U.S. patent application number 16/343796 was filed with the patent office on 2019-08-15 for treatment with anti-kir3dl2 agents.
The applicant listed for this patent is INNATE PHARMA. Invention is credited to CARINE PATUREL, HELENE SICARD.
Application Number | 20190248895 16/343796 |
Document ID | / |
Family ID | 60268349 |
Filed Date | 2019-08-15 |
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United States Patent
Application |
20190248895 |
Kind Code |
A1 |
PATUREL; CARINE ; et
al. |
August 15, 2019 |
TREATMENT WITH ANTI-KIR3DL2 AGENTS
Abstract
This disclosure relates to the use of KIR3DL2-targeting agents
for the treatment of CTCL. The invention provides advantageous
treatment regimens using anti-KIR3DL2 antibodies for the treatment
of CTCL, notably in first-line CTCL.
Inventors: |
PATUREL; CARINE; (MARCY
L'ETOILE, FR) ; SICARD; HELENE; (MARSEILLE,
FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INNATE PHARMA |
MARSEILLE |
|
FR |
|
|
Family ID: |
60268349 |
Appl. No.: |
16/343796 |
Filed: |
October 19, 2017 |
PCT Filed: |
October 19, 2017 |
PCT NO: |
PCT/EP2017/076751 |
371 Date: |
April 22, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62410880 |
Oct 21, 2016 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/3061 20130101;
C07K 2317/34 20130101; A61K 2039/545 20130101; A61K 2039/505
20130101; C07K 2317/24 20130101; A61P 35/00 20180101; C07K 2317/732
20130101; C07K 2317/92 20130101; C07K 16/2803 20130101; C07K
2317/77 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61P 35/00 20060101 A61P035/00 |
Claims
1-41. (canceled)
42. A method of treating a T cell malignancy with tissue
manifestation in both individuals having blood involvement and in
individuals lacking blood involvement, the method comprising
administering to an individual having a T cell malignancy an agent
that binds a KIR3DL2 polypeptide and is capable of causing effector
cell-mediated lysis of a KIR3DL2-expressing cell, for at least one
administration cycle in which the agent is administered at least
twice in an amount that maintains a concentration in blood of at
least the EC.sub.60 for NK lytic capacity, between two successive
administrations of the agent, wherein the agent is an antibody that
binds specifically to a KIR3DL2 polypeptide and is administered in
an amount of 10 mg/kg body weight, and wherein the treatment
regimen comprises: an induction period in which said amount of the
antibody is administered in a plurality of successive intravenous
administrations at a frequency of one administration per week, and
a treatment period in which said amount of the antibody is
administered in a plurality of successive intravenous
administrations at a frequency of one or two administrations per
month, and wherein the agent is an antibody comprising: a heavy
chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) comprising SEQ ID NO: 2
(HCDR1), SEQ ID NO: 3 (HCDR2) and SEQ ID NO: 4 (HCDR3)
respectively, and a light chain CDR 1, 2 and 3 (LCDR1, LCDR2,
LCDR3) comprising SEQ ID NO: 5 (LCDR1), 6 (LCDR2) and 7 (LCDR3),
respectively.
43. The method of claim 42, wherein the treatment is effective in
both individuals having high blood tumor burden and in individuals
having low blood tumor burden.
44. The method of claim 42, wherein treatment is used as first-line
treatment.
45. The method of claim 42, wherein the individual has not received
bone marrow transplantation or hematopoietic stem cell
transplantation.
46. The method of claim 42, wherein the agent is administered
intravenously.
47. The method of claim 42, wherein the T cell malignancy with
tissue manifestation is a CTCL.
48. The method of claim 47, wherein the CTCL is an indolent
CTCL.
49. The method of claim 42, wherein the treatment or method is used
for the treatment or prevention of T cell proliferative disease in
individuals substantially lacking detectable KIR3DL2-expressing
malignant cells in circulation.
50. The method of claim 42, wherein the treatment or method is used
for the treatment of T cell proliferative disease in individuals
having high blood tumor burden, optionally B2 peripheral blood
involvement.
51. The method of claim 42, wherein the same administration regimen
is used for the treatment or prevention of T cell proliferative
disease in individuals having stage 2 or 3 Mycosis fungoides.
52. The method of claim 42, wherein the agent is an antibody that
binds specifically to a KIR3DL2 polypeptide and comprises an Fc
domain of human IgG isotype that binds to a human CD16
polypeptide.
53. The method of claim 42, wherein the agent is an antibody that
binds specifically to a KIR3DL2 polypeptide and is capable of
causing an increase of cell surface KIR3DL2 polypeptide available
for binding by an anti-KIR3DL2 antibody.
54. The method of claim 42, wherein the agent is an antibody that
binds specifically to a KIR3DL2 polypeptide, comprising an Fc
region derived from a human IgG1 isotype, characterized by an
EC.sub.50 in a .sup.51Cr-release assay for HuT78 tumor lysis by
PBMC from healthy volunteers, that is (a) less than or within 1-log
of the EC.sub.50 of an antibody comprising a heavy chain variable
region comprising SEQ ID NO: 31, a light chain variable region
comprising SEQ ID NO: 25 or 26, and an Fc region of human IgG1
isotype, and/or (b) less than 100 ng/ml, optionally between 1 and
100 ng/ml.
55. The method of claim 42, wherein the agent is an antibody that
binds specifically to a KIR3DL2 polypeptide is administered in an
amount of 10 mg/kg body weight, and wherein the treatment regimen
comprises: an induction period (or cycle) in which said amount of
the antibody is administered in a plurality of successive
intravenous administrations at a frequency of one administration
per week, and a treatment period in which said amount of the
antibody is administered in a plurality of successive intravenous
administrations at a frequency of two administrations per
month.
56. The method claim 42, wherein the agent is administered in an
amount of 10 mg/kg body weight, and wherein the treatment regimen
comprises: an induction period (or cycle) in which said amount of
the antibody is administered in a plurality of successive
intravenous administrations at a frequency of one administration
per week, and a treatment period in which said amount of the
antibody is administered in a plurality of successive intravenous
administrations at a frequency of one administration per month.
57. The method of claim 42, wherein the individual presents
pathogenic cells that express KIR3DL2 in skin.
58. The method of claim 42, wherein the disease is a Sezary
Sydrome, Mycosis fungoides or NK/T lymphoma.
59. The method of claim 42, characterized by the absence of a step
of detecting KIR3DL2-expressing malignant cells in blood prior to
treatment with the agent.
60. The method of claim 42, wherein the agent is an antibody
selected from the group consisting of: (a) an antibody comprising a
heavy chain variable region comprising SEQ ID NO: 31; and a light
chain variable region comprising SEQ ID NO: 25; and (b) an antibody
comprising a heavy chain variable region comprising SEQ ID NO: 31;
and a light chain variable region comprising SEQ ID NO: 26.
61. The method of claim 60, wherein the antibody is a full length
antibody comprising a heavy chain variable region (VH) fused to a
human gamma 1 constant region and a light chain variable region
(VL) fused to a human kappa constant region.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/410,880 filed 21 Oct. 2016; which is
incorporated herein by reference in its entirety; including any
drawings.
REFERENCE TO SEQUENCE LISTING
[0002] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled "KIR-7.sub._ST25", created 18 Oct. 2017, which is 53
KB in size. The information in the electronic format of the
Sequence Listing is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0003] This invention relates to the use of KIR3DL2-targeting
agents for the treatment of CTCL.
BACKGROUND OF THE INVENTION
[0004] A variety of T- and B-cell neoplasms can involve the skin,
either primarily or secondarily. Primary cutaneous lymphomas
present in the skin with no evidence of extracutaneous disease at
the time of diagnosis. Primary cutaneous lymphomas often have a
completely different clinical behavior and prognosis from
histologically similar systemic lymphomas, which may involve the
skin secondarily, and therefore require different types of
treatment. Cutaneous T-cell lymphoma (CTCL) is a group of
lymphoproliferative disorders characterized by localization of
neoplastic T lymphocytes to the skin. Collectively, CTCL is
classified as a type of non-Hodgkin lymphoma (NHL). Treatment
selection for CTCL typically depends on the extent of skin
involvement, the type of skin lesion, and whether the cancer has
spread to the lymph nodes or other internal organs. For mycosis
fungoides, treatment can be directed to the skin or to the entire
body. Sezary syndrome is generally characterized by blood
involvement and it is usually not treated with skin-directed
therapies alone. Treatments may be prescribed alone or in
combination to achieve the best long-term benefit. Skin-directed
therapies in CTCL are useful for patch and limited plaque disease
and include, inter alia, topical treatments such as
corticosteroids, retinoids, or imiquimod, topical chemotherapy,
local radiation, methotrexate, photopheresis, ultraviolet light
(phototherapy).
[0005] More recently, several antibody therapeutics targeting
proteins expressed at the surface of malignant cells have shown
promise for the treatment of CTCL.
[0006] Alemtuzumab is a humanized IgG1 kappa monoclonal antibody
specific for CD52, an antigen expressed by most T and B lymphocytes
that has been used in treatment of CTCLs and PTCLs, with a usual
protocol of administration is 30 mg three times/week. However,
while several retrospective and prospective studies have shown a
good efficacy in Sezary syndrome, alemtuzumab treatment causes a
broad depletion of NK and T cells, and leads to cytopenia and
immune depletion. Moreover, Clark et al., 2012 Sci. Trans. Med.
4(117): 117ra7 (DOI: 10.1126/scitranslmed.3003008) reported that
alemtuzumab treatment nevertheless does not fully deplete T cells
in skin. Alemtuzumab depleted all T cells in blood, but a diverse
population of skin resident T effector memory cells remained in
skin after therapy. T cell depletion with alemtuzumab required the
presence of neutrophils, a cell type frequent in blood but rare in
normal skin, suggesting that central memory T cells were depleted
because they recirculate between the blood and the skin, whereas
skin resident effector memory T cells were spared because they are
sessile and non-recirculating.
[0007] Even more recently, mogamulizumab (KW-0761) has emerged as a
treatment for relapsed/refractory CTCL and PTCL. Mogamulizumab is a
humanized anti-CCR4 monoclonal antibody that depletes CCR4 and has
been approved for use in Japan for the treatment of CCR4+ ATLL,
PTCL or CTCL. Mogalizumab, however, also leads to depletion of
healthy CCR4 expressing cells, resulting in a depletion of healthy
regulatory T (TReg) cells. Depletion of healthy TReg cells has the
consequence that it pre-excludes subsequent or combined
hematopoietic stem cell transplants due to risk of
Graft-versus-host disease, or other therapeutic agents that require
a properly functioning immune system notably for their safety.
[0008] A further immunotherapeutic agent showing promising efficacy
in treating CTCL is brentuximab vedotin, an antibody-drug conjugate
that targets the CD30 antigen and depletes CD30-expressing cells.
Adcetris.TM. (Brentuximab vedotin) is anti-CD30 monoclonal antibody
(clone cAC10) attached by a protease-cleavable linker to a
microtubule disrupting agent, monomethyl auristatin E (MMAE). Once
bound to CD30, brentuximab vedotin is internalized and MMAE is
released with the action of lysosomal enzymes on the linker,
leading to cell death. While brentuximab vedotin has shown high
efficacy with manageable toxicity, the treatment may also target
healthy CD30-expressing immune cells, notably activated B- and
T-cells. Some authors have also suggested that MMAE released in the
tumor environment may contribute to the mechanism of action by
depletion of regulatory T (TReg) cells.
[0009] Finally, KIR3DL2 has been proposed as a target for CTCL
(see, e.g., Ortonne et al. (2006) Blood 107(10):4030-4038; and PCT
publication no. WO02/50122). KIR3DL2/CD158k is a cell surface
receptor expressed on healthy circulating NK and CD8+T lymphocytes.
KIR3DI2 has also been found on the surface of CTCL cell lines and
freshly isolated CD4+PBL from SS patients, and in circulating
malignant tumor cells in CTCL patients (Nikolova et al. (2002) Leuk
Lymphoma. 43(4):741-746). Poszepczynska-Guigne J Invest Dermatol.
(2004) 122(3):820-3 report a strong positive correlation between
the percentage of CD158k+ blood lymphocytes analyzed by flow
cytometry and the percentage of atypical circulating cells (Sezary
cells) determined by cytomorphology in a large group of patients
with Sezary syndrome, and that circulating CD4+CD158k+ lymphocytes
correspond to the malignant clonal cell population. KIR3DL2 has
therefore been proposed as a marker for the evaluation of the
circulating tumoral burden and the follow-up of patients with
Sezary syndrome. PCT publication no. WO2014/044686 reports
anti-KIR3DL2 antibodies, in particular antibodies efficient in
mediating ADCC towards circulating KIR3DL2-expressing tumor cells
or tumor cell lines.
[0010] While many treatments for CTCL are available, many or most
of them have side effects that limit their use, including
antibodies that target proteins expressed at the surface of tumor
cells. Notably, both CD52 and CC4 respectively targeted by
alemtuzumab and mogamulizumab are also expressed on healthy cells
leading to side effects linked to depletion of healthy T and NK
cells, and additionally limiting the range of subsequent or
combined use of other available anti-CTCL treatments. Improved
treatments for CTCL are therefore needed.
SUMMARY OF THE INVENTION
[0011] The present disclosure provides use of depleting
anti-KIR3DL2 agents as immunomodulating agents, in an amount
effective to induce immune responses at extravascular, notably
cutaneous, sites of T cell proliferative disorders. The treatment
is, in particular, capable of depleting malignant
KIR3DL2-expressing cells without causing the depletion of healthy
KIR3DL2-expressing NK and T cells. The agents can be used in
treatment, notably, of cutaneous T cell lymphoma (CTCL)
irrespective of presence of detectable malignant cells in
circulation. The agents can advantageously be used in patients
having overt or advanced disease yet lacking detectable malignant
KIR3DL2-expressing cells in circulation. The agents can
advantageously be used in treatment of patients having indolent or
early stage T cell lymphomas (e.g., CTCL) characterized by having
low or no significant malignant cells in circulation. In one
embodiment, the agents can advantageously be used as first line
treatment in a T cell lymphoma (e.g., a CTCL). Optionally the
subject has not yet been treated with a chemotherapeutic agent.
Optionally the subject has not yet been treated with an
immunotherapeutic agent (e.g. mogamulizumab, alemtuzumab and/or
brentuximab vedotin). Optionally the subject has not yet been
treated with a bone marrow transplant or hematopoietic stem cell
transplant. Optionally the subject has progressing disease. In one
embodiment, the agents can advantageously be used to treat a
patient prior to bone marrow transplantation or hematopoietic stem
cell transplantation.
[0012] In a clinical trial of relapsed/refractory CTCL in human
patients with an ADCC-inducing anti-KIR3DL2 antibody the inventors
surprisingly observed a strong anti-tumor effect in skin lesions in
patients who received very low amounts of anti-KIR3DL2
antibody--amounts sufficient to reach but a very small number of
malignant cells in skin, and in certain dose levels, only a portion
of malignant cells in blood (if any were present).
[0013] Furthermore, a strong anti-tumor effect in skin disease
(erythroderma, plaque or patches in skin) was observed in a patient
who lacked detectable malignant KIR3DL2-expressing cells in
circulation.
[0014] Additionally, analysis from the clinical trials revealed
more generally that patients experienced a strong amelioration of
disease in skin, including restoration of normal skin structure and
strong reduction of KIR3DL2-expressing cells at cutaneous sites of
disease, upon treatment with a depleting anti-KIR3DL2 agent at
doses administered so as to provide as little as partial/minimal NK
lytic activity towards KIR3DL2+ cells in circulation, and far lower
than the amount that would provide significant occupation of
KIR3DL2 receptors on cells in skin tumors (e.g. including in
patients' with high tumor burden).
[0015] The results suggest that KIR3DL2 binding agents, when
administered so as to provide activity in circulation over a
sufficiently long period (e.g. 10 weeks or more), can be effective
in treating disease in tissues (e.g. skin) despite low amounts
therapeutic agent expected to act at the disease sites in tissues.
Moreover, the treatment advantageously can avoid depletion of
healthy KIR3DL2-expressing NK and/or T cells in circulation, unlike
that which is observed with other treatments. The KIR3DL2 binding
agents can, accordingly, be particularly advantageously used in
first-line treatment of CTCL, including but not limited to
individuals having early and/or indolent disease. In one
embodiment, the KIR3DL2 binding agent is used in combination with
(e.g., prior to) hematopoietic stem cell or bone marrow
transplantation, in both early and later stages of disease. In one
embodiment, the KIR3DL2 binding agent is used to treat first-line
CTCL In one embodiment, the KIR3DL2 binding agent is used to treat
CTCL in a subject who is not eligible (e.g. due to high blood
and/or skin tumor burden) for hematopoietic stem cell or bone
marrow transplantation. In one embodiment, the KIR3DL2 binding
agent achieves a decrease in blood and/or skin tumor burden without
depletion of healthy NK and/or T cells, and renders the subject
eligible for hematopoietic stem cell or bone marrow
transplantation.
[0016] In our studies, we used a new paradigm for determining
dosing of anti-KIR3DL2 antibodies. Rather than seek to maintain
full KIR3DL2 occupancy on the malignant cells in solid tumors in
skin which are believed to require particularly high blood
concentrations in individuals with higher tumor burden associated
with advanced disease, anti-KIR3DL2 antibodies dosed in an amount
and frequency that were lower, but sufficient to maintain
concentration in blood (e.g., blood serum) that provides for
example of a NK % lytic capacity of at least 60%, 80%, 90% or 100%,
led to remarkable anti-tumor responses while permitting a single
dosing regimen for all patients. These treatment regimens can be
used for an extended treatment period and/or several treatment
cycles, optionally preceded by an induction or loading period in
which higher administrations frequencies (or higher doses) are
employed. In certain embodiments, a single treatment regimen (e.g.
same dosage and same frequency of administration) can
advantageously be employed in both individuals having low blood
and/or cutaneous disease burden and in individuals having high
blood and/or cutaneous disease burden.
