U.S. patent application number 10/336672 was filed with the patent office on 2004-03-04 for novel antibodies reactive with human carcinomas.
This patent application is currently assigned to Bristol-Myers Squibb Company. Invention is credited to Bruce, Kim Folger, Hellstrom, Ingegerg, Hellstrom, Karl Erik, Schreiber, George J..
Application Number | 20040043029 10/336672 |
Document ID | / |
Family ID | 46252863 |
Filed Date | 2004-03-04 |
United States Patent
Application |
20040043029 |
Kind Code |
A1 |
Hellstrom, Ingegerg ; et
al. |
March 4, 2004 |
Novel antibodies reactive with human carcinomas
Abstract
The present invention relates to novel antibodies, antibody
fragments and antibody conjugates and single-chain immunotoxins
reactive with human carcinoma cells. More particularly, the
antibodies, conjugates and single-chain immunotoxins of the
invention include: a murine monoclonal antibody, BR96; a
human/murine chimeric antibody, ChiBR96; a F(ab').sub.2 fragment of
BR96; ChiBR96-PE, ChiBR96-LysPE40, ChiBR96 F(ab').sub.2-LysPE40 and
ChiBR96 Fab'-LysPE40 conjugates and recombinant BR96 sFv-PE40
immunotoxin. These molecules are reactive with a cell membrane
antigen on the surface of human carcinomas. The BR96 antibody and
its functional equivalents, displays a high degree of selectivity
for carcinoma cells and possess the ability to mediate
antibody-dependent cellular cytotoxicity and complement-dependent
cytotoxicity activity. In addition, the antibodies of the invention
internalize within the carcinoma cells to which they bind and are
therefore particularly useful for therapeutic applications, for
example, as the antibody component of antibody-drug or
antibody-toxin conjugates. The antibodies also have a unique
feature in that they are cytotoxic when used in the unmodified
form, at specified concentrations.
Inventors: |
Hellstrom, Ingegerg;
(Seattle, WA) ; Hellstrom, Karl Erik; (Seattle,
WA) ; Bruce, Kim Folger; (Seattle, WA) ;
Schreiber, George J.; (Redmond, WA) |
Correspondence
Address: |
Pennie & Edmonds LLP
1155 Avenue of the Americas
New York
NY
10036-2711
US
|
Assignee: |
Bristol-Myers Squibb
Company
New York
NY
10156
|
Family ID: |
46252863 |
Appl. No.: |
10/336672 |
Filed: |
January 2, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10336672 |
Jan 2, 2003 |
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09782504 |
Feb 12, 2001 |
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09782504 |
Feb 12, 2001 |
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09290798 |
Apr 13, 1999 |
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09290798 |
Apr 13, 1999 |
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08077253 |
Jun 14, 1993 |
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5980896 |
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08077253 |
Jun 14, 1993 |
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08057444 |
May 5, 1993 |
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5491088 |
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08057444 |
May 5, 1993 |
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07544246 |
Jun 26, 1990 |
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07544246 |
Jun 26, 1990 |
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07374947 |
Jun 30, 1989 |
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Current U.S.
Class: |
424/155.1 ;
435/344; 530/388.8 |
Current CPC
Class: |
C07K 2319/00 20130101;
G01N 33/57484 20130101; A61K 49/0058 20130101; A61K 51/1087
20130101; C07K 2317/732 20130101; C07K 2317/77 20130101; C12N 15/10
20130101; C07K 2317/54 20130101; C07K 16/30 20130101; C07K 2317/24
20130101; C07K 14/415 20130101; A61K 47/6809 20170801; A61K 47/6851
20170801; C07K 2317/56 20130101; A61K 2123/00 20130101; C07K
2317/55 20130101; A61K 47/6811 20170801; C07K 2317/622 20130101;
C07K 2317/734 20130101; A61K 51/1045 20130101; A61K 38/00 20130101;
A61K 47/6829 20170801 |
Class at
Publication: |
424/155.1 ;
530/388.8; 435/344 |
International
Class: |
A61K 039/395; C12N
005/06; C07K 016/30 |
Claims
What is claimed is:
1. A monoclonal antibody BR96 produced by hybridoma ATCC HB10036,
or fragments thereof, and functional equivalents thereof having an
antigen-binding region that competitively inhibits the
immunospecific binding of monoclonal antibody BR96, having specific
immunological reactivity with human carcinoma cells, said antibody
characterized by being capable of internalizing within the
carcinoma cells with which it reacts, mediating antibody-dependent
cellular cytotoxicity and complement-dependent cytotoxicity
activity, and/or killing of said human carcinoma cells in the
absence of host effector cells or complement.
2. Hybridoma HB 10036 as deposited with the ATCC.
3. An Fab, F(ab').sub.2 or Fv fragment of the antibody of claim
1.
4. An immunoconjugate comprising a molecule containing the
antigen-binding region of the BR96 monoclonal antibody joined to a
cytotoxic agent.
5. The immunoconjugate of claim 4, wherein the molecule comprises
BR96 monoclonal antibody or fragments thereof.
6. The immunoconjugate of claim 4, wherein the molecule comprises
chimeric human/murine BR96 antibody or fragments thereof.
7. The immunoconjugate of claim 5, wherein the fragments are
selected from the group consisting of Fv, F(ab') and F(ab').sub.2
fragments.
8. The immunoconjugate of claim 6, wherein the fragments are
selected from the group consisting of Fv, F(ab)' and F(ab').sub.2
fragments.
9. A method for selectively killing tumor cells expressing the
antigen that immunospecifically binds to BR96 monoclonal antibody
comprising reacting the immunoconjugate of claim 4 with said tumor
cells.
10. A recombinant single-chain immunotoxin molecule comprising a
cloned heavy chain Fv portion and a cloned light chain Fv portion
of the BR96 monoclonal antibody joined to a cytotoxic agent.
11. A bispecific antibody with a binding specificity for two
different antigens, one of the antigens being that with which the
monoclonal antibody of claim 1 binds.
12. The bispecific antibody of claim 11, wherein one of the
antigens comprises a variant of Le.sup.y determinant which includes
an epitopic site containing fucose .alpha.1-3.
13. A monoclonal antibody, the antigen-binding region of which
competitively inhibits the immunospecific binding of monoclonal
antibody BR96 produced by hybridoma HB 10036 to its target
antigen.
14. The antibody of claim 13, wherein said antigen comprises a
variant of Le.sup.y determinant which includes an epitopic site
containing fucose .alpha.1-3.
15. A human/murine recombinant antibody, the antigen-binding region
of which competitively inhibits the immunospecific binding of
monoclonal antibody BR96 produced by hybridoma HB 10036 to its
target antigen.
16. The antibody of claim 1, wherein the antibody is conjugated to
a therapeutic agent to form an antibody conjugate.
17. The antibody of claim 16, wherein the therapeutic agent is an
anti-tumor drug, a cytotoxin, a radioactive agent, a second
antibody or an enzyme.
18. The antibody of claim 17, wherein the cytotoxin is a ribosome
binding toxin.
19. The antibody of claim 18, wherein the ribosome binding toxin is
ricin A.
20. A composition comprising a combination of an immunoconjugate
comprising the antibody of claim 1 linked to an enzyme capable of
converting a prodrug into a cytotoxic drug, and said prodrug.
21. A pharmaceutical composition useful in the treatment of
carcinomas comprising a pharmaceutically effective amount of the
antibody of claim 1 and an acceptable carrier.
22. A pharmaceutical composition useful in the treatment of
carcinomas comprising a pharmaceutically effective amount of at
least one antibody conjugate according to claim 16 and an
acceptable carrier.
23. A method of treating carcinomas in vivo comprising
administering to a patient a pharmaceutically effective amount of a
composition containing the antibody of claim 1.
24. A method for determining the presence of carcinoma in human
tissue comprising contacting a specimen of said tissue with the
antibody of claim 1 and detecting the binding of said antibody to
said tissue.
25. The method of claim 24, wherein said antibody is labeled so as
to directly or indirectly produce a detectable signal with a
compound selected from the group consisting of a radiolabel, an
enzyme, a chromophore and a fluorescer.
26. A method for imaging carcinoma comprising administering to a
patient intravenously the antibody of claim 1 in an amount
effective for detection of the carcinoma, allowing the antibody to
bind to carcinoma cells and to localize to the site of carcinoma
cells and detecting said antibody bound to the carcinoma cells.
27. The method of claim 26, wherein said antibody is labeled so as
to directly or indirectly produce a detectable signal with a label
selected from the group consisting of a radiolabel, an enzyme, a
chromophore, and a fluorescer.
28. A monoclonal anti-idiotypic antibody reactive with an idiotope
on the antibody of claim 1.
29. A diagnostic kit comprising: a) the antibody of claim 1; and b)
a conjugate of a detectable label and a specific binding partner of
the antibody-of (a) above.
30. The diagnostic kit of claim 29, wherein the label is selected
from the group consisting of enzymes, radiolabels, chromophores and
fluorescers.
31. The immunoconjugate of claim 4, wherein the cytotoxic agent is
selected from a group consisting of antimetabolites, alkylating
agents, anthracyclines, antibiotics, anti-mitotic agents, and
chemotherapeutic agents.
32. The immunoconjugate of claim 4, wherein the cytotoxic agent is
selected from a group consisting of ricin, doxorubicin,
daunorubicin, taxol, ethidium bromide, mitomycin, etoposide,
tenoposide, vincristine, vinblastine, colchicin, dihydroxy
anthracin dione, actinomycin D, 1-dehydrotestosterone, and
glucocorticoid.
33. A method for curing a subject suffering from a cancer, the
cancer being characterized as a group of cells having a tumor
associated antigen on the cell surface, which method comprises
administering to the subject a cancer killing amount of a tumor
targeted antibody joined to a cytotoxic agent under conditions
which permit the antibody so joined to bind the tumor associated
antigen on the cell surface so as to kill the cells so bound
thereby curing the subject.
34. The method of claim 33, wherein the tumor targeted antibody is
an internalizing antibody.
35. The method of claim 33, wherein the tumor targeted antibody is
an internalizing antibody which recognizes and binds to a Le.sup.y
determinant.
36. A method of inhibiting the proliferation of mammalian tumor
cells which comprises contacting the mammalian tumor cells with a
proliferation inhibiting amount of a tumor targeted antibody joined
to doxorubicin so as to inhibit proliferation of the mammalian
tumor cells.
37. The method of claim 36, wherein the tumor targeted antibody is
the monoclonal antibody BR96 produced by hybridoma ATCC
HB10036.
38. The method claim 36, wherein the tumor targeted antibody is a
chimeric antibody ChiBR96 produced by the hybridoma having the
identifying characteristics of HB 10460 as deposited with the
ATCC.
39. The method of claim 36, wherein the tumor targeted antibody is
the bispecific antibody with a binding specificity for two
different antigens, one of the antigens being that with which the
monoclonal antibody BR96 produced by hybridoma ATCC HB10036
binds.
40. The method of claim 36, wherein the tumor targeted antibody is
the monoclonal antibody, the antigen-binding region of which
competitively inhibits the immunospecific binding of monoclonal
antibody BR96 produced by hybridoma HB 10036 to its target
antigen.
41. The method of claim 36, wherein the tumor targeted antibody is
the human/murine recombinant antibody, the antigen-binding region
of which competitively inhibits the immunospecific binding of
monoclonal antibody BR96 produced by hybridoma HB 10036 to its
target antigen.
42. A method for selectively killing tumor cells expressing the
antigen that immunospecifically binds to BR96 monoclonal antibody
comprising reacting an immunoconjugate comprising a molecule
containing the antigen-binding region of the BR96 monoclonal
antibody joined to doxorubicin with the tumor cells so as to obtain
a BR96/doxorubicin-tumor cell complex thereby permitting the
doxorubicin to kill the tumor cells so complexed.
43. A method of inhibiting the proliferation of mammalian tumor
cells which comprises contacting the mammalian tumor cells with a
sufficient concentration of an immunoconjugate comprising a
molecule containing the antigen-binding region of the BR96
monoclonal antibody joined to doxorubicin so as to obtain a
BR96/doxorubicin-tumor cell complex thereby inhibiting
proliferation of the mammalian tumor cells so complexed.
44. A method for treating a subject suffering from a proliferative
type disease characterized by cells having the BR96 antigen on the
cell surface which comprises administering to the subject an
effective amount of an immunoconjugate comprising the
antigen-binding region of the BR96 monoclonal antibody joined to
doxorubicin such that the immunoconjugate binds the BR96 antigen
and kills said cells thereby treating the subject.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. Ser. No.
08/057,444, filed May 5, 1993, which is a file wrapper continuation
application of U.S. Ser. No: 07/544,246 filed Jun. 26, 1990, which
was a continuation-in-part of U.S. Ser. No: 07/374,947, filed Jun.
30, 1989, now abandoned, the entire disclosure of these
applications being incorporated by reference herein.
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention relates to novel antibodies reactive
with carcinoma cells. More particularly, the invention relates to a
murine monoclonal antibody and a chimeric monoclonal antibody,
including immunoconjugates and recombinant immunotoxins made
therefrom, that react with cell membrane antigens associated with a
large variety of carcinomas including carcinomas of the colon,
breast, ovary and lung. The murine monoclonal antibody is highly
specific for carcinomas, showing no to very low reactivity with
normal animal tissues or other types of tumors such as lymphomas or
sarcomas.
BACKGROUND OF THE INVENTION
[0003] 1. Monoclonal Antibodies Directed Against Cell Membrane
Antigens.
[0004] Monoclonal antibodies (MAbs) to human tumor-associated
differentiation antigens offer promises for the "targeting" of
various antitumor agents such as radioisotopes, chemotherapeutic
drugs, and toxins. (Order, in "Monoclonal Antibodies for Cancer
Detection and Therapy," Baldwin and Byers, (eds.), London, Academic
Press (1985)).
[0005] In addition, some monoclonal antibodies have the advantage
of killing tumor cells via antibody-dependent cellular cytotoxicity
(ADCC) or complement-dependent cytotoxicity (CDC) in the presence
of human effector cells or serum (Hellstrom et al., Proc. Natl.
Acad. Sci. USA 83:7059-7063 (1986)), and there are a few monoclonal
antibodies that have a direct antitumor activity which does not
depend on any host component (Drebin et al., Oncogene 2:387-394
(1988)).
[0006] Many monoclonal antibodies reactive with
carcinoma-associated antigens are known (see, e.g., Papsidero,
"Recent Progress In The Immunological Monitoring of Carcinomas
Using Monoclonal Antibodies, Semin. Surg. Oncol. 1(4):171-81
(1985); Schlom et al., "Potential Clinical Utility Of Monoclonal
Antibodies In The Management Of Human Carcinomas," Important Adv.
Oncol., 170-92 (1985); Allum et al., "Monoclonal Antibodies In The
Diagnosis And Treatment of Malignant Conditions," Surg. Ann.
18:41-64 (1986); and Houghton et al., "Monoclonal Antibodies:
Potential Applications To The Treatment Of Cancer," Semin. Oncol.,
13(2):165-79 (1986)).
[0007] These known monoclonal antibodies can bind to a variety of
different carcinoma-associated antigens including glycoproteins,
glycolipids and mucins (see, e.g., Fink et al., "Monoclonal
Antibodies As Diagnostic Reagents for The Identification And
Characterization Of Human Tumor Antigens," Prog. Clin. Pathol.,
9:121-33 (1984)).
[0008] For example, monoclonal antibodies that bind to glycoprotein
antigens on specific types of carcinomas include those described in
U.S. Pat. No. 4,737,579 (monoclonal antibodies to non-small cell
lung carcinomas), U.S. Pat. No. 4,753,894 (monoclonal antibodies to
human breast cancer), U.S. Pat. No. 4,579,827 (monoclonal
antibodies to human gastrointestinal cancer), and U.S. Pat. No.
4,713,352 (monoclonal antibodies to human renal carcinoma).
[0009] Monoclonal antibody B72.3, which is one of the antibodies
studied the most, recognizes a tumor-associated mucin antigen of
greater than 1,000 kd molecular weight that is selectively
expressed on a number of different carcinomas. Thus, B72.3 has been
shown to react with 84% of breast carcinomas, 94% of colon
carcinomas, 100% of ovarian carcinomas and 96% of non-small cell
lung carcinomas (see Johnston, "Applications of Monoclonal
Antibodies In Clinical Cytology As Exemplified By Studies With
Monoclonal Antibody B72.3," Acta Cytol. 1(5):537-56 (1987) and U.S.
Pat. No. 4,612,282, issued to Schlom et al.). Another patented
monoclonal antibody, KC-4, (see U.S. Pat. No. 4,708,930),
recognizes an approximately 400-500 kd protein antigen expressed on
a number of carcinomas, such as colon, prostate, lung and breast
carcinoma. It appears that neither the B72.3 nor KC-4 antibodies
internalize within the carcinoma cells with which they react.
[0010] Monoclonal antibodies reactive with glycolipid antigens
associated with tumor cells have been disclosed. For example, Young
et al., "Production Of Monoclonal Antibodies Specific For Two
Distinct Steric Portions of The Glycolipid
Ganglio-N-Triosylceramide (Asialo GM.sub.2)." J. Exp. Med.,
150:1008-1019 (1979) disclose the production of two monoclonal
antibodies specific for asialo GM.sub.2, a cell surface
glycosphingolipid antigen that was established as a marker for
BALB/c V3T3 cells transformed by Kirsten murine sarcoma virus. See,
also, Kniep et al., "Gangliotriasylceramide (Asialo GM.sub.2) A
Glycosphingolipid Marker For Cell Lines Derived From Patients With
Hodgkin's Disease," J. Immunol. 131(3):1591-94 (1983) and U.S. Pat.
No. 4,507,391 (monoclonal antibody to human melanoma).
[0011] Other monoclonal antibodies reactive with glycolipid
antigens on carcinoma cells include those described by Rosen et
al., "Analysis Of Human Small Cell Lung Cancer Differentiation
Antigens Using A Panel Of Rat Monoclonal Antibodies," Cancer
Research, 44:2052-61 (1984) (monoclonal antibodies to human small
cell lung cancer), Varki et al., "Antigens Associated with a Human
Lung Adenocarcinoma Defined by Monoclonal Antibodies," Cancer
Research 44:681-87 (1984); (monoclonal antibodies to human
adenocarcinomas of the lung, stomach and colon and melanoma), and
U.S. Pat. No. 4,579,827 (monoclonal antibodies to human colon
adenocarcinoma). See, also, Hellstrom et al., "Antitumor Effects Of
L6, An IgG2a Antibody That Reacts With Most Human Carcinomas,"
Proc. Natl. Acad. Sci. USA, 83:7059-63 (1986) which describes the
L6 monoclonal antibody that recognizes a carbohydrate antigen
expressed on the surface of human non-small cell lung carcinomas,
breast carcinomas and colon carcinomas.
[0012] Antibodies to tumor-associated antigens which are not able
to internalize within the tumor cells to which they bind are
generally not useful to prepare conjugates with antitumor drugs or
toxins, since these would not be able to reach their site of action
within the cell. Other approaches would then be needed so as to use
such antibodies therapeutically.
[0013] Additional monoclonal antibodies exhibiting a high specific
reactivity to the majority of cells from a wide range of carcinomas
are greatly needed. This is so because of the antigenic
heterogeneity of many carcinomas which often necessitates, in
diagnosis or therapy, the use of a number of different monoclonal
antibodies to the same tumor mass. There is a further need,
especially for therapy, for so called "internalizing" antibodies,
i.e., antibodies that are easily taken up by the tumor cells to
which they bind. Antibodies of this type find use in therapeutic
methods for selective cell killing utilizing antibody-drug or
antibody-toxin conjugates ("immunotoxins") wherein a therapeutic
antitumor agent is chemically or biologically linked to an antibody
or growth factor for delivery to the tumor, where the antibody
binds to the tumor-associated antigen or receptor with which it is
reactive and "delivers" the antitumor agent inside the tumor cells
(see, e.g., Embleton et al., "Antibody Targeting Of Anti-Cancer
Agents," in Monoclonal Antibodies For Cancer Detection and Therapy,
pp. 317-44 (Academic Press, 1985)).
[0014] 2. Immunotoxins.
[0015] Immunotoxins have been investigated as a new approach for
treating metastatic tumors in man (Pastan and FitzGerald, Science
254:1173-1177 (1991); FitzGerald and Pastan, Seminars in Cell
Biology 2:31-37 (1991) and Vitetta et al., Science 644:650 (1987)).
Pseudomonas exotoxin A ("PE") is a cytotoxic agent produced by
Pseudomonas aeruginosa that kills cells by ADP-ribosylating
elongation factor 2, thereby inhibiting protein synthesis (Iglewski
et al., Proc. Natl. Acad. Sci. USA 72:2284-2285 (1975)).
[0016] PE is a polypeptide comprising three domains (Allured et
al., Proc. Natl. Acad. Sci. USA 83:1320-1324 (1986)).
[0017] Domain I encodes the cell-binding ability; domain II encodes
the proteolytic sensitivity site and the membrane translocation
ability; and domain III encodes the ADP-ribosylation activity of
the toxin (Hwang et al., Cell 48:129-136 (1987), Siegall et al., J.
Biol. Chem. 264:14256-14261 (1989)). By removing domain I from PE,
a truncated 40 kDa toxin is formed ("PE40") (Kondo et al., J. Biol.
Chem. 263:9470-9475 (1988)).
[0018] PE40 is weakly toxic to cells because it lacks the cell
binding domain for the PE receptor (Id.) For conjugation of this
molecule to an antibody, the amino terminus of PE40 is modified to
include a lysine residue to form "LysPE40" (Batra et al., supra).
Immunotoxins using PE, have shown promise in preclinical models
using human tumor xenografts in nude mice (Batra et al., Proc.
Natl. Acad. Sci. USA 86:8545-8549 (1989); and Pai et al., Proc.
Natl. Acad. Sci. USA 88:3358-3362 (1991)).
[0019] Several internalizing antibodies reacting with lymphocyte
antigens are known. In contrast, such antibodies are rare when
dealing with solid tumors. One of the few examples of an
internalizing antibody reacting with carcinomas is an antibody
disclosed in Domingo et al., "Transferrin Receptor As A Target For
Antibody-Drug Conjugates," Methods Enzymol. 112:238-47 (1985). This
antibody is reactive with the human transferrin-receptor
glycoprotein expressed on tumor cells. However, because the
transferrin-receptor is also expressed on many normal tissues, and
often at high levels, the use of an anti-transferrin-recepto- r
antibody in an antibody-drug or antibody-toxin conjugate may have
significant toxic effects on normal cells. The utility of this
antibody for selective killing or inhibition of tumor cells is
therefore questionable. Another internalizing antibody is BR64
(disclosed in co-pending patent applications U.S. Ser. No. 289,635,
filed Dec. 22, 1988, and Ser. No. 443,696 filed Nov. 29, 1989, and
incorporated by reference herein), which binds to a large spectrum
of human carcinomas.
[0020] 3. Chimeric Antibodies.
[0021] The cell fusion technique for the production of monoclonal
antibodies (Kohler and Milstein, Nature (London) 256:495 (1975))
has permitted the development of a number of murine monoclonal
antibodies reactive with antigens, including previously unknown
antigens.
[0022] However, murine monoclonal antibodies may be recognized as
foreign substances by the human immune system and neutralized such
that their potential in human therapy is not realized. Therefore,
recent efforts have focused on the production of so-called
"chimeric" antibodies by the introduction of DNA into mammalian
cells to obtain expression of immunoglobulin genes (Oi et al.,
Proc. Natl. Acad. Sci. USA 80:825 (1983); Potter et al., Proc.
Natl. Acad. Sci. USA 81:7161; Morrison et al., Proc. Natl. Acad.
Sci. USA 81:6581 (1984); Sahagan et al., J. Immunol. 137:1066
(1986); Sun et al., Proc. Natl. Acad. Sci. 84:214 (1987)).
[0023] Chimeric antibodies are immunoglobulin molecules comprising
a human and non-human portion. More specifically, the antigen
combining region (variable region) of a chimeric antibody is
derived from a non-human source (e.g., murine) and the constant
region of the chimeric antibody which confers biological effector
function to the immunoglobulin is derived from a human source. The
chimeric antibody should have the antigen binding specificity of
the non-human antibody molecule and the effector function conferred
by the human antibody molecule.
[0024] In general, the procedures used to produce chimeric
antibodies involve the following steps:
[0025] a) identifying and cloning the correct gene segment encoding
the antigen binding portion of the antibody molecule; this gene
segment (known as the VDJ, variable, diversity and joining regions
for heavy chains or VJ, variable, joining regions for light chains
or simply as the V or variable region) may be in either the cDNA or
genomic form;
[0026] b) cloning the gene segments encoding the constant region or
desired part thereof;
[0027] c) ligating the variable region with the constant region so
that the complete chimeric antibody is encoded in a form that can
be transcribed and translated;
[0028] d) ligating this construct into a vector containing a
selectable marker and gene control regions such as promoters,
enhancers and poly (A) addition signals;
[0029] e) amplifying this construct in bacteria;
[0030] f) introducing this DNA into eukaryotic cells (transfection)
most often mammalian lymphocytes;
[0031] g) selecting for cells expressing the selectable marker;
[0032] h) screening for cells expressing the desired chimeric
antibody; and
[0033] i) testing the antibody for appropriate binding specificity
and effector functions.
[0034] Antibodies of several distinct antigen binding specificities
have been manipulated by these protocols to produce chimeric
proteins (e.g., -anti-TNP: Boulianne et al., Nature 312:643 (1984);
and anti-tumor antigens: Sahagan et al., J. Immunol. 137:1066
(1986)). Likewise, several different effector functions have been
achieved by linking new sequences to those encoding the antigen
binding region. Some of these include enzymes (Neuberger et al.,
Nature 312:604 (1984)), immunoglobulin constant regions from
another species and constant regions of another immunoglobulin
chain (Sharon et al., Nature 309:364 (1984); Tan et al., J.
Immunol. 135:3565-3567 (1985)).
[0035] 4. Modifying Genes In Situ Encoding Monoclonal
Antibodies.
[0036] The discovery of homologous recombination in mammalian cells
permits the targeting of new sequences to specific chromosomal
loci. Homologous recombination occurs when cultured mammalian cells
integrate exogenous DNA into chromosomal DNA at the chromosome
location which contains sequences homologous to the plasmid
sequences (Folger et al., Mol. Cell. Biol. 2:1372-1387 (1982);
Folger et al., Symp. Quant. Biol. 49:123-138 (1984); Kucherlapati
et al., Proc. Natl. Acad. Sci. USA 81:3153-3157 (1984); Lin et al.,
Proc. Natl. Acad. Sci. USA 82:1391-1395 (1985); de Saint Vincent et
al., Proc. Natl. Acad. Sci. USA 80:2002-2006 (1983); Shaul et al.,
Proc. Natl. Acad. Sci. USA 82:3781-3784 (1985)).
[0037] The potential for homologous recombination within cells
permits the modification of endogenous genes in situ. Conditions
have been found where the chromosomal sequence can be modified by
introducing into the cell a plasmid DNA which contains a segment of
DNA homologous to the target locus and a segment of new sequences
with the desired modification (Thomas et al., Cell 44:419-428
(1986); Smithies et al., Nature 317:230-234 (1985); Smith et al.,
Symp. Quant. Biol. 49:171-181 (1984)). Homologous recombination
between mammalian cell chromosomal DNA and the exogenous plasmid
DNA can result in the integration of the plasmid or in the
replacement of some of the chromosomal sequences with homologous
plasmid sequences. This can result in placing a desired new
sequence at the endogenous target locus.
[0038] The process of homologous recombination has been evaluated
using genes which offer dominant selection such as NEO and HPRT for
a few cell types (Song et al., Proc. Natl. Acad. Sci. USA
84:6820-6824 (1987); Rubinitz-and Subramani, Mol. Cell Biol.
6:1608-1614 (1986); and Liskay, Cell 35:157-164 (1983)). Recently,
procedures for modifying antibody molecules and for producing
chimeric antibody molecules using homologous recombination to
target gene modification have been described (Fell et al., Proc.
Natl. Acad. Sci. USA 86:8507-8511 (1989); and co-pending U.S.
patent applications Ser. No. 243,873 filed Sep. 14, 1988, and Ser.
No. 468,035 filed Jan. 22, 1990, assigned to the same assignee as
the present application, all of which are incorporated by reference
herein).
[0039] 5. Monoclonal Antibodies in Therapy.
[0040] The most direct way to apply antitumor monoclonal antibodies
clinically is to administer them in unmodified form, using
monoclonal antibodies which display antitumor activity in vitro and
in animal (such as humans, dogs, cows, pigs, horses, cats, rats,
and mice) models. Most monoclonal antibodies to tumor antigens do
not appear to have any antitumor activity by themselves, but
certain monoclonal antibodies are known which mediate
complement-dependent cytotoxicity (complement-dependent
cytotoxicity), i.e., kill human tumor cells in the presence of
human serum as a source of complement (see, e.g., Hellstrom et al.,
Proc. Natl. Acad. Sci. USA 82:1499-1502 (1985)), or
antibody-dependent cellular cytotoxicity (antibody-dependent
cellular cytotoxicity) together with effector cells such as human
NK cells or macrophages.
[0041] To detect antibody-dependent cellular cytotoxicity and
complement-dependent cytotoxicity activity monoclonal antibodies
are tested for lysing cultured .sup.51Cr-labeled tumor target cells
over a 4-hour incubation period.
[0042] Target cells are labeled with .sup.51Cr and then exposed for
4 hours to a combination of effector cells (in the form of human
lymphocytes purified by the use of a lymphocyte-separation medium)
and antibody, which is added in concentrations varying between 0.1
.mu.g/ml and 10 .mu.g/ml. The release of .sup.51Cr from the target
cells is measured as evidence of tumor-cell lysis (cytotoxicity).
Controls include the incubation of target cells alone or with
either lymphocytes or monoclonal antibody separately.
[0043] The total amount of .sup.51Cr that can be released is
measured and antibody-dependent cellular cytotoxicity is calculated
as the percent killing of target cells observed with monoclonal
antibody plus effector cells as compared to target cells being
incubated alone. The procedure for complement-dependent
cytotoxicity is identical to the one used to detect
antibody-dependent cellular cytotoxicity except that human serum,
as a source of complement, (diluted 1:3 to 1:6) is added in place
of the effector cells.
[0044] Monoclonal antibodies with antibody-dependent cellular
cytotoxicity and complement-dependent cytotoxicity activity are
considered for therapeutic use because they often have anti-tumor
activities in vivo. Antibodies lacking antibody-dependent cellular
cytotoxicity and complement-dependent cytotoxicity activity in
vitro, on the other hand, are commonly ineffective in vivo unless
used as carriers of antitumor agents.
[0045] The ability of a monoclonal antibody to activate the host's
complement may prove to be therapeutically beneficial not only
because tumor cells may be killed, but also because the blood
supply to tumors may increase, thus facilitating the uptake of
drugs (see Hellstrom et al., "Immunological Approaches to Tumor
Therapy: Monoclonal Antibodies, Tumor Vaccines, and Anti-Idiotypes,
in Covalently Modified Antigens and Antibodies in Diagnosis and
Therapy, Quash & Rodwell, eds., Marcel Dekker, pp. 15-18
(1989)).
[0046] Among mouse monoclonal antibodies, the IgG2a and IgG3
isotypes are most commonly associated with antibody-dependent
cellular cytotoxicity and complement-dependent cytotoxicity.
Antibodies having both antibody-dependent cellular cytotoxicity and
complement-dependent cytotoxicity activity have high selectivity
for killing only the tumor cells to which they bind and would be
unlikely to lead to toxic effects if non-specifically trapped in
lung, liver or other organs. This may give such antibodies an
advantage over radiolabeled antibodies or certain types of
immunoconjugates.
[0047] Therapeutic modalities directed to treating tumors are
commonly available. For example, chemotherapy is an effective
treatment for selected human tumors. However, with chemotherapy
only modest progress has been made for treating the majority of
carcinomas, including carcinomas of breast, lung, and colon.
[0048] The introduction of monoclonal antibody (MAb) technology in
the 1970s raised hopes that tumor-specific MAbs could be used to
target anti-tumor agents and provide more effective therapy (K. E.
Hellstrom, and I. Hellstrom, in Biologic Therapy of Cancer:
Principles and Practice, V. T. DeVita, S. Hellman, and S. A.
Rosenberg, Eds. (J. P. Lippincott Company, Philadelphia, Pa., 1991,
pp. 35-52).
[0049] 6. Immunoconjugates in Therapy.
[0050] Various immunoconjugates in which antibodies were used to
target chemotherapeutic drugs (P. N. Kularni, A. H. Blair, T. I.
Ghose, Cancer Res. 41, 2700 (1981); R. Arnon, R. and M. Sela,
Immunol Rev. 62, 5 (1982); H. M. Yang and R. A. Resifeld, Proc.
Natl. Acad. Sci. U.S.A., 85, 1189 (1988); R. O. Dilman, D. E.
Johnson, D. L. Shawler, J. A. Koziol, Cancer Res. 48, 6097 (1988);
L. B. Shih, R. M. Sharkey, F. J. Primus, D. M. Goldenberg, Int. J.
Cancer 41, 832 (1988); P. A. Trail et al., Cancer Res. 52, 5693
(1992)), or plant and bacterial toxins (I. Pastan, M. C.
Willingham, D. J. Fitzgerald, Cell 47, 641 (1986); D. C. Blakey, E.
J. Wawrzynczak, P. M. Wallace, P. E. Thorpe, in Monoclonal Antibody
Therapy Prog. Allergy, H. Waldmann, Ed. (Karger, Basel, 1988), pp.
50-90) have been evaluated in preclinical models and found to be
active in vitro and in vivo.
[0051] However, activity of these MAbs was usually assessed against
newly implanted rather than established tumors and was typically
superior to matching, but not optimal, doses of the unconjugated
drug.
[0052] Although conjugates have been described with anti-tumor
activity against established tumors that were superior to that of
an optimal dose of unconjugated drug, the therapeutic index was low
and superior activity was achieved only at or near the maximum
tolerated dose (MTD) of the conjugate (P. A. Trail et al., Cancer
Res. 52, 5693 (1992)).
[0053] The results of clinical studies of drug and toxin conjugates
(i.e., immunoconjugates) have also been disappointing, particularly
for solid tumors (E. S. Vitetta, R. J. Fulton, R. D. May, M. Till,
J. W. Uhr, Science 238, 1098 (1987); H. G. Eichler, Biotherapy 3,
11 (1991); E. Wawrzynczak, Br. J. Cancer 64, 624 (1991); G. A.
Pietersz and I. F. C. McKenzie, Immunol. Rev. 129, 57 (1992)).
[0054] Very few antibodies are able to kill tumor cells by
themselves, that is, in the absence of effector cells or complement
as in antibody-dependent cellular cytotoxicity or
complement-dependent cytotoxicity. BR96 is such an antibody,
because it can kill cells by itself at an antibody concentration of
approximately 10 .mu.g/ml or higher. Such antibodies are of
particular interest since they can interfere with some key event in
the survival of neoplastic cells.
[0055] Presently, chemotherapeutic agents, by themselves, do not
distinguish between malignant and normal cells. They are absorbed
by both cell types. Tumors that are detected early on such as acute
lymphocytic leukemia and lymphomas are highly susceptible to
drugs.
[0056] Tumors that are hidden until growth has reached a plateau,
such as cancer of the lung and colon, have little sensitivity to
drugs. Normal cells with high growth fraction are inevitably
attacked by today's anti-cancer drugs, explaining the prevalence of
severe side effects in the gastrointestinal tract and of hair loss.
This holds true whether the cytotoxicity of the drug is due to
alkylation, intercalation, or disruption of
biosynthesis/antimetabolites.
[0057] The molecules of the invention, e.g., the immunotoxins, are
homogeneous molecules that retain the specificity of the cell
binding portion with the cytotoxic potential of the toxin.
[0058] It is thus apparent that antibodies, antibody conjugates and
immunotoxins hat display a high degree of selectivity to a wide
range of carcinomas, have anti-tumor activity, and are capable of
being readily internalized by tumor cells, may be of great benefit
in tumor therapy. SUMMARY OF THE INVENTION
[0059] The present invention provides internalizing antibodies,
antibody conjugates and recombinant, single-chain-immunotoxins that
are highly selective for a range of human carcinomas. More
specifically, the novel antibodies of the invention, designated as
BR96 antibodies, are a murine monoclonal antibody and a chimeric
antibody that bind to a cell membrane antigen found on human
carcinoma cells.
[0060] The novel conjugates and single-chain immunotoxins contain
an exotoxin such as PE and bind to the antigen on tumor cells. The
antibodies, conjugates and single-chain immunotoxins are highly
reactive with carcinoma cells, such as those derived from breast,
lung, colon and ovarian carcinomas, showing no or limited
reactivity with normal human cells or other types of tumors such as
lymphomas or sarcomas. In addition, the antibodies of the invention
internalize within the carcinoma cells to which they bind and they
are capable of killing tumor cells by themselves, i.e., not in
conjugated form, and without effector cells or complement.
[0061] Thus the BR96 antibodies are of particular use in
therapeutic applications, for example to react with tumor cells,
and in conjugates and single-chain immunotoxins as target-selective
carriers of various agents which have antitumor effects including
chemotherapeutic drugs, toxins, immunological response modifiers,
enzymes and radioisotopes. The antibodies can thus be used as a
component of various immunoconjugates including antibody-drug and
antibody-toxin conjugates, including ricin and PE-antibodies and
ricin and PE-antibody fragment immunotoxins, where internalization
of the conjugate is favored, and after radiolabeling to deliver
radioisotope to tumors. The BR96 antibodies can also be
therapeutically beneficial even in the unmodified form.
Furthermore, the antibodies are useful for in vitro or in vivo
diagnostic methods designed to detect carcinomas.
BRIEF DESCRIPTION OF THE FIGURES
[0062] FIG. 1 depicts the percent inhibition of thymidine
incorporation into the DNA of 3396 breast carcinoma cells treated
with a BR96-RA immunotoxin at varying concentrations as described
in Example 3, infra. BR6-RA is an internalizing antibody which is
used as a negative control because it does not bind to the 3396
cells.
[0063] FIG. 2 depicts the percent inhibition of thymidine
incorporation into the DNA of 2707 lung carcinoma cells treated
with a BR96-RA immunotoxin at varying concentrations as described
in Example 3, infra. BR6-RA is an internalizing antibody which also
binds to the 2707 cells.
[0064] FIG. 3 depicts the percent inhibition of thymidine
incorporation into the DNA of HCT116 colon carcinoma cells treated
with a BR96-RA immunotoxin at varying concentrations as described
in Example 3, infra. BR96 does not bind to HCT 116 cells.
[0065] FIG. 4 depicts the percent inhibition of thymidine
incorporation into the DNA of C colon carcinoma cells treated with
a BR96-RA immunotoxin at varying concentrations as described in
Example 3, infra. BR6-RA does not bind to the C cells; L6-RA binds
to the C cells but does not internalize.
[0066] FIG. 5 depicts the percent inhibition of thymidine
incorporation into the DNA of 3347 colon carcinoma cells treated
with a BR96-RA immunotoxin at varying concentrations as described
in Example 3, infra. BR96 does not bind to these cells while BR6
does.
[0067] FIG. 6 depicts the results of FACS analysis of the
cytotoxicity of propidium iodide stained 3396 breast carcinoma
cells, 2987 lung carcinoma cells and 3619 colon carcinoma cells, as
described in Example 4, infra.
[0068] FIG. 7 depicts the effects of BR96 on cell proliferation of
various cell lines as described in Example 4, infra.
[0069] FIG. 8 illustrates the effect of BR96 on cell growth of
various cell lines, measured by a staining method as described in
Example 4, infra.
[0070] FIG. 9 illustrates the results of tests to determine
antibody-dependent cellular cytotoxicity activity of BR96 as
described in Example 5, infra.
[0071] FIG. 10 describes the results of tests to determine
complement-dependent cytotoxicity activity of BR 96 as described in
Example 6, infra.
[0072] FIG. 11 is a bar graph of the results of testing the
reactivity of BR96 against glycolipids as described in Example 7,
infra.
[0073] FIG. 12 is a bar graph of the results of testing the
reactivity of BR96 against neoglycoproteins as described in Example
7, infra.
[0074] FIG. 13 is a graph of the binding activity of BR96
F(ab').sub.2 fragments compared to that of whole BR96 monoclonal
antibody in an ELISA using goat anti-K light chain detecting
reagent, as described in Example 8, infra.
[0075] FIG. 14 is a graph of the binding activity of BR96
F(ab').sub.2 fragments as compared to that of whole BR96 monoclonal
antibody in an ELISA using peroxidase conjugated protein A
detecting reagent, as described in Example 8, infra.
[0076] FIG. 15 is a diagram of vector ph.gamma..sub.1HC-D used in
the electroporation procedure, as described in Example 9,
infra.
[0077] FIG. 16 is a diagram of vector pSV.sub.2gpt/C.sub.K used in
the electroporation procedure, as described in Example 9,
infra.
[0078] FIG. 17 is a graph depicting the results of the competition
binding assay comparing the binding of the murine. BR96 monoclonal
antibody of the invention with binding of the chimeric BR96
antibody of the invention, as described in Example 9, infra.
[0079] FIG. 18 depicts the results of FACS analysis of the
cytotoxicity of the antibodies of the invention on 3396 breast
carcinoma cells as described in Example 10, infra.
[0080] FIG. 19 depicts the results of FACS analysis of the
cytotoxicity of the antibodies of the invention on 2987 human lung
adenocarcinoma cells as described in Example 10, infra.
[0081] FIG. 20 depicts the results of FACS analysis of the
cytotoxicity of the ntion on MCF-7 cells as described in Example
10, infra.
[0082] FIG. 21 depicts the percent inhibition of thymidine
incorporation into the carcinoma cells treated with a murine
BR96-RA immunotoxin and RA at varying concentrations as described
in Example 10, infra.
[0083] FIG. 22 depicts the percent inhibition of thymidine
incorporation into the carcinoma cells treated with a murine
BR96-RA immunotoxin and ng concentrations, as described in Example
10, infra.
[0084] FIG. 23 is a graph depicting the antitumor effects of
unmodified BR96 on 987, as described in Example 11, infra.
[0085] FIG. 24 is a bar graph illustrating the absence of tumors at
the end of treated with BR96, as described in Example 11,
infra.
[0086] FIG. 25 depicts the dose effects of BR96 antibody after
implantation of determined by tumor volume, as described in Example
11, infra.
[0087] FIG. 26 illustrates the effects of treatment with
F(ab').sub.2 fragments and implantation of 2707 cells as determined
by tumor volume, as 11, infra.
