U.S. patent application number 13/069705 was filed with the patent office on 2011-10-27 for novel anti-cd38 antibodies for the treatment of cancer.
This patent application is currently assigned to SANOFI-AVENTIS. Invention is credited to Laura BARTLE, Veronique BLANC, Jutta DECKERT, Viktor GOLMAKHER, Vincent MILOL, Peter U. PARK, Anna SKALETSKAYA, Daniel TAVARES.
Application Number | 20110262454 13/069705 |
Document ID | / |
Family ID | 37831721 |
Filed Date | 2011-10-27 |
United States Patent
Application |
20110262454 |
Kind Code |
A1 |
PARK; Peter U. ; et
al. |
October 27, 2011 |
NOVEL ANTI-CD38 ANTIBODIES FOR THE TREATMENT OF CANCER
Abstract
Antibodies, humanized antibodies, resurfaced antibodies,
antibody fragments, derivatized antibodies, and conjugates of same
with cytotoxic agents, which specifically bind to CD38, are capable
of killing CD38.sup.+ cells by apoptosis, antibody-dependent
cell-mediated cytotoxicity (ADCC), and/or complement-dependent
cytotoxicity (CDC). Said antibodies and fragments thereof may be
used in the treatment of tumors that express CD38 protein, such as
multiple myeloma, chronic lymphocytic leukemia, chronic myelogenous
leukemia, acute myelogenous leukemia, or acute lymphocytic
leukemia, or the treatment of autoimmune and inflammatory diseases
such as systemic lupus, rheumatoid arthritis, multiple sclerosis,
erythematosus, and asthma. Said derivatized antibodies may be used
in the diagnosis and imaging of tumors that express elevated levels
of CD38. Also provided are cytotoxic conjugates comprising a cell
binding agent and a cytotoxic agent, therapeutic compositions
comprising the conjugate, methods for using the conjugates in the
inhibition of cell growth and the treatment of disease, and a kit
comprising the cytotoxic conjugate. In particular, the cell binding
agent is a monoclonal antibody, and epitope-binding fragments
thereof, that recognizes and binds the CD38 protein.
Inventors: |
PARK; Peter U.; (Somerville,
MA) ; BARTLE; Laura; (Arlington, MA) ;
SKALETSKAYA; Anna; (Cambridge, MA) ; GOLMAKHER;
Viktor; (Newton, MA) ; TAVARES; Daniel;
(Natick, MA) ; DECKERT; Jutta; (Lexington, MA)
; MILOL; Vincent; (Charenton-Pont, FR) ; BLANC;
Veronique; (Daumesnil, FR) |
Assignee: |
SANOFI-AVENTIS
PARIS
FR
|
Family ID: |
37831721 |
Appl. No.: |
13/069705 |
Filed: |
March 23, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12441466 |
Aug 10, 2009 |
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PCT/IB2007/004172 |
Oct 16, 2007 |
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13069705 |
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Current U.S.
Class: |
424/158.1 ;
424/178.1; 435/320.1; 435/344; 435/7.23; 530/387.3; 530/387.9;
530/388.26; 530/389.6; 530/391.7; 536/23.53 |
Current CPC
Class: |
A61K 47/6867 20170801;
C07K 2317/30 20130101; A61P 25/00 20180101; A61P 37/06 20180101;
A61P 43/00 20180101; A61K 47/6803 20170801; A61P 19/02 20180101;
A61P 7/04 20180101; A61P 9/00 20180101; A61P 37/00 20180101; A61P
37/02 20180101; A61P 13/12 20180101; A61P 1/00 20180101; A61P 1/04
20180101; A61P 35/00 20180101; A61P 35/02 20180101; A61P 29/00
20180101; A61P 17/00 20180101; A61P 17/04 20180101; G01N 2333/924
20130101; A61P 1/16 20180101; C07K 2317/56 20130101; G01N 33/57484
20130101; G01N 33/57426 20130101; A61K 2039/505 20130101; A61P
27/02 20180101; C07K 2317/34 20130101; C07K 2317/565 20130101; A61P
11/00 20180101; A61P 11/06 20180101; C07K 2317/73 20130101; C07K
16/2896 20130101; C07K 2317/732 20130101; C07K 2317/734 20130101;
A61P 21/00 20180101; C07K 16/462 20130101; C07K 2317/24
20130101 |
Class at
Publication: |
424/158.1 ;
435/7.23; 435/320.1; 530/389.6; 435/344; 530/388.26; 530/387.3;
530/391.7; 424/178.1; 536/23.53; 530/387.9 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12N 15/85 20060101 C12N015/85; C12N 5/10 20060101
C12N005/10; C07K 16/40 20060101 C07K016/40; A61P 35/00 20060101
A61P035/00; C07H 21/04 20060101 C07H021/04; A61P 19/02 20060101
A61P019/02; A61P 1/00 20060101 A61P001/00; A61P 1/04 20060101
A61P001/04; A61P 25/00 20060101 A61P025/00; A61P 11/06 20060101
A61P011/06; G01N 33/574 20060101 G01N033/574; A61P 35/02 20060101
A61P035/02 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 19, 2006 |
EP |
06291628.3 |
Claims
1) An antibody or epitope-binding fragment thereof that
specifically binds CD38, characterized in that said antibody or
epitope-binding fragment thereof is capable of killing a CD38.sup.+
cell by apoptosis, antibody-dependent cell-mediated cytotoxicity
(ADCC), and complement-dependent cytoxicity (CDC).
2) The antibody or epitope-binding fragment thereof according to
claim 1, characterized in that said antibody or epitope-binding
fragment thereof is capable of killing said CD38.sup.+ cell by
apoptosis in the absence of stroma cells or stroma-derived
cytokines.
3) The antibody or epitope-binding fragment thereof according to
claim 1, characterized in that said antibody or epitope-binding
fragment thereof is a monoclonal antibody.
4) The antibody or epitope-binding fragment thereof according to
claim 1, characterized in that said CD38.sup.+ cell is a lymphoma
cell, a leukemia cell, or a multiple myeloma cell.
5) The antibody or epitope-binding fragment thereof of claim 4,
characterized in that said CD38.sup.+ cell is a non-Hodgkin's
lymphoma (NHL) cell, a Burkitt's lymphoma (BL) cell, a multiple
myeloma (MM) cell, a B chronic lymphocytic leukemia (B-CLL) cell, a
B and T acute lymphocytic leukemia (ALL) cell, a T cell lymphoma
(TCL) cell, an acute myeloid leukemia (AML) cell, a hairy cell
leukemia (HCL) cell, a Hodgkin's Lymphoma (HL) cell, or a chronic
myeloid leukemia (CML) cell.
6) The antibody or epitope-binding fragment thereof according to
claim 1, characterized in that said antibody or epitope-binding
fragment thereof is capable of killing at least 24% of Daudi
lymphoma cells by apoptosis in the absence of stroma cells or
stroma-derived cytokines.
7) The antibody or epitope-binding fragment thereof according to
claim 1, characterized in that said antibody or epitope-binding
fragment thereof is capable of killing more than 7% of Ramos
lymphoma cells by apoptosis in the absence of stroma cells or
stroma-derived cytokines.
8) The antibody or epitope-binding fragment thereof according to
claim 1, characterized in that said antibody or epitope-binding
fragment thereof is capable of killing more than 11% of MOLP-8
multiple myeloma cells by apoptosis in the absence of stroma cells
or stroma-derived cytokines.
9) The antibody or epitope-binding fragment thereof according to
claim 1, characterized in that said antibody or epitope-binding
fragment thereof is capable of killing more than 36% of SU-DHL-8
lymphoma cells by apoptosis in the absence of stroma cells or
stroma-derived cytokines.
10) The antibody or epitope-binding fragment thereof according to
claim 1, characterized in that said antibody or epitope-binding
fragment thereof is capable of killing more than 62% of DND-41
leukemia cells by apoptosis in the absence of stroma cells or
stroma-derived cytokines.
11) The antibody or epitope-binding fragment thereof according to
claim 1, characterized in that said antibody or epitope-binding
fragment thereof is capable of killing more than 27% NU-DUL-1
lymphoma cells by apoptosis in the absence of stroma cells or
stroma-derived cytokines.
12) The antibody or epitope-binding fragment thereof according to
claim 1, characterized in that said antibody or epitope-binding
fragment thereof is capable of killing more than 9% of JVM-13
leukemia cells by apoptosis in the absence of stroma cells or
stroma-derived cytokines.
13) The antibody or epitope-binding fragment thereof according to
claim 1, characterized in that said antibody or epitope-binding
fragment thereof is capable of killing more than 4% of HC-1
leukemia cells by apoptosis in the absence of stroma cells or
stroma-derived cytokines.
14) The antibody or epitope-binding fragment thereof according to
claim 1, characterized in that said antibody or epitope-binding
fragment thereof binds CD38 with a k.sub.D of 3.times.10.sup.-9 M
or lower.
15) The antibody or epitope-binding fragment thereof according to
claim 1, characterized in that said antibody or epitope-binding
fragment thereof comprises one or more complementarity-determining
region having an amino acid sequence selected from the group
consisting of SEQ ID NOS: 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, and 36.
16) The antibody or epitope-binding fragment thereof according to
claim 15, characterized in that said antibody or epitope-binding
fragment thereof comprises at least one heavy chain and at least
one light chain, and said heavy chain comprises three sequential
complementarity-determining regions having amino acid sequences
selected from the group consisting of SEQ ID NOs:1, 2, 3, 7, 8, 9,
13, 14, 15, 19, 20, 21, 25, 26, 27, 31, 32, and 33, and said light
chain comprises three sequential complementarity-determining
regions having amino acid sequences selected from the group
consisting of SEQ ID NOs: 4, 5, 6, 10, 11, 12, 16, 17, 18, 22, 23,
24, 28, 29, 30, 34, 35, and 36.
17) The antibody or epitope-binding fragment thereof according to
claim 16, characterized in that said antibody or epitope-binding
fragment thereof comprises a light chain variable region having an
amino acid sequence selected from the group consisting of SEQ ID
NOS: 38, 40, 42, 44, 46, and 48.
18) The antibody or epitope-binding fragment thereof according to
claim 16, characterized in that said antibody or epitope-binding
fragment thereof comprises a heavy chain variable region having an
amino acid sequence selected from the group consisting of SEQ ID
NOS: 50, 52, 54, 56, 58, 60.
19) The antibody or epitope-binding fragment thereof according to
claim 16, characterized in that said antibody or epitope-binding
fragment thereof comprises at least one heavy chain and at least
one light chain, wherein said heavy chain comprises three
sequential complementarity-determining regions having amino acid
sequences represented by SEQ ID NOS: 1, 2, and 3, and wherein said
light chain comprises three sequential complementarity-determining
regions having amino acid sequences represented by SEQ ID NOS: 4,
5, and 6.
20) The antibody or epitope-binding fragment thereof according to
claim 19, characterized in that said antibody or epitope-binding
fragment thereof comprises a light chain variable region having an
amino acid sequence consisting of SEQ ID NO: 38.
21) The antibody or epitope-binding fragment thereof according to
claim 19, characterized in that said antibody or epitope-binding
fragment thereof comprises a heavy chain variable region having an
amino acid sequence consisting of SEQ ID NO: 50.
22) The antibody or epitope-binding fragment thereof according to
claim 16, characterized in that said antibody or epitope-binding
fragment thereof comprises at least one heavy chain and at least
one light chain, wherein said heavy chain comprises three
sequential complementarity-determining regions having amino acid
sequences represented by SEQ ID NOS: 7, 8, and 9, and wherein said
light chain comprises three sequential complementarity-determining
regions having amino acid sequences represented by SEQ ID NOS: 10,
11, and 12.
23) The antibody or epitope-binding fragment thereof according to
claim 22, characterized in that said antibody or epitope-binding
fragment thereof comprises a light chain variable region having an
amino acid sequence consisting of SEQ ID NO: 40.
24) The antibody or epitope-binding fragment thereof according to
claim 22, characterized in that said antibody or epitope-binding
fragment thereof comprises a heavy chain variable region having an
amino acid sequence consisting of SEQ ID NO: 52.
25) The antibody or epitope-binding fragment thereof according to
claim 16, characterized in that said antibody or epitope-binding
fragment thereof comprises at least one heavy chain and at least
one light chain, wherein said heavy chain comprises three
sequential complementarity-determining regions having amino acid
sequences represented by SEQ ID NOS: 13, 14, and 15, and wherein
said light chain comprises three sequential
complementarity-determining regions having amino acid sequences
represented by SEQ ID NOS: 16, 17, and 18.
26) The antibody or epitope-binding fragment thereof according to
claim 25, characterized in that said antibody or epitope-binding
fragment thereof comprises a light chain variable region having an
amino acid sequence consisting of SEQ ID NO: 42.
27) The antibody or epitope-binding fragment thereof according to
claim 25, characterized in that said antibody or epitope-binding
fragment thereof comprises a heavy chain variable region having an
amino acid sequence consisting of SEQ ID NO: 54.
28) The antibody or epitope-binding fragment thereof according to
claim 16, characterized in that said antibody or epitope-binding
fragment thereof comprises at least one heavy chain and at least
one light chain, wherein said heavy chain comprises three
sequential complementarity-determining regions having amino acid
sequences represented by SEQ ID NOS: 19, 20, and 21, and wherein
said light chain comprises three sequential
complementarity-determining regions having amino acid sequences
represented by SEQ ID NOS: 22, 23, and 24.
29) The antibody or epitope-binding fragment thereof according to
claim 28, characterized in that said antibody or epitope-binding
fragment thereof comprises a light chain variable region having an
amino acid sequence consisting of SEQ ID NO: 44.
30) The antibody or epitope-binding fragment thereof according to
claim 28, characterized in that said antibody or epitope-binding
fragment thereof comprises a heavy chain variable region having an
amino acid sequence consisting of SEQ ID NO: 56.
31) The antibody or epitope-binding fragment thereof according to
claim 16, characterized in that said antibody or epitope-binding
fragment thereof comprises at least one heavy chain and at least
one light chain, wherein said heavy chain comprises three
sequential complementarity-determining regions having amino acid
sequences represented by SEQ ID NOS: 25, 26, and 27, and wherein
said light chain comprises three sequential
complementarity-determining regions having amino acid sequences
represented by SEQ ID NOS: 28, 29, and 30.
32) The antibody or epitope-binding fragment thereof according to
claim 31, characterized in that said antibody or epitope-binding
fragment thereof comprises a light chain variable region having an
amino acid sequence consisting of SEQ ID NO: 46.
33) The antibody or epitope-binding fragment thereof according to
claim 31, characterized in that said antibody or epitope-binding
fragment thereof comprises a heavy chain variable region having an
amino acid sequence consisting of SEQ ID NO: 58.
34) The antibody or epitope-binding fragment thereof according to
claim 16, characterized in that said antibody or epitope-binding
fragment thereof comprises at least one heavy chain and at least
one light chain, wherein said heavy chain comprises three
sequential complementarity-determining regions having amino acid
sequences represented by SEQ ID NOS: 31, 32, and 33, and wherein
said light chain comprises three sequential
complementarity-determining regions having amino acid sequences
represented by SEQ ID NOS: 34, 35, and 36.
35) The antibody or epitope-binding fragment thereof according to
claim 34, characterized in that said antibody or epitope-binding
fragment thereof comprises a light chain variable region having an
amino acid sequence consisting of SEQ ID NO: 48.
36) The antibody or epitope-binding fragment thereof according to
claim 34, characterized in that said antibody or epitope-binding
fragment thereof comprises a heavy chain variable region having an
amino acid sequence consisting of SEQ ID NO: 60.
37) The antibody or epitope-binding fragment thereof according to
claim 16, characterized in that said antibody or epitope-binding
fragment thereof is produced by a hybridoma cell line selected from
the group of hybridoma cell lines deposited at the American Type
Culture Collection (10801 University Bld, Manassas, Va.,
20110-2209, USA), on Jun. 21, 2006, under the deposit numbers
PTA-7667, PTA-7669, PTA-7670, PTA-7666, PTA-7668, and PTA-7671.
38) The antibody or epitope-binding fragment thereof according to
claim 16, characterized in that said antibody or epitope-binding
fragment thereof comprises at least one human constant region.
39) The antibody or epitope-binding fragment thereof according to
claim 38, characterized in that said constant region is the human
IgG1/IgKappa constant region.
40) The antibody or epitope-binding fragment thereof according to
claim 16, characterized in that said antibody or epitope-binding
fragment thereof is a humanized or resurfaced antibody.
41) The humanized or resurfaced antibody or epitope-binding
fragment thereof according to claim 40, characterized in that said
humanized or resurfaced antibody or epitope-binding fragment
thereof comprises at least one heavy chain and at least one light
chain, wherein said heavy chain comprises three sequential
complementarity-determining regions having amino acid sequences
represented by SEQ ID NOS: 1, 2, and 3, and wherein said light
chain comprises three sequential complementarity-determining
regions having amino acid sequences represented by SEQ ID NOS: 4,
5, and 6.
42) The humanized or resurfaced antibody or epitope-binding
fragment thereof according to claim 40, characterized in that said
humanized or resurfaced antibody or epitope-binding fragment
thereof comprises at least one heavy chain and at least one light
chain, wherein said heavy chain comprises three sequential
complementarity-determining regions having amino acid sequences
represented by SEQ ID NOS: 7, 8, and 9, and wherein said light
chain comprises three sequential complementarity-determining
regions having amino acid sequences represented by SEQ ID NOS: 10,
11, and 12.
43) The humanized or resurfaced antibody or epitope-binding
fragment thereof according to claim 40, characterized in that said
humanized or resurfaced antibody or epitope-binding fragment
thereof comprises at least one heavy chain and at least one light
chain, wherein said heavy chain comprises three sequential
complementarity-determining regions having amino acid sequences
represented by SEQ ID NOS: 13, 14, and 15, and wherein said light
chain comprises three sequential complementarity-determining
regions having amino acid sequences represented by SEQ ID NOS: 16,
17, and 18.
44) The humanized or resurfaced antibody or epitope-binding
fragment thereof according to claim 43, characterized in that said
humanized or resurfaced antibody or epitope-binding fragment
thereof comprises a heavy chain variable region having an amino
acid sequence represented by SEQ ID NO: 66.
45) The humanized or resurfaced antibody or epitope-binding
fragment thereof according to claim 43, characterized in that said
humanized or resurfaced antibody or epitope-binding fragment
thereof comprises a light chain variable region having an amino
acid sequence selected from the group of SEQ ID NOS: 62 and 64.
46) The humanized or resurfaced antibody or epitope-binding
fragment thereof according to claim 40, characterized in that said
humanized or resurfaced antibody or epitope-binding fragment
thereof comprises at least one heavy chain and at least one light
chain, wherein said heavy chain comprises three sequential
complementarity-determining regions having amino acid sequences
represented by SEQ ID NOS: 19, 20, and 21, and wherein said light
chain comprises three sequential complementarity-determining
regions having amino acid sequences represented by SEQ ID NOS: 22,
23, and 24.
47) The humanized or resurfaced antibody or epitope-binding
fragment thereof according to claim 40, characterized in that said
humanized or resurfaced antibody or epitope-binding fragment
thereof comprises at least one heavy chain and at least one light
chain, wherein said heavy chain comprises three sequential
complementarity-determining regions having amino acid sequences
represented by SEQ ID NOS: 25, 26, and 27, and wherein said light
chain comprises three sequential complementarity-determining
regions having amino acid sequences represented by SEQ ID NOS: 28,
29, and 30.
48) The humanized or resurfaced antibody or epitope-binding
fragment thereof according to claim 47, characterized in that said
humanized or resurfaced antibody or epitope-binding fragment
thereof comprises a heavy chain variable region having an amino
acid sequence represented by SEQ ID NO: 72.
49) The humanized or resurfaced antibody or epitope-binding
fragment thereof according to claim 47, characterized in that said
humanized or resurfaced antibody or epitope-binding fragment
thereof comprises a light chain variable region having an amino
acid sequence selected from the group of SEQ ID NOS: 68 and 70.
50) The humanized or resurfaced antibody or epitope-binding
fragment thereof according to claim 40, characterized in that said
humanized or resurfaced antibody or epitope-binding fragment
thereof comprises at least one heavy chain and at least one light
chain, wherein said heavy chain comprises three sequential
complementarity-determining regions having amino acid sequences
represented by SEQ ID NOS: 31, 32, and 33, and wherein said light
chain comprises three sequential complementarity-determining
regions having amino acid sequences represented by SEQ ID NOS: 34,
35, and 36.
51) The antibody or epitope-binding fragment thereof according to
claim 16, characterized in that said antibody or epitope-binding
fragment thereof is a Fab, Fab', F(ab')2 or Fv fragment.
52) The antibody or epitope-binding fragment thereof according to
claim 1 wherein the antibody or epitope-binding fragment is linked
to a cytotoxic agent thereby forming a conjugate.
53) The conjugate of claim 52, characterized in that said cytotoxic
agent is selected from the group consisting of a maytansinoid, a
small drug, a tomaymycin derivative, a leptomycin derivative, a
prodrug, a taxoid, CC-1065 and a CC-1065 analog.
54) The conjugate of claim 53, characterized in that said cytotoxic
agent is the maytansine DM1 of formula: ##STR00019##
55) The conjugate of claim 53, characterized in that said cytotoxic
agent is the maytansine DM4 of formula: ##STR00020##
56) The conjugate of claim 53, characterized in that said cytotoxic
agent is a tomaymycin derivative selected from the group consisting
of:
8,8'-[1,3-benzenediylbis(methyleneoxy)]-bis[(S)-2-eth-(E)-ylidene-7-metho-
xy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]
8,8'-[5-methoxy-1,3-benzenediylbis(methyleneoxy)]-bis[(S)-2-eth-(E)-ylide-
ne-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-on-
e]
8,8'-[1,5-pentanediylbis(oxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,-
3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]
8,8'-[1,4-butanediylbis(oxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,1-
1a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]
8,8'-[3-methyl-1,5-pentanediylbis(oxy)]-bis[(S)-2-eth-(E)-ylidene-7-metho-
xy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]
8,8'-[2,6-pyridinediylbis(oxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3-
,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]
8,8'44-(3-tert-butoxycarbonylaminopropyloxy)-2,6-pyridinediylbis-(methyle-
neoxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrol-
o[2,1-c][1,4]benzodiazepin-5-one]
8,8'-[5-(3-aminopropyloxy)-1,3-benzenediylbis(methyleneoxy)]-bis[(S)-2-et-
h-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodi-
azepin-5-one]
8,8'-[5-(N-methyl-3-tert-butoxycarbonylaminopropyl)-1,3-benzenediylbis-(m-
ethyleneoxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H--
pyrrolo[2,1-c][1,4]benzodiazepin-5-one]
8,8'-{5-[3-(4-methyl-4-methyldisulfanyl-pentanoylamino)propyloxy]-1,3-ben-
zenediylbis(methyleneoxy)}-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-t-
etrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]
8,8'-[5-acetylthiomethyl-1,3-benzenediylbis(methyleneoxy)]-bis[(S)-2-meth-
ylene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-
-one]
bis-{2-[(S)-2-methylene-7-methoxy-5-oxo-1,3,,11a-tetrahydro-5H-pyrro-
lo[2,1-c][1,4]benzodiazepin-8-yloxy]-ethyl}-carbamic acid
tert-butyl ester
8,8'-[3-(2-acetylthioethyl)-1,5-pentanediylbis(oxy)]-bis[(S)-2-methylene--
7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]
8,8'-[5-(N-4-mercapto-4,4-dimethylbutanoyl)amino-1,3-benzenediylbis(methy-
leneoxy)]-bis[7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c]-
[1,4]benzodiazepin-5-one]
8,8'-[5-(N-4-methyldithio-4,4-dimethylbutanoyl)-amino-1,3-benzenediylbis(-
methyleneoxy)]-bis[7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2-
,1-c][1,4]benzodiazepin-5-one]
8,8'-[5-(N-methyl-N-(2-mercapto-2,2-dimethylethyl)amino-1,3-benzenediyl(m-
ethyleneoxy)]-bis[7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,-
1-c][1,4]benzodiazepin-5-one]
8,8'-[5-(N-methyl-N-(2-methyldithio-2,2-dimethylethyl)amino-1,3-benzenedi-
yl(methyleneoxy)]-bis[7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrol-
o[2,1-c][1,4]benzodiazepin-5-one]
8,8'-[(4-(2-(4-mercapto-4-methyl)-pentanamido-ethoxy)-pyridin-2,6-dimethy-
l)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrro-
lo[2,1-c][1,4]benzodiazepin-5-one]
8,8'-[(1-(2-(4-methyl-4-methyldisulfanyl)-pentanamido-ethoxy)-benzene-3,5-
-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahyd-
ro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]
8,8'-[(4-(3-(4-methyl-4-methyldisulfanyl)-pentanamido-propoxy)-pyridin-2,-
6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahy-
dro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]
8,8'-[(4-(4-(4-methyl-4-methyldisulfanyl)-pentanamido-butoxy)-pyridin-2,6-
-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahyd-
ro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]
8,8'-[(4-(3-[4-(4-methyl-4-methyldisulfanyl-pentanoyl)-piperazin-1-yl]-pr-
opyl)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1-
,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]
8,8'-[(1-(3-[4-(4-methyl-4-methyldisulfanyl-pentanoyl)-piperazin-1-yl]-pr-
opyl)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1-
,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]
8,8'-[(4-(2-{2-[2-(4-methyl-4-methyldisulfanyl-pentanoylamino)-ethoxy]-et-
hoxy}-ethoxy)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dim-
ethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]
8,8'-[(1-(2-{2-[2-(2-{2-[2-(4-methyl-4-methyldisulfanyl-pentanoylamino)-e-
thoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-benzene-3,5-dimethyl)-dioxy-
]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c-
][1,4]benzodiazepin-5-one]
8,8'-[(1-(2-{2-[2-(4-methyl-4-methyldisulfanyl-pentanoylamino)-ethoxy]-et-
hoxy}-ethoxy)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dim-
ethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]
8,8'-[(4-(2-{2-[2-(2-{2-[2-(4-methyl-4-methyldisulfanyl-pentanoylamino)-e-
thoxy]-ethoxy}-ethoxy)-ethoxy]ethoxy}-ethoxy)-pyridin-2,6-dimethyl)-dioxy]-
-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c]-
[1,4]benzodiazepin-5-one]
8,8'-[(1-(2-[methyl-(2-methyl-2-methyldisulfanyl-propyl)-amino]-ethoxy)-b-
enzene-3,5-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11-
a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]
8,8'-[(4-(3-[methyl-(4-methyl-4-methyldisulfanyl-pentanoyl)-amino]-propyl-
)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3-
,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]
8,8'-[(4-(3-[methyl-(2-methyl-2-methyldisulfanyl-propyl)-amino]-propyl)-p-
yridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11-
a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]
8,8'-[(1-(4-methyl-4-methyldisulfanyl)-pentanamido)-benzene-3,5-dimethyl)-
-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo-
[2,1-c][1,4]benzodiazepin-5-one].
57) The conjugate of claim 53, characterized in that the cytotoxic
agent is a leptomycin derivative selected from the group consisting
of: (2-Methylsulfanyl-ethyl)-amid of
(2E,10E,12E,16Z,18E)-(R)-6-Hydroxy-3,5,
7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran--
2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoic acid
(2-methylsulfanyl-ethyl)-amid Bis-[(2-mercaptoethyl)-amid of
(2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,
11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl-
)-8-oxo-nonadeca-2,10,12,16,18-pentaenoic acid]
(2-Mercapto-ethyl)-amid of
(2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,11,
15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-
-oxo-nonadeca-2,10,12,16,18-pentaenoic acid
(2-Methyldisulfanyl-ethyl)-amid of
(2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptamethyl-19-((2S,3-
S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18--
pentaenoic acid (2-Methyl-2-methyldisulfanyl-propyl)-amid of
(2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptamethyl-19-((2S,3-
S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18--
pentaenoic acid (2-Mercapto-2-methyl-propyl)-amid of
(2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptamethyl-19-((2S,3-
S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18--
pentaenoic acid.
58) The antibody or epitope-binding fragment thereof according to
claim 1 or the conjugate according to claim 52, for use as a
medicament.
59) A pharmaceutical composition comprising the antibody or
epitope-binding fragment thereof according to claim 1 or the
conjugate according to claim 52, and a pharmaceutically acceptable
carrier or excipients.
60) The pharmaceutical composition of claim 59, characterized in
that said composition contains a further therapeutic agent.
61) The pharmaceutical composition of claim 60, characterized in
that the further therapeutic agent is an antagonist of
epidermal-growth factor (EGF), fibroblast-growth factor (FGF),
hepatocyte growth factor (HGF), tissue factor (TF), protein C,
protein S, platelet-derived growth factor (PDGF), heregulin,
macrophage-stimulating protein (MSP) or vascular endothelial growth
factor (VEGF), or an antagonist of a receptor for epidermal-growth
factor (EGF), fibroblast-growth factor (FGF), hepatocyte growth
factor (HGF), tissue factor (TF), protein C, protein S,
platelet-derived growth factor (PDGF), heregulin,
macrophage-stimulating protein (MSP), or vascular endothelial
growth factor (VEGF), including HER2 receptor, HER3 receptor,
c-MET, and other receptor tyrosine kinases.
62) The pharmaceutical composition of claim 60, characterized in
that the further therapeutic agent is an antibody directed against
a cluster of differentiation antigen selected from a group
comprising CD3, CD14, CD19, CD20, CD22, CD25, CD28, CD30, CD33,
CD36, CD40, CD44, CD52, CD55, CD59, CD56, CD70, CD79, CD80, CD103,
CD134, CD137, CD138, and CD152.
63) A method of treating cancer or autoimmune disease comprising
administering a medicament comprising a pharmaceutical composition
comprising the antibody or epitope-binding fragment thereof
according to claim 1 or the conjugate according to claim 52, to
make a medicament to treat cancer or autoimmune disease.
64) The method of claim 63, characterized in that said cancer is
selected from the group consisting of carcinoma, including that of
the bladder, breast, colon, kidney, liver, lung, ovary, pancreas,
stomach, cervix, thyroid and skin; including squamous cell
carcinoma; tumors of mesenchymal origin, including fibrosarcoma and
rhabdomyoscarcoma; other tumors, including melanoma, seminoma,
tetratocarcinoma, neuroblastoma and glioma; tumors of the central
and peripheral nervous system, including astrocytoma,
neuroblastoma, glioma, and schwannomas; tumors of mesenchymal
origin, including fibrosarcoma, rhabdomyoscarama, and osteosarcoma;
and other tumors, including melanoma, xeroderma pigmentosum,
keratoactanthoma, seminoma, thyroid follicular cancer and
teratocarcinoma.
65) The method of claim 63, characterized in that said cancer is
hematopoietic tumors of lymphoid lineage, including leukemia,
non-Hodgkin's lymphoma, acute lymphocytic leukemia, acute
lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkitt's
lymphoma, Hodgkin's lymphoma, hairy cell leukemia, multiple
myeloma, chronic lymphocytic leukemia; hematopoietic tumors of
myeloid lineage, including acute and chronic myeloid leukemias and
promyelocytic leukemia.
66) The method of claim 63, characterized in that said autoimmune
or inflammatory disease is selected from the group consisting of
systemic lupus erythematosus, rheumatoid arthritis, multiple
sclerosis, Crohn's diasease, ulcerative colitis, gastritis,
Hashimoto's thyroiditis, ankylosing spondylitis, hepatitis
C-associated cryoglobulinemic vasculitis, chronic focal
encephalitis, bullous pemphigoid, hemophilia A,
membranoproliferative glomerulnephritis, Sjogren's syndrome, adult
and juvenile dermatomyositis, adult polymyositis, chronic
urticaria, primary biliary cirrhosis, idiopathic thrombocytopenic
purpura, neuromyelitis optica, Graves' dysthyroid disease, bullous
pemphigoid, membranoproliferative glonerulonephritis, Churg-Strauss
syndrome, and asthma.
67) The method according to claim 63, further comprising the use of
a further therapeutic agent in the manufacture of the same or
different composition.
68) The method according to claim 67, characterized in that the
further therapeutic agent is an antagonist of fibroblast-growth
factor (FGF), hepatocyte growth factor (HGF), tissue factor (TF),
protein C, protein S, platelet-derived growth factor (PDGF),
heregulin, macrophage-stimulating protein (MSP) or vascular
endothelial growth factor (VEGF), or an antagonist of a receptor
for epidermal-growth factor (EGF), fibroblast-growth factor (FGF),
hepatocyte growth factor (HGF), tissue factor (TF), protein C,
protein S, platelet-derived growth factor (PDGF), heregulin,
macrophage-stimulating protein (MSP), or vascular endothelial
growth factor (VEGF), including HER2 receptor, HER3 receptor,
c-MET, and other receptor tyrosine kinases.
69) The method according to claim 67, characterized in that the
further therapeutic agent is an antibody directed against a cluster
of differentiation antigen selected from a group comprising CD3,
CD14, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD36, CD40, CD44,
CD52, CD55, CD59, CD56, CD70, CD79, CD80, CD103, CD134, CD137,
CD138, and CD152.
70) A method of diagnosing a cancer in a subject known to or
suspected to have a cancer, said method comprising: a) Contacting
cells of said patient with an antibody or epitope-binding fragment
thereof according to claim 1, b) Measuring the binding of said
antibody or epitope-binding fragment thereof to said cells, and c)
comparing the expression in part (b) with that of a normal
reference subject or standard.
71) The method of claim 70, characterized in that said cancer is
selected from the group consisting of carcinoma, including that of
the bladder, breast, colon, kidney, liver, lung, ovary, pancreas,
stomach, cervix, thyroid and skin; including squamous cell
carcinoma; tumors of mesenchymal origin, including fibrosarcoma and
rhabdomyoscarcoma; other tumors, including melanoma, seminoma,
tetratocarcinoma, neuroblastoma and glioma; tumors of the central
and peripheral nervous system, including astrocytoma,
neuroblastoma, glioma, and schwannomas; tumors of mesenchymal
origin, including fibrosarcoma, rhabdomyoscarama, and osteosarcoma;
and other tumors, including melanoma, xeroderma pigmentosum,
keratoactanthoma, seminoma, thyroid follicular cancer and
teratocarcinoma.
72) The method of claim 70, wherein said cancer is hematopoietic
tumors of lymphoid lineage, including leukemia, non-Hodgkin's
lymphoma, acute lymphocytic leukemia, acute lymphoblastic leukemia,
B-cell lymphoma, T-cell lymphoma, Burkitt's lymphoma, Hodgkin's
lymphoma, hairy cell leukemia, multiple myeloma, chronic
lymphocytic leukemia; hematopoietic tumors of myeloid lineage,
including acute and chronic myeloid leukemias and promyelocytic
leukemia.
73) The method of claim 70, characterized in that the said cells
are in frozen or fixed tissue or cells from said patient.
74) A polynucleotide encoding a polypeptide selected from the group
consisting of SEQ ID NOS: 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, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,
58, 60, 62, 64, 66, 68, 70, and 72.
75) The polynucleotide according to claim 74, characterized in that
said polynucleotide has a sequence sharing at least 80% homology
with a polynucleotide selected from the group consisting of SEQ ID
NOs: 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65,
67, 69, and 71.
76) A recombinant vector comprising a nucleic acid of any of claim
74 or 75.
77) A host cell comprising a vector of claim 76.
78) An antibody or epitope-binding fragment thereof according to
claim 1 or the conjugate according to claim 52, characterized in
that said antibody or epitope-binding fragment thereof binds to an
epitope of the human CD38 extracellular domain that comprises
residues at least 10 residues selected among Pro75, Glu76, His79,
Gln107, Pro108, Met110, Lys111, Leu112, Gly113, Thr114, Gln115,
Thr116, Val117, Pro118, Cys119, Thr148, Leu150, Arg194, Arg195,
Glu198, Ala199, Cys201 and Glu233 or a conservatively substituted
form thereof.
