U.S. patent application number 11/364732 was filed with the patent office on 2006-09-21 for vegf-conjugate combined methods for tumor vasculature targeting and tumor treatment.
This patent application is currently assigned to Board of Regents, The University of Texas System. Invention is credited to Francis J. Burrows, Philip E. Thorpe.
Application Number | 20060210473 11/364732 |
Document ID | / |
Family ID | 46252831 |
Filed Date | 2006-09-21 |
United States Patent
Application |
20060210473 |
Kind Code |
A1 |
Thorpe; Philip E. ; et
al. |
September 21, 2006 |
VEGF-conjugate combined methods for tumor vasculature targeting and
tumor treatment
Abstract
The present invention relates generally to methods and
compositions for targeting the vasculature of solid tumors using
immunological- and growth factor-based reagents. In particular
aspects, antibodies carrying diagnostic or therapeutic agents are
targeted to the vasculature of solid tumor masses through
recognition of tumor vasculature-associated antigens, such as, for
example, through endoglin binding, or through the specific
induction of endothelial cell surface antigens on vascular
endothelial cells in solid tumors.
Inventors: |
Thorpe; Philip E.; (Dallas,
TX) ; Burrows; Francis J.; (San Diego, CA) |
Correspondence
Address: |
PEREGRINE PHARMACEUTICALS, INC.
5353 WEST ALABAMA
SUITE 306
HOUSTON
TX
77056
US
|
Assignee: |
Board of Regents, The University of
Texas System
|
Family ID: |
46252831 |
Appl. No.: |
11/364732 |
Filed: |
February 28, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10376194 |
Feb 27, 2003 |
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11364732 |
Feb 28, 2006 |
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09738970 |
Dec 14, 2000 |
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10376194 |
Feb 27, 2003 |
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09207277 |
Dec 8, 1998 |
6261535 |
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09738970 |
Dec 14, 2000 |
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08350212 |
Dec 5, 1994 |
5965132 |
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09207277 |
Dec 8, 1998 |
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08205330 |
Mar 2, 1994 |
5855866 |
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08350212 |
Dec 5, 1994 |
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07846349 |
Mar 5, 1992 |
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08205330 |
Mar 2, 1994 |
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Current U.S.
Class: |
424/1.49 ;
424/9.34; 424/9.6; 530/388.22; 530/388.8; 530/391.1 |
Current CPC
Class: |
C07K 2317/34 20130101;
C07K 16/2818 20130101; A61K 2123/00 20130101; C07K 16/28 20130101;
C07K 2317/55 20130101; C07K 16/2833 20130101; A61K 47/6855
20170801; C07K 2317/73 20130101; A61K 47/6849 20170801; C07K
2319/55 20130101; A61K 51/1045 20130101; Y10S 530/807 20130101;
A61K 49/16 20130101; Y10S 530/828 20130101; C07K 2317/31 20130101;
C07K 2317/77 20130101; A61K 47/6827 20170801; C07K 16/30 20130101;
C07K 16/46 20130101; C07K 2319/33 20130101; A61K 47/6817 20170801;
C07K 16/3015 20130101; A61K 2039/505 20130101; A61K 38/00 20130101;
A61P 35/00 20180101; A61K 47/6851 20170801; C07K 16/2896 20130101;
A61K 49/085 20130101; C07K 16/22 20130101 |
Class at
Publication: |
424/001.49 ;
424/009.34; 424/009.6; 530/388.22; 530/391.1; 530/388.8 |
International
Class: |
A61K 51/00 20060101
A61K051/00; A61K 49/00 20060101 A61K049/00; C07K 16/46 20060101
C07K016/46; C07K 16/30 20060101 C07K016/30 |
Goverment Interests
[0002] The U.S. government owns rights in the present invention
pursuant to NIH Grant CA-28149 and NIH Grant CA54168.
Claims
1-93. (canceled)
94. A method for treating an animal having a vascularized tumor,
comprising: administering to said animal an amount of a construct
effective to treat said vascularized tumor, said construct
comprising a selected therapeutic agent linked to VEGF, wherein
said VEGF binds to a VEGF receptor expressed on the cell surfaces
of intratumoral blood vessels of said vascularized tumor; and
administering to said animal a chemotherapeutic agent or an
antitumor drug.
95. The method of claim 94, wherein said construct is a fusion
protein produced using recombinant DNA techniques.
96. The method of claim 94, wherein said selected therapeutic agent
is an anticellular agent that kills or suppresses the growth or
cell division of tumor-associated endothelial cells of intratumoral
blood vessels.
97. The method of claim 96, wherein said anticellular agent is a
chemotherapeutic agent, a radioisotope or a cytotoxin.
98. The method of claim 96, wherein said anticellular agent is a
steroid, an antimetabolite, an anthracycline, a vinca alkaloid, an
antibiotic, an alkylating agent or an epipodophyllotoxin.
99. The method of 96, wherein said anticellular agent is a plant-,
fungus- or bacteria-derived toxin.
100. The method of claim 99, wherein said toxin is an A chain
toxin, a ribosome inactivating protein, gelonin, .alpha.-sarcin,
aspergillin, restrictocin, a ribonuclease, diphtheria toxin,
Pseudomonas exotoxin, a bacterial endotoxin or the lipid A moiety
of a bacterial endotoxin.
101. The method of claim 99, wherein said toxin is ricin A
chain.
102. The method of claim 101, wherein said toxin is deglycosylated
ricin A chain.
103. The method of claim 99, wherein said toxin is gelonin.
104. The method of claim 94, wherein said chemotherapeutic agent is
an antimetabolite, an anthracycline, a vinca alkaloid or an
antitumor alkylating agent.
105. The method of claim 94, wherein said chemotherapeutic agent is
selected from the group consisting of cytosine arabinoside,
fluorouracil, methotrexate, aminopterin, mitomycin C, demecolcine,
etoposide, mithramycin, chlorambucil, melphalan, doxorubicin,
daunomycin, vinblastine, neocarzinostatin, macromycin, trenimon and
.alpha.-amanitin.
106. The method of claim 94, further comprising treating said
animal with radiotherapy.
107. The method of claim 94, wherein said animal has a squamous
cell carcinoma, adenocarcinoma, small cell carcinoma, glioma or
neuroblastoma.
108. The method of claim 94, wherein said animal has a carcinoma of
the lung, breast, ovary, stomach, liver, colon, rectum, cervix,
uterus, endometrium, bladder or prostate.
109. The method of claim 94, wherein said animal is a human cancer
patient.
Description
[0001] The present application is a continuation of co-pending U.S.
patent application Ser. No. 10/376,194, filed Feb. 27, 2003; which
is a divisional of co-pending U.S. patent application Ser. No.
09/738,970, filed Dec. 14, 2000; which is a continuation of U.S.
patent application Ser. No. 09/207,277, filed Dec. 8, 1998, now
issued as U.S. Pat. No. 6,261,535; which is a continuation of U.S.
patent application Ser. No. 08/350,212, filed Dec. 5, 1994, now
issued as U.S. Pat. No. 5,965,132; which is a continuation-in-part
of U.S. patent application Ser. No. 08/205,330, filed Mar. 2, 1994,
now issued as U.S. Pat. No. 5,855,866; which is a
continuation-in-part of U.S. patent application Ser. No.
07/846,349, filed Mar. 05, 1992. The entire text and figures of
which disclosures are specifically incorporated by reference herein
without disclaimer.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates generally to methods and
compositions for targeting the vasculature of solid tumors using
immunological and growth factor-based reagents. In particular
aspects, antibodies carrying diagnostic or therapeutic agents are
targeted to the vasculature of solid tumor masses through
recognition of tumor vasculature-associated antigens, such as
endoglin, or through the specific induction of other antigens on
vascular endothelial cells in solid tumors.
[0005] 2. Description of Related Art
[0006] Over the past 30 years, fundamental advances in the
chemotherapy of neoplastic disease have been realized. While some
progress has been made in the development of new chemotherapeutic
agents, the more startling achievements have been made in the
development of effective regimens for concurrent administration of
drugs and our knowledge of the basic science, e.g., the underlying
neoplastic processes at the cellular and tissue level, and the
mechanism of action of basic antineoplastic agents. As a result of
the fundamental achievement, we can point to significant advances
in the chemotherapy of a number of neoplastic diseases, including
choriocarcinoma, Wilm's tumor, acute leukemia, rhabdomyosarcoma,
retinoblastoma, Hodgkin's disease and Burkitt's lymphoma, to name
just a few. Despite the impressive advances that have been made in
a few tumors, though, many of the most prevalent forms of human
cancer still resist effective chemotherapeutic intervention.
[0007] The most significant underlying problem that must be
addressed in any treatment regimen is the concept of "total cell
kill." This concept holds that in order to have an effective
treatment regimen, whether it be a surgical or chemotherapeutic
approach or both, there must be a total cell kill of all so-called
"clonogenic" malignant cells, that is, cells that have the ability
to grow uncontrolled and replace any tumor mass that might be
removed. Due to the ultimate need to develop therapeutic agents and
regimens that will achieve a total cell kill, certain types of
tumors have been more amenable than others to therapy. For example,
the soft tissue tumors (e.g., lymphomas), and tumors of the blood
and blood-forming organs (e.g., leukemias) have generally been more
responsive to chemotherapeutic therapy than have solid tumors such
as carcinomas. One reason for this is the greater physical
accessibility of lymphoma and leukemic cells to chemotherapeutic
intervention. Simply put, it is much more difficult for most
chemotherapeutic agents to reach all of the cells of a solid tumor
mass than it is the soft tumors and blood-based tumors, and
therefore much more difficult to achieve a total cell kill. The
toxicities associated with most conventional antitumor agents then
become a limiting factor.
[0008] A key to the development of successful antitumor agents is
the ability to design agents that will selectively kill tumor
cells, while exerting relatively little, if any, untoward effects
against normal tissues. This goal has been elusive to achieve,
though, in that there are few qualitative differences between
neoplastic and normal tissues. Because of this, much research over
the years has focused on identifying tumor-specific "marker
antigens" that can serve as immunological targets both for
chemotherapy and diagnosis. Many tumor-specific, or
quasi-tumor-specific ("tumor-associated"), markers have been
identified as tumor cell antigens that can be recognized by
specific antibodies. Unfortunately, it is generally the case that
tumor specific antibodies will not in and of themselves exert
sufficient antitumor effects to make them useful in cancer
therapy.
[0009] Over the past fifteen years, immunotoxins have shown great
promise as a means of selectively targeting cancer cells.
Immunotoxins are conjugates of a specific targeting agent typically
a tumor-directed antibody or fragment, with a cytotoxic agent, such
as a toxin moiety. The targeting agent directs the toxin to, and
thereby selectively kills, cells carrying the targeted antigen.
Although early immunotoxins suffered from a variety of drawbacks,
more recently, stable, long-lived immunotoxins have been developed
for the treatment of a variety of malignant diseases. These "second
generation" immunotoxins employ deglycosylated ricin A chain to
prevent entrapment of the immunotoxin by the liver and
hepatotoxicity (Blakey et al., 1987). They employ new crosslinkers
which endow the immunotoxins with high in vivo stability (Thorpe et
al., 1988) and they employ antibodies which have been selected
using a rapid indirect screening assay for their ability to form
highly potent immunotoxins (Till et al., 1988).
[0010] Immunotoxins have proven highly effective at treating
lymphomas and leukemias in mice (Thorpe et al., 1988; Ghetie et
al., 1991; Griffin et al., 1988) and in man (Vitetta et al., 1991).
Lymphoid neoplasias are particularly amenable to immunotoxin
therapy because the tumor cells are relatively accessible to
blood-borne immunotoxins; also, it is possible to target normal
lymphoid antigens because the normal lymphocytes which are killed
along with the malignant cells during therapy are rapidly
regenerated from progenitors lacking the target antigens. In Phase
I trials where patients had large bulky tumor masses, greater than
50% tumor regressions were achieved in approximately 40% of the
patients (Vitetta et al., 1991). It is predicted that the efficacy
of these immunotoxins in patients with less bulky disease will be
even better.
[0011] In contrast with their efficacy in lymphomas, immunotoxins
have proved relatively ineffective in the treatment of solid tumors
such as carcinomas (Weiner et al., 1989; Byers et al., 1989). The
principal reason for this is that solid tumors are generally
impermeable to antibody-sized molecules: specific uptake values of
less than 0.001% of the injected dose/g of tumor are not uncommon
in human studies (Sands et al., 1988; Epenetos et al., 1986).
Furthermore, antibodies that enter the tumor mass do not distribute
evenly for several reasons. Firstly, the dense packing of tumor
cells and fibrous tumor stromas present a formidable physical
barrier to macromolecular transport and, combined with the absence
of lymphatic drainage, create an elevated interstitial pressure in
the tumor core which reduces extravasation and fluid convection
(Baxter et al., 1991; Jain, 1990). Secondly, the distribution of
blood vessels in most tumors is disorganized and heterogeneous, so
some tumor cells are separated from extravasating antibody by large
diffusion distances (Jain, 1990). Thirdly, all of the antibody
entering the tumor may become adsorbed in perivascular regions by
the first tumor cells encountered, leaving none to reach tumor
cells at more distant sites (Baxter et al., 1991; Kennel et al.,
1991). Finally, antigen-deficient mutants can escape being killed
by the immunotoxin and regrow (Thorpe et al., 1988).
[0012] Thus, it is quite clear that a significant need exists for
the development of novel strategies for the treatment of solid
tumors. One approach would be to target cytotoxic agents or
coagulants to the vasculature of the tumor rather than to the
tumor. Indeed, it has been observed that many existing therapies
may already have, as part of their action, a vascular-mediated
mechanism of action (Denekamp, 1990). The present inventors propose
that this approach offers several advantages over direct targeting
of tumor cells. Firstly, the target cells are directly accessible
to intravenously administered therapeutic agents, permitting rapid
localization of a high percentage of the injected dose (Kennel et
al., 1991). Secondly, since each capillary provides oxygen and
nutrients for thousands of cells in its surrounding `cord` of
tumor, even limited damage to the tumor vasculature could produce
an avalanche of tumor cell death (Denekamp, 1990; Denekamp, 1984).
Finally, the outgrowth of mutant endothelial cells lacking the
target antigen is unlikely because they are normal cells.
[0013] For tumor vascular targeting to succeed, antibodies are
required that recognize tumor endothelial cells but not those in
normal tissues. Although several antibodies have been raised
(Duijvestijn et al., 1987; Hagemeier et al., 1986; Bruland et al.,
1986; Murray et al., 1989; Schlingemann et al., 1985) none has
shown a high degree of specificity.
[0014] The antibodies termed TP-1 and TP-3, which were raised
against human osteosarcoma cells, have been reported to react with
the same antigen present on proliferating osteoblasts in normal
degenerating bone tissue. They also cross-react with capillary buds
in a number of tumor types and in placenta, but apparently not with
capillaries in any of the normal adult tissues examined (Bruland et
al., 1986). It remains to be seen whether the TP-1/TP-3 antigen is
present on the surface of endothelial cells or whether the
antibodies cross-react with gut endothelial cells, as was found
with another antibody against proliferating endothelium (Hagemeier
et al., 1986). This antibody described by Hagemeier and colleagues
(1986), termed EN7/44, reacts with a predominantly intracellular
antigen whose expression appears to be linked to migration rather
than proliferation (Hagemeier et al., 1986).
[0015] Immunotoxins in which the antibody portion is directed
against the fibronectin receptor have also been proposed for use in
killing proliferating vascular endothelial cells (Thorpe et al.,
1990). However, intravenous administration of an immunotoxin
containing dgA linked to the anti-fibronectin receptor antibody
termed PB1 did not result in reduced vascularization of tumors
(Thorpe et al., 1990). Unfortunately, further studies also revealed
that fibronectin receptors were too ubiquitous to enable good
targeting of tumor vasculature.
[0016] Other molecular markers have been described that are
specific for endothelial cells, although not for tumor endothelial
cells. For example, an endothelial-leukocyte adhesion molecule,
termed ELAM-1, has been identified that can be induced on the
surface of endothelial cells through the action of cytokines such
as IL-1, TNF, lymphotoxin or bacterial endotoxin (Bevilacqua et
al., 1987). However, the art currently lacks methods by which such
inducible molecules could be effectively employed in connection
with an anti-cancer strategy. Thus, unfortunately, while vascular
targeting presents promising theoretical advantages, no effective
strategies incorporating these advantages have been developed.
SUMMARY OF THE INVENTION
[0017] The present invention addresses one or more of the foregoing
or other disadvantages in the prior art, by providing a series of
novel approaches for the treatment and/or diagnosis (imaging) of
vascularized solid tumors. The invention rests in a general and
overall sense on the use of reagents, particularly immunological
reagents, to target therapeutic or diagnostic agents to
tumor-associated vascular endothelial cells, alone or in
combination with the direct targeting of tumor cells.
[0018] Such antibodies or growth factors will be referred to herein
as "targeting agents". Thus, the targeting compounds of the
invention may be either targeting agent/therapeutic agent compounds
or targeting agent/diagnostic agent compounds. Further, a targeting
agent/therapeutic agent compound comprises a targeting agent
operatively attached to a therapeutic agent, wherein the targeting
agent recognizes and binds to a tumor-associated endothelial cell
marker. A targeting agent/diagnostic agent compound comprises a
targeting agent operatively attached to a diagnostic agent, wherein
the targeting agent recognizes and binds to a tumor-associated
endothelial cell marker. The targeting agent/therapeutic agent
compounds of the invention may be produced using either standard
recombinant DNA techniques or standard synthetic chemistry
techniques, both of which are well known to those of skill in the
art.
[0019] In the case of diagnostic agents, the constructs will have
the ability to provide an image of the tumor vasculature, for
example, through magnetic resonance imaging, x-ray imaging,
computerized emission tomography and the like.
[0020] In the case of therapeutic agents, constructs are designed
to have a cytotoxic or otherwise anticellular effect against the
tumor vasculature, by suppressing the growth or cell division of
the vascular endothelial cells. This attack is intended to lead to
a tumor-localized vascular collapse, depriving the tumor cells,
particularly those tumor cells distal of the vasculature, of oxygen
and nutrients, ultimately leading to cell death and tumor necrosis.
In animal model systems, the inventors have achieved truly dramatic
tumor regressions, with some cures being observed in combination
therapy with anti-tumor directed therapy.
[0021] It is proposed that the various methods and compositions of
the invention will be broadly applicable to the treatment or
diagnosis of any tumor mass having a vascular endothelial
component. Typical vascularized tumors are the solid tumors,
particularly carcinomas, which require a vascular component for the
provision of oxygen and nutrients. Exemplary solid tumors to which
the present invention is directed include but are not limited to
carcinomas of the lung, breast, ovary, stomach, pancreas, larynx,
esophagus, testes, liver, parotid, biliary tract, colon, rectum,
cervix, uterus, endometrium, kidney, bladder, prostate, thyroid,
squamous cell carcinomas, adenocarcinomas, small cell carcinomas,
melanomas, gliomas, neuroblastomas, and the like.
[0022] A preferred method of the invention includes preparing an
antibody that recognizes an antigen or other ligand associated with
the vascular endothelial cells of the vascularized tumor mass,
linking, or operatively attaching the antibody to the selected
agent to form an antibody-agent conjugate, and introducing the
antibody-agent conjugate into the bloodstream of an animal, such as
a human cancer patient or a test animal in an animal model system.
As used however, the term "antibody" is intended to refer broadly
to any immunologic binding agent such as IgG, IgM, IgA, IgE,
F(ab').sub.2, a univalent fragment such as Fab', Fab, Dab, as well
as engineered antibodies such as recombinant antibodies, humanized
antibodies, bispecific antibodies, and the like.
[0023] Alternatively, growth factors, rather than antibodies, may
be utilized as the reagents to target therapeutic or diagnostic
agents to tumor-associated vascular endothelial cells, alone, or in
combination with the direct targeting of tumor cells. Any growth
factor may be used for such a targeting purpose, so long as it
binds to a tumor-associated endothelial cell, generally by binding
to a growth factor receptor present on the surface of such a
tumor-associated endothelial cell. Suitable growth factors for
targeting include, but are not limited to, VEGF/VPF (vascular
endothelial growth factor/vascular permeability factor), FGF
(which, as used herein, refers to the fibroblast growth factor
family of proteins), TFGB (transforming growth factor .beta.), and
pleitotropin. Preferably, the growth factor receptor to which the
targeting growth factor binds should be present at a higher
concentration on the surface of tumor-associated endothelial cells
than on non-tumor associated endothelial cells. Most preferably,
the growth factor receptor to which the targeting growth factor
binds should, further, be present at a higher concentration on the
surface of tumor-associated endothelial cells than on any non-tumor
associated cell type.
[0024] The agent that is linked to the antibody or growth factor
targeting agent will, of course, depend on the ultimate application
of the invention. Where the aim is to provide an image of the
tumor, one will desire to use a diagnostic agent that is detectable
upon imaging, such as a paramagnetic, radioactive or fluorogenic
agent. Many diagnostic agents are known in the art to be useful for
imaging purposes, as are methods for their attachment to antibodies
(see, e.g., U.S. Pat. Nos. 5,021,236 and 4,472,509, both
incorporated herein by reference). In the case of paramagnetic
ions, one might mention by way of example ions such as chromium
(III), manganese (II), iron (III), iron (II), cobalt (II), nickel
(II), copper (II), neodymium (III), samarium (III), ytterbium
(III), gadolinium (III), vanadium (II), terbium (III), dysprosium
(III), holmium (III) and erbium (III), with gadolinium being
particularly preferred. Ions useful in other contexts, such as
X-ray imaging, include but are not limited to lanthanum (III), gold
(III), lead (II), and especially bismuth (III). Moreover, in the
case of radioactive isotopes for therapeutic and/or diagnostic
application, one might mention iodine.sup.131, iodine.sup.123,
technicium.sup.99m, indium.sup.111, rhenium.sup.188,
rhenium.sup.186, gallium.sup.67, copper.sup.67, yttrium.sup.90,
iodine.sup.125, or astatine.sup.211 (for a review of the use of
monoclonal antibodies in the diagnosis and therapy of cancer, see
Vaickus et al., 1991).
[0025] For certain applications, it is envisioned that the
therapeutic agents will be pharmacologic agents will serve as
useful agents for attachment to antibodies or growth factors,
particularly cytotoxic or otherwise anticellular agents having the
ability to kill or suppress the growth or cell division of
endothelial cells. In general, the invention contemplates the use
of any pharmacologic agent that can be conjugated to a targeting
agent, preferably an antibody, and delivered in active form to the
targeted endothelium. Exemplary anticellular agents include
chemotherapeutic agents, radioisotopes as well as cytotoxins. In
the case of chemotherapeutic agents, the inventors propose that
agents such as a hormone such as a steroid; an antimetabolite such
as cytosine arabinoside, fluorouracil, methotrexate or aminopterin;
an anthracycline; mitomycin C; a vinca alkaloid; demecolcine;
etoposide; mithramycin; or an antitumor alkylating agent such as
chlorambucil or melphalan, will be particularly preferred. Other
embodiments may include agents such as a coagulant, a cytokine,
growth factor, bacterial endotoxin or the lipid A moiety of
bacterial endotoxin. In any event, it is proposed that agents such
as these may, if desired, be successfully conjugated to a targeting
agent, preferably an antibody, in a manner that will allow their
targeting, internalization, release or presentation to blood
components at the site of the targeted endothelial cells as
required using known conjugation technology (see, e.g., Ghose et
al., 1983 and Ghose et al., 1987).
[0026] In certain preferred embodiments, therapeutic agents will
include generally a plant-, fungus- or bacteria-derived toxin, such
as an A chain toxins, a ribosome inactivating protein,
.alpha.-sarcin, aspergillin, restrictocin, a ribonuclease,
diphtheria toxin or pseudomonas exotoxin, to mention just a few
examples. The use of toxin-antibody constructs is well known in the
art of immunotoxins, as is their attachment to antibodies. Of
these, a particularly preferred toxin for attachment to antibodies
will be a deglycosylated ricin A chain. Deglycosylated ricin A
chain is preferred because of its extreme potency, longer
half-life, and because it is economically feasible to manufacture
it a clinical grade and scale.
[0027] The present invention contemplates two separate and distinct
approaches to the targeting of targeting agents, preferably
antibodies, to the tumor vasculature. The first approach involves a
targeting agent having a binding affinity for a marker found,
expressed, accessible to binding, or otherwise localized on the
cell surfaces of tumor-associated vascular endothelial cells as
compared to normal, non-tumor associated vasculature. Further,
certain markers for which a targeting agent has a binding affinity
may be associated with the tumor-associated vasculature rather than
on the tumor-associated endothelial cells, themselves. For example,
such markers may be located on basement membranes or
tumor-associated connective tissue. It is preferred that targeting
agents specific for such non-endothelial cell markers be
operatively attached to agents such as radioisotopes.
[0028] In the case of antibody targeting agents, such an approach
involves the preparation of an antibody having a binding affinity
for antigenic markers found, expressed, accessible to binding or
otherwise localized on the cell surfaces of tumor-associated
vascular endothelium as compared to normal vasculature. Such a
targeting agent, preferably an antibody, is then employed to
deliver the selected diagnostic or therapeutic agent to the tumor
vasculature.
[0029] Naturally, where a therapeutic as opposed to diagnostic
application is envisioned it will be desirable to prepare and
employ an antibody having a relatively high degree of tumor
vasculature selectivity, which might be expressed as having little
or no reactivity with the cell surface of normal endothelial cells
as assessed by immunostaining of tissue sections. Of course, with
certain agents such as DNA synthesis inhibitors, and, more
preferably, antimetabolites, the requirement for selectivity is not
as necessary as it would be, for example, with a toxin, because a
DNA synthesis inhibitor would have relatively little effect on the
vascularation of normal tissues because the capillary endothelial
cells are not dividing. Further, such a degree of selectivity is
not a requirement for imaging purposes since cell death, and hence
toxicity, is not the ultimate goal. In the case of diagnostic
application, it is proposed that targeting agents, such as
antibodies, having a reactivity for the tumor vasculature of at
least two-fold higher than for normal endothelial cells, as
assessed by immunostaining, will be useful.
[0030] This aspect of the invention rests on the proposition that
because of their proximity to the tumor itself, tumor-associated
vascular endothelial cells are constantly exposed to many
tumor-derived products such as cytokines (including lymphokines,
monokines, colony-stimulating factors and growth factors),
angiogenic factors, and the like, that will bind to and serve to
selectively elicit the expression of tumor endothelium-specific
cell surface markers. For use in the present invention, the
anti-tumor vasculature antibodies may be directed to any of the
tumor-derived antigens which bind to the surface of vascular
endothelial cells, and particularly to tumor-derived ligands, such
as growth factors, which bind to specific cell surface receptors of
the endothelial cells.
[0031] In connection with certain aspects of the invention,
antibodies directed against tumor vasculature may be prepared by
using endothelial cells isolated from a tumor of an animal, or by
"mimicking" the tumor vasculature phenomenon in vitro. As such,
endothelial cells may be subjected to tumor-derived products, such
as might be obtained from tumor-conditioned media. Thus, this
method involves generally stimulating endothelial cells with
tumor-conditioned medium and employing the stimulated endothelial
cells as immunogens to prepare a collection of antibodies, for
example, by utilizing conventional hybridoma technology or other
techniques, such as combinatorial immunoglobulin phagemid libraries
prepared from RNA isolated from the spleen of the immunized animal.
One will then select from the antibody collection an antibody that
recognizes the tumor-stimulated vascular endothelium to a greater
degree than it recognizes non-tumor-stimulated vascular
endothelium, and reacts more strongly with tumor-associated
endothelial cells in tissue sections than with those in normal
adult human tissues, and producing the antibody, e.g., by culturing
a hybridoma to provide the antibody.
[0032] Stimulated endothelial cells contemplated to be of use in
this regard include, for example, human umbilical vein endothelial
cells (HUVE), human dermal microvascular endothelial cells (HDEMC),
human saphenous vein endothelial cells, human omental fat
endothelial cells, other human microvascular endothelial cells,
human brain capillary endothelial cells, and the like. It is also
contemplated that even endothelial cells from another species may
stimulated by tumor-conditioned media and employed as immunogens to
generate hybridomas to produce an antibodies in accordance
herewith, i.e., to produce antibodies which crossreact with
tumor-stimulated human vascular endothelial cells, and/or
antibodies for use in pre-clinical models.
[0033] As used herein, "tumor-conditioned medium" is defined as a
composition or medium, such as a culture medium, which contains one
or more tumor-derived cytokines, lymphokines or other effector
molecules. Most typically, tumor-conditioned medium is prepared
from a culture medium in which selected tumor cells have been
grown, and will therefore be enriched in such tumor-derived
products. The type of medium is not believed to be particularly
important, so long as it at least initially contains appropriate
nutrients and conditions to support tumor cell growth. It is also,
of course, possible to extract and even separate materials from
tumor-conditioned media and employ one or more of the extracted
products for application to the endothelial cells.
[0034] As for the type of tumor used for the preparation of the
media, one will, of course, prefer to employ tumors that mimic or
resemble the tumor that will ultimately be subject to analysis or
treatment using the present invention. Thus, for example, where one
envisions the development of a protocol for the treatment of breast
cancer, one will desire to employ breast cancer cells such as
ZR-75-1, T47D, SKBR3, MDA-MB-231. In the case of colorectal tumors,
one may mention by way of example the HT29 carcinoma, as well as
DLD-1, HCT116 or even SW48 or SW122. In the case of lung tumors,
one may mention by way of example NCI-H69, SW2, NCI H23, NCI H460,
NCI H69, or NCI H82. In the case of melanoma, good examples are
DX.3, A375, SKMEL-23, HMB-2, MJM, T8 or indeed VUP. In any of the
above cases, it is further believed that one may even employ cells
produced from the tumor that is to be treated, i.e., cells obtained
from a biopsy.
[0035] Once prepared, the tumor-conditioned media is then employed
to stimulate the appearance of tumor endothelium-specific marker(s)
on the cell surfaces of endothelial cells, e.g., by culturing
selected endothelial cells in the presence of the tumor-conditioned
media (or products derived therefrom). Again, it is proposed that
the type of endothelial cell that is employed is not of critical
importance, so long as it is generally representative of the
endothelium associated with the vasculature of the particular tumor
that is ultimately to be treated or diagnosed. The inventors prefer
to employ human umbilical vein endothelial cells (HUVE), or human
dermal microvascular endothelial cells (HDMEC, Karasek, 1989), in
that these cells are of human origin, respond to cytokine growth
factors and angiogenic factors and are readily obtainable. However,
it is proposed that any endothelial cell that is capable of being
cultured in vitro may be employed in the practice of the invention
and nevertheless achieve benefits in accordance with the invention.
One may mention by way of example, cells such as EA.hy9.26, ECV304,
human saphenous vein endothelial cells, and the like.
[0036] Once stimulated using the tumor-derived products, the
endothelial cells are then employed as immunogens in the
preparation of monoclonal antibodies. The technique for preparing
monoclonal antibodies against antigenic cell surface markers is
quite straightforward, and may be readily carried out using
techniques well known to those of skill in the art, as exemplified
by the technique of Kohler & Milstein (1975). Generally
speaking, the preparation of monoclonal antibodies using stimulated
endothelial cells involves the following procedures. Cells or cell
lines derived from human tumors are grown in tissue culture for
.gtoreq.4 days. The tissue culture supernatant (`tumor-conditioned
medium`) is removed from the tumor cell cultures and added to
cultures of HUVEC at a final concentration of 50% (v/v). After 2
days culture the HUVEC are harvested non-enzymatically and
1-2.times.10.sup.6 cells injected intraperitoneally into mice. This
process is repeated three times at two-weekly intervals, the final
immunization being by the intravenous route. Three days later the
spleen cells are harvested and fused with SP2/0 myeloma cells by
standard protocols (Kohler & Milstein, 1975): Hybridomas
producing antibodies with the appropriate reactivity are cloned by
limiting dilution.
[0037] From the resultant collection of hybridomas, one will then
desire to select one of more hybridomas that produce an antibody
that recognizes the activated vascular endothelium to a greater
extent than it recognizes non-activated vascular endothelium. Of
course, the ultimate goal is the identification of antibodies
having virtually no binding affinity for normal endothelium.
However, for imaging purposes this property is not so critical. In
any event, one will generally identify suitable antibody-producing
hybridomas by screening using, e.g., an ELISA, RIA, IRMA, IIF, or
similar immunoassay, against one or more types of tumor-activated
endothelial cells. Once candidates have been identified, one will
desire to test for the absence of reactivity for non-activated or
"normal" endothelium or other normal tissue or cell type. In this
manner, hybridomas producing antibodies having an undesirably high
level of normal cross-reactivity for the particular application
envisioned may be excluded.
[0038] The inventors have applied the foregoing technique
successfully, in that antibodies having relative specificity for
tumor vascular endothelium have been prepared and isolated. In one
particular example, the inventors employed the HT29 carcinoma to
prepare the conditioned medium, which was then employed to
stimulate HUVE cells in culture. The resultant HT29-activated HUVE
cells were then employed as immunogens in the preparation of a
hybridoma bank, which was ELISA-screened using HT29-activated HUVE
cells and by immunohistologic analysis of sections of human tumors
and normal tissues. From this bank, the inventors have selected
antibodies that recognized a tumor vascular endothelial cell
antigen.
[0039] The two most preferred monoclonal antibodies prepared by the
inventors using this technique are referred to as tumor endothelial
cell antibody 4 and 11 (TEC4 and TEC11). The antigen recognized by
TEC4 and TEC11 was initially believed to migrate as a doublet of
about 43 kilodaltons (kD), as assessed by SDS/PAGE. However, as
detailed herein, the present inventors subsequently determined this
antigen to be the molecule endoglin, which migrates as a 95 kD
species on SDS/PAGE under reducing conditions. The epitopes on
endoglin recognized by TEC4 and TEC11 are present on the cell
surface of stimulated HUVE cells, and only minimally present (or
immunologically accessible) on the surface of non-stimulated
cells.
[0040] Monoclonal antibodies have previously been raised against
endoglin (Gougos and Letarte, 1988; Gougos et al., 1992; O'Connel
et al., 1992; Buhring et al., 1991). However, analyzing the
reactivity with HUVEC or TCM-activated HUVEC cell surface
determinants by FACS or indirect immunofluorescence shows the
epitopes recognized by TEC-4 and TEC-11 to be distinct from those
of a previous antibody termed 44G4 (Gougos and Letarte, 1988).
[0041] The TEC-4 and TEC-11 mAbs are envisioned to be particularly
suitable for targeting human tumor vasculature as they label
capillary and venular endothelial cells moderately to strongly in a
broad range of solid tumors (and in several chronic inflammatory
conditions and fetal placenta), but display relatively weak
staining of vessels in the majority of normal, healthy adult
tissues. TEC-11 is particularly preferred as it shows virtually no
reactivity with non-endothelial cells. Furthermore, both TEC-4 and
TEC-11 are complement-fixing, which imparts to them the potential
to also induce selective lysis of endothelial cells in the tumor
vascular bed.
[0042] In addition to their use in therapeutic embodiments, TEC-4
and TEC-11 antibodies may also be used for diagnostic, prognostic
and imaging purposes. For example, TEC-4 and TEC-11 may be employed
to identify tumors with high vessel density, which is known to
correlate with metastatic risk and poor prognosis. This is a marked
advance over the laborious enumeration of capillaries labelled with
pan-endothelial cell markers or the use of complex and subjective
in vivo assays of angiogenesis. Indeed, studies are disclosed
herein which indicate that TEC-4 and TEC-11 can distinguish between
intraductal carcinoma in situ (CIS), an aggressive preneoplastic
lesion and lobular CIS, which is associated with a more indolent
clinical course.
[0043] TEC-4 or TEC-11 antibodies may be linked to a paramagnetic,
radioactive or fluorogenic ion and employed in tumor imaging in
cancer patients, where it is contemplated that they will result in
rapid imaging due to the location of endoglin on the luminal face
of endothelial cells. Furthermore, TEC-4 and TEC-11 are of the IgM
isotype, which limits extravasation and enables more specific
imaging of antigens in the intravascular compartment. This is in
contrast to 44G4 which is an IgG1 antibody.
[0044] The present invention therefore encompasses anti-endoglin
antibodies and antibody-based compositions, including antibody
conjugates linked to paramagnetic, radioactive or fluorogenic ions
and anti-cellular agents such as anti-metabolites, toxins and the
like, wherein the antibodies bind to endoglin at the same epitope
as either of the MAbs TEC-4 and TEC-11. Such antibodies may be of
the polyclonal or monoclonal type, with monoclonals being generally
preferred, especially for use in preparing endoglin-directed
antibody conjugates, immunotoxins and compositions thereof.
[0045] The identification of an antibody or antibodies that bind to
endoglin at the same epitopes as TEC-4 or TEC-11 is a fairly
straightforward matter. This can be readily determined using any
one of variety of immunological screening assays in which antibody
competition can be assessed. For example, where the test antibodies
to be examined are obtained from a different source to that of
TEC-4 or TEC-11 , e.g., a rabbit, or are even of a different
isotype, for example, IgG1 or IgG3, a competition ELISA may be
employed. In one such embodiment of a competition ELISA one would
pre-mix TEC-4 or TEC-11 with varying amounts of the test antibodies
prior to applying to the antigen-coated wells in the ELISA plate.
By using either anti-murine or anti-IgM secondary antibodies one
will be able to detect only the bound TEC-4 or TEC-11
antibodies--the binding of which will be reduced by the presence of
a test antibody which recognizes the same epitope as either TEC-4
or TEC-11.
[0046] To conduct an antibody competition study between TEC-4 or
TEC-11 and any test antibody, one may first label TEC-4 or TEC-11
with a detectable label, such as, e.g., biotin or an enzymatic or
radioactive label, to enable subsequent identification. In these
cases, one would incubate the labelled antibodies with the test
antibodies to be examined at various ratios (e.g., 1:1, 1:10 and
1:100) and, after a suitable period of time, one would then assay
the reactivity of the labelled TEC-4 or TEC-11 antibodies and
compare this with a control value in which no potentially competing
antibody (test) was included in the incubation.