[0088] FIG. 27 illustrates the absence of tumors after treatment
with various antibody, as compared to the effects of F(ab').sub.2
fragments and chimeric Example 11, infra.
[0089] FIG. 28 is a photograph of the gel obtained from
non-reducing SDS-PAGE and unconjugated ChiBR96 IgG, Fab' and
F(ab').sub.2 immunotoxins as 13, infra (Lane 1: ChiBR96 IgG; Lane
2: ChiBR96 Fab'; Lane 3: E40; Lane 4: Native PE; Lane 5: LysPE40;
Lane 6: ChiBR96 7: ChiBR96(Fab').sub.2; Lane 8: ChiBR96
F(ab').sub.2-LysPE40; Lane 9: 40; Lane 10: ChiBR96 IgG).
[0090] FIG. 29 is a graph depicting the results of competition of
ChiBR96-PE and binding as described in Example 13, infra (ChiBR96
(closed circle); square); ChiBR96-LysPE40 (open triangle)).
[0091] FIGS. 30A, B, C are graphs of the direct binding of intact
ChiBR96-LysPE40, F(ab').sub.2-LysPE40 and Fab'-LysPE40 to L2987
cells, as described in Example 13, infra (ChiBR96 (closed circle);
ChiBR96-LysPE40 (open triangle); ChiBR96 F(ab').sub.2 (open
square); ChiBR96 F(ab').sub.2 LysPE40 (closed circle); ChiBR96 Fab'
(open circle); ChiBR96 Fab'-LysPE40 (open circle)).
[0092] FIGS. 31A and B are graphs showing the determination of
endocytosis of cell-surface immunotoxin after modulation with
ChiBR96-PE or ChiBR96-LysPE40 immunotoxins as described in Example
13, infra (31A: loss of cell surface immunotoxin under modulating
and non-modulating conditions; 31B: internalization of cell-bound
immunotoxin using immunotoxin plus radiolabeled M-40/1 complex;
ChiBR96-PE coated cells were incubated at 4.degree. C. (open
circle) or 37.degree. C. (closed circle); ChiBR96-LysPE40 coated
cells were incubated at 4.degree. C. (open triangle) or 37.degree.
C. (closed triangle).
[0093] FIG. 32 is a graph of the cytotoxic effects of various
ChiBR96 forms conjugated to LysPE40 against MCF-7 cells as
described in Example 13, infra (ChiBR96-PE40 (closed square);
ChiBR96 F(ab').sub.2-PE40 (closed circle); ChiBR96 Fab'-PE40
(closed triangle); PE40 open circle).
[0094] FIG. 33 is a bar graph depicting the results of competition
analysis of ChiBR96-PE40 cytotoxic activity against MCF-7 cells as
described in Example 13, infra.
[0095] FIGS. 34A and B are graphs showing the results of protein
synthesis inhibition analysis of ChiBR96-(PE/LysPE40) vs. PE
against MCF-7 cells as described in Example 13, infra (34A: 1 hr;
34B: 20 hr; ChiBR96-PE (closed square); BR96-PE40 (closed circle);
PE (closed triangle).
[0096] FIG. 35 (SEQ ID NO: 3) is the DNA and amino acid sequence
for BR96 sFv encoded by plasmid pBR96 Fv, as described in Example
14, infra.
[0097] FIG. 36 is a schematic illustration of the construction of
expression plasmid pBW 7.0 encoding BR96 sFv-PE40 as described in
Example 14, infra (E, Eco RI; H, Hind III; K, KPNI; N, NDe I; S,
Sal I; (Gly.sub.4Ser).sub.3 represents a 15 amino acid linker).
[0098] FIGS. 37A, B, C illustrate the purification of BR96 sFv-PE40
by gel filtration as described in Example 14, infra (FIG. 37A:
profile of gel filtration column chromatography of renatured BR96
sFv-PE40 after initial purification over Q-Sepharose; FIG. 37B: 12%
denaturing SDS-polyacrylamide gel stained with Coomassie brilliant
blue; FIG. 37C: immunoblot of a 4-12% non-denaturing
SDS-polyacrylamide gel probed with BR96 anti-idiotypic antibody;
lanes 1-15 correspond to fractions 7-21 on the gel filtration
profile shown in FIG. 37A; lane M represents molecular weight
marker proteins in kilodaltons. Molecular weight standards
correspond to 670 kDa, 158 kDa, 44 kDa and 17 kDa eluted in
fractions 10, 15, 21 and 30, respectively).
[0099] FIG. 38 is a graph depicting the results of a direct binding
assay on ELISA plates coated with Lewis-Y antigen and probed with
BR96 anti-idiotype antibody, and comparing the binding of BR96 IgG
(open square), BR96 sFv-PE40 monomers (closed circle), BR96
sFv-PE40 aggregates (closed triangle) and L6 IgG (open circle), as
described in Example 14, infra.
[0100] FIG. 39 is a graph showing the results of binding analysis
of BR96 sFv-PE40, with competition of .sup.125I-labeled BR96 IgG
with BR96 sFv-PE40 (closed circle), BR96 IgG (open square) and L6
IgG (open circle), as described in Example 14, infra.
[0101] FIG. 40 is a graph showing the results of cytotoxicity
analysis of BR96 sFv-PE40 inhibition of protein synthesis in MCF-7
cells as described in Example 14, infra (BR96 sFv-PE40 (closed
circle) and ChiBR96-LysPE40 (closed square)).
[0102] FIG. 41 are histograms of FACS analysis of five human
carcinoma lines as described in Example 14, infra (data is
displayed in each histogram as the mean channel number for BR96 IgG
or a human IgG control antibody. Fluorescence intensity for each
cell line is determined by subtracting the human IgG mean channel
number from the BR96 mean channel number).
[0103] FIG. 42 is a bar graph showing the results of competitive
cytotoxic analysis of BR96 sFv-PE40 inhibition of protein synthesis
in L2987 cells by BR96 sFv-PE40 (50 ng/ml) alone or in the presence
of either BR96 IgG or L6 IgG (100 .mu.g/ml) as described in Example
14, infra.
[0104] FIG. 43 is a graph showing anti-tumor effects of
BR96-sFvPE40 in vivo against MCF-7 human breast tumor xenografts,
as described in Example 15, infra (ADM 6 mg/kg (closed square),
BR96-sFvPE40 0.50 mg/kg (closed circle), BR96-sFvPE40 0.75 mg/kg
(open circle), control (closed triangle)).
[0105] FIG. 44 is a graph showing anti-tumor activity of
BR96-immunotoxins against L2987 human lung tumor xenografts as
described in Example 15, infra (sFv-PE40 0.125 mg/kg (closed
triangle), sFv-PE40 0.25 mg/kg (closed circle), sFv-PE40 0.375
mg/kg (open circle), IgG Lys-PE40 conjugate 1.25 mg/kg (closed
square), Interleukin 6-PE40 0.375 mg/kg (open square), control
(closed triangle)).
[0106] FIG. 45 is a drawing of the structure of BR96-DOX.
[0107] FIGS. 46A-D are line graphs showing the antigen-specific
antitumor activity of BR96-DOX.
[0108] (A) Control animals (closed square); animals treated with
BR96-DOX (5 mg/kg DOX) (closed circle), IgG-DOX (5 mg/kg DOX)
(closed triangle); or optimized DOX (8 mg/kg) (open square).
[0109] (B) Control animals (closed square); animals treated with
2BR96-DOX (10 mg/kg) (closed circle), IgG-DOX (10 mg/kg) (closed
triangle), or optimized DOX (8 mg/kg) (open square).
[0110] (C) Control animals (closed square); animals treated with
BR96-DOX (5 mg/kg) (closed circle), IgG-DOX (5 mg/kg) (closed
triangle), or DOX (6 mg/kg) (open square).
[0111] (D) Control animals (closed square) animals treated with
BR96-DOX (8 mg/kg) (closed circle), or DOX (8 mg/kg) (open
triangle).
[0112] FIG. 47 is a line graph showing that BR96-DOX cures athymic
mice of large disseminated tumors. Untreated controls (closed
triangle), BR96-DOX treated (8 mg/kg) (closed circle) or DOX
treated (8 mg/kg) (closed square) 82, 86 and 90 days after
inoculation of tumor cells.
[0113] FIG. 48 is a line graph showing that BR96-DOX cures human
lung tumors implanted in athymic rats. Control animals (closed
square), animals treated with BR96-DOX (4 mg/kg) (open circle),
BR96-DOX (2 mg/kg) (closed triangle), or DOX (4 mg/kg) (open
square).
[0114] FIG. 49(a) provides a synthetic scheme for preparing a
thiolated antibody using SPDP as the thiolation agent.
[0115] FIG. 49(b) provides a synthetic scheme for preparing an
immunoconjugate of the invention in which the ligand is a
SPDP-thiolated antibody.
[0116] FIG. 49(c) provides a synthetic scheme for preparing an
immunoconjugate of the invention in which the ligand is an
iminothiolane-thiolated antibody.
[0117] FIG. 50 shows a process for reducing with DTT an antibody to
prepare a "relaxed" antibody and synthesis of an immunoconjugate of
the invention.
[0118] FIG. 51 provides in vitro cytotoxic activity data for
BR64-Adriamycin conjugates of the invention against L2987
tumors.
[0119] FIG. 52 provides in vivo cytotoxic activity data for
BR64-Adriamycin conjugates of the invention against L2987
tumors.
[0120] FIGS. 53A/B/C provides comparative in vivo cytotoxic data
for combination therapy using BR64, Adriamycin and non-binding
conjugate (SN7-Adriamycin).
[0121] FIG. 54 provides in vivo cytotoxic activity data for
Bombesin-Adriamycin conjugates of the invention against H345
tumors.
[0122] FIG. 55 provides in vitro cytotoxic activity data for
Adriamycin conjugates of relaxed chimeric BR96 and SPDP-thiolated
chimeric BR96.
[0123] FIG. 56 provides in vivo cytotoxic activity data for
Adriamycin conjugates of relaxed BR64 and relaxed chimeric L6
against L2987 tumors.
[0124] FIGS. 57 to 59 provide in vivo cytotoxic activity data
against L2987 tumors for Adriamycin conjugates of relaxed chimeric
BR96 compared to free Adriamycin and non-binding conjugates.
[0125] FIG. 60 provides in vivo cytotoxic activity data for
Adriamycin conjugates of relaxed chimeric BR96 against RCA Human
Breast Tumors.
[0126] FIG. 61 provides in vivo cytotoxic activity data for
Adriamycin conjugates of relaxed chimeric BR96 against RCA Human
Colon Tumors.
[0127] FIG. 62 provides a graph of the effect on --SH titer as a
function of mole ratio of DTT to antibody in the preparation, under
an inert atmosphere, of a relaxed antibody.
DETAILED DESCRIPTION OF THE INVENTION
[0128] Definitions
[0129] As used in this application, the following words or phrases
have the meanings specified.
[0130] As used herein, "functional equivalent" means being capable
of (1) binding the antigen binding site to which BR96 is directed
(i.e., competitively inhibit the antigen binding site), (2) binding
carcinoma cells, (3) being internalized within the carcinoma cells
to which they bind, and/or (4) mediating ADCC and CDC effector
functions.
[0131] As used herein, "joined" means to couple directly or
indirectly one molecule with another by whatever means, e.g., by
covalent bonding, by non-covalent bonding, by ionic bonding, or by
non-ionic bonding. Covalent bonding includes bonding by various
linkers such as thioether linkers or thioester linkers. Direct
coupling involves one molecule attached to the molecule of
interest. Indirect coupling involves one molecule attached to
another molecule not of interest which in turn is attached directly
or indirectly to the molecule of interest.
[0132] As used herein, "recombinant molecule" means a molecule
produced by genetic engineering methods.
[0133] As used herein, "fragment" is defined as at least a portion
of the variable region of the immunoglobulin molecule which binds
to its target, i.e., the antigen binding region. Some of the
constant region of the immunoglobulin may be included.
[0134] As used herein, an "immunoconjugate" means any molecule or
ligand such as an antibody or growth factor chemically or
biologically linked to a cytotoxin, a radioactive agent, an
anti-tumor drug or a therapeutic agent. The antibody or growth
factor may be linked to the cytotoxin, radioactive agent,
anti-tumor drug or therapeutic agent at any location along the
molecule so long as it is able to bind its target. Examples of
immunoconjugates include immunotoxins and antibody conjugates.
[0135] As used herein, "selectively killing" means killing those
cells to which the antibody binds.
[0136] As used herein, examples of "carcinomas" include bladder,
breast, colon, liver, lung, ovarian, and pancreatic carcinomas.
[0137] As used herein, "immunotoxin" means an antibody or growth
factor chemically or biologically linked to a cytotoxin or
cytotoxic agent.
[0138] As used herein, an "effective amount" is an amount of the
antibody, immunoconjugate, recombinant molecule which kills cells
or inhibits the proliferation thereof.
[0139] As used herein, "competitively inhibits" means being capable
of binding to the same target as another molecule. With regard to
an antibody, competitively inhibits mean that the antibody is
capable of recognizing and binding the same antigen binding region
to which another antibody is directed.
[0140] As used herein, "antigen-binding region" means that part of
the antibody, recombinant molecule, the fusion protein, or the
immunoconjugate of the invention which recognizes the target or
portions thereof.
[0141] As used herein, "therapeutic agent" means any agent useful
for therapy including anti-tumor drugs, cytotoxins, cytotoxin
agents, and radioactive agents.
[0142] As used herein, "anti-tumor drug" means any agent useful to
combat cancer including, but not limited to, cytotoxins and agents
such as antimetabolites, alkylating agents, anthracyclines,
antibiotics, antimitotic agents, procarbazine, hydroxyurea,
asparaginase, corticosteroids, mytotane (0,P'-(DDD)), interferons
and radioactive agents.
[0143] As used herein, "a cytotoxin or cytotoxic agent" means any
agent that is detrimental to cells. Examples include taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof.
[0144] As used herein, "a radioactive agent" includes any
radioisotope which is effective in destroying a tumor. Examples
include, but are not limited to, cobalt-60 and X-rays.
Additionally, naturally occurring radioactive elements such as
uranium, radium, and thorium which typically represent mixtures of
radioisotopes, are suitable examples of a radioactive agent.
[0145] As used herein, "administering" means oral administration,
administration as a suppository, topical contact, intravenous,
intraperitoneal, intramuscular or subcutaneous administration, or
the implantation of a slow-release device such as a miniosmotic
pump, to the subject.
[0146] As used herein, "directly" means the use of antibodies
coupled to a label. The specimen is incubated with the labeled
antibody, unbound antibody is removed by washing, and the specimen
may be examined.
[0147] As used herein, "indirectly" means incubating the specimen
with an unconjugated antibody, washing and incubating with a
fluorochrome-conjugated antibody. The second or "sandwich" antibody
thus reveals the presence of the first.
[0148] As used herein "reacting" means to recognize and bind the
target. The binding may be non-specific. Specific binding is
preferred.
[0149] As used herein, "curing" means to provide substantially
complete tumor regression so that the tumor is not palpable for a
period of time, i.e., .gtoreq.10 tumor volume doubling delays
(TVDD=the time in days that it takes for control tumors to double
in size).
[0150] As used herein, "tumor associated antigens" means any cell
surface antigen which is generally associated with tumor cells,
i.e., occurring to a greater extent as compared with normal cells.
Such antigens may be tumor specific. Alternatively, such antigens
may be found on the cell surface of both tumorigenic and
non-tumorigenic cells. These antigens need not be tumor specific.
However, they are generally more frequently associated with tumor
cells than they are associated with normal cells.
[0151] As used herein, "tumor targeted antibody" means any antibody
which recognizes cell surface antigens on tumor (i.e., cancer)
cells. Although such antibodies need not be tumor specific, they
are tumor selective, i.e., bind tumor cells more so than it does
normal cells.
[0152] As used herein, "internalizing tumor targeted antibody"
includes any tumor targeted antibody which is easily taken up by
the tumor cells to which they bind.
[0153] As used herein, "internalizing tumor targeted antibody which
recognizes the Le.sup.y determinant" includes internalizing tumor
targeted antibody which specifically recognizes at least a portion
of the Le.sup.y determinant.
[0154] As used herein, "inhibit proliferation" means to interfere
with cell growth by whatever means.
[0155] As used herein, "mammalian tumor cells" include cells from
animals such as human, ovine, porcine, murine, bovine animals.
[0156] As used herein, "pharmaceutically acceptable carrier"
includes any material which when combined with the antibody retains
the antibody's immunogenicity and non-reactive with the subject's
immune systems. Examples include, but are not limited to, any of
the standard pharmaceutical carriers such as a phosphate buffered
saline solution, water, emulsions such as oil/water emulsion, and
various types of wetting agents. Other carriers may also include
sterile solutions, tablets including coated tablets and
capsules.
[0157] Typically such carriers contain excipients such as starch,
milk, sugar, certain types of clay, gelatin, stearic acid or salts
thereof, magnesium or calcium stearate, talc, vegetable fats or
oils, gums, glycols, or other known excipients. Such carriers may
also include flavor and color additives or other ingredients.
Compositions comprising such carriers are formulated by well known
conventional methods.
[0158] In order that the invention herein described may be more
fully understood, the following description is set forth.
[0159] 1. Novel Antibodies of the Invention.
[0160] The present invention relates to novel antibodies that are
highly specific for carcinoma cells. More particularly, the
antibodies react with a range of carcinomas such as breast, lung,
ovary and colon carcinomas, while showing none or limited
reactivity with normal human tissues or other types of tumors such
as sarcomas or lymphomas.
[0161] One type of novel antibodies of the invention is designated
BR96. The BR96 antibodies can be used to isolate and characterize
the antigen to which they bind. Thus, the BR96 antibodies can be
used as a probe to identify and characterize the epitope recognized
and to further define the cell membrane antigen with which they
react (see, e.g., Nudelman et al., "Characterization of Human
Melanoma-Associated Ganglioside Antigen Defined By A Monoclonal
Antibody, 4.2," J. Biol. Chem., 257(1):12752-56 (1982) and
Hakomori, "Tumor Associated Carbohydrate Antigens," Ann. Rev.
Immunol. 2:103-262(1984)).
[0162] BR96 recognizes as at least part of its binding site a
portion of an epitope of a Le.sup.y carbohydrate determinant which
is a portion of an antigen abundantly expressed on carcinomas of
the colon, breast, ovary, and lung and, to a lesser extent, on
epithelial cells from the gastrointestinal tract. Further, BR96 in
the absence of effector cells or complement can inhibit tumor cell
DNA synthesis.
[0163] Results of preliminary epitope screens conducted on
monoclonal antibody BR96 have indicated that the epitope which is a
portion of the antigen on the carcinoma cells to which BR96
antibody binds is a fucosylated variant of Lewis Y (Le.sup.y).
Le.sup.y has been described by Abe et al., J. Biol. Chem. 258:8934
(1983); Lloyd et al., Immunogenetics 17:537 (1983); Brown et al.,
Biosci. Rep. 3:163 (1983); and Hellstrom et al., Cancer Res.
46:3917 (1986). Certain fucosylated variants of Lewis Y have been
described by Abe et al., Cancer Res. 46:2639-2644 (1986).
[0164] The monoclonal antibody of the invention can be produced
using well-established hybridoma techniques first introduced by
Kohler and Milstein (see, Kohler and Milstein, "Continuous Cultures
Of Fused Cells Secreting Antibody Of Pre-Defined Specificity,"
Nature, 256:495-97 (1975). See, also, Brown et al., "Structural
Characterization Of Human Melanoma-Associated Antigen p97 with
Monoclonal Antibodies," J. Immunol. 127(2):539-46 (1981)); Brown et
al., "Protein Antigens Of Normal And Malignant Human Cells
Identified By Immunoprecipitation With Monoclonal Antibodies," J.
Biol. Chem. 255:4980-83 (1980); Yeh et al., "Cell Surface Antigens
Of Human Melanoma Identified By Monoclonal Antibody," Proc. Natl.
Acad. Sci. USA, 76(6):297-31 (1979); and Yeh et al., "A
Cell-Surface Antigen Which is Present In the Ganglioside Fraction
And Shared By Human Melanomas," Int. J. Cancer. 29:269-75
(1982).
[0165] These techniques involve the injection of an immunogen
(e.g., cells or cellular extracts carrying the antigen or purified
antigen) into an animal (e.g., a mouse) so as to elicit a desired
immune response (i.e., antibodies) in that animal. After a
sufficient time, antibody-producing lymphocytes are obtained from
the animal either from the spleen, lymph nodes or peripheral blood.
Preferably, the lymphocytes are obtained from the spleen. The
splenic lymphocytes are then fused with a myeloma cell line,
usually in the presence of a fusing agent such as polyethylene
glycol (PEG). Any of a number of myeloma cell lines may be used as
a fusion partner according to standard techniques; for example, the
P3-NS1/1Ag4-1, P3-x63-Ag8.653 or Sp2/O Ag14 myeloma lines. These
myeloma lines are available from the American Type Culture
Collection ("ATCC") in Rockville, Md.
[0166] The resulting cells, which include the desired hybridomas,
are then grown in a selective medium, such as HAT medium, in which
unfused parental myeloma or lymphocyte cells eventually die. Only
the hybridoma cells survive and can be grown under limiting
conditions to obtain isolated clones. The supernatants of the
hybridomas are screened for the presence of antibody of that
desired specificity, e.g., by immunoassay techniques using the
antigen that had been used for immunization. Positive clones can
then be subcloned under limiting dilution conditions and the
monoclonal antibody produced can be isolated. Hybridomas produced
according to these methods can be propagated in vitro or in vivo
(in ascites fluid) using techniques known in the art (see,
generally, Fink et al., supra at page 123, FIGS. 6-11). Commonly
used methods for purifying monoclonal antibodies include ammonium
sulfate precipitation, ion exchange chromatography, and affinity
chromatography (see, e.g., Zola et al., "Techniques For The
Production And Characterization Of Monoclonal Hybridoma
Antibodies," in Monoclonal Hybridoma Antibodies: Techniques And
Applications, Hurell (ed.), pp. 51-52 (CRC Press 1982)).
[0167] According to a preferred embodiment, a monoclonal antibody
of this invention, designated BR96, was produced via the hybridoma
techniques described hereinbelow using a breast cancer cell line
3396 as the immunogen. The BR96 hybridoma, prepared as described
hereinbelow and producing the BR96 antibody, was deposited on Feb.
22, 1989 with the American Type Culture Collection (ATCC), 12301
Parklawn Drive, Rockville, Md. 20852 and has there been identified
as follows:
[0168] BR96 ATCC Accession No.: HB 10036
[0169] The BR96 antibody is of the IgG3 subclass. The antibody
displays a high specificity for carcinoma cells of different organ
types, for example, tumors of the breast, lung, colon and ovary as
well as cultured cell lines established from various breast, lung
and colon carcinomas. Furthermore, the BR96 antibody shows no
binding to other types of tumor cells such as the T-cell lymphoma
cells lines, CEM and MOLT-4, the B cell lymphoma cell line P3HR-1
or melanoma cell lines. The BR96 antibody is able to be
internalized in antigen-positive tumor cells, is toxic to
antigen-positive tumor cells, mediates antibody-dependent cellular
cytotoxicity and complement-dependent cytotoxicity activity, and
surprisingly, is cytotoxic alone, i.e., in unmodified form. The
BR96 antibodies appear to recognize a novel epitope of the Le.sup.y
determinant.
[0170] The present invention provides an immunoconjugate comprising
a molecule having the antigen-binding region of the BR96 monoclonal
antibody joined to doxorubicin. It would be clear that doxorubicin
may be joined at any location along the molecule so long as it
retains its ability to bind its target. Doxorubicin may be joined
by any means including chemical and biological means.
[0171] Clearly analogs and homologs of doxorubicin are encompassed
by the invention. For example, an improved analog of doxorubicin is
Fe-chelate.
[0172] 2. Fragments of the Monoclonal Antibodies of the
Invention.
[0173] According to another embodiment, F(ab').sub.2 fragments of
the BR96 monoclonal antibody were produced by pepsin digestion of
purified BR96 (Lamoyi, "Preparation of F(ab').sub.2 Fragments from
Mouse IgG of Various Subclasses," Methods of Enzymol. 121:652-663
(1986)), as described hereinbelow. The binding of the F(ab').sub.2
fragments to tumor (3396) and MCF7 cells was shown to be comparable
to the binding of the whole BR96 monoclonal antibody.
[0174] 3. Chimeric Antibodies of the Invention.
[0175] In another preferred embodiment, a chimeric (murine/human)
antibody of the invention was produced using a two-step homologous
recombination procedure as described by Fell et al., in Proc. Natl.
Acad. Sci. USA 86:8507-8511 (1989) and in co-pending patent
application U.S. Ser. No. 243,873, filed Sep. 14, 1988, and Ser.
No. 468,035, filed Jun. 22, 1990, assigned to the same assignee as
the present application; the disclosures of all of these documents
are incorporated in their entirety by reference herein. This
two-step protocol involves use of a target vector encoding human
IgG.gamma.1 heavy chain to transfect a mouse hybridoma cell line
expressing murine BR96 monoclonal antibody (hybridoma ATCC No. HB
10036) to produce a hybridoma expressing a BR96 chimeric antibody
containing human IgG.gamma.1 heavy chain. This hybridoma is then
transfected with a target vector containing DNA encoding human
kappa (K) light chain to produce a murine hybridoma expressing a
BR96 chimeric antibody containing human IgG.gamma.1 heavy chain and
human K light chain. The target vectors used to transfect the
hybridomas are the ph.gamma.1HC-D vector digested with Xbal enzyme
(Bristol-Myers Squibb Co., Seattle, Wash., NRRL No. B 18599) and
the HindIII digested pSV.sub.2gpt/C.sub.K vector (Bristol-Myers
Squibb Co., Seattle, Wash., NRRL No. B 18507).
[0176] The chimeric BR96 hybridoma, identified herein as ChiBR96,
prepared as described hereinbelow and producing the chimeric
human/murine BR96 antibody, was deposited on May 23, 1990, with the
ATCC, 12301 Parklawn Drive, Rockville, Md. 20852 and has there been
identified as follows.
[0177] ChiBR96 ATCC Accession No.: HB 10460
[0178] Once the hybridoma that expresses the chimeric antibody is
identified, the hybridoma is cultured and the desired chimeric
molecules are isolated from the cell culture supernatant using
techniques well known in the art for isolating monoclonal
antibodies.
[0179] The term "BR96 antibody" as used herein includes whole,
intact polyclonal and monoclonal antibody materials such as the
murine BR96 monoclonal antibody produced by hybridoma ATCC No. HB
10036, and chimeric antibody molecules such as chimeric BR96
antibody produced by hybridoma ATCC No. 10460. The BR96 antibody
described above includes any fragments thereof containing the
active antigen-binding region of the antibody such as Fab,
F(ab').sub.2 and Fv fragments, using techniques well established in
the art (see, e.g., Rousseaux et al., "Optimal Conditions For The
Preparation of Proteolytic Fragments From Monoclonal IgG of
Different Rat IgG Subclasses," in Methods Enzymol., 121:663-69
(Academic Press 1986)). The BR96 antibody of the invention also
includes fusion proteins.
[0180] In addition, the BR96 antibody of this invention does not
display any immunohistologically detectable binding to normal human
tissues from major organs, such as kidney, spleen, liver, skin,
lung, breast, colon, brain, thyroid, heart, lymph nodes or ovary.
Nor does the antibody react with peripheral blood leukocytes. BR96
antibody displays limited binding to some cells in the tonsils and
testes, and binds to acinar cells in the pancreas, and to
epithelial cells in the stomach and esophagus. Thus, the BR96
antibody is superior to most known antitumor antibodies in the high
degree of specificity for tumor cells as compared to normal cells
(see, e.g., Helltrom et al., "Immunological Approaches To Tumor
Therapy: Monoclonal Antibodies, Tumor Vaccines, And
Anti-Idiotypes," in Covalently Modified Antigens And Antibodies In
Diagnosis And Therapy, Quash/Rodwell (eds.), pp. 1-39 (Marcel
Dekker, Inc., 1989) and Bagshawe, "Tumour Markers--Where Do We Go
From Here," Br. J. Cancer. 48:167-75 (1983)).
[0181] Also included within the scope of the invention are
anti-idiotypic antibodies to the BR96 antibody of the invention.
These anti-idiotypic antibodies can be produced using the BR96
antibody and/or the fragments thereof as immunogen and are useful
for diagnostic purposes in detecting humoral response to tumors and
in therapeutic applications, e.g., in a vaccine, to induce an
anti-tumor response in patients (see, e.g., Nepom et al.,
"Anti-Idiotypic Antibodies And The Induction Of Specific Tumor
Immunity," in Cancer And Metastasis Reviews, 6:487-501(1987)).
[0182] In addition, the present invention encompasses antibodies
that are capable of binding to the same antigenic determinant as
the BR96 antibodies and competing with the antibodies for binding
at that site. These include antibodies having the same antigenic
specificity as the BR96 antibodies but differing in species origin,
isotype, binding affinity or biological functions (e.g.,
cytotoxicity). For example, class, isotype and other variants of
the antibodies of the invention having the antigen-binding region
of the BR96 antibody can be constructed using recombinant
class-switching and fusion techniques known in the art (see, e.g.,
Thammana et al., "Immunoglobulin Heavy Chain Class Switch From IgM
to IgG In A Hybridoma," Eur. J. Immunol. 13:614 (1983); Spira et
al., "The Identification Of Monoclonal Class Switch Variants By
Subselection And ELISA Assay," J. Immunol. Meth. 74:307-15 (1984);
Neuberger et al., "Recombinant Antibodies Possessing Novel Effector
Functions," Nature. 312:604-608 (1984); and Oi et al., "Chimeric
Antibodies," Biotechniques, 4(3):214-21 (1986)). Thus, other
chimeric antibodies or other recombinant antibodies (e.g., fusion
proteins wherein the antibody is combined with a second protein
such as a lymphokine or a tumor inhibitory growth factor) having
the same binding specificity as the BR96 antibodies fall within the
scope of this invention.
[0183] Genetic engineering techniques known in the art are used as
described herein to prepare recombinant immunotoxins produced by
fusing antigen binding regions of antibody BR96 to a therapeutic or
cytotoxic agent at the DNA level and producing the cytotoxic
molecule as a chimeric protein.
[0184] Examples of therapeutic agents include, but are not limited
to, antimetabolites, alkylating agents, anthracyclines,
antibiotics, and anti-mitotic agents.
[0185] Antimetabolites include methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine.
[0186] Alkylating agents include mechlorethamine, thiotepa
chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin,
mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)
cisplatin.
[0187] Anthracyclines include daunorubicin (formerly daunomycin)
and doxorubicin (also referred to herein as adriamycin). Additional
examples include mitozantrone and bisantrene.
[0188] Antibiotics include dactinomycin (formerly actinomycin),
bleomycin, mithramycin, and anthramycin (AMC).
[0189] Antimytotic agents include vincristine and vinblastine
(which are commonly referred to as vinca alkaloids).
[0190] Other cytotoxic agents include procarbazine, hydroxyurea,
asparaginase, corticosteroids, mytotane (O, P'-(DDD)),
interferons.
[0191] Further examples of cytotoxic agents include, but are not
limited to, ricin, doxorubicin, taxol, cytochalasin B, gramicidin
D, ethidium bromide, etoposide, tenoposide, colchicin, dihydroxy
anthracin dione, 1-dehydrotestosterone, and glucocorticoid.
[0192] Clearly analogs and homologs of such therapeutic and
cytotoxic agents are encompassed by the present invention. For
example, the chemotherapeutic agent aminopterin has a correlative
improved analog namely methotrexate.
[0193] Further, the improved analog of doxorubicin is an
Fe-chelate. Also, the improved analog for 1-methylnitrosourea is
lomustine. Further, the improved analog of vinblastine is
vincristine. Also, the improved analog of mechlorethamine is
cyclophosphamide.
[0194] 4. Immunotoxins of the Invention.
[0195] Recombinant immunotoxins, particularly single-chain
immunotoxins, have an advantage over drug/antibody conjugates in
that they are more readily produced than these conjugates, and
generate a population of homogenous molecules, i.e., single
peptides composed of the same amino acid residues.
[0196] The techniques for cloning and expressing DNA sequences
encoding the amino acid sequences corresponding to the single-chain
immunotoxin BR96 sFv-PE40, e.g., synthesis of oligonucleotides,
PCR, transforming cells, constructing vectors, expression systems,
and the like are well-established in the art, and most
practitioners are familiar with the standard resource materials for
specific conditions and procedures (see, e.g., Sambrook et al.,
eds., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold
Spring Harbor Laboratory Press (1989)).
[0197] Details of the construction of the single-chain recombinant
immunotoxin of the invention, BR96 sFv-PE40 are provided in Example
13, infra. Briefly, polymerase chain reaction (PCR) (Mullis et al.,
U.S. Pat. Nos. 4,683,195 and 4,683,202; Mullis and Faloona, Methods
Enzymol. 154:335-350 (1987)) is used to amplify a 550 bp BR96 sFv
sequence (FIG. 35) encoded by plasmid pBR96 Fv using the selected
primers.
[0198] After PCR amplification and enzymatic digestion the 550 bp
fragment is ligated using standard procedures into a 4220 bp
fragment from vector pMS8 (Covell et al., Cancer Res. 46:3969-3978
(1986)) encoding the gene for PE40 to form intermediate vector pBW
7.01.
[0199] A fragment from pBR96 Fv is then subcloned into pBW 7.01 to
form plasmid pBW 7.0 encoding the BR96 sFv-PE40 gene fusion.
Correct ligations for vector construction are confirmed by DNA
sequence analysis using known procedures (Sanger et al., Proc.
Natl. Acad. Sci. USA 74:5463 (1977) and Messing et al., Nucleic
Acids Res. 9:309 (1981)). Colonies are then screened by restriction
enzyme digestion for the appropriate plasmids.
[0200] The following include preferred embodiments of the
immunoconjugates of the invention. Other embodiments which are
known in the art are encompassed by the invention. Specific
embodiments are set forth in the Examples which follow.
[0201] The invention is not limited to these specific
immunoconjugates, but also includes other immunoconjugates
incorporating antibodies and/or antibody fragments according to the
present invention.
[0202] The conjugates comprise at least one drug molecule connected
by a linker of the invention to a targeting ligand molecule that is
reactive with the desired target cell population. The ligand
molecule can be an immunoreactive protein such as an antibody, or
fragment thereof, a non-immunoreactive protein or peptide ligand
such as bombesin or, a binding ligand recognizing a cell associated
receptor such as a lectin or steroid molecule.
[0203] As previously noted, a conjugate of the invention is
represented by general Formula (I): 1
[0204] in which D is a drug molecule;
[0205] n is 1 to 10;
[0206] p is 1 to 6;
[0207] Y is O or NH.sub.2.sup.+C17;
[0208] z is 0 or 1;
[0209] q is about 1 to about 10;
[0210] X is a ligand; and,
[0211] A is Michael Addition Adduct.
[0212] For a better understanding of the invention, the drugs and
ligands will be discussed individually. The intermediates used for
the preparation of the conjugates and the synthesis of the
conjugates then will be explained.
[0213] The Drug
[0214] One skilled in the art understands that the present
invention requires the drug and ligand to be linked by means of an
acylhydrazone linkage, through a Michael Addition Adduct and
thioether-containing linker. Neither the specific drug nor the
specific ligand is to be construed as a limitation on the present
invention. The linkers of the present invention may be used with
any drug having any desired therapeutic, biological
activity-modifying or prophylactic purpose, limited only in that
the drug used in preparing the conjugate be able to form an
hydrazone bond. Preferably, to prepare the hydrazone, the drug
should have a reactively available carbonyl group, such as, for
example, a reactive aldehyde or ketone moiety (represented herein
as "[D--(C.dbd.O)]") which is capable of forming a hydrazone (i.e.,
a --C.dbd.N--NH-- linkage). The drug hydrazone linkage is
represented herein as "[D.dbd.N--NH--." In addition, the reaction
of that reactively available group with the linker preferably must
not destroy the ultimate therapeutic activity of the conjugate,
whether that activity is the result of the drug being released at
the desired site of action or whether the intact conjugate, itself,
is responsible for such activity.
[0215] One skilled in the art understands that for those drugs
which lack a reactively available carbonyl group, a derivative
containing such a carbonyl group may be prepared using procedures
known in the art. As can be appreciated, the conjugate prepared
from such derivatized drug must retain therapeutic activity when
present at the active site, whether this is due to the intact
conjugate, or otherwise. Alternatively, the derivatized drug or,
for example, a prodrug, must be released in such a form that a
therapeutically active form of the drug is present at the active
site.
[0216] The present linker invention may be used in connection with
drugs of substantially all therapeutic classes including, for
example, antibacterials, antivirals, antifungals, anticancer drugs,
antimycoplasmals, and the like. The drug conjugates so constructed
are effective for the usual purposes for which the corresponding
drugs are effective, and have superior efficacy because of the
ability, inherent in the ligand, to transport the drug to the
desired cell where it is of particular benefit.
[0217] Further, because the conjugates of the invention can be used
for modifying a given biological response, the drug moiety is not
to be construed as limited to classical chemical therapeutic
agents. For example, the drug moiety may be a protein or
polypeptide possessing a desired biological activity. Such proteins
may include, for example, a toxin such as abrin, ricin A,
pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor
necrosis factor, .alpha.-interferon, .beta.-interferon, nerve
growth factor, platelet derived growth factor, tissue plasminogen
activator; or, biological response modifiers such as, for example,
lymphokines, interleukin-1, ("IL-1"), interleukin-2 ("IL-2"),
interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating
factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"),
or other growth factors.
[0218] The preferred drugs for use in the present invention are
cytotoxic drugs, particularly those which are used for cancer
therapy. Such drugs include, in general, alkylating agents,
anti-proliferative agents, tubulin binding agents and the like.
Preferred classes of cytotoxic agents include, for example, the
anthracycline family of drugs, the vinca drugs, the mitomycins, the
bleomycins, the cytotoxic nucleosides, the pteridine family of
drugs, diynenes, and the podophyllotoxins. Particularly useful
members of those classes include, for example, adriamycin,
carminomycin, daunorubicin, aminopterin, methotrexate, methopterin,
dichloromethotrexate, mitomycin C, porfiromycin, 5-fluorouracil,
6-mercaptopurine, cytosine arabinoside, podophyllotoxin, or
podophyllotoxin derivatives such as etoposide or etoposide
phosphate, melphalan, vinblastine, vincristine, leurosidine,
vindesine, leurosine and the like. As noted previously, one skilled
in the art may make chemical modifications to the desired compound
in order to make reactions of that compound more convenient for
purposes of preparing conjugates of the invention.
[0219] A highly preferred group of cytotoxic agents for use as
drugs in the present invention include drugs of the following
formulae:
[0220] The Methotrexate Group of Formula (2): 2
[0221] in which R.sup.12 is amino or hydroxy;
[0222] R.sup.7 is hydrogen or methyl;
[0223] R.sup.8 is hydrogen, fluoro, chloro, bromo or iodo;
[0224] R.sup.9 is hydroxy or a moiety which completes a salt of the
carboxylic acid;
[0225] The Mitomycin Group of Formula (3): 3
[0226] in which R.sup.10 is hydrogen or methyl;
[0227] The Bleomycin Group of Formula (4): 4
[0228] in which R"is hydroxy, amino, C.sub.1-C.sub.3 alkylamino,
di(C.sub.1-C.sub.3 alkyl)amino, C.sub.4-C.sub.6 polymethylene
amino, 5
[0229] Melphalan of Formula (5): 6
[0230] 6-Mercaptopurine of Formula (6): 7
[0231] A Cytosine Arabinoside of Formula (7): 8
[0232] The Podophyllotoxins of Formula (8): 9
[0233] in which R.sup.13 is hydrogen or methyl;
[0234] R.sup.14 is methyl or thienyl;
[0235] or a phosphate salt thereof;
[0236] The Vinca Alkaloid Group of Drugs of Formula (9): 10
[0237] in which R.sup.15 is H, CH.sub.3 or CHO; when R.sup.17 and
R.sup.18 are taken singly, R.sup.18 is H, and one of R.sup.16 and
R.sup.17 is ethyl and the other is H or OH; when R.sup.17 and
R.sup.18 are taken together with the carbons to which they are
attached, they form an oxirane ring in which case R.sup.16 is
ethyl, R.sup.19 is hydrogen, (C.sub.1-C.sub.3 alkyl)-CO, or
chlorosubstituted (C.sub.1-C.sub.3 alkyl)-CO;
[0238] Difluoronucleosides of Formula (10): 11
[0239] in which R.sup.21 is a base of one of the formulae: 12
[0240] in which R.sup.22 is hydrogen, methyl, bromo, fluoro, chloro
or iodo;
[0241] R.sup.23 is --OH or --NH.sub.2;
[0242] R.sup.24 is hydrogen, bromo, chloro or iodo;
[0243] or,
[0244] The Anthracyclines Antibiotics of Formula (11): 13
[0245] wherein R.sub.1 is --CH.sub.3, --CH.sub.2OH,
--CH.sub.2OCO(CH.sub.2).sub.3CH.sub.3 or
--CH.sub.2OCOCH(OC.sub.2H.sub.5)- .sub.2;
[0246] R.sub.3 is --OCH.sub.3, --OH or --H
[0247] R.sub.4 is --NH.sub.2, --NHCOCF.sub.3, 4-morpholinyl,
3-cyano-4-morpholinyl, 1-piperidinyl, 4-methoxy-1-piperidinyl,
benzylamine, dibenzylamine, cyanomethylamine, or
1-cyano-2-methoxyethyl amine
[0248] R.sub.5 is --OH, --OTHP, or --H; and,
[0249] R.sub.6 is --OH or --H provided that R.sub.6 is not --OH
when R.sub.5 is --OH or --OTHP.
[0250] The most highly preferred drugs are the anthracycline
antibiotic agents of Formula (11), described previously. One
skilled in the art understands that this structural formula
includes compounds which are drugs, or are derivatives of drugs,
which have acquired in the art different generic or trivial names.
Table 14, which follows, represents a number of anthracycline drugs
and their generic or trivial names and which are especially
preferred for use in the present invention.