79) An antibody or epitope-binding fragment thereof, characterized
in that said antibody or epitope-binding fragment thereof that
specifically binds to the same epitope bound by a monoclonal
antibody produced by a hybridoma cell line selected from the group
of hybridoma cell lines deposited at the American Type Culture
Collection (10801 University Bld, Manassas, Va., 20110-2209, USA),
on Jun. 21, 2006, under the deposit numbers PTA-7667, PTA-7669,
PTA-7670, PTA-7666, PTA-7668, and PTA-7671.
Description
BACKGROUND OF THE INVENTION
[0001] CD38 is a 45 kD type II transmembrane glycoprotein with a
long C-terminal extracellular domain and a short N-terminal
cytoplasmic domain. The CD38 protein is a bifunctional ectoenzyme
that can catalyze the conversion of NAD.sup.+ into cyclic
ADP-ribose (cADPR) and also hydrolyze cADPR into ADP-ribose. During
ontogeny, CD38 appears on CD34.sup.+ committed stem cells and
lineage-committed progenitors of lymphoid, erythroid and myeloid
cells. CD38 expression persists mostly in the lymphoid lineage with
varying expression levels at different stages of T and B cell
development.
[0002] CD38 is upregulated in many hematopoeitic malignancies and
in cell lines derived from various hematopoietic malignancies,
including non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL),
multiple myeloma (MM), B chronic lymphocytic leukemia (B-CLL), B
and T acute lymphocytic leukemia (ALL), T cell lymphoma (TCL),
acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's
Lymphoma (HL), and chronic myeloid leukemia (CML). On the other
hand, most primitive pluripotent stem cells of the hematopoietic
system are CD38.sup.-. CD38 expression in hematopoietic
malignancies and its correlation with disease progression makes
CD38 an attractive target for antibody therapy.
[0003] CD38 has been reported to be involved in Ca.sup.2+
mobilization (M. Morra et al., 1998, FASEB J., 12: 581-592; M. T.
Zilber et al., 2000, Proc Natl Acad Sci USA, 97: 2840-2845) and in
the signal transduction through tyrosine phosphorylation of
numerous signaling molecules, including phospholipase C-.gamma.,
ZAP-70, syk, and c-cbl, in lymphoid and myeloid cells or cell lines
(A. Funaro et al., 1993, Eur J Immunol, 23: 2407-2411; M. Morra et
al., 1998, FASEB J., 12: 581-592; A. Funaro et al., 1990, J
Immunol, 145: 2390-2396; M. Zubiaur et al., 1997, J Immunol, 159:
193-205; S. Deaglio et al., 2003, Blood 102: 2146-2155; E. Todisco
et al., 2000, Blood, 95: 535-542; M. Konopleva et al., 1998, J
Immunol, 161: 4702-4708; M. T. Zilber et al., 2000, Proc Natl Acad
Sci USA, 97: 2840-2845; A. Kitanaka et al., 1997, J Immunol, 159:
184-192; A. Kitanaka et al., 1999, J Immunol, 162: 1952-1958; R.
Mallone et al., 2001, Int Immunol, 13: 397-409). On the basis of
these observations, CD38 was proposed to be an important signaling
molecule in the maturation and activation of lymphoid and myeloid
cells during their normal development.
[0004] The exact role of CD38 in signal transduction and
hematopoiesis is still not clear, especially since most of these
signal transduction studies have used cell lines ectopically
overexpressing CD38 and anti-CD38 monoclonal antibodies, which are
non-physiological ligands. Because the CD38 protein has an
enzymatic activity that produces cADPR, a molecule that can induce
Ca.sup.2+ mobilization (H. C. Lee et al., 1989, J Biol Chem, 264:
1608-1615; H. C. Lee and R. Aarhus, 1991, Cell Regul, 2: 203-209),
it has been proposed that CD38 ligation by monoclonal antibodies
triggers Ca.sup.2+ mobilization and signal transduction in
lymphocytes by increasing production of cADPR(H. C. Lee et al.,
1997, Adv Exp Med Biol, 419: 411-419). Contrary to this hypothesis,
the truncation and point-mutation analysis of CD38 protein showed
that neither its cytoplasmic tail nor its enzymatic activity is
necessary for the signaling mediated by anti-CD38 antibodies (A.
Kitanaka et al., 1999, J Immunol, 162: 1952-1958; F. E. Lund et
al., 1999, J Immunol, 162: 2693-2702; S. Hoshino et al., 1997, J
Immunol, 158, 741-747).
[0005] The best evidence for the function of CD38 comes from
CD38.sup.-/.sup.- knockout mice, which have a defect in their
innate immunity and a reduced T-cell dependent humoral response due
to a defect in dendritic cell migration (S. Partida-Sanchez et al.,
2004, Immunity, 20: 279-291; S. Partida-Sanchez et al., 2001, Nat
Med, 7: 1209-1216). Nevertheless, it is not clear if the CD38
function in mice is identical to that in humans since the CD38
expression pattern during hematopoiesis differs greatly between
human and mouse: a) unlike immature progenitor stem cells in
humans, similar progenitor stem cells in mice express a high level
of CD38 (T. D. Randall et al., 1996, Blood, 87: 4057-4067; R. N.
Dagher et al., 1998, Biol Blood Marrow Transplant, 4: 69-74), b)
while during the human B cell development, high levels of CD38
expression are found in germinal center B cells and plasma cells
(F. M. Uckun, 1990, Blood, 76: 1908-1923; M. Kumagai et al., 1995,
J Exp Med, 181: 1101-1110), in the mouse, the CD38 expression
levels in the corresponding cells are low (A. M. Oliver et al.,
1997, J Immunol, 158: 1108-1115; A. Ridderstad and D. M. Tarlinton
1998, J Immunol, 160: 4688-4695).
[0006] Several anti-human CD38 antibodies with different
proliferative properties on various tumor cells and cell lines have
been described in the literature. For example, a chimeric OKT10
antibody with mouse Fab and human IgG1 Fc mediates
antibody-dependent cell-mediated cytotoxicity (ADCC) very
efficiently against lymphoma cells in the presence of peripheral
blood mononuclear effector cells from either MM patients or normal
individuals (F. K. Stevenson et al. 1991, Blood, 77: 1071-1079). A
CDR-grafted humanized version of the anti-CD38 antibody AT13/5 has
been shown to have potent ADCC activity against CD38-positive cell
lines (U.S. Ser. No. 09/797,941 A1). Human monoclonal anti-CD38
antibodies have been shown to mediate the in vitro killing of
CD38-positive cell lines by ADCC and/or complement-dependent
cytotoxicity (CDC), and to delay the tumor growth in SCID mice
bearing MM cell line RPMI-8226 (WO2005/103083 A2). On the other
hand, several anti-CD38 antibodies, IB4, SUN-4B7, and OKT10, but
not IB6, AT1, or AT2, induced the proliferation of peripheral blood
mononuclear cells (PBMC) from normal individuals (C. M. Ausiello et
al. 2000, Tissue Antigens, 56: 539-547).
[0007] Some of the antibodies of the prior art have been shown to
be able to trigger apoptosis in CD38.sup.+ B cells. However, they
can only do so in the presence of stroma cells or stroma-derived
cytokines. An agonistic anti-CD38 antibody (IB4) has been reported
to prevent apoptosis of human germinal center (GC) B cells (S. Zupo
et al. 1994, Eur J Immunol, 24: 1218-1222), and to induce
proliferation of KG-1 and HL-60 AML cells (M. Konopleva et al.
1998, J Immunol, 161: 4702-4708), but induces apoptosis in Jurkat T
lymphoblastic cells (M. Morra et al. 1998, FASEB J, 12: 581-592).
Another anti-CD38 antibody T16 induced apoptosis of immature
lymphoid cells and leukemic lymphoblast cells from an ALL patient
(M. Kumagai et al. 1995, J Exp Med, 181: 1101-1110), and of
leukemic myeloblast cells from AML patients (E. Todisco et al.
2000, Blood, 95: 535-542), but T16 induced apoptosis only in the
presence of stroma cells or stroma-derived cytokines (IL-7, IL-3,
stem cell factor).
[0008] On the other hand, some prior art antibodies induce
apoptosis after cross-linking, but are totally devoid of any
apoptotic activity when incubated alone (WO 2006/099875).
[0009] Because CD38 is an attractive target for antibody therapy
for various hematopoietic malignancies, we generated and screened a
large number of anti-human CD38 antibodies for high potency in the
following three cytotoxic activities against CD38-positive
malignant hematopoietic cells: induction of apoptosis, ADCC, and
CDC. The present invention describes novel anti-CD38 antibodies
capable of killing CD38.sup.+ cells by three different cytotoxic
mechanisms: induction of apoptosis, ADCC, and CDC. Remarkably, the
present invention discloses the first anti-CD38 antibodies that are
able to directly induce apoptosis of CD38.sup.+ cells, even without
the presence of stroma cells or stroma-derived cytokines.
SUMMARY OF THE INVENTION
[0010] It is an object of the invention to provide antibodies
specifically binding CD38, and capable of killing CD38.sup.+ cells
by apoptosis. Whereas some prior art antibodies are able to trigger
apoptosis only when crosslinked, but are otherwise devoid of any
apoptotic activity, the antibodies of the invention are capable of
inducing apoptotic cell death of CD38+ cells even when incubated
alone. In one aspect of the invention, the antibodies of the
invention are capable of killing CD38.sup.+ B cells by ADCC or CDC.
In yet another aspect, the antibodies of the invention are capable
of killing CD38.sup.+ cell by at least two of the aforementioned
mechanisms, i.e. apoptosis, ADCC, and CDC. Remarkably, the
antibodies of the invention are the first anti-CD38 antibodies that
have been demonstrated to kill CD38.sup.+ B cells by all three
different mechanisms: apoptosis, ADCC, and CDC. In a further
embodiment of the invention, said antibodies are capable of killing
CD38.sup.+ B cells by apoptosis even in the absence of stroma cells
or stroma-derived cytokines.
[0011] The antibodies of the invention are capable in particular of
killing malignant CD38.sup.+ B cells, including lymphoma cells,
leukemia cells, and multiple myeloma cells. In some embodiments,
the CD38.sup.+ B cell is a NHL, BL, MM, B-CLL, ALL, TCL, AML, HCL,
HL, or CML cell.
[0012] In one aspect of the invention, the antibodies of the
invention are capable of killing at least 24% of Daudi lymphoma
cells and/or at least 7% of Ramos lymphoma cells and/or 11% of
MOLP-8 multiple myeloma cells and/or 36% of SU-DHL-8 lymphoma cells
and/or 62% of DND-41 leukemia cells and/or 27% of NU-DUL-1 lymphoma
cells and/or 9% of JVM-13 leukemia cells and/or 4% of HC-1 leukemia
cells by apoptosis in the absence of stroma cells or stroma-derived
cytokines.
[0013] Antibodies of the invention can be polyclonal or monoclonal.
Preferred are monoclonal anti-CD38 antibodies. In a more preferred
embodiment, there are provided murine antibodies selected from
38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39, which are fully
characterized herein with respect to the amino acid sequences of
both their light and heavy chain variable regions, the cDNA
sequences of the genes for the light and heavy chain variable
regions, the identification of their CDRs
(complementarity-determining regions), the identification of their
surface amino acids, and means for their expression in recombinant
form.
[0014] The present invention includes chimeric versions of the
murine anti-CD38 monoclonal antibody selected from 38SB13, 38SB18,
38SB19, 38SB30, 38SB31, and 38SB39. Also included are resurfaced or
humanized versions of the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31,
and 38SB39 antibodies wherein surface-exposed residues of the
variable region frameworks of the antibodies, or their
epitope-binding fragments, are replaced in both light and heavy
chains to more closely resemble known human antibody surfaces. The
humanized antibodies and epitope-binding fragments thereof of the
present invention have improved properties in that they are less
immunogenic (or completely non-immunogenic) than murine versions in
human subjects to which they are administered. Thus, the different
versions of humanized 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and
38SB39 antibodies and epitope-binding fragments thereof of the
present invention specifically recognize CD38 while not being
immunogenic to a human.
[0015] The humanized versions of the 38SB13, 38SB18, 38SB19,
38SB30, 38SB31, and 38SB39 antibodies of the present invention are
fully characterized herein with respect to their respective amino
acid sequences of both light and heavy chain variable regions, the
DNA sequences of the genes for the light and heavy chain variable
regions, the identification of the complementarity determining
regions (CDRs), the identification of their variable region
framework surface amino acid residues, and disclosure of a means
for their expression in recombinant form.
[0016] This invention also contemplates the use of conjugates
between cytotoxic conjugates comprising (1) a cell binding agent
that recognizes and binds CD38, and (2) a cytotoxic agent. In the
cytotoxic conjugates, the cell binding agent has a high affinity
for CD38 and the cytotoxic agent has a high degree of cytotoxicity
for cells expressing CD38, such that the cytotoxic conjugates of
the present invention form effective killing agents.
[0017] In a preferred embodiment, the cell binding agent is an
anti-CD38 antibody (e.g., 38SB13, 38SB18, 38SB19, 38SB30, 38SB31,
and 38SB39) or an epitope-binding fragment thereof, more preferably
a humanized anti-CD38 antibody (e.g., 38SB13, 38SB18, 38SB19,
38SB30, 38SB31, and 38SB39) or an epitope-binding fragment thereof,
wherein a cytotoxic agent is covalently attached, directly or via a
cleavable or non-cleavable linker, to the antibody or
epitope-binding fragment thereof. In more preferred embodiments,
the cell binding agent is the humanized 38SB13, 38SB18, 38SB19,
38SB30, 38SB31, and 38SB39 antibodies or an epitope-binding
fragment thereof, and the cytotoxic agent is a taxol, a
maytansinoid, a tomaymycin derivative, a leptomycin derivative,
CC-1065 or a CC-1065 analog.
[0018] More preferably, the cell binding agent is the humanized
anti-CD38 antibody 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and
38SB39, and the cytotoxic agent is a maytansine compound, such as
DM1 or DM4.
[0019] The present invention also encompasses the use of fragments
of anti-CD38 antibodies which retain the ability to bind CD38. In
another aspect of the invention, the use of functional equivalents
of anti-CD38 antibodies is contemplated.
[0020] The present invention also includes a method for inhibiting
the growth of a cell expressing CD38. In preferred embodiments, the
method for inhibiting the growth of the cell expressing CD38 takes
place in vivo and results in the death of the cell, although in
vitro and ex vivo applications are also included.
[0021] The present invention also provides a therapeutic
composition comprising an anti-CD38 antibody or an anti-CD38
antibody-cytotoxic agent conjugate, and a pharmaceutically
acceptable carrier or excipients. In some embodiments, the
therapeutic composition comprises a second therapeutic agent. This
second therapeutic agent can be chosen from the group comprising
the antagonists of epithermal-growth factor (EGF),
fibroblast-growth factor (FGF), hepatocyte growth factor (HGF),
tissue factor (TF), protein C, protein S, platelet-derived growth
factor (PDGF), heregulin, macrophage-stimulating protein (MSP) or
vascular endothelial growth factor (VEGF), or an antagonist of a
receptor for epidermal-growth factor (EGF), fibroblast-growth
factor (FGF), hepatocyte growth factor (HGF), tissue factor (TF),
protein C, protein S, platelet-derived growth factor (PDGF),
heregulin, macrophage-stimulating protein (MSP), or vascular
endothelial growth factor (VEGF), including HER2 receptor, HER3
receptor, c-MET, and other receptor tyrosine kinases. This second
therapeutic agent can be also chosen from the group comprising of
antibodies targeting clusters of differentiation (CD) antigens,
including CD3, CD14, CD19, CD20, CD22, CD25, CD28, CD30, CD33,
CD36, CD40, CD44, CD52, CD55, CD59, CD56, CD70, CD79, CD80, CD103,
CD134, CD137, CD138, and CD152. This second therapeutic agent can
be also chosen from the group of chemotherapeutic or
immunomodulatory agents.
[0022] The present invention further includes a method of treating
a subject having a cancer or an inflammatory disease, including
autoimmune disease using the therapeutic composition. In some
embodiments, the cancer is selected from a group consisting of NHL,
BL, MM, B-CLL, ALL, TCL, AML, HCL, HL, and CML. In another
embodiment, the autoimmune disease is selected from a group
consisting of systemic lupus erythematosus, multiple sclerosis,
rheumatoid arthritis, Crohn's disease, ulcerative colitis,
gastritis, Hashimoto's thyroiditis, ankylosing spondylitis,
hepatitis C-associated cryoglobulinemic vasculitis, chronic focal
encephalitis, bullous pemphigoid, hemophilia A,
membranoproliferative glomerulnephritis, Sjogren's syndrome, adult
and juvenile dermatomyositis, adult polymyositis, chronic
urticaria, primary biliary cirrhosis, idiopathic thrombocytopenic
purpura, neuromyelitis optica, Graves' dysthyroid disease, bullous
pemphigoid, membranoproliferative glonerulonephritis, Churg-Strauss
syndrome, and asthma. In preferred embodiments, the cytotoxic
conjugate comprises an anti-CD38 antibody and a cytotoxic agent. In
more preferred embodiments, the cytotoxic conjugate comprises a
humanized 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39
antibody-DM1 conjugate, humanized 38SB13, 38SB18, 38SB19, 38SB30,
38SB31, and 38SB39 antibody-DM4 or a humanized 38SB13, 38SB18,
38SB19, 38SB30, 38SB31, and 38SB39 antibody-taxane conjugate, and
the conjugate is administered along with a pharmaceutically
acceptable carrier or excipients.
[0023] In another aspect of the invention, anti-CD38 antibodies are
used to detect the CD38 protein in a biological sample. In a
preferred embodiment, said antibodies are used to determine CD38
levels in tumor tissue.
[0024] The present invention also includes a kit comprising an
anti-CD38 antibody or an anti-CD38 antibody-cytotoxic agent
conjugate and instructions for use. In preferred embodiments, the
anti-CD38 antibodies are the humanized 38SB13, 38SB18, 38SB19,
38SB30, 38SB31, and 38SB39 antibodies, the cytotoxic agent is a
maytansine compound, such as DM1 or DM4, a tomaymycin derivative, a
leptomycin derivative, or a taxane, and the instructions are for
using the conjugates in the treatment of a subject having cancer.
The kit may also include components necessary for the preparation
of a pharmaceutically acceptable formulation, such as a diluent if
the conjugate is in a lyophilized state or concentrated form, and
for the administration of the formulation.
[0025] Unless otherwise stated, all references and patents cited
herein are incorporated by reference.
BRIEF DESCRIPTION OF THE FIGURES
[0026] FIG. 1A shows a FACS analysis of the specific binding of
purified murine anti-CD38 antibodies, 38SB13, 38SB18, 38SB19, 38 to
the 300-19 cells expressing human CD38 and CD38-positive Ramos
lymphoma cells.
[0027] FIG. 1B shows a FACS analysis of the specific binding of
purified murine anti-CD38 antibodies, 38SB30, 38SB31, 38SB39 and
the control anti-CD38 antibody AT13/5 to the 300-19 cells
expressing human CD38 and CD38-positive Ramos lymphoma cells.
[0028] FIG. 2 shows the binding titration curves of 38SB13, 38SB18,
38SB19, 38SB30, 38SB31, and 38SB39 established with Ramos
cells.
[0029] FIG. 3 shows FACS dot plots (FL4-H; TO-PRO-3 staining;
y-axis and FL1-H; Annexin V-FITC staining; x-axis) of Ramos cells
undergoing apoptosis after incubation with 38SB13, 38SB19, or
AT13/5 (10 nM) for 24 h.
[0030] FIG. 4A shows the average percentages of Ramos cells
undergoing apoptosis after a 24-h incubation with 38SB13, 38SB18,
38SB19, 38SB30, 38SB31, 38SB39, 38SB7, 38SB23, IB4, AT13/5, OKT10,
or SUN-4B7. The average percentage of Annexin V-positive cells
(y-axis; includes both TO-PRO-3 positive and negative cells) from
duplicate samples were plotted.
[0031] FIG. 4B shows the average percentages of Daudi cells
undergoing apoptosis after a 24-h incubation with the same set of
antibodies as in FIG. 4A.
[0032] FIG. 4C shows the average percentages of Molp-8 cells
undergoing apoptosis after a 24-h incubation with the same set of
antibodies as in FIG. 4A.
[0033] FIG. 5A shows a diagram of the human expression vector used
to express hu38SB19-LC.
[0034] FIG. 5B shows a diagram of the human expression vector used
to express hu38SB19-HC.
[0035] FIG. 5C shows a diagram of the human expression vector used
to express both hu38SB19LC and hu38SB19HC.
[0036] FIG. 6A shows ADCC activities mediated by the antibodies,
ch38SB13, ch38SB18, and ch38SB19, towards Ramos cells.
[0037] FIG. 6B shows ADCC activities mediated by the antibodies,
ch38SB30, ch38SB31, and ch38SB39 towards Ramos cells.
[0038] FIG. 7 A) shows ADCC activities mediated by the antibodies
ch38SB18, ch38SB19, ch38SB31, and non-binding chimeric human IgG1
control antibody towards LP-1 multiple myeloma cells.
[0039] FIG. 7 B) compares ADCC activities mediated by the
antibodies ch38SB19 and murine 38SB19 towards Daudi cells.
[0040] FIG. 8 A shows ADCC activities mediated by the ch38SB19
antibody and by non-binding chimeric human IgG1 control antibody
towards NALM-6 B-ALL cells.
[0041] FIG. 8 B shows ADCC activities mediated by the ch38SB19
antibody and by non-binding chimeric human IgG1 control antibody
towards MOLT-4 T-ALL cells.
[0042] FIG. 9 A shows CDC activities mediated by the antibodies
ch38SB13, ch38SB18, ch38SB19, ch38SB30, and ch38SB39 towards
Raji-IMG cells.
[0043] FIG. 9 B shows CDC activities mediated by the antibodies
ch38SB19 and ch38SB31 towards Raji-IMG cells.
[0044] FIG. 10 shows CDC activities mediated by the antibodies
ch38SB18, ch38SB19, ch38SB31, and by non-binding chimeric human
IgG1 control antibody towards LP-1 multiple myeloma cells.
[0045] FIG. 11A shows CDC activities mediated by the antibodies
ch38SB13, cch38SB19, and ch38SB39 towards Daudi cells.
[0046] FIG. 11B shows CDC activities mediated by the antibodies
ch38SB18 and ch38SB30 towards Daudi cells.
[0047] FIG. 11C shows CDC activities mediated by the antibodies
ch38SB19 and ch38SB31 towards Daudi cells
[0048] FIG. 12 A shows the binding titration curves of ch38SB19,
hu38SB19 v1.00, and hu38SB19 v1.20 for binding to Ramos cells.
[0049] FIG. 12B shows the binding curves that compare ch38SB19,
hu38SB19 v1.00, and hu38SB19 v1.00 for their ability to compete
with binding of biotinylated murine 38SB19 antibody to Ramos
cells.
[0050] FIG. 13 shows the average percentages of Daudi cells
undergoing apoptosis after 24 h of incubation with ch38SB19,
hu38SB19 v1.00, or hu38SB19 v1.20 antibody.
[0051] FIG. 14 shows ADCC activities mediated by the antibodies
ch38SB19, hu38SB19 v1.00, hu38SB19 v1.20, and by non-binding
chimeric human IgG1 control antibody towards LP-1 multiple myeloma
cells.
[0052] FIG. 15A shows CDC activities mediated by antibodies
ch38SB19, hu38SB19 v1.00, and hu38SB19 v1.20 towards Raji-IMG
lymphoma cells.
[0053] FIG. 15B shows CDC activities mediated by antibodies
ch38SB19, hu38SB19 v1.00, and hu38SB19 v1.20 towards LP-1 multiple
myeloma cells.
[0054] FIG. 15C shows CDC activities mediated by antibodies
ch38SB19, hu38SB19 v1.00, and hu38SB19 v1.20 towards DND-41 T-cell
acute lymphoblastic leukemia cells.
[0055] FIG. 16 shows the average percentages of Annexin V positive
cells after 24 h of incubation with hu38SB19 v1.00 antibody for
SU-DHL-8 diffuse large B cell lymphoma cells, NU-DUL-1 B-cell
lymphoma cells, DND-41 T-cell acute lymphoblastic leukemia cells,
JVM-13 B-cell chronic lymphocytic leukemia cells and HC-1 hairy
cell leukemia cells.
[0056] FIG. 17 shows the percent survival of SCID mice bearing
established disseminated human Ramos tumors. Mice were treated with
murine 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, or 38SB39 antibody
or PBS as indicated.
[0057] FIG. 18 shows the percent survival of SCID mice bearing
established disseminated human Daudi tumors. Mice were treated with
hu38SB19 or mu38SB19 antibody or PBS as indicated.
[0058] FIG. 19 shows the mean tumor volume of SCID mice bearing
NCl-H929 multiple myeloma xenograft tumors. Mice were treated with
hu38SB19, a non-binding control IgG1 antibody or PBS as
indicated.
[0059] FIG. 20 shows the mean tumor volume of SCID mice bearing
MOLP-8 multiple myeloma xenograft tumors. Mice were treated with
hu38SB19, mu38SB19, a non-binding control IgG1 antibody or PBS as
indicated.
[0060] FIG. 21A is the hu38SB19-light chain nucleotide sequence
from pBH3045 (SEQ ID No. 81).
[0061] FIG. 21B is the hu38SB19 light chain deduced protein
sequence from pBH3045 (signal peptide in bold) (SEQ ID No 82).
[0062] FIG. 21C is the hu38SB19 heavy chain-1-247-His6 nucleotide
sequence from pBH3093 (SEQ ID No 83).
[0063] FIG. 21D is the hu38SB19-heavy chain-1-247-His6 deduced
protein sequence from pBH303 (signal peptide in bold) (SEQ ID NO
84).
[0064] FIG. 21E is the nucleotide sequence of the
huCD38-unglycosylated mutant-N100D-N164A-N209D-N219D from pBH3133
(SEQ ID No 85).
[0065] FIG. 21F is the CD38-unglycosylated
mutant-N100D-N164A-N209D-N219D, deduced protein sequence from
pBH3133 (corresponding mutations are underlined; signal peptide is
in bold) (SEQ ID No 86).
[0066] FIGS. 22A and 22B illustrate the mapping of the human CD38
(epitope residues are defined as residues which contain atoms that
lie within 4 .ANG. from any atom of the CDR residues of the Fab
fragment of hu38SB19).
[0067] FIG. 23 shows the sequence SEQ ID N.degree. 87 of the
R45-1300 CD38 extracellular domain where the residues that are part
of the epitope are underlined. Mutations that are introduced to
silence the four glycosylation sites of CD38 are shown in bold in
the sequence but are not represented in FIG. 22 because they are
out of the interface huCD38/hu38SB19.
[0068] FIG. 24 represents the overall structure of the complex in
surface representation. Heavy and light chain are coloured in black
and grey, respectively. Human CD38 is coloured in white.
[0069] FIGS. 25A and 25B represent the structure of the
paratope.
DETAILED DESCRIPTION OF THE INVENTION
[0070] New antibodies capable of specifically binding CD38 are
herein provided. In particular, the present inventors have
discovered novel antibodies that specifically bind to CD38 on the
cell surface and kill CD38.sup.+ cells by apoptosis. In one aspect
of the invention, the anti-CD38 antibodies are also capable of
killing a CD38.sup.+ cell by antibody-dependent cytotoxicity
(ADCC). In another aspect, the anti-CD38 antibodies of the
invention are capable of killing a CD38.sup.+ cell by
complement-dependent cytotoxicity (CDC). In yet another aspect, the
anti-CD38 antibodies of the invention are capable of killing a
CD38.sup.+ cell by at least two of the above mentioned mechanisms,
apoptosis, ADCC, and CDC. In particular, in a preferred embodiment,
the anti-CD38 antibodies of the invention are capable of killing a
CD38.sup.+ cell by apoptosis, ADCC, and CDC. The invention thus
provides the first anti-CD38 antibodies capable of killing a
CD38.sup.+ cell by three different mechanisms.
[0071] Antibodies capable of binding CD38 and triggering apoptotic
cell death in CD38.sup.+ cells have been previously described (M.
Kumagai et al., 1995, J Exp Med, 181: 1101-1110; E. Todisco et al.
2000, Blood, 95: 535-542), but the antibodies of the invention are
the first for which an apoptotic activity in the absence of stroma
cells or stroma-derived cytokines is demonstrated. The term
"stroma" as used herein refers to the nonmalignant supporting
tissue of a tumor which includes connective tissue, blood vessels,
and inflammatory cells. Stromal cells produce growth factors and
other substances, including cytokines, that can influence the
behavior of cancer cells. The term "cytokine", as used herein,
refers to small secreted proteins (e.g. IL-1, IL-2, IL-4, IL-5, and
IL-6, IFNg, IL-3, IL-7 and GM-CSF) which mediate and regulate
immunity, inflammation, and hematopoiesis. It is shown herein that
the antibodies of the prior art are unable to trigger apoptotic
cell death in the absence of stroma cells or stroma-derived
cytokines. By contrast, the anti-CD38 antibodies of the invention
display under the same conditions potent apoptotic activities.
[0072] In another aspect, the antibodies of the invention are
capable of binding the CD38 protein with a k.sub.D of
3.times.10.sup.-9 M or lower.
[0073] The term "CD38" as used herein refers to a type II
transmembrane protein, comprising, for example, an amino acid
sequence as in Genbank accession number NP.sub.--001766. A
"CD38.sup.+ cell" is a cell expressing the CD38 protein.
Preferably, the CD38.sup.+ cell is a mammalian cell.
[0074] In one embodiment of this invention, the CD38.sup.+ cell is
a malignant cell. In another embodiment, the CD38.sup.+ cell is a B
cell. In a preferred embodiment, the CD38.sup.+ cell is a tumor
cell derived from a hemapoietic malignancy. In a more preferred
embodiment, the CD38.sup.+ cell is a lymphoma cell, a leukemia
cell, or a multiple myeloma cell. In a further preferred
embodiment, the CD38.sup.+ cell is a NHL, BL, MM, B-CLL, ALL, TCL,
AML, HCL, HL, or CML cell.
[0075] Thus, in one embodiment, this invention provides anti-CD38
antibodies capable of killing at least 24% of Daudi lymphoma cells
in the absence of stroma cells or stroma-derived cytokines. In
another embodiment, the anti-CD38 antibodies of the invention are
capable of killing at least 7% of Ramos lymphoma cells in the
absence of stroma cells or stroma-derived cytokines. In another
embodiment, the anti-CD38 antibodies of the invention are capable
of killing at least 11% of MOLP-8 multiple myeloma cells in the
absence of stroma cells or stroma-derived cytokines. In another
embodiment, the anti-CD38 antibodies of the invention are capable
of killing at least 36% of SU-DHL-8 lymphoma cells in the absence
of stroma cells or stroma-derived cytokines. In another embodiment,
the anti-CD38 antibodies of the invention are capable of killing at
least 27% of NU-DUL-1 lymphoma cells in the absence of stroma cells
or stroma-derived cytokines. In another embodiment, the anti-CD38
antibodies of the invention are capable of killing at least 62% of
DND-41 leukemia cells in the absence of stroma cells or
stroma-derived cytokines. In another embodiment, the anti-CD38
antibodies of the invention are capable of killing at least 9% of
JVM-13 leukemia cells in the absence of stroma cells or
stroma-derived cytokines. In another embodiment, the anti-CD38
antibodies of the invention are capable of killing at least 4% of
HC-1 leukemia cells in the absence of stroma cells or
stroma-derived cytokines.
[0076] Antibodies
[0077] The term "antibody" is used herein in the broadest sense and
specifically covers monoclonal antibodies (including full length
monoclonal antibodies) of any isotype such as IgG, IgM, IgA, IgD
and IgE, polyclonal antibodies, multispecific antibodies, chimeric
antibodies, and antibody fragments. An antibody reactive with a
specific antigen can be generated by recombinant methods such as
selection of libraries of recombinant antibodies in phage or
similar vectors, or by immunizing an animal with the antigen or an
antigen-encoding nucleic acid.
[0078] A typical IgG antibody is comprised of two identical heavy
chains and two identical light chains that are joined by disulfide
bonds. Each heavy and light chain contains a constant region and a
variable region. Each variable region contains three segments
called "complementarity-determining regions" ("CDRs") or
"hypervariable regions", which are primarily responsible for
binding an epitope of an antigen. They are usually referred to as
CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus.
The more highly conserved portions of the variable regions are
called the "framework regions".
[0079] As used herein, "V.sub.H" or "VH" refers to the variable
region of an immunoglobulin heavy chain of an antibody, including
the heavy chain of an Fv, scFv, dsFv, Fab, Fab' or F(ab')2
fragment. Reference to "V.sub.L " or "VL" refers to the variable
region of the immunoglobulin light chain of an antibody, including
the light chain of an Fv, scFv, dsFv, Fab, Fab' or F(ab')2
fragment.
[0080] A "polyclonal antibody" is an antibody which was produced
among or in the presence of one or more other, non-identical
antibodies. In general, polyclonal antibodies are produced from a
B-lymphocyte in the presence of several other B-lymphocytes
producing non-identical antibodies. Usually, polyclonal antibodies
are obtained directly from an immunized animal.
[0081] A "monoclonal antibody", as used herein, is an antibody
obtained from a population of substantially homogeneous antibodies,
i.e. the antibodies forming this population are essentially
identical except for possible naturally occurring mutations which
might be present in minor amounts. These antibodies are directed
against a single epitope and are therefore highly specific.
[0082] An "epitope" is the site on the antigen to which an antibody
binds. If the antigen is a polymer, such as a protein or
polysaccharide, the epitope can be formed by contiguous residues or
by non-contiguous residues brought into close proximity by the
folding of an antigenic polymer. In proteins, epitopes formed by
contiguous amino acids are typically retained on exposure to
denaturing solvents, whereas epitopes formed by non-contiguous
amino acids are typically lost under said exposure.
[0083] As used herein, the term "K.sub.D" refers to the
dissociation constant of a particular antibody/antigen
interaction.
[0084] The present invention proceeds from novel murine anti-CD38
antibodies, herein 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and
38SB39 which are fully characterized with respect to the amino acid
sequences of both light and heavy chains, the identification of the
CDRs, the identification of surface amino acids, and means for
their expression in recombinant form. The primary amino acid and
DNA sequences of antibodies 38SB13, 38SB18, 38SB19, 38SB30, 38SB31,
and 38SB39 light and heavy chains, and of humanized versions, are
disclosed herein.
[0085] The hybridoma cell lines producing the 38SB13, 38SB18,
38SB19, 38SB30, 38SB31, and 38SB39 murine anti-CD38 antibodies have
been deposited at the American Type Culture Collection (10801
University Bld, Manassas, Va., 20110-2209, USA), on Jun. 21, 2006,
under the deposit numbers PTA-7667, PTA-7669, PTA-7670, PTA-7666,
PTA-7668, and PTA-7671, respectively.
[0086] The scope of the present invention is not limited to
antibodies and fragments comprising these sequences. Instead, all
antibodies and fragments that specifically bind to CD38 and capable
of killing CD38.sup.+ cells by apoptosis, ADCC, and/or CDC, fall
within the scope of the present invention. Thus, antibodies and
antibody fragments may differ from antibody 38SB13, 38SB18, 38SB19,
38SB30, 38SB31, and 38SB39 or the humanized derivatives in the
amino acid sequences of their scaffold, CDRs, light chain and heavy
chain, and still fall within the scope of the present
invention.
[0087] In one embodiment, this invention provides antibodies or
epitope-binding fragment thereof comprising one or more CDRs having
an amino acid sequence selected from the group consisting of SEQ ID
NOS: 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,
and 36. In a preferred embodiment, the antibodies of the invention
comprise at least one heavy chain and at least one light chain, and
said heavy chain comprises three sequential CDRs having amino acid
sequences selected from the group consisting of SEQ ID NOS: 1, 2,
3, 7, 8, 9, 13, 14, 15, 19, 20, 21, 25, 26, 27, 31, 32, and 33, and
said light chain comprises three sequential CDRs having amino acid
sequences selected from the group consisting of SEQ ID NOS: 4, 5,
6, 10, 11, 12, 16, 17, 18, 22, 23, 24, 28, 29, 30, 34, 35, and
36.
