U.S. patent application number 10/206361 was filed with the patent office on 2002-12-12 for polyspecific immunoconjugates and antibody compositiesfor targeting the multidrug resistant phenotype.
This patent application is currently assigned to IMMUNOMEDICS, INC.. Invention is credited to Goldenberg, David M..
Application Number | 20020187141 10/206361 |
Document ID | / |
Family ID | 23098570 |
Filed Date | 2002-12-12 |
United States Patent
Application |
20020187141 |
Kind Code |
A1 |
Goldenberg, David M. |
December 12, 2002 |
Polyspecific immunoconjugates and antibody compositiesfor targeting
the multidrug resistant phenotype
Abstract
Polyspecific immunoconjugates and antibody composites that bind
a multidrug transporter protein and an antigen associated with a
tumor or infectious agent are used to overcome the multidrug
resistant phenotype. These immunoconjugates and composites also can
be used diagnostically to determine whether the failure of
traditional chemotherapy is due to the presence of multidrug
resistant tumor cells, multidrug resistant HIV-infected cells or
multidrug resistant infectious agents.
Inventors: |
Goldenberg, David M.;
(Mendham, NJ) |
Correspondence
Address: |
FOLEY AND LARDNER
SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
IMMUNOMEDICS, INC.
|
Family ID: |
23098570 |
Appl. No.: |
10/206361 |
Filed: |
July 29, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10206361 |
Jul 29, 2002 |
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08629388 |
Apr 8, 1996 |
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6468530 |
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08629388 |
Apr 8, 1996 |
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08286403 |
Aug 5, 1994 |
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5532889 |
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Current U.S.
Class: |
424/130.1 ;
424/1.49; 424/9.6; 530/388.1; 530/391.1 |
Current CPC
Class: |
A61P 35/00 20180101;
B82Y 5/00 20130101; A61P 31/18 20180101; C07K 16/28 20130101; C07K
2317/31 20130101; A61P 31/00 20180101; A61K 51/1084 20130101; C07K
16/3007 20130101; A61K 2039/505 20130101; A61K 51/1093 20130101;
A61K 47/68 20170801; A61K 47/6898 20170801 |
Class at
Publication: |
424/130.1 ;
424/1.49; 424/9.6; 530/388.1; 530/391.1 |
International
Class: |
A61K 051/00; A61K
049/00; A61K 039/395; C07K 016/46 |
Claims
What is claimed is:
1. A polyspecific immunoconjugate comprising: (a) at least one
antibody component that binds with a first epitope of a multidrug
transporter protein; (b) at least one antibody component that binds
with a first epitope of an antigen, wherein said antigen is
associated with a tumor or an infectious agent; and (c) at least
one diagnostic or therapeutic agent.
2. The polyspecific immunoconjugate of claim 1, wherein said
antibody components are selected from the group consisting of: (a)
a murine monoclonal antibody; (b) a humanized antibody derived from
(a); (c) a human monoclonal antibody; (d) a subhuman primate
antibody; and (e) an antibody fragment derived from (a), (b), (c)
or (d).
3. The polyspecific immunoconjugate of claim 2, wherein said
antibody fragment is selected from the group consisting of
F(ab').sub.2, F(ab).sub.2, Fab', Fab, Fv, sFv and minimal
recognition unit.
4. The polyspecific immunoconjugate of claim 3, wherein said
multidrug transporter protein is selected from the group consisting
of P-glycoprotein, OtrB, Tel(L), Mmr, ActII, TcmA, NorA, QacA,
CmlA, Bcr, EmrB, EmrD, AcrE, EnvD, MexB, Smr, QacE, MvrC, MsrA,
DrrA, DrrB, TlrC, Bmr, TetA and OprK.
5. The polyspecific immunoconjugate of claim 4, wherein said
diagnostic agent is selected from the group consisting of
radioactive label, photoactive agent or dye, florescent label,
enzyme label, bioluminescent label, chemiluminescent label,
colloidal gold and paramagnetic ion.
6. The polyspecific immunoconjugate of claim 5, wherein said
radioactive label is selected from the group consisting of
.gamma.-emitters and positron-emitters.
7. The polyspecific immunoconjugate of claim 6, wherein said
.gamma.-emitters have a gamma radiation emission peak in the range
of 50-500 Kev.
8. The polyspecific immunoconjugate of claim 7, wherein said
.gamma.-emitters with a gamma radiation emission peak in the range
of 50-500 Kev are selected from the group consisting of .sup.99mTc,
.sup.67Ga, .sup.123I, .sup.125I and .sup.131I.
9. The polyspecific immunoconjugate of claim 4, wherein said
therapeutic agent is selected from the group consisting of
radioisotope, boron addend, immunomodulator, toxin, photoactive
agent or dye, cancer chemotherapeutic drug, antiviral drug,
antifungal drug, antibacterial drug, antiprotozoal drug and
chemosensitizing agent.
10. The polyspecific immunoconjugate of claim 9, wherein said
radioisotope is selected from the group consisting of
.alpha.-emitters, .beta.-emitters, .gamma.-emitters, Auger electron
emitters, neutron capturing agents that emit a particles and
radioisotopes that decay by electron capture.
11. The polyspecific immunoconjugate of claim 9, wherein said
radioisotope is selected from the group consisting of .sup.198Au,
.sup.32P, .sup.125I, .sup.131I, .sup.90Y, .sup.186Re, .sup.188Re,
.sup.67Cu and .sup.211At.
12. The polyspecific immunoconjugate of claim 4, further comprising
an antibody component that binds with a second epitope of said
multidrug transporter protein.
13. The polyspecific immunoconjugate of claim 12, further
comprising an antibody component that binds with a second epitope
of said tumor or infectious agent associated antigen, or with an
epitope of a second antigen associated with said tumor or said
infectious agent.
14. The polyspecific immunoconjugate of any one of claims 4, 12 or
13, further comprising an immunomodulator, wherein said
immunomodulator is selected from the group consisting of cytokines,
stem cell growth factors and hematopoietic factors.
15. A method for treating a mammal having either a multidrug
resistant tumor that expresses a tumor associated antigen or a
multidrug resistant disease caused by an infectious agent, said
method comprising the step of administering a polyspecific
immunoconjugate to the mammal, wherein said polyspecific
immunoconjugate comprises: (a) at least one antibody component that
binds with a first epitope of a multidrug transporter protein, (b)
at least one antibody component that binds with a first epitope of
an antigen, wherein said antigen is associated with said tumor or
said infectious agent, and (c) at least one therapeutic agent.
16. The method of claim 15, wherein said antibody components are
selected from the group consisting of: (a) a murine monoclonal
antibody; (b) a humanized antibody derived from (a); (c) a human
monoclonal antibody; (d) a subhuman primate antibody; and (e) an
antibody fragment derived from (a), (b), (c) or (d).
17. The method of claim 16, wherein said antibody fragment is
selected from the group consisting of F(ab').sub.2, F(ab).sub.2,
Fab', Fab, Fv, sFv and minimal recognition unit.
18. The method of claim 15, wherein said multidrug transporter
protein is selected from the group consisting of P-glycoprotein,
OtrB, Tel(L), Mmr, ActII, TcmA, NorA, QacA, CmlA, Bcr, EmrB, EmrD,
AcrE, EnvD, MexB, Smr, QacE, MvrC, MsrA, DrrA, DrrB, TlrC, Bmr,
TetA and OprK.
19. The method of claim 18, wherein said therapeutic agent is
selected from the group consisting of radioisotope, boron addend,
toxin, immunomodulator, photoactive agent or dye, cancer
chemotherapeutic drug, antiviral drug, antifungal drug,
antibacterial drug, antiprotozoal drug and a chemosensitizing
agent.
20. The method of claim 19, wherein said radioisotope is selected
from the group consisting of .alpha.-emitters, .beta.-emitters,
.gamma.-emitters, Auger electron emitters, neutron capturing agents
that emit .alpha.-particles and radioisotopes that decay by
electron capture.
21. The method of claim 19, wherein said radioisotope is selected
from the group consisting of .sup.198Au, .sup.32P, .sup.125I,
.sup.131I, .sup.90Y, .sup.186Re, .sup.188Re, .sup.67Cu and
.sup.211At.
22. The method of claim 19, further comprising the step of
administering a chemosensitizing agent to said mammal.
23. The method of claim 19, wherein said therapeutic agent is a
chemosensitizing agent.
24. The method of claim 23, further comprising the step of
administering a chemotherapeutic agent selected from the group
consisting of cancer chemotherapeutic drug, antibacterial drug,
antiviral drug, antifungal drug and antiprotozoal drug.
25. The method of claim 19, further comprising the step of
administering an immunomodulator, wherein said immunomodulator is
selected from the group consisting of cytokine, stem cell growth
factor and hematopoietic factor.
26. The method of claim 25, wherein said cytokine is
granulocyte-colony stimulating factor.
27. The method of claim 25, wherein said hematopoietic factor is
thrombopoietin.
28. The method of claim 25, wherein said immunomodulator is
administered prior to or simultaneously with said administration of
said polyspecific immunoconjugate.
29. The method of claim 25, wherein said immunomodulator is
administered subsequent to said administration of said polyspecific
immunoconjugate.
30. A method for detecting the location of multidrug resistant
(MDR) tumor cells, MDR HIV-infected cells or MDR infectious agents
in a mammal having a multidrug resistant disease caused by a tumor
or infectious agent, said method comprising the steps of: (a)
parenterally injecting the mammal with an antibody composite
comprising (1) at least one antibody component that binds a first
epitope of a multidrug transporter protein, and (2) at least one
antibody component that binds a first epitope of an antigen that is
associated with the tumor or the infectious agent, wherein said
antibody composite is conjugated with a biotin-binding-molecule or
with biotin; (b) parenterally injecting a clearing composition
comprised of: (i) biotin, when said antibody composite is
conjugated with a biotin-binding molecule, or (ii) a biotin-binding
molecule, when said antibody composite is conjugated with biotin,
and allowing said clearing composition to substantially clear said
antibody composite from sites that do not contain MDR tumor cells,
MDR HIV-infected cells or MDR infectious agents; and (c)
parenterally injecting a diagnostic composition comprised of: (i)
biotin, when said antibody composite is conjugated with a
biotin-binding molecule, or (ii) a biotin-binding molecule, when
said antibody composite is conjugated with biotin, and a diagnostic
agent which is conjugated with said biotin or said biotin-binding
molecule.
31. The method of claim 30, wherein said antibody components are
selected from the group consisting of: (a) a murine monoclonal
antibody; (b) a humanized antibody derived from (a); (c) a human
monoclonal antibody; (d) a subhuman primate antibody; and (e) an
antibody fragment derived from (a), (b), (c) or (d).
32. The method of claim 31, wherein said antibody fragment is
selected from the group consisting of F(ab').sub.2, F(ab).sub.2,
Fab', Fab, Fv, sFv and minimal recognition unit.
33. The method of claim 32, wherein said multidrug X transporter
protein is selected from the group consisting of P-glycoprotein,
OtrB, Tel(L), Mmr, ActII, TcmA, NorA, QacA, CmlA, Bcr, EmrB, EmrD,
AcrE, EnvD, MexB, Smr, QacE, MvrC, MsrA, DrrA, DrrB, TlrC, Bmr,
TetA and OprK.
34. The method of claim 33, wherein said diagnostic agent is
selected from the group consisting of radioactive label,
photoactive agent or dye, fluorescent label and paramagnetic
ion.
35. The method of claim 34, wherein said radioactive label is
selected from the group consisting of .gamma.-emitters and
positron-emitters.
36. The method of claim 35, wherein said .gamma.-emitters have a
gamma radiation emission peak in the range of 50-500 Kev.
37. The method of claim 36, wherein said .gamma.-emitters with a
gamma radiation emission peak in the range of 50-500 Kev are
selected from the group consisting of .sup.99mTc, .sup.67Ga,
.sup.123I, .sup.125I and .sup.131I.
38. The method of claim 37, wherein said biotin-binding molecule is
avidin or streptavidin.
39. A method for treating a mammal having a multidrug resistant
disease caused by a tumor or infectious agent, said method
comprising the steps of: (a) parenterally injecting the mammal with
an antibody composite comprising (1) at least one antibody
component that binds a first epitope of a multidrug transporter
protein, and (2) at least one antibody component that binds a first
epitope of an antigen that is associated with the tumor or the
infectious agent, wherein said antibody composite is conjugated
with a biotin-binding molecule or with biotin; (b) parenterally
injecting a clearing composition comprised of: (i) biotin, when
said antibody composite is conjugated with a biotin-binding
molecule, or (ii) a biotin-binding molecule, when said antibody
composite is conjugated with biotin, and allowing said clearing
composition to substantially clear said antibody composite from
sites that do not contain multidrug resistant (MDR) cells or MDR
infectious agents; and (c) parenterally injecting, a therapeutic
composition comprised of: (i) biotin, when said antibody composite
is conjugated with a biotin-binding molecule, or (ii) a
biotin-binding molecule, when said antibody composite is conjugated
with biotin, and a therapeutic agent which is conjugated with said
biotin or said biotin-binding molecule.
40. The method of claim 39, wherein said biotin-binding molecule is
avidin or streptavidin.
41. The method of claim 40, wherein said antibody components are
selected from the group consisting of: (a) a murine monoclonal
antibody; (b) a humanized antibody derived from (a); (c) a human
monoclonal antibody; (d) a subhuman primate antibody; and (e) an
antibody fragment derived from (a), (b), (c) or (d).
42. The-method of claim 41, wherein said antibody fragment is
selected from the group consisting of F(ab').sub.2, F(ab).sub.2,
Fab', Fab, Fv, sFv and minimal recognition unit.
43. The method of claim 42, wherein said multidrug transporter
protein is selected from the group consisting of P-glycoprotein,
OtrB, Tel(L), Mmr, ActII, TcmA, NorA, QacA, CmlA, Bcr, EmrB, EmrD,
AcrE, EnvD, MexB, Smr, QacE, MvrC, MsrA, DrrA, DrrB, TlrC, Bmr,
TetA and OprK.
44. The method of claim 43, wherein said therapeutic agent is
selected from the group consisting of radioisotope, boron addend,
toxin, immunomodulator, photoactive agent or dye, cancer
chemotherapeutic drug, antiviral drug, antifungal drug,
antibacterial drug, antiprotozoal drug and a chemosensitizing
agent.
45. The method of claim 44, wherein said radioisotope is selected
from the group consisting of .alpha.-emitters, .beta.-emitters,
.gamma.-emitters, Auger electron emitters, neutron capturing agents
that emit .alpha.-particles and radioisotopes that decay by
electron capture.
46. The method of claim 44, wherein said radioisotope is selected
from the group consisting of .sup.198Au, .sup.32P, .sup.125I,
.sup.131I, .sup.90Y, .sup.186Re, .sup.188Re, .sup.67Cu and
.sup.211At.
47. A method for detecting the presence of multidrug resistant
(MDR) tumor cells, MDR HIV-infected cells or MDR infectious agents
in a mammal, said method comprising: (a) removing from the mammal a
biological sample that is suspected of containing MDR tumor cells,
MDR HIV-infected cells or MDR infectious agents; (b) contacting
said biological sample with an antibody composite which comprises
(1) at least one antibody component that binds with a first epitope
of a multidrug transporter protein, and (2) at least one antibody
component that binds with a first epitope of an antigen that is
associated with said tumor or said infectious agent, wherein said
contacting is performed under conditions which allow the binding of
said antibody composite to said biological sample; and (c)
detecting any of said bound antibody composite.
48. The method of claim 47, wherein said antibody components are
selected from the group consisting of: (a) a murine monoclonal
antibody; (b) a humanized antibody derived from (a); (c) a human
monoclonal antibody; (d) a subhuman primate antibody; and (e) an
antibody fragment derived from (a), (b), (c) or (d).
49. The method of claim 48, wherein said antibody fragment is
selected from the group consisting of F(ab').sub.2, F(ab).sub.2,
Fab', Fab, Fv, sFv and minimal recognition unit.
50. The method of claim 49, wherein said multidrug transporter
protein is selected from the group consisting of P-glycoprotein,
OtrB, Tel(L), Mmr, ActII, TcmA, NorA, QacA, CmlA, Bcr, EmrB, EmrD,
AcrE, EnvD, MexB, Smr, QacE, MvrC, MsrA, DrrA, DrrB, TlrC, Bmr,
TetA and OprK.
51. The method of claim 50, wherein said antibody composite further
comprises a diagnostic agent selected from the group consisting of
radioisotope, fluorescent label, chemiluminescent label, enzyme
label, bioluminescent label and colloidal gold.
52. The method of claim 50, wherein said antibody composite further
comprises biotin or a biotin-binding molecule.
53. A method for detecting the location of multidrug resistant
(MDR) tumor cells, MDR HIV-infected cells or MDR infectious agents
in a mammal having a multidrug resistant disease caused by a tumor
or infectious agent, said method comprising the steps of: (a)
parenterally injecting the mammal with a polyspecific
immunoconjugate that comprises (1) at least one antibody component
that binds with a first epitope of a multidrug transporter protein,
(2) at least one antibody component that binds with a first epitope
of an antigen that is associated with the tumor or infectious
agent, and (3) a diagnostic agent; (b) parenterally injecting said
mammal with an antibody or antibody fragment that binds with said
polyspecific immunoconjugate in an amount that is sufficient to
decrease the level of circulating polyspecific immunoconjugate by
about 10-85% within 2 to 72 hours; (c) scanning said mammal with a
detector to locate the site or sites of uptake of said polyspecific
immunoconjugate.
54. The method of claim 53, wherein said antibody components are
selected from the group consisting of: (a) a murine monoclonal
antibody; (b) a humanized antibody derived from (a); (c) a human
monoclonal antibody; (d) a subhuman primate antibody; and (e) an
antibody fragment derived from (a), (b), (c) or (d).
