U.S. patent application number 10/755382 was filed with the patent office on 2005-04-28 for cdr grafted type iii anti-cea humanized mouse monoclonal antibodies.
This patent application is currently assigned to IMMUNOMEDICS, INC.. Invention is credited to Armour, Kathryn L., Hansen, Hans J..
Application Number | 20050089538 10/755382 |
Document ID | / |
Family ID | 23236916 |
Filed Date | 2005-04-28 |
United States Patent
Application |
20050089538 |
Kind Code |
A1 |
Hansen, Hans J. ; et
al. |
April 28, 2005 |
CDR grafted type III anti-CEA humanized mouse monoclonal
antibodies
Abstract
A Class III, anti-CEA monoclonal antibody, preferably a
monoclonal antibody comprising the complementarity-determining
regions (CDRs) of a parental murine Class III, anti-CEA monoclonal
antibody is disclosed. This monoclonal antibody is preferably a
humanized monoclonal antibody in which the CDRs are engrafted to
the framework regions of a heterologous antibody, wherein the
humanized monoclonal antibody retains the binding specificity of
the parental murine monoclonal antibody. A preferred Class III,
anti-CEA monoclonal antibody is one in which the preferred
heterologous antibody is from a human. Also provided are DNA
constructs, vectors, cells and methods for producing the Class III,
anti-CEA monoclonal antibodies, and diagnostic and therapeutic
conjugates using same. Methods of treatment with a Class III,
anti-CEA monoclonal antibody and with the conjugate containing this
monoclonal antibody are provided as well as methods of diagnosis
with the conjugate containing this monoclonal antibody.
Inventors: |
Hansen, Hans J.; (Picayune,
MS) ; Armour, Kathryn L.; (Scotland, GB) |
Correspondence
Address: |
Heller Ehrman & McAuliffe
Suite 300
1666 K Street Northwest
Washington
DC
20006
US
|
Assignee: |
IMMUNOMEDICS, INC.
|
Family ID: |
23236916 |
Appl. No.: |
10/755382 |
Filed: |
January 13, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10755382 |
Jan 13, 2004 |
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09253794 |
Feb 22, 1999 |
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6676924 |
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09253794 |
Feb 22, 1999 |
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08318157 |
Oct 5, 1994 |
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5874540 |
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Current U.S.
Class: |
424/401 |
Current CPC
Class: |
C07K 2319/00 20130101;
A61K 47/6853 20170801; C07K 16/3007 20130101; A61P 35/00 20180101;
A61K 38/00 20130101; C07K 2317/565 20130101; C07K 2317/56 20130101;
C07K 2317/24 20130101 |
Class at
Publication: |
424/401 |
International
Class: |
A61K 006/00 |
Claims
What is claimed is:
1. A method for treating a patient comprising administering a
humanized Class III, anti-CEA, monoclonal antibody (mAb) to said
patient in an effective amount for treatment.
2. The method of claim 1, wherein the constant regions of said mAb
are from a human IgG1 antibody.
3. The method of claim 1, wherein said mAb comprises a
complementarity-determining region (CDR) selected from the group
consisting of KASQDVGTSVA (SEQ ID NO:20), WTSTRHT (SEQ ID NO:21),
QQYSLYRS (SEQ ID NO:22), TYWMS (SEQ.ID NO:23), EIHPDSSTINYAPSLKD
(SEQ ID NO:24), LYFGFPWFAY (SEQ ID NO:25), and a combination
thereof, wherein said mAb is unreactive with meconium antigen by
enzyme immunoassay.
4. The method of claim 3, wherein said mAb retains the binding
specificity of a parental murine Class III, anti-CEA mAb which
comprises said CDRs.
5. The method of claim 1, wherein said treatment comprises
administering said mAb to patients with a CEA-producing cancer.
6. The method of claim 5, wherein said CEA-producing cancer is
selected from the group consisting of colon cancer, breast cancer
and lung cancer.
7. The method of claim 1, wherein said mAb further comprises
framework regions (FRs) of light and heavy chain variable regions
from a human antibody.
8. The method of claim 1, wherein said mAb further comprises
framework regions (FRs) in the light and heavy chain variable
regions, wherein each FR is separately a FR of a human
antibody.
9. The method of claim 8, wherein each of said light and heavy
chain variable regions chains comprise FRs from at least two human
antibodies.
10. The method of claim 7, wherein the light chain variable region
comprises a framework region (FR) selected from the group
consisting of DIQLTQSPSSLSASVGDRVTITC (SEQ ID NO:26);
WYQQKPGKAPKLLIY (SEQ ID NO:27); GVP(S or D)RFSGS(G or V)
SGTDFTFTISSLQPEDIATYYC (SEQ ID NO:28); FGQGTKVEIK (SEQ ID NO:29) or
a combination thereof; and the heavy chain variable region
comprises a framework region (FR) selected from the group
consisting of EVQLVESGGGVVQPG RSLRLSCSSSGFDFT (SEQ ID NO:30),
EVQLVESGGG VVQPGRSLRLSCSASGFDFT (SEQ ID NO:31); WVRQAPGKGLEWVA (SEQ
ID NO:33); RFTIS RDNSKNTLFLQMDSLRPEDTGVYFCAS (SEQ ID NO:36),
RFTISRDNAKNTLFLQMDSLRPEDTGVYF- CAS (SEQ ID NO:37); WGQGTPVTVSS (SEQ
ID NO:39); and a combination thereof; and wherein C may be in the
sulfhydryl or disulfide form.
11. The method of claim 10, wherein the light chain and heavy chain
variable regions comprise FRs comprising:
5 FRL1 comprises DIQLTQSPSSLSASVGDRVTITC; (SEQ ID NO:26) FRL2
comprises WYQQKPGKAPKLLIY; (SEQ ID NO:27) FRL3 comprises GVP(S or
D)RFSGS(G or V) (SEQ ID NO:28) SGTDFTFTISSLQPEDIATYYC; FRL4
comprises FGQGTKVEIK; (SEQ ID NO:29) FRH1 comprises EVQLVESGGGVVQPG
RSLRLSCSSSGFDFT, (SEQ ID NO:30) or EVQLVESGGG VVQPGRSLRLSCSASGFDFT;
(SEQ ID NO:31) FRH2 comprises WVRQAPGKGLEWVA; (SEQ ID NO:33) FRH3
comprises RFTIS RDNSKNTLFLQMDSLRPEDTGVYFCAS, (SEQ ID NO:36) or
RFTISRDNAKNTLFLQMDSLRPEDTGVYFCAS; (SEQ ID NO:37) and FRH4 comprises
WGQGTPVTVSS; (SEQ ID NO:39)
and wherein C may be in the sulfhydryl or disulfide form.
12. A method for treating a patient comprising administering a
conjugate to said patient in an effective amount for treatment,
wherein said conjugate comprises: a therapeutic agent bound to a
humanized Class III, anti-CEA, monoclonal antibody (mAb) or a
fragment thereof, wherein said mAb or fragment thereof comprises a
complementarity-determining region (CDR) selected from the group
consisting of KASQDVGTSVA (SEQ ID NO:20), WTSTRHT (SEQ ID NO:21),
QQYSLYRS (SEQ ID NO:22), TYWMS (SEQ.ID NO:23), EIHPDSSTINYAPSLKD
(SEQ ID NO:24), LYFGFPWFAY (SEQ ID NO:25), and a combination
thereof, wherein said mAb is unreactive with meconium antigen by
enzyme immunoassay.
13. The method of claim 12, wherein said mAb retains the binding
specificity of a parental murine Class III, anti-CEA mAb which
comprises said CDRs.
14. The method of claim 12, wherein said therapeutic agent
comprises a cytotoxic agent or an immunomodulator.
15. The method of claim 14, wherein said cytotoxic agent comprises
doxorubicin, methotrexate, taxol, ricin A or a radionuclide.
16. The method of claim 15, wherein said radionuclide comprises
.sup.131I.
17. The method of claim 12, further comprising administering a
second therapeutic agent to said patient.
18. The method of claim 17, wherein said second therapeutic agent
is doxorubicin, methotrexate, taxol, ricin A, or a
radionuclide.
19. The method of claim 14, wherein said cytotoxic agent is a
radionuclide, and said method further comprises administering a
second therapeutic agent or performing a treatment regimen to said
patient.
20. The method of claim 19, wherein said treatment regimen is a
bone marrow reinfusion.
21. The method of claim 19, wherein said radionuclide is
.sup.131I.
22. The method of claim 12, wherein said mAb retains the binding
specificity of a parental murine Class III, anti-CEA mAb which
comprises said CDRs.
23. The method of claim 12, wherein said treatment comprises
administering said conjugate to a patient with a CEA-producing
cancer.
24. The method of claim 23, wherein said CEA-producing cancer is
selected from the group consisting of colon cancer, breast cancer
and lung cancer.
25. The method of claim 12, said mAb or fragment thereof further
comprises framework regions (FRs) of light and heavy chain variable
regions from a human antibody.
26. The method of claim 12, wherein said mAb further comprises
framework regions (FRs) in the light and heavy chain variable
regions, wherein each FR is separately a FR of a human
antibody.
27. The method of claim 26, wherein each of said light and heavy
chain variable regions chains comprise FRs from at least two human
antibodies.
28. The method of claim 25, wherein the light chain variable region
comprises a framework region (FR) selected from the group
consisting of DIQLTQSPSSLSASVGDRVTITC (SEQ ID NO:26);
WYQQKPGKAPKLLIY (SEQ ID NO:27); GVP(S or D)RFSGS(G or V)
SGTDFTFTISSLQPEDIATYYC (SEQ ID NO:28); FGQGTKVEIK (SEQ ID NO:29)
and a combination thereof; and the heavy chain variable region
comprises a framework region (FR) selected from the group
consisting of EVQLVESGGGVVQPG RSLRLSCSSSGFDFT (SEQ ID NO:30),
EVQLVESGGG VVQPGRSLRLSCSASGFDFT (SEQ ID NO:31); WVRQAPGKGLEWVA (SEQ
ID NO:33); RFTIS RDNSKNTLFLQMDSLRPEDTGVYFCAS (SEQ ID NO:36),
RFTISRDNAKNTLFLQMDSLRPEDTGVYF- CAS (SEQ ID NO:37); WGQGTPVTVSS (SEQ
ID NO:39); and a combination thereof; and wherein C may be in the
sulfhydryl or disulfide form.
29. The method of claim 28, wherein the light chain and heavy chain
variable regions comprise FRs comprising:
6 FRL1 comprises DIQLTQSPSSLSASVGDRVTITC; (SEQ ID NO:26) FRL2
comprises WYQQKPGKAPKLLIY; (SEQ ID NO:27) FRL3 comprises GVP(S or
D)RFSGS(G or V) (SEQ ID NO:28) SGTDFTFTISSLQPEDIATYYC; FRL4
comprises FGQGTKVEIK; (SEQ ID NO:29) FRH1 comprises EVQLVESGGGVVQPG
RSLRLSCSSSGFDFT, (SEQ ID NO:30) or EVQLVESGGG VVQPGRSLRLSCSASGFDFT;
(SEQ ID NO:31) FRH2 comprises WVRQAPGKGLEWVA; (SEQ ID NO:33) FRH3
comprises RFTIS RDNSKNTLFLQMDSLRPEDTGVYFCAS, (SEQ ID NO:36) or
RFTISRDNAKNTLFLQMDSLRPEDTGVYFCAS; (SEQ ID NO:37) and FRH4 comprises
WGQGTPVTVSS; (SEQ ID NO:39)
and wherein C may be in the sulfhydryl or disulfide form.
