Note: Descriptions are shown in the official language in which they were submitted.
CA 02010164 1999-10-25
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CT-1922A
ANTHRACYCLINE IMMUNOCONJUGATES HAVING A NOVEL LINKER
AND METHODS FOR THEIR PRODUCTION
TECHNICAL FIELD OF THE INVENTION
The present invention relates to novel anthracycline
immunoconjugates and methods for their production. More
particularly, the present invention relates to immunocon-
jugates comprised of an antibody reactive with a selected
cell population to be eliminated, the antibody having a
number of cytotoxic anthracycline molecules covalently
linked to its structure. Each anthracycline molecule is
conjugated to the antibody via a linker arm, the
anthracycline being bound to that linker via an
acid-sensitive acylhydrazone bond at the 13-keto position of
the anthracycline. A preferred embodiment of the invention
relates to an adriamycin immunoconjugate wherein adriamycin
~s attached to the linker arm through an acylhydrazone bond
at the 13-keto position. The linker additionally contains a
~~.i~.~.~,~.~i ~~
disulfide or thioether linkage as part of the antibody
attachment to the immunoconjugate. In addition, according
to this invention, new acylhydrazone derivatives of the
anthracycline are synthesized and used in the preparation of
the immunoconjugates of this invention.
The acid-sensitive acylhydrazone bond of the immuno-
conjugates of this invention allows for the release of
anthracycline from the immunoconjugate in the acidic
external or internal environment of the target cell. The
immunoconjugates and methods of the invention are therefore
useful in antibody-mediated drug delivery systems for the
preferential killing of a selected population of cells in
the treatment of diseases such as cancers and other tumors,
non-cytocidal viral or other pathogenic infections, and
autoimmune disorders.
BACKGROUND OF THE INVENTION
Anthracyclines are antibiotic compounds that exhibit
cytotoxic activity. Studies have indicated that anthra-
cyclines may operate to kill cells by a number of different
mechanisms including: 1) intercalation of the drug
molecules into the DNA of a cell thereby inhibiting
DNA-dependent nucleic acid synthesis; 2) production by the
drug of free radicals which then react with cellular
macromolecules to cause damage to the cells or 3)
-2-
I. n I
interactions of the drug molecules with the cell membrane
[see, e.g., C. Peterson et al., "Transport And Storage Of
Anthracyclines In Experimental Systems And Human Leukemia",
in Anthracycline Antibiotics In Cancer Therapy, F.M. Muggia
et al. (ed.s), p. 132 (Martinus Nijhoff Publishers 1982);°
see also, N.R. Bachur, "Free Radical Damage", id. at pp.
97-102). Because of their cytotoxic potential, anthra-
cyclines have been used in the treatment of numerous cancers
such as leukemia, breast carcinoma, lung carcinoma, ovarian
adenocarcinoma, and sarcomas [see, e.g., P.H. Wiernik,
"Current Status Of Adriamycin And Daunomycin In Cancer
Treatment", in Anthracvclines: Current Status And New
Developments, S.T. Crooke et al. (eds.), pp. 273-94
(Academic Press 1980)). Commonly used anthracyclines
include adriamycin and daunomycin.
Although these compounds may be useful in the treatment
of neoplasms and other disease states wherein a selected
cell population is sought to be eliminated, their
therapeutic efficacy is often limited by the dose-dependent
toxicity associated with their administration. For example,
in the treatment of tumors, myelosuppression and
cardiotoxicity are typical adverse side effects [see S.T.
Crooke, "Goals For Anthracycline Analog Development At
Bristol Laboratories", Anthra~clines: Current Status And
New Developments, s-u~ra, at p, Z1). Attempts have therefore
been made in the treatment of tumors to improve the
-3-
~~f,~''~°1.~ ~.~.~a''~
therapeutic effects of these compounds by linking the
anthracycline to antibodies directed against
tumor-associated antigens. In this way, the drug'can be
delivered or "targeted" to the tumor site and its toxic side
effects on normal cells in the body may be diminished. a
Immunoconjugates comprised of the anthracyclines, adriamycin
(ADM) or daunomycin (DAU), linked to polyclonal or
monoclonal antibodies to tumor-associated antigens are known
in the art [see, e.g., J. Gallego et al., '°Preparation Of
Four Daunomycin-Monoclonal Antibody 791T/36 Conjugates With
Anti-Tumour Activity", Int. J. Cancer, 33, pp. 737-44 (1984)
and R. Arnon et ai., "In Vitro And In Vivo Efficacy Of
Conjugates Of Daunomycin With Anti-Tumor Antibodies",
Immunolocrical Rev., 62, pp. 5-27 (1982)J.
The most frequently used approaches for the attachment
of an anthracycline to an antibody have utilized a linkage
at the amino sugar moiety of the anthracycline. For
example, the amino sugar has been oxidized by sodium
periodate treatment and directly attached to lysine residues
on the antibody via Schiff base formation [see, e.g., E.
Hurwitz et al., "The Covalent Binding Of Daunomycin And
Adriamycin To Antibodies, With Retention Of Both Drug And
Antibody Activities", Cancer Rea._, 35, pp. 1182-86 (1975)].
Alternatively, anthracyclines have been linked to antibodies
through carbodiimide-mediated linkage of the amino sugar of
the anthracycline to carboxyl groups on the antibody (see,
-4-
~ ~~.~1~~~
e.g., E. Hurwitz et al., su ra]. And, anthracyclines have
also been linked to antibodies by cross-linking the amino
sugar of the drug and amino groups on the antibody with
glutaraldehyde [see, e.g., M. Belles-Isles et al., "In'Vitro
Activity Of Daunomycin-Anti-AlphaFetoprotein Conjugate On-
Mouse Hepatoma Cells", Br. J. Cancer, 41, pp. 841-42
(1980)]. However, studies with immunoconjugates in which
the amino_sugar portion of the anthracycline molecule was
modified by linkage to the antibody indicate a loss of
cytotoxic activity of the conjugated drug [see, e.g., R.
Arnon et al., supra, at pp. 7-8]. In addition, studies of
anthracycline analogs indicate that modifications of
anthra~yclines at their amino sugars result in a decrease in
the eytotoxic activity of the drug analog relative to the .
parent drug [see, e.g., K. Yamamoto et al., '°Antitumor
Activity Of Some Derivatives Of Daunomycin At The Amino And
Methyl Ketone Functions", J. Med. Chem._, 15, pp. 872-75
(1972)].
Still other immunoconjugates have been prepared wherein
the anthracycline, daunomycin, has been linked directly to
an antibody at the 14-carbon (C-14) position of the drug.
However, the selective cytotoxic activity of these
immunoconjugates toward tumor cells was not easily
reproducible and was revealed consistently only at a
concentration of 20 ug/ml [see J. Gallego et al., supra].
Japanese patent application 274658 discloses the
-5-
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conjugation of an anthracycline to an antibody via a 13-keto
acylhydrazone linkage. This conjugation was accomplished
using methods that involve derivatization of the antibody
and subsequent reaction of that derivative with ,
anthracycline. These methods are disfavored because
derivatization of the antibody involves undesirable
non-specific reactions and very low anthracycline:antibody
ratios are obtained.
According to the first method, the antibody was treated
with carbodiimide in the presence of hydrazine to yield a
hydrazido antibody derivative which was then reacted with
the anthracycline such that the anthracycline was linked
directly to the antibody structure. The resulting
immunoconjugates, however, are prone to aggregation of the
antibody molecules. Furthermore, because this method
requires carboxylic acid groups on the antibody molecule
which are limited in number, these immunoconjugates have low
anthracycline:antibody ratios (approximately 1.1-1.3).
The second method involves reacting the antibody with
succinic anhydride to yield an amide acid derivative of the
antibody. This derivative was next reacted with hydrazine
to yield an antibody hydrazid derivative which was then
reacted with the anthracycline, daunomycin. This second
approach is flawed in that the reaction of the antibody
derivative with hydrazine is non-specific, leading to the
production of a mixture of different antibody derivatives in
_6_
if:o~"l.~b.~ ~i;-~
addition to the desired hydrazid derivative. Thus, as
indicated in the 274658 application, the molar ratio of
anthracycline to antibody was very low {approximately 1, see
Japanese application, page 264, column 1). ,
Finally, other anthracycline hydrazones are disclosed
in G.L. Tong et al., J. Med. Chem., 21, pp. 732-37 (1978);
T. Smith et al., J. Med. Chem., 21, pp. 280-83 (1978); and
R.T.C. Brownlee et al., J. Chem. Soc., pp. 659-61 (1986).
See also United States Patent 4,112,217, which discloses
bis-hydrazones of daunomycin and adriamycin.
In other studies, anthracyclines have been linked to
high molecular weight carriers, such as dextran or
polyglutamic acid, in order to potentiate the cytotoxic
activity and reduce the toxicity of 'the drug (see, e.g., R..
Arnon et al., supra, at p.. 5 and E. Hurwitz et al., "Soluble
Macromolecules As Carriers For Daunorubicin", J. Appl.
Biochem., 2, pp.. 25-35 (1980)). These carrier-linked
anthracyclines have also been covalently bound to
antibodies directed against tumor-associated antigens to
form immunoconjugates for targeting of the cytotoxic drug
specifically to tumor cells. For example, adriamycin has
been linked to such an "anti-tumor" antibody via a carboxy-
methyl-dextran hydrazide bridge wherein the adriamycin
molecule was linked to a hydrazine derivative of
carboxymethyl dextran at 'the C-13 carbonyl side chain of the
tetracycline ring of the adriamycin to form a hydrazone.
-7-
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tGr~~d'I ~~~)~
The antibody was then linked to the dextran hydrazide
derivative with glutaraldehyde to form an adriamycin-dex-
antibody conjugate (see R. Arnon et al., ."Monoclonal
Antibodies As Carriers For Immunotargeting Of Drugs", in
Monoclonal Antibodies For Cancer Detection And Therapy, R.W.
Baldwin et al. (eds.), pp. 365-83 (1985) and E. Hurwitz et
al., "A Conjugate Of Adriamycin And Monoclonal Antibodies To
Thy-1 Antigen Inhibits Human Neuroblastoma Cells In Vitro",
Ann. N.Y. Acad. Sci., 417, pp. 125-36 (1983)].
However, the use of carriers entails certain disadvan-
tages. For example, carrier-containing immunoconjugates are
quite large in size and are removed rapidly by the
reticuloendothelial system in vivo [see, e.g., R.O. Dillman
et al., "Preclinical Trials With Combinations And Conjugates
Of T101 Monoclonal Antibody And Doxorubicin'°, Cancer Res.,
46, pp. 4886-91 (1986)J. This rapid removal of the
carrier-containing immunoconjugates may not be advantageous
for therapy because the conjugated drug may never reach its
intended site of action, i.e., the selected group of cells
to be killed. In addition, the presence of the high
molecular weight carrier may negatively affect the stability
of the immunoconjugate and has been shown to reduce the
binding activity of the antibody of the conjugate [see,
e.g., M.J. Embleton et al.., "Antibody Targeting Of
Anti-Cancer Agents", in Monoclonal Antibodies For Cancer
Detection And Therapy, R.W. Baldwin et al. (eds.), pp.
_g_
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~~~i~~~~~~
323-24 (1985)]. Furthermore, in studies with tumor cells,
there is no evidence that high molecular weight
carrier-containing immunoconjugates are able to localize to
the tumor cells in vivo. Compare C.H.J. Ford et al., .
"Localization And Toxicity Study Of A Vindesine-Anti-CEA _
Conjugate In Patients With Advanced Cancer", Br. J. Cancer,
47, 35-42 (1983), which demonstrates localization of
directly-conjugated drug-antibody conjugates to tumor cells
in vivo.
Thus, the conjugation of anthracyclines to antibodies
by the use of specific linkages and carriers has been
disclosed. As outlined above, the use of these immunocon-
jugates entails distinct disadvantages depending upon the
specific linkage or carrier used.
