U.S. patent application number 14/847428 was filed with the patent office on 2016-03-10 for combination therapies with anti-cd38 antibodies.
The applicant listed for this patent is Janssen Biotech, Inc.. Invention is credited to Henk M. Lokhorst, Tuna Mutis, Inger S Nijhof, Niels Van de Donk.
Application Number | 20160067205 14/847428 |
Document ID | / |
Family ID | 55436472 |
Filed Date | 2016-03-10 |
United States Patent
Application |
20160067205 |
Kind Code |
A1 |
Lokhorst; Henk M. ; et
al. |
March 10, 2016 |
Combination Therapies with Anti-CD38 Antibodies
Abstract
The present invention relates to combination therapies with
anti-CD38 antibodies and all-trans retinoic acid.
Inventors: |
Lokhorst; Henk M.; (De
Boelelaan, NL) ; Mutis; Tuna; (Zoeterwoude, NL)
; Nijhof; Inger S; (Utrecht, NL) ; Van de Donk;
Niels; (Amsterdam, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Janssen Biotech, Inc. |
Horsham |
PA |
US |
|
|
Family ID: |
55436472 |
Appl. No.: |
14/847428 |
Filed: |
September 8, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62047877 |
Sep 9, 2014 |
|
|
|
62087287 |
Dec 4, 2014 |
|
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Current U.S.
Class: |
424/139.1 |
Current CPC
Class: |
A61K 39/39558 20130101;
C07K 16/2896 20130101; C07K 2317/732 20130101; C07K 2317/56
20130101; C07K 2317/51 20130101; C07K 2317/92 20130101; A61K 31/203
20130101; C07K 2317/734 20130101; A61P 35/00 20180101; C07K 2317/34
20130101; C07K 2317/565 20130101; A61K 2039/505 20130101; A61K
39/39558 20130101; A61K 2300/00 20130101; A61K 31/203 20130101;
A61K 2300/00 20130101 |
International
Class: |
A61K 31/203 20060101
A61K031/203; A61K 39/395 20060101 A61K039/395; C07K 16/40 20060101
C07K016/40 |
Claims
1. A method of treating a subject having a CD38-positive
hematological malignancy, comprising administering to the subject
in need thereof an anti-CD38 antibody in combination with all-trans
retinoic acid (ATRA).
2. The method of claim 1, wherein the anti-CD38 antibody induces
killing of CD38-expressing cells in vitro by antibody-dependent
cell-mediated cytotoxicity (ADCC) or complement dependent
cytotoxicity (CDC).
3. The method of claim 2, wherein the anti-CD38 antibody induces
killing of the CD38-expressing cells by CDC in vitro.
4. The method of claim 2, wherein the anti-CD38 antibody induces
killing of the CD38-expressing cells by ADCC in vitro.
5. The method of any one of claims 1, 2 3 or 4, wherein the
CD38-positive hematological malignancy is multiple myeloma (MM),
acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL),
diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma (BL),
follicular lymphoma (FL) or mantle-cell lymphoma (MCL).
6. The method of claim 5, wherein the CD38-positive hematological
malignancy is MM.
7. The method of claim 6, wherein the subject is resistant to or
has acquired resistance to treatment with at least one
chemotherapeutic agent, an anti-CD38 antibody, or a combination of
at least one chemotherapeutic agent and an anti-CD38 antibody.
8. The method of claim 7, wherein the at least one chemotherapeutic
agent is lenalidomide, bortezomib, melphalan, dexamethasone or
thalidomide.
9. The method of claim 8, wherein the at least one chemotherapeutic
agent is lenalidomide or bortezomib.
10. The method of claim 1, 2, 3 or 4, wherein the anti-CD38
antibody is of IgG1, IgG2, IgG3 or IgG4 isotype.
11. The method of claim 10, wherein the anti-CD38 antibody is of
IgG1 isotype.
12. The method of claim 1, wherein the anti-CD38 antibody competes
for binding to CD38 with an antibody comprising a heavy chain
variable region (VH) of SEQ ID NO: 4 and a light chain variable
region (VL) of SEQ ID NO: 5.
13. The method of claim 12, wherein the antibody binds to the
region SKRNIQFSCKNIYR (SEQ ID NO: 2) and the region EKVQTLEAWVIHGG
(SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1).
14. The method of claim 13, wherein the anti-CD38 antibody
comprises the heavy chain complementarity determining regions
(HCDR) 1 (HCDR1), 2 (HCDR2) and 3 (HCDR3) sequences of SEQ ID NOs:
6, 7 and 8, respectively.
15. The method of claim 14, wherein the anti-CD38 antibody
comprises the light chain complementarity determining regions
(LCDR) 1 (LCDR1), 2 (LCDR2) and 3 (LCDR3) sequences of SEQ ID NOs:
9, 10 and 11, respectively.
16. The method of claim 15, wherein the anti-CD38 antibody
comprises the heavy chain variable region (VH) of SEQ ID NO: 4 and
the light chain variable region (VL) of SEQ ID NO: 5.
17. The method of claim 1, wherein the anti-CD38 antibody comprises
a heavy chain comprising an amino acid sequence that is 95%, 96%,
97%, 98% or 99% identical to that of SEQ ID NO: 12 and a light
chain comprising an amino acid sequence that is 95%, 96%, 97%, 98%
or 99% identical to that of SEQ ID NO: 13.
18. The method of claim 1, wherein the anti-CD38 antibody comprises
the heavy chain of SEQ ID NO: 12 and the light chain of SEQ ID NO:
13.
19. The method of claim 1, wherein the anti-CD38 antibody comprises
the VH of SEQ ID NO: 14 and the VL of SEQ ID NO: 15.
20. The method of claim 1, wherein the anti-CD38 antibody comprises
the VH of SEQ ID NO: 16 and the VL of SEQ ID NO: 17.
21. The method of claim 1, wherein the anti-CD38 antibody comprises
the VH of SEQ ID NO: 18 and the VL of SEQ ID NO: 19.
22. The method of claim 1, wherein the anti-CD38 antibody comprises
the VH of SEQ ID NO: 20 and the VL of SEQ ID NO: 21.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 62/087,287 filed 4 Dec. 2014 and U.S.
Provisional Application Ser. No. 62/047,877, filed 9 Sep. 2014, the
entire contents of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to combination therapies with
anti-CD38 antibodies and all-trans retinoic acid.
BACKGROUND OF THE INVENTION
[0003] B-cell malignancies include B-cell chronic lymphocytic
leukemia, mantle cell lymphoma, Burkitt lymphoma, follicular
lymphoma, diffuse large B-cell lymphoma, multiple myeloma,
Hodgkin's lymphoma, hairy cell leukemia, primary effusion lymphoma
and AIDS-related Non-Hodgkin's Lymphoma. B-cell malignancies
comprise more than 85% of diagnosed lymphomas.
[0004] Multiple myeloma (MM) is a B cell malignancy characterized
by the latent accumulation of secretory plasma cells in bone marrow
with a low proliferative index and an extended life span. The
disease ultimately attacks bones and bone marrow, resulting in
multiple tumors and lesions throughout the skeletal system.
Approximately 1% of all cancers, and slightly more than 10% of all
hematologic malignancies, can be attributed to MM. Incidence of MM
increases in the aging population, with the median age at time of
diagnosis being about 61 years.
[0005] CD38 is a type II membrane protein having function in
receptor-mediated adhesion and signaling as well as mediating
calcium mobilization via its ecto-enzymatic activity, catalyzing
formation of cyclic ADP-ribose (cADPR) from NAD.sup.+ and also
hydrolyzing cADPR into ADP-ribose (ADPR). CD38 mediates cytokine
secretion and activation and proliferation of lymphocytes (Funaro
et al., J Immunology 145:2390-6, 1990; Terhorst et al., Cell
771-80, 1981; Guse et al., Nature 398:70-3, 1999), and via its NAD
glycohydrolase activity regulates extracellular NAD.sup.+ levels
which have been implicated in modulating the regulatory T-cell
compartment (Adriouch et al., 14:1284-92, 2012; Chiarugi et al.,
Nature Reviews 12:741-52, 2012).
[0006] CD38 is expressed on MM malignant plasma cells, and is
implicated in various hematological malignancies.
[0007] Currently available therapies for MM include chemotherapy,
stem cell transplantation, Thalomid.RTM. (thalidomide),
Revlimid.RTM. (lenalidomide), Velcade.RTM. (bortezomib),
Aredia.RTM. (pamidronate), and Zometa.RTM. (zoledronic acid).
Current treatment protocols, which include a combination of
chemotherapeutic agents such as vincristine, BCNU, melphalan,
cyclophosphamide, adriamycin, and prednisone or dexamethasone,
yield a complete remission rate of only about 5%. Median survival
is approximately 36-48 months from the time of diagnosis. Recent
advances using high dose chemotherapy followed by autologous bone
marrow or peripheral blood mononuclear cell transplantation have
increased the complete remission rate and remission duration, yet
overall survival has only been slightly prolonged, and no evidence
for a cure has been obtained. Ultimately, all MM patients relapse,
even under maintenance therapy with interferon-alpha (IFN-.alpha.)
alone or in combination with steroids. Thus, there is a need for
additional therapies for the treatment of multiple myeloma and
other B-cell malignancies.
SUMMARY OF THE INVENTION
[0008] One embodiment of the invention is a method of treating a
subject having a CD38-positive hematological malignancy, comprising
administering to a patient in need thereof an anti-CD38 antibody in
combination with all-trans retinoic acid (ATRA), wherein the
anti-CD38 antibody induces killing of CD38-expressing cells in
vitro by antibody-dependent cell-mediated cytotoxicity (ADCC) or
complement dependent cytotoxicity (CDC).
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1A shows that all-trans retinoic acid (ATRA) enhances
CD38 expression on multiple myeloma (MM) cell lines in a dose
dependent manner. MM cell lines RPMI8226, UM9 and XG1 were
incubated with RPMI-1640 medium alone or with 0-25 nM ATRA for 48
hours and then harvested to determine CD38 expression by flow
cytometry. The graph shows results of one representative
experiment. The Y axis shows the fold increase of mean fluorescent
intensity (MFI) of CD38 surface expression.
[0010] FIG. 1B shows that ATRA enhances CD38 expression on MM cell
lines in a time dependent manner. MM cell lines RPMI8226, UM9 and
XG1 were incubated with RPMI-1640 medium alone or with 10 nM ATRA
for 24, 48, 72 or 96 hours and then harvested to determine CD38
expression by flow cytometry. The graph shows results of one
representative experiment. The Y axis shows the fold increase of
mean fluorescent intensity (MFI) of CD38 surface expression.
[0011] FIG. 2 shows that ATRA enhances CD38 expression on bone
marrow mononuclear cells (BM-MNCs) from MM patients ex vivo.
BM-MNCs from 26 MM patients were incubated with RPMI-1640 medium
alone or with 10 nM ATRA for 48 hours and then harvested to
determine CD38 expression by flow cytometry. The Y axis shows the
MFI of CD38 surface expression. Medium: medium at 0 hours. ns: not
significant. ***p<0.001; ****p<0.0001.
[0012] FIG. 3A shows daratumumab-induced complement-dependent
cytotoxicity (CDC) (top panel) and antibody-dependent cell mediated
cytotoxicity (ADCC) (bottom panel) in MM XG1 cell line pretreated
with or without 10 nM ATRA for 48 hours prior to performing CDC or
ADCC in the presence of 10 .mu.g/ml daratumumab. The Y axis shows
the percent (%) CDC or ADCC. Data show the mean and SEM of at least
three experiments. p-values between the indicated groups were
calculated using a paired student's t test. Dara: daratumumab; *
p<0.05; ** p<0.01.
[0013] FIG. 3B shows daratumumab-induced CDC (top panel) and ADCC
(bottom panel) in MM RPMI8226 cell line pretreated with or without
10 nM ATRA for 48 hours prior to performing CDC or ADCC in the
presence of 10 .mu.g/ml daratumumab. The Y axis shows the percent
(%) CDC or ADCC. Data show the mean and SEM of at least three
experiments. P-values between the indicated groups were calculated
using a paired student's t test. Dara: daratumumab; ns: not
significant.
[0014] FIG. 3C shows daratumumab-induced CDC (top panel) and ADCC
(bottom panel) in MM UM9 cell line pretreated with or without 10 nM
ATRA for 48 hours prior to performing CDC or ADCC in the presence
of 10 .mu.g/ml daratumumab. The Y axis shows the percent (%) CDC or
ADCC. Data show the mean and SEM of at least three experiments.
P-values between the indicated groups were calculated using a
paired student's t test. Dara: daratumumab; * p<0.05; ns: not
significant.
[0015] FIG. 4A shows that pretreatment of primary MM cells for 48
hours with 10 nM ATRA potentiates daratumumab-mediated CDC of the
primary MM cells. MM cells were pretreated for 48 hours with or
without 10 nM ATRA as indicated in the Figure at daratumumab
concentrations ranging from 0-10 .mu.g/ml. The graph shows pooled
results of 16 patient samples. *** p<0.001, **** p<0.0001.
DARA: daratumumab.
[0016] FIG. 4B shows that pretreatment of primary MM cells for 48
hours with 10 nM ATRA potentiates daratumumab-mediated ADCC of the
primary MM cells. MM cells were pretreated for 48 hours with or
without 10 nM ATRA as indicated in the Figure at daratumumab
concentrations ranging from 0-10 .mu.g/ml. The graph shows pooled
results of 13 patient samples. * p<0.05. DARA: daratumumab.
[0017] FIG. 5A shows the results of in vitro CDC of primary MM
cells isolated from patient 1 and patient 2 pretreated for 48 hours
with or without 10 nM ATRA as indicated in the Figure at
daratumumab concentrations ranging from 1-10 .mu.g/ml.
[0018] FIG. 5B shows the results of in vitro CDC of primary MM
cells isolated from patient 3 and patient 4 pretreated for 48 hours
with or without 10 nM ATRA as indicated in the Figure at
daratumumab concentrations ranging from 1-10 .mu.g/ml.
[0019] FIG. 5C shows the results of in vitro CDC of primary MM
cells isolated from patient 5 and patient 6 pretreated for 48 hours
with or without 10 nM ATRA as indicated in the Figure at
daratumumab concentrations ranging from 1-10 .mu.g/ml.
[0020] FIG. 5D shows the results of in vitro CDC of primary MM
cells isolated from patient 7 and patient 8 pretreated for 48 hours
with or without 10 nM ATRA as indicated in the Figure at
daratumumab concentrations ranging from 1-10 .mu.g/ml.
[0021] FIG. 5E shows the results of in vitro CDC of primary MM
cells isolated from patient 9 and patient 10 pretreated for 48
hours with or without 10 nM ATRA as indicated in the Figure at
daratumumab concentrations ranging from 1-10 .mu.g/ml.
[0022] FIG. 5F shows the results of in vitro CDC of primary MM
cells isolated from patient 11 and patient 12 pretreated for 48
hours with or without 10 nM ATRA as indicated in the Figure at
daratumumab concentrations ranging from 1-10 .mu.g/ml.
