U.S. patent application number 17/550288 was filed with the patent office on 2022-06-30 for antibodies against human cd38.
The applicant listed for this patent is GENMAB A/S. Invention is credited to Michel DE WEERS, Paul PARREN, Jan VAN DE WINKEL, Tom VINK, Tim WALSETH.
Application Number | 20220204636 17/550288 |
Document ID | / |
Family ID | 1000006200403 |
Filed Date | 2022-06-30 |
United States Patent
Application |
20220204636 |
Kind Code |
A1 |
DE WEERS; Michel ; et
al. |
June 30, 2022 |
ANTIBODIES AGAINST HUMAN CD38
Abstract
Isolated monoclonal antibodies which bind to human CD38 and
related antibody-based compositions and molecules, are disclosed.
Also disclosed are pharmaceutical compositions comprising the
antibodies and therapeutic and diagnostic methods for using the
antibodies.
Inventors: |
DE WEERS; Michel; (Houten,
NL) ; WALSETH; Tim; (Roseville, MN) ; VAN DE
WINKEL; Jan; (Zeist, NL) ; VINK; Tom; (Alphen
aan den Rijn, NL) ; PARREN; Paul; (Odijk,
NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GENMAB A/S |
Copenhagen V |
|
DK |
|
|
Family ID: |
1000006200403 |
Appl. No.: |
17/550288 |
Filed: |
December 14, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15909540 |
Mar 1, 2018 |
11230604 |
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17550288 |
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14990869 |
Jan 8, 2016 |
9944711 |
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15909540 |
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13702857 |
Dec 17, 2012 |
9249226 |
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PCT/EP2011/059507 |
Jun 8, 2011 |
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14990869 |
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61353082 |
Jun 9, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/2896 20130101;
C07K 2317/734 20130101; A61K 39/3955 20130101; C07K 2317/565
20130101; C07K 2317/732 20130101; C07K 16/2878 20130101; C07K
2317/34 20130101; C07K 2317/33 20130101; C07K 2317/56 20130101;
C07K 2317/21 20130101; C07K 2317/51 20130101; C07K 2317/515
20130101; C07K 2317/75 20130101; G01N 2333/91148 20130101; G01N
33/573 20130101; C07K 2317/76 20130101; A61K 45/06 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 39/395 20060101 A61K039/395; A61K 45/06 20060101
A61K045/06; G01N 33/573 20060101 G01N033/573 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 9, 2010 |
DK |
PA201000498 |
Claims
1-74. (canceled)
75. A method for detecting the presence of CD38 antigen, or a cell
expressing CD38, in a sample comprising: (a) contacting the sample
with an antibody that binds to human CD38 (SEQ ID NO: 52), wherein
the antibody does not bind to a variant of human CD38 wherein Asp
in position 202 has been substituted with Gly to the same degree
that it binds to human CD38 under conditions that allow for
formation of a complex between the antibody and CD38; and (b)
analyzing whether a complex has been formed.
76. The method of claim 75, wherein the sample is a human tissue
sample.
77. The method of claim 75, which is performed in vitro.
78. The method of claim 75, which is performed in vivo.
79. The method of claim 75, wherein the cell expressing CD38 is a
tumor cell.
80. The method of claim 75, wherein the method is used to diagnose
the level of invasive cells in a tissue, wherein the formation of a
complex between the antibody and CD38 correlates with the presence
of invasive cells in the tissue.
81. The method of claim 75, wherein the formation of the complex is
detected using a method selected from the group consisting of:
enzyme linked immunosorbent assay, RIA, FACS assay, plasma
resonance assay, chromatography, tissue immunohistochemistry,
Western blot, and immunoprecipitation.
82. The method of claim 75, wherein the antibody comprises a
detectable label selected form the group consisting of an enzyme,
prosthetic group, fluorescent label, luminescent label, and
radioactive label.
83. The method of claim 75, wherein the formation of the complex is
detected using a secondary antibody comprising a detectable
label.
84. The method of claim 83, wherein the detectable label is
selected from the group consisting of an enzyme, prosthetic group,
fluorescent label, luminescent label, and radioactive label.
85. The method of claim 75, wherein the antibody comprises: (i) a
VH CDR1 having the sequence as set forth in any of the sequences
SEQ ID NOs: 3, 8, 13, 18, and 23, a VH CDR2 having the sequence as
set forth in any of the sequences SEQ ID NOs: 4, 9, 14, 19, and 24,
a VH CDR3 having the sequence as set forth in any of the sequences
SEQ ID NOs: 5, 10, 15, 20, and 25, a VL CDR1 having the sequence as
set forth in any of the sequences SEQ ID NOs: 28, 33, 38, 43, and
48, a VL CDR2 having the sequence as set forth in any of the
sequences SEQ ID NOs: 29, 34, 39, 44, and 49, a VL CDR3 having the
sequence as set forth in any of the sequences SEQ ID NOs: 30, 35,
40, 45, and 50, (ii) a VH CDR1 having the sequence as set forth in
SEQ ID NO: 3, a VH CDR2 having the sequence as set forth in SEQ ID
NO: 4, a VH CDR3 having the sequence as set forth in SEQ ID NO: 5,
a VL CDR1 having the sequence as set forth in SEQ ID NO: 28, a VL
CDR2 having the sequence as set forth in SEQ ID NO: 29, and a VL
CDR3 having the sequence as set forth in SEQ ID NO: 30, (iii) a VH
CDR1 having the sequence as set forth in SEQ ID NO: 8, a VH CDR2
having the sequence as set forth in SEQ ID NO: 9, a VH CDR3 having
the sequence as set forth in SEQ ID NO: 10, a VL CDR1 having the
sequence as set forth in SEQ ID NO: 33, a VL CDR2 having the
sequence as set forth in SEQ ID NO: 34, and a VL CDR3 having the
sequence as set forth in SEQ ID NO: 35, (iv) a VH CDR1 having the
sequence as set forth in SEQ ID NO: 13, a VH CDR2 having the
sequence as set forth in SEQ ID NO: 14, a VH CDR3 having the
sequence as set forth in SEQ ID NO: 15, a VL CDR1 having the
sequence as set forth in SEQ ID NO: 38, a VL CDR2 having the
sequence as set forth in SEQ ID NO: 39, and a VL CDR3 having the
sequence as set forth in SEQ ID NO: 40, (v) a VH CDR1 having the
sequence as set forth in SEQ ID NO: 18, a VH CDR2 having the
sequence as set forth in SEQ ID NO: 19, a VH CDR3 having the
sequence as set forth in SEQ ID NO: 20, a VL CDR1 having the
sequence as set forth in SEQ ID NO: 43, a VL CDR2 having the
sequence as set forth in SEQ ID NO: 44, and a VL CDR3 having the
sequence as set forth in SEQ ID NO: 45, (vi) a VH CDR1 having the
sequence as set forth in SEQ ID NO: 23, a VH CDR2 having the
sequence as set forth in SEQ ID NO: 24, a VH CDR3 having the
sequence as set forth in SEQ ID NO: 25, a VL CDR1 having the
sequence as set forth in SEQ ID NO: 48, a VL CDR2 having the
sequence as set forth in SEQ ID NO: 49, and a VL CDR3 having the
sequence as set forth in SEQ ID NO: 50, or (vii) a variant of any
of the antibodies defined above, wherein the variant preferably has
at most 1, 2, or 3 amino acid modifications, more preferably
amino-acid substitutions, such as conservative amino acid
substitutions in one or more of the sequences.
86. The method of claim 75, wherein the antibody comprises a VH
CDR1 having the sequence as set forth in SEQ ID NO: 3, a VH CDR2
having the sequence as set forth in SEQ ID NO: 4, a VH CDR3 having
the sequence as set forth in SEQ ID NO: 5, a VL CDR1 having the
sequence as set forth in SEQ ID NO: 28, a VL CDR2 having the
sequence as set forth in SEQ ID NO: 29, and a VL CDR3 having the
sequence as set forth in SEQ ID NO: 30.
87. The method of claim 75, wherein the antibody comprises a VH
region comprising any one of the sequences as set forth in SEQ ID
NOs: 2, 7, 12, 17, and 22, and a VL region comprising any one of
the sequences as set forth in SEQ ID NOs: 27, 32, 37, 42, and
47.
88. The method of claim 75, wherein the antibody comprises: (i) a
VH region comprising the sequence as set forth in SEQ ID NO: 2, and
a VL region comprising the sequence as set forth in SEQ ID NO: 27,
(ii) a VH region comprising the sequence as set forth in SEQ ID NO:
7, and a VL region comprising the sequence as set forth in SEQ ID
NO: 32, (iii) a VH region comprising the sequence as set forth in
SEQ ID NO: 12, and a VL region comprising the sequence as set forth
in SEQ ID NO: 37, (iv) a VH region comprising the sequence as set
forth in SEQ ID NO: 17, and a VL region comprising the sequence as
set forth in SEQ ID NO: 42, or (v) a VH region comprising the
sequence as set forth in SEQ ID NO: 22, and a VL region comprising
the sequence as set forth in SEQ ID NO: 47.
89. The method of claim 75, wherein the antibody comprises a VH
region comprising the sequence as set forth in SEQ ID NO: 2, and a
VL region comprising the sequence as set forth in SEQ ID NO:
27.
90. The method of claim 75, wherein the antibody possesses the
following binding characteristics: (i) it does not bind to a
variant of human CD38 wherein Asp in position 202 has been
substituted with Gly to the same degree that it binds to human
CD38, (ii) it binds to a variant of human CD38 wherein Gln in
position 272 has been substituted with Arg to the same degree that
it binds to human CD38, and (iii) it binds to a variant of human
CD38 wherein the Ser in position 274 has been substituted with Phe
to the same degree that it binds to human CD38.
91. The method of claim 75, wherein the antibody binds to human
CD38 with a K.sub.D of 10.sup.-8 M or less.
92. The method of claim 75, wherein the antibody is a full length
IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody.
93. The method of claim 75, wherein the antibody is an antibody
fragment or a single-chain antibody.
94. The method of claim 75, wherein the antibody is an effector
function-deficient antibody.
95. The method of claim 75, wherein the antibody is a monovalent
antibody.
96. The method of claim 75, wherein the antibody is a human
monovalent antibody.
97. The method of claim 75, wherein the antibody is a bivalent
antibody.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to antibodies directed to
human CD38 and to uses of such antibodies, in particular
therapeutic uses.
BACKGROUND OF THE INVENTION
[0002] CD38 is a type II transmembrane glycoprotein which is
normally found on hemopoietic cells and in solid tissues. With
regard to hemopoietic cells, the majority of medullary thymocytes
are CD38.sup.+, resting and circulating T- and B-cells are
CD38.sup.- and activated cells are CD38.sup.+. CD38 is also
expressed on approximately 80% of resting NK cells and monocytes
and on lymph node germinal center lymphoblasts, plasma B cells and
some intrafollicular cells. CD38 can also be expressed by dendritic
cells. A significant proportion of normal bone marrow cells,
particular precursor cells, express CD38. In addition, 50-80% of
umbilical cord blood cells is CD38.sup.+ and remains so in human
blood for the first two to three years of life. In addition to
lymphoid precursor cells, CD38 is also expressed on erythrocytes
and on platelets. With regard to solid tissues, CD38 is expressed
in the gut by intra-epithelial cells and lamina propria
lymphocytes, by Purkinje cells and neurofibrillary tangles in the
brain, by epithelial cells in the prostate, .beta.-cells in the
pancreas, osteoclasts in the bone, retinal cells in the eye, and
sarcolemma of smooth and striated muscle.
[0003] CD38 is also expressed in a variety of malignant
hematological diseases, including multiple myeloma, B-cell chronic
lymphocytic leukemia, B-cell acute lymphocytic leukemia,
Waldenstrom macroglobulinemia, primary systemic amyloidosis,
mantle-cell lymphoma, pro-lymphocytic/myelocytic leukemia, acute
myeloid leukemia, chronic myeloid leukemia, follicular lymphoma,
NK-cell leukemia and plasma-cell leukemia. Expression of CD38 has
been described on epithelial/endothelial cells of different origin,
including glandular epithelium in prostate, islet cells in
pancreas, ductal epithelium in glands, including parotid gland,
bronchial epithelial cells, cells in testis and ovary and tumor
epithelium in colorectal adenocarcinoma. Other diseases, where CD38
expression could be involved, include, e.g. broncho-epithelial
carcinomas of the lung, breast cancer (evolving from malignant
proliferation of epithelial lining in ducts and lobules of the
breast), pancreatic tumors, evolving from the b-cells
(insulinomas), tumors evolving from epithelium in the gut (e.g.
adenocarcinoma and squamous cell carcinoma), carcinoma in the
prostate gland, seminomas in testis and ovarian cancers. In CNS,
neuroblastomas express CD38.
[0004] Other disclosures also suggest the role of CD38 in
autoimmunity such as Graves disease and thyroiditis (Antonelli A,
et. al., Clin. Exp. Immunol. 126, 426-431, 2001), and type 1 and 2
Diabetes (Mallone R and Perin P C, Diabetes Metab Res Rev 2006; 22:
284-294) and inflammation of airway smooth muscle cells during
asthma (Desphande et al. 2004 am J Respir Cell Mol Biol 31:
36-42)
[0005] CD38 is a multifunctional protein. Functions ascribed to
CD38 include both receptor mediation in adhesion and signaling
events and (ecto-) enzymatic activity. As an ectoenzyme, CD38 uses
NAD.sup.+ as substrate for the formation of cyclic ADP-ribose
(cADPR) and ADPR, but also of nicotinamide and nicotinic
acid-adenine dinucleotide phosphate (NAADP). cADPR has been shown
to act as second messenger for Ca.sup.2+ mobilization from the
endoplasmatic reticulum. The CD38/cyclic ADP ribose system: 1) in
lung, contributes to airway smooth muscle tone and responsiveness
through its effects on agonist induced elevation of intra-cellular
Ca.sup.2+ (Desphande et al. 2005 Am J physiol Lung cell Mol Physiol
288: L773-L788), 2) regulates migration of neutrophil chemotaxis to
bacterial chemoattractants, migration of DC precursors from blood
to peripheral sites and migration of mature DCs from sites of
inflammation to lymph nodes (Partida-Sanchez et al. Nat Med 7:
1209-121, 2001; Morita et al. 2008 J Pharmacol Sci. 2008 March;
106(3):492-504; Partida-Sanchez et al. Immunity 20: 279-291, 2004),
3) is involved in astrocyte calcium signaling which has
implications for neuroinflammation and HIV-1-associated dementia
(Banerjee S. et. al., J. Neurimmune Pharmacol., 3, 154-164 (2008)),
4) regulates Fc.gamma.R-mediated phagocytosis in murine macrophages
(Song E., et. al., Biochem. and Biophys. Res. Comm., 367, 156-161,
(2008), 5) is linked to insulin secretion Okamoto, Molecular and
Cellular Biochemistry, 193, 115-118, 1999 and 6) has a key role in
neuropeptide release and regulating maternal and social behaviors
(Jin D et al. Nature 446: 41-45, 2007). In addition to signaling
via Ca.sup.2+, CD38 signaling occurs via cross-talk with
antigen-receptor complexes on T and B cells or other types of
receptor complexes, e.g. MHC molecules, and is in this way involved
in several cellular responses, but also in switching and secretion
of IgG1.
[0006] Several anti-CD38 antibodies are described in the
literature, for instance in Lande R, et al., Cell Immunol. 220(1),
30-8 (2002), Ausiello C M, et al., Tissue Antigens. 56(6), 539-47
(2000), and Cotner T, et al., Int J Immunopharmacol. 3(3), 255-68
(1981). Antibody binding to CD38 can have different effects on the
functions of CD38. For instance, mouse anti-CD38 antibody IB4 has
been shown to induce T cell activation as indicated by Ca.sup.2+
mobilization in Jurkat cells (Zubiaur M, et al., J Immunol. 159(1),
193-205 (1997), to induce significant proliferation of peripheral
blood mononuclear cells (PBMCs), to induce release of significant
IL-6 levels and to induce release of detectable IFN-.gamma. levels
(Lande, Zubiaur Morra, Ansiello supra). Hara-Yokoyama et al. Int
Immunopharmacol 8, 59-70 (2008) described one anti-mouse CD38
antibody (CS/2) which inhibits the NAD.sup.+ glycohydrolase
activity of CD38 and another anti-mouse CD38 antibody (clone 90)
which stimulates the NAD.sup.+ glycohydrolase activity of an
isolated extracellular domain of CD38, but has little effect on the
NAD.sup.+ glycohydrolase activity of cell-surface CD38. As it can
be seen from data presented below, the antibodies of the present
invention provide activity on the surface of CD38 positive
cells.
[0007] WO2006099875 (Genmab) describes several human anti-CD38
antibodies, including 003 and 005. Antibody 005 was shown to
inhibit the production of cGDPR from NGD.sup.+ by CD38.
[0008] In view of the multiple functions of human CD38, there is a
need for new therapeutic antibodies that more specifically modulate
particular functions of CD38.
SUMMARY OF THE INVENTION
[0009] The present invention provides a new class of anti-CD38
antibodies which through interacting with particular amino acids of
human CD38 have a strong stimulating effect on the cADPR hydrolase
activity of CD38 leading to decreased levels of cADPR. Furthermore,
the anti-CD38 antibodies inhibit the ability of CD38 to catalyze
the formation, via a base-exchange reaction, of nicotinic acid
adenine dinucleotide 2'-phosphate (NAADP).
[0010] These antibodies are useful for the treatment of several
diseases, including autoimmune and (chronic) inflammatory diseases,
such as Type 1 and 2 diabetes, thyroiditis, Graves disease,
arthritis, neuroinflammation and asthma.
[0011] Recent scientific work suggests that cADPR synthesized
extracellularly by CD38, may be transported into cells through
nucleoside transporters and then mobilize Ca(2+) through a
FK506-binding protein-dependent process. This process may be
involved in fMLP-induced intracellular Ca(2+) signaling and
migration in human neutrophils (Morita et al. 2008 3 Pharmacol Sci.
2008 March; 106(3):492-504), migration of DC precursors from blood
to peripheral sites and migration of mature DCs from sites of
inflammation to lymph nodes (Partida-Sanchez et al. Immunity 20:
279-291, 2004). Without being bound by any particular theory, the
reduction of cADPR levels obtained by treatment with an antibody of
the present invention may thus reduce migration of neutrophils and
dendritic cells and have anti-inflammatory effects. Accordingly,
while the antibodies of the invention may be useful for a number of
purposes, they may be particularly useful for the treatment of
inflammation, e.g. in connection with autoimmune disease, because
of their unique effects on the enzymatic activities of CD38,
through binding at a particular site on CD38.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 shows cross-block studies of antibodies of the
invention. More particularly, the figure shows the binding of
005-FITC to CHO-CD38 cells treated with excess unlabelled
CD38-specific antibodies 025, 026, 028, 049 and 056.
[0013] FIG. 2 shows binding of the anti-CD38 antibodies of the
invention to wt and mutant CD38. FIG. 2(A) shows binding of the
anti-CD38 antibodies 025, 026, 028 and 049 to wild type (WT) and
mutant (T237A, Q272R, and S274F) CD38. FIG. 2(B) shows binding of
the anti-CD38 antibodies 025, 028 and 049 to wild type (WT) and
mutant (D202G) CD38.
[0014] FIG. 3 shows binding of antibodies of the invention to
Daudi-luc cells and CHO-CD38 cells.
[0015] FIG. 4 shows ADCC mediated lysis of Daudi-luc cells caused
by the anti-CD38 antibodies of the invention and as isotype control
anti-KLH antibody (HuMab-KLH.
[0016] FIG. 5 shows CDC mediated lysis of CHO CD38 cells caused by
the anti-CD38 antibodies of the invention.
[0017] FIG. 6 shows inhibition of cGDPR production by His-tagged
CD38 protein and cellular expressed CD38 in the presence of the
anti-CD38 antibodies of the invention. FIG. 6(A) shows the
percentage inhibition of cGDPR production (by recombinant human
CD38 protein) in the presence of CD38 specific antibodies 025, 026,
028, 049 and 056 (3 .mu.g/mL). FIG. 6(B) shows the effect of the
anti-CD38 antibodies on cGDPR production in time. The anti-CD38
antibodies were used at a final concentration of 10 .mu.g/ml. FIG.
6(C) shows the effect of the anti-CD38 antibodies on cGDPR
production using serial dilutions (0.01-30 .mu.g/mL) of 028 or
isotype control HuMab-KLH. FIG. 6(D) shows the percentage
inhibition of cGDPR production (by cellular expressed CD38
(CHO-CD38 cells)) in the presence of serial dilutions (0.01-30
.mu.g/mL) of 028 or IgG1 isotype control HuMab-KLH.
[0018] FIG. 7 shows the effect of antibody 028 of the invention on
8NH2-cADPR production. Products of each reaction were analyzed by
HPLC. FIG. 7(A) indicates the elution position of the products and
substrates. FIG. 7(B) shows the antibody concentration dependence
on 8NH2-cADPR production. HuMab-KLH (open circles), mAb-028 (closed
circles).
[0019] FIG. 8 shows the effect of antibody 028 of the invention on
cADPR hydrolase and NADa se activity, more particularly the effect
of mAb-028 on cADPR hydrolase (Figure A, left figure, B and C) and
NADse (Figure A, right figure) activity. FIG. 8(A) shows the
results of incubating CD38 recombinant protein with cADPR or NAD in
the presence of 10 .mu.g HuMab-KLH (CD38+10 .mu.g HuMab-KLH), 10
.mu.g Ab 028 (CD38+10 .mu.g Ab 028), or no antibody (CD38 control).
Products of each reaction were analyzed by HPLC. FIG. 8(B) shows
CD38 antibody titration at different concentrations on cADPR
hydrolase activity as analyzed by HPLC. FIG. 8(C) shows the results
of incubating CD38 recombinant protein with .sup.32P-cADPR in the
presence of mAb-003, mAb-028, daratumumab (005), or HuMab-KLH.
Products were analyzed by thin layer chromatography. HuMab-KLH
(open circles), mAb-028 (closed circles).
[0020] FIG. 9 shows the effect of the anti-CD38 antibodies of the
invention on the base-exchange activity of CD38. FIG. 9(A) shows
the effect of the antibodies on NAADP production at the indicated
concentrations. FIG. 9(B) shows the effect of mAb-028 titration on
the rate of NAADP formation.
TABLE-US-00001 [0021] SEQUENCE LIST VH-region SEQ ID VH 028 DNA NO:
1 caggtccaac tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc
tcctgcaagg cttttggagg caccttcagc agctacgcta tcagctgggt gcgacaggcc
cctggacaag ggcttgagtg gatgggaagg atcatccgtt tccttggtat agcaaactac
gcacagaagt tccagggcag agtcacgctt atcgcggaca aatccacgaa cacagcctac
atggagctga gcagcctgag atctgaggac acggccgttt attactgtgc gggggaacct
ggggagcggg accccgatgc tgttgatatc tggggccaag ggacaatggt caccgtctct
tca SEQ ID VH 028 NO: 2 QVQLVQSGAE VKKPGSSVKV SCKAFGGTFS SYAISWVRQA
PGQGLEWMGR IIRFLGIANY AQKFQGRVTL IADKSTNTAY MELSSLRSED TAVYYCAGEP
GERDPDAVDI WGQGTMVTVSS SEQ ID VH 028 CDR1 NO: 3 GGTSFSSYA SEQ ID VH
028 CDR2 NO: 4 IIRFLGIA SEQ ID VH 028 CDR3 NO: 5 AGEPGERDPDAVDI SEQ
ID VH 025 DNA NO: 6 caggtccaactggtgcagtctggggctgaggtga
agaagcctgggtcctcggtgaaggtctcctgca aggcttttggaggcaccttcagcagctatgcta
tcagctgggtacgacaggcccctggacaagggc ttgagtggatgggaaggatcatccgtttccttg
gtaaagcaaatcacgcacagaagttccagggca gagtcacgcttaccgcggacaaatccacgaaca
cagcctacatggagctgagcagcctgagatctg aggacacggccgtttattactgtgcgggggaac
ctggggatcgggaccccgatgctgttgatatct ggggccaagggacaatggtcaccgtctcttcag
SEQ ID VH 025 NO: 7 QVQLVQSGAEVKKPGSSVKVSCKAFGGTFSSYA
ISWVRQAPGQGLEWMGRIIRFLGKANHAQKFQG RVTLTADKSTNTAYMELSSLRSEDTAVYYCAGE
PGDRDPDAVDIWGQGTMVTVSS SEQ ID VH 025 CDR1 NO: 8 GGTFSSYA SEQ ID VH
025 CDR2 NO: 9 IIRFLGKA SEQ ID VH 025 CDR3 NO: 10 AGEPGDRDPDAVDI
SEQ ID VH 026 DNA NO: 11 caggtccaactggtgcagtctggggctgaggt
gaagaagcctgggtcctcggtgaaggtctcct gcaaggcttttggaggcaccttcagcagttat
gctattagctgggtgcgacaggcccctggaca agggcttgagtggatgggaaggatcatccgtt
tccttggtaaaacaaatcacgcacagaagttc cagggcagagtcacacttaccgcggacaaatc
cacgaacacagcctacatggagctgagcagcc tgagatctgaggacacggccgtttattactgt
gcgggggaacctggggatcgggaccccgatgc tgttgatatctggggccaagggacaatggtca
ccgtctcttcag SEQ ID VH 026 NO: 12 QVQLVQSGAEVKKPGSSVKVSCKAFGGTFSSYA
ISWVRQAPGQGLEWMGRIIRFLGKTNHAQKFQG RVTLTADKSTNTAYMELSSLRSEDTAVYYCAGE
PGDRDPDAVDIWGQGTMVTVSS SEQ ID VH 026 CDR1 NO: 13 GGTFSSYA SEQ ID VH
026 CDR2 NO: 14 IIRFLGKT SEQ ID VH 026 CDR3 NO: 15 AGEPGDRDPDAVDI
SEQ ID VH 049 DNA NO: 16 caggtccagctggtgcagtctggggctgaggtg
atgaagcctgggtcctcggtgaaggtctcctgc aaggcttccggaggcaccttccgcagctatgct
atcagttgggtgcgacaggcccctggacaaggg cttgagtggatgggaaggatcatcgttttcctt
ggtaaaacaaactacgcacagaagttccagggc agagtcacgcttaccgcggacaaatccacgacc
acagcctacatggagctgagcagcctgagatct gaggacacggccgtgtattactgtacgggggaa
cctggggctcgggaccccgacgcttttgatatc tggggccaagggacaatggtcaccgtctcttca
g SEQ ID VH 049 NO: 17 QVQLVQSGAEVMKPGSSVKVSCKASGGTFRSYA
ISWVRQAPGQGLEWMGRIIVFLGKTNYAQKFQG RVTLTADKSTTTAYMELSSLRSEDTAVYYCTGE
PGARDPDAFDIWGQGTMVTVSS SEQ ID VH 049 CDR1 NO: 18 GGTFRSYA SEQ ID VH
049 CDR2 NO: 19 IIVFLGKT SEQ ID VH 049 CDR3 NO: 20 TGEPGARDPDAFDI
SEQ ID VH 056 DNA NO: 21 caggtccagctggtgcagtctggggctgaggtg
aagaagcctgggtcctcggtgaaggtctcctgc aagccttccggaggcaccttcaggagctacgct
atcagctgggtacgacaggcccctggacaaggg cttgagtggatgggaaggatcatcgttttcctt
ggtaaagtaaactacgcacagaggtttcagggc agagtcacgcttaccgcggacaaatccacgacc
acagcctacatggagctgagcagcctgagatct gaggacacggccgtgtattactgtacgggggaa
cctggggctcgggaccccgacgcttttgatatc tggggccaagggacaatggtcaccgtctcttca
g SEQ ID VH 056 NO: 22 QVQLVQSGAEVKKPGSSVKVSCKPSGGTFRSYA
ISWVRQAPGQGLEWMGRIIVFLGKVNYAQRFQG RVTLTADKSTTTAYMELSSLRSEDTAVYYCTGE
PGARDPDAFDIWGQGTMVTVSS SEQ ID VH 056 CDR1 NO: 23 GGTFRSYA SEQ ID VH
056 CDR2 NO: 24 IIVFLGKV SEQ ID VH 056 CDR3 NO: 25 TGEPGARDPDAFDI
SEQ ID VL 028 DNA NO: 26 gacatccagatgacccagtctccatcctcactg
tctgcatctgtaggagacagagtcaccatcact tgtcgggcgagtcagggtattcgcagctggtta
gcctggtatcagcagaaaccagagaaagcccct aagtccctgatctatgctgcatccagtttgcaa
agtggggtcccatcaaggttcagcggcagtgga tctgggacagatttcactctcaccatcagcagc
ctgcagcctgaagattttgcaacttattactgc caacagtataatagttacccgctcactttcggc
ggagggaccaaggtggagatcaaa SEQ ID VL 028 NO: 27
DIQMTQSPSSLSASVGDRVTITCRASQGIRSWL AWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSG
SGTDFTLTISSLQPEDFATYYCQQYNSYPLTFG GGTKVEIK SEQ ID VL 028 CDR1 NO:
28 GGIRSW SEQ ID VL 028 CDR2 NO: 29 AAS SEQ ID VL 028 CDR3 NO: 30
QQYNSYPLT SEQ ID VL 025 DNA NO: 31
gacatccagatgacccagtctccatcctcactg tctgcatctgtaggagacagagtcaccatcact
tgtcgggcgagtcagggtattcgcagctggtta gcctggtatcagcagaaaccagagaaagcccct
aagtccctgatctatgctgcatccagtttgcaa agtggggtcccatcaaggttcagcggcagtgga
tctgggacagatttcactctcaccatcagcagc ctgcagcctgaagattttgcaacttattactgc
caacagtataatagttacccgctcactttcggc ggagggaccaaggtggagatcaaac SEQ ID
VL 025 NO: 32 DIQMTQSPSSLSASVGDRVTITCRASQGIRSWL
AWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQYNSYPLTFG
GGTKVEIK SEQ ID VL 025 CDR1 NO: 33 QGIRSW SEQ ID VL 025 CDR2 NO: 34
AAS SEQ ID VL 025 CDR3 NO: 35 QQYNSYPLT SEQ ID VL 026 DNA NO: 36
gacatccagatgacccagtctccatcctcactg tctgcatctgtaggagacagagtcaccatcact
tgtcgggcgagtcagggtattcgcagctggtta gcctggtatcagcagaaaccagagaaagcccct
aagtccctgatctatgctgcatccagtttgcaa agtggggtcccatcaaggttcagcggcagtgga
tctgggacagatttcactctcaccatcagcagc ctgcagcctgaagattttgcaacttattactgc
caacagtataatagttacccgctcactttcggc ggagggaccaaggtggagatcaaac SEQ ID
VL 026 NO: 37 DIQMTQSPSSLSASVGDRVnTCRASQGIRSWLA
WYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQYNSYPLTFGG
GTKVEIK SEQ ID VL 026 CDR1 NO: 38 QGIRSW SEQ ID VL 026 CDR2 NO: 39
AAS SEQ ID VL 026 CDR3 NO: 40 QQYNSYPLT SEQ ID VL 049 DNA NO: 41
gacatccagatgacccagtctccatcctcactg tctgcatctgtaggagacagagtcaccatcact
tgtcgggcgagtcagggtattcgcagctggtta gcctggtatcagcagaaaccagagaaagcccct
aagtccctgatctatgctgcatccagtttgcaa agtggggtcccatcaaggttcagcggcagtgga
tctgggacagatttcactctcaccatcagcagc ctgcagcctgaagattttgcaacttattactgc
caacagtataataattatccgctcactttcggc ggagggaccaaggtggagatcaaac SEQ ID
VL 049 NO: 42 DIQMTQSPSSLSASVGDRVTITCRASQGIRSWL
AWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQYNNYPLTFG
GGTKVEIK SEQ ID VL 049 CDR1
NO: 43 QGIRSW SEQ ID VL 049 CDR2 NO: 44 AAS SEQ ID VL 049 CDR3 NO:
45 QQYNNYPLT SEQ ID VL 056 DNA NO: 46
gacatccagatgacccagtctccatcctcactg tctgcatctgtaggagacagagtcaccatcact
tgtcgggcgagtcagggtattcgcagctggtta gcctggtatcagcagaaaccagagaaagcccct
aagtccctgatctatgctgcatccagtttgcaa agtggggtcccatcaaggttcagcggcagtgga
tctgggacagatttcactctcaccatcagcagc ctgcagcctgaagattttgcaacttattactgc
caacagtataataattatccgctcactttcggc ggagggaccaaggtggagatcaaac SEQ ID
VL 056 NO: 47 DIQMTQSPSSLSASVGDRVTITCRASQGIRSWL
AWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQYNNYPLTFG
GGTKVEIK SEQ ID VL 056 CDR1 NO: 48 QGIRSW SEQ ID VL 056 CDR2 NO: 49
AAS SEQ ID VL056CDR3 NO: 50 QQYNNYPLT SEQ ID Mutant human CD38 NO:
51 MANCEFSPVSGDKPCCRLSRRAQLCLGVSILVL
ILVVVLAVVVPRWRQQWSGPGTTKRFPETVLAR CVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPC
NITEEDYQPLMKLGTQTVPCNKILLWSRIKDLA HQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNT
SKINYQSCPDWRKDCSNNPVSVFWKTVSRRFAE AACGVVHVMLNGSRSKIFDKNSTFGSVEVHNLQ
PEKVQTLEAWVIHGGREDSRDLCQDPTIKELES IISKRNIQFSCKNIYRPDKFLQCVKNPEDSSCT
SEI SEQ ID Human CD38 NO: 52 MANCEFSPVSGDKPCCRLSRRAQLCLGVSILVL
ILVVVLAVVVPRWRQQWSGPGTTKRFPETVLAR CVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPC
NITEEDYQPLMKLGTQTVPCNKILLWSRIKDLA HQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNT
SKINYQSCPDWRKDCSNNPVSVFWKTVSRRFAE AACDVVHVMLNGSRSKIFDKNSTFGSVEVHNLQ
PEKVQTLEAWVIHGGREDSRDLCQDPTIKELES IISKRNIQFSCKNIYRPDKFLQCVKNPEDSSCT
SEI
[0022] CDR regions are indicated according to IMGT.
[0023] The sequence of human CD38 is described in sequence 52. A
mutant of human CD38 wherein S was mutated to F at position 274 was
described in WO2006099875 as SEQ. ID NO: 34, and a mutation wherein
Q was mutated to R at position 272 was described in WO2006099875 as
SEQ. ID NO: 33. A mutant of human CD38 wherein D was mutated to G
at position 202 is described above as SEQ. ID NO: 51.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0024] The term "human CD38" when used herein includes any
variants, isoforms and species homologs of human CD38 (Swissprot:
locus CD38 HUMAN, accession P28907) which are naturally expressed
by cells or are expressed on cells transfected with the human CD38
gene.
[0025] The term "immunoglobulin" refers to a class of structurally
related glycoproteins consisting of two pairs of polypeptide
chains, one pair of light (L) low molecular weight chains and one
pair of heavy (H) chains, all four inter-connected by disulfide
bonds. The structure of immunoglobulins has been well
characterized. See for instance Fundamental Immunology Ch. 7 (Paul,
W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, each heavy
chain typically is comprised of a heavy chain variable region
(abbreviated herein as V.sub.H or VH) and a heavy chain constant
region. The heavy chain constant region typically is comprised of
three domains, C.sub.H1, C.sub.H2, and C.sub.H3. Each light chain
typically is comprised of a light chain variable region
(abbreviated herein as V.sub.L or VL) and a light chain constant
region. The light chain constant region typically is comprised of
one domain, C.sub.L. The V.sub.H and V.sub.L regions may be further
subdivided into regions of hypervariability (or hypervariable
regions which may be hypervariable in sequence and/or form of
structurally defined loops), also termed complementarity
determining regions (CDRs), interspersed with regions that are more
conserved, termed framework regions (FRs). Each V.sub.H and V.sub.L
is typically composed of three CDRs and four FRs, arranged from
amino-terminus to carboxy-terminus in the following order: FR1,
CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol.
Biol. 196, 901-917 (1987)). Typically, the numbering of amino acid
residues in this region is performed by the method described in
Kabat et al., Sequences of Proteins of Immunological Interest, 5th
Ed. Public Health Service, National Institutes of Health, Bethesda,
Md. (1991) (phrases such as variable domain residue numbering as in
Kabat or according to Kabat herein refer to this numbering system
for heavy chain variable domains or light chain variable domains).
Using this numbering system, the actual linear amino acid sequence
of a peptide may contain fewer or additional amino acids
corresponding to a shortening of, or insertion into, a FR or CDR of
the variable domain. For example, a heavy chain variable domain may
include a single amino acid insert (residue 52a according to Kabat)
after residue 52 of V.sub.H CDR2 and inserted residues (for
instance residues 82a, 82b, and 82c, etc. according to Kabat) after
heavy chain FR residue 82. The Kabat numbering of residues may be
determined for a given antibody by alignment at regions of homology
of the sequence of the antibody with a "standard" Kabat numbered
sequence.