[0017] In one embodiment, a common treatment regimen (e.g. same
dosage and same frequency of administration) that does not result
in healthy NK and/or T cell depletion can advantageously be
employed in individuals irrespective of initial tumor burden and/or
disease stage, wherein the common treatment regimen is preceded by
an induction regimen or loading period in which anti-KIR3DL2
antibody is administered to an individual (e.g. an individual
having a high tumor burden) at a higher administration frequency
(optionally wherein the doses at each administration of antibody in
the common treatment regimen and the induction regimen are the
same.
[0018] In one aspect, the KIR3DL2 receptor, rather than being
clonally expanded from circulating malignant and non-malignant
CD4(+) T cell populations, may be arising from skin manifestations
of disease. Accordingly, KIR3DL2 may actually be sufficiently
expressed in skin T cell malignancies in cases where patients lack
blood involvement (lacking detectable KIR3DL2-expressing malignant
cells in circulation), including in indolent or early-stage CTCL,
to permit therapeutic targeting by an anti-KIR3DL2 binding agent.
Additionally or alternatively, tumor cells from skin lesions may
enter (or re-enter) circulation, such that lysis of a small number
of tumor cells in circulation helps to contribute to a broader
anti-tumor response in skin.
[0019] In one aspect, provided is an agent that binds a KIR3DL2
polypeptide and is capable of causing effector cell-mediated lysis
of a KIR3DL2-expressing cell, for use in treatment of a CTCL. In
one embodiment, the CTLC has tissue manifestation of disease, e.g.,
pruritis, erythroderma and/or skin tumors. In one embodiment, the
agent is used as first-line treatment. In one embodiment, provided
is a method comprising administering to an individual a T cell
malignancy (e.g. a CTCL) an agent that binds a KIR3DL2 polypeptide
and is capable of causing effector cell-mediated lysis of a
KIR3DL2-expressing cell. In one aspect, provided is an agent that
binds a KIR3DL2 polypeptide and is capable of causing effector
cell-mediated lysis of a KIR3DL2-expressing cell, for use preparing
an individual having a T cell malignancy (e.g. a CTCL) for a
subsequent bone marrow transplant or hematopoietic stem cell
transplant. In one aspect, provided is an anti-KIR3DL2 binding
agent for use in treatment of an individual having a T cell
malignancy with tissue manifestation of disease (e.g., a CTCL with
pruritis, erythroderma and/or skin tumors) but lacking detectable
KIR3DL2-expressing malignant cells in circulation. In one aspect,
provided is an anti-KIR3DL2 binding agent for use in treatment of
an individual having an indolent or early-stage CTCL.
[0020] In one aspect, an anti-KIR3DL2 binding agent can be
administered in an amount effective to achieve as little as the
EC.sub.10 for NK lytic capacity in circulation can suffice to
induce a strong anti-tumor effect in patients having CTCL with skin
manifestations of disease e.g., pruritis, erythroderma and/or skin
tumors. Doses that maintained as little as EC.sub.10 for NK lytic
capacity in circulation reduced blood tumor burden and doses that
maintained as little as the EC.sub.60, for NK lytic capacity in
circulation restored normal skin structure and reduces
KIR3DL2-expressing cells at cutaneous sites of disease.
Accordingly, in one embodiment, provided is an anti-KIR3DL2 binding
agent for use in treatment of an individual having a skin
manifestation of CTCL but no or low levels of detectable malignant
cells in circulation, wherein an anti-KIR3DL2 binding agent is
administered in an amount effective to maintain as little as the
EC.sub.10 for NK lytic capacity in circulation can suffice to
induce a strong anti-tumor effect. In another embodiment, provided
is an anti-KIR3DL2 binding agent for use in treatment of an
individual having a skin manifestation of CTCL and detectable (e.g.
higher levels of) malignant cells in circulation, wherein the
treatment comprises a plurality of administrations of an
anti-KIR3DL2 binding agent in an amount effective to maintain as
little the EC.sub.10 for NK lytic capacity.
[0021] Targeting KIR3DL2 by an anti-KIR3DL2 binding agent can
therefore be advantageous in several therapeutic settings, moreover
without requiring prior testing of KIR3DL2 expression on malignant
cells in circulation and/or skin. Furthermore, use of an
anti-KIR3DL2 binding agent does not require dosage that would
maintain full receptor occupancy on tumor cells in skin disease
(e.g. erythroderma, skin lesions) on all patients within a
population having a diverse range of tumor burden, and can benefit
from the ability of healthy immune cells (e.g. NK cells, CD8 T
cells, gamma-delta T cells) to contribute to a broader anti-tumor
response through depletion of a small number of KIR3DL2-expressing
tumor cells in circulation (e.g., below the detection limit), for
example tumor cells that enter circulation from skin lesions,
and/or through the induction of antibody-dependent cellular
phagocytosis (ADCP) in skin lesions.
[0022] In one aspect, provided are therapeutic regimens for
administration of anti-KIR3DL2 agents capable of inducing such
anti-tumor responses.
[0023] In one aspect, the therapeutic regimens disclosed herein
have the advantage of being adapted to treating individuals with T
cell lymphomas (e.g. CTCL) having detectable malignant cells in
circulation (e.g. KIR3DL2-expressing malignant cells) as well as
individuals with T cell lymphomas lacking such detectable malignant
cells in circulation. Notably, a single administration dose and/or
dosing regimen can be used to treat such patients, avoiding the
need for different treatments as a function of levels (or lack of)
malignant cells in circulation. Advantageously, the therapeutic
regimens can be used to treat patients having high tumor burden,
optionally by repeatedly and/or continuously over a period of time,
to generate a broader response against skin manifestations.
[0024] In one embodiment, an advantageous treatment comprises
administering to an individual an amount and frequency of
anti-KIR3DL2 agent that provides a concentration in blood (e.g.,
blood serum) that corresponds to at least the EC.sub.10, the
EC.sub.60, the EC.sub.80, the EC.sub.90, or the EC.sub.100 for NK
lytic capacity. Optionally, the amount and frequency of
anti-KIR3DL2 agent is less than that which would maintain the
EC.sub.90, or the EC.sub.100 for receptor saturation in skin (or
within skin lesions or tumors, e.g. advanced disease stages, high
tumor burden or erythema).
[0025] In one aspect of any embodiment herein, an advantageous
treatment comprises a plurality of administrations of an amount and
frequency of anti-KIR3DL2 agent that provides a concentration in
blood (e.g., blood serum) that is at least the EC.sub.10, the
EC.sub.60, the EC.sub.80, the EC.sub.90, or the EC.sub.100 for NK
lytic capacity. Optionally, the therapy is administered for a
duration of at least 10 weeks, 12 weeks, 3 months, 4 months or 6
months. Optionally, the administrations are separated by a period
of time between about one week and about two months. Optionally,
the anti-KIR3DL2 agent is administered at least 4, 6, 8, 10 or 20
times. Optionally, the amount and frequency of anti-KIR3DL2 agent
is less than that which would provide the EC.sub.90, or the
EC.sub.100 for receptor saturation in skin (or within skin lesions
or tumors, e.g. advanced disease stages, high tumor burden or
erythema).
[0026] In one embodiment, an advantageous treatment comprises
administering to an individual an amount of anti-KIR3DL2 agent that
maintains, between two successive administrations, a concentration
in blood (e.g., blood serum) that provides a NK % lytic capacity of
at least 10%, optionally at least 60%, optionally at least 80%,
optionally at least 90% or optionally 100%).
[0027] In one embodiment, an advantageous treatment comprises
administering to an individual an amount of anti-KIR3DL2 agent that
maintains, between two successive administrations, a concentration
in blood (e.g., blood serum) that is at least the EC.sub.10, the
EC.sub.60, the EC.sub.80, the EC.sub.90, or the EC.sub.100 for NK
lytic capacity. In one embodiment, the treatment maintains a trough
concentration of at least the concentration in blood (e.g., blood
serum) that is at least the EC.sub.10, the EC.sub.60, the
EC.sub.80, the EC.sub.90, or the EC.sub.100 for NK lytic
capacity.
[0028] Optionally, the treatment is administered for a duration of
at least 10 weeks, 12 weeks, 3 months, 4 months or 6 months.
[0029] Optionally, the administrations are separated by a period of
time between about one week and about two months.
[0030] Optionally, the treatment comprises at least 4, 6, 8, 10 or
20 successive administrations of the anti-KIR3DL2 agent.
[0031] In one embodiment, the individual is (remains eligible or is
made eligible) eligible for hematopoietic stem cell transplantation
or bone marrow transplantation prior to treatment with the
anti-KIR3DL2 agent.
[0032] In one embodiment, the individual is not eligible for
hematopoietic stem cell transplantation or bone marrow
transplantation prior to treatment with the anti-KIR3DL2 agent, and
is made eligible for hematopoietic stem cell transplantation or
bone marrow transplantation after treatment with the anti-KIR3DL2
agent.
[0033] Optionally, the treatment with the anti-KIR3DL2 agent is
prior to hematopoietic stem cell transplant or bone marrow
transplant. Optionally, the treatment is in combination with
hematopoietic stem cell transplant or bone marrow transplant. In
any embodiment, a treatment method further comprises administering
to the individual a hematopoietic stem cell transplant or bone
marrow transplant following treatment with the anti-KIR3DL2
agent.
[0034] In one embodiment, provided is an agent that binds a KIR3DL2
polypeptide and is capable of causing effector cell-mediated lysis
of a KIR3DL2-expressing cell, for use in treating a T cell
malignancy with tissue manifestations, wherein the treatment is
effective in both individuals having blood involvement and in
individuals lacking blood involvement.
[0035] In one embodiment, provided is a method comprising
administering to an individual a T cell malignancy (e.g. a CTCL) an
agent that binds a KIR3DL2 polypeptide and is capable of causing
effector cell-mediated lysis of a KIR3DL2-expressing cell, for at
least one administration cycle in which the agent is administered
at least twice in an amount that maintains a % lytic capacity in
circulation of at least 10%, optionally at least 60%, 80%, or 90%,
or optionally 100%, between two successive administrations of the
agent. E.g., the agent is administered in an amount that maintains
a concentration in blood (e.g., blood serum) of at least the
EC.sub.10, the EC.sub.60, the EC.sub.80, the EC.sub.90, or the
EC.sub.100 for NK lytic capacity. In one embodiment, the agent is
administered intravenously. In one embodiment, the agent is
administered at least 4, 6, 8 or 10 times, optionally wherein
successive administrations are separated by a period of between one
week and one month. In one embodiment, the agent that binds a
KIR3DL2 polypeptide is administered such that the concentration in
circulation that provides said % lytic capacity (or the EC value)
in circulation is maintained for at least 10 weeks, optionally at
least 3 months, optionally at least 6 months. In one embodiment,
the method is a method of treating a T cell malignancy with tissue
manifestations that is effective in both individuals having blood
involvement and in individuals lacking blood involvement. In one
embodiment, the method is a method for preparing an individual
having a T cell malignancy (e.g. a CTCL) for a subsequent bone
marrow transplant or hematopoietic stem cell transplant.
[0036] Optionally, in any embodiment, the treatment regimen is
preceded by an induction or loading period in which the
anti-KIR3DL2 binding agent is administered in a higher amount
and/or frequency. Optionally, in any embodiment, the treatment
regimen is preceded by an induction or loading period in which the
anti-KIR3DL2 binding agent is administered (e.g. in a plurality of
successive administrations) in the same amount but at a higher
frequency.
[0037] Optionally, the amount of anti-KIR3DL2 agent is less than
that which would provide the EC.sub.80, the EC.sub.90, or the
EC.sub.100 for receptor saturation in tissues (e.g. extravascular
tissues, disease tissue, skin, within skin lesions or tumors,
including e.g. advanced disease stages, high tumor burden or
erythema).
[0038] In one embodiment, the anti-KIR3DL2 binding agent is an
agent that mediates effector-cell mediated lysis of a
KIR3DL2-expressing cell (e.g. a tumor cell). Optionally, the agent
is an antigen binding polypeptide, optionally an antibody or
fragment thereof (e.g. a protein comprising a VH and/or a VL
domain), that binds a KIR3DL2 polypeptide, or an immune effector
cell that expresses such a polypeptide (e.g. a chimeric antigen
receptor immune effector cell), antibody or other compound.
Optionally, the antibody is a depleting polypeptide (antibody).
Optionally, the antibody is monospecific or a multispecific (e.g.
bispecific) antibody that directs ADCC and/or ADCP toward a
KIR3DL2-expressing cell.
[0039] In one aspect of any embodiment herein, a KIR3DL2-binding
agent comprises an anti-KIR3DL2 antibody of human IgG isotype
capable of mediating ADCC, and is administered to an individual at
least twice, in an amount effective to achieve (and/or to maintain
for a specified period of time or between two successive
administrations) a blood (serum) concentration of anti-KIR3DL2
antibody of at least 0.1 .mu.g/ml (or, optionally at least 0.4, 1,
2, 10 .mu.g/mL), optionally less than 60 or less than 100 .mu.g/mL,
optionally between 2 and 30 .mu.g/mL, optionally between 2 and 60
.mu.g/mL. In one embodiment, the antibody is administered once per
week, once every two weeks, once every month (or four weeks),
optionally between once per month and once every two months,
intravenously.
[0040] These aspects are more fully described in, and additional
aspects, features, and advantages will be apparent from, the
description of the invention provided herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1A shows while incubation at 4.degree. C. which
inhibits receptor internalization/cycling was expected to result in
an at least equal level of cell-surface KIR3DL2, staining with
antibody 2B12 (human IgG1) was higher at 37.degree. C. than at
4.degree. C. Furthermore, higher median fluorescence was observed
with increasing duration of incubation, the greatest KIR3DL2
expression was observed after 24 hours of incubation.
[0042] FIG. 1B shows the effect of antibody 2B12 (dark
line/squares) and isotype control (light line/circles) of KIR3DL2
levels. It can be seen that free receptors and 2B12-bound KIR3DL2
receptors read-outs were correlated, with similar EC.sub.50. The
rightmost panel shows that a 20 hour incubation with 2B12 increases
total KIR3DL2 receptor level at cell surface as detected by
non-competing anti-KIR3DL2 (mAb2) linked to APC.
[0043] FIG. 1C shows that incubation at 37.degree. C. with antibody
2B12 increases surface expression of KIR3DL2 (as detected by
non-competing anti-KIR3DL2 (mAb2) or by 2B12 itself+secondary Ab),
in a dose-dependent manner. This increase is already observed after
1 h at 37.degree. C., and seems to reach its maximum after 24 h.
Staining is optimal after 24 h (in terms of total staining and of
detected Ab-bound receptors).
[0044] FIG. 2 shows the PK simulation model for IPH4102, a
two-compartment model with parallel first order and saturable
elimination pathways.
[0045] FIG. 3 shows that IPH4102 did not result in depletion of NK
cells, as illustrated by the % change from baseline (day 1 of week
1) in patients' NK cells over a period of up to 50 weeks.
[0046] FIG. 4 shows that IPH4102 did not result in depletion of NK
cells, as illustrated by the number of patients' NK cells (NK cells
per .mu.l) over a period of up to 50 weeks.
DESCRIPTION OF THE INVENTION
[0047] As used herein, "a" or "an" may mean one or more. As used in
the claim(s), when used in conjunction with the word "comprising",
the words "a" or "an" may mean one or more than one. As used herein
"another" may mean at least a second or more.
[0048] Where "comprising" is used, this can optionally be replaced
by "consisting essentially of" or by "consisting of".
[0049] Whenever "treatment" is mentioned with reference to a
disease and an anti-KIR3DL2 binding agent (e.g. antibody), there is
meant: (a) method of treatment of a disease, said method comprising
the step of administering (for at least one treatment) an
anti-KIR3DL2 binding agent, (e.g., in a pharmaceutically acceptable
carrier material) to a warm-blooded animal, especially a human, in
need of such treatment, in a dose that allows for the treatment of
disease, (a therapeutically effective amount), e.g., in a dose
(amount) as specified hereinabove and herein below; (b) the use of
an anti-KIR3DL2 binding agent for the treatment of disease, or an
anti-KIR3DL2 binding agent, for use in said treatment (especially
in a human); (c) the use of an anti-KIR3DL2 binding agent for the
manufacture of a pharmaceutical preparation for the treatment of
disease, a method of using an anti-KIR3DL2 binding agent for the
manufacture of a pharmaceutical preparation for the treatment of
disease, comprising admixing an anti-KIR3DL2 binding agent with a
pharmaceutically acceptable carrier, or a pharmaceutical
preparation comprising an effective dose of an anti-KIR3DL2 binding
agent that is appropriate for the treatment of disease; or (d) any
combination of a), b), and c), in accordance with the subject
matter allowable for patenting in a country where this application
is filed.
[0050] The term "biopsy" as used herein is defined as removal of a
tissue for the purpose of examination, such as to establish
diagnosis. Examples of types of biopsies include by application of
suction, such as through a needle attached to a syringe; by
instrumental removal of a fragment of tissue; by removal with
appropriate instruments; by surgical excision, such as of the whole
lesion; and the like.
[0051] The term "antibody," as used herein, refers to polyclonal
and monoclonal antibodies. Depending on the type of constant domain
in the heavy chains, antibodies are assigned to one of five major
classes: IgA, IgD, IgE, IgG, and IgM. Several of these are further
divided into subclasses or isotypes, such as IgG1, IgG2, IgG3,
IgG4, and the like. An exemplary immunoglobulin (antibody)
structural unit comprises a tetramer. Each tetramer is composed of
two identical pairs of polypeptide chains, each pair having one
"light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The
N-terminus of each chain defines a variable region of about 100 to
110 or more amino acids that is primarily responsible for antigen
recognition. The terms variable light chain (V.sub.L) and variable
heavy chain (V.sub.H) refer to these light and heavy chains
respectively. The heavy-chain constant domains that correspond to
the different classes of immunoglobulins are termed "alpha,"
"delta," "epsilon," "gamma" and "mu," respectively. The subunit
structures and three-dimensional configurations of different
classes of immunoglobulins are well known. IgG are the exemplary
classes of antibodies employed herein because they are the most
common antibodies in the physiological situation and because they
are most easily made in a laboratory setting. In one embodiment, an
antibody is a monoclonal antibody. Provided are humanized,
chimeric, human, or otherwise-human-suitable antibodies.