55. The method of claim 54, wherein said antibody fragment is
selected from the group consisting of F(ab').sub.2, F(ab).sub.2,
Fab', Fab, Fv, sFv and minimal recognition unit.
56. The method of claim 55, wherein said multidrug transporter
protein is selected from the group consisting of P-glycoprotein,
OtrB, Tel(L), Mmr, ActII, TcmA, NorA, QacA, CmlA, Bcr, EmrB, EmrD,
AcrE, EnvD, MexB, Smr, QacE, MvrC, MsrA, DrrA, DrrB, TlrC, Bmr,
TetA and OprK.
57. The method of claim 56, wherein said diagnostic agent is
selected from the group consisting of radioactive label,
photoactive agent or dye, fluorescent label and paramagnetic
ion.
58. The method of claim 57, wherein said radioactive label is
selected from the group consisting of .gamma.-emitters and
positron-emitters.
59. The method of claim 58, wherein said .gamma.-emitters have a
gamma radiation emission peak in the range of 50-500 Kev.
60. The method of claim 59, wherein said .gamma.-emitters with a
gamma radiation emission peak in the range of 50-500 Kev are
selected from the group consisting of .sup.99mTc, .sup.67Ga,
.sup.123I, .sup.125I and .sup.131I.
61. A method for treating a mammal having a multidrug resistant
disease caused by a tumor or infectious agent, said method
comprising the steps of: (a) parenterally injecting the mammal with
a polyspecific immunoconjugate comprising (1) at least one antibody
component that binds with a first epitope of a multidrug
transporter protein, (2) at least one antibody component that binds
with a first epitope of an antigen that is associated with the
tumor or infectious agent, and (3) a therapeutic agent; and (b)
parenterally injecting said mammal with an antibody or antibody
fragment that binds with said polyspecific immunoconjugate in an
amount that is sufficient to decrease the level of circulating
polyspecific immunoconjugate by about 10-85% within 2 to 72
hours.
62. The method of claim 61, wherein said antibody components are
selected from the group consisting of: (a) a murine monoclonal
antibody; (b) a humanized antibody derived from (a); (c) a human
monoclonal antibody; (d) a subhuman primate antibody; and (e) an
antibody fragment derived from (a), (b), (c) or (d).
63. The method of claim 62, wherein said antibody fragment is
selected from the group consisting of F(ab').sub.2, F(ab).sub.2,
Fab', Fab, Fv, sFv and minimal recognition unit.
64. The method of claim 63, wherein said multidrug transporter
protein is selected from the group consisting of P-glycoprotein,
OtrB, Tel(L), Mmr, ActII, TcmA, NorA, QacA, CmlA, Bcr, EmrB, EmrD,
AcrE, EnvD, MexB, Smr, QacE, MvrC, MsrA, DrrA, DrrB, TlrC, Bmr,
TetA and OprK.
65. The method of claim 64, wherein said therapeutic agent is
selected from the group consisting of radioisotope, boron addend,
toxin, immunomodulator, photoactive agent or dye, cancer
chemotherapeutic drug, antiviral drug, antifungal drug,
antibacterial drug, antiprotozoal drug and a chemosensitizing
agent.
66. The method of claim 65, wherein said radioisotope is selected
from the group consisting of .alpha.-emitters, .beta.-emitters,
.gamma.-emitters, Auger electron emitters, neutron capturing agents
that emit .alpha.-particles and radioisotopes that decay by
electron capture.
67. The method of claim 65, wherein said radioisotope is selected
from the group consisting of .sup.198Au, .sup.32P, .sup.125I,
.sup.131I, .sup.90Y, .sup.186Re, .sup.188Re, .sup.67Cu and
.sup.211At.
68. A method for detecting the location of multidrug resistant
(MDR) tumor cells, MDR HIV-infected cells or MDR infectious agents
in a subject having a multidrug resistant disease caused by a tumor
or infectious agent, said method comprising the steps of: (a)
parenterally injecting the subject with a polyspecific
immunoconjugate comprising (1) at least one antibody component that
binds with a first epitope of a multidrug transporter protein, (2)
at least one antibody component that binds with a first epitope of
an antigen that is associated with a tumor or infectious agent, and
(3) a diagnostic agent; (b) surgically exposing or endoscopically
accessing the interior of the body cavity of said subject; and (c)
scanning said interior body cavity with a detection probe to detect
the sites of accretion of said polyspecific immunoconjugate.
69. The method of claim 68, wherein said antibody components are
selected from the group consisting of: (a) a murine monoclonal
antibody; (b) a humanized antibody derived from (a); (c) a human
monoclonal antibody; (d) a subhuman primate antibody; and (e) an
antibody fragment derived from (a), (b), (c) or (d).
70. The method of claim 69, wherein said antibody fragment is
selected from the group consisting of F(ab').sub.2, F(ab).sub.2,
Fab', Fab, Fv, sFv and minimal recognition unit.
71. The method of claim 70, wherein said multidrug transporter
protein is selected from the group consisting of P-glycoprotein,
OtrB, Tel(L), Mmr, ActII, TcmA, NorA, QacA, CmlA, Bcr, EmrB, EmrD,
AcrE, EnvD, MexB, Smr, QacE, MvrC, MsrA, DrrA, DrrB, TlrC, Bmr,
TetA and OprK.
72. The method of claim 71, wherein said diagnostic agent is a
radioisotope.
73. The method of claim 72, wherein said radioisotope is a
.gamma.-emitter or a positron-emitter.
74. The method of claim 72, wherein said diagnostic agent is a
photoactive agent or dye.
75. The method of claim 74, wherein said photoactive agent or dye
is detected by laser-induced fluorescence.
76. An antibody composite comprising: (a) at least one antibody
component that binds with a first epitope of a multidrug
transporter protein; and (b) at least one antibody component that
binds with a first epitope of an antigen, wherein said antigen is
associated with a tumor or an infectious agent.
77. The antibody composite of claim 76, wherein said antibody
components are selected from the, group consisting of: (a) a murine
monoclonal antibody; (b) a humanized antibody derived from (a); (c)
a human monoclonal antibody; (d) a subhuman primate antibody; and
(e) an antibody fragment derived from (a), (b), (c) or (d).
78. The antibody composite of claim 77, wherein said antibody
fragment is selected from the group consisting of F(ab').sub.2,
F(ab).sub.2, Fab', Fab, Fv, sFv and minimal recognition unit.
79. The antibody composite of claim 78, wherein said multidrug
transporter protein is selected from the group consisting of
P-glycoprotein, OtrB, Tel(L), Mmr, ActII, TcmA, NorA, QacA, CmlA,
Bcr, EmrB, EmrD, AcrE, EnvD, MexB, Smr, QacE, MvrC, MsrA, DrrA,
DrrB, TlrC, Bmr, TetA and OprK.
80. The antibody composite of claim 79, further comprising an
antibody component that binds with a second epitope of said
multidrug transporter protein.
81. The antibody composite of claim 80, further comprising an
antibody component that binds with a second epitope of said tumor
or infectious agent associated antigen, or with an epitope of a
second antigen associated with said tumor or said infectious
agent.
82. A method for treating a mammal having either a multidrug
resistant tumor that expresses a tumor associated antigen or a
multidrug resistant disease caused by an infectious agent, said
method comprising the step of administering an antibody composite
to the mammal, wherein said antibody composite comprises: (a) at
least one antibody component that binds with a first epitope of a
multidrug transporter protein, and (b) at least one antibody
component that binds with a first epitope of an antigen, wherein
said antigen is associated with said tumor or said infectious
agent.
83. The method of claim 82, wherein said antibody components are
selected from the group consisting of: (a) a murine monoclonal
antibody; (b) a humanized antibody derived from (a); (c) a human
monoclonal antibody; (d) a subhuman primate antibody; and (e) an
antibody fragment derived from (a), (b), (c) or (d).
84. The method of claim 83, wherein said antibody fragment is
selected from the group consisting of F(ab').sub.2, F(ab).sub.2,
Fab', Fab, Fv, sFv and minimal recognition unit.
85. The method of claim 84, wherein said multidrug transporter
protein is selected from the group consisting of P-glycoprotein,
OtrB, Tel(L), Mmr, ActII, TcmA, NorA, QacA, CmlA, Bcr, EmrB, EmrD,
AcrE, EnvD, MexB, Smr, QacE, MvrC, MsrA, DrrA, DrrB, TlrC, Bmr,
TetA and OprK.
86. The method of claim 85, further comprising the step of
administering a therapeutic agent to said mammal, wherein said
therapeutic agent is selected from the group consisting of cancer
chemotherapeutic drug, antiviral drug, antifungal drug,
antibacterial drug and antiprotozoal drug.
87. The method of claim 86, further comprising the step of
administering an immunomodulator, wherein said immunomodulator is
selected from the group consisting of cytokine, stem cell growth
factor and hematopoietic factor.
88. The method of claim 87, wherein said cytokine is
granulocyte-colony stimulating factor.
89. The method of claim 87, wherein said hematopoietic factor is
thrombopoietin.
90. The method of claim 87, wherein said immunomodulator is
administered prior to or simultaneously with said administration of
said antibody composite.
91. The method of claim 87, wherein said immunomodulator is
administered subsequent to said administration of said antibody
composite.
92. A method for treating a subject having a multidrug resistant
disease caused by a tumor or infectious agent, said method
comprising the steps of: (a) parenterally injecting the subject
with a polyspecific immunoconjugate comprising (1) at least one
antibody component that binds with a first epitope of a multidrug
transporter protein, (2) at least one antibody component that binds
with a first epitope of an antigen that is associated with a tumor
or infectious agent, and (3) a photoactive agent or dye; (b)
surgically exposing or endoscopically accessing the interior of the
body cavity of said subject;, and (c) treating sites of accretion
of said polyspecific immunoconjugate to light, wherein said
treatment activates said photoactive agent or dye.
93. The method of claim 92, wherein said antibody components are
selected from the group consisting of: (a) a murine monoclonal
antibody; (b) a humanized antibody derived from (a); (c) a human
monoclonal antibody; (d) a subhuman primate antibody; and (e) an
antibody fragment derived from (a), (b), (c) or (d).
94. The method of claim 93, wherein said antibody fragment is
selected from the group consisting of F(ab').sub.2, F(ab).sub.2,
Fab', Fab, Fv, sFv and minimal recognition unit.
95. The method of claim 94, wherein said multidrug transporter
protein is selected from the group consisting of P-glycoprotein,
OtrB, Tel(L), Mmr, ActII, TcmA, NorA, QacA, CmlA, Bcr, EmrB, EmrD,
AcrE, EnvD, MexB, Smr, QacE, MvrC, MsrA, DrrA, DrrB, TlrC, Bmr,
TetA and OprK.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to novel polyspecific
immunoconjugates that are useful for diagnosis and therapy of
diseases caused by cells that are multidrug resistant. In
particular, this invention relates to polyspecific immunoconjugates
that comprise at least one moiety that binds with a multidrug
transporter protein, at least one moiety that binds with a tumor
associated antigen or infectious agent antigen, and a therapeutic
or diagnostic agent. This invention also relates to methods of
diagnosis and therapy using the polyspecific immunoconjugates. This
invention further relates to diagnostic and therapeutic uses of
antibody composites comprising at least one moiety that binds with
a multidrug transporter protein, and at least one moiety that binds
with a tumor associated antigen or infectious agent antigen.
[0003] 2. Background
[0004] One of the major limitations of cancer chemotherapy is the
development of drug resistance by cancer cells. Despite initial
sensitivity to a particular chemotherapeutic agent, some tumors
become progressively unresponsive to the particular agent, or to
various chemotherapeutic agents. This phenomenon of acquired drug
resistance is believed to be due to the selection and growth of
drug resistant mutant tumor cells. See, for example, Deuchars et
al., Sem. Oncol. 16: 156 (1989).
[0005] Cultured cell lines and transplantable tumors have been used
to study the mechanism of acquired drug resistance in vitro. These
studies have shown that under certain selection conditions, cells
may acquire simultaneous resistance to a diverse group of drugs
that are unrelated to the selecting agent in structure, cellular
target and mode of action. See, for example, Bradley et al.,
Biochim. Biophys. Acta 948: 87 (1988); Deuchars et al., supra. Many
of the drugs affected by this "multidrug-resistance" (MDR)
phenotype are important in current treatment protocols, such as
vincristine, actinomycin D, and adriamycin. Id.
[0006] The MDR phenotype is consistently associated with
over-expression of a 170 kilodalton membrane glycoprotein,
designated "gp170" or "P-glycoprotein." Endicott et al., Ann. Rev.
Biochem. 58: 137 (1989); Kane et al., J. Bioenerg. Biomembr. 22:
593 (1990); Efferth et al., Urol. Res. 18: 309 (1990). Studies
indicate that P-glycoprotein is a transmembrane protein responsible
for an ATP-dependent efflux of a broad spectrum of structurally and
functionally distinct drugs from multidrug-resistant cells. Riordan
et al., Pharmacol. Ther. 28: 51 (1985). In fact, expression of
P-glycoprotein has been shown to be predictive of a poor response
to chemotherapy in a number of neoplasms. See, for example, Pearson
et al., J. Nat'l Cancer Inst. 83: 1386 (1991).
[0007] Recent observations indicate that infectious agents can
induce the MDR phenotype in noncancerous cells. For example,
prolonged treatment with 3'-azido-3'-deoxythymidine (AZT) for human
immunodeficiency virus (HIV) infection is associated with an
acquired resistance to AZT. Gollapudi et al., Biochem. Biophys.
Res. Commun. 171: 1002 (1990); Antonelli et al., AIDS Research and
Human Retroviruses 8: 1839 (1992). In vitro studies demonstrate
that HIV-infected human cells have an increased expression of
P-glycoprotein and accumulate less AZT, compared with non-infected
control cells. Id.; Gupta et al., J. Clin. Immunol. 13: 289 (1993).
Thus, overexpression of P-glycoprotein and the accompanying MDR
phenotype can impair chemotherapy with anti-viral drugs.
[0008] Considerable effort has been employed to overcome the
multidrug-resistant phenotype and thus, improve the efficacy of
chemotherapy. Most of these strategies have involved
pharmacological agents that enhance the intracellular accumulation
of the cancer drugs by biochemically inhibiting the multidrug
transporter. See, for example, Ford et al., Pharmacol. Rev. 42: 155
(1990). Examples of agents that modulate P-glycoprotein activity
include calcium channel blockers, calmodulin inhibitors,
antiarrythmics, antimalarials, various lysoosmotropic agents,
steroids, antiestrogens, and cyclic peptide antibiotics.
Rittmann-Grauer et al., Cancer Res. 52: 1810 (1992).
[0009] However, multidrug-resistant reversing drugs used in early
clinical trials have shown major side effects unrelated to the
inhibition of P-glycoprotein, such as cardiac toxicity (verapamil)
or immunosuppression (cyclosporin A), which limit the dosage of
drug that can be administered. See, for example, Ozols et al., J.
Clin. Oncol. 5: 641 (1987); Dalton et al., J. Clin. Oncol. 7: 415
(1989); Cano-Gauci et al., Biochem. Pharmacol. 36: 2115 (1987);
Ford et al., supra. Thus, there has been limited success in
reversing MDR in vivo due to the toxicity of many of these small
modulators. See, for example, Rittmann-Grauer et al., supra.
[0010] The use of antibody-drug conjugates provides an alternative
approach to overcoming the MDR phenotype. For example, in vitro
studies have shown that MDR can be partially overcome by
conjugating the resistant drug to an antitumor antibody to increase
uptake and subsequent cell death. Durrant et al., Brit. J. Cancer
56: 722 (1987); Sheldon et al., Anticancer Res. 9: 637 (1989). This
approach, however, lacks specificity for tumor cells that express
the MDR phenotype.
[0011] A more targeted approach to overcoming the MDR phenotype is
to use antibodies or antibody conjugates that bind with
P-glycoprotein. For example, the administration of an
anti-P-glycoprotein monoclonal antibody and a resistant drug can
increase the survival time of nude mice that carry human tumor
cells. Pearson et al., J. Nat'l Cancer Inst. 83: 1386 (1991);
Iwahashi et al., Cancer Res. 53: 5475 (1993). Also, see Grauer et
al., international publication No. WO 93/02105 (1993). In addition,
an anti-P-glycoprotein monoclonal antibody-Pseudomonas toxin
conjugate has been shown to kill multidrug-resistant human cells in
vitro. FitzGerald et al., Proc. Nat'l Acad. Sci. USA 84: 4288
(1987). Also, see Efferth et al., Med. Oncol. & Tumor
Pharmacother. 9: 11 (1992), and Mechetner et al., international
publication No. WO 93/19094 (1993).
[0012] Similarly, investigators have produced bispecific antibodies
comprising a P-glycoprotein binding moiety and a moiety that binds
with a cytotoxic cell. van Dijk et al., Int. J. Cancer 44: 738
(1989); Ring et al., international Publication No. WO 92/08802
(1992). The theory behind this approach is that the bispecific
antibodies can be used to direct cytotoxic cells to
multidrug-resistant cells that express P-glycoprotein.
[0013] However, studies have shown that P-glycoprotein is expressed
in normal human tissues, such as liver, kidney, adrenal gland,
pancreas, colon and jejunum. See, for example, Endicott et al.,
Ann. Rev. Biochem. 58: 137 (1989). Consequently, investigators have
warned that "blocking P-glycoprotein action in order to circumvent
MDR will also affect the normally expressed P-glycoprotein and this
may cause unacceptable side toxic effects." Childs et al., "The MDR
Superfamily of Genes and Its Biological Implications," in IMPORTANT
ADVANCES IN ONCOLOGY 1994, DeVita et al., (eds.), pages 21-36 (J.B.
Lippincott Co. 1994). This admonition particularly applies to
therapeutic methods that use antibody conjugates consisting of a
P-glycoprotein binding moiety and a cytotoxic agent. Therefore, the
success of an antibody-directed treatment of MDR tumors will mainly
depend upon the ability to kill drug-resistant tumor cells with
tolerable side effects to normal tissues of the patient. Efferth et
al., Med. Oncol. & Tumor Pharmacother. 9: 11 (1992).