[0026] The term "antibody" (Ab) in the context of the present
invention refers to an immunoglobulin molecule, a fragment of an
immunoglobulin molecule, or a derivative of either thereof, which
has the ability to specifically bind to an antigen under typical
physiological conditions with a half life of significant periods of
time, such as at least about 30 minutes, at least about 45 minutes,
at least about one hour, at least about two hours, at least about
four hours, at least about 8 hours, at least about 12 hours, about
24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or
more days, etc., or any other relevant functionally-defined period
(such as a time sufficient to induce, promote, enhance, and/or
modulate a physiological response associated with antibody binding
to the antigen and/or time sufficient for the antibody to recruit
an Fc-mediated effector activity). The variable regions of the
heavy and light chains of the immunoglobulin molecule contain a
binding domain that interacts with an antigen. The constant regions
of the antibodies (Abs) may mediate the binding of the
immunoglobulin to host tissues or factors, including various cells
of the immune system (such as effector cells) and components of the
complement system such as C1q, the first component in the classical
pathway of complement activation. An anti-CD38 antibody may also be
a bispecific antibody, diabody, or similar molecule (see for
instance PNAS USA 90(14), 6444-8 (1993) for a description of
diabodies). Indeed, bispecific antibodies, diabodies, and the like,
provided by the present invention may bind any suitable target in
addition to a portion of CD38. As indicated above, the term
antibody herein, unless otherwise stated or clearly contradicted by
context, includes fragments of an antibody that retain the ability
to specifically bind to the antigen. It has been shown that the
antigen-binding function of an antibody may be performed by
fragments of a full-length antibody. Examples of binding fragments
encompassed within the term "antibody" include (i) a Fab' or Fab
fragment, a monovalent fragment consisting of the V.sub.L, V.sub.H,
C.sub.L and C.sub.H1 domains, or a monovalent antibody as described
in WO2007059782 (Genmab); (ii) F(ab').sub.2 fragments, bivalent
fragments comprising two Fab fragments linked by a disulfide bridge
at the hinge region; (iii) a Fd fragment consisting essentially of
the V.sub.H and C.sub.H1 domains; (iv) a Fv fragment consisting
essentially of the V.sub.L and V.sub.H domains of a single arm of
an antibody, (v) a dAb fragment (Ward et al., Nature 341, 544-546
(1989)), which consists essentially of a VH domain and also called
domain antibodies (Holt et al; Trends Biotechnol. 2003 November;
21(11):484-90); (vi) camelid or nanobodies (Revets et al; Expert
Opin Biol Ther. 2005 January; 5(1):111-24) and (vii) an isolated
complementarity determining region (CDR). Furthermore, although the
two domains of the Fv fragment, V.sub.L and V.sub.H, are coded for
by separate genes, they may be joined, using recombinant methods,
by a synthetic linker that enables them to be made as a single
protein chain in which the V.sub.L and V.sub.H regions pair to form
monovalent molecules (known as single chain antibodies or single
chain Fv (scFv), see for instance Bird et al., Science 242, 423-426
(1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such
single chain antibodies are encompassed within the term antibody
unless otherwise noted or clearly indicated by context. Although
such fragments are generally included within the meaning of
antibody, they collectively and each independently are unique
features of the present invention, exhibiting different biological
properties and utility. These and other useful antibody fragments
in the context of the present invention are discussed further
herein. It also should be understood that the term antibody, unless
specified otherwise, also includes polyclonal antibodies,
monoclonal antibodies (mAbs), antibody-like polypeptides, such as
chimeric antibodies and humanized antibodies, and antibody
fragments retaining the ability to specifically bind to the antigen
(antigen-binding fragments) provided by any known technique, such
as enzymatic cleavage, peptide synthesis, and recombinant
techniques. An antibody as generated can possess any isotype. As
used herein, "isotype" refers to the immunoglobulin class (for
instance IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) that is
encoded by heavy chain constant region genes. An "anti-CD38
antibody" is an antibody which binds to the antigen CD38.
[0027] The term "human antibody", as used herein, is intended to
include antibodies having variable and constant regions derived
from human germline immunoglobulin sequences. The human antibodies
of the invention may include amino acid residues not encoded by
human germline immunoglobulin sequences (e.g., mutations introduced
by random or site-specific mutagenesis in vitro or by somatic
mutation in vivo). However, the term "human antibody", as used
herein, is not intended to include antibodies in which CDR
sequences derived from the germline of another mammalian species,
such as a mouse, have been grafted onto human framework
sequences.
[0028] In a preferred embodiment, the antibody of the invention is
isolated. An "isolated antibody," as used herein, is intended to
refer to an antibody which is substantially free of other
antibodies having different antigenic specificities (for instance
an isolated antibody that specifically binds to CD38 is
substantially free of antibodies that specifically bind antigens
other than CD38). An isolated antibody that specifically binds to
an epitope, isoform or variant of human CD38 may, however, have
cross-reactivity to other related antigens, for instance from other
species (such as CD38 species homologs). Moreover, an isolated
antibody may be substantially free of other cellular material
and/or chemicals. In one embodiment of the present invention, a
combination of "isolated" monoclonal antibodies having different
specificities is combined in a well-defined composition.
[0029] The terms "monoclonal antibody" or "monoclonal antibody
composition" as used herein refer to a preparation of antibody
molecules of single molecular composition. A monoclonal antibody
composition displays a single binding specificity and affinity for
a particular epitope. Accordingly, the term "human monoclonal
antibody" refers to antibodies displaying a single binding
specificity which have variable and constant regions derived from
human germline immunoglobulin sequences. The human monoclonal
antibodies may be generated by a hybridoma which includes a B cell
obtained from a transgenic or transchromosomal nonhuman animal,
such as a transgenic mouse, having a genome comprising a human
heavy chain transgene and a light chain transgene, fused to an
immortalized cell.
[0030] As used herein, the term "binding" in the context of the
binding of an antibody to a predetermined antigen typically is a
binding with an affinity corresponding to a K.sub.D of about
10.sup.-7 M or less, such as about 10.sup.-8 M or less, such as
about 10.sup.-9 M or less, about 10.sup.-10 M or less, or about
10.sup.-11 M or even less when determined by for instance surface
plasmon resonance (SPR) technology in a BIAcore 3000 instrument
using the antigen as the ligand and the antibody as the analyte,
and binds to the predetermined antigen with an affinity
corresponding to a K.sub.D that is at least ten-fold lower, such as
at least 100 fold lower, for instance at least 1,000 fold lower,
such as at least 10,000 fold lower, for instance at least 100,000
fold lower than its affinity for binding to a non-specific antigen
(e.g., BSA, casein) other than the predetermined antigen or a
closely-related antigen. The amount with which the affinity is
lower is dependent on the K.sub.D of the antibody, so that when the
K.sub.D of the antibody is very low (that is, the antibody is
highly specific), then the amount with which the affinity for the
antigen is lower than the affinity for a non-specific antigen may
be at least 10,000 fold.
[0031] The term "k.sub.d" (sec.sup.-1), as used herein, refers to
the dissociation rate constant of a particular antibody-antigen
interaction. Said value is also referred to as the k.sub.off
value.
[0032] The term "k.sub.a" (M.sup.-1.times.sec.sup.-1), as used
herein, refers to the association rate constant of a particular
antibody-antigen interaction.
[0033] The term "K.sub.D" (M), as used herein, refers to the
dissociation equilibrium constant of a particular antibody-antigen
interaction.
[0034] The term "K.sub.A" (M.sup.-1), as used herein, refers to the
association equilibrium constant of a particular antibody-antigen
interaction and is obtained by dividing the k.sub.a by the
k.sub.d.
[0035] The antibodies of the present invention have an effect on
enzymatic systems as described in the examples section. The
antibodies are described by stimulatory effects or inhibitory
effects on different parameters. The stimulatory and inhibitory
effects may be measured as disclosed in the examples herein.
[0036] An antibody as described and claimed herein may also be a
functional variant of any of the specific antibodies described
herein. Such a variant antibody is an antibody that differs from a
specific antibody described herein by one or more suitable amino
acid residue alterations, that is substitutions, deletions,
insertions, or terminal sequence additions, for instance in the
constant domain, and/or the variable regions (or any one or more
CDRs thereof) in a single variant antibody. A functional variant of
a V.sub.L, V.sub.H, or CDR region used in the context of an
anti-CD38 antibody still allows the antibody to retain at least a
substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95%
or more) of the affinity/avidity and/or the specificity/selectivity
of the parent antibody and in some cases such an anti-CD38 antibody
may be associated with greater affinity, selectivity and/or
specificity than the parent antibody.
[0037] Such functional variants typically retain significant
sequence identity to the parent antibody. The percent identity
between two sequences is a function of the number of identical
positions shared by the sequences (i.e., % homology=# of identical
positions/total # of positions.times.100), taking into account the
number of gaps, and the length of each gap, which need to be
introduced for optimal alignment of the two sequences. The
comparison of sequences and determination of percent identity
between two sequences may be accomplished using a mathematical
algorithm, as described in the non-limiting examples below.
[0038] The percent identity between two nucleotide sequences may be
determined using the GAP program in the GCG software package
(available at http://www.gcg.com), using a NWSgapdna.CMP matrix and
a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2,
3, 4, 5, or 6. The percent identity between two nucleotide or amino
acid sequences may also be determined using the algorithm of E.
Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988)) which
has been incorporated into the ALIGN program (version 2.0), using a
PAM120 weight residue table, a gap length penalty of 12 and a gap
penalty of 4. In addition, the percent identity between two amino
acid sequences may be determined using the Needleman and Wunsch, J.
Mol. Biol. 48, 444-453 (1970)) algorithm which has been
incorporated into the GAP program in the GCG software package
(available at http://www.gcg.com), using either a Blossum 62 matrix
or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4
and a length weight of 1, 2, 3, 4, 5, or 6.
[0039] The sequence of CDR variants may differ from the sequence of
the CDR of the parent antibody sequences through mostly
conservative substitutions; for instance at least about 35%, about
50% or more, about 60% or more, about 70% or more, about 75% or
more, about 80% or more, about 85% or more, about 90% or more,
about 95% or more (e.g., about 65-99%) of the substitutions in the
variant are conservative amino acid residue replacements. In the
context of the present invention, conservative substitutions may be
defined by substitutions within the classes of amino acids
reflected in one or more of the following three tables:
[0040] Amino Acid Residue Classes for Conservative
Substitutions
TABLE-US-00002 Acidic Residues Asp (D) and Glu (E) Basic Residues
Lys (K), Arg (R), and His (H) Hydrophilic Uncharged Residues Ser
(S), Thr (T), Asn (N), and Gln (Q) Aliphatic Uncharged Residues Gly
(G), Ala (A), Val (V), Leu (L), and Ile (I) Non-polar Uncharged
Residues Cys (C), Met (M), and Pro (P) Aromatic Residues Phe (F),
Tyr (Y), and Trp (W)
[0041] Alternative Conservative Amino Acid Residue Substitution
Classes
TABLE-US-00003 1 A S T 2 D E 3 N Q 4 R K 5 I L M 6 F Y W
[0042] Alternative Physical and Functional Classifications of Amino
Acid Residues
TABLE-US-00004 Alcohol group-containing residues S and T Aliphatic
residues I, L, V, and M Cycloalkenyl-associated residues F, H, W,
and Y Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W, and
Y Negatively charged residues D and E Polar residues C, D, E, H, K,
N, Q, R, S, and T Positively charged residues H, K, and R Small
residues A, C, D, G, N, P, S, T, and V Very small residues A, G,
and S Residues involved in turn A, C, D, E, G, H, K, N, Q, R, S, P,
and formation T Flexible residues Q, T, K, S, G, P, D, E, and R
[0043] More conservative substitutions groupings include:
valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,
alanine-valine, and asparagine-glutamine.
[0044] Additional groups of amino acids may also be formulated
using the principles described in, e.g., Creighton (1984) Proteins:
Structure and Molecular Properties (2d Ed. 1993), W.H. Freeman and
Company.
[0045] As explained above, typically, amino acid sequence
alterations, desirably do not substantially change the structural
characteristics of the parent sequence (e.g., a replacement amino
acid should not tend to disrupt secondary structure that
characterizes the function of the parent sequence), but may be
associated with advantageous properties, such as changing the
functional or pharmacokinetic properties of the antibodies, for
example increasing the half-life, altering the immunogenicity,
providing a site for covalent or non-covalent binding to another
molecule, reducing susceptibility to proteolysis, reducing
susceptibility to oxidation, or altering the glycosylation
pattern.
[0046] Examples of functional properties of antibodies, which may
be altered or retained in variant anti-CD38 antibodies of the
present invention compared to antibodies of prior art are for
example:
(1) high affinity binding to CD38 and/or (2) binding to transfected
cells, e.g. CHO or HEK293 cells expressing CD38 and/or (3)
induction of CDC and/or (4) induction of ADCC and/or (5) alteration
of enzymatic activity and/or (6) induction of apoptosis after
secondary cross-linking and/or (7) phagocytosis
[0047] The term "epitope" means a protein determinant capable of
specific binding to an antibody. Epitopes usually consist of
surface groupings of molecules such as amino acids or sugar side
chains and usually have specific three dimensional structural
characteristics, as well as specific charge characteristics.
Conformational and nonconformational epitopes are distinguished in
that the binding to the former but not the latter is lost in the
presence of denaturing solvents. The epitope may comprise amino
acid residues directly involved in the binding (also called
immunodominant component of the epitope) and other amino acid
residues, which are not directly involved in the binding, such as
amino acid residues which are effectively blocked by the
specifically antigen binding peptide (in other words, the amino
acid residue is within the footprint of the specifically antigen
binding peptide).
[0048] As used herein, a human antibody is "derived from" a
particular germline sequence if the antibody is obtained from a
system using human immunoglobulin sequences, for instance by
immunizing a transgenic mouse carrying human immunoglobulin genes
or by screening a human immunoglobulin gene library, and wherein
the selected human antibody is at least 90%, such as at least 95%,
for instance at least 96%, such as at least 97%, for instance at
least 98%, or such as at least 99% identical in amino acid sequence
to the amino acid sequence encoded by the germline immunoglobulin
gene. Typically, outside the heavy chain CDR3, a human antibody
derived from a particular human germline sequence will display no
more than 20 amino acid differences, e.g. no more than 10 amino
acid differences, such as no more than 5, for instance no more than
4, 3, 2, or 1 amino acid difference from the amino acid sequence
encoded by the germline immunoglobulin gene.
[0049] As used herein, the term "inhibits growth" (e.g. referring
to cells, such as tumor cells) is intended to include any
measurable decrease in the cell growth when contacted with an
anti-CD38 antibody as compared to the growth of the same cells not
in contact with an anti-CD38 antibody, e.g., the inhibition of
growth of a cell culture by at least about 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, 99%, or 100%. Such a decrease in cell growth
can occur by a variety of mechanisms, e.g. effector cell
phagocytosis, ADCC, CDC, and/or apoptosis.
[0050] The term "bispecific antibody" is intended to include any
antibody which has two different binding specificities. The term
"bispecific antibodies" also includes diabodies (see for instance
Holliger, P. et al., PNAS USA 90, 6444-6448 (1993), Poljak, R. J.
et al., Structure 2, 1121-1123 (1994)).
[0051] An "antibody deficient in effector function" or an
"effector-function-deficient antibody" refers to an antibody which
has a significantly reduced or no ability to activate one or more
immune effector mechanisms, such as complement activation or Fc
receptor binding. Thus, effector-function deficient antibodies have
significantly reduced or no ability to mediate antibody-dependent
cell-mediated cytotoxicity (ADCC) and/or complement-dependent
cytotoxicity (CDC).
[0052] The term "monovalent antibody" means in the context of the
present invention that an antibody molecule is capable of binding a
single molecule of the antigen, and thus is not able of antigen
crosslinking.
[0053] As used herein, the term "effector cell" refers to an immune
cell which is involved in the effector phase of an immune response,
as opposed to the cognitive and activation phases of an immune
response. Exemplary immune cells include a cell of a myeloid or
lymphoid origin, for instance lymphocytes (such as B cells and T
cells including cytolytic T cells (CTLs)), killer cells, natural
killer cells, macrophages, monocytes, eosinophils,
polymorphonuclear cells, such as neutrophils, granulocytes, mast
cells, and basophils. Some effector cells express specific Fc
receptors and carry out specific immune functions. In some
embodiments, an effector cell is capable of inducing
antibody-dependent cellular cytotoxicity (ADCC), such as a natural
killer cell, capable of inducing ADCC. For example, monocytes,
macrophages, which express FcR are involved in specific killing of
target cells and presenting antigens to other components of the
immune system, or binding to cells that present antigens. In some
embodiments, an effector cell may phagocytose a target antigen or
target cell.
[0054] The term "vector," as used herein, is intended to refer to a
nucleic acid molecule capable of transporting another nucleic acid
to which it has been linked. Various types of vectors are
well-known in the art. One type of vector is a plasmid.
[0055] The term "recombinant host cell" (or simply "host cell"), as
used herein, is intended to refer to a cell into which an
expression vector has been introduced. It should be understood that
such terms are intended to refer not only to the particular subject
cell, but also to the progeny of such a cell. Because certain
modifications may occur in succeeding generations due to either
mutation or environmental influences, such progeny may not, in
fact, be identical to the parent cell, but are still included
within the scope of the term "host cell" as used herein.
Recombinant host cells include, for example, transfectomas, such as
CHO cells, HEK293 cells, NS/0 cells, and lymphocytic cells.
[0056] The term "transgenic non-human animal" refers to a non-human
animal having a genome comprising one or more human heavy and/or
light chain transgenes or transchromosomes (either integrated or
non-integrated into the animal's natural genomic DNA) and which is
capable of expressing fully human antibodies. For example, a
transgenic mouse can have a human light chain transgene and either
a human heavy chain transgene or human heavy chain transchromosome,
such that the mouse produces human anti-CD38 antibodies when
immunized with CD38 antigen and/or cells expressing CD38. The human
heavy chain transgene may be integrated into the chromosomal DNA of
the mouse, as is the case for transgenic mice, for instance HuMAb
mice, such as HCo7 or HCo12 mice, or the human heavy chain
transgene may be maintained extrachromosomally, as is the case for
transchromosomal KM mice as described in WO02/43478. Such
transgenic and transchromosomal mice (collectively referred to
herein as "transgenic mice") are capable of producing multiple
isotypes of human monoclonal antibodies to a given antigen (such as
IgG, IgA, IgM, IgD and/or IgE) by undergoing V-D-J recombination
and isotype switching. Transgenic, nonhuman animal can also be used
for production of antibodies against a specific antigen by
introducing genes encoding such specific antibody, for example by
operatively linking the genes to a gene which is expressed in the
milk of the animal.
[0057] The terms "B-cell neoplasms" or "mature B-cell neoplasms" in
the context of the present invention include small lymphocytic
lymphoma, B-cell prolymphocytic lymphoma, B-cell chronic
lymphocytic leukemia, mantle cell lymphoma, Burkitt's lymphoma,
follicular lymphoma, diffuse large B-cell lymphoma, multiple
myeloma, lymphoplasmacytic lymphoma, splenic margina zone lymphoma,
plasma cell neoplasms, such as plasma cell myeloma, plasmacytoma,
monoclonal immunoglobulin deposition disease, heavy chain disease,
MALT lymphoma, nodal marginal B cell lymphoma, intravascular large
B cell lymphoma, primary effusion lymphoma, lymphomatoid
granulomatosis, non-Hodgkins lymphoma, Hodgkins lymphoma, hairy
cell leukemia, primary effusion lymphoma and AIDS-related
non-Hodgkins lymphoma.
[0058] "Treatment" refers to the administration of an effective
amount of a therapeutically active compound of the present
invention with the purpose of easing, ameliorating, arresting or
eradicating (curing) symptoms or disease states.
[0059] An "effective amount" refers to an amount effective, at
dosages and for periods of time necessary, to achieve a desired
therapeutic result. A therapeutically effective amount of an
anti-CD38 antibody may vary according to factors such as the
disease state, age, sex, and weight of the individual, and the
ability of the anti-CD38 antibody to elicit a desired response in
the individual. A therapeutically effective amount is also one in
which any toxic or detrimental effects of the antibody or antibody
portion are outweighed by the therapeutically beneficial
effects.
[0060] An "anti-idiotypic" (Id) antibody is an antibody which
recognizes unique determinants generally associated with the
antigen-binding site of an antibody.
Antibodies of the Invention
[0061] The invention relates to an antibody that binds to human
CD38 (SEQ ID NO: 52), wherein the antibody does not bind to a
variant of human CD38 wherein Asp in position 202 has been
substituted with Gly to the same degree that it binds to human
CD38. In one embodiment, the EC50 of the binding of the antibody to
the variant of human CD38 wherein Asp in position 202 has been
substituted with Gly is less than 50%, such as less than 10%, less
than 5%, or less than 1% of the EC50 of the binding of the antibody
to human CD38.
[0062] In one embodiment, the antibody as defined above binds to a
variant of human CD38 wherein Gln in position 272 has been
substituted with Arg to the same degree that it binds to human
CD38. In one embodiment, the EC50 of the binding of the antibody to
the variant of human CD38 wherein Gln in position 272 has been
substituted with Arg is at least 80%, such as at least 90%, such as
at least 95%, such as at least 98% of the EC50 of the binding of
the antibody to human CD38.
[0063] In one embodiment, the antibody as defined in any of the
embodiments above binds to a variant of human CD38 wherein the Ser
in position 274 has been substituted with Phe to the same degree
that it binds to human CD38. In one embodiment, the EC50 of the
binding of the antibody to a variant of human CD38 is at least 75%,
such as at least 80%, such as at least 90%, such as at least 95%,
such as at least 98% of the EC50 of the binding of the antibody to
human CD38.
[0064] In one embodiment, the antibody as defined above possesses
the following binding characteristics: (i) it does not bind to a
variant of human CD38 wherein Asp in position 202 has been
substituted with Gly to the same degree that it binds to human
CD38, (ii) it binds to a variant of human CD38 wherein Gln in
position 272 has been substituted with Arg to the same degree that
it binds to human CD38, (iii) it binds to a variant of human CD38
wherein the Ser in position 274 has been substituted with Phe to
the same degree that it binds to human CD38.
[0065] In one embodiment, the antibody as defined in any of the
embodiments above binds human CD38 and has an inhibitory effect on
the CD38 cyclase activity and a stimulatory effect on the CD38
hydrolase activity as measured in the assays of Example 8, such as
wherein the inhibitory effect is at least 50-66% compared to the
inhibitory effect on the CD38 cyclase activity in the absence of
antibody.
[0066] In one embodiment, the antibody as defined in any of the
embodiments above is encoded by a human heavy chain nucleic acid
comprising a nucleotide sequence in its variable region as set
forth in SEQ ID NO: 1, 6, 11, 16 or 21, and a human light chain
nucleic acid comprising a nucleotide sequence in its variable
region as set forth in SEQ ID NOs: 26, 31, 36, 41 or 46.
[0067] In one embodiment, the antibody as defined in any of the
embodiments above is encoded by a human heavy chain and a human
light chain nucleic acid comprising nucleotide sequences in their
variable regions as set forth in SEQ ID NOs: 1 and 26, 6 and 31, 11
and 36, 16 and 41, or 21 and 46, respectively.
[0068] In one embodiment, the antibody as defined in any of the
embodiments above comprises a VH CDR3 comprising [0069] a) the
sequence as set forth in SEQ ID NOs: 5, 10, 15, 20 or 25, or [0070]
b) a variant of said sequence, such as a variant having at most 1,
2 or 3 amino acid modifications, preferably substitutions, such as
conservative substitutions.
[0071] In one embodiment, the antibody as defined in any of the
embodiments above comprises a VH CDR3 having the sequence set forth
in SEQ ID NOs: 5, 10, 15, 20 or 25, and comprising a VL CDR3 having
the sequence set forth in SEQ ID NO: 30, 35, 40, 45 or 50.
[0072] In one embodiment, the antibody as defined in any of the
embodiments above comprises SEQ ID NO: 5 and SEQ ID NO: 30, or SEQ
ID NO: 10 and SEQ ID NO: 35, or SEQ ID NO: 15 and SEQ ID NO: 40, or
SEQ ID NO: 20 and SEQ ID NO: 45, or SEQ ID NO: 25 and SEQ ID NO: 50
as the VH CDR3 and VL CDR3 respectively.
[0073] In one embodiment, the antibody as defined in any of the
embodiments above comprises
[0074] (i) a VH CDR1 having the sequence as set forth in any of the
sequences SEQ ID NOs: 3, 8, 13, 18 and 23, a VH CDR2 having the
sequence as set forth in any of the sequences SEQ ID NOs: 4, 9, 14,
19 and 24, a VH CDR3 having the sequence as set forth in any of the
sequences SEQ ID NOs: 5, 10, 15, 20 and 25, a VL CDR1 having the
sequence as set forth in any of the sequences SEQ ID NO: 28, 33,
38, 43 and 48, a VL CDR2 having the sequence as set forth in any of
the sequences SEQ ID NOs: 29, 34, 39, 44 and 49, a VL CDR3 having
the sequence as set forth in any of the sequences SEQ ID NOs: 30,
35, 40, 45 and 50,
[0075] (ii) a VH CDR1 having the sequence as set forth in SEQ ID
NO: 3, a VH CDR2 having the sequence as set forth in SEQ ID NOs: 4,
a VH CDR3 having the sequence as set forth in SEQ ID NO: 5, a VL
CDR1 having the sequence as set forth in SEQ ID NO: 28, a VL CDR2
having the sequence as set forth in SEQ ID NO: 29, a VL CDR3 having
the sequence as set forth in SEQ ID NO: 30,
[0076] (iii) a VH CDR1 having the sequence as set forth in SEQ ID
NO: 8, a VH CDR2 having the sequence as set forth in SEQ ID NOs: 9,
a VH CDR3 having the sequence as set forth in SEQ ID NO: 10, a VL
CDR1 having the sequence as set forth in SEQ ID NO 33, a VL CDR2
having the sequence as set forth in SEQ ID NO: 34, a VL CDR3 having
the sequence as set forth in SEQ ID NO: 35,
[0077] (iv) a VH CDR1 having the sequence as set forth in SEQ ID
NO: 13, a VH CDR2 having the sequence as set forth in SEQ ID NO:
14, a VH CDR3 having the sequence as set forth in SEQ ID NO: 15, a
VL CDR1 having the sequence as set forth in SEQ ID NO: 38, a VL
CDR2 having the sequence as set forth in SEQ ID NO: 39, a VL CDR3
having the sequence as set forth in SEQ ID NO: 40,
[0078] (v) a VH CDR1 having the sequence as set forth in SEQ ID NO:
18, a VH CDR2 having the sequence as set forth in SEQ ID NOs: 19, a
VH CDR3 having the sequence as set forth in SEQ ID NO: 20, a VL
CDR1 having the sequence as set forth in SEQ ID NO: 43, a VL CDR2
having the sequence as set forth in SEQ ID NO: 44, a VL CDR3 having
the sequence as set forth in SEQ ID NO: 45,
[0079] (vi) a VH CDR1 having the sequence as set forth in SEQ ID
NO: 23, a VH CDR2 having the sequence as set forth in SEQ ID NOs:
24, a VH CDR3 having the sequence as set forth in SEQ ID NO: 25, a
VL CDR1 having the sequence as set forth in SEQ ID NO 48, a VL CDR2
having the sequence as set forth in SEQ ID NO: 49, a VL CDR3 having
the sequence as set forth in SEQ ID NO: 50, or
[0080] (vii) a variant of any of the antibodies defined above,
wherein said variant preferably has at most 1, 2 or 3 amino acid
modifications, more preferably amino-acid substitutions, such as
conservative amino acid substitutions in one or more of said
sequences.
[0081] In one embodiment, the antibody as defined in any of the
embodiments above comprises a VH region
[0082] (i) comprising the sequence of SEQ ID NOs: 2, 7, 12, 17 or
22, or
[0083] (ii) having at least 80% identity, such as 90%, or 95%, or
97%, or 98%, or 99% or 100% identity to the VH region sequence set
forth in SEQ ID NOs: 2, 7, 12, 17 or 22.
[0084] In one embodiment, the antibody as defined in any of the
embodiments above comprises a VL region
[0085] (i) comprising the sequence of SEQ ID NOs: 27, 32, 37, 42 or
47, or
[0086] (ii) having at least 80% identity, such as 90%, or 95%, or
97%, or 98%, or 99% or 100% identity to a VL region sequence
selected from the group consisting of: SEQ ID NOs: 27, 32, 37, 42
or 47.
[0087] In one embodiment, the antibody as defined in any of the
embodiments above comprises a VH region comprising any of the
sequences of SEQ ID NOs: 2, 7, 12, 17 and 22, and a VL region
comprising any of the sequences of SEQ ID NOs: 27, 32, 37, 42 and
47.
[0088] In one embodiment, the antibody as defined in any of the
embodiments above comprises
[0089] (i) a VH region comprising the sequence as set forth in SEQ
ID NO: 2, and a VL region comprising any the sequence as set forth
in SEQ ID NO: 27,
[0090] (ii) a VH region comprising the sequence as set forth in SEQ
ID NO: 7, and a VL region comprising any the sequence as set forth
in SEQ ID NO: 32,
[0091] (iii) a VH region comprising the sequence as set forth in
SEQ ID NO: 12, and a VL region comprising any the sequence as set
forth in SEQ ID NO: 37,
[0092] (iv) a VH region comprising the sequence as set forth in SEQ
ID NO: 17, and a VL region comprising any the sequence as set forth
in SEQ ID NO: 42, or
[0093] (v) a VH region comprising the sequence as set forth in SEQ
ID NO: 22, and a VL region comprising any the sequence as set forth
in SEQ ID NO: 47.
[0094] In one embodiment, the invention relates to an anti-CD38
antibody which binds to the same epitope on CD38 as an anti-CD38
antibody as described in any one of the embodiments above.
[0095] In one embodiment, the invention relates to an anti-CD38
antibody which has substantially the same specific binding
characteristics for binding human CD38 as described in any one of
the embodiments above.
[0096] In one embodiment, the antibody as defined in any of the
embodiments above is capable of inducing antibody-dependent
cellular cytotoxicity (ADCC), such as in Daudi cells, preferably
with an EC.sub.50 value of 5 nM or less, e.g. 1 nM or less, such as
0.2 nM or less, as determined by the method described in Example 6
herein.
[0097] In one embodiment, the antibody as defined in any of the
embodiments above is not capable of inducing ADCC in Daudi cells
according to the method described in Example 6 herein.
[0098] In one embodiment, the antibody as defined in any of the
embodiments above is not capable of inducing complement-dependent
cytotoxicity (CDC) in CHO-CD38 cells.
[0099] In one embodiment, the antibody as defined in any of the
embodiments above binds to human CD38 with a K.sub.D of 10.sup.-8 M
or less, preferably with a K.sub.D of 10.sup.-9 M or less.
[0100] In one embodiment, the antibody as defined in any of the
embodiments above is a human monovalent antibody.
[0101] In one embodiment, the antibody as defined in any of the
embodiments above is a full length IgG1, IgG2, IgG3, IgG4, IgD,
IgA, IgE, or IgM antibody, such as an IgG1 antibody, preferably an
IgG1,.kappa. antibody or an IgM antibody, preferably an IgM,.kappa.
antibody.
[0102] In one embodiment, the antibody as defined in any of the
embodiments above is an antibody fragment or a single-chain
antibody.
[0103] In one embodiment, the antibody as defined in any of the
embodiments above is an effector-function-deficient antibody, such
as a stabilized human IgG4 antibody.
[0104] In one embodiment, such stabilized IgG4 antibody is an
antibody wherein arginine at position 409 in the heavy chain
constant region of human IgG4 is substituted with lysine,
threonine, methionine, or leucine, preferably lysine. In one
embodiment, such antibody comprises a Lys residue at the position
corresponding to 409 or the CH3 region of the antibody has been
replaced by the CH3 region of human IgG1, of human IgG2 or of human
IgG3. In one embodiment, such antibody does not comprise a
Cys-Pro-Pro-Cys sequence in the hinge region. In another
embodiment, such antibody does comprise a Cys-Pro-Pro-Cys sequence
in the hinge region.
[0105] In one embodiment, the antibody as defined in any of the
embodiments above is a monovalent antibody.
[0106] In one embodiment, such monovalent antibody is constructed
by a method comprising:
[0107] i) providing a nucleic acid construct encoding the light
chain of said monovalent antibody, said construct comprising a
nucleotide sequence encoding the VL region of SEQ ID NO: 27, 32,
37, 42 or 47 and a nucleotide sequence encoding the constant CL
region of an Ig, wherein said nucleotide sequence encoding the VL
region of a selected antigen specific antibody and said nucleotide
sequence encoding the CL region of an Ig are operably linked
together, and wherein, in case of an IgG1 subtype, the nucleotide
sequence encoding the CL region has been modified such that the CL
region does not contain any amino acids capable of forming
disulfide bonds or covalent bonds with other peptides comprising an
identical amino acid sequence of the CL region in the presence of
polyclonal human IgG or when administered to an animal or human
being;
[0108] ii) providing a nucleic acid construct encoding the heavy
chain of said monovalent antibody, said construct comprising a
nucleotide sequence encoding the VH region of SEQ ID NO: 2, 7, 12
17 or 22 and a nucleotide sequence encoding a constant CH region of
a human Ig, wherein the nucleotide sequence encoding the CH region
has been modified such that the region corresponding to the hinge
region and, as required by the Ig subtype, other regions of the CH
region, such as the CH3 region, does not comprise any amino acid
residues which participate in the formation of disulphide bonds or
covalent or stable non-covalent inter-heavy chain bonds with other
peptides comprising an identical amino acid sequence of the CH
region of the human Ig in the presence of polyclonal human IgG or
when administered to an animal human being, wherein said nucleotide
sequence encoding the VH region of a selected antigen specific
antibody and said nucleotide sequence encoding the CH region of
said Ig are operably linked together;
[0109] iii) providing a cell expression system for producing said
monovalent antibody;
[0110] iv) producing said monovalent antibody by co-expressing the
nucleic acid constructs of (i) and (ii) in cells of the cell
expression system of (iii).
[0111] In one embodiment, the CH region comprising the CH2 and CH3
regions has been modified such that the region corresponding to the
hinge region and, if the immunoglobulin is not an IgG4 subtype,
other regions of the CH region, such as the CH3 region, do not
comprise any amino acid residues, which are capable of forming
disulfide bonds with an identical CH region or other covalent or
stable non-covalent inter-heavy chain bonds with an identical CH
region in the presence of polyclonal human IgG.
[0112] In one embodiment, such monovalent antibody is of the IgG4
subtype, but the CH3 region has been modified so that one or more
of the following amino acid substitutions have been made: Thr (T)
in position 366 has been replaced by Ala (A); Leu (L) in position
368 has been replaced by Ala (A); Leu (L) in position 368 has been
replaced by Val (V); Phe (F) in position 405 has been replaced by
Ala (A); Phe (F) in position 405 has been replaced by Leu (L); Tyr
(Y) in position 407 has been replaced by Ala (A); Arg (R) in
position 409 has been replaced by Ala (A).
[0113] In one embodiment, the heavy chain of such monovalent
antibody has been modified such that the entire hinge has been
deleted.
[0114] In one embodiment, the sequence of said monovalent antibody
has been modified so that it does not comprise any acceptor sites
for N-linked glycosylation.
[0115] In one embodiment, the antibody as defined in any of the
embodiments above inhibits the CD38 catalyzed synthesis of cGDPR by
at least 25%, such as at least 30% after 90 minutes at a
concentration of 3 .mu.g/ml as determined by the spectophotometric
method described in Example 8 of the specification.
[0116] In one embodiment, the antibody as defined in any of the
embodiments above inhibits the CD38 catalyzed synthesis of cADPR by
at least 25%, such as at least 30% after 90 minutes at a
concentration of 3 .mu.g/ml as determined by the HPLC method
described in Munshi et al., 3. Biol. Chem. 275, 21566-21571
(2000).
[0117] In one embodiment, the antibody stimulates the hydrolase
activity of CD38 by at least 25%.
[0118] In one embodiment, the antibody stimulates the NAD hydrolase
activity of CD38 by at least 25%.
[0119] In one embodiment, the antibody as defined in any of the
embodiments above stimulates the cADPR-hydrolase activity of CD38
by at least 25%.
[0120] In one embodiment, the antibody as defined in any of the
embodiments above inhibits the ability of CD38 to catalyze the
formation, via a base-exchange reaction, of NAADP with an IC50 of
below 0.5 .mu.g/mL, such as of below 0.2 .mu.g/mL by the method
described in Example 8 of the specification.
[0121] In one embodiment, the invention relates to an antibody drug
conjugate comprising an antibody as defined in any of the
embodiments above, wherein the antibody has been conjugated to a
cytotoxic agent, a radioisotope, or a drug. In one embodiment, the
antibody has been conjugated to an auristatin or a functional
peptide analog or derivate thereof via a linker.