SUMMARY OF THE INVENTION
The present invention therefore provides a novel
chemistry for linking a number of cytotoxic anthracycline
molecules via a linker arm, to an antibody directed against
a selected target cell population to be killed. According
to this invention, each anthracycline is linked to the
antibody via a linker arm, the anthracycline being bound to
that linker through an acylhydrazone bond at the 13-keto
position of the anthracycline, to form the novel
immunoconjugates of the invention. For example, a preferred
_g-
embodiment of the invention involves the synthesis of a
novel adriamycin hydrazone derivative (ADM-HZN~ that was
then condensed with a thiolated antibody, resulting in the
attachment of the anthracycline to the antibody via a linker
arm. An acylhydrazone bond formed at the C-13 position o~
the ADM serves as the site of attachment of the ADM to 'the
linker. Additionally, a disulfide bond is present within
the linker as the site of attachment of the antibody.
According to another preferred embodiment, the ADM-HZN was
reduced to generate a sulfhydryl group and the resulting
novel hydrazone derivative was condensed with a maleimide-
derivatized antibody. This led to the formation of a linker
arm having an acylhydrazone bond as the site of the linker
attachment to the C-13 position of ADM and a thioether bond
within the linker as part of the linker attachment to the
antibody. As is evident from these embodiments, the present
invention provides novel acylhydrazone derivatives of
anthracyclines useful in the preparation of the
immunoconjugates of this invention.
The immunoconjugates c~f the present invention have
anthracycline:antibody molar ratios of approximately 4-10
and retain both antibody and cytotoxic drug activity for the
killing of selected target cells. The acid-sensitive
hydrazone bond that is present at the site of attachment of
the anthracycline to the linker arm of the immunoconjugate,
and additionally the disulfide or thioether linkages within
-10-
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' I
the linker arm of the preferred embodiments of this
invention, are ideally suited for the release of active drug
under reducing and acidic conditions such as those-typically
encountered within a cell, e.g., in lysosomal vesicles:
The immunoconjugates of this invention may be used in
pharmaceutical compositions, such as those comprising a
pharmaceutically effective amount of at least one
immunoconjugate of the invention and a pharmaceutically
acceptable carrier. The present invention also encompasses
methods for the selective delivery of cytotoxic drugs to a
selected population of target cells desired to be elimina-
ted, as well as methods for treating a mammal in a
pharmaceutically acceptable manner with a pharmaceutically
effective amount of the compositions of the invention.
Advantageously, the immunoconjugates, pharmaceutical
compositions, and methods disclosed herein provide a useful
approach to the targeting of cytotoxic anthracycline drugs
to a selected population of cells for the preferential
killing of those target cells in the treatment of diseases
such as cancers and other tumors, non-cytocidal viral or
other pathogenic infections, and autoimmune disorders.
BRIEF DESCRIPTION OF THE~DRAWINGS
Figure 1 depicts in schematic form the synthesis of the
novel AI7M-HZN hydrazone derivative used in the preparation
-11-
t.r".~. ~~.~i 0.~
of the immunoconjugates of this invention.
Figure 2 depicts in schematic form the synthesis of the
immunoconjugates of one embodiment of this inventi'an wherein
a monoclonal antibody (MAB) was first thiolated using either
SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate) or 2-IT
(2-iminothiolane) and the thiolated antibody was then
reacted with ADM-HZN to form an immunoconjugate of the
invention, with a hydrazone bond at the 13-keto position of
the ADM and a disulfide bond within the linker arm.
Figure 3 depicts a scattergram that compares the number
of reactive thiol groups substituted on the monoclonal
antibody (SH/MAB ratio) to the final ADM/MAB molar ratio
achieved in imrnunoconjugates produced by the condensation of
the SPDP-thiolated SE9 and 3A1 monoclonal antibodies with
ADM-HZN.
Figure 4 depicts a scattergram comparing the SH/MAB
ratio with the final ADM/MAB molar ratio achieved in
immunoconjugates produced by the reaction of 2-IT-thiolated
SE9 and 3A1 antibodies with ADM-HZN.
Figure 5 depicts a scattergram showing the relation-
ship between ADM/MAB molar ratio and the protein yield of
immunoconjugates of the invention prepared using either
- SPDP-thiolated antibodies or 2-IT-thiolated antibodies.
Figure 6 depicts a scattergram comparing the ADM/MAB
molar ratio vs. protein yield obtained in immunoconjugate
preparations using monoclonal antibodies of either the IgGl
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1
isotype (e. g., 5E9 and 3X11) or the IgG2 isotype (e. g., L6).
These antibodies had been thiolated with SPDP.
Figure 7 also depicts a scattergram comparing the
ADM/MAB molar ratio vs. protein yield of immunoconjugates
having antibodies of the IgGl vs. IgG2 isotypes (as in
Figure 6) except that these antibodies had been thiolated
using 2-IT.
Figure 8 depicts in graph form the binding curves of
two immunoconjugates of the invention compared to the
binding curves of the respective unconjugated monoclonal
antibodies.
Figure 9 is an HPLC chromatograph depicting the
stability of an immunoconjugate of this invention over the
pH range ~of 4-7. This chromatograph demonstrates the
acid-sensitivity of the acylhydrazone bond of the invention
as indicated by the increased release of free ADM from the
immunoconjugate as the pH became more acidic.
Figure 10 is an HPLC chromatograph showing the release
of an X~DM moiety from an immunoconjugate of this invention
after treatment with DTT.
Figure 11 depicts in graph form the selective
cytotoxicity of immunoconjugates of this invention toward
the Daudi cell line using a soft agar colony formation
essay. These immunoconjugates had been prepared using
2-IT-thiolated antibodies.
Figure 12 depicts in graph form the selective
-13-
e_..
P~
cytotoxicity of immunoconjugates of this invention toward
Namalwa cells and the increased potency of the immunocon-
jugates compared to free ADM, using a limiting dilution
assay. ,
Figure l3 depicts in graph form the selective
cytotoxicity of immunoconjugates of the invention toward
Daudi cells using a soft agar colony formation assay. In
this instance, the immunoconjugates had been prepared using
SPDP-thiolated antibodies.
Figure 14 depicts in graph form the selective
cytotoxicity of another immunoconjugate of this invention
prepared with SPDP as the thiolating agent. This
immunoconjugate was cytotoxic toward antigen-positive Daudi
and Namalwa cells, but not antigen-negative HSB-2 cells,
using the soft agar colony formation assay.
Figure 15 depicts the selective cytotoxicity of 5E9 and
3A1 immunoconjugates of this invention toward a human colon
carcinoma cell line (5E9+, 3A1-), using a colony formation
assay.
Figure 16 depicts in graph form the lack of
cytotoxicity towards Daudi cells of immunoconjugates
prepared by attaching ADM to monoclonal antibodies at the
amino sugar residue of the ADM through a leu-ala dipeptide
linker.
Figure 17 depicts in schematic form the synthesis of an
immunoconjugate of this invention wherein the novel ADM-FiZN
-14-
I ;
~~:~~fi.~.~i ~
derivative of this invention is reduced and then reacted
with a SMPB (succinimidyl-4-(p-maleimidophenyl)butyrate)-
treated antibody to form an immunoconjugate having a linker
arm with a thioether linkage within its structure.
Figure 18 depicts in graph form the cytotoxicity of an
immunoconjugate of the invention having, additional to the
13-keto acylhydrazone linkage, a thioether bond within its
linker arm. The immunoconjugate showed greater potency
relative to free ADM toward Namalwa cells, using a
3H-thymidine incorporation assay.
Figure 19 depicts in graph form the cytotoxicity toward
HSB-2 cells of the immunoconjugate of Figure 18, using the
same 3H-thymidine incorporation assay.
Figure 20 depicts in graph form the selective
cytotoxicity of an immunoconjugate of the invention toward
antigen-positive cells vs. antigen-negative cells using the
3H-thymidine incorporation assay, the immunoconjugate
having, additional to the 13-keto acylhydrazone linkage, a
thioether bond within its linker arm.
Figure 21 depicts in graph form the in vivo anti-tumor
activity of an immunoconjugate of the invention on human
Daudi tumor xenografts in mice. The immunoconjugate showed
a greater anti-tumor activity than that seen using an
equivalent dose of free ADM.
Figure 22 depicts in graph form the in vivo anti-tumor
activity of ADM on human Daudi tumor xenografts in mice over
-15-
- ._
~.~'~.~~3
time and at varying dosages of ADM, using a Q7Dx3 treatment
schedule and i.v. administration.
Figure 2~ depicts in graph form the in vivo anti-tumor
activity of an immunoconjugate of the invention on human
Daudi tumor xenografts in mice compared to the anti-tumor_
activity of optimized free ADM (given i.v. on a Q7Dx3
treatment schedule at a dose of 10-11 mg/kg/inj). The
immunoconjugate showed a greater anti-tumor activity.
Figure 24 depicts in table form the in vivo anti-tumor
activity of ADM on human Ramos tumor xenografts in mice
using i.v. administration but varying the treatment
schedules and dosages.
Figure 25 depicts in graph form the in vivo anti-tumor
activity. of ADM on human Ramos tumor xenografts in mice over
time and at varying dosages of ADM, using a single injection
treatment schedule and i.v. administration.
Figure 26A depicts in graph form the in vivo anti-tumor
activity of an immunoconjugate of this invention on human
Ramos tumor xenografts in mice compared to the anti-tumor
activity of optimized free ADM (given i.v. on a QlDx1
treatment schedule at 16-18 mg/kg/inj), The immunoconjugate
showed a greater anti-tumor activity than the free ADM.
Figure 26S depicts the i~n vivo anti-tumor activity of the
immunoconjugate over time at different dosages of the
conjugate, demonstrating the dosage-dependent nature of the
conjugate's anti-tumor effect. The dosages of the
-16-
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immunoconjugates tested in Figures 26A and B are given as
the input of conjugated anthracycline, with the antibody
input given in parenthesis. -
DETAILED DESCRIPTION OF THE INVENTION
Tn order that the invention herein disclosed may be
more fully understood, the following detailed description is
set forth.
The present invention relates to novel anthracycline
immunoconjugates, novel anthracycline acylhydrazone
derivatives, methods for their production, pharmaceutical
compositions and methods for delivering cytotoxic
anthracyclines to a selected population of cells desired to
be eliminated, in the treatment of diseases such as cancers
and other tumors, non-cytocidal viral or other pathogenic
infections, and autoimmune disorders. More particularly,
the invention relates to immunoconjugates comprised of an
antibody directed against a selected cell population, the
antibody having a number of anthracycline molecules linked
to its structure. The anthracycline molecules are
covalently bound to the antibody such that a linker arm is
formed between each drug molecule and the antibody, the
linker being attached to the anthracycline by an
acylhydrazone bond at the 13-keto position of the
anthracycline. This can be accomplished in a stepwise
-17-
fashion by the initial formation of a novel anthracycline-
hydrazone derivative which is then reacted with an antibody
of the appropriate specificity (see, e.g., R.R. Hardy,
"Purification And Coupling Of Fluorescent Proteins For'Use
In Flow Cytometry", in Handbook Of Experimental Immunol~,
Volume 1: Immunochemistry, D.M. Weir et al. (eds.), pp.
31.4-31.12 (4th Ed. 1986) for a discussion of conventional
antibody coupling techniques]. The length of the linker arm
that connects the anthracycline and antibody components of
the immunoconjugate may vary as long as the point of
attachment of the linker to the anthracycline is in the~form
of an acylhydrazone at the C-13 position of the
anthracycline. The linker arm may additionally eontain
another bond, such as a disulfide, thioether, amide,
carbamate, ether or ester bond, along its length between the
points of attachment from the drug to the antibody.