1TABLE 14 Formula (11) 14 Compound R.sub.1 R.sub.3 R.sub.4 R.sub.5
R.sub.6 Daunorubicin.sup.a CH.sub.3 OCH.sub.3 NH.sub.2 OH H
Adriamycin.sup.b CH.sub.2OH OCH.sub.3 NH.sub.2 OH H Detorubicin
CH.sub.2OCOCH(OC.sub.2H.sub.5).sub.2 OCH.sub.3 NH.sub.2 OH H
Carminomycin CH.sub.3 OH NH.sub.2 OH H Idarubicin CH.sub.3 H
NH.sub.2 OH H Epirubicin CH.sub.2OH OCH.sub.3 NH.sub.2 H OH
Esorubicin CH.sub.2OH OCH.sub.3 NH.sub.2 H H THP CH.sub.2OH
OCH.sub.3 NH.sub.2 OTHP H AD-32 CH.sub.2OCO(CH.sub.2).sub.3CH.sub.-
3 OCH.sub.3 NHCOCF.sub.3 OH H .sup.a"Daunomycin" is an alternate
name for daunorubicin .sup.b"Doxorubicin" is an alternate name for
adriamycin
[0251] Of the compounds shown in Table 14, the most highly
preferred drug is adriamycin. Adriamycin (also referred to herein
as "ADM") is that anthracycline of Formula (11) in which R.sub.1 is
--CH.sub.2OH, R.sub.3 is --OCH.sub.3, R.sub.4 is --NH.sub.2,
R.sub.5 --OH, and R.sub.6 is --H.
[0252] The Ligands
[0253] One skilled in the art understands that "ligand" includes
within its scope any molecule that specifically binds or reactively
associates or complexes with a receptor or other receptive moiety
associated with a given target cell population. This cell reactive
molecule, to which the drug reagent is linked via the linker in the
conjugate, can be any molecule that binds to, complexes with or
reacts with the cell population sought to be therapeutically or
otherwise biologically modified and, which possesses a free
reactive sulfhydryl (--SH) group or can be modified to contain such
a sulfhydryl group. The cell reactive molecule acts to deliver the
therapeutically active drug moiety to the particular target cell
population with which the ligand reacts. Such molecules include,
but are not limited to, large molecular weight proteins (generally
greater than 10,000 daltons) such as, for example, antibodies,
smaller molecular weight proteins (generally, less than 10,000
daltons), polypeptide or peptide ligands, and non-peptidyl
ligands.
[0254] The non-immunoreactive protein, polypeptide, or peptide
ligands which can be used to form the conjugates of this invention
may include, but are not limited to, transferrin, epidermal growth
factors ("EGF"), bombesin, gastrin, gastrin-releasing peptide,
platelet-derived growth factor, IL-2, IL-6, tumor growth factors
("TGF"), such as TGF-.alpha. and TGF-.beta., vaccinia growth factor
("VGF"), insulin and insulin-like growth factors I and II.
Non-peptidyl ligands may include, for example, steroids,
carbohydrates and lectins.
[0255] The immunoreactive ligands comprise an antigen-recognizing
immunoglobulin (also referred to as "antibody"), or
antigen-recognizing fragment thereof. Particularly preferred
immunoglobulins are those immunoglobulins which can recognize a
tumor-associated antigen. As used, "immunoglobulin" may refer to
any recognized class or subclass of immunoglobulins such as IgG,
IgA, IgM, IgD, or IgE. Preferred are those immunoglobulins which
fall within the IgG class of immunoglobulins. The immunoglobulin
can be derived from any species. Preferably, however, the
imunoglobulin is of human, murine, or rabbit origin. Further, the
immunoglobulin may be polyclonal or monoclonal, preferably
monoclonal.
[0256] As noted, one skilled in the art will appreciate that the
invention also encompasses the use of antigen recognizing
immunoglobulin fragments. Such imunoglobulin fragments may include,
for example, the Fab', F(ab').sub.2, F.sub.v or Fab fragments, or
other antigen recognizing immunoglobulin fragments. Such
immunoglobulin fragments can be prepared, for example, by
proteolytic enzyme digestion, for example, by pepsin or papain
digestion, reductive alkylation, or recombinant techniques. The
materials and methods for preparing such immunoglobulin fragments
are well-known to those skilled in the art. See generally, Parham,
J. Immunology, 131, 2895 (1983); Lamoyi et al., J. Immunological
Methods, 56, 235 (1983); Parham, Id., 53, 133 (1982); and Matthew
et al., Id., 50, 239 (1982).
[0257] The immunoglobulin can be a "chimeric antibody" as that term
is recognized in the art. Also, the immunoglobulin may be a
"bifunctional" or "hybrid" antibody, that is, an antibody which may
have one arm having a specificity for one antigenic site, such as a
tumor associated antigen while the other arm recognizes a different
target, for example, a hapten which is, or to which is bound, an
agent lethal to the antigen-bearing tumor cell. Alternatively, the
bifunctional antibody may be one in which each arm has specificity
for a different epitope of a tumor associated antigen of the cell
to be therapeutically or biologically modified. In any case, the
hybrid antibodies have a dual specificity, preferably with one or
more binding sites specific for the hapten of choice or one or more
binding sites specific for a target antigen, for example, an
antigen associated with a tumor, an infectious organism, or other
disease state.
[0258] Biological bifunctional antibodies are described, for
example, in European Patent Publication, EPA 0 105 360, to which
those skilled in the art are referred. Such hybrid or bifunctional
antibodies may be derived, as noted, either biologically, by cell
fusion techniques, or chemically, especially with cross-linking
agents or disulfide bridge-forming reagents, and may be comprised
of whole antibodies and/or fragments thereof. Methods for obtaining
such hybrid antibodies are disclosed, for example, in PCT
application WO83/03679, published Oct. 27, 1983, and published
European Application EPA 0 217 577, published Apr. 8, 1987, both of
which are incorporated herein by reference. Particularly preferred
bifunctional antibodies are those biologically prepared from a
"polydoma" or "quadroma" or which are synthetically prepared with
cross-linking agents such as bis-(maleimido)-methyl ether ("BMME"),
or with other cross-linking agents familiar to those skilled in the
art.
[0259] In addition the immunoglobin may be a single chain antibody
("SCA"). These may consist of single chain Fv fragments ("scFv") in
which the variable light ("V.sub.L") and variable heavy ("V.sub.H")
domains are linked by a peptide bridge or by disulfide bonds. Also,
the immunoglobulin may consist of single V.sub.H domains (dAbs)
which possess antigen-binding activity. See, e.g., G. Winter and C.
Milstein, Nature, 349, 295 (1991); R. Glockshuber et al.,
Biochemistry 29, 1362 (1990); and E. S. Ward et al., Nature 341,
544 (1989).
[0260] Especially preferred for use in the present invention are
chimeric monoclonal antibodies, preferably those chimeric
antibodies having specificity toward a tumor associated antigen. As
used herein, the term "chimeric antibody" refers to a monoclonal
antibody comprising a variable region, i.e., binding region, from
one source or species and at least a portion of a constant region
derived from a different source or species, usually prepared by
recombinant DNA techniques. Chimeric antibodies comprising a murine
variable region and a human constant region are especially
preferred in certain applications of the invention, particularly
human therapy, because such antibodies are readily prepared and may
be less immunogenic than purely murine monoclonal antibodies. Such
murine/human chimeric antibodies are the product of expressed
immunoglobulin genes comprising DNA segments encoding murine
immunoglobulin variable regions and DNA segments encoding human
immunoglobulin constant regions. Other forms of chimeric antibodies
encompassed by the invention are those in which the class or
subclass has been modified or changed from that of the original
antibody. Such "chimeric" antibodies are also referred to as
"class-switched antibodies." Methods for producing chimeric
antibodies involve conventional recombinant DNA and gene
transfection techniques now well known in the art. See, e.g.,
Morrison, S. L., et al., Proc. Nat'l Acad. Sci., 81, 6851
(1984).
[0261] Encompassed by the term "chimeric antibody" is the concept
of "humanized antibody," that is those antibodies in which the
framework or "complementarity determining regions ("CDR") have been
modified to comprise the CDR of an immunoglobulin of different
specificity as compared to that of the parent immunoglobulin. In a
preferred embodiment, a murine CDR is grafted into the framework
region of a human antibody to prepare the "humanized antibody."
See, e.g., L. Riechmann et al., Nature 332, 323 (1988); M. S.
Neuberger et al., Nature 314, 268 (1985). Particularly preferred
CDR's correspond to those representing sequences recognizing the
antigens noted above for the chimeric and bifunctional antibodies.
The reader is referred to the teaching of EPA 0 239 400 (published
Sep. 30, 1987), incorporated herein by reference, for its teaching
of CDR modified antibodies.
[0262] One skilled in the art will recognize that a
bifunctional-chimeric antibody can be prepared which would have the
benefits of lower immunogenicity of the chimeric or humanized
antibody, as well as the flexibility, especially for therapeutic
treatment, of the bifunctional antibodies described above. Such
bifunctional-chimeric antibodies can be synthesized, for instance,
by chemical synthesis using cross-linking agents and/or recombinant
methods of the type described above. In any event, the present
invention should not be construed as limited in scope by any
particular method of production of an antibody whether
bifunctional, chimeric, bifunctional-chimeric, humanized, or an
antigen-recognizing fragment or derivative thereof
[0263] In addition, the invention encompasses within its scope
immunoglobulins (as defined above) or immunoglobulin fragments to
which are fused active proteins, for example, an enzyme of the type
disclosed in Neuberger et al., PCT application, WO86/01533,
published Mar. 13, 1986. The disclosure of such products is
incorporated herein by reference.
[0264] As noted, "bifunctional," "fused," "chimeric" (including
humanized), and "bifunctional-chimeric" (including humanized)
antibody constructions also include, within their individual
contexts constructions comprising antigen recognizing fragments. As
one skilled in the art will recognize, such fragments could be
prepared by traditional enzymatic cleavage of intact bifunctional,
chimeric, humanized, or chimeric-bifunctional antibodies. If,
however, intact antibodies are not susceptible to such cleavage,
because of the nature of the construction involved, the noted
constructions can be prepared with immunoglobulin fragments used as
the starting materials; or, if recombinant techniques are used, the
DNA sequences, themselves, can be tailored to encode the desired
"fragment" which, when expressed, can be combined in vivo or in
vitro, by chemical or biological means, to prepare the final
desired intact immunoglobulin "fragment." It is in this context,
therefore, that the term "fragment" is used.
[0265] Furthermore, as noted above, the immunoglobulin (antibody),
or fragment thereof, used in the present invention may be
polyclonal or monoclonal in nature. Monoclonal antibodies are the
preferred immunoglobulins, however. The preparation of such
polyclonal or monoclonal antibodies now is well known to those
skilled in the art who, of course, are fully capable of producing
useful immunoglobulins which can be used in the invention. See,
e.g., G. Kohler and C. Milstein, Nature 256, 495 (1975). In
addition, hybridomas and/or monoclonal antibodies which are
produced by such hybridomas and which are useful in the practice of
the present invention are publicly available from sources such as
the American Type Culture Collection ("ATCC") 12301 Parklawn Drive,
Rockville, Md. 20852 or, commercially, for example, from
Boehringer-Mannheim Biochemicals, P.O. Box 50816, Indianapolis,
Ind. 46250.
[0266] Particularly preferred monoclonal antibodies for use in the
present invention are those which recognize tumor associated
antigens. Such monoclonal antibodies, are not to be so limited,
however, and may include, for example, the following:
2 Antigen Site Monoclonal Recognized Antibodies Reference Lung
Tumors KS1/4 N. M. Varki et al., Cancer Res. 44: 681 (1984). 534,
F8, F. Cuttitta et al., in: G. L. Wright (ed) Monoclonal 604A9
Antibodies and Cancer, Marcel Dekker, Inc., NY., p. 161, 1984.
Squamous Lung G1, LuCa2, Kyoizumi et al., Cancer Res. 45: 327
(1985). LuCA3, LuCA4 Small Cell Lung TFS-2 Okabe et al., Cancer
Res. 45: 1930 (1985). Cancer Colon Cancer 11.285.14 G. G. Rowland
et al., Cancer Immunol. Immunother. 14.95.55 19: 1 (1985).
NS-3a-22, Z. Steplewski et al., Cancer Res. 41: 2723 (1981). NS-10
NS-19-9, NS-33a NS-52a, 17- 1A Carcinoembryonic moAb 35 or Acolla,
R. S. et al., Proc. Natl. Acad. Sci. (USA) 77: 563 ZCE025 (1980).
Melanoma 9.2.27 T. F. Bumol and R. A. Reisfeld, Proc. Natl. Acad.
Sci. (USA) 79: 1245 (1982). p97 96.5 K. E. Hellstrom et al.,
Monoclonal Antibodies and Cancer, loc. cit. p. 31. Antigen T65 T101
Boehringer-Mannheim P. O. Box 50816 Indianapolis, IN 46250 Ferritin
Antiferrin Boehringer-Mannheim P. O. Box 50816 Indianapolis, IN
46250 R24 W. G. Dippold et al., Proc. Natl. Acad. Sci (USA) 77:
6114 (1980). Neuroblastoma P1 153/3 R. H. Kennet and F. Gilbert,
Science 203: 1120 (1979). MIN 1 J. T. Kemshead in Monoclonal
Antibodies and Cancer, loc. cit. p. 49. UJ13A Goldman et al.,
Pediatrics 105: 252 (1984). Glioma BF7, GE2, N. de Tribolet et al.,
in Monoclonal Antibodies and CG12 Cancer, loc. cit. p. 81.
Ganglioside L6 I. Hellstrom et al., Proc. Natl Acad. Sci. (USA) 83:
7059 (1986); U.S. Pat. Nos. 4,906,562, issued Mar. 6, 1990 and
4,935,495, issued Jun. 19, 1990. Chimeric L6 U.S. Ser. No.
07/923,244, filed Oct. 27, 1986, equivalent to PCT Patent
Publication, WO 88/03145, published May 5, 1988. Lewis Y BR64 U.S.
Ser. Nos. 07/289,635, filed Dec. 22, 1988, and U.S. Ser. No.
07/443,696, filed Nov. 29, 1989, equivalent to European Patent
Publication, EP A 0 375 562, published Jun. 27, 1990. fucosylated
Lewis Y BR96, U.S. Ser. Nos. 07/374,947, filed Jun. 30, 1989, and
Chimeric U.S. Ser. No. 07/544,246, filed Jun. 26, 1990, BR96
equivalent to PCT Patent Publication, WO 91/00295, published Jan.
10, 1991. Breast Cancer B6.2, B72.3 D. Colcher et al., in
Monoclonal Antibodies and Cancer, loc. cit. p. 121. Osteogenic
792T/48, M. J. Umbleton, ibid, p. 181. carcoma 792T/36 Leukemia
CALL 2 C. T. Teng et al., Lancet 1: 01 (1982). anti-idiotype R. A.
Miller et al., N. Eng. J. Med. 306: 517 (1982). Ovarian Cancer OC
125 R. C. Bast et al., J. Clin. Invest. 68: 1331 (1981). Prostate
Cancer D83.21, J. J. Starling et al., in Monoclonal Antibodies and
P6.2, Turp- Cancer, loc. cit., p. 253. 27 Renal Cancer A6H, D5D P.
H. Lang et al., Surgery 98: 143 (1985).
[0267] In the most preferred embodiment, the ligand containing
conjugate is derived from chimeric antibody BR96, "ChiBR96,"
disclosed in U.S. Ser. No. 07/544,246, filed Jun. 26, 1990, and
which is equivalent to PCT Published Application, WO 91,00295,
published Jan. 10, 1991. ChiBR96 is an internalizing murine/human
chimeric antibody and is reactive, as noted, with the fucosylated
Lewis Y antigen expressed by human carcinoma cells such as those
derived from breast, lung, colon and ovarian carcinomas. The
hybridoma expressing chimeric BR96 and identified as ChiBR96 was
deposited on May 23, 1990, under the terms of the Budapest Treaty,
with the American Type Culture Collection ("ATCC"), 12301 Parklawn
Drive, Rockville, Md. 20852. Samples of this hybridoma are
available under the accession number ATCC HB 10460. ChiBR96 is
derived, in part, from its source parent, BR96. The hybridoma
expressing BR96 was deposited, on Feb. 21, 1989, at the ATCC, under
the terms of the Budapest Treaty, and is available under the
accession number HB 10036. The desired hybridoma is cultured and
the resulting antibodies are isolated from the cell culture
supernatant using standard techniques now well known in the art.
See, e.g., "Monoclonal Hybridoma Antibodies: Techniques and
Applications," Hurell (ed.) (CRC Press, 1982).
[0268] In another highly preferred embodiment the immunoconjugate
is derived from the BR64 murine monoclonal antibody disclosed in
U.S. Ser. Nos. 07/289,635, filed Dec. 22, 1988, and 07/443,696,
filed Nov. 29, 1989, equivalent to European Published Application
EP A 0 375 562, published Jun. 27, 1990. As noted above, this
antibody also is internalizing and is reactive with the Lewis Y
antigen expressed by carcinoma cells derived from the human colon,
breast, ovary and lung. The hybridoma expressing antibody BR64 and
is identified as BR64 was deposited on Nov. 3, 1988, under the
terms of the Budapest Treaty, with the ATCC and is available under
the accession number HB 9895. The hybridoma is cultured and the
desired antibody is isolated using standard techniques well known
in the art, such as those referenced above.
[0269] In a third highly preferred embodiment, an immunoconjugate
of the invention is derived from the L6 murine monoclonal antibody
disclosed in U.S. Pat. Nos. 4,906,562, issued Mar. 6, 1990, and
4,935,495, issued Jun. 19, 1990. L6 is a non-internalizing antibody
active against a ganglioside antigen expressed by human carcinoma
cells derived from human non-small cell lung, breast, colon or
ovarian carcinomas. The hybridoma expressing L6 and identified as
L6 was deposited under the terms of the Budapest Treaty on Dec. 6,
1984 at the ATCC and is available under the accession number HB
8677. The hybridoma is cultured and the desired antibody is
isolated using the standard techniques referenced above. A chimeric
form of the L6 antibody, if desired, is described in U.S. Ser. No.
07/923,244, equivalent to PCT Published Application, WO 88/03145,
published May 5, 1988.
[0270] Thus, as used "immunoglobulin" or "antibody" encompasses
within its meaning all of the immunoglobulin/antibody forms or
constructions noted above.
[0271] The Intermediates and the Conjugates
[0272] The invention provides as intermediates a Michael Addition
Receptor- and acylhydrazone-containing drug derivative of Formula
(IIa): 15
[0273] in which D is a drug moiety, n is an integer from 1 to 10
and R is a Michael Addition Receptor, all of which are as defined
above.
[0274] An especially preferred intermediate encompassed by Formula
(IIa) and which is useful for preparation of a conjugate of the
invention is one defined by Formula (IIb): 16
[0275] in which R.sub.1 is --CH.sub.3, --CH.sub.2OH,
--CH.sub.2OCO(CH.sub.2).sub.3CH.sub.3 or
--CH.sub.2OCOCH(OC.sub.2H.sub.5)- .sub.2;
[0276] R.sub.3 is --OCH.sub.3, --OH or --H
[0277] R.sub.4 is --NH.sub.2, --NHCOCF.sub.3, 4-morpholinyl,
3-cyano-4-morpholinyl, 1-piperidinyl, 4-methoxy-1-piperidinyl,
benzylamine, dibenzylamine, cyanomethylamine, or
1-cyano-2-methoxyethyl amine
[0278] R.sub.5 is --OH, --OTHP, or --H; and,
[0279] R.sub.6 is --OH or --H provided that R.sub.6 is not --OH
when R.sub.5 is --OH or --OTHP.
[0280] N is an integer from 1 to 10; and,
[0281] R is a Michael Addition receptor moiety.
[0282] The most preferred intermediate for use in the present
invention is defined by Formula (IIc): 17
[0283] in which R.sub.1, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are
as defined above for Formula (IIb).
[0284] Also used as an intermediate in the invention is a targeting
ligand which contains a freely reactive sulfhydryl group. The
sulfhydryl group can be contained within the native targeting
ligand or can be derived directly from the ligand or, from a
derivatized form of the ligand. In the preferred method for
preparing the conjugates of the invention, a sulfhydryl group on
the ligand or modified ligand reacts directly with the Michael
Addition Receptor of intermediate of Formula (IIa) to form the
final conjugate. Using this process, generally between about one
and about ten drug molecules may be linked to each ligand. Thus, in
Formula (I), g may be from about 1 to about 10.
[0285] When the conjugate is formed, the Michael Addition Receptor
portion becomes a "Michael Addition Adduct," as used herein. Thus,
for example, as one skilled in the art will appreciate, if the
Michael Addition receptor moiety in the Formulae (Ia) or (IIb)
compound is a maleimido moiety, the corresponding "Michael Addition
Adduct" portion of the final conjugate of Formula (I) will be a
succinimido moiety. Thus, a "Michael Addition Adduct" refers to a
moiety which would be obtained had a Michael Addition Receptor, as
defined in more detail below, undergone a Michael Addition
reaction.
[0286] One skilled in the art understands that in the synthesis
of-compounds of the invention, one may need to protect or block
various reactive functionalities on the starting compounds and
intermediates while a desired reaction is carried out on other
portions of the molecule. After the desired reactions are complete,
or at any desired time, normally such protecting groups will be
removed by, for example, hydrolytic or hydrogenolytic means. Such
protection and deprotection steps are conventional in organic
chemistry. One skilled in the art is referred to Protective Groups
in Organic Chemistry, McOmie, ed., Plenum Press, N.Y., N.Y. (1973);
and Protective Groups in Organic Synthesis, Greene, ed., John Wiley
& Sons, New York, N.Y., (1981) for the teaching of protective
groups which may be useful in the preparation of compounds of the
present invention.
[0287] By way of example only, useful amino-protecting groups may
include, for example, C.sub.1-C.sub.10 alkanoyl groups such as
formyl, acetyl, dichloroacetyl, propionyl, hexanoyl,
3,3-diethylhexanoyl, .gamma.-chlorobutyryl, and the like;
C.sub.1-C.sub.10 alkoxycarbonyl and C.sub.5-C.sub.15
aryloxy-carbonyl groups such as tert-butoxycarbonyl,
benzyloxycarbonyl, allyloxycarbonyl, 4-nitro-benzyloxycarbonyl and
cinnamoyloxycarbonyl; halo-(C.sub.2-C.sub.10)-alkoxycarbonyl such
as 2,2,2-trichloroethoxy-carbonyl; and C.sub.1-C.sub.15 arylalkyl
and alkenyl groups such as benzyl, phenethyl, allyl, trityl, and
the like. Other commonly used amino-protecting groups are those in
the form of enamines prepared with .beta.-keto-esters such as
methyl or ethyl acetoacetate.
[0288] Useful carboxy-protecting groups may include, for example,
C.sub.1-C.sub.10 alkyl groups such as methyl, tert-butyl, decyl;
halo-C.sub.1-C.sub.10 alkyl such as 2,2,2-trichloroethyl, and
2-iodoethyl; C.sub.5-C.sub.15 arylalkyl such as benzyl,
4-methoxybenzyl, 4-nitrobenzyl, triphenylmethyl, diphenylmethyl,
C.sub.1-C.sub.10 alkanoyloxymethyl such as acetoxymethyl,
propionoxymethyl and the like; and groups such as phenacyl,
4-halophenacyl, allyl, dimethylallyl, tri-(C.sub.1-C.sub.3
alkyl)silyl, such as trimethylsilyl, .beta.-p-toluenesulfonylethyl,
.beta.-p-nitrophenyl-thioethyl, 2,4,6,-trimethylbenzyl,
.beta.-methylthioethyl, -pthalimidomethyl,
2,4-dinitrophenylsulphenyl, 2-nitrobenzhydryl and related
groups.
[0289] Similarly, useful hydroxy protecting groups may include, for
example, the formyl group, the chloroacetyl group, the benzyl
group, the benzhydryl group, the trityl group, the 4-nitrobenzyl
group, the trimethylsilyl group, the phenacyl group, the tert-butyl
group, the methoxymethyl group, the tetrahydropyranyl group, and
the like.
[0290] In general, the intermediate Michael Addition Receptor
containing hydrazone drug derivative of Formulae (IIa), (IIb), or
(IIc) may be prepared, depending on the Michael Addition Receptor
moiety used, by reaction of the drug (or derivatized drug) with a
hydrazide containing a Michael Addition Receptor in the general
manner described in Method A: 18
[0291] As noted below, Method A is the preferred method when the
Michael Addition Receptor is a maleimido moiety.
[0292] Alternatively, the Formula (IIa) compound may be prepared by
reaction of the drug with a hydrazide to form an intermediate
hydrazone drug derivative followed by reaction of this compound
with a Michael Addition Receptor containing moiety according to the
general process described in Method B: 19
[0293] In Method A and Method B, D, n and R have the meanings
previously noted. In Method B, L represents a leaving group, such
as for example, halogen, mesylate or tosylate, capable of
undergoing nucleophilic displacement while C represents a group
which renders the Michael Addition Receptor, R, a good nucleophilic
reagent. Particularly useful groups represented by C may include,
for example, alkali metal ions such as Na.sup.+, K.sup.+ or
Li.sup.+.
[0294] A "Michael Addition Receptor," as one skilled in the art
will understand, is a moiety capable of reacting with a
nucleophilic reagent so as to undergo a nucleophilic addition
reaction characteristic of a Michael Addition reaction. As noted,
after the nucleophilic addition occurs, the Michael Addition
Receptor moiety is referred to as a "Michael Addition Adduct."
[0295] Michael Addition Receptors generally used in the Method A
process may include, for example, .alpha.,.beta.-ethylenic acids or
.alpha.,.beta.-thioacids such as those containing a
--C.dbd.C--COOH, --C.dbd.C--C(O)SH, --C.dbd.C--C(S)SH, or a
--C.dbd.C--C(S)OH moiety; .alpha.,.beta.-ethylenic esters or
thio-esters where the alkyl moiety is other than methyl or ethyl,
for example, those which contain a --C.dbd.C--COOR,
--C.dbd.C--C(S)OR, --C.dbd.C--C(S)SR, or --C.dbd.C--C(O)--SR
moiety, wherein R is an ester forming group other than methyl or
ethyl; .alpha.,.beta.-ethylenic amides, imides, thioamides and
thioimides (whether cyclic or acylic), for example, those which
contain a moiety such as --C.dbd.C--CONR.sub.2,
--C.dbd.C--CONHCO--, --C.dbd.C--CSNR.sub.2, --C.dbd.C--CSNHCO--, or
--C.dbd.C--CSNHCS--, whether cyclic or acyclic and in which
--CONR.sub.2 or --CSNR.sub.2 represents a primary, secondary, or
tertiary amide or thioamide moiety; .alpha.,.beta.-acetylenic acids
or thioacids, for example, those containing a moiety such as
--C.ident.--C--COOH, --C.ident.C--C(S)OH, --C.ident.C--C(S)SH, or
--C.ident.C--C(O)--SH; .alpha.,.beta.-acetylenic esters, for
example those which contain a moiety such as --C.ident.C--COOR,
--C.ident.C--C(S)OR, --C.ident.C--C(S)SR, or --C.ident.C--C(O)--SR
in which R is an ester forming group other than methyl or ethyl;
.alpha.,.beta.-ethylenic nitriles, for example those containing a
moiety such as --C.dbd.C--C.ident.N; Michael Addition reactive
cyclopropane derivatives, for example, 1-cyano-1-ethoxycarbonyl
cyclopropane 20
[0296] a vinyl dimethyl-sulphonium bromide, for example, one
containing a --C.dbd.C--S.sup.+(Me).sub.2Br.sup.- moiety; an
.alpha.,.beta.-ethylenic sulfone, for example, one containing a
21
[0297] moiety; .alpha.,.beta.-ethylenic nitro compounds, for
example, one containing --C.dbd.C--NO.sub.2 moiety;
.alpha.,.beta.-ethylenic phosphonium compounds, for example one
containing a 22
[0298] group; a compound containing a grouping such as
C.dbd.C--C.dbd.N, as would be found, for example, in an aromatic
heterocyle such as a 2- or 4-vinyl pyridine; or a compound
containing an .alpha.,.beta.-unsaturated thionium ion moiety, such
as 23
[0299] Michael Addition Receptors used in Method B may include
.alpha.,.beta.-ethylenic aldehydes, for example those compounds
containing a --C.dbd.C--CHO moiety; .alpha.,.beta.-ethylenic
ketones, for example those compounds containing a 24
[0300] moiety; .alpha.,.beta.-ethylenic esters or thio-esters such
as compounds containing a --C.dbd.C--COOR, --C.dbd.C--C(S)OR,
--C.dbd.C--C(S)SR, or --C.dbd.C--C(O)--SR moiety in which R is a
ester-forming moiety which is methyl or ethyl, e.g., 25
[0301] .alpha.,.beta.-acetylenic aldehydes or ketones, for example
compounds containing a --C.ident.C--CHO or --C.ident.C--CO--
moiety; .alpha.,.beta.-acetylenic esters or thio-esters that have
methkyl or ethyl as their alkyl moiety, for example a compound
containing a --C.ident.C--COOR, --C.ident.C--C(S)OR,
--C.ident.C--C(O)SR or --C.ident.C--CSSR group in which R is an
ester forming moiety which is methyl or ethyl.
[0302] One skilled in the art may be familiar with other Michael
Addition Receptors which may be used in the present invention. For
a general discussion of the Michael Addition Reaction, the reader
is referred to E. D. Bergman, D. Ginsberg, and R. Pappo, Org.
React. 10, 179-555 (1959); and, D. A. Oare and C. H. Heathcock,
Topics in Stereochemistry, Vol. 20, eds., E. L. Eliel and S. H.
Wilen, John Wiley and Sons, Inc. (1991), and references cited
therein.
[0303] The precise reaction conditions used to prepare the
intermediates of Formulae (IIa), (IIb), or (IIc) will depend upon
the nature of the drug and the Michael Addition Receptor used in
the reaction. The most preferred intermediate of the invention is
that represented by Formula (IIc), above, in which the drug moiety
is an anthracycline drug and the Michael Addition Receptor is a
maleimido group. As noted earlier, for this reaction, Method A,
described above, is used. Upon reaction with the ligand (thiolated,
modified or otherwise), the maleimido Michael Addition Receptor of
the intermediate becomes a succinimido group (the Michael Addition
Adduct") in the final conjugate.
[0304] The sulfhydryl containing ligands exist naturally (i.e., the
ligand has not been modified) or may be produced, for example, (a)
by thiolation of the ligand by reaction with a thiolating reagent
such as SMCC or N-succinimid-yl-3-(2-pyridyldithio) propionate
("SPDP") followed by reduction of the product; (b) thiolation of
the native ligand by reaction with iminothiolane ("IMT"); (c)
addition of a sulfhydryl containing amino acid residue, for
example, a cysteine residue, to the ligand should the ligand, for
example, a protein peptide or polypeptide, fail to have a reactive
and available sulfhydryl moiety; or, (d) reduction of a disulfide
bond in a native molecule using a reducing agent useful for such
purposes, for example, dithiothreitol ("DTT"). Method (d) is the
most preferred method for production of sulfhydryl groups in
antibody molecules used in the conjugates of the invention.
[0305] If a thiolating reagent such as SPDP or iminothiolane is
used to prepare a conjugate of the invention, one skilled in the
art will appreciate that a short "spacer" residue will be inserted
between the Michael Addition Receptor moiety and the ligand in the
conjugate of Formula (I). In such a case, z will be 1 in the
Formula (I) compound. In the situation in which a free sulfhydryl
group on the ligand is used directly, for example by use of a DTT
reduced ligand (particularly a "relaxed" antibody prepared using
for example, DTT), or in which a reactive residue, for example,
cysteine is inserted into the ligand portion of the molecule, z in
Formula (I) will be 0 and a direct thioether bond will exist
between the binding ligand and the Michael Addition portion of the
molecule.
[0306] To form a conjugate, the thiolated ligand, or ligand having
a freely reactive sulfhydryl group, is reacted with the Michael
Addition receptor containing hydrazone of Formula (IIa). In
general, the reaction conditions must be chosen with regard to the
stability of the ligand, the drug and the desired number of drug
moieties to be linked to the ligand. For example, one skilled in
the art will appreciate that the average number of drug molecules
linked to the ligand can be varied by (1) modifying the amount of
the intermediate drug-hydrazone of Formula (IIa) relative to the
number of reactive sulfhydryl groups on the ligand moiety of the
conjugate; or (2)(a) modifying the number of reactive sulfhydryl
groups on the ligand by, for example, only partially reducing the
ligand (in the case of a protein, peptide or polypeptide), (b)by
inserting a limited number of, for example, cysteine residues to a
protein, peptide or polypeptide, or (c)by limiting the degree of
thiolation using less than maximal amounts of thiolation agents,
for example, SPDP or iminothiolane. Although the --SH titer can be
varied, the preferred level of free sulfhydryl groups, particularly
for a relaxed antibody, is the maximum obtainable using the
particular reagents in question. The degree of variation in the
--SH titer is easily controlled in the relaxed antibody process.
For example, FIG. 62 shows the effect on --SH titer for antibodies
BR64 and chimeric BR96 depending on the mole ratio of DTT to
ligand, at 37.degree. C., for a 1.5 hour reaction. One skilled in
the art will appreciate that different classes or subclasses of
immnoglobulins can have different numbers of disulfide bridges
susceptible to reduction by reagents such as DTT. Thus, a further
consideration in determining the desired level of conjugation of an
antibody or antibody fragment is the number of disulfide groups
available for reduction to free --SH groups. In general, however,
the preferred conjugate of Formula (I) will have, on the average
from a given reaction, from about 1 to about 10 drug molecules per
ligand molecule. An especially preferred average drug to ligand
molar ratio ("MR") is about 4 to about 8.
[0307] After the reaction of the conjugate is complete, the
conjugate may be isolated and purified using commonly known
dialysis, chromatographic and/or filtration methods. A final
solution containing the conjugate customarily may be lyophilized to
provide the conjugate in a dry, stable form which can be safely
stored and shipped. The lyophilized product eventually can be
reconstituted with sterile water or another suitable diluent for
administration. Alternatively, the ultimate product may be frozen,
for example under liquid nitrogen, and thawed and brought to
ambient temperature prior to administration.
[0308] In a first preferred embodiment, the anthracyclic hydrazone
of Formula (IIa) is made by reacting the anthracycline with a
maleimido-(C.sub.1-C.sub.10)-alkyl hydrazide, or a salt thereof.
The reaction is outlined in Method A, described earlier. The
reaction generally is carried out in two steps. First the
maleimido-(C.sub.1-C.sub- .10)-alkyl hydrazide, or its salt, is
prepared. After purification by, for example, chromatography and/or
crystallization, either the free base of the hydrazide or the salt
are reacted with the desired anthracycline or anthracyline salt.
After concentration of the reaction solution, the
maleimido-containing hydrazone reaction product of Formula (IIa) is
collected, and if desired, purified by standard purification
techniques.
[0309] The Formula (IIa) hydrazone then is reacted with a
sulfhydryl-containing antibody as described earlier. If the
antibody is thiolated using, for example,
N-succinimidyl-3-(2-pyridyldithio)propionat- e ("SPDP"), the
thiolation reaction generally is performed in two steps: (1)
Reaction of a free amino group on the antibody with SPDP; and, (2)
DTT reduction of the SPDP disulfide to yield a free --SH group. In
a preferred procedure, in Step (1) of the thiolation reaction, the
SPDP/antibody molar ratio ranges between about 7.5:1 to about 60:1,
depending upon the number of sulfhydryl groups desired, with a
preferred range of about 7.5:1 to about 30:1, especially for BR64,
and preferably about 20:1 for BR96. The reaction is carried out
between about 0.degree. C. and about 50.degree. C., with a most
preferred temperature of about 30.degree. C. The reaction may be
carried out at a pH range of between about 6 and about 8 with the
most preferred pH being about 7.4. The reduction in Step (2), using
preferably DTT, is performed using a DTT/SPDP molar ratio of
between about 2.5:1 to about 10:1. The most preferred DTT/SPDP
molar ratio is about 5:1 and the number of moles of SPDP is that
which is added in Step (1) of the reaction. The reaction generally
is carried out at about 0.degree. C. to about 40.degree. C.,
preferably 0.degree. C. and is usually complete after about 20
minutes. After dialysis and concentration of the solution of
thiolated ligand (an antibody in the most preferred embodiment),
the molar concentration of sulfhydryl groups on the ligand is
determined and the thiolated ligand is reacted with the desired
molar ratio of the hydrazone derivative of Formula (IIa) relative
to the molar amount of reactive sulfhydryl groups on the ligand.
Preferably, the ratio is at least about 1:1. This reaction
generally is performed at a temperature of about 0.degree. C. to
about 25.degree. C., preferably about 4.degree. C. The resulting
conjugate then may be purified by standard methods. This reaction
scheme is outlined in FIGS. 49a and 49b.
[0310] In a second preferred embodiment, the hydrazone of Formula
(IIa) is made as described above. The hydrazone then is reacted, as
outlined in FIG. 49c, with an antibody which previously has been
thiolated with iminothiolane ("IMT"). Thiolation of the ligand
(preferably an antibody) with IMT generally is a one step reaction.
The IMT/antibody ratio may range from between about 30:1 to about
80:1, preferably about 50:1. The reaction is performed for about 30
minutes to about 2 hours, preferably about 30 minutes, at a pH of
about 7 to about 9.5, preferably at a pH of about 9, at a
temperature of about 20.degree. C. to about 40.degree. C.,
preferably about 30.degree. C. The reaction product then is reacted
with the hydrazone of Formula (IIa) at a temperature of about
0.degree. C. to about 25.degree. C., preferably at about 4.degree.
C. and at a pH of about 7 to about 9.5, preferably about 7.4. The
conjugate then is purified using methods standard in the art, for
example, dialysis, filtration, or chromatography.
[0311] In a third especially preferred embodiment the intermediate
hydrazone of Formula (IIa) is made as described above. The
hydrazone then is reacted with a ligand, most preferably, an
antibody, in which at least one disulfide group has been reduced to
form at least one sulfhydryl group. An especially preferred ligand
is a "relaxed antibody," as described below. The preferred reducing
agent for preparing a free sulfhydryl group is DTT although one
skilled in the art will understand that other reducing agents may
be suitable for this purpose.
[0312] A "relaxed" antibody, is one in which one or more, or
preferably, three or more, disulfide bridges have been reduced.
Most preferably, a relaxed antibody is one in which at least four
disulfide bridges have been reduced. In a preferred process for
preparing a relaxed (i.e., reduced) antibody, the reduction,
especially with DTT, and the purification of the reaction product,
is carried out in the absence of oxygen, under an inert atmosphere,
for example, under nitrogen or argon. This process, as described in
detail below, allows one to carefully control the degree of
reduction. Thus, this process allows one skilled in the art to
reproduce at any time the desired level of reduction of a ligand
and, therefore, the number of free --SH groups available for
preparing a conjugate of the invention.
[0313] In an alternative procedure, the reaction is carried out
under ambient conditions, however, a sufficiently large amount of
the reducing agent, preferably DTT, is used to overcome any
reoxidation of the reduced disulfide bonds which may occur. In
either case, purification of the product, is carried out as soon as
possible after the reaction is complete and most preferably under
an inert atmosphere such as an argon or nitrogen blanket. The
preferred method for preparing the free sulfhydryl containing
ligand, however, is the process in which atmospheric oxygen is
excluded from the reaction. An antibody produced by either method
is referred to as a "relaxed" antibody. The product, however
prepared, should be used for subsequent reaction as quickly as
possible or stored under conditions which avoid exposure to oxygen,
preferably under an inert atmosphere.
[0314] In the process in which oxygen is excluded from the reaction
(i.e., the reaction is performed under an inert atmosphere), the
ligand is incubated, for a period of about 30 minutes to about 4
hours, preferably about 3 hours, with a molar excess of DTT. The
DTTl/ligand ratios may range between about 1:1 to about 20: 1,
preferably about 1:1 to about 10:1, most preferably about 7:1 to
about 10:1, depending upon the number of sulfhydryl groups desired.
For a reduction performed in the presence of oxygen, the mole ratio
of DTT to ligand ranges from about 50:1 to about 400:1, preferably
from about 200:1 to about 300:1. This latter reaction is carried
out for about 1 to about 4 hours, preferably 1.5 hours, at a
temperature of between about 20.degree. C. and about 50.degree. C.,
with a preferred temperature being about 37.degree. C. The reaction
is carried out at a pH of between about 6 and about 8, preferably
between about 7 to 7.5 The product then is purified using standard
purification techniques such as dialysis, filtration and/or
chromatography. A preferred purification method is diafiltration.
To prevent reoxidation of --SH groups, during purification and
storage, the product preferably is maintained under an inert
atmosphere to exclude exposure to oxygen.
[0315] One skilled in the art will appreciate that different
ligands, particularly an antibody, may possess different degrees of
susceptibility to reduction and/or reoxidation. Consequently, the
conditions for reduction described above may need to be modified in
order to obtain a given reduced ligand such as that described
above. Furthermore, alternate means for preparing a reduced
antibody useful in the conjugation process will be evident to one
skilled in the art. Thus, however prepared, a reduced ligand used
in the preparation of a conjugate of Formula (I) is meant to be
encompassed by the present invention.
[0316] To prepare a conjugate of Formula (I), as noted earlier, the
reduced antibody reaction product is reacted with the hydrazone
intermediate of Formula (IIC). The reaction preferably is performed
under an inert atmosphere at a temperature of about 0.degree. C. to
about 10.degree. C., preferably at about 4.degree. C. and at a pH
of about 6 to about 8, preferably about 7.4 The immunoconjugate is
purified using standard techniques such as dialysis, filtration, or
chromatography.
[0317] In another embodiment of the invention, an anthracycline of
Formula (11) is joined to a ligand to which is added a moiety
carrying a free sulfhydryl group. In one such embodiment, the
ligand is a non-antibody ligand, for example, bombesin. The
sulfhydryl may be, for example, part of a cysteine residue added to
the native bombesin molecule. The anthracycline is joined through a
hydrazone moiety to a Michael Addition Receptor containing moiety
which then reacts with the modified bombesin to form a conjugate of
Formula (I). The product then is purified with standard techniques
such as dialysis, centrifugation, or chromatography.
[0318] Preparation 1
[0319] 2,5-Dihydro-2,5-Dioxo-1H-Pyrrolo-1-Hexanoic Acid Hydrazide
and its Trifluoroacetic Acid Salt ("Maleimidocaproyl
Hydrazide")
[0320] Maleimidocaproic acid (2.11 g, 10 mmol) (See, e.g., D. Rich
et al., J. Med. Chem. 18:1004 (1975); and, O. Keller et al., Helv.
