U.S. patent application number 10/975434 was filed with the patent office on 2006-03-02 for immunoconjugates targeting syndecan-1 expressing cells and use thereof.
Invention is credited to Viktor S. Goldmakher.
Application Number | 20060045877 10/975434 |
Document ID | / |
Family ID | 35997729 |
Filed Date | 2006-03-02 |
United States Patent
Application |
20060045877 |
Kind Code |
A1 |
Goldmakher; Viktor S. |
March 2, 2006 |
Immunoconjugates targeting syndecan-1 expressing cells and use
thereof
Abstract
Immunoconjugates comprising a targeting agent selectively
targeting cell-surface expressed syndecan-1 and at least one
effector molecule as well as in vitro and in vivo methods of using
those immunocomjugates are disclosed. The effector molecule may
have, in its native form, high non-selective cytotoxicity, but
substantially no non-selective cytotoxicity when part of said
immunoconjugate. Targeting agents include the antibody B-B4 as well
as other agents that bind cell-surface expressed syndecan-1.
Inventors: |
Goldmakher; Viktor S.;
(Newton, MA) |
Correspondence
Address: |
JOYCE VON NATZMER
4615 NORTH PARK AVENUE, SUITE 919
CHEVY CHASE
MD
20815
US
|
Family ID: |
35997729 |
Appl. No.: |
10/975434 |
Filed: |
October 29, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60605394 |
Aug 30, 2004 |
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Current U.S.
Class: |
424/133.1 ;
424/178.1; 530/391.1; 540/462 |
Current CPC
Class: |
A61K 47/6803 20170801;
A61K 47/6849 20170801; A61K 31/5365 20130101; A61K 45/06 20130101;
A61K 47/6851 20170801; A61P 35/00 20180101 |
Class at
Publication: |
424/133.1 ;
424/178.1; 530/391.1; 540/462 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/46 20060101 C07K016/46 |
Claims
1. An immunoconjugate comprising: at least one targeting agent
selectively targeting cell-surface expressed syndecan-1, at least
one effector molecule, wherein said effector molecule has, in its
native form, high non-selective cytotoxicity, wherein said
targeting agent is functionally attached to said effector molecule
to form said immunoconjugate, and wherein said effector molecule
has substantially no non-selective cytotoxicity when part of said
immunoconjugate.
2. The immunoconjugate of claim 1, wherein the cytotoxicity of said
effector molecule, in its native form, on cells targeted by said
targeting agent is higher or about the same as the cytotoxicity of
said immunoconjugate on said targeted cells.
3. The immunoconjugate of claim 1, wherein said effector molecule
has, in its native form, a potency of about 10.sup.-11-10.sup.-8
M.
4. The immunoconjugate of claim 1, wherein said effector molecule
is a maytansinoid, a CC1065 analogue, a calicheamicin or a
taxane.
5. The immunoconjugate of claim 4, wherein said effector molecule
is a maytansinoid.
6. The immunoconjugate of claim 5, wherein said maytansinoid is
DM1.
7. The immunoconjugate of claim 5, wherein said maytansinoid is
DM3.
8. The immunoconjugate of claim 5, wherein said maytansinoid is
DM4.
9. The immunoconjugate of claim 1, wherein said effector molecule
has a molecular weight of less than 5 kDa.
10. The immunoconjugate of claim 9, wherein said effector molecule
has a molecular weight of between about 600 and about 800 Da.
11. The immunoconjugate of claim 1, wherein said targeting agent is
a targeting antibody or non-immunoglobulin targeting molecule.
12. The immunoconjugate of claim 11, wherein said targeting agent
is a targeting antibody.
13. The immunoconjugate of claim 12, wherein said targeting
antibody is an antibody fragment.
14. The immunoconjugate of claim 12, wherein said targeting
antibody is derived from an antibody that internalizes poorly.
15. The immunoconjugate of claim 12, wherein said targeting
antibody is derived from the antibody B-B4.
16. A pharmaceutical composition comprising an effective amount of
the immunoconjugate according to claim 1 and one or more
pharmaceutically acceptable excipients.
17. A kit comprising, in separate containers, pharmaceutical
compositions for use in combination to inhibit, delay and/or
prevent the growth of tumors and/or spread of tumor cells, wherein
one container comprises an effective amount of the pharmaceutical
composition of claim 16, and wherein, a separate container
comprises a second pharmaceutical composition comprising an
effective amount of an agent for the inhibition, delay and/or
prevention of the growth of tumors and/or spread of tumor cells,
and one or more pharmaceutically acceptable excipients.
18. The kit of claim 17, wherein said agent in said second
pharmaceutical composition is a chemotherapeutic agent or another
immunoconjugate.
19. A method for treating inhibiting, delaying and/or preventing
the growth of tumor cells in a cell culture containing syndecan-1
expressing tumor cells and non-tumor cells comprising administering
a growth of syndecan-1 expressing tumor cells inhibiting, delaying
and/or preventing effective amount of the immunoconjugate of claim
1.
20. The method of claim 19, wherein said effective amount induces
cell death or continuous cell cycle arrest in said syndecan-1
expressing tumor cells.
21. The method of claim 19, wherein said cells in said cell culture
are obtained from a cancer patient and wherein, after
administration of said effective amount of said immunoconjugate,
the cells of said cell culture are reimplanted into said cancer
patient.
22. The method of claim 19, wherein the cells in said cell culture
were isolated from a patient suffering from an hematologic
malignancy and/or a solid tumor comprising syndecan-1 expressing
cells.
23. The method of claim 22, wherein said patient is suffering from
one of the following: multiple myeloma, ovarian carcinoma, kidney
carcinoma, gall bladder carcinoma, breast carcinoma, prostate
cancer, lung cancer, colon carcinoma, Hodgkin's and non-Hodgkin's
lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic
leukemia (ALL), acute myeloblastic leukemia (AML), a solid tissue
sarcoma or a colon carcinoma.
24. The method of claim 23, wherein said patient is suffering from
multiple myeloma.
25. A method for inhibiting, delaying and/or preventing the growth
of a tumor comprising syndecan-1 expressing tumor cells and/or
spread of syndecan-1 expressing tumor cells in a patient in need
thereof, comprising administering to said patient at least one
immunoconjugate in a growth of said tumor and/or spreading of said
tumor cells inhibiting or reducing amount, wherein said
immunoconjugate comprises at least one effector molecule and at
least one targeting agent, and wherein said immunoconjugate
selectively inhibits, delays or prevents the growth and/or spread
of syndecan-1 expressing cells.
26. The method of claim 25, wherein said patient suffers from an
hematologic malignancy and/or a solid tumor comprising syndecan-1
expressing cells.
27. The method of claim 26, wherein said patient suffers from one
of the following: multiple myeloma, ovarian carcinoma, kidney
carcinoma, gall bladder carcinoma, breast carcinoma, prostate
cancer, lung cancer, colon carcinoma, Hodgkin's and non-Hodgkin's
lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic
leukemia (ALL), acute myeloblastic leukemia (AML), solid tissue
sarcoma or colon carcinoma.
28. The method of claim 27, wherein the disease is multiple
myeloma.
29. The method of claim 25, wherein said effector molecule
exhibits, in its native form, high non-selective cytotoxicity.
30. The method of claim 25, wherein said effector molecule of said
immunoconjugate is a toxin, cytotoxic enzyme, low molecular weight
cytotoxic drug, a pore-forming agent, biological response modifier,
prodrug activating enzyme, an antibody, cytokine or a
radionuclide.
31. A method for inhibiting, delaying and/or preventing the growth
of a tumor and/or spread of malignant tumor cells in a patient in
need thereof, comprising (a) administering to said patient one or
more cancer drugs and/or radiation in an amount effective to reduce
tumor load; and (b) administering to said patient at least one
immunoconjugate in a growth of a tumor and/or spreading of tumor
cells inhibiting, delaying or preventing amount, wherein said
immunoconjugate selectively inhibits, delays or prevents the growth
and/or spread of syndecan-1 expressing cells.
32. The method of claim 31, wherein (a) and (b) are performed
consecutively in two consecutive treatment regimes.
33. The method of claim 31, wherein the at least one drug of (a)
and the immunoconjugate of (b) are administered in a single
administration step.
34. A method for treating a subject having a condition that would
benefit from the selective suppression of myeloma cell survival,
the method comprising: (a) providing at least one immunoconjugate
that selectively binds to syndecan-1 expressed on myeloma cells;
and (b) administering the immunoconjugate to the subject to
selectively decrease survival or growth of said myeloma cells of
said subject.
35. The method of claim 34, wherein the immunoconjugate comprises a
B-B4 targeting antibody.
36. The method of claim 34, wherein the immunoconjugate comprises a
maytansinoid effector molecule.
37. The method of claim 34, wherein said selective suppression of
myeloma cell survival induces growth arrest or cell death in
myeloma cells.
Description
BACKGROUND
[0001] This invention pertains to immunoconjugates and their use in
different indications. In particular, the present invention relates
to immunoconjugates, the delivery of their effector molecule(s) to
target sites and the site specific release of the effector
molecule(s) in, at or near target cells, tissues and organs. More
particularly, the present invention relates to immunoconjugates
comprising one or more syndecan-1 targeting agent and highly potent
effector molecules, which are attached to the targeting agent. The
effector molecule is activated by cleavage/dissociation from the
targeting agent portion of the immunoconjugate in, at or near the
target cells, tissues or organs.
[0002] The publications and other materials, including patents,
used herein to illustrate the invention and, in particular, to
provide additional details respecting the practice are incorporated
by reference. For convenience, the publications are referenced in
the following text by author and date and are listed alphabetically
by author in the appended bibliography.
[0003] A substantial body of research has concentrated on the
development of systems in which an effector agent can be
selectively delivered to a desired location or cell population,
i.e., a system for a more targeted treatment of ailments with fewer
toxic side effects. In spite of considerable progress that has been
achieved, many of those delivery systems for the treatment of
various diseases, for example, the treatment of cancer, are still
often ineffective or subject the patient to considerable risk.
