U.S. patent application number 11/742725 was filed with the patent office on 2007-08-30 for antibody therapy.
This patent application is currently assigned to IMMUNOMEDICS, INC.. Invention is credited to David M. Goldenberg, Hans J. Hansen.
Application Number | 20070202044 11/742725 |
Document ID | / |
Family ID | 34830287 |
Filed Date | 2007-08-30 |
United States Patent
Application |
20070202044 |
Kind Code |
A1 |
Goldenberg; David M. ; et
al. |
August 30, 2007 |
ANTIBODY THERAPY
Abstract
The present invention provides a composition comprising naked
humanized, chimeric, and human anti-CEA antibodies and a
therapeutic agent, which is useful for treatment of CEA expressing
cancers and other diseases, and methods of use in treatment using
this composition.
Inventors: |
Goldenberg; David M.;
(Morris Plains, NJ) ; Hansen; Hans J.; (Morris
Plains, NJ) |
Correspondence
Address: |
FAEGRE & BENSON LLP;PATENT DOCKETING
2200 WELLS FARGO CENTER
90 SOUTH SEVENTH STREET
MINNEAPOLIS
MN
55402-3901
US
|
Assignee: |
IMMUNOMEDICS, INC.
300 American Road
Morris Plains
NJ
07950
|
Family ID: |
34830287 |
Appl. No.: |
11/742725 |
Filed: |
May 1, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10680734 |
Oct 8, 2003 |
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11742725 |
May 1, 2007 |
|
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60467161 |
May 2, 2003 |
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Current U.S.
Class: |
424/1.49 ;
424/155.1; 424/178.1; 424/649; 424/85.1; 424/85.4; 424/94.1;
514/13.3; 514/150; 514/19.3; 514/2.1; 514/251; 514/256; 514/263.31;
514/27; 514/283; 514/34; 514/449; 514/492; 514/588; 514/672;
514/7.9; 514/8.1; 514/8.5; 514/9.3 |
Current CPC
Class: |
A61K 31/437 20130101;
A61K 31/4545 20130101; A61K 38/164 20130101; A61K 38/193 20130101;
A61K 2039/505 20130101; A61P 31/00 20180101; C07K 16/3007 20130101;
A61K 31/675 20130101; A61K 38/168 20130101; A61K 38/193 20130101;
A61K 39/39558 20130101; A61K 51/1096 20130101; C07K 2317/24
20130101; A61K 38/191 20130101; A61K 51/1048 20130101; A61K 38/196
20130101; A61K 31/555 20130101; A61K 38/20 20130101; A61P 35/00
20180101; A61P 37/04 20180101; A61K 38/191 20130101; A61K 38/217
20130101; A61K 31/4745 20130101; A61K 38/1816 20130101; A61K
38/1816 20130101; A61K 31/519 20130101; A61K 31/475 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 38/164 20130101; A61K 38/196 20130101; A61K
38/217 20130101; A61K 31/655 20130101; A61K 31/513 20130101; A61K
38/20 20130101; A61K 39/39558 20130101; A61K 51/1045 20130101; A61K
38/168 20130101; A61K 31/704 20130101 |
Class at
Publication: |
424/001.49 ;
424/155.1; 424/178.1; 424/085.4; 424/085.1; 514/012; 514/034;
514/150; 514/251; 514/283; 514/449; 514/027; 424/094.1; 424/649;
514/263.31; 514/256; 514/492; 514/588; 514/672 |
International
Class: |
A61K 51/00 20060101
A61K051/00; A61K 39/395 20060101 A61K039/395; A61K 38/43 20060101
A61K038/43; A61K 38/21 20060101 A61K038/21; A61K 38/19 20060101
A61K038/19; A61K 38/18 20060101 A61K038/18; A61K 31/7076 20060101
A61K031/7076; A61K 31/7072 20060101 A61K031/7072; A61K 31/7048
20060101 A61K031/7048; A61K 31/704 20060101 A61K031/704; A61K
31/655 20060101 A61K031/655; A61K 31/525 20060101 A61K031/525; A61K
31/522 20060101 A61K031/522; A61K 31/505 20060101 A61K031/505 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 11, 2002 |
US |
PCT/US02/32307 |
Claims
1. A method of treating cancer comprising administering to a
subject with cancer at least one humanized, chimeric or human
anti-CEA antibody, followed by administering to the subject at
least one therapeutic agent, wherein said anti-CEA antibody does
not bind to human granulocytes and has a human IgG1 hinge and human
IgG1 constant region domains.
2. The method of claim 1, wherein administering said anti-CEA
antibody to said subject sensitizes the cancer to the therapeutic
agent.
3. The method of claim 1, wherein said anti-CEA antibody comprises
the complementarity-determining regions (CDRs) of a murine MN-14
monoclonal antibody, comprising the light chain variable region
CDR1 (KASQDVGTSVA, SEQ ID NO: 20); CDR2 (WTSTRHT, SEQ ID NO: 21);
and CDR3 (QQYSLYRS, Q ID NO: 22); and the heavy chain variable
region CDR1 (TYWMS, SEQ ID NO: 23); CDR2 (EIHPDSSTINYAPSLKD, SEQ ID
NO: 24); and CDR3 (LYFGFPWFAY, SEQ ID NO: 25).
4. The method of claim 3, wherein the anti-CEA antibody is a
humanized MN-14 antibody.
5. The method of claim 4, wherein the framework regions (FRs) of
the light and heavy chain variable regions of said humanized MN-14
antibody comprises at least one amino acid substituted from the
corresponding FRs of a murine MN-14 monoclonal antibody.
6. The method of claim 1, wherein said fragment is selected from
the group consisting of F(ab').sub.2, Fab', Fab, Fv and scFv.
7. The method of claim 1, wherein said therapeutic agent is a naked
non-CEA antibody or fragment or a non-CEA antibody or fragment
conjugated to another therapeutic agent.
8. The method of claim 7, wherein said non-CEA antibody or fragment
is selected from the group consisting of a humanized, chimeric,
human or murine antibody or fragment reactive with EGP-1, EGP-2,
IL-6, MUC-1, MUC-2, MUC-3, MUC-4, PAM-4, KC4, TAG-72, EGFR,
HER2/neu, BrE3, Le-Y, A3, A33, Ep-CAM, AFP, Tn,
Thomson-Friedenreich antigens, tumor necrosis antigens, VEGF, IL-6,
insulin-like growth factor, placenta growth factor, tenascin,
fibronectin, a tumor angiogenesis antigen, Ga 733, and a
combination thereof.
9. The method of claim 1, wherein said therapeutic agent is
selected from the group consisting of a naked antibody, a cytotoxic
agent, a drug, a radionuclide, an immunomodulator, a photoactive
therapeutic agent, an immunoconjugate, a hormone, an antisense
oligonucleotide or a combination thereof.
10. The method of claim 1, wherein said therapeutic agent is not
DTIC.
11. The method of claim 9, wherein said cytotoxic agent is a drug
or a toxin.
12. The method of claim 11, wherein said drug possesses a
pharmaceutical property selected from the group consisting of
antimitotic, alkylating, antimetabolite, antiangiogenic, apoptotic,
alkaloid, COX-2, and antibiotic agents and combinations
thereof.
13. The method of claim 11, wherein said drug is selected from the
group consisting of nitrogen mustards, ethylenimine derivatives,
alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs,
anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs,
purine analogs, antimetabolites, antibiotics, enzymes,
epipodophyllotoxins, platinum coordination complexes, vinca
alkaloids, substituted ureas, methyl hydrazine derivatives,
adrenocortical suppressants, antagonists, endostatin, taxols,
camptothecins, doxorubicins, and a combination thereof.
14. The method of claim 1, wherein the therapeutic agent is
CPT-11.
15. The method of claim 11, wherein said toxin is a microbial,
plant or animal toxin selected from the group consisting of ricin,
abrin, alpha toxin, saporin, ribonuclease (RNase), DNase I,
Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,
diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas
endotoxin.
16. The method of claim 9, wherein said immunomodulator is selected
from the group consisting of a cytokine, a stem cell growth factor,
a lymphotoxin, a hematopoietic factor, a colony stimulating factor
(CSF), an interferon (IFN), a stem cell growth factor,
erythropoietin, thrombopoietin and a combination thereof.
17. The method of claim 16, wherein said lymphotoxin is tumor
necrosis factor (TNF), said hematopoietic factor is an interleukin
(IL), said colony stimulating factor is granulocyte-colony
stimulating factor (G-CSF) or granulocyte macrophage-colony
stimulating factor (GM-CSF), said interferon is interferon-.alpha.,
-.beta., or -.gamma., and said stem cell growth factor is
designated "S1 factor."
18. The method of claim 9, wherein said immunomodulator comprises
IL-1, IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, interferon-.gamma.,
TNF-.alpha. or a combination thereof.
19. The method of claim 9, wherein said radionuclide has an energy
between 20 and 10,000 keV.
20. The method of claim 19, wherein said radionuclide is selected
from the group consisting of .sup.125I, .sup.131I, .sup.90Y,
.sup.88Y, .sup.225Ac, .sup.177Lu, .sup.188Re, .sup.86Re, and
combinations thereof.
21. The method of claim 1, wherein said therapeutic agent is
dacarbazine.
22. The method of claim 4, wherein said humanized MN-14 antibody is
administered in a dosage of 100 to 600 milligrams protein per dose
per injection.
23. The method of claim 22, wherein said humanized MN-14 antibody
is administered in a dosage of 300 to 400 milligrams protein per
dose per injection.
24. The method of claim 1, wherein said therapeutic agent is
selected from the group consisting of vincristine, doxorubicin,
DTIC, oxaliplatin, 5-fluorouracil/leucovorin and
cyclophosphamide.
25. The method of claim 14, wherein the anti-CEA antibody is
administered 3 days prior to administration of CPT-11.
26. The method of claim 1, wherein treatment with antibody prior to
treatment with therapeutic agent is more effective than treatment
with either antibody or therapeutic agent alone, without increased
toxicity.
27. The method of claim 4, wherein the therapeutic agent is CPT-11,
and administration of humanized MN-14 antibody 3 days prior to
administration of CPT-11 results in a 58% increase in survival of a
subject with cancer over CPT-11 alone.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 10/680,734 filed on Oct. 11, 2002, entitled "Antibody
Therapy" which is a continuation-in-part of U.S. Provisional
Application No. 60/467,161, filed May 2, 2003. This application
also claims priority to International Application No.
PCT/US/02/32307, filed Oct. 11, 2002, which in turn claims priority
to U.S. Provisional Application No. 60/416,531, filed Oct. 8, 2002,
the full text of each of which are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] A. Field of the Invention
[0003] The invention relates to methods of treating cancers that
express carcinoembryonic antigen ("CEA"), particularly medullary
thyroid cancer (MTC), non-medullary thyroid cancers (non-MTC),
colorectal cancers, hepatocellular carcinoma, gastric cancer, lung
cancer, breast cancer and other cancers, in which CEA is expressed,
by administering an immunological reagent comprising an antibody in
combination with at least one other therapeutic agent, such as
another antibody, a chemotherapeutic agent, a radioactive agent, an
antisense oligonucleotide, an immunomodulator, an immunoconjugate
or a combination thereof. The invention further relates to
pharmaceutical compositions comprising the immunological reagent
and at least one therapeutic agent in an unconjugated form. In
particular, the invention relates to methods of treating cancers
that express CEA by administering, prior to, with or after
administering the therapeutic agent, a Class III
anticarcinoembryonic antigen ("anti-CEA") monoclonal antibody
("MAb"), particularly a MAb that has the binding affinity
characteristics and specificities of corresponding murine Class III
anti-CEA MAb, and more particularly humanized, chimeric or human
MAbs, that possess more of the antigenic and effector properties of
a human antibody. Particularly useful MAbs in the method of
treatment are humanized MAbs in which the
complementarity-determining regions ("CDRs") of an anti-CEA murine
MAb are grafted into the framework regions of a human antibody.
[0004] B. Background
[0005] CEA is an oncofetal antigen commonly expressed in a number
of epithelial cancers, most commonly those arising in the colon but
also in the breast, lung, pancreas, thyroid (medullary type) and
ovary (Goldenberg et al., J. Natl. Cancer Inst. 57: 11-22 (1976),
Shively, et al., Crit. Rev. Oncol. Hematol. 2:355-399 (1985)). CEA
was originally thought to be a tumor-specific antigen of colorectal
cancer (Gold et al., J. Exper. Med., 122:467 (1965)). However, it
was later found to be present in a diverse number of carcinomas,
benign tumors, and diseased tissues, as well as in normal human
colon (Shively et al., Crit. Rev. Oncol. Hematol., 2:355 (1985);
von Kleist et al., Proc. Natl. Acad. Sci. U.S.A., 69:2492 (1972)).
CEA has been shown to mediate cell-cell adhesion through homotypic
and heterotypic interactions, which in turn have implicated a role
for CEA in various aspects of tumorigenesis.
[0006] Medullary thyroid cancer (MTC) confined to the thyroid gland
is potentially curable by total thyroidectomy and central lymph
node dissection. However, disease recurs in approximately 50% of
these patients. In addition, the prognosis of patients with
unresectable disease or distant metastases is poor, less than 30%
survive 10 years (Rossi et al., Amer. J. Surgery, 139:554 (1980);
Samaan et al., J. Clin. Endocrinol. Metab., 67:801 (1988); Schroder
et al., Cancer, 61:806 (1988). These patients are left with few
therapeutic choices (Principles and Practice of Oncology, DeVita,
Hellman and Rosenberg (eds.), New York: JB Lippincott Co. 1333-1435
(1989); Cancer et al., Current Problems Surgery, 22: 1 (1985)).
Chemotherapy has been of little value and radiation therapy may
only be used to control local disease (Cancer et al.; Tubiana et
al., Cancer, 55:2062 (1985)). Thus, new therapeutic modalities are
needed to control this disease.
[0007] A useful approach to cancer therapy and diagnosis involves
the use of targeting antibodies to deliver therapeutic and
diagnostic 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 therapeutic agents, such as drugs,
toxins, radionuclides, immunomodulators, such as cytokines or other
agents, and to administer the conjugates to patients that target
the tumor. However, patients treated with drugs or radionuclides
complexed with murine monoclonal antibodies (which have been the
most commonly used targeting antibodies for humans) develop
circulating human anti-mouse antibodies (HAMAs) and sometimes a
generalized immediate type-III hypersensitivity reaction to the
antibody moiety of the conjugate. But these problems have been
minimized by making these murine antibodies less immunogenic by a
number of different methods, which include making humanized,
chimeric or human antibodies, by chemically modifying the targeting
antibody, such as by conjugating to polyethylene glycol to the
targeting antibody (PEGylation), or by characterizing the situs of
antigenicity in an antibody and then removing it; e.g., 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.
[0008] Regardless of these treatment advances, there still exists a
need to provide more effective methods of treating CEA-expressing
cancers. The present invention provides an effective therapy
utilizing anti-CEA antibodies, such as a Class III anti-CEA MAb,
the murine MN-14 MAb as defined in U.S. Pat. No. 5,874,540 and
Hansen et at., Cancer, 71 :3478 (1993), and a Class III anti-CEA
MAb, the chimeric and humanized MN-14 MAbs as also defined in U.S.
Pat. No. 5,874,540, and the NP-4 as defined in U.S. Pat. No.
4,818,709 by Primus et at., for example, all incorporated herein in
their entirety by reference. Preferably, the Class III anti-CEA MAb
is humanized, and used in combination with a therapeutic agent,
particularly a chemotherapeutic agent, to yield an effective
therapeutic treatment for CEA expressing cancers with minimal
toxicity. Additionally, other anti-CEA antibodies, such Class II
MAbs, for example, MN-6 (see Hansen et al., above, and NP-3 (se
U.S. Pat. No. 4,818,709), and Class I MAbs, such MN-3 and MN-15
(see also Hansen et al., above) provide effective methods of
treating CEA expressing cancers. Further, the separate
administration of these two components provides enhanced results
and the versatility and the flexibility to tailor individual
treatment methods.
SUMMARY OF THE INVENTION
[0009] Contemplated in the present invention are compositions and
methods of treating medullary and non-medullary thyroid
carcinomas.
[0010] The first embodiment of the present invention is a
composition comprising at least one anti-CEA monoclonal antibody
(MAb) or fragment thereof, which is preferably a Class III anti-CEA
MAb or fragment, and at least one therapeutic agent. Preferably,
the antibody fragment is selected from the group consisting of
F(ab').sub.2, Fab', Fab, Fv and scFv. Also preferred, the Class III
anti-CEA MAb or fragment thereof is humanized, and wherein the
humanized MAb retains substantially the Class III anti-CEA binding
specificity of a murine Class III anti-CEA MAb. Also preferred, the
Class III anti-CEA MAb or fragment thereof is a chimeric MAb, and
wherein the chimeric MAb retains substantially the Class III
anti-CEA binding specificity of murine Class III anti-CEA MAb.
Still preferred, the Class III anti-CEA MAb or fragment thereof is
a fully human MAb, and wherein said fully human MAb retains
substantially the Class III anti-CEA binding specificity of murine
Class III anti-CEA MAb. Other preferred anti-CEA Mabs for this
purpose include Class II Mabs or fragments thereof, that are not
CD66a-d cross-reactive which are discussed in greater detail
herein. Another embodiment includes Class II anti-CEA Mabs or
fragments thereof, that may react with CD66a, b and d but not CD66c
or Class I Mabs or fragments thereof, that react with CD66a, b, or
d as well as CD66c (by definition a Class I Mab binds with
CD66c).
[0011] In one embodiment of the present invention, the Class III
anti-CEA monoclonal antibody or fragment thereof is preferably a
MN-14 antibody or fragment thereof. More preferably, the MN-14
monoclonal antibody or fragment thereof comprises the
complementarity-determining regions (CDRs) of a murine MN-14
monoclonal antibody, wherein the CDRs of the light chain variable
region of the MN14 antibody comprises CDR1 comprising the amino
acid sequence KASQDVGTSVA (SEQ ID NO:20); CDR2 comprising the amino
acid sequence WTSTRHT (SEQ ID NO: 21); and CDR3 comprising the
amino acid sequence QQYSLYRS (SEQ ID NO: 22); and the CDRs of the
heavy chain variable region of the Class III anti-CEA antibody
comprises CDR1 comprising TYWMS (SEQ ID NO: 23); CDR2 comprising
EIHPDSSTINYAPSLKD (SEQ ID NO: 24); and CDR3 comprising LYFGFPWFAY
(SEQ ID NO: 25). Also preferred, the MN-14 monoclonal antibody
reacts with CEA and is unreactive with normal cross-reactive
antigen (NCA) and meconium antigen (MA). Most preferably, the MN-14
monoclonal antibody or fragment thereof is a humanized, chimerized
or fully human MN-14 antibody or fragment thereof.
[0012] In a preferred embodiment, the framework regions (FRs) of
the light and heavy chain variable regions of the humanized MN-14
antibody or fragment thereof comprise at least one amino acid
substituted from the corresponding FRs of a murine MN-14 monoclonal
antibody. Specifically, the humanized MN-14 antibody or fragment
thereof preferably comprises at least one amino acid from the
corresponding FR of the murine MN-14 antibody is selected from the
group consisting of amino acid residue 24 (A), 28 (D), 30 (T), 48
(I), 49 (G), 74 (A) and 94 (S) of the murine heavy chain variable
region (KLHuVhAIGA) of FIG. 14A-C. Likewise, the humanized MN-14
antibody or fragment thereof may also comprise at least one amino
acid from said corresponding FR of the murine MN-14 light chain
variable region. Still preferred, the humanized MN-14 antibody or
fragment thereof comprises the light chain variable region as set
forth in FIG. 13A, and the heavy chain variable region set forth in
FIG. 14A-C designated as KLHuVhAIGA.
[0013] In the first embodiment of the present invention, the
therapeutic agent is selected from the group consisting of a naked
antibody, a cytotoxic agent, a drug, a radionuclide, an
immunomodulator, a photoactive therapeutic agent, an
immunoconjugate, a hormone, or a combination thereof, optionally
formulated in a pharmaceutically acceptable vehicle. It is also
contemplated herein that the therapeutic agent is not dacarbazine
(DTIC).
[0014] The second embodiment of the present invention describes a
method for treating medullary as well as non-medullary thyroid
carcinoma comprising administering to a subject, either
concurrently or sequentially, a therapeutically effective amount a
Class III anti-CEA monoclonal antibody or fragment thereof and at
least one therapeutic agent, and optionally formulated in a
pharmaceutically acceptable vehicle. Preferably, the antibody
fragment is selected from the group consisting of F(ab').sub.2,
Fab', Fab, Fv and scFv. Also preferred, the Class III anti-CEA MAb
or fragment thereof is humanized, wherein said humanized MAb
retains substantially the Class III anti-CEA binding specificity of
a murine Class III anti-CEA MAb. It is also contemplated that the
Class III anti-CEA MAb or fragment thereof is a chimeric MAb, and
wherein said chimeric MAb retains substantially the Class III
anti-CEA binding specificity of murine Class III anti-CEA MAb.
[0015] In a preferred embodiment, the Class III anti-CEA monoclonal
antibody or fragment thereof is a MN-14 antibody or fragment
thereof. Preferably, the MN-14 monoclonal antibody or fragment
thereof comprises the complementarity-determining regions (CDRs) of
a murine MN-14 monoclonal antibody, wherein the CDRs of the light
chain variable region of said MN-14 antibody comprises CDR1
comprising the amino acid sequence KASQDVGTSVA (SEQ ID NO: 20);
CDR2 comprising the amino acid sequence WTSTRHT (SEQ ID NO: 21);
and CDR3 comprising the amino acid sequence QQYSLYRS (SEQ ID NO:
22); and the CDRs of the heavy chain variable region of said Class
III anti-CEA antibody comprises CDR1 comprising TYWMS (SEQ ID NO:
23); CDR2 comprising EIHPDSSTINYAPSLKD (SEQ ID NO: 24); and CDR3
comprising LYFGFPWFAY (SEQ ID NO: 25). Also preferred, the MN-14
monoclonal antibody is humanized, chimerized or fully human, and
reacts with CEA and is unreactive with normal cross-reactive
antigen (NCA) and meconium antigen. Also preferred, the MN-14
antibody or fragment thereof is administered in a dosage of 100 to
600 milligrams protein per dose per injection. Most preferably, the
MN-14 antibody or fragment thereof is administered in a dosage of
300-400 milligrams protein per dose per injection.