"Antibodies" also includes any fragment or derivative of any of the
herein described antibodies. "Fragments" comprise a portion of the
intact antibody, generally the antigen binding site or variable
region. Examples of antibody fragments include Fab, Fab', Fab'-SH,
F (ab') 2, and Fv fragments; diabodies; any antibody fragment that
is a polypeptide having a primary structure consisting of one
uninterrupted sequence of contiguous amino acid residues (referred
to herein as a "single-chain antibody fragment" or "single chain
polypeptide"), including without limitation (1) single-chain Fv
molecules (2) single chain polypeptides containing only one light
chain variable domain, or a fragment thereof that contains the
three CDRs of the light chain variable domain, without an
associated heavy chain moiety and (3) single chain polypeptides
containing only one heavy chain variable region, or a fragment
thereof containing the three CDRs of the heavy chain variable
region, without an associated light chain moiety; and multispecific
(e.g. bispecific) antibodies formed from antibody fragments.
Included, inter alia, are a nanobody, domain antibody, single
domain antibody or a "dAb".
[0052] The term "specifically binds to" means that an antibody can
bind in a competitive binding assay to the binding partner, e.g.
KIR3DL2, as assessed using either recombinant forms of the
proteins, epitopes therein, or native proteins present on the
surface of isolated target cells. Competitive binding assays and
other methods for determining specific binding are further
described below and are well known in the art.
[0053] When an antibody is said to "compete with" a particular
monoclonal antibody, it means that the antibody competes with the
monoclonal antibody in a binding assay using either recombinant
KIR3DL2 molecules or surface expressed KIR3DL2 molecules. For
example, if a test antibody reduces the binding of 19H12, 12B11,
10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or20E9 to a
KIR3DL2 polypeptide or KIR3DL2-expressing cell in a binding assay,
the antibody is said to "compete" respectively with 19H12, 12B11,
10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or20E9.
[0054] The term "affinity", as used herein, means the strength of
the binding of an antibody to an epitope. The affinity of an
antibody is given by the dissociation constant Kd, defined as
[Ab].times.[Ag]/[Ab-Ag], where [Ab-Ag] is the molar concentration
of the antibody-antigen complex, [Ab] is the molar concentration of
the unbound antibody and [Ag] is the molar concentration of the
unbound antigen. The affinity constant K.sub.a is defined by 1/Kd.
Methods for determining the affinity of mAbs can be found in
Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan et al.,
eds., Current Protocols in Immunology, Greene Publishing Assoc. and
Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol.
92:589-601 (1983), which references are entirely incorporated
herein by reference. One standard method well known in the art for
determining the affinity of mAbs is the use of surface plasmon
resonance (SPR) screening (such as by analysis with a BIAcore.TM.
SPR analytical device).
[0055] The term "epitope" refers to an antigenic determinant, and
is the area or region on an antigen to which an antibody binds. A
protein epitope may comprise amino acid residues directly involved
in the binding as well as amino acid residues which are effectively
blocked by the specific antigen binding antibody or peptide, i.e.,
amino acid residues within the "footprint" of the antibody. It is
the simplest form or smallest structural area on a complex antigen
molecule that can combine with e.g., an antibody or a receptor.
Epitopes can be linear or conformational/structural. The term
"linear epitope" is defined as an epitope composed of amino acid
residues that are contiguous on the linear sequence of amino acids
(primary structure). The term "conformational or structural
epitope" is defined as an epitope composed of amino acid residues
that are not all contiguous and thus represent separated parts of
the linear sequence of amino acids that are brought into proximity
to one another by folding of the molecule (secondary, tertiary
and/or quaternary structures). A conformational epitope is
dependent on the 3-dimensional structure. The term `conformational`
is therefore often used interchangeably with `structural`.
[0056] The term "intracellular internalization", or
"internalization" when referring to a KIR3DL2 polypeptide and/or
antibody that binds such, refers to the molecular, biochemical and
cellular events associated with the process of translocating a
molecule from the extracellular surface of a cell to the
intracellular surface of a cell. The processes responsible for
intracellular internalization of molecules are well-known and can
involve, inter alia, the internalization of extracellular molecules
(such as hormones, antibodies, and small organic molecules);
membrane-associated molecules (such as cell-surface receptors); and
complexes of membrane-associated molecules bound to extracellular
molecules (for example, a ligand bound to a transmembrane receptor
or an antibody bound to a membrane-associated molecule). Thus,
"inducing and/or increasing intracellular internalization"
comprises events wherein intracellular internalization is initiated
and/or the rate and/or extent of intracellular internalization is
increased.
[0057] The term "depleting", "deplete" or "depletion", with respect
to KIR3DL2-expressing cells means a process, method, or composition
that can kill, eliminate, lyse or induce such killing, elimination
or lysis, so as to negatively affect the number of
KIR3DL2-expressing cells present in a sample or in a subject.
Depleting of cells can occur, for example, via ADCC.
[0058] The term "agent" is used herein to denote a chemical
compound, a mixture of chemical compounds, a biological
macromolecule, a cell, or an extract made from biological
materials. The term "therapeutic agent" refers to an agent that has
biological activity.
[0059] A "humanized" or "human" antibody refers to an antibody in
which the constant and variable framework region of one or more
human immunoglobulins is fused with the binding region, e.g. the
CDR, of an animal immunoglobulin. Such antibodies are designed to
maintain the binding specificity of the non-human antibody from
which the binding regions are derived, but to avoid an immune
reaction against the non-human antibody. Such antibodies can be
obtained from transgenic mice or other animals that have been
"engineered" to produce specific human antibodies in response to
antigenic challenge (see, e.g., Green et al. (1994) Nature Genet
7:13; Lonberg et al. (1994) Nature 368:856; Taylor et al. (1994)
Int Immun 6:579, the entire teachings of which are herein
incorporated by reference). A fully human antibody also can be
constructed by genetic or chromosomal transfection methods, as well
as phage display technology, all of which are known in the art
(see, e.g., McCafferty et al. (1990) Nature 348:552-553). Human
antibodies may also be generated by in vitro activated B cells
(see, e.g., U.S. Pat. Nos. 5,567,610 and 5,229,275, which are
incorporated in their entirety by reference).
[0060] A "chimeric antibody" is an antibody molecule in which (a)
the constant region, or a portion thereof, is altered, replaced or
exchanged so that the antigen binding site (variable region) is
linked to a constant region of a different or altered class,
effector function and/or species, or an entirely different molecule
which confers new properties to the chimeric antibody, e.g., an
enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the
variable region, or a portion thereof, is altered, replaced or
exchanged with a variable region having a different or altered
antigen specificity.
[0061] The terms "Fc domain," "Fc portion," and "Fc region" refer
to a C-terminal fragment of an antibody heavy chain, e.g., from
about amino acid (aa) 230 to about aa 450 of human .gamma. (gamma)
heavy chain or its counterpart sequence in other types of antibody
heavy chains (e.g., .alpha., .delta., .epsilon. and .mu. for human
antibodies), or a naturally occurring allotype thereof. Unless
otherwise specified, the commonly accepted Kabat amino acid
numbering for immunoglobulins is used throughout this disclosure
(see Kabat et al. (1991) Sequences of Protein of Immunological
Interest, 5th ed., United States Public Health Service, National
Institute of Health, Bethesda, Md.).
[0062] The term "NK % lytic capacity" refers to the ability of NK
cells from healthy donors to lyse tumor cells (e.g. HUT78 cells) in
an in vitro cytotoxicity assay, as measured in a .sup.51Cr release
assay, by the percentage of maximal tumor cell lysis obtained
(=Tumor cell lysis/Max tumor cell lysis at saturation.times.100).
Examples of suitable assays employing PBMC and HUT78 cells as
effector and target cells are described in the Examples herein. The
"EC.sub.10" (or "EC.sub.60", "EC.sub.80", "EC.sub.90", or
"EC.sub.100") with respect to NK lytic capacity refers to the
efficient concentration of anti-KIR3DL2 agent which produces 10%
(or 60%, 80%, 90% or 100% when referring respectively to the
"EC.sub.60", "EC.sub.80", "EC.sub.90", or "EC.sub.100") of its
maximum response or effect with respect to such NK lytic
capacity.
[0063] The term "antibody-dependent cell-mediated cytotoxicity" or
"ADCC" is a term well understood in the art, and refers to a
cell-mediated reaction in which non-specific cytotoxic cells that
express Fc receptors (FcRs) recognize bound antibody on a target
cell and subsequently cause lysis of the target cell. Non-specific
cytotoxic cells that mediate ADCC include natural killer (NK)
cells, macrophages, monocytes, neutrophils, and eosinophils.
[0064] The terms "isolated", "purified" or "biologically pure"
refer to material that is substantially or essentially free from
components which normally accompany it as found in its native
state. Purity and homogeneity are typically determined using
analytical chemistry techniques such as polyacrylamide gel
electrophoresis or high performance liquid chromatography. A
protein that is the predominant species present in a preparation is
substantially purified.
[0065] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers and non-naturally occurring
amino acid polymer.
[0066] The term "recombinant" when used with reference, e.g., to a
cell, or nucleic acid, protein, or vector, indicates that the cell,
nucleic acid, protein or vector, has been modified by the
introduction of a heterologous nucleic acid or protein or the
alteration of a native nucleic acid or protein, or that the cell is
derived from a cell so modified. Thus, for example, recombinant
cells express genes that are not found within the native
(non-recombinant) form of the cell or express native genes that are
otherwise abnormally expressed, under expressed or not expressed at
all.
[0067] The term "modification" when referring to a sequence of
amino acids (e.g., "amino acid modification"), is meant an amino
acid substitution, insertion, and/or deletion in a polypeptide
sequence. By "modification" or "amino acid modification" is meant
an amino acid substitution, insertion, and/or deletion in a
polypeptide sequence. By "amino acid substitution" or
"substitution" herein is meant the replacement of an amino acid at
a given position in a protein sequence with another amino acid. For
example, the substitution P14S refers to a variant of a parent
polypeptide, in which the proline at position 14 is replaced with
serine. A "variant" of a polypeptide refers to a polypeptide having
an amino acid sequence that is substantially identical to a
reference polypeptide, typically a native or "parent" polypeptide.
The polypeptide variant may possess one or more amino acid
substitutions, deletions, and/or insertions at certain positions
within the native amino acid sequence.
[0068] Within the context herein, the term antibody that "binds" a
polypeptide or epitope designates an antibody that binds said
determinant with specificity and/or affinity.
[0069] The term "identity" or "identical", when used in a
relationship between the sequences of two or more polypeptides,
refers to the degree of sequence relatedness between polypeptides,
as determined by the number of matches between strings of two or
more amino acid residues. "Identity" measures the percent of
identical matches between the smaller of two or more sequences with
gap alignments (if any) addressed by a particular mathematical
model or computer program (i.e., "algorithms"). Identity of related
polypeptides can be readily calculated by known methods. Such
methods include, but are not limited to, those described in
Computational Molecular Biology, Lesk, A. M., ed., Oxford
University Press, New York, 1988; Biocomputing: Informatics and
Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;
Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and
Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence
Analysis in Molecular Biology, von Heinje, G., Academic Press,
1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J.,
eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM
J. Applied Math. 48, 1073 (1988).
[0070] Methods for determining identity are designed to give the
largest match between the sequences tested. Methods of determining
identity are described in publicly available computer programs.
Computer program methods for determining identity between two
sequences include the GCG program package, including GAP (Devereux
et al., Nucl. Acid. Res. 12, 387 (1984); Genetics Computer Group,
University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA
(Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX
program is publicly available from the National Center for
Biotechnology Information (NCBI) and other sources (BLAST Manual,
Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al.,
supra). The well-known Smith Waterman algorithm may also be used to
determine identity.
Treatment of Disease
[0071] Anti-KIR3DL2 agents and the administrations regimens
disclosed herein can be used advantageously to treat
KIR3DL2-expression T cell lymohomas, notably CTCL, optionally as
first-line treatment, optionally Sezary Syndrome (SS), optionally
Mycosis fungoides (MF), optionally transformed MF, optionally
advanced stage disease (e.g. stage IIB, III, IIIA, IIIB, IVA1, IVA2
or IVB), optionally disease with peripheral blood involvement,
optionally disease with detectable or high levels of
KIR3DL2-expressing malignant cells in peripheral blood, optionally
indolent or early stage disease, optionally stage IA, IB or, IIA,
disease, optionally disease without peripheral blood involvement,
optionally disease without detectable or with low levels of
KIR3DL2-expressing malignant cells in peripheral blood. In another
aspect, provided is a method of preventing a lymphoma in an
individual having a CTCL. In another aspect, provided is a method
of reducing the risk of disease progression, reducing the risk of
lymphoma in a cell population that has undergone initiation, in an
individual having a CTCL. In another aspect, provided is a method
of preparing a subject for, or making a subject eligible for, a
hematopoietic stem cell or bone marrow transplant.
[0072] Cutaneous T-cell lymphoma (CTCL) (see the image below) is a
group of lymphoproliferative disorders characterized by
localization of neoplastic T lymphocytes to the skin. Collectively,
CTCL is classified as a type of non-Hodgkin lymphoma (NHL). The
World Health Organization-European Organization for Research and
Treatment of Cancer (WHO-EORTC) classification of CTCLs is reported
in Willemze et al. (2005) Blood 105:3768-3785. The WHO-EORTC
divides CTCL into those with indolent clinical behavior and those
with aggressive subtypes. A third category is that of precursor
hematologic neoplasms that are not T-cell lymphomas (CD4+/CD56+
hematodermic neoplasm, blastic natural killer (NK)-cell lymphoma or
B-cell derived primary cutaneous neoplasms). CTCLs which can have
indolent clinical behavior include Mycosis fungoides (MF) and its
variants, primary cutaneous CD30+ lymphoproliferative disorder
(e.g., primary cutaneous anaplastic large cell lymphoma,
lymphomatoid papulosis), subcutaneous panniculitis-like T-cell
lymphoma (provisional) and primary cutaneous CD4+
small/medium-sized pleomorphic T-cell lymphoma (provisional). CTCLs
with aggressive clinical behavior include Sezary syndrome (SS),
Adult T-cell leukemia/lymphoma, Extranodal NK/T-cell lymphoma,
nasal type, Primary cutaneous peripheral T-cell lymphoma,
unspecified, Primary cutaneous aggressive epidermotropic CD8+
T-cell lymphoma (provisional) and Cutaneous gamma/delta-positive
T-cell lymphoma (provisional).
[0073] The most common CTLCs are MF and SS. Their features are
reviewed, e.g. in Willemze et al. (2005) Blood 105:3768-3785, the
disclosure of which is incorporated herein by reference. In most
cases of MF, the diagnosis is reached owing to its clinical
features, disease history, and histomorphologic and cytomorphologic
findings. An additional diagnostic criterion to distinguish CTCL
from inflammatory dermatoses is demonstration of a dominant T-cell
clone in skin biopsy specimens by a molecular assay (e.g., Southern
blot, polymerase chain reaction (PCR)). Genetic testing may also be
considered. Classic mycosis fungoides is divided into three stages:
(1) Patch (atrophic or non-atrophic): Nonspecific dermatitis,
patches on lower trunk and buttocks; minimal/absent pruritus; (2)
Plaque: Intensely pruritic plaques, lymphadenopathy and (3) Tumor:
Prone to ulceration. Sezary syndrome is defined by erythroderma and
leukemia. Signs and symptoms include edematous skin,
lymphadenopathy, palmar and/or plantar hyperkeratosis, alopecia,
nail dystrophy, ectropion and hepatosplenomegaly. For a diagnosis
of Sezary syndrome, criteria typically include absolute Sezary cell
count, immunophenotypic abnormalities, loss of T-cell antigens
and/or a T-cell clone in the peripheral blood shown by molecular or
cytogenetic methods.
[0074] CTCL stages include I, II, III and IV, according to TNM
classification, and as appropriate, peripheral blood involvement.
Peripheral blood involvement with mycosis fungoides or Sezary
syndrome (MF/SS) cells is correlated with more advanced skin stage,
lymph node and visceral involvement, and shortened survival. MF and
SS have a formal staging system proposed by the International
Society for Cutaneous Lymphomas (ISCL) and the European
Organization of Research and Treatment of Cancer (EORTC). See,
Olsen et al., (2007) Blood. 110(6):1713-1722; and Agar et al.
(2010) J. Clin. Oncol. 28(31):4730-4739, the disclosures of which
are incorporated herein by reference. In SS and MF, stage IV (IVA1,
IVA2 and IVB) can include B2 peripheral blood involvement (high
blood-tumor burden: 1,000/.mu.L Sezary cells with positive clone).
SS and MF stages include I (IA and IB), II (IIA and IIB), Ill (III,
IIIA and IIIB) and IV (IVA1, IVA2 and IVB).
[0075] A treatment designed to effectuate effector cell-mediated
lysis of malignant KIR3DL2+ cells in circulation may, through lysis
of as little as a small number of circulating cells (compared to
the malignant cells overall, e.g. in extracellular manifestations
of disease), through activation of a limited number of effector
cells in circulation, and/or through the induction of
antibody-dependent cellular phagocytosis (ADCP) toward a limited
number of malignant cells in skin lesions, of generating an
anti-tumor response that leads to elimination of malignant cells
and generally disease improvement in skin lesions. Responses were
obtained through repeated dosing of a KIR3DL2-binding antibody, via
different treatment regimens designed to maintain a particular
amount of anti-KIR3DL2 binding agent in circulation effective to
provide the EC.sub.10 for NK lytic capacity. While the
administration regimens ameliorated skin lesions in patients having
low or no blood involvement, the repeated administration regimens
also ameliorated skin lesions in patients having high blood
involvement.
[0076] Upon diagnosis of a CTCL, a subject can be treated with an
anti-KIR3DL2 binding agent. The treatment, irrespective of tumor
burden, can be used to eliminate malignant cells while maintaining
healthy NK and T cells. This treatment is consequently compatible
with subsequent BMT or HSCT. In one embodiment, the disclosure
provides use of anti-KIR3DL2 binding agent as a first line therapy
to treat a subject having a CTCL. The term "first-line therapy" as
used herein refers to the first type of systemic drug therapy given
for the treatment of CTCL. This can be a single-agent, combination
or maintenance therapy offered initially following diagnosis.
