ OF whole body dose during therapy ~are markedly reduced as
compared to~ that~from presendy employed methods of radiation therapy
such as isotope implflnts, external radiation therapy, and
immunoradiotherapy employing iodine-131 labeled polyclonal or autologus
antibodies. Additionally, both biological and physical half-lives of the
targeting radiobiological may now be controlled, minimizing whole body
radiation effects. Since radiation is targeted specifically to cell types
(e.g., ne~plastic cells) a therapeutic dose is delivered specifically to
m~lign~nt cells, either localized or met~stasized. The ability of
radiomet~l ahelate conjugated monoclonal antibody to provide an effective
dose of therapeutic radiation specifically to metastasized cells is also
unique and singldarly useful for cancer therapy.
The present invention employs the metal chelate conjugated
monoclonal antibody containing an alpha emitting radiometal to treat
;
~ 15 ~ 122~i~30
cellular disorders. It is desiraWe in most applications that the radiometal
have a half-life of less than ~bout 4 days and decay rapidly to a stable
isotope once the alpha particle is emitted. The preferred isotopes
employed in the present invention ~re bismuth-211, bismut~212, bismut~
213 and bismut~214. Bismuth-212, with a half-life of 60.6 minutes, is
particuarly pref erred.
The monoclonal antibody employed is specific Ior the diseased
cell which is to be killed. Cell death is caused by decay of the
radiometal and can occur in one of two ways. First, if the alpha
particle is emitted in the direction of the diseased cell, a single hit in
the cell nucleus can be cytotoxic. The isotope to w~ch the radiometal
decays after emitting the alpha particle is ejected from the chelate on
a trajectory opposite that of the alpha particle. The bound cell,
therefore, can still be hit even when the alpha particle is emitted on
a trajectory away from the cell. A single hit in the cell membrane
by the decayed isotope can cause irreparable cell injury leading to cell
death. The relatively high effectiveness of the alpha particle means
that less radioactive material c~n be employed. Selectivity of the
monoclonal antibody and the short range (a few cell diameters) of the
alpha particles minimizes the destruction of healthy tissue on a cellular
level.
Bismuth-212 decays by one of two different pathways.
Approximately 64% of the bismuth-al2 decays via beta emission to
polonium-212 which has a half-life of 0.3 microseconds. The polonium-
212 decays to stable lead-208 after emitting an alpha particle with a
range of approximately 90 microns. The other 36% of the bismuth-212
decays to thallium-208 by emitting an alpha particle with a range of
approximately 35 to 50 microns. The thallium-208, with a half-life of
3 minutes, then decays via beta emission to stable lead-208.
Generators f or Bi-212 have been described in the literature by
Gleu, et al., Z.Anorg. Alleg. Chem. 290:a70 (1957), and by Zucchini, et
al., Int. J. Nucl. Med. Sc Biol. (June, 198a), (the abstract of the manuscript
was distributed at the August, 1981 ACS meeting in New York3. A
useful generator consists of Th-228 in the tetravalent state absorbed on
a 3 x 5 mm bed of sodium titanate contained in a quartz column above
- 16 - 12~593~
a coarse fritted glass disc sealed in the column. The titanate tigmly
retains both T~228 and its Ra-224 daughter. When water is passed
through the tit~nate, the Rn-220 daughter of the Ra-224 isotope dissolves
into the water and passes through the fritted disc and is ~ollecte~ in
a 10 cc glass reservoir filled with water. The aqueous Rn~220 solution
flows from the reservoir into a 10 mm diameter column containing
approximately 1 ml of a strong~y acidic ion exchange resin such as Bio-
Rad A~50 WX8 cation exchange resin. Rn-220 decays substantially
within 5 minutes in the reservoir to Pb-212 which is absorbed upon
passage through the resin. At flow rate of appro~matelg 1.5 ml/min.
through the resin, about B5% of the Pb-212 produced is collected in the
column where it decays to its Bi-21? daughter.