[0088] In a more preferred embodiment, the antibodies of the
invention comprise three CDRs having amino acid sequences selected
from the group of SEQ ID NOS: 1, 2, 3, 4, 5, and 6. In a further
more preferred embodiment, there is provided a 38SB13 antibody,
which comprises at least one heavy chain and at least one light
chain, and said heavy chain comprises three sequential CDRs having
amino acid sequences consisting of SEQ ID NOS: 1, 2, and 3, and
said light chain comprises three sequential CDRs having amino acid
sequences consisting of SEQ ID NOS: 4, 5, and 6.
[0089] In another more preferred embodiment, the antibodies of the
invention comprise three CDRs having amino acid sequences selected
from the group of SEQ ID NOS: 7, 8, 9, 10, 11, and 12. In a further
more preferred embodiment, there is provided a 38SB18 antibody,
which comprises at least one heavy chain and at least one light
chain, and said heavy chain comprises three sequential CDRs having
amino acid sequences consisting of SEQ ID NOS: 7, 8, and 9, and
said light chain comprises three sequential CDRs having amino acid
sequences consisting of SEQ ID NOS: 10, 11, and 12.
[0090] In another more preferred embodiment, the antibodies of the
invention comprise three CDRs having amino acid sequences selected
from the group of SEQ ID NOS: 13, 14, 15, 16, 17, and 18. In a
further more preferred embodiment, there is provided a 38SB19
antibody, which comprises at least one heavy chain and at least one
light chain, and said heavy chain comprises three sequential CDRs
having amino acid sequences consisting of SEQ ID NOS: 13, 14, and
15, and said light chain comprises three sequential CDRs having
amino acid sequences consisting of SEQ ID NOS: 16, 17, and 18.
[0091] In another more preferred embodiment, the antibodies of the
invention comprise three CDRs having amino acid sequences selected
from the group of SEQ ID NOS: 19, 20, 21, 22, 23, 24. In a further
more preferred embodiment, there is provided a 38SB30 antibody,
which comprises at least one heavy chain and at least one light
chain, and said heavy chain comprises three sequential CDRs having
amino acid sequences consisting of SEQ ID NOS: 19, 20, and 21, and
said light chain comprises three sequential CDRs having amino acid
sequences consisting of SEQ ID NOS: 22, 23, and 24.
[0092] In another more preferred embodiment, the antibodies of the
invention comprise three CDRs having amino acid sequences selected
from the group of SEQ ID NOS: 25, 26, 27, 28, 29, and 30. In a
further more preferred embodiment, there is provided a 38SB31
antibody, which comprises at least one heavy chain and at least one
light chain, and said heavy chain comprises three sequential CDRs
having amino acid sequences consisting of SEQ ID NOS: 25, 26, and
27, and said light chain comprises three sequential CDRs having
amino acid sequences consisting of SEQ ID NOS: 28, 29, and 30.
[0093] In another more preferred embodiment, the antibodies of the
invention comprise three CDRs having amino acid sequences selected
from the group of 31, 32, 33, 34, 35, and 36. In a further more
preferred embodiment, there is provided a 38SB39 antibody, which
comprises at least one heavy chain and at least one light chain,
and said heavy chain comprises three sequential CDRs having amino
acid sequences consisting of SEQ ID NOS: 31, 32, and 33, and said
light chain comprises three sequential CDRs having amino acid
sequences consisting of SEQ ID NOS: 34, 35, and 36.
[0094] In another embodiment, the anti-CD38 antibodies of the
invention comprise a V.sub.L having an amino acid sequence selected
from the group consisting of SEQ ID NOS: V.sub.L for 38, 40, 42,
44, 46, and 48. In a more preferred embodiment, there is provided a
38SB13 antibody comprising a V.sub.L having an amino acid sequence
consisting of SEQ ID NO: 38. In a more preferred embodiment, there
is provided a 38SB18 antibody comprising a V.sub.L having an amino
acid sequence consisting of SEQ ID NO: 40. In a more preferred
embodiment, there is provided a 38SB19 antibody comprising a
V.sub.L having an amino acid sequence consisting of SEQ ID NO: 42.
In a more preferred embodiment, there is provided a 38SB30 antibody
comprising a V.sub.L having an amino acid sequence consisting of
SEQ ID NO: 44. In a more preferred embodiment, there is provided a
38SB31 antibody comprising a V.sub.L having an amino acid sequence
consisting of SEQ ID NO: 46. In a more preferred embodiment, there
is provided a 38SB39 antibody comprising a V.sub.L having an amino
acid sequence consisting of SEQ ID NO: 48.
[0095] In another embodiment, the antibodies of the invention
comprise a V.sub.H having an amino acid sequence selected from the
group consisting of SEQ ID NOS: 50, 52, 54, 56, 58, and 60. In a
more preferred embodiment, there is provided a 38SB13 antibody
comprising a V.sub.H having an amino acid sequence consisting of
SEQ ID NO: 50. In a more preferred embodiment, there is provided a
38SB18 antibody comprising a V.sub.H having an amino acid sequence
consisting of SEQ ID NO: 52. In a more preferred embodiment, there
is provided a 38SB19 antibody comprising a V.sub.H having an amino
acid sequence consisting of SEQ ID NO: 54. In a more preferred
embodiment, there is provided a 38SB30 antibody comprising a
V.sub.H having an amino acid sequence consisting of SEQ ID NO: 56.
In a more preferred embodiment, there is provided a 38SB31 antibody
comprising a V.sub.H having an amino acid sequence consisting of
SEQ ID NO: 58. In a more preferred embodiment, there is provided a
38SB39 antibody comprising a V.sub.H having an amino acid sequence
consisting of SEQ ID NO: 60.
[0096] Chimeric and Humanized 38SB13, 38SB18, 38SB19, 38SB30,
38SB31, and 38SB39 Antibodies
[0097] As used herein, a "chimeric antibody" is an antibody in
which the constant region, or a portion thereof, is altered,
replaced, or exchanged, so that the variable region is linked to a
constant region of a different species, or belonging to another
antibody class or subclass. "Chimeric antibody" also refers to an
antibody in which the variable region, or a portion thereof, is
altered, replaced, or exchanged, so that the constant region is
linked to a variable region of a different species, or belonging to
another antibody class or subclass. Methods for producing chimeric
antibodies are known in the art. See, e.g., Morrison, 1985,
Science, 229: 1202; Oi et al., 1986, BioTechniques, 4: 214; Gillies
et al., 1989, J. Immunol. Methods, 125: 191-202; U.S. Pat. Nos.
5,807,715; 4,816,567; and 4,816,397, which are incorporated herein
by reference in their entireties.
[0098] In one embodiment of the invention, chimeric versions of
38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 are provided. In
particular, said chimeric versions contain at least one human
constant region. In a more preferred embodiment, this human
constant region is the human IgG1/Kappa constant region.
[0099] The term "humanized antibody", as used herein, refers to a
chimeric antibody which contain minimal sequence derived from
non-human immunoglobulin. The goal of humanization is a reduction
in the immunogenicity of a xenogenic antibody, such as a murine
antibody, for introduction into a human, while maintaining the full
antigen binding affinity and specificity of the antibody. Humanized
antibodies, or antibodies adapted for non-rejection by other
mammals, may be produced using several technologies such as
resurfacing and CDR grafting. As used herein, the resurfacing
technology uses a combination of molecular modeling, statistical
analysis and mutagenesis to alter the non-CDR surfaces of antibody
variable regions to resemble the surfaces of known antibodies of
the target host. The CDR grafting technology involves substituting
the complementarity determining regions of, for example, a mouse
antibody, into a human framework domain, e.g., see W0 92/22653.
Humanized chimeric antibodies preferably have constant regions and
variable regions other than the complementarity determining regions
(CDRs) derived substantially or exclusively from the corresponding
human antibody regions and CDRs derived substantially or
exclusively from a mammal other than a human.
[0100] Strategies and methods for the resurfacing of antibodies,
and other methods for reducing immunogenicity of antibodies within
a different host, are disclosed in U.S. Pat. No. 5,639,641, which
is hereby incorporated in its entirety by reference. Briefly, in a
preferred method, (1) position alignments of a pool of antibody
heavy and light chain variable regions is generated to give a set
of heavy and light chain variable region framework surface exposed
positions wherein the alignment positions for all variable regions
are at least about 98% identical; (2) a set of heavy and light
chain variable region framework surface exposed amino acid residues
is defined for a rodent antibody (or fragment thereof); (3) a set
of heavy and light chain variable region framework surface exposed
amino acid residues that is most closely identical to the set of
rodent surface exposed amino acid residues is identified; (4) the
set of heavy and light chain variable region framework surface
exposed amino acid residues defined in step (2) is substituted with
the set of heavy and light chain variable region framework surface
exposed amino acid residues identified in step (3), except for
those amino acid residues that are within 5 .ANG. of any atom of
any residue of the complementarity-determining regions of the
rodent antibody; and (5) the humanized rodent antibody having
binding specificity is produced.
[0101] Antibodies can be humanized using a variety of other
techniques including CDR-grafting (EP 0 239 400; WO 91/09967; U.S.
Pat. Nos. 5,530,101; and 5,585,089), veneering or resurfacing (EP 0
592 106; EP 0 519 596; Padlan E. A., 1991, Molecular Immunology
28(4/5): 489-498; Studnicka G. M. et al., 1994, Protein
Engineering, 7(6): 805-814; Roguska M. A. et al., 1994, PNAS, 91:
969-973), and chain shuffling (U.S. Pat. No. 5,565,332). Human
antibodies can be made by a variety of methods known in the art
including phage display methods. See also U.S. Pat. Nos. 4,444,887,
4,716,111, 5,545,806, and 5,814,318; and international patent
application publication numbers WO 98/46645, WO 98/50433, WO
98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741
(said references incorporated by reference in their
entireties).
[0102] The present invention provides humanized antibodies or
fragments thereof, which recognize CD38 and kill CD38.sup.+ cells
by apoptosis, ADCC, and/or CDC. In a further embodiment, the
humanized antibodies or epitope-binding fragments thereof have the
ability to kill said CD38.sup.+ cells by all three mechanisms. In
yet another further embodiment, the humanized antibodies or
epitope-binding fragments thereof of the invention are capable of
killing said CD38.sup.+ cells by apoptosis even in the absence of
stroma cells or stroma-derived cytokines.
[0103] A preferred embodiment of such a humanized antibody is a
humanized 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, or 38SB39
antibody, or an epitope-binding fragment thereof.
[0104] In more preferred embodiments, there are provided resurfaced
or humanized versions of the 38SB13, 38SB18, 38SB19, 38SB30,
38SB31, and 38SB39 antibodies wherein surface-exposed residues of
the antibody or its fragments are replaced in both light and heavy
chains to more closely resemble known human antibody surfaces. The
humanized 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39
antibodies or epitope-binding fragments thereof of the present
invention have improved properties. For example, humanized 38SB13,
38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 antibodies or
epitope-binding fragments thereof specifically recognize the CD38
protein. More preferably, the humanized 38SB13, 38SB18, 38SB19,
38SB30, 38SB31, and 38SB39 antibodies or epitope-binding fragments
thereof have the additional ability to kill a CD38.sup.+ cell, by
apoptosis, ADCC, and/or CDC.
[0105] The humanized versions of the 38SB13, 38SB18, 38SB19,
38SB30, 38SB31, and 38SB39 antibodies are also fully characterized
herein with respect to their respective amino acid sequences of
both light and heavy chain variable regions, the DNA sequences of
the genes for the light and heavy chain variable regions, the
identification of the CDRs, the identification of their surface
amino acids, and disclosure of a means for their expression in
recombinant form. However, the scope of the present invention is
not limited to antibodies and fragments comprising these sequences.
Instead, all antibodies and fragments that specifically bind to
CD38 and are capable of killing CD38.sup.+ cells by apoptosis, ADCC
and/or CDC fall within the scope of the present invention.
Preferably, such antibodies are capable of killing CD38.sup.+ cells
by all three mechanisms. Thus, antibodies and epitope-binding
antibody fragments of the present invention may differ from the
38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 antibodies or
the humanized derivatives thereof, in the amino acid sequences of
their scaffold, CDRs, and/or light chain and heavy chain, and still
fall within the scope of the present invention.
[0106] The CDRs of the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and
38SB39 antibodies are identified by modeling and their molecular
structures have been predicted. Again, while the CDRs are important
for epitope recognition, they are not essential to the antibodies
and fragments of the invention. Accordingly, antibodies and
fragments are provided that have improved properties produced by,
for example, affinity maturation of an antibody of the present
invention.
[0107] The sequences of the heavy chain and light chain variable
regions of the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39
antibodies, and the sequences of their CDRs were not previously
known and are set forth in this application. Such information can
be used to produce humanized versions of the 38SB13, 38SB18,
38SB19, 38SB30, 38SB31, and 38SB39 antibodies. These humanized
anti-CD38 antibodies or their derivatives may also be used as the
cell binding agent of the present invention.
[0108] Thus, in one embodiment, this invention provides humanized
antibodies or epitope-binding fragment thereof comprising one or
more CDRs having an amino acid sequence selected from the group
consisting of SEQ ID NOS: 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, and 36. In a preferred embodiment, the
humanized antibodies of the invention comprise at least one heavy
chain and at least one light chain, and said heavy chain comprises
three sequential CDRs having amino acid sequences selected from the
group consisting of SEQ ID NOS: 1, 2, 3, 7, 8, 9, 13, 14, 15, 19,
20, 21, 25, 26, 27, 31, 32, and 33, and said light chain comprises
three sequential CDRs having amino acid sequences selected from the
group consisting of SEQ ID NOS: 4, 5, 6, 10, 11, 12, 16, 17, 18,
22, 23, 24, 28, 29, 30, 34, 35, and 36. In a further preferred
embodiment, a humanized version of 38SB13 is provided, which
comprises at least one heavy chain and at least one light chain,
wherein said heavy chain comprises three sequential
complementarity-determining regions having amino acid sequences
represented by SEQ ID NOS: 1, 2, and 3, and wherein said light
chain comprises three sequential complementarity-determining
regions having amino acid sequences represented by SEQ ID NOS: 4,
5, and 6. In another further preferred embodiment, a humanized
version of 38SB18 is provided, which comprises at least one heavy
chain and at least one light chain, wherein said heavy chain
comprises three sequential complementarity-determining regions
having amino acid sequences represented by SEQ ID NOS: 7, 8, and 9,
and wherein said light chain comprises three sequential
complementarity-determining regions having amino acid sequences
represented by SEQ ID NOS: 10, 11, and 12. In another further
preferred embodiment, a humanized version of 38SB19 is provided,
which comprises at least one heavy chain and at least one light
chain, wherein said heavy chain comprises three sequential
complementarity-determining regions having amino acid sequences
represented by SEQ ID NOS: 13, 14, and 15, and wherein said light
chain comprises three sequential complementarity-determining
regions having amino acid sequences represented by SEQ ID NOS: 16,
17, and 18. In another further preferred embodiment, a humanized
version of 38SB30 is provided, which comprises at least one heavy
chain and at least one light chain, wherein said heavy chain
comprises three sequential complementarity-determining regions
having amino acid sequences represented by SEQ ID NOS: 19, 20, and
21, and wherein said light chain comprises three sequential
complementarity-determining regions having amino acid sequences
represented by SEQ ID NOS: 22, 23, and 24. In another further
preferred embodiment, a humanized version of 38SB31 is provided,
which comprises at least one heavy chain and at least one light
chain, wherein said heavy chain comprises three sequential
complementarity-determining regions having amino acid sequences
represented by SEQ ID NOS: 25, 26, and 27, and wherein said light
chain comprises three sequential complementarity-determining
regions having amino acid sequences represented by SEQ ID NOS: 28,
29, and 30. In another further preferred embodiment, a humanized
version of 38SB39 is provided, which comprises at least one heavy
chain and at least one light chain, wherein said heavy chain
comprises three sequential complementarity-determining regions
having amino acid sequences represented by SEQ ID NOS: 31, 32, and
33, and wherein said light chain comprises three sequential
complementarity-determining regions having amino acid sequences
represented by SEQ ID NOS: 34, 35, and 36.
[0109] In one embodiment, this invention provides humanized
antibodies or fragments thereof which comprise a V.sub.H having an
amino acid sequence selected from the group of SEQ ID NOS: 66 and
72. In a preferred embodiment, a humanized 38SB19 antibody is
provided which comprises a V.sub.H having an amino acid sequence
represented by SEQ ID NO: 66. In another preferred embodiment, a
humanized 38SB31 antibody is provided which comprises a V.sub.H
having an amino acid sequence represented by SEQ ID NO: 72.
[0110] In another embodiment, this invention provides humanized
antibodies or fragments thereof which comprise a V.sub.L having an
amino acid sequence selected from the group of SEQ ID NOS: 62, 64,
68, and 70. In a preferred embodiment, a humanized 38SB19 antibody
is provided which comprises a V.sub.L having an amino acid sequence
chosen from the group of SEQ ID NOS: 62 and 64. In another
preferred embodiment, a humanized 38SB31 antibody is provided which
comprises a V.sub.L having an amino acid sequence chosen from the
group of SEQ ID NOS: 68 and 70.
[0111] The humanized 38SB19 antibodies and epitope-binding
fragments thereof of the present invention can also include
substitutions in light and/or heavy chain amino acid residues at
one or more positions defined by the grey residues in Table 1A and
1B which represent the murine surface framework residues that have
been changed from the original murine residue to the corresponding
framework surface residue in the human antibody, 28E4. The starred
(*) residues in Table 1B correspond to the murine back mutations in
the humanized 38SB19 heavy chain variant (SEQ ID NO:65). The
residues for back mutations are proximal to CDR's and were chosen
as described in U.S. Pat. No. 5,639,641 or in analogy to the
selection of residues that had in previous humanization efforts
resulted in a decrease in antigen binding affinity (Roguska et al.,
1996, U.S. patent application publications 2003/0235582 and
2005/0118183).
[0112] Likewise, the humanized 38SB13, 38SB18, 38SB30, 38SB31, and
38SB39 antibodies and epitope-binding fragments thereof of the
present invention can also include substitution in light and/or
heavy chain amino acid residues.
[0113] Polynucleotides, Vectors, and Host Cells
[0114] Nucleic acids encoding anti-CD38 antibodies of the invention
are provided. In one embodiment, the nucleic acid molecule encodes
a heavy and/or a light chain of an anti-CD38 immunoglobulin. In a
preferred embodiment, a single nucleic acid encodes a heavy chain
of an anti-CD38 immunoglobulin and another nucleic acid molecule
encodes the light chain of an anti-CD38 immunoglobulin.
[0115] In another aspect of this invention, there are provided
polynucleotides encoding polypeptides having an amino acid sequence
selected from the group of SEQ ID NOS: 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, 38, 40, 42, 44, 46, 48, 50,
52, 54, 56, 58, 60, 62, 64, 66, 68, 70, and 72. In a preferred
embodiment, the polynucleotide of the invention is selected from
the group consisting of SEQ ID NOs: 37, 39, 41, 43, 45, 47, 49, 51,
53, 55, 57, 59, 61, 63, 65, 67, 69, and 71. The invention is not
limited to said polynucleotides per se but also includes all
polynucleotides displaying at least 80% identity with said
polynucleotides.
[0116] The invention provides vectors comprising the
polynucleotides of the invention. In one embodiment, the vector
contains a polynucleotide encoding a heavy chain of an anti-CD38
immunoglobulin. In another embodiment, said polynucleotide encodes
the light chain of an anti-CD38 immunoglobulin. The invention also
provides vectors comprising polynucleotide molecules encoding,
fusion proteins, modified antibodies, antibody fragments, and
probes thereof.
[0117] In order to express the heavy and/or light chain of the
anti-CD38 antibodies of the invention, the polynucleotides encoding
said heavy and/or light chains are inserted into expression vectors
such that the genes are operatively linked to transcriptional and
translational sequences. Expression vectors include plasmids, YACs,
cosmids, retrovirus, EBV-derived episomes, and all the other
vectors that the skilled man will know to be convenient for
ensuring the expression of said heavy and/or light chains. The
skilled man will realize that the polynucleotides encoding the
heavy and the light chains can be cloned into different vectors or
in the same vector. In a preferred embodiment, said polynucleotides
are cloned in the same vector.
[0118] Polynucleotides of the invention and vectors comprising
these molecules can be used for the transformation of a suitable
mammalian host cell. Transformation can be by any known method for
introducing polynucleotides into a cell host. Such methods are well
known of the man skilled in the art and include dextran-mediated
transformation, calcium phosphate precipitation, polybrene-mediated
transfection, protoplast fusion, electroporation, encapsulation of
the polynucleotide into liposomes, biolistic injection and direct
microinjection of DNA into nuclei.
[0119] Antibody Fragments
[0120] The antibodies of the present invention include both the
full length antibodies discussed above, as well as epitope-binding
fragments thereof. As used herein, "antibody fragments" include any
portion of an antibody that retains the ability to bind to the
epitope recognized by the full length antibody, generally termed
"epitope-binding fragments." Examples of antibody fragments
include, but are not limited to, Fab, Fab' and F(ab')2, Fd,
single-chain Fvs (scFv), single-chain antibodies, disulfide-linked
Fvs (dsFv) and fragments comprising either a VL or VH region.
Epitope-binding fragments, including single-chain antibodies, may
comprise the variable region(s) alone or in combination with the
entirety or a portion of the following: hinge region, CH1, CH2, and
CH3 domains.
[0121] Such fragments may contain one or both Fab fragments or the
F(ab')2 fragment. Preferably, the antibody fragments contain all
six CDRs of the whole antibody, although fragments containing fewer
than all of such regions, such as three, four or five CDRs, are
also functional. Further, the fragments may be or may combine
members of any one of the following immunoglobulin classes: IgG,
IgM, IgA, IgD, or IgE, and the subclasses thereof.
[0122] Fab and F(ab')2 fragments may be produced by proteolytic
cleavage, using enzymes such as papain (Fab fragments) or pepsin
(F(ab')2 fragments).
[0123] The "single-chain FVs" ("scFvs") fragments are
epitope-binding fragments that contain at least one fragment of an
antibody heavy chain variable region (V.sub.H) linked to at least
one fragment of an antibody light chain variable region (V.sub.L).
The linker may be a short, flexible peptide selected to assure that
the proper three-dimensional folding of the V.sub.L and V.sub.H
regions occurs once they are linked so as to maintain the target
molecule binding-specificity of the whole antibody from which the
single-chain antibody fragment is derived. The carboxyl terminus of
the V.sub.L or V.sub.H sequence may be covalently linked by a
linker to the amino acid terminus of a complementary V.sub.L or
V.sub.H sequence.
[0124] Single-chain antibody fragments of the present invention
contain amino acid sequences having at least one of the variable or
complementarity determining regions (CDRs) of the whole antibodies
described in this specification, but lack some or all of the
constant domains of those antibodies. These constant domains are
not necessary for antigen binding, but constitute a major portion
of the structure of whole antibodies. Single-chain antibody
fragments may therefore overcome some of the problems associated
with the use of antibodies containing a part or all of a constant
domain. For example, single-chain antibody fragments tend to be
free of undesired interactions between biological molecules and the
heavy-chain constant region, or other unwanted biological activity.
Additionally, single-chain antibody fragments are considerably
smaller than whole antibodies and may therefore have greater
capillary permeability than whole antibodies, allowing single-chain
antibody fragments to localize and bind to target antigen-binding
sites more efficiently. Also, antibody fragments can be produced on
a relatively large scale in prokaryotic cells, thus facilitating
their production. Furthermore, the relatively small size of
single-chain antibody fragments makes them less likely to provoke
an immune response in a recipient than whole antibodies.
[0125] Single-chain antibody fragments may be generated by
molecular cloning, antibody phage display library or similar
techniques well known to the skilled artisan. These proteins may be
produced, for example, in eukaryotic cells or prokaryotic cells,
including bacteria. The epitope-binding fragments of the present
invention can also be generated using various phage display methods
known in the art. In phage display methods, functional antibody
domains are displayed on the surface of phage particles which carry
the polynucleotide sequences encoding them. In particular, such
phage can be utilized to display epitope-binding domains expressed
from a repertoire or combinatorial antibody library (e.g., human or
murine). Phage expressing an epitope-binding domain that binds the
antigen of interest can be selected or identified with antigen,
e.g., using labeled antigen bound or captured to a solid surface or
bead. Phage used in these methods are typically filamentous phage
including fd and M13 binding domains expressed from phage with Fab,
Fv or disulfide-stabilized Fv antibody domains recombinantly fused
to either the phage gene III or gene VIII protein.
[0126] Examples of phage display methods that can be used to make
the epitope-binding fragments of the present invention include
those disclosed in Brinkman et al., 1995, J. Immunol. Methods, 182:
41-50; Ames et al., 1995, J. Immunol. Methods, 184: 177-186;
Kettleborough et al., 1994, Eur. J. Immunol., 24: 952-958; Persic
et al., 1997, Gene, 187: 9-18; Burton et al., 1994, Advances in
Immunology, 57: 191-280; WO/1992/001047; WO 90/02809; WO 91/10737;
WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401;
and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717;
5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637;
5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is
incorporated herein by reference in its entirety.
[0127] After phage selection, the regions of the phage encoding the
fragments can be isolated and used to generate the epitope-binding
fragments through expression in a chosen host, including mammalian
cells, insect cells, plant cells, yeast, and bacteria, using
recombinant DNA technology, e.g., as described in detail below. For
example, techniques to recombinantly produce Fab, Fab' and F(ab')2
fragments can also be employed using methods known in the art such
as those disclosed in WO 92/22324; Mullinax et al., 1992, Bio
Techniques, 12(6): 864-869; Sawai et al., 1995, AJRI, 34: 26-34;
and Better et al., 1988, Science, 240:1041-1043; said references
incorporated by reference in their entireties. Examples of
techniques which can be used to produce single-chain Fvs and
antibodies include those described in U.S. Pat. Nos. 4,946,778 and
5,258,498; Huston et al., 1991, Methods in Enzymology, 203: 46-88;
Shu et al., 1993, PNAS, 90: 7995-7999; Skerra et al., 1988,
Science, 240:1038-1040.
[0128] Functional Equivalents
[0129] Also included within the scope of the invention are
functional equivalents of the anti-CD38 antibody and the humanized
anti-CD38 receptor antibody. The term "functional equivalents"
includes antibodies with homologous sequences, chimeric antibodies,
artificial antibodies and modified antibodies, for example, wherein
each functional equivalent is defined by its ability to bind to the
CD38 protein. The skilled artisan will understand that there is an
overlap in the group of molecules termed "antibody fragments" and
the group termed "functional equivalents." Methods of producing
functional equivalents are known to the person skilled in the art
and are disclosed, for example, in WO 93/21319, EP 239,400; WO
89/09622; EP 338,745; and EP 332,424, which are incorporated in
their respective entireties by reference.
[0130] Antibodies with homologous sequences are those antibodies
with amino acid sequences that have sequence homology with amino
acid sequence of an anti-CD38 antibody and a humanized anti-CD38
antibody of the present invention. Preferably homology is with the
amino acid sequence of the variable regions of the anti-CD38
antibody and humanized anti-CD38 antibody of the present invention.
"Sequence homology" as applied to an amino acid sequence herein is
defined as a sequence with at least about 90%, 91%, 92%, 93%, or
94% sequence homology, and more preferably at least about 95%,
96%.sub., 97%, 98%, or 99% sequence homology to another amino acid
sequence, as determined, for example, by the FASTA search method in
accordance with Pearson and Lipman, 1988, Proc. Natl. Acad. Sci.
USA, 85: 2444-2448.
[0131] Artificial antibodies include scFv fragments, diabodies,
triabodies, tetrabodies and mru (see reviews by Winter, G. and
Milstein, C., 1991, Nature, 349: 293-299; Hudson, P. J., 1999,
Current Opinion in Immunology, 11: 548-557), each of which has
antigen-binding ability. In the single chain Fv fragment (scFv),
the V.sub.H and VL domains of an antibody are linked by a flexible
peptide. Typically, this linker peptide is about 15 amino acid
residues long. If the linker is much smaller, for example 5 amino
acids, diabodies are formed, which are bivalent scFv dimers. If the
linker is reduced to less than three amino acid residues, trimeric
and tetrameric structures are formed that are called triabodies and
tetrabodies. The smallest binding unit of an antibody is a CDR,
typically the CDR2 of the heavy chain which has sufficient specific
recognition and binding that it can be used separately. Such a
fragment is called a molecular recognition unit or mru. Several
such mrus can be linked together with short linker peptides,
therefore forming an artificial binding protein with higher avidity
than a single mru.
[0132] The functional equivalents of the present application also
include modified antibodies, e.g., antibodies modified by the
covalent attachment of any type of molecule to the antibody. For
example, modified antibodies include antibodies that have been
modified, e.g., by glycosylation, acetylation, pegylation,
phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, linkage to a
cellular ligand or other protein, etc. The covalent attachment does
not prevent the antibody from generating an anti-idiotypic
response. These modifications may be carried out by known
techniques, including, but not limited to, specific chemical
cleavage, acetylation, formylation, metabolic synthesis of
tunicamycin, etc. Additionally, the modified antibodies may contain
one or more non-classical amino acids.
[0133] Functional equivalents may be produced by interchanging
different CDRs on different chains within different frameworks.
Thus, for example, different classes of antibody are possible for a
given set of CDRs by substitution of different heavy chains,
whereby, for example, IgG1-4, IgM, IgA1-2, IgD, IgE antibody types
and isotypes may be produced. Similarly, artificial antibodies
within the scope of the invention may be produced by embedding a
given set of CDRs within an entirely synthetic framework.
[0134] Functional equivalents may be readily produced by mutation,
deletion and/or insertion within the variable and/or constant
region sequences that flank a particular set of CDRs, using a wide
variety of methods known in the art. The antibody fragments and
functional equivalents of the present invention encompass those
molecules with a detectable degree of binding to CD38, when
compared to the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, or 38SB39
antibody. A detectable degree of binding includes all values in the
range of at least 10-100%, preferably at least 50%, 60% or 70%,
more preferably at least 75%, 80%, 85%, 90%, 95% or 99% of the
binding ability of the murine 38SB13, 38SB18, 38SB19, 38SB30,
38SB31, or 38SB39 antibody to CD38.
[0135] Improved Antibodies
[0136] The CDRs are of primary importance for epitope recognition
and antibody binding. However, changes may be made to the residues
that comprise the CDRs without interfering with the ability of the
antibody to recognize and bind its cognate epitope. For example,
changes that do not affect epitope recognition, yet increase the
binding affinity of the antibody for the epitope may be made.
[0137] Thus, also included in the scope of the present invention
are improved versions of both the murine and humanized antibodies,
which also specifically recognize and bind CD38, preferably with
increased affinity.
[0138] Several studies have surveyed the effects of introducing one
or more amino acid changes at various positions in the sequence of
an antibody, based on the knowledge of the primary antibody
sequence, on its properties such as binding and level of expression
(Yang, W. P. et al., 1995, J. Mol. Biol., 254 : 392-403; Rader, C.
et al., 1998, Proc. Natl. Acad. Sci. USA, 95 : 8910-8915; Vaughan,
T. J. et al., 1998, Nature Biotechnology, 16 : 535-539).
[0139] In these studies, equivalents of the primary antibody have
been generated by changing the sequences of the heavy and light
chain genes in the CDR1, CDR2, CDR3, or framework regions, using
methods such as oligonucleotide-mediated site-directed mutagenesis,
cassette mutagenesis, error-prone PCR, DNA shuffling, or
mutator-strains of E. coli (Vaughan, T. J. et al., 1998, Nature
Biotechnology, 16: 535-539; Adey, N. B. et al., 1996, Chapter 16,
pp. 277-291, in "Phage Display of Peptides and Proteins", Eds. Kay,
B. K. et al., Academic Press). These methods of changing the
sequence of the primary antibody have resulted in improved
affinities of the secondary antibodies (Gram, H. et al., 1992,
Proc. Natl. Acad. Sci. USA, 89 : 3576-3580; Boder, E. T. et al.,
2000, Proc. Natl. Acad. Sci. USA, 97: 10701-10705; Davies, J. and
Riechmann, L., 1996, Immunotechnolgy, 2: 169-179; Thompson, J. et
al., 1996, J. Mol. Biol., 256: 77-88; Short, M. K. et al., 2002, J.
Biol. Chem., 277: 16365-16370; Furukawa, K. et al., 2001, J. Biol.
Chem., 276: 27622-27628).
[0140] By a similar directed strategy of changing one or more amino
acid residues of the antibody, the antibody sequences described in
this invention can be used to develop anti-CD38 antibodies with
improved functions, including improved affinity for CD38.
[0141] Preferred amino acid substitutions are those which: (1)
reduce susceptibility to proteolysis, (2) reduce susceptibility to
oxidation, (3) alter binding affinity for forming protein
complexes, and (4) confer or modify other physico-chemical or
functional properties of such analogs. Analogs can include various
muteins of a sequence other than the naturally-occurring peptide
sequence. For example, single or multiple amino acid substitutions
(preferably conservative amino acid substitutions) may be made in
the naturally-occurring sequence (preferably in the portion of the
polypeptide outside the domain (s) forming intermolecular contacts.
A conservative amino acid substitution should not substantially
change the structural characteristics of the parent sequence (e.g.,
a replacement amino acid should not tend to break a helix that
occurs in the parent sequence, or disrupt other types of secondary
structure that characterizes the parent sequence). Examples of
art-recognized polypeptide secondary and tertiary structures are
described in Proteins, Structures and Molecular Principles
(Creighton, Ed., W. H. Freeman and Company, New York (1984));
Introduction to Protein Structure (C. Branden and J. Tooze, eds.,
Garland Publishing, New York, N.Y. (1991)); and Thornton et al.,
1991, Nature, 354: 105, which are each incorporated herein by
reference.
[0142] Improved antibodies also include those antibodies having
improved characteristics that are prepared by the standard
techniques of animal immunization, hybridoma formation and
selection for antibodies with specific characteristics.
[0143] Improved antibodies according to the invention include in
particular antibodies with enhanced functional properties. Of
special interest are those antibodies with enhanced ability to
mediate cellular cytotoxic effector functions such as ADCC. Such
antibodies may be obtained by making single or multiple
substitutions in the constant framework of the antibody, thus
altering its interaction with the Fc receptors. Methods for
designing such mutants can be found for example in Lazar et al.
(2006, Proc. Natl. Acad. Sci. U.S.A. 103(11): 4005-4010) and
Okazaki et al. (2004, J. Mol. Biol. 336(5):1239-49). See also WO
03/074679, WO 2004/029207, WO 2004/099249, WO2006/047350, WO
2006/019447, WO 2006/105338, WO 2007/041635. It is also possible to
use cell lines specifically engineered for production of improved
antibodies. In particular, these lines have altered regulation of
the glycosylation pathway, resulting in antibodies which are poorly
fucosylated or even totally defucosylated. Such cell lines and
methods for engineering them are disclosed in e.g. Shinkawa et al.
(2003, J. Biol. Chem. 278(5): 3466-3473), Ferrara et al. (2006, J.
Biol. Chem. 281(8): 5032-5036; 2006, Biotechnol. Bioeng. 93(5):
851-61), EP 1331266, EP 1498490, EP 1498491, EP 1676910, EP
1792987, and WO 99/54342.
[0144] Mapping of the epitope and identification of the paratope
was achieved by structure determination of the crystal structure of
the huCD38 in complex with the Fab fragment from hu38SB19 at 1.53
.ANG. resolution.
