U.S. patent application number 15/340214 was filed with the patent office on 2017-02-16 for immune modulation and treatment of solid tumors with antibodies that specifically bind cd38.
The applicant listed for this patent is Janssen Biotech, Inc.. Invention is credited to Tahamtan Ahmadi, Tineke Casneuf, Henk Lokhorst, Tuna Mutis, Amy Sasser, Niels van de Donk.
Application Number | 20170044265 15/340214 |
Document ID | / |
Family ID | 57994571 |
Filed Date | 2017-02-16 |
United States Patent
Application |
20170044265 |
Kind Code |
A1 |
Ahmadi; Tahamtan ; et
al. |
February 16, 2017 |
Immune Modulation and Treatment of Solid Tumors with Antibodies
that Specifically Bind CD38
Abstract
The present invention relates to methods of immunomodulation and
treating patients having solid tumors with antibodies that
specifically bind CD38.
Inventors: |
Ahmadi; Tahamtan; (Spring
House, PA) ; Casneuf; Tineke; (Beerse, BE) ;
Lokhorst; Henk; (De Boelelaan, NL) ; Mutis; Tuna;
(De Boelelaan, NL) ; Sasser; Amy; (Doylestown,
PA) ; van de Donk; Niels; (De Boelelaan, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Janssen Biotech, Inc. |
Horsham |
PA |
US |
|
|
Family ID: |
57994571 |
Appl. No.: |
15/340214 |
Filed: |
November 1, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15191808 |
Jun 24, 2016 |
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15340214 |
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62184018 |
Jun 24, 2015 |
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62249546 |
Nov 2, 2015 |
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62331489 |
May 4, 2016 |
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62250566 |
Nov 4, 2015 |
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62263307 |
Dec 4, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/47 20130101;
C12Y 204/99 20130101; A61K 31/454 20130101; C07K 2317/56 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 39/39558 20130101; A61K 2039/545 20130101; A61K
2039/55 20130101; C07K 16/2896 20130101; C07K 16/40 20130101; A61K
45/06 20130101; A61K 31/454 20130101; C12Y 302/01035 20130101; A61K
39/39558 20130101; A61K 31/573 20130101; A61K 31/573 20130101; A61K
2039/505 20130101; C07K 2317/565 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 38/47 20060101 A61K038/47; A61K 39/395 20060101
A61K039/395; C07K 16/40 20060101 C07K016/40; C07K 16/30 20060101
C07K016/30 |
Claims
1) A method of treating a patient having a solid tumor, comprising
administering to the patient in need thereof a therapeutically
effective amount of an antibody that specifically binds CD38 for a
time sufficient to treat the solid tumor.
2) The method of claim 1, wherein the antibody that specifically
binds CD38 a) elicits an immune response in the patient; b) elicits
an immune response in the patient that is an effector T cell (Teff)
response; c) elicits an immune response in the patient that is an
effector T cell (Teff) response mediated by CD4.sup.+ T cells or
CD8.sup.+ T cells; d) elicits an immune response in the patient
that is an effector T cell (Teff) response mediated by CD8.sup.+ T
cells; e) increases the number of CD8.sup.+ T cells, increases
CD8.sup.+ T cell proliferation, increased T cell clonal expansion,
increases CD8.sup.+ memory cell formation, increases
antigen-dependent antibody production, increases cytokine
production, increases chemokine production or increases interleukin
production; f) inhibits function of an immune suppressor cell; g)
inhibits function of a regulatory T cell (Treg); h) inhibits
function of a CD3.sup.+CD4.sup.+CD25.sup.+CD127.sup.dim Treg; i)
inhibits function of CD38.sup.+ Treg; j) kills the Treg is by
antibody-dependent cell cytotoxicity (ADCC); k) inhibits function
of a myeloid-derived suppressor cell (MDSC); l) inhibits function
of a CD11b.sup.+HLADR.sup.-CD14.sup.-CD33.sup.+CD15.sup.+ cell
MDSC; m) inhibits function of CD38.sup.+ MDSC; n) kills the MDSC by
ADCC; o) inhibits function of a regulatory B cell (Breg); p)
inhibits function of a CD19.sup.+CD24.sup.+CD38.sup.+ Breg; q)
kills the Breg by ADCC; or r) inhibits function of an immune
suppressor cell that resides in bone marrow or in peripheral
blood.
3) The method of claim 1, wherein the solid tumor is a melanoma, a
lung cancer, a squamous non-small cell lung cancer (NSCLC), a
non-squamous NSCLC, a colorectal cancer, a prostate cancer, a
castration-resistant prostate cancer, a stomach cancer, an ovarian
cancer, a gastric cancer, a liver cancer, a pancreatic cancer, a
thyroid cancer, a squamous cell carcinoma of the head and neck, a
carcinoma of the esophagus or gastrointestinal tract, a breast
cancer, a fallopian tube cancer, a brain cancer, an urethral
cancer, a genitourinary cancer, an endometriosis, a cervical cancer
or a metastatic lesion of the cancer.
4) The method of claim 3, wherein the solid tumor lacks detectable
CD38 expression.
5) The method of claim 1, wherein the antibody that specifically
binds CD38 is a non-agonistic antibody.
6) The method of claim 5, wherein the antibody that specifically
binds CD38 a) 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; b) binds CD38 at
least to the region SKRNIQFSCKNIYR (SEQ ID NO: 2) and the region
EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1); c)
comprises a heavy chain complementarity determining region (HCDR)
1, a HCDR2, a HCDR3, a light chain complementarity determining
region (LCDR) 1, a LCDR2 and a LCDR3 amino acid sequences of SEQ ID
NOs: 6, 7, 8, 9, 10 and 11, respectively; d) comprises the VH of
SEQ ID NO: 4 and the VL of SEQ ID NO: 5; e) comprises the HCDR1,
the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of: i) the
VH of SEQ ID NO: 14 and the VL of SEQ ID NO: 15; ii) the VH of SEQ
ID NO: 16 and the VL of SEQ ID NO: 17; iii) the VH of SEQ ID NO: 18
and the VL of SEQ ID NO: 19; or iv) the VH of SEQ ID NO: 20 and the
VL of SEQ ID NO: 21; f) comprises the VH of SEQ ID NO: 14 and the
VL of SEQ ID NO: 15; g) comprises the VH of SEQ ID NO: 16 and the
VL of SEQ ID NO: 17; h) comprises the VH of SEQ ID NO: 18 and the
VL of SEQ ID NO: 19; or i) comprises the VH of SEQ ID NO: 20 and
the VL of SEQ ID NO: 21.
7) The method of claim 5, wherein the antibody that specifically
binds CD38 is administered in combination with a second therapeutic
agent.
8) The method of claim 7, wherein the second therapeutic agent is
a) a chemotherapeutic agent, a targeted anti-cancer therapy, a
standard of care drug for treatment of solid tumor, or an immune
checkpoint inhibitor; b) an anti-PD-1 antibody, an anti-PD-L1
antibody, an anti-PD-L2 antibody, an anti-LAG3 antibody, an
anti-TIM3 antibody, or an anti-CTLA-4 antibody; c) an anti-PD-1
antibody comprising i) the VH of SEQ ID NO: 22 and the VL of SEQ ID
NO: 23; ii) the VH of SEQ ID NO: 24 and the VL of SEQ ID NO: 25;
iii) the VH of SEQ ID NO: 32 and the VL of SEQ ID NO: 33; or iv)
the VH of SEQ ID NO: 34 and the VL of SEQ ID NO:35; d) an
anti-PD-L1 antibody; e) anti-PD-L1 antibody comprising i) the VH of
SEQ ID NO: 26 and the VL of SEQ ID NO: 27; ii) the VH of SEQ ID NO:
28 and the VL of SEQ ID NO: 29; or iii) the VH of SEQ ID NO: 30 and
the VL of SEQ ID NO: 31; f) an anti-PD-L2 antibody; g) an anti-LAG3
antibody; h) an anti-TIM-3 antibody; i) anti-TIM-3 antibody
comprising i) the VH of SEQ ID NO: 36 and the VL of SEQ ID NO: 37;
or ii) the VH of SEQ ID NO: 38 and the VL of SEQ ID NO: 39; j)
radiation therapy; or k) surgery.
9) The method of claim 7, wherein the second therapeutic agent is
administered simultaneously, sequentially or separately.
10) The method of claim 5, wherein the antibody that specifically
binds CD38 is administered subcutaneously in a pharmaceutical
composition comprising the antibody that specifically binds CD38
and a hyaluronidase.
11) The method of claim 10, wherein the hyaluronidase is rHuPH20 of
SEQ ID NO: 40.
12) The method of claim 5, wherein the antibody that specifically
binds CD38 is administered intravenously in a pharmaceutical
composition.
13) A method of suppressing activity of an immune suppressor cell,
comprising contacting the immune suppressing cell with an antibody
that specifically binds CD38.
14) The method of claim 13, wherein a) the immune suppressor cell
is a Treg; b) the immune suppressor cell is a
CD3.sup.+CD4.sup.+CD25.sup.+CD127.sup.dim Treg; c) the immune
suppressor cell is a MDSC; d) the immune suppressor cell is a
CD11b.sup.+HLADR.sup.-CD14.sup.-CD33.sup.+CD15.sup.+ MDSC; e) the
immune suppressor cell is a Breg; f) the immune suppressor cell is
a CD19.sup.+CD24.sup.+CD38.sup.+ Breg;
15) The method of claim 14, wherein the antibody that specifically
binds CD38 is a non-agonistic antibody.
16) The method of claim 15, wherein the antibody that specifically
binds CD38 a) competes for binding to CD38 with an antibody
comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5; b)
binds CD38 at least to the region SKRNIQFSCKNIYR (SEQ ID NO: 2) and
the region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO:
1); c) comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the
LCDR2 and the LCDR3 amino acid sequences of SEQ ID NOs: 6, 7, 8, 9,
10 and 11, respectively; d) comprises the VH of SEQ ID NO: 4 and
the VL of SEQ ID NO: 5; e) comprises the HCDR1, the HCDR2, the
HCDR3, the LCDR1, the LCDR2 and the LCDR3 of: i) the VH of SEQ ID
NO: 14 and the VL of SEQ ID NO: 15; ii) the VH of SEQ ID NO: 16 and
the VL of SEQ ID NO: 17; iii) the VH of SEQ ID NO: 18 and the VL of
SEQ ID NO: 19; or iv) the VH of SEQ ID NO: 20 and the VL of SEQ ID
NO: 21; f) comprises the VH of SEQ ID NO: 14 and the VL of SEQ ID
NO: 15; g) comprises the VH of SEQ ID NO: 16 and the VL of SEQ ID
NO: 17; h) comprises the VH of SEQ ID NO: 18 and the VL of SEQ ID
NO: 19; or i) comprises the VH of SEQ ID NO: 20 and the VL of SEQ
ID NO: 21.
17) A method of enhancing an immune response in a patient,
comprising administering to the patient an antibody that
specifically binds CD38.
18) The method of claim 17, wherein the patient has a cancer or a
viral infection.
19) The method of claim 18, wherein the antibody that specifically
binds CD38 is a non-agonistic antibody.
20) The method of claim 19, wherein the antibody that specifically
binds CD38 a) competes for binding to CD38 with an antibody
comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5; b)
binds CD38 at least to the region SKRNIQFSCIYR (SEQ ID NO: 2) and
the region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO:
1); c) comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the
LCDR2 and the LCDR3 amino acid sequences of SEQ ID NOs: 6, 7, 8, 9,
10 and 11, respectively; d) comprises the VH of SEQ ID NO: 4 and
the VL of SEQ ID NO: 5; e) comprises the HCDR1, the HCDR2, the
HCDR3, the LCDR1, the LCDR2 and the LCDR3 of: i) the VH of SEQ ID
NO: 14 and the VL of SEQ ID NO: 15; ii) the VH of SEQ ID NO: 16 and
the VL of SEQ ID NO: 17; iii) the VH of SEQ ID NO: 18 and the VL of
SEQ ID NO: 19; or iv) the VH of SEQ ID NO: 20 and the VL of SEQ ID
NO: 21; or f) comprises the VH of SEQ ID NO: 14 and the VL of SEQ
ID NO: 15; g) comprises the VH of SEQ ID NO: 16 and the VL of SEQ
ID NO: 17; h) comprises the VH of SEQ ID NO: 18 and the VL of SEQ
ID NO: 19; or i) 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 is a continuation-in-part of U.S. patent
application Ser. No. 15/191,808, filed 24 Jun. 2016, which claims
the benefit of U.S. Provisional Application No. 62/331,489 filed 4
May 2016, and U.S. Provisional Application No. 62/263,307, filed 4
Dec. 2015, and U.S. Provisional Application No. 62/250,566, filed 4
Nov. 2015, and U.S. Provisional Application No. 62/249,546, filed 2
Nov. 2015, and U.S. Provisional Application No. 62/184,018, filed
24 Jun. 2015, the entire contents of which are incorporated herein
by reference.
SEQUENCE LISTING
[0002] This application contains a Sequence Listing submitted via
EFS-Web, the entire content incorporated herein by reference in its
entirety. The ASCII text file, created on 29 Oct. 2016, is named
JBI5067USCIP_ST25.txt and is 45 kilobytes in size.
FIELD OF THE INVENTION
[0003] The present invention relates to methods of immune
modulation and treatment of solid tumors with antibodies that
specifically bind CD38.
BACKGROUND OF THE INVENTION
[0004] The immune system is tightly controlled by a network of
costimulatory and co-inhibitory ligands and receptors. These
molecules provide secondary signals for T cell activation and
provide a balanced network of positive and negative signals to
maximize immune responses against infection and tumors, while
limiting immunity to self (Wang et al., (Epub Mar. 7, 2011) J Exp
Med 208(3):577-92; Lepenies et al., (2008) Endocr Metab Immune
Disord Drug Targets 8:279-288).
[0005] Immune checkpoint therapy to treat solid tumors, targeting
co-inhibitory pathways in T cells to promote antitumor immune
responses, has led to advances in clinical care of cancer patients
with approval of anti-CTLA-4 and anti-PD-1 antibodies YERVOY.RTM.
(ipilimumab), KEYTRUDA.RTM. (pembrolizumab) and OPDIVO.RTM.
(nivolumab). While anti-PD-1/PD-L1 antibodies are demonstrating
encouraging clinical responses in patients with multiple solid
tumors, the response rates are still fairly low, about 15%-20% in
pretreated patients (Swaika et al., (2015) Mol Immunol doi:
10.1016/j.molimm 2015.02.009).
[0006] While natural killer cells (NK), dendritic cells (DC) and
effector T cells are capable of driving potent anti-tumor
responses, tumor cells often induce an immunosuppressive
microenvironment, favoring the development of immunosuppressive
populations of immune cells, such as myeloid-derived suppressor
cells (MDSC), regulatory T-cells (Treg) or regulatory B-cells
(Breg), which contribute to tumor immune tolerance and the failure
of immunotherapy regimens in cancer patients and experimental tumor
models.
[0007] Thus, there remains a need to develop new cancer
immunotherapies that induce adaptive immune response against tumors
or target the immunosuppressive immune cells.
SUMMARY OF THE INVENTION
[0008] The invention provides a method of treating a patient having
a solid tumor comprising administering to the patient in need
thereof a therapeutically effective amount of an antibody that
specifically binds CD38.
[0009] The invention also provides a method for treating a patient
having a regulatory T-cell (Treg) mediated disease comprising
administering to the patient in need thereof a therapeutically
effective amount of an antibody that specifically binds CD38.
[0010] The invention also provides a method for treating a patient
having a myeloid-derived suppressor cell (MDSC) mediated disease
comprising administering to the patient in need thereof a
therapeutically effective amount of an antibody that specifically
binds CD38.
[0011] The invention also provides a method for treating a patient
having a regulatory B-cell (Breg) mediated disease comprising
administering to the patient in need thereof a therapeutically
effective amount of an antibody that specifically binds CD38.
[0012] The invention also provides a method of suppressing activity
of a regulatory T-cell (Treg), comprising contacting the Treg with
an antibody that specifically binds CD38.
[0013] The invention also provides method of suppressing activity
of a myeloid-derived suppressor cell (MDSC), comprising contacting
the MDSC with an antibody that specifically binds CD38.
[0014] The invention also provides a method of suppressing activity
of a regulatory B-cell (Breg), comprising contacting the Breg with
an antibody that specifically binds CD38.
[0015] The invention also provides a method of enhancing an immune
response in a patient, comprising administering to the patient an
antibody that specifically binds CD38.
[0016] The invention also provides a method of treating a patient
having a solid tumor comprising reducing the number of Tregs cells
in the patient by administering to the patient an antibody that
specifically binds CD38.
[0017] The invention also provides a method of treating a patient
having a solid tumor, comprising reducing the number of
myeloid-derived suppressor cells (MDSC) in the patient by
administering to the patient an antibody that specifically binds
CD38.
[0018] The invention also provides a method of suppressing activity
of an immune suppressor cell, comprising contacting the immune
suppressing cell with an antibody that specifically binds CD38.
[0019] The invention also provides a method of treating a patient
having a viral infection, comprising administering to the patient
in need thereof an antibody that specifically binds CD38.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 shows that the median number of lymphocytes was
increased in patients responding to DARZALEX.TM. (daratumumab)
treatment at 8 mg/kg (upper line) or 16 mg/kg (lower line) doses
over time, and that the lymphocyte numbers returned to baseline
after end of treatment. Study: SIRIUS. X-axis indicates time
expressed as treatment cycle and days of dosing within each
treatment cycle (C1D1: cycle 1, day 1; C1D4; cycle 1, day 4, etc).
SCR: baseline; EOT: end of treatment; WK: week; POST-WK:
post-treatment at indicated weeks; post-PD FU: follow-up after
progression. The highlighted areas in gray shades indicate the
25-27% Interquartile Range (IQR) for the data points for each visit
for responders.
[0021] FIG. 2 shows the percent (%) change of absolute counts of
CD3.sup.+ T cells to baseline in peripheral blood in patients
treated with DARZALEX.TM. (daratumumab) for each individual patient
(light gray lines). Study: SIRIUS (MMY2002). The X-axis indicates
time expressed as treatment cycle and days of dosing within each
treatment cycle (C1D1: cycle 1, day 1; C1D4; cycle 1, day 4, etc).
WK: week; POST-WK: post-treatment at indicated weeks; POST-PD FU:
follow-up after progression. The black line shows the median %
change for all patients.
[0022] FIG. 3 shows the percent (%) change of absolute counts of
CD4.sup.+ T cells to baseline in peripheral blood in patients
treated with DARZALEX.TM. (daratumumab) for each individual patient
(light gray lines). Study: SIRIUS. The X-axis indicates time
expressed as treatment cycle and days of dosing within each
treatment cycle (C1D1: cycle 1, day 1; C1D4; cycle 1, day 4, etc).
WK: week; POST-TMT: post-treatment. The black line shows the median
% change for all patients.
[0023] FIG. 4 shows the percent (%) change of absolute counts of
CD8.sup.+ T cells to baseline in peripheral blood in patients
treated with DARZALEX.TM. (daratumumab) for each individual patient
(light gray lines). Study: SIRIUS. The X-axis indicates time
expressed as treatment cycle and days of dosing within each
treatment cycle (C1D1: cycle 1, day 1; C1D4; cycle 1, day 4, etc).
WK: week; Pre-PD FU: follow-up before progression; Post-PD FU:
follow-up after progression. The black line shows the median %
change for all patients.
[0024] FIG. 5 shows that the number of CD45.sup.+CD3.sup.+ cells
(measured as percentage of lymphocytes) in bone marrow aspirates
was increased during DARZALEX.TM. (daratumumab) treatment over time
at doses 8 mg/kg or 16 mg/kg. The graph includes both responders
and non-responders as indicated. Study: SIRIUS. The X-axis
indicates time expressed as treatment cycle and days of dosing
within each treatment cycle (C2D22: cycle 2, day 22; etc). SCR:
baseline; Post-PD follow-up after progression. The highlighted
areas in gray shade indicate the 25-27% Interquartile Range (IQR)
for the data points for each visit for the non-responders dosed at
8 mg/kg, the responders dosed at 16 mg/kg, or the non-responders
dosed at 16 mg/kg, respectively. NR: no-responder; R:
responder.
[0025] FIG. 6 shows that the number of CD45.sup.+CD3.sup.+CD8.sup.+
cells (measured as percentage of lymphocytes) in bone marrow
aspirates was increased during DARZALEX.TM. (daratumumab) treatment
over time at doses 8 mg/kg or 16 mg/kg. The graph includes both
responders and non-responders as indicated. Study: SIRIUS. The
X-axis indicates time expressed as treatment cycle and days of
dosing within each treatment cycle (C2D22: cycle 2, day 22; etc).
SCR: baseline; Post-PD follow-up after progression. The highlighted
areas in gray shade indicate the 25-27% Interquartile Range (IQR)
for the data points for each visit for the non-responders dosed at
8 mg/kg, the responders dosed at 16 mg/kg, or the non-responders
dosed at 16 mg/kg, respectively. NR: no-responder; R:
responder.
[0026] FIG. 7A shows that the ratio of CD8.sup.+/Treg and
CD8.sup.+/CD4.sup.+ cells in peripheral blood expressed as median
values of all treated patients increased over time during
DARZALEX.TM. (daratumumab) treatment. Time points: C1D1: cycle 1
day; C3D1: cycle 3 day 1; C4D1: cycle 4 day 1. Study: SIRIUS. SRC:
baseline.
[0027] FIG. 7B shows that the ratio of CD8.sup.+/Treg cells in bone
marrow aspirates expressed as median values of all treated patients
increased over time during DARZALEX.TM. (daratumumab) treatment.
Time points: C1D1: cycle 1 day; C3D1: cycle 3 day 1; C4D1: cycle 4
day 1. Study: SIRIUS.
[0028] FIG. 8A shows that responders had increased CD8.sup.+ T-cell
clonality when compared to non-responders, as measured using %
change in abundance (CIA) of particular clonal cells. Study: GEN501
17 patient subset.
[0029] FIG. 8B shows the fold change in CD8.sup.+ T-cell clonality
in individual patients pre- vs. post DARZALEX.TM. (daratumumab)
treatment. Responders are indicated with the star. Clonality was
measured as fold change in abundance (CIA) of particular clonal
cells. Study: GEN501 17 patient subset.
[0030] FIG. 8C shows that responders (Group A) had greater total
expansion in the TCR repertoire, measured using CIA (change in
abundance) when compared to non-responders (Group B). P=0.037.
Study: GEN501 17 patient subset.
[0031] FIG. 8D shows the sum of absolute change in abundance (CIA)
in responders and non-responders for each expanded T cell clone.
P=0.035 between responders (Group A) and non-responders (Group B).
Study: GEN501 17 patient subset.
[0032] FIG. 8E shows the maximum CIA of a single T-cell clone in
responders (Group A) and non-responders (Group B). Study: GEN501 17
patient subset.
[0033] FIG. 8F shows that responders (Group A) had greater maximum
expansion of a single clone, measured using maximum % CIA, when
compared to non-responders (Group B). P=0.0477. Study: GEN501 17
patient subset.
[0034] FIG. 9A shows the percentage (%) of CD8.sup.+ naive cells in
peripheral blood in non-responders (NR, black squares) and in
patients having at least minimal response (MR, white squares) to
DARZALEX.TM. (daratumumab) at baseline, or at 2 weeks, 4 weeks or 8
weeks of treatment, or after relapse. Study: GEN501 17 patient
subset. **p=1.82.times.10.sup.-4.
[0035] FIG. 9B shows the percentage of CD8.sup.+ central memory
cells (Tem) in peripheral blood in non-responders (NR, black
squares) and in patients having at least minimal response (MR,
white squares) to DARZALEX.TM. (daratumumab) at baseline, or at 2
weeks, 4 weeks or 8 weeks of treatment, or after relapse. Study:
GEN501 17 patient subset. *p=4.88.times.10.sup.-2.