[0122] In one embodiment, the invention relates to a bispecific
antibody comprising an antibody as defined in any of the
embodiments above and a second binding specificity for a human
effector cell or a cancer antigen. In one embodiment, the second
binding specificity is for a human Fc receptor or for a T cell
receptor, such as CD3.
[0123] In one embodiment, the invention relates to an isolated
nucleic acid encoding an antibody as defined in any of the
embodiments above.
[0124] In one embodiment, the invention relates to an expression
vector comprising a nucleotide sequence encoding one or more of the
amino acid sequences as defined in any of the embodiments
above.
[0125] In one embodiment, the expression vector further comprises a
nucleotide sequence encoding the constant region of a light chain,
a heavy chain or both light and heavy chains of a human
antibody.
[0126] In one embodiment, the invention relates to a recombinant
eukaryotic or prokaryotic host cell which produces an antibody as
defined in any of the embodiments above.
[0127] In one embodiment, the invention relates to a pharmaceutical
composition comprising an antibody, an immunoconjugate, a
bispecific antibody, or an expression vector as defined in any of
the embodiments above and a pharmaceutically acceptable
carrier.
[0128] In one embodiment, the invention relates to an antibody as
defined in any of the embodiments above for use as a
medicament.
[0129] In one embodiment, the invention relates to an antibody as
defined in any of the embodiments above for use in inhibiting
growth and/or proliferation, migration or inducing phagocytosis of
a tumor cell expressing CD38.
[0130] In one embodiment, the invention relates to an antibody as
defined in any of the embodiments above for use in treating
rheumatoid arthritis.
[0131] In one embodiment, the invention relates to an antibody as
defined in any of the embodiments above for use in treating a
disorder selected from chronic lymphocytic leukemia (CLL), acute
lymphoblastic leukemia (ALL), acute myelogenous leukemia (adults)
(AML), mantle cell lymphoma, follicular lymphoma, and diffuse large
B-cell lymphoma.
[0132] In one embodiment, the invention relates to an antibody as
defined in any of the embodiments above for use in treating
multiple myeloma.
[0133] In one embodiment, the invention relates to a method for
producing an anti-CD38 antibody as defined in any of the
embodiments above, said method comprising the steps of
[0134] a) culturing a host cell as defined in any of the
embodiments above, and
[0135] b) purifying the anti-CD38 antibody from the culture
media.
[0136] In one embodiment, the invention relates to diagnostic
composition comprising an antibody as defined in any of the
embodiments above.
[0137] In one embodiment, the invention relates to a method for
detecting the presence of CD38 antigen, or a cell expressing CD38,
in a sample comprising: [0138] contacting the sample with an
anti-CD38 antibody as defined in any of the embodiments above under
conditions that allow for formation of a complex between the
antibody or bispecific molecules and CD38; and [0139] analyzing
whether a complex has been formed.
[0140] In one embodiment, the invention relates to a kit for
detecting the presence of CD38 antigen, or a cell expressing CD38,
in a sample comprising an anti-CD38 antibody as defined in any of
the embodiments above and instructions for use of the kit.
[0141] In one embodiment, the invention relates to an
anti-idiotypic antibody which binds to an anti-CD38 antibody as
defined in any of the embodiments above.
[0142] In one embodiment, the invention relates to a method of
inhibiting growth and/or proliferation migration or inducing
phagocytosis of a cell expressing CD38, comprising administration
of an antibody, an immunoconjugate, a bispecific antibody, an
expression vector or a pharmaceutical composition as defined in any
of the embodiments above, such that the growth and/or
proliferation, migration or phagocytosis of the cell is
inhibited.
[0143] In one embodiment, the invention relates to a method of
treating a disease or disorder involving cells expressing CD38 in a
subject, which method administration of an antibody, an
immunoconjugate, a bispecific antibody, an expression vector or a
pharmaceutical composition as defined in any of the embodiments
above to a subject in need thereof.
[0144] In one embodiment, the disease or disorder is rheumatoid
arthritis.
[0145] In another embodiment, the disease or disorder is selected
from chronic lymphocytic leukemia (CLL), acute lymphoblastic
leukemia (ALL), acute myelogenous leukemia (adults) (AML), mantle
cell lymphoma, follicular lymphoma, and diffuse large B-cell
lymphoma.
[0146] In yet another embodiment, the disease or disorder is
multiple myeloma.
[0147] In one embodiment, the method as defined in any of the
embodiments above comprises administration of one or more further
therapeutic agents to the subject, such as one or more further
therapeutic agents are selected from a chemotherapeutic agent, an
anti-inflammatory agent, or an immunosuppressive and/or
immunomodulatory agent. In one embodiment, the one or more further
therapeutic agents are selected from a group consisting of
cisplatin, gefitinib, cetuximab, rituximab, ofatumumab,
bevacizumab, erlotinib, bortezomib, thalidomide, pamidronate,
zoledronic acid, clodronate, risendronate, ibandronate, etidronate,
alendronate, tiludronate, arsenic trioxide, lenalidomide,
dexamethasone, prednisolone, filgrastim, pegfilgrastim,
sargramostim, suberoylanilide hydroxamic acid, and SCIO-469.
[0148] An embodiment of the invention provides an antibody that
binds to human CD38, wherein the antibody does not bind to a
variant of human CD38 wherein Asp in position 202 has been
substituted with Gly.
[0149] An embodiment of the invention provides an antibody
according to the embodiment above, wherein the EC50 of the binding
of the antibody to a variant of human CD38 is less than 50%, such
as less than 10%, less than 5%, or less than 1% of the EC50 of the
binding of the peptide to human CD38.
[0150] An embodiment of the invention provides an antibody
according to any of the above embodiments, wherein the antibody
binds to a variant of human CD38 wherein the Gln in position 272
has been substituted with Arg to the same degree that it binds to
human CD38.
[0151] An embodiment of the invention provides an antibody
according to the above embodiment, wherein the EC50 of the binding
of the antibody to a variant of human CD38 is at least 80%, such as
at least 90%, such as at least 95%, such as at least 98% of the
EC50 of the binding of the peptide to human CD38.
[0152] An embodiment of the invention provides an antibody
according to any of the above embodiments, wherein the antibody
binds to a variant of human CD38 wherein the Ser in position 274
has been substituted with Phe to the same degree that it binds to
human CD38.
[0153] An embodiment of the invention provides an antibody
according to the above embodiment, wherein the EC50 of the binding
of the antibody to a variant of human CD38 is at least 75%, such as
at least 80%, such as at least 90%, such as at least 95%, such as
at least 98% of the EC50 of the binding of the peptide to human
CD38.
[0154] An embodiment of the invention provides an antibody
according to any of the above embodiments, wherein the antibody
possesses the following binding characteristics: (i) it does not
bind to a variant of human CD38 wherein Asp in position 202 has
been substituted with Gly to the same degree that it binds to human
CD38 (ii) it binds to a variant of human CD38 wherein the Gln in
position 272 has been substituted with Arg to the same degree that
it binds to human CD38 (iii) it binds to a variant of human CD38
wherein the Ser in position 274 has been substituted with Phe to
the same degree that it binds to human CD38.
[0155] An embodiment of the invention provides an antibody
according to any of the above embodiments, that binds human CD38
and has an inhibitory effect on the CD38 cyclase activity and a
stimulatory effect on the CD38 hydrolase activity as measured in
the assays of Example 8.
[0156] An embodiment of the invention provides an antibody
according to the above embodiment, wherein the inhibitory effect is
at least 50-66% compared to CD38 alone.
[0157] An embodiment of the invention provides an antibody binding
to human CD38 encoded by a human heavy chain nucleic acids
comprising nucleotide sequences in their variable regions as set
forth the in seq id no.: 1, 6, 11, 16 or 21, and a human light
chain comprising nucleotide sequences in their variable regions as
set forth in seq id no. 26, 31, 36, 41 or 46, and comprising
conservative sequence modifications of the sequences set forth
above.
[0158] An embodiment of the invention provides an antibody
according to the above embodiment, encoded by a human heavy chain
and a human light chain nucleic acids comprising nucleotide
sequences in their variable regions as set forth the in seq id no.:
1 and 26, 6 and 31, 11 and 36, 16 and 41 or 21 and 46,
respectively, and comprising conservative sequence modifications of
the sequences set forth above.
[0159] An embodiment of the invention provides an antibody binding
to human CD38 comprising a VH CDR3 region having [0160] a) the
sequence as set forth in SEQ ID NOs: 5, 10, 15, 20 or 25 30 [0161]
or [0162] b) a variant of said sequence, such as a variant having
at most 1, 2 or 3 amino-acid modifications, preferably
substitutions, such as conservative substitutions.
[0163] An embodiment of the invention provides an antibody binding
to human CD38 comprising a VH CDR3 region having the sequence as
set forth in SEQ ID NOs: 5, 10, 15, 20, 25 or 30 and comprising a
VL CDR3 region having the sequence set forth in SEQ ID NO: 30, 35,
40, 45 or 50;
[0164] An embodiment of the invention provides an antibody binding
to human CD38 comprising a VH CDR3 region having the sequence as
set forth in SEQ ID NO: 5 and a VL CDR3 region comprising SEQ ID
NO: 30, or SEQ ID NO: 10 and SEQ ID NO: 35, or SEQ ID NO: 15 and
SEQ ID NO: 40, or SEQ ID NO: 20 and SEQ ID NO: 45, or SEQ ID NO: 25
and SEQ ID NO: 45, or SEQ ID NO: 30 and SEQ ID NO: 50, as VH CDR3
region and VL CDR3 region respectively.
[0165] An embodiment of the invention provides an anti-CD38
antibody according to any of the above embodiments, wherein the
antibody comprises a VH CDR1 region having the sequence as set
forth in any of the sequences SEQ ID NOs: 3, 8, 13, 18 or 23, a VH
CDR2 region having the sequence as set forth in any of the
sequences SEQ ID NOs: 4, 9, 14, 19 or 24, a VL CDR3 region having
the sequence as set forth in any of the sequences SEQ ID NOs: 30,
35, 40, 45 or 50, and a VH CDR3 region having the sequence as set
forth in SEQ ID NOs: 5, 10, 15, 20 or 25.
[0166] An embodiment of the invention provides an antibody which
binds to CD38, wherein the antibody comprises a VH CDR1 region
having the sequence as set forth in any of the sequences SEQ ID
NOs: 3, 8, 13, 18 or 23, a VH CDR2 region having the sequence as
set forth in any of the sequences SEQ ID NOs: 4, 9, 14, 19 or 24, a
VH CDR3 region having the sequence as set forth in SEQ ID NOs: 5,
10, 15, 20 or 25, a VL CDR1 region as set forth in SEQ ID NOs: 28,
33, 38, 43 or 48, a VL CDR2 region as set forth in SEQ ID NOs: 29,
34, 39, 44 or 49, a VL CDR3 region having the sequence as set forth
in any of the sequences SEQ ID NOs: 30, 35, 40, 45 or 50 or
[0167] a variant of said antibody, wherein said variant preferably
has at most 1, 2 or 3 amino-acid modifications, more preferably
amino-acid substitutions, such as conservative amino-acid
substitutions in said sequences.
[0168] An embodiment of the invention provides an antibody which
binds to CD38, wherein the antibody comprises a VH CDR1 region
having the sequence as set forth in any of the sequences SEQ ID
NOs: 3, 8, 13, 18 or 23, a VH CDR2 region having the sequence as
set forth in any of the sequences SEQ ID NOs: 4, 9, 14, 19 or 24, a
VH CDR3 region having the sequence as set forth in SEQ ID NOs: 5,
10, 15, 20 or 25, a VL CDR1 region as set forth in SEQ ID NOs: 28,
33, 38, 43 or 48, a VL CDR2 region as set forth in SEQ ID NOs: 29,
34, 39, 44 or 49, a VL CDR3 region having the sequence as set forth
in any of the sequences SEQ ID NOs: 30, 35, 40, 45 or 50;
[0169] An embodiment of the invention provides an anti-CD38
antibody according to any of the above embodiments, comprising a VH
having at least 80% identity, such as 90%, or 95%, or 97%, or 98%,
or 99% or 100% identity to the VH region sequence set forth in SEQ
ID NOs: 2, 7, 12, 17, or 22.
[0170] An embodiment of the invention provides an anti-CD38
antibody according to any of the above embodiments, comprising a VL
having at least 80% identity, such as 90%, or 95%, or 97%, or 98%,
or 99% or 100% identity to a VL region sequence selected from the
group consisting of: SEQ ID NOs: 27, 32, 37, 42 or 47.
[0171] An embodiment of the invention provides an anti-CD38
antibody according to any of the above embodiments, comprising a VH
region comprising the sequence of SEQ ID NOs: 2, 7, 12, 17 or 22
and a VL region comprising the sequence of SEQ ID NOs: SEQ ID NOs:
27, 32, 37, 42 or 47.
[0172] according to any of the above embodiments according to any
of the above embodiments according to any of the above
embodiments
[0173] An embodiment of the invention provides an antibody which
competes with an antibody according to any of the above
embodiments, for binding to CD38.
[0174] An embodiment of the invention provides an anti-CD38
antibody, which competes for CD38 binding with an anti-CD38
antibody comprising a VH region comprising any of the sequences of
SEQ ID NOs: 2, 7, 12, 17 or 22 and a VL region comprising any of
the sequences of SEQ ID NO: 27, 32, 37, 42 or 47.
[0175] An embodiment of the invention provides an anti-CD38
antibody according to any of the above embodiments, wherein the
antibody binds to the same epitope on CD38 as an anti-CD38 antibody
as described in any of the above embodiments.
[0176] An embodiment of the invention provides an antibody having
substantially the same specific binding characteristics for binding
human CD38 has an antibody according to any of the above
embodiments.
[0177] An embodiment of the invention provides an anti-CD38
antibody according to any of the above embodiments, wherein the
antibody is capable of inducing complement-dependent cytotoxicity
(CDC) in CHO-CD38 cells.
[0178] An embodiment of the invention provides an anti-CD38
antibody according to any of the above embodiments, wherein the
antibody is capable of inducing antibody-dependent cellular
cytotoxicity (ADCC).
[0179] An embodiment of the invention provides an anti-CD38
antibody of claim 25, wherein said antibody induces ADCC in Daudi
cells, preferably with an EC.sub.50 value of 5 nM or less, e.g. 1
nM or less, such as 0.2 nM or less, as determined by the method
described in Example 6 herein.
[0180] An embodiment of the invention provides an anti-CD38
antibody according to any of the above embodiments, wherein the
antibody is not capable of inducing ADCC.
[0181] An embodiment of the invention provides an anti-CD38
antibody according to any of the above embodiments, wherein the
antibody is not capable of inducing complement-dependent
cytotoxicity (CDC).
[0182] An embodiment of the invention provides an anti-CD38
antibody according to any of the above embodiments, wherein the
antibody binds to human CD38 with a K.sub.D of 10.sup.-8 M or less,
preferably with a K.sub.D of 10.sup.-9 M or less.
[0183] An embodiment of the invention provides an anti-CD38
antibody according to any of the above embodiments, wherein the
antibody comprises: [0184] a heavy chain variable region derived
from a human germline VH sequence selected from the group
consisting of: IGHV1-69*04, and/or IGHJ3*02 [0185] a light chain
variable region derived from a human germline V.kappa. sequence
selected from the group consisting of: IGKV1D-16*01, and/or
IGKJ4*01.
[0186] An embodiment of the invention provides an anti-CD38
antibody according to any of the above embodiments, which is a
human antibody.
[0187] An embodiment of the invention provides an antibody
according to any of the above embodiments, characterized in that it
is a full length IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM
antibody, such as an IgG1 antibody, preferably an IgG1,.kappa.
antibody or an IgM antibody, preferably an IgM,.kappa.
antibody.
[0188] An embodiment of the invention provides an anti-CD38
antibody according to any of the above embodiments, wherein the
antibody is an antibody fragment or a single-chain antibody.
[0189] An embodiment of the invention provides an anti-CD38
antibody according to any of the above embodiments, wherein the
antibody is conjugated to another moiety, such as a cytotoxic
moiety, a radioisotope or a drug.
[0190] An embodiment of the invention provides an anti-CD38
antibody according to any of the above embodiments, wherein the
antibody is an effector-function-deficient antibody.
[0191] An embodiment of the invention provides an anti-CD38
antibody of the above embodiment, wherein the
effector-function-deficient anti-CD38 antibody is a stabilized
human IgG4 antibody.
[0192] An embodiment of the invention provides an anti-CD38
antibody of the above embodiment, wherein the stabilized IgG4
antibody is an antibody wherein arginine at position 409 in the
heavy chain constant region of human IgG4 is substituted with
lysine, threonine, methionine, or leucine, preferably lysine.
[0193] An embodiment of the invention provides an anti-CD38
antibody of the above embodiment, wherein said antibody comprises a
Lys residue at the position corresponding to 409 or the CH3 region
of the antibody has been replaced by the CH3 region of human IgG1,
of human IgG2 or of human IgG3.
[0194] An embodiment of the invention provides an anti-CD38
antibody of the above embodiments, wherein said antibody does not
comprise a Cys-Pro-Pro-Cys sequence in the hinge region.
[0195] An embodiment of the invention provides an anti-CD38
antibody of the above embodiments, wherein said antibody does
comprise a Cys-Pro-Pro-Cys sequence in the hinge region.
[0196] An embodiment of the invention provides an anti-CD38
antibody according to any of the above embodiments, wherein the
antibody is a monovalent antibody.
[0197] An embodiment of the invention provides an anti-CD38
antibody of the above embodiment, wherein said monovalent antibody
is constructed by a method comprising:
[0198] i) providing a nucleic acid construct encoding the light
chain of said monovalent antibody, said construct comprising a
nucleotide sequence encoding the VL region of a selected antigen
specific antibody and a nucleotide sequence encoding the constant
CL region of an Ig, wherein said nucleotide sequence encoding the
VL region of a selected antigen specific antibody and said
nucleotide sequence encoding the CL region of an Ig are operably
linked together, and wherein, in case of an IgG1 subtype, the
nucleotide sequence encoding the CL region has been modified such
that the CL region does not contain any amino acids capable of
forming disulfide bonds or covalent bonds with other peptides
comprising an identical amino acid sequence of the CL region in the
presence of polyclonal human IgG or when administered to an animal
or human being;
[0199] ii) providing a nucleic acid construct encoding the heavy
chain of said monovalent antibody, said construct comprising a
nucleotide sequence encoding the VH region of a selected antigen
specific antibody and a nucleotide sequence encoding a constant CH
region of a human Ig, wherein the nucleotide sequence encoding the
CH region has been modified such that the region corresponding to
the hinge region and, as required by the Ig subtype, other regions
of the CH region, such as the CH3 region, does not comprise any
amino acid residues which participate in the formation of
disulphide bonds or covalent or stable non-covalent inter-heavy
chain bonds with other peptides comprising an identical amino acid
sequence of the CH region of the human Ig in the presence of
polyclonal human IgG or when administered to an animal human being,
wherein said nucleotide sequence encoding the VH region of a
selected antigen specific antibody and said nucleotide sequence
encoding the CH region of said Ig are operably linked together;
[0200] iii) providing a cell expression system for producing said
monovalent antibody;
[0201] iv) producing said monovalent antibody by co-expressing the
nucleic acid constructs of (i) and (ii) in cells of the cell
expression system of (iii).
[0202] An embodiment of the invention provides an anti-CD38
antibody of the above embodiment, wherein the monovalent antibody
comprises
[0203] (i) a variable region of an antibody according to any of the
above embodiments, or an antigen binding part of the said region,
and
[0204] (ii) a C.sub.H region of an immunoglobulin or a fragment
thereof comprising the C.sub.H2 and C.sub.H3 regions, wherein the
C.sub.H region or fragment thereof has been modified such that the
region corresponding to the hinge region and, if the immunoglobulin
is not an IgG4 subtype, other regions of the C.sub.H region, such
as the CH3 region, do not comprise any amino acid residues, which
are capable of forming disulfide bonds with an identical C.sub.H
region or other covalent or stable non-covalent inter-heavy chain
bonds with an identical C.sub.H region in the presence of
polyclonal human IgG.
[0205] An embodiment of the invention provides an anti-CD38
antibody of the above embodiments wherein said monovalent antibody
is of the IgG4 subtype, but the C.sub.H3 region has been modified
so that one or more of the following amino acid substitutions have
been made: Thr (T) in position 366 has been replaced by Ala (A);
Leu (L) in position 368 has been replaced by Ala (A); Leu (L) in
position 368 has been replaced by Val (V); Phe (F) in position 405
has been replaced by Ala (A); Phe (F) in position 405 has been
replaced by Leu (L); Tyr (Y) in position 407 has been replaced by
Ala (A); Arg (R) in position 409 has been replaced by Ala (A).
[0206] An embodiment of the invention provides an anti-CD38
antibody of any of the above embodiments, the heavy chain has been
modified such that the entire hinge has been deleted.
[0207] An embodiment of the invention provides an anti-CD38
antibody of any of the above embodiments, wherein the sequence of
said monovalent antibody has been modified so that it does not
comprise any acceptor sites for N-linked glycosylation.
[0208] An embodiment of the invention provides an antibody
according to any of the above embodiments, which inhibits the
synthesis of cGDPR by at least 25%, such as at least 30% after 90
minutes as determined by spectophotometric method described in
Example 8 of the specification.
[0209] An embodiment of the invention provides an antibody
according to any of the above embodiments which inhibits the
synthesis of cADPR by at least 25%, such as at least 30% after 90
minutes at a concentration of 3 .mu.g/ml as determined by the HPLC
method described in Munshi et al., J. Biol. Chem. 275, 21566-21571
(2000).
[0210] An embodiment of the invention provides an antibody
according to any of the above embodiments, which stimulate the
hydrolase activity of CD38 by at least 25%.
[0211] An embodiment of the invention provides an antibody
according to any of the above embodiments, which stimulate the NAD
hydrolase activity by at least 25%.
[0212] An embodiment of the invention provides an antibody
according to any of the above embodiments, which stimulate the
cADPR-hydrolase activity by at least 25%.
[0213] An embodiment of the invention provides an isolated nucleic
acid encoding a peptide according to the any of the above
embodiments.
[0214] An embodiment of the invention provides an expression vector
comprising a nucleotide sequence encoding one or more of the amino
acid sequences according to any of the above embodiments.
[0215] An embodiment of the invention provides an expression vector
according to the above embodiment, further comprising a nucleotide
sequence encoding the constant region of a light chain, a heavy
chain or both light and heavy chains of a human antibody.
[0216] An embodiment of the invention provides a recombinant
eukaryotic or prokaryotic host cell which produces an antibody as
defined in any of the above embodiments.
[0217] An embodiment of the invention provides a hybridoma which
produces an antibody as defined in any of the above
embodiments.
[0218] An embodiment of the invention provides a pharmaceutical
composition comprising an antibody as defined in any of the above
embodiments, and a pharmaceutically acceptable carrier.
[0219] An embodiment of the invention provides an antibody as
defined in any of the embodiments above for use as a
medicament.
[0220] An embodiment of the invention provides an antibody as
defined in any of the embodiments above for use in inhibiting
growth and/or proliferation, migration or inducing phagocytosis of
a tumor cell expressing CD38.
[0221] An embodiment of the invention provides an antibody as
defined in any of the embodiments above for use in treating
rheumatoid arthritis.
[0222] An embodiment of the invention provides an antibody as
defined in any of the embodiments above for use in treating
multiple myeloma.
[0223] An embodiment of the invention provides an antibody as
defined in any of the embodiments above for use in treating
multiple sclerosis.
[0224] An embodiment of the invention provides an antibody as
defined in any of the embodiments above for use in treating B-cell
neoplasms such as any one of the following: small lymphocytic
lymphoma, B-cell prolymphocytic lymphoma, B-cell chronic
lymphocytic leukemia, mantle cell lymphoma, Burkitt's lymphoma,
follicular lymphoma, diffuse large B-cell lymphoma, multiple
myeloma, lymphoplasmacytic lymphoma, splenic margina zone lymphoma,
plasma cell neoplasms, such as plasma cell myeloma, plasmacytoma,
monoclonal immunoglobulin deposition disease, heavy chain disease,
MALT lymphoma, nodal marginal B cell lymphoma, intravascular large
B cell lymphoma, primary effusion lymphoma, lymphomatoid
granulomatosis, non-Hodgkins lymphoma, Hodgkins lymphoma, hairy
cell leukemia, primary effusion lymphoma or AIDS-related
non-Hodgkins lymphoma
[0225] An embodiment of the invention provides a method for
inhibiting growth and/or proliferation, migration or inducing
phagocytosis of a tumor cell expressing CD38, comprising
administration, to an individual in need thereof, of an antibody of
any of the above embodiments.
[0226] An embodiment of the invention provides a method for
producing an anti-CD38 antibody of any of the above embodiments,
said method comprising the steps of
[0227] a) culturing a host cell of claim 52 or a hybridoma of the
above embodiment, and
[0228] b) purifying the anti-CD38 antibody from the culture
media.
[0229] An embodiment of the invention provides a diagnostic
composition comprising an antibody as defined in any of the above
embodiments.
[0230] An embodiment of the invention provides a method for
detecting the presence of CD38 antigen, or a cell expressing CD38,
in a sample comprising: [0231] contacting the sample with an
anti-CD38 antibody of any of the above embodiments under conditions
that allow for formation of a complex between the antibody or
bispecific molecules and CD38; and [0232] analyzing whether a
complex has been formed.
[0233] An embodiment of the invention provides a kit for detecting
the presence of CD38 antigen, or a cell expressing CD38, in a
sample comprising [0234] an anti-CD38 antibody of any of the above
embodiments or and [0235] instructions for use of the kit.
[0236] An embodiment of the invention provides an anti-idiotypic
antibody which binds to an anti-CD38 antibody of any of the above
embodiments.
[0237] An embodiment of the invention provides a method of
inhibiting growth and/or proliferation of a cell expressing CD38,
comprising administration of a peptide according to any of the
above embodiments, an immunoconjugate according to the above
embodiment, a pharmaceutical composition according to the above
embodiments or an expression vector mentioned in the above
embodiments, such that the growth and/or proliferation, migration
or phagocytosis of the cell is inhibited.
[0238] An embodiment of the invention provides a method of treating
a disease or disorder involving cells expressing CD38 in a subject,
which method comprises administration of a peptide according to any
of the above embodiments, an immunoconjugate according to an
embodiment above, a pharmaceutical composition according to an
embodiment above, or an expression vector according to any one the
embodiments above to a subject in need thereof.
[0239] An embodiment of the invention provides a method of
preventing a disease or disorder involving cells expressing CD38 in
a subject, which method comprises administration of a peptide
according to any of the above embodiments, an immunoconjugate
according to an embodiment above, a pharmaceutical composition
according to an embodiment above, or an expression vector according
to any one the embodiments above to a subject in need thereof.
[0240] An embodiment of the invention provides a method according
to the above embodiments wherein the disease or disorder is
rheumatoid arthritis.
[0241] An embodiment of the invention provides a method according
to the above embodiments wherein the disease or disorder is B-cell
neoplasms such as any one of the following: small lymphocytic
lymphoma, B-cell prolymphocytic lymphoma, B-cell chronic
lymphocytic leukemia, mantle cell lymphoma, Burkitt's lymphoma,
follicular lymphoma, diffuse large B-cell lymphoma, multiple
myeloma, lymphoplasmacytic lymphoma, splenic margina zone lymphoma,
plasma cell neoplasms, such as plasma cell myeloma, plasmacytoma,
monoclonal immunoglobulin deposition disease, heavy chain disease,
MALT lymphoma, nodal marginal B cell lymphoma, intravascular large
B cell lymphoma, primary effusion lymphoma, lymphomatoid
granulomatosis, non-Hodgkins lymphoma, Hodgkins lymphoma, hairy
cell leukemia, primary effusion lymphoma and AIDS-related
non-Hodgkins lymphoma.
[0242] An embodiment of the invention provides a method according
to the above embodiments wherein the disease or disorder is
multiple myeloma An embodiment of the invention provides a method
according to the above embodiments wherein the disease or disorder
is autoimmune disease.
[0243] An embodiment of the invention provides a method according
to the above embodiments wherein the disease or disorder is
diabetes.
[0244] An embodiment of the invention provides a method according
to the above embodiments wherein the disease or disorder is
multiple sclerosis.
[0245] An embodiment of the invention provides a method according
to the above embodiments wherein the disease or disorder is Grave's
disease.
[0246] An embodiment of the invention provides a method according
to the above embodiments wherein the disease or disorder is
neuroinflammation.
[0247] An embodiment of the invention provides a method according
to the above embodiments wherein the disease or disorder is
inflammation of airway smooth muscle cells during asthma.
[0248] An embodiment of the invention provides a method according
to the above embodiments, wherein the method comprises
administration of one or more further therapeutic agents to the
subject.
[0249] An embodiment of the invention provides a method according
to the above embodiment, wherein the one or more further
therapeutic agents are selected from a chemotherapeutic agent, an
anti-inflammatory agent, or an immunosuppressive and/or
immunomodulatory agent.
[0250] An embodiment of the invention provides a method according
to the above embodiment, wherein the one or more further
therapeutic agents are selected from a group consisting of
cisplatin, gefitinib, cetuximab, rituximab, bevacizumab, erlotinib,
bortezomib, thalidomide, pamidronate, zoledronic acid, clodronate,
risendronate, ibandronate, etidronate, alendronate, tiludronate,
arsenic trioxide, lenalidomide, filgrastim, pegfilgrastim,
sargramostim, suberoylanilide hydroxamic acid, and SCIO-469.
[0251] Monoclonal antibodies of the present invention may e.g. be
produced by the hybridoma method first described by Kohler et al.,
Nature 256, 495 (1975), or may be produced by recombinant DNA
methods. Monoclonal antibodies may also be isolated from phage
antibody libraries using the techniques described in, for example,
Clackson et al., Nature 352, 624-628 (1991) and Marks et al., J.
Mol. Biol. 222, 581-597 (1991). Monoclonal antibodies may be
obtained from any suitable source. Thus, for example, monoclonal
antibodies may be obtained from hybridomas prepared from murine
splenic B cells obtained from mice immunized with an antigen of
interest, for instance in form of cells expressing the antigen on
the surface, or a nucleic acid encoding an antigen of interest.
[0252] In one embodiment, the antibody of the invention is a human
antibody. Human monoclonal antibodies directed against CD38 may be
generated using transgenic or transchromosomal mice carrying parts
of the human immune system rather than the mouse system. Such
transgenic and transchromosomic mice include mice referred to
herein as HuMAb mice and KM mice, respectively, and are
collectively referred to herein as "transgenic mice".
[0253] The HuMAb mouse contains a human immunoglobulin gene
miniloci that encodes unrearranged human heavy (.mu. and .gamma.)
and .kappa. light chain immunoglobulin sequences, together with
targeted mutations that inactivate the endogenous .mu. and .kappa.
chain loci (Lonberg, N. et al., Nature 368, 856-859 (1994)).
Accordingly, the mice exhibit reduced expression of mouse IgM or
.kappa. and in response to immunization, the introduced human heavy
and light chain transgenes, undergo class switching and somatic
mutation to generate high affinity human IgG,.kappa. monoclonal
antibodies (Lonberg, N. et al. (1994), supra; reviewed in Lonberg,
N. Handbook of Experimental Pharmacology 113, 49-101 (1994),
Lonberg, N. and Huszar, D., Intern. Rev. Immunol. Vol. 13 65-93
(1995) and Harding, F. and Lonberg, N. Ann. N.Y. Acad. Sci 764
536-546 (1995)). The preparation of HuMAb mice is described in
detail in Taylor, L. et al., Nucleic Acids Research 20, 6287-6295
(1992), Chen, J. et al., International Immunology 5, 647-656
(1993), Tuaillon et al., J. Immunol. 152, 2912-2920 (1994), Taylor,
L. et al., International Immunology 6, 579-591 (1994), Fishwild, D.
et al., Nature Biotechnology 14, 845-851 (1996). See also U.S. Pat.
Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,789,650,
5,877,397, 5,661,016, 5,814,318, 5,874,299, 5,770,429, 5,545,807,
WO 98/24884, WO 94/25585, WO 93/1227, WO 92/22645, WO 92/03918 and
WO 01/09187.
[0254] The HCo7 mice have a JKD disruption in their endogenous
light chain (kappa) genes (as described in Chen et al., EMBO J. 12,
821-830 (1993)), a CMD disruption in their endogenous heavy chain
genes (as described in Example 1 of WO 01/14424), a KCo5 human
kappa light chain transgene (as described in Fishwild et al.,
Nature Biotechnology 14, 845-851 (1996)), and a HCo7 human heavy
chain transgene (as described in U.S. Pat. No. 5,770,429).
[0255] The HCo12 mice have a JKD disruption in their endogenous
light chain (kappa) genes (as described in Chen et al., EMBO J. 12,
821-830 (1993)), a CMD disruption in their endogenous heavy chain
genes (as described in Example 1 of WO 01/14424), a KCo5 human
kappa light chain transgene (as described in Fishwild et al.,
Nature Biotechnology 14, 845-851 (1996)), and a HCo12 human heavy
chain transgene (as described in Example 2 of WO 01/14424).
[0256] In the KM mouse strain, the endogenous mouse kappa light
chain gene has been homozygously disrupted as described in Chen et
al., EMBO J. 12, 811-820 (1993) and the endogenous mouse heavy
chain gene has been homozygously disrupted as described in Example
1 of WO 01/09187. This mouse strain carries a human kappa light
chain transgene, KCo5, as described in Fishwild et al., Nature
Biotechnology 14, 845-851 (1996). This mouse strain also carries a
human heavy chain transchromosome composed of chromosome 14
fragment hCF (SC20) as described in WO 02/43478.
[0257] Splenocytes from these transgenic mice may be used to
generate hybridomas that secrete human monoclonal antibodies
according to well known techniques.
[0258] Human monoclonal or polyclonal antibodies of the present
invention, or antibodies of the present invention originating from
other species may also be generated transgenically through the
generation of another non-human mammal or plant that is transgenic
for the immunoglobulin heavy and light chain sequences of interest
and production of the antibody in a recoverable form therefrom. In
connection with the transgenic production in mammals, antibodies
may be produced in, and recovered from, the milk of goats, cows, or
other mammals. See for instance U.S. Pat. Nos. 5,827,690,
5,756,687, 5,750,172 and 5,741,957.
[0259] Further, human antibodies of the present invention or
antibodies of the present invention from other species may be
generated and identified through display-type technologies,
including, without limitation, phage display, retroviral display,
ribosomal display, and other techniques, using techniques well
known in the art and the resulting molecules may be subjected to
additional maturation, such as affinity maturation, as such
techniques are well known in the art (see for instance Hoogenboom
et al., J. Mol. Biol. 227, 381 (1991) (phage display), Vaughan et
al., Nature Biotech 14, 309 (1996) (phage display), Hanes and
Plucthau, PNAS USA 94, 4937-4942 (1997) (ribosomal display),
Parmley and Smith, Gene 73, 305-318 (1988) (phage display), Scott
TIBS 17, 241-245 (1992), Cwirla et al., PNAS USA 87, 6378-6382
(1990), Russel et al., Nucl. Acids Research 21, 1081-1085 (1993),
Hogenboom et al., Immunol. Reviews 130, 43-68 (1992), Chiswell and
McCafferty TIBTECH 10, 80-84 (1992), and U.S. Pat. No. 5,733,743).
If display technologies are utilized to produce antibodies that are
not human, such antibodies may be humanized.
[0260] Competition for binding to CD38 or a portion of CD38 by two
or more anti-CD38 antibodies may be determined by any suitable
technique. Competition in the context of the present invention
refers to any detectably significant reduction in the propensity
for a particular molecule to bind a particular binding partner in
the presence of another molecule that binds the binding partner.
Typically, competition means an at least about 10% reduction, such
as an at least about 15%, or an at least about 20% reduction in
binding between an anti-CD38 antibody and [0261] (a) a form of CD38
(e.g. "processed", "mature", "unprocessed", "not processed" or
"immature" CD38); [0262] (b) a form of free CD38 (e.g., a CD38
fragment produced by in vivo processing); [0263] (c) a
heterodimeric peptide composed of another peptide associated with
CD38, such as CD31 associated with CD38; [0264] (d) a complex of
CD38 and one or more substrates, such as cAMP, NAD+ and/or cADPR;
[0265] (e) a dimerized, associated and/or processed dimer of CD38
with a soluble ligand, such as CD31; or [0266] (f) a portion of
CD38, caused by the presence of another anti-CD38 antibody as
determined by, e.g., ELISA analysis or FACS analysis (as described
in the examples section) using sufficient amounts of the two or
more competing anti-CD38 antibodies and CD38 molecule. It may also
be the case that competition may exist between anti-CD38 antibodies
with respect to more than one form of CD38, and/or a portion of
CD38, e.g. in a context where the antibody-binding properties of a
particular region of CD38 are retained in fragments thereof, such
as in the case of a well-presented linear epitope located in
various tested fragments or a conformational epitope that is
presented in sufficiently large CD38 fragments as well as in
CD38.