[0145] In another embodiment the antibody of the invention
specifically binds to an epitope of the human CD38 extracellular
domain (R45-I300 herein specified huCD38) carrying amino acid
substitutions N100D, N164A, N209D and N219D to avoid
N-glycosylation. The identified epitope comprises residues Pro75,
Glu76, His79, Gln107, Pro108, Met110, Lys111, Leu112, Gly113,
Thr114, Gln115, Thr116, Val117, Pro118, Cys119, Thr148, Leu150,
Arg194, Arg195, Glu198, Ala199, Cys201 and Glu233 or a
conservatively substituted form thereof.
[0146] The term "conservatively substituted form" refers to a
peptide having an amino acid residue sequence substantially
identical to a sequence of a reference peptide in which one or more
residues have been conservatively substituted with a functionally
similar residue such that the "conservatively substituted variant"
will bind to the same binding partner with substantially the same
affinity as the parental variant and will prevent binding of the
parental variant. In one embodiment, a conservatively substituted
variant displays a similar binding specificity when compared to the
reference peptide. The phrase "conservatively substituted variant"
also includes peptides wherein a residue is replaced with a
chemically derivatized residue.
[0147] Examples of conservative substitutions include the
substitution of one non-polar (hydrophobic) residue such as
isoleucine, valine, leucine or methionine for another; the
substitution of one aromatic residue such as tryptophan, tyrosine,
or phenylalanine for another; the substitution of one polar
(hydrophilic) residue for another such as between arginine and
lysine, between glutamine and asparagine, between glycine, alanine,
threonine and serine; the substitution of one basic residue such as
lysine, arginine or histidine for another; or the substitution of
one acidic residue such as aspartic acid or glutamic acid for
another.
[0148] The present invention also includes cytotoxic conjugates.
These cytotoxic conjugates comprise two primary components, a
cell-binding agent and a cytotoxic agent.
[0149] As used herein, the term "cell binding agent" refers to an
agent that specifically recognizes and binds the CD38 proteins on
the cell surface. In one embodiment, the cell binding agent
specifically recognizes CD38 such that it allows the conjugates to
act in a targeted fashion with little side-effects resulting from
non-specific binding.
[0150] In another embodiment, the cell binding agent of the present
invention also specifically recognizes the CD38 protein so that the
conjugates will be in contact with the target cell for a sufficient
period of time to allow the cytotoxic drug portion of the conjugate
to act on the cell, and/or to allow the conjugates sufficient time
in which to be internalized by the cell.
[0151] In a preferred embodiment, the cytotoxic conjugates comprise
an anti-CD38 antibody as the cell binding agent, more preferably
the murine 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, or 38SB39
anti-CD38 monoclonal antibody. In another preferred embodiment, the
cell binding agent is a chimeric version of said anti-CD38
antibody. In a more preferred embodiment, the cytotoxic conjugate
comprises a humanized 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and
38SB39 antibody or an epitope-binding fragment thereof. The 38SB13,
38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 antibody is able to
specifically recognize CD38, and directs the cytotoxic agent to an
abnormal cell or a tissue, such as cancer cells, in a targeted
fashion.
[0152] The second component of the cytotoxic conjugates of the
present invention is a cytotoxic agent. The term "cytotoxic agent"
as used herein refers to a substance that reduces or blocks the
function, or growth, of cells and/or causes destruction of
cells.
[0153] In preferred embodiments, the cytotoxic agent is a small
drug, a prodrug, a taxoid, a maytansinoid such as DM1 or DM4, a
tomaymycin derivative, a leptomycin derivative, CC-1065 or a
CC-1065 analog. In preferred embodiments, the cell binding agents
of the present invention are covalently attached, directly or via a
cleavable or non-cleavable linker, to the cytotoxic agent.
[0154] The cell binding agents, cytotoxic agents, and linkers are
discussed in more detail below.
[0155] Cell Binding Agents
[0156] The effectiveness of the compounds of the present invention
as therapeutic agents depends on the careful selection of an
appropriate cell binding agent. Cell binding agents may be of any
kind presently known, or that become known, and includes peptides
and non-peptides. The cell binding agent may be any compound that
can bind a cell, either in a specific or non-specific manner.
Generally, these can be antibodies (especially monoclonal
antibodies), lymphokines, hormones, growth factors, vitamins,
nutrient-transport molecules (such as transferrin), or any other
cell binding molecule or substance.
[0157] More specific examples of cell binding agents that can be
used include: [0158] a) polyclonal antibodies; [0159] b) monoclonal
antibodies; [0160] c) fragments of antibodies such as Fab, Fab',
and F(ab')2, Fv (Parham, 1983, J. Immunol., 131: 2895-2902; Spring
et al., 1974, J. Immunol., 113: 470-478; Nisonoff et al., 1960,
Arch. Biochem. Biophys., 89: 230-244);
[0161] In particular, an anti-CD38 monoclonal antibody selected
from 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 can be used
as a cell binding agent according to the present invention.
Likewise, said cell binding agent can be a chimeric version of one
of the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39
monoclonal antibodies. Preferably, a humanized anti-CD38 antibody
is used as the cell binding agent of the present invention. More
preferably the humanized anti-CD38 antibody is selected from
humanized or resurfaced 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and
38SB39 antibodies.
[0162] Cytotoxic Agents
[0163] In another embodiment, the humanized antibody or an
epitope-binding fragment thereof can be conjugated to a drug, such
as a maytansinoid, to form a prodrug having specific cytotoxicity
towards antigen-expressing cells by targeting the drug to the CD38
protein. Cytotoxic conjugates comprising such antibodies and a
small, highly toxic drug (e.g., maytansinoids, taxanes, tomaymycin
derivatives, leptomycin derivatives, and CC-1065 analogs) can be
used as a therapeutic for treatment of tumors, such as lymphoma,
leukemia, and multiple myeloma.
[0164] The cytotoxic agent used in the cytotoxic conjugate of the
present invention may be any compound that results in the death of
a cell, or induces cell death, or in some manner decreases cell
viability. Preferred cytotoxic agents include, for example,
maytansinoids and maytansinoid analogs, taxoids, tomaymycin
derivatives, leptomycin derivatives, CC-1065 and CC-1065 analogs,
dolastatin and dolastatin analogs, defined below. These cytotoxic
agents are conjugated to the antibodies, antibodies fragments,
functional equivalents, improved antibodies and their analogs as
disclosed herein
[0165] The cytotoxic conjugates may be prepared by in vitro
methods. In order to link a drug or prodrug to the antibody, a
linking group is used. Suitable linking groups are well known in
the art and include disulfide groups, thioether groups, acid labile
groups, photolabile groups, peptidase labile groups and esterase
labile groups. Preferred linking groups are disulfide groups and
thioether groups. For example, conjugates can be constructed using
a disulfide exchange reaction or by forming a thioether bond
between the antibody and the drug or prodrug.
[0166] Maytansinoids
[0167] Among the cytotoxic agents that may be used in the present
invention to form a cytotoxic conjugate, are maytansinoids and
maytansinoid analogs. Examples of suitable maytansinoids include
maytansinol and maytansinol analogs. Maytansinoids are drugs that
inhibit microtubule formation and that are highly toxic to
mammalian cells.
[0168] Examples of suitable maytansinol analogues include those
having a modified aromatic ring and those having modifications at
other positions. Such suitable maytansinoids are disclosed in U.S.
Pat. Nos. 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946;
4,315,929; 4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254;
4,322,348; 4,371,533; 6,333,410; 5,475,092; 5,585,499; and
5,846,545.
[0169] Specific examples of suitable analogues of maytansinol
having a modified aromatic ring include:
(1) C-19-dechloro (U.S. Pat. No. 4,256,746) (prepared by LAH
reduction of ansamytocin P2); (2) C-20-hydroxy (or
C-20-demethyl)+/-C-19-dechloro (U.S. Pat. Nos. 4,361,650 and
4,307,016) (prepared by demethylation using Streptomyces or
Actinomyces or dechlorination using LAH); and (3) C-20-demethoxy,
C-20-acyloxy (--OCOR), +/-dechloro (U.S. Pat. No. 4,294,757)
(prepared by acylation using acyl chlorides).
[0170] Specific examples of suitable analogues of maytansinol
having modifications of other positions include:
(1) C-9-SH (U.S. Pat. No. 4,424,219) (prepared by the reaction of
maytansinol with H2S or P2S5); (2) C-14-alkoxymethyl
(demethoxy/CH2OR) (U.S. Pat. No. 4,331,598); (3) C-14-hydroxymethyl
or acyloxymethyl (CH2OH or CH2OAc) (U.S. Pat. No. 4,450,254)
(prepared from Nocardia); (4) C-15-hydroxy/acyloxy (U.S. Pat. No.
4,364,866) (prepared by the conversion of maytansinol by
Streptomyces); (5) C-15-methoxy (U.S. Pat. Nos. 4,313,946 and
4,315,929) (isolated from Trewia nudiflora); (6) C-18-N-demethyl
(U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared by the
demethylation of maytansinol by Streptomyces); and (7) 4,5-deoxy
(U.S. Pat. No. 4,371,533) (prepared by the titanium trichloride/LAH
reduction of maytansinol).
[0171] In a preferred embodiment, the cytotoxic conjugates of the
present invention utilize the thiol-containing maytansinoid (DM1),
formally termed
N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine, as the
cytotoxic agent. DM1 is represented by the following structural
formula (I):
##STR00001##
[0172] In another preferred embodiment, the cytotoxic conjugates of
the present invention utilize the thiol-containing maytansinoid
N2'-deacetyl-N-2'(4-methyl-4-mercapto-1-oxopentyl)-maytansine as
the cytotoxic agent. DM4 is represented by the following structural
formula (II):
##STR00002##
[0173] In further embodiments of the invention, other maytansines,
including thiol and disulfide-containing maytansinoids bearing a
mono or di-alkyl substitution on the carbon atom bearing the sulfur
atom, may be used. These include a maytansinoid having, at C-3,
C-14 hydroxymethyl, C-15 hydroxy, or C-20 desmethyl, an acylated
amino acid side chain with an acyl group bearing a hindered
sulfhydryl group, wherein the carbon atom of the acyl group bearing
the thiol functionality has one or two substituents, said
substituents being CH.sub.3, C.sub.2H.sub.5, linear or branched
alkyl or alkenyl having from 1 to 10 carbon atoms, cyclic alkyl or
alkenyl having from 3 to 10 carbon atoms, phenyl, substituted
phenyl, or heterocyclic aromatic or heterocycloalkyl radical, and
further wherein one of the substituents can be H, and wherein the
acyl group has a linear chain length of at least three carbon atoms
between the carbonyl functionality and the sulfur atom.
[0174] Such additional maytansines include compounds represented by
formula (III):
##STR00003##
wherein: Y' represents
(CR.sub.7R.sub.8).sub.l(CR.sub.9.dbd.CR.sub.10).sub.p(C.ident.C).sub.qA.s-
ub.r(CR.sub.5R.sub.6).sub.mD.sub.u(CR.sub.11.dbd.CR.sub.12).sub.r(C.ident.-
C).sub.sB.sub.t(CR.sub.3R.sub.4).sub.nCR.sub.1R.sub.2SZ, wherein:
[0175] R.sub.1 and R.sub.2 are each independently CH.sub.3,
C.sub.2H.sub.5, linear alkyl or alkenyl having from 1 to 10 carbon
atoms, branched or cyclic alkyl or alkenyl having from 3 to 10
carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic
or heterocycloalkyl radical, and in addition R.sub.2 can be H;
[0176] A, B, D are cycloalkyl or cycloalkenyl having 3-10 carbon
atoms, simple or substituted aryl or heterocyclic aromatic or
heterocycloalkyl radical; R.sub.3, R.sub.4, R.sub.5, R.sub.6,
R.sub.7, R.sub.8, R.sub.9, R.sub.11, and R.sub.12 are each
independently H, CH.sub.3, C.sub.2H.sub.5, linear alkyl or alkenyl
having from 1 to 10 carbon atoms, branched or cyclic alkyl or
alkenyl having from 3 to 10 carbon atoms, phenyl, substituted
phenyl or heterocyclic aromatic or heterocycloalkyl radical; [0177]
l, m, n, o, p, q, r, s, and t are each independently 0 or an
integer of from 1 to 5, provided that at least two of l, m, n, o,
p, q, r, s and t are not zero at any one time; and [0178] Z is H,
SR or --COR, wherein R is linear alkyl or alkenyl having from 1 to
10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3
to 10 carbon atoms, or simple or substituted aryl or heterocyclic
aromatic or heterocycloalkyl radical.
[0179] Preferred embodiments of formula (III) include compounds of
formula (III) wherein:
R.sub.1 is H, R.sub.2 is methyl and Z is H. R.sub.1 and R.sub.2 are
methyl and Z is H. R.sub.1 is H, R.sub.2 is methyl, and Z is
--SCH.sub.3. R.sub.1 and R.sub.2 are methyl, and Z is
--SCH.sub.3.
[0180] Such additional maytansines also include compounds
represented by formula (IV-L), (IV-D), or (IV-D,L):
##STR00004##
wherein: Y represents
(CR.sub.7R.sub.8).sub.l(CR.sub.5R.sub.6).sub.m(CR.sub.3R.sub.4).sub.nCR.s-
ub.1R.sub.2SZ, wherein: [0181] R.sub.1 and R.sub.2 are each
independently CH.sub.3, C.sub.2H.sub.5, linear alkyl or alkenyl
having from 1 to 10 carbon atoms, branched or cyclic alkyl or
alkenyl having from 3 to 10 carbon atoms, phenyl, substituted
phenyl, or heterocyclic aromatic or heterocycloalkyl radical, and
in addition R.sub.2 can be H; [0182] R.sub.3, R.sub.4, R.sub.5,
R.sub.6, R.sub.7 and R.sub.8 are each independently H, CH.sub.3,
C.sub.2H.sub.5, linear alkyl or alkenyl having from 1 to 10 carbon
atoms, branched or cyclic alkyl or alkenyl having from 3 to 10
carbon atoms, phenyl, substituted phenyl, or heterocyclic aromatic
or heterocycloalkyl radical; [0183] l, m and n are each
independently an integer of from 1 to 5, and in addition n can be
0; [0184] Z is H, SR or --COR wherein R is linear or branched alkyl
or alkenyl having from 1 to 10 carbon atoms, cyclic alkyl or
alkenyl having from 3 to 10 carbon atoms, or simple or substituted
aryl or heterocyclic aromatic or heterocycloalkyl radical; and
[0185] May represents a maytansinoid which bears the side chain at
C-3, C-14 hydroxymethyl, C-15 hydroxy or C-20 desmethyl.
[0186] Preferred embodiments of formulas (IV-L), (IV-D) and
(IV-D,L) include compounds of formulas (IV-L), (IV-D) and (IV-D,L)
wherein:
R.sub.1 is H, R.sub.2 is methyl, R.sub.5, R.sub.6, R.sub.7, and
R.sub.8 are each H, l and m are each 1, n is 0, and Z is H. R.sub.1
and R.sub.2 are methyl, R.sub.5, R.sub.6, R.sub.7, R.sub.8 are each
H, l and m are 1, n is 0, and Z is H. R.sub.1 is H, R.sub.2 is
methyl, R.sub.5, R.sub.6, R.sub.7, R.sub.8 are each H, l and m are
each 1, n is 0, and Z is --SCH.sub.3. R.sub.1 and R.sub.2 are
methyl, R.sub.5, R.sub.6, R.sub.7, R.sub.8 are each H, l and m are
1, n is 0, and Z is --SCH.sub.3.
[0187] Preferably the cytotoxic agent is represented by formula
(IV-L).
[0188] Such additional maytansines also include compounds
represented by formula (V):
##STR00005##
wherein: Y represents
(CR.sub.7R.sub.8).sub.l(CR.sub.5R.sub.6).sub.m(CR.sub.3R.sub.4).sub.nCR.s-
ub.1R.sub.2SZ, wherein: [0189] R.sub.1 and R.sub.2 are each
independently CH.sub.3, C.sub.2H.sub.5, linear alkyl or alkenyl
having from 1 to 10 carbon atoms, branched or cyclic alkyl or
alkenyl having from 3 to 10 carbon atoms, phenyl, substituted
phenyl or heterocyclic aromatic or heterocycloalkyl radical, and in
addition R.sub.2 can be H; [0190] R.sub.3, R.sub.4, R.sub.5,
R.sub.6, R.sub.7, and R.sub.8 are each independently H, CH.sub.3,
C.sub.2H.sub.5, linear alkyl or alkenyl having from 1 to 10 carbon
atoms, branched or cyclic alkyl or alkenyl having from 3 to 10
carbon atoms, phenyl, substituted phenyl, or heterocyclic aromatic
or heterocycloalkyl radical; [0191] l, m and n are each
independently an integer of from 1 to 5, and in addition n can be
0; and [0192] Z is H, SR or --COR, wherein R is linear alkyl or
alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl
or alkenyl having from 3 to 10 carbon atoms, or simple or
substituted aryl or heterocyclic aromatic or heterocycloalkyl
radical.
[0193] Preferred embodiments of formula (V) include compounds of
formula (V) wherein:
R.sub.1 is H, R.sub.2 is methyl, R.sub.5, R.sub.6, R.sub.7, and
R.sub.8 are each H; l and m are each 1; n is 0; and Z is H. R.sub.1
and R.sub.2 are methyl, R.sub.5, R.sub.6, R.sub.7, and R.sub.8 are
each H, l and m are 1; n is 0; and Z is H. R.sub.1 is H, R.sub.2 is
methyl, R.sub.5, R.sub.6, R.sub.7, and R.sub.8 are each H, l and m
are each 1, n is 0, and Z is --SCH.sub.3. R.sub.1 and R.sub.2 are
methyl, R.sub.5, R.sub.6, R.sub.7, and R.sub.8 are each H, l and m
are 1, n is 0, and Z is --SCH.sub.3.
[0194] Such additional maytansines further include compounds
represented by formula (VI-L), (VI-D), or (VI-D,L):
##STR00006##
wherein: Y.sub.2 represents
(CR.sub.7R.sub.8).sub.l(CR.sub.5R.sub.6).sub.m(CR.sub.3R.sub.4).sub.nCR.s-
ub.1R.sub.2SZ.sub.2, wherein: [0195] R.sub.1 and R.sub.2 are each
independently CH.sub.3, C.sub.2H.sub.5, linear alkyl or alkenyl
having from 1 to 10 carbon atoms, branched or cyclic alkyl or
alkenyl having from 3 to 10 carbon atoms, phenyl, substituted
phenyl or heterocyclic aromatic or heterocycloalkyl radical, and in
addition R.sub.2 can be H; [0196] R.sub.3, R.sub.4, R.sub.5,
R.sub.6, R.sub.7 and R.sub.8 are each independently H, CH.sub.3,
C.sub.2H.sub.5, linear cyclic alkyl or alkenyl having from 1 to 10
carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to
10 carbon atoms, phenyl, substituted phenyl or heterocyclic
aromatic or heterocycloalkyl radical; [0197] l, m and n are each
independently an integer of from 1 to 5, and in addition n can be
0; [0198] Z.sub.2 is SR or COR, wherein R is linear alkyl or
alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl
or alkenyl having from 3 to 10 carbon atoms, or simple or
substituted aryl or heterocyclic aromatic or heterocycloalkyl
radical; and [0199] May is a maytansinoid.
[0200] Such additional maytansines also include compounds
represented by formula (VII):
##STR00007##
wherein: Y.sub.2' represents
(CR.sub.7R.sub.8).sub.l(CR.sub.9.dbd.CR.sub.10).sub.p(C.ident.C).sub.qA.s-
ub.r(CR.sub.5R.sub.6).sub.mD.sub.u(CR.sub.11.dbd.CR.sub.12).sub.r(C.ident.-
C).sub.sB.sub.t(CR.sub.3R.sub.4).sub.nCR.sub.1R.sub.2SZ.sub.2,
wherein: [0201] R.sub.1 and R.sub.2 are each independently
CH.sub.3, C.sub.2H.sub.5, linear branched or alkyl or alkenyl
having from 1 to 10 carbon atoms, cyclic alkyl or alkenyl having
from 3 to 10 carbon atoms, phenyl, substituted phenyl or
heterocyclic aromatic or heterocycloalkyl radical, and in addition
R.sub.2 can be H; [0202] A, B, and D each independently is
cycloalkyl or cycloalkenyl having 3 to 10 carbon atoms, simple or
substituted aryl, or heterocyclic aromatic or heterocycloalkyl
radical; [0203] R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7,
R.sub.8, R.sub.9, R.sub.11, and R.sub.12 are each independently H,
CH.sub.3, C.sub.2H.sub.5, linear alkyl or alkenyl having from 1 to
10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3
to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic
aromatic or heterocycloalkyl radical; [0204] l, m, n, o, p, q, r,
s, and t are each independently 0 or an integer of from 1 to 5,
provided that at least two of l, m, n, o, p, q, r, s and t are not
zero at any one time; and [0205] Z.sub.2 is SR or --COR, wherein R
is linear alkyl or alkenyl having from 1 to 10 carbon atoms,
branched or cyclic alkyl or alkenyl having from 3-10 carbon atoms,
or simple or substituted aryl or heterocyclic aromatic or
heterocycloalkyl radical.
[0206] Preferred embodiments of formula (VII) include compounds of
formula (VII) wherein: R.sub.1 is H and R.sub.2 is methyl.
[0207] The above-mentioned maytansinoids can be conjugated to
anti-CD38 antibody 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, or
38SB39 or a homologue or fragment thereof, wherein the antibody is
linked to the maytansinoid using the thiol or disulfide
functionality that is present on the acyl group of an acylated
amino acid side chain found at C-3, C-14 hydroxymethyl, C-15
hydroxy or C-20 desmethyl of the maytansinoid, and wherein the acyl
group of the acylated amino acid side chain has its thiol or
disulfide functionality located at a carbon atom that has one or
two substituents, said substituents being CH.sub.3, C.sub.2H.sub.5,
linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched
or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms,
phenyl, substituted phenyl or heterocyclic aromatic or
heterocycloalkyl radical, and in addition one of the substituents
can be H, and wherein the acyl group has a linear chain length of
at least three carbon atoms between the carbonyl functionality and
the sulfur atom.
[0208] A preferred conjugate of the present invention is the one
that comprises the anti-anti-CD38 antibody 38SB13, 38SB18, 38SB19,
38SB30, 38SB31, or 38SB39 or a homologue or fragment thereof,
conjugated to a maytansinoid of formula (VIII):
##STR00008##
wherein: Y.sub.1' represents
(CR.sub.7R.sub.8).sub.l(CR.sub.9.dbd.CR.sub.10).sub.p(C.ident.C).sub.qA.s-
ub.r(CR.sub.5R.sub.6).sub.mD.sub.u(CR.sub.11.dbd.CR.sub.12).sub.r(C.ident.-
C).sub.sB.sub.t(CR.sub.3R.sub.4).sub.nCR.sub.1R.sub.2S--, wherein:
A, B, and D, each independently is cycloalkyl or cycloalkenyl
having 3-10 carbon atoms, simple or substituted aryl, or
heterocyclic aromatic or heterocycloalkyl radical; R.sub.3,
R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, R.sub.11, and
R.sub.12 are each independently H, CH.sub.3, C.sub.2H.sub.5, linear
alkyl or alkenyl having from 1 to 10 carbon atoms, branched or
cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl,
substituted phenyl or heterocyclic aromatic or heterocycloalkyl
radical; and l, m, n, o, p, q, r, s, and t are each independently 0
or an integer of from 1 to 5, provided that at least two of l, m,
n, o, p, q, r, s and t are non-not zero at any one time.
[0209] Preferably, R.sub.1 is H and R.sub.2 is methyl or R.sub.1
and R.sub.2 are methyl.
[0210] An even more preferred conjugate of the present invention is
the one that comprises the anti-CD38 antibody 38SB13, 38SB18,
38SB19, 38SB30, 38SB31, or 38SB39 or a homologue or fragment
thereof, conjugated to a maytansinoid of formula (IX-L), (IX-D), or
(IX-D,L):
##STR00009##
wherein: Y.sub.1 represents
(CR.sub.7R.sub.8).sub.l(CR.sub.5R.sub.6).sub.m(CR.sub.3R.sub.4).sub.nCR.s-
ub.1R.sub.2S--, wherein: R.sub.1 and R.sub.2 are each independently
CH.sub.3, C.sub.2H.sub.5, linear alkyl or alkenyl having from 1 to
10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3
to 10 carbon atoms, phenyl, substituted phenyl, heterocyclic
aromatic or heterocycloalkyl radical, and in addition R.sub.2 can
be H; R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7 and R.sub.8 are
each independently H, CH.sub.3, C.sub.2H.sub.5, linear alkyl or
alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl
or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted
phenyl or heterocyclic aromatic or heterocycloalkyl radical; l, m
and n are each independently an integer of from 1 to 5, and in
addition n can be 0; and May represents a maytansinol which bears
the side chain at C-3, C-14 hydroxymethyl, C-15 hydroxy or C-20
desmethyl.
[0211] Preferred embodiments of formulas (IX-L), (IX-D) and
(IX-D,L) include compounds of formulas (IX-L), (IX-D) and (IX-D,L)
wherein:
R.sub.1 is H and R.sub.2 is methyl or R.sub.1 and R.sub.2 are
methyl, [0212] R.sub.1 is H, R.sub.2 is methyl, R.sub.5, R.sub.6,
R.sub.7 and R.sub.8 are each H; l and m are each 1; n is 0, [0213]
R.sub.1 and R.sub.2 are methyl; R.sub.5, R.sub.6, R.sub.7 and
R.sub.8 are each H; l and m are 1; n is 0. Preferably the cytotoxic
agent is represented by formula (IX-L).
[0214] An further preferred conjugate of the present invention is
the one that comprises the anti-CD38 antibody 38SB13, 38SB18,
38SB19, 38SB30, 38SB31, or 38SB39 or a homologue or fragment
thereof, conjugated to a maytansinoid of formula (X):
##STR00010##
wherein the substituents are as defined for formula (IX) above.
[0215] Especially preferred are any of the above-described
compounds, wherein R.sub.1 is H, R.sub.2 is methyl, R.sub.5,
R.sub.6, R.sub.7 and R.sub.8 are each H, l and m are each 1, and n
is 0.
[0216] Further especially preferred are any of the above-described
compounds, wherein R.sub.1 and R.sub.2 are methyl, R.sub.5,
R.sub.6, R.sub.7, R.sub.8 are each H, l and m are 1, and n is 0
[0217] Further, the L-aminoacyl stereoisomer is preferred.
[0218] Each of the maytansinoids taught in pending U.S. patent
application Ser. No. 10/849,136, filed May 20, 2004, may also be
used in the cytotoxic conjugate of the present invention. The
entire disclosure of U.S. patent application Ser. No. 10/849,136 is
incorporated herein by reference.
[0219] Disulfide-Containing Linking Groups
[0220] In order to link the maytansinoid to a cell binding agent,
such as the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, or 38SB39
antibody, the maytansinoid comprises a linking moiety. The linking
moiety contains a chemical bond that allows for the release of
fully active maytansinoids at a particular site. Suitable chemical
bonds are well known in the art and include disulfide bonds, acid
labile bonds, photolabile bonds, peptidase labile bonds and
esterase labile bonds. Preferred are disulfide bonds.
[0221] The linking moiety also comprises a reactive chemical group.
In a preferred embodiment, the reactive chemical group can be
covalently bound to the maytansinoid via a disulfide bond linking
moiety.
[0222] Particularly preferred reactive chemical groups are
N-succinimidyl esters and N-sulfosuccinimidyl esters.
[0223] Particularly preferred maytansinoids comprising a linking
moiety that contains a reactive chemical group are C-3 esters of
maytansinol and its analogs where the linking moiety contains a
disulfide bond and the chemical reactive group comprises a
N-succinimidyl or N-sulfosuccinimidyl ester.
[0224] Many positions on maytansinoids can serve as the position to
chemically link the linking moiety. For example, the C-3 position
having a hydroxyl group, the C-14 position modified with
hydroxymethyl, the C-15 position modified with hydroxy and the C-20
position having a hydroxy group are all expected to be useful.
However the C-3 position is preferred and the C-3 position of
maytansinol is especially preferred.
[0225] While the synthesis of esters of maytansinol having a
linking moiety is described in terms of disulfide bond-containing
linking moieties, one of skill in the art will understand that
linking moieties with other chemical bonds (as described above) can
also be used with the present invention, as can other
maytansinoids. Specific examples of other chemical bonds include
acid labile bonds, photolabile bonds, peptidase labile bonds and
esterase labile bonds. The disclosure of U.S. Pat. No. 5,208,020,
incorporated herein, teaches the production of maytansinoids
bearing such bonds.
[0226] The synthesis of maytansinoids and maytansinoid derivatives
having a disulfide moiety that bears a reactive group is described
in U.S. Pat. Nos. 6,441,163 and 6,333,410, and U.S. application
Ser. No. 10/161,651, each of which is herein incorporated by
reference.
[0227] The reactive group-containing maytansinoids, such as DM1,
are reacted with an antibody, such as the 38SB13, 38SB18, 38SB19,
38SB30, 38SB31, or 38SB39 antibody, to produce cytotoxic
conjugates. These conjugates may be purified by HPLC or by
gel-filtration.
[0228] Several excellent schemes for producing such
antibody-maytansinoid conjugates are provided in U.S. Pat. No.
6,333,410, and U.S. application Ser. Nos. 09/867,598, 10/161,651
and 10/024,290, each of which is incorporated herein in its
entirety.
[0229] In general, a solution of an antibody in aqueous buffer may
be incubated with a molar excess of maytansinoids having a
disulfide moiety that bears a reactive group. The reaction mixture
can be quenched by addition of excess amine (such as ethanolamine,
taurine, etc.). The maytansinoid-antibody conjugate may then be
purified by gel-filtration.
[0230] The number of maytansinoid molecules bound per antibody
molecule can be determined by measuring spectrophotometrically the
ratio of the absorbance at 252 nm and 280 nm. An average of 1-10
maytansinoid molecules/antibody molecule is preferred.
[0231] Conjugates of antibodies with maytansinoid drugs can be
evaluated for their ability to suppress proliferation of various
unwanted cell lines in vitro. For example, cell lines such as the
human lymphoma cell line Daudi, the human lymphoma cell line Ramos,
the human multiple myeloma cell line MOLP-8, and the human T acute
lymphocytic leukemia line MOLT-4 can easily be used for the
assessment of cytotoxicity of these compounds. Cells to be
evaluated can be exposed to the compounds for 24 hours and the
surviving fractions of cells measured in direct assays by known
methods. IC50 values can then be calculated from the results of the
assays.
[0232] Peg-Containing Linking Groups
[0233] Maytansinoids may also be linked to cell binding agents
using PEG linking groups, as set forth in U.S. application Ser. No.
10/024,290. These PEG linking groups are soluble both in water and
in non-aqueous solvents, and can be used to join one or more
cytotoxic agents to a cell binding agent. Exemplary PEG linking
groups include hetero-bifunctional PEG linkers that bind to
cytotoxic agents and cell binding agents at opposite ends of the
linkers through a functional sulfhydryl or disulfide group at one
end, and an active ester at the other end.
[0234] As a general example of the synthesis of a cytotoxic
conjugate using a PEG linking group, reference is again made to
U.S. application Ser. No. 10/024,290 for specific details.
Synthesis begins with the reaction of one or more cytotoxic agents
bearing a reactive PEG moiety with a cell-binding agent, resulting
in displacement of the terminal active ester of each reactive PEG
moiety by an amino acid residue of the cell binding agent, to yield
a cytotoxic conjugate comprising one or more cytotoxic agents
covalently bonded to a cell binding agent through a PEG linking
group.
[0235] Taxanes
[0236] The cytotoxic agent used in the cytotoxic conjugates
according to the present invention may also be a taxane or
derivative thereof.
[0237] Taxanes are a family of compounds that includes paclitaxel
(Taxol), a cytotoxic natural product, and docetaxel (Taxotere), a
semi-synthetic derivative, two compounds that are widely used in
the treatment of cancer. Taxanes are mitotic spindle poisons that
inhibit the depolymerization of tubulin, resulting in cell death.
While docetaxel and paclitaxel are useful agents in the treatment
of cancer, their antitumor activity is limited because of their
non-specific toxicity towards normal cells. Further, compounds like
paclitaxel and docetaxel themselves are not sufficiently potent to
be used in conjugates of cell binding agents.
[0238] A preferred taxane for use in the preparation of cytotoxic
conjugates is the taxane of formula (XI):
##STR00011##
[0239] Methods for synthesizing taxanes that may be used in the
cytotoxic conjugates of the present invention, along with methods
for conjugating the taxanes to cell binding agents such as
antibodies, are described in detail in U.S. Pat. Nos. 5,416,064,
5,475,092, 6,340,701, 6,372,738 and 6,436,931, and in U.S.
application Ser. Nos. 10/024,290, 10/144,042, 10/207,814,
10/210,112 and 10/369,563.
[0240] Tomaymycin Derivatives
[0241] The cytotoxic according to the present invention may also a
tomaymycin derivative. Tomaymycin derivatives are
pyrrolo[1,4]benzodiazepines (PBDs), a known class of compounds
exerting their biological properties by covalently binding to the
N2 of guanine in the minor groove of DNA. PBDs include a number of
minor groove binders such as anthramycin, neothramycin and
DC-81.
[0242] Novel tomaymycin derivatives that retain high cytotoxicity
and that can be effectively linked to cell binding agents are
described in the International Application No. PCT/IB2007/000142,
whose content is herein incorporated by reference. The cell binding
agent-tomaymycin derivative complexes permit the full measure of
the cytotoxic action of the tomaymycin derivatives to be applied in
a targeted fashion against unwanted cells only, therefore avoiding
side effects due to damage to non-targeted healthy cells.
[0243] The cytotoxic agent according to the present invention
comprises one or more tomaymycin derivatives, linked to a cell
binding agent, such as the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31,
or 38SB39 antibody, via a linking group. The linking group is part
of a chemical moiety that is covalently bound to a tomaymycin
derivative through conventional methods. In a preferred embodiment,
the chemical moiety can be covalently bound to the tomaymycin
derivative via a disulfide bond.
[0244] The tomaymycin derivatives useful in the present invention
have the formula (XII) shown below:
##STR00012##
wherein - - - represents an optional single bond; represents either
a single bond or a double bond; provided that when represents a
single bond, U and U', the same or different, independently
represent H, and W and W', the same or different, are independently
selected from the group consisting of OH, an ether such as --OR, an
ester (e.g. an acetate), such as --OCOR, a carbonate such as
--OCOOR, a carbamate such as --OCONRR', a cyclic carbamate, such
that N10 and C11 are a part of the cycle, a urea such as
--NRCONRR', a thiocarbamate such as --OCSNHR, a cyclic
thiocarbamate such that N10 and C11 are a part of the cycle, --SH,
a sulfide such as --SR, a sulphoxide such as --SOR, a sulfone such
as --SOOR, a sulphonate such as --SO3-, a sulfonamide such as
--NRSOOR, an amine such as --NRR', optionally cyclic amine such
that N10 and C11 are a part of the cycle, a hydroxylamine
derivative such as --NROR', an amide such as --NRCOR, an azido such
as --N3, a cyano, a halo, a trialkyl or triarylphosphonium, an
aminoacid-derived group; Preferably W and W' are the same or
different and are OH, Ome, Oet, NHCONH.sub.2, SMe; and when
represents a double bond, U and U' are absent and W and W'
represent H; [0245] R1, R2, R1', R2' are the same or different and
independently chosen from Halide or Alkyl optionally substituted by
one or more Hal, CN, NRR', CF.sub.3, OR, Aryl, Het, S(O).sub.qR, or
R1 and R2 and R1' and R2' form together a double bond containing
group .dbd.B and .dbd.B' respectively.
[0246] Preferably, R1 and R2 and R1' and R2' form together a double
bond containing group .dbd.B and .dbd.B' respectively. [0247] B and
B' are the same or different and independently chosen from Alkenyl
being optionally substituted by one or more Hal, CN, NRR',
CF.sub.3, OR, Aryl, Het, S(O).sub.qR or B and B' represent an
oxygen atom.
[0248] Preferably, B.dbd.B'.