[0047] The assay may be any one of a range of immunological assays
based upon antibody binding and the TEC-4 or TEC-11 antibodies
would be detected by means of detecting their label, e.g., using
streptavidin in the case of biotinylated antibodies or by using a
chromogenic substrate in connection with an enzymatic label or by
simply detecting the radiolabel. An antibody that binds to the same
epitope as TEC-4 or TEC-11 will be able to effectively compete for
binding and thus will significantly reduce TEC-4 or TEC-11 binding,
as evidenced by a reduction in labelled antibody binding. In the
present case, after mixing the labelled TEC-4 or TEC-11 antibodies
with the test antibodies, suitable assays to determine the
remaining reactivity include, e.g., ELISAs, RIAs or western blots
using human endoglin; immunoprecipitation of endoglin; ELISAs, RIAs
or immunofluorescent staining of recombinant cells expressing human
endoglin; indirect immunofluorescent staining of tumor vasculature
endothelial cells; reactivity with HUVEC or TCM-activated HUVEC
cell surface determinants indirect immunofluorescence and FACS
analysis. This latter method is most preferred and was employed to
show that the epitopes recognized by TEC-4 and TEC-11 are distinct
from that of 44G4 (Gougos and Letarte, 1988).
[0048] The reactivity of the labelled TEC-4 or TEC-11 antibodies in
the absence of any test antibody is the control high value. The
control low value is obtained by incubating the labelled antibodies
with unlabelled antibodies of the same type, when competition would
occur and reduce binding of the labelled antibodies. A significant
reduction in labelled antibody reactivity in the presence of a test
antibody is indicative of a test antibody that recognizes the same
epitope, i.e., one that "cross-reacts" with the labelled antibody.
A "significant reduction" in this aspect of the present application
may be defined as a reproducible (i.e., consistently observed)
reduction in binding of at least about 10%-50% at a ratio of about
1:1, or more preferably, of equal to or greater than about 90% at a
ratio of about 1:100.
[0049] The present invention further encompasses antibodies which
are specific for epitopes present only on growth factor/growth
factor receptor complexes, while being absent from either the
individual growth factor or growth factor receptor. Thus, such
antibodies will recognize and bind a growth factor/growth factor
receptor complex while not recognizing or binding either the growth
factor molecule or growth factor receptor molecule while these
molecules are not in growth factor/growth factor receptor complex
form. A "growth factor/growth factor receptor complex" as used
herein refers to a growth factor ligand bound specifically to its
growth factor receptor, such as, by way of example only, a
VEGF/VEGF receptor complex.
[0050] As it is envisioned that the growth factor/growth factor
receptor complexes to which these antibodies specifically bind are
present in significantly higher number on tumor-associated
endothelial cells than on non-tumor associated endothelial cells,
such antibodies, when used as targeting agents, serve to further
increase the targeting specificity of the agents of the invention.
Such antibodies may be of the polyclonal or monoclonal type, with
monoclonals being generally preferred.
[0051] The second overall general approach presented by the present
invention involves the selective elicitation of vascular
endothelial antigen targets on the surface of tumor-associated
vasculature. This approach targets known endothelial antigens that
are present, or inducible, on the cell surface of endothelial
cells. The key to this aspect of the invention is the successful
manipulation of antigenic expression or surface presentation such
that the target antigen is expressed or otherwise available on the
surface of tumor associated vasculature and not expressed or
otherwise available for binding, or at least to a lesser extent, on
the surface of normal endothelium.
[0052] A variety of endothelial cell markers are known that can be
employed as inducible targets for the practice of this aspect of
the invention, including endothelial-leukocyte adhesion molecule
(ELAM-1; Bevilacqua et al., 1987); vascular cell adhesion
molecule-1 (VCAM-1; Dustin et al., 1986); intercellular adhesion
molecule-1 (ICAM-1; Osborn et al., 1989); the agent for leukocyte
adhesion molecule-1 (LAM-1 agent), or even a major
histocompatibility complex (MHC) Class II antigen, such as HLA-DR,
HLA-DP or HLA-DQ (Collins et al., 1984). Of these, the targeting of
ELAM-1 or an MHC Class II antigen will likely be preferred for
therapeutic application, with ELAM-1 being particularly preferred,
since the expression of these antigens will likely be the most
direct to promote selectively in tumor-associated endothelium.
[0053] The targeting of an antigen such as ELAM-1 is the most
straightforward since ELAM-1 is not expressed on the surfaces of
normal endothelium. ELAM-1 is an adhesion molecule that can be
induced on the surface of endothelial cells through the action of
cytokines such as IL-1, TNF, lymphotoxin or bacterial endotoxin
(Bevilacqua et al., 1987). In the practice of the present
invention, the expression of ELAM-1 is selectively induced in tumor
endothelium through the use of a bispecific antibody having the
ability to cause the selective release of one or more of the
foregoing or other appropriate cytokines in the tumor environment,
but not elsewhere in the body. This bispecific antibody is designed
to cross-link cytokine effector cells, such as cells of
monocyte/macrophage lineage, T cells and/or NK cells or mast cells,
with tumor cells of the targeted solid tumor mass. This
cross-linking is intended to effect a release of cytokine that is
localized to the site of cross-linking, i.e., the tumor.
[0054] Bispecific antibodies useful in the practice of this aspect
of the invention, therefore, will have a dual specificity,
recognizing a selected tumor cell surface antigen on the one hand,
and, on the other hand, recognizing a selected "cytokine
activating" antigen on the surface of a selected leukocyte cell
type. As used herein, the term "cytokine activating" antigen is
intended to refer to any one of the various known molecules on the
surfaces of leukocytes that, when bound by an effector molecule
such as an antibody or a fragment thereof or a naturally-occurring
agent or synthetic analog thereof, be it a soluble factor or
membrane-bound counter-receptor on another cell, will promote the
release of a cytokine by the leukocyte cell. Examples of cytokine
activating molecules include CD14 and FcR for IgE, which will
activate the release of IL-1 and TNF.alpha.; and CD16, CD2 or CD3
or CD28, which will activate the release of IFN.gamma. and
TNF.beta., respectively.
[0055] Once introduced into the bloodstream of an animal bearing a
tumor, such a bispecific construct will bind to tumor cells within
the tumor, cross-link those tumor cells with, e.g.,
monocytes/macrophages that have infiltrated the tumor, and
thereafter effect the selective release of cytokine within the
tumor. Importantly, however, without cross-linking of the tumor and
leukocyte, the bispecific antibody will not effect the release of
cytokine. Thus, no cytokine release will occur in parts of the body
removed from the tumor and, hence, expression of ELAM-1 will occur
only within the tumor endothelium.
[0056] A number of useful "cytokine activating" antigens are known,
which, when cross-linked with an appropriate bispecific antibody,
will result in the release of cytokines by the cross-linked
leukocyte. The most preferred target for this purpose is CD14,
which is found on the surface of monocytes and macrophages. When
CD14 is cross linked it will stimulate the monocyte/macrophage to
release IL-1, and possibly other cytokines, which will, in turn
stimulate the appearance of ELAM-1 on nearby vasculature. Other
possible targets for cross-linking in connection with ELAM-1
targeting includes FcR for IgE, found on Mast cells; FcR for IgG
(CD16), found on NK cells; as well as CD2, CD3 or CD28, found on
the surfaces of T cells. Of these, CD14 targeting will be the most
preferred due to the relative prevalence of monocyte/macrophage
infiltration of solid tumors as opposed to the other leukocyte cell
types.
[0057] In that MHC Class II antigens are expressed on "normal"
endothelium, their targeting is not quite so straightforward as
ELAM-1. However, the present invention takes advantage of the
discovery that immunosuppressants such as Cyclosporin A (CsA) have
the ability to effectively suppress the expression of Class II
molecules in the normal tissues. There are various other
cyclosporins related to CsA, including cyclosporins A, B, C, D, G,
and the like, which have immunosuppressive action, and will likely
also demonstrate an ability to suppress Class II expression. Other
agents that might be similarly useful include FK506 and
rapamycin.
[0058] Thus, the practice of the MHC Class II targeting embodiment
requires a pretreatment of the tumor-bearing animal with a dose of
CsA or other Class II immunosuppressive agent that is effective to
suppress Class II expression. In the case of CsA, this will
typically be on the order of about 10 to 30 mg/kg. Once suppressed
in normal tissues, Class II antigens can be selectively induced in
the tumor endothelium through the use of a bispecific antibody,
this one having specificity for the tumor cell as well as an
activating antigen found on the surface of helper T cells. Note
that in this embodiment, it is necessary that T cells, or NK cells
if CD16 is used, be present in the tumor to produce the cytokine
intermediate in that Class II antigen expression is achieved using
IFN-.gamma., but is not achieved with the other cytokines. Thus,
for the practice of this aspect of the invention, one will desire
to select CD2, CD3 or CD28 (most preferably CD28) as the cytokine
activating antigen.
[0059] An alternative approach to using "cytokine-activating"
bispecific antibodies might be to activate the patients peripheral
blood leukocytes or tumor-infiltrating lymphocytes in vitro (using
IL-2 or autologous tumor cells for instance), reinfuse them into
the patient and then localize them in the tumor with a bispecific
antibody against any reliable leukocyte-specific marker, including
CD5, CD8, CD11/CD18, CD15, CD32, CD44, CD45 or CD64. In order to
selectively localize those leukocytes that had become activated
from within a mixed population, it is recommended that the
anti-leukocyte arm of the bispecific antibody should recognize a
marker restricted to activate cells, such as CD25, CD30, CD54 or
CD71. Neither of these approaches is favored as much as the
`cytokine-activating` antibody approach because cross-linking to
tumor cells is not a prerequisite for cytokine secretion and thus
the resultant induction of cytokine-induced endothelial cell
antigens may not be confined to the tumor.
[0060] The targeting of the other adhesion molecules, ICAM-1,
VCAM-1 and LAM-1 agent, will typically not be preferred for the
practice of therapeutic embodiments, in that these targets are
constitutively expressed in normal endothelium. Thus, these
adhesion molecules will likely only be useful in the context of
diagnostic embodiments. Furthermore, it is unlikely that ICAM-1 or
VCAM-1 expression by normal endothelial cells would be inhibited in
vivo by CsA because low levels of expression of both markers are
constitutive properties of human endothelial cells (Burrows et al.,
1991). However, it may still be possible to utilize one of these
molecules in diagnostic or even therapeutic embodiments because
their level of expression on the endothelial cell surface is
increased 10-50 fold by cytokines. As a consequence, there may be a
therapeutic or diagnostic `window` enabling use of anti-ICAM-1 or
anti-VCAM-1 conjugates in an analogous way to the proven clinical
utility of some antibodies against `tumor-associated` antigens
whose expression differs quantitatively but not qualitatively from
normal tissues.
[0061] The tumor antigen recognized by the bispecific antibodies
employed in the practice of the present invention will be one that
is located on the cell surfaces of the tumor being targeted. A
large number of solid tumor-associated antigens have now been
described in the scientific literature, and the preparation and use
of antibodies are well within the skill of the art (see, e.g.,
Table II hereinbelow). Of course, the tumor antigen that is
ultimately selected will depend on the particular tumor to be
targeted. Most cell surface tumor targets will only be suitable for
imaging purposes, while some will be suitable for therapeutic
application. For therapeutic application, preferred tumor antigens
will be TAG 72 or the HER-2 proto-oncogene protein, which are
selectively found on the surfaces of many breast, lung and
colorectal cancers (Thor et al., 1986; Colcher et al., 1987;
Shepard et al., 1991). Other targets that will be particularly
preferred include milk mucin core protein, human milk fat globule
(Miotti et al., 1985; Burchell et al., 1983) and even the high Mr
melanoma antigens recognized by the antibody 9.2.27 (Reisfeld et
al., 1982).
[0062] In still further embodiments, the inventors contemplate an
alternative approach for suppressing the expression of Class II
molecules, and selectively eliciting Class II molecule expression
in the locale of the tumor. This embodiment takes advantage of the
fact that the expression of Class II molecules can be effectively
inhibited by suppressing IFN-.gamma. production by T-cells, e.g.,
through use of an anti-CD4 antibody (Street et al., 1989). Thus, in
this embodiment, one will desire to pretreat with a dose of
anti-CD4 that is effective to suppress IFN-.gamma. production and
thereby suppress the expression of Class II molecules (for example,
on the order of 4 to 10 mg/kg). After Class II expression is
suppressed, one will then prepare and introduce into the
bloodstream an IFN-.gamma.-producing T-cell clone (e.g., T.sub.h1
or CTL) specific for an antigen expressed on the surface of the
tumor cells.
[0063] A preferred means of producing the IFN-.gamma.-producing
T-cell clone is by a method that includes removing a portion of the
tumor mass from the patient, extracting tumor infiltrating
leukocytes from the tumor, and expanding the tumor infiltrating
leukocytes in vitro to provide the IFN-.gamma. producing clone.
This clone will necessarily be immunologically compatible with the
patient, and therefore should be well tolerated by the patient. It
is proposed that particular benefits will be achieved by further
selecting a high IFN-.gamma. producing T-cell clone from the
expanded leukocytes by determining the cytokine secretion pattern
of each individual clone every 14 days. To this end, rested clones
will be mitogenically or antigenically-stimulated for 24 hours and
their culture supernatants assayed by a specific sandwich ELISA
technique (Cherwinski et al., 1989) for the presence of IL-2,
IFN-.gamma., IL-4, IL-5 and IL-10. Those clones secreting high
levels of IL-2 and IFN-.gamma., the characteristic cytokine
secretion pattern of T.sub.H1 clones, will be selected. Tumor
specificity will be confirmed using proliferation assays.
Furthermore, one will prefer to employ as the anti-CD4 antibody an
anti-CD4 Fab, because it will be eliminated from the body within 24
hours after injection and so will not cause suppression of the
tumor recognizing T cell clones that are subsequently administered.
The preparation of T-cell clones having tumor specificity is
generally known in the art, as exemplified by the production and
characterization of T cell clones from lymphocytes infiltrating
solid melanoma tumors (Maeda et al., 1991).
[0064] The invention contemplates that still further advantages
will be realized through combination regimens wherein both the
tumor endothelial vasculature and the tumor itself are targeted.
Combination regimens may thus include targeting of the tumor
directly with either conventional antitumor therapy, such as with
radiotherapy or chemotherapy, or through the use of a second
immunological reagent such as an antitumor immunotoxin. In fact,
dramatic, synergistic antitumor effects were seen by the inventors
when solid tumors were targeted with both an antitumor endothelial
cell immunotoxin and an antitumor cell immunotoxin. Such
combination therapy is founded theoretically on 1) the use of the
endothelial-directed immunotoxin to kill those tumor cells that
depend upon vascular oxygen and nutrients, and 2) the use of the
tumor-directed immunotoxin to kill those tumor cells that may have
an alternate source of oxygen and nutrients (i.e., those tumor
cells lining the vasculature and those forming the outer boundary
of the tumor mass). Thus, it is proposed that particular advantages
will be realized through the targeting of agents both to tumor cell
targets as well as to tumor endothelial cell targets.
[0065] The invention further contemplates the selected combinations
of agents particularly adapted for use in connection with the
methods of the present invention, defined as including a first
pharmaceutical composition which includes a bispecific antibody
recognizing an activating antigen on the cell surface of a
leukocyte cell and a tumor antigen on the cell surface of tumor
cells of a vascularized solid tumor, together with a second
pharmaceutical composition comprising a second antibody or fragment
thereof linked to a selected therapeutic or diagnostic agent that
recognizes the induced endothelial antigen. In accordance with one
aspect of the invention, these agents may be conveniently packaged
together, being suitably aliquoted into separate containers, and
the separate containers dispensed in a single package.
[0066] In particular embodiments, the activating antigen induced by
the bispecific antibody will be CD2, CD3, CD14, CD16, FcR for IgE,
CD28 or the T-cell receptor antigen, as may be the case. However,
preferably, the bispecific antibody will recognize CD14, and induce
the expression of IL-1 by monocyte/macrophage cells in the tumor,
or recognize CD28 and induce the expression of IFN-.gamma. by
T-cells in the tumor. Where IL-1 is the cytokine intermediate, the
second antibody will preferably be one that recognizes ELAM-1,
since this adhesion molecule will be induced on the surface of
endothelial cells by IL-1. In contrast, where IFN-.gamma. is the
intermediate, the second antibody will preferably be one that
recognizes an MHC Class II antigen. In the later case, one might
desire to include with the combination a third pharmaceutical
composition comprising one of the cyclosporins, or another
immunosuppressive agent useful for suppressing Class II
expression.
[0067] Furthermore, in that the invention contemplates combination
regimens as discussed above, particular embodiments of the
invention will involve the inclusion of a third pharmaceutical
composition comprising an antitumor antibody conjugated to a
selected agent, such as an anti-tumor immunotoxin. In these
embodiments, particularly preferred will be the targeting of tumor
antigens such as p185.sup.HER2, milk mucin core protein, TAG-72,
Lewis a, carcinoembryonic antigen (CEA), the high Mr melanoma
antigens recognized by the 9.2.27 antibody, or the
ovarian-associated antigens recognized by OV-TL3 or MOV18. These
same antigens will also be preferred as the target for the
bispecific antibody. Of course, where such a bispecific antibody is
employed in combination with an antitumor antibody, it may be
desirable to target different tumor antigens with the bispecific
and antitumor antibody.
BRIEF DESCRIPTION OF THE DRAWINGS
[0068] FIG. 1a. Induction of I-E.sup.k on SVEC cells by IFN-.gamma.
in regular medium, r.IFN-.gamma., or r.IFN-.gamma. plus excess
neutralizing anti-IFN-.gamma. antibody. SVEC cells were cultured
for 72 hours in regular medium (-----), r.IFN-.gamma. (.....) or
r.IFN-.gamma. plus excess neutralizing anti-IFN-.gamma. antibody (.
. . .). Their expression of I-E.sup.k was then measured by M5/114
antibody binding by indirect immunofluorescence using the FACS
(fluorescence-activated cell sorter). Other cultures were treated
with r.IFN-.gamma. and stained with an isotype-matched control
antibody (- - - -).
[0069] FIG. 1b. Induction of I-E.sup.k on SVEC cells by IFN-.gamma.
in C1300-conditioned media. SVEC cells were cultured for 72 hours
in C1300-conditioned medium (-----), C1300(Mu.gamma.)-conditioned
medium (.....) or C1300(Mu.gamma.)-conditioned medium plus excess
neutralizing anti-IFN-.gamma. antibody (. . . .). Their expression
of I-E.sup.k was then measured as in FIG. 1a. Other cultures were
treated with C1300(Mu.gamma.)-conditioned medium and stained with
an isotype-matched control antibody (- - -).
[0070] FIG. 2a. Expression of I-E.sup.k and H-2K.sup.k by pure and
mixed populations of C1300 and C1300(Mu.gamma.) cells stained with
anti-I-E.sup.k antibody. C1300 cells (. . . .), C1300(Mu.gamma.)
cells (- - -), a mixture of C1300 and C1300(Mu.gamma.) cells in the
ratio 7:3 cocultured in vitro (.....) or cells recovered from a
mixed subcutaneous tumor in a BALB/c nu/nu mouse (-----) were
stained with anti-I-E.sup.k antibody by indirect immunofluorescence
using the FACS. No staining of any tumor cell population was seen
with the isotype-matched control antibodies.
[0071] FIG. 2b. Expression of I-E.sup.k and H-2K.sup.k by pure and
mixed populations of C1300 and C1300(Mu.gamma.) cells stained with
anti-H-2K.sup.k antibody. C1300 cells (. . . .), C1300(Mu.gamma.)
cells (- - -), a mixture of C1300 and C1300(Mu.gamma.) cells in the
ratio 7:3 cocultured in vitro (.....) or cells recovered from a
mixed subcutaneous tumor in a BALB/c nu/nu mouse (-----) were
stained with anti-H-2K.sup.k antibody by indirect
immunofluorescence using the FACS. No staining of any tumor cell
population was seen with the isotype-matched control
antibodies.
[0072] FIG. 3. Tumorigenicity, growth, and tumor endothelial cell
Ia.sup.d expression in pure and mixed subcutaneous C1300 and
C1300(Mu.gamma.) tumors. BALB/c nu/nu mice were injected with a
total of 2.times.10.sup.7 tumor cells in which the ratios of
C1300:C1300(Mu.gamma.) cells were 10:0 (.DELTA.), 9:1
(.largecircle.), 8:2 (.circle-solid.), 7:3 (.diamond.), 5:5
(.box-solid.), 3:7 (.quadrature.) or 0:10 (.tangle-solidup.). The
vertical axis shows the mean diameter of the tumors at various
times after injection. Also shown are the percentage of animals in
each group which developed tumors. The proportion of Ia.sup.d
positive vascular endothelial cells was categorized as follows: +,
75-100%; +/-, 25-75%; -, 0-5%; n.d., not determined because no
intact blood vessels were visible. Standard deviations were <15%
of mean diameters and are not shown.
[0073] FIG. 4a. Killing activity of anti-Class II immunotoxin
(M5/114 dgA) against unstimulated SVEC mouse endothelial cells. The
data shown are for treatment of cells with varying concentrations
of ricin (.quadrature.); M5/114dgA (.circle-solid.); and the
control immunotoxin CAMPATH-2 dgA (.largecircle.).
[0074] FIG. 4b. Killing activity of anti-Class II immunotoxin
(M5/114 dgA) against SVEC mouse endothelial cells stimulated with
conditioned medium from the IFN-.gamma.-secreting tumor C1300
(Mu-.gamma.). The data shown are for treatment of cells with
varying concentrations of ricin (.quadrature.); M5/114dgA
(.circle-solid.); and the control immunotoxin CAMPATH-2dgA
(.largecircle.).
[0075] FIG. 5. This FIG. also shows the killing of SVEC cells under
various conditions by the anti-Class II immunotoxin, M5/114dgA. The
data shown are for treatment of cells with varying concentrations
of the immunotoxin following treatment with IFN-.gamma. TCM
(.largecircle.); C1300 TCM (.tangle-solidup.); C1300(Mu-.gamma.)
TCM (.box-solid.); and C1300(Mu-.gamma.) treated with
anti-IFN-.gamma. (.quadrature.).
[0076] FIG. 6a. Shows a comparison of killing activity of an
anti-Class I (antitumor) immunotoxin (11-4.1-dgA, which recognized
H-2K.sup.k) and an anti-Class II (anti-tumor endothelial cell)
immunotoxin (M5/114-dgA) against a 70:30 mixed population of C1300
and C1300(Mu-.gamma.) cells. Data was obtained through treatment of
the cells with ricin (.circle-solid.); the 11-4.1-dgA immunotoxin
(.largecircle.); the M5/114-dgA immunotoxin (.box-solid.) and a
control immunotoxin (.quadrature.).
[0077] FIG. 6b. Shows killing of cells freshly recovered from
subcutaneous tumors in mice. Data was obtained through treatment of
the cells with ricin (.circle-solid.); the 11-4.1-dgA immunotoxin
(.largecircle.); the M5/114-dgA immunotoxin (.box-solid.) and a
control immunotoxin (.quadrature.).
[0078] FIG. 7a. Killing of pure populations of C1300
(.circle-solid.) and C1300(Mu-.gamma.) (.largecircle.) by the
antitumor cell immunotoxin, 11-4.1-dgA. Also shown are 70:30 mixed
populations mixed in vitro or in vivo (i.e., recovered from S/C
tumors). Also shown are controls, including ricin
(.tangle-solidup.) and a control immunotoxin (.DELTA.).
[0079] FIG. 7b. Killing of pure populations of C1300
(.circle-solid.) and C1300(Mu-.gamma.) (.largecircle.) by the
antitumor cell immunotoxin, 11-4.1-dgA, as shown in FIG. 7a, with
the controls, ricin (.tangle-solidup.) and a control immunotoxin
(.DELTA.), for comparison.
[0080] FIG. 8. This FIG. shows the in vivo antitumor effects of the
anti-endothelial cell immunotoxin, M5/114-dgA, at various doses,
including 20 .mu.g (.largecircle.) and 40 .mu.g (.box-solid.) .
These studies involved the administration of the immunotoxin
intravenously 14 days after injection of tumor cells. Controls
included the use of a control immunotoxin, CAMPATH-2-dgA (.DELTA.)
and PBS+BSA (.tangle-solidup.).
[0081] FIG. 9. This FIG. is a histological analysis of 1.2 cm
H&E-stained tumor sections 72 hours after treatment with 20
.mu.g of the anti-Class II immunotoxin, M5/114-dgA.
[0082] FIG. 10. This FIG. is a histological analysis of 1.2 cm
H&E-stained tumor sections 72 hours after treatment with 100
.mu.g of the anti-Class II immunotoxin, M5/114-dgA.
[0083] FIG. 11. This FIG. is a representation of the appearance of
a solid tumor 48-72 hours after intravenous immunotoxin treatment,
and compares the effect achieved with anti-tumor immunotoxin, to
that achieved with anti-endothelial cell immunotoxin therapy.
[0084] FIG. 12. This FIG. shows the antitumor effects of single and
combined treatments with anti-Class I and anti-Class II
immunotoxins in SCID mice bearing large solid C1300(Mu-.gamma.)
tumors. SCID mice bearing 1.0-1.3 cm diameter tumors were injected
intravenously 14 days after tumor inoculation with 20 .mu.g of
Class II immunotoxin (.largecircle.), 100 .mu.g Class I immunotoxin
(.circle-solid.), or diluent alone (.tangle-solidup.) . Other
animals received the anti-Class II immunotoxin followed by two days
later by the anti-Class I immunotoxin (.box-solid.), or vice versa
(.quadrature.). Tumor size was measured at regular intervals and is
expressed as mean tumor diameter +/- SEM. Each treatment group
consisted of 4-8 mice.
[0085] FIG. 13a. Gel electrophoretic analysis of proteins
immunoprecipitated from .sup.35S-labelled human umbilical vein
endothelial cells (HUVEC), showing that TEC-4 and TEC-11 recognize
endoglin. 12.5% SDS-PAGE gel of proteins immunoprecipitated under
reducing (lanes 2-4) or non-reducing (lanes 5-7) conditions with
TEC-4 (lanes 2,5), TEC-11 (lanes 3,6) or TEPC-183 (lanes 4,7). Lane
1: Position of the .sup.14C-labelled standards of the molecular
weights indicated. Lane 8: Positions of 95 kDa and 180 kDa
species.
[0086] FIG. 13b. Reactivity of TEC-4 and TEC-11 with human endoglin
transfectants, showing that TEC-4 and TEC-11 recognize endoglin.
Parental murine L cells and L cell transfectants expressing human
endoglin were incubated with purified MAb TEC-11, TEC-4 and 44G4
followed by FITC-conjugated F(ab').sub.2 goat anti-mouse IgG (H+L).
The staining observed on the parental L cells with the MAb (white
histograms) was indistinguishable from that observed with IgM and
IgG1 controls. The L cell endoglin transfectants (black histograms)
were specifically reactive with all 3 antibodies as revealed by the
percentage of cells included within the gate and shown in
parentheses.
[0087] FIG. 14. Crossblocking of TEC-4 and TEC-11 antibodies.
Biotinylated antibodies (10 .mu.g/ml) were mixed with an equal
volume of unlabelled TEC-4 antibody at 10 .mu.g/ml (.quadrature.),
100 .mu.g/ml () or 1000 .mu.g/ml (.box-solid.) or with unlabelled
TEC-11 antibody at 10 .mu.g/ml (), 100 .mu.g/ml () and were added
to HUVEC in PBS-BSA-N.sub.3. Indirect immunofluorescence staining
was carried out as described in Example V with the exception that
labelled antibody binding was detected with a
streptavidin-phycoerythrin conjugate. Each group of histograms
shows the percent blocking of the biotinylated antibody by the
different concentrations of unlabelled antibodies. Bar: SD of
triplicate determinations.
[0088] FIG. 15. Complement fixation by TEC-4 and TEC-11 antibodies.
HUVEC were incubated with TEC-4 (), TEC-11 (.box-solid.) or MTSA
(.tangle-solidup.) antibodies, washed and subsequently incubated
with guinea-pig complement. Cell number and viability were
determined by trypan blue dye exclusion.
[0089] FIG. 16a. Correlation between TEC-11 binding and cellular
proliferation in HUVEC. HUVEC from sparse cultures (hatched
histogram) or post-confluent cultures (open histogram) were stained
with TEC-11 by indirect immunofluorescence. Also shown, confluent
HUVEC stained with negative control antibody MTSA (stippled
histogram). Endoglin.sup.lo and endoglin.sup.hi populations of
post-confluent HUVEC were separated on a FACStar Plus cell sorter
as indicated and subsequently analyzed for RNA and DNA content.
[0090] FIG. 16b. Lack of correlation between LM142 binding and
cellular proliferation in HUVEC. HUVEC from sparse (hatched
histogram) or post-confluent (open histogram) were stained with the
anti-vitronectin receptor antibody LM142. Also shown, sparse HUVEC
stained with negative control antibody MTSA (stippled
histogram).
[0091] FIG. 16c. Correlation between TEC-11 binding and cellular
proliferation in HUVEC. Endoglin.sup.lo HUVEC assayed for acridine
orange interrelation into cellular DNA (x-axes) and DNA (y-axes).
Essentially all cells contained low levels of RNA and DNA and were
located within the lower left (G.sub.0) quadrant.
[0092] FIG. 16d. Correlation between TEC-11 binding and cellular
proliferation in HUVEC. Endoglin.sup.hi HUVEC assayed for RNA and
DNA as described in the above legend. Significant numbers of cells
contain increased RNA levels (G.sub.1 phase, lower right quadrant)
and increased RNA and DNA levels (S+G.sub.2M phases, upper right
quadrant).
[0093] FIG. 17a. Immunohistochemical detection of TEC-4 binding to
malignant human parotid tumor tissue. Numerous blood vessels are
stained strongly by TEC-4 in a parotid tumor whereas only a single
stained vessel is present in the adjacent normal glandular tissue
(b,arrow). Antibody binding was detected with biotinylated
F(ab').sub.2 rabbit anti-mouse Ig and SABC-HRP with AEC substrate
and hematoxylin counterstaining; .times.40.
[0094] FIG. 17b. Immunohistochemical detection of TEC-4 binding to
normal human glandular tissue. Only a single vessel is stained by
TEC-4 in this normal glandular tissue (b,arrow). Antibody binding
was detected with biotinylated F(ab').sub.2 rabbit anti-mouse Ig
and SABC-HRP with AEC substrate and hematoxylin counterstaining;
.times.40.
[0095] FIG. 17c. Immunohistochemical detection of TEC-4 binding to
human breast carcinoma tissues. Under high power, endothelial cells
in a breast carcinoma are strongly stained with TEC-4 whereas
normal umbilical vein endothelial cells are weak-negative. Antibody
binding was detected with biotinylated F(ab').sub.2 rabbit
anti-mouse Ig and SABC-HRP with AEC substrate and hematoxylin
counterstaining; .times.64.
[0096] FIG. 17d. Immunohistochemical detection of TEC-4 binding to
normal umbilical vein endothelial cells. Antibody binding was
detected with biotinylated F(ab').sub.2 rabbit anti-mouse Ig and
SABC-HRP with AEC substrate and hematoxylin counterstaining;
.times.20.
[0097] FIG. 18a. Differential TEC-4 binding to endothelial cells in
normal breast tissue, control. Sections of normal mammary gland
were stained with a control anti-endothelial cell antibody, F8/86
(anti-von Willebrands Factor). Antibody binding was detected as in
the legend to FIG. 16 except that DAB substrate was used and a
light hematoxylin counterstaining was applied; .times.20.
[0098] FIG. 18b. Differential TEC-4 binding to endothelial cells in
normal breast tissue, TEC-4 control. Sections of normal mammary
gland were stained with TEC-4. Binding of TEC-4 to normal
endothelial cells is not seen. Antibody binding was detected as in
the legend to FIG. 16 except that DAB substrate was used and a
light hematoxylin counterstaining was applied; .times.20.
[0099] FIG. 18c. Differential TEC-4 binding to endothelial cells in
malignant breast tissue, control. Sections of breast carcinoma were
stained with a control anti-endothelial cell antibody, F8/86
(anti-von Willebrands Factor). Antibody binding was detected as in
the legend to FIG. 16 except that DAB substrate was used and a
light hematoxylin counterstaining was applied; .times.20.
[0100] FIG. 18d. Differential TEC-4 binding to endothelial cells in
malignant breast tissue, TEC-4. Sections of breast carcinoma were
stained with TEC-4. Binding of TEC-4 to endothelial cells is only
seen in the malignant breast sample. Antibody binding was detected
as in the legend to FIG. 16 except that DAB substrate was used and
a light hematoxylin counterstaining was applied; .times.20.
[0101] FIG. 19a. No significant killing of proliferating HUVEC by
MTSA-dgA. Quiescent (.circle-solid.), confluent (.box-solid.) or
subconfluent (.tangle-solidup.) HUVEC cultures were incubated for
48 hours with a negative control immunotoxin (MTSA-dgA). Protein
synthesis was estimated from the uptake of .sup.3H-leucine during
the last 24 hours of culture. Points and bars: mean and standard
error of 6 individual studies.
[0102] FIG. 19b. Significant killing of proliferating HUVEC by
UV-3-dgA. Quiescent (.circle-solid.), confluent (.box-solid.) or
subconfluent (.tangle-solidup.) HUVEC cultures were incubated for
48 hours with a positive control immunotoxin (UV-3-dgA). Protein
synthesis was estimated from the uptake of .sup.3H-leucine during
the last 24 hours of culture. Points and bars: mean and standard
error of 6 individual studies.
[0103] FIG. 19c. Significant killing of proliferating HUVEC by
ricin. Quiescent (.circle-solid.), confluent (.box-solid.) or
subconfluent (.tangle-solidup.) HUVEC cultures were incubated for
48 hours with ricin. Protein synthesis was estimated from the
uptake of .sup.3H-leucine during the last 24 hours of culture.
Points and bars: mean and standard error of 6 individual
studies.
[0104] FIG. 19d. Significant killing of proliferating HUVEC by
TEC-11dgA. Quiescent (.circle-solid.), confluent (.box-solid.) or
subconfluent (.tangle-solidup.) HUVEC cultures were incubated for
48 hours with TEC-11dgA. Protein synthesis was estimated from the
uptake of .sup.3H-leucine during the last 24 hours of culture.
Points and bars: mean and standard error of 6 individual
studies.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0105] Although they show great promise in the therapy of lymphomas
and leukemias (Lowder et al., 1987; Vitetta et al., 1991),
monoclonal antibodies (mAbs), immunotoxins (ITs) and other
immunoconjugates have thus far proved relatively ineffective in
clinical trials against carcinomas and other solid tumors (Byers
and Baldwin, 1988; Abrams and Oldham, 1985), which account for more
than 90% of all cancers in man (Shockley et al., 1991). A principal
reason for this is that macromolecules do not readily extravasate
into solid tumors (Sands, 1988; Epenetos et al., 1986) and, once
within the tumor mass, fail to distribute evenly due to the
presence of tight junctions between tumor cells (Dvorak et al.,
1991), fibrous stroma (Baxter and Jain, 1991), interstitial
pressure gradients (Jain, 1990) and binding site barriers (Juweid
et al., 1992).
[0106] A solution to the problem of poor penetration of antibodies
into solid tumors is to attack the endothelial cells (EC) lining
the blood vessels in the tumor. This approach offers several
advantages over direct targeting of tumor cells. Firstly, the
target cells are directly accessible to intravenously administered
therapeutic agents, permitting rapid localization of a high
percentage of the injected dose (Burrows et al., 1990; Kennel, et
al., 1991). Secondly, since each capillary provides oxygen and
nutrients for thousands of cells in its surrounding `cord` of
tumor, even limited damage to the tumor vasculature could produce
an avalanche of tumor cell death (Denekamp, 1984; 1986; 1990;
Burrows and Thorpe, 1993). The outgrowth of mutant endothelial
cells lacking the target antigen is unlikely because endothelial
cells are normal and not neoplastic cells. Finally, endothelial
cells are similar in different tumors, making it feasible to
develop a single reagent for treating numerous types of cancer.
[0107] For tumor vascular targeting to succeed, targeting agents,
such as antibodies, are required that recognize tumor endothelial
cells but not those in normal tissues. Differences between tumor
blood vessels and normal ones have been documented (reviewed in
Denekamp, 1990; Dvorak et al., 1991; and Jain, 1988) which
suggested to the inventors that antigenic differences might exist.
For example, tumors elaborate angiogenic factors (Kandel et al.,
1991; Folkman, 1985) and cytokines (Burrows et al., 1991; Ruco et
al., 1990; Borden et al., 1990) which alter the behavior and
phenotype of local endothelial cells. Vascular endothelial cells in
tumors proliferate at a rate 30-fold greater than those in
miscellaneous normal tissues (Denekamp et al., 1982), suggesting
that proliferation-linked determinants could serve as markers for
tumor vascular endothelial cells.
[0108] Nevertheless, despite fairly intensive efforts in several
laboratories (Duijvestijn et al., 1987, Hagemeier et al., 1986;
Schlingmann et al., 1985), antibodies have not yet been obtained
which clearly distinguish tumor from normal vasculature.
Migration-linked endothelial markers have been described, but as
yet none has been found to be reliably and selectively expressed in
the tumor vasculature (Gerlach et al., 1989; Hagemeier et al.,
1986; Sarma et al., 1992). For example, the antigen recognized by
the antibody termed EN7/44 (Hagemeier et al., 1986) appears to be
linked to migration rather than to proliferation, and, since it is
almost entirely cytoplasmically located, is not believed to be a
good candidate for tumor vasculature targeting.