[0023] FIG. 5G shows the results of in vitro CDC of primary MM
cells isolated from patient 13 and patient 14 pretreated for 48
hours with or without 10 nM ATRA as indicated in the Figure at
daratumumab concentrations ranging from 1-10 .mu.g/ml.
[0024] FIG. 5H shows the results of in vitro CDC of primary MM
cells isolated from patient 15 and patient 16 pretreated for 48
hours with or without 10 nM ATRA as indicated in the Figure at
daratumumab concentrations ranging from 1-10 .mu.g/ml.
[0025] FIG. 6A shows the results of in vitro ADCC of primary MM
cells isolated from patient 3 and patient 4 pretreated for 48 hours
with or without 10 nM ATRA as indicated in the Figure at
daratumumab concentrations ranging from 1-10 .mu.g/ml.
[0026] FIG. 6B shows the results of in vitro ADCC of primary MM
cells isolated from patient 7 and patient 8 pretreated for 48 hours
with or without 10 nM ATRA as indicated in the Figure at
daratumumab concentrations ranging from 1-10 .mu.g/ml.
[0027] FIG. 6C shows the results of in vitro ADCC of primary MM
cells isolated from patient 9 and patient 10 pretreated for 48
hours with or without 10 nM ATRA as indicated in the Figure at
daratumumab concentrations ranging from 1-10 .mu.g/ml.
[0028] FIG. 6D shows the results of in vitro ADCC of primary MM
cells isolated from patient 14 and patient 15 pretreated for 48
hours with or without 10 nM ATRA as indicated in the Figure at
daratumumab concentrations ranging from 1-10 .mu.g/ml.
[0029] FIG. 6E shows the results of in vitro ADCC of primary MM
cells isolated from patient 16 and patient 17 pretreated for 48
hours with or without 10 nM ATRA as indicated in the Figure at
daratumumab concentrations ranging from 1-10 .mu.g/ml.
[0030] FIG. 6F shows the results of in vitro ADCC of primary MM
cells isolated from patient 18 pretreated for 48 hours with or
without 10 nM ATRA as indicated in the Figure at daratumumab
concentrations ranging from 1-10 .mu.g/ml.
[0031] FIG. 7 shows CD38 expression levels in BM-MNCs isolated from
MM patients before and after incubation of cells with (black bars)
or without (white bars) in the presence of 10 nM ATRA. The same
patient samples were used in ADCC and CDC assays as shown in FIGS.
4A, 4B, 5 and 6.
[0032] FIG. 8A shows ATRA-induced reduction of CD55, CD59 and CD46
expression on RPMI8226 cells after 48 hour incubation of cells with
0-25 nM ATRA. MFI; mean fluorescent intensity. Expression of CD55,
CD59 and CD46 were assessed using flow cytometry. Top panel: MFI;
bottom panel: MFI fold change when compared to control.
[0033] FIG. 8B shows ATRA-induced reduction of CD55, CD59 and CD46
expression on UM9 cells after 48 hour incubation of cells with 0-25
nM ATRA. MFI; mean fluorescent intensity. Expression of CD55, CD59
and CD46 were assessed using flow cytometry. Top panel: MFI; bottom
panel: MFI fold change when compared to control.
[0034] FIG. 8C shows ATRA-induced reduction of CD55, CD59 and CD46
expression on XG1 cells after 48 hour incubation of cells with 0-25
nM ATRA. MFI; mean fluorescent intensity. Expression of CD55, CD59
and CD46 were assessed using flow cytometry. Top panel: MFI; bottom
panel: MFI fold change when compared to control.
[0035] FIG. 9A shows ATRA-induced reduction of CD55 expression on
primary MM cells after 48 hour incubation of cells with (grey bars)
or without (black bars) in 10 nM ATRA as indicated. * p=0.019.
[0036] FIG. 9B shows ATRA-induced reduction of CD59 expression on
primary MM cells after 48 hour incubation of cells with (grey bars)
or without (black bars) in 10 nM ATRA as indicated. **
p=0.0047.
[0037] FIG. 9C shows effect of ATRA on CD46 expression on primary
MM cells after 48 hour incubation of cells with (grey bars) or
without (black bars) in 10 nM ATRA as indicated. ns: not
significant.
[0038] FIG. 10A shows CD55 expression on primary MM cells isolated
from 16 MM patients after 48 hour incubation of cells with (black
bars) or without (white bars) 10 nM ATRA. The same patient samples
were used in CDC assays as shown in FIG. 5.
[0039] FIG. 10B shows CD59 expression on primary MM cells isolated
from 16 MM patients after 48 hour incubation of cells with (black
bars) or without (white bars) 10 nM ATRA. The same patient samples
were used in CDC assays as shown in FIG. 5.
[0040] FIG. 10C shows CD46 expression on primary MM cells isolated
from 16 MM patients after 48 hour incubation of cells with (black
bars) or without (white bars) 10 nM ATRA. The same patient samples
were used in CDC assays as shown in FIG. 5.
[0041] FIG. 11 shows that ATRA improves response to daratumumab in
a humanized multiple myeloma mouse model.
Rag2.sup.-/-.gamma..sub.e.sup.-/- mice carrying mesenchymal stem
cell (MSC)-coated scaffolds were inoculated with
luciferase-transduced XG1 cells. Mice were treated with control,
ATRA plus T-cell depleted PBMCs as effector cells (PBMC-T),
daratumumab plus PBMC-T, or daratumumab plus ATRA plus PBMC-T, and
monitored weekly by bioluminescent imaging (BLI) for growth of the
transduced XG1 cells. The Figure shows tumor load per treatment
group with 4 mice per group and each mouse with 4 scaffolds.
Statistical differences between mice treated with daratumumab and
mice treated with daratumumab plus ATRA were calculated using the
Mann-Whitney U-test. * P<0.05, ** P<0.01, *** P<0.001; ns:
not significant.
DETAILED DESCRIPTION OF THE INVENTION
[0042] "CD38" refers to the human CD38 protein (synonyms:
ADP-ribosyl cyclase 1, cADPr hydrolase 1, Cyclic ADP-ribose
hydrolase 1). Human CD38 has the amino acid sequence shown in SEQ
ID NO: 1
[0043] "Antibodies" as used herein is meant in a broad sense and
includes immunoglobulin molecules including, monoclonal antibodies
including murine, human, human-adapted, humanized and chimeric
monoclonal antibodies, antibody fragments, bispecific or
multispecific antibodies, dimeric, tetrameric or multimeric
antibodies, and single chain antibodies.
[0044] Immunoglobulins can be assigned to five major classes,
namely IgA, IgD, IgE, IgG and IgM, depending on the heavy chain
constant domain amino acid sequence. IgA and IgG are further
sub-classified as the isotypes IgA.sub.1, IgA.sub.2, IgG.sub.1,
IgG.sub.2, IgG.sub.3 and IgG.sub.4. Antibody light chains of any
vertebrate species can be assigned to one of two clearly distinct
types, namely kappa (.kappa.) and lambda (.lamda.), based on the
amino acid sequences of their constant domains.
[0045] "Antibody fragments" as used herein refers to a portion of
an immunoglobulin molecule that retains the heavy chain and/or the
light chain antigen binding site, such as heavy chain
complementarity determining regions (HCDR) 1, 2 and 3, light chain
complementarity determining regions (LCDR) 1, 2 and 3, a heavy
chain variable region (VH), or a light chain variable region (VL).
Antibody fragments include a Fab fragment, a monovalent fragment
consisting of the VL, VH, CL and CHI domains, a F(ab).sub.2
fragment, a bivalent fragment comprising two Fab fragments linked
by a disulfide bridge at the hinge region, a Fd fragment consisting
of the VH and CHI domains; a Fv fragment consisting of the VL and
VH domains of a single arm of an antibody, a domain antibody (dAb)
(Ward et al., Nature 341:544-546, 1989), which consists of a VH
domain. VH and VL domains can be engineered and linked together via
a synthetic linker to form various types of single chain antibody
designs where the VH/VL domains pair intramolecularly, or
intermolecularly in those cases when the VH and VL domains are
expressed by separate single chain antibody constructs, to form a
monovalent antigen binding site, such as single chain Fv (scFv) or
diabody; described for example in Intl. Pat. Publ. Nos.
WO1998/44001, WO1988/01649, WO1994/13804, and WO1992/01047. These
antibody fragments are obtained using well known techniques known
to those of skill in the art, and the fragments are screened for
utility in the same manner as are full length antibodies.
[0046] "Isolated antibody" as used herein refers to an antibody or
antibody fragment that is substantially free of other antibodies
having different antigenic specificities (e.g., an antibody that
specifically binds CD38). An isolated antibody that specifically
binds CD38, however, may have cross-reactivity to other antigens,
such as orthologs of human CD38 such as Macaca fascicularis
(cynomolgus) CD38. Moreover, an isolated antibody may be
substantially free of other cellular material and/or chemicals.
[0047] An antibody variable region consists of a "framework" region
interrupted by three "antigen binding sites". The antigen binding
sites are defined using various terms: Complementarity Determining
Regions (CDRs), three in the VH (HCDR1, HCDR2, HCDR3), and three in
the VL (LCDR1, LCDR2, LCDR3) are based on sequence variability (Wu
and Kabat J Exp Med 132:211-50, 1970; Kabat et al Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md., 1991); "Hypervariable
regions", "HVR", or "HV", three in the VH (H1, H2, H3) and three in
the VL (L1, L2, L3) refer to the regions of an antibody variable
domains which are hypervariable in structure as defined by Chothia
and Lesk (Chothia and Lesk Mol Biol 196:901-17, 1987). Other terms
include "IMGT-CDRs" (Lefranc et al., Dev Comparat Immunol 27:55-77,
2003) and "Specificity Determining Residue Usage" (SDRU) (Almagro,
Mol Recognit 17:132-43, 2004). The International ImMunoGeneTics
(IMGT) database (http://www_imgt_org) provides a standardized
numbering and definition of antigen-binding sites. The
correspondence between CDRs, HVs and IMGT delineations is described
in Lefranc et al., Dev Comparat Immunol 27:55-77, 2003.
[0048] "Chothia residues" as used herein are the antibody VL and VH
residues numbered according to Al-Lazikani (Al-Lazikani et al., J
Mol Biol 273:927-48, 1997).
[0049] "Framework" or "framework sequences" are the remaining
sequences of a variable region other than those defined to be
antigen binding sites.
[0050] "Humanized antibody" refers to an antibody in which the
antigen binding sites are derived from non-human species and the
variable region frameworks are derived from human immunoglobulin
sequences. Humanized antibodies may include substitutions in the
framework so that the framework may not be an exact copy of
expressed human immunoglobulin or germline gene sequences.
[0051] "Human-adapted" antibodies or "human framework adapted
(HFA)" antibodies refers to humanized antibodies adapted according
to methods described in U.S. Pat. Publ. No. US2009/0118127.
Human-adapted antibodies are humanized by selecting the acceptor
human frameworks based on the maximum CDR and FR similarities,
length compatibilities and sequence similarities of CDR1 and CDR2
loops and a portion of light chain CDR3 loops.
[0052] "Human antibody" refers to an antibody having heavy and
light chain variable regions in which both the framework and the
antigen binding sites are derived from sequences of human origin.
If the antibody contains a constant region, the constant region
also is derived from sequences of human origin.
[0053] A human antibody comprises heavy or light chain variable
regions that are "derived from" sequences of human origin where the
variable regions of the antibody are obtained from a system that
uses human germline immunoglobulin or rearranged immunoglobulin
genes. Such systems include human immunoglobulin gene libraries
displayed on phage, and transgenic non-human animals such as mice
carrying human immunoglobulin loci as described herein. A human
antibody may contain amino acid differences when compared to the
human germline or rearranged immunoglobulin sequences due to for
example naturally occurring somatic mutations or intentional
introduction of substitutions in the framework or antigen binding
sites. Typically, a human antibody is at least about 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or 100% identical in amino acid sequence to an
amino acid sequence encoded by a human germline or rearranged
immunoglobulin gene. In some cases, a human antibody may contain
consensus framework sequences derived from human framework sequence
analyses, for example as described in Knappik et al., J Mol Biol
296:57-86, 2000), or synthetic HCDR3 incorporated into human
immunoglobulin gene libraries displayed on phage, for example as
described in Shi et al., J Mol Biol 397:385-96, 2010 and Intl. Pat.
Publ. No. WO2009/085462). Antibodies in which antigen binding sites
are derived from a non-human species are not included in the
definition of human antibody.
[0054] Isolated humanized antibodies may be synthetic. Human
antibodies may be generated using systems such as phage display
incorporating synthetic CDRs and/or synthetic frameworks, or can be
subjected to in vitro mutagenesis to improve antibody
properties.
[0055] "Recombinant antibody" as used herein includes all
antibodies that are prepared, expressed, created or isolated by
recombinant means, such as antibodies isolated from an animal
(e.g., a mouse or a rat) that is transgenic or transchromosomal for
human immunoglobulin genes or a hybridoma prepared therefrom
(described further below), antibodies isolated from a host cell
transformed to express the antibody, antibodies isolated from a
recombinant combinatorial antibody library, and antibodies
prepared, expressed, created or isolated by any other means that
involve splicing of human immunoglobulin gene sequences to other
DNA sequences, or antibodies that are generated in vitro using Fab
arm exchange such as bispecific antibodies.
[0056] "Monoclonal antibody" as used herein refers to a preparation
of antibody molecules of single molecular composition. A monoclonal
antibody composition displays a single binding specificity via its
VH, VL and/or VH/VL pair and affinity for a particular epitope, or
in a case of a bispecific monoclonal antibody, a dual binding
specificity to two distinct epitopes.
[0057] "Epitope" as used herein means a portion of an antigen to
which an antibody specifically binds. Epitopes usually consist of
chemically active (such as polar, non-polar or hydrophobic) surface
groupings of moieties such as amino acids or polysaccharide side
chains and can have specific three-dimensional structural
characteristics, as well as specific charge characteristics. An
epitope may be composed of contiguous and/or noncontiguous amino
acids that form a conformational spatial unit. For a noncontiguous
epitope, amino acids from differing portions of the linear sequence
of the antigen come in close proximity in 3-dimensional space
through the folding of the protein molecule.
[0058] "Variant" as used herein refers to a polypeptide or a
polynucleotide that differs from a reference polypeptide or a
reference polynucleotide by one or more modifications for example,
substitution, insertion or deletion.
[0059] "Synergy", "synergism" or "synergistic" mean more than the
expected additive effect of a combination.
[0060] "In combination with" as used herein means that two or more
therapeutics maybe administered to a subject together in a mixture,
concurrently as single agents or sequentially as single agents in
any order.
[0061] The terms "treat" or "treatment" refer to therapeutic
treatment wherein the object is to slow down (lessen) an undesired
physiological change or disease, or provide a beneficial or desired
clinical outcome during treatment, such as the development, growth
or spread of tumor or tumor cells. Beneficial or desired clinical
outcomes include alleviation of symptoms, diminishment of extent of
disease, stabilized (i.e., not worsening) state of disease, delay
or slowing of disease progression, amelioration or palliation of
the disease state, and remission (whether partial or total),
whether detectable or undetectable. "Treatment" can also mean
prolonging survival as compared to expected survival if a subject
was not receiving treatment. Those in need of treatment include
those subjects already with the undesired physiological change or
diseaseas well as those subjects prone to have the physiological
change or disease.