[0014] Thus, an need exists for a method to overcome the MDR
phenotype but that also minimizes toxicity to normal tissue.
[0015] The emergence of the MDR phenotype also is the major cause
of failure in the treatment of infectious diseases. Davies, Science
264: 375 (1994). In particular, pathogenic bacteria have active
drug efflux systems of very broad substrate specificity. Nikaido,
Science 264: 382 (1994), which is incorporated by reference. For
example, studies indicate that a drug efflux system plays a major
role in the intrinsic resistance of Psuedomonas aeruginosa, a
common opportunistic pathogen. Poole et al., Mol. Microbiol. 10:
529 (1993); Poole et al., J. Bacteriol. 175: 7363 (1993).
[0016] Recent studies indicate that bacterial drug efflux systems
are functionally similar to the mammalian MDR efflux pump. As an
illustration, both the Bacillus subtilis and the mammalian
multidrug transporters can be inhibited by reserpine and verapamil.
Neyfakh et al., Proc. Nat'l Acad. Sci. 88: 4781 (1991). Moreover,
investigators have recognized a superfamily of ATP-dependent
membrane transporters that includes prokaryotic permeases and
mammalian P-glycoprotein. Doige et al., Ann. Rev. Microbiol. 47:
291 (1993).
[0017] Active drug efflux as a mechanism for drug resistance is
significant in nonbacterial infectious agents. For instance, a
Plasmodium falciparum protein is involved in imparting resistance
to quinoline-containing drugs used for prophylaxis and treatment of
malaria. Id.; Bray, FEMS Microbiol. Lett. 113: 1 (1993). In
addition, drug resistance has been linked to active efflux in the
fungus, Aspergillus nidulans. de Waard et al., Pestic. Biochem.
Physiol. 13: 255 (1980).
[0018] Historically, the pharmaceutical industry has concentrated
on designing drugs to overcome specific mechanisms of MDR in
infectious agents, such as increased degradation of particular
drugs and inactivation of drugs by enzymatic modification of
specific groups. Nikaido et al., supra. However, in the future,
general mechanisms of MDR, such as active drug efflux, are likely
to become more important in the clinical setting.
[0019] Thus, a need exists for methods that can be used to inhibit
the function of multidrug transporter proteins expressed by
infectious agents.
SUMMARY OF THE INVENTION
[0020] Accordingly, it is an object of the present invention to
provide a method for overcoming the multidrug-resistant phenotype
that has a therapeutic index superior to conventional methods.
[0021] Another object of this invention is to provide methods for
selectively targeting diagnostic and therapeutic agents to
multidrug-resistant cells, while avoiding major toxic side effects
to normal organs.
[0022] Another object of this invention is to provide antibody
composites that bind a multidrug transporter protein and an antigen
associated with a tumor or infectious agent.
[0023] A further object of this invention is to provide
polyspecific immunoconjugates which are conjugates of antibody
composites and diagnostic or therapeutic agents.
[0024] These and other objects are achieved, in accordance with one
embodiment of the present invention by the provision of a
polyspecific immunoconjugate comprising:
[0025] (a) at least one antibody component that binds with a first
epitope of a multidrug transporter protein;
[0026] (b) at least one antibody component that binds with a first
epitope of an antigen, wherein the antigen is associated with a
tumor or an infectious agent; and
[0027] (c) at least one diagnostic or therapeutic agent.
[0028] The antibody components of such a polyspecific
immunoconjugate are selected from the group consisting of (a) a
murine monoclonal antibody; (b) a humanized antibody derived from
(a); (c) a human monoclonal antibody; (d) a subhuman primate
antibody; and (e) an antibody fragment derived from (a), (b), (c)
or (d), wherein the antibody fragment is selected from the group
consisting of F(ab').sub.2, F(ab).sub.2, Fab', Fab, Fv, sFv and
minimal recognition unit. The multidrug transporter protein of such
a polyspecific immunoconjugate is selected from the group
consisting of P-glycoprotein, OtrB, Tel(L), Mmr, ActII, TcmA, NorA,
QacA, CMlA, Bcr, EmrB, EmrD, AcrE, EnvD, MexB, Smr, QacE, MvrC,
MsrA, DrrA, DrrB, TlrC, Bmr, TetA and OprK.
[0029] As stated above, the polyspecific immunoconjugate comprises
a diagnostic or therapeutic agent. A suitable diagnostic agent is
selected from the group consisting of radioactive label,
photoactive agent or dye, florescent label, enzyme label,
bioluminescent label, chemiluminescent label, colloidal gold and
paramagnetic ion. Moreover, a suitable radioactive label may be a
.gamma.-emitter or a positron-emitter. Preferably, .gamma.-emitters
have a gamma radiation emission peak in the range of 50-500 Kev,
such as a radioisotope selected from the group consisting of
.sup.99mTc, .sup.67Ga, .sup.123I, .sup.125I and .sup.131I.
[0030] A suitable therapeutic agent is selected from the group
consisting of radioisotope, boron addend, immunomodulator, toxin,
photoactive agent or dye, cancer chemotherapeutic drug, antiviral
drug, antifungal drug, antibacterial drug, antiprotozoal drug and
chemosensitizing agent. Moreover a suitable therapeutic
radioisotope is selected from the group consisting of
.alpha.-emitters, .beta.-emitters, .gamma.-emitters, Auger electron
emitters, neutron capturing agents that emit .alpha.-particles and
radioisotopes that decay by electron capture. Preferably, the
radioisotope is selected from the group consisting of .sup.198Au,
.sup.32P, .sup.125I, .sup.131I, .sup.9OY, .sup.186Re, .sup.188Re,
.sup.67Cu and .sup.211At.
[0031] The present invention also contemplates polyspecific
immunoconjugates which further comprise an antibody component that
binds with a second epitope of the multidrug transporter protein.
Moreover, polyspecific immunoconjugates may additionally comprise
an antibody component that binds with a second epitope of the tumor
or infectious agent associated antigen, or with an epitope of a
second antigen associated with the tumor or the infectious
agent.
[0032] The present invention also is directed to a method for
treating a mammal having either a multidrug resistant tumor that
expresses a tumor associated antigen or a multidrug resistant
disease caused by an infectious agent, the method comprising the
step of administering a polyspecific immunoconjugate to the mammal,
wherein the polyspecific immunoconjugate comprises:
[0033] (a) at least one antibody component that binds with a first
epitope of a multidrug transporter protein,
[0034] (b) at least one antibody component that binds with a first
epitope of an antigen, wherein the antigen is associated with the
tumor or the infectious agent, and
[0035] (c) at least one therapeutic agent.
[0036] Moreover, the present invention contemplates methods further
comprising the administration of a chemosensitizing agent or
immunomodulator to the mammal.
[0037] In addition, the present invention is directed to a method
for detecting the location of multidrug resistant (MDR) tumor
cells, MDR HIV-infected cells or MDR infectious agents in a mammal
having a multidrug resistant disease caused by a tumor or
infectious agent, the method comprising the steps of:
[0038] (a) parenterally injecting the mammal with an antibody
composite comprising (1) at least one antibody component that binds
a first epitope of a multidrug transporter protein, and (2) at
least one antibody component that binds a first epitope of an
antigen that is associated with the tumor or the infectious agent,
wherein the antibody composite is conjugated with a biotin-binding
molecule or with biotin;
[0039] (b) parenterally injecting a clearing composition comprised
of:
[0040] (i) biotin, when the antibody composite is conjugated with a
biotin-binding molecule, or
[0041] (ii) a biotin-binding molecule, when the antibody composite
is conjugated with biotin,
[0042] and allowing the clearing composition to substantially clear
the antibody composite from sites that do not contain MDR tumor
cells, MDR HIV-infected cells or MDR infectious agents;
[0043] and
[0044] (c) parenterally injecting a diagnostic composition
comprised of:
[0045] (i) biotin, when the antibody composite is conjugated with a
biotin-binding molecule, or
[0046] (ii) a biotin-binding molecule, when the antibody composite
is conjugated with
[0047] and a diagnostic agent which is conjugated with the biotin
or the biotin-binding molecule.
[0048] In such a detection method, the diagnostic agent is selected
from the group consisting of radioactive label, photoactive agent
or dye, fluorescent label and paramagnetic ion. Moreover, the
biotin-binding molecule is avidin or streptavidin.
[0049] The present invention also contemplates a method for
treating a mammal having a multidrug resistant disease caused by a
tumor or infectious agent, the method comprising the steps of:
[0050] (a) parenterally injecting the mammal with an antibody
composite comprising (1) at least one antibody component that binds
a first epitope of a multidrug transporter protein, and (2) at
least one antibody component that binds a first epitope of an
antigen that is associated with the tumor or the infectious agent,
wherein the antibody composite is conjugated with a biotin-binding
molecule or with biotin;
[0051] (b) parenterally injecting a clearing composition comprised
of:
[0052] (i) biotin, when the antibody composite is conjugated with a
biotin-binding molecule, or
[0053] (ii) a biotin-binding molecule, when the antibody composite
is conjugated with biotin,
[0054] and allowing the clearing composition to substantially clear
the antibody composite from sites that do not contain multidrug
resistant (MDR) cells or MDR infectious agents; and
[0055] (c) parenterally injecting a therapeutic composition
comprised of:
[0056] (i) biotin, when the antibody composite is conjugated with a
biotin-binding molecule, or
[0057] (ii) a biotin-binding molecule, when the antibody composite
is conjugated with biotin,
[0058] and a therapeutic agent which is conjugated with the biotin
or the biotin-binding molecule.
[0059] A suitable therapeutic agent is selected from the group
consisting of radioisotope, boron addend, toxin, immunomodulator,
photoactive agent or dye, cancer chemotherapeutic drug, antiviral
drug, antifungal drug, antibacterial drug, antiprotozoal drug and a
chemosensitizing agent. Again, the biotin-binding molecule is
avidin or streptavidin.
[0060] The present invention also is directed to a method for
detecting the presence of multidrug resistant (MDR) tumor cells,
MDR HIV-infected cells or MDR infectious agents in a mammal, the
method comprising:
[0061] (a) removing from the mammal a biological sample that is
suspected of containing MDR tumor cells, MDR HIV-infected cells or
MDR infectious agents;
[0062] (b) contacting the biological sample with an antibody
composite which comprises (1) at least one antibody component that
binds with a first epitope of a multidrug transporter protein, and
(2) at least one antibody component that binds with a first epitope
of an antigen that is associated with the tumor or the infectious
agent, wherein the contacting is performed under conditions which
allow the binding of the antibody composite to the biological
sample; and
[0063] (c) detecting any of the bound antibody composite.
[0064] Here, a suitable diagnostic agent selected from the group
consisting of radioisotope, fluorescent label, chemiluminescent
label, enzyme label, bioluminescent label and colloidal gold.
Moreover, the antibody composite can further comprise biotin or a
biotin-binding molecule.
[0065] The present invention is further directed to a method for
detecting the location of multidrug resistant (MDR) tumor cells,
MDR HIV-infected cells or MDR infectious agents in a mammal having
a multidrug resistant disease caused by a tumor or infectious
agent, the method comprising the steps of:
[0066] (a) parenterally injecting the mammal with a polyspecific
immunoconjugate that comprises (1) at least one antibody component
that binds with a first epitope of a multidrug transporter protein,
(2) at least one antibody component that binds with a first epitope
of an antigen that is associated with the tumor or infectious
agent, and (3) a diagnostic agent;
[0067] (b) parenterally injecting the mammal with an antibody or
antibody fragment that binds with the polyspecific immunoconjugate
in an amount that is sufficient to decrease the level of
circulating polyspecific immunoconjugate by about 10-85% within 2
to 72 hours;
[0068] (c) scanning the mammal with a detector to locate the site
or sites of uptake of the polyspecific immunoconjugate.
[0069] A suitable diagnostic agent is selected from the group
consisting of radioactive label, photoactive agent or dye,
fluorescent label and paramagnetic ion.
[0070] The present invention also contemplates a method for
treating a mammal having a multidrug resistant disease caused by a
tumor or infectious agent, the method comprising the steps of:
[0071] (a) parenterally injecting the mammal with a polyspecific
immunoconjugate comprising (1) at least one antibody component that
binds with a first epitope of a multidrug transporter protein, (2)
at least one antibody component that binds with a first epitope of
an antigen that is associated with the tumor or infectious agent,
and (3) a therapeutic agent; and
[0072] (b) parenterally injecting the mammal with an antibody or
antibody fragment that binds with the polyspecific immunoconjugate
in an amount that is sufficient to decrease the level of
circulating polyspecific immunoconjugate by about 10-85% within 2
to 72 hours.
[0073] In addition, the present invention is directed to a method
for detecting the location of multidrug resistant (MDR) tumor
cells, MDR HIV-infected cells or MDR infectious agents in a subject
having a multidrug resistant disease caused by a tumor or
infectious agent, the method comprising the steps of:
[0074] (a) parenterally injecting the subject with a polyspecific
immunoconjugate comprising (1) at least one antibody component that
binds with a first epitope of a multidrug transporter protein, (2)
at least one antibody component that binds with a first epitope of
an antigen that is associated with a tumor or infectious agent, and
(3) a diagnostic agent;
[0075] (b) surgically exposing or endoscopically accessing the
interior of the body cavity of the subject; and
[0076] (c) scanning the interior body cavity with a detection probe
to detect the sites of accretion of the polyspecific
immunoconjugate.
[0077] Suitable diagnostic agents include radioisotopes, such as a
.gamma.-emitter or a positron-emitter, and a photoactive agent or
dye that is detected by laser-induced fluorescence.
[0078] The present invention also contemplates a method for
treating a subject having a multidrug resistant disease caused by a
tumor or infectious agent, the method comprising the steps of:
[0079] (a) parenterally injecting the subject with a polyspecific
immunoconjugate comprising (1) at least one antibody component that
binds with a first epitope of a multidrug transporter protein, (2)
at least one antibody component that binds with a first epitope of
an antigen that is associated with a tumor or infectious agent, and
(3) a photoactive agent or dye;
[0080] (b) surgically exposing or endoscopically accessing the
interior of the body cavity of the subject; and
[0081] (c) treating sites of accretion of the polyspecific
immunoconjugate to light, wherein the treatment activates the
photoactive agent or dye.
[0082] In addition, the present invention is directed to an
antibody composite comprising:
[0083] (a) at least one antibody component that binds with a first
epitope of a multidrug transporter protein; and
[0084] (b) at least one antibody component that binds with a first
epitope of an antigen, wherein the antigen is associated with a
tumor or an infectious agent.
[0085] Suitable antibody components of antibody composites are
selected from the group consisting of (a) a murine monoclonal
antibody; (b) a humanized antibody derived from (a); (c) a human
monoclonal antibody; (d) a subhuman primate antibody; and (e) an
antibody fragment derived from (a), (b), (c) or (d), where an
antibody fragment is selected from the group consisting of
F(ab').sub.2, F(ab).sub.2, Fab', Fab, Fv, sFv and minimal
recognition unit. Moreover, a suitable multidrug transporter
protein is selected from the group consisting of P-glycoprotein,
OtrB, Tel(L), Mmr, ActII, TcmA, NorA, QacA, CmlA, Bcr, EmrB, EmrD,
AcrE, EnvD, MexB, Smr, QacE, MvrC, MsrA, DrrA, DrrB, TlrC, Bmr,
TetA and OprK.
[0086] The present invention also contemplates an antibody
composite further comprising an antibody component that binds with
a second epitope of the multidrug transporter protein. An antibody
composite can additionally include an antibody component that binds
with a second epitope of the tumor or infectious agent associated
antigen, or with an epitope of a second antigen associated with the
tumor or the infectious agent.
[0087] The present invention is further directed to a method for
treating a mammal having either a multidrug resistant tumor that
expresses a tumor associated antigen or a multidrug resistant
disease caused by an infectious agent, the method comprising the
step of administering an antibody composite to the mammal, wherein
the antibody composite comprises:
[0088] (a) at least one antibody component that binds with a first
epitope of a multidrug transporter protein, and
[0089] (b) at least one antibody component that binds with a first
epitope of an antigen, wherein the antigen is associated with the
tumor or the infectious agent.
[0090] Moreover, the present invention contemplates a method
further comprising the step of administering a therapeutic agent to
the mammal, wherein the therapeutic agent is selected from the
group consisting of cancer chemotherapeutic drug, antiviral drug,
antifungal drug, antibacterial drug and antiprotozoal drug.
Finally, the present invention also is directed to a method which
further comprises the step of administering an immunomodulator,
wherein the immunomodulator is selected from the group consisting
of cytokine, stem cell growth factor and hematopoietic factor.
DETAILED DESCRIPTION
[0091] 1. Definitions
[0092] In the description that follows, a number of terms are used
extensively. The following definitions are provided to facilitate
understanding of the invention.
[0093] A structural gene is a DNA sequence that is transcribed into
messenger RNA (mRNA) which is then translated into a sequence of
amino acids characteristic of a specific polypeptide.
[0094] A promoter is a DNA sequence that directs the transcription
of a structural gene. Typically, a promoter is located in the 5'
region of a gene, proximal to the transcriptional start site of a
structural gene. If a promoter is an inducible promoter, then the
rate of transcription increases in response to an inducing agent.
In contrast, the rate of transcription is not regulated by an
inducing agent if the promoter is a constitutive promoter.
[0095] An isolated DNA molecule is a fragment of DNA that is not
integrated in the genomic DNA of an organism. For example, a cloned
T cell receptor gene is a DNA fragment that has been separated from
the genomic DNA of a mammalian cell. Another example of an isolated
DNA molecule is a chemically-synthesized DNA molecule that is not
integrated in the genomic DNA of an organism.