When the desired amount of Bi212 has been formed on the resin,
it may be eluted by acid according to procedures entirely f&milar to
those skilled in the art. A useful method of elution for both P~212
and Bi-21a is to pass S ml of a N HCl through the resin. Alternatively,
if only Bi-il2 is desired, 1.5 ml of 0.5 M HCl can be passed through
the resin.
While metals that emit beta particles or Auger electrons can be
employed far therapy, alpha emitting radiomet~ls are preferred for
several reasons. First, alpha nucleotide radiation characteristically has
a short range in tissue and a very high linear energy transfer vi~a-vis
beta or Auger radiation. Alpha radiation can kill a cell with only one
hit to the nucleus and will kill substantially any cell with lO hits or
less. In addition, the decay also emits an isotope (e.g., Tl-208 or P~
aos in the case of Bi-212) which can also cause cell death. The range
of alpha particles is usually less than about 150 microns in tissue. In
contrast, beta and Auger particles require hundreds of hits in the nucleus
before causing cell death and have ranges in tissue on the order of
tenths of millimeters to centimeters. When employing beta particles,
a higher dose is required and the decay of substantially more radiolabeled
antibodies will be needed to achieve cell death Thus, specific~lly bound
antibodies will be catabolized releasing beta emitting radiometals into
the blood. Alpha-emitting radiometals kill relatively quicldy so that
fewer antibodies are catabolized.
- 17 - ~2593~
The metal chelate conjugated antibodies of this invention can be
administered in vivo in any suitable pharmaceutic~ carrier. As noted
earlier, a physiologic normal saline solution can appropriately be
employed. Often the carrier will include a minor amount of carrier
protein such as human serum albumin to stabilize the antibody. The
concentration of metal chelate conjugated antibodies within the solution
will be a matter of ~hoice. Levels of 0.5 mg per ml are readily
attainable but the concentrations may vary considerably depending upon
the specifics of any given application. Appropriate concentrations of
biologically active materials in a carrier are routinely determined in the
art.
The effective dose of radiation or metal content to be utilized
for any application will also depend upon the partic~ars of that
application. In treating tumors, for example, the dose will depend, inter
upon tumor burden, accessability and the like. Somewhat similarly,
the use of metal chelate conjugated antibodies for diagnostic purposes
will depend, inter ~ upon the sensing apparatus employed, the location
of the site to be examined ~nd the like. In the event that the patient
has circulating antigen in addition to those located at the site, the
circulating antigens can be removed prior to treatment. Such removal
of antigens can be accomplished, for example, by the use of unlabeled
antibodies, or by plasmaphoresis in which the patient~s serum is treated
to rem ove antigens.
The following examples are in~uded to better illustrate the
practice of this invention. These examples are included for illustrative
purposes only and are not intended in any way to limit the scope of
the`invention.
EXAMPLE I
One hundred miligrams of DTPA was weighed into a flask and to
this was added 1 ml of water. This solution was reacted with 0.125 g
redistilled triethylamine. The reaction solution was warmed to complete
the reaction and a solid product was collected by freeze dryin~.
The freeze dried solid was dissolved in G.5 ml of pure, dry
acetonitrile and 35 ul isobutylctdoroformate added at a temperature of
.::
- l~ 122S93~
approximately -20 C and brought down to about -70 C. After about
45 minutes, the solution was centrifuged in an Eppendorf vi~l. The
supernatant liquid was collected which contained the desired mixed
carboxycarbonic anhydride of DTPA at a concentration of approximately
0.5 M.
The monoclonal antibody employed was designated 103A5 and was
obtained by fusing P3X63Ag8 mouse myeloma cells with the isolated
spleen cells of C56BV6 mice which had been imm~ized with purified
retro~irus glycoprotein of 70,000 daltons (gp70) obtained ~s described by
M. Strand and J.T. August, 251 J.Biol. Chem. 559 (1976). The fusion
was carried out QS described by M. 5trand, 77 Proc. Natl. Acad. Sci.
USA 3234 (1980).