[0016] In the methods of the instant invention, the framework
regions (FRs) of the light and heavy chain variable regions of said
humanized MN-14 antibody or fragment thereof preferably comprise at
least one amino acid substituted from the corresponding FRs of a
murine MN-14 monoclonal antibody. More preferred, the humanized
MN-14 antibody or fragment thereof comprising at least one amino
acid from said corresponding FR of said murine MN-14 antibody is
selected from the group consisting of amino acid residue 24, 28,
30, 48, 49, 74 and 94 of the murine heavy chain variable region of
FIG. 14A-C, as noted above. Also preferred, the humanized MN-14
antibody or fragment thereof comprising at least one amino acid
from said corresponding FR of said murine MN-14 light chain
variable region. Most preferably, the humanized MN-14 antibody or
fragment thereof comprises the light chain variable region as set
forth in FIG. 13A (middle sequence) or FIG. 22A (hMN-14) or FIG.
23A and the heavy chain variable region set forth in FIG. 14A-C
designated as KLHuVhAIGA or FIG. 22B (hMn-14) or FIG. 23B.
[0017] The methods of the instant invention may further comprise
administering to a subject, either concurrently or sequentially, a
therapeutically effective amount of a second humanized, chimeric,
human or murine monoclonal antibody or fragment thereof selected
from the group consisting of a monoclonal antibody or fragment
thereof reactive with EGP-1, EGP-2 (e.g., 17-1A), 1L-6, insulin
like growth factor-1, MUC-1, MUC-2, MUC-3, MUC-4, PAM-4, KC4,
TAG-72, EGFR, HER2/neu, BrE3, Le-Y, A3, A33, Ep-CAM, AFP, Tn,
Thomson-Friedenreich antigens, tumor necrosis antigens, VEGF,
placenta growth factor (PlGF) or other tumor angiogenesis antigens,
Ga 733, tenascin, fibronectin and a combination thereof. Similarly,
the methods may comprise administering to a subject, either
concurrently or sequentially, a therapeutically effective amount of
a second humanized, chimeric, human or murine monoclonal antibody
or fragment thereof selected from the group consisting of a Class I
or Class II or Class III anti-CEA monoclonal antibody or fragment
thereof as described above. Preferably, the second antibody or
fragment thereof is either naked or conjugated to a therapeutic
agent.
[0018] In a preferred embodiment of the methods described herein,
the therapeutic agent is selected from the group consisting of a
naked antibody, cytotoxic agent, a drug, a radionuclide, an
immunomodulator, a photoactive therapeutic agent, an
immunoconjugate of a CEA or non-CEA antibody, a hormone, or a
combination thereof, optionally formulated in a pharmaceutically
acceptable vehicle. It is also contemplated that the therapeutic
agent is not dacarbazine (DTIC).
[0019] Preferably, the therapeutic agent is a cytotoxic agent
selected from the group consisting of a drug or a toxin. For
example, it is contemplated that the drug possesses the
pharmaceutical property selected from the group consisting of
antimitotic, alkylating, antimetabolite, antiangiogenic, apoptotic,
alkaloid, COX-2, and antibiotic agents and combinations thereof.
Preferably, the drug is selected from the group consisting of
nitrogen mustards, ethylenimine derivatives, alkyl sulfonates,
nitrosoureas, triazenes, folic acid analogs, anthracyclines,
taxanes, COX-2 inhibitors, pyrimidine analogs, purine analogs,
antimetabolites, antibiotics, enzymes, epipodophyllotoxins,
platinum coordination complexes, vinca alkaloids, substituted
ureas, methyl hydrazine derivatives, adrenocortical suppressants,
antagonists, endostatin, taxols, camptothecins, oxaliplatin,
doxorubicins and their analogs, and a combination thereof.
[0020] When the therapeutic agent is a microbial, plant or animal
toxin, the agent can be selected from the group consisting of
ricin, abrin, alpha toxin, saporin, ribonuclease (RNase), DNase I,
Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,
diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas
endotoxin.
[0021] It is also contemplated in the methods of the instant
invention that the therapeutic agent is an immunomodulator is
selected from the group consisting of a cytokine, a stem cell
growth factor, a lymphotoxin, a hematopoietic factor, a colony
stimulating factor (CSF), an interferon (IFN), a stem cell growth
factor, erythropoietin, thrombopoietin and a combination thereof.
Preferably, the lymphotoxin is tumor necrosis factor (TNF), said
hematopoietic factor is an interleukin (IL), said colony
stimulating factor is granulocyte-colony stimulating factor (G-CSF)
or granulocyte macrophage-colony stimulating factor (GM-CSF)), said
interferon is interferons-.alpha., -.beta. or -.gamma., and said
stem cell growth factor is designated "S1 factor." Also preferred,
the immunomodulator comprises IL-1, IL-2, IL-3, IL-6, IL-10, IL-12,
IL-18, IL-21, interferon-.gamma., TNF-.alpha. or a combination
thereof. Administration of a cytokine prior to, simultaneous with,
or subsequent to exposure to a cytotoxic agent that results in
myeloid or hematopoietic toxicity is described in U.S. Pat. No.
5,120,525, which is incorporated herein by reference in its
entirety.
[0022] Also preferred the therapeutic agent is a photoactive
therapeutic agent that is a chromogen or dye or an alkylating agent
that is dacarbazine.
[0023] Also preferred, the therapeutic agent is a radionuclide that
has an energy between 20 and 10,000 keV. Preferably, the
radionuclide is selected from the group consisting of .sup.125I,
.sup.131I, .sup.90Y, .sup.88Y, .sup.225Ac, .sup.177Lu, .sup.188Re,
.sup.186Re, and combinations thereof.
[0024] In another embodiment, an immunomodulator, as described
herein, is administered prior to the administration of a
therapeutically effective amount of a anti-CEA monoclonal antibody
or fragment thereof alone or an immunomodulator is administered
prior to the administration of a therapeutically effective amount
of a anti-CEA monoclonal antibody and at least one therapeutic
agent, wherein any of these components described herein are
optionally formulated in a pharmaceutically acceptable vehicle.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1. Graphs comparing tumor volume after treatment with
hMN-14 alone, DTIC alone or the combination of hMN-14 and DTIC.
FIG. 1A shows DTIC administered alone at 25 and 100 .mu.g/dose or
with 250g hMN-14 antibody and FIG. 1B shows DTIC administered alone
at 50 and 75 .mu.g/dose or with 100g hMN-14 antibody.
[0026] FIG. 2. Graph comparing tumor volume after
radioimmunotherapy (RAIT) with .sup.131I and .sup.90Y-MN-14.
[0027] FIG. 3. Graph comparing the therapeutic efficacy of several
chemotherapeutic drugs on tumor volume in TT bearing mice. TT
bearing mice were given doxorubicin @ 20 mg/m.sup.2; days 0, 1, and
2, (70 .mu.g/dose) (0); DTIC @ 300 mg/m.sup.2; days 0, 1, and 2,
(1.08 mg/dose) (.quadrature.); doxorubicin and DTIC as above
(.circle-solid.); cyclophosphamide @ 600 mg/m.sup.2; day 0 (2.16
mg/dose) (.DELTA.); vincristine @ 4.2 .mu.g/dose; day 0 (x); all 4
drugs (doxorubicin, DTIC, cyclophosphamide, and vincristine, at the
doses described for each above (.box-solid.); or left untreated
(.diamond-solid.). Groups consisted of 6-9 nude mice bearing
established TT tumors. Mean tumor volume at time of treatment was
0.51+/-0.33 cm.sup.3. Points: mean tumor size. Error bars: std dev,
and are shown only above the symbol for clarity.
[0028] FIG. 4. Graph comparing the therapeutic efficacy of
combination therapy of RAIT with .sup.90Y-labeled anti CEA MAb
MN-14 and a 4-drug combination initiated 24 hours after RAIT on
tumor volume in mice. Tumor bearing animals were either left
untreated (.diamond-solid.); given the 4-drug regimen described in
FIG. 1, administered on days 1, 2, and 3 (.box-solid.); 52.5 .mu.Ci
.sup.90Y-MN-14 day on 0, followed by the 4-drug regimen
administered on days 1, 2, and 3 (.tangle-solidup.); 52.5 .mu.Ci
.sup.90Y-MN-14 on day 0 (.DELTA.); or 105 .mu.Ci .sup.90Y-MN-14 on
day 0 (O). N=5 for the untreated group, and n=9-10 in the treatment
groups. Mean tumor volume at time of treatment was 0.28+/-0.12
cm.sup.3. Points: mean tumor size. Error bars: std dev, and are
shown only above the symbol for clarity.
[0029] FIG. 5. Graph comparing the efficacy of RAIT plus DTIC and
RAIT plus doxorubicin and DTIC in TT bearing mice. TT bearing mice
were either left untreated (.diamond-solid.); given 105 .mu.Ci
.sup.90Y-MN-14 day on 0 (O); 105 .mu.Ci .sup.90Y-MN-14 day on 0,
followed by the doxorubicin and DTIC regimen administered at 50%
the full dosage on days 1, 2, and 3 (.tangle-solidup.); 105 .mu.Ci
.sup.90Y-MN-14 on day 0 followed by DTIC at 75% of the full dosage
on days 1, 2, and 3 (x); or the full dosage of DTIC, 300 mg/m.sup.2
on days 1, 2, and 3, (1.08 mg/dos) (.quadrature.). N=5 for the
untreated group, and n=8-9 in the treatment groups. Mean tumor
volume at time of treatment ws 0.29+/-0.20 cm.sup.3. Points: mean
tumor size. Error bars: std dev, and are shown only above the
symbol for clarity.
[0030] FIG. 6. Graph comparing the effectiveness of naked hMN-14
with treatment regimens in mice bearing TT xenografts. Animals were
given s.c. injections of TT cells, and either left untreated (A) or
given an i.v. injection of 0.5 mg hMN-14 1 day (B) or 11 days (C)
later. Lines in panels A, B, and C represent tumor volumes of
individual animals. Means of respective treatment groups are shown
in panel D. Error bars represent standard error of the mean and are
shown only in one direction for clarity. .diamond-solid.,
untreated; .quadrature., day-1 treated; .DELTA., day-11
treated.
[0031] FIG. 7. Graph comparing the effectiveness of humanized and
murine MN-14 antibodies in treating medullary thyroid carcinoma.
Animals were given s.c. injections of TT cells, and either left
untreated or given an i.v. injection of MAb (0.5 mg) 1 day later.
Means of respective treatment groups are shown. Error bars
represent standard error of the mean and are shown only in one
direction for clarity. .diamond-solid., untreated; .quadrature.,
hMN-14; A, murine MN-14; x, hLL2.
[0032] FIG. 8. Graph comparing the effectiveness of different
hMN-14 doses in treating medullary thyroid carcinoma. Animals were
given i.v. injections of increasing doses of hMN-14 1 day after
s.c. injection of TT cells. Means of respective treatment groups
are shown. .diamond-solid., untreated; .circle-solid., 0.125 mg; O,
0.25 mg; .box-solid., 0.50 mg; x, 1.0 mg, .tangle-solidup., 2.0 mg.
Error bars represent standard error of the mean and are shown only
for the untreated group and the group that received 0.50 mg
hMN-14/mouse for clarity.
[0033] FIG. 9. Graph comparing the effectiveness of different
treatment times in TT bearing nude mice. Animals were given i.v.
injections of 0.25 hMN-14 either 1 (.circle-solid.), 3
(.tangle-solidup.), or 7 (.box-solid.) days after s.c. injection of
TT cells, or left untreated (.diamond-solid.). Means of respective
treatment groups are shown. Error bars represent standard error of
the mean and are shown only in one direction for clarity.
[0034] FIG. 10. Graph comparing treatment of TT bearing nude mice
with hMN-14 plus DTIC, DTIC alone, hMN-14 alone, and untreated
mice. Animals were given i.p. injections of hMN-14 at 100
.mu.g/dose on days 2, 3, 4, 5, 7, 8, 9, 10 and 11, 15, and 22, then
every 7 days (O); DTIC, 75 .mu.g/dose on days 2, 3, and 4,
(.tangle-solidup.); the combination of these hMN-14 and DTIC
regimens (.DELTA.); or left untreated (.diamond-solid.). Means of
respective treatment groups are shown. 10 animals/group.
[0035] FIG. 11. FIGS. 11A and 11 B show the consensus DNA sequence
of the murine MN-14 variable region heavy chain (VH) (SEQ ID NO: 1)
and the amino acid sequence encoded by the DNA (SEQ ID NO: 2)
sequence. The CDRs are enclosed in boxes.
[0036] FIG. 12. FIGS. 12A and 12 B show the consensus DNA sequence
of the murine MN-14 variable region light chain (VK) (SEQ ID NO: 3)
and the amino acid sequence (SEQ ID NO: 4) encoded by the DNA
sequence. The CDRs are enclosed in boxes.
[0037] FIG. 13. FIGS. 13A and 13B show the alignment of the murine
MN-14 variable region (MN14VH is shown in SEQ ID NO:2, MN14VK is
shown in SEQ ID NO:4) with the human variable regions NEWM VH (SEQ
ID NO:5) and REI VK (SEQ ID NO:6) (FIG. 13A) and with the human KOL
VH region (SEQ ID NO: 7) (FIG. 13B). 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.
[0038] FIG. 14. FIGS. 14A-14C show a comparison of the amino acid
sequence between murine and humanized MN-14 VH framework residues
(FR) (SEQ ID NOS 2, 26, 8-11, 27 and 12-15, respectively in order
of appearance). Only human FR residues different from the mouse are
shown. CDRs for NEWM and KOL are also not shown. The areas of amino
acid substitutions in the respective FRs are highlighted in bold,
and the position of the substitution indicated according to the
Kabat et al., numbering system. The 3 CDRs are boxed.
[0039] FIG. 15. FIG. 15 shows the effects of naked hMN-14 CEA Mab
and DTIC treatment in a human medullary thyroid cancer model.
[0040] FIG. 16. FIG. 16 shows the effects of naked hMN-14 CEA Mab
and CPT-11 treatment in an advanced human colon cancer model.
[0041] FIG. 17. FIG. 17 shows the effects of naked hMN-14 CEA Mab
and CPT-11 treatment in a low tumor burden human colon cancer
model.
[0042] FIG. 18. FIG. 18 shows the effects of pre-treatment with
naked hMN-14 CEA Mab given 3 days prior to CPT-11 treatment in a
human colon cancer model.
[0043] FIG. 19. FIG. 19 shows a comparison of various
administration sequences of naked hMN-14 CEA Mab and CPT-11 in a
human colon cancer model.
[0044] FIG. 20. FIG. 20 shows the effects of GM-CSF pre-treatment
on naked hMN-14 CEA Mab therapy in a human colon cancer model.
[0045] FIG. 21. FIG. 21 shows a comparison of the effects of naked
hMN-14 CEA Mab therapy on both low CEA expression and high
(interferon-induced) CEA expression tumor cells in a human colon
cancer model.
[0046] FIG. 22. FIGS. 22A and 22B show the comparison of the human,
murine and humanized sequences of the Vk and VH regions of the
human REI and KOL antibodies, respectively with murine and
humanized MN-14. The human sequences of the REI Vk (SEQ ID NO:6) in
FIG. 22A are compared with the murine (SEQ ID NO:4) and humanized
(SEQ ID NO:19) MN-14 Vk sequences. The closed circles indicate
sequences retained from the human REI Vk sequences. The CDRs are
boxed. The human sequences of the KOL VH (SEQ ID NO:7) in FIG. 22B
are compared with the murine (SEQ ID NO:2) and humanized (SEQ ID
NO:14) MN-14 VH sequences. The closed circles indicate sequences
retained from the human KOL VH sequences. The CDRs are boxed.
[0047] FIG. 23. FIGS. 23A and 23B show the Vk, the variable light
chain, and the VH, the variable heavy chain sequences of hMN-14, a
humanized Class III anti-CEA antibody. The CDR region sequences are
shown in bold and underlined. The amino acid residues and the
nucleotides are numbered sequentially. The light chain variable
region is shown in FIG. 23A (Nucleotide and encoded protein are
disclosed as SEQ ID NOS 18 and 19, respectively) and the heavy
chain variable region is shown in FIG. 23B (Nucleotide and encoded
protein are disclosed as SEQ ID NOS 16 and 17, respectively).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
1. Overview
[0048] The present invention provides methods of treatment in which
a naked anti-CEA antibody or fragment thereof and at least one
therapeutic agent are administered either sequentially or
concurrently over a treatment period. The method is particularly
useful for treating medullary thyroid carcinoma but is surprisingly
useful for treating non-medullary thyroid cancers, colorectal
cancers, hepatocellular carcinoma, pancreatic, breast, lung,
head-and-neck, bladder, uterine and ovarian cancers, and even
cancers that do not express CEA at very high levels. For example,
treatment is contemplated in cancers that express CEA at levels of
at least 100 ng/g of tissue. The present method further provides
compositions comprising the anti-CEA antibody, which is preferably
a Class III anti-CEA antibody or antibody fragment in which the
antibody and the therapeutic agent are not conjugated or linked to
each other. As used herein, the phrase "Class III anti-CEA"
antibody or antibody fragment means an antibody or fragment that
binds the CEA antigen (or CD66e) and is unreactive with normal
cross-reactive antigen (NCA), meconium antigen (MA), granulocytes
and CD66a-d (see, Primus et al., U.S. Pat. No. 4,818,709,
incorporated by reference). The naked Class III anti-CEA antibody
or fragment thereof may be a humanized, chimeric, human or murine
antibody. In a preferred embodiment, the naked Class III anti-CEA
antibody or fragment thereof is a humanized MN-14 antibody or
fragment thereof.
[0049] Also contemplated for use in the present invention are Class
II Mabs that are not CD66a-d cross-reactive. These are Mabs that
are reactive with CEA domains N-A1B1, A2B2, which do react with
Meconium Antigen, but not with NCA, and do not react with
granulocytes. For example, NP-3 and MN-6 are Class II anti-CEA
antibodies useful in the present invention. Additionally
contemplated for use in the present invention are Class II anti-CEA
Mabs or fragments thereof, that may react with CD66a, b and d but
not CD66c or Class I Mabs or fragments thereof, that react with
CD66a, b, or d as well as CD66c (by definition a Class I Mab binds
with CD66c).
[0050] Surprisingly, the compositions and methods described herein
are also useful for treating non-medullary thyroid carcinoma,
including colorectal cancer, pancreatic cancer, breast cancer, lung
cancers, hepatocellular carcinoma, urinary bladder cancer,
head-and-neck cancers, and ovarian cancer. Because such forms of
cancer express less CEA than medullary thyroid cancers, it was
unexpected that a naked Class III anti-CEA antibody, in combination
with a therapeutic agent, would be useful for treating
non-medullary thyroid carcinomas.
[0051] The mechanism of tumor cell killing by the naked Class III
anti-CEA antibody is not known with certainty and is likely
involves several mechanisms. It is hypothesized that the naked
antibody alone or in combination with the therapeutic agent may
affect tumor growth by blocking biological activities of their
respective antigens or by stimulating natural immunological
functions, such as antibody-dependent cell-mediated cytotoxicity
(ADCC) or complement-mediated lysis. Additionally, the naked
antibody alone or in combination with the therapeutic agent may
treat and control the cancer by inhibiting cell growth and cell
cycle progression, inducing apoptosis, inhibiting angiogenesis,
inhibiting metastatic activity, and/or affecting tumor cell
adhesion. In fact, the anti-CEA antibody or fragment thereof of the
present invention may be more effective in treating metastases than
primary cancers, since the metastases may be more susceptible to
antagonists of tumor cell adhesion. The present treatment method
provides a treatment plan that may be optimized to provide the
maximum anti-tumor activity for individual patients by allowing the
titration of the antibody and one or more different therapeutic
agents to provide an effective treatment regimen.
[0052] In one aspect of the present invention, the naked Class III
anti-CEA antibody or fragment thereof and therapeutic agent may be
supplemented with at least one additional therapeutic agent, such
as a naked or conjugated humanized, murine, chimeric or human
antibody, fusion protein, or fragment thereof. For example, another
class III CEA antibody or antibody fragment that is non-blocking
and does not bind granulocytes or CD66a-d; a Class II anti-CEA
antibody or antibody fragment that is non-blocking and does not
bind granulocytes or CD66a-d; a Class II anti-CEA Mabs or fragments
thereof, that may react with CD66a, b and d but not CD66c, Class I
Mabs or fragments thereof, that react with CD66a, b, or d as well
as CD66c (by definition a Class I Mab binds with CD66c) or an
antibody against a different carcinoma-associated epitope or
antigen, may be used as the therapeutic agent for combination
therapy with the preferred humanized MN-14 antibody. Such an
additional antibody, fusion protein or fragment thereof may bind
CEA or another cancer or tumor-associated antigen, as described in
more detail below.
2. Definitions
[0053] In the description that follows, a number of terms are used
and the following definitions are provided to facilitate
understanding of the present invention.
[0054] An antibody, as described herein, refers to a full-length
(i.e., naturally occurring or formed by normal immunoglobulin gene
fragment recombinatorial processes) immunoglobulin molecule (e.g.,
an IgG antibody) or an immunologically active (i.e., specifically
binding) portion of an immunoglobulin molecule, like an antibody
fragment.
[0055] An antibody fragment is a portion of an antibody such as
F(ab').sub.2, F(ab).sub.2, Fab', Fab, Fv, scFv (single chain Fv)
and the like. Regardless of structure, an antibody fragment binds
with the same antigen that is recognized by the intact
antibody.