[0096] An enhancer is a DNA regulatory element that can increase
the efficiency of transcription, regardless of the distance or
orientation of the enhancer relative to the start site of
transcription.
[0097] Complementary DNA (cDNA) is a single-stranded DNA molecule
that is formed from an mRNA template by the enzyme reverse
transcriptase. Typically, a primer complementary to portions of
mRNA is employed for the initiation of reverse transcription. Those
skilled in the art also use the term "cDNA" to refer to a
double-stranded DNA molecule consisting of such a single-stranded
DNA molecule and its complementary DNA strand.
[0098] The term expression refers to the biosynthesis of a gene
product. For example, in the case of a structural gene, expression
involves transcription of the structural gene into mRNA and the
translation of mRNA into one or more polypeptides.
[0099] A cloning vector is a DNA molecule, such as a plasmid,
cosmid, or bacteriophage, that has the capability of replicating
autonomously in a host cell. Cloning vectors typically contain one
or a small number of restriction endonuclease recognition sites at
which foreign DNA sequences can be inserted in a determinable
fashion without loss of an essential biological function of the
vector, as well as a marker gene that is suitable for use in the
identification and selection of cells transformed with the cloning
vector. Marker genes typically include genes that provide
tetracycline resistance or ampicillin resistance.
[0100] An expression vector is a DNA molecule comprising a gene
that is expressed in a host cell. Typically, gene expression is
placed under the control of certain regulatory elements, including
constitutive or inducible promoters, tissue-specific regulatory
elements, and enhancers. Such a gene is said to be "operably linked
to" the regulatory elements.
[0101] A recombinant host may be any prokaryotic or eukaryotic cell
that contains either a cloning vector or expression vector. This
term also includes those prokaryotic or eukaryotic cells that have
been genetically engineered to contain the cloned gene(s) in the
chromosome or genome of the host cell.
[0102] A tumor associated antigen is a protein normally not
expressed, or expressed at very low levels, by a normal
counterpart. Examples of tumor associated antigens include
.alpha.-fetoprotein and carcinoembryonic antigen (CEA). Many other
illustrations of tumor associated antigens are known to those of
skill in the art. See, for example, Urban et al., Ann. Rev.
Immunol. 10: 617 (1992).
[0103] As used herein, an infectious agent denotes both microbes
and parasites. A "microbe" includes viruses, bacteria, rickettsia,
mycoplasma, protozoa, fungi and like microorganisms. A "parasite"
denotes infectious, generally microscopic or very small
multicellular invertebrates, or ova or juvenile forms thereof,
which are susceptible to antibody-induced clearance or lytic or
phagocytic destruction, such as malarial parasites, spirochetes,
and the like.
[0104] A multidrug transporter Protein is a membrane-associated
protein which transports diverse cytotoxic compounds out of a cell
in an energy-dependent manner. Examples of multidrug transporter
proteins include P-glycoprotein, OtrB, Tel(L), Mmr, ActII, TcmA,
NorA, QacA, CmlA, Bcr, EmrB, EmrD, AcrE, EnvD, MexB, Smr, QacE,
MvrC, MsrA, DrrA, DrrB, TlrC, Bmr, TetA, OprK, and the like.
[0105] An antibody fragment is a portion of an antibody such as
F(ab').sub.2, F(ab).sub.2, Fab', Fab, and the like. Regardless of
structure, an antibody fragment binds with the same antigen that is
recognized by the intact antibody.
[0106] The term "antibody fragment" also includes any synthetic or
genetically engineered protein that acts like an antibody by
binding to a specific antigen to form a complex. For example,
antibody fragments include isolated fragments consisting of the
light chain variable region, "Fv" fragments consisting of the
variable regions of the heavy and light chains, recombinant single
chain polypeptide molecules in which light and heavy variable
regions are connected by a peptide linker ("sFv proteins"), and
minimal recognition units consisting of the amino acid residues
that mimic the hypervariable region.
[0107] Humanized antibodies are recombinant proteins in which
murine complementary determining regions of monoclonal antibodies
have been transferred from heavy and light variable chains of the
murine immunoglobulin into a human variable domain.
[0108] As used herein, the term antibody component includes both an
entire antibody and an antibody fragment.
[0109] As used herein, a diagnostic or therapeutic agent is a
molecule or atom which is conjugated to an antibody moiety to
produce a conjugate which is useful for diagnosis or for therapy.
Examples of diagnostic or therapeutic agents include drugs, toxins,
immunomodulators, chelators, boron compounds, photoactive agents or
dyes, radioisotopes, fluorescent agents, paramagnetic ions or
molecules and marker moieties.
[0110] An antibody composite is a polyspecific antibody composition
comprising at least two substantially monospecific antibody
components, wherein at least one antibody component binds with an
epitope of a multidrug transporter protein, and wherein at least
one antibody component binds with an antigen that is associated
with either a tumor or an infectious agent.
[0111] A polyspecific immunoconjugate is a conjugate of an antibody
composite with a diagnostic or therapeutic agent.
[0112] 2. Production of Rodent Monoclonal Antibodies, Humanized
Antibodies, Primate Antibodies and Human Antibodies
[0113] An antibody composite of the present invention may be
derived from a rodent monoclonal antibody (MAb) Rodent monoclonal
antibodies to specific antigens may be obtained by methods known to
those skilled in the art. See, for example, Kohler and Milstein,
Nature 256: 495 (1975), and Coligan et al. (eds.), CURRENT
PROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley
& Sons 1991) [hereinafter "Coligan"]. Briefly, monoclonal
antibodies can be obtained by injecting mice with a composition
comprising an antigen, verifying the presence of antibody
production by removing a serum sample, removing the spleen to
obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells
to produce hybridomas, cloning the hybridomas, selecting positive
clones which produce antibodies to the antigen, culturing the
clones that produce antibodies to the antigen, and isolating the
antibodies from the hybridoma cultures.
[0114] MAbs can be isolated and purified from hybridoma cultures by
a variety of well-established techniques. Such isolation techniques
include affinity chromatography with Protein-A Sepharose,
size-exclusion chromatography, and ion-exchange chromatography.
See, for example, Coligan at pages 2.7.1-2.7.12 and pages
2.9.1-2.9.3. Also, see Baines et al., "Purification of
Immunoglobulin G (IgG)," in METHODS IN MOLECULAR BIOLOGY, VOL. 10,
pages 79-104 (The Humana Press, Inc. 1992).
[0115] A wide variety of monoclonal antibodies against tumor
associated antigens or infectious agents have been developed. See,
for example, Goldenberg et al., international application
publication No. WO 91/11465 (1991), Hansen et al., international
application publication No. Wo 93/23062, and Goldenberg,
international application publication No. WO 94/04702 (1994), each
of which is incorporated by reference in its entirety.
[0116] Furthermore, such antibodies are readily available from
commercial sources. For example, rodent monoclonal antibodies that
bind with adenocarcinoma-associated antigen (Cat. No. 121730),
human chorionic gonadotropin (Cat. No. 230740), carcinoembryonic
antigen (Cat. Nos. 215920 and 215922), human alpha-fetoprotein
(Cat. No. 341646), and the like can be obtained from
Calbiochem-Novabiochem Corp. (San Diego, Calif.). Moreover, rodent
monoclonal antibodies that bind with antigenic determinants of
infectious agents such as Escherichia coli (HB 8178), Legionella
pneumophila (CRL 1770), Schistosoma mansoni (HB 8088),
Streptococcus, Group A (HB 9696), Treponema pallidum (HB 8134),
hepatitis B (CRL 8017), herpes simplex (HB 8181), human
immunodeficiency virus (HB 9101), among others, can be obtained
from American Type Culture Collection (Rockville, Md.).
Furthermore, murine monoclonal antibodies against merozoites and
sporozoites of Plasmodium falciparum can be prepared as described
by Goldenberg, U.S. Pat. No. 5,332,567 (1994), which is
incorporated by reference.
[0117] Methods for producing P-glycoprotein antibodies are
well-known to those of skill in the art. See, for example, Lathan
et al., Cancer Res. 45: 5064 (1985); Kartner et al., Nature 316:
820 (1985); Hamada et al., Proc. Nat'l Acad. Sci 83: 7785 (1986);
Scheper et al., Int. J. Cancer 42: 389 (1988); Rittmann-Grauer et
al., Cancer Res. 52: 1810 (1992); Ling et al., U.S. Pat. No.
4,837,306 (1989); Ring et al., international publication No. WO
92/08802; Grauer et al., international publication No. WO 93/02105;
and Mechetner et al., international publication No. WO 93/19094,
which are incorporated by reference. Since P-glycoprotein retains
its structural identity across different mammalian species (Rubin,
U.S. Pat. No. 5,005,588; Kane et al., J. Bioenergetics and
Biomembranes 22: 593 (1990)), antibodies raised against
P-glycoprotein from non-human cells can be used for diagnosis and
therapy in humans. Conversely, antibodies raised against human
P-glycoprotein should be suitable for veterinary uses.
[0118] Preferred P-glycoprotein antibodies bind with the
extracellular domain of P-glycoprotein, and can be produced against
cells that express the MDR phenotype as described, for example, by
Mechetner et al., supra, and Rittmann-Grauer et al., supra.
Alternatively, such antibodies can be obtained using peptides that
contain an extracellular epitope P-glycoprotein. See, for example,
Cianfriglia et al., international publication No. WO 93/25700,
which is incorporated by reference.
[0119] Those of skill in the art can readily apply standard
techniques to produce antibodies against multidrug transporter
proteins of infectious agents. Suitable antigens include multidrug
transporter proteins such as Bmr, TetA, EmrB, OprK, Smr, and the
like. See, for example, Nikaido et al., supra; Poole et al., J.
Bacteriol. 175: 7363 (1993); and Childs et al., "The MDR
Superfamily of Genes and Its Biological Implications," in IMPORTANT
ADVANCES IN ONCOLOGY 1994, DeVita et al., (eds.), pages 21-36 (J.B.
Lippincott Co. 1994), which are incorporated by reference. One
approach for preparing antibodies against infectious agent
multidrug transporter proteins is illustrated in Example 6.
[0120] An antibody composite of the present invention may also be
derived from a subhuman primate antibody. General techniques for
raising therapeutically useful antibodies in baboons may be found,
for example, in Goldenberg et al., international patent publication
No. WO 91/11465 (1991), and in Losman et al., Int. J. Cancer 46:
310 (1990), which is incorporated by reference.
[0121] Alternatively, an antibody composite may be derived from a
"humanized" monoclonal antibody. Humanized monoclonal antibodies
are produced by transferring mouse complementary determining
regions from heavy and light variable chains of the mouse
immunoglobulin into a human variable domain, and then, substituting
human residues in the framework regions of the murine counterparts.
The use of antibody components derived from humanized monoclonal
antibodies obviates potential problems associated with the
immunogenicity of murine constant regions. General techniques for
cloning murine immunoglobulin variable domains are described, for
example, by the publication of Orlandi et al., Proc. Nat'l Acad.
Sci. USA 86: 3833 (1989), which is incorporated by reference in its
entirety., Techniques for producing humanized MAbs are described,
for example, by Jones et al., Nature 321: 522 (1986), Riechmann et
al., Nature 332: 323 (1988), Verhoeyen et al., Science 239: 1534
(1988), Carter et al., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992),
Sandhu, Crit. Rev. Biotech. 12: 437 (1992), and Singer et al., J.
Immun. 150: 2844 (1993), each of which is hereby incorporated by
reference.
[0122] As an alternative, an antibody composite of the present
invention may be derived from human antibody fragments isolated
from a combinatorial immunoglobulin library. See, for example,
Barbas et al., METHODS: A Companion to Methods in Enzymology 2: 119
(1991), and Winter et al., Ann. Rev. Immunol. 12: 433 (1994), which
are incorporated by reference. Cloning and expression vectors that
are useful for producing a human immunoglobulin phage library can
be obtained, for example, from STRATAGENE Cloning Systems (La
Jolla, Calif.).
[0123] In addition, an antibody composite of the present invention
may be derived from a human monoclonal antibody. Such antibodies
are obtained from transgenic mice that have been "engineered" to
produce specific human antibodies in response to antigenic
challenge. In this technique, elements of the human heavy and light
chain locus are introduced into strains of mice derived from
embryonic stem cell lines that contain targeted disruptions of the
endogenous heavy chain and light chain loci. The transgenic mice
can synthesize human antibodies specific for human antigens, and
the mice can be used to produce human antibody-secreting
hybridomas. Methods for obtaining human antibodies from transgenic
mice are described by Green et al., Nature Genet. 7: 13 (1994),
Lonberg et al., Nature 368: 856 (1994), and Taylor et al., Int.
Immun. 6: 579 (1994), which are incorporated by reference.
[0124] 3. Production of Antibody Fragments
[0125] The present invention contemplates the use of antibody
fragments to produce antibody composites. Antibody fragments can be
prepared by proteolytic hydrolysis of the antibody or by expression
in E. coli of the DNA coding for the fragment. Antibody fragments
can be obtained by pepsin or papain digestion of whole antibodies
by conventional methods. For example, antibody fragments can be
produced by enzymatic cleavage of antibodies with pepsin to provide
a 5S fragment denoted F(ab').sub.2. This fragment can be further
cleaved using a thiol reducing agent, and optionally a blocking
group for the sulfhydryl groups resulting from cleavage of
disulfide linkages, to produce 3.5S Fab' monovalent fragments.
Alternatively, an enzymatic cleavage using pepsin produces two
monovalent Fab fragments and an Fc fragment directly. These methods
are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945
and 4,331,647 and references contained therein, which patents are
incorporated herein in their entireties by reference. Also, see
Nisonoff et al., Arch Biochem. Biophys. 89: 230 (1960); Porter,
Biochem. J. 73: 119 (1959), Edelman et al., in METHODS IN
ENZYMOLOGY VOL. 1, page 422 (Academic Press 1967), and Coligan at
pages 2.8.1-2.8.10 and 2.10.-2.10.4.
[0126] Other methods of cleaving antibodies, such as separation of
heavy chains to form monovalent light-heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical or
genetic techniques may also be used, so long as the fragments bind
to the antigen that is recognized by the intact antibody.
[0127] For example, Fv fragments comprise an association of V.sub.H
and V.sub.L chains. This association can be noncovalent, as
described in Inbar et al., Proc. Nat'l Acad. Sci. USA 69: 2659
(1972). Alternatively, the variable chains can be linked by an
intermolecular disulfide bond or cross-linked by chemicals such as
glutaraldehyde. See, for example, Sandhu, supra.
[0128] Preferably, the Fv fragments comprise V.sub.H and V.sub.L
chains which are connected by a peptide linker. These single-chain
antigen binding proteins (sFv) are prepared by constructing a
structural gene comprising DNA sequences encoding the V.sub.H and
V.sub.L domains which are connected by an oligonucleotide. The
structural gene is inserted into an expression vector which is
subsequently introduced into a host cell, such as E. coli. The
recombinant host cells synthesize a single polypeptide chain with a
linker peptide bridging the two V domains. Methods for producing
sFvs are described, for example, by Whitlow et al., Methods: A
Companion to Methods in Enzymology 2: 97 (1991). Also see Bird et
al., Science 242:423-426 (1988), Ladner et al., U.S. Pat. No.
4,946,778, Pack et al., Bio/Technology 11:1271-1277 (1993), and
Sandhu, supra.
[0129] Another form of an antibody fragment is a peptide coding for
a single complementarity.gamma.-determining region (CDR). CDR
peptides ("minimal recognition units") can be obtained by
constructing genes encoding the CDR of an antibody of interest.
Such genes are prepared, for example, by using the polymerase chain
reaction to synthesize the variable region from RNA of
antibody-producing cells. See, for example, Larrick et al.,
Methods: A Companion to Methods in Enzymology 2: 106 (1991).
[0130] 4. Production of Antibody composites
[0131] Antibody composites can be prepared by a variety of
conventional procedures, ranging from glutaraldehyde linkage to
more specific linkages between functional groups. The antibodies
and/or antibody fragments are preferably covalently bound to one
another, directly or through a linker moiety, through one or more
functional groups on the antibody or fragment, e.g., amine,
carboxyl, phenyl, thiol, or hydroxyl groups. Various conventional
linkers in addition to glutaraldehyde can be used, e.g.,
disiocyanates, diiosothiocyanates, bis(hydroxysuccinimide) esters,
carbodiimides, maleimidehydroxysuccinimde esters, and the like. The
optimal length of the linker may vary according to the type of
target cell. The most efficacious linker size can be determined by
using antibody composites with various linker lengths for the
immunochemical staining of a patient tissue sample that contains
cells expressing a multidrug transporter protein and the target
antigen. Immunochemical techniques are described below.
[0132] A simple method to produce antibody composites is to mix the
antibodies or fragments in the presence of glutaraldehyde to form
an antibody composite. The initial Schiff base linkages can be
stabilized, e.g., by borohydride reduction to secondary amines. A
diiosothiocyanate or carbodiimide can be used in place of
glutaraldehyde as a non-site-specific linker.
[0133] The simplest form of an antibody composite is a bispecific
antibody comprising binding moieties for a multidrug transporter
protein and an antigen that is associated with a tumor cell or
infectious agent. Bispecific antibodies can be made by a variety of
conventional methods, e.g., disulfide cleavage and reformation of
mixtures of whole IgG or, preferably F(ab').sub.2 fragments,
fusions of more than one hybridoma to form polyomas that produce
antibodies having more than one specificity, and by genetic
engineering. Bispecific antibody composites have been prepared by
oxidative cleavage of Fab' fragments resulting from reductive
cleavage of different antibodies. This is advantageously carried
out by mixing two different F(ab').sub.2 fragments produced by
pepsin digestion of two different antibodies, reductive cleavage to
form a mixture of Fab' fragments, followed by oxidative reformation
of the disulfide linkages to produce a mixture of F(ab').sub.2
fragments including bispecific antibody composites containing a
Fab' potion specific to each of the original epitopes. General
techniques for the preparation of antibody composites may be found,
for example, in Nisonhoff et al., Arch Biochem. Biophys. 93: 470
(1961), Hammerling et al., J. Exp. Med. 128: 1461 (1968), and U.S.