30. A method for diagnosing a patient comprising administering a
conjugate to said patient in an effective amount for diagnosis,
wherein said conjugate comprises: a diagnostic agent bound to a
humanized Class III, anti-CEA, monoclonal antibody (mAb) or a
fragment thereof, wherein said mAb or fragment thereof comprises a
complementarity-determining region (CDR) selected from the group
consisting of KASQDVGTSVA (SEQ ID NO:20), WTSTRHT (SEQ ID NO:21),
QQYSLYRS (SEQ ID NO:22), TYWMS (SEQ.ID NO:23), EIHPDSSTINYAPSLKD
(SEQ ID NO:24), LYFGFPWFAY (SEQ ID NO:25), and a combination
thereof, wherein said mAb is unreactive with meconium antigen by
enzyme immunoassay.
31. The method of claim 30, wherein said mAb retains the binding
specificity of a parental murine Class III, anti-CEA mAb which
comprises said CDRs.
32. The method of claim 30, wherein said diagnostic agent comprises
an imaging agent.
33. The method of claim 32, wherein said imaging agent is a
radionuclide or a metal chelator complexed radionuclide.
34. The method of claim 33, wherein said radionuclide is iodine,
yttrium or technetium.
35. The method of claim 34, wherein said radionuclide is
.sup.131I.
36. The method of claim 30, wherein said diagnosis comprises
administering said conjugate to a patient with a CEA-producing
cancer.
37. The method of claim 36, wherein said CEA-producing cancer is
selected from the group consisting of colon cancer, breast cancer
and lung cancer.
38. The method of claim 30, said mAb or fragment thereof further
comprises framework regions (FRs) of light and heavy chain variable
regions from a human antibody.
39. The method of claim 30, wherein said mAb further comprises
framework regions (FRs) in the light and heavy chain variable
regions, wherein each FR is separately a FR of a human
antibody.
40. The method of claim 39, wherein each of said light and heavy
chain variable regions chains comprise FRs from at least two human
antibodies.
41. The method of claim 38, wherein the light chain variable region
comprises a framework region (FR) selected from the group
consisting of DIQLTQSPSSLSASVGDRVTITC (SEQ ID NO:26);
WYQQKPGKAPKLLIY (SEQ ID NO:27); GVP(S or D)RFSGS(G or V)
SGTDFTFTISSLQPEDIATYYC (SEQ ID NO:28); FGQGTKVEIK (SEQ ID NO:29)
and a combination thereof; and the heavy chain variable region
comprises a framework region (FR) selected from the group
consisting of EVQLVESGGGVVQPG RSLRLSCSSSGFDFT (SEQ ID NO:30),
EVQLVESGGG VVQPGRSLRLSCSASGFDFT (SEQ ID NO:31); WVRQAPGKGLEWVA (SEQ
ID NO:33); RFTIS RDNSKNTLFLQMDSLRPEDTGVYFCAS (SEQ ID NO:36),
RFTISRDNAKNTLFLQMDSLRPEDTGVYF- CAS (SEQ ID NO:37); WGQGTPVTVSS (SEQ
ID NO:39); and a combination thereof; and wherein C may be in the
sulfhydryl or disulfide form.
42. The method of claim 41, wherein the light chain and heavy chain
variable regions comprise FRs comprising:
7 FRL1 comprises DIQLTQSPSSLSASVGDRVTITC; (SEQ ID NO:26) FRL2
comprises WYQQKPGKAPKLLIY; (SEQ ID NO:27) FRL3 comprises GVP(S or
D)RFSGS(G or V) (SEQ ID NO:28) SGTDFTFTISSLQPEDIATYYC; FRL4
comprises FGQGTKVEIK; (SEQ ID NO:29) FRH1 comprises EVQLVESGGGVVQPG
RSLRLSCSSSGFDFT, (SEQ ID NO:30) or EVQLVESGGG VVQPGRSLRLSCSASGFDFT;
(SEQ ID NO:31) FRH2 comprises WVRQAPGKGLEWVA; (SEQ ID NO:33) FRH3
comprises RFTIS (SEQ ID NO:36) RDNSKNTLFLQMDSLRPEDTGVYFCAS, or
RFTISRDNAKNTLFLQMDSLRPEDTGVYFCAS; (SEQ ID NO:37) and FRH4 comprises
WGQGTPVTVSS; (SEQ ID NO:39)
and wherein C may be in the sulfhydryl or disulfide form.
43. A Class III, anti-CEA monoclonal antibody (mAb) or
antigen-binding fragment thereof comprising a
complementarity-determining region (CDR) selected from the group
consisting of KASQDVGTSVA (SEQ ID NO:20), WTSTRHT (SEQ ID NO:21),
QQYSLYRS (SEQ ID NO:22), TYWMS (SEQ.ID NO:23), EIHPDSSTINYAPSLKD
(SEQ ID NO:24), LYFGFPWFAY (SEQ ID NO:25), and a combination
thereof, wherein said mAb is unreactive with meconium antigen by
enzyme immunoassay.
44. The mAb or fragment thereof of claim 43, wherein said mAb
retains the binding specificity of a parental murine Class III,
anti-CEA mAb which comprises said CDRs.
45. The mAb or fragment thereof of claim 44, wherein the light
chain variable region of said mAb or fragment thereof comprises
CDRL1 comprising KASQDVGTSVA (SEQ ID NO:20), CDRL2 comprising
WTSTRHT (SEQ ID NO:21), and CDRL3 comprising QQYSLYRS (SEQ ID
NO:22), and the heavy chain variable region of said mAb or fragment
thereof comprises CDRH1 comprising TYWMS (SEQ.ID NO:23), CDRH2
comprising EIHPDSSTINYAPSLKD (SEQ ID NO:24) and CDRH3 comprising
LYFGFPWFAY (SEQ ID NO:25).
46. The mAb of claim 45, said mAb or fragment thereof is a
humanized Class III, anti-CEA mAb and further comprises framework
regions (FRs) of light and heavy chain variable regions from a
human antibody.
47. The mAb of claim 46, wherein: (a) the light chain variable
regions are characterized by the formula:
FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4, wherein each FR is a
different framework region of a human antibody, and FRL1 comprises
a region of about 23 amino acids that occurs naturally in the FRL1
of a human antibody; FRL2 comprises a region of about 15 amino
acids that occurs naturally in the FRL2 of a human antibody; FRL3
comprises a region of about 32 amino acids that occurs naturally in
the FRL3 of a human antibody; FRL4 comprises a region of about 10
amino acids that occurs naturally in the FRL4 of a human antibody;
and (b) the heavy chain variable regions are characterized by the
formula: FRH1--CDRH1--FRH2-CDRH2--FRH3-CDRH3--FRH4, wherein each FR
is a different framework region of a human antibody, and FRH1
comprises a region of 28-32 amino acids that occurs naturally in
the FRH1 of a human antibody; FRH2 comprises a region of 12-16
amino acids that occurs naturally in the FRH2 of a human antibody;
FRH3 comprises a region of 30-34 amino acids that occurs naturally
in the FRH3 of a human antibody; and FRH4 comprises a region of
9-13 amino acids that occurs naturally in the FRH4 of a human
antibody.
48. The mAb of claim 47, wherein
8 FRL1 comprises DIQLTQSPSSLSASVGDRVTITC; (SEQ ID NO:26) FRL2
comprises WYQQKPGKAPKLLIY; (SEQ ID NO:27) FRL3 comprises GVP(S or
D)RFSGS(G or V) (SEQ ID NO:28) SGTDFTFTISSLQPEDIATYYC; FRL4
comprises FGQGTKVEIK; (SEQ ID NO:29) FRH1 comprises EVQLVESGGG
VQPGRSLRLSCSSSGFDFT, (SEQ ID NO:30) or
EVQLVESGGGVVQPGRSLRLSCSASGFDFT; (SEQ ID NO:31) FRH2 comprises
WVRQAPGKGLEWVA; (SEQ ID NO:33) FRH3 comprises
RFTISRDNSKNTLFLQMDSLRPEDTGVYFCAS, (SEQ ID NO:36) or
RFTISRDNAKNTLFLQMDSLRPEDTGVYFC AS; (SEQ ID NO:37) and FRH4
comprises WGQGTPVTVSS; (SEQ ID NO:39) and wherein C may be in the
sulfhydryl or disulfide form.
49. A humanized Class III, anti-CEA mAb comprising framework
regions (FRs) of light and heavy chain variable regions from a
human antibody, wherein the light chain variable region comprises a
framework region (FR) selected from the group consisting of
DIQLTQSPSSLSASVGDRVTITC (SEQ ID NO:26); WYQQKPGKAPKLLIY (SEQ ID
NO:27); GVP(S or D)RFSGS(G or V) SGTDFTFTISSLQPEDIATYYC (SEQ ID
NO:28); FGQGTKVEIK (SEQ ID NO:29) and a combination thereof; and
the heavy chain variable region comprises a framework region (FR)
selected from the group consisting of EVQLVESGGGVVQPG
RSLRLSCSSSGFDFT (SEQ ID NO:30), EVQLVESGGG VVQPGRSLRLSCSASGFDFT
(SEQ ID NO:31); WVRQAPGKGLEWVA (SEQ ID NO:33); RFTIS
RDNSKNTLFLQMDSLRPEDTGVYFCAS (SEQ ID NO:36),
RFTISRDNAKNTLFLQMDSLRPEDTGVYF- CAS (SEQ ID NO:37); WGQGTPVTVSS (SEQ
ID NO:39); and a combination thereof; and wherein C may be in the
sulfhydryl or disulfide form; and wherein said mAb is unreactive
with meconium antigen by enzyme immunoassay and unreactive with
normal tissue.
50. The mAb of claim 49, wherein the light chain and heavy chain
variable regions comprise FRs comprising:
9 FRL1 comprises DIQLTQSPSSLSASVGDRVTITC; (SEQ ID NO:26) FRL2
comprises WYQQKPGKAPKLLIY; (SEQ ID NO:27) FRL3 comprises GVP(S or
D)RFSGS(G or V) (SEQ ID NO:28) SGTDFTFTISSLQPEDIATYYC; FRL4
comprises FGQGTKVEIK; (SEQ ID NO:29) FRH1 comprises EVQLVESGGGVVQPG
RSLRLSCSSSGFDFT, (SEQ ID NO:30) or EVQLVESGGG VVQPGRSLRLSCSASGFDFT;
(SEQ ID NO:31) FRH2 comprises WVRQAPGKGLEWVA; (SEQ ID NO:33) FRH3
comprises RFTIS RDNSKNTLFLQMDSLRPEDTGVYFCAS, (SEQ ID NO:36) or
RFTISRDNAKNTLFLQMDSLRPEDTGVYFCAS; (SEQ ID NO:37) and FRH4 comprises
WGQGTPVTVSS; (SEQ ID NO:39)
and wherein C may be in the sulfhydryl or disulfide form.
51. An isolated polynucleotide comprising a nucleic acid sequence
encoding the mAb or fragment thereof of claim 43.
52. An expression vector comprising the isolated polynucleotide of
claim 51.
53. A transformed cell comprising the isolated polynucleotide of
claim 51.
54. The transformed cell of claim 53, wherein said cell is a
mammalian cell.
55. A method of producing a Class III, anti-CEA mAb comprising (a)
transforming a cell with the isolated polynucleotide of claim 51;
and (b) expressing said polynucleotide by culturing said cell; and
(c) producing said mAb.