Chim. Acta. 58:531 (1975)) was dissolved in dry tetrahydrofuran
(200 mL). The solution was stirred under nitrogen, cooled to
4.degree. C. and treated with N-methylmorpholine (1.01 g, 10 mmol)
followed by dropwise addition of a solution of isobutyl
chloroformate (1.36 g, 10 mmol) in THF (10 mL). After 5 min a
solution of t-butyl carbazate (1.32 g, 10 mmol) in THF (10 mL) was
added dropwise. The reaction mixture was kept at 4.degree. C. for a
half hour and at room temperature for 1 hour. The solvent was
evaporated and the residue partitioned between ethyl acetate and
water. The organic layer was washed with dilute HCl solution, water
and dilute bicarbonate solution, dried over anhydrous sodium
sulfate and the solvent evaporated. The material was purified by
flash chromatography using a gradient solvent system of methylene
chloride:methanol (100:1-2). The protected hydrazide was obtained
in 70% yield (2.24 g).
[0321] This material (545 mg, 2.4 mmol) was dissolved and stirred
in trifluoroacetic acid at 0.degree.-4.degree. C. for 8 min. The
acid was removed under high vacuum at room temperature. The residue
was triturated with either to yield a crystalline trifluoroacetic
acid salt of maleimidocaproyl hydrazide (384 mg, 70%). An
analytical sample was prepared by crystallization from
methanol-ether, to prepare the product, mp 102.degree.-105.degree.
C. The NMR and MS were consistent with structure. Anal: Calc'd. for
C.sub.10H.sub.15N.sub.3O.sub.3.0.8CF.sub.3CO- OH: C, 44.02; H,
4.99; N, 13.28. Found (duplicate analyses): C, 44.16, 44.13; H,
4.97, 5.00; N, 12.74, 12.75.
[0322] The salt (220 mg) was converted to the free base by
chromatography over silica using a methylene
chloride:methanol:concentrated NH.sub.4OH (100:5:0.5) solvent
system. The material obtained (124 mg, 80%) was crystallized from
methylene chloride-ether to prepare a final product, mp
92-93.degree. C. NMR and MS were consistent with the structure.
Anal: Calc'd. for C.sub.10H.sub.15N.sub.3O.sub.3: C, 53.33; H,
6.67; N, 18.67. Found: C, 53.12; H, 6.67; N, 18.44.
[0323] Preparation 2
[0324] Maleimidocaproylhydrazone of Adriamycin
[0325] A mixture of adriamycin hydrochloride (44 mg, 0.075 mmol),
maleimidocaproyl hydrazide (23 mg, 0.102 mmol), prepared according
to the procedure outlined in Preparation 1, and 2-3 drops of
trifluoroacetic acid in absolute methanol (25 mL) was stirred for
15 hours under nitrogen and protected from light. At the end of
this period no free adriamycin was detected by HPLC (mobile phase
0.01 molar ammonium acetate:acetonitrile, (70:30)). The solution
was concentrated at room temperature under vacuum to 10 mL and
diluted with acetonitrile. The clear solution was concentrated to a
small volume, the solid was collected by centrifugation, and the
product was dried under high vacuum to yield the title compound.
The NMR was consistent with structure. High Resolution MS, calc'd.
for C.sub.31H.sub.42N.sub.4O.sub.13: 751.2827; Found 751.2804.
[0326] The hydrazone also was formed by using adriamycin and the
trifluoroacetic acid salt of the hydrazide. Thus, the salt (40 mg,
0.12 mmol), prepared according to the process outlined in Procedure
1, and adriamycin hydrochloride (50 mg, 0.086 mmol) were stirred in
methanol (30 mL) for 15 hrs. The solution was concentrated to 2 mL
and diluted with acetonitrile. The red solid was collected by
centrifugation and dried under vacuum. The product (28 mg, 43%) was
identical in NMR and TLC to the one described above. High
Resolution MS calc'd. for C.sub.31H.sub.42N.sub.4O.sub.13:
751.2827; found 751.2819.
[0327] 5. Expression and Purification of Coding Sequences for BR96
sFv-PE40
[0328] The DNA sequences encoding the single-chain immunotoxin may
be expressed in a variety of systems as set forth below. The DNA
may be excised from pBW 7.0 by suitable restriction enzymes and
ligated into suitable prokaryotic or eukaryotic expression vectors
for such expression.
[0329] To propagate the cloned DNA, the expression plasmid pBW 7.0,
encoding the single-chain immunotoxin, is first transformed into
suitable host cells, such as the bacterial cell line E. coli strain
BL21 (lambdaDE3) (provided by Dr. Studier, Brookhaven National
Laboratories, New York, described by Chaudhary et al., Proc. Natl.
Acad. Sci. USA 84:4538-4542 (1987)) using standard procedures
appropriate to such cells. The treatment employing calcium
chloride, as described by Cohen, Proc. Natl. Acad. Sci. USA 69:2110
(1972) or the CaCl.sub.2 method described in Sambrook et al.
(eds.), Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold
Spring Harbor Press, (1989), may be used for prokaryotes or other
cells which contain substantial cell wall barriers.
[0330] Depending on the host cell used, transformation or
transfection is performed using standard techniques appropriate to
such cells. For example, transfection into mammalian cells is
accomplished using DEAE-dextran mediated transfection, CaPO.sub.4
co-precipitation, lipofection, electroporation, or protoplast
fusion, and other methods known in the art including: lysozyme
fusion or erythrocyte fusion, scraping, direct uptake, osmotic or
sucrose shock, direct microinjection, indirect microinjection such
as via erythrocyte-mediated techniques, and/or by subjecting host
cells to electric currents. The above list of transfection
techniques is not considered to be exhaustive, as other procedures
for introducing genetic information into cells will no doubt be
developed.
[0331] Expression in prokaryotic cells is preferred. Prokaryotes
most frequently are represented by various strains of E. coli;
however, other microbial strains may also be used. Commonly used
prokaryotic control sequences which are defined herein to include
promoters for transcription initiation, optionally with an
operator, along with ribosome binding site sequences, include such
commonly used promoters as the beta-lactamase (penicillinase) and
lactose (lac) promoter systems (Chang et al., Nature 198:1056
(1977)), the tryptophan (trp) promoter system (Goeddel et al.,
Nucleic Acids Res. 8:4057 (1980)) and the lambda derived P.sub.L
promoter and N-gene ribosome binding site (Shimatake et al., Nature
292:128 (1981)).
[0332] Expression of the single-chain immunotoxin is detected by
Coomassie stained SDS-PAGE and immunoblotting using both
anti-idiotypic antibodies that bind to BR96, and anti-PE antibodies
to bind to the PE40-portion of the fusion protein.
[0333] 6. Recovery of Products
[0334] The recombinant immunotoxin may be produced along with a
signal sequence in cells capable of processing this sequence for
secretion. When secreted into the medium, the immunotoxin is
recovered using standard protein purification techniques such as
anion-exchange and gel-filtration chromatography. Purification may
also be performed using antibodies reactive with the
anti-immunoglobulin portion of the immunotoxin. However, while the
procedures are more laborious, it is within the means known in the
art to purify the molecule from sonicates or lysates of cells in
which it is produced intracellularly in fused or mature form.
[0335] In the preferred embodiment described herein, BR96 sFV-PE40
was purified using anion-exchange and gel-filtration
chromatographies with fast protein liquid chromatography (FPLC) as
described by Siegall et al., Proc. Natl. Acad. Sci. USA
85:9738-9742 (1988).
[0336] 7. Uses
[0337] The BR96 antibody of the invention is useful for diagnostic
applications, both in vitro and in vivo, for the detection of human
carcinomas that possess the antigen for which the antibodies are
specific. In vitro diagnostic methods include immunohistological
detection of tumor cells (e.g., on human tissue, cells or excised
tumor specimens) or serologic detection of tumor-associated
antigens (e.g., in blood samples or other biological fluids).
[0338] Immunohistochemical techniques involve staining a biological
specimen such as a tissue specimen with the BR96 antibody of the
invention and then detecting the presence on the specimen of the
antibody complexed to its antigen. The formation of such
antibody-antigen complexes with the specimen indicates the presence
of carcinoma cells in the tissue. Detection of the antibody on the
specimen can be accomplished using techniques known in the art such
as immunoenzymatic techniques, e.g., the immunoperoxidase staining
technique or the avidin-biotin (ABC) technique, or
immunofluorescence techniques (see, e.g., Ciocca et al.,
"Immunohistochemical Techniques Using Monoclonal Antibodies," Meth.
Enzymol. 121:562-79 (1986); Hellstrom et al., "Monoclonal Mouse
Antibodies Raised Against Human Lung Carcinoma," Cancer Research
46:3917-23 (1986); and Kimball (ed.), Introduction To Immunology
(2nd Ed.), pp. 113-117 (Macmillan Pub. Co. 1986)). For example,
immunoperoxidase staining was used as described in Example 2,
infra, to demonstrate the reactivity of the BR96 antibody with
lung, breast, colon, and ovary carcinomas and the low reactivity of
the antibody with normal human tissue specimens.
[0339] 8. Diagnostic Techniques.
[0340] Serologic diagnostic techniques involve the detection and
quantitation of tumor-associated antigens that have been secreted
or "shed" into the serum or other biological fluids of patients
thought to be suffering from carcinoma. Such antigens can be
detected in the body fluids using techniques known in the art such
as radioimmunoassays (RIA) or enzyme-linked immunosorbent assays
(ELISA) wherein an antibody reactive with the "shed" antigen is
used to detect the presence of the antigen in a fluid sample (see,
e.g., Uotila et al., "Two-Site Sandwich ELISA With Monoclonal
Antibodies To Human AFP," J. Immunol. Methods 42:11 (1981) and
Allum et al., supra at pp. 48-51). These assays, using the BR96
antibodies disclosed herein, can therefore be used for the
detection in biological fluids of the antigen with which the BR96
antibodies react and thus the detection of human carcinoma in
patients. Thus, it is apparent from the foregoing that the BR96
antibodies of the invention can be used in most assays involving
antigen-antibody reactions. These assays include, but are not
limited to, standard RIA techniques, both liquid and solid phase,
as well as ELISA assays, immunofluorescence techniques, and other
immunocytochemical assays (see, e.g., Sikora et al. (eds.),
Monoclonal Antibodies, pp. 32-52 (Blackwell Scientific Publications
1984)).
[0341] The invention also encompasses diagnostic kits for carrying
out the assays described above. In one embodiment, the diagnostic
kit comprises the BR96 monoclonal antibody, fragments thereof,
fusion proteins or chimeric antibody of the invention, and a
conjugate comprising a specific binding partner for the BR96
antibody and a label capable of producing a detectable signal. The
reagents can also include ancillary agents such as buffering agents
and protein stabilizing agents (e.g., polysaccharides). The
diagnostic kit can further comprise, where necessary, other
components of the signal-producing system including agents for
reducing background interference, control reagents or an apparatus
or container for conducting the test. In another embodiment, the
diagnostic kit comprises a conjugate of the BR96 antibodies of the
invention and a label capable of producing a detectable signal.
Ancillary agents as mentioned above can also be present.
[0342] The BR96 antibody of the invention is also useful for in
vivo diagnostic applications for the detection of human carcinomas.
One such approach involves the detection of tumors in vivo by tumor
imaging techniques. According to this approach, the BR96 antibody
is labeled with an appropriate imaging reagent that produces a
detectable signal. Examples of imaging reagents that can be used
include, but are not limited to, radiolabels such as .sup.131,
.sup.111In, .sup.123I, .sup.99mTc, .sup.32P, .sup.125I, .sup.3H,
and .sup.14C, fluorescent labels such as fluorescein and rhodamine,
and chemiluminescers such as luciferin. The antibody can be labeled
with such reagents using techniques known in the art. For example,
see Wensel and Meares, Radioimmunoimaging and Radioimmunotherapy,
Elsevier, N.Y. (1983) for techniques relating to the radiolabeling
of antibodies (see also, Colcher et al., "Use Of Monoclonal
Antibodies As Radiopharmaceuticals For The Localization Of Human
Carcinoma Xenografts In Athymic Mice," Meth. Enzymol., 121:802-16
(1986)).
[0343] In the case of radiolabeled antibody, the antibody is
administered to the patient, localizes to the tumor bearing the
antigen with which the antibody reacts, and is detected or "imaged"
in vivo using known techniques such as radionuclear scanning using,
e.g., a gamma camera or emission tomography (see, e.g., Bradwell et
al., "Developments In Antibody Imaging," in Monoclonal Antibodies
For Cancer Detection and Therapy, Baldwin et al. (eds.), pp. 65-85
(Academic Press 1985)). The antibody is administered to the patient
in a pharmaceutically acceptable carrier such as water, saline,
Ringer's solution, Hank's solution or nonaqueous carriers such as
fixed oils. The carrier may also contain substances that enhance
isotonicity and chemical stability of the antibody such as buffers
or preservatives. The antibody formulation is administered, for
example, intravenously, at a dosage sufficient to provide enough
gamma emission to allow visualization of the tumor target site.
Sufficient time should be allowed between administration of the
antibody and detection to allow for localization to the tumor
target. For a general discussion of tumor imaging, see Allum et
al., supra at pp. 51-55.
[0344] 9. Therapeutic Applications of the Antibodies of the
Invention and Fragments Thereof.
[0345] The properties of the BR96 antibody: a) very high
specificity for tumor cells; b) internalization; c) toxicity to
antigen-positive tumor cells alone, i.e., in unmodified form, when
used at appropriate concentrations; and d) complement-dependent
cytotoxicity and antibody-dependent cellular cytotoxicity activity,
suggest a number of in vivo therapeutic applications. First, the
BR96 antibody can be used alone to target and kill tumor cells in
vivo.
[0346] The antibody can also be used in conjunction with an
appropriate therapeutic agent to treat human carcinoma. For
example, the antibody can be used in combination with standard or
conventional treatment methods such as chemotherapy, radiation
therapy or can be conjugated or linked to a therapeutic drug, or
toxin, as well as to a lymphokine or a tumor-inhibitory growth
factor, for delivery of the therapeutic agent to the site of the
carcinoma.
[0347] Techniques for conjugating such therapeutic agents to
antibodies are well known (see, e.g., Arnon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
for Drug Delivery," in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review," in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985); and
Thorpe et al., "The Preparation And Cytotoxic Properties Of
Antibody-Toxin Conjugates," Immunol. Rev., 62:119-58 (1982)).
[0348] The BR96 antibody of the invention is particularly suited
for use in a therapeutic conjugate because it is readily
internalized within the carcinoma cells to which it binds and thus
can deliver the therapeutic agent to intracellular sites of
action.
[0349] Alternatively, the BR96 antibody can be coupled to
high-energy radiation, e.g., a radioisotope such as .sup.131I;,
which, when localized at the tumor site, results in a killing of
several cell diameters (see, e.g., Order, "Analysis, Results, And
Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody
In Cancer Therapy," in Monoclonal Antibodies For Cancer Detection
And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press
1985)). According to yet another embodiment, the BR96 antibody can
be conjugated to a second antibody to form an antibody
heteroconjugate for the treatment of tumor cells as described by
Segal in U.S. Pat. No. 4,676,980.
[0350] Still other therapeutic applications for the BR96 antibody
of the invention include conjugation or linkage, e.g., by
recombinant DNA techniques, to an enzyme capable of converting a
prodrug into a cytotoxic drug and the use of that antibody-enzyme
conjugate in combination with the prodrug to convert the prodrug to
a cytotoxic agent at the tumor site (see, e.g., Senter et al.,
"Anti-Tumor Effects Of Antibody-alkaline Phosphatase," Proc. Natl.
Acad. Sci. USA, 85:4842-46 (1988); "Enhancement of the in vitro and
in vivo Antitumor Activities of Phosphorylated Mitomycin C and
Etoposide Derivatives by Monoclonal Antibody-Alkaline Phosphatase
Conjugates," Cancer Research 49:5789-5792 (1989); and Senter,
"Activation of Prodrugs by Antibody-Enzyme Conjugates: A New
Approach to Cancer Therapy," FASEB J. 4:188-193 (1990)).
[0351] Still another therapeutic use for the BR96 antibody involves
use, either in the presence of complement or as part of an
antibody-drug or antibody-toxin conjugate, to remove tumor cells
from the bone marrow of cancer patients. According to this
approach, autologous bone marrow may be purged ex vivo by treatment
with the antibody and the marrow infused back into the patient
(see, e.g., Ramsay et al., "Bone Marrow Purging Using Monoclonal
Antibodies," J. Clin. Immunol. 8(2):81-88 (1988)).
[0352] Furthermore, chimeric BR96, recombinant immunotoxins and
other recombinant constructs of the invention containing the
specificity of the antigen-binding region of the BR96 monoclonal
antibody, as described earlier, may be used therapeutically. For
example, the single-chain immunotoxin of the invention, BR96
sFv-PE40 may be used to treat human carcinoma in vivo.
[0353] Similarly, a fusion protein comprising at least the
antigen-binding region of the BR96 antibody joined to at least a
functionally active portion of a second protein having anti-tumor
activity, e.g., a lymphokine or oncostatin can be used to treat
human carcinoma in vivo. Furthermore, recombinant techniques known
in the art can be used to construct bispecific antibodies wherein
one of the binding specificities of the antibody is that of BR96
(see, e.g., U.S. Pat. No. 4,474,893), while the other binding
specificity of the antibody is that of a molecule other than
BR96.
[0354] Finally, anti-idiotypic antibodies of the BR96 antibody may
be used therapeutically in active tumor immunization and tumor
therapy (see, e.g., Hellstrom et al., "Immunological Approaches To
Tumor Therapy: Monoclonal Antibodies, Tumor Vaccines, And
Anti-Idiotypes," in Covalently Modified Antigens And Antibodies In
Diagnosis And Therapy, supra at pp. 35-41).
[0355] The present invention provides a method for selectively
killing tumor cells expressing the antigen that specifically binds
to the BR96 monoclonal antibody or functional equivalent. This
method comprises reacting the immunoconjugate (e.g., the
immunotoxin) of the invention with said tumor cells. These tumor
cells may be from a human carcinoma.
[0356] Additionally, this invention provides a method of treating
carcinomas (for example human carcinomas) in vivo. This method
comprises administering to a subject a pharmaceutically effective
amount of a composition containing at least one of the
immunoconjugates (e.g., the immunotoxin) of the invention.
[0357] In accordance with the practice of this invention, the
subject may be a human, equine, porcine, bovine, murine, canine,
feline, and avian subjects. Other warm blooded animals are also
included in this invention.
[0358] The present invention also provides a method for curing a
subject suffering from a cancer. The subject may be a human, dog,
cat, mouse, rat, rabbit, horse, goat, sheep, cow, chicken. The
cancer may be identified as a retinoblastoma, papillary
cystadenocarcinoma of the ovary, Wilm's tumor, or small,cell lung
carcinoma and is generally characterized as a group of cells having
tumor associated antigens on the cell surface. This method
comprises administering to the subject a cancer killing amount of a
tumor targeted antibody joined to a cytotoxic agent. Generally, the
joining of the tumor targeted antibody with the cytotoxic agent is
made under conditions which permit the antibody so joined to bind
its target on the cell surface. By binding its target, the tumor
targeted antibody acts directly or indirectly to cause or
contribute to the killing of the cells so bound thereby curing the
subject.
[0359] In accordance with the practice of the invention, the tumor
targeted antibody is an internalizing tumor targeted antibody.
Examples include BR96, fragments of BR96, and functional
equivalents thereof. Functional equivalents of BR96 include any
molecule which binds the antigen binding site to which BR6 is
directed and is characterized by (1) binding carcinoma cells, (2)
internalizing within the carcinoma cells to which they bind, and
(3) mediating ADCC and CDC effector functions.
[0360] Further, in accordance with the practice of the invention,
the tumor targeted antibody may be an internalizing tumor targeted
antibody which recognizes and binds to the Le.sup.y determinant.
Although, antibodies directed against the Le.sup.y determinant are
known, such antibodies were not known to internalize within the
carcinoma cells to which they bind and/or mediate ADCC and CDC
effector functions.
[0361] Further, Le.sup.y is a fairly common determinant which is
overexpressed in many cancer and some normal cells. Because its
presence is widely found and thus common in both some tumor and
non-tumorigenic cells others have questioned whether such
antibodies which recognize Le.sup.y may be therapeutically
useful.
[0362] The claimed invention also provides a method of inhibiting
the proliferation of mammalian tumor cells. This method comprises
contacting the mammalian tumor cells with a proliferation
inhibiting amount (i.e., effective amount) of a tumor targeted
antibody joined to a cytotoxic or therapeutic agent or anti-tumor
drug so as to inhibit proliferation of the mammalian tumor
cells.
[0363] In one example, the tumor targeted antibody is the
monoclonal antibody BR96 produced by hybridoma ATCC HB10036. Other
examples include functional equivalents of BR96 such as ChiBR96;
fragments of BR96; bispecific antibodies with a binding specificity
for two different antigens, one of the antigens being that with
which the monoclonal antibody BR96 produced by hybridoma ATCC
HB10036 binds; and a human/murine recombinant antibody, the
antigen-binding region of which competitively inhibits the
immunospecific binding of monoclonal antibody BR96 produced by
hybridoma HB10036 to its target antigen.
[0364] Also provided is a method of inhibiting the proliferation of
mammalian tumor cells which comprises contacting the mammalian
tumor cells with a sufficient concentration of the immunoconjugate
of the invention so as to inhibit proliferation of the mammalian
tumor cells.
[0365] Examples of such immunoconjugates include, but are not
limited to, BR96-PE, PE-BR96 fragment, BR96-RA, BR96 (Fab)-lysPE40,
BR96 F(ab').sub.2-lysPE40, ChiBR96-LysPE40, IL-6-PE40,
BR96-DOX.
[0366] The subject invention further provides methods for
inhibiting the growth of human tumor cells, treating a tumor in a
subject, and treating a proliferative type disease in a subject.
These methods comprise administering to the subject an effective
amount of the composition of the invention.
[0367] It is apparent therefore that the present invention
encompasses pharmaceutical compositions, combinations and methods
for treating human carcinomas. For example, the invention includes
pharmaceutical compositions for use in the treatment of human
carcinomas comprising a pharmaceutically effective amount of a BR96
antibody and a pharmaceutically acceptable carrier.
[0368] The compositions may contain the BR96 antibody or antibody
fragments, either unmodified, conjugated to a therapeutic agent
(e.g., drug, toxin, enzyme or second antibody) or in a recombinant
form (e.g., chimeric BR96, fragments of chimeric BR96, bispecific
BR96 or single-chain immunotoxin BR96). The compositions may
additionally include other antibodies or conjugates for treating
carcinomas (e.g., an antibody cocktail).
[0369] The antibody, antibody conjugates and immunotoxin
compositions of the invention can be administered using
conventional modes of administration including, but not limited to,
intravenous, intraperitoneal, oral, intralymphatic or
administration directly into the tumor. Intravenous administration
is preferred.
[0370] The compositions of the invention may be in a variety of
dosage forms which include, but are not limited to, liquid
solutions or suspensions, tablets, pills, powders, suppositories,
polymeric microcapsules or microvesicles, liposomes, and injectable
or infusible solutions. The preferred form depends upon the mode of
administration and the therapeutic application.
[0371] The compositions of the invention also preferably include
conventional pharmaceutically acceptable carriers and adjuvants
known in the art such as human serum albumin, ion exchangers,
alumina, lecithin, buffer substances such as phosphates, glycine,
sorbic acid, potassium sorbate, and salts or electrolytes such as
protamine sulfate.
[0372] The most effective mode of administration and dosage regimen
for the compositions of this invention depends upon the severity
and course of the disease, the patient's health and response to
treatment and the judgment of the treating physician. Accordingly,
the dosages of the compositions should be titrated to the
individual patient. Nevertheless, an effective dose of the
compositions of this invention may be in the range of from about 1
to about 2000 mg/m.sup.2.
[0373] The molecules described herein may be in a variety of dosage
forms which include, but are not limited to, liquid solutions or
suspensions, tablets, pills, powders, suppositories, polymeric
microcapsules or microvesicles, liposomes, and injectable or
infusible solutions. The preferred form depends upon the mode of
administration and the therapeutic application.
[0374] The most effective mode of administration and dosage regimen
-for the molecules of the present invention depends upon the
location of the tumor being treated, the severity and course of the
cancer, the subject's health and response to treatment and the
judgment of the treating physician. Accordingly, the dosages of the
molecules should be titrated to the individual subject.
[0375] The interrelationship of dosages for animals of various
sizes and species and humans based on mg/m.sup.2 of surface area is
described by Freireich, E. J., et al. Cancer Chemother., Rep.
50(4):219-244 (1966). Adjustments in the dosage regimen may be made
to optimize the tumor cell growth inhibiting and killing response,
e.g., doses may be divided and administered on a daily basis or the
dose reduced proportionally depending upon the situation (e.g.,
several divided doses may be administered daily or proportionally
reduced depending on the specific therapeutic situation).
[0376] It would be clear that the dose of the composition of the
invention required to achieve cures may be further reduced with
schedule optimization.
[0377] In accordance with the practice of the invention, the
pharmaceutical carrier may be a lipid carrier. The lipid carrier
may be a phospholipid. Further, the lipid carrier may be a fatty
acid. Also, the lipid carrier may be a detergent. As used herein, a
detergent is any substance that alters the surface tension of a
liquid, generally lowering it.
[0378] In one example of the invention, the detergent may be a
nonionic detergent. Examples of nonionic detergents include, but
are not limited to, polysorbate 80 (also known as Tween 80 or
(polyoxyethylenesorbitan monooleate), Brij, and Triton (for
example, Triton WR-1339 and Triton A-20).
[0379] Alternatively, the detergent may be an ionic detergent. An
example of an ionic detergent includes, but is not limited to,
alkyltrimethylammonium bromide.
[0380] Additionally, in accordance with the invention, the lipid
carrier may be a liposome. As used in this application, a
"liposome" is any membrane bound vesicle which contains any
molecules of the invention or combinations thereof.
[0381] In order that the invention described herein may be more
fully understood, the following examples are set forth. It should
be understood that these examples are for illustrative purposes
only and are not to be construed as limiting the scope of this
invention in any manner.
[0382] ADVANTAGES OF THE INVENTION: Initial studies with various
previously known immunoconjugates have been disappointing
particularly with solid tumors. In our effort to improve antibody
based therapy of carcinomas, we have developed and examined novel
immunoconjugates and the anti-cancer drug doxorubicin (DOX).
[0383] BR96 is important for several reasons. It can trigger
irreversible changes in membrane structure which leads to tumor
cell death, most likely through the loss of osmotic control (J.
Garrigues, U. Garrigues, I. Hellstrom, K. E. Hellstrom, Am. J
Pathol. 142, 607 (1993)). Further, it is an internalizing MAb that
cycles in a nondegraded form between the intracellular compartment
and the medium for extended periods of time. The latter
characteristic makes BR96 an attractive candidate for targeting to
tumors various agents for selective concentration in antigen
positive cells.
[0384] The antigen for BR96 is abundantly expressed (>200,000
molecules/cell) on human carcinoma lines. BR96 binds, according to
immunohistology, the majority of human carcinomas of the breast,
lung and colon. Although BR96, like essentially all MAbs to human
tumors, is not truly tumor-specific, it offers advantages over most
other antibodies which recognize the Le.sup.y determinant (K.
Lloyd, G. Larson, N. Stromberg, J. Thurin, K. A. Karlsson,
Immunogenetics 17, 537 (1983); P. M. Pour, V. E. Tempero, C.
Cordon-Cardo, P. Avner, Cancer Res. 48, 5422 (1988); J. Sakamoto et
al., ibid. 49, 745 (1989); T. F. Orntoft, H. Wolf, H. Clausen, E.
Dabelsteen, S. I. Hakomori, Int. J. Cancer 43, 774 (1989)).
[0385] BR96 is more tumor selective and the normal tissues to which
it binds primarily comprise differentiated cells of the esophagus,
stomach, and intestine as well as acinar cells of the pancreas (I.
Hellstrom, H. J. Garrigues, U. Garrigues, K. E. Hellstrom, Cancer
Res. 50, 2183 (1990)).
[0386] BR96 is rapidly internalized into lysosomes and endosomes
after binding to cells expressing the antigen (J. Garrigues et al.
1993).
[0387] The antibodies mediate antibody-dependent cellular
cytotoxicity "antibody-dependent cellular cytotoxicity,"
"complement-mediated cytotoxicity," and "complement-dependent
cytotoxicity."
[0388] The antibodies can kill antigen-positive tumor cells in the
unconjugated form if present at a sufficient concentration. The
antibody conjugates and recombinant immunotoxins are useful as
reagents for killing tumor cells. The antibodies are also useful in
diagnostic methods, such as the detection of carcinomas by in vitro
or in vivo technology.
EXAMPLE 1
[0389] Preparation of the BR96 Monoclonal Antibody
[0390] The BR96 monoclonal antibody of the invention was produced
using hybridoma fusion techniques as described previously by M. Yeh
et al., Proc. Natl. Acad. Sci. USA, (1979), supra and Yeh et al.,
Int. J. Cancer (1982), supra. Briefly, a three-month old BALB/c
mouse was immunized using as the immunogen explanted cultured cells
from a human breast adenocarcinoma, designated 3396 or H3396 (from
adenocarcinoma of the breast from a patient which had been
established in culture at Bristol-Myers Squibb Co., Seattle,
Wash.). Methods for establishing and maintaining cell lines from
carcinomas isolated from patients are fully described in Yeh et
al., Proc. Natl. Acad. Sci. USA 76:2927-2931 (1979). The mouse
received injections on five occasions: on the first four occasions,
the mouse received one intraperitoneal injection and 1 subcutaneous
injection split between 4 sites on the mouse. On the fifth
occasion, the mouse was given only one intraperitoneal injection.
The total number of cells injected on each occasion was
approximately 10.sup.7 cells. Three days after the last
immunization, the spleen was removed and spleen cells were
suspended in RPMI culture medium. The spleen cells were then fused
with P3-x63-Ag8.653 mouse myeloma cells in the presence of
polyethylene glycol (PEG) and the cell suspension grown in
microtiter wells in selective HAT medium as described by Yeh et
al., supra (see also Kohler and Milstein, Nature 256:495-97 (1975)
and Eur. J. Immunol. 6:511-19 (1976)). The mixture was seeded to
form low density cultures originating from single fused cells or
clones.
[0391] The supernatants from these hybridoma cultures were then
screened for direct binding activity on the breast cancer cell
line, 3396, and a fibroblast cell line obtained from a skin biopsy
using an ELISA assay similar to that described by Douillard et al.,
"Enzyme-Linked Immunosorbent Assay For Screening Monoclonal
Antibody Production Using Enzyme-Labeled Second Antibody," Meth.
Enzymol. 92:168-74 (1983).
[0392] According to this assay, the antigen (with which the
antibody being screened for is reactive) is immobilized on
microtiter plates and then incubated with hybridoma supernatants.
If a supernatant contains the desired antibody, the antibody will
bind to the immobilized antigen and is detected by addition of an
anti-immunoglobulin antibody-enzyme conjugate and a substrate for
the enzyme which leads to a measurable change in optical density.
In the present studies, breast cancer cells or control fibroblast
cells were dispensed into a 96-well tissue culture plate (Costar
Cambridge, Mass.) and incubated overnight in a humid 37.degree. C.
incubator (5% CO.sub.2). The cells were then fixed with 100 .mu.l
of freshly prepared 1.0% glutaraldehyde to a final well
concentration of 0.5% and incubated for 15 min at room temperature,
followed by washing three times with 1.times. phosphate buffered
saline (PBS). The cells were next blocked for 30 min with 5% bovine
serum albumin (BSA) in PBS and washed again three times with PBS.
The supernatants from the hybridoma cultures were then added at 100
.mu.l/well, the wells incubated for 1 h at room temperature, and
the cells washed three times with PBS. Next, goat anti-mouse
horseradish peroxidase (Zymed, Calif.) diluted in 0.1% BSA and PBS
was added to a concentration of 100 .mu.l/well. The reaction
mixture was incubated for either 1 h at room temperature or 30 min
at 37.degree. C. and the cells were then washed three times with
PBS. o-Phenylenediamine (OPD) was then added at 100 .mu.l/well and
the plates incubated in the dark at room temperature for 5-45 min.
Antibody binding to the cells was detected by a color change in the
wells that occurred within 10-20 min. The reaction was stopped by
adding 100 .mu.l/well H.sub.2SO.sub.4 and the absorbance read in a
Dynatech (Alexandria, Va.) Microelisa autoreader at 490 nm.
[0393] It should be noted that this assay can be performed using
intact cells or purified soluble antigen or cellular extracts as
the immobilized antigen. When soluble antigen or cell extracts were
used as antigen, the antigen was initially plated at 50 .mu.l/well
in PBS and the plates were incubated overnight at room temperature
before beginning the assay. When using intact cells as antigen,
they may be used fresh or after fixation. In either case, the cells
were initially plated at 10.sup.4 cells in 100 .mu.l/well in
culture medium and incubated overnight in a 37.degree. C. incubator
(5% CO.sub.2).
[0394] Hybridomas which produced antibodies binding to the breast
cancer cell line and not to the human fibroblast cells were thus
selected, and tested in a FACS cell sorter on peripheral blood
leukocytes (PBLs), as described in Example 2, infra. Hybridomas
that were negative on PBLs were cloned, expanded in vitro, and
further tested for antibody specificity. Those hybridomas producing
antibody reactive with human breast cancer were recloned, expanded,
and injected into pristane-primed 3-month old BALB/c mice, where
they grew as ascites tumors.
[0395] Following this procedure, hybridoma cell line BR96 was
obtained, cloned and injected into mice to develop as an ascites
tumor. As disclosed above, the BR96 hybridoma has been deposited
with the ATCC. Monoclonal BR96 antibody was purified from ascites
by affinity chromatography on immobilized recombinant protein A
(Repligen, Cambridge, Mass.). Clarified ascites was diluted with an
equal volume of binding buffer (1 M potassium phosphate, pH 8) and
applied to a protein A column previously equilibrated with binding
buffer. The column was extensively washed with binding buffer and
then the antibody was eluted with 50 mM phosphoric acid, pH 3. The
purified antibody fraction was neutralized with 1 M Tris, pH 9 and
then dialyzed against phosphate buffered saline. Purified BR96 was
finally sterile filtered and stored refrigerated or frozen.
EXAMPLE 2
[0396] Characterization of the BR96 Monoclonal Antibody
[0397] Isotype Determination
[0398] To determine the class of immunoglobulin produced by the
BR96 hybridoma, the following techniques were utilized:
[0399] (a) Ouchterlony Immunodiffusion
[0400] An aliquot of supernatant of the hybridoma cells was placed
into the center well of a 25% agar plate. Monospecific rabbit
anti-mouse Ig isotype antibodies (Southern Biotechnology,
Birmingham, Ala.) were placed in the outer wells and the plate was
incubated for 24-28 h at room temperature. Precipitation lines were
then read.
[0401] (b) ELISA Isotyping
[0402] Dynatech Immulon 96-well plates were coated with goat
anti-mouse Ig antibodies at 1 .mu.g/ml concentration, 50 .mu.l/well
in PBS and left covered overnight at 4.degree. C. The plates were
washed with PBS/Tween 20, 0.05% and blocked with medium at 100
.mu.l/well for 1 h at room temperature. After washing the plates,
supernatants from the BR96 hybridoma were added and incubated at
room temperature for 1 h. After washing with PBS containing 2%
bovine serum albumin (BSA), plates were incubated at 37.degree. C.
for 30 min with monospecific rabbit anti-mouse Ig isotype
antibodies coupled to peroxidase (Zymed, South San Francisco,
Calif.). After further washing, the plates were incubated with 1
mg/ml OPD and 0.03% H.sub.2O.sub.2 in 0.1 M citrate buffer, pH 4.5.
Optical density at 630 nm was determined on a Dynatec ELISA plate
reader.
[0403] Based on these procedures, it was determined that the BR96
monoclonal antibody is of the IgG3 isotype.
[0404] Characteristics of the BR96 Monoclonal Antibody
[0405] The BR96 antibody shows a high degree of reactivity with a
wide range of carcinomas and displays only limited reactivity with
normal cells. This was shown by experiments involving
immunohistological studies on frozen tissue sections as well as
binding studies using intact cultured cells.
[0406] Immunohistology
[0407] The peroxidase-antiperoxidase (PAP) technique of L. A.
Sternberger as described in Immunochemistry, pp. 104-69 (John Wiley
& Sons, New York, 1979) and as modified by J. Garrigues et al.,
"Detection Of A Human Melanoma-Associated Antigen, p97, In
Histological Sections Of Primary Human Melanomas," Int. J. Cancer.
29:511-15 (1982), was used for the immunohistological studies. The
target tissues for these tests were obtained at surgery and frozen
within 4 h of removal using isopentane precooled in liquid
nitrogen. Tissues were then stored in liquid nitrogen or at
-70.degree. C. until used. Frozen sections were prepared, air
dried, treated with acetone and dried again (see Garrigues et al.,
supra). Sections to be used for histologic evaluation were stained
with hematoxylin. To decrease non-specific backgrounds sections
were preincubated with normal human serum diluted 1/5 in PBS (see
Garrigues et al., supra). Mouse antibodies, rabbit anti-mouse IgG,
and mouse PAP were diluted in a solution of 10% normal human serum
and 3% rabbit serum. Rabbit anti-mouse IgG (Sternberger-Meyer
Immunochemicals, Inc., Jarettsville, Md.), was used at a dilution
of {fraction (1/50)}. Mouse PAP complexes (Sternberger-Meyer
Immunochemicals, Inc.) containing 2 mg/ml of specifically purified
PAP was used at a dilution of {fraction (1/80)}.
[0408] The staining procedure consisted of treating serial sections
with either specific antibody, i.e., BR96, or a control antibody
for 2.5 h, incubating the sections for 30 min at room temperature
with rabbit anti-mouse IgG diluted {fraction (1/50)} and then
exposing the sections to mouse PAP complexes diluted {fraction
(1/80)} for 30 min at room temperature. After each treatment with
antibody, the slides were washed twice in PBS.
[0409] The immunohistochemical reaction was developed by adding
freshly prepared 0.5% 3,3'-diaminobenzidine tetrahydrochloride
(Sigma Chermical Co., St. Louis, Mo.) and 0.01% H.sub.2O.sub.2 in
0.05 M Tris buffer, pH 7.6, for 8 min (see Hellstrom et al., J.
Immunol. 127:157-60 (1981)). Further exposure to a 1% OsO.sub.4
solution in distilled water for 20 min intensified the stain. The
sections were rinsed with water, dehydrated in alcohol, cleared in
xylene, and mounted on slides. Parallel sections were stained with
hematoxylin.
[0410] The slides were each evaluated under code and coded samples
were checked by an independent investigator. Typical slides were
photographed by using differential interference contrast optics
(Zeiss-Nomarski). The degree of antibody staining was evaluated as
0 (no reactivity), + (a few weakly positive cells), ++ (at least
one third of the cells positive), +++ (most cells positive), ++++
(approximately all cells strongly positive). Because differences
between + and 0 staining were less clear cut than between + and ++
staining, a staining graded as ++ or greater was considered
"positive." Both neoplastic and stroma cells were observed in tumor
samples. The staining recorded is that of the tumor cells because
the stroma cells were not stained at all or were stained much more
weakly than the tumor cells.
[0411] Table 1 below demonstrates the immunohistological staining
of various tumor and normal tissue specimens using the BR96
monoclonal antibody. As the table clearly demonstrates, the BR96
antibody reacts with a wide range of human carcinoma specimens,
does not react with sarcoma and displays only infrequent reactivity
with melanoma. Furthermore, it shows only limited reactivity with
any of the large number of normal human tissues tested. The only
reactivity detected with normal cells was binding to a small
subpopulation of cells in the tonsils and in the testis, and to
acinar cells in the pancreas, and to certain epithelial cells of
the stomach and esophagus.
3TABLE 1 IMMUNOPEROXIDASE STAINING OF HUMAN TUMORS AND NORMAL
TISSUE SPECIMENS WITH BR96 MONOCLONAL ANTIBODY NUMBER POSITIVE/
TISSUE TYPE NUMBER TESTED Tumors Lung carcinoma (non-small cell)
14/17 Breast carcinoma 17/19 Colon carcinoma 15/18 Ovary carcinoma
4/4 Endometrial carcinoma 2/2 Melanoma 2/5 Sarcoma 0/5 Stomach
carcinoma 2/2 Pancreatic carcinoma 2/2 Esophagus carcinoma 2/2
Cervical carcinoma 2/2 Normal Tissues Lung 0/7 Spleen 0/5 Breast
0/2 Colon 0/7 Kidney 0/7 Liver 0/5 Brain 0/2 Heart 0/3 Skin 0/2
Thyroid 0/2 Adrenal 0/1 Ovary 0/2 Lymph nodes 0/2 Lymphocyte pellet
0/4 Pancreas 2/2 (only acinar cells were positive) Uterus 0/7
Retina 0/1 Testis 2/2 (only small sub-population of cells were
positive) Tonsil 2/2 (only small sub-population of cells were
positive) Stomach 2/2 (epithelial cells positive) Esophagus 2/2
(epithelial cells positive)
[0412] The binding of the BR96 antibody to various cultured cell
lines was also evaluated. Antibody binding to the cell surface of
intact cultured cells was identified either by a direct binding
assay with .sup.125I-labeled antibody as described in Brown et al.,
"Quantitative Analysis Of Melanoma-Associated Antigen p97 In Normal
And Neoplastic Tissues," Proc. Natl. Acad. Sci. USA, 78:539-43
(1981), or by direct immunofluorescence using a Coulter Epics C
fluorescence activated cell sorter (FACS) II (Hellstrom et al.,
Cancer Res. 46:3917-3923 (1986)).
[0413] For binding analyses using a FACS cell sorter,
2.times.10.sup.5 to 1.times.10.sup.6 cultured cells were aliquoted
in 15% fetal bovine serum (FBS) in IMDM media (Gibco, N.Y.) to a
total volume of 500 .mu.l/tube. The cells were centrifuged for 1.5
min on a Serofuge and the supernatant removed. 100 .mu.l of the
BR96 monoclonal antibody at 10 .mu.l/ml was added to each tube, the
contents of which was then mixed and incubated on ice for 30 mm.
The reaction mixture was washed three times with 500 .mu.l of 15%
FBS/IMDM by centrifugation for 1.5 min on the Serofuge (tubes were
blotted after the third wash). Then, 50 .mu.l of optimized
FITC-conjugated goat anti-mouse IgG antibody (Tago, Burlingame,
Calif.) diluted 1:25 in 15% FBS/IMDM was added to each tube and the
reaction mixture was mixed and incubated for 30 min. The wash step
was then repeated and after blotting of the tubes, each pellet was
resuspended in 200-500 .mu.l of PBS. Each sample was run on a
Coulter Epics C FACS and the mean fluorescence intensity (MFI) was
determined. From the MFI, the linear fluorescent equivalent (LFE)
was determined. The LFE of each test sample divided by the LFE of a
negative control gave a ratio between the brightness of cells
stained by specific versus control antibody. The binding data is
shown in Table 2 below.