Pat. No. 4,331,647.
[0134] More selective linkage can be achieved by using a
heterobifunctional linker such as maleimide-hydroxysuccinimide
ester. Reaction of the ester with an antibody or fragment will
derivatize amine groups on the antibody or fragment, and the
derivative can then be reacted with, e.g., an antibody Fab fragment
having free sulfhydryl groups (or, a larger fragment or intact
antibody with sulfhydryl groups appended thereto by, e.g., Traut's
Reagent). Such a linker is less likely to crosslink groups in the
same antibody and improves the selectivity of the linkage.
[0135] It is advantageous to link the antibodies or fragments at
sites remote from the antigen binding sites. This can be
accomplished by, e.g., linkage to cleaved interchain sulfydryl
groups, as noted above. Another method involves reacting an
antibody having an oxidized carbohydrate portion with another
antibody which has at lease one free amine function. This results
in an initial Schiff base (imine) linkage, which is preferably
stabilized by reduction to a secondary amine, e.g., by borohydride
reduction, to form the final composite. Such site-specific linkages
are disclosed, for small molecules, in U.S. Pat. No. 4,671,958, and
for larger addends in U.S. Pat. No. 4,699,784.
[0136] In the present context, a bispecific antibody comprises
binding moieties for a multidrug transporter protein and an antigen
that is associated with a tumor cell or infectious agent. For
example, the multidrug transporter protein-binding moiety can be
derived from anti-multidrug transporter protein Mab, while a
carcinoembryonic antigen (CEA) binding moiety can be derived from a
Class III Mab. Methods for preparing multidrug transporter protein
Mab are described above, while methods for preparing Class III
anti-CEA Mab are described by Primus et al., Cancer Research 43:
686 (1983), and by Primus et al., U.S. Pat. No. 4,818,709, which
are incorporated by reference.
[0137] For example, a bispecific antibody can be prepared by
obtaining an F(ab').sub.2 fragment from an anti-CEA Class III Mab,
using the techniques described above. The interchain disulfide
bridges of the anti-CEA Class III F(ab').sub.2 fragment are gently
reduced with cysteine, taking care to avoid light-heavy chain
linkage, to form Fab'-SH fragments. The SH group(s) is(are)
activated with an excess of bis-maleimide linker
(1,1'-(methylenedi-4,1-phenylene)bis-malemide). The multidrug
transporter protein Mab is converted to Fab'-SH and then reacted
with the activated anti-CEA Class III Fab'-SH fragment to obtain a
bispecific antibody.
[0138] Alternatively, such bispecific antibodies can be produced by
fusing two hybridoma cell lines that produce anti-multidrug
transporter protein Mab and anti-CEA Class III Mab. Techniques for
producing tetradomas are described, for example, by Milstein et
al., Nature 305: 537 (1983) and Pohl et al., Int. J. Cancer 54: 418
(1993).
[0139] Finally, such bispecific antibodies can be produced by
genetic engineering. For example, plasmids containing DNA coding
for variable domains of an anti-CEA Class III Mab can be introduced
into hybridomas that secrete anti-multidrug transporter protein
antibodies. The resulting "transfectomas" produce bispecific
antibodies that bind CEA and the multidrug transporter protein.
Alternatively, chimeric genes can be designed that encode both
anti-multidrug transporter protein, and anti-CEA binding domains.
General techniques for producing bispecific antibodies by genetic
engineering are described, for example, by Songsivilai et al.,
Biochem. Biophys. Res. Commun. 164: 271 (1989); Traunecker et al.,
EMBO J. 10: 3655 (1991); and Weiner et al., J. Immunol. 147: 4035
(1991)
[0140] A polyspecific antibody composite can be obtained by adding
various antibody components to a bispecific antibody composite. For
example, a bispecific antibody can be reacted with 2-iminothiolane
to introduce one or more sulfhydryl groups for use in coupling the
bispecific antibody to an antibody component that binds an epitope
of a multidrug transporter protein that is distinct from the
epitope bound by the bispecific antibody, using the bis-maleimide
activation procedure described above. These techniques for
producing antibody composites are well known to those of skill in
the art. See, for example, U.S. Pat. No. 4,925,648, and Goldenberg,
international publication No. WO 92/19273, which are incorporated
by reference.
[0141] 5. Preparation of Polyspecific Immunoconjugates
[0142] Polyspecific immunoconjugates can be prepared by indirectly
conjugating a diagnostic or therapeutic agent to an antibody
composite. General techniques are described in Shih et al., Int. J.
Cancer 41:832-839 (1988); Shih et al., Int. J. Cancer 46:1101-1106
(1990); and Shih et al., U.S. Pat. No. 5,057,313. The general
method involves reacting an antibody component having an oxidized
carbohydrate portion with a carrier polymer that has at least one
free amine function and that is loaded with a plurality of drug,
toxin, chelator, boron addends, or other diagnostic or therapeutic
agent. This reaction results in an initial Schiff base (imine)
linkage, which can be stabilized by reduction to a secondary amine
to form the final conjugate.
[0143] The carrier polymer is preferably an aminodextran or
polypeptide of at least 50 amino acid residues, although other
substantially equivalent polymer carriers can also be used.
Preferably, the final polyspecific immunoconjugate is soluble in an
aqueous solution, such as mammalian serum, for ease of
administration and effective targeting for use in diagnosis or
therapy. Thus, solubilizing functions on the carrier polymer will
enhance the serum solubility of the final polyspecific
immunoconjugate. Solubilizing functions also are important for use
of polyspecific immunoconjugates for immunochemical detection, as
described below. In particular, an aminodextran will be
preferred.
[0144] The process for preparing a polyspecific immunoconjugate
with an aminodextran carrier typically begins with a dextran
polymer, advantageously a dextran of average molecular weight of
about 10,000-100,000. The dextran is reacted with an oxidizing
agent to effect a controlled oxidation of a portion of its
carbohydrate rings to generate aldehyde groups. The oxidation is
conveniently effected with glycolytic chemical reagents such as
NaIO.sub.4, according to conventional procedures.
[0145] The oxidized dextran is then reacted with a polyamine,
preferably a diamine, and more preferably, a mono- or polyhydroxy
diamine. Suitable amines include ethylene diamine, propylene
diamine, or other like polymethylene diamines, diethylene triamine
or like polyamines, 1,3-diamino-2-hydroxypropane, or other like
hydroxylated diamines or polyamines, and the like. An excess of the
amine relative to the aldehyde groups of the dextran is used to
insure substantially complete conversion of the aldehyde functions
to Schiff base groups.
[0146] A reducing agent, such as NaBH.sub.4, NaBH.sub.3CN or the
like, is used to effect reductive stabilization of the resultant
Schiff base intermediate. The resultant adduct can be purified by
passage through a conventional sizing column to remove cross-linked
dextrans.
[0147] Other conventional methods of derivatizing a dextran to
introduce amine functions can also be used, e.g., reaction with
cyanogen bromide, followed by reaction with a diamine.
[0148] The aminodextran is then reacted with a derivative of the
particular drug, toxin, chelator, paramagnetic ion, boron addend,
or other diagnostic or therapeutic agent to be loaded, in an
activated form, preferably, a carboxyl-activated derivative,
prepared by conventional means, e.g., using
dicyclohexylcarbodiimide (DCC) or a water soluble variant thereof,
to form an intermediate adduct.
[0149] Alternatively, polypeptide toxins such as pokeweed antiviral
protein or ricin A-chain, and the like, can be coupled to
aminodextran by glutaraldehyde condensation or by reaction of
activated carboxyl groups on the protein with amines on the
aminodextran.
[0150] Chelators for radiometals or magnetic resonance enhancers
are well-known in the art. Typical are derivatives of
ethylenediaminetetraace- tic acid (EDTA) and
diethylenetriaminepentaacetic acid (DTPA). These chelators
typically have groups on the side chain by which the chelator can
be attached to a carrier. Such groups include, e.g.,
benzylisothiocyanate, by which the DTPA or EDTA can be coupled to
the amine group of a carrier. Alternatively, carboxyl groups or
amine groups on a chelator can be coupled to a carrier by
activation or prior derivatization and then coupling, all by
well-known means.
[0151] Labels such as enzymes, fluorescent compounds, electron
transfer agents, and the like can be linked to a carrier by
conventional methods well known to the art. These labeled carriers
and the polyspecific immunoconjugates prepared from them can be
used for immunochemical detection, as described below.
[0152] Boron addends, such as carboranes, can be attached to
antibody components by conventional methods. For example,
carboranes can be prepared with carboxyl functions on pendant side
chains, as is well known in the art. Attachment of such carboranes
to a carrier, e.g., aminodextran, can be achieved by activation of
the carboxyl groups of the carboranes and condensation with amines
on the carrier to produce an intermediate conjugate. Such
intermediate conjugates are then attached to. antibody components
to produce therapeutically useful polyspecific immunoconjugates, as
described below.
[0153] A polypeptide carrier can be used instead of aminodextran,
but the polypeptide carrier must have at least 50 amino acid
residues in the chain, preferably 100-5000 amino acid residues. At
least some of the amino acids should be lysine residues or
glutamate or aspartate residues. The pendant amines of lysine
residues and pendant carboxylates of glutamine and aspartate are
convenient for attaching a drug, toxin, chelator, boron addend or
other diagnostic or therapeutic agent. Examples of suitable
polypeptide carriers include polylysine, polyglutamic acid,
polyaspartic acid, copolymers thereof, and mixed polymers of these
amino acids and others, e.g., serines, to confer desirable
solubility properties on the resultant loaded carrier and
polyspecific immunoconjugate.
[0154] Conjugation of the intermediate conjugate with the antibody
component is effected by oxidizing the carbohydrate portion of the
antibody component and reacting the resulting aldehyde (and ketone)
carbonyls with amine groups remaining on the carrier after loading
with a drug, toxin, chelator, boron addend, or other diagnostic or
therapeutic agent. Alternatively, an intermediate conjugate can be
attached to an oxidized antibody component via amine groups that
have been introduced in the intermediate conjugate after loading
with the diagnostic or therapeutic agent. Oxidation is conveniently
effected either chemically, e.g., with NaIO.sub.4 or other
glycolytic reagent, or enzymatically, e.g., with neuraminidase and
galactose oxidase. In the case of an aminodextran carrier, not all
of the amines of the aminodextran are typically used for loading a
diagnostic or therapeutic agent. The remaining amines of
aminodextran condense with the oxidized antibody component to form
Schiff base adducts, which are then reductively stabilized,
normally with a borohydride reducing agent.
[0155] Analogous procedures are used to produce other polyspecific
immunoconjugates according to the invention. Loaded polypeptide
carriers preferably have free lysine residues remaining for
condensation with the oxidized carbohydrate portion of an antibody
component. Carboxyls on the polypeptide carrier can, if necessary,
be converted to amines by, e.g., activation with DCC and reaction
with an excess of a diamine.
[0156] The final polyspecific immunoconjugate is purified using
conventional techniques, such as sizing chromatography on Sephacryl
S-300.
[0157] Alternatively, polyspecific immunoconjugates can be prepared
by directly conjugating an antibody component with a diagnostic or
therapeutic agent. The general procedure is analogous to the
indirect method of conjugation except that a diagnostic or
therapeutic agent is directly attached to an oxidized antibody
component.
[0158] It will be appreciated that other diagnostic or therapeutic
agents can be substituted for the chelators described herein. Those
of skill in the art will be able to devise conjugation schemes
without undue experimentation.
[0159] In addition, those of skill in the art will recognize
numerous possible variations of the conjugation methods. For
example, the carbohydrate moiety can be used to attach
polyethyleneglycol in order to extend the half-life of an intact
antibody, or antigen-binding fragment thereof, in blood, lymph, or
other extracellular fluids. Moreover, it is possible to construct a
"divalent immunoconjugate" by attaching a diagnostic or therapeutic
agent to a carbohydrate moiety and to a free sulfhydryl group. Such
a free sulfhydryl group may be located in the hinge region of the
antibody component.
[0160] 6. Use of Polyspecific Immunoconjugates and Antibody
Composites for Diagnosis
[0161] A. In Vitro Diagnosis
[0162] The present invention contemplates the use of polyspecific
immunoconjugates and antibody composites to screen biological
samples in vitro for the expression of P-glycoprotein by tumor
cells. For example, the polyspecific immunoconjugates and antibody
composites of the present invention can be used to detect the
presence of P-glycoprotein and tumor associated antigen in tissue
sections prepared from a biopsy specimen. Such immunochemical
detection can be used to determine the abundance of P-glycoprotein
and to determine the distribution of P-glycoprotein in the examined
tissue. General immunochemistry techniques are well-known to those
of ordinary skill. See, for example, Ponder, "Cell Marking
Techniques and Their Application," in MAMMALIAN DEVELOPMENT: A
PRACTICAL APPROACH, Monk (ed.), pages 115-38 (IRL Press 1987), Volm
et al., Eur. J. Cancer Clin. Oncol. 25: 743 (1989), Coligan at
pages 5.8.1-5.8.8, and Ausubel et al. (eds.), CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, pages 14.6.1 to 14.6.13 (Wiley Interscience
1990). Also, see generally, Manson (ed.), METHODS IN MOLECULAR
BIOLOGY, VOL.10: IMMUNOCHEMICAL PROTOCOLS (The Humana Press, Inc.
1992). Moreover, methods for the immunochemical detection of
P-glycoprotein are described, for example, by Dalton et al., Blood
73: 747 (1989), and Volm et al., Eur. J. Cancer Clin. Oncol. 25:
743 (1989).
[0163] In addition, the present invention contemplates the use of
polyspecific immunoconjugates and antibody composites to screen
biological samples in vitro for the expression of a multidrug
transporter protein by an infectious agent. For example, the
polyspecific immunoconjugates and antibody composites of the
present invention can be used to detect the presence of OprK
protein in clinical isolates. The presence of this particular
multidrug transporter protein would indicate that the tissue was
infected with multidrug resistant Psuedomonas aeruginosa.
[0164] Moreover, immunochemical detection techniques can be used to
optimize antibody composites for subsequent in vivo diagnosis and
therapy in the form of antibody composites per se or as
polyspecific immunoconjugates. Accordingly, immunochemical
detection can be performed with a battery of antibody composites to
identify the most appropriate combination of antibody components
for subsequent in vivo diagnosis and therapy. For example, an
antibody moiety that binds the c-erb B2 proto-oncogene product may
be more suitable for a particular breast cancer than an antibody
moiety that binds carcinoembryonic antigen. After a suitable
combination of antibody components have been identified, further in
vitro testing can be used to delineate the most efficacious linker
size in the antibody composite, as discussed above.
[0165] Immunochemical detection can be performed by contacting a
biological sample with an antibody composite and then contacting
the biological sample with a detectably labeled molecule which
binds to the antibody composite. For example, the detectably
labeled molecule can comprise an antibody moiety that binds the
antibody composite. Alternatively, the antibody composite can be
conjugated with avidin/streptavidin (or biotin) and the detectably
labeled molecule can comprise biotin (or avidin/streptavidin).
Numerous variations of this basic technique are well-known to those
of skill in the art.
[0166] Alternatively, an antibody composite can be conjugated with
a diagnostic agent to form a polyspecific immunoconjugate. Antibody
composites can be detectably labeled with any appropriate marker
moiety, for example, a radioisotope, a fluorescent label, a
chemiluminescent label, an enzyme label, a bioluminescent label or
colloidal gold. Methods of making and detecting such
detectably-labeled polyspecific immunoconjugates are well-known to
those of ordinary skill in the art, and are described in more
detail below.
[0167] The marker moiety can be a radioisotope that is detected by
autoradiography. Isotopes that are particularly useful for the
purpose of the present invention are .sup.3H, .sup.125I, .sup.131I,
.sup.35S and .sup.14C .
[0168] Polyspecific immunoconjugates also can be labeled with a
fluorescent compound. The presence of a fluorescently-labeled
antibody component is determined by exposing the polyspecific
immunoconjugate to light of the proper wavelength and detecting the
resultant fluorescence. Fluorescent labeling compounds include
fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin,
allophycocyanin, o-phthaldehyde and fluorescamine.
[0169] Alternatively, polyspecific immunoconjugates can be
detectably labeled by coupling an antibody component to a
chemiluminescent compound. The presence of the
chemiluminescent-tagged polyspecific immunoconjugate is determined
by detecting the presence of luminescence that arises during the
course of a chemical reaction. Examples of chemiluminescent
labeling compounds include luminol, isoluminol, an aromatic
acridinium ester, an imidazole, an acridinium salt and an oxalate
ester.
[0170] Similarly, a bioluminescent compound can be used to label
polyspecific immunoconjugates of the present invention.
Bioluminescence is a type of chemiluminescence found in biological
systems in which a catalytic protein increases the efficiency of
the chemiluminescent reaction. The presence of a bioluminescent
protein is determined by detecting the presence of luminescence.
Bioluminescent compounds that are useful for labeling include
luciferin, luciferase and aequorin.
[0171] Alternatively, polyspecific immunoconjugates can be
detectably labeled by linking an antibody component to an enzyme.
When the polyspecific immunoconjugates-enzyme conjugate is
incubated in the presence of the appropriate substrate, the enzyme
moiety reacts with the substrate to produce a chemical moiety which
can be detected, for example, by spectrophotometric, fluorometric
or visual means. Examples of enzymes that can be used to detectably
label polyspecific immunoconjugates include .beta.-galactosidase,
glucose oxidase, peroxidase and alkaline phosphatase.
[0172] Those of skill in the art will know of other suitable labels
which can be employed in accordance with the present invention. The
binding of marker moieties to antibody components can be
accomplished using standard techniques known to the art. Typical
methodology in this regard is described by Kennedy et al., Clin.