The anthracyclines that comprise the immunoconjugates
of this invention may be any anthracycline containing a keto
group at the 13-carbon (C-13) position. Such anthracyclines
include, but are not limited to, adriamycin, daunomycin,
detorubicin, carminomycin, idarubicin, epirubicin,
esorubicin, 4'-THP-adriamycin, AD-32, and 3'-deamino-3'-
(3-cyano-4-morpholinyl)-doxorubicin (see A.M. Casazza,
';Experimental Studies On New Anthracyclines", in Adriamycin:
Its Expanding Role In Cancer Treatment, M. Ogawa et al.
(eds.), pp, 439-52 (Excerpts Medics 1984)J.
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The antibodies that comprise the immunoconjugates of
this invention may be any antibody reactive with a specific
cell population desired to be eliminated or killed.
Examples of such antibodies include, but are not limited to,
antibodies that bind to tumor-associated antigens such as
antigens found on carcinomas, melanomas, lymphomas, bone or
soft tissue sarcomas, as well as other tumors, antibodies
that bind to virus- or other pathogen-associated antigens,
and antibodies that bind to abnormal cell surface antigens.
These antibodies may be polyclonal or preferably, monoclonal
and can be produced using techniques well established in the
art [see, e.g., R. A. DeWeger et al., "Eradication Of Marine
Lymphoma And Melanoma Cells By Chlorambucil-Antibody
Complexes", Immunoloaical Rev., 62, pp. 29-45 (1982)
(tumor-specific polyclonal antibodies produced and used in
conjugates) and M. Yeh et al., °'Cell Surface Antigens Of
Human Melanoma Identified By Monoclonal Antibody," Proc.
Natl. Acad. Sci., 76, pp. 2927-31 (1979) and J. P. Brown et
al., "Structural Characterization Of Human Melanoma-
Associated Antigen p97 With Monoclonal Antibodies," J.
Immunol., 127 (No.2), pp. 539-46 (1981) (tumor-specific
monoclonal antibodies produced)). For example, the
monoclonal antibody, L6, specific for human lung carcinoma
cells or the monoclonal antibody, 791T/36, specific for
osteogenic sarcoma cells, can be used. Furthermore,
non-internalizing or preferably, internalizing antibodies
-19-
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may be used. The term "antibody" as used in this
application includes intact antibody molecules or fragments
containing the active binding region of the antibody
molecule, e.g., Fab or F(ab~)2 . If monoclonal antibodies
are used, the antibodies may be of, but are not limited tp,
mouse or human origin or chimeric antibodies.
Thus, the antibodies of the immunoconjugates of this
invention act to deliver the anthracycline molecules to the
particular cell population with which the antibody is
reactive. For example, an antibody directed against an
antigen found on the surface of tumor cells will bind to and
deliver its anthracyclines to those tumor cells or an
antibody directed against a protein of the Human Immuno-
deficiency Virus (HIV) that causes AIDS will deliver its
cytotoxic anthracyclines to HIV-infected cells. Release of
the drug within or at the site of the particular cell
population with which the antibody reacts results in the
preferential killing of those particular cells. Thus, it is
apparent that the immunoconjugates of this invention are
useful in the treatment of any disease wherein a specific
cell population is sought to be eliminated, the cell
population having a cell surface antigen which allows
binding of the immunoconjugate. Diseases for which the
present immunoconjugates are useful include, but are not
limited to, cancers and other tumors, non-cytocidal viral or
other pathogenic infections such as AIDS, herpes, CMV
-zo-
-
(cytomegalovirus), EBV (Epstein Barr Virus), and SSPE
(subacute schlerosis panencephalitis), and rheumatoid
arthritis. -
Without being bound by theory, it is believed that the
antibody-linked anthracycline molecules, i.e., in the form
of the immunoconjugate of the invention, are delivered to
the target cells to be killed via the antibody specificity
and may then enter the cell via the same endocytic pathway
that leads to internalization of membrane-bound unconjugated
antibodies and ligands [see, e.g., I. Pastan et al.,
"Pathway Of Endocytosis", in Endocytosis, I. Pastan et al.
(eds.), pp. 1-44 (Plenum Press 1985)]. Once inside the
cell, the endocytic vesicles containing the immunoconjugate
fuse with primary lysosomes to .form secondary lysosomes
[see, e.g., M.J. Embleton et al., supra, at p. 334J. '
Because the anthracycline molecules are bound to the
antibody of the immunoconjugate via acid-sensitive
acylhydrazone bonds, exposure of the immunoconjugate to the
acid environment of the endocytic vesicles and lysosomes
results in the release of the anthracycline from the
immunoconjugate. Furthermore, the anthracycline released is
believed to be a relatively unmodified drug capable of full
cytotoxic activity. Thus, the acid-sensitive hydrazone bond
of the immunoconjugate is highly advantageous for the
release of the cytotoxic drug within target cells, enhancing
the cytotoxicity of the immunoconjugate toward those cells.
-21-
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Alternatively, the hydrazone bond may be cleaved under
acidic and reducing conditions in the immediate environment
external to or surrounding the target cells, e.g.,- at the
site of a tumor, and the released drug may be taken up, by
the tumor cells.
The immunoconjugates of the invention and the methods
for their production are exemplified by preferred
embodiments in which the anthracycline, adriamycin, was
conjugated to various antibodies.
First, a novel adriamycin hydrazone derivative was
synthesized in a two-step reaction. The heterobifunctional
reagent SPDP (N-succii-iimidyl-3-(2-pyridyldithio)propionate)
was allowed to react with hydrazine to form a 3-(2-pyrid-
yldithio) propionyl hydrazide and~the hydrazide was then
reacted with adriamycin hydrochloride (ADM-HC1) to form a
novel acylhydrazone derivative of ADM, containing a
pyridyl-protected disulfide moiety. An acid catalyst such
as trifluoroacetic acid m'ay be employed to facilitate the
formation of hydrazone. The derivative formed was
designated adriamycin 13-(3-(2-pyridyldithio) propionylJ-
hydrazone hydrochloride (ADM-HZN) (see Figure 1).
This novel ADM-hydrazone derivative was then reacted
with a monoclonal antibody that had been previously
thiolated with SPDP and then reduced or thiolated with 2-IT
(2-iminothiolane) (see Figure 2). The resulting
immunoconjugate was comprised of ADM molecules conjugated to
-22-
the monoclonal antibody by means of a linker arm attached to
the C-13 position of each ADM through an acylhydrazone bond,
the linker arm additionally containing a disulfide-bond
through which it was attached to the antibody (see Figure
2).
Another embodiment of the invention involved the
synthesis of another novel adriamycin hydrazone derivative
wherein the ADM-FiZN described above was further treated with
the reducing agents, DTT (dithiotreitol) or tributyl-
phosphine, to produce 13-(3-(mercaptopropionyl)jadriamycin
hydrazone (see Figure 17). This derivative was then reacted
with a monoclonal antibody to which maleimide groups had
been attached, for example, by reaction of the antibody with
SMPB (succinimidyl-4-(p-maleimidophenyl)butyrate). As shown
in Figure 17, an immunoconjugate was formed having a linker
arm attached by a hydrazone bond to the C-13 position of
each ADM and also having a thioether linkage as part of its
attachment to the antibody. Thus, it is apparent that the
linker arm connecting the drug and antibody may be comprised
of a number of constituents and linkages as long as these
linkages include the acid-sensitive hydrazone bond at the
13-keto position of the anthracycline.
It is also apparent that the present invention provides
-23-
' -~ (1 ~
~~.~~.~~:&
novel acylhydrazone derivatives of 13-keto-containing
anthracyclines having formulae I, II or III:
HK'C <CH~)" -$ -$ -RZ
0 OH H/
~~ OH Rl
3 ~ ~H
cH F'ormul a 1
R'
a
0
wherein:
R1 is CH3, CH20H, CH20C0(CH2)3CH3, CH~OCOCH(OC2H5)2'
R2 is
x
or -x.
wherein X : H, N02 or halogen;
R3 is OCH3, OH or hydrogen;
R4 is NH2, NHCOCF3, 4-morpholinyl, 3-cyano-4-
morpholinyl, 1-piperidinyl, 4-methoxy-1-piperdinyl,
benzyl amine, dibenzyl amine, cyanomethyl amine or
1-cyano-2-methoxyethyl amine;
R5 is OH, O~THP or hydrogen;
R6 is OH or hydrogen, provided that R6 is not OH when
R5 is OH or O-THP; and
n is an integer from 1 to 10, inclusive;
-24-
' - il ~
a
a
NN'~C <CN°)" _g -H
0 ON N/
e° ~ I ~ . ~str
ON
N
D
eN Formula 11
Na
wherein:
R1 is CH3, CHaOH, CH20C0(CH2)3CH3, CH20COCH(OC2H5)2'
R3 is OCH3, OH or hydrogen;
R4 is NH2, NHCOCE3, 4-morpholinyl, 3-cyano-4-
morpholinyl, 1-piperidinyl, 4-methoxy-1-piperdinyl,
benzyl amine, dibenzyl amine, cyanomethyl amine or
1-cyano-2-methoxyethyl amine;
R5 is OH, O-THP or hydrogen;
R6 is OH or.hydrogen, provided that R6 is not OH when
RS is OH or O-THP; and
n is an integer from 1 to 10, inclusive; and
0
Nu'e -ICNt)"_° _s-a°
0 ON w~
\ ~.
ON
\ ~/
N
0
eN ~orwla III
11
° °enl
wherein:
R1 is CH3, CH20H, CH20C0(CH2)3CH3, CHZOCOCH(OC2H5)2'
-25-
1~
RZ i s
X or X
r
wherein X = M, NOZ or halogen;
R3 is OCH3, OH or hydrogen;
R~ and R~ axe independently hydrogen, alkyl,
substituted alkyl, cycloalkyl, substituted cycloalkyl,
aryl, substituted aryl, aralkyl or substituted
aralkyl; or R~, R~ and N together form a 4-7 membered
ring, wherein said ring may be optionally substituted;
RS is OH, 0-THP or hydrogen;
R6 is OH or hydrogen, pravided that R6 is not OH when
RS is OH or 0-THP; and
n is an integer from 1 to 10, inclusive.
The above-disclosed anthracycline acylhydrazones represent
novel intermediates in the preparation of the
immunoconjugates of the invention and are exemplified by
ADM-HZN and 13-~(3-(mercaptopropionyl)jadriamycin hydrazone,
respectively, as described in the preferred embodiments
discussed herein.
As can be seen from the above formulae, the
~cylhydrazone intermediates of the invention include
hydrazones of any of a number of known anthracyclines such
as adriamycin, daunomycin and carminomycin. In addition,
-26-
' 1 ~r
~~~;,',~.~.~tp=~
the intermediates include acylhydrazones derivatized at
specific sites on the anthracycline structure (e. g.,
4'-THP-adriamycin hydrazone and 3'-deamino-3'-(3-cyano-4-
morpholinyl)adriamycin hydrazone). These latter ,
intermediates can be synthesized by first derivatizing the
anthracycline to form a desired analog and then using that
analog to prepare the hydrazone intermediate of the
invention. Known anthracycline analogs include those
described in U.S. Patents 4,464,529 and 4,301,277
(3'-deamino-3'-(4-morpholinyl) or 3'-deamino-3'-(3-
cyano-4-morpholinyl) anthracycline analogs), U.S. Patents
4,202,967 and 4,314,054 (3'-deamino-3'-(1-piperdinyl) or
3'-deamino-3'-(4-methoxy-1-piperdinyl) anthracyline
analogs), U.S. Patent 4,250,303 (N-benzyl or N,N-dibenzyl
~anthracycline analogs), U.S. Patent 4,591,637 '
(N-methoxymethyl or N-cyanomethyl anthracycline analogs) and
U.S. Patent 4,303,785 (acetal analogs of anthracyclines).
Thus, these known anthracycline analogs can be reacted as
described hereinabove (see Figure 1) to produce novel
acylhydrazones which can then be conjugated to an antibody
of a desired specificity as described herein.