4TABLE 2 FACS ANALYSIS OF THE BINDING OF BR96 TO VARIOUS TYPES OF
SUSPENDED CELLS Ratio Cell line (10 .mu.g/ml) Breast carcinoma 3396
54 Breast carcinoma MCF-7 38 Breast carcinoma 3630 22 Breast
carcinoma 3680 22 Lung carcinoma 2987 15 Lung carcinoma 2707 30
Lung carcinoma 2964 2 Lung carcinoma 3655-3 18 Colon carcinoma RCA
34 Colon carcinoma 3619 22 Colon carcinoma 3347 5 Colon carcinoma
HCT116 1 Colon carcinoma CB5 27 Colon carcinoma C 30 Colon
carcinoma 3600 16 Ovary carcinoma 3633-3 11 Melanoma 2669 1
Melanoma 3606 1 Melanoma 3620 1 T cell lymphoma line CEM 1 T cell
lymphoma line MOLT-4 1 B cell lymphoma line P3HR1 1 Peripheral
blood leukocytes 1
[0414] As Table 2 demonstrates, the BR96 monoclonal antibody
reacted with most breast, lung and colon carcinoma cell lines but
did not react with melanoma lines or with T or B lymphoma lines nor
with normal peripheral blood leukocytes. Scatchard analysis using
radiolabeled antibody indicated that the approximate association
constant (K.sub.a) of BR96 was calculated to be 3.6.times.10.sup.6
antigen sites/cell for the 3396 line which binds BR96.
[0415] These data demonstrate that monoclonal antibody BR96
recognize cell surface antigens abundantly expressed (up to
10.sup.6 molecules/cell) on the majority of human carcinomas.
EXAMPLE 3
[0416] Internalization of the BR96 Monoclonal Antibody within
Carcinoma Cells
[0417] Studies were conducted to measure internalization of the
BR96 monoclonal antibody within antigen-positive carcinoma cells.
According to one procedure, BR96 was conjugated to the ricin A
chain toxin to form an immunotoxin, BR96-RA, whose internalization
by carcinoma cells was then determined. Uptake of the conjugate by
the carcinoma cells was assessed by determining to what extent the
tumor cells were killed by ricin A chain.
[0418] Conjugation of the antibody to the toxin was carried out as
follows: Deglycosylated ricin-A chain (Inland Labs, Austin, Tex.)
(see, also, Blakey et al., Cancer Res., 47:947-952 (1987)) was
treated with dithiothreitol (5 mM) prior to gel filtration on G-25
Sephadex using PBS, pH 7.2 as eluant. This was added in a 2:1 molar
ratio to the antibody in PBS, the antibody having been previously
modified with N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP)
(Pierce, Rockford, Ill.) according to the procedure of Lambert et
al., J. Biol. Chem., 260:12035-12041 (1985). Reaction was allowed
to proceed for 12-24 h at room temperature, and the solution was
then diluted with 1 volume of H.sub.20. Removal of unconjugated
antibody was achieved using Blue Sepharose CL-6B (Pharmacia,
Uppsala, Sweden) (see Knowles et al., Anal. Biochem., 160:440-443
(1987)).
[0419] The conjugate and excess ricin-A chain were eluted with high
salt (10.times. PBS) and subjected to further purification on
Sephacryl-300 (Pharmacia) using PBS as eluant. The resulting
conjugate was free of unbound monoclonal antibody or ricin A-chain
and consisted mostly of 1:1 adducts.
[0420] The internalization of BR96-RA by various carcinoma cell
lines was then measured using a thymidine uptake inhibition assay.
According to this assay, the inhibition of .sup.3H-thymidine
incorporation into the DNA of the carcinoma cells (i.e., the
inhibition of cell proliferation) is a measure of the cytotoxic
effect of BR96-RA on the cells and thus a measure of the
internalization of the immunotoxin within the cell.
[0421] For the assay, carcinoma cells were plated into a 96-well
microtiter plate at 1.times.10.sup.4 cells/well in 100 .mu.l of
IMDM medium with 15% fetal calf serum FCS). The plates were
incubated for 12-18 h at 37.degree. C. to let the cells adhere.
Then the media was removed. Plates were kept on ice. The BR96-RA
immunotoxin (100 .mu.l) was then added in log 10 serial dilutions,
starting at 10 .mu.g/ml final concentration down to 0.01 .mu.g/ml.
The reaction mixture was incubated for 4 h on ice. The plates were
washed and 200 .mu.l/ml media was added and further incubated at
37.degree. C. for 18 h. At this point, 50 .mu.l of
.sup.3H-thymidine was added at 1 .mu.Ci/well and the plates
incubated for 6 h at 37.degree. C. in a 5% CO.sub.2 incubator. The
assay plates were then frozen at -70.degree. C. for at least I h
and thawed in a gel dryer for 15 min. The cells were harvested onto
glass fiber filters (Filter Strips, No. 240-1, Cambridge
Technology) in plastic scintillation vials using a PHD cell
harvester. 3 ml of scintillation counting liquid was added to the
vials and the vials were counted on a Beckman LS3891 beta
scintillation counter at 1 minute per sample.
[0422] Graphs of the percent inhibition of thymidine incorporation
vs. immunotoxin concentration for each cell line tested were
plotted and are shown in FIGS. 1-5. In each assay, a control was
run. The results of the assay are expressed as a percentage of the
.sup.3[H] thymidine incorporated by untreated control cells.
[0423] FIG. 1 depicts the percent inhibition of thymidine
incorporation by cells from the 3396 breast carcinoma cell line
caused by internalization of BR96-RA. Similar results were obtained
with the 2707 lung carcinoma cell line (FIG. 2) and C colon
carcinoma cell line (see FIG. 4). The BR96-RA was not internalized
by HCT 116 cell line (ATCC No. CCL 247), a human colon carcinoma
cell line that does not bind BR96 (see FIG. 3). FIG. 5 shows no
internalization of BR96-RA on 3347, a colon carcinoma cell line to
which BR96 does not bind; BR6-RA, on the other hand, which binds to
the 3347 cells, does internalize. This study, therefore,
demonstrated not only internalization of the BR96 antibody but the
selectivity of the internalization of the BR96 antibody for antigen
positive carcinoma cells.
EXAMPLE 4
[0424] Cytotoxicity of Unmodified BR96 Monoclonal Antibody
[0425] Three types of experiments were performed to follow up on
the unexpected observation that monoclonal antibody BR96 appeared
to be cytotoxic by itself (i.e., in unmodified state) when tested
in a FACS assay. So as to avoid an effect of complement in serum,
all sera used were heat inactivated (56.degree. C. for 30 min); in
addition, some of the experiments with FACS analysis (as described
below) were performed on cells which were grown in serum-free
medium and tested in the absence of serum.
[0426] First, living suspended cells from a variety of antigen
positive carcinoma lines (3396, 2987, 3619) were treated with
monoclonal antibody BR96. Cells (5.times.10.sup.5) were incubated
on ice for 30 min with 100 .mu.l of BR96 or control monoclonal
antibody at a concentration of 60, 30, 15, 7.5 and 3.8 .mu.g/ml in
culture medium (IMDM, 15% FBS). After washing the cells twice with
culture medium, the cells were suspended in 500 .mu.l medium and
stained by adding the dye propidium iodide which stains dead cells
(Krishan, Cell Biol. 66:188 (1975); and Yeh, J. Immunol. Methods,
43:269 (1981)). Out of a 1 mg/ml stock solution (in 70% alcohol) 5
.mu.l dye was added to cell samples, incubated on ice for 15 min,
washed once and finally suspended in 500 .mu.l medium. The cells
were evaluated on a Coulter Epics C FACS, with dead cells being
identified by their red fluorescence. The analysis was done on a
two-parameter display with log forward lightscatter in the
horizontal and log red fluorescence in the vertical display.
Computations of cell size versus cell viability were obtained by
applying the Coulter Epics C Quadstat program. Tumor cells which
could bind BR96 as well as tumor cells not binding BR96 were
studied in parallel. The results are shown in FIG. 6. FIG. 6
demonstrates that incubation of cells from any of three
antigen-positive carcinomas with BR96 rapidly killed them.
Untreated or antigen-negative cells were not killed.
[0427] Second, tumor cells (3396, 3630, 2987, 3619 and HCT 116)
were exposed to BR96 (or the control monoclonal antibody) for 18 h
at 37.degree. C. in a 96-well microtiter plate at 3.times.10.sup.3
cells/well in 150 .mu.l of IMDM medium containing FBS for 66 h
after which 50 .mu.l of .sup.3[H]-thymidine was added at 1
.mu.Ci/well and the plate was incubated for another 6 h at
37.degree. C. Subsequently, it was frozen at -70.degree. C. for at
least 1 h and thawed in a gel dryer for 15 min, and the cells
harvested onto glass fiber filters. The tritiated thymidine assay
was then performed as described in the preceding example, except
that the cells and antibodies were incubated at 37.degree. C. FIG.
7 illustrates the results. BR96 caused an inhibition of [.sup.3H]
thymidine incorporation into antigen-positive cell lines, and this
effect was dose dependent. The antigen-negative cell line HCT116
was not affected by any concentration of BR96 examined.
[0428] Third, using a modification of a procedure described by
Linsley et al. (Linsley et al., "Identification and
characterization of cellular receptors for growth regulator,
Oncostatin M," J. Biol. Chem. 264:4282-4289 (1989)) a growth
inhibition assay was performed. Cells from four different cell
lines (HCT116, 2987, 3396 and 3630) were seeded (3.times.10.sup.3)
in a volume of 0.1 ml of IMDM with 15% fetal bovine serum (FBS) in
96-well microtiter plates and allowed to attach for 3 h at
37.degree. C. Various concentrations of whole BR96 monoclonal were
then added in a volume of 0.1 ml, after which incubation at
37.degree. C. was continued for 72 h. Subsequently, the culture
medium was removed and the cells were stained by crystal violet
(0.1% in 20% methanol) for 30 min. and washed three times with PBS.
The bound dye was eluted by the addition of 0.1 ml of a solution of
0.1 M sodium citrate, pH 4.2, in 50% ethanol. Samples were assayed
in triplicate on an ELISA reader measuring the absorbance in the
presence of BR96 with the absorbance in untreated samples. The
results of this procedure are expressed as percentage inhibition of
cell growth. FIG. 8 illustrates the results. The results of this
assay were in agreement with those presented above for the
thymidine incorporation assay (FIG. 7).
EXAMPLE 5
[0429] Antibody-Dependent Cellular Cytotoxicity Activity of BR96
Antibody
[0430] Determination of antibody-dependent cellular cytotoxicity
activity of BR96 monoclonal antibody was performed as described by
Hellstrom et al., Proc. Natl. Acad. Sci. (USA) 82:1499-1502 (1985).
Briefly, a short-term .sup.51Cr-release test that measures the
release of .sup.51Cr as described by Cerrotini et al., Adv.
Immunol. 18:67-132 (1974) was used as evidence of tumor-cell lysis
(cytotoxicity). Peripheral blood lymphocytes from healthy human
subjects were separated on Ficoll-Hypaque (Hellstrom et al., Int.
J. Cancer 27:281-285 (1981)) to provide effector cells equal to 5%
natural killer cell reactivity against SK-MEL-28 cells (ATCC No.
HTB 72); 10.sup.6 cells were labeled by incubation with 100 .mu.Ci
(1 Ci=37 Gbq) of .sup.51Cr for 2 h at 37.degree. C., after which
they were washed three times and resuspended in medium. The labeled
cells were seeded (2.times.10.sup.4 cells per well in 20 .mu.l)
into Microtiter V-bottom plates (Dynatech Laboratories, Alexandria,
Va.). Purified antibody BR96 (10 .mu.g/ml, 1 .mu.g/ml, and 0.1
.mu.g/ml) was then added, followed by 2.times.10.sup.5 lymphocytes
per well in 100 .mu.l. The mixtures were incubated for 2 to 4 h
after which the plates were centrifuged at 400.times.g. The
supernatants were removed and the radioactivity in 100 .mu.l
samples was measured with a gamma-counter. There were two
replicates per group; the variation between replicates was less
than 10%. Several "criss-cross" experiments were done, in which
lung (or colon) carcinoma and melanoma targets were tested in
parallel with monoclonal antibody BR96 and with the antimelanoma
monoclonal antibody MG-22 (Hellstrom et al., Proc. Natl. Acad. Sci.
USA, 82:1499-1502 (1985)) which do not bind to most carcinoma
cells. Controls included the incubation of target cells alone or
with either lymphocytes or monoclonal antibody separately.
[0431] Spontaneous release was defined as the counts per minute
(cpm) released into the medium from target cells exposed to neither
antibodies nor lymphocytes, and total release, as the number of
counts released from target cells that were osmotically lysed at
the end of the assay. Percent cytotoxicity was calculated as: 1
experimental group release - spontaneous release total release -
spontaneous release .times. 100
[0432] Effector cells were characterized by assessing their
sensitivity to incubation with anti-serum to the Leu-11b surface
marker and guinea pig complement, using procedures described by
Hellstrom et al., in Monoclonal Antibodies and Cancer Therapy, UCLA
Symposia on Molecular and Cellular Biology, New Series, eds.
Reisfeld & Sell, Liss, N.Y., Vol 27, pp. 149-164 (1985),
incorporated herein by reference. This was done to measure the
expression of the Leu-11b marker, which characterizes natural
killer (NK) cells and is expressed by lymphocytes mediating
antibody-dependent cellular cytotoxicity against human melanoma
cells in the presence of monoclonal antibody BR96. The cytotoxicity
by effector cells alone ("natural killer effect") was subtracted
from the data provided in FIG. 9.
[0433] The results shown in FIG. 9 for an antibody concentration of
10 .mu.g/ml indicate that BR96 mediates antibody-dependent cellular
cytotoxicity activity if present in sufficient concentrations and
if the target cells express sufficient concentrations of the
epitope. The antibody-dependent cellular cytotoxicity activity can
be seen at antibody concentrations lower than those at which the
antibody is cytotoxic by itself (usually around 20 .mu.g/ml). When
antibody BR96 was used alone as a control it produced 0% killing at
the concentrations tested and using the .sup.51Cr assay.
Antibody-dependent cellular cytotoxicity activity was only found
with BR96 antibody-binding cell lines. Thus, cells from five
different carcinoma lines, which all bound BR96, were killed via
antibody-dependent cellular cytotoxicity at monoclonal antibody
concentrations down to 0.1 .mu.g/ml, while cells from a sixth line,
2964, which did not bind BR96, were not killed. The requirement for
antibody binding to obtain antibody-dependent cellular cytotoxicity
was further demonstrated by the fact that both of the two
carcinomas which could bind a different antibody, L6 (lines 3619
and 2987), were killed by L6 via antibody-dependent cellular
cytotoxicity, while the others were not. Under the conditions of
the assay, BR96 alone caused the release of only 1% of the label,
even when tested at a concentration of 10 .mu.g/ml.
EXAMPLE 6
[0434] Ability of BR96 to Mediate Complement-Mediated Cytotoxicity
(Complement-Dependent Cytotoxicity)
[0435] Tests to evaluate the ability of monoclonal antibody BR96 to
kill tumor cells in the presence of human serum as a source of
complement (complement-mediated cytotoxicity or
complement-dependent cytotoxicity) were performed similarly to
those for the antibody-dependent cellular cytotoxicity tests
described in Example 5, supra, except that 100 .mu.l of human serum
from normal human subjects as the source of complement diluted 1:3
to 1:6 was added per microtest well in place of a suspension of
effector cells.
[0436] As shown in FIG. 10, complement-dependent cytotoxicity
against cells binding BR96 was seen at an antibody concentration of
0.1-5.0 .mu.g/ml, while there was no complement-dependent
cytotoxicity against the BR96 antigen-negative lines HCT116 and
3347. The 3347 cells could, however, be killed when using the L6
monoclonal antibody, which binds to these cells. Controls were
always included in which BR96 was tested in the absence of
complement. No killing by BR96 alone was detected by the .sup.51Cr
release assay. These data show that BR96 gave a cytotoxic effect in
the presence of human serum at concentrations where it is not
cytotoxic by itself. (Control antibody gave no complement-dependent
cytotoxicity).
EXAMPLE 7
Determination of Reactivity of BR96 to Glycolipids and
Glycoproteins
[0437] BR96 antibody was tested for reactivity to a variety of
immobilized glycolipid antigens having known carbohydrate
structures and synthetic glycoproteins (so called
"neoglycoproteins") using an ELISA assay in which purified
glycolipids and glycoproteins and antibody were used in excess (Dr.
John Magnani, Biocarb, Gaithersburg, Md.; Lloyd et al.,
Immunogenetics 17:537-541 (1983)). Glycolipids were dried from
methanol in microtiter wells at 100 ng/well. Synthetic
glycoproteins were coated on the surface of the wells by incubation
of glycoprotein diluted to 200 ng in phosphate buffered saline
(PBS), at pH 7.4/well. Purified BR96 was assayed at a concentration
of 10 .mu.g/ml in 0.01 M Tris-HCl, pH 7.4, containing 1% BSA
containing antibodies from ascites were assayed at a dilution of
1:100 in the same buffer. At these high concentrations most binding
interactions are readily detected. Absorbance values were
calculated as the average of duplicate wells. The results of this
analysis are summarized in FIGS. 11 and 12 showing that BR96
reacted with Le.sup.y and Lex determinant.
[0438] These findings indicate that BR96 can bind to a variant form
of the Lewis Y (Fuc .alpha.1-2Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc)
antigen and that fucose .alpha.1-3 attached to GlcNAc forms a
portion of the Le.sup.y-related epitope recognized by BR96. The
high tumor specificity of BR96 and ability to internalize (not
previously described for monoclonal antibodies reactive with the
other Le.sup.y determinant) suggests that the antibody recognizes a
complex epitope, a portion of which includes at least a part of a
Le.sup.y determinant.
EXAMPLE 8
[0439] Preparation and Characterization of BR96 F(ab').sub.2
Fragments
[0440] Murine BR96 (IgG.sub.3) was purified by Protein A affinity
chromatography from murine ascites fluid. Briefly, delipidated
ascites was passed over a column containing a matrix of immobilized
Protein A (RepliGen Corp., Cambridge, Mass.) previously
equilibrated with 1 M potassium phosphate, pH 8.0. Following the
passage of ascites, the column was washed with equilibration buffer
until no further protein was spectrophotometrically detected. The
bound BR96 was then eluted from the column using 0.1 M citrate
buffer, pH 3.0. Immediately after elution, the eluate was
neutralized with 1.0 M Tris buffer, pH 9.0, until the pH was
approximately 7.0. The monoclonal antibody was then dialyzed into
PBS and concentrated prior to storage or use.
[0441] F(ab').sub.2 fragments were then generated by digesting
purified BR96 monoclonal antibody with pepsin according to Lamoyi,
"Preparation of F(ab').sub.2 Fragments from Mouse IgG of Various
Subclasses," Meth. Enzymol. 121:652-663 (1986). Residual whole
antibody and Fc fragments were adsorbed from the reaction mixture
by passage over a protein A affinity column. The resulting
F(ab').sub.2 fragment preparations were dialyzed extensively
against PBS and sterile filtered.
[0442] The BR96 F(ab').sub.2 fragments preparations were
characterized by gel permeation HPLC, SDS-PAGE and by ELISA on the
human breast tumor line 3396 (Bristol-Myers Squibb Co., Seattle,
Wash.). Gel permeation HPLC was used to assess the molecular sizes
of the proteins comprising the F(ab').sub.2 preparation.
Reproducible chromatograms from different preparations indicated
that 75-80% of the protein was F(ab').sub.2. No protein was
detected at the positions representing higher molecular weight
material, such as whole BR96 or protein aggregates. The remaining
20-25% of the protein eluted at positions corresponding to
inactivated pepsin and to other smaller non-protein A-binding
digestion products.
[0443] Nonreducing and reducing SDS-PAGE was used to examine the
denatured molecular sizes and structural arrangement of the
proteins in the F(ab').sub.2 preparations. A single major band at
the position of F(ab').sub.2 (approximately 100 kdal) was typically
observed, with no visible contaminating whole monoclonal antibody
band (160 kdal). Lower molecular weight bands (i.e., less than 100
kdal) representing inactivated pepsin and small digestion products
were minimal. Under reducing conditions the expected results were
obtained with the only major bands occurring as a doublet at
approximately 25 kdal representing the light chain and the
remaining fragmented portion of the heavy chain. No whole heavy
chain band was observed.
[0444] Functional (binding) activity of the BR96 F(ab').sub.2
fragments was compared to that of whole BR96 in an ELISA with 3396
cells supplying the antigen. Binding of BR96 whole antibody or
F(ab').sub.2 fragments to the cells was detected with an
HRP-conjugated goat anti-murine K light chain reagent as shown in
FIG. 13. On a duplicate plate, binding of whole BR96 was
distinguished from binding of F(ab').sub.2 fragments by using
HRP-conjugated protein A which binds to the whole antibody but not
the F(ab').sub.2 fragments (FIG. 14).
[0445] These results indicate that BR96 F(ab').sub.2 (lot
R0201-1663-03, lot 2) contained a trace amount of whole BR96
antibody. The level of contaminating whole antibody can be
estimated to be approximately 8 trifold dilutions away from the
amount of F(ab').sub.2 present, or about 0.01%. The other
F(ab').sub.2 preparation (lot R9011-1481-48, lot 1) showed no
detectable level of contaminating whole BR96, indicating that any
effect of BR96 can be explained by binding of the Fab region and
not the Fe region.
[0446] In summary, the BR96 F(ab').sub.2 preparations appear to be
completely free of contaminating whole BR96 IgG by HPLC and by
SDS-PAGE. In only one instance, when a very sensitive ELISA method
was used were detectable levels of contaminating whole BR96
antibody found and this represented only approximately 0.01% by
weight compared to the amount of F(ab').sub.2 fragments
present.
EXAMPLE 9
[0447] Preparation and Characterization of Chimeric BR96 Antibody
(ChiBr96)
[0448] The murine/human chimeric BR96 antibody of the invention
("ChiBR96") was produced using a two-step homologous recombination
protocol as described by Fell et al., in Proc. Natl. Acad. Sci. USA
86:8507-8511 (1989) and in co-pending patent application by Fell
and Folger-Bruce, U.S. Ser. No. 243,873, filed Sep. 14, 1988, and
Ser. No. 468,035, filed Jan. 22, 1990, and assigned to the same
assignee as the present application; the disclosures of all of
these documents are incorporated in their entirety by reference
herein.
[0449] Human Heavy Chain DNA Transfection
[0450] The murine hybridoma cell line BR96, ATCC No. HB10036,
obtained as described above was transfected (8.times.10.sup.6
cells) with h.gamma.1/HC-D (deposited at Agricultural Research
Service Culture Collection, Peoria, Ill., NRRL No. B 18599) (FIG.
15) by electroporation (Gene Pulser; Biorad Laboratories, Richmond,
Calif.) at 250 V, 960 .mu.Fd capacitance setting, in isotonic
phosphate buffered saline (PBS) and 30 .mu.g/ml of the purified 6.2
kb Xbal restricted fragment of the vector hg.gamma.1HC-D. After 48
hr cells were seeded in 96-well plates at 10.sup.4 cells/well.
Selection for Neo.sup.R was carried out in IMDM medium (GIBCO,
Grand Island, N.Y.) containing 10% (vol/vol) fetal bovine serum
(FBS) and the antibiotic aminoglycoside G418. (GIBCO) at 2.0
mg/ml.
[0451] Detection of Secreted Human IaG (Hu .gamma.1) Antibody by
ELISA
[0452] Culture supernatants were screened using a sandwich ELISA
assay 2 weeks after transfection. Goat anti-human IgG, Fc specific
(CALTAG, San Francisco, Calif.) was used as the capture antibody
and goat anti-human IgG, Fc specific conjugated to horseradish
peroxidase HRPO, (CALTAG) was the antibody used to detect bound
human IgG. Cells from the HuIgG positive wells were subcloned by
dilution and dilution clones were screened by ELISA to detect human
IgG.gamma.1 by the previously described method. The clones
containing human IgG.gamma.1 were also screened by ELISA to detect
murine IgG3 heavy chain. Goat anti-mouse IgG3 (Southern
Biotechnology Assoc., Inc., Birmingham, Ala.) was used as the
capture antibody and goat anti-mouse conjugated to HRPO (Southern
Biotechnology Assoc., Inc.) was the antibody used to detect the
mouse IgG3.
[0453] One of the human IgG.gamma.1 positive murine IgG3 negative
(Hu.gamma.1.sup.+, MuG3.sup.-) clones was chosen and designated
ChiHBR96. This heavy chain chimeric hybridoma cell line, ChiHBR96
was characterized for antigen specificity on MCF-7 cells and for
expression levels by a quantitative ELISA for human IgG expression
on MCF7 cells. The cell line ChiHBR96 expressed approximately 20
.mu.g/ml of antigen-specific human IgG antibody.
[0454] Limit Chain DNA Transfection
[0455] The ChiHBR96 hybridoma (8.times.10.sup.6 cells) was
transfected by electroporation as described above but using 30
.mu.g/ml of the human light chain recombination vector
pSV.sub.2gpt/C.sub.K (NRRL No. B 18507) containing the human light
chain K immunoglobulin sequence shown in FIG. 16, linearized with
HindIII. After 48 hr cells were seeded in 96-well plates at
10.sup.4 cells/well. Selection for gpt was carried out in IMDM
medium containing 10% (vol/vol) FES, 15 .mu.g/ml hypoxanthine, 250
.mu.g/ml xanthine and 2.25 .mu.g/ml mycophenolic acid (Mass.).
[0456] Detection of Secreted Human Kappa (Hu K) Antibody by
ELISA
[0457] Culture supernatants were screened using a sandwich ELISA
assay as described above, 2 weeks after transfection. Goat
.alpha.-human K (CALTAG) was the capture antibody and goat
anti-human K HRPO (CALTAG) was the antibody used to detect bound
human K. Wells containing human K antibody were subcloned by
dilution and the clones were screened by ELISA to detect human K or
murine K chain. Goat anti-mouse K (Fisher Scientific, Pittsburgh,
Pa.) was used as the capture antibody and goat anti-mouse K
conjugated to HRPO (Fisher Scientific) was the antibody used to
detect the presence of the mouse K chain. One of the human K
positive, murine K negative clones (HuK.sup.+, MuK.sup.-) was
chosen to analyze antigen specificity on MCF-7 cells and for
expression levels by a quantitative ELISA for human IgG expression
on MCF-7 cells. A cell line that was antigen specific for MCF-7
cells and HuIgG.sup.+, MuIgG3.sup.-, HuK.sup.+, MuK.sup.- was
chosen and designated Chimeric BR96 (ChiBR96).
[0458] The original expression of the heavy and light chain antigen
specific chimeric BR96 (ChiBR96) antibody was approximately 25
.mu.g/ml. Through four sequential rounds of cloning the line in
soft agarose with a rabbit a HuIgG antibody overlay to detect cells
secreting the highest amount of chimeric antibody (Coffino et al.,
J. Cell. Physiol. 79:429-440 (1972)), a hybridoma cell line
(ChiBR96) was obtained secreting approximately 130 .mu.g/ml of
chimeric antibody. Hybridoma ChiBR96 was deposited with the ATCC on
May 23, 1990, and there provided with the deposit number, ATCC No.
HB 10460.
[0459] Binding of ChiBR96
[0460] The relative affinity of the ChiBR96 antibody and murine
BR96 antibody of the invention for the tumor associated antigen on
MCF-7 cells was determined by an ELISA competition binding assay
(Hellstrom et al., Cancer Res. 50:2449-2454 (1990)). Briefly,
adherent antigen bearing cell line MCF-7 was plated in a 96-well
microtiter dish at 3.times.10.sup.4 cells/well and allowed to grow
to confluency for about 3-4 days. The growth media was discarded
and the cells are fixed with 0.5% glutaraldehyde in PBS (Sigma
Chemical Co., St. Louis, Mo.), at 100 .mu.l/well for 30 min. The
glutaraldehyde was discarded and the plate was washed gently with
PBS three times. The plate was then blocked with binding buffer
(0.1% BSA in DMEM) 200 .mu.l/well for 1 hr or was stored
indefinitely at -20.degree. C. Binding buffer was discarded and
samples and standards were added to the wells. The plates were
covered and incubated overnight at 4.degree. C. Samples and
standards were discarded and the plates were washed three times
with PBS. HRP-conjugate diluted in 1% horse serum in PBS was added
to wells, 100 .mu.l/well and incubated for 1 hr at 37.degree. C.
The ELISA was developed with 3,3',5,5'-tetramethyl benzidine (TMB)
chromagen (Genetic Systems, -Seattle, Wash.) in a citrate buffer.
Color development was arrested with 3N H.sub.2SO.sub.4 and the
plate was read on a Titertek Microplate reader at 450 nm. This
assay determined how well 0.3 .mu.g/ml of biotinylated ChiBR96
antibody competes with either unlabeled ChiBR96 or unlabeled murine
BR96 monoclonal antibody for the antigen. The bound biotinylated
ChiBR96 antibody was detected with avidin-HRPO and developed with
standard ELISA reagents.
[0461] As shown in FIG. 17, the overlap of the two binding curves
indicates that the two antibodies have the same specificity and
relative affinity for the tumor antigen.
EXAMPLE 10
[0462] Characterization of the ChiBR96 Antibidy and BR96
F(ab').sub.2 Fragments
[0463] Cytotoxicity of Unmodified ChiBR96 and BR96 F(ab').sub.2
Fragments
[0464] Living suspended cells from the BR96 antigen positive
carcinoma lines 3396, 2987 and MCF-7, were treated with ChiBR96 and
BR96 F(ab').sub.2 fragments prepared as described in Examples 8 and
9, above, to determine cytotoxicity of these antibodies as compared
to the BR96 monoclonal antibody of the invention. The cytotoxicity
tests were performed by FACS assay as described above in Example 4.
The results of these experiments are shown in FIGS. 18-20 as
percentage dead cells vs. antibody concentration in .mu.g/ml.
[0465] FIGS. 18 and 20 show that the chimeric BR96 antibody and
F(ab').sub.2 fragments of BR96 IgG3 are similar to BR96 monoclonal
antibody with respect to cytotoxicity to 3396 and MCF-7 cells. FIG.
19 demonstrates that the cytotoxic effect on 2987 cells is much
lower than on the other breast carcinoma cells (FIGS. 18 and 20).
These results suggest that a higher binding ratio (Table 2) is
important for killing by these antibodies and/or that different
tumor cells might have different sensitivity to killing by these
antibodies. These results illustrate that the ChiBR96 antibody and
the F(ab').sub.2 fragments are cytotoxic by themselves, i.e., in
unconjugated form, and also illustrate that the cytotoxicity of the
BR96 antibodies is not dependent on the Fe region.
[0466] Internalization of ChiBR96
[0467] The internalization of the ChiBR96 antibody within carcinoma
cells was evaluated in comparison to internalization of the BR96
monoclonal antibody. The antibodies were conjugated to ricin A
chain toxin to form immunotoxins ChiBR96-RA (1-4 Ricin A chains per
antibody molecule) and BR96-RA (1-2 Ricin A chains per antibody
molecule) and internalization by carcinoma cell lines 3396 and 3630
was measured using a thymidine uptake inhibition assay, as
described in Example 3, above.
[0468] Graphs of the percent inhibition of thymidine incorporation
vs. immunotoxin concentration for each cell line tested are shown
in FIGS. 21 and 22. FIG. 21 depicts the percent inhibition of
thymidine incorporation by cells from the 3396 breast carcinoma
cell line caused by internalization of ChiBR96-RA and BR96-RA. As
shown in the graph, ChiBR96 is internalized similarly to BR96, and
appears to be at least as efficient as BR96 at killing tumor cells.
Similar results were obtained with the 3630 breast carcinoma cell
line (FIG. 22).
[0469] Antibody-dependent Cellular Cytotoxicity Activity of ChiBR96
Antibody
[0470] Determination of antibody-dependent cellular cytotoxicity
activity of ChiBR96 was conducted as described in Example 5, above
using the following cell lines: breast cancer lines 3396, 3630 and
3680 (Bristol-Myers Squibb Co., Seattle, Wash.) and MCF-7 (ATCC No.
HTB22); ovarian cancer line 3633-3 (Bristol-Myers Squibb Co.,
Seattle, Wash.); and lung cancer lines 2987; 3655-3 and 2981
(Bristol-Myers Squibb Co., Seattle, Wash.). The results are shown
in Table 3 for various antibody concentrations.
5TABLE 3 ANTIBODY-DEPENDENT CELLULAR CYTOTOXICITY ACTIVITY OF
CHIBR96 Antibody Concentration (.mu.g/ml) Cell Lines Antibody NK 10
1 0.1 0.01 0.001 Breast Cancer 3396 BR96 28 86 74 58 27 25 ChiBR96
88 79 60 34 26 MCF-7 BR96 16 82 69 54 17 15 ChiBR96 90 82 57 25 17
MCF-7 BR96 22 73 69 48 22 22 ChiBR96 76 70 57 33 26 3630 BR96 30 69
64 42 30 34 ChiBR96 69 56 42 36 36 3680 BR96 13 73 67 58 34 38
ChiBR96 70 71 61 39 30 Ovarian Cancer 3633-3 BR96 20 92 90 64 28 23
ChiBR96 88 88 54 43 29 Lung Cancer 2987 BR96 11 51 57 41 9 7
ChiBR96 69 65 51 28 15 3655-3 BR96 4 49 37 0 0 0 ChiBR96 39 35 12 6
5 2981 BR96 3 4 3 3 4 5 ChiBR96 5 4 3 4 4
[0471] The results shown in Table 3 for various antibody
concentrations indicate that ChiBR96 mediates antibody-dependent
cellular cytotoxicity activity to a similar extent as BR96. The
antibody-dependent cellular cytotoxicity activity can be seen at
antibody concentrations lower than those at which the ChiBR96
antibody is cytotoxic by itself. When antibody BR96 was used alone
as a control it produced 0% killing at the concentrations tested.
Antibody-dependent cellular cytotoxicity activity was only found
with the BR96 antibody-binding cell lines.
[0472] Ability of ChiBR96 to Mediate Complement-Mediated
Cytotoxicity
[0473] Determination of the ability of ChiBR96 to kill tumor cells
in the presence of human serum as a source of complement
(complement-dependent cytotoxicity) were performed as described in
Example 6, using breast cell lines 3396; MCF-7, 3630 and 3680;
ovarian cancer cell line 3633-3; and lung cancer cell lines 3655-3,
2987 and 2981. Table 4 presents the results.
6TABLE 4 COMPLEMENT-DEPENDENT CYTOTOXICITY ACTIVITY OF CHIBR96
ANTIBODY CONCENTRATION (.mu.G/ML) Cell Lines Antibody 10 1 0.1 0.01
Breast Cancer 3396 BR96 100 99 78 13 ChiBR96 86 92 13 2 MCF-7 BR96
94 100 63 2 ChiBR96 92 83 1 0 3630 BR96 94 100 82 9 ChiBR96 86 86
33 9 3680 BR96 100 100 19 7 ChiBR96 87 100 5 9 Ovarian Cancer
3633-3 BR96 98 98 21 0 ChiBR96 100 100 26 1 Lung Cancer 3655-3 BR96
91 22 0 0 ChiBR96 46 3 0 0 2987 BR96 100 100 1 0 ChiBR96 100 43 0 0
2981 BR96 0 3 3 2 ChiBR96 1 1 2 10
[0474] As shown in Table 4, ChiBR96 gave a cytotoxic effect
(complement-dependent cytotoxicity) similar to that of BR96, in the
presence of human serum containing complement. BR96 and ChiBR96
were not cytotoxic in any concentration. Human serum was also not
cytotoxic.
[0475] The above results demonstrate that the whole BR96 antibody
and chimeric antibody of the invention are internalized within
carcinoma cells to which they bind, are cytotoxic alone in
unmodified form and have antibody-dependent cellular cytotoxicity
and complement-dependent cytotoxicity activity for cells expressing
a higher amount of epitopes.
EXAMPLE 11
[0476] Evaluation of BR96 Antibodies in vivo
[0477] The therapeutic potential of the unmodified BR96 antibody of
the invention for treatment of tumors was examined in a series of
experiments using human tumor xenografts in nude mice. In addition,
certain parameters were examined that might influence the efficacy
of BR96 as an antitumor agent. These parameters include level of
antigen expression on the target tumor line, time from tumor
implantation to initiation of therapy and effects of dose.
[0478] In all the in vivo experiments of this example, the required
number of Balb/c nu/nu mice (Harlan Sprague Dawley, Indianapolis,
Ind.) were implanted with either the human lung adenocarcinoma cell
line H2987 or H2707 tumor line. Cells from these tumor lines were
grown in vitro, harvested, washed and resuspended in PBS prior to
subcutaneous (s.c.) implantation of 10 million cells into the rear
flank of each mouse. These groups of mice were then randomized and
separated into smaller equal groups of 8 or 10 mice each.
[0479] To increase the chance of observing any antitumor effects of
BR96 while still requiring the antibody to actually localize to the
tumor implant site for any effect to occur, therapy was initiated
24 hours after tumor implantation on day 2. Both the BR96 and
control monoclonal antibodies were administered at the same dose
and schedule, although initiation of therapy in some cases varied.
The treatment dose was administered in 0.2 ml PBS intravenously
(i.v.) through the tail vein of the mouse. Normally the schedule
was once every three days for five injections (Q3DX5). However, two
extra injections were given on days 19 and 21 after H2987 tumor
implantation in the initial experiment.
[0480] Antitumor Effects of BR96 Antibody in 2987 and 2707
Tumors
[0481] Tumor volumes were determined for each animal weekly with
measurement usually beginning on the eighth day after implantation.
Tumor volumes were calculated from the measurements of tumor length
and perpendicular width using the formula:
Tumor volume=longest length.times.(perpendicular width
squared/2)
[0482] Group mean values were then calculated and plotted against
time after tumor implantation.
[0483] In the initial experiment depicted in FIG. 23 treatment with
BR96 resulted in highly significant anti-tumor effects against the
H2987 cell line. BR64, which also binds and is internalized by
these cells, was used as a negative control, and showed little if
any effect compared to the PBS treated controls.
[0484] Table 5 summarizes the effects on the individual tumors at
the end of treatment in this first experiment.
7TABLE 5 EFFECTS OF TREATMENT WITH UNMODIFIED BR96 INITIATED AT
DIFFERENT TIMES AFTER H2987 IMPLANTATION EXPERIMENT 1 DAY 28 TUMOR
RESPONSE GROUP MAb COMPLETE PARTIAL STABLE PROGRESSION 1 BR96 2 0 3
5 2 BR64 0 0 1 9 3 PBS 0 0 0 10
[0485] Only treatment with BR96 antibody resulted in complete
absence of tumor. Two animals in this group were tumor free and an
additional 3 animals showed cessation of growth of their tumors
following treatment with BR96 antibody. The two mice showing no
signs of tumor remained tumor free throughout the course of the
experiment.
[0486] Antitumor Effects of BR96 Antibody on Established Tumors
[0487] One of the ultimate goals of tumor therapy is the effective
treatment of established and growing tumors. To examine whether
BR96 could have an antitumor effect on established tumors the H2987
or H2707 lung adenocarcinoma tumor lines were used as xenografts in
nude mice. Because both of these tumor lines result in palpable
tumors eight days after administration of 10 million cells s.c.,
delaying initiation of treatment provided a method to examine
antitumor effects on established tumors.
[0488] Therefore, to further examine the efficacy of unmodified
BR96, several experiments were performed where treatment was
withheld for either 5 or 8 days following s.c. tumor implantation.
The delay in treatment initiation allowed the tumor cells to become
established tumors. This results in an animal model that is more
difficult to treat but resembles the clinical situation in a more
realistic manner.
[0489] The treatment protocol is summarized in Table 6. Three
groups of 10 mice each were treated with BR96 antibody initiated at
different times as described in this Table. Control mice received
either FA6 or PBS beginning on DAY 2. FA6 is a murine IgG.sub.3
directed against a bacterial antigen not found in mammalian
species, and acted as an isotype matched nonbinding negative
control monoclonal antibody.
8TABLE 6 EFFECTS OF TREATMENT WITH UNMODIFIED BR96 INITIATED AT
DIFFERENT TIMES AFTER H2987 OR H2707 IMPLANTATION TREATMENT
PROTOCOL DAYS GROUP MAb SCHEDULE/ROUTE DOSE INJECTED 1 BR96 Q3DX5
i.v. 1 mg, 2, 5, 8, 11, 14 2 BR96 Q3DX5 i.v. 1 mg 5, 8, 11, 14, 17
3 BR96 Q3DX5 i.v. 1 mg 8, 11, 14, 17, 20 4 FA6 Q3DX5 i.v. 1 mg 2,
5, 8, 11, 14 5 PBS Q3DX5 i.v. 0.2 ml 2, 5, 8, 11, 14
[0490] The results of this treatment protocol for both H2987 and
H2707 tumor cell lines are shown in FIG. 24, where the number of
animals without tumors versus when initiation of treatment after
tumor implantation occurred are plotted. Absence of tumor, as
defined by the absence of a palpable tumor, was assessed at the end
of treatment for each group. The day used for the determination of
tumor absence varied since treatment was initiated at different
times post tumor implant. Early initiation of treatment was clearly
more effective and efficacy decrease as onset of treatment
increased from time of tumor implant. Since delay in initiation of
treatment allows greater growth and establishment of the tumor,
decreased efficacy at later treatment initiation times reflects the
increasing difficulty of treating larger and more established
tumors.
[0491] These results demonstrate that BR96 has antitumor effects
against two different tumor cell lines. Antitumor effects were only
observed in the three groups treated with BR96 antibody while those
animals treated with either the control FA6 or PBS showed no
antitumor effects.
[0492] It is significant that the differences in efficacy with more
established tumors are greater with the higher antigen expressing
tumor line, H2707. The observation that H2707 has a greater
response to BR96 therapy than H2987 is consistent with the
assumption that the amount of antigen expressed by a tumor cell may
influence the efficacy of BR96 treatment. From the data above it is
clear that BR96 has antitumor effects against staged tumors.