[0077] In one aspect, provided is method for treating a CTCL in an
individual, the method comprising administering to the individual
an anti-KIR3DL2 binding agent without a step of prior testing of
KIR3DL2 expression on malignant cells from a blood sample.
[0078] In one aspect, provided is method for treating a CTCL in an
individual, the method comprising administering to the individual
an anti-KIR3DL2 binding agent without a step of prior testing of
KIR3DL2 expression on malignant cells from a skin biopsy.
[0079] In one aspect, provided is method for treating a CTCL in an
individual lacking detectable KIR3DL2-expressing malignant cells
(e.g. KIR3DL2-expressing Sezary cells) in circulation, the method
comprising administering to the individual an anti-KIR3DL2 binding
agent. In one aspect, provided is method for treating a CTCL in an
individual having low levels of detectable KIR3DL2-expressing
malignant cells (e.g. KIR3DL2-expressing Sezary cells) in
circulation, the method comprising administering to the individual
an anti-KIR3DL2 binding agent.
[0080] In one aspect, provided is method for treating a CTCL in an
individual having less than B2 stage peripheral blood involvement,
comprising administering to the individual an anti-KIR3DL2 binding
agent. Optionally the individual has blood-tumor burden of less
than 1,000/.mu.L Sezary cells, and/or without positive clone.
[0081] In one aspect, provided is method for treating an indolent
CTCL, comprising administering to the individual an anti-KIR3DL2
binding agent.
[0082] In embodiment, provided is a method of treating CTCL, the
method comprising: (a) assessing the stage and/or disease prognosis
of CTCL in an individual having a CTCL; and (b) if the individual
has a stage II or III disease, optionally IIB, IIIA or IIIB,
administering to the individual an anti-KIR3DL2 binding agent.
[0083] In one aspect, provided is method for treating a stage I
CTLC, comprising administering to the individual an anti-KIR3DL2
binding agent. In one aspect, provided is method for treating a
stage II CTLC, comprising administering to the individual an
anti-KIR3DL2 binding agent. In one aspect, provided is method for
treating a stage III CTLC, comprising administering to the
individual an anti-KIR3DL2 binding agent.
[0084] In one aspect, provided is method for treating a CTCL in an
individual having less than B2 stage peripheral blood involvement,
comprising administering to the individual an anti-KIR3DL2 binding
agent. Optionally, the individual lacks or has low blood tumor
burden, optionally wherein the individual has BO (absence of
significant blood involvement, e.g. 5% of peripheral blood
lymphocytes are atypical (Sezary) cells) or B1 (low blood-tumor
burden, e.g. >5% of peripheral blood lymphocytes are atypical
(Sezary) cells, does not meet the criteria of B2) peripheral blood
involvement.
[0085] In one aspect of any of the above, an individual having a
CTCL has a skin lesion, optionally significant or advanced skin
disease, optionally T2 (patches, papules, or plaques covering 10%
of the skin surface, optionally further T2a (patch only) or T2b
(plaque.+-.patch), T3 (at least one tumor (1 cm diameter) or T4
stage skin involvement erythema covering E30% of body surface
area). In one embodiment, the individual has multiple and/or high
skin tumor burden. In one embodiment, the individual has one or
multiple skin tumors greater than 1 cm diameter.
[0086] In one aspect of any of the above, an individual having a
CTCL has patches, papules, or plaques covering 10% of the skin
surface. In one aspect of any of the above, an individual having a
CTCL has least one tumor >1 cm diameter. In one aspect of any of
the above, an individual having a CTCL has erythema covering E30%
of body surface area.
[0087] In embodiment, provided is a method of treating CTCL, the
method comprising: (a) assessing the stage and/or disease prognosis
of CTCL in an individual having a CTCL; and (b) if the individual
has a stage IV disease, optionally IVA1 or IVA2 disease, optionally
IVB disease, administering to the individual an anti-KIR3DL2
binding agent.
[0088] It will be appreciated that a treatment method of the
disclosure may or may not involve a step of characterizing the CTCL
prior to treatment. In embodiment, provided is a method of treating
CTCL, the method comprising: (a) determining whether an individual
has a CTCL comprising skin manifestation of CTCL (e.g.
erythroderma, skin lesions or tumors), optionally a skin
manifestation characterized by pathogenic KIR3DL2-expressing cells;
and (b) if the individual has skin manifestations of CTCL,
optionally a skin manifestation characterized by pathogenic
KIR3DL2-expressing cells, administering to the individual an
anti-KIR3DL2 binding agent. Optionally, the step of determining
whether an individual has a CTCL comprising skin manifestation of
CTCL comprises characterizing the extent of skin lesions;
optionally, if the individual has plaques and/or ulcerating tumors,
administering to the individual an anti-KIR3DL2 binding agent.
Optionally, the step of determining whether an individual has a
CTCL comprising skin manifestation of CTCL comprises characterizing
the stage of skin disease, e.g. T2, T3, or T4 disease. Optionally,
if the individual has advanced skin manifestations of CTCL, e.g.,
T2, T3, or T4 disease, administering to the individual an
anti-KIR3DL2 binding agent.
[0089] In embodiment, provided is a method of treating CTCL, the
method comprising: (a) assessing the stage and/or disease prognosis
of CTCL in an individual having a CTCL; and (b) if the individual
has an indolent CTCL, administering to the individual an
anti-KIR3DL2 binding agent.
[0090] It will be appreciated that a treatment method of the
disclosure may or may not involve a step of characterizing tumor
cells for KIR3DL2-expression prior to treatment. In embodiment,
provided is a method of treating CTCL, the method comprising: (a)
determining whether skin manifestation of CTCL in an individual
comprise pathogenic KIR3DL2-expressing cells (e.g.
KIR3DL2-expressing cells in erythroderma and/or skin lesions); and
(b) if the individual has skin manifestations of CTCL comprising
pathogenic KIR3DL2-expressing cells, administering to the
individual an anti-KIR3DL2 binding agent.
[0091] It will be appreciated that a treatment method of the
disclosure may or may not involve a step of characterizing tumor
cells for KIR3DL2-expression prior to treatment. In embodiment,
provided is a method of treating CTCL, the method comprising: (a)
obtaining a blood sample or biopsy (e.g. skin biopsy) from an
individual and determining whether the sample comprises pathogenic
KIR3DL2-expressing cells (KIR3DL2+ tumor cells); and (b) if the
sample comprises pathogenic KIR3DL2-expressing cells, administering
to the individual an anti-KIR3DL2 binding agent. In another
embodiment, provided is a method of treating CTCL, the method
comprising: (a) obtaining a blood sample from an individual and
determining whether the sample comprises pathogenic
KIR3DL2-expressing cells (KIR3DL2+ tumor cells); and (b) if the
sample does not comprise detectable pathogenic KIR3DL2-expressing
cells, administering to the individual an anti-KIR3DL2 binding
agent.
[0092] Optionally the method further comprises determining whether
disease cells also express other markers of abnormal lymphocytes at
their surface, for example determining whether cells are CD4, CD30,
CD3, CD8 cells.
[0093] In some aspects, an anti-KIR3DL2 binding agent can be
administered to an individual who is in remission following
treatment for CTCL or who has having otherwise responded positively
to a first (one or more) anti-CTCL therapy (i.e. with a
non-KIR3DL2), optionally having a low blood-tumor burden.
[0094] In some aspects, the anti-KIR3DL2 binding agent can be
administered to an individual having a poor disease prognosis
and/or who has relapsed, is resistant or is not responsive to
therapy with a first (one or more) therapeutic agent.
[0095] Provided herein are treatment regimens that can be used for
treatment of both CTCL with low or no blood tumor burden (and/or
without detectable KIR3DL2+ tumor cells), or in CTCL with blood
involvement or with high blood tumor burden (and/or with detectable
KIR3DL2+ tumor cells). It will be appreciated, however, that the
regimens can also be used for one or the other subgroup
separately.
[0096] In one embodiment, optionally an anti-KIR3DL2 binding agent
is administered in low doses, optionally in an amount that is
designed to be below that would maintain full receptor occupancy on
tumor cells in skin disease (e.g. erythroderma, skin lesions or
tumors) in all patients, including those with high blood and skin
tumor burden; such a dose may have the advantageous properties of
giving rise to a broader anti-tumor response through depletion of a
small number of KIR3DL2-expressing tumor cells in circulation
(e.g., below the detection limit), for example tumor cells that
enter circulation from skin tumors, and/or of giving rise to a
broader anti-tumor response through the induction of
antibody-dependent cellular phagocytosis (ADCP) in skin tumors. In
one embodiment, the doses of anti-KIR3DL2 binding agent are
repeated, in particular, the treatment comprises a first, second,
and optionally further administration of an anti-KIR3DL2 binding
agent. Optionally, the schedule of administration (e.g. the time
between two successive administrations) and dose are chosen so as
to maintain a trough level of anti-KIR3DL2 binding agent that
provides a concentration in blood (e.g., blood serum) that
corresponds to at least the EC.sub.10, the EC.sub.60, the
EC.sub.80, the EC.sub.90, or the EC.sub.100 for NK lytic
capacity.
[0097] In some embodiments, an anti-KIR3DL2 agent is administered
in an dose and frequency so as to obtain and/or maintain a
concentration in blood (e.g., blood serum) that corresponds to at
least the EC.sub.10 for NK lytic capacity, optionally at about or
at least about, the EC.sub.60, the EC.sub.80, the EC.sub.90, or the
EC.sub.100.
[0098] Optionally, in any embodiment herein, the amount and
frequency of anti-KIR3DL2 agent is less than that which provides a
concentration in blood (e.g., blood serum) that corresponds to that
provided by 25 mg/kg, 20 mg/kg, 15 mg/kg, 10 mg/kg, 7.5 mg/kg or 6
mg/kg body weight, when administered weekly. Optionally, in any
embodiment herein, an amount or dose of anti-KIR3DL2 agent
administered can be specified to be less than 25 mg/kg, 20 mg/kg or
15 mg/kg body weight.
[0099] In another embodiment of any aspect herein, a treatment
method is a method of reducing or preventing progression,
maintaining remission, or preventing relapse of CTCL or preventing
relapse of lymphoma in CTCL. In another embodiment of any aspect
herein, a treatment method is a method of increasing the likelihood
of survival over a relevant period. In another embodiment of any
aspect herein, a treatment method is a method of improving the
quality of life in an individual. In another embodiment of any
aspect herein, a treatment method is a method of reducing the
number of circulating lymphoma cells (e.g. Sezary cells) in an
individual. In another embodiment of any aspect herein, a treatment
method is a method of reducing blood tumor burden in an
individual.
[0100] In another embodiment of any aspect herein, a treatment
method is a method of preventing progression of an early stage CTCL
to a more advanced stage of CTCL. In another embodiment of any
aspect herein, a treatment method is a method of preventing
progression of an early stage I, II or III CTCL to a stage IV CTCL.
In another embodiment of any aspect herein, a treatment method is a
method of preventing progression of a CTCL without blood tumors or
with low blood tumor burden to a CTCL with blood tumor burden or
high blood tumor burden. In another embodiment of any aspect
herein, a treatment method is a method of preventing progression of
a CTCL with BO or B1 blood tumor burden to a CTCL with B2 blood
tumor burden.
[0101] Delivering an anti-KIR3DL2 agent (e.g. an antibody or
fragment thereof) to a subject (either by direct administration as
an isolated proteinaceous binding agent, by administration as a
cell such as a CAR effector cell that expresses an anti-KIR3DL2
binding protein at its surface, or by expression of a proteinaceous
binding agent from a nucleic acid therein, such as from a pox viral
gene transfer vector comprising anti-KIR3DL2 antibody-encoding
nucleic acid sequence(s)) and practicing the other methods herein
can be used to reduce, treat, prevent, or otherwise ameliorate any
suitable aspect of CTCL as disclosed herein. The treatments can be
administered parenterally, e.g. intravenously, and can be
particularly useful in the reduction and/or amelioration of
proliferation of abnormal lymphocytes in skin lesions, restoration
of normal skin structure and strong reduction of pathogenic T
cells.
[0102] In certain embodiment herein, a KIR3DL2-binding agent is
administered to an individual for at least one administration cycle
in which the agent is administered at least twice in an amount that
provides a concentration in blood (e.g., blood serum) that
corresponds to at least the EC.sub.10, the EC.sub.60, the
EC.sub.80, the EC.sub.90, or the EC.sub.100 for NK lytic capacity.
Optionally, the agent is administered in an amount effective to
achieve, and/or to maintain between two successive administrations
of the agent, a concentration that provides a concentration in
blood (e.g., blood serum) that corresponds to at least the
EC.sub.10, the EC.sub.60, the EC.sub.80, the EC.sub.90, or the
EC.sub.100 for NK lytic capacity. Optionally, the administration
cycle comprises at least a first and second (and optionally a
3.sup.rd, 4.sup.th, 5.sup.th, 6.sup.th, 7.sup.th and/or 8.sup.th or
further) administration of the agent. Optionally, the agent is
administered intravenously. Optionally the treatment has a duration
of at least 10 weeks, 2 months, 3 months, 4 months or 6 months.
[0103] In one aspect of any embodiment herein, a KIR3DL2-binding
agent is administered to an individual in an amount that provides
(e.g. achieves and/or maintains) a concentration in blood (e.g.,
blood serum) that is at least the EC.sub.10, the EC.sub.60, the
EC.sub.80, the EC.sub.90, or the EC.sub.100 for NK lytic
capacity.
[0104] In one aspect of any embodiment herein, a KIR3DL2-binding
agent is administered to an individual in an amount that maintains
for at least 1 week, at least 2 weeks, at least 1 month or at least
2 months, a concentration in blood (e.g., blood serum) that
corresponds to between the EC.sub.10 and the EC.sub.70, between
EC.sub.10 and the EC.sub.80, between EC.sub.10 and the EC.sub.90,
or between EC.sub.60 and the EC.sub.100 for NK lytic capacity.
[0105] In one aspect of any embodiment herein, a KIR3DL2-binding
agent is administered to an individual in an amount that is less
than the amount that maintains substantially full KIR3DL2 occupancy
on CTCL cells in skin (e.g. in skin lesions or tumors) between two
successive administrations of the agent. In one aspect of any
embodiment herein, a KIR3DL2-binding agent is administered to an
individual in an amount that is less than the amount that maintains
between two successive administrations of the agent a concentration
in skin (e.g. in skin lesions or tumors) that corresponds to at
least the EC.sub.50, the EC.sub.70, the EC.sub.80, the EC.sub.90,
or the EC.sub.100 for NK lytic capacity. In one aspect of any
embodiment herein, an anti-KIR3DL2 antibody of human IgG isotype,
optionally an antibody characterized by EC.sub.50 in
.sup.51Cr-release assay for HuT78 tumor lysis by PBMC from healthy
volunteers, of less than 100 ng/ml, optionally between 1 and 100
ng/ml, optionally between 1 and 50 ng/ml, optionally about 50
ng/ml, and is administered to an individual in an amount (e.g.
administered weekly) that is less than 15, 20 or 30 mg/kg body
weight.
[0106] In one aspect of any embodiment herein, a KIR3DL2-binding
agent comprises an anti-KIR3DL2 antibody of human IgG isotype,
optionally an antibody characterized by EC.sub.50 in
.sup.51Cr-release assay for HuT78 tumor lysis by PBMC from healthy
volunteers, of less than 100 ng/ml, optionally between 1 and 100
ng/ml, optionally between 1 and 50 ng/ml, optionally about 50
ng/ml, and is administered to an individual in an amount effective
to achieve (and/or to maintain for a specified period of time or
between two successive administrations) a blood (serum)
concentration of anti-KIR3DL2 antibody of at least 0.1 .mu.g/ml
(or, optionally at least 0.4, 1, 2 or 10 .mu.g/mL). In one
embodiment, the antibody is administered once per week, once every
two weeks, once every three weeks, once per month, optionally
between once per month and once every two months intravenously. In
one embodiment, the antibody is administered to an individual in an
amount effective to maintain between two successive
administrations) a blood (serum) concentration of anti-KIR3DL2
antibody of at least 7 ng/ml (e.g. 10% lytic capacity), optionally
at least 70 ng/ml (e.g. 60% lytic capacity), optionally at least
0.4 .mu.g/ml (e.g. 80% lytic capacity), optionally at least 2
.mu.g/ml (e.g. 90% lytic capacity), optionally at least 10 .mu.g/ml
(e.g. 100% lytic capacity), or optionally at least 20 .mu.g/ml, 50
.mu.g/ml or 80 .mu.g/ml.
[0107] In one aspect of any embodiment herein, a KIR3DL2-binding
agent comprises an anti-KIR3DL2 antibody of human IgG isotype and
is administered to an individual in an amount effective to maintain
for a specified period of time or between two successive
administrations) a minimum (trough) blood (serum) concentration of
anti-KIR3DL2 antibody of between 0.1-0.5 .mu.g/ml, optionally
between 0.4-2 .mu.g/ml, optionally between 2-7 .mu.g/ml, optionally
between 2-10 .mu.g/ml, optionally between 2-50 .mu.g/ml, optionally
between 10 and 20 .mu.g/ml, optionally between 20 and 50 .mu.g/ml,
or optionally between 50 and 100 .mu.g/ml. In one embodiment, the
antibody is administered once per month, optionally between once
per month and once every two months intravenously.
[0108] The amount of antibody required to achieve a particular
blood concentration can be determined based on the properties of
the particular antibody. In one aspect of any embodiment herein, a
KIR3DL2-binding agent comprises an anti-KIR3DL2 antibody of human
IgG isotype, optionally an antibody characterized by EC.sub.50 in
.sup.51Cr-release assay for HuT78 tumor lysis by PBMC from healthy
volunteers comparable to that of an anti-KIR3DL2 antibody disclosed
herein (e.g., having an EC.sub.50 that is lower or within 1-log or
0.5-log of the EC.sub.50 of that of an antibody disclosed herein
(e.g. a 2B12 antibody), optionally an EC.sub.50 of less than 100
ng/ml, optionally between 1 and 100 ng/ml, optionally between 1 and
50 ng/ml, optionally about 50 ng/ml. In one aspect of any
embodiment herein, the KIR3DL2-binding agent is administered to an
individual intravenously at a dose of between 0.1-0.75 mg/kg,
optionally 0.2-0.75 mg/kg, optionally 0.4-1 mg/kg, optionally
0.75-1.5 mg/kg, optionally about 0.01 mg/kg, optionally about 0.2
mg/kg, optionally about 0.75 mg/kg, or optionally about 1.5 mg/kg
body weight. In one embodiment, the antibody is administered once
per month, optionally between once per month and once every two
months intravenously, at a dose of between 0.1-0.75 mg/kg,
optionally 0.2-0.75 mg/kg, optionally 0.4-1 mg/kg, optionally
0.75-1.5 mg/kg, optionally about 0.01 mg/kg, optionally about 0.2
mg/kg, optionally about 0.75 mg/kg, optionally about 1 mg/kg, or
optionally about 1.5 mg/kg body weight.