[0004] Immunoconjugates comprise at least one targeting agent
attached to at least one effector molecule. Such immunoconjugates
can be categorized according to their effector molecules into, for
example, drug immunoconjugates, immunotoxin conjugate and
radioimmunoconjugates (Payne, 2003).
[0005] Efficiency in killing cells is one key factor in the
usefulness of an immunoconjugate. Efficiency can be influenced by
the potency of the effector molecule (Blattler and Chari, 2001), by
the ability of the effector to retain its potency (Chari et al.,
1995; Liu et al., 1996; Ojima et al., 2002; Senter et al., 2002 and
Sievers and Linenberger, 2001), By the tumor accessibility
(Charter, 2001), by the level of expression of the target antigen
on the target cell, targeting agent affinity, and by the ability of
the target cell to internalize the immunoconjugate (Wargalla,
1989). In the initial development period of immunoconjugates, the
efficiencies of conjugates having a drug as an effector molecule
often were disappointing compared to the free drug.
[0006] In response, immunoconjugates with highly cytotoxic effector
toxin molecules were constructed. However, while the efficiencies
of this new generation of immunoconjugates were much improved, they
were often immunogenic in humans, inducing neutralizing antibodies
both to the toxin protein and to the mouse monoclonal antibody. In
response, "humanized" antibodies conjugated to nonimmunogenic
effector molecules were developed (Payne, 2003).
[0007] In the context of both highly cytotoxic drugs and toxins
conjugated to a targeting agent, systemic toxicity has to be
considered. If the cytotoxic drug or the toxin is highly cytotoxic,
the immunoconjugate has to reach its target site without adversely
affecting the host on its way. Accordingly, if the immunoconjugate
circulates, for example, in the bloodstream to reach its target
site, then this should occur without a substantial release of
active drug. Thus, ideally, a highly cytotoxic drug or toxin of an
immunoconjugate is only activated upon reaching its target.
[0008] Specificity is another factor critical for the usability of
an immunoconjugate. The immunoconjugate has to be able to
selectively interact with the target cells. Particularly for in
vivo applications, it is critical that the immunoconjugate does not
have substantial adverse effects on essential non-target cells.
Thus, both the cellular target of the immunoconjugate and the
targeting agent of the immunoconjugate have to be carefully
selected to ensure specificity (Blattler and Chari, 2001).
[0009] It has also been considered important that immunoconjugates
comprising targeting antibodies demonstrate pharmacokinetic and
tissue distribution characteristics similar to those of
corresponding antibodies (Xie, 2003).
[0010] First successes have been achieved with immunoconjugates.
For example, MYLOTARG, a conjugate of an anti-CD33 humanized
monoclonal antibody and the highly cytotoxic DNA-damaging agent
calicheamicin, has been recently approved by the FDA as the first
drug immunoconjugate for clinical treatment of certain indications
of myeloid leukemia (Bross, 2001; Hamann, 2002; Dowell, 2001).
[0011] However, there remains a need to develop effective
immunoconjugates for a wide array of indications.
SUMMARY OF THE INVENTION
[0012] The present invention pertains in one embodiment to an
immunoconjugate comprising
[0013] at least one targeting agent selectively targeting
cell-surface expressed syndecan-1,
[0014] at least one effector molecule, [0015] wherein the effector
molecule has, in its native form, high non-selective cytotoxicity,
[0016] wherein the targeting agent is functionally attached to said
effector molecule to form the immunoconjugate, and [0017] wherein
the effector molecule has substantially no non-selective
cytotoxicity when part of said immunoconjugate.
[0018] The cytotoxicity of the effector molecule, in its native
form, on cells targeted by said targeting antibody may be higher or
about the same as the cytotoxicity of the immunoconjugate on said
targeted cells. The effector molecule may have, in its native form,
a potency of about 10.sup.-14-10.sup.-7, preferably a potency of
about 10.sup.-13-10.sup.-7M, of about 10.sup.-12-10.sup.-7M, of
about 10.sup.-12-10.sup.-8M, most preferably of about
10.sup.-11-10.sup.-10 M, which includes any narrower potency ranges
encompassed by the ranges specified above, such as, but not limited
to, a potency of about 10.sup.-11-10.sup.-10 M. The effector
molecule may be a maytansinoid, in particular DM1, DM3 or DM4, a
CC1065 analogue, a calicheamicin or a taxane. In certain
embodiments, the effector molecule may have a molecular weight of
less than 5 kDa, in particular less than 2 kDa, more in particular
less than 1 kDa and in between about 600 and about 800 Da.
[0019] The targeting agent may be a targeting antibody, which
includes fragments of antibodies, or non-immunoglobulin targeting
molecule.
[0020] The targeting antibody may be derived from an antibody that
internalizes poorly. In certain embodiments, the targeting antibody
may be derived from the antibody B-B4.
[0021] The present invention is also directed to a pharmaceutical
composition comprising an effective amount of the immunoconjugate
described above and one or more pharmaceutically acceptable
excipients.
[0022] The present invention is also directed to a kit comprising,
in separate containers, pharmaceutical compositions for use in
combination to inhibit, delay and/or prevent the growth of tumors
and/or spread of tumor cells, wherein one container comprises an
effective amount of above described pharmaceutical composition, and
wherein, a separate container comprises a second pharmaceutical
composition comprising an effective amount of an agent for the
inhibition, delay and/or prevention of the growth of tumors and/or
spread of tumor cells, and one or more pharmaceutically acceptable
excipients. The agent in said second pharmaceutical composition may
be a chemotherapeutic agent or another immunoconjugate.
[0023] In one embodiment, the invention is directed to a method for
treating, inhibiting, delaying and/or preventing the growth of
tumor cells in a cell culture containing syndecan-1 expressing
tumor cells and non-tumor cells, comprising administering an
effective amount of the above described immunoconjugate. The
effective amount induces, in certain embodiments, cell death or
continuous cell cycle arrest of said syndecan-1 expressing tumor
cells. The cells in said cell culture may be obtained from a cancer
patient and may, after administration of said effective amount of
said immunoconjugate, be reimplanted into said cancer patient. The
cells in said cell culture may be isolated from a patient suffering
from an hematologic malignancy and/or a solid tumor comprising
syndecan-1 expressing cells, in particular from a patient suffering
from one of the following: multiple myeloma, ovarian carcinoma,
kidney carcinoma, gall bladder carcinoma, breast carcinoma,
prostate cancer, lung cancer, colon carcinoma, Hodgkin's and
non-Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute
lymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML), a
solid tissue sarcoma or a colon carcinoma.
[0024] The present invention is also directed to a method of
inhibiting, delaying and/or preventing the growth of a tumor
comprising syndecan-1 expressing tumor cells and/or spread of
syndecan-1 expressing tumor cells in a patient in need thereof,
comprising [0025] administering to the patient at least one
immunoconjugate in a growth of the tumor and/or spreading of the
tumor cells inhibiting or reducing amount, wherein the
immunoconjugate selectively inhibits, delays or prevents the growth
and/or spread of syndecan-1 expressing cells. The patient may, in
this embodiment of the invention, suffer from an hematologic
malignancy and/or a solid tumor comprising syndecan-1 expressing
cells, in particular from one of the following: multiple myeloma,
ovarian carcinoma, kidney carcinoma, gall bladder carcinoma, breast
carcinoma, prostate cancer, lung cancer, colon carcinoma, Hodgkin's
and non-Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL),
acute lymphoblastic leukemia (ALL), acute myeloblastic leukemia
(AML), a solid tissue sarcoma or a colon carcinoma. An effector
molecule of the immunoconjugate may, in this embodiment, exhibit,
in its native form, high non-selective cytotoxicity.
[0026] The invention is also directed to a method for inhibiting,
delaying and/or preventing the growth of a tumor and/or spread of
malignant tumor cells in a patient in need thereof, comprising
[0027] (a) administering to the patient one or more cancer drugs
and/or radiation in an amount effective to reduce tumor load; and
[0028] (b) administering to the patient at least one
immunoconjugate in a growth of a tumor and/or spreading of tumor
cells inhibiting, delaying or preventing amount, wherein the
immunoconjugate selectively inhibits, delays or prevents the growth
and/or spread of syndecan-1 expressing cells; (a) and (b) may
hereby be performed consecutively in two consecutive treatment
regimes. The drug of (a) and the immunoconjugate of (b) may also be
administered in a single administration step.
[0029] The present invention is also directed to a method for
treating a subject having a condition that would benefit from the
selective suppression of myeloma cell survival, the method
comprising: [0030] (a) providing at least one immunoconjugate that
selectively binds to syndecan-1 expressed on myeloma cells; and
[0031] (b) administering the immunoconjugate to the subject to
selectively decrease survival or growth of said myeloma cells of
the subject. The immunoconjugate may comprise a B-B4 targeting
antibody. The immunoconjugate may, in this embodiment, comprise a
maytansinoid effector molecule. The selective suppression of
myeloma cell survival may also induce growth arrest or apoptosis in
myeloma cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIGS. 1A-1F show the expression of CD138 in multiple myeloma
(MM) cells.
[0033] FIGS. 2A-2C show the effect of B-B4-DM1 in comparison with
that produced by the naked antibody or by non-conjugated drug on
survival of CD138.sup.+ and CD138.sup.- MM cells.
[0034] FIGS. 3A to 3C show the inhibitory effect of B-B4-DM1 on
proliferation of CD138.sup.+ and CD138.sup.- cells adherent to bone
marrow stromal cells (BMSCs).
[0035] FIGS. 4A and 4B show the survival and cell cycle effects of
B-B4-DM1 on CD138.sup.+ MM cells.
[0036] FIGS. 5A to 5D show the activity of B-B4-DM1 in a tumor
xenograft model of human CD138.sup.+ multiple myeloma.
[0037] FIGS. 6A to F show the activity of B-B4-DM1 on large tumor
xenografts of human CD138.sup.+ OPM multiple myeloma.
[0038] FIG. 7A shows the expression of GFP in the cells (GFP stands
for Green Fluorescent Protein). FIGS. 7B to 7F show the activity of
B-B4-DM1 on GFP.sup.+ human MM xenografts.
[0039] FIGS. 8A to 8C show that B-B4-DM1 reduces MM tumor burden in
SCID-hu hosts implanted with patient MM cells.