[0493] Dose Effects of BR96 Antibody
[0494] In another experiment, the dose effects of BR96 against the
H2707 tumor line were examined. In this experiment, BR96 was
administered in decreasing half log amounts from 1 mg/dose to 0.032
mg/dose. The mean tumor volumes versus time post tumor implant of
the groups are presented in FIG. 25. The control treated animals
were given only the highest dose of monoclonal antibody, 1 mg/dose
FA6. These control animals showed no antitumor effects while there
was a dose dependent response when BR96 antibody was administered
over the chosen dose range.
[0495] Antitumor Effects of F(ab').sub.2 Fragment and Chimeric
BR96
[0496] In addition, antitumor effects of the F(ab').sub.2 BR96
fragment were examined to determine if the antitumor effects seen
in vivo were due to the Fc portion or if actual binding to the
tumor with its subsequent internalization was sufficient for cell
death, as indicated by in vitro assays. The dose of F(ab').sub.2
fragment was 0.66 mg/dose using the same schedule as the whole
BR96. This dose corresponds to an approximate molar equivalent of
binding regions compared to the 1.0 mg/dose whole IgG.sub.3 BR96.
Mean tumor volume values versus time post tumor implantation for
this group treated with the antibody fragment are shown in FIG. 26.
There were clearly some antitumor effects although the effects were
not as strong as with whole antibody. These effects were most
pronounced at the earlier time points during and immediately
following treatment.
[0497] Chimeric BR96 was also examined for antitumor effects in
this experiment. An intermediate dose of 0.32 mg/dose for the
chimeric monoclonal antibody was chosen. The mean tumor volume
values for this group of mice is also shown in FIG. 26. Treatment
with chimeric antibody BR96 was more efficacious than a comparable
dose of the murine BR96 IgG.sub.3. This is further demonstrated in
FIG. 27 which shows that 6 of the 8 mice treated with chimeric BR96
were free of palpable tumors at the end of treatment.
[0498] Examination of the individual tumors depicted in FIG. 27
shows that at completion of treatment a clear dose effect was
evident by the number of animals without tumors after treatment
with decreasing amounts of whole IgG3 BR96 antibody from 1.0 to 0.1
mg/dose. Surprisingly, treatment with 0.032 mg/dose resulted in an
antitumor effect similar to the 0.32 mg/dose. This may reflect that
the level of cells killed in the tumor from the treatment was very
close to the minimum amount necessary for the tumor to continue to
grow.
[0499] Three of the eight animals treated with the F(ab').sub.2
fragment were free of palpable tumors after treatment. Therefore
the Fe portion is not entirely necessary for the antibody to have
antitumor effects in vivo although it should enhance the tumorcidal
properties of BR96, particularly in immunocompetent animals.
[0500] The above results demonstrate that unmodified BR96
antibodies are effective antitumor agents against tumor lines in
vivo. Moreover, the BR96 antibodies have an effect on staged or
established growing tumors. There is an indication that higher
antigen density on the tumor line may increase the ability of BR96
to kill these cells. It has been shown that any of the forms of the
monoclonal antibody, i.e., chimeric, marine whole IgG.sub.3 or
F(ab').sub.2 fragments, are effective as antitumor agents. Earlier
treatment and higher doses are preferred.
EXAMPLE 12
[0501] Localization and Biodistribution of BR96 Antibodies
[0502] Radioiodinated BR96 monoclonal antibodies administered at
doses used in the therapy experiments described above in Example 11
were used to determine biodistribution characteristics.
Specifically, the whole IgG.sub.3 BR96, chimeric or F(ab').sub.2
fragments together with the appropriate control (whole monoclonal
antibody FA6, chimeric 96.5 and 96.5 F(ab').sub.2, respectively)
were used to localize in the tumor and various other organs.
[0503] Prior to the localization experiments, animals were injected
with tumor cells as described above in Example 11, for the therapy
studies. However, the tumors were allowed to grow in the animals
for approximately 2 weeks. At this time, 100 .mu.g of BR96 antibody
or fragment was radiolabeled with .sup.125I using 10 .mu.g Iodogen
for 10 minutes at room temperature as directed by the manufacturer.
Control antibody or fragments were labeled with .sup.131I using the
same method. These radioiodinated antibodies were diluted with the
appropriate unlabeled antibodies to reach the doses used in the
therapy experiments. Both the specific and nonspecific antibodies
were then mixed and administered together, i.v., through the tail
vein of the mouse. At selected times mice were randomly pulled from
the group, anesthetized, bled through the orbital plexus and
sacrificed. Various tissues were removed, weighed and counted on a
gamma counter capable of differentiating between the two
radioisotopes of iodine. From this data, percent injected dose and
percent injected dose per gram were calculated.
[0504] The accumulated data from the 24 post administration time
point in the localization experiments are summarized in Table
7.
9TABLE 7 SUMMARY OF BIODISTRIBUTION EXPERIMENTS % INJECTED
DOSE/GRAM 24 HRS. POST ADMINISTRATION DOSE TUMOR ANTIBODY (mg) CELL
BLOOD TUMOR LIVER SPLEEN KIDNEY LUNG 1) BR96-G.sub.3 1.0 H2987 10.2
6.8 2.2 1.9 3.4 4.7 FA6 1.0 6.3 2.1 2.1 1.6 2.4 3.2 2) BR96-G.sub.3
0.3 H2707 9.0 7.0 1.8 1.6 2.7 3.7 FA6 0.3 5.9 2.7 2.0 1.8 2.2 2.8
3) ChiBR96 0.32 H2707 7.2 8.2 1.4 1.6 2.0 3.5 Chi96.5 0.32 7.5 2.3
1.8 1.6 1.9 3.5 4) F(ab').sub.2 BR96 0.65 H2707 <0.3 <0.3
<0.3 <0.3 <0.3 <0.3 96.5 0.65 <0.3 <0.3 <0.3
<0.3 <0.3 <0.3
[0505] The only tissue showing significant differences between
specific and nonspecific antibody is the tumor. All other tissues
examined show approximately equal uptake between the specific and
nonspecific antibodies. One possible exception is the lower blood
levels for the nonspecific antibody, FA6. This indicates
accelerated blood clearance of this antibody. However, the
difference between the specific and nonspecific antibody in the
tumor is greater than the difference in blood levels between the
FA6 and BR96 antibodies.
[0506] The data in Table 7 also demonstrate that the percent of the
dose present in a particular organ is constant regardless of the
dose administered. This would therefore indicate there is
quantitatively more antibody present at the tumor site when higher
doses are administered. In addition, there are no apparent
differences between the two tumor lines with respect to specific vs
nonspecific uptake.
[0507] Table 7 also demonstrates that the F(ab').sub.2 was cleared
from the animal at a much faster rate than either the IgG.sub.3 or
chimeric BR96. This could explain the reduction in efficacy of the
fragment compared to whole antibody in the therapeutic experiments.
Any antitumor effects from the fragment must therefore be rapid and
occur during the short time span prior to being cleared.
[0508] ChiBR96 localized at a comparable level to the IgG.sub.3
BR96. Higher amounts were present only in the tumor compared to the
control chimeric antibody. This suggests that any increase in
efficacy of the chimeric antibody compared to the murine BR96
IgG.sub.3 is due to the human constant region substitution. Of
equal importance, the human constant region substitution does not
appear to affect the ability of the chimeric antibody to localize
to the tumor or adversely affect its biodistribution.
[0509] In summary, the IgG.sub.3 and chimeric forms of BR96 are
capable of specifically localizing to the tumor site. Moreover,
both localization and therapeutic effects have been shown in these
preliminary experiments at comparable doses. Indirect evidence of
localization of the F(ab').sub.2 fragments was shown by the
antitumor activity of the fragments in the therapy experiments.
This activity must occur before 24 hours.
EXAMPLE 13
[0510] Preparation and Cytotoxicity of Chimeric Monoclonal Antibody
F(ab').sub.2 and Fab' Fragments Conjugated to Pseudomonas
Exotoxin
[0511] Cell-specific cytotoxic reagents were prepared by chemically
combining the chimeric antibody BR96 (ChiBR96) with Pseudomonas
exotoxin A (PE) using either native PE or a truncated form
(LysPE40) devoid of the cell recognition region (Domain I). A
variety of chimeric BR96-immunotoxins were constructed by chemical
conjugation of PE and LysPE40 with Fab, or F(ab').sub.2 enzymatic
digest products, or BR96 antibody, by thiolation with
2-iminothiolane or by direct attachment to intact BR96 antibody by
reduction with DTT as described below.
[0512] Reagents
[0513] Succinimidyl 4-(N-maleimido-methyl) cyclohexane
1-carboxylate (SMCC) and 2-iminothiolane (2-IT) were purchased from
the Pierce Chemical Corporation (Rockford, Ill.). Soluble pepsin
was purchased from Sigma Chemical Co. (St. Louis, Mo.).
Na(.sup.125I) and (.sup.3H)-leucine were purchased from New England
Nuclear (Boston, Mass.). Native PE was purchased from Berna
Products (Coral Gables, Fla.). Mono Q columns were purchased from
Pharmacia (Uppsala, Sweden). TSK-3000 columns were purchased from
TosoHaas, Inc. (Philadelphia, Pa.). Immunoblots were performed
using mouse (anti-id BR96) and rabbit (anti-PE) ABC kits (Vector
Laboratories, Burlingame, Calif.). Rabbit polyclonal anti-PE
antibody and mouse anti-PE monoclonal antibody M40/1 were supplied
by Drs. Ira Pastan and David FitzGerald, National Institutes of
Health (Bethesda, Md.). Anti-idiotypic BR96 antibody 757-4-1 was
prepared using the BR96 antibody of the invention and standard
procedures for preparing anti-id antibodies (see, Kahn et al.,
Cancer Res. 49:3159-3162 (1989) and Hellstrom et al., Cancer Res.
50:2449-2454 (1990)) by Dr. Bruce Mixan, Bristol-Myers Squibb
(Seattle, Wash.)).
[0514] Cell Culture and Plasmids
[0515] All cells were cultured in RPMI 1640 supplemented with 10%
fetal bovine serum, except L929, which was cultured in DMEM
supplemented with 10% fetal bovine serum. Plasmid pMS8 (FIG. 36),
which encodes the gene for LysPE40 under control of the T7
promoter, was constructed by Dr. Clay Siegall (provided by Dr. Ira
Pastan, NIH, Bethesda, Md.) from the vector pVC85 (Kondo et al., J.
Biol. Chem. 263:7470-7475 (1988)) by inserting at the amino
terminus a lysine residue and also inserting a multiple cloning
site.
[0516] Expression and Purification of LysPE40
[0517] The plasmid pMS8 encoding LysPE40 was transformed into E.
coli BL21 .lambda.DE3) cells and cells were cultured in Super Broth
(Digene, Inc., Silver Spring, Md.) containing 75 .mu.g of
ampicillin per ml at 37.degree. C. When absorbance at 650 was 2.0
or greater, isopropyl 1-thio-.beta.-D-galactopyranoside was added
(1 mM) and cells were harvested 90 minutes later. The bacteria were
washed in sucrose buffer (20% sucrose, 30 mM Tris-HCl pH 7.4), 1 mM
EDTA), and osmotically shocked in ice-cold H.sub.2O to isolate the
periplasm. LysPE40 protein was purified from the periplasm by
successive anion-exchange and gel-filtration chromatographies using
a Pharmacia fast protein liquid chromatography (FPLC) system as
described previously (Batra et al., Proc. Natl. Acad. Sci. USA
86:8545-8549 (1989) and Siegall et al., Proc. Natl. Acad. Sci. USA
85:9738-9742 (1988)).
[0518] Generation of BR96 F(ab').sub.2 and Fab' Fragments
[0519] F(ab').sub.2 fragments were generated from ChiBR96 (4 mg/ml)
by pepsin digestion (25 .mu.g/ml in 0.1 M citrate buffer, pH 4.0,
(Parham, in Handbook of Experimental Immunology, Weir, Ed.,
Blackwell Scientific Publishers, p. 1-23 (1986)). After 6 hours
incubation at 37.degree. C., digestion was terminated by adjusting
the pH to 7.2 with PBS. Purity of the digest preparation was 90-95%
F(ab').sub.2 determined by SDS-PAGE (4-20% gradient gels) and
Coomassie blue staining.
[0520] Fab' was prepared from the ChiBR96 F(ab').sub.2 by reduction
with cysteine to break the remaining interchain disulfide bonds
(Parham et al., supra). Briefly, F(ab').sub.2 molecules (2-4 mg/ml)
in 0.1 M Tris-HCl (pH 7.5) were incubated at 37.degree. C. for 2
hours with cysteine (0.01 M final concentration). Free sulfhydryl
groups on the Fab' molecule were alkylated with 0.02 M
iodoacetamide (CalBiochem, San Diego, Calif.) for 30 minutes at
room temperature to prevent recombination of the Fab' to
F(ab').sub.2. The reaction mixture was dialyzed against PBS. Purity
was greater than 85% as assessed by SDS-PAGE.
[0521] Immunotoxin Construction and Purification
[0522] Chimeric BR96 (6-10 mg/ml) was thiolated by addition of a
3-fold molar excess of 2-iminothiolane (2-IT) in 0.2 M sodium
phosphate (pH 8.0), 1 mM EDTA for 1 hour at 37.degree. C. (Batra et
al., Proc. Natl. Acad. Sci. USA 86:8545-8549 (1989)), which
introduces sulfhydryl groups by reaction of 2-iminothiolate with
primary amines. Unreacted 2-IT was removed by PD-10 column
chromatography (Pharmacia). Alternatively, free thiol groups were
generated by reduction with dithiothreitol (DTT). Chimeric BR96 was
incubated with a 20-fold molar excess of DTT for 2.5 hours at
42.degree. C. Excess DTT was removed by overnight dialysis against
PBS under nitrogen. The number of thiol groups on the monoclonal
antibody was determined by DTNB reduction (Ellman's reagent, Sigma
Chem. Co.) as described by Deakin et al., Biochem. J. 89:296-304
(1963), incorporated by reference herein. This procedure routinely
gave 4 thiol groups per BR96 antibody, with no reduction in
antibody binding reactivity or protein concentration. The procedure
was not used with F(ab').sub.2 or Fab' fragments.
[0523] Thiolated BR96 antibody was condensed with
maleimide-modified PE or LysPE40. Anon-cleavable maleimide group
was attached to lysine residues on the toxin (PE or LysPE40; 6-8
mg/mil) by mixing with 3-fold molar excess of SMCC in 0.2 M sodium
phosphate (pH 7.0), 1 mM EDTA at room temperature for 30 minutes
and purified on a PD-10 column. Modified toxin and thiolated
antibody were mixed in a 4:1 molar ratio and incubated at room
temperature for 14-16 hours to allow a thioether linkage to form.
Immunotoxins were purified by anion-exchange (Mono Q) to remove
unreacted antibody and gel-filtration chromatography (TSK-3000) to
remove unconjugated toxin as previously described by Kondo et al.,
J. Biol. Chem. 263:9470-9475 (1988); and Batra et al., Proc. Natl.
Acad. Sci. USA 86:8545-8549 (1989), all incorporated by reference
herein.
[0524] Chimeric BR96 IgG-LysPE40 (190 kDa), Fab'-LysPE40 (96 kDa)
and F(ab').sub.2-LysPE40 (145 kDa) conjugates were additionally
analyzed by non-reducing SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) to determine the size of the native conjugate (FIG. 28).
From the Coomassie blue stained gels, it was determined that there
was less than 5% unconjugated antibody after purification.
[0525] Binding Studies
[0526] For competition binding studies, L2987 cells (Bristol-Myers
Squibb Co., Seattle, Wash.) were removed from monolayer culture
using 0.2% trypsin and washed with RPMI 1640 containing 2% FCS
(wash buffer). Cell suspensions (1.0.times.10.sup.6 cells/0.1 ml)
were incubated with 0.1 ml fluorescein isothiocyanate
(FITC)-labeled ChiBR96 (13.3 .mu.g/ml final concentration) and 0.1
ml of diluted antibody or immunotoxin at 4.degree. C. for 1 hour,
washed, and the amount of cell-bound FITC labeled-ChiBR96 was
quantified on an EPICS V model 753 Flow Cytometer (Coulter Corp.,
Hialeah, Fla.).
[0527] For direct binding studies, two-fold serially diluted
immunotoxins or antibody was incubated for 1 hour at 4.degree. C.
in 0.2 ml wash buffer containing 1.times.10.sup.6 L2987 cells.
Cells were washed and then incubated in wash buffer containing 1:40
diluted FITC labelled goat anti-human kappa antibody (Bethyl Labs,
Montgomery, Tex.) for an additional 30 min at 4.degree. C. to
quantitate cell-bound antibody. Cells were washed and analyzed for
cell surface fluorescence on a flow cytometer to determine the
amount of immunotoxin or antibody remaining on the cell
surface.
[0528] Two methods were used to determine whether there was an
alteration in antibody binding activity after conjugation to PE or
LysPE40. Competition binding analysis showed that both immunotoxins
competed with FITC-labeled ChiBR96 as efficiently as unconjugated
ChiBR96 antibody (FIG. 29), indicating that binding affinity for
the BR96 antigen was not perturbed after chemical conjugation.
Similar results were obtained using the direct binding assay for
both PE and LysPE40 conjugates.
[0529] Binding activities of LysPE40 conjugated and unconjugated
IgG, F(ab').sub.2 and Fab' were also compared by direct binding to
L2987 tumor cells. Cell-bound antibody protein was quantitated
using FITC-labeled goat anti-human kappa light chain antibody.
Binding of the LysPE40 immunotoxin was similar to that obtained
using unconjugated ChiBR96 antibody (FIG. 30A) and agreed with
results obtained using the competition binding assay (FIG. 29).
FIG. 30B compares the binding activity of intact IgG to
F(ab').sub.2 and F(ab').sub.2-LysPE40. There was no loss in
immunoreactivity with the F(ab').sub.2 and F(ab').sub.2-immunotoxin
as compared to ChiBR96 IgG. Conjugation of PE40 to Fab' did not
affect immunoreactivity (FIG. 30C), however, binding of the Fab'
was significantly decreased as compared to intact IgG (FIG. 30C),
most likely because of the monovalency of the Fab' molecule.
[0530] Antigenic Modulation and Internalization
[0531] Modulation of intact ChiBR96, F(ab').sub.2 or Fab'
immunotoxins was assayed on L2987 cells propagated as 90-95%
confluent monolayer cultures in 96 well microtiter plates as
described above. Target cells were pulsed for 1 hour at 4.degree.
C. with 0.1 ml of two-fold serially diluted immunotoxin ranging
from 5-800.times.10.sup.7 M antibody protein in binding buffer.
Monolayer cultures were washed free from unbound material using
growth medium and individual plates were incubated in complete
medium under either non-modulating (4.degree. C.) or modulating
(37.degree. C.) conditions.
[0532] The amount of membrane-associated immunotoxin bound to
target cell populations at each time point shown in FIG. 31 was
quantified using (.sup.125I)M40/1 (anti-PE) antibody (provided by
Dr. D. Fitzgerald, NCI, Bethesda, Md., (Ogata et al., Inf. and
Immun. 59:407-414 (1991))). Epitope mapping of M40/1 antibody
determined that it binds to a 44 amino acid region in the PE domain
II (Ogata et al., supra). Monoclonal antibody M40/1 was
radioiodinated using Na[.sup.125I] (New England Nuclear, Boston,
Mass.) and chloramine T (Kodak Chemical Co., Rochester, N.Y.) as
described by McConahey and Dixon, Arch. Allergy Appl. 29:185-188
(1966), incorporated by reference herein. Radioiodinated M40/1 was
separated from unbound iodine by PD10 column chromatography
(Pharmacia). Specific activities ranged from 2 to 5.times.10.sup.5
CPM/.mu.g.
[0533] At various times during incubation at 37.degree. C. or
4.degree. C., a triplicate set of wells were twice washed with wash
buffer and pulse-labeled with 0.1 ml (.sup.125I)-labeled M40/1
antibody (0.5 .mu.g/ml in wash buffer containing 0.2% sodium azide)
to determine membrane bound conjugate. After 15 minutes, monolayers
were washed free of unbound label, and cell-bound cpm was
determined by solubilization of the cell monolayer with 0.5 N NaOH.
Cell-bound radioactivity was determined using a LKB model 1272
gamma counter. Non-specific binding was determined by incubation of
target cells with a similar concentration of unconjugated ChiBR96.
In certain experiments, unconjugated PE was used to determine
background binding levels. (.sup.125I)-labeled M40/1 antibody did
not react with membrane bound antibody or PE.
[0534] The ability of ChiBR96-PE and ChiBR96-tysPE40 to induce
antigenic modulation was initially measured by determining the loss
of immunotoxin from the cell surface membrane (FIG. 31). There was
no difference in modulation kinetics between PE or LysPE40
immunotoxins with approximately 50% of the original cell-bound
immunotoxin modulated from the surface membrane 30 minutes after
warming to 37.degree. C. Cells incubated under conditions which do
not allow antigenic modulation (4.degree. C.), showed essentially
no loss of cell surface toxin within 6 hours (FIG. 31A).
[0535] In order to confirm that the loss of cell-surface
immunotoxin was due to endocytosis, cells were incubated with a
[.sup.125I]-labeled immunotoxin complex for 1 hour at 4.degree. C.
to permit binding, washed and were subsequently modulated at
37.degree. C. As shown in FIG. 31B, essentially all the
radiolabeled immunotoxin remained cell-associated, despite the
concomitant loss from the cell-surface membrane (FIG. 31A). These
findings confirm that most if not all of the membrane-associated
BR96 immunotoxins were rapidly internalized, and that
internalization rates were similar for PE and LysPE40
immunotoxins.
[0536] The capacity of ChiBR96 F(ab').sub.2-LysPE40 and
Fab'-LysPE40 immunotoxins to internalize was also determined by
measuring the loss of cell-surface immunotoxin using radiolabeled
anti-PE antibody. Essentially all of the ChiBR96 immunotoxins were
completely internalized after 4.5 hours including the
Fab'-immunotoxin (Table 8). However, rate differences were
observed. At 2.5 hours, when 76% of the intact IgG toxin and 72% of
the F(ab').sub.2 were internalized, only 12% of the Fab'
immunotoxin was internalized. Therefore, both IgG, F(ab').sub.2 and
Fab'-LysPE40 immunotoxins were modulated from the cell surface
membrane, but at different rates.
10TABLE 8 INTERNALIZATION OF BR96-IMMUNOTOXINS FROM THE CELL
SURFACE MEMBRANE OF L2987 CELLS % INTERNALIZATION 2.5 hr 4.5 hr
BR96-LysPE40 74.0 85.0 F(ab`).sub.2-LysPE40 72.0 91.6 Fab`-PE40
12.0 89.6
[0537] Inhibition of Protein Synthesis Assay
[0538] Cytotoxicity of various forms of ChiBR96 antibody conjugated
to LysPE40 against tumor cells was determined by measuring
inhibition of protein synthesis as follows: Tumor cells
(1.times.10.sup.5 cells/ml) in growth media were added to 96 well
flat bottom tissue culture plates (0.1 ml/well) and incubated at
37.degree. C. for 16 hours. Dilutions of toxin or toxin-conjugates
were made in growth media and 0.1 ml added to each well (3
wells/dilution) for 1 hour or 20 hours at 37.degree. C. After the
appropriate incubation time, unreacted material was removed by
washing the monolayer with growth media. Cells were incubated in
0.2 ml growth media for a total of 20 hours and pulse-labeled with
[.sup.3H]-leucine (1 .mu.Ci/well) for an additional 4 hours at
37.degree. C. The cells were lysed by freezing, thawing at
37.degree. C. and harvested using a Tomtec cell harvester (Orange,
Conn.). Cellular protein labeled with [.sup.3H]-leucine was
determined by counting the radioisotope using a LKB Beta Plate
(LKB, Piscataway, N.J.) liquid scintillation counter.
[0539] Analysis of Competition of Immunotoxin Cytotoxic
Activity
[0540] Chimeric BR96-PE40 was added at 0.8, 4 and 20 pM
concentrations to MCF-7 cells in the presence or absence of 50
.mu.g (333 pM) ChiBR96 antibody. Cytotoxicity was determined as
described in the inhibition of protein synthesis assay as described
above.
[0541] In vitro Cytotoxicity of Intact IgG-PE Immunotoxins
[0542] The in vitro cytotoxic activity of the immunotoxins against
cancer cells was assayed by comparing inhibition of protein
synthesis on antigen positive and antigen negative cells (Table 9).
BR96 antigen-positive cell lines MCF-7, L2987, and RCA were the
most sensitive to ChiBR96-PE with EC.sub.50 values of 0.14, 0.28,
and 1.4 pM, respectively. The immunotoxin was also more inhibitory
than native PE which had EC.sub.50 values of 200, 140 and 380 pM.
When tested on antigen-negative cell lines, little difference in
EC.sub.50 values between PE and the immunotoxin was observed.
Specificity, (antibody-directed cell-killing), must take into
account the different sensitivities of the various cell lines to
native PE. Thus, the ChiBR96 immunotoxins were 100-500 fold more
potent than native PE against antigen-positive cell lines.
11TABLE 9 CYTOTOXICITY OF CHIBR96-PE ON HUMAN TUMOR CELLS
EC.sub.50, pM Cell BR96 DTT Reduced 2-IT-Treated Native Line Type
Antigen ChiBR96-PE ChiBR96-PE PE MCF-7.sup.1 Breast Ca. + 0.10 0.14
200.0 L2987 Lung Ca. + 0.25 0.28 140.0 RCA Colon Ca. + 1.2 1.4
380.0 A2780 Ovarian Ca. - 23.0 23.0 60.0 L929 Mouse Fblst - 13.5
14.0 3.0 KB.sup.2 Epidermoid - 220.0 231.0 227.0 .sup.1ATCC No. HTB
22 .sup.2ATCC No. CCL 17
[0543] EC.sub.50 represents the amount of immunotoxin or toxin
required to inhibit 50% of the protein synthesis as determined by
[.sup.3H]-leucine incorporation in cellular protein. (BR96
antigen+=Epitope density of>1.times.10.sup.4
molecules/cell).
[0544] Cytotoxicity of ChiBR96 Mab and Enzymatic Fragments Linked
to LysPE40 Against MCF-7 Cells
[0545] Smaller immunotoxin molecules may be beneficial in tumor
penetration, therefore, the cytotoxic activity of ChiBR96 as Fab',
F(ab').sub.2 fragments and as an IgG linked to LysPE40 was compared
(Table 10). As with the ChiBR96-PE immunotoxin (Table 9), MCF-7 and
L2987 cells were the most sensitive cell lines tested. The IgG and
F(ab').sub.2-LysPE40 molecules showed similar cytotoxic activity
against MCF-7 cells (EC.sub.50=8-14 pM) while the Fab'-LysPE40
conjugate was much less active (EC.sub.50=780 pM) (FIG. 32).
Specificity of protein synthesis inhibition activity of Fab' and
F(ab').sub.2 conjugates was also preserved, with little or no
inhibitory activity observed against the antigen-negative cell
lines A2780.
12TABLE 10 CYTOTOXICITY OF 2-IMINOTHIOLANE SUBSTITUTED CHIMERIC
BR96-PE40 ON HUMAN TUMOR CELLS EC.sub.50 pM Cell BR96 BR96
F(ab').sub.2- Fab'- Line Type Antigen PE40 PE40 PE40 PE40 MCF-7
Breast Ca. ++ 8 14 780 15,000 L2987 Lung Ca. + 37 70 2700 17,500
RCA Colon Ca. + 84 110 5000 15,000 A2780 Ovarian Ca. - 650 2500
11,000 15,000 KB Epidermoid Ca - >5000 N.D. N.D. >25,000
EC.sub.50 is described in Table 8 legend. N.D. = not
determined.
[0546] Specificity of Growth Inhibition By ChiBR96-LysPE40
[0547] Specificity was confirmed by abrogating the protein
synthesis inhibition by ChiBR96-LysPE40 with unconjugated ChiBR96.
Addition of excess ChiBR96 antibody (50 .mu.g) with ChiBR96-LysPE40
immunotoxin, resulted in a decrease of in vitro potency (FIG. 33).
At 20 pM of ChiBR96-LysPE40, approximately 50% of its cytotoxic
effect was blocked by the addition of excess unconjugated antibody,
while at 4 pM, the excess ChiBR96 completely blocked the cytotoxic
activity of ChiBR96-LysPE40.
[0548] Kinetics of Cytotoxicity of ChiBR96 Immunotoxins and Native
PE
[0549] In part, the effectiveness of immunotoxins may depend on the
rate of internalization after binding to antigen-positive cells. To
determine the cytotoxic activity of ChiBR96-PE, ChiBR96-LysPE40 and
PE, a time course analysis was performed where cells were incubated
with toxin for up to 20 hours as described above.
[0550] After 1 hour incubation, MCF-7 cells were sensitive to
ChiBR96-PE and ChiBR96-LysPE40 (EC.sub.50 values of 1 and 60 pm,
respectively) but not to the native toxin (EC.sub.50>10,000 pM).
After 20 hours MCF-7 cells were slightly more sensitive to
ChiBR96-PE and ChiBR96-PE40, but much more sensitive to PE;
EC.sub.50=200 pM (FIG. 34). This assay was repeated at 2, 4, and 6
hour time points. At each time point, PE was considerably less
cytotoxic against MCF-7 cells than ChiBR96-immunotoxins. This may
be due in part to the mechanism by which the toxin molecule is
delivered to target cells.
[0551] This example demonstrates the production of immunotoxins
containing the carcinoma-associated monoclonal antibody ChiBR96 and
Pseudomonas exotoxin A. The antibody was used in forms including
native IgG, reduced IgG, F(ab').sub.2 and Fab'. The toxin component
of the immunotoxin was either native PE or LysPE40, a truncated
form containing a genetically modified amino terminus that includes
a lysine residue for conjugation purposes. Chimeric BR96-toxin
conjugates were found to be cytotoxic to cells which display Lewis
Y, a determinant recognized by the BR96 monoclonal antibody of the
invention. The most cytotoxic of the conjugates produced was
ChiBR96-PE which was 1000-fold more potent than PE itself against
MCF-7 breast carcinoma cells. Chimeric BR96-LysPE40 was also
extremely cytotoxic towards BR96 antigen positive cells (1000-fold
more potent than LysPE40). Both ChiBR96-PE and ChiBR96-LysPE40 were
produced using two procedures which generated sulfhydryl groups on
the antibody, by mild reduction of the antibody or by derivatizing
the antibody with 2-iminothiolane. The former procedure produced a
greater yield of conjugate, but conjugates produced by both
procedures resulted in chimeric molecules of identical
activities.
[0552] Chimeric BR96-PE and ChiBR96-LysPE40 were almost fully
active with 1 hour incubation, while PE was relatively inactive
(FIG. 34). With continued incubation, ChiBR96-immunotoxins increase
cytotoxic activity only slightly while PE becomes cytotoxic to the
MCF-7 cells at later time points. This rapid efficacy of
ChiBR96-immunotoxins is evidence of the utility of ChiBR96 in
targeting cell populations for killing.
[0553] The binding and internalization activities of
ChiBR96-immunotoxins were also examined. Immunoconjugates prepared
with intact IgG or its F(ab').sub.2 or Fab' enzymatic digest
products were not affected in terms of binding by chemical
conjugation to LysPE40 (FIG. 30) or PE. Differences in binding
activity between Fab' and F(ab').sub.2 or IgG conjugates may be
attributed to differences in avidity due to the monovalence of the
Fab' molecule. We also cannot exclude the possibility that
enzymatic digestion used to generate the Fab' fragments contributed
to the decreased avidity. Of most interest was the comparison
between ChiBR96-LysPE40 and the enzymatic fragment immunotoxins
ChiBR96 F(ab').sub.2-LysPE40 and ChiBR96 Fab'-LysPE40. This finding
is also reflected in the cytotoxicity data (Table 10).
[0554] The results presented in this example demonstrate that both
intact PE and LysPE40 immunotoxins as well as F(ab').sub.2 and Fab'
immunotoxins demonstrate cytotoxic activity in vitro.
EXAMPLE 14
[0555] Preparation of Single-Chain BR96 sFv-PE40 Immunotoxin
[0556] This example describes the preparation and characterization
of cytotoxicity of a single-chain immunotoxin, BR96 sFV-PE40,
consisting of the cloned heavy and light chain Fv portions of the
BR96 monoclonal antibody of the invention linked to PE40.
[0557] Q Sepharose was purchased from Pharmacia (Uppsala, Sweden).
TSK-3000 columns were purchased from TosoHaas, Inc. (Philadelphia,
Pa.). Immunoblots were performed using mouse anti-idiotypic BR96
antibody 757-4-1 as described above in Example 13, and ABC
immunoblot kits (Vector Laboratories, Burlingame, Calif.).
Chloramine T was purchased from Sigma Chemical Co. (St. Louis,
Mo.). MCF-7 human breast carcinoma cells were originally obtained
from the ATCC (Rockville, Md.) and have been maintained by
Bristol-Myers Squibb Company, Seattle, Wash. RCA colon carcinoma
cells were obtained from M. Brattain, Baylor University, Texas.
L2987 lung adenocarcinoma cells were obtained from Dr. I.
Hellstrom, Bristol-Myers Squibb Co., Seattle, Wash. A2780 ovarian
carcinoma cells were obtained from K. Scanlon, N I H, Bethesda, M
D, and K B epidermoid carcinoma cells were obtained from Dr. Ira
Pastan, N I H, Bethesda, Md. Cells were cultured in RPMI 1640
supplemented with 10% fetal bovine serum.
[0558] Construction of BR96 sFv-PE40
[0559] In order to produce a single-chain recombinant immunotoxin,
the Fv domains of the light and heavy chains of BR96 IgG were
isolated from plasmid pBR96 Fv (FIG. 36) containing the BR96 Fv
sequences using PCR amplification.
[0560] Identification of Primers for PCR Amplification
[0561] Two sets of PCR primers:
[0562] Primer 1: 5'-GCTAGACATATGGAGGTGCAGCTGGTGGAGTCT-3' (SEQ ID
NO: 1) and primer 2: 5'-GCTGTGGAGACTGGCCTGGTTTCTGCAGGTACC-3' (SEQ
ID NO: 2) were devised for the amplification of the V.sub.L and
V.sub.H of murine chimeric BR96 monoclonal antibody. The V.sub.L
and V.sub.H-5' PCR primers were based on the N-terminal amino acid
sequence of the BR96 light and heavy chains (FIG. 35, SEQ ID NO:
3), respectively, while the 3' primers were designed according to
the frequency of the most common nucleotide at each position of
joining (J)-region segments after alignment of V.sub.H and V.sub.k
genes (Kabat et al., in Sequences of Proteins Of Immunological
Interest, U.S. Dept. Health and Human Services, Washington, D.C.
(1987)). The V.sub.L-5' primer was comprised of 24 nucleotides
encoding the N-terminal amino acids of the variable light chain and
a Hind III site 5' to these nucleotides while the V.sub.L-3' primer
consisted of 22 nucleotides which were complementary to the J
region of mouse kappa light chain mRNA and a Sph I site 5' to these
nucleotides. The V.sub.H-5' primer contained 30 nucleotides
encoding the N-terminal amino acids of the heavy chain and an Eco
RI site 5' to these nucleotides while the V.sub.H-3' primer
contained 22 nucleotides with J region complimentarity and a BamHI
site 5' to these nucleotides. In designing each primer, additional
nucleotides were incorporated at the 5' end in order to optimize
restriction site digestion and subsequent cloning of the PCR
reaction products. The 5' PCR primer (primer 1) was designed to
encode a unique Nde I restriction site.
[0563] RNA Isolation, cDNA Synthesis and Amplification
[0564] RNA was prepared from about 1.times.10.sup.8 BR96 hybridoma
cells grown in IMDM supplemented with 10% fetal calf serum (FCS).
Total RNA was used for first strand cDNA synthesis using random
hexamers at 23.degree. C. for 10 minutes in a 20 .mu.l reaction
mixture containing 1 .mu.g of total RNA, 4 MM MgCl.sub.2, 1 mM of
each dNTP, 1 unit of RNAase inhibitor (recombinant RNAase inhibitor
originally isolated from human placenta, Perkin-Elmer/Cetus,
Norwalk, Conn.), 1.times.PCR buffer (10.times.PCR Reaction
buffer=500 mM KCl, 100 mM Tris-HCl, pH 8.3), 2.5 .mu.M random
hexanucleotide (Perkin-Elmer/Cetus) and 2.5 units of reverse
transcriptase (cloned Moloney murine leukemia virus (M-MLV) reverse
transcriptase, 2.5 units/.mu.l from Perkin=Elmer/fetus). Subsequent
to hexameric primer extension with reverse transcriptase, the
reaction mixtures were incubated successively in a thermal cycler
(Eppendorf MicroCycler) at 42.degree. C. for 15 minutes, 99.degree.
C. for 5 minutes and 5.degree. C. for 5 minutes.
[0565] Amplification of V.sub.L and V.sub.H cDNAs was performed
with 35 cycles of PCR using reagents according to manufacturer's
instructions (GeneAmp RNA-PCR, Perkin-Elmer/Cetus) in two separate
tubes with 0.15 .mu.M each of either V.sub.L-5' and V.sub.L-3 or
V.sub.H-5' and V.sub.H-3' primers. Each PCR cycle consisted of
denaturation at 95.degree. C. for 1 minute followed by annealing
and extension at 60.degree. C. for 1 minute. In order to fully
extend all cDNAs, a single held extension was performed at
60.degree. C. for 7 minutes.
[0566] Cloning of Amplified cDNA
[0567] The amplified PCR products were purified on ion-exchange
mini-columns (Elutip-D, Schleicher & Schuell, Keene, N H),
concentrated by ethanol precipitation and digested with either
EcoRI and BamHI (V.sub.H gene) or Hind III and Sph I (V.sub.L
gene). Subsequently, the digested PCR reaction products were
further purified on 1.5% agarose gels (SeaKem, FMC Corp. Rockland,
Me.) and the V.sub.H or V.sub.L gene fragments separately cloned
into pEV3-SM2 (Crowl et al., Gene 38:31-38 (1985)) digested with
either EcoRI and BamHI or with Hind III and Sph I respectively.
Clones containing V gene inserts were identified by colony
hybridization using either random-primed radiolabeled V.sub.L or
V.sub.H cDNA fragments as probes. The nucleotide sequence was then
determined for several cloned V.sub.L or V.sub.H cDNA inserts,
employing primers based upstream within the lambda P.sub.L promoter
or downstream of Sal I within pBR322 sequences.
[0568] Construction of Plasmid pBW 7.0
[0569] Starting with the BR96 sFv sequence encoded by plasmid
pBR96Fv (FIG. 35, prepared by Dr. McAndrew, Bristol-Myers Squibb
Co.) a 550 bp sequence corresponding to the variable heavy and
variable light chains connected with a synthetic
(Gly.sub.4Ser).sub.3 hinge region up to the Kpn I restriction site
in the light chain, was used to PCR-amplify with primer 1 and
primer 2 described above. After PCR-amplification and digestion
with Nde I and Kpn I the 550 bp Nde I-Kpn I fragment was ligated
into a 4220 bp Nde I-Kpn I vector fragment prepared from plasmid
pMS8 described above, (supplied by Dr. Ira Pastan, N I H, Bethesda,
M D), which encodes the gene for PE40 under the transcriptional
control of the T7 promoter (Studier et al., J. Mol. Biol.
189:113-130 (1986)). The product of this ligation was an
intermediate vector designated pBW 7.01 (FIG. 35). Subsequently,
the 227 bp Kpn I fragment from pBR96 Fv was subcloned into the
unique Kpn I site of pBW 7.01. The resulting plasmid pBW 7.0 (FIG.
36), encoding the BR96 sFv-PE40 gene fusion, was confirmed by DNA
sequence analysis.
[0570] Expression and Purification of BR96 sFv-PE40
[0571] The plasmid pBW 7.0 encoding BR96 sFv-PE40 obtained as
described above was transformed into E. coli BL21 (.lambda. DE3)
cells cultured in Super Broth (Digene, Inc., Silver Springs, Md.)
containing 75 .mu.g of ampicillin per ml at 37.degree. C. When
absorbance at 650 nm reached 1.0, isopropyl
1-thiol-B-D-galactopyranoside (IPTG) was added to a final
concentration of 1 mM, and cells were harvested 90 minutes later.
Upon induction with IPTG, the E. coli cells transformed with pBW
7.0 expressed large amounts of fusion protein that was localized to
the inclusion bodies. The bacteria were washed in sucrose buffer
(20% sucrose, 30 mM Tris-HCl (pH 7.4), 1 mm EDTA) and were
osmotically shocked in ice-cold H.sub.2O to isolate the periplasm.
Subsequently, inclusion bodies were isolated away from the
spheroplast membrane proteins by extensive treatment with Tergitol
(Sigma) to remove excess bacterial proteins, followed by
denaturation in 7 M guanidine-HCl (pH 7.4), refolding in PBS
supplemented with 0.4 M L-Arginine and extensive dialysis against
0.02 M Tris, pH 7.4. Protein was purified using anion-exchange on a
Q-Sepharose column and fractions containing BR96 sFv-PE40 were then
pooled and separated by gel-filtration (on a TSK-3000 column)
chromatographies with a Pharmacia fast protein liquid chromatograph
(FLPC) system as described by Siegall et al., Proc. Natl. Acad.
Sci. USA 85:9738-9742 (1988).
[0572] The chromatographic profile of the size exclusion column
indicated the presence of two major species (FIG. 37A). The first
species eluted between gel filtration standards of 660 kD and 158
kD and represents an aggregated form of the recombinant protein
(fractions 9-14). The second species (fractions 15-21) represents
the 67 kDa monomeric form of BR96 sFv-PE40 which, as expected,
eluted between the 158 kDa and 44 kDa standards. These results were
confirmed by reducing (FIG. 37B) and non-reducing SDS-PAGE analysis
(FIG. 37C). Whereas FIG. 37B shows the purification profile based
on Coomassie staining, FIG. 37C shows immunoblot analysis using
anti-idiotypic BR96 antibody. The above data demonstrate that
purification yielded two forms of recombinant protein, monomers and
aggregates.
[0573] Direct Lewis Y Determinant Binding_ELISA
[0574] Because BR96 sFv-PE40 is monovalent it provides only one
antigen-binding site per molecule. In order to test the relative
binding activities of monovalent BR96 sFv-PE40 compared to the
bivalent BR96 antibody, a direct binding assay was performed in
which purified Lewis Y was coated on ELISA plates and the
recombinant BR96 sFv-PE40 molecule was compared, in its ability to
bind to the antigen, with several antibodies and antibody
fragments. Lewis-Y (ChemBiomed, Alberta, Canada) was diluted to 0.2
.mu.g/ml in Coating Buffer (100 mM sodium carbonate/bicarbonate, pH
9.4) prior to coating Dynatech Immunon II plates and incubating for
16 hours at 4.degree. C. Excess antigen was removed and the plates
were blocked with PTB buffer (PBS containing 0.05% Tween 20 and 1%
BSA) for 1 hour at room temperature followed by 3 washes with PTB.