[0109] In one aspect of any embodiment herein, the KIR3DL2-binding
agent is administered to an individual intravenously at a dose of
between 0.75 and 10 mg/kg, optionally between 0.75-1.5 mg/kg,
optionally between 1-3 mg/kg, optionally 1.5-3 mg/kg, optionally
3-6 mg/kg, optionally 6-10 mg/kg, optionally about 1 mg/kg,
optionally about 1.5 mg/kg, optionally about 3 mg/kg, optionally
about 6 mg/kg, or optionally about 10 mg/kg body weight. In one
embodiment, the antibody is administered once per week (optionally
once per 2 weeks), or between once per week and once per month (or
every 4 weeks), intravenously, at a dose of between 1-3 mg/kg,
optionally 1.5-3 mg/kg, optionally 3-6 mg/kg, optionally 1.5-8
mg/kg, optionally 6-10 mg/kg, optionally about 1 mg/kg, optionally
about 1.5 mg/kg, optionally about 3 mg/kg, optionally about 4
mg/kg, optionally about 6 mg/kg, optionally less than 10 mg/kg body
weight, or optionally about 10 mg/kg body weight.
[0110] In one aspect of any embodiment herein, the KIR3DL2-binding
agent is administered to an individual intravenously at a dose of
between 1-3 mg/kg, optionally 1.5-3 mg/kg, optionally 3-6 mg/kg,
optionally 6-10 mg/kg, optionally about 1 mg/kg, optionally about
1.5 mg/kg, optionally about 3 mg/kg, optionally about 6 mg/kg, or
optionally about 10 mg/kg body weight. In one embodiment, the
antibody is administered between once per month and once every two
months intravenously at a dose of between 1-3 mg/kg, optionally
1.5-3 mg/kg, optionally 3-6 mg/kg, optionally 6-10 mg/kg,
optionally about 1 mg/kg, optionally about 1.5 mg/kg, optionally
about 3 mg/kg, optionally about 6 mg/kg, optionally less than 10
mg/kg body weight, or optionally about 10 mg/kg body weight.
[0111] In any embodiment, a mg/kg dose can be expressed as a fixed
dose equivalent of any of the doses using for example a body weight
of 65 kg or 75 kg, e.g., a fixed dose equivalent of 10 mg/kg can be
defined as 750 mg.
[0112] In one embodiment, provided is a method of treating a CTCL
in an individual (e.g. an individual having a CTCL as described
herein), the method comprising administering to the individual a
KIR3DL2-binding agent for at least one administration cycle in
which the agent is administered at least twice in an amount that
provides a concentration in blood (e.g., blood serum) that is at
least the EC.sub.10, the EC.sub.60, the EC.sub.80, the EC.sub.90,
or the EC.sub.100 for NK lytic capacity. Optionally, the agent is
administered in an amount effective to achieve, and/or to maintain
between two successive administrations of the agent, a
concentration that provides a concentration in blood (e.g., blood
serum) that is at least the EC.sub.10, the EC.sub.60, the
EC.sub.80, the EC.sub.90, or the EC.sub.100 for NK lytic capacity.
Optionally, the administration cycle comprises at least a first and
second (and optionally a 3.sup.rd, 4.sup.th, 5.sup.th, 6.sup.th,
7.sup.th and/or 8.sup.th or further) administration of the agent.
Optionally, the agent is administered intravenously.
[0113] Optionally, treatment regimens can comprise an induction
cycle. For example, a regimen for an anti-KIR3DL2 antibody of human
IgG isotype, optionally an antibody characterized by EC.sub.50 in
.sup.51Cr-release assay for HuT78 tumor lysis by PBMC from healthy
volunteers comparable to that of an anti-KIR3DL2 antibody disclosed
herein (e.g., having an EC.sub.50 that is lower or within 1-log or
0.5-log of the EC.sub.50 of that of a 2B12 antibody disclosed
herein; optionally, an antibody characterized by EC.sub.50 in
.sup.51Cr-release assay for HuT78 tumor lysis by PBMC from healthy
volunteers of less than 100 ng/ml, optionally between 1 and 100
ng/ml, optionally between 1 and 50 ng/ml, optionally about 50
ng/ml, can comprise: [0114] (a) an induction treatment cycle
comprising a plurality of administrations of the antibody, wherein
the antibody is administered to an individual intravenously in an
amount effective to maintain for a specified period of time or
between two successive administrations) a minimum (trough) blood
(serum) concentration of anti-KIR3DL2 antibody of at least 50, 80,
90, 100, 200 or 300 .mu.g/ml, optionally between 50 and 200
.mu.g/ml, optionally between 50 and 100 .mu.g/ml, followed by:
[0115] (b) a treatment cycle comprising a plurality of
administrations of the antibody, wherein the antibody is
administered to the individual intravenously in an amount effective
to maintain for a specified period of time or between two
successive administrations) a minimum (trough) blood (serum)
concentration of anti-KIR3DL2 antibody of less than 100 .mu.g/ml,
optionally less than 50 .mu.g/ml, optionally at least 0.1-0.5
.mu.g/ml, optionally between 0.4-2 .mu.g/ml, optionally between 2-7
.mu.g/ml, optionally between 2-10 .mu.g/ml, optionally between 2-50
.mu.g/ml, optionally between 10 and 20 .mu.g/ml, optionally between
20 and 50 .mu.g/ml. In one embodiment, the amount administered in
the treatment cycle (b) is the same amount administered in the
treatment cycle (a) but at lesser frequency of administration.
[0116] In another exemplary treatment regimen for an anti-KIR3DL2
antibody of human IgG isotype, the treatment comprises: [0117] (a)
an induction treatment cycle comprising a plurality (e.g. at least
2, 4, 8, or 10) of administrations of the antibody, wherein the
antibody is administered to an individual intravenously at a dose
of between 1-20 mg/kg, optionally 1-10 mg/kg, optionally 1-3 mg/kg,
optionally 1.5-3 mg/kg, optionally 3-6 mg/kg, optionally 6-10
mg/kg, optionally about 1 mg/kg, optionally about 1.5 mg/kg,
optionally about 3 mg/kg, optionally about 6 mg/kg, or optionally
about 10 mg/kg body weight at a frequency of about 2, 3 or 4 times
per month, optionally once per week, followed by: [0118] (b) a
treatment cycle (e.g. maintenance cycle) comprising a plurality of
(e.g. at least 2, 4, 8, or 10) administrations of the antibody,
wherein the antibody is administered to the individual
intravenously at a dose of between 1-20 mg/kg, optionally 1-10
mg/kg, optionally 1-3 mg/kg, optionally 1.5-3 mg/kg, optionally 3-6
mg/kg, optionally 6-10 mg/kg, optionally about 1 mg/kg, optionally
about 1.5 mg/kg, optionally about 3 mg/kg, optionally about 6
mg/kg, or optionally about 10 mg/kg body weight at a frequency of
about once every 1-3 months, optionally about once per month. In
one embodiment, the dose (e.g. 1, 1.5, 3, 6 or 10 mg/kg)
administered in the treatment cycle (b) is the same dose
administered in the treatment cycle (a).
[0119] In one embodiment, a common treatment regimen (e.g. same
dosage and same frequency of administration) that does not result
in healthy NK and/or T cell depletion can advantageously be
employed in individuals irrespective of initial tumor burden and/or
disease stage, wherein the common treatment regimen is preceded by
an induction regimen or loading period in which anti-KIR3DL2
antibody is administered to an individual (e.g. an individual
having a high tumor burden) at a higher administration frequency
(optionally wherein the doses at each administration of antibody in
the common treatment regimen and the induction regimen are the
same.
[0120] In one embodiment, provided is a method of treating an
individual having a cancer (e.g. a solid tumor), the method
comprising administering to the individual an anti-KIR3DL2 antibody
of human IgG isotype, for at least one administration cycle,
wherein the method comprises: [0121] a. an induction period (or
cycle) in which antibody is administered in a plurality of
successive intravenous administrations, in a dose of between 0.75
and 10 mg/kg body weight, at a frequency of 2-4 administrations per
month (e.g. one administration per week), and [0122] b. a
maintenance period (or cycle) in which the antibody is administered
in a plurality of successive intravenous administrations, in a dose
of between 0.75 and 10 mg/kg body weight, at a frequency of one
administration every one or two months (e.g. one administration per
week). In one embodiment, the first administration within the
maintenance period occurs no more than one month after the last
dose of the loading period. In one embodiment, the dose at each
administration in the induction cycle of (a) and at each
administration in the maintenance period of (b) is the same (e.g.
0.75 mg/kg, 1.5 mg/kg, 6 mg/kg or 10 mg/kg are used both in the
induction cycle and the maintenance period),
[0123] In one embodiment of any of the treatments comprising an
induction cycle or period, the induction period comprises 4, 5, 6,
7, 8 or more administrations. In one embodiment, the following
(e.g. maintenance) period comprises at least 2, 3, 4, 5, 6, 7 or 8
administrations. In one embodiment, the antibody is administered at
the same dose in both the loading period and the maintenance
period. In one embodiment, the induction period and the maintenance
period each comprise administering the antibody in a dose of 0.75
mg/kg body weight. In one embodiment, the induction period and the
maintenance period each comprise administering the antibody in a
dose of 1.5 mg/kg body weight. In one embodiment, the induction
period and the maintenance period each comprise administering the
antibody in a dose of 3 mg/kg body weight. In one embodiment, the
induction period and the maintenance period each comprise
administering the antibody in a dose of 6 mg/kg body weight. In one
embodiment, the induction period and the maintenance period each
comprise administering the antibody in a dose of 10 mg/kg body
weight. In one embodiment, the antibody comprises a heavy chain
variable region comprising an amino acid sequence of SEQ ID NO: 31;
and a light chain variable region comprising an amino acid sequence
of SEQ ID NO: 25. In one embodiment, the antibody comprises a heavy
chain variable region comprising an amino acid sequence of SEQ ID
NO: 31; and a light chain variable region comprising an amino acid
sequence of SEQ ID NO: 26.
[0124] Optionally, the treatment of the disclosure does not cause
depletion of KIR3DL2-expressing healthy immune cells (e.g. NK
cells, CD8 T cells, gammadelta T cells). Optionally, the amount of
agent is an amount effective to give rise to a broader anti-tumor
response through depletion of KIR3DL2-expressing tumor cells in
circulation (e.g., where such cells are few or below the detection
limit, e.g. in an individual having low/no blood tumor burden), for
example tumor cells that enter circulation from skin lesions.
Optionally, the amount of agent is an amount effective to give rise
to a broader anti-tumor response through the induction of
antibody-dependent cellular phagocytosis (ADCP) in skin
lesions.
[0125] In one aspect, any of the treatment regimens herein are used
for the treatment of individuals having CTCL, wherein the treatment
regimen (e.g. the same dose of anti-KIR3DL2 agent and frequency of
administration) is used in individuals having SS and in individuals
having MF.
[0126] In one aspect, any of the treatment regimens herein are used
for the treatment of individuals having CTCL, wherein the treatment
regimen (e.g. the same dose of anti-KIR3DL2 agent and frequency of
administration) is used in individuals having indolent disease and
in individuals having aggressive disease.
[0127] In one aspect, any of the treatment regimens herein are used
for the treatment of individuals having CTCL, wherein the treatment
regimen (e.g. the same dose of anti-KIR3DL2 agent and frequency of
administration) is used in individuals lacking detectable
KIR3DL2-expressing malignant cells (e.g. KIR3DL2-expressing Sezary
cells) in circulation, and in individuals having detectable
KIR3DL2-expressing malignant cells (e.g. KIR3DL2-expressing Sezary
cells) in circulation.
[0128] In one aspect, any of the treatment regimens herein are used
for the treatment of individuals having CTCL, wherein the treatment
regimen (e.g. the same dose of anti-KIR3DL2 agent and frequency of
administration) is used in individuals having low numbers of
detectable KIR3DL2-expressing malignant cells (e.g.
KIR3DL2-expressing Sezary cells) in circulation, and in individuals
having high numbers of detectable KIR3DL2-expressing malignant
cells (e.g. KIR3DL2-expressing Sezary cells) in circulation.
[0129] In one aspect, any of the treatment regimens herein are used
for the treatment of individuals having CTCL, wherein the treatment
regimen (e.g. the same dose of anti-KIR3DL2 agent and frequency of
administration) is used in individuals having low or no blood tumor
burden, and in individuals having (or having high) blood tumor
burden. In one embodiment, no or low tumor burden is BO (absence of
significant blood involvement, e.g. .ltoreq.5% of peripheral blood
lymphocytes are atypical (Sezary) cells) or B1 (low blood-tumor
burden, e.g. >5% of peripheral blood lymphocytes are atypical
(Sezary) cells. In one embodiment, having blood tumor burden or
having high blood tumor burden is B2 (high blood-tumor burden:
1,000/.mu.L Sezary cells with positive clone).
[0130] In one aspect, any of the treatment regimens herein are used
for the treatment of individuals having CTCL, wherein the treatment
regimen (e.g. the same dose of anti-KIR3DL2 agent and frequency of
administration) is used in individuals having early stage CTCL
(e.g. stage I, II and/or III), and in individuals having late stage
CTCL (e.g., stage IV).
[0131] In one aspect, any of the treatment regimens herein are used
for the treatment of individuals having CTCL having skin lesions,
optionally significant or advanced skin disease, optionally T2
(patches, papules, or plaques covering .gtoreq.10% of the skin
surface, optionally further T2a (patch only) or T2b
(plaque.+-.patch), T3 (at least one tumor (1 cm diameter) or T4
stage skin involvement erythema covering 80% of body surface area).
In one embodiment, the individual has multiple and/or high skin
tumor burden. In one embodiment, the individual has one or multiple
skin tumors greater than 1 cm diameter.
[0132] The anti-KIR3DL2 binding agent may be used in combined
treatments with one or more other treatments or therapeutic agents,
including treatments and agents normally utilized for the
particular therapeutic purpose for which the agent is being
administered. The additional treatment or agent will normally be
administered in amounts and treatment regimens typically used for
that treatment or agent in a monotherapy for the particular disease
or condition being treated. In the treatment methods, the
KIR3DL2-binding compound and the second therapeutic agent or
treatment can be administered sequentially. The KIR3DL2-binding
compound can be administered prior to the administration of the
second therapeutic agent or treatment. For example, the
KIR3DL2-binding compound can be administered approximately 0 to 30
days prior to the administration of the second therapeutic agent or
treatment. In some embodiments, an KIR3DL2-binding compound is
administered from about 30 minutes to about 2 weeks, from about 30
minutes to about 1 week, from about 1 hour to about 2 hours, from
about 2 hours to about 4 hours, from about 4 hours to about 6
hours, from about 6 hours to about 8 hours, from about 8 hours to 1
day, or from about 1 to 5 days prior to the administration of the
second therapeutic agent or treatment. In one embodiment, the
treatment is a bone marrow transplant or hematopoietic stem cell
transplant. In some embodiments, a KIR3DL2-binding compound is
administered concurrently with the administration of the
therapeutic agents.
[0133] In one embodiment, a subject receives treatment with the
anti-KIR3DL2 agent prior to treatment with a bone marrow transplant
or hematopoietic stem cell transplant. For example, a transplant
can be administered within 1, 2 or 3 months following the end of
treatment with the anti-KIR3DL2 agent.
[0134] In one embodiment with the anti-KIR3DL2 agent precedes
treatment with an additional therapeutic agent or treatment for
CTCL selected from the group consisting of: a corticosteroid,
nitrogen mustard, carmustine, topical tacrolimus (Protopic.RTM.),
imiquimod (Aldara.RTM.; 3M Inc.), topical retinoids, and rexinoids
(bexarotene; Targretin.RTM.; Ligand Pharmaceuticals, San Diego,
Calif.)), mogamulizumab, alemtuzumab, brentuximab vedotin, as well
as ultraviolet light therapy (Psoralen+ UVA (PUVA), narrowband UVB,
and UVB), Photodynamic therapy (PDT) and body irradiation, histone
deacetylase inhibitors such as vorinostat (suberoylanilide
hydroxamic acid, Zolinza.RTM.) and Romidepsin (depsipeptide,
FK-228, Istodax.RTM.), a cyclic peptide that selectively inhibits
histone deacetylase isotypes 1, 2, 4 and 6, chemotherapy or
combination chemotherapy, gemcitabine, antifolate analogues such as
Pralatrexate (Folotyn.RTM.), IMiDs (immunomodulatory drugs),
CC-5013 (lenalidomide; Revlimid.RTM.), CC-4047 (Actimid), and
ENMD-0995, proteosome inhibitors and bortezomib (Velcade.RTM.).
Anti-KIR3DL2 agent can be advantageously used in individuals who
have not received one or more of (or any of) the above
treatments.
[0135] In one embodiment, the anti-KIR3DL2 agent compositions
optionally do not comprise a further therapeutic agent. In one
embodiment, the anti-KIR3DL2 agent compositions may be employed as
monotherapy, e.g., without the combined administration of another
therapeutic agent for the particular therapeutic purpose for which
the anti-KIR3DL2 agent is being administered, notably for the
treatment of a CTCL.
KIR3DL2 Binding Agents
[0136] An agent that binds a KIR3DL2 polypeptide (used
interchangeable with the terms Anti-KIR3DL2 agent, KIR3DL2-binding
agent, anti-KIR3DL2 binding agent and the like) can be any agent
suitable to bind KIR3DL2 and have the functionality in accordance
with the disclosure.