Alternatively, an underivatized acylhydrazone
intermediate of this invention can first be produced as
described herein from the underivatized anthracycline, such
as adriamycin, daunomycin or carminomycin, and this novel
intermediate then derivatized to produce a novel
-27-
~ r:
~ ~~~~Z~~v
acylhydrazone substituted as desired. For example, ADM-HZN
can be derivatized at its amino sugar moiety by reductive
amination with 2,2'-oxydiacetaldehyde using the procedure
described in U.S. Patent 4,464,529, to produce 3'-deamino-
3'-(3-cyano-4-morpholinyl)adriamycin hydrazone. Similarl~r,
ADM-HZN can be derivatized at the amino sugar moiety to
produce novel acylhydrazone derivatives such as 3'-deamino-
3'-(4-morpholinyl) ADM hydrazone (see U.S. Patent
4,301,277), 3'-deamino-3'-(1-piperdinyl) ADM hydrazone (see
U.S. Patent 4,202,967), 3'-deamino-3'-(4-methoxy-1-
piperdinyl) ADM hydrazone (see U.S. Patent 4,314,054),
N-benzyl ADM hydrazone and N,N-dibenzyl ADM hydrazone (see
U.S. Patent 4,250,303) or N-methyoxymethyl ADM hydrazone and
N-cyanomethyl ADM hydrazone (see U.S. Patent 4,591,637). In
addition, ADM-HZN can be derivatized at the R5 position of
Formulae I-III as described in U.S. Patent 4,303,785 to
produce acetal derivatives of the hydrazone such as
4'-THP-ADM hydrazone.
It should be understood that these novel procedures for
derivatizing the acylhydrazones of the invention can utilize
as starting materials hydrazones of anthracyclines other
than ADM, such as daunomycin or carminomycin, to produce
novel compounds such as N-benzyl daunomycin hydrazone or
3'-deamino-3'-(4-morpholinyl)carminomycin hydrazone, which
are also within the scope of this invention.
Evaluation of the anthracycline-antibody
-28-
as ~~~.~~~3v
immunoconjugates prepared according to this invention showed
that the immunoconjugates retained antibody binding activity
and exhibited antibody-directed cell killing for both
lymphoma and carcinoma cells under various assay conditions.
Thus, cells possessing the antigen to which the antibody of
the conjugate was directed were efficiently killed by the
anthracycline whereas cells that did not possess the
appropriate antigen were not killed. In fact, in several
experiments, antibody-delivered anthracycline was found to
be more potent than equivalent amounts of unconjugated
anthracycline. Differences in uptake mechanisms into the
tumor cell and intracellular transport mechanisms may be
respansible for the potency differences observed between the
free drug and the antibody-conjugated drug.
Furthermore,' studies using human tumor xenografts in
mice have demonstrated the ability of the immunoconjugates
of this invention to inhibit tumor growth in vivo, leading
in some cases to complete tumor regression. The immuno-
conjugates were shown to possess a greater potency and
inhibited tumor growth to a greater extent than the
unconjugated anthracycline. Furthermore, the
immunoconjugates were tolerated by the animals to a much
greater extent than the free drug, being at least 10 times
less toxic than the unconjugated anthracycline alone.
The binding and cytotoxicity properties of the
immunoconjugates of this invention appear to represent an
-29-
'1
~~.~~.~l.f~=~
improvement over immunoconjugates reported in the literature
in which anthracyclines were directly linked to antibody
through the amino sugar portion of the anthracycline. Those
amino sugar-linked immunoconjugates often contained loNer
anthracycline to antibody molar ratios and exhibited reduced
cytotoxicity relative to the free drug and reduced antibody
binding properties [see, e.g., R. Arnon et al.,
Immunolocaical Rev., 62, supra; E. Hurwitz et al., Cancer
Res., 35, supra; and R. Yamamato et al., su raJ.
Furthermore, stability studies performed on the
immunoconjugates of this invention indicated that the
anthracycline was released from the immunoconjugates under
reducing and acidic conditions similar to those found in a
cellular environment. Thus, the retention of high cytotoxic
drug activity observed with the immunoconjugates described
herein may be explained by the fact that a relatively
unmodified drug is delivered to the target Bells.
In addition, we were able to optimize reaction
conditions such that anthracycline:antibody molar ratios of
approximately ~k-IO were reached, using several antibodies of
different isotypes. The amount of protein recovered after
condensation with the ADM-HZN derivative dropped off
dramatically when molar ratios greater than 10 were
attempted. It appeared that the major limitation in
obtaining immunoconjugates with molar ratios greater
than 10 was due to the reduced solubility of the conjugates
-30-
in aqueous solution and the physical association of
anthracycline with protein.
Our in yivo studies showing the enhanced anti-tumor
activity of the immunoconjugates of this invention over the
free drug as well as their reduced systemic toxicity,
indicate an increased therapeutic index for the conjugates.
Thus, the present invention also encompasses pharmaceutical
compositions, combinations and methods for treating diseases
such as cancers and other tumors, non-cytocidal viral or
other pathogenic infections, and autoimmune diseases. More
particularly, the invention includes methods for treating
disease in mammals wherein a pharmaceutically effective
amount of at least one anthracycline-containing
immunoconjugate is administered in a pharmaceutically
acceptable manner to the host mammal.
Alternative embodiments of the methods of this
invention include the administration, either simultaneously
or sequentially, of a number of different immunoconjugates,
i.e., bearing different anthracyclines or different
antibodies, for use in methods of combination chemotherapy.
E'or example, an embodiment of this invention may involve the
use of a number of anthracycline-immunoconjugates wherein
the specificity of the antibody component of the conjugate
' varies, i.e., a number of immunoconjugates are used, each
one having an antibody that binds specifically to a
different antigen or to different sites or epitopes on the
-31-
' . I':
~~~.a~..l.~i-'
same antigen present on the cell population of interest.
The anthracycline component of these immunoconjugates may be
the same or may vary. For example, this embodiment may be
especially useful in the treatment of certain tumors were
the amounts of the various antigens on the surface of a
tumor is unknown or the tumor cell population is
heterogenous in'antigen expression and one wants to be
certain that a sufficient amount of drug is targeted to all
of the tumor cells at the tumor site. The use of a number
of conjugates bearing different antigenic or epitope
specificities for the tumor increases the likelihood of
obtaining sufficient drug at the tumor site. Additionally,
this embodiment is important for achieving a high degree of
specificity for the tumor because the likelihood that normal
tissue will possess all of the same tumor-associated
antigens is small (cf., I. ~ellstrom et al., "Monoclonal
Antibodies To Two Determinants Of Melanoma--Antigen p97 Act
Synergistically In Complement-Dependent Cytotoxicity", J.
Immunol., 127 (No. 1), pp. 157-60 (1981)).
Alternatively, a number of different immunoconjugates
can be used, wherein only the anthracycline component of the
conjugate varies. For example, a particular antibody can be
linked to adriamycin to form one immunoconjugate and can be
linked to daunomycin to form a second immunoconjugate. Both
conjugates can then be administered to a host to be treated
and will localize, due to the antibody specificity, at the
-32-
W:
site of the selected cell population caught to be
eliminated. Both drugs will then be released at that site.
This embodiment may be important where there is some
uncertainty as to the drug resistance of a particular dell
population such as a tumor because this method allows the
release of a number of different drugs at the site of or
within the target cells. An additional embodiment includes
the conjugation of more than one anthracycline to a
particular antibody to form an immunoconjugate bearing a
variety of different anthracycline molecules along its
surface -- all linked to the antibody via a 13-keto
acylhydrazone bond. Administration of the immunoconjugate
of this embodiment results in the release of a number of
different drugs at the site of or within the target cells.
The anthracycline immunoconjugates of the invention can
be administered in the form of pharmaceutical compositions
using conventional modes of administration including, but
not limited to, intravenous, intraperitoneal, oral,
intralymphatic, or administration directly into the site of
a selected cell population such as a tumor. Intravenous
administration is preferred. Furthermore, for in vivo
treatment, it may be useful to use immunoconjugates
comprising antibody fragments such as Fab or F(ab')2 or
chimeric antibodies.
The pharmaceutical compositions of the invention -
comprising the anthracycline immunoconjugates -- may be in a
-33-
~~3~~~~y
variety of dosage forms which include, but are not limited
to, solid, semi-solid and liquid dosage forms such as
tablets, pills, powders, liquid solutions or suspeTisions,
suppositories, polymeric microcapsules or microvesicles,
liposomes, and injectable or infusible solutions. The
preferred form depends upon the mode of administration and
the therapeutic application.
The pharmaceutical compositions may also include
conventional pharmaceutically acceptable carriers known in
the art such as serum proteins such as human serum albumin,
buffer substances such as phosphates, water or salts or
electrolytes.
The most effective mode of administration and dosage
regimen for the immunoconjugate compositions of this
invention depends upon the severity and course of the
disease, the patient's health and response to treatment and
the judgment of the treating physician. Accordingly, the
dosages of the immunoconjugates and any accompanying
compounds should be titrated to the individual patient.
Nevertheless, an effective dose of the anthracycline
immunoconjugate of this invention may be in the range of
from about 1 to about 100 mg/m2 anthracycline or from about
500-5000 mg/m2 antibody.
In order that the invention described herein may be
more fully understood, the following examples are set forth.
It should be understood that these examples are for
-34-
~a~.~.~.~i ~~
illustrative purposes only and are not to be construed as
limiting the scope of this invention in any manner.
EXAMPLE 1
The following example demonstrates the production of a
novel anthracycline immunoconjugate according to the present
invention wherein the drug is linked directly to a
monoclonal antibody via a hydrazone bond at the 13-keto
position of the drug.
The particular embodiment described in this example
involves the conjugation of ADM to a monoclonal antibody to
form an immunoconjugate having a linker arm with an
acylhydrazone bond as its point of attachment to the ADM
molecule of the immunoconjugate, the linker additionally
having a disulfide bond as part of its attachment to the
antibody. This embodiment also provides a novel
acylhydrazone derivative of ADM (ADM-HZN).
~nthesis Of An Adriam~in Hydrazone
As the initial step in the preparation of the
immunoconjugate of this embodiment, an ADM-hydxazone
derivative was first synthesized as follows: 0.3 ml of 1 M
h drazine i.e., NH NH
y ' 2 2, solution in isopropyl alcohol was
added to a cooled solution of SPDP (70 mg, 0.22 mmol) in 3
-35-
y:,
~~~~.~.~.f~~~
ml of THF (tetrahydrofuran). After stirring 20 min at 0°C,
the product was extracted with CH2C12, washed with brine and
dried over K2C03. The residue obtained after evaporation of
the solvents was chromatographed on neutral alumina (5%
MeOH, 95% CH2C12) to give 21 mg (41%) of 3-(2-pyridyldithio)
propionyl hydrazide (compound 2 in Figure 1). This
hydrazide and adriamycin HC1 (obtained from Sanraku Tnc.,
Japan) (48 mg, 0.083 mmol) were dissolved in 5 ml of MeOH
and then stirred in the dark at room temperature for 6 days.
The reaction was followed by reverse phase thin layer
chromatography (TLC) (MeOH:H20 = 2:1, containing 3% w/v
NH40Ac). After this period, the solvent was evaporated and
the residue was chromatographed on a C18 column (MeOH:H20 =
3:2, containing 3% w/v NH40Ac). The fractions were combined
and lyophilized and excess NH40Ac was removed under reduced
pressure. The residue was dissolved in MeOH and
precipitated by addition of acetonitrile to give 45 mg (72%)
of adriamycin 13-J3-{2-pyridyldithio) propionylJ-hydrazone
hydrochloride, referred to hereinafter as ADM-HZN (compound
4 in Figure 1). The ADM-HZN was characterized as follows:
mp > 125° darkens its color and not well-defined; NMR
(acetone - d6, d) 1.25 (s,3H,J=6Hz), 1.77 (m,lH), 2.06
(m,lH), 2.30 (m,lH), 2.53 (d,lH,J=l5Hz), 2.89-3.18 (m,6H),
3.71 (m,lH), 3.85 (m,lH), 3.97 (m,lH), 4.07 (s,3H), 4.78
(s,2H), 5.21 (m,lH), 5.58 (t,lH,J=7Hz), 7.12 (m,lH), 7.64
(d,lH,J=8Hz), 7.75 (m,2H), 7.90 (t,lH,J=8Hz), 7.98
-36-
,. y
~~~.~,~.l~i~
(d,lH,J=8Hz), 8.37 (d,IH,J=4Hz), 10.50 (s,lH), 10.52 (s,lH),
14.19 (bs,lH); IR (KBr) 3438, 1674 1618, 1579, 1419, 1286,
1016, 988, 698 cm-1; FABMS (glycerol) m/e 755 (M+1), 737,
645, 625, 609.