The antibody samples were serially diluted in PTB to a final
concentration ranging from 1.25 .mu.g/ml to 80 .mu.g/ml and
incubated overnight at 4.degree. C. on the plate in a volume of 50
.mu.l/well. The plates were washed 3 times with PTB buffer and each
well was incubated with 100 .mu.l/well of biotinylated BR96
anti-idiotypic antibodies (2.56 .mu.g/ml) in PTB for 1 hour at room
temperature. The plates were then washed 4 additional times with
PTB. Alkaline phosphatase-conjugated streptavidin (Kirkegaard &
Perry Labs, Gaithersburg, Md.) was added to each well (100 .mu.l of
0.5 .mu.g/ml in PBS containing 1% BSA) and incubated for 1 hour at
37.degree. C. Plates were washed 3 times with PTB and 3 times with
phosphatase buffer (75 M Tris, 0.1 M NaCl, 5 mM MgCl.sub.2, pH 9.4)
and reacted with p-nitrophenyl phosphate (1 mM in phosphatase
buffer) for 30 to 60 minutes at 37.degree. C. The reaction was
stopped by the addition of 2 N NaOH. The plates were read at 405 nm
on a Molecular Device, Inc. (Menlo Park, Calif.) microplate
Reader.
[0575] In comparison with BR96 IgG, monomeric BR96 sFv-PE40 bound
approximately 5-fold less well (FIG. 38). In contrast, the
aggregated form of BR96 sFv-PE40 was unable to bind to the Lewis-Y
determinant. L6 IgG, an antibody that does not bind the BR96
antigen, was used as a negative control.
[0576] In addition, the competitive binding ability of BR96
sFv-PE40 was compared with BR96 IgG. Microtiter plates were coated
with Lewis-Y antigen as described above. Antibody samples were
diluted in PBS containing 1% BSA to final concentrations ranging
from 1.36 .mu.g/ml to 175 .mu.g/ml. .sup.125I-BR96 IgG was added to
each sample (5 .mu.Ci/ml) along with antibody competitor to a final
volume of 100 .mu.l. The entire mixture of radiolabeled BR96 IgG
and antibody competitor were added to the Lewis-Y coated plates and
incubated for 2 hours at 37.degree. C. The plates were washed five
times with PBS containing 0.05% Tween-20 and the wells counted on a
gamma counter. This assay, which also used Lewis-Y coated plates,
measured the amount of bound radioiodinated BR96 IgG when compared
with various amounts of BR96 sFv-PE40 or BR96 IgG.
[0577] The results of the competitive binding assay were that BR96
sFv-PE40 competed 5-fold less well than BR96 IgG (FIG. 39) which
correlates with the direct binding data in FIG. 38. The addition of
L6 IgG, which did not compete for binding, demonstrates the
specificity of this assay.
[0578] Cytotoxicity of BR96 sFv-PE40 Against Cancer Cells
[0579] To determine the cytotoxic potential of monomeric BR96
sFv-PE40 the effect of the single-chain immunotoxin was compared to
that of the chemical conjugate, ChiBR96-LysPE40 on MCF-7 breast
carcinoma cells measured as inhibition of protein synthesis (FIG.
40). Determination of inhibition of protein synthesis was as
follows:
[0580] All cell lines were cultured as monolayers at 37.degree. C.
in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM
L-glutamine and 50 units/ml penicillin/streptomycin. Tumor cells
were plated onto 96-well flat bottom tissue culture plates
(1.times.10.sup.4 cells/well) and kept at 37.degree. C. for 16
hours. Dilutions of immunotoxin were made in growth media and 0.1
ml added to each well for 20 hours at 37.degree. C. Each dilution
was done in triplicate. The cells were pulsed with
[.sup.3H]-leucine (1 .mu.Ci/well) for an additional 4 hours at
37.degree. C. The cells were lysed by freeze-thawing and harvested
using a Tomtec cell harvester (Orange, Conn.). Incorporation of
[.sup.3H]-leucine was determined by a LKB Beta-Plate liquid
scintillation counter.
[0581] For competition experiments, tumor cells were prepared as
described above. BR96 IgG or, as a control, L6 IgG was diluted to
100 .mu.g/ml in growth media before addition to the cell monolayer
(0.1 ml/well). After incubation at 37.degree. C. for 1 hour,
dilutions of BR96 sFv-PE40 were added, incubated an additional
hour, cell supernatants were removed, and cells were washed with
complete RPMI growth media. Growth media (0.2 ml) was added to each
well, cells were incubated at 37.degree. C. for 20 hours and were
labelled with [.sup.3H]-leucine as described above.
[0582] The results indicate that the single-chain immunotoxin was
3-fold more potent than the conjugate, with ID.sub.50 values of 4
and 12 pM, respectively.
[0583] Next, in order to correlate the cytotoxicity with the
presence of the BR96 antigen, the relative antigen density was
determined on five tumor cell lines by FACS analysis (FIG. 41).
Assays were performed by fluorescence as described by Hellstrom et
al., Cancer Res. 50:2183-2190 (1990). Briefly, target cells were
harvested in logarithmic phase with EDTA (0.02%) in calcium- and
magnesium-free PBS. The cells were washed twice in PBS containing
1% BSA and resuspended to 1.times.10.sup.7 cells/ml in PBS
containing 1% BSA and 0.02% NaN.sub.3. Cells (0.1 ml) were mixed
with BR96 or a human IgG control (0.2 ml at 50 .mu.g/ml) and
incubated for 45 minutes at 4.degree. C. The cells were washed 2
times and resuspended in 0.1 ml of an appropriate concentration of
FITC labelled rabbit anti-human IgG (Cappel, Malvern, Pa.). Cells
were incubated for 30 minutes at 4.degree. C., washed 2 times in
PBS containing 0.02% NaN.sub.3 and analyzed on a Coulter EPICS 753
fluorescence-activated cell sorter. Data are expressed as the
fluorescence intensity of cells reacted with BR96 minus cells
reacted with control antibody. On a logarithmic scale, 25 units of
fluorescence intensity represents a doubling of antigen density.
FACS analysis of a non-specific human IgG antibody was performed
for each cell line to determine non-specific fluorescence and a
fluorescence intensity was calculated (Table 11).
13TABLE 11 CYTOTOXICITY OF BR96 sFv-PE40 ON VARIOUS CELL LINES BR96
ID.sub.50 Fluorescence ID.sub.50 Cell Line Cancer Type Intensity
ng/ml (pM) MCF-7 Breast 177.8 0.3 (4.4) L2987 Lung 172.8 5.0 (75)
RCA Colon 138.5 8.0 (119) A2780 Ovarian 103.2 50.0 (750) KB
Epidertnoid 33.6 500.0 (7,462) ID.sub.50 is the amount of BR96
sFv-PE40 required to inhibit 50% of protein synthesis as determined
by [.sup.3H]-leucine incorporation. BR96 Fluorescence intensity is
the specific BR96 fluorescence intensity minus non-specific human
IgG fluorescence.
[0584] When the cytotoxic potential of BR96 sFv-PE40 was tested on
the cell lines, it was found that inhibition of protein synthesis
correlated with BR96 antigen density (Table 11). For example, MCF-7
cells were the most sensitive to BR96 sFv-PE40 (ID.sub.50 of 4.4
pM) of the cell lines tested. In contrast, KB cells which display
negligible amounts of the BR96 antigen were much less sensitive to
BR96 sFv-PE40 (ID.sub.50 of 7,462 pM). The cytotoxic activity of
monomeric and aggregated BR96 sFv-PE40 was also compared, and the
monomer was demonstrated to be approximately 50-60 times more
effective at inhibiting protein synthesis than the aggregate
population with ID.sub.50 values on L2987 cells of 75 pM and 2920
pM, respectively.
[0585] The competitive cytotoxicity experiments were conducted to
confirm the specificity of the immunotoxin for its antigen binding
site (FIG. 42). The cytotoxic effect of BR96 sFv-PE40 is due to
specific antigen binding, because the effect is severely reduced by
excess BR96 IgG, but not by L6 IgG, which does not recognize the
BR96 antigen,.
[0586] Comparative Blood-Level Lifetime Analysis of
BR96-Immunotoxins
[0587] BR96 sFv-PE40 is approximately one-third the size of the
immunotoxin conjugate, ChiBR96-LysPE40. Because protein size can
affect biological kinetics, the difference in blood half-life
between BR96 sFv-PE40 and ChiBR96-LysPE40 was measured. Both
immunotoxins were radioiodinated and administered to athymic mice
via their tail vein using the following procedures.
[0588] BR96 sFv-PE.sup.40 and ChiBR96-LysPE40 were labelled with Na
.sup.125I using Chloramine T (McConahey et al., Arch. Allergy Appl.
29:185-188 (1966)). Each reaction contained 100 .mu.g of
immunotoxin in PBS, 1 .mu.Ci of Na .sup.125I, and 10 ng/ml
chloramine T in a total reaction volume of 100 .mu.l.
[0589] After a five minute incubation at room temperature, the
reaction was terminated by addition of 20 ng/ml Na-metabisulfide.
The free Na .sup.125I was separated from the radiolabeled
immunotoxin by gel filtration through PD-10 columns (Pharmacia).
The specific activity of both immunotoxins was approximately 10
.mu.Ci/.mu.g.
[0590] Female athymic mice (nu/nu) were purchased from Harlan
Sprague Dawley (Indianapolis, Ind.) at 4-6 weeks of age. The
animals were intravenously injected via the tail vein with 10
.mu.Ci of .sup.125I-BR96 sFv-PE40 or .sup.125I-ChiBR96-LysPE40. The
animal (2-4/data point) were sacrificed at various time points and
the blood was collected and counted in a gamma counter. The percent
(%) ID for the blood was determined as (CPM detected/CPM
injected).times.100. ID/ml was calculated assuming a 1.6 ml total
blood volume. Results are shown in Table 12.
14TABLE 12 SINGLE-CHAIN IMMUNOTOXIN VS. CHEMICAL CONJUGATE
IMMUNOTOXIN COMPARATIVE BLOOD LEVEL ANALYSIS BR96 sFv-PE40
ChiBR96-LysPE40 Time % ID/ml Blood % ID/ml Blood 5 minutes 49.8
57.5 15 minutes 43.3 54.8 30 minutes 28.2 46.3 60 minutes 15.5 41.5
2 hours 8.6 23.4 4 hours 5.2 22.0 6 hours 2.5 20.5 24 hours 0.2 7.2
48 hours 0.1 3.6
[0591] BR96 sFv-PE40 clears from the blood faster than
ChiBR96-LysPE40. The estimated half-life in the blood for the
single-chain immunotoxin is approximately 30 minutes as compared to
almost 2 hours for the chimeric BR96 immunotoxin conjugate. In this
experiment, the measurement of .sup.125I-labelled BR96 immunotoxin
in the blood determined how much of the molecule was present.
[0592] In order to measure the amount of detectable single-chain
immunotoxin that was biologically active, the blood was assayed for
BR96 sFv-PE40 directed cytotoxic activity at the various times
indicated in Table 12.
[0593] The results in this Example provide an expression plasmid
for the production of a single-chain immunotoxin composed of the
carcinoma-reactive antibody of the invention, BR96 and a truncated
form of Pseudomonas exotoxin. The chimeric molecule, BR96 sFv-PE40,
purified from E. coli exists in both a monomeric and an aggregated
form. The specificity of the monomeric BR96 sFv-PE40 for its
antigen was confirmed through a competition analysis with BR96 IgG.
The FACS analysis of five different cell lines demonstrates the
distribution of the BR96 antigen, and the cytotoxic potential of
BR96 sFv-PE40 was correlated with the relative number of antigen
expressed on the surface of the target cells. BR96 sFv-PE40 is
extremely potent against cancer cells displaying the BR96 antigen,
with MCF-7 cells being the most sensitive cell line examined. BR96
sFv-PE40 was shown to be more potent than the BR96 IgG chemical
conjugate against the tumor cell lines tested. To assess the
potential anti-tumor activity of BR96 sFv-PE40, the chimeric toxin
was intravenously administered to mice and found to have a serum
half-life of 30 minutes, as compared to that of ChiBR96-LysPE40,
which was almost 2 hours. It may be an advantage that the
single-chain immunotoxin is cleared so rapidly from the blood. The
immununotoxin molecules are stable and retain biological activity
following administration into animals.
EXAMPLE 15
[0594] In vivo Effects of BR96 sFv-PE40
[0595] Anti-tumor Activity of BR 96 sFv-PE40 Against Human Tumor
Xenographs
[0596] L2987 and MCF-7 tumor fragments were implanted into female
athymic mice (nu/nu) (Harlan Sprague Dawley, Indianapolis, Ind.) at
4-6 weeks of age. They were implanted with L2987 and MCF-7 tumor
fragments from established tumor xenografts that were approximately
4 weeks old (800 cu mm). Tumor sections were implanted
subcutaneously using a trocar onto the back hind quarter of the
mice. Two weeks after implantation the animals were randomized and
their tumors measured.
[0597] For the anti-tumor experiments, we only used animals that
had tumors ranging from 50-100 cubic mm in size. The animals were
intravenously injected via the tail vein with the BR96 sFv-PE40
inmunotoxin according to the administration schedule indicated in
FIGS. 43 and 44. Each treatment group consisted of five to ten
animals.
[0598] Regression of MCF-7 breast carcinoma xenografts was observed
with doses up to 0.75 mg/kg using administration schedules of Q4DX3
(FIG. 43). Using an administration schedule of Q2DX5, the L2987
lung tumors were observed to regress upon treatment with BR96
sFv-PE40 (FIG. 44). Complete regression of the tumor xenografts was
observed at doses ranging from 0.375 mg/kg to 0.125 mg/kg.
[0599] The effect of the tumor xenografts was dose-dependent, as at
lower doses 5 the tumors were able to grow back after being
regressed for a seven day period while at higher doses the complete
regression lasted for over twenty days.
[0600] In untreated animals, the tumors grew rapidly and the
animals were sacrificed approximately 30 days post implantation. No
apparent toxicity was observed at the doses used in this
experiment.
[0601] The tumor xenografts used in this study emanated from a
small piece of a solid tumor excised from another animal. The tumor
tissue was subcutaneously implanted and allowed to vascularize and
grow before treatment was initiated. In this manner, the data
presented herein demonstrate a tumor model of tumors found in
humans.
EXAMPLE 16
[0602] Materials and Methods
[0603] Animals
[0604] Athymic mice and athymic Rowett rats (Harlan Sprague Dawley)
were used in this study.
[0605] The binding of BR96 to normal rat tissues was similar to
BR96 binding to normal human tissues, i.e., BR96 bound to cells in
the esophagus, stomach, intestine and acinar cells of pancreas.
[0606] In contrast to rats, normal tissues from athymic mice did
not bind BR96.
[0607] BR96-DOX
[0608] The conjugates were prepared by linking the DOX derivative
maleimidocaproyl doxorubicin hydrazone to BR96 or control
immunoglobulin (FIG. 45). For more detail, see Examples 20, 22 and
26.
[0609] Implanted Carcinoma Cells
[0610] L2987 lung carcinoma cells were selected in vitro for the
ability to grow as multicellular spheroids. When injected IV into
athymic mice or rats, tumors developed at various sites, including
lymph nodes, lung, spleen, liver, brain, subcutaneously, and
ascites was formed in some animals.
[0611] Athymic rats were transplanted subcutaneously with human
lung adenocarcinoma L2987, colon carcinoma RCA, or breast carcinoma
MCF7 and permitted to grow. Therapy (3 treatments 4 days apart)
started 14 to 28 days after tumor transplantation when tumors were
well established, i.e., when the tumors were 50 to 100 mm.sup.3 in
size.
[0612] Administration of BR96 Into The Animals
[0613] Mice and rats were administered with BR96-DOX by three
injections, each injection being about four days apart. Mice which
were injected intraperitoneally (IP) were given 20 mg/kg of
BR96-DOX. Mice which were injected intravenously were given 10
mg/kg of BR96. Intravenous (IV) injection involved less volume of
BR96 because of the constraints of injection volume, namely, only
10 mg/kg was administrable by IV.
[0614] At the doses tested, there was no difference in the
anti-tumor activity of BR96-DOX whether administered IP or IV.
[0615] Controls
[0616] One set of controls included untreated mice and mice that
received (1) DOX (at doses optimized to produce maximal antitumor
activity in each model); (2) unconjugated BR96; (3) mixtures of
BR96 and DOX; and (4) DOX conjugated to either normal human IgG or
the control MAb SN7. Doses of DOX and MAb are presented as
mg/kg/infection.
[0617] Another set included rats using the protocol used in control
mice.
[0618] Results of Treatment of Mice
[0619] Treatment with BR96-DOX consistently cured most mice bearing
L2987 (FIG. 46A) or RCA (FIG. 46B) tumors. Further, mice treated
with BR96-DOX exhibited complete and partial tumor regressions
against MCF7 tumors (FIG. 46C). Complete tumor regression (CR)
refers to a tumor that for a period of time is not palpable.
Partial tumor regression (PR) means a decrease in tumor volume to
.ltoreq.50% of the initial tumor volume.
[0620] Specifically, BR96-DOX cured 78% of the treated mice. In
contrast, DOX alone was not active against established RCA tumors
either in terms of tumor growth delay or regressions.
[0621] The MTD of free DOX (4 mg/kg administered as 3 injections 4
days apart) resulted in a delay in tumor growth and 25% cures.
However, BR96-DOX given at a matching DOX dose (4 mg/kg DOX, 140
mg/kg BR96) cured 100% of the animals.
[0622] FIGS. 46A-D are line graphs showing the antigen-specific
anti-tumor activity of BR96-DOX. FIG. 46A shows mice transplanted
with L2987 lung tumor xenografts which have grown to about 50 to
100 mm.sup.3 at the initiation of therapy. Treatment with BR96-DOX
consistently cured most mice bearing L2987 (FIG. 46A).
[0623] FIG. 46B shows mice transplanted with colon carcinoma RCA
which have grown to tumor xenografts of about 50 to 100 mm.sup.3 at
the initiation of therapy. Treatment with BR96-DOX consistently
cured most mice bearing colon carcinoma RCA (FIG. 46B).
[0624] FIG. 46C shows the efficacy of BR96-DOX in mice transplanted
with MCF7 tumors. These mice treated with BR96-DOX consistently
exhibited complete and partial tumor regressions against MCF7
tumors (FIG. 46C).
[0625] Equivalent doses of non-binding IgG-DOX or SN7-DOX had no
effect against these tumors. Although optimal doses of DOX delayed
the growth of small L2987 tumors (50 to 100 mm.sup.3) and MCF7
tumors; regressions or cures were not observed.
[0626] FIG. 46D shows mice transplanted with L2987 lung tumor
xenografts which grew to about 250 to 800 mm.sup.3 at the
initiation of therapy. Such mice which were treated with BR96-DOX
also exhibited 56% cures, 22% complete and 22% partial regressions
of lung tumors. In contrast, antitumor activity was not observed
after treatment with an optimal dose of DOX.
[0627] FIG. 47 shows that BR96 is efficacious in curing athymic
mice having large disseminated tumors.
[0628] Mice were inoculated IV with L2987 spheroids. Approximately
twelve weeks later mice (14 mice/group) were selected for treatment
with BR96 (8 mg/kg equivalent DOX administered as 3 injections 4
days apart) or DOX on the basis of visible tumor burden, i.e.,
therapy was delayed until mice displayed extensive disseminated
disease, .gtoreq.0.5 g of visible tumor burden.
[0629] The burden of disseminated disease in these animals was so
far advanced that 50% of control animals died during the first 6
days of the experiment. The median survival time (MST) of the
control group was 90 days and 100% of the mice were dead by day
102. Surviving mice were sacrificed 200 days after cell inoculation
and sections of lung, lymph nodes, spleen, colon, jejunum, kidney,
liver, brain, and heart were examined by histology.
[0630] Mice inoculated with L2987 spheroids and treated had an
increased MST (MST of >200 days) relative to that of control
mice (MST of 85 days) or mice treated with an optimal dose of DOX
(MST of 140 days).
[0631] According to immunohistology, the degree of binding of BR96
to cells from these carcinoma lines was similar to that of biopsy
material from human carcinomas of the same respective types
(21).
[0632] Table 13 summarizes the tumor regression rates following
treatment with various doses of (1) BR96-DOX, (2) DOX, and (3)
mixtures of MAb and DOX against established L2987 and RCA tumor
xenografts in mice.
[0633] BR96-DOX administered at equivalent DOX doses of .gtoreq.5
mg/kg (3 injections 4 days apart) produced long-term cures in 72 to
100% of mice (n=291) bearing L2987 tumors.
[0634] In the RCA colon tumor model, which was not sensitive to
unconjugated DOX, BR96-DOX administered at equivalent DOX doses of
.gtoreq.10 mg/kg (3 injections 4 days apart) cured 72 to 100% of
mice (n=48).
[0635] Mice cured of L2987 or RCA tumors remained alive and tumor
free for more than 1 year with no indication of side effects.
15TABLE 13 ANTITUMOR ACTIVITY OF BR96-DOX AGAINST ESTABLISHED HUMAN
TUMOR XENOGRAFTS Dose (mg/kg/injection) PERCENT TUMOR REGRESSIONS
No.: Treatment Schedule DOX MAb BR96* Tumor Cures Complete Partial
MICE L2987 BR96-DOX q4dx3.sup.+ 20.0 689 100 0 0 8 15.0 711 .+-. 36
83.0 .+-. 0.8 3.3 .+-. 0.9 7.0 .+-. 0.9 29 10.0 513 .+-. 12 83.0
.+-. 1.1 8.0 .+-. 0.7 2.0 .+-. 0.4 100 8.0 317 .+-. 3 88.5 .+-. 0.1
3.7 .+-. 1.0 0 27 5.0 246 .+-. 5 72.3 .+-. 2.2 17.9 .+-. 1.5 5.6
.+-. 0.7 117 2.5 109 .+-. 3 30.4 .+-. 3.4 33.7 .+-. 2.4 21.3 .+-.
2.6 62 1.25 49 .+-. 1 6.9 .+-. 0.9 11.6 .+-. 1.2 11.9 .+-. 2.1 44
BR96-DOX q1dx1.noteq. 30.0 1078 50.0 50.0 0 10 25.0 930 30.0 30.0
40.0 10 20.0 735 60.0 20.0 10.0 10 15.0 540 11.0 22.0 44.0 9
IgG-DOX q4dx3 10.0 403 .+-. 5.2 0 0 0 19 5.0 202 .+-. 3.2 0 0 3.7
.+-. 1.0 27 DOX q4dx3 8.0 -- 0 0 0.8 .+-. 0.8 125 Mab BR96 q4dx3 --
400 0 0 0 8 -- 200 0 0 0 8 -- 100 0 0 0 8 BR96 + DOX q4dx3 8.0 400
0 0 0 9 8.0 200 0 0 0 9 8.0 100 0 0 0 9 RCA BR96-DOX q4dx3 20.0 903
100 0 0 10 15.0 625 80.0 10.0 10.0 10 10.0 376 .+-. 5.4 71.7 .+-.
0.9 0 10.7 .+-. 0.1 28 5.0 176 11.0 22.0 11.0 9 2.5 90 0 0 5.5 .+-.
1.3 18 BR96-DOX q7dx3.sup.@ 20.0 900 100 0 0 10 15.0 625 100 0 0 10
10.0 420 80 10 0 10 5.0 210 10 0 10 10 IgG-DOX q4dx3 10.0 405 0 0 0
10 DOX q4dx3 8.0 -- 0 0 0 29 DOX q7dx3 10.0 -- 0 0 0 10 *Mean .+-.
SEM .sup.+3 injections administered with a 4-day interval
.noteq.single injection .sup.@3 injections administered with a
7-day interval
[0636] BR96 administered at equivalent doses was not active against
established tumors (either in terms of tumor growth delay or
regressions) and the tumor growth delay produced by mixtures of
BR96 and DOX was equivalent to that of DOX administered alone.
[0637] Contrary to our expectations, cells lacking BR96 expression
were not detected after treatment with BR96-DOX. Also, cells
obtained from tumors that grew back after BR96-DOX induced
regression were as sensitive in vitro to DOX as the parental cell
line.
[0638] The IC.sub.50 (concentration required to produce 50%
inhibition of .sup.3[H]-thymidine incorporation) was 0.4.+-.0.1
.mu.M and 0.3.+-.0.2 .mu.M DOX for treatment and parental,
respectively. These cells were also as sensitive to BR96-DOX as the
parental cell line with IC.sub.50 values of 2.7.+-.0.5 .mu.M and
2.6.+-.0.8 .mu.M equivalent DOX for parental and treated,
respectively.
[0639] These data suggest that it may be possible to successfully
retreat tumors with several rounds of BR96-DOX therapy.
[0640] The maximum tolerated dose (MTD) (equivalent DOX dose) of
the BR96-DOX conjugate (administered as 3 injections 4 days apart)
was 20 mg/kg administered intraperitoneally (IP). When administered
intravenously (IV) the MTD was .gtoreq.10 mg/kg. This was the
maximum dose that could be administered IV because of the
constraints of injection volume.
[0641] At the doses tested, there was no difference in the
antitumor activity of BR96-DOX whether administered IP or IV. At
doses of BR96-DOX (Table 1) equivalent to .gtoreq.mg/kg of DOX
(.gtoreq.250 mg/kg BR96) more than 70% of treated animals were
cured of established L2987 tumors.
[0642] In fact, the BR96-DOX (Table 13) equivalent to .gtoreq.5
mg/kg of DOX (>250 mg/kg BR96) more than 70% of treated animals
were cured of established L2987 tumors. The BR96-DOX conjugate was
active at doses as low as 1 mg/kg equivalent DOX. Therefore, the
BR96-DOX conjugate was active at a dose equivalent to {fraction
(1/20)}th of its MTD.
[0643] These data demonstrate the broad range of therapeutic doses
which were achieved with BR96-DOX. The MTD of unconjugated DOX (8
mg/kg IV and 4 mg/kg TP) was lower than that of the BR96-DOX
conjugate. Unconjugated DOX administered IV at the MTD produced a
delay in tumor growth but no tumor regressions and if the dose was
reduced to 50% of the MTD, DOX had no effect.
[0644] In contrast, activity equivalent to that of an optimal dose
of DOX (8 mg/kg) was achieved at a dose of 1 mg/kg of BR96-DOX. The
BR96-DOX conjugate produced antitumor activity comparable to that
of an optimal dose of unconjugated DOX at 1/8th of the equivalent
DOX dose. In summary, the BR96-DOX conjugate was more active,,had a
much broader range of therapeutic doses, and was more potent than
unconjugated DOX.
[0645] Seven of the 8 surviving mice were free of detectable tumor
(70% cures by combined life span and histologic examination).
[0646] The BR96-DOX conjugate demonstrated strong antitumor
activity in all preclinical models evaluated. The efficacy and
potency of BR96-DOX conjugates is likely due to several factors.
The antigen to which BR96 binds is abundantly expressed at the
tumor cell surface and active drug is released following
antigen-specific binding and internalization of the conjugate into
the acidic environment of lysosomes/endosomes.
[0647] Acid-labile immunoconjugates, in which a less stable
disulfide linker was used, have been investigated previously (7,
26). Although these conjugates were active in an antigen-specific
manner, they had poor potency in vivo (7). The use of a more stable
thioether linker, and a MAb with higher avidity and more rapid
rates of internalization, improved the activity and potency of
BR96-DOX conjugates and also increased the range of therapeutic
doses.
[0648] We showed that administration of BR96-DOX conjugate at
cumulative doses of at least 15 mg/kg DOX and 700 mg/kg MAb
(equivalent to 45 Mg/M.sup.2 DOX and 2100 mg/M.sup.2 MAb) resulted
in greater than 70% cures of established lung tumors. This dose of
MAb in mice is approximately equivalent to a cumulative dose of 3 g
of MAb per patient and is only slightly higher than that required
to achieve saturation of human carcinomas in patients given L6,
another anticarcinoma MAb (G. Goodman et al., J. Clin. Oncol., 8,
1083 (1990).
[0649] It would be clear to those skilled in the art that the
optimal schedule for administering BR96-DOX will vary based upon
the subject, the subject's height and weight, the severity of the
disease.
[0650] The demonstration of tumor cures in animals in which BR96
binds to normal tissues highlights the fact that the appropriate
combination of MAb, drug, and linker chemistry are critical aspects
to successful antibody-directed therapy. The toxic effects of DOX
are dose related and it is likely that increasing the intra-tumoral
concentration of DOX will produce a significant increase in
antitumor activity (S. K. Carter, J. Natl. Cancer Inst. 55, 1265
(1975); R. C. Young, R. F. Ozols, C. E. Myers, N. Eng. J. Med. 305,
139 (1981)).
[0651] BR96-DOX induced complete regressions and cures of
xenografted human lung, breast and colon carcinomas growing
subcutaneously in athymic mice and cured 70% of mice bearing
extensive metastases of a human lung carcinoma.
[0652] Results of Treatment with Rats
[0653] The MTD of free DOX (4 mg/kg administered as 3 injections 4
days apart) resulted in a delay in tumor growth and 25% cures.
[0654] The MTD of free DOX (4 mg/kg administered as 3 injections 4
days apart) resulted in a delay in tumor growth and 25% cures.
However, BR96-DOX given at a matching DOX dose (4 mg/kg DOX, 140
mg/kg BR96) cured 100% of the animals, and a dose equivalent to 2
mg/kg DOX (70 mg/kg BR96) cured 88% of the rats.
[0655] BR96-DOX given at a matching DOX dose (4 mg/kg DOX, 140
mg/kg BR96) cured 100% of the animals, and a dose equivalent to 2
mg/kg GOX (70 mg/kg BR96) cured 88% of the rats. Of the rats
treated with BR96-DOX, 94% (15/16) remained alive and tumor free
with no evidence of toxicity 150 days after the last dose of
BR96-DOX.
[0656] It is surprising that BR96-DOX also cured 94% of athymic
rats with subcutaneous human lung carcinoma, even though the rats,
like humans, in contrast to mice, express the BR96 target antigen
in some normal tissues.
[0657] The BR96-DOX conjugate demonstrated antigen-specific
activity in vitro and was 8 to 25 fold more potent than non-binding
(IgG-DOX or SN7-DOX) conjugates against carcinoma lines that
expressed the BR96 antigen. BR96-DOX was much less active against
cells that did not bind BR96.
[0658] Optimal doses of DOX (8 mg/kg) had no effect on the large
disseminated tumors; the MST was 94 days and 100% of the mice were
dead by day 140. In contrast, mice treated with BR96-DOX (8 mg/kg)
had a MST of .gtoreq.200 days and 8 of the 10 animals survived for
the duration of the experiment.
EXAMPLE 17
[0659] Conjugate of SPDP Thiolated Monoclonal Antibidy BR64 with
the Maleimidocaproyl Hydrazone of Adriamycin
[0660] A solution of the BR64 antibody (25 mL, 10.37 mg/mL;
determined by UV at 280 nm, 1.4 absorbance units equal 1 mg
protein) was treated with SPDP solution in absolute ethanol (1.3 mL
of 10 mmol solution). The solution was incubated for 1 hour at
31.degree.-32.degree. C., then chilled in ice and treated with a
solution of DTT in phosphate buffered saline ("PBS") (1.3 mL of a
50 mmol solution). The solution was kept in ice for 1 hour then
transferred to a dialysis tube and dialyzed three times against PBS
(2 L per dialysis) for a period of at least 8 hours. After the
dialysis, the concentration of protein was measured, as above,
followed by a determination of molar concentration of free
sulfhydryl groups by the Ellman method.
[0661] The thiolated protein (3 mL) was treated with an equivalent
thiol molar amount of maleimidocaproyl hydrazone of adriamycin,
prepared as in Preparation 2, dissolved in dimethylformamide (DMF)
(5 mg/mL, 0.131 mL) and the mixture was incubated at 4.degree. C.
for 24 hours. The solution was dialyzed three times against PBS
(1000 mL) for a period of at least 8 hours. The solution was
centrifuged and the supernatant was shaken for a few hours with
Bio-beads.TM. SM-2 (non-polar neutral macroporous polystyrene
polymer beads, Bio-Rad Laboratories, Richmond, Calif. 94804) and
finally filtered through a Millex-GV (Millipore Corporation,
Bedford, Mass. 01730) 0.22 .mu.m filter unit. The overall average
number of molecules of adriamycin per molecule of antibody ("MR")
was determined by measuring the amount of adriamycin from the
absorption at 495 nm (.epsilon.=8030 cm.sup.-1M.sup.-1) and the
amount of protein from the absorption at 280 nm after correcting
for the absorption of adriamycin at 280 nm according to the
formula: 2 Antibody ( mg / mL ) = A 280 - ( 0.724 .times. A 495 )
1.4
[0662] The MR found for the product was 5.38; free adriamycin
0.14%; protein yield 60%.
EXAMPLE 18
[0663] Conjugate of SPDP Thiolated BR64 with the Maleimidocaproyl
Hydrazone of Adriamycin
[0664] A solution of the BR64 antibody (405 mL, 11.29 mg/mL) was
stirred and treated with SPDP solution in absolute ethanol (22.3 mL
of 10 mmol solution). The solution was incubated for 1 hour at
31.degree.-32.degree. C. while being gently shaken, then cooled in
ice to 4.degree. C., stirred and treated with a solution of DTT in
PBS (22.3 mL of a 50 mmol solution). The solution was kept in ice
for 1 hour then divided into 2 equal parts, each transferred to a
dialysis tube and dialyzed six times against PBS (6 L per dialysis)
for a period of at least 8 hours. After that the contents of the
tubes were combined (400 mL) and the concentration of protein and
free thiol groups was determined (molar ratio of --SH groups to
protein is 8.5).
[0665] The solution of thiolated protein was stirred and treated
with an equivalent thiol molar amount of maleimidocaproyl hydrazone
of adriamycin dissolved in DMF (5 mg/mL, 35.7 mL) and the mixture
was incubated at 4.degree. C. for 24 hours. The solution was
divided into 2 equal parts, transferred to dialysis tubes and
dialyzed five times against PBS (6 L per dialysis) for a period of
at least 8 hours. The contents of the dialysis tubes were combined,
filtered through a 0.22.mu. cellulose acetate filter, and the
filtrate was shaken for 24 hours with Bio-beads.TM. SM-2 (Bio-Rad
Laboratories, Richmond, Calif. 94804) the solution was filtered
through a 0.22.mu. cellulose acetate filter. The concentration of
protein and adriamycin was determined (6.26 mg/mL and 162.4
.mu.g/mL, respectively) yielding a molar ratio (MR) of 7.18. The
protein yield was 77%. Unconjugated adriamycin present was
0.07%.
EXAMPLE 19
[0666] Conjugate of STDP Thiolated SN7 with the Maleimidocaproyl
Hydrazone of Adriamycin
[0667] In a manner analogous to that described in Examples 17 and
18, monoclonal antibody SN7, an antibody which does not bind to the
antigen recognized by BR64, was thiolated with SPDP and reacted
with the maleimidocaproyl hydrazone of adriamycin to yield a
conjugate with a molar ratio (MR) of 4. Protein yield was 51%.
Unconjugated adriamycin present was 0.36%.
EXAMPLE 20
[0668] Conjugate of STDP Thiolated ChiBR96 with the
Maleimidocaproyl Hydrazone of Adriamycin
[0669] A solution of chimeric BR96 antibody, ChiBR96, (27.5 mL,
12.53 mg/mL) was treated with a 10 mM solution of SPDP in absolute
ethanol (1.7 mL). The solution was incubated at 31.degree. C. for
35 minutes, chilled in ice and treated with a 0.50 mM solution of
DTT in PBS (1.7 mL) for 15 min at 4.degree. C. The solution was
transferred to a dialysis tube and dialyzed four times in PBS-0.1 M
histidine buffer (4.5 L per dialysis) for a period of at least 8
hours. The amount of protein and molar concentration of thiol
groups was determined (9.29 mg/mL and 2.06.times.10.sup.-4 M;
respectively). The solution (17 mL) was treated with an equivalent
molar amount of the maleimidocaproyl hydrazone of adriamycin in DMF
(5 mg/mL, 0.59 mL) and the reaction mixture incubated at 4.degree.
C. for 24 hours. The reaction mixture was dialyzed three times, in
the same buffer (4.5 L per dialysis), for at least 8 hours. The
dialyzed solution was centrifuged and the supernatant shaken gently
with BioBeads.TM. SM-2 (Bio-Rad Laboratories, Richmond, Calif.
94804) for a few hours at 4.degree. C. The solution was centrifuged
and the concentration of protein and adriamycin in the supernatant
(19 mL) was determined (6.5 mg/mL and 67.86 .mu.g/mL,
respectively). The molar ratio of drug to protein is 2.9. Protein
yield is 72%; unconjugated adriamycin present is 1.2%.
EXAMPLE 21
[0670] Conjugation of Modified Bombesin with the Maleimidocaproyl
Hydrazone of Adriamycin
[0671] Bombesin does not contain a free reactive sulfhydryl group
which can be used to link the drug through the Michael Addition
Receptor-containing linker. Thus, there was prepared a modified
bombesin which contains an additional cysteine residue at the amino
terminus of native bombesin. In addition, residue-3 of the native
bombesin has been changed to a lysine residue. The modified
bombesin, therefore, is designated
"Cys.sup.0-lys.sup.3-bombesin."
[0672] Cys.sup.0-lys.sup.3-bombesin (11.3 mg) was dissolved in 1.1
mL of deionized water and adjusted to pH 7-7.5 with 10 .mu.l 1.5 M
Tris-HCl, pH 8.8 and then reacted with 0.45 mL maleimidocaproyl
adriamycin hydrazone (15 mg/mL in deionized water) at ambient
temperature for several hours. The reaction mixture was dialyzed
against water overnight in dialysis tubing (molecular weight
cutoff: 1000). The precipitate was removed by centrifugation
(12,000.times.g) and the supernatant was saved. Adriamycin ("ADM")
content of the bombesin-adriamycin conjugate was measured by
diluting 1:50 in acetate buffer, pH 6.0. The adriamycin ("ADM")
content was calculated using the formula:
[O.D..sub.495/8030].times.50=ADM(M)
[0673] For this preparation O.D..sub.495=0.116 thus the adriamycin
content was 7.2.times.10.sup.-4 M.
[0674] The product was chromatographed by HPLC using a C.sub.18
(Beckman Instruments, Ultrasphere 5.mu., 4.6 mm.times.25 cm)
column. Buffer A: 10 mM NH.sub.4OAc pH 4.5; Buffer B: 90%
acetonitrile/10% Buffer A. The column was equilibrated with 90%
Buffer A/10% Buffer B and the chromatography conditions were: 90%
buffer A/10% buffer B to 60% Buffer A/60% buffer B for 2 minutes,
gradient to 50% buffer A/50% buffer B for 15 minutes. The product
had a retention time of 9.3 minutes under these conditions.
EXAMPLE 22
[0675] A Conjugare of Iminothiolane Thiolated Chimeric BR96 and
Maleimidocaproyl Hydraxone pf Adriamycin
[0676] Chimeric BR96 (15 mL, 9.05 mg/mL) was dialyzed two times
against 4 liters of 0.1 M sodium carbonate/bicarbonate buffer, pH
9.1. The antibody solution then was heated with iminothiolane (0.75
mL, 20 mM) at 32.degree. C. for 45 minutes. The solution was then
dialyzed against 4 liters of sodium carbonate/bicarbonate buffer,
pH 9.1 followed by dialysis against 4 liters of 0.0095 M PBS-0.1 M
L-histidine, pH 7.4. This solution had a molar ratio of
--SH/protein of 1.35. The protein then was re-thiolated as
described above to yield a solution with a molar ratio of
--SH/protein of 5.0.
[0677] The maleimidocaproyl hydrazone of adriamycin (3.2 mg in
0.640 mL DMF) was added with stirring at 4.degree. C. to the
thiolated protein solution. The conjugate was incubated at
4.degree. C. for 16 hours then it was dialyzed against 4 liters of
0.0095 M PBS-0.1 M L-histidine, pH 7.4. The dialyzed conjugate was
filtered through a 0.22.mu. cellulose acetate membrane into a
sterile tube to which a small quantity (>5% (v/v)) of
BioBeads.TM. SM-2 (Bio-Rad Laboratories, Richmond, Calif. 94804)
were added. After 24 hours of gentle agitation, the beads were
filtered off and the conjugate was frozen in liquid nitrogen and
stored at -80.degree. C. The resulting conjugate had a molar ratio
of 3.4 adriamycin molecules to 1 molecule of protein and was
obtained in 24% yield from chimeric BR96.
EXAMPLE 23
[0678] Conjugate of Maleimidocaproyl Hydrozone of Adriamycin with
DTT Reduced Human IgG ("Relaxes Human IGG")
[0679] Human IgG (obtained from Rockland, Gilbertsville, Pa.) was
diluted with 0.0095 M PBS to a protein concentration of 10.98
mg/mL. This solution (350 mL) was heated to 37.degree. C. in a
water bath under a nitrogen atmosphere. Dithiothreitol (16.8 mL, 10
mM) in PBS was added and the solution was stirred for 3 hours at
37.degree. C. The solution was divided equally between two Amicon
(Amicon Division of W. R. Grace and Co., Beverly, Mass. 01915)
Model 8400 Stirred Ultrafiltration Cells, each fitted with an
Amicon YM 30 Ultrafilter membrane (MW cutoff 30,000, 76 mm diam.)
and connected via an Amicon Model CDS10 concentration/dialysis
selector to an Amicon Model RC800 mini-reservoir. Each reservoir
contained 700 mL of 0.0095 M PBS-0.1 M L-histidine. The protein
solutions were dialyzed until concentration of free thoil in the
filtrate was 41 .mu.M. the molar ratio of --SH/protein in the
retentate was determined to be 8.13.
[0680] The retentate was transferred from the cells to a sterile
container maintained under a nitrogen atmosphere and a solution of
maleimidocaproyl hydrazone of adriamycin (36.7 mL, 5 mg/mL in
water) was added with stirring. The conjugate was incubated at
4.degree. C. for 48 hours after which it was filtered through a
0.22.mu. cellulose acetate membrane. A Bio-Rad Econocolumn.TM. (2.5
cm.times.50 cm, Bio-Rad Laboratories, Richmond, Calif. 94804) was
packed with slurry of 100 g of BioBeads.TM. SM-2 (Bio-Rad
Laboratories, Richmond, Calif. 94804) in 0.00095 M-0.1 M
L-histidine buffer. The beads had been prepared by washing in
methanol, followed by water and then several volumes of buffer. The
filtered conjugate was percolated through this column at 2 mL/min.