56. A complementarity determining region (CDR) comprising
KASQDVGTSVA (SEQ ID NO:20).
57. A complementarity determining region (CDR) comprising WTSTRHT
(SEQ ID NO:21).
58. A complementarity determining region (CDR) comprising QQYSLYRS
(SEQ ID NO:22).
59. A complementarity determining region (CDR) comprising TYWMS
(SEQ.ID NO:23).
60. A complementarity determining region (CDR) comprising
EIHPDSSTINYAPSLKD (SEQ ID NO:24).
61. A complementarity determining region (CDR) comprising
LYFGFPWFAY (SEQ ID NO:25).
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This application is a continuation of application Ser. No.
09/253,794, filed Feb. 22, 1999, now pending, which is a divisional
of application Ser. No. 08/318,157, filed Oct. 5, 1994, now U.S.
Pat. No. 5,874,540, which are both incorporated by reference in
their entirety. This application claims only subject matter
disclosed in the parent applications and therefore presents no new
matter.
BACKGROUND OF THE INVENTION
[0002] The invention relates to immunological reagents for
diagnostic and therapeutic use in colon and other cancers. In
particular, the invention relates to humanized
anti-carcinoembryonic antigen ("CEA") monoclonal antibodies
("mAbs") that have the binding affinity characteristics of
corresponding mouse anti-CEA mAb (MN14) and the antigenic and
effector properties of a human antibody. Further, the invention
relates to humanized mAbs in which the complementarity determining
regions ("CDRs") of an anti-CEA murine mAb is grafted into the
framework regions of a human antibody, to DNAs that encode such
CDR-grafted antibodies, to vectors and transformed hosts for
propagating and expressing the DNAs, and to conjugates of the
antibodies useful in diagnostic and therapeutic applications.
[0003] A promising approach to cancer diagnosis and therapy
involves the use of targeting antibodies to deliver diagnostic and
therapeutic agents directly to the site of a malignancy. Over the
past decade, a wide variety of tumor-specific antibodies and
antibody fragments have been developed, as have methods to
conjugate the antibodies to drugs, toxins, radionuclides or other
agents, and to administer the conjugates to patients. These efforts
have produced great progress, but a variety of largely
unanticipated problems have limited the diagnostic and therapeutic
utility of some of the reagents thus far developed.
[0004] Among the most intractable problems is that which is caused
by the human immune system itself, which may respond to the
targeting conjugate as a foreign antigen. For instance, patients
treated with drugs or radionuclides complexed with murine
monoclonal antibodies (which have been the most commonly used
targeting antibodies for human) develop circulating human
anti-mouse antibodies (HAMAs) and a generalized immediate type-III
hypersensitivity reaction to the antibody moiety of the conjugate.
Furthermore, even when adverse side effects are minimal (for
example, as in a single administration), circulating HAMAs decrease
the effective concentration of the targeting agent in the patient
and therefore limiting the diagnostic or therapeutic agent from
reaching the target site.
[0005] Several approaches have been developed to overcome or avoid
this problem, with only limited success. One strategy has been to
chemically modify the targeting antibody to suppress its
antigenicity. For example, conjugation of polyethylene glycol to
the targeting antibody (PEGylation) is reported to reduce
antigenicity of antibodies. Another approach has been to
characterize the situs of antigenicity in an antibody and then
remove it. In this vein, Fab', F(ab).sub.2 and other antibody
fragments have been used in place of whole IgG. In addition,
attempts have been made to reduce the adverse effects of HAMA by
plasmaphoretically removing HAMA from blood. Immunosuppressive
techniques also have been used to ameliorate the adverse effect of
the foreign antibody sufficiently to permit multiple treatments
with the targeting agent.
[0006] None of these approaches has proven altogether satisfactory.
An important need persists for a means to reduce or eliminate the
adverse immune response to targeting antibody and antibody
conjugates in order to gain the full benefit of these diagnostic
and therapeutic agents.
[0007] This goal has been achieved with the CDR-grafted humanized
murine anti-human CEA mAbs that are described below.
SUMMARY OF THE INVENTION
[0008] It is an object of the present invention to provide a
humanized Class III anti-CEA mAb in which the CDRs of a murine
Class III anti-CEA mAb (MN14) are functionally engrafted to the
amino acid sequence of a human antibody or antibody fragment to
provide an immunological reagent with the anti-CEA binding
properties of the murine Class III, anti-CEA mAb and the
immunogenic properties of a human mAb in a human patient.
[0009] It is another object of the present invention to provide DNA
constructs encoding such antibodies. Particular objects in this
regard are substrate DNAs that facilitate genetic manipulation to
produce improved antibodies and DNAs encoding the antibodies with
advantageous properties in cell culture and antibody
production.
[0010] Yet another object of the invention is to provide vectors
for propagating the DNA and for expressing the antibody. A related
object of the invention is to provide cells containing a vector for
the purposes of storage, propagation, antibody production and
therapeutic applications.
[0011] Still another object of the invention is to provide
compositions comprising the antibodies for use in diagnosis and
therapy. In this regard it is an object of the invention to provide
conjugates comprising the antibodies complexed with imaging agents
and therapeutic agents for ex vivo and in vivo imaging, diagnosis,
prognosis and therapy, among others.
[0012] In accomplishing the foregoing objects, there has been
provided, in accordance with one aspect of the present invention, a
humanized mouse mAb, comprising the CDRs of a murine Class III,
anti-CEA mAb (MN-14) engrafted to the framework regions of a
heterologous (human) antibody, wherein the thus humanized mAb
antibody retains the Class III, anti-CEA binding specificity of the
murine mAb but in the patient is less immunogenic than is the
parent MN-14 murine monoclonal antibody.
[0013] In a highly preferred embodiment, the light chain variable
regions of the humanized antibody are characterized by the
formula:
FR.sub.L1-CDR.sub.L1-FR.sub.L2-CDR.sub.L2-FR.sub.L3-CDR.sub.L3-FR.sub.L4
[0014] wherein each FR is separately a framework region of a human
antibody, and each CDR is separately in a
complementarity-determining region of the light chains of MN-14,
and the subscripts refer to light ("L") chain regions. The heavy
chain variable regions are characterized by the formula:
FR.sub.H1-CDR.sub.H1-FR.sub.H2-CDR.sub.H2-FR.sub.H3-CDR.sub.H3-FR.sub.H4
[0015] wherein FR and CDR have the same meanings as above, and
wherein the subscripts "H" refer to heavy chain regions.
[0016] In one embodiment, CDR.sub.L1 has the amino acid sequence
KASQDVGTSVA (SEQ. ID NO. 20); CDR.sub.L2 has the amino acid
sequence WTSTRHT (SEQ. ID NO. 21); CDR.sub.L3 has the amino acid
sequence QQYSLYRS (SEQ. ID NO. 22); CDR.sub.H1 has the amino acid
sequence TYWMS (SEQ. ID. NO. 23); CDR.sub.H2 has the amino acid
sequence EIHPD SSTINYAPSLKD (SEQ. ID NO. 24); and, CDR.sub.H3 has
the amino acid sequence LYFGFPWFAY (SEQ. ID NO. 25).
[0017] In another embodiment, FR.sub.L1 has the amino acid sequence
DIQLTQSPSS LSASVGDRVTITC (SEQ. ID NO. 26); FR.sub.L2 has the amino
acid sequence WYQQKPGKAP KLLIY (SEQ. ID NO. 27); FR.sub.L3 has the
amino acid sequence GVP(S or D)RFSGS(G or V) SGTDFTFTISSLQPEDIATYYC
(SEQ. ID NO. 28); FR.sub.L4 has the amino acid sequence FGQGTKVEIK
(SEQ. ID NO. 29); FR.sub.H1 has the amino acid sequence EVQLVESGGG
VVQPGRSLRLSCSSSGFDFT (SEQ. ID NO. 30), EVQLVESGGGVVQPGRSLRLSCSAS
GFDFT (SEQ. ID NO. 31), or QVQLQ ESGPG LVRPS QTLSL TCTSS GFDFT
(SEQ. ID NO. 32); FR.sub.H2 has the amino acid sequence
WVRQAPGKGLEWVA (SEQ. ID NO. 33), WVRQAPGKGLEWIA (SEQ. ID NO. 34),
or WVRQPPGRGLEWIA (SEQ. ID NO. 35); FR.sub.H3 has the amino acid
sequence RFTISRDNSKNTLFLQMDSLRPEDTGVYFCAS (SEQ. ID NO. 36),
RFTISRDNAKNTLFLQMDSLRPEDTGVYFC AS (SEQ. ID NO. 37), or
RVTMLRDTSKNGSFLRLSSVTAADTAVYYCAS (SEQ. ID NO. 38); and FR.sub.H4
has the amino acid sequence WGQGTPVTVSS (SEQ. ID NO. 39), or
WGQGTTVTVSS (SEQ. ID NO. 40); and wherein C may be in the
sulfhydryl or disulfide form.
[0018] Another preferred embodiment comprises a diagnostic or
therapeutic agent complexed to Class III, anti-CEA humanized mAb in
which the CDRs of the antibody are derived from those of the MN-14
murine mAb and the FRs are derived from those of the heterologous
(human) antibody, wherein the conjugate retains the Class III,
anti-CEA binding specificity of MN-14, but is in humans less
immunogenic than is murine MN-14. In one such embodiment the light
chain and heavy chain variable regions are characterized as shown
above and have amino acid sequences also as described above.
[0019] In yet another preferred embodiment, a method for diagnosing
or treating a patient comprises the step of administering in an
appropriate regimen the conjugate of the previous preferred
embodiment.
[0020] Another preferred embodiment comprises an isolated, purified
DNA that encodes the light chain, the heavy chain or both chains of
the humanized antibody described above.
[0021] Another preferred embodiment comprises the DNA sequence of
the CDRs and FRs described above.
[0022] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description and appended claims.
Illustrative Glossary
[0023] The following terms or abbreviations are used in the present
application. The meanings set out in this glossary are for
illustrative purposes only. The full meaning of the terms will be
apparent to those of skill in the art.
[0024] "CDR" is used as an abbreviation for Complementarity
Determining Region. These are the regions within the variable
regions of an antibody that are primarily, but not exclusively,
responsible for antigen-antibody binding.
[0025] "FR" is an abbreviation for Framework Region. Broadly
speaking, these are the portions of the variable regions of an
antibody which lie adjacent to or flank the CDRs. In general, these
regions have more of a structural function that affects the
conformation of the variable region and are less directly
responsible for the specific binding of antigen to antibody,
although, nonetheless, the framework regions can affect the
interaction.
[0026] "Chimeric" refers to an antibody in which the variable
region is derived from a mouse antibody and the constant region is
derived from an antibody from a heterologous (other) species.
[0027] "Humanized" refers to a chimeric antibody as defined above,
but one in which the FR variable regions are derived from a human
antibody.
[0028] "HAMA" refers to human antibodies directed to a mouse
antibody, that are produced when a mouse antibody is administered
to a human subject.
[0029] "HAHA" refers to human antibodies directed to a humanized
mouse antibody.
[0030] "CEA" refers to carcinoembryonic antigen, a 180 kDa
glycoprotein that is expressed in most adenocarcinomas of
endodermally-derived digestive system epithelia and in some other
cancers such as breast cancer and non-small cell lung cancer.
[0031] The letter "h" as a prefix means "humanized".