[0056] 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
variable regions, such as the "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 ("scFv proteins"), and
minimal recognition units consisting of the amino acid residues
that mimic the hypervariable region. The Fv fragments may be
constructed in different ways as to yield multivalent and/or multi
specific binding forms. In the former case of multivalent, they
react with more than one binding site against the CEA epitope,
whereas with multispecific forms, more than one epitope (either of
CEA or even against CEA and a different antigen) is bound.
[0057] As used herein, the term antibody component includes both an
entire antibody, a fusion protein, and fragments thereof.
[0058] A naked antibody is generally an entire antibody which is
not conjugated to a therapeutic agent. This is so because the Fc
portion of the antibody molecule provides effector or immunological
functions, such as complement fixation and ADCC (antibody dependent
cell cytotoxicity), which set mechanisms into action that may
result in cell lysis. However, the Fc portion may not be required
for therapeutic function of the antibody, but rather other
mechanisms, such as apoptosis, anti-angiogenesis, anti-metastatic
activity, anti-adhesion activity, such as inhibition of heterotypic
or homotypic adhesion, and interference in signaling pathways, may
come into play and interfere with the disease progression. Naked
antibodies include both polyclonal and monoclonal antibodies, and
fragments thereof, that include murine antibodies, as well as
certain recombinant antibodies, such as chimeric, humanized or
human antibodies and fragments thereof. As defined in the present
invention, "naked" is synonymous with "unconjugated," and means not
linked or conjugated to the therapeutic agent with which it
administered.
[0059] A chimeric antibody is a recombinant protein that contains
the variable domains of both the heavy and light antibody chains,
including the complementarity determining regions (CDRs) of an
antibody derived from one species, preferably a rodent antibody,
while the constant domains of the antibody molecule are derived
from those of a human antibody. For veterinary applications, the
constant domains of the chimeric antibody may be derived from that
of other species, such as a cat or dog.
[0060] A humanized antibody is a recombinant protein in which the
CDRs from an antibody from one species; e.g., a rodent antibody, is
transferred from the heavy and light variable chains of the rodent
antibody into human heavy and light variable domains. The constant
domains of the antibody molecule are derived from those of a human
antibody.
[0061] A human antibody is an antibody 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). A fully human
antibody also can be constructed by genetic or chromosomal
transfection methods, as well as phage display technology, all of
which are known in the art. See for example, McCafferty et al.,
Nature 348:552-553 (1990) for the production of human antibodies
and fragments thereof in vitro, from immunoglobulin variable domain
gene repertoires from unimmunized donors. In this technique,
antibody variable domain genes are cloned in-frame into either a
major or minor coat protein gene of a filamentous bacteriophage,
and displayed as functional antibody fragments on the surface of
the phage particle. Because the filamentous particle contains a
single-stranded DNA copy of the phage genome, selections based on
the functional properties of the antibody also result in selection
of the gene encoding the antibody exhibiting those properties. In
this way, the phage mimics some of the properties of the B cell.
Phage display can be performed in a variety of formats, for their
review, see e.g. Johnson and Chiswell, Current Opinion in
Structural Biology 3:5564-571 (1993).
[0062] Human antibodies may also be generated by in vitro activated
B cells. See U.S. Pat. Nos. 5,567,610 and 5,229,275, which are
incorporated in their entirety by reference.
[0063] A therapeutic agent is a molecule or atom which is
administered separately, concurrently or sequentially with an
antibody component, i.e., an antibody or antibody fragment, or a
subfragment thereof, and is useful in the treatment of a disease.
Examples of therapeutic agents include antibodies, antibody
fragments, immunoconjugates, drugs, cytotoxic agents, toxins,
nucleases, hormones, immunomodulators, chelators, boron compounds,
photoactive agents or dyes, radioisotopes or radionuclides,
antisense oligonucleotides, immunoconjugates or combinations
thereof.
[0064] An immunoconjugate is an antibody component conjugated to a
therapeutic agent. Suitable therapeutic agents are described
above.
[0065] As used herein, the term antibody fusion protein is a
recombinantly produced antigen-binding molecule in which two or
more of the same or different natural antibody, single-chain
antibody or antibody fragment segments with the same or different
specificities are linked. A Class III anti-CEA fusion protein
comprises at least one CEA binding site. Preferably, the Class III
anti-CEA fusion protein is a MN-14 fusion protein. Valency of the
fusion protein indicates the total number of binding arms or sites
the fusion protein has to antigen(s) or epitope(s); i.e.,
monovalent, bivalent, trivalent or multivalent. The multivalency of
the antibody fusion protein means that it can take advantage of
multiple interactions in binding to an antigen, thus increasing the
avidity of binding to the antigen, or to different antigens.
Specificity indicates how many different types of antigen or
epitope an antibody fusion protein is able to bind; i.e.,
monospecific, bispecific, trispecific, multispecific. Using these
definitions, a natural antibody, e.g., an IgG, is bivalent because
it has two binding arms but is monospecific because it binds to one
type of antigen or epitope. A monospecific, multivalent fusion
protein has more than one binding site for the same antigen or
epitope. For example, a mono-specific diabody is a fusion protein
with two binding sites reactive with the same antigen. The fusion
protein may comprise a multivalent or multispecific combination of
different antibody components or multiple copies of the same
antibody component. For example, the fusion protein of the present
invention may be multispecific, wherein one arm of the fusion
protein (e.g., scFv or Fab) is a Class III, anti-CEA mAb that
targets CD66e and another arm of the fusion protein is from another
CEA crossreactive antibody that targets CD66a-d.
[0066] A preferred bispecific fusion protein according to the
invention has an arm against a Class III CEA epitope, and a second
arm against CD66a-d epitopes (Class II) expressed on granulocytes.
In these embodiments, the CD66a-d binding portion should not be
able to fix complement or bind to Fc-receptors to effect ADCC
(which would result in release of cytokines from granulocytes).
Though complement fixation and effecting ADCC are preferred
properties for the naked therapy embodiments of the present
invention, they should be avoided in the context of the instant
embodiments relating to bispecific fusion proteins. On normal colon
cells NCA-50/90 and CEA are both expressed, but they are restricted
to the apical face of the normal epithelial cell, and this face is
presented only to the colon lumen, and not accessible to injected
antibody. CEA released from these normal cells as CEA, or bound to
dead normal cells is eliminated in the feces. This polarization is
lost when a colon cancer develops, and both NCA-50/90 and CEA are
then expressed on the cancer cell membrane that is invading the
underlying normal basement membrane which anchors the normal
epithelial cells. A bispecific antibody such as hMN3/hMN14 is
expected to react with both CEA and NCA-50/90 on these invading
cells. Furthermore, as NCA50/90 is present on granulocytes this
bispecific is expected to direct granulocytes to kill the invading
colon cancer cells. An even more preferred construct according to
this embodiment is a bispecific, trivalent protein with one arm
reactive with NCA50/90 and two arms reactive with only CEA. Another
embodiment would be a bispecific protein with two arms that bind to
NCA50/90.
[0067] A preferred fusion protein also reactive with granulocytes
would be a diabody, having one arm against NCA-50/90 (example
hMN-3), and one arm against a Class III epitope on CEA (hMN14).
These fusion proteins do not have an Fc-domain so they will not
activate cytokine release from granulocytes. An even more preferred
fusion protein would be a triabody with one hMN-3 arm and two hMN14
arms. The construction of such diabodies and triabodies is
disclosed in U.S. application Ser. Nos. 60/404,919 (filed Aug. 22,
2002), 60/345,641 (filed Jan. 8, 2002), 60/328,835 (filed Oct. 15,
2001), and 60/341,881 (filed Dec. 21, 2001).
[0068] Any kind of multispecific antibody made with MAbs of the
hMN14/NP-3 specificities are also preferred and can have an
Fc-domain able to fix complement/activate ADCC. For example, a
hMN14-IgG1/[NP-3-scFv]2 fusion protein could be used; the making of
which is taught in U.S. application Ser. No. 09/337,756.
[0069] Yet another preferred type of multispecific antibody
according to the present invention is an hMN-3 MAb which has an
Fc-domain lacking the ability to fix complement/effect-ADCC.
[0070] The fusion protein may additionally comprise a therapeutic
agent. For example, where at least one of the antibodies or
fragments thereof, such as the Class III, anti-CEA MAb that targets
CD66e or its scFv or Fab may be conjugated to cytokines, such as
interferon or a colony-stimulating factor, such as GM-CSF or GCSF
or an interleukin, all of which are described herein.
[0071] An immunomodulator is a therapeutic agent as defined in the
present invention that when present, alters, suppresses or
stimulates the body's immune system. Typically, the immunomodulator
useful in the present invention stimulates immune cells to
proliferate or become activated in an immune response cascade, such
as macrophages, B-cells, and/or T-cells. An example of an
immunomodulator as described herein is a cytokine, which is a
soluble small protein of approximately 5-20 kDa that are released
by one cell population (e.g., primed T-lymphocytes) on contact with
specific antigens, and which act as intercellular mediators between
cells. As the skilled artisan will understand, examples of
cytokines include lymphokines, monokines, interleukins, and several
related signalling molecules, such as tumor necrosis factor (TNF)
and interferons. Chemokines are a subset of cytokines. Certain
interleukins and interferons are examples of cytokines that
stimulate T cell or other immune cell proliferation.
Preparation of Monoclonal Antibodies, Including Chimeric, Humanized
and Human Antibodies
[0072] Monoclonal antibodies (MAbs) are a homogeneous population of
antibodies to a particular antigen and the antibody comprises only
one type of antigen binding site and binds to only one epitope on
an antigenic determinant. 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.
[0073] 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).
[0074] Abs to peptide backbones are generated by well-known methods
for Ab production. For example, injection of an immunogen, such as
(peptide) N-KLH, wherein KLH is keyhole limpet hemocyanin, and
n=1-30, in complete Freund's adjuvant, followed by two subsequent
injections of the same immunogen suspended in incomplete Freund's
adjuvant into immunocompetent animals. The animals are given a
final i.v. boost of antigen, followed by spleen cell harvesting
three days later. Harvested spleen cells are then fused with
Sp2/0-Ag14 myeloma cells and culture supernatants of the resulting
clones analyzed for anti-peptide reactivity using a direct binding
ELISA. Fine specificity of generated Abs can be analyzed for by
using peptide fragments of the original immunogen. These fragments
can be prepared readily using an automated peptide synthesizer. For
Ab production, enzyme-deficient hybridomas are isolated to enable
selection of fused cell lines. This technique also can be used to
raise antibodies to one or more of the chelates comprising the
linker, e.g., In(III)-DTPA chelates. Monoclonal mouse antibodies to
an In(III)-di-DTPA are known (U.S. Pat. No. 5,256,395 to
Barbet).
[0075] Another method for producing antibodies is by production in
the milk of transgenic livestock. See, e.g., Colman, A., Biochem.
Soc. Symp., 63: 141-147, 1998; U.S. Pat. No. 5,827,690, both of
which are incorporated in their entirety by reference. Two DNA
constructs are prepared which contain, respectively, DNA segments
encoding paired immunoglobulin heavy and light chains. The DNA
segments are cloned into expression vectors that contain a promoter
sequence that is preferentially expressed in mammary epithelial
cells. Examples include, but are not limited to, promoters from
rabbit, cow and sheep casein genes, the cow .alpha.lactoglobulin
gene, the sheep .beta.-lactoglobulin gene and the mouse whey acid
protein gene. Preferably, the inserted fragment is flanked on its
3' side by cognate genomic sequences from a mammary-specific gene.
This provides a polyadenylation site and transcript-stabilizing
sequences. The expression cassettes are coinjected into the
pronuclei of fertilized, mammalian eggs, which are then implanted
into the uterus of a recipient female and allowed to gestate. After
birth, the progeny are screened for the presence of both transgenes
by Southern analysis. In order for the antibody to be present, both
heavy and light chain genes must be expressed concurrently in the
same cell. Milk from transgenic females is analyzed for the
presence and functionality of the antibody or antibody fragment
using standard immunological methods known in the art. The antibody
can be purified from the milk using standard methods known in the
art.
[0076] After the initial raising of antibodies to the immunogen,
the variable genes of the monoclonal antibodies can be cloned from
the hybridoma cells, sequenced and subsequently prepared by
recombinant techniques. 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.
Humanization and chimerization of murine antibodies and antibody
fragments are well known to those skilled in the art. A chimeric
antibody is a recombinant protein that contains the variable
domains including the CDRs derived from one species of animal, such
as a rodent antibody, while the remainder of the antibody molecule;
i.e., the constant domains, is derived from a human antibody. The
use of antibody components derived from humanized and chimerized
monoclonal antibodies alleviates potential problems associated with
the immunogenicity of murine constant regions. Techniques for
constructing chimeric antibodies are well known to those of skill
in the art. As an example, Leung et al., Hybridoma 13:469 (1994),
describe how they produced an LL2 chimera by combining DNA
sequences encoding the V.sub.K and V.sub.H domains of LL2
monoclonal antibody, an anti-CD22 antibody, with respective human K
and IgG.sub.1 constant region domains.
[0077] A chimeric monoclonal antibody (MAb) can also be humanized
by replacing the sequences of the murine FR in the variable domains
of the chimeric MAb with one or more different human FR.
Specifically, 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. As simply
transferring mouse CDRs into human FRs often results in a reduction
or even loss of antibody affinity, additional modification might be
required in order to restore the original affinity of the murine
antibody. This can be accomplished by the replacement of one or
more human residues in the FR regions with their murine
counterparts to obtain an antibody that possesses good binding
affinity to its epitope. See, for example, Tempest et al.,
Biotechnology 9:266 (1991) and Verhoeyen et al., Science 239: 1534
(1988).
[0078] In a preferred embodiment, some human residues in the
framework regions of the humanized anti-CEA antibody or fragments
thereof are replaced by their murine counterparts. Additionally,
knowing that chimeric anti-CEA exhibits a binding affinity
comparable to that of its murine counterpart, defective designs, if
any, in the original version of the humanized anti-CEA MAb can be
identified by mixing and matching the light and heavy chains of the
chimeric anti-CEA to those of the humanized version. Preferably,
the humanized anti-CEA antibody is a humanized MN-14 antibody, and
its preparation and sequences are disclosed in U.S. Pat. No.
5,874,540, which is incorporated in its entirety by reference.
Although the two human antibodies are REI and NEWM are the
preferred antibodies for preparing both humanized and chimeric
MN-14 antibodies, a combination of framework sequences from 2 or
more different human antibodies can be used for V.sub.H and
V.sub.K. The production of 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. Further, the affinity of humanized, chimeric and human
MAbs to a specific epitope can be increased by mutagenesis of the
CDRs, so that a lower dose of antibody may be as effective as a
higher dose of a lower affinity MAb prior to mutagenesis. See for
example, WO0029584A1.
[0079] In another embodiment, an antibody of the present invention
is a human Class III anti-CEA monoclonal antibody. The anti-CEA
MAb, or another human antibody, can be obtained from a transgenic
non-human animal. See, e.g., Mendez et al., Nature Genetics, 15:
146-156 (1997) and U.S. Pat. No. 5,633,425, which are incorporated
in their entirety by reference. For example, a human antibody can
be recovered from a transgenic mouse possessing human
immunoglobulin loci. Preferably, the anti-CEA antibody is an MN-14
antibody. The mouse humoral immune system is humanized by
inactivating the endogenous immunoglobulin genes and introducing
human immunoglobulin loci. The human immunoglobulin loci are
exceedingly complex and comprise a large number of discrete
segments which together occupy almost 0.2% of the human genome. To
ensure that transgenic mice are capable of producing adequate
repertoires of antibodies, large portions of human heavy- and
light-chain loci must be introduced into the mouse genome. This is
accomplished in a stepwise process beginning with the formation of
yeast artificial chromosomes (YACs) containing either human heavy-
or light-chain immunoglobulin loci in germline configuration. Since
each insert is approximately 1 Mb in size, YAC construction
requires homologous recombination of overlapping fragments of the
immunoglobulin loci. The two YACs, one containing the heavy-chain
loci and one containing the light-chain loci, are introduced
separately into mice via fusion of YAC containing yeast
spheroblasts with mouse embryonic stem cells. Embryonic stem cell
clones are then microinjected into mouse blastocysts. Resulting
chimeric males are screened for their ability to transmit the Y AC
through their germline and are bred with mice deficient in murine
antibody production. Breeding the two transgenic strains, one
containing the human heavy-chain loci and the other containing the
human light-chain loci, creates progeny which produce human
antibodies in response to immunization.
[0080] Unrearranged human immunoglobulin genes also can be
introduced into mouse embryonic stem cells via microcell-mediated
chromosome transfer (MMCT). See, e.g., Tomizuka et al., Nature
Genetics, 16: 133 (1997). In this methodology microcells containing
human chromosomes are fused with mouse embryonic stem cells.
Transferred chromosomes are stably retained, and adult chimeras
exhibit proper tissue-specific expression.
[0081] As an alternative, an antibody or antibody fragment of the
present invention may be derived from human antibody fragments
isolated from a combinatorial immunoglobulin library. See, e.g.,
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. Many of the difficulties associated
with generating monoclonal antibodies by B-cell immortalization can
be overcome by engineering and expressing antibody fragments in E.
coli, using phage display. To ensure the recovery of high affinity,
monoclonal antibodies a combinatorial immunoglobulin library must
contain a large repertoire size. A typical strategy utilizes mRNA
obtained from lymphocytes or spleen cells of immunized mice to
synthesize cDNA using reverse transcriptase. The heavy- and
light-chain genes are amplified separately by PCR and ligated into
phage cloning vectors. Two different libraries are produced, one
containing the heavy-chain genes and one containing the light-chain
genes. Phage DNA is isolated from each library, and the heavy- and
light-chain sequences are ligated together and packaged to form a
combinatorial library. Each phage contains a random pair of heavy-
and light-chain cDNAs and upon infection of E. coli directs the
expression of the antibody chains in infected cells. To identify an
antibody that recognizes the antigen of interest, the phage library
is plated, and the antibody molecules present in the plaques are
transferred to filters. The filters are incubated with
radioactively labeled antigen and then washed to remove excess
unbound ligand. A radioactive spot on the autoradiogram identifies
a plaque that contains an antibody that binds the antigen. 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.).
[0082] In one embodiment, the antibodies of the present invention
are produced as described in Hansen et al., U.S. Pat. No.
5,874,540; Hansen et al., Cancer, 71:3478 (1993); Primus et al.,
U.S. Pat. No. 4,818,709, and Shively et al., U.S. Pat. No.
5,081,235, which have been incorporated by reference in their
entirety.
Production of Antibody Fragments.
[0083] The present invention contemplates the use of fragments of a
Class III anti-CEA antibody, preferably a MN-14 antibody. The Class
III anti-CEA antibody or fragment thereof of the present invention
does not bind granulocytes or CD66a-d. Antibody fragments which
recognize specific epitopes can be generated by known techniques.
For example, antibody fragments can be prepared by proteolytic
hydrolysis of an antibody or by expression in E. coli of the DNA
coding for the fragment. The antibody fragments are antigen binding
portions of an antibody, such as F(ab').sub.2, Fab', Fab, Fv, scFv
and the like, and can be obtained by pepsin or papain digestion of
whole antibodies by conventional methods.
[0084] For example, an antibody fragment can be produced by
enzymatic cleavage of antibodies with pepsin to provide a 100 Kd
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 50 Kd Fab' monovalent fragments.
Alternatively, an enzymatic cleavage using papain 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.
[0085] 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.
[0086] 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. U.S.A. 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, Crit. Rev. Biotech.
12:437 (1992);
[0087] 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 that 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 (1988), Ladner et al., U.S. Pat. No.
4,946,778; Pack et al., Bio Technology 11: 1271 (1993) and Sandhu,
supra.
[0088] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). A CDR is a
segment of the variable region of an antibody that is complementary
in structure to the epitope to which the antibody binds and is more
variable than the rest of the variable region. Accordingly, a CDR
is sometimes referred to as hypervariable region. A variable region
comprises three CDRs. CDR peptides 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 at., Methods: A Companion to
Methods in Enzymology 2: 106 (1991); Courtenay-Luck, "Genetic
Manipulation of Monoclonal Antibodies," in MONOCLONAL ANTIBODIES:
PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al.
(eds.), pages 166-179 (Cambridge University Press 1995); and Ward
et al., "Genetic Manipulation and Expression of Antibodies," in
MONOCLONAL ANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al.,
(eds.), pages 137-185 (Wiley-Liss, Inc. 1995).
[0089] 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.
Humanized, Chimeric and Human Anti-CEA Antibodies for
Treatment.
[0090] Described in the present invention are compositions and
methods using murine, chimeric, humanized and human Class III
anti-CEA antibodies and fragments thereof for treatment.
Preferably, the Class III anti-CEA antibody or fragment thereof is
a MN-14 antibody or fragment thereof. The antibodies of the present
invention can be used to treat medullary thyroid carcinoma (MTC),
as well as non-MTC CEA-expressing carcinomas. Exemplary non-MTC CEA
expressing carcinomas include colorectal cancer, pancreatic cancer,
hepatocellular carcinoma, gastric cancer, lung cancer, head- and
neck cancers, urinary bladder cancer, uterine cancer, breast
cancer, and ovarian cancer.
Compositions
[0091] Contemplated herein is a composition comprising at least one
Class III anti-CEA monoclonal antibody (MAb) or fragment thereof
and at least one therapeutic agent, which are not conjugated to
each other, and thus are present in the composition as unconjugated
forms of each of the components. In compositions comprising more
than one antibody or antibody fragments, such as a second Class III
anti-CEA antibody, the second antibody is non-blocking (i.e., does
not block binding of the first Class III anti-CEA antibody or
antibody fragment).
[0092] In one embodiment, the Class III anti-CEA monoclonal
antibody or fragment thereof is humanized, chimeric, or fully
human, wherein the humanized, chimeric, or fully human MAb retains
substantially the Class III anti-CEA binding specificity of a
murine Class III anti-CEA MAb.
