U.S. patent application number 09/921290 was filed with the patent office on 2002-04-11 for immunotherapy of malignant and autoimmune disorders in domestic animals using naked antibodies, immunoconjugates and fusion proteins.
Invention is credited to Goldenberg, David M..
Application Number | 20020041847 09/921290 |
Document ID | / |
Family ID | 23191283 |
Filed Date | 2002-04-11 |
United States Patent
Application |
20020041847 |
Kind Code |
A1 |
Goldenberg, David M. |
April 11, 2002 |
Immunotherapy of malignant and autoimmune disorders in domestic
animals using naked antibodies, immunoconjugates and fusion
proteins
Abstract
49. B-cell, T-cell, myeloid-cell, mast-cell, and plasma-cell
disorders are significant contributors to illness and mortality in
domestic animals, especially in companion animals such as dogs and
cats. These disorders include both autoimmune disorders and
malignancies, such as the B-cell subtype of non-Hodgkin's lymphoma,
acute and chronic lymphocytic or myeloid leukemias, multiple
myeloma, and mastocytomas. Antibody components that bind with
B-cell or T-cell antigens or epitopes, as well as antigens or
epitopes of myeloid, plasma and mast cells provide an effective
means to treat these disorders in domestic animals. The
immunotherapy uses naked antibodies, immunoconjugates and fusion
proteins, alone or in combination with standard therapeutic
regimens.
Inventors: |
Goldenberg, David M.;
(Mendham, NJ) |
Correspondence
Address: |
Bernhard D. Saxe
FOLEY & LARDNER
Washington Harbour
3000 K Street, N.W., Suite 500
Washington
DC
20007-5109
US
|
Family ID: |
23191283 |
Appl. No.: |
09/921290 |
Filed: |
August 3, 2001 |
Related U.S. Patent Documents
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Application
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Filing Date |
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09921290 |
Aug 3, 2001 |
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09038995 |
Mar 12, 1998 |
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6134982 |
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09038995 |
Mar 12, 1998 |
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09307816 |
May 10, 1999 |
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6306393 |
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Current U.S.
Class: |
424/1.49 ;
424/154.1; 424/178.1 |
Current CPC
Class: |
A61P 35/02 20180101;
A61K 2039/505 20130101; C07K 16/2887 20130101; A61K 39/395
20130101; A61K 38/00 20130101; A61P 35/00 20180101; A61K 39/39558
20130101; A61K 2039/507 20130101; C07K 16/2803 20130101; A61K
39/395 20130101; A61K 2300/00 20130101; A61K 39/39558 20130101;
A61K 2300/00 20130101 |
Class at
Publication: |
424/1.49 ;
424/178.1; 424/154.1 |
International
Class: |
A61K 039/395; A61K
051/00 |
Claims
What is claimed is:
1. A method for treating a B-cell, T-cell, myeloid-cell, mast-cell,
or plasma-cell disorder in a domestic animal, comprising
administering to a domestic animal having said disorder a
therapeutic composition comprising a pharmaceutically acceptable
carrier and at least one antibody component that is specific to a
B-cell, T-cell, myeloid, mast cell, or plasma cell antigen or
epitope in said domestic animal.
2. The method of claim 1, wherein said antibody component is a
naked antibody.
3. The method of claim 1, wherein said antibody component is an
immunoconjugate.
4. The method of claim 1, wherein said antibody is combined with
the administration of another antibody that shows higher
specificity for tumors of these cells as compared to their normal
counterparts.
5. The method of claim 3, wherein said immunoconjugate is a
radiolabeled immunoconjugate.
6. The method of claim 3, wherein said immunoconjugate comprises a
cytokine.
7. The method of claim 3, wherein said immunoconjugate comprises a
drug or toxin.
8. The method of claim 1, wherein said antibody component is part
of a fusion protein.
9. The method of claim 1, wherein said B-cell, T-cell, myeloid,
mast cell, or plasma cell disorder is a malignancy.
10. The method of claim 1, wherein said B-cell, T-cell, myeloid,
mast cell, or plasma cell disorder is an autoimmune disease and
said antibody component is specific to a B-cell or T-cell.
11. The method of claim 1, for treating a B-cell or T-cell
disorder, wherein said antibody component binds to both a B-cell
and a T-cell antigen.
12. The method of claim 11, wherein said disorder is a B- or T-cell
malignancy.
13. The method of claim 11, wherein said disorder is an autoimmune
disease.
14. The method of claim 1, additionally comprising administering a
cytokine.
15. The method of claim 1, additionally comprising administering a
chemotherapeutic agent.
16. The method of claim 1, wherein said domestic animal is a
companion animal.
17. The method of claim 16, wherein said companion animal is a dog
or a cat.
18. The method of claim 1, wherein said domestic animal is a
horse.
19. The method of claim 1, wherein said antibody component is an
antibody against a domestic animal equivalent of the human CD4,
CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD25, CD33, CD38,
CD52, CD54, CD74, CD126 MUC1, Ia, HM1.24, or HLA-DR antigen.
20. The method of claim 19, wherein said antibody component is a
radiolabeled antibody component.
21. The method of claim 19, wherein said antibody component is a
naked antibody.
22. The method of claim 19, wherein said antibody component is a
naked antibody that is specific for a malignancy of B or T cells,
myeloid cells, plasma cells, or mast cells.
23. The method of claim 19, wherein said therapeutic composition
comprises a combination of an antibody component and a
chemotherapeutic agent or immunomodulator.
24. The method of claim 19, wherein said therapeutic composition
comprises a combination of a naked antibody and an immunoconjugate
or fusion protein.
25. The method of claim 19, wherein said therapeutic composition
comprises a combination of two or more naked antibodies against
different epitopes of the same antigen or against different
antigens associated with one cell type.
26. The method of claim 19, wherein said therapeutic composition
comprises a combination of a naked antibody and a radiolabled
immunoconjugate.
27. The method of claim 19, wherein said therapeutic composition
comprises a combination of a naked antibody and a toxin
immunoconjugate.
28. The method of claim 27, wherein said toxin immunoconjugate
comprises an RNase.
29. The method of claim 28, wherein said RNase is a recombinant
RNase.
30. The method of claim 29, wherein the antibody component
comprises a neutron-capturing boron addend.
31. The method of claim 29, wherein the antibody component
comprises a photoactive agent or dye.
32. The method of claim 1, wherein the antibody component comprises
a multispecific antibody.
33. The method of claim 1, wherein the antibody component comprises
a bispecific antibody.
34. The method of claim 33, wherein said antibody component
comprises an arm that is specific for a low-molecular weight hapten
and wherein a low-molecular weight hapten with an attached
therapeutic agent is administered after the antibody component that
is specific to a B-cell, T-cell, myeloid, mast cell, or plasma cell
antigen or epitope is administered and has bound to the antigen or
epitope.
35. The method of claim 34, wherein the therapeutic agent is a
radionuclide.
36. The method of claim 34, wherein the therapeutic agent is a
drug.
37. The method of claim 1, wherein said therapeutic composition
comprises a combination of a chemotherapeutic agent and an antibody
component labeled with a therapeutic radionuclide.
38. The method of claim 1, wherein said therapeutic composition
comprises a combination of antibody components which are labeled
with different radionuclides.
39. The method of claim 1, additionally comprising comprising first
administering to said domestic animal a diagnostic composition
comprising a pharmaceutically acceptable carrier and at least one
antibody component that is specific to a B-cell, T-cell, myeloid,
mast cell, or plasma cell antigen or epitope in said domestic
animal, wherein said antibody component is coupled to a diagnostic
agent.
40. The method of claim 39, wherein said antibody component
comprises an arm that is specific for a low-molecular weight hapten
to which the diagnostic agent is conjugated or fused.
41. The method of claim 1, wherein said therapeutic composition
comprises a combination of naked antibodies.
42. The method of claim 41, wherein said therapeutic composition
comprises a fusion protein of said combination of antibodies.
43. The method of claim 1, wherein said therapeutic composition
comprises a combination of naked antibodies and
immunoconjugates.
44. The method claim 1, wherein said therapeutic composition
comprises a hybrid antibody that binds to more than one B-cell,
T-cell, myeloid-cell, mast-cell or plasma-cell antigen.
45. The method of claim 1, additionally comprising administering at
least one chemotherapeutic drug.
46. The method of claim 1, additionally comprising administering
radiation therapy.
47. The method of claim 1, additionally comprising administering
cytokine therapy.
48. The method claim 1, additionally comprising administering an
immunosuppressive agent.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to immunotherapeutic methods
for treating B-cell and T-cell, myeloid, mast-cell and plasma-cell
disorders in domestic animals, particularly in companion animals
such as dogs, cats, and horses. In particular, this invention is
directed to methods for treating B-cell and T-cell, myeloid, mast
cell, and plasma-cell disorders by administering comparatively low
doses of naked antibodies against antigens associated with these
cell types, which are equivalent to the respective normal lineage
and non-lineage antigens present in similar normal cells of humans,
by antibody given alone, by antibody combinations in which each
antibody binds to a different target antigen or antigen epitope, or
by administering an immunoconjugate in which at least one antibody
component is conjugated to a therapeutic agent. The present
invention also is directed to multimodal therapeutic methods in
which naked antibody or immunoconjugate administration is
supplemented with chemotherapy, radiotherapy, cytokines, or by
administration of therapeutic proteins, such as antibody fusion
proteins. The present invention also contemplates the combination
of such antibodies and antibody conjugates with lymphoma-,
leukemia-, or myeloma-specific antibodies, which bind more
selectively to such malignant cells than to their normal cell
counterparts. Examples of such lymphoma-specific antibodies are
those described, in U.S. Pat. No. 5,169,775, issued to Steplewski
et al., which are preferentially reactive with canine lymphoma
cells and insignificantly reactive with normal lymphocytes, and do
not react with DR antigens of the dog, which are equivalent with
HLA-DR antigens in humans and Ia antigens in humans and mice.
Although the antibodies of this invention react with both normal
and malignant cells of domestic animals, administration of these
antibodies in animals having malignancies of these cell populations
results in tumor responses and only minimal side effects because of
the concomitant transient depletion of target normal cells.
[0003] 2. Background
[0004] B- and T-cell lymphomas and leukemias, such as the B-cell
subtype of non-Hodgkin's lymphoma (NHL) and T-cell leukemias, are
significant contributors to cancer mortality in domestic animals
and are on the increase, particularly in companion animals such as
dogs and cats. Significant similarities between human and canine
NHL have been reported. See, for example, Fournel-Fleury, et al.,
J. Comp. Pathol., 117(1):35-59 (1997); Ruslander et al., In Vivo,
11(2):169-72 (1997). In particular, fine-needle aspirates from 21
dogs with peripheral lymphadenopathy (18 with lymphoma and three
with lymph node hyperplasia) showed that 14 of the lymphomas were
B-cell lymphomas. Caniatti et al., Vet. Pathol., 33(2):204-12
(1996). Ruslander et al. reported that 76% (134/175) of dogs with
lymphoma were determined to be derived from B-cells. Similarly, Day
et al. reported in a study based on eight cats that the clinical,
histological and immunophenotypic findings in cats were identical
with those of NHL in humans. J. Comp. Pathol., 120(2):155-67
(1999).
[0005] The response of B-cell and T-cell malignancies to various
forms of treatment is mixed in both humans and animals. For
example, in humans in cases in which adequate clinical staging of
non-Hodgkin's lymphoma is possible, field radiation therapy can
provide satisfactory treatment. Still, about one-half of the
patients die from the disease. In dogs, standard treatment involves
chemotherapy with a combination of vincristine, cyclophosphamide,
prednisolone, doxorubicin, and L-asparaginase.
[0006] The majority of chronic lymphocytic leukemias in humans are
of B-cell lineage. Freedman, Hematol. Oncol. Clin. North Am. 4:405
(1990). This type of B-cell malignancy is the most common leukemia
in the Western world. Goodman et al., Leukemia and Lymphoma 22:1
(1996). The natural history of chronic lymphocytic leukemia falls
into several phases. In the early phase, chronic lymphocytic
leukemia is an indolent disease, characterized by the accumulation
of small mature functionally-incompeten- t malignant B-cells having
a lengthened life span. Eventually, the doubling time of the
malignant B-cells decreases and patients become increasingly
symptomatic. While treatment can provide symptomatic relief, the
overall survival of the patients is only minimally affected. The
late stages of chronic lymphocytic leukemia are characterized by
significant anemia and/or thrombocytopenia. At this point, the
median survival is less than two years. Foon et al., Annals Int.