Chim. Acta 70: 1 (1976), Schurs et al., Clin. Chim. Acta 81: 1
(1977), Shih et al., Int'l J. Cancer 46: 1101 (1990), Stein et al.,
Cancer Res. 50: 1330 (1990), supra, and Stein et al., Int. J.
Cancer 55: 938 (1993). Also, see generally, Coligan.
[0173] In addition, the convenience and versatility of
immunochemical detection can be enhanced by using antibody
components that have been conjugated with avidin, streptavidin, and
biotin. See, for example, Wilchek et al. (eds.), Avidin-Biotin
Technology, METHODS IN ENZYMOLOGY, VOL. 184 (Academic Press 1990),
and Bayer et al., "Immunochemical Applications of Avidin-Biotin
Technology," in METHODS IN MOLECULAR BIOLOGY, VOL. 10, Manson
(ed.), pages 149-162 (The Human Press, Inc. 1992).
[0174] Thus, the above-described immunochemical detection methods
can be used to assist in the diagnosis or staging of a pathological
condition. These techniques also can be used to identify the most
suitable composition of antibody composite or polyspecific
immunoconjugate for subsequent in vivo diagnosis and therapy.
[0175] B. In Vivo Diagnosis
[0176] The present invention also contemplates the use of antibody
composites and polyspecific immunoconjugates for in vivo diagnosis.
The method of diagnostic imaging with radiolabeled MAbs is
well-known. In the technique of immunoscintigraphy, for example,
antibodies are labeled with a gamma-emitting radioisotope and
introduced into a patient. A gamma camera is used to detect the
location and distribution of gamma-emitting radioisotopes. See, for
example, Srivastava (ed.), RADIOLABELED MONOCLONAL ANTIBODIES FOR
IMAGING AND THERAPY (Plenum Press 1988), Chase, "Medical
Applications of Radioisotopes," in REMINGTON'S PHARMACEUTICAL
SCIENCES, 18th Edition, Gennaro et al. (eds.), pp. 624-652 (Mack
Publishing Co., 1990), Brown, "Clinical Use of Monoclonal
Antibodies," in BIOTECHNOLOGY AND PHARMACY 227-49, Pezzuto et al.
(eds.) (Chapman & Hall 1993), and Goldenberg, Calif.--A Cancer
Journal for Clinicians 44: 43 (1994). For diagnostic imaging,
radioisotopes may be bound to an antibody composite either
directly, or indirectly by using an intermediary functional group.
Useful intermediary functional groups include chelators such as
ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic
acid. For example, see Shih et al., supra, and U.S. Pat. No.
5,057,313. Also, see Griffiths, U.S. Pat. No. 5,128,119 (1992).
[0177] The radiation dose delivered to the patient is maintained at
as low a level as possible through the choice of isotope for the
best combination of minimum half-life, minimum retention in the
body, and minimum quantity of isotope which will permit detection
and accurate measurement. Examples of radioisotopes that can be
bound to antibody composites and are appropriate for 30, diagnostic
imaging include .gamma.-emitters and positron-emitters such as
.sup.99mTc, .sup.67Ga, .sup.111In, .sup.123I, .sup.124I, .sup.125I,
.sup.131I, .sup.51Cr, .sup.89Zr, .sup.18F and .sup.68Ga. Other
suitable radioisotopes are known to those of skill in the art.
[0178] Preferred .gamma.-emitters have a gamma radiation emission
peak in the range of 50-500 Kev, primarily because the state of the
art for radiation detectors currently favors such labels. Examples
of such .gamma.-emitters include .sup.99mTc, .sup.67Ga, .sup.123I,
.sup.125I and .sup.131I.
[0179] Antibody composites also can be labeled with paramagnetic
ions for purposes of in vivo diagnosis. Elements that are
particularly useful for magnetic resonance imaging include Gd, Mn,
Dy and Fe ions.
[0180] A high background level of non-targeted antibody provides a
major impediment to in vivo diagnosis methodology. However, the
ratio of target to nontarget radiolabeled antibody can be enhanced
through the use of a nonlabeled second antibody which scavenges and
promotes the clearance of the nontargeted circulating radiolabeled
antibody. The second antibody may be whole IgG or IgM, or a
fragment of IgG or IgM, so long as it is capable of binding the
radiolabeled antibody to form a complex which is cleared from the
circulation and nontarget spaces more rapidly than the radiolabeled
antibody alone. In the present context, suitable second antibodies
may bind with either the Fc portion or variable region of a
radiolabeled polyspecific immunoconjugate. See, for example,
Goldenberg, U.S. Pat. No. 4,624,846, Goldenberg, international
publication No. WO 92/19273, and Sharkey et al., Int. J. Cancer 51:
266 (1992), which are incorporated by reference.
[0181] For example, the location of multidrug resistant (MDR) tumor
cells, MDR HIV-infected cells or MDR infectious agents in a mammal
having a multidrug resistant disease caused by a tumor or
infectious agent can be determined by parenterally injecting the
mammal with a polyspecific immunoconjugate comprising (1) at least
one antibody component that binds with a first epitope of a
multidrug transporter protein, (2) at least one antibody component
that binds with a first epitope of an antigen that is associated
with the tumor or infectious agent, and (3) a diagnostic agent.
Subsequently, the mammal is injected with an antibody or antibody
fragment that binds with the polyspecific immunoconjugate in an
amount that is sufficient to decrease the level of circulating
polyspecific immunoconjugate by about 10-85% within 2 to 72 hours.
The mammal is then scanned with a detector to locate the site or
sites of uptake of the polyspecific immunoconjugate. See
Goldenberg, U.S. Pat. No. 4,624,846.
[0182] In an alternate approach, detection methods are improved by
taking advantage of the binding between avidin/streptavidin and
biotin. Avidin, found in egg whites, has a very high binding
affinity for biotin, which is a B-complex vitamin. Streptavidin,
isolated from Streptomyces avidinii, is similar to avidin, but has
lower non-specific tissue binding and therefore, streptavidin often
is used in place of avidin. A basic diagnostic method comprises
administering an antibody composite conjugated with
avidin/streptavidin (or biotin), injecting a clearing composition
comprising biotin (or avidin/streptavidin), and administering a
conjugate of a diagnostic agent and biotin (or
avidin/streptavidin). Preferably, the biotin (or
avidin/streptavidin) component of the clearing composition is
coupled with a carbohydrate moiety (such as dextran) or a polyol
group (e.g., polyethylene glycol) to decrease immunogenicity and
permit repeated applications.
[0183] A modification of the basic method is performed by
parenterally injecting a mammal with an antibody composite which
has been conjugated with avidin/streptavidin (or biotin), injecting
a clearing composition comprising biotin (or avidin/streptavidin),
and parenterally injecting a polyspecific immunoconjugate according
to the present invention, which further comprises
avidin/streptavidin (or biotin). See Goldenberg, international
publication No. WO 94/04702, which is incorporated by
reference.
[0184] In a further variation of this method, improved detection
can be achieved by conjugating multiple avidin/streptavidin or
biotin moieties to a polymer which, in turn, is conjugated to an
antibody component. Adapted to the present invention, antibody
composites or polyspecific immunoconjugates can be produced which
contain multiple avidin/streptavidin or biotin moieties. Techniques
for constructing and using multiavidin/multistreptavidin and/or
multibiotin polymer conjugates to obtain amplification of targeting
are disclosed by Griffiths, international application No.
PCT/US94/04295, which is incorporated by reference.
[0185] In another variation, improved detection is achieved by
injecting a targeting antibody composite conjugated to biotin (or
avidin/streptavidin), injecting at least one dose of an
avidin/streptavidin (or biotin) clearing agent, and injecting a
diagnostic composition comprising a conjugate of biotin (or
avidin/streptavidin) and a naturally occurring metal atom chelating
protein which is chelated with a metal detection agent. Suitable
targeting proteins according to the present invention would be
ferritin, metallothioneins, ferredoxins, and the like. This
approach is disclosed by Goldenberg et al., international
application No. PCT/US94/05149, which is incorporated by
reference.
[0186] Polyspecific immunoconjugates which comprise a radiolabel
also can be used to detect multidrug resistant (MDR) tumor cells,
MDR HIV-infected cells or MDR infectious agents in the course of
intraoperative and endoscopic examination using a small radiation
detection probe. See Goldenberg U.S. Pat. No. 4,932,412, which is
incorporated by reference. As an illustration of the basic
approach, a surgical or endoscopy subject is injected parenterally
with a polyspecific immunoconjugate comprising (1) at least one
antibody component that binds with a first epitope of a multidrug
transporter protein, (2) at least one antibody component that binds
with a first epitope of an antigen that is associated with a tumor
or infectious agent, and (3) a radioisotope. Subsequently, the
surgically exposed or endoscopically accessed interior of the body
cavity of the subject is scanned at close range with a radiation
detection probe to detect the sites of accretion of the
polyspecific immunoconjugate.
[0187] In a variation of this method, a photoactive agent or dye,
such as dihematoporphyrin ether (Photofrin II), is injected
systemically and sites of accretion of the agent or dye are
detected by laser-induced fluorescence and endoscopic imaging. See
Goldenberg, international application No. PCT/US93/04098, which is
incorporated by reference. The prior art discloses imaging
techniques using certain dyes that are accreted by lesions, such as
tumors, and which are in turn activated by a specific frequency of
light. These methods are described, for example, in Dougherty et
al., Cancer Res. 38: 2628 (1978); Dougherty, Photochem. Photobiol.
45: 879 (1987); Doiron et al. (eds.), PORPHYRIN LOCALIZATION AND
TREATMENT OF TUMORS (Alan Liss, 1984); and van den Bergh, Chem.
Britain 22: 430 (1986), which are incorporated herein in their
entirety by reference.
[0188] In a basic technique, a subject is injected parenterally
with a polyspecific immunoconjugate comprising (1) at least one
antibody component that binds with a first epitope of a multidrug
transporter protein, (2) at least one antibody component that binds
with a first epitope of an antigen that is associated with a tumor
or infectious agent, and (3) a photoactive agent or dye. Sites of
accretion are detected using a light source provided by an
endoscope or during a surgical procedure.
[0189] The detection of polyspecific immunoconjugate during
intraoperative or endoscopic examination can be enhanced through
the use of second antibody or avidin/streptavidin/biotin clearing
agents, as discussed above.
[0190] In these endoscopic techniques the detection means can be
inserted into a body cavity through an orifice, such as, the mouth,
nose, ear, anus, vagina or incision. As used herein, the term
"endoscope" is used generically to refer to any scope introduced
into a body cavity, e.g., an anally introduced endoscope, an orally
introduced bronchoscope, a urethrally introduced cystoscope, an
abdominally introduced laparoscope or the like. Certain of these
may benefit greatly from further progress in miniaturization of
components and their utility to practice the method of the present
invention will be enhanced as a function of the development of
suitably microminiaturized components for this type of
instrumentation. Highly miniaturized probes which could be
introduced intravascularly, e.g., via catheters or the like, are
also suitable for use in the embodiments of the invention for
localizing MDR tumor cells, MDR HIV-infected cells or MDR
infectious agents.
[0191] 7. Use of Polyspecific Immunoconjugates and Antibody
Composites for Therapy
[0192] The present invention also contemplates the use of antibody
composites and polyspecific immunoconjugates for immunotherapy. An
objective of immunotherapy is to deliver cytotoxic doses of
radioactivity, toxin, or drug to target cells, while minimizing
exposure to non-target tissues. The polyspecific immunoconjugates
and antibody composites of the present invention are expected to
have a greater binding specificity than multidrug transporter
protein MAbs, since the polyspecific immunoconjugates and antibody
composites comprise moieties that bind to at least one multidrug
transporter protein epitope and an antigen associated with either a
tumor or an infectious agent.
[0193] For example, a therapeutic polyspecific immunoconjugate may
comprise an .alpha.-emitting radioisotope, a .beta.-emitting
radioisotope, a .gamma.-emitting radioisotope, an Auger electron
emitter, a neutron capturing agent that emits .alpha.-particles or
a radioisotope that decays by electron capture. Suitable
radioisotopes include .sup.198Au, .sup.32P, .sup.125I, .sup.131I,
.sup.90Y, .sup.186Re, .sup.188Re, .sup.67Cu, .sup.211At, and the
like.
[0194] As discussed above, a radioisotope can be attached to an
antibody composite directly or indirectly, via a chelating agent.
For example, .sup.67Cu, considered one of the more promising
radioisotopes for radioimmunotherapy due to its 61.5 hour half-life
and abundant supply of beta particles and gamma rays, can be
conjugated to an antibody composite using the chelating agent,
p-bromoacetamido-benzyl-tetraethylaminetetraac- etic acid (TETA).
Chase, supra. Alternatively, .sup.90Y, which emits an energetic
beta particle, can be coupled to an antibody composite using
diethylenetriaminepentaacetic acid (DTPA). Moreover, a method for
the direct radiolabeling of the antibody composite with .sup.131I
is described by Stein et al., Antibody Immunoconj. Radiopharm. 4:
703 (1991).
[0195] Alternatively, boron addends such as carboranes can be
attached to antibody composites. Carboranes can be prepared with
carboxyl functions on pendant side chains, as is well-known in the
art. Attachment of carboranes to a carrier, such as aminodextran,
can be achieved by activation of the carboxyl groups of the
carboranes and condensation with amines on the carrier. The
intermediate conjugate is then conjugated to the antibody
composite. After administration of the polyspecific
immunoconjugate, a boron addend is activated by thermal neutron
irradiation and converted to radioactive atoms which decay by
.alpha.-emission to produce highly toxic, short-range effects.
[0196] In addition, therapeutically useful polyspecific
immunoconjugates can be prepared in which an antibody composite is
conjugated to a toxin or a chemotherapeutic drug. Illustrative of
toxins which are suitably employed in the preparation of such
conjugates are ricin, abrin, human ribonuclease, pokeweed antiviral
protein, gelonin, diphtherin toxin, and Pseudomonas endotoxin. See,
for example, Pastan et al., Cell 47: 641 (1986), and Goldenberg,
CA--A Cancer Journal for Clinicians 44: 43 (1994). Other suitable
toxins are known to those of skill in the art.
[0197] Useful cancer chemotherapeutic drugs for the preparation of
polyspecific immunoconjugates include nitrogen mustards, alkyl
sulfonates, nitrosoureas, triazenes, folic acid analogs, pyrimidine
analogs, purine analogs, antibiotics, epipodophyllotoxins, platinum
coordination complexes, hormones, and the like. Chemotherapeutic
drugs that are useful for treatment of infectious agents include
antiviral drugs (such as AZT, 2',3'-dideoxyinosine and
2',3'-dideoxycytidine), antimalarial drugs (such as chloroquine and
its congeners, diaminopyrimidines, mefloquine), antibacterial
agents, antifungal agents, antiprotozoal agents, and the like.
Suitable chemotherapeutic agents are described in REMINGTON'S
PHARMACEUTICAL SCIENCES, 18th Ed. (Mack Publishing Co. 1990), and
in GOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS,
7th Ed. (MacMillan Publishing Co. 1985), which are incorporated by
reference. Other suitable chemotherapeutic agents, such as
experimental drugs, are known to those of skill in the art.
[0198] In addition, therapeutically useful polyspecific
immunoconjugates can be obtained by conjugating photoactive agents
or dyes to an antibody composite. Fluorescent and other chromogens,
or dyes, such as porphyrins sensitive to visible light, have been
used to detect and to treat lesions by directing the suitable light
to the lesion (cited above). In therapy, this has been termed
photoradiation, phototherapy, or photodynamic therapy (Jori et al.
(eds.), PHOTODYNAMIC THERAPY OF TUMORS AND OTHER DISEASES (Libreria
Progetto 1985); van den Bergh, Chem. Britain 22: 430 (1986)).
Moreover, monoclonal antibodies have been coupled with
photoactivated dyes for achieving phototherapy (Mew et al., J.
Immunol. 130: 1473 (1983); idem., Cancer Res. 45: 4380 (1985);
Oseroff et al., Proc. Natl. Acad. Sci. USA 83: 8744 (1986); idem.,
Photochem. Photobiol. 46: 83 (1987); Hasan et al., Prog. Clin.
Biol. Res. 288: 471 (1989); Tatsuta et al., Lasers Surg. Med. 9:
422 (1989); Pelegrin et al., Cancer 67: 2529 (1991)--all
incorporated in their entirety herein by reference). However, these
earlier studies did not include use of endoscopic therapy
applications, especially with the use of antibody fragments or
subfragments. Thus, the present invention contemplates the
therapeutic use of polyspecific immunoconjugates comprising
photoactive agents or dyes. The general methodology is described
above in relation to the use of such polyspecific immunoconjugates
for diagnosis.
[0199] Moreover, therapeutically useful polyspecific
immunoconjugates can be prepared in which an antibody composite is
conjugated to a compound that reverses multidrug resistance. Such
"chemosensitizing agents" include verapamil and its analogs,
calmodulin antagonists, anthracycline and Vinca alkaloid analogs,
and the like. See, for example, Endicott et al., Ann. Rev. Biochem.
58: 137 (1989), Ford et al., Pharmacol. Rev. 42: 155 (1990) and
Calabresi et al., PPO Updates 8:1 (1994). See also Sarkadi et al.,
FASEB J. 8: 766 (1994), which provides methods to identify
hydrophobic peptide derivatives that reverse multidrug resistance.
These polyspecific immunoconjugates may be administered prior to,
or concurrent with, the administration of appropriate
chemotherapeutic drugs.
[0200] As an alternative, unconjugated chemosensitizing agents may
be administered with polyspecific immunoconjugates comprising a
toxin or chemotherapeutic drug. Typical modes of administration and
dosages of chemosensitizing agents are described, for example, by
Presant et al., Am. J. Clin. Oncol. 9: 355 (1986), Cairo et al.,
Cancer Res. 49: 1063 (1989), Miller et al., J. Clin. Oncol. 9: 37
(1991) and Calabresi et al., supra, Rubin, U.S. Pat. No. 5,005,588,
and Levy, U.S. Pat. No. 5,258,372, which are incorporated by
reference.