[0032] Other abbreviations are used in accordance with Roitt et al,
IMMUNOLOGY, 3rd ed. Mosby Year Book Europe Ltd. (1993), the
entirety of which is herein incorporated by reference.
[0033] These and other terms used in the present disclosure are
used in the same sense as ordinarily they are employed in the arts
to which this invention pertains.
BRIEF DESCRIPTION OF THE FIGURES
[0034] FIGS. 1A and 1B (SEQ. ID. NOS. 1 and 2) shows the consensus
DNA sequence of murine NEWM MN-14 variable region heavy chain
("VH") and its protein translation product. The CDRs are enclosed
in boxes.
[0035] FIGS. 2A and 2B (SEQ. ID. NOS. 3 and 4) shows the consensus
DNA sequence of murine MN-14 variable region light chain ("VK") and
its protein translation product. The CDRs are enclosed in
boxes.
[0036] FIG. 3 shows a vector for the expression of chimeric or
humanized MN-14 heavy chain gene. The schematic diagram shows both
the chimeric and a reshaped heavy chain immunoglobulin (Ig) gene
and the pSVgpt expression vector. The diagram at top, labeled
"CHIMERIC," is a map showing DNA encoding the MN-14 mouse VH region
joined to DNA encoding a human IgG1 constant region. In the MN-14
VH region the three CDRs are indicated by the three dark areas. The
FRs are indicated by the four stippled areas. The middle diagram,
labeled "RESHAPED" shows the humanization of the MN-14 VH region in
which the mouse FRs have been replaced by human FRs, indicated by
the four clear areas in the "Human" VH region. The circular map of
the expression vector pSVgpt at bottom shows the HindIII/BamHI
insertion site for the reshaped MN-14 antibody gene just downstream
from an Igh enhancer element. The map also indicates some important
functional domains in the vector, including the replication origins
for propagation in E. coli (colEl ori) and in mammalian cells (SV40
promoter region), and genes encoding selective markers for
culturing bacterial (Ap.sup.r) and mammalian (gpt) cells
transformed with the vector. Expression of the antibody gene in
this case is mediated by the Ig promoter indicated by the solid
circles near the HindIII site in the maps of the antibody
genes.
[0037] FIG. 4 shows a vector for the expression of chimeric or
humanized MN-14 kappa chain gene. The diagram shows a chimeric and
a reshaped MN-14 kappa light chain gene and the pSVhyg expression
vector. The diagram at top, labelled "CHIMERIC," is a map showing
DNA encoding the MN-14 mouse kappa light chain variable region
("VK") joined to DNA encoding a human kappa constant ("CK") region.
In the mouse VK region the three CDRs are indicated by the three
dark areas and the FRs are indicated by the four stippled areas.
The middle diagram, labelled "RESHAPED" shows the humanization of
the MN-14 VK region in which the mouse FRs have been replaced by
human FRs, which are indicated by the four clear areas in the
"Human" VK region. The circular map of the expression vector pSVhyg
at bottom shows the HindIII/BamH1 insertion site for the reshaped
MN-14 antibody gene just downstream from an Igh enhancer element.
The map indicates some important functional domains in the vector,
including the replication origins for E. coli (col E1 ori) and
mammalian propagation (SV40 promoter region), and genes encoding
selective markers for culturing bacterial (Ap.sup.r) and mammalian
(gpt) cells transformed with the vector. Expression of the antibody
gene in this case is mediated by the Ig promoter schematized by the
solid circles near the HindIII site in the maps of the antibody
genes.
[0038] FIGS. 5A and 5B shows the alignments of the murine MN-14
variable regions (SEQ. ID. NOS. 2 and 4) with the human variable
regions NEWM VH (SEQ. ID NO. 5) and REI VK (SEQ. ID NO. 6) (FIG.
5A). and with the human KOL VH region (SEQ. ID NO. 7) (FIG. 5B).
CDRs are boxed, and the murine VH FRs, which are incorporated into
the humanized VH, are marked with their positions according to the
numbering system of Kabat et al. SEQUENCES OF PROTEINS OF
IMMUNOLOGICAL INTEREST, U.S. Government Printing Office,
Washington, D.C., 1987. Murine residues outside the CDRs that were
included in the KLHuVH are indicated by a filled circle.
[0039] FIGS. 6A, 6B and 6C shows a comparison of the amino acid
sequence between murine (SEQ. ID NO. 2) and humanized (SEQ. ID NOS.
57, 8-11, 58 and 12-15). MN-14 VH framework residues (FR). Only
human FR residues different from the mouse are shown. CDAs for NEWM
and KOL are also not shown. The position of the substitution is
indicated according to the Kabat et al. numbering system. The 3
CDRs are boxed.
[0040] FIG. 7 shows the DNA sequence and corresponding amino acid
sequence (SEQ. ID NOS. 16 and 17) of the MN-14HuVH (KLHuVh) region.
CDRs are boxed.
[0041] FIG. 8 shows the DNA sequence and corresponding amino acid
sequence (SEQ. ID NOS. 18 and 19) of the MN-14HuVK region. CDRs are
boxed.
[0042] FIG. 9 is a graph of MN-14 blocking (i.e., competition)
assays comparing relative binding affinities of KLHuVH variants,
including: (-.smallcircle.-) KLHuVH/HuVK; (-.box-solid.-)
KLHuVHAIG/HuVK; (-.circle-solid.-) KLHuVHAIGAY/HuVK;
(-.quadrature.-) KLHuVHAIGA/HuVK; (-.tangle-solidup.-) chimeric
control; and, (-.DELTA.-) murine control.
[0043] FIG. 10 shows MN-14 blocking assays comparing hMN-14 with
HMN-14-NVT (glycosylated in FR 1 region).
[0044] FIG. 11 is a radioautogram of the abdomen of a colon cancer
patient following administration of .sup.131I-labelled hMN14IgG
(left panel) or mMN14IgG (right panel) in the same patient.
DETAILED DESCRIPTION OF THE INVENTION
[0045] Notwithstanding past failures to develop an effective
non-HAMA-inducing anti-CEA antibody having the CEA-binding
characteristics of MN-14, it has been discovered that the CDRs of
the MN-14 mAb can be grafted onto the FRs of a human antibody to
provide antibodies and antibody-derived reagents that have the
antigen binding properties of the MN-14 anti-CEA mAb, while also
exhibiting reduced induction of HAMA and augmented effector
activities.
[0046] The murine anti-CEA IgG1 monoclonal antibody MN-14, and its
production, have been described previously. Hansen et al., Cancer,
71: 3478 (1993); Primus et al., U.S. Pat. No. 4,818,709. MN-14
meets all of the criteria of a Class III, anti-CEA monoclonal
antibody, being unreactive with meconium antigen by EIA and not
reacting with normal tissues.
[0047] Blocking studies are carried out according to Hansen et al.
1993, above, Losman et al., Int. Cancer, 56: 580 (1994); Hansen et
al., Clin. Chem., 35: 146 (1989). Using the same conditions as
described in those references for quantification of CEA, binding of
humanized MN-14 may be assessed relative to a labeled MN-14 probe.
A typical probe is MN-14 conjugated to horse radish peroxidase
(HRP). Both labeled and unlabeled MN-14 are added to a CEA sample
fixed to a solid support such as microtitre plate wells. The degree
of "blocking" of labeled MN-14 binding to CEA is a direct
reflection of unlabeled MN-14 activity. Using standard MN-14, the
relative activity of an unknown sample of humanized MN-14 or
derivatives thereof can be determined. Typically, the reactions are
performed in the wells of a microtitre plate where wells are
charged directly with CEA at a level of, for example, 25 .mu.g/well
or indirectly where the wells are precharged with an antibody
reactive with CEA but to an epitope different than that to which
MN-14 interacts; such an antibody may be the MN-15 mAb. CEA can
thus be indirectly fixed to the well. A competitive binding EIA
assay can then be performed with such a charged plate.
[0048] Alternate to the aforementioned HRP-labeled mAbs, antibodies
can be radioiodinated conventionally with, for example, .sup.131I
by the chloramine-T method to a specific activity of about 10
mCi/.mu.g, and free radioisotopes removed by chromatography on
acrylamide gel columns (see Hansen et al., 1993, above).
[0049] Molecular biological techniques suitable to carrying out the
invention as herein described also are known to those skilled in
the art. Suitable teachings are described in numerous manuals and
primary publications, including inter alia, Sambrook et al.,
MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1989), PROTOCOLS IN
MOLECULAR BIOLOGY, Ausubel et al., Eds., Green Publishing
Associates and Wiley-Interscience, John Wiley and Sons, New York
(1987, 1988, 1989), which are herein incorporated by reference in
their entirety including supplements.
[0050] MN-14 light and heavy chain CDRs disclosed herein, and
modified MN-14 CDRs can be integrated into other antibodies using
well-known recombinant techniques, such as those described in the
above references.
[0051] Specific methods suitable to this end are shown below in the
examples. Based on the amino acid sequences set forth herein,
oligonucleotides encoding MN-14 CDRs can be synthesized.
Oligonucleotides that encode modified CDRs may be made, as well as
those that encode exactly the amino acid sequences herein set
forth. Also, the oligonucleotides may contain nucleotides in
addition to those of an MN-14 CDRs, to facilitate cloning, for
instance. Oligonucleotide synthesis techniques are well known, and
can be carried out on automated equipment available from a number
of manufacturers. Moreover, oligonucleotides of any specified
sequence can be obtained commercially.
[0052] Oligonucleotides encoding the MN-14 CDRs and/or specific FR
residues. or representing the complementary strand thereof, may be
used to introduce codons for these residues into VH or VK DNA by
site-directed mutagenesis provided that the ends of the
oligonucleotides, generally 12 nucleotides, are designed to anneal
perfectly to the template DNA. The template DNAs are typically
single-stranded DNAs representing M13 vectors that carry a variable
region DNA encoding the required FRs. In one method, the mutagenic
oligonucleotides are phosphorylated at their 5' ends and, together
with an oligonucleotide priming 5' to variable region DNA, are
annealedd to the ssDNA template. The oligonucleotides are extended
using T7 polymerase and the fragments linked together by T4 DNA
ligase to give a complete mutant strand covering the whole variable
region. Using the mutant strand as a template, multiple copies of
its complementary strand can be synthesized from a suitable primer
using Taq DNA polymerase in a thermal cycling reaction. Once the
mutant strand has been preferentially amplified in this manner, the
DNA can be amplified by conventional PCR for cloning, sequencing
and expression.
[0053] Suitable antibody-encoding DNAs are illustrated by the
disclosure herein, but include practically any such DNA. A variety
of human antibody genes are available in the form of publically
accessible deposits. Many sequences of antibodies and
antibody-encoding genes have been published and suitable antibody
genes can be synthesized from these sequences much as described
above.
[0054] The scope of this invention encompasses all alleles,
variants and mutations of the DNA sequences described herein.
[0055] CDR grafting in accordance with the present disclosure may
be carried out using established techniques. Antibody-producing
cell lines may be selected and cultured using techniques well known
to the skilled artisan. Such techniques are described in a variety
of laboratory manuals and primary publications. For instance,
techniques suitable for use in the invention as described below are
described in CURRENT PROTOCOLS IN IMMUNOLOGY, Coligan et al., Eds.,
Green Publishing Associates and Wiley-Interscience, John Wiley and
Sons, New York (1991) which is herein incorporated by reference in
its entirety, including supplements.