[0040] FIGS. 9A to 9C show that B-B4-DM1 increases survival in
SCID-hu hosts implanted with the Ocy-My5 MM cell line.
DETAILED DESCRIPTION OF VARIOUS AND PREFERRED EMBODIMENTS
[0041] The present invention relates to immunoconjugates and the
delivery of their effector molecule(s) to target sites and the site
specific release of effector(s) molecule in, at or near target
cells, tissues and organs. More particularly, the present invention
relates to immunoconjugates comprising syndecan-1 targeting agents
and potent effector molecules. The effector molecules are
covalently linked, chelated or otherwise associated with the
targeting agent. The effector molecules may be activated by
cleavage/dissociation from the targeting agent portion of the
immunoconjugate at the target site.
[0042] The immunoconjugates according to the present invention are
administered to a subject in need of therapeutic treatment or to
cells isolated from such a subject in need of therapeutic
treatment. The effector molecule or molecules may be released from
the immunoconjugate by cleavage/dissociation in, at or close to the
target cell, tissue or organ.
[0043] As one example, the immunoconjugate comprises the antibody
B-B4 and at least one highly cytotoxic drug or toxin as an effector
molecule and is administered to a patient with cancer. In this
example, a therapeutically effective amount of the immunoconjugate
is administered intravenously to a patient so that it concentrates
in the cancer cells. The effector molecule or molecules are
released from the antibody target by natural means.
[0044] As a second example, the immunoconjugate comprises the
antibody B-B4 and at least one highly cytotoxic drug and is
administered to a cell population isolated from a patient with
cancer. In this example, a cell death or continuous cell cycle
arrest inducing amount of the immunoconjugate is administered to
the cell population so that it concentrates in the cancerous cells.
The effector molecule or molecules are released from the targeting
antibody by natural means or external means to induce cell death or
continuous cell cycle arrest in the cancer cells.
[0045] As a third example, the immunoconjugate comprises the
antibody B-B4 and at least one highly cytotoxic drug or an
immunotoxin as an effector molecule and is administered to a
patient with cancer. In this example, a therapeutically effective
amount of the immunoconjugate is administered intravenously to a
patient so that it concentrates in the cancerous cells. The
effector molecule or molecules are released from the antibody
target by an external means to induce cell death or continuous cell
cycle arrest in the cancer cells.
[0046] Targeting agent: A targeting agent according to the present
invention is able to associate with a molecule expressed by a
target cell and includes peptides and non-peptides. In particular,
targeting agents according to the present invention include
targeting antibodies and non-immunoglobulin targeting molecules,
which may be based on non-immunoglobulin proteins, including, but
not limited to, AFFILIN.RTM. molecules, ANTICALINS.RTM. and
AFFIBODIES.RTM.. Non-immunoglobulin targeting molecules also
include non-peptidic targeting molecules including targeting DNA
and RNA oligonucleotides (aptamers).
[0047] Targeting antibody: A targeting antibody according to the
present invention is or is based on a natural antibody or is
produced synthetically or by genetic engineering and binds to an
antigen on a cell or cells (target cell(s)) of interest. A
targeting antibody according to the present invention includes a
monoclonal antibody, a polyclonal antibody, a multispecific
antibody (for example, a bispecific antibody), or an antibody
fragment. The targeting antibody may be engineered to, for example,
improve its affinity to the target cells (Ross, 2003) or diminish
its immunogenicity. The targeting antibody may be attached to a
liposomal formulation including effector molecules (Carter, 2003).
An antibody fragment comprises a portion of an intact antibody,
preferably the antigen binding or variable region of the intact
antibody. Examples of antibody fragments according to the present
invention include Fab, Fab', F(ab').sub.2, and Fv fragments, but
also diabodies; domain antibodies (dAb) (Ward, 1989; U.S. Pat. No.
6,005,079); linear antibodies; single-chain antibody molecules; and
multispecific antibodies formed from antibody fragments. In a
single chain variable fragment antibody (scFv) the heavy and light
chains (VH and VL) can be linked by a short amino acid linker
having, for example, the sequence (glycine.sub.4serine).sub.n,
which has sufficient flexibility to allow the two domains to
assemble a functional antigen binding pocket. Addition of various
signal sequences may allow for more precise targeting of the
targeting antibody. Addition of the light chain constant region
(CL) may allow dimerization via disulphide bonds, giving increased
stability and avidity. Variable regions for constructing the scFv
can, if a mAb against a target of interest is available, be
obtained by RT-PCR which clones out the variable regions from mRNA
extracted from the parent hybridoma. Alternatively, the scFv can be
generated de novo by phage display technology (Smith, 2001). A
bispecific antibody according to the present invention may, for
example, have at least one arm that is reactive against a target
tissue and one arm that is reactive against a linker moiety (U.S.
Patent Publication 20020006379). A bispecific antibody according to
the present invention may also bind to more than one antigen on a
target cell (Carter, 2003). An antibody according to the present
invention may be modified by, for example, introducing cystein
residues to introduce thiol groups (Olafsen, 2004).
[0048] In accordance with the present invention, the targeting
antibody may be derived from any source and may be, but is not
limited to, a camel antibody, a murine antibody, a chimeric
human/mouse antibody or a chimeric human/monkey antibody, in
particular, a chimeric human/monkey antibody with the monkey
portion stemming from a cynomolgus monkey. Humanized antibodies are
antibodies that contain sequences derived from a human-antibody and
from a non-human antibody and are also within the scope of the
present invention. Suitable methods for humanizing antibodies
include CDR-grafting (complementarity determining region grafting)
(EP 0 239 400; WO 91/09967; U.S. Pat. Nos. 5,530,101; and
5,585,089), veneering or resurfacing (EP 0 592 106; EP 0 519 596;
Padian, 199; Studnicka et al., 1994; Roguska et al., 1994), chain
shuffling (U.S. Pat. No. 5,565,332) and Delmmunosation.TM.
(Biovation, LTD). In CDR-grafting, the mouse
complementarity-determining regions (CDRs) from, for example, mAb
B-B4 are grafted into human variable frameworks, which are then
joined to human constant regions, to create a human B-B4 antibody.
Several antibodies humanized by CDR-grafting are now in clinical
use, including MYLOTARG (Sievers et al., 2001) and HECEPTIN (Pegram
et al, 1998).
[0049] The resurfacing technology uses a combination of molecular
modeling, statistical analysis and mutagenesis to alter the non-CDR
surfaces of antibody variable regions to resemble the surfaces of
known antibodies of the target host. Strategies and methods for the
resurfacing of antibodies, and other methods for reducing
immunogenicity of antibodies within a different host, are
disclosed, for example, in U.S. Pat. No. 5,639,641. Human
antibodies can be made by a variety of methods known in the art
including phage display methods. See also U.S. Pat. Nos. 4,444,887,
4,716,111, 5,545,806, and 5,814,318; and international patent
application publications WO 98/46645, WO 98/50433, WO 98/24893, WO
98/16654, WO 96/34096, WO 96/33735, and WO 91/10741.
[0050] Fully human antibodies may also been used. Those antibodies
can be selected by the phage display approach, where CD138 or an
antigenic determinant thereof is used to selectively bind phage
expressing, for example, B-B4 variable regions (see, Krebs, 2001).
This approach is advantageously coupled with an affinity maturation
technique to improve the affinity of the antibody.
[0051] In one embodiment, the targeting antibody is, in its
unconjugated form, moderately or poorly internalizable. Moderate
internalization constitutes about 30% to about 75% internalization
of antibody, poor internalization constitutes about 0.01% to up to
about 30% internalization after 3 hours incubation at 37.degree. C.
In another preferred embodiment the targeting antibody binds to
CD138, for example, antibodies B-B4, BC/B-B4, B-B2, DL-101, 1 D4,
MI15, 1.BB.210, 2Q1484, 5F7, 104-9, 281-2 in particular B-B4.
Preferably the targeting antibody binds primarily to cell-surface
expressed CD138. In another embodiment, the targeting antibody does
not substantially bind non-cell-surface expressed CD138. When, in
the context of the present invention, the name of a specific
antibody is combined with the term "targeting antibody" such as
"B-B4 targeting antibody," this means that this targeting antibody
has the binding specificity of the antibody B-B4. If a targeting
antibody is said to be "derived from" a specified antibody, this
means that this targeting antibody has the binding specificity of
this antibody, but might take any form consistent with the above
description of a targeting antibody. If, in the context of the
present invention, for example, a targeting antibody is said to do
something "selectively" such as "selectively targeting cell-surface
expressed syndcan-1" or, to be "selective" for something, this
means that there is a significant selectivity (i.e. a higher
affinity towards CD138-positive cells compared with CD138-negative
cells) for, in case of the example provided, cell-surface expressed
syndecan-1, compared to any other antigens and adverse side effects
in a given environment are substantially avoided due to this
selectivity.
[0052] Non-immunoglobulin targeting molecules: Non-immunoglobulin
targeting molecules according to the present invention include
targeting molecues derived from non-immunoglobulin proteins as well
as non-peptidic targeting molecules. Small non-immunoglobulin
proteins which are included in this definition are designed to have
specific affinities towards, in particular surface expressed CD138.
These small non-immunoglobulin proteins include scaffold based
engineered molecules such as Affilin.RTM. molecules that have a
relatively low molecular weight such as between 10 kDa and 20 kDa.
Appropriate scaffolds include, for example, gamma crystalline.