[0201] In addition, therapeutic polyspecific immunoconjugates can
comprise an immunomodulator moiety. As used herein, the term
"immunomodulator" includes cytokines, stem cell growth factors,
tumor necrosis factors (TNF) and hematopoietic factors, such as
interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6 and
IL-10), colony stimulating factors (e.g., granulocyte-colony
stimulating factor (G-CSF) and granulocyte macrophage-colony
stimulating factor (GM-CSF)), interferons (e.g.,
interferons-.alpha., -.beta. and -.gamma.), the stem cell growth
factor designated "S1 factor," erythropoietin and thrombopoietin.
Examples of suitable immunomodulator moieties include IL-2, IL-6,
IL-10, interferon-', TNF-.alpha., and the like.
[0202] Such polyspecific immunoconjugates provide a means to
deliver an immunomodulator to a target cell and are particularly
useful against tumor cells and mammalian cells that express an
infectious agent antigen on the cell surface, such as HIV-infected
cells. The cytotoxic effects of immunomodulators are well known to
those of skill in the art. See, for example, Klegerman et al.,
"Lymphokines and Monokines," in BIOTECHNOLOGY AND PHARMACY, Pessuto
et al. (eds.), pages 53-70 (Chapman & Hall 1993). As an
illustration, type I interferons and interferon-.gamma. induce an
antiviral state in various cells by activating 2',5'-oligoadenylate
synthetase and protein kinase. Moreover, interferons can inhibit
cell proliferation by inducing increased expression of class I
histocompatibility antigens on the surface of various cells and
thus, enhance the rate of destruction of cells by cytotoxic T
lymphocytes. Furthermore, tumor necrosis factors, such as
TNF-.alpha., are believed to produce cytotoxic effects by inducing
DNA fragmentation.
[0203] The present invention also contemplates two-, three- or
four-step targeting strategies to enhance antibody therapy. General
techniques include the use of antibody components conjugated with
avidin, streptavidin or biotin, and the use of second antibodies
that bind with the primary immunoconjugate, as discussed above.
See, for example, Goodwin et al., Eur. J. Nucl. Med. 9:209 (1984),
Goldenberg et al., J. Nucl. Med. 28:1604 (1987), Hnatowich et al.,
J. Nucl. Med. 28: 1294 (1987), Paganelli et al., Cancer Res. 51:
5960 (1991), Goldenberg, international publication No. WO 92/19273,
Sharkey et al., Int. J. Cancer 51: 266 (1992), and Goldenberg,
international application No. WO 94/04702, which are incorporated
by reference. Also, see Griffiths, international application No.
PCT/US94/04295, which describes a method using multiavidin and/or
multibiotin polymer conjugates, and Goldenberg et al.,
international application No. PCT/US94/05149, which discloses
improved methods for therapy with chelatable radiometals.
[0204] For example, a mammal having a multidrug resistant disease
caused by a tumor or infectious agent may be treated by
parenterally injecting the mammal with a polyspecific
immunoconjugate comprising (1) at least one antibody component that
binds with a first epitope of a multidrug transporter protein, (2)
at least one antibody component that binds with a first epitope of
an antigen that is associated with the tumor or infectious agent,
and (3) a therapeutic agent. Subsequently, the mammal is injected
with an antibody or antibody fragment that binds with the
polyspecific immunoconjugate in an amount that is sufficient to
decrease the level of circulating polyspecific immunoconjugate by
about 10-85% within 2 to 72 hours.
[0205] In an alternative approach to enhancing the therapeutic
index comprises administering an antibody composite conjugated with
avidin/streptavidin (or biotin), injecting a clearing composition
comprising biotin (or avidin/streptavidin), and administering a
conjugate of a therapeutic agent and avidin/streptavidin (or
biotin), as discussed above.
[0206] The present invention also contemplates a method of therapy
using unconjugated antibody composites. Investigators have found
that P-glycoprotein antibodies can restore drug sensitivity in
multidrug resistant cultured cells and multidrug resistant human
tumor xenografts in nude mice. Grauer et al., European patent
application No. EP-0 569 141, Rittmann-Grauer et al., Cancer Res.
52: 1810 (1992), Pearson et al., J. Nat'l Cancer Inst. 83: 1386
(1991), and Iwahashi et al., Cancer Res. 53: 5475 (1993).
P-glycoprotein antibodies also can inhibit the growth of multidrug
resistant human xenografts in nude mice. Grauer et al., European
patent application No. EP-0 569,141. Accordingly, the more specific
antibody composites of the present invention provide an improved
method to treat a mammal having a multidrug resistant disease
caused by a tumor or infectious agent in which the multidrug
resistant cells overexpress P-glycoprotein. Moreover, the antibody
composites of the present invention can be used to inhibit active
drug efflux in infectious agents and thus, restore sensitivity to
chemotherapy.
[0207] Antibody composites may be administered alone, or in
conjugation with the conventional chemotherapeutic agents described
above. Modes of chemotherapeutic administration and suitable
dosages are well known to those of skill in the art. See, for
example, REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Ed. (Mack
Publishing Co. 1990), and GOODMAN AND GILMAN'S THE PHARMACOLOGICAL
BASIS OF THERAPEUTICS, 7th Ed. (MacMillan Publishing Co. 1985).
[0208] In general, the dosage of administered polyspecific
immunoconjugates and antibody composites will vary depending upon
such factors as the patient's age, weight, height, sex, general
medical condition and previous medical history. Typically, it is
desirable to provide the recipient with a dosage of polyspecific
immunoconjugate or antibody composite which is in the range of from
about 1 pg/kg to 10 mg/kg (amount of agent/body weight of patient),
although a lower or higher dosage also may be administered as
circumstances dictate.
[0209] Administration of polyspecific immunoconjugates or antibody
composites to a patient can be intravenous, intraarterial,
intraperitoneal, intramuscular, subcutaneous, intrapleural,
intrathecal, by perfusion through a regional catheter, or by direct
intralesional injection. When administering polyspecific
immunoconjugates or antibody composites by injection, the
administration may be by continuous infusion or by single or
multiple boluses.
[0210] Polyspecific immunoconjugates having a boron addend-loaded
carrier for thermal neutron activation therapy will normally be
effected in similar ways. However, it will be advantageous to wait
until non-targeted polyspecific immunoconjugate clears before
neutron irradiation is performed. Clearance can be accelerated
using an antibody that binds to the polyspecific immunoconjugate.
See U.S. Pat. No. 4,624,846 for a description of this general
principle.
[0211] The polyspecific immunoconjugates and antibody composites of
the present invention can be formulated according to known methods
to prepare pharmaceutically useful compositions, whereby
polyspecific immunoconjugates or antibody composites are combined
in a mixture with a pharmaceutically acceptable carrier. A
composition is said to be a "pharmaceutically acceptable carrier"
if its administration can be tolerated by a recipient patient.
Sterile phosphate-buffered saline is one example of a
pharmaceutically acceptable carrier. Other suitable carriers are
well-known to those in the art. See, for example, REMINGTON'S
PHARMACEUTICAL SCIENCES, 18th Ed. (1990).
[0212] For purposes of therapy, a polyspecific immunoconjugate (or
antibody composite) and a pharmaceutically acceptable carrier are
administered to a patient in a therapeutically effective amount. A
combination of a polyspecific immunoconjugate (or antibody
composite) and a pharmaceutically acceptable carrier is said to be
administered in a "therapeutically effective amount" if the amount
administered is physiologically significant. An agent is
physiologically significant if its presence results in a detectable
change in the physiology of a recipient patient. In the present
context, an agent is physiologically significant if its presence
results in the inhibition of the growth of target cells, or in the
increased susceptibility of target cells to a chemotherapeutic
agent.
[0213] Additional pharmaceutical methods may be employed to control
the duration of action of a polyspecific immunoconjugate or
antibody composite in a therapeutic application. Control release
preparations can be prepared through the use of polymers to complex
or adsorb the polyspecific immunoconjugate or antibody composite.
For example, biocompatible polymers include matrices of
poly(ethylene-co-vinyl acetate) and matrices of a polyanhydride
copolymer of a stearic acid dimer and sebacic acid. Sherwood et
al., Bio/Technology 10: 1446 (1992). The rate of release of a
polyspecific immunoconjugate (or antibody composite) from such a
matrix depends upon the molecular weight of the polyspecific
immunoconjugate (or antibody composite), the amount of polyspecific
immunoconjugate (or antibody composite) within the matrix, and the
size of dispersed particles. Saltzman et al., Biophys. J. 55: 163
(1989); Sherwood et al., supra. Other solid dosage forms are
described in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th ed.
(1990).
[0214] The present invention also contemplates a method of
treatment in which immunomodulators are administered to prevent,
mitigate or reverse radiation-induced or drug-induced toxicity of
normal cells, and especially hematopoietic cells. Adjunct
immunomodulator therapy allows the administration of higher doses
of cytotoxic agents due to increased tolerance of the recipient
mammal. Moreover, adjunct immunomodulator therapy can prevent,
palliate, or reverse dose-limiting marrow toxicity. Examples of
suitable immunomodulators for adjunct therapy include G-CSF,
GM-CSF, thrombopoietin, IL-1, IL-3, and the like. The method of
adjunct immunomodulator therapy is disclosed by Goldenberg, U.S.
Pat. No. 5,120,525, which is incorporated by reference.
[0215] Those of skill in the art are aware that an antibody
component is just one example of a moiety that can be used to
target particular cells. Other useful targeting moieties include
non-antibody proteins, peptides, polypeptides, glycoproteins,
lipoproteins, or the like, e.g., growth factors, enzymes, receptor
proteins, immunomodulators and hormones. For example, Sarkadi et
al., The FASEB Journal 8: 766 (1994), which is incorporated by
reference, provides methods for identifying hydrophobic peptides
that interact with P-glycoprotein. As an illustration, a
polyspecific conjugate suitable for diagnosis and/or treatment of
certain multidrug resistant breast cancers would comprise a
hydrophobic peptide that binds with P-glycoprotein, an epidermal
growth factor moiety that binds with the c-erb B2 proto-oncogene
product, and a diagnostic or therapeutic agent.
[0216] The present invention, thus generally described, will be
understood more readily by reference to the following examples,
which are provided by way of illustration and are not intended to
be limiting of the present invention.
EXAMPLE 1
Production of Antibody Components: Murine P-Glycoprotein MAb and
Anti-CEA MAb
[0217] 1. Production of Monoclonal Antibodies
[0218] Methods for producing anti-P-glycoprotein murine monoclonal
antibodies are well-known to those of skill in the art, as
discussed above. One approach is to immunize mice with cells, or
cellular membranes, that contain an abundance of P-glycoprotein.
Cells that over-express P-glycoprotein can be obtained by selecting
and enriching cells that express the MDR phenotype from a human
continuous cell line. See, for example, Gottesman, "Drug-Resistant
Mutants: Selection and Dominance Analysis," in METHODS IN
ENZYMOLOGY, VOL. 151, Colowick et al., (eds.), pages 113-121
(Academic Press 1987), and Clynes et al., Cytotechnology 12: 231
(1993).
[0219] A general method for using MDR cells to produce
anti-P-glycoprotein monoclonal antibodies is described, for
example, by Rittmann-Grauer et al., Cancer Res. 52: 1810 (1992),
which is incorporated by reference. Briefly, six week old female
BALB/c mice are injected intraperitoneally (i.p.) with
5.times.10.sup.6 MDR cells that have been scraped from the surface
of tissue culture flasks. Three weeks later, mice receive a second
i.p. injection of 5.times.10.sup.6 MDR cells. Four days prior to
fusion, mice receive a final intravenous boost of 5.times.10.sup.6
MDR cells. Splenocytes from the immunized mice are fused with
murine myeloma cells, SP2/0-Ag 14, according to the method of
Gerhard, "Fusion of Cells in Suspension and Outgrowth of Hybrids in
Conditioned Medium," in MONOCLONAL ANTIBODIES, Kennet et al.
(eds.), pages 370-371 (Plenum Publishing Corp. 1981).
[0220] Anti-P-glycoprotein hybridoma cultures are initially
screened using an indirect ELISA with a horseradish peroxidase
conjugate of goat anti-mouse immunoglobulin. Monolayers of the MDR
cells and the drug-sensitive parental cell line are cultured in
96-well microtiter plates. Cells are fixed with 0.01%
glutaraldehyde for 45 minutes at room temperature, the fixative is
removed, cells are washed three times with phosphate-buffered
saline (PBS), and the microtiter wells are blocked with 10% bovine
serum albumin for at least 45 minutes. Fifty microliters of
hybridoma supernatants are added to the microtiter wells and
allowed to incubate for one hour at 37.degree. C. Plates are then
washed with PBS and incubated with 50 .mu.l of
peroxidase-conjugated goat anti-mouse immunoglobulin diluted 1:1000
in PBS with 10% horse serum. Following five washes with PBS,
positive clones are identified by the addition of 100 .mu.l of a
solution containing 1 mg/ml O-phenylenediamine, 0.1% hydrogen
peroxide, 50 mM citrate, and 100 mM sodium phosphate buffer (pH
5.0). The reaction is quenched by the addition of 50 .mu.l 4N
sulfuric acid, and the plates are read at 490 nm.
[0221] Clones that produce a five-fold or greater ELISA signal for
the MDR cells, compared with drug-sensitive cells, are expanded.
Hybridoma cells that produce anti-P-glycoprotein antibodies are
injected into BALB/c mice for ascites production according to the
procedure of Hoogenraad et al., J. Immunol. Methods 61: 317 (1983).
Anti-P-glycoprotein antibodies are purified from the ascites fluid
using protein A chromatography. See, for example, Langone et al.,
J. Immunol. Methods 51: 3 (1982).
[0222] The production of highly specific anti-CEA MAb, has been
described by Hansen et al., Cancer 71: 3478 (1993), which is
incorporated by reference. Briefly, a 20 gram BALB/c female mouse
was immunized subcutaneously with 7.5 .mu.g of partially-purified
CEA in complete Freund adjuvant. On day 3, the mouse was boosted
subcutaneously with 7.5 .mu.g of CEA in incomplete Freund adjuvant
and then, the mouse was boosted intravenously with 7.5 .mu.g of CEA
in saline on days 6 and 9. On day 278, the mouse was given 65 .mu.g
of CEA intravenously in saline and 90 .mu.g of CEA in saline on day
404. On day 407, the mouse was sacrificed, a cell suspension of the
spleen was prepared, the spleen cells were fused with murine
myeloma cells, SP2/0-Ag 14 (ATCC CRL 1581) using polyethylene
glycol, and the cells were cultured in medium containing
8-azaguanine. Hybridoma supernatants were screened for CEA-reactive
antibody using an .sup.125I-CEA radioimmunoassay (Roche; Nutley,
N.J.). Positive clones were recloned.
[0223] 2. The Production of Antibody Fragments
[0224] As described above, proteolysis provides one method for
preparing antibody fragments. This technique is well-known to those
of skill in the art. For example, see Coligan et al., supra, at pp.
2.8.1-2.8.10. Also see Stanworth et al. "Immunochemical Analysis of
Human and Rabbit Immunoglobulins and Their Subunits," in HANDBOOK
OF EXPERIMENTAL IMMUNOLOGY, Vol. 1, Weir (ed.), pages 12.1-12.46
(Blackwell Scientific 1986), and Parham, "Preparation and
Purification of Active Fragments from Mouse Monoclonal Antibodies,"
Id. at pages 14.1-14.23.
[0225] As an example, preactivated papain can be used to prepare
F(ab).sub.2 fragments from IgG1 or Fab fragments from IgG2a and
IgG2b, as follows. Papain is activated by incubating 2 mg/ml papain
(2.times.recrystallized suspension, Sigma #P3125) and 0.05 M
cysteine (free-base, crystalline; Sigma #C7755) for 30 minutes in a
37.degree. C. water bath. To remove cysteine, the papain/cysteine
mixture is applied to a PD-10 column (Pharmacia #G-25), which has
been equilibrated with 20 ml of acetate/EDTA buffer (0.1 M acetate
with 3 mM EDTA, pH 5.5). Fractions are assayed by measuring
absorbance at 280 nm, and the two or three fractions that contain
protein are pooled. The concentration of preactivated papain is
determined by using the formula: (absorbance at 280 nm)/2.5=mg.
preactivated papain/ml.
[0226] To prepare antibody for digestion, 10 mg of antibody in 2 to
5 ml of PBS are dialyzed against acetate/EDTA buffer. Five hundred
micrograms of preactivated papain are added to the dialyzed
antibody solution, and the mixture is vortexed. After a 6-12 hour
incubation in a 37.degree. C. water bath, papain is inactivated by
adding crystalline iodoacetamide (Sigma #I6125) to a final
concentration of 0.03 M. The mixture is then dialyzed against 1
liter of PBS (pH 8.0) at 4.degree. C. for 6-12 hours.
[0227] To remove undigested antibody and Fc fragments, the mixture
is applied to a protein A-Sepharose column which has been
equilibrated in PBS (pH 8.0). Unbound fractions are collected in 2
ml aliquots and pooled. After concentrating the pool to a total
volume of 5 ml or less, protein is fractionated by size-exclusion
chromatography and the results are analyzed by SDS-PAGE.