[0267] Assessing competition typically involves an evaluation of
relative inhibitory binding using a first amount of a first
molecule; a second amount of a second molecule; and a third amount
of a third molecule (or a standard determined by binding studies
that may be reasonably compared to new binding data with respect to
the first and second molecules as a surrogate for actual
contemporaneous data), wherein the first, second, and third amounts
all are sufficient to make a comparison that imparts information
about the selectivity and/or specificity of the molecules at issue
with respect to the other present molecules. The first, second, and
third amounts may vary with the nature of the anti-CD38 antibody
and potential targets therefore at issue. For instance, for ELISA
assessments, similar to those described in the Examples section,
about 5-50 .mu.g (e.g., about 10-50 .mu.g, about 20-50 .mu.g, about
5-20 .mu.g, about 10-20 .mu.g, etc.) of anti-CD38 antibody and/or
CD38 targets are required to assess whether competition exists.
Conditions also should be suitable for binding. Typically,
physiological or near-physiological conditions (e.g., temperatures
of about 20-40.degree. C., pH of about 7-8, etc.) are suitable for
anti-CD38 antibody:CD38 binding. Often competition is marked by a
significantly greater relative inhibition than about 5% as
determined by ELISA and/or FACS analysis. It may be desirable to
set a higher threshold of relative inhibition as a
criteria/determinant of what is a suitable level of competition in
a particular context (e.g., where the competition analysis is used
to select or screen for new antibodies designed with the intended
function of blocking the binding of another peptide or molecule
binding to CD38 (e.g., the natural binding partners of CD38 such as
CD31, also called CD31 antigen, EndoCAM, GPIIA', PECAM-1,
platelet/endothelial cell adhesion molecule or naturally occurring
anti-CD38 antibody)). Thus, for example, it is possible to set a
criterion for competitiveness wherein at least about 10% relative
inhibition is detected; at least about 15% relative inhibition is
detected; or at least about 20% relative inhibition is detected
before an antibody is considered sufficiently competitive. In cases
where epitopes belonging to competing antibodies are closely
located in an antigen, competition may be marked by greater than
about 40% relative inhibition of CD38 binding (e.g., at least about
45% inhibition, such as at least about 50% inhibition, for instance
at least about 55% inhibition, such as at least about 60%
inhibition, for instance at least about 65% inhibition, such as at
least about 70% inhibition, for instance at least about 75%
inhibition, such as at least about 80% inhibition, for instance at
least about 85% inhibition, such as at least about 90% inhibition,
for instance at least about 95% inhibition, or higher level of
relative inhibition).
[0268] Competition may be considered the inverse of
cross-reactivity between a molecule and two potential binding
partners. In certain embodiments, a anti-CD38 antibody of the
present invention specifically binds to one or more residues or
regions in CD38 but also does not cross-react with other peptides,
peptide regions, or molecules, e.g., the present invention provides
an anti-CD38 antibody that does not cross-react with proteins with
homology to CD38, such as BST-1 (bone marrow stromal cell
antigen-1) and Mo5, also called CD157; or anti-CD38 antibodies that
do not cross-react with CD38 in the context of normal tissue, such
as tissues not involved in multiple myeloma. Typically, a lack of
cross-reactivity means less than about 5% relative competitive
inhibition between the molecules when assessed by ELISA and/or FACS
analysis using sufficient amounts of the molecules under suitable
assay conditions.
[0269] In one embodiment, the present invention provides an
anti-CD38 antibody that competes with an antibody having a V.sub.L
sequence of SEQ ID NO: 27 and a V.sub.H sequence of SEQ ID NO: 2,
such as the antibody 028, for binding to CD38 or a portion
thereof.
[0270] In one embodiment, the present invention provides an
anti-CD38 antibody that competes with an antibody having a V.sub.L
sequence of SEQ ID NO: 32 and a V.sub.H sequence of SEQ ID NO: 7,
such as the antibody 025, for binding to CD38 or a portion
thereof.
[0271] In one embodiment, the present invention provides an
anti-CD38 antibody that competes with an antibody having a V.sub.L
sequence of SEQ ID NO: 37 and a V.sub.H sequence of SEQ ID NO: 12,
such as the antibody 026, for binding to CD38 or a portion
thereof.
[0272] In one embodiment, the present invention provides an
anti-CD38 antibody that competes with an antibody having a V.sub.L
sequence of SEQ ID NO: 42 and a V.sub.H sequence of SEQ ID NO: 17,
such as the antibody 049, for binding to CD38 or a portion
thereof.
[0273] In one embodiment, the present invention provides an
anti-CD38 antibody that competes with an antibody having a V.sub.L
sequence of SEQ ID NO: 47 and a V.sub.H sequence of SEQ ID NO: 22,
such as the antibody 056, for binding to CD38 or a portion
thereof.
[0274] As discussed elsewhere herein, unless otherwise stated or
clearly contradicted by context, references to binding of an
anti-CD38 antibody to CD38 are intended to refer to binding in any
suitable context, such as in a conformational context where the
structure of CD38 is present; or in a linear epitope context. Of
course, binding in a limited subset of such context(s) may be an
important characteristic with respect to any anti-CD38 antibody
provided by the present invention.
[0275] Additional methods for determining anti-CD38 antibody
specificity by competitive inhibition may be found in for instance
Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1988), Colligan et al.,
eds., Current Protocols in Immunology, Greene Publishing Assoc. and
Wiley InterScience N.Y., (1992, 1993), and Muller, Meth. Enzymol.
92, 589-601 (1983)).
[0276] Human CD38 comprises a number of different epitopes, which
may include (1) peptide antigenic determinants that are comprised
within single peptide chains within human CD38; (2) conformational
antigenic determinants which consist of one or more noncontiguous
amino acids on a particular chain and/or amino acids present on
spatially contiguous but separate peptide chains (typically where
the respective amino acid sequences of the chains are located
disjointedly along the human CD38 polypeptide sequence); (3)
post-translational antigenic determinants which consist, either in
whole or part, of molecular structures covalently attached to human
CD38, such as carbohydrate groups; or (4) combinations of
(1)-(3).
[0277] An epitope in the context of the present invention includes
any peptide or peptide-derivative determinant capable of specific
binding to an immunoglobulin. An epitope may comprise any suitable
number of amino acids, in any suitable position (with respect to
the linear sequence of CD38), orientation (with respect to folded
CD38, or a fragment thereof), amino acid composition (and
consequently, at least in part, charge). Thus, for example, an
epitope may be composed of about 3-10 amino acids, typically 3-8
amino acids, in one or more contiguous or noncontiguous locations
with respect to the primary sequence of CD38 (for instance an
epitope may consist essentially of 2, 3, 4, 5, 6, 7, or 8 amino
acid residues distributed in 1, 2, 3, 4, or 5 noncontiguous
locations in CD38). Alternatively, for example, an epitope may be
considered to be defined by a region of about 5-40 contiguous amino
acid residues (e.g., about 7-30 amino acid residues, about 5-20
amino acid residues, or about 3-15 amino acid residues) in CD38
(solely or in combination with a portion of an adjacent CD38
domain). In some epitopes it may be the case that just one amino
acid residue or only a few amino acid residues are critical to CDR
or CDR(s) recognition (and thereby most important to anti-CD38
antibody:CD38 antigen affinity and avidity). As such, an epitope
may be characterized on the basis of one or more of such critical
residues, with the recognition that other residues may also make
some lesser contribution to the epitope. In the case of an epitope
defined by a region of amino acids, it may be that one or more
amino acids in the region make only a minor contribution or even
negligible contribution to antibody binding, such that the residue
may be subject to substitution with an appropriate different
residue without resulting in "a loss" of the epitope to at least
some anti-CD38 antibodies specific for it.
[0278] In one embodiment, the present invention provides a
anti-CD38 antibody, such as an anti-CD38 antibody, that
specifically binds to a CD38 epitope that also is specifically
bound by an antibody having a V.sub.L sequence of SEQ ID NO: 27 and
a V.sub.H sequence of SEQ ID NO: 2 (such as antibody 028), or an
antibody having a V.sub.L sequence of SEQ ID NO: 32 and a V.sub.H
sequence of SEQ ID NO: 7 (such as antibody 025), or an antibody
having a V.sub.L sequence of SEQ ID NO: 37 and a V.sub.H sequence
of SEQ ID NO: 12 (such as antibody 026), or an antibody having a
V.sub.L sequence of SEQ ID NO: 42 and a V.sub.H sequence of SEQ ID
NO: 17 (such as antibody 049), or an antibody having a V.sub.L
sequence of SEQ ID NO: 47 and a V.sub.H sequence of SEQ ID NO: 22
(such as antibody 056).
[0279] It is possible that anti-CD38 antibodies having one or more
CDRs that differ from the CDRs of an antibody having a V.sub.L
sequence of SEQ ID NO: 27 and a V.sub.H sequence of SEQ ID NO: 2,
or the CDRs of an antibody having a V.sub.L sequence of SEQ ID NO:
32 and a V.sub.H sequence of SEQ ID NO: 7, or the CDRs of an
antibody having a V.sub.L sequence of SEQ ID NO: 37 and a V.sub.H
sequence of SEQ ID NO: 12, or the CDRs of an antibody having a
V.sub.L sequence of SEQ ID NO: 42 and a V.sub.H sequence of SEQ ID
NO: 17, or the CDRs of an antibody having a V.sub.L sequence of SEQ
ID NO: 47 and a V.sub.H sequence of SEQ ID NO: 22, may still be
specific for the same epitope as an antibody having the CDRs of an
antibody having a V.sub.L sequence of SEQ ID NO: 27 and a V.sub.H
sequence of SEQ ID NO: 2, or the CDRs of an antibody having a
V.sub.L sequence of SEQ ID NO: 32 and a V.sub.H sequence of SEQ ID
NO: 7, or the CDRs of an antibody having a V.sub.L sequence of SEQ
ID NO: 37 and a V.sub.H sequence of SEQ ID NO: 12, or the CDRs of
an antibody having a V.sub.L sequence of SEQ ID NO: 42 and a
V.sub.H sequence of SEQ ID NO: 17, or the CDRs of an antibody
having a V.sub.L sequence of SEQ ID NO: 47 and a V.sub.H sequence
of SEQ ID NO: 22, respectively. In such cases, the anti-CD38
antibody in question may recognize or be more specific/selective
for particular structures or regions of the epitope than the
antibody having the CDRs of an antibody having a V.sub.L sequence
of SEQ ID NO: 27 and a V.sub.H sequence of SEQ ID NO: 2, or the
CDRs of an antibody having a V.sub.L sequence of SEQ ID NO: 32 and
a V.sub.H sequence of SEQ ID NO: 7, or the CDRs of an antibody
having a V.sub.L sequence of SEQ ID NO: 37 and a V.sub.H sequence
of SEQ ID NO: 12, or the CDRs of an antibody having a V.sub.L
sequence of SEQ ID NO: 42 and a V.sub.H sequence of SEQ ID NO: 17,
or the CDRs of an antibody having a V.sub.L sequence of SEQ ID NO:
47 and a V.sub.H sequence of SEQ ID NO: 22 respectively.
[0280] A CD38 epitope bound by an antibody having a V.sub.L
sequence of SEQ ID NO: 27 and a V.sub.H sequence of SEQ ID NO: 2
(such as antibody 028), or an antibody having a V.sub.L sequence of
SEQ ID NO: 32 and a V.sub.H sequence of SEQ ID NO: 7 (such as
antibody 025), or an antibody having a V.sub.L sequence of SEQ ID
NO: 37 and a V.sub.H sequence of SEQ ID NO: 12 (such as antibody
026), or an antibody having a V.sub.L sequence of SEQ ID NO: 42 and
a V.sub.H sequence of SEQ ID NO: 17 (such as antibody 049), or an
antibody having a V.sub.L sequence of SEQ ID NO: 47 and a V.sub.H
sequence of SEQ ID NO: 22 (such as antibody 056), may be identified
via standard mapping and characterization techniques, further
refinement of which may be identified by any suitable technique,
numerous examples of which are available to the skilled
artisan.
[0281] These techniques may also be used to identify and/or
characterize epitopes for anti-CD38 antibodies generally. As one
example of such mapping/characterization methods, an epitope for an
anti-CD38 antibody may be determined by epitope "foot-printing"
using chemical modification of the exposed amines/carboxyls in the
CD38 protein. One specific example of such a foot-printing
technique is the use of HXMS (hydrogen-deuterium exchange detected
by mass spectrometry) wherein a hydrogen/deuterium exchange of
receptor and ligand protein amide protons, binding, and back
exchange occurs, wherein the backbone amide groups participating in
protein binding are protected from back exchange and therefore will
remain deuterated. Relevant regions may be identified at this point
by peptic proteolysis, fast microbore high-performance liquid
chromatography separation, and/or electrospray ionization mass
spectrometry. See, e.g., Ehring H, Analytical Biochemistry, 267(2)
252-259 (1999) and/or Engen, J. R. and Smith, D. L. (2001) Anal.
Chem. 73, 256A-265A. Another example of a suitable epitope
identification technique is nuclear magnetic resonance epitope
mapping (NMR), where typically the position of the signals in
two-dimensional NMR spectres of the free antigen and the antigen
complexed with the antigen binding peptide, such as an antibody,
are compared. The antigen typically is selectively isotopically
labeled with .sup.15N so that only signals corresponding to the
antigen and no signals from the antigen binding peptide are seen in
the NMR-spectrum. Antigen signals originating from amino acids
involved in the interaction with the antigen binding peptide
typically will shift position in the spectres of the complex
compared to the spectres of the free antigen, and the amino acids
involved in the binding may be identified that way. See for
instance Ernst Schering Res Found Workshop. (44), 149-67 (2004),
Huang et al., Journal of Molecular Biology 281(1), 61-67 (1998) and
Saito and Patterson, Methods. 9(3), 516-24 (1996).
[0282] Epitope mapping/characterization may also be performed using
mass spectrometry methods. See for instance Downward, J Mass
Spectrom. 35(4), 493-503 (2000) and Kiselar and Downard, Anal Chem.
71(9), 1792-801 (1999).
[0283] Protease digestion techniques may also be useful in the
context of epitope mapping and identification. Antigenic
determinant-relevant regions/sequences may be determined by
protease digestion, e.g. by using trypsin in a ratio of about 1:50
to CD38 overnight (0/N) digestion at 37.degree. C. and pH 7-8,
followed by mass spectrometry (MS) analysis for peptide
identification. The peptides protected from trypsin cleavage by the
CD38BP may subsequently be identified by comparison of samples
subjected to trypsin digestion and samples incubated with CD38BP
and then subjected to digestion by e.g. trypsin (thereby revealing
a foot-print for the binder). Other enzymes like chymotrypsin,
pepsin, etc. may also or alternatively be used in a similar epitope
characterization method.
[0284] An anti-CD38 antibody which gives the significantly same
result as an antibody having a V.sub.L sequence of SEQ ID NO: 27
and a V.sub.H sequence of SEQ ID NO: 2 (such as antibody 028), or
an antibody having a V.sub.L sequence of SEQ ID NO: 32 and a
V.sub.H sequence of SEQ ID NO: 7 (such as antibody 025), or an
antibody having a V.sub.L sequence of SEQ ID NO: 37 and a V.sub.H
sequence of SEQ ID NO: 12 (such as antibody 026), or an antibody
having a V.sub.L sequence of SEQ ID NO: 42 and a V.sub.H sequence
of SEQ ID NO: 17 (such as antibody 049), or an antibody having a
V.sub.L sequence of SEQ ID NO: 47 and a V.sub.H sequence of SEQ ID
NO: 22 (such as antibody 056), in these measurements are deemed to
be an antibody that bind the same epitope as an antibody having a
V.sub.L sequence of SEQ ID NO: 27 and a V.sub.H sequence of SEQ ID
NO: 2 (such as antibody 028), or an antibody having a V.sub.L
sequence of SEQ ID NO: 32 and a V.sub.H sequence of SEQ ID NO: 7
(such as antibody 025), or an antibody having a V.sub.L sequence of
SEQ ID NO: 37 and a V.sub.H sequence of SEQ ID NO: 12 (such as
antibody 026), or an antibody having a V.sub.L sequence of SEQ ID
NO: 42 and a V.sub.H sequence of SEQ ID NO: 17 (such as antibody
049), or an antibody having a V.sub.L sequence of SEQ ID NO: 47 and
a V.sub.H sequence of SEQ ID NO: 22 (such as antibody 056),
respectively. See for instance Manca, Ann Ist Super Sanita. 27(1),
15-9 (1991) for a discussion of similar techniques.
[0285] Epitope mapping by competitive binding to CD38 with two
antibodies where one is biotinylated is another method for
identifying relevant antigenic determinant regions. The binding of
antibodies to linear and looped peptides of CD38 by a PEPSCAN-based
enzyme-linked immuno assay is another method for identifying
relevant antigenic determinant regions, see for instance
Slootstra-J W et al. Mol-Divers. 1, 87-96 (1996).
[0286] Site directed mutagenesis is another method for identifying
relevant antigenic determinant regions, see for instance Polyak and
Deans, Blood 99, 3956-3962 (2002). Various phage display techniques
may also be used to identify epitopes. See for instance Wang and
Yu, Curr Drug Targets. 5(1), 1-15 (2004), Burton, Immunotechnology.
1(2), 87-94 (1995 August), Cortese et al., Immunotechnology. 1(2),
87-94 (1995) and Irving et al., Curr Opin Chem Biol. 5(3), 314-24
(2001). Consensus epitopes may also be identified through modified
phage display-related techniques (see,
http://www.cs.montana.edu/.about.mumey/papers/jcb03.pdf) for
discussion.
[0287] Other methods potentially helpful in mapping epitopes
include crystallography techniques, X-ray diffraction techniques
(such as the X-ray diffraction/sequence study techniques developed
by Poljak and others in the 1970s-1980s), and the application of
Multipin Peptide Synthesis Technology. Computer-based methods such
as sequence analysis and three dimensional structure analysis and
docking may also be used to identify antigenic determinants. For
example, an epitope may also be determined by molecular modeling
using a structure of CD38 with docking of the structure of the Fab
fragment of the individual monoclonal antibody. These and other
mapping methods are discussed in Epitope Mapping A Practical
Approach (Westwood and Hay Eds.) 2001 Oxford University Press.
[0288] In one embodiment, the present invention provides an
anti-CD38 antibody having substantially the same specific
CD38-binding characteristics of one or more mAbs selected from an
antibody having a V.sub.L sequence of SEQ ID NO: 27 and a V.sub.H
sequence of SEQ ID NO: 2 (such as antibody 028), or an antibody
having a V.sub.L sequence of SEQ ID NO: 32 and a V.sub.H sequence
of SEQ ID NO: 7 (such as antibody 025), or an antibody having a
V.sub.L sequence of SEQ ID NO: 37 and a V.sub.H sequence of SEQ ID
NO: 12 (such as antibody 026), or an antibody having a V.sub.L
sequence of SEQ ID NO: 42 and a V.sub.H sequence of SEQ ID NO: 17
(such as antibody 049), or an antibody having a V.sub.L sequence of
SEQ ID NO: 47 and a V.sub.H sequence of SEQ ID NO: 22 (such as
antibody 056).
[0289] Mapping studies have indicated that several monoclonal
antibodies raised against human CD38 bind to epitopes in the
C-terminal region of CD38 (220-296) (Hoshino et al. and Ferrero et
al.). Within this region three amino acid differences have been
found between the human and the cynomolgus CD38 sequence: T237,
Q272 and S274 in humans correspond to A238, R273 and F275 in
cynomolgus. A limited number of amino acid differences exist
between the human and the monkey CD38 sequence, for instance in the
carboxyterminal part to the protein, for instance the following
three amino acid differences between the human and the cynomolgus
CD38 sequence: T237, Q272 and S274 in human CD38s correspond to
A238, R273 and F275 in cynomolgus monkey CD38 (compare SEQ ID No.21
and SEQ ID No.22).
[0290] The antibodies of the present invention do not bind to human
CD38 mutants wherein aspartic acid in position 202 has been
substituted with a glycine to the same degree that it binds to
human CD38. The present invention provides antibodies, which bind
to human CD38 and which binds to a mutant human CD38, wherein the
serine residue in position 274 has been substituted with a
phenylalanine residue. The antibodies of the present invention also
bind to human CD38 mutants wherein glutamine in position 272 has
been substituted with an arginine. The antibodies of the present
invention also bind to human CD38 mutants wherein the threonine in
position 237 has been substituted with an alanine.
[0291] The term "do not bind to the same degree" should be
interpreted so that the binding of the antibody to the mutant human
CD38 is significantly lower than the binding of the antibody to the
wild type human CD38. The term "bind to the same degree" should be
interpreted so that the binding of the antibody to the mutant human
CD38 is substantially of the same order as the binding of the
antibody to the wild type human CD38. The binding of a peptide to
the CD38 molecules (wild type and mutant) may be determined in a
number of ways and it is within the common general knowledge of a
person skilled in the art to determine whether the binding to the
mutant is "significantly lower" than the binding to the wild type.
A large number of different techniques for determining the binding
of a peptide to another peptide are available to the person skilled
in the art, for example ELISA, radioimmunoassay, BIAcore or flow
cytometry.
[0292] One method of determining the binding is by determining the
EC.sub.50 of the binding of the antibody to the mutant protein and
to the wild type protein and then comparing the values obtained.
Another method of determining the binding is by examining the
magnitude of binding at saturating concentration (for instance the
plateau of binding signal), or by determining kinetic rate
constants k.sub.on and k.sub.off for example by BIAcore.
[0293] In one embodiment, the binding of the antibody in question
to the CD38 proteins (mutant or wild type) is by use of an ELISA as
described in Example 4.
[0294] In a further embodiment, the antibody of the invention
comprises a human heavy chain variable region (VH) CDR3 sequence
comprising: [0295] an amino acid sequence selected from the group
consisting of: SEQ ID NOs: 5, 10, 15, 20 and 25, or [0296] a
variant of any of said sequences, wherein said variant preferably
only has conservative amino acid modifications.
[0297] In one embodiment, said variant consists essentially of a
sequence having at least about 50%, such as at least 60%, for
instance at least about 70%, such as at least about 75%, for
instance at least about 80%, such as at least about 85%, for
instance at least about 90%, such as at least about 95% amino acid
sequence identity to a sequence according to any one of SEQ ID Nos:
5, 10, 15, 20 and 25.
[0298] In a further embodiment, said variant has at most 1, 2 or 3
amino-acid modifications, e.g. amino acid substitutions, preferably
conservative substitutions as compared to said sequence.
[0299] In a preferred embodiment, said antibody comprises a human
heavy chain variable region CDR3 sequence comprising an amino acid
sequence selected from the group consisting of: SEQ ID NOs: 5, 10,
15, 20 and 25.
[0300] In an even further embodiment, the antibody of the invention
comprises: [0301] a VH region comprising the CDR1, 2 and 3
sequences of SEQ ID NO: 3, 4 and 5; or [0302] a VH region
comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 8, 9 and 10;
or [0303] a VH region comprising the CDR1, 2 and 3 sequences of SEQ
ID NO: 13, 14 and 15; or [0304] a VH region comprising the CDR1, 2
and 3 sequences of SEQ ID NO: 18, 19 and 20; or [0305] a VH region
comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 23, 24 and 25;
or [0306] a variant of any said VH regions, wherein said variant
preferably only has conservative amino-acid substitutions.
[0307] In one embodiment, said variant comprises a VH CDR1 which
consists essentially of a sequence having at least about 50%, such
as at least 60%, for instance at least about 70%, such as at least
about 75%, for instance at least about 80%, such as at least about
85%, for instance at least about 90%, such as at least about 95%
amino acid sequence identity to a sequence according to any one of
SEQ ID Nos:3, 8, 13, 18 or 23.
[0308] In one embodiment, said variant comprises a VH CDR2 which
consists essentially of a sequence having at least about 50%, such
as at least 60%, for instance at least about 70%, such as at least
about 75%, for instance at least about 80%, such as at least about
85%, for instance at least about 90%, such as at least about 95%
amino acid sequence identity to a sequence according to any one of
SEQ ID Nos:4, 9, 14, 19 or 24.
[0309] In one embodiment, said variant comprises a VH CDR3 which
consists essentially of a sequence having at least about 50%, such
as at least 60%, for instance at least about 70%, such as at least
about 75%, for instance at least about 80%, such as at least about
85%, for instance at least about 90%, such as at least about 95%
amino acid sequence identity to a sequence according to any one of
SEQ ID Nos: 5, 10, 15, 20 or 25.
[0310] In another embodiment, the antibody the antibody comprises
[0311] a) a VL CDR3 region having the sequence as set forth in SEQ
ID NO: 30 and a VH CDR3 region having a sequence selected from the
group consisting of SEQ ID NO: 5, [0312] b) a VL CDR3 region having
the sequence as set forth in SEQ ID NO: 35 and a VH CDR3 region
having the sequence as set forth in SEQ ID NO: 10, [0313] c) a VL
CDR3 region having the sequence as set forth in SEQ ID NO: 40 and a
VH CDR3 region having the sequence as set forth in SEQ ID NO: 15,
[0314] d) a VL CDR3 region having the sequence as set forth in SEQ
ID NO: 45 and a VH CDR3 region having the sequence as set forth in
SEQ ID NO: 20, [0315] e) a VL CDR3 region having the sequence as
set forth in SEQ ID NO: 50 and a VH CDR3 region having the sequence
as set forth in SEQ ID NO: 25, [0316] f) a variant of any of the
above, wherein said variant preferably only has conservative
substitutions in said sequences
[0317] In one embodiment, said variant comprises a VH CDR3 which
consists essentially of a sequence having at least about 50%, such
as at least 60%, for instance at least about 70%, such as at least
about 75%, for instance at least about 80%, such as at least about
85%, for instance at least about 90%, such as at least about 95%
amino acid sequence identity to a sequence according to any one of
SEQ ID Nos: 5, 10, 15, 20 or 25. In one embodiment, said variant
comprises a VL CDR3 which consists essentially of a sequence having
at least about 50%, such as at least 60%, for instance at least
about 70%, such as at least about 75%, for instance at least about
80%, such as at least about 85%, for instance at least about 90%,
such as at least about 95% amino acid sequence identity to a
sequence according to any one of SEQ ID Nos: 30, 35, 40, 45 or
50;
In a further embodiment, the antibody of the invention comprises:
[0318] a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID
NO: 3, 4 and 5, and a VL region comprising the CDR 3 sequence of
SEQ ID NO: 30; or [0319] a VH region comprising the CDR1, 2 and 3
sequences of SEQ ID NO: 8, 9 and 10, and a VL region comprising the
CDR3 sequence of SEQ ID NO: 35; or [0320] a VH region comprising
the CDR1, 2 and 3 sequences of SEQ ID NO: 13, 14 and 15, and and a
VL region comprising the CDR3 sequence of SEQ ID NO: 40; or [0321]
a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:
18, 19 and 20, and a VL region comprising the CDR3 sequence of SEQ
ID NO: 45; or [0322] a VH region comprising the CDR1, 2 and 3
sequences of SEQ ID NO: 23, 24 and 25, and a VL region comprising
the CDR3 sequence of SEQ ID NO: 50; or [0323] a variant of any of
said antibodies, wherein said variant preferably only has
conservative amino-acid substitutions in said sequences.
[0324] In one embodiment, said variant comprises a VH CDR1 which
consists essentially of a sequence having at least about 50%, such
as at least 60%, for instance at least about 70%, such as at least
about 75%, for instance at least about 80%, such as at least about
85%, for instance at least about 90%, such as at least about 95%
amino acid sequence identity to a sequence according to any one of
SEQ ID Nos: 3, 8, 13, 18 or 23.
[0325] In one embodiment, said variant comprises a VH CDR2 which
consists essentially of a sequence having at least about 50%, such
as at least 60%, for instance at least about 70%, such as at least
about 75%, for instance at least about 80%, such as at least about
85%, for instance at least about 90%, such as at least about 95%
amino acid sequence identity to a sequence according to any one of
SEQ ID Nos: 4, 9, 14, 19 or 24.
[0326] In one embodiment, said variant comprises a VH CDR3 which
consists essentially of a sequence having at least about 50%, such
as at least 60%, for instance at least about 70%, such as at least
about 75%, for instance at least about 80%, such as at least about
85%, for instance at least about 90%, such as at least about 95%
amino acid sequence identity to a sequence according to any one of
SEQ ID Nos:5, 10, 15, 20 or 25.
[0327] In one embodiment, said variant comprises a VL CDR1 which
consists essentially of a sequence having at least about 50%, such
as at least 60%, for instance at least about 70%, such as at least
about 75%, for instance at least about 80%, such as at least about
85%, for instance at least about 90%, such as at least about 95%
amino acid sequence identity to a sequence according to any one of
SEQ ID Nos:28, 33, 38, 43 or 48.
[0328] In one embodiment, said variant comprises a VL CDR2 which
consists essentially of a sequence having at least about 50%, such
as at least 60%, for instance at least about 70%, such as at least
about 75%, for instance at least about 80%, such as at least about
85%, for instance at least about 90%, such as at least about 95%
amino acid sequence identity to a sequence according to any one of
SEQ ID Nos:29, 34, 39, 44 or 49.
[0329] In one embodiment, said variant comprises a VL CDR3 which
consists essentially of a sequence having at least about 50%, such
as at least 60%, for instance at least about 70%, such as at least
about 75%, for instance at least about 80%, such as at least about
85%, for instance at least about 90%, such as at least about 95%
amino acid sequence identity to a sequence according to any one of
SEQ ID Nos:30, 35, 40, 45, or 50.
[0330] In a further embodiment, the antibody of the invention
comprises: [0331] a VH region comprising the CDR1, 2 and 3
sequences of SEQ ID NO: 3, 4 and 5 and a VL region comprising the
CDR1, 2 and 3 sequences of SEQ ID NO: 28, 29 and 30; or [0332] a VH
region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 8, 9
and 10 and a VL region comprising the CDR1, 2 and 3 sequences of
SEQ ID NO: 33, 34 and 35; or [0333] a VH region comprising the
CDR1, 2 and 3 sequences of SEQ ID NO: 13, 14 and 15 and a VL region
comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 38, 39 and 40;
or [0334] a VH region comprising the CDR1, 2 and 3 sequences of SEQ
ID NO: 18, 19 and 20 and a VL region comprising the CDR1, 2 and 3
sequences of SEQ ID NO: 43, 44 and 45; or [0335] a VH region
comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 23, 24 and 25
and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID
NO: 48, 49 and 50; or [0336] a variant of any of said antibodies,
wherein said variant preferably only has conservative amino acid
modifications in said sequences.
[0337] In one embodiment, said variant comprises a VH CDR1 which
consists essentially of a sequence having at least about 50%, such
as at least 60%, for instance at least about 70%, such as at least
about 75%, for instance at least about 80%, such as at least about
85%, for instance at least about 90%, such as at least about 95%
amino acid sequence identity to a sequence according to any one of
SEQ ID Nos: 3, 8, 13, 18 or 23;
[0338] In one embodiment, said variant comprises a VH CDR2 which
consists essentially of a sequence having at least about 50%, such
as at least 60%, for instance at least about 70%, such as at least
about 75%, for instance at least about 80%, such as at least about
85%, for instance at least about 90%, such as at least about 95%
amino acid sequence identity to a sequence according to any one of
SEQ ID Nos:4, 9, 14, 19 or 24.
[0339] In one embodiment, said variant comprises a VH CDR3 which
consists essentially of a sequence having at least about 50%, such
as at least 60%, for instance at least about 70%, such as at least
about 75%, for instance at least about 80%, such as at least about
85%, for instance at least about 90%, such as at least about 95%
amino acid sequence identity to a sequence according to any one of
SEQ ID Nos:5, 10, 15, 20 or 25.
[0340] In one embodiment, said variant comprises a VL CDR1 which
consists essentially of a sequence having at least about 50%, such
as at least 60%, for instance at least about 70%, such as at least
about 75%, for instance at least about 80%, such as at least about
85%, for instance at least about 90%, such as at least about 95%
amino acid sequence identity to a sequence according to any one of
SEQ ID Nos:28, 33, 38, 43 or 48.
[0341] In one embodiment, said variant comprises a VL CDR2 which
consists essentially of a sequence having at least about 50%, such
as at least 60%, for instance at least about 70%, such as at least
about 75%, for instance at least about 80%, such as at least about
85%, for instance at least about 90%, such as at least about 95%
amino acid sequence identity to a sequence according to any one of
SEQ ID Nos:29, 34, 39, 44 or 49.
[0342] In one embodiment, said variant comprises a VL CDR3 which
consists essentially of a sequence having at least about 50%, such
as at least 60%, for instance at least about 70%, such as at least
about 75%, for instance at least about 80%, such as at least about
85%, for instance at least about 90%, such as at least about 95%
amino acid sequence identity to a sequence according to any one of
SEQ ID Nos:30, 35, 40, 45 or 50.
[0343] In an even further embodiment, the antibody of the invention
comprises: [0344] a VH region comprising the sequence of SEQ ID NO:
2 and a VL region comprising the sequence of SEQ ID NO: 27; or
[0345] a VH region comprising the sequence of SEQ ID NO: 7 and a VL
region comprising the sequence of SEQ ID NO: 32; or [0346] a VH
region comprising the sequence of SEQ ID NO: 12 and a VL region
comprising the sequence of SEQ ID NO: 37; or [0347] a VH region
comprising the sequence of SEQ ID NO: 17 and a VL region comprising
the sequence of SEQ ID NO: 42; or [0348] a VH region comprising the
sequence of SEQ ID NO: 22 and a VL region comprising the sequence
of SEQ ID NO: 47; or [0349] a variant of any of the above, wherein
said variant preferably only has conservative modifications.
[0350] In one embodiment, said variant comprises a VH region which
consists essentially of a sequence having at least about 50%, such
as at least 60%, for instance at least about 70%, such as at least
about 75%, for instance at least about 80%, such as at least about
85%, for instance at least about 90%, such as at least about 95%
amino acid sequence identity to a sequence according to any one of
SEQ ID Nos:2, 7, 12, 17 or 22.
[0351] In one embodiment, said variant comprises a VL region which
consists essentially of a sequence having at least about 50%, such
as at least 60%, for instance at least about 70%, such as at least
about 75%, for instance at least about 80%, such as at least about
85%, for instance at least about 90%, such as at least about 95%
amino acid sequence identity to a sequence according to any one of
SEQ ID NO: 27, 32, 37, 42 or 47.
[0352] In a further embodiment the antibody of the present
invention comprises a VH having at least 80% identity, such as 90%,
or 95%, or 97%, or 98%, or 99% identity to a VH region sequence
selected from the group consisting of: SEQ ID NO: 2, 7, 12, 17 or
22.
[0353] In a further embodiment the antibody of the present
invention comprises a VL having at least 80% identity, such as 90%,
or 95%, or 97%, or 98%, or 99% identity to a VL region sequence
selected from the group consisting of: SEQ ID NO: 27, 32, 37, 42 or
47.
[0354] In an even further embodiment, the antibody of the invention
comprises a VH region selected from the group consisting of: SEQ ID
NO: 2, 7, 12, 17 or 22.
[0355] In an even further embodiment, the antibody of the invention
comprises a VL region selected from the group consisting of: SEQ ID
NO: 27, 32, 37, 42 or 47.
[0356] In an even further embodiment, the antibody of the invention
comprises: [0357] a VH region comprising the sequence of SEQ ID NO:
2 and a VL region comprising the sequence of SEQ ID NO: 27; or
[0358] a VH region comprising the sequence of SEQ ID NO: 7 and a VL
region comprising the sequence of SEQ ID NO: 32; or [0359] a VH
region comprising the sequence of SEQ ID NO: 12 and a VL region
comprising the sequence of SEQ ID NO: 37; or [0360] a VH region
comprising the sequence of SEQ ID NO: 17 and a VL region comprising
the sequence of SEQ ID NO: 42; or [0361] a VH region comprising the
sequence of SEQ ID NO: 22 and a VL region comprising the sequence
of SEQ ID NO: 47; or
[0362] The present invention also, in one aspect, provides
anti-CD38 antibodies which are characterized with respect to their
ability to compete with an antibody having: [0363] a VH region
comprising the sequence of SEQ ID NO: 2 and a VL region comprising
the sequence of SEQ ID NO: 27; or [0364] a VH region comprising the
sequence of SEQ ID NO: 7 and a VL region comprising the sequence of
SEQ ID NO: 32; or [0365] a VH region comprising the sequence of SEQ
ID NO: 12 and a VL region comprising the sequence of SEQ ID NO: 37;
or [0366] a VH region comprising the sequence of SEQ ID NO: 17 and
a VL region comprising the sequence of SEQ ID NO: 42; or [0367] a
VH region comprising the sequence of SEQ ID NO: 22 and a VL region
comprising the sequence of SEQ ID NO: 47.