[0036] FIG. 9C shows the percentage increase of HLA Class I
restricted .sup.CD8+ T cells in peripheral blood at baseline, or at
week 1, 4 or 8 of treatment, or after relapse. Study: GEN501 17
patient subset.
[0037] FIG. 9D shows that CD38 is expressed at low levels in
CD8.sup.+ naive T cells and in CD8.sup.+ central memory cells (Tem)
in peripheral blood at bassline or on treatment. Study: GEN501 17
patient subset. MFI: Mean fluorescent intensity.
[0038] FIG. 10A shows a histogram of FACS analyses showing
frequency of Tregs (CD3.sup.+
CD3.sup.+CD4.sup.+CD25.sup.+CD127.sup.dim (top histogram, P4 cell
population) and the frequency of CD38.sup.+ Tregs within the Treg
population (bottom histogram, P5 cell population) in multiple
myeloma patients at baseline. Study: GEN501 17 patient subset.
[0039] FIG. 10B shows a histogram of FACS analyses showing
frequency of Tregs (CD3.sup.+
CD3.sup.+CD4.sup.+CD25.sup.+CD127.sup.dim (top histogram, P4 cell
population) and the frequency of CD38.sup.+ Tregs within the Treg
population (bottom histogram, P5 cell population) in multiple
myeloma patients after DARZALEX.TM. (daratumumab) treatment.
DARZALEX.TM. (daratumumab) treatment depleted CD38.sup.+ Tregs.
Study: GEN501 17 patient subset.
[0040] FIG. 10C shows that frequency of the CD38.sup.high
CD3.sup.+CD4.sup.+CD25.sup.+CD127.sup.dim Tregs in patients treated
with DARZALEX.TM. (daratumumab) at baseline, or at 1 week, 4 week,
8 weeks, after relapse or at end of treatment at 6 months (EOT).
CD38.sup.high Treg frequency was reduced with DARZALEX.TM.
(daratumumab) treatment and returned to baseline at EOT. Y-axis: %
of CD38.sup.highCD3.sup.+CD4.sup.+CD25.sup.+CD127.sup.dim Tregs
from CD3 T-cells. Study: GEN501 17 patient subset.
[0041] FIG. 10D shows the CD8.sup.+/Treg cell ratio in responders
and non-responders at baseline, at 1 week, 4 weeks and 8 weeks of
treatment. The CD8.sup.+/Treg cell ratio was significantly higher
in responders vs. non-responders (p=0.00955) at Week 8 of
treatment. Study: GEN501 17 patient subset.
[0042] FIG. 10E shows that effector cell proliferation is inhibited
more efficiently in the presence of CD38.sup.+ Tregs when compared
to the CD38.sup.- Tregs or negative controls. Error bars represent
standard error. Asterisks denote significant changes. Samples were
obtained from multiple healthy donors. Cell proliferation was
assessed through the dilution of carboxyfluorescein succinimidyl
ester (CFSE).
[0043] FIG. 11 shows that Myeloid-derived suppressor cells (MDSC)
are present in multiple myeloma patients (top graph, boxed cells)
and that about half of the cells expressed CD38 (middle graph,
boxed cells). The CD38high MDSC population was depleted in patients
treated with one infusion of DARZALEX.TM. (daratumumab) (bottom
graph, boxed cells). Study: GEN501 17 patient subset.
[0044] FIG. 12 shows that the number of CD38high MDSCs
(CD11b.sup.+HLADR.sup.-CD14.sup.-CD33.sup.+CD15.sup.+) was reduced
in patients after 1 week, 4 week or 8 week treatment with
DARZALEX.TM. (daratumumab) when compared to the baseline, and
returned to close to baseline after end of treatment (EOT).
Relapsed patients still demonstrated reduced CD38high MDSCs. Black
squares: non-responders; white squares: patients with at least
Minimal Response to DARZALEX.TM. (daratumumab) treatment. The
vertical lines indicate the median values in each group. Patients
2, 4, 15, 16 and 17 demonstrated high initial CD38high MDSCs
population. Study: GEN501 17 patient subset.
[0045] FIG. 13 shows that the patients with highest CD38high MDSCs
(patients 2, 4, 15, 16 and 17) had the highest Progression-Free
Survival (PFS). These patients had either partial Response (PR) or
Minimal Response (MR) to DARZALEX.TM. (daratumumab) treatment. SD:
Stable Disease; PD: Progressive Disease. X-axis shows the PFS for
each individual numbered patient.
[0046] FIG. 14 shows that MDSC are sensitive to DARZALEX.TM.
(daratumumab)-induced ADCC. Daudi cells were used as positive
control for target cells in the assays. % cell lysis was
measured.
[0047] FIG. 15A shows that CD38.sup.+ Bregs were depleted in
patients treated with DARZALEX.TM. (daratumumab) at Week 1, Week 4
and Week 8 of treatment.
[0048] FIG. 15B shows that CD38.sup.+ Bregs secrete IL-10 upon
stimulation.
[0049] FIG. 16A shows the anti-viral response measured through CMV,
EBV and influenza virus specific (CEF) IFN-.gamma. production in
PBMCs from DARZALEX.TM. (daratumumab) treated patient with VGPR at
baseline and at indicated times during treatment. OD: optical
density. White bar: negative control; black bar: CEF added; dashed
bar: allogeneic PBMCs only. Asterix indicates a statistically
significant change. Pre 4, 8, 10=Week 4, 8 or 10 of treatment.
[0050] FIG. 16B shows the anti-viral response measured through CMV,
EBV and influenza virus specific (CEF) IFN-.gamma. production in
PBMCs from DARZALEX.TM. (daratumumab) treated patient with CR at
baseline and at indicated times during treatment. OD: optical
density. White bar: negative control; black bar: CEF added; dashed
bar: allogeneic PBMCs only. Asterix indicates a statistically
significant change. Pre 4, 8, 10=Week 4, 8 or 10 of treatment.
[0051] FIG. 16C shows the anti-viral response measured through CMV,
EBV and influenza virus specific (CEF) IFN-.gamma. production in
PBMCs from DARZALEX.TM. (daratumumab) treated patient with PD at
baseline and at indicated times during treatment. OD: optical
density. White bar: negative control; black bar: CEF added; dashed
bar: allogeneic PBMCs only. Ns: not significant. Pre 4, 8=Week 4 or
8 of treatment.
[0052] FIG. 16D shows the anti-viral response measured through CMV,
EBV and influenza virus specific (CEF) IFN-.gamma. production in
PBMCs from DARZALEX.TM. (daratumumab) treated patient with MR at
baseline and at indicated times during treatment. OD: optical
density. White bar: negative control; black bar: CEF added; dashed
bar: allogeneic PBMCs only. Ns: not significant. Pre 4, 8=Week 4 or
8 of treatment.
[0053] FIG. 16E shows the percentage (%) of proliferating
virus-reactive T cells in PBMCs from DARZALEX.TM. (daratumumab)
treated patient with VGPR at baseline and at indicated times during
treatment. White bar: negative control; black bar: CEF added.
Asterix indicates a statistically significant change. Pre 4, 8,
10=Week 4, 8 or 10 of treatment.
[0054] FIG. 16F shows the percentage (%) of proliferating
virus-reactive T cells in PBMCs from DARZALEX.TM. (daratumumab)
treated patient with CR at baseline and at indicated times during
treatment. White bar: negative control; black bar: CEF added.
Asterix indicates a statistically significant change. Pre 4, 8,
10=Week 4, 8 or 10 of treatment.
[0055] FIG. 17A shows a histogram of FACS analyses showing CD38
expression levels on natural killer cells (NK), monocytes, B cells
and T cells from a healthy donor.
[0056] FIG. 17B shows a histogram of FACS analyses showing CD38
expression levels on plasma cells, natural killer cells (NK),
monocytes, B cells and T cells from a multiple myeloma patient.
[0057] FIG. 17C shows a comparison of the mean fluorescent
intensity (MFI) of CD38 in CD38+ Tregs, Bregs, NK, B cells and T
cells from relapsed and refractory multiple myeloma patients. CD38
was expressed at lower level in B cells and T cells when compared
to the CD38+Tregs, Bregs and NK cells.
[0058] FIG. 18 shows that PD-L1 protein is downregulated in PBMC
samples from responders (R) and upregulated in non-responders (NR)
over time. SD: stable disease. C1D1: cycle 1 day 1; C3D1, cycle 3,
day 1. Y axis shows the log 2 protein concentration values.
DETAILED DESCRIPTION OF THE INVENTION
[0059] As used in this specification and the appended claims, the
singular forms "a," "an," and "the" include plural referents unless
the content clearly dictates otherwise. Thus, for example,
reference to "a cell" includes a combination of two or more cells,
and the like.
[0060] "CD38" refers to the human CD38 protein (synonyms:
ADP-ribosyl cyclase 1, cADPr hydrolase 1, cyclic ADP-ribose
hydrolase 1). Human CD38 has an amino acid sequence shown in
GenBank accession number NP_001766 and in SEQ ID NO: 1. It is well
known that CD38 is a single pass type II membrane protein with
amino acid residues 1-21 representing the cytosolic domain, amino
acid residues 22-42 representing the transmembrane domain, and
residues 43-300 representing the extracellular domain of CD38.
TABLE-US-00001 SEQ ID NO: 1
MANCEFSPVSGDKPCCRLSRRAQLCLGVSILVLILVVVLAVVVPRWRQQW
SGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCN
ITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLL
GYLADDLTWCGEFNTSKINYQSCPDWRKDCSNNPVSVFWKTVSRRFAEAA
CDVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQTLEAWVIHGGREDS
RDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKNPEDSSCTSEI
[0061] "Antibodies" as used herein is meant in a broad sense and
includes immunoglobulin molecules including monoclonal antibodies
including murine, human, humanized and chimeric monoclonal
antibodies, antibody fragments, bispecific or multispecific
antibodies, dimeric, tetrameric or multimeric antibodies, single
chain antibodies, domain antibodies and any other modified
configuration of the immunoglobulin molecule that comprises an
antigen binding site of the required specificity.
[0062] Immunoglobulins may 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 IgA1, IgA2, IgG1, IgG2, IgG3 and
IgG4. 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.
[0063] "Antibody fragments" 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 CH1 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 CH1 domains; a Fv fragment consisting of the VL and
VH domains of a single arm of an antibody; a domain antibody (dAb)
fragment (Ward et al., Nature 341:544-6, 1989), which consists of a
VH domain. VH and VL domains may be engineered and linked together
via a synthetic linker to form various types of single chain
antibody designs in which 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.
[0064] "Isolated antibody" refers to an antibody or antibody
fragment that is substantially free of other antibodies having
different antigenic specificities (e.g., an isolated antibody
specifically binding CD38 is substantially free of antibodies that
specifically bind antigens other than human CD38). An isolated
antibody that specifically binds CD38, however, may have
cross-reactivity to other antigens, such as orthologues of human
CD38, such as Macaca fascicularis (cynomolgus) CD38. In case of a
bispecific antibody, the bispecific antibody specifically binds two
antigens of interest, and is substantially free of antibodies that
specifically bind antigens other that the two antigens of interest.
Moreover, an isolated antibody may be substantially free of other
cellular material and/or chemicals. "Isolated antibody" encompasses
antibodies that are isolated to a higher purity, such as antibodies
that are 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure.
[0065] "Specific binding" or "specifically binds" or "binds" refers
to an antibody binding to an antigen or an epitope within the
antigen with greater affinity than for other antigens. Typically,
the antibody binds to the antigen or the epitope within the antigen
with an equilibrium dissociation constant (K.sub.D) of about
1.times.10.sup.-8 M or less, for example about 1.times.10.sup.-9 M
or less, about 1.times.10.sup.-10 M or less, about
1.times.10.sup.-11 M or less, or about 1.times.10.sup.-12 M or
less, typically with the K.sub.D that is at least one hundred fold
less than its K.sub.D for binding to a nonspecific antigen (e.g.,
BSA, casein). The dissociation constant may be measured using
standard procedures. Antibodies that specifically bind to the
antigen or the epitope within the antigen may, however, have
cross-reactivity to other related antigens, for example to the same
antigen from other species (homologs), such as human or monkey, for
example Macaca fascicularis (cynomolgus, cyno), Pan troglodytes
(chimpanzee, chimp) or Callithrix jacchus (common marmoset,
marmoset). While a monospecific antibody specifically binds one
antigen or one epitope, a bispecific antibody specifically binds
two distinct antigens or two distinct epitopes.
[0066] 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 (1970) J Exp Med 132:211-50; 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 antibody variable
domains which are hypervariable in structure as defined by Chothia
and Lesk (Chothia and Lesk (1987) Mol Biol 196:901-17). Other terms
include "IMGT-CDRs" (Lefranc et al., (2003) Dev Comparat Immunol
27:55-77) and "Specificity Determining Residue Usage" (SDRU)
(Almagro (2004) Mol Recognit 17:132-43). 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., (2003) Dev Comparat Immunol 27:55-77.
[0067] "Chothia residues" as used herein are the antibody VL and VH
residues numbered according to Al-Lazikani (Al-Lazikani et al.,
(1997) J Mol Biol 273:927-48).
[0068] "Framework" or "framework sequences" are the remaining
sequences of a variable region other than those defined to be
antigen binding sites. Because the antigen binding sites can be
defined by various terms as described above, the exact amino acid
sequence of a framework depends on how the antigen-binding site was
defined.
[0069] "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 regions so that the framework may not be an exact copy of
expressed human immunoglobulin or germline gene sequences.
[0070] "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.
[0071] A human antibody comprises heavy or light chain variable
regions that are "derived from" sequences of human origin wherein
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. A "human antibody"
may contain amino acid differences when compared to the human
germline immunoglobulin or rearranged immunoglobulin genes due to
for example naturally occurring somatic mutations or intentional
introduction of substitutions in the framework or antigen binding
site, or both. Typically, "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 human germline
immunoglobulin or rearranged immunoglobulin genes. In some cases,
"human antibody" may contain consensus framework sequences derived
from human framework sequence analyses, for example as described in
Knappik et al., (2000) J Mol Biol 296:57-86, or synthetic HCDR3
incorporated into human immunoglobulin gene libraries displayed on
phage, for example as described in Shi et al., (2010) J Mol Biol
397:385-96, and Intl. Pat. Publ. No. WO2009/085462.
[0072] Human antibodies derived from human immunoglobulin sequences
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, resulting in
antibodies that do not naturally exist within the human antibody
germline repertoire in vivo.
[0073] Antibodies in which antigen binding sites are derived from a
non-human species are not included in the definition of human
antibody.
[0074] "Recombinant antibody" includes all antibodies that are
prepared, expressed, created or isolated by recombinant means, such
as antibodies isolated from an animal, for example 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.
[0075] "Monoclonal antibody" refers to a preparation of antibody
molecules of single molecular composition. A monoclonal antibody
composition displays a single binding specificity and affinity for
a particular epitope, or in a case of a bispecific monoclonal
antibody, a dual binding specificity to two distinct epitopes.
"Monoclonal antibody" therefore refers to an antibody population
with single amino acid composition in each heavy and each light
chain, except for possible well known alterations such as removal
of C-terminal lysine from the antibody heavy chain Monoclonal
antibodies may have heterogeneous glycosylation within the antibody
population. Monoclonal antibody may be monospecific or
multispecific, or monovalent, bivalent or multivalent. A bispecific
antibody is included in the term monoclonal antibody.
[0076] "Epitope" 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 may
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.
[0077] "Variant" refers to a polypeptide or a polynucleotide that
differs from a reference polypeptide or a reference polynucleotide
by one or more modifications for example, substitutions, insertions
or deletions.
[0078] "In combination with" means that two or more therapeutics
are administered to a subject together in a mixture, concurrently
as single agents or sequentially as single agents in any order. In
general, each agent will be administered at a dose and/or on a time
schedule determined for that agent.
[0079] "Treat" or "treatment" refers to therapeutic treatment
wherein the object is to slow down (lessen) an undesired
physiological change or disease, such as the development or spread
of tumor or tumor cells, or to provide a beneficial or desired
clinical outcome during treatment. 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, lack of metastasis, amelioration
or palliation of the disease state, and remission (whether partial
or total), whether detectable or undetectable. "Treatment" may 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 disease as well as those subjects prone to have the
physiological change or disease.
[0080] "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 include, for example, improved
well-being of the patient, reduction in a tumor burden, arrested or
slowed growth of a tumor, and/or absence of metastasis of cancer
cells to other locations in the body.
[0081] "Inhibits growth" (e.g. referring to tumor cells) refers to
a measurable decrease or delay in the tumor cell growth or tumor
tissue in vitro or in vivo when contacted with a therapeutic or a
combination of therapeutics or drugs, when compared to the decrease
or delay in the growth of the same tumor cells or tumor tissue in
the absence of the therapeutic or the combination of therapeutic
drugs. Inhibition of growth of a tumor cell or tumor tissue in
vitro or in vivo may be at least about 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, 99%, or 100%.
[0082] "Regulatory T cells" or "Tregs" or "Treg" refers to T
lymphocytes that regulates the activity of other T cell(s) and/or
other immune cells, usually by suppressing their activity. The
Tregs may be CD3.sup.+CD4.sup.+CD25.sup.+CD127.sup.dim T cells. It
is appreciated that the Tregs may not be fully restricted to this
phenotype, and may express Foxp3.
[0083] "Effector T cells" or "Teffs" or "Teff" refers to T
lymphocytes that carry out a function of an immune response, such
as killing tumor cells and/or activating an anti-tumor
immune-response which can result in clearance of the tumor cells
from the body. The Teffs may be CD3.sup.+ with CD4.sup.+ or
CD8.sup.+. The Teffs may secrete, contain or express markers such
as IFN-.gamma., granzyme B and ICOS. It is appreciated that the
Teffs may not be fully restricted to these phenotypes.
[0084] "Function of Tregs" or "Treg function" refers to a
suppressive function of the Tregs that relates to regulation of
host immune responses and/or prevention of autoimmunity. Function
of Tregs may be suppression of an anti-tumor response elicited by
CD8.sup.+ T cells, natural killer (NK) cells, MO cells, B cells, or
dendritic cells (DCs), or suppression of proliferation of effector
T cells.
[0085] "Inhibit function of Tregs" or "inhibit Treg function"
refers to decreasing the level of function of Tregs in vitro or in
vivo in an animal or human subject, which may be determined by
conventional techniques known in the art. The level of the function
of Tregs may be decreased by, for example, at least about 1%, 5%,
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%.
"Inhibit function of Tregs" include reducing the number of Tregs,
for example by killing the Tregs via antibody effector functions
such as antibody-dependent cellular cytotoxicity (ADCC).
[0086] "Myeloid-derived suppressor cells" or "MDSCs" or "MDSC"
refers to a specialized population of cells that are of the
hematopoietic lineage and express the macrophage/monocyte marker
CD11b and the granulocyte marker Gr-1/Ly-6G. Phenotype of the MDSCs
may be for example
CD11b.sup.+HLA-DR.sup.-CD14.sup.-CD33.sup.+CD15.sup.+. The MDSCs
express low or undetectable expression of the mature antigen
presenting cell markers MHC Class II and F480. The MDSCs are
immature cells of the myeloid lineage and may further differentiate
into several cell types, including macrophages, neutrophils,
dendritic cells, monocytes or granulocytes. The MDSCs may be found
naturally in normal adult bone marrow of human and animals or in
sites of normal hematopoiesis, such as the spleen.
[0087] "Inhibit function of MDSCs" or "inhibit MDSC function"
refers to decreasing the level of function of MDSCs in vitro or in
vivo in an animal or human subject, which may be determined by
conventional techniques known in the art. The level of the function
of MDSC may be decreased by, for example, at least about 1%, 5%,
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%.
"Inhibit function of MDSC" include reducing the number of MDSC, for
example by killing the MDSC via antibody effector functions, such
as ADCC. The MDSCs may suppress T cell responses such as
proliferation, clonal expansion or cytokine production by various
mechanisms such as production of reactive oxygen species,
peroxynitrites, increased arginase metabolism due to high levels of
arginase, and increased nitrous oxide synthase. The MDSCs may
response to IFN-.gamma. and several cytokines such as IL-4 and
IL-13. IFN-.gamma. may activate MDSCs which induces the activity of
nitric-oxide synthase 2 (NOS2). Alternately, Th2 cytokines such as
interleukin-4 (IL-4) and IL-13 may activate MDSCs which may lead to
the induction of arginase-1 (ARG1) activity. The metabolism of
L-arginine by either NOS2 or ARG1 may lead to the inhibition of
T-cell proliferation, and the activity of both enzymes together can
result in T-cell apoptosis through the production of reactive
nitrogen-oxide species.
[0088] "Treg related disease" refers to a disease or disorder
linked to T regulatory cells (Tregs). Treg related disease may be
caused by Treg function, for example, suppression of an anti-tumor
response or suppression of effector T cell proliferation. The Treg
mediated disease may be cancer. "Treg related disease" and "Treg
mediated disease" are used exchangeably herein.
[0089] "Enhance response of effector T cells" or "enhance T cell
responses" refers to enhancement or stimulation of effector T cells
in vitro or in vivo in an animal or human subject to have a
sustained or amplified biological function, or renew or reactivate
exhausted or inactive T-cells. Exemplary T-cell responses are
proliferation, secretion of .gamma.-interferon from CD 8.sup.+
T-cells, antigen responsiveness, or clonal expansion. The manner of
measuring this enhancement is known to one of ordinary skill in the
art.
[0090] "MDSC related disease" refers to a disease or disorder
linked to myeloid-derived suppressor cells (MDSCs). MDSC related
disease may be caused by a MDSC function, for example, suppression
of an anti-tumor response or effector T cell proliferation. The
MDSC mediated disease may be cancer. "MDSC related disease" and
"MDSC mediated disease" are used exchangeably herein.
[0091] "Regulatory B cell" or "Breg" or "Bregs" refers to B
lymphocytes that suppress immune responses. The Bregs may be
CD19.sup.+CD24.sup.+CD38.sup.+ cells, and may suppress immune
responses by inhibiting T cell proliferation mediated by IL-10
secreted by the Bregs. It is appreciated that other Breg subsets
exists, and are described in for example Ding et al., (2015) Human
Immunology 76: 615-621.
[0092] "Breg related disease" refers to a disease or disorder
linked to regulatory B cells. Breg related disease may be caused by
for example Breg mediated suppression of an anti-tumor response or
effector T cell proliferation. The Breg mediated disease may be
cancer. "Breg related disease" and "Breg mediated disease" are used
exchangeably herein.
[0093] "Patient" includes any human or nonhuman animal "Nonhuman
animal" includes all vertebrates, e.g., mammals and non-mammals,
such as nonhuman primates, sheep, dogs, cats, horses, cows
chickens, amphibians, reptiles, etc. "Patient" and "subject" are
used interchangeably herein.
[0094] The invention provides a method for treating a patient
having a solid tumor with an antibody that specifically binds CD38
regardless whether the tumor cells express CD38 or not. The
invention further provides methods for treating a patient having
regulatory T cell (Treg), myeloid-derived suppressor cell (MDSC) or
regulatory B cell (Breg) mediated disease. The invention further
provides methods for modulating Treg, MDSC or Breg activity to
treat solid tumors that are CD38 positive and/or associated with
high levels of these immune suppressive cells.
[0095] The invention is based, at least in part, on the discovery
that the anti-CD38 antibody DARZALEX.TM. (daratumumab) has an
immunomodulatory activity in patients, reducing the number of
immune suppressive Tregs, MDSCs and Bregs, increasing the number of
CD8.sup.+ T cells and the ratio of CD8.sup.+ to Tregs, promoting
CD8.sup.+ central memory cell formation and increasing T cell
clonality.
[0096] DARZALEX.TM. (daratumumab) and other anti-CD38 antibodies
are being evaluated in the clinic for their efficacy to treat heme
malignancies and plasma cell disorders, including multiple myeloma,
by the ability of the antibody to eliminate CD38-positive cells by
antibody effector functions, such as ADCC, CDC, ACDP and apoptosis,
but their immunomodulatory activity in promoting adaptive immune
responses has not been recognized Other immune modulatory
antibodies (anti-PD1, anti-CTLA4) function through targeting
components of the immune system that suppress anti-tumor responses.