[0137] KIR3DL2 (CD158k) is a disulphide-linked homodimer of
three-Ig domain molecules of about 140 kD, described in Pende et
al. (1996) J. Exp. Med. 184: 505-518, the disclosure of which is
incorporated herein by reference. Several allelic variants have
been reported for KIR3DL2 polypeptides, each of these are
encompassed by the term KIR3DL2. The amino acid sequence of the
mature human KIR3DL2 (allele *002) is shown in SEQ ID NO: 1, below,
corresponding to Genbank accession no. AAB52520 in which the 21
amino acid residue leader sequence has been omitted:
TABLE-US-00001 (SEQ ID NO: 1) LMGGQDKPF LSARPSTVVP RGGHVALQCH
YRRGFNNFML YKEDRSHVPI FHGRIFQESF IMGPVTPAHA GTYRCRGSRP HSLTGWSAPS
NPLVIMVTGN HRKPSLLAHP GPLLKSGETV ILQCWSDVMF EHFFLHRDGI SEDPSRLVGQ
IHDGVSKANF SIGPLMPVLA GTYRCYGSVP HSPYQLSAPS DPLDIVITGL YEKPSLSAQP
GPTVQAGENV TLSCSSWSSY DIYHLSREGE AHERRLRAVP KVNRTFQADF PLGPATHGGT
YRCFGSFRAL PCVWSNSSDP LLVSVTGNPS SSWPSPTEPS SKSGICRHLH VLIGTSVVIF
LFILLLFFLL YRWCSNKKNA AVMDQEPAGD RTVNRQDSDE QDPQEVTYAQ LDHCVFIQRK
ISRPSQRPKT PLTDTSVYTE LPNAEPRSKV VSCPRAPQSG LEGVF.
[0138] Also encompassed are any nucleic acid or protein that
represent allelic variants of KIR3DL2 shown in SEQ ID NO: 1--for
example KIR3DL2 proteins sharing at least 95%, 97%, 98%, 99%, or
higher amino acid identity.
[0139] Closely related KIR3DL1 (CD158e1) is a monomeric molecule of
about 70 kD, described in Colonna and Samaridis (1995) Science 268
(5209), 405-408. The cDNA encoding a KIR3DL1 (CD158e2) polypeptide
(allele *00101) is shown in Genbank accession no. L41269; the
encoded amino acid sequence is shown in Genbank accession no.
AAA69870. In one embodiment, a KIR3DL1 polypeptide referred to
herein is allele *00101.
[0140] KIR3DL2 binding agents can be readily derived from any
suitable source, for example KIR3DL2 binding agents can be made
from a variety of immunoglobulin or non-immunoglobulin scaffolds,
for example affibodies based on the Z-domain of staphylococcal
protein A, engineered Kunitz domains, monobodies or adnectins based
on the 10th extracellular domain of human fibronectin III,
anticalins derived from lipocalins, DARPins (designed ankyrin
repeat domains, multimerized LDLR-A module, avimers or
cysteine-rich knottin peptides. See, e.g., Gebauer and Skerra
(2009) Current Opinion in Chemical Biology 13:245-255, the
disclosure of which is incorporated herein by reference. In certain
embodiments, a KIR3DL2 binding agent comprises an antibody (or an
antibody fragment).
[0141] A KIR3DL2 binding agent (e.g. an antibody, an antibody
fragment) for use in treating CTCL may for example be in the form
of an isolated protein, or it can be present on the surface of a
cell (e.g. a CAR effector cell such as a T cell, NK cell or NKT
cell) or encoded by a nucleic acid therein, such as from a pox
viral gene transfer vector comprising anti-KIR3DL2
antibody-encoding nucleic acid sequence(s). A cell expressing a
chimeric antigen receptor (CAR) can be constructed. Examples of
CARs are engineered to comprise an extracellular single chain
antibody (scFv) fused to the intracellular signaling domain of the
T cell antigen receptor complex zeta chain, and have the ability,
when expressed in effector cells such as T cells, NKT cells or NK
cells, to redirect antigen recognition (i.e. KIR3DL2 recognition)
based on the monoclonal antibody's specificity. In one aspect,
provided are genetically engineered immune cells which express and
bear on the cell surface membrane a KIR3DL2-specific chimeric
immune receptor comprising an intracellular signaling domain, a
transmembrane domain (TM) and a KIR3DL2-specific extracellular
domain (e.g., a domain derived from variable heavy and light chain
regions of the a monoclonal antibody that binds specifically to
KIR3DL2, e.g. one of antibodies disclosed herein). Also provided
are the KIR3DL2 specific chimeric immune receptors, DNA constructs
encoding the receptors, and plasmid expression vectors containing
the constructs in proper orientation for expression.
[0142] In one embodiment, the KIR3DL2-binding antibody is an
antibody that directs ADCC and optionally further ADCP toward a
KIR3DL2-expressing cell.
[0143] In one embodiment, the antibody used in any embodiment
herein binds a KIR3DL2 polypeptide, optionally wherein the antibody
does not substantially bind to a KIR3DL1 polypeptide, is
characterized by binding affinity (K.sub.D) for a human KIR3DL2
polypeptide of less than (better than) 100 ng/ml, optionally
between 1 and 100 ng/ml.
[0144] The antibody is optionally characterized by an EC.sub.50 in
.sup.51Cr-release assay for HuT78 tumor lysis by PBMC from healthy
volunteers, of less than 100 ng/ml, optionally between 1 and 100
ng/ml, optionally between 1 and 50 ng/ml, optionally between 25 and
75 ng/ml, optionally about 50 ng/ml. The antibody is optionally
characterized by an EC.sub.50 in .sup.51Cr-release assay for HuT78
tumor lysis by PBMC from healthy volunteers comparable to that of
an anti-KIR3DL2 antibody disclosed herein (e.g., having an EC50
that is lower or within 1-log or 0.5-log of the EC50 of that of a
2B12 antibody disclosed herein having a VH of SEQ ID NO 31: and a
VL of SEQ ID NOS: 25 or 26, comprising an Fc domain of wild type or
modified human IgG1 isotype, and that mediates ADCC.
[0145] An exemplary anti-KIR3DL2 antibody can be characterized by
an average disassociation constant (K.sub.D) of less than
1.times.10.sup.-9 M with respect to KIR3DL2, as determined by,
e.g., surface plasmon resonance (SPR) screening (such as by
analysis with a BIAcore.TM. SPR analytical device). Optionally, the
anti-KIR3DL2 antibody has a KD of about 1.times.10.sup.-8 M to
about 1.times.10.sup.-10 M, or about 1.times.10.sup.-9 M to about
1.times.10.sup.-11 M, for KIR3DL2. In one aspect, an antibody that
specifically binds KIR3DL2 can be characterized by having one or
more (including any combination thereof, to the extent that such
combination is not contradictory) of the following properties:
[0146] (a) has a Kd of less than 10.sup.-8 M, preferably less than
10.sup.-9 M, or preferably less than 10-10 M for binding to a
KIR3DL2 polypeptide; [0147] (b) binds to at least one residue in
the segment corresponding to residues 1-98 or residues 193-292 of
the KIR3DL2 polypeptide; [0148] (c) competes for binding to a
KIR3DL2 polypeptide with antibody 10F6, 2B12, 18C6, 9E10, 10G5,
13H1, 5H1, 1E2, 1C3 and/or 20E9; [0149] (d) competes with a natural
ligand of KIR3DL2 (e.g. a HLA polypeptide, optionally HLA-B27) for
binding to a KIR3DL2 polypeptide (e.g. in a polypeptide interaction
assay); [0150] (e) does not substantially increase or induce
internalization of KIR3DL2 polypeptides in KIR3DL2-expressing cells
and/or is not internalized into KIR3DL2-expressing cells; [0151]
(f) does or does not inhibit KIR3DL2 signaling induced by a natural
ligand of KIR3DL2 (e.g. a HLA polypeptide; HLA-B27); [0152] (g)
does not substantially bind to a KIR3DL1, KIR3DS1, KIR3DL3,
KIR2DS1, KIR2DS2, KIR2DL3, KIR2DL1 and/or KIR2DS4 polypeptide;
[0153] (h) binds to an epitope comprising any one or more of amino
acid residues R13, P14, S15, H23, A25, Q27, H32, G33, 160, G62,
R78, L82, W226, 1231 and/or R246 of a KIR3DL2 polypeptide; and
[0154] (i) has reduced binding to a KIR3DL2 polypeptide having a
mutation at one or more of residues R13, P14, S15, H23, A25, Q27,
H32, G33, 160, G62, R78, L82, W226, 1231 and/or R246 of a KIR3DL2
polypeptide.
[0155] In any of the embodiments herein, an antibody may be
characterized by any one or more features of (a)-(i), above. In any
of the embodiments herein, an antibody may be characterized by the
features of (a), (b), (c), and (g), further in combination with the
features of (d) or (f), and optionally further the features of (e),
above. Optionally, the antibody is further characterized by
features (h) and/or (i).
[0156] In one embodiment, the antibody is human-suitable. In one
embodiment the antibody is chimeric, e.g. contains variable regions
of non-human or murine origin, and constant regions of human or
non-murine origin. In one embodiment, the antibody is human or
humanized.
[0157] In one embodiment the antibody comprises an Fc domain or is
of an isotype that is bound by Fc.gamma.R (e.g. Fc.gamma.RIIIA),
e.g. an antibody of IgG1 or IgG3 isotype.
[0158] Exemplary of antibodies that bind human KIR3DL2 include
antibodies 19H12, 12B11, 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1,
1E2, 1C3 or 20E9. These and further antibodies are provided in
PCT/EP2013/069302 and PCT/EP2013/069293, both filed 17 Sep. 2013,
the disclosures of which antibodies are incorporated herein by
reference. These antibodies bind selectively to KIR3DL2 and do not
bind KIR3DL1 (or KIR3DS1). While antibody 10F6, 2B12, 18C6, 9E10,
10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 can be used, for example, as
therapeutic agent administered to an individual for the depleting
of a KIR3DL2 expressing target, e.g. by induction of ADCC toward a
pathogenic KIR3DL2-expressing cell, antibody 12B11 and 19H12 will
be advantageous for use in detection (e.g. in vitro assays) of
KIR3DL2 expression on the surface of cells because 12B11 and 19H12
are particularly efficient in the detection of KIR3DL2-positive
cells in detection assays, 12B11 is advantageous for
immunohistochemistry assays using frozen tissue sections, while
19H12 is advantageous for flow cytometry detection.
[0159] The amino acid sequence of the heavy and light chain
variable regions of antibodies 10F6, 2B12, 18C6, 9E10, 10G5, 13H1,
5H1, 1E2, 1C3 or 20E9 are listed in Table C. In a specific
embodiment, an anti-KIR3DL2 antibody binds essentially the same
epitope or determinant as any of monoclonal antibodies 10F6, 2B12,
18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9; optionally the
antibody comprises an antigen binding region of antibody 10F6,
2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9. In any of the
embodiments herein, antibody 10F6, 2B12, 18C6, 9E10, 10G5, 13H1,
5H1, 1E2, 1C3 or 20E9 can be characterized by its amino acid
sequence and/or nucleic acid sequence encoding it. In one
embodiment, the monoclonal antibody comprises the Fab or
F(ab').sub.2 portion of 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1,
1E2, 1C3 or 20E9. A monoclonal antibody can comprise the heavy
chain variable region of 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1,
1E2, 1C3 or 20E9. According to one embodiment, the monoclonal
antibody comprises the three CDRs of the heavy chain variable
region of 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or
20E9. A monoclonal antibody can further comprise the variable light
chain variable region of 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1,
1E2, 1C3 or 20E9 or one, two or three of the CDRs of the light
chain variable region of 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1,
1E2, 1C3 or 20E9. Optionally any one or more of said light or heavy
chain CDRs may contain one, two, three, four or five or more amino
acid modifications (e.g. substitutions, insertions or deletions).
Optionally, any of the light and/or heavy chain variable regions
comprising part or all of an antigen binding region of antibody
10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 are fused
to an immunoglobulin constant region of the human IgG type,
optionally a human constant region, optionally a human IgG1 or IgG3
isotype.
[0160] In another aspect, an antibody comprises: a HCDR1 region of
10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9
comprising an amino acid sequence as set forth in Table A, or a
sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids
thereof, optionally wherein one or more of these amino acids may be
substituted by a different amino acid; a HCDR2 region of 10F6,
2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 comprising an
amino acid sequence as set forth in Table A, or a sequence of at
least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof,
optionally wherein one or more of these amino acids may be
substituted by a different amino acid; a HCDR3 region of 10F6,
2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 comprising an
amino acid sequence as set forth in Table A, or a sequence of at
least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof,
optionally wherein one or more of these amino acids may be
substituted by a different amino acid; a LCDR1 region of 10F6,
2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 comprising an
amino acid sequence as set forth in Table B, or a sequence of at
least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof,
optionally wherein one or more of these amino acids may be
substituted by a different amino acid; a LCDR2 region of 10F6,
2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 comprising an
amino acid sequence as set forth in Table B, or a sequence of at
least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof,
optionally wherein one or more of these amino acids may be
substituted by a different amino acid; a LCDR3 region of 10F6,
2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 comprising an
amino acid sequence as set forth in Table B, or a sequence of at
least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof,
optionally wherein one or more of these amino acids may be deleted
or substituted by a different amino acid. The HCDR1, 2, 3 and
LCDR1, 2, 3 sequences can optionally be specified as all (or each,
independently) being those of the Kabat numbering system (as
indicated in Tables A and/or B for each CDR), those of the Chotia
numbering system as indicated in Table A for each CDR), those of
the IMGT numbering system as indicated in Table A for each CDR), or
any other suitable numbering system.