[0368] The present invention also relates to provides anti-CD38
antibodies which bind to the same epitope as an antibody having:
[0369] a VH region comprising the sequence of SEQ ID NO: 2 and a VL
region comprising the sequence of SEQ ID NO: 27; or [0370] a VH
region comprising the sequence of SEQ ID NO: 7 and a VL region
comprising the sequence of SEQ ID NO: 32; or [0371] a VH region
comprising the sequence of SEQ ID NO: 12 and a VL region comprising
the sequence of SEQ ID NO: 37; or [0372] a VH region comprising the
sequence of SEQ ID NO: 17 and a VL region comprising the sequence
of SEQ ID NO: 42; or [0373] a VH region comprising the sequence of
SEQ ID NO: 22 and a VL region comprising the sequence of SEQ ID NO:
47.
[0374] The antibody of the invention may be of any isotype. The
choice of isotype typically will be guided by the desired effector
functions, such as ADCC induction. Exemplary isotypes are IgG1,
IgG2, IgG3, and IgG4. Either of the human light chain constant
regions, kappa or lambda, may be used. If desired, the class of an
anti-CD38 antibody of the present invention may be switched by
known methods. For example, an antibody of the present invention
that was originally IgM may be class switched to an IgG antibody of
the present invention. Further, class switching techniques may be
used to convert one IgG subclass to another, for instance from IgG1
to IgG2. Thus, the effector function of the antibodies of the
present invention may be changed by isotype switching to, e.g., an
IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody for various
therapeutic uses. In one embodiment an antibody of the present
invention is an IgG1 antibody, for instance an IgG1,.kappa..
[0375] In one embodiment, the antibody of the invention is a
full-length antibody. In another embodiment, the antibody of the
invention is an antibody fragment or a single-chain antibody.
[0376] Antibody fragments may e.g. be obtained by fragmentation
using conventional techniques, and the fragments screened for
utility in the same manner as described herein for whole
antibodies. For example, F(ab').sub.2 fragments may be generated by
treating antibody with pepsin. The resulting F(ab').sub.2 fragment
may be treated to reduce disulfide bridges to produce Fab'
fragments. Fab fragments may be obtained by treating an IgG
antibody with papain; Fab' fragments may be obtained with pepsin
digestion of IgG antibody. A F(ab') fragment may also be produced
by binding Fab' described below via a thioether bond or a disulfide
bond. A Fab' fragment is an antibody fragment obtained by cutting a
disulfide bond of the hinge region of the F(ab').sub.2. A Fab'
fragment may be obtained by treating a F(ab').sub.2 fragment with a
reducing agent, such as dithiothreitol. Antibody fragments may also
be generated by expression of nucleic acids encoding such fragments
in recombinant cells (see for instance Evans et al., J. Immunol.
Meth. 184, 123-38 (1995)). For example, a chimeric gene encoding a
portion of a F(ab')2 fragment could include DNA sequences encoding
the CH1 domain and hinge region of the H chain, followed by a
translational stop codon to yield such a truncated antibody
fragment molecule.
[0377] In one embodiment, the anti-CD38 antibody is a monovalent
antibody, preferably a monovalent antibody as described in
WO2007059782 (Genmab) (incorporated herein by reference).
Accordingly, in one embodiment, the antibody is a monovalent
antibody, wherein said anti-CD38 antibody is constructed by a
method comprising:
i) providing a nucleic acid construct encoding the light chain of
said monovalent antibody, said construct comprising a nucleotide
sequence encoding the VL region of a selected antigen specific
anti-CD38 antibody and a nucleotide sequence encoding the constant
CL region of an Ig, wherein said nucleotide sequence encoding the
VL region of a selected antigen specific antibody and said
nucleotide sequence encoding the CL region of an Ig are operably
linked together, and wherein, in case of an IgG1 subtype, the
nucleotide sequence encoding the CL region has been modified such
that the CL region does not contain any amino acids capable of
forming disulfide bonds or covalent bonds with other peptides
comprising an identical amino acid sequence of the CL region in the
presence of polyclonal human IgG or when administered to an animal
or human being; ii) providing a nucleic acid construct encoding the
heavy chain of said monovalent antibody, said construct comprising
a nucleotide sequence encoding the VH region of a selected antigen
specific antibody and a nucleotide sequence encoding a constant CH
region of a human Ig, wherein the nucleotide sequence encoding the
CH region has been modified such that the region corresponding to
the hinge region and, as required by the Ig subtype, other regions
of the CH region, such as the CH3 region, does not comprise any
amino acid residues which participate in the formation of
disulphide bonds or covalent or stable non-covalent inter-heavy
chain bonds with other peptides comprising an identical amino acid
sequence of the CH region of the human Ig in the presence of
polyclonal human IgG or when administered to an animal human being,
wherein said nucleotide sequence encoding the VH region of a
selected antigen specific antibody and said nucleotide sequence
encoding the CH region of said Ig are operably linked together;
iii) providing a cell expression system for producing said
monovalent antibody; iv) producing said monovalent antibody by
co-expressing the nucleic acid constructs of (i) and (ii) in cells
of the cell expression system of (iii).
[0378] Similarly, in one embodiment, the anti-CD38 antibody is a
monovalent antibody, which comprises
(i) a variable region of an antibody of the invention as described
herein or an antigen binding part of the said region, and (ii) a
C.sub.H region of an immunoglobulin or a fragment thereof
comprising the C.sub.H2 and C.sub.H3 regions, wherein the C.sub.H
region or fragment thereof has been modified such that the region
corresponding to the hinge region and, if the immunoglobulin is not
an IgG4 subtype, other regions of the C.sub.H region, such as the
C.sub.H3 region, do not comprise any amino acid residues, which are
capable of forming disulfide bonds with an identical C.sub.H region
or other covalent or stable non-covalent inter-heavy chain bonds
with an identical C.sub.H region in the presence of polyclonal
human IgG.
[0379] In a further embodiment, the heavy chain of the monovalent
anti-CD38 antibody has been modified such that the entire hinge has
been deleted.
[0380] In a further embodiment, said monovalent antibody is of the
IgG4 subtype, but the C.sub.H3 region has been modified so that one
or more of the following amino acid substitutions have been made:
Thr (T) in position 366 has been replaced by Ala (A); Leu (L) in
position 368 has been replaced by Ala (A); Leu (L) in position 368
has been replaced by Val (V); Phe (F) in position 405 has been
replaced by Ala (A); Phe (F) in position 405 has been replaced by
Leu (L); Tyr (Y) in position 407 has been replaced by Ala (A); Arg
(R) in position 409 has been replaced by Ala (A).
[0381] In another further embodiment, the sequence of said
monovalent antibody has been modified so that it does not comprise
any acceptor sites for N-linked glycosylation.
[0382] Anti-CD38 antibodies of the invention also include single
chain antibodies. Single chain antibodies are peptides in which the
heavy and light chain Fv regions are connected. In one embodiment,
the present invention provides a single-chain Fv (scFv) wherein the
heavy and light chains in the Fv of an anti-CD38 antibody of the
present invention are joined with a flexible peptide linker
(typically of about 10, 12, 15 or more amino acid residues) in a
single peptide chain. Methods of producing such antibodies are
described in for instance U.S. Pat. No. 4,946,778, Pluckthun in The
Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and
Moore eds. Springer-Verlag, New York, pp. 269-315 (1994), Bird et
al., Science 242, 423-426 (1988), Huston et al., PNAS USA 85,
5879-5883 (1988) and McCafferty et al., Nature 348, 552-554 (1990).
The single chain antibody may be monovalent, if only a single
V.sub.H and V.sub.L are used, bivalent, if two V.sub.H and V.sub.L
are used, or polyvalent, if more than two V.sub.H and V.sub.L are
used.
[0383] In one embodiment, the anti-CD38 antibody of the invention
is an effector-function-deficient antibody. Such antibodies are
particularly useful when the antibody is for use in stimulation and
demping of the immune system through blocking of the inhibitory
effects of CD38. For such applications, it may be advantageous that
the antibody has no effector functions, such as ADCC, as this may
lead to undesired cytotoxicity.
[0384] In one embodiment, the effector-function-deficient anti-CD38
antibody is a stabilized IgG4 antibody. Examples of suitable
stabilized IgG4 antibodies are antibodies, wherein arginine at
position 409 in a heavy chain constant region of human IgG4, which
is indicated in the EU index as in Kabat et al., is substituted
with lysine, threonine, methionine, or leucine, preferably lysine
(described in WO2006033386 (Kirin)). Preferably, said antibody
comprises a Lys or Ala residue at the position corresponding to 409
or the CH3 region of the antibody has been replaced by the CH3
region of human IgG1, of human IgG2 or of human IgG3.
[0385] In a further embodiment. the stabilized IgG4 anti-CD38
antibody is an IgG4 antibody comprising a heavy chain and a light
chain, wherein said heavy chain comprises a human IgG4 constant
region having a residue selected from the group consisting of: Lys,
Ala, Thr, Met and Leu at the position corresponding to 409 and/or a
residue selected from the group consisting of: Ala, Val, Gly, Ile
and Leu at the position corresponding to 405, and wherein said
antibody optionally comprises one or more further substitutions,
deletions and/or insertions, but does not comprise a
Cys-Pro-Pro-Cys sequence in the hinge region. Preferably, said
antibody comprises a Lys or Ala residue at the position
corresponding to 409 or the CH3 region of the antibody has been
replaced by the CH3 region of human IgG1, of human IgG2 or of human
IgG3.
[0386] In an even further embodiment. the stabilized IgG4 anti-CD38
antibody is an IgG4 antibody comprising a heavy chain and a light
chain, wherein said heavy chain comprises a human IgG4 constant
region having a residue selected from the group consisting of: Lys,
Ala, Thr, Met and Leu at the position corresponding to 409 and/or a
residue selected from the group consisting of: Ala, Val, Gly, Ile
and Leu at the position corresponding to 405, and wherein said
antibody optionally comprises one or more further substitutions,
deletions and/or insertions and wherein said antibody comprises a
Cys-Pro-Pro-Cys sequence in the hinge region. Preferably, said
antibody comprises a Lys or Ala residue at the position
corresponding to 409 or the CH3 region of the antibody has been
replaced by the CH3 region of human IgG1, of human IgG2 or of human
IgG3.
[0387] In a further embodiment, the effector-function-deficient
anti-CD38 antibody is an antibody of a non-IgG4 type, e.g. IgG1,
IgG2 or IgG3 which has been mutated such that the ability to
mediate effector functions, such as ADCC, has been reduced or even
eliminated. Examples of such mutations have e.g. been described in
Dall'Acqua W F et al., J Immunol. 177(2):1129-1138 (2006) and
Hezareh M, J Virol.; 75(24):12161-12168 (2001).
Conjugates
[0388] In a further embodiment. the antibody of the invention is
conjugated to another moiety, such as a cytotoxic moiety, a
radioisotope or a drug.
[0389] Such antibodies may be produced by chemically conjugating
the other moiety to the N-terminal side or C-terminal side of the
anti-CD38 antibody or fragment thereof (e.g., an anti-CD38 antibody
H chain, L chain, or anti-CD38 specific/selective fragment thereof)
(see, e.g., Antibody Engineering Handbook, edited by Osamu
Kanemitsu, published by Chijin Shokan (1994)). Such conjugated
antibody derivatives may also be generated by conjugation at
internal residues or sugars, where appropriate.
[0390] Anti-CD38 antibodies described herein may also be modified
by inclusion of any suitable number of modified amino acids.
Suitability in this context is generally determined by the ability
to at least substantially retain CD38 selectivity and/or
specificity associated with the non-derivatized parent anti-CD38
antibody. The inclusion of one or more modified amino acids may be
advantageous in, for example, increasing polypeptide serum
half-life, reducing polypeptide antigenicity, or increasing
polypeptide storage stability. Amino acid(s) are modified, for
example, co-translationally or post-translationally during
recombinant production (e.g., N-linked glycosylation at N-X-S/T
motifs during expression in mammalian cells) or modified by
synthetic means. Non-limiting examples of a modified amino acid
include a glycosylated amino acid, a sulfated amino acid, a
prenylated (e.g., farnesylated, geranylgeranylated) amino acid, an
acetylated amino acid, an acylated amino acid, a PEGylated amino
acid, a biotinylated amino acid, a carboxylated amino acid, a
phosphorylated amino acid, and the like. References adequate to
guide one of skill in the modification of amino acids are replete
throughout the literature. Example protocols are found in Walker
(1998) Protein Protocols On Cd-Rom, Humana Press, Towata, N.J.
[0391] Anti-CD38 antibodies may also be chemically modified by
covalent conjugation to a polymer to for instance increase their
circulating half-life. Exemplary polymers, and methods to attach
them to peptides, are illustrated in for instance U.S. Pat. Nos.
4,766,106, 4,179,337, 4,495,285 and 4,609,546.
[0392] In one embodiment, the present invention provides an
anti-CD38 antibody that is conjugated to a second molecule that is
selected from a radionuclide, an enzyme, an enzyme substrate, a
cofactor, a fluorescent marker, a chemiluminescent marker, a
peptide tag, or a magnetic particle. In one embodiment, an
anti-CD38 antibody may be conjugated to one or more antibody
fragments, nucleic acids (oligonucleotides), nucleases, hormones,
immunomodulators, chelators, boron compounds, photoactive agents,
dyes, and the like. These and other suitable agents may be coupled
either directly or indirectly to an anti-CD38 antibody of the
present invention. One example of indirect coupling of a second
agent is coupling by a spacer moiety.
[0393] In one embodiment, anti-CD38 antibodies comprising one or
more radiolabeled amino acids are provided. A radiolabeled
anti-CD38 antibody may be used for both diagnostic and therapeutic
purposes (conjugation to radiolabeled molecules is another possible
feature). Nonlimiting examples of labels for polypeptides include,
but are not limited to 3H, 14C, 15N, 35S, 90Y, 99Tc, and 125I,
131I, and 186Re. Methods for preparing radiolabeled amino acids and
related peptide derivatives are known in the art (see for instance
Junghans et al., in Cancer Chemotherapy and Biotherapy 655-686 (2d
edition, Chafner and Longo, eds., Lippincott Raven (1996)) and U.S.
Pat. Nos. 4,681,581, 4,735,210, 5,101,827, 5,102,990 (U.S.
RE35,500), 5,648,471 and 5,697,902.
[0394] In one embodiment, the anti-CD38 antibody of the invention
is conjugated to a radioisotope or to a radioisotope-containing
chelate. For example, the anti-CD38 antibody can be conjugated to a
chelator linker, e.g. DOTA, DTPA or tiuxetan, which allows for the
anti-CD38 antibody to be complexed with a radioisotope. The
anti-CD38 antibody may also or alternatively comprise or be
conjugated to one or more radiolabeled amino acids or other
radiolabeled molecule. A radiolabeled anti-CD38 antibody may be
used for both diagnostic and therapeutic purposes. Non-limiting
examples of radioisotopes include 3H, 14C, 15N, 35S, 90Y, 99Tc,
125I, 111In, 131I, 186Re, 213Bs, 225Ac and 227Th.
[0395] In one embodiment, the anti-CD38 antibody of the invention
is conjugated to auristatins or auristatin peptide analogs and
derivates (U.S. Pat. Nos. 5,635,483; 5,780,588). Auristatins have
been shown to interfere with microtubule dynamics, GTP hydrolysis
and nuclear and cellular division (Woyke et al (2001) Antimicrob.
Agents and Chemother. 45(12): 3580-3584) and have anticancer (U.S.
Pat. No. 5,663,149) and antifungal activity (Pettit et al., (1998)
Antimicrob. Agents and Chemother. 42:2961-2965. The auristatin drug
moiety may be attached to the antibody, via an linker, through the
N (amino) terminus or the C (terminus) of the peptidic drug
moiety.
[0396] Exemplary auristatin embodiments include the
N-terminus-linked monomethyl auristatin drug moieties DE and DF,
disclosed in Senter et al., Proceedings of the American Association
for Cancer Research. Volume 45, abstract number 623, presented Mar.
28, 2004 and described in US 2005/0238648).
[0397] An exemplary auristatin embodiment is MMAE (monomethyl
auristatin E), wherein the wavy line indicates the covalent
attachment to the linker (L) of an antibody drug conjugate:
##STR00001##
[0398] Another exemplary auristatin embodiment is MMAF (monomethyl
auristatin F), wherein the wavy line indicates the covalent
attachment to a linker (L) of an antibody drug conjugate
(US2005/0238649):
##STR00002##
[0399] The anti-CD38 antibody drug conjugates according to the
invention comprise a linker unit between the cytostatic drug unit
and the antibody unit. In some embodiments, the linker is cleavable
under intracellular conditions, such that the cleavage of the
linker releases the drug unit from the antibody in the
intracellular environment. In yet another embodiment, the linker
unit is not cleavable and the drug is for instance released by
antibody degradation. In some embodiments, the linker is cleavable
by a cleavable agent that is present in the intracellular
environment (e.g. within a lysosome or endosome or caveola). The
linker can be, e.g. a peptidyl linker that is cleaved by an
intracellular peptidase or protease enzyme, including but not
limited to, a lysosomal or endosomal protease. In some embodiments,
the peptidyl linker is at least two amino acids long or at least
three amino acids long. Cleaving agents can include cathepsins B
and D and plasmin, all of which are known to hydrolyze dipeptide
drug derivatives resulting in the release of active drug inside the
target cells (see e.g. Dubowchik and Walker, 1999, Pharm.
Therapeutics 83:67-123). In a specific embodiment, the peptidyl
linker cleavable by an intracellular protease is a Val-Cit
(valine-citrulline) linker or a Phe-Lys (phenylalanine-lysine)
linker (see e.g. U.S. Pat. No. 6,214,345, which describes the
synthesis of doxorubicin with the Val-Cit linker). An advantage of
using intracellular proteolytic release of the therapeutic agent is
that the agent is typically attenuated when conjugated and the
serum stabilities of the conjugates are typically high.
[0400] In yet another embodiment, the linker unit is not cleavable
and the drug is released by antibody degradation (see US
2005/0238649). Typically, such a linker is not substantially
sensitive to the extracellular environment. As used herein, "not
substantially sensitive to the extracellular environment" in the
context of a linker means that no more than 20%, typically no more
than about 15%, more typically no more than about 10%, and even
more typically no more than about 5%, no more than about 3%, or no
more than about 1% of the linkers, in a sample of antibody drug
conjugate compound, are cleaved when the antibody drug conjugate
compound presents in an extracellular environment (e.g. plasma).
Whether a linker is not substantially sensitive to the
extracellular environment can be determined for example by
incubating with plasma the antibody drug conjugate compound for a
predetermined time period (e.g. 2, 4, 8, 16 or 24 hours) and then
quantitating the amount of free drug present in the plasma.
[0401] Additional exemplary embodiments comprising MMAE or MMAF and
various linker components have the following structures (wherein Ab
means antibody and p, representing the drug-loading (or average
number of cytostatic drugs per molecule), is 1 to about 8).
[0402] Examples where a cleavable linker is combined with an
auristatin include vcMMAF and vcMMAE (vc is the abbreviation for
the Val-Cit (valine-citruline) based linker):
##STR00003##
[0403] Other examples include auristatins combined with a
non-cleavable linker, such as mcMMAF. (mc is an abbreviation of
maleimido caproyl):
##STR00004##
[0404] The cytostatic drug loading is represented by p and is the
average number of cytostatic drug moieties per antibody in a
molecule (also designated as the drug to antibody ratio, DAR). The
cytostatic drug loading may range from 1 to 20 drug moieties per
antibody and may occur on amino acids with useful functional groups
such as, but not limited to, amino or sulfhydryl groups, as in
lysine or cysteine.
[0405] Depending on the way of conjugation, p may be limited by the
number of attachment sites on the antibody, for example where the
attachment is a cysteine thiol, as in the present invention.
Generally, antibodies do not contain many free and reactive
cysteine thiol groups which may be linked to a drug moiety as most
cysteine thiol residues in antibodies exist as disulfide bridges.
Therefore, in certain embodiments, an antibody may be reduced with
reducing agent such as dithiothreitol (DTT) or
tricarbonylethylphosphine (TCEP), under partial or fully reducing
conditions, to generate reactive cysteine thiol groups. In certain
embodiments, the drug loading for an ADC of the invention ranges
from 1 to about 8, as a maximum of 8 free cysteine thiol groups
becomes available after (partial) reduction of the antibody (there
are 8 cysteines involved in inter-chain disulfide bonding).
[0406] In one embodiment, the drug linker moiety is vcMMAE. The
vcMMAE drug linker moiety and conjugation methods are disclosed in
WO2004010957, U.S. Pat. Nos. 7,659,241, 7,829,531, 7,851,437 and
U.S. Ser. No. 11/833,028 (Seattle Genetics, Inc.), (which are
incorporated herein by reference), and the vcMMAE drug linker
moiety is bound to the anti-CD38 antibodies at the cysteines using
a method similar to those disclosed in therein.
In one embodiment, the drug linker moiety is mcMMAF. The mcMMAF
drug linker moiety and conjugation methods are disclosed in U.S.
Pat. No. 7,498,298, U.S. Ser. No. 11/833,954, and WO2005081711
(Seattle Genetics, Inc.) (which are incorporated herein by
reference), and the mcMMAF drug linker moiety is bound to the
anti-CD38 antibodies at the cysteines using a method similar to
those disclosed in therein.
[0407] Upon purifying the anti-CD38 antibody drug conjugates they
may be formulated into pharmaceutical compositions using well known
pharmaceutical carriers or excipients.
[0408] In one embodiment, an anti-CD38 antibody is conjugated to a
functional nucleic acid molecule. Functional nucleic acids include
antisense molecules, interfering nucleic acid molecules (e.g.,
siRNA molecules), aptamers, ribozymes, triplex forming molecules,
and external guide sequences. External guide sequences (EGSs) are
molecules that bind a target nucleic acid molecule forming a
complex that is recognized by RNase P, which cleaves the target
molecule. The functional nucleic acid molecules may act as
effectors, inhibitors, modulators, and stimulators of a specific
activity possessed by a target molecule, or the functional nucleic
acid molecules may possess a de novo activity independent of any
other molecules. A representative sample of methods and techniques
which aid in the design and use of antisense molecules may be found
in the following non-limiting list of US patents: U.S. Pat. Nos.
5,135,917, 5,294,533, 5,627,158, 5,641,754, 5,691,317, 5,780,607,
5,786,138, 5,849,903, 5,856,103, 5,919,772, 5,955,590, 5,990,088,
5,994,320, 5,998,602, 6,005,095, 6,007,995, 6,013,522, 6,017,898,
6,018,042, 6,025,198, 6,033,910, 6,040,296, 6,046,004, 6,046,319
and 6,057,437.
[0409] Any method known in the art for conjugating the anti-CD38
antibody to the conjugated molecule(s), such as those described
above, may be employed, including those methods described by Hunter
et al., Nature 144, 945 (1962), David et al., Biochemistry 13, 1014
(1974), Pain et al., J. Immunol. Meth. 40, 219 (1981) and Nygren,
J. Histochem. and Cytochem. 30, 407 (1982). Linkage/conjugation may
be accomplished in any suitable way. For example, a covalent
linkage may take the form of a disulfide bond (if necessary and
suitable, an anti-CD38 antibody could be engineered to contain an
extra cysteine codon. A toxin molecule, derivatized with a
sulfhydryl group reactive with the cysteine of the modified
anti-CD38 antibody, may form an immunoconjugate with the anti-CD38
antibody. Alternatively, a sulfhydryl group may be introduced
directly to an anti-CD38 antibody using solid phase polypeptide
techniques. For example, the introduction of sulfhydryl groups into
peptides is described by Hiskey, Peptides 3, 137 (1981). The
introduction of sulfhydryl groups into proteins is described in
Maasen et al., Eur. J. Biochem. 134, 32 (1983).
[0410] Numerous types of cytotoxic compounds may be joined to
proteins through the use of a reactive group on the cytotoxic
compound or through the use of a cross-linking agent. A common
reactive group that will form a stable covalent bond in vivo with
an amine is isothiocyanate (Means et al., Chemical modifications of
proteins (Holden-Day, San Francisco 1971) pp. 105-110). This group
preferentially reacts with the .epsilon.-amine group of lysine.
Maleimide is a commonly used reactive group to form a stable in
vivo covalent bond with the sulfhydryl group on cysteine (Ji.,
Methods Enzymol 91, 580-609 (1983)). Monoclonal antibodies
typically are incapable of forming covalent bonds with radiometal
ions, but they may be attached to the antibody indirectly through
the use of chelating agents that are covalently linked to the
antibodies. Chelating agents may be attached through amines (Meares
et al., Anal. Biochem. 142, 68-78 (1984)) and sulfhydral groups
(Koyama, Chem. Abstr. 120, 217262t (1994)) of amino acid residues
and also through carbohydrate groups (Rodwell et al., PNAS USA 83,
2632-2636 (1986), Quadri et al., Nucl. Med. Biol. 20, 559-570
(1993)). A therapeutic or diagnostic agent may also or
alternatively be attached at the hinge region of a reduced antibody
component via disulfide bond formation.
[0411] In one embodiment, the present invention provides an
anti-CD38 antibody, such as a human anti-CD38 antibody, conjugated
to a therapeutic moiety, such as a cytotoxin, a chemotherapeutic
drug, an immunosuppressant, or a radioisotope. Such conjugates are
referred to herein as "immunoconjugates". Immunoconjugates which
include one or more cytotoxins are referred to as "immunotoxins". A
cytotoxin or cytotoxic agent includes any agent that is detrimental
to (e.g., kills) cells. For a description of these classes of drugs
which are well known in the art, and their mechanisms of action,
see Goodman et al., Goodman and Gilman's The Pharmacological Basis
Of Therapeutics, 8th Ed., Macmillan Publishing Co., 1990.
Additional techniques relevant to the preparation of antibody
immunotoxins are provided in for instance Vitetta, Immunol. Today
14, 252 (1993) and U.S. Pat. No. 5,194,594.
[0412] Suitable therapeutic agents for forming immunoconjugates of
the present invention include taxol, cytochalasin B, gramicidin D,
ethidium bromide, emetine, mitomycin, etoposide, tenoposide,
vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,
dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin
D, 1-dehydro-testosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin, antimetabolites (such as
methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,
fludarabin, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase,
gemcitabine, cladribine), alkylating agents (such as
mechlorethamine, thioepa, chlorambucil, melphalan, carmustine
(BSNU), lomustine (CCNU), cyclophosphamide, busulfan,
dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine,
mitomycin C, cisplatin and other platinum derivatives, such as
carboplatin), antibiotics (such as dactinomycin (formerly
actinomycin), bleomycin, daunorubicin (formerly daunomycin),
doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone,
plicamycin, anthramycin (AMC)), diphtheria toxin and related
molecules (such as diphtheria A chain and active fragments thereof
and hybrid molecules), ricin toxin (such as ricin A or a
deglycosylated ricin A chain toxin), cholera toxin, a Shiga-like
toxin (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin,
pertussis toxin, tetanus toxin, soybean Bowman-Birk protease
inhibitor, Pseudomonas exotoxin, alorin, saporin, modeccin,
gelanin, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites
fordii proteins, dianthin proteins, Phytolacca americana proteins
(PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin,
crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin,
restrictocin, phenomycin, enomycin toxins, calicheamicins and
duocarmycins. Therapeutic agents, which may be administered in
combination with a an anti-CD38 antibody of the present invention
as described elsewhere herein, may also be candidates for
therapeutic moieties useful for conjugation to an anti-CD38
antibody of the present invention.
[0413] As indicated above, the drug moiety need not be construed as
limited to classical chemical therapeutic agents. For example, the
drug moiety may be a protein or polypeptide possessing a desired
biological activity. In one embodiment, the anti-CD38 antibody of
the present invention is attached to a chelator linker, e.g.
tiuxetan, which allows for the antibody to be conjugated to a
radioisotope.
Bispecific Antibodies
[0414] In a further aspect, the invention relates to a bispecific
molecule comprising a first antigen binding site from an anti-CD38
antibody of the invention as described herein above and a second
antigen binding site with a different binding specificity, such as
a binding specificity for a human effector cell, a human Fc
receptor, a T cell receptor, a B cell receptor or a binding
specificity for a non-overlapping epitope of CD38, i.e. a
bispecific antibody wherein the first and second antigen binding
sites do not cross-block each other for binding to CD38, e.g. when
tested as described in Example 3.
[0415] Exemplary bispecific antibody molecules of the invention
comprise (i) two antibodies, one with a specificity to CD38 and
another to a second target that are conjugated together, (ii) a
single antibody that has one chain or arm specific to CD38 and a
second chain or arm specific to a second molecule, (iii) a single
chain antibody that has specificity to CD38 and a second molecule,
e.g., via two scFvs linked in tandem by an extra peptide linker;
(iv) a dual-variable-domain antibody (DVD-Ig), where each light
chain and heavy chain contains two variable domains in tandem
through a short peptide linkage (Wu et al., Generation and
Characterization of a Dual Variable Domain Immunoglobulin
(DVD-Ig.TM.) Molecule, In: Antibody Engineering, Springer Berlin
Heidelberg (2010)); (v) a chemically-linked bispecific (Fab').sub.2
fragment; (vi) a Tandab, which is a fusion of two single chain
diabodies resulting in a tetravalent bispecific antibody that has
two binding sites for each of the target antigens; (vii) a
flexibody, which is a combination of scFvs with a diabody resulting
in a multivalent molecule; (viii) a so called "dock and lock"
molecule, based on the "dimerization and docking domain" in Protein
Kinase A, which, when applied to Fabs, can yield a trivalent
bispecific binding protein consisting of two identical Fab
fragments linked to a different Fab fragment; (ix) a so-called
Scorpion molecule, comprising, e.g., two scFvs fused to both
termini of a human Fc-region; and (x) a diabody. In one embodiment,
the bispecific antibody of the present invention is a diabody, a
cross-body, or a bispecific obtained via a controlled Fab arm
exchange as those described in the present invention.
[0416] Examples of platforms useful for preparing bispecific
antibodies include but are not limited to BITE (Micromet), DART
(MacroGenics), Fcab and Mab.sup.2 (F-star), Fc-engineered IgG1
(Xencor) or DuoBody (based on Fab arm exchange, Genmab, this
application).
[0417] Examples of different classes of bispecific antibodies
include but are not limited to [0418] asymmetric IgG-like
molecules, wherein the one side of the molecule contains the Fab
region or part of the Fab region of at least one antibody, and the
other side of the molecule contains the Fab region or parts of the
Fab region of at least one other antibody; in this class, asymmetry
in the Fc region could also be present, and be used for specific
linkage of the two parts of the molecule; [0419] symmetric IgG-like
molecules, wherein the two sides of the molecule each contain the
Fab region or part of the Fab region of at least two different
antibodies; [0420] IgG fusion molecules, wherein full length IgG
antibodies are fused to extra Fab regions or parts of Fab regions;
[0421] Fc fusion molecules, wherein single chain Fv molecules or
stabilized diabodies are fused to Fc.gamma. regions or parts
thereof; [0422] Fab fusion molecules, wherein different
Fab-fragments are fused together; [0423] ScFv- and diabody-based
molecules wherein different single chain Fv molecules or different
diabodies are fused to each other or to another protein or carrier
molecule.
[0424] Examples of asymmetric IgG-like molecules include but are
not limited to the Triomab/Quadroma (Trion Pharma/Fresenius
Biotech), the Knobs-into-Holes (Genentech), CrossMAbs (Roche) and
the electrostatically-matched (Amgen), the LUZ-Y (Genentech), the
Strand Exchange Engineered Domain body (EMD Serono), the Biclonic
(Merus) and the DuoBody (Genmab A/S).
[0425] Example of symmetric IgG-like molecules include but are not
limited to Dual Targeting (DT)-Ig (GSK/Domantis), Two-in-one
Antibody (Genentech), Cross-linked Mabs (Karmanos Cancer Center),
mAb.sup.2 (F-Star) and CovX-body (CovX/Pfizer).
[0426] Examples of IgG fusion molecules include but are not limited
to Dual Variable Domain (DVD)-Ig (Abbott), IgG-like Bispecific
(ImClone/Eli Lilly), Ts2Ab (MedImmune/AZ) and BsAb (Zymogenetics),
HERCULES (Biogen Idec) and TvAb (Roche).
[0427] Examples of Fc fusion molecules include but are not limited
to ScFv/Fc Fusions (Academic Institution), SCORPION (Emergent
BioSolutions/Trubion, Zymogenetics/BMS), Dual Affinity Retargeting
Technology (Fc-DART) (MacroGenics) and Dual(ScFv).sub.2-Fab
(National Research Center for Antibody Medicine--China).
[0428] Examples of class V bispecific antibodies include but are
not limited to F(ab).sub.2 (Medarex/Amgen), Dual-Action or Bis-Fab
(Genentech), Dock-and-Lock (DNL) (ImmunoMedics), Bivalent
Bispecific (Biotecnol) and Fab-Fv (UCB-Celltech).
Examples of ScFv- and diabody-based molecules include but are not
limited to Bispecific T Cell Engager (BITE) (Micromet9, Tandem
Diabody (Tandab) (Affimed), Dual Affinity Retargeting Technology
(DART) (MacroGenics), Single-chain Diabody (Academic), TCR-like
Antibodies (AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion
(Merrimack) and COMBODY (Epigen Biotech).
[0429] In a further aspect, the invention relates to a bispecific
molecule comprising an anti-CD38 antibody of the invention as
described herein above and a second binding specificity such as a
binding specificity for human cytokines. In one embodiment, said
cytokine is an anti-inflammatory cytokine such as IL-1ra, IL-4,
IL-6, IL-10, IL-11, IL-13, IL-16, IFN-alpha and TGF-beta. In
another embodiment said cytokine is a pro-inflammatory cytokine
such as IL-1alpha, IL-1beta and IL-6. In an embodiment the binding
specificity is for a human effector cell, a human Fc receptor or a
T cell receptor. In one embodiment, said T cell receptor is CD3. In
another embodiment, said human Fc receptor is human Fc.gamma.RI
(CD64), human Fc.gamma.RII (CD32), Fc.gamma.RIII (CD16) or a human
Fc.alpha. receptor (CD89). Bispecific molecules of the present
invention may further include a third binding specificity, in
addition to an anti-CD38 binding specificity and a binding
specificity for a human effector cell, a human Fc receptor or a T
cell receptor.
[0430] Exemplary bispecific antibody molecules of the invention
comprise (i) two antibodies one with a specificity to CD38 and
another to a second target that are conjugated together, (ii) a
single antibody that has one chain specific to CD38 and a second
chain specific to a second molecule, and (iii) a single chain
antibody that has specificity to CD38 and a second molecule. In one
embodiment, the second molecule is a cancer
antigen/tumor-associated antigen such as CD20, carcinoembryonic
antigen (CEA), prostate specific antigen (PSA), RAGE (renal
antigen), .alpha.-fetoprotein, CAMEL (CTL-recognized antigen on
melanoma), CT antigens (such as MAGE-B5, -B6, -C2, -C3, and D;
Mage-12; CT10; NY-ESO-1, SSX-2, GAGE, BAGE, MAGE, and SAGE), mucin
antigens (e.g., MUC1, mucin-CA125, etc.), ganglioside antigens,
tyrosinase, gp75, C-myc, Marti, MelanA, MUM-1, MUM-2, MUM-3,
HLA-B7, and Ep-CAM. In one embodiment, the second molecule is a
cancer-associated integrin, such as .alpha.5.beta.3 integrin. In
one embodiment, the second molecule is an angiogenic factor or
other cancer-associated growth factor, such as a vascular
endothelial growth factor (VEGF), a fibroblast growth factor (FGF),
epidermal growth factor (EGF), epidermal growth factor receptor
(EGFR), angiogenin, and receptors thereof, particularly receptors
associated with cancer progression (for instance one of the
HER1-HER4 receptors). Other cancer progression-associated proteins
discussed herein may also be suitable second molecules.
[0431] In an embodiment of the invention, the antibody is a single
antibody that has one chain specific to the CD38 epitope described
in this invention comprising aspartic acid at position 202 and a
second chain specific for a CD38 specific epitope that does not
comprise the aspartic acid at position 202 (a non-competing
antibody). Such antibody is described for example in WO2006099875
as antibody 003.
[0432] In one embodiment, a bispecific antibody of the present
invention is a diabody.
Generation of Bispecific Antibodies by 2-MEA-Induced Fab-Arm
Exchange
[0433] An in vitro method for producing bispecific antibodies is
described in WO 2008119353 (Genmab) and reported by van der
Neut-Kolfschoten et al. (Science. 2007 Sep. 14; 317(5844):1554-7).
Herein, a bispecific antibody is formed by "Fab-arm" or
"half-molecule" exchange (swapping of a heavy chain and attached
light chain) between two monospecific IgG4- or IgG4-like antibodies
upon incubation under mildly reducing conditions. This Fab-arm
exchange reaction is the result of a disulfide-bond isomerization
reaction wherein the inter heavy-chain disulfide bonds in the hinge
regions of monospecific antibodies are reduced and the resulting
free cysteines form a new inter heavy-chain disulfide bond with
cysteine residues of another antibody molecule with a different
specificity. The resulting product is a bispecific antibody having
two Fab arms with different sequences.