[0093] In a preferred embodiment, the Class III anti-CEA monoclonal
antibody or fragment thereof is a MN-14 antibody or fragment
thereof. Preferably, the MN-14 monoclonal antibody or fragment
thereof comprises the complementarity determining regions (CDRs) of
a murine MN-14 monoclonal antibody, wherein the CDRs of the light
chain variable region of said MN-14 antibody comprises CDR1
comprising the amino acid sequence KASQDVGTSVA (SEQ ID NO:20); CDR2
comprising the amino acid sequence WTSTRHT (SEQ ID NO:21); and CDR3
comprising the amino acid sequence QQYSLYRS (SEQ ID NO:22); and the
CDRs of the heavy chain variable region of said Class III anti-CEA
antibody comprises CDR1 comprising TYWMS (SEQ ID NO:23); CDR2
comprising EIHPDSSTINYAPSLKD (SEQ ID NO:24); and CDR3 comprising
LYFGFPWFAY (SEQ ID NO:25). Also preferred, the MN-14 monoclonal
antibody reacts with CEA and is unreactive with normal
cross-reactive antigen (NCA) and meconium antigen (MA). However,
antibodies against these cross-reactive determinants may be used in
combination therapy with CEA-specific antibodies, such as combined
with the MN-14 monoclonal antibody.
[0094] In another embodiment of the present invention, the MN-14
monoclonal antibody or fragment thereof is a humanized or fully
human MN-14 antibody or fragment thereof. The framework regions
(FRs) of the light and heavy chain variable regions of the
humanized MN-14 antibody or fragment thereof preferably comprise at
least one amino acid substituted from the corresponding FRs of a
murine MN-14 monoclonal antibody. Still preferred, the humanized
MN-14 antibody or fragment thereof comprises at least one amino
acid from the corresponding FR of the murine MN-14 antibody
selected from the group consisting of amino acid residue 24, 28,
30, 48, 49, 74 and 94 of the murine heavy chain variable region
(KLHuVhAIGA) of FIG. 14A-C as noted above. The amino acid sequence
of a preferred humanized heavy chain variable region is also set
forth in Hansen et al., U.S. Pat. No. 5,874,540, which is
incorporated by reference in its entirety. Also preferred, the
humanized heavy chain variable region comprises the amino acid
sequence set forth in FIGS. 14A-C, designated as KLHuVhAIG and
KLHuVhAIGAY. In another embodiment, the humanized MN-14 antibody or
fragment thereof comprises at least one amino acid from the
corresponding FR of the murine MN-14 light chain variable region.
Most preferably, the humanized MN-14 antibody or fragment thereof
comprises the light chain variable region of FIG. 13A or FIG. 22A
or FIG. 23A. Another embodiment of the present invention is a
composition comprising a chimeric MN-14 monoclonal antibody or
fragment thereof and at least one therapeutic agent, which are not
conjugated to each other, and thus are present in the composition
as unconjugated forms of each of the components. Preferably, the
chimeric MN-14 antibody or fragment thereof comprises the CDRs of
the murine MN14 light chain variable region set forth in FIG. 13A
or FIG. 22A or FIG. 23A and the CDRs of the murine MN14 heavy chain
variable region as set forth in FIGS. 14A-C or FIG. 22B or FIG.
23B.
[0095] Also described herein is a composition comprising a naked
murine, humanized, chimeric or human Class III anti-CEA antibody or
fragment thereof and a therapeutic agent, and a second naked or
conjugated Class III anti-CEA antibody or antibody fragment
thereof, that is non-blocking, i.e., does not block binding of the
first Class III anti-CEA antibody or fragment thereof, and
formulated in a pharmaceutically acceptable vehicle. In other
words, both Class III anti-CEA antibodies or fragments thereof are
non-blocking to each other, thus, allowing both antibodies or
fragments thereof to bind to CEA (CD66e). Additionally, the Class
III CEA antibody or antibody fragment of the present invention, as
well as those for use in combination therapy, do not bind
granulocytes or CD66a-d. Other Class III antibodies suitable for
combination therapy as a naked antibody or as a component of an
immunoconjugate, with the naked Class III anti-CEA antibody of
antibody fragment of the present invention include the non-blocking
antibodies or fragments thereof described in Kuroki et al., JP J.
Cancer Res., 78(4):386 (1987) and Hammarstrom (Cancer Res.
52(8):2329 (1992), that also do not bind granulocytes or
CD66a-d.
[0096] Additionally, other anti-CEA antibodies, such as Class II or
Class I anti-CEA antibodies, can be used in combination with the
Class III anti-CEA antibody of the present invention, in either a
naked or conjugated form. Such Class II antibodies or antibody
fragments that can be used for combination therapy are nonblocking
and do not bind granulocytes or CD66a-d but are reactive with
meconium antigen (MA) and CEA. For example, one or more chimeric or
humanized Class II anti-CEA antibody or fragment thereof, such as
MN-6 or NP-3, may be combined with a Class III anti-CEA antibody or
fragment thereof of the present invention. These two antibodies do
not react with CD66a-d or with granulocytes (Hansen et al., Cancer
1993 Jun. 1; 71(11):3478-85). A number of publications disclose
MAbs that recognize CEA and different members of the CEA gene
family, such as Thompson et al., J. Clin. Lab. Anal. 5:344 (1991);
Kuroki et al.: J. Bioi. Chem. 266:11810 (1991); Nagel et al., Eur.
J. Biochem. 214:27 (1993); Skubitz et al., J. Immunol. 155:5382
(1995); Skubitz et al., J. Leukoc. Bioi. 60:106 (1996); and Chen et
al., Proc. Natl. Acad. Sci. 93: 14851 (1996).
[0097] Moreover, the second antibody or antibody fragment is either
unconjugated (naked) or conjugated to at least one therapeutic
agent (immunocougate). Immunoconjugates can be prepared by
indirectly conjugating a therapeutic agent to an antibody
component. General techniques are described in Shih et al., Int. J.
Cancer, 41 :832 (1988); Shih et al., Int. J. Cancer, 46:1101
(1990); and Shih et ai., 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 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. Preferably, the anti-CEA antibody or fragment
thereof in the composition for treatment is a MN-14 antibody or
fragment thereof. More preferred, the MN-14 antibody or fragment
thereof is humanized.
[0098] Also contemplated in the present invention is a composition
comprising a naked humanized, chimeric, murine or human Class III
anti-CEA antibody or fragment thereof and a therapeutic agent, and
a conjugated or unconjugated second antibody or antibody fragment
thereof. In one embodiment, the second antibody or fragment thereof
is unconjugated (naked) or conjugated to at least one therapeutic
agent. Non Class I, Class II or Class III anti-CEA antibodies and
fragments thereof that are suitable for combination therapy
include, but are not limited to, carcinoma-associated antibodies
and fragments thereof. Examples of carcinoma associated antibodies
and antibody fragments bind EGP-1, EGP-2 (e.g., 17-1A), MUC-I,
MUC-2, MUC-3, MUC-4, PAM-4, KC4, TAG-72, EGFR, HER2/neu, BrE3,
Le-Y, A3, A33, Ep-CAM, AFP, Tn, Thomson-Friedenreich antigens,
tumor necrosis antigens, VEGF, PlGF or other tumor angiogenesis
antigens, Ga 733, IL-6, insulin-like growth factor-1, tenascin,
fibronectin or a combination thereof. As discussed supra,
non-blocking Class II and Class III anti-CEA MAbs that do not bind
CD66a-d or granulocytes or alternatively, Class II anti-CEA MAbs
that do bind CD66a, b and d or Class I anti-CEA MAbs that bind
CD66a, b and d, as well as CD66c, may also be used in combination
with Class III CEA antibodies. Other antibodies and antibody
fragments suitable for combination therapy also include those
targeted against oncogene markers or products, or antibodies
against tumorvasculature markers, such as the angiogenesis factor,
Placental Growth Factor (PlGF), and antibodies against certain
immune response modulators, such as antibodies to CD40.
Methods
[0099] Also described in the present invention are methods for
treating medullary thyroid carcinoma and non-medullary thyroid
carcinomas. Non-medullary thyroid carcinomas include colorectal
cancer and any other CEA expressing tumor, such as pancreatic
cancer, breast cancer, hepatocellular carcinoma, ovarian cancer,
certain kinds of lung, head-and-neck, endometrial, bladder, and
liver cancers that express variable quantities of CEA. The CEA
levels in these types of cancers are much lower than present in
medullary thyroid carcinomas but all that is necessary is that the
CEA levels be sufficiently high so that the Class III anti-CEA
therapy provides an effective treatment. Normal colon mucosa has
about 100-500 ng/gram but carcinomas expressing CEA at levels of
about 5 mcg/gram of tissue are suitable for treatment with the
methods described in the instant invention.
[0100] For example, contemplated herein is a method for treating
medullary thyroid carcinoma or non-medullary thyroid carcinoma
comprising administering to a subject, either concurrently or
sequentially, a therapeutically effective amount of a Class III
anti-CEA monoclonal antibody or fragment thereof and at least one
therapeutic agent, and optionally formulated in a pharmaceutically
acceptable vehicle. Preferably, the Class III anti-CEA monoclonal
antibody or fragment thereof is chimeric, murine, humanized or
human, wherein the chimeric, humanized, murine, or human Class III
anti-CEA MAb retains substantially the Class III anti-CEA binding
specificity of the murine MAb. More preferably, the Class III
anti-CEA antibody is humanized, and most preferably, the humanized
MN-14 monoclonal antibody, as described herein and in U.S. Pat. No.
5,874,540. Preferably the therapeutic agent is a cytotoxic agent,
more preferably an alkylating agent, and most preferably,
dacarbazine (DTIC). But in another embodiment, the therapeutic
agent may also not be DTIC. Other classes of anti-cancer cytostatic
and cytotoxic agents, such as 5-fluorouracil, CPT-11 (which is also
known as irinotecan and camptosar) and oxaliplatin can also be used
in combinations with these antibodies, especially in the therapy of
colorectal cancers. In other cancer types, cancer drugs that are
known to be effective are also good candidates for combining with
the antibody therapies proposed herein.
[0101] Also contemplated herein is a method for treating medullary
thyroid carcinoma and non-medullary thyroid carcinoma comprising
administering to a subject, either concurrently or sequentially, a
therapeutically effective amount of a first Class III anti-CEA
monoclonal antibody or fragment thereof and at least one
therapeutic agent, and a naked or conjugated second humanized,
chimeric, human or murine monoclonal antibody or fragment thereof,
and optionally formulated in a pharmaceutically acceptable vehicle.
Preferably, the first Class III anti-CEA MAb is a humanized MN-14
antibody or fragment thereof. In one embodiment, the second
antibody or fragment thereof is a carcinoma-associated antibody or
fragment thereof selected from the group consisting of a monoclonal
antibody or fragment thereof reactive with TAG-72, EGFR, HER2/neu,
MUC1, MUC2, MUC3, MUC4, EGP-1, EGP-2, AFP, Tn, IL-6, insulin growth
factor-1, or another such tumor-associated antigen, as described
above. In another embodiment, the second antibody or fragment
thereof can be a different Class III anti-CEA antibody or fragment
thereof that is non-blocking and does not bind granulocytes or
CD66a-d.
[0102] In another embodiment, the second anti-CEA antibody is a
Class II antibody or fragment thereof, such as those described in
Hammarstrom and Kuroki, provided that they do not bind granulocytes
or CD66a-d. In another embodiment, this antibody includes Class I
Mabs or fragments thereof, that react with CD66a, b, or d as well
as CD66c. The antibodies and fragments thereof may be administered
either concurrently or sequentially with each other or the
therapeutic agent. In one embodiment, the second antibody or
fragment thereof is either naked or conjugated to a therapeutic
agent.
[0103] Accordingly, the present invention contemplates the
administration of naked murine, humanized, chimeric and human
anti-CEA antibodies and fragments thereof sequentially or
concurrently with one or more therapeutic agents, or administered
as a multimodal therapy. A Class III, anti-CEA antibody is
preferred but any anti-CEA antibody that targets tumor cells is
useful in the present invention. A naked Class III anti-CEA
antibody as described herein can significantly increase the
chemosensitivity of cancer cells to one or more therapeutic agents.
For example, treatment of colon cancer cells with a naked Class
III, anti-CEA antibody, MN-14 as described herein, either before or
concurrently with a therapeutic agent, such as CPT-11,
5'-fluorouracil (5-FU) or oxaliplatin, improves a cell's response
to a therapeutic agent, such as a cytotoxic drug. Further, these
therapeutic methods of treatment with a naked Class III, anti-CEA
antibody alone or in combination with a therapeutic agent can be
further enhanced by administering an immunomodulator as described
herein, prior to the administration of the naked antibody or the
administration of the naked antibody and at least one of the
therapeutic agents.
[0104] Multimodal therapies of the present invention include
immunotherapy with a Class III anti-CEA antibody or fragment
thereof, and a therapeutic agent, supplemented with administration
of an unconjugated or conjugated antibody, unconjugated or
conjugated fusion protein, or fragment thereof. For example, an
unconjugated humanized, chimeric, murine or human MN-14 MAb or
fragment thereof may be combined with another naked humanized,
murine, chimeric or human Class III anti-CEA antibody (such as an
antibody against a different epitope on CEA and also does not bind
granulocytes or CD66a-d), or a humanized, chimeric, murine or human
Class III anti-CEA antibody immunoconjugate conjugated to a
radioisotope, chemotherapeutic agent, cytokine, enzyme,
enzyme-inhibitor, hormone or hormone antagonist, metal, toxin,
antisense oligonucleotide (e.g., anti-bcl-2), or a combination
thereof. A naked Class III anti-CEA antibody or fragment thereof
may also be combined with a conjugated or unconjugated fusion
protein of a murine, humanized, chimeric or human Class III
anti-CEA antibody. However, the Class III anti-CEA antibodies for
combination therapy are non-blocking to each other and unable to
bind granulocytes or CD66a-d. Preferably, the naked Class III
anti-CEA antibody is administered sequentially or concurrently with
the second naked or conjugated antibody, fusion protein, or
fragment thereof. Also preferred, one of the antibodies or antibody
fragments for use in combination therapy is a naked humanized MN-14
antibody or fragment thereof. Additionally, the second antibody
used as a naked or conjugated antibody, fusion protein, or fragment
thereof, may be a human, humanized, chimeric or murine Class II CEA
antibody or fragment thereof that is non-blocking and does not bind
granulocytes or CD66a-d. A preferred combination of antibodies
according to this embodiment would include a naked cross reactive
anti-CD66a-d antibody which lacks an effector function, which does
not activate complement and does not induce cytokine release. In
addition, a cross reactive anti-CD66a-d Fab' or even a F(ab').sub.2
would likely not damage granulocytes and could be used with a Class
III anti-CEA MAb or a Class II anti-CEA MAb of the NP-3 type.
[0105] In the methods described herein, subjects receive at least
one naked Class III anti-CEA antibody or fragment thereof,
administered before, after or in conjunction with a therapeutic
agent. In one embodiment, a Class III anti-CEA antibody is used for
pretreating cells, i.e., administered before a therapeutic agent.
Preferably, the class III anti-CEA antibody is an MN-14 antibody,
such as a humanized MN-14 (hMN-14) that is administered at least
one hour before a therapeutic agent, such as 5-FU or CPT-11.
[0106] Preferably, the therapeutic agent is a drug used in standard
cancer chemotherapy, such as taxane or platinum drugs in ovarian
cancer, fluorouracil, CPT-11, and oxaliplatin drugs in colorectal
cancer, gemcitabine in pancreatic and other cancers, or taxane
derivatives in breast cancers. COX-2 inhibitors represent still
another class of agents that show activity in combination with
typical cytotoxic agents in cancer chemotherapy, and can be used in
this invention in the same way, but combined in addition with CEA
antibodies alone and in combination with other cancer-associated
antibodies. Optionally, these drugs can be used in combination with
radiolabeled antibodies, either CEA antibody conjugates or
radioconjugates with other carcinoma-associated antibodies, of the
kinds described above. Also preferred, the Class III anti-CEA
antibody or fragment thereof is a MN-14 antibody or fragment
thereof. Still preferred, the MN-14 antibody or fragment thereof is
humanized.
[0107] In a preferred embodiment, a naked Class III anti-CEA
antibody or fragment thereof is administered sequentially (either
prior to or after) or concurrently with dacarbazine (DTIC),
doxorubin, cyclophosphamide or vincristine, or any combination of
these. For example, DTIC and cyclophosphamide may be administered
sequentially or concurrently with a naked Class III anti-CEA
antibody or fragment thereof. Preferably, the anti-CEA antibody or
fragment thereof is a humanized MN-14 antibody or fragment thereof.
Similarly, 5-fluorouracil in combination with folinic acid, alone
or in combination irinotecan (CPT-11) or in combination with
oxaliplatin, is a regimen used to treat colorectal cancer. Other
suitable combination chemotherapeutic regimens are well known, such
as with oxaliplatin alone, or in combination with these other
drugs, to those of skill in the art. Accordingly, combination
therapy with any of these chemotherapeutic agents and a naked Class
III anti-CEA antibody or fragment thereof can be used to treat MTC
or non-MTC, depending on the regimen used. In medullary thyroid
carcinoma, still other chemotherapeutic agents may be preferred,
such as one of the alkylating agents (e.g., DTIC), as well as
gemcitabine and other more recent classes of cytotoxic drugs. The
chemotherapeutic drugs and a naked Class III anti-CEA antibody or
fragment thereof, can be administered in any order, or together. In
other words, the antibody and therapeutic agent may be administered
concurrently or sequentially. In a preferred multimodal therapy,
both chemotherapeutic drugs and naked Class III anti-CEA antibodies
or fragments thereof are administered before, after, or
co-administered with a conjugated or unconjugated anti-CEA
antibody, fusion protein, or fragment thereof, according to the
present invention. Preferably, the Class III anti-CEA antibody or
fragment thereof is a humanized MN-14 antibody or fragment
thereof.
[0108] A preferred treatment schedule of multimodal treatment is
administering both hMN-14 and DTIC for 3 days, and administering
only hMN-14 on days 7, 14, 21 and then every 21 days for a
treatment duration of 12 months. The doses of hMN-14 are 0.5-15
mg/kg body weight per infusion, more preferably 2-8, and still more
preferably 3-5 mg/kg per infusion, and the doses of DTIC are as
currently applied at the preferred dose clinically, but could also
be given at two-thirds or less of the maximum preferred dose in
use, thereby decreasing drug-related adverse events. Repeated drug
cycles can be given, such as every 1-6 months, with continuation of
the naked antibody therapy, or with different schedules of
radiolabeled antibody, drug-conjugated antibody, and inclusion of
certain cytokines, such as G-CSF and/or GM-CSF, each dose adjusted
so that toxicity to the patient is not enhanced by the therapeutic
combination. The application of a cytokine growth factor, such as
G-CSF, may enable even higher doses of myelosuppressive agents,
such as radiolabeled antibody or cytotoxic drugs, to be
administered, and these schedules and doses will be adjusted for
the patients individually, depending on their disease status and
prior therapy, all influence bone marrow status and tolerability to
additional cytotoxic therapies. In a preferred embodiment, the
MN-14 antibody or fragment thereof is administered in a dosage of
100-600 milligrams protein per dose per injection. Still preferred,
the MN-14 antibody or fragment thereof is administered in a dosage
of 300-400 milligrams of protein per dose per injection, with
repeated doses preferred. The preferred antibody schedule is
infusing once weekly or even less frequently, such as once every
other week or even every third week, depending on a number of
factors, including the extent of the disease and the amount of CEA
circulating in the patient's blood.
Therapeutic Agents
[0109] The therapeutic agents recited here are those agents that
also are useful for administration separately with a naked
antibody, as described herein. Suitable therapeutic agents can be
selected from the group consisting of a cytotoxic agent, a toxin, a
hormone, a radionuclide, an immunomodulator, a photoactive
therapeutic agent (such as a chromagen or dye), an antisense
oligonucleotide, an immunoconjugate, another naked antibody, a
hormone, or a combination thereof. Therapeutic agents include, for
example, chemotherapeutic drugs such as vinca alkaloids and other
alkaloids, anthracyclines, epidophyllotoxins, taxanes,
antimetabolites, alkylating agents, antibiotics, COX-2 inhibitors,
antimitotics, antiangiogenic and apoptotoic agents, particularly
doxorubicin, methotrexate, taxol, CPT-11, camptothecans, and others
from these and other classes of anticancer agents, and the like.
Other useful cancer chemotherapeutic drugs for the preparation of
immunoconjugates and antibody fusion proteins include nitrogen
mustards, alkyl sulfonates, nitrosoureas, triazenes, oxaliplatin,
folic acid analogs, COX-2 inhibitors, pyrimidine analogs, purine
analogs, platinum coordination complexes, hormones, toxins (e.g.,
RNAse, Pseudomonas exotoxin), and the like. Preferred therapeutic
agents include DTIC, CPT-11, 5-fluorouracil, taxol, oxaliplatin,
doxorubicin, cyclophosphamide and vincristine, or a combination
thereof, depending on the malignancy to be treated. Suitable
chemotherapeutic agents are described in REMINGTON'S PHARMACEUTICAL
SCIENCES, 19th Ed. (Mack Publishing Co. 1995), and in GOODMAN AND
GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed.
(MacMillan Publishing Co. 1985), as well as revised editions of
these publications. Other suitable chemotherapeutic agents, such as
experimental drugs, are known to those of skill in the art.
[0110] A toxin, such as Pseudomonas exotoxin, may also be
administered with a naked Class III anti-CEA antibody or fragment
thereof. Preferably, the Class III anti-CEA antibody or fragment
thereof is a humanized MN-14 antibody or fragment thereof. Other
suitable microbial, plant or animal toxins to be administered
unconjugated to, but before, after, or simultaneously with the
naked Class III anti-CEA antibody or fragment thereof include
ricin, abrin, ribonuclease (RNase), DNase I, Staphylococcal
enterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin
toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin. See, for
example, Pastan et al., Cell 47:641 (1986), and Goldenberg, C A--A
Cancer Journal for Clinicians 44:43 (1994). Additional toxins
suitable for use in the present invention are known to those of
skill in the art and are disclosed in U.S. Pat. No. 6,077,499,
which is incorporated in its entirety by reference. These can be
derived, for example, from animal, plant and microbial sources, or
chemically or recombinantly engineered. The toxin can be a plant,
microbial, or animal toxin, or a synthetic variation thereof.