Thiolation Of Monoclonal Antibodies
Before reacting the ADM-HZN compound prepared as
described above with a monoclonal antibody of interest, the
antibody had to be thiolated, i.e., reactive sulfhydryl
groups had to be introduced onto the antibody molecule.
The monoclonal antibodies utilized were: 1) 5E9, an
IgGl antibody reactive with the transferrin receptor on all
dividing human cells and cross-reactive with various
histological types of cancer cells; 2) T33A1 (hereinafter
referred to as "3A1"), an IgGl antibody reactive with the 40
Kd human T cell antigen and also found on a number of T cell
leukemias; 3) 628.5, an IgGl antibody reactive with the 50
Kd human B cell antigen and also reactive with human B cell
lymphomas; 4) 628.1, an IgGl antibody reactive with the 39
Kd human B cell antigen and also reactive with H cell
lymphomas; and 5) L6, an IgG2a antibody reactive with a
glycolipid antigen an human non-small cell lung carcinomas.
Hybridomas secreting the 5E9 and T33A1 monoclonal
antibodies were obtained from the American Type Culture
Collection (ATCC). The respective antibodies were purified
-37-
~~ I~\
~s4?~
from ascitic fluid produced in BALB/c mice according to the
procedure of C. Bruck et al, '°One-Step Purification Of Mouse
Monoclonal Antibodies From Ascitic Fluid By DEAE-A,ffigel
Blue Chromatography", J. Immun. Methods, 5b, pp. 313-19
(1982). Purified 628.5, 628.1, and L6 were provided by Drs.
J. Ledbetter and I. Hellstrom (Oncogen, Seattle, WA).
Hybridomas secreting the L6 and 628.5 monoclonal antibodies
were deposited with the ATCC on December 6, 1984 and May 22,
1986, respectively, under ATCC accession nos. HB 8677 and HB
9110. The 628.1 monoclonal antibody is one of a number of
antibodies known in the art to be reactive with a major
epitope of the CD37 antigen and has been characterized in
A.J. Michael (ed.), Leukocyte Typing III, Oxford University
Press (U. K. 1987). A number of these anti-CD37 antibodies
are commercially available.
Thiolation of any of these antibodies with SPDP was
carried out as fellows: SPDP (Pierce Chemical Co., IL) (50
mM), dissolved in ethanol, was added to the monoclonal
antibody of choice, e.g., 5E9 (5-10 mg/ml), in PBS
(phosphate buffered saline, pH 7.2) to give a final
concentration of between 5-10 mM. The reaction mixture was
incubated for 30 min at 30°C. Unreacted SPDP was separated
from SPDP-derivatized antibody by gel filtration
chromatography using a PD-10 column (Pharmacia). The
thiopyridyl protecting groups were removed by reduction with
excess DTT. The reduced antibodies were passed through a
-38-
,, r. ,., ,
o~~s .di..~,.~.~l.~i'~
PD-10 column and the free thiol-containing antibodies were
used for condensation with the ADM-HZN derivative (see
Figure 2). -
Reactive thiol groups were also introduced onto the
antibody protein using 2-IT: the antibody (5-10 mg/ml in_SO
mM triethylamine, 50 mM NaCl, 1 mM EDTA at pH 8.0) was mixed
with 2-IT (Pierce Chemical Co., IL) at a final concentration
of 5-10 mM. The reaction was allowed to proceed for 90 min
at 4oC and thiolated antibodies were separated on a PD-10
column equilibrated with 2 M NaCl/PBS.
The number of reactive thiol groups incorporated onto
the antibodies was determined using DTNB (5,5'-dithiobis-
(2-nitrobenzoic acid) (E412 = 14150), according to the
procedure of G.L. Ellman, Arch. Biochem. Biophys., 82, pp.
70-77 (1959).
ConiuQation Of Thiolated Monoclonal Antibodies With ADM-HZN
A number of conjugations were next performed wherein
monoclonal antibodies thiolated as described above were each
linked to ADM-HZN (see Figure 2).
ADM-HZN was dissolved in methanol and added to
SPDP-thiolated antibodies in PBS or to 2-IT-thiolated
antibodies in 2 M NaCl/PBS. In a typical experiment, 10
equivalents of ADM-HZN were added to monoclonal antibodies
containing 10-20 reactive thiol groups. The conjugation
-39-
CA 02010164 1999-10-25
reaction was allowed to incubate overnight at 4°C. The
reaction mixture was centrifuged at 10,000 x g and
conjugated ADM was then separated from unreacted ADM by
passage through a PD-10 column. The amount of conjugated
anthracycline bound to antibody was determined by absorbance
at 495 nm (E495=8030). The amount of antibody protein was
determined by absorbance at 280 nm (1 mg/ml = 1.4 OD units).
To correct for the overlap of ADM absorbance at 280 nm, the
following formula was used:
A280 - (0.72 x A495)
Antibody (mg/ml) -
1.4
Immunoconjugates were analyzed for the presence of
unconjugated ADM or ADM derivatives using HPLC analysis.
HPLC was done using a Phenomenex column packed with 5 micron
IB-SIL C18 beads. Unconjugated ADM-HC1, ADM-HZN (0.1 a
moles), or immunoconjugates containing 0.5-5 ymoles drug
equivalents were applied to the column and eluted with
methanol and 10 mM ammonium phosphate, pH 4.5 (70:30) at 1.5
ml/min. All of the immunoconjugates produced contained no
significant amount (<1%) of unconjugated drug by HPLC
analysis.
Characterization Of The Immunoconiugates Of The Invention
'The immunoconjugates so produced were comprised of ADM
* Trademark -40-
i~~~..~b:.~.~;~:
molecules conjugated at the 13-keto position to a linker arm
that formed a bridge between the drug and the respective
monoclonal antibody. Furthermore, the addition of the
monoclonal antibody with free thiol groups to the ADM-HZN
derivative which contained a thiopyridyl protected disulfide
bond led to the formation of a disulfide bond in the linker
joining ADM to the antibodies (see Figure 2). Immunoconju-
gates produced according to this embodiment include, but are
not limited to, 5E9-ADM-7.5, 3A1-ADM-7.0, L6-ADM-9.0 and
628.1-ADM-9.0, wherein the first part of the designation
represents the monoclonal antibody used to form the
conjugate, the second part of the designation represents the
anthracycline linked to the antibody and the numeral in the
designation represents the molar ratio'of ADM/antibody in
the particular xmmunoconjugate.~
The ADM/antibody molar ratios achieved according to
this embodiment depended upon the number of thiol groups
introduced onto the monoclonal antibody and the amount of
ADM-HZN derivative added to the thiolated antibody. The
scattergrams in Figures 3 and 4 show that ADM/antibody
ratios of 3-4 were achieved when ADM-HZN was condensed with
either the 5E9 or 3A1 monoclonal antibody containing
- approximately eight thiol groups. Typically, a 10-fold
molar excess of ADM-HZN to protein was added in these
reactions. ADM/antibody ratios increased to 8-10 when those
antibodies having 18-25 thiol groups were used. No
-41-
a~
~~~~g~~
significant differences in the ADM/antibody ratios were
observed when using SPDP vs. 2-IT to thiolate the monoclonal
antibody (compare Figures 3 and 4). However, final protein
yields following conjugation of drug to antibody appeased to
be somewhat higher with SPDP-thiolated antibodies as
compared to 2-IT-thiolated antibodies (see Figure 5).
Protein yields of 50-80% were commonly obtained for
SPDP-thiolated antibodies such as 5E9 and 3A1 whereas yields
of 20-50% were obtained for the same antibodies thiolated
using 2-IT. Additionally, somewhat better immunoconjugate
yields were obtained using monoclonal antibodies of the IgG1
isotype such as 5E9 and ~A1 for conjugations carried out
with SPDP or 2-IT (see Figures 6 and 7).
The binding activity of the immunoconjugates of the
invention was determined using a competition assay involving
the use of 1125-labeled antibody. Respective antigen-
positive and antigen-negative cells (1 x 106) were suspended
in 0.1 ml of RPMI 1640 containing 2% FBS and mixed with 0.1
ml of 2-fold serially diluted unconjugated monoclonal
antibody or immunoconjugate at concentrations starting at 50
~g/ml. The cell suspensions, in duplicate, were incubated
while mixing at 4oC for 1 hour. The cells were then washed
two times and suspended in 0.1 ml containing 5 ug/ml of
125I-labelled homologous antibody (specific activity from
1-50 X 104 c m ug antibod
p / y protein). Samples were incubated
at 4°C for 1 hour and overlaid onto 0.15 mls 1:1 mixture of
-42-
i 1
s~.~.~a~~
dibutyl:dinonyl phthalate that was cooled to 4oC. The
samples were centrifuged at 10,000 x g for 1 min at 4°C and
the cell-bound counts (pellet) determined using an LKB gamma
counter. ,
The retention of binding activity by the imm~anocon- _
jugates of this invention is demonstrated in Table 1 below.
-43-
4 ~e
~r~3~~~)'~
Table 1
Relative Binding Affinity ADM-HZN
Estimates
After
Conjugation
(Itla (Tt]b =K conj
c
Inhibitor Molar Ratio Trac e x10-9M x10 9M x107 L/M
- - 5E9- 1251_ - 3.2
- - 3A1- 1251- - 5.1
SPDP Linker
SE9-ADM 3.5 5E9- 125I4.2 3.4 2.6
4.0 6.7 2.1 1.0
4.9 4.2 4.0 3.0
6.8 4.2 2.1 1.6
8.5 0.1 2.1 0.7
3A1-~ADM 2.6 3A1- 12514.2 1.3 1.5
3.3 1.3 1.3 5.1
4.2 8.3 1.3 0.8
6.7 7.3 1.3 0.9
2-IT Linker
5E9-ADM 5.6 5E9- 12514.1 4.0 3.1
6.7 4.7 4.0 2.7
3A1-ADM 6.0 3A1-.125I4.0 1.3 1.6
a(It) = Molar concentration of antibody conjugate giving 50%
inhibition of tracer antibody.
b(Tt] = Molar concentration of antibody giving 50%
inhibition of tracer antibody.
_,~4_
1
<3~~~D~~
cKconj - Relative (K) affinities were calculated using the
formula:
K conj = jTt]K~
(It)
KAb is the equilibrium constant of the unconjugated MAb as
determined by Scatchard analysis.
-45-
CA 02010164 1999-10-25
As shown in the table, the 5E9 immmunoconjugates
prepared using SPDP and having molar ratios ranging between
3.5 and 8.5 retained over 80% of their original binding
activity as compared to unconjugated 5E9. 5E9 ~
immunoconjugates prepared using 2-IT also retained high
binding activities. 3A1 immunoconjugates prepared using
SPDP showed some loss in antibody binding activity. In
general, conjugation of ADM to these and other antibodies
resulted in loss of relatively small degrees of antibody
binding activity.