[0062] "Inhibits growth" (e.g., referring to cells, such as tumor
cells) refers to a measurable decrease in the cell growth in vitro
or in vivo when contacted with a therapeutic or a combination of
therapeutics or drugs when compared to the growth of the same cells
grown in appropriate control conditions well known to the skilled
in the art. Inhibition of growth of a cell in vitro or in vivo may
be at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%,
or 100%. Inhibition of cell growth may occur by a variety of
mechanisms, for example by antibody-dependent cell-mediated
toxicity (ADCC), antibody dependent cellular phagocytosis (ADCP),
complement dependent cytotoxicity (CDC), apoptosis, necrosis, or
inhibition of cell proliferation.
[0063] A "therapeutically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
a desired therapeutic result. A therapeutically effective amount
may vary according to factors such as the disease state, age, sex,
and weight of the individual, and the ability of a therapeutic or a
combination of therapeutics to elicit a desired response in the
individual. Exemplary indicators of an effective therapeutic or
combination of therapeutics that include, for example, improved
well-being of the patient, reduction of a tumor burden, arrested or
slowed growth of a tumor, and/or absence of metastasis of cancer
cells to other locations in the body.
[0064] The invention provides methods for treating patients having
CD38-positive hematological malignancy with the combination of a
CD38 antibody and all-trans retinoic acid (ATRA). The invention is
based, at least in part, on the discovery that ATRA augments
anti-CD38 antibody daratumumab-mediated lysis by ADCC and/or CDC of
primary MM cells expressing low, intermediate or high levels of
CD38 by enhancing CD38 expression on MM cells. ATRA is also able to
induce daratumumab-mediated ADCC and/or CDC in primary MM samples
which were resistant to daratumumab-mediated CDC and/or ADCC in
vitro or were obtained from heavily pretreated multiple myeloma
patients having double-refractory (lenalidomide- and
bortezomib-refractory) disease. ATRA augmented daratumumab-mediated
CDC to a higher extent than ADCC, which may be explained by the
findings that ATRA also down-regulates complement-inhibitory
proteins CD55 and CD59.
[0065] ATRA (CAS 302-79-4) has a well-known molecular
structure.
[0066] One embodiment of the invention disclosed herein, including
in the numbered embodiments listed below, is a method of treating a
subject having a CD38-positive hematological malignancy, comprising
administering to the subject in need thereof an anti-CD38 antibody
in combination with all-trans retinoic acid (ATRA).
[0067] One embodiment of the invention disclosed herein, including
in the numbered embodiments listed below, is a method of treating a
subject having a CD38-positive hematological malignancy, comprising
administering to the subject in need thereof an anti-CD38 antibody
in combination with all-trans retinoic acid (ATRA), wherein the
anti-CD38 antibody induces killing of CD38-expressing cells in
vitro by antibody-dependent cell-mediated cytotoxicity (ADCC) or
complement dependent cytotoxicity (CDC).
[0068] The methods of the invention may be used to treat an animal
subject belonging to any classification. Examples of such animals
include mammals such as humans, rodents, dogs, cats and farm
animals.
[0069] In some embodiments of the invention disclosed herein,
including in the numbered embodiments listed below, the anti-CD38
antibody induces killing of the CD38-expressing cells by CDC in
vitro.
[0070] "CD38-positive hematological malignancy" refers to a
hematological malignancy characterized by the presence of tumor
cells expressing CD38 including leukemias, lymphomas and myeloma.
Examples of such CD38-positive hematological malignancies are
precursor B-cell lymphoblastic leukemia/lymphoma and B-cell
non-Hodgkin's lymphoma, acute promyelocytic leukemia, acute
lymphoblastic leukemia and mature B-cell neoplasms, such as B-cell
chronic lymphocytic leukemia(CLL)/small lymphocytic lymphoma (SLL),
B-cell acute lymphocytic leukemia, B-cell prolymphocytic leukemia,
lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular
lymphoma (FL), including low-grade, intermediate-grade and
high-grade FL, cutaneous follicle center lymphoma, marginal zone
B-cell lymphoma (MALT type, nodal and splenic type), hairy cell
leukemia, diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma
(BL), plasmacytoma, multiple myeloma (MM), plasma cell leukemia,
post-transplant lymphoproliferative disorder, Waldenstrom's
macroglobulinemia, plasma cell leukemias and anaplastic large-cell
lymphoma (ALCL).
[0071] CD38 is expressed in a variety of malignant hematological
diseases, including multiple myeloma, leukemias and lymphomas, such
as B-cell chronic lymphocytic leukemia, T- and B-cell acute
lymphocytic leukemia, Waldenstrom macroglobulinemia, primary
systemic amyloidosis, mantle-cell lymphoma,
pro-lymphocytic/myelocytic leukemia, acute myeloid leukemia,
chronic myeloid leukemia, follicular lymphoma, Burkitt's lymphoma,
large granular lymphocytic (LGL) leukemia, NK-cell leukemia and
plasma-cell leukemia. Expression of CD38 has been described on
epithelial/endothelial cells of different origin, including
glandular epithelium in prostate, islet cells in pancreas, ductal
epithelium in glands, including parotid gland, bronchial epithelial
cells, cells in testis and ovary and tumor epithelium in colorectal
adenocarcinoma. Other diseases, where CD38 expression could be
involved, include, e.g., broncho-epithelial carcinomas of the lung,
breast cancer (evolving from malignant proliferation of epithelial
lining in ducts and lobules of the breast), pancreatic tumors,
evolving from the .beta.-cells (insulinomas), tumors evolving from
epithelium in the gut (e.g. adenocarcinoma and squamous cell
carcinoma), carcinoma in the prostate gland, and seminomas in
testis and ovarian cancers. In the central nervous system,
neuroblastomas express CD38.
[0072] In one embodiment of the invention disclosed herein,
including in the numbered embodiments listed below, the
CD38-positive hematological malignancy is multiple myeloma.
[0073] In one embodiment of the invention disclosed herein,
including in the numbered embodiments listed below, the
CD38-positive hematological malignancy is diffuse large B-cell
lymphoma (DLBCL).
[0074] In one embodiment of the invention disclosed herein,
including in the numbered embodiments listed below, the
CD38-positive hematological malignancy is non-Hodgkin's
lymphoma.
[0075] In one embodiment of the invention disclosed herein,
including in the numbered embodiments listed below, the
CD38-positive hematological malignancy is acute lymphoblastic
leukemia (ALL).
[0076] In one embodiment of the invention disclosed herein,
including in the numbered embodiments listed below, the
CD38-positive hematological malignancy is follicular lymphoma
(FL).
[0077] In one embodiment of the invention disclosed herein,
including in the numbered embodiments listed below, the
CD38-positive hematological malignancy is Burkitt's lymphoma
(BL).
[0078] In one embodiment of the invention disclosed herein,
including in the numbered embodiments listed below, the
CD38-positive hematological malignancy is mantle cell lymphoma
(MCL).
[0079] In one embodiment of the invention disclosed herein,
including in the numbered embodiments listed below, the
CD38-positive hematological malignancy is multiple myeloma, acute
lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma, diffuse large
B-cell lymphoma (DLBCL), Burkitt's lymphoma (BL), follicular
lymphoma (FL) or mantle-cell lymphoma (MCL).
[0080] Examples of B-cell non-Hodgkin's lymphomas are lymphomatoid
granulomatosis, primary effusion lymphoma, intravascular large
B-cell lymphoma, mediastinal large B-cell lymphoma, heavy chain
diseases (including .gamma., .mu., and a disease), lymphomas
induced by therapy with immunosuppressive agents, such as
cyclosporine-induced lymphoma, and methotrexate-induced
lymphoma.
[0081] In one embodiment of the present invention, including in the
numbered embodiments listed below the disorder involving cells
expressing CD38 is Hodgkin's lymphoma.
[0082] Other examples of disorders involving cells expressing CD38
include malignancies derived from T and NK cells including mature T
cell and NK cell neoplasms including T-cell prolymphocytic
leukemia, T-cell large granular lymphocytic leukemia, aggressive NK
cell leukemia, adult T-cell leukemia/lymphoma, extranodal NK/T cell
lymphoma, nasal type, enteropathy-type T-cell lymphoma,
hepatosplenic T-cell lymphoma, subcutaneous panniculitis-like
T-cell lymphoma, blastic NK cell lymphoma, Mycosis Fungoides/Sezary
Syndrome, primary cutaneous CD30 positive T-cell
lymphoproliferative disorders (primary cutaneous anaplastic large
cell lymphoma C-ALCL, lymphomatoid papulosis, borderline lesions),
angioimmunoblastic T-cell lymphoma, peripheral T-cell lymphoma
unspecified, and anaplastic large cell lymphoma.
[0083] Examples of malignancies derived from myeloid cells include
acute myeloid leukemia, including acute promyelocytic leukemia, and
chronic myeloproliferative diseases, including chronic myeloid
leukemia.
[0084] Any anti-CD38 antibody may be used in the methods of the
invention as disclosed herein, including in the numbered
embodiments listed below.
[0085] In some embodiments, the anti-CD38 antibody induces in vitro
killing of CD38-expressing cells by antibody-dependent
cell-mediated cytotoxicity (ADCC) and/or complement dependent
cytotoxicity (CDC).
[0086] The variable regions of the anti-CD38 antibodies may be
obtained from existing anti-CD38 antibodies, and cloned as full
length antibodies or into various antibody formats and fragments
using standard methods. Exemplary variable regions binding CD38
that may be used are described in Intl. Pat. Publ. Nos.
WO05/103083, WO06/125640, WO07/042309, WO08/047242, WO12/092612,
WO06/099875 and WO11/154453A1.
[0087] An exemplary anti-CD38 antibody that may be used is
daratumumab. Daratumumab comprises the heavy chain variable region
(VH) and the light chain variable region (VL) amino acid sequences
shown in SEQ ID NO: 4 and 5, respectively, heavy chain CDRs HCDR1,
HCDR2 and HCDR3 of SEQ ID NOs: 6, 7 and 8, respectively, and light
chain CDRs LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 9, 10 and 11,
respectively, and is of IgG1/.kappa. subtype and described in U.S.
Pat. No. 7,829,693. Daratumumab heavy chain amino acid sequence is
shown in SEQ ID NO: 12 and light chain amino acid sequence shown in
SEQ ID NO: 13.
TABLE-US-00001 SEQ ID NO: 1
MANCEFSPVSGDKPCCRLSRRAQLCLGVSILVLILVVVLAVVV
PRWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVW
DAFKGAFISKHPCNITEEDYQPLMKLGTQTVPCNKILLWSRIK
DLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNTSKINYQS
CPDWRKDCSNNPVSVFWKTVSRRFAEAACDVVHVMLNGSRSKI
FDKNSTFGSVEVHNLQPEKVQTLEAWVIHGGREDSRDLCQDPT
IKELESIISKRNIQFSCKNIYRPDKFLQCVKNPEDSSCTSEI SEQ ID NO: 2
SKRNIQFSCKNIYR SEQ ID NO: 3 EKVQTLEAWVIHGG SEQ ID NO: 4
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGK
GLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSL
RAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS SEQ ID NO: 5
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQA
PRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVY YCQQRSNWPPTFGQGTKVEIK
SEQ ID NO: 6 SFAMS SEQ ID NO: 7 AISGSGGGTYYADSVKG SEQ ID NO: 8
DKILWFGEPVFDY SEQ ID NO: 9 RASQSVSSYLA SEQ ID NO: 10 DASNRAT SEQ ID
NO: 11 QQRSNWPPTF SEQ ID NO: 12
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGK
GLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSL
RAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSSASTKGPS
VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
VDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE
SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 13 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQA
PRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVY
YCQQRSNWPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGT
ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST
YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
[0088] Another exemplary anti-CD38 antibody that may be used is
mAb003 comprising the VH and VL sequences of SEQ ID NOs: 14 and 15,
respectively and described in U.S. Pat. No. 7,829,693. The VH and
the VL of mAb003 may be expressed as IgG1/.kappa..
TABLE-US-00002 SEQ ID NO: 14
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQ
GLEWMGRVIPFLGIANSAQKFQGRVTITADKSTSTAYMDLSSL
RSEDTAVYYCARDDIAALGPFDYWGQGTLVTVSSAS SEQ ID NO: 15
DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKA
PKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY
YCQQYNSYPRTFGQGTKVEIK
[0089] Another exemplary anti-CD38 antibody that may be used is
mAb024 comprising the VH and VL sequences of SEQ ID NOs: 16 and 17,
respectively, described in U.S. Pat. No. 7,829,693. The VH and the
VL of mAb024 may be expressed as IgG1/.kappa..
TABLE-US-00003 SEQ ID NO: 16
EVQLVQSGAEVKKPGESLKISCKGSGYSFSNYWIGWVRQMPGK
GLEWMGIIYPHDSDARYSPSFQGQVTFSADKSISTAYLQWSSL
KASDTAMYYCARHVGWGSRYWYFDLWGRGTLVTVSS SEQ ID NO: 17
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQA
PRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVY
YCQQRSNWPPTFGQGTKVEIK
[0090] Another exemplary anti-CD38 antibody that may be used is
MOR-202 (MOR-03087) comprising the VH and VL sequences of SEQ ID
NOs: 18 and 19, respectively, described in U.S. Pat. No. 8,088,896.
The VH and the VL of MOR-202 may be expressed as IgG1/.kappa..
TABLE-US-00004 SEQ ID NO: 18
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMNWVRQAPGK
GLEWVSGISGDPSNTYYADSVKGRFTISRDNSKNTLYLQMNSL
RAEDTAVYYCARDLPLVYTGFAYWGQGTLVTVSS SEQ ID NO: 19
DIELTQPPSVSVAPGQTARISCSGDNLRHYYVYWYQQKPGQAP
VLVIYGDSKRPSGIPERFSGSNSGNTATLTISGTQAEDEADYY
CQTYTGGASLVFGGGTKLTVLGQ
[0091] Another exemplary anti-CD38 antibody that may be used is
Isatuximab comprising the VH and VL sequences of SEQ ID NOs: 20 and
21, respectively, described in U.S. Pat. No. 8,153,765. The VH and
the VL of Isatuximab may be expressed as IgG1/.kappa..
TABLE-US-00005 SEQ ID NO 20:
QVQLVQSGAEVAKPGTSVKLSCKASGYTFTDYWMQWVKQRPGQ
GLEWIGTIYPGDGDTGYAQKFQGKATLTADKSSKTVYMHLSSL
ASEDSAVYYCARGDYYGSNSLDYWGQGTSVTVSS SEQ ID NO: 21:
DIVMTQSHLSMSTSLGDPVSITCKASQDVSTVVAWYQQKPGQS
PRRLIYSASYRYIGVPDRFTGSGAGTDFTFTISSVQAEDLAVY
YCQQHYSPPYTFGGGTKLEIK
[0092] Other exemplary anti-CD38 antibodies that may be used in the
methods of the invention include those described in Int. Pat. Publ.