[0109] VEGF receptors are known to be upregulated on tumor
endothelial cells as opposed to endothelial cells in normal
tissues, both in rodents and man. Possibly, this is a consequence
of hypoxia--a characteristic of the tumor microenvironment. FGI
receptors are also upregulated three-fold on endothelial cells
exposed to hypoxia, and so are probably upregulated in tumors.
[0110] Both VEGF and bFGF are concentrated in or on tumor
vasculature and potentially provide a target for attack on tumor
vasculature. TGF .beta. receptor (endoglin) on endothelial cells is
upregulated on dividing cells (as shown herein), explaining the
greater binding on TEC-11 to tumor vasculature where endothelial
cells are dividing.
[0111] Tumor endothelial markers could potentially be induced
directly by tumor-derived cytokines (Burrows et al., 1991; Ruco et
al., 1990) or angiogenic factors (Mignatti et al., 1991). In
support of the existence of tumor vasculature markers, two
antibodies against unrelated antigens of unknown function that are
expressed in the vasculature of human tumors but not in most normal
tissues have been described subsequent to the present invention
(Rettig et al., 1992; Wang et al., 1993).
[0112] The present inventors have developed a variety of strategies
for specifically targeting the targeting agents, such as
antibodies, to tumor vasculature, which strategies address the
shortcomings in the prior approaches. One strategy for vascular
targeting presented by the invention involves the use of a
targeting agent, such as an antibody directed against a tumor
vasculature-associated antigen, whether specifically bound to the
vasculature surface or expressed by an endothelial cell, to target
or deliver a selected therapeutic or diagnostic agent to the tumor.
In addition to an antibody, the targeting agent may be a growth
factor which specifically binds a growth factor receptor present on
the surface of a tumor-associated endothelial cell. A second
approach involves the selective induction of MHC Class II molecules
on the surfaces of tumor-associated endothelia which can then serve
as endothelial cell targets. A third, related but distinct approach
involves the selective elicitation of an endothelial marker in the
tumor vascular endothelium and the targeting of such an antigen
with an appropriate antibody. Naturally, the existence of
endothelial markers, such as ELAM-1, VCAM-1, ICAM-1 and the like,
has been documented, however exploiting such molecules by selective
induction and subsequent targeting has not been described
previously.
A. Identification of Existing Tumor Vasculature Markers
[0113] The present inventors developed a novel approach to identify
tumor vascular antigens which employs tumor-conditioned cell
culture media to induce specific antigens on vascular endothelial
cells. The conditioned media, which undoubtedly includes numerous
cytokines, growth factors and tumor-specific products, mimics the
solid tumor vascular environment and thereby promotes the
appearance of specific tumor vascular antigen markers. This
approach allows specific markers of tumor vasculature to be
identified and then targeted with a targeting agent, such as a
specific antibody, linked, or operatively attached to, a selected
therapeutic or diagnostic agent.
[0114] The generation of antibodies against specific markers of
tumor vasculature generally involves using the stimulated
endothelial cells as immunogens in an animal system. The methods of
generating polyclonal antibodies in this manner are well known, as
are the techniques of preparing monoclonal antibodies via standard
hybridoma technology. The inventors also contemplate the use of a
molecular cloning approach to generate monoclonals. For this,
combinatorial immunoglobulin phagemid libraries are prepared from
RNA isolated from the spleen of the immunized animal, and phagemids
expressing appropriate antibodies are selected by panning on
normal-versus-tumor endothelium. The advantages of this approach
over conventional hybridoma techniques are that approximately
10.sup.4 times as many antibodies can be produced and screened in a
single round, and that new specificities are generated by H and L
chain combination which further increases the chance of finding
appropriate antibodies.
[0115] In one of the studies disclosed herein, the inventors report
the development and characterization of two murine IgM monoclonal
antibodies, TEC-4 and TEC-11, which are envisioned to be suitable
for targeting the tumor vasculature of humans. TEC-4 and TEC-11
were initially believed to recognize an antigen that migrated as a
43 kD doublet on SDS/PAGE. However, as detailed herein, the present
inventors subsequently determined this antigen to be the molecule
endoglin, which is a dimeric glycoprotein consisting of 95 kDa
disulfide-linked subunits whose primary sequence is known (Gougos,
1990). Monoclonal antibodies have previously been raised against
endoglin (Gougos and Letarte, 1988; Gougos et al., 1992; O'Connel
et al., 1992; Buhring et al., 1991). However, TEC-4 and TEC-11 are
believed to recognize distinct epitopes, as shown, for example, by
the failure of 44G4 to block TEC-4 or TEC-11, even at high
ratios.
[0116] Endoglin is expressed on human endothelial cells, fetal
syncytiotrophoblast (Gougos et al., 1992), some macrophages
(O'Connell et al., 1992), immature erythroid cells (Buhring et al.,
1991), and some leukemic and hemopoietic cell lines (Gougos and
Letarte, 1988; Gougos et al., 1992; O'Connel et al., 1992; Buhring
et al., 1991). Its expression on dermal endothelium has recently
been demonstrated to be upregulated in several chronic inflammatory
skin lesions (Westphal et al., 1993).
[0117] Using the TEC-4 and TEC-11 antibodies, the inventors report
herein that endoglin is upregulated on activated and dividing HUVEC
in culture, and is strongly expressed in human tissues on
endothelial cells at sites of neovascularization, including a broad
range of solid tumors and fetal placenta. In contrast, endothelial
cells in the majority of miscellaneous non-malignant adult tissues,
including preneoplastic lesions, were only weakly positive or not
stained by TEC-4 and TEC-11. Importantly, TEC-4 and TEC-11 antibody
binding is also shown to correlate with neoplastic progression in
the breast: benign fibroadenomas, and early carcinoma-in-situ bound
low levels of TEC-4 and TEC-11 whereas late stage intraductal
carcinomas and invasive carcinomas bound high levels of the
antibodies.
[0118] HUVEC in sections of umbilical vein react weakly with TEC-4
and TEC-11, whereas proliferating HUVEC in tissue culture react
strongly and uniformly. HUVEC cultures grown to confluence and then
rested contain two subpopulations having high and low levels of
endoglin expression. Multiparameter analysis by FACS revealed that
a significant proportion of cells with high endoglin expression are
cycling, having markedly increased levels of cellular protein, RNA
and DNA by comparison with low endoglin-expressing cells, which
appear all to be non-cycling. Taken together, the increased binding
of TEC-4 and TEC-11 to tumor vasculature and to dividing as opposed
to non-cycling HUVEC in vitro indicates that endoglin is an
endothelial cell proliferation-associated marker. Anti-endoglin
antibodies are thus proposed to have broad-based applicability in
the diagnosis, imaging and therapy of solid tumors in man.
Furthermore, the uniformity of staining of vessels in different
tumors and within any individual tumor indicates that TEC-4 and
TEC-11 compare favorably with various other antibodies, such as,
e.g., FB-5 (Rettig et al., 1992) and E9 (Wang et al., 1993).
[0119] An additional concept of one of the present inventors is the
use of "tumor-derived endothelial cell binding factors" as a means
of distinguishing between tumor vasculature and the vasculature of
normal tissues. That is, if tumors secrete factors for which
vascular endothelial cells have receptors, the endothelial cells in
the tumor will capture that factor and display it on their surface.
In contrast, endothelial cells in normal tissues will bind
relatively little of the factor because it is diluted within the
blood pool or because the receptors on normal endothelial cells are
not upregulated as they are on tumor endothelial cells. Thus,
operationally, the tumor-derived factor will also constitute a
tumor endothelial cell marker.
[0120] Such tumor-derived endothelial cell binding factors can be
manufactured by the tumor cells themselves, by cells (e.g.
macrophages, mast cells) which have infiltrated tumors or by
platelets which become activated within the tumor. It is proposed
that an antibody or other ligand which recognizes that factor will
home selectively to tumor vasculature after injection. Such an
antibody or ligand should thus enable the imaging or targeting of
drugs or other agents to solid tumors. Further, such an antibody
may be specific for a factor/factor receptor complex present on the
surface of the tumor vasculature, so that the antibody recognizes
only a factor/factor receptor complex, while not binding to either
the factor or the factor receptor individually.
[0121] Various candidate factors include, for example, vascular
endothelial cell growth factor (VEGF), also called vascular
permeability factor (VPF); members of the fibroblast growth factor
(FGF) family, e.g., basic FGF; Tumor necrosis factor-.alpha.
(TNF-.alpha.); transforming growth factor-.alpha. (TGF-.alpha.) and
transforming growth factor-.gamma. (TGF-.gamma.); angiogenic;
angiotropin; and platelet-derived endothelial cell growth factor
(PD-ECGF).
B. Selective Induction of Other Tumor Vasculature Markers
[0122] Another approach to targeting tumor vasculature involves the
selective induction of a molecule that is capable of acting as a
marker for subsequent tumor endothelial cell targeting. Within this
general strategy, the inventors have focused on both the induction
of MHC Class II molecules and the induction of endothelial cell
adhesion molecules. The MHC Class II approach, however, requires
that MHC Class II expression be effectively inhibited in normal
tissues. It is known that CsA and related immunosuppressants have
this capability via inhibition of T cell activation, and can
therefore be employed to pretreat the patient or animal to inhibit
Class II expression. Alternatively, it is proposed that inhibition
of Class II expression can be achieved using anti-CD4 in that CD4
directed antibodies are known to additionally suppress T cell
function (Street et al., 1989). Then, Class II targets are
selectively induced in the tumor-associated vascular endothelium
through a locally released cytokine intermediate (IFN-.gamma.).
[0123] To use the related approach of selectively eliciting an
endothelial marker in tumor vascular endothelium, one may exploit
one or more of the various endothelial adhesion molecules. The
expression of an endothelial adhesion molecule, such as ELAM-1,
VCAM-1, ICAM-1, LAM-1 ligand etc., may thus be selectively induced
and then targeted with an appropriate antibody. Of these, ELAM-1 is
the preferred target in that it is quite clear that this antigen is
not expressed in normal endothelial vasculature (Cotran et al.,
1986). The other adhesion molecules appear to be expressed to
varying degrees in other normal tissues, generally in lymphoid
organs and on endothelium, making their targeting perhaps
appropriate only in diagnostic embodiments.
[0124] In either case, the key is the use of a bispecific
"cytokine-inducing" antibody that will selectively induce the
release of the appropriate cytokine in the locale of the tumor.
This specifically localized release of cytokine is achieved through
a bispecific antibody having the ability to "cross-link"
cytokine-producing leukocytes to cells of the tumor mass. The
preparation and use of bispecific antibodies such as these is
predicated in part on the fact that cross-linking antibodies
recognizing CD3, CD14, CD16 and CD28 have previously been shown to
elicit cytokine production selectively upon cross-linking with the
second antigen (Qian et al., 1991). In the context of the present
invention, since only successfully tumor cell-crosslinked
leukocytes will be activated to release the cytokine, cytokine
release will be restricted to the locale of the tumor. Thus,
expression of ELAM-1 will be similarly limited to the endothelium
of the tumor vasculature.
[0125] An overview of various exemplary inducible vascular
endothelial targets, as well as the mechanisms for their induction,
is set forth in Table I. This Table lists various potential
endothelial cell targets, such as ELAM-1, VCAM-1, etc., the
inducing intermediate cytokine, such as IL-1, IFN-.gamma., etc.,
and the leukocyte cell type and associated cytokine activating
molecule whose targeting will result in the release of the
cytokine. Thus, for example, a bispecific antibody targeted to an
appropriate solid tumor antigen and CD14, will promote the release
of IL-1 by tumor-localized monocytes and macrophages, resulting in
the selective expression of the various adhesion molecules in the
tumor vascular endothelium. Alternatively, the bispecific antibody
may be targeted to FcR for IgE, FcR for IgG (CD16), CD2, CD3, or
CD28, and achieve a similar result, with the cytokine intermediate
and cytokine-producing leukocyte being different or the same.
TABLE-US-00001 TABLE I POSSIBLE INDUCIBLE VASCULAR TARGETS
LEUKOCYTE MOLECULES WHICH, WHEN CROSSLINKED INDUCIBLE LEUKOCYTES
WHICH BY MONOCLONAL ANTIBODIES ENDOTHELIAL CELL SUBTYPES/ALIASES
INDUCING PRODUCE THOSE ACTIVATE THE CELLS TO MOLECULES ACRONYM
(MOLECULAR FAMILY) CYTOKINES CYTOKINS PRODUCE CYTOKINES
Endothelial- ELAM-1 -- IL-1, TNF-.alpha., monocytes CD14 Leukocyte
(Selectin) (TNF-.beta.) macrophages CD14 Adhesion Molecule-1
(Bacterial mast cells FcR for IgE Endotoxin) Vascular Cell VCAM-1
Inducible Cell (Bacterial monocytes CD14 Adhesion Molecule-1
Adhesion Molecule-110 Endotoxin) macrophages CD14 (INCAM-110) IL-1,
TNF-.alpha. mast cells FcR for IgE (Immunoglobulin TNF-.beta., IL-4
helper T cells CD2, CD2, CD28 Family) TNF NK cells FcR for IgG
(CD16) Intercellular ICAM-1 -- IL-1, TNF.alpha. monocytes CD14
Adhesion Molecule-1 (Immunoglobulin (Bacterial macrophages CD15
Family) Endotoxin) mast cells FcR for IgE TNF-.beta., IFN.gamma. T
helper cells CD2, CD3, CD28 NK cells FcR for IgG (CD16) The Agent
for LAM-1 MEL-14 Agent (Mouse) Il-1, TNF.alpha. monocytes CD14
Leukocyte Adhesion Agent (Bacterial Molecule-1 Endotoxin)
macrophages CD14 mast cells FcR for IgE Major MHC Class HLA-DR
IFN-.gamma. helper T cells CD2, CD3, CD28 Histocompatability II
HLA-DP - Human Complex Class II HLA-DQ Antigen I-A - Mouse I-E NK
cells FcR for IgG (CD16)
[0126] As pointed out, the distinction between the selective
activation of ELAM-1 and the MHC Class II molecules rests on the
fact that ELAM-1 is not normally expressed in normal epithelium,
whereas Class II molecules are normally expressed in normal
endothelium. Thus, when one seeks to target MHC Class II antigens,
it will be important to first inhibit their expression in normal
tissues using CsA or a similar immunosuppressant agent having the
ability to suppress MHC Class II expression. Then, MHC Class II
molecules can be selectively induced in the tumor vasculature
using, e.g., a bispecific antibody against a solid tumor antigen
that activates T.sub.h1 cells in the tumor in a CsA-independent
fashion, such as CD28. Such an antibody will trigger the release of
IFN-.gamma. which, in turn, will result in the selective expression
of Class II molecules in the tumor vasculature.
[0127] An alternative approach that avoids both the use of CsA and
a bispecific activating antibody involves the use of anti-CD4 to
suppress IFN-.gamma. production, followed by introduction of an
IFN-.gamma.-producing T-cell clone (e.g., T.sub.h1 or cytotoxic
T-lymphocytes (CTLs)) that is specific for a selected tumor
antigen. In this embodiment, the T-cell clone itself localizes to
the tumor mass due to its antigen recognition capability, and only
upon such recognition will the T-cell clone release IFN-.gamma.. In
this manner, cytokine release is again restricted to the tumor,
thus limiting the expression of Class II molecules to the tumor
vasculature.
[0128] T lymphocytes from the peripheral blood (Mazzocchi et al.,
1990) or within the tumor mass (Fox et al., 1990) will be isolated
by collagenase digestion where necessary, and density gradient
centrifugation followed by depletion of other leukocyte subsets by
treatment with specific antibodies and complement. In addition,
CD4.sup.+ or CD8.sup.+ T cell subsets may be further isolated by
treatment with anti-CD8 or anti-CD4 and complement, respectively.
The remaining cells will be plated at limiting dilution numbers in
96-well (round bottom) plates, in the presence of 2.times.10.sup.5
irradiated (2500 rad) tumor cells per well. Irradiated syngeneic
lymphocytes (2.times.10.sup.5 per well) and interleukin-2 (10 U/ml)
will also be included. Generally, clones can be identified after 14
days of in vitro culture. The cytokine secretion pattern of each
individual clone will be determined every 14 days. To this end,
rested clones will be mitogenically or antigenically-stimulated for
24 hours and their culture supernatants assayed for the presence of
IL-2, IFN-.gamma., IL-4, IL-5 and IL-10. Those clones secreting
high levels of IL-2 and IFN-.gamma., the characteristic cytokine
secretion pattern of T.sup.H1 clones, will be selected. Tumor
specificity will be confirmed utilizing proliferation assays.
[0129] Supernatants obtained after 24 hour mitogen or
antigen-stimulation will be analyzed in the following cytokine
assays: IL-2, IFN-.gamma., IL-4, IL-5 and IL-10. The levels of IL-2
and IL-4 will be assayed using the HT-2 bioassay in the presence of
either anti-IL-2, anti-IL-4 antibodies or both. The remaining
cytokines will be assayed using specific two-site sandwich ELISAs
(Cherwinski et al., 1989). Cytokines in the unknown samples will be
quantitated by comparison with standard curves, by using either
linear or four-parameter curve-fitting programs.
[0130] A few generalizations can be made as to which approach would
be the more appropriate for a given solid tumor type. Generally
speaking, the more "immunogenic" tumors would be more suitable for
the MHC Class II approach involving, e.g., the cross-linking of
T-cells in the tumor through an anti-CD28/anti-tumor bispecific
antibody, because these tumors are more likely to be infiltrated by
T cells, a prerequisite. Examples of immunogenic solid tumors
include renal carcinomas, melanomas, a minority of breast and colon
cancers, as well as possibly pancreatic, gastric, liver, lung and
glial tumor cancers. These tumors are referred to as "immunogenic"
because there is evidence that they elicit immune responses in the
host and they have been found to be amenable to cellular
immunotherapy (Yamaue et al., 1990). In the case of melanomas and
large bowel cancers, the most preferred antibodies for use in these
instances would be B72.3 (anti-TAG-72) and PRSC5/PR4C2 (anti-Lewis
a) or 9.2.27 (anti-high Mr melanoma antigen).
[0131] For the majority of solid tumors of all origins, an
anti-CD14 approach that employs a macrophage/monocyte intermediate
would be more suitable. This is because most tumors are rich in
macrophages. Examples of macrophage-rich tumors include most
breast, colon and lung carcinomas. Examples of preferred anti-tumor
antibodies for use in these instances would be anti-HER-2, B72.3,
SM-3, HMFG-2, and SWA11 (Smith et al., 1989).
[0132] The inventors have recently developed a model system in the
mouse in which to demonstrate and investigate immunotoxin-mediated
targeting of vascular endothelial cells in solid tumors. A
neuroblastoma transfected with the murine interferon-.gamma.
(IFN-.gamma.) is grown in SCID or BALB/c nude mice reared under
germ-free conditions. The IFN-.gamma. secreted by the tumor cells
induces the expression of MHC Class II antigens on the vascular
endothelial cells in the tumor. Class II antigens are absent from
the vasculature of normal tissues in germ-free SCID and nude mice
although they are present on certain non-life-sustaining normal
cells (such as on B lymphocytes and monocytes) and some epithelial
cells.
[0133] When mice with large (1.2 cm diameter) tumors were injected
with anti-Class II-ricin A chain immunotoxin, dramatic anti-tumor
effects were observed. Histological examination of tumors taken
from mice at various times after injecting the immunotoxin revealed
that vascular endothelial cell degeneration was the first
discernable event followed by platelet deposition on the injured
vessels and coagulation of the tumor blood supply. This was
followed by extensive tumor cell degeneration which occurred within
24 hours after injection of the immunotoxin. By 72 hours, no viable
tumor cells remained apart from a few cells on the edge of the
tumor where it penetrated into the normal tissues. These surviving
tumor cells could be killed by administering an immunotoxin
directed against the tumor cells themselves, resulting in lasting
complete tumor regressions in a third of the animals.
[0134] These background studies have demonstrated the feasibility
of targeting tumor vasculature through targeting of MHC Class II or
adhesion molecules such as ELAM-1.
C. MHC Class II
[0135] Class II antigens are expressed on vascular endothelial
cells in most normal tissues in several species, including man.
Studies in vitro (Collins et al., 1984; Daar et al., 1984;
O'Connell et al., 1990) and in vivo (Groenewegen et al., 1985) have
shown that the expression of Class II antigens by vascular
endothelial cells requires the continuous presence of IFN-.gamma.
which is elaborated by T.sub.H1 cells and, to a lesser extent, by
NK cells and CD8.sup.+ T cells. As shown in the dog (Groenewegen et
al., 1985) and as confirmed by the inventors in normal mice, Class
II expression through the vasculature is abolished when CsA is
administered. The CsA acts by preventing the activation of T cells
and NK cells (Groenewegen et al., 1985; DeFranco, 1991), thereby
reducing the basal levels of IFN-.gamma. below those needed to
maintain Class II expression on endothelium.
[0136] A strategy for confining Class II expression to tumor
vasculature is to suppress IFN-.gamma. production through out the
animal by administering CsA and then to induce IFN-.gamma.
production specifically in the tumor by targeting a CsA-resistant T
cell activator to the tumor. A bispecific (Fab'-Fab') antibody
having one arm directed against a tumor antigen and the other arm
directed against CD28 should localize in the tumor and then
crosslink CD28 antigens on T cells in the tumor. Crosslinking of
CD28, combined with a second signal (provided, for example, by IL-1
which is commonly secreted by tumor cells (Burrows et al., 1991;
Ruco et al., 1990) has been shown to activate T cells through a
CA.sup.2+-independent non-CsA-inhibitable pathway (Hess et al.,
1991; June et al., 1987; Bjorndahl et al., 1989). The T cells that
should be activated in the tumor are those adjacent to the
vasculature since this is the region most accessible to cells and
is also where the bispecific antibody will be most concentrated.
The activated T cells should then secrete IFN-.gamma. which induces
Class II antigens on the adjacent tumor vasculature.
[0137] MHC Class II antigens are not unique to vascular endothelial
cells. They are expressed constitutively on B cells, activated T
cells, cells of monocyte/macrophage linage and on certain
epithelial cells both in mice (Hammerling, 1976) and in man (Daar
et al., 1984). It would therefore be anticipated that damage to
these normal tissues would result if anti-Class II immunotoxin were
to be administered. However this presumption is not correct, at
least in mice. Anti-Class II immunotoxins administered
intravenously to germ-free SCID or BALB/c nude mice are no more
toxic to the mice than are immunotoxins having no reactivity with
mouse tissues. There are a number of possible explanations for this
surprising result. First, anti-Class II antibodies injected
intravenously do not appear to reach the epithelial cells or the
monocytes/macrophages in organs other than the liver and spleen.
Presumably this is because the vascular endothelium in most organs
is tight, not fenestrated as it is in the liver and spleen, and so
the antibodies must diffuse across basement membranes to reach the
Class Ii-positive cells.
[0138] Secondly, hepatic Kupffer cells and probably other cells of
monocyte/macrophage lineage are not killed by the anti-Class II
immunotoxin even though it binds to them. No morphological changes
in the Kupffer cells are visible even several days after
administration of the immunotoxin. This is probably because cells
of monocyte/macrophage linage are generally resistant to
immunotoxin-mediated killing (Engert et al., 1991). Cells of
monocyte/macrophage lineage appear to bind and internalize
immunotoxins but route them to the lysosomes where they are
destroyed, unlike other cell types which route immunotoxins to the
trans-Golgi region or the E.R. which are thought to be site(s) from
which ricin A chain enters the cytosol (Van Deurs et al., 1986; Van
Deurs et al., 1988).
[0139] Finally, there were little morphological evidence of splenic
damage despite the fact that the immunotoxin bound to the B cells
and that the cells are sensitive to anti-Class II immunotoxins
(Lowe et al., 1986; Wu et al., 1990). It is possible that the B
cells were killed, but, being metabolically inactive, they
degenerated very slowly. In any event, B cell elimination is
unlikely to be a significant problem in mice or in man because the
cells would be replenished from Class II negative progenitors (Lowe
et al., 1986); indeed, in B lymphoma patients and normal monkeys
treated with anti-B cell immunotoxins, B cell killing definitely
occurs but causes no obvious harm (Vitetta et al., 1991).
D. ELAM-1
[0140] In contrast to Class II, ELAM-1 is not found on the
vasculature of normal tissues in humans and is absent from any
other cell types (Cotran et al., 1986). It is induced on vascular
endothelial cells by IL-1 and TNV but not by IFN-.gamma. (Wu et
al., 1990). Its induction is rapid, peaking at 4-6 hours and,
thereafter, it is rapidly lost, being hardly detectable by 24 hours
(Bevilacqua et al., 1987).
[0141] With ELAM-1, the strategy is to induce its expression
selectively on tumor vasculature using a bispecific antibody that
will home to the tumor and activate monocytes/macrophages within
the tumor. The bispecific antibody will have one Fab' arm directed
against a tumor antigen and the other directed against CD14 (the
LPS receptor). After localizing in the tumor, the bispecific
antibody should crosslink CD14 receptors on monocytes and the
macrophages within the tumor. This should result in powerful
activation of these cells (Schutt et al., 1988; Chen et al., 1990)
and the production of IL-1 and TNF which will induce ELAM-1 on
tumor vascular endothelial cells.
E. Preparation of Targeting Agent Antibodies
[0142] The origin or derivation of the targeting agent antibody or
antibody fragment (e.g., Fab', Fab or F(ab').sub.2) is not believed
to be particularly crucial to the practice of the invention, so
long as the antibody or fragment that is actually employed for the
preparation of bispecific antibodies otherwise exhibit the desired
activating or binding properties. Thus, where monoclonal antibodies
are employed, they may be of human, murine, monkey, rat, hamster,
chicken or even rabbit origin. The invention contemplates that the
use of human antibodies, "humanized` or chimeric antibodies from
mouse, rat, or other species, bearing human constant and/or
variable region domains, single domain antibodies (e.g., DABs), Fv
domains, as well as recombinant antibodies and fragments thereof.
Of course, due to the ease of preparation and ready availability of
reagents, murine monoclonal antibodies will typically be
preferred.
[0143] In general, the preparation of bispecific antibodies is also
well known in the art, as exemplified by Glennie et al. (1987), as
is their use in the activation of leukocytes to release cytokines
(Qian et al., 1991). Bispecific antibodies have even been employed
clinically, for example, to treat cancer patients (Bauer et al.,
1991). Generally speaking, in the context of the present invention
the most preferred method for their preparation involves the
separate preparation of antibodies having specificity for the
targeted tumor cell antigen, on the one hand, and the targeted
activating molecule on the other. While numerous methods are known
in the art for the preparation of bispecific antibodies, the
Glennie et al. method preferred by the inventors involves the
preparation of peptic F(ab'.gamma.).sub.2 fragments from the two
chosen antibodies (e.g., an antitumor antibody.and an anti-CD14 or
anti-CD28 antibody), followed by reduction of each to provide
separate Fab'.gamma.SH fragments. The SH groups on one of the two
partners to be coupled are then alkylated with a cross-linking
reagent such as o-phenylenedimaleimide to provide free maleimide
groups on one partner. This partner may then be conjugated to the
other by means of a thioether linkage, to give the desired
F(ab'.gamma.).sub.2 heteroconjugate.
[0144] While, due to ease of preparation and high yield and
reproducibility, the Glennie et al. (1987) method is preferred for
the preparation of bispecific antibodies, there are of course
numerous other approaches that can be employed and that are
envisioned by the inventors. For example, other techniques are
known wherein crosslinking with SPDP or protein A is carried out,
or a trispecific construct is prepared (Titus et al., 1987; Tutt et
al., 1991). Furthermore, recombinant technology is now available
for the preparation of antibodies in general, allowing the
preparation of recombinant antibody genes encoding an antibody
having the desired dual specificity (Van Duk et al., 1989). Thus,
after selecting the monoclonal antibodies having the most preferred
binding and activation characteristics, the respective genes for
these antibodies can be isolated, e.g., by immunological screening
of a phage expression library (Oi & Morrison, 1986; Winter
& Milstein, 1991). Then, through rearrangement of Fab coding
domains, the appropriate chimeric construct can be readily
obtained.
[0145] The preparation of starting antibodies against the various
cytokine activating molecules is also well known in the art. For
example, the preparation and use of anti-CD14 and anti-CD28
monoclonal antibodies having the ability to induce cytokine
production by leukocytes has now been described by several
laboratories (reviewed in Schutt et al., 1988; Chen et al., 1990,
and June et al., 1990, respectively). Moreover, the preparation of
monoclonal antibodies that will stimulate leukocyte release of
cytokines through other mechanisms and other activating antigens is
also known (Clark et al., 1986; Geppert et al., 1990).
[0146] Similarly, there is a very broad array of antibodies known
in the art that have immunological specificity for the cell surface
of virtually any solid tumor type, as a vast number of solid tumor
assorted antigens have been identified (see, e.g., Table II).
Methods for the development of antibodies that are
"custom-tailored" to the patient's tumor are likewise known
(Stevenson et al., 1990). Of course, not all antibodies will have
sufficient selectivity, specificity, affinity and toxin-delivering
capability to be of use in the practice of the invention. These
properties can be readily evaluated using conventional
immunological screening methodology. TABLE-US-00002 TABLE II MARKER
ANTIGENS OF SOLID TUMORS AND CORRESPONDING MONOCLONAL ANTIBODIES
Antigen Identity/ Monoclonal Tumor Site Characteristics Antibodies
Reference A: Gynecological `CA 125` >200 kD OC 125 Kabawat et
al., Int. J. Gynecol. Pathol, mucin GP 4: 265, 1983; Szymendera,
Tumour Biology, 7: 333, 1986 GY ovarian 80 Kd GP OC 133 Masuko et
al, Cancer Res., 44: 2813, 1984 ovarian `SGA` 360 Kd GP OMI de
Krester et al., Int. J. Cancer, 37: 705, 1986 ovarian High M.sub.r
mucin Mo v1 Miotti et al, Cancer Res., 65: 826, 1985 ovarian High
M.sub.r mucin/ Mo v2 Miotti et al, Cancer Res., 65: 826, 1985
glycolipid ovarian NS 3C2 Tsuji et al., Cancer Res., 45: 2358, 1985
ovarian NS 4C7 Tsuji et al., Cancer Res., 45: 2358, 1985 ovarian
High M.sub.r mucin ID.sub.3 Gangopadhyay et al., Cancer Res., 45:
1744, 1985 ovarian High M.sub.r mucin DU-PAN-2 Lan et al, Cancer
Res., 45: 305, 1985 GY 7700 Kd GP F 36/22 Croghan et al., Cancer
Res., 44: 1954, 1984 ovarian `gp 68` 48 Kd 4F.sub.7/7A.sub.10
Bhattacharya et al., Cancer Res., 44: 4528, GP 1984 GY 40, 42 kD GP
OV-TL3 Poels et al., J. Natl. Cancer, 76: 781, 1986 GY `TAG-72`
High B72.3 Thor et al., Cancer Res., 46: 3118, 1986 M.sub.r mucin
ovarian 300-400 Kd GP DF.sub.3 Kufe et al., Hybridoma, 3: 223, 1984
ovarian 60 Kd GP 2C.sub.8/2F.sub.7 Bhattacharya et al., Hybridoma,
4: 153, 1985 GY 105 Kd GP MF 116 Mattes et al., PNAS, 81: 568, 1984
ovarian 38-40 kD GP MOv18 Miotti et al., Int. J. Cancer 39: 297,
1987 GY `CEA` 180 Kd GP CEA 11-H5 Wagener et al., Int. J. Cancer,
33: 469, 1984 ovarian CA 19-9 or GICA CA 19-9 (1116NS 19- Atkinson
et al., Cancer Res., 62: 6820, 1982 9) ovarian `PLAP` 67 Kd GP
H17-E2 McDicken et al., Br. J. Cancer, 52: 59, 1985 ovarian 72 Kd
791T/36 Perkins et al., Eur. J. Nucl. Med., 10: 296, 1985 ovarian
69 Kd PLAP NDOG.sub.2 Sunderland et al., Cancer Res., 44: 4496,
1984 ovarian unknown M.sub.r PLAP H317 Johnson et al., Am. J.
Reprod. Immunol., 1: 246, 1981 ovarian p185.sup.HER2 4D5, 3H4, 7C2,
6E9, Shepard et al., J. Clin. Immunol., 11(3): 117, 2C4, 7F3, 2H11,
1991 3E8, 5B8, 7D3, SB8 uterus ovary HMFG-2 HMFG2 Epenetos et al.,
Lancet, Nov. 6, 1000-1004, 1982 GY HMFG-2 3.14.A3 Burchell et al.,
J. Immunol., 131: 508, 1983 B: BREAST 330-450 Kd GP DF3 Hayes et
al., J. Clin. Invest., 75: 1671, 1985 NS NCRC-11 Ellis et al.,
Histopathol., 8: 501, 1984 37 kD 3C6F9 Mandeville et al., Cancer
Detect. Prev., 10: 89, 1987 NS MBE6 Teramoto et al., Cancer, 50:
241, 1982 NS CLNH5 Glassy et al., PNAS, 80: 63227, 1983 47 Kd GP
MAC 40/43 Kjeldsen et al, 2nd Int. Wkshop of MAbs & Breast
Cancer, San Fran., Nov. 1986 High M.sub.r GP EMA Sloane et al.,
Cancer, 17: 1786, 1981 High M.sub.r GP HMFG1 HFMG2 Arklie et al.,
Int. J. Cancer, 28: 23, 1981 NS 3.15.C3 Arklie et al., Int. J.
Cancer, 28: 23, 1981 NS M3, M8, M24 Foster et al., Virchows Arch.
(Pathol. Anat. Histopathol.), 394: 295, 1982 1 (Ma) blood M18
Foster et al., HumanPathol., 15: 502, 1984 group Ags NS 67-D-11
Rasmussen et al., Breast Cancer Res. Treat., 2: 401, 1982 oestrogen
D547Sp, D75P3, H222 Kinsel et al., Cancer Res., 49: 1052, 1989
receptor EGF Receptor Anti-EGF Sainsbury et al, Lancet, 1: 364,
1985 Laminin LR-3 Horan Hand et al., Cancer Res., 45: 2713, 1985
Receptor erb B-2 p185 TA1 Gusterson et al., Br. J. Cancer, 58: 453,
1988 NS H59 Hendler et al., Trans. Assoc. Am. Physicians, 94: 217,
1981 126 Kd GP 10-3D-2 Soule et al., PNAS, 80: 1332, 1983 NS
HmAB1,2 Imam et al., cited in Schlom et al., Adv. Cancer Res., 43:
143, 1985 NS MBR 1, 2, 3 Menard et al., Cancer Res., 63: 1295, 1983
95 Kd 24.17.1 Thompson et al., J. Natl. Cancer Inst., 70: 409, 1983
100 Kd 24.17.2 (3E1.2) Croghan et al., Cancer Res., 43: 4980, 1983
NS F36/22.M7/105 Croghan et al., Cancer Res., 44: 1954, 1984 24 Kd
C11, G3, H7 Adams et al., Cancer Res., 43: 6297, 1983 90 Kd GP B6.2
Colcher et al., PNAS, 78: 3199, 1981 CEA & 180 Kd GP B1.1
Colcher et al., Cancer Invest., 1: 127, 1983 colonic & Cam 17.1
Imperial Cancer Research Technology MAb pancreatic listing mucin
similar to Ca 19-9 milk mucin core SM3 Imperial Cancer Research
Technology Mab protein listing milk mucin core SM4 Imperial Cancer
Research Technology Mab protein listing affinity- C-Mul (566)
Imperial Cancer Research Technology Mab purified milk listing mucin
p185.sup.HER2 4D5 3H4, 7C2, 6E9, Shepard et al., J. Clin. Immunol.,
11(3): 117, 2C4, 7F3, 2H11, 1991 3E8, 5B8, 7D3, 5B8 CA 125 >200
Kd OC 125 Kabawat et al., Int. J. Gynecol. Pathol., GP 4: 245, 1985
High M.sub.r mucin/ MO v2 Miotti et al., Cancer Res., 45: 826, 1985
glycolipid High M.sub.r mucin DU-PAN-2 Lan et al., Cancer Res., 44:
1954, 1984 `gp48` 48 Kd GP 4F.sub.7/7A.sub.10 Bhattacharya et al.,
Cancer Res., 44: 4528, 1984 300-400 Kd GP DF.sub.3 Kufe et al.,
Hybridoma, 3: 223, 1984 `TAG-72` high B72.3 Thor et al., Cancer
Res., 46: 3118, 1986 M.sub.r mucin `CEA` 180 Kd GP cccccCEA 11
Wagener et al., Int. J. Cancer, 33: 469, 1984 `PLAP` 67 Kd GP
H17-E2 McDicken et al., Br. J. Cancer, 52: 59, 1985 HMFG-2 >400
Kd 3.14.A3 Burchell et al., J. Immunol., 131: 508, 1983 GP NS
FO23C5 Riva et al., Int. J. Cancer, 2: 114, 1988 (Suppl.) C:
COLORECTAL TAG-72 High M.sub.r B72.3 Colcher et al., Cancer Res.,
47: 1185 & 4218, mucin 1987 GP37 (17-1A) 1083-17-1A Paul et
al., Hybridoma, 5: 171, 1986 Surface GP CO17-1A LoBugilo et al.,
JNCl, 80: 932, 1988 CEA ZCE-025 Patt et al., Cancer Bull., 40: 218,
1988 CEA AB2 Griffin et al., Proc. 2nd Conf. on
Radioimmunodetection & Therapy of Cancer, 82, 1988 cell surface
AG HT-29-15 Cohn et al., Arch. Surg. 122: 1425, 1987 secretory
250-30.6 Leydem et al., Cancer, 57: 1135, 1986 epithelium surface
44X14 Gallagher et al., J. Surg. Res., 40: 159, 1986 glycoprotein
NS A7 Takahashi et al., Cancer, 61: 881, 1988 NS GA73.3 Munz et
al., J. Nucl, Med., 27: 1739, 1986 NS 791T/36 Farrans et al.,
Lancet, 2: 397, 1982 cell membrane & 28A32 Smith et al., Proc.