A 114 ul solution containing 2 mg of monoc~onal antibody 103A5
in 0.1 M NaHCO3 at a pH of approximately 7.2 and 150 mM sodium
chloride was prepared and pipetted into a Nunc vial. Then, 33 ul of
a 0.1 M NaHCO3 solution at a pH of 7.0 was added to the vial. Finally,
26.4 ul of the mixed carboxycarbonic anhydride of DTPA (0.5 M in
acetonitrile) was added after cooling the chelate and antibody solutions
to approxmately 0 C. The reaction was allowed to proceed overnight.
The product was first dialyzed at 4C, against one liter of 30
mM ascorbic acid, 5 mM EDrA, 200 mM NaCI and 20 mM of sodium
citrate (pH 7.0). The res~ting solution was dialyzed at 4C against
three one liter changes of 50 mM citrate, 200 mM sodium chloride at
pH 6.0, and ~ 1 ml Chelex 100 resin (Bio-Rad) over a 48 hour period.
Finally, the resulting solution was dialyzed for 8 hours against one liter
of a solution that had a concentration of 10 mM MES and 200 mM
sodium chloride at pH 6Ø Approximately 1.7 mg of chelate conjugated
monoclonal antibody was recovered. Analogous experiments employing
C-14 l~bled DTPA were analyzed by scintillation counting and shown to
contain approximately 1.5 chelates per antibody molecule.
Forty microliters of Indium-lll cMoride solution (New England
Nuclear Corp.) was adjusted to pH 3.0 by the addition of 11.4 ul of 0.4
M citric acid at pH 5Ø A separate solution was prepared containing
250 micrograms of chelate conjugated monoclonal antibody in a total
volume of 21.6 microliters. The solution hQd a concentration of 200
.~
-- ~Z2~i930
-- 19 --
mM sodium chloride and 10 mM MES at a pH of 6Ø The solution was
adjusted to pH 4.6 by the addition of 6 ul of 0.25 M citric acid at a
pEI of 3Ø
The met~l chelete conjugated monoclonal antibody was prepared
by combining the indium chloride and chelate conjugated antibody
solutions and allowing them to react for ~pproximately 30 minutes at
ambient temperature. The reaction was terminated by adding 25 ul of
a saturated solution of trisodium citrate to adjust the pH to about 6.
The chelate conjugated antibody was purified by chromatography
on 9 cm long column containing 1.0 ml of an ion retarda~ion resin (A~
l}-A8 available from Bio-Rad) above ~.0 ml of a cation exchange resin
(AG-50-WX8, H form, 200-400 mesh available from Bio-Rad) above 7
ml of Sephadex G-50 gel (Pharmacia). A solution with concentrations
of 200 mM sodium chloride and 10 mM MES at a pH of 6.0 was used
as the eluant and was used pre-equilibrate the column.
The eluate was collected in 0.5 ml fractions~ The two fractions
with most of the protein were shown to contain 150 ug of monoclonal
antibody labeled with 157.1 microcuries Indium-lll. Dialysis at 4C against
one liter of an aqueous solution of 20 mM MES and 200 mM sodium
chloride at pH 6.0 showed less than 6% loss of Indium. The antibody
was shown to retain ~substantially 100% of its biological activity and
specificity by in vitro tests. In vivo imaging in leukemic mice hig~ighted
the tumor site in the spleen. When administered to normal mice there
was no uptake by the spleen.
EXAMPLE II
A hybridoma was obtained by fusing P3 653 mouse myeloma cells
With the isdated spleen cells of C56BV6 mice which had been immunized
with purified tumor-associated ferritin isolated from the human spleen.
A hybridoma was isolated that produced an anti-ferritin antibody
designated 263D5. The antibody was specific for human ferritin and
did not react with ferritin of other mammalian species.