No. WO05/103083, Intl. Pat. Publ. No. WO06/125640, Intl. Pat. Publ.
No. WO07/042309, Intl. Pat. Publ. No. WO08/047242 or Intl. Pat.
Publ. No. WO14/178820.
[0093] Anti-CD38 antibodies used in the methods of the invention
disclosed herein, including in the numbered embodiments listed
below, may also be selected de novo from a phage display library,
where the phage is engineered to express human immunoglobulins or
portions thereof such as Fabs, single chain antibodies (scFv), or
unpaired or paired antibody variable regions (Knappik et al., J Mol
Biol 296:57-86, 2000; Krebs et al., J Immunol Meth 254:67-84, 2001;
Vaughan et al., Nature Biotechnology 14:309-314, 1996; Sheets et
al., PITAS (USA) 95:6157-6162, 1998; Hoogenboom and Winter, J Mol
Biol 227:381, 1991; Marks et al., J Mol Biol 222:581, 1991). CD38
binding variable domains may be isolated from for example phage
display libraries expressing antibody heavy and light chain
variable regions as fusion proteins with bacteriophage pIX coat
protein as described in Shi et al., J. Mol. Biol. 397:385-96, 2010
and PCT Intl. Publ. No. WO09/085462). The antibody libraries may be
screened for binding to human CD38 extracellular domain, obtained
positive clones further characterized, Fabs isolated from the clone
lysates, and subsequently cloned as full length antibodies. Such
phage display methods for isolating human antibodies are
established in the art. See for example: U.S. Pat. No. 5,223,409;
U.S. Pat. No. 5,403,484; and U.S. Pat. No. 5,571,698, U.S. Pat. No.
5,427,908, U.S. Pat. No. 5,580,717, U.S. Pat. No. 5,969,108, U.S.
Pat. No. 6,172,197, U.S. Pat. No. 5,885,793; U.S. Pat. No.
6,521,404; U.S. Pat. No. 6,544,731; U.S. Pat. No. 6,555,313; U.S.
Pat. No. 6,582,915; and U.S. Pat. No. 6,593,081.
[0094] The Fc portion of the antibody may mediate antibody effector
functions such as antibody-dependent cell-mediated cytotoxicity
(ADCC), antibody-dependent cellular phagocytosis (ADCP) or
complement dependent cytotoxicity (CDC). Such functions may be
mediated by binding of an Fc effector domain(s) to an Fc receptor
on an immune cell with phagocytic or lytic activity or by binding
of an Fc effector domain(s) to components of the complement system.
Typically, the effect(s) mediated by the Fe-binding cells or
complement components result in inhibition and/or depletion of
target cells, e.g., CD38-expressing cells. Human IgG isotypes IgG1,
IgG2, IgG3 and IgG4 exhibit differential capacity for effector
functions. ADCC may be mediated by IgG1 and IgG3, ADCP may be
mediated by IgG1, IgG2, IgG3 and IgG4, and CDC may be mediated by
IgG1 and IgG3.
[0095] In the methods described herein, and in some embodiments of
each and every one of the numbered embodiments listed below, the
anti-CD38 antibody is of IgG1, IgG2, IgG3 or IgG4 isotype.
[0096] In the methods described herein, and in some embodiments of
each and every one of the numbered embodiments listed below, the
anti-CD38 antibody is of IgG1 or IgG3 isotype.
[0097] In the methods described herein, and in some embodiments of
each and every one of the numbered embodiments listed below, the
anti-CD38 antibody induces in vitro killing of CD38-expressing
cells by ADCC.
[0098] In the methods described herein, and in some embodiments of
each and every one of the numbered embodiments listed below, the
anti-CD38 antibody induces in vitro killing of CD38-expressing
cells by CDC.
[0099] In the methods described herein, and in some embodiments of
each and every one of the numbered embodiments listed below, the
anti-CD38 antibody induces killing of CD38-expressing cells by ADCC
and CDC in vitro.
[0100] "Antibody-dependent cellular cytotoxicity," or
"antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a
mechanism for inducing cell death that depends upon the interaction
of antibody-coated target cells with effector cells possessing
lytic activity, such as natural killer cells, monocytes,
macrophages and neutrophils via Fc gamma receptors (Fc.gamma.R)
expressed on effector cells. For example, NK cells express
Fc.gamma.RIIIa, whereas monocytes express Fc.gamma.RI, Fc.gamma.RII
and Fc.gamma.RIIIa. Death of the antibody-coated target cell, such
as CD38-expressing cells, occurs as a result of effector cell
activity through the secretion of membrane pore-forming proteins
and proteases. To assess ADCC activity of an anti-CD38 antibody in
vitro, the antibody may be added to CD38-expressing cells in
combination with immune effector cells, which may be activated by
the antigen antibody complexes resulting in cytolysis of the target
cell. Cytolysis is generally detected by the release of label
(e.g., radioactive substrates, fluorescent dyes or natural
intracellular proteins) from the lysed cells. For example, primary
BM-MNC cells isolated from a patient with a B-cell malignancy such
as MM may be used for the assay. In an exemplary assay, BM-MNCs may
be treated with an anti-CD38 antibody for 1 hour at a concentration
of 0.3-10 .mu.g/ml, and the survival of primary CD138.sup.+ MM
cells may be determined by flow cytometry using techniques
described in van der Veer et al., Haematologica 96:284-290, 2001 or
in van der Veer et al., Blood Cancer J 1(10):e41, 2011. The
percentage of MM cell lysis may be determined relative to an
isotype control as described herein. Anti-CD38 antibodies used in
the methods of the invention may induce ADCC by about 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95% or 100% of control.
[0101] "Complement-dependent cytotoxicity", or "CDC", refers to a
mechanism for inducing cell death in which an Fc effector domain of
a target-bound antibody binds and activates complement component
C1q which in turn activates the complement cascade leading to
target cell death. Activation of complement may also result in
deposition of complement components on the target cell surface that
facilitate ADCC by binding complement receptors (e.g., CR3) on
leukocytes. In an exemplary assay, primary BM-MNC cells isolated
from a patient with a B-cell malignancy may be treated with an
anti-CD38 antibody and complement derived from 10% pooled human
serum for 1 hour at a concentration of 0.3-10 .mu.g/ml, and the
survival of primary CD138.sup.+ MM cells may be determined by flow
cytometry using techniques described in van der Veer et al.,
Haematologica 96:284-290, 2011; van der Veer et al., Blood Cancer J
1(10):e41, 2011. The percentage of MM cell lysis may be determined
relative to an isotype control as described herein. Anti-CD38
antibodies used in the methods of the invention may induce CDC by
about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95% or 100%
[0102] The ability of monoclonal antibodies to induce ADCC may be
enhanced by engineering their oligosaccharide component. Human IgG1
or IgG3 are N-glycosylated at Asn297 with the majority of the
glycans in the well-known biantennary G0, G0F, G1, G1F, G2 or G2F
forms. Antibodies produced by non-engineered CHO cells typically
have a glycan fucose content of about at least 85%. The removal of
the core fucose from the biantennary complex-type oligosaccharides
attached to the Fc regions enhances the ADCC of antibodies via
improved Fc.gamma.RIIIa binding without altering antigen binding or
CDC activity. Such antibodies may be achieved using different
methods reported to lead to the expression of relatively high
defucosylated antibodies bearing the biantennary complex-type of Fc
oligosaccharides such as control of culture osmolality (Konno et
al., Cytotechnology 64:249-65, 2012), application of a variant CHO
line Lec13 as the host cell line (Shields et al., J Biol Chem
277:26733-40, 2002), application of a variant CHO line EB66 as the
host cell line (Olivier et al., MAbs; 2(4), 2010; Epub ahead of
print; PMID:20562582), application of a rat hybridoma cell line
YB2/0 as the host cell line (Shinkawa et al., J Biol Chem
278:3466-3473, 2003), introduction of small interfering RNA
specifically against the .alpha. 1,6-fucosyltrasferase (FUT8) gene
(Mori et al., Biotechnol Bioeng 88:901-908, 2004), or co-expression
of .beta.-1,4-N-acetylglucosaminyltransferase III and Golgi
.alpha.-mannosidase II or a potent alpha-mannosidase I inhibitor,
kifunensine (Ferrara et al., J Biol Chem 281:5032-5036, 2006,
Ferrara et al., Biotechnol Bioeng 93:851-861, 2006; Xhou et al.,
Biotechnol Bioeng 99:652-65, 2008). ADCC elicited by anti-CD38
antibodies used in the methods of the invention, and in some
embodiments of each and every one of the numbered embodiments
listed below, may also be enhanced by certain substitutions in the
antibody Fc. Exemplary substitutions are, for example,
substitutions at amino acid positions 256, 290, 298, 312, 356, 330,
333, 334, 360, 378 or 430 (residue numbering according to the EU
index) as described in U.S. Pat. No. 6,737,056. CDC elicited by
anti-CD38 antibodies used in the methods of the invention, and in
some embodiments of each and every one of the numbered embodiments
listed below, may also be enhanced by certain substitutions in the
antibody Fc. Exemplary substitutions are, for example,
substitutions at amino acid positions 423, 268, 267 and/or 113
(residue numbering according to the EU index) as described in Moore
et al., Mabs 2:181-189, 2010.
[0103] In some methods described herein, and in some embodiments of
each and every one of the numbered embodiments listed below, the
anti-CD38 antibodies comprise a substitution in the antibody
Fc.
[0104] In some methods described herein, and in some embodiments of
each and every one of the numbered embodiments listed below, the
anti-CD38 antibodies comprise a substitution in the antibody Fc at
amino acid positions 256, 290, 298, 312, 356, 330, 333, 334, 360,
378 and/or 430 (residue numbering according to the EU index).
[0105] In some methods described herein, and in some embodiments of
each and every one of the numbered embodiments listed below, the
anti-CD38 antibodies comprise a substitution in the antibody Fc at
amino acid position 113, 267, 268 and/or 423 (residue numbering
according to the EU index).
[0106] Another embodiment of the invention, including in the
numbered embodiments listed below, is a method of treating a
subject having a CD38-positive hematological malignancy, comprising
administering to the subject in need thereof an anti-CD38 antibody
in combination with all-trans retinoic acid (ATRA), wherein the
anti-CD38 antibody competes for binding to CD38 with an antibody
comprising a heavy chain variable region (VH) of SEQ ID NO: 4 and a
light chain variable region (VL) of SEQ ID NO: 5 (daratumumab).
[0107] Another embodiment of the invention, including in the
numbered embodiments listed below, is a method of treating a
subject having a CD38-positive hematological malignancy, comprising
administering to the subject in need thereof an anti-CD38 antibody
in combination with all-trans retinoic acid (ATRA), wherein the
anti-CD38 antibody induces killing of CD38-expressing cells in
vitro by antibody-dependent cell-mediated cytotoxicity (ADCC) or
complement dependent cytotoxicity (CDC), wherein the anti-CD38
antibody competes for binding to CD38 with an antibody comprising a
heavy chain variable region (VH) of SEQ ID NO: 4 and a light chain
variable region (VL) of SEQ ID NO: 5 (daratumumab).
[0108] Antibodies may be evaluated for their competition with
daratumumab having the VH of SEQ ID NO: 4 and the VL of SEQ ID NO:
5 for binding to CD38 using well known in vitro methods. In an
exemplary method, CHO cells recombinantly expressing CD38 may be
incubated with unlabeled daratumumab for 15 min at 4.degree. C.,
followed by incubation with an excess of fluorescently labeled test
antibody for 45 min at 4.degree. C. After washing in PBS/BSA,
fluorescence may be measured by flow cytometry using standard
methods. In another exemplary method, extracellular portion of
human CD38 may be coated on the surface of an ELISA plate. Excess
of unlabeled daratumumab may be added for about 15 minutes and
subsequently biotinylated test antibodies may be added. After
washes in PBS/Tween, binding of the test biotinylated antibodies
may be detected using horseradish peroxidase (HRP)-conjugated
streptavidine and the signal detected using standard methods. It is
readily apparent that in the competition assays, daratumumab may be
labelled and the test antibody unlabeled. The test antibody
competes with daratumumab when daratumumab inhibits binding of the
test antibody, or the test antibody inhibits binding of daratumumab
by 80%, 85%, 90%, 95% or 100%. The epitope of the test antibody can
further be defined, for example, by peptide mapping or
hydrogen/deuterium protection assays using known methods.
[0109] Another embodiment of the invention disclosed herein,
including in the numbered embodiments listed below, is a method of
treating a subject having a CD38-positive hematological malignancy,
comprising administering to the subject in need thereof an
anti-CD38 antibody that binds to the region SKRNIQFSCKNIYR (SEQ ID
NO: 2) and the region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38
(SEQ ID NO: 1) in combination with all-trans retinoic acid
(ATRA).
[0110] Another embodiment of the invention disclosed herein,
including in the numbered embodiments listed below, is a method of
treating a subject having a CD38-positive hematological malignancy,
comprising administering to the subject in need thereof an
anti-CD38 antibody that binds to the region SKRNIQFSCKNIYR (SEQ ID
NO: 2) and the region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38
(SEQ ID NO: 1) in combination with all-trans retinoic acid (ATRA),
wherein the anti-CD38 antibody induces killing of CD38-expressing
cells in vitro by antibody-dependent cell-mediated cytotoxicity
(ADCC) or complement dependent cytotoxicity (CDC). The antibody
"binds to the region SKRNIQFSCKNIYR (SEQ ID NO: 2) and the region
EKVQTLEAWVIHGG (SEQ ID NO: 3)" when the antibody binds at least one
amino acid residue within each region. The antibody may bind for
example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid
residues within each region of SEQ ID NO:2 and SEQ ID NO: 3. The
antibody may also optionally bind one or more residues outside of
the regions of SEQ ID NO: 2 and SEQ ID NO: 3. Binding may be
assessed by known methods such as mutagenesis studies or by
resolving the crystal structure of CD38 in complex with the
antibody. In some embodiments disclosed herein, including in the
numbered embodiments listed below, the antibody epitope comprises
at least one amino acid in the region SKRNIQFSCKNIYR (SEQ ID NO: 2)
and at least one amino acid in the region EKVQTLEAWVIHGG (SEQ ID
NO: 3) of human CD38 (SEQ ID NO: 1). In some embodiments disclosed
herein, including in the numbered embodiments listed below, the
antibody epitope comprises at least two amino acids in the region
SKRNIQFSCKNIYR (SEQ ID NO: 2) and at least two amino acids in the
region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1).
In some embodiments disclosed herein, including in the numbered
embodiments listed below, the antibody epitope comprises at least
three amino acids in the region SKRNIQFSCKNIYR (SEQ ID NO: 2) and
at least three amino acids in the region EKVQTLEAWVIHGG (SEQ ID NO:
3) of human CD38 (SEQ ID NO: 1). In some embodiments disclosed
herein, including in the numbered embodiments listed below, the
anti-CD38 antibody binds to an epitope comprising at least KRN in
the region SKRNIQFSCKNIYR (SEQ ID NO: 2) and comprising at least
VQLT (SEQ ID NO: 22) in the region EKVQTLEAWVIHGG (SEQ ID NO: 3) of
human CD38 (SEQ ID NO: 1).