Am. Soc. Clin. O. col., cytoplasmic Ag 6: 250, 1987 CEA &
vindesine 28.19.8 Corvalen, Cancer Immuno., 24: 133, 1987 gp72 X
MMCO-791 Byers et al., 2nd Int. Conf. Mab Immunocon. Cancer, 41:
1987 high M.sub.r mucin DU-PAN-2 Lan et al., Cancer Res., 45: 305,
1985 high M.sub.r mucin ID.sub.3 Gangopadhyay et al., Cancer Res.,
45: 1744, 1985 CEA 180 Kd GP CEA 11-H5 Wagener et al., Int. J.
Cancer, 33: 469, 1984 60 Kd GP 2C.sub.8/2F.sub.7 Bhattacharya et
al., Hybridoma, 4: 153, 1985 CA-19-9 (or CA-19-9 (1116NS 19-
Atkinson et al., Cancer Res., 62: 6820, 1982 GICA) 9) Lewis a PR5C5
Imperial Cancer Research Technology Mab Listing Lewis a PR4D2
Imperial Cancer Research Technology Mab Listing colonic mucus PR4D1
Imperial Cancer Research Technology Mab Listing D: MELANOMA
p97.sup.a 4.1 Woodbury et al., PNAS, 77: 2183, 1980 p97.sup.a 8.2
M.sub.17 Brown, et al., PNAS, 78: 539, 1981 p97.sup.b 96.5 Brown,
et al., PNAS, 78: 539, 1981 p97.sup.c 118.1, 133.2, (113.2) Brown,
et al., PNAS, 78: 539, 1981 p97.sup.c L.sub.1, L.sub.10,
R.sub.10(R.sub.19) Brown et al., J. Immunol., 127: 539, 1981
p97.sup.d I.sub.12 Brown et al., J. Immunol., 127: 539, 1981
p97.sup.e K.sub.5 Brown et al., J. Immunol., 127: 539, 1981 p155
6.1 Loop et al., Int. J. Cancer, 27: 775, 1981 G.sub.D3 disialogan-
R24 Dippold et al., PNAS, 77: 6115, 1980 glioside p210, p60, p250
5.1 Loop et al., Int. J. Cancer, 27: 775, 1981 p280 p440 225.28S
Wilson et al., Int. J. Cancer, 28: 293, 1981 GP 94, 75, 70 &
465.12S Wilson et al., Int. J. Cancer, 28: 293, 1981 25 P240-P250,
P450 9.2.27 Reisfeld et al., Melanoma Ags & Abs, 1982 pp. 317-
100, 77, 75 Kd F11 Chee et al., Cancer Res., 42: 3142, 1982 94 Kd
376.96S Imai et al., JNCI, 68: 761, 1982 4 GP chains 465.12S Imai
et al., JNCI, 68: 761, 1982; Wilson et al., Int. J. Cancer, 28:
293, 1981 GP 74 15.75 Johnson & Reithmuller, Hybridoma, 1: 381,
1982 GP 49 15.95 Johnson & Reithmuller, Hybridoma, 1: 381, 1982
230 Kd Mel-14 Carrel et al., Hybridoma, 1: 387, 1982 92 Kd Mel-12
Carrel et al., Hybridoma, 1: 387, 1982 70 Kd Me3-TB7 Carrel et al.,
Hybridoma, 1: 387, 1982 HMW MAA similar 225.28SD Kantor et al.,
Hybridoma, 1: 473, 1982 to 9.2.27 AG HMW MAA similar 763.24TS
Kantor et al., Hybridoma, 1: 473, 1982 to 9.2.27 AG GP95 similar to
705F6 Stuhlmiller et al., Hybridoma, 1: 447, 1982 376.96S 465.12S
GP125 436910 Saxton et al., Hybridoma, 1: 433, 1982 CD41 M148
Imperial Cancer Research Technology Mab listing E: high M.sub.r
mucin ID3 Gangopadhyay et al., Cancer Res., 45: 1744,
GASTROINTESTINAL 1985 pancreas, stomach gall bladder, high M.sub.r
mucin DU-PAN-2 Lan et al., Cancer Res., 45: 305, 1985 pancreas,
stomach pancreas NS OV-TL3 Poels et al., J. Natl. Cancer Res., 44:
4528, 1984 pancreas, stomach, `TAG-72` high B72.3 Thor et al.,
Cancer Res., 46: 3118, 1986 oesophagus M.sub.r mucin stomach `CEA`
180 Kd GP CEA 11-H5 Wagener et al., Int. J. Cancer, 33: 469, 1984
pancreas HMFG-2 >400 Kd 3.14.A3 Burchell et al., J. Immunol.,
131: 508, 1983 GP G.I. NS C COLI Lemkin et al., Proc. Am. Soc.
Clin. Oncol., 3: 47, 1984 pancreas, stomach CA 19-9 (or CA-19-9
(1116NS 19- Szymendera, Tumour Biology, 7: 333, 1986 GICA) 9) and
CA50 pancreas CA125 GP OC125 Szymendera, Tumour Biology, 7: 333,
1986 F: LUNG p185.sup.HER2 4D5 3H4, 7C2, 6E9, Shepard et al., J.
Clin. Immunol., 11(3): 117, non-small cell 2C4, 7F3, 2H11, 1991
lung carcinoma 3E8, 5B8, 7D3, SB8 high M.sub.r mucin/ MO v2 Miolti
et al., Cancer Res., 65: 826, 1985 glycolipid `TAG-72` high B72.3
Thor et al., Cancer Res., 46: 3118, 1986 M.sub.r mucin high M.sub.r
mucin DU-PAN-2 Lan et al., Cancer Res., 45: 305, 1985 `CEA` 180 kD
GP CEA 11-H5 Wagener et al, Int. J. Cancer., 33: 469, 1984
Malignant Gliomas cytoplasmic MUC 8-22 Stavrou, Neurosurg. Rev.,
13: 7, 1990 antigen from 85HG-22 cells cell surface Ag MUC 2-63
Stavrou, Neurosurg. Rev., 13: 7, 1990 from 85HG-63 cells cell
surface Ag MUC 2-39 Stavrou, Neurosurg. Rev., 13: 7, 1990 from
86HG-39
cells cell surface Ag MUC 7-39 Stavrou, Neurosurg. Rev., 13: 7,
1990 from 86HG-39 cells G: MISCELLANEOUS p53 PAb 240 Imperial
Cancer Research Technology MaB PAb 246 Listing PAb 1801 small round
cell neural cell ERIC.1 Imperial Cancer Research Technology MaB
tumors adhesion Listing molecule medulloblastoma M148 Imperial
Cancer Research Technology MaB neuroblastoma Listing
rhabdomyosarcoma neuroblastoma FMH25 Imperial Cancer Research
Technology MaB Listing renal cancer & p155 6.1 Loop et al.,
Int. J. Cancer, 27: 775, 1981 glioblastomas bladder & "Ca
Antigen" CA1 Ashall et al., Lancet, Jul. 3, 1, 1982 laryngeal
cancers 350-390 kD neuroblastoma GD2 3F8 Cheung et al., Proc. AACR,
27: 318, 1986 Prostate gp48 48 kD GP 4F.sub.7/7A.sub.10
Bhattacharya et al., Cancer Res. 44: 4528, 1984 Prostate 60 kD GP
2C.sub.8/2F.sub.7 Bhattacharya et al., Hybridoma, 4: 153, 1985
Thyroid `CEA` 180 kD GP CEA 11-H5 Wagener et al., Int. J. Cancer,
33: 469, 1984 abbreviations: Abs, antibodies; Ags, antigens; EGF,
epidermal growth factor; GI, gastrointestinal; GICA,
gastrointestinal-associated antigen; GP, glycoprotein; GY,
gynecological; HMFG, human milk fat globule; Kd, kilodaltons; Mabs,
monoclonal antibodies; M.sub.r, molecular weight; NS, not
specified; PLAP, placental alkaline phosphatase; TAG,
tumor-associated glycoprotein; CEA, carcinoembryonic antigen.
footnotes: the CA 19-9 Ag (GICA) is
sialosylfucosyllactotetraosylceramide, also termed sialylated Lewis
pentaglycosyl ceramide or sialyated lacto-N-fucopentaose II; p97
Ags are believed to be chondroitin sulphate proteoglycan; antigens
reactive with Mab 9.2.27 are believed to be sialylated
glycoproteins associated with chondroitin sulphate proteoglycan;
unless specified, GY can include cancers of the cervix, endocervix,
endometrium, fallopian tube, ovary, vagina or mixed Mullerian
tumor; unless specified GI can include cancers of the liver, small
intestine, spleen, pancreas, stomach and oesophagus.
[0147] Generally speaking, antibodies of the present invention will
preferably exhibit properties of high affinity, such as exhibiting
a K.sub.d of <200 nM, and preferably, of <100 nM, and will
not show significant reactivity with life-sustaining normal
tissues, such as one or more tissues selected from heart, kidney,
brain, liver, bone marrow, colon, breast, prostate, thyroid, gall
bladder, lung, adrenals, muscle, nerve fibers, pancreas, skin, or
other life-sustaining organ or tissue in the human body. The
"life-sustaining" tissues that are the most important for the
purposes of the present invention, from the standpoint of low
reactivity, include heart, kidney, central and peripheral nervous
system tissues and liver. The term "significant reactivity", as
used herein, refers to an antibody or antibody fragment, which,
when applied to the particular tissue under conditions suitable for
immunohistochemistry, will elicit either no staining or negligible
staining with only a few positive cells scattered among a field of
mostly negative cells. Many of the antibodies listed in Table II
will not be of sufficient tumor specificity to be of use
therapeutically. An example is MUC8-22 which recognizes a
cytoplasmic antigen. Antibodies such as these will be of use only
in diagnostic or investigational embodiments such as in model
systems or screening assays.
[0148] Particularly promising antibodies from the stand point of
therapeutic application of the present invention are those having
high selectivity for the solid tumor, such as B72.3, PR5C5 or PR4D2
for colorectal tumors; HMFG-2, TAG 72, SM-3, or anti-p 185.sup.Her2
for breast tumors; anti-p 185.sup.Her2 for lung tumors; 9.2.27 for
melanomas; and MO v18 and OV-TL3 for ovarian tumors.
[0149] The listing of potential solid tumor cell surface antigen
targets in Table II is intended to be illustrative rather than
exhaustive. Of course, in the practice of the invention, one will
prefer to ensure in advance that the clinically-targeted tumor
expresses the antigen ultimately selected. This is a fairly
straightforward assay, involving antigenically testing a tumor
tissue sample, for example, a surgical biopsy, or perhaps testing
for circulating shed antigen. This can readily be carried out in an
immunological screening assay such as an ELISA (enzyme-linked
immunosorbent assay), wherein the binding affinity of antibodies
from a "bank" of hybridomas are tested for reactivity against the
tumor. Antibodies demonstrating appropriate tumor selectivity and
affinity are then selected for the preparation of bispecific
antibodies of the present invention. Antitumor antibodies will also
be useful in the preparation of antitumor antibody conjugates for
use in combination regimens, wherein tumor endothelium and the
solid tumor itself are both targeted (see, e.g., FIG. 12).
F. Preparation of Targeting Agent/Toxin Compounds; Including
Immunotoxins
[0150] Methods for the production of the target agent/toxin agent
compounds of the invention are described herein. The targeting
agents, such as antibodies, of the invention may be linked, or
operatively attached, to the toxins of the invention by either
crosslinking or via recombinant DNA techniques, to produce, for
example, targeted immunotoxins.
[0151] While the preparation of immunotoxins is, in general, well
known in the art (see, e.g., U.S. Pat. No. 4,340,535, and EP 44167,
both incorporated herein by reference), the inventors are aware
that certain advantages may be achieved through the application of
certain preferred technology, both in the preparation of the
immunotoxins and in their purification for subsequent clinical
administration. For example, while IgG based immunotoxins will
typically exhibit better binding capability and slower blood
clearance than their Fab' counterparts, Fab' fragment-based
immunotoxins will generally exhibit better tissue penetrating
capability as compared to IgG based immunotoxins.
[0152] Additionally, while numerous types of disulfide-bond
containing linkers are known which can successfully be employed to
conjugate the toxin moiety with the targeting agent, certain
linkers will generally be preferred over other linkers, based on
differing pharmacologic characteristics and capabilities. For
example, linkers that contain a disulfide bond that is sterically
"hindered" are to be preferred, due to their greater stability in
vivo, thus preventing release of the toxin moiety prior to binding
at the site of action. Furthermore, while certain advantages in
accordance with the invention will be realized through the use of
any of a number of toxin moieties, the inventors have found that
the use of ricin A chain, and even more preferably deglycosylated A
chain, will provide particular benefits.
[0153] A wide variety of cytotoxic agents are known that may be
conjugated to anti-endothelial cell antibodies. Examples include
numerous useful plant-, fungus- or even bacteria-derived toxins,
which, by way of example, include various A chain toxins,
particularly ricin A chain, ribosome inactivating proteins such as
saporin or gelonin, .alpha.-sarcin, aspergillin, restrictocin,
ribonucleases such as placental ribonuclease, angiogenic,
diphtheria toxin, and pseudomonas exotoxin, to name just a few. The
most preferred toxin moiety for use in connection with the
invention is toxin A chain which has been treated to modify or
remove carbohydrate residues, so called deglycosylated A chain. The
inventors have had the best success through the use of
deglycosylated ricin A chain (dgA) which is now available
commercially from Inland Laboratories, Austin, Tx.
[0154] However, it may be desirable from a pharmacologic standpoint
to employ the smallest molecule possible that nevertheless provides
an appropriate biological response. One may thus desire to employ
smaller A chain peptides which will provide an adequate
anti-cellular response. To this end, it has been discovered by
others that ricin A chain may be "truncated" by the removal of 30
N-terminal amino acids by Nagarase (Sigma), and still retain an
adequate toxin activity. It is proposed that where desired, this
truncated A chain may be employed in conjugates in accordance with
the invention.
[0155] Alternatively, one may find that the application of
recombinant DNA technology to the toxin A chain moiety will provide
additional significant benefits in accordance the invention. In
that the cloning and expression of biologically active ricin A
chain has now been enabled through the publications of others
(O'Hare et al., 1987; Lamb et al., 1985; Halling et al., 1985), it
is now possible to identify and prepare smaller or otherwise
variant peptides which nevertheless exhibit an appropriate toxin
activity. Moreover, the fact that ricin A chain has now been cloned
allows the application of site-directed mutagenesis, through which
one can readily prepare and screen for A chain derived peptides and
obtain additional useful moieties for use in connection with the
present invention.
[0156] The cross-linking of the toxin A chain region of the
conjugate with the targeting agent region is an important aspect of
the invention. In certain cases, it is required that a cross-linker
which presents disulfide function be utilized for the conjugate to
have biological activity. The reason for this is unclear, but is
likely due to a need for certain toxin moieties to be readily
releasable from the targeting agent once the agent has "delivered"
the toxin to the targeted cells. Each type of cross-linker, as well
as how the cross-linking is performed, will tend to vary the
pharmacodynamics of the resultant conjugate. Ultimately, in cases
where a releasable toxin is contemplated, one desires to have a
conjugate that will remain intact under conditions found everywhere
in the body except the intended site of action, at which point it
is desirable that the conjugate have good "release"
characteristics. Therefore, the particular cross-linking scheme,
including in particular the particular cross-linking reagent used
and the structures that are cross-linked, will be of some
significance.
[0157] Cross-linking reagents are used to form molecular bridges
that tie together functional groups of two different proteins
(e.g., a toxin and a binding agent). To link two different proteins
in a step-wise manner, heterobifunctional cross-linkers can be used
which eliminate the unwanted homopolymer formation. An exemplary
heterobifunctional cross-linker contains two reactive groups: one
reacting with primary amine group (e.g., N-hydroxy succinimide) and
the other reacting with a thiol group (e.g., pyridyl disulfide,
maleimides, halogens, etc.). Through the primary amine reactive
group, the cross-linker may react with the lysine residue(s) of one
protein (e.g., the selected antibody or fragment) and through the
thiol reactive group, the cross-linker, already tied up to the
first protein, reacts with the cysteine residue (free sulfhydryl
group) of the other protein (e.g., dgA).
[0158] The spacer arm between these two reactive groups of any
cross-linkers may have various length and chemical composition. A
longer spacer arm allows a better flexibility of the conjugate
components while some particular components in the bridge (e.g.,
benzene group) may lend extra stability to the reactive group or an
increased resistance of the chemical link to the action of various
aspects (e.g., disulfide bond resistant to reducing agents).
[0159] The most preferred cross-linking reagent is SMPT, which is a
bifunctional cross-linker containing a disulfide bond that is
"sterically hindered" by an adjacent benzene ring and methyl
groups. It is believed that stearic hindrance of the disulfide bond
serves a function of protecting the bond from attack by thiolate
anions such as glutathione which can be present in tissues and
blood, and thereby help in preventing decoupling of the conjugate
prior to its delivery to the site of action by the binding agent.
The SMPT cross-linking reagent, as with many other known
cross-linking reagents, lends the ability to cross-link functional
groups such as the SH of cysteine or primary amines (e.g., the
epsilon amino group of lysine). Another possible type of
cross-linker includes the heterobifunctional photoreactive
phenylazides containing a cleavable disulfide bond such as
sulfosuccinimidyl-2-(p-azido salicylamido)
ethyl-1,3'-dithiopropionate. The N-hydroxy-succinimidyl group
reacts with primary amino groups and the phenylazide (upon
photolysis) reacts non-selectively with any amino acid residue.
[0160] Although the "hindered" cross-linkers will generally be
preferred in the practice of the invention, non-hindered linkers
can be employed and advantages in accordance herewith nevertheless
realized. Other useful cross-linkers, not considered to contain or
generate a protected disulfide, include SATA, SPDP and
2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of such
cross-linkers is well understood in the art.
[0161] Once conjugated, it will be important to purify the
conjugate so as to remove contaminants such as unconjugated toxin
or targeting agent. It is important to remove unconjugated A chain
because of the possibility of increased toxicity. Moreover, it is
important to remove unconjugated targeting agent to avoid the
possibility of competition for the antigen between conjugated and
unconjugated species. In any event, a number of purification
techniques are disclosed in the Examples below which have been
found to provide conjugates to a sufficient degree of purity to
render them clinically useful.
[0162] In general, the most preferred technique will incorporate
the use of Blue-Sepharose with a gel filtration or gel permeation
step. Blue-Sepharose is a column matrix composed of Cibacron Blue
3GA and agarose, which has been found to be useful in the
purification of immunoconjugates (Knowles & Thorpe, 1987). The
use of Blue-Sepharose combines the properties of ion exchange with
A chain binding to provide good separation of conjugated from
unconjugated binding.
[0163] The Blue-Sepharose allows the elimination of the free (non
conjugated) targeting agent (e.g., the antibody or fragment) from
the conjugate preparation. To eliminate the free (unconjugated)
toxin (e.g., dgA) a molecular exclusion chromatography step is
preferred using either conventional gel filtration procedure or
high performance liquid chromatography. One may also use the
methods disclosed in U.S. patent application Ser. No. 08/147,768,
incorporated herein by reference, which enables the production of
immunotoxins bearing one, two or three toxin chains per antibody
molecule.
[0164] Standard recombinant DNA techniques that are well known to
those of skill in the art may be utilized to express nucleic acids
encoding the targeting agent/toxin compounds of the invention.
These methods include, for example, in vitro recombinant DNA
techniques, synthetic techniques and in vivo recombination/genetic
recombination. DNA and RNA synthesis may, additionally, be
performed using an automated synthesizers (see, for example, the
techniques described in Sambrook et al., 1989; and Ausubel et al.,
1989).
[0165] When produced via recombinant DNA techniques such as those
described herein, the targeting agent/toxin compounds of the
invention may be referred to herein as "fusion proteins" . It is to
be understood that such fusion proteins contain at least a
targeting agent and a toxin of the invention, operatively attached.
The fusion proteins may also include additional peptide sequences,
such as peptide spacers which operatively attach the targeting
agent and toxin compound, as long as such additional sequences do
not appreciably affect the targeting or toxin activities of the
fusion protein.
[0166] Depending on the specific toxin compound used as part of the
fusion protein, it may be necessary to provide a peptide spacer
operatively attaching the targeting agent and the toxin compound
which is capable of folding into a disulfide-bonded loop structure.
Proteolytic cleavage within the loop would then yield a
heterodimeric polypeptide wherein the targeting agent and the toxin
compound are linked by only a single disulfide bond. See, for
example, Lord et al. (1992). An example of such a toxin is a Ricin
A-chain toxin.
[0167] When certain other toxin compounds are utilized, a
non-cleavable peptide spacer may be provided to operatively attach
the targeting agent and the toxin compound of the fusion protein.
Toxins which may be used in conjunction with non-cleavable peptide
spacers are those which may, themselves, be converted by
proteolytic cleavage, into a cytotoxic disulfide-bonded form (see
for example, Ogata et al., 1990). An example of such a toxin
compound is a Pseudonomas exotoxin compound.
[0168] Nucleic acids that may be utilized herein comprise nucleic
acid sequences that encode a targeting agent of interest and
nucleic acid sequences that encode a toxin agent of interest. Such
target agent-encoding and toxin agent-encoding nucleic acid
sequences are attached in a manner such that translation of the
nucleic acid yields the targeting agent/toxin compounds of the
invention.
[0169] Standard techniques, such as those described above may be
used to construct expression vectors containing the above-described
nucleic acids and appropriate transcriptional/translational control
sequences. A variety of host-expression vector systems may be
utilized. These include but are not limited to microorganisms such
as bacteria (e.g., E. coli, B. subtilis) transformed with
recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression
vectors containing targeting agent/toxin coding sequences; yeast
(e.g., Saccharomyces, Pichia) transformed with recombinant yeast
expression vectors containing targeting agent/toxin coding
sequences; insect cell systems infected with recombinant virus
expression vectors (e.g., baculovirus) containing the targeting
agent/toxin coding sequences; plant cell systems infected with
recombinant virus expression vectors (e.g., cauliflower mosaic
virus, CaMV; tobacco mosaic virus, TMV) or transformed with
recombinant plasmid expression vectors (e.g., Ti plasmid)
containing the targeting agent/toxin coding sequences coding
sequence; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3)
harboring recombinant expression constructs containing promoters
derived from the genome of mammalian cells (e.g., metallothionein
promoter) or from mammalian viruses (e.g., the adenovirus late
promoter; the vaccinia virus 7.5K promoter).
[0170] In bacterial systems a number of expression vectors may be
advantageously selected depending upon the use intended for the
targeting agent/toxin compound being expressed. For example, when
large quantities of targeting agent/toxin compound are to be
produced for the generation of antibodies or to screen peptide
libraries, vectors which direct the expression of high levels of
fusion protein products that are readily purified may be desirable.
Such vectors include but are not limited to the E. coli expression
vector pUR278 (Ruther et al., 1983), in which the targeting
agent/toxin coding sequence may be ligated individually into the
vector in frame with the lac Z coding region so that a fusion
protein additionally containing a portion of the lac Z product is
provided; pIN vectors (Inouye et al., 1985; Van Heeke et al.,
1989); and the like. pGEX vectors may also be used to express
foreign polypeptides, such as the targeting agent/toxin compounds
as fusion proteins additionally containing glutathione
S-transferase (GST). In general, such fusion proteins are soluble
and can easily be purified from lysed cells by adsorption to
glutathione-agarose beads followed by elution in the presence of
free glutathione. The pGEX vectors are designed to include thrombin
or factor Xa protease cleavage sites so that the targeting
agent/toxin protein of the fusion protein can be released from the
GST moiety.
[0171] In an insect system, Autograph californica nuclear
polyhidrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. The
targeting agent/toxin coding sequences may be cloned into
non-essential regions (for example the polyhedrin gene) of the
virus and placed under control of an AcNPV promoter (for example
the polyhedrin promoter). Successful insertion of the targeting
agent/toxin coding sequences will result in inactivation of the
polyhedrin gene and production of non-occluded recombinant virus
(i.e., virus lacking the proteinaceous coat coded for by the
polyhedrin gene). These recombinant viruses are then used to infect
Spodoptera frugiperda cells in which the inserted gene is expressed
(e.g., see Smith et al., 1983; Smith U.S. Pat. No. 4,215,051).
[0172] In mammalian host cells, a number of viral based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, the targeting agent/toxin coding sequences may
be ligated to an adenovirus transcription/translation control
complex, e.g., the late promoter and tripartite leader sequence.
This chimeric gene may then be inserted in the adenovirus genome by
in vitro or in vivo recombination. Insertion in a non-essential
region of the viral genome (e.g., region E1 or E3) will result in a
recombinant virus that is viable and capable of expressing
targeting agent/toxin proteins in infected hosts (e.g., see Logal
et al., 1984). Specific initiation signals may also be required for
efficient translation of inserted targeting agent/toxin coding
sequences. These signals include the ATG initiation codon and
adjacent sequences. Exogenous translational control signals,
including the ATG initiation codon, may additionally need to be
provided. One of ordinary skill in the art would readily be capable
of determining this and providing the necessary signals.
Furthermore, the initiation codon must be in phase with the reading
frame of the desired coding sequence to ensure translation of the
entire insert. These exogenous translational control signals and
initiation codons can be of a variety of origins, both natural and
synthetic. The efficiency of expression may be enhanced by the
inclusion of appropriate transcription enhancer elements,
transcription terminators, etc. (see Bittner et al., 1987).
[0173] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the
protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins. Appropriate cells lines or host systems can be chosen
to ensure the correct modification and processing of the foreign
protein expressed. To this end, eukaryotic host cells which possess
the cellular machinery for proper processing of the primary
transcript, glycosylation, and phosphorylation of the gene product
may be used. Such mammalian host cells include, but are not limited
to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, etc. For
long-term, high-yield production of recombinant proteins, stable
expression is preferred. For example, cell lines which stably
express constructs encoding the targeting agent/toxin compounds may
be engineered. Rather than using expression vectors which contain
viral origins of replication, host cells can be transformed with
targeting agent/toxin DNA controlled by appropriate expression
control elements (e.g., promoter, enhancer, sequences,
transcription terminators, polyadenylation sites, etc.), and a
selectable marker. Following the introduction of foreign DNA,
engineered cells may be allowed to grow for 1-2 days in an enriched
media, and then are switched to a selective media. The selectable
marker in the recombinant plasmid confers resistance to the
selection and allows cells to stably integrate the plasmid into
their chromosomes and grow to form foci which in turn can be cloned
and expanded into cell lines.
[0174] A number of selection systems may be used, including, but
not limited, to the herpes simplex virus thymidine kinase (Wigler
et al., 1977), hypoxanthine-guanine phosphoribosyltransferase
(Szybalska et al., 1962), and adenine phosphoribosyltransferase
genes (Lowy et al., 1980) can be employed in tk-, hgprt- or
aprt-cells, respectively. Also, antimetabolite resistance can be
used as the basis of selection for dhfr, which confers resistance
to methotrexate (Wigler et al., 1980; O'Hare et al., 1981); gpt,
which confers resistance to mycophenolic acid (Mulligan et al.,
1981); neo, which confers resistance to the aminoglycoside G-418
(Colberre-Garapin et al., 1981); and hygro, which confers
resistance to hygromycin (Santerre et al., 1984).
[0175] After a sufficiently purified compound has been prepared,
one will desire to prepare it into a pharmaceutical composition
that may be administered parenterally. This is done by using for
the last purification step a medium with a suitable pharmaceutical
composition.
[0176] Suitable pharmaceutical compositions in accordance with the
invention will generally comprise from about 10 to about 100 mg of
the desired conjugate admixed with an acceptable pharmaceutical
diluent or excipient, such as a sterile aqueous solution, to give a
final concentration of about 0.25 to about 2.5 mg/ml with respect
to the conjugate. Such formulations will typically include buffers
such as phosphate buffered saline (PBS), or additional additives
such as pharmaceutical excipients, stabilizing agents such as BSA
or HSA, or salts such as sodium chloride. For parenteral
administration it is generally desirable to further render such
compositions pharmaceutically acceptable by insuring their
sterility, non-immunogenicity and non-pyrogenicity. Such techniques
are generally well known in the art as exemplified by Remington's
Pharmaceutical Sciences, 16th Ed. Mack Publishing Company, 1980,
incorporated herein by reference. It should be appreciated that
endotoxin contamination should be kept minimally at a safe level,
for example, less that 0.5 ng/mg protein. Moreover, for human
administration, preparations should meet sterility, pyrogenicity,
general safety and purity standards as required by FDA Office of
Biological Standards.
[0177] A preferred parenteral formulation of the targeting
agent/toxin compounds, including immunotoxins, in accordance with
the present invention is 0.25 to 2.5 mg conjugate/ml in 0.15M NaCl
aqueous solution at pH 7.5 to 9.0. The preparations may be stored
frozen at -10.degree. C. to -70.degree. C. for at least 1 year.
G. Attachment of Other Agents to Targeting Agents
[0178] It is contemplated that most therapeutic applications of the
present invention will involve the targeting of a toxin moiety to
the tumor endothelium. This is due to the much greater ability of
most toxins to deliver a cell killing effect as compared to other
potential agents. However, there may be circumstances, such as when
the target antigen does not internalize by a route consistent with
efficient intoxication by targeting agent/toxin compounds, such as
immunotoxins, where one will desire to target chemotherapeutic
agents such as antitumor drugs, other cytokines, antimetabolites,
alkylating agents, hormones, and the like. The advantages of these
agents over their non-targeting agent conjugated counterparts is
the added selectivity afforded by the targeting agent, such as an
antibody. One might mention by way of example agents such as
steroids, cytosine arabinoside, methotrexate, aminopterin,
anthracyclines, mitomycin C, vinca alkaloids, demecolcine,
etoposide, mithramycin, and the like. This list is, of course,
merely exemplary in that the technology for attaching
pharmaceutical agents to targeting agents, such as antibodies, for
specific delivery to tissues is well established (see, e.g., Ghose
& Blair, 1987).
[0179] It is proposed that particular benefits may be achieved
through the application of the invention to tumor imaging. Imaging
of the tumor vasculature is believed to provide a major advantage
when compared to present imaging techniques, in that the cells are
readily accessible. Moreover, the technology for attaching
paramagnetic, radioactive and even fluorogenic ions to targeting
agents, such as antibodies, is well established. Many of these
methods involve the use of a metal chelate complex employing, for
example, an organic chelating agent such a DTPA attached to the
antibody (see, e.g., U.S. Pat. No. 4,472,509). In the context of
the present invention the selected ion is thus targeted to the
tumor endothelium by the targeting agent, such as an antibody,
allowing imaging to proceed by means of the attached ion.
[0180] A variety of chemotherapeutic and other pharmacologic agents
have now been successfully conjugated to antibodies and shown to
function pharmacologically (see, e.g., Vaickus et al., 1991).
Exemplary antineoplastic agents that have been investigated include
doxorubicin, daunomycin, methotrexate, vinblastine, and various
others (Dillman et al., 1988; Pietersz et al., 1988). Moreover, the
attachment of other agents such as neocarzinostatin (Kimura et al.,
1983), macromycin (Manabe et al., 1984), trenimon (Ghose, 1982) and
.alpha.-amanitin (Davis & Preston, 1981) has been
described.
[0181] In addition to chemotherapeutic agents, the inventors
contemplate that the invention will be applicable to the specific
delivery of a wide variety of other agents to tumor vasculature.
For example, under certain circumstances, one may desire to deliver
a coagulant such as Russell's Viper Venom, activated Factor IX,
activated Factor X or thrombin to the tumor vasculature. This will
result in coagulation of the tumor's blood supply. One can also
envisage targeting a cell surface lytic agent such as phospholipase
C (Flickinger & Trost, 1976) or cobra venom factor (CVF) (Vogel
& Muller-Eberhard, 1981) which should lyse the tumor
endothelial cells directly. The operative attachment of such
structures to targeting agents, such as antibodies, may be readily
accomplished, for example, by protein-protein coupling agents such
as SMPT. Moreover, one may desire to target growth factors, other
cytokines or even bacterial endotoxin or the lipid A moiety of
bacterial endotoxin to a selected cell type, in order, e.g., to
achieve modulation of cytokine release. The attachment of such
substances is again well within the skill in the art as exemplified
by Ghose & Blair (1987).
[0182] Thus, it is generally believed to be possible to conjugate
to antibodies any pharmacologic agent that has a primary or
secondary amine group, hydrazide or hydrazine group, carboxyl
alcohol, phosphate, or alkylating group available for binding or
cross-linking to the amino acids or carbohydrate groups of the
antibody. In the case of protein structures, this is most readily
achieved by means of a cross linking agent (see preceding section
on immunotoxins). In the case of doxorubicin and daunomycin,
attachment may be achieved by means of an acid labile acyl
hydrazone or cis aconityl linkage between the drug and the
antibody. Finally, in the case of methotrexate or aminopterin,
attachment is achieved through a peptide spacer such as
L-Leu-L-Ala-L-Leu-L-Ala, between the .gamma.-carboxyl group of the
drug and an amino acid of the antibody. For a general overview of
linking technology, one may wish to refer to Ghose & Blair
(1987).
[0183] Alternatively, any such structures which are nucleic
acid-encoded structures may be operatively attached to the
targeting agents of the invention by standard recombinant DNA
techniques, such as, for example, those discussed above, in the
previous section.
[0184] The following examples are representative of techniques
employed by the inventors in carrying out aspects of the present
invention. It should be appreciated that while these techniques are
exemplary of preferred embodiments for the practice of the
invention, those of skill in the art, in light of the present
disclosure, will recognize that numerous modifications can be made
without departing from the spirit and intended scope of the
invention.
EXAMPLE I
A Murine Model for Antibody-Directed Targeting of Vascular
Endothelial Cells in Solid Tumors
[0185] This example describes the development of a model system in
which to investigate the antibody-directed targeting of vascular
endothelial cells in solid tumors in mice. A neuroblastoma
transfected with the mouse interferon-.gamma. (IFN-.gamma.) gene,
C1300(Mu.gamma.), was grown in SCID and antibiotic-treated BALB/c
nude mice. The INF-.gamma. secreted by the tumor induces the
expression of MHC Class II antigens on the tumor vascular
endothelium. Class II antigens are absent from the vasculature of
normal tissues, although they are present on B-lymphocytes, cells
of monocyte/macrophage lineage and some epithelial cells.
Intravenously-administered anti-Class II antibody strongly stains
the tumor vasculature whereas an anti-tumor antibody, directed
against a MHC Class I antigen of the tumor allograft, produces
classical perivascular tumor cell staining.
A. Materials and Methods
[0186] 1. Animals
[0187] BALB/c nu/nu mice were purchased from Simonsen (Gilroy,
Calif.). SCID mice were from the UT Southwestern Medical Center
breeding colony. All animals were maintained in microisolation
units on sterilized food and water. Where indicated,
tetracycline--HCI (Vedeo, St. Joseph, Mo.) was added to drinking
water to a final concentration of 1.1 mg/ml (Harkness et al.,
1983). Both strains carry the H-2.sup.d haplotype.
[0188] 2. Cells and Culture Conditions
[0189] All cell lines used in this study were cultured in modified
Eagle's medium (MEM) supplemented with 10% (v/v) fetal calf serum,
2.4 mM L-glutamine, 200 units/ml penicillin and 100 .mu.g/ml
streptomycin. Cultures were maintained at 37.degree. C. in a
humidified atmosphere of 90% air/10 CO.sub.2. The C1300
neuroblastoma line was established from a spontaneous tumor which
arose in an A/Jax mouse in 1940 (Dunham et al., 1953). The
C1300(Mu.gamma.)12 line, hereafter abbreviated to C1300
(Mu.gamma.), was derived by transfection of C1300 cells with murine
IFN-.gamma. gene using the IFN-.gamma. expression retrovirus pSVX
(Mu.gamma.A.sub.s) (Watanabe et al., 1988; Watanabe et al., 1989),
and was cultured in MEM as above containing 1 mg/ml G418
(Geneticin, Sigma). Both lines carry the MHC haplotype H-2K.sup.k,
I-A.sup.k, I-E.sup.k, D.sup.d. C1300 and C1300(Mu.gamma.) cells
were grown in regular tissue culture flasks or, when large
quantities were required for in vivo studies, in cell factories
(Baxter, Grand Prairie, Tex.). Cells from subcutaneous tumors were
recovered for in vitro analysis by gentle mincing in MEM. After
tumor cells had adhered overnight the monolayers were washed twice
with MEM to remove nonadherent contaminant host cells.
[0190] Tumor conditioned media were prepared by seeding C1300 and
C1300(MuA.gamma.) cells at 25% of confluent density and culturing
them for four days. Conditioned media were dialyzed for 16 hours
against MEM without FCS to remove G418, filtered through a 0.22
.mu.M membrane and stored at 4.degree. C. for no more than one week
before assay. Aliquots of anti-IFN-.gamma. antibodies (see
`Monoclonal Antibodies`) sufficient to neutralize 200 international
units (I.U.) of murine IFN-.gamma./ml of conditioned medium were
added to some samples 24 hours before assay. The SVEC-10 murine
endothelial cell line, hereafter abbreviated to SVEC, was kindly
provided to Dr. M. Edidin, Department of Biology, Johns Hopkins
University, Baltimore, Md. and was derived by immortalization of
lymph node endothelial cells from a C3H (H-2.sup.k) mouse with SV40
(O'Connell et al., 1990). For some studies, SVEC cells were
cultured for 72 hours with 100 I.U./ml recombinant murine
IFN-.gamma., (r.IFN-.gamma., a generous gift from Dr. F. Balkwill,
Imperial Cancer Research Fund, London, England) or
tumor-conditioned medium. In addition, 200 I.U./ml anti-IFN-.gamma.
antibody was added to some flasks at the beginning of the 72 hour
culture period.