Figure 8 shows the binding curves of two immuno-
conjugates of the invention, SE9-ADM-7.5 and 3A1-ADM-7.0,
compared to the binding curves of the unconjugated 5E9 and
3A1 monoclonal antibodies. To obtain these curves, the
immunoconjugates were incubated at 4°C in 0.1 ml complete
growth medium containing 1 x 106 antigen-positive HSB-2
target cells. After 1 hour, the cells were washed twice in
the medium and incubated for an additional 30 min in 0.1 ml
of medium containing 1:40 dilution of FITC-labeled goat
anti-mouse IgG (Boehringer-Mannheim). The cells were
analyzed on a Coulter Epics V fluorescence cell analyzer.
Cell surface fluorescence was compared to the fluorescence
obtained using similarly diluted unconjugated monoclonal
,antibody. As the figure indicates, the binding activity of
each of the immunoconjugates was preserved as demonstrated
by the fact that the concentration of immunoconjugate that
was required to saturate the antigen-positive cells was at
* Trademark -46-
1
~ i~~.~6.~~
most one doubling dilution greater than the concentration
required for the unconjugated antibody. The differences in
the plateau levels of fluorescence intensity between
unconjugated antibodies and the immunoconjugates was found
to be due to reduced binding of the secondary FITC-goat _
anti-mouse reagent to the immunoconjugate as compared to the
unconjugated antibody.
The stability of an immunoconjugate of this embodi-
ment -- an L6-ADM conjugate -- at various pH's, ranging from
4.0 to 7.0, was studied using HPLC analysis. The L6-ADM-9.0
conjugate was incubated in phosphate buffers at each of the
indicated pAs for 24 hours at 37°. Each solution was then
applied to an HPLC column and the amount of unconjugated
drug was determined. As shown in Figure 9, the single
product detected after 24 hour incubation at the different
pH's had a column retention time similar to that of the
ADM-HC1 standard. The amount of material released from the
immunoconjugate after 24 hours increased as the pH was
lowered from 7 to 4. The "untreated" control represents the
chromatograph of the conjugate stored at -20° in pH 7.4
phosphate buffer. It thus appears that the immunoconjugate
has an acid-sensitive linkage group which resulted in the
_ release of ADM from the antibady protein. These results are
consistent with the existence of a hydrazone bond joining
the ADM to the linker arm as described in Figure 2.
According to the embodiment of this example, ADM was
attached to the antibody through a linker arm that also
-47-
a~
~a:.~~~~~a~~
contained a disulfide bond (see Figure 2). It should thus
be possible to release an ADM moiety by reduction of an
immunoconjugate of this embodiment with DTT. Thex-efore, the
L6-ADM conjugate, L6-ADM-9.0, was treated with a 10-fold
excess DTT, incubated at room temperature for 15 min and
applied to an HPLC column. ADM-HC1 and ADM-HZN standards
were run at the same time and the peaks from the
chromatographs are indicated in Figure 10. L6-ADM-9.0 had
no detectable peak of unconjugated drug prior to 'the
addition of DTT. HPLC analysis showed the appearance of a
single peak that had a column retention time similar to that
of ADM-HC1 rather than the ADM-HZN derivative (see Figure
10). The amount of ADM released after DTT treatment was
approximately 99% of the starting ADM equivalents bound to
the antibody. The experimental data of Figures 9 and 10
demonstrate that an ADM-like moiety is released from the
immunoconjugates of this invention under "physiologic"
conditions, i.e., acidic and reducing conditions, typical of
the cellular environment.
Cytotoxic Activity Of The Immunoconiugates Of The Invention
The immunoconjugates of the invention were tested in
vitro for cytotoxicity using a number of assay systems.
According to a soft agar colony formation assay, Daudi
(Burk3 tt's lymphoma) cells (phenotype: 5E9+, 3A1-) obtained
from the ATCC were grown in complete medium (RPMI 1640
medium plus 10% fetal calf serum). 1 x 105 cells in 1 ml of
-48-
- J
s R,~~.~i
medium were exposed for 1.5 hours to serially diluted
5E9-ADM or 3A1-ADM immunoconjugates or unconjugated ADM.
Triplicate determinations were done for each dilution.
Controls consisted of similarly treated cells not exposed to
drugs. The cells were then washed and suspended in RPMI
1640 medium containing 15% FBS and 0.3% agarose (Marine
Colloid). One ml of the cell suspension (1 X 103 cells) was
then overlayed onto a 0.4% agarose layer in 6-well
microtiter plates (Costar). Samples were incubated for 7-10
days at 37o and the resulting colonies stained with 0.5 ml
of 1 mg/ml of p-iodonitrotetrazolium violet (Sigma) for 48
hours. Colonies were counted using an Optimax 40-10 image
analyzer and the inhibition of colony formation determined
by comparing drug-treated or immunoconjugate-treated cells
to the untreated control. '
Figure 11 compares the cytotoxic activity of the SE9-
ADM conjugate, 5E9-ADM-7.5, and the 3A1-ADM conjugate,
3A1-ADM-7.0, after- 1.5 hours exposure on the 5E9 antigen-
positive and 3A1 antigen-negative Burkitt's lymphoana cell
line, Daudi. Both of these immunoconjugates had been
prepared via thiolation with 2-IT. Comparison of the dose
response curves shows that the 5E9-ADM-7.5 conjugate which
retained 93% of the original binding activity for antigen-
bearing target cells (see Figure 8) was significantly more
potent than 3A1-ADM-7.0, the non-binding control conjugate.
A limiting dilution assay, which provides a measure of
the log cell kill, was used to test for the cytotoxic drug
-49-
~ ~~ ~'.l~iv
activity of the above-mentioned two immunoconjugates, using
a longer exposure format (24 hours). This assay was
performed using Namalwa cells (phenotype: 5E9+, 3A3-)
essentially as described by M. Colombatti et al., "Selective
Killing Of Target Cells By Antibody-Ricin A Chain Or
Antibody-Gelonin Hybrid Molecules: Comparison Of Cytotoxic
Potency And Use In Immunoselection Procedures", J. Immunol.,
131, pp. 3091-95 (1983). The cells, obtained from the ATCC,
were incubated with the immunoconjugates for 22 hours,
washed and log cell kill was determined. Log cell kill was
calculated based on the plating efficiencies that were
estimated by the portion of wells without growth at limiting
cell concentrations.
As shown in Figure 12, SE9-ADM-7.5 produced 1-2 logs
greater cell kill at concentrations tested as compared with
the non-binding 3A1-ADM-7.0 conjugate. Up to 5 logs of cell
kill was measured at the highest dose of 5E9-ADM-7.5.
Additionally, while cytotoxic activity for the non-binding
3A1 immunoconjugate was detected, the level of cytotoxicity
was less than an equivalent amount of unconjugated ADM.
However, the activity of the 5E9 immunoconjugate was, at
several concentrations, greater than an equivalent dose of
free ADM.
As stated above, the 5E9-ADM-7.5 and 3A1-ADM-7.0
immunoconjugates were synthesized using 2-IT as the
thiolating agent. Immunospecific cytotoxicity was also
observed with immunoconjugates that, were prepared using SPDP
-50-
r~
2~~.~:~.
as the thiolating agent. Figure 13 shows selective
cytotoxic activity of 5E9-ADM and 3A1-ADM immunoconjugates,
made using SPDP as the thiolating agent, on Daudi cells
using the soft agar colony formation assay described above.
Further evidence for selective cytotoxicity of _
immunoconjugates prepared using SPDP is shown in Figure 14,
where the 628.1-ADM-9.0 immunoconjugate was tested on two
628.1 antigen-positive Bell lines, Daudi and Namalwa, and on
one 628.1 antigen-negative human T cell leukemia cell linQ,
HSB-2, using the soft agar colony formation assay. The
HSB-2 cells were obtained from the ATCC. As shown in the
figure, the immunoconjugate was cytotoxic toward the two
antigen-positive cell lines but not toward the antigen-
negative cell line.
Preferential killing of antigen-positive cells by the
immunoconjugate, 5E9-ADM-7.5, was also observed in a colony
formation assay using the anchorage-dependent. human colon
carcinoma cell line, HCT116, obtained as a gift from Dr. M.
Brattain Bristol-Baylor Labs, Houston, TxJ.
Monolayer cultures of the carcinoma cells were removed
from culture flasks with trypsin-EDTA (GIBCO), washed and
passed through a 22-gauge needle to obtain a single cell
- suspension. 5E9-ADM-7.5, 3A1-ADM-7.0 ox unconjugated ADM
was serially diluted in 0.2 ml of medium containing 1 X 105
carcinoma cells. Each dilution was done in triplicate.
Controls included untreated or antibody-treated cells.
Cells were incubated for 3 hours, washed one time in medium
-51-
CA 02010164 1999-10-25
_. ~ w
and 1 X 103 cells in one ml were plated in 12-well
microtiter plates (Costar). Plates were incubated for 7-10
days at 37° and fixed with absolute methanol for ten min.
The colonies were stained with crystal violet and coun~.ed on
an Optimax 40-10 image analyzer. As shown in Figure 15,
greater cytotoxicity was observed when the carcinoma cells
were exposed to SE9-ADM-7.5 than when they were exposed to
3A1-ADM-7Ø
Because many reports have shown that ADM linked to
antibody at the amino sugar of the drug resulted in
immunoconjugates showing a significant loss of drug
activity, we tested the cytotoxicity of immunoconjugates
prepared by attaching ADM to the antibody at the amino sugar
of the drug thru a leu-ala dipeptide linker, using our soft
agar colony formation assay system. As shown in Figure 16,
neither the SE9-ADM-4.0 nor 3A1-ADM-3.9 peptide-linked
conjugates were cytotoxic on Daudi cells. The ADM-leu-ala
derivative used to make conjugates was about 2 logs less
potent than equivalent amounts of unconjugated ADM.
EXAMPLE 2
This example describes the preparation of an
anthracycline immunoconjugate according to the present
invention wherein ADM is conjugated to a monoclonal antibody
via a linker arm having an acylhydrazone bond as its site of
attachment to the ADM molecule and additionally having a
* Trademark -52-
i as
thioether linkage as part of its attachment to the antibody.
This embodiment also provides a novel acylhydrazide
derivative of ADM.
Preparation Of ImmunoconiuQates Having A Thioether Bond
Within The Linker Arm
Monoclonal antibody, 5E9, (2.5 mg in 2.5 ml phosphate
buffered saline) was reacted with SMPB (succinimidyl-4-
(p-maleimidophenyl)butyrate) (59.5 ug in 100 ul tetrahy-
drofuran) at 30° for 30 min. The pH was adjusted to 6.0
with sodium citrate buffer. The mixture was passed through
a' PD-10 gel filtration column (Pharmacia) to separate
maleimide-containing antibody from unreacted materials. The
ADM-HZN derivative (1 mg) prepared as described in Example 1
was then dissolved in 1 ml MeOH/H20 (9:1) and 0.5 umoles of
the ADM-HZN was reacted with 0.5 umoles of tri-n-butyl-
phosphine in 4:1 acetone: H20 to prepare a novel reduced
ADM-HZN (see Figure 17). After 10 min, 0.1 M sulfur in
toluene was added to destroy remaining phosphine. The
reduced ADM-HZN was then mixed with the 5E9 maleimide-
containing antibody. Immunoconjugates so produced were
purified by passage through a PD-10 gel filtration column.
~n some instances, when removal of toluene solvent had not
been complete, an organic solvent layer separated, floating
some protein from the reaction mixture. A gentle stream of
air was used to remove the solvent and the denatured protein
-53-
~ ~ ~.:~~,.~
was removed by spinning of the mixture for 2 min at 16,000 x
g. The clear supernatant containing the immunoconjugates
was then gel filtered and analyzed in PBS at pH 7.4. The
ADM/antibody molar ratio was determined spectrophotometri-
tally using OD280 and OD495 as described in Example 1. A-
typical reaction yielded immunoconjugates with molar ratios
of between 3 and 4.