[0111] An immunomodulator, such as a cytokine may also be
administered unconjugated to the chimeric, murine, humanized or
human Class III anti-CEA antibody or fragment thereof of the
present invention. As used herein, the term "immunomodulator"
includes cytokines, stem cell growth factors, lymphotoxins, such as
tumor necrosis factor (TNF), and hematopoietic factors, such as
interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10,
IL-12, IL-18, and IL-21), 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, IL-12, IL-18, IL-21, interferon-.gamma., TNF-.alpha., and
the like. Therefore, subjects can receive a naked Class III
anti-CEA antibody or fragment thereof and a separately administered
cytokine, which can be administered before, concurrently or after
administration of the naked Class III anti-CEA antibody or fragment
thereof. Since some antigens may also be immunomodulators, CD40
antigen, for example, may also be administered in combination with
a naked Class III anti-CEA antibody or fragment thereof either
together, before or after the naked antibody or antibody
combinations are administered. Additionally, radionuclides suitable
for treating a diseased tissue include, but are not limited to,
.sup.32P, .sup.33P, .sup.47Sc, .sup.59Fe, .sup.64Cu, .sup.67Cu,
.sup.75Se, .sup.77As, .sup.89Sr, .sup.90Y, .sup.99Mo, .sup.105Rh,
.sup.109Pd, .sup.111Ag, .sup.125I, .sup.131I, .sup.142Pr,
.sup.143Pr, .sup.149 Pm, .sup.153Sm, .sup.161Tb, .sup.166Ho,
.sup.169Er, .sup.177Lu, .sup.186Re, .sup.188Re, .sup.189R,
.sup.194Ir, .sup.198Au, .sup.199Au, .sup.211Pb, .sup.212Pb, and
.sup.213Bi, .sup.58Co, .sup.67Ga, .sup.80mBr, .sup.99mTc,
.sup.103mRh, .sup.109Pt, .sup.111In, .sup.119Sb, .sup.161Ho,
.sup.189mOs, .sup.192Ir, .sup.152Dy, .sup.211At, .sup.212Bi
.sup.223Ra, .sup.219Rn, .sup.215Po, .sup.211Bi, .sup.225Ac,
.sup.221Fr, .sup.217At, .sup.213Bi, .sup.88Y and .sup.255Fm.
Preferred radionuclides are .sup.125I, .sup.131I, .sup.90Y,
.sup.177Lu, and .sup.225Ac. Also preferred, the radionuclide has an
energy between 20 and 10,000 keV.
Pharmaceutically Acceptable Vehicles
[0112] The naked murine, humanized, chimeric and human Class III
anti-CEA MAbs to be delivered to a subject can comprise one or more
pharmaceutically acceptable vehicles, one or more additional
ingredients, or some combination of these.
[0113] The unconjugated Class III anti-CEA antibodies and fragments
thereof of the present invention can be formulated according to
known methods to prepare pharmaceutically useful compositions.
Preferably, the Class III anti-CEA antibody or fragment thereof is
a MN-14 antibody or fragment thereof. Sterile phosphate-buffered
saline is one example of a pharmaceutically acceptable vehicle.
Other acceptable vehicles are well-known to those in the art. See,
for example, Ansel et at., PHARMACEUTICAL DOSAGE FORMS AND DRUG
DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro
(ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack
Publishing Company 1990), and revised editions thereof.
[0114] The unconjugated Class III anti-CEA antibody or fragment
thereof of the present invention can be formulated for intravenous
administration via, for example, bolus injection or continuous
infusion. Preferably, the Class III anti-CEA antibody or fragments
is a MN-14 antibody or fragment thereof. Formulations for injection
can be presented in unit dosage form, e.g., in ampules or in
multi-dose containers, with an added preservative. The compositions
can take such forms as suspensions, solutions or emulsions in oily
or aqueous vehicles, and can contain formulatory agents such as
suspending, stabilizing and/or dispersing agents. Alternatively,
the active ingredient can be in powder form for constitution with a
suitable vehicle, e.g., sterile pyrogen-free water, before use.
[0115] Additional pharmaceutical methods may be employed to control
the duration of action of the agent and naked antibody or fragment
thereof. Control release preparations can be prepared through the
use of polymers to complex or adsorb the naked antibody. 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 an
antibody or fragment thereof from such a matrix depends upon the
molecular weight of the immunoconjugate or antibody, the amount of
antibody 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 Ansel et al.,
PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition
(Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'S
PHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company
1990), and revised editions thereof.
[0116] The unconjugated Class III anti-CEA antibody or fragment
thereof may also be administered to a mammal subcutaneously or even
by other parenteral routes. Moreover, the administration may be by
continuous infusion or by single or multiple boluses. In general,
the dosage of an administered naked antibody or fragment thereof
for humans 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 naked antibody or fragment thereof that
is in the range of from about 0.5 mg/kg to 20 mg/kg as a single
intravenous infusion, although a lower or higher dosage also may be
administered as circumstances dictate. This dosage may be repeated
as needed, for example, once per month for 4-10 months, preferably
once per every other week for 16 weeks, and more preferably, once
per week for 8 weeks. It may also be given less frequently, such as
every other week for several months or given more frequently and/or
over a longer duration. The dosage may be given through various
parenteral routes, with appropriate adjustment of the dose and
schedule.
[0117] For purposes of therapy, the Class III anti-CEA antibody or
fragment thereof is administered to a mammal in a therapeutically
effective amount to reduce the size of the tumor as compared to
untreated controls. Preferably, the Class III anti CEA antibody or
fragment thereof is a humanized MN-14 antibody or fragment thereof.
A suitable subject for the present invention is usually a human,
although a non-human mammal or animal subject is also contemplated.
An antibody preparation 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 mammal. In particular, an antibody
preparation of the present invention is physiologically significant
if its presence invokes an antitumor response. A physiologically
significant effect could also be the evocation of a humoral and/or
cellular immune response in the recipient mammal.
[0118] The present invention further includes the following
numbered embodiments:
[0119] 1. A composition comprising at least one anti-CEA monoclonal
antibody (MAb) or fragment thereof and at least one therapeutic
agent. The composition of embodiment 1, wherein said anti-CEA MAb
is a Class I, Class II or Class III anti-CEA mAb, and when said MAb
is a Class I or Class II MAb and is reactive with granulocytes,
said MAb is a monovalent form of the MAb.
[0120] 2. The composition of embodiment 1, wherein said anti-CEA
MAb or fragment thereof is humanized, wherein said humanized MAb
retains substantially the anti-CEA binding specificity of a murine
anti-CEA MAb.
[0121] 3. The composition of embodiment 1, wherein said anti-CEA
MAb or fragment thereof is a chimeric MAb, and wherein said
chimeric MAb retains substantially the anti-CEA binding specificity
of murine anti-CEA MAb.
[0122] 4. The composition of embodiment 1, wherein said anti-CEA
MAb or fragment thereof is a fully human MAb, and wherein said
fully human MAb retains substantially the anti-CEA binding
specificity of murine anti-CEA MAb.
[0123] 5. The composition of embodiment 1, wherein said anti-CEA
monoclonal antibody or fragment thereof is a MN-14 antibody or
fragment thereof.
[0124] 6. The composition of embodiment 5, wherein said MN-14
monoclonal antibody or fragment thereof comprises the
complementarity-determining regions (CDRs) of a murine MN-14
monoclonal antibody, wherein the CDRs of the light chain variable
region of said MN-14 antibody comprises CDR1 comprising the amino
acid sequence KASQDVGTSVA (SEQ ID NO:20); CDR2 comprising the amino
acid sequence WTSTRHT (SEQ ID NO:21); and CDR3 comprising the amino
acid sequence QQYSLYRS (SEQ ID NO:22); and the CDRs of the heavy
chain variable region of said anti-CEA antibody comprises CDR1
comprising TYWMS (SEQ ID NO:23); CDR2 comprising EIHPDSSTINYAPSLKD
(SEQ ID NO:24); and CDR3 comprising LYFGFPWFAY (SEQ ID NO:25).
[0125] 7. The composition of embodiment 1, wherein said anti-CEA
monoclonal antibody reacts with CEA and is unreactive with normal
cross-reactive antigen (NCA) and meconium antigen (MA).
[0126] 8. The composition of embodiment 7, wherein said MN-14
monoclonal antibody or fragment thereof is a humanized MN-14
antibody or fragment thereof.
[0127] 9. The composition of embodiment 7, wherein said MN-14
monoclonal antibody or fragment thereof is a chimeric MN-14
antibody or fragment thereof.
[0128] 10. The composition of embodiment 7, wherein said MN-14
monoclonal antibody or fragment thereof is a fully human MN-14
antibody or fragment thereof.
[0129] 11. The composition of embodiment 8, wherein the framework
regions (FRs) of the light and heavy chain variable regions of said
humanized MN-14 antibody or fragment thereof comprise at least one
amino acid substituted from the corresponding FRs of a murine MN-14
monoclonal antibody.
[0130] 12. The composition of embodiment 11, wherein said humanized
MN-14 antibody or fragment thereof comprises at least one amino
acid from said corresponding FR of said murine MN-14 antibody is
selected from the group consisting of amino acid residue 24, 28,
30, 48, 49, 74 and 94 of the murine heavy chain variable region
(KLHuVhAIGA) of FIG. 14A-C or FIG. 22B (hMn14) or FIG. 23B.
[0131] 13. The composition of embodiment 11, wherein said humanized
MN-14 antibody or fragment thereof comprises at least one amino
acid from said corresponding FR of said murine MN-14 light chain
variable region.
[0132] 14. The composition of embodiment 8, wherein said humanized
MN-14 antibody or fragment thereof comprises the light chain
variable region as set forth in FIG. 13A or FIG. 22A or FIG. 23A,
and the heavy chain variable region set forth in FIG. 14A-C
designated as KLHuVhAIGA or FIG. 22B (hMN-14) or FIG. 23B.
[0133] 15. The composition of embodiment 9, wherein said chimeric
MN-14 antibody or fragment thereof comprises the light chain
variable region as set forth in FIG. 13A designated as murine MN-14
VK and the heavy chain variable region set forth in FIG. 14A-C
designated as murine MN-14 VH.
[0134] 16. The composition of any of embodiments 1-15, wherein said
fragment is selected from the group consisting of F(ab').sub.2,
Fab', Fab, Fv and scFv.
[0135] 17. The composition of any of embodiments 1-15, wherein said
therapeutic agent is selected from the group consisting of a naked
antibody, a cytotoxic agent, a drug, a radionuclide, an
immunomodulator, a photoactive therapeutic agent, an
immunoconjugate, a hormone, a toxin, an antisense oligonucleotide,
or a combination thereof, optionally formulated in a
pharmaceutically acceptable vehicle.
[0136] 18. The composition of embodiment 17, wherein said
combination thereof comprises vincristine, doxorubicin,
oxaliplatin, CPT-11, fluorouracil, DTIC and cyclophosphamide.
[0137] 19. The composition of embodiment 17, wherein said
therapeutic agent is a naked antibody or an immunoconjugate.
[0138] 20. The composition of embodiment 19, wherein said naked
antibody or an antibody portion of said immunoconjugate comprises a
humanized, chimeric, human or murine monoclonal antibody or
fragment thereof selected from the group consisting of a monoclonal
antibody or fragment thereof reactive with EGP-1, EGP-2 (e.g.,
17-1A), MUC-1, MUC-2, MUC-3, MUC-4, PAM-4, KC4, TAG-72, EGFR,
HER2/neu, BrE3, Le-Y, A3, A33, Ep-CAM, AFP, Tn,
Thomson-Friedenreich antigens, tumor necrosis antigens, VEGF, PlGF,
or other tumor angiogenesis antigens, Ga 733, IL-6, insulin-like
growth factor-1, tenascin, fibronectin or a combination
thereof.
[0139] 21. The composition of embodiment 20, wherein said fragment
is selected from the group consisting of F(ab).sub.2, F(ab').sub.2,
Fab', Fab, Fv and scFv.
[0140] 22. The composition of any of embodiments 1-15, wherein said
therapeutic agent is not DTIC.
[0141] 23. A method for treating non-medullary thyroid carcinoma
comprising administering to a subject, either concurrently or
sequentially, a therapeutically effective amount of an anti-CEA
antibody or fragment thereof and at least one therapeutic agent,
and optionally formulated in a pharmaceutically acceptable
vehicle.
[0142] 24. The method of embodiment 23, wherein said anti-CEA MAb
or fragment thereof is humanized, wherein said humanized MAb
retains substantially the anti-CEA binding specificity of a murine
anti-CEA MAb.
[0143] 25. The method of embodiment 23, wherein said anti-CEA MAb
or fragment thereof is a chimeric MAb, and wherein said chimeric
MAb retains substantially the anti-CEA binding specificity of
murine anti-CEA MAb.
[0144] 26. The method of embodiment 23, wherein said anti-CEA
monoclonal antibody or fragment thereof is a MN-14 antibody or
fragment thereof.
[0145] 27. The method of embodiment 23, wherein said MN-14
monoclonal antibody or fragment thereof comprises the
complementarity-determining regions (CDRs) of a murine MN-14
monoclonal antibody, wherein the CDRs of the light chain variable
region of said MN-14 antibody comprises CDR1 comprising the amino
acid sequence KASQDVGTSVA (SEQ ID NO:20); CDR2 comprising the amino
acid sequence WTSTRHT (SEQ ID NO:21); and CDR3 comprising the amino
acid sequence QQYSLYRS (SEQ ID NO:22); and the CDRs of the heavy
chain variable region of said anti-CEA antibody comprises CDR1
comprising TYWMS (SEQ ID NO:23); CDR2 comprising EIHPDSSTINYAPSLKD
(SEQ ID NO:24); and CDR3 comprising LYFGFPWFAY (SEQ ID NO:25).
[0146] 28. The method of embodiment 27, wherein said MN-14
monoclonal antibody reacts with CEA and is unreactive with normal
cross-reactive antigen (NCA) and meconium antigen (MA).
[0147] 29. The method of embodiments 28, wherein said MN-14
monoclonal antibody or fragment thereof is a humanized MN-14
antibody or fragment thereof.
[0148] 30. The method of embodiments 28, wherein said MN-14
monoclonal antibody or fragment thereof is a chimeric MN-14
antibody or fragment thereof.
[0149] 31. The method of embodiments 28, wherein said MN-14
monoclonal antibody or fragment thereof is a fully human MN-14
antibody or fragment thereof.
[0150] 32. The method of embodiment 29, wherein the framework
regions (FRs) of the light and heavy chain variable regions of said
humanized MN-14 antibody or fragment thereof comprise at least one
amino acid substituted from the corresponding FRs of a murine MN-14
monoclonal antibody.
[0151] 33. The method of embodiment 32, wherein said humanized
MN-14 antibody or fragment thereof comprising at least one amino
acid from said corresponding FR of said murine MN-14 antibody is
selected from the group consisting of amino acid residue 24, 28,
30, 48, 49, 74 and 94 of the murine heavy chain variable region of
FIG. 14A-C designated as KLHuVhAIGA or 22B (hMN-14) or 23B.
[0152] 34. The method of embodiment 32, wherein said humanized
MN-14 antibody or fragment thereof comprising at least one amino
acid from said corresponding FR of said murine MN-14 light chain
variable region.
[0153] 35. The method of embodiment 32, wherein said humanized
MN-14 antibody or fragment thereof comprises the light chain
variable region as set forth in FIG. 13A or FIG. 22A (hMN-14) or
FIG. 23A and the heavy chain variable region set forth in FIG.
14A-C designated as KLHuVhAIGA or FIG. 22B (hMN-14) or FIG.
23B.
[0154] 36. The method of any of embodiments 23-35, wherein said
fragment is selected from the group consisting of F(ab).sub.2,
F(ab).sub.2, Fab', Fab, Fv and sFv.
[0155] 37. The method of any of embodiments 23-35, wherein said
therapeutic agent is selected from the group consisting of
humanized, chimeric, human or murine monoclonal antibody or
fragment thereof selected from the group consisting of a Class I
anti-CEA monoclonal antibody, Class II anti-CEA monoclonal
antibody, Class III anti-CEA monoclonal antibody, and a fragment
thereof, and is administered either concurrently or sequentially in
a therapeutically effective amount.
[0156] 38. The method of embodiment 37, wherein said antibody or
fragment thereof is either naked or conjugated to another
therapeutic agent.
[0157] 39. The method of any of embodiments 23-35, wherein said
therapeutic agent is selected from the group consisting of a naked
antibody, cytotoxic agent, a drug, a radionuclide, an
immunomodulator, a photoactive therapeutic agent, an antisense
oligonucleotide, an immunoconjugate of a CEA or non-CEA antibody, a
hormone, or a combination thereof, optionally formulated in a
pharmaceutically acceptable vehicle.
[0158] 40. The method of embodiment 39, wherein said therapeutic
agent is selected from the group consisting of a humanized,
chimeric, human or murine monoclonal antibody or fragment thereof
reactive with EGP-1, EGP-2 (e.g., 17-1A), IL-6, MUC-1, MUC-2,
MUC-3, MUC-4, PAM-4, KC4, TAG-72, EGFR, EGP-2, HER2/neu, BrE3,
Le-Y, A3, A33, Ep-CAM, AFP, Tn, Thomson-Friedenreich antigens,
tumor necrosis antigens, VEGF, PlGF or other tumor angiogenesis
antigens, Ga 733, IL-6, insulin-like growth factor-1, and a
combination thereof, and is administered to said subject either
concurrently or sequentially in a therapeutically effective
amount.
[0159] 41. The method of embodiment 40, wherein said antibody or
fragment thereof is either naked or conjugated to another
therapeutic agent.
[0160] 42. The method of any of embodiments 23-35, wherein said
therapeutic agent is not DTIC.
[0161] 43. The method of embodiment 39, wherein said cytotoxic
agent is a drug or a toxin.
[0162] 44. The method of embodiment 43, wherein said drug possesses
the pharmaceutical property selected from the group consisting of
antimitotic, alkylating, antimetabolite, antiangiogenic, apoptotic,
alkaloid, COX-2, and antibiotic agents and combinations
thereof.
[0163] 45. The method of embodiment 43, wherein said drug is
selected from the group consisting of nitrogen mustards,
ethylenimine derivatives, alkyl sulfonates, nitrosoureas,
triazenes, folic acid analogs, anthracyclines, taxanes, COX-2
inhibitors, pyrimidine analogs, purine analogs, antimetabolites,
antibiotics, enzymes, epipodophyllotoxins, platinum coordination
complexes, vinca alkaloids, substituted ureas, methyl hydrazine
derivatives, adrenocortical suppressants, antagonists, endostatin,
taxols, camptothecins, doxorubicins and their analogs, and a
combination thereof.
[0164] 46. The method of embodiment 43, wherein said toxin is a
microbial, plant or animal toxin selected from the group consisting
of ricin, abrin, alpha toxin, saporin, ribonuclease (RNase), DNase
I, Staphylococcal enterotoxin-A, pokeweed antiviral protein,
gelonin, diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas
endotoxin.
[0165] 47. The method of embodiment 39, wherein said
immunomodulator is selected from the group consisting of a
cytokine, a stem cell growth factor, a lymphotoxin, a hematopoietic
factor, a colony stimulating factor (CSF), an interferon (IFN), a
stem cell growth factor, erythropoietin, thrombopoietin and a
combination thereof.
[0166] 48. The method of embodiment 47, wherein said lymphotoxin is
tumor necrosis factor (TNF), said hematopoietic factor is an
interleukin (IL), said colony stimulating factor is
granulocyte-colony stimulating factor (G-CSF) or granulocyte
macrophage-colony stimulating factor (GM-CSF), said interferon is
interferons-.alpha., -.beta. or -.gamma., and said stem cell growth
factor is designated "S1 factor".
[0167] 49. The method of embodiment 47, wherein said
immunomodulator comprises IL-1, IL-2, IL-3, IL-6, IL-10, IL-12,
IL-18, IL-21, interferon-.gamma., TNF-.alpha. or a combination
thereof.
[0168] 50. The method of embodiment 39, wherein said radionuclide
has an energy between 20 and 10,000 keV.
[0169] 51. The method of embodiment 50, wherein said radionuclide
is selected from the group consisting of .sup.125I, .sup.131I,
.sup.90Y, .sup.88Y, .sup.225Ac, .sup.177Lu, .sup.188Re, .sup.186Re,
and combinations thereof.
[0170] 52. The method of embodiment 39, wherein said photoactive
therapeutic agent is a chromogen or dye.
[0171] 53. The method of embodiment 44, wherein said alkylating
agent is dacarbazine.
[0172] 54. The method of embodiment 53, wherein said MN-14 antibody
or fragment thereof is administered in a dosage of 100 to 600
milligrams protein per dose per injection.
[0173] 55. The method of embodiment 54, wherein said MN-14 antibody
or fragment thereof is administered in a dosage of 300-400
milligrams protein per dose per injection.
[0174] 56. A method for treating medullary thyroid carcinoma
comprising administering to a subject, either concurrently or
sequentially, a therapeutically effective amount of an anti-CEA
monoclonal antibody or fragment thereof and at least one
therapeutic agent, and optionally formulated in a pharmaceutically
acceptable vehicle.
[0175] 57. The method of embodiment 56, wherein said anti-CEA MAb
or fragment thereof is humanized, wherein said humanized MAb
retains substantially the anti-CEA binding specificity of a murine
anti-CEA MAb.
[0176] 58. The method of embodiment 56, wherein said anti-CEA MAb
or fragment thereof is a chimeric MAb, and wherein said chimeric
MAb retains substantially the anti-CEA binding specificity of
murine anti-CEA MAb.