Medicine 113:525 (1990). Due to the very low rate of cellular
proliferation, chronic lymphocytic leukemia is resistant to
treatment.
[0007] B-cell leukemias have also been identified in canines.
Nakaichi et al., J. Vet. Med. Sci. 58(5):469-71 (1996). Canines and
felines also are afflicted with numerous lymphoproliferative
disorders. In one study of 175 dogs with lymphoma, 134 were
determined to be derived from B-cells and 38 were derived from
T-cells. Ruslander et al., In Vivo 11(2):169-172 (1997). Day et al.
reported on cases of T-cell rich, B-cell lymphoma in cats.
Treatment options for domestic animals generally are limited to
chemotherapy, however.
[0008] Canine lymphoma has been found to be similar to human
non-Hodgkin's lymphoma in pathological types, response to the same
chemotherapeutic agents, correlation of immunophenotyping of cell
surface markers to histological classification and
chemosensitivity, and distribution of B, T, and non-T, non-B cell
lymphomas (Macewen et al., J. Am. Vet. Med. Assoc. 178:1178,
(1981); Applebaum et al., Hematol. Oncol. 2:151, (1984); Carter et
al. Canad. J. Vet. Res. 50:154, (1986)). It is thus reasonable to
postulate that equivalent markers on B- and T-cells might serve as
useful targets for therapeutic approaches, as found in humans.
Similar markers on myeloid-, plasma-, and mast-cells, which
transform to malignancies in domestic animals, as in humans, may
also serve as targets for therapeutic agents. In contrast to
humans, however, mastocytomas are the most frequent neoplasm in
dogs, and thus may have a different pathogenesis and etiology than
in humans. Although lineage and non-lineage antigens associated
with these cell types will result in depletion of these cells when
specific antibodies are given, there will be an advantageous
suppression of the malignant cells without intolerable side effects
to the host animals by immunotherapeutic use of these antibodies.
Thus, we have discovered that an absolute or even high specificity
of such antibodies for malignant as compared to normal cells is not
required for achieving therapeutic responses in these domestic
animals. Indeed, the binding of such antibodies to the malignant
cells can be less than double that observed in their normal cell
counterparts, thus being markedly different from the invention of
Steplewski et al., infra.
[0009] Traditional methods of treating B-cell and T-cell
malignancies, including chemotherapy and radiotherapy, have limited
utility due to toxic side effects. The use of monoclonal antibodies
to direct radionuclides, toxins, or other therapeutic agents offers
the possibility that such agents can be delivered selectively to
tumor sites, thus limiting toxicity to normal tissues.
[0010] Antibodies against the CD20 antigen have been investigated
for the therapy of B-cell lymphomas in humans. For example, a
chimeric anti-CD20 antibody, designated as "IDEC-C2B8," has
activity against B-cell lymphomas when provided as unconjugated
antibodies at repeated injections of doses exceeding 500 mg per
injection. Maloney et al., Blood 84:2457 (1994); Longo, Curr. Opin.
Oncol. 8:353 (1996). About 50 percent of non-Hodgkin's patients,
having the low-grade indolent form, treated with this regimen
showed responses. Therapeutic responses have also been obtained
using .sup.131I-labeled B1 anti-CD-20 murine monoclonal antibody
when provided as repeated doses exceeding 600 mg per injection.
Kaminski et al., N. Engl. J. Med. 329:459 (1993); Press et al., N.
Engl. J. Med. 329:1219 (1993); Press et al., Lancet 346:336 (1995).
However, these antibodies, whether provided as unconjugated forms
or radiolabeled forms, have shown less impressive responses in
patients with the more prevalent and lethal form of B-cell
lymphoma, the intermediate or aggressive type.
[0011] A need exists to develop an immunotherapy for B-cell and
T-cell malignancies, as well as myeloid, mast-cell, and plasma-cell
(myeloma) malignancies, in domestic animals, particularly in
companion animals such as dogs and cats, that allows repeated
administration of comparatively low doses of an antibody, and that
is not limited by the necessity of adding a toxic agent for
achieving a therapeutic response of significant duration.
[0012] Autoimmune diseases are a class of diseases associated with
a B-cell or T-cell disorder. Examples include immune-mediated
autoimmune hemolytic anemia, canine granulomatous
meningoencephalitis, rheumatoid arthritis, chronic superficial
keratitis, systemic lupus erythematosus, bullous pemphigoid,
pemphigus, and thrombocytopenias. The most common treatments are
corticosteroids and cytotoxic drugs, which can be very toxic. These
drugs also suppress the entire immune system, can result in serious
infection, and have adverse affects on the liver and kidneys. Other
therapeutics that have been used to treat Class III autoimmune
diseases to date have been directed against T-cells and
macrophages. A need also exists for more effective methods of
treating autoimmune diseases, particularly Class III autoimmune
diseases.
SUMMARY OF THE INVENTION
[0013] The present invention provides methods of treating a B-cell,
T-cell, myeloid, mast-cell, or plasma-cell disorder in a domestic
animal, particularly in a companion animal such as a dog or cat.
The method entails the administration of antibodies directed
against antigen determinants found on cells of these malignant
neoplasms or which generate the autoimmune disease cells or
antibodies. These antibodies recognize antigens that are equivalent
to similar lineage and non-lineage antigens expressed by normal
human B-, T-, myeloid-, plasma-, and mast-cells. A complete listing
of normal human lineage and non-lineage antigens can be found in
the 5th CD Leucocyte Typing Workshop (1995), the contents of which
are incorporated herein in their entirety by reference. Animal
equivalents of these antigens, which may vary by species, are
readily identifiable and their use is preferred in methods
according to the present invention.
[0014] These antibodies can be either given by themselves as
unconjugated immunoglobulins, in antibody combinations, or as
antibody conjugates with isotopes, drugs, or other therapeutic
modalities, or still other applications of such monoclonal
antibodies in combination with other therapy modalities, such as
chemotherapy, radiation therapy, cytokine therapy, or bispecific
antibody fusion proteins involving other therapeutic agents or
multiple targets. Many of these antigen targets on suitable
malignant cells or normal hematopoietic or tissue cells involved in
generating automimmune disease are similar to or counterparts of
equivalent structures on human cells, such as B-cell complex,
T-cell, myeloid, mast-cell, plasma cell, and HLA-DR antigens.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0015] 1. Overview
[0016] Immunoconjugates as well as unconjugated or so-called
"naked" antibodies to any known antigen that is characteristic of a
B-cell, T-cell, myeloid-, plasma-, or mast-cell disorder can be
used in accordance with the present invention to treat B-cell,
T-cell, myeloid-, plasma-, or mast-cell disorder in domestic
animals, particularly in dogs and cats. Preferred B-cell antigens
include those equivalent to human CD19, CD20, CD21, CD22, CD52,
CD74, and CD5 antigens. Preferred T-cell antigens include those
equivalent to human CD4, CD8 and CD25 (the IL-2 receptor) antigens.
An equivalent to HLA-DR antigen can be used in treatment of both
B-cell and T-cell disorders. Particularly preferred B-cell antigens
are those equivalent to human CD19, CD20, CD21 and HLA-DR)
antigens. Particularly preferred T-cell antigens are those
equivalent to human CD4, CD8 and CD25 antigens.
[0017] The human CD33, CD14 and CD15 antigens are markers for
myeloid leukemias, and antibodies to equivalent antigens in
domestic animals are preferred in the treatment of such disorders
in domestic animals. MUC1 and Ia (HLA-DR) antigens are markers for
myeloma and other plasma-cell disorders, and their equivalents in
domestic animals can be used to treat corresponding disorders in
these animals.
[0018] The foregoing are exemplary, and not all-inclusive for
lineage and non-lineage markers of B-, T-, myeloid-, plasma-, and
mast-cells. Antibodies to equivalent antigens in domestic animals
to such lineage and non-lineage antigens associated with these cell
types can be used to treat such disorders in these domestic
species. It has been reported that some of these antigens present
on human cells are also detected in domestic animals when using
suitable antibodies against the human cell antigens (Jacobsen et
al., Vet. Immunol. Immunopathol. 39:461, (1993); Cobbold and
Metcalfe, Tissue Antigens 43:137, (1994); Moore et al., Tissue
Antigens 40:75, (1992); Greenlee et al., Vet. Immunol.
Immunopathol. 15:285, (1987); Aasted et al., Vet. Immunol.
Immunopathol. 19:31, (1988); Caniatti et al., Vet. Pathol. 33:204,
(1996); Darbes et al., J. Vet. Diagn. Invest. 9:94, (1997);
Grindem, Vet. Clin. N. Amer. 26:1043, (1996); Fournel-Fleury et
al., J. Comp. Pathol. 117:35, (1997)), all of which are
incorporated herein in their entirety. Corresponding canine
antibodies against such lineage antigens have been described by
Teske et al., Exper. Hematol. 22:1179, (1994). Ladiges et al., Am.
J. Vet. Res. 49:870, (1988), describes both canine and human
antibodies reacting with lineage antigens in canine lymphoma. Both
of these documents also are incorporated herein in their
entirety.
[0019] Bispecific antibody fusion proteins which bind to these
various antigens can be used according to the present invention,
including hybrid antibodies which bind to more than one B-cell,
T-cell, myeloid-cell, mast-cell or plasma-cell antigen. Preferably
the bispecific and hybrid antibodies target either a B-cell
antigen, a T-cell, a myeloid-cell, a mast-cell, a plasma-cell or a
macrophage antigen.
[0020] In accordance with the teaching of the present invention,
B-cell, T-cell, myeloid, mast-cell or plasma-cell disorders can be
treated with antibody therapy. The B-cell, T-cell, myeloid, mast
cell, or plasma cell disorder can be treated with its respective
normal-cell antibodies directed against the equivalent cell
antigens in domestic animals, or an autoimmune disease associated
with B-cells or T-cells, particularly a Class III autoimmune
disease, can be treated with equivalent B-cell, T-cell or HLA-DR
antigens in domestic animals.
[0021] In a preferred embodiment, an antibody component is used
that comprises an arm that is specific for a low-molecular weight
hapten to which a therapeutic agent is conjugated or fused. In this
case, the antibody pretargets B-cells, T-cells, myeloid cells, mast
cells or plasma cells, and the low-molecular weight hapten with the
attached therapeutic agent is administered after the antibody has
bound to the targets. Examples of recognizable haptens include, but
are not limited to, chelators, such as DTPA, fluorescein
isothiocyanate, vitamin B-12, polymers (such as polymeric proteins
or carbohydrates), and other moieties to which specific antibodies
can be raised.
[0022] Malignancies that can be treated include B-cell lymphomas,
B-cell leukemias, T-cell lymphomas, T-cell leukemias, and myeloid
leukemias, mastocytomas, and myelomas, as well as certain
premalignant conditions related thereto, such as myelodysplasia
syndrome (MDS). The therapeutic compositions described herein are
particularly useful for treatment of indolent forms of B-cell
lymphomas, aggressive forms of B-cell lymphomas, chronic lymphatic
or myeloid leukemias, acute lymphatic or myeloid leukemias, diverse
plasma cell dyscrasias (such as multiple myeloma), and
mastocytomas.
[0023] Plasma cell tumors consist principally of Waldenstrom's
macroglobulinemia (WM) and multiple myeloma (MM). CD20 is expressed
more in the former than in the latter, since multiple myeloma is a
more differentiated cell type proceeding from B-cell lymphomas to
Waldenstrom's and then to multiple myeloma (Treon et al., Ann.
Oncol. 11 (Suppl 1):107, 2000). Antibodies against a number of
human markers, including the immunoglobulin idiotype, can be used
as targets for antibodies used in the treatment of plasma cell
dyscrasias, including WM and MM, such as CD20, CD38, CD54, CD126,
HM1.24, and MUC1 (Treon et al., Semin. Oncol. 26 (Suppl 14):97,
1999). A high affinity Mab (AT13/5) against CD38, made as a
chimeric and CDR-grafted humanized IgG1, as promising antibodies
for the therapy of MM was described by Ellis et al. (J. Immunol.