For example, anti-PD1 antibodies have been demonstrated to increase
T-cell proliferation, stimulate antigen-specific memory responses,
and partially relieve Treg-mediated suppression of effector T cells
in vitro (for example, see U.S. Pat. No. 8,779,105). Two anti-PD-1
antibodies are currently approved for treatment of melanoma,
OPDIVO.RTM. (nivolumab) and KEYTRUDA.RTM. (pembrolizumab) and these
antibodies are in clinical development for various solid tumors,
such as lung non-small cell carcinoma, prostate, head and neck,
gastrointestinal, stomach, prostate, fallopian tube, ovarian,
pancreatic, breast and brain cancer, renal, bladder, urethral,
oesophageal and colorectal cancer. Anti-CTLA-4 antibody YERVOY.RTM.
(ipilimumab) has been approved for treatment of melanoma.
YERVOY.RTM. (ipilimumab) and another anti-CTLA-4 antibody,
tremelimumab are also being developed for prostate, non-small cell
lung cancer, ovarian, gastrointestinal, stomach, colorectal, renal,
oesophageal, and genitourinary cancer.
[0097] Without wishing to be bound by any particular theory, based
on the immunomodulatory effects observed with DARZALEX.TM.
(daratumumab) described herein, DARZALEX.TM. (daratumumab) and
other anti-CD38 antibodies may be efficacious in treatment of solid
tumors. Due to the general activation of immune response observed
in patients treated with DARZALEX.TM. (daratumumab), patients
having CD38-negative solid tumors may respond to anti-CD38 antibody
therapies as well.
[0098] The invention provides for a method of treating a patient
having a solid tumor, comprising administering to the patient in
need thereof a therapeutically effective amount of an antibody that
specifically binds CD38 for a time sufficient to treat the solid
tumor.
[0099] The invention also provides for a method of treating a
patient having a regulatory T cell (Treg) mediated disease,
comprising administering to the patient in need thereof a
therapeutically effective amount of an antibody that specifically
binds CD38 for a time sufficient to treat the Treg mediated
disease.
[0100] The invention also provides for a method of treating a
patient having a myeloid-derived suppressor cell (MDSC) mediated
disease, comprising administering to the patient in need thereof a
therapeutically effective amount of an antibody that specifically
binds CD38 for a time sufficient to treat the MDSC mediated
disease.
[0101] The invention also provides for a method of treating a
patient having a regulatory B cell (Breg) mediated disease,
comprising administering to the patient in need thereof a
therapeutically effective amount of an antibody that specifically
binds CD38 for a time sufficient to treat the Breg mediated
disease.
[0102] The invention also provides for a method of suppressing
activity of a regulatory T cell (Treg), comprising contacting the
regulatory T cell with an antibody that specifically binds
CD38.
[0103] The invention also provides for a method of suppressing
activity of a myeloid-derived suppressor cell (MDSC), comprising
contacting the MDSC with an antibody that specifically binds
CD38.
[0104] The invention also provides for a method of suppressing
activity of a regulatory B cell (Breg), comprising contacting the
Breg with an antibody that specifically binds CD38.
[0105] The invention also provides for a method of treating a
patient having a solid tumor, comprising reducing the number of
regulatory T cells (Treg) in the patient by administering to the
patient an antibody that specifically binds CD38.
[0106] The invention also provides for a method of treating a
patient having a solid tumor, comprising reducing the number of
myeloid-derived suppressor cells (MDSC) in the patient by
administering to the patient an antibody that specifically binds
CD38.
[0107] The invention also provides for a method of treating a
patient having a solid tumor, comprising reducing the number of
regulatory B cells (Breg) in the patient by administering to the
patient an antibody that specifically binds CD38.
[0108] The invention also provides for a method of enhancing an
immune response in a patient, comprising administering to the
patient in need thereof an antibody that specifically binds CD38
for a time sufficient to enhance the immune response.
[0109] In some embodiments, the patient has a viral infection.
[0110] The invention also provides for a method of treating a viral
infection in a patient, comprising administering to the patient in
need therefor an antibody that specifically binds CD38 for a time
sufficient to treat the viral infection.
[0111] In some embodiments, the immune response is an effector T
cell (Teff) response.
[0112] In some embodiments, the Teff response is mediated by
CD4.sup.+ T cells or CD8.sup.+ T cells.
[0113] In some embodiments, the Teff response is mediated by
CD4.sup.+ T cells.
[0114] In some embodiments, the Teff response is mediated by
CD8.sup.+ T cells.
[0115] In some embodiments, the Teff response is an increase in the
number of CD8.sup.+ T cells, increased CD8.sup.+ T cell
proliferation, increased T cell clonal expansion, increased
CD8.sup.+ memory cell formation, increased antigen-dependent
antibody production, or increased cytokine, chemokine or
interleukin production.
[0116] Proliferation of T cells may be assessed for example by
measuring the rate of DNA synthesis using tritiated thymidine or
measuring production of interferon-.gamma. (IFN-.gamma.) in vitro,
or measuring absolute number or percentage of T cells in a
population of cells from patient samples using known methods.
[0117] Clonal expansion may be assessed by for example sequencing
TCR from a pool of T cells using know methods.
[0118] Memory cell formation may be assessed by measuring the ratio
of naive T cells (CD45RO.sup.-/CD62L.sup.+) to memory T cells
(CD45RO.sup.+/CD62L.sup.high) using for example FACS.
[0119] Cytokine, chemokine or interleukin production, such as
production of interferon-.gamma. (IFN-.gamma.), tumor necrosis
factor-alpha (TNF-.alpha.), IL-1, IL-2, IL-3, IL-4, IL-6, IL-8,
IL-10, IL-12, IL-13, IL-16, IL-18 and IL-23, MIP-1.alpha.,
MIP-1.beta., RANTES, CCL4 may be assessed using standard methods
such as ELISA or ELLISPOT assay.
[0120] Antigen-specific antibody production may be assessed from
samples derived from patient using standard methods, such as ELISA
or radioimmunoassay (RIA).
[0121] The meaning of "increase" or "increasing" various Teff
responses is readily understood. The increase may be increase of at
least about 5%, at least about 10%, 25%, 50%, 51%, 52%, 53%, 54%,
55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,
68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%,
200%, 250%, 300%, 350%, 400% or more in a test sample or in a
subject when compared to control, e.g., for example in a patient
treated with an anti-CD38 antibody when compared to the same
patient before treatment, or in a patient or group of patients that
are responsive to anti-CD38 antibody treatment when compared to a
patient or a group of patients that are non-responsive to the same
treatment. Typically, the increase is statistically
significant.
[0122] Similarly, the meaning of "reduce" or "reducing" or
"decreasing" or "decrease" the number of Tregs, MDSCs and/or Bregs
is readily understood. The decrease may be at least about 10%, 25%,
50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,
63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,
76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%,
120%, 130%, 140%, 150%, 200%, 250%, 300%, 350%, 400% or more in a
test sample or in a subject when compared to control, e.g., for
example in a patient treated with an anti-CD38 antibody when
compared to the same patient before treatment, or in a patient or
group of patients that are responsive to anti-CD38 antibody
treatment when compared to a patient or a group of patients that
are non-responsive to the same treatment. Typically, the decrease
is statistically significant.
[0123] In some embodiments, the antibody that specifically binds
CD38 inhibits function of immune suppressor cells.
[0124] In some embodiments, the immune suppressor cells are
regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSC)
or regulatory B cells (Bregs).
[0125] In some embodiments, the Tregs are
CD3.sup.+CD4.sup.+CD25.sup.+CD127.sup.dim T cells.
[0126] In some embodiments, the
CD3.sup.+CD4.sup.+CD25.sup.+CD127.sup.dim cells express Foxp3.
[0127] In some embodiments, the
CD3.sup.+CD4.sup.+CD25.sup.+CD127.sup.dim T cells express CD38.
[0128] Treg function, such as their ability to suppress Teff cells,
may be assessed using known methods, such as assessing the ability
of Tregs to suppress Teff proliferation in mixed lymphocyte
reaction (MLR).
[0129] Treg function may be inhibited by for example reducing the
relative number of Tregs when compared to Teffs (e.g. increasing
the ratio of CD8.sup.+/Treg cells) by direct killing of Tregs or a
sub-population of Tregs, such as CD38.sup.+ Tregs.
[0130] In some embodiments, the Treg function is inhibited by
killing the Treg cells.
[0131] In some embodiments, the Treg killing is mediated by
antibody-induced antibody-dependent cell cytotoxicity (ADCC),
antibody-dependent cell phagocytosis (ADCP), complement-dependent
cytotoxicity (CDC) or apoptosis induced by an antibody specifically
binding CD38.
[0132] In some embodiment, the Treg killing is mediated by
ADCC.
[0133] In some embodiments, the CD38.sup.+ Tregs are killed.
[0134] In some embodiments, 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%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%,
49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58% or 60% of Tregs
are killed.
[0135] As CD38 is expressed only in a portion of Tregs and MDSCs,
it is expected that treatment of patients with solid tumors will
not result in systemic depletion of Tregs and MDSCs, therefore
likely providing an improved safety profile.
[0136] In some embodiments, the MDSCs are
CD11b.sup.+HLA-DR.sup.-CD14.sup.-CD33.sup.+CD15.sup.+ cells.
[0137] In some embodiments, the
CD11b.sup.+HLA-DR.sup.-CD14.sup.-CD33.sup.+CD15.sup.+ MDSCs express
CD38.
[0138] MDSC function may be inhibited for example by reducing the
number of MDSCs by direct killing of the cells.
[0139] In some embodiments, the MDSC function is inhibited by
killing the CD38.sup.+ MDSC.
[0140] In some embodiments, the MDSC killing is mediated by
antibody-induced antibody-dependent cell cytotoxicity (ADCC),
antibody-dependent cell phagocytosis (ADCP), complement-dependent
cytotoxicity (CDC) or apoptosis induced by the antibody that
specifically binds CD38.
[0141] In some embodiments, the MDSC killing is mediated by
ADCC.
[0142] In some embodiments, 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%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%,
49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58% or 60% of the
MDSCs are killed.
[0143] In some embodiments, the Bregs are
CD19.sup.+CD24.sup.+CD38.sup.+ cells.
[0144] The Breg function may be inhibited for example by reducing
the number of Bregs by direct killing of the Bregs.
[0145] In some embodiments, the Breg function is inhibited by
killing the CD38.sup.+ Bregs.
[0146] In some embodiments, the Breg killing is mediated by
antibody-induced antibody-dependent cell cytotoxicity (ADCC),
antibody-dependent cell phagocytosis (ADCP), complement-dependent
cytotoxicity (CDC) or apoptosis induced by the antibody that
specifically binds CD38.
[0147] In some embodiments, the Breg killing is mediated by
ADCC.
[0148] In some embodiments, 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%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%,
49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58% or 60% of the
Bregs are killed.
[0149] Tregs play a critical role in the maintenance of peripheral
self-tolerance. Naturally occurring CD4.sup.+CD25.sup.hi Tregs are
produced in the thymus and express Foxp3, a transcriptional factor
required for establishment and maintenance of Treg lineage identity
and suppressor function. Tregs can accumulate at a disease site
(e.g. within tumor), where they suppress the effector function of
tumor antigen specific T cells, resulting in insufficient
anti-tumor responses. Increased densities of tumor-infiltrating
Foxp3.sup.+ Tregs have been associated with poor prognosis in
various solid tumors, including pancreatic, ovarian, and
hepatocellular carcinoma. Depletion of Tregs results in enhanced
antitumor immunity and tumor rejection in murine models but may
also result in the development of autoimmune diseases.
[0150] Myeloid-derived suppressor cells (MDSC) are a heterogeneous
population of early myeloid progenitors, immature granulocytes,
macrophages, and dendritic cells at different stages of
differentiation. They accumulate in large numbers in cancer
patients and they have potent immunosuppressive functions,
suppressing both the cytotoxic activities of natural killer cells
(NK) and natural killer T cells (NKT), and the adaptive immune
response mediated by CD8.sup.+ T cells. While the mechanism of NK
cell inhibition is currently not well-understood, multiple pathways
are responsible for MDSC-mediated T cell suppression including
production of arginase 1/ARG1 and upregulation of nitric oxide
synthase 2 (NOS2). ARG1 and NOS2 metabolize L-arginine and either
together or separately blocks the translation of the T cell CD3
zeta chain, inhibits T cell proliferation, and promotes T cell
apoptosis. Additionally, MDSCs secrete immunosuppressive cytokines
and induce regulatory T cell development.
[0151] MDSC are induced by pro-inflammatory cytokines and are found
in increased numbers in infectious and inflammatory pathological
conditions. They accumulate in the blood, bone marrow, and
secondary lymphoid organs of tumor-bearing mice and their presence
in the tumor microenvironment has been suggested to have a
causative role in promoting tumor-associated immune
suppression.
[0152] MDSC have been described in patients with colon carcinoma,
melanoma, hepatocellular carcinoma, head and neck squamous cell
carcinoma, non-small cell lung carcinoma, renal cell carcinoma,
pancreatic adenocarcinoma and breast carcinoma (Mandruzzato et al.,
(2009) J Immunol 182: 6562-6568; Liu et al., (2009) J Cancer Res
Clin Oncol 136: 35-45; Ko et al., (2009) Clin Cancer Res 15:
2148-2157; Morse et al., (2009) Expert Opin Biol Ther 9: 331-339;
Diaz-Montero et al., (2009) Cancer Immunol Immunother 58: 49-59;
Corzo et al., (2009) J Immunol 182: 5693-5701). In cancer patients,
Diaz et al (Diaz-Montero et al., (2009) Cancer Immunol Immunother
58: 49-59) propose that accumulation of MDSC correlates with more
advanced disease and poor prognosis.
[0153] Tumor-infiltrating Bregs have been identified in solid
tumors, and the Bregs may promote tumor growth and metastasis by
various mechanisms such as suppressing the anti-tumor activity of
CD8.sup.+ T cells and NK cells, as described in for example Ding et
al., (2015) Human Immunology 76:615-62.
[0154] "Antibody-dependent cellular cytotoxicity",
"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 FcvRIIIa. 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 antibody that specifically
binds CD38, 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. Exemplary effector cells for such
assays include peripheral blood mononuclear cells (PBMC) and NK
cells. Exemplary target cells include Tregs or MDSCs expressing
CD38. In an exemplary assay, target cells are labeled with 20
.mu.Ci of .sup.51Cr for 2 hours and washed extensively. Cell
concentration of the target cells may be adjusted to
1.times.10.sup.6 cells/ml, and anti-CD38 antibodies at various
concentrations are added. Assays are started by adding target cells
at an effector:target cell ratio of 40:1. After incubation for 3 hr
at 37.degree. C. assays are stopped by centrifugation and .sup.51Cr
release from lysed cells are measured in a scintillation counter.
Percentage of cellular cytotoxicity may be calculated as % maximal
lysis which may be induced by adding 3% perchloric acid to target
cells.
[0155] "Antibody-dependent cellular phagocytosis" ("ADCP") refers
to a mechanism of elimination of antibody-coated target cells by
internalization by phagocytic cells, such as macrophages or
dendritic cells. ADCP may be evaluated by using Tregs or MDSCs
expressing CD38 as target cells engineered to express GFP or other
labeled molecule. Effctor:target cell ratio may be for example 4:1.
Effector cells may be incubated with target cells for 4 hours with
or without anti-CD38 antibody. After incubation, cells may be
detached using accutase. Macrophages may be identified with
anti-CD11b and anti-CD14 antibodies coupled to a fluorescent label,
and percent phagocytosis may be determined based on % GFP
fluorescent in the CD11.sup.+CD14.sup.+ macrophages using standard
methods.
[0156] "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.
[0157] 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 mAbs may be achieved using different methods
reported to lead to the successful expression of relatively high
defucosylated antibodies bearing the biantennary complex-type of Fc
oligosaccharides such as control of culture osmolality (Konno et
al., (2012) Cytotechnology 64:249-65), application of a variant CHO
line Lec13 as the host cell line (Shields et al., (2002) J Biol
Chem 277:26733-26740), application of a variant CHO line EB66 as
the host cell line (Olivier et al., (2010) MAbs 2(4), Epub ahead of
print; PMID: 20562582), application of a rat hybridoma cell line
YB2/0 as the host cell line (Shinkawa et al., (2003) J Biol Chem
278:3466-3473), introduction of small interfering RNA specifically
against the .alpha. 1,6-fucosyltransferase (FUT8) gene (Mori et
al., (2004) Biotechnol Bioeng 88:901-908), or coexpression of
.beta.-1,4-N-acetylglucosaminyltransferase III and Golgi
.alpha.-mannosidase II or a potent alpha-mannosidase I inhibitor,
kifunensine (Ferrara et al., (2006) J Biol Chem 281:5032-5036;
Ferrara et al., (2006) Biotechnol Bioeng 93:851-861; Xhou et al.,
(2008) Biotechnol Bioeng 99:652-65). 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.
[0158] In some embodiments, the antibody that specifically binds
CD38 comprises a substitution in the antibody Fc.
[0159] In some embodiments, the antibody that specifically binds
CD38 comprises a substitution in the antibody Fc at amino acid
positions 256, 290, 298, 312, 356, 330, 333, 334, 360, 378 or 430
(residue numbering according to the EU index).
[0160] In some embodiments, the antibody that specifically binds
CD38 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%.
[0161] In some embodiments, the antibody that specifically binds
CD38 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%
[0162] Substitutions in the Fc and reduced fucose content may
enhance the ADCC activity of the antibody that specifically binds
CD38.
[0163] "Fucose content" means 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. These 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 Intl. 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).
[0164] "Low fucose" or "low fucose content" as used herein refers
to antibodies with fucose content of about 0%-15%.
[0165] "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%.
[0166] In some embodiments, the antibody that specifically binds
CD38 may induce killing of Tregs, MDSCs and/or Bregs by apoptosis.
Methods for evaluating apoptosis are well known, and include for
example annexin IV staining using standard methods. The anti-CD38
antibodies used in the methods of the invention may induce
apoptosis in about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of cells.
[0167] In some embodiments, the Teffs or the immune suppressor
cells reside in bone marrow or in peripheral blood.
[0168] In some embodiments, the Teffs or the immune suppressor
cells reside in bone marrow.
[0169] In some embodiments, the Teffs or the immune suppressor
cells reside in peripheral blood.
[0170] In some embodiments, the antibody that specifically binds
CD38 increases the ratio of CD8.sup.+ T cells to Tregs.
[0171] In some embodiments, the antibody that specifically binds
CD38 increases the ratio of CD8.sup.+ central memory cells to
CD8.sup.+ naive cells. CD8.sup.+ central memory cells can be
identified as CD45RO.sup.+/CD62L.sup.+high cells. CD8.sup.+ naive
cells can be identified as CD45RO-/CD62L.sup.+ cells.
[0172] In some embodiments, the antibody that specifically binds
CD38 is a non-agonistic antibody.
[0173] A non-agonistic antibody that specifically binds CD38 refers
to an antibody which upon binding to CD38 does not induce
significant proliferation of a sample of peripheral blood
mononuclear cells in vitro when compared to the proliferation
induced by an isotype control antibody or medium alone.
[0174] In some embodiments, the non-agonistic antibody that
specifically binds CD38 induces proliferation of peripheral blood
mononuclear cells (PBMCs) in a statistically insignificant manner
PBMC proliferation may be assessed by isolating PBMCs from healthy
donors and culturing the cells at 1.times.10.sup.5 cells/well in
flat bottom 96-well plates in the presence or absence of a test
antibody in 200 .mu.l RPMI After four day incubation at 37.degree.
C., 30 .mu.l .sup.3H-thymidine (16.7 .mu.Ci/ml) may be added, and
culture may be continued overnight. .sup.3H-thymidine incorporation
may be assessed using a Packard Cobra gamma counter (Packard
Instruments, Meriden, DT, USA), according to the manufacturer's
instructions. Data may be calculated as the mean cpm (.+-.SEM) of
PBMCs obtained from several donors. Statistical significance or
insignificance between samples cultured in the presence or absence
of the test antibody is calculated using standard methods.
[0175] An exemplary anti-CD38 antibody that may be used in the
methods of the invention is DARZALEX.TM. (daratumumab).
DARZALEX.TM. (daratumumab) comprises a heavy chain variable region
(VH) and a light chain variable region (VL) amino acid sequences
shown in SEQ ID NO: 4 and 5, respectively, a heavy chain
complementarity determining region 1 (HCDR1), a HCDR2 and a HCDR3
of SEQ ID NOs: 6, 7 and 8, respectively, and a light chain
complementarity determining region 1 (LCDR1), a LCDR2 and a 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. DARZALEX.TM.
(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.
[0176] In some embodiments, the antibody that specifically binds
CD38 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.
[0177] In some embodiments, the antibody that specifically binds
CD38 binds at least to the region SKRNIQFSCKNIYR (SEQ ID NO: 2) and
the region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO:
1).
TABLE-US-00002 SEQ ID NO: 1
MANCEFSPVSGDKPCCRLSRRAQLCLGVSILVLILVVVLAVVVPRWRQQW
SGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCN
ITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLL
GYLADDLTWCGEFNTSKINYQSCPDWRKDCSNNPVSVFWKTVSRRFAEAA
CDVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQTLEAWVIHGGREDS
RDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKNPEDSSCTSEI SEQ ID NO: 2
SKRNIQFSCKNIYR SEQ ID NO: 3 EKVQTLEAWVIHGG SEQ ID NO: 4
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSA
ISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDK
ILWFGEPVFDYWGQGTLVTVSS SEQ ID NO: 5
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYD
ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQ GTKVEIK 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
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSA
ISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDK
ILWFGEPVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK SEQ ID NO: 13
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYD
ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC
[0178] Antibodies may be evaluated for their competition with a
reference antibody such as DARZALEX.TM. (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 an
unlabeled reference antibody 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 the unlabeled
reference antibody 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 antibody may
be detected using horseradish peroxidase (HRP)-conjugated
streptavidin and the signal detected using standard methods. It is
readily apparent that in the competition assays, the reference
antibody may be labelled and the test antibody unlabeled. The test
antibody competes with the reference antibody when the reference
antibody inhibits binding of the test antibody, or the test
antibody inhibits binding of the reference antibody to CD38 by at
least 80%, for example 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. The
epitope of the test antibody may further be defined for example by
peptide mapping or hydrogen/deuterium protection assays using known
methods, or by crystal structure determination.
[0179] Antibodies binding 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 those described herein, and
characterizing the obtained antibodies for binding to the peptides
using for example ELISA or mutagenesis studies.
[0180] The invention also provides for a method of treating a
patient having a solid tumor, comprising administering to the
patient 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). The epitope of the
antibody used in the methods of the invention includes some or all
of the residues having the sequences shown in SEQ ID NO: 2 or SEQ
ID NO: 3. In some embodiments, 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, 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, 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).
[0181] In some embodiments, the antibody that specifically binds
CD38 comprises the HCDR1, the HCDR2 and the HCDR3 amino acid
sequences of SEQ ID NOs: 6, 7 and 8, respectively.
[0182] In some embodiments, the antibody that specifically binds
CD38 comprises the LCDR1, the LCDR2 and the LCDR3 amino acid
sequences of SEQ ID NOs: 9, 10 and 11, respectively.
[0183] In some embodiments, the antibody that specifically binds
CD38 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the
LCDR2 and the LCDR3 amino acid sequences of SEQ ID NOs: 6, 7, 8, 9,
10 and 11, respectively.
[0184] In some embodiments, the antibody that specifically binds
CD38 comprises the VH that is 95%, 96%, 97%, 98%, 99% or 100%
identical to SEQ ID NO: 4 and the VL that is 95%, 96%, 97%, 98%,
99% or 100% identical to SEQ ID NO: 5.
[0185] In some embodiments, the antibody that specifically binds
CD38 comprises the VH of SEQ ID NO: 4 and the VL of SEQ ID NO:
5.