[0177] 59. The method of embodiment 56, wherein said anti-CEA
monoclonal antibody or fragment thereof is a MN-14 antibody or
fragment thereof.
[0178] 60. The method of embodiment 59, wherein said MN-14
monoclonal antibody or fragment thereof comprises the
complementarity-determining regions (CDRs) of a murine MN-14
monoclonal antibody, wherein the CDRs of the light chain variable
region of said MN-14 antibody comprises CDR1 comprising the amino
acid sequence KASQDVGTSVA (SEQ ID NO:20); CDR2 comprising the amino
acid sequence WTSTRHT (SEQ ID NO:21); and CDR3 comprising the amino
acid sequence QQYSLYRS (SEQ ID NO:22); and the CDRs of the heavy
chain variable region of said Class III anti-CEA antibody comprises
CDR1 comprising TYWMS (SEQ ID NO:23); CDR2 comprising
EIHPDSSTINYAPSLKD (SEQ ID NO:24); and CDR3 comprising LYFGFPWFAY
(SEQ ID NO:25).
[0179] 61. The method of embodiment 60, wherein said MN-14
monoclonal antibody reacts with CEA and is unreactive with normal
cross-reactive antigen (NCA) and meconium antigen (MA).
[0180] 62. The method of embodiments 61, wherein said MN-14
monoclonal antibody or fragment thereof is a humanized MN-14
antibody or fragment thereof.
[0181] 63. The method of embodiments 61, wherein said MN-14
monoclonal antibody or fragment thereof is a chimeric MN-14
antibody or fragment thereof.
[0182] 64. The method of embodiments 61, wherein said MN-14
monoclonal antibody or fragment thereof is a fully human MN-14
antibody or fragment thereof.
[0183] 65. The method of embodiment 62, wherein the framework
regions (FRs) of the light and heavy chain variable regions of said
humanized MN-14 antibody or fragment thereof comprise at least one
amino acid substituted from the corresponding FRs of a murine MN-14
monoclonal antibody.
[0184] 66. The method of embodiment 65, wherein said humanized
MN-14 antibody or fragment thereof comprising at least one amino
acid from said corresponding FR of said murine MN-14 antibody is
selected from the group consisting of amino acid residue 24, 28,
30, 48, 49, 74 and 94 of the murine heavy chain variable region of
FIG. 14A-C or 22B.
[0185] 67. The method of embodiment 65, wherein said humanized
MN-14 antibody or fragment thereof comprising at least one amino
acid from said corresponding FR of said murine MN-14 light chain or
heavy chain variable region.
[0186] 68. The method of embodiment 65, wherein said humanized
MN-14 antibody or fragment thereof comprises the light chain
variable region as set forth in FIG. 13A or 22A (hMN-14) or 23A and
the heavy chain variable region set forth in FIG. 14A-C or 22B
(hMN-14) or 23B.
[0187] 69. The method of any of embodiments 56-68, wherein said
fragment is selected from the group consisting of F(ab).sub.2,
F(ab').sub.2, Fab', Fab, Fv and sFv.
[0188] 70. The method of any of embodiments 56-68, wherein said
therapeutic agent is selected from the group consisting of
humanized, chimeric, human or murine monoclonal antibody or
fragment thereof selected from the group consisting of a Class I
anti-CEA monoclonal antibody, Class II anti-CEA monoclonal
antibody, Class III anti-CEA monoclonal antibody, and a fragment
thereof, and is administered either concurrently or sequentially in
a therapeutically effective amount.
[0189] 71. The method of embodiment 70, wherein said antibody or
fragment thereof is either naked or conjugated to another
therapeutic agent.
[0190] 72. The method of any of embodiments 56-68, wherein said
therapeutic agent is selected from the group consisting of a naked
antibody, cytotoxic agent, a drug, a toxin, a radionuclide, an
immunomodulator, an antisense oligonucleotide, a photoactive
therapeutic agent, an immunoconjugate of a CEA or non-CEA antibody,
a hormone, or a combination thereof, optionally formulated in a
pharmaceutically acceptable vehicle.
[0191] 73. The method of embodiment 72, wherein said therapeutic
agent is selected from the group consisting of a humanized,
chimeric, human or murine monoclonal antibody or fragment thereof
reactive with EGP-1, EGP-2 (e.g., 17-IA), MUC-1, MUC-2, MUC-3,
MUC-4, PAM-4, KC4, TAG-72, EGFR, HER2/neu, BrE3, Le-Y, A3, A33,
Ep-CAM, AFP, Tn, Thomson-Friedenreich antigens, tumor necrosis
antigens, VEGF, PlGF or other tumor angiogenesis antigens, Ga 733,
IL-6, insulin-like growth factor-1, and a combination thereof, and
is administered to said subject either concurrently or sequentially
in a therapeutically effective amount.
[0192] 74. The method of embodiment 73, wherein said antibody or
fragment thereof is either naked or conjugated to another
therapeutic agent.
[0193] 75. The method of any of embodiments 56-68, wherein said
therapeutic agent is not DTIC.
[0194] 76. The method of embodiment 72, wherein said cytotoxic
agent is a drug or a toxin.
[0195] 77. The method of embodiment 72, wherein said drug possesses
the pharmaceutical property selected from the group consisting of
antimitotic, alkylating, antimetabolite, antiangiogenic apoptotic,
alkaloid, COX-2, and antibiotic agents and combinations
thereof.
[0196] 78. The method of embodiment 76, wherein said drug is
selected from the group consisting of nitrogen mustards,
ethylenimine derivatives, alkyl sulfonates, nitrosoureas,
triazenes, folic acid analogs, anthracyclines, taxanes, COX-2
inhibitors, pyrimidine analogs, purine analogs, antimetabolites,
antibiotics, enzymes, epipodophyllotoxins, platinum coordination
complexes, vinca alkaloids, substituted ureas, methyl hydrazine
derivatives, adrenocortical suppressants, antagonists, endostatin,
taxols, camptothecins, doxorubicins and their analogs, and a
combination thereof.
[0197] 79. The method of embodiment 76, wherein said microbial,
plant or animal toxin is selected from the group consisting of
ricin, abrin, alpha toxin, saporin, ribonuclease (RNase), DNase I,
Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,
diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas
endotoxin.
[0198] 80. The method of embodiment 72, wherein said
immunomodulator is selected from the group consisting of a
cytokine, a stem cell growth factor, a lymphotoxin, a hematopoietic
factor, a colony stimulating factor (CSF), an interferon (IFN), a
stem cell growth factor, erythropoietin, thrombopoietin and a
combination thereof.
[0199] 81. The method of embodiment 80, wherein said lymphotoxin is
tumor necrosis factor (TNF), said hematopoietic factor is an
interleukin (IL), said colony stimulating factor is
granulocyte-colony stimulating factor (G-CSF) or granulocyte
macrophage-colony stimulating factor (GM-CSF)), said interferon is
interferons-.alpha., -.beta. or -.gamma., and said stem cell growth
factor is designated "S1 factor".
[0200] 82. The method of embodiment 72, wherein said
immunomodulator comprises IL-1, IL-2, IL-3, IL-6, IL-10, IL-12,
IL-18, IL-21, interferon-.gamma., TNF-.alpha. or a combination
thereof.
[0201] 83. The method of embodiment 72, wherein said radionuclide
has an energy between 20 and 10,000 keV.
[0202] 84. The method of embodiment 83, wherein said radionuclide
is selected from the group consisting of .sup.125I, .sup.131I,
.sup.90Y, .sup.88Y, .sup.225Ac, .sup.177Lu, .sup.188Re, .sup.186Re,
and combinations thereof.
[0203] 85. The method of embodiment 72, wherein said photoactive
therapeutic agent is a chromogen or dye.
[0204] 86. The method of embodiment 77, wherein said alkylating
agent is dacarbazine.
[0205] 87. The method of embodiment 86, wherein said MN-14 antibody
or fragment thereof is administered in a dosage of 100 to 600
milligrams protein per dose per injection.
[0206] 88. The method of embodiment 87, wherein said MN-14 antibody
or fragment thereof is administered in a dosage of 300-400
milligrams protein per dose per injection.
[0207] 89. A method for treating cancer comprising administering to
a subject, either concurrently or sequentially, a therapeutically
effective amount of an anti-CEA antibody or fragment thereof and at
least one therapeutic agent, and optionally formulated in a
pharmaceutically acceptable vehicle.
[0208] 90. The method of embodiment 89, wherein the therapeutic
agent is CPT-11.
[0209] 91. The method of embodiment 90, wherein the anti-CEA
antibody. or fragment is administered prior to administration of
CPT-11.
[0210] 92. The method of embodiment 91, wherein the anti-CEA
antibody or fragment is administered around 3 days prior to
administration of CPT-11.
[0211] 93. The method of embodiment 89, wherein the therapeutic
agent is DTIC.
[0212] 94. The method of embodiment 89, wherein the therapeutic
agent is oxaliplatin.
[0213] 95. The method of embodiment 89, wherein the therapeutic
agent is 5-fluorouracil/leucovorin.
[0214] 96. In a method of treating cancer with a non-antibody
therapeutic agent, the improvement comprising pre-treating a
subject suffering from cancer with an anti-CEA antibody or a
fragment thereof prior to administration of the non-antibody
therapeutic agent.
[0215] 97. The method of embodiment 96, wherein the anti-CEA
antibody is hMN-14.
[0216] 98. The method of embodiment 96, wherein the therapeutic
agent is CPT-11.
[0217] 99. A method of treating cancer with an antibody comprising
administering to a subject suffering from cancer, prior to
administration of the antibody, an agent that activates
granulocytes and/or NK cells in order to increase effector function
of the antibody.
[0218] 100. The method of embodiment 99, wherein the agent is
GM-CSF.
[0219] 101. The method of embodiment 99, wherein the antibody is an
anti-CEA antibody.
[0220] 102. The method of embodiment 101, wherein the antibody is
hMN-14.
[0221] 103. A method of treating cancer with an anti-CEA antibody
or fragment, comprising administering to a subject suffering from
cancer, prior to administration of the anti-CEA antibody or
fragment, an amount of interferon effective to upregulate CEA
expression in tumor cells.
[0222] 104. The method of embodiment 103, wherein the anti-CEA
antibody is hMN-14.
[0223] 105. An antibody fusion protein comprising at least one CEA
binding site and at least one other binding site for the same or
different antigen.
[0224] 106. The antibody fusion protein according to embodiment
105, wherein the CEA binding site binds to the same site as an
MN-14 antibody.
[0225] 107. The antibody fusion protein according to embodiment
105, which is bivalent and trivalent.
[0226] 108. The antibody fusion protein according to embodiment
105, wherein one arm of the fusion protein is a Class III, anti-CEA
mAb that targets CD66e and another arm of the fusion protein is
from another CEA crossreactive antibody that targets CD66a-d.
[0227] 109. The antibody fusion protein according to embodiment
105, wherein the binding arms are scFv or Fab regions.
[0228] 110. An antibody fusion protein according to embodiment 105,
which is a bispecific, trivalent protein comprising one arm
reactive with CD66a-d and two arms reactive with only CEA
(CD66e).
[0229] 111. An antibody fusion protein according to embodiment 105,
which is a bispecific protein comprising two arms that bind to
NCA50/90.
[0230] 112. An antibody fusion protein according to embodiment 105,
which is a diabody comprising one arm that binds to NCA50/90 and a
second arm that binds to a Class III epitope of CEA.
[0231] 113. An antibody fusion protein according to embodiment 112,
wherein the NCA-50/90 arm is obtained from an hMN-3 antibody and
the second arm that binds to a Class III epitope of CEA is obtained
from hMN-14.
[0232] 114. An antibody fusion protein according to embodiment 113,
wherein the fusion protein lacks an Fc-domain to prevent activation
of cytokine release from granulocytes or which has an Fc-domain
that has been modified to prevent complement fixation and ADCC.
[0233] 115. An antibody fusion protein according to embodiment 104,
which is a triabody comprising one hMN-3 arm and two hMN-14 arms.
115a. An antibody fusion protein according to embodiment 104, which
is a triabody comprising one hMN-15 arm and two hMN-14 arms.
[0234] 116. An antibody fusion protein according to embodiment 104,
comprising at least one hMN-14 arm and at least one NP-3 arm.
[0235] 117. An antibody fusion protein according to embodiment 116,
which comprises an Fc-domain to enable complement fixation and
activation of ADCC.
[0236] 118. An antibody fusion protein according to embodiment 104,
further comprising a therapeutic agent.
[0237] 119. An antibody fusion protein according to embodiment 118,
wherein the therapeutic agent is a cytokine.
[0238] 120. An antibody fusion protein according to embodiment 119,
wherein the cytokine is interferon, a colony-stimulating factor, or
an interleukin.
[0239] 121. An antibody fusion protein according to embodiment 120,
wherein the colony-stimulating factor is GM-CSF or G-CSF.
[0240] The invention is further illustrated by, though in no way
limited to, the following examples.
EXAMPLE 1
Materials and Methods
Monoclonal Antibodies and Cell Lines
[0241] TT, a human medullary thyroid cell line, was purchased from
the American Type Culture Collection. The cells were grown as
monolayers in DMEM (Life Technologies, Gaithersburg, Md.)
supplemented with 10% fetal bovine serum, penicillin (100 u/ml),
streptomycin (100 .mu.g/ml), and L-glutamine (2 mM). The cells were
routinely passaged after detachment with trypsin, 0.2% EDTA.
[0242] MN-14 is a Class III anti-CEA MAb, reacting with CEA and
unreactive with the normal cross reactive antigen, NCA, and
meconium antigen (Hansen et al., Cancer, 71:3478 (1993)). The
construction and characterization of the humanized forms of MN-14
and LL2, the anti-CD22 MAb used here as a negative control, have
been previously described. (Sharkey et al., Cancer Res., 55:5935s
(1995); Leung et al., Mol. Immunol., 32:1416 (1995)). P3x63Ag8
(MOPC-21) is an irrelevant mouse myeloma IgG.sub.1 obtained from
the American Type Culture collection (Rockville, Md.). The
antibodies were purified by protein A chromatography.
In Vivo Studies
[0243] Tumors were propagated in female nu/nu mice (Taconic Farms,
Germantown, N.Y.) at 6-8 weeks of age by s.c. injection of
2.times.10.sup.8 washed TT cells, which had been propagated in
tissue culture. Antibodies were injected i.v., via the lateral tail
vein, into the tumor-bearing animals. Details on the quantities of
antibodies injected and the time of administration are indicated in
the Results section for each study. Results are given as tumor
volumes of individual animals as well as the mean I:SE. Tumor size
was monitored by weekly measurements of the length, width, and
depth of the tumor using a caliper. Tumor volume was calculated as
the product of the three measurements. Statistical comparisons were
made using the Student's T-test to compare tumor volumes and area
under the growth curves.
EXAMPLE 2
Combination Therapy of Naked hMN-14 and DTIC Delivered 2 Days after
Injection of TT (Human Medullary Thyroid) Tumor Cells
[0244] In a previous study, naked hMN-14 and dacarbazine (DTIC)
were given in combination to TT 2 days after tumor implantation,
using 100 .mu.g and 25 .mu.g doses of DTIC (days 2, 3, and 4) and
250 .mu.g doses of hMN-14 given on day 2, then weekly. The 100
.mu.g DTIC dose combined with hMN-14 was more effective than either
treatment alone (FIG. 1A). However, the 100 .mu.g DTIC dose yielded
too strong a response, while the 25 .mu.g dose was not effective.
Surprisingly, the effects of MN-14 alone and DTIC alone were not
additive. In other words, given the results of treatment with 250
.mu.g hMN-14 alone and 100 .mu.g DTIC alone, one would not predict
that the combination of 250 .mu.g hMN-14 and 100 .mu.g DTIC would
have such a pronounced effect. See FIG. 1A.
[0245] In this study, treatment began 2 days after IT cell
injection, as in the previous study. hMN-14 was given at 100
.mu.g/dose on days 2, 3, 4, 5, 7, 8, 9, 10, 11, 15 and 22, then
every 7 days until the animal died, the tumor attained a volume of
2.0 cm.sup.3 or the study terminated for humane reasons. Doses of
DTIC were 50 and 75 .mu.g per dose, which is between the doses
given in the previous study. TT cells were injected subcutaneously
in 60 nude mice. The day of injection was Monday, day 0. See FIG.
1B.
[0246] Results demonstrate that significant delays in tumor growth
were caused by either MAb therapy alone or chemotherapy alone (FIG.
1B). The 75 .mu.g dose of DTIC in combination with this schedule of
hMN-14 antibody was significantly more effective than either
treatment alone (p<0.02). Unexpectedly, the results of combined
DTIC and MAb therapy were not additive. At 7 weeks, 8/10 mice in
the 75 .mu.g DTIC and MAb group had no palpable tumor, compared to
1/10 mice in the 75 .mu.g DTIC only group and 0/10 mice in the
untreated and MAb group.
[0247] Mean tumor volumes at 7 weeks were 0.018.+-.0.039 cm.sup.3
(75 .mu.g DTIC plus MN-14), 0.284+0.197 cm.sup.3 (75 .mu.g DTIC
only), 0.899.+-.0.545 cm.sup.3 (hMN-14 only) and 1.578.+-.0.959
cm.sup.3 (untreated). Combined therapy of the naked anti-CEA
antibody with DTIC augments the anti-tumor effects of antibody or
chemotherapy alone, without increased toxicity. The superiority of
the combined modality treatment was surprising.
[0248] Dosing Summary: (1) hMN-14 was given daily (i.p.), except
Sundays, at 100 .mu.g/dose/mouse on days 2 through 11. The antibody
treatment was initiated on the same day as DTIC treatment. (2) DTIC
was given on days 2, 3, and 4 at 50 and 75 .mu.g/dose, which
corresponded to 5% and 7.5% of the MTD. Only one course of DTIC was
given.
[0249] Groups: 6 groups of mice, each group containing 10 mice.
[0250] Group 1: Untreated.
[0251] Group 2. DTIC at 50 .mu.g/dose, days 2, 3, and 4 (Wednesday,
Thursday, and Friday).
[0252] Group 3. DTIC at 75 .mu.g/dose, days 2, 3, and 4.
[0253] Group 4. DTIC at 50 .mu.g/dose, days 2, 3, and 4, plus
hMN-14 (100 .mu.g/dose) day 2, 3, 4, 5, 7, 8, 9, 10, 11, 15 and 22,
then every 7 days until the animal died, the tumor attained a
volume of 2.0 cm.sup.3, or the study terminated.
[0254] Group 5. DTIC at 25 .mu.g/dose, days 2, 3, and 4, plus
hMN-14 (100 .mu.g/dose) day 2, 3, 4, 5, 7, 8, 9, 10, 11, 15 and 22,
then every 7 days until the animal died, the tumor attained a
volume of 2.0 cm.sup.3, or the study terminated.
[0255] Group 6. hMN-14 (confirm 100 .mu.g/dose), days 2, 3, 4, 5,
7, 8, 9, 10, 11, 15 and 22, then every 7 days until the animal
died, the tumor attained a volume of 2.0 cm.sup.3, or the study
terminated.
[0256] Animals were monitored for survival. Tumor and body weight
were measured weekly.
[0257] Protocol: On day 2, 200 mg/vial DTIC was reconstituted with
19.7 ml sterile water for injection. The resulting solution
contained 10 mg/ml of dacarbazine with a pH range of 3.0-4.0. The
solution was used as needed for the dilutions described below and
the remainder was frozen in 1 ml aliquots for subsequent use.
[0258] Groups 2 and 4: 5 ml of 0.5 mg/ml solution was prepared. 100
.mu.l of 0.5 mg/ml/mouse was injected i.v.
[0259] Groups 3 and 5: 5 ml of 0.75 mg/ml solution was prepared.
100 .mu.l of 0.75 mg/ml/mouse was injected i.v.
[0260] Quantity of hMN-14 was estimated. 100 .mu.l of 1 mg/ml
hMN-14 was injected i.p. in mice in Groups 4, 5 and 6.
EXAMPLE 3
Radioimmunotherapy Studies in a Human MTC Xenograft Model
[0261] Applicants developed a model for experimental
radioimmunotherapy of MTC with radiolabeled anti-CEA MAbs using
human MTC xenografts of the CEA- and calcitonin producing human MTC
cell line designated TT ([Stein, 1999 #82], see Appendix). MTC
tumors were established in nude mice by a s.c. inoculation of
2.times.10.sup.8 cells and allowed to grow for 2-5 weeks before
injection of MAbs. Biodistribution and RAIT studies were then
carried out with MN-14, which was shown by flow cytometry to react
with TT cells. Both Ag8 and Mu-9 were used as negative control MAbs
in these studies. Preliminary studies using smaller tumors of
.about.0.08 g showed that 7 days after the injection of
.sup.131I-MN-14, the percent of injected dose per gram of tumor (%
ID/g) was 68.9% compared with only 12.6% ID/g for the co-injected
.sup.125I-Ag8 control. Using larger tumors, (grown for five weeks
in nude mice; mean tumor weight=0.404 g), the % ID/g of tumor
observed at seven days post injection of .sup.125I-MN-14 was 12.4%.
However, the % ID/g of the co-injected .sup.88Y-MN-14 was 50.5%, or
4.1-fold higher than .sup.125I-MN-14. The tumor-to-blood, lungs,
liver, spleen, and kidneys were also higher with .sup.88Y-MN-14
than with .sup.125I-MN-14, while the tumor-to-bone ratios were
equal with both agents. When .sup.125I-MN-14 and .sup.88Y-MN-14
biodistribution data were used to predict the tumor dosimetry with
.sup.131I-MN-14 and .sup.90Y-MN-14, respectively, the radiation
absorbed dose delivered at the MTD of .sup.90Y-MN-14 (115 .mu.Ci)
was 1.75-fold higher than that delivered at the MTD of
.sup.131I-MN-14 (275 .mu.Ci) (4900 cGy vs. 2800 cGy).