[0111] In some embodiments of the invention described herein, and
in some embodiments of each and every one of the numbered
embodiments listed below, the anti-CD38 antibody binds to an
epitope comprising at least KRN in the region SKRNIQFSCKNIYR (SEQ
ID NO: 2) and comprising at least VQLT (SEQ ID NO: 22) in the
region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO:
1).
[0112] An exemplary antibody that binds to the region
SKRNIQFSCKNIYR (SEQ ID NO: 2) and the region EKVQTLEAWVIHGG (SEQ ID
NO: 3) of human CD38 (SEQ ID NO: 1) or minimally to residues KRN
and VQLT (SEQ ID NO: 22) as shown above is daratumumab having
certain VH, VL and CDR sequences as described above. Antibodies
that bind to the region SKRNIQFSCKNIYR (SEQ ID NO: 2) and the
region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1)
may be generated, for example, by immunizing mice with peptides
having the amino acid sequences shown in SEQ ID NOs: 2 and 3 using
standard methods and as described herein. Antibodies may be further
evaluated, for example, by assaying competition between daratumumab
and a test antibody for binding to CD38 as described above.
[0113] In the methods described herein, and in some embodiments of
each and every one of the numbered embodiments listed below, the
anti-CD38 antibody may bind human CD38 with a range of affinities
(K.sub.D). In one embodiment according to the invention, and in
some embodiments of each and every one of the numbered embodiments
listed below, the anti-CD38 antibody binds to CD38 with high
affinity, for example, with a K.sub.D equal to or less than about
10.sup.-7 M, such as about 1, 2, 3, 4, 5, 6, 7, 8, or
9.times.10.sup.-8M, 1.times.10.sup.-9 M, about 1.times.10.sup.-10
M, about 1.times.10.sup.-11 M, about 1.times.10.sup.-12 M, about
1.times.10.sup.-13 M, about 1.times.10.sup.-14 M, about
1.times.10.sup.-15 M or any range or value therein, as determined
by surface plasmon resonance or the Kinexa method, as practiced by
those of skill in the art. One exemplary affinity is equal to or
less than 1.times.10.sup.-8 M. Another exemplary affinity is equal
to or less than 1.times.10.sup.-9 M.
[0114] In some methods described herein, and in some embodiments of
each and every one of the numbered embodiments listed below, the
anti-CD38 antibody has a biantennary glycan structure with fucose
content of about between 0% to about 15%, for example 15%, 14%,
13%, 12%, 11% 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0%.
[0115] In some methods described herein, and in some embodiments of
each and every one of the numbered embodiments listed below, the
anti-CD38 antibody has a biantennary glycan structure with fucose
content of about 50%, 40%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%,
13%, 12%, 11% 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0%
[0116] Substitutions in the Fc and reduced fucose content may
enhance the ADCC activity of the anti-CD38 antibody.
[0117] "Fucose content" refers to the amount of the fucose
monosaccharide within the sugar chain at Asn297. The relative
amount of fucose is the percentage of fucose-containing structures
related to all glycostructures. Glycostructures may be
characterized and quantified by multiple methods, for example: 1)
using MALDI-TOF of N-glycosidase F treated sample (e.g. complex,
hybrid and oligo- and high-mannose structures) as described in Int.
Pat. Publ. No. WO2008/077546; 2) by enzymatic release of the Asn297
glycans with subsequent derivatization and detection/quantitation
by HPLC (UPLC) with fluorescence detection and/or HPLC-MS
(UPLC-MS); 3) intact protein analysis of the native or reduced mAb,
with or without treatment of the Asn297 glycans with Endo S or
other enzyme that cleaves between the first and the second GlcNAc
monosaccharides, leaving the fucose attached to the first GlcNAc;
4) digestion of the mAb to constituent peptides by enzymatic
digestion (e.g., trypsin or endopeptidase Lys-C), and subsequent
separation, detection and quantitation by HPLC-MS (UPLC-MS); or 5)
separation of the mAb oligosaccharides from the mAb protein by
specific enzymatic deglycosylation with PNGase F at Asn 297. The
oligosaccharides released may be labeled with a fluorophore,
separated and identified by various complementary techniques which
allow fine characterization of the glycan structures by
matrix-assisted laser desorption ionization (MALDI) mass
spectrometry by comparison of the experimental masses with the
theoretical masses, determination of the degree of sialylation by
ion exchange HPLC (GlycoSep C), separation and quantification of
the oligosacharride forms according to hydrophilicity criteria by
normal-phase HPLC (GlycoSep N), and separation and quantification
of the oligosaccharides by high performance capillary
electrophoresis-laser induced fluorescence (HPCE-LIF).
[0118] "Low fucose" or "low fucose content" as used in the
application refers to antibodies with fucose content of about
0%-15%.
[0119] "Normal fucose" or `normal fucose content" as used herein
refers to antibodies with fucose content of about over 50%,
typically about over 60%, 70%, 80% or over 85%.
[0120] In some methods of the invention described herein, and in
some embodiments of each and every one of the numbered embodiments
listed below, the anti-CD38 antibody comprises the heavy chain
complementarity determining regions (HCDR) 1 (HCDR1), 2 (HCDR2) and
3 (HCDR3) sequences of SEQ ID NOs: 6, 7 and 8, respectively.
[0121] In some methods of the invention described herein, and in
some embodiments of each and every one of the numbered embodiments
listed below, the anti-CD38 antibody comprises the light chain
complementarity determining regions (LCDR) 1 (LCDR1), 2 (LCDR2) and
3 (LCDR3) sequences of SEQ ID NOs: 9, 10 and 11, respectively.
[0122] In some methods of the invention described herein, and in
some embodiments of each and every one of the numbered embodiments
listed below, the anti-CD38 antibody comprises the heavy chain
complementarity determining regions (HCDR) 1 (HCDR1), 2 (HCDR2) and
3 (HCDR3) sequences of SEQ ID NOs: 6, 7 and 8, respectively and the
light chain complementarity determining regions (LCDR) 1 (LCDR1), 2
(LCDR2) and 3 (LCDR3) sequences of SEQ ID NOs: 9, 10 and 11,
respectively.
[0123] In some methods of the invention described herein, and in
some embodiments of each and every one of the numbered embodiments
listed below, the anti-CD38 antibody comprises the heavy chain
variable region (VH) of SEQ ID NO: 4 and the light chain variable
region (VL) of SEQ ID NO: 5.
[0124] In some methods of the invention described herein, and in
some embodiments of each and every one of the numbered embodiments
listed below, the anti-CD38 antibody comprises the heavy chain of
SEQ ID NO: 12 and the light chain of SEQ ID NO: 13.
[0125] In some methods of the invention described herein, and in
some embodiments of each and every one of the numbered embodiments
listed below, the anti-CD38 antibody comprises the heavy chain
variable region (VH) of SEQ ID NO: 14 and the light chain variable
region (VL) of SEQ ID NO: 15.
[0126] In some methods of the invention described herein, and in
some embodiments of each and every one of the numbered embodiments
listed below, the anti-CD38 antibody comprises the heavy chain
variable region (VH) of SEQ ID NO: 16 and the light chain variable
region (VL) of SEQ ID NO: 17.
[0127] In some methods of the invention described herein, and in
some embodiments of each and every one of the numbered embodiments
listed below, the anti-CD38 antibody comprises the heavy chain
variable region (VH) of SEQ ID NO: 18 and the light chain variable
region (VL) of SEQ ID NO: 19.
[0128] In some methods of the invention described herein, and in
some embodiments of each and every one of the numbered embodiments
listed below, the anti-CD38 antibody comprises the heavy chain
variable region (VH) of SEQ ID NO: 20 and the light chain variable
region (VL) of SEQ ID NO: 21.
[0129] In some methods of the invention described herein, and in
some embodiments of each and every one of the numbered embodiments
listed below, the anti-CD38 antibody comprises a heavy chain
comprising an amino acid sequence that is 95%, 96%, 97%, 98% or 99%
identical to that of SEQ ID NO: 12 and a light chain comprising an
amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to
that of SEQ ID NO: 13.
[0130] Antibodies that are substantially identical to the antibody
comprising the heavy chain of SEQ ID NO: 12 and the light chain of
SEQ ID NO: 13 may be used in the methods of the invention, and in
some embodiments of each and every one of the numbered embodiments
listed below. The term "substantially identical" as used herein
means that the two antibody heavy chain or light chain amino acid
sequences being compared are identical or have "insubstantial
differences." Insubstantial differences are substitutions of 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in an
antibody heavy chain or light chain that do not adversely affect
antibody properties. Percent identity can be determined for example
by pairwise alignment using the default settings of the AlignX
module of Vector NTI v.9.0.0 (Invitrogen, Carlsbad, Calif.). The
protein sequences of the present invention can be used as a query
sequence to perform a search against public or patent databases to,
for example, identify related sequences. Exemplary programs used to
perform such searches are the XBLAST or BLASTP programs
(http_//www_ncbi_nlm/nih_gov), or the GenomeQuest.TM. (GenomeQuest,
Westborough, Mass.) suite using the default settings. Exemplary
substitutions that can be made to the anti-CD38 antibodies used in
the methods of the invention are for example conservative
substitutions with an amino acid having similar charge,
hydrophobic, or stereochemical characteristics. Conservative
substitutions may also be made to improve antibody properties, for
example stability or affinity, or to improve antibody effector
functions. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15
amino acid substitutions may be made for example to the heavy or
the light chain of the anti-CD38 antibody. Furthermore, any native
residue in the heavy or light chain may also be substituted with
alanine, as has been previously described for alanine scanning
mutagenesis (MacLennan et al., Acta Physiol Scand Suppl 643:55-67,
1998; Sasaki et al., Adv Biophys 35:1-24, 1998). Desired amino acid
substitutions may be determined by those skilled in the art at the
time such substitutions are desired. Amino acid substitutions may
be done for example by PCR mutagenesis (U.S. Pat. No. 4,683,195).
Libraries of variants may be generated using well known methods,
for example using random (NNK) or non-random codons, for example
DVK codons, which encode 11 amino acids (Ala, Cys, Asp, Glu, Gly,
Lys, Asn, Arg, Ser, Tyr, Trp) and screening the libraries for
variants with desired properties. The generated variants may be
tested for their binding to CD38, their ability to induce ADCC,
ADCP or apoptosis in vitro using methods described herein.
[0131] In some embodiments, and in some embodiments of each and
every one of the numbered embodiments listed below, the anti-CD38
antibody is a bispecific antibody. The VL and/or the VH regions of
the existing anti-CD38 antibodies or the VL and VH regions
identified de novo as described above may be engineered into
bispecific full length antibodies. Such bispecific antibodies may
be made by modulating the CH3 interactions between the two
monospecific antibody heavy chains to form bispecific antibodies
using technologies such as those described in U.S. Pat. No.
7,695,936; Int. Pat. Publ. No. WO04/111233; U.S. Pat. Publ. No.
US2010/0015133; U.S. Pat. Publ. No. US2007/0287170; Int. Pat. Publ.
No. WO2008/119353; U.S. Pat. Publ. No. US2009/0182127; U.S. Pat.
Publ. No. US2010/0286374; U.S. Pat. Publ. No. US2011/0123532; Int.
Pat. Publ. No. WO2011/131746; Int. Pat. Publ. No. WO2011/143545; or
U.S. Pat. Publ. No. US2012/0149876. Additional bispecific
structures into which the VL and/or the VH regions of the
antibodies of the invention can be incorporated are for example
Dual Variable Domain Immunoglobulins (Int. Pat. Publ. No.
WO2009/134776), or structures that include various dimerization
domains to connect the two antibody arms with different
specificity, such as leucine zipper or collagen dimerization
domains (Int. Pat. Publ. No. WO2012/022811, U.S. Pat. No.
5,932,448; U.S. Pat. No. 6,833,441).
[0132] Another embodiment of the invention is a method of treating
a subject having a CD38-positive hematological malignancy,
comprising administering to the subject in need thereof an
anti-CD38 antibody in combination with all-trans retinoic acid
(ATRA), wherein the CD38-positive hematological malignancy is
multiple myeloma (MM), acute lymphoblastic leukemia (ALL),
non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL),
Burkitt's lymphoma (BL), follicular lymphoma (FL) or mantle-cell
lymphoma (MCL).
[0133] Another embodiment of the invention is a method of treating
a subject having a CD38-positive hematological malignancy,
comprising administering to the subject in need thereof an
anti-CD38 antibody in combination with all-trans retinoic acid
(ATRA), wherein the anti-CD38 antibody induces killing of
CD38-expressing cells in vitro by antibody-dependent cell-mediated
cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC),
wherein the CD38-positive hematological malignancy is multiple
myeloma (MM), acute lymphoblastic leukemia (ALL), non-Hodgkin's
lymphoma, diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma
(BL), follicular lymphoma (FL) or mantle-cell lymphoma (MCL).
[0134] Another embodiment of the invention is a method of treating
a subject having a CD38-positive hematological malignancy,
comprising administering to the subject in need thereof an
anti-CD38 antibody in combination with all-trans retinoic acid
(ATRA), wherein the CD38-positive hematological malignancy is
multiple myeloma (MM).
[0135] Another embodiment of the invention is a method of treating
a subject having a CD38-positive hematological malignancy,
comprising administering to the subject in need thereof an
anti-CD38 antibody in combination with all-trans retinoic acid
(ATRA), wherein the anti-CD38 antibody induces killing of
CD38-expressing cells in vitro by antibody-dependent cell-mediated
cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC),
wherein the CD38-positive hematological malignancy is multiple
myeloma (MM).
[0136] The invention also provides for a method of treating a
subject having a CD38-positive hematological malignancy, comprising
administering to the subject in need thereof an anti-CD38 in
combination with all-trans retinoic acid (ATRA), wherein the
subject is resistant to or has acquired resistance to treatment
with the anti-CD38 antibody.
[0137] The invention also provides for a method of treating a
subject having a CD38-positive hematological malignancy, comprising
administering to the subject in need thereof an anti-CD38 in
combination with all-trans retinoic acid (ATRA), wherein the
anti-CD38 antibody induces killing of CD38-expressing cells in
vitro by antibody-dependent cell-mediated cytotoxicity (ADCC) or
complement dependent cytotoxicity (CDC), wherein the subject is
resistant to or has acquired resistance to treatment with the
anti-CD38 antibody.
[0138] The invention also provides for a method of treating a
subject having a CD38-positive hematological malignancy, comprising
administering to the subject in need thereof an anti-CD38 in
combination with all-trans retinoic acid (ATRA), wherein the
subject is resistant to or has acquired resistance to treatment
with at least one chemotherapeutic agent.
[0139] The invention also provides for a method of treating a
subject having a CD38-positive hematological malignancy, comprising
administering to the subject in need thereof an anti-CD38 in
combination with all-trans retinoic acid (ATRA), wherein the
anti-CD38 antibody induces killing of CD38-expressing cells in
vitro by antibody-dependent cell-mediated cytotoxicity (ADCC) or
complement dependent cytotoxicity (CDC), wherein the subject is
resistant to or has acquired resistance to treatment with at least
one chemotherapeutic agent.