Cytotoxic Activity Of Immunoconiugates Having A Thioether
Linkage
A number of immunoconjugates prepared according to the
embodiment of this example were tested for cytotoxicity
toward antigen-positive vs. antigen-negative tumor cell
lines using a 3H-thymidine incorporation assay which
measures inhibition of DNA synthesis. According to this
assay, dilutions of the immunoconjugates or unconjugated ADM
were made in complete medium and 100 u1 of each dilution was
added to wells in 96-well microtiter plates. Each dilution
was done in triplicate. Tumor cells were suspended in
medium and 100 ul containing 1x105 cells were then added to
each well. Cells were incubated for 24 hours at 37°C in a
5% C02 humid atmosphere. Fifty microliters containing 1 a
Ci(6-3HJ-thymidine (New England Nuclear, l5Ci/mmole) was
added to each well and incubated for four hours at 37oC.
Cells were transferred to Millititer sv plates (Millipore)
and precipitated with 25% cold trichlo.roacetic acid (TCA).
-54-
CA 02010164 1999-10-25
.,
The precipitates were washed ten times with 5% cold TCA.
Filters were dried, punched and counted in Econofluor liquid
scintillation fluid (New England Nuclear). All counts were
corrected by subtraction of background counts. ,
An immunoconjugate according to this embodiment --
SE9-ADM-3.9-- was highly cytotoxic toward 5E9 antigen-
positive Namalwa and HSB-2 cells (see Figure 18). The
immunoconjugate was more potent than equivalent
concentrations of unconjugated ADM. In another experiment,
a 3A1-ADM-6.0 immunoconjugate at concentrations below 0.1
yg/ml ADM was found to be cytotoxic toward 3A1-antigen-
positive HSB-2 cells but not toward 3A1 antigen-negative
Namalwa cells (see Figure 20). At higher concentrations,
the cytotoxicity of the immunoconjugate was about the same
toward both cell lines.
EXAMPLE 3
In Vivo Anti-Tumor Activity Of The_Immunoconjugates Of The
Invention
Immunoconjugates of the invention were next tested for
their anti-tumor activity in vivo. More particularly, the
immunoconjugates were tested for their ability to inhibit
the growth of human B lymphoma tumors in mice.
* Trademark -55-
t'.~ i ~. ~~ .~ Vii'
Primary Daudi and Ramos (Burkitt's lymphoma) solid
tumors were established in BALB/c nude mice by subcutaneous
(s. c.) inoculation of tissue culture-maintained lymphoid
cells. The Ramos cell line is available from the ATCC. The
Daudi and Ramos tumors were then serially passaged in vivo
in 4-6 week old female BALB/c (nu/nu) mice weighing from
20-25 gm (Harlan Sprague-Dawley), using 1 x 10~ tumor
cells/0.1 ml in PBS for implantation subcutaneously into the
flank of the mice. Both tumor lines showed a linear growth
rate between 200 and 4000 mm3. The median tumor volume
doubling time during exponential growth was 6.9 + 0.8 days
for Daudi tumors and 4.4 ~ 0.6 days for Ramos tumors. Tumor
volumes (V) were calculated using the formula:
V = L x W2
2
where L = length (mm) and W = width (mm).
When tumor volumes reached 400-600 mm3 for Daudi tumors
and 250-400 mm3 for Ramos tumors, the mice were randomized
into groups of 5 - 10 animals for treatment with ADM-HC1
(i.e., free drug), ADM-immunoconjugates of the invention,
unconjugated monoclonal antibody or a mixture of the
monoclonal antibody plus ADM. Specificity of cell killing
was demonstrated by comparing the anti-tumor activity
obtained with the tested immunoconjugates (i.e.,, whose
antibody component is reactive with the tumor cells to be
killed) vs. that obtained using non-binding conjugates
(i.e., conjugates that are not reactive with that tumor
-56-
~~~.~1.~~1~
population). The mixture of antibody plus free drug was a
control demonstrating the need for a covalent coupling of
drug to antibody.
Results were expressed as inhibitian of tumor growth
(T-G) or tumor doubling delay (TDD) which were estimated
from the delay in the tumor volume doubling time (TVDT) when
growth curves from treated groups were compared to
uninoculated controls. TDD was calculated using the formula
TDD - T - C
TVDT x 3.3
where T = time (days for the tumors in a treated group to
reach 3000 mm3, C = time (days) for tumors in a control
group to reach 3000 mm3, and TVDT (tumor volume doubling
time) = time (days) for the tumor volume in control
(non-treated) mice to increase from 1500 - 3000 mm3. Each
point represents the median tumor volume in the experimental
group.
In these studies, the anti-tumor activity of the
ADM-immunoconjugates on Daudi or Ramos tumors was compared
to: a) that obtained using free drug at an equivalent dose,
route of administration, and schedule and b) the activity
obtained using the free drug given at its optimal dose,
route and schedule.
In all of the studies described herein, drug treatment
with ADM-HCl was performed by adding 50-100 a DMSO to the
powdezed drug, diluting the dissolved drug in PBS to a
particular dosage of my/kg/inj on the day of injection
-57-
and inoculating it into the tumor-bearing mice either
intravenously (i.v., tail vein) or intraperitoneally (i.p.).
The ADM-immunoconjugates used in these studies were prepared
as described in Example 1 and all retained greater than 90%
of the original antibody binding activity. Specifically,_
the monoclonal antibodies 5E9 and 628.1 were used as the
antibody component of the immunoconjugates in these studies.
The ADM-immunoconjugates were stored at 4°C in PBS and used
no later than two weeks after their preparation. All of the
immunoconjugates tested as well as unconjugated antibody
controls were administered i.p.
Furthermore, as used in this application, the treatment
schedule notation "Q7Dx3" connotes a treatment schedule
wherein each mouse in that drug group was given 3
injections, each injection spaced 7 days apart, i.e.,.an
injection weekly for three weeks. Likewise, "Q5Dx2" refers
to a treatment schedule wherein the mice in that group were
given a total of 2 injections of drug or conjugate spaced 5
days apart. QlDx1 refers to a single injection. Thus, the
treatment schedule notations are defined wherein the first
numeral of the notation represents the spacing (in days) of
injections and the last numeral represents the total number
of injections per schedule.
The anti-tumor activity of the ADM-immunoconjugates of
the invention was therefore first evaluated as compared to
the free ADM-HC1 drug at a matching or equivalent dose,
route of administration, and schedule. The anti-tumor
-58-
W:
~'~t ~ ~.~.~~v
activity on Daudi tumors of a 5E9-ADM immunoconjugate,
5E9-ADM-1.8 (mole ratio = MR = 1.8 ADM molecules/MAB), was
compared to the activity of a) unconjugated ADM-HCi at a
matching drug dose (~4.1 mg/kg/inj), b) the 5E9 monoclonal
antibody at a matching antibody dose (630 mg/kg/inj), c) a
mixture of the 5E9 antibody plus ADM-HC1 (4.1 mg ADM + 630
mg 5E9), and d) a non-binding immunoconjugate, L6-ADM-8.6
(4.1 mg/kg/inj ADM) as a control.
Mice (5 mice/group) were dosed, i.p., on days 20 and 25
after tumor implantation (i.e:, a Q5Dx2 schedule) when
initial tumor sizes ranged from between 800 to 1100 mm3.
The dose used in this experiment represented the maximum
tolerated dose (MTD), i.p., for free drug, that is, the dose
of the drug administered via any given route or schedule
that results in an LD10 (death of 10% of the animals) (see
Table 1 below).
As Figure 21 indicates, significant anti-tumor activity
was obtained with the 5E9-ADM conjugate. Furthermore, this
anti-tumor activity was greater than that observed at an
equivalent dose of free drug. And, as Table 4 below
indicates, three of the five mice treated with the conjugate
had complete tumor regressions (cures), which corresponded
_ to a >1.5 TDD. Tn cantrast, ADM-HC1 and unconjugated 5E9 as
well as the L6-ADM non-binding conjugate exhibited no
anti-tumor activity. Same tumor growth inhibition was
observed using the ADM-HC1 plus 5E9 mixture, but this effect
-59-
"..' r.
was transitory and represented only a statistically
insignificant 0.2 TDD.
In this experiment, the free drug was dosed at 4.1
mg/kg/inj due to the toxicity associated with i.p.-
administered free drug at doses above 4-5 mg/kg. At these
low doses, both free ADM and mixtures of ADM plus monoclonal
antibody were inactive. However, as Figure 21 makes clear,
even at this low dose, the ADM immunoconjugate was still
active in inhibiting tumor growth.
Next, we sought to determine the anti-tumor activity of
the ADM-immunoconjugates on Daudi tumors as compared to the
anti-tumor activity obtained using the unconjugated drug
given at its optimal dose, route of administration, and
schedule. Initially therefore, we had to determine the
dase, route, and schedule of free ADM-HC1 that led to a
maximal anti-tumor activity on Daudi cells. E'or this
optimization study, mice were treated with ADM-HC1 using
different routes of administration, dosages and schedules.
The spacing of inoculations was dependent on the treatment
schedule employed. TDD values were then determined as
described above.
The results of this optimization study are summarized
in Table 2 below. As can be seen from Table 2, the Q7Dx3
schedule, given i.v., gave an optimal anti-tumor response,
both in terms of tumor growth delay and in tumor regression
rates at a dose of 11 mg/kg/inj, which was also the MTD for
the drug using the Q7Dx3 schedule, i.v.
-60-
,.. , J 1
~a..Y~ u~~8
Table 2
Anti-Tumor Activity Of ADM-HC1 On Daudi Tumor Xenografts
Dose /ka)a Tumor Toxicityc
(ma Inhibitionb
Schedule inj cum T-C CR Cures TDD D/T (%)
'
A) i.v. Route
QlDx1 20 20 - - - - 7/7 (100)
18 18 - _ - - 6/8 (
75)
15 15 - - - - 7/7 (100)
12 12 - - - - 7/7 (100)
Q7Dx3 12 36 >62 1 3 >2.0 4/8 (
50)
11 33 28 0 3 1.1 2/8 (
25)
11 33 >42 0 4 >1.3 2/10 (
20)
11 33 21 1 0 0.7 0/8
10 30 18 0 1 0.7 0/8
10 30 13 0 1 0.8 0/8
10 30 27 0 3 0.8 0/7
9 27 23 0 1 0.9 0/10
5 15 6.2 2 0 0.18 1/9 (
11)
$) i.p. Route
Q5Dx2 4.5 9 0 0 0 0/5
4.1 8.2 0 0 0 0 1/5
4.5 9 2.2 0 0 .1 0/8
5.5 11 2.2 0 0 .1 0/8
Q8Dx2 13 26 - - - - 7/7 (100)
10 20 - - - - g/8
5 10 - - - - 3/8 (
37)
Q4Dx3 5 15 - - - - 6/9 (
67)
Q7Dx3 10 30 - - - - 8/8 (100)
5 15 _ _ _ - 6/9 (
67)
aResults from groups.
individual
drug
_ bT-CC: presentsthe time lay in3days drug..
re de for the
treated group to reach 000 mm as to
(T) 3 compared
untreate d controls ).
(C
Complete Regressions (CR): temporary in
reduction tumor
volume tumor size.
below
palpable
Cures: completeregressionwith no evidence tumor
of
regrowth.
nu mber eaths overtotal number als thin
of d of anim wi
a group. Drug athswere ecorded up 55 s
de r to day after
the
last dru g dose.
-61-
d
~e~3~'~.~~~;~~
The anti-tumor activity of free drug on Daudi tumor
cells is further depicted in Figure 22. Using the Q7Dx3
schedule, given i.v., the growth of Daudi tumor xenografts
was significantly inhibited in a dose dependent fashion at
9, 10 or 11 mg/kg/injection, respectively, after treatment
with ADM-HC1. Control mice were untreated. Inhibition of
tumor growth (T-C) at the MTD, which was 11 mg/kg/inj, was
28 days which corresponded to a 1.1 TDD.