[0056] RNA may be isolated from the original hybridoma cells by
standard techniques, such as guanidinium isothiocyanate extraction
and precipitation followed by centrifugation or chromatography.
Where desirable, mRNA may be isolated from total RNA by standard
techniques such as chromatography on oligodT cellulose. Techniques
suitable to these purposes are well known in the art as described
in the foregoing references.
[0057] cDNAs that encode the light and the heavy chains of the
antibody may be made, either simultaneously or separately, using
reverse transcriptase and DNA polymerase in accordance with well
known methods. It may be initiated by consensus constant region
primers or by more specific primers based on the published heavy
and light chain DNA and amino acid sequences.
[0058] PCR also may be used to isolate DNA clones encoding the
antibody light and heavy chains. In this case the libraries may be
screened by consensus primers or larger homologous probes, such as
mouse constant region probes. The necessary techniques are well
known to those of skill in the art, are set forth in the foregoing
Sambrook and Ausubel references and are illustrated by the examples
set forth below.
[0059] cDNAs that encode the light and the heavy chain of an
antibody can be propagated in any suitable vector in any suitable
host prior to isolation of the CDR. Often the clones will most
conveniently be propagated for this purpose in E. coli as
illustrated in the examples below. However, a variety of other
vectors and host cells well knowN to those of skill profitably may
be employed in this aspect of the invention. A variety of such
vectors are described in the foregoing references.
[0060] DNA, typically plasmid DNA, may be isolated from the cells,
restriction mapped and sequenced in accordance with standard, well
known techniques set forth in detail in the foregoing references
relating to recombinant DNA techniques.
[0061] DNAs encoding antibody heavy and light chains and fragments
thereof in accordance with the vector are used to construct
chimeric and CDR-grafted humanized MN-14 antibodies.
[0062] The CDRs of the MN-14 anti-CEA mAb are herein identified and
described, and illustrated in FIGS. 1A and 1B and and 2A and 2B
(SEQ. ID NOS. 2 and 4, respectively) Using these sequences, CDRs of
the MN-14 heavy and light chain can be synthesized for use in the
present invention. It is not necessary to redone MN-14 CDRs from a
natural source. The DNA and amino acid sequences are set forth
herein. Oligonucleotide synthesis techniques suitable to this
aspect of the invention are well known to the skilled artisan and
may be carried out using any of several commercially available
automated synthesizers. In addition, DNAs encoding the CDRs set
forth herein can be obtained through the services of commercial DNA
synthesis vendors.
[0063] Polynucleotides synthesized in accordance with this aspect
of the invention may include those not derived from an MN-14 CDR as
well as those that make up the CDR. The additional bases may be
included to facilitate joining the CDR to the FRs from a
heterologous source. They may comprise restriction sites or
overlapping complementary regions for this purpose. The synthesis
of longer, double-stranded DNAs from shorter, overlapping,
single-stranded DNAs is well known to those of skill in the art.
Likewise, well known is the end-to-end joining of DNAs, including
blunt-ended DNAs and those with at least partially overlapping
complementary termini. These techniques are illustrated in the
foregoing references on recombinant DNA techniques, for
instance.
[0064] The CDRs of the MN-14 heavy and light chains may also be
modified particularly after incorporation into a chimeric or
humanized antibody using well-known recombinant DNA techniques for
deleting, inserting and altering bases in a cloned or synthetic DNA
or RNA. Site-specific mutagenesis techniques suitable to this end
are well known to those of skill in the art, and are illustrated in
the foregoing references on recombinant DNA techniques. Also
illustrated are deletional and insertional techniques. These
methods can be used to introduce practically any desired alteration
into polynucleotides that encode the MN-14 CDRs or into other
regions of a closed heavy or light chain gene.
[0065] MN-14 CDRs and modified MN-14 CDRs can be introduced into
practically any set of FRs in accordance with the present
invention. It will be appreciated by those of skill in the art that
a variety of well known techniques for cloning and manipulating
polynucleotides may be effectively employed in this regard. Such
techniques are illustrated by the methods set forth in the
foregoing recombinant DNA-related references.
[0066] In a particularly preferred embodiment of the present
invention, MN-14 CDRs are grafted into a human antibody. It will be
understood that human antibody in this context refers to any
antibody that occurs in a human or an engineered antibody that has
been designed, in some respect, to be compatible with the human
immune system. Particularly preferred for this purpose are
antibodies that, broadly, do not engender an adverse immune
response in a patient. More particularly, the expression "human
antibody" is intended to mean an antibody encoded by a gene
actually occurring in a human, or an allele, variant or mutant
thereof.
[0067] Once DNA encoding an MN-14-derived CDR-grafted antibody has
been assembled from MN-14 VH and VK region DNAs and the variable
regions thus formed combined with their respective light and heavy
chains of human constant domains, it may be inserted into a vector
for propagation and expression by conventional techniques. In this
manner desired amounts of the antibody may be obtained.
[0068] The MN-14 CDR-grafted human antibody can be used in imaging
applications by administrating to a subject the humanized antibody
or Fab' thereof conjugated with an imaging compound or isotope.
[0069] The antibody is conjugated to a label for imaging using
conventional methods. Such conventional methods include, but are
not restricted to: 1) direct radioiodination of the antibody
protein or fragments thereof or 2) direct attachment to the
antibody or fragments thereof of metallic nuclides (see, e.g.,
Hansen et al., Cancer, 73: 761 (1994)). The use of bifunctional
chelates that can be used to bind various diagnostic or therepeutic
metals to the antibody or fragment thereof is also within the scope
of the present invention (see, Antibodies in Radiodiagnosis and
Therapy, ed. M. R. Zalutsky, 1989, CRC Press, Boca Raton, Fla., and
Cancer Therapy with Radiolabeled Antibodies, ed. D. M. Goldenberg,
1994, CRC Press, Boca Raton, Fla.). Following the conjugation
procedure and characterization of the product, satisfactorily
labelled conjugates are purified to homogeneity under conditions
that conform to Good Manufacturing Procedures ("GMP") appropriate
to the production of diagnostic compositions for use in human
patients.
[0070] The reaction of serum antibody with the MN-14 CDR-grafted
antibody and imaging agent portions of the conjugate can be
determined over the course of the diagnostic procedures, including
the reaction of control sera obtained prior to administration of
conjugate. Similar determinations are made in other patients
treated with similar conjugates of MN-14 itself. The sera antibody
reactive with CDR-grafted MN-14 human antibodies detected by these
tests is much less than the antibody reactive with antibody portion
of the conjugate in patients treated with the murine
MN-14-containing conjugates.
[0071] Humanized MN-14 antibodies conjugated to aminodextran and to
boron may be used for diagnostic purposes. MN-14 and CDR-grafted
MN-14 antibodies can be prepared as set forth above for conjugation
to an aminodextran-boron adduct. Amino-dextran-boron adducts can be
prepared by reaction of a suitable boron cage compound (e.g., a
12-boron carborane suitably derivatized with an amino-dectran
functional group). In a preferred embodiment, the amino-dextran is
reacted with an excess of a haloacetyl acid ester or anhydride
(such as iodoacetic anhydride), thereby producing an amino-dextran
with a desired number of haloacetyl groups, usually ranging from
10-1000 groups, depending on the reaction conditions and the size
of the amino-dextran. A suitable boron derivative such as
mercaptocarborane-B12 is reacted, in a desired molar excess, with
the haloacetyl-amino-dextran via an alkylation reaction. In a
preferred embodiment, a number of haloacetyl groups on the
boronated haloacetyl amino-dextran remain unreacted, and can be
used as a "handle" to attach the adduct to protein thiol
groups.
[0072] MN-14 CDR-grafted humanized antibodies and their
derivatives, because of their reduced immunogenicity, are useful in
therapy, for passive immunization without negative immune reactions
such as serum sickness or anaphylactic shock, for localization and
in vivo imaging of tumors as described above, for specific
treatment of disease cells, e.g., site directed delivery of
cytotoxins, immunomodulators or other pharmaceutically active
molecules where local concentration of the active agent is an
important factor, or the like, thereby establishing the practical
utility of these humanized antibodies. As described above, for in
vivo imaging, the humanized, CDR-grafted MN-14 monoclonal antibody
is radiolabeled or conjugated with a metal chelator complexed with
a radionuclide, e.g., iodine, ytrium, technetium, or the like, and
radio-scanning techniques may be used to detect primary and
metastatic CEA tumors. To that end, the radioactive antibody is
injected, e.g., intravenously, and the patient scanned with a gamma
imager at regular intervals. Tumors expressing CEA will take up
more radioantibodies than other tissues and will be easily
recognized by the imaging camera. Preferentially, monoclonal
antibodies labelled with .sup.131I are used in amounts of 3 to 10
.mu.g representing 15 to 30 .mu.Ci per kg body weight. For therapy
with cytotoxic agents, the antibodies are conjugated to any of a
variety of known therapeutic agents such as doxorubicin,
methotrexate, taxol, ricin A, radioactive atoms, cytoxic agents,
and the like, formulating such conjugate in a pharmaceutically
acceptable sterile vehicle, and administering the formulation by
conventional means. The therapeutic dosages can be readily
determined conventionally by the user of average skills in these
arts. The therapeutic dose for mammals is between about 1 mg and 5
mg per kg body weight for the monoclonal antibodies themselves, and
between 0.1 mg and 5 mg per kg body weight for conjugates with
cytotoxic drugs, depending on the status of the patient and the
mode of administration. Alternately, the humanized antibodies can
be used in combination with components of the host's immune system,
e.g., complement or cell mediated responses, in order to remove
from the subject CEA-presenting cancer cells. The immune responses
of patients may be monitored in accordance with the foregoing
procedures. For additional procedures for radioimaging and therapy,
see EP 0 323,806, Hansen et al., Cancer 71: 3478-85 (1993), and
U.S. Pat. No. 4,818,709 and references contained therein, all of
which are incorporated by reference.
[0073] Preferred are pharmaceutical preparations for parenteral
administration, such as are described in Remington's Pharmaceutical
Sciences, Mack Publishing Co., Easton, Pa., 1989. The final
preparations contain from 0.01% to 50% of active ingredients.
Methods for the production of such conjugates and their use in
diagnostics and therapeutics are provided in, for example, Shih et
al., U.S. Pat. No. 5,057,313; Shih et al., Int. J. Cancer 41:832
(1988); copending, commonly owned U.S. Ser. No. 08/162,912, now
U.S. Pat. No. 5,443,953; and, McKearn et al., U.S. Pat. No.
5,156,840, the contents of which are incorporated by reference.
[0074] As noted above, for purposes of therapy, a humanized
antibody conjugate and a pharmaceutically acceptable carrier are
administered to a patient in a therapeutically effective amount. A
combination of a conjugate 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. A
targeted therapeutic agent is "therapeutically effective" if it
delivers a higher proportion of the administered dose to the
intended target than accretes at the target upon systemic
administration of the equivalent untargeted agent.
[0075] To be therapeutically effective the conjugate and carrier
may need to be administered in combination with other therapeutic
agents or as part of a broader treatment regimen. Physicians are
currently of the opinion that the effectiveness of targeted
therapeutics can often be greatly increased when used in a
combination therapy approach. For example, high-dose
radioimmunotherapy for B-cell lymphomas, which causes severe
hematologic toxicity when used alone, has been shown to be highly
effective when used in combination with autologous bone marrow
reinfusion. Press et al., "Treatment of Relapsed B Cell Lymphomas
with High Dose Radioimmunotherapy and Bone Marrow Transplantation"
in CANCER THERAPY WITH RADIOLABELED ANTIBODIES, Goldenberg, ed.