The procedure of Example 2 was repeated to provide an indium-
111 containing DTPA conjugated monoclonal antibody. A physiologic
normal saline solution containing the met~l chelate conjugated monoclonal
- 20 - ~2Z593~
antibody was injected into normal and leukemic mice. In both the
leukemic and normal mice, radio imaging showed that there was no
concen~ation of radio labeled metal. These tests demonstrated that
the chelate was stable in vivo both with respect to the chelat~antibody
conjugation and with respect to the retention of the radioactive metal.
Neither the s~leen nor the liver was highlighted in the images.
EXAMPLE m
The f~illowing example employs a gamma particle emitting isotope
to demonstrate the selective localization of radiometals (including alpha
particle emitting isotopes) achieved by the present invention.
Indium-lll chelate conjugated monoclonal antibodies were prepared
from an antibody specific for human breast tumor. The hybridoma that
produced the ~ntibody was prepared from a fusion of mouse myeloma
and mouse spleen cells The hybridoma and antibody are described in
78 Proc. Natl. Acad. Sci. 3199 (1981).
The procedure employed was substantially the same as the
procedures of Examples I and II, except for the following. First, the
step of dialyzing the chelate conjugated monoclonal antibody against
ascorbate-ED~A was omitted. Second, lO~microliters of 0.1 M ascorbate
at pH 4 was added to the indium-lll solution prior its addition to the
aqueous saline solution of the chelate conjugated monoclonal antibody.
The labeling efficiency e~ibited a three-fold increase over the
methods of Example I and II. The final product was labeled with
approximately 2.1 microcuries per microgram.
Ten micrograms of the indium-lll chelated conjugated monoclonal
antibody collected from the purification column was diluted to 100
microliters with an aqueous solution of phosphate buffered s~line. The
diluted indium-lll conjugated antibody was injected into the tail vein of
a nude, athymic mouse in which a human breast tumor had been grown.
The human breast tumor cells expressed an antigen for the antibody.
Seventy-two hours after injection, a cle~r and well-defined gamma camera
image demonstrated high localization of indium-lll in the tumor tissue.
No similar localization of the indium-lll in the liver or spleen was
observed.
".J
- '
- 21- 1225g30
EXAMPIE IV
The following tables demonstrate that a radiomet~l-DTPA chelate,
as opposed to free radiometal, does not l~cplize in the liver and spleen
and is rapidly excreted through ~he kidheys and stomach. The upt~ke
of radiometal into the organs of normal and leukemic mice was
determined by the following procedure. Six-week old normal mice and
mice made leukemic eight days previou~ly by the injection of Rauscher
leukemia virus were injected intraperitoneally with 5 micrograms (5
microcurie per microgram) of free Bi-207, DTPA chelated Bi-207 and
DTPA chelated Sc-46. Eighteen and forty-two hours later, mice were
sacraficed, their organs weighed, and the amount of radioactivity
associated with the organs determined. In order to normalize for
differences in the injections, in body weights, and in times of excising
the organs, the amount of radioactivity per gram of tissue was divided
by the amount the radioactivity per gram of blood, and results are
expressed as this ratio. The results are shown in Tables 1, 2 and 3.
TABLE l
MEANS AND STANDARD ERRORS OF THE MEAN OF RATIO/BLOOD
OF S ORGANS OF 14-DAY LEUKEMIC AND NORMAL2duICE
AT 18 AND 42 HOURS AFTER INJECTION OF FREE Bi .
18 HOURS 42 HOURS
_
TISSUE LEUKEMI_ NORMAL LEUKEMIC
HEART 2.35 + 0.65b 4.0 + 0.30 1.48 + 0.12
LIVER 31.3 + 7-50d 29.0 + 0.35 43.3 + 2.65
SPLEEN 7.18 ~ 2.03 21.I + 0.65 8.70 + 0.66
KIDNEY 69.4 + 43.7 612.6 + 47.8 89.3 ~ 9.18
STOMACH 1.35 + 0.35 6.95 + 0.65 5.01 + 0.43
a = injected dose = 7 X 106 cpm per mouse
b = approximately 0.13% of total cpm in heart
c = approximately 40% of total cpm in liver
d = approXimately 12% of total cpm in spleen
- 22- ~;22~i93~
TABLE 2
.,.