155:925, 1995). It has been shown that the humanized anti-HM1.24
antibody effectively kills multiple myeloma cells by human effector
cell-mediated cytotoxicity (Ono et al., Mol. Immunol. 36:387, 1999;
Ozaki et al., Blood 93:3922, 1999). Regression of murine leukemias
and multiple myeloma has also been accomplished with CD25
antibodies (interleukin-2 receptor alpha) (Onizuka et al., Cancer
Res. 59:3128, 1999). Two human plasma-cell reactive antibodies,
B-B2 and B-B4, which are suggested as candidates for immunotherapy
of multiple myeloma, were reported by Vooijs et al., Cancer
Immunol. Immunother, 42:319 (1996), when these were tested as
immunotoxins with saporin.
[0024] Preferred plasma-cell targets in domestic animals are those
equivalent to the human CD20, CD22, CD38, HM1.24, and MUC1. WM has
also been shown to express CD22 (Behr et al. in Clin. Cancer Res.
Suppl. 5 (Suppl 10):3304s-2214s (1999) and unpublished results of
the inventor). CD20 and CD22 are expressed more in WM, while CD38,
HM1.24, and MUC1 are expressed in both WM and MM.
[0025] The most common malignant neoplasms in the dog are mast cell
tumors. It has been estimated that this tumor type represents
between 7% and 21% of all tumors occurring in dogs, which is much
higher in frequency than is found in humans. These tumors
frequently spread to local lymph nodes, liver, spleen, and bone
marrow (London et al., Exp. Hematol. 27:689, 1999).
[0026] The c-kit gene encodes a receptor tyrosine kinase for stem
cell factor (SCF; c-kit ligand, KL), both of which play a critical
role in the differentiation and growth of hematopoietic stem cells
(Teyssier-Le Discorde et al., Leukemia 11 (Suppl 3):396, 1997).
Stem cell factor receptor (SCFR, c-kit) is normally expressed on
hematopoietic and mast cells, where it is believed to play a
regulatory role in cellular growth and differentiation (London et
al., J. Comp. Pathol. 115:399, 1996). It has been found that canine
mast cell tumors express SCFR, or KIT (c-kit protein), which is a
type III transmembrane receptor kinase. The ligand of the KIT, stem
cell factor, is a cytokine that stimulates mast cell growth and
differentiation. KIT has been detected in both human and canine
mast cell malignancies, as can be shown by immunohistochemical
methods, such that KIT has been reported as a reliable marker for
canine mast cells and undifferentiated mast cell tumors (London et
al., J Comp. Pathol. 115:399, 1996; Reguera et al., Am. J.
Dennatopathol. 22:49, 2000). Antibodies are already available
against KIT, and have been used in the immunohistochemical methods
described. These kind of antibodies can serve as immunotherapeutics
for controlling the growth of mast cell tumors, while the KIT
ligand, stem cell factor, can also be used either to deliver
cytotoxic agents to its receptor, or to serve as a hapten or hapten
component in a bispecific antibody/pretargeting methodology, in
which one arm is directed against the tumor (e.g., KIT or stem cell
factor receptor) and the other arm is made against the ligand or a
peptide linked to the ligand (stem cell factor). The latter is for
improved delivery of drugs, toxins and isotopes, whereas the naked
antibody to KIT can be used by itself or in suitable combinations
with other antibodies targeting mast cell tumors or with
immunoconjugates consisting of antibodies to SCFR linked to a
cytotoxic agent.
[0027] Other potential targets for antibodies against mast cell
tumors are thioflavine T (Brunnert and Altman, J. Vet. Diagn.
Invest. 3:245, 1991), and chymotrypsin-like proteinase (Schechter
et al., Arch. Biochem. Biophys. 262:232, 1988). Antibodies have
been made against these substances, and used to detect them in mast
cell tumor tissues. These antibodies can then serve as
immunotherapeutics and as the basis for immunoconjugates made for
the therapy of mast cell tumors.
[0028] A number of myeloid and lymphatic cancers have been
documented in dogs and cats, including both chronic lymphatic or
myeloid leukemias and acute lymphatic or myeloid leukemias, e.g.,
acute lymphoid leukemia, acute myelogenous leukemia, acute
myelomonocytic leukemia, monocytic leukemia, lymphocytic leukemia,
myelocytic leukemia, and chronic granulocytic leukemia. The term
"myelocytic leukemia" is synonymous with the terms granulocytic
leukemia, myelogenic leukemia, myelogenous leukemia and myeloid
leukemia. The etiology of each of these disorders has been well
characterized, in particular the cell type(s) with which the
disorder is associated. For example, chronic myelocytic leukemia is
characterized by an uncontrolled proliferation of myelopoietic
cells in the bone marrow and extramedullary sites in which the
malignant myeloblast is able to differentiate and give rise to
myelocytes, metamyelocytes, band cells and granulocytes, while
acute myelocytic leukemia is characterized by an uncontrolled
proliferation of myeloblasts which are unable to differentiate into
more mature cell-types. The present methods use naked antibodies or
antibody conjugates against these cell types to treat the
disorder.
[0029] Recent studies suggest that immunotherapy utilizing naked
antibodies can be an effective tool for treating these cancers. For
example, the use of naked, humanized, anti-CD33 antibodies has
proved effective in treating acute myelocytic leukemia and in
reducing the residual disease in patients. See Caron et al., Clin.
Cancer Res., 4:1421-1428 (1998); Jurcic et al., Clin. Cancer Res.,
6:372-380 (2000). U.S. Provisional Appln. 60/223,698 (Goldenberg et
al.), which is incorporated herein by reference, describes the use
of naked antibodies for treating acute myelocytic leukemia (AML),
acute promyelocytic leukemia (APML) and chronic myelocytic leukemia
(CML). The method uses naked granulocyte-specific antibodies that
recognize an antigen that is present on two or more cell-types of
the granulocyte/myelocyte lineage, alone or in conjunction with
immunoconjugates or other therapeutics to destroy myeloid leukemia
cells. The present invention contemplates the use of
myelocyte-specific, metamyelocyte-specific, band-specific
antibodies to treat myeloid leukemias, and anti-granulocytic
antibodies directed to antigens present on a single granulocyte
precursor, such as anti-CD33 or anti-CD15 antibodies, or on two or
more cell-types in the granulocyte/myelocyte lineage. The
antibodies may be in the form of naked antibodies or
immunoconjugates.
[0030] The present invention is also useful in the treatment of
autoimmune diseases. B-cell clones that bear autoantibody
Ig-receptors are present in normal individuals. Autoimmunity
results when these B-cells become overactive, and mature to plasma
cells that secrete autoantibody. Other autoimmune disorders are
tied to a T-cell disorder. In accordance with the present
invention, autoimmune disorders can be treated by administering an
antibody that binds to a B-cell or T-cell receptor or antigen, or
to an Ia or HLA-DR determinant or equivalent in domestic animals.
In one embodiment, comparatively low doses of an entire, naked
antibody or combinations of entire, naked antibodies are used. In
other embodiments, immunoconjugates or fusion proteins of antibody
components with drugs, toxins or therapeutic radioisotopes are
useful.
[0031] The antibodies described in this invention can be antibodies
from the same species as the animal to be treated. Preferred
monoclonal antibodies can be made by immunizing an animal of the
species to be treated, and fusing the resulting antibodies with a
myeloma cell line, either from the same species or from another
species, such as a mouse. In a preferred embodiment, molecular
engineering methods can be used to graft mouse or other species'
CDRs to dog or cat (or other domestic species) framework regions.
Humanized antibodies can also be used in accordance with the
present invention. Alternatively, mouse, mouse/human chimeric, and
fully human monoclonal antibodies, as well as dog, mouse/dog
chimeric, or caninized monoclonal antibodies, as described above,
can also be used. Fully canine, feline, equine or other domestic
species antibodies, as may be preferred for a particular animal
species to be treated, may be made by methods similar to those
employed to construct or isolate fully human antibodies, such as by
phage display and gene or chromosome transfer techniques.
[0032] The antibodies can be infused into a domestic animal
diagnosed with a B-cell, T-cell, myeloid-, plasma-, or mast-cell
disorder, in order to treat or control the disorder. Where the
disorder is a malignancy, the malignancy will be decreased in size
as a result of the treatment, and the animal may even enter a state
of total remission. Where the disorder is an autoimmune disease,
treatment is generally an ongoing treatment of a chronic
condition.
[0033] In a preferred embodiment, naked antibodies are used alone
in treatment. Combinations of naked antibodies may be used, i.e.,
naked antibodies to more than one epitope of a B-cell antigen or a
T-cell antigen, or even combinations against distinctly different
B-cell or T-cell antigens, may be used. In some cases, it may be
preferable to use a naked antibody that binds to both B-cell and
T-cell antigens. For example, certain HLA antibodies may bind to
both. Other examples include human-equivalent lymphocyte
antibodies, such as against CD21 in the dog.
[0034] In another embodiment, naked antibody therapy can be
enhanced by supplementing the naked antibodies with
immunoconjugates comprising an antibody component and a therapeutic
agent, or with fusion proteins, conventional chemotherapeutic
drugs, cytokines, and other forms of supplemental therapy. In such
multimodal regimens, the naked antibodies are the primary
therapeutic, and the supplemental therapeutic compositions can be
administered before, concurrently or after administration of one or
more naked antibodies.
[0035] Immunoconjugates also may be used alone for treatment. In
one embodiment, the immunoconjugate is a radiolabeled
immunoconjugate. The radionuclide may be an alpha emitter, a
beta-emitter, and/or an Auger emitter. Suitable radionuclides
include .sup.198Au, .sup.32p, .sup.125I, .sup.131I, .sup.90Y,
.sup.186Re, .sup.188Re, .sup.67Cu, .sup.177Lu, .sup.211At,
.sup.213Bi, .sup.111In, .sup.67Ga, and .sup.225Ac. In another
embodiment, the immunoconjugate comprises a drug or toxin. Suitable
drugs or toxins include ricin, abrin, ribonuclease, DNase I,
Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,
diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.
The immunoconjugate therapy may be supplemented with conventional
chemotherapy, radiation therapy, and/or cytokine therapy.
[0036] In a further embodiment, imaging is performed prior to
therapy to confirm the presence and/or location of the disease.
Examples of diagnostic agents include, but are not limited to,
radioisotopes, coloring agents (such as the biotin-streptavidin
complex), contrasting agents, fluorescent compounds or molecules
and enhancing agents for magnetic resonance imaging (MRI).
Preferably, the diagnostic agents are selected from the group
consisting of radioisotopes, enhancing agents for use in magnetic
resonance imaging, and fluorescent compunds.
[0037] In order to load an antibody component with radioactive
metals or paramagnetic ions, it may be necessary to react it with a
reagent having a long tail to which are attached a multiplicity of
chelating groups for binding the ions. Such a tail can be a polymer
such as a polylysine, polysaccharide, or other derivatized or
derivatizable chain having pendant groups to which can be bound
chelating groups such as, e.g. ethylenediaminetetraacetic acid
(EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins,
polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and
like groups known to be useful for this purpose. Chelates are
coupled to the peptide antigens using standard chemistries. The
chelate is normally linked to the antibody by a group which enables
formation of a bond to the molecule with minimal loss of
immunoreactivity and minimal aggregation and/or internal
cross-linking. other, more unusual, methods and reagents for
conjugating chelates to antibodies are disclosed in U.S. Pat.
4,824,659 to Hawthorne, entitled "Antibody Conjugates", issued Apr.
25, 1989, the disclosure of which is incorporated herein in its
entirety by reference.
[0038] Particularly useful metal-chelate combinations include
2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, used with
scandium-47, iron-52, cobalt-55, gallium-67, gallium-68,
indium-111, zirconium-89, yttrium-90, terbium-161, lutetium-177,
bismuth-212, bismuth-213, and actinium-225 for radio-imaging and
RAIT. The same chelates, when complexed with non-radioactive
metals, such as manganese, iron and gadolinium are useful for MRI,
when used along with the antibodies of the invention. Macrocyclic
chelates such as NOTA, DOTA, and TETA are of use with a variety of
metals and radiometals, most particularly with radionuclides of
gallium, ytrrium and copper, respectively. Such metal-chelate
complexes can be made very stable by tailoring the ring size to the
metal of interest. Other ring-type chelates such as macrocyclic
polyethers, which are of interest for stably binding nuclides such
as radium-223 for RAIT are encompassed by the invention.