[0191] 3. Monoclonal Antibodies
[0192] The M5/114.15.2 (hereafter abbreviated to M5/114) and 11-4.1
hybridomas were purchased from the American Type Collection
(Rockville, Md.) and were grown in MEM-10% FCS. The antibodies were
purified from culture supernatant by precipitation in 50% ammonium
sulphate and affinity chromatography on Protein A. The rat IgG2b
antibody, M5/114, detects an Ia specificity on I-A.sup.b,
I-A.sup.q, I-A.sup.d, I-E.sup.d and I-E.sup.k molecules
(Bhattacharya et al., 1981). Thus, the antibody recognizes
I-E.sup.k molecules on SVEC (H-2.sup.k) cells and I-A.sup.d and
I-E.sup.d, hereafter referred to collectively as Ia.sup.d, on cells
from BALB/C nu/nu or SCID mice (both H-2.sup.d) . The anti-Ia.sup.d
reactivity of M5/114 was confirmed in this study by FACS analyses
with the Ia.sup.d expressing B-lymphoma line, A20/25 (Kim, 1978).
The mouse IgG2a antibody 11-4.1 recognizes H-2K.sup.k but not
H-2K.sup.d molecules (Oi et al., 1978) and so binds to H-2K.sup.k
on C1300 and C1300(Mu.gamma.) cells but is unreactive with MHC
antigens from BALB/c nu/nu or SCID mice. Isotype-matched control
antibodies of irrelevant specificity were CAMPATH-2 (rat IgG2b,
anti-human CD7 (Bindon et al., 1988) and WT-1 (mouse IgG2a,
anti-human CD7 (Tax et al., 1984). Purified preparations of
CAMPATH-2 and WT-1 were generous gifts from Dr. G. Hale (Department
of Pathology, Cambridge, England) and Dr. W. Tax (Sint
Radboudzeikenhuis, Nijmegen, the Netherlands) respectively.
[0193] Rat anti-mouse endothelial cell antibody MECA-20
(Duijvestijn et al., 1987) was donated as a concentrated culture
supernatant by Dr. A. Duijvestijn (University of Limburg, the
Netherlands) and used at a dilution of 1/200 for indirect
immunoperoxidase staining. Rat antibodies against mouse macrophages
(M1) and mouse CD3 (KT 31.1) were generously provided by Dr. P.
Beverley (Imperial Cancer Research Fund, London, England). Hamster
anti-mouse IFN-.gamma. antibody 1222-00 (Sanchez-Madrid, 1983),
used for specific neutralization of IFN-.gamma. in vitro, was
purchased from Genzyme (Boston, Mass.). Anti-mouse IFN-.gamma.
antibodies, XMG1.2 and R46A2, used in IFN-.gamma. ELISAs, were
kindly provided by Dr. N. Street (U.T. Southwestern Medical Center,
Dallas, Tex.). Purified 11-4.1, WT-1 and XMG1.2 antibodies were
biotinylated by incubation with a 12.5 fold molar excess of
N-hydroxysuccinimidobiotin amidocaproate (Sigma) for one hour at
room temperature followed by dialysis against two changes of
PBS.
[0194] 4. ELISA for Murine IFN-.gamma.
[0195] Sandwich ELISAs for murine IFN-.gamma. were carried out as
described by (Cherwinski et al., 1989). The wells of flexible PVC
microtiter plates (Dynatech, Alexandria, Va.) were coated with 50
.mu.l/well of a 2 .mu.g/ml solution of capture anti-IFN-.gamma.
antibody, R46A2, in PBS for 2 hours at room temperature.
Non-specific protein binding sites were blocked with 20% FCS in PBS
for 15 minutes at 37.degree. C. The plates were washed three times
in PBS containing 0.05% (v/v) Tween 20 (Sigma) (PBS-T) and 25
.mu.l/well control and test samples in MEM-10% FCS were added.
After incubating for 1 hour at 37.degree. C., the wells were washed
as before and 50 .mu.l/well of a 1 .mu.g/ml solution of
biotinylated anti-IFN-.gamma. antibody XMG1.2 in PBS-T containing
1% BSA were added. After incubation for 30 minutes at 37.degree. C.
the wells were washed as before and incubated with 75 .mu.l of a
1:2000 dilution of horseradish peroxidase-conjugated streptavidin
(DAKO) for one hour at room temperature. After thorough washing in
PBS-T the wells were incubated for 30 minutes with 100 .mu.l/well
of a 1 mg/ml solution of
2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS,
Sigma) in citrate/phosphate butter containing 0.003% (v/v)
H.sub.2O.sub.2. Reaction product was measured as Abs.405 nm-Abs.490
nm. IFN-.gamma. levels in test samples were calculated by reference
to a recombinant murine IFN-.gamma. standard solution in MEM-10%
FCS.
[0196] 5. Indirect Immunofluorescence
[0197] SVEC, C1300 and C1300(Mu.gamma.) cells were prepared for
FACS analyses as described by Burrows et al. (1991). All
manipulations were carried out at room temperature. 50 .mu.l of a
cell suspension at 2-3.times.10.sup.6 cells/ml in PBS containing
0.2% (w/v) BSA and 0.2% (w/v) NaN.sub.3 (PBS-BSA-N.sub.3) were
added to the wells of round-bottomed 96-well microtiter plates
(Falcon 3910). Optimal dilutions of rat or mouse antibodies were
distributed in 50 .mu.l volumes, and the plates sealed. After 15
minutes, the cells were washed four times by centrifuging the
plates at 800.times.g for 30 seconds, removing the supernatants,
and resuspending the cells in 150 .mu.l/well PBS-BSA-N3.
Fluorescein isothiocyanate-conjugated rabbit antibodies against rat
or mouse IgG (ICN, High Wycombe, England), diluted 1:20 in
PBS-BSA-N.sub.3 were distributed in 50 .mu.l volumes into the
appropriate wells. The cells were incubated for a further 15
minutes and washed as before. Cell-associated fluorescence was
measured on a FACScan (Becton Dickenson, Fullerton, Calif.). Data
were analyzed using the CONSORT 30 program.
[0198] 6. Preparation of Tissues and Immunohistochemistry
[0199] For the establishment of solid tumors, a total of
2.times.10.sup.7 C1300 or C1300(Mu.gamma.) cells, or a mixture of
the two, in 200 .mu.l MEM-30% FCS were injected subcutaneously into
the right anterior flank of BALB/c nu/nu or SCID mice. Tumor
diameters were measured at regular intervals and the animals were
euthanized after 16 days (rapidly-growing tumors) or 20 days
(slowly-growing tumors). Tumors and normal tissues were excised
immediately and snap-frozen over liquid nitrogen. Normal tissues
were also harvested from non-tumor-bearing animals. Antibody
localization studies were performed in animals bearing 1 cm
subcutaneous tumors induced by injection of C1300 and
C1300(Mu.gamma.) in the ratio 7:3. One hundred micrograms of
unconjugated M5/114 or CAMPATH-2 antibodies or 100 .mu.g
biotinylated 11-4.1 or WT-1 antibodies in 100 .mu.l PBS were
injected intravenously. At various times thereafter the animals
were euthanized and their circulation was flushed with PBS for 5
minutes before removal and freezing of tumors and normal tissues as
before. 8 .mu.M frozen sections were cut on a Tissuetek 2 cryostat
(Baxter) and air-dried for 2 hours at room temperature. Slides were
stored at -20 C. for up to 3 months before assay.
[0200] Indirect immunoperoxidase staining for rat IgG was adapted
from a method described by Billington et al. (1986). Sections were
allowed to return to room temperature, air dried for 30 minutes and
fixed in acetone for 15 minutes. After rehydration in PBS for 5
minutes, sections were incubated in a humidified chamber for 45-60
minutes with primary antibodies, diluted optimally in PBS-0.2% BSA,
(PBS-BSA). After two washes in PBS, the sections were incubated for
30-45 minutes with horseradish peroxidase-conjugated rabbit
anti-mouse IgG (Dakopatts, Carpinteria, Calif.) diluted 1:10 in
PBS-BSA supplemented with 20% normal mouse serum (ICN, High
Wycombe, UK) to block antibodies cross-reacting with mouse
immunoglobulins. After a further two washes in PBS, the reaction
product was developed using 0.5 mg/ml 3',3'-diaminobenzidine
(Sigma) containing 0.01% (v/v) hydrogen peroxide for 8 minutes. The
sections were counterstained with Mayer's hematoxylin (Sigma) for
15 seconds, dehydrated in absolute ethanol, cleared in xylene and
mounted with Accumount 60 medium (Baxter). Indirect
immunoperoxidase staining with biotinylated mouse antibodies was
carried out in the same manner, except that peroxidase-conjugated
streptavidin-biotin complex, diluted 1:50 in PBS with no blocking
serum, was used as the second layer.
B. Results
[0201] 1. Murine IFN-.gamma. Levels in C1300(Mu.gamma.)
Conditioned-medium
[0202] C1300(Mu.gamma.)-conditioned medium contained 50.2-63.5
I.U./ml murine IFN-.gamma., in accordance with previous reports
(Watanabe et al., 1989). By contrast, less than 5 I.U./ml
IFN-.gamma. was detected in C1300-conditioned medium or
C1300(Mu.gamma.)-conditioned medium to which an excess of
neutralizing anti-IFN-.gamma. antibody had been added 24 hours
before assay.
[0203] 2. Induction of MHC Class II (I-E.sup.k) on SVEC Cells by
r.IFN-.gamma. in C1300(Mu.gamma.)-conditioned-medium
[0204] As shown in FIG. 1a, unstimulated SVEC cells did not express
I-E.sup.k. By contrast, a large majority of cells preincubated with
r.IFN-.gamma. (FIG. 1a) or with C1300(Mu.gamma.)-conditioned medium
(FIG. 1b) expressed significant levels of I-E.sup.k, and this
induction was almost completely blocked by anti-IFN-.gamma..
Treatment of SVEC cells with r.IFN-.gamma. or
C1300(Mu.gamma.)-conditioned medium did not cause non-specific
antibody binding since the isotype-matched control antibody did not
bind to the cells. These results were confirmed by indirect
immunoperoxidase staining of cytospin preparations.
[0205] These findings suggested that vascular endothelial cells in
tumors containing sufficient quantities of IFN-.gamma.-secreting
C1300(Mu.gamma.) cells should be induced to express high cell
surface levels of MHC Class II molecules.
[0206] 3. Expression of MHC Class I (H-2K.sup.k) and Class II
(I-E.sup.k) by C1300 and C1300(Mu.gamma.) Cells
[0207] Since IFN-.gamma. can induce MHC Class II antigen expression
in diverse cell types (Capobianchi et et al. 1985; Collins et al.,
1984; Hokland et al., 1988) and since the M5/114 antibody
crossreacts with I-E.sup.k, we determined whether the M5/114
antibody--intended for use to target tumor endothelial cells in
vivo--would also bind to the tumor cells themselves. As shown in
FIG. 2a, C1300(Mu.gamma.) cells expressed I-E.sup.k, but at levels
10-20 fold lower than those on SVEC cells stimulated with
IFN-.gamma..
[0208] Similarly, C1300 cells expressed detectable but low levels
of H-2K.sup.k whereas C1300(Mu.gamma.) cells displayed uniformly
high levels, approximately 20-fold greater than on the parental
line (FIG. 2b). This result was expected from the known autocrine
Class I-inducing activity of IFN-.gamma. and is in keeping with a
previous report (Watanabe et al., 1989). Coculture of
C1300(Mu.gamma.) cells and C1300 cells induced homogeneous
expression of I-Ek and H-2Kk on both populations (FIG. 2).
Induction of these antigens on C1300 cells appears to be caused by
IFN-.gamma. released into the culture medium by the
C1300(Mu.gamma.) cells since the effect was centralized by
anti-IFN-.gamma. antibodies.
[0209] 4. Growth of C1300 and C1300 (Mu.gamma.) Tumors in
Immunodeficient Mice and Induction of Ia.sup.d on Tumor Vascular
Endothelial Cells
[0210] The inventors first attempted to grow subcutaneous
C1300(Mu.gamma.) tumors in BALB/c nu/nu and SCID mice because both
strains carry the MHC haplotype (H-2.sup.d) with which the anti-MHC
Class II antibody M5/114 reacts, and because neither strain would
be expected to reject the tumors, as do syngeneic immunocompetent
A/J animals (Watanabe et al., 1989). For unknown reasons inocula
composed entirely of C1300(Mu.gamma.) cells failed to produce
progressively-growing tumors in BALB/c nu/nu or SCID mice.
Conversely, pure C1300 inocula displayed 100% tumorigenicity but,
as expected, did not contain Ia.sup.d-positive endothelial
cells.
[0211] In order to identify a combination which would yield a high
percentage of tumor takes, reliable growth kinetics and cause
Ia.sup.d induction of a large majority of intratumoral endothelial
cells, several ratios of C1300 and C1300(Mu.gamma.) cells were
inoculated into BALB/c nu/nu mice. As shown in FIG. 3, mixtures
containing C1300 and C1300(Mu.gamma.) cells in the ratio 9:1
produced rapidly-growing tumors but, when sections of the tumors
were stained with anti-Ia.sup.d antibody by the indirect
immunoperoxidase technique, none of the endothelial cells in the
tumor were found to be stained. Dropping the ratio of
C1300:C1300(Mu.gamma.) to 8:2 gave rapidly-growing tumors in which
approximately 50% of blood vessels were Ia.sup.d-positive. Dropping
the ratio further to 7:3 or 5:5 produced tumors which grew quite
rapidly and contained a large majority of Ia.sup.d-positive
vessels. Dropping the ratio still further to 3:7 produced tumors in
no more than half of the animals and those tumors that became
palpable failed to grow beyond 6 mm in diameter. Histological
analyses of the latter revealed no morphologically recognizable
intact blood vessels and, hence, it was not possible to ascertain
their level of Ia.sup.d expression.
[0212] Of the two usable C1300:C1300(Mu.gamma.) ratios identified,
7:3 and 5:5, the ratio of 7:3 was adopted for the remainder of this
study because the take rate was higher (100% vs. 80%) and the
variability in tumor growth rate between individual animals was
lower.
[0213] 5. Distribution of Ia.sup.d in BALB/c Nude and SCID Mice
[0214] The distribution of M5/114 binding in tissues from
tumor-bearing BALB/c nu/nu mice is shown in Table III. In
subcutaneous tumors, most or all vascular endothelial cells and
numerous interstitial macrophages were stained. In most organs, the
binding of M5/114 reflected the classical distribution of MHC Class
II antigens, being restricted to B cells in lymphoid organs,
resident macrophages in all tissues studied except brain and to
tissue-specific elements of the reticuloendothelial system, such as
liver Kupffer cells and Langerhans cells of the skin. In addition,
staining was occasionally seen in some kidney tubules. When
sections of small and large intestine from BALB/c nu/nu mice were
examined, heavy labeling of both epithelial and endothelial cells
was seen in both regions. By contrast, very little staining with
M5/114 was seen in sections of intestine from SCID mice maintained
in germ-free conditions. The staining of nu/nu mouse intestine was
found to be related to the microbiological status of the animals
and is discussed below. Apart from in the gut, no staining of
endothelial cells with M5/114 was seen in any tissues examined in
either nu/nu or SCID mice. The distribution of Ia.sup.d antigens in
normal tissues was not affected by the presence of the tumor
because the staining pattern of M5/114 was identical in
non-tumor-bearing mice. TABLE-US-00003 TABLE III Localization of
Intravenously Administered anti-la.sup.d Antibody in
C1300(MU.gamma.) Tumor-Bearing Mice..sup.(a) Localization in vivo
Tissue Antigen Expression 1 hour 4 hours 24 hours Tumor.sup.(b)
Endothelial cells (EC), M.phi. EC.sup.(c) EC EC.sup.(d) Brain None
None None None Colon.sup.(a) Minority of epithelium & EC,
M.phi. None None None Duodenum Some epithelial cells & EC,
M.phi. None None None Heart Interstitial M.phi. None None None
Kidney Occasional proximal tubule, M.phi. None None None Liver
Kupffer cells (KC), numerous M.phi. in KC.sup.(c) KC KC.sup.(d)
parenchyma some M.phi. Lung Numerous M.phi. in parenchyma None None
None Pancreas Numerous M.phi. in parenchyma None None None
Skin.sup.(e) Langerhans cells None None None Spleen Red pulp (RP)
M.phi., marginal zone (MZ) MZ MZ MZ, RP B cells & M.phi., some
T/B cells in PALS PALS PALS .sup.(a)Studies performed with SCID or
antibiotic-treated BALB/c nu/nu mice. .sup.(b)Mixed tumor of 7:3
C1300:C1300(Mu.gamma.) cells grown subcutaneously. .sup.(c)Strong
staining, including discernable labelling of luminal membranes.
.sup.(d)Weaker staining, entirely intracellular. .sup.(e)Either
adjacent to, or distant from tumor. PALS: Periarteriolar lymphatic
sheath, M.phi.: Macrophages
[0215] 6. Attenuation of Expression of Ia.sup.d on Colonic
Endothelium and Epithelium of Nude Mice by Administration of
Antibiotics
[0216] In BALB/c nu/nu mice, most epithelial cells from all regions
of the gut were intensely stained with anti-Ia.sup.d antibody. In
addition, some endothelial cells in both upper and lower bowel
bound M5/114 antibody, particularly those associated with colonic
villi. When the animals were treated with oral tetracycline-Hcl, a
broad-spectrum antibiotic, for 1-3 weeks there was a progressive
diminution of Ia.sup.d expression in the colon and elsewhere in the
gut, so that binding of M5/114 was in most sections restricted to
the luminal membranes of a minority of epithelial cells. Light
cytoplasmic staining of occasional endothelial cells was observed
in some antibiotic-treated animals. The pattern of epithelial and
endothelial Ia.sup.d expression was not homogeneous and the
intensity of M5/114 staining correlated with the frequency of CD3+
T lymphocytes in the adjacent lamina propria. Antibiotic treatment
was associated with a dramatic decrease in the numbers of
intravillous CD3-positive cells: after three weeks practically all
had disappeared from the underlying parenchyma and associated
lymphoid deposits and there was a coincident decline in Iad
expression on surrounding epithelial and endothelial cells.
[0217] In SCID mice, epithelial and endothelial cell Ia.sup.d
expression and T-cell infiltration of the colon resembled that of
antibiotic-treated BALB/c nu/nu animals.
[0218] 7. Specific Localization of Intravenously Administered
Anti-Ia.sup.d Antibody to Tumor Vasculature, B Cells and
Macrophages in SCID and Antibiotic-treated Nude Mice
[0219] Tumor-bearing BALB/c nu/nu and SCID mice were given
intravenous injections of anti-Ia.sup.d or the isotype-matched
control antibody and euthanized 1, 4 or 24 hours later. The in vivo
localization of anti-Ia.sup.d antibody in tumor and normal tissues
is shown in Table III. Anti-Ia.sup.d antibody was found on the
luminal membrane and in the cytoplasm of most or all tumor vascular
endothelial cells one hour after injection. A similar pattern was
seen at four hours after injection, but by 24 hours the labeling of
tumor endothelial cells was weaker and entirely intracellular,
consistent with the progressive internalization and metabolism of
the antibody by endothelial cells (Table III). Also, at 24 hours
small amounts of antibody were detectable in the immediate
perivascular regions of the tumor.
[0220] Anti-Ia.sup.d antibody was bound to Kupffer cells in the
intravascular compartment of the liver within one hour of
injection. At later times after injection, internalization and
degradation of the antibody was apparent (Table III). Adjacent
sinusoidal endothelial cells were not stained. The high
permeability of hepatic fenestrated endothelia was indicated by the
penetrance of the antibody to reach some hepatic parenchymal
macrophages (Table III). In the spleen, perivascular B cells and
macrophages in white pulp marginal zones were stained within one
hour, showing that the vasculature of this organ was particularly
permeable to antibody. At later stages the antibody penetrated
throughout the splenic lymphoid compartment and also labelled a
minority of red pulp macrophages (Table III). In organs other than
the liver and spleen, macrophages and related cells such as the
Langerhans cells of the skin were unstained probably because their
vascular endothelium contains tight junctions and is relatively
impermeable to antibodies.
[0221] Anti-Ia.sup.d antibody was bound to some endothelial cells
in the colon of BALB/c nu/nu mice, but not elsewhere in the
intestine, one hour after injection. Antibiotic treatment for 1-3
weeks before injection of anti-Ia.sup.d antibody completely
abolished localization to gut endothelial cells. No intravenously
injected anti-Ia.sup.d antibody homed to gut endothelia in SCID
mice. The isotype-matched control antibody was not detected in
tumor or normal tissues at any time after injection.
[0222] Taken together, these results strongly indicate that, when
injected into appropriate tumor-bearing animals anti-Ia.sup.d
antibody or immunoconjugates will localize effectively to most or
all tumor endothelial cells while sparing life-sustaining normal
tissues.
[0223] 8. Perivascular Staining of Tumor Cells in Mice Injected
with Anti-tumor (H-2K.sup.k) Antibody
[0224] When frozen sections of subcutaneous tumors deriving from
inocula of mixed C1300 and C1300(Mu.gamma.) cells (7:3) were
stained with biotinylated anti-H-2K.sup.k antibody, a homogeneous
staining pattern was obtained. The levels of IFN-.gamma. secreted
by the C1300(Mu.gamma.) cells in the tumor were therefore
sufficient to induce increased H-2K.sup.k expression by the C1300
component of the tumor, in accordance with the in vitro co-culture
studies described above. The staining was specific because no
staining was seen with the isotype-matched control antibody. No
specific labeling of any normal tissue by anti-H-2K.sup.k antibody
was found, as expected since this antibody was raised in an
H-2.sup.d mouse strain.
[0225] In contrast with the rapid binding of
intravenously-administered anti-Ia.sup.d antibody to tumor
vasculature, no significant accumulation of anti-H-2K.sup.k
antibody was apparent one hour after injection. After four hours,
however, anti-H-2K.sup.k antibody was detected in small islands of
tumor cells surrounding central capillaries. After 24 hours, the
antibody was bound to larger discrete areas of tumor cells but
staining intensity was diminished relative to the earlier time
points. Each with localization times of up to 72 hours, homogenous
labeling of all tumor cells was not achieved.
[0226] No localization of anti-H-2K.sup.k antibody was found in any
normal tissues and binding of the isotype-matched control antibody
was not detectable in tumor or normal tissues.
C. Discussion
[0227] This example describes a murine model for studying the
antibody-directed targeting of vascular endothelial cells in solid
tumors. In this model, IFN-.gamma. gene-transfected tumor cells
growing in SCID or antibiotic-treated nude mice release IFN-.gamma.
which induces the de novo expression of MHC Class II antigens on
the tumor vasculature. MHC Class II is absent from the vasculature
in the normal tissues of these mice and hence the Class II induced
on the tumor vascular endothelial cells serves as a specific
marker. Class II is present on B-lymphocytes, Kupffer cells and
other cells of monocyte/macrophage lineage but these cells are not
life-sustaining so their temporary absence after targeting with
cytotoxic immunoconjugates should be tolerable. IFN-.gamma. also
induces the tumor cells themselves to express high levels of the
MHC Class I antigen, H-2K.sup.k, which can serve as a tumor
cell-specific marker in BALB/c nu/nu or SCID mice, which both carry
the H-2K.sup.d haplotype. Thus, anti-Ia.sup.d and anti-H-2K.sup.k
antibodies injected systemically localize selectively to tumor
vascular endothelial cells and tumor cells respectively, which
enables the approaches of targeting the tumor vasculature and the
tumor cells to be compared in this model, or used in
combination.
[0228] It was necessary to dilute the C1300(Mu.gamma.) cells with
C1300 parental cells in the ratio 3:7 to establish
progressively-growing subcutaneous tumors in which the vascular
endothelial cells were Class II (Ia.sup.d)--positive. Undiluted
C1300(Mu.gamma.) cells were poorly tumorigenic in BALB/c nu/nu
mice, in contrast with a prior report (Watanabe et al., 1989).
Vascular dysfunction appeared to be the reason why pure
C1300(Mu.gamma.) tumors would not grow beyond a diameter of 5-6 mm.
Staining of sections of tumors with the anti-endothelial cell
antibody MECA 20 revealed that the vessels were morphologically
atypical with no visible lumens. It is possible that excessively
high intratumoral IFN-.gamma. levels in pure C1300(Mu.gamma.)
tumors caused direct vascular toxicity or activated macrophages in
the tumor to become cytotoxic for endothelial cells (Peri et al.,
1990).
[0229] Intravenously injected anti-Ia.sup.d antibody bound rapidly
and homogeneously to vascular endothelial cells in the tumor,
confirming the immediate accessibility of intravascular targets
(Kennel et al., 1991). Remarkably, the inductive influence of
IFN-.gamma. from C1300(Mu.gamma.) cells was completely restricted
to the tumor mass: endothelial cells in the overlying area of skin
expressed no detectable Ia.sup.d and did not bind any
intravenously-injected anti-Ia.sup.d antibody. It is likely that
IFN-.gamma. entering the systemic circulation is neutralized by a
specific binding protein, perhaps a soluble form of the IFN-.gamma.
receptor (Novick et al., 1989), whose normal role may be to
down-regulate cytokine activity (Fernandez-Botran et al., 1991) or
to restrict it to the immediate locale of secretion.
[0230] Ia.sup.d antigens are not restricted solely to tumor
endothelial cells. MHC Class II antigens are expressed
constitutively by B cells, activated T cells and cells of the
monocyte/macrophage lineage in humans and rodents (Daar et al.,
1984; Hammerling et al, 1976) and were found in this study also to
be present on occasional proximal tubules in the kidney and on some
epithelial cells in the intestine of SCID and antibiotic-treated
BALB/c nu/nu mice. However, when injected intravenously, only the
hepatic Kupffer cells, splenic B cells and macrophages in the liver
and spleen bound detectable amounts of the anti-Ia.sup.d antibody:
the potentially life-sustaining Class II--positive renal and gut
epithelial cells were unstained. Localization of
intravenously-injected anti-Ia.sup.d antibody to hepatic Kupffer
cells and splenic marginal zone B cells occurred within one hour,
in accordance with the report of Kennel et al. (1991). Presumably,
the extreme permeability of the discontinuous splenic endothelium
permits rapid extravasation of antibodies into the parenchyma of
this organ and staining of the marginal zone B-cells (Kennel et
al., 1991).
[0231] The reason for the lack of staining of renal and gut
epithelial cells is probably that these cells are not readily
accessible to intravenously-administered antibody because the
antibody would have to diffuse across basement membranes and
several tissue layers to reach these cells. In addition, it is
likely that all the remaining anti-Ia.sup.d antibody in the
circulation was absorbed by more accessible splenic white pulp
lymphocytes before significant extravasation into the red pulp
(Kennel et al., 1991; Fujimori et al., 1989) or other normal
tissues could occur. This is important because it illustrates a
potentially critical pharmacokinetic difference between vascular
targeting and tumor cell targeting. Because the tumor endothelial
cells are so accessible to intravenously-administered antibody, the
presence of a large `sink` of competing antigen in the blood or
lymphoid organs should not prevent the antibody from reaching the
target cells but should protect antigen-positive cells in most
extravascular compartments. It is conceivable that an antibody
recognizing a tumor vascular endothelial cell antigen that is
shared by epithelial cells, for instance, might be targeted without
the toxic side-effects which have complicated therapy with
anti-tumor cell immunoconjugates (Spitler, 1988). Furthermore, even
in the absence of such a sink, it is possible that operative
specificity for tumor endothelial cells could be achieved in the
face of cross-reactivity with extravascular normal tissues by
decreasing the dose or by using rapidly-cleared antibody fragments
in the construction of the immunoconjugate.
[0232] Although anti-Ia.sup.d antibody did not localize to
life-sustaining Ia.sup.d+ extravascular tissues such as kidney
tubules and gut epithelium, it did bind to colonic endothelial
cells in non-antibiotic-treated BALB/c nu/nu mice. These cells were
as accessible as tumor endothelial cells and were required for
survival since regular BALB/c nu/nu mice treated with high doses of
M5/114 immunotoxins died from intestinal damage. Murine endothelial
cells do not express MHC Class II antigens in vitro (O'Connell et
al., 1990; Duijvestijn et al., 1986) or in vivo (de Waal et al.,
1983) unless stimulated with IFN-.gamma. so it is likely that
induction of Ia.sup.d on intestinal endothelial and epithelial
cells was a result of local secretion of IFN-.gamma. by helper T
cells (Cherwinski, 1987) or activated NK cells (Anegon, 1988;
Kasahara, 1983) in response to gut flora. In accordance with this
view, numerous CD3+, CD8+ T cells were observed in the villous
stroma and their frequency correlated with the intensity of
staining of endothelial and epithelial cells with anti-Ia.sup.d
antibody. Furthermore, oral administration of tetracycline-Hcl (a
broad spectrum antibiotic) reversed T cell infiltration, diminished
Ia.sup.d expression and abolished localization of
intravenously-injected anti-Ia.sup.d antibody to colonic
endothelial cells.
[0233] Antibiotic treatment had no effect on Ia.sup.d expression by
tumor endothelial cells. In subsequent studies it was found that
SCID mice had little Ia.sup.d on colonic epithelial or endothelial
cells and that intravenously-administered anti-Ia.sup.d antibody
did not localize to their colonic endothelium. Furthermore, high
doses of M5/114 immunotoxins were non-toxic in these animals. Given
the possibility of antibiotic resistance arising in the gut flora
of tetracycline-treated BALB/c nu/nu mice, we believe that SCID
mice may be more suitable for these types of studies.
[0234] Consistent with the findings of others (Baxter et al., 1991;
Kennel et al., 1991; Jones et al., 1988; Pervez et al., 1988), an
anti-tumor antibody directed against the H-2K.sup.k antigen on
C1300 and C1300(Mu.gamma.) cells showed perivascular staining of
tumor cells after intravenous administration. In view of the
homogeneous expression of H-2K.sup.k by tumor cells in vitro and in
sections of subcutaneous tumors, it is likely that the uneven
intratumoral distribution of intravenously-injected anti-H-2K.sup.k
antibody was related to the vascular and interstitial physiology of
the tumor (Jain, 1990; Fujimori et al., 1989). This nicely
demonstrates, in a single system, the limitations of using
antitumor antibodies for targeting and the virtues of tumor
vascular targeting. It may be possible to combine both approaches
to advantage because the tumor cells that survive destruction of
intratumoral blood vessels are likely to be those at the periphery
of the tumor mass, close to the tumor-host interface. These areas
are likely to be well vascularized by capillaries in adjacent
normal tissues and have low interstitial pressure (Jain, 1990), so
the surviving cells should be amenable to attack by antitumor
immunoconjugates.
[0235] In summary, the inventors describe a murine model with which
to test the feasibility of targeting the vasculature of solid
tumors. The model permits the antitumor effects of immunoconjugates
directed against tumor vasculature to be compared with those of
immunoconjugates directed against the tumor cells themselves.
EXAMPLE II
Solid Tumor Therapy Using A Vascular Targeted Immunotoxin
[0236] This example describes the successful therapy of the solid
tumor model described in Example I, using the anti-tumor
endothelial cell immunotoxin, MS/114dgA, and the anti-tumor cell
immunotoxin, 11-4.1dgA, alone as well as in combination
therapy.
A. Materials and Methods
[0237] 1. Animals
[0238] BALB/c nu/nu mice were purchased from Simonsen (Gilroy,
Calif.). SCID mice were from the National Cancer Institute
(Bethesda, Md.). Germ-free SCID mice were from the University of
Wisconsin (Madison, Wis.). All animals were maintained in
microisolation units on sterilized food and water.
[0239] 2. Cells and Culture Conditions
[0240] All cell lines used in this study were cultured in modified
Eagle's medium (MEM) supplemented with 10% (v/v) fetal calf serum,
2.4 mM L-glutamine, 200 units/ml penicillin and 100 .mu.g/ml
streptomycin. Cultures were maintained at 37.degree. C. in a
humidified atmosphere of 90% air/10% CO.sub.2. The C1300
neuroblastoma line was established from a spontaneous tumor which
arose in an A/Jax mouse in 1940 (Dunham et al., 1953). The
C1300(Mu.gamma.)12 line, hereafter abbreviated to C1300
(Mu.gamma.), was derived by transfection of C1300 cells with murine
IFN-.gamma. gene using the IFN-.gamma. expression retrovirus PSVX
(Mu.gamma..delta.A.sub.s) (Watanabe et al., 1988), and was cultured
in MEM as above containing 1 mg/ml G418 (Geneticin, Sigma). Both
lines carry the MHC haplotype H-2K.sup.k, I-A.sup.k, I-E.sup.k,
D.sup.d. C1300 and C1300(Mu.gamma.) cells were grown in regular
tissue culture flasks or, when large quantities were required for
in vivo studies in cell factories (Baxter, Grand Prairie, Tex.).
Cells from subcutaneous tumors were recovered for in vitro analysis
by gentle mincing in MEM. After tumor cells had adhered overnight
the monolayers were washed twice with MEM to remove nonadherent
contaminant host cells.
[0241] Tumor conditioned media were prepared by seeding C1300 and
C1300(MuA.gamma.) cells at 25% of confluent density and culturing
them for four days. Conditioned media were dialyzed for 16 hours
against MEM without FCS to remove G418, filtered through a 0.22
.mu.M membrane and stored at 4.degree. C. for no more than one week
before assay. Aliquots of anti-IFN-.gamma. antibodies (see
`Monoclonal Antibodies`) sufficient to neutralize 200 international
units (I.U.) of murine IFN-.gamma./ml of conditioned medium were
added to some samples 24 hours before assay. The SVEC-10 murine
endothelial cell line, hereafter abbreviated to SVEC, was kindly
provided to Dr. M. Edidin, Department of Biology, Johns Hopkins
University, Baltimore, Md. and was derived by immortalization of
lymph node endothelial cells from a C3H (H-2.sup.k) mouse with SV40
(O'Connell et al., 1990). For some studies, SVEC cells were
cultured for 72 hours with 100 I.U./ml recombinant murine
IFN-.gamma., (r.IFN-.gamma., obtained from Dr. F. Balkwill,
Imperial Cancer Research Fund, London, England) or
tumor-conditioned medium. In addition, 200 I.U./ml anti-IFN-.gamma.
antibody was added to some flasks at the beginning of the 72 hour
culture period.
[0242] 3. Monoclonal Antibodies
[0243] The M5/114.15.2 (hereafter abbreviated to M5/114) and 11-4.1
hybridomas were purchased from the American Type Collection
(Rockville, Md.) and were grown in MEM-10% FCS. The antibodies were
purified from culture supernatant by precipitation in 50% ammonium
sulphate and affinity chromatography on Protein A. The rat IgG2b
antibody, M5/114, detects an Ia specificity on I-A.sup.b,
I-A.sup.q, I-A.sup.d, I-E.sup.d and I-E.sup.k molecules
(Bhattacharya et al., 1981). Thus, the antibody recognizes
I-E.sup.k molecules on SVEC (H-2.sup.k) cells and I-A.sup.d and
I-E.sup.d, hereafter referred to collectively as Ia.sup.d, on cells
from BALB/C nu/nu or SCID mice (both H-2.sup.d). The mouse IgG2a
antibody 11-4.1 recognizes H-2K.sup.k but not H-2K.sup.d molecules
(Oi et al., 1978) and so binds to H-2K.sup.k on C1300 and
C1300(Mu.gamma.) cells but is unreactive with MHC antigens from
BALB/c nu/nu or SCID mice. Isotype-matched control antibodies of
irrelevant specificity were CAMPATH-2 (rat IgG2b, anti-human CD7
(Bindon, 1988) and WT-1 (mouse IgG2a, anti-human CD7 (Tax et al.,
1984). Purified preparations of CAMPATH-2 and WT-1 were obtained
from Dr. G. Hale (Department of Pathology, Cambridge, England) and
Dr. W. Tax (Sint Radboudzeikenhuis, Nijmegen, the Netherlands)
respectively.
[0244] 4. Preparation of dgA
[0245] The ricin A chain was purified by the method of Fulton et
al. (Fulton et al., 1986). Deglycosylated ricin A was prepared as
described by Thorpe et al. (1985). For conjugation with antibodies,
the A chain was reduced with 5 Mm DTT and subsequently separated
from DTT by gel filtration on a column of Sephadex G-25 in PBS, pH
7.5.
[0246] 5. Preparation of Immunotoxins
[0247] IgG immunotoxins were prepared using the
4-succinimidyloxycarbonyl-.alpha.methyl(1-pyridyldithio)toluene
linking agent described by Thorpe et al. (1987).
4-succinimidyloxycarbonyl-.alpha.-methyl(2-pyridyldithio)toluene
dissolved in dimethylformamide was added to the antibody solution
(7.5 mg/ml in borate buffer, pH 9.0) to give a final concentration
of 0.11 Mm. After 1 hour the derivatized protein was separated from
unreacted material by gel chromatography on a Sephadex G-25 column
and mixed with freshly reduced ricin A chain. The solution was
concentration to about 3 mg/ml and allowed to react for 3 days.
Residual thiol groups were inactivated by treating the immunotoxin
with 0.2 Mm cysteine for 6 hours. The solution was then filtered
through a Sephacryl S-200 HR column in 0.1 M phosphate buffer, pH
7.5, to remove unreacted ricin A, cysteine, and aggregates.
Finally, the immunotoxin was separated from free antibody by
chromatography on a Blue Sepharose CL-6B column equilibrated in 0.1
M sodium phosphate buffer, pH 7.5, according to the method of
Knowles and Thorpe (1987).
[0248] 6. Cytotoxicity Assays
[0249] C1300, C1300(Mu.tau.) and SVEC cells suspended at 10.sup.5
cells/ml in MEM-10% FCS were distributed in 100 .mu.l volumes into
the wells of flat-bottomed microtiter plates. For some assays, SVEC
cells were suspended in C1300- or C1300(Mu.tau.)-conditioned medium
or MEM supplemented with 100 I.U./ml r.IFN-.tau. as indicated.