TABLE-US-00002 TABLE A CDR HCDR1 HCDR2 HCDR3 defini- SEQ SEQ SEQ
mAb tion ID Sequence ID Sequence ID Sequence 10F6 Kabat 51 IAGMQ 54
WINTHSGVPKYAEDFKG 20 GGDEGVMDY Chotia 52 GYTFTI 55 WINTHSGVPK 20
GGDEGVMDY AbM 53 GYTFTIAG WINTHSGVPK 20 GGDEGVMDY MQ 2B12 Kabat 18
TAGMQ 19 WINSHSGVPKYAEDFK 20 GGDEGVMDY Chotia 56 GYTFTT 58
WINSHSGVP 59 GGDEGVMDYW AbM 57 GYTFTTAG 58 WINSHSGVP 59 GGDEGVMDYW
MQ 10G5 Kabat 2 SYTMH 3 YINPSSGYTENNRKF 4 RLGKGLLPPF DY Chotia 60
GYTFTS 62 YINPSSGY 63 RLGKGLLPPF DY AbM 61 GYTFTSYT YINPSSGY
RLGKGLLPPF MH DY 13H1 Kabat 64 GYTMN 67 LINPYNGDTTYNQKFKG 69
ENWGYPYAMD Y Chotia 65 HYSFIG 68 LINPYNGDTT 69 ENWGYPYAMD Y AbM 66
HYSFIGYTM 68 LINPYNGDTT 69 ENWGYPYAMD N Y 1E2 Kabat 70 DYAMN 73
VISTYYGDANYNQKFKG 75 IYYDYDGSY Chotia 71 GYTFTD 74 VISTYYGDAN 75
IYYDYDGSY AbM 72 GYTFTDYA 74 VISTYYGDAN 75 IYYDYDGSY MN 9E10 Kabat
76 SYTMH 79 YINPSSGYTDYNQKFKD 81 LGKGLLPPFD Y Chotia 77 GYTFTS 80
YINPSSGYTD 81 LGKGLLPPFD Y AbM 78 GYTFTSYT 80 YINPSSGYTD 81
LGKGLLPPFD MH Y 1C3 Kabat 82 SYWMQ 85 AIYPGDGDTRYTQKFKG 87
RYDGYYHFDY Chotia 83 GYTFTS 86 AIYPGDGDTR 87 RYDGYYHFDY AbM 84
GYTFTSYW 86 AIYPGDGDTR 87 RYDGYYHFDY MQ 20E9 Kabat 88 TYWMQ 91
AIYPGDGDTRYTQKFKG 93 RGDYGNYGMD Y Chotia 89 GFTFTT 92 AIYPGDGDTR 93
RGDYGNYGMD Y AbM 90 GFTFTTYW 92 AIYPGDGDTR 93 RGDYGNYGMD MQ Y
TABLE-US-00003 TABLE B LCDR1 LCDR2 LCDR3 mAb Sequence Sequence
Sequence 10F6 21 KASQDVSTAVA 22 WASTRHT 94 QQHYNTPWT 2B12 21
KASQDVSTAVA 22 WTSTRHT 23 QQHYSTPWT 10G5 5 RASENIYSNLA 6 AATNLAD 7
QHFWGTPYT 13H1 95 RASESVDNFGISFMN 96 AASNQGS 97 QQSKEVPYT 1E2 98
RSSQSLVHSNGNTYLH 99 KVSNRFS 100 SQSTHVPPYT 9E10 101
KSNQNLLWSGNQRYCLV 102 WTSDRYS 103 QQHLHIPYT 1C3 104
KSSQSLLWSVNQKNYLS 105 GASIRES 106 QHNHGSFLPLT 20E9 107
RSSQSIVHSNGNTYLE 108 KVSNHFS 109 FQGSHVPPT
TABLE-US-00004 TABLE C Antibody portion Amino acid sequence 10F6 VH
34 Q I Q L V Q S G P E L K K P G E T V R I S C K A S G Y T F T I A
G M Q W V Q K M P G K G L K W I G W I N T H S G V P K Y A E D F K G
R F A F S L E T S A N I A Y L Q I S N L K N E D T A T Y F C A R G G
D E G V M D Y W G Q G T S V T V S 10F6 VL 35 D I V M T Q S H K F M
S T S V G D R V S I T C K A S Q D V S T A V A W Y H Q K P G Q S P K
L L I Y W A S T R H T G V P D R F S G S G S G T D Y T L T I S A L Q
A E D L A L Y Y C Q Q H Y N T P W T F G G G T K L E I K 2B12 VH 36
Q I Q L V Q S G P E L K K P G E T V R I S C K A S G Y T F T T A G M
Q W V Q K T P G K G L K W I G W I N S H S G V P K Y A E D F K G R F
A F S L E T S A S T A Y L Q I S T L K N E D T A T Y F C A R G G D E
G V M D Y W G Q G T S V T V S 2B12 VL 37 D I V M T Q S H K F M S T
S L G D R V S F T C K A S Q D V S T A V A W Y Q Q K P G Q S P K L L
I Y W T S T R H T G V P D R F T G S G S G T D Y T L T I S S V Q A E
D L A L Y Y C Q Q H Y S T P W T F G G G T K L E I K 10G5 VH 38 Q V
Q L Q Q S A A E L A R P G A S V K M S C K A S G Y T F T S Y T M H W
V K Q R P G Q G L E W I G Y I N P S S G Y T E N N R K F K D K T T L
T A D K S S S T A Y M Q L S S L T S E D S A V Y Y C A R L G K G L L
P P F D Y W G Q G T T L T V S S A K T T P P S V Y P L A P G S A A Q
T 10G5 VL 39 D I Q M T Q S P A S L S V S V G E T V T I T C R A S E
N I Y S N L A W Y Q Q K Q G K S P Q L L V Y A A T N L A D G V P S R
F S G S G S G T Q Y S L K I N S L Q S E D F G S Y Y C Q H F W G T P
Y T F G G G T K L E I K 13H1 VH 40 E V Q L Q Q S G P E L V K P G A
S M K I S C K A S H Y S F I G Y T M N W V K Q R H G K N L E W I G L
I N P Y N G D T T Y N Q K F K G K A S L T V D K S S S T A Y M E I L
S L T S E D S A V Y Y C A R E N W G Y P Y A M D Y W G Q G T S V T V
S 13H1 VL 41 D I V L T Q S P A S L A V S L G Q R A T I S C R A S E
S V D N F G I S F M N W F Q Q K P G Q P P K L L I Y A A S N Q G S G
V P A R F S G S R S G T D F S L N I H P M E E D D T A M Y F C Q Q S
K E V P Y T F G G G T K L E I K 1E2 VH 42 Q V Q L Q Q S G A E L V R
P G V S V K I S C K G S G Y T F T D Y A M N W V K Q S H A K S L E W
I G V I S T Y Y G D A N Y N Q K F K G K A T M T V D K S S S T A Y M
E L A R L T S E D S A I Y Y C A L I Y Y D Y D G S Y W G Q G T T L T
V S 1E2 VL 43 D V V M T Q T P L S L P V S L G D Q A S I S C R S S Q
S L V H S N G N T Y L H W Y L Q K P G Q S P K L L I Y K V S N R F S
G V P D R F S G S G S G T D F T L K I S R V E A E D L G V Y F C S Q
S T H V P P Y T F G G G T K L E I K 9E10 VH 44 Q V Q L Q Q S A A E
L A R P G A S V K M S C K A S G Y T F T S Y T M H W V K Q R P G Q G
L E W I G Y I N P S S G Y T D Y N Q K F K D K T T L T A D R S S S T
A Y M Q L S S L T S E D S A V Y Y C A R L G K G L L P P F D Y W G Q
G S T L T V S S 9E10 VL1 45 E I V L T Q S I P S L T V S A G E R V T
I S C K S N Q N L L W S G N Q R Y C L V W H Q W K P G Q T P T P L I
T W T S D R Y S G V P D R F I G S G S V T D F T L T I S S V Q A E D
V A V Y F C Q Q H L H I P Y T F G G G T K L E I K 9E10 VL2 46 D I Q
M T Q S P A S L S V S V G E T V T I T C R A S E N I Y S N L A W Y Q
Q K Q G K S P Q L L V Y A A T N L A D G V P S R F S G S G S G T Q Y
S L K I N S L Q S E D F G S Y Y C Q H F W G T P Y T F G G G T K L E
I K 1C3 VH 47 Q V Q L Q Q S G A E L A R P G A S V K L S C K A S G Y
T F T S Y W M Q W V K Q R P G Q G L E W I G A I Y P G D G D T R Y T
Q K F K G K A T L T A D K S S S T A Y M Q L S S L A S E D S A V Y Y
C A R R Y D G Y Y H F D Y W G Q G T T L T V S 1C3 VL 48 D I V M T Q
S P S S L A V T A G E K V T M S C K S S Q S L L W S V N Q K N Y L S
W Y Q Q K Q R Q P P K L L I Y G A S I R E S W V P D R F T G S G S G
T D F T L T I S N V H A E D L A V Y Y C Q H N H G S F L P L T F G S
G T K L E I K 20E9 VH 49 Q V Q L Q Q S G A E V A R P G A S V K L S
C K S S G F T F T T Y W M Q W V K Q R P G Q G L E W I G A I Y P G D
G D T R Y T Q K F K G K A T L T A D K S S I T A Y M Q L S S L A S E
D S A V Y Y C A R R G D Y G N Y G M D Y W G Q G T S V T V S S 20E9
VL 50 D V L M T Q T P L S L P V S L G D Q A S I S C R S S Q S I V H
S N G N T Y L E W Y L Q K P G Q S P K L L I Y K V S N H F S G V P D
R F S G S G S G T D F T L K I S R V E A E D L G V Y Y C F Q G S H V
P P T F G G G T K L E I K
[0161] Examples of humanized VH and VL amino acid sequences of
antibody 10G5 are shown in Table D and in SEQ ID NOS: 13-17 and
8-12, respectively. In one aspect, provided is an isolated
humanized antibody that binds a human KIR3DL2 polypeptide, wherein
the antibody comprises: a HCDR1 region comprising an amino acid
sequence SYTMH as set forth in SEQ ID NO: 2, or a sequence of at
least 3 or 4 amino acids thereof; a HCDR2 region comprising an
amino acid sequence YINPSSGYTENNRKF as set forth in SEQ ID NO: 3,
or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino
acids thereof; a HCDR3 region comprising an amino acid sequence
LGKGLLPPFDY as set forth in SEQ ID NO: 4, or a sequence of at least
4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof; a LCDR1
region comprising an amino acid sequence RASENIYSNLA as set forth
in SEQ ID NO: 5, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10
contiguous amino acids thereof; a LCDR2 region comprising an amino
acid sequence AATNLAD as set forth in SEQ ID NO: 6, or a sequence
of at least 3, 4 or 5 contiguous amino acids thereof; a LCDR3
region comprising an amino acid sequence QHFWGTPYT as set forth in
SEQ ID NO: 7, or a sequence of at least 4, 5, 6, 7, or 8 contiguous
amino acids thereof.
[0162] In one aspect, a humanized 10G5 antibody that binds a human
KIR3DL2 polypeptide comprises: [0163] (a) a CDR-H1 comprising the
amino acid sequence of SEQ ID NO:2; [0164] (b) a CDR-H2 comprising
the amino acid sequence of SEQ ID NO:3; [0165] (c) a CDR-H3
comprising the amino acid sequence of SEQ ID NO:4; [0166] (d) a
CDR-L1 comprising the amino acid sequence of SEQ ID NO:5; [0167]
(e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:6;
[0168] (f) a CDR-L3 comprising the amino acid sequence of SEQ ID
NO:7; and [0169] (g) human framework sequences.
[0170] In one embodiment, the humanized antibody comprises a heavy
chain framework from the human subgroup VH1 together with JH6,
optionally the antibodies comprises IGHV1-46*03, together with
IGHJ6*01. In one embodiment, the humanized antibody comprises a
light chain framework from the human subgroup VK1, optionally
IGKV1-NL1*01.
[0171] Optionally the human framework comprises one or more
mutations, e.g. back mutations that show a retain ability to bind
KIR3DL2. Embodiments of the invention thus include the back-mutated
10G5 heavy chain variants having back mutations at any one or more
(or any combination of) the following residues, using Abnum
numbering: [0172] 10G5 VH: 5, 11, 12, 13, 20, 38, 40, 48, 66, 67,
69, 71, 72a, 75.
[0173] Abnum amino acid numbering nomenclature is described in
Abhinandan and Martin, (2008) Molecular Immunology 45: 3832-3839,
the disclosure of which is incorporated by reference). Sequence
numbering using the Abnum system can also be automatically
generated at http://www.bioinfo.org.uk/abs/abnum. However it will
be appreciated that the person of skill in the art can use an
alternative numbering system and identify positions corresponding
to Abnum numbering, for example the Kabat numbering system can be
used (Kabat et al. (1991) Sequences of Protein of Immunological
Interest, 5th ed., United States Public Health Service, National
Institute of Health, Bethesda, Md.).
[0174] Further embodiments of the invention thus include the
back-mutated 10G5 light chain variants having back mutations at any
one or more (or any combination of) the following residues: [0175]
10G5 VL: 17, 18, 40, 45, 48, 70, 76, 100.
[0176] The humanized antibody may further comprise one or more
additional mutations (e.g. back-mutations) in the human framework
sequences, to, e.g., enhance affinity, stability, or other
properties of the humanized antibody.
[0177] In one aspect, a humanized 10G5 antibody that binds human
KIR3DL2 polypeptide comprises: [0178] (a) a CDR-H1 comprising the
amino acid sequence of SEQ ID NO: 2; [0179] (b) a CDR-H2 comprising
the amino acid sequence of SEQ ID NO: 3; [0180] (c) a CDR-H3
comprising the amino acid sequence of SEQ ID NO: 4; [0181] (d) a
CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5; [0182]
(e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6;
[0183] (f) a CDR-L3 comprising the amino acid sequence of SEQ ID
NO: 7; and [0184] (g) human framework sequences, wherein a
glutamine (Q) residue is present at position 39 of the VH domain
and at position 38 of the VL domain. Optionally, the human
framework sequences further comprise one or more
back-mutations.
[0185] The glutamine (Q) residue at position 39 may exist naturally
in the human VH framework sequence, or may be introduced by amino
acid substitution or other modification of the sequence.
[0186] In another aspect, a humanized antibody may comprise a VH
domain having at least about 80% sequence identity (e.g., at least
about 85%, 90%, 95%, 97%, 98%, or more identity) to the VH domain
of 10G5 of SEQ ID NOS: 13-17. In another particular aspect, a
humanized antibody may comprise (a) a VH domain that comprises
non-human CDR residues incorporated into a human VH domain, wherein
the VH domain is at least about 80% (such as at least 90%, 95%,
97%, 98%) identical to a humanized 10G5 VH of SEQ ID NOS: 13-17,
and (b) a VL domain that comprises non-human CDR residues
incorporated into a human VL domain, wherein the VL domain is at
least about 80% (such as at least 90%, 95%, 97%, 98%) identical to
humanized 10G5 VL of SEQ ID NOS: 8-12.
[0187] Examples of humanized VH and VL amino acid sequences of
antibody 2B12 are shown in Table D and in SEQ ID NOS: 24-28 and
30-33, respectively. In one aspect, a humanized antibody comprises:
a HCDR1 region comprising an amino acid sequence TAGMQ as set forth
in SEQ ID NO: 18, or a sequence of at least 3 or 4 contiguous amino
acids thereof; a HCDR2 region comprising an amino acid sequence
WINSHSGVPKYAEDFK as set forth in SEQ ID NO: 19, or a sequence of at
least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof; a
HCDR3 region comprising an amino acid sequence GGDEGVMDY as set
forth in SEQ ID NO: 20, or a sequence of at least, 5, 6, 7, or 8
contiguous amino acids thereof; a LCDR1 region comprising an amino
acid sequence KASQDVSTAVA as set forth in SEQ ID NO: 21, or a
sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids
thereof; a LCDR2 region comprising an amino acid sequence WTSTRHT
as set forth in SEQ ID NO: 22, or a sequence of at least 3, 4 or 5
contiguous amino acids thereof; and/or a LCDR3 region comprising an
amino acid sequence QQHYSTPWT as set forth in SEQ ID NO: 23, or a
sequence of at least 4, 5, 6, 7, or 8 contiguous amino acids
thereof.
[0188] In any of the embodiments herein, any of the CDRs 1, 2 and 3
of the heavy and light chains may be characterized by a sequence of
at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof,
and/or as having an amino acid sequence that shares at least 70%,
80%, 85%, 90% or 95% sequence identity with the particular CDR or
set of CDRs listed in the corresponding SEQ ID NO.
[0189] In one aspect, a humanized 2B12 antibody comprises: [0190]
(a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18;
[0191] (b) a CDR-H2 comprising the amino acid sequence of SEQ ID
NO: 19; [0192] (c) a CDR-H3 comprising the amino acid sequence of
SEQ ID NO: 20; [0193] (d) a CDR-L1 comprising the amino acid
sequence of SEQ ID NO: 21; [0194] (e) a CDR-L2 comprising the amino
acid sequence of SEQ ID NO: 22; [0195] (f) a CDR-L3 comprising the
amino acid sequence of SEQ ID NO: 23; and [0196] (g) human
framework sequences.
[0197] In one embodiment, a humanized antibody comprises a heavy
chain framework from the human subgroup VH1 and/or VH7 together
with JH6, optionally the antibodies comprises IGHV7-4-1*02 and/or
IGHV1-c*01, together with IGHJ6*01. In one embodiment, a humanized
antibody comprises a light chain framework from the human subgroup
VK1 and/or VK4, optionally IGKV4-1*01 and/or IGKV1-39*01, together
with JH4, optionally IGKJ4*01.
[0198] Optionally a human framework comprises one or more
mutations, e.g. back mutations, for example. Optionally, a 2B12
heavy chain variant of the amino acid sequence below (SEQ ID NO:
29) can have back mutations at any one or more (or any combination
of) the following residues, using Abnum numbering: [0199] 2B12 VH:
2, 38, 39, 40, 43, 48, 68, 72c, 91, 108.
TABLE-US-00005 [0199] (SEQ ID NO: 29)
QVQLVQSGSELKKPGASVKVSCKASGYTFTTAGMQWVRQAPGQGLEWMGW
INSHSGVPKYAEDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARGG
DEGVMDYWGQGTTVTVSS.
[0200] Further embodiments of the invention thus include the
back-mutated 2B12 light chain variants having back mutations at any
one or more (or any combination of) the following residues: [0201]
2B12 VL: 3, 8, 9, 21, 43, 71, 78, 104.
[0202] The humanized antibody may further comprise one or more
additional mutations (e.g. back-mutations) in the human framework
sequences, to, e.g., enhance affinity, stability, or other
properties of the humanized antibody.
[0203] In one aspect, a humanized 2B12 antibody comprises: [0204]
(a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18;
[0205] (b) a CDR-H2 comprising the amino acid sequence of SEQ ID
NO: 19; [0206] (c) a CDR-H3 comprising the amino acid sequence of
SEQ ID NO: 20; [0207] (d) a CDR-L1 comprising the amino acid
sequence of SEQ ID NO: 21; [0208] (e) a CDR-L2 comprising the amino
acid sequence of SEQ ID NO: 22; [0209] (f) a CDR-L3 comprising the
amino acid sequence of SEQ ID NO: 23; and [0210] (g) human
framework sequences, wherein a glutamine (Q) residue is present at
position 39 of the VH domain and at position 38 of the VL domain.
Optionally, the human framework sequences further comprise one or
more back-mutations.
[0211] In another aspect, humanized antibodies comprise a VH domain
having at least about 80% sequence identity (e.g., at least about
85%, 90%, 95%, 97%, 98%, or more identity) to the VH domain of 2B12
or humanized 2B12 of SEQ ID NOS: 30-33. In another particular
aspect, a humanized antibody comprises: (a) a VH domain that
comprises non-human CDR residues incorporated into a human VH
domain, wherein the VH domain is at least about 80% (such as at
least.sub.90%, 95%, 97%, 98%) identical to humanized 2B12 VH of SEQ
ID NOS: 30-33, and (b) (a) a VL domain that comprises non-human CDR
residues incorporated into a human VL domain, wherein the VL domain
is at least about 80% (such as at least 90%, 95%, 97%, 98%)
identical to humanized 2B12 VL of SEQ ID NOS: 24-28.
[0212] The glutamine (Q) residue at position 39 may exist naturally
in the human VH framework sequence, or may be introduced by amino
acid substitution or other modification of the sequence.
[0213] The 10G5 or 2B12 antibody may further comprise a native or
engineered human IgG constant domain. Optionally the constant
domain is an IgG1 domain, optionally further comprising a
modification to increase Fc receptor binding.