[0140] The invention also provides for a method of treating a
subject having multiple myeloma, comprising administering to the
subject in need thereof an anti-CD38 in combination with all-trans
retinoic acid (ATRA), wherein the subject is resistant to or has
acquired resistance to treatment with at least one chemotherapeutic
agent.
[0141] The invention also provides for a method of treating a
subject having multiple myeloma, comprising administering to the
subject in need thereof an anti-CD38 in combination with all-trans
retinoic acid (ATRA), wherein the anti-CD38 antibody induces
killing of CD38-expressing cells in vitro by antibody-dependent
cell-mediated cytotoxicity (ADCC) or complement dependent
cytotoxicity (CDC), wherein the subject is resistant to or has
acquired resistance to treatment with at least one chemotherapeutic
agent.
[0142] In some embodiments of the invention described herein, and
in some embodiments of each and every one of the numbered
embodiments listed below, the subject is resistant to or has
acquired resistance to treatment with at least one chemotherapeutic
agent, wherein the at least one chemotherapeutic agent is
lenalidomide, bortezomib, melphalan, dexamethasone or
thalidomide.
[0143] In some embodiments of the invention described herein, and
in some embodiments of each and every one of the numbered
embodiments listed below, the subject is resistant to or has
acquired resistance to treatment with at least one chemotherapeutic
agent, wherein the at least one chemotherapeutic agent is
lenalidomide, bortezomib, melphalan, dexamethasone, thalidomide,
cyclophosphamide, hydroxydaunorubicin (doxorubicin), vincristine or
prednisone.
[0144] In some embodiments of the invention described herein, and
in some embodiments of each and every one of the numbered
embodiments listed below, the subject is resistant to or has
acquired resistance to treatment with at least one chemotherapeutic
agent, wherein the at least one chemotherapeutic agent is
lenalidomide and/or bortezomib.
[0145] Various qualitative and/or quantitative methods may be used
to determine if a subject is resistant, has developed or is
susceptible to developing a resistance to treatment with an
anti-CD38 antibody or other therapeutic agent. Symptoms that may be
associated with resistance include, for example, a decline or
plateau of the well-being of the patient, an increase in the size
of a tumor, increase in the number of cancer cells, arrested or
slowed decline in growth of a tumor or tumor cells, and/or the
spread of cancerous cells in the body from one location to other
organs, tissues or cells. Re-establishment or worsening of various
symptoms associated with tumor may also be an indication that a
subject has developed or is susceptible to developing resistance to
an anti-CD38 antibody or other therapeutic agent. The symptoms
associated with cancer may vary according to the type of cancer.
For example, symptoms associated with B-cell malignancies may
include swollen lymph nodes in neck, groin or armpits, fever, night
sweats, coughing, chest pain, unexplained weight loss, abdominal
swelling or pain, or a feeling of fullness. Remission in malignant
lymphomas is standardized using the Cheson criteria (Cheson et al.,
J Clin Oncology 25:579-586, 2007), which guidelines can be used to
determine if a subject has developed a resistance to an anti-CD38
antibody or other therapeutic agent.
[0146] In some embodiments of the invention described herein, and
in some embodiments of each and every one of the numbered
embodiments listed below, the subject having a CD38-positive
hematological malignancy is homozygous for phenylalanine at
position 158 of CD16 (Fc.gamma.RIIIa-158F/F genotype) or
heterozygous for valine and pheynylalanine at position 158 of CD16
(Fc.gamma.RIIIa-158F/V genotype). CD16 is also known as the Fc
gamma receptor Ma (Fc.gamma.RIIIa) or the low affinity
immunoglobulin gamma Fc region receptor III-A isoform.
Valine/phenylalanine (V/F) polymorphism at Fc.gamma.RIIIa protein
residue position 158 has been shown to affect Fc.gamma.RIIIa
affinity to human IgG. Receptor with Fc.gamma.RIIIa-158F/F or
Fc.gamma.RIIIa-158F/V polymorphisms demonstrates reduced Fc
engagement and therefore reduced ADCC when compared to the
Fc.gamma.RIIIa-158V/V. The lack of or low amount of fucose on human
N-linked oligosaccharides improves the ability of the antibodies to
induce ADCC due to improved binding of the antibodies to human
Fc.gamma.RIIIa (CD16) (Shields et al., J Biol Chem 277:26733-40,
2002). Patients can be analyzed for their Fc.gamma.RIIIa
polymorphism using routine methods.
[0147] The invention also provides for the method of treating a
subject having a CD38-positive hematological malignancy, comprising
administering to the subject in need thereof an anti-CD38 in
combination with all-trans retinoic acid (ATRA), wherein the
subject is homozygous for phenylalanine at position 158 of CD16 or
heterozygous for valine and pheynylalanine at position 158 of
CD16.
[0148] The invention also provides for the method of treating a
subject having a CD38-positive hematological malignancy, comprising
administering to the subject in need thereof an anti-CD38 in
combination with all-trans retinoic acid (ATRA), wherein the
anti-CD38 antibody induces killing of CD38-expressing cells in
vitro by antibody-dependent cell-mediated cytotoxicity (ADCC) or
complement dependent cytotoxicity (CDC), wherein the subject is
homozygous for phenylalanine at position 158 of CD16 or
heterozygous for valine and pheynylalanine at position 158 of
CD16.
Administration/Pharmaceutical Compositions
[0149] In the methods of the invention, and in some embodiments of
each and every one of the numbered embodiments listed below, the
anti-CD38 antibodies may be provided in suitable pharmaceutical
compositions comprising the anti-CD38 antibody and a
pharmaceutically acceptable carrier. The carrier may be diluent,
adjuvant, excipient, or vehicle with which the anti-CD38 antibody
is administered. Such vehicles may be liquids, such as water and
oils, including those of petroleum, animal, vegetable or synthetic
origin, such as peanut oil, soybean oil, mineral oil, sesame oil
and the like. For example, 0.4% saline and 0.3% glycine may be
used. These solutions are sterile and generally free of particulate
matter. They may be sterilized by conventional, well-known
sterilization techniques (e.g., filtration). The compositions may
contain pharmaceutically acceptable auxiliary substances as
required to approximate physiological conditions such as pH
adjusting and buffering agents, stabilizing, thickening,
lubricating and coloring agents, etc. The concentration of the
molecules or antibodies of the invention in such pharmaceutical
formulation may vary widely, i.e., from less than about 0.5%,
usually to at least about 1% to as much as 15 or 20% by weight and
will be selected primarily based on required dose, fluid volumes,
viscosities, etc., according to the particular mode of
administration selected. Suitable vehicles and formulations,
inclusive of other human proteins, e.g., human serum albumin, are
described, for example, in e.g. Remington: The Science and Practice
of Pharmacy, 21.sup.st Edition, Troy, D. B. ed., Lipincott Williams
and Wilkins, Philadelphia, Pa. 2006, Part 5, Pharmaceutical
Manufacturing pp 691-1092, see especially pp. 958-989.
[0150] The mode of administration of the anti-CD38 antibody in the
methods of the invention may be any suitable route such as
parenteral administration, e.g., intradermal, intramuscular,
intraperitoneal, intravenous or subcutaneous, pulmonary,
transmucosal (oral, intranasal, intravaginal, rectal) or other
means appreciated by the skilled artisan, as well known in the
art.
[0151] The anti-CD38 antibody in the methods of the invention, and
in some embodiments of each and every one of the numbered
embodiments listed below, may be administered to a patient by any
suitable route, for example parentally by intravenous (i.v.)
infusion or bolus injection, intramuscularly or subcutaneously or
intraperitoneally. i.v. infusion may be given over for, example,
15, 30, 60, 90, 120, 180, or 240 minutes, or from 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11 or 12 hours.
[0152] The dose given to a patient having a CD38-positive
hematological malignancy is sufficient to alleviate or at least
partially arrest the disease being treated ("therapeutically
effective amount") and may be sometimes 0.005 mg/kg to about 100
mg/kg, e.g. about 0.05 mg/kg to about 30 mg/kg or about 5 mg to
about 25 mg/kg, or about 4 mg/kg, about 8 mg/kg, about 16 mg/kg or
about 24 mg/kg, or, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10
mg/kg, but may even higher, for example about 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90 or 100
mg/kg.
[0153] A fixed unit dose may also be given, for example, 50, 100,
200, 500 or 1000 mg, or the dose may be based on the patient's
surface area, e.g., 500, 400, 300, 250, 200, or 100 mg/m.sup.2.
Usually between 1 and 8 doses, (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) may
be administered to treat a CD38-positive B-cell malignancy, but 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more doses may be
given.
[0154] The administration of the anti-CD38 antibody in the methods
of the invention and in some embodiments of each and every one of
the numbered embodiments listed below, may be repeated after one
day, two days, three days, four days, five days, six days, one
week, two weeks, three weeks, one month, five weeks, six weeks,
seven weeks, two months, three months, four months, five months,
six months or longer. Repeated courses of treatment are also
possible, as is chronic administration. The repeated administration
may be at the same dose or at a different dose. For example, the
anti-CD38 antibody in the methods of the invention may be
administered at 8 mg/kg or at 16 mg/kg at weekly interval for 8
weeks, followed by administration at 8 mg/kg or at 16 mg/kg every
two weeks for an additional 16 weeks, followed by administration at
8 mg/kg or at 16 mg/kg every four weeks by intravenous
infusion.
[0155] The anti-CD38 antibodies may be administered in the methods
of the invention and in some embodiments of each and every one of
the numbered embodiments listed below, by maintenance therapy, such
as, e.g., once a week for a period of 6 months or more.
[0156] For example, anti-CD38 antibodies in the methods of the
invention and in some embodiments of each and every one of the
numbered embodiments listed below, may be provided as a daily
dosage in an amount of about 0.1-100 mg/kg, such as 0.5, 0.9, 1.0,
1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60,
70, 80, 90 or 100 mg/kg, per day, on at least one of day 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39, or 40, or alternatively, at least one of week 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after
initiation of treatment, or any combination thereof, using single
or divided doses of every 24, 12, 8, 6, 4, or 2 hours, or any
combination thereof.
[0157] Anti-CD38 antibodies in the methods of the invention and in
some embodiments of each and every one of the numbered embodiments
listed below, may also be administered prophylactically in order to
reduce the risk of developing cancer, delay the onset of the
occurrence of an event in cancer progression, and/or reduce the
risk of recurrence when a cancer is in remission. This may be
especially useful in patients wherein it is difficult to locate a
tumor that is known to be present due to other biological
factors.
[0158] The anti-CD38 antibody in the methods of the invention and
in some embodiments of each and every one of the numbered
embodiments listed below, may be lyophilized for storage and
reconstituted in a suitable carrier prior to use. This technique
has been shown to be effective with conventional protein
preparations and well known lyophilization and reconstitution
techniques can be employed.
[0159] The anti-CD38 antibody in the methods of the invention and
in some embodiments of each and every one of the numbered
embodiments listed below may be administered in combination with
all-trans retinoic acid (ATRA).
[0160] ATRA may be provided as a dosage of 45 mg/m.sup.2/day PO or
25 mg/m.sup.2/day PO.
[0161] The anti-CD38 antibody in the methods of the invention and
in some embodiments of each and every one of the numbered
embodiments listed below may be administered in combination with
all-trans retinoic acid (ATRA) and a third therapeutic agent.
[0162] In the methods of the invention, and in some embodiments of
each and every one of the numbered embodiments listed below, the
third therapeutic agent may be melphalan, mechlorethamine, thioepa,
chlorambucil, carmustine (BSNU), lomustine (CCNU),
cyclophosphamide, busulfan, dibromomannitol, streptozotocin,
dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other
platinum derivatives, such as carboplatin, thalidomide or a
thalidomide analog, lenalidomide or CC4047, a proteasome inhibitor,
such as bortezomib or vinca alkaloid, such as vincristine or an
anthracycline, such as doxorubicin.
[0163] While having described the invention in general terms, the
embodiments of the invention will be further disclosed in the
following examples that should not be construed as limiting the
scope of the claims.
Further Embodiments of the Invention
[0164] Set out below are certain further embodiments of the
invention according to the disclosures elsewhere herein. Features
from embodiments of the invention set out above described as
relating to the invention disclosed herein also relate to each and
every one of these further numbered embodiments. [0165] 1. An
anti-CD38 antibody for use in treating a subject having a
CD38-positive hematological malignancy, in combination with
all-trans retinoic acid (ATRA). [0166] 2. ATRA for use in treating
a subject having a CD38-positive hematological malignancy, in
combination with an anti-CD38 antibody. [0167] 3. The combination
of an anti-CD38 antibody) and ATRA for use in treating a subject
having a CD38-positive hematological malignancy. [0168] 4. The
anti-CD38 antibody for use according to embodiment 1, ATRA for use
according to embodiment 2, or the combination for use according to
embodiment 3, wherein the anti-CD38 antibody induces killing of the
CD38-expressing cells by [0169] a. antibody-dependent cell-mediated
cytotoxicity (ADCC); [0170] b. complement dependent cytotoxicity
(CDC); or [0171] c. both ADCC and CDC in vitro. [0172] 5. The
anti-CD38 antibody for use according to embodiment 1, ATRA for use
according to embodiment 2, or the combination for use according to
embodiment 3, wherein the anti-CD38 antibody induces killing of the
CD38-expressing cells by ADCC in vitro. [0173] 6. The anti-CD38
antibody for use according to embodiment 1, 4 or 5, ATRA for use
according to embodiment 2, 4 or 5, or the combination for use
according to embodiment 3-5, wherein the CD38-positive
hematological malignancy is multiple myeloma (MM), acute
lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), diffuse
large B-cell lymphoma (DLBCL), Burkitt's lymphoma (BL), follicular
lymphoma (FL) or mantle-cell lymphoma (MCL). [0174] 7. The
anti-CD38 antibody for use according to embodiment 1, 4-6, ATRA for
use according to embodiment 2, 4-6, or the combination for use
according to embodiment 3-6, wherein the CD38-positive
hematological malignancy is MM. [0175] 8. The anti-CD38 antibody
for use according to embodiment 1, 4-7, ATRA for use according to
embodiment 2, 4-7, or the combination for use according to
embodiment 3-7, wherein the subject is resistant to or has acquired
resistance to treatment with at least one chemotherapeutic agent,
and anti-CD38 antibody, or a combination of at least one
chemotherapeutic agent and an anti-CD38 antibody. [0176] 9. The
anti-CD38 antibody for use according to embodiment 1, 4-8, ATRA for
use according to embodiment 2, 4-8, or the combination for use
according to embodiment 3-8, wherein the at least one
chemotherapeutic agent is lenalidomide, bortezomib, melphalan,
dexamethasone or thalidomide. [0177] 10. The anti-CD38 antibody for
use according to embodiment 1, 4-9, ATRA for use according to
embodiment 2, 4-9, or the combination for use according to
embodiment 3-9, wherein the at least one chemotherapeutic agent is
lenalidomide or bortezomib. [0178] 11. The anti-CD38 antibody for
use according to embodiment 1, 4-10, ATRA for use according to
embodiment 2, 4-10, or the combination for use according to
embodiment 3-10, wherein [0179] a. the anti-CD38 antibody is of
IgG1, IgG2, IgG3 or IgG4 isotype; [0180] b. the anti-CD38 antibody
competes for binding to CD38 with an antibody comprising a heavy
chain variable region (VH) of SEQ ID NO: 4 and a light chain
variable region (VL) of SEQ ID NO: 5; [0181] c. the anti-CD38
antibody binds to the region SKRNIQFSCKNIYR (SEQ ID NO: 2) and the
region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1);
[0182] d. the anti-CD38 antibody comprises the heavy chain
complementarity determining regions (HCDR) 1 (HCDR1), 2 (HCDR2) and
3 (HCDR3) sequences of SEQ ID NOs: 6, 7 and 8, respectively; [0183]
e. the anti-CD38 antibody comprises the light chain complementarity
determining regions (LCDR) 1 (LCDR1), 2 (LCDR2) and 3 (LCDR3)
sequences of SEQ ID NOs: 9, 10 and 11, respectively; [0184] f. the
anti-CD38 antibody comprises the heavy chain variable region (VH)
of SEQ ID NO: 4 and the light chain variable region (VL) of SEQ ID
NO: 5; [0185] g. the anti-CD38 antibody comprises a heavy chain
comprising an amino acid sequence that is 95%, 96%, 97%, 98% or 99%
identical to that of SEQ ID NO: 12 and a light chain comprising an
amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to
that of SEQ ID NO: 13; [0186] h. the anti-CD38 antibody comprises
the heavy chain of SEQ ID NO: 12 and the light chain of SEQ ID NO:
13 [0187] i. the anti-CD38 antibody comprises th VH of SEQ ID NO:
14 and the VL of SEQ ID NO: 15; [0188] j. the anti-CD38 antibody
comprises th VH of SEQ ID NO: 16 and the VL of SEQ ID NO: 17;
[0189] k. the anti-CD38 antibody comprises th VH of SEQ ID NO: 18
and the VL of SEQ ID NO: 19; or [0190] l. the anti-CD38 antibody
comprises th VH of SEQ ID NO: 20 and the VL of SEQ ID NO: 21.