[0434] In a novel invention the knowledge of this natural IgG4
Fab-arm exchange is adapted to generate a method to produce stable
IgG1-based bispecific antibodies. The bispecific antibody product
generated by this method described below will no longer participate
in IgG4 Fab-arm exchange. The basis for this method is the use of
complimentary CH3 domains, which promote the formation of
heterodimers under specific assay conditions. To enable the
production of bispecific antibodies by this method, IgG1 molecules
carrying certain mutations in the CH3 domain were generated: in one
of the parental IgG1 antibody T350I, K370T and F405L mutations in
the other parental IgG1 antibody the K409R mutation.
[0435] To generate bispecific antibodies, these two parental
antibodies, each antibody at a final concentration of 0.5 mg/mL
(equimolar concentration), were incubated with 25 mM
2-mercaptoethylamine-HCl (2-MEA) in a total volume of 100 .mu.L TE
at 37.degree. C. for 90 min. The reduction reaction is stopped when
the reducing agent 2-MEA is removed by using spin columns (Microcon
centrifugal filters, 30 k, Millipore) according to the
manufacturer's protocol. By this method the following bispecific
antibodies may be generated:
[0436] A bispecfic antibody wherein the anti-CD38 antibody is 025,
026, 028, 049 or 056, and the second binding moiety is an anti-CD3
antibody.
[0437] A bispecfic antibody wherein the anti-CD38 antibody is 025,
026, 028, 049 or 056, and the second binding moiety is an anti-CD20
antibody, such as ofatumumab.
[0438] A bispecfic antibody wherein the anti-CD38 antibody is 025,
026, 028, 049 or 056, and the second binding moiety is an anti-CD16
antibody.
[0439] A bispecfic antibody wherein the anti-CD38 antibody is 025,
026, 028, 049 or 056, and the second binding moiety is an anti-CD32
antibody.
[0440] A bispecfic antibody wherein the anti-CD38 antibody is 025,
026, 028, 049 or 056, and the second binding moiety is an anti-CD64
antibody.
Nucleic Acids, Vectors, Host Cells and Method for Producing
Antibodies of the Invention
[0441] In a further aspect, the invention relates to nucleic acids
encoding (parts of) an antibody of the invention and to expression
vectors comprising such nucleic acids.
[0442] In one embodiment, the expression vector of the invention
comprises a nucleotide sequence encoding one or more of the amino
acid sequences selected from the group consisting of: SEQ ID NO: 1
and SEQ ID NO: 5.
[0443] In a further embodiment, the expression vector further
comprises a nucleotide sequence encoding the constant region of a
light chain, a heavy chain or both light and heavy chains of an
antibody, e.g. a human antibody.
[0444] Such expression vectors may be used for recombinant
production of antibodies of the invention.
[0445] An expression vector in the context of the present invention
may be any suitable vector, including chromosomal, non-chromosomal,
and synthetic nucleic acid vectors (a nucleic acid sequence
comprising a suitable set of expression control elements). Examples
of such vectors include derivatives of SV40, bacterial plasmids,
phage DNA, baculovirus, yeast plasmids, vectors derived from
combinations of plasmids and phage DNA, and viral nucleic acid (RNA
or DNA) vectors. In one embodiment, an anti-CD38 antibody-encoding
nucleic acid is comprised in a naked DNA or RNA vector, including,
for example, a linear expression element (as described in for
instance Sykes and Johnston, Nat Biotech 17, 355-59 (1997)), a
compacted nucleic acid vector (as described in for instance U.S.
Pat. No. 6,077,835 and/or WO 00/70087), a plasmid vector such as
pBR322, pUC 19/18, or pUC 118/119, a "midge" minimally-sized
nucleic acid vector (as described in for instance Schakowski et
al., Mol Ther 3, 793-800 (2001)), or as a precipitated nucleic acid
vector construct, such as a CaP04-precipitated construct (as
described in for instance WO 00/46147, Benvenisty and Reshef, PNAS
USA 83, 9551-55 (1986), Wigler et al., Cell 14, 725 (1978), and
Coraro and Pearson, Somatic Cell Genetics 7, 603 (1981)). Such
nucleic acid vectors and the usage thereof are well known in the
art (see for instance U.S. Pat. Nos. 5,589,466 and 5,973,972).
[0446] In one embodiment, the vector is suitable for expression of
the anti-CD38 antibody in a bacterial cell. In another embodiment,
the expression vector may be a vector suitable for expression in a
yeast system. Most typically, the vector will be a vector suitable
for expression of the antibody of the invention in a mammalian
cell, such as a CHO, HEK or PER.C6.RTM. cell (human cell line
developed by DSM and Crucell N.V., the Netherlands). Another
suitable vector system is the glutamine synthetase (GS) vector
system developed by Lonza Biologics (see e.g. EP216846, U.S. Pat.
No. 5,981,216, WO8704462, EP323997, U.S. Pat. Nos. 5,591,639,
5,658,759, EP338841, U.S. Pat. Nos. 5,879,936, and 5,891,693).
[0447] In an expression vector of the invention, anti-CD38
antibody-encoding nucleic acids may comprise or be associated with
any suitable promoter, enhancer, and other expression-facilitating
elements. Examples of such elements include strong expression
promoters (e.g., human CMV IE promoter/enhancer as well as RSV,
SV40, SL3-3, MMTV, and HIV LTR promoters), effective poly (A)
termination sequences, an origin of replication for plasmid product
in E. coli, an antibiotic resistance gene as selectable marker,
and/or a convenient cloning site (e.g., a polylinker). Nucleic
acids may also comprise an inducible promoter as opposed to a
constitutive promoter such as CMV IE.
[0448] In one embodiment, the anti-CD38-antibody-encoding
expression vector may be positioned in and/or delivered to the host
cell or host animal via a viral vector.
[0449] In an even further aspect, the invention relates to a
recombinant eukaryotic or prokaryotic host cell, such as a
transfectoma, which produces an antibody of the invention as
defined herein. Examples of host cells include yeast, bacterial,
and mammalian cells, such as a CHO, HEK or PER.C6.RTM. cells. For
example, in one embodiment, the present invention provides a cell
comprising a nucleic acid stably integrated into the cellular
genome that comprises a sequence coding for expression of an
anti-CD38 antibody of the present invention. In another embodiment,
the present invention provides a cell comprising a non-integrated
nucleic acid, such as a plasmid, cosmid, phagemid, or linear
expression element, which comprises a sequence coding for
expression of an anti-CD38 antibody of the invention.
[0450] In a further aspect, the invention relates to a hybridoma
which produces an antibody of the invention as defined herein. In
an even further aspect, the invention relates to a transgenic
non-human animal comprising nucleic acids encoding a human heavy
chain and a human light chain, wherein the animal or plant produces
an antibody of the invention. Generation of such hybridomas and
transgenic animals has been described above.
[0451] In a further aspect, the invention relates to a method for
producing an anti-CD38 antibody of the invention, said method
comprising the steps of:
a) culturing a hybridoma or a host cell of the invention as
described herein above, and b) purifying the antibody of the
invention from the culture media.
Pharmaceutical Compositions
[0452] In an even further aspect, the invention relates to a
pharmaceutical composition comprising: [0453] an anti-CD38 antibody
as defined herein, and [0454] a pharmaceutically-acceptable
carrier.
[0455] The pharmaceutical compositions may be formulated with
pharmaceutically acceptable carriers or diluents as well as any
other known adjuvants and excipients in accordance with
conventional techniques such as those disclosed in Remington: The
Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack
Publishing Co., Easton, Pa., 1995. A pharmaceutical composition of
the present invention may also include diluents, fillers, salts,
buffers, detergents (e.g., a nonionic detergent, such as Tween-20
or Tween-80), stabilizers (e.g., sugars or protein-free amino
acids), preservatives, tissue fixatives, solubilizers, and/or other
materials suitable for inclusion in a pharmaceutical
composition.
[0456] The pharmaceutically acceptable carriers or diluents as well
as any other known adjuvants and excipients should be suitable for
the chosen compound of the present invention and the chosen mode of
administration. Suitability for carriers and other components of
pharmaceutical compositions is determined based on the lack of
significant negative impact on the desired biological properties of
the chosen compound or pharmaceutical composition of the present
invention (e.g., less than a substantial impact (10% or less
relative inhibition, 5% or less relative inhibition, etc.)) on
antigen binding.
[0457] The actual dosage levels of the active ingredients in the
pharmaceutical compositions of the present invention may be varied
so as to obtain an amount of the active ingredient which is
effective to achieve the desired therapeutic response for a
particular patient, composition, and mode of administration,
without being toxic to the patient. The selected dosage level will
depend upon a variety of pharmacokinetic factors including the
activity of the particular compositions of the present invention
employed, or the amide thereof, the route of administration, the
time of administration, the rate of excretion of the particular
compound being employed, the duration of the treatment, other
drugs, compounds and/or materials used in combination with the
particular compositions employed, the age, sex, weight, condition,
general health and prior medical history of the patient being
treated, and like factors well known in the medical arts.
[0458] The pharmaceutical composition may be administered by any
suitable route and mode. Suitable routes of administering a
compound of the present invention in vivo and in vitro are well
known in the art and may be selected by those of ordinary skill in
the art.
[0459] In one embodiment, a pharmaceutical composition of the
present invention is administered parenterally.
[0460] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
include epidermal, intravenous, intramuscular, intraarterial,
intrathecal, intracapsular, intraorbital, intracardiac,
intradermal, intraperitoneal, intratendinous, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal, intracranial, intrathoracic, epidural
and intrasternal injection and infusion.
[0461] In one embodiment that pharmaceutical composition is
administered by intravenous or subcutaneous injection or
infusion.
[0462] In one embodiment the compounds of the present invention are
administered in crystalline form by subcutaneous injection, cf.
Yang et al., PNAS USA 100(12), 6934-6939 (2003).
[0463] Pharmaceutically acceptable carriers include any and all
suitable solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonicity agents, antioxidants and absorption
delaying agents, and the like that are physiologically compatible
with a compound of the present invention.
[0464] Examples of suitable aqueous and nonaqueous carriers which
may be employed in the pharmaceutical compositions of the present
invention include water, saline, phosphate buffered saline,
ethanol, dextrose, polyols (such as glycerol, propylene glycol,
polyethylene glycol, and the like), and suitable mixtures thereof,
vegetable oils, carboxymethyl cellulose colloidal solutions,
tragacanth gum and injectable organic esters, such as ethyl oleate,
and/or various buffers. Other carriers are well known in the
pharmaceutical arts.
[0465] Pharmaceutical compositions of the present invention may
also comprise pharmaceutically acceptable antioxidants for instance
(1) water soluble antioxidants, such as ascorbic acid, cysteine
hydrochloride, sodium bisulfate, sodium metabisulfite, sodium
sulfite and the like; (2) oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol,
and the like; and (3) metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0466] Pharmaceutical compositions of the present invention may
also comprise isotonicity agents, such as sugars, polyalcohols,
such as mannitol, sorbitol, glycerol or sodium chloride.
[0467] The pharmaceutical compositions of the present invention may
also contain one or more adjuvants appropriate for the chosen route
of administration such as preservatives, wetting agents,
emulsifying agents, dispersing agents, preservatives or buffers,
which may enhance the shelf life or effectiveness of the
pharmaceutical composition. The compounds of the present invention
may be prepared with carriers that will protect the compound
against rapid release, such as a controlled release formulation,
including implants, transdermal patches, and microencapsulated
delivery systems. Methods for the preparation of such formulations
are generally known to those skilled in the art. See e.g.,
Sustained and Controlled Release Drug Delivery Systems, J. R.
Robinson, ed., Marcel Dekker, Inc., New York, 1978.
[0468] The pharmaceutical composition of the present invention may
contain one antibody of the present invention or a combination of
two or more antibodies of the present invention.
Therapeutic Uses
[0469] In another aspect, the invention relates to the antibody of
the invention as defined herein for use as a medicament.
[0470] The anti-CD38 antibodies of the present invention have
numerous therapeutic utilities involving the treatment of disorders
involving cells expressing CD38. For example, the antibodies may be
administered to cells in culture, e.g., in vitro or ex vivo, or to
human subjects, e.g., in vivo, to treat or prevent a variety of
disorders. As used herein, the term "subject" is intended to
include human and non-human animals which respond to the antibody.
Subjects may for instance include human patients having disorders
that may be corrected or ameliorated by modulating CD38 function,
such as enzymatic activity, signal transduction, induction of
cytokine expression, induction of proliferation or differentiation,
and/or induction of lysis and/or eliminating/reducing the number of
CD38 expressing cells.
[0471] For example, the anti-CD38 antibodies may be used to elicit
in vivo or in vitro one or more of the following biological
activities: modulating CD38 function (such as enzymatic activity,
signal transduction, induction of cytokine expression, induction of
proliferation or differentiation, and/or induction of lysis),
killing a cell expressing CD38, mediating phagocytosis or ADCC of a
cell expressing CD38 in the presence of human effector cells, and
by mediating CDC of a cell expressing CD38 in the presence of
complement or by killing CD38 expressing cells by apoptosis.
[0472] The present invention provides methods for treating or
preventing a disorder involving cells expressing CD38 in a subject,
which method comprises administration of a therapeutically
effective amount of an anti-CD38 antibody of the present invention
to a subject in need thereof. Such a method involves administering
to a subject an anti-CD38 antibody of the present invention in an
amount effective to treat or prevent the disorder.
[0473] In one embodiment of the present invention, the disorder
involving cells expressing CD38 may be cancer, i.e. a tumorigenic
disorder, such as a disorder characterized by the presence of tumor
cells expressing CD38 including, for example, B cell lymphoma,
plasma cell malignancies, T/NK cell lymphoma and myeloid
malignancies.
[0474] Examples of such tumorigenic diseases include B cell
lymphomas/leukemias including precursor B cell lymphoblastic
leukemia/lymphoma and B cell non-Hodgkin's lymphomas; acute
promyelocytic leukemia, acute lymphoblastic leukemia and mature B
cell neoplasms, such as B cell chronic lymhocytic leukemia
(CLL)/small lymphocytic lymphoma (SLL), B cell acute lymphocytic
leukemia, B cell prolymphocytic leukemia, lymphoplasmacytic
lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL),
including low-grade, intermediate-grade and high-grade FL,
cutaneous follicle center lymphoma, marginal zone B cell lymphoma
(MALT type, nodal and splenic type), hairy cell leukemia, diffuse
large B cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasma
cell myeloma, plasma cell leukemia, post-transplant
lymphoproliferative disorder, Waldenstrom's macroglobulinemia,
plasma cell leukemias and anaplastic large-cell lymphoma
(ALCL).
[0475] In one embodiment, the disorder involving cells expressing
CD38 is multiple myeloma.
[0476] In one embodiment, the disorder involving cells expressing
CD38 is selected from chronic lymphocytic leukemia (CLL), acute
lymphoblastic leukemia (ALL), acute myelogenous leukemia (adults)
(AML), mantle cell lymphoma, follicular lymphoma, and diffuse large
B-cell lymphoma.
[0477] In one embodiment the disorder involving cells expressing
CD38 is non-small cell lung cancer (NSCLC).
[0478] Examples of B cell non-Hodgkin's lymphomas are lymphomatoid
granulomatosis, primary effusion lymphoma, intravascular large B
cell lymphoma, mediastinal large B cell lymphoma, heavy chain
diseases (including .gamma., .mu., and .alpha. disease), lymphomas
induced by therapy with immunosuppressive agents, such as
cyclosporine-induced lymphoma, and methotrexate-induced
lymphoma.
[0479] In one embodiment of the present invention, the disorder
involving cells expressing CD38 is Hodgkin's lymphoma.
[0480] Other examples of disorders involving cells expressing CD38
include malignancies derived from T and NK cells including: mature
T cell and NK cell neoplasms including T cell prolymphocytic
leukemia, T cell large granular lymphocytic leukemia, aggressive NK
cell leukemia, adult T cell leukemia/lymphoma, extranodal NK/T cell
lymphoma, nasal type, enteropathy-type T cell lymphoma,
hepatosplenic T cell lymphoma, subcutaneous panniculitis-like T
cell lymphoma, blastic NK cell lymphoma, Mycosis Fungoides/Sezary
Syndrome, primary cutaneous CD30 positive T cell
lymphoproliferative disorders (primary cutaneous anaplastic large
cell lymphoma C-ALCL, lymphomatoid papulosis, borderline lesions),
angioimmunoblastic T cell lymphoma, peripheral T cell lymphoma
unspecified, and anaplastic large cell lymphoma.
[0481] Examples of malignancies derived from myeloid cells include
acute myeloid leukemia, including acute promyelocytic leukemia, and
chronic myeloproliferative diseases, including chronic myeloid
leukemia.
[0482] In another embodiment of the present invention, the disorder
involving cells expressing CD38 is an immune disorder in which CD38
expressing B cells, macrophages, plasma cells, monocytes and T
cells are involved, such as an inflammatory and/or autoimmune
disease. Examples of immune disorders in which CD38 expressing B
cells, plasma cells, monocytes and T cells are involved include
autoimmune disorders, such as psoriasis, psoriatic arthritis,
dermatitis, systemic scleroderma and sclerosis, inflammatory bowel
disease (IBD), Crohn's disease, ulcerative colitis, respiratory
distress syndrome, meningitis, encephalitis, uveitis,
glomerulonephritis, eczema, asthma, atherosclerosis, leukocyte
adhesion deficiency, multiple sclerosis, Raynaud's syndrome,
Sjogren's syndrome, juvenile onset diabetes, Reiter's disease,
Behcet's disease, immune complex nephritis, IgA nephropathy, IgM
polyneuropathies, immune-mediated thrombocytopenias, such as acute
idiopathic thrombocytopenic purpura and chronic idiopathic
thrombocytopenic purpura, hemolytic anemia, myasthenia gravis,
lupus nephritis, systemic lupus erythematosus, rheumatoid arthritis
(RA), atopic dermatitis, pemphigus, Graves' disease, Hashimoto's
thyroiditis, Wegener's granulomatosis, Omenn's syndrome, chronic
renal failure, acute infectious mononucleosis, multiple sclerosis,
HIV, and herpes virus associated diseases. Further examples are
severe acute respiratory distress syndrome and choreoretinitis.
Furthermore, other diseases and disorders are included such as
those caused by or mediated by infection of B-cells with virus,
such as Epstein-Barr virus (EBV).
[0483] In one embodiment, the disorder involving cells expressing
CD38 is rheumatoid arthritis.
[0484] Further examples of inflammatory, immune and/or autoimmune
disorders in which autoantibodies and/or excessive B and T
lymphocyte activity are prominent and which may be treated
according to the present invention include the following:
vasculitides and other vessel disorders, such as microscopic
polyangiitis, Churg-Strauss syndrome, and other ANCA-associated
vasculitides, polyarteritis nodosa, essential cryoglobulinaemic
vasculitis, cutaneous leukocytoclastic angiitis, Kawasaki disease,
Takayasu arteritis, giant cell arthritis, Henoch-Schonlein purpura,
primary or isolated cerebral angiitis, erythema nodosum,
thrombangiitis obliterans, thrombotic thrombocytopenic purpura
(including hemolytic uremic syndrome), and secondary vasculitides,
including cutaneous leukocytoclastic vasculitis (e.g., secondary to
hepatitis B, hepatitis C, Waldenstrom's macroglobulinemia, B-cell
neoplasias, rheumatoid arthritis, Sjogren's syndrome, or systemic
lupus erythematosus); further examples are erythema nodosum,
allergic vasculitis, panniculitis, Weber-Christian disease, purpura
hyperglobulinaemica, and Buerger's disease; skin disorders, such as
contact dermatitis, linear IgA dermatosis, vitiligo, pyoderma
gangrenosum, epidermolysis bullosa acquisita, pemphigus vulgaris
(including cicatricial pemphigoid and bullous pemphigoid), alopecia
areata (including alopecia universalis and alopecia totalis),
dermatitis herpetiformis, erythema multiforme, and chronic
autoimmune urticaria (including angioneurotic edema and urticarial
vasculitis); immune-mediated cytopenias, such as autoimmune
neutropenia, and pure red cell aplasia; connective tissue
disorders, such as CNS lupus, discoid lupus erythematosus, CREST
syndrome, mixed connective tissue disease,
polymyositis/dermatomyositis, inclusion body myositis, secondary
amyloidosis, cryoglobulinemia type I and type II, fibromyalgia,
phospholipid antibody syndrome, secondary hemophilia, relapsing
polychondritis, sarcoidosis, stiff man syndrome, and rheumatic
fever; a further example is eosinophil fasciitis; arthritides, such
as ankylosing spondylitis, juvenile chronic arthritis, adult
Still's disease, and SAPHO syndrome; further examples are
sacroileitis, reactive arthritis, Still's disease, and gout;
hematologic disorders, such as aplastic anemia, primary hemolytic
anemia (including cold agglutinin syndrome), hemolytic anemia
secondary to CLL or systemic lupus erythematosus; POEMS syndrome,
pernicious anemia, and Waldemstrom's purpura hyperglobulinaemica;
further examples are agranulocytosis, autoimmune neutropenia,
Franklin's disease, Seligmann's disease, gamma heavy chain disease,
paraneoplastic syndrome secondary to thymoma and lymphomas, an,
paraneoplastic syndrome secondary to thymoma and lymphomas, and
factor VIII inhibitor formation; endocrinopathies, such as
polyendocrinopathy, and Addison's disease; further examples are
autoimmune hypoglycemia, autoimmune hypothyroidism, autoimmune
insulin syndrome, de Quervain's thyroiditis, and insulin receptor
antibody-mediated insulin resistance; hepato-gastrointestinal
disorders, such as celiac disease, Whipple's disease, primary
biliary cirrhosis, chronic active hepatitis, and primary sclerosing
cholangiitis; a further example is autoimmune gastritis;
nephropathies, such as rapid progressive glomerulonephritis,
post-streptococcal nephritis, Goodpasture's syndrome, membranous
glomerulonephritis, and cryoglobulinemic nephritis; a further
example is minimal change disease; neurological disorders, such as
autoimmune neuropathies, mononeuritis multiplex, Lambert-Eaton's
myasthenic syndrome, Sydenham's chorea, tabes dorsalis, and
Guillain-Barre's syndrome; further examples are myelopathy/tropical
spastic paraparesis, myasthenia gravis, acute inflammatory
demyelinating polyneuropathy, and chronic inflammatory
demyelinating polyneuropathy; multiple sclerosis; cardiac and
pulmonary disorders, such as COPD, fibrosing alveolitis,
bronchiolitis obliterans, allergic aspergillosis, cystic fibrosis,
Loffler's syndrome, myocarditis, and pericarditis; further examples
are hypersensitivity pneumonitis, and paraneoplastic syndrome
secondary to lung cancer; allergic disorders, such as bronchial
asthma and hyper-IgE syndrome; a further example is amaurosis
fugax; ophthalmologic disorders, such as idiopathic
chorioretinitis; infectious diseases, such as parvovirus B
infection (including hands-and-socks syndrome);
gynecological-obstretical disorders, such as recurrent abortion,
recurrent fetal loss, and intrauterine growth retardation; a
further example is paraneoplastic syndrome secondary to
gynaecological neoplasms; male reproductive disorders, such as
paraneoplastic syndrome secondary to testicular neoplasms; and
transplantation-derived disorders, such as allograft and xenograft
rejection, and graft-versus-host disease.
[0485] Dosage regimens in the above methods of treatment and uses
are adjusted to provide the optimum desired response (e.g., a
therapeutic response). For example, a single bolus may be
administered, several divided doses may be administered over time
or the dose may be proportionally reduced or increased as indicated
by the exigencies of the therapeutic situation.
[0486] The efficient dosages and the dosage regimens for the
anti-CD38 antibodies depend on the disease or condition to be
treated and may be determined by the persons skilled in the art. An
exemplary, non-limiting range for a therapeutically effective
amount of a compound of the present invention is about 0.005-100
mg/kg, such as 0.05-100 mg/kg or 1-100 mg/kg, such as about 0.1-50
mg/kg, for example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg,
for instance about 0.1, 0.3, about 0.5, about 1, 2, 3, 4, 8, 16 or
24 mg/kg.
[0487] Administration may e.g. be intravenous, intramuscular,
intraperitoneal, or subcutaneous, and for instance administered
proximal to the site of the target. If desired, the effective daily
dose of a pharmaceutical composition may be administered as two,
three, four, five, six or more sub-doses administered separately at
appropriate intervals throughout the day, optionally, in unit
dosage forms.
[0488] In one embodiment, the anti-CD38 antibodies may be
administered by infusion in a weekly dosage of from 10 to 500
mg/m.sup.2, such as of from 200 to 400 mg/m.sup.2. Such
administration may be repeated, e.g., 1 to 8 times, such as 3 to 5
times. The administration may be performed by continuous infusion
over a period of from 2 to 24 hours, such as of from 2 to 12
hours.
[0489] In one embodiment, the anti-CD38 antibodies may be
administered by slow continuous infusion over a long period, such
as more than 24 hours, in order to reduce toxic side effects.
[0490] In one embodiment the anti-CD38 antibodies may be
administered in a weekly dosage of from 250 mg to 2000 mg, such as
for example 300 mg, 500 mg, 700 mg, 1000 mg, 1500 mg or 2000 mg,
for up to 8 times, such as from 4 to 6 times. The administration
may be performed by continuous infusion over a period of from 2 to
24 hours, such as of from 2 to 12 hours. Such regimen may e.g. be
repeated one or more times as necessary, for example, after 6
months or 12 months.
[0491] In one embodiment, the anti-CD38 antibodies may be
administered by maintenance therapy, such as, e.g., once a week for
a period of 6 months or more.
[0492] In another embodiment, the anti-CD38 antibodies may be
administered by a regimen including one infusion of an anti-CD38
antibody of the present invention followed by an infusion of an
anti-CD38 antibody of the present invention conjugated to a
radioisotope. The regimen may be repeated, e.g., 7 to 9 days
later.
[0493] As non-limiting examples, treatment according to the present
invention may be provided as a daily dosage of a compound of the
present invention in an amount of about 0.1-100 mg/kg, such as 0.5,
0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45,
50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of day 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, or 40, or alternatively, at least one of week 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after
initiation of treatment, or any combination thereof, using single
or divided doses of every 24, 12, 8, 6, 4, or 2 hours, or any
combination thereof.
[0494] An "effective amount" for tumor therapy may also be measured
by its ability to stabilize the progression of disease. The ability
of a compound to inhibit cancer may be evaluated in an animal model
system predictive of efficacy in human tumors. Alternatively, this
property of a composition may be evaluated by examining the ability
of the compound to inhibit cell growth or to induce apoptosis by in
vitro assays known to the skilled practitioner. A therapeutically
effective amount of a therapeutic compound may decrease tumor size,
or otherwise ameliorate symptoms in a subject.
[0495] A "therapeutically effective amount" for rheumatoid
arthritis may result in an at least ACR.sub.20 Preliminary
Definition of Improvement in the patients, such as in at least an
ACR.sub.50 Preliminary Definition of Improvement, for instance at
least an ARC.sub.70 Preliminary Definition of Improvement.
ACR.sub.20 Preliminary Definition of Improvement is defined as:
.gtoreq.20% improvement in: Tender Joint Count (TJC) and Swollen
Joint Count (SJC) and .gtoreq.20% improvement in 3 of following 5
assessments: Patient Pain Assessment (VAS), Patient Global
assessment (VAS), Physician Global Assessment (VAS), Patent
Self-Assessed Disability (HAQ), Acute Phase Reactant (CRP or ESR).
ACR.sub.50 and ACR.sub.70 are defined in the same way with
.gtoreq.50% and .gtoreq.70% improvements, respectively. For further
details see Felson et al., in American College of Rheumatology
Preliminary Definition of Improvement in Rheumatoid Arthritis;
Arthritis Rheumatism 38, 727-735 (1995).
[0496] Alternatively, a therapeutically effective amount for
rheumatoid arthritis can be measured by DAS (disease activity
score), including DAS28 and/or DAS56, as defined by EU LAR.
[0497] An anti-CD38 antibody may also be administered
prophylactically in order to reduce the risk of developing cancer,
delay the onset of the occurrence of an event in cancer
progression, and/or reduce the risk of recurrence when a cancer is
in remission. This may be especially useful in patients wherein it
is difficult to locate a tumor that is known to be present due to
other biological factors.
Combination Therapy
[0498] The anti-CD38 antibodies of the present invention may also
be administered in combination therapy, i.e., combined with other
therapeutic agents relevant for the disease or condition to be
treated. Such administration may be simultaneous, separate or
sequential. For simultaneous administration the agents may be
administered as one compositions or as separate compositions, as
appropriate.
[0499] Accordingly, the present invention provides methods for
treating a disorder involving cells expressing CD38 as described
above, which methods comprise administration of an anti-CD38
antibody of the present invention combined with one or more
additional therapeutic agents as described below.
[0500] In an embodiment of the invention the antibodies of the
present invention are administered as a combination with other
anti-CD38 antibodies. Such antibodies are described in the present
invention and in prior art. Specifically, antibodies are described
in WO2006099875. More specifically, a combination of the present
anti-CD38 antibodies with anti-CD38 antibodies which are
non-cross-blocking, such as antibody 003 described in WO2006099875
are embodiments of the present invention.
[0501] In one embodiment, the combination therapy may include
administration of a composition of the present invention together
with at least one cytotoxic agent, at least one chemotherapeutic
agent, at least one anti-angiogenic agent, at least one
anti-inflammatory agent, and/or at least one immunosuppressive
and/or immunomodulatory agent.
[0502] In one embodiment, the present invention provides a method
for treating a disorder involving cells expressing CD38, such as
cancer, in a subject, which method comprises administration of a
therapeutically effective amount of an anti-CD38 antibody of the
present invention and at least one chemotherapeutic agent to a
subject in need thereof
[0503] In one embodiment, the present invention provides a method
for treating multiple myeloma, which method comprises
administration of a therapeutically effective amount of an
anti-CD38 antibody of the present invention and at least one
chemotherapeutic agent to a subject in need thereof.
[0504] In one embodiment, such a chemotherapeutic agent may be
selected from an antimetabolite, such as methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine,
5-fluorouracil, decarbazine, hydroxyurea, asparaginase,
gemcitabine, cladribine and similar agents.
[0505] In one embodiment, such a chemotherapeutic agent may be
selected from an alkylating agent, such as mechlorethamine,
thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine
(CCNU), cyclophosphamide, busulfan, dibromomannitol,
streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C,
cisplatin and other platinum derivatives, such as carboplatin, and
similar agents.
[0506] In one embodiment, such a chemotherapeutic agent may be
selected from an antibiotic, such as dactinomycin (formerly
actinomycin), bleomycin, daunorubicin (formerly daunomycin),
doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone,
plicamycin, anthramycin (AMC) and similar agents.
[0507] In one embodiment, such a chemotherapeutic agent may be
selected from an anti-mitotic agent, such as taxanes, for instance
docetaxel, and paclitaxel, and vinca alkaloids, for instance
vindesine, vincristine, vinblastine, and vinorelbine.
[0508] In one embodiment, such a chemotherapeutic agent may be
selected from a topoisomerase inhibitor, such as topotecan.
[0509] In one embodiment, such a chemotherapeutic agent may be
selected from a growth factor inhibitor, such as an inhibitor of
ErbB1 (EGFR) (such as gefitinib (Iressa.RTM.), cetuximab
(Erbitux.RTM.), erlotinib (Tarceva.RTM.), HuMax-EGFr (zalutumumab,
2F8 disclosed in WO 2002/100348) and similar agents), an inhibitor
of ErbB2 (Her2/neu) (such as trastuzumab (Herceptin.RTM.) and
similar agents) and similar agents. In one embodiment, such a
growth factor inhibitor may be a farnesyl transferase inhibitor,
such as SCH-66336 and R115777. In one embodiment, such a growth
factor inhibitor may be a vascular endothelial growth factor (VEGF)
inhibitor, such as bevacizumab (Avastin.RTM.).
[0510] In one embodiment, such a chemotherapeutic agent may be a
tyrosine kinase inhibitor, such as imatinib (Glivec, Gleevec
ST1571), lapatinib, PTK787/ZK222584 and similar agents.
[0511] In one embodiment, such a chemotherapeutic agent may be a
histone deacetylase inhibitor. Examples of such histone deacetylase
inhibitors include hydroxamic acid-based hybrid polar compounds,
such as SAHA (suberoylanilide hydroxamic acid).
[0512] In one embodiment, such a chemotherapeutic agent may be a
P38a MAP kinase inhibitor, such as SCIO-469.
[0513] In one embodiment, the present invention provides a method
for treating a disorder involving cells expressing CD38 in a
subject, which method comprises administration of a therapeutically
effective amount of an anti-CD38 antibody of the present invention
and at least one inhibitor of angiogenesis, neovascularization,
and/or other vascularization to a subject in need thereof
[0514] In one embodiment, the present invention provides a method
for treating multiple myeloma, which method comprises
administration of a therapeutically effective amount of an
anti-CD38 antibody of the present invention and at least one
inhibitor of angiogenesis, neovascularization, and/or other
vascularization to a subject in need thereof.
[0515] Examples of such angiogenesis inhibitors are urokinase
inhibitors, matrix metalloprotease inhibitors (such as marimastat,
neovastat, BAY 12-9566, AG 3340, BMS-275291 and similar agents),
inhibitors of endothelial cell migration and proliferation (such as
TNP-470, squalamine, 2-methoxyestradiol, combretastatins,
endostatin, angiostatin, penicillamine, SCH66336 (Schering-Plough
Corp, Madison, N.J.), R115777 (Janssen Pharmaceutica, Inc,
Titusville, N.J.) and similar agents), antagonists of angiogenic
growth factors (such as ZD6474, SU6668, antibodies against
angiogenic agents and/or their receptors (such as VEGF, bFGF, and
angiopoietin-1), thalidomide (Thalomid.RTM.), thalidomide analogs
(such as CC-5013 (lenalidomide, Revlimid.TM.) and CC4047
(Actimid.TM.), Sugen 5416, SU5402, antiangiogenic ribozyme (such as
angiozyme), interferon .alpha. (such as interferon .alpha.2a),
suramin and similar agents), VEGF-R kinase inhibitors and other
anti-angiogenic tyrosine kinase inhibitors (such as SU011248),
inhibitors of endothelial-specific integrin/survival signaling
(such as vitaxin and similar agents), copper antagonists/chelators
(such as tetrathiomolybdate, captopril and similar agents),
carboxyamido-triazole (CAI), ABT-627, CM101, interleukin-12
(IL-12), IM862, PNU145156E as well as nucleotide molecules
inhibiting angiogenesis (such as antisense-VEGF-cDNA, cDNA coding
for angiostatin, cDNA coding for p53 and cDNA coding for deficient
VEGF receptor-2) and similar agents.
[0516] Other examples of such inhibitors of angiogenesis,
neovascularization, and/or other vascularization are
anti-angiogenic heparin derivatives and related molecules (e.g.,
heperinase III), temozolomide, NK4, macrophage migration inhibitory
factor (MIF), cyclooxygenase-2 inhibitors, inhibitors of
hypoxia-inducible factor 1, anti-angiogenic soy isoflavones,
oltipraz, fumagillin and analogs thereof, somatostatin analogues,
pentosan polysulfate, tecogalan sodium, dalteparin, tumstatin,
thrombospondin, NM-3, combrestatin, canstatin, avastatin,
antibodies against other relevant targets (such as
anti-alpha-v/beta-3 integrin and anti-kininostatin mAbs) and
similar agents.
[0517] In one embodiment, the present invention provides the use of
an anti-CD38 antibody of the present invention for the preparation
of a pharmaceutical composition to be administered with thalidomide
(Thalomid.RTM.), thalidomide analogs (such as CC-5013
(lenalidomide, Revlimid.TM.) and/or CC4047 (Actimid.TM.). In a
further embodiment, the present invention provides the use of an
anti-CD38 antibody of the present invention for the preparation of
a pharmaceutical composition to be administered with
thalidomide.
[0518] In one embodiment, the present invention provides the use of
an anti-CD38 antibody of the present invention for the preparation
of a pharmaceutical composition to be administered with an
anti-CD20 antibody, such as rituximab (Rituxan.RTM.,
Mabthera.RTM.), a human monoclonal anti-CD20 antibody as disclosed
in WO 2004/035607, such as 11B8, 2F2 (ofatumumab, Arzerra.RTM.) or
7D8.
[0519] In one embodiment, a therapeutic agent for use in
combination with the anti-CD38 antibody of the present invention
for treating the disorders as described above may be a proteasome
inhibitor, such as bortezomib (Velcade.RTM.).
[0520] In one embodiment, a therapeutic agent for use in
combination with the anti-CD38 antibody of the present invention
for treating the disorders as described above may be a
corticosteroid, such as prednisone, prednisolone, dexamethasone,
etc.
[0521] In one embodiment, the anti-CD38 antibody of the present
invention is used in combination with lenalidomide and
dexamethasone for treating the disorders as described above, such
as multiple myeloma, e.g. relapsed multiple myeloma.
[0522] In one embodiment, the anti-CD38 antibody of the present
invention is used in combination with bortezomib and dexamethasone
for treating the disorders as described above, such as multiple
myeloma, e.g. relapsed multiple myeloma.