Various schedules using i.p. administration were also
tested. As discussed above, the MTD of ADM-HC1, i.p., was
determined to be between 4-5 mg/kg/inj. As can be seen from
Table 2B, the free drug is inactive on Daudi cells at its
MTD via the i.p route. Thus, we determined that optimal
anti-tumor activity for the free drug is obtainable via i.v.
administration where the MTD is 11 mg/kg/inj, a drug dose
that shows growth inhibition of Daudi tumor cells.
From these experiments, therefore, it was determined
that the optimal ADM-HC1 dosage for anti-tumor activity on
Daudi tumors was approximately 11 mg/kg/inj, the optimal
schedule was Q7Dx3 and the optimal route of administration
was i.v.
We next compared the anti-tumor activity on Daudi
tumors of the ADM-immunoconjugates of this invention to the
anti-tumor activity of the free ADM-HC1 drug given under
optimal conditions as deterrnined above. A 628.1-ADM
immurroconjugate, 628.1-ADM-7.6 (MR = 7.6 drugs/MAB), dosed
-62-
~ ~~.~~,~i~
using a Q5Dx2 schedule, i.p., was compared to ADM-HCl dosed
at 10, 11 and 12 mg/kg/inj on a Q7Dx3 schedule, i.v.,
schedule. As shown in Figure 23 and Table 3 belowt the free
drug was active, giving a 28 day delay in tumor growth,at 11
mg/kg (its MTD) with two of eight mice showing complete
tumor regression (cures). The immunoconjugate at the
highest tested dose (18.7 mg ADM, Q5Dx2, i.p.) was well
tolerated (no deaths, no weight loss) and gave an anti-tumor
activity somewhat higher than the free drug with three of
the eight treated animals showing complete tumor regression.
Again, there was no anti-tumor activity associated with
non-binding L6-immunoconjugate, unconjugated 628.1 or a
mixture of unconjugated 628.1 plus ADM-HC1. Thus, we
determined that the ADM-immunoconjugates inhibited tumor
growth to a greater extent than could be achieved using the
unconjugated drug at its optimal dose and schedule, i.v. or
i.p.
-63-
I E.
~1 y~~~~i~
Table 3*
Anti-Tumor Activity Of MAB Conjugated ADM (Q5Dx2; i.p.)
Compared To Optimized ADM-HC1 (Q7Dx3; i.v.) On Daudi Tumor
Xenografts
Dose a/kg)a Tumor Inbibitionb Toxicityc
(m
ADM MAB T-C CR Cures TDD D/T (%)
ADM-HC1 Q7Dx3; i.v.
12 >33 3 3 >1.5 2/8
11 28 0 2 >1.1 0/8
la 18 0 1 o.e o/e
628.1-ADM O5Dx2
(7.6) p
i
18.7 700 >31 0 3 >1.5 0/8
8.1 300 3 0 0 0.1 0/8
4.4 165 1 0 0 0 0/8
L6-ADM 5.5) OSDx2 i
( p
18.7 965 7 0 0 0.3 0/8
8.1 415 0 0 0 0 0/8
.
628.1 ADM O5Dx2 i
* p
4.5 700 6 0 0 0.2 0/8
G28. QSDx2 i~_
1
700 2 0 0 0.1 0/8
*See Table 2 for legend.
-64-
1
~~.~1.~~
Table 4 summarizes the anti-tumor activity obtained
using different preparations of 5E9 and 628.1 immuno-
conjugates on Daudi tumor xenografts in athymic mice. The
highest response rate was consistently obtained at antibody
doses of 500 mg/kg or greater. At these doses, anti-tumor
activity was obtained using conjugates having molar ratios
of 1.8 to 8.6. The anti-tumor activity appeared to be
dependent upon the antibody dose rather than the conjugated
drug dose as evidenced by the fact that as the monoclonal
antibody dose increased, there was a corresponding increase
in both TDD, i.e., inhibition of tumor growth, and tumor
regression rates. In all experiments, no anti-tumor
activity was observed with the non-binding L6-ADM conjugates
that were tested in parallel at equivalent antibody and
conjugated drug doses (results not shown). Tn addition,
this table illustrates the increased potency of the
immunoconjugates of the invention as compared to free drug,
which was inactive at equivalent drug doses (compare Table 2
above).
-65-
l
s.. ~.~ .~. ~i=~
Table 4
Anti-Tumor Activity Of ADM-Immunoconjugates 0n Daudi Tumor
Xenograftsa
Cum. Dose (mg/kg) T-Cb
Conjugate - MR MAB ADM (Days) Cures TDD
5E9-ADM-4.2d 200 4 10 0/7 0.5
5E9-ADM-8.6 260 8.2 8 0/5 0.3
d
5E9-ADM-4.2 500 5 17 1/7 0.8
5E9-ADM-5.4 1110 22.8 31 2/5 1.4
d f
5E9-ADM-4.2 1200 18.3 >61 2/7 >1.5
5E9-ADM-1.8 1260 8.2 >39 3/5 >1.5
628.1-ADM-4.9 200 4 8 0/7 0.4
628.1-ADM-7.6e 330 8.8 1 0/7 0
628.1-ADM-7.6e 600 16 3 0/7 0.1
628.1-ADM-4.2 1110 16.8 >37 2/3 >1.7f
628.1-ADM-4.9 1200 21.4 >56 2/7 >1.5
628.1-ADM-7.6e 1400 37.4 31 3/8 1.3
aSchedule: Q5Dx2Route:i.p. MR: mole ratio drug
of
molecules/MAB
bT-C: representsthe
time
delay
in3days
for
the
drug-
treated group to as
(T) reach compared
3000 to
mm
untreated controls (C).
cCures: cures/number animals
of treated.
d5E9-ADM-4.2 d at
teste three
doses.
eG28.1-ADM-7.6 ted three doses.
tes at
(Death in controlgroup.
Table 5 below demonstrates the reduced toxicity
achieved using the ADM-immunoconju gates of the invention vs.
the unconjugated drug. As can be seen, the immunoconjugates
were at least 10 times less toxic than free ADM dosed i.p.
-66-
~~~.~~.~i~~
Table
5
Toxi cityof Free and MAB-ADMin or-Bearing Micea
ADM Tum Nude
ADM (mg/kg)c - %
b
CompoundN Schedule Route inj Cumulative D/T Deaths
ADM 4 QlDxi i.v. 18 18 14/3244
ADM 3 QlDx1 i.v. 16 16 3/31 10
ADM 1 QlDx1 i.v. 14 14 0/8 0
ADM 1 Q2Dx2 i.v. 15 30 7/7 100
ADM 2 Q2Dx2 i.v. 12 24 10/1283
ADM 1 Q2Dx2 i.v. 10 20 3/5 60
ADM 1 Q2Dx2 i.v. 8 16 1/5 20
ADM 1 Q3Dx2 i.v. 16 32 8/8 100
ADM 1 Q3Dx2 i.v. 14 28 7/g gg
ADM 1 Q3Dx2 i.v. 12 24 6/8 75
ADM 1 Q4Dx2 i.v. 14 28 6/7 86
ADP1 2 Q4Dx2 i.v. 1?. 24 7/15 47
ADM i Q4Dx2 i.v. 10 20 2/8 25
ADM 1 Q7Dx3 i.v. 12 36 4/8 50
ADM 3 Q7Dx3 i.v. 11 33 4/26 15
ADM 3 Q7Dx3 i.v. 10 30 0/24 0
ADM 1 Q8Dx2 i.p. 13 , 26 7/7 100
ADM 1 Q8Dx2 i.p. 10 20 8/8 100
ADM 1 Q8Dx2 i.p. 5 10 3/8 38
ADM 1 Q4Dx3 i.p. 5 15 6/9 67
ADM 1 Q5Dx2 i.p. 5.5 11 0/8 0
ADM 1 Q5Dx2 i.p. 4.1 8,2 1/5 20
629.1-ADM1 Q5Dx2 i.p. 27.2 55.4 p/g p
1 Q5Dx2 i.p. 18.7 37.4 0/8 0
628.1-ADM1 QlDx4 i.p. 24 96 3/8 38
1 QlDx4 i.p. 14 64 0/8 0
GE28.1-ADM1 QlDx4 i,p. 10.5 42 0/5 0
L6-ADM 1 QlDx4 i.p. 31 124 1/8 13
1 QlDx4 i.p. 18.6
74 0/8 0
aMice Bearing Daudi or Ramos Tumors
bN = Number of Experiments
cADM = Amount Given Free or Conjugated to MAB
~/T = # Deaths/Total Treated
-67-
~ ~
Finally, Figure 26A and Table 6 depict the anti-tumor
activity of 628.1-ADM conjugates on human Ramos tumors.
Again, the anti-tumor effect of the immunoconjugates on
Ramos tumors was compared to that observed using free ,
ADM-HC1 under conditions which gave optimal results, which
was previously determined to be a single dose injection at a
dosage of 16-18 mg/kg/inj (see Figures 24 and 25). At the
highest immunoconjugate dose tested (10.6 mg/kg), the
anti-tumor activity of the conjugate was superior to the
activity obtained using free drug at 18 mg/kg (25%
lethality) by 0.5 TDD and to the activity of ADM-HG1 at 16
mg/kg (12% lethality) by 1.0 TDD. The conjugate at this
dose was~well tolerated with all of the treated animals
showing no weight loss or deaths. The anti-tumor activity
of 628.1-ADM was also found to be dose dependent as shown in
Figure 26B. Thus, decreasing the conjugate dose resulted in
decreases in the TDD and number of complete regressions.
The L6-ADM (non-binding) conjugate at a comparable dose
(10.6 mg/kg) was inactive.
-68-
a m
~~~~~f
Table 6
Anti-Tumor Activity Of MAB Conjugated ADM (QlDx4; i.p.)
To Optimized ADM-HC1 (QlDxl; i.v.) Using Ramos Tumor
Xenografts
Dose (mg/k4)a Tumor Inhibitionb Toxicityc
ADM MAB T-C CR TDD D/T (%)
Cures
ADM-HC1 Q7Dx3~i v
18 8 0 0 0.5 2/8 ( 25)
16 5 C 0 0.3 1/8 ( 12)
628.1-ADM (4.8)1Dx4;
9 i.p.
10.6 600 10.5 0 1 1.1 0/5
5.3 300 5 0 0 0.5 0/5
2.6 150 3.5 0 1 0.4 0/5
L6-ADM (7.9) QlDx4~i p
18.2 600 - - - - 2/5 ( 40)
10.6 360 0 0 1 0 0/5
aDose per injection.
bSee Table 2 for legend.
_69_
i~
~v.Y~~~~~~)l~
The above examples therefore demonstrate the
preparation of novel anthracycline immunoconjugates in which
a cytotoxic anthracycline drug is eonjugated to an antibody
via a novel acid-sensitive acylhydrazone linkage. Thea
immunoconjugates retain both antibody binding activity
(i.e., target cell specificity) and cytotoxic drug activity
and allow the release of free unmodified drug under acidic
and reducing conditions typical of the cellular environment
of the target cells. The anti-tumor activity of these
conjugates has been demonstrated both in vitro and in vivo
and has been shown to be greater than the activity obtained
with the free unconjugated anthracycline. ~ Furthermore, the
immunoconjugates were tolerated in vivo to a much greater
extent than the unconjugated drug. Thus, the immuno-
conjugates of this invention show an enhanced therapeutic
index (of anti-tumor activity vs. toxicity) and are
therefore particularly useful in delivering cytotoxic drugs
to a selected cell population for the preferential killing
of those cells in the treatment of diseases such as cancers
and other tumors, non-cytocidal viral or other pathogenic
infections and autoimmune disorders.
While we have hereinbefore presented a number of
- embodiments of this invention, it is apparent that our basic
construction can be altered to provide other embodiments
which utilize the immunoconjugates and methods of this
invention. Therefore, it will be appreciated that the scope
-70-
of this invention is to be defined by the claims appended
hereto rather than by the specific embodiments which have
been presented hereinbefore by way of example. _
--71-