Those molecules have, in their natural state, no specific binding
activity towards the target molecules. By engineering the protein
surfaces through locally defined randomization of solvent exposed
amino acids, completely new binding sites are created. Former
non-binding proteins are thereby transformed into specific binding
proteins. Such molecules can be specifically designed to bind a
target, such as CD138, and allow for specific delivery of one or
more effector molecules (see, scil Proteins GmbH at
www.scilproteins.com, 2004). Another kind of non-immunoglobulin
targeting molecules are derived from lipocalins, and include, for
example ANTICALINS.RTM., which resemble in structure somewhat
immunoglobulins. However, lipocalins are composed of a single
polypeptide chain with 160 to 180 amino acid residues. The binding
pocket of lipocalins can be reshaped to recognize a molecule of
interest with high affinity and specificity (see, for example,
Beste et al., 1999). Artificial bacterial receptors such as those
marketed under the trademark Affibody.RTM. (Affibody AB) are also
within the scope of the present invention. These artificial
bacterial receptor molecules are small, simple proteins and may be
composed of a three-helix bundle based on the scaffold of one of
the IgG-binding domains of Protein A (Staylococcus aureus). These
molecules have binding properties similar to many immunoglobulins,
but are substantially smaller, having a molecular weight often not
exceeding 10 kDa and are also comparatively stable. Suitable
artificial bacterial receptor molecules are, for example, described
in U.S. Pat. Nos. 5,831,012; 6,534,628 and 6,740,734. Non-peptidic
targeting molecules include, but are limited to, to DNA and RNA
oligonucleotides that bind to CD138 (aptamers).
[0053] Effector molecule: An effector molecule according to the
present invention is a molecule or a derivative, or an analogue
thereof that is attached to a targeting agent and exerts a desired
effect, for example apoptosis, or another type of cell death, or a
continuous cell cycle arrest on the target cell or cells. Effector
molecules according to the present invention include molecules that
can exert desired effects in a target cell and include, but are not
limited to, toxins, drugs, in particular low molecular weight
cytotoxic drugs, radionuclides, biological response modifiers,
pore-forming agents, cytotoxic enzymes, prodrug activating enzymes,
antisense oligonucleotides, antibodies or cytokines as well as
functional derivatives or analogues/fragments thereof.
[0054] In a preferred embodiment, the effector increases internal
effector delivery of the immunoconjugate, in particular when the
natural form of the antibody on which the targeting antibody of the
immunoconjugate is based is poorly internalizable. In another
preferred embodiment the effector is, in its native form,
non-selective. In certain embodiments the effector has high
non-selective toxicity, including systemic toxicity, when in its
native form. The "native form" of an effector molecule of the
present invention is an effector molecule before being attached to
the targeting agent to form an immunoconjugate. In another
preferred embodiment, the non-selective toxicity of the effector
molecule is substantially eliminated upon conjugation to the
targeting agent. In another preferred embodiment, the effector
molecule causes, upon reaching the target cell, death or continuous
cell cycle arrest in the target cell. A drug-effector molecule
according to the present invention includes, but is not limited to,
a drug including, for example, small highly cytotoxic drugs that
act as inhibitors of tubulin polymerization such as maytansinoids,
dolastatins, auristatin and crytophycin; DNA alkylating agents like
CC-1065 analogues or derivatives (U.S. Pat. Nos. 5,475,092;
5,585,499; 6,716,821) and duocarmycin; enediyne antibiotics such as
calicheamicin and esperamicin; and potent taxoid (taxane) drugs
(Payne, 2003). Maytansinoids and calicheamicins are particularly
preferred. An effector maytansinoid includes maytansinoids of any
origin, including, but not limited to synthetic maytansinol and
maytansinol analogue and derivative. Doxorubicin, daunomycin,
methotrexate, vinblastine, neocarzinostatin, macromycin, trenimon
and .alpha.-amanitin are some other effector molecules within the
scope of the present invention. Also within the scope of the
present invention are antisense DNA molecules as effector
molecules. When the name of, for example, a specific drug or class
of drugs is combined herein with the term "effector" or "effector
molecule," reference is made to an effector of an immunoconjugate
according to the present invention that is based on the specified
drug or class of drugs.
[0055] Maytansine is a natural product originally derived from the
Ethiopian shrub Maytenus serrata (Remillard, 1975; U.S. Pat. No.
3,896,111). This drug inhibits tubulin polymerization, resulting in
mitotic block and cell death (Remillard, 1975; Bhattacharyya, 1977;
Kupchan, 1978). The cytotoxicity of maytansine is 200-1000-fold
higher than that of anti-cancer drugs in clinical use that affect
tubulin polymerization, such as Vinca alkaloids or taxol. However,
clinical trials of maytansine indicated that it lacked a
therapeutic window due to its high systemic toxicity. Maytansine
and maytansinoids are highly cytotoxic but their clinical use in
cancer therapy has been greatly limited by their severe systemic
side-effects primarily attributed to their poor selectivity for
tumors. Clinical trials with maytansine showed serious adverse
effects on the central nervous system and gastrointestinal
system.
[0056] Maytansinoids have also been isolated from other plants
including seed tissue of Trewia nudiflora (U.S. Pat. No.
4,418,064)
[0057] Certain microbes also produce maytansinoids, such as
maytansinol and C-3 maytansinol esters (U.S. Pat. No.
4,151,042).
[0058] The present invention is directed to maytansinoids of any
origin, including synthetic maytansinol and maytansinol analogues
which are disclosed, for example, in U.S. Pat. Nos. 4,137,230;
4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016;
4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821;
4,322,348; 4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,371,533;
4,424,219 and 4,151,042.
[0059] In a preferred embodiment, the maytansinoid is a
thiol-containing maytansinoid and is more preferably produced
according to the processes disclosed in U.S. Pat. No. 6,333,410 to
Chari et al or in Chari et al. (Chari, 1992).
[0060] DM-1
(N.sup.2-deacetyl-N.sup.2-(3-mercapto-1-oxopropyl)-maytansine) is a
preferred effector molecule in the context of the present
invention. DM1 is 3- to 10-fold more cytotoxic than maytansine, and
has been converted into a pro-drug by linking it via disulfide
bond(s) to a monoclonal antibody directed towards a
tumor-associated antigen. Certain of these conjugates (sometimes
called "tumor activated prodrugs" (TAPs)) are not cytotoxic in the
blood compartment, since they are activated upon associating with a
target cells and internalized, thereby releasing the drug
(Blattler, 2001). Several antibody-DM1 conjugates have been
developed (Payne, 2003), and been evaluated in clinical trials. For
example, huC242-DM1 treatment in colorectal cancer patients was
well tolerated, did not induce any detectable immune response, and
had a long circulation time (Tolcher, 2003).
[0061] Other particularly preferred maytansinoids comprise a side
chain that contains a sterically hindered thiol bond such as, but
not limited to, maytansinoids
N.sup.2'-deacetyl-N.sup.2'-(4-mercapto-1-oxopentyl)-maytansine,
also referred to as "DM3," and
N.sup.2'-deacetyl-N.sup.2'-(4-methyl-4-mercapto-1-oxopentyl)-maytansine,
also referred to as "DM4."
[0062] DNA alkylating agents are also particularly preferred as
effector molecules and include, but are not limited to, CC-1065
analogues or derivatives. CC-1065 is a potent antitumor-antibiotic
isolated from cultures of Streptomyces zelensis and has been shown
to be exceptionally cytotoxic in vitro (U.S. Pat. No. 4,169,888).
Within the scope of the present invention are, for examples the
CC-1065 analogues or derivatives described in U.S. Pat. Nos.
5,475,092, 5,585,499 and 5,739,350. As the person skilled in the
art will readily appreciate, modified CC-1065 analogues or
derivatives as described in U.S. Pat. No. 5,846,545 and prodrugs of
CC-1065 analogues or derivatives as described, for example, in U.S.
Pat. No. 6,756,397 are also within the scope of the present
invention. In certain embodiments of the invention, CC-1065
analogues or derivatives may, for example, be synthesized as
described in U.S. Pat. No. 6,534,660.
[0063] Another group of compounds that make preferred effector
molecules are taxanes, especially highly potent ones and those that
contain thiol or disulfide groups. Taxanes are mitotic spindle
poisons that inhibit the depolymerization of tubulin, resulting in
an increase in the rate of microtubule assembly and cell death.
Taxanes that are within the scope of the present invention are, for
example, disclosed in U.S. Pat. Nos. 6,436,931; 6,340,701;
6,706,708 and U.S. Patent Publications 20040087649; 20040024049 and
20030004210. Other taxanes are disclosed, for example, in U.S. Pat.
No. 6,002,023, U.S. Pat. No. 5,998,656, U.S. Pat. No. 5,892,063,
U.S. Pat. No. 5,763,477, U.S. Pat. No. 5,705,508, U.S. Pat. No.
5,703,247 and U.S. Pat. No. 5,367,086. As the person skilled in the
art will appreciate, PEGylated taxanes such as the ones described
in U.S. Pat. No. 6,596,757 are also within the scope of the present
invention.
[0064] Calicheamicin effector molecules according to the present
invention include gamma 1I, N-acetyl calicheamicin and other
derivatives of calicheamicin. Calicheamicin binds in a
sequence-specific manner to the minor groove of DNA, undergoes
rearrangement and exposes free radicals, leading to breakage of
double-stranded DNA, resulting in cell apoptosis and death. One
example of a calicheamicin effector molecule that can be used in
the context of the present invention is described in U.S. Pat. No.
5,053,394.
[0065] Immunoconjugate: An immunoconjugate according to the present
invention comprises at least one targeting agent, in particular
targeting antibody, and one effector molecule. The immunoconjugate
might comprise further molecules for example for stabilization. For
immunoconjugates, the term "conjugate" is generally used to define
the operative association of the targeting agent with one or more
effector molecules and is not intended to refer solely to any type
of operative association, and is particularly not limited to
chemical "conjugation". So long as the targeting agent is able to
bind to the target site and the attached effector functions
sufficiently as intended, particularly when delivered to the target
site, any mode of attachment will be suitable. The conjugation
methods according to the present invention include, but are not
limited to, direct attachment of the effector molecule to the
targeting antibody, with or without prior modification of the
effector molecule and/or the targeting antibody or attachment via
linkers. Linkers can be categorized functionally into, for example,
acid labile, photosensitive, enzyme cleavable linkers etc. Other
suitable linkers may include disulfide bonds and non-cleavable
bonds, such as, but not limited to Sulfosuccinimidyl
maleimidomethyl cyclohexane carboxylate (SMCC), which is a
heterobifunctional linker capable of linking compounds with
SH-containing compounds. Bifunctional and heterobifunctional linker
molecules, such as carbohydrate-directed heterobifunctional linker
molecules, such as S-(2-thiopyridyl)-L-cysteine hydrazide (TPCH),
are also within the scope of the present invention (Vogel, 2004).