Immunotoxins in the same medium were added (100 .mu.l/well) and the
plates were incubated for 24 hours at 37.degree. C. in an
atmosphere of 10% CO.sub.2 in humidified air. After 24 hours, the
cells were pulsed with 2.5 .mu.Ci/well [.sup.3H] leucine for
another 24 hours. The cells were then harvested onto glass fiber
filters using a Titertek harvester and the radioactivity on the
filters was measured using a liquid scintillation spectrometer
(LKB; Rackbeta). The percentage of reduction in [.sup.3H] leucine
incorporation, as compared with untreated control cultures, was
used as the assessment of killing.
[0250] 7. Antitumor Studies
[0251] For the establishment of solid tumors, a mixture of
1.4.times.10.sup.7 C1300 cells and 6.times.10.sup.6 C1300 (Mu.tau.)
cells in 200 .mu.l MEM-30% FCS were injected subcutaneously into
the right anterior flank of BALB/c nu/nu or SCID mice. Fourteen
days later, when the tumors had grown to 0.8-1.2 cm in diameter,
the mice were separated into groups of 5-10 animals and injected
intravenously with 200 .mu.l of immunotoxins, antibodies or
diluent. Perpendicular tumor diameters were measured at regular
intervals and tumor volumes were estimated according to the
following equation (Steel, 1977). volume - Smaller .times. .times.
diameter 2 .times. larger .times. .times. diameter .times. .pi. 6
##EQU1## For histopathological analyses, animals were euthanized at
various times after treatment and the tumors were excised
immediately into 4% (v/v) formalin. Paraffin sections were cut and
stained with hematoxylin and eosin or Massons trichrome. B.
Results
[0252] The first studies carried out involved a comparison of
killing activity of anti-Class II immunotoxin (M5/114 dgA) against
unstimulated SVEC mouse endothelial cells with those stimulated
with conditioned medium from IFN-.gamma.-secreting tumor cells
(C1300 Mu.gamma.). These studies were carried out in order to
demonstrate that the anti-Class II immunotoxin, M5/114 dgA exerts a
selective toxicity against IFN-.gamma. stimulated endothelial
cells, and not against unstimulated cells. The results are shown in
FIGS. 4a and b. In FIG. 4a, SVEC mouse endothelial cells were
cultured in regular medium and the cultured cells subjected to
varying immunotoxin concentrations as indicated. As will be
appreciated, while ricin effected a 50% inhibition of leucine
incorporation at about 3.times.10.sup.-11, neither the anti-Class
II immunotoxin (M5/114 dgA) nor the control immunotoxin (CAMPATH-2
dgA) exerted a significant toxic effect within the concentration
ranges tested. In contrast, when the SVEC mouse endothelial cells
were stimulated by culturing in the presence of
C1300(Mu.gamma.)-conditioned medium, the mouse endothelial cells
became quite sensitive to the anti-Class II immunotoxin, with 50%
of stimulated cells being killed by the anti-Class II immunotoxin
at a concentration of about 3.times.10.sup.-10M. Thus, these
studies demonstrate that .gamma. interferon, which is produced by
the C1300(Mu.gamma.) and present in the conditioned media
effectively promote the appearance of Class II targets on the
surface of the SVEC cells.
[0253] FIG. 5 illustrates similar studies, which confirm the
finding that the C1300(Mu.gamma.) conditioned media effectively
promotes the expression of Class II molecules on endothelial cells.
In particular, the data shown in FIG. 5 demonstrate that both
recombinant IFN-.gamma. as well as conditioned media from
C1300(Mu.gamma.) sensitize endothelial cells to the anti-tumor
endothelial cell immunotoxin, M5/114 dgA. FIG. 5 also demonstrates
that conditioned media from C1300 cells that do not secrete
interferon (C1300 TCM), as well as interferon-producing C1300 cells
(Mu.gamma.) pretreated with anti-IFN-.gamma., both do not promote
an anti-Class II immunotoxin sensitizing effect.
[0254] Next, a series of studies were carried out wherein the
killing activity of an anti-Class I (anti-tumor) immunotoxin
(11-4.1-dgA) and that of the anti-Class II immunotoxin (M5/114-dgA)
are compared against a 70:30 mixed population of C1300 and
C1300(Mu.gamma.) cells. FIG. 6a simply demonstrates that in a 70:30
culture of C1300 and C1300(Mu.gamma.), that only the anti-Class I
immunotoxin, 11-4.1-dgA, and ricin, exert a cytotoxic effect. FIG.
6b shows killing of cells freshly recovered from subcutaneous
tumors in mice. Taken together, these FIGS. demonstrate that the
anti-tumor immunotoxin kills tumor cells well, but that the
anti-tumor endothelial cell immunotoxin does not. Thus, any
anti-tumor effect of M5/114-dgA would not likely be due to direct
tumor cell killing. Therefore, these studies serve as a control for
later studies wherein it is demonstrated that M5/114-dgA can have a
profound anti-tumor effect in the solid tumor model system
described in Example I, through an anti vascular effect.
[0255] FIG. 7 also shows a comparison of killing of pure and mixed
populations of C1300 and C1300(Mu.gamma.) by the anti-tumor cell
immunotoxin 11-4.1 dgA (anti-H-2K.sup.k) . Both FIGS. show the
effects of the anti-tumor cell immunotoxin against four different
tumor populations. Again, in each case the anti-tumor cell
immunotoxin demonstrate significant anti-tumor activity, at a
concentration of on the order of about 10.sup.-10M. Thus, these
data show that mixed tumors should be highly sensitive to the
anti-tumor immunotoxin, a control that is needed in order to
demonstrate the anti-vascular attributes of the anti-Class II
immunotoxin.
[0256] The next series of studies involved the application of one
or both of the foregoing anti-Class I and anti-Class II
immuno-toxins, in the model tumor system disclosed in Example I.
FIG. 8 illustrates the anti-tumor effects of the anti-tumor
endothelial cell immunotoxin, M5-114 dgA. As can be seen, dosages
as low as 20 .mu.g exhibited a noticeable antitumor effect. While
the change in mean tumor volume in the 20 .mu.g-treated population
does not, in FIG. 8, appear to be particularly dramatic, sections
of the tumor, when H & E-stained, illustrated surviving
"islands" of tumor cells in a "sea" of necrotic cells. This can be
seen in FIG. 9, wherein the surviving islands of tumor cells are
the darker staining areas, and the necrotic tissue the more
lightly-staining areas.
[0257] Importantly, treatment with 40 .mu.g of M5/115-dgA resulted
in dramatic anti-tumor effects, as can be seen in FIG. 8. Here, 30
days after tumor inoculation the mean tumor volume equated with the
16 day figure in the controls. The dotted line in FIG. 8 represents
the results that were expected with the use of 100 .mu.g of M5/114
dgA, with a possible reoccurrence of tumor cell indicated at the 26
day position being the partial result of a surviving rim of viable
cells observed in the treated solid tumor.
[0258] FIG. 10 is a section through a 1.2 cm tumor 72 hours after
treatment with 100 .mu.g of the anti-Class II immunotoxin M5/114
dgA, followed by H & E staining. As can be seen, this pattern
is similar to the 20 .mu.g data shown in FIG. 9, but certainly much
more dramatic in that virtually no "islands" of tumor cells remain.
It is estimated that this pattern represents a complete necrosis of
greater than 95% of the tumor diameter, leaving only a thin cuff of
surviving tumor cells, presumably nourished by vessels in overlying
skin.
[0259] To address this potential source of recurrence, i.e., the
potential for a cuff of surviving tumor cells, combined therapy
with both an antitumor (anti-Class I) and an anti-endothelial
(anti-Class II) immunotoxin was undertaken. The theory for this
combined therapeutic approach can be seen in FIG. 11, which
illustrates the appearance of the solid tumor following 48-72 hours
of intravenous immunotoxin treatment. At the left hand side of the
FIG. is represented a tumor following anti-tumor immunotoxin
therapy alone. As illustrated, only those areas immediately
surrounding the blood vessels become necrotic following treatment
with the anti-tumor immunotoxin, due to the inability of the
immunotoxin to sufficiently infiltrate the tumor and reach the
tumor cells that are distal of the blood vessels. In stark
contrast, shown in the middle panel of FIG. 11 is a representation
of the low dose treatment with anti-endothelial cell immunotoxin.
Here is illustrated the effects of a low dose of the
anti-endothelial cell immunotoxin, which results in necrosis of the
tumor in those parts distal of the blood vessels, except for the
outer rim of the tumor which is presumably fed by associated normal
tissues. At low dosages, only those areas of the tumor closest to
the blood vessels will receive sufficient oxygen and nutrients.
Next, the high dose anti-endothelial immunotoxin results are
illustrated on the right hand side of FIG. 11. Here, the only
living tumor remaining is that associated with the outer rim of the
tumor. It was a goal of combined therapy studies to demonstrate an
additive or even synergistic effect when both an anti-tumor
immunotoxin and anti-endothelial cell immunotoxin were employed in
combination. This effect is illustrated in the panel at the right
hand side of FIG. 11.
[0260] The results of this combination therapy are shown in FIG.
12. FIG. 12 shows the anti-tumor effects of the anti-tumor
immunotoxin (11-4.1-dgA) alone at a high dose, the anti-tumor
endothelial cell immunotoxin (M5/114-dgA) alone at a low dose, as
well as combinations of both. The results demonstrate that both
immunotoxins had a transient but noticeable effect in and of
themselves, with the anti-tumor immunotoxin showing a slightly
greater anti-tumor effect than the anti-tumor endothelial cell
immunotoxin, although this might be a dosing effect.
[0261] Truly dramatic synergistic results were seen when both were
used in combination. When 100 .mu.g of the anti-tumor immunotoxin
was given on day 14, followed by 20 .mu.g of the anti-tumor
endothelial cell immunotoxin on day 16, one out of four cures were
observed. When the order of administration was reversed, i.e., the
anti-tumor endothelial cell immunotoxin given first, even more
dramatic results were observed, with two out of four cures
realized. The latter approach is the more logical in that the
initial anti-endothelial cell therapy serves to remove tumor mass
by partial necrosis, allowing better penetration into the tumor of
the anti-tumor immunotoxin.
[0262] Therapeutic doses (.gtoreq.40 .mu.g) of M5/114-dgA did not
cause detectable damage to Class II-positive epithelial cells or to
hepatic Kupffer cells, as assessed by histopathological analysis at
various times after treatment. Any lymphoid cells destroyed by
M5/114-dgA were apparently replaced from bone marrow precursors
because, 20 days after treatment, all mature bone marrow cell
populations and splenic B cell compartments were normal.
C. Discussion
[0263] The findings from this model validate the concept of tumor
vascular targeting and, in addition, demonstrate that this strategy
is complimentary to that of direct tumor targeting. The theoretical
superiority of vascular targeting over the conventional approach
was established by comparing the in vivo antitumor effects of two
immunotoxins, one directed against tumor endothelium, the other
against the tumor cells themselves, in the same model. The
immunotoxins were equally potent against their respective target
cells in vitro but, while 100 .mu.g of the tumor-specific
immunotoxin had practically no effect against large solid
C1300(Mu.gamma.) tumors, as little as 40 .mu.g of the anti-tumor
endothelial cell immunotoxin caused complete occlusion of the tumor
vasculature and dramatic tumor regressions.
[0264] Despite causing thrombosis of all blood vessels within the
tumor mass, the anti-tumor endothelial cell immunotoxin was not
curative because a small population of malignant cells at the
tumor-host interface survived and proliferated to cause the
observed relapses 7-10 days after treatment. The proximity of these
cells to intact capillaries in adjacent skin and muscle suggests
that they derived nutrition from the extratumoral blood supply, but
the florid vascularization and low interstitial pressure in those
regions of the tumor rendered the surviving cells vulnerable to
killing by the anti-tumor immunotoxin (Jain, 1990; Weinstein &
van Osdol, 1992), so that combination therapy produced some
complete remissions.
[0265] The time course study demonstrated that the anti-Class II
immunotoxin exerted its antitumor activity via the tumor
vasculature since endothelial cell detachment and diffuse
intravascular thrombosis clearly preceded any changes in tumor cell
morphology. In contrast with the anti-tumor immunotoxin, the onset
of tumor regression in animals treated with the anti-tumor
endothelial cell immunotoxin was rapid. Massive necrosis and tumor
shrinkage were apparent in 48-72 hours after injection. Focal
denudation of the endothelial living was evident within 2-3 hours,
in keeping with the fast and efficient in vivo localization of
M5/114 antibody and the endothelial cell intoxication kinetics of
the immunotoxin (t 1/10=2 hours, t 1/2=12.6 hours.
[0266] As only limited endothelial damage is required to upset the
hemostatic balance and initiate irreversible coagulation, many
intratumoral vessels were quickly thrombosed with the result that
tumor necrosis began within 6-8 hours of administration of the
immunotoxin. This illustrates several of the strengths of vascular
targeting in that an avalanche of tumor cell death swiftly follows
destruction of a minority of tumor vascular endothelial cells.
Thus, in contrast to conventional tumor cell targeting,
anti-endothelial immunotoxins could be effective even if they have
short serum half lives and only bind to a subset of tumor
endothelial cells.
[0267] MHC Class II antigens are also expressed by B-lymphocytes,
some bone marrow cells, myeloid cells and some renal and gut
epithelia in BALB/c nu/nu mice, however, therapeutic doses of
anti-Class II immunotoxin did not cause any permanent damage to
these cell populations. Splenic B cells and bone marrow myelocytes
bound intravenously injected anti-Class II antibody but early bone
marrow progenitors do not express Class II antigens and mature bone
marrow subsets and splenic B cell compartments were normal 3 weeks
after therapy, so it is likely that any Ia.sup.+ myelocytes and B
cells killed by the immunotoxin were replaced from the stem cell
pool. It is contemplated that the existence of large numbers of
readily accessible B cells in the spleen prevented the anti-Class
II immunotoxin from reaching the relatively inaccessible Ia.sup.+
epithelial cells but hepatic Kupffer cells were not apparently
damaged by M5/114-dgA despite binding the immunotoxin. Myeloid
cells are resistant to ricin A-chain immunotoxins, probably due to
unique endocytic pathways related to their degradative physiologic
function (Engert et al., 1991).
[0268] No severe vascular-mediated toxicity was seen in the studies
reported here because mice were maintained on oral antibiotics
which minimized immune activity in the small intestine.
[0269] The findings described in this example demonstrate the
therapeutic potential of the vascular targeting strategy against
large solid tumors. As animal models for cancer treatment are
widely accepted in the scientific community for their predictive
value in regard to clinical treatment, the invention is also
intended for use in man. Numerous differences between tumor blood
vessels and normal ones have been documented (Denekamp, 1990; Jain,
1988) and are envisioned to be of use in practicing this invention.
Tumor endothelial markers may be induced directly by tumor-derived
angiogenic factors or cytokines (Ruco et al., 1990; Burrows et al.,
1991) or could relate to the rapid proliferation (Denekamp &
Hobson, 1982) and migration (Folkman, 1985a) of endothelial cells
during neovascularization. Candidate anti-tumor endothelial cell
antibodies include FB-5, against endosialin (Rettig et al., 1992),
and E9 (Kumar et al., 1993) which are reportedly highly selective
for tumor vascular endothelial cells. Two related antibodies
developed by the present inventors, TEC-4 and TEC-11, against
carcinoma-stimulated human endothelial cells, show strong
reactivity against vascular endothelial cells in a wide range of
malignant tumors but little or no staining of vessels in benign
tumors or normal tissues. Vascular targeting is therefore
envisioned to be a valuable new approach to the therapy of
disseminated solid cancers for which there are currently no
effective treatments.
EXAMPLE III
Targeting the Vasculature of Breast Tumors
[0270] This example describes an approach for targeting the
vasculature of breast cancer and other solid tumors in humans. This
approach is exemplified through the use of bispecific antibodies to
selectively induce the activation antigens, Class II and ELAM-1, on
the vascular endothelial cells of syngeneic breast tumors in mice
and then targeting these antigens with immunotoxins.
[0271] Murine models may first be employed. The results from such
studies will be understood to parallel the situation in humans, as
mouse models are well accepted and routinely employed for such
purposes. Following successful vascular targeting in the mouse,
success in man is likely as highly specific anti-breast cancer
antibodies are available (Denekamp, 1984; Girling et al., 1989;
Griffin et al., 1989; Lan et al., 1987; Boyer et al., 1989).
[0272] In the case of clinical (as opposed to diagnostic
applications), the central issue is to confine the expression of
the induced target antigen to tumor vasculature. In the case of
Class II, which is present on the vasculature of normal tissues in
mice and humans (Natali et al., 1981, Daar et al., 1984;
Hammerling, 1976), the objective is to suppress its expression
throughout the vasculature and then selectively induce it on tumor
vasculature. In the case of ELAM-1, which is absent from the
vasculature of normal tissues (Cotran et al., 1976), the objective
is to induce its expression selectively on tumor vasculature.
A. Overview
[0273] 1. Selective Induction of Class II Expression on Tumor
Vasculature
[0274] C3H/He mice will be injected subcutaneously with syngeneic
MM102 mammary tumor cells. The tumor cells express Ly6.2 which is a
unique marker in C3H mice (Ly6.1 positive). Mice bearing solid
MM102 mammary tumors will be treated with CsA to reduce or abolish
Class II expression throughout the vasculature. As originally shown
in the dog (Groenewegen et al., 1985), and, as recently confirmed
by the inventors in the mouse, CsA inhibits T cell and NK cell
activation and lowers the basal levels of IFN-.gamma. to the extent
that Class II disappears from the vasculature. The mice will then
be injected with a bispecific (Fab'-Fab') anti-CD28/anti-Ly6A.2
antibody, which should localize to the tumor by virtue of its
Ly6.2-binding activity. The bispecific antibody should then bind to
T cells which are present in (or which subsequently infiltrate
(Blanchard et al., 1988) the tumor. Crosslinking of CD28 antigens
on the T cells by multiple molecules of bispecific antibody
attached to the tumor cells should activate the T cells via the
CsA-resistant CD28 pathway (Hess et al., 1991; June et al., 1987;
Bjorndahl et al., 1989). Activation of T cells should not occur
elsewhere because the crosslinking of CD28 antigens which is
necessary for activation (Thompson et al., 1989; Koulova et al.,
1991) should not occur with soluble, non-tumor cell bound,
bispecific antibody. T cells which become activated in the tumor
should release IFN-.gamma. which should induce Class II antigens on
the tumor vascular endothelium (Collins et al., 1984; Pober et al.,
1983) and probably on the tumor cells themselves (Boyer et al.,
1989). Animals will then be treated with anti-Class II immunotoxins
to destroy the tumor blood supply.
[0275] 2. Induction of ELAM-1 Expression on Tumor Vasculature
[0276] Mice bearing solid MM102 mammary tumors will be injected
with bispecific (Fab'-Fab') anti-CD14/anti-Ly6A.2 antibody. The
antibody should localize in the tumor by virtue of its
Ly6.2-binding activity. It should then activate monocytes and
macrophages in the tumor by crosslinking their CD14 antigens
(Schutt et al., 1988; Chen et al., 1990). The activated
monocytes/macrophages should have tumoricidal activity (Palleroni
et al., 1991) and release IL-1 and TNF which should rapidly induce
ELAM-1 antigens on the tumor vascular endothelial cells (Bevilacqua
et al., 1987; Pober et al., 1991). A monoclonal antibody to mouse
ELAM-1 will be generated and used as an immunotoxin to destroy the
tumor blood supply.
B. Study Design and Methods
[0277] 1. Suppression of Class II Expression [0278] a) Mouse
Mammary Tumors, MM102 and MM48
[0279] The tumors preferred for use are the mouse mammary (MM)
tumors which have been extensively characterized by Dr. Reiko Irie
and colleagues (Irie, 1971; Irie et al., 1970). The inventors have
obtained from Dr. Irie (UCLA School of Medicine, CA) two
transplantable tumors, MM102 and MM48. The MM102 line derives from
a spontaneous mammary tumor which originated in a C3H/He mouse. The
MM102 tumor carries an antigen which is closely related to, or
identical to, Ly6A.2 (Seto, et al., 1982). Since the C3H/He mouse
expresses Ly6.1 and not Ly6.2, this marker is tumor-specific in
syngeneic mice. The MM48 tumor, a variant of the original tumor,
lacks Ly6A.2 and provides a specificity control in the proposed
studies. Both tumors form continuously-growing solid tumors when
injected into the subcutaneous site. [0280] b) Monoclonal
Antibodies
[0281] For targeting the Ly6A.2 antigen, the inventors have
obtained the anti-Ly6A.2 hybridoma, S8.106, from Dr. Ulrich
Hammerling (Memorial Sloan-Kettering Cancer Center, N.Y.). This
hybridoma secretes a mouse IgG.sub.2a antibody (Kimura, et al.,
1980) which has been shown to react specifically with MM102 and
other Ly6A.2 expressing MM tumors (Seto, et al., 1982).
[0282] An appropriate anti-mouse CD28 antibody (Gross, et al.,
1990) is that obtainable from Dr. James Allison (University of
California, CA). Ascitic fluid from hybridoma-bearing animals is
also available for synthesizing the bispecific antibody. The
antibody is a hamster IgG.
[0283] Isotype-matched negative control antibodies will be the WT1
antibody (anti-human CD7) which is a mouse IgG.sub.2a and a hamster
IgG of irrelevant specificity from the ATCC.
[0284] Antibodies will be purified on staphylococcal Protein A
coupled to Sepharose, or by ion exchange and size exclusion
chromatography on Sepharose 4B as described by Ghetie, et al.
(1988). The ability of the purified anti-Ly6A.2 antibody to bind to
MM102 cells and of the anti-CD28 antibody to bind mouse T cells
will be confirmed by FACS analyses as described by Burrows et al.,
(1991).
[0285] Purified antibodies will be filtered through 0.22 .mu.m
membranes, aliquotted, and stored at -70.degree. C. [0286] c)
Preparation of Fab' Fragments
[0287] F(ab').sub.2 fragments of purified anti-Ly6A.2 and anti-CD28
antibodies will be prepared by pepsin digestion, as described by
Glennie et al. (1987). Purified antibodies (5-10 mg) will be
dialyzed against 0.1 M sodium acetate, pH 4.1, and digested with 4%
(w/w) pepsin at 37.degree. C. The digestion will be followed by
SDS-PAGE and terminated by raising the pH when optimal digestion is
achieved. Undigested IgG will be removed by chromatography on a
Sephacryl S-200 column equilibrated with PBS. The F(ab').sub.2
fragments will be analyzed by SDS-PAGE and, if detectable levels of
undigested antibody should remain, the F(ab').sub.2 fragments will
be further purified by removal of undigested antibody on a Protein
A-Sepharose column. Fab' fragments will be prepared from
F(ab').sub.2 fragments by reduction with 5 mM DTT for 1 hr at
25.degree. C., followed by removal of free DTT on a Sephadex G-25
column equilibrated against phosphate-EDTA buffer (Glennie et al.,
1987) [0288] d) Preparation of Anti-Ly6A.2/Anti-CD28 Bispecific
Antibodies
[0289] For the production of anti-Ly6A.2-anti-CD28 bispecific
antibodies, Fab' fragments of each antibody will be initially
prepared as above and will be left unalkylated. Heterodimer
molecules will be prepared as described by Glennie et al. (1987).
Fab' fragments will be reduced with DTT in 0.2 M Tris-HCl buffer,
pH 8.0, containing 10 Mm EDTA for 60 min. at room temperature. One
of the Fab' fragments will be then reacted with Ellman's reagent (2
mM) for 1 hour at room temperature in acetate buffer, pH 5.0. The
free Ellman's reagent will be separated using a Sephadex G-25
column. The derivatized Fab' fragment will be then mixed with the
other reduced Fab' and allowed to react at room temperature for 24
hours. Bispecific antibodies will be separated from remaining Fab'
fragments by gel filtration over Sephacryl S-200 columns. [0290] e)
Confirmation of Cell Binding-capacity of Anti-Ly6A.2/Anti-CD28
Bispecific-antibody
[0291] FACS analyses will be performed to verify the dual
cell-binding capacity of the bispecific antibody. MM102 tumor cells
(grown as an ascites) will be treated for 30 minutes at 4.degree.
C. with the bispecific antibody (10 .mu.g/10.sup.6 cells) and
washed. The tumor cells will then be incubated with fluoresceinated
goat anti-hamster immunoglobulin for 30 minutes at 4.degree. C. and
washed again. The fluorescence associated with the cells will then
be measured using the FACS. Positive staining of tumor cells coated
with bispecific antibody and lack of staining of cells coated with
anti-Ly6A.2 antibody alone will confirm that the bispecific
antibody is intact and is capable of binding tumor cells. The study
will be repeated using a CD28 positive mouse T cell lymphoma line
(e.g., EL4) and with fluoresceinated goat anti-mouse immunoglobulin
as the detecting antibody to confirm that the bispecific antibody
has CD28-binding capacity. [0292] f) Activation of T Cells by
Anti-Ly6A.2/Anti-CD28 Bispecific Antibody Plus MM102 Tumor
Cells
[0293] It will be important to confirm that tumor cells coated with
the bispecific antibody, but not free bispecific antibody, are able
to activate T cells in a CsA-resistant fashion. T cells will be
enriched from the spleens of C3H/He mice by depleting B-cells and
macrophages according to the procedure of Lee and colleagues 1990
(Lee, et al., 1990). Spleen cells are treated with mouse anti-Class
II antibody and the Class II-expressing cells are removed by
treating them with goat anti-mouse IgG-coupled magnetic beads and
withdrawing them with a strong magnet. The non-adherent cells are
decanted and are treated further to remove residual B cells and
macrophages by successive rounds of treatment with anti-J11D plus
BRC and anti-MAC-1 antibody plus goat anti-rat serum. After these
procedures, the remaining cells are .gtoreq.95% T cells and <3%
Ig positive.
[0294] T cells will be cultured (0.5 to 1.times.10.sup.5 cells/0.2
ml) in medium in the wells of 96-well plates. Various
concentrations of anti-CD28 IgG, anti-CD28 Fab' or
anti-Ly6A.2/anti-CD28 bispecific antibody will be added together
with various concentrations of one of the following costimulants:
PMA, IL1 or anti-CD3 IgG. CsA (0.5 .mu.g/ml) will be added to an
identical set of cultures. The cultures will be incubated at
37.degree. C. for 3 days, .sup.3H-thymidine (1 .mu.Ci/culture) will
be added and the plates harvested 24 hours later. These studies
should confirm that bivalent anti-CD28, but not monovalent Fab'
anti-CD28 or the bispecific antibody, stimulate T cells and that
the stimulation is not CsA inhibitable.
[0295] Next, MM102 and MM48 cells, obtained from ascitic tumors of
C3H/He mice, will be treated with mitomycin C (25 .mu.g/ml) for 20
minutes at 37.degree. C. The cells will then be washed and the
above study repeated with the inclusion of 0.5 to 1.times.10.sup.5
mitomycin-treated MM102 or MM48 cells along with the T cells in the
cultures. The MM102 cells, but not the MM48 cells, should present
the bispecific antibody to the T cells and, together with the
costimulant, induce their stimulation. [0296] g) Confirmation that
Injection of Anti-Ly6A.2/Anti-CD28 Bispecific Antibody into
CsA-treated MM102 Tumor-bearing Mice Results in Induction of Class
II Selectively on Tumor Vasculature
[0297] C3H/He mice will be injected subcutaneously with 10.sup.6
MM102 or MM48 tumor cells. One day later they will start daily
treatments with CsA (60 mg/kg/day) given either orally dissolved in
olive oil or injected intraperitoneally. After 10-14 days, when the
tumors will have reached 1.0-1.3 cm in diameter, and when Class II
will have disappeared from the vasculature, mice will be injected
with 50-100 .mu.g of anti-Ly6A.2/anti-CD28 bispecific antibody.
Other mice will receive various control treatments, including
unconjugated anti-Ly6A.2 or anti-CD28 (Fab' and IgG) or diluent
alone. Two or three days later, the mice will be sacrificed and the
tumors and various normal tissues will be removed for
immunohistochemical examination. Frozen sections will be cut and
stained for the presence of Class II antigens and for the presence
of hamster immunoglobulin using indirect immunoperoxidase
techniques, as presented in the foregoing examples.
[0298] Upon demonstration that Class II antigens are strongly and
selectively expressed on the vasculature of MM102 tumors but not on
MM48 tumors, the tumor therapy studies below will be carried out.
If Class II antigens are absent from tumor vasculature but hamster
immunoglobulin is present, this would indicate that the bispecific
antibody had localized to the tumor, as anticipated from prior
studies with analogous bispecific antibodies (Perez, et al., 1985;
Garrido, et al., 1990), but that T cell activation had not occurred
sufficiently for IFN-.gamma. secretion to ensue. If so, the
presence of T cells will be verified by staining frozen tumor
sections with anti-CD28 and anti-CD3 antibodies. If T cells are
present, again as would be anticipated from prior studies (Koulova,
et al., 1991; Perez, et al., 1985), the failure to get Class II
induction might be attributable to the need for two signals for T
cell activation, i.e. a 2nd signal might be missing. This will be
checked by coadministering an anti- Ly6A.2/anti-CD3 bispecific
antibody, which together with the anti-Ly6A.2/anti-CD28 bispecific,
should provide the signalling needed for T cell activation. [0299]
h) Synthesis of Anti-class II-SMPT-dgA Immunotoxin
[0300] Immunotoxins directed against murine class II MHC molecules
will be prepared by linking the rat monoclonal anti-murine
I-A.sup.k antibody to deglycosylated ricin A (dgA) using the
disulfide cleavable crosslinker, SMPT (Thorpe, et al., 1988).
Affinity purified antibody molecules will be derivatized by
reaction with a five-fold molar excess of SMPT in borate buffer, pH
9.0, for 60 min. at room temperature. Free SMPT will be removed by
passage through a Sephadex G-25 column equilibrated against
phosphate buffered saline containing EDTA (1 mM, PBSE). Under these
conditions, an average of 1.7 molecules of SMPT are introduced per
immunoglobulin molecule. Next, the derivatized antibody will be
allowed to react with reduced dgA for 72 hrs. at room temperature.
Under these conditions, immunoconjugates form through formation of
disulfide linkage between sulfhydryl groups on dgA molecules and
SMPT. Immunoconjugates will be separated from free dgA by gel
filtration in Sephacryl S-200 columns and from unreacted antibody
by passage through Blue-Sepharose and elution with phosphate buffer
containing 0.5M NaCl. Purity of immunoconjugates will be assessed
by SDS-PAGE. [0301] i) Tumor Therapy Studies
[0302] C3H/He mice will be injected subcutaneously with 10.sup.6
MM102 or MM48 tumor cells and, one day later, will start daily
treatments with CsA (60 mg/kg/d). When the tumors have grown to
1.0-1.3 cm diameter, the mice will receive an intravenous injection
of 50-100 .mu.g anti-Ly6A.2/anti-CD28 bispecific antibody (perhaps
together with anti-Ly6A.2/anti-CD3 bispecific antibody if indicated
by the studies in Section (h) above). Two or three days later, 100
.mu.g of the anti-Class II immunotoxin will be administered
intravenously. Anti-tumor effects will be monitored by measuring
the size of the tumors at regular intervals and by histological
examination as in Section C. The specificity of any anti-tumor
effects will be established by comparing the anti-tumor effects
with those in mice which receive various control treatments,
including unconjugated anti-Ly6A.2 Fab' and IgG, unconjugated
anti-CD28 Fab' and IgG, and anti-Class II immunotoxin alone. [0303]
2. Induction of ELAM-1 on Tumor Vasculature by
Anti-Ly6A.2/Anti-CD14 Bispecific Antibody [0304] a) Raising of
Anti-ELAM-1 Monoclonal Antibodies [0305] i) Induction of ELAM-1 on
SVEC Cells for Immunization
[0306] Expression of cytokine-induced adhesion molecules on SVEC
murine endothelial cells will be induced by stimulation of SVEC
cell monolayers with a cocktail of rMuIL-1.beta. (50 I.U./ml),
rMuTNF.alpha. (100 IU/ml) and bacterial endotoxin (100 ng/ml) for 4
hrs at 37.degree. C., as described for the induction of human
ELAM-1 (Bevilacqua, et al., 1987). Preliminary evidence suggests
that SVEC cells activated in this manner express murine ELAM-1,
since radiolabeled U937 cells, which bear the ELAM-1 agent, display
increased adhesion to activated SVEC cells within 2 hrs of the
addition of the cytokines. The increased endothelial cell
adhesiveness peaked at 4-6 hrs., as previously reported for human
ELAM-1. Although cytokine-activated SVEC cells also displayed
long-term (up to 48 hrs.) adhesiveness to U937 cells, this was
probably not due to ICAM-1/LFA-1 or VCAM-1/VLA-4 interactions,
since the assays were carried out at 4.degree. C., under shear
stress conditions, which inhibits any adhesive interactions other
than those between selections and their carbohydrate agents
(Spertini, et al., 1991). Increased adhesiveness at the later time
points was probably mediated by the selection LAM-1 (Mel-14) on
U937 cells and its agent (the MECA 79 antigen) on SVEC cells (81).
In subsequent studies, this pathway will be blocked by the
inclusion of Mel-14 and/or MECA 79-specific antibodies in the
adhesion assays (Imai, et al., 1991). [0307] ii) Immunization
[0308] Rat monoclonal antibodies will be raised against inducible
proteins on mouse endothelial cells. SVEC cells will be stimulated
for 6 hrs., as previously described, before immunization of Wistar
rats. The rats will be boosted three weeks following the initial
injection with identically-prepared SVEC cells. Serum from the
injected rats will be tested for the presence of antibodies
specific for induced proteins on endothelial cells 7-10 days after
the second boost, using FACS analysis of induced and non-induced
SVEC cells. Additional boosting and screening will be repeated as
necessary.
[0309] Once antibody levels have been detected in acceptable
titers, rats will be given a final boost with induced SVEC cells
and their spleens removed after 3 days. Splenocytes will be fused
with Y3 Ag1.2.3 rat myeloma cells according to standard protocols
using poly-ethylene glycol 4000 (82). Hybridomas will be selected
using HAT medium and the supernatants screened using FACS analysis.
[0310] iii) Screening
[0311] Those hybridomas secreting antibodies reacting with
cytokine-induced but not with resting SVEC cells will be selected
for further characterization. Reactivity will be assessed by
indirect immunofluorescence with hybridoma supernatants and
FITC-labeled mouse anti-rat immunoglobulin antibodies. The selected
hybridomas will be expanded, and the immunoglobulin purified from
their culture supernatants. Confirmation of ELAM-1 reactivity will
be carried out as described below. [0312] iv) Characterization of
Antigens
[0313] The physicochemical properties of the precipitated antigens
will be investigated after activating SVEC cells with cytokines in
the presence of cycloheximide or tunicamycin and by
immunoprecipitation of antibody-reactive molecules from lysates of
.sup.35S-methionine-labeled SVEC cells, using the selected MAbs.
Immunoprecipitates will be subsequently analyzed by SDS-PAGE.
Confirmation of murine ELAM-1 reactivity will be carried out by
comparison of the precipitated material and human ELAM-1 using
SDS-PAGE, one-dimensional proteolytic maps with staphylococcal V8
protease and NH.sub.2-terminal sequences. [0314] b) Preparation of
Anti-Ly6A.2-anti-CD14 Bispecific Antibodies
[0315] Bispecific antibodies will be constructed using Fab'
fragments derived from anti-Ly6A.2 and anti-CD14 monoclonal
antibodies, essentially as described in the previous section.
Several anti-mouse CD14 monoclonal antibodies have been raised
(23). We feel it is premature to approach these workers with a view
to establishing a collaboration until we have raised anti-mouse
ELAM-1 monoclonals and verified their performance as immunotoxins.
[0316] c) Synthesis and Cytotoxicity Testing of Anti-ELAM-1
Immunotoxins
[0317] Immunotoxins directed against murine ELAM-1 will be
constructed by cross-linking monoclonal anti-mouse ELAM-1
antibodies (as characterized above) to dgA using SMPT. The
procedure involved will be identical to that described in the
previous sections. Activity will be assessed by toxicity studies
with cytokine-activated SVEC cells. [0318] d) Confirmation of
ELAM-1 Induction on Tumor Vasculature and Not on Normal
Vasculature
[0319] C3H/He mice bearing 1.0-1.3 cm MM102 or MM48 tumors will be
injected i.v with anti-Ly6A.2/anti-CD14 bispecific antibody or with
various control materials including unconjugated anti-Ly6A.2 and
anti-CD14 antibodies (Fab' and IgG) and diluent alone. Tumors will
be removed at various times and cryostat sections will be cut and
stained with rat monoclonal antibodies to murine ELAM-1, using
standard indirect immunoperoxidase techniques. The presence of the
bispecific antibody on tumor cells will be verified by staining for
rat immunoglobulin. Resident macrophages and infiltrating monocytes
will be detected by indirect immunoperoxidase staining with
anti-Mac-1 (CD 11b/CD 18) antibodies. Cytokine-producing cells will
be identified in serial cryostat sections of tumors by in situ
hybridization with .sup.35S-labeled antisense asymmetric RNA probes
for murine IL-1.beta. and TNF.alpha. mRNA. [0320] e) Tumor Therapy
Studies
[0321] C3H/He mice bearing 1.0-1.3 cm MM102 or MM48 tumors will be
injected with 50-100 .mu.g anti-Ly6A.2/anti-CD14 bispecific
antibody or with various control materials including unconjugated
anti-Ly6A.2 and anti-CD14 antibodies (Fab' and IgG) and diluent
alone. One to three days later, the mice will receive intravenous
injections of anti-ELAM-1 immunotoxin, an isotype-matched
immunotoxin of irrelevant specificity or unconjugated anti-ELAM-1
antibody. Anti-tumor effects will be monitored by measuring the
size of the tumors at regular intervals and by histological
examination, as in the preceding examples.