[0039] As in the case of therapeutic antibodies, an antibody
component can be used that comprises an arm that is specific for a
low-molecular weight hapten to which the diagnostic agent is
conjugated or fused. In this case, the antibody component
pretargets B-cells, T-cells, myeloid cells, mast cells or plasma
cells, and the low-molecular weight hapten with the attached
diagnostic agent is administered after the antibody has bound to
the target.
[0040] MRI contrast agents are well known in the art and include,
for example, Gadolinium, Iron, Manganese, Rhenium, Europium,
Lanthanium, Holmium, and Ferbium. Where a radionuclide is use, the
antibody component comprises a gamma-emitting diagnostic
radionuclide. The diagnostic radionuclide is selected from the
group consisting of .sup.95Ru, .sup.97Ru, .sup.103Ru, .sup.105Ru,
.sup.99Tc, .sup.197Hg, .sup.67Ga, .sup.68Ga, .sup.119Os,
.sup.111In, .sup.113In and .sup.203Pb. Some therapeutic
radionuclides have a companion gamma energy component which can be
used for diagnostic imaging. In this case, imaging occurs in
conjunction with therapy.
[0041] Definitions
[0042] In the description that follows, and in the documents
incorporated by reference herein, a number of terms are used
extensively. The following definitions are provided to facilitate
understanding of the invention.
[0043] A structural gene is a DNA sequence that is transcribed into
messenger RNA (mRNA) which is then translated into a sequence of
amino acids characteristic of a specific polypeptide.
[0044] A promoter is a DNA sequence that directs the transcription
of a structural gene. Typically, a promoter is located in the 5'
region of a gene, proximal to the transcriptional start site of a
structural gene. If a promoter is an inducible promoter, then the
rate of transcription increases in response to an inducing agent.
In contrast, the rate of transcription is not regulated by an
inducing agent if the promoter is a constitutive promoter.
[0045] An isolated DNA molecule is a fragment of DNA that is not
integrated in the genomic DNA of an organism. For example, a cloned
antibody gene is a DNA fragment that has been separated from the
genomic DNA of a mammalian cell. Another example of an isolated DNA
molecule is a chemically-synthesized DNA molecule that is not
integrated in the genomic DNA of an organism.
[0046] An enhancer is a DNA regulatory element that can increase
the efficiency of transcription, regardless of the distance or
orientation of the enhancer relative to the start site of
transcription.
[0047] Complementary DNA (cDNA) is a single-stranded DNA molecule
that is formed from an mRNA template by the enzyme reverse
transcriptase. Typically, a primer complementary to portions of
mRNA is employed for the initiation of reverse transcription. Those
skilled in the art also use the term "cDNA" to refer to a
double-stranded DNA molecule consisting of such a single-stranded
DNA molecule and its complementary DNA strand.
[0048] The term expression refers to the biosynthesis of a gene
product. For example, in the case of a structural gene, expression
involves transcription of the structural gene into mRNA and the
translation of mRNA into one or more polypeptides.
[0049] A cloning vector is a DNA molecule, such as a plasmid,
cosmid, or bacteriophage, that has the capability of replicating
autonomously in a host cell. Cloning vectors typically contain one
or a small number of restriction endonuclease recognition sites at
which foreign DNA sequences can be inserted in a determinable
fashion without loss of an essential biological function of the
vector, as well as a marker gene that is suitable for use in the
identification and selection of cells transformed with the cloning
vector. Marker genes typically include genes that provide
tetracycline resistance or ampicillin resistance.
[0050] An expression vector is a DNA molecule comprising a gene
that is expressed in a host cell. Typically, gene expression is
placed under the control of certain regulatory elements, including
constitutive or inducible promoters, tissue-specific regulatory
elements, and enhancers. Such a gene is said to be "operably linked
to" the regulatory elements.
[0051] A recombinant host may be any prokaryotic or eukaryotic cell
that contains either a cloning vector or expression vector. This
term also includes those prokaryotic or eukaryotic cells that have
been genetically engineered to contain the cloned gene(s) in the
chromosome or genome of the host cell.
[0052] An antibody fragment is a portion of an antibody such as
F(ab').sub.2, F(ab).sub.2, Fab', Fab, and the like. Regardless of
structure, an antibody fragment binds with the same antigen that is
recognized by the intact antibody. For example, an anti-CD22
monoclonal antibody fragment binds with an epitope of CD22.
[0053] The term "antibody fragment" also includes any synthetic or
genetically engineered protein that acts like an antibody by
binding to a specific antigen to form a complex. For example,
antibody fragments include isolated fragments consisting of the
light chain variable region, "Fv" fragments consisting of the
variable regions of the heavy and light chains, recombinant single
chain polypeptide molecules in which light and heavy variable
regions are connected by a peptide linker ("sFv proteins"), and
minimal recognition units consisting of the amino acid residues
that mimic the hypervariable region.
[0054] A chimeric antibody is a recombinant protein that contains
the variable domains and complementary determining regions derived
from one species while the remainder of the antibody molecule is
derived from another species antibody.
[0055] Humanized antibodies are recombinant proteins in which
murine complementarity determining regions of a monoclonal antibody
have been transferred from heavy and light variable chains of the
murine immunoglobulin into a human variable domain.
[0056] Caninized or felinized antibodies are recombinant proteins
in which rodent (or another species) complementarity determining
regions of a monoclonal antibody have been transferred from heavy
and light variable chains of rodent (or another species)
immunoglobulin into a dog or cat, respectively, variable
domain.
[0057] As used herein, a therapeutic agent is a molecule or atom
that is conjugated to an antibody moiety to produce a conjugate
that is useful for therapy. Examples of therapeutic agents include
drugs, toxins, antagonists, enzymes, immunomodulators, hormones,
cytokines, chelators, boron compounds, photoactive agents or dyes,
and radioisotopes.
[0058] A naked antibody is an entire antibody, as opposed to an
antibody fragment, which is not conjugated with a therapeutic
agent. Naked antibodies include both polyclonal and monoclonal
antibodies, as well as certain recombinant antibodies, such as
chimeric and humanized, caninized, or felinized antibodies, as well
as fully human, canine, or feline antibodies derived from
recombinant engineering or other genetic or chromosomal
manipulation technologies.
[0059] As used herein, the term antibody component includes both an
entire antibody and an antibody fragment.
[0060] An immunoconjugate is a conjugate of an antibody component
with a therapeutic agent.
[0061] A fusion protein is a recombinantly produced antigen-binding
molecule in which two or more different single-chain antibody or
antibody fragment segments with the same or different specificities
are linked. A variety of bispecific fusion proteins can be produced
using molecular engineering. In one form, the bispecific fusion
protein is monovalent, consisting of, for example, a scFv with a
single binding site for one antigen and a Fab fragment with a
single binding site for a second antigen. In another form, the
bispecific fusion protein is divalent, consisting of, for example,
an IgG with two binding sites for one antigen and two scFv with two
binding sites for a second antigen. The fusion protein additionally
comprises a therapeutic agent. Examples of therapeutic agents
suitable for such fusion proteins include immunomodulators
("antibody-immunomodulator fusion protein") and toxins
("antibody-toxin fusion protein"). One preferred toxin comprises a
ribonuclease (RNase), preferably a recombinant RNase. The fusion
protein may comprise a single antibody component, a multivalent
combination of different antibody components or multiple copies of
the same antibody component.
[0062] A multispecific antibody is an antibody that can bind
simultaneously to at least two targets that are of different
structure, e.g., two different antigens, two different epitopes on
the same antigen, or a hapten and/or an antigen or epitope. One
specificity would be for a B-cell, T-cell, myeloid-, plasma-, and
mast-cell antigen or epitope. Another specificity could be to a
different antigen on the same cell type, such as CD19 and CD20 on
B-cells.
[0063] A bispecific antibody is an antibody that can bind
simultaneously to two targets which are of different structure.
Bispecific antibodies (bsAb) and bispecific antibody fragments
(bsFab) have at least one arm that specifically binds to, for
example, a B-cell, T-cell, myeloid-, plasma-, and mast-cell antigen
or epitope and at least one other arm that specifically binds to a
targetable conjugate that bears a therapeutic or diagnostic
agent.
[0064] Domestic animals include large animals such as horses,
cattle, sheep, goats, llamas, alpacas, and pigs, as well as
companion animals. In a preferred embodiment, the domestic animal
is a horse.
[0065] Companion animals include animals kept as pets. These are
primarily dogs and cats, although small rodents, such as guinea
pigs, hamsters, rats, and ferrets, are also included, as are
subhuman primates such as monkeys. In a preferred embodiment the
companion animal is a dog or a cat.
Production of Naked Antibodies
[0066] Naked antibodies to any known antigen that is
characteristics of a B-cell or T-cell disorder can be used in
accordance with the present invention. Many monoclonal antibodies
to B-cell and T-cell antigens are available, such as 1F5, L243,
Leu-16 and ACT1. A clone to make 1F5 anti-CD20 mAb can be obtained
from ATCC (HB-96450). A clone to make L243, an MHC class II
antibody, can be obtained from ATCC (HB-55). Leu-16, an anti-CD3
antibody, can be obtained from Becton-Dickinson. Clones to make
anti-IL-2 receptor mAbs include 2A3A1H from ATCC (HB8555) and 7G7B6
from ATCC (HB8784). The ACT1 antibody is described in Galkowska et
al., Vet. Immunol. Immunopathol., 53;329-324 (1996), which further
reports that many anti-human antibodies are species cross-reactive.
Thus, anti-human antibodies can be used in methods according to the
invention. See, also, Shienvold et al, ("Antibodies against Ia-like
antigens have shown cross-species properties" Transplantation
41:364 (1986)) which shows that monoclonal antibodies directed
against Ia show crossreactivity among different species. For
example, the B1F6 antibody shows reactivity with Ia in the dog,
rat, pig and human, and B2E8 shows reactivity with Ia in the dog,
pig and human.
[0067] Rodent monoclonal antibodies to CD22 or CD19 can be obtained
by methods known to those skilled in the art. See generally, 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) ["Coligan"]. The
production of CD22 and CD19 according to the following protocol is
exemplary of techniques for the production of antibodies to any
lineage or non-lineage human antigen or its cross-species
equivalent. Briefly, monoclonal antibodies can be obtained by
injecting mice with a composition comprising CD22 or CD19,
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 anti-CD22 or
anti-CD19 antibodies, culturing the clones that produce antibodies
to the antigen, and isolating the antibodies from the hybridoma
cultures.
[0068] Monoclonal antibodies 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).
[0069] Suitable amounts of the well-characterized CD22 or CD19
antigen for production of antibodies can be obtained using standard
techniques. As an example, CD22 can be immunoprecipitated from
B-lymphocyte protein using the deposited antibodies described by
Tedder et al., U.S. Pat. No. 5,484,892 (1996).
[0070] Alternatively, CD22 protein or CD19 protein can be obtained
from transfected cultured cells that overproduce CD22 or CD19.
Expression vectors that comprise DNA molecules encoding CD22 or
CD19 proteins can be constructed using published CD22 and CD19
nucleotide sequences. See, for example, Wilson et al., J. Exp. Med.
173:137 (1991); Wilson et al, J. Immunol. 150:5013 (1993). As an
illustration, DNA molecules encoding CD22 or CD19 can be obtained
by synthesizing DNA molecules using mutually priming long
oligonucleotides. See, for example, Ausubel et al., (eds.), CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, pages 8.2.8 to 8.2.13 (1990)
["Ausubel"]. Also, see Wosnick et al., Gene 60:115 (1987); and
Ausubel et al. (eds.), SHORT PROTOCOLS IN MOLECULAR BIOLOGY, 3rd
Edition, pages 8-8 to 8-9 (John Wiley & Sons, Inc. 1995).
Established techniques using the polymerase chain reaction provide
the ability to synthesize genes as large as 1.8 kilobases in
length. Adang et al., Plant Molec. Biol. 21:1131 (1993); Bambot et
al., PCR Methods and Applications 2:266 (1993); Dillon et al., "Use
of the Polymerase Chain Reaction for the Rapid Construction of
Synthetic Genes," in METHODS IN MOLECULAR BIOLOGY, Vol. 15: PCR
PROTOCOLS: CURRENT METHODS AND APPLICATIONS, White (ed.), pages
263-268, (Humana Press, Inc. 1993).