The effector molecule, such as a maytansinoid, may be conjugated to
the targeting antibody via a two reaction step process, including
as a first step modification of the targeting antibody with a
cross-linking reagent such as N-succinimidyl
pyridyidithiopropionate (SPDP) to introduce dithiopyridyl groups
into the targeting antibody. In a second step, a reactive
maytansinoid having a thiol group, such as DM1, may be added to the
modified antibody, resulting in the displacement of the thiopyridyl
groups in the modified antibody, and the production of
disulfide-linked cytotoxic maytansinoid/antibody conjugate (U.S.
Pat. No. 5,208,020). However, one-step conjugation processes such
as the one disclosed in U.S. Patent Publication 20030055226 to
Chari et al are also within the scope of the present invention. In
one embodiment of the present invention multiple effector molecules
of the same or different kind are attached to a targeting
antibody.
[0066] CC-1065 analogues or derivatives may be conjugated to the
targeting agent via for example PEG linking groups as described in
U.S. Pat. No. 6,716,821.
[0067] Calicheamicins may be conjugated to the targeting antibodies
via linkers (U.S. Pat. No. 5,877,296 and U.S. Pat. No. 5,773,001)
or according to the conjugation methods disclosed in U.S. Pat. No.
5,712,374 and U.S. Pat. No. 5,714,586. Another preferred method for
preparing calicheamicin conjugates is disclosed in U.S. Patent
Publication 20040082764.
[0068] The immunoconjugates of the present invention also include
recombinant fusion proteins.
[0069] The present invention takes advantage of the property of
antibodies, in particular monoclonal antibodies, to bind to
specific antigen targets, in particular, the property of certain
antibodies to bind to CD138.
[0070] CD138 or sydecan-1 (also described as SYND1; SYNDECAN; SDC;
SCD1; CD138 ANTIGEN, SwissProt accession number: P18827 human) is a
membrane glycoprotein that was originally described to be present
on cells of epithelial origin, and subsequently found on
hematopoietic cells (Sanderson, 1989). In malignant hematopoiesis,
CD138 is highly expressed on the majority of MM cells, ovarian
carcinoma, kidney carcinoma, gall bladder carcinoma, breast
carcinoma, prostate cancer, lung cancer, colon carcinoma cells and
cells of Hodgkin's and non-Hodgkin's lymphomas, chronic lymphocytic
leukemia (CLL) (Horvathova, 1995), acute lymphoblastic leukemia
(ALL), acute myeloblastic leukemia (AML) (Seftalioglu, 2003 (a);
Seftalioglu, 2003 (b)), solid tissue sarcomas, colon carcinomas as
well as other hematologic malignancies and solid tumors that
express syndecan-1 (Carbone et al., 1999; Sebestyen et al., 1999;
Han et al., 2004; Charnaux et al., 2004; O'Connell et al., 2004;
Orosz and Kopper, 2001).
[0071] Other cancers that have been shown to be positive for CD138
expression are many ovarian adenocarcinomas, transitional cell
bladder carcinomas, kidney clear cell carcinomas, squamous cell
lung carcinomas; breast carcinomas and uterine cancers (see, for
example, Davies et al., 2004; Barbareschi et al., 2003; Mennerich
et al., 2004; Anttonen et al., 2001; Wijdenes, 2002).
[0072] In the normal human hematopoietic compartment, CD138
expression is restricted to plasma cells (Wijdenes, 1996; Chilosi,
1999) and is not expressed on peripheral blood lymphocytes,
monocytes, granulocytes, and red blood cells. In particular,
CD34.sup.+ stem and progenitor cells do not express CD138 and
anti-CD138 mAbs do not affect the number of colony forming units in
hematopoietic stem cell cultures (Wijdenes, 1996). In
non-hematopoietic compartments, CD138 is mainly expressed on simple
and stratified epithelia within the lung, liver, skin, kidney and
gut. Only a weak staining was seen on endothelial cells (Bernfield,
1992; Vooijs, 1996). It has been reported that CD138 exists in
polymorphic forms in human lymphoma cells (Gattei, 1999).
[0073] Monoclonal antibodies antibodies B-B4, BC/B-B4, B-B2,
DL-101, 1 D4, MI15, 1.BB.210, 2Q1484, 5F7, 104-9, 281-2 in
particular B-B4 have been reported to be specific to CD138. Of
those B-B4, 1D4 and MI15 recognized both the intact molecule and
the core protein of CD138 and were shown to recognize either the
same or closely related epitopes (Gattei, 1999). B-B4 has the
advantage of not recognizing soluble CD138, but only CD138 in
membrane bound form (Wijdenes, 2002).
[0074] B-B4, a murine IgG1 mAb, binds to a linear epitope between
residues 90-95 of the core protein on human syndecan-1 (CD138)
(Wijdenes, 1996; Dore, 1998). Consistent with the expression
pattern of CD138, B-B4 was shown to strongly react with plasma cell
line RPM18226, but not to react with endothelial cells. Also
consistent with the expression pattern of CD138, B-B4 also reacted
with epithelial cells lines A431 (keratinocyte derived) and HepG2
(hepatocyte derived). An immunotoxin B-B4-saporin was also highly
toxic towards the plasma cell line RPM18226, in fact considerably
more toxic than free saporin. However, from the two epithelial cell
lines tested, B-B4-saporin showed only toxicity towards cell line
A431, although in a clonogenic assay B-B4 saporin showed no
inhibitory effect on the outgrowth of A431 cells (Vooijs, 1996).
Other researchers reported lack of specificity of MM-associated
antigens against tumors (Couturier, 1999).
[0075] The reactivity of B-B4 with tissue of various organs is
shown in Table 1, the reactivity of B-B4 with cell lines of
different origins is shown in Table 2. The reactivity was
determined by immunohistochemistry (Table 1) and cytofluorography
(Table 2). The number of (+) signs indicate the intensity of the
reaction. TABLE-US-00001 TABLE 1 Reactivity of B-B4 with tissues of
various organs (immunohistochemistry) Organ Tissue B-B4 Blood
Normal plasma cells +++ Blood MM patient cells +++ Kidney Tubular
epithelium - Kidney Glomerular - Kidney Urothelium ++ Kidney Smooth
muscle of hilus - Liver Sinusoid endothelium - Liver Biliary
epithelium - Liver Hepatocytes ++ Lung Alveolar epithelium ++ Lung
Bronchial epithelium + Lung Blood vessel - Lung Bronchial gland ++
Duodenum Crypts epithelium ++ Duodenum Glands ++ Duodenum Chorion
lymphocytes ++ Duodenum Smooth muscle - Duodenum Blood vessels -
Heart Myocytes ++ (cytoplasmic) Spleen Red pulp + Various organs
Muscle - Various organs Connective tissue - Various organs Nervous
tissue - Various organs Epithelium +++ Various organs Endothelium +
Cell lines MM cell lines +++
[0076] TABLE-US-00002 TABLE 2 Reactivity of B-B4 with cell lines of
different origins (Cytofluorography) Cell line Cell type B-B4 RPMI
8226 Multiple myeloma +++ U266 Multiple myeloma +++ UM-1 Multiple
myeloma +++ XG-1 Multiple myeloma +++ Daudi EBV-infected LCL -
Ramos EBV-infected LCL - Jijoye EBV-infected LCL - BJAB Burkitt
lymphoma - Raji Burkitt lymphoma - BTL-1 LCL - BTL-6 LCL - KM-3
Pre-B + REH Pre-B - NALM-6 Pre-B + ROS Pre-B - 697 Pre-B - CEM
T-cell - Jurkat T-cell - HL-60 Myeloid - U937 Myeloid + HEL Myeloid
- KG1A Myeloid - K562 Erythroid ++ A341 Epithelial +++ HepG
Hepatocytic ++ HUVEC Endothelial + Peripheral blood Monocyte -
Peripheral blood B-cell (CD19+) - Peripheral blood T-cell (CD3+) -
Peripheral blood Granulocytes - Bone marrow (CD34+, CD33+, CD19+,
CD20+, CD10+, - CD3+, CD19+, CD14+, CD38+) cells Bone marrow Plasma
cells ++ Bone marrow Myeloma cells/CD38 high +++ Tonsil (CD19+,
CD38+) cells - Patient sample ALL B-cell - Patient sample CLL
B-cell - Patient sample Reed-Sternberg cell ++ Hodgkin
[0077] The activity of immunoconjugates on a cellular level has
been described, for example, for huC242-DM1 (Immunogen, Inc.), an
immunoconjugate comprising the antibody huC242 and the maytansinoid
DM1, an inhibitor of tubulin polymerization described above. The
activity of this immunoconjugate at the cellular level was
described to include the following steps: (1) binding of the
immunoconjugate to the antigen expressed on a cancer cell, (2) the
internalization of the conjugate-antigen complex by the cancer
cell, and (3) release of DM1, thereby allowing DM1 to reach its
intracellular target tubulin and to inhibit tubulin polymerization
(Xie, 2003). This multi-step attachment, internalization and
release model forms the rationale behind the development of tumor
activated prodrugs (TAPs) (Immunogen, 2003). Similar uptake
mechanisms have been described for immunoconjugates based on
anti-PSCA antibodies, which were reported to be internalized via
caveolae (Ross, 2002).
[0078] The present invention is useful in the treatment of, but is
not limited to, cancers, in particular, multiple myeloma, ovarian
carcinoma, kidney carcinoma, gall bladder carcinoma, breast
carcinoma, prostate cancer, lung cancer, colon carcinoma, Hodgkin's
and non-Hodgkin's lymphomas, chronic lymphocytic leukemia (CLL)
(Horvathova, 1995), acute lymphoblastic leukemia (ALL), acute
myeloblastic leukemia (AML) (Seftalioglu, 2003 (a); Seftalioglu,
2003 (b)), solid tissue sarcomas, colon carcinomas as well as other
hematologic malignancies and solid tumors that express syndecan-1
(Carbone et al., 1999; Sebestyen et al., 1999; Han et al., 2004;
Charnaux et al., 2004; O'Connell et al., 2004; Orosz and Kopper,
2001).