MEANS AND STANDARD ERRORS OF THE MEAN O~ RATIO/BLOOD
OF 5 ORGANS OF LEUKEMIC AND ~pRMAL MICE AT 18 HOURS
AFTER INJECTION WITH 2 E~l-DTPA CHELAT~
207BI_DTpAa
TISSIJE LEUKEMIC NORMAL
HEART 2.60 + 0.33b 2.45 + 0.21
LIVER :I9.12 + 6.3~c10.93 ~ 1.24
SPLEEN 13.1 + 2.6 12.4 + 2.0
KIDNEY 294.0 + 37.0 281.0 t 26.0
STOMACH ~.52 + 2.44 7.20 + 2.90
a = injected dose 3.8 X 106 cpm per mouse
b = approximately 0.003% of total cpm in the heart
c = approximately 0.2196 of ~total cpm in the liver
d = approximately 0.06% of total cpm în the spleen
TABLE 3
::
MEANS AND STANDARD ERRORS OF THE MEAN OF RATIO/BLOOD
OF 5 ORGANS OF: LEUKEMIC AND gORMAL MIGE AT 18: HOURS
APTER INJECTiON WITH 4 SC-DTPA: CHELATE
46BC-DTPAa
TISSUE LEUKEMIC NORMAL
HEART 3.66 + 0.21b 4.49 + 0.84
LIVER 9.96 + 0.57d 7.23 + 1.04
SPLEEN 3.17 + 0.39 5.69 ~ 0.28
KIDNEY 42.1 + 0.9 29.5 + 5.5
STOMACH 12.45 ~ 5.43 7.76 + 2.06
a = injected ~dose 5.4 X 106 cpm per mouse
b = approximately 0.006% of total cpm in the heart
c = approximately 0.14% of total cpm in the liver
d = approximstely 0.08% of total cpm in the spleen
..
~.
- 23- ~Z;~593(~
The data in the above tables demonstrates that DTP-A chelated
bismuth and scandium do not concentrate in the liver or spleen of mice
as opposed to free bismuth. High concentrations of chelated metal in
the kidneys demonstrates that it is being voided through the urine. The
variation in kidney concen~ations between leukemic and normal mice is
attributable ~o fr~quent voiding by the leukemic mice due to stress
EXAMPLE V
Tests were conducted to determine the effect of bismuth alpha
radiation on mammalian cells. ~-46 leukemic cells were grown ~n vitro
in Dulbacco's Modified Eagle medium containing 10% heat inactivated
fetal calf serum to provide a cell population of 1 x 105 in each well.
The cell populations were exposed to bismuth-212 by ~dding serial dilutions
(as indicated in Table 4) in the growth medium. The cells were then
grown for 96 hours and the number of surviving cells was determined.
The results are shown in Table 4 below.
TABLE 4
Dose Mean Number Standard %
(Rads) of Survi~ing Deviation Survival
cells (10 )
0 6.9 1.4 100
0.2 8.0 0.5 116
0.4 6.1 1.7 88
0.8 6.7 1.4 97
1.5 6.0 2.8 87
3.1 6.0 0.6 87
6.2 5.0 0.6 72
12.3 2.9 0.4 42
24.6 2.2 0.4 32
49.2 1.1 0.4 16
98.4 0.6 0.1 9
From the Qbove data of Table 4, employing standard calculation methods,Do (37% survival) is 38.5 rads. This demonstrates that bismuth-212
emits highly cytotoxic, densely ionizing radiation. By comparison, 900
rads of sparsely ionizing radiation from a cobalt-60 source was required
.
3LZ2~i930
- 24 -
to achieYe the same results. For ~ discussion of radiation doses see;
nm/mird pamphlets No's 1 (revised) and 10.
Since modific~tions will be apparent to those skilled in the art,
it is intended that this invention be limited only by the scope of the
appended cl~ims.