[0249] More preferably, B.dbd.B'.dbd..dbd.CH.sub.2 or
.dbd.CH--CH.sub.3, [0250] X, X' are the same or different and
independently chosen from one or more --O--, --NR--, --(C.dbd.O)--,
--S(O).sub.q--.
[0251] Preferably, X.dbd.X'.
[0252] More preferably, X.dbd.X'.dbd.O. [0253] A, A' are the same
or different and independently chosen from Alkyl or Alkenyl
optionally containing an oxygen, a nitrogen or a sulfur atom, each
being optionally substituted by one or more Hal, CN, NRR',
CF.sub.3, OR, S(O).sub.qR, Aryl, Het, Alkyl, Alkenyl.
[0254] Preferably, A=A'.
[0255] More preferably, A=A'=linear unsubstituted alkyl. [0256] Y,
Y' are the same or different and independently chosen from H,
OR;
Preferably, Y.dbd.Y'.
[0257] More preferably, Y.dbd.Y'.dbd.OAlkyl, more preferably
OMethyl. [0258] T is --NR--, --O--, --S(O).sub.q--, or a 4 to
10-membered aryl, cycloalkyl, heterocyclic or heteroaryl, each
being optionally substituted by one or more Hal, CN, NRR',
CF.sub.3, R, OR, S(O).sub.qR, and/or linker(s), or a branched
Alkyl, optionally substituted by one or more Hal, CN, NRR',
CF.sub.3, OR, S(O).sub.qR and/or linker(s), or a linear Alkyl
substituted by one or more Hal, CN, NRR', CF.sub.3, OR, S(O).sub.qR
and/or linker(s).
[0259] Preferably, T is a 4 to 10-membered aryl or heteroaryl, more
preferably phenyl or pyridyl, optionally substituted by one or more
linker(s).
[0260] Said linker comprises a linking group. Suitable linking
groups are well known in the art and include thiol, sulfide,
disulfide groups, thioether groups, acid labile groups, photolabile
groups, peptidase labile groups and esterase labile groups.
Preferred are disulfide groups and thioether groups.
[0261] When the linking group is a thiol-, sulfide (or so-called
thioether --S--) or disulfide (--S--S--)-containing group, the side
chain carrying the thiol, the sulfide or disulfide group can be
linear or branched, aromatic or heterocyclic. One of ordinary skill
in the art can readily identify suitable side chains.
[0262] Preferably, said linker is of formula:
-G-D-(Z)p-S--Z'
where G is a single or double bond, --O--, --S-- or --NR--; D is a
single bond or -E-, -E-NR--, -E-NR--F--, -E-O--, -E-O--F--,
-E-NR--CO--, -E-NR--CO--F--, -E-CO--, --CO-E-, -E-CO--F, -E-S--,
-E-S--F--, -E-NR--C--S--, -E-NR--CS--F--; where E and F are the
same or different and are independently chosen from linear or
branched --(OCH2CH2)iAlkyl(OCH2CH2)j-, -Alkyl(OCH2CH2)i-Alkyl-,
--(OCH2CH2)i-, --(OCH2CH2)iCycloalkyl(OCH2CH2)j-,
--(OCH2CH2)iHeterocyclic(OCH2CH2)j-, --(OCH2CH2)iAryl(OCH2CH2)j-,
--(OCH2CH2)iHeteroaryl(OCH2CH2)j-,
-Alkyl-(OCH2CH2)iAlkyl(OCH2CH2)j-, -Alkyl-(OCH2CH2)i-,
-Alkyl-(OCH2CH2)iCycloalkyl(OCH2CH2)j-,
-Alkyl(OCH2CH2)iHeterocyclic(OCH2CH2)j-,
-Alkyl-(OCH2CH2)iAryl(OCH2CH2)j-,
-Alkyl(OCH2CH2)iHeteroaryl(OCH2CH2)j-, -Cycloalkyl-Alkyl-,
-Alkyl-Cycloalkyl-, -Heterocyclic-Alkyl-, -Alkyl-Heterocyclic-,
-Alkyl-Aryl-, -Aryl-Alkyl-, -Alkyl-Heteroaryl-, -Heteroaryl-Alkyl-;
where i and j, identical or different are integers and
independently chosen from 0, 1 to 2000; Z is linear or branched
-Alkyl-; p is 0 or 1; Z' represents H, a thiol protecting group
such as COR, R20 or SR20, wherein R20 represents H, methyl, Alkyl,
optionally substituted Cycloalkyl, aryl, heteroaryl or
heterocyclic, provided that when Z' is H, said compound is in
equilibrium with the corresponding compound formed by
intramolecular cyclisation resulting from addition of the thiol
group --SH on the imine bond --NH.dbd. of one of the PBD moieties.
[0263] n, n', equal or different are 0 or 1. [0264] q is 0, 1 or 2.
[0265] R, R' are equal or different and independently chosen from
H, Alkyl, Aryl, each being optionally substituted by Hal, CN, NRR',
CF3, R, OR, S(O).sub.qR, Aryl, Het; or their pharmaceutically
acceptable salts, hydrates, or hydrated salts, or the polymorphic
crystalline structures of these compounds or their optical isomers,
racemates, diastereomers or enantiomers.
[0266] The compounds of the general formula (XII) having
geometrical and stereoisomers are also a part of the invention.
[0267] The N-10, C-11 double bond of tomaymycin derivatives of
formula (XII) is known to be readily convertible in a reversible
manner to corresponding imine adducts in the presence of water, an
alcohol, a thiol, a primary or secondary amine, urea and other
nucleophiles. This process is reversible and can easily regenerate
the corresponding tomaymycin derivatives in the presence of a
dehydrating agent, in a non-protic organic solvent, in vacuum or at
high temperatures (Z. Tozuka, 1983, J. Antibiotics, 36: 276).
[0268] Thus, reversible derivatives of tomaymycin derivatives of
general formula (XIII) can also be used in the present
invention:
##STR00013##
where A, X, Y, n, T, A', X', Y', n', R1, R2, R1, R2' are defined as
in formula (XII) and W, W' are the same or different and are
selected from the group consisting of OH, an ether such as --OR, an
ester (e.g. an acetate), such as --OCOR, --COOR, a carbonate such
as --OCOOR, a carbamate such as --OCONRR', a cyclic carbamate, such
that N10 and C11 are a part of the cycle, a urea such as
--NRCONRR', a thiocarbamate such as --OCSNHR, a cyclic
thiocarbamate such that N10 and C11 are a part of the cycle, --SH,
a sulfide such as --SR, a sulphoxide such as --SOR, a sulfone such
as --SOOR, a sulphonate such as --SO3-, a sulfonamide such as
--NRSOOR, an amine such as --NRR', optionally cyclic amine such
that N10 and C11 are a part of the cycle, a hydroxylamine
derivative such as --NROR', an amide such as --NRCOR, --NRCONRR',
an azido such as --N3, a cyano, a halo, a trialkyl or
triarylphosphonium, an aminoacid-derived group. Preferably, W and
W' are the same or different and are OH, Ome, Oet, NHCONH2,
SMe.
[0269] Compounds of formula (XIII) may thus be considered as
solvates, including water when the solvent is water; these solvates
can be particularly useful.
[0270] In a preferred embodiment, the tomaymycin derivatives of the
invention are selected from the group consisting in: [0271]
8,8'-[1,3-benzenediylbis(methyleneoxy)]-bis[(S)-2-eth-(E)-ylidene-7-metho-
xy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]
[0272]
8,8'-[5-methoxy-1,3-benzenediylbis(methyleneoxy)]-bis[(S)-2-eth-(E)-ylide-
ne-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-on-
e] [0273]
8,8'-[1,5-pentanediylbis(oxy)]-bis[(S)-2-eth-(E)-ylidene-7-metho-
xy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]
[0274]
8,8'-[1,4-butanediylbis(oxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,1-
1a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one] [0275]
8,8'-[3-methyl-1,5-pentanediylbis(oxy)]-bis[(S)-2-eth-(E)-ylidene-7-metho-
xy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]
[0276]
8,8'-[2,6-pyridinediylbis(oxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3-
,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one] [0277]
8,8'-[4-(3-tert-butoxycarbonylaminopropyloxy)-2,6-pyridinediylbis-(methyl-
eneoxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrro-
lo[2,1-c][1,4]benzodiazepin-5-one] [0278]
8,8'-[5-(3-aminopropyloxy)-1,3-benzenediylbis(methyleneoxy)]-bis[(S)-2-et-
h-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodi-
azepin-5-one] [0279] 8,
8'-[5-(N-methyl-3-tert-butoxycarbonylaminopropyl)-1,3-benzenediylbis-(met-
hyleneoxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-py-
rrolo[2,1-c][1,4]benzodiazepin-5-one] [0280]
8,8'-{5-[3-(4-methyl-4-methyldisulfanyl-pentanoylamino)propyloxy]-1,3-ben-
zenediylbis(methyleneoxy)}-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-t-
etrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one] [0281]
8,8'-[5-acetylthiomethyl-1,3-benzenediylbis(methyleneoxy)]-bis[(S)-2-meth-
ylene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-
-one] [0282]
bis-{2-[(S)-2-methylene-7-methoxy-5-oxo-1,3,,11a-tetrahydro-5H-pyrrolo[2,-
1-c][1,4]benzodiazepin-8-yloxy]-ethyl}-carbamic acid tert-butyl
ester [0283]
8,8'-[3-(2-acetylthioethyl)-1,5-pentanediylbis(oxy)]-bis[(S)-2-met-
hylene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin--
5-one] [0284]
8,8'-[5-(N-4-mercapto-4,4-dimethylbutanoyl)amino-1,3-benzenediylbis(methy-
leneoxy)]-bis[7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c]-
[1,4]benzodiazepin-5-one] [0285]
8,8'-[5-(N-4-methyldithio-4,4-dimethylbutanoyl)-amino-1,3-benzenediylbis(-
methyleneoxy)]-bis[7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2-
,1-c][1,4]benzodiazepin-5-one] [0286]
8,8'-[5-(N-methyl-N-(2-mercapto-2,2-dimethylethyl)amino-1,3-benzenediyl(m-
ethyleneoxy)]-bis[7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,-
1-c][1,4]benzodiazepin-5-one] [0287]
8,8'-[5-(N-methyl-N-(2-methyldithio-2,2-dimethylethyl)amino-1,3-benzenedi-
yl(methyleneoxy)]-bis[7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrol-
o[2,1-c][1,4]benzodiazepin-5-one] [0288]
8,8'-[(4-(2-(4-mercapto-4-methyl)-pentanamido-ethoxy)-pyridin-2,6-dimethy-
l)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrro-
lo[2,1-c][1,4]benzodiazepin-5-one] [0289]
8,8'-[(1-(2-(4-methyl-4-methyldisulfanyl)-pentanamido-ethoxy)-benzene-3,5-
-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahyd-
ro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one] [0290]
8,8'-[(4-(3-(4-methyl-4-methyldisulfanyl)-pentanamido-propoxy)-pyridin-2,-
6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahy-
dro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one] [0291]
8,8'-[(4-(4-(4-methyl-4-methyldisulfanyl)-pentanamido-butoxy)-pyridin-2,6-
-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahyd-
ro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one] [0292]
8,8'-[(4-(3-[4-(4-methyl-4-methyldisulfanyl-pentanoyl)-piperazin-1-yl]-pr-
opyl)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1-
,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one] [0293]
8,8'-[(1-(3-[4-(4-methyl-4-methyldisulfanyl-pentanoyl)-piperazin-1-yl]-pr-
opyl)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1-
,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one] [0294]
8,8'-[(4-(2-{2-[2-(4-methyl-4-methyldisulfanyl-pentanoylamino)-ethoxy]-et-
hoxy}-ethoxy)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dim-
ethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]
[0295]
8,8'-[(1-(2-{2-[2-(2-{2-[2-(4-methyl-4-methyldisulfanyl-pentanoylamino)-e-
thoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-benzene-3,5-dimethyl)-dioxy-
]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c-
][1,4]benzodiazepin-5-one] [0296]
8,8'-[(1-(2-{2-[2-(4-methyl-4-methyldisulfanyl-pentanoylamino)-ethoxy]-et-
hoxy}-ethoxy)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dim-
ethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]
[0297]
8,8'-[(4-(2-{2-[2-(2-{2-[2-(4-methyl-4-methyldisulfanyl-pentanoylamino)-e-
thoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-pyridin-2,6-dimethyl)-dioxy-
]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c-
][1,4]benzodiazepin-5-one] [0298]
8,8'-[(1-(2-[methyl-(2-methyl-2-methyldisulfanyl-propyl)-amino]-ethoxy)-b-
enzene-3,5-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11-
a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one] [0299]
8,8'-[(4-(3-[methyl-(4-methyl-4-methyldisulfanyl-pentanoyl)-amino]-propyl-
)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3-
,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one] [0300]
8,8'-[(4-(3-[methyl-(2-methyl-2-methyldisulfanyl-propyl)-amino]-propyl)-p-
yridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11-
a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one] [0301]
8,8'-[(1-(4-methyl-4-methyldisulfanyl)-pentanamido)-benzene-3,5-dimethyl)-
-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo-
[2,1-c][1,4]benzodiazepin-5-one] as well as the corresponding
mercapto derivatives, or their pharmaceutically acceptable salts,
hydrates, or hydrated salts, or the polymorphic crystalline
structures of these compounds or their optical isomers, racemates,
diastereomers or enantiomers.
[0302] Preferred compounds are those of formula:
##STR00014##
where X, X', A, A', Y, Y', T, n, n' are defined as above.
[0303] The compounds of formula (XII) may be prepared in a number
of ways well known to those skilled in the art. The compounds can
be synthesized, for example, by application or adaptation of the
methods described below, or variations thereon as appreciated by
the skilled artisan. The appropriate modifications and
substitutions will be readily apparent and well known or readily
obtainable from the scientific literature to those skilled in the
art. In particular, such methods can be found in R.C. Larock,
Comprehensive Organic Transformations, Wiley-VCH Publishers,
1999.
[0304] Methods for synthesizing the tomaymycin derivatives which
may be used in the invention are described in the International
Application No. PCT/IB2007/000142. Compounds of the present
invention may be prepared by a variety of synthetic routes. The
reagents and starting materials are commercially available, or
readily synthesized by well-known techniques by one of ordinary
skill in the arts (see, for example, WO 00/12508, WO 00/12507, WO
2005/040170, WO 2005/085260, FR1516743, M. Mori et al., 1986,
Tetrahedron, 42: 3793-3806).
[0305] The conjugate molecules of the invention may be formed using
any techniques. The tomaymycin derivatives of the invention may be
linked to an antibody or other cell binding agent via an acid
labile linker, or by a photolabile linker. The derivatives can be
condensed with a peptide having a suitable sequence and
subsequently linked to a cell binding agent to produce a peptidase
labile linker. The conjugates can be prepared to contain a primary
hydroxyl group, which can be succinylated and linked to a cell
binding agent to produce a conjugate that can be cleaved by
intracellular esterases to liberate free derivative. Preferably,
the derivatives are synthesized to contain a free or protected
thiol group, and then one or more disulfide or thiol-containing
derivatives are each covalently linked to the cell binding agent
via a disulfide bond or a thioether link.
[0306] Numerous methods of conjugation are taught in U.S. Pat. No.
5,416,064 and U.S. Pat. No. 5,475,092. The tomaymycin derivatives
can be modified to yield a free amino group and then linked to an
antibody or other cell binding agent via an acid labile linker or a
photolabile linker. The tomaymycin derivatives with a free amino or
carboxyl group can be condensed with a peptide and subsequently
linked to a cell binding agent to produce a peptidase labile
linker. The tomaymycin derivatives with a free hydroxyl group on
the linker can be succinylated and linked to a cell binding agent
to produce a conjugate that can be cleaved by intracellular
esterases to liberate free drug. Most preferably, the tomaymycin
derivatives are treated to create a free or protected thiol group,
and then the disulfide- or thiol containing tomaymycin dimers are
linked to the cell binding agent via disulfide bonds.
[0307] Preferably, monoclonal antibody- or cell binding
agent-tomaymycin derivative conjugates are those that are joined
via a disulfide bond, as discussed above, that are capable of
delivering tomaymycin derivatives. Such cell binding conjugates are
prepared by known methods such as by modifying monoclonal
antibodies with succinimidyl pyridyl-dithiopropionate (SPDP)
(Carlsson et al., 1978, Biochem. J., 173: 723-737). The resulting
thiopyridyl group is then displaced by treatment with
thiol-containing tomaymycin derivatives to produce disulfide linked
conjugates. Alternatively, in the case of the aryldithio-tomaymycin
derivatives, the formation of the cell binding conjugate is
effected by direct displacement of the aryl-thiol of the tomaymycin
derivative by sulfhydryl groups previously introduced into antibody
molecules. Conjugates containing 1 to 10 tomaymycin derivative
drugs linked via a disulfide bridge are readily prepared by either
method.
[0308] More specifically, a solution of the dithio-nitropyridyl
modified antibody at a concentration of 2.5 mg/ml in 0.05 M
potassium phosphate buffer, at pH 7.5 containing 2 mM EDTA is
treated with the thiol-containing tomaymycin derivative (1.3 molar
eq./dithiopyridyl group). The release of thio-nitropyridine from
the modified antibody is monitored spectrophotometrically at 325 nm
and is complete in about 16 hours. The antibody-tomaymycin
derivative conjugate is purified and freed of unreacted drug and
other low molecular weight material by gel filtration through a
column of Sephadex G-25 or Sephacryl S300. The number of tomaymycin
derivative moieties bound per antibody molecule can be determined
by measuring the ratio of the absorbance at 230 nm and 275 nm. An
average of 1-10 tomaymycin derivative molecules/antibody molecule
can be linked via disulfide bonds by this method.
[0309] The effect of conjugation on binding affinity towards the
antigen-expressing cells can be determined using the methods
previously described by Liu et al., 1996, Proc. Natl. Acad. Sci.
U.S.A., 93: 8618-8623. Cytotoxicity of the tomaymycin derivatives
and their antibody conjugates to cell lines can be measured by
back-extrapolation of cell proliferation curves as described in
Goldmacher et al., 1985, J. Immunol., 135: 3648-3651. Cytotoxicity
of these compounds to adherent cell lines can be determined by
clonogenic assays as described in Goldmacher et al., 1986, J. Cell
Biol., 102: 1312-1319.
[0310] Leptomycin Derivatives
[0311] The cytotoxic according to the present invention may also a
leptomycin derivative. According to the present invention,
"leptomycin derivatives" refer to members of the leptomycin family
as defined in Kalesse et al. (2002, Synthesis 8: 981-1003), and
includes: leptomycins, such as leptomycin A and leptomycin B,
callystatins, ratjadones such as ratjadone A and ratjadone B,
anguinomycins such as anguinomycin A, B, C, D, kasusamycins,
leptolstatin, leptofuranins, such as leptofuranin A, B, C, D.
Derivatives of leptomycin A and B are preferred.
[0312] More specifically, the derivatives of the invention are of
formula (I):
##STR00015##
wherein Ra and Ra' are H or -Alk; preferably Ra is -Alk, preferably
methyl and Ra' is H; R17 is alkyl optionally substituted by OR, CN,
NRR', perfluoroalkyl; preferably, R17 is alkyl, more preferably
methyl or ethyl; R9 is alkyl optionally substituted by OR, CN,
NRR', perfluoroalkyl; preferably, R9 is alkyl, more preferably
methyl; X is --O-- or --NR--; preferably, X is --NR--;
Y is --U--, --NR--U--, --O--U--, --NR--CO--U--, --U--NR--CO--,
--U--CO--, --CO--U--;
[0313] preferably, when X is --O--, Y is --U--, --NR--U--,
--U--NR--CO--; where U is chosen from linear or branched -Alk-,
-Alk(OCH.sub.2CH.sub.2).sub.m--, --(OCH.sub.2CH.sub.2).sub.m-Alk-,
-Alk(OCH.sub.2CH.sub.2).sub.m-Alk-, --(OCH.sub.2CH.sub.2).sub.m--,
-Cycloalkyl-, -Heterocyclic-, -Cycloalkyl-Alk-, -Alk-Cycloalkyl-,
-Heterocyclic-Alk-, -Alk-Heterocyclic-; where m is an integer
chosen from 1 to 2000; preferably, U is linear or branched
-Alk-,
Z is -Alk-;
[0314] n is 0 or 1; preferably n is 0; T represents H, a thiol
protecting group such as Ac, R.sub.1 or SR.sub.1, wherein R.sub.1
represents H, methyl, Alk, Cycloalkyl, optionally substituted aryl
or heterocyclic, or T represents
##STR00016##
where: Ra, Ra', R17, R9, X, Y, Z, n are defined as above;
preferably, T is H or SR.sub.1, wherein R.sub.1 represents Alk,
more preferably methyl; R, R' identical or different are H or
alkyl; Alk represents a linear or branched alkyl; preferably Alk
represents (--(CH.sub.2-q (CH.sub.3).sub.q).sub.p where p
represents an integer from 1 to 10; and q represents an integer
from 0 to 2; preferably, Alk represents --(CH.sub.2)-- ou
--C(CH.sub.3).sub.2--. or their pharmaceutically acceptable salts,
hydrates, or hydrated salts, or the polymorphic crystalline
structures of these compounds or their optical isomers, racemates,
diastereomers or enantiomers.
[0315] Preferred compounds may be chosen from:
(2-Methylsulfanyl-ethyl)-amid of
(2E,10E,12E,16Z,18E)-(R)-6-Hydroxy-3,5,
7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran--
2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoic acid
Bis-[(2-mercaptoethyl)-amid of (2E,10
E,12E,16Z,18E)-(R)-6-hydroxy-3,5,
7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran--
2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoic acid]
(2-Mercapto-ethyl)-amid of
(2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptamethyl-19-((2S,3-
S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18--
pentaenoic acid (2-Methyldisulfanyl-ethyl)-amid of
(2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,
7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran--
2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoic acid
(2-Methyl-2-methyldisulfanyl-propyl)amid of
(2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptamethyl-19-((2S,3-
S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18--
pentaenoic acid (2-Mercapto-2-methyl-propyl)-amid of
(2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptamethyl-19-((2S,3-
S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18--
pentaenoic acid or their pharmaceutically acceptable salts,
hydrates, or hydrated salts, or the polymorphic crystalline
structures of these compounds or their optical isomers, racemates,
diastereomers or enantiomers.
[0316] In order to link the derivative to a cell-binding agent, the
derivative must include a moiety (linking group) that allows the
derivatives to be linked to a cell binding agent via a linkage such
as a disulfide bond, a sulfide (or called herein thioether) bond,
an acid-labile group, a photo-labile group, a peptidase-labile
group, or an esterase-labile group. The derivatives are prepared so
that they contain a moiety necessary to link the leptomycin
derivative to a cell binding agent via, for example, a disulfide
bond, a thioether bond, an acid-labile group, a photo-labile group,
a peptidase-labile group, or an esterase-labile group. In order to
further enhance solubility in aqueous solutions, the linking group
can contain a polyethylene glycol spacer. Preferably, a sulfide or
disulfide linkage is used because the reducing environment of the
targeted cell results in cleavage of the sulfide or disulfide and
release of the derivatives with an associated increase in
cytotoxicity.
[0317] Compounds of the present invention may be prepared by a
variety of synthetic routes. The reagents and starting materials
are commercially available, or readily synthesized by well-known
techniques by one of ordinary skill in the art. Methods for
synthesizing leptomycin derivatives that may be used in the
cytotoxic conjugates of the present invention, along with methods
for conjugating said leptomycin derivatives to cell binding agents
such as antibodies, are described in detail in European Patent
Application No. 06290948.6, whose content is incorporated herein by
reference.
[0318] CC-1065 Analogues
[0319] The cytotoxic agent used in the cytotoxic conjugates
according to the present invention may also be CC-1065 or a
derivative thereof.
[0320] CC-1065 is a potent anti-tumor antibiotic isolated from the
culture broth of Streptomyces zelensis. CC-1065 is about 1000-fold
more potent in vitro than are commonly used anti-cancer drugs, such
as doxorubicin, methotrexate and vincristine (B. K. Bhuyan et al.,
1982, Cancer Res., 42, 3532-3537). CC-1065 and its analogs are
disclosed in U.S. Pat. Nos. 6,372,738, 6,340,701, 5,846,545 and
5,585,499.
[0321] The cytotoxic potency of CC-1065 has been correlated with
its alkylating activity and its DNA-binding or DNA-intercalating
activity. These two activities reside in separate parts of the
molecule. Thus, the alkylating activity is contained in the
cyclopropapyrroloindole (CPI) subunit and the DNA-binding activity
resides in the two pyrroloindole subunits.
[0322] Although CC-1065 has certain attractive features as a
cytotoxic agent, it has limitations in therapeutic use.
Administration of CC-1065 to mice caused a delayed hepatotoxicity
leading to mortality on day 50 after a single intravenous dose of
12.5 .mu.g/kg (V. L. Reynolds et al., 1986, J. Antibiotics, XXIX:
319-334). This has spurred efforts to develop analogs that do not
cause delayed toxicity, and the synthesis of simpler analogs
modeled on CC-1065 has been described (M. A. Warpehoski et al.,
1988, J. Med. Chem., 31: 590-603).
[0323] In another series of analogs, the CPI moiety was replaced by
a cyclopropabenzindole (CBI) moiety (D. L. Boger et al., 1990, J.
Org. Chem., 55: 5823-5833; D. L. Boger et al., 1991, BioOrg. Med.
Chem. Lett., 1: 115-120). These compounds maintain the high in
vitro potency of the parental drug, without causing delayed
toxicity in mice. Like CC-1065, these compounds are alkylating
agents that bind to the minor groove of DNA in a covalent manner to
cause cell death. However, clinical evaluation of the most
promising analogs, Adozelesin and Carzelesin, has led to
disappointing results (B. F. Foster et al., 1996, Investigational
New Drugs, 13: 321-326; I. Wolff et al., 1996, Clin. Cancer Res.,
2: 1717-1723). These drugs display poor therapeutic effects because
of their high systemic toxicity.
[0324] The therapeutic efficacy of CC-1065 analogs can be greatly
improved by changing the in vivo distribution through targeted
delivery to the tumor site, resulting in lower toxicity to
non-targeted tissues, and thus, lower systemic toxicity. In order
to achieve this goal, conjugates of analogs and derivatives of
CC-1065 with cell-binding agents that specifically target tumor
cells have been described (U.S. Pat. Nos. 5,475,092; 5,585,499;
5,846,545). These conjugates typically display high target-specific
cytotoxicity in vitro, and exceptional anti-tumor activity in human
tumor xenograft models in mice (R. V. J. Chari et al., 1995, Cancer
Res., 55: 4079-4084).
[0325] Recently, prodrugs of CC-1065 analogs with enhanced
solubility in aqueous medium have been described (European Patent
Application No. 06290379.4). In these prodrugs, the phenolic group
of the alkylating portion of the molecule is protected with a
functionality that renders the drug stable upon storage in acidic
aqueous solution, and confers increased water solubility to the
drug compared to an unprotected analog. The protecting group is
readily cleaved in vivo at physiological pH to give the
corresponding active drug. In the prodrugs described in EP
06290379.4, the phenolic substituent is protected as a sulfonic
acid containing phenyl carbamate which possesses a charge at
physiological pH, and thus has enhanced water solubility. In order
to further enhance water solubility, an optional polyethylene
glycol spacer can be introduced into the linker between the indolyl
subunit and the cleavable linkage such as a disulfide group. The
introduction of this spacer does not alter the potency of the
drug.
[0326] Methods for synthesizing CC-1065 analogs that may be used in
the cytotoxic conjugates of the present invention, along with
methods for conjugating the analogs to cell binding agents such as
antibodies, are described in detail in EP 06290379.4 (whose content
is incorporated herein by reference) and U.S. Pat. Nos. 5,475,092,
5,846,545, 5,585,499, 6,534,660 and 6,586,618 and in U.S.
application Ser. Nos. 10/116,053 and 10/265,452.
[0327] Other Drugs
[0328] Drugs such as methotrexate, daunorubicin, doxorubicin,
vincristine, vinblastine, melphalan, mitomycin C, chlorambucil,
calicheamicin, tubulysin and tubulysin analogs, duocarmycin and
duocarmycin analogs, dolastatin and dolastatin analogs are also
suitable for the preparation of conjugates of the present
invention. The drug molecules can also be linked to the antibody
molecules through an intermediary carrier molecule such as serum
albumin. Doxarubicin and Danorubicin compounds, as described, for
example, in U.S. Ser. No. 09/740,991, may also be useful cytotoxic
agents.
[0329] Therapeutic Composition
[0330] The invention also relates to a therapeutic composition for
the treatment of a hyperproliferative disorder or inflammatory
disease or an autoimmune disease in a mammal which comprises a
therapeutically effective amount of a compound of the invention and
a pharmaceutically acceptable carrier. In one embodiment said
pharmaceutical composition is for the treatment of cancer,
including (but not limited to) the following: carcinoma, including
that of the bladder, breast, colon, kidney, liver, lung, ovary,
pancreas, stomach, cervix, thyroid and skin; including squamous
cell carcinoma; hematopoietic tumors of lymphoid lineage, including
leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia,
B-cell lymphoma, T-cell lymphoma, Burkitt's lymphoma; hematopoietic
tumors of myeloid lineage, including acute and chronic myelogenous
leukemias and promyelocytic leukemia; tumors of mesenchymal origin,
including fibrosarcoma and rhabdomyoscarcoma; other tumors,
including melanoma, seminoma, tetratocarcinoma, neuroblastoma and
glioma; tumors of the central and peripheral nervous system,
including astrocytoma, neuroblastoma, glioma, and schwannomas;
tumors of mesenchymal origin, including fibrosarcoma,
rhabdomyoscarama, and osteosarcoma; and other tumors, including
melanoma, xeroderma pigmentosum, keratoactanthoma, seminoma,
thyroid follicular cancer and teratocarcinoma, and other cancers
yet to be determined in which CD38 is expressed predominantly. In a
preferred embodiment, the pharmaceutical compositions of the
invention are used for the treatment of a cancer such as
non-Hodgkin's lymphoma, Hodgkin's lymphoma, hairy cell leukemia,
multiple myeloma, chronic lymphocytic leukemia, chronic myeloid
leukemia, acute myeloid leukemia, or acute lymphocytic leukemia, in
which CD38 is expressed, and other cancers yet to be determined in
which CD38 is expressed predominantly. In another embodiment, the
pharmaceutical composition of the invention can be used to treat
autoimmune diseases, such as systemic lupus erythematosus,
rheumatoid arthritis, multiple sclerosis, Crohn's diasease,
ulcerative colitis, gastritis, Hashimoto's thyroiditis, ankylosing
spondylitis, hepatitis C-associated cryoglobulinemic vasculitis,
chronic focal encephalitis, bullous pemphigoid, hemophilia A,
membranoproliferative glomerulnephritis, Sjogren's syndrome, adult
and juvenile dermatomyositis, adult polymyositis, chronic
urticaria, primary biliary cirrhosis, idiopathic thrombocytopenic
purpura, neuromyelitis optica, Graves' dysthyroid disease, bullous
pemphigoid, membranoproliferative glonerulonephritis, Churg-Strauss
syndrome, and asthma. In another embodiment, said pharmaceutical
composition relates to other disorders such as, for example, graft
rejections, such as renal transplant rejection, liver transplant
rejection, lung transplant rejection, cardiac transplant rejection,
and bone marrow transplant rejection; graft versus host disease;
viral infections, such as mV infection, HIV infection, AIDS, etc.;
and parasite infections, such as giardiasis, amoebiasis,
schistosomiasis, and others as determined by one of ordinary skill
in the art.
[0331] The instant invention provides pharmaceutical compositions
comprising: [0332] a) an effective amount of an antibody, antibody
fragment or antibody conjugate of the present invention, and;
[0333] b) a pharmaceutically acceptable carrier, which may be inert
or physiologically active.
[0334] As used herein, "pharmaceutically-acceptable carriers"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, and the like that are
physiologically compatible. Examples of suitable carriers, diluents
and/or excipients include one or more of water, saline, phosphate
buffered saline, dextrose, glycerol, ethanol, and the like, as well
as combination thereof. In many cases, it will be preferable to
include isotonic agents, such as sugars, polyalcohols, or sodium
chloride in the composition. In particular, relevant examples of
suitable carrier include: (1) Dulbecco's phosphate buffered saline,
pH.about.7.4, containing or not containing about 1 mg/ml to 25
mg/ml human serum albumin, (2) 0.9% saline (0.9% w/v sodium
chloride (NaCl)), and (3) 5% (w/v) dextrose; and may also contain
an antioxidant such as tryptamine and a stabilizing agent such as
Tween 20.
[0335] The compositions herein may also contain a further
therapeutic agent, as necessary for the particular disorder being
treated. Preferably, the antibody, antibody fragment or antibody
conjugate of the present invention, and the supplementary active
compound will have complementary activities, that do not adversely
affect each other. In a preferred embodiment, the further
therapeutic agent is an antagonist of epidermal-growth factor
(EGF), fibroblast-growth factor (FGF), hepatocyte growth factor
(HGF), tissue factor (TF), protein C, protein S, platelet-derived
growth factor (PDGF), heregulin, macrophage-stimulating protein
(MSP) or vascular endothelial growth factor (VEGF), or an
antagonist of a receptor for epidermal-growth factor (EGF),
fibroblast-growth factor (FGF), hepatocyte growth factor (HGF),
tissue factor (TF), protein C, protein S, platelet-derived growth
factor (PDGF), heregulin, macrophage-stimulating protein (MSP), or
vascular endothelial growth factor (VEGF), including HER2 receptor,
HER3 receptor, c-MET, and other receptor tyrosine kinases. In a
preferred embodiment, the further therapeutic agent is an agent
targeting clusters of differentiation (CD) antigens, including CD3,
CD14, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD36, CD40, CD44,
CD52, CD55, CD59, CD56, CD70, CD79, CD80, CD103, CD134, CD137,
CD138, and CD152. In a preferred embodiment, the further
therapeutic agent is a chemotherapeutic or immunomodulatory
agent.
[0336] The compositions of the invention may be in a variety of
forms. These include for example liquid, semi-solid, and solid
dosage forms, but the preferred form depends on the intended mode
of administration and therapeutic application. Typical preferred
compositions are in the form of injectable or infusible solutions.
The preferred mode of administration is parenteral (e.g.
intravenous, intramuscular, intraperinoneal, subcutaneous). In a
preferred embodiment, the compositions of the invention are
administered intravenously as a bolus or by continuous infusion
over a period of time. In another preferred embodiment, they are
injected by intramuscular, subcutaneous, intra-articular,
intrasynovial, intratumoral, peritumoral, intralesional, or
perilesional routes, to exert local as well as systemic therapeutic
effects.
[0337] Sterile compositions for parenteral administration can be
prepared by incorporating the antibody, antibody fragment or
antibody conjugate of the present invention in the required amount
in the appropriate solvent, followed by sterilization by
microfiltration. As solvent or vehicle, there may be used water,
saline, phosphate buffered saline, dextrose, glycerol, ethanol, and
the like, as well as combination thereof. In many cases, it will be
preferable to include isotonic agents, such as sugars,
polyalcohols, or sodium chloride in the composition. These
compositions may also contain adjuvants, in particular wetting,
isotonizing, emulsifying, dispersing and stabilizing agents.
Sterile compositions for parenteral administration may also be
prepared in the form of sterile solid compositions which may be
dissolved at the time of use in sterile water or any other
injectable sterile medium.
[0338] The antibody, antibody fragment or antibody conjugate of the
present invention may also be orally administered. As solid
compositions for oral administration, tablets, pills, powders
(gelatine capsules, sachets) or granules may be used. In these
compositions, the active ingredient according to the invention is
mixed with one or more inert diluents, such as starch, cellulose,
sucrose, lactose or silica, under an argon stream. These
compositions may also comprise substances other than diluents, for
example one or more lubricants such as magnesium stearate or talc,
a coloring, a coating (sugar-coated tablet) or a glaze.