[0071] In a variation of this approach, anti-CD22 or anti-CD19
monoclonal antibody can be obtained by fusing myeloma cells with
spleen cells from mice immunized with a murine pre-B cell line
stably transfected with CD22 cDNA or CD19 cDNA. See Tedder et al.,
U.S. Pat. No. 5,484,892 (1996).
[0072] One example of a suitable murine anti-CD22 monoclonal
antibody is the LL2 (formerly EPB-2) monoclonal antibody, which was
produced against human Raji cells derived from a Burkitt lymphoma.
Pawlak-Byczkowska et al., Cancer Res. 49:4568 (1989). This
monoclonal antibody has an IgG.sub.2.alpha. isotype, and the
antibody is rapidly internalized into lymphoma cells. Shih et al,
Int. J. Cancer 56:538 (1994). Immunostaining and in vivo
radioimmunodetection studies have demonstrated the excellent
sensitivity of LL2 in detecting B-cell lymphomas. Pawlak-Byczkowska
et al., Cancer Res. 49:4568 (1989); Murthy et al., Eur. J. Nucl.
Med. 19:394 (1992). Moreover, .sup.99mTc-labeled LL2-Fab' fragments
have been shown to be useful in following upstaging of B-cell
lymphomas, while .sup.131I-labeled intact LL2 and labeled LL2
F(ab').sub.2 fragments have been used to target lymphoma sites and
to induce therapeutic responses. Murthy et al., Eur. J. Nucl. Med.
19:394 (1992); Mills et al., Proc. Am. Assoc. Cancer Res. 34:479
(1993) [Abstract 2857]; Baum et al., Cancer 73 (Suppl. 3):896
(1994); Goldenberg et al., J. Clin. Oncol. 9:548 (1991).
Furthermore, Fab' LL2 fragments conjugated with a derivative of
Pseudomonas exotoxin has been shown to induce complete remissions
for measurable human lymphoma xenografts growing in nude mice.
Kreitman et al., Cancer Res. 53:819 (1993).
[0073] In an additional embodiment, an antibody of the present
invention is a chimeric antibody in which the variable regions of a
target species antibody have been replaced by the variable regions
of a rodent anti-CD20 or anti-CD19 antibody. The advantages of
chimeric antibodies include decreased immunogenicity and increased
in vivo stability.
[0074] 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 with respective human K and IgG.sub.1
constant region domains. This publication also provides the
nucleotide sequences of the LL2 light and heavy chain variable
regions, V.sub.K and V.sub.H, respectively.
[0075] In yet another embodiment, an antibody of the present
invention comprises a monoclonal antibody in which mouse
complementarity determining regions are transferred from heavy and
light variable chains of the mouse immunoglobulin into the variable
domain of the target species, followed by the replacement of some
residues of the target species in the framework regions of their
murine counterparts. These antibodies are particularly suitable for
use in therapeutic methods in the target species. 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). Techniques for producing
monoclonal antibodies that are humanized, i.e., where a human is
the target species, 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). The
publication of Leung et al., Mol. Immunol. 32:1413 (1995),
describes the construction of humanized LL2 antibody. These
techniques can be extended to the construction of antibodies with
the variable domain of the horse, dog, cat, or other targeted
species.
[0076] In another embodiment, an antibody of the present invention
is a target species monoclonal antibody, obtained from transgenic
mice that have been "engineered" to produce specific target species
antibodies in response to antigenic challenge. In this technique,
elements of the target species 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
target species antibodies specific for target species antigens, and
the mice can be used to produce target species 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). These techniques can be extended to the
engineering of antibodies with the variable domain of the horse,
dog, cat, or other targeted species.
[0077] In another embodiment, an antibody of the present invention
is raised in another individual of the same species as the species
that is the target of the therapy. General techniques for raising
therapeutically useful antibodies in baboons may be found, for
example, in Goldenberg et al., international patent publication No.
WO 91/11465 (1991), and in Losman et al., Int. J. Cancer 46: 310
(1990), and these techniques can be extended to the production of
antibodies in other species.
Production of Antibody Fragments
[0078] The present invention contemplates the use of fragments of
antibodies or other therapeutically useful antibody components.
Antibody fragments can be prepared by proteolytic hydrolysis of an
antibody or by expression in E. coli of the DNA coding for the
fragment.
[0079] Antibody fragments can be obtained by pepsin or papain
digestion of whole antibodies by conventional methods. For example,
antibody fragments can be produced by enzymatic cleavage of
antibodies with pepsin to provide a 5S fragment denoted
F(ab').sub.2. This fragment can be further cleaved using a thiol
reducing agent, and optionally a blocking group for the sulfhydryl
groups resulting from cleavage of disulfide linkages, to produce
3.5S Fab' monovalent fragments. Alternatively, an enzymatic
cleavage using pepsin produces two monovalent Fab fragments and an
Fc fragment directly. These methods are described, for example, by
Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647 and references
contained therein. 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.
[0080] 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.
[0081] For example, Fv fragments comprise an association of V.sub.H
and V.sub.L chains. This association can be noncovalent, as
described in Inbar et al., Proc. Nat'l Acad. Sci. USA 69:2659
(1972). Alternatively, the variable chains can be linked by an
intermolecular disulfide bond or cross-linked by chemicals such as
glutaraldehyde. See, for example, Sandhu, supra.
[0082] Preferably, the Fv fragments comprise V.sub.H and V.sub.L
chains which are connected by a peptide linker. These single-chain
antigen binding proteins (sFv) are prepared by constructing a
structural gene comprising DNA sequences encoding the V.sub.H and
V.sub.L domains which are connected by an oligonucleotide. The
structural gene is inserted into an expression vector which is
subsequently introduced into a host cell, such as E. coli. The
recombinant host cells synthesize a single polypeptide chain with a
linker peptide bridging the two V domains. Methods for producing
sFvs are described, for example, by Whitlow et al., Methods: A
Companion to Methods in Enzymology 2:97 (1991). See also 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.
[0083] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing
cells. See, for example, Larrick et al., Methods: A Companion to
Methods in Enzymology 2:106 (1991); 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).
Preparation of Immunoconjugates
[0084] The present invention contemplates the use of "naked"
antibodies, as well as the use of immunoconjugates to effect
treatment of B-cell, T-cell, myeloid-, plasma-, and mast-cell
disorders. Such 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 41:832-839
(1988); Shih et al., Int. J. Cancer 46:1101-1106 (1990); and Shih
et al., U.S. Pat. No. 5,057,313. The general method involves
reacting an antibody component having an oxidized carbohydrate
portion with a carrier polymer that has at least one free amine
function and that is loaded with a plurality of drug, toxin,
chelator, boron addends, or other 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.
[0085] The carrier polymer is preferably an aminodextran or
polypeptide of at least 50 amino acid residues, although other
substantially equivalent polymer carriers can also be used.
Preferably, the final immunoconjugate is soluble in an aqueous
solution, such as mammalian serum, for ease of administration and
effective targeting for use in therapy. Thus, solubilizing
functions on the carrier polymer will enhance the serum solubility
of the final immunoconjugate. In particular, an aminodextran will
be preferred.
[0086] The process for preparing an immunoconjugate with an
aminodextran carrier typically begins with a dextran polymer,
advantageously a dextran of average molecular weight of about
10,000-100,000. The dextran is reacted with an oxidizing agent to
effect a controlled oxidation of a portion of its carbohydrate
rings to generate aldehyde groups. The oxidation is conveniently
effected with glycolytic chemical reagents such as NaIO.sub.4,
according to conventional procedures.
[0087] The oxidized dextran is then reacted with a polyamine,
preferably a diamine, and more preferably, a mono- or polyhydroxy
diamine. Suitable amines include ethylene diamine, propylene
diamine, or other like polymethylene diamines, diethylene triamine
or like polyamines, 1,3-diamino-2-hydroxypropane, or other like
hydroxylated diamines or polyamines, and the like. An excess of the
amine relative to the aldehyde groups of the dextran is used to
insure substantially complete conversion of the aldehyde functions
to Schiff base groups.
[0088] A reducing agent, such as NaBH.sub.4, NaBH.sub.3CN or the
like, is used to effect reductive stabilization of the resultant
Schiff base intermediate. The resultant adduct can be purified by
passage through a conventional sizing column to remove cross-linked
dextrans.
[0089] Other conventional methods of derivatizing a dextran to
introduce amine functions can also be used, e.g., reaction with
cyanogen bromide, followed by reaction with a diamine.
[0090] The aminodextran is then reacted with a derivative of the
particular drug, toxin, chelator, immunomodulator, boron addend, or
other therapeutic agent to be loaded, in an activated form,
preferably, a carboxyl-activated derivative, prepared by
conventional means, e.g., using dicyclohexylcarbodiimide (DCC) or a
water soluble variant thereof, to form an intermediate adduct.
[0091] Alternatively, polypeptide toxins such as pokeweed antiviral
protein or ricin A-chain, and the like, can be coupled to
aminodextran by glutaraldehyde condensation or by reaction of
activated carboxyl groups on the protein with amines on the
aminodextran.
[0092] Chelators for radiometals or magnetic resonance enhancers
are well-known in the art. Typical are derivatives of
ethylenediaminetetraace- tic acid (EDTA) and
diethylenetriaminepentaacetic acid (DTPA). These chelators
typically have groups on the side chain by which the chelator can
be attached to a carrier. Such groups include, e.g.,
benzylisothiocyanate, by which the DTPA or EDTA can be coupled to
the amine group of a carrier. Alternatively, carboxyl groups or
amine groups on a chelator can be coupled to a carrier by
activation or prior derivatization and then coupling, all by
well-known means.
[0093] Boron addends, such as carboranes, can be attached to
antibody components by conventional methods. For example,
carboranes can be prepared with carboxyl functions on pendant side
chains, as is well known in the art. Attachment of such carboranes
to a carrier, e.g., aminodextran, can be achieved by activation of
the carboxyl groups of the carboranes and condensation with amines
on the carrier to produce an intermediate conjugate. Such
intermediate conjugates are then attached to antibody components to
produce therapeutically useful immunoconjugates, as described
below.
[0094] A polypeptide carrier can be used instead of aminodextran,
but the polypeptide carrier must have at least 50 amino acid
residues in the chain, preferably 100-5000 amino acid residues. At
least some of the amino acids should be lysine residues or
glutamate or aspartate residues. The pendant amines of lysine
residues and pendant carboxylates of glutamine and aspartate are
convenient for attaching a drug, toxin, immunomodulator, chelator,
boron addend or other therapeutic agent. Examples of suitable
polypeptide carriers include polylysine, polyglutamic acid,
polyaspartic acid, co-polymers thereof, and mixed polymers of these
amino acids and others, e.g., serines, to confer desirable
solubility properties on the resultant loaded carrier and
immunoconjugate.
[0095] Conjugation of the intermediate conjugate with the antibody
component is effected by oxidizing the carbohydrate portion of the
antibody component and reacting the resulting aldehyde (and ketone)
carbonyls with amine groups remaining on the carrier after loading
with a drug, toxin, chelator, immunomodulator, boron addend, or
other therapeutic agent. Alternatively, an intermediate conjugate
can be attached to an oxidized antibody component via amine groups
that have been introduced in the intermediate conjugate after
loading with the therapeutic agent. Oxidation is conveniently
effected either chemically, e.g., with NaIO.sub.4 or other
glycolytic reagent, or enzymatically, e.g., with neuraminidase and
galactose oxidase. In the case of an aminodextran carrier, not all
of the amines of the aminodextran are typically used for loading a
therapeutic agent. The remaining amines of aminodextran condense
with the oxidized antibody component to form Schiff base adducts,
which are then reductively stabilized, normally with a borohydride
reducing agent.
[0096] Analogous procedures are used to produce other
immunoconjugates according to the invention, as well as other
procedures known in the art. Loaded polypeptide carriers preferably
have free lysine residues remaining for condensation with the
oxidized carbohydrate portion of an antibody component. Carboxyls
on the polypeptide carrier can, if necessary, be converted to
amines by, e.g., activation with DCC and reaction with an excess of
a diamine.
[0097] The final immunoconjugate is purified using conventional
techniques, such as sizing chromatography on Sephacryl S-300.
[0098] Alternatively, immunoconjugates can be prepared by directly
conjugating an antibody component with a therapeutic agent. The
general procedure is analogous to the indirect method of
conjugation except that a therapeutic agent is directly attached to
an oxidized antibody component.
[0099] It will be appreciated that other therapeutic agents can be
substituted for the chelators described herein. Those of skill in
the art will be able to devise conjugation schemes without undue
experimentation.
[0100] As a further illustration, a therapeutic agent can be
attached at the hinge region of a reduced antibody component via
disulfide bond formation. For example, the tetanus toxoid peptides
can be constructed with a single cysteine residue that is used to
attach the peptide to an antibody component. As an alternative,
such peptides can be attached to the antibody component using a
heterobifunctional cross-linker, such as N-succinyl
3-(2-pyridyldithio)proprionate (SPDP). Yu et al., Int. J. Cancer
56:244 (1994). General techniques for such conjugation are well
known in the art. See, for example, Wong, CHEMISTRY OF PROTEIN
CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis et al.,
"Modification of Antibodies by Chemical Methods," in MONOCLONAL
ANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages
187-230 (Wiley-Liss, Inc. 1995); Price, "Production and
Characterization of Synthetic Peptide-Derived Antibodies," in
MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL
APPLICATION, Ritter et al. (eds.), pages 60-84 (Cambridge
University Press 1995).
[0101] As described above, carbohydrate moieties in the Fc region
of an antibody can be used to conjugate a therapeutic agent.
However, the Fc region is absent if an antibody fragment is used as
the antibody component of the immunoconjugate. Nevertheless, it is
possible to introduce a carbohydrate moiety into the light chain
variable region of an antibody or antibody fragment. See, for
example, Leung et al., J. Immunol. 154:5919 (1995); Hansen et al.,
U.S. Pat. No. 5,443,953 (1995). The engineered carbohydrate moiety
is then used to attach a therapeutic agent.
[0102] In addition, those of skill in the art will recognize
numerous possible variations of the conjugation methods. For
example, the carbohydrate moiety can be used to attach
polyethyleneglycol in order to extend the half-life of an intact
antibody, or antigen-binding fragment thereof, in blood, lymph, or
other extracellular fluids. Moreover, it is possible to construct a
"divalent immunoconjugate" by attaching therapeutic agents to a
carbohydrate moiety and to a free sulfhydryl group. Such a free
sulfhydryl group may be located in the hinge region of the antibody
component.
Preparation of Fusion Proteins
[0103] The present invention further contemplates the use of fusion
proteins comprising one or more antibody moieties. In addition to
antibody components that bind with a first B-cell, T-cell, myeloid,
mast-cell, or plasma-cell antigen or epitope, useful antibody
moieties may include antibody components that bind with other
B-cell, T-cell, myeloid, mast cell or plasma cell antigens or
epitopes, and a fusion protein may comprise one, two, three, four
or even more antibody types. Bivalent, trivalent, tetravalent and
quatravalent constructs can be used in accordance with the
invention. These fusion proteins optionally comprise an
immunomodulator or toxin moiety.
[0104] Methods of making antibody-immunomodulator fusion proteins
are known to those of skill in the art. For example, antibody
fusion proteins comprising an interleukin-2 moiety are described by
Boleti et al., Ann. Oncol. 6:945 (1995), Nicolet et al., Cancer
Gene Ther. 2:161 (1995), Becker et al., Proc. Nat'l Acad. Sci. USA
93:7826 (1996), Hank et al., Clin. Cancer Res. 2:1951 (1996), and
Hu et al., Cancer Res. 56:4998 (1996). In addition, Yang et al.,
Hum. Antibodies Hybridomas 6:129 (1995), describe a fusion protein
that includes an F(ab').sub.2 fragment and a tumor necrosis
factor-alpha moiety.
[0105] Methods of making antibody-toxin fusion proteins in which a
recombinant molecule comprises one or more antibody components and
a toxin or chemotherapeutic agent also are known to those of skill
in the art. For example, antibody-Pseudomonas exotoxin A fusion
proteins have been described by Chaudhary et al., Nature 339:394
(1989), Brinkmann et al., Proc. Nat'l Acad. Sci. USA 88:8616
(1991), Batra et al., Proc. Nat'l Acad. Sci. USA 89:5867 (1992),
Friedman et al., J. Immunol. 150:3054 (1993), Wels et al., Int. J.
Can. 60:137 (1995), Fominaya et al., J. Biol. Chem. 271:10560
(1996), Kuan et al., Biochemistry 35:2872 (1996), and Schmidt et
al., Int. J. Can. 65:538 (1996). Antibody-toxin fusion proteins
containing a diphtheria toxin moiety have been described by
Kreitman et al., Leukemia 7:553 (1993), Nicholls et al., J. Biol.
Chem. 268:5302 (1993), Thompson et al, J. Biol. Chem. 270:28037
(1995), and Vallera et al., Blood 88:2342 (1996). Deonarain et al.,
Tumor Targeting 1:177 (1995), have described an antibody-toxin
fusion protein having an RNase moiety, while Linardou et al., Cell
Biophys. 24-25:243 (1994), produced an antibody-toxin fusion
protein comprising a DNase I component. Gelonin was used as the
toxin moiety in the antibody-toxin fusion protein of Wang et al.,
Abstracts of the 209th ACS National Meeting, Anaheim, Calif., 2-6
April, 1995, Part 1, BIOT005. As a further example, Dohlsten et
al., Proc. Nat'l Acad. Sci. USA 91:8945 (1994), reported an
antibody-toxin fusion protein comprising Staphylococcal
enterotoxin-A.
[0106] Illustrative of toxins which are suitably employed in the
preparation of such conjugates are 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, CA--A Cancer Journal for Clinicians 44:43
(1994). Other suitable toxins and conjugation methods are known to
those of skill in the art.
Coupling of Antibodies, Immunoconjugates and Fusion Proteins to
Lipid Emulsions
[0107] Long-circulating sub-micron lipid emulsions, stabilized with
poly(ethylene glycol)-modified phosphatidylethanolamine (PEG-PE),
can be used as drug carriers for the naked antibodies,
immunoconjugates, and fusion proteins of the present invention. The
emulsions are composed of two major parts: an oil core, e.g.,
triglyceride, stabilized by emulsifiers, e.g., phospholipids. The
poor emulsifying properties of phospholipids can be enhanced by
adding a biocompatible co-emulsifier such as polysorbate 80. In a
preferred embodiment, the naked antibodies, immunoconjugates and/or
fusion proteins are conjugated to the surface of the lipid emulsion
globules with a poly(ethylene glycol)-based, heterobifunctional
coupling agent, poly(ethylene glycol)-vinylsulfone-N-h-
ydroxy-succinimidyl ester (NHS-PEG-VS).
[0108] The submicron lipid emulsion is prepared and characterized
as described. Lundberg, J. Pharm. Sci., 83:72 (1993); Lundberg et
al., Int. J. Pharm., 134:119 (1996). The basic composition of the
lipid emulsion is triolcin:DPPC:polysorbate 80, 2:1:0.4 (w/w). When
indicated, PEG-DPPE is added into the lipid mixture at an amount of
2-8 mol % calculated on DPPC.
[0109] The coupling procedure starts with the reaction of the NHS
ester group of NHS-PEG-VS with the amino group of distearoyl
phosphatidyl-ethanolamine (DSPE). Twenty-five .mu.mol of NHS-PEG-VS
are reacted with 23 .mu.mol of DSPE and 50 .mu.mol triethylamine in
1 ml of chloroform for 6 hours at 40.degree. C. to produce a
poly(ethylene glycol) derivative of phosphatidyl-ethanolamine with
a vinylsulfone group at the distal terminus of the poly(ethylene
glycol) chain (DSPE-PEG-VS). For antibody conjugation, DSPE-PEG-VS
is included in the lipid emulsion at 2 mol % of DPPC. The
components are dispersed into vials from stock solutions at
-20.degree. C., the solvent is evaporated to dryness under reduced
pressure. Phosphate-buffered saline (PBS) is added, the mixture is
heated to 50.degree. C., vortexed for 30 seconds and sonicated with
a MSE probe sonicator for 1 minute. Emulsions can be stored at
4.degree. C., and preferably are used for conjugation within 24
hours.
[0110] Coupling of antibodies to emulsion globules is performed via
a reaction between the vinylsulfone group at the distal PEG
terminus on the surface of the globules and free thiol groups on
the antibody. Vinylsulfone is an attractive derivative for
selective coupling to thiol groups. At approximately neutral pH, VS
will couple with a half life of 15-20 minutes to proteins
containing thiol groups. The reactivity of VS is slightly less than
that of maleimide, but the VS group is more stable in water and a
stable linkage is produced from reaction with thiol groups.
[0111] Before conjugation, the antibody is reduced by 50 mM
2-mercaptoethanol for 10 minutes at 4.degree. C. in 0.2 M Tris
buffer (pH 8.7). The reduced antibody is separated from excess
2-mercaptoethanol with a Sephadex G-25 spin column, equilibrated in
50 mM sodium acetate buffered 0.9% saline (pH 5.3). The product is
assayed for protein concentration by measuring its absorbance at
280 nm (and assuming that a 1 mg/ml antibody solution of 1.4) or by
quantitation of .sup.125I-labeled antibody. Thiol groups are
determined with Aldrithiol.TM. following the change in absorbance
at 343 nm and with cystein as standard.
[0112] The coupling reaction is performed in HEPES-buffered saline
(pH 7.4) overnight at ambient temperature under argon. Excess
vinylsulfone groups are quenched with 2 mM 2-mercaptoethanol for 30
minutes, excess 2-mercaptoethanol and antibody are removed by gel
chromatography on a Sepharose CL-48 column. The immunoconjugates
are collected near the void volume of the column, sterilized by
passage through a 0.45 .mu.m sterile filter, and stored at
4.degree. C.
[0113] Coupling efficiency is calculated using .sup.125I-labeled
antibody. Recovery of emulsions is estimated from measurements of
[.sup.14C]DPPC in parallel experiments. The conjugation of reduced
LL2 to the VS group of surface-grafted DSPE-PEG-VS is very
reproducible with a typical efficiency of near 85%.
Therapeutic Use of Antibodies in Simple and Multimodal Regimens
[0114] The present invention contemplates the use of naked
antibodies, or immunoconjugates or fusion proteins, as the primary
therapeutic composition for treatment of B-cell, T-cell, myeloid,
mast-cell, and plasma-cell disorders. Such a composition can
contain polyclonal antibodies or monoclonal antibodies.
[0115] A therapeutic composition of the present invention can
contain a monoclonal antibody directed to non-blocking B-cell,
T-cell, myeloid-, plasma-, and mast-cell antigens or epitopes, or
it can contain a mixture of monoclonal antibodies directed to
different, non-blocking B-cell or T-cell antigens or epitopes. For
example, monoclonal antibody cross-inhibition studies have
identified five epitopes on CD22, designated as epitopes A-E. See,
for example, Schwartz-Albiez et al., "The Carbohydrate Moiety of
the CD22 Antigen Can Be Modulated by Inhibitors of the
Glycosylation Pathway," in LEUKOCYTE TYPING IV. WHITE CELL
DIFFERENTIATION ANTIGENS, Knapp et al. (eds.), p. 65 (Oxford
University Press 1989). As an illustration, the LL2 antibody binds
with epitope B. Stein et al., Cancer Immunol. Immunother. 37:293
(1993). Accordingly, therapeutic compositions comprising a mixture
of monoclonal anti-CD22 antibodies that bind at least two CD22
epitopes could be used. For example, such a mixture can contain
monoclonal antibodies that bind with at least two CD22 epitopes
selected from the group consisting of epitope A, epitope B, epitope
C, epitope D and epitope E. Similarly, the present invention
contemplates therapeutic compositions comprising a mixture of
monoclonal anti-CD 19 antibodies that bind at least two CD19
epitopes, and so forth.
[0116] Methods for determining the binding specificity of a
particular antibody are well known to those of skill in the art.
General methods for anti-CD22 antibodies are provided, for example,
by Mole, "Epitope Mapping," in METHODS IN MOLECULAR BIOLOGY, VOLUME
10: IMMUNOCHEMICAL PROTOCOLS, Manson (ed.), pages 105-116 (The
Humana Press, Inc. 1992). More specifically, competitive blocking
assays to determine CD22 epitope specificity are described by Stein
et al., Cancer Immunol. Immunother. 37:293 (1993), and by Tedder et
al., U.S. Pat. No. 5,484,892 (1996).
[0117] The Tedder patent also describes the production of CD22
mutants which lack one or more immunoglobulin-like domains. These
mutant proteins were used to determine that immunoglobulin-like
domains 1, 2, 3, and 4 correspond with epitopes A, D, B, and C,
respectively. Thus, CD22 epitope specificity can also be identified
by binding a test antibody with a panel of CD22 proteins lacking
particular immunoglobulin-like domain. These techniques may be
extended to other antibodies.
[0118] Naked antibodies can be used as the primary therapeutic
compositions for treatment of B-cell, T-cell, myeloid, mast cell,
or plasma cell malignancies, although the efficacy of such antibody
therapy can be enhanced by supplementing naked antibodies with
immunoconjugates, fusion proteins, and other forms of supplemental
therapy described herein. Multimultimodal regimens with naked
antibodies, the supplemental therapeutic compositions can be
administered before, concurrently or after administration of the
naked antibodies. Alternatively, the B-cell or T-cell, myeloid,
mast cell or plasma cell disorder can be treated with
immunoconjugates or fusion proteins, without the use of naked
antibodies.
[0119] The therapeutic compositions described herein are
particularly useful for treatment of indolent forms of B-cell
lymphomas, aggressive forms of B-cell lymphomas, chronic and acute
lymphatic leukemias, chronic and acute myeloid leukemias,
mastocytomas, and multiple myeloma. For example,
anti-CD20-equivalent antibody components and immunoconjugates can
be used to treat both indolent and aggressive forms of
non-Hodgkin's lymphoma. The therapeutic compositions are also
particularly useful for treatment of autoimmune disorders, such as
immune-mediated autoimmune hemolytic anemia, canine granulomatous
meningoencephalitis, rheumatoid arthritis, chronic superficial
keratitis, systemic lupus erythematosus, bullous pemphigoid,
pemphigus, and thrombocytopenias.
[0120] A radiolabeled antibody, immunoconjugate or fusion protein
may comprise an .alpha.-emitting radioisotope, a .beta.-emitting
radioisotope, an Auger electron emitter, or a neutron capturing
agent that emits .alpha.-particles or a radioisotope that decays by
electron capture. Suitable radioisotopes include .sup.198Au,
.sup.32P, 125I, .sup.131I, .sup.90Y, .sup.186Re, .sup.188Re,
.sup.67Cu, .sup.211At, .sup.213Bi, .sup.111In, .sup.67Ga,
.sup.177Lu, and .sup.225Ac, and the like.
[0121] As discussed above, a radioisotope can be attached to an
antibody component directly or indirectly, via a chelating agent.
For example, .sup.67Cu, considered one of the more promising
radioisotopes for radioimmunotherapy due to its 61.5 hour half-life
and abundant supply of beta particles and gamma rays, can be
conjugated to an antibody component using the chelating agent,
p-bromoacetamido-benzyl-tetraethylaminetetraac- etic acid (TETA).
Chase, "Medical Applications of Radioisotopes," in REMINGTON'S
PHARMACEUTICAL SCIENCES, 18th Edition, Gennaro et al. (eds.), pages
624-652 (Mack Publishing Co. 1990). Alternatively, .sup.90Y, which
emits an energetic beta particle, can be coupled to an antibody
component using diethylenetriaminepentaacetic acid (DTPA).
Moreover, a method for the direct radiolabeling of the antibody
component with .sup.131I is described by Stein et al., Antibody
Immunoconj. Radiopharm. 4:703 (1991).
[0122] Alternatively, boron addends such as carboranes can be
attached to antibody components, as discussed above.
[0123] Preferred immunoconjugates and fusion proteins to be used in
conjunction with a naked antibody or an immunoconjugate or fusion
protein that includes a drug, toxin or therapeutic radionuclide
include conjugates of an antibody component and an immunomodulator.
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-l (IL-1), IL-2, IL-3, IL-6, IL-10 and IL-12), colony
stimulating factors (e.g., granulocyte-colony stimulating factor
(G-CSF) and granulocyte macrophage-colony stimulating factor
(GM-CSF)), interferons (e.g., interferons-.alpha., -62 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,
interferon-.gamma., TNF-.alpha., and the like.
[0124] Subjects also can receive naked antibodies, immunoconjugates
and fusion proteins with a separately administered cytokine, which
can be administered before, concurrently or after administration of
the naked antibodies, immunoconujugates or fusion protein. The
cytokines enhance the activity of ADCC/NK, the effector cells that
effect kill of tumor cells by binding to the Fc domain of human
IgG1 antibodies, a domain that is present in C2B8 (CD20
antibody).
[0125] Antibody-immunomodulator immunoconjugates and
antibody-immunomodulator fusion proteins provide a means to deliver
an immunomodulator to a target cell and are particularly useful
against tumor cells. The cytotoxic effects of immunomodulators are
well known to those of skill in the art. See, for example,
Klegerman et al., "Lymphokines and Monokines," in BIOTECHNOLOGY AND
PHARMACY, Pessuto et al. (eds.), pages 53-70 (Chapman & Hall
1993). As an illustration, interferons can inhibit cell
proliferation by inducing increased expression of class I
histocompatibility antigens on the surface of various cells and
thus, enhance the rate of destruction of cells by cytotoxic T
lymphocytes. Furthermore, tumor necrosis factors, such as
TNF-.alpha., are believed to produce cytotoxic effects by inducing
DNA fragmentation.
[0126] Useful cancer chemotherapeutic drugs for the preparation of
immunoconjugates and fusion proteins include nitrogen mustards,
camptothecins, doxorubicin and analogs, alkyl sulfonates,
nitrosoureas, triazenes, folic acid analogs, pyrimidine analogs,
purine analogs, antibiotics, epipodophyllotoxins, platinum
coordination complexes, hormones, and the like. 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). Other suitable chemotherapeutic
agents, such as experimental drugs, are known to those of skill in
the art.
[0127] In addition, therapeutically useful immunoconjugates can be
obtained by conjugating photoactive agents or dyes to an antibody
composite. Fluorescent and other chromogens, or dyes, such as
porphyrins sensitive to visible light, have been used to detect and
to treat lesions by directing the suitable light to the lesion. In
therapy, this has been termed photoradiation, phototherapy, or
photodynamic therapy (Jori et al. (eds.), PHOTODYNAMIC THERAPY OF
TUMORS AND OTHER DISEASES (Libreria Progetto 1985); van den Bergh,
Chem. Britain 22:430 (1986)). Moreover, monoclonal antibodies have
been coupled with photoactivated dyes for achieving phototherapy.
Mew et al., J. Immunol. 130:1473 (1983); idem., Cancer Res. 45:4380
(1985); Oseroff et al., Proc. Natl. Acad. Sci. USA 83:8744 (1986);
idem., Photochem. Photobiol. 46:83 (1987); Hasan et al., Prog.
Clin. Biol. Res. 288:471 (1989); Tatsuta et al., Lasers Surg. Med.
9:422 (1989); Pelegrin et al., Cancer 67:2529 (1991). However,
these earlier studies did not include use of endoscopic therapy
applications, especially with the use of antibody fragments or
subfragments. Thus, the present invention contemplates the
therapeutic use of immunoconjugates comprising photoactive agents
or dyes.
[0128] Multimodal therapies of the present invention further
include immunotherapy with naked anti-CD20 and naked anti-CD19
antibodies supplemented with administration of anti-CD21 and
anti-CD22 antibodies, respectively, as well as with the
co-administration of anti-HLA-DR, CD52 and/or CD74 antibodies in
the form of naked antibodies or as therapeutic immunoconjugates.
Anti-CD19 and anti-CD20 antibodies are known to those of skill in
the art. See, for example, Ghetie et al., Cancer Res. 48:2610
(1988); Hekman et al., Cancer Immunol. Immunother. 32:364 (1991);
Kaminski et al., N. Engl. J. Med. 329:459 (1993); Press et al., N.
Engl. J. Med. 329:1219 (1993); Maloney et al., Blood 84:2457
(1994); Press et al., Lancet 346:336 (1995); Longo, Curr. Opin.
Oncol. 8:353 (1996).
[0129] In another form of multimodal therapy, subjects receive
naked antibodies, and/or immunoconjugates or fusion proteins, in
conjunction with standard cancer chemotherapy. For example, "CVB"
(1.5 g/m.sup.2 cyclophosphamide, 200-400 mg/m.sup.2 etoposide, and
150-200 mg/m.sup.2 carmustine) is a regimen used to treat
non-Hodgkin's lymphoma. Patti et al., Eur. J Haematol. 51:18
(1993). Other suitable combination chemotherapeutic regimens are
well known to those of skill in the art. See, for example, Freedman
et al., "Non-Hodgkin's Lymphomas," in CANCER MEDICINE, VOLUME 2,
3rd Edition, Holland et al. (eds.), pages 2028-2068 (Lea &
Febiger 1993). As an illustration, first generation
chemotherapeutic regimens for treatment of intermediate-grade
non-Hodgkin's lymphoma include C-MOPP (cyclophosphamide,
vincristine, procarbazine and prednisone) and CHOP
(cyclophosphamide, doxorubicin, vincristine, and prednisone). A
useful second-generation chemotherapeutic regimen is m-BACOD
(methotrexate, bleomycin, doxorubicin, cyclophosphamide,
vincristine, dexamethasone and leucovorin), while a suitable third
generation regimen is MACOP-B (methotrexate, doxorubicin,
cyclophosphamide, vincristine, prednisone, bleomycin and
leucovorin). Additional useful drugs include phenyl butyrate and
bryostatin-1. In a preferred multimodal therapy, both
chemotherapeutic drugs and cytokines are co-administered with a
naked antibody, immunoconjugate or fusion protein according to the
present invention. The cytokines, chemotherapeutic drugs and
antibody, immunoconjugate or fusion protein can be administered in
any order, or together.
[0130] Subjects being treated for autoimmune diseases also may
receive a multimodal therapy. In this case, naked antibodies,
immunoconjugates and/or fusion proteins are administered in
conjunction with standard agents used to treat autoimmune diseases.
Multimodal therapy of Class III autoimmune diseases may comprise
co-administration of therapeutics that are targeted against
T-cells, plasma cells or macrophages, such as antibodies directed
against T-cell epitopes, more particularly against the CD4 and CD5
epitopes. Gamma globulins also may be co-administered. In some
cases, it may be desirable to co-administer immunosupproessive
drugs such as corticosteroids and possibly also cytotoxic drugs. In
this case, lower doses of the corticosteroids and cytotoxic drugs
can be used as compared to the doses used in conventional
therapies, thereby reducing the negative side effects of these
therapeutics. The supplemental therapeutic compositions can be
administered before, concurrently or after administration of the
naked antibodies, immunoconjugates and/or fusion proteins.
[0131] Drugs which are known to act on B-cells, myeloid cells, mast
cells, plasma cells and/or T-cells are particularly useful in
accordance with the present invention, whether conjugated to a
targeting antibody, or administered as a separate component in
combination with naked antibodies, immunoconjugates and/or fusion
proteins. These include methotrexate, phenyl butyrate, bryostatin,
cyclophosphamide, etoposide, bleomycin, doxorubicin, carmustine,
vincristine, procarbazine, dexamethasone, leucovorin, prednisone,
maytansinoids such as DM1, calicheamicin, rapamycin, leflunomide,
FK506, immuran, fludarabine, azathiopine, mycophenolate, and
cyclosporin. Drugs such as immuran, methotrexate, and fludarabine,
which act on both B-cells and T-cells, are particularly preferred.
Illustrative of toxins which are suitably employed in accordance
with the present invention are ricin, abrin, ribonuclease, DNase I,
Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,
diphtherin toxin, Pseudomonas exotoxin, Pseudomonas endotoxin and
RNases, such as onconase. See, for example, Pastan et al., Cell
47:641 (1986), and Goldenberg, CA--A Cancer Journal for Clinicians
44:43 (1994). Other suitable drugs and toxins are known to those of
skill in the art.
[0132] In general, the dosage of administered naked antibodies,
antibody components, immunoconjugates, and fusion proteins will
vary depending upon such factors as the patient's age, species,
breed, weight, sex, general medical condition and previous medical
history. Typically, it is desirable to provide the recipient with a
dosage of naked antibody, immunoconjugate or fusion protein which
is in the range of from about 1 to 20, preferably 1 to 10, mg/kg.