[0523] In one embodiment, the anti-CD38 antibody of the present
invention is used in combination with bortezomib and prednisolone
for treating the disorders as described above, such as multiple
myeloma, e.g. relapsed multiple myeloma.
[0524] In one embodiment, a therapeutic agent for use in
combination with the anti-CD38 antibody of the present invention
for treating the disorders as described above may be an anti-cancer
immunogen, such as a cancer antigen/tumor-associated antigen (e.g.,
epithelial cell adhesion molecule (EpCAM/TACSTD1), mucin 1 (MUC1),
carcinoembryonic antigen (CEA), tumor-associated glycoprotein 72
(TAG-72), gp100, Melan-A, MART-1, KDR, RCAS1, MDA7,
cancer-associated viral vaccines (e.g., human papillomavirus
vaccines), tumor-derived heat shock proteins, and similar agents. A
number of other suitable cancer antigens/tumor-associated antigens
described elsewhere herein and similar molecules known in the art
may also or alternatively be used in such embodiment. Anti-cancer
immunogenic peptides also include anti-idiotypic "vaccines" such as
BEC2 anti-idiotypic antibodies, Mitumomab, CeaVac and related
anti-idiotypic antibodies, anti-idiotypic antibody to MG7 antibody,
and other anti-cancer anti-idiotypic antibodies (see for instance
Birebent et al., Vaccine. 21(15), 1601-12 (2003), Li et al., Chin
Med J (Engl). 114(9), 962-6 (2001), Schmitt et al., Hybridoma.
13(5), 389-96 (1994), Maloney et al., Hybridoma. 4(3), 191-209
(1985), Raychardhuri et al., J Immunol. 137(5), 1743-9 (1986), Pohl
et al., Int J Cancer. 50(6), 958-67 (1992), Bohlen et al.,
Cytokines Mol Ther. 2(4), 231-8 (1996) and Maruyama, J Immunol
Methods. 264(1-2), 121-33 (2002)). Such anti-idiotypic Antibodies
may optionally be conjugated to a carrier, which may be a synthetic
(typically inert) molecule carrier, a protein (for instance keyhole
limpet hemocyanin (KLH) (see for instance Ochi et al., Eur J
Immunol. 17(11), 1645-8 (1987)), or a cell (for instance a red
blood cell--see for instance Wi et al., J Immunol Methods. 122(2),
227-34 (1989)).
[0525] In one embodiment, a therapeutic agent for use in
combination with the anti-CD38 antibody of the present invention
for treating the disorders as described above may be a
bisphosphonate. Examples of potentially suitable biphosphonates are
pamidronate (Aredia.RTM.), zoledronic acid (Zometa.RTM.),
clodronate (Bonefos.RTM.), risendronate (Actonel.RTM.), ibandronate
(Boniva.RTM.), etidronate (Didronel.RTM.), alendronate
(Fosamax.RTM.), tiludronate (Skelid.RTM.), incadronate (Yamanouchi
Pharmaceutical) and minodronate (YM529, Yamanouchi).
[0526] In one embodiment, a therapeutic agent for use in
combination with the anti-CD38 antibody of the present invention
for treating the disorders as described above may be a colony
stimulating factor. Examples of suitable colony stimulating factors
are granulocyte-colony stimulating factors (G-CSF), such as
filgrastim (Neupogen.RTM.) and pegfilgrastim (Neulasta.RTM.), and
granulocyte macrophage-colony stimulating factors (GM-CSF) such as
sargramostim (Leukine.RTM.).
[0527] In one embodiment, a therapeutic agent for use in
combination with the anti-CD38 antibody of the present invention
for treating the disorders as described above may be a
erythropoietic agent. Examples of suitable erythropoietic agents
are erythropoietin (EPO), such as epoetin alfa (for instance
Procrit.RTM., Epogen.RTM., and Eprex.RTM.) and epoetin beta (for
instance NeoRecormon.RTM.) and erythropoiesis-stimulating proteins
(for instance Aranesp.RTM.).
[0528] In one embodiment, a therapeutic agent for use in
combination with the anti-CD38 antibody of the present invention
for treating the disorders as described above may be an anti-cancer
cytokine, chemokine, or combination thereof. Examples of suitable
cytokines and growth factors include IFN.gamma., IL-2, IL-4, IL-6,
IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27,
IL-28a, IL-28b, IL-29, KGF, IFN.alpha. (e.g., INF.alpha.2b),
IFN.beta., GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim,
and TNF.alpha.. Suitable chemokines may include Glu-Leu-Arg
(ELR)-negative chemokines such as IP-10, MCP-3, MIG, and
SDF-1.alpha. from the human CXC and C-C chemokine families.
Suitable cytokines include cytokine derivatives, cytokine variants,
cytokine fragments, and cytokine fusion proteins. These and other
methods or uses involving naturally occurring peptide-encoding
nucleic acids herein may alternatively or additionally be performed
by "gene activation" and homologous recombination gene upregulation
techniques, such as are described in U.S. Pat. Nos. 5,968,502,
6,063,630 and 6,187,305 and EP 0505500.
[0529] In one embodiment, a therapeutic agent for use in
combination with the anti-CD38 antibody of the present invention
for treating the disorders as described above may be an agent that
modulates, e.g., enhances or inhibits, the expression or activity
of Fc.alpha. or Fc.gamma. receptors. Examples of agents suitable
for this use include interleukin-1 (IL-1), interleukin-2 (IL-2),
interleukin-6 (IL-6), granulocyte colony-stimulating factor
(G-CSF), such as filgrastim (Neupogen.RTM.) and pegfilgrastim
(Neulasta.RTM.), and granulocyte macrophage-colony stimulating
factors (GM-CSF) such as sargramostim (Leukine.RTM.),
interferon-.gamma. (IFN-.gamma.), and tumor necrosis factor
(TNF).
[0530] In one embodiment, a therapeutic agent for use in
combination with the anti-CD38 antibody of the present invention
for treating the disorders as described above may be a cell cycle
control/apoptosis regulator (or "regulating agent"). A cell cycle
control/apoptosis regulator may include molecules (i) that target
and modulate cell cycle control/apoptosis regulators such as cdc-25
(such as NSC 663284), (ii) cyclin-dependent kinases that
overstimulate the cell cycle (such as flavopiridol (L868275,
HMR1275), 7-hydroxy-staurosporine (UCN-01, KW-2401), and
roscovitine (R-roscovitine, CYC202)), and (iii) telomerase
modulators (such as BIBR1532, SOT-095, GRN163 and compositions
described in for instance U.S. Pat. Nos. 6,440,735 and 6,713,055).
Non-limiting examples of molecules that interfere with apoptotic
pathways include TNF-related apoptosis-inducing ligand
(TRAIL)/apoptosis-2 ligand (Apo-2L), agents inducing NF-.kappa.B
blockade leading to inhibition of IL-6 production, antibodies that
activate TRAIL receptors, IFNs, anti-sense Bcl-2, and
As.sub.2O.sub.3 (arsenic trioxide, Trisenox.RTM.).
[0531] In one embodiment, a therapeutic agent for use in
combination with the anti-CD38 antibody of the present invention
for treating the disorders as described above may be a hormonal
regulating agent, such as agents useful for anti-androgen and
anti-estrogen therapy. Examples of such hormonal regulating agents
are tamoxifen, idoxifene, fulvestrant, droloxifene, toremifene,
raloxifene, diethylstilbestrol, ethinyl estradiol/estinyl, an
antiandrogene (such as flutaminde/eulexin), a progestin (such as
such as hydroxy-progesterone caproate, medroxyprogesterone/provera,
megestrol acepate/megace), an adrenocorticosteroid (such as
hydrocortisone, prednisone), luteinizing hormone-releasing hormone
(and analogs thereof and other LHRH agonists such as buserelin and
goserelin), an aromatase inhibitor (such as anastrazole/arimidex,
aminoglutethimide/cytraden, exemestane), a hormone inhibitor (such
as octreotide/sandostatin) and similar agents.
[0532] In one embodiment, a therapeutic agent for use in
combination with the anti-CD38 antibody of the present invention
for treating the disorders as described above may be an
anti-anergic agents (for instance small molecule compounds,
proteins, glycoproteins, or antibodies that break tolerance to
tumor and cancer antigens). Examples of such compounds are
molecules that block the activity of CTLA-4, such as MDX-010 (Phan
et al., PNAS USA 100, 8372 (2003)).
[0533] In one embodiment, a therapeutic agent for use in
combination with the anti-CD38 antibody of the present invention
for treating the disorders as described above may be a tumor
suppressor gene-containing nucleic acid or vector such as a
replication-deficient adenovirus encoding human recombinant
wild-type p53/SCH58500, etc.; antisense nucleic acids targeted to
oncogenes, mutated, or deregulated genes; or siRNA targeted to
mutated or deregulated genes. Examples of tumor suppressor targets
include, for example, BRCA1, RB1, BRCA2, DPC4 (Smad4), MSH2, MLH1,
and DCC.
[0534] In one embodiment, a therapeutic agent for use in
combination with the anti-CD38 antibody of the present invention
for treating the disorders as described above may be an anti-cancer
nucleic acid, such as genasense (augmerosen/G3139), LY900003 (ISIS
3521), ISIS 2503, OGX-011 (ISIS 112989), LE-AON/LEraf-AON (liposome
encapsulated c-raf antisense oligonucleotide/ISIS-5132), MG98, and
other antisense nucleic acids that target PKC.alpha., clusterin,
IGFBPs, protein kinase A, cyclin D1, or Bcl-2h.
[0535] In one embodiment, a therapeutic agent for use in
combination with the anti-CD38 antibody of the present invention
for treating the disorders as described above may be an anti-cancer
inhibitory RNA molecule (see for instance Lin et al., Curr Cancer
Drug Targets. 1(3), 241-7 (2001), Erratum in: Curr Cancer Drug
Targets. 3(3), 237 (2003), Lima et al., Cancer Gene Ther. 11(5),
309-16 (2004), Grzmil et al., Int J Oncol. 4(1), 97-105 (2004),
Collis et al., Int J Radiat Oncol Biol Phys. 57(2 Suppl), S144
(2003), Yang et al., Oncogene. 22(36), 5694-701 (2003) and Zhang et
al., Biochem Biophys Res Commun. 303(4), 1169-78 (2003)).
[0536] Compositions and combination administration methods of the
present invention also include the administration of nucleic acid
vaccines, such as naked DNA vaccines encoding such cancer
antigens/tumor-associated antigens (see for instance U.S. Pat. Nos.
5,589,466, 5,593,972, 5,703,057, 5,879,687, 6,235,523, and
6,387,888). In one embodiment, the combination administration
method and/or combination composition comprises an autologous
vaccine composition. In one embodiment, the combination composition
and/or combination administration method comprises a whole cell
vaccine or cytokine-expressing cell (for instance a recombinant
IL-2 expressing fibroblast, recombinant cytokine-expressing
dendritic cell, and the like) (see for instance Kowalczyk et al.,
Acta Biochim Pol. 50(3), 613-24 (2003), Reilly et al., Methods Mol
Med. 69, 233-57 (2002) and Tirapu et al., Curr Gene Ther. 2(1),
79-89 (2002). Another example of such an autologous cell approach
that may be useful in combination methods of the present invention
is the MyVax.RTM. Personalized Immunotherapy method (previously
referred to as GTOP-99) (Genitope Corporation--Redwood City,
Calif., USA).
[0537] In one embodiment, the present invention provides
combination compositions and combination administration methods
wherein an anti-CD38 antibody is combined or co-administered with
an oncolytic virus.
[0538] Combination compositions and combination administration
methods of the present invention may also involve "whole cell and
"adoptive" immunotherapy methods. For instance, such methods may
comprise infusion or re-infusion of immune system cells (for
instance tumor-infiltrating lymphocytes (TILs), such as CD4.sup.+
and/or CD8.sup.+ T cells (for instance T cells expanded with
tumor-specific antigens and/or genetic enhancements),
antibody-expressing B cells or other antibody producing/presenting
cells, dendritic cells (e.g., anti-cytokine expressing recombinant
dendritic cells, dendritic cells cultured with a DC-expanding agent
such as GM-CSF and/or Flt3-L, and/or tumor-associated
antigen-loaded dendritic cells), anti-tumor NK cells, so-called
hybrid cells, or combinations thereof. Cell lysates may also be
useful in such methods and compositions. Cellular "vaccines" in
clinical trials that may be useful in such aspects include
Canvaxin.TM., APC-8015 (Dendreon), HSPPC-96 (Antigenics), and
Melacine.RTM. cell lysates. Antigens shed from cancer cells, and
mixtures thereof (see for instance Bystryn et al., Clinical Cancer
Research Vol. 7, 1882-1887, July 2001), optionally admixed with
adjuvants such as alum, may also be components in such methods and
combination compositions.
[0539] In one embodiment, an anti-CD38 antibody of the present
invention may be delivered to a patient in combination with the
application of an internal vaccination method. Internal vaccination
refers to induced tumor or cancer cell death, such as drug-induced
or radiation-induced cell death of tumor cells, in a patient, that
typically leads to elicitation of an immune response directed
towards (i) the tumor cells as a whole or (ii) parts of the tumor
cells including (a) secreted proteins, glycoproteins or other
products, (b) membrane-associated proteins or glycoproteins or
other components associated with or inserted in membranes, and/or
(c) intracellular proteins or other intracellular components. An
internal vaccination-induced immune response may be humoral (i.e.
antibody--complement-mediated) or cell-mediated (e.g., the
development and/or increase of endogenous cytotoxic T lymphocytes
that recognize the internally killed tumor cells or parts thereof).
In addition to radiotherapy, non-limiting examples of drugs and
agents that may be used to induce said tumor cell-death and
internal vaccination are conventional chemotherapeutic agents,
cell-cycle inhibitors, anti-angiogenesis drugs, monoclonal
antibodies, apoptosis-inducing agents, and signal transduction
inhibitors.
[0540] Examples of other anti-cancer agents, which may be relevant
as therapeutic agents for use in combination with the anti-CD38
antibody of the present invention for treating the disorders as
described above are differentiation inducing agents, retinoic acid
and retinoic acid analogues (such as all trans retinoic acid,
13-cis retinoic acid and similar agents), vitamin D analogues (such
as seocalcitol and similar agents), inhibitors of ErbB3, ErbB4,
IGF-IR, insulin receptor, PDGFRa, PDGFRbeta, Flk2, Flt4, FGFR1,
FGFR2, FGFR3, FGFR4, TRKA, TRKC, c-met, Ron, Sea, Tie, Tie2, Eph,
Ret, Ros, Alk, LTK, PTK7 and similar agents.
[0541] Examples of other anti-cancer agents, which may be relevant
as therapeutic agents for use in combination with the anti-CD38
antibody of the present invention for treating the disorders as
described above are cathepsin B, modulators of cathepsin D
dehydrogenase activity, glutathione-S-transferase (such as
glutacylcysteine synthetase and lactate dehydrogenase),
estramustine, epirubicin, HSP90 inhibitor like 17-allyl amino
geld-anamycin, antibodies directed against a tumor antigen such as
PSA, CA125, KSA, etc., integrins like integrin .beta.1, inhibitors
of VCAM and similar agents.
[0542] Examples of other anti-cancer agents, which may be relevant
as therapeutic agents for use in combination with the anti-CD38
antibodies of the present invention for treating the disorders as
described above are calcineurin-inhibitors (such as valspodar, PSC
833 and other MDR-1 or p-glycoprotein inhibitors), TOR-inhibitors
(such as sirolimus, everolimus and rapamcyin). and inhibitors of
"lymphocyte homing" mechanisms (such as FTY720), and agents with
effects on cell signaling such as adhesion molecule inhibitors (for
instance anti-LFA, etc.).
[0543] In one embodiment, the present invention provides a method
for treating a disorder involving cells expressing CD38, such as
cancer, in a subject, which method comprises administration of a
therapeutically effective amount of an anti-CD38 antibody of the
present invention and radiotherapy to a subject in need
thereof.
[0544] In one embodiment, the present invention provides a method
for treating multiple myeloma, which method comprises
administration of a therapeutically effective amount of an
anti-CD38 antibody of the present invention and radiotherapy to a
subject in need thereof.
[0545] Radiotherapy may comprise radiation or associated
administration of radiopharmaceuticals to a patient is provided.
The source of radiation may be either external or internal to the
patient being treated (radiation treatment may, for example, be in
the form of external beam radiation therapy (EBRT), brachytherapy
(BT) or skeletal targeted radiotherapy). Radioactive elements that
may be used in practicing such methods include, e.g., radium,
cesium-137, iridium-192, americium-241, gold-198, cobalt-57,
copper-67, technetium-99, iodide-123, iodide-131, and
indium-111.
[0546] In one embodiment, the present invention provides a method
for treating a disorder involving cells expressing CD38 in a
subject, which method comprises administration of a therapeutically
effective amount of an anti-CD38 antibody of the present invention
to a subject in need thereof combined with autologous peripheral
stem cell or bone marrow transplantation.
[0547] In one embodiment, the present invention provides a method
for treating multiple myeloma, which method comprises
administration of a therapeutically effective amount of an
anti-CD38 antibody of the present invention to a subject in need
thereof combined with autologous peripheral stem cell or bone
marrow transplantation.
[0548] In one embodiment, the present invention provides a method
for treating a disorder involving cells expressing CD38 in a
subject, which method comprises administration of a therapeutically
effective amount of an anti-CD38 antibody of the present invention
to a subject in need thereof combined with orthopedic
intervention.
[0549] In one embodiment, the present invention provides a method
for treating a disorder involving cells expressing CD38 in a
subject, which method comprises administration of a therapeutically
effective amount of an anti-CD38 antibody of the present invention
and at least one anti-inflammatory agent to a subject in need
thereof.
[0550] In one embodiment such an anti-inflammatory agent may be
selected from a steroidal drug and a NSAID (nonsteroidal
anti-inflammatory drug).
[0551] In one embodiment such an anti-inflammatory agent may be
selected from aspirin and other salicylates, Cox-2 inhibitors (such
as rofecoxib and celecoxib), NSAIDs (such as ibuprofen, fenoprofen,
naproxen, sulindac, diclofenac, piroxicam, ketoprofen, diflunisal,
nabumetone, etodolac, oxaprozin, and indomethacin), anti-IL6R
antibodies, anti-IL8 antibodies (e.g. antibodies described in
WO2004058797, e.g. 10F8), anti-IL15 antibodies (e.g. antibodies
described in WO03017935 and WO2004076620), anti-IL15R antibodies,
anti-CD4 antibodies (e.g. zanolimumab), anti-CD11a antibodies
(e.g., efalizumab), anti-alpha-4/beta-1 integrin (VLA4) antibodies
(e.g. natalizumab), CTLA4-Ig for the treatment of inflammatory
diseases, prednisolone, prednisone, disease modifying antirheumatic
drugs (DMARDs) such as methotrexate, hydroxychloroquine,
sulfasalazine, pyrimidine synthesis inhibitors (such as
leflunomide), IL-1 receptor blocking agents (such as anakinra),
TNF-.alpha. blocking agents (such as etanercept, infliximab, and
adalimumab) and similar agents.
[0552] In one embodiment, the present invention provides a method
for treating a disorder involving cells expressing CD38 in a
subject, which method comprises administration of a therapeutically
effective amount of an anti-CD38 antibody of the present invention
and at least one immunosuppressive and/or immunomodulatory agent to
a subject in need thereof.
[0553] In one embodiment, such an immunosuppressive and/or
immunomodulatory agent may be selected from cyclosporine,
azathioprine, mycophenolic acid, mycophenolate mofetil,
corticosteroids such as prednisone, methotrexate, gold salts,
sulfasalazine, antimalarials, brequinar, leflunomide, mizoribine,
15-deoxyspergualine, 6-mercaptopurine, cyclophosphamide, rapamycin,
tacrolimus (FK-506), OKT3, anti-thymocyte globulin, thymopentin,
thymosin-.alpha. and similar agents.
[0554] In one embodiment, such an immunosuppressive and/or
immunomodulatory agent may be selected from immunosuppressive
antibodies, such as antibodies binding to p75 of the IL-2 receptor,
or antibodies binding to for instance MHC, CD2, CD3, CD4, CD7,
CD28, B7, CD40, CD45, IFN.gamma., TNF-.alpha., IL-4, IL-5, IL-6R,
IL-6, IGF, IGFR1, IL-7, IL-8, IL-10, CD11a, or CD58, or antibodies
binding to their ligands.
[0555] In one embodiment, such an immunosuppressive and/or
immunomodulatory agent may be selected from soluble IL-15R, IL-10,
B7 molecules (B7-1, B7-2, variants thereof, and fragments thereof),
ICOS, and OX40, an inhibitor of a negative T cell regulator (such
as an antibody against CTLA4) and similar agents.
[0556] In one embodiment, the anti-CD38 antibody of the present
invention may be administered in combination with two or more
immunosuppressive and/or immunomodulatory agents, such as in
combination with prednisone and cyclosporine; prednisone,
cyclosporine and azathioprine; or prednisone, cyclosporine and
mycophenolate mofetil.
[0557] In one embodiment, the present invention provides a method
for treating a disorder involving cells expressing CD38 in a
subject, which method comprises administration of a therapeutically
effective amount of an anti-CD38 antibody of the present invention
and an anti-C3b(i) antibody to a subject in need thereof.
[0558] In one embodiment, the present invention provides a method
for treating a disorder involving cells expressing CD38 in a
subject, which method comprises administration of a therapeutically
effective amount of an anti-CD38 antibody of the present invention
and an anti-CD32b antibody to a subject in need thereof. In one
embodiment of the present invention, the anti-CD32b antibody is
selected from HuMab-016, -020, -022, -024, 026, 028, -034, -038 or
-053 all disclosed in WO2009/083009.
[0559] In one embodiment, a therapeutic agent for use in
combination with the anti-CD38 antibody of the present invention
for treating the disorders as described above may be selected from
histone deacetylase inhibitors (for instance phenylbutyrate) and/or
DNA repair agents (for instance DNA repair enzymes and related
compositions such as dimericine).
[0560] Methods of the present invention for treating a disorder as
described above comprising administration of a therapeutically
effective amount of an anti-CD38 antibody of the present invention
may also comprise anti-cancer directed photodynamic therapy (for
instance anti-cancer laser therapy--which optionally may be
practiced with the use of photosensitizing agent, see, for instance
Zhang et al., J Control Release. 93(2), 141-50 (2003)), anti-cancer
sound-wave and shock-wave therapies (see for instance Kambe et al.,
Hum Cell. 10(1), 87-94 (1997)), and/or anti-cancer nutraceutical
therapy (see for instance Roudebush et al., Vet Clin North Am Small
Anim Pract. 34(1), 249-69, viii (2004) and Rafi, Nutrition. 20(1),
78-82 (2004). Likewise, an anti-CD38 antibody of the present
invention may be used for the preparation of a pharmaceutical
composition for treating a disorder as described above to be
administered with anti-cancer directed photodynamic therapy (for
instance anti-cancer laser therapy--which optionally may be
practiced with the use of photosensitizing agent, anti-cancer
sound-wave and shock-wave therapies, and/or anti-cancer
nutraceutical therapy.
[0561] In a further embodiment, the anti-CD38 antibody of the
present invention is administered together with complement.
[0562] As described above, a pharmaceutical composition of the
present invention may be administered in combination therapy, i.e.,
combined with one or more agents relevant for the disease or
condition to be treated either as separate pharmaceutical
compositions or with a compound of the present invention
coformulated with one or more additional therapeutic agents as
described above. Such combination therapies may require lower
dosages of the compound of the present invention and/or the
co-administered agents, thus avoiding possible toxicities or
complications associated with the various monotherapies.
[0563] In one embodiment, the present invention provides a method
for treating a disorder involving cells expressing CD38 in a
subject, which method comprises administration of a therapeutically
effective amount of an anti-CD38 antibody of the present invention
and at least one immunosuppressive and/or immunomodulatory agent to
a subject in need thereof
Diagnostic Uses
[0564] The anti-CD38 antibodies of the invention may also be used
for diagnostic purposes. Thus, in a further aspect, the invention
relates to a diagnostic composition comprising an anti-CD38
antibody as defined herein.
[0565] In one embodiment, the anti-CD38 antibodies of the present
invention may be used in vivo or in vitro for diagnosing diseases
wherein activated cells expressing CD38 play an active role in the
pathogenesis, by detecting levels of CD38, or levels of cells which
contain CD38 on their membrane surface. This may be achieved, for
example, by contacting a sample to be tested, optionally along with
a control sample, with the anti-CD38 antibody under conditions that
allow for formation of a complex between the antibody and CD38.
Complex formation is then detected (e.g., using an ELISA). When
using a control sample along with the test sample, complex is
detected in both samples and any statistically significant
difference in the formation of complexes between the samples is
indicative of the presence of CD38 in the test sample.
[0566] Thus, in a further aspect, the invention relates to a method
for detecting the presence of CD38 antigen, or a cell expressing
CD38, in a sample comprising: [0567] contacting the sample with an
anti-CD38 antibody of the invention or a bispecific molecule of the
invention, under conditions that allow for formation of a complex
between the antibody and CD38; and [0568] analyzing whether a
complex has been formed.
[0569] In one embodiment, the method is performed in vitro.
[0570] More specifically, the present invention provides methods
for the identification of, and diagnosis of invasive cells and
tissues, and other cells targeted by anti-CD38 antibodies of the
present invention, and for the monitoring of the progress of
therapeutic treatments, status after treatment, risk of developing
cancer, cancer progression, and the like.
[0571] In one example of such a diagnostic assay, the present
invention provides a method of diagnosing the level of invasive
cells in a tissue comprising forming an immunocomplex between an
anti-CD38 antibody and potential CD38-containing tissues, and
detecting formation of the immunocomplex, wherein the formation of
the immunocomplex correlates with the presence of invasive cells in
the tissue. The contacting may be performed in vivo, using labeled
isolated antibodies and standard imaging techniques, or may be
performed in vitro on tissue samples.
[0572] Examples of conventional immunoassays provided by the
present invention include, without limitation, an ELISA, an RIA,
FACS assays, plasmon resonance assays, chromatographic assays,
tissue immunohistochemistry, Western blot, and/or
immunoprecipitation using an anti-CD38 antibody. Suitable labels
for the anti-CD38 antibody and/or secondary antibodies used in such
techniques include, without limitation, various enzymes, prosthetic
groups, fluorescent materials, luminescent materials, and
radioactive materials.
[0573] The anti-CD38 antibodies are particularly useful in the in
vivo imaging of tumors. In vivo imaging of tumors associated with
CD38 may be performed by any suitable technique. For example,
.sup.99Tc-labeling or labeling with another gamma-ray emitting
isotope may be used to label anti-CD38 antibodies in tumors or
secondary labeled (e.g., FITC labeled) anti-CD38 antibody:CD38
complexes from tumors and imaged with a gamma scintillation camera
(e.g., an Elscint Apex 409ECT device), typically using low-energy,
high resolution collimator or a low-energy all-purpose collimator.
Stained tissues may then be assessed for radioactivity counting as
an indicator of the amount of CD38-associated peptides in the
tumor. The images obtained by the use of such techniques may be
used to assess biodistribution of CD38 in a patient, mammal, or
tissue, for example in the context of using CD38 or CD38-fragments
as a biomarker for the presence of invasive cancer cells.
Variations on this technique may include the use of magnetic
resonance imaging (MRI) to improve imaging over gamma camera
techniques. Similar immunoscintigraphy methods and principles are
described in, e.g., Srivastava (ed.), Radiolabeled Monoclonal
Antibodies For Imaging And Therapy (Plenum Press 1988), Chase,
"Medical Applications of Radioisotopes," in Remington's
Pharmaceutical Sciences, 18th Edition, Gennaro et al., (eds.), pp.
624-652 (Mack Publishing Co., 1990), and Brown, "Clinical Use of
Monoclonal Antibodies," in Biotechnology And Pharmacy 227-49,
Pezzuto et al., (eds.) (Chapman & Hall 1993).
[0574] In a further aspect, the invention relates to a kit for
detecting the presence of CD38 antigen, or a cell expressing CD38,
in a sample comprising [0575] an anti-CD38 antibody of the
invention or a bispecific molecule of the invention; and [0576]
instructions for use of the kit.
[0577] In one embodiment, the present invention provides a kit for
diagnosis of cancer comprising a container comprising an anti-CD38
antibody, and one or more reagents for detecting binding of the
anti-CD38 antibody to a CD38 peptide. Reagents may include, for
example, fluorescent tags, enzymatic tags, or other detectable
tags. The reagents may also include secondary or tertiary
antibodies or reagents for enzymatic reactions, wherein the
enzymatic reactions produce a product that may be visualized. In
one embodiment, the present invention provides a diagnostic kit
comprising one or more anti-CD38 antibodies of the present
invention in labeled or unlabeled form in suitable container(s),
reagents for the incubations for an indirect assay, and substrates
or derivatizing agents for detection in such an assay, depending on
the nature of the label. Control reagent(s) may also be
included.
[0578] In a further aspect, the invention relates to an
anti-idiotypic antibody which binds to an anti-CD38 antibody of the
invention as described herein.
[0579] An anti-idiotypic (Id) antibody is an antibody which
recognizes unique determinants generally associated with the
antigen-binding site of an antibody. An Id antibody may be prepared
by immunizing an animal of the same species and genetic type as the
source of an anti-CD38 mAb with the mAb to which an anti-Id is
being prepared. The immunized animal typically can recognize and
respond to the idiotypic determinants of the immunizing antibody by
producing an antibody to these idiotypic determinants (the anti-Id
antibody). Such antibodies are described in for instance U.S. Pat.
No. 4,699,880.
[0580] An anti-Id antibody may also be used as an "immunogen" to
induce an immune response in yet another animal, producing a
so-called anti-anti-Id antibody. An anti-anti-Id may be
epitopically identical to the original mAb, which induced the
anti-Id. Thus, by using antibodies to the idiotypic determinants of
a mAb, it is possible to identify other clones expressing
antibodies of identical specificity. Anti-Id antibodies may be
varied (thereby producing anti-Id antibody variants) and/or
derivatized by any suitable technique, such as those described
elsewhere herein with respect to anti-CD38 antibodies of the
present invention.
[0581] The present invention is further illustrated by the
following examples which should not be construed as further
limiting.
EXAMPLES
Example 1
Generation of Antibodies
[0582] HCo12 mice were immunized every fortnight with 20 .mu.g
purified HA-CD38 alternating with NIH-3T3-CD38 transfected cells.
The first immunization was performed with 5.times.10.sup.6 cells in
100 .mu.l PBS, mixed with 100 .mu.l CFA, i.p., the second and
following immunizations with HA-CD38 s.c., in the presence of 100
.mu.l PBS, mixed with 100 .mu.l IFA. The following immunizations
with transfected cells were performed in the presence of 200 .mu.l
PBS. After titer development, mice were boosted with 20 .mu.g
HA-CD38 in PBS, i.v.
[0583] Spleens were harvested from these mice, splenocytes were
isolated and fused with PEG to a mouse myeloma cell line based upon
standard protocols. The resulting hybridomas were then screened for
human antibody production by ELISA and for CD38 specificity using
human CD38-transfected NS/0 cells by FACS analysis and recombinant
HA-CD38 protein binding by ELISA.
Sequence Analysis of the Anti-CD38 HuMab Variable Domains and
Cloning in Expression Vectors
[0584] Total RNA of the anti-CD38 HuMabs was prepared from
5.times.10.sup.6 hybridoma cells and 5'-RACE-Complementary DNA
(cDNA) was prepared from 100 ng total RNA, using the SMART RACE
cDNA Amplification kit (Clontech), according to the manufacturer's
instructions.
[0585] VH and VL coding regions were amplified by PCR and cloned
into the pCR-Blunt II-TOPO vector (Invitrogen) using the Zero Blunt
PCR cloning kit (Invitrogen). For each HuMab, 16 VL clones and 8 VH
clones were sequenced.
The VL and VH encoding regions were cloned into the pKappa and pG1f
vectors.
[0586] CDR regions are indicated according to IMGT.
(http://imgt.cines.fr/IMGT_vquest/vquest?livret=0&Option=humanIg)
The following IgG1,.kappa. human monoclonal antibodies were
identified:
TABLE-US-00005 VH VL 025 SEQ ID NO: 2 SEQ ID NO: 27 026 SEQ ID NO:
7 SEQ ID NO: 27 028 SEQ ID NO: 12 SEQ ID NO: 37 049 SEQ ID NO: 17
SEQ ID NO: 42 056 SEQ ID NO: 22 SEQ ID NO: 47
Example 2
Electrospray Ionisation-Quadrupole-Time of Flight Mass Spectrometry
of Anti-CD38 Antibodies
[0587] Intact molecular weight data for anti-CD38 antibodies 025,
057 (same amino acid sequence as antibody 026), 028, 049 and 056
were obtained using nanospray Electrospray-MS on a Q-TOF mass
spectrometer. Aliquots of each antibody sample were desalted
offline using C.sub.4 micro-tap cartridge and eluted in
propanol/trifluoroacetic acid solvent. The instrument was
calibrated using glu-fibrinopeptide fragment ions in MS/MS mode.
MassLynx 4.0 software was used to de-convolute the multiply-charged
data obtained.
[0588] Information on the molecular weight of light and heavy chain
components of these antibodies was obtained following reduction
using dithiothreitol and analysis as described above.
TABLE-US-00006 TABLE 1 Mass of CD38 antibodies (in Dalton) Anti-
intact MW Light Heavy Chain body K0 K1 K2 Chain K0 K1 -025 144742.8
144874.1 144999.8 23357.8 49017.4 49145.8 -057 144828.2 144946.4
145071.2 23357.8 49047.4 49175.8 -028 144818.3 144946.4 145074.7
23357.8 49054.5 49182.8 -049 144864.0 144990.6 145117.8 23384.8
49049.3 49177.9 -056 145100.7 145222.7 23384.8 49099.5 49226.4
Example 3
Cross-Block Studies Using FACS
[0589] CHO-CD38 cells were incubated with an excess of unlabelled
CD38-specific antibodies (4.degree. C., 15 min). Then, cells were
incubated with FITC-labeled 005 antibody (concentration
approximates EC.sub.90, 4.degree. C., 45 min) (005 is disclosed in
WO2006099875). After washing the cells twice with PBS-BSA,
fluorescence was measured by flow cytometry. 005-FITC labeled
antibody binding was blocked by excess unlabelled antibodies 025,
026, 028, 049 and 056, indicating that these antibodies have
overlapping epitopes. Binding of 005-FITC was not blocked by excess
unlabelled 003 (disclosed in WO2006099875) providing evidence for
binding to a different epitope.
Cross-Blocking Studies Using ELISA
[0590] Soluble human CD38 was coated on the surface of an ELISA
plate. Coated CD38 was incubated with an excess of unlabelled CD38
specific antibodies for about 15 minutes and subsequently
biotinylated CD38-specific antibodies were added (concentration
approximates EC.sub.90, RT, 1 hour). After washing three times with
PBS/Tween, horseradish peroxidase (HRP)-conjugated streptavidine
was added and the mixture was incubated for 1 hour at RT. The
complex was detected by addition of an ABTS-solution and the HRP
mediated substrate conversion was measured using an ELISA reader at
OD 405 nm.
Cross-Blocking Studies Using Sandwich-ELISA
[0591] Anti-CD38 antibodies were coated on the surface of an ELISA
plate. Plate-bound antibodies were incubated with biotinylated
soluble CD38 in the presence of an excess of anti-CD38 antibodies
in fluid phase. After washing with PBS/Tween, bound biotinylated
CD38 was detected with HRP-conjugated streptavidine for 1 hr at RT.
This complex was detected by addition of an ABTS-solution (after
washing with PBS/Tween) and the HRP mediated substrate conversion
was measured using an ELISA reader at OD 405 nm.
Example 4
Epitope Mapping
Construction of HA-CD38 and His-CD38 Expression Vectors.
[0592] The encoding sequences for the extracellular domain of human
CD38 (identical to amino acids 45-300 from Genbank entry AAA68482)
were amplified from plasmid pCIpuroCD38 (obtained from Prof. M.
Glennie, Tenovus Research Laboratory, Southampton General Hospital,
Southampton, UK) using PCR, introducing, restriction sites, an
ideal Kozak sequence (GCCGCCACC), and sequences encoding a signal
peptide and a N-terminal HA tag (ypydvpdya). The construct was
cloned in the mammalian expression vector pEE13.4 (Lonza
Biologics). This construct was named pEE13.4HACD38.
[0593] A similar construct was made synthetically and fully codon
optimized (GeneArt, Regensburg, Germany), replacing the HA tag
encoding part by a His tag (HHHHHH) encoding part. The construct
was cloned in pEE13.4 and named pEE13.4HisCD38.
Site Directed Mutagenesis
[0594] Several Mutations were Introduced in the Putative Antibody
Binding Site on the CD38 Molecule.
[0595] DNA substitutions leading to T237A, Q272R or S274F amino
acid substitutions were generated using the QuickChange II XL
Site-directed Mutagenesis Kit (Stratagene, Amsterdam, The
Netherlands) in the pEE13.4HACD38 vector. Similarly a D202G
encoding substitution was introduced in the pEE13.4HisCD38
vector.