[0339] As liquid compositions for oral administration, there may be
used pharmaceutically acceptable solutions, suspensions, emulsions,
syrups and elixirs containing inert diluents such as water,
ethanol, glycerol, vegetable oils or paraffin oil. These
compositions may comprise substances other than diluents, for
example wetting, sweetening, thickening, flavoring or stabilizing
products.
[0340] The doses depend on the desired effect, the duration of the
treatment and the route of administration used; they are generally
between 5 mg and 1000 mg per day orally for an adult with unit
doses ranging from 1 mg to 250 mg of active substance. In general,
the doctor will determine the appropriate dosage depending on the
age, weight and any other factors specific to the subject to be
treated.
[0341] Therapeutic Methods of Use
[0342] In another embodiment, the present invention provides a
method for killing a CD38.sup.+ cell by administering to a patient
in need thereof an antibody which binds said CD38 and is able to
kill said CD38.sup.+ cell by apoptosis, ADCC, and/or CDC. Any of
the type of antibodies, antibody fragments, or cytotoxic conjugates
of the invention, may be used therapeutically. The invention thus
includes the use of anti-CD38 monoclonal antibodies, fragments
thereof, or cytotoxic conjugates thereof as medicaments.
[0343] In a preferred embodiment, antibodies, antibody fragments,
or cytotoxic conjugates of the invention are used for the treatment
of a hyperproliferative disorder or inflammatory disease or
autoimmune disease in a mammal. In a more preferred embodiment, one
of the pharmaceutical compositions disclosed above, and which
contains an antibody, antibody fragment, or cytotoxic conjugate of
the invention, is used for the treatment of a hyperproliferative
disorder in a mammal. In one embodiment, the disorder is a cancer.
In particular, the cancer is a metastatic cancer.
[0344] Accordingly, the pharmaceutical compositions of the
invention are useful in the treatment or prevention of a variety of
cancers, including (but not limited to) the following: carcinoma,
including that of the bladder, breast, colon, kidney, liver, lung,
ovary, pancreas, stomach, cervix, thyroid and skin; including
squamous cell carcinoma; hematopoietic tumors of lymphoid lineage,
including leukemia, acute lymphocytic leukemia, acute lymphoblastic
leukemia, B-cell lymphoma, T-cell lymphoma, Burkitt's lymphoma;
hematopoietic tumors of myeloid lineage, including acute and
chronic myelogenous leukemias and promyelocytic leukemia; tumors of
mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma;
other tumors, including melanoma, seminoma, tetratocarcinoma,
neuroblastoma and glioma; tumors of the central and peripheral
nervous system, including astrocytoma, neuroblastoma, glioma, and
schwannomas; tumors of mesenchymal origin, including fibrosarcoma,
rhabdomyoscarama, and osteosarcoma; and other tumors, including
melanoma, xeroderma pigmentosum, keratoactanthoma, seminoma,
thyroid follicular cancer and teratocarcinoma, and other cancers
yet to be determined in which CD38 is expressed. Preferrably, the
disorder is NHL, BL, MM, B-CLL, ALL, TCL, AML, HCL, HL, or CML, in
which CD38 is expressed, and other cancers yet to be determined in
which CD38 is expressed predominantly. In another embodiment, the
pharmaceutical composition of the invention can be used to treat
autoimmune diseases, such as systemic lupus erythematosus,
rheumatoid arthritis, multiple sclerosis, Crohn's diasease,
ulcerative colitis, gastritis, Hashimoto's thyroiditis, ankylosing
spondylitis, hepatitis C-associated cryoglobulinemic vasculitis,
chronic focal encephalitis, bullous pemphigoid, hemophilia A,
membranoproliferative glomerulnephritis, Sjogren's syndrome, adult
and juvenile dermatomyositis, adult polymyositis, chronic
urticaria, primary biliary cirrhosis, idiopathic thrombocytopenic
purpura, neuromyelitis optica, Graves' dysthyroid disease, bullous
pemphigoid, membranoproliferative glonerulonephritis, Churg-Strauss
syndrome, and asthma. In another embodiment, said pharmaceutical
composition relates to other disorders such as, for example, graft
rejections, such as renal transplant rejection, liver transplant
rejection, lung transplant rejection, cardiac transplant rejection,
and bone marrow transplant rejection; graft versus host disease;
viral infections, such as mV infection, HIV infection, AIDS, etc.;
and parasite infections, such as giardiasis, amoebiasis,
schistosomiasis, and others as determined by one of ordinary skill
in the art.
[0345] Similarly, the present invention provides a method for
inhibiting the growth of selected cell populations comprising
contacting target cells, or tissue containing target cells, with an
effective amount of an antibody, antibody fragment or antibody
conjugate of the present invention, or an antibody, antibody
fragment or a therapeutic agent comprising a cytotoxic conjugate,
either alone or in combination with other cytotoxic or therapeutic
agents. In a preferred embodiment, the further therapeutic agent is
an antagonist of epidermal-growth factor (EGF), fibroblast-growth
factor (FGF), hepatocyte growth factor (HGF), tissue factor (TF),
protein C, protein S, platelet-derived growth factor (PDGF),
heregulin, macrophage-stimulating protein (MSP) or vascular
endothelial growth factor (VEGF), or an antagonist of a receptor
for epidermal-growth factor (EGF), fibroblast-growth factor (FGF),
hepatocyte growth factor (HGF), tissue factor (TF), protein C,
protein S, platelet-derived growth factor (PDGF), heregulin,
macrophage-stimulating protein (MSP), or vascular endothelial
growth factor (VEGF), including HER2 receptor, HER3 receptor,
c-MET, and other receptor tyrosine kinases. In a preferred
embodiment, the further therapeutic agent is an agent targeting
clusters of differentiation (CD) antigens, including CD3, CD14,
CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD36, CD40, CD44, CD52,
CD55, CD59, CD56, CD70, CD79, CD80, CD103, CD134, CD137, CD138, and
CD152. In a preferred embodiment, the further therapeutic agent is
a chemotherapeutic or immunomodulatory agent.
[0346] The method for inhibiting the growth of selected cell
populations can be practiced in vitro, in vivo, or ex vivo. As used
herein, "inhibiting growth" means slowing the growth of a cell,
decreasing cell viability, causing the death of a cell, lysing a
cell and inducing cell death, whether over a short or long period
of time.
[0347] Examples of in vitro uses include treatments of autologous
bone marrow prior to their transplant into the same patient in
order to kill diseased or malignant cells; treatments of bone
marrow prior to its transplantation in order to kill competent T
cells and prevent graft-versus-host-disease (GVHD); treatments of
cell cultures in order to kill all cells except for desired
variants that do not express the target antigen; or to kill
variants that express undesired antigen.
[0348] The conditions of non-clinical in vitro use are readily
determined by one of ordinary skill in the art.
[0349] Examples of clinical ex vivo use are to remove tumor cells
or lymphoid cells from bone marrow prior to autologous
transplantation in cancer treatment or in treatment of autoimmune
disease, or to remove T cells and other lymphoid cells from
autologous or allogeneic bone marrow or tissue prior to transplant
in order to prevent graft versus host disease (GVHD). Treatment can
be carried out as follows. Bone marrow is harvested from the
patient or other individual and then incubated in medium containing
serum to which is added the cytotoxic agent of the invention.
Concentrations range from about 10 .mu.M to 1 .mu.M, for about 30
minutes to about 48 hours at about 37.degree. C. The exact
conditions of concentration and time of incubation, i.e., the dose,
are readily determined by one of ordinary skill in the art. After
incubation the bone marrow cells are washed with medium containing
serum and returned to the patient by i.v. infusion according to
known methods. In circumstances where the patient receives other
treatment such as a course of ablative chemotherapy or total-body
irradiation between the time of harvest of the marrow and
reinfusion of the treated cells, the treated marrow cells are
stored frozen in liquid nitrogen using standard medical
equipment.
[0350] For clinical in vivo use, the antibody, the epitope-binding
antibody fragment, or the cytotoxic conjugate of the invention will
be supplied as solutions that are tested for sterility and for
endotoxin levels. Examples of suitable protocols of cytotoxic
conjugate administration are as follows. Conjugates are given
weekly for 4 weeks as an i.v. bolus each week. Bolus doses are
given in 50 to 100 ml of normal saline to which 5 to 10 ml of human
serum albumin can be added. Dosages will be 10 .mu.g to 100 mg per
administration, i.v. (range of 100 ng to 1 mg/kg per day). More
preferably, dosages will range from 50 .mu.g to 30 mg. Most
preferably, dosages will range from 1 mg to 20 mg. After four weeks
of treatment, the patient can continue to receive treatment on a
weekly basis. Specific clinical protocols with regard to route of
administration, excipients, diluents, dosages, times, etc., can be
determined by one of ordinary skill in the art as the clinical
situation warrants.
[0351] Diagnostic
[0352] The antibodies or antibody fragments of the invention can
also be used to detect CD38 in a biological sample in vitro or in
vivo. In one embodiment, the anti-CD38 antibodies of the invention
are used to determine the level of CD38 in a tissue or in cells
derived from the tissue. In a preferred embodiment, the tissue is a
diseased tissue. In a preferred embodiment of the method, the
tissue is a tumor or a biopsy thereof. In a preferred embodiment of
the method, a tissue or a biopsy thereof is first excised from a
patient, and the levels of CD38 in the tissue or biopsy can then be
determined in an immunoassay with the antibodies or antibody
fragments of the invention. In another preferred embodiment, the
level of CD38 is determined on a sample of a tissue or biopsy
thereof, which can be frozen or fixed. The same method can be used
to determine other properties of the CD38 protein, such as its cell
surface levels, or its cellular localization.
[0353] The above-described method can be used to diagnose a cancer
in a subject known to or suspected to have a cancer, wherein the
level of CD38 measured in said patient is compared with that of a
normal reference subject or standard. Said method can then be used
to determine whether a tumor expresses CD38, which may suggest that
the tumor will respond well to treatment with the antibodies,
antibody fragments or antibody conjugates of the present invention.
Preferrably, the tumor is a NHL, BL, MM, B-CLL, ALL, TCL, AML, HCL,
HL, or CML, in which CD38 is expressed, and other cancers yet to be
determined in which CD38 is expressed predominantly.
[0354] The present invention further provides for monoclonal
antibodies, humanized antibodies and epitope-binding fragments
thereof that are further labeled for use in research or diagnostic
applications. In preferred embodiments, the label is a radiolabel,
a fluorophore, a chromophore, an imaging agent or a metal ion.
[0355] A method for diagnosis is also provided in which said
labeled antibodies or epitope-binding fragments thereof are
administered to a subject suspected of having a cancer or an
inflammatory disease or an autoimmune disease, and the distribution
of the label within the body of the subject is measured or
monitored.
[0356] Kit
[0357] The present invention also includes kits, e.g., comprising a
described cytotoxic conjugate and instructions for the use of the
cytotoxic conjugate for killing of particular cell types. The
instructions may include directions for using the cytotoxic
conjugates in vitro, in vivo or ex vivo.
[0358] Typically, the kit will have a compartment containing the
cytotoxic conjugate. The cytotoxic conjugate may be in a
lyophilized form, liquid form, or other form amendable to being
included in a kit. The kit may also contain additional elements
needed to practice the method described on the instructions in the
kit, such a sterilized solution for reconstituting a lyophilized
powder, additional agents for combining with the cytotoxic
conjugate prior to administering to a patient, and tools that aid
in administering the conjugate to a patient.
EXAMPLES
[0359] The invention is now described by reference to the following
examples, which are illustrative only, and are not intended to
limit the present invention.
Example 1
Mouse CD38 Antibodies
[0360] 300-19 cells, a pre-B cell line derived from a Balb/c mouse
(M. G. Reth et al. 1985, Nature, 317: 353-355), stably expressing a
high level of human CD38 were used for immunization of Balb/c VAF
mice. Mice were subcutaneously immunized with about
5.times.10.sup.6 CD38-expressing 300-19 cells per mouse every 2-3
weeks by standard immunization protocols used at ImmunoGen, Inc.
The immunized mice were boosted with another dose of antigen three
days before being sacrificed for hybridoma generation. The spleen
from the mouse was collected according to standard animal protocols
and was ground between two sterile, frosted microscopic slides to
obtain a single cell suspension in RPMI-1640 medium. The spleen
cells were pelleted, washed, and fused with murine myeloma
P3X63Ag8.653 cells (J. F. Kearney et al. 1979, J Immunol, 123:
1548-1550) by using polyethylene glycol-1500 (Roche 783 641). The
fused cells were resuspended in RPMI-1640 selection medium
containing hypoxanthine-aminopterin-thymidine (HAT) (Sigma H-0262)
and selected for growth in 96-well flat-bottomed culture plates
(Corning-Costar 3596, 200 .mu.L of cell suspension per well) at
37.degree. C. (5% CO2). After 5 days of incubation, 100 .mu.L of
culture supernatant were removed from each well and replaced with
100 .mu.L of RPMI-1640 medium containing hypoxanthine-thymidine
(HT) supplement (Sigma H-0137). Incubation at 37.degree. C. (5%
CO2) was continued until hydridoma clones were ready for antibody
screening. Other techniques of immunization and hybridoma
production can also be used, including those described in J.
Langone and H. Vunakis (Eds., Methods in Enzymology, Vol. 121,
"Immunochemical Techniques, Part I"; Academic Press, Florida) and
E. Harlow and D. Lane ("Antibodies: A Laboratory Manual"; 1988;
Cold Spring Harbor Laboratory Press, New York).
[0361] By fluorescence activated cell sorting (FACS) using a Becton
Dickinson FACSCalibur or a FACSArray machine, culture supernatants
from the hybridoma were screened (with FITC or PE-conjugated
anti-mouse IgG antiserum) for secretion of mouse monoclonal
antibodies that bind to the CD38-expressing 300-19 cells, but not
to the parental 300-19 cells. The hybridoma clones that tested
positive were subcloned, and the isotype of each secreted anti-CD38
antibody was identified using commercial isotyping reagents (Roche
1493027). A total of 29 antibodies that were positive for CD38
binding were purified by Protein A or G chromatography using a
standard protocol and then characterized further.
Example 2
Binding Characterization of Anti-CD38 Antibodies
[0362] FACS histograms demonstrating the binding of anti-CD38
antibodies, 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 to
CD38-expressing 300-19 cells and the absence of binding to the
parental 300-19 cells are shown in FIG. 1. 38SB13, 38SB18, 38SB19,
38SB30, 38SB31, or 38SB39 antibody (10 nM) was incubated for 3 h
with either CD38-expressing 300-19 cells or the parental 300-19
cells (1-2.times.10.sup.5 cells per sample) in 100 .mu.L ice-cold
RPMI-1640 medium supplemented with 2% normal goat serum. Then, the
cells were pelleted, washed, and incubated for 1 h on ice with
FITC-conjugated goat anti-mouse IgG-antibody (Jackson Laboratory,
100 .mu.L, 6 .mu.g/mL in cold RPMI-1640 medium supplemented with 2%
normal goat serum). The cells were pelleted again, washed,
resuspended in 200 .mu.L of PBS containing 1% formaldehyde, and
analyzed using a FACSCalibur flow cytometer with CellQuest software
(BD Biosciences).
[0363] The FACS histograms of CD38-expressing 300-19 cells
incubated with 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, or 38SB39
showed a strong fluorescence shift, compared to that of the
corresponding negative control (cells incubated only with
FITC-conjugated, goat anti-mouse IgG-antibody) (FIG. 1). Also, no
significant fluorescence shift was detected when parental 300-19
cells were incubated with any of these antibodies. Similar results
were obtained when the positive control anti-CD38 antibody, AT13/5
(Serotec, MCA1019) was used.
[0364] A strong fluorescence shift was also observed when Ramos
(ATCC CRL 1596) lymphoma cells were incubated with 38SB13, 38SB18,
38SB19, 38SB30, 38SB31, or 38SB39 (FIG. 1). The values for the
apparent dissociation constants (KD) of 38SB13, 38SB18, 38SB19,
38SB30, 38SB31, and 38SB39 for the binding to Ramos cells were
estimated from the FACS analysis curves shown in FIG. 2, using the
non-linear regression method for sigmoidal dose response curves
(GraphPad Prizm, version 4, software, San Diego, Calif.). The
values are as follows: 0.10 nM, 0.10 nM, 0.12 nM, 0.16 nM, 0.11 nM,
and 3.03 nM, respectively.
Example 3
Induction of Apoptosis of Ramos and Daudi Lymphoma Cells, by
38Sb13, 38Sb18, 38SB19, 38SB30, 38SB31, and 38SB39 Antibodies
[0365] The anti-CD38 antibodies, 38SB13, 38SB18, 38SB19, 38SB30,
38SB31, and 38SB39 induced apoptosis of Ramos and Daudi (ATCC
CCL-213) lymphoma cell lines and the MOLP-8 multiple myeloma cell
line (DSMZ ACC 569). The degree of apoptosis was measured by FACS
analysis after staining with FITC conjugates of Annexin V
(Biosource PHN1018) and with TO-PRO-3 (Invitrogen T3605). Annexin V
binds phosphatidylserine on the outside but not on the inside of
the cell membrane bilayer of intact cells. In healthy, normal
cells, phosphatidylserine is expressed on the inside of the
membrane bilayer, and the transition of phosphatidylserine from the
inner to the outer leaflet of the plasma membrane is one of the
earliest detectable signals of apoptosis. Binding of Annexin V is
thus a signal for the induction of apoptosis. TO-PRO-3 is a
monomeric cyanine nucleic acid stain that can only penetrate the
plasma membrane when the membrane integrity is breached, as occurs
in the later stages of apoptosis.
[0366] Exponentially growing cells were plated at about
2.times.10.sup.5 cells/mL in 24-well plates in RMPI-1640 medium
supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine,
and 50 .mu.g/mL gentamycin (denoted below as complete RMPI-1640
medium). Cells were generally grown in complete RMPI-1640 medium,
unless stated otherwise. Cells were incubated with anti-CD38
antibodies (10 nM) for 24 h at 37.degree. C. in a humidified
atmosphere containing 5% CO.sub.2. The cells were then pelleted,
washed twice with 500 .mu.L PBS, resuspended in 100 .mu.L binding
buffer (provided in the Annexin V-FITC kit), containing 5 .mu.L of
Annexin V-FITC, and incubated for 15 min on ice. Then, 400 .mu.L of
binding buffer and TO-PRO-3 (to a final concentration of 1 .mu.M)
was added to the mix, and the cell-associated fluorescence of FITC
and TO-PRO-3 was immediately measured by FACS. Four thousand events
were collected for each sample. The dot plots for fluorescence of
TO-PRO-3 (FL4-H; y-axis) and fluorescence of Annexin V-FITC (FL1-H;
x-axis) were generated using CellQuest software.
[0367] The results are shown in FIGS. 3 and 4. FIG. 3 gives an
example of such a dot plot for Daudi cells after a 24-h incubation
with various anti-CD38 antibodies. The average percentages of
Annexin V-positive cells (includes both TO-PRO-3 positive and
negative cells) from duplicate samples were determined from these
plots and are shown in FIG. 4. Unexpectedly, 38SB13, 38SB18,
38SB19, 38SB30, 38SB31, and 38SB39 showed strong apoptotic
activities. Greater than 30% of Daudi cells exposed to any of these
antibodies were Annexin V-positive, compared to only about 6% of
untreated cells (FIGS. 3 and 4A). 38SB13, 38SB18, 38SB19, 38SB30,
38SB31, and 38SB39 showed at least 2.4-fold stronger apoptotic
activities (24% after subtraction of the non-treated control value)
than prior art murine CD38 antibodies tested at the same
concentration of 10 nM, (AT13/5, OKT10, IB4, and SUN-4B7, less than
10% Annexin V-positive after subtraction of the non-treated control
value) and two other anti-CD38 antibodies generated in our
laboratory, (38SB7 and 38SB23, not higher than non-treated control,
i.e. about 6% Annexin V-positive) (FIG. 4A). AT13/5 was purchased
from Serotec (MCA1019), and OKT10 was produced and purified from
hybridoma (ATCC CRL-8022). IB4 and SUN-4B7 was a gift from Prof. F.
Malavasi, University of Turin, Italy. Similarly, 38SB13, 38SB18,
38SB19, 38SB30, 38SB31, and 38SB39 anti-CD38 antibodies displayed
at least 3.5-fold stronger pro-apoptotic activity on another
lymphoma cell line, Ramos (7% or more Annexin-V-positive after
subtraction of the non-treated control value) than either prior art
murine CD38 antibodies, AT13/5, OKT10, IB4, and SUN-4B7, or two
other new anti-CD38 antibodies, 38SB7 and 38SB23 (less than 2%
Annexin V-positive after subtracting the non-treated control value)
(FIG. 4B). Finally, 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and
38SB39 anti-CD38 antibodies displayed strong pro-apoptotic activity
on the multiple myeloma cell line MOLP-8 (FIG. 4C). Approximately
50% of MOLP-8 cells treated with these antibodies were Annexin
V-positive, compared to about 39% of untreated cells. In contrast,
treatment with any of the prior art murine CD38 antibodies, AT13/5,
OKT10, IB4, and SUN-4B7, or two other new anti-CD38 antibodies,
38SB7 and 38SB23 resulted on no increase in the portion of
apoptotic cells.
Example 4
Cloning and Sequencing of the Light and Heavy Chains of Anti-CD38
Antibodies
[0368] 38SB19 Antibody
[0369] RNA Preparation from Hybridoma Cells that Produces the
38SB19 Antibody
[0370] Preparations of total RNA were obtained from
5.times.10.sup.6 hybridoma cells, which produce 38SB19 antibody,
using Qiagen's RNeasy miniprep kit. Briefly, 5.times.10.sup.6 cells
were pelleted and resuspended in 350 .mu.L RLT buffer (containing
1% .beta.-mercaptoethanol). The suspension was homogenized by
passing it through a 21.5 gauge needle and syringe roughly 10-20
times or until it was no longer viscous. Ethanol (350 .mu.L of 70%
aqueous ethanol) was added to the homogenate, which was mixed well.
The solution was transferred to a spin column, placed in a 2-mL
collection tube and spun at >8000.times.g for 15 seconds. The
column was washed twice with 500 .mu.L RPE buffer, then transferred
to a fresh tube and eluted with 30 .mu.L RNase free water and a
1-minute spin. The eluate (30 .mu.L) was placed back on the column
for a second 1-minute elution spin. An aliquot of the 30 .mu.L
eluate was diluted with water and used to measure the UV absorption
at 260 nm for RNA quantitation. cDNA Preparation with Reverse
Transcriptase (RT) reaction
[0371] The variable region 38SB19 antibody cDNA was generated from
the total RNA using Invitrogen's SuperscriptII kit. The kit
protocols were followed closely, utilizing up to 5 .mu.g of total
RNA from the Qianeasy mini preps. Briefly, the RNA, 1 .mu.L random
primers, and 1 .mu.L dNTP mix were brought up to 12 .mu.L with
RNase free sterile distilled water and incubated at 65.degree. C.
for 5 minutes. The mix was then put on ice for at least 1 minute.
Next 4 .mu.L of 5.times. reaction buffer, 2 .mu.L 0.1 M DTT, and 1
.mu.L RNaseOUT were added and the mix was incubated at 25.degree.
C. for 2 minutes in an MJ Research thermalcycler. The thermalkylcer
was paused so that 1 .mu.L of SuperscriptII enzyme could be added
and then restarted for an additional 10 minutes at 25.degree. C.
before shifting to 55.degree. C. for 50 minutes. The reaction was
heat inactivated by heating to 70.degree. C. for 15 min and the RNA
was removed by adding 1 .mu.L RNase H and incubating at 37.degree.
C. for 20 minutes.
[0372] Degenerate PCR Reactions
[0373] The procedure for the first round degenerate PCR reaction on
the cDNA derived from hybridoma cells was based on methods
described in Wang et al. (2000) and Co et al. (1992). The primers
for this round (Table 2) contain restriction sites to facilitate
cloning into the pBluescriptII plasmids.
[0374] The PCR reaction components (Table 3) were mixed on ice in
thin walled PCR tubes and then transferred to an MJ research
thermalcycler preheated and paused at 94.degree. C. The reactions
were performed using a program derived from Wang et al., 2000 as
follows:
Name: Wang45
94.degree. C. 3:00 min
94.degree. C. 0:15 sec
45.degree. C. 1:00 min
72.degree. C. 2:00 min
[0375] Goto 2 29 times
72.degree. C. 6:00 min
[0376] 4.degree. C. for ever end
[0377] The PCR reaction mixtures were then run on a 1% low melt
agarose gel, the 300 to 400 bp bands were excised, purified using
Zymo DNA mini columns, and sent to Agencourt biosciences for
sequencing. The respective 5' and 3' PCR primers were used as
sequencing primers to generate the 38SB19 variable region cDNAs
from both directions.
[0378] Cloning the 5' End Sequence
[0379] Since the degenerate primers used to clone the 38SB19
variable region light chain and heavy chain cDNA sequences alters
the 5' end sequences, additional sequencing efforts were needed to
decipher the complete sequences. The preliminary cDNA sequence from
the methods described above were used to search the NCBI IgBlast
site (http://www.ncbi.nlm.nih.gov/igblast/) for the murine germline
sequences from which the 38SB19 sequence is derived. PCR primers
were designed (Table 3) to anneal to the leader sequence of the
murine antibody so that a new PCR reaction could yield the complete
variable region cDNA, unaltered by the PCR primers. The PCR
reactions, band purifications, and sequencing were performed as
described above.
[0380] Peptide Analysis for Sequence Confirmation
[0381] The cDNA sequence information for the variable region was
combined with the germline constant region sequence to obtain full
length antibody cDNA sequences. The molecular weights of the heavy
chain and light chain were then calculated and compared with the
molecular weights obtained by LC/MS analyses of the murine 38SB19
antibody.
[0382] Table 4 gives the calculated mass from the cDNA sequences
for 38SB19 LC and HC together with the values measured by LC/MS.
The molecular weight measurements are consistent with the cDNA
sequences for both the 38SB19 light and heavy chain.
Example 5
Recombinant Expression of hu38SB19 Antibodies
[0383] The variable region sequences for hu38SB19 were
codon-optimized and synthesized by Blue Heron Biotechnology. The
sequences are flanked by restriction enzyme sites for cloning
in-frame with the respective constant sequences in both single
chain and the tandem dual chain mammalian expression plasmids. The
light chain variable region is cloned into EcoRI and BsiWI sites in
both the ps38SB19LCZv1.0 and ps38SB19v1.00 plasmids (FIGS. 5A and
5C). The heavy chain variable region is cloned into the HindIII and
Apa1 sites in both the ps38SB19HCNv1.0 and ps38SB19v1.00 plasmids
(FIGS. 5B and 5C). These plasmids can be used to express hu38SB19
in either transient or stable transfections in mammalian cells.
Similar expression vector constructs were used to produce other
chimeric and humanized antibodies.
[0384] Transient transfections to express hu38SB19 in HEK-293T
cells were performed using CaPO.sub.4 reagents from BD biosciences.
The supplied protocols were slightly modified for enhanced
expression yields. Briefly, 2.times.10.sup.6 HEK-293T cells were
plated on 10 cm tissue culture plates coated with polyethyleneimine
(PEI) 24 h prior to transfection. The transfection began by washing
the cells with PBS and replacing the media with 10 mL DMEM
(Invitrogen) with 1% Ultra Low IgG FBS (Hyclone). Solution A (10
.mu.g DNA, 86.8 .mu.L Ca.sup.2+ solution, and up to 500 .mu.L with
H.sub.2O) was added drop wise to Solution B while vortexing. The
mixture was incubated at RT for 1 min and 1 mL of the mixture was
added drop wise to each 10 cm plate. Approximately 16 h post
transfection, media was replaced with 10 mL fresh DMEM with 1%
Ultra Low IgG FBS. Approximately 24 hours later 2 mM sodium
butyrate was added to each 10 cm plate. The transfection was
harvested 4 days later.
[0385] Supernatant was prepared for Protein A affinity
chromatography by the addition of 1/10 volume of 1 M Tris/HCl
buffer, pH 8.0. The pH-adjusted supernatant was filtered through a
0.22 .mu.m filter membrane and loaded onto a Protein A Sepharose
column (HiTrap Protein A HP, 1 mL, Amersham Biosciences)
equilibrated with binding buffer (PBS, pH 7.3). A Q-Sepharose
precolumn (10 mL) was connected upstream of the Protein A column
during sample loading to reduce contamination from cellular
material such as DNA. Following sample loading, the precolumn was
removed and the Protein A column orientation was reversed for wash
and elution. The column was washed with binding buffer until a
stable baseline was obtained with no absorbance at 280 nm. Antibody
was eluted with 0.1 M acetic acid buffer containing 0.15 M NaCl, pH
2.8, using a flow rate of 0.5 mL/min. Fractions of approximately
0.25 mL were collected and neutralized by the addition of 1/10
volume of 1M Tris/HCl, pH 8.0. The peak fraction(s) was dialysed
overnight twice against PBS and purified antibody was quantitated
by absorbance at OD.sub.280. Humanized and chimeric antibodies can
also be purified using a Protein G column with slightly different
procedures.
[0386] All the described chimeric and humanized anti-CD38
antibodies were expressed and purified in similar procedures as
described above.
Example 6
Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Activities of
Chimeric Anti-CD38 Antibodies
[0387] Since some anti-CD38 antibodies have been previously shown
to have ADCC and/or CDC activity as chimeric or humanized
antibodies with human IgG1 constant regions (J. H. Ellis et al.
1995, J Immunol, 155: 925-937; F. K. Stevenson et al. 1991, Blood,
77: 1071-1079; WO 2005/103083), the chimeric versions of 38SB13,
38SB18, 38SB19, 38SB30, 38SB31, and 38SB39, consisting of murine
variable regions and the human IgG1/IgKappa constant region, were
made and tested for ADCC and/or CDC activities.
[0388] Ch38SB13, ch38SB18, ch38SB19, ch38SB30, ch38SB31, and
ch38SB39 were first tested for ADCC using Ramos cells as target
cells and human natural killer (NK) cells as effector cells. A
lactate dehydrogenase (LDH) release assay was used to measure cell
lysis (R. L. Shields et al., 2001, J Biol Chem, 276:
6591-6604).
[0389] The NK cells were first isolated from human blood (from a
normal donor; purchased from Research Blood Components, Inc.,
Brighton, Mass.) using a modified protocol for NK Isolation Kit II
(Miltenyi Biotech). Blood was diluted 2-3-fold with Hank's Balanced
Salt Solution (HBSS). Twenty five mL of diluted blood was carefully
layered over 25 mL of Ficoll Paque in a 50 mL conical tube and
centrifuged at 400 g for 45 min at 19.degree. C. The peripheral
blood mononuclear cells (PBMC) were collected from the interface,
transferred into a new conical 50 mL tube, and washed once with
HBSS. The PBMC were resuspended in 2 mL of NK-isolation buffer, and
then 500 .mu.L of Biotin-Antibody Cocktail (from the NK-isolation
kit, 130-091-152, Miltenyi Biotech) were added to the cell
suspension. The Biotin-Antibody Cocktail contains biotinylated
antibodies that bind to the lymphocytes, except for NK cells. The
mixture was incubated at 4.degree. C. for 10 min, and then 1.5 mL
of NK-isolation buffer (PBS, 0.1% BSA, 1 mM EDTA) and 1 mL of
Anti-Biotin Micro Beads was added. The cell-antibody mixture was
incubated for another 15 min at 4.degree. C. Next, cells were
washed once with 50 mL of NK-isolation buffer and resuspended in 3
mL of NK-isolation buffer. Then, MACS LS column (on the MACS
separator, Miltenyi Biotech) was pre-washed with 3 mL of
NK-isolation Buffer. The cell suspension was then applied onto the
LS column. The effluent (fraction with unlabeled cells) was
collected into a new 50-mL conical tube. The column was washed 3
times with 3 mL of NK-isolation Buffer. The entire effluent was
collected into the same tube and washed once with 50 mL of
NK-isolation Buffer. NK cells were plated into 30 mL of RPMI-1640
supplemented with 5% fetal bovine serum, 50 .mu.g/mL
gentamycin.
[0390] Various concentrations of ch38SB13, ch38SB18, ch38SB19,
ch38SB30, ch38SB31, and ch38SB39 antibodies in RPMI-1640 medium
supplemented with 0.1% BSA, 20 mM HEPES, pH 7.4, and 50 .mu.g/mL
gentamycin (denoted below as RHBP medium) were aliquoted (50
.mu.L/well) into a round bottom 96-well plate. The target Ramos
cells were resuspended at 10.sup.6 cells/mL in RHBP medium and
added to each well (100 .mu.L/well) containing antibody dilutions.
The plate containing target cells and antibody dilutions was
incubated for 30 min at 37.degree. C. NK cells (50 .mu.L/well) were
then added to the wells containing the target cells typically at a
ratio of 1 target cell to 3-6 NK cells ratio. RHBP medium (50
.mu.L/well) was added to the control wells with NK cells. Also, 20
.mu.L of Triton X-100 solution (RPMI-1640 medium, 10% Triton X-100)
was added to the 3 wells containing only target cells without
antibody, to determine the maximum possible LDH release. The
mixtures were incubated at 37.degree. C. for 4 h, then centrifuged
for 10 min at 1200 rpm, and 100 .mu.L of the supernatant was
carefully transferred to a new flat-bottom 96-well plate. LDH
reaction mixture (100 .mu.L/well) from Cytotoxicity Detection Kit
(Roche 1 644 793) was added to each well and incubated at room
temperature for 5-30 min. The optical density of samples was
measured at 490 nm (OD.sub.490). The percent specific lysis of each
sample was determined by ascribing 100% lysis to the OD.sub.490
value of Triton X-100-treated samples and 0% lysis to the
OD.sub.490 value of the untreated control sample containing only
target cells. The samples containing only NK cells gave negligible
OD.sub.490 readings.
[0391] When tested with Ramos target cells and NK effector cells,
chimeric anti-CD38 antibodies showed very potent ADCC activities
(FIG. 6). The EC.sub.50 values were estimated by a non-linear
regression method with sigmoidal dose response curves and found to
be as follows: 0.0013 .mu.g/mL for ch38SB13, 0.0013 .mu.g/mL for
ch38SB18, 0.0018 .mu.g/mL for ch38SB19, 0.0022 .mu.g/mL for
ch38SB30, 0.0012 .mu.g/mL for ch38SB31, 0.1132 .mu.g/mL for
ch38SB39. Chimeric anti-CD38 antibodies also showed potent ADCC
activity on LP-1 (DSMZ ACC 41) multiple myeloma cells (EC.sub.50
values: 0.00056 .mu.g/mL for ch38SB18; 0.00034 .mu.g/mL for
ch38SB19; 0.00024 .mu.g/mL for ch38SB31) (FIG. 7A). Ch38SB19 also
efficiently killed Daudi lymphoma cells (FIG. 7B), NALM-6 B-ALL
cells (DSMZ ACC 128) (FIG. 8A), and MOLT-4 T-ALL cells (ATTC
CRL-1582) (FIG. 8B) by ADCC, suggesting anti-CD38 antibodies with
unusually potent apoptotic activity also have potent ADCC activity
against various tumor cells derived from various hematopoietic
malignancies. Also, a non-binding IgG1 control antibody (rituximab,
BiogenIdec) (FIGS. 7A, 8A, and 8B) or mu38SB19 (FIG. 7B) in the
same experiment had no significant ADCC activity.