[0186] In some embodiments, the antibody that specifically binds
CD38 comprises the heavy chain of SEQ ID NO: 12 and the light chain
of SEQ ID NO: 13.
[0187] Other exemplary anti-CD38 antibodies that may be used in any
embodiment of the invention are:
mAb003 comprising the VH and the 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-00003 SEQ ID NO: 14
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGR
VIPFLGIANSAQKFQGRVTITADKSTSTAYMDLSSLRSEDTAVYYCARDD
IAALGPFDYWGQGTLVTVSSAS SEQ ID NO: 15 DIQMTQSP
SSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIY
AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPRTFG QGTKVEIK;
mAb024 comprising the VH and the 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-00004 SEQ ID NO: 16
EVQLVQSGAEVKKPGESLKISCKGSGYSFSNYWIGWVRQMPGKGLEWMGH
YPHDSDARYSPSFQGQVTFSADKSISTAYLQWSSLKASDTAMYYCARHVG
WGSRYWYFDLWGRGTLVTVSS SEQ ID NO: 17
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPGLLIYD
ASNRASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGG GTKVEIK
MOR-202 (MOR-03087) comprising the VH and the 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-00005 SEQ ID NO: 18
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMNWVRQAPGKGLEWVSG
ISGDPSNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDL
PLVYTGFAYWGQGTLVTVSS SEQ ID NO: 19
DIELTQPPSVSVAPGQTARISCSGDNLRHYYVYWYQQKPGQAPVLVIYGD
SKRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCQTYTGGASLVFGG GTKLTVLGQ;
Isatuximab; comprising the VH and the 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-00006 SEQ ID NO 20:
QVQLVQSGAEVAKPGTSVKLSCKASGYTFTDYWMQWVKQRPGQGLEWIGT
IYPGDGDTGYAQKFQGKATLTADKSSKTVYMHLSSLASEDSAVYYCARGD
YYGSNSLDYWGQGTSVTVSS SEQ ID NO: 21:
DIVMTQSHLSMSTSLGDPVSITCKASQDVSTVVAWYQQKPGQSPRRLIYS
ASYRYIGVPDRFTGSGAGTDFTFTISSVQAEDLAVYYCQQHYSPPYTFGG GTKLEIK
[0188] 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.
[0189] The invention also provides for a method of treating a
patient having a solid tumor, comprising administering to the
patient in need thereof a therapeutically effective amount of an
antibody that specifically binds CD38 comprising the VH of SEQ ID
NO: 4 and the VL of SEQ ID NO: 5 for a time sufficient to treat the
solid tumor.
[0190] The invention also provides for a method of treating a
patient having a solid tumor, comprising administering to the
patient in need thereof a therapeutically effective amount of an
antibody that specifically binds CD38 comprising the VH of SEQ ID
NO: 14 and the VL of SEQ ID NO: 15 for a time sufficient to treat
the solid tumor.
[0191] The invention also provides for a method of treating a
patient having a solid tumor, comprising administering to the
patient in need thereof a therapeutically effective amount of an
antibody that specifically binds CD38 comprising the VH of SEQ ID
NO: 16 and the VL of SEQ ID NO: 17 for a time sufficient to treat
the solid tumor.
[0192] The invention also provides for a method of treating a
patient having a solid tumor, comprising administering to the
patient in need thereof a therapeutically effective amount of an
antibody that specifically binds CD38 comprising the VH of SEQ ID
NO: 18 and the VL of SEQ ID NO: 19 or a time sufficient to treat
the solid tumor.
[0193] The invention also provides for a method of treating a
patient having a solid tumor, comprising administering to the
patient in need thereof a therapeutically effective amount of an
antibody that specifically binds CD38 comprising the VH of SEQ ID
NO: 20 and the VL of SEQ ID NO: 21 or a time sufficient to treat
the solid tumor.
[0194] In some embodiments, the solid tumor is a melanoma.
[0195] In some embodiments, the solid tumor is a lung cancer.
[0196] In some embodiments, the solid tumor is a squamous non-small
cell lung cancer (NSCLC).
[0197] In some embodiments, the solid tumor is a non-squamous
NSCLC.
[0198] In some embodiments, the solid tumor is a lung
adenocarcinoma.
[0199] In some embodiments, the solid tumor is a renal cell
carcinoma (RCC) (e.g., a kidney clear cell carcinoma or a kidney
papillary cell carcinoma), or a metastatic lesion thereof.
[0200] In some embodiments, the solid tumor is a mesothelioma.
[0201] In some embodiments, the solid tumor is a nasopharyngeal
carcinoma (NPC).
[0202] In some embodiments, the solid tumor is a colorectal
cancer.
[0203] In some embodiments, the solid tumor is a prostate cancer or
castration-resistant prostate cancer.
[0204] In some embodiments, the solid tumor is a stomach
cancer.
[0205] In some embodiments, the solid tumor is an ovarian
cancer.
[0206] In some embodiments, the solid tumor is a gastric
cancer.
[0207] In some embodiments, the solid tumor is a liver cancer.
[0208] In some embodiments, the solid tumor is pancreatic
cancer.
[0209] In some embodiments, the solid tumor is a thyroid
cancer.
[0210] In some embodiments, the solid tumor is a squamous cell
carcinoma of the head and neck.
[0211] In some embodiments, the solid tumor is a carcinomas of the
esophagus or gastrointestinal tract.
[0212] In some embodiments, the solid tumor is a breast cancer.
[0213] In some embodiments, the solid tumor is a fallopian tube
cancer.
[0214] In some embodiments, the solid tumor is a brain cancer.
[0215] In some embodiments, the solid tumor is an urethral
cancer.
[0216] In some embodiments, the solid tumor is a genitourinary
cancer.
[0217] In some embodiments, the solid tumor is an
endometriosis.
[0218] In some embodiments, the solid tumor is a cervical
cancer.
[0219] In some embodiments, the solid tumor is a metastatic lesion
of the cancer.
[0220] In some embodiments, the solid tumor lacks detectable CD38
expression.
[0221] The solid tumor lacks detectable CD38 expression when CD38
expression in the solid tumor tissue or on cells isolated from the
solid tumor is statistically insignificant when compared to a
control, e.g. expression detected with anti-CD38 antibody vs
expression detected with an isotype control antibody using well
known methods.
[0222] Anti-CD38 antibodies used in the methods of the invention
may also be selected de novo from, e.g., 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.,
(2000) J Mol Biol 296:57-86; Krebs et al., (2001) J Immunol Meth
254:67-84; Vaughan et al., (1996) Nature Biotechnology 14:309-314;
Sheets et al., (1998) PITAS (USA) 95:6157-6162; Hoogenboom and
Winter, (1991) J Mol Biol 227:381; Marks et al., (1991) J Mol Biol
222:581). CD38 binding variable domains may be isolated from e.g.,
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., (2010) J Mol Biol 397:385-96,
and Intl. Pat. Publ. No. WO09/085462. The antibody libraries may be
screened for binding to human CD38 extracellular domain, the
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, 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.
[0223] In some embodiments, the anti-CD38 antibody is of IgG1,
IgG2, IgG3 or IgG4 isotype.
[0224] 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 function 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 Fc-binding cells or
complement components result in inhibition and/or depletion of
target cells, for example 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.
[0225] Antibodies that are substantially identical to the antibody
comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 may be
used in the methods of the invention. The term "substantially
identical" as used herein means that the two antibody VH or VL
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 may 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
may 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 may be made to the
antibodies that specifically bind CD38 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 VH or the VL 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, or modulate CD38 enzymatic activity in vitro
using methods described herein.
[0226] In some embodiments, the antibody that specifically binds
CD38 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
antibody that specifically binds CD38 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 but not limited to, 1-9.9 (or any range or
value therein, such as 1, 2, 3, 4, 5, 6, 7, 8, or
9).times.10.sup.-8M, 10.sup.-9 M, 10.sup.-10 M, 10.sup.-11 M,
10.sup.-12 M, 10.sup.-13 M, 10.sup.-14 M, 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.
[0227] In some embodiments, the antibody that specifically binds
CD38 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 herein may be engineered into bispecific full
length antibodies. Such bispecific antibodies may be made by
modulating the CH3 interactions between the monospecific antibody
heavy chains to form bispecific antibodies using technologies such
as those described in U.S. Pat. No. 7,695,936; Intl. Pat. Publ. No.
WO04/111233; U.S. Pat. Publ. No. US2010/0015133; U.S. Pat. Publ.
No. US2007/0287170; Intl. 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; Intl. 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 may be
incorporated are for example Dual Variable Domain Immunoglobulins
(Inlt. 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).
[0228] For example, bispecific antibodies may be generated in vitro
in a cell-free environment by introducing asymmetrical mutations in
the CH3 regions of two monospecific homodimeric antibodies and
forming the bispecific heterodimeric antibody from two parental
monospecific homodimeric antibodies in reducing conditions to allow
disulfide bond isomerization according to methods described in
Intl. Pat. Publ. No. WO2011/131746. In the methods, the first
monospecific bivalent antibody (e.g., anti-CD38 antibody) and the
second monospecific bivalent antibody are engineered to have
certain substitutions at the CH3 domain that promote heterodimer
stability; the antibodies are incubated together under reducing
conditions sufficient to allow the cysteines in the hinge region to
undergo disulfide bond isomerization; thereby generating the
bispecific antibody by Fab arm exchange. The incubation conditions
may optimally be restored to non-reducing. Exemplary reducing
agents that may be used are 2-mercaptoethylamine (2-MEA),
dithiothreitol (DTT), dithioerythritol (DTE), glutathione,
tris(2-carboxyethyl)phosphine (TCEP), L-cysteine and
beta-mercaptoethanol, preferably a reducing agent selected from the
group consisting of: 2-mercaptoethylamine, dithiothreitol and
tris(2-carboxyethyl)phosphine. For example, incubation for at least
90 min at a temperature of at least 20.degree. C. in the presence
of at least 25 mM 2-MEA or in the presence of at least 0.5 mM
dithiothreitol at a pH of from 5-8, for example at pH of 7.0 or at
pH of 7.4 may be used.
[0229] Exemplary CH3 mutations that may be used in a first heavy
chain and in a second heavy chain of the bispecific antibody are
K409R and/or F405L.
[0230] The methods of the invention may be used to treat an animal
patient belonging to any classification. Examples of such animals
include mammals such as humans, rodents, dogs, cats and farm
animals.
Administration/Pharmaceutical Compositions
[0231] The antibodies that specifically bind CD38 may be provided
in the methods of the invention in suitable pharmaceutical
compositions comprising the antibody that specifically bind CD38
and a pharmaceutically acceptable carrier. The carrier may be
diluent, adjuvant, excipient, or vehicle with which the antibodies
that specifically bind CD38 are 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 antibodies that specifically bind CD38 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%,
25%, 30%, 35%, 40%, 45% or 50% 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, 20 Edition,
Troy, D. B. ed., Lipincott Williams and Wilkins, Philadelphia, Pa.
2006, Part 5, Pharmaceutical Manufacturing pp 691-1092, see
especially pp. 958-989.
[0232] The mode of administration of the antibodies that
specifically bind CD38 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. The antibodies that specifically
bind CD38 may be administered intratumorally, to a lymph node
draining site for local delivery into the tumor using known
methods.
[0233] The antibodies that specifically bind CD38 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.
[0234] The dose given to a patient is sufficient to alleviate or at
least partially arrest the disease being treated ("therapeutically
effective amount") and may be sometimes 0.005 mg to about 100
mg/kg, e.g. about 0.05 mg 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 for example 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.
[0235] 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, but 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20
or more doses may be given.
[0236] The administration of the antibodies that specifically bind
CD38 in the methods of the invention 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 antibodies that
specifically bind CD38 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.
[0237] The antibodies that specifically bind CD38 may be
administered in the methods of the invention by maintenance
therapy, such as, e.g. once a week for a period of 6 months or
more.
[0238] For example, the antibodies that specifically bind CD38 in
the methods of the invention may be provided as a daily dosage in
an amount of about 0.1-100 mg/kg, such as 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.
[0239] The antibodies that specifically bind CD38 in the methods of
the invention 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.
[0240] The antibodies that specifically bind CD38 in the methods of
the invention 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.
[0241] The antibodies that specifically bind CD38 in the methods of
the invention may be administered in combination with a second
therapeutic agent.
[0242] In the methods of the invention, the antibodies that
specifically bind CD38 may be administered together with any one or
more of the chemotherapeutic drugs or other anti-cancer
therapeutics known to those of skill in the art. Chemotherapeutic
agents are chemical compounds useful in the treatment of cancer and
include growth inhibitory agents or other cytotoxic agents and
include alkylating agents, anti-metabolites, anti-microtubule
inhibitors, topoisomerase inhibitors, receptor tyrosine kinase
inhibitors, angiogenesis inhibitors and the like. Examples of
chemotherapeutic agents include alkylating agents such as thiotepa
and cyclosphosphamide (CYTOXAN.RTM.); alkyl sulfonates such as
busulfan, improsulfan and piposulfan; aziridines such as benzodopa,
carboquone, meturedopa, and uredopa; ethylenimines and
methylamelamines including altretamine, triethylenemelamine,
trietylenephosphoramide, triethylenethiophosphaoramide and
trimethylolomelamine; nitrogen mustards such as chlorambucil,
chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitrosureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, ranimustine; antibiotics such as
aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,
cactinomycin, calicheamicin, carabicin, carminomycin,
carzinophilin, chromomycins, dactinomycin, daunorubicin,
detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin,
esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic
acid, nogalamycin, olivomycins, peplomycin, potfiromycin,
puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin,
tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such
as methotrexate and 5-FU; folic acid analogues such as denopterin,
methotrexate, pteropterin, trimetrexate; purine analogues such as
fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine
analogues such as ancitabine, azacitidine, 6-azauridine, carmofur,
cytarabine, dideoxyuridine, doxifluridine, enocitabine,
floxuridine; androgens such as calusterone, dromostanolone
propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals
such as aminoglutethimide, mitotane, trilostane; folic acid
replenisher such as frolinic acid; aceglatone; aldophosphamide
glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene;
edatraxate; defofamine; demecolcine; diaziquone; eflornithine;
elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol;
nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid;
2-ethylhydrazide; procarbazine; PSK.RTM.; razoxane; sizofiran;
spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; urethan; vindesine; dacarbazine;
mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;
arabinoside ("Ara-C"); cyclophosphamide; thiotepa; members of
taxoid or taxane family, such as paclitaxel (TAXOL.RTM. docetaxel
(TAXOTERE.RTM.) and analogues thereof; chlorambucil; gemcitabine;
6-thioguanine; mercaptopurine; methotrexate; platinum analogues
such as cisplatin and carboplatin; vinblastine; platinum; etoposide
(VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine;
vinorelbine; navelbine; novantrone; teniposide; daunomycin;
aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor
RFS 2000; difluoromethylornithine (DMFO); retinoic acid;
esperamicins; capecitabine; inhibitors of receptor tyrosine kinases
and/or angiogenesis, including NEXAVAR.RTM. (sorafenib),
SUTENT.RTM. (sunitinib), VOTRIENT.TM. (pazopanib), PALLADIA.TM.
(toceranib), ZACTIMA.TM. (vandetanib), RECENTIN.RTM. (cediranib),
regorafenib (BAY 73-4506), axitinib (AG013736), lestaurtinib
(CEP-701), TARCEVA.RTM. (erlotinib), IRESSA.TM. (gefitinib),
Gilotrif.RTM. (afatinib), TYKERB.RTM. (lapatinib), neratinib, and
the like, and pharmaceutically acceptable salts, acids or
derivatives of any of the above. Also included in this definition
are anti-hormonal agents that act to regulate or inhibit hormone
action on tumors such as anti-estrogens including for example
tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles,
4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone,
and FARESTON.RTM. (toremifene); and anti-androgens such as
flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and
pharmaceutically acceptable salts, acids or derivatives of any of
the above. Other conventional cytotoxic chemical compounds as those
disclosed in Wiemann et al., 1985, in Medical Oncology (Calabresi
et aL, eds.), Chapter 10, McMillan Publishing, are also applicable
to the methods of the present invention.
[0243] Exemplary agents that may be used in combination with the
antibody that specifically binds CD38 in the methods of the
invention include tyrosine kinase inhibitors and targeted
anti-cancer therapies such as IRESSA.TM. (gefitinib) and
Tarceva.RTM. (erlotinib) and other antagonists of HER2, HER3, HER4
or VEGF. Exemplary HER2 antagonists include CP-724-714,
HERCEPTIN.TM. (trastuzumab), OMNITARG.TM. (pertuzumab), TAK-165,
TYKERB.RTM. (lapatinib) (EGFR and HER2 inhibitor), and GW-282974.
Exemplary HER3 antagonists include anti-Her3 antibodies (see e.g.,
U.S. Pat. Publ. No. 2004/0197332). Exemplary HER4 antagonists
include anti-HER4 siRNAs (see e.g., Maatta et al., Mol Biol Cell
17: 67-79, 2006. An exemplary VEGF antagonist is (Avastin.TM.
(Bevacizumab).
[0244] Exemplary agents that may be used in combination with the
antibody that specifically binds CD38 in the methods of the
invention include standard of care drugs for solid tumors, or an
immune checkpoint inhibitor.
[0245] The second therapeutic agent in the methods of the invention
may be an immune checkpoint inhibitor.
[0246] In some embodiments, the immune checkpoint inhibitor is an
anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody,
an anti-LAG3 antibody, an anti-TIM3 antibody, or an anti-CTLA-4
antibody.
[0247] In some embodiments, the immune checkpoint inhibitor is an
antagonistic anti-PD-1 antibody, an antagonistic anti-PD-L1
antibody, an antagonistic anti-PD-L2 antibody, an antagonistic
anti-LAG3 antibody, or an antagonistic anti-TIM3 antibody.
[0248] In some embodiments, the immune checkpoint inhibitor is an
anti-PD-1 antibody.
[0249] In some embodiments, the immune checkpoint inhibitor is an
anti-PD-L1 antibody.
[0250] In some embodiments, the immune checkpoint inhibitor is an
anti-PD-L2 antibody.
[0251] In some embodiments, the immune checkpoint inhibitor is an
anti-LAG3 antibody.
[0252] In some embodiments, the immune checkpoint inhibitor is an
anti-TIM3 antibody.
[0253] In some embodiments, the immune checkpoint inhibitor is an
anti-CTLA-4 antibody.
[0254] Any antagonistic anti-PD-1 antibodies may be used in the
methods of the invention. Exemplary anti-PD-1 antibodies that may
be used are OPVIDO.RTM. (nivolumab) and KEYTRUDA.RTM.
(pembrolizumab). OPVIDO.RTM. (nivolumab) is described in for
example U.S. Pat. No. 8,008,449 (antibody 5C4) and comprises the VH
of SEQ ID NO: 24 and the VL of SEQ ID NO: 25. KEYTRUDA.RTM.
(pembrolizumab) is described in for example U.S. Pat. No. 8,354,509
and comprises the VH of SEQ ID NO: 22 and the VL of SEQ ID NO: 23.
The amino acid sequences of nivolumab and pembrolizumab are also
available through the CAS registry. Additional PD-1 antibodies that
may be used are described in U.S. Pat. No. 7,332,582, U.S. Pat.
Publ. No. 2014/0044738, Int. Pat. Publ. No. WO2014/17966 and U.S.
Pat. Publ. No. 2014/0356363.
[0255] "Antagonist" refers to a molecule that, when bound to a
cellular protein, suppresses at least one reaction or activity that
is induced by a natural ligand of the protein. A molecule is an
antagonist when the at least one reaction or activity is suppressed
by at least about 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, or 100% more than the at least one reaction or
activity suppressed in the absence of the antagonist (e.g.,
negative control), or when the suppression is statistically
significant when compared to the suppression in the absence of the
antagonist. Antagonist may be an antibody, a soluble ligand, a
small molecule, a DNA or RNA such as siRNA. A typical reaction or
activity that is induced for example by PD-1 binding to its
receptor PD-L1 or PD-L2 may be reduced antigen-specific CD4.sup.+
or CD8.sup.+ cell proliferation or reduced interferon-.gamma.
(IFN-.gamma.) production by T cells, resulting in suppression of
immune responses against for example tumor. A typical reaction or
activity that is induced by TIM-3 binding to its receptor, such as
galectin-9, may be reduced antigen specific CD4.sup.+ or CD8.sup.+
cell proliferation, reduced IFN-.gamma. production by T cells, or
reduced CD137 surface expression on CD4.sup.+ or CD8.sup.+ cells,
resulting in suppression of immune responses against for example
tumor. Hence, an antagonistic PD-1 antibody specifically binding
PD-1, an antagonistic PD-L2, an antagonistic antibody specifically
binding TIM-3 induces immune responses by inhibiting the inhibitory
pathways.
TABLE-US-00007 SEQ ID NO: 22
QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMG
GINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCAR
RDYRFDMGFDYWGQGTTVTVSS SEQ ID NO: 23
EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPR
LLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDL PLTFGGGTKVEIK SEQ
ID NO: 24 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVA
VIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCAT NDDYWGQGTLVTVSS
SEQ ID NO: 25 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIY
DASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTF GQGTKVEIK
[0256] Anti-PD-L1 antibodies that enhance immune response may be
used in the methods of the invention (e.g. antagonistic anti-PD-L1
antibodies). Exemplary anti-PD-L1 antibodies that may be used are
durvalumab, atezolizumab and avelumab, and those described in, for
example, U.S. Pat. Publ. No. 2009/0055944, U.S. Pat. No. 8,552,154,
U.S. Pat. No. 8,217,149 and U.S. Pat. No. 8,779,108.
[0257] Durvalumab comprises the VH of SEQ ID NO: 26 and the VL of
SEQ ID NO: 27.
[0258] Atezolizumab comprises the VH of SEQ ID NO: 28 and the VL of
SEQ ID NO: 29.
Avelumab comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:
31.
TABLE-US-00008 SEQ ID NO: 26
EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVAN
IKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREG
GWFGELAFDYWGQGTLVTVSS SEQ ID NO: 27
EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIY
DASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFG QGTKVEIK SEQ ID
NO: 28 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAW
ISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRH
WPGGFDYWGQGTLVTVSS SEQ ID NO: 29
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYS
ASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQ GTKVEIK SEQ ID
NO: 30 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSS
IYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIK
LGTVTTVDYWGQGTLVTVSS SEQ ID NO: 31
QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMI
YDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRV FGTGTKVTVL
[0259] The invention also provides for a method of treating a
patient having a solid tumor, comprising administering to the
patient in need thereof a therapeutically effective amount of an
antibody that specifically binds CD38 comprising the VH of SEQ ID
NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-1
antibody comprising the VH of SEQ ID NO: 24 and the VL of SEQ ID
NO: 25 for a time sufficient to treat the solid tumor.
[0260] The invention also provides for a method of treating a
patient having a solid tumor, comprising administering to the
patient in need thereof a therapeutically effective amount of an
antibody that specifically binds CD38 comprising the VH of SEQ ID
NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-1
antibody comprising the VH of SEQ ID NO: 22 and the VL of SEQ ID
NO: 23 for a time sufficient to treat the solid tumor.
[0261] The invention also provides for a method of treating a
patient having a solid tumor, comprising administering to the
patient in need thereof a therapeutically effective amount of an
antibody that specifically binds CD38 comprising the VH of SEQ ID
NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-L1
antibody comprising the VH of SEQ ID NO: 26 and the VL of SEQ ID
NO: 27 for a time sufficient to treat the solid tumor.
[0262] The invention also provides for a method of treating a
patient having a solid tumor, comprising administering to the
patient in need thereof a therapeutically effective amount of an
antibody that specifically binds CD38 comprising the VH of SEQ ID
NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-L1
antibody comprising the VH of SEQ ID NO: 28 and the VL of SEQ ID
NO: 29 for a time sufficient to treat the solid tumor.