[0079] The immunoconjugates according to the present invention can
be administered by any route, including intravenously,
parenterally, orally, intramuscularly, intrathecally or as an
aerosol. The mode of delivery will depend on the desired effect. A
skilled artisan will readily know the best route of administration
for a particular treatment in accordance with the present
invention. The appropriate dosage will depend on the route of
administration and the treatment indicated, and can readily be
determined by a skilled artisan in view of current treatment
protocols.
[0080] Pharmaceutical compositions containing an immunoconjugate of
the present invention as the active ingredient can be prepared
according to conventional pharmaceutical compounding techniques.
See, for example, Remington's Pharmaceutical Sciences, 17th Ed.
(1985, Mack Publishing Co., Easton, Pa.). Typically, an
antagonistic amount of active ingredient will be admixed with a
pharmaceutically acceptable carrier. The carrier may take a wide
variety of forms depending on the form of preparation desired for
administration, for example, intravenous, oral, parenteral,
intrathecal, transdermal, or by aerosol.
[0081] For oral administration, the immunoconjugate can be
formulated into solid or liquid preparations such as capsules,
pills, tablets, lozenges, melts, powders, suspensions or emulsions.
In preparing the compositions in oral dosage form, any of the usual
pharmaceutical media may be employed, such as, for example, water,
glycols, oils, alcohols, flavoring agents, preservatives, coloring
agents, suspending agents, and the like in the case of oral liquid
preparations (such as, for example, suspensions, elixirs and
solutions); or carriers such as starches, sugars, diluents,
granulating agents, lubricants, binders, disintegrating agents and
the like in the case of oral solid preparations (such as, for
example, powders, capsules and tablets). Because of their ease in
administration, tablets and capsules represent the most
advantageous oral dosage unit form, in which case solid
pharmaceutical carriers are obviously employed. If desired, tablets
may be sugar-coated or enteric-coated by standard techniques. The
active agent must be stable to passage through the gastrointestinal
tract. If necessary, suitable agents for stable passage can be
used, and may include phospholipids or lecithin derivatives
described in the literature, as well as liposomes, microparticles
(including microspheres and macrospheres).
[0082] For parenteral administration, the immunoconjugate may be
dissolved in a pharmaceutical carrier and administered as either a
solution or a suspension. Illustrative of suitable carriers are
water, saline, phosphate buffer solution (PBS), dextrose solutions,
fructose solutions, ethanol, or oils of animal, vegetative or
synthetic origin. The carrier may also contain other ingredients,
for example, preservatives, suspending agents, solubilizing agents,
buffers and the like. When the immunoconjugate are being
administered intracerebroventricularly or intrathecally, they may
also be dissolved in cerebrospinal fluid.
[0083] In accordance with the present invention, MM is treated as
follows, with use of the B-B4-DM1 conjugate as an example. This
example is not intended to limit the present invention in any
manner, and a skilled artisan could readily determine other
immunoconjugates of the present invention and other treatment
regimes which could be utilized for the treatment of diseases such
as MM. Due to the selective expression of CD138 on patient MM cells
on via the blood stream accessible cells, the specificity of B-B4
and the stability of the B-B4-DM1 conjugate in the bloodstream, the
immunoconjugate removes the systemic toxicity of DM1 and provides
an opportunity to target the delivery of the DM1-effector
molecule(s). The immunoconjugates of this invention provide a means
for the effective administration of the effector molecules to cell
sites where the effector molecules can be released from the
immunoconjugates. This targeted delivery and release provides a
significant advance in the treatment of multiple myeloma, for which
current chemotherapy methods sometimes provide incomplete
remission.
[0084] In accordance with the present invention, in particular
solid tumors may also be treated as follows with use of B-B4-DM1,
as an example. This example is not intended to limit the present
invention in any manner, and a skilled artisan could readily
determine other immunoconjugates of the present invention and other
treatment regimes which could be utilized for the treatment of
solid tumors. The tumor is first treated to reduce the size of the
tumor, for example chemotherapeutically or radioactively.
Subsequent administration of the immunoconjugates of this invention
provides a means for eliminating residual cancer cells. The
administration of the immunoconjugate allows specific targeting of
these residual cells and release of the effector molecules at the
target site. This targeted delivery and release provides a
significant advance in the treatment of residual cancer cells of
solid tumors, for which current chemotherapy methods sometimes
provide incomplete remission.
[0085] The present invention is further described by reference to
the following Examples, which are offered by way of illustration
and are not intended to limit the invention in any manner. Standard
techniques well known in the art or the techniques specifically
described below were utilized.
Materials and Methods
[0086] Preparation of mAb-DM1 Conjugate
[0087] The thiol-containing maytansinoid DM1 was synthesized from
the microbial fermentation product ansamitocin P-3, as previously
described by Chari (Chari et al, 1992). Characterization of murine
B-B4 (Wijdenes, 1996) and preparation of humanized C242 (huC242)
(Roguska, 1994) have been previously described. Antibody-drug
conjugates were prepared as described by Liu et al (Liu, 1996). An
average of 3.5 DM1 molecules was linked per antibody molecule.
Cell Lines and Patient Cells
[0088] CD138.sup.+ dexamethasone (Dex)-sensitive MM.1S and
Dex-resistant MM.1R, Ocy-My5, OPM1 and OPM2 human MM cell lines and
CD138.sup.- Waldenstrom Macroglobulinemia (WM) WSU-WM and the
lymphoma (LB) SUDHL4 cell lines were used. Cell lines were cultured
in RPMI-1640 medium (GIBCO) supplemented with 10% fetal bovine
serum (FBS; Hyclone, Logan, Utah), L-glutamine, penicillin, and
streptomycin (GIBCO) (denoted below as RPMI complete medium).
Plasma cells (PC) and bone marrow (BM) cells were isolated using
Ficoll-Hypaque density gradient sedimentation from BM aspirates,
obtained from MM patients following informed consent. BM cells were
separated. BMSCs were obtained by long-term cultures of BM cells
(4-8 weeks) in RPMI 1640 medium supplemented with 20% FBS.
Gene Expression Analysis and Data Analysis: Expression of CD138 in
MM Patients
[0089] Expression of CD138 on normal plasma cells and patient MM
cells was evalutated. BM aspirate samples from normal donors and
patients with MM were treated with 0.86% ammonium chloride to lyse
red blood cells. PC were then isolated by positive immunomagnetic
bead selection using anti-CD138 antibodies and Magnet Assisted Cell
Sorting ("MACS," Miltenyi Biotech). Purity of plasma cells
(>95%) was assessed by flow cytometric (Becton-Dickinson
"FACSort") monitoring for CD38.sup.+/CD45.sup.lo phenotype as well
as forward and side scatter and morphological characteristics.
[0090] Total RNA was isolated from 5.times.10.sup.6 cells utilizing
an "RNeasy.RTM. kit" (Qiagen Inc., Valencia, Calif.). Total RNA
(10-15 .mu.g) was reverse-transcribed to get cDNA using the
"Superscript.RTM. II RT kit" (Invitrogen Life Technologies,
Carlsbad, Calif.). cDNA was used in an in vitro transcription
reaction to synthesize biotin-labeled cRNA utilizing "ENZO.RTM. RNA
labeling kit" (Enzo Diagnostics, Inc., Farmingdale, N.Y.). Labeled
cRNA was purified with the "RNeasy.RTM. Mini-kit" (Qiagen Inc.,
Valencia, Calif.) and quantitated. Purified cRNA (15 .mu.g) was
hybridized to Human Genome U133 (HG-U133) GeneChip.RTM. arrays
(Affymetrix, Inc.) representing approximately 33,000 human genes,
and GeneChip.RTM. arrays were scanned on a GeneArray.RTM. Scanner
(Affymetrix, Inc., Santa Clara, Calif.). Normalization of arrays
and calculation of expression values was performed using the
DNA-Chip Analyzer ("dChip") program. Arrays were normalized based
on relative signal produced for an invariant subset of genes. This
model-based method was used for probe selection and computing
expression values. By pooling hybridization information across
multiple arrays, it was possible to assess standard errors for the
expression level indexes. This approach also allowed automatic
probe selection in the analysis stage to reduce errors due to
cross-hybridizing probes and image contamination.
Antibody Internalization
[0091] Internalization of B-B4 antibody was examined with a
cultured CD138.sup.+ cell line by flow cytometry and under a
fluorescent microscope. The antibody was modified by Alexa 488 dye
(Molecular Probes), and the fluorescence of the non-internalized
antibody bound to cells was quenched by exposure to an "anti-Alexa
antibody" (Molecular Probes). Thus, semi-quantitatively
discrimination between surface-bound and internalized antibody was
possible. B-B4 was poorly internalized.
Colorimetric Survival Assay
[0092] Survival of CD138.sup.+ and CD138.sup.- cells upon
administration of B-B4-DM1, B-B4 and DM1 was examined using a
tetrazolium colorimetric assay (CellTiter 96.RTM. Non-Radioactive
Cell Proliferation Assay; Promega, WI), as previously described
(Mossmann, 1983). Cells (1.times.10.sup.4) were plated in 24-well
plates in 1 ml RPMI complete medium and then treated as indicated.
At the end of each treatment, cells were incubated with 150 .mu.l
of Dye Solution and then incubated for 4 h at 37.degree. C. A
solubilization/stop solution was then added to each well under
vigorous pipetting to dissolve the formazan crystals. Absorbance
was measured at 570 nm, and cell viability was estimated as
percentage of untreated controls. All experiments were repeated 3
times, and each experimental condition was repeated in triplicate
wells in each experiment. Data reported are average values.+-.SD of
3 representative experiments.
Cell Proliferation Assay
[0093] The effect of B-B4-DM1 on cell proliferation was measured by
the extent of [3H]-thymidine (NEN Life Science Products, Boston,
Mass.) incorporation. Cells (2.times.10.sup.4 cells/well) were
incubated in 96-well culture plates in the presence of 70%-80%
confluent BMSCs at 37.degree. C. with or without a test-agent (in
triplicate wells). [.sup.3H]-thymidine (0.5 .mu.Ci) was then added
to each well for the last 8 h. Cells were harvested onto glass
filters with an automatic cell harvester (Cambridge Technology,
Cambridge, Mass.) and counted using a Micro-Beta.RTM. Trilux
counter (Wallac, Gaithersburgh, Md.).