[0262] Therapy studies in this model confirmed that .sup.90Y-MN-14
is a better therapeutic agent than .sup.131I-MN-14. In 5-week-old
tumors, a 5-week complete inhibition of tumor growth was seen at
the MTD of .sup.90Y-MN14 compared to only a tumor growth delay with
.sup.131I-MN14 (FIG. 2). Moreover, when smaller 2-week old tumors
were treated, an average of 60% tumor volume reduction, with some
complete tumor regressions, was seen at the MTD of .sup.90Y-MN14.
These anti-tumor effects were very significant compared with the
relatively rapid tumor growth in untreated animals or those treated
at the MTD of control MAbs. Thus, our preclinical studies
demonstrated that this animal model is exquisitely suitable for
experimental RAIT with anti-CEA MAbs.
[0263] The longer path length and higher energy of .sup.90Y
compared with .sup.131I, in addition to the fact that .sup.90Y is
retained longer by target cells led to delivery of an increased
radiation dose to tumor and thus more effective therapy at
equitoxic doses. If our results with residualizing .sup.131I (refs)
can be generalized to MN-14 in MTC, we would expect that
residualizing .sup.131I would be at least equally effective to
.sup.90Y in tumors of the size studied here, and most likely
superior in the setting of micrometastatic disease or as adjuvant
therapy following surgery.
EXAMPLE 4
Chemotherapy
[0264] Four drugs, doxorubicin, DTIC (dacarbazine),
cyclophosphamide, and vincristine, were evaluated, singly and in
combination, for their effect on the growth of TT MTC xenografts in
nude mice. Doses were selected based on the doses of each drug
given clinically to humans on a mg/m.sup.2 basis. Animals were
monitored for survival, and tumor volumes and body weights were
measured weekly. FIG. 3 shows the tumor growth curve for animals in
this study. Given individually, doxorubicin, DTIC and
cyclophosphamide, but not vincristine, yielded significant growth
inhibition, although the growth delay caused by DTIC was markedly
longer than that of the other drugs. Approximate mean time to
doubling for each group was: untreated, 1 week; doxorubicin, 2.5
weeks; DTIC, 7.5 weeks; cyclophosphamide, 3 weeks; and vincristine,
1.5 weeks. Combining doxorubicin and DTIC improved the efficacy
compared to either drug alone, increasing the mean time to doubling
to 10 weeks. However, the increased efficacy of doxorubicin and
DTIC combination did not reach the 95% confidence level in
comparison to DTIC alone. The P values for AUC comparisons were as
follows: P<0.01 for doxorubicin+DTIC versus doxorubicin, and
P<0.1 for doxorubicin+DTIC versus DTIC. The 4-drug regimen
extended the mean time to doubling to 12 weeks; P<0.01 for
comparisons to both doxorubicin and DTIC.
[0265] Log rank analysis of survival data for the individual drugs
versus the untreated group indicated a significant difference only
for DTIC and cyclophosphamide. Mean survival time for the untreated
control group was 4 weeks compared to 11 weeks and 8 weeks for DTIC
and cyclophosphamide treatment groups, respectively, and greater
than 12 weeks for the drug combinations. Toxicity, as measured by
body weight loss, was within the acceptable range for all study
groups. Maximum weight loss was observed 1 week after treatment in
the mice treated with all 4 drugs, ranging from 3-12% loss of body
weight.
EXAMPLE 5
Combining Radioimmunotherapy and Chemotherapy for Treatment of
MTC
[0266] RAIT Plus 4-drug Combination. The effect of combining RAIT
with .sup.90Y-anti CEA MAb MN-14 and the 4-drug combination was
evaluated by comparing the growth of TT in untreated mice to those
treated with the 4-drug regimen described above (doxorubicin, DTIC,
cyclophosphamide, and vincristine), 100% of the maximum tolerated
dose (MTD) of RAIT (105 .mu.Ci), 50% of the MTD of RAIT, and 50% of
the MTD of RAIT combined with the 4 drugs. FIG. 4 shows the growth
curves of IT tumors in mice given the various treatment regimens.
All four of the treatment groups yielded significant improvement in
efficacy compared to the untreated animals. Whereas the approximate
mean time to doubling in the untreated animals was 1.5 weeks,
chemotherapy with the 4 drugs extended the mean doubling time to 10
weeks and RAIT alone yielded 4-week and 8-week doubling times at
50% and 100% of the MTD, respectively. As expected, both the 100%
RAIT group and the 4-drug therapy regimen were significantly better
than the 50% RAIT group. Most importantly, combining 50% RAIT and
the 4-drug regimen yielded improved results, compared to either
therapy alone, further extending the mean doubling time to
approximately 12.5 weeks. For the comparison of the combined
treatment to the 4-drug regimen, P<0.02, and for the comparison
to 100% RAIT, P<0.01.
[0267] Mean weight loss 1 week post treatment (nadir) was 9% for
the 100% RAIT and the 4-drug regimens, but 15% for combined 50%
RAIT plus 4-drug treatment. In addition, in the combined therapy
group, one animal died three weeks post treatment and a second
animal had a weight loss greater than 20%. Thus, this treatment
exceeded the maximum tolerated dose.
RAIT Plus Chemotherapy with 2-Drug Regimens
[0268] The effect of combining RAIT with .sup.90Y-anti CEA MAb
MN-14 and chemotherapy with a 2-drug combination, consisting of
doxorubicin and DTIC, was also evaluated in this MTC xenograft
model. Approximate doubling times for the groups were: untreated,
1.5 weeks; doxorubicin plus DTIC, 8 weeks; the MTD of RAIT, 10
weeks; and the MID of RAIT combined with 25-75% of the 2-drug
regimen, greater than 12 weeks. Thus, RAIT alone was more effective
than the 2-drug regimen and, most significantly, combining RAIT and
the 2-drug regimen yielded improved results compared to either
therapy alone. For the comparison of the combined treatment to the
2-drug regimen, P<0.005, for the comparison to RAIT alone,
P<0.02.
[0269] Mean weight loss 1-2 weeks post treatment (nadir) was 2-8%
for all groups, except the 100% RAIT plus 75% 2-drug chemotherapy
group, where a 13% loss was observed at 2 weeks. In addition, in
this combined therapy group, two animals died 3-4 weeks post
treatment and one experienced a weight loss greater than 20%. Thus,
addition of the 75% dose level of doxorubicin and DTIC to 100% RAIT
treatment exceeded the MTD, whereas 50% of this 2-drug combination
can be tolerated in combination with 100% RAIT.
RAIT Plus Doxorubicin
[0270] Because previous publications have reported the combination
of RAIT with doxorubicin in this model (Stein et al., Clin Cancer
Res., 5:3199s (1999); Behr et al., Cancer Res. 57:5309 (1997)), a
direct comparison was made to the RAIT plus doxorubicin regimen. A
direct comparison was also made to RAIT plus the 4-drug regimen.
All treatments yielded significant efficacy compared to the
untreated animals. The mean doubling time for the RAIT plus
doxorubicin group was 12 weeks. In this study combining the full
MTD of RAIT with either 50% of doxorubicin and DTIC or the 4-drug
regimen extended the mean doubling time to greater than 15 weeks,
with no statistically significant difference between these two
groups. A substantial number of objective responses were observed
in these studies. Following treatment with RAIT plus doxorubicin
there were 3 complete responses, 2 partial responses, and 5 animals
with stable disease for at least 4 weeks, out of a total of 10
mice. The RAIT plus 2-drug protocol increased the objective
responses to 10 complete responses and 2 partial responses of 12
animals, and the RAIT plus 4-drug treatment protocol led to 7
complete responses and 2 partial responses out of 9 mice.
RAIT Plus DTIC
[0271] Because DTIC was the most effective chemotherapeutic agent
when administered alone, the efficacy of RAIT plus DTIC was
evaluated in comparison to that of RAIT plus doxorubicin and DTIC.
Omitting doxorubicin from the treatment protocol will be important
for clinical application in order to avoid the added toxicity of
this drug, especially the known cardiac toxicity. As shown in FIG.
5, the two study groups which received the chemotherapy in
combination with RAIT, either doxorubicin and DTIC or DTIC only,
are approximately equal to each other, and both are more effective
than the single modality treatments. P values for AVC comparisons
were as follows: P<0.01 for RAIT+DTIC versus DTIC, and P<0.05
for RAIT+DTIC versus RAIT. The mean doubling time for the RAIT plus
DTIC, and RAIT plus doxorubicin and DTIC groups were 15.5 weeks and
14 weeks, respectively, compared to 7.5 weeks and 9 weeks for DTIC
and RAIT alone, respectively. Thus, the combined modality treatment
of RAIT plus DTIC extended the mean time to doubling by 100% over
the DTIC chemotherapy. No significant difference was observed by
either AUC or log rank analyses between the RAIT plus DTIC, and
RAIT plus doxorubicin and DTIC groups.
EXAMPLE 6
Studies with Naked Anti-CEA Alone
Therapy with Naked hMN-14
[0272] To study the effect of unlabeled hMN-14 on the growth of TT
tumors in nude mice, a single injection of hMN-14 was administered
i.v. either one day or eleven days post tumor cell injection. FIG.
6 shows the tumor growth curves of animals treated with 0.5 mg
hMN-14/mouse compared to untreated controls. The untreated group
contained 16 animals; the two treatment groups contained 10 animals
each. A significant growth delay was observed between the untreated
group and the group treated on day-1 post tumor injection.
Significant differences in the mean tumor sizes (p<0.05) were
observed from day-32 through day-93. Between day-32 and day-60
there was a 64-70% inhibition of tumor size in the MN-14 treated
group compared to the untreated animals. There were no significant
differences between the mean tumor sizes in the day-11 group and
untreated animals. Significant delay in tumor growth was also seen
by t-test analysis of the area under the growth curves. P<0.05
for the untreated group compared to the group treated one day
following tumor injection, but not for the group treated eleven
days following tumor injection.
Specificity of Treatment
[0273] FIG. 7 summarizes the results of a study on the specificity
of the anti-tumor response. The effect of unlabeled hMN-14 on the
growth of TT tumors in nude mice was compared to that of a negative
control humanized MAb, hLL2 (antiCD22), and the murine MN-14. MAbs
(0.5 mg/mouse) were administered (i.v.) one day after TT cells,
then three additional weekly doses of 0.5 mglmouse were given.
Groups of 15 animals were studied. The growth inhibition observed
in the first study from treatment with 0.5 mg hMN-14 was confirmed
in this study. Significant differences in mean tumor sizes
(p<0.05) between the hMN-14 and the untreated group were
observed starting at day-23. At day-37 the mean tumor volume in the
group treated with hMN-14 was 42.7% of the untreated control
animals. Treatment with murine MN-14 yielded results similar to the
hMN-14. Treatment with hLL2 did not slow tumor growth; instead
there was a small (not significant) increase in growth rate. For
example, at day-37 87% of the tumors treated with hMN-14 were less
than 0.5 cm3, compared to 40% of the untreated and 29% of the hLL2
treated group. T-test analysis of the area under the growth curves
demonstrated significant differences (p<0.05) between the
untreated group and the groups treated with either hMN-14 or murine
MN-14, but not the group treated with hLL2. In addition, the hMN-14
group was significantly different from the hLL2 group but not the
murine MN-14 treated animals.
Effect of Dose
[0274] To study the effect of dose of unlabeled hMN-14 on the
growth of TT tumors in nude mice, increasing doses of hMN-14 were
evaluated. Antibody doses were administered 1 day after TT cells,
then weekly until the termination of the study. Weekly doses ranged
from 0.125 mg to 2.0 mg hMN-14/mouse in groups of six mice.
Significant differences in mean tumor sizes and area under the
growth curves between the untreated group and all treatment groups
were observed (FIG. 8). For example, between day-21 and day-49 mean
tumor volume in the 2 lowest hMN-14 treatment groups were 27-40% of
the size of tumors in the untreated animals. Treatment with the
lower doses, 0.125 mg and 0.25 mg, appeared to be more effective
than treatment with the higher doses, although the difference did
not reach statistical significance.
Timing
[0275] The effect of time between TT injection and initial dose of
hMN-14 on the growth of TT tumors in nude mice was evaluated by
varying the day of administration of MAb hMN-14 (0.25 mg) was
administered either 1, 3, or 7 days after TT cells, then weekly
until termination of the study. Groups of 7-8 animals were studied.
Results are summarized in FIG. 9. Significant differences in mean
tumor sizes (p<0.05) between the untreated group and all three
treatment groups were observed. However, the difference in mean
tumor size between the untreated mice and the day-7 treatment group
was only significant at one time point, day-28. Day-1 treated mice
yielded significant differences from 21-77 days, and day-3 treated
mice yielded significant differences from 21-70 days. T-test
analysis of the area under the growth curves indicated significant
growth inhibition for the groups treated with hMN-14 either 1 or 3
days after TT cell administration compared to untreated group.
[0276] This analysis did not reach the 95% confidence limit for
difference between the untreated group and the group treated on
day-7 (p=0.057 at 5 weeks).
EXAMPLE 7
Combined Naked Anti-CEA Plus DTIC Therapy of MTC
[0277] To study whether naked hMN-14 can add to the efficacy of
DTIC, TT bearing nude mice were given DTIC (75 .mu.g/dose) in
combination with a course of treatment of the unlabeled MAb. DTIC
was administered for 3 consecutive days at 75 .mu.g/dose as one
course, beginning 2 days after s.c. injection of TT cells. hMN-14
MAb treatment was initiated on the same day as the first dose of
DTIC, at 100 .mu.g/dose/day for 5 days in the first two weeks, then
twice weekly. Significant delays in tumor growth were caused by
these schedules of either MAb therapy or chemotherapy alone (FIG.
10). The 75-.mu.g dose of DTIC in combination with this schedule of
hMN-14 was significantly more effective than either treatment alone
(P<0.02). At 7 weeks, 8/10 mice in the 75 .mu.g DTIC+MAb group
had no palpable tumor, compared to 1/10 in the 75 .mu.g DTIC-only
group and 0/10 in the untreated and MAb-only groups. Mean tumor
volumes at 7 weeks were 0.018+0.039 cm.sup.3 (75 .mu.g
DTIC+hMN-14), 0.284+0.197 cm.sup.3 (75 .mu.g DTIC), 0.899+0.545
cm.sup.3 (hMN-14) and 1.578+0.959 cm.sup.3 (untreated).
[0278] The anti-CEA MAb MN-14 has shown unexpected anti-tumor
efficacy in MTC without conjugation to a cytotoxic agent.
Differences in mean tumor sizes between the hMN-14 treated and the
untreated groups were observed beginning at 3 weeks and lasting at
least 2 months. Treatment with isotype matched negative control
MAbs did not slow tumor growth. This is the first evidence of tumor
suppression with a "naked" anti-CEA MAb. However, combined therapy
of the naked anti-CEA MAb with DTIC augments the anti-tumor effects
of antibody or chemotherapy alone, without increased toxicity. The
superiority of the combined modality treatment argues for the
integration of CEA-MAb therapy into chemotherapeutic regimens for
MTC management.
EXAMPLE 8
[0279] FIG. 15 shows the effects of naked hMN-14 CEA Mab and DTIC
treatment in a medullary thyroid cancer model. Treatment was
initiated 2-days after tumor transplantation. DTIC was administered
at 75 .mu.g on days 2, 3, and 4 at 7.5% of the MTD to mice. hMN-14
was administered at 100 .mu.g/day on days 2-5, 7-10, 11, 15, 22,
and then once per week. The results show a statistically
significant difference (P<0.05) between the areas under the
curve for all groups. Naked hMN-14 CEA Mab treatment showed a
significant effect on inhibiting tumor growth. When combined with
DTIC, a surprisingly enhanced level of inhibition of tumor growth
occurred relative to either treatment alone.
EXAMPLE 9
Naked Anti-CEA Antibody Treatment Plus CPT-11 or 5-FU in Colon
Cancer Cells
[0280] The present experiment discloses the in vitro and in vivo
effect of a humanized, naked anti-CEA, hMN-14 antibody (hMN-14)
alone, and in combination with chemotherapy on colon cancer
growth.
Methods and Materials
[0281] Antibody Production. The CDR-grafted (humanized) MN-14
(hMN-14) anti-carcinoembryonic antigen (CEA) (Sharkey, R. M., et
al., Cancer Res, 55: 5935-5945, 1995) along with the murine MN-14
and other antibodies targeting different CEA epitopes (NP1, NP3,
MN3, MN15; (Sharkey, R. M., et al., Cancer Res, 50: 2823-2831,
1990) were purified by protein A and ion-exchange chromatography
(Q-Sepharose; Pharmacia, Piscataway, N.J.). Purity was tested by
immunoelectrophoresis, polyacrylamide gel electrophoresis using
reducing and nonreducing conditions and size-exclusion
high-pressure liquid chromatography.
[0282] In Vivo Therapy Studies. Survival therapy studies were
performed using a CEA-positive GW-39 intrapulmonary micrometastasis
model (Sharkey, R. M., et. al., J. Natl. Cancer Inst., 83: 627-632,
1991; Blumenthal, R. D., et al., Cancer Res, 52: 6036-6044, 1992).
Stock subcutaneous GW-39 human colorectal tumors were used to
prepare a 10% or 5% cell suspension. Cells (30 .mu.l) were injected
i.v. into the caudal vein. HuMN-14 IgG was initiated on either day
0 or day 3 after cell implantation and administered daily.times.14
days and twice weekly thereafter for the duration of the study at a
dose of 100 .mu.g/d. CPT-11 was administered at a dose of 160 .mu.g
daily for 5 days i.p. (20% of the MID) starting on day 0 or day 3
after cell implantation. For some studies, the stock GW-39 tumor
came from mice that received 100,000 U of IFN.gamma. twice daily
for 4 days to upregulate CEA expression (Greiner, J. W., et al.,
16: 2129-2133, 1996), which was confirmed by immunohistology as
previously described (Blumenthal, R. D., et al., Int. J. Cancer,
51: 935-941, 1992). Body weight was monitored weekly and animal
survival recorded. Results were analyzed with the Kaplan-Meir test
and median survival time determined.
In Vivo Effects of Antibody-Induced Chemosensitization of Cancer
Cells.
[0283] The effect of hMN14-induced chemosensitization was apparent
in vivo as well as in vitro. Survival curves for mice bearing GW-39
intrapulmonary micrometastases, as described above, and untreated
or treated with hMN14 alone (100 .mu.g/d.times.14 d and twice
weekly for the duration of the study), a 10% MTD of CPT-11 (80
.mu.g/d.times.5 days) alone or both modalities together. Treatment
was initiated the day of cell implantation (30 .mu.l of a 10% GW-39
cell suspension). Each treatment group started with 10 mice and the
study was repeated twice. The results show that coadministration of
hMN-14 and CPT-11 to nude mice bearing GW-39 lung micrometastases
increases survival beyond the effect of either modality alone.
Administration of a 10% MTD of CPT-11 resulted in a 1-week increase
in median survival from 56 days to 63 days (p<0.05). Median
survival time of animals dosed with both hMN-14 and CPT-11 on day 0
increased by an additional 2 weeks to 77 days (p<0.005 compared
with untreated mice). Since maximal antibody accretion occurs 3
days post injection, hMN-14 treatments were initiated 3-days before
CPT-11 to determine whether such dosing would further enhance the
therapeutic effect of the combined modality treatment approach by
allowing high antibody uptake and chemosensitization in vivo. The
results demonstrate that the 3-day pretreatment with hMN-14
followed by CPT-11 increased median survival to 105 days
(p<0.001), compared with CPT-11 alone on day 3, with a median
survival of 70 days. In this study, co-treatment of hMN-14 and
CPT-11 was superior, as evidenced by a median survival of 70 days
vs. CPT-11 alone on day 0, with a median survival of 63 days or
untreated mice with a median survival of 35 days. The results were
similar for a further experiment where a 5% GW-39 cell suspension
was used instead of the 10% GW-39 cell suspension.
EXAMPLE 10
In Vivo Effect of Pretreatment with an Immunomodulator Prior to
Treatment with hMN-14 and CPT-11 on Tumor Cell Chemosensitivity
[0284] A further experiment evaluated the combined treatment of
hMN-14 with CPT-11, initiated together in mice with GW-39 tumors
expressing higher CEA levels, as a result of pretreatment of GW-39
stock tumors (10% GW-39 cell suspension) with interferon-.gamma.
(IFN.gamma.). The experiments involving interferon-gamma enhancing
the antitumor effects of naked CEA antibody (hMN-14) were conducted
as follows.
[0285] First, GW-39 human colon cancer was grown subcutaneously in
a mouse that received 100,000 units of IFN-gamma twice a day for 4
days. A control mouse with GW-39 tumor was not given IFN.
Experimental mice were injected i.v. with a 5% suspension of GW-39
(w/v) from either of the two mice (i.e., with or without IFN
treatment) into two groups of eight. Four of each received tumor
from the IFN-treated mice and four from the untreated mice. One
group of 8 mice then received hMN-14 (100 ug per day.times.14 days
and then twice weekly thereafter until expt was ended), another
group CPT-11 at 160 ug/day.times.5 days (=20% of maximum tolerated
dose), a third group received the same doses of antibody+drug
combined, and a fourth group that was not treated at all. Animal
weights were measured and survival determined weekly. Also, samples
of stock tumor treated with IFN in the mice that were later
implanted were also processed for immunohistology to assess
increase in CEA expression in the tumors from mice treated with
IFN-gamma, and this was controlled by also treating the suspensions
by immunohistology with an irrelevant IgG, such as Ag8, which
showed no CEA staining.
EXAMPLE 11
[0286] A comparison was performed of the effects of naked hMN-14
CEA Mab on low and high (induced by interferon-gamma, as described
earlier) CEA-expressing tumor cells in an animal model. The results
demonstrate that increased expression of CEA antigen on tumor cells
correlates with improved efficacy of anti-CEA antibody. The results
of the comparison study are shown in FIG. 21. Thus,
interferon-gamma pre-treatment is useful to boost the efficacy of
anti-CEA antibody therapy in the treatment of cancer.