[0263] The invention also provides for a method of treating a
patient having a solid tumor, comprising administering to the
patient in need thereof a therapeutically effective amount of an
antibody that specifically binds CD38 comprising the VH of SEQ ID
NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-L1
antibody comprising the VH of SEQ ID NO: 30 and the VL of SEQ ID
NO: 31 for a time sufficient to treat the solid tumor.
[0264] The invention also provides for a method of enhancing an
immune response in a patient, comprising administering to the
patient in need thereof a therapeutically effective amount of an
antibody that specifically binds CD38 comprising the VH of SEQ ID
NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-1
antibody comprising the VH of SEQ ID NO: 24 and the VL of SEQ ID
NO: 25 for a time sufficient to enhance the immune response.
[0265] The invention also provides for a method of enhancing an
immune response in a patient, comprising administering to the
patient in need thereof a therapeutically effective amount of an
antibody that specifically binds CD38 comprising the VH of SEQ ID
NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-1
antibody comprising the VH of SEQ ID NO: 22 and the VL of SEQ ID
NO: 23 for a time sufficient to enhance the immune response.
[0266] The invention also provides for a method of enhancing an
immune response in a patient, comprising administering to the
patient in need thereof a therapeutically effective amount of an
antibody that specifically binds CD38 comprising the VH of SEQ ID
NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-L1
antibody comprising the VH of SEQ ID NO: 26 and the VL of SEQ ID
NO: 27 for a time sufficient to enhance the immune response.
[0267] The invention also provides for a method of enhancing an
immune response in a patient, comprising administering to the
patient in need thereof a therapeutically effective amount of an
antibody that specifically binds CD38 comprising the VH of SEQ ID
NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-L1
antibody comprising the VH of SEQ ID NO: 28 and the VL of SEQ ID
NO: 29 for a time sufficient to enhance the immune response.
[0268] The invention also provides for a method of enhancing an
immune response in a patient, comprising administering to the
patient in need thereof a therapeutically effective amount of an
antibody that specifically binds CD38 comprising the VH of SEQ ID
NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-L1
antibody comprising the VH of SEQ ID NO: 30 and the VL of SEQ ID
NO: 31 for a time sufficient to enhance the immune response.
[0269] The invention also provides for a method of treating a
patient having a colorectal cancer, comprising administering to the
patient in need thereof a therapeutically effective amount of an
antibody that specifically binds CD38 in combination with an
antagonistic anti-PD-1 antibody for a time sufficient to treat the
colorectal cancer.
[0270] The invention also provides for a method of treating a
patient having a colorectal cancer, comprising administering to the
patient in need thereof a therapeutically effective amount of an
antibody that specifically binds CD38 in combination with an
antagonistic anti-PD-L1 antibody for a time sufficient to treat the
colorectal cancer.
[0271] The invention also provides for a method of treating a
patient having a colorectal cancer, comprising administering to the
patient in need thereof a therapeutically effective amount of an
antibody that specifically binds CD38 in combination with an
antagonistic anti-PD-L2 antibody for a time sufficient to treat the
colorectal cancer.
[0272] The invention also provides for a method of treating a
patient having a lung cancer, comprising administering to the
patient in need thereof a therapeutically effective amount of an
antibody that specifically binds CD38 in combination with an
antagonistic anti-PD-1 antibody for a time sufficient to treat the
lung cancer.
[0273] The invention also provides for a method of treating a
patient having a lung cancer, comprising administering to the
patient in need thereof a therapeutically effective amount of an
antibody that specifically binds CD38 in combination with an
antagonistic anti-PD-L1 antibody for a time sufficient to treat the
lung cancer.
[0274] The invention also provides for a method of treating a
patient having a lung cancer, comprising administering to the
patient in need thereof a therapeutically effective amount of an
antibody that specifically binds CD38 in combination with an
antagonistic anti-PD-L2 antibody for a time sufficient to treat the
lung cancer.
[0275] The invention also provides for a method of treating a
patient having a prostate cancer, comprising administering to the
patient in need thereof a therapeutically effective amount of an
antibody that specifically binds CD38 in combination with an
antagonistic anti-PD-1 antibody for a time sufficient to treat the
prostate cancer.
[0276] The invention also provides for a method of treating a
patient having a prostate cancer, comprising administering to the
patient in need thereof a therapeutically effective amount of an
antibody that specifically binds CD38 in combination with an
antagonistic anti-PD-L1 antibody for a time sufficient to treat the
prostate cancer.
[0277] The invention also provides for a method of treating a
patient having a prostate cancer, comprising administering to the
patient in need thereof a therapeutically effective amount of an
antibody that specifically binds CD38 in combination with an
antagonistic anti-PD-L2 antibody for a time sufficient to treat the
prostate cancer.
[0278] Anti-LAG-3 antibodies that enhance immune response may be
used in the methods if the invention. Exemplary anti-LAG-3
antibodies that may be used are those described in, for example,
Int. Pat. Publ. No. WO2010/019570.
[0279] Anti-CTLA-4 antibodies that enhance immune response may be
used in the methods if the invention. An exemplary anti-CTLA-4
antibody that may be used is ipilimumab.
[0280] Anti-PD-1, anti-PD-L1, anti-PD-L2, anti-LAG3, anti-TIM3 and
anti-CTLA-4 antibodies that may be used in the methods of the
invention may also be generated de novo using methods described
herein.
[0281] In some embodiments, anti-PD1 antibodies comprising the VH
of SEQ ID NO: 32 and the VL of SEQ ID NO: 33 may be used.
[0282] In some embodiments, anti-PD1 antibodies comprising the VH
of SEQ ID NO: 34 and the VL of SEQ ID NO: 35 may be used.
[0283] In some embodiments, anti-TIM-3 antibodies comprising the VH
of SEQ ID NO: 36 and the VL of SEQ ID NO: 37 may be used.
[0284] In some embodiments, anti-TIM-3 antibodies comprising the VH
of SEQ ID NO: 38 and the VL of SEQ ID NO: 39 may be used.
TABLE-US-00009 SEQ ID NO: 32
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG
IIPIFDTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARPG
LAAAYDTGSLDYWGQGTLVTVSS SEQ ID NO: 33
EIVLTQSPATLSLSPGERATLSCRASQSVRSYLAWYQQKPGQAPRLLIYD
ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRNYWPLTFGQ GTKVEIK SEQ ID
NO: 34 EVQLVESGGGLVQPGGSLRLSCAASGFAFSRYDMSWVRQAPGKGLESVAY
ISGGGANTYYLDNVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPY
LSYFDVWGQGTLVTVSS SEQ ID NO: 35
EIVMTQSPATLSVSPGERATLSCRASQSLSDYLHWYQQKPGQAPRLLIKS
ASQSISGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQNGHSFPYTFGQ GTKLEIK SEQ ID
NO: 36 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSA
ISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSP
YAPLDYWGQGTLVTVSS SEQ ID NO: 37
EIVLTQSPATLSLSPGERATLSCRASQSVNDYLAWYQQKPGQAPRLLIYD
ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQGGHAPITFGQ GTKVEIK SEQ ID
NO: 38 EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMQWVRQMPGKGLEWMGA
IYPGDGDIRYTQNFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARWE
KSTTVVQRNYFDYWGQGTTVTVSS SEQ ID NO: 39
DIQMTQSPSSLSASVGDRVTITCKASENVGTFVSWYQQKPGKAPKLLIYG
ASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGQSYSYPTFGQG TKLEIK
[0285] The invention also provides for a method of treating a
patient having a solid tumor, comprising administering to the
patient in need thereof a therapeutically effective amount of an
antibody that specifically binds CD38 comprising the VH of SEQ ID
NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-1
antibody comprising the VH of SEQ ID NO: 32 and the VL of SEQ ID
NO: 33 for a time sufficient to treat the solid tumor.
[0286] The invention also provides for a method of treating a
patient having a solid tumor, comprising administering to the
patient in need thereof a therapeutically effective amount of an
antibody that specifically binds CD38 comprising the VH of SEQ ID
NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-1
antibody comprising the VH of SEQ ID NO: 34 and the VL of SEQ ID
NO: 35 for a time sufficient to treat the solid tumor.
[0287] The invention also provides for a method of treating a
patient having a solid tumor, comprising administering to the
patient in need thereof a therapeutically effective amount of an
antibody that specifically binds CD38 comprising the VH of SEQ ID
NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-TIM-3
antibody comprising the VH of SEQ ID NO: 36 and the VL of SEQ ID
NO: 37 for a time sufficient to treat the solid tumor.
[0288] The invention also provides for a method of treating a
patient having a solid tumor, comprising administering to the
patient in need thereof a therapeutically effective amount of an
antibody that specifically binds CD38 comprising the VH of SEQ ID
NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-TIM-3
antibody comprising the VH of SEQ ID NO: 38 and the VL of SEQ ID
NO: 39 for a time sufficient to treat the solid tumor.
[0289] In the methods of the invention, the combination of the
antibody that specifically binds CD38 and the second therapeutic
agent may be administered over any convenient timeframe. For
example, the antibody that specifically binds CD38 and the second
therapeutic agent may be administered to a patient on the same day,
and even in the same intravenous infusion. However, the antibody
that specifically binds CD38 and the second therapeutic agent may
also be administered on alternating days or alternating weeks or
months, and so on. In some methods, the antibody that specifically
binds CD38 and the second therapeutic agent may be administered
with sufficient proximity in time that they are simultaneously
present (e.g., in the serum) at detectable levels in the patient
being treated. In some methods, an entire course of treatment with
the antibody that specifically binds CD38 consisting of a number of
doses over a time period is followed or preceded by a course of
treatment with the second therapeutic agent, consisting of a number
of doses. A recovery period of 1, 2 or several days or weeks may be
used between administration of the antibody that specifically binds
CD38 and the second therapeutic agent.
[0290] The antibody that specifically binds CD38 or a combination
of the antibody that specifically binds CD38 and the second
therapeutic agent may be administered together with any form of
radiation therapy including external beam radiation, intensity
modulated radiation therapy (IMRT), focused radiation, and any form
of radiosurgery including Gamma Knife, Cyberknife, Linac, and
interstitial radiation (e.g. implanted radioactive seeds, GliaSite
balloon), and/or with surgery.
[0291] Focused radiation methods that may be used include
stereotactic radiosurgery, fractionated stereotactic radiosurgery,
and intensity-modulated radiation therapy (IMRT). It is apparent
that stereotactic radiosurgery involves the precise delivery of
radiation to a tumorous tissue, for example, a brain tumor, while
avoiding the surrounding non-tumorous, normal tissue. The dosage of
radiation applied using stereotactic radiosurgery may vary,
typically from 1 Gy to about 30 Gy, and may encompass intermediate
ranges including, for example, from 1 to 5, 10, 15, 20, 25, up to
30 Gy in dose. Because of noninvasive fixation devices,
stereotactic radiation need not be delivered in a single treatment.
The treatment plan may be reliably duplicated day-to-day, thereby
allowing multiple fractionated doses of radiation to be delivered.
When used to treat a tumor over time, the radiosurgery is referred
to as "fractionated stereotactic radiosurgery" or FSR. In contrast,
stereotactic radiosurgery refers to a one-session treatment.
Fractionated stereotactic radiosurgery may result in a high
therapeutic ratio, i.e., a high rate of killing of tumor cells and
a low effect on normal tissue. The tumor and the normal tissue
respond differently to high single doses of radiation vs. multiple
smaller doses of radiation. Single large doses of radiation may
kill more normal tissue than several smaller doses of radiation
may. Accordingly, multiple smaller doses of radiation can kill more
tumor cells while sparing normal tissue. The dosage of radiation
applied using fractionated stereotactic radiation may vary from
range from 1 Gy to about 50 Gy, and may encompass intermediate
ranges including, for example, from 1 to 5, 10, 15, 20, 25, 30, 40,
up to 50 Gy in hypofractionated doses. Intensity-modulated
radiation therapy (IMRT) may also be used. IMRT is an advanced mode
of high-precision three-dimensional conformal radiation therapy
(3DCRT), which uses computer-controlled linear accelerators to
deliver precise radiation doses to a malignant tumor or specific
areas within the tumor. In 3DCRT, the profile of each radiation
beam is shaped to fit the profile of the target from a beam's eye
view (BEV) using a multileaf collimator (MLC), thereby producing a
number of beams. IMRT allows the radiation dose to conform more
precisely to the three-dimensional (3-D) shape of the tumor by
modulating the intensity of the radiation beam in multiple small
volumes. Accordingly, IMRT allows higher radiation doses to be
focused to regions within the tumor while minimizing the dose to
surrounding normal critical structures. IMRT improves the ability
to conform the treatment volume to concave tumor shapes, for
example, when the tumor is wrapped around a vulnerable structure,
such as the spinal cord or a major organ or blood vessel.
Subcutaneous Administration of Pharmaceutical Compositions
Comprising an Antibody that Specifically Binds CD38 and a
Hyaluronidase
[0292] The antibody that specifically binds CD38 may be
administered as a pharmaceutical composition comprising the
antibody that specifically binds CD38 and a hyaluronidase
subcutaneously.
[0293] The concentration of the antibody that specifically binds
CD38 in the pharmaceutical composition administered subcutaneously
may be about 20 mg/ml.
[0294] The pharmaceutical composition administered subcutaneously
may comprise between about 1,200 mg-1,800 mg of the antibody that
specifically binds CD38.
[0295] The pharmaceutical composition administered subcutaneously
may comprise about 1,200 mg of the antibody that specifically binds
CD38.
[0296] The pharmaceutical composition administered subcutaneously
may comprise about 1,600 mg of the antibody that specifically binds
CD38.
[0297] The pharmaceutical composition administered subcutaneously
may comprise about 1,800 mg of the antibody that specifically binds
CD38.
[0298] The pharmaceutical composition administered subcutaneously
may comprise between about 30,000 U-45,000 U of the
hyaluronidase.
[0299] The pharmaceutical composition administered subcutaneously
may comprise about 1,200 mg of the antibody that specifically binds
CD38 and about 30,000 U of the hyaluronidase.
[0300] The pharmaceutical composition administered subcutaneously
may comprise about 1,800 mg of the antibody that specifically binds
CD38 and about 45,000 U of the hyaluronidase.
[0301] The pharmaceutical composition administered subcutaneously
may comprise about 1,600 mg of the antibody that specifically binds
CD38 and about 30,000 U of the hyaluronidase.
[0302] The pharmaceutical composition administered subcutaneously
may comprise about 1,600 mg of the antibody that specifically binds
CD38 and about 45,000 U of the hyaluronidase.
[0303] The pharmaceutical composition administered subcutaneously
may comprise the hyaluronidase rHuPH20 having the amino acid
sequence of SEQ ID NO: 40.
[0304] rHuPH20 is a recombinant hyaluronidase (HYLENEX.RTM.
recombinant) and is described in Int. Pat. Publ. No.
WO2004/078140.
[0305] Hyaluronidase is an enzyme that degrades hyaluronic acid (EC
3.2.1.35) and lowers the viscosity of hyaluronan in the
extracellular matrix, thereby increasing tissue permeability.
TABLE-US-00010 SEQ ID NO: 40
MGVLKFKHIFFRSFVKSSGVSQIVFTFLLIPCCLTLNFRAPPVIPNVPFL
WAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYP
YIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEW
RPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFL
VETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLS
WLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPV
FAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKS
CLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHL
NPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVK
DTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSATMFIVSILF LIISSVASL
[0306] The administration of the pharmaceutical composition
comprising the antibody that specifically binds CD38 and the
hyaluronidase may be repeated after one day, two days, three days,
four days, five days, six days, one week, two weeks, three weeks,
four weeks, 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 pharmaceutical composition
comprising the antibody that specifically binds CD38 and the
hyaluronidase may be administered once weekly for eight weeks,
followed by once in two weeks for 16 weeks, followed by once in
four weeks. The pharmaceutical compositions to be administered may
comprise about 1,200 mg of the antibody that specifically binds
CD38 and about 30,000 U of hyaluronidase, wherein the concentration
of the antibody that specifically binds CD38 in the pharmaceutical
composition is about 20 mg/ml. The pharmaceutical compositions to
be administered may comprise about 1,800 mg of the antibody that
specifically binds CD38 and about 45,000 U of hyaluronidase. The
pharmaceutical compositions to be administered may comprise about
1,600 mg of the antibody that specifically binds CD38 and about
30,000 U of hyaluronidase. The pharmaceutical compositions to be
administered may comprise about 1,600 mg of the antibody that
specifically binds CD38 and about 45,000 U of hyaluronidase.
[0307] The pharmaceutical composition comprising the antibody that
specifically binds CD38 and the hyaluronidase may be administered
subcutaneously to the abdominal region.
[0308] The pharmaceutical composition comprising the antibody that
specifically binds CD38 and the hyaluronidase may be administered
in a total volume of about 80 ml, 90 ml, 100 ml, 110 ml or 120
ml.
[0309] For administration, 20 mg/ml of the antibody that
specifically binds CD38 in 25 mM sodium acetate, 60 mM sodium
chloride, 140 mM D-mannitol, 0.04% polysorbate 20, pH 5.5 may be
mixed with rHuPH20, 1.0 mg/mL (75-150 kU/mL) in 10 mM L-Histidine,
130 mM NaCl, 10 mM L-Methionine, 0.02% Polysorbate 80, pH 6.5 prior
to administration of the mixture to a subject.
[0310] 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.
Example 1
General Materials and Methods
Sample Collection and Processing
[0311] Peripheral blood and bone marrow aspirates were collected in
heparinized tubes at baseline immediately prior to the first
infusion and at specified time points during treatment. The
majority of samples were evaluated using real-time flow cytometry,
as they arrived at a central laboratory, 24-48 hours after
collection. Peripheral blood mononuclear cells (PBMCs) were
obtained from whole blood, isolated by density-gradient
centrifugation, and stored frozen until analysis. For the T-cell
activation, clonality, and CD38.sup.+ Treg suppression assays, pre-
and post-treatment samples were analyzed at the same time, using
frozen PBMC samples.
[0312] Flow cytometric analysis of these samples was performed at
BARC global central laboratory for evaluation of NK, T, B, myeloma
cells (CD138.sup.+) and CD38 expression using a pre-validated
immunophenotyping assay. Briefly, blood samples and bone marrow
samples were stained with the following multifluorochrome antibody
panels: cell lineage panel: PerCPCy5.5.alpha.-CD19 (cloneHIB19;
Becton Dickinson [BD]), APC.alpha.-CD24 (SN3; eBioscience),
PC7.alpha.-CD3 (UCHT-1; Beckman Coulter), V500.alpha.-CD16 (3G8;
BD), and PE.alpha.-CD56 (MY; BD); regulatory T cell (T.sub.red
panel: APC.alpha.-CD25 (2A3; BD), PE.alpha.-CD127 (HIL-7R-M21; BD),
APC-H7.alpha.-HLA-DR (G46-6; BD), and PerCP.alpha.-CD4 (L200; BD);
naive/memory T-cell panel: APC-H7.alpha.-CD4 (RPA-T4; BD),
PerCP-Cy5.5.alpha.-CD8 (RPA-T4 BD), PE.alpha.-CD62L (SK11; BD), and
APC.alpha.-CD45RA (HI100; BD). CD38 expression was evaluated using
Alexa 647 labeled antibody mAb 003 described in U.S. Pat. No.
7,829,693 having the VH and the VL sequences of SEQ ID NO: 14 and
SEQ ID NO: 15. The blood samples were prepared using different
Lyse-wash methods. For bone marrow aspirate samples either membrane
or intracellular staining was performed with various antibodies.
Becton Dickinson FACSLysing solution was used for lysing red blood
cells in peripheral blood samples and Fix and Perm cell
permeabilization reagents from Invitrogen were used for
intracellular staining of bone marrow aspirate samples. Stained
samples were acquired on FACS Canto II flow cytometers and data was
analyzed using FacsDiva software. Absolute counts of immune cell
populations in the blood samples and as percent of lymphocytes in
bone marrow samples were determined at all the time points
tested.
T-Cell Receptor (TCR) Sequencing
[0313] T-cell diversity was analyzed by deep sequencing of TCR
rearrangements to assess CD8.sup.+ T-cell clonality using genomic
DNA from PBMC samples. TCR sequencing was performed using Adaptive
Biotechnologies commercial Immunoseq.TM. assay, and analysis was
performed using prequalified multiplex polymerase chain reaction
(PCR) assays (TR2015CRO-V-019), which were composed of forward and
reverse primers that directly targeted the family of variable (V)
genes (forward primers) and joining (J) genes (reverse primers).
Each V and J gene primer acted as priming pairs to amplify
somatically recombined TCRs, and each primer contained a specific
universal DNA sequence. Following the initial PCR amplification,
each amplicon was amplified a second time with forward and reverse
primers containing the universal sequence and adaptor sequence
needed for DNA sequencing by Illumina.
T-Cell Responses to Viral- and Alloantigens
[0314] Patient PBMCs were seeded on 96 well plates
(2.times.10.sup.5 cells/well) and stimulated for 5 days with a
cocktail of 23 major histocompatibility complex (MHC) class
I-restricted viral peptides from human cytomegalovirus (CMV),
Epstein-Barr virus (EBV), and influenza virus (2 .mu.g/ml; CEF
peptide pool; PANATecs.RTM.) or an equivalent number of 25-Gy
irradiated allogeneic PBMCs from healthy donors. Unstimulated PBMCs
and PBMCs stimulated with anti-CD3/CD28-coated beads served as
negative and positive controls, respectively. On day 5, interferon
.gamma. (IFN-.gamma.) from cell-free supernatant was measured by
sandwich enzyme-linked immunosorbent assay (ELISA; Human IFN gamma
ELISA Ready-SET-Go; eBioscience) and served as a surrogate marker
for T-cell activation.
Regulatory T-Cell (Treg) Suppression of Effector Cell Functions:
Carboxyfluorescein Succinimidyl (CFSE) Dilution Assay
[0315] PBMCs from healthy donors were labelled with
PerCP-Cy5.5.alpha.-CD3 (5K7; BD), KO.alpha.-CD45, (J33; Beckman
Coulter), V450.alpha.-CD4 (5K3; BD), PE.alpha.-CD25 (M-A251, BD),
PE Cy7.alpha.-CD127 (HIL-7R-M21; BD), and APC.alpha.-CD38 (HB-7;
BD) and sorted by FACS Aria (BD). Sorted effector cells were
labelled with carboxyfluorescein succinimidyl ester (CFSE;
eBioscience) and stimulated with anti-CD3/CD28-coated beads in the
presence or absence of CD38.sup.+Tregs or CD38.sup.-Tregs (1:1 Treg
to effector cell ratio) in RPMI plus 10% fetal calf serum. After 72
hours, flow cytometry was performed and the percent dilution of
CFSE was used as a surrogate for T-cell proliferation.
Myeloid Derived Suppressor Cell (MDSC) Phenotyping and DARZALEX.TM.
(Daratumumab)-Mediated ADCC
[0316] PBMC from three normal healthy donors were co-cultured with
myeloma tumor cell lines (RPMI8226, U266, H929) for six days, and
evaluated for the production of granulocytic MDSC (G-MDSC)
(CD11b.sup.+CD14.sup.-HLA.sup.-DR.sup.-CD15.sup.+CD33.sup.+) as
described in Gorgun et al., Blood 121:2975-87, 2013. G-MDSC were
not present in normal healthy PBMC, however following co-culture
with all three myeloma cells lines G-MDSC were present as 5-25% of
total PBMC population (data not shown). Gating strategy for flow
cytometric evaluation of G-MDSC included CD11b.sup.+ as the first
gate, followed by CD14.sup.- and HLA.sup.-DR.sup.- gating, and then
followed by CD15.sup.+ and CD33.sup.+ gating. G-MDSCs were cell
sorted and evaluated for CD38 expression levels and sensitivity to
DARZALEX.TM. (daratumumab) mediated ADCC. To evaluate the effect of
DARZALEX.TM. (daratumumab) on ADCC/CDC of MDSCs, serum containing
complement or an isotype control was added to ADCC assays.