Detection of Apoptosis
[0094] Dual staining with FITC-labeled Annexin V and propidium
iodide (PI) was carried out to detect induction of apoptotic cell
death by B-B4-DM1. After treatment of 1.times.10.sup.6 tumor cells
for 48 h, cells were washed with PBS and re-suspended in 100
.quadrature.l of HEPES buffer containing Annexin V-FITC and
propidium iodide (PI) (Annexin V-FLUOS staining kit; Roche
Diagnostic, Indianapolis, Ind.). Following 15 min incubation at
room temperature, cells were analyzed using a Coulter Epics XL flow
cytometer for the presence of an Annexin
V-FITC-positive/PI-negative apoptotic cell population.
Cell Cycle Analysis
[0095] 1.times.10.sup.6 MM cells were incubated with or without a
test-agent for 48 h, washed with PBS, permeabilized by a 30 min
exposure to 70% ethanol at 4.degree. C., incubated with PI
(50-.mu.g/mL) in 0.5 ml PBS containing 20 U/mL Rnase A (Roche) for
30 min at room temperature, and analyzed for DNA content by
cell-associated fluorescence using a flow cytometer and
CellQuest.TM. software.
In Vivo Activity
Human MM Xenograft Murine Model
[0096] In this model, CB-17 SCID mice were subcutaneously (s.c.)
inoculated in the interscapular area with 5.times.10.sup.6 OPM1 or
OPM2 cells in 100 .mu.l of RPMI-1640 medium. Treatment was
initiated after the detection of palpable tumors. Tumor growth was
measured weekly in two dimensions using a caliper, and volume was
expressed in mm.sup.3 using the formula: V=0.5a.times.b.sup.2,
where a and b are the long and short diameter of the tumor,
respectively. Tumor size was evaluated from the first day of
treatment until day of first sacrifice. The survival time is
defined as the time interval between start of the experiment and
either death or day of sacrifice. Mice were treated intavenously
(i.v.) with vehicle alone (PBS), unconjugated B-B4 (13.3 .mu.g/ml),
B-B4-DM1 (conjugate containing 75 or 150 .mu.g DM1/Kg per day), or
control huC242-DM1 (150 .mu.g DM1/Kg per day), for a total of 3
days. In addition, two mice bearing very large tumors (average size
of 1309.+-.60 mm.sup.3) were treated with B-B4-DM1 (150 .mu.g
DM1/kg per day) for a total of 3 days and observed for changes in
the tumor size.
Autofluorescencent GFP.sup.+ Human MM Xenograft Model
[0097] Procedures for stably transfection of green fluorescent
protein (GFP) in tumor cells and use have been previously described
(Yang, 1999; Yang 2000). Five mice were injected s.c. with
GFP.sup.+ OPM1 cells as described above. Mice were monitored by
whole-body fluorescence imaging using "Illumatool Bright Light
System LT-9900" (Lightools Research, Encinitas, Calif.). After
accurate cutaneous shave of tumor area, fluorescence imaging
results were digitally captured by a Sony.RTM. DSC-P5.TM. digital
camera (Sony, New York, N.Y.) and analyzed with Adobe
PhotoShop.RTM. 4.0.
SCID-hu Mouse Model
[0098] Human fetal bones were obtained from products of conceptions
of second trimester abortions in compliance with state and federal
regulations (Advanced Bioscience Resourses, ABR; Alameda, Calif.).
The implantation of human fetal long bone grafts into SCID mice to
produce SCID-hu mice has been previously described (McCune et al,
1988; Namikawa et al, 1988; Kyoizumi et al, 1993; Akkina et al,
1994; Chen et al, 1994; Sandhu et al, 1996; Urashima, 1997). In
brief, the femurs or tibias of 19 to 23 gestational week fetuses
were cut into fragments and implanted s.c. into SCID mice. After
approximately 8 weeks, 2 to 5.times.10.sup.6 BM cells from a MM
patient or Ocy-My5 MM cells were injected in 50 .mu.l PBS directly
into human bone of SCID-hu hosts. Production and level of human
paraprotein in mouse serum was an indicator of myeloma engrafinent
and growth. At least 2 consecutive measurements, of increasing
levels of circulating human immunoglobulin (huIg), signified human
MM cell growth.
Measurement of Serum Paraprotein Concentration
[0099] Blood (50-100 .mu.l) was withdrawn from the tail vein for
measurement of human paraprotein in murine serum using ELISA
(Bethyl, Montgomery, Tex.). Goat anti-human .lamda. and .kappa.
antisera were used for capture and goat anti-human .lamda. or
.kappa. HRP conjugates were used for detection.
Histopatological Analysis
[0100] Excised bone grafts were fixed in 10% buffered formalin;
skeletal tissues were decalcified with 14% EDTA and embedded in
paraffin by previously described standard techniques (Sasaki,
1995). Sections were then stained with H & E (Hematoxylin and
eosin) for histopathological examination. Immunoperoxidase studies
were performed on paraffin sections using an indirect technique as
described (Urashima, 1997). Rabbit anti-human .lamda. and .kappa.
antisera were used for detection of MM cells in fetal bone.
Statistical Analysis
[0101] Statistical significance of differences was determined using
Student's t-test. Differences were considered significant when
p<0.05.
Results and Discussion
[0102] CD138 is expressed on patient MM cells and is the most
important target Ag for identification and selection of these
cells. However previous reports show heterogenous CD138 expression
on MM cells (Wijdenes, 1996; Dhodapkar, 1998; Witzig, 1996;
Schneider, 1997; Rawstron, 1997). The expression of CD138 on
patient MM cells was measured by gene profiling and flow cytometry.
FIGS. 1A to 1C show the CD138 gene expression profiles of normal
plasma cells (n=3) and patient MM cells (n=15) measured utilizing
HG-U133 GeneChip.RTM. array (Affymetrix) data. FIG. 1A shows the
individual fold increase in intensity of CD138 gene expression
compared to normal PC; FIG. 1B shows the mean of intensity of CD138
gene expression in normal PC (n=3) and patient MM cells (n=15) and
FIG. 1C the mean fold increase intensity of CD138 gene expression
in MM cells (n=15) compared to normal PC (n=3). As can be seen,
CD138 was expressed in all 15 MM specimens (100%) examined at a
95.+-.8-fold mean increase in intensity relative to normal plasma
cells.
[0103] Furthermore, flow cytometry was used to assess cell surface
expression of CD138 on MM cells from 25 patients. Expression of
CD138 on the CD38.sup.brightCD45.sup.lo cell population was
assessed both by percentage of positive cells, and by mean
fluorescence intensity (MFI). FIG. 1D shows the percentage of
patients expressing CD138.sup.+ MM cells, as determined by flow
cytometry on fresh BM aspirate samples. FIG. 1E shows the
percentage of CD138.sup.+ MM cells in CD138.sup.+ patients on fresh
BM aspirates and FIG. 1F shows MFI of CD138.sup.+ or CD138.sup.- MM
cells within CD38.sup.brightCD45.sup.lo population. As can be seen
from FIG. 1D 18 of 25 patients (72%) expressed CD138, with a mean
of 68.+-.31% CD138.sup.+ cells (FIG. 1E) and MFI of 1234.+-.539
(range: 166-2208) (FIG. 1F). Taken together, these results indicate
that CD138 is highly expressed in patient MM cells.
[0104] These results were consistent with previous reports showing
CD138 expression on 60% and 100% of cases (Horvathova, 1995;
Wijdenes, 1996). Possible explanations for the variability in CD138
detection by flow cytometry are the rapid shedding of protein
during flow cytometric manipulation of specimens, the high turnover
rate of the molecule on the cell membrane, lack of cell surface
antigen (Ag) in pre-apoptotic plasma cells or Ag expression
dependent on stage of the cell cycle (Clement, 1995). By
immunohistochemistry, CD138 has been reported to be highly
sensitive and specific marker of MM cells in 100% of BM biopsies
(Chilosi, 1999). These data support the potential value of CD138 as
a target for immunotherapeutic approaches in MM.
[0105] The effects of B-B4-DM1 on survival of CD138.sup.+ (MM-1S,
MM-1R, Ocy-My5) and CD138.sup.- cells (SUDHL-4 and WSU-WM) were
determined using an MTT assay. MM cell lines were exposed to
unconjugated B-B4 mAb (FIG. 2A), immunoconjugate B-B4-DM1 (FIG.
2B), or free DM1 drug at equimolar concentrations (FIG. 2C). Cell
survival was measured using an MTT assay. Data (mean.+-.SD of
triplicate experiments) are shown in FIGS. 2A to 2C as percentage
of untreated controls. CD138.sup.+ MM cell lines MM-1S, MM-1R and
Ocy-My5 were evaluated as well as CD138.sup.- cell lines including
the lymphoma cell line SUDHL4 and the Waldestrom's
Macroglobulinemia cell line WSU-WM.
[0106] As can be seen from FIG. 2B, treatment with B-B4-DM1 (1-50
nM) induced growth inhibition in CD138.sup.+ tumor cells in a time-
and dose-dependent manner. This effect was clearly detected after
72 h in all CD138.sup.+ cells. B-B4-DM1 treatment of CD138.sup.+
OPM1 and OPM2 MM cells further confirmed these observations (data
not shown). In contrast, B-B4-DM1 (1-50 nM) was not toxic to
CD138.sup.- cells, even after treatment for 96 h. To confirm that
inhibitory activity of the immunoconjugate is specifically related
to mAb-delivered cytotoxicity, the effect of equimolar
concentrations of B-B4 antibody or unconjugated drug DM1 were
tested. Even the highest concentrations of B-B4 did not affect the
growth of cells at 96 h, (FIG. 2A), whereas free DM1 was equally
and highly cytotoxic in both CD138.sup.+ and CD138.sup.- cell lines
(FIG. 2C). These data indicate that activity of the immunoconjugate
is not related to the differential sensitivity of cells to the drug
nor the intrinsic properties of the antibody.