Example 7
CDC Activities of Chimeric Anti-CD38 Antibodies
[0392] The CDC activities of ch38SB13, ch38SB18, ch38SB19,
ch38SB30, ch38SB31, and ch38SB39 were measured based on a method
modified from (H. Gazzano-Santoro et al. 1997, J. Immunol. Methods,
202: 163-171). Human complement was lyophilized human complement
serum (Sigma-Aldrich S1764) that was reconstituted with sterile
purified water as indicated by the manufacturer and then diluted
five-fold with RHBP media immediately before the experiment. Target
cells suspended at 10.sup.6 cells/mL in RHBP medium were aliquoted
into wells of a flat-bottom 96-well tissue culture plate (50
.mu.L/well). Then, 50 .mu.L of various concentrations (from 10 nM
to 0.001 nM) of the anti-CD38 antibodies in RHBP medium were added
(one antibody per sample), which was followed by 50 .mu.L/well of
complement solution. The plate was then incubated for 2 h at
37.degree. C. in a humidified atmosphere containing 5% CO.sub.2,
after which time 50 .mu.L of 40% Alamar Blue reagent (Biosource
DAL1100) diluted in RHBP (10% final) was added to each well to
measure the viability of the cells. Alamar Blue monitors the
reducing capacity of the viable cells. The plate was incubated for
5-18 h at 37.degree. C. before measuring the fluorescence (in
relative fluorescence units, RFU) at 540/590 nm. The percentage of
specific cell viability for each sample was determined by first
correcting the experimental values for background fluorescence by
subtracting the background RFU value (wells with medium only,
without any cells) from the RFU values for each sample, and then,
dividing the corrected RFU values by the corrected RFU value of
untreated cell samples.
[0393] When the CDC activities of the chimeric anti-CD38 antibody
samples were tested with Raji-IMG cells using human complement at a
final dilution of 5%, chimeric anti-CD38 antibodies showed very
potent CDC activities (FIG. 9). Raji-IMG are cells derived from
Raji cells (ATCC CCL-86) and express lower levels of the membrane
complement inhibitors CD55 and CD59. The EC.sub.50 values were
estimated by non-linear regression from the sigmoidal dose response
curve shown in FIG. 8. and are as follows: 0.005 .mu.g/mL for
ch38SB13, 0.0101 .mu.g/mL for ch38SB18, 0.028 .mu.g/mL for
ch38SB19, 0.020 .mu.g/mL for ch38SB30, 0.010 .mu.g/mL for ch38SB31,
and 0.400 .mu.g/mL for ch38SB39. Chimeric anti-CD38 antibodies also
showed potent CDC activity towards LP-1 multiple myeloma cells
(EC.sub.50 value: 0.032 .mu.g/mL for ch38SB18; 0.030 .mu.g/mL for
ch38SB19; 0.043 .mu.g/mL for ch38SB31), while a non-binding
chimeric control IgG1 (rituximab, BiogenIdec) did not have any CDC
activity (FIG. 10). When chimeric CD38 antibodies were tested on
Daudi lymphoma cells, different anti-CD38 antibodies differed in
their CDC activities (FIG. 11). While the specific viability of
Daudi cells was less than 15% following their incubation with 1.25
.mu.g/mL of ch38SB19 in the presence of complement, there was only
a marginal decrease in the specific viability of these cells
following their incubation with ch38SB18 or ch38SB39 (1.25 .mu.g/mL
or higher concentration) in the presence of complement (the
specific viability was 85% and 91%, respectively). Also, only a
modest reduction of specific viability was observed when the Daudi
cells were incubated with 1.25 .mu.g/mL or higher concentration of
ch38SB13, ch38SB30, and ch38SB31 in the presence of complement (the
specific viability was 65%, 45%, and 53%, respectively).
Example 8
Binding Affinity and Apoptotic, ADCC. and CDC Activities of
Humanized Anti-CD38 Antibodies
[0394] The two versions of humanized 38SB19 (hu38SB19 v1.00 and
v1.20) and the chimeric 38SB19 showed similar binding affinities
when tested with Ramos cells with K.sub.D values of 0.23 nM, 0.25
nM, and 0.18 nM, respectively (FIG. 12A). The binding affinities of
chimeric and humanized 38SB19 antibodies were also compared in a
competiton binding assay, where their ability to compete with the
binding of biotinylated murine 38SB19 antibody is measured.
Biotin-labeled murine 38SB19 antibody (3.times.10.sup.-10 M) was
mixed with various concentrations of ch38SB19, hu38SB19 v1.00, or
hu38SB19 v1.20. The antibody mixture was incubated with Ramos
cells, and the amount of the biotinylated murine 38SB19 bound to
the cells was measured with FITC-conjugated streptavidin by FACS
analysis. Hu38SB19 v1.00, hu38SB19 v1.20, and ch38SB19 competed
with the binding of biotinylated murine 38SB19 equally well (FIG.
12B), again indicating that the binding affinity was unaffected by
the humanization. When ch38SB19, hu38SB19 v1.00 and hu38SB19 v1.20
(10.sup.-8 to 10.sup.-11 M) were compared for their ability to
induce apoptosis of Daudi cells, they showed similar apoptotic
activities (FIG. 13). Moreover, hu38SB19 v1.00 and v1.20 also
showed similar ADCC as ch38SB19 in LP-1 cells (FIG. 14) and similar
CDC potencies as ch38SB19 in Raji-IMG and LP-1 cells (FIG. 15).
Hu38SB19 v1.00 also showed similar CDC activity as ch38SB19 in the
T-cell acute lymphoblastic leukemia cell line DND-41 (DSMZ 525)
(FIG. 15). Hu38SB19 v1.00 was further tested for its ability to
induce apoptosis in a diverse set of cell lines (FIG. 16).
Treatment with hu38SB19 v1.00 (10.sup.-8 M) resulted in a dramatic
increase of Annexin V-positive cells in the B cell lymphoma cell
lines SU-DHL-8 (DSMZ ACC 573) (from 7% in untreated control to 97%
in hu38SB19-treated cells) and NU-DUL-1 (DSMZ ACC 579) (from 10% in
untreated control to 37% in hu38SB19-treated cells) and the T-ALL
cell line DND-41 (from 7% in untreated control to 69% in
hu38SB19-treated cells). In addition, treatment with hu38SB19 v1.00
(10.sup.-8 M) increased the portion of Annexin V-positive cells in
the B-cell lymphocytic leukemia cell line JVM-13 (DSMZ ACC 19)
(from 8% in untreated control to 17% in hu38SB19-treated cells) and
in the hairy cell leukemia cell line HC-1 (DSMZ ACC 301) (from 6%
in untreated control to 10% in hu38SB19-treated cells).
[0395] Similarily, two versions of humanized 38SB31 (hu38SB31 v1.1
and v1.2) and the chimeric 38SB31 showed similar binding affinities
when tested with Ramos cells with K.sub.D values of 0.13 nM, 0.11
nM, and 0.12 nM, respectively. The binding affinities of chimeric
and humanized 38SB31 antibodies were also compared in a competition
binding assay, as described above and performed equally well.
Hu38SB31v1.1 was further tested for its ability to induce apoptosis
in several cell lines. The humanized antibody showed similar
apoptotic activities as ch38SB31 towards Ramos, Daudi, Molp-8 and
SU-DHL-8 cells. Moreover, hu38SB31 v1.1 also showed similar ADCC
and CDC activities as ch38SB31 in these cell lines.
Example 9
In Vivo Efficacy of 38Sb13, 38Sb18, 38Sb19, 38Sb30, 38Sb31, and
38Sb39
[0396] In vivo anti-tumor activities of 38SB13, 38SB18, 38SB19,
38SB30, 38SB31, and 38SB39 were investigated in a survival human
xenograft tumor model in immunodeficient mice (female CB.17 SCID)
established with Ramos lymphoma cells. Female CB.17 SCID mice were
inoculated with 2.times.10.sup.6 Ramos cells in 0.1 mL serum-free
medium through a lateral tail vein. Seven days after tumor cell
inoculation, mice were randomized into seven groups based on body
weight. There were 10 mice per group, except for the 38SB31-treated
group, which had 6 mice, and the 38SB39-treated group, which had 8
mice. Antibodies were given to mice intravenously at a dose of 40
mg/kg, twice per week, in three successive weeks, starting seven
days after cell inoculation. Mice were sacrificed if one or both
hind legs were paralyzed, the loss of body weight was more than 20%
from the pre-treatment value, or the animal was too sick to reach
food and water. The treatment with 38SB13, 38SB18, 38SB19, 38SB30,
38SB31, or 38SB39 significantly extended the survival of mice
compared to that of PBS-treated mice (FIG. 17). The median survival
of PBS-treated mice was 22 days and that of antibody-treated groups
ranged from 28 to 33 days.
[0397] In vivo anti-tumor activities of mu38SB19 and hu38SB19 were
further investigated in additional human xenograft tumor models in
immunodeficient mice. For a Daudi lymphoma survival model, SCID
mice were inoculated with 5.times.10.sup.6 Daudi cells in 0.1 mL
serum-free medium through a lateral tail vein. The study was
carried out as described above. The treatment with either mu38SB19
or hu38SB19 significantly extended the survival of mice compared to
that of PBS-treated mice (FIG. 18). The median survival of
PBS-treated mice was 22 days, while the median survival of
antibody-treated mice was 47 days.
[0398] For a NCl-H929 multiple myeloma tumor model, SCID mice were
inoculated subcutaneously with 10.sup.7 cells. When tumors were
palpable on day 6, the animals were randomized into groups of 10
according to body weight and antibody treatment was started. The
hu38SB19 antibody or a non-binding chimeric IgG1 control antibody
(rituximab, BiogenIdec) were given to mice intravenously at a dose
of 40 mg/kg, twice per week, in three successive weeks. Tumor
volume was monitored and animals were sacrificed if tumors reached
2000 mm.sup.3 in size or became necrotic. The PBS treated group
reached a mean tumor volume of 1000 mmm.sup.3 on day 89, the
chimeric IgG1 control antibody group on day 84 (FIG. 19). Treatment
with hu38SB19 completely prevented tumor growth in all 10 animals.
In contrast, only two animals in the PBS treated group and three
animals in the chimeric IgG1 control antibody showed tumor
regression.
[0399] For a MOLP-8 multiple myeloma tumor model, SCID mice were
inoculated subcutaneously with 10.sup.7 cells. When tumors were
palpable on day 4, the animals were randomized into groups of 10
according to body weight and antibody treatment was started. The
hu38SB19 and mu38SB19 antibodies or a chimeric IgG1 control
antibody were given to mice intravenously at a dose of 40 mg/kg,
twice per week, in three successive weeks. Tumor volume was
monitored and animals were sacrificed if tumors reached 2000
mm.sup.3 in size or became necrotic. The PBS treated group reached
a mean tumor volume of 500 mmm.sup.3 on day 22, the chimeric IgG1
control antibody group on day 23 (FIG. 20). None of the tumors in
these groups regressed. In contrast, treatment with hu38SB19 or
mu38SB19 led to tumor regression in 8 of 10 or 6 of 10 animals,
respectively.
Example 10
Epitope Mapping Using Co-Crystallization Assay
[0400] The CDRs of the Fab fragment of hu38SB19 have been
determined by solving the crystal structure of the Fab fragment in
complex with huCD38. The residues from the Fab fragment of the
hu38SB19 antibody which interact with huCD38 have been
identified.
[0401] Mapping of the epitope and identification of the paratope
was achieved by structure determination of the crystal structure of
the huCD38 in complex with the Fab fragment from hu38SB19 at 1.53
.ANG. resolution.
[0402] Material and Methods
[0403] Generation of the DNA Constructs for Expression of the
hu38SB19 Fab Fragment in HEK293-FS.TM. Cells
[0404] In order to allow transient expression of the Fab fragment
of the hu38SB19 antibody in HEK293-FS.TM. cells, two expression
vectors encoding respectively the light chain and a CH2 and CH3 Fc
deleted form of the heavy chain were generated.
[0405] The DNA sequence encoding the light chain of hu38SB19 was
cloned, in frame and downstream of a signal peptide (i.e.
MGWSCIILFVATATGVHS from the mouse Igh-VJ558 gene), into a mammalian
expression vector, resulting in the light chain expression vector
pBH3045. The coding sequence was verified by DNA sequencing using
the forward and reverse primers 5'pXL_C31592 and 3'pXL_C31593
presented in table 66, respectively SEQ ID N.degree. 91 and 92. The
corresponding nucleotide and protein sequences are listed as SEQ ID
No.81 and No.82 and represented on FIG. 21.
[0406] The DNA sequence encoding the VH--CH1 region of the heavy
chain (or Fc deleted heavy chain) of hu38SB19 was PCR amplified
from a vector previously obtained, using a set of primers
SB19H601_L, SB19H601_c (forward) and SB19H551_c, SB19H551_L
(reverse), represented in table 6 respectively SEQ ID N.degree. 88
to 91. The His.times.6 tag coding sequence was introduced in frame
in the reverse primers to allow expression of the
hu38SB19HC-His.times.6 fusion protein. Partial NheI and HindIII
sites were added at 5' extremities of forward and reverse primers
respectively, which allow cloning PCR fragments directly into the
mammalian expression vector digested by NheI and HindIII. The
coding sequence was verified by DNA sequencing, using primers
5'pXL_C31592 (forward) or 3'pXL_C31593 (reverse) respectively SEQ
ID N.degree. 92 and 93. The resulting expression vector was named
pBH3093. It was used together with pBH3045 to express the hu38SB19
Fab fragment in HEK293-FS.TM. cells. The corresponding nucleotide
and resulting protein sequences are listed as SEQ ID No.83 and
No.84 and represented on FIG. 21.
[0407] Generation of the DNA Constructs for Expression of Soluble
huCD38
[0408] Based on the crystal structure of the human CD38
extracellular domain (Liu et al. 2005, Structure, 13:1331-1339),
the DNA sequence encoding a signal peptide (MGWSCIILFVATATGVHS,
coded by mouse IgH-VJ558 gene), followed by the huCD38
extracellular domain R45-I300 (UniProt: P28907) was generated by a
synthetic gene approach. Four mutations were introduced to prevent
potential N-glycosylation: N100D, N164A, N209D and N219D. In
addition, a NheI and a HindIII site were also added respectively at
5' and 3' of the synthetic DNA fragment. The fragment was cloned
into the mammalian expression vector between the NheI and HindIII
sites to obtain the final expression vector pBH3133. The coding
sequence was verified by DNA sequencing, using primers 5'pXL_C31592
(forward) or 3'pXL_C31593 (reverse). The corresponding nucleotide
and protein sequences are listed as SEQ ID No. 85 and No.86 and
represented on FIG. 21.
[0409] Protein Purification
[0410] The hu38SB19 Fab fragment was expressed in
suspension-cultured HEK293-FS.TM. cells by transient transfection
of the two expression plasmids, pBH3045 and pBH3093, complexed with
293fectin.TM. (Invitrogen). Culture supernatant containing the
secreted protein was harvested ten days post-transfection,
centrifuged and filtered on a 0.22 .mu.m membrane. The His-tagged
Fab fragment (Fab-His) was purified by affinity chromatography on
IMAC (HisTrap, GE Healthcare) using imidazole gradient in PBS.
Then, the pool of fractions containing Fab-His was purified by size
exclusion chromatography (Superdex 200, GE Healthcare) equilibrated
with PBS.
[0411] The soluble human CD38 was produced in HEK293-FS.TM. cells
cultured in a 8.5 L-bioreactor by transient transfection of the
expression plasmid pBH3133, encoding the unglycosylated mutant of
huCD38. Culture supernatant containing the secreted protein was
harvested seven days post-transfection, centrifuged and filtered on
0.22 .mu.m membrane. Ammonium sulphate was added to the supernatant
to a final concentration of 2 M and the pH was adjusted to 7.6.
This solution was loaded onto a Phenyl Sepahrose HP column
equilibrated with ammonium sulphate 2 M, Tris 50 mM pH=7.6. The
column was washed with the same buffer then the bound protein was
eluted with a linear gradient of ammonium sulphate (from 2 to 0 M).
The elution fractions containing unglycosylated-CD38 were pooled
and dialysed against sodium acetate buffer 50 mM and pH 4.05. The
dialysed sample was loaded onto a SP Sepharose HP column
equilibrated with the same buffer. The column was eluted with a
linear salt gradient (0-1 M NaCl). Then the pooled elution fraction
was purified by SEC (Superdex 200, GE Healthcare) equilibrated with
PBS.
[0412] After that, unglycosylated-CD38 was mixed to the hu38SB19
Fab fragment (1.5 moles of antigen/1 mole of Fab) and incubated 30
minutes at room temperature. Antigen/Fab-complex was concentrated
by spin-ultrafiltration and purified by SEC as described above.
[0413] All purifications were monitored by measuring the UV
absorbance at 280 nm. Eluted fractions were analysed by SDS-PAGE
and analytical SEC. Protein concentration was determined by using
calculated epsilon absorbance of each protein or complex at 280
nm.
[0414] Co-Crystallization Assay
[0415] Crystallization trials were made on huCD38 in complex with
the Fab fragment of hu38SB19. Human CD38 (pdb code 1YH3) was used
as search models for molecular replacement calculation; models of
the variable and constant domains of the Fab were also produced and
used, (pdb code 1578-E and 2OSL-A) to solve the crystal
structure.
[0416] A single crystallisation condition was tested and a 1.53
.ANG. dataset collected at the ESRF was used to solve the structure
of the complex. It has enabled us to analyze the interface between
the human CD38 and the Fab of the hu38SB19 anti-CD38 antibody.
[0417] Results
[0418] The mapping of the human CD38 (epitope residues are defined
as residues which contain atoms that lie within 4 .ANG. from any
atom of the CDR residues of the Fab fragment of hu38SB19) is
illustrated on FIGS. 22 A and 22B
[0419] The epitope of huCD38 when bound to the Fab fragment of
hu38SB19 includes residues Pro75, Glu76, His79, Gln107, Pro108,
Met110, Lys111, Leu112, Gly113, Thr114, Gln115, Thr116, Val117,
Pro118, Cys119, Thr148, Leu150, Arg194, Arg195, Glu198, Ala199,
Cys201 and Glu233.
[0420] The residues that are part of the epitope are represented in
dark grey on FIG. 23. This figure shows the sequence of the
R45-I300 CD38 extracellular domain. Mutations that are introduced
to silence the four glycosylation sites of CD38 are shown in light
grey in the sequence but are not represented in FIG. 22 because
they are out of the interface huCD38/hu38SB19.
[0421] The overall structure of the complex in surface
representation is represented on FIG. 24. Heavy and light chain are
coloured in black and grey, respectively. Human CD38 is coloured in
white.
[0422] Paratope Analysis
[0423] The interface between Human CD38 and the Fab fragment of
hu38SB19 does not involve all the residues in the CDR loop.
[0424] As can be seen from FIGS. 25A and B, the heavy chain CDRs
are mainly involved in binding.
[0425] The paratope of the Fab part of hu38SB19 for human CD38
involves the following residues of the light chain: Ser30, Thr31
and Val32 of Loop L1, Tyr49, Ser50, Tyr53, Tyr55 and Ile56 of L2
and His91, Tyr92, Ser93, Pro94 and Tyr96 of loop L3.
[0426] It involves in the heavy chain the following residues:
Gly26, Tyr27, Thr28, Thr30, Asp31, Tyr32 and Trp33 of Loop H1,
Tyr52, Gly54, Asp55 and Asp57 of H2 and Asp100, Tyr101, Tyr102,
Gly103, Ser104, Asn105 and Tyr110 of H3. A sequential numbering
scheme is used for the light and heavy chains.
[0427] Tables
TABLE-US-00001 TABLE 1A mu38SB19 Light Chain Framework Surface
Residues And Corresponding Residues In The Human 1.69 Antibody
##STR00017## The mu38SB19 light chain framework surface residues
and corresponding residues at the same Kabat position in the human
1.69 antibody. The residues that are different and therefore
changed in the hu38SB19 antibody are in grayed boxes. The starred
(*) residues are back mutated to the mu38SB19 residue in one or
more hu38SB19 variants.
TABLE-US-00002 TABLE 1B mu38SB19 Heavy Chain Framework Surface
Residues And Corresponding Residues In The Human 1.69 Antibody
##STR00018## The mu38SB19 heavy chain framework surface residues
and corresponding residues at the same Kabat position in the human
1.69 antibody. The residues that are different and therefore
changed in the hu38SB19 antibody are in grayed boxes.
TABLE-US-00003 TABLE 2 Primer Sequence BamIgG1
GGAGGATCCATAGACAGATGGGGGTGTCGT (SEQ ID NO. 73) TTTGGC IgG2Abam
GGAGGATCCCTTGACCAGGCATCCTAGAGT (SEQ ID NO. 74) CA EcoMH1
CTTCCGGAATTCSARGTNMAGCTGSAGSAG (SEQ ID NO. 75) TC EcoMH2
CTTCCGGAATTCSARGTNMAGCTGSAGSAG (SEQ ID NO. 76) TCWGG SacIMK
GGAGCTCGAYATTGTGMTSACMCARWCTMC (SEQ ID NO. 77) A HindKL
TATAGAGCTCAAGCTTGGATGGTGGGAAGA (SEQ ID NO. 78) TGGATACAGTTGGTGC
Primers used for the degenerate PCR reactions are based on those in
Wang et al., 2000 except HindKL which is based on Co et al. 1992.
Mixed bases are defined as follows: H = A + T + C, S = g + C, Y = C
+ T, K = G + T, M = A + C, R = A + g, W = A + T, V = A + C + G.
TABLE-US-00004 TABLE 3 The light and heavy chain PCR reaction mixes
for cloning of the 38SB19 variable region cDNA sequences. Light
Chain Reaction Mix Heavy Chain Reaction Mix 5 .mu.l 10 X PCR
reaction buffer 5 .mu.l 10 X PCR reaction buffer (Roche) (Roche) 4
.mu.l 10 mM dNTP mix 4 .mu.l 10 mM dNTP mix (2.5 mM each) (2.5 mM
each) 2 .mu.l Template (RT reaction) 2 .mu.l Template (RT reaction)
5 .mu.l 10 .mu.M Sac1MK left primer 2.5 .mu.l 10 .mu.M EcoMH1 left
primer 5 .mu.l 10 .mu.M HindKL right primer 2.5 .mu.l 10 .mu.M
EcoMH2 left primer 5 .mu.l 10 .mu.M BamIgG1 right primer 5 .mu.l
DMSO 5 .mu.l DMSO 0.5 .mu.l Taq Polymerase (Roche) 0.5 .mu.l Taq
Polymerase (Roche) 23.5 .mu.l sterile distilled H.sub.2O 23.5 .mu.l
sterile distilled H.sub.2O 50 .mu.l Total 50 .mu.l Total
TABLE-US-00005 TABLE 4 Primer Sequence Light Chain
ATGGAGTCACAGATTCAGGTC 38SB19 LC Leader (SEQ ID NO. 79) Heavy Chain
TTTTGAATTCCAGTAACTTCA 38-19HCLead1 GGTGTCCACTC (SEQ ID NO. 80) The
5' end murine leader sequence primers used for the 38SB19 second
round PCR reactions. The 3' end primers are identical to those used
in the first round reactions since they prime to the respective
constant region sequences.
TABLE-US-00006 TABLE 5 cDNA calculated and LC/MS measured molecular
weights of the murine 38SB19 antibody light and heavy chains. Light
Chain Heavy Chain Dif- Dif- cDNA LC/MS ference cDNA LC/MS ference
Mu38SB19 23735 23736 1 48805 48826 21
TABLE-US-00007 TABLE 6 Primers used in the generation of the DNA
constructs for expression of the hu38SB19 Fab Name SEQ ID N.sup.o
Sequence SB19H601_L 88 ctagCCACCATGGGTTGGAGCTGTAT TATCCTTTTTTTG
SB19H601_c 89 CCACCATGGGTTGGAGCTGTATTATC CTTTTTTTG SB19H551_c 90
TTTAGTGGTGATGGTGATGGTGTGTG TGAGTTTTGTCACAAGATTTGGG SB19H551_L 91
agctTTTAGTGGTGATGGTGATGG TGTGTGTGAGTTTTGTCACAAGAT TTGGG
5'pXL_C31592 92 CTGGCCATACACTTGAGTGACA 3'pXL_C31593 93
GCATTCTAGTTGTGGTTTGTCC
Sequence CWU 1
1
9315PRTArtificialsequence SEQ ID NO. 87 of the R45-I300 CD38
extracellular domain 1Ser Tyr Gly Met Asn1 5217PRTMus sp. 2Trp Ile
Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys1 5 10
15Gly35PRTMus sp. 3Arg Gly Phe Ala Tyr1 5415PRTMus sp. 4Arg Ala Ser
Glu Ser Val Glu Ile Tyr Gly Asn Gly Phe Met Asn1 5 10 1557PRTMus
sp. 5Arg Ala Ser Asn Leu Glu Ser1 569PRTMus sp. 6Gln Gln Ile Asn
Glu Asp Pro Phe Thr1 575PRTMus sp. 7Asn Ser Gly Met Asn1 5817PRTMus
sp. 8Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe
Lys1 5 10 15Gly95PRTMus sp. 9Arg Gly Phe Val Tyr1 51015PRTMus sp.
10Arg Ala Ser Glu Ser Val Ala Ile Tyr Gly Asn Ser Phe Leu Lys1 5 10
15117PRTMus sp. 11Arg Ala Ser Asn Leu Glu Ser1 5129PRTMus sp. 12Gln
Gln Ile Asn Glu Asp Pro Tyr Thr1 5135PRTMus sp. 13Asp Tyr Trp Met
Gln1 51417PRTMus sp. 14Thr Ile Tyr Pro Gly Asp Gly Asp Thr Gly Tyr
Ala Gln Lys Phe Lys1 5 10 15Gly1511PRTMus sp. 15Gly Asp Tyr Tyr Gly
Ser Asn Ser Leu Asp Tyr1 5 101611PRTMus sp. 16Lys Ala Ser Gln Asp
Val Ser Thr Val Val Ala1 5 10177PRTMus sp. 17Ser Ala Ser Tyr Arg
Tyr Ile1 5189PRTMus sp. 18Gln Gln His Tyr Ser Pro Pro Tyr Thr1
5195PRTMus sp. 19Gly Ser Trp Met Asn1 52017PRTMus sp. 20Arg Ile Tyr
Pro Gly Asp Gly Asp Ile Ile Tyr Asn Gly Asn Phe Arg1 5 10
15Asp2110PRTMus sp. 21Trp Gly Thr Phe Thr Pro Ser Phe Asp Tyr1 5
102211PRTMus sp. 22Lys Ala Ser Gln Asp Val Val Thr Ala Val Ala1 5
10237PRTMus sp. 23Ser Ala Ser His Arg Tyr Thr1 5249PRTMus sp. 24Gln
Gln His Tyr Thr Thr Pro Thr Thr1 5255PRTMus sp. 25Ser Tyr Thr Leu
Ser1 52617PRTMus sp. 26Thr Ile Ser Ile Gly Gly Arg Tyr Thr Tyr Tyr
Pro Asp Ser Val Glu1 5 10 15Gly278PRTMus sp. 27Asp Phe Asn Gly Tyr
Ser Asp Phe1 52811PRTMus sp. 28Lys Ala Ser Gln Val Val Gly Ser Ala
Val Ala1 5 10297PRTMus sp. 29Trp Ala Ser Thr Arg His Thr1
5309PRTMus sp. 30Gln Gln Tyr Asn Ser Tyr Pro Tyr Thr1 5315PRTMus
sp. 31Asn Phe Gly Met His1 53217PRTMus sp. 32Tyr Ile Arg Ser Gly
Ser Gly Thr Ile Tyr Tyr Ser Asp Thr Val Lys1 5 10 15Gly3311PRTMus
sp. 33Ser Tyr Tyr Asp Phe Gly Ala Trp Phe Ala Tyr1 5 103411PRTMus
sp. 34Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala1 5 10357PRTMus
sp. 35Ser Ala Ser Ser Arg Tyr Ser1 5369PRTMus sp. 36Gln Gln Tyr Asn
Ser Tyr Pro Leu Thr1 537336DNAMus sp.CDS(1)..(336) 37aac att gtg
ctg acc caa tct cca gct tct ttg gct gtg tct ctt ggg 48Asn Ile Val
Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly1 5 10 15cag agg
gcc acc ata tcc tgc aga gcc agt gaa agt gtt gag att tat 96Gln Arg
Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Glu Ile Tyr 20 25 30ggc
aat ggt ttt atg aac tgg ttc cag cag aaa cca gga cag cca ccc 144Gly
Asn Gly Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro 35 40
45aaa ctc ctc atc tat cgt gca tcc aac cta gaa tct ggg atc cct gcc
192Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala
50 55 60agg ttc agt ggc agt ggg tct agg aca gag ttc acc ctc acc att
gat 240Arg Phe Ser Gly Ser Gly Ser Arg Thr Glu Phe Thr Leu Thr Ile
Asp65 70 75 80cct gtg gag gct gat gat gtt gca acc tat tac tgt caa
caa att aat 288Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln
Gln Ile Asn 85 90 95gag gat cca ttc acg ttc ggc tcg ggg aca aag ttg
gaa ata aaa cgg 336Glu Asp Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu
Glu Ile Lys Arg 100 105 11038112PRTMus sp. 38Asn Ile Val Leu Thr
Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr
Ile Ser Cys Arg Ala Ser Glu Ser Val Glu Ile Tyr 20 25 30Gly Asn Gly
Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu
Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60Arg
Phe Ser Gly Ser Gly Ser Arg Thr Glu Phe Thr Leu Thr Ile Asp65 70 75
80Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ile Asn
85 90 95Glu Asp Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
Arg 100 105 11039336DNAMus sp.CDS(1)..(336) 39gac att gta ctg acc
caa tct cca gct tct ttg gct gtg tct cta ggg 48Asp Ile Val Leu Thr
Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly1 5 10 15cag agg gcc acc
ata tcc tgc aga gcc agt gag agt gtt gct att tat 96Gln Arg Ala Thr
Ile Ser Cys Arg Ala Ser Glu Ser Val Ala Ile Tyr 20 25 30ggc aat agt
ttt ctg aaa tgg ttc cag cag aaa ccg gga cag cca ccc 144Gly Asn Ser
Phe Leu Lys Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45aaa ctc
ctc atc tat cgt gca tcc aac cta gaa tct ggg atc cct gcc 192Lys Leu
Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60agg
ttc agt ggc agt ggg tct ggg aca gac ttc acc ctc acc att aat 240Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn65 70 75
80cct gtg gag gct gat gat gtt gca acc tat tac tgt cag caa att aat
288Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ile Asn
85 90 95gag gat ccg tac acg ttc gga ggg ggg acc aag ctg gaa ata aaa
cgg 336Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
Arg 100 105 11040112PRTMus sp. 40Asp Ile Val Leu Thr Gln Ser Pro
Ala Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys
Arg Ala Ser Glu Ser Val Ala Ile Tyr 20 25 30Gly Asn Ser Phe Leu Lys
Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr
Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn65 70 75 80Pro Val
Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ile Asn 85 90 95Glu
Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105
11041324DNAMus sp.CDS(1)..(324) 41gac att gtg atg gcc cag tct cac
aaa ttc atg tcc aca tca gtt gga 48Asp Ile Val Met Ala Gln Ser His
Lys Phe Met Ser Thr Ser Val Gly1 5 10 15gac agg gtc agc atc acc tgc
aag gcc agt cag gat gtg agt act gtt 96Asp Arg Val Ser Ile Thr Cys
Lys Ala Ser Gln Asp Val Ser Thr Val 20 25 30gtg gcc tgg tat caa cag
aaa cca gga caa tct cct aaa cga ctg att 144Val Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ser Pro Lys Arg Leu Ile 35 40 45tac tcg gca tcc tat
cgg tat att gga gtc cct gat cgc ttc act ggc 192Tyr Ser Ala Ser Tyr
Arg Tyr Ile Gly Val Pro Asp Arg Phe Thr Gly 50 55 60agt gga tct ggg
acg gat ttc act ttc acc atc agc agt gtg cag gct 240Ser Gly Ser Gly
Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala65 70 75 80gaa gac
ctg gca gtt tat tac tgt cag caa cat tat agt cct ccg tac 288Glu Asp
Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Pro Pro Tyr 85 90 95acg
ttc gga ggg ggg acc aag ctg gaa ata aaa cgg 324Thr Phe Gly Gly Gly
Thr Lys Leu Glu Ile Lys Arg 100 10542108PRTMus sp. 42Asp Ile Val
Met Ala Gln Ser His Lys Phe Met Ser Thr Ser Val Gly1 5 10 15Asp Arg
Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Val 20 25 30Val
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Arg Leu Ile 35 40
45Tyr Ser Ala Ser Tyr Arg Tyr Ile Gly Val Pro Asp Arg Phe Thr Gly
50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln
Ala65 70 75 80Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser
Pro Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 10543324DNAMus sp.CDS(1)..(324) 43gac att gtg atg acc cag tct
cac aaa ttc ttg tcc aca tca gtt gga 48Asp Ile Val Met Thr Gln Ser
His Lys Phe Leu Ser Thr Ser Val Gly1 5 10 15gac agg gtc agt atc acc
tgc aag gcc agt cag gat gtg gtt act gct 96Asp Arg Val Ser Ile Thr
Cys Lys Ala Ser Gln Asp Val Val Thr Ala 20 25 30gtt gcc tgg ttt caa
cag aaa cca gga caa tct cca aaa cta ctg att 144Val Ala Trp Phe Gln
Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45tat tcg gca tcc
cac cgg tac act gga gtc cct gat cgc ttc act ggc 192Tyr Ser Ala Ser
His Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60agt gga tct
ggg aca gat ttc act ttc acc atc atc agt gtg cag gct 240Ser Gly Ser
Gly Thr Asp Phe Thr Phe Thr Ile Ile Ser Val Gln Ala65 70 75 80gaa
gac ctg gca gtt tat tac tgt caa caa cat tat act act ccc acg 288Glu
Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Thr 85 90
95acg ttc ggt gga ggc acc aag ctg gac ttc aga cgg 324Thr Phe Gly
Gly Gly Thr Lys Leu Asp Phe Arg Arg 100 10544108PRTMus sp. 44Asp
Ile Val Met Thr Gln Ser His Lys Phe Leu Ser Thr Ser Val Gly1 5 10
15Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Val Thr Ala
20 25 30Val Ala Trp Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu
Ile 35 40 45Tyr Ser Ala Ser His Arg Tyr Thr Gly Val Pro Asp Arg Phe
Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ile Ser
Val Gln Ala65 70 75 80Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His
Tyr Thr Thr Pro Thr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Asp Phe
Arg Arg 100 10545324DNAMus sp.CDS(1)..(324) 45gac act gtg atg acc
cag tct cac aaa ttc ata tcc aca tca gtt gga 48Asp Thr Val Met Thr
Gln Ser His Lys Phe Ile Ser Thr Ser Val Gly1 5 10 15gac agg gtc agc
atc acc tgc aag gcc agt cag gtt gtg ggt agt gct 96Asp Arg Val Ser
Ile Thr Cys Lys Ala Ser Gln Val Val Gly Ser Ala 20 25 30gta gcc tgg
tat caa cag aaa cca ggg caa tct cct aaa cta ctg att 144Val Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45tac tgg
gca tcc acc cgg cac act gga gtc cct gat cgc ttc aca ggc 192Tyr Trp
Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60agt
gga tct ggg aca gat ttc act ctc acc att agc aat gtg cag tct 240Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser65 70 75
80gaa gac ttg gca gat tat ttc tgt cag caa tat aac agc tat ccg tac
288Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr
85 90 95acg ttc gga ggg ggg acc aag ctg gaa ata aaa cgg 324Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 10546108PRTMus sp.