Naive and Memory T-Cell Analysis
[0317] Heparinized peripheral-blood samples were obtained from
patients prior to each infusion of DARZALEX.TM. (daratumumab).
Peripheral-blood mononuclear cells (PBMCs) were isolated by
Ficoll-Hypaque density-gradient centrifugation and stored in
cryopreservation medium (RPMI supplemented with 10% human serum and
10% dimethyl sulfoxide) in liquid nitrogen. For FACS analysis,
PBMCs were thawed and 2.times.10.sup.6 cells/panel was resuspended
in phosphate-buffered saline (PBS) with 0.05% azide and 0.1%
HAS.
Data Analysis
[0318] All data analysis and generation of relevant graphs were
performed exclusively using R software (R: A Language and
Environment for Statistical Computing, R Development Core Team, R
Foundation for Statistical Computing, Vienna, Austria, 2011, ISBN
3-900051-07-0 http_//_www_R-project_org/). All treated subjects
with an evaluable response were included in the data analysis.
Consistently throughout, responders are defined as subjects with a
Best Response per IRC of sCR, VGPR and PR, and non-responders are
defined as subjects with a Best Response per IRC of MR, SD and
PD.
[0319] Different statistical comparisons included (i) baseline
levels between responders and non-responders, (ii) baseline versus
on treatment for responders and for non-responders, (iii) percent
changes between responders and non-responders, (iv) ratio changes
of baseline versus on treatment. Each comparison included first a
test for normality with a Shapiro-Wilk test (Royston (1995) Remark
AS R94: A remark on Algorithm AS 181: The W test for normality
Applied Statistics, 44, 547-551). Almost exclusively, the data was
found to not have a normal distribution. The differential level
testing included conducting both a non-parametric Wilcox rank sum
test (Hollander and Wolfe (1973), Nonparametric Statistical
Methods. New York: John Wiley & Sons. Pages 27-33 (one-sample),
68-75 (two-sample), and a t-test following a Box Cox transformation
(Weisberg, S. (2014) Applied Linear Regression, Fourth Edition,
Wiley Wiley, Chapter 7). For the Box Cox transformation, a small
number (1e-07) was added to values equal to zero. In all cases, the
two tests agreed. The Wilcox rank sum test p-values are shown in
the tables throughout the specification unless otherwise indicated.
When testing for differences of the baseline versus on treatment
for responders and non-responders a two-group paired test per
subject was ran, in all other cases a two group unpaired test was
conducted.
[0320] As samples to analyze various lymphocyte populations were
not taken at identical time points for the different dosing
schedules, population modeling was conducted. Model fitting was
done on the rank of the visits. Population modeling on total and
activated NK cells involved fitting a broken stick model (Lutz et
al., "Statistical model to estimate a threshold dose and its
confidence limits for the analysis of sublinear doseresponse
relationships, exemplified for mutagenicity data." Mutation
Research/Genetic Toxicology and Environmental Mutagenesis 678.2
(2009): 118-122.). Linear mixed effect models with random intercept
and slope were fit on the B-cell, T-cell subpopulations, and
leukocytes, monocytes, neutrophils and lymphocytes patient
population data (Bates et al., (2014). "lme4: Linear mixed-effects
models using Eigen and S4." ArXiv e-print; submitted to Journal of
Statistical Software, http:_//_arxiv_org/abs/_1406.5823). This
linear mixed modeling was done on the relative day since treatment
start (ADY). The linear mixed model fitting were done on log
transformed response variables. In case of response variable values
equal to zero, 0.1 was added to all response variable values to
allow for modeling on log scale.
Example 2
Study 54767414MMY2002 Design (SIRIUS)
[0321] The target population for Study 54767414MMY2002 (SIRIUS) is
patients with advanced multiple myeloma who received at least 3
prior lines of therapy including a proteasome inhibitor (PI) and an
immunomodulatory drug (IMiD) or double refractory to a PI and an
IMiD. Response evaluations for the primary endpoint/final analysis
were based on assessments from an independent review committee
(IRC) and computerized algorithm, using 2011 IMWG Guidelines
(Clinical Study Report: An Open-label, Multicenter, Phase 2 Trial
Investigating the Efficacy and Safety of DARZALEX.TM. (daratumumab)
in Subjects With Multiple Myeloma Who Have Received at Least 3
Prior Lines of Therapy (Including a Proteasome Inhibitor and IMiD)
or are Double Refractory to a Proteasome Inhibitor and an IMiD
(EDMS-ERI-92399922; de Weers et al., (2011) J Immunol
186(3):1840-1848).
[0322] These assessments included: overall response rate (ORR),
duration of response, time to response and best response, clinical
benefit rate, time to progression (TTP), progression free survival
(PFS), and overall survival (OS).
[0323] A total of 124 subjects were treated with DARZALEX.TM.
(daratumumab) in this study (de Weers et al., (2011) J Immunol
186(3):1840-1848). 18 subjects were treated with 8 mg/kg and 106
subjects were treated with 16 mg/kg. The dosing schedule was as
follows:
[0324] Group A: DARZALEX.TM. (daratumumab) 16 mg/kg: Cycles 1 and
2: Days 1, 8, 15, and 22 (weekly), Cycle 3 to 6: Days 1 and 15
(every other week), Cycles 7+: Day 1 (every 4 weeks). Each cycle
was 4 weeks.
[0325] Group B: DARZALEX.TM. (daratumumab) 8 mg/kg: Cycle 1+: Day 1
(every 4 weeks).
[0326] The primary objective of the study was to determine the
efficacy of 2 treatment regimens of DARZALEX.TM. (daratumumab), as
measured by the ORR (CR+PR), in subjects with multiple myeloma who
have received at least 3 prior lines of therapy including a PI and
an IMiD or whose disease is double refractory to both a PI and an
IMiD (Clinical Study Report: An Open-label, Multicenter, Phase 2
Trial Investigating the Efficacy and Safety of DARZALEX.TM.
(daratumumab) in Subjects With Multiple Myeloma Who Have Received
at Least 3 Prior Lines of Therapy (Including a Proteasome Inhibitor
and IMiD) or are Double Refractory to a Proteasome Inhibitor and an
IMiD. EDMS-ERI-92399922).
[0327] The secondary objectives of this study included evaluation
of the safety and tolerability of DARZALEX.TM. (daratumumab),
demonstration of additional measures of efficacy (e.g, clinical
benefit, TTP, PFS, and OS) along with assessment of
pharmacokinetics, immunogenicity, pharmacodynamics, and to explore
biomarkers predictive of response to DARZALEX.TM. (daratumumab).
Additional study related information is available from the clinical
study protocol (Clinical Study Report: An Open-label, Multicenter,
Phase 2 Trial Investigating the Efficacy and Safety of DARZALEX.TM.
(daratumumab) in Subjects With Multiple Myeloma Who Have Received
at Least 3 Prior Lines of Therapy (Including a Proteasome Inhibitor
and IMiD) or are Double Refractory to a Proteasome Inhibitor and an
IMiD. EDMS-ERI-92399922).
[0328] In Stage 1 of Part 1, 1 subject (6%) responded in the 8
mg/kg group, and 5 subjects (31%) responded in the 16 mg/kg group.
Therefore, only the 16 mg/kg group was expanded in Stage 2 of Part
1 and in Part 2.
[0329] In the 16 mg/kg group, 31 subjects achieved response of PR
or better based on IRC assessment; the ORR was 29% (95% CI: 21%,
39%). Three subjects (3%) achieved sCR, and 13 subjects (12%)
achieved VGPR or better.
Example 3
Effect of DARZALEX.TM. (Daratumumab) on T-Cell Expansion and
Activity in Patients Enrolled in the 54767414MMY2002 Study
(SIRIUS)
[0330] CD38 is expressed on a variety of immune and hematopoietic
cells. Broad immune profiling by flow cytometry was performed to
examine the effect of DARZALEX.TM. (daratumumab) on immune cell
subsets and the association of baseline levels of these cells to
clinical response. Various cell populations, including T-cells
(CD3.sup.+, CD4.sup.+, CD8.sup.+ and regulatory T-cells (Treg)),
B-cells (CD19.sup.+), NK cells, monocytes (CD14.sup.+), leukocytes,
and neutrophils were evaluated by flow cytometry in peripheral
blood and bone marrow aspirates at baseline and following
DARZALEX.TM. (daratumumab) treatment to monitor for changes in
these cellular populations in responders and non-responders.
Lymphocytes, Leukocytes, Monocytes and Neutrophils
[0331] Leukocyte, lymphocyte, monocyte, and neutrophil counts were
studied in peripheral blood in responders and non-responders. Total
lymphocytes were found increased with DARZALEX.TM. (daratumumab)
treatment in responders with both 8 mg/kg and 16 mg/kg dose (FIG.
1). Linear mixed effect modeling revealed an increase of
0.8.times.10.sup.6 cells/.mu.L on log scale per 100 days (CI=0.06,
0.11). Slight increases were found for monocytes and leukocytes
with significant increase of 0.03.times.10.sup.6 cells/.mu.L
(CI=0.01, 0.04) and 0.03.times.10.sup.6 cells/.mu.L on log scale
(CI=0.01, 0.05) for each 100 days respectively. Median neutrophil
counts were consistent with baseline and did not vary
significantly, although there was neutropenia noted for some
patients.
[0332] Baseline levels of each of these cellular populations were
compared between response groups. No evidence was found for
baseline levels to be different for any of these cell types across
response groups using Wilcoxon signed-rank test (Table 1).
TABLE-US-00011 TABLE 1 Peripheral blood cell counts in responders
vs non-responders at baseline P- N Median Mean (SD) Range value*
Leukocytes: R 33 4.3 4.32 (1.65) (1.6; 8.8) Leukocytes: NR 82 4.19
4.77 (2.26) (2.13; 13.8) 0.60987 Lymphocytes: R 33 0.9 1.09 (0.59)
(0.27; 2.67) Lymphocytes: NR 82 1 1.05 (0.55) (0.3; 2.8) 0.85028
Monocytes: R 33 0.43 0.5 (0.25) (0.2; 0.97) Monocytes: NR 82 0.5
0.51 (0.25) (0.04; 1.3) 0.72803 Neutrophils: R 33 2.47 2.54 (1.23)
(1.06; 5.94) Neutrophils: NR 82 2.44 3.05 (2.08) (1; 11.7) 0.40373
N: number of samples per group R: responder NR: non-responder
*non-responder vs responder SD: standard deviation
NK Cells
[0333] Total NK cells (CD16.sup.+CD56) and activated NK cells
(CD16.sup.+CD56.sup.dim) were reduced with DARZALEX.TM.
(daratumumab) treatment over time (data not shown).
B-Cells
[0334] Absolute counts of B-cells (CD45.sup.+CD3.sup.-CD19.sup.+)
were measured in peripheral blood or bone marrow aspirates during
DARZALEX.TM. (daratumumab) treatment over time in responders and
non-responders. B-cells slightly increased in the whole blood and
were maintained in the bone marrow aspirates. Linear mixed modeling
of B-cells in peripheral blood revealed a minimal increase of
0.1.times.10.sup.6 cells/.mu.1 [CI=0.04, 0.16 for each 100 days on
log scale over the course of DARZALEX.TM. (daratumumab) treatment.
There were no changes to the percentages of B-cells
(CD45.sup.+CD3.sup.-CD19.sup.+/Lymphocytes) in the bone marrow
aspirates during daratumumab treatment, in either responders or
non-responders (p=0.1 and 0.4, respectively). Further, no evidence
was found for B-cell counts to be different at baseline between
responders and non-responders (p=0.5).
T-Cells
[0335] Lymphocytes were noted to increase with DARZALEX.TM.
(daratumumab) treatment (FIG. 1) even though B cells showed only a
minimal increase (see above). To investigate further, various
T-cell populations were studied (CD3.sup.+, CD4.sup.+, CD8.sup.+ T
cells, regulatory T cells) in both peripheral blood and bone
marrow.
[0336] CD3.sup.+, CD4.sup.+ and CD8.sup.+ T-cells were increased in
peripheral blood (both absolute counts/.mu.1 and percentage of
lymphocytes) following DARZALEX.TM. (daratumumab) treatment. FIG. 2
shows the percent change of absolute counts of CD3 T-cells
(CD45.sup.+CD3.sup.+) from baseline in peripheral blood over time
for every patient. The black line in the Figure shows the median
absolute counts.times.10.sup.6 cells/.mu.L for all patients. Only
visits with more than 2 observations were included into the Figure.
FIG. 3 shows the % change of absolute counts of CD4.sup.+ T-cells
(CD45.sup.+CD3.sup.+CD4.sup.+) from baseline in peripheral blood
over time for every patient. The black line in the Figure shows the
median for all patients. Only visits with more than 2 observations
were included into the Figure. FIG. 4 shows the % change of
absolute counts of CD8.sup.+ T-cells (CD45.sup.+CD3.sup.+CD8.sup.+)
from baseline in peripheral blood over time for every patient. The
black line in the Figure shows the median for all patients. Only
visits with more than 2 observations were included into the Figure.
Linear mixed modeling on absolute counts in peripheral blood
revealed on average total T-cell (CD45.sup.+CD3.sup.+) increase of
0.13.times.10.sup.6 cells/.mu.l on log scale for each 100 days
(CI=0.1, 0.15) following DARZALEX.TM. (daratumumab) treatment.
CD8.sup.+ T-cells were found to significantly increase by
0.16.times.10.sup.6 cells/.mu.l on log scale for each 100 days
(CI=0.13, 0.19). CD4.sup.+ cells were found to have a moderate
increase of 0.11.times.10.sup.6 cells/.mu.l on log scale for each
100 days (CI=0.09, 0.13).
[0337] For each of the T-cell subpopulations, responders showed a
higher maximum percent change of absolute counts to baseline than
non-responders (CD3.sup.+ p=3.2993e-05; CD4.sup.+ p=3.486e-05;
CD8.sup.+ p=2.7172e-05; regulatory T cell p=0.002). Table 2 shows
the Wilcoxon signed-rank test results for the comparison of each
T-cell subpopulation in peripheral blood between responders and
non-responders for percent change of absolute counts to
baseline.
TABLE-US-00012 TABLE 2 Percent change of absolute cell counts;
peripheral blood P- Sample N Median Mean (SD) Range value*
CD45.sup.+CD3.sup.+: R 33 86.76 118.91 (104.07) (-16.1; 398.71)
CD45.sup.+CD3.sup.+: NR 80 28.08 43.02 (69.55) (-67.11; 286.67)
3.30E-05 CD45.sup.+CD3.sup.+CD4.sup.+: R 33 72.08 77.74 (60.99)
(-21.05; 233.21) CD45.sup.+ CD3.sup.+CD4.sup.+: NR 80 19.48 29.36
(59.58) (-68; 298.89) 3.49E-05 CD45.sup.+ CD3.sup.+CD8.sup.+: R 33
106.6 180.81 (192.37) (-7.07; 760.51) CD45.sup.+
CD3.sup.+CD8.sup.+: NR 80 32.24 63.96 (112.44) (-66.22; 588.89)
2.72E-05 N: number of samples per group R: responder NR:
non-responder *responder vs. non-responder SD: standard
deviation
[0338] Similarly in bone marrow, total T-cells (CD45.sup.+CD3.sup.+
as a percentage of lymphocytes) and CD8.sup.+ T-cells
(CD45.sup.+CD3.sup.+CD8.sup.+ as a percentage of lymphocytes) were
found to significantly increase during DARZALEX.TM. (daratumumab)
treatment, for both responders and non-responders (CD3.sup.+
responders p=3.8147e-06, non-responders p=9.8225e-05; CD8.sup.+
responders p=3.8147e-06, non-responders p=0.0003). There was no
change in median CD4.sup.+ T-cells in either clinical response
group in bone marrow. Table 3 shows the Wilcoxon signed-rank test
results for the various T cells as % lymphocytes in bone marrow.
FIG. 5 shows the percentage (%) of CD45.sup.+CD3.sup.+ cells over
time during DARZALEX.TM. (daratumumab) treatment (both responders
and non-responders included in the graph). FIG. 6 shows the %
CD45.sup.+CD3.sup.+CD8.sup.+ cells over time during DARZALEX.TM.
(daratumumab) treatment (both responders and non-responders
included in the graph).
TABLE-US-00013 TABLE 3 T cell populations (% lymphocytes) in bone
marrow NR: NR: On R: R: On Sample Baseline treatment Baseline
treatment CD45.sup.+CD3.sup.+/ N 29 29 19 19 Lymphocytes Median
72.2 83.6 77.9 91.4 Mean (SD) 68.57 (13.64) 80.93 (11.57) 71.82
(14.92) 87.67 (9.49) Range (36.3; 94.5) (50.9; 97.4) (42.2; 94.8)
(63.3; 97.2) P-value* 9.8225e-05 3.8147e-06
CD45.sup.+CD3.sup.+CD4.sup.+/ N 29 29 19 19 Lymphocytes Median 33.7
29.2 22.7 22.8 Mean (SD) 31.24 (12.14) 32.96 (12.57) 24.18 (7.37)
24.29 (9.58) Range (6.3; 54.2) (9.6; 60.9) (8.1; 36.6) (12.5; 45.4)
P-value* 0.18351 0.98432 CD45.sup.+CD3.sup.+CD8.sup.+/ N 29 29 19
19 Lymphocytes Median 36.3 43.3 49.4 66.9 Mean (SD) 37.39 (13.64)
47.74 (18.14) 46.91 (14.89) 62.82 (12.79) Range (15.9; 67.2) (18.5;
81) (24.5; 79.6) (33.1; 83.3) P-value* 0.00026883 3.8147e-06 N:
number of samples per group R: responder NR: non-responder
*baseline vs. on treatment for responder or non-responder group SD:
standard deviation
[0339] While both responders and non-responders demonstrated T-cell
increases in the peripheral blood and bone marrow, responders had
the largest percentage change from baseline. To distinguish whether
responders or non-responders had different levels of CD3.sup.+,
CD4.sup.+ and CD8.sup.+ T-cells prior to DARZALEX.TM. (daratumumab)
treatment, baseline measurements of each subgroup were compared in
the peripheral blood.
[0340] There were no statistically significant differences between
responders and non-responders in absolute T-cell counts at baseline
in peripheral blood (Table 4) or in percentage of T cells from
total lymphocytes in bone marrow (Table 5), Wilcoxon signed-rank
test.
TABLE-US-00014 TABLE 4 Absolute cell counts in peripheral blood
prior to treatment at baseline P- Sample N Median Mean (SD) Range
value* CD45.sup.+CD3.sup.+: R 33 574 715.91 (472.54) (186; 2096)
CD45.sup.+CD3.sup.+: NR 80 638 672.5 (426.36) (85; 2407) 0.81527
CD4.sup.5+CD3.sup.+CD4.sup.+: R 33 190 276.91 (207.39) (77; 1085)
CD45.sup.+CD3.sup.+CD4.sup.+: NR 80 214 251.61 (146.13) (21; 766)
0.94965 CD45.sup.+CD3.sup.+CD8.sup.+: R 33 332 424.55 (324.49) (93;
1238) CD45.sup.+CD3.sup.+CD8.sup.+: NR 80 318 398.14 (354.52) (43;
2221) 0.56555 N: number of samples per group R: responder NR:
non-responder *responder vs non-responder per cell type SD:
standard deviation
TABLE-US-00015 TABLE 5 T cells (% lymphocytes) in bone marrow prior
to treatment P- Sample N Median Mean (SD) Range value
CD45.sup.+CD3.sup.+: R 23 78.4 73.66 (14.43) (42.2; 94.8)
CD45.sup.+CD3.sup.+: NR 65 76.5 73.5 (13.93) (36.3; 94.5) 0.81232
CD45.sup.+CD3.sup.+CD4.sup.+: R 23 25 25.57 (8.32) (8.1; 41.4)
CD45.sup.+CD3.sup.+CD4.sup.+: NR 65 25.3 27.53 (12.76) (6.3; 55.2)
0.76482 CD45.sup.+CD3.sup.+CD8.sup.+: R 23 50 47.7 (13.73) (24.5;
79.6) CD45.sup.+CD3.sup.+CD8.sup.+: NR 65 44.3 44.73 (15.49) (15.9;
76.1) 0.41678 N: number of samples per group R: responder NR:
non-responder *responder vs non-responder per cell type SD:
standard deviation
T Regulatory Cells
[0341] Treg cells were identified as the
CD3.sup.+CD4.sup.+CD25.sup.+CD127.sup.dim cell population in a
sample. The ratio of CD8.sup.+ T cells to Tregs was assessed in the
peripheral blood and bone marrow in patients treated with
DARZALEX.TM. (daratumumab) over time. The ratio increased in both
the periphery and bone marrow. FIG. 7A shows the median values of
the CD8.sup.+/Treg and CD8.sup.+/CD4.sup.+ cell ratios of all
patients per time point in peripheral blood. FIG. 7B shows the
median values of the CD8.sup.+/Treg and CD8.sup.+/CD4.sup.+ T-cell
ratios of all patients per time point in bone marrow. The changes
in the ratios of absolute counts of CD8.sup.+ Tregs and
CD8.sup.+/CD4.sup.+ were significant in peripheral blood over time
of treatment (Table 6) and in bone marrow (Table 7), Wilcoxon
signed-rank test.
[0342] In a combined data analyses of SIRIUS and GEN501 study
(Example 6), median ratios of CD8.sup.+/CD4.sup.+ and
CD8.sup.+/Treg cells in peripheral blood were increased at week 8
(p=5.1.times.10.sup.-5 for CD8.sup.+/CD4.sup.+ and
p=1.8.times.10.sup.-7 for CD8.sup.+/Treg) and at week 16 (p=0.00017
for CD8.sup.+/CD4.sup.+ and p=4.1.times.10.sup.-7 for
CD8.sup.+/Treg). Similarly, in bone marrow, median ratios of
CD8.sup.+/CD4.sup.+ and CD8.sup.+/Treg cells were increased on
treatment (week 12.+-.1 cycle) compared to baseline (p=0.00016 for
CD8.sup.+/CD4.sup.+ and p=2.8.times.10.sup.-7 for CD8.sup.+/Treg).
No significant differences were observed between responders and
nonresponders.
TABLE-US-00016 TABLE 6 T cell ratios in peripheral blood P- Sample
N Median Mean (SD) Range value* CD8.sup.+/CD4.sup.+: Baseline 66
119.75 191.78 (231.09) (24.17; 1461.18) CD8.sup.+/CD4.sup.+: C3D1
66 204.86 222.96 (167.44) (25.53; 867.58) 0.00046409
CD8.sup.+/CD4.sup.+: C4D1 66 210.05 215.15 (151.31) (25.86; 798.83)
0.00042154 CD8.sup.+/Tregs: Baseline 66 1258.33 2338.46 (3465.12)
(206.82; 18550).sup. CD8.sup.+/Tregs: C3D1 66 2326.74 3361.87
(3661.61) (155; 23066.67) 5.25E-06 CD8.sup.+/Tregs: C4D1 66 2763.16
3382.86 (3629.69) (316.67; 22087.5) 9.95E-08 *comparison to
baseline; N: number of samples per group; SD: standard
deviation
TABLE-US-00017 TABLE 7 T cell ratios in bone marrow P- Sample N
Median Mean (SD) Range value* CD8.sup.+/CD4.sup.+(/Lymphocytes): 31
163.18 184.4 (129.5) (32.58; 674.58) Baseline
CD8.sup.+/CD4.sup.+(/Lymphocytes): 31 221.89 240.85 (155.57)
(30.38; 666.4) 0.0038599 On treatment
CD8.sup.+/Tregs(/Lymphocytes): 30 1219.58 1802.73 (1582.7) (306.41;
7960).sup. Baseline CD8.sup.+/Tregs(/Lymphocytes): 30 2273.56
3905.72 (4232.73) (451.22; 20825) 3.15E-07 On treatment *comparison
to baseline; N: number of samples per group; SD: standard
deviation
Example 4
Study Design (GEN501)
[0343] Study GEN501 (NCT00572488) evaluated DARZALEX.TM.
(daratumumab) as monotherapy in double-refractory MM patients.