[0191] m.
Example 1
General Methods
Antibodies and Reagents
[0192] A human mAb against an innocuous antigen (HIV-1 gp120) was
used as an isotype control as described previously (van der Veers
et al., Haematologica 96:284-290, 2011; van der Veers et al., Blood
Cancer J 1:e41, 2011). All-trans retinoic acid (ATRA) was purchased
from Sigma-Aldrich and diluted in DMSO.
Bioluminescence Imaging (BLI)-Based ADCC Assays Using Luciferase
(LUC)-Transduced MM Cell Lines
[0193] LUC-transduced MM cell lines were co-cultured with effector
cells (freshly isolated PBMCs from healthy donors) at an effector
to target ratio of 1:25 in white opaque 96-well flat bottom plates
(Costar) in the presence of daratumumab (0.001, 0.01, 0.1, and 1.0
.mu.g/mL) for four hours. The survival of LUC.sup.+-MM cells was
then determined by BLI, 10 minutes after addition of the substrate
luciferin (125 .mu.g/mL; Promega). Lysis of MM cells was determined
using the following formula: % lysis=1-(mean BLI signal in the
presence of effector cells and daratumumab/mean BLI signal in the
presence of effector cells and control antibody).times.100%.
BLI-Based CDC Assays Using LUC-Transduced MM Cell Lines
[0194] Daratumumab (0, 0.03, 0.1, 0.3, 1.0 and 3.0 .mu.g/mL) was
added to MM cell lines in medium supplemented with pooled human
serum (10%; Sanquin) or heat-inactivated human serum. After a
1-hour incubation at 37.degree. C., cell lysis was determined by
BLI, 10 minutes after addition of luciferin (125 .mu.g/ml), and
calculated using the following formula: % lysis=1-(mean BLI signal
in the presence of native human serum/mean BLI signal in the
presence of heat-inactivated serum).times.100%.
Flow Cytometry-Based Ex Vivo ADCC and CDC Assays in BM-MNC
[0195] Freshly isolated BM-MNCs, containing 2-57% malignant plasma
cells as determined by flow cytometry, were immediately used in ex
vivo experiments. For ADCC experiments, BM-MNCs, containing the
malignant plasma cells, as well as the patient's own effector
cells, were incubated in RPMI+10% fetal bovine serum with
daratumumab (0.01-10 .mu.g/mL) in 96-well flat-bottom plates in
fully humidified incubators at 37.degree. C., 5% CO.sub.2-air
mixture for 48 h. Sample viability at incubation was more than 98%,
as assessed by using ToPro-3 (Invitrogen/Molecular Probes). For CDC
assays, BM-MNCs were treated with daratumumab (0.3-10 .mu.g/mL) and
complement for 1 hour prior to flow cytometric analysis. Pooled
human serum (10%) was used as a source of complement. The survival
of primary CD138.sup.+ MM cells in the BM-MNCs was determined by
flow-cytometry as previously described (van der Veers et al.,
Haematologica 96:284-290, 2011; van der Veers et al., Blood Cancer
J 1:e41, 2011). Surviving MM cells were enumerated by single
platform flow-cytometric analysis of CD138.sup.+ cells (with
CD138-PE (Beckman Coulter, Miami, Fla., USA)) in the presence of
Flow-Count Fluorospheres (Beckman Coulter) to determine absolute
numbers of cells. The percentage of MM cell lysis in the different
treated conditions was determined relative to MM survival of wells
treated with the control antibody (IgG1-b12 as IgG1 control
antibody for daratumumab) using the following formula: % lysis
cells=1-(absolute number of surviving CD138.sup.+ cells in treated
wells/absolute number of surviving CD138.sup.+ cells in control
wells).times.100%.
Immunophenotyping by Flow Cytometry
[0196] Expression of several cell surface proteins was determined
by flow cytometric analysis using FITC-, PE-, Per-CP-, or
APC-conjugated monoclonal antibodies. Anti-CD38, anti-CD138, and
anti-CD56 were purchased from Beckman Coulter; anti-CD3, anti-CD16,
anti-CD55, anti-CD59 from BD Biosciences; and anti-CD46 from
Biolegend. Flow cytometry was done using a FACS-Calibur device
(Becton Dickinson); the data were analyzed using the CellQuest
software.
Statistics
[0197] Statistical analyses were performed using Prism software
(Graphpad Software Inc, version 5). Comparisons between variables
were performed using two-tailed paired Student's t test.
Correlations between variables were made using the Spearman's rank
correlation coefficient. p-values below 0.05 were considered
significant.
Example 2
ATRA Increases CD38 Expression on MM Cell Lines and in Primary MM
Cells
[0198] An increase in CD38 expression levels may enhance the
efficacy of daratumumab to kill MM cells via ADCC or CDC.
Interaction of ATRA with nuclear retinoic acid receptors results in
altered expression of target genes including induction of CD38
expression (Malavasi F. J Leukoc Biol 90:217-219, 2011; Drach et
al., Cancer Res 54:1746-1752, 1994). Therefore, effect of ATRA on
MM cell lines RPMI8226, UM9, and XG1 was studied. MM cells were
incubated with RPMI-1640 medium alone or with ATRA ranging from
0-25 nM for 48 hours (FIG. 1A) or were incubated with 10 nM ATRA
for 24, 48, 72 or 96 hours (FIG. 1B) and then harvested to
determine CD38 expression by flow cytometry using a FACS-Calibur
device (Becton Dickinson) and anti-CD38 antibody (Beckman Coulter).
The data were analyzed using the CellQuest software.
[0199] Minimum of 10 nM ATRA was sufficient to induce a
1.9-4.4-fold increase in CD38 expression on the MM cell lines
RPMI8226, UM9, and XG1. Higher doses of ATRA did not further
enhance CD38 expression (FIG. 1A). Maximum enhancement of CD38
expression occurred at 48 hours (FIG. 1B). Therefore 10 nM ATRA for
48 hours was used in all subsequent experiments.
[0200] Ex vivo ATRA exposure (10 nM, 48 hours) of primary MM cells
from 26 patients was also studied. In these experiments, BM-MNCs
from 26 MM patients were incubated with RPMI-1640 medium alone or
with 10 nM ATRA for 48 hours, incubated at 4.degree. C. for 20 min
with FITC-conjugated CD38 antibody (Beckman Coulter) and then
harvested to determine CD38 expression by flow cytometry. Flow
cytometric analyses were performed using a FACS-Calibur device
(Becton Dickinson); the data were analyzed using the CellQuest
software.
[0201] ATRA induced CD38 expression (median increase 1.7-fold,
range 1.0-26.5-fold) (FIG. 2). There was also a significant
upregulation of CD138 expression levels (median increase:
2.0-fold), which is characteristic of MM cell differentiation. In
contrast, no significant changes in the expression of other plasma
cell antigens, such as HLA A/B/C or CD56 were observed in response
to ATRA.
Example 3
ATRA-Mediated Upregulation of CD38 Enhances Both
Daratumumab-Mediated ADCC and CDC Against MM Cells
[0202] Possible effect of ATRA-induced upregulation of CD38
expression on daratumumab-induced ADCC and CDC was tested in MM
cell lines XG-1, RPMI8226 and UM9 and in primary MM cells.
[0203] For MM cell lines, CDC and ADCC were assessed using
bioluminescence imaging (BLI) based ADCC and CDC assays as
described above. For primary MM cells, CDC and ADCC were assessed
using Flow cytometry-based ex vivo ADCC and CDC assays in BM-MNC as
described above. In the assays, cells were pre-treated with 10 nM
ATRA or solvent control for 48 hours, followed by incubation with
or without daratumumab in the presence of PBMCs as effector cells
for assessment of ADCC or in the presence of human serum as
complement source for analysis of CDC. Isotype control was added at
10 .mu.g/ml, and 10% heat-inactivated serum was used as control for
CDC.
[0204] FIG. 3A, FIG. 3B and FIG. 3C show the results of
daratumumab-induced CDC and ADCC in the XG1, RPMI8226 and UM9 cell
lines, respectively.
[0205] 10 nM ATRA alone induced no MM cell lysis. Pretreatment of
MM cell lines with 10 nM ATRA significantly increased
daratumumab-mediated CDC in XG-1 cells (FIG. 3A), and ADCC in XG-1
(FIG. 3A) and UM9 (FIG. 3C) cells, compared with solvent control
(FIG. 3A). In RPMI8226 cells there was no significant improvement
in daratumumab-mediated ADCC and CDC. These differences in ATRA
responsiveness may be partly explained by the fact that ATRA
enhanced CD38 expression 2.9-fold in XG-1 and 4.4-fold in UM9,
while the upregulation was only 1.9-fold in RPMI8226 cells (FIGS.
1A and 1B).
Example 4
ATRA-Mediated Upregulation of CD38 Enhances Both
Daratumumab-Mediated ADCC and CDC Against Primary MM Cells
[0206] Primary MM cells were evaluated to further explore the
effect of ATRA-mediated induction of CD38 expression on daratumumab
sensitivity.
[0207] FIG. 4A and FIG. 4B show results of daratumumab-induced CDC
and ADCC, respectively, in primary MM cells pretreated for 48 hours
with or without 10 nM ATRA. The graphs in FIG. 4A and FIG. 4B
represent pooled results of 16 or 13 patient samples,
respectively.
[0208] In primary MM cells, pretreatment with ATRA for 48 hours
resulted in a significant increase in their susceptibility to
daratumumab-mediated CDC in 13 out of 16 patients (data not shown)
and ADCC in 8 out of 11 patients (data not shown). Pooled results
of these patients show that ATRA improved CDC mediated by 10
.mu.g/mL daratumumab median from 16.1% to 43.9% (P<0.0001) (FIG.
4A), and ADCC mediated by 10 .mu.g/mL daratumumab improved median
from 25.1% to 39.5% (P=0.0315) by ATRA (FIG. 4B).
[0209] FIG. 5 shows results of daratumumab-induced CDC in primary
MM cells from each patient. FIG. 5A shows daratumumab-induced CDC
in primary MM cells form patient 1 and patient 2. FIG. 5B shows
daratumumab-induced CDC in primary MM cells form patient 3 and
patient 4. FIG. 5C shows daratumumab-induced CDC in primary MM
cells form patient 5 and patient 6. FIG. 5D shows
daratumumab-induced CDC in primary MM cells form patient 7 and
patient 8. FIG. 5E shows daratumumab-induced CDC in primary MM
cells form patient 9 and patient 10. FIG. 5F shows
daratumumab-induced CDC in primary MM cells form patient 11 and
patient 12. FIG. 5G shows daratumumab-induced CDC in primary MM
cells form patient 13 and patient 14. FIG. 5h shows
daratumumab-induced CDC in primary MM cells form patient 15 and
patient 16. ATRA induced daratumumab-mediated CDC in primary MM
cells that were not responsive to daratumumab alone in vitro (for
example patients 1, 4, 8, 12, 13, 15 and 16). These primary MM
cells were isolated from patients with refractory or double
refractory disease as indicated in Table 1. In some patient primary
MM cell samples, ATRA had no additional effect enhancing
daratumumab-mediated CDC (for example see patients 6, 7 and
14).
[0210] FIG. 6 shows results of daratumumab-induced ADCC in primary
MM cells from each patient. FIG. 6A shows daratumumab-induced CDC
in primary MM cells form patient 3 and patient 4. FIG. 6B shows
daratumumab-induced CDC in primary MM cells form patient 7 and
patient 8. FIG. 6C shows daratumumab-induced CDC in primary MM
cells form patient 9 and patient 10. FIG. 6D shows
daratumumab-induced CDC in primary MM cells form patient 14 and
patient 15. FIG. 6E shows daratumumab-induced CDC in primary MM
cells form patient 16 and patient 17. FIG. 6f shows
daratumumab-induced CDC in primary MM cells form patient 18. ATRA
induced daratumumab-mediated ADCC most primary MM cells tested.
These primary MM cells were isolated from patients with refractory
or double refractory disease as indicated in Table 1.
[0211] Surface expression of CD38 was also assessed in all these
tested primary MM cells in BM-MNCs incubated with RPMI-1640 medium
alone or with ATRA 10 nM for 48 hours (FIG. 7).
[0212] Overall the results suggest that ATRA is an attractive
strategy to improve CD38 expression and daratumumab activity in MM
cell lines and in primary MM cells, including MM cells that are
refractory to daratumumab-mediated CDC and/or ADCC.
[0213] Table 1 shows the baseline characteristics of the BM-MNC of
the tested 19 MM patients. In the table, * lenalidomide- and/or
bortezomib-refractory disease is defined as progressive disease on
lenalidomide- and bortezomib-therapy, no response (less than
partial response) to lenalidomide- and bortezomib-therapy, or
progressive disease within 60 days of stopping a lenalidomide- and
bortezomib-containing regimen, according to the International
Uniform Response Criteria for Multiple Myeloma.