[0322] Mice will be injected subcutaneously with 10.sup.6 MM102 or
MM48 tumor cells (in 0.1 ml saline) either on the abdominal wall or
on the flank. In some studies, cyclosporin A (60 mg/kg/day) will be
injected intraperitoneally or given in the drinking water. The mice
will be observed daily thereafter and the dimensions of the tumor
will be measured. When the tumor reaches a diameter of 1.0-1.3 cm,
the mice will receive an injection of bispecific antibody (0.1 ml
in saline) into a tail vein and then 2-3 days later will receive an
intravenous injection of immunotoxin, again into the tail vein. The
study is terminated by euthanizing the mice when their tumors reach
1.5-2 cm in diameter in any dimension.
[0323] Each group will comprise 8-10 animals, and there will
generally be 5 or 6 treatment groups making a total of 40-60 mice
per study. One such study will be performed per month. [0324] f)
Raising Monoclonal Antibodies
[0325] Adult Wistar rats will be used. Rats will be immunized by
injecting them i.m with mouse endothelial cells (SVECs) homogenized
in 0.1 ml of a 50:50 mixture of Freund's incomplete adjuvant and
saline. Rats will be boosted 1 month and 2 months later in the same
manner. 7-10 days after the second boost, 0.1 ml blood will be
removed from tail vein and the serum will be analyzed for the
presence of antibody. If sufficiently positive, the rats will be
given a final i.m boost with SVEC cells and 3 days later, the rats
will be euthanized their spleens dissected out for monoclonal
antibody production. [0326] g) Raising Ascites
[0327] BALB/c nude mice will be injected intraperitoneally with 0.5
ml Pristane (2, 6, 10, 14-tetramethylpentadecane) 2 to 4 weeks
before being injected intraperitoneally with rat hybridoma cells.
The mice will be weighed daily and euthanized when their body
weight increases by 20% or more due to hybridoma growth in the
peritoneal cavity. The contents of the peritoneal cavity will then
be drained and monoclonal antibodies purified from the ascitic
fluid. [0328] h) Choice of Species and Number of Animals
[0329] Mice: The antitumor effects of immunotoxins in animals
cannot be predicted from tissue culture studies. Such factors as
hepatic entrapment, blood clearance rates and binding to serum
components makes it essential that intact animal systems are used
for evaluation. The choice of mice as the test animal is determined
by the fact that inbred strains exist in which mammary tumors will
grow reproducibly. The number of animals (8-10) per treatment group
is the minimum for statistically significant differences between
different treatment groups to become apparent. The number of
treatment groups (5-6) per study is the minimum for an effect of a
specific immunotoxin to be distinguished from an effect of its
components (antibody alone, ricin A chain alone, or mixtures of the
two) and for superiority over control immunotoxin of irrelevant
specificity to be demonstrated.
[0330] Rats: Since antibodies are to be raised to mouse endothelial
cell antigens, it is best to use another species for immunization.
Rats are preferred for these studies because they are inbred and
respond consistently to the immunogen.
EXAMPLE IV
Identification and Characterization of the Tumor Endothelial Cell
Marker, Endoglin, and Antibodies thereto
[0331] This example describes the generation of two new monoclonal
antibodies, TEC-4 and TEC-11, directed against a tumor vasculature
antigen. TEC-4 and TEC-11 are shown to recognize endoglin, which is
shown to be associated with growth and proliferation of human tumor
endothelial cells in vitro and in vivo. Endoglin is selectively
upregulated on vascular endothelial cells in a broad range of
malignant tumors and is envisioned to provide a suitable marker for
use in the diagnosis and therapy of miscellaneous solid tumors.
A. Materials and Methods
[0332] 1. Cells and Culture Conditions
[0333] Cell lines were obtained from the Imperial Cancer Research
Fund, London (U.K.) Tissue Bank unless otherwise indicated. SP2/0
murine myeloma cells, SAOS osteosarcoma cells and the ECV-304 human
endothelial cell line were obtained from the American Type Culture
Collection, Rockville, Md. A375M and T8 human melanoma lines were
obtained from Dr. I. R. Hart (Imperial Cancer Research Fund,
London, U.K.). NCI-H146 and SCC-5 human lung cancer cell lines were
obtained from Dr. J. D. Minna (U.T. Southwestern Medical Center,
Dallas, Tex.). L428 and L540 human Hodgkins/Sternberg-Reed cells
were obtained from Dr. A. Engert (Department of Medicine, Cologne,
Germany).
[0334] The murine endothelial cell lines used were SV40-transformed
mouse lymph node endothelial cells (SVEC, O'Connell et al., 1990),
obtained from Dr. M. Edidin (Johns-Hopkins, Baltimore, Md.); LEII,
obtained from Dr. W. Risau (Max-Planck Institute, Martinsried,
Germany); and mouse pulmonary capillary endothelial cells (MPCE)
obtained from Prof. A. Curtis (Department of Cell Biology, Glasgow,
U.K.).
[0335] Primary endothelial cell cultures from chinese hamster
epididymal fat pad and bovine heart were established using the
methods described by Bjorntorp et al. (Bjorntorp et al., 1983) and
Revtyak et al. (Revtyak et al., 1988).
[0336] Human umbilical vein endothelial cells (HUVEC) were isolated
from fresh tissue by the method of Jaffe et al. (Jaffe et al.,
1973) or were purchased from Clonetics Corp., San Diego, Calif.
Cultures were maintained in gelatin-coated flasks in Medium 199
(Gibco-Biocult, Ltd., Paisley, U.K.) supplemented with Earle's
salts, 20% (v/v) fetal calf serum, endothelial cell growth
supplement (ECGS, 0.12 mg/ml), 0.09 mg/ml heparin, glutamine and
antibiotics at 37.degree. C. in 5% CO.sub.2 in air or in
Endothelial Growth Medium (Clonetics) at 37.degree. C. in 10%
CO.sub.2 in air. All other cell lines were maintained in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal
calf serum, 2.4 mM L-glutamine, 200 units/ml penicillin, 10
.mu.g/ml streptomycin, 100 .mu.M non-essential amino acids, 1 .mu.M
Na Pyruvate and 18 .mu.M HEPES at 37.degree. C. in 10% CO.sub.2 in
air.
[0337] Murine L-cell transfectants expressing human endoglin were
produced as described by Bellon et al. (1993). [0338] 2.
Antibodies
[0339] The F8/86 mouse IgG1 anti-human von-Willebrands Factor
antibody (Naieum et al., 1982) was purchased from DAKO Ltd (High
Wycombe, U.K.) and used at 1:50 dilution. A mouse IgG1 anti-human
vitronectin receptor antibody, LM142 (Cheresh, 1987) was obtained
from by Dr. D. Cheresh (Scripps Clinic, La Jolla, Calif.). The
mouse IgG1 anti-rat Thy 1.2 antibody (Mason and Williams, 1980) was
used as an isotype matched control for F8/86 and LM142. The mouse
myeloma proteins TEPC-183 (IgM.sub.k) and antibody MTSA
(IgM.sub.k), which do not react with human tissues, were used as
negative controls for TEC-4 and TEC-11. The mouse IgG1
anti-endoglin antibody 44G4 (Gougos and Letarte, 1988) has been
described previously. [0340] 3. Immunoprecipitation
[0341] HUVEC were metabolically labelled by overnight incubation in
0.1 mCi/10.sup.6 cells .sup.35S-methionine in methionine-free RPMI
supplemented with 20% (v/v) fetal calf serum, ECGS, glutamine
heparin and antibiotics as described above. Monolayers were washed
twice in PBS and adherent cells were lysed in NET buffer (50 mM
Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5% NP-40) supplemented
with 0.1 M iodoacetamide to inhibit disulfide exchange.
[0342] For immunoprecipitation, preformed immunocomplexes were used
as described by Li et al. (1989). 0.2 mg of purified MAb in 200
.mu.l of NET buffer was mixed with 250 .mu.l of GAMIg (goat
anti-mouse immunoglobulin) serum and incubated for 30 min. at room
temperature. Immunocomplexes were pelleted by centrifugation and
the pellet was washed two times in 300 .mu.l of NET buffer. One ml
of lysate from 5.times.10.sup.7 125I-labeled cells was mixed with
immunocomplexes (prepared as above) on a rotator at 4.degree. C.
for 1 hr. The lysate containing the immunocomplexes was layered on
top of a discontinuous sucrose gradient consisting of 750 .mu.l
each of 40 (bottom), 30, 20, and 10% (top) sucrose in
12.times.75-mm tubes. Tubes were centrifuged at 600 g for 20 min.
at room temperature. The sucrose was decanted and the pellets were
resuspended in a small amount of PBS. Samples were transferred to
new tubes and then centrifuged. The pellet was boiled for 1 min. in
sample buffer with or without 2-mercaptoethanol (2-ME) and
electrophoresed on 12.5% slab gels for 16 hr at 15 mA. The gels
were dried and exposed to film for 24 hr at -70.degree. C. [0343]
4. Complement Fixation
[0344] The ability of TEC-4 and TEC-11 to fix complement was
assessed as follows. Primary antibody incubations and washing were
performed as for indirect immunofluorescence. After the third wash,
HUVEC were resuspended in 50 .mu.l of a 1/10 dilution of guinea pig
complement (ICN, High Wyecombe, U.K.) for 20 min. at 37.degree. C.,
at which point 50 .mu.l of 0.25% (w/v) trypan blue was added and
cell number and plasma membrane integrity were estimated visually.
[0345] 5. Indirect Immunofluorescence
[0346] Tumor cells and endothelial cells were prepared for FACS
analyses as described by Burrows et al. (1991). All manipulations
were carried out at room temperature. 50 .mu.l of cell suspension
at 2-3.times.10.sup.6 cells/ml in PBS-BSA-N.sub.3 were added to the
wells of round-bottomed 96 well microtiter plates (Falcon 3910).
Primary antibodies (10 .mu.g/ml) were added in 50 .mu.l volumes,
and the plates sealed. After 15 min., the cells were washed 3 times
by centrifuging the plates at 800.times.g for 30 s, removing the
supernatants by flicking and patting dry on absorbent tissue, and
resuspending the cells in 150 .mu.l/well PBS-BSA-N.sub.3.
Fluorescein isothiocyanate (FITC)-conjugated rabbit antibodies
against mouse immunoglobulins (DAKO Corp., Carpinteria, Calif.),
diluted 1:20 in PBS-BSA-N.sub.3, were distributed in 50 .mu.l
volumes into the wells. The cells were incubated for a further 15
min. and washed as before. Cell-associated fluorescence was
measured on a FACScan (Becton-Dickenson, Fullerton, Calif.). Data
were analyzed using the LYSYSII program. The mean fluorescence
intensity (MFI) of cells treated with the control antibody, MTSA,
was subtracted from that of cells treated with TEC-4 or TEC-11 to
obtain the specific MFI attributable to antigen binding.
[0347] For competitive binding inhibition assays, biotinylated
TEC-4 or TEC-11 antibodies were mixed with unlabelled TEC-4, TEC-11
or control antibodies at ratios of 1:1, 1:10 and 1:100. Indirect
immunofluorescent staining of HUVEC was carried as before except
that biotinylated antibodies bound to the cells were detected with
a 1:50 dilution of streptavidin-phycoerythrin (Tago, Inc.,
Burlingame, Calif.). Percent blocking of biotinylated antibodies
was calculated as follows: % .times. .times. blocking = MFI .times.
.times. in .times. .times. presence .times. .times. of .times.
.times. blocking .times. .times. Ab ( background .times. .times.
MFI ) MFI .times. .times. in .times. .times. presence .times.
.times. of .times. .times. non .times. - .times. specific AB
.function. ( background .times. .times. MFI ) ##EQU2##
[0348] Indirect immunofluorescence of L-endoglin transfectants was
carried out as described by Bellon et al. (1993). Parental L cells
and L cell transfectants expressing human endoglin
(1.times.10.sup.6 cells) were incubated for 45 min. at 4.degree. C.
with MAb 44G4 IgG (10 ug/ml), TEC-4 (10 .mu.g/ml) or TEC-11 (10
.mu.g/ml). Cells were then washed and incubated with
FITC-conjugated F(ab').sub.2 goat anti-mouse IgG (H+L)(Tago).
[0349] 6. Cellular Protein and Nucleic Acid Analyses
[0350] HUVEC were stained with TEC-11 at 20 .mu.g/ml as described
in `Indirect Immunofluorescence`, and sorted into TEC-11.sup.lo and
TEC-11.sup.hi populations using a FACStar Plus cell sorter.
[0351] Total cellular protein content was estimated from the uptake
of free FITC as described by Stout and Suttles (1992). Sorted cells
were centrifuged at 150.times.g, 5 min., and fixed in cold 70%
ethanol for 15 min. The cells were pelleted as before, resuspended
in PBS containing 20 .mu.g/ml FITC and incubated on ice for 60 min.
before being washed and analyzed on the FACStar Plus. The ethanol
fixation effectively removed any prior fluorescent label as
evidenced by the lack of detectable fluorescence of controls which
were not incubated with FITC.
[0352] Acridine orange staining of DNA and RNA was performed
according to a method adapted from that of Darzynkiewicz et al.
(Darzynkiewicz et al., 1976). Sorted cells were centrifuged at
150.times.g for 5 min. and resuspended at 1.5.times.10.sup.6
cells/ml in 200 .mu.l of DMEM/10% FCS. 0.5 ml 0.15 M
citrate/phosphate buffer, pH 3.0, containing 0.1% (v/v)
Triton-X-100 (Sigma), 0.2 M sucrose and 0.1 mM EDTA was added and
the cells were maintained at 4.degree. C. Immediately before
analysis, 0.5 ml 0.15 M citrate/phosphate buffer, pH 3.8,
containing 1 M NaCl and 0.002% (w/v) acridine orange (Polysciences
Inc., Warrington, Pa.) was added. After 5 min. at room temperature,
the fluorescence intensities of individual cells were measured in
the FACStar Plus. Free nuclei and cell doublets were excluded from
the data collection. Green and red fluorescence were plotted
against each other in a 2-dimensional dot plot that enables cells
to be assigned to different stages of the cell cycle (G.sub.0,
G.sub.1, S, G.sub.2+M) according to relative DNA and RNA content
(Darzynikiewicz et al., 1976). [0353] 7. Immunohistochemistry
[0354] Human and animal tissue samples were snap-frozen over liquid
nitrogen, mounted in OCT Compound (Miles, Inc., Elkhart, Ind.) and
8 .mu.M sections were cut in a Tissuetek 2 cryostat (Baxter) onto
slides precoated with 3-aminopropyltriethoxysilane (Sigma).
Sections were stored at -80.degree. C. until required. Indirect
streptavidin-biotin immunoperoxidase staining was carried out as
follows. Sections were air-dried at room temperature for 30 min.,
fixed in acetone for 15 min., rehydrated in PBS for 5 min. and
incubated in a humidified chamber for 45-60 min. with purified
primary antibodies at 10 .mu.g/ml in PBS-0.2% BSA or with undiluted
culture supernatant. After 2 washes in PBS, the sections were
incubated for 30-45 min. with biotinylated F(ab').sub.2 sheep
anti-mouse IgG (H+L) (Sigma #B-6774) diluted 1:200 in PBS-0.2% BSA.
After a further 2 washes in PBS, the sections were incubated for
30-45 min. with streptavidin-biotin-horseradish peroxidase complex
(Strept ABC Complex, DAKO #K377) diluted 1:100 in PBS-0.2% BSA.
After a final 2 washes, the reaction product was developed using
0.5 mg/ml 3',3'-diaminobenzidine (DAB, Sigma) or 1 mM
3-amino-9-ethylcarbazole (AEC, Sigma) containing 0.01% (v/v)
hydrogen peroxide. The sections were counterstained with Mayer's
hematoxylin (Sigma) for 10-30 s, washed in tap water and mounted in
CrystalMount (Biomeda Corp., Foster City, Calif.).
B. Results
[0355] 1. Production of TEC-4 and TEC-11 Antibodies
[0356] The TEC4 and TEC11 monoclonal antibodies were raised and
selectively screened in the following manner. HT-29 human colonic
adenocarcinoma cells were obtained from the Imperial Cancer
Research Fund Central Tissue Bank and seeded at 25% of confluent
density in tissue culture flasks in Dulbeccos Modified Eagles
Medium (DMEM) supplemented with 10% v/v fetal calf serum (FCS), 2.4
mM L-glutamine, 200 units/ml penicillin and 100 .mu.g/ml
streptomycin. The cells were allowed to grow to full confluence
over 4-5 days incubation at 37.degree. C. in a humidified
atmosphere of 90% air/10% CO.sub.2 before the supernatant tissue
culture medium (hereafter referred to as HT-29 tumor-conditioned
medium, HT-29 TCM) was removed, filtered through a 0.22 .mu.M
filter to ensure sterility and to remove any particulate matter,
and stored at 4.degree. C. for no more than one week before use.
HT-29 human adenocarcinoma cells were used to prepare TCM because
they had previously been shown to secrete angiogenic into their
culture medium (Rybak et al., 1987) and angiogenic has been found
to induce neovascularization (i.e., profound alteration of
endothelial cell behavior) in an in vivo assay (Fett et al.,
1987).
[0357] Human umbilical vein endothelial cells (HUVEC) were
incubated in Medium 199 supplemented with 20% w/v FCS, glutamine
and antibiotics and mixed 1:1 with HT-29 TCM. After 48-72 hours at
37.degree. C. the endothelial cells were harvested
non-enzymatically, using a rubber policeman, and 1-2.times.10.sup.6
cells were injected intraperitoneally into a BALB/c mouse. This
entire procedure was repeated three times at two to three week
intervals, the final injection being by the intravenous route.
[0358] Three days later, splenocytes from the immunized animal were
fused with SP2/O murine myeloma cells at a ratio of 1:2 using
PEG2000 (Kohler and Milstein, 1975; Tazzari et al., 1987). The cell
mixture was introduced into the wells of 96-well flat bottomed
microtiter plates along with 3.times.10.sup.4 syngeneic peritoneal
feeder cells per well. Twenty-four hours later 100 .mu.l of medium
containing hypoxanthine, ammopterin and thymidine (HAT Medium) was
added to select for fused cells (hybridomas). The cultures were fed
with additional HAT Medium at 3 day intervals.
[0359] When hybridomas had grown to high density, 50 .mu.l samples
of supernatant were taken and screened by galactosidase
anti-galactosidase (GAG) ELISA (Burrows et al., 1991) for
antibodies reactive with HT-29-activated HUVEC. All positive wells
were seeded into 24-well plates, expanded by further culture in HAT
medium, and retested 7-10 days later by the same technique.
Antibodies that bound to HUVEC in the ELISA were further tested for
reactivity with HUVEC cell surface determinants by FACS (see
`Indirect Immunofluorescence`). All positive wells were harvested
and samples stored in liquid nitrogen. The remaining cells from
each positive well were cloned in 96 well plates by the limiting
dilution method (Kohler and Milstein, 1975).
[0360] When the clones had grown to high density, 50 .mu.l samples
of supernatant were taken and assayed by GAG ELISA against HT-29
TCM-activated HUVEC and `resting` HUVEC grown in the absence of
tumor-derived factors. Any wells which showed significantly greater
reactivity with HT-29 TCM-activated HUVEC than with control HUVEC
were recloned and expanded to culture flasks to provide adequate
supernatant for subsequently screening for lack of reactivity with
quiescent HUVEC in frozen sections of human umbilical vein (see
`Immunohistochemistry`).
[0361] Supernatants from these expanded clones were screened by
standard indirect immunoperoxidase techniques (Billington and
Burrows, 1987) by sequential incubation with F(ab).sub.2 sheep
anti-mouse IgG (1:200, Sigma) and streptavidin-biotin-horseradish
peroxidase complex (1:100, Dakopatts) against a small panel of
normal and malignant human tissues on cryostat sections. After this
round of screening two clones, 1G4 (TEC4) and 2G11 (TEC11), were
selected for further study on the basis of significantly greater
reactivity with endothelial cells in sections of solid tumors than
with those in sections of normal tissues.
[0362] Both TEC4 and TEC11 antibodies were isotyped as IgM using
the Immunotype.TM. mouse monoclonal antibody isotyping kit (Sigma
Chemical Co., St. Louis, Mo.). TEC-4 and TEC-11 were purified from
tissue culture supernatant by ammonium sulfate precipitation
followed by Sephacryl S-300 size-exclusion chromatography and
affinity chromatography on a Mannose-Binding Protein column (Pierce
Chemical Co., Rockford, Ill.). TEC-4 and TEC-11 antibodies were
biotinylated by incubation with a 40-fold molar excess of
N-hydroxy-succinimidobiotin amidocaproate (Sigma) for 1 h at room
temperature followed by dialysis against 2 changes of PBS. [0363]
2. Evidence That TEC-4 and TEC-11 Recognize Endoglin
[0364] TEC-4 and TEC-11 immunoprecipitated a molecule that migrated
as a 95 kDa species when analyzed on SDS-polyacrylamide gels under
reducing conditions (FIG. 13(a), lanes 2-4). When isolated and
analyzed under non-reducing conditions, the molecule moved
predominantly as a 180 kDa homodimer (FIG. 13(a), lanes 5-7). These
biochemical characteristics were consistent with those described
previously for endoglin; indirect immunofluorescence analysis of
the binding of TEC-4 and TEC-11 to murine L-cells transfected with
human endoglin was therefore performed to determine whether the
antibodies indeed recognize endoglin. As shown in FIG. 13(b),
TEC-4, TEC-11 and the reference anti-endoglin antibody, 44G4, all
reacted strongly with endoglin-transfectant L-cells but were
unreactive with parental L-cells. [0365] 3. Cross-blocking of TEC-4
and TEC-11 Antibodies
[0366] Binding to L-endoglin transfectants (FIG. 13) and reciprocal
preclearing before immunoprecipitation confirmed that TEC-4 and
TEC-11 react with the same protein. Competitive inhibition of
binding to HUVEC was carried out to determine whether TEC-4 and
TEC-11 recognize the same or different epitopes on the endoglin
molecule (FIG. 14).
[0367] TEC-4 and TEC-11 blocked themselves in a dose dependent
manner, with TEC-11 being slightly more efficient that TEC-4 (FIG.
14). TEC-11 partially inhibited binding of TEC-4 by 51% at a ratio
of 1:1, but increasing the ratio of TEC-4:TEC-11 to 1:10 did not
increase the blocking. By contrast, TEC-4 inhibited TEC-11 binding
by only 1% at 1:1, but this blocking increased in a dose-dependent
fashion to reach 20% at a TEC-11:TEC-4 ratio of 1:100 (FIG. 14).
The blocking effects were specific since neither TEC-4 nor TEC-11
blocked the binding of the anti-vitronectin receptor antibody LM142
to HUVEC, nor did LM142 interfere with TEC-4 or TEC-11 binding.
[0368] The epitopes recognized by TEC-4 and TEC-11 were distinct
from that of 44G4, because 44G4 did not block TEC-4 or TEC-11, even
at a ratio of 100:1.
[0369] Taken together, the above results indicate that (i) TEC-4
and TEC-11 recognize distinct but spatially close epitopes on the
endoglin molecule, because neither antibody inhibited the binding
of the other as efficiently or as completely as it blocked itself,
and (ii) TEC-11 antibody has a greater affinity for endoglin than
does TEC-4, because blocking of TEC-4 binding by TEC-11 was more
efficient than the reciprocal event. [0370] 4. Complement Fixation
by TEC-4 and TEC-11 Antibodies
[0371] TEC-11 fixed complement approximately 100-fold more
efficiently than did TEC-4, inducing lysis of >50% of HUVEC at a
concentration of 1 .mu.g/ml (FIG. 15). TEC-4 displayed no
complement fixation activity at 1 .mu.g/ml and lysed only 48% of
HUVEC at a concentration of 100 .mu.g/ml under these conditions.
The superior complement-fixing activity of TEC-11 was due, at least
in part, to its greater affinity, which resulted in greater binding
of TEC-11 than TEC-4 to HUVEC at low antibody concentrations. The
concentration of TEC-11 which produced half-maximal fluorescence in
FACS analyses was 0.4 .mu.g/ml as compared with 4 .mu.g/ml for
TEC-4. [0372] 5. Reactivity of TEC-4 and TEC-11 with Normal and
Malignant Human Cell Lines
[0373] Binding of TEC-4 and TEC-11 to endothelial cells from
several species and a panel of non-endothelial human cell lines was
determined by indirect immunofluorescence and cytofluorimetry. The
results are shown in Table IV. Essentially identical results were
obtained with TEC-4 and TEC-11. Substantially higher levels of
staining were detected on HUVEC than on any other cell tested in
this study. The specific mean fluorescence intensity (MFI) of TEC-4
and TEC-11 on HUVEC was around 200.
[0374] A human endothelial cell line, ECV-304, which was derived
from HUVEC (Kobayashi et al., 1991), gave lower MFI values of
35-45. ECV-304 cells also displayed diminished expression of
several other endothelial cell markers, including EN4 antigen,
angiotensin-converting enzyme and CD34. Endothelial cells of
bovine, murine and chinese hamster origin displayed no detectable
reactivity with either TEC-4 or TEC-11 antibodies. U937 cells were
weakly labelled. Among the tumor cells, all lymphoma/leukemia lines
were negative, as were the majority (5/7) of carcinoma lines.
Interestingly, all 4 melanoma and sarcoma lines tested were weakly
stained by TEC-4/TEC-11, with MFI values of 12-26. In addition, 2/4
breast cancer lines bound the antibodies weakly. TABLE-US-00004
TABLE IV Endoglin expression in tissue culture Endoglin Cell type
Cell line(s) expression.sup.1 Endothelial HUVEC ++++ Endothelial
ECV-304 ++ Endothelial BCA, CHEC, LE II, - MPCE, SVEC Myeloid U937
+ Myeloid U266 - Leukemia/lymphoma ARH-77, CEM, Daudi, - Gaynor
L428, L540, Nalm-6, K562 Sarcoma HT-1080, SAOS-2 + Melanoma A375M,
T8 + Carcinoma LOVO, NCI-H146, SCC-5 - Breast tumor MDA-MB-231,
SKBR3 + Breast tumor MCF-7, T 47D - Fibroblast L-endoglin +++
transfectant .sup.1Expressed as the corrected fluorescence
intensity (CFI), calculated as described in the Materials and
Methods where a CFT of <5 is represented by (-), 5-25 (+), 25-50
(++), 50-100 (+++) and >100 (++++).
[0375] 6. Correlation Between Endoglin Expression and Endothelial
Cell Proliferation in vitro
[0376] TEC-4 and TEC-11 bound only weakly to quiescent HUVEC in
situ in frozen sections of human umbilical vein (see following
section). Endoglin expression reached high levels within 16 hours
after the HUVEC were removed from the umbilical cord and placed
into tissue culture and was not abolished in HUVEC cultures grown
to confluence or deprived of serum or growth factors. However,
confluent cultures displayed a bimodal distribution of TEC-4/TEC-11
binding by FACS (FIG. 16).
[0377] To characterize HUVEC populations expressing low and high
levels of endoglin, confluent HUVEC cultures were stained with
TEC-11 and sorted on a FACStar Plus cell sorter. The sorted
populations were analyzed for total cellular protein content and
relative DNA and RNA levels. The results of these studies are shown
in FIG. 16 and Table V.
[0378] HUVEC from sparse cultures expressed uniformly high endoglin
levels, as shown in FIG. 16a, hatched histogram, but when the same
cells were grown to confluence and allowed to become partially
quiescent over an additional 3-6 days, a bimodal distribution of
TEC-11 binding was seen (FIG. 16a, open histogram). This pattern of
TEC-11 binding was not simply a reflection of variations in cell
size among the postconfluent HUVEC, because the expression of a
control marker (the vitronectin receptor) was not significantly
altered between HUVEC from sparse and postconfluent cultures (FIG.
16b). Postconfluent cells stained with TEC-11 were sorted into two
fractions as indicated in FIG. 16a. Endoglin.sup.hi cells had a
specific MFI 4.4 times greater than that of endoglin.sup.lo cells
(Table V). Similarly, there was a 1.6-fold increase in binding of
free FITC to cellular proteins in permeabilized endoglin.sup.hi
cells by comparison to low endoglin expressors (Table V).
Upregulated protein synthesis in the endoglin.sup.hi population
reflected similar increases in cellular transcription, as indicated
by acridine orange staining. While endoglin.sup.lo cells formed a
single population with low relative RNA and DNA content (FIG. 16c),
significant numbers of endoglin.sup.hi cells showed evidence of RNA
and DNA synthesis consistent with cellular activation and
proliferation (FIG. 16d). Indeed, when the sorted populations were
separated into zones in the dot plots (FIG. 16c,d) according to
standard criteria (Darzynkiewicz et al., 1976), virtually all
endoglin.sup.lo cells were assigned to the non-cycling (G.sub.0)
population but, by contrast, 15% and 5% of endoglin.sup.hi cells
were located in the G.sub.1 (activated) and S+G.sub.2/M
(proliferating) fractions respectively (Table V). TABLE-US-00005
TABLE V Correlation between endoglin expression and endothelial
cell proliferation in vitro. Relative Stage of cell cycle (%).sup.3
Fraction MFI.sup.1 protein.sup.2 G.sub.0 G.sub.1 S + G.sub.2/M
Endoglin.sup.lo 61.6(2.4) 100 95.8(2.4) 3.1(1.4) 1.1(1.4)
Endoglin.sup.hi 270.2(3.8) 156 79.1(5.8.sup. 15.5(2.9) 5.4(1.2)
.sup.1Mean fluorescence intensity of cells stained with 20 .mu.g/ml
TEC-11. Mean and standard deviation (in parentheses) of 2 studies.
.sup.2Estimated from non-specific binding of FITC to cellular
proteins in permeabilized cells. .sup.3Estimated from 2-dimensional
dot-plot after acridine orange staining as described in Materials
and Methods and shown in FIG. 13. Mean and standard deviation (in
parentheses) of 2 studies.
[0379] 7. TEC-4 and TEC-11 Binding to Malignant and Normal Human
Tissues [0380] a) Endothelial Cells in Miscellaneous Tumors
[0381] TEC-4 and TEC-11 binding to vascular endothelial cells was
assessed by immunoperoxidase staining of a panel of 51
miscellaneous human tumors. The results are shown in Table VI and
FIGS. 17 and 18.
[0382] Both antibodies clearly stained the cytoplasm and luminal
plasma membranes of vascular endothelial cells in a large majority
of the tumors examined, including extensive series of breast and
colorectal carcinomas. TEC-4 and TEC-11 reacted with capillaries
and venules but not with arterioles in 20/22 evaluable cases. The
reactivity patterns of the two antibodies were very similar,
although TEC-11 tended to produce more intense staining than TEC-4
in some tissues (Table VI). In most tumor samples, a large majority
(80-100%) of vessels which stained with the positive control
anti-endothelial cell antibody (anti-von Willebrands Factor) also
stained moderately to strongly with TEC-4 and TEC-11. Often, TEC-4
and TEC-11 gave more uniform staining of capillaries than did the
anti-von Willebrands antibody. TEC-4 and 11 binding was variable
both between and within histological tumor types.
[0383] Vascular endothelial cells were most strongly stained by
TEC-4 and TEC-11 in sections of angiosarcoma, Hodgkins disease and
colon, cecum and rectosigmoid carcinoma (Table VI). Parotid tumors
(FIG. 17) and some breast carcinomas (FIGS. 17 and 18) also
contained heavily-labelled vessels. Moderate staining of vascular
endothelium was characteristic of pharyngeal, lung and ovarian
carcinomas (Table VI). Vessels in soft tissue tumors (melanoma and
osteosarcoma) were stained moderately by TEC-11 but only weakly by
TEC-4. Lymphoma samples examined showed little or no staining with
either antibody. TEC-4 and TEC-11 reactivity was restricted to
human tissues--neither antibody stained endothelial cells in a
variety of mouse, rat, guinea pig and hamster tumors, nor were
vessels labelled in human tumor xenografts in nude mice.
TABLE-US-00006 TABLE VI Endoglin expression on endothelial cells in
miscellaneous tumors Antibody Tumor type n anti-vWF TEC-4 TEC-11
Angiosarcoma 1 +++.sup.1 +++ +++ Benign breast 6 +++ +/- - tumor
Breast carcinoma 12 +++ ++ ++/+++ Cecum carcinoma 1 +++ +++ +++
Colon carcinoma 3 +++ ++ +++ Hodgkins disease 11 +++ ++ +++
Lymphoma 2 +++ +/- + Lung carcinoma 1 +++ ++ ++ Melanoma 1 +++ + ++
Osteosarcoma 1 +++ + ++ Ovarian 1 +++ ++ ++ carcinoma Parotid tumor
3 +++ +++ +++ Pharyngeal 2 +++ ++ ++ carcinoma Rectosigmoid 6 +++
++/+++ +++ carcinoma .sup.1Staining intensity was strong (+++),
moderate (++), weak (+) or negative (-).
[0384] b) Endothelial Cells in Normal Tissues
[0385] A panel of 27 normal human tissues was used to assess the
reactivity of TEC-4 and TEC-11 with vascular endothelial cells in
non-malignant settings. The staining conditions used were ones that
gave distinct staining of tumor vasculature. As shown in Table VII,
the staining of normal endothelium obtained with both antibodies
was usually weak or negative, but there was moderate staining of
capillary endothelium in adrenal gland and placenta (both
antibodies), parathyroid (TEC-4) and lung, cervix, testis, kidney
and lymphoid organs (TEC-11). In addition, TEC-4 displayed strong
binding to skin vessels (Table VII). Endothelial cells in numerous
tissues showed no detectable staining with one or both antibodies,
including bladder, brain, cranial nerve, mammary gland (FIG. 18b),
ovary, pancreas, stomach, thymus and umbilical vein (FIG. 17d).
[0386] Several `normal` tissue samples were in fact adjacent
non-malignant tissue from cancer biopsies, enabling staining of
endothelial cells in normal and malignant areas of the same organ
to be compared in the same section. FIG. 17 (a and b) shows a
sample of parotid tumor and associated histologically normal
parotid gland where a marked difference in the staining of vascular
endothelial cells by TEC-4 antibody is visible. In the stroma
between the nests of malignant cells, all endothelial cells were
heavily labelled (FIG. 17a). By contrast, only light staining of a
single vessel in the normal glandular tissue was discernible (FIG.
17b). TABLE-US-00007 TABLE VII Endoglin expression on endothelial
cells in non-neoplastic tissues Antibody anti-vWF TEC4 TEC11 Normal
tissues Adrenal +++.sup.1 ++ ++ Bladder ++ - - Brain cortex ++ - -
Brain stem ++ - - Cerebellum +++ - - Colon +++ ++ + Cranial nerve
+++ - - Gall bladder +++ .+-. + Kidney +++ .+-. .+-. Liver +++ + +
Lung +++ .+-. ++ Mammary gland +++ - - Ovary +++ .+-. - Pancreas +
- - Parathyroid ++++ ++ - Parotid gland +++ .+-. .+-. Placenta +++
++ ++ Prostate +++ .+-. .+-. Salivary gland +++ .+-. .+-. Skin +++
+++ + Stomach +++ +/- + Stomach muscle +++ - + Testis +++ - .+-.
Thymus +++ - - Thyroid +++ + .+-. Tonsil +++ + + Umbilical vein +++
+/- +/- Inflammatory tissues Tonsilitis +++ ++ +++ Reactive +++ +/-
+ hyperplasia Cat-scratch +++ + ++ fever Ulcerative +++ ++ +++
colitis .sup.1Staining intensity was strong (+++), moderate (++),
weak (+) or negative (-).
[0387] c) Endothelial Cells During Tumor Progression in the
Breast
[0388] Endothelial cells in normal mammary glands were not stained
by either TEC-4 or TEC-11 (FIG. 18b) whereas endothelial cells in
malignant breast tumors were stained moderately to strongly by both
antibodies (FIG. 17c and Table VI). A series of abnormal breast
tissues was therefore examined to determine at which stage of
neoplastic progression endoglin expression was initiated. Vessels
in 6/6 benign fibroadenomas displayed little or no reactivity with
TEC-4 and TEC-11 (Table VI), as did those in low-grade hyperplastic
lesions and early carcinoma-in-situ. Moderate staining was apparent
in late-stage intraductal carcinomas whereas all vessels in frank
malignant carcinomas were heavily labelled with both TEC-4 and
TEC-11 (FIG. 17c, FIG. 18d, Table VI). [0389] d) Endothelial Cells
in Inflammatory Sites
[0390] TEC-4 and TEC-11 staining of endothelial cells was strong in
3/4 types of inflammatory tissues examined (Table VII). TEC-11
staining was somewhat stronger than TEC-4 staining in all cases. In
6/8 cases of tonsillitis, cat-scratch fever and ulcerative colitis,
which are all associated with neovascularization, endothelial cells
were stained moderately to strongly with TEC-11. By contrast,
vessels were weakly stained in 2 cases of reactive hyperplasia, a
non-angiogenic condition. [0391] e) Non-endothelial Cells
[0392] Both TEC-4 and TEC-11 showed highly restricted binding to
cryostat sections of normal and malignant human tissues, reacting
primarily with vascular endothelial cells. However, both antibodies
displayed cross-reactivities, typically quite weak, with certain
non-endothelial cell types (Table VIII). Both antibodies bound
weakly to stromal components in the prostate, the basal layer of
seminiferous tubules, and to follicular dendritic cells in lymphoid
organs and in small lymphoid deposits associated with colorectal
tumors (Table VIII). Strong staining of syncytiotrophoblast in
placenta was also seen. TEC-11 gave a more restricted staining
pattern than did TEC-4 (Table VIII), which also bound to
myoepithelial cells in the breast, smooth muscle cells, especially
in the gut, and to miscellaneous epithelial tissues including some
rectal glandular epithelium, epidermis and breast carcinoma cells
in a minority of samples. [0393] 7. Selective Cytotoxic Effects of
TEC-11 Immunotoxin
[0394] An immunotoxin was prepared by chemical linkage of TEC-11
antibody with deglycosylated ricin A-chain, as described in detail
in Example II. The immunotoxin was tested for cytotoxicity against
quiescent, confluent and subconfluent populations of human
endothelial cells in a standard protein synthesis inhibition assay,
as described in Example II. For comparison, the same endothelial
cell cultures were also treated with an isotype-matched non-binding
immunotoxin (MTSA-dgA), an immunotoxin against an endothelial cell
antigen (ICAM-1) whose expression does not vary according to the
growth status of the cells (UV-3-dgA) and native ricin.