EXAMPLE 12--SIGMOID COLON CANCER THERAPY WITH CEA ANTIBODY AND
GM-CSF
[0287] JR is a 62-year-old man who is refractive to chemotherapy
with 5-fluorouracil and leukovorin to reduce his metastases to the
liver found at the time of discovery and removal of his sigmoid
colon cancer. His plasma titer of carcinoembryonic antigen (CEA) at
presentation is 34 ng/mL, and computed tomography of the liver
shows several small lesions measuring between 2 and 4 cm in
diameter in the right lobe; other radiological studies appear to be
normal. Immunotherapy with humanized anti-CEA IgG1, hMN-14,
monoclonal antibody is begun on a weekly basis for 4 weeks, at an
intravenous dose of 300 mg/m.sup.2) infused over 2 hours. One week
prior to hMN-14 therapy, the patient receives 2 subcutaneous
injections of 200 mcg/m2 GM-CSF (sargamostim, Leukine.RTM.), 3 days
apart, and continued twice weekly during the 4 weeks of hMN-14
therapy. After these four weeks, both hMN-14 and GM-CSF are given
at the same doses every other week for an additional 3 months, but
the dose of GM-CSF is increased to 250 mcg/m.sup.2. Prior to each
administration of the humanized CEA antibody, the patient is given
diphenhydramine (Benadryl.RTM., 50 mg orally), and acetaminophen
(Tylenol.RTM., 500 mg orally). At this time, the patient is
restaged, with CT measurements made of the liver metastases and
diverse radiological scans of the rest of the body. Blood is also
taken for chemistries and for determination of his blood CEA titer.
No areas of disease outside of the liver are noted, but the sum of
the diameters of the measurable tumors in the liver appear to
decrease by 40 percent, and the patient's blood CEA titer decreases
to 18 ng/mL, thus indicating a therapeutic response. Immunotherapy
with hMN-14 and GM-CSF, given once every other week at 200
mg/m.sup.2 for hMN-14 and 250 mcg/m.sup.2 for GM-CSF, are
administered for another 2 months, and restaging shows additional
decrease in the sum of the diameters of the liver tumors and a fall
in the CEA titer to 10 ng/mL. Since tumor decrease is measured as
being >65% over the pre-therapy baseline, the therapy is
considered to have provided a partial response. After this, the
doses were made less frequent, once every month for the next six
months, and all studies indicate no change in disease. The patient
is then followed for another 10 months, and remains in a partial
remission, with no adverse reactions to the therapy, and generally
without any symptoms of disease.
EXAMPLE 13--COMBINED IMMUNOTHERAPY AND CHEMOTHERAPY OF METASTATIC
COLON CANCER
[0288] ST is a 52-year-old woman presenting with liver and lung
metastases of colon cancer following resection of the primary
tumor. She is placed on a combined chemotherapy and immunotherapy
protocol based on the Gramont schedule (A. de Gramont et al, J Clin
Oncol. 2000; 18:2938-1947), but with the addition of humanized
anti-CEA monoclonal antibody IgG.sub.1. Prior to infusions of the
antibody, she receives 50 mg orally of diphenhydramine
(Benadryl.RTM.) and 500 mg orally of acetaminophen (Tylenol.RTM.).
She receives a 2-hr infusion of leucovorin (200 mg/m.sup.2/day)
followed by a bolus of 5-fluorouracil (400 mg/m.sup.2/day) and
22-hour continuous infusion of 5-fluorouracil (600 mg/m.sup.2/day)
for 2 consecutive days every 2 weeks, together with oxaliplatin at
85 mg/m.sup.2 as a 2-hr infusion in 250 mL of dextrose 5%,
concurrent with leukocorin on day 1 (FOLFOX4 schedule). The patient
also receives anti-emetic prophylaxis with a
5-hydroxytryptamine-3-receptor antagonist. One week prior to this
2-week chemotherapy cycle, hMN-14 monoclonal anti-CEA antibody is
infused over 2 hrs at a dose of 200 mg/m.sup.2, and repeated each
week of the 2-week chemotherapy cycle, and every week thereafter
for the next month with another chemotherapy cycle. Also, a
subcutaneous dose of 5 mcg/kg/day of G-CSF (filgrastim,
Neupogen.RTM.) is administered once weekly beginning with the
second chemotherapy cycle, and continued at this dose for the
duration of immunotherapy with hMN-14 antibody, over the next 3
months. A total of 5 cycles of chemotherapy with continued
administration of hMN-14 antibody and filgrastim. Thereafter, hMN14
and filgrastim therapy is given, at the same doses, every other
week for the next 3 months, without chemotherapy. The patient is
staged 2 months later, and her liver and lung metastases show
shrinkage by computed tomography measurements of >80 percent of
disease measured in the liver and lungs, as compared to the
measurements made prior to therapy. Her blood CEA titer also shows
a drop from the pre-therapy level of 63 ng/mL to 9 ng/mL. She is
followed over the next 6 months, and her disease appears to be
stable, with no new lesions found and no increase in the disease
remaining in the liver and lungs. The patient's predominant
toxicity is peripheral sensory neuropathy, which consists of
laryngeopharyngeal dysesthesia. The patient also experiences
diarrhea, mucositis, nausea and vomiting during the chemotherapy
cycles, but these are not excessive. She does not experience any
adverse events when only immunotherapy is administered, and is able
to return to full-time activities without any significant
restrictions.
EXAMPLE 14
[0289] FIG. 16 shows the effects of naked hMN-14 CEA Mab and CPT-11
treatment in an advanced colon cancer model. hMN-14 was given to
mice at a dose of 100 .mu.g/day over 14 days and then 2 times/week
thereafter, starting on day 0 after tumor implantation. CPT-11 was
given at 60 .mu.g/day over 5 days. No effect of hMN-14 by itself is
apparent under these conditions, and only a modest effect of CPT-11
(p<0.05) was observed. However, hMN-14 increases the effect of
CPT-11, as seen by comparing the CPT-11 median survival of 63 days
vs. combination therapy median survival of 77 days (p<0.005).
Combination therapy with hMN-14 CEA Mab and CPT-11 significantly
prolongs survival of an animal with advanced human colonic tumor
metastasis.
EXAMPLE 15
[0290] FIG. 17 shows the effects of naked hMN-14 CEA Mab and CPT-11
treatment in a low tumor burden cancer model. In a reduced tumor
burden model utilizing a 5% tumor cell suspension, CPT-11, hMN-14
alone, and combination therapy of hMN-14+CPT-11 were compared.
Dosages were as indicated Example 14. There was no apparent effect
of hMN-14 alone under these conditions. CPT-11 alone resulted in a
median survival time of 70 days. By contrast, the combination
therapy produced a median survival time of 91 days (p<0.025).
The combination of hMN-14 and CPT-11 significantly prolongs
survival of animals with low tumor burden in a metastatic model of
human colonic cancer.
EXAMPLE 16
[0291] FIG. 18 shows the effects of pre-treatment with naked hMN-14
CEA Mab given 3 days prior to CPT-11 treatment in a cancer model.
In a reduced tumor burden model utilizing a 5% tumor cell
suspension, CPT-11, hMN-14 alone, and combination therapy of
hMN-14+CPT-11 where the hMN-14 was administered 3 days prior to the
CPT-11 were compared. Dosages were as indicated Example 14. hMN-14
alone increased median survival time by 21% (p<0.05) under these
conditions. CPT 11 alone increased survival by 76% (p<0.001). By
contrast, the combination therapy where hMN-14 is administered 3
days prior to CPT-11 produced a median survival time increase of an
additional 58% above CPT-11 alone (p<<0.001 compared with
CPT-11 alone). Pre-treatment with hMN-14 significantly prolongs
survival of animals with low tumor burden in a metastatic model of
human colonic cancer.
EXAMPLE 17
[0292] FIG. 19 shows a comparison of various administration
schedules of naked hMN-14 CEA Mab and CPT-11 in a human colon
cancer model. Giving hMN14 3 days before CPT-11 is the most
effective. Dosages were as indicated Example 14. When the order is
reversed (CPT-11 is given 3 days before hMN-14) or when both are
given together at the same time, median survival time of 70 days
was an increase over the untreated control group (35 days) but was
still significantly less than the median survival time of 105 days
with the hMN-14 pre-treatment 3 days before CPT-11.
EXAMPLE 18
[0293] FIG. 20 shows the effects of GM-CSF pre-treatment on naked
hMN-14 CEA Mab therapy in a human colon cancer model. GM-CSF was
administered at a dose of 1 .mu.g/mouse/day on days -4, -3, -2, and
-1. Tumor cells were implanted at day 0 along with hMN-14
treatments. Other dosages were as indicated Example 14. The GM-CSF
pre-treatment resulted in a statistically significant increase in
median survival time (p<0.002) over either GM-CSF alone or
hMN-14 alone.
[0294] 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.
[0295] All of the publications and patent applications and patents
cited in this specification are herein incorporated in their
entirety by reference.
Sequence CWU 1
1
27 1 357 DNA Mus sp. CDS (1)..(357) 1 gag gtg aag ctt ctc gag tct
gga ggt ggc ctg gtg cag tct gga gga 48 Glu Val Lys Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Ser Gly Gly 1 5 10 15 tcc ctg aaa ctc tcc
tgt gca gcc tca gga ttc gat ttt act aca tat 96 Ser Leu Lys Leu Ser
Cys Ala Ala Ser Gly Phe Asp Phe Thr Thr Tyr 20 25 30 tgg atg agt
tgg gtc cgg cag gct cca ggg aaa ggc cta gaa tgg att 144 Trp Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 gga
gaa att cat cca gat agc agt acg att aac tat gcg ccg tct cta 192 Gly
Glu Ile His Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu 50 55
60 aag gat aaa ttc atc gtc tcc aga gac aac gcc aaa aat acg ctg tac
240 Lys Asp Lys Phe Ile Val Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80 ctg caa atg agc aaa gtg aga tct gag gac aca gcc ctt tat
tac tgt 288 Leu Gln Met Ser Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr
Tyr Cys 85 90 95 gca agc ctt tac ttc ggc ttc ccc tgg ttt gct tat
tgg ggc caa ggg 336 Ala Ser Leu Tyr Phe Gly Phe Pro Trp Phe Ala Tyr
Trp Gly Gln Gly 100 105 110 act ccg gtc act gtc tct gca 357 Thr Pro
Val Thr Val Ser Ala 115 2 119 PRT Mus sp. 2 Glu Val Lys Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Ser Gly Gly 1 5 10 15 Ser Leu Lys Leu
Ser Cys Ala Ala Ser Gly Phe Asp Phe Thr Thr Tyr 20 25 30 Trp Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45
Gly Glu Ile His Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu 50
55 60 Lys Asp Lys Phe Ile Val Ser Arg Asp Asn Ala Lys Asn Thr Leu
Tyr 65 70 75 80 Leu Gln Met Ser Lys Val Arg Ser Glu Asp Thr Ala Leu
Tyr Tyr Cys 85 90 95 Ala Ser Leu Tyr Phe Gly Phe Pro Trp Phe Ala
Tyr Trp Gly Gln Gly 100 105 110 Thr Pro Val Thr Val Ser Ala 115 3
318 DNA Mus sp. CDS (1)..(318) 3 gaa att cag ctg acc cag tct cac
aaa atg atg tcc aca tca gtg gga 48 Glu Ile Gln Leu Thr Gln Ser His
Lys Met Met Ser Thr Ser Val Gly 1 5 10 15 gac agg gtc agc atc acc
tgc aag gcc agt cag gat gtg ggt act tct 96 Asp Arg Val Ser Ile Thr
Cys Lys Ala Ser Gln Asp Val Gly Thr Ser 20 25 30 gta gcc tgg tat
caa cag aga cca gga caa tct cct aaa cta ctg att 144 Val Ala Trp Tyr
Gln Gln Arg Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 tac tgg
aca tcc acc cgg cac act gga gtc cct gat cgc ttc aca ggc 192 Tyr Trp
Thr Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60
agt gtg tct ggg aca gat ttc act ctc acc att acc aat gtg cag tct 240
Ser Val Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Val Gln Ser 65
70 75 80 gaa gac ttg gca gat tat ttc tgt cag caa tat agc ctc tat
cgg tcg 288 Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Leu Tyr
Arg Ser 85 90 95 ttc ggt gga ggc acc aaa ctg gag atc aaa 318 Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 4 106 PRT Mus sp. 4 Glu
Ile Gln Leu Thr Gln Ser His Lys Met Met Ser Thr Ser Val Gly 1 5 10
15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ser
20 25 30 Val Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Lys Leu
Leu Ile 35 40 45 Tyr Trp Thr Ser Thr Arg His Thr Gly Val Pro Asp
Arg Phe Thr Gly 50 55 60 Ser Val Ser Gly Thr Asp Phe Thr Leu Thr
Ile Thr Asn Val Gln Ser 65 70 75 80 Glu Asp Leu Ala Asp Tyr Phe Cys
Gln Gln Tyr Ser Leu Tyr Arg Ser 85 90 95 Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys 100 105 5 117 PRT Homo sapiens 5 Gln Val Gln Leu
Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu
Ser Leu Thr Cys Thr Val Ser Gly Ser Thr Phe Ser Asn Asp 20 25 30
Tyr Tyr Thr Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile 35
40 45 Gly Tyr Val Phe Tyr His Gly Thr Ser Asp Asp Thr Thr Pro Leu
Arg 50 55 60 Ser Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn Gln
Phe Ser Leu 65 70 75 80 Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys Ala 85 90 95 Arg Asn Leu Ile Ala Gly Cys Ile Asp
Val Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 6
107 PRT Homo sapiens 6 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala
Ser Gln Asp Ile Ile Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Ser Asn
Leu Gln Ala Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Gln Ser Leu Pro Tyr 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 7 126 PRT
Homo sapiens 7 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln
Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ser Ser Gly Phe
Ile Phe Ser Ser Tyr 20 25 30 Ala Met Tyr Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ile Ile Trp Asp Asp Gly
Ser Asp Gln His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met
Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys 85 90 95 Ala
Arg Asp Gly Gly His Gly Phe Cys Ser Ser Ala Ser Cys Phe Gly 100 105
110 Pro Asp Tyr Trp Gly Gln Gly Thr Pro Val Thr Val Ser Ser 115 120
125 8 119 PRT Homo sapiens 8 Gln Val Gln Leu Gln Glu Ser Gly Pro
Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr
Ala Ser Gly Phe Asp Phe Thr Thr Tyr 20 25 30 Trp Met Ser Trp Val
Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile
His Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu 50 55 60 Lys
Asp Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70
75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Ser Leu Tyr Phe Gly Phe Pro Trp Phe Ala Tyr Trp
Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 9 119 PRT
Homo sapiens 9 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg
Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe
Asp Phe Thr Thr Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Pro Pro
Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile His Pro Asp Ser
Ser Thr Ile Asn Tyr Ala Pro Ser Leu 50 55 60 Lys Asp Arg Val Thr
Met Leu Arg Asp Thr Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Arg Leu
Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Ser Leu Tyr Phe Gly Phe Pro Trp Phe Ala Tyr Trp Gly Gln Gly 100 105
110 Thr Thr Val Thr Val Ser Ser 115 10 119 PRT Homo sapiens 10 Gln
Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10
15 Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Asp Phe Thr Thr Tyr
20 25 30 Trp Met Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu
Trp Ile 35 40 45 Gly Glu Ile His Pro Asp Ser Ser Thr Ile Asn Tyr
Ala Pro Ser Leu 50 55 60 Lys Asp Arg Val Thr Met Leu Arg Asp Thr
Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Lys Val Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Leu Tyr Phe Gly
Phe Pro Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr
Val Ser Ser 115 11 119 PRT Homo sapiens 11 Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu
Thr Cys Thr Ala Ser Gly Phe Asp Phe Thr Thr Tyr 20 25 30 Trp Met
Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45
Gly Glu Ile His Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu 50
55 60 Lys Asp Lys Phe Ile Val Ser Arg Asp Thr Ser Lys Asn Gln Phe
Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Ser Leu Tyr Phe Gly Phe Pro Trp Phe Ala
Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 12
119 PRT Homo sapiens 12 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val
Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ser Ser
Gly Phe Asp Phe Thr Thr Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Glu Ile His Pro
Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu 50 55 60 Lys Asp Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu
Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys 85 90
95 Ala Ser Leu Tyr Phe Gly Phe Pro Trp Phe Ala Tyr Trp Gly Gln Gly
100 105 110 Thr Pro Val Thr Val Ser Ser 115 13 119 PRT Homo sapiens
13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Asp Phe Thr
Thr Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Ile 35 40 45 Gly Glu Ile His Pro Asp Ser Ser Thr Ile
Asn Tyr Ala Pro Ser Leu 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asp Ser Leu
Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys 85 90 95 Ala Ser Leu Tyr
Phe Gly Phe Pro Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Pro
Val Thr Val Ser Ser 115 14 119 PRT Homo sapiens 14 Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ser Ala Ser Gly Phe Asp Phe Thr Thr Tyr 20 25 30
Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35
40 45 Gly Glu Ile His Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser
Leu 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Thr Leu Phe 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr
Gly Val Tyr Phe Cys 85 90 95 Ala Ser Leu Tyr Phe Gly Phe Pro Trp
Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Pro Val Thr Val Ser Ser
115 15 119 PRT Homo sapiens 15 Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser
Ala Ser Gly Phe Asp Phe Thr Thr Tyr 20 25 30 Trp Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile
His Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu 50 55 60 Lys
Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe
Cys 85 90 95 Ala Ser Leu Tyr Phe Gly Phe Pro Trp Phe Ala Tyr Trp
Gly Gln Gly 100 105 110 Thr Pro Val Thr Val Ser Ser 115 16 357 DNA
Homo sapiens CDS (1)..(357) 16 gag gtc caa ctg gtg gag agc ggt gga
ggt gtt gtg caa cct ggc cgg 48 Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg 1 5 10 15 tcc ctg cgc ctg tcc tgc tcc
gca tct ggc ttc gat ttc acc aca tat 96 Ser Leu Arg Leu Ser Cys Ser
Ala Ser Gly Phe Asp Phe Thr Thr Tyr 20 25 30 tgg atg agt tgg gtg
aga cag gca cct gga aaa ggt ctt gag tgg att 144 Trp Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 gga gaa att
cat cca gat agc agt acg att aac tat gcg ccg tct cta 192 Gly Glu Ile
His Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu 50 55 60 aag
gat aga ttt aca ata tcg cga gac aac gcc aag aac aca ttg ttc 240 Lys
Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe 65 70
75 80 ctg caa atg gac agc ctg aga ccc gaa gac acc ggg gtc tat ttt
tgt 288 Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe
Cys 85 90 95 gca agc ctt tac ttc ggc ttc ccc tgg ttt gct tat tgg
ggc caa ggg 336 Ala Ser Leu Tyr Phe Gly Phe Pro Trp Phe Ala Tyr Trp
Gly Gln Gly 100 105 110 acc ccg gtc acc gtc tcc tca 357 Thr Pro Val
Thr Val Ser Ser 115 17 119 PRT Homo sapiens 17 Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ser Ala Ser Gly Phe Asp Phe Thr Thr Tyr 20 25 30 Trp
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40
45 Gly Glu Ile His Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu
50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
Leu Phe 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly
Val Tyr Phe Cys 85 90 95 Ala Ser Leu Tyr Phe Gly Phe Pro Trp Phe
Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Pro Val Thr Val Ser Ser 115
18 318 DNA Homo sapiens CDS (1)..(318) 18 gac atc cag ctg acc cag
agc cca agc agc ctg agc gcc agc gtg ggt 48 Asp Ile Gln Leu Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 gac aga gtg acc
atc acc tgt aag gcc agt cag gat gtg ggt act tct 96 Asp Arg Val Thr
Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ser 20 25 30 gta gct
tgg tac cag cag aag cca ggt aag gct cca aag ctg ctg atc 144 Val Ala
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45
tac tgg aca tcc acc cgg cac act ggt gtg cca agc aga ttc agc ggt 192
Tyr Trp Thr Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 agc ggt agc ggt acc gac ttc acc ttc acc atc agc agc ctc cag
cca 240 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln
Pro 65 70 75 80 gag gac atc gcc acc tac tac tgc cag caa tat agc ctc
tat cgg tcg 288 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Leu
Tyr Arg Ser 85 90 95 ttc ggc caa ggg acc aag gtg gaa atc aaa 318
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 19 106 PRT
Homo sapiens 19 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln
Asp Val Gly Thr Ser 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Thr Ser Thr Arg His
Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile
Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Leu Tyr Arg Ser 85 90 95 Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 20 11 PRT Mus sp. 20
Lys Ala Ser Gln Asp Val Gly Thr Ser Val Ala 1 5 10 21 7 PRT Mus sp.
21 Trp Thr Ser Thr Arg His Thr 1 5 22 8 PRT Mus sp. 22 Gln Gln Tyr
Ser Leu Tyr Arg Ser 1 5 23 5 PRT Mus sp. 23 Thr Tyr Trp Met Ser 1 5
24 17 PRT Mus sp. 24 Glu Ile His Pro Asp Ser Ser Thr Ile Asn Tyr
Ala Pro Ser Leu Lys 1 5 10 15 Asp 25 10 PRT Mus sp. 25 Leu Tyr Phe
Gly Phe Pro Trp Phe Ala Tyr 1 5 10 26 119 PRT Homo sapiens 26 Gln
Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10
15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Thr Phe Ser Thr Tyr
20 25 30 Trp Met Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu
Trp Ile 35 40 45 Gly Glu Ile His Pro Asp Ser Ser Thr Ile Asn Tyr
Ala Pro Ser Leu 50 55 60 Lys Asp Arg Val Thr Met Leu Val Asp Thr
Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Tyr Phe Gly
Phe Pro Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr
Val Ser Ser 115 27 119 PRT Homo sapiens 27 Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ser Ser Ser Gly Phe Ile Phe Ser Thr Tyr 20 25 30 Trp Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Glu Ile His Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu 50
55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Phe 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val
Tyr Phe Cys 85 90 95 Ala Arg Leu Tyr Phe Gly Phe Pro Trp Phe Ala
Tyr Trp Gly Gln Gly 100 105 110 Thr Pro Val Thr Val Ser Ser 115
* * * * *