EXAMPLE 2
Preparation of Antibody Composite: Anti-P-Glycoprotein/anti-CEA
Bispecific Antibody
[0228] A bispecific F(ab').sub.2 antibody composite is prepared
from an Fab' fragment of an anti-P-glycoprotein monoclonal antibody
and an Fab' fragment of a monoclonal antibody specific for CEA,
using the methods described above. Also, see Goldenberg,
international publication No. WO 92/19273, which is incorporated by
reference. Briefly, the interchain disulfide bridges are reduced
carefully with cysteine, taking care to avoid light-heavy chain
cleavage, to form Fab'-SH fragments. The SH group(s) of one
antibody fragment is(are) activated with an excess of bis-maleimide
linker (1,1'-(methylenedi-1,4-phenylene) bismaleimide (Aldrich
Chemical Co.; Milwaukee, Wis.). The second antibody fragment is
also converted to Fab'-SH and then reacted with the activated first
antibody fragment to obtain a bispecific antibody composite.
EXAMPLE 3
Preparation of Polyspecific Immunoconjugate
[0229] A polyspecific immunoconjugate can be prepared by binding a
therapeutic or diagnostic agent to the bispecific antibody
composite, described in Example 2. As an example, the
anti-P-glycoprotein/anti-CEA composite can be conjugated with
doxorubicin via dextran, using the method of Shih et al., Int. J.
Cancer 41:832-839 (1988). Briefly, amino dextran is prepared by
dissolving one gram of dextran (m.w. 18 kD; Sigma Chemical Co.; St.
Louis, Mo.) in 70 ml of water. The dextran is partially oxidized to
form polyaldehyde dextran by adding 0.5 gram of sodium
metaperiodate, and stirring the solution at room temperature
overnight. After concentrating the mixture with an Amicon cell
(YM10 membrane; MWCO=10,000), the polyaldehyde dextran is purified
by Sephadex G-25 chromatography and lyophilized to give about 900
grams of white powder. Polyaldehyde dextran is then treated with
two equivalents of 1,3-diamino-2-hydroxypropane in aqueous phase
for 24 hours at room temperature. The resultant Schiff base is
stabilized by addition of sodium borohydride (0.311 mmol per 2.15
mmol of 1,3-diamino-2-hydroxyprop- ane) to the mixture. The mixture
is allowed to incubate at room temperature for six hours. Amino
dextran is purified using a Sephadex G-25 column.
[0230] Doxorubicin (Sigma Chemical Co.; St. Louis, Mo.) is
activated by adding one milliliter of anhydrous DMF to 0.1 mmole of
doxorubicin in a dried Reacti-vial, followed by a solution of
N-hydroxysuccinimide (23 mg, 0.2 mmole; Sigma) in 750 .mu.l of
anhydrous DMF and a solution of 1,3-dicyclohexylcarbodiimide (41.5
mg, 0.2 mmol; Sigma) in 750 .mu.l of anhydrous DMF. The reaction
mixture is stirred in the dark at room temperature for 16 hours
under anhydrous conditions. The precipitate is then centrifuged and
the solution is stored in a sealed bottle at -20.degree. C.
[0231] Doxorubicin-dextran intermediate conjugate is prepared by
dissolving aminodextran (18 kD; 10 mg) in two milliliters of PBS
(pH 7.2) and gradually adding 0.7 ml of the above
N-hydroxy-succinimide-activated doxorubicin solution. Thus, 50
moles of doxorubicin are present per mole of aminodextran. The
solution is stirred at room temperature for five hours and after
removing any precipitate, the conjugate is purified using a
Sephadex G-25 column. Doxorubicin-dextran conjugate is typically
characterized by a doxorubicin/dextran ratio of 14.
[0232] Alternatively, doxorubicin-dextran conjugate is prepared by
reacting doxorubicin with
1-ethyl-3(3-dimethylaminopropyl)-carbodiimide, as described by Shih
et al., Int. J. Cancer 41:832-839 (1988). Also, see Shih et al.,
Cancer Research 51:4192-4198 (1991).
[0233] The bispecific antibody conjugate (25 mg) in 5 ml of PBS (pH
5.5) is oxidized in the dark by treatment with sodium metaperiodate
(800 .mu.l of a 21.5 mg/ml solution) at room temperature for 60
minutes. The reaction mixture is then treated with ethylene glycol
(50 .mu.l) to decompose the unreacted periodate and the oxidized
antibody fragment is purified using a Sephadex G-25 column
equilibrated in 0.05 M HEPES (pH 7.4). Subsequently, the oxidized
fragment is concentrated to 5 mg/ml in 0.05 M HEPES (pH 7.4) and
reacted with the doxorubicin-dextran conjugate (22 mg). After 24
hours at room temperature, the Schiff base is reduced by
NaBH.sub.3CN. Conjugated antibody is purified using a Sepharose
CL-6B column.
EXAMPLE 4
Preparation of an Polyspecific Immunoconjugate Comprising a
Radioisotope
[0234] A polyspecific immunoconjugate can be prepared in which a
radioisotope is bound to one or more antibody components via a
chelator. As an illustration, the antibody composite of Example 2
may be conjugated with either aminobenzyl
diethylenetriaminepentaacetic acid (DTPA) or a derivative of DTPA
containing the long-chain linker, --CSNH(CH.sub.2).sub.10NH.sub.2
(LC-DTPA). Briefly, the antibody composite (2.5 mg in about one
milliliter of 50 mM acetate-buffered 0.9% saline [ABS; pH 5.3]) is
oxidized in the dark by treatment with sodium metaperiodate (210
.mu.l of a 5.68 mg/ml solution) at 0.degree. C. for one hour. The
reaction mixture is treated with ethylene glycol (20 .mu.l) to
decompose the unreacted periodate and the oxidized antibody
fragment is purified using a Sephadex G-50/80 column (Pharmacia;
Piscataway, N.J.) equilibrated in PBS (pH 6.1). The oxidized
fragment is then reacted with excess DTPA or LC-DTPA. After 40
hours at room temperature, the Schiff base is reduced by
NaBH.sub.3CN. Conjugated antibody composite is then purified using
a centrifuged size-exclusion column (Sephadex G-50/80) equilibrated
in 0.1 M acetate (pH 6.5). The concentrations of antibody
conjugates are determined by measuring absorbance at 280 nm.
[0235] The ratio of chelator molecules per molecule of antibody
composite is determined by a metal-binding assay. The assay is
performed by mixing an aliquot of the antibody conjugate with 0.1 M
ammonium acetate (pH 7) and 2 M triethanolamine, and incubating the
mixture at room temperature with a known excess of cobalt acetate
spiked with .sup.57cobalt acetate. After 30 minutes, EDTA (pH 7) is
added to a final concentration of 10 mM. After a further 10 minute
incubation, the mixture is analyzed by instant thin layer
chromatography (ITLC) using 10 mM EDTA for development. The
fraction of radioactivity bound to antibody is determined by
counting sections of ITLC strips on a gamma counter. Typically, the
results will show that there are about 6 molecules of DTPA per
antibody component and about 5 molecules of LC-DTPA per antibody
component.
[0236] Antibody conjugates are labeled with .sup.90yttrium, as
follows. Briefly, commercially available .sup.90yttrium chloride
(DuPont NEN; 17.68 .mu.l; 5.63 mCi) is buffered with 35.4 .mu.l of
0.5 M acetate (pH 6.0). The solution is allowed to stand for 5-10
minutes at room temperature, and then used for radiolabeling.
[0237] .sup.90Yttrium-labeled antibody composite-DTPA is prepared
by mixing .sup.90yttrium acetate (128.7 .mu.Ci) with antibody
composite-DTPA (30 .mu.g; 8.3 .mu.l), incubating at room
temperature for one hour, and diluting with 90 .mu.l of 0.1 M
acetate (pH 6.5). .sup.90 Yttrium-labeled antibody
composite-LC-DTPA is prepared by mixing .sup.90yttrium acetate
(109.5 .mu.Ci) with antibody composite-LC-DTPA (30 .mu.g; 7.6
.mu.l), incubating at room temperature for one hour, and diluting
with 90 .mu.l of 0.1 M acetate (pH 6.5).
[0238] The extent of .sup.90yttrium incorporation can be analyzed
by incubating the labeling mixture with 10 mM EDTA for ten minutes,
followed by ITLC examination using 10 mM EDTA for development. In
this assay, unbound .sup.90yttrium migrates with the solvent front,
while antibody-bound .sup.90yttrium remains at the origin. The
presence of any colloidal .sup.90yttrium is assayed by ITLC
(co-spotted with human serum albumin) using a water:ethanol:ammonia
(5:2:1) solution for development. In this system, the fraction of
radioactivity at the origin represents colloidal .sup.90yttrium. In
addition, all labeling mixtures may be analyzed using radio-high
pressure liquid chromatography. Typically, 90 to 96% of
.sup.90yttrium is incorporated into the resultant polyspecific
immunoconjugate.
EXAMPLE 5
Treatment of Colon Cancer with .sup.90Yttrium-labeled Polyspecific
Immunoconjugate and G-CSF
[0239] A patient has a carcinoembryonic antigen (CEA) blood titer
of 55 ng/ml due to peritoneal spread of a colon cancer which had
been resected two years earlier and found to be a Dukes' C lesion.
Since previous chemotherapy with fluorouracil had been
unsuccessful, the patient presents for experimental therapy. The
patient is given a 35 mCi dose of the .sup.90yttrium-labeled
polyspecific immmunoconjugate prepared in Example 4, by
intraperitoneal injection. Two days later, an infusion of 5
.mu.g/kg G-CSF (such as NEUPOGEN [Amgen, Inc.; Thousand Oaks,
Calif.]) is instituted intravenously, and the patient's hematologic
values are monitored thereafter. No significant drop in white blood
cell count is noted, thus permitting a repetition of the
radioimmunotherapy three weeks later, followed again by G-CSF
therapy. A third treatment is given two months later, and
radiological evidence of some tumor and ascites reduction is noted
four weeks later. Thus, the patient is able to tolerate higher and
more frequent doses of the radioimmunotherapy agent.
EXAMPLE 6
Preparation of an Antibody Composite Targeted to Multidrug
Resistant Psuedomonas Aeruginosa
[0240] Those of skill in the art can use standard methods to
produce antibodies against a multidrug transporter protein of an
infectious agent. As an illustration, a bispecific antibody can be
constructed which is targeted to multidrug resistant Psuedomonas
aeruginosa. Antibody components that bind to OprK, a multidrug
transporter protein of Psuedomonas aeruginosa, can be obtained
using OprK protein that is overexpressed by bacterial cells. For
example, the OprK gene can be synthesized using mutually priming
long oligonucleotides which are based upon the nucleotide sequence
disclosed in Poole et al., J. Bacteriol. 175: 7363 (1993). See, for
example, Ausubel et al. (eds.), CURRENT PROTOCOLS IN MOLECULAR
BIOLOGY, pages 8.2.8 to 8.2.13 (Wiley Interscience 1990). Also, see
Wosnick et al., Gene 60:115 (1987). Moreover, current techniques
using the polymerase chain reaction provide the ability to
synthesize genes as large as 1.8 kilobases in length. Adang et al.,
Plant Molec. Biol. 21:1131 (1993); Bambot et al., PCR Methods and
Applications 2:266 (1993).
[0241] The OprK gene is then cloned into a prokaryotic expression
vector which is subsequently introduced into competent E. coli
cells, using standard techniques. See, for example, Ausubel et al.,
supra, at pages 16.1.1-16.7.8. OprK protein is isolated from the
host cells using standard techniques. (Id.)
[0242] Alternatively, OprK protein can be isolated from Psuedomonas
aeruginosae which have been selected for the multidrug resistant
phenotype, as described by Poole et al., supra.
[0243] Isolated OprK protein is used to generate anti-OprK MAb, as
described above. Also, see Mole et al., "Production of Monoclonal
Antibodies Against Fusion Proteins Produced in Eschericia coli," in
DNA CLONING, VOLUME III: A PRACTICAL APPROACH, Glover (ed.), pages
113-139 (IRL Press 1987), and Dean "Preparation and Testing of
Monoclonal Antibodies to Recombinant Proteins,.sub.1" in METHODS IN
MOLECULAR BIOLOGY, VOLUME 10: IMMUNOCHEMICAL PROTOCOLS, Manson
(ed.) pages 43-63 (The Humana Press, Inc. 1992).
[0244] Thus, anti-OprK MAb, or a fragment thereof, provides one
antibody component of a bispecific antibody. The second antibody
component, which binds with a different antigen associated with the
exterior surface of Psuedomonas aeruginosa, may be obtained using
the general techniques described above. Alternatively, suitable
monoclonal antibodies can be purchased from American Type Culture
Collection (Rockville, Md.), such as antibodies against Psuedomonas
aeruginosa lipopolysaccharide (ATCC CRL Nos. 8753, 8754, 8795, 8796
and 8797), lipoprotein H2 of the outer envelop of Psuedomonas
aeruginosa (ATCC CRL 1783), Psuedomonas aeruginosa type a flagella
(ATCC HB 9130), and Psuedomonas aeruginosa type b flagella (ATCC HB
9129).
[0245] An antibody composite comprising a moiety that binds OprK
and a moiety that binds an exterior surface antigen of P.
aeruginosa can be prepared using the methods described in Example
2.
EXAMPLE 7
Preparation and Use of an .sup.111lndium-labeled Polyspecific
Immunoconjugate Targeted to Multidrug Resistant Psuedomonas
Aeruginosa
[0246] Antibody composite-chelator conjugates are prepared as
described in Example 4. The conjugates are labeled with
.sup.111Indium, as follows. Briefly, .sup.111Indium chloride is
buffered at pH 5.5 using ammonium acetate such that the final
acetate concentration is about 0.2 M. .sup.111Indium acetate is
added to a solution of the antibody composite-chelator conjugate in
0.1 M acetate (pH 6.5), and the mixture is incubated for about one
hour. Typically, reaction mixtures contain either 10 .mu.g of
antibody composite-DTPA and 73 .mu.Ci of .sup.111Indium, or 10
.mu.g of antibody composite-LC-DTPA and 126.7 .mu.Ci of
.sup.111Indium. The extent of .sup.111Indium incorporation is
analyzed using ITLC, as described above.
[0247] A patient with granulocytopenia has Pseudomonas aeruginosa
pneumonia which is no longer responsive to carbenicillin treatment.
Four millicuries of .sup.111Indium-labeled polyspecific
immmunoconjugate are injected intravenously and after waiting at
least 24 hours, the patient is scanned with a gamma camera. Foci of
increased radioactivity appear as nodes in the lower lobes of the
lung, indicating the presence of pneumonic infiltrates with
multidrug resistant Pseudomonas aeruginosa. A course of therapy is
designed in which an aminoglycoside and carbenicillin are
administered with nonradioactive polyspecific immunoconjugate that
comprises an OprK-binding moiety, a moiety that binds an exterior
surface antigen of Pseudomonas aeruginosa and a chemosensitizing
agent.
EXAMPLE 8
Preparation of a .sup.99mTc-labeled Polyspecific Immunoconjugate
Targeted to Multidrug Resistant Psuedomonas Aeruginosa
[0248] An antibody composite is prepared which binds oprK and E87
antigen, an exterior surface antigen of Psuedomonas aeruginosa.
General techniques for preparing the antibody composite are
described in Example 6, and preparation of anti-E87 monoclonal
antibodies is described by Sawada et al., U.S. Pat. No.
5,089,262.
[0249] The antibody composite is labeled with .sup.99mTc using
methods that are well-known to those of skill in the art. See, for
example, Crockford et al., U.S. Pat. No. 4,424,200, Paik et al.,
U.S. Pat. No. 4,652,440, Baidoo et al., Cancer Research (Suppl.)
50: 799s (1990), Griffiths et al., Cancer Research 51: 4594 (1991),
Pak et al., U.S. Pat. No. 5,053,493, Griffiths et al., U.S. Pat.
No. 5,128,119, Lever et al., U.S. Pat. No. 5,095,111, and Dean et
al., U.S. Pat. No. 5,180,816.
[0250] As an illustration, .sup.99mTc-labeled polyspecific
immunoconjugate can be obtained as described by Hansen et al., U.S.
Pat. No. 5,328,679. Briefly, a solution of 0.075 M SnCl.sub.2
(solution I) is prepared by dissolving 3350 mg SnCl.2H.sub.2O in
one milliliter of 6 N HCl and diluting the resultant solution with
sterile H.sub.2O which has been purged with argon. A solution of
0.1 M NaK tartrate in 0.05 M NaAc (pH 5.5) [solution II] is
prepared with sterile H.sub.2O purged with argon. One volume of
solution I is mixed with 26 volumes of solution II, and the
resultant solution III is filter sterilized and purged with
argon.
[0251] A solution of antibody composite is reduced with 20 mM
cysteine, and excess cysteine is removed by gel filtration. The
reduced antibody composite (2 mg/ml) is stabilized at pH 4.5 in
0.05 M NaOAc buffer containing 0.15 M saline. The resultant
solution IV is filter sterilized and purged with argon. Solution IV
is mixed with a sufficient amount of solution III to obtain a final
concentration of 123 .mu.g Sn per mg of reduced antibody composite.
The resultant solution V is adjusted to a pH of 4.5-4.8.
[0252] A sterile solution of sodium pertechnetate (10 mCi) in
saline is added to an aliquot of solution V which contains 1.25 mg
antibody composite and stable stannous ions, and the mixture is
gently agitated. Labeling is quantitative within 5 minutes. The
resultant solution of .sup.99mTc-labeled polyspecific
immunoconjugate is ready for immediate injection.
[0253] The .sup.99mTc-labeled polyspecific immunoconjugate is
administered to a subject, and sites of infection caused by
multidrug resistant Psuedomonas aeruginosa are localized using
single-photon emission computed tomography.
[0254] Although the foregoing refers to particular preferred
embodiments, it will be understood that the present invention is
not so limited. It will occur to those of ordinary skill in the art
that various modifications may be made to the disclosed embodiments
and that such modifications are intended to be within the scope of
the present invention, which is defined by the following
claims.
[0255] All publications and patent applications mentioned in this
specification are indicative of the level of skill of those in the
art to which the invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application were
specifically and individually indicated to be incorporated by
reference in its entirety.
* * * * *