[0395] The results of several such assays are combined and shown in
FIG. 19 and Table IX. The negative and positive controls, MTSA-dgA
(FIG. 19a) and ricin (FIG. 19c), respectively, gave essentially
identical cytoxicity profiles against all three endothelial cell
populations. Ricin inhibited protein synthesis by 50% (IC.sub.50)
in quiescent, confluent and subconfluent cultures at concentrations
of 0.15-0.27 pM (FIG. 19c; Table IX) and MTSA-dgA displayed no
cytotoxicity to any endothelial cell population at concentrations
below 0.1 .mu.m (FIG. 19a and Table IX).
[0396] The anti-ICAM-1 immunotoxin, UV-3-dgA, was weakly but
clearly cytotoxic to endothelial cells at all stages of growth,
with IC.sub.50 values ranging from 9 to 80 nM (FIG. 19b and Table
IX). As expected from their higher metabolic rate and consequent
requirements for protein synthesis, the proliferating subconfluent
cultures were almost 9-fold more sensitive to UV-3dgA than were the
quiescent cells (Table IX). The confluent cultures were fully
metabolically active but were 4-fold less sensitive to UV-3-dgA
than were the subconfluent cells (Table IX), probably reflecting
decreased accessibility of the target antigen due to the dense
packing of the cells in confluent cultures.
[0397] By contrast, TEC-11-dgA showed striking differences in
cytotoxicity towards quiescent, confluent and subconfluent
endothelial cells (FIG. 19d and Table IX). TEC-11-dgA at a
concentration of 10-8 M inhibited protein synthesis in quiescent
cultures by only 10% and in confluent cultures by only 20%, (FIG.
19d) and yet inhibited protein synthesis in subconfluent cultures
by over 60% at concentrations as low as 10.sup.-10 M (FIG. 19d).
When the IC.sub.50 values for TEC-11-dgA towards the different
endothelial cell cultures were compared, it was found that the
immunotoxin was 2400-fold more toxic to subconfluent cells than to
confluent cultures. When sparse and quiescent cells were compared,
this ratio rose to over 3000 (Table IX).
[0398] Taken together, these results demonstrate that an
immunotoxin prepared from the TEC-11 antibody displays highly
selectively cytotoxicity towards subconfluent, actively
proliferating human endothelial cells. TABLE-US-00008 TABLE VIII
Reactivity of TEC-4 and TEC-11 with non-endothelial cells Antibody
Cell type TEC-4 TEC-11 Tumor tissue Angiosarcoma Sarcoma ++ ++
Breast carcinoma Carcinoma + - Myoepithelium ++ - Colorectal
carcinoma Smooth muscle +/++ - Hodgkins disease Follicular
dendritic + + cells Various Stroma +/- +/- Normal tissue Gut Smooth
muscle +/++ - Liver Bile duct + - Lymphoid tissues Follicular
dendritic + + cells Mammary gland Myoepithelium ++ - Placenta
Syncitiotrophoblast ++ ++ Prostate Fibromuscular stroma ++ + Skin
Epidermis +/- - Testis Basal layer of + + seminiferous tubules 1.
Staining intensity was strong (+++), moderate (++), weak (+) or
negative (-).
[0399] TABLE-US-00009 TABLE IX Immunotoxin cytotoxicity to
endothelial cells at different stages of growth. IC.sub.50 (nm)
Treat- Quiescent Confluent Subcon- IC.sub.50 ratio ment (Q) (C)
fluent (S) Q/S C/S MTSA- 80 160.sup.1 45 1.8 3.6 dgA Ricin 0.00027
0.00018 0.00015 1.8 1.2 UV-3- 37 80 9 4.1 8.9 dgA TEC- 230.sup.2
180.sup.2 0.075 3067 2400 11-dgA .sup.1Extrapolated by fitting an
exponential curve to the graph shown in FIG. 19a.
.sup.2Extrapolated by fitting an exponential curve to the graph
shown in FIG. 19d.
C. Discussion
[0400] In this example, the inventors describe two new monoclonal
antibodies, TEC-4 and TEC-11, directed against a marker that is
upregulated in tumor-associated vascular endothelial cells. TEC-4
and TEC-11 specifically react with endoglin, which is known to be a
proliferation-linked endothelial cell marker that is upregulated on
dividing endothelial cells in vitro and on vascular endothelial
cells in solid tumors, sites of chronic inflammation and fetal
placenta in vivo.
[0401] HUVEC rapidly express endoglin after removal from the
umbilical cord and establishment in tissue culture. Induction of
the antigen was not diminished by depriving the cells of serum or
growth factors or increased by addition of cytokines, such as IL-1,
TNF-.alpha. and IFN-.gamma., known to activate endothelial cells
(Dustin et al., 1986; Rice et al., 1990), in accordance with the
findings of Westphal et al. (Westphal et al., 1993). The level of
endoglin expression appeared to correlate with entry into or
progression through the cell cycle. In HUVEC which had been grown
to confluence, two subpopulations were present, one with low
endoglin levels and the other with high expression. All
endoglin.sup.lo cells were in G.sub.0, but the endoglin.sup.hi
population contained significant proportions of activated (G.sub.1)
and dividing (S+G.sub.2M) cells, as indicated by increased levels
of protein/RNA and DNA, respectively. The majority of
endoglin.sup.hi cells were also in G.sub.0, suggesting that cell
surface endoglin is long-lived and is maintained at high levels in
cells that have divided and subsequently enter a non-cycling
state.
[0402] An association between increased endoglin expression and
endothelial cell proliferation is also suggested by strong
TEC-4/TEC-11 staining of blood vessels in sites of
neovascularization. Moderate to strong labelling of most or all
capillaries and venules was observed in cryostat sections of solid
tumors of diverse histological types. The only malignant tumors
that did not show significant endothelial cell staining were
B-lymphomas, in keeping with the fact that lymphomas, unlike
carcinomas and non-lymphoid sarcomas, grow by infiltrating existing
vascular tracts rather than by inducing de novo blood vessel growth
(Denekamp and Hobson, 1982). Endothelial cell staining was absent
or weak in all normal healthy tissues examined other than placenta,
where endothelial cells proliferate even faster than they do in
tumors (Denekamp, 1986; Denekamp and Hobson, 1982). The inventors
also observed endoglin upregulation in several cases of ulcerative
colitis, a chronic inflammatory condition, and in cat-scratch fever
and tonsillitis, which are associated with marked vascular
proliferation (Garcia et al., 1990; Fujihara, 1991). By contrast,
endoglin levels in reactive hyperplasia, which is not associated
with angiogenesis (Jones et al., 1984), were not higher than in
normal lymph nodes. Increased endoglin expression by vascular
endothelial cells has also been reported in angiogenesis-dependent
chronic inflammatory skin lesions, such as psoriasis, dermatitis
and granulation tissue, and on one case of cutaneous malignant
melanoma (Westphal et al., 1993). Increased endoglin expression
appeared to be related to endothelial proliferation rather than to
inflammation per se, because a range of inflammatory cytokines had
little or no effect on endoglin levels in HUVEC in vitro (Westphal
et al., 1993).
[0403] The reactivity patterns of TEC-4 and TEC-11 in frozen
sections of human tissues and on human cells in vitro were similar
to those of other anti-endoglin antibodies (Gougos and Letarte,
1988; Gougos et al., 1992; O'Connel et al., 1992; Buhring et al.,
1991; Westphal et al., 1993). TEC-4 and TEC-11 bound to HUVEC and
U937 cells in vitro, but were unreactive with the human lymphoma
lines K562, CEM and Daudi, in accordance with previous reports
(Gougos and Letarte, 1988; Westphal et al., 1993). All the
antibodies labelled endothelial cells in miscellaneous human
tissues and gave strong staining of fetal endothelium and
syncytiotrophoblast in the placenta (Gougos et al., 1992; Westphal
et al., 1993).
[0404] Several authors have reported stronger staining of
endothelial cells in normal organs, especially kidney and liver
(Gougos and Letarte, 1988; Westphal et al., 1993) and umbilical
cord (Gougos and Letarte, 1988), than the inventors observed with
TEC-4 and TEC-11. These discrepancies probably reflect differences
in the sensitivity of the immunohistochemical staining techniques
employed in different laboratories. In the present study, staining
conditions were selected which produced clear staining of tumor
endothelium whereas in previous studies (Gougos and Letarte, 1988;
Gougos et al., 1992), more sensitive staining conditions were
previously used in order to visualize normal endothelium. Evidence
that this is indeed true is provided by our finding that 44G4,
previously reported to stain normal endothelium, produced identical
endothelial staining patterns to that described herein for TEC-4
and TEC-11 when the current techniques were employed. However,
TEC-4 and TEC-11 recognize a distinct epitopes from that of 44G4,
as shown by the failure of 44G4 to block TEC-4 or TEC-11, even at
high ratios.
[0405] TEC-11 showed almost complete specificity for endothelial
cells whereas TEC-4 also reacted moderately strongly with certain
non-endothelial cells, particularly smooth muscle. The inventors
interpret these additional reactivities of TEC-4 as being
cross-reactivities possibly related to the low affinity of TEC-4.
Similar staining of smooth muscle was frequently seen during the
screening of the original hybridoma supernatants on tissue
sections.
[0406] Upregulation of endoglin on vascular endothelial cells in
solid tumors and chronic inflammatory disorders might be involved
functionally in the regulation of angiogenesis in these
pathological conditions. Recent evidence indicates that endoglin is
an essential component of the TGF-.beta. (transforming growth
factor-.beta.) receptor complex of human endothelial cells. It
binds TGF-.beta.1 and TGF-.beta.3 with a K.sub.D=50 pM (Cheifetz et
al., 1992). TGF-.beta. inhibits endothelial cell proliferation in
vitro (Madri et al., 1992) but, paradoxically, is a potent
angiogenic agent in vivo (Enenstein et al., 1992). Increased
expression of endoglin by proliferating endothelial cells could
modulate their response to TGF-.beta. and hence regulate the
angiogenic process (Cheifetz et al., 1992).
[0407] Antibodies to antigens other than endoglin have been
reported to stain endothelial cells in miscellaneous neoplasms but
not those in normal tissues. EN7/44 reacts with a predominantly
intracellular antigen (Mr 30.5 kDa) in budding capillary sprouts in
solid tumors and other neovascular sites whose expression appears
to be linked to migration rather than proliferation (Hagemeier et
al., 1986). FB-5 recognizes a heavily sialylated glycoprotein (Mr
170 kDa) on reactive fibroblasts and in a proportion of blood
vessels in various human tumors (Rettig et al., 1992). E9 reacts
with a 95 kD homodimer that is upregulated in tumors, fetal organs
and regenerating tissues, but which can be distinguished from
endoglin on the basis of lack of reactivity with placental
endothelium and different staining of tumor-derived endothelial
cells in vitro (Wang et al., 1993). Taken overall, the uniformity
of staining of vessels in different tumors and within any
individual tumor suggest that TEC-4 and TEC-11 compare favorably
with these antibodies.
[0408] The TEC-4 and TEC-11 antibodies have important potential for
diagnosis and therapy of human cancer. Firstly, the antibodies
could be used to distinguish between histologically indistinct
benign and malignant lesions. Studies in breast (Weidner et al.,
1992; Horak et al., 1992), prostate (Bigler et al., 1993), bladder
(Bigler et al., 1993), and cervical (Sillman et al., 1981)
carcinomas have established that high vessel density or tumor
angiogenic activity is strongly correlated with risk of metastasis
and poor prognosis and so could be used to determine when
aggressive post-operative therapy is appropriate (Weidner et al.,
1992; Horak et al., 1992). Diagnosis in these studies required
laborious enumeration of capillaries labelled with pan-endothelial
cell markers (Weidner et al., 1992; Horak et al., 1992; Bigler et
al., 1993) or the use of complex and subjective in vivo assays of
angiogenesis (Chodak et al., 1980), both of which might be
supplanted by a simple immunohistochemical procedure employing
TEC-4 or TEC-11. Indeed, the studies of breast tumors reported in
this Example indicate that vascular endothelial cell expression of
endoglin, as determined using TEC-4 or TEC-11, may distinguish
between intraductal carcinoma in situ (CIS), an aggressive
preneoplastic lesion and lobular CIS, which is associated with a
more indolent clinical course.
[0409] Secondly, the antibodies could be used for imaging tumors in
cancer patients. Being present on the luminal face of the
endothelial cell, endoglin is ideally situated for antibody binding
and should therefore permit rapid imaging. The antibodies would not
be subject to the major limitation of imaging procedures against
the tumor cells themselves, since they need not penetrate into
solid tumor masses. In addition, being of the IgM isotype,
extravasation of TEC-4 and TEC-11 should be minimal and their
specific imaging of antigens in the intravascular compartment
should be superior.
[0410] Thirdly, the antibodies could be used for therapy. The
highly accessible location of endoglin on the luminal surface of
the tumor vasculature is especially advantageous for therapeutic
application, because all of the target endothelial cells are able
to bind the therapeutic antibody, as shown in Example I. Both TEC-4
and TEC-11 are complement-fixing and so might induce selective
lysis of endothelial cells in the tumor vascular bed. Also, the
antibodies could be used to deliver therapeutic quantities of
radioisotopes, toxins, chemotherapeutic drugs or coagulants to the
tumor vasculature. Animal studies indicates that anti-tumor
endothelial cell immunotoxins are most effective when combined with
anti-tumor cell immunotoxins, which kill those tumor cells that
have invaded surrounding normal host tissue (as disclosed
hereinabove and in Burrows and Thorpe, 1993, Thus, TEC-4 or TEC-11
could be used clinically in combination with antibodies against
well-characterized tumor markers such as p185.sup.HER-2, TAG-72,
and CO17-1A (Shepard et al., 1991; Greiner et al., 1991; Kaplan,
1989) or indeed with conventional chemotherapeutic drugs.
EXAMPLE V
Preparation, Characterization and Use of Antibodies Directed
Against Tumor-Derived Endothelial Cell Binding Factors
[0411] This example describes the generation of polyclonal and
monoclonal antibodies directed against tumor-derived endothelial
cell "binding factors" for use in distinguishing between tumor
vasculature and the vasculature of normal tissues. Particularly
described is the generation of antibodies directed against vascular
permeability factor (VPF), also termed vascular endothelial cell
growth factor (VEGF), and against bFGF (basic fibroblast growth
factor).
[0412] For further details concerning FGF one may refer to
Gomez-Pinilla and Cotman (1992); Nishikawa et al. (1992), that
describe the localization of basic fibroblast growth factor; Xu et
al. (1992), that relates to the expression and immunochemical
analysis of FGF; Reilly et al. (1989), that concerns monoclonal
antibodies; Dixon et al. (1989), that relates to FGF detection and
characterization; Matsuzaki et al. (1989), that concerns monoclonal
antibodies against heparin-binding growth factor; and Herbin and
Gross (1992), that discuss the binding sites for bFGF on solid
tumors associated with the vasculature.
[0413] In these studies, rabbits were hyperimmunized with
N-terminal peptides of human VEGF, mouse VEGF, guinea pig VEGF,
human bFGF, mouse bFGF or guinea pig bFGF coupled to tuberculin
(purified protein derivative, PPD) or thyroglobulin carriers. The
peptides were 25 to 26 amino acids in length and were synthesized
on a peptide synthesizer with cysteine as the C-terminal residue.
Antisera were affinity purified on columns of the peptides coupled
to Sephraose matrices.
[0414] Antibodies to VEGF were identified by ELISA and by their
staining patterns on frozen sections of guinea pig tumors and
normal tissues. Polyclonal antibodies to guinea pig VEGF and human
VEGF reacted with the majority of vascular endothelial cells on
frozen sections of guinea pig L10 tumors and a variety of human
tumors (parotid, ovarian, mammary carcinomas) respectively. The
anti-human VEGF antibody stained mesangial cells surrounding the
endothelial cells in normal human kidney glomerulae and endothelial
cells in the liver, but did not stain blood vessels in normal human
stomach, leg muscle and spleen. The anti-guinea pig VEGF antibody
did not stain endothelial cells in any normal tissues, including
kidney, brain, spleen, heart, seminal vesicle, lung, large
intestine, thymus, prostrate, liver, testicle and skeletal
muscle.
[0415] Polyclonal antibodies to human FGF stained endothelial cells
in parotid and ovarian carcinomas, but not those in mammary
carcinomas. Anti-human FGF antibodies stained glomerular
endothelial cells in human kidney, but not endothelial cells in
normal stomach, leg muscle and spleen.
[0416] Monoclonal antibodies to guinea pig VEGF, human VEGF and
guinea pig bFGF were prepared by immunizing BALB/c mice with the
N-terminal sequence peptides (with cysteine at the C-terminus of
the peptide) coupled to PPD or to thyroglobulin. The synthetic
peptides immunogens of defined sequence are shown below and are
represented by SEQ ID NO:1, SEQ ID NO:2 AND SEQ ID NO:3,
respectively: TABLE-US-00010 guinea pig VEGF A P M A E G E Q K P R
E V V K F M D V Y K R S Y C human VEGF A P M A E G G G Q N H H E V
V K F M D V Y Q R S Y C guinea pig bFGF M A A G S I T T L P A L P E
G G D G G A F A P G C
[0417] The peptides were conjugated to thyroglobulin or to PPD by
derivatizing the thyroglobulin with succimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and reacting
the derivative with the peptide. This yields a conjugate having one
or more peptide sequences linked via a thioether bond to
thyroglobulin.
[0418] Specifically, the generation of monoclonal antibodies
against the above sequences was achieved using the following
procedure: BALB/c mice were immunized by serial injections with
peptide-PPD or peptide-thyroglobulin into several sites. Four or
five days after the last injection, the spleens were removed and
splenocytes were fused with P3xG3Ag8.653 myeloma cells using
polyethyleneglycol according to the procedures published in Morrow,
et al. (1991).
Individual hybridoma supernatants were screened as follows:
[0419] First screen: ELISA on peptide-thyroglobulin-coated plates.
[0420] Second screen: ELISA on cysteine linked via SMCC to
thyroglobulin. [0421] Third screen: Indirect immunoperoxidase
staining of frozen sections of guinea pig line 10 tumor or human
parotid carcinoma. [0422] Fourth screen: Indirect immunoperoxidase
staining of frozen sections of miscellaneous malignant and normal
guinea pig and human tissues.
[0423] Antibodies were selected that bound to peptide-thyroglobulin
but not to cysteine-thyroglobulin, and which bound to endothelial
cells in malignant tumors more strongly than they did to
endothelial cells in normal tissues (Table X). TABLE-US-00011 TABLE
X Reactivity of Monoclonal Antibodies Reactivity with Tumor Tumor
Endothelium Reactivity MoAB Immunogen.sup.+ Class g. pig human
Pattern* GV14 gp VEGF IgM + + BV + some tumor cells GV35 gp VEGF
IgM .+-. .+-. Tumor cells, weak on BV GV39 gp VEGF IgM + + BV and
some tumor cells GV59 gp VEGF IgM + + BV and some tumor cells GV97
gp VEGF IgM + + BV, weak on tumor cells HV55 hu VEGF IgG ? +
Basement membrane, some BV GF67 gp FGF IgM + + BV and tumor cells
GF82 gp FGF IgM + + BV and tumor cells *BV = blood vessels .sup.+gp
= guinea pig hu = human
[0424] The GV39 and GV97 antibodies against guinea pig VEGF
N-terminus bound to endothelial cells in miscellaneous human
malignant (Table XI) and normal (Table XII) tissues. The GV39 and
GV97 antibodies were deposited Dec. 12, 1997 with the American Type
Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, USA
(now 10801 University Blvd., Manassas, Va. 20110-2209, USA) and
given the ATCC Accession numbers ATCC HB-12450 and ATCC HB-12451,
respectively.
[0425] Binding to endothelial cells in malignant tumors tended to
exceed that to endothelial cells in normal tissues; however, this
difference was not striking.
[0426] The staining of endothelial cells in guinea pig tumor (line
10 hepatocellular carcinoma) and normal tissues was similar in
distribution and intensity to that observed with human tissues
(Table XIII).
[0427] In Tables XI through XV, + indicates a positive, as opposed
to a negative, result. The numbers 2+, 3+ and 4+ refer to a
positive signal of increasing strength, as is routinely understood
in this field of study. TABLE-US-00012 TABLE XI Anti-GPVEGF on
Human Tumors Purified GV97 1 ug/ml or GV59 Tumor TISSUE 20 ug/ml 10
ug/ml 5 ug/ml 2 ug/ml 0.5 ug/ml GV97 supt. GV14 GV39 supt.
DIGESTIVE TRACT 92-01-A073 2+ 1+ +/- -ve 4+ 4+ esophagus carcinoma
M4 Parotid 4+ 87-07-A134 Parotid 3+ 2+ +/- -ve 3+ 4+ carcinoma M5
Parotid 4+ 88-04-A010 parotid 1-2+ 1+ -ve -ve 1-3+ adenoca.
90-11-B319 Adeno. Ca. of 3-4+ 3-4+ colon to liver 94-02-B021C 3-4+
3-4+ Adenocarcinoma of colon 93-10-A333 Adeno. Ca. of 4+ 2-4+ 1-4+
-ve-1+ 4+ 3+ colon with normal 93-02-B004 Villous and 4+ 3-4+ 2-4+
1-2+ 3-4+ 2-3+ Adenomatous polyp of colon 93-02-A130 3+ 2+ +/--1+
-ve 4+ 4+ 3-4+ Leiomyosarcoma in colon 93-02-B020 Gastric Ca. 4+ 2+
2-3+ -ve-1+ 1-2+ 4+ 93-04-A221 Pancreas 3-4+ 2-3+ 1-2+ -ve-0.5+ 4+
4+ Adenoca. 94-04-A390 rectum 4+ 3+ 1-2+ 1+ 3+ adenoca. 93-12-A160
tongue 1-2+ +/- -ve -ve 3+ 3+ carcinomaadenoca. 101-84a Stomach
signetring 3+ 2+ -ve-1+ -ve most 1-2+ 3+ Ca. (101-84b pair) but a
few 3-4+ 90-05-A172 Stomach 4+ 3+ 1-2+ -ve-1+ -ve 3+ Adenoca.
REPRODUCTIVE TRACT 91-10-A115 Squam. cell 1-4+ 1-3+ 1-2+ 1-2+ 1-4+
1-3+ Ca. of vulva 93-03-A343 Prostate +/--3-4+ +/- to 2-3+ +/- to
1-2+ +/- 3-4+ 3-4+ Adenoca. MUSCLE IMMUNE SYSTEM URINARY SYSTEM
93-10-B002 Renal cell 2+ 3+ Ca. 90-01-A225 Renal cell 4+ 4+ 3-4+ of
most 1-3+ of 3-4+ 3+ 3-4+ Ca. some 93-01-A257 Transit. cell 3-4+
2-3+ 1-2+ +/- 2-3+ 2-3+ Ca. of bladder ENDOCRINE SYSTEM 94-01-A246
4+ 4+ 3-4+ 3+ 4+ 3-4+ Pheochromocytoma of adrenal 93-11-A074
Adrenal Cort. 3-4+ 3-4+ 2-3+ 1+ 3-4+ 4+ Ca. RESPIRATORY SYSTEM
93-08-N009 Lung Adenoca. 3-4+ 3-4+ 3-4+ 92-10-A316 Sq. cell lung 4+
3-4+ 1-2+ -ve-0.5+ 4+ 4+ Ca. 03-05-A065 Lung adenoca. 4+ 3-4+
-ve-1+ 1+ 3+ 3+ CENTRAL NERVOUS SYSTEM 94-01-A299 malig. 4+ 4+ 4+
3-4+ 4+ 3-4+ metast. schwanoma to Lymph node 92-10-A139 Meningioma
4+ 3-4+ 2-3+ 1-2+ 4+ 3-4+ 91-12-A013 Meningioma 4+ 2-3+ -ve-3+ +/-
4+ 3+ 93-03-A361 Atypical 4+ 4+ 3+ 2+ 4+ 3+ meningioma
INTEGUMENTARY SYSTEM 94-04-V037 Skin Sq. cell -ve to 4+ -ve to 3+
-ve to 1+ -ve 2-3+ 2-3+ Ca. w/normal 89-02-225 leg sarcoma 4+ 3-4+
1+ 1+ 4+ 2+ MISC. TUMORS
[0428] TABLE-US-00013 TABLE XII Anti-GPVEGF on Human Normal Tissues
Purified GV97 1 ug/ml or GV59 Tumor TISSUE 20 ug/ml 10 ug/ml 5
ug/ml 2 ug/ml 0.5 ug/ml GV97 supt. GV14 GV39 supt. DIGESTIVE SYSTEM
91-01-A128 Bladder w/ 3+ 2+ 1+ -ve 2-3+ 2-3+ cystitis 94-02-B020
uninvolved 2-3+ 2-3+ colon 92-01-A292 N. Colon 4+ 4+ 4+ 3-4+ 4+
3-4+ 93-10-A116 N. Colon Z-4+ 1-4+ 1-3+ -ve-2+ -ve 3-4+ 2-3+ 3-4+
90-06-A116 N. colon 3+ of many 2+ 93-02-A350 N. esophagus 3-4+ 3+
1+ +/- 4+ 4+ 93-05-A503 N. Ileum 4+ 4+ 94-03-A244 N. Liver 4+ 1-3+
-ve-1+ -ve 4+ 4+ 90-02-B132 N. Liver 1+ of a +/- -ve -ve -ve 1-3+
2-3+ 2-3+ 2-3+ few 94-01-A181 N. Pancreas 1-4+ 1-3+ 1-3+ of -ve
3-4+ a few 90-05-D008 N. Pancreas 2-4+ 1-3+ +/- -ve 2-3+ 2-3+
93-05-A174 N. Parotid 2+ of a 1-2+ of a few 1+ of a few -ve -ve 3+
of 2-3+ few a few 94-04-A391 N. Small 1-3+ -ve-2+ -ve -ve 3+ bowel
88-06-107 N. Stomach 3+ 2+ +/- -ve 3-4+ 3+ 101-84b N. Stomach 3-4+
in 2-3+ in main +/- in main -ve in 3+ 3-4+ (101 = 84a pair) main
and 3-4+ in and 2+ in main and and periphery periphery 1+in
periphery periphery 90-11-B337 N. Stomach 2-3+ +/--1+ -ve -ve 3+ 3+
REPRODUCTIVE TRACT 93-04-A041 N. Breast 4+ 3+ 94-02-A197 N. Breast
4+ 3+ w/fibrocystic change 93-02-A051 Breast -ve-1+ -ve -ve -ve +/-
+/--2+ w/fibrocystic change 93-02-A103 Breast 4+ 3+ 2+ 1+
w/fibrocyst. change 92-11-A006 N. ectocervix 2+ of 1-2+ of 0.5+ -ve
-ve 1-2+ of 3+ of most most some most 91-03-A207 N. ectocervix 2.5+
1.5+ 1+ .5+ 2-3+ 92-02-A139 N. ovary 1+ in -ve in -ve -ve -ve in
most -ve in w/corp. lusteum most most but 3-4+ in most but 2+ but
1+ one area bet 3-4 in one in one in area area one area 93-06-A11B
N. Prostate 1+ of a -ve -ve -ve 3+ few 93-11-A317d Prostate 3-4+
2-3+ -ve-3+ -ve-1+ 3-4+ 3-4+ chip 93-02-A315 Seminal 0.5-1+ 0.5+
-ve -ve 1+ 1.2+ Vesicle 92-04-A069 N. testis 1+ +/- +/- +/- 1-2+
91-04-A117 Ureter 1+ +/- -ve -ve +/--1+ 3-4+ w/inflammation MUSCLE
94-01-A065 N. Heart 3-4+ 2+ +/- -ve 3-4+ 4+ 91-07-D007 N. skeletal
1-4+ 1-3+ 1-2+ -ve -ve 1-3+ 1-3+ muscle 95-03-A395 N. Skeletal 4+
3-4+ 1-2+ 0.5-1+ 4+ 4+ muscle IMMUNE SYSTEM 90-01-A077 N. lymph
2-3+ 2+ 1+ of some -ve -ve 2-3+ 3-4+ node 90-08-A022 N. lymph most
1+ most 0.5+ but most -ve but most -ve 3+ 3+ node but a a few 2+ a
few 2+ but a few few 4+ 0.5-1+ 91-03-A057 N. lymph 2+ 1+ +/- -ve
3-4+ 3-4+ node 91-09-B017E uninvolved 3+ 2+ +/--1+ -ve 2-3+ 2-3+
lymph node 93-07-A236 N. Spicen 3-4+ 3-4+ -ve-3+ -ve 2-4+ 93-07-252
N. spicen 3+ 1+ +/- -ve 2-3+ ENDOCRINE SYSTEM 94-04-A252 N. adrenal
4+ 4+ 3-4+ 1-2+ 4+ 3+ w/medulia and cortex 93-05-A086 N. Adrenal
most -ve most -ve a -ve -ve 2-3+ 3-4+ medulla a few few 1-2+ 1-2+
92-03-A157 Hyperplasic 1+ +/- +/- -ve 4+ 4+ thyroid 91-03-B019 N.
Thyroid -ve-3+ -ve-2+ -ve-1+ -ve 2-3+ 2-3+ URINARY SYSTEM
93-09-A048 N. Kidney 4+ 2-3+ 91-11-A075 N. Kidney 4+ 3+ 2+ 1+ 4+ on
4+ on 4+ on glomeruli glomeruli glomeruli 93-10-B001 N. Kidney 4+
3+ +/- -ve 4+ on 4+ on 4+ on glomeruli glomeruli glomeruli
INTEGUMENTARY SYSTEM 92-08-A029 N. Breast +/- to +/- to 3+ +/- to
1+ +/- 2+ 2+ skin 4+ 89-02-257 Cartiledge 4+ 3-4+ 2-3+ 1-2+ 1+ 3-4+
marches 2SS RESPIRATORY SYSTEM 93-05-A203 N. Lung -ve-2+ -ve-1+ +/-
-ve 2+ 3+ 92-12-A263 N. Bronchus 2-3+ 1-2+ w/ducts -ve -ve 2-3+
w/ducts staining 2-3+ staining 3-4+
[0429] TABLE-US-00014 TABLE XIII Staining Pattern of 9F7 anti-VEGF
by direct immunohistochemical staining on 6-8 week old GP tissues
Purified GV97 1 ug/ml or TISSUE 20 ug/ml 10 ug/ml 5 ug/ml 2 ug/ml
0.5 ug/ml 9F7 supt. 3F9 supt. 5F9 supt. DIGESTIVE SYSTEM LIVER 2+
1-2+ +/- +/- 1-2+ 1-2+ INTESTINE 4+ 3+ 2+ 1+ 4+ m 4+ m lymphoid,
lymphoid, rest diff. rest diff. than than PANCREAS 1+ of many and
3+ in islands of cells SMALL INTESTINE 4+ of many 2-3+ of 1-2+ of
+/- of many 3+ of some 3+ of some and 4+ in many and many and 4+
and 4+ in and 4+ in and 4+ in lymphoid 4+ in in lymphoid, lymphoid
lymphoid lymphoid, lymphoid, rest diff. rest rest diff. than fVIII
diff. than fVIII than fVIII STOMACH 3-4+ 1-2+ on +/- on most +/- on
most 3-4+ (some 3-4+ (some most occasional occasional 1+ fVIII-ve)
fVIII-ve) occasional 2+ 3+ REPRODUCTIVE SYSTEM TESTIS MUSCLE AND
INTEGUMENTARY SYSTEM HEART -ve -ve -ve -ve 3-4+ (some 3-4+ (some
fVIII-ve) fVIII-ve) MUSCLE SKIN 1-2+ in 1+ in +/- in +/- in fatty
3+ 3+ fatty layer fatty fatty layer layer and 1- and 3-4+ in layer
and 3-4+ of 2+ of a few cellular and 3-4+ a few in in cellular
layer in cellular layer cellular layer layer IMMUNE SYSTEM SPLEEN
4+ 3+ 2+ -ve 4+ 4+ THYMUS URINARY SYSTEM KIDNEY glomeruli glomeruli
glomeruli glomeruli 1-2+ glomeruli glomeruli 4+ 3-4+ 2-3+ 3-4+ 3-4+
ENDOCRINE SYSTEM ADRENAL RESPIRATORY SYSTEM LUNG NERVOUS SYSTEM
CEREBELLUM 4+ 2+ +/- of most +/- of most 4+ 4+ and 1+ of a and 1+
of a few few TUMORS TUMOR 4+ 4+ 3-4+ 2-3+ (2) 4+ 3+
Lack of Reactivity of GV97 With Soluble Human VEGF
[0430] GV97 (ATCC HB-12451) did not bind to recombinant VEGF-coated
ELISA plates, nor did recombinant human VEGF bind to GV97 coated
ELISA plates. Soluble recombinant human VEGF did not block the
binding of 5 .mu.g/ml GV97 to tumor endothelium in histological
sections even when added at 50 .mu.g/ml.
[0431] These data suggest that GV97 (ATCC HB-12451) recognizes an
etoposide of VEGF that is concealed in recombinant human VEGF but
which becomes accessible when VEGF binds to its receptor on
endothelial cells.
GV97 Localization After Injection in Line 10-Bearing Guinea
Pigs
[0432] In contrast with staining data obtained from histological
sections, GV97 antibody localized selectively to tumor endothelial
cells after injection into line 10 tumor-bearing guinea pigs (Table
XIV). Staining of endothelial cells in the tumor was moderately
strong whereas staining of normal endothelium in miscellaneous
organs was undetectable.
Anti-bFGF Antibodies Selectively Bind to Tumor Endothelial
Cells
[0433] GF67 and GF82, which had been raised against guinea pig bFGF
N-terminus, bound strongly to endothelial cells in frozen sections
of guinea pig line 10 tumor and to endothelial cells in two types
of human malignant tumors (Table XV). By contrast, relatively weak
staining of endothelial cells in miscellaneous guinea pig normal
tissues was observed. TABLE-US-00015 TABLE XIV GV97 injected into
tumor bearing GP GV 97 20 ug/ml serum volume TISSUE GV97 10 ug/ml
injected DIGESTIVE SYSTEM LIVER 2+ -ve INTESTINE 3+ possible 0.5-1+
of a few PANCREAS +/- of many and 2+ in possible 0.5-1+ of islands
of cells a few SMALL INTESTINE 2-3+ of many and 4+ in +/- lymphoid,
rest diff. than fVIII STOMACH 1-2+ on most occasional possibly 0.5+
of 3+ a few REPRODUCTIVE SYSTEM TESTIS +/- MUSCLE AND INTEGUMENTARY
SYSTEM HEART -ve -ve MUSCLE -ve SKIN 1+ in fatty layer and 3-4+ in
cellular layer IMMUNE SYSTEM SPLEEN 3+ possibly a few 1+ THYMUS
URINARY SYSTEM KIDNEY glomeruli 3-4+ ENDOCRINE SYSTEM ADRENAL 4+
-ve RESPIRATORY SYSTEM LUNG 2+ -ve NERVOUS SYSTEM CEREBELLUM 2+ -ve
TUMORS TUMOR 4+ 2-3+
[0434] TABLE-US-00016 TABLE XV Anti-GP FGF Antibody Staining on GP
Tissues GP TISSUE GF 67 GF 82 DIGESTIVE SYSTEM LIVER ND ND
INTESTINE +/- +/- PANCREAS 2-3+ 2+ SMALL INTESTINE +/- +/- STOMACH
ND ND REPRODUCTIVE SYSTEM TESTIS ND ND MUSCLE AND INTEGUMENTARY
SYSTEM HEART 2-3+ 1+ MUSCLE +/- 1+ SKIN ND ND IMMUNE SYSTEM SPLEEN
3+ -ve THYMUS URINARY SYSTEM KIDNEY 1-2+ -ve ENDOCRINE SYSTEM
ADRENAL 1-2+ +/- RESPIRATORY SYSTEM LUNG 1-2+ 2-3+ NERVOUS SYSTEM
CEREBELLUM 1+ -1+ TUMORS LINE 1 TUMOR 4+ 4+ HUMAN TUMORS PHEOCHROMO
CYTOMA 4+ 4+ SCHWANOMA 4+ 4+
EXAMPLE VI
Thrombosis of Line 10 Tumor Vasculature by Administration of
GV97-ricin A-chain Immunotoxin to Guinea Pigs
[0435] This example describes the effects observed following the
administration of GV97-ricin A-chain immunotoxin to guinea pigs in
vivo.
[0436] GV97 (ATCC HB-12451) was conjugated to deglycosylated ricin
A-chain using previously published methods (Thorpe et al., 1988).
Guinea pigs having a 2 cm diameter subcutaneous line 10 tumor were
injected intravenously with 700 .mu.g/kg of this immunotoxin. The
tumor and various normal tissues were removed 24 hours after
injection. Killing of endothelial cells throughout the tumor plus
thrombosis of the tumor vasculature was apparent. No damage to any
normal tissues was observed. The effect was specific; no damage to
tumor endothelium was observed in recipients of a class-matched
(IgM) immunotoxin of irrelevant specificity.
[0437] It is important to note that no toxicity to the guinea pigs
was observed. The treated animals maintained normal appearance,
activity, and body weight.
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Sequence CWU 1
1
* * * * *