After chromatography the conjugate was filtered through a 0.22.mu.
cellulose acetate membrane and frozen in liquid nitrogen and stored
at -80.degree. C. The conjugate obtained had an average molar ratio
of 7.45 molecules of adriamycin per molecule of protein and was
obtained in 99% yield from human IgG.
EXAMPLE 24
[0681] Conjugate of Relaxed BR64 with Maleimidocaproyl Hydrazone of
Adriamycin
[0682] A solution of BR64 (435 mL; 11.31 mg/mL,
7.07.times.10.sup.-5 M) was treated with DTT (947 mg) and heated at
42.degree.-43.degree. C. with gentle stirring for 2 hours. The
solution was cooled in ice, transferred into 2 dialysis tubes and
each tube was dialyzed 5 times against PBS (14 L per dialysis) for
8 hours at 4.degree. C. The contents of the tubes were combined
(400 mL) and the protein and --SH content determined (10.54 mg/mL,
6.58.times.10.sup.-5 M, 5.14.times.10.sup.-4 M, respectively). The
molar ratio of --SH to protein was 7.8.
[0683] A solution of maleimidocaproyl hydrazone of adriamycin in
DMF (5 mg/mL, 32.6 mL) was added to the antibody solution with
gentle stirring and then incubated at 4.degree. C. for 24 hours.
The solution was filtered through a 0.22.mu. cellulose acetate
filter and then transferred to two dialysis tubes and dialyzed as
described above. After dialysis, the contents of the tubes were
combined, filtered and shaken with BioBeads.TM. SM-2 (Bio-Rad
Laboratories, Richmond, Calif. 94804) for 24 hours at 4.degree. C.
The beads were filtered off using a cellulose acetate filter to
yield the conjugate solution. The concentration of protein and
adriamycin were determined (8.66 mg/mL, 5.42.times.10.sup.-5 M; 168
.mu.g/mL, 2.89.times.10.sup.-4 M, respectively). The protein yield
is 97%. The molar ratio of adriamycin to protein is 5.33; and,
unconjugated adriamycin is 0.5%.
EXAMPLE 25
[0684] Genera; Procedure for Conjugating the Maleimidocaproyl
Hydrazone of Adriamycin to a Relaxed Antibody
[0685] 1. A solution (300 mL) of antibody (3 g, 10 mg/mL) in BPS
buffer (note 1) is continuously blanketed with nitrogen, immersed
in a 37.degree. C. water bath and stirred gently with a magnetic
stirrer. The solution is treated with 7 molar equivalents of DTT
(notes 2, 3) for 3 hours. The --SH group molar ratio ("MR") to
protein is determined initially and hourly and, for a maximally
conjugated product, should remain constant at about 14 (notes 2,
4).
[0686] 2. The solution is transferred as quickly as possible to an
Amicon diafiltration cell (Amicon, Division of W. R. Grace and Co.,
Beverly, Mass. 01 915) (note 5) maintained at about 4.degree. C. to
about 7.degree. C. The system is pressurized with argon or nitrogen
and diafiltration is started using PBS buffer containing 0.1 M
histidine which has been precooled to about 4.degree. C. to about
7.degree. C. The initial temperature of the effluent, immediately
after starting the diafiltration, is 16.degree.-18.degree. C. and
drops to 8.degree.-9.degree. C. within about 90 minutes. The
effluent is monitored for a MR of --SH to protein and, when this
value is <1, the diafiltration is complete (note 6).
[0687] 3. The solution is transferred back to a round bottom flask
equipped with a magnetic stirrer and kept in ice. The solution
continuously is blanketed by nitrogen. The volume of the solution
is noted. Aliquots of 0.1 mL are taken out and diluted with PBS
buffer to 1.0 mL to determine the amount of protein in mg/mL (and
also the molar equivalent of protein and the molarity of the --SH
groups (and hence the MR of the --SH to protein)). A solution of
maleimidocaproyl hydrazone of adriamycin in distilled water (5
mg/mL, 6.3.times.10.sup.-3 M) is prepared (notes 7, 8). The amount
(in mL) of this solution needed for the conjugation is determined
by the formula: 3 ( molarity of --SH ) .times. ( volume of protein
solution ) .times. 1.05 6.3 .times. 10 - 3
[0688] (note 9) and this amount is added slowly to the protein
solution which is stirred gently. The solution is kept at 4.degree.
C. for 30 minutes.
[0689] 4. A column of BioBeads.TM. SM-2, mesh 20-50 (Bio-Rad
Laboratories, Richmond, Calif. 94804) is prepared (note 10) at
4.degree. C. The red protein solution is filtered through a
0.22.mu. cellulose acetate filter, then passed through the column
at a rate of 2.5 mL/min and the red effluent collected. Finally
PBS-0.1 M histidine buffer is poured on top of the column and the
effluent collected until it is colorless. The volume of the
collected red solution is noted. An aliquot of 0.1 mL is diluted to
1 mL with PBS buffer and the amount of protein and adriamycin is
measure. The amount of conjugated adriamycin is determined by
absorbance at 495 nm (.epsilon.=8030 cm.sup.-1M.sup.-1) and
expressed in micromoles and micrograms per mL. The amount of
protein, expressed in mg per mL and micromoles, is determined as
above by reading the absorbance at 280 nm with a correction for the
absorbance of adriamycin at the same wavelength according to the
general formula: 4 Antibody ( mg / mL ) = A 280 - ( 0.724 .times. A
495 ) 1.4
[0690] where A is the observed absorbance at the noted wavelength.
The MR of adrianycin to protein then is calculated.
[0691] 5. An aliquot of 5 mL of conjugate is passed over an
Econo-Pac.TM. 10 SM-2 column (a prepacked Bio-Beads.TM. SM-2 column
(Bio-Rad Laboratories, Richmond, Calif. 94804), volume 10 mL, that
has been washed and equilibrated with PBS-0.1 M histidine buffer)
in the manner described above. The amount of protein and conjugated
adriamycin is determined and the MR determined. This value should
be the same as that of the bulk of the solution (note 11).
[0692] 6. The conjugate is frozen in liquid nitrogen and stored at
-80.degree. C. Aliquots can be taken for determining cytotoxicity,
binding and presence of free adriamycin (note 12) concentration by
the molar protein concentration. Should this value be less than 14
during the reaction an appropriate additional amount of DTT is
added.
[0693] 7. On a scale of 3 g/300 mL, two Amicon cells of 350 mL each
are used, dividing the solution into two portions of 150 mL per
cell.
[0694] 8. On the reaction scale provided, the diafiltration usually
takes 2-4 hours. The duration will depend on factors such as the
age of the membrane, rate of stirring of solution and pressure in
cell.
[0695] 9. The hydrazone is not very soluble in PBS and a
precipitate is formed in a short while.
[0696] 10. Brief applications of a sonicator will facilitate
dissolution in distilled water. The resulting solution is
stable.
[0697] 11. This amount provides for a 5% excess of the hydrazone.
The process described generally takes about 15-20 minutes.
[0698] 12. The Bio-Beads.TM. are prepared for chromatography by
swelling them in methanol for at least one hour, preferably
overnight, washing them with distilled water and finally
equilibrating them with PBS-0.1 M histidine buffer. For 3 g of
protein 100 g of beads are used to form a column of 2.5 cm.times.30
cm.
[0699] 13. Because of the inherent error of the spectroscopic
methods used, a variation of 1 MR unit is accepted to be a
satisfactory result. Generally, however, the MR varies less than
0.5 MR units.
[0700] 14. The values of free adriamycin in the conjugate are
generally much less than 1%.
EXAMPLE 26
[0701] Conjugate of Relaxed Chimeric BR96 with Maleimidocaproyl
Hydrazone of Adriamycin
[0702] Chimeric BR96, prepared in the manner previously described,
was diluted with 0.0095 M PBS to a protein concentration of 10.49
mg/mL. This solution (500 mL) was heated to 37.degree. C., under a
nitrogen atmosphere, in a water bath. Dithiothreitol (26.2 mL, 10
mM) in PBS was added and the solution was stirred for 3 hours at
37.degree. C. The solution was divided equally between two Amicon
Model 8400 stirred ultrafiltration cells each fitted with a YM 30
ultrafilter (MW cutoff 30,000, 76 mm diam.) and connected via a
Model CDS10 concentration/dialysis selector to a Model RC800
mini-reservoir (Amicon, Division of W. R. Grace and Co., Beverley,
Mass. 01915). Each reservoir contained 800 mL of 0.0095 M PBS-0.1 M
L-histidine. The protein solutions were dialyzed until the
concentration of free thiol in the filtrate was 60 .mu.M. The molar
ratio of --SH/protein in the retentate was determined to be 8.16.
The retentate was transferred from the cells to a sterile container
under nitrogen and a solution of maleimidocaproyl hydrazone of
adriamycin (42.6 mL, 5 mg/mL in water) was added with stirring. The
conjugate was incubated at 4.degree. C. for 48 hours after which it
was filtered through a 0.22.mu. cellulose acetate membrane. A 2.5
cm.times.50 cm Bio-Rad Econocolumn was packed with slurry of 100 g
of BioBeads.TM. SM-2 (Bio-Rad Laboratories, Richmond, Calif. 94804)
in 0.00095 M-0.1 M L-histidine buffer. The beads had been prepared
by washing in methanol, followed by water then several volumes of
buffer. The filtered conjugate was percolated through this column
at 2 mL/min. After chromatography the conjugate was filtered
through a 0.22.mu. cellulose acetate membrane, frozen in liquid
nitrogen and stored at -80.degree. C. The conjugate obtained had a
molar ratio of 6.77 adriamycin to protein and was obtained in 95%
yield from chimeric BR96.
EXAMPLE 27
[0703] Conjugate of Relaxed Murine Antibody L6 with
Maleimidocaproyl Hydrazone of Adriamycin
[0704] Murine antibody L6, prepared as defined earlier, was diluted
with 0.0095 M PBS to a protein concentration of 11.87 mg/mL. This
solution (350 mL) was heated to 37.degree. C., under a nitrogen
atmosphere, in a water bath. Dithiothreitol (18.2 mL, 10 mM) in PBS
was added and the solution was stirred for 3 hours at 37.degree. C.
The solution was divided equally between two Amicon Model 8400
stirred ultrafiltration cells each fitted with a YM 30 ultrafilter
(MW cutoff 30,000, 76 mm diam.) and connected via a Model CDS10
concentration/dialysis selector to a Model RC800 min--reservoir
(Amicon, Division of W. R. Grace and Co., Beverly, Mass. 01915).
Each reservoir contained 800 mL of 0.0095 M PBS-0.1 M L-histidine.
The protein solutions were dialyzed until concentration of free
thiol in the filtrate was 14 .mu.M. The molar ratio of --SH/protein
in the retentate was determined to be 9.8. The retentate was
transferred from the cells to a sterile container under nitrogen
and a solution of maleimidocaproyl hydrazone of adriamycin (40.4
mL, 5 mg/mL in water) was added with stirring. The conjugate was
incubated at 4.degree. C. for 48 hours after which it was filtered
through a 0.22.mu. cellulose acetate membrane. A 2.5 cm.times.50 cm
Bio-Rad Econocolumn was packed with a slurry of 100 g of
BioBeads.TM. SM-2 (Bio-Rad Laboratories, Richmond, Calif. 94804) in
0.00095 M-0.1 M L-histidine buffer. The beads had been prepared by
washing in methanol, followed by water then several volumes of
buffer. The filtered conjugate was percolated through this column
at 2 mL/min. After chromatography the conjugate was filtered
through a 0.22.mu. cellulose acetate membrane, frozen in liquid
nitrogen and stored at -80.degree. C. The conjugate obtained had a
molar ratio of 7.39 Adriamycin to protein and was obtained in 100%
yield from murine L6.
[0705] Biological Activity
[0706] Representative conjugates of the present invention were
tested in both in vitro and in vivo systems to determine biological
activity. In these tests, the potency of conjugates of cytotoxicity
drugs was determined by measuring the cytotoxicity of the
conjugates against cells of human cancer origin. The following
describes representative tests used and the results obtained.
Throughout the data presented, the conjugates are referred to using
the form ligand-drug-molar ratio of ligand to drug. Thus, for
example, "BR64-ADM-5.33" refers to a conjugate between antibody
BR64 and adriamycin and the mole ratio of drug to antibody is 5.33.
One skilled in the art will recognize that any tumor line
expressing the desired antigen could be used in substitution of the
specific tumor lines used in the following analyses.
[0707] Test I
[0708] In vitro Activity of BR64-Adriamycin Conjugates
[0709] The immunoconjugates of Examples 18 and 19 were tested in
vitro against a human lung carcinoma line, L2987 (obtained from I.
Hellstrom, Bristol-Myers Squibb Seattle; see also I. Hellstrom, et
al., Cancer Research 50:2183 (1990)), which expresses the antigens
recognized by monoclonal antibodies BR64, L6 and BR96. Monolayer
cultures of L2987 cells were harvested using trypsin-EDTA (GIBCO,
Grand Island, N.Y.), and the cells counted and resuspended to
1.times.10.sup.5/mL in RPMI-1640 containing 10% heat inactivated
fetal calf serum ("RPMI-10% FCS"). Cells (0.1 mL/well) were added
to each well of 96-well flat bottom microtiter plates and incubated
overnight at 37.degree. C. in a humidified atmosphere of 5%
CO.sub.2. Media was removed from the plates and serial dilutions of
adriamycin or the antibody conjugates of adriamycin were added to
the wells. All dilutions were performed in quadruplicate. Following
a 2 hour drug or conjugate exposure, the plates were centrifuged
(100.times.g, 5 min.), the drug or conjugate removed, and the
plates washed three times with RPMI-10% FCS. The cells were
cultured in RPMI-10% FCS for an additional 48 hours. At this time
the cells were pulsed for 2 hours with 1.0 .mu.Ci/well of
.sup.3H-thymidine (New England Nuclear, Boston, Mass.). The plates
were harvested and the counts per minute ("CPM") determined.
Inhibition of proliferation was determined by comparing the mean
CPM for treated samples with that of the untreated controls. The
data presented in FIG. 51 demonstrates the cytotoxicity against
L2987 lung cells of binding immunoconjugate (MR of adriamycin to
BR64 equal to 7.18, designated BR64-THADMHZN-7.18") compared to a
non-binding immunoconjugate of SN7 and adriamycin (MR of adriamycin
to SN7 equal to 4, designated "SN7-THADMHZN-4"). The BR64
conjugates prepared by the method described in Example 18 are
active and demonstrate antigen-specific cytotoxicity in this in
vitro screen.
[0710] Test II
[0711] In vivo Activity of BR64-Adriamycin Conjugates
[0712] The immunoconjugates of Examples 18 and 19 were evaluated in
vivo for antigen-specific antitumor activity. Congenitally athymic
female mice of BALB/c background (BALB/c nu/nu; Harlan
Sprague-Dawley, Indianapolis, Ind.) were used in these studies.
Mice were housed in Thoren caging units on sterile bedding with
controlled temperature and humidity. Animals received sterile food
and water ad libitum. The L2987 human lung tumor line, described
above, was used in these studies. This line has been shown to
maintain expression of the BR64, BR96 and L6 antigens following
repeated passage in vivo. The tumor lines were maintained by serial
passage in athymic mice as described previously (P. A. Trail, et
al., in vivo 3 319-324 (1989)). Tumors were measured, using
calipers, in 2 perpendicular directions at weekly or biweekly
intervals.
[0713] Tumor volume was calculated according to the equation: 5 V (
mm 3 ) = ( L .times. W 2 ) 2
[0714] in which
[0715] V=volume (mm.sup.3)
[0716] L=measurement of longest axis (mm)
[0717] W =measurement (mm) of axis perpendicular to L
[0718] Data are presented as the median tumor size for treated and
control groups. Each treatment or control group contained 8-10
animals. Therapy was initiated when tumors had reached a median
size of 50-100 mm.sup.3. Therapy was administered by the ip or iv
route on various schedules as denoted. Adriamycin was diluted in
normal saline and native antibody and adriamycin conjugates were
diluted in phosphate buffered saline ("PBS") for administration.
All dosages were administered on a weight basis (mg/kg) and were
calculated for each animal. In these studies the antitumor activity
of binding BR64 immunoconjugates was compared to that of optimized
dosages of adriamycin, mixtures of native BR64 and adriamycin, and
non-binding conjugates. Unconjugated adriamycin was administered
according to the route, dosage, and schedule demonstrated to be
optimal for the L2987 human xenograft model. The unconjugated
adriamycin, therefore, was administered at a dose of 8 mg/kg by the
iv route every fourth day for a total of 3 injections (denoted "8
mg/kg, q4dx3, iv"). The binding (BR64) and non-binding (SN7)
immunoconjugates were administered at several doses by the ip route
every fourth day for a total of 3 injections (denoted "q4dx3, ip").
As shown in FIG. 52, significant antitumor activity was observed
following the administration of tolerated doses (10 and 15
mg/kg/injection) of the BR64-adriamycin conjugate. The antitumor
activity observed following therapy with the BR64 conjugate was
significantly better than that observed for therapy with optimized
adriamycin and matching doses of non-binding (SN7) conjugate.
[0719] In this experiment, complete tumor regressions were observed
in 66% of the animals following treatment with 15 mg/kg/injection
of the BR64 conjugate and 50% complete tumor regressions were
observed following treatment with 10 mg/kg/injection of the BR64
conjugate. Partial or complete regressions of established L2987
tumors have not been observed following therapy with optimized
adriamycin, mixtures of native BR64 and adriamycin, or equivalent
doses of non-binding conjugates.
[0720] To demonstrate that the observed activity required the
covalent coupling of the antibody to adriamycin, several control
experiments using mixtures of native BR64 and adriamycin were
performed. Representative data for several types of combined
therapy are shown in FIGS. 5a-c. The antitumor activity observed
for various modes of combined therapy with MAb and adriamycin was
not significantly different from that observed for therapy with
optimized adriamycin alone. Taken together these data indicate that
the covalent coupling of BR64 to adriamycin is required to observe
the antitumor activity described in FIG. 21.
[0721] Test III
[0722] In vivo Activity of Bombesin Conjugates P The conjugate of
Example 21 was evaluated in vivo for antitumor activity. BALB/c
athymic nude mice were implanted with H345 human small cell lung
carcinoma tumor pieces (obtained from Dr. D. Chan, University of
Colorado Medical School, Colo.), subcuntaneously, using trocars.
Tumors were allowed to grow to 50-100 mm.sup.3 before initiation of
treatment. Mice were treated i.v. on 23, 26, 28, and 30 days
post-implant with adriamycin alone (1.6 mg/kg), or the conjugates
bombesin-adriamycin ("BN-ADM(TH)", in an amount equivalent to 1.6
mg/kg adriamycin) or P77-adriamycin conjugate (P77-ADM (TH)", in an
amount equivalent to 1.6 mg/kg of adriamycin). P77 is a 12 amino
acid peptide with an internal cysteine residue
(sequence=KKLTCVQTRLKI) that does not bind to H345 cells and was
conjugated to the maleimidocaproyl hydrazone of adriamycin
according to the procedure outlined in Example 21. Thus, the
conjugate represents a non-binding conjugate with respect to H345
cells. Tumors were measured with calipers and tumor volume was
calculated using formula: 6 V ( mm 3 ) = ( L .times. W 2 ) 2
[0723] in which V, L, and W are as defined in Test II.
[0724] The median tumor volumes were determined and the observed
results are shown in FIG. 54.
[0725] Test IV
[0726] In vitro Cytotoxicity Data for Relaxed ChiBR96 Antibody
Conjugates
[0727] Immunoconjugates of adriamycin and ChiBR96 antibody are
prepared using the general method for preparing relaxed antibodies
as described in Example 25. The conjugates were tested, using the
protocol below, for in vitro cytotoxicity and their cytotoxicity
was compared to that of free adriamycin, and SPDP-thiolated
immunoconjugates prepared by the method described in Example 18.
The results of these tests are provided in FIG. 55.
[0728] Monolayer cultures of L2987 human lung cells were maintained
in RPMI-1640 media containing 10% heat inactivated fetal calf serum
(RPMI-10% FCS). The cells were harvested using trypsin-EDTA (GIBCO,
Grand Island, N.Y.), and the cells counted and resuspended to
1.times.10.sup.5/mil in RPMI-10% FCS. Cells (0.1 ml/well) were
added to each well of 96 well microtiter plates and incubated
overnight at 37.degree. C. in a humidified atmosphere of 5%
CO.sub.2. Media was removed from the plates and serial dilutions of
adriamycin or antibody/ADM conjugates added to the wells. All
dilutions were performed in quadruplicate. Following a 2 hour drug
or conjugate exposure, the plates were centrifuged (200.times.g, 5
min.), the drug or conjugate removed, and the plates washed
3.times. with RPMI-10% FCS. The cells were cultured in RPMI-10% FCS
for an additional 48 hours. At this time the cells were pulsed for
2 hours with 1.0 .mu.Ci/well of .sup.3H-thymidine (New England
Nuclear, Boston, Mass.). The plates were harvested and the counts
per minute ("CPM") were determined. Inhibition of proliferation was
determined by comparing the mean CPM for treated samples with that
of the untreated control. IC.sub.50 values are reported as .mu.M of
equivalent adriamycin.
[0729] Test V
[0730] In Vivo Antitumor Activity of BR64 and Murine L6
Conjugates
[0731] The in vivo antitumor activity of immunoconjugates of
adriamycin and relaxed BR64 or relaxed L6 was evaluated. The
observed data are provided in FIG. 56.
[0732] Congenitally athymic female mice of BALB/c background
(BALB/C nu/nu; Harlan Sprague-Dawley, Indianapolis, Ind.) were
used. Mice were housed in Thoren caging units on sterile bedding
with controlled temperature and humidity. Animals received sterile
food and water ad libitum.
[0733] The L2987 human tumor line was established as tumor
xenograft models in athymic mice. The tumor line was maintained by
serial passage in vivo. Tumors were measured in 2 perpendicular
directions at weekly or biweekly intervals using calipers. Tumor
volume was calculated according to the equation: 7 V ( mm 3 ) = L
.times. W 2 2
[0734] in which:
[0735] V=volume (mm.sup.3)
[0736] L=measurement of longest axis (mm)
[0737] W=measurement of axis perpendicular to L
[0738] In general, there were 8-10 mice per control or treatment
group. Data are presented as median tumor size for control or
treated groups. Antitumor activity is expressed in terms of gross
log cell kill ("LCK") where: 8 LCK = T - C 3.3 .times. TVDT
[0739] T-C is defined as the median time (days) for treated tumors
to reach target size minus the median time for control tumors to
reach target size and TVDT is the time (days) for control tumors to
double in volume (250-500 mm.sup.3) Partial tumor regression ("PR")
refers to a decrease in tumor volume to .ltoreq.50% of the initial
tumor volume; complete tumor regression ("CR") refers to a tumor
which for a period of time is not palpable; and cure is defined as
an established tumor which is not palpable for a period of time
.gtoreq.10 TVDTs.
[0740] For animals bearing the L2987 human lung tumor, therapy was
typically initiated when the median tumor size was 75 mm.sup.3
(12-14 days after tumor implant). The average TVDT was 4.8.+-.0.9
days and antitumor activity was assessed at a tumor size of 500
mm.sup.3. In several experiments (described below in Test VI)
therapy was initiated when L2987 tumors were 225 mm.sup.3in
size.
[0741] Materials under investigation were administered by the ip or
iv route. Adriamycin was diluted in normal saline; antibody and
antibody/adriamycin conjugates were diluted in phosphate buffered
saline. Compounds were administered on a mg/kg basis calculated for
each animal, and doses are presented as mg/kg of equivalent
adriamycin/injection. Immunoconjugates were administered on a q4dx3
schedule. The maximum tolerated dose ("MTD") for a treatment
regimen is defined as the highest does on a given schedule which
resulted in .ltoreq.20% lethality.
[0742] In the data shown in FIG. 56, injection of optimized doses
of adriamycin produced antitumor activity equivalent to 1.1 LCK and
tumor regressions were not observed. The BR64-ADM conjugate
produced antitumor activity equivalent to >10 LCK at all doses
tested and 89%, 78%, and 100% cures were observed at doses of 5
mg/kg, 8 mg/kg, and 10 mg/kg of BR64-ADM, respectively. At doses of
8 mg/kg or 10 mg/kg the L6-ADM conjugate produced antitumor
activity (1.8 and 3.5 LCK, respectively) which was significantly
better than that of optimized adriamycin but less than that of
equivalent doses of internalizing BR64-ADM conjugates. Thus, the
data show that the antitumor activity of binding non-internalizing
L6-ADM conjugates is superior to that of unconjugated adriamycin.
Treatment with L6-adriamycin conjugate results in lower antitumor
activity than is observed with matching doses of the internalizing
BR64-adriamycin conjugate.
[0743] Test VI
[0744] In vivo Antitumor Activity of ChiBR96-ADM Conjugates
[0745] The antitumor activity of ChiBR96-ADM conjugates was
evaluated against binding established human lung ("L2987"), breast
("MCF7," obtainable from the ATCC under the accession number ATCC
HTB 22; see also I. Hellstrom, et al., Cancer Research 50:2183
(1990)), and colon ("RCA" from M. Brattain, Baylor University; see
also I. Hellstrom, et al., Cancer Research 50:2183 (1990))
tumors.
[0746] Animals were maintained and tumor xenograft models were
established for the MCF7 and RCA and the L2987 human tumor lines as
described for the L2987 in Test V. Materials were administered as
described in Test V.
[0747] For animals bearing the L2987 human lung tumor, therapy
typically was initiated when the median tumor size was 75 mm.sup.3
(12-14 days after tumor implant). The average TVDT was 4.8.+-.0.9
days and antitumor activity was assessed at a tumor size of 500
mm.sup.3. In several experiments therapy was initiated when L2987
tumors were 225 mm.sup.3 in size.
[0748] The MCF7 tumor is an estrogen-dependent human breast tumor
line. Athymic mice were implanted with 0.65 mg (65 day release
rate) estradiol pellets (Innovative Research of America, Toledo,
Ohio) on the day of tumor implant. Therapy was initiated when the
median tumor size was 100 mm.sup.3 (typically 13 days after tumor
implant). The MCF7 tumor had an average TVDT of 6.4.+-.2.0 days and
antitumor activity was assessed at 400 mm.sup.3.
[0749] For animals bearing the RCA colon tumor, therapy was
initiated 15 days after tumor implant when the median tumor size
was 75 mm.sup.3. The average TVDT for RCA tumor xenografts was
9.5.+-.1.5 days and antitumor activity was assessed at 400
mm.sup.3. Data for the antitumor activity of optimized adriamycin
in the L2987, MCF7, and RCA xenograft models is summarized in the
following Tables and referenced Figures.
[0750] The antitumor activity of the ChiBR96-ADM conjugates was
compared to that of optimized adriamycin and equivalent doses of
non-binding (IgG) immunoconjugates. In each model, complete tumor
regressions and/or cures of established tumors were observed
following the administration of tolerated doses of ChiBR96-ADM
conjugate.
[0751] Representative data demonstrating the antigen-specific
antitumor activity of ChiBR96-ADM conjugates is presented in FIGS.
57 and 48. As shown in FIG. 57, the ip administration of
ChiBR96-ADM conjugate (MR=4.19) at a dose of 10 mg/kg equivalent of
adriamycin produced antitumor activity equivalent to >10 LCK. At
this does of ChiBR96-ADM conjugate, 78% of the mice were cured of
the tumor and an additional 11% of the mice demonstrated a complete
tumor regression. The administration of 5 mg/kg of the ChiBR96-ADM
conjugate also produced antitumor activity equivalent to >10 LCK
with 88% tumor cures and 12% complete tumor regressions. The
antitumor activity observed following administration of ChiBR96-ADM
conjugates (>10 LCK) was substantially better than that observed
for optimized adriamycin (1.0 LCK). The ChiBR96-ADM conjugate was
also more potent than optimized adriamycin; that is, the antitumor
activity of the ChiBR96-ADM conjugate tested at a dose of 5 mg/kg
equivalent adriamycin was superior to that of adriamycin tested at
a dose of 8 mg/kg. The non-binding human IgG conjugate (MR=7.16)
was not active against L2987 xenografts when tested at a dose of 10
mg/kg equivalent of adriamycin indicating that the superior
activity of the ChiBR96-ADM conjugate was due to antigen specific
binding of the immunoconjugate to L2987 tumor cells.
[0752] Similar data are presented in FIG. 58. As shown, the
ChiBR96-ADM conjugate (MR=5.8) tested at a dose equivalent of 10
mg/kg adriamycin resulted in antitumor activity equivalent to
>10 LCK. At this dose, 90% tumor cures and 10% complete tumor
regressions were observed. The administration of 5 mg/kg of the
ChiBR96-ADM conjugate resulted in 4.8 LCK with 10% cures, 50%
complete and 10% partial tumor regressions. The antitumor activity
of ChiBR96-ADM conjugate greatly exceeded that of optimized
adriamycin (1.6 LCK) and, as described above, the ChiBR96-ADM
conjugate was more potent than unconjugated adriamycin. The
non-binding IgG-ADM conjugate (MR=7.16) was not active at a dose of
10 mg/kg.
[0753] The antitumor activity of various preparations of
ChiBR96-ADM conjugates prepared by the "relaxed" antibody technique
and evaluated against established L2987 lung tumor xenograft is
presented in Table 15.
16TABLE 15 Antitumor Activity of ChiBR96-ADM Conjugates Against
Established L2987 Human Lung Tumor Xenografts* % Tumor Dose (mg/kg)
Regressions Conjugate ADM Antibody Route LCK PR CR Cure No. of Mice
ChiBR96-ADM-6.85 15 615 ip >10 10 0 80 10 10 410 ip >10 0 0
89 9 8 328 iv >10 0 0 100 9 5 205 iv >10 0 22 78 9
ChiBR96-ADM-4.19 15 980 ip >10 0 11 89 9 10 654 ip >10 11 11
66 9 5 327 iv >10 0 11 89 9 2.5 164 iv >10 0 22 78 9
ChiBR96-ADM-6.85 10 410 ip >10 11 11 78 9 8 328 iv >10 0 0
100 9 5 205 iv >10 0 11 89 9 ChiBR96-ADM-4.19 10 654 ip >10 0
0 100 9 5 327 iv >10 0 0 100 9 ChiBR96-ADM-4.19 10 654 ip >8
0 22 78 9 5 327 ip >8 0 11 89 9 ChiBR96-ADM-5.80 10 500 ip
>10 0 10 90 10 5 250 ip >4.8 10 50 10 10 ChiBR96-ADM-6.82 5
204 iv >10 22 22 55 9 2 82 iv 3.5 44 33 0 9 1 41 iv 2.0 0 22 0 9
ChiBR96-ADM-6.82 10 400 ip >5.3 11 11 56 9 5 200 ip 4.8 30 10 40
10 2.5 100 ip 2.9 30 0 30 10 1.25 50 ip 1.1 11 0 11 9 0.62 25 ip 0
0 0 0 9 5 200 iv >5.3 10 20 70 10 2.5 100 iv 2.9 22 33 0 9 1.25
50 iv 1.5 11 11 0 9 0.62 25 iv 0.6 0 0 0 9 Adriamycin 8 -- iv 1-1.8
3.6 0 0 55 *All treatment administered on a q4dx3 schedule
[0754]
17TABLE 17 Summary of Antitumor Activity of ChiBR96-ADM Thioether
Conjugates Evaluated Against Established MCF7 Human Breast Tumor
Xenografts % Tumor Dose (mg/kg).sup.a Regressions Conjugate ADM
ChiBR96 Route LCK PR CR Cure No. of Mice ChiBR96-ADM-7.88 10 350 ip
--.sup.b -- -- -- 10 5 175 ip 4.2 30 0 0 10 5 175 iv 4.2 50 10 0 10
IgG-ADM-7.16 5 225 ip 1.1 0 0 0 10 2.5 112 ip 0.6 0 0 0 10 2.5 112
iv 0.8 0 0 0 10 Adriamycin 6 0 iv 1.4 0 0 0 10 .sup.aAll therapy
administered q4dx3 .sup.b40% lethality occurred at this dose of
immunoconjugate
[0755] As shown, the antitumor activity of ChiBR96-ADM conjugates
is superior to that of optimized adriamycin and the ChiBR96-ADM
conjugates are 6-8 fold more potent than unconjugated
adriamycin.
[0756] The antitumor activity of ChiBR96-ADM conjugates was also
evaluated against large (225 mm.sup.3) established L2987 tumors
(FIG. 49). The administration of the ChiBR96-ADM conjugate
(MR=6.85) at a dose of 10 mg/kg equivalent to >10 LCK and 70%
cures and 30% partial tumor regressions were observed.
[0757] The antitumor activity of unconjugated ChiBR96 antibody was
evaluated using established (50-100 mm.sup.3) L2987 human lung
tumor xenografts. As shown in Table 10, ChiBR96 antibody
administered at doses of 100, 200 or 400 mg/kg was not active
against established L2987 tumors. The antitumor activity of
mixtures of ChiBR96 and adriamycin was not different from that of
adriamycin administered alone. Therefore, the antitumor activity of
the ChiBR96-ADM conjugates reflects the efficacy of the conjugate
itself rather than a synergistic antitumor effect of antibody and
adriamycin.
18TABLE 16 Antitumor Activity of Adriamycin, ChiBR96, and Mixtures
of ChiBR96 and Adriamycin Against Established L2987 Human Lung
Tumor Xenografts % Tumor Dose (mg/kg).sup.a Regressions No. of
Treatment ADM ChiBR96 LCK PR CR Cure Mice Adriamycin 8 -- 1.5 0 0 0
9 ChiBR96 -- 400 0 0 0 0 8 -- 200 0 0 0 0 8 -- 100 0 0 0 0 8
Adriamycin + 8 400 1.8 11 0 0 9 ChiBR96 8 200 1.6 0 0 0 9 8 100 1.9
0 0 0 8 .sup.aTreatment administered iv on a q4dx3 schedule
[0758] In summary ChiBR96-ADM conjugates demonstrated
antigen-specific antitumor activity when evaluated against
established L2987 human lung tumors. The antitumor activity of
ChiBR96-ADM conjugates was superior to that of optimized
adriamycin, mixtures of ChiBR96 and adriamycin, and equivalent
doses of non-binding conjugates. The ChiBR96-ADM conjugates were
approximately 6 fold more potent than unconjugated adriamycin.
Cures or complete regressions of established tumors were observed
in 50% of animals treated with doses of .gtoreq.2.5 mg/kg of
ChiBR96-ADM conjugate.
[0759] As shown in FIG. 60, ChiBR96-ADM conjugates (MR-7.88)
demonstrated antigen-specific antitumor activity against
established (75-125 mm.sup.3) MCF7 tumors. The activity of
ChiBR96-ADM conjugate administered at a dose of 5 mg/kg by either
the ip or iv route (4.2 LCK) was superior to that of optimized
adriamycin (1.4 LCK) or equivalent doses of non-binding IgG
conjugate (1.2 LCK). The antitumor activity of ChiBR96-ADM and
non-binding IgG-ADM conjugates is summarize in Table 17. The MTD of
ChiBR96-ADM conjugates like that of free adriamycin is lower in the
MCF7 model due to the estradiol supplementation required for tumor
growth.
[0760] The antigen-specific antitumor activity and dose response of
ChiBR96-ADM conjugates was also evaluated in the RCA human colon
carcinoma model. RCA tumors are less sensitive to unconjugated
adriamycin than are L2987 and MCF7 tumors. In addition, as
described previously, RCA tumors have a longer tumor volume
doubling time than L2987 or MCF7 tumors, are more poorly
vascularized, and the localization of radiolabelled BR64 antibody
is lower in RCA tumors than in L2987 tumors. As Shown in FIG. 61,
the antitumor activity of the ChiBR96-ADM conjugate (MR=7.88)
administered at a dose of 10 mg/kg was superior to that of
adriamycin and an equivalent dose of non-binding IgG conjugate
(MR-7.16). As shown in table 18, the ChiBR96-ADM conjugate tested
at a dose of 10 mg/kg produced antitumor activity equivalent to
>3 LCK. At this dose of ChiBR96-ADM conjugate, 80% cures and 11%
partial tumor regressions occurred. In this experiment,
unconjugated adriarnycin showed antitumor activity, equivalent to
0.4 LCK. Thus, in this experiment, the BR96-ADM conjugate produced
89% cures of established tumors whereas unconjugated adriamycin was
inactive.
19TABLE 18 Summary of Antitumor Activity of ChiBR96-ADM Thioether
Conjugates Evaluated Against Established RCA Human Colon Tumor
Xenografts % Tumor Dose (mg/kg).sup.a Regressions Conjugate ADM
ChiBR96 Route LCK PR CR Cure No. of Mice ChiBR96-ADM-7.88 10 350 ip
>3 11 0 89 9 5 175 ip 0.6 11 22 11 9 2.5 85 ip 0.2 0 0 0 9
IgG-ADM-7.16 2.5 85 iv 0.6 11 0 0 9 Adriamycin 10 405 ip 0 0 0 0 9
8 0 iv 0.4 0 0 0 9 .sup.aAll therapy administered q4dx3
[0761] In summary, the ChiBR96-ADM conjugate demonstrated
antigen-specific antitumor activity in the RCA human colon tumor
model. Cures and complete regressions of established RCA tumors
were observed following the administration of ChiBR96-ADM conjugate
at doses of 5-10 mg/kg.
[0762] The invention has been described with reference to specific
examples, materials and data. As one skilled in the art will
appreciate, alternate means for using or preparing the various
aspects of the invention may be available. Such alternate means are
to be construed as included within the intent and spirit of the
present invention as defined by the following claims.
Sequence CWU 1
1
4 1 33 DNA Artificial Description of Artificial Sequence Plasmid 1
gctagacata tggaggtgca gctggtggag tct 33 2 33 DNA Artificial
Description of Artificial Sequence Plasmid 2 gctgtggaga ctggcctggt
ttctgcaggt acc 33 3 720 DNA Mus musculus CDS (1)..(720) 3 atg gag
gtg cag ctg gtg gag tct ggg gga ggc tta gtg cag cct ggg 48 Met Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15
tcc ctg aaa gtc tcc tgt gta acc tct gga ttc act ttc agt gac tat 96
Ser Leu Lys Val Ser Cys Val Thr Ser Gly Phe Thr Phe Ser Asp Tyr 20
25 30 tac atg tgg gtt cgc cag act cca gag aag agg ctg gag tgg gtc
gca 144 Tyr Met Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val
Ala 35 40 45 tac att agt caa ggt gat ata acc gac tat cca gac act
gta aag ggt 192 Tyr Ile Ser Gln Gly Asp Ile Thr Asp Tyr Pro Asp Thr
Val Lys Gly 50 55 60 cga ttc acc atc tcc aga gac aat aag aac acc
ctg tac ctg caa atg 240 Arg Phe Thr Ile Ser Arg Asp Asn Lys Asn Thr
Leu Tyr Leu Gln Met 65 70 75 80 agc cgt ctg aag tct gag gac aca gcc
atg tat tgt gca aga ggc ctg 288 Ser Arg Leu Lys Ser Glu Asp Thr Ala
Met Tyr Cys Ala Arg Gly Leu 85 90 95 gac gac ggg gcc tgg ttt gct
tac tgg ggc caa ggg acc acg acc gtc 336 Asp Asp Gly Ala Trp Phe Ala
Tyr Trp Gly Gln Gly Thr Thr Thr Val 100 105 110 tcc tca gga tcc gga
ggt gga ggt tct ggt gga ggt gga tct gga ggt 384 Ser Ser Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 gga tct aag
ctt gat gtt ttg atg acc caa att cca gtc tcc ctg cct 432 Gly Ser Lys
Leu Asp Val Leu Met Thr Gln Ile Pro Val Ser Leu Pro 130 135 140 gtc
agt ctt gga caa gcg tcc atc tct tgc aga tct agt cag atc att 480 Val
Ser Leu Gly Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ile Ile 145 150
155 160 gta cat aat aat ggc aac acc tta gaa tgg tac ctg cag aaa cca
ggc 528 Val His Asn Asn Gly Asn Thr Leu Glu Trp Tyr Leu Gln Lys Pro
Gly 165 170 175 cag tct cca cag ctc ctg atc tac aaa gtt aac cga ttt
tct ggg gtc 576 Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Asn Arg Phe
Ser Gly Val 180 185 190 cca gac agg ttc agc ggc agt gga tca ggg aca
gat ttc ctc aag atc 624 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Leu Lys Ile 195 200 205 agc aga gtg gag gct gag gat ctg gga
gtt tat tac tgc ttt caa gtt 672 Ser Arg Val Glu Ala Glu Asp Leu Gly
Val Tyr Tyr Cys Phe Gln Val 210 215 220 cat gtt cca ttc acg ttc ggc
tcg ggg acc aag ctg gag atc aaa cgc 720 His Val Pro Phe Thr Phe Gly
Ser Gly Thr Lys Leu Glu Ile Lys Arg 225 230 235 240 4 240 PRT Mus
musculus 4 Met Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly 1 5 10 15 Ser Leu Lys Val Ser Cys Val Thr Ser Gly Phe Thr
Phe Ser Asp Tyr 20 25 30 Tyr Met Trp Val Arg Gln Thr Pro Glu Lys
Arg Leu Glu Trp Val Ala 35 40 45 Tyr Ile Ser Gln Gly Asp Ile Thr
Asp Tyr Pro Asp Thr Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg
Asp Asn Lys Asn Thr Leu Tyr Leu Gln Met 65 70 75 80 Ser Arg Leu Lys
Ser Glu Asp Thr Ala Met Tyr Cys Ala Arg Gly Leu 85 90 95 Asp Asp
Gly Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Thr Thr Val 100 105 110
Ser Ser Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115
120 125 Gly Ser Lys Leu Asp Val Leu Met Thr Gln Ile Pro Val Ser Leu
Pro 130 135 140 Val Ser Leu Gly Gln Ala Ser Ile Ser Cys Arg Ser Ser
Gln Ile Ile 145 150 155 160 Val His Asn Asn Gly Asn Thr Leu Glu Trp
Tyr Leu Gln Lys Pro Gly 165 170 175 Gln Ser Pro Gln Leu Leu Ile Tyr
Lys Val Asn Arg Phe Ser Gly Val 180 185 190 Pro Asp Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Leu Lys Ile 195 200 205 Ser Arg Val Glu
Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Val 210 215 220 His Val
Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 225 230 235
240
* * * * *