Sample isolation, processing and statistical analyses was as
described in Example 1 and Example 2. The study has been described
in Lokhorst et al., N Eng J Med 373:1207-19, 2005.
[0344] Briefly, Study GEN501 was the first-in-human clinical study
of DARZALEX.TM. (daratumumab) in subjects with MM. It is a Phase
1/2, dose-escalation, safety study divided into 2 parts. Part 1 is
an open-label, dose-escalation study; Part 2 is an open-label,
single-arm study with multiple cohorts, based on the dose levels
established in Part 1
[0345] In Part 1, 10 dose levels of DARZALEX.TM. (daratumumab) were
evaluated: 0.005, 0.05, 0.10, 0.50, 1, 2, 4, 8, 16, and 24 mg/kg.
The 2 lowest dose cohorts were allocated 1 (+3) subject(s) each,
and a standard 3 (+3) subject allocation was applied to the
remaining 8 dose cohorts. Part 2 was an open-label, single study
including two dose levels, 8 mg/kg and 16 mg/kg. Part 1 included 32
subjects and Part 2 included 72 subjects.
Example 5
DARZALEX.TM. (Daratumumab) Treatment Induces T Cell Clonality in
Patients
[0346] Given the expansion of CD8.sup.+ T-cells noted in both the
periphery and the bone marrow in the MY2002 study, high throughput
next-generation sequencing of the T-cell receptor (TCR) was
performed using the Immunoseq.TM. assay to determine whether
expanding CD8.sup.+ T-cells were clonal in nature, indicative of an
adaptive immune response. Total of 17 patient samples of subjects
who were enrolled in the GEN501 study were evaluated (n=6
responders i.e., .gtoreq.PR; n=11 non-responders i.e., MR, SD,
PD).
[0347] TCR sequencing revealed that DARZALEX.TM. (daratumumab)
treatment significantly increased clonality across patients. FIG.
8A shows the correlation between T cell clonality pre- vs.
post-DARZALEX.TM. (daratumumab) treatment (p=0.0056). FIG. 8B shows
the fold change in clonality in individual patients. Responders are
marked with the star. This data suggests that the T cell expansion
noted with DARZALEX.TM. (daratumumab) treatment may be clonal in
nature.
[0348] Responders had a greater total expansion in the TCR
repertoire (as measured by change in abundance; CIA) when compared
to non-responders. FIG. 8C shows the % CIA for individual patients.
Group A: responders, Group B: non-responders. Statistically
significant difference was observed between responders and
non-responders (p=0.037). FIG. 8D shows the sum of absolute change
in abundance (CIA) in responders and non-responders for each
expanded T cell clone. FIG. 8E shows the maximum % CIA for each
individual patient. Group A: responders, Group B: non-responders.
Statistically significant difference was observed between
responders and non-responders (p=0.048). FIG. 8F shows the maximum
CIA of a single T-cell clone in responders (Group A) and
non-responders (Group B).
[0349] CIA was obtained by identifying significant differences in
clonal abundance between two samples using Fisher's exact test
(DeWitt et al. J. Virol. 2015) and summing the absolute change in
abundance for each expanded clone.
Example 6
Immunomodulatory Effects of DARZALEX.TM. (Daratumumab) in Patients
Enrolled in the GEN501 Study
[0350] Various T and B cell populations were evaluated in
responders and non-responders enrolled in the GEN501.
Lymphocytes
[0351] Similar to SIRIUS (MMY2002) study, lymphocytes were
increased in both peripheral blood and bone marrow during
DARZALEX.TM. (daratumumab) treatment. This increment was attributed
to increased numbers of both CD4.sup.+ and CD8.sup.+ cells.
CD8.sup.+ Central Memory Cells
[0352] CD8.sup.+ T-cell phenotype was studied in patients treated
with DARZALEX.TM. (daratumumab) over time in a subset of 17
patients enrolled in the GEN 501 study. CD8.sup.+ cells from
patients were identified as naive (CD45RO-/CD62L.sup.+) (T.sub.N)
or central memory (T.sub.CM) (CD45RO.sup.+/CD62L.sup.+high) cells
using standard protocols.
[0353] FIG. 9A shows the % of CD8.sup.+ naive cells (% of CD8.sup.+
cells) and FIG. 9B shows the % of CD8.sup.+ central memory cells.
DARZALEX.TM. (daratumumab) treatment significantly decreased the
quantity of naive CD8.sup.+ T cells (p=1.82.times.10.sup.-4 at Week
8) and increased the quantity of CD8.sup.+ memory T cells
(p=4.88.times.10.sup.-2 at Week 8). This would suggest a transition
from naive cytotoxic T cells to memory T cells which may be
activated against a specific antigen. White squares indicate
patients that achieved at least a minimal response (.gtoreq.MR) and
black squares indicate patients that had stable disease or
progressive disease. A significantly greater decrease in CD8.sup.+
naive T cells was apparent in patients who responded to treatment
(data not shown). FIG. 9C shows that DARZALEX.TM. (daratumumab)
treatment increased the percentage in HLA Class I-restricted T
cells, which partially drive the virus-specific and alloreactive T
cell responses. FIG. 9D shows that the expanding effector memory T
cells expressed low levels of CD38. It is important to note that
these T cells display normal and even increased functional activity
against viral peptides and alloantigens (see Example 8). From these
functional results we concluded that there is an expansion of, or
improved activity of, antigen-experienced T cells against viral and
alloantigens during DARZALEX.TM. (daratumumab) treatment. These
data suggest that, unlike regulatory cell subsets, effector T cells
do not need CD38 expression to properly function and expand.
CD38-Positive Regulatory T-Cells
[0354] The observation of the robust expansion and increased
activity of cytotoxic T-cells together with recent literature
indicating that several immune-suppressive cell subsets express
CD38 prompted examination of the effects of DARZALEX.TM.
(daratumumab) on regulatory cell populations regulatory T-cells
(Tregs), myeloid derived suppressor cells (MDSCs) and regulatory
B-cells (Bregs).
[0355] Regulatory T-cells (Tregs)
(CD3.sup.+CD4.sup.+CD25.sup.+CD127.sup.dim) were isolated using
standard protocols. The frequency of the Tregs was analyzed using
flow cytometery.
[0356] A subpopulation of peripheral Tregs (10%.+-.10%) expressed
high levels of CD38 prior to Treg activation. FIG. 10A, top panel
shows the frequency of the Tregs in the CD3.sup.+CD4.sup.+ cell
population (P4 cell population) at baseline. FIG. 10A, bottom panel
shows the subset of Tregs expressing high CD38 (P5 cell
population). These CD38.sup.+ Tregs were highly sensitive to
DARZALEX.TM. (daratumumab) treatment and exhibited a significant
and almost immediate decline following the first dose of
DARZALEX.TM. (daratumumab) (n=17 patients; P=8.88.times.10.sup.-16
at Week 1 versus baseline). The frequency of Tregs after
DARZALEX.TM. (daratumumab) treatment is shown in FIG. 10B, top
panel (P4 cell population). FIG. 10B, bottom panel shows that the
CD38.sup.high Tregs (P5 cells) was the most significantly depleted
Treg population after 1.sup.st DARZALEX.TM. (daratumumab) infusion.
These CD38.sup.+ Tregs remained depleted throughout DARZALEX.TM.
(daratumumab) treatment (p=8.88.times.10.sup.-16,
1.11.times.10.sup.-15, and 1.50.times.10.sup.-11 at Weeks 1, 4, and
8, respectively, versus baseline. FIG. 10C shows the % of
CD38.sup.high Tregs from total CD3.sup.+ cells at baseline, week 1,
week 4, week 8, relapse, and 6 months after the end of treatment
(EOT). The CD38.sup.high Tregs were recovered to the baseline at
that time point. Changes in CD38.sup.+ Tregs were similar between
patients who did and did not respond to treatment however, the
CD8.sup.+ T-cell:Treg ratio was significantly higher at Week 8 in
patients who showed a response to DARZALEX.TM. (daratumumab)
(P=0.00955; FIG. 10D).
[0357] To assess the possible biological relevance of depletion of
CD38.sup.+ Tregs with DARZALEX.TM. (daratumumab) treatment, the
suppressive capacity of CD38.sup.+ Tregs versus CD38.sup.- Tregs on
autologous CD3.sup.+ T cells was assessed. In a series of
experiments performed with sample from multiple healthy donors,
CD38.sup.+ Tregs suppressed T-cell proliferation more robustly
(9.9% cell proliferation observed) than CD38.sup.- Tregs (53.2%
cell proliferation observed) or the negative control (74.9% cell
proliferation observed) (FIG. 10E).
[0358] Since MDSCs were not readily detectable in frozen PBMC
samples, CD38.sup.+ granulocytic MDSCs
(CD11b.sup.+CD14.sup.-HLA-DR.sup.-CD15.sup.+CD33.sup.+) were
generated in vitro from PBMCs isolated from patients at baseline
and from patients who had received one infusion of DARZALEX.TM.
(daratumumab). FIG. 11 shows the flow cytometry histogram of
identified MDSCs (FIG. 11, top histogram, boxed cell population).
Approximately half of the MDSCs expressed CD38 (FIG. 11, middle
graph; circled P7 cell population). The CD38.sup.high MDSCs were
nearly depleted in patients treated with DARZALEX.TM. (daratumumab)
(FIG. 11, bottom graph; circled P7 cell population).
[0359] The CD38.sup.high lineage nonspecific MDSCs were depleted
with DARZALEX.TM. (daratumumab) treatment over time in both
non-responders and patients who have at least Minimal Repose to
treatment. FIG. 12 shows that the percentage of the CD38.sup.high
MDSCs was reduced to nearly 0% in patients at 1 week, 4 weeks or 8
weeks of treatment. The CD38.sup.high lineage nonspecific MDSCs
returned to baseline after the end of treatment.
[0360] Patients with the largest CD38.sup.+ populations within
lineage nonspecific MDSC's demonstrated the best and most durable
responses to DARZALEX.TM. (daratumumab) treatment. FIG. 13 shows
that the patients 2, 4, 15, 16 and 17 having the highest percentage
of CD38.sup.high MDSC (as shown in FIG. 11) and classified as
patients with PR or MR, had a Progression-Free Survival (PFS) of at
least 8 months.
[0361] The CD38.sup.high lineage nonspecific MDSCs were also
sensitive to DARZALEX.TM. (daratumumab)-induced ADCC in vitro. ADCC
assays were performed using CD38.sup.high MDSCs from two donors and
Daudi cells as control target cells with effector:target cell ratio
of 50:1. FIG. 14 shows the results of the experiment from one
donor. DARZALEX.TM. (daratumumab) induced lysis of MDSC cells.
[0362] CD38.sup.+ Bregs were measured in DARZALEX.TM.
(daratumumab)-treated patients (n=16) and, similar to CD38.sup.+
Tregs, were depleted following the first dose of DARZALEX.TM.
(daratumumab) (p=0.0018 at Week 1 compared with baseline; paired
Wilcoxon rank test) and remained low while patients were on
treatment (FIG. 15A). The FACS sorted Bregs, when stimulated,
produced IL-10 (FIG. 15B).
[0363] Collectively, these observations suggest that the depletion
of immunosuppressive CD38.sup.+ MDSCs, Bregs, and Tregs is a
significant contributory mechanism to DARZALEX.TM.
(daratumumab)-induced changes in T-cell populations and
clonality.
Example 7
CD38.sup.+ MDSC Cells are Present in Cancer Patients
[0364] Percentage of MDSC (Lin.sup.-CD14.sup.-HLADR.sup.low/-) and
their CD38 expression was studied in the peripheral blood of
patients with NSCLC or prostate cancer using flow cytometry.
[0365] The percentage of MDSCs was between about 10%-37% and
between about 10%-27% of PBMCs in the analyzed samples from the
NSCLC and prostate cancer patients, respectively. CD38 expression
was identified in 80-100% of Lin.sup.-CD14.sup.+HLADR.sup.-/low
MDSCs from PBMCs from NSCLC patients and in 70-100% of MDSCs from
PBMCs from prostate cancer patients.
Example 8
DARZALEX.TM. (Daratumumab) Enhances Antiviral T-Cell Responses
[0366] To further assess the effect of DARZALEX.TM. (daratumumab)
on T-cell activation and functionality, IFN-.gamma. production from
peripheral T cells in response to viral and alloantigens was
measured in DARZALEX.TM. (daratumumab)-treated patients (n=7) with
a range of clinical outcomes. Patients with a PR or better
demonstrated significant increases in IFN-.gamma. secretion in
response to viral and alloantigens following DARZALEX.TM.
(daratumumab) treatment, compared with baseline, for at least one
time point during treatment, suggesting that T cell function is not
impaired by low CD38 expression (see Example 6, FIG. 9C) Similar to
the TCR clonality data, this increase was more marked in patients
who responded to DARZALEX.TM. (daratumumab) than those who did not.
FIG. 16A shows the anti-viral response of one representative
patient with VGPR. FIG. 16B shows the anti-viral response of one
representative patient with CR. FIG. 16C shows the anti-viral
response of one representative patient with PD. FIG. 16D shows the
anti-viral response of one representative patient with MR. In the
Figures, error bars represent standard error of the mean of
duplicate cultures. Asterisk denotes statistically significant
changes between the indicated comparisons. Best response per
Independent Review Committee is shown. Consistent with these
results, virus-reactive T-cells in patients with VGPR (FIG. 16E) or
CR (FIG. 16F) demonstrated an increase in proliferative capacity
during DARZALEX.TM. (daratumumab) treatment.
Example 9
Mechanism of Sensitivity of CD38 Expressing Immune Cell Subtypes to
DARZALEX.TM. (Daratumumab)
[0367] Data from both GEN501 and SIRIUS studies indicated that some
immune cells that express CD38 are depleted (NK cells, regulatory
T-cells (Tregs), regulatory B-cells (Bregs), and myeloid derived
suppressor cells (MDSCs)), while others that express CD38 increase
in number (cytotoxic and helper T cells) with DARZALEX.TM.
(daratumumab) therapy.
[0368] To address the mechanism of sensitivity, expression levels
of CD38 were assessed in various subpopulations of immune cells in
healthy donors and in multiple myeloma patients enrolled in either
GEN501 or SIRIUS study. FIG. 17A shows a histogram of expression of
CD38 in immune cells from a healthy donor, and FIG. 17B shows a
histogram of expression of CD38 in immune cells from a multiple
myeloma patient. In a healthy donor, CD38 expression was highest on
NK cells, followed by monocytes, B and T cells. In a multiple
myeloma patient, CD38 expression was highest on plasma cells,
followed by a subset of B cells, NK cells, monocytes, B-cells and
T-cells. FIG. 17C shows a comparison of the mean fluorescent
intensity (MFI) of CD38 across NK cells, Tregs, Bregs, B- and
T-cells cells from relapsed and refractory myeloma patients,
demonstrating that after plasma cells, NK cells expressed the
highest levels of CD38, followed by regulatory T-cells (Tregs) and
regulatory B-cells (Bregs).
[0369] In addition to CD38 expression, other cell surface proteins
such as complement inhibitory proteins (CIPs; CD46, CD55, CD59) may
contribute to sensitivity or resistance to DARZALEX.TM.
(daratumumab). In vitro evaluation of CIPs across immune cell
subpopulations found that NK cells express very low levels of CD59
and CD55, while other T and B cell populations express much higher
levels. This could also contribute to the variability of
DARZALEX.TM. (daratumumab) sensitivity across immune cell subtypes
(data not shown).
Discussion
[0370] This study describes previously unknown immunomodulatory
effects of DARZALEX.TM. (daratumumab) through reduction of
CD38.sup.+ immune suppressive cellular populations and concomitant
induction of helper and cytotoxic T-cell expansion, production of
IFN-.gamma. in response to viral peptides, and increased TCR
clonality, indicating an improved adaptive immune response.
[0371] This study demonstrates that MDSCs and Bregs express CD38
and were susceptible to DARZALEX.TM. (daratumumab) treatment. These
cells are known to be present in the tumor microenvironment and
contribute to tumor growth, immune evasion, angiogenesis,
metastasis, and production of suppressive cytokines. In addition to
these CD38.sup.+ suppressive cellular subsets, a novel
subpopulation of regulatory T cells
(CD4.sup.+CD25.sup.+CD127.sup.dim) was identified that also
expressed high levels of CD38 and demonstrated superior autologous
T-cell suppressive capacities. These cells were also sensitive to
DARZALEX.TM. (daratumumab) and were significantly reduced in
patients receiving treatment. DARZALEX.TM. (daratumumab)-mediated
elimination of these CD38.sup.+ immune-regulatory cells may reduce
local immune suppression within the myeloma microenvironment and
allow positive immune effector cells to expand and contribute to
antitumor response.
[0372] Indeed, significant increases in broad T-cells populations,
including both CD4.sup.+ and CD8.sup.+, were observed in both
peripheral blood and within bone marrow (i.e., the tumor). Specific
CD8.sup.+ subpopulations were altered with DARZALEX.TM.
(daratumumab) therapy, including significant decreases in naive
T-cells and concomitant significant increases in effector memory
CD8.sup.+ T-cells, indicating a shift in effector T-cells towards
an antigenic experienced phenotype that retained immunological
memory and may be reactive against tumor antigens. Ratios of
CD8.sup.+:CD4.sup.+ and CD8.sup.+:Tregs also increased
significantly with treatment, demonstrating a shift in positive
versus negative immune regulators.
[0373] To evaluate whether expanded CD4.sup.+ and CD8.sup.+ T-cells
were clonal in nature, the T-cell repertoire was examined in a
subset of patients. T-cell clonality significantly increased with
DARZALEX.TM. (daratumumab) treatment, even in patients who had a
best response of SD or who progressed. Therefore, increased T-cell
clonality cannot be due simply to reduction in tumor burden.
However, the skew in T-cell clonality was greater in patients with
a good clinical response, and was correlated with the increase in
CD8.sup.+ T-cells, suggesting the observed T-cell expansion with
DARZALEX.TM. (daratumumab) treatment was antigen-driven. This is
remarkable in this patient population, which was heavily pretreated
(median of 5 prior lines of therapy) and not expected to be able to
mount a strong antitumor immune response. In addition to increased
TCR clonality, patients with a response to DARZALEX.TM.
(daratumumab) demonstrated increased T-cell responses to
preexisting viral- and alloantigens, suggesting the rescue of the
immune system from an immunosuppressive state.
[0374] Treatment with DARZALEX.TM. (daratumumab) caused a reduction
in immune suppressive MDSC and regulatory T- and B-cells. These
reductions were concomitant with an expansion of CD4.sup.+ T-helper
cells and CD8.sup.+ cytotoxic T-cells. T-cell clonality and
functional anti-viral responses as measured by IFN-.gamma.
production also increased with DARZALEX.TM. (daratumumab)
treatment. These observations indicate that T-cells continued to
function properly, despite low CD38 expression, and suggest that
increased T-cell response may be due to depletion of regulatory
cells. Further, these changes in T-cell expansion, activity, and
clonality were more pronounced in patients who responded to
DARZALEX.TM. (daratumumab) compared with those who did not. Relapse
from DARZALEX.TM. (daratumumab) therapy was associated with
reversal of many of these changes. This suggests an additional,
previously-uncharacterized mechanism of action of DARZALEX.TM.
(daratumumab) through immunomodulation that may contribute to
clinical responses and its efficacy.
[0375] Recently, antibodies that promote antitumor immune
responses, rather than targeting the cancer directly, have
demonstrated efficacy in a range of settings. Antibodies inhibiting
CTLA-4 and PD-1 promote T-cell expansion and enhance T-cell
activation, resulting in prolonged survival and delayed disease
recurrence in patients with advanced solid tumors and hematologic
malignancies such as Hodgkin lymphoma. By enhancing anticancer
immunity, these immunomodulatory antibodies may not only induce
clinical responses, but also prevent disease recurrence.
Example 10
Serum Proteomic Analysis of Multiple Myeloma Subjects Traded with
Single-Agent DARZALEX.TM. (Daratumumab) in 54767414MMY2002 (SIRIUS)
Part 2 Clinical Study
Biomarker Sample Collection and Processing
[0376] Peripheral blood samples were collected in standard serum
separator tubes (2.5 mL to 5 mL) and serum aliquots were shipped
frozen SomaLogic, Inc (Boulder, Colo.) for multianalyte serum
protein profiling.
[0377] The serum protein profiling was performed at SomaLogic using
a pre-validated SOMAscan assay that measures 1129 protein analytes
by use of SOMAmer affinity based molecules. SOMAmer reagents are
single stranded DNA-based protein affinity reagents. The assay uses
small amounts of input sample (150 .mu.L plasma) and converts the
protein signal to a SOMAmer signal that is quantified by custom DNA
microarray.
Each SOMAmer contains 4 functional moieties: 1. A unique protein
recognition sequence 2. Biotin for capture 3. Photocleavable linker
4. Fluorescent molecule for detection
[0378] The unique protein recognition sequence uses DNA and
incorporates chemically modified nucleotides that mimic amino acid
side chains, expanding the diversity of standard aptamers and
enhancing the specificity and affinity of protein-nucleic acid
interactions (Gold et al., PLoS One 5:e15004, 2010). The aptamers
are selected for by SELEX. SOMAmer reagents are selected using
proteins in their native conformations. As such SOMAmer reagents
require an intact, tertiary protein structures for binding.
Unfolded or denatured presumably inactive proteins are not detected
by SOMAmer reagents.
[0379] Master mixes of SOMAmer reagents are grouped for sample type
and dilution. The reagents are pre-bound to streptavidin beads
prior to sample incubation. Proteins in the samples are bound to
the cognate SOMAmers during equilibrium, washed, incubated with
NHS-biotin, washed and then the beads are exposed to UV light to
cleave the photocleavable linker. The elution contains the SOMAmer
reagents bound to their biotin labeled proteins. A streptavidin
capture and subsequent washes removes the unbound SOMAmer reagents.
In the final elution the SOMAmer molecules are released from their
cognate proteins through denaturing conditions. The final eluate is
hybridized to custom Agilent DNA microarrays and the fluorophore
from the SOMAmer molecules it quantified by relative fluorescent
units (RFU). The RFU is proportional to the amount of protein in
the sample.
[0380] Samples from the MMY2002 study were tested in two primary
batches. A first batch of 180 samples contained paired Cycle 1 Day
1 (C1D1, baseline) and C3D1 (Cycle 3 Day 1) serum samples from 90
subjects. The 180 samples were analyzed together on 3 separate
SomaScan plates. The second batch of samples includes 50 C1D1
samples, including 35 repeated samples from batch 1.
Data Analyses
Input Datasets and Definitions
[0381] Treated subjects with an evaluable response were included in
the data analysis. Consistently throughout the report, responders
are defined as subjects with an overall best response (per IRC, for
MMY2002) of sCR, VGPR, and PR, stable disease (SD) subjects as a
subject with minimal response (MR) or SD, and non-responders are
defined as subjects with an overall best response (per IRC, for
MMY2002) of progressive disease (PD).
Somalogic Data Pre-Processing
Batch Alignment
[0382] Batches 1 and 2 of MMY2002 samples were tested on two
different versions of the SOMAscan platform. Differences between
the two versions were minor, and included three SOMAmer sequences
that changed between the versions (CTSE: 3594-6_1->3594-6_5,
FCN1: 3613-62_1->3613-62_5, BMPER: 3654-27_1->3654-27_4).
These were removed from the analysis.
[0383] The measurements of the three batch 1 plates were aligned
according to SomaLogic's standard inter-plate calibration workflow,
by defining plate-wide calibration scaling factors for each SOMAmer
by calculating the ratio of a Master-mix specific global reference
value to the median of 7 in-plate control calibrator measurements.
The plate-specific scaling factor for each SOMAmer reagent was
applied to each sample on the plate equivalently.