[0107] Since adhesion of MM cells to BMSC (bone marrow stromal
cells) protects MM cells against drug-induced apoptosis, the effect
of B-B4-DM1 on proliferation of CD138.sup.+ (Ocy-My5) MM and
CD138.sup.- (SUDHL-4) LB cells adherent to BMSC was evaluated.
[0108] Ocy-My5 (FIG. 3A) or SUDHL-4 (FIG. 3B) cells
(2.times.10.sup.4) were seeded on 70%-80% confluent BMSC for 24 h.
Cell proliferation was measured by [.sup.3H]thymidine incorporation
following 72 h treatment with B-B4-DM1 (10 nM). Values represent
the mean [.sup.3H]-TdR incorporation (cpm) of triplicate cultures.
As seen in FIGS. 3A and 3B, B-B4-DM1 (10 nM) significantly
inhibited the proliferation of CD138.sup.+ Ocy-My5 cells, but had
no significant effect on CD138.sup.- SUDHL-4 cells. Unconjugated
B-B4 did not exert any significant effect, whereas free DM1 (10 nM)
was cytotoxic to both cell lines.
[0109] CD56, another CD associated with MM, and CD138 expression
was evaluated by flow cytometry. Previous experiments established
that B-B4-DM1, even at a concentrations as high as 240 .mu.M, did
not affect binding of FITC-labeled anti-CD138 antibody to
CD138-expressing cells. FIG. 3C shows the cytotoxic activity of
B-B4-DM1 (10 nM) on CD138.sup.+/CD56.sup.+ patient MM cells
cultured with BMSCs using flow cytometry. Following 72 h of
treatment with the immunoconjugate, >90% reduction in the MM
cells was observed. Taken together, these results indicate that
B-B4-DM1 overcomes cell adhesion mediated drug resistance
(CAM-DR).
[0110] To determine whether apoptotic cell death occurs in cells
exposed to the immunoconjugate, CD138.sup.+ Ocy-My5 MM cells were
incubated with B-B4-DM1 (10 nM) for 72 h. Apoptotic cell death was
then measured by staining with annexin V and PI and flow cytometric
analysis.
[0111] FIG. 4A shows the induction of apoptotic cell death in
CD138.sup.+ Ocy-My5 MM cells after 48 h exposure to B-B4-DM1 (10
nM). Percentages of stained cells are reported in each quadrant.
FIG. 4A shows a significant increase in both annexin
V.sup.+/PI.sup.- and annexin V.sup.+/PI.sup.+ fractions in
CD138.sup.+ cells exposed to B-B4-DM1 and free DM1, whereas no
significant differences were detected in cells treated with
unconjugated mAb alone. FIG. 4B shows the effects of B-B4-DM1
treatment on the cell cycle. Ocy-My5 MM cells were exposed to B-B4
mAb (13.3 .mu.g/ml) or B-B4-DM1 (10 nM) for 48 h, labeled with PI,
and analyzed using flow cytometry. Percentages of cells in the
S-phase (S) and G2/M phase (G2) are indicated. As shown in FIG. 4B,
B-B4 mAb alone had no significant effect on the proportion of cells
in G2/M phase compared to untreated cells (15% vs 16%), whereas
exposure of MM cells to B-B4-DM1 induced a majority (88%) of cells
into the G2/M phase.
[0112] A human MM s.c. xenograft model in SCID mice was used to
study the in vivo activity of B-B4-DM1 against CD138.sup.+ OPM1
cells. In this model, the therapeutic efficacy of B-B4-DM1 was
measured in mice bearing large palpable tumors (average size
453.+-.74 mm.sup.3). Animals were treated daily i.v. for 3
consecutive days with vehicle alone (PBS 9phosphate buffered
saline); n=5), unconjugated B-B4 (13.3 .mu.g/ml; n=5), B-B4-DM1
(150 .mu.g DM1/kg; n=5), or control huC242-DM1 (150 .mu.g DM1/kg;
n=5) which does not bind OPM1 cells. Tumor size and overall
survival were monitored serially in this cohorts.
[0113] FIG. 5 shows the results obtained after CB-17 SCID mice were
inoculated s.c. in the interscapular area with 5.times.10.sup.6
OPM1 (A and B) or OPM2 (C and D) MM cells. Mice were treated i.v.
with B-B4-DM1 or control mAbs for 3 consecutive days. Tumor volume
was assessed in two dimensions using an caliper eletronic, and the
volume was expressed in mm.sup.3 using the formula:
V=0.5a.times.b.sup.2, where a and b are the long and short diameter
of the tumor, respectively. Tumor volume and survival were
calculated as described previously.
[0114] As shown in FIGS. 5A and 5B, vehicle alone, unconjugated
B-B4 and huC242-DM1, had no significant effect on tumor growth
(panel A) or survival (panel B). Importantly, treatment with 150
.mu.g/kg of B-B4-DM1 induced tumor regression and a significant
increase in survival (p<0.001). We also studied the effect
induced by B-B4-DM1 (75 or 150 .mu.g DM1/kg; n=10) against OPM2 MM
cells. As shown in FIGS. 5C and D, treatment with 75 .mu.g/kg of
B-B4-DM1 induced a significant delay in tumor growth, and 150
.mu.g/kg of B-B4-DM1 completely inhibited tumor growth. A
significant increase in survival was also observed in mice treated
at both dose levels (p<0.05) relative to animals treated with
vehicle or huC242-DM1 alone. To confirm the activity of B-B4-DM1
(150 .mu.g DM1/kg), animals bearing a significant burden of disease
(average tumor size was 1309.+-.60 mm.sup.3) were treated. FIGS. 6A
to 6F also shows the results obtained when CB-17 SCID mice were
inoculated s.c. in the interscapular area with 5.times.10.sup.6
OPM1 MM cells. Again, mice were treated i.v. with B-B4-DM1 (150
.mu.g DM1/kg) for a total of 3 consecutive days. Tumor volume was
measured in two dimensions using a caliper, and the volume was
expressed in mm.sup.3 using the formula: V=0.5a.times.b.sup.2,
where a and b are the long and short diameter of the tumor,
respectively.
[0115] As shown in FIGS. 6A to 6F, significant tumor regression was
induced by B-B4-DM1 treatment. Taken together, these results
indicate that B-B4-DM1 is highly active in controlling tumor growth
in a murine xenograft model of human MM.
[0116] Since expression of reporter genes encoding fluorescent
proteins are sensitive method for in vivo detection of localized
tumor growth as well as distant metastasis, the OPM1 MM cells were
transfected with green fluorescent protein (GFP) and B-B4-DM1
activity was further characterized. In particular, mice were
injected s.c. with GFP.sup.+ OPM1 cells, followed by serial
whole-body fluorescence imaging to assess development of GFP.sup.+
tumors. Mice were then treated with B-B4-DM1 (150 .mu.g DM1/kg;
n=5). FIG. 7A shows a flow cytometry analysis of GFP.sup.+ OPM1
cells, indicating a .about.2-log difference in MFI of transfected
cells. FIG. 7B shows results from five animals being injected with
5.times.10.sup.6 GFP.sup.+ cells, monitored with whole-body
fluorescence imaging for tumor development, and then treated with
B-B4-DM1 (150 .mu.g DM1/kg). Tumor sizes were determined directly
by imaging the GFP-expressing tumor. FIGS. 7 C and D are
representative whole-body fluorescence imaging from a mouse treated
with B-B4-DM1. FIGS. 7E and 7F are negative images of the
representative mouse. As seen in FIGS. 7B and 7C to 7F, B-B4-DM1
induced significant regressions of GFP.sup.+ tumors, confirming
high activity of the immunoconjugate against CD138.sup.+ MM
cells.
[0117] Since the SCID-hu model of MM accurately reproduces the
pathological behaviours of the disease, the efficacy of B-B4-DM1
treatment was tested in (i) SCID-hu mice injected with patient MM
cells and (ii) SCID-hu mice injected with Ocy-My5 MM cell line
(Urashima, 1997). The activity of the immunoconjugate on disease
confined to the human fetal bone chip implanted s.c. in mice was
studied. Four mice with patient MM cells growing in human bone
environment increasing serum huIg levels, were treated with either
B-B4-DM1 (150 .mu.g DM1/kg) or the control huC242-DM1 (150 .mu.g
DM1/kg).
[0118] In FIGS. 8A and 8B the results of monitoring mice for
changes in levels of human .kappa. chain as an indicator of disease
burden are shown. The Figure shows a significant reduction of
.kappa. levels after treatment with B-B4-DM1. FIG. 8C shows final
human .kappa. chain levels (mean.+-.SD) (n=4) after treatment. As
seen in FIGS. 8A to 8C, treatment with B-B4-DM1 induced a
significant reduction of human paraprotein, whereas human
paraprotein continued to rise in mice treated with control
antibody.
[0119] The activity of B-B4-DM1 after injection of Ocy-My5 cells in
human fetal bone, tumor cell growth in bone (FIGS. 9A and 9B) and
subsequent spread to surrounding tissues was also studied
(Urashima, 1997) were treated with either B-B4-DM1 (150 .mu.g
DM1/kg) or the control huC242-DM1 (150 .mu.g DM1/kg). FIGS. 9A and
9B show representative human bone sections after implantation of
Ocy-My5 cells and before treatment. Sections are respectively
stained by H & E and with anti-.lamda. mAb. Finally, the
activity of B-B4-DM1 on survival of tumor bearing mice was studied.
FIG. 9C shows the survival of mice measured from the first day of
treatment to the day of death or sacrifice. Figure shows a
significant prolongation of survival after treatment with B-B4-DM1
As seen in FIG. 9C, treatment with B-B4-DM1 (150 .mu.g DM1/kg)
induced a significant prolongation in survival compared with
control huC242-DM1 (150 .mu.g DM1/kg) therapy. Taken together,
these results confirm in vivo B-B4-DM1 activity in preclinical
models which mimic many features of human MM.
[0120] It will be appreciated that the methods and compositions of
the instant invention can be incorporated in the form of a variety
of embodiments, only a few of which are disclosed herein. It will
be apparent to the artisan that other embodiments exist and do not
depart from the spirit of the invention. Thus, the described
embodiments are illustrative and should not be construed as
restrictive.
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