TABLE-US-00006 TABLE 1 Patient 1 2 3 4 5 6 Parameter: Age (years)
71 43 71 64 64 55 Sex M M F M M F Type of monoclonal heavy IgG --
-- IgD -- IgG chain Type of light chain K K L K L L Previous
therapy Prior lines of therapy 10 4 4 6 3 0 (number) Prior stem
cell transplantation yes yes yes yes yes no Autologous yes yes yes
yes yes no Allogeneic no no no no no no Prior lenalidomide
treatment, yes yes yes yes yes no lenalidomide refractory yes yes
yes yes yes no status* Prior bortezomib treatment yes yes yes yes
yes no bortezomib refractory status* yes yes yes yes yes no CD38
expression on MM cells 1258 1346 764 1275 2642 1134 (MFI) CD46
expression on MM cells 1165 264 866 1346 661 1124 (MFI) CD55
expression on MM cells 610 119 552 227 1 594 (MFI) CD59 expression
on MM cells 235 62 228 108 7 90 (MFI) Patient 7 8 9 10 11 12
Parameter: Age (years) 55 64 75 63 56 59 Sex F M M F M M Type of
monoclonal heavy IgA -- -- IgA IgA -- chain Type of light chain L K
L K K K Previous therapy Prior lines of therapy 2 2 5 6 2 4
(number) Prior stem cell transplantation yes yes no yes yes yes
Autologous yes yes no yes yes yes Allogeneic no no no no no no
Prior lenalidomide treatment, no yes yes yes yes yes lenalidomide
refractory no yes yes yes no yes status* Prior bortezomib treatment
yes yes yes yes no yes bortezomib refractory status* yes no yes yes
no no CD38 expression on MM cells 1999 578 1252 1310 843 64 (MFI)
CD46 expression on MM cells 2288 4870 1700 196 368 264 (MFI) CD55
expression on MM cells 655 528 813 4 362 60 (MFI) CD59 expression
on MM cells 92 151 241 7 74 47 (MFI) Patient 13 14 15 16 17 18
Parameter: Age (years) 71 72 67 64 63 53 Sex F M M M M M Type of
monoclonal heavy -- -- IgG -- IgG IgA chain Type of light chain L K
K K L K Previous therapy Prior lines of therapy 4 5 2 3 4 2
(number) Prior stem cell transplantation yes no no yes yes yes
Autologous yes no no yes yes yes Allogeneic no no no no no no Prior
lenalidomide treatment, yes yes yes yes no yes lenalidomide
refractory yes yes yes yes no yes status* Prior bortezomib
treatment yes yes yes yes yes yes bortezomib refractory status* yes
yes no yes no yes CD38 expression on MM cells 173 241 78 1000 667
11 (MFI) CD46 expression on MM cells 300 492 362 491 538 557 (MFI)
CD55 expression on MM cells 379 1275 59 176 231 519 (MFI) CD59
expression on MM cells 188 75 9 107 70 52 (MFI) BM-MNCs; bone
marrow mononuclear cells. MM; multiple myeloma. M; male. F; female.
K; kappa. L; lambda
Example 5
ATRA Downregulates CD55 and CD59 Expression in Primary MM Cells
[0214] The experiments conducted revealed that the pretreatment of
MM cells with ATRA rendered these cells more susceptible to
daratumumab-mediated ADCC and CDC. The improvement in CDC was more
pronounced than the enhancement of ADCC. The molecular basis for
the observation was assessed.
[0215] The effect of ATRA on effector cells was evaluated. ATRA had
no effect or minimal effect on the ability of PBMCs from healthy
donors to induce ADCC on human MM cell lines L363-CD38, LME-1,
RPMI8226 and UM9 (data not shown). On the contrary, ATRA reduced
expression levels of complement-inhibitory proteins CD55, CD59 and
CD46 on MM cell lines and primary MM cells. In RPMI8226 (FIG. 8A),
L363 (FIG. 8B) and XG-1 (FIG. 8C) cells, ATRA reduced expression
levels of CD55, CD59, and CD46. In primary MM cells derived from 16
patients, ATRA significantly reduced the expression of CD55 (mean
reduction 21.3%, P=0.019) (FIG. 9A) and CD59 (mean reduction 37.5%,
P=0.0047) (FIG. 9B), while ATRA did not significantly affect CD46
expression levels (data not shown). The CD46, CD55 and CD59
expression levels from the tested 16 patients' samples are shown in
FIG. 10A (CD55), FIG. 10B (CD59) and FIG. 10C (CD46). In the
experiments, cells were cultured at 37.degree. C. with RPMI-1640
medium with or without 10 nM ATRA 10 nM for 48 h. Cells were then
incubated at 4.degree. C. for 20 min with the appropriate
conjugated antibodies panel. Flow cytometric analyses were
performed using a FACS-Calibur device (Becton Dickinson); the data
were analyzed using the CellQuest software.
Example 6
In Vivo Efficacy of the Combination of ATRA and Daratumumab Against
MM Tumors Growing in a Humanized Microenvironment
[0216] Hybrid scaffolds consisting of three 2-3 mm biphasic calcium
phosphate particles were coated in vitro with human mesenchymal
stromal cells (MSCs; 2.times.10.sup.5 cells/scaffold). After a week
of in vitro culture in a osteogenic medium, humanized scaffolds
were implanted subcutaneously into RAG2.sup.-/- .gamma.c.sup.-/-
mice, as described previously (Groen et al., Blood. 19; 120:e9-e16,
2012; de Haart et al., Clin. Cancer Res. 19:5591-5601, 2013).
[0217] Eight weeks after implantation, mice received a sublethal
irradiation dose (3 Gy, 200 kV, 4 mA) and luciferase-transduced XG1
cells were injected directly into the scaffold (1.times.10.sup.6
cells/scaffold). Three weeks after inoculation, when there was
visible tumor growth in the scaffolds by bioluminescent imaging
(BLI), different groups of mice were treated with 1) vehicle, 2)
ATRA plus T-cell depleted PBMC as effector cells (PBMC-T), 3)
daratumumab plus PBMC-T, and 4) daratumumab plus ATRA plus PBMC-T.
Daratumumab (8 mg/kg) was given intraperitoneally on days 23, 30,
and 37; PBMC-T (8.times.10.sup.6 cells/mouse) were given
intravenously on days 24, 31, and 38; and ATRA (10 mg/kg) was given
via intraperitoneal injection on days 21-24, 28-31, and 35-38.
PBMC-T were prepared by Ficoll-Hypaque density-gradient
centrifugation of buffy coats, and subsequent depletion of T cells
by CD3-beads using the EasySep.TM.-technology (STEMCELL
Technologies). Tumor growth was monitored by weekly BLI
measurements as described previously (Groen et al., Blood. 19;
120:e9-e16, 2012). All animal experiments were conducted after
acquiring permission from the local ethical committee for animal
experimentation and were in compliance with the Dutch Animal
Experimentation Act. The statistical differences between the
different treatment groups in the mice experiments were calculated
using a Mann-Whitney test. P-values below 0.05 were considered
significant.
[0218] Luciferase-transduced XG1 multiple myeloma cells developed
into aggressive tumors in immunodeficient RAG2.sup.-/-
.gamma..sub.c.sup.-/- mice in a humanized bone marrow
microenvironment generated by subcutaneous implantation of
MSC-coated ceramic scaffolds. To optimally evaluate the effects of
daratumumab and ATRA, mice were co-injected with NK cell-enriched
(T cell-depleted) PBMCs of a healthy donor in combination with
daratumumab and/or ATRA, as RAG2.sup.-/- .gamma..sub.c.sup.-/- mice
are devoid of NK cells. To follow the outgrowth of the tumor, BLI
was performed weekly for 5 weeks. As shown in FIG. 11, daratumumab
markedly slowed tumor progression, whereas ATRA as single agent had
no effect. ATRA also significantly enhanced the anti-MM effect of
daratumumab in this model.
Sequence CWU 1
1
221300PRTHomo sapiens 1Met Ala Asn Cys Glu Phe Ser Pro Val Ser Gly
Asp Lys Pro Cys Cys 1 5 10 15 Arg Leu Ser Arg Arg Ala Gln Leu Cys
Leu Gly Val Ser Ile Leu Val 20 25 30 Leu Ile Leu Val Val Val Leu
Ala Val Val Val Pro Arg Trp Arg Gln 35 40 45 Gln Trp Ser Gly Pro
Gly Thr Thr Lys Arg Phe Pro Glu Thr Val Leu 50 55 60 Ala Arg Cys
Val Lys Tyr Thr Glu Ile His Pro Glu Met Arg His Val 65 70 75 80 Asp
Cys Gln Ser Val Trp Asp Ala Phe Lys Gly Ala Phe Ile Ser Lys 85 90
95 His Pro Cys Asn Ile Thr Glu Glu Asp Tyr Gln Pro Leu Met Lys Leu
100 105 110 Gly Thr Gln Thr Val Pro Cys Asn Lys Ile Leu Leu Trp Ser
Arg Ile 115 120 125 Lys Asp Leu Ala His Gln Phe Thr Gln Val Gln Arg
Asp Met Phe Thr 130 135 140 Leu Glu Asp Thr Leu Leu Gly Tyr Leu Ala
Asp Asp Leu Thr Trp Cys 145 150 155 160 Gly Glu Phe Asn Thr Ser Lys
Ile Asn Tyr Gln Ser Cys Pro Asp Trp 165 170 175 Arg Lys Asp Cys Ser
Asn Asn Pro Val Ser Val Phe Trp Lys Thr Val 180 185 190 Ser Arg Arg
Phe Ala Glu Ala Ala Cys Asp Val Val His Val Met Leu 195 200 205 Asn
Gly Ser Arg Ser Lys Ile Phe Asp Lys Asn Ser Thr Phe Gly Ser 210 215
220 Val Glu Val His Asn Leu Gln Pro Glu Lys Val Gln Thr Leu Glu Ala
225 230 235 240 Trp Val Ile His Gly Gly Arg Glu Asp Ser Arg Asp Leu
Cys Gln Asp 245 250 255 Pro Thr Ile Lys Glu Leu Glu Ser Ile Ile Ser
Lys Arg Asn Ile Gln 260 265 270 Phe Ser Cys Lys Asn Ile Tyr Arg Pro
Asp Lys Phe Leu Gln Cys Val 275 280 285 Lys Asn Pro Glu Asp Ser Ser
Cys Thr Ser Glu Ile 290 295 300 214PRTHomo sapiens 2Ser Lys Arg Asn
Ile Gln Phe Ser Cys Lys Asn Ile Tyr Arg 1 5 10 314PRTHomo sapiens
3Glu Lys Val Gln Thr Leu Glu Ala Trp Val Ile His Gly Gly 1 5 10
4122PRTArtificial SequenceVH of anti-CD38 antibody 4Glu Val Gln Leu
Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Asn Ser Phe 20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Ala Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Phe Cys 85 90 95 Ala Lys Asp Lys Ile Leu Trp Phe Gly
Glu Pro Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120 5107PRTArtificial SequenceVL of anti-CD38
antibody 5Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser
Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr
Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95 Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 65PRTArtificial
SequenceHCDR1 of anti-CD38 antibody 6Ser Phe Ala Met Ser 1 5
717PRTArtificial SequenceHCDR2 of anti-CD38 antibody 7Ala Ile Ser
Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
813PRTArtificial SequenceHCDR3 of anti-CD38 antibody 8Asp Lys Ile
Leu Trp Phe Gly Glu Pro Val Phe Asp Tyr 1 5 10 911PRTArtificial
SequenceLCDR1 of anti-CD38 antibody 9Arg Ala Ser Gln Ser Val Ser
Ser Tyr Leu Ala 1 5 10 107PRTArtificial SequenceLCDR2 of anti-CD38
antibody 10Asp Ala Ser Asn Arg Ala Thr 1 5 1110PRTArtificial
SequenceLCDR3 of anti-CD38 antibody 11Gln Gln Arg Ser Asn Trp Pro
Pro Thr Phe 1 5 10 12452PRTArtificial Sequenceheavy chain of
anti-CD38 antibody 12Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser
Gly Phe Thr Phe Asn Ser Phe 20 25 30 Ala Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly
Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90
95 Ala Lys Asp Lys Ile Leu Trp Phe Gly Glu Pro Val Phe Asp Tyr Trp
100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His
Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 210 215
220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340
345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro
Gly Lys 450 13214PRTArtificial Sequencelight chain of anti-CD38
antibody 13Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser
Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr
Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95 Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115
120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly
Glu Cys 210 14120PRTArtificial SequenceVH of anti-CD38 antibody
14Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30 Ala Phe Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Arg Val Ile Pro Phe Leu Gly Ile Ala Asn
Ser Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp
Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Asp Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Asp Ile
Ala Ala Leu Gly Pro Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu
Val Thr Val Ser Ser 115 120 15107PRTArtificial SequenceVL of
anti-CD38 antibody 15Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Arg 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
16122PRTArtificial SequenceVH of anti-CD38 antibody 16Glu Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser
Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Ser Asn Tyr 20 25
30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
35 40 45 Gly Ile Ile Tyr Pro His Asp Ser Asp Ala Arg Tyr Ser Pro
Ser Phe 50 55 60 Gln Gly Gln Val Thr Phe Ser Ala Asp Lys Ser Ile
Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp
Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg His Val Gly Trp Gly Ser
Arg Tyr Trp Tyr Phe Asp Leu Trp 100 105 110 Gly Arg Gly Thr Leu Val
Thr Val Ser Ser 115 120 17107PRTArtificial SequenceVL of anti-CD38
antibody 17Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser
Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr
Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95 Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 18120PRTArtificial
SequenceVH of anti-CD38 antibody 18Gln Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Tyr Met Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly
Ile Ser Gly Asp Pro Ser Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Asp Leu Pro Leu Val Tyr Thr Gly Phe Ala
Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120
19109PRTArtificial SequenceVL of anti-CD38 antibody 19Asp Ile Glu
Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr
Ala Arg Ile Ser Cys Ser Gly Asp Asn Leu Arg His Tyr Tyr Val 20 25
30 Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr
35 40 45 Gly Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser
Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly
Thr Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Tyr
Thr Gly Gly Ala Ser Leu 85 90 95 Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Gly Gln 100 105 20120PRTArtificial SequenceVH of
anti-CD38 antibody 20Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Ala Lys Pro Gly Thr 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Trp Met Gln Trp Val Lys Gln
Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Thr Ile Tyr Pro
Gly Asp Gly Asp Thr Gly Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Lys
Ala Thr Leu Thr Ala Asp Lys Ser Ser Lys Thr Val Tyr 65 70 75 80 Met
His Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Asp Tyr Tyr Gly Ser Asn Ser Leu Asp Tyr Trp Gly Gln
100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120
21107PRTArtificial SequenceVL of anti-CD38 antibody 21Asp Ile Val
Met Thr Gln Ser His Leu Ser Met Ser Thr Ser Leu Gly 1 5 10 15 Asp
Pro Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Val 20 25
30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Arg Leu Ile
35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Ile Gly Val Pro Asp Arg Phe
Thr Gly 50 55 60 Ser Gly Ala Gly Thr Asp Phe Thr Phe Thr Ile Ser
Ser
Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His
Tyr Ser Pro Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys 100 105 224PRTHomo sapiens 22Val Gln Leu Thr 1
* * * * *
References