(CRC Press, Boca Raton, 1994) ch. 17. In another example a
five-fold enhancement of tumor uptake of a radiolabeled antibody is
observed when the tumor is preirradiated. Leichner et al., Int. J.
Radiat. Oncol. Biol. Phys. 14:1033 (1987). Mechanisms which have
been shown to have the potential for improving the clinical
efficacy of radioimmunotherapy are also discussed in DeNardo et
al., "Overview of Obstacles and Opportunities for
Radioimmunotherapy of Cancer" in CANCER THERAPY WITH RADIOLABELED
ANTIBODIES, Goldenberg, Ed. (CRC Press, Boca Raton, 1994) ch. 11.
Methods of developing such combination protocols, as well as to
investigate dose-limiting side effects and to potentiate and
amplify targeting, uptake, and beneficial side effects, are well
known to skilled clinical artisans in this field and would not
require undue experimentation to develop.
[0076] In vivo experiments using conjugates of the humanized MN-14
with diagnostic and therapeutic agents have been carried out with
animal models and with human patients (see Example 11 below). The
CDR-grafted humanized antibody conjugate exhibited a better
therapeutic profile and could be used in longer treatment regimens
than the parental MN-14 antibody conjugate. The CDR-grafted
antibody conjugate provided a better therapeutic effect and fewer
deleterious side effects than the control murine antibody
conjugates.
[0077] For example, the antibody was covalently complexed to
aminodextran-conjugated methotrexate using the method described by
Shih et al., above using carbohydrate hydroxyl groups for
derivatization purposes. In order to determine the contribution of
antibody carbohydrate groups on immunoactivity, a mutation can be
introduced at position 18-20 in the VK FR1 region of hMN-14 (the
prefix "h" is intended to mean "humanized") so as to introduce a
glycosylation site, NVT, prior to expression of the blocking gene
in mammalian cells. Comparison of hMN-14 antibody with mutated
hMN-14-NVT in a blocking cell binding assay (FIG. 8) has
demonstrated that the carbohydrate moiety at position 18 is without
influence on immunoreactivity of this humanized antibody.
[0078] Aminodextran, average molecular mass 40 kDa, is oxidized by
NaIO.sub.4 to form aldehydes (by the oxidation of hydroxyl groups).
About 50 to 150 moles of aldehydic groups are introduced per mole
of aminodextran by careful control of the reaction conditions and
timing. The aldehydes then are reacted with an excess of
1,3-diamino-2-hydroxypro- pane to form Schiff bases with virtually
all of the aldehydes. The Schiff bases are then reduced by
treatment with excess NaBH.sub.4. The amine-derivatized dextran
then is purified by gel-exclusion chromatography.
[0079] The cytotoxic drug methotrexate (MTX) is activated by
treatment with dicyclocarbodiamide, followed by reaction with
N-hydroxysuccinimide, both in dimethylformamide. Activated MTX is
mixed in a 50:1 ratio with the amino derivatized dextran in aqueous
solution. The product provides, after purification, MTX-derivatized
dextran having about 35 MTX moles per mole. The MTX adduct thus
obtained is conjugated to a MN-14 CDR-grafted antibody using
methods described in Shih, et al., supra. For example, the antibody
carbohydrates are oxidized and the resultant aldehydes are reacted
with the remaining amines on the dextran in the adduct. The
Schiff-base product obtained thereby is reduced by treatment with
sodium cyanoborohydride in 10-fold molar excess over antibody. The
reduced antibody-dextran-MTX product is thoroughly purified prior
to assay, and formulated for administration to patients.
[0080] Parental MN-14 antibody is conjugated to dextran-MTX in the
same way, as a control.
[0081] The purified CDR-grafted antibody conjugate can be
administered to patients with a CEA-producing cancer (see above).
The response to therapy is monitored, including adverse side
effects, particularly those which are mediated by the patient's
immune systems. Patients treated with the CDR-grafted antibody
conjugate show improved therapeutic results, decreased immune
response to the agent and notably decreased immune-mediated adverse
effects of therapy. Therapy with the CDR-grafted antibody conjugate
can be carried out at higher dosages and for longer periods of time
then with the parental murine MN-14 antibody, allowing more
aggressive therapies and improved responses.
[0082] The present invention is further described by reference to
the following, illustrative examples. It will be appreciated that
the techniques related to isolating DNA clones encoding MN-14 light
and heavy chain genes are illustrated by cloning techniques useful
to isolate light and heavy chain genes of any antibody from
producing cells. There is no necessity, given the disclosed
sequence, to reisolate MN-14 heavy and light chain genes to carry
out the invention.
[0083] It should be understood that the detailed description and
the specific examples, while indicating preferred embodiments of
the invention, are given by way of illustration only, since various
changes and modifications within the spirit and scope of the
invention will become apparent to those skilled in the art from the
following illustrative description.
EXAMPLE 1
Culturing Antibody Producer Cells
[0084] A mouse/mouse hybridoma cell line producing Class III,
anti-CEA monoclonal antibodies was established according to Hansen
et al. (1993) above and Primus et al. (1983) above.
[0085] Cells were selected for secretion of kappa IgG1 by testing
conditioned medium using standard isotyping techniques. A variety
of kits for this purpose are commercially available. Such cells
were screened for production of antibody by testing conditioned
medium using a standard blocking assay described above. Stocks of
producer cells that proved out in the assay were expanded and
frozen in liquid nitrogen.
EXAMPLE 2
Isolating RNA from Producing Cell Lines
[0086] MN-14-producing cells were expanded in culture, collected by
centrifugation and washed. Total RNA was isolated from the cells in
the pellet according to Favaloro et al., Methods in Enzymology 65:
718 (1980) and Orlandi et al., Proc. Nat'l Acad. Sci., USA 86: 3833
(1989), which are incorporated by reference.
EXAMPLE 3
cDNA Synthesis and Amplification of the Heavy Chain Variable
Region
[0087] mRNA from MN-14 producing cells was used to synthesize cDNA
using standard techniques of cDNA synthesis and DNA amplification
by PCR, as described below. In general, the primers used for PCR
included a restriction endonuclease cleavage site at their 5' ends
to facilitate cloning of the amplification product. An
oligonucleotide complementary to the end of the sense strand of the
DNA encoding the first constant region domain of the murine
IgG.sub.1 heavy chain ("CH1") was used to prime first strand cDNA
synthesis by reverse transcriptase. The sequence of this primer,
CG1FOR, is shown in Table 1. Table 1 below provides other
oligonucleotide sequences used herein.
1TABLE 1 OLIGONUCLEOTIDE SEQUENCES SEQ. ID NO. CG1FOR 41 5'
GGAAGCTTAGACAGATGGGGGTGTCGTTTTG 3' VH1FOR 42 5'
TGAGGAGACGGTGACCGTGGTCCCTTGGCCCCAG 3' VH1BACK 43 5'
AGGTSMARCTGCAGSAGTCWGG 3' SH1BACK 44 5'
TGGAATTCATGGRATGGAGCTGGRTCWTBHTCTT 3' SH2BACK 45 5'
TGGAATTCATGRACTTCDGGYTCAACTKRRTTT CK2FOR 46 5'
GGAAGCTTGAAGATGGATACAGTTGGTGCAGC 3' VK1FOR 47 5'
GTTAGATCTCCAGCTTGGTCCC 3' VK3FOR 48 5' GTTAGATCTCCAGTTTGGTGCCT 3'
VK1BACK 49 5' GACATTCAGCTGACCCAGTCTCCA 3' VK2BACK 50 5'
GACRTTCAGCTGACCCAGGMTGMA 3' VK3BACK 51 5' GACATTCAGCTGACCCA 3'
VK4BACK 52 5' GACATTGAGCTCACCCAGTCTCCA 3' VK5BACK 53 5'
TTGAATTCGGTGCCAGAKCWSAHATYGTKATG 3' VK6BACK 54 5'
TTGAATTCGGTGCCAGAKCWSAHATYGTKCTC 3' VK7BACK 55 5'
TTGAATTCGGAGCTGATGGGAACATTGTAATG 3' VK8BACK 56 5'
CWGAGAAATTCAGCTGACCCAGTCTC 3'
[0088] Restriction sites incorporated in primers to facilitate
cloning are underlined.
[0089] The variable region of the heavy chain ("VH") cDNA then was
amplified by the PCR using the same primer, CG1FOR, and a primer
based on the consensus sequence of the 5' end of VH genes
(VH1BACK), as described in Orlandi et al. (1989) cited above. The
PCR product of this reaction was analyzed by agarose gel
electrophoresis, which, upon ethidium bromide staining and
fluorescence illumination, revealed one major band of about 400 bp,
as expected.
[0090] For confirmatory sequences from a second cDNA preparation,
signal sequence primers were used in the PCR to allow determination
of the authentic amino acids of the N-terminus. SH1BACK and
SH2BACK, degenerate oligonucleotides based on heavy chain signal
sequence coding regions, were used in separate reactions in concert
with CG1FOR. A diffuse product band was obtained from CG1FOR,
SH1BACK amplification.
[0091] In order to increase the VH content of the product it was
excised from low melting point agarose and amplified using SH1BACK
and an oligonucleotide complementary to a fourth framework region
consensus sequence, VH1FOR. This product of this reaction was a
discrete band when analyzed by agarose gel electrophoresis.
EXAMPLE 4
Cloning and Sequencing DNA Encoding the MN-14 Heavy Chain Variable
Region Obtained by PCR
[0092] The amplification product obtained using the CG1FOR, VH1BACK
primer pair was digested with HindIII and PstI separately. The
cleavage sites of these enzymes are included in the PCR primers. It
was preferable to determine whether there were also sites internal
to the VH. Agarose gel analysis of the restriction fragments
indicated the presence of an internal PstI site close to one end of
the DNA. The PCR product was digested with HindIII and PstI, cloned
into M13mp18 and 19 and the DNA sequence of the inserts of
representative clones determined. The majority of the clones
contained inserts of the same VH DNA.
[0093] The sequencing confirmed the presence of this additional,
unexpected PstI site, which was close to the 3' end of the sequence
of the CG1FOR primer partially encoding the final two amino acids
of the VH. Although several full length VH clones were obtained by
this method, further PCR product DNA was cloned as PstI-PstI
fragments. These clones thus contained full VH sequences but none
of the constant region given by CG1FOR. A total of 16 full-length
clones were obtained from the VK1BACK, CG1FOR product. In these
experiments about 25% of the clones that were analyzed contained
inserts unrelated to the VH region.
[0094] In order to confirm the VH sequence from a second cDNA
preparation and, at the same time, to obtain the authentic, rather
than primer-dictated, DNA sequence corresponding to the N-terminus
of the VH, the PCR product from VH1FOR and SH1BACK primers was
cloned. These primers contain BstEII and EcoR1 restriction sites,
rather than the PstI and HindIII sites of CG1FOR and VH1BACK
described above. The PCR product of this reaction was cloned by
digesting with BstEII, filling in the BstEII ends, digesting with
EcoRI, and ligating the EcoRI and blunt ends to the vector, which
had been digested with EcoRI and HindIII. The sequences of the
cloned fragments were determined. The yield of VH fragments was
relatively low, perhaps reflecting a lack of specificity in the PCR
caused by degeneracy of SH1BACK. However, four of the 18 clones
that were sequenced contained DNA encoding the MN-14 VH region as
previously sequenced. The other inserts did not derive from
VH-encoding DNA.