[0384] Given the different SOMAscan platform versions of batch 1
and 2, systematic inter-batch variability correction was done with
a modified implementation of SomaLogic's standard inter-plate
calibration workflow, by leveraging the repeated measurement of 35
samples across batches. For each SOMAmer the ratio of the batch 1
post-calibration measurement divided by the batch 2 pre-calibration
measurement was calculated for each of the 35 repeated samples
(ri,j). The median of these 35 ratios was used to define the
revised SOMAmer-specific calibration scaling factor for the batch 2
samples (r.sub.i). These calibration scaling factors were then
implemented identically to the standard SOMAscan procedure.
r i , j = ( Post - Calib . Conc Batch 1 , i , j Pre - Calib . Conc
Batch 2 , i , j ) , r i .fwdarw. = ( r i , 1 , r i , 2 , , r i , j
) for all repeated samples j ##EQU00001## Calibration Scaling
Factor i = median ( r i .fwdarw. ) = r i ~ ##EQU00001.2##
[0385] Once the revised calibration scaling factors were
calculated, the distributions of all the scaling factors for each
batch of the analysis were plotted to assess the presence of
outliers. 9 SOMAmers with extremely large or small calibrators
(>0.25 and <3) were removed from the analyses due to poor
reproducibility.
[0386] After batch alignment and SOMAmer filtering was complete for
MMY2002, a log 2 transformation was applied to all protein
concentration values of MMY2002 to bring the data more in line with
a normal distribution and to improve the performance of parametric
statistical tests.
Confounding Variable Correction
[0387] Estimation of the portion of dataset variance explained by
meta-variables (like demographics, response class, and sample time
point) and identification of potential confounding factors was
performed by principal component analysis on the centered and
scaled dataset. Simple linear models were fit to identify the
highest ranked PC that was significantly associated with each of
the variables of interest. The significance of these associations
was determined using a Wald test and the fraction of the PC
variability explained by the model was estimated by the R2 of the
fit. For the MMY2002 data, site ID was found to be correlated with
PC1 and explained the largest portion of dataset variability
(.gtoreq.7.37%, p-value=3.71.times.10-9). In order reduce the
impact of sample acquisition site related effects within the data,
ComBat28 was utilized to correct for site ID effects.
Repeated Sample Merging
[0388] The data of the 35 samples repeated between MMY2002 batches
1 and 2 was merged by calculating the mean for each protein.
Differential Protein Concentration Analysis
Responders Versus Non-Responders
[0389] Statistical comparison of protein concentration
distributions in DARZALEX.TM. (daratumumab) responders versus
non-responders was performed at both baseline and on treatment
using two complementary methods (i) Wilcoxon rank-sum test
(Hollander and Wolfe, Ninparametirc Statistical Methods. New York:
John Wiley & Sons. 1973. 27-33 (one-sample), 68-75 (two-sample)
on each individual SOMAmer and (ii) Limma analysis (Ritchie, M. E.,
et al., Nucleic Acids Res. 2015; 20:43(7):e47) on all SOMAmers
simultaneously. All p-values were adjusted using the
Benjamini-Hochberg (BH) method for multiple hypothesis correction
(Benjamini and Hochberg, (1995) J. R. Statist. Soc. B.57: 289-300;
R: A Language and Environment for Statistical Computing, R
Development Core Team, R Foundation for Statistical Computing,
Vienna, Austria. 2011; ISBN 3-900051-07-0). The null hypothesis of
no differential expression was rejected when the adjusted p-value
<0.05.
On Treatment Versus Baseline
[0390] Baseline versus on treatment protein levels were compared
using three alternative statistical methods: (i) two-way
repeated-measures ANOVA6 (ii) the Wilcoxon signed-rank test and
(iii) the Friedman test (Johnson et al., (2007) Biostatistics
8(1):118-127). All p-values were adjusted to control FDR using the
BH method for multiple hypothesis correction (Benjamini and
Hochberg, J. R. Statist. Soc. B.57:289-300, 1995). In addition to
the treatment significance, the two-way repeated-measures ANOVA
(Chambers et al., Analysis of variance; designed experiments:
Chapter 5. Statistical Models in S, Editors J. M Chambers and T. J
Hastie. Wadsworth & Brookes/Cole. 1992) was also applied to
determine if significant time-point:response-class interaction
occurred for each SOMAmer. A modified Wilcoxon rank-sum test was
applied as a post-hoc test to specifically determine if responders
and non-responders showed different treatment effects, by
calculating the difference between every subject's on treatment and
baseline protein concentration values and performing a Wilcoxon
rank-sum test. Significance values were adjusted using the BH
method and the null hypothesis was rejected when the adjusted
p-value <0.05.
Classifier Training
[0391] Baseline protein level MMY2002 data was used to build a
response prediction classifier. A nested-loop stratified 10-fold
cross-validation approach repeated 30 times, using 4 different
machine learners: Support Vector Machines (SVM), Random Forests
(RF), Naive Bayes (NB), and j48 decision trees. For each learner,
the training procedure began with creating 10 balanced folds of the
dataset (outer loop). One of these folds was held out as a test
cohort while the remaining 9 were passed to an inner loop as the
training cohort. Within the inner loop, the training cohort was
once again split into 10 balanced folds, creating inner-training
and inner-test sets. Learners were trained on each of these
inner-training sets and this process was repeated 30 times for each
cohort within the outer loop. The accuracy of each inner loop
learner at predicting the inner-test sets was used to select
features and optimize model parameters. Once the 30.times. inner
looping was complete for each training cohort, the performance of
the outer loop (using the optimized parameters and features) was
assessed on each corresponding test cohort. The entire outer
looping procedure was then repeated 30 times, producing 30 response
predictions for every sample within the dataset. The AUC,
Sensitivity, and Specificity statistics obtained from this looping
approach were an approximation of how well the final model, trained
on the full original dataset, will perform on new test cases.
Results from MMY2002 Study
[0392] Various comparisons were conducted including treatment
induced response dependent changes in protein expression. One of
the proteins that showed decreased expression in responders over
time was PD-L1, whereas PD-L1 protein expression increased in
non-responders over time. The engagement of PD-L1 on T cells leads
to reduced T cell function and increased Treg development. FIG. 18
shows protein expression profile of PD-L1 in responders,
non-responders and in patients with stable disease at cycle 1 and
cycle 3.
[0393] PD-L1 engagement with its receptor PD-1 suppresses
anti-tumor responses and drives T cell anergy and exhaustion. While
not wishing to be bound by any particular theory, downregulation of
PD-L1 upon anti-CD38 treatment may also result in improved
potentiation of anti-tumor immune responses in solid tumors.
Example 11
Daratumumab in Combination with Lenalidomide Plus Dexamethasone
Induces Clonality Increase and T-Cell Expansion: Results from a
Phase 3 Randomized Study (POLLUX)
[0394] To further explore the ability of daratumumab to promote
adaptive T-cell responses, T-cell repertoires (TCR) were profiled
to evaluate T-cell clonality, expansion, and diversity from samples
collected in POLLUX (MMY3003), a phase 3, randomized, open-label,
multicenter study for patients with relapsed/refractory MM, in
which daratumumab was tested in combination with lenalidomide plus
dexamethasone versus lenalidomide plus dexamethasone alone (DRd vs.
Rd; Dimopoulos M A et al, N Engl J Med. 2016 Oct. 6;
375(14):1319-1331). Clinical Trial number NCT02076009.
POLLUX Trial Treatments
[0395] Patients were randomly assigned in a 1:1 ratio to receive
daratumumab, lenalinomide and dexamehtasone (DRd) or lenalinomide
and dexamethasone (Rd). Randomization was stratified by
International Staging System (ISS), number of prior treatment
programs (1 vs. 2 or 3 vs. >3), and prior lenalinomide treatment
("no" vs. "yes").
[0396] Daratumumab was administered as an IV infusion at a dose of
16 mg/kg weekly (on days 1, 8, 15, and 22) for 8 weeks during
cycles 1 and 2, every 2 weeks (on days 1 and 15) for 16 weeks
(cycles 3 through 6), and every 4 weeks thereafter. Both groups
received lenalidomide at a dose of 25 mg orally on days 1 to 21 of
each cycle if the creatinine clearance was more than 60 ml per
minute (or a dose of 10 mg daily if the creatinine clearance was 30
to 60 ml per minute) and dexamethasone at a dose of 40 mg weekly.
For the daratumumab group, the dose of dexamethasone was split:
dexamethasone was administered at a dose of 20 mg before infusion
as prophylaxis for infusion-related reactions and 20 mg was
administered the next day.
Efficacy
[0397] At a median follow-up of 13.5 months, a total of 169 events
of disease progression or death (in 53 patients [18.5%] in the
daratumumab group vs. 116 [41.0%] in the control group) were
reported. The hazard ratio for disease progression or death in the
daratumumab group versus the control group was 0.37 (95% confidence
interval [CI], 0.27 to 0.52; P<0.001 by stratified log-rank
test). The Kaplan-Meier rate of progression-free survival at 12
months was 83.2% (95% CI, 78.3 to 87.2) in the daratumumab group
and 60.1% (95% CI, 54.0 to 65.7) in the control group. The median
progression-free survival was not reached (95% CI, could not be
estimated) in the daratumumab group, as compared with 18.4 months
(95% CI, 13.9 to could not be estimated) in the control group.
Similarly, in the time-to event analysis of disease progression, a
total of 148 events (in 44 patients [15.4%] in the daratumumab
group vs. 104 [36.7%] in the control group) were observed (hazard
ratio, 0.34; 95% CI, 0.23 to 0.48; P<0.001). The rate of
progression-free survival at 12 months was 85.7% (95% CI, 80.9 to
89.4) in the daratumumab group, as compared with 63.2% (95% CI,
57.1 to 68.8) in the control group.
Methods
[0398] T-cell receptor beta (TCR.beta.) sequencing for repertoire
profiling was conducted on whole blood samples collected at
baseline and eight weeks after daratumumab treatment (cycle 3 [C3])
from subjects on both arms using the ImmunoSEQ assay (Adaptive
Biotechnologies. Seattle, Wash., USA). 133 subjects in DRd and 124
subjects in Rd treatment groups were included in this analysis and
represented a balanced subgroup of the POLLUX clinical trial
subjects. T-cell metric changes were compared between arms with
ANOVA, including the treatment arm and visit interaction term.
Within treatment-arm changes were evaluated with a Wilcoxon
signed-rank test comparing baseline to on-treatment values per
patient.
Results
[0399] Consistent with the randomized treatment groups, no baseline
differences were observed in T-cell repertoire metrics between the
treatment arms, including T-cell clonality, diversity (or
richness), and T-cell fraction. Similar to prior findings from
daratumumab monotherapy studies, significantly larger increase of
TCR.beta. clonality was observed in the DRd arm (median of 0.166 at
baseline to 0.263 at C3). Interestingly, there was no increase in
TCR.beta. clonality in the Rd arm (median of 0.175 at baseline to
0.175 at C3). The change in TCR.beta. clonality between C1
(baseline) and C3 was significantly different between DRd and Rd
(p=3.26E-10), demonstrating that the addition of daratumumab to Rd
induces a specific clonal expansion of T cells. Estimated richness
(diversity), on the other hand, slightly decreased with DRd
treatment but not with Rd treatment (median of 503,951 at baseline
to 427,096 at C3 [p=1.01E-04] vs 572,182 to 532,806 [p=3.58-01]).
Among patients in both treatment groups, a bigger increase in
T-cell fraction was observed in DRd vs Rd (median of 0.231 at
baseline to 0.278 at C3 [p=2.62E-3] vs 0.228 to 0.249
[p=1.91E-01]). Although there were no significant differences in
baseline characteristics in T-cell clonality, richness, and T-cell
fraction, quartile analysis demonstrated that high baseline TCR
richness predicted for better PFS with DRd but not for Rd.
Conclusion
[0400] Daratumumab in combination with lenalidomide plus
dexamethasone specifically induced robust increases in T-cell
clonality, which was not observed within the control lenalidomide
plus dexamethasone arm. Interestingly, baseline TCR richness was
associated with improved PFS in DRd subjects. This observation is
similar to results with immune checkpoint inhibitors (Postow M A et
al, J Immunother Cancer 2015; 3:23), and together with the
significant increase in T-cell clonality, provides further evidence
for the immunomodulatory activity of daratumumab, even in
combination therapy. These data support daratumumab's
immune-modulatory MOA and provide additional insights into
daratumumab's effect on the TCR in combination with standard of
care treatment.
Example 12
High-Parameter Mass Cytometry (CyTOF) Evaluation of
Relapsed/Refractory Multiple Myeloma (MM) Patients Treated with
Daratumumab Supports Immune Modulation as a Novel Mechanism of
Action
[0401] Next-generation mass cytometry (CyTOF), which allows high
parameter evaluation of the immune system, was used to assess the
effects of daratumumab alone or in combination on a more
comprehensive profile of immune cell subpopulations.
Methods
[0402] Relapsed/refractory MM patient samples from a subset of
single agent studies; SIRIUS (32 patients; whole blood [WB] only)
and GEN501 (5 patients; WB and bone marrow [BM]) along with GEN503,
a study of daratumumab plus lenalidomide and dexamethasone (9 pts;
WB and BM) were analyzed. Fluorochrome or metal-conjugated antibody
panel stained samples were evaluated by flow cytometry or cytometry
by time-of-flight (CyTOF.RTM.) platforms, respectively. FACS
analyses were performed and analyzed by FACS Canto II flow
cytometers and FACSDiva software. For CyTOF analysis, events were
clustered by phenotype by a spanning tree progression of density
normalized events (SPADE) algorithm, and each cluster was
associated with an immune population via Cytobank.RTM. software.
Differential analysis of population fractions and marker intensity,
over time and between response groups, derived raw P values from
t-tests and single cell level bootstrap adjusted P values corrected
for multiple dependent hypothesis testing. Results were visualized
using SPADE trees and Radviz projections, a new method that allows
for the comparison of populations and conditions while preserving
the relation to original dimensions.
Results
[0403] Flow cytometry and high-dimensional CyTOF analyses confirmed
previous findings including higher CD38 expression on plasma cells
compared with other immune populations of natural killer (NK),
monocytes, B and T cells, and depletion of both plasma cells and NK
cells upon daratumumab treatment. Interestingly, while NK cells
were significantly reduced with DARA treatment, remaining active NK
cells (CD16.sup.+CD56.sup.dim) demonstrated increased expression of
activation markers CD69, CD25 and CD137 while also decreasing
granzyme B and increasing naive marker CD27. Though functionality
tests weren't performed, the ability to evaluate several markers
simultaneously suggests these cells possess limited cytotoxicity.
Additionally, these studies indicated depletion of CD38 positive
immune suppressive subsets of Tregs and Bregs. CD38.sup.+ basophil
reductions occurred independent of response and may provide insight
to short-lived infusion related reactions. Several observations
within the T-cell compartment were indicative of a DARA-mediated
adaptive response in both WB and BM samples. T cells displayed
increases in total numbers and shifted towards higher CD8:CD4 and
effector:naive ratios after 2 months of DARA treatment. Responders
had higher expression levels of several activation markers
including CD69 and HLA-DR along with increased production of
cytolytic enzyme granzyme B in CD8.sup.+ T cells following DARA
treatment. Interestingly, in the GEN503 sample set, patients who
achieved a complete response presented with a distinct BM CD4
T-cell phenotype of high granzyme B positivity versus those that
achieved a partial response or very good partial response. This
observation suggests patients with an active immune phenotype may
achieve deeper responses to daratumumab in combination with
standard of care agents lenalidomide and dexamethasone.
CONCLUSION
[0404] CyTOF analysis of patient samples from both single agent and
combination daratumumab studies agree with flow cytometry and
support the pharmacodynamics and immune modulatory mechanism of
action of daratumumab while providing additional insight into
changes in T-cell subtypes and activation status.
Sequence CWU 1
1
401300PRTHomo 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-D38
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 14122PRTArtificial SequenceVH of anti-CD38 antibody 003
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 Ala Ser 115 120 15107PRTArtificial SequenceVL
of anti-CD38 antibody 003 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 024 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 024 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 Gly Leu Leu Ile 35 40 45 Tyr Asp Ala Ser
Asn Arg Ala Ser 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 Leu 85
90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
18120PRTArtificial SequenceVH of anti-CD38 antibody MOR202 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 MOR202 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 mAb isatuximab 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 mAb
isatuximab 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 22120PRTArtificial SequenceVH of anti-PD-1 mAb Keytruda 22Gln
Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala 1 5 10
15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30 Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45 Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe
Asn Glu Lys Phe 50 55 60 Lys Asn Arg Val Thr Leu Thr Thr Asp Ser
Ser Thr Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Lys Ser Leu Gln Phe
Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Asp Tyr Arg
Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val
Thr Val Ser Ser 115 120 23111PRTArtificial SequenceVL of anti-PD-1
mAb Keytruda 23Glu 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 Lys
Gly Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Leu His Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro 35 40 45 Arg Leu Leu Ile Tyr Leu Ala
Ser Tyr Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Glu
Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg 85 90 95 Asp
Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110
24113PRTArtificial SequenceVH of anti-PD-1 mAb Opdivo 24Gln Val Gln
Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser
Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser 20 25
30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asn Asp Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser 25107PRTArtificial
SequenceVL of anti-PD-1 mAb Opdivo 25Glu 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 Ser Ser Asn Trp
Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105 26121PRTArtificial SequenceVH of anti-PD-L1 mAb durvalumab
26Glu 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 Arg
Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr
Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser 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 Glu Gly Gly
Trp Phe Gly Glu Leu Ala Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr
Leu Val Thr Val Ser Ser 115 120 27108PRTArtificial SequenceVL of
anti-PD-L1 mAb durvalumab 27Glu Ile Val Leu Thr Gln Ser Pro Gly Thr
Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg
Ala Ser Gln Arg Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Asp Ala
Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80
Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Leu Pro 85
90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
28118PRTArtificial SequenceVH of anti-PD-L1 mAb atezolizumab 28Glu
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 Asp Ser
20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr
Ser Lys Asn Thr Ala 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 Arg His Trp Pro
Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val
Ser Ser 115 29107PRTArtificial SequenceVL of anti-PD-L1 mAb
atezolizumab 29Asp 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
Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr
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 Leu Tyr His Pro Ala 85 90 95 Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 30120PRTArtificial
SequenceVH of anti-PD-L1 mAb avelumab 30Glu 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 Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ile Met Met
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr 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 Ile Lys Leu Gly Thr Val Thr Thr Val Asp
Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120
31110PRTArtificial SequenceVL of anti-PD-L1 mAb avelumab 31Gln Ser
Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15
Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20
25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys
Leu 35 40 45 Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser
Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu
Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr
Cys Ser Ser Tyr Thr Ser Ser 85 90 95 Ser Thr Arg Val Phe Gly Thr
Gly Thr Lys Val Thr Val Leu 100 105 110 32123PRTArtificial
SequenceVH of anti-PD-1 mAb 32Gln 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 Ile Ser Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile
Ile Pro Ile Phe Asp Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln
Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70
75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Pro Gly Leu Ala Ala Ala Tyr Asp Thr Gly Ser
Leu Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 33107PRTArtificial SequenceVL of anti-PD-1 mAb 33Glu 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 Arg 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 Asn Tyr Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys 100 105 34117PRTArtificial SequenceVH of anti-PD-1
mAb 34Glu 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 Ala Phe
Ser Arg Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Ser Val 35 40 45 Ala Tyr Ile Ser Gly Gly Gly Ala Asn
Thr Tyr Tyr Leu Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Ser 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 Ser Pro
Tyr Leu Ser Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 100 105 110 Val
Thr Val Ser Ser 115 35107PRTArtificial SequenceVH of anti-PD-1 mAb
35Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1
5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Leu Ser Asp
Tyr 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Leu Ile 35 40 45 Lys Ser Ala Ser Gln Ser Ile Ser Gly Ile Pro
Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu
Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr
Cys Gln Asn Gly His Ser Phe Pro Tyr 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys 100 105 36117PRTArtificial SequenceVH of
anti-TIM-3 mAb 36Glu 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 Ala Ser Gly
Phe Thr Phe Ser Ser Tyr 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 Ser 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 Lys Ser Pro Tyr Ala Pro Leu Asp Tyr Trp Gly Gln Gly Thr Leu 100
105 110 Val Thr Val Ser Ser 115 37107PRTArtificial SequenceVL of
anti-TIM-3 mAb 37Glu 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 Asn Asp 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 Gly Gly His Ala Pro Ile 85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
38124PRTArtificial SequenceVH of anti-TIM-3 mAb 38Glu 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 Thr Ser Tyr 20 25 30
Trp Met Gln Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35
40 45 Gly Ala Ile Tyr Pro Gly Asp Gly Asp Ile Arg Tyr Thr Gln Asn
Phe 50 55 60 Lys Gly Gln Val Thr Ile 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 Trp Glu Lys Ser Thr Thr Val
Val Gln Arg Asn Tyr Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Thr
Val Thr Val Ser Ser 115 120 39106PRTArtificial SequenceVL of
anti-TIM-3 mAb 39Asp 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 Lys Ala Ser
Glu Asn Val Gly Thr Phe 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg
Tyr Thr 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 Gly Gln Ser Tyr Ser Tyr Pro Thr 85 90 95
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 40509PRTArtificial
SequenceRecombinant hyaluronidase 40Met Gly Val Leu Lys Phe Lys His
Ile Phe Phe Arg Ser Phe Val Lys 1 5 10 15 Ser Ser Gly Val Ser Gln
Ile Val Phe Thr Phe Leu Leu Ile Pro Cys 20 25 30 Cys Leu Thr Leu
Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro 35 40 45 Phe Leu
Trp Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe 50 55 60
Asp Glu Pro Leu Asp Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg 65
70
75 80 Ile Asn Ala Thr Gly Gln Gly Val Thr Ile Phe Tyr Val Asp Arg
Leu 85 90 95 Gly Tyr Tyr Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr
Val Asn Gly 100 105 110 Gly Ile Pro Gln Lys Ile Ser Leu Gln Asp His
Leu Asp Lys Ala Lys 115 120 125 Lys Asp Ile Thr Phe Tyr Met Pro Val
Asp Asn Leu Gly Met Ala Val 130 135 140 Ile Asp Trp Glu Glu Trp Arg
Pro Thr Trp Ala Arg Asn Trp Lys Pro 145 150 155 160 Lys Asp Val Tyr
Lys Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn 165 170 175 Val Gln
Leu Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe 180 185 190
Glu Lys Ala Gly Lys Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys 195
200 205 Leu Leu Arg Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp
Cys 210 215 220 Tyr Asn His His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser
Cys Phe Asn 225 230 235 240 Val Glu Ile Lys Arg Asn Asp Asp Leu Ser
Trp Leu Trp Asn Glu Ser 245 250 255 Thr Ala Leu Tyr Pro Ser Ile Tyr
Leu Asn Thr Gln Gln Ser Pro Val 260 265 270 Ala Ala Thr Leu Tyr Val
Arg Asn Arg Val Arg Glu Ala Ile Arg Val 275 280 285 Ser Lys Ile Pro
Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr 290 295 300 Arg Ile
Val Phe Thr Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu 305 310 315
320 Leu Val Tyr Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile
325 330 335 Val Ile Trp Gly Thr Leu Ser Ile Met Arg Ser Met Lys Ser
Cys Leu 340 345 350 Leu Leu Asp Asn Tyr Met Glu Thr Ile Leu Asn Pro
Tyr Ile Ile Asn 355 360 365 Val Thr Leu Ala Ala Lys Met Cys Ser Gln
Val Leu Cys Gln Glu Gln 370 375 380 Gly Val Cys Ile Arg Lys Asn Trp
Asn Ser Ser Asp Tyr Leu His Leu 385 390 395 400 Asn Pro Asp Asn Phe
Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr 405 410 415 Val Arg Gly
Lys Pro Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys 420 425 430 Phe
Tyr Cys Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp 435 440
445 Val Lys Asp Thr Asp Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys
450 455 460 Ile Asp Ala Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro
Gln Ile 465 470 475 480 Phe Tyr Asn Ala Ser Pro Ser Thr Leu Ser Ala
Thr Met Phe Ile Val 485 490 495 Ser Ile Leu Phe Leu Ile Ile Ser Ser
Val Ala Ser Leu 500 505
* * * * *
References