46Asp Thr Val Met Thr Gln Ser His Lys Phe Ile Ser Thr Ser Val Gly1
5 10 15Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Val Val Gly Ser
Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu
Leu Ile 35 40 45Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg
Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Asn Val Gln Ser65 70 75 80Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln
Tyr Asn Ser Tyr Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys Arg 100 10547324DNAMus sp.CDS(1)..(324) 47gac att gtg atg
acc cag tct caa aaa ttc atg tcc aca tca gta gga 48Asp Ile Val Met
Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly1 5 10 15gac agg gtc
agc gtc acc tgc aag gcc agt cag aat gtg ggt act aat 96Asp Arg Val
Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn 20 25 30gtt gcc
tgg tat caa cac aaa cca gga caa tcc cct aaa ata atg att 144Val Ala
Trp Tyr Gln His Lys Pro Gly Gln Ser Pro Lys Ile Met Ile 35 40 45tat
tcg gcg tcc tcc cgg tac agt gga gtc cct gat cgc ttc aca ggc 192Tyr
Ser Ala Ser Ser Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly 50 55
60agt gga tct ggg aca ctt ttc act ctc acc atc aac aat gtg cag tct
240Ser Gly Ser Gly Thr Leu Phe Thr Leu Thr Ile Asn Asn Val Gln
Ser65 70 75 80gaa gac ttg gca gag tat ttc tgt cag caa tat aac agc
tat cct ctc 288Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ser
Tyr Pro Leu 85 90 95acg ttc ggc tcg ggg aca aag ttg gaa ata aaa cgg
324Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 100
10548108PRTMus sp. 48Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met
Ser Thr Ser Val Gly1 5 10 15Asp Arg Val Ser Val Thr Cys Lys Ala Ser
Gln Asn Val Gly Thr Asn 20 25 30Val Ala Trp Tyr Gln His Lys Pro Gly
Gln Ser Pro Lys Ile Met Ile 35 40 45Tyr Ser Ala Ser Ser Arg Tyr Ser
Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Leu Phe
Thr Leu Thr Ile Asn Asn Val Gln Ser65 70 75 80Glu Asp Leu Ala Glu
Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95Thr Phe Gly Ser
Gly Thr Lys Leu Glu Ile Lys Arg 100 10549342DNAMus sp.CDS(1)..(342)
49cag atc cag ttg gtg cag tct gga cct gag ctg aag aag cct gga gag
48Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu1
5 10 15aca gtc aag atc tcc tgc aag gct tct ggg tat acc ctc aca agc
tac 96Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Leu Thr Ser
Tyr 20 25 30gga atg aac tgg gtg aag cag gct cca gga aag ggt tta aag
tgg atg 144Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys
Trp Met 35 40 45ggc tgg ata aac acc tac act gga gaa cca aca tat gct
gat gac ttt 192Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala
Asp Asp Phe 50 55 60aag gga cgt ttt gcc ttc tct ttg gaa acc tct gcc
agc act gcc ttt 240Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala
Ser Thr Ala Phe65 70 75 80ttg cag atc aac aac ctc aaa aat gag gac
acg gct aca tat ttc tgt 288Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp
Thr Ala Thr Tyr Phe Cys 85 90 95gta aga cgc ggg ttt gct tac tgg ggc
caa ggg act ctg gtc act gtc 336Val Arg Arg Gly Phe Ala Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110tct gca 342Ser
Ala50114PRTMus sp. 50Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu
Lys Lys Pro Gly Glu1 5 10 15Thr Val Lys Ile Ser Cys Lys Ala Ser Gly
Tyr Thr Leu Thr Ser Tyr 20 25 30Gly Met Asn Trp Val Lys Gln Ala Pro
Gly Lys Gly Leu Lys Trp Met 35 40 45Gly Trp Ile Asn Thr Tyr Thr Gly
Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60Lys Gly Arg Phe Ala Phe Ser
Leu Glu Thr Ser Ala Ser Thr Ala Phe65 70 75 80Leu Gln Ile Asn Asn
Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95Val Arg Arg Gly
Phe Ala Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val 100 105 110Ser Ala51342DNAMus sp.CDS(1)..(342)
51cag atc cag ttg gtg cag tct gga cct gag ctg aag aag cct gga gag
48Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu1
5 10 15aca gtc aag atc tcc tgc aag gct tct ggg tat acc ttc aca aac
tct 96Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn
Ser 20 25 30gga atg aac tgg gtg aag cag gct cca gga aag ggt tta aag
tgg atg 144Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys
Trp Met 35 40 45ggc tgg ata aac acc tac act gga gag ccg aca tat gct
gat gac ttc 192Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala
Asp Asp Phe 50 55 60aag gga cgg ttt gcc ttc tct ttg gaa acc tct gcc
agc tct gcc tat 240Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala
Ser Ser Ala Tyr65 70 75 80ttg cag atc agt aac ctc aaa aat gag gac
acg gct aca tat ttc tgt 288Leu Gln Ile Ser Asn Leu Lys Asn Glu Asp
Thr Ala Thr Tyr Phe Cys 85 90 95gca aga agg ggt ttt gtt tac tgg ggc
caa ggg act ctg gta act gtc 336Ala Arg Arg Gly Phe Val Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110tct gca 342Ser
Ala52114PRTMus sp. 52Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu
Lys Lys Pro Gly Glu1 5 10 15Thr Val Lys Ile Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Asn Ser 20 25 30Gly Met Asn Trp Val Lys Gln Ala Pro
Gly Lys Gly Leu Lys Trp Met 35 40 45Gly Trp Ile Asn Thr Tyr Thr Gly
Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60Lys Gly Arg Phe Ala Phe Ser
Leu Glu Thr Ser Ala Ser Ser Ala Tyr65 70 75 80Leu Gln Ile Ser Asn
Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95Ala Arg Arg Gly
Phe Val Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 100 105 110Ser
Ala53360DNAMus sp.CDS(1)..(360) 53cag gtt cag ctc cag cag tct ggg
gct gag ctg gca aga cct ggg act 48Gln Val Gln Leu Gln Gln Ser Gly
Ala Glu Leu Ala Arg Pro Gly Thr1 5 10 15tca gtg aag ttg tcc tgt aag
gct tct ggc tac acc ttt act gac tac 96Ser Val Lys Leu Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30tgg atg cag tgg gta aaa
cag agg cct gga cag ggt ctg gag tgg att 144Trp Met Gln Trp Val Lys
Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45ggg act att tat cct
gga gat ggt gat act ggg tac gct cag aag ttc 192Gly Thr Ile Tyr Pro
Gly Asp Gly Asp Thr Gly Tyr Ala Gln Lys Phe 50 55 60aag ggc aag gcc
aca ttg act gcg gat aaa tcc tcc aaa aca gtc tac 240Lys Gly Lys Ala
Thr Leu Thr Ala Asp Lys Ser Ser Lys Thr Val Tyr65 70 75 80atg cac
ctc agc agt ttg gct tct gag gac tct gcg gtc tat tac tgt 288Met His
Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95gca
aga ggg gat tac tac ggt agt aat tct ttg gac tat tgg ggt caa 336Ala
Arg Gly Asp Tyr Tyr Gly Ser Asn Ser Leu Asp Tyr Trp Gly Gln 100 105
110gga acc tca gtc acc gtc tcc tca 360Gly Thr Ser Val Thr Val Ser
Ser 115 12054120PRTMus sp. 54Gln Val Gln Leu Gln Gln Ser Gly Ala
Glu Leu Ala Arg Pro Gly Thr1 5 10 15Ser Val Lys Leu Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Trp Met Gln Trp Val Lys Gln
Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Thr Ile Tyr Pro Gly
Asp Gly Asp Thr Gly Tyr Ala Gln Lys Phe 50 55 60Lys Gly Lys Ala Thr
Leu Thr Ala Asp Lys Ser Ser Lys Thr Val Tyr65 70 75 80Met His Leu
Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Gly Asp Tyr Tyr Gly Ser Asn Ser Leu Asp Tyr Trp Gly Gln 100 105
110Gly Thr Ser Val Thr Val Ser Ser 115 12055357DNAMus
sp.CDS(1)..(357) 55cag gtc cag tta cag caa tct gga cct gaa ctg gtg
agg cct ggg gcc 48Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val
Arg Pro Gly Ala1 5 10 15tca gtg aag att tcc tgc aaa act tct ggc tac
gca ttc agt ggc tcc 96Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr
Ala Phe Ser Gly Ser 20 25 30tgg atg aac tgg gtg aag cag agg cct gga
cag ggt cta gag tgg att 144Trp Met Asn Trp Val Lys Gln Arg Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45gga cgg att tat ccg gga gat gga gat
atc att tac aat ggg aat ttc 192Gly Arg Ile Tyr Pro Gly Asp Gly Asp
Ile Ile Tyr Asn Gly Asn Phe 50 55 60agg gac aag gtc aca ctg tct gca
gac aaa tcc tcc aac aca gcc tac 240Arg Asp Lys Val Thr Leu Ser Ala
Asp Lys Ser Ser Asn Thr Ala Tyr65 70 75 80atg cag ctc agc agc ctg
acc tct gtg gac tct gcg gtc tat ttt tgt 288Met Gln Leu Ser Ser Leu
Thr Ser Val Asp Ser Ala Val Tyr Phe Cys 85 90 95tcg aga tgg ggg aca
ttt acg ccg agt ttt gac tat tgg ggc caa ggc 336Ser Arg Trp Gly Thr
Phe Thr Pro Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110acc act ctc
aca gtc tcc tca 357Thr Thr Leu Thr Val Ser Ser 11556119PRTMus sp.
56Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Ala1
5 10 15Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Ala Phe Ser Gly
Ser 20 25 30Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu
Trp Ile 35 40 45Gly Arg Ile Tyr Pro Gly Asp Gly Asp Ile Ile Tyr Asn
Gly Asn Phe 50 55 60Arg Asp Lys Val Thr Leu Ser Ala Asp Lys Ser Ser
Asn Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Val Asp
Ser Ala Val Tyr Phe Cys 85 90 95Ser Arg Trp Gly Thr Phe Thr Pro Ser
Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr Leu Thr Val Ser Ser
11557351DNAMus sp.CDS(1)..(351) 57gac gtg aag ctg gtg gag tct ggg
gga ggc tta gtg aag cct gga ggg 48Asp Val Lys Leu Val Glu Ser Gly
Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15tcc ctg aaa ctc tcc tgt gaa
gcc tct gga ttc act ttc agt agc tat 96Ser Leu Lys Leu Ser Cys Glu
Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30acc ctg tct tgg gtt cgc
cag act ccg gag acg agg ctg gag tgg gtc 144Thr Leu Ser Trp Val Arg
Gln Thr Pro Glu Thr Arg Leu Glu Trp Val 35 40 45gca acc att agt att
ggt ggt cgc tac acc tat tat cca gac agt gtg 192Ala Thr Ile Ser Ile
Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60gag ggc cga ttc
acc atc tcc aga gac aat gcc aag aac acc ctg tac 240Glu Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80ctg caa
atg aac agt ctg aag tct gag gac aca gcc atg tat tac tgt 288Leu Gln
Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95aca
aga gat ttt aat ggt tac tct gac ttc tgg ggc caa ggc acc act 336Thr
Arg Asp Phe Asn Gly Tyr Ser Asp Phe Trp Gly Gln Gly Thr Thr 100 105
110ctc aca gtc tcc tca 351Leu Thr Val Ser Ser 11558117PRTMus sp.
58Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1
5 10 15Ser Leu Lys Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30Thr Leu Ser Trp Val Arg Gln Thr Pro Glu Thr Arg Leu Glu
Trp Val 35 40 45Ala Thr Ile Ser Ile Gly Gly Arg Tyr Thr Tyr Tyr Pro
Asp Ser Val 50 55 60Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Ser Glu Asp
Thr Ala Met Tyr Tyr Cys 85 90 95Thr Arg Asp Phe Asn Gly Tyr Ser Asp
Phe Trp Gly Gln Gly Thr Thr 100 105 110Leu Thr Val Ser Ser
11559360DNAMus sp.CDS(1)..(360) 59aat gta cag ctg gta gag tct ggg
gga ggc tta gtg cag cct gga ggg 48Asn Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15tcc cgg aaa ctc tcc tgt gca
gcc tct gga ttc act ttc agt aac ttt 96Ser Arg Lys Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Asn Phe 20 25 30gga atg cac tgg gtt cgt
cag gct cca gag aag ggt ctg gag tgg gtc 144Gly Met His Trp Val Arg
Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45gca tac att cgt agt
ggc agt ggt acc atc tac tat tca gac aca gtg 192Ala Tyr Ile Arg Ser
Gly Ser Gly Thr Ile Tyr Tyr Ser Asp Thr Val 50 55 60aag ggc cga ttc
acc atc tcc aga gac aat ccc aag aac acc ctg ttc 240Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Leu Phe65 70 75 80ctg caa
atg acc agt cta agg tct gag gac acg gcc atg tat tac tgt 288Leu Gln
Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95gca
aga tcc tac tat gat ttc ggg gcc tgg ttt gct tac tgg ggc caa 336Ala
Arg Ser Tyr Tyr Asp Phe Gly Ala Trp Phe Ala Tyr Trp Gly Gln 100 105
110ggg act ctg gtc act gtc tct gca 360Gly Thr Leu Val Thr Val Ser
Ala 115 12060120PRTMus sp. 60Asn Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Arg Lys Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asn Phe 20 25 30Gly Met His Trp Val Arg Gln
Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Ala Tyr Ile Arg Ser Gly
Ser Gly Thr Ile Tyr Tyr Ser Asp Thr Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Pro Lys Asn Thr Leu Phe65 70 75 80Leu Gln Met
Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg
Ser Tyr Tyr Asp Phe Gly Ala Trp Phe Ala Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ala 115 12061324DNAHomo
sapiensCDS(1)..(324) 61gat atc gta atg acc cag tcc cac ctg agt atg
agt acc tcc ctg gga 48Asp Ile Val Met Thr Gln Ser His Leu Ser Met
Ser Thr Ser Leu Gly1 5 10 15gat cct gtg tca atc act tgc aag gcc tca
cag gat gtg agc acc gtc 96Asp Pro Val Ser Ile Thr Cys Lys Ala Ser
Gln Asp Val Ser Thr Val 20 25 30gtt gct tgg tat cag cag aag ccc ggg
caa tca ccc aga cgt ctc atc 144Val Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ser Pro Arg Arg Leu Ile 35 40 45tac tca gca tca tac cgt tac atc
ggg gtg cct gac cga ttt act ggc 192Tyr Ser Ala Ser Tyr Arg Tyr Ile
Gly Val Pro Asp Arg Phe Thr Gly 50 55 60tct ggc gct ggc aca gat ttc
acc ttt aca att agt tcc gtc cag gcc 240Ser Gly Ala Gly Thr Asp Phe
Thr Phe Thr Ile Ser Ser Val Gln Ala65 70 75 80gaa gac ctg gcc gtg
tac tac tgc cag cag cac tac agt ccc cca tac 288Glu Asp Leu Ala Val
Tyr Tyr Cys Gln Gln His Tyr Ser Pro Pro Tyr 85 90 95act ttc ggg gga
ggg act aag ctc gaa atc aaa cgt 324Thr Phe Gly Gly Gly Thr Lys Leu
Glu Ile Lys Arg 100 10562108PRTHomo sapiens 62Asp Ile Val Met Thr
Gln Ser His Leu Ser Met Ser Thr Ser Leu Gly1 5 10 15Asp Pro Val Ser
Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Val 20 25 30Val Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Arg Leu Ile 35 40 45Tyr Ser
Ala Ser Tyr Arg Tyr Ile Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser
Gly Ala Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala65 70 75
80Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Pro Pro Tyr
85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100
10563324DNAHomo sapiensCDS(1)..(324) 63gac att gtt atg gct caa agc
cat ctg tct atg agc aca tct ctg gga 48Asp Ile Val Met Ala Gln Ser
His Leu Ser Met Ser Thr Ser Leu Gly1 5 10 15gat cct gtg tcc atc act
tgc aaa gcc agt caa gac gtg tct aca gtt 96Asp Pro Val Ser Ile Thr
Cys Lys Ala Ser Gln Asp Val Ser Thr Val 20 25 30gtt gca tgg tat caa
cag aag cca ggc cag tca ccc aga cgg ctc att 144Val Ala Trp Tyr Gln
Gln Lys Pro Gly Gln Ser Pro Arg Arg Leu Ile 35 40 45tac tca gct tct
tac cga tac atc ggg gtc cct gac aga ttt aca ggt 192Tyr Ser Ala Ser
Tyr Arg Tyr Ile Gly Val Pro Asp Arg Phe Thr Gly 50 55 60agt ggg gcc
ggt act gac ttc act ttt act atc tca tcc gta caa gcc 240Ser Gly Ala
Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala65 70 75 80gaa
gac ctg gca gta tat tac tgc cag caa cat tat tcc cca ccc tac 288Glu
Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Pro Pro Tyr 85 90
95aca ttc ggc ggg ggt act aag ctg gaa att aaa cgt 324Thr Phe Gly
Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 10564108PRTHomo sapiens
64Asp Ile Val Met Ala Gln Ser His Leu Ser Met Ser Thr Ser Leu Gly1
5 10 15Asp Pro Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr
Val 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Arg
Leu Ile 35 40 45Tyr Ser Ala Ser Tyr Arg Tyr Ile Gly Val Pro Asp Arg
Phe Thr Gly 50 55 60Ser Gly Ala Gly Thr Asp Phe Thr Phe Thr Ile Ser
Ser Val Gln Ala65 70 75 80Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln
His Tyr Ser Pro Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys Arg 100 10565360DNAHomo sapiensCDS(1)..(360) 65cag gta cag
ctc gtt cag tcc ggc gcc gag gta gct aag cct ggt act 48Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Ala Lys Pro Gly Thr1 5 10 15tcc gta
aaa ttg tcc tgt aag gct tcc ggg tac aca ttt aca gac tac 96Ser Val
Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30tgg
atg cag tgg gta aaa cag cgg cca ggt cag ggc ctg gag tgg att 144Trp
Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40
45gga aca ata tat ccc ggc gac ggc gac aca ggc tat gcc cag aag ttt
192Gly Thr Ile Tyr Pro Gly Asp Gly Asp Thr Gly Tyr Ala Gln Lys Phe
50 55 60caa ggc aag gca acc ctt act gct gat aaa tct tcc aag act gtc
tac 240Gln Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Lys Thr Val
Tyr65 70 75 80atg cat ctg tct tcc ttg gca tct gag gat agc gct gtc
tat tac tgt 288Met His Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val
Tyr Tyr Cys 85 90 95gct agg ggg gac tac tat ggg tca aat tcc ctg gat
tac tgg ggc cag 336Ala Arg Gly Asp Tyr Tyr Gly Ser Asn Ser Leu Asp
Tyr Trp Gly Gln 100 105 110ggc acc agt gtc acc gtg agc agc 360Gly
Thr Ser Val Thr Val Ser Ser 115 12066120PRTHomo sapiens 66Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Ala Lys Pro Gly Thr1 5 10 15Ser
Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25
30Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45Gly Thr Ile Tyr Pro Gly Asp Gly Asp Thr Gly Tyr Ala Gln Lys
Phe 50 55 60Gln Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Lys Thr
Val Tyr65 70 75
80Met His Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Asp Tyr Tyr Gly Ser Asn Ser Leu Asp Tyr Trp Gly
Gln 100 105 110Gly Thr Ser Val Thr Val Ser Ser 115 12067324DNAHomo
sapiensCDS(1)..(324) 67gac acc gtg atg acc cag tcc ccc tcc acc atc
tcc acc tct gtg ggc 48Asp Thr Val Met Thr Gln Ser Pro Ser Thr Ile
Ser Thr Ser Val Gly1 5 10 15gac cgg gtg tcc atc acc tgt aag gcc tcc
cag gtg gtg ggc tcc gcc 96Asp Arg Val Ser Ile Thr Cys Lys Ala Ser
Gln Val Val Gly Ser Ala 20 25 30gtg gcc tgg tat cag cag aag cct ggc
cag tcc cct aag ctg ctg atc 144Val Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ser Pro Lys Leu Leu Ile 35 40 45tac tgg gcc tcc acc cgg cat acc
ggc gtg cct gac cgg ttc acc ggc 192Tyr Trp Ala Ser Thr Arg His Thr
Gly Val Pro Asp Arg Phe Thr Gly 50 55 60tcc ggc agc ggc acc gac ttc
acc ctg acc atc tcc aac gtg cag tcc 240Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Asn Val Gln Ser65 70 75 80gac gac ctg gcc gac
tac ttc tgc cag cag tac aac tcc tac cct tac 288Asp Asp Leu Ala Asp
Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr 85 90 95acc ttt ggc ggc
gga aca aag ctg gag atc aag cgt 324Thr Phe Gly Gly Gly Thr Lys Leu
Glu Ile Lys Arg 100 10568108PRTHomo sapiens 68Asp Thr Val Met Thr
Gln Ser Pro Ser Thr Ile Ser Thr Ser Val Gly1 5 10 15Asp Arg Val Ser
Ile Thr Cys Lys Ala Ser Gln Val Val Gly Ser Ala 20 25 30Val Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Trp
Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser65 70 75
80Asp Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr
85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100
10569324DNAHomo sapiensCDS(1)..(324) 69gac acc gtg atg acc cag tcc
ccc tcc tcc atc tcc acc tcc atc ggc 48Asp Thr Val Met Thr Gln Ser
Pro Ser Ser Ile Ser Thr Ser Ile Gly1 5 10 15gac cgg gtg tcc atc acc
tgt aag gcc tcc cag gtg gtg ggc tcc gcc 96Asp Arg Val Ser Ile Thr
Cys Lys Ala Ser Gln Val Val Gly Ser Ala 20 25 30gtg gcc tgg tat cag
cag aag cct ggc cag tcc cct aag ctg ctg atc 144Val Ala Trp Tyr Gln
Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45tac tgg gcc tcc
acc cgg cat acc ggc gtg cct gcc cgg ttc acc ggc 192Tyr Trp Ala Ser
Thr Arg His Thr Gly Val Pro Ala Arg Phe Thr Gly 50 55 60tcc ggc agc
ggc acc gac ttc acc ctg acc atc tcc aac gtg cag tcc 240Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser65 70 75 80gag
gac ctg gcc gac tac ttc tgc cag cag tac aac tcc tac cct tac 288Glu
Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr 85 90
95acc ttt ggc ggc gga aca aag ctg gag atc aag cgt 324Thr Phe Gly
Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 10570108PRTHomo sapiens
70Asp Thr Val Met Thr Gln Ser Pro Ser Ser Ile Ser Thr Ser Ile Gly1
5 10 15Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Val Val Gly Ser
Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu
Leu Ile 35 40 45Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Ala Arg
Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Asn Val Gln Ser65 70 75 80Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln
Tyr Asn Ser Tyr Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys Arg 100 10571351DNAHomo sapiensCDS(1)..(351) 71gag gtg cag
ctg gtg gag tct ggc ggc gga ctg gtg aag cct ggc ggc 48Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15tcc ctg
agg ctg tcc tgt gag gcc tcc ggc ttc acc ttc tcc tcc tac 96Ser Leu
Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30acc
ctg tcc tgg gtg agg cag acc cct ggc aag ggc ctg gag tgg gtg 144Thr
Leu Ser Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val 35 40
45gcc acc atc tcc atc ggc ggc agg tac acc tac tac cct gac tcc gtg
192Ala Thr Ile Ser Ile Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Ser Val
50 55 60aag ggc cgg ttc acc atc tcc cgg gac aac gcc aag aac acc ctg
tac 240Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu
Tyr65 70 75 80ctg cag atg aac tcc ctg aag tcc gag gac acc gcc atg
tac tac tgt 288Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Met
Tyr Tyr Cys 85 90 95acc cgg gac ttc aac ggc tac tcc gac ttc tgg ggc
cag ggc acc aca 336Thr Arg Asp Phe Asn Gly Tyr Ser Asp Phe Trp Gly
Gln Gly Thr Thr 100 105 110ctg acc gtg tcc tcc 351Leu Thr Val Ser
Ser 11572117PRTHomo sapiens 72Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Glu Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Thr Leu Ser Trp Val Arg Gln
Thr Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Ile Gly
Gly Arg Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Thr Arg
Asp Phe Asn Gly Tyr Ser Asp Phe Trp Gly Gln Gly Thr Thr 100 105
110Leu Thr Val Ser Ser 1157336DNAMus sp. 73ggaggatcca tagacagatg
ggggtgtcgt tttggc 367432DNAMus sp. 74ggaggatccc ttgaccaggc
atcctagagt ca 327532DNAMus sp.misc_feature(1)..(32)mixed bases are
defined as follows H=A+T+C, S=G+C, Y=C+T, K= G+T, M=A+C, R=A+G,
W=A+T, V = A+C+G, N = A+C+G+T 75cttccggaat tcsargtnma gctgsagsag tc
327635DNAMus sp.misc_feature(1)..(35)mixed bases are defined as
follows H=A+T+C, S=G+C, Y=C+T, K= G+T, M=A+C, R=A+G, W=A+T, V =
A+C+G, N = A+C+G+T 76cttccggaat tcsargtnma gctgsagsag tcwgg
357731DNAMus sp.misc_feature(1)..(31)mixed bases are defined as
follows H=A+T+C, S=G+C, Y=C+T, K= G+T, M=A+C, R=A+G, W=A+T, V =
A+C+G, N = A+C+G+T 77ggagctcgay attgtgmtsa cmcarwctmc a
317846DNAMus sp. 78tatagagctc aagcttggat ggtgggaaga tggatacagt
tggtgc 467921DNAMus sp. 79atggagtcac agattcaggt c 218032DNAMus sp.
80ttttgaattc cagtaacttc aggtgtccac tc
3281702DNAArtificialhu38SB19-light chain nucleotide sequence from
pBH3045 81atggggtggt catgtattat tctttttctc gttgccaccg ctactggagt
gcacagtgat 60atcgtaatga cccagtccca cctgagtatg agtacctccc tgggagatcc
tgtgtcaatc 120acttgcaagg cctcacagga tgtgagcacc gtcgttgctt
ggtatcagca gaagcccggg 180caatcaccca gacgtctcat ctactcagca
tcataccgtt acatcggggt gcctgaccga 240tttactggct ctggcgctgg
cacagatttc acctttacaa ttagttccgt ccaggccgaa 300gacctggccg
tgtactactg ccagcagcac tacagtcccc catacacttt cgggggaggg
360actaagctcg aaatcaaacg tacggtggct gcaccatctg tcttcatctt
cccgccatct 420gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc
tgctgaataa cttctatccc 480agagaggcca aagtacagtg gaaggtggat
aacgccctcc aatcgggtaa ctcccaggag 540agtgtcacag agcaggacag
caaggacagc acctacagcc tcagcagcac cctgacgctg 600agcaaagcag
actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg
660agctcgcccg tcacaaagag cttcaacagg ggagagtgtt ag
70282233PRTArtificialhu38SB19 light chain deduced protein sequence
from pBH3045 82Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr
Ala Thr Gly1 5 10 15Val His Ser Asp Ile Val Met Thr Gln Ser His Leu
Ser Met Ser Thr 20 25 30Ser Leu Gly Asp Pro Val Ser Ile Thr Cys Lys
Ala Ser Gln Asp Val 35 40 45Ser Thr Val Val Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ser Pro Arg 50 55 60Arg Leu Ile Tyr Ser Ala Ser Tyr Arg
Tyr Ile Gly Val Pro Asp Arg65 70 75 80Phe Thr Gly Ser Gly Ala Gly
Thr Asp Phe Thr Phe Thr Ile Ser Ser 85 90 95Val Gln Ala Glu Asp Leu
Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser 100 105 110Pro Pro Tyr Thr
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr 115 120 125Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 130 135
140Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro145 150 155 160Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly 165 170 175Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr 180 185 190Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His 195 200 205Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val 210 215 220Thr Lys Ser Phe
Asn Arg Gly Glu Cys225 23083762DNAArtificialhu38SB19 heavy
chain-1-247-His6 nucleotide sequence from pBH3093 83atgggttgga
gctgtattat cctttttttg gtggccaccg ctactggcgt acacagccag 60gtacagctcg
ttcagtccgg cgccgaggta gctaagcctg gtacttccgt aaaattgtcc
120tgtaaggctt ccgggtacac atttacagac tactggatgc agtgggtaaa
acagcggcca 180ggtcagggcc tggagtggat tggaacaata tatcccggcg
acggcgacac aggctatgcc 240cagaagtttc aaggcaaggc aacccttact
gctgataaat cttccaagac tgtctacatg 300catctgtctt ccttggcatc
tgaggatagc gctgtctatt actgtgctag gggggactac 360tatgggtcaa
attccctgga ttactggggc cagggcacca gtgtcaccgt gagcagcgcc
420tccaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac
ctctgggggc 480acagcggccc tgggctgcct ggtcaaggac tacttccccg
aaccggtgac ggtgtcgtgg 540aactcaggcg ccctgaccag cggcgtgcac
accttcccgg ctgtcctaca gtcctcagga 600ctctactccc tcagcagcgt
ggtgaccgtg ccctccagca gcttgggcac ccagacctac 660atctgcaacg
tgaatcacaa gcccagcaac accaaggtgg acaagaaagt tgagcccaaa
720tcttgtgaca aaactcacac acaccatcac catcaccact aa
76284253PRTArtificialhu38SB19-heavy chain-1-247-His6 deduced
protein sequence from pBH3093 84Met Gly Trp Ser Cys Ile Ile Leu Phe
Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Ala Lys 20 25 30Pro Gly Thr Ser Val Lys Leu
Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45Thr Asp Tyr Trp Met Gln
Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 50 55 60Glu Trp Ile Gly Thr
Ile Tyr Pro Gly Asp Gly Asp Thr Gly Tyr Ala65 70 75 80Gln Lys Phe
Gln Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Lys 85 90 95Thr Val
Tyr Met His Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val 100 105
110Tyr Tyr Cys Ala Arg Gly Asp Tyr Tyr Gly Ser Asn Ser Leu Asp Tyr
115 120 125Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr
Lys Gly 130 135 140Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly145 150 155 160Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val 165 170 175Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys225 230
235 240Ser Cys Asp Lys Thr His Thr His His His His His His 245
25085828DNAArtificialNucleotide sequence of the
huCD38-unglycosylated mutant-N100D-N164A-N209D-N219D from pBH3133
85atgggctgga gttgtataat tctctttctt gtggctactg ctaccggtgt gcattcccgg
60tggcggcagc agtggagcgg acccggcact acaaagcgct ttccagaaac cgttctggct
120agatgcgtga agtacacaga aattcatccc gagatgaggc atgtagactg
tcagagcgtg 180tgggatgcct ttaagggcgc ttttatttcc aaacatccat
gcgatatcac agaggaagac 240tatcagccat tgatgaaact cggcacgcag
acagtgccat gcaataagat actgctttgg 300tcacgaatca aggacctcgc
tcaccagttt acacaagttc agcgagatat gttcacactg 360gaggatacac
tgctcggata tctcgccgac gatctcactt ggtgcggtga atttgcgacc
420agcaaaatta actaccagtc ttgtcccgat tggcgcaaag attgctccaa
caatcccgtg 480tcagtatttt ggaagaccgt aagtaggagg ttcgctgagg
cagcctgtga tgtggtgcac 540gtgatgttgg atggaagccg atcaaaaatc
ttcgacaagg actctacctt tggcagtgtg 600gaggtgcaca acctccagcc
tgaaaaggtc cagacattgg aagcctgggt gatacacggc 660ggccgggagg
attcccgaga tctgtgccag gatcctacta ttaaagaact ggaatccatc
720atctccaaaa ggaacataca gtttagctgt aaaaacattt acaggcctga
caagtttctc 780cagtgtgtga agaatccaga ggattcctcc tgcaccagcg aaatctaa
82886275PRTArtificialCD38-unglycosylated
mutant-N100D-N164A-N209D-N219D, deduced protein sequence from
pBH3133 86Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala
Thr Gly1 5 10 15Val His Ser Arg Trp Arg Gln Gln Trp Ser Gly Pro Gly
Thr Thr Lys 20 25 30Arg Phe Pro Glu Thr Val Leu Ala Arg Cys Val Lys
Tyr Thr Glu Ile 35 40 45His Pro Glu Met Arg His Val Asp Cys Gln Ser
Val Trp Asp Ala Phe 50 55 60Lys Gly Ala Phe Ile Ser Lys His Pro Cys
Asp Ile Thr Glu Glu Asp65 70 75 80Tyr Gln Pro Leu Met Lys Leu Gly
Thr Gln Thr Val Pro Cys Asn Lys 85 90 95Ile Leu Leu Trp Ser Arg Ile
Lys Asp Leu Ala His Gln Phe Thr Gln 100 105 110Val Gln Arg Asp Met
Phe Thr Leu Glu Asp Thr Leu Leu Gly Tyr Leu 115 120 125Ala Asp Asp
Leu Thr Trp Cys Gly Glu Phe Ala Thr Ser Lys Ile Asn 130 135 140Tyr
Gln Ser Cys Pro Asp Trp Arg Lys Asp Cys Ser Asn Asn Pro Val145 150
155 160Ser Val Phe Trp Lys Thr Val Ser Arg Arg Phe Ala Glu Ala Ala
Cys 165 170 175Asp Val Val His Val Met Leu Asp Gly Ser Arg Ser Lys
Ile Phe Asp 180 185 190Lys Asp Ser Thr Phe Gly Ser Val Glu Val His
Asn Leu Gln Pro Glu 195 200 205Lys Val Gln Thr Leu Glu Ala Trp Val
Ile His Gly Gly Arg Glu Asp 210 215 220Ser Arg Asp Leu Cys Gln Asp
Pro Thr Ile Lys Glu Leu Glu Ser Ile225 230 235 240Ile Ser Lys Arg
Asn Ile Gln Phe Ser Cys Lys Asn Ile Tyr Arg Pro 245 250 255Asp Lys
Phe Leu Gln Cys Val Lys Asn Pro Glu Asp Ser Ser Cys Thr 260 265
270Ser Glu Ile 27587256PRTArtificialsequence SEQ ID NO. 87 of the
R45-I300 CD38 extracellular domain 87Arg Trp Arg Gln Gln Trp Ser
Gly Pro Gly Thr Thr Lys Arg Phe Pro1 5 10 15Glu Thr Val Leu Ala Arg
Cys Val Lys Tyr Thr Glu Ile His Pro Glu 20 25 30Met Arg His Val Asp
Cys Gln Ser Val Trp Asp Ala Phe Lys Gly Ala 35 40 45Phe Ile Ser Lys
His Pro Cys Asp Ile Thr Glu Glu Asp Tyr Gln Pro 50 55 60Leu Met Lys
Leu Gly Thr Gln Thr Val Pro Cys Asn Lys Ile Leu Leu65 70 75 80Trp
Ser Arg Ile Lys Asp Leu Ala His Gln Phe Thr Gln Val Gln Arg 85 90
95Asp Met Phe Thr Leu Glu Asp Thr Leu Leu Gly Tyr Leu Ala Asp Asp
100 105 110Leu Thr Trp Cys Gly Glu Phe Ala Thr Ser Lys Ile Asn Tyr
Gln Ser 115 120
125Cys Pro Asp Trp Arg Lys Asp Cys Ser Asn Asn Pro Val Ser Val Phe
130 135 140Trp Lys Thr Val Ser Arg Arg Phe Ala Glu Ala Ala Cys Asp
Val Val145 150 155 160His Val Met Leu Asp Gly Ser Arg Ser Lys Ile
Phe Asp Lys Asp Ser 165 170 175Thr Phe Gly Ser Val Glu Val His Asn
Leu Gln Pro Glu Lys Val Gln 180 185 190Thr Leu Glu Ala Trp Val Ile
His Gly Gly Arg Glu Asp Ser Arg Asp 195 200 205Leu Cys Gln Asp Pro
Thr Ile Lys Glu Leu Glu Ser Ile Ile Ser Lys 210 215 220Arg Asn Ile
Gln Phe Ser Cys Lys Asn Ile Tyr Arg Pro Asp Lys Phe225 230 235
240Leu Gln Cys Val Lys Asn Pro Glu Asp Ser Ser Cys Thr Ser Glu Ile
245 250 2558839DNAArtificialPrimer SB19H601_L 88ctagccacca
tgggttggag ctgtattatc ctttttttg 398935DNAArtificialPrimer
SB19H601_c 89ccaccatggg ttggagctgt attatccttt ttttg
359049DNAArtificialPrimer SB19H551_c 90tttagtggtg atggtgatgg
tgtgtgtgag ttttgtcaca agatttggg 499153DNAArtificialPrimer
SB19H551_L 91agcttttagt ggtgatggtg atggtgtgtg tgagttttgt cacaagattt
ggg 539222DNAArtificialPrimer 5'pXL_C31592 92ctggccatac acttgagtga
ca 229322DNAArtificialPrimer 3'pXL_C31593 93gcattctagt tgtggtttgt
cc 22
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References