[0095] In all, 20 full-length MN-14 VH clones were obtained. Five
transition mutations were observed amongst the sequences in the
MN-14 VH region clones. These mutations are likely to have been
introduced during amplification as a result of misincorporation by
Taq polymerase.
EXAMPLE 5
Analysis of the Amino Acid Sequence of the Heavy Chain Variable
Region of MN-14
[0096] The amino acid sequence of murine MN-14 heavy chain variable
region, translated from the VH DNA sequence, is shown in FIGS. 1A
and 1B (SEQ. ID. NOS. 1 and 2). Comparison of this sequence with
sequences representing the murine VH subgroups indicated that the
variable heavy region of MN-14 belongs to subgroup IIIB (see, Kabat
et al. SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, U.S.
Government Printing Office, 1987).
[0097] The MN-14 CDR sequences are different from any of those
reported by Kabat et al. (1987), supra. Furthermore, the amino
acids at four positions in the MN-14 heavy chain VH framework
regions are different from those in the framework regions of other
subgroup IIIB VH sequences. These four substitutions (Ser 14, Thr
30, Ser 98 and Pro 108) have been observed in other murine VH
regions outside the IIIB VH subgroup, however, with the proline in
Kabat position 108 being the most unusual.
[0098] Any unusual residues in the VH or VK may represent somatic
mutations which proved advantageous to the binding of murine
MN-14.
EXAMPLE 6
cDNA Synthesis and Amplification of DNA Encoding the MN-14 VK
[0099] cDNA encoding the kappa light chain of MN-14 was cloned in
much the same fashion as the cDNA encoding the variable region of
the heavy chain, as described above. Several primers were used to
prime reverse transcriptase for synthesis of the first strand of
the kappa chain cDNA. The sequence of one primer, CK2FOR, was
derived from the sequence of the 5' end of the constant region of
kappa light chain genes ("CK"). The sequences of two other primers,
VK1FOR and VK3FOR, were based on the sequence of the 3' end of the
variable region of kappa light chain genes ("VK").
[0100] The first strand DNA product was amplified by PCR using a
number of primer pairs. Synthesis in one direction was primed by
the primers used to make the first strand. Polymerization in the
other, "backward," direction initiated from a series of kappa light
chain-specific primers which had sequences based on either the
sequence at the 5' end of the VK region, VK1BACK, VK2BACK, VK3BACK,
VK4BACK and VK8BACK, or the sequence encoding the last four amino
acids of the signal peptide and the first four amino acids of the
variable region, VK5BACK, VK6BACK and VK7BACK. In addition, cDNA
primed by CK2FOR also was amplified using VK1FOR and VK8BACK.
[0101] The amplification products were analyzed by gel
electrophoresis in the manner described in the Examples above. The
products from the reactions primed by VK1BACK, VK3BACK, VK5BACK,
VK7BACK and VK8BACK gave rise to the expected 350 bp band.
EXAMPLE 7
Cloning and Sequencing the MN-14 Kappa Light Chain Variable Region
Obtained by PCR
[0102] Selected PCR products were cloned into M13mp18 and 19 using
the restriction sites included in the amplification primers in a
manner similar to that described for VH in Example 4 above.
Nucleotide sequencing revealed that most inserts were not
VK-related. This is not uncommon when attempting to clone VK cDNAs
and it appears to be more difficult to design VK-specific primers
than VH specific primers.
[0103] From the VK1FOR/VK8BACK combination, a VK cDNA insert was
obtained, but this did not yield a functional VK due to a
frameshift within the cDNA encoding CDR.sub.L3 and absence of the
invariant Cys at position 23. This VK cDNA has been isolated from
other hybridoma cells and it is derived from the Sp2/0 fusion
partner. CK2FOR/VK1BACK product yielded a further four different
aberrant VK cDNA inserts, in this case lacking the conserved
residues of framework 4. A fifth VK insert obtained using this
primer pair was that of a functional VK with the exception of a
frameshift at the 3'end of VK1BACK, a phenomenon apparently due to
mismatch-induced slippage of the primer. This problem may be
avoided by the use of VK8BACK which does not extend as far into the
VK gene. However, analysis of further clones from VK1FOR/VK8BACK
product did not yield the desired insert.
[0104] In order to amplify preferentially the putative VK, VK3FOR
was designed from its genuine fourth framework sequence, and
synthesized as an alternative to VK1FOR. This strategy proved
successful when amplification of VK3FOR-primed cDNA with VK3FOR and
VK8BACK yielded 4 clones containing the desired VK.
[0105] The DNA and amino acid sequence of the murine MN-14 kappa
light chain variable region is set out in FIGS. 2A and 2B (SEQUENCE
ID. NOS. 3 and 4). This MN-14 VK can be placed in Kabat's VK
subgroup V. Only 3 residues in the MN-14 VK framework regions (Met
10, Val 66 and Thr 76) do not appear in other members of this
subgroup. Met 10 and Val 66 are the most unusual of the three. They
are not found in any murine VK listed in Kabat. None of the MN-14
VK CDRs are previosuly-reported sequences in Kabat.
EXAMPLE 8
Grafting of the MN-14 VH and VK CDRs into Variable Regions of Human
Antibody
8.1. Construction of Chimeric Anitbody Expression Vectors
[0106] In the production of a chimeric antibody consisting of
murine variable regions and human constant regions, testing
alongside the parent mouse antibody served to check that the
correct variable region cDNAs have been isolated. A successful
chimeric antibody also acts as a useful control when assessing the
binding of humanized versions. The scheme used in cloning the
variable regions for expression is described by Orlandi et al.
(1989) supra, and is illustrated in FIGS. 3 and 4.
[0107] VH DNA was amplified from the M13 clone MNVH41 using the PCR
with oligonucleotides VH1BACK and VH1FOR. The PstI and BstEII
restriction sites in the primers allowed the VH to be inserted into
M13VHPCR1 in the correct context for expression. At this point, the
internal BamHI restriction site of the VH was removed by site
directed mutagenesis. The reaction product, which encompassed the
entire HindIII-BamHI fragment of M13VHPCR1 was cloned into pSVgpt
and the VH sequence confirmed.
[0108] The human IgG1 constant region gene, published by Takahashi
et al., Cell, 29: 671-679 (1962) above then was added to the
construct as a BamHI fragment, which yielded the vector referred to
as pSVgptMN14MuVHHuIgG1.
[0109] VK DNA was similarly obtained from the M13 clone MNVK154 by
PCR amplification with the primers VK8BACK and VK3FOR and the
PvuII, BglII-digested product cloned into M13VKPCR1, whereupon the
sequence of the variable region was checked. The HindIII-BamHI
fragment was excised from RF DNA and transferred to the plasmid
pSVhyg. The construct already contains a human kappa constant
region gene as described in Hieter et al., Cell, 22: 197-207
(1980). The final vector thus obtained was designated
pSVhygMN14MuVKHuCK.
8.2 Expression and Testing of the Hybrid Antibody
[0110] The HindIII-BamHI fragment of M13KLHuVHAIGA was inserted
into a plasmid pSVgpt to yield the expression vector
pSVgptKLHuAIGAHuIgG1. Similarly, the HindIII-BamHI fragment of
M13HuVK was inserted into the plasmid pSVhyg to yield the
expression vector pSVhygMN14REIHuVKHuCK. About 5 .mu.g
pSVgptMN14MuVHHuIgG1 and 10 .mu.g pSVhygMN14MuVKHuCK DNAs were
linearized with Pvul and transferred into about 10.sup.7
subconfluent SP2/0 myeloma cells by electroporation conventionally
using a BioRad Model 165BR1160 Gene Pulser Electroporator with a
single pulse of 170 V, 960.degree. F. Cells were selected for the
expression of the gpt gene in 24-well plates by addition of
mycophenolic acid and xanthine to the DMEM+10% FCS growth
medium.
[0111] Wells which contained colonies of surviving cells were
identified. The supernatant medium was removed from these wells and
assayed for human antibody. Colonies that secreted antibodies were
expanded to give 0.5 L of conditioned medium for isolation of
larger amounts of antibody.
[0112] Antibody was purified conventionally from the medium by
protein-A agarose affinity chromatography, initially. The purified
antibody was characterized further with reference to native MN-14
antibody, human antibodies and other controls.
[0113] The antibody was also characterized by its reaction profile
in a MN-14 blocking assay, which provided an informative comparison
of CEA binding affinities of the hybrid antibodies with CEA binding
by the MN-14 murine antibody which served as a positive
control.
8.3 Humanization of the MN-14 Antibody
[0114] The human NEWM VH, KOL VH and REI VK frameworks were chosen
as the basis for reshaping the antibody, as they are likely to be
tolerated in humans. Alignments of the MN-14 VH (SEQ. ID NO. 2) and
VK (SEQ. ID NO. 4) with these human variable regions are shown in
FIGS. 5A and 5B (SEQ. ID. NOS. 5-7).
[0115] A. NEWM Based Humanization.
[0116] The starting points for the introduction of MN-14 CDRs are
DNAs encoding the required FRs and irrelevant CDRs. These template
variable regions are in a form compatable with the expression
vectors used, that is, within HindIII-BamHI fragments, that also
include promotor regions, signal peptide and intron DNA (FIGS. 3
and 4). For the NEWM VH version, the template is M13VHPCR1 (Orlandi
et al, above, and section 8.3 below). A derivative of this
template, containing KOL FRs and irrelevant CDRs, was used to
generate the KOL coding region. A derivative of M13VKPCR1 (Orlandi
et al. above) was used in the creation of the HuVK vector. The
resulting vectors were termed M13NMHuVH, M13KLHuVH and M13HuVK. The
HindIII-BamHI fragments containing the humanized MN-14 variable
region DNAs were transferred from these M13 vectors to the
expression vectors essentially as described for the construction of
the chimeric MN-14 expression vectors in Example 8.1.
[0117] The NEWM FR is described in Poljak et al. Biochemistry 16:
3412-20 (1977). Construction of a hMN-14 with an affinity for CEA
comparable to that of its murine counterpart was achieved in a
stepwise approach. Production of the chimeric antibody provided a
useful control when assessing the binding of the humanized
versions. The human NEWM VH and REI VK frameworks were initially
chosen as the basis for reshaping the antibody as they are known to
be tolerated in man. MN-14 residues Phe27, Asp28 and Thr30 were
retained because, although not part of the Kabat's hypervariable
region, CDR1 residues 31-35, those amino acids are part of the CDR1
structural loop (Chothia et al., J. Mol. Biol. 176: 901-917
(1987)). In addition, the following residues were also selected for
incorporation into the humanized VH for the following reasons:
Ala24, this residue contacts CDR1; Arg71, the side chain of this
residue pokes through the center of the domain to interact with
CDRs 1 and 2; substitution of the smaller Val may alter the
conformation of these CDR loops; and, Ser94, the majority of
antibodies have Arg in this position where it is thought to
interact with an Asp residue on CDR3, and the inclusion of the Arg
of NEWM could create an unwanted interaction with the murine CDR.
Other changes were made to this version (NMHuVH) in three areas
corresponding to regions which had been proved important in other
reshaped molecules. These changes were:
2 (i) Gln77Phe78Ser79 to ThrLeuTyr (NMHuVhHTLY, SEQ. ID NO. 9) (ii)
Ser82Thr83Ala84Ala85 to LysArgSerGlu (NMHuVhHKRSE, SEQ. ID NO. 10)
(iii) Arg66Val67Thr68Met69Leu70 to LysPhelleValSer (NMHuVhKFIVS,
SEQ. ID NO. 11)
[0118] The alignment of the different versions of the NEWM VH
frameworks (SEQ. ID NOS. 5 and 8-11) with the MN-14 VH (SEQ. ID NO.