TABLE-US-00006 TABLE D Antibody domain Amino acid sequence (SEQ ID
NO) 10G5-L0
DIQMTQSPSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKAPKLLLYAATNLADGVPS
RFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPYTFGQGTKLEIK (SEQ ID NO: 8)
10G5-L2
DIQMTQSPSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKAPQLLVYAATNLADGVPS
RFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPYTFGGGTKLEIK (SEQ ID NO: 9)
10G5-L3
DIQMTQSPSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKAPQLLVYAATNLADGVPS
RFSGSGSGTQYTLTISSLQPEDFATYYCQHFWGTPYTFGGGTKLEIK (SEQ ID NO: 10)
10G5-L4
DIQMTQSPSSLSASVGDRVTITCRASENIYSNLAWYQQKQGKAPQLLVYAATNLADGVPS
RFSGSGSGTQYTLTINSLQPEDFATYYCQHFWGTPYTFGGGTKLEIK (SEQ ID NO: 11)
10G5-L5
DIQMTQSPSSLSASVGETVTITCRASENIYSNLAWYQQKQGKAPQLLVYAATNLADGVPS
RFSGSGSGTQYTLTINSLQPEDFATYYCQHFWGTPYTFGGGTKLEIK (SEQ ID NO: 12)
10G5-H0
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYTMHWVRQAPGQGLEWMGYINPSSGYTEN
NRKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARLGKGLLPPFDYWGQGTTVTVSS (SEQ
ID NO: 13) 10G5-H3
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYTMHWVRQAPGQGLEWIGYINPSSGYTEN
NRKFKDKTTMTADTSTSTAYMELSSLRSEDTAVYYCARLGKGLLPPFDYWGQGTTVTVSS (SEQ
ID NO: 14) 10G5-H4
QVQLQQSGAEVKKPGASVKMSCKASGYTFTSYTMHWVRQAPGQGLEWIGYINPSSGYTEN
NRKFKDKTTLTADTSTSTAYMELSSLRSEDTAVYYCARLGKGLLPPFDYWGQGTTLTVSS (SEQ
ID NO: 15) 10G5-H5
QVQLVQSGAELARPGASVKVSCKASGYTFTSYTMHWVRQAPGQGLEWIGYINPSSGYTEN
NRKFKDKTTLTADKSTSTAYMELSSLRSEDTAVYYCARLGKGLLPPFDYWGQGTTVTVSS (SEQ
ID NO: 16) 10G5-H6
QVQLQQSGAEVKKPGASVKMSCKASGYTFTSYTMHWVKQRPGQGLEWIGYINPSSGYTEN
NRKFKDKTTLTADKSTSTAYMELSSLRSEDTAVYYCARLGKGLLPPFDYWGQGTTLTVSS (SEQ
ID NO: 17) 2B12-L0
DIQMTQSPSFLSASVGDRVTITCKASQDVSTAVAWYQQKPGQPPKLLIYWTSTRHTGVPD
RFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPWTFGGGTKVEIK (SEQ ID NO: 24)
2B12-L1
DIQMTQSPSFLSASVGDRVTITCKASQDVSTAVAWYQQKPGQPPKLLIYWTSTRHTGVPD
RFSGSGSGTDYTLTISSLQAEDVAVYYCQQHYSTPWTFGGGTKVEIK (SEQ ID NO: 25)
2B12-L2
DIVMTQSPSFLSASVGDRVTITCKASQDVSTAVAWYQQKPGQPPKLLIYWTSTRHTGVPD
RFSGSGSGTDYTLTISSVQAEDVAVYYCQQHYSTPWTFGGGTKVEIK (SEQ ID NO: 26)
2B12-L3
DIVMTQSPSFLSASVGDRVTFTCKASQDVSTAVAWYQQKPGQSPKLLIYWTSTRHTGVPD
RFSGSGSGTDYTLTISSVQAEDVAVYYCQQHYSTPWTFGGGTKVEIK (SEQ ID NO: 27)
2B12-L4
DIVMTQSHKFLSASVGDRVTFTCKASQDVSTAVAWYQQKPGQSPKLLIYWTSTRHTGVPD
RFSGSGSGTDYTLTISSVQAEDVAVYYCQQHYSTPWTFGGGTKLEIK (SEQ ID NO: 28)
2B12-H1
QVQLVQSGSELKKPGASVKVSCKASGYTFTTAGMQWVQKSPGQGLEWMGWINSHSGVPKY
AEDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYFCARGGDEGVMDYWGQGTTVTVSS (SEQ ID
NO: 30) 2B12-H2
QIQLVQSGSELKKPGASVKVSCKASGYTFTTAGMQWVRQAPGQGLEWIGWINSHSGVPKY
AEDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYFCARGGDEGVMDYWGQGTTVTVSS (SEQ ID
NO: 31) 2B12-H3
QIQLVQSGSELKKPGASVKVSCKASGYTFTTAGMQWVQKSPGQGLEWIGWINSHSGVPKY
AEDFKGRFAFSLDTSVSTAYLQISSLKAEDTAVYFCARGGDEGVMDYWGQGTTVTVSS (SEQ ID
NO: 32) 2B12-H4
QIQLVQSGSELKKPGASVKVSCKASGYTFTTAGMQWVQKTPGKGLEWIGWINSHSGVPKY
AEDFKGRFAFSLDTSASTAYLQISSLKAEDTAVYFCARGGDEGVMDYWGQGTSVTVSS (SEQ ID
NO: 33)
[0214] In one embodiment, a humanized 2B12 monoclonal antibody
comprises:
(a) a heavy chain variable region comprising an amino acid sequence
of SEQ ID NO: 31, and (b) a light chain variable region comprising
an amino acid sequence of SEQ ID NO: 25.
[0215] In one embodiment, a humanized 2B12 monoclonal antibody
comprises:
(a) a heavy chain variable region comprising an amino acid sequence
of SEQ ID NO: 31, and (b) a light chain variable region comprising
an amino acid sequence of SEQ ID NO: 26.
[0216] In one embodiment, a humanized 2B12 monoclonal antibody
comprises:
(a) a heavy chain variable region comprising an amino acid sequence
of SEQ ID NO: 32, and (b) a light chain variable region comprising
an amino acid sequence of SEQ ID NO: 26.
[0217] In one embodiment, a humanized 2B12 monoclonal antibody
comprises:
(a) a heavy chain variable region comprising an amino acid sequence
of SEQ ID NO: 33, and (b) a light chain variable region comprising
an amino acid sequence of SEQ ID NO: 26.
[0218] In one embodiment, a humanized 10G5 monoclonal antibody
comprises:
(a) a heavy chain variable region comprising an amino acid sequence
of SEQ ID NO: 13, and (b) a light chain variable region comprising
an amino acid sequence of SEQ ID NO: 8.
[0219] In one embodiment, a humanized 10G5 monoclonal antibody
comprises:
(a) a heavy chain variable region comprising an amino acid sequence
of SEQ ID NO: 14, and (b) a light chain variable region comprising
an amino acid sequence of SEQ ID NO: 9.
[0220] In one embodiment, a humanized 10G5 monoclonal antibody
comprises:
(a) a heavy chain variable region comprising an amino acid sequence
of SEQ ID NO: 15, and (b) a light chain variable region comprising
an amino acid sequence of SEQ ID NO: 9.
[0221] In one aspect, an anti-KIR3DL2 agent used in accordance with
the methods of treatment herein binds to an epitope on a KIR3DL2
polypeptide that at least partially overlaps, or includes at least
one residue in the segment corresponding to residues 1-192,
residues 1-98, or residues 99-192 of the KIR3DL2 polypeptide of SEQ
ID NO: 1 (or a subsequence thereof). In one embodiment, all key
residues of the epitope is in a segment corresponding to residues
1-192, residues 1-98 or residues 99-192 of the KIR3DL2 polypeptide
of SEQ ID NO: 1. In one embodiment, the antibodies bind an epitope
comprising 1, 2, 3, 4, 5, 6, 7 or more residues in the segment
corresponding to residues 1-192, 1-98 or 99-192 of the KIR3DL2
polypeptide of SEQ ID NO: 1. Preferably the residues bound by the
antibody are present on the surface of the KIR3DL2 polypeptide.
[0222] In one aspect, an anti-KIR3DL2 agent used in accordance with
the methods of treatment herein binds an epitope comprising one,
two, three, four, five or more of residues selected from the group
consisting of: R13, P14, S15, H23, A25, Q27, 160 and G62 (with
reference to SEQ ID NO: 1), and/or has reduced binding to a KIR3DL2
polypeptide having a mutation at a residue selected from the group
consisting of: R13, P14, S15, H23, A25, Q27, 160 and G62 (with
reference to SEQ ID NO: 1).
[0223] The shorthand notation used for mutations herein is: wild
type residue: position in polypeptide, with numbering of residues
as indicated in SEQ ID NO: 1: mutant residue.
[0224] In one aspect, an anti-KIR3DL2 agent binds an epitope
comprising residues R13, A25 and/or Q27 of the KIR3DL2 polypeptide,
and/or has reduced binding to a KIR3DL2 polypeptide having a
mutation at residues R13, A25 and/or Q27 (with reference to SEQ ID
NO: 1). For example, an antibody can have reduced binding to a
KIR3DL2 polypeptide having the mutations R13W, A25T and/or Q27R.
Optionally, the epitope additionally comprises one or more of
residues 160 and/or G62 (with reference to SEQ ID NO: 1), and/or
the antibodies have reduced binding to a KIR3DL2 polypeptide having
a mutation at residues 160 and/or G62 (with reference to SEQ ID NO:
1, e.g. 160N, G62S). Optionally, the epitope additionally or
alternatively comprises one or more of residues P14, S15 and/or H23
(with reference to SEQ ID NO: 1), and/or the antibodies have
reduced binding to a KIR3DL2 polypeptide having a mutation at
residues P14, S15 and/or H23 (with reference to SEQ ID NO: 1, e.g.
P14S, 515A, H23S). Optionally, the epitope does not comprise
residues R32 and/or G33 (with reference to SEQ ID NO: 1), and/or
the antibodies do not have reduced binding to a KIR3DL2 polypeptide
having a mutation at residues R32 and/or G33 (with reference to SEQ
ID NO: 1, e.g., R32H and/or G33R). Optionally, the epitope does not
comprise residues F50 and/or R53 (with reference to SEQ ID NO: 1),
and/or the antibodies do not have reduced binding to a KIR3DL2
polypeptide having a mutation at residues F50 and/or R53 (with
reference to SEQ ID NO: 1, e.g., F50A, R53S). The antibody may
(e.g. antibodies that block the KIR3DL2-HLA B27 and -HLA A3
interactions) or may not (e.g. non-internalizing antibodies) bind
to residues Q56 and/or E57, and/or residues F9 and/or S11; thus, in
one embodiment, optionally, the epitope does not comprise residues
F9, S11, Q56 and/or E57 (with reference to SEQ ID NO: 1), and/or
the antibodies do not have reduced binding to a KIR3DL2 polypeptide
having a mutation at residues F9, S11, Q56 and/or E57 (with
reference to SEQ ID NO: 1, e.g., F9S and S11A, Q56S and E57A); in
another embodiment, optionally, the epitope comprises residues F9,
S11, Q56 and/or E57 (with reference to SEQ ID NO: 1), and/or the
antibodies have reduced binding to a KIR3DL2 polypeptide having a
mutation at residues F9, S11, Q56 and/or E57 (with reference to SEQ
ID NO: 1, e.g., F9S and S11A, Q56S and E57A). Optionally, the
epitope does not comprise residues H29 and/or F34 (with reference
to SEQ ID NO: 1), and/or the antibodies do not have reduced binding
to a KIR3DL2 polypeptide having a mutation at residues H29 and/or
F34 (with reference to SEQ ID NO: 1, e.g., H29S, F34A). Optionally,
the epitope does not comprises one or more of residues F9 and/or
S11 (with reference to SEQ ID NO: 1), and/or the antibodies do not
have reduced binding to a KIR3DL2 polypeptide having a mutation at
residues F9 and/or S11 (with reference to SEQ ID NO: 1, e.g., F9S,
S11A).
[0225] In one aspect, an anti-KIR3DL2 agent binds an epitope
comprising residues 160 and/or G62 of the KIR3DL2 polypeptide of
SEQ ID NO: 1, and/or has reduced binding to a KIR3DL2 polypeptide
having a mutation at residues 160 and/or G62 (with reference to SEQ
ID NO: 1). For example, an antibody can have reduced binding to a
KIR3DL2 polypeptide having the mutations 160N and/or G62S.
Optionally, the epitope additionally or alternatively comprises one
or more of residues P14, S15 and/or H23 (with reference to SEQ ID
NO: 1), and/or the antibodies have reduced binding to a KIR3DL2
polypeptide having a mutation at residues P14, 515 and/or H23 (with
reference to SEQ ID NO: 1, e.g. P14S, S15A, H23S). Optionally, the
antibodies do not bind residues R13, A25 and/or Q27 of the KIR3DL2
polypeptide, and/or do not have reduced binding to a KIR3DL2
polypeptide having a mutation at residues R13, A25 and/or Q27
(e.g., a KIR3DL2 polypeptide having the mutations R13W, A25T and/or
Q27R).
[0226] In one aspect, an anti-KIR3DL2 agent binds an epitope
comprising residues P14, S15 and/or H23 of the KIR3DL2 polypeptide
of SEQ ID NO: 1, and/or has reduced binding to a KIR3DL2
polypeptide having a mutation at residues P14, S15 and/or H23 (with
reference to SEQ ID NO: 1, e.g. P14S, S15A, H23S).
[0227] In one aspect, an anti-KIR3DL2 agent displays reduced
binding to (1) a KIR3DL2 polypeptide having a mutation at residues
160 and/or G62 (with reference to SEQ ID NO: 1, e.g. 160N, G62S),
and (2) a KIR3DL2 polypeptide having a mutation at residues P14,
S15 and/or H23 (with reference to SEQ ID NO: 1, e.g. P14S, S15A,
H23S).
[0228] In one aspect, an anti-KIR3DL2 agent binds an epitope
comprising: (a) 1, 2 or 3 of residues R13, A25 and/or Q27 and (b)
one or both of residues 160 and/or G62 of the KIR3DL2 polypeptide.
In one aspect antibodies have reduced binding to a KIR3DL2
polypeptide having: (a) a mutation at 1, 2 or 3 of residues R13,
A25 and/or Q27, and (b) a mutation at one or both of residues 160
and/or G62.
[0229] In one aspect, an anti-KIR3DL2 agent binds an epitope
comprising residues R78 and/or L82 of the KIR3DL2 polypeptide of
SEQ ID NO: 1, and/or has reduced binding to a KIR3DL2 polypeptide
having a mutation at residues R78 and/or L82 (with reference to SEQ
ID NO: 1). For example, an antibody can have reduced binding to a
KIR3DL2 polypeptide having the mutations R78H and L82P. Optionally,
the epitope additionally comprises, or excludes, one or more of
residues K7, Y30, R31, P79, H80, S81, T83, G84, W85, S86 and/or A87
(with reference to SEQ ID NO: 1), and/or the antibodies have
reduced binding to, or does not have reduced binding to, a KIR3DL2
polypeptide having a mutation at residues K7, Y30, R31, P79, H80,
S81, T83, G84, W85, S86 and/or A87 (with reference to SEQ ID NO:
1). In one embodiment, the antibodies bind an epitope comprising 1,
2, 3, 4, 5, 6, 7 or more residues in the segment corresponding to
residues 1 to 98 of the KIR3DL2 polypeptide (with reference to SEQ
ID NO: 1), optionally further wherein the epitope comprises one or
more (e.g. 1, 2, 3, 4, 5) of residues K7, Y30, R31, R78, P79, H80,
S81, L82, T83, G84, W85, S86 and/or A87.
[0230] In one aspect, an anti-KIR3DL2 agent binds an epitope
comprising residues W226 of the KIR3DL2 polypeptide of SEQ ID NO:
1, and/or has reduced binding to a KIR3DL2 polypeptide having a
mutation at residues W226 (with reference to SEQ ID NO: 1).
Optionally, the epitope additionally comprises one or more of
residues I231 and/or R246 (with reference to SEQ ID NO: 1), and/or
the antibodies have reduced binding to a KIR3DL2 polypeptide having
a mutation at residues I231 and/or R246 (with reference to SEQ ID
NO: 1, e.g., I231M, R246P). Optionally, the epitope additionally
comprises residue E239 (with reference to SEQ ID NO: 1), and/or the
antibodies have reduced binding to a KIR3DL2 polypeptide having a
mutation at residue E239 (with reference to SEQ ID NO: 1, e.g.,
E239G).
[0231] In one aspect, an anti-KIR3DL2 agent binds an epitope
comprising residues I231 and/or R246 of the KIR3DL2 polypeptide of
SEQ ID NO: 1, and/or has reduced binding to a KIR3DL2 polypeptide
having a mutation at residues I231 and/or R246 (with reference to
SEQ ID NO: 1).
[0232] In one aspect, an anti-KIR3DL2 agent binds an epitope
comprising residue W226 and one or both of residues I231 and/or
R246 of the KIR3DL2 polypeptide.
[0233] In one aspect, an anti-KIR3DL2 agent has reduced binding to
a KIR3DL2 polypeptide having a mutation at residues W226 and a
mutation at one or both of residues I231 and/or R246.
[0234] Binding of anti-KIR3DL2 antibody to cells transfected with
the KIR3DL2 mutants was measured and compared to the ability of
anti-KIR3DL2 antibody to bind wild-type KIR3DL2 polypeptide (SEQ ID
NO:1) (see International patent publication no. WO2014/044686, the
disclosure of which is incorporated herein by reference). A
reduction in binding between an anti-KIR3DL2 antibody and a mutant
KIR3DL2 polypeptide as used herein means that there is a reduction
in binding affinity (e.g., as measured by known methods such FACS
testing of cells expressing a particular mutant, or by Biacore
testing of binding to mutant polypeptides) and/or a reduction in
the total binding capacity of the anti-KIR3DL2 antibody (e.g., as
evidenced by a decrease in Bmax in a plot of anti-KIR3DL2 antibody
concentration versus polypeptide concentration). A significant
reduction in binding indicates that the mutated residue is directly
involved in binding to the anti-KIR3DL2 antibody or is in close
proximity to the binding protein when the anti-KIR3DL2 antibody is
bound to KIR3DL2. An antibody epitope will may thus include such
residue and may include additional residues spatially adjacent to
such residue.
[0235] In some embodiments, a significant reduction in binding
means that the binding affinity and/or capacity between an
anti-KIR3DL2 antibody and a mutant KIR3DL2 polypeptide is reduced
by greater than 40%, greater than 50%, greater than 55%, greater
than 60%, greater than 65%, greater than 70%, greater than 75%,
greater than 80%, greater than 85%, greater than 90% or greater
than 95% relative to binding between the antibody and a wild type
KIR3DL2 polypeptide (e.g., the polypeptide shown in SEQ ID NO:1).
In certain embodiments, binding is reduced below detectable limits.
In some embodiments, a significant reduction in binding is
evidenced when binding of an anti-KIR3DL2 antibody to a mutant
KIR3DL2 polypeptide is less than 50% (e.g., less than 45%, 40%,
35%, 30%, 25%, 20%, 15% or 10%) of the binding observed between the
anti-KIR3DL2 antibody and a wild-type KIR3DL2 polypeptide (e.g.,
the extracellular domain shown in SEQ ID NO:1). Such binding
measurements can be made using a variety of binding assays known in
the art. A specific example of one such assay is described in the
Example section.
[0236] In some embodiments, anti-KIR3DL2 antibodies exhibit
significantly lower binding for a mutant KIR3DL2 polypeptide in
which a residue in a wild-type KIR3DL2 polypeptide (e.g., SEQ ID
NO:1) is substituted, e.g. the mutants as described in Example 1.
In the shorthand notation used here, the format is: Wild type
residue: Position in polypeptide: Mutant residue, with the
numbering of the residues as indicated in SEQ ID NO: 1.
[0237] Optionally, the antibodies have reduced binding to a KIR3DL2
polypeptide having a substitