U.S. patent application number 14/707174 was filed with the patent office on 2015-09-10 for anti-cd19 antibodies.
The applicant listed for this patent is IMMUNOMEDICS, INC.. Invention is credited to David M. Goldenberg, Hans J. Hansen, Zhengxing Qu.
Application Number | 20150252110 14/707174 |
Document ID | / |
Family ID | 42153175 |
Filed Date | 2015-09-10 |
United States Patent
Application |
20150252110 |
Kind Code |
A1 |
Hansen; Hans J. ; et
al. |
September 10, 2015 |
Anti-CD19 Antibodies
Abstract
The present invention provides humanized, chimeric and human
anti-CD19 antibodies, anti-CD19 antibody fusion proteins, and
fragments thereof that bind to a human B cell marker. Such
antibodies, fusion proteins and fragments thereof are useful for
the treatment and diagnosis of various B-cell disorders, including
B-cell malignancies and autoimmune diseases. In more particular
embodiments, the humanized anti-CD19 antibodies may comprise one or
more framework region amino acid substitutions designed to improve
protein stability, antibody binding and/or expression levels. In a
particularly preferred embodiment, the substitutions comprise a
Ser9lPhe substitution in the hA19 VH sequence.
Inventors: |
Hansen; Hans J.; (Picayune,
MS) ; Qu; Zhengxing; (Warren, NJ) ;
Goldenberg; David M.; (Mendham, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
IMMUNOMEDICS, INC. |
MORRIS PLAINS |
NJ |
US |
|
|
Family ID: |
42153175 |
Appl. No.: |
14/707174 |
Filed: |
May 8, 2015 |
Related U.S. Patent Documents
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Application
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14087799 |
Nov 22, 2013 |
9056917 |
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14707174 |
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13919512 |
Jun 17, 2013 |
8624001 |
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14087799 |
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13680713 |
Nov 19, 2012 |
8486395 |
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13919512 |
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13398214 |
Feb 16, 2012 |
8337840 |
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13680713 |
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12907262 |
Oct 19, 2010 |
8147831 |
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13398214 |
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12266999 |
Nov 7, 2008 |
7902338 |
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12907262 |
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11445410 |
Jun 1, 2006 |
7462352 |
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12266999 |
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10903858 |
Aug 2, 2004 |
7109304 |
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11445410 |
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Current U.S.
Class: |
424/85.2 ;
424/136.1; 424/178.1; 424/183.1; 424/85.1; 424/85.5; 424/85.6;
424/85.7 |
Current CPC
Class: |
A61K 2039/505 20130101;
C07K 2317/567 20130101; C07K 16/2809 20130101; A61K 51/1027
20130101; A61P 3/10 20180101; A61P 37/06 20180101; A61K 47/6867
20170801; A61K 2039/507 20130101; A61K 39/3955 20130101; C07K
16/2833 20130101; C07K 2317/52 20130101; A61K 47/6849 20170801;
A61K 39/39558 20130101; A61K 51/1093 20130101; C07K 2317/565
20130101; A61K 45/06 20130101; C07K 16/30 20130101; C07K 16/3061
20130101; C07K 2317/56 20130101; A61P 37/02 20180101; A61K 51/1069
20130101; C07K 16/2803 20130101; A61P 43/00 20180101; C07K 2317/92
20130101; C07K 16/283 20130101; A61P 37/00 20180101; A61K 38/00
20130101; C07K 16/468 20130101; A61P 35/02 20180101; C07K 2317/24
20130101; C07K 2317/732 20130101; C07K 16/2887 20130101; C07K
2317/31 20130101; A61P 29/00 20180101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 47/48 20060101 A61K047/48; A61K 39/395 20060101
A61K039/395; C07K 16/46 20060101 C07K016/46; A61K 45/06 20060101
A61K045/06 |
Claims
1. A method of treating an autoimmune disease comprising
administering a bispecific antibody comprising: a) a first
anti-CD19 antibody or antigen-binding fragment thereof, wherein the
antibody or antigen-binding fragment thereof comprises (i) the
light chain complementarity determining region CDR sequences CDR1
of SEQ ID NO: 16 (KASQSVDYDGDSYLN); CDR2 of SEQ ID NO: 17
(DASNLVS); and CDR3 of SEQ ID NO: 18 (QQSTEDPWT); (ii) the heavy
chain CDR sequences CDR1 of SEQ ID NO: 19 (SYWMN); CDR2 of SEQ ID
NO: 20 (QIWPGDGDTNYNGKFKG) and CDR3 of SEQ ID NO: 21
(RETTTVGRYYYAMDY), and (iii) human antibody framework (FR) and
constant region sequences with one or more framework region amino
acid residues substituted from the corresponding framework region
sequences of the parent murine antibody, wherein said substituted
FR residues comprise the substitution of serine for phenylalanine
at Kabat residue 91 of the heavy chain variable region; and b) a
second antibody or humanized antibody or antigen-binding fragment
thereof, wherein the second antibody binds to an antigen selected
from the group consisting of CD3, CD5, CD16, CD20, CD22, CD32b,
CD33, CD37, CD38, CD40, and HLA-DR.
2. The method of claim 1, wherein the autoimmune disease is
selected from the group consisting of acute idiopathic
thrombocytopenic purpura, chronic idiopathic thrombocytopenic
purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis,
systemic lupus erythematosus, lupus nephritis, rheumatic fever,
polyglandular syndromes, bullous pemphigoid, diabetes mellitus,
Henoch-Schonlein purpura, post-streptococcal nephritis, erythema
nodosum, Takayasu's arteritis, Addison's disease, rheumatoid
arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis,
erythema multiforme, IgA nephropathy, polyarteritis nodosa,
ankylosing spondylitis, Goodpasture's syndrome, thromboangitis
ubiterans, Sjogren's syndrome, primary biliary cirrhosis,
Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic
active hepatitis, polymyositis, dermatomyositis, polychondritis,
pemphigus vulgaris, Wegener's granulomatosis, membranous
nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant
cell arteritis/polymyalgia, pernicious anemia, rapidly progressive
glomerulonephritis, psoriasis, and fibrosing alveolitis.
3. The method of claim 1, wherein the bispecific antibody is not
conjugated to any therapeutic or diagnostic agent.
4. The method of claim 1, wherein the second antibody or fragment
thereof binds to CD3.
5. The method of claim 1, wherein the second antibody or fragment
thereof binds to CD16.
6. The method of claim 1, wherein the second antibody or fragment
thereof binds to HLA-DR.
7. The method of claim 3, further comprising administering to said
subject a drug selected from the group consisting of doxorubicin,
methotrexate, paclitaxel, cyclophosphamide, etoposide, carmustine,
vincristine, procarbazine, prednisone, bleomycin, leucovorin,
phenyl butyrate, bryostatin-1 and CPT-11.
8. The method of claim 1, wherein the anti-CD19 antibody or
antigen-binding fragment thereof comprises the sequences of SEQ ID
NO:7 (hA19VK) and SEQ ID NO:10 (hA19VH).
9. The method of claim 1, wherein the bispecific antibody is
conjugated to at least one therapeutic agent.
10. The method of claim 9 wherein said therapeutic agent is a drug
selected from the group consisting of a vinca alkaloid, an
anthracycline, a camptothecan, an epipodophyllotoxin, a taxane, a
proteosome inhibitor, a nitrogen mustard, an alkyl sulfonate, a
nitrosourea, an antimetabolite, an alkylating agent, a triazene, a
folic acid analog, a COX-2 inhibitor, a pyrimidine analog, a purine
analog, a platinum coordination complex, an antibiotic, a COX-2
inhibitor, an anti-mitotic agent, an anti-angiogenic agent and a
pro-apoptotic agent.
11. The method of claim 10, wherein said therapeutic agent is a
drug selected from the group consisting of doxorubicin,
methotrexate, paclitaxel, cyclophosphamide, etoposide, carmustine,
vincristine, procarbazine, prednisone, bleomycin, leucovorin,
phenyl butyrate, bryostatin-1 and CPT-11.
12. The method of claim 9, wherein said therapeutic agent is a
toxin selected from the group consisting of ricin, abrin, alpha
toxin, saporin, onconase, ribonuclease (RNase), DNase I,
Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,
diphtheria toxin, Pseudomonas exotoxin and Pseudomonas
endotoxin.
13. The method of claim 9, wherein said therapeutic agent is
selected from the group consisting of tumor necrosis factor (TNF),
an interleukin (IL), granulocyte-colony stimulating factor (G-CSF),
granulocyte macrophage-colony stimulating factor (GM-CSF),
interferon-alpha, interferon-beta, and interferon-gamma.
14. A method of treating an autoimmune disease comprising
administering a bispecific antibody comprising: a) a first
anti-CD19 antibody or antigen-binding fragment thereof, wherein the
antibody or antigen-binding fragment thereof comprises (i) the
light chain complementarity determining region CDR sequences CDR1
of SEQ ID NO: 16 (KASQSVDYDGDSYLN); CDR2 of SEQ ID NO: 17
(DASNLVS); and CDR3 of SEQ ID NO: 18 (QQSTEDPWT); (ii) the heavy
chain CDR sequences CDR1 of SEQ ID NO: 19 (SYWMN); CDR2 of SEQ ID
NO: 20 (QIWPGDGDTNYNGKFKG) and CDR3 of SEQ ID NO: 21
(RETTTVGRYYYAMDY), and (iii) human antibody framework (FR) and
constant region sequences with one or more framework region amino
acid residues substituted from the corresponding framework region
sequences of the parent murine antibody, wherein said substituted
FR residues comprise the substitution of serine for phenylalanine
at Kabat residue 91 of the heavy chain variable region; and b) a
second antibody or humanized antibody or antigen-binding fragment
thereof, wherein the second antibody binds to CD3.
15. A method of treating an autoimmune disease comprising
administering a bispecific antibody comprising: a) a first
anti-CD19 antibody or antigen-binding fragment thereof, wherein the
antibody or antigen-binding fragment thereof comprises (i) the
light chain complementarity determining region CDR sequences CDR1
of SEQ ID NO: 16 (KASQSVDYDGDSYLN); CDR2 of SEQ ID NO: 17
(DASNLVS); and CDR3 of SEQ ID NO: 18 (QQSTEDPWT); (ii) the heavy
chain CDR sequences CDR1 of SEQ ID NO: 19 (SYWMN); CDR2 of SEQ ID
NO: 20 (QIWPGDGDTNYNGKFKG) and CDR3 of SEQ ID NO: 21
(RETTTVGRYYYAMDY), and (iii) human antibody framework (FR) and
constant region sequences with one or more framework region amino
acid residues substituted from the corresponding framework region
sequences of the parent murine antibody, wherein said substituted
FR residues comprise the substitution of serine for phenylalanine
at Kabat residue 91 of the heavy chain variable region; and b) a
second antibody or humanized antibody or antigen-binding fragment
thereof, wherein the second antibody binds to CD16.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 14/087,799, filed Nov. 22, 2013, which was a divisional of
U.S. patent application Ser. No. 13/919,512 (now U.S. Pat. No.
8,624,001), filed Jun. 17, 2013, which was a divisional of U.S.
patent application Ser. No. 13/680,713 (now U.S. Pat. No.
8,486,395), filed Nov. 19, 2012, which was a divisional of U.S.
patent application Ser. No. 13/398,214 (now U.S. Pat. No.
8,337,840), filed Feb. 16, 2012, which was a divisional of U.S.
patent application Ser. No. 12/907,262 (now issued U.S. Pat. No.
8,147,831), filed Oct. 19, 2010, which was a divisional of U.S.
patent application Ser. No. 12/266,999 (now issued U.S. Pat. No.
7,902,338), filed Nov. 7, 2008, which was a continuation-in-part of
U.S. patent application Ser. No. 11/445,410 (now issued U.S. Pat.
No. 7,462,352), filed Jun. 1, 2006, which was a divisional of U.S.
patent application Ser. No. 10/903,858 (now issued U.S. Pat. No.
7,109,304), filed Aug. 2, 2004, which claimed priority to a
provisional U.S. Patent Application No. 60/491,282, filed Jul. 31,
2003, the contents of each of which are incorporated herein by
reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to anti-CD19 antibodies,
particularly humanized, chimeric and human anti-CD19 antibodies,
particularly monoclonal antibodies (MAbs) and fragments thereof,
either naked or conjugated to at least one therapeutic and/or
diagnostic agent, and methods of use thereof. In particular, the
anti-CD19 antibodies can be used for treating B cell disease such
as, for example, a malignancy, an inflammatory disease or disorder,
or an autoimmune disease. In more particular embodiments, the
anti-CD19 antibodies may comprise one or more substituted amino
acids designed to optimize a physical and/or physiological
characteristic of the antibody.
[0004] 2. Description of Related Art
[0005] The immune system of vertebrates consists of a number of
organs and cell types which have evolved to accurately recognize
foreign antigens, specifically bind to, and eliminate/destroy such
foreign antigens. Lymphocytes, among other cell types, are critical
to the immune system. Lymphocytes are divided into two major
sub-populations, T cells and B cells. Although inter-dependent, T
cells are largely responsible for cell-mediated immunity and B
cells are largely responsible for antibody production (humoral
immunity).
[0006] In humans, each B cell can produce an enormous number of
antibody molecules. Such antibody production typically ceases (or
substantially decreases) when a foreign antigen has been
neutralized. Occasionally, however, proliferation of a particular B
cell will continue unabated and may result in a cancer known as a B
cell lymphoma or leukemia. B-cell lymphomas, such as the B-cell
subtype of non-Hodgkin's lymphoma, are significant contributors to
cancer mortality.
[0007] The response of B-cell malignancies to various forms of
treatment is mixed. For example, 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. Devesa et al., J.
Nat'l Cancer Inst. 79:701 (1987).
[0008] The majority of chronic lymphocytic leukemias are of the
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-incompetent 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
cytotoxic drug treatment. Traditional methods of treating B-cell
malignancies, including chemotherapy and radiotherapy, have limited
utility due to toxic side effects.
[0009] B cells comprise cell surface proteins which can be utilized
as markers for differentiation and identification. One such human
B-cell marker is a CD19 antigen and is found on mature B cells but
not on plasma cells. CD19 is expressed during early pre-B cell
development and remains until plasma cell differentiation. CD19 is
expressed on both normal B cells and malignant B cells whose
abnormal growth can lead to B-cell lymphomas. For example, CD19 is
expressed on B-cell lineage malignancies, including, but not
limited to non-Hodgkin's lymphoma, chronic lymphocytic leukemia,
and acute lymphoblastic leukemia.
[0010] A potential problem with using non-human monoclonal
antibodies (e.g., murine monoclonal antibodies) is typically lack
of human effector functionality. In other words, such antibodies
may be unable to mediate complement-dependent lysis or lyse human
target cells through antibody-dependent cellular toxicity or
Fc-receptor mediated phagocytosis. Furthermore, non-human
monoclonal antibodies can be recognized by the human host as a
foreign protein and, therefore, repeated injections of such foreign
antibodies can lead to the induction of immune responses leading to
harmful hypersensitivity reactions. For murine-based monoclonal
antibodies, this is often referred to as a Human Anti-Mouse
Antibody (HAMA) response.
[0011] The use of chimeric antibodies is more preferred because
they do not elicit as strong a HAMA response as murine antibodies.
Chimeric antibodies are antibodies which comprise portions from two
or more different species. For example, Liu, A. Y. et al,
"Production of a Mouse-Human Chimeric Monoclonal Antibody to CD20
with Potent Fc-Dependent Biologic Activity" J. Immun.
139/10:3521-3526 (1987), describe a mouse/human chimeric antibody
directed against the CD20 antigen. See also, PCT Publication No. WO
88/04936. However, no information is provided as to the ability,
efficacy or practicality of using such chimeric antibodies for the
treatment of B cell disorders in the reference. It is noted that in
vitro functional assays (e.g., complement-dependent lysis (CDC);
antibody dependent cellular cytotoxicity (ADCC), etc.) cannot
inherently predict the in vivo capability of a chimeric antibody to
destroy or deplete target cells expressing the specific antigen.
See, for example, Robinson, R. D. et al., "Chimeric mouse-human
anti-carcinoma antibodies that mediate different anti-tumor cell
biological activities," Hum. Antibod. Hybridomas 2:84-93 (1991)
(chimeric mouse-human antibody having undetectable ADCC activity).
Therefore, the potential therapeutic efficacy of a chimeric
antibody can only truly be assessed by in vivo experimentation,
preferably in the species of interest for the specific therapy.
[0012] One approach that has improved the ability of murine
monoclonal antibodies to be effective in the treatment of B-cell
disorders has been to conjugate a radioactive label or
chemotherapeutic agent to the antibody, such that the label or
agent is localized at the tumor site. For example, studies indicate
that .sup.90Y labeled anti-CD19 antibodies can be used to reduce
lymphoma in mice (McDevitt et al., Leukemia 16:60, 2002), anti-CD19
antibodies conjugated to idarubicin result in tumor regression in
an experimental model (Rowland et al., Cancer Immunol. Immunother.,
37:195, 1993), and .sup.125I and .sup.111In radiolabeled anti-CD19
is specifically taken up in tumor bearing organs (Mitchell et al.,
J. Nucl. Med., 44: 1105, 2003). Combination therapy with an
anti-CD19 antibody is also disclosed in Ek et al., Leuk. Lymphoma
31: 143 (1998) and Uckun et al., Blood, 79:3116 (1992). Treatment
of human B cell lymphoma with an anti-CD19 antibody and
anti-CD3.times. anti-CD19 diabody is disclosed in Hekman et al.,
Cancer Immunol. Immunother., 32:364 (1991) and Cochlovius et al.,
J. Immunol., 165:888 (2000), respectively.
[0013] However, these approaches have not eliminated the obstacles
associated with using murine antibodies, despite the fact that many
patients with lymphoma who have received prior aggressive cytotoxic
chemotherapy are immune suppressed, thus having lower HAMA rates
than lymphoma patients who have not been heavily pretreated.
[0014] Inflammatory diseases, including autoimmune diseases are
also a class of diseases associated with B-cell disorders. 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 bone marrow, liver and kidneys. Other therapeutics that have
been used to treat Class III autoimmune diseases have been directed
against T-cells and macrophages. There is a need for more effective
methods of treating autoimmune diseases, particularly Class III
autoimmune diseases.
SUMMARY OF THE INVENTION
[0015] The present invention provides humanized, chimeric and human
anti-CD19 monoclonal antibodies and fragments thereof, and antibody
fusion proteins and fragments thereof for the treatment of B cell
lymphomas and leukemias and autoimmune disorders in humans and
other mammals. The antibodies, fusion proteins and fragments
thereof can be used alone, conjugated to at least one diagnostic
and/or therapeutic agent or in combination with other treatment
modalities.
[0016] Methods of use of the claimed antibodies may include
treatment of mammalian subjects, such as humans or domestic
animals, with one or more humanized, chimeric or human anti-CD19
antibodies, alone, as an antibody fusion protein, as a therapeutic
conjugate alone or as part of an antibody fusion protein, in
combination, or as a multimodal therapy, with other antibodies,
other therapeutic agents or immunomodulators or as an
immunoconjugate linked to at least one therapeutic agent,
therapeutic radionuclide or immunomodulator. These humanized,
chimeric and human anti-CD19 antibodies can also be used as a
diagnostic imaging agent alone, in combination with other
diagnostic imaging agents, and/or in conjunction with therapeutic
applications. Disease states that may be treated include
neoplasias, preferably B cell related lymphomas and leukemias, such
as non-Hodgkin's lymphoma, chronic lymphocytic leukemia, acute
lymphoblastic leukemia, or multiple myeloma. Other disease states
that may be treated include autoimmune diseases, such as acute
idiopathic thrombocytopenic purpura, chronic idiopathic
thrombocytopenic purpura, dermatomyositis, Sydenham's chorea,
myasthenia gravis, systemic lupus erythematosus, lupus nephritis,
rheumatic fever, polyglandular syndromes, bullous pemphigoid,
diabetes mellitus, Henoch-Schonlein purpura, post-streptococcal
nephritis, erythema nodosurn, Takayasu's arteritis, Addison's
disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis,
ulcerative colitis, erythema multiforme, IgA nephropathy,
polyarteritis nodosa, ankylosing spondylitis, Goodpasture's
syndrome, thromboangitis ubiterans, Sjogren's syndrome, primary
biliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis,
scleroderma, chronic active hepatitis,
polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris,
Wegener's granulomatosis, membranous nephropathy, amyotrophic
lateral sclerosis, tabes dorsalis, giant cell
arteritis/polymyalgia, pernicious anemia, rapidly progressive
glomerulonephritis, psoriasis, and fibrosing alveolitis. The
skilled artisan will realized that these are not limiting and any
disease state in which CD19 expressing cells play a role may
potentially be treated with the claimed anti-CD19 antibodies, alone
or in combination.
[0017] Various embodiments concern antibody fusion proteins and
fragments thereof comprising at least two anti-CD19 MAbs or
fragments thereof, or at least one anti-CD19 MAb or fragment
thereof and at least one second MAb or fragment thereof, other than
the anti-CD19 MAb or fragment thereof. Second antibodies of use may
include antibodies against other B cell associated or B cell
specific antigens, such as CD20, CD22, CD23, CD80 or HLA-DR. The
multispecific and/or fusion proteins can be either naked or
conjugated to at least one therapeutic and/or diagnostic agent.
[0018] The humanized, chimeric and human anti-CD19 MAbs and
fragments thereof, and antibody fusion proteins and fragments
thereof may be administered alone, either naked or conjugated to a
therapeutic or diagnostic agent, or in combination with another
naked antibody, fragment or immunoconjugate. Also, naked or
conjugated anti-CD19 antibodies and fragments thereof, and antibody
fusion proteins and fragments thereof may be administered in
combination with at least one therapeutic agent or diagnostic agent
that is not conjugated to an anti-CD19 antibody or fragment
thereof, or fusion protein or fragment thereof.
[0019] Other embodiments relate to DNA sequences encoding a
humanized, chimeric or human anti-CD19 antibody and fragment
thereof, and antibody fusion protein and fragment thereof.
Likewise, a vector and host cell containing the DNA sequence is
also contemplated. The claimed methods also include methods of
making the humanized, chimeric and human anti-CD19 antibodies and
fragments thereof, and fusion proteins and fragments thereof.
[0020] Particular embodiments relate to anti-CD19 MAbs or fragments
thereof that contain specific murine CDRs that have specificity for
CD19. These MAbs can be humanized, chimeric or human anti-CD19
MAbs. In preferred embodiments, the antibodies may comprise one or
more substituted amino acid residues, such as the substitution of a
corresponding murine framework region amino acid residue in a human
framework region sequence of a humanized antibody. Preferred
framework region amino acids that are candidates for substitution
include those that are located close or adjacent to one or more CDR
amino acid side chains, or that otherwise affect the stability
and/or expression levels of the encoded protein. In a most
preferred embodiment, the substitution includes replacement of a
serine residue with a phenylalanine residue at Kabat residue 91 of
the VH sequence of a humanized A19 (hA19) antibody.
BRIEF DESCRIPTION OF THE FIGURES
[0021] FIG. 1A discloses the Vk (variable light chain) sequences of
cA19, a chimeric anti-CD19 antibody. The light chain variable
region sequences are shown (SEQ ID NO:1 and SEQ ID NO:2). The CDR
region sequences are shown in bold and underlined. The nucleotides
are numbered sequentially. Kabat's Ig molecule numbering is used
for amino acid residues as shown by the numbering above the amino
acid residues. The amino acid residues numbered by letters are the
insertion residues defined by Kabat numbering scheme. The insertion
residues have the same preceding digits as that of the previous
residue.
[0022] FIG. 1B discloses the V.sub.H (variable heavy chain)
sequences of cA19, a chimeric anti-CD19 antibody. The heavy chain
variable region sequences are shown (SEQ ID NO:3 and SEQ ID NO:4).
The CDR region sequences are shown in bold and underlined. The
nucleotides are numbered sequentially. Kabat's Ig molecule
numbering is used for amino acid residues as shown by the numbering
above the amino acid residues. The amino acid residues numbered by
letters are the insertion residues defined by Kabat numbering
scheme. The insertion residues have the same preceding digits as
that of the previous residue. For example, residues 82, 82A, 82B,
and 82C in FIG. 1B are indicated as 82, A, B, and C,
respectively.
[0023] FIG. 2 shows the results of cell surface competitive binding
assay to compare the binding specificity of the cA19 antibody with
that of other anti-CD19 antibodies, BU12 and B4. Increasing
concentrations of cA19 blocked the binding of radiolabeled BU12 to
Raji cells in a similar fashion as the unlabeled BU12 and B4,
indicating these antibodies recognize similar or overlap epitopes
of the CD19 molecule.
[0024] FIG. 3A compares the amino acid sequences of the variable
light chain (Vk) regions of human antibodies, the chimeric and the
humanized anti-CD19 antibodies. The amino acid sequences of the
variable light chain (Vk) of the human antibody, (REIVk, SEQ ID
NO:5), a chimeric antibody, (cA19Vk, SEQ ID NO:6), and a humanized
antibody, (hA19Vk, SEQ ID NO:7).
[0025] FIG. 3B compares the amino acid sequences of the variable
heavy chain (V.sub.H) regions of human antibodies, the chimeric and
the humanized anti-CD19 antibodies. The amino acid sequences of the
variable heavy chain (VH) of the human antibodies, EU (SEQ ID NO:8)
and NEWM (FR4 only, SEQ ID NO:11), the chimeric antibody, (cA19VH,
SEQ ID NO:9) and a humanized antibody (hA19VH, SEQ ID NO:10).
[0026] FIG. 4A discloses the DNA and amino acid sequences (SEQ ID
NO:12 and SEQ ID NO:13) of the light chain of the humanized
anti-CD19 antibody, hA19. The nucleotide sequences are shown in
lowercase. Numbering of Vk amino acid residues is the same as that
in FIG. 1.
[0027] FIG. 4B discloses the DNA and amino acid sequences (SEQ ID
NO:14 and SEQ ID NO:15) of the heavy chain of the humanized
anti-CD19 antibody, hA19. The nucleotide sequences are shown in
lowercase. Numbering of V.sub.H amino acid residues is the same as
that in FIG. 1.
[0028] FIG. 5A shows the results of cell surface competitive
binding assay to compare the binding specificity and activity of
the humanized A19 antibody, hA19, with that of cA19. Both
unconjugated hA19 (closed triangles) and cA19 (closed diamonds)
blocked the binding of .sup.125I-hA19 to Raji cells.
[0029] FIG. 5B shows the results of cell surface competitive
binding assay to compare the binding specificity and activity of
the humanized A19 antibody, hA19, with that of cA19. Both hA19
(closed triangles) and cA19 (closed diamonds) competed equally well
for the binding of .sup.125I-cA19 to Raji cells. Increasing
concentrations of either cA19 or hA19 blocked the binding of
radiolabeled hA19 or cA19 to Raji cells respectively.
[0030] FIG. 6 shows the determination of the Ag-binding affinity
(avidity) of the anit-CD19 Ab by the direct cell surface binding
and Scatchard plot analysis. Varying concentrations of
.sup.125I-hA19 (diamonds) or .sup.112In-cA19 (squares) were
incubated with Raji cells at 40.degree. C. for 1 h. Total and bound
radioactivities were counted and analyzed by Scatchard plot as
shown in the inset. hA19 showed virtually same binding affinity as
cA19. As shown the apparent dissociation constant values were
calculated to be 1.1 and 1.2 nM for hA19 and cA19,
respectively.
DETAILED DESCRIPTION OF THE INVENTION
[0031] Definitions
[0032] Unless otherwise specified, "a" or "an" as used herein means
"one or more."
[0033] Unless otherwise specified, terms are used in accordance
with their plain and ordinary meaning
[0034] An "antibody," as described herein, refers to a full-length
(i.e., naturally occurring or formed by normal immunoglobulin gene
fragment recombinatorial processes) immunoglobulin molecule (e.g.,
an IgG antibody) or an immunologically active (i.e., specifically
binding) portion of an immunoglobulin molecule, like an antibody
fragment.
[0035] An "antibody fragment" is a portion of an antibody such as
F(ab).sub.2, F(ab').sub.2, Fab, Fab', Fv, sFv 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-CD19 monoclonal antibody fragment binds with an epitope of
CD19. Antibody fragments include isolated fragments consisting of
the variable regions, such as "Fv" fragments consisting of the
variable regions of the heavy and light chains or recombinant
single chain polypeptide molecules in which light and heavy
variable regions are connected by a peptide linker ("scFv
proteins").
[0036] A "naked antibody" is generally an entire antibody which is
not conjugated to a therapeutic agent. This is so because the Fc
portion of the antibody molecule provides effector functions, such
as complement fixation and ADCC (antibody dependent cell
cytotoxicity), which set mechanisms into action that may result in
cell lysis. However, it is possible that the Fc portion is not
required for therapeutic function, with other mechanisms, such as
apoptosis, coming into play. "Naked antibodies" include both
polyclonal and monoclonal antibodies, as well as certain
recombinant antibodies, such as chimeric, humanized or human
antibodies.
[0037] A "chimeric antibody" is a recombinant protein that contains
the variable domains including the complementarity determining
regions (CDRs) of an antibody derived from one species, preferably
a rodent antibody, while the constant domains of the antibody
molecule are derived from those of a human antibody. For veterinary
applications, the constant domains of the chimeric antibody may be
derived from that of other species, such as a cat or dog.
[0038] A "humanized antibody" is a recombinant protein in which the
CDRs from an antibody from one species; e.g., a rodent antibody,
are transferred from the heavy and light variable chains of the
rodent antibody into human heavy and light variable domains. The
constant domains of the antibody molecule are derived from those of
a human antibody.
[0039] A "human antibody" is an antibody that contains human
variable and constant region sequences. For example, human
antibodies may be obtained from transgenic mice that have been
engineered to produce human antibodies in response to antigenic
challenge. In this technique, elements of the human heavy and light
chain loci are introduced into strains of mice derived from
embryonic stem cell lines that contain targeted disruptions of the
murine endogenous heavy chain and light chain loci. The transgenic
mice can synthesize human antibodies specific for human antigens,
and the mice can be used to produce human antibody-secreting
hybridomas. Methods for obtaining human antibodies from transgenic
mice are described by Green et al., Nature Genet. 7:13 (1994),
Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int.
Immun. 6:579 (1994). A fully human antibody also can be constructed
by genetic or chromosomal transfection methods, as well as phage
display technology, all of which are known in the art. See for
example, McCafferty et al., Nature 348:552-553 (1990) for the
production of human antibodies and fragments thereof in vitro, from
immunoglobulin variable domain gene repertoires from unimmunized
donors. In this technique, antibody variable domain genes are
cloned in-frame into either a major or minor coat protein gene of a
filamentous bacteriophage, and displayed as functional antibody
fragments on the surface of the phage particle. Because the
filamentous particle contains a single-stranded DNA copy of the
phage genome, selections based on the functional properties of the
antibody also result in selection of the gene encoding the antibody
exhibiting those properties. In this way, the phage mimics some of
the properties of the B cell. Phage display can be performed in a
variety of formats, for their review, see e.g. Johnson and
Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993).
Human antibodies may also be generated by in vitro activated B
cells. See U.S. Pat. Nos. 5,567,610 and 5,229,275, which are
incorporated in their entirety by reference.
[0040] A "therapeutic agent" is a molecule or atom which is
administered separately, concurrently or sequentially with an
antibody moiety or conjugated to an antibody moiety, i.e., antibody
or antibody fragment, or a sub fragment, and is useful in the
treatment of a disease. Examples of therapeutic agents include
antibodies, antibody fragments, drugs, toxins, enzymes,
oligonucleotides, antisense and RNAi oligonucleotides, nucleases,
hormones, immunomodulators, chelators, boron compounds, photoactive
agents or dyes and radioisotopes.
[0041] A "diagnostic agent" is a molecule or atom which may be
administered conjugated to an antibody moiety, i.e., antibody or
antibody fragment, or subfragment, and is useful in diagnosing a
disease by locating the cells containing a target antigen. Useful
diagnostic agents include, but are not limited to, radioisotopes,
dyes, contrast agents, ultrasound-enhancing agents,
optical-enhancing agents, fluorescent compounds or molecules and
enhancing agents (e.g. paramagnetic ions) for magnetic resonance
imaging (MRI). U.S. Pat. No. 6,331,175 describes MRI technique and
the preparation of antibodies conjugated to a MRI enhancing agent
and is incorporated in its entirety by reference. Preferably, the
diagnostic agents are selected from the group consisting of
radioisotopes, enhancing agents for use in magnetic resonance
imaging, ultrasound, and fluorescent compounds. 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), DOTA, NOTA, porphyrins,
polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and
like groups known to be useful for this purpose. Chelates are
coupled to the peptides or proteins 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. No.
4,824,659 to Hawthorne, entitled "Antibody Conjugates", issued Apr.
25, 1989, the disclosure of which is incorporated herein in its
entirety by reference. Particularly useful metal-chelate
combinations include 2-benzyl-DTPA and its monomethyl and
cyclohexyl analogs, used with diagnostic isotopes in the general
energy range of 60 to 4,000 keV, such as .sup.125I, .sup.131I,
.sup.123I, .sup.124I, .sup.62Cu, .sup.18F, .sup.111In, .sup.67Ga,
.sup.68Ga, .sup.99mTc, .sup.94mTc, .sup.11C, .sup.13N, .sup.15O,
.sup.76Br, for radio-imaging. 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, yttrium 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 macro cyclic polyethers, which are of interest for stably
binding nuclides, such as .sup.223Ra for RAIT are encompassed.
[0042] An "immunoconjugate" is a conjugate of an antibody component
with a therapeutic or diagnostic agent.
[0043] An "expression vector" is a nucleic acid molecule,
preferably a double-stranded 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.
[0044] A "recombinant host cell" 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, as well as a transgenic animal, that have been genetically
engineered to contain the cloned gene(s) in the chromosome or
genome of the host cell or cells of the host animal. Suitable
mammalian host cells include myeloma cells, such as SP2/0 cells,
and NS0 cells, as well as Chinese Hamster Ovary (CHO) cells,
hybridoma cell lines and other mammalian host cells useful for
expressing antibodies. Also useful to express MAbs and other fusion
proteins, is a human cell line, PER.C6 disclosed in WO 0063403 A2.
Most preferred host cells are cells that have been engineered to
contain a Bcl-EEE gene or other apoptosis inhibitor, that have been
pre-adapted to grow and be transfected in serum-free or low-serum
media. Examples of such host cells are disclosed in U.S. patent
application Ser. No. 11/187,863, filed Jul. 25, 2005 and Ser. No.
11/487,215, filed Jul. 14, 2006, the text of each of which is
incorporated herein by reference in its entirety. Special
transgenic animals with a modified immune system are particularly
useful for making fully human antibodies.
[0045] As used herein, the term "antibody fusion protein" refers to
a recombinantly produced antigen-binding molecule comprising one or
more of the same or different single-chain antibody or antibody
fragment segments with the same or different specificities.
Antibody fusion proteins may comprise an antibody or fragment
thereof attached to another protein or peptide, such as a
therapeutic agent, toxin, cytokine, hormone or other protein or
peptide. In other embodiments, antibody fusion proteins may
comprise at least a first and second antibody or antibody fragment.
Where fusion proteins comprise two or more antibodies or fragments,
the valency of the fusion protein indicates how many binding arms
or sites the fusion protein has to a single antigen or epitope;
i.e., monovalent, bivalent, trivalent or multivalent. Specificity
indicates how many antigens or epitopes an antibody fusion protein
is able to bind; i.e., monospecific, bispecific, trispecific,
multispecific. Using these definitions, a natural antibody, e.g.,
an IgG, is bivalent because it has two binding arms but is
monospecific because it binds to one epitope.
[0046] 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 CD20, CD19, CD20,
CD21, CD23, CD46, CD80, HLA-DR, CD74, and CD22 on B-cells.
Multispecific, multivalent antibodies are constructs that have more
than one binding site, and the binding sites are of different
specificity. For example, a diabody, where one binding site reacts
with one antigen and the other with another antigen.
[0047] 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.
A variety of bispecific fusion proteins can be produced using
molecular engineering.
[0048] 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. 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.
[0049] Overview
[0050] As discussed above, anti-CD19 antibodies that are
unconjugated or labeled with a therapeutic radionuclide, have
failed to provide high rates of objective and lasting responses in
patients with intermediate or aggressive forms of B-cell lymphoma.
The present invention provides humanized, chimeric or human
anti-CD19 antibodies and antibody fusion proteins useful for
treatment of mammalian subjects, humans and domestic animals,
alone, as a conjugate or administered in combination with other
therapeutic agents, including other naked antibodies and antibody
therapeutic conjugates.
[0051] The anti-CD19 MAbs preferably contain specific murine CDRs
from one or more murine or chimeric anti-CD19 MAbs that have
specificity for the CD19 antigen. The anti-CD19 MAbs are humanized,
chimeric or human MAbs. The CDRs of the light chain variable region
of the anti-CD19 MAb preferably comprises CDR1 comprising amino
acids KASQSVDYDGDSYLN (SEQ ID NO:16); CDR2 comprising amino acids
DASNLVS (SEQ ID NO:17); and CDR3 comprising amino acids QQSTEDPWT
(SEQ ID NO:18); and the heavy chain variable region CDR1 comprising
amino acids SYWMN (SEQ ID NO:19); CDR2 comprising amino acids
QIWPGDGDTNYNGKFKG (SEQ ID NO:20) and CDR3 comprising amino acids
RETTTVGRYYYAMDY (SEQ ID NO:21).
[0052] In a preferred embodiment, the humanized anti-CD19 MAb or
fragment thereof comprises the CDRs of a murine anti-CD19 MAb and
the framework (FR) regions of the light and heavy chain variable
regions of a human antibody and the light and heavy chain constant
regions of a human antibody, while retaining substantially the
B-cell, and B-cell lymphoma and leukemia cell targeting of the
parent murine anti-CD19 MAb, and wherein the CDRs of the light
chain variable region of the anti-CD19 MAb comprise CDR1 comprising
amino acids KASQSVDYDGDSYLN (SEQ ID NO:16); CDR2 comprising amino
acids DASNLVS (SEQ ID NO:17); and CDR3 comprising amino acids
QQSTEDPWT (SEQ ID NO:18); and the CDRs of the heavy chain variable
region of the anti-CD19 MAb comprise CDR1 comprising amino acids
SYWMN (SEQ ID NO:19); CDR2 comprising amino acids QIWPGDGDTNYNGKFKG
(SEQ ID NO:20) and CDR3 comprising amino acids RETTTVGRYYYAMDY (SEQ
ID NO:21). The humanized anti-CD19 MAb or fragment thereof may
further contain in the FRs of the light and heavy chain variable
regions of the antibody at least one amino acid from the
corresponding FRs of the murine MAb. Specifically, the humanized
anti-CD19 MAb or fragment thereof may contain at least one amino
acid residue selected from residues 5, 27, 28, 40, 48, 91, 94, 107
and 108 of the murine heavy chain variable region of FIG. 4A and at
least one amino acid residue selected from residues 4, 39, 58, 60,
87, 100, and 107 of the murine light chain variable region FIG. 4B.
In a more preferred embodiment, the humanized A19 antibody (hA19)
contains each of the substituted murine FR amino acid residues
listed above. One or more of the murine amino acid sequences can be
maintained in the human FR regions of the light and heavy variable
chains if necessary to maintain proper binding or to enhance
binding to the CD19 antigen. More preferably the humanized
anti-CD19 MAb or fragment thereof comprises the hA19Vk (SEQ ID
NO:7) of FIG. 3A and the hA19VH (SEQ ID NO:10) of FIG. 3B. Most
preferably, the humanized anti-CD19 MAb comprises an additional FR
substitution of a serine residue with a phenylalanine residue at
Kabat residue 91 of the hA19VH (SEQ ID NO:10) sequence.
[0053] The preferred chimeric anti-CD19 (cA19) MAb or fragment
thereof comprises the CDRs of a murine anti-CD19 MAb and the FR
regions of the light and heavy chain variable regions of the murine
anti-CD19 MAb, i.e., the Fvs of the parental murine MAb, and the
light and heavy chain constant regions of a human antibody, wherein
the chimeric anti-CD19 MAb or fragment thereof retains
substantially the B-cell, and B-cell lymphoma and leukemia cell
targeting of the murine anti-CD19 MAb, wherein the CDRs of the
light chain variable region of the anti-CD19 MAb comprises CDR1
comprising amino acids KASQSVDYDGDSYLN (SEQ ID NO:16); CDR2
comprising amino acids DASNLVS (SEQ ID NO:17); and CDR3 comprising
amino acids QQSTEDPWT (SEQ ID NO:18); and the CDRs of the heavy
chain variable region of the anti-CD19 MAb comprises CDR1
comprising amino acids SYWMN (SEQ ID NO:19); CDR2 comprising amino
acids Q1WPGDGDTNYNGKFKG and CDR3 comprising amino acids
RETTTVGRYYYAMDY (SEQ ID NO:21). More preferably the chimeric
anti-CD19 MAb or fragment thereof comprises the light and heavy
chain variable region sequences of the chimeric anti-CD19 MAb shown
in FIGS. 1A and 1B, respectively, designated cA19Vk (SEQ ID NO:6)
and cA19VH (SEQ ID NO:9).
[0054] Various embodiments also encompass a human anti-CD19 MAb or
fragment thereof comprising the light and heavy chain variable and
constant regions of a human antibody, wherein said human anti-CD19
MAb retains substantially the B-cell, and B-cell lymphoma and
leukemia cell targeting and cell binding characteristics of a
murine anti-CD19 MAb, wherein the CDRs of the light chain variable
region of the human anti-CD19 MAb comprises the same CDRs (SEQ ID
NOs:16-21) as set forth above for the chimeric and humanized
anti-CD19 MAbs and as shown in FIGS. 1A and 1B, and 3A and 3B,
respectively.
[0055] Certain embodiments are also intended to encompass antibody
fusion proteins or fragments thereof comprising at least two
anti-CD19 MAbs or fragments thereof, as described above. The
antibody fusion protein or fragment thereof is also intended to
encompass an antibody fusion protein or fragment thereof comprising
at least one first anti-CD19 MAb or fragment thereof as described
above and at least one second MAb or fragment thereof, other than
the anti-CD19 MAb or fragment described above. More preferably this
second MAb is a MAb reactive with CD4, CD5, CD8, CD14, CD15, CD19,
CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD4OL, CD46,
CD52, CD54, CD74, CD80, CD126, CD138, B7, MUC-1, Ia, HM1.24,
HLA-DR, tenascin, an angiogenesis factor, VEGF, PIGF, ED-B
fibronectin, an oncogene, an oncogene product, CD66a-d, necrosis
antigens, Ii, IL-2, T101, TAC, IL-6, TRAIL-R1 (DR4) and TRAIL-R2
(DR5) or a combination thereof, and even an anti-CD19 MAb that is
directed to a different epitope than the anti-CD19 MAb described
herein. The antibody fusion proteins may be composed of one
anti-CD19 MAb and one or more of the second MAbs to provide
specificity to different antigens, and are described in more detail
below.
[0056] The humanized, chimeric and human anti-CD19 antibody may
possess enhanced affinity binding with the epitope, as well as
antitumor and anti-B-cell activity, as a result of amino acid
mutation and manipulation of the sequences in the variable region
to obtain a superior therapeutic agent for the treatment of B-cell
disorders, including B-cell lymphomas and leukemias and autoimmune
diseases. Modification to the binding specificity, affinity or
avidity of an antibody is known and described in WO 98/44001, as
affinity maturation, and this application summarizes methods of
modification and is incorporated in its entirety by reference.
[0057] It may also be desirable to modify the antibodies to improve
effector function, e.g., so as to enhance antigen-dependent
cell-mediated cytotoxicity (ADCC) and/or complement dependent
cytotoxicity (CDC) of the antagonist. One or more amino acid
substitutions or the introduction of cysteine in the Fc region may
be made, thereby improving internalization capability and/or
increased complement-mediated cell killing and ADCC. See Caron et
al., J. Ex. Med. 176:1191-1195 (1991) and Shopes, Brit. J. Immunol.
148:2918-2022 (1992), incorporated herein by reference in their
entirety. An antibody fusion protein may be prepared that has dual
Fc regions with both enhanced complement lysis and ADCC
capabilities.
[0058] Certain embodiments are also directed to DNA sequences
comprising a nucleic acid encoding a MAb or fragment thereof
selected from the group consisting of: (a) an anti-CD19 MAb or
fragment thereof as described herein, (b) an antibody fusion
protein or fragment thereof comprising at least two of the
anti-CD19 MAbs or fragments thereof, (c) an antibody fusion protein
or fragment thereof comprising at least one first MAb or fragment
thereof comprising the anti-CD19 MAb or fragment thereof as
described herein and at least one second MAb or fragment thereof,
other than the antiCD19 MAb or fragment thereof, and (d) an
antibody fusion protein or fragment thereof comprising at least one
first MAb or fragment thereof comprising the anti-CD19 MAb or
fragment thereof and at least one second MAb or fragment thereof,
wherein the second MAb is a MAb reactive with CD4, CD5, CD8, CD14,
CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40,
CD4OL, CD46, CD52, CD54, CD74, CD80, CD126, CD138, B7, MUC1, la,
HM1.24, HLA-DR, tenescin, ED-B fibronectin, IL-6, VEGF, PIGF,
TRAIL-R1 (DR4) and TRAIL-R2 (DR5) or a combination thereof.
[0059] Also encompassed are expression vectors comprising the DNA
sequences. These vectors contain the light and heavy chain constant
regions and the hinge region of the human immunoglobulin, in the
case of vectors for use in preparing the humanized, chimeric and
human anti-CD19 MAbs or antibody fusion proteins thereof or
fragments thereof. These vectors additionally contain, where
required, promoters that express the MAbs in the selected host
cell, immunoglobulin enhancers and signal or leader sequences.
Vectors that are particularly useful in the present invention are
pdHL2 or GS, particularly when used to express a chimeric,
humanized or human antibody, such as IgGs, where the vector codes
for the heavy and light chain constant regions and hinge region of
IgG1. More preferably, the light and heavy chain constant regions
and hinge region are from a human ED myeloma immunoglobulin, where
optionally at least one of the amino acids in the allotype
positions is changed to that found in a different IgG1 allotype,
and wherein optionally amino acid 253 of the heavy chain of EU
based on the EU number system may be replaced with alanine See
Edelman et al., Proc. Natl. Acad. Sci USA 63: 78-85 (1969),
incorporated herein in its entirety by reference.
[0060] Host cells containing the DNA sequences encoding the
anti-CD19 MAbs or fragments thereof or antibody fusion proteins or
fragments thereof or host cells containing the vectors that contain
these DNA sequences are encompassed by the present invention.
Particularly useful host cells are mammalian cells, more
specifically lymphocytic cells, such as myeloma cells, discussed in
more detail below.
[0061] Also encompassed are methods of expressing the anti-CD19 MAb
or fragment thereof or antibody fusion protein or fragment thereof
comprising: (a) transfecting a mammalian cell with a DNA sequence
encoding the anti-CD19 MAbs or fragments thereof or antibody fusion
proteins or fragments thereof, and (b) culturing the cell
transfected with the DNA sequence that secretes the anti-CD19 or
fragment thereof or antibody fusion protein or fragment thereof.
Known techniques may be used that include a selectable marker on
the vector so that host cells that express the MAbs and the marker
can be easily selected.
[0062] The present invention particularly encompasses B-lymphoma
cell, leukemia cell and/or autoimmune cell targeting diagnostic or
therapeutic conjugates comprising an antibody component comprising
an anti-CD19 MAb or fragment thereof or an antibody fusion protein
or fragment thereof that binds to the target cell that is
conjugated or otherwise attached to at least one diagnostic or at
least one therapeutic agent.
[0063] The diagnostic conjugate comprises an antibody component
comprising an anti-CD19 MAb or fragment thereof or an antibody
fusion protein or fragment thereof, wherein the diagnostic agent
comprises at least one photoactive diagnostic agent, more
preferably wherein the label is a radioactive label with an energy
between 60 and 4,000 keV or a non-radioactive label. The
radioactive label is preferably a gamma-, beta- or
positron-emitting isotope and is selected from the group consisting
of .sup.125I, .sup.131I, .sup.123I, .sup.124I, .sup.86Y,
.sup.186Re, .sup.188Re, .sup.62Cu, .sup.64Cu, .sup.111In,
.sup.67Ga, .sup.68Ga, .sup.99mTc, .sup.94mTc, .sup.18F, .sup.11C,
.sup.13N, .sup.15O, .sup.76Br and combinations thereof.
[0064] The diagnostic conjugate may utilize a diagnostic agent,
such as a contrast agent, for example, such as manganese, iron or
gadolinium, or including an ultrasound-enhancing agent. In one
embodiment, the ultrasound-enhancing agent is a liposome that
comprises a chimerized or humanized anti-CD19 antibody or fragment
thereof. Also preferred, the ultrasound enhancing agent is a
liposome that is gas filled. Similarly, a bispecific antibody can
be conjugated to a contrast agent. For example, the bispecific
antibody may comprise more than one image enhancing agent for use
in ultrasound imaging. The ultrasound enhancing agent can be a
liposome, and preferably, the liposome comprises a bivalent DTP A
peptide covalently attached to the outside surface of the liposome.
Also preferred, the liposome is gas filled.
[0065] The therapeutic conjugate comprises an antibody component,
such as an antibody fusion protein or fragment thereof, wherein
each of said MAbs or fragments thereof are bound to at least one
therapeutic agent. The therapeutic conjugate preferably is selected
from the group consisting of a radioactive label, an
immunomodulator, a hormone, an enzyme, an oligonucleotide, a
photoactive therapeutic agent, a cytotoxic agent, which may be a
drug or a toxin, and a combination thereof. Useful drugs include
those drugs that possess a pharmaceutical property selected from
the group consisting of antimitotic, alkylating, antimetabolite,
antibiotic, alkaloid, antiangiogenic, apoptotic agents and
combinations thereof, as well as antisense oligonucleotides and RNA
molecules, such as short double stranded RNA molecules that
activate the RNA interference pathway. More specifically, the drugs
may be selected from the group consisting of nitrogen mustards,
ethylenimine derivatives, alkyl sulfonates, nitrosoureas,
triazenes, folic acid analogs, COX-2 inhibitors, pyrimidine
analogs, purine analogs, antibiotics, enzymes, epipodophyllotoxins,
platinum coordination complexes, vinca alkaloids, substituted
ureas, methyl hydrazine derivatives, adrenocortical suppressants,
thalidomide and its derivatives, antagonists, endostatin, taxols,
camptothecins, anthracyclines, taxanes, and their analogs, and a
combination thereof. The toxins may be selected from the group
consisting of ricin, abrin, alpha toxin, saporin, onconase, i.e.,
ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A,
pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas
exotoxin, and Pseudomonas endotoxin.
[0066] Other therapeutic agents suitable for use include
anti-angiogenic agents (or angiogenesis inhibitors). These agents
are suitable for use in combination therapy or for conjugating
antibodies to, for example, angiostatin, endostatin, vasculostatin,
canstatin and maspin, as well as the use of antibodies against
angiogenesis factors, such as vascular endothelium growth factor
(VEGF), placental growth factor (PlGF), ED-B fibronectin, and
against other vascular growth factors. Single and double stranded
oligonucleotides are a new class of therapeutic agents, and
include, for example, antisense oligonucleotides, such as antisense
bc1-2, and molecules, such as double stranded RNA molecules, that
activate the RNA interference pathway and cause highly specific
inhibition of gene expression, such as inhibition of bc1-2.
Inhibition of bc1-2 (and related bc1 family molecules) in a cell
inhibits the anti-apoptotic activity of bc1-2 and promotes
apoptosis of the cell. See Zangemeister-Wittke, Ann N Y Acad Sci.
1002:90-4 (2003).
[0067] Useful therapeutic conjugates are immunomodulators selected
from the group consisting of a cytokine, a stem cell growth factor,
a lymphotoxin, a hematopoietic factor, a colony stimulating factor
(CSF), an interferon (IFN), erythropoietin, thrombopoietin and a
combination thereof. Specifically useful are lymphotoxins, such as
tumor necrosis factor (TNF), hematopoietic factors, such as
interleukin (IL), colony stimulating factor, such as
granulocyte-colony stimulating factor (G-CSF) or granulocyte
macrophage-colony stimulating factor (GM-CSF), interferon, such as
interferons-alpha, -beta or -gamma, and stem cell growth factor,
such as "S1 factor". More specifically, immunomodulators, such as
IL-1, IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, IL-21, interferon,
TNF-alpha or -beta or a combination thereof may be of use.
[0068] Particularly useful therapeutic conjugates include one or
more radioactive labels that have an energy between 60 and 700 keV.
Such radioactive labels may be selected from the group consisting
of .sup.225Ac, .sup.67Ga, .sup.90Y, .sup.111In, .sup.131I,
.sup.125I, .sup.186Re, .sup.188Re, .sup.177Lu, .sup.32P, .sup.64Cu,
.sup.67Cu, .sup.212Bi, .sup.213Bi, .sup.211At and combinations
thereof. Other useful therapeutic conjugates are photoactive
therapeutic agents, such as a chromogen or dye.
[0069] The claimed methods encompass methods of treating a B-cell
disease in a subject, such as a mammal, including humans, domestic
or companion pets, such as dogs and cats. B cell diseases that can
be treated by the methods include any disease which involves
unwanted or undesirable B cell growth or activity, and includes
malignancies such as lymphoma or leukemia or an autoimmune disease.
The methods involve administering to the subject a therapeutically
effective amount of an anti-CD19 MAb or a fragment thereof,
formulated in a pharmaceutically acceptable vehicle. This therapy
may utilize a "naked antibody" that does not have a therapeutic
agent bound to it. The administration of the "naked anti-CD19
antibody" can be supplemented by administering to the subject
concurrently or sequentially a therapeutically effective amount of
another therapeutic agent, such as a second "naked antibody" that
binds to or is reactive with another antigen on the surface of the
target cell or that has other functions, such as effector functions
in the Fc portion of the MAb, that is therapeutic and which is
discussed herein. Preferred additional MAbs are at least one
humanized, chimeric, human or murine (in the case of non-human
animals) MAb selected from the group consisting of a MAb reactive
with CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25,
CD30, CD33, CD37, CD38, CD40, CD4OL, CD45, CD46, CD52, CD54, CD74,
CD80, CD126, CD138, B7, HM1.24, HLA-DR, an angiogenesis factor,
tenascin, VEGF, PIGF, ED-B fibronectin, an oncogene, an oncogene
product, CD66a-d, necrosis antigens, Ii, IL-2, MUC-1, T01, TAC,
IL-6, TRAIL-R1 (DR4) and TRAIL-R2 (DR5) formulated in a
pharmaceutically acceptable vehicle. In other embodiments, the
anti-CD19 antibody may be conjugated to one or more therapeutic or
diagnostic agents.
[0070] Both the naked anti-CD19 therapy alone or in combination
with other naked MAbs as discussed above can be further
supplemented with the administration, either concurrently or
sequentially, of a therapeutically effective amount of at least one
therapeutic agent, formulated in a pharmaceutically acceptable
vehicle. As discussed herein the therapeutic agent may comprise a
cytotoxic agent, a radioactive label, an immunomodulator, a
hormone, an oligonucleotide (such as an antisense or RNAi
oligonucleotide), an enzyme, a photoactive therapeutic agent or a
combination thereof, formulated in a pharmaceutically acceptable
vehicle.
[0071] In another therapeutic method, both the naked anti-CD19
therapy alone or in combination with other naked MAbs, as discussed
above, can be further supplemented with the administration, either
concurrently or sequentially, of a therapeutically effective amount
of at least one therapeutic conjugate, described herein and
formulated in a pharmaceutically acceptable vehicle. The antibody
component of the therapeutic conjugate comprises at least one
humanized, chimeric, human or murine (for non-human subjects) MAb
selected from the group consisting of a MAb reactive with CD4, CD5,
CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37,
CD38, CD40, CD4OL, CD46, CD52, CD54, CD74, CD80, CD126, CD138, B7,
HM1.24, HLA-DR, an angiogenesis factor, tenascin, VEGF, PIGF, ED-B
fibronectin, an oncogene, an oncogene product, CD66a-d, necrosis
antigens, Ii, IL-2, T101, TAC, IL-6, MUC-1, TRAIL-R1 (DR4) and
TRAIL-R2 (DR5), formulated in a pharmaceutically acceptable
vehicle. As discussed herein the therapeutic agent may comprise a
cytotoxic agent, a radioactive label, an immunomodulator, a
hormone, a photoactive therapeutic agent or a combination thereof,
formulated in a pharmaceutically acceptable vehicle.
[0072] Other embodiments concern methods of treating a B-cell
lymphoma or leukemia or an autoimmune disease in a subject
comprising administering to a subject a therapeutically effective
amount of an antibody fusion protein or fragment thereof comprising
at least two anti-CD19 MAbs or fragments thereof or comprising at
least one anti-CD19 MAb or fragment thereof and at least one
additional MAb, preferably selected from the group consisting of
MAbs reactive with CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21,
CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD4OL, CD46, CD52, CD54,
CD74, CD80, CD126, CD138, B7, HM1.24, HLA-DR, tenascin, VEGF, PIGF,
ED-B fibronectin, MUC-1, an oncogene, an oncogene product, CD66a-d,
necrosis antigens, Ii, IL-2, T101, TAC, IL-6, TRAIL-R1 (DR4) and
TRAIL-R2 (DR5) formulated in a pharmaceutically acceptable
vehicle.
[0073] In other methods based on pretargeting techniques, a
multispecific antibody or fusion protein may comprise an anti-CD19
antibody as described herein, attached to at least one other
antibody or fragment that binds to a hapten, such as an HSG hapten.
The hapten may be incorporated into a targetable conjugate that
comprises one or more therapeutic and/or diagnostic agents. The
multispecific antibody or fusion protein may be administered to a
subject and allowed to bind to a target antigen, such as CD19,
expressed on a target cell, such as a B cell. After allowing a
sufficient amount of time for non-bound circulating antibody to be
removed from circulation, the targetable conjugate may be
administered, which then binds to the multispecific antibody or
fusion protein localized to the target cell or tissue. Such
pretargeting methods improve the therapeutic index by preferential
delivery of therapeutic agent to the target cell or tissue compared
to normal cells or tissues. Methods of pretargeting are well known
in the art (see, e.g., U.S. Pat. Nos. 6,361,774; 6,962,702;
7,074,403; 7,201,890; 7,230,084; 7,230,085 and 7,429,381, each
incorporated herein by reference in its entirety.)
[0074] The therapeutic methods can further be supplemented with the
administration to the subject concurrently or sequentially of a
therapeutically effective amount of at least one therapeutic agent,
formulated in a pharmaceutically acceptable vehicle, wherein the
therapeutic agent is preferably a cytotoxic agent, a radioactive
label, an immunomodulator, a hormone, a photoactive therapeutic
agent or a combination thereof, formulated in a pharmaceutically
acceptable vehicle.
[0075] Further, the antibody fusion proteins can be administered to
a subject concurrently or sequentially with a therapeutically
effective amount of a therapeutic conjugate comprising at least one
MAb bound to at least one therapeutic agent, wherein said MAb
component of the conjugate preferably comprises at least one
humanized, chimeric, human or murine (for non-human subjects) MAb
selected from the group consisting of a MAb reactive with CD4, CD5,
CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37,
CD38, CD40, CD4OL, CD46, CD52, CD54, CD74, CD80, CD126, CD138, B7,
MUC1, Ia, HM1.24, HLA-DR, tenascin, VEGF, PIGF, ED-B fibronectin,
an oncogene, an oncogene product, CD66a-d, necrosis antigens, Ii,
IL-2, IL-6, T101, TAC, IL-6, TRAIL-R1 (DR4) and TRAIL-R2 (DR5)
formulated in a pharmaceutically acceptable vehicle. The antibody
fusion protein itself can be conjugated to a therapeutic agent and
thus provides a vehicle to attach more than one therapeutic agent
to an antibody component and these therapeutic agents can be a
combination of different recited agents or combinations of the same
agents, such as two different therapeutic radioactive labels.
[0076] Also encompassed are methods of diagnosing or detecting a
B-cell lymphoma or leukemia or autoimmune disease in a subject
comprising administering to the subject, such as a mammal,
including humans and domestic and companion pets, such as dogs,
cats, rabbits, guinea pigs, a diagnostic conjugate comprising an
anti-CD19 MAb or fragment thereof or an antibody fusion protein or
fragment thereof that binds to the lymphoma, leukemia or autoimmune
cell, wherein the anti-CD19 MAb or fragment thereof or antibody
fusion protein or fragment thereof is bound to at least one
diagnostic agent, formulated in a pharmaceutically acceptable
vehicle. Useful diagnostic agents are described herein.
[0077] Antibody Preparation
[0078] Monoclonal antibodies (MAbs) are a homogeneous population of
antibodies to a particular antigen wherein the antibody comprises
only one type of antigen binding site and binds to only one epitope
on an antigenic determinant. Rodent monoclonal antibodies to
specific antigens may be obtained by methods known to those skilled
in the art. See, for example, Kohler and Milstein, Nature 256: 495
(1975), and Coligan et al. (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY,
VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991) [hereinafter
"Coligan"]. Briefly, monoclonal antibodies can be obtained by
injecting mice with a composition comprising an antigen, verifying
the presence of antibody production by removing a serum sample,
removing the spleen to obtain B-lymphocytes, fusing the
B-lymphocytes with myeloma cells to produce hybridomas, cloning the
hybridomas, selecting positive clones which produce antibodies to
the antigen, culturing the clones that produce antibodies to the
antigen, and isolating the antibodies from the hybridoma
cultures.
[0079] MAbs can be isolated and purified from hybridoma cultures by
a variety of well-established techniques. Such isolation techniques
include affinity chromatography with Protein-A Sepharose,
size-exclusion chromatography, and ion-exchange chromatography.
See, for example, Coligan at pages 2.7.1-2.7.12 and pages
2.9.1-2.9.3. Also, see Baines et al., "Purification of
Immunoglobulin G (IgG)," in METHODS IN MOLECULAR BIOLOGY, VOL. 10,
pages 79-104 (The Humana Press, Inc. 1992).
[0080] After the initial raising of antibodies to the immunogen,
the antibodies can be sequenced and subsequently prepared by
recombinant techniques. Humanization and chimerization of murine
antibodies and antibody fragments are well known to those skilled
in the art. For example, humanized monoclonal antibodies are
produced by transferring mouse complementary determining regions
from heavy and light variable chains of the mouse immunoglobulin
into human variable domains attached to human constant region
sequences, and then, substituting selected human residues in the
framework regions with their murine counterparts. Preferred FR
residues for substitution include those FR residues that are
located near or touching the CDR residues, as well as residues that
affect the stability and/or expression levels of the antibody. The
use of antibody components derived from humanized monoclonal
antibodies obviates potential problems associated with the
immunogenicity of murine constant regions.
[0081] General techniques for cloning murine immunoglobulin
variable domains are disclosed, for example, by the publication of
Orlandi et al., Proc. Natl Acad. Sci. USA 86: 3833 (1989), which is
incorporated by reference in its entirety. Techniques for
constructing chimeric antibodies are well known to those of skill
in the art. As an example, Leung et at., Hybridoma 13:469 (1994),
produced an LL2 chimera by combining DNA sequences encoding the Vk
and VH domains of LL2 monoclonal antibody, an anti-CD22 antibody,
with respective human and IgG1 constant region domains. This
publication also provides the nucleotide sequences of the LL2 light
and heavy chain variable regions, Vk and VH, respectively.
Techniques for producing humanized MAbs are described, for example,
by Jones et al., Nature 321: 522 (1986), Riechmann et al., Nature
332: 323 (1988), Verhoeyen et al., Science 239: 1534 (1988), Carter
et al., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu, Crit.
Rev. Biotech. 12: 437 (1992), and Singer et al., J. Immun. 150:
2844 (1993), each of which is incorporated herein by reference.
[0082] A chimeric antibody is a recombinant protein that contains
the variable domains including the CDRs derived from one species of
animal, such as a rodent antibody, while the remainder of the
antibody molecule; i.e., the constant domains, is derived from a
human antibody. Accordingly, a chimeric monoclonal antibody can
also be humanized by replacing the sequences of the murine FR in
the variable domains of the chimeric MAb with one or more different
human FR. Specifically, mouse CDRs are transferred from heavy and
light variable chains of the mouse immunoglobulin into the
corresponding variable domains of a human antibody. As simply
transferring mouse CDRs into human FRs often results in a reduction
or even loss of antibody affinity, additional modification might be
required in order to restore the original affinity of the murine
antibody. This can be accomplished by the replacement of one or
more human residues in the FR regions with their murine
counterparts to obtain an antibody that possesses good binding
affinity to its epitope. See, for example, Tempest et al.,
Biotechnology 9:266 (1991) and Verhoeyen et al., Science 239: 1534
(1988).
[0083] Another method for producing the antibodies is by production
in the milk of transgenic livestock. See, e.g., Colman, A.,
Biochem. Soc. Symp., 63: 141-147, 1998; U.S. Pat. No. 5,827,690,
both of which are incorporated in their entirety by reference. Two
DNA constructs are prepared which contain, respectively, DNA
segments encoding paired immunoglobulin heavy and light chains. The
DNA segments are cloned into expression vectors which contain a
promoter sequence that is preferentially expressed in mammary
epithelial cells. Examples include, but are not limited to,
promoters from rabbit, cow and sheep casein genes, the
cow-lactoglobulin gene, the sheep-lactoglobulin gene and the mouse
whey acid protein gene. Preferably, the inserted fragment is
flanked on its 3' side by cognate genomic sequences from a
mammary-specific gene. This provides a polyadenylation site and
transcript-stabilizing sequences. The expression cassettes are
coinjected into the pronuclei of fertilized, mammalian eggs, which
are then implanted into the uterus of a recipient female and
allowed to gestate. After birth, the progeny are screened for the
presence of both transgenes by Southern analysis. In order for the
antibody to be present, both heavy and light chain genes must be
expressed concurrently in the same cell. Milk from transgenic
females is analyzed for the presence and functionality of the
antibody or antibody fragment using standard immunological methods
known in the art. The antibody can be purified from the milk using
standard methods known in the art.
[0084] A fully human antibody, i.e., human anti-CD19 MAbs or other
human antibodies, such as anti-CD22, anti-CD23, anti-CD20,
anti-CD74 or anti-CD21 MAbs, can be obtained from a transgenic
non-human animal. See, e.g., Mendez et al., Nature Genetics, 15:
146-156 (1997); U.S. Pat. No. 5,633,425, which are incorporated in
their entirety by reference. For example, a human antibody can be
recovered from a transgenic mouse possessing human immunoglobulin
loci. The mouse humoral immune system is humanized by inactivating
the endogenous immunoglobulin genes and introducing human
immunoglobulin loci. The human immunoglobulin loci are exceedingly
complex and comprise a large number of discrete segments which
together occupy almost 0.2% of the human genome. To ensure that
transgenic mice are capable of producing adequate repertoires of
antibodies, large portions of human heavy- and light-chain loci
must be introduced into the mouse genome. This is accomplished in a
stepwise process beginning with the formation of yeast artificial
chromosomes (YACs) containing either human heavy or light-chain
immunoglobulin loci in germline configuration. Since each insert is
approximately 1 Mb in size, YAC construction requires homologous
recombination of overlapping fragments of the immunoglobulin loci.
The two YACs, one containing the heavy-chain loci and one
containing the light-chain loci, are introduced separately into
mice via fusion of YAC-containing yeast spheroblasts with mouse
embryonic stem cells. Embryonic stem cell clones are then
microinjected into mouse blastocysts. Resulting chimeric males are
screened for their ability to transmit the Y AC through their
germline and are bred with mice deficient in murine antibody
production. Breeding the two transgenic strains, one containing the
human heavy-chain loci and the other containing the human
light-chain loci, creates progeny which produce human antibodies in
response to immunization.
[0085] Further recent methods for producing bispecific MAbs include
engineered recombinant MAbs which have additional cysteine residues
so that they crosslink more strongly than the more common
immunoglobulin isotypes. See, e.g., FitzGerald et al., Protein Eng.
10(10):1221-1225, 1997. Another approach is to engineer recombinant
fusion proteins linking two or more different single-chain antibody
or antibody fragment segments with the needed dual specificities.
See, e.g., Coloma et al., Nature Biotech. 15:159-163, 1997. A
variety of bispecific fusion proteins can be produced using
molecular engineering. See, for example, Alt et al., FEBS Lett.
454:90-4 (1999), which is incorporated herein by reference in its
entirety. In one form, the bispecific fusion protein consists 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 consists of, for
example, an IgG with two binding sites for one antigen and two scFv
with two binding sites for a second antigen.
[0086] Bispecific fusion proteins linking two or more different
single-chain antibodies or antibody fragments are produced in
similar manner. Recombinant methods can be used to produce a
variety of fusion proteins. For example a fusion protein comprising
a Fab fragment derived from a humanized monoclonal anti-CD19
antibody and a scFv derived from a murine anti-diDTPA can be
produced. A flexible linker, such as GGGS connects the scFv to the
constant region of the heavy chain of the anti-CD19 antibody.
Alternatively, the scFv can be connected to the constant region of
the light chain of another humanized antibody. Appropriate linker
sequences necessary for the in-frame connection of the heavy chain
Fd to the scFv are introduced into the VL and Vk domains through
PCR reactions. The DNA fragment encoding the scFv is then ligated
into a staging vector containing a DNA sequence encoding the CH1
domain. The resulting scFv-CH1 construct is excised and ligated
into a vector containing a DNA sequence encoding the VH region of
an anti-CD19 antibody. The resulting vector can be used to
transfect an appropriate host cell, such as a mammalian cell for
the expression of the bispecific fusion protein.
[0087] More recently, a novel technique known as dock-and-lock
(DNL) has been developed to provide for the highly efficient
formation of constructs comprising virtually any combination of
peptide or protein effectors (see, e.g., U.S. Patent Application
Publ. Nos. 20060228357; 20060228300; 20070086942; 20070140966 and
20070264265, each incorporated herein by reference in its
entirety). In various permutations, the constructs are not limited
to protein or peptide effectors, but may comprise other types of
effector agents that may be attached to proteins or peptides, such
as chemotherapeutic agents. The technique utilizes complementary
protein binding domains, referred to as anchoring domains and
dimerization and docking domains, which bind to each other and
allow the assembly of complex structures, ranging from dimers,
trimers, tetramers, quintamers and hexamers. These form stable
complexes in high yield without requirement for extensive
purification. The DNL technique allows the assembly of
monospecific, bispecific or multispecific antibodies, either as
naked antibody moieties or in combination with a wide range of
other effector molecules such as immunomodulators, enzymes,
chemotherapeutic agents, chemokines, cytokines, diagnostic agents,
therapeutic agents, radionuclides, imaging agents, anti-angiogenic
agents, growth factors, oligonucleotides, hormones, peptides,
toxins, pro-apoptotic agents, or a combination thereof. Any of the
techniques known in the art for making bispecific or multispecific
antibodies may be utilized in the practice of the presently claimed
methods.
[0088] Known Antibodies
[0089] In certain embodiments, for example those involving
bispecific or multispecific antibodies incorporating an anti-CD19
antibody and another antibody against a different antigenic target,
it may be preferred to utilize a commercially available or publicly
known antibody, rather than making one de novo. Antibodies of use
may be commercially obtained from a wide variety of known sources.
For example, a variety of antibody secreting hybridoma lines are
available from the American Type Culture Collection (ATCC,
Manassas, Va.). A large number of antibodies against various
disease targets, including but not limited to tumor-associated
antigens, have been deposited at the ATCC and/or have published
variable region sequences and are available for use in the claimed
methods and compositions. See, e.g., U.S. Pat. Nos. 7,312,318;
7,282,567; 7,151,164; 7,074,403; 7,060,802; 7,056,509; 7,049,060;
7,045,132; 7,041,803; 7,041,802; 7,041,293; 7,038,018; 7,037,498;
7,012,133; 7,001,598; 6,998,468; 6,994,976; 6,994,852; 6,989,241;
6,974,863; 6,965,018; 6,964,854; 6,962,981; 6,962,813; 6,956,107;
6,951,924; 6,949,244; 6,946,129; 6,943,020; 6,939,547; 6,921,645;
6,921,645; 6,921,533; 6,919,433; 6,919,078; 6,916,475; 6,905,681;
6,899,879; 6,893,625; 6,887,468; 6,887,466; 6,884,594; 6,881,405;
6,878,812; 6,875,580; 6,872,568; 6,867,006; 6,864,062; 6,861,511;
6,861,227; 6,861,226; 6,838,282; 6,835,549; 6,835,370; 6,824,780;
6,824,778; 6,812,206; 6,793,924; 6,783,758; 6,770,450; 6,767,711;
6,764,688; 6,764,681; 6,764,679; 6,743,898; 6,733,981; 6,730,307;
6,720,155; 6,716,966; 6,709,653; 6,693,176; 6,692,908; 6,689,607;
6,689,362; 6,689,355; 6,682,737; 6,682,736; 6,682,734; 6,673,344;
6,653,104; 6,652,852; 6,635,482; 6,630,144; 6,610,833; 6,610,294;
6,605,441; 6,605,279; 6,596,852; 6,592,868; 6,576,745; 6,572;856;
6,566,076; 6,562,618; 6,545,130; 6,544,749; 6,534,058; 6,528,625;
6,528,269; 6,521,227; 6,518,404; 6,511,665; 6,491,915; 6,488,930;
6,482,598; 6,482,408; 6,479,247; 6,468,531; 6,468,529; 6,465,173;
6,461,823; 6,458,356; 6,455,044; 6,455,040, 6,451,310; 6,444,206,
6,441,143; 6,432,404; 6,432,402; 6,419,928; 6,413,726; 6,406,694;
6,403,770; 6,403,091; 6,395,276; 6,395,274; 6,387,350; 6,383,759;
6,383,484; 6,376,654; 6,372,215; 6,359,126; 6,355,481; 6,355,444;
6,355,245; 6,355,244; 6,346,246; 6,344,198; 6,340,571; 6,340,459;
6,331,175; 6,306,393; 6,254,868; 6,187,287; 6,183,744; 6,129,914;
6,120,767; 6,096,289; 6,077,499; 5,922,302; 5,874,540; 5,814,440;
5,798,229; 5,789,554; 5,776,456; 5,736,119; 5,716,595; 5,677,136;
5,587,459; 5,443,953, 5,525,338, incorporated herein by reference
with respect to antibody variable region and/or CDR sequences
and/or ATCC Accession Numbers for antibody-producing hybridoma cell
lines. These are exemplary only and a wide variety of other
antibodies and their hybridomas are known in the art. The skilled
artisan will realize that antibody sequences or antibody-secreting
hybridomas against almost any disease-associated antigen may be
obtained by a simple search of the ATCC, NCBI and/or USPTO
databases for antibodies against a selected disease-associated
target of interest. The antigen binding domains of the cloned
antibodies may be amplified, excised, ligated into an expression
vector, transfected into an adapted host cell and used for protein
production, using standard techniques well known in the art.
[0090] Production of Antibody Fragments
[0091] Antibody fragments which recognize specific epitopes can be
generated by known techniques. The antibody fragments are antigen
binding portions of an antibody, such as F(ab').sub.2, Fab',
F(ab).sub.2, Fab, Fv, sFv and the like. Other antibody fragments
include, but are not limited to: the F(ab').sub.2 fragments which
can be produced by pepsin digestion of the antibody molecule and
the Fab' fragments, which can be generated by reducing disulfide
bridges of the F(ab').sub.2 fragments. Alternatively, Fab'
expression libraries can be constructed (Huse et al., 1989,
Science, 246:1274-1281) to allow rapid and easy identification of
monoclonal Fab' fragments with the desired specificity.
[0092] A single chain Fv molecule (scFv) comprises a VL domain and
a VH domain. The VL and VH domains associate to form a target
binding site. These two domains are further covalently linked by a
peptide linker (L). A scFv molecule is denoted as either VL-L-VH if
the VL domain is the N-terminal part of the scFv molecule, or as
VH-L-VL if the VH domain is the N-terminal part of the scFv
molecule. Methods for making scFv molecules and designing suitable
peptide linkers are described in U.S. Pat. No. 4,704,692, U.S. Pat.
No. 4,946,778, R. Raag and M. Whitlow, "Single Chain Fvs." FASEB
Vol 9:73-80 (1995) and R. E. Bird and B. W. Walker, "Single Chain
Antibody Variable Regions," TIBTECH, Vol 9: 132-137 (1991), each
incorporated herein by reference.
[0093] An antibody fragment can be prepared by proteolytic
hydrolysis of the full-length antibody or by expression in E. coli
or another host of the DNA coding for the fragment. An antibody
fragment can be obtained by pepsin or papain digestion of full
length antibodies by conventional methods. For example, an antibody
fragment 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 papain produces two
monovalent Fab fragments and an Fc fragment directly. These methods
are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945
and 4,331,647 and references contained therein, incorporated herein
in their entireties by reference. Also, see Nisonoff et al., Arch
Biochem. Biophys. 89: 230 (1960); Porter, Biochem. J. 73: 119
(1959), Edelman et al., in METHODS IN ENZYMOLOGY VOL. 1, page 422
(Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and
2.10.-2.10.4.
[0094] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). A CDR is a
segment of the variable region of an antibody that is complementary
in structure to the epitope to which the antibody binds and is more
variable than the rest of the variable region. Accordingly, a CDR
is sometimes referred to as hypervariable region. A variable region
comprises three CDRs. CDR peptides can be obtained by constructing
genes encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing
cells. See, for example, Larrick et 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).
[0095] 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 same antigen that is recognized by the intact antibody.
[0096] Multispecific and Multivalent Antibodies
[0097] The anti-CD19 antibodies, as well as other antibodies with
different specificities for use in combination therapy, can also be
made as multispecific antibodies, comprising at least one binding
site to a CD19 epitope or antigen and at least one binding site to
another epitope on CD19 or another antigen, or multivalent
antibodies, comprising multiple binding sites to the same epitope
or antigen.
[0098] A bispecific antibody or antibody fragment may have at least
one binding region that specifically binds a targeted cell marker
and at least one other binding region that specifically binds a
targetable conjugate. The targetable conjugate comprises a carrier
portion which comprises or bears at least one epitope recognized by
at least one binding region of the bispecific antibody or antibody
fragment. A variety of recombinant methods can be used to produce
bispecific antibodies and antibody fragments as described
above.
[0099] An anti-CD19 multivalent antibody is also contemplated. This
multivalent target binding protein is constructed by association of
a first and a second polypeptide. The first polypeptide comprises a
first single chain Fv molecule covalently linked to a first
immunoglobulin-like domain which preferably is an immunoglobulin
light chain variable region domain. The second polypeptide
comprises a second single chain Fv molecule covalently linked to a
second immunoglobulin-like domain which preferably is an
immunoglobulin heavy chain variable region domain. Each of the
first and second single chain Fv molecules forms a target binding
site, and the first and second immunoglobulin-like domains
associate to form a third target binding site.
[0100] A single chain Fv molecule with the VL-L-VH configuration,
wherein L is a linker, may associate with another single chain Fv
molecule with the VH-L-VL configuration to form a bivalent dimer.
In this case, the VL domain of the first scFv and the VH domain of
the second scFv molecule associate to form one target binding site,
while the VH domain of the first scFv and the VL domain of the
second scFv associate to form the other target binding site.
[0101] Another embodiment is an anti-CD19 bispecific, trivalent
targeting protein comprising two heterologous polypeptide chains
associated noncovalently to form three binding sites, two of which
have affinity for one target and a third which has affinity for a
hapten that can be made and attached to a carrier for a diagnostic
and/or therapeutic agent. Preferably, the binding protein has two
CD19 binding sites and one CD22 binding site. The bispecific,
trivalent targeting agents have two different scFvs, one scFv
contains two VH domains from one antibody connected by a short
linker to the VL domain of another antibody and the second scFv
contains two VL domains from the first antibody connected by a
short linker to the VH domain of the other antibody. The methods
for generating multivalent, multispecific agents from VH and VL
domains provide that individual chains synthesized from a DNA
plasmid in a host organism are composed entirely of VH domains (the
VH-chain) or entirely of VL domains (the VL-chain) in such a way
that any agent of multivalency and multispecificity can be produced
by non-covalent association of one VH-chain with one VL-chain. For
example, forming a trivalent, trispecific agent, the VH-chain will
consist of the amino acid sequences of three VH domains, each from
an antibody of different specificity, joined by peptide linkers of
variable lengths, and the VL-chain will consist of complementary VL
domains, joined by peptide linkers similar to those used for the
VH-chain. Since the VH and VL domains of antibodies associate in an
anti-parallel fashion, the preferred method in this invention has
the VL domains in the VL-chain arranged in the reverse order of the
VH domains in the VH-chain.
[0102] Diabodies, Triabodies and Tetrabodies
[0103] The anti-CD19 antibodies can also be used to prepare
functional bispecific single-chain antibodies (bsAb), also called
diabodies, and can be produced in mammalian cells using recombinant
methods. See, e.g., Mack et al., Proc. Natl. Acad. Sci., 92:
7021-7025, 1995, incorporated herein by reference. For example,
bsAb are produced by joining two single-chain Fv fragments via a
glycine-serine linker using recombinant methods. The V light-chain
(VL) and V heavy-chain (VH) domains of two antibodies of interest
are isolated using standard PCR methods. The VL and VH cDNAs
obtained from each hybridoma are then joined to form a single-chain
fragment in a two-step fusion PCR. The first PCR step introduces
the (Gly4-Ser1).sub.3 linker, and the second step joins the VL and
VH amplicons. Each single chain molecule is then cloned into a
bacterial expression vector. Following amplification, one of the
single-chain molecules is excised and sub-cloned into the other
vector, containing the second single-chain molecule of interest.
The resulting bsAb fragment is subcloned into a eukaryotic
expression vector. Functional protein expression can be obtained by
transfecting the vector into CHO cells, Sp2/0 cells or Sp-EEE
cells. Bispecific fusion proteins are prepared in a similar
manner.
[0104] For example, a humanized, chimeric or human anti-CD19
monoclonal antibody can be used to produce antigen specific
diabodies, triabodies, and tetrabodies. The monospecific diabodies,
triabodies, and tetrabodies bind selectively to targeted antigens
and as the number of binding sites on the molecule increases, the
affinity for the target cell increases and a longer residence time
is observed at the desired location. For diabodies, the two chains
comprising the VH polypeptide of the humanized anti-CD19 MAb
connected to the VK polypeptide of the humanized anti-CD19 MAb by a
five amino acid residue linker are utilized. Each chain forms one
half of the humanized anti-CD19 diabody. In the case of triabodies,
the three chains comprising VH polypeptide of the humanized
anti-CD19 MAb connected to the VK polypeptide of the humanized
anti-CD19 MAb by no linker are utilized. Each chain forms one third
of the hCD19 triabody.
[0105] The ultimate use of the bispecific diabodies described
herein is for pretargeting CD19 positive tumors for subsequent
specific delivery of diagnostic or therapeutic agents. These
diabodies bind selectively to targeted antigens allowing for
increased affinity and a longer residence time at the desired
location. Moreover, non-antigen bound diabodies are cleared from
the body quickly and exposure of normal tissues is minimized.
Bispecific antibody point mutations for enhancing the rate of
clearance can be found in U.S. Provisional Application No.
60/361,037, which is incorporated herein by reference in its
entirety. Bispecific diabodies for affinity enhancement are
disclosed in U.S. application Ser. No. 10/270,071, Ser. No.
10/270,073 and Ser. No. 10/328,190, which are incorporated herein
by reference in their entirety.
[0106] The diagnostic and therapeutic agents can include isotopes,
drugs, toxins, cytokines, hormones, enzymes, oligonucleotides,
growth factors, conjugates, radionuclides, and metals. For example,
gadolinium metal is used for magnetic resonance imaging (MRI).
Examples of radio nuclides are .sup.225Ac, .sup.18F, .sup.68Ga,
.sup.67Ga, .sup.90Y, .sup.86Y, .sup.111In, .sup.131I, .sup.125I,
.sup.123I, .sup.99mTc, .sup.94mTc, .sup.186Re, .sup.188Re,
.sup.177Lu, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.212Bi,
.sup.213Bi, .sup.32P, .sup.11C, .sup.13N, .sup.15O, .sup.76Br, and
.sup.211At. Other radionuclides are also available as diagnostic
and therapeutic agents, especially those in the energy range of 60
to 4,000 keV.
[0107] More recently, a tetravalent tandem diabody (termed tandab)
with dual specificity has been reported (Cochlovius et al., Cancer
Research (2000) 60: 4336-4341). The bispecific tandab is a dimer of
two identical polypeptides, each containing four variable domains
of two different antibodies (VH1, VL1, VH2, VL2) linked in an
orientation to facilitate the formation of two potential binding
sites for each of the two different specificities upon
self-association.
[0108] Conjugated Anti-CD19 Antibodies
[0109] Another embodiment concerns conjugated anti-CD19 antibodies.
Compositions and methods for multivalent, multispecific agents are
described in U.S. Patent Application Ser. No. 60/436,359, filed
Dec. 24, 2002, and U.S. Patent Application Ser. No. 60/464,532,
filed Apr. 23, 2003, which are incorporated herein by reference in
their entirety.
[0110] Additional amino acid residues may be added to either the N-
or C-terminus of the polypeptide. The additional amino acid
residues may comprise a peptide tag, a signal peptide, a cytokine,
an enzyme (for example, a pro-drug activating enzyme), a hormone, a
peptide toxin, such as pseudomonas exotoxin, a peptide drug, a
cytotoxic protein or other functional proteins. As used herein, a
functional protein is a protein which has a biological
function.
[0111] In one embodiment, drugs, toxins, radioactive compounds,
enzymes, hormones, oligonucleotides, cytotoxic proteins, chelates,
cytokines and other functional agents may be conjugated to the
target binding protein, preferably through covalent attachments to
the side chains of the amino acid residues of the target binding
protein, for example amine, carboxyl, phenyl, thiol or hydroxyl
groups. Various conventional linkers may be used for this purpose,
for example, diisocyanates, diisothiocyanates,
bis(hydroxysuccinimide) esters, carbodiimides,
maleimide-hydroxysuccinimide esters, glutaraldehyde and the like.
Conjugation of agents to the binding protein preferably does not
significantly affect the protein's binding specificity or affinity
to its target. As used herein, a functional agent is an agent which
has a biological function. A preferred functional agent is a
cytotoxic agent.
[0112] As discussed above, enzymes are also useful therapeutic
agents. For example, alkaline phosphatase for use in combination
with phosphate-containing prodrugs (U.S. Pat. No. 4,975,278);
arylsulfatase for use in combination with sulfate-containing
prodrugs (U.S. Pat. No. 5,270,196); peptidases and proteases, such
as serratia protease, thermolysin, subtilisin, carboxypeptidase
(U.S. Pat. Nos. 5,660,829; 5,587,161; 5,405,990) and cathepsins
(including cathepsin B and L), for use in combination with
peptide-based prodrugs; D-alanylcarboxypeptidases for use in
combination with D-amino acid-modified prodrugs;
carbohydrate-cleaving enzymes such as beta-galactosidase and
neuraminidase for use in combination with glycosylated prodrugs
(U.S. Pat. Nos. 5,561,119; 5,646,298); beta-lactamase for use in
combination with beta-lactam-containing prodrugs; penicillin
amidases, such as penicillin-V-amidase (U.S. Pat. No. 4,975,278) or
penicillin-G-amidase, for use in combination with drugs derivatized
at their amino nitrogens with phenoxyacetamide or phenylacetamide
groups; and cytosine deaminase (U.S. Pat. Nos. 5,338,678;
5,545,548) for use in combination with 5-fluorocytosine-based
prodrugs (U.S. Pat. No. 4,975,278), are suitable therapeutic
agents.
[0113] In still other embodiments, bispecific antibody-directed
delivery of therapeutics or prodrug polymers to in vivo targets can
be combined with bispecific antibody delivery of radionuclides,
such that combination chemotherapy and radioimmunotherapy is
achieved. Each therapy can be conjugated to the targetable
conjugate and administered simultaneously, or the nuclide can be
given as part of a first targetable conjugate and the drug given in
a later step as part of a second targetable conjugate.
[0114] In another embodiment, cytotoxic agents may be conjugated to
a polymeric carrier, and the polymeric carrier may subsequently be
conjugated to the multivalent target binding protein. For this
method, see Ryser et al., Proc. Natl. Acad. Sci. USA, 75:3867-3870,
1978, U.S. Pat. No. 4,699,784 and U.S. Pat. No. 4,046,722, which
are incorporated herein by reference. Conjugation preferably does
not significantly affect the binding specificity or affinity of the
binding protein.
[0115] Humanized, Chimeric and Human Antibodies for Treatment and
Diagnosis
[0116] Humanized, chimeric and human monoclonal antibodies, i.e.,
anti-CD19 MAbs and other MAbs described herein, are suitable for
use in therapeutic methods and diagnostic methods. Accordingly, the
present invention contemplates the administration of the humanized,
chimeric and human antibodies alone as a naked antibody or
administered as a multimodal therapy, temporally according to a
dosing regimen, but not conjugated to, a therapeutic agent. The
efficacy of the naked anti-CD19 MAbs can be enhanced by
supplementing naked antibodies with one or more other naked
antibodies, i.e., MAbs to specific antigens, such as CD4, CD5, CD8,
CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38,
CD40, CD40L, CD46, CD52, CD54, CD74, CD80, CD126, CD138, B7, MUC1,
Ia, HM1.24, HLA-DR, tenascin, VEGF, PlGF, ED-B fibronectin, an
oncogene, an oncogene product, CD66a-d, necrosis antigens, Ii,
IL-2, T101, TAC, IL-6, TRAIL-R1 (DR4) and TRAIL-R2 (DR5) with one
or more immunoconjugates of anti-CD19, or antibodies to theses
recited antigens, conjugated with therapeutic agents, including
drugs, toxins, immunomodulators, hormones, enzymes,
oligonucleotides, therapeutic radionuclides, etc., with one or more
therapeutic agents, including drugs, toxins, enzymes,
oligonucleotides, immunomodulators, hormones, therapeutic
radionuclides, etc., administered concurrently or sequentially or
according to a prescribed dosing regimen, with the MAbs.
[0117] Preferred B-cell antigens include those equivalent to human
CD19, CD20, CD21, CD22, CD23, CD46, CD52, CD74, CD80, 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, CD22, CD21, CD23, CD74, CD80, and
HLA-DR antigens. Particularly preferred T-cell antigens are those
equivalent to human CD4, CD8 and CD25 antigens. CD46 is an antigen
on the surface of cancer cells that block complement-dependent
lysis (CDC).
[0118] Further, the present invention contemplates the
administration of an immunoconjugate for diagnostic and therapeutic
uses in B cell lymphomas and other disease or disorders. An
immunoconjugate, as described herein, is a molecule comprising an
antibody component and a therapeutic or diagnostic agent, including
a peptide which may bear the diagnostic or therapeutic agent. An
immunoconjugate retains the immunoreactivity of the antibody
component, i.e., the antibody moiety has about the same or slightly
reduced ability to bind the cognate antigen after conjugation as
before conjugation.
[0119] A wide variety of diagnostic and therapeutic reagents can be
advantageously conjugated to the claimed antibodies. The
therapeutic agents recited here are those agents that also are
useful for administration separately with the naked antibody as
described above. Therapeutic agents include, for example,
chemotherapeutic drugs such as vinca alkaloids, anthracyclines,
epidophyllotoxins, taxanes, antimetabolites, alkylating agents,
antibiotics, COX-2 inhibitors, antimitotics, antiangiogenic and
apoptotic agents, particularly doxorubicin, methotrexate, taxol,
CPT-11, camptothecans, proteosome inhibitors, and others from these
and other classes of anticancer agents, thalidomide and derivates,
oligonucleotides, particularly antisense and RNAi oligonucleotides
(e.g., against bc1-2), and the like. Other useful cancer
chemotherapeutic drugs for the preparation of immunoconjugates and
antibody fusion proteins include nitrogen mustards, alkyl
sulfonates, nitrosoureas, triazenes, folic acid analogs, COX-2
inhibitors, pyrimidine analogs, purine analogs, platinum
coordination complexes, enzymes, 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), as well as revised editions of
these publications. Other suitable chemotherapeutic agents, such as
experimental drugs, are known to those of skill in the art.
[0120] Additionally, a chelator such as DTPA, DOTA, TETA, or NOTA
or a suitable peptide, to which a detectable label, such as a
fluorescent molecule, or cytotoxic agent, such as a heavy metal or
radionuclide, can be conjugated to the claimed antibodies. For
example, a therapeutically useful immunoconjugate can be obtained
by conjugating a photoactive agent or dye to an antibody composite.
Fluorescent compositions, such as fluorochrome, 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 (Joni et al.
(eds.), PHOTODYNAMIC THERAPY OF TUMORS AND OTHER DISEASES (Libreria
Progetto 1985); van den Bergh, Chem. Britain 22:430 (1986).
[0121] 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 photo active agents or dyes.
[0122] Also contemplated by the present invention are the use of
radioactive and non-radioactive agents as diagnostic agents. A
suitable non-radioactive diagnostic agent is a contrast agent
suitable for magnetic resonance imaging, computed tomography or
ultrasound. Magnetic imaging agents include, for example,
non-radioactive metals, such as manganese, iron and gadolinium,
complexed with metal-chelate combinations that include 2-benzyl-DTP
A and its monomethyl and cyclohexyl analogs, when used along with
the antibodies of the invention. See U.S. Ser. No. 09/921,290 filed
on Oct. 10, 2001, which is incorporated in its entirety by
reference.
[0123] Furthermore, a radiolabeled antibody or immunoconjugate may
comprise a gamma-emitting radioisotope or a positron-emitter useful
for diagnostic imaging. Suitable radioisotopes, particularly in the
energy range of 60 to 4,000 keV, include .sup.131I, .sup.123I,
.sup.124I, .sup.86Y, .sup.62Cu, .sup.64Cu, .sup.111In, .sup.67Ga,
.sup.68Ga, .sup.99mTc, .sup.94mTc, .sup.18F, .sup.11C, .sup.13N,
.sup.15O, .sup.75Br, and the like. See for example, U.S.
Provisional Application No. 60/342,104, which discloses positron
emitters, such as .sup.18F, .sup.68Ga, .sup.94mTc and the like, for
imaging purposes and which is incorporated in its entirety by
reference.
[0124] A toxin, such as Pseudomonas exotoxin, may also be complexed
to or form the therapeutic agent portion of an antibody fusion
protein of an anti-CD19 antibody. Other toxins suitably employed in
the preparation of such conjugates or other fusion proteins,
include ricin, abrin, ribonuclease (RNase), DNase I, Staphylococcal
enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria
toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin. See, for
example, Pastan et al., Cell 47:641 (1986), and Goldenberg, C A--A
Cancer Journal for Clinicians 44:43 (1994). Additional toxins
suitable for use in the present invention are known to those of
skill in the art and are disclosed in U.S. Pat. No. 6,077,499,
which is incorporated in its entirety by reference.
[0125] An immunomodulator, such as a cytokine may also be
conjugated to, or form the therapeutic agent portion of an antibody
fusion protein or be administered with the humanized anti-CD19
antibodies . Suitable cytokines for the present invention include,
but are not limited to, interferons and interleukins, as described
below.
[0126] Preparation of Immunoconjugates
[0127] Any of the antibodies or antibody fusion proteins can be
conjugated with one or more therapeutic or diagnostic agents.
Generally, one therapeutic or diagnostic agent is attached to each
antibody or antibody fragment but more than one therapeutic agent
or diagnostic agent can be attached to the same antibody or
antibody fragment. The antibody fusion proteins may comprise two or
more antibodies or fragments thereof and each of the antibodies
that compose this fusion protein can contain a therapeutic agent or
diagnostic agent. Additionally, one or more of the antibodies of
the antibody fusion protein can have more than one therapeutic of
diagnostic agent attached. Further, the therapeutic agents do not
need to be the same but can be different therapeutic agents. For
example, one can attach a drug and a radioisotope to the same
fusion protein. Particularly, an IgG can be radiolabeled with
.sup.131I and attached to a drug. The .sup.131I can be incorporated
into the tyrosine of the IgG and the drug attached to the epsilon
amino group of the IgG lysines. Both therapeutic and diagnostic
agents also can be attached to reduced SH groups and to the
carbohydrate side chains.
[0128] Bispecific antibodies are useful in pretargeting methods and
provide a preferred way to deliver therapeutic agents or diagnostic
agents to a subject. U.S. Ser. No. 09/382,186 discloses a method of
pretargeting using a bispecific antibody, in which the bispecific
antibody is labeled with .sup.125I and delivered to a subject,
followed by a divalent peptide labeled with .sup.99mTc.
Pretargeting methods are also described in U.S. Ser. No. 09/823,746
(Hansen et al.) and Ser. No. 10/150,654 (Goldenberg et al.), which
are incorporated herein by reference in their entirety. The
delivery results in excellent tumor/normal tissue ratios for
.sup.125I and .sup.99mTc. Any combination of known therapeutic
agents or diagnostic agents can be used to label the antibodies and
antibody fusion proteins. The binding specificity of the antibody
component of the MAb conjugate, the efficacy of the therapeutic
agent or diagnostic agent and the effector activity of the Fc
portion of the antibody can be determined by standard testing of
the conjugates.
[0129] A therapeutic or diagnostic agent can be attached at the
hinge region of a reduced antibody component via disulfide bond
formation. As an alternative, such peptides can be attached to the
antibody component using a heterobifunctional crosslinker, 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).
[0130] Alternatively, the therapeutic or diagnostic agent can be
conjugated via a carbohydrate moiety in the Fc region of the
antibody. The carbohydrate group can be used to increase the
loading of the same peptide that is bound to a thiol group, or the
carbohydrate moiety can be used to bind a different peptide.
[0131] Methods for conjugating peptides to antibody components via
an antibody carbohydrate moiety are well-known to those of skill in
the art. See, for example, Shih et al., Int. J. Cancer 41: 832
(1988); Shih et al., Int. J. Cancer 46: 1101 (1990); and Shih et
al., U.S. Pat. No. 5,057,313, all of which are incorporated in
their entirety by reference. 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 peptide. 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.
[0132] The Fc region is absent if the antibody used as the antibody
component of the immunoconjugate is an antibody fragment. However,
it is possible to introduce a carbohydrate moiety into the light
chain variable region of a full length 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), Leung et
al., U.S. Pat. No. 6,254,868, all of which are incorporated in
their entirety by reference. The engineered carbohydrate moiety is
used to attach the therapeutic or diagnostic agent.
[0133] Pharmaceutically Acceptable Excipients
[0134] The humanized, chimeric and human anti-CD19 MAbs to be
delivered to a subject can consist of the MAb alone,
immunoconjugate, fusion protein, or can comprise one or more
pharmaceutically suitable excipients, one or more additional
ingredients, or some combination of these.
[0135] The immunoconjugate or naked antibody can be formulated
according to known methods to prepare pharmaceutically useful
compositions, whereby the immunoconjugate or naked antibody are
combined in a mixture with a pharmaceutically suitable excipient.
Sterile phosphate-buffered saline is one example of a
pharmaceutically suitable excipient. Other suitable excipients are
well-known to those in the art. See, for example, Ansel et al.,
PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition
(Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'S
PHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company
1990), and revised editions thereof.
[0136] The immunoconjugate or naked antibody can be formulated for
intravenous administration via, for example, bolus injection or
continuous infusion. Formulations for injection can be presented in
unit dosage form, e.g., in ampules or in multi-dose containers,
with an added preservative. The compositions can take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and can contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient can
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use.
[0137] Additional pharmaceutical methods may be employed to control
the duration of action of the therapeutic or diagnostic conjugate
or naked antibody. Control release preparations can be prepared
through the use of polymers to complex or adsorb the
immunoconjugate or naked antibody. For example, biocompatible
polymers include matrices of poly(ethylene-co-vinyl acetate) and
matrices of a polyanhydride copolymer of a stearic acid dimer and
sebacic acid. Sherwood et al., Bio/Technology 10: 1446 (1992). The
rate of release of an immunoconjugate or antibody from such a
matrix depends upon the molecular weight of the immunoconjugate or
antibody, the amount of immunoconjugate, antibody within the
matrix, and the size of dispersed particles. Saltzman et al.,
Biophys. J. 55: 163 (1989); Sherwood et al., supra. Other solid
dosage forms are described in Ansel et al., PHARMACEUTICAL DOSAGE
FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger
1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th
Edition (Mack Publishing Company 1990), and revised editions
thereof.
[0138] The immunoconjugate, antibody fusion proteins, or naked
antibody may also be administered to a mammal subcutaneously or
even by other parenteral routes. Moreover, the administration may
be by continuous infusion or by single or multiple boluses. In
general, the dosage of an administered immunoconjugate, fusion
protein or naked antibody for humans will vary depending upon such
factors as the patient's age, weight, height, sex, general medical
condition and previous medical history. Typically, it is desirable
to provide the recipient with a dosage of immunoconjugate, antibody
fusion protein or naked antibody that is in the range of from about
1 mg/kg to 20 mg/kg as a single intravenous infusion, although a
lower or higher dosage also may be administered as circumstances
dictate. This dosage may be repeated as needed, for example, once
per week for 4-10 weeks, preferably once per week for 8 weeks, and
more preferably, once per week for 4 weeks. It may also be given
less frequently, such as every other week for several months. The
dosage may be given through various parenteral routes, with
appropriate adjustment of the dose and schedule.
[0139] For purposes of therapy, the immunoconjugate, fusion
protein, or naked antibody is administered to a mammal in a
therapeutically effective amount. A suitable subject is usually a
human, although a non-human animal subject is also contemplated. An
antibody preparation is said to be administered in a
"therapeutically effective amount" if the amount administered is
physiologically significant. An agent is physiologically
significant if its presence results in a detectable change in the
physiology of a recipient mammal. In particular, an antibody
preparation is physiologically significant if its presence invokes
an antitumor response or mitigates the signs and symptoms of an
autoimmune disease state. A physiologically significant effect
could also be the evocation of a humoral and/or cellular immune
response in the recipient mammal.
[0140] Methods of Treatment
[0141] The present invention contemplates the use of naked or
conjugated anti-CD19 antibodies as the primary composition for
treatment of B cell disorders and other diseases. In particular,
the compositions described herein are particularly useful for
treatment of various autoimmune diseases as well as indolent forms
of B-cell lymphomas, aggressive forms of B-cell lymphomas, chronic
lymphatic leukemias, acute lymphatic leukemias, and Waldenstrom's
macroglobulinemia. For example, the humanized anti-CD19 antibody
components and immunoconjugates can be used to treat both indolent
and aggressive forms of non-Hodgkin's lymphoma.
[0142] As discussed above, the antibodies are also suitable for
diagnosis and treatment of various autoimmune diseases. Such
diseases include acute idiopathic thrombocytopenic purpura, chronic
idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's
chorea, myasthenia gravis, systemic lupus erythematosus, lupus
nephritis, rheumatic fever, polyglandular syndromes, bullous
pemphigoid, diabetes mellitus, Henoch-Schonlein purpura,
post-streptococcal nephritis, erythema nodosurn, Takayasu's
arteritis, Addison's disease, rheumatoid arthritis, multiple
sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme,
IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis,
Goodpasture's syndrome, thromboangitis ubiterans, Sjogren's
syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,
thyrotoxicosis, scleroderma, chronic active hepatitis,
polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris,
Wegener's granulomatosis, membranous nephropathy, amyotrophic
lateral sclerosis, tabes dorsalis, giant cell
arteritis/polymyalgia, pernicious anemia, rapidly progressive
glomerulonephritis, psoriasis, and fibrosing alveolitis. The most
common treatments for such diseases 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 effects on the bone marrow, 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.
[0143] The compositions for treatment contain at least one
humanized, chimeric or human monoclonal anti-CD19 antibody alone or
in combination with other antibodies, such as other humanized,
chimeric, or human antibodies, therapeutic agents or
immunomodulators. In particular, combination therapy with a fully
human antibody is contemplated.
[0144] Naked or conjugated antibodies to the same or different
epitope or antigen may also be used in combination. For example, a
humanized, chimeric or human naked anti-CD19 antibody may be
combined with another naked humanized, chimeric or human anti-CD19
or with a naked anti-CD20, anti-CD22 or other B-cell lineage
antibody. A humanized, chimeric or human naked anti-CD19 antibody
may be combined with an anti-CD19 immunoconjugate or anti-CD22
radioconjugate. An anti-CD22 naked antibody may be combined with a
humanized, chimeric or human anti-CD19 antibody conjugated to an
isotope, one or more chemotherapeutic agents, cytokines, toxins or
a combination thereof. A fusion protein of a humanized, chimeric or
human anti-CD19 antibody and a toxin or immunomodulator, or a
fusion protein of at least two different B-cell antibodies (e.g.,
an anti-CD19 and an anti-CD22 MAb or an anti-CD19 and an anti-CD20
MAb) may also be used. Many different antibody combinations,
targeting at least two different antigens associated with B-cell
disorders, as listed above, may be constructed, either as naked
antibodies or conjugated with a therapeutic agent or
immunomodulator, or merely in combination with another therapeutic
agent, such as a cytotoxic drug, a cytokine or radionuclide.
[0145] As used herein, the term "immunomodulator" includes
cytokines, stem cell growth factors, lymphotoxins, such as tumor
necrosis factor (TNF), and hematopoietic factors, such as
interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-I0,
IL-12, IL-21 and IL-18), colony stimulating factors (e.g.,
granulocyte-colony stimulating factor (G-CSF) and granulocyte
macrophage-colony stimulating factor (GM-CSF)), interferons (e.g.,
interferons-alpha, -beta and -gamma), the stem cell growth factor
designated "S1 factor," erythropoietin and thrombopoietin. Examples
of suitable immunomodulator moieties include IL-2, IL-6, IL-10,
IL-12, IL-18, IL-21, interferon, TNF, and the like. Alternatively,
subjects can receive naked anti-CD19 antibodies and a separately
administered cytokine, which can be administered before,
concurrently with or after administration of the naked anti-CD19
antibodies. As discussed supra, the anti-CD19 antibody may also be
conjugated to the immunomodulator. The immunomodulator may also be
conjugated to a hybrid antibody consisting of one or more
antibodies binding to different antigens.
[0146] Multimodal therapies further include immunotherapy with
naked anti-CD19 antibodies supplemented with administration of
antibodies that bind CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21,
CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD4OL, CD46, CD52, CD54,
CD74, CD80, CD126, CD138, B7, MUC1, Ia, HM1.24, HLA-DR (including
the invariant chain), tenascin, VEGF, PlGF, ED-B fibronectin, an
oncogene, an oncogene product, CD66a-d, necrosis antigens, Ii,
IL-2, T101, TAC, IL-6, TRAIL-R1 (DR4) and TRAIL-R2 (DR5) in the
form of naked antibodies, fusion proteins, or as immunoconjugates.
These antibodies include polyclonal, monoclonal, chimeric, human or
humanized antibodies that recognize at least one epitope on these
antigenic determinants. Anti-CD19 and anti-CD22 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); Longo, Curr. Opin. Oncol. 8:353 (1996)
and U.S. Pat. Nos. 5,798,554 and 6,187,287, incorporated in their
entirety by reference. Immunotherapy of autoimmune disorders with
B-cell antibodies is described in the art. See, for example,
W00074718A1, which is incorporated herein by reference in its
entirety.
[0147] In another form of multimodal therapy, subjects receive
naked anti-CD19 antibodies, and/or immunoconjugates, 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 2027-2068 (Lea &
Febiger 1993). As an illustration, first generation
chemotherapeutic regimens for treatment of intermediate-grade
non-Hodgkin's lymphoma (NHL) 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. Antisense bc1-2 oligonucleotide is also in clinical
trials as a therapeutic for certain malignancies, including B-cell
tumors. In a preferred multimodal therapy, both chemotherapeutic
drugs and cytokines are co-administered with an antibody,
immunoconjugate or fusion protein. The cytokines, chemotherapeutic
drugs and antibody or immunoconjugate can be administered in any
order, or together.
[0148] In a preferred embodiment, NHL is treated with 4 weekly
infusions of the humanized anti-CD19 antibody at a dose of 200-400
mg/m.sup.2 weekly for 4 consecutive weeks (iv over 2-8 hours),
repeated as needed over next months/yrs. Also preferred, NHL is
treated with 4 weekly infusions as above, but combined with
epratuzumab (anti-CD22 humanized antibody) on the same days, at a
dose of 360 mg/m.sup.2, given as iv infusion over 1 hour, either
before, during or after the anti-CD19 monoclonal antibody infusion.
Still preferred, NHL is treated with 4 weekly infusions of the
anti-CD19 antibody as above, combined with one or more injections
of anti-CD22 MAb radiolabeled with a therapeutic isotope such as
yttrium-90 (at dose of Y-90 between 5 and 35 mCi/m.sup.2 as one or
more injections over a period of weeks or months. Anti-CD19 MAb may
also be combined, in similar regimens, with anti-CD20 MAbs, such as
the hA20 humanized MAb (U.S. application Ser. No. 10/366,709, filed
Feb. 14, 2003), whereby a weekly dose.times.4 weeks per cycle, with
optional repeated cycles, is given of each antibody at an
individual dose of 250 mg/m.sup.2 i.v. in combination. Either or
both antibodies can also be given by s.c. injection, whereby a
similar dose is given every other week, particularly for the
therapy of patients with autoimmune disease.
[0149] In addition, a therapeutic composition can contain a mixture
or hybrid molecules of monoclonal naked anti-CD19 antibodies
directed to different, non-blocking CD19 epitopes. Accordingly, the
present invention contemplates therapeutic compositions comprising
a mixture of monoclonal anti-CD19 antibodies that bind at least two
CD19 epitopes.
[0150] Although anti-CD19 antibodies are the primary therapeutic
compositions for treatment of B cell lymphoma and autoimmune
diseases, the efficacy of such antibody therapy can be enhanced by
supplementing the naked antibodies, with supplemental agents, such
as immunomodulators, like interferons, including IFN-alpha,
IFN-beta and IFN-gamma, interleukins including IL-1, IL-2, IL-6,
IL-12, IL-15, IL-18, IL-21, and cytokines including G-CSF and
GM-CSF. Accordingly, the anti-CD19 antibodies can be combined not
only with antibodies and cytokines, either as mixtures (given
separately or in some predetermined dosing regimen) or as
conjugates or fusion proteins to the anti-CD19 antibody, but also
can be given as a combination with drugs. For example, the
anti-CD19 antibody may be combined with CHOP as a 4-drug
chemotherapy regimen. Additionally, a naked anti-CD19 antibody may
be combined with a naked anti-CD22 antibody and/or naked anti-CD20
antibodies and CHOP or Fludarabine as a drug combination for NHL
therapy. The supplemental therapeutic compositions can be
administered before, concurrently or after administration of the
anti-CD19 antibodies.
[0151] As discussed supra, the antibodies can be used for treating
B cell lymphoma and leukemia, and other B cell diseases or
disorders. The antibodies may be used for treating any disease or
syndrome which involves unwanted or undesirable B-cell activity or
proliferation. For example, anti-CD19 antibodies can be used to
treat B-cell related autoimmune diseases, including Class III
autoimmune diseases. The antibodies can also be used to treat
B-cell diseases such as graft versus host disease, or for
transplant immunosuppressive therapy.
[0152] Anti-CD19 antibodies may also induce apoptosis in cells
expressing the CD19 antigen. Evidence of this induction is
supported in the literature. For example, it was demonstrated that
apoptosis could be induced using lymphoid cells that have
Fc-receptors reactive with the IgG1-Fc of anti-CD19 MAbs that
crosslinked. See Shan et al., Cancer Immunol. Immunother.
48(12):673-683 (2000). Further, it was reported that aggregates of
a chimeric anti-CD19 MAb, i.e., homopolymers, induced apoptosis.
See Ghetie et al., Blood 97(5): 1392-1398 (2000) and Ghetie et al.,
Proc. Natl. Acad. Sci USA 94(14): 7509-7514 (1997). Enhancement of
the pro-apoptotic activity of the antibodies may be achieved by
simultaneous use of a pro-apoptotic agent, such as an agent that
inhibits the activity of one or more members of the anti-apoptosis
gene family bc1-2. Antisense and RNAi agents are particularly
useful in this regard and can be directed to B cells by conjugation
with anti-CD19 antibodies as described herein.
[0153] Antibodies specific to the CD19 surface antigen of B cells
can be injected into a mammalian subject, which then bind to the
CD19 cell surface antigen of both normal and malignant B cells. A
mammalian subject includes humans and domestic animals, including
pets, such as dogs and cats. The anti-CD19 MAbs, i.e., humanized,
chimeric, human, and even murine anti-CD19 MAbs, can be used to
treat the non-human mammalian subjects when there is a species
crossreactivity for the CD19 antigen. See Examples 10 and 11,
below. The murine MAbs, which are immunogenic in humans, are
usually less immunogenic in non-human mammalian subjects. The
anti-CD19 antibody bound to the CD19 surface antigen leads to the
destruction and depletion of neoplastic B cells. Because both
normal and malignant B cells express the CD19 antigen, the
anti-CD19 antibody will result in B cell death. However, only
normal B cells will repopulate and the malignant B cells will be
eradicated or significantly reduced. Additionally, chemical agents
or radioactive labels having the potential to destroy the tumor can
be conjugated to the anti-CD19 antibody such that the agent is
specifically targeted to the neoplastic B cells.
[0154] Expression Vectors
[0155] The DNA sequence encoding a humanized, chimeric or human
anti-CD19 MAb can be recombinantly engineered into a variety of
known host vectors that provide for replication of the nucleic
acid. These vectors can be designed, using known methods, to
contain the elements necessary for directing transcription,
translation, or both, of the nucleic acid in a cell to which it is
delivered. Known methodology can be used to generate expression
constructs that have a protein-coding sequence operably linked with
appropriate transcriptional/translational control signals. These
methods include in vitro recombinant DNA techniques and synthetic
techniques. For example, see Sambrook et al., 1989, MOLECULAR
CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory (New
York); Ausubel et al., 1997, CURRENT PROTOCOLS IN MOLECULAR
BIOLOGY, John Wiley & Sons (New York).
[0156] Vectors suitable for use can be viral or non-viral.
Particular examples of viral vectors include adenovirus, AAV,
herpes simplex virus, lentivirus, and retrovirus vectors. An
example of a non-viral vector is a plasmid. In a preferred
embodiment, the vector is a plasmid.
[0157] An expression vector, as described herein, is a
polynucleotide 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.
[0158] Preferably, the expression vector comprises the DNA sequence
encoding a humanized, chimeric or human anti-CD19 MAb, which
includes both the heavy and the light chain variable and constant
regions. However, two expression vectors may be used, with one
comprising the heavy chain variable and constant regions and the
other comprising the light chain variable and constant regions.
Still preferred, the expression vector further comprises a
promoter, a DNA sequence encoding a secretion signal peptide, a
genomic sequence encoding a human IgG1 heavy chain constant region,
an Ig enhancer element and at least one DNA sequence encoding a
selectable marker.
[0159] Also contemplated herein is a method for expressing a
humanized anti-CD19 MAb, comprising (i) linearizing at least one
expression vector comprising a DNA sequence encoding a humanized,
chimeric, or human anti-CD19 MAb, (ii) transfecting mammalian cells
with at least one of said linearized vector, (iii) selecting
transfected cells which express a marker gene, and (iv) identifying
the cells secreting the humanized anti-CD19 MAb from the
transfected cells.
[0160] Methods of Making Anti-CD19 Antibodies
[0161] In general, the Vk and VH sequences encoding an anti-CD19
MAb can be obtained by a variety of molecular cloning procedures,
such as RT-PCR, 5'-RACE, and cDNA library screening. Specifically,
the V genes of an anti-CD19 MAb can be cloned by PCR amplification
from a cell that expresses a murine or chimeric anti-CD19 MAb and
then sequenced. To confirm their authenticity, the cloned VL and VH
genes can be expressed in cell culture as a chimeric Ab as
described by Orlandi et al., (Proc. Natl. Acad. Sci., USA, 86: 3833
(1989)) which is incorporated by reference. Based on the V gene
sequences, a humanized anti-CD19 MAb can then be designed and
constructed as described by Leung et al. (Mol. Immunol., 32: 1413
(1995)), which is incorporated herein by reference. cDNA can be
prepared from any known hybridoma line or transfected cell line
producing a murine or chimeric anti-CD19 MAb by general molecular
cloning techniques (Sambrook et al., Molecular Cloning, A
laboratory manual, 2nd Ed (1989)). The VK sequence for the MAb may
be amplified using the primers Vk1BACK and Vk1FOR (Orlandi et al.,
1989) or the extended primer set described by Leung et al.
(BioTechniques, 15: 286 (1993)), which is incorporated herein by
reference, while VH sequences can be amplified using the primer
pair VH1BACK/VH1FOR (Orlandi et al., 1989), or the primers
annealing to the constant region of murine IgG described by Leung
et al. (Hybridoma, 13:469 (1994)), which is incorporated herein by
reference.
[0162] The PCR reaction mixtures containing 10 .mu.l of the first
strand cDNA product, 10 .mu.l of 10.times. PCR buffer [500 mM KCl,
100 mM Tris-HCl (pH 8.3), 15 mM MgCl.sub.2, and 0.01% (w/v)
gelatin] (Perkin Elmer Cetus, Norwalk, Conn.), 250 .mu.M of each
dNTP, 200 nM of the primers, and 5 units of Taq DNA polymerase
(Perkin Elmer Cetus) can be subjected to 30 cycles of PCR. Each PCR
cycle preferably consists of denaturation at 94.degree. C. for 1
min, annealing at 50.degree. C. for 1.5 min, and polymerization at
72.degree. C. for 1.5 min. Amplified VK and VH fragments can be
purified on 2% agarose (BioRad, Richmond, Calif.). Similarly, the
humanized V genes can be constructed by a combination of long
oligonucleotide template syntheses and PCR amplification as
described by Leung et al. (Mol. Immunol., 32: 1413 (1995)). See
Example 3 for a method for the synthesis of an oligo A and an oligo
B on an automated RNA/DNA synthesizer (Applied Biosystems, Foster
City, Calif.) for use in constructing humanized V genes.
[0163] PCR products for VK can be subcloned into a staging vector,
such as a pBR327-based staging vector, VkpBR, that contains an Ig
promoter, a signal peptide sequence and convenient restriction
sites to facilitate in-frame ligation of the VK PCR products. PCR
products for VH can be subcloned into a similar staging vector,
such as the pBluescript-based VHpBS. Individual clones containing
the respective PCR products may be sequenced by, for example, the
method of Sanger et al. (Proc. Natl. Acad. Sci., USA, 74: 5463
(1977)).
[0164] The expression cassettes containing the VK and VH, together
with the promoter and signal peptide sequences can be excised from
VKpBR and VHpBS, respectively, by double restriction digestion as
HindIII-BamHI fragments. The VK and VH expression cassettes can
then be ligated into appropriate expression vectors, such as pKh
and pG1g, respectively (Leung et al., Hybridoma, 13:469 (1994)).
The expression vectors can be co-transfected into an appropriate
cell, e.g., myeloma Sp2/0-Ag14 or Sp-EEE, colonies selected for
hygromycin resistance, and supernatant fluids monitored for
production of a chimeric or humanized anti-CD19 MAb by, for
example, an ELISA assay. Alternatively, the VK and VH expression
cassettes can be assembled in the modified staging vectors, YKpBR2
and VHpBS2, excised as XbaI/BamHI and XhoI/BamHI fragments,
respectively, and subcloned into a single expression vector, such
as pdHL2, as described by Gilles et al. (J. Immunol. Methods
125:191 (1989)) and also shown in Losman et al., (Cancer, 80:2660
(1997)) for expression in cells. Another vector that is useful is
the GS vector, as described in Barnes et al., Cytotechnology 32:
109-123 (2000), which is preferably expressed in the NSO cell line
and CHO cells. Other appropriate mammalian expression systems are
described in Werner et al., Arzneim.-Forsch./Drug Res. 48(11), Nr.
8, 870-880 (1998).
[0165] Co-transfection and assay for antibody secreting clones by
ELISA, can be carried out as follows. About 10 .mu.g of VkpKh
(light chain expression vector) and 20 .mu.g of VHpG1g (heavy chain
expression vector) can be used for the transfection of
5.times.10.sup.6 SP2/0 myeloma cells by electroporation (BioRad,
Richmond, Calif.) according to Co et al., J. Immunol., 148: 1149
(1992). Following transfection, cells may be grown in 96-well
microtiter plates in complete HSFM medium (Life Technologies, Inc.,
Grand Island, N.Y.) at 37.degree. C., 5% CO.sub.2. The selection
process can be initiated after two days by the addition of
hygromycin selection medium (Calbiochem, San Diego, Calif.) at a
final concentration of 500 units/ml of hygromycin. Colonies
typically emerge 2-3 weeks post-electroporation. The cultures can
then be expanded for further analysis.
[0166] Transfectoma clones that are positive for the secretion of
chimeric or humanized heavy chain can be identified by ELISA assay.
Briefly, supernatant samples (about.100 .mu.l) from transfectoma
cultures are added in triplicate to ELISA microtiter plates
precoated with goat anti-human (GAH)-IgG, F(ab').sub.2
fragment-specific antibody (Jackson ImmunoResearch, West Grove,
Pa.). Plates are incubated for 1 h at room temperature. Unbound
proteins are removed by washing three times with wash buffer (PBS
containing 0.05% polysorbate 20). Horseradish peroxidase (HRP)
conjugated GAH-IgG, Fc fragment-specific antibodies (Jackson
ImmunoResearch) are added to the wells, (100 .mu.l of antibody
stock diluted.times.10.sup.4, supplemented with the unconjugated
antibody to a final concentration of 1.0 .mu.g/ml). Following an
incubation of 1 h, the plates are washed, typically three times. A
reaction solution, [100 .mu.l, containing 167 .mu.g of
orthophenylene-diamine (OPD) (Sigma, St. Louis, Mo.), 0.025%
hydrogen peroxide in PBS], is added to the wells. Color is allowed
to develop in the dark for 30 minutes. The reaction is stopped by
the addition of 50 .mu.l of 4 N HCl solution into each well before
measuring absorbance at 490 nm in an automated ELISA reader
(Bio-Tek instruments, Winooski, Vt.). Bound chimeric antibodies are
than determined relative to an irrelevant chimeric antibody
standard (obtainable from Scotgen, Ltd., Edinburg, Scotland).
[0167] Antibodies can be isolated from cell culture media as
follows. Transfectoma cultures are adapted to serum-free medium.
For production of chimeric antibody, cells are grown as a 500 ml
culture in roller bottles using HSFM. Cultures are centrifuged and
the supernatant filtered through a 0.2 .mu.m membrane. The filtered
medium is passed through a protein A column (1.times.3 cm) at a
flow rate of 1 ml/min. The resin is then washed with about 10
column volumes of PBS and protein A-bound antibody is eluted from
the column with 0.1 M glycine buffer (pH 3.5) containing 10 mM
EDTA. Fractions of 1.0 ml are collected in tubes containing 10
.mu.l of 3 M Tris (pH 8.6), and protein concentrations determined
from the absorbance at 280/260 nm. Peak fractions are pooled,
dialyzed against PBS, and the antibody concentrated, for example,
with the Centricon 30 (Amicon, Beverly, Mass.). The antibody
concentration is determined by ELISA, as before, and its
concentration adjusted to about 1 mg/ml using PBS. Sodium azide,
0.01% (w/v), is conveniently added to the sample as
preservative.
[0168] The following are the nucleotide sequences of the primers
used to prepare the anti-CD19 antibodies:
TABLE-US-00001 hA19VkA (SEQ ID NO: 24) 5'-ATCACTTGTA AGGCCAGCCA
AAGTGTTGAT TATGATGGTG ATAGTTATTT GAACTGGTAC CAGCAGATTC CAGGGAAAGC
ACCTAAATTG TTGATCTACG ATGCTTCGAA TCTAGTTTCT GGTATC-3' hA19VkB (SEQ
ID NO: 25) 5'-TGCTGACAGT GATATGTTGC AATGTCTTCT GGTTGAAGAG
AGCTGATGGT GAAAGTGTAA TCTGTCCCAG ATCCGCTGCC AGAGAATCGA GGAGGGATAC
CAGAAACTAG ATTCGAAGCA TCGTA-3' hA19VkBack (SEQ ID NO: 26)
5'-TCCGACATCC AGCTGACCCA GTCTCCATCA TCTCTGAGCG CATCTGTTGG
AGATAGGGTC ACTATCACTT GTAAGGCCAG CCAAAG-3' hA19VkFor (SEQ ID NO:
27) 5'-GCTCCTTGAG ATCTGTAGCT TGGTCCCTCC ACCGAACGTC CACGGATCTT
CAGTACTTTG CTGACAGTGA TATGTTGCAA-3' hA19VHA (SEQ ID NO: 28)
5'-CTGGCTACGC TTTCAGTAGC TACTGGATGA ACTGGGTGAG GCAGAGGCCT
GGACAGGGTC TTGAGTGGAT TGGACAGATT TGGCCTGGAG ATGGTGATAC TAACTACAAT
GGAAAGTTCA AGGGGCGCGC CACTATT-3' hA19VHB (SEQ ID NO: 29)
5'-CGTAGTCTCC CGTCTTGCAC AAGAATAGAA CGCTGTGTCC TCAGATCGTA
GGCTGCTGAG TTCCATGTAG GCTGTATTAG TGGATTCGTC GGCAGTAATA GTGGCGCGCC
CCTTGAACTT TCCATTGTA-3' hA19VHBack (SEQ ID NO: 30) 5'-CAGGTCCAAC
TGCAGCAATC AGGGGCTGAA GTCAAGAAAC CTGGGTCATCG GTGAAGGTCTC CTGCAAGGCT
TCTGGCTACG CTTTCAGTAG C-3' hA19VHFor (SEQ ID NO: 31) 5'-TGAGGAGACG
GTGACCGTGG TCCCTTGGCC CCAGTAGTCC ATAGCATAGT AATAACGGCC TACCGTCGTA
GTCTCCCGTC TTGCACAAG-3'
EXAMPLES
[0169] The invention is further described by reference to the
following examples, which are provided for illustration only. The
invention is not limited to the examples but rather includes all
variations that are evident from the teachings provided herein.
Example 1
Construction of a Humanized Anti-CD19 Antibody
[0170] A chimeric A19 (cA19) antibody was constructed and expressed
in Sp2/0 cell. The Vk (SEQ ID NO:1 and SEQ ID NO:3) and VH (SEQ ID
NO:3 and SEQ ID NO:4) sequences of cA19 are shown in FIG. 1. The
cA19 antibody was shown to bind to CD19+ human lymphoma cell lines,
such as Raji, Daudi, and Ramos. The Ag-binding specificity of
purified cA19 was evaluated by a cell surface competitive binding
assay against other anti-CD19 antibodies, e.g. B4 (Coulter) and
BU12 (Chembiochem). Briefly, varying concentrations of cA19 were
incubated with Raji cells in the presence of a constant amount of
an 1-125 radiolabeled anti-CD19 antibody for 1 h. After washing to
remove the unbound antibodies, the cell surface-bound radiolabeled
antibody was quantitated by counting the cell pellets in a gamma
counter. As shown in FIG. 2, cA19 competed with BU12 (Chembiochem)
for cell surface binding, indicating these antibodies share similar
or overlapping epitopes of the CD19 molecule.
[0171] The light chain and heavy chain variable region sequences
encoding the humanized anti-hCD19 antibody (hA19) were designed and
constructed. Comparison of the variable (V) region framework (FR)
sequences of the cA19 (FIGS. 1A and 1B) to registered human
antibodies in the Kabat database showed that the FRs of cA19 VK
exhibited the highest degree of sequence homology to that of the
human antibody REI (VK), while the VH sequence was most closely
related with that of the human antibody EU (VH). The VH FR4
sequence of the human antibody NEWM, however, was better aligned
with that of cA19 and used to replace the EU FR4 sequence for the
humanization of the A19 heavy chain (FIG. 3B). Therefore, human REI
framework sequences were used for Vk (FIG. 3A), and a combination
of EU and NEWM framework sequences were used for VH (FIG. 3B).
There are a number of amino acid changes in each chain outside of
the CDR regions when compared to the starting human antibody
frameworks. These residues are 4L, 39I, 58I, 60P, 87H, 100G, and
107K of VK (FIG. 3A) and 5Q, 27Y, 28A, 40R, 91S, 94R, 107T, and
108T of VH (FIG. 3B). The DNA and amino acid sequences of hA19 VK
and VH are shown in FIGS. 4A and 4B, respectively.
Example 2
Method of hA19 Antibody Construction
[0172] To engineer the CDR-grafted hA19VH and VK genes, a modified
strategy as described by Leung et al. (1995) was used to construct
the designed VK and VH genes for hA19 using a combination of long
oligonucleotide syntheses and PCR. Briefly, two long synthetic
oligonucleotides (ca. 130 mer in length) representing the 5'-(sense
strand, designated as A) and 3'-half (anti-sense strand, designated
as B) of a V sequence are used as the templates in a PCR reaction.
The 3'-terminal sequences of the long oligonucleotides A and B are
designed to overlap and be complementary to each other. PCR is
initiated by annealing of the 3'-termini of A and B to form a short
double strand DNA flanked by the rest of long oligonucleotides
(single strand). Each annealed end serves as a primer for the
replication of the single stranded DNA, resulting in elongation of
A and B to form the double-strand DNA. In the presence of two short
oligonucleotide primers, V gene segment is generated by PCR
amplification of the double strand DNA.
[0173] Heavy Chain
[0174] For the construction of hA19 VH domain, the long
oligonucleotides, hA19VHA (SEQ ID NO:28, 126-mer) and hA19VHB (SEQ
ID NO:29, 128-mer) were synthesized on an automated DNA synthesizer
(Applied Biosystems). hA19VHA represents nt 74 to 126 of the hA19
VH domain, and hA19VHB represents the minus strand of the hA19VH
domain complementary to nt 178 to 306. The 3'-terminal sequences
(33 nt residues) of hA19VHA and VHB are complementary to each
other. A minimal amount of hA19VHA and VHB (determined empirically)
was amplified in the presence of 10 .mu.L of 10.times. PCR Buffer
(500 mM KCl, 100 mM Tris-HCl buffer, pH 8.3, 15 mM MgCl.sub.2), 2
.mu.mol of hA19VHBack (5'-CAGGTCCAAC TGCAGCAATC AGGGGCTGAA
GTCAAGAAAC CTGGGTCATCG GTGAAGGTCTC CTGCAAGGCT TCTGGCTACG CTTTCAGTAG
C-3' SEQ ID NO:30) and hA19VHFor (5'-TGAGGAGACG GTGACCGTGG
TCCCTTGGCC CCAGTAGTCC ATAGCATAGT AATAACGGCC TACCGTCGTA GTCTCCCGTC
TTGCACAAG-3' SEQ ID NO:31), and 2.5 units of Taq DNA polymerase
(Perkin Elmer Cetus, Norwalk, Conn.). The underlined portions are
the restriction sites for subcloning as shown in FIG. 4B. This
reaction mixture was subjected to three cycles of polymerase chain
reaction (PCR) consisting of denaturation at 94.degree. C. for 1
minute, annealing at 45.degree. C. for 1 minute, and polymerization
at 72.degree. C. for 1.5 minutes. This procedure was followed by 27
cycles of PCR reaction consisting of denaturation at 94.degree. C.
for 1 minute, annealing at 55.degree. C. for 1 minute, and
polymerization at 72.degree. C. for 1 minute. The resulting DNA
fragment showed an expected molecular size in agarose gel
electrophoresis. The double-stranded PCR-amplified product for
hA19VH was gel-purified, restriction-digested with PstI and BstEII
restriction enzymes and cloned into the complementary PstI/BstEII
restriction sites of the heavy chain staging vector, VHpBS2, in
which the VH sequence was fully assembled with the DNA sequence
encoding the translation initiation codon and a secretion signal
peptide in-frame ligated at the 5'-end and an intron sequence at
the 3'-end. VHpBS2 is a modified staging vector of VHpBS (Leung et
al., Hybridoma, 13:469 (1994)), into which a XhoI restriction site
was introduced sixteen bases upstream of the translation initiation
codon to facilitate the next subcloning step. The assembled VH gene
was subcloned as a XhoI-BamHI restriction fragment into the
expression vector, pdHL2, which contains the expression cassettes
for both human IgG heavy and light chains under the control of IgH
enhancer and MT1 promoter, as well as a mouse dhfr gene as a marker
for selection and amplification (FIG. 4B). Since the heavy chain
region of pdHL2 lacks a BamHI restriction site, this ligation
requires use of a linker to provide a bridge between the BamHI site
of the variable chain and the HindIll site present in the pdHL2
vector. The resulting expression vector was designated as
hAI9VHpdHL2.
[0175] For constructing the full length DNA of the humanized VK
sequence, hA19VkA (SEQ ID NO:24, 126-mer, represents nt 61 to 186
of the hA19 VK domain) and hA19VkB (SEQ ID NO:25, 124-mer,
represents the minus strand of the hA19 VK domain complementary to
nt 157 to 281) were synthesized as described above. hA19VkA and VkB
were amplified by two short oligonucleotides hA19VkBack
(5'-CAGGTCCAAC TGCAGCAATC AGGGGCTGAA GTCAAGAAAC CTGGGTCATCG
GTGAAGGTCTC CTGCAAGGCT TCTGGCTACG CTTTCAGTAG C-3' SEQ ID NO:26) and
hA19VkFor (5'-TGAGGAGACG GTGACCGTGG TCCCTTGGCC CCAGTAGTCC
ATAGCATAGT AATAACGGCC TACCGTCGTA GTCTCCCGTC TTGCACAAG-3' SEQ ID
NO:27) as described above. The underlined portions are restriction
sites for subcloning as described below. Gel-purified PCR products
for hA19 VK were restriction-digested with PvulI and BglII and
cloned into the complementary PvulI/BclI sites of the light chain
staging vector, VkpBR2. VkpBR2 is a modified staging vector of
VkpBR (Leung et at, Hybridoma, 13:469 (1994)), into which a XbaI
restriction site was introduced at sixteen bases upstream of the
translation initiation codon. The assembled VK genes were subcloned
as XbaI-BamHI restriction fragments into the expression vector
containing the VH sequence, hA19VHpdHL2. The resulting expression
vectors were designated as hA19pdHL2.
Example 3
Transfection and Expression of hA19 Antibodies
[0176] Approximately 30 .mu.g of the expression vectors for hA19
were linearized by digestion with SalIand transfected into
Sp2/0-Ag14 cells by electroporation (450V and 25 J-.mu.F). The
transfected cells were plated into 96-well plates for 2 days and
then selected for drug-resistance by adding MTX into the medium at
a final concentration of 0.075 .mu.M. MTX-resistant colonies
emerged in the wells after 2-3 weeks. Supernatants from colonies
surviving selection were screened for human MAb secretion by ELISA
assay. Briefly, 100 .mu.l supernatants were added into the wells of
a microtiter plate precoated with GAH-IgG, F(ab').sub.2
fragment-specific Ab and incubated for 1 h at room temperature.
Unbound proteins were removed by washing three times with wash
buffer (PBS containing 0.05% polysorbate 20). HRP-conjugated
GAH-IgG, Fc fragment-specific Ab was added to the wells. Following
an incubation of 1 h, the plate was washed. The bound
HRP-conjugated Ab was revealed by reading A490 nm after the
addition of a substrate solution containing 4 mM OPD and 0.04%
H202. Positive cell clones were expanded and hB43 were purified
from cell culture supernatant by affinity chromatography on a
Protein A column.
Example 4
Determination of the Antigen-Binding Specificity and Affinity of
anti-CD19 Antibodies
[0177] The Ag-binding specificity of cA19 and hA19 purified by
affinity chromatography on a Protein A column were evaluated and
compared by a cell surface competitive binding assay. Briefly, a
constant amount (100,000 cpm, .about.10 .mu.Ci/.mu.g) of
.sup.125I-labeled cA19 or hA19 was incubated with Raji cells in the
presence of varying concentrations (0.2-700 nM) of cA19 or hA19 at
4.degree. C. for 1-2 h. Unbound Abs were removed by washing the
cells in PBS. The radioactivity associated with cells was
determined after washing. As shown in FIG. 2, the purified hA19
competed with .sup.125I-labeled cA19 for cell surface binding and
vice versa, indicating the apparent binding avidities are
comparable between these two Abs.
[0178] The antigen-binding affinity (avidity) constant of hA19 was
determined by direct cell surface binding assay of the radiolabeled
Ab and Scatchard plot analysis, in comparison to that of cA19.
Briefly, hA19 and cA19 were labeled with .sup.125I by the
chloramines-T method. Varying amounts of either .sup.1251-hA19 or
.sup.1251-cA19 were incubated with 2.times.10.sup.5 Raji cells at
4.degree. C. for 2 h and unbound antibodies were removed by
washing. The cell-associated radioactivity was counted and
Scatchard plot analysis was performed to determine the maximum
number of hA19 and cA19 binding sites per cell and the apparent
dissociation constants of the equilibrium binding. As shown in FIG.
5, hA19 showed virtually the same binding affinity as cA19. The
apparent dissociation constant values for these two antibodies were
calculated to be 1.1 and 1.2 nM, respectively.
Example 5
Sequence Variants of hA19
[0179] The humanized anti-CD19 MAb (hA19) described in Example 2
above was expressed in Sp2/0 cells, but the productivity of
resulting clones was low. Numerous transfections (a total of 15)
were performed and hundreds of clones were screened. The
productivities of positive clones remained between 0.5-3 .mu.g/ml
and amplification with methotrexate did not improve the
productivities of subjected clones.
[0180] Modifying the Vk Gene Sequence to Reduce the AT Contents
[0181] To generate a higher producing hA19 clone, two approaches
were used. The first approach (hA19VkpdHL2) was to re-design the
hA19Vk gene sequence to reduce the AT content, which presumably has
a negative impact on the Ab gene expression. The new hA19Vk gene
was synthesized, assembled, and used to reconstruct the expression
vector for hA19.
[0182] After construction of hA19VkpdHL2 was completed and DNA
sequencing confirmed the sequences to be correct, a maxiprep was
performed to prepare the plasmids for transfection. Three
transfections were performed: #739 (hA19VkpdHL2#1), #740
(hA19VkpdHL2#5) and #741 (hB43pdHL2#1). SP-E26 cells and 10 .mu.g
of SalI-linearized DNA were used. MTX selection (0.075 .mu.M)
started 48 h post electroporation. An ELISA was used to screen
transfections for positive clones. Transfection #739 yielded 31
positive clones (13 were selected), #740 yielded 49 positive clones
(13 were selected), and #741 yielded 41 positive clones (19 were
selected). The productivities were determined and monitored by
ELISA and ranged from 0.5-3 .mu.g/ml, the same as observed for the
original hA19 construct.
[0183] Two serum-free transfections were performed: #746
(hA19VkpdHL2#1) and #747 (hB43pdHL2#1). SP-ESF cells and 10 .mu.g
of SalI-linearized DNA were used for both transfections. MTX
selection (0.075 .mu.M) started 48 h post electroporation.
Transfection #746 yielded 9 positive clones and #747 yielded 6
positives. The positives were expanded to a 48 well plate and
amplified to 0.2 .mu.M MTX. The 45 clones from transfections #739,
#740, and #741 were narrowed down to 12 (based on an ELISA reading
above 1.0 .mu.g/ml) and the MTX was increased to 0.15 .mu.M. The
initial p.sub.max of the serum-free clones was determined and the
productivities were similar to the clones in serum medium (0.5-1.5
.mu.g/ml).
[0184] Two more serum transfections were performed: #756
(hA19VkpdHL2#5) and #757 (hB43pdHL2#1). The transfection conditions
were the same as before, however, the slow growing clones were
given time to develop to see if the p.sub.max would be higher.
Transfection #756 yielded 19 positive clones and #757 yielded 8
positive clones. The p. of transfections #756 and #757 were
compared with transfections #739, #740, #741, #746, and #747. The
productivities were similar (.about.0.5-3 .mu.g/ml). The best
producers were selected (739.1A9, 740.1B1, and 756.2G7) and
amplified to 0.2 .mu.M MTX. Results reported in Table 2 show an
average antibody productivity of between 2 and 3 .mu.g/ml.
TABLE-US-00002 TABLE 1 Comparison of initial positives and
p.sub.max values in both 10% FBS and serum-free conditions. Initial
P.sub.max Positives (ug/ml) SP-E26 #739 31 0.5-3 #740 49 0.5-3 #741
41 0.5-3 #756 19 0.5-3 #757 8 0.5-3 SP-ESF #746 9 0.5-1.5 #747 6
0.5-1.5
TABLE-US-00003 TABLE 2 Average p.sub.max and standard deviation
values from the three best hA19VkpdHL2 clones. 0.2 .mu.M Avg. MTX
(ug/ml) St. Dev. 739.1A9 2.96 .+-.1.40 740.1B1 2.30 .+-.0.30
756.2G7 2.95 .+-.0.70
[0185] Cell-Based Antibody Dependent Cytotoxicity Assay
[0186] To determine whether or not the redesigned vector produced
active antibody, two liters of clone 756.2G7 were purified by
protein-A purification. The purification yielded 7.3 mgs of
anti-CD-19 Ab. HPLC and SDS-PAGE showed the purified protein to be
pure (not shown). The purified protein was used to examine the
effects of antibody treatment on cell proliferation and death. A
cell based antibody dependent cytotoxicity assay was performed to
compare hA19, hA20, and hLL2 with and without GAH IgG and Fc
fragment. Daudi D1-1 and SP-E26 cells were plated in two 48 well
plates at a final density of 150,000 cells/ml. hA19, hA20 and hLL2
were diluted (final concentration of 10 .mu.g/ml) in complete
medium (RPMI for D1-1 and SFM for SP-E26) both with and without GAH
(final concentration of 40 .mu.g/ml). The Ab mixtures were added to
the plate and placed on a shaker for a few minutes and then put in
the incubator. An MTT was performed at dayS 3 and 4. The results
from day 3 showed that there was no difference in cell growth in
SP-E26 cells. The D1-1 cells show inhibition of cell growth in
hA19+GAH and hA20+GAH. These results showed that the redesigned
hA19 is active.
[0187] hA19FpdHL2
[0188] The second approach (hA19FpdHL2) was to re-design the hA19VH
gene to replace the heavy chain (VH) framework region amino acid
residue serine 91 with the consensus phenylalanine residue. The TCT
codon for S91 in hA19VHpdHL2 was changed to a TTC codon for F91 in
hA19FVHpdHL2. The new hA19F gene was synthesized, assembled, and
used to re-construct the expression vector for hA19.
[0189] After construction of hA19FpdHL2 was completed and DNA
sequencing confirmed the sequences to be correct a maxiprep was
performed to prepare the plasmids for transfection. Two
transfections were performed: #762 (hA19FpdHL2#2) and #763
(hA19FpdHL2#3). Transfection conditions were the same as
transfections #739, #740, and #741. An ELISA was used to screen
both transfections for positive clones. Transfection #762 yielded
13 positive clones (8 were selected), and #743 yielded 18 positive
clones (16 were selected). The initial productivities were
determined by ELISA, shown in Table 3.
TABLE-US-00004 TABLE 3 Average p.sub.max and standard deviation
values from the hA19FpdHL2 clones that were selected. 0.075 .mu.M
Avg. MTX (ug/ml) St. Dev. 762.2D6 42.42 762.2H9 11.34 762.2B10
14.86 762.2D4 12.69 762.2A10 15.96 762.2C11 6.34 762.2F11 9.17
763.2B2 11.57 3.39 763.2G2 11.18 1.74 763.2B4 9.08 5.27 763.2D4
10.88 13.64 763.2E11 15.33 4.32 763.2C4 11.57 8.13 763.2B5 2.94
1.13 763.2H5 7.46 5.39 763.2C5 18.91 10.70 763.2H6 6.54 4.09
763.2A6 14.17 12.64 763.2A9 2.35 763.2A10 2.95 0.95 763.2B12 15.76
6.95 763.2C12 6.28 5.50 763.2D3 6.65
[0190] Transfection of host cells with the new vector resulted in
more than 100 positive clones. We randomly picked 30 clones for
evaluation. Most of these clones were estimated to have yields of
antibody production in cell culture of between 5-25 mg/L of IgG.
This contrasts with the cell culture productivity of the clones
generated previously, which were in the range of 1-2 mg/L.
Therefore, we conclude that the substitution Ser91Phe resulted in a
significant increase in the expression level of the hA19 antibody,
with about a 10-fold increase in antibody production. This
surprising and unexpected result allows substantially greater
amounts of the antibody protein to be produced in cell culture
using expression in mammalian cell lines.
Example 6
Therapy of Non-Hodgkin's Lymphoma
[0191] A patient with indolent, follicular-cell NHL relapses after
chemotherapy including dexamethasone, and has disease in the chest
(para-aortic lymph nodes), an enlarged and involved spleen, and
enlarged cervical lymph nodes. The patient is given a course of 300
mg/m.sup.2 each of hA19 MAb combined with humanized anti-CD20 MAb
(hA20) sequentially on the same day by i.v. infusion, weekly for 4
weeks, each time being premedicated with TYLENOL.RTM. and
BENADRYL.RTM. according to standard, published doses for
suppressing infusion-related reactions. Four weeks later, the
patient returns for the first follow-up examination and the only
observation is that some of the palpable lymph nodes feel softer.
Upon returning 3 months following the first therapy cycle, the
patient's chest disease appears to have become reduced by 40% on CT
scan, the spleen is about half the pre-therapy size, and the
cervical lymph nodes are almost gone. The patient is then given a
retreatment cycle, and another three months later appears to have a
normal-sized spleen, no cervical lymph nodes palpable or measurable
on CT scan, and only a small, 1.5-cm lesion in the chest. The
patient continues to appear almost free of disease for another 4
months.
[0192] 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.
[0193] All of the publications and patent applications and patents
cited in this specification are herein incorporated in their
entirety by reference.
Sequence CWU 1
1
311336DNAArtificial SequenceDescription of Artificial Sequence
Synthetic anti-CD19 antibody, cA19 1gac atc cag ctg acc cag tct cca
gct tct ttg gct gtg tct cta ggg 48Asp Ile Gln Leu Thr Gln Ser Pro
Ala Ser Leu Ala Val Ser Leu Gly1 5 10 15cag agg gcc acc atc tcc tgc
aag gcc agc caa agt gtt gat tat gat 96Gln Arg Ala Thr Ile Ser Cys
Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30ggt gat agt tat ttg aac
tgg tac caa cag att cca gga cag cca ccc 144Gly Asp Ser Tyr Leu Asn
Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro 35 40 45aaa ctc ctc atc tat
gat gca tcc aat cta gtt tct ggc atc cca ccc 192Lys Leu Leu Ile Tyr
Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro 50 55 60agg ttt agt ggc
agt ggg tct ggg aca gac ttc acc ctc aac atc cat 240Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His65 70 75 80cct gtg
gag aag gtg gat gct gca acc tat cac tgt cag caa agt act 288Pro Val
Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr 85 90 95gaa
gat ccg tgg acg ttc ggt gga ggg acc aag ctg gag atc aaa cgt 336Glu
Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105
1102112PRTArtificial SequenceDescription of Artificial Sequence
Synthetic anti-CD19 antibody, cA19 2Asp Ile Gln Leu Thr Gln Ser Pro
Ala Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys
Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30Gly Asp Ser Tyr Leu Asn
Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr
Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro 50 55 60Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His65 70 75 80Pro Val
Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr 85 90 95Glu
Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105
1103372DNAArtificial SequenceDescription of Artificial Sequence
Synthetic anti-CD19 antibody, cA19 3cag gtc caa ctg cag gag tct ggg
gct gag ctg gtg agg cct ggg tcc 48Gln Val Gln Leu Gln Glu Ser Gly
Ala Glu Leu Val Arg Pro Gly Ser1 5 10 15tca gtg aag att tcc tgc aag
gct tct ggt tat gca ttc agt agc tac 96Ser Val Lys Ile Ser Cys Lys
Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30tgg atg aac tgg gtg aag
cag agg cct gga cag ggt ctt gag tgg att 144Trp Met Asn Trp Val Lys
Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45gga cag att tgg cct
gga gat ggt gat act aac tac aat gga aag ttc 192Gly Gln Ile Trp Pro
Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60aag ggt aaa gcc
act ctg act gcc gac gaa tcc tcc agc aca gcc tac 240Lys Gly Lys Ala
Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr65 70 75 80atg caa
ctc agc agc cta cga tct gag gac tct gcg gtc tat tct tgt 288Met Gln
Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Ser Cys 85 90 95gca
aga cgg gag act acg acg gta ggc cgt tat tac tat gct atg gac 336Ala
Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp 100 105
110tac tgg ggc caa ggg acc acg gtc acc gtc tcc tca 372Tyr Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser 115 1204124PRTArtificial
SequenceDescription of Artificial Sequence Synthetic anti-CD19
antibody, cA19 4Gln Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Val Arg
Pro Gly Ser1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala
Phe Ser Ser Tyr 20 25 30Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln
Gly Leu Glu Trp Ile 35 40 45Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr
Asn Tyr Asn Gly Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Ala Asp
Glu Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Leu Arg
Ser Glu Asp Ser Ala Val Tyr Ser Cys 85 90 95Ala Arg Arg Glu Thr Thr
Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp 100 105 110Tyr Trp Gly Gln
Gly Thr Thr Val Thr Val Ser Ser 115 1205108PRTHomo sapiens 5Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ile Lys Tyr 20 25
30Leu Asn Trp Tyr Gln Gln Thr Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45Tyr Glu Ala Asn Ser Leu Gln Ala Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Gln
Ser Leu Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Gln Ile Thr
Arg 100 1056112PRTArtificial SequenceDescription of Artificial
Sequence Chimeric cA19Vk antibody 6Asp Ile Gln Leu Thr Gln Ser Pro
Ala Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys
Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30Gly Asp Ser Tyr Leu Asn
Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr
Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro 50 55 60Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His65 70 75 80Pro Val
Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr 85 90 95Glu
Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Gln Ile Lys Arg 100 105
1107112PRTArtificial SequenceDescription of Artificial Sequence
Synthetic humanized hA19Vk antibody 7Asp Ile Gln Leu Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30Gly Asp Ser Tyr Leu
Asn Trp Tyr Gln Gln Ile Pro Gly Lys Ala Pro 35 40 45Lys Leu Leu Ile
Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro 50 55 60Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser65 70 75 80Ser
Leu Gln Pro Glu Asp Ile Ala Thr Tyr His Cys Gln Gln Ser Thr 85 90
95Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Gln Ile Lys Arg
100 105 1108117PRTHomo sapiens 8Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Trp Leu His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Val Pro Met
Phe Gly Pro Pro Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Ala Thr
Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr65 70 75 80Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Phe Tyr Phe Cys 85 90 95Ala Gly
Gly Tyr Gly Ile Tyr Ser Pro Glu Trp Gly Gln Gly Ser Leu 100 105
110Val Thr Val Ser Ser 1159124PRTArtificial SequenceDescription of
Artificial Sequence Chimeric cA19VH antibody 9Gln Val Gln Leu Gln
Glu Ser Gly Ala Glu Leu Val Arg Pro Gly Ser1 5 10 15Ser Val Lys Ile
Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30Trp Met Asn
Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Gln
Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60Lys
Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr65 70 75
80Met Gln Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Ser Cys
85 90 95Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met
Asp 100 105 110Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115
12010124PRTArtificial SequenceDescription of Artificial Sequence
Synthetic humanized hA19VH antibody 10Gln Val Gln Leu Gln Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30Trp Met Asn Trp Val
Arg Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Gln Ile Trp
Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60Lys Gly Arg
Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr65 70 75 80Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Phe Tyr Ser Cys 85 90
95Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp
100 105 110Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115
1201111PRTHomo sapiens 11Trp Gly Gln Gly Ser Leu Val Thr Val Ser
Ser1 5 1012336DNAArtificial SequenceDescription of Artificial
Sequence Synthetic humanized anti-CD19 antibody, hA19Vk 12gac atc
cag ctg acc cag tct cca tca tct ctg agc gca tct gtt gga 48Asp Ile
Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15gat
agg gtc act atc act tgt aag gcc agc caa agt gtt gat tat gat 96Asp
Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25
30ggt gat agt tat ttg aac tgg tac cag cag att cca ggg aaa gca cct
144Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly Lys Ala Pro
35 40 45aaa ttg ttg atc tac gat gct tcg aat cta gtt tct ggt atc cct
cct 192Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro
Pro 50 55 60cga ttc tct ggc agc gga tct ggg aca gat tac act ttc acc
atc agc 240Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr
Ile Ser65 70 75 80tct ctt caa cca gaa gac att gca aca tat cac tgt
cag caa agt act 288Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr His Cys
Gln Gln Ser Thr 85 90 95gaa gat ccg tgg acg ttc ggt gga ggg acc aag
cta cag atc aaa cgt 336Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys
Leu Gln Ile Lys Arg 100 105 11013112PRTArtificial
SequenceDescription of Artificial Sequence Synthetic humanized
anti-CD19 antibody, hA19Vk 13Asp Ile Gln Leu Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys
Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30Gly Asp Ser Tyr Leu Asn Trp
Tyr Gln Gln Ile Pro Gly Lys Ala Pro 35 40 45Lys Leu Leu Ile Tyr Asp
Ala Ser Asn Leu Val Ser Gly Ile Pro Pro 50 55 60Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser65 70 75 80Ser Leu Gln
Pro Glu Asp Ile Ala Thr Tyr His Cys Gln Gln Ser Thr 85 90 95Glu Asp
Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Gln Ile Lys Arg 100 105
11014372DNAArtificial SequenceDescription of Artificial Sequence
Synthetic humanized anti-CD19 antibody, hA19VH 14cag gtc caa ctg
cag caa tca ggg gct gaa gtc aag aaa cct ggg tca 48Gln Val Gln Leu
Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15tcg gtg aag
gtc tcc tgc aag gct tct ggc tac gct ttc agt agc tac 96Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30tgg atg
aac tgg gtg agg cag agg cct gga cag ggt ctt gag tgg att 144Trp Met
Asn Trp Val Arg Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45gga
cag att tgg cct gga gat ggt gat act aac tac aat gga aag ttc 192Gly
Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55
60aag ggg cgc gcc act att act gcc gac gaa tcc act aat aca gcc tac
240Lys Gly Arg Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala
Tyr65 70 75 80atg gaa ctc agc agc cta cga tct gag gac aca gcg ttc
tat tct tgt 288Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Phe
Tyr Ser Cys 85 90 95gca aga cgg gag act acg acg gta ggc cgt tat tac
tat gct atg gac 336Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr
Tyr Ala Met Asp 100 105 110tac tgg ggc caa ggg acc acg gtc acc gtc
tcc tca 372Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115
12015124PRTArtificial SequenceDescription of Artificial Sequence
Synthetic humanized anti-CD19 antibody, hA19VH 15Gln Val Gln Leu
Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30Trp Met
Asn Trp Val Arg Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly
Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55
60Lys Gly Arg Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr65
70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Phe Tyr Ser
Cys 85 90 95Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala
Met Asp 100 105 110Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 1201615PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 16Lys Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser
Tyr Leu Asn1 5 10 15177PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 17Asp Ala Ser Asn Leu Val
Ser1 5189PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 18Gln Gln Ser Thr Glu Asp Pro Trp Thr1
5195PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 19Ser Tyr Trp Met Asn1 52017PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 20Gln
Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys1 5 10
15Gly2115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 21Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala
Met Asp Tyr1 5 10 15224PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide linker 22Gly Gly Gly
Ser12315PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide linker 23Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser1 5 10 1524126DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 24atcacttgta aggccagcca
aagtgttgat tatgatggtg atagttattt gaactggtac 60cagcagattc cagggaaagc
acctaaattg ttgatctacg atgcttcgaa tctagtttct 120ggtatc
12625125DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 25tgctgacagt gatatgttgc aatgtcttct ggttgaagag
agctgatggt gaaagtgtaa 60tctgtcccag atccgctgcc agagaatcga ggagggatac
cagaaactag attcgaagca 120tcgta 1252686DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
26tccgacatcc agctgaccca gtctccatca tctctgagcg catctgttgg agatagggtc
60actatcactt gtaaggccag ccaaag 862781DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
27gctccttgag atctgtagct tggtccctcc accgaacgtc cacggatctt cagtactttg
60ctgacagtga tatgttgcaa t 8128137DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 28ctggctacgc tttcagtagc
tactggatga actgggtgag gcagaggcct ggacagggtc 60ttgagtggat tggacagatt
tggcctggag
atggtgatac taactacaat ggaaagttca 120aggggcgcgc cactatt
13729129DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 29cgtagtctcc cgtcttgcac aagaatagaa cgctgtgtcc
tcagatcgta ggctgctgag 60ttccatgtag gctgtattag tggattcgtc ggcagtaata
gtggcgcgcc ccttgaactt 120tccattgta 1293093DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
30caggtccaac tgcagcaatc aggggctgaa gtcaagaaac ctgggtcatc ggtgaaggtc
60tcctgcaagg cttctggcta cgctttcagt agc 933189DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
31tgaggagacg gtgaccgtgg tcccttggcc ccagtagtcc atagcatagt aataacggcc
60taccgtcgta gtctcccgtc ttgcacaag 89
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