2) is shown in FIGS. 6A, 6B, and 6C. Each of these versions has
been paired with the same HuVK. The inclusion of either the TLY or
KFIVS motifs gave about a two-fold improvement.
[0119] There are 2 differences from the NEWN framework sequences
given in Kabat et. al (1987) above: S107 to T and L108 to T. Kabat
lists residue 1 as PCA and residues 5 and 6 as E or Q.
[0120] B. KOL Based Humanization.
[0121] In parallel to the use of NEWM VH, we have also reshaped the
human KOL VH. KOL VH is described in Schmidt et al., Z. physiol.
Chem, 364: 713-747 (1987). The FR used for humanizing the antibody
is as given in Kabat. The original version of the KOL-based VH
contained the MN-14 CDRs, as defined in terms of residue
variability (Kabat et al., above) and three additional murine
residues. As with the NEWM VH, two of these substitutions were made
because the actual peptide CDR1 structural loop, which extends from
the .beta.-sheet framework, consists of residues 25 to 32 (Chothia
et al. 1987, above). Changes at positions 28 and 30 allowed this
loop to be transplanted as a whole from the murine antibody. MN-14
residue 94 was included because the Arg residue of the KOL VH is
involved in a salt bridge with Asp 110 and, like with the NEWM VH,
it was felt that retention of Arg 94 might perturb the MN-14 CDR3
structure. The side-chain of residue 94 may also interact with
residues of CDR1. Other changes made to this basis MN-14 KOLHuVH
(SEQ. ID NO. 12) were as follows:
3 (i) Ser24 to Ala24 and Val48Ala49 to IleGly (KLHuVhAIG, SEQ. ID
NO. 13). (ii) Ser24 to Ala24, Val48Ala49 to IleGly and Ser74 to Ala
(KLHuVhAIGA, SEQ. ID NO. 14). (iii) Ser 24 to Ala24, VAl48Ala49 to
IleGly, Ser74 to Ala and Phe79 to Tyr (KLHuVhAIGAY, SEQ. ID NO.
15).
[0122] Mutation Rationale
[0123] Ala24--The loop of CDR1 is anchored by the penetration of
the side chain of residue 29 into the framework. Residue 24 is one
of those with which it interacts (Chothia et al., J. Mol. Biol.
227: 799-817 (1992)).
[0124] Ile48Gly49--Although both these residues are adjacent to the
CDR2 hypervariable region they are far removed from the actual
structural loop. Both residues are completely buried (Padlan, Mol.
Immunolog. 28: 489-498, 1991) and it was considered possible that
these would effect binding via their packing interaction.
[0125] Ala74--This residue is part of the fourth loop found at the
VH antigen-binding surface and its side chain is almost completely
exposed to solvent (Padlan, 1991 above). Direct interaction of this
residue with antigen could be envisaged.
[0126] Tyr79--Like residue 74, this residue is close to the
antigen-binding site and could effect binding.
[0127] The alignment of the different versions of KOL VH frameworks
(SEQ. ID NOS. 7 and 12-15) with NEWM based versions (SEQ. ID NOS. 5
and 8-11) and the murine MN14 (SEQ. ID NO. 2) is shown in FIGS. 6A,
6B, and 6C.
[0128] The DNA sequences and translation products of MN14HuVH and
MN14HuVL are shown in FIGS. 7 and 8, 0respectively (SEQ. ID NOS.
16-19, respectively).
[0129] The humanized KLHuVH varients, such as antibody
KLHuVHAIGA/HuVK, were purified and tested in a blocking assay
carried out as follows. Antibodies were added at the indicated
concentrations together with HRP-labeled MN-14 to a final volume of
0.1 ml. Following 30 mins. of incubation at 37.sub.1C, and washing
to remove unbound antibodies, the relative affinities of the
antibodies were determined from the remaining bound peroxidase
activity. A shown by the assay data of FIG. 9, the activity of the
"reshaped" (i.e., CDR-engrafted on FR) humanized antibody was
similar to that of chimeric and murine positive controls.
[0130] Results obtained using supernatant fluids from cells
secreting KLHuVHAIG/HuVK and KLHuVHAIGAY/HuVK antibodies suggests
that these have blocking activities that are similar to that of the
KLHuVHAIGA/HuVK antibody.
[0131] C. REI Based Humanization
[0132] REI VL is described in Epp et al., Eur. J. Biochem., 45:
513-24 (1974). No change4s were made in REI framework to improve
binding. The changes from the sequences given in Kabat et al.
(1987) are: M4 to L; T39 to K: Y71 to F; L104 to V; Q105 to E: and
T107 to K. These changes preexisted in the template used when
grafting the MN-14 CDRs and were not made specifically to improve
binding. The M4 to L change incorporates a restriction site but the
remaining differences eliminate unusual residues in the REI
framework. A similar framework has been referred to as a consensus
of huma kappa subgroup I by Foote et al., J. Mol. Biol. 224: 487-9
(1992).
EXAMPLE 9
Expression of MN-14 CDR-Grafted Humanized Antibodies
[0133] Cells that were stably transformed for expression of the
MN-14 CDR-grafted human antibodies were selected in the manner
described above and cloned out to establish individual producer
lines. Each of the lines was assayed to determine production of the
correct antibody and to assess the efficiency of production. The
antibody class was determined and the anti-CEA binding affinity
assessed.
[0134] The best producers were further characterized for the
overall amount of the antibody produced and, for the best of these,
sequences were obtained from the mRNA to insure that the mutation
has not occurred in the antibody genes during transfection,
integration, propagation or selection.
EXAMPLE 10
Purification of MN-14 CDR-Grafted Humanized Antibodies Expressed in
Cell Culture
[0135] The best producer lines of the MN-14 CDR-grafted human
antibody were cultured, the growth medium collected and filtered
through a 0.2 micron membrane. The antibody was then purified by
protein A chromatography followed by other conventional
purification steps such as ion exchange and size exclusion
chromatography. The cells were pelleted and from the supernatant by
conventional centrifugation. The antibody was purified from the
supernatant fluid as described above.
EXAMPLE 11
Uses of Humanized MN-14 Monoclonal Antibody in Diagnoses
[0136] A. Animal Studies
[0137] The biodistribution of labeled humanized MN-14 IgG in nude
mice bearing human colon cancer was determined. For
radiolocalization studies, at 4-5 weeks female athymic mice (nu/nu,
Harlan, Indianapolis, Ind.) were given s.c. 0.2 ml of a 10%
suspension of LS174T human colon adenocarcinoma prepared from a
xenograft serially propagated in an athymic mouse (Sharkey et al.,
Cancer Res., 50: 828-34 (1990)). After waiting 2 weeks for tumor
development, the mice were injected i.v. with 20 .mu.Ci (about 2
.mu.g) of .sup.131-labelled humanized MN-14 monoclonal antibodies.
Groups of 4-5 mice were sacrificed at intervals thereafter, and
radioactivity localized in tissues according to Sharkey et al.,
above. The data of Table 2 show the % injected dose/g tissue and
tumor:nontumor ratios.
[0138] The results show excellent tumor accretion of the antibody,
with maximum accretion occuring within 2 days. Blood clearance of
the hMN-14 antibody was more rapid than the parental mMN-14
antibody. In addition, there was higher uptake of hMN-14 by the
spleen than there was of mMN-14, reflecting the fact that the
former antibody is "foreign" to the mouse. Tumor:nontumor ratios
were excellent. These results demonstrate that the inventive hMN-14
mAb is capable of targeting CEA-producing tumors.
4 TABLE 2 Time Post-Injection .sup.131I-hMN-14 IgG Tissue 4 hour 1
day 2 days 5 days 7 days 14 days Percent Injected Dose Per Gram
Tissue (N = 4 to 5 animals) LS174T 11.8 .+-. 2.9 18.1 .+-. 14.9
32.6 .+-. 17.2 30.2 .+-. 13.4 10.6 .+-. 15.2 11.6 .+-. 5.6 Weight
0.31 .+-. 0.07 0.37 .+-. 0.02 0.27 .+-. 0.08 0.31 .+-. 0.9 0.3 .+-.
0.2 0.40 .+-. 0.07 Liver 10.8 .+-. 2.1 6.0 .+-. 3.3 2.8 .+-. 0.5
0.8 .+-. 0.4 0.5 .+-. 0.7 0.08 .+-. 0.05 Spleen 17.0 .+-. 4.8 10.5
.+-. 8.8 4.9 .+-. 0.4 1.3 .+-. 0.6 0.6 .+-. 0.8 0.14 .+-. 0.09
Kidney 7.0 .+-. 0.8 3.1 .+-. 0.7 2.3 .+-. 0.9 0.9 .+-. 0.4 0.4 .+-.
0.6 0.07 .+-. 0.04 Lungs 8.7 .+-. 0.4 3.7 .+-. 1.4 3.7 .+-. 1.4 1.4
.+-. 0.6 0.6 .+-. 1.0 0.11 .+-. 0.06 Blood 15.4 .+-. 6.6 5.5 .+-.
4.7 6.8 .+-. 4.1 2.3 .+-. 1.2 0.9 .+-. 1.8 0.14 .+-. 0.13 Tumor
(LS174T)/Nontumor Ratios (N = 4 to 5 amimals) Liver 1.1 .+-. 0.3
5.0 .+-. 5.0 11.1 .+-. 4.7 39.5 .+-. 7.5 24.6 .+-. 4.8 160 .+-. 28
Spleen 0.7 .+-. 0.3 4.2 .+-. 4.6 6.6 .+-. 3.4 24.7 .+-. 7.8 15.8
.+-. 4.4 91 .+-. 21 Kidney 1.7 .+-. 0.5 5.3 .+-. 3.5 13.3 .+-. 3.6
36.1 .+-. 4.0 38.4 .+-. 12.9 173 .+-. 41 Lungs 1.4 .+-. 0.4 4.2
.+-. 2.2 8.2 .+-. 2.1 22.3 .+-. 2.1 25.4 .+-. 7.3 112 .+-. 15 Blood
0.9 .+-. 0.5 3.7 .+-. 0.9 5.4 .+-. 1.4 14.2 .+-. 2.9 26.0 .+-. 12.9
111 .+-. 35
B. Clinical Studies with .sup.131I-Labeled Humanized MN-14 IgG
[0139] Patients were entered into an Institutional Review
Board-approved protocol at the Center for Molecular Medicine and
Immunology, Newark, N.J. for a pilot investigation of the targeting
and pharmacokinetic behavior of the humanized MN-14 IgG. In the
case the results of which are shown in FIG. 12, the male patient
had colorectal cancer that had metastasized to the liver. He was
injected i.v. with .sup.131I-hMN-14 IgG (8 mCi, 0.6 mg antibody)
and images were taken over a six day period. The patient was
subsequently injected with an identical dose of mMN-14 IgG. The
images shown in FIG. 12 shaw the anterior abdominal view about 140
h after each injection. The images are adjusted to exactly the same
intensity so that they are directly comparable. The results
indicate that the humanized antibody is taken up by the
CEA-producing tumor as well as the parental murine antibody. These
experiments establish the practical utility of diagnosing human
CEA-producing colon cancers with the inventive humanized MN-14 mAb.
Sequence CWU 1
1
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