Transient Expression in HEK-293F Cells
[0596] Freestyle.TM. 293-F (a HEK-293 subclone adapted to
suspension growth and chemically defined Freestyle medium,
(HEK-293F)) cells were obtained from Invitrogen and transfected
with pEE13.4HACD38, pEE13.4HisCD38 or the four constructs carrying
the mutations, according to the manufacturer's protocol using
293fectin (Invitrogen). Cell culture supernatants of transfected
cells were used in ELISA for anti-CD38 binding studies.
Anti-CD38 Antibody Binding
[0597] Mutations T237A, Q272R, and S274F: ELISA plates (Greiner,
#655092) were coated 0/N at 4.degree. C. with 1 .mu.g anti-HA
antibody (Sigma, #H-9658) and subsequently blocked with 2% chicken
serum. Culture supernatants of transfected HEK293F cells were
diluted, applied to the ELISA plates and incubated for 1 hr at RT.
After washing, serial dilutions of anti-CD38 antibodies were added
and incubated for 1 hr at RT. Bound antibodies were detected with
HRP-conjugated goat-anti-human IgG antibodies. The assay was
developed with ABTS (Roche, #1112597) and the absorbance was
measured at 405 nm using a spectrophotometer.
[0598] Mutation D202G: ELISA plates (Greiner, #655092) were coated
0/N at 4.degree. C. with 1 .mu.g penta-His (Qiagen #34660) and
subsequently blocked with 2% PBS/BSA. Culture supernatants of
transfected HEK293F cells were diluted, applied to the ELISA plates
and incubated for 2 hr at RT. After washing, serial dilutions of
anti-CD38 antibodies were added and incubated for 1 hr at RT. Bound
antibodies were detected with HRP-conjugated goat-anti-human IgG
antibodies. The assay was developed with ABTS (Roche, #1112597) and
the absorbance was measured at 405 nm using a
spectrophotometer.
[0599] This study revealed that binding of 025, 026, 028 and 049
was not sensitive to mutations T237A, Q272R, S274F, and A199T, but
was seriously affected by D202G (025, 028, 049) (FIG. 2).
Example 5
Binding of Anti-CD38 Antibodies to CD38-Transfected CHO (CHO-CD38)
Cells and to Daudi-Luc Cells
[0600] After harvesting and counting, Daudi-luc cells, CHO cells
transfected with CD38 and control CHO cells, were resuspended in
PBS (1.times.10.sup.6 cells/mL). Cells were transferred to 96-well
V-bottom plates (100 .mu.L/well) and washed twice in PBS-BSA (PBS
supplemented with 0.1% BSA and 0.02% Na-azide). 50 .mu.L antibody
in PBS-BSA was added in three-fold dilutions ranging from 0.3 to 30
.mu.g/mL (4.degree. C., 30 min). After washing three times in
PBS-BSA, 50 .mu.L (1:400 dilution) of rabbit anti-human IgG-FITC in
PBS-BSA was added (4.degree. C. in the dark, 30 minutes). Cells
were washed three times and specific binding of CD38-antibodies to
CHO-CD38 and Daudi-luc cells was detected by flow cytometry.
[0601] FIG. 3 shows that 025, 026, 028, 049, and 056 bind to
CHO-CD38 cells and to Daudi-luc cells.
Example 6
Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)
[0602] The ability of anti-CD38 antibodies to perform ADCC of
Daudi-luc cells was determined as explained below. As effector
cells, peripheral blood mononuclear cells from healthy volunteers
(UMC Utrecht, The Netherlands) were used.
[0603] Daudi-luc cells were collected (5.times.10.sup.6 cells) in
RPMI.sup.++ (RPMI 1640 culture medium supplemented with 10% cosmic
calf serum (HyClone, Logan, Utah, USA)), to which 100 .mu.Ci
.sup.51Cr (Chromium-51; Amersham Biosciences Europe GmbH,
Roosendaal, The Netherlands) was added, and the mixture was
incubated in a 37.degree. C. water bath for 1 hr. After washing of
the cells (twice in PBS, 1500 rpm, 5 min), the cells were
resuspended in RPMI.sup.++ and counted by trypan blue exclusion.
Cells were brought at concentration of 1.times.10.sup.5
cells/mL.
Preparation of Effector Cells
[0604] Fresh peripheral blood mononuclear cells (healthy
volunteers, UMC Utrecht, Utrecht, The Netherlands) were isolated
from 40 ml of heparin blood by Ficoll (Bio Whittaker; lymphocyte
separation medium, cat 17-829E) according to the manufacturer's
instructions. After resuspension of cells in RPMI.sup.++, cells
were counted by trypan blue exclusion and brought at concentration
of 1.times.10.sup.7 cells/ml.
ADCC Set Up
[0605] 50 .mu.l of .sup.51Cr-labeled targets cells were pipetted
into 96-well plates, and 50 .mu.l of antibody was added, diluted in
RPMI.sup.++ (final concentrations 10, 1, 0.1, 0.01 .mu.g/ml). Cells
were incubated (RT, 15 min), and 50 .mu.l effector cells were
added, resulting in an effector to target ratio of 100:1 (for
determination of maximal lysis, 100 .mu.l 5% Triton-X100 was added
instead of effector cells; for determination of spontaneous lysis,
50 .mu.L target cells and 100 .mu.L RPMI.sup.++ were used). Cells
were spun down (500 rpm, 5 min), and incubated (37.degree. C., 5%
CO.sub.2, 4 hr). After spinning down cells (1500 rpm, 5 min), 100
.mu.L of supernatant was harvested into micronic tubes, and counted
in gamma counter. The percentage specific lysis was calculated as
follows:
(cpm sample-cpm target cells only)/(cpm maximal lysis-cpm target
cells only)
wherein cpm is counts per minute. 025, 026, 028, 049, and 056
induced ADCC mediated lysis in Daudi cells (FIG. 4).
Example 7
Complement-Dependent Cytotoxicity (CDC)
[0606] After harvesting and counting of Daudi-luc cells, the
viability of the cells should be .gtoreq.90%. After washing (PBS),
cells are resuspended at 2.0.times.10.sup.6 cells/ml in RPMI-B
(RPMI supplemented with 1% BSA). Thereafter, cells are put in
96-well round-bottom plates at 1.times.10.sup.5 cells/well (50
.mu.L/well). Then, 50 .mu.L antibodies is added to the wells (final
concentration range between 0-100 .mu.g/ml (three-fold dilutions in
RPMI-B)). After incubation (RT, 15 min), 11 .mu.L of pooled human
serum (pool of 18 healthy donors) was added to each well
(37.degree. C., 45 min). Wells were resuspended once and 120 .mu.L
was transferred to FACS tubes (Greiner). Then, 10 .mu.L propidium
iodide (PI; Sigma-Aldrich Chemie B.V.) was added (10 .mu.g/ml
solution) to this suspension. Lysis was detected by flow cytometry
(FACScalibur.TM., Becton Dickinson, San Diego, Calif., USA) by
measurement of the percentage of dead cells (corresponds to
PI-positive cells).
[0607] FIG. 5 presents CDC-mediated CHO-CD38 cell lysis caused by
anti-CD38 antibodies 025, 026, 028, 049 and 056. These anti-CD38
antibodies failed to induce CDC of Daudi-luc cells.
Example 8
Enzymatic Activity
[0608] The effects of anti-CD38 antibodies on the enzymatic
activities of CD38 were determined. CD38 is known to catalyze
several different enzymatic reactions, including a cyclase reaction
converting NAD into cyclic ADP ribose (cADPR), a hydrolase reaction
converting NAD or cADPR into ADP ribose, and a base-exchange
reaction in which nicotinic acid adenine dinucleotide 2'-phosphate
(NAADP) is produced.
Cyclase Activity
NGD Assay
[0609] The ability of anti-CD38 antibodies to interfere with the
cyclase activity of CD38 using NGD as a substrate was measured in
an assay essentially as described in Graeff et al., J. Biol. Chem.
269, 30260-30267 (1994):
[0610] Briefly, substrate NOD.sup.+ (80 .mu.M) was incubated with
CD38 (0.6 .mu.g/ml His-tagged extracellular domain of human CD38,
see Example 3 of WO2006099875 regarding purification of His-CD38 in
a buffer containing 20 mM Tris-HCl, pH 7.0). The production of
cGDPR can be monitored spectrophotometrically at the emission
wavelength of 410 nm (excitation at 300 nm). In this example an
excitation filter of 340.+-.60 nm and an emission filter of
430.+-.8 nm were used.
[0611] To test the effect of 025, 026, 028, 049 and 056 on the
enzymatic activity of CD38, recombinant His-CD38 protein was
pre-incubated for 15 minutes at room temperature with 3 .mu.g/ml of
the antibodies before adding the substrate NGD.sup.+. The
production of cyclic GDP-ribose (cGDPR) was recorded after 90
minutes.
[0612] FIG. 6A shows that antibodies 025, 026, 028, 049 and 056
have a pronounced inhibitory effect on the production of cGDPR.
After 90 minutes, 3 .mu.g/ml of antibody (025, 026, 028, 049 or
056) resulted in a 53-66% reduced production of cGDPR. In a time
course experiment it was shown that the rate of cGDPR production
was reduced in samples treated with the CD38-specific antibody mAb
028 compared to the cGDPR production in the presence of HuMab-KLH
or in the untreated CD38 control (FIG. 6B). FIG. 6C shows a dose
response curve (0.01-30 .mu.g/ml) for antibody 028. In this
experiment a maximum reduction of cGDPR production of 41% is
observed.
[0613] To test the effect of 028 on the enzymatic activity of
cellular expressed CD38, CHO-CDC38 cells were pre-incubated for 30
minutes at room temperature with a serial dilution of 028 (0.01-30
.mu.g/ml) before adding the substrate NGD.sup.+. The production of
cyclic GDP-ribose (cGDPR) was recorded after 90 min. As shown in
FIG. 6D antibody 028 inhibits the production of cGDPR in a
concentration dependent fashion.
Reverse Cyclase Reaction
[0614] The effect of mAb 028 on cADPR production from NAD by CD38
was determined by the reverse cyclase reaction. This assay is based
on the reversibility of the reaction catalyzed by CD38. In the
presence of high concentrations of nicotinamide and cADPR, the
ADP-ribosyl cyclases can produce NAD. Antibodies were diluted to 10
.mu.g/ml in 20 mM Tris-HCl, 0.01% (v/v) BSA, pH 7.2 (Tris/BSA).
Human recombinant CD38 was diluted to 2 .mu.g/ml with Tris/BSA. The
antibodies were preincubated for 10 minutes with CD38 by mixing
equal volumes (50 .mu.L) of the diluted antibodies with the diluted
CD38. The preincubation was done at room temperature. The reaction
was initiated by transferring 25 .mu.L of CD38/antibody mixture to
25 .mu.L of a solution containing 1 mM cADPR and 10 mM
nicotinamide. The reaction was allowed to proceed at room
temperature for 1 to 20 minutes and was stopped at the appropriate
time by filtering the entire sample through a Millipore
MultiScreen-IP Filter 96-well plate to remove protein. The
resulting NAD produced was measured by the method of Graeff and Lee
(1). Controls containing nicotinamide without cADPR were run to
estimate the amount NAD contaminating the reagents. In these
experiments there was undetectable contaminating NAD.
[0615] Table 2 shows that 1 .mu.g/ml mAb-028 reduced cADPR
production from NAD by 67%. mAb-KLH had no effect on cADPR
production from NAD.
TABLE-US-00007 TABLE 2 The effect of antibody 028 on cADPR
production from NAD Condition pmol cADPR/min CD38 control 4.3
mAb-KLH 4.3 mAb-028 1.4
8-amino-NAD (8NH2-NAD) Assay
[0616] As cADPR production only accounts for approximately 1% of
the product generated from NAD by CD38 (ADPR accounts for the
rest), ribosyl cyclase activity was also assessed using 8-amino-NAD
(8NH2-NAD) as a substrate. Unlike NAD, a considerably larger amount
(approximately 8%) of the 8NH2-NAD substrate is cyclized to
8-amino-cADPR (8NH2-cADPR) and is detectable by HPLC analysis.
Briefly, antibodies were diluted to 10 .mu.g/mL in 20 mM Tris-HCl,
0.01% (v/v) BSA, pH 7.2 (Tris/BSA). Human recombinant CD38 was
diluted to 2 .mu.g/ml with Tris/BSA. The antibodies were
preincubated for 10 minutes with CD38 by mixing equal volumes (50
.mu.L) of the diluted antibodies with the diluted CD38. The
preincubation was done at room temperature. The reaction was
initiated by transferring 25 .mu.L of CD38/antibody mixture to 75
.mu.L of 0.5 mM 8NH2-NAD. The reaction was allowed to proceed at
room temperature for 10 minutes and was stopped at the appropriate
time by filtering the entire sample through a Millipore
MultiScreen-IP Filter 96-well plate to remove protein. The reaction
products (8NH2-cADPR and 8NH2-ADPR) were analyzed by reverse phase
HPLC as follows. The column was a 0.46.times.15 cm LC18-T reverse
phase column from Supelco. Solvent A consisted of 20 mM KH2PO4, 5
mM tetrabutylammonium phosphate, pH 6 and solvent B consisted of
50% A and 50% methanol. The flow rate was 1 mL/min and the initial
composition of solvents was 15% B. Separation of substrates and
products was accomplished using the following gradient: 0 to 3.5
minutes (15% B), 3.5 to 5.5 minutes (15 to 32.5% B), 5.5 to 9
minutes (32.5 to 40% B), 9 to 11.5 minutes (40 to 50% B) and 16 to
18 minutes (50 to 15% B) gradient was used to elute the substrates
and products. Samples were prepared by adding 400 .mu.L of solvent
A to 100 .mu.L of filtered sample. The entire sample was injected.
The flow rate and buffer composition were controlled by Beckman 125
HPLC pumps and System Gold software and peaks were detected with a
Beckman 166 UV detector. The areas of the 8NH2-NAD, 8NH2-cADPR and
8NH2-ADPR peaks were used to calculate the amount of 8NH2-cADPR
produced in the assay. The HPLC system is based on a system
described by Schweitzer et al. (2).
[0617] FIG. 7A shows that mAb-028 inhibits 8NH2-cADPR by 78%.
mAb-028 inhibits 8NH2-cADPR production in a concentration dependent
manner (FIG. 7B)
[0618] Thus mAb-028 inhibits the ADP-ribosyl cyclase reaction of
CD38 as assayed by three different methods.
Hydrolase Activity
Hydrolase Activity Analysis by HPLC
[0619] The hydrolase activity was measured by determining the
amount of ADPR produced from cADPR or NAD by HPLC. Antibodies were
diluted to 10 .mu.g/mL or titrated in 20 mM Tris-HCl, 0.01% (v/v)
BSA, pH 7.2 (Tris/BSA). Human recombinant CD38 was diluted to 2
.mu.g/mL with Tris/BSA. The antibodies were preincubated for 10
minutes with CD38 by mixing equal volumes (50 .mu.L) of the diluted
antibodies with the diluted CD38. The preincubation was done at
room temperature. For the HPLC-based method the cADPR hydrolase
reaction was initiated by transferring 40 .mu.L of CD38/antibody
mixture to 10 .mu.L of 4.3 mM cADPR and the NADase reaction was
initiated by transferring 40 .mu.L of CD38/antibody mixture to 10
.mu.L 1 mM NAD. The reaction was allowed to proceed at room
temperature and was stopped at the appropriate time by adding 25
.mu.L of 1 M HCl. Protein was removed by filtering the entire
sample through a Millipore MultiScreen-IP Filter 96-well plate.
Each filtrate was neutralized by adding 15 .mu.L of 2M Tris-base
and kept on ice until analyzed by HPLC. The analysis of hydrolase
activity is based on the HPLC assay developed by Lee and Aarhus
(3). The samples were analyzed on a 0.5.times.5 cm column of AG
MP-1 (trifluoroacetate form) eluted at 3 mL/min with a 0 to 150 mM
concave upward gradient of trifluoroacetic acid (TFA) over 10
minutes. The flow rate and buffer composition were controlled by
Beckman 125 HPLC pumps and System Gold software and peaks were
detected with a Beckman 166 UV detector. The areas of NAD, cADPR
and ADPR were used to calculate the amount of ADPR produced in the
assay.
[0620] At concentrations of 10 .mu.g/mL mAb-028, but not mAb-KLH,
stimulated the cADPR hydrolase activity by 62% and the NAD
hydrolase activity by 37% compared to the CD38 control (FIG. 8A).
FIG. 8B shows that mAb-028 stimulated cADPR hydrolysis in a
dose-dependent manner. At concentrations of 30 .mu.g/mL, mAb-028
stimulated hydrolase activity by 78%.
Hydrolase Activity Analysis by Thin Layer Chromatography (TLC)
[0621] The hydrolase activity was measured by measuring the amount
of .sup.32P-ADPR produced from .sup.32P-cADPR by thin layer
chromatography (4). The .sup.32P-based cADPR hydrolase reaction was
initiated by adding 20 .mu.L of CD38/antibody mixture (as above) to
5 .mu.L of a mixture containing 0.5 mM cADPR and approximately 0.1
.mu.Ci of .sup.32P-cADPR. The reaction was allowed to proceed at
room temperature and at the appropriate times, 5 .mu.L of the
reaction was added to 5 .mu.L of 150 mM TFA to stop the reaction.
The reaction was analyzed by PEI-cellulose thin layer
chromatography (TLC). One (1) .mu.L of each stopped reaction sample
was spotted on the origin of a PEI-cellulose TLC plate (10.times.20
cm). The plates were developed with 0.2 M NaCl in 30% (v/v)
ethanol. The plates were dried and exposed to phosphoimage screens.
The screens were analyzed on a Packard Cyclone Phosphorimager to
determine the amount of .sup.32P-ADPR produced.
[0622] FIG. 8C shows that mAb-028 stimulated .sup.32P-cADPR
hydrolysis in a dose-dependent manner. These results were similar
to the results of the cADPR hydrolase activity measured by HPLC
(see FIG. 8B).
Base-Exchange Activity
[0623] The effect of CD38 antibodies on nicotinic acid adenine
dinucleotide 2'-phosphate (NAADP) synthesis by the base-exchange
activity of CD38 was assessed. Antibodies (mAb-KLH and 028) were
diluted to 40 .mu.g/mL in 20 mM Hepes, pH 7.3, 0.01% (v/v) BSA
(Hepes/BSA). Human recombinant CD38 was diluted to 2 .mu.g/mL with
Hepes/BSA. The antibodies were preincubated for 10 minutes with
CD38 by mixing equal volumes (90 .mu.L) of the diluted antibodies
with the diluted CD38. The preincubation was performed at room
temperature. The base-exchange reaction was initiated by
transferring 50 .mu.L of CD38/antibody mixture to 50 .mu.L of a
reaction mixture containing 200 mM sodium acetate, pH 4.0, 25 mM
nicotinic acid and 2 mM nicotinamide adenine dinucleotide
2'-phosphate (NADP). The reaction was allowed to proceed at room
temperature for 30 minutes and was stopped by filtering the entire
sample through a Millipore MultiScreen-IP Filter 96-well plate to
remove protein. The reaction products in the filtrates were
determined by anion-exchange HPLC on a 0.5.times.5 cm column of AG
MP-1 (trifluoroacetate form) eluted at 1 mL/min with a 0 to 150 mM
concave upward gradient of trifluoroacetic acid (TFA) over 30
minutes (5). The filtrates (50 .mu.L) were neutralized by adding 5
.mu.L of 2 M Tris-base just prior to injection. The flow rate and
buffer composition were controlled by Beckman 125 HPLC pumps and
System Gold software and peaks were detected with a Beckman 166 UV
detector. The areas of the NADP, NAADP (base-exchange product) and
adenosine diphospho-ribose 2'-phosphate (ADPR-P, hydrolytic
product) peaks were used to calculate the rates of NAADP synthesis
and NADP hydrolysis.
[0624] FIG. 9 shows that mAb-028 inhibits the ability of CD38 to
catalyze the formation of NAADP. The inhibition of NAADP production
by mAb-028 is concentration dependent (FIG. 9B) with an IC50 of
0.14 .mu.g/mL.
LIST OF REFERENCES
[0625] 1. Graeff, R., and H. C. Lee. 2002. A novel cycling assay
for cellular cADP-ribose with nanomolar sensitivity. Biochem J
361:379-384. [0626] 2. Schweitzer, K., G. W. Mayr, and A. H. Guse.
2001. Assay for ADP-ribosyl cyclase by reverse-phase
high-performance liquid chromatography. Anal Biochem 299:218-226.
[0627] 3. Lee, H. C., and R. Aarhus. 1993. Wide distribution of an
enzyme that catalyzes the hydrolysis of cyclic ADP-ribose. Biochim
Biophys Acta 1164:68-74. [0628] 4. White, T. A., S. Johnson, T. F.
Walseth, H. C. Lee, R. M. Graeff, C. B. Munshi, Y. S. Prakash, G.
C. Sieck, and M. S. Kannan. 2000. Subcellular localization of
cyclic ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase
activities in porcine airway smooth muscle. Biochim Biophys Acta
1498:64-71. [0629] 5. Aarhus, R., R. M. Graeff, D. M. Dickey, T. F.
Walseth, and H. C. Lee. 1995. ADP-ribosyl cyclase and CD38 catalyze
the synthesis of a calcium-mobilizing metabolite from NADP. J Biol
Chem 270:30327-30333.
Sequence CWU 1
1
551363DNAhomo sapiens 1caggtccaac tggtgcagtc tggggctgag gtgaagaagc
ctgggtcctc ggtgaaggtc 60tcctgcaagg cttttggagg caccttcagc agctacgcta
tcagctgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggaagg
atcatccgtt tccttggtat agcaaactac 180gcacagaagt tccagggcag
agtcacgctt atcgcggaca aatccacgaa cacagcctac 240atggagctga
gcagcctgag atctgaggac acggccgttt attactgtgc gggggaacct
300ggggagcggg accccgatgc tgttgatatc tggggccaag ggacaatggt
caccgtctct 360tca 3632121PRThomo sapiens 2Gln 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 Phe Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile
Ile Arg Phe Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly
Arg Val Thr Leu Ile Ala Asp Lys Ser Thr Asn Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Gly Glu Pro Gly Glu Arg Asp Pro Asp Ala Val Asp Ile Trp
Gly 100 105 110Gln Gly Thr Met Val Thr Val Ser Ser 115 12039PRThomo
sapiens 3Gly Gly Thr Ser Phe Ser Ser Tyr Ala1 548PRThomo sapiens
4Ile Ile Arg Phe Leu Gly Ile Ala1 5514PRThomo sapiens 5Ala Gly Glu
Pro Gly Glu Arg Asp Pro Asp Ala Val Asp Ile1 5 106364DNAhomo
sapiens 6caggtccaac tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc
ggtgaaggtc 60tcctgcaagg cttttggagg caccttcagc agctatgcta tcagctgggt
acgacaggcc 120cctggacaag ggcttgagtg gatgggaagg atcatccgtt
tccttggtaa agcaaatcac 180gcacagaagt tccagggcag agtcacgctt
accgcggaca aatccacgaa cacagcctac 240atggagctga gcagcctgag
atctgaggac acggccgttt attactgtgc gggggaacct 300ggggatcggg
accccgatgc tgttgatatc tggggccaag ggacaatggt caccgtctct 360tcag
3647121PRThomo sapiens 7Gln 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 Phe Gly
Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile Ile Arg Phe Leu Gly
Lys Ala Asn His Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr
Ala Asp Lys Ser Thr Asn Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Gly Glu Pro
Gly Asp Arg Asp Pro Asp Ala Val Asp Ile Trp Gly 100 105 110Gln Gly
Thr Met Val Thr Val Ser Ser 115 12088PRThomo sapiens 8Gly Gly Thr
Phe Ser Ser Tyr Ala1 598PRThomo sapiens 9Ile Ile Arg Phe Leu Gly
Lys Ala1 51014PRThomo sapiens 10Ala Gly Glu Pro Gly Asp Arg Asp Pro
Asp Ala Val Asp Ile1 5 1011364DNAhomo sapiens 11caggtccaac
tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60tcctgcaagg
cttttggagg caccttcagc agttatgcta ttagctgggt gcgacaggcc
120cctggacaag ggcttgagtg gatgggaagg atcatccgtt tccttggtaa
aacaaatcac 180gcacagaagt tccagggcag agtcacactt accgcggaca
aatccacgaa cacagcctac 240atggagctga gcagcctgag atctgaggac
acggccgttt attactgtgc gggggaacct 300ggggatcggg accccgatgc
tgttgatatc tggggccaag ggacaatggt caccgtctct 360tcag 36412121PRThomo
sapiens 12Gln 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 Phe Gly Gly Thr Phe
Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Arg Ile Ile Arg Phe Leu Gly Lys Thr Asn
His Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Lys
Ser Thr Asn Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Gly Glu Pro Gly Asp Arg
Asp Pro Asp Ala Val Asp Ile Trp Gly 100 105 110Gln Gly Thr Met Val
Thr Val Ser Ser 115 120138PRThomo sapiens 13Gly Gly Thr Phe Ser Ser
Tyr Ala1 5148PRThomo sapiens 14Ile Ile Arg Phe Leu Gly Lys Thr1
51514PRThomo sapiens 15Ala Gly Glu Pro Gly Asp Arg Asp Pro Asp Ala
Val Asp Ile1 5 1016364DNAhomo sapiens 16caggtccagc tggtgcagtc
tggggctgag gtgatgaagc ctgggtcctc ggtgaaggtc 60tcctgcaagg cttccggagg
caccttccgc agctatgcta tcagttgggt gcgacaggcc 120cctggacaag
ggcttgagtg gatgggaagg atcatcgttt tccttggtaa aacaaactac
180gcacagaagt tccagggcag agtcacgctt accgcggaca aatccacgac
cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtgt
attactgtac gggggaacct 300ggggctcggg accccgacgc ttttgatatc
tggggccaag ggacaatggt caccgtctct 360tcag 36417121PRThomo sapiens
17Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Met Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Ser
Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Arg Ile Ile Val Phe Leu Gly Lys Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr
Thr Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Thr Gly Glu Pro Gly Ala Arg Asp Pro
Asp Ala Phe Asp Ile Trp Gly 100 105 110Gln Gly Thr Met Val Thr Val
Ser Ser 115 120188PRThomo sapiens 18Gly Gly Thr Phe Arg Ser Tyr
Ala1 5198PRThomo sapiens 19Ile Ile Val Phe Leu Gly Lys Thr1
52014PRThomo sapiens 20Thr Gly Glu Pro Gly Ala Arg Asp Pro Asp Ala
Phe Asp Ile1 5 1021364DNAhomo sapiens 21caggtccagc tggtgcagtc
tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60tcctgcaagc cttccggagg
caccttcagg agctacgcta tcagctgggt acgacaggcc 120cctggacaag
ggcttgagtg gatgggaagg atcatcgttt tccttggtaa agtaaactac
180gcacagaggt ttcagggcag agtcacgctt accgcggaca aatccacgac
cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtgt
attactgtac gggggaacct 300ggggctcggg accccgacgc ttttgatatc
tggggccaag ggacaatggt caccgtctct 360tcag 36422121PRThomo sapiens
22Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Pro Ser Gly Gly Thr Phe Arg Ser
Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Arg Ile Ile Val Phe Leu Gly Lys Val Asn Tyr Ala
Gln Arg Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr
Thr Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Thr Gly Glu Pro Gly Ala Arg Asp Pro
Asp Ala Phe Asp Ile Trp Gly 100 105 110Gln Gly Thr Met Val Thr Val
Ser Ser 115 120238PRThomo sapiens 23Gly Gly Thr Phe Arg Ser Tyr
Ala1 5248PRThomo sapiens 24Ile Ile Val Phe Leu Gly Lys Val1
52514PRThomo sapiens 25Thr Gly Glu Pro Gly Ala Arg Asp Pro Asp Ala
Phe Asp Ile1 5 1026321DNAhomo sapiens 26gacatccaga tgacccagtc
tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgtc gggcgagtca
gggtattcgc agctggttag cctggtatca gcagaaacca 120gagaaagccc
ctaagtccct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca
180aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag
cctgcagcct 240gaagattttg caacttatta ctgccaacag tataatagtt
acccgctcac tttcggcgga 300gggaccaagg tggagatcaa a 32127107PRThomo
sapiens 27Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile
Arg Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro
Lys Ser Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys 100 105286PRThomo sapiens 28Gly Gly Ile Arg Ser
Trp1 5293PRThomo sapiens 29Ala Ala Ser1309PRThomo sapiens 30Gln Gln
Tyr Asn Ser Tyr Pro Leu Thr1 531322DNAhomo sapiens 31gacatccaga
tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgtc
gggcgagtca gggtattcgc agctggttag cctggtatca gcagaaacca
120gagaaagccc ctaagtccct gatctatgct gcatccagtt tgcaaagtgg
ggtcccatca 180aggttcagcg gcagtggatc tgggacagat ttcactctca
ccatcagcag cctgcagcct 240gaagattttg caacttatta ctgccaacag
tataatagtt acccgctcac tttcggcgga 300gggaccaagg tggagatcaa ac
32232107PRThomo sapiens 32Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Arg Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95Thr Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys 100 105336PRThomo sapiens 33Gln Gly
Ile Arg Ser Trp1 5343PRThomo sapiens 34Ala Ala Ser1359PRThomo
sapiens 35Gln Gln Tyr Asn Ser Tyr Pro Leu Thr1 536322DNAhomo
sapiens 36gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga
cagagtcacc 60atcacttgtc gggcgagtca gggtattcgc agctggttag cctggtatca
gcagaaacca 120gagaaagccc ctaagtccct gatctatgct gcatccagtt
tgcaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagat
ttcactctca ccatcagcag cctgcagcct 240gaagattttg caacttatta
ctgccaacag tataatagtt acccgctcac tttcggcgga 300gggaccaagg
tggagatcaa ac 32237107PRThomo sapiens 37Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Gly Ile Arg Ser Trp 20 25 30Leu Ala Trp Tyr Gln
Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr Ala Ala Ser
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90
95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105386PRThomo
sapiens 38Gln Gly Ile Arg Ser Trp1 5393PRThomo sapiens 39Ala Ala
Ser1409PRThomo sapiens 40Gln Gln Tyr Asn Ser Tyr Pro Leu Thr1
541322DNAhomo sapiens 41gacatccaga tgacccagtc tccatcctca ctgtctgcat
ctgtaggaga cagagtcacc 60atcacttgtc gggcgagtca gggtattcgc agctggttag
cctggtatca gcagaaacca 120gagaaagccc ctaagtccct gatctatgct
gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg gcagtggatc
tgggacagat ttcactctca ccatcagcag cctgcagcct 240gaagattttg
caacttatta ctgccaacag tataataatt atccgctcac tttcggcgga
300gggaccaagg tggagatcaa ac 32242107PRThomo sapiens 42Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Ser Trp 20 25 30Leu
Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40
45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Asn
Tyr Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
105436PRThomo sapiens 43Gln Gly Ile Arg Ser Trp1 5443PRThomo
sapiens 44Ala Ala Ser1459PRThomo sapiens 45Gln Gln Tyr Asn Asn Tyr
Pro Leu Thr1 546322DNAhomo sapiens 46gacatccaga tgacccagtc
tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgtc gggcgagtca
gggtattcgc agctggttag cctggtatca gcagaaacca 120gagaaagccc
ctaagtccct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca
180aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag
cctgcagcct 240gaagattttg caacttatta ctgccaacag tataataatt
atccgctcac tttcggcgga 300gggaccaagg tggagatcaa ac 32247107PRThomo
sapiens 47Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile
Arg Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro
Lys Ser Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Tyr Asn Asn Tyr Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys 100 105486PRThomo sapiens 48Gln Gly Ile Arg Ser
Trp1 5493PRThomo sapiens 49Ala Ala Ser1509PRThomo sapiens 50Gln Gln
Tyr Asn Asn Tyr Pro Leu Thr1 551300PRThomo sapiens 51Met Ala Asn
Cys Glu Phe Ser Pro Val Ser Gly Asp Lys Pro Cys Cys1 5 10 15Arg Leu
Ser Arg Arg Ala Gln Leu Cys Leu Gly Val Ser Ile Leu Val 20 25 30Leu
Ile Leu Val Val Val Leu Ala Val Val Val Pro Arg Trp Arg Gln 35 40
45Gln Trp Ser Gly Pro Gly Thr Thr Lys Arg Phe Pro Glu Thr Val Leu
50 55 60Ala Arg Cys Val Lys Tyr Thr Glu Ile His Pro Glu Met Arg His
Val65 70 75 80Asp Cys Gln Ser Val Trp Asp Ala Phe Lys Gly Ala Phe
Ile Ser Lys 85 90 95His Pro Cys Asn Ile Thr Glu Glu Asp Tyr Gln Pro
Leu Met Lys Leu 100 105 110Gly Thr Gln Thr Val Pro Cys Asn Lys Ile
Leu Leu Trp Ser Arg Ile 115 120 125Lys Asp Leu Ala His Gln Phe Thr
Gln Val Gln Arg Asp Met Phe Thr 130 135 140Leu Glu Asp Thr Leu Leu
Gly Tyr Leu Ala Asp Asp Leu Thr Trp Cys145 150 155 160Gly Glu Phe
Asn Thr Ser Lys Ile Asn Tyr Gln Ser Cys Pro Asp Trp 165 170 175Arg
Lys Asp Cys Ser Asn Asn Pro Val Ser Val Phe Trp Lys Thr Val 180 185
190Ser Arg Arg Phe Ala Glu Ala Ala Cys Gly Val Val His Val Met Leu
195 200 205Asn Gly Ser Arg Ser Lys Ile Phe Asp Lys Asn Ser Thr Phe
Gly Ser 210 215 220Val Glu Val His Asn Leu Gln Pro Glu Lys Val Gln
Thr Leu Glu Ala225 230 235 240Trp Val Ile His Gly Gly Arg Glu Asp
Ser Arg Asp Leu Cys Gln Asp 245 250 255Pro Thr Ile Lys Glu Leu Glu
Ser Ile Ile Ser Lys Arg Asn Ile Gln 260 265 270Phe Ser Cys Lys Asn
Ile Tyr Arg Pro Asp Lys Phe Leu Gln Cys Val 275 280 285Lys Asn Pro
Glu Asp Ser Ser Cys Thr Ser Glu Ile 290 295 30052300PRTHomo sapiens
52Met Ala Asn Cys Glu Phe Ser Pro Val Ser Gly Asp Lys Pro Cys Cys1
5 10 15Arg Leu Ser Arg Arg Ala Gln Leu Cys Leu Gly Val Ser Ile Leu
Val 20 25 30Leu Ile Leu Val
Val Val Leu Ala Val Val Val Pro Arg Trp Arg Gln 35 40 45Gln Trp Ser
Gly Pro Gly Thr Thr Lys Arg Phe Pro Glu Thr Val Leu 50 55 60Ala Arg
Cys Val Lys Tyr Thr Glu Ile His Pro Glu Met Arg His Val65 70 75
80Asp Cys Gln Ser Val Trp Asp Ala Phe Lys Gly Ala Phe Ile Ser Lys
85 90 95His Pro Cys Asn Ile Thr Glu Glu Asp Tyr Gln Pro Leu Met Lys
Leu 100 105 110Gly Thr Gln Thr Val Pro Cys Asn Lys Ile Leu Leu Trp
Ser Arg Ile 115 120 125Lys Asp Leu Ala His Gln Phe Thr Gln Val Gln
Arg Asp Met Phe Thr 130 135 140Leu Glu Asp Thr Leu Leu Gly Tyr Leu
Ala Asp Asp Leu Thr Trp Cys145 150 155 160Gly Glu Phe Asn Thr Ser
Lys Ile Asn Tyr Gln Ser Cys Pro Asp Trp 165 170 175Arg Lys Asp Cys
Ser Asn Asn Pro Val Ser Val Phe Trp Lys Thr Val 180 185 190Ser Arg
Arg Phe Ala Glu Ala Ala Cys Asp Val Val His Val Met Leu 195 200
205Asn Gly Ser Arg Ser Lys Ile Phe Asp Lys Asn Ser Thr Phe Gly Ser
210 215 220Val Glu Val His Asn Leu Gln Pro Glu Lys Val Gln Thr Leu
Glu Ala225 230 235 240Trp Val Ile His Gly Gly Arg Glu Asp Ser Arg
Asp Leu Cys Gln Asp 245 250 255Pro Thr Ile Lys Glu Leu Glu Ser Ile
Ile Ser Lys Arg Asn Ile Gln 260 265 270Phe Ser Cys Lys Asn Ile Tyr
Arg Pro Asp Lys Phe Leu Gln Cys Val 275 280 285Lys Asn Pro Glu Asp
Ser Ser Cys Thr Ser Glu Ile 290 295 300534PRTArtificial
SequenceSynthetic peptide 53Cys Pro Pro Cys1549PRTArtificial
SequenceSynthetic peptide 54Tyr Pro Tyr Asp Val Pro Asp Tyr Ala1
5556PRTArtificial SequenceSynthetic peptide 55His His His His His
His1 5
* * * * *
References