The naked antibodies are administered parenterally, and may given
once, or more preferably, repeatedly. In a preferred embodiment,
the animal initially is treated repeatedly over the course of
several weeks. After the initial regimen of repeated treatments,
the animal may be placed on a maintenance regimen which involves
treatments on a monthly, bimonthly or quarterly basis, as needed,
for example.
[0133] Administration of antibody components, immunoconjugates or
fusion proteins to a patient can be intravenous, intraarterial,
intraperitoneal, intramuscular, subcutaneous, intrapleural,
intrathecal, by perfusion through a regional catheter, or by direct
intralesional injection. When administering therapeutic proteins by
injection, the administration may be by continuous infusion or by
single or multiple boluses.
[0134] Those of skill in the art are aware that intravenous
injection provides a useful mode of administration due to the
thoroughness of the circulation in rapidly distributing antibodies.
Intravenous administration, however, is subject to limitation by a
vascular barrier comprising endothelial cells of the vasculature
and the subendothelial matrix. Still, the vascular barrier is a
more notable problem for the uptake of therapeutic antibodies by
solid tumors. Lymphomas have relatively high blood flow rates,
contributing to effective antibody delivery. Intralymphatic routes
of administration, such as subcutaneous or intramuscular injection,
or by catheterization of lymphatic vessels, also provide a useful
means of treating lymphomas.
[0135] As described above, the present invention contemplates
therapeutic methods in which naked antibody components are
supplemented with immunoconjugate or fusion protein administration.
In one variation, naked antibodies are administered with low-dose
radiolabeled antibodies or fragments or fusion proteins. As a
second alternative, naked antibodies are administered with cytokine
immunoconjugates. As a third alternative, naked antibodies are
administered with immunoconjugates comprising a drug or toxin as a
therapeutic agent. As a fourth alternative, naked antibodies are
administered in conjunction with cytokine or chemotherapeutic
agents that are not conjugated to any antibody component.
[0136] For an .sup.131I-labeled immunoconjugate, a preferable
dosage is in the range of 0.25 to 2 mCi/kg, which can be repeated.
In contrast, a preferred dosage of .sup.90Y-labeled
immunoconjugates is in the range from 0.1 to 0.6 mCi/kg, which also
can be repeated.
[0137] Where the targeted B-cell antigen is the CD20 antigen, the
preferred antibody component is an antibody derived from a B1
antibody, including murine B1 monoclonal antibody, chimeric C2B8
antibody (rituximab), and humanized or caninized CD20 antibody.
[0138] Immunoconjugates having a boron addend-loaded carrier for
thermal neutron activation therapy will normally be effected in
similar ways. However, it will be advantageous to wait until
non-targeted immunoconjugate clears before neutron irradiation is
performed. Clearance can be accelerated using an antibody that
binds to the immunoconjugate. See U.S. Pat. No. 4,624,846 for a
description of this general principle.
[0139] The naked antibodies, immunoconjugates and fusion proteins
alone, or conjugated to liposomes, can be formulated according to
known methods to prepare pharmaceutically useful compositions,
whereby the therapeutic proteins are combined in a mixture with a
pharmaceutically acceptable carrier. A composition is said to be a
"pharmaceutically acceptable carrier" if its administration can be
tolerated by a recipient patient. Sterile phosphate-buffered saline
is one example of a pharmaceutically acceptable carrier. Other
suitable carriers are well-known to those in the art. See, for
example, REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (1995).
[0140] For purposes of therapy, naked antibodies, immunoconjugates
and/or fusion proteins are administered to a patient in a
pharmaceutically acceptable carrier in a therapeutically effective
amount. A "therapeutically effective amount" is an amount that is
physiologically significant. An amount is physiologically
significant if its presence results in a detectable change in the
physiology of a recipient patient. In the present context, an
amount is physiologically significant if its presence results in
the inhibition of proliferation, inactivation, or killing of
targeted cells.
[0141] Additional pharmaceutical methods may be employed to control
the duration of action of a naked antibody, immunoconjugate or
fusion protein in a therapeutic application. Control release
preparations can be prepared through the use of polymers to complex
or adsorb the antibody component, immunoconjugate or fusion
protein. 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 component (or immunoconjugate) from such a matrix depends
upon the molecular weight of the protein, the amount of antibody
component/immunoconjugate/fusion protein within the matrix, and the
size of dispersed particles. Saltzman et al., Biophys. J. 55:163
(1989); Sherwood et al., supra. Other solid dosage forms are
described in REMINGTON'S PHARMACEUTICAL SCIENCES, 19th ed.
(1995).
[0142] The present invention also contemplates a method of
treatment in which immunomodulators are administered to prevent,
mitigate or reverse radiation-induced or drug-induced toxicity of
normal cells, and especially hematopoietic cells. Adjunct
immunomodulator therapy allows the administration of higher doses
of cytotoxic agents due to increased tolerance of the recipient
mammal. Moreover, adjunct immunomodulator therapy can prevent,
palliate, or reverse dose-limiting marrow toxicity. Examples of
suitable immunomodulators for adjunct therapy include G-CSF,
GM-CSF, thrombopoietin, IL-1, IL-3, IL-12, and the like. The method
of adjunct immunomodulator therapy is disclosed by Goldenberg, U.S.
Pat. No. 5,120,525.
[0143] For example, recombinant IL-2 may be administered
intravenously as a bolus at 6.times.10.sup.5 IU/kg or as a
continuous infusion at a dose of 18.times.10.sup.6 IU/m.sup.2/d.
Weiss et al., J. Clin. Oncol. 10:275 (1992). Alternatively,
recombinant IL-2 may be administered subcutaneously at a dose of
12.times.10.sup.6 IU. Vogelzang et al., J. Clin. Oncol. 11:1809
(1993). Moreover, INF-.gamma. may be administered subcutaneously at
a dose of 1.5.times.10.sup.6 U. Lienard et al., J. Clin. Oncol.
10:52 (1992). Furthermore, Nadeau et al., J. Pharmacol. Exp. Ther.
274:78 (1995), have shown that a single intravenous dose of
recombinant IL-12 (42.5 .mu.g/kilogram) elevated IFN-.gamma. levels
in rhesus monkeys.
[0144] Suitable IL-2 formulations include PROLEUKIN (Chiron
Corp./Cetus Oncology Corp.; Emeryville, Calif.) and TECELEUKIN
(Hoffmann-La Roche, Inc.; Nutley, N.J.). ACTIMMUNE (Genentech,
Inc.; South San Francisco, Calif.) is a suitable INF-.gamma.
preparation.
[0145] The present invention, thus generally described, will be
understood more readily by reference to the following examples,
which are provided by way of illustration and are not intended to
be limiting of the present invention.
EXAMPLE 1
[0146] Peripheral blood was collected from a dog to test
crossreactivity with dog lymphocytes. Murine MAbs against a panel
of human leukocyte antigens were included in the assay. A human
blood sample was tested at the same time as a control. Single color
indirect flow cytometry analysis was performed.
[0147] Reactivity of the MAb panel with the human lymphocytes was
within the expected range. Mabs 1F5 (anti-CD20), an anti-CD20 MAb
from Biogenex, and L243 (MHC class II) all reacted with the dog
lymphocytes. LL2, LL1, A103, Leu 4 (Becton-Dickinson anti-CD3),
Leu-16 (Becton-Dickinson anti-CD20) and H-Le-1 (Becton-Dickinson
anti-CD45) did not show high crossreactivity with the dog
lymphocytes.
1 Human Canine Lymphocytes Lymphocytes Mab % Positive % Positive
Ag8 3.20 1.19 A103 33.75 2.91 LL2 10.19 3.48 LL1 10.06 0.62 Leu-4
(CD3, T-cells) 70.60 0.44 1F5 (CD20, B-cells) 11.29 24.70 Biogenex
(CD20, B-cells) 6.64 23.48 Leu-16 (CD20, B-cells) 9.15 3.17 H-Le-1
(CD45, all WBCs) 95.91 1.77 L243 (MHC class II) 16.80 79.12
EXAMPLE 2
Treatment of a Dog with Aggressive Non-Hodgkin's B-cell Lymphoma in
Lymph Nodes and Bone Marrow
[0148] A 65-pound, 7-year old male Golden Retriever is diagnosed
with diffuse large cell aggressive lymphoma. The dog is placed on
combination chemotherapy with vincristine, cyclophosphamide,
prednisolone, and doxorubicin, with good response. However, the dog
subsequently is characterized as having progressive
lymphadenopathy, and seven months after this is found to have
extensive lymphoma infiltration of bone marrow, extensive
lymphoadenopathy of neck, chest, abdomen, pelvis, and
hepatosplenomegaly (Day 0).
[0149] The dog is given therapy with 1F5 monoclonal antibody. The
dog is infused intravenously with 120 mg of 1F5 antibody, and the
treatment is repeated weekly for 4 weeks following this initial
treatment. Four months after the final dose of 1F5, a computerized
tomography scan of the patient shows no evidence of lymphoma, and
all signs and symptoms of the disease were not evident.
EXAMPLE 3
Treatment of a Dog with Relapsed Intermediate-Grade Non-Hodgkin's
Lymphoma
[0150] A 78-pound, 9-year old, German Shepherd Dog with
intermediate grade non-Hodgkin's lymphoma receives chemotherapy,
which initially leads to a complete remission for five months,
followed by another course of chemotherapy which results in stable
disease for six months. The dog then presents with recurrent
lymphoma in the chest and in a neck lymph node, both measurable by
computerized tomography and palpation, respectively.
[0151] The patient is infused with a .sup.90Y-labeled
immunoconjugate of L243 monoclonal antibody weekly for two weeks,
at a radiation dose of 8 mCi in 50 mg of antibody protein. Three
weeks later, palpation of the neck node enlargement shows a
measurable decrease, while a repeat computerized tomography scan of
the chest shows a marked reduction in tumor. Follow-up measurements
made at ten weeks post therapy show evidence of the disease in the
neck or the chest being reduced by a about 60 percent. Since new
disease is not detected elsewhere, the patient is considered to be
in partial remission. Follow-up studies every 10-12 weeks confirms
a partial remission for at least 7 months post therapy.
EXAMPLE 4
Treatment of a Cat with Relapsed Lymphoma
[0152] A 10-pound, 12-year-old, domestic short hair presents with
enlargement of a single submandibular lymph node. After excision,
there is recurrence of the lesion at 6 months. The lesion is again
excised, but then reappears 6 months later. The cat is then given
weekly treatments for 4 weeks with an .sup.131-labeled
immunoconjugate of anti-CD20 B1 monoclonal antibody, at a radiation
dose of 15 mCi in 45 mg antibody protein. The treatment is repeated
3 months later. When examined 3 months after the last treatment, a
marked decrease can be palpated. No recurrence of the disease is
observed for over one year.
EXAMPLE 5
Combination Therapy of a Dog with Autoimmune Hemolytic Anemia
[0153] A 35-pound cocker spaniel presents with immune-mediated
autoimmune hemolytic anemia. The dog is given weekly treatments
with a combination of 1F5 and L243, and is given 10 mg prednisone
twice a day. Red blood cell counts done following treatment reveal
values within normal ranges, and other abnormal signs and symptoms
disappear for 2 months.
EXAMPLE 6
Combination Therapy of a Dog with Myeloid Leukemia
[0154] A 25-pound, 9-year-old female Beagle is diagnosed with a
chronic myeloid leukemia. The dog is placed on combination
chemotherapy with vincristine, cyclophosphamide, and doxorubicin.
After an initial period of remission, the disease recurs, and the
dog is again treated with vincristine, cyclophosphamide, and
doxorubicin, but additionally is given 50 mg of CD33 (M195)
monoclonal antibody. The dog is infused intravenously, and the
treatment is repeated weekly for 4 weeks following this initial
treatment. Three months after the final treatment, a peripheral
blood cell count, as well as a bone marrow biopsy, show evidence of
a reduction of the leukemic cells by about 75 percent.
[0155] Although the foregoing refers to particular preferred
embodiments, it will be understood that the present invention is
not so limited. It will occur to those of ordinary skill in the art
that various modifications may be made to the disclosed embodiments
and that such modifications are intended to be within the scope of
the present invention, which is defined by the following
claims.
[0156] All publications and patent applications mentioned in this
specification are indicative of the level of skill of those in the
art to which the invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application were
specifically and individually indicated to be incorporated by
reference in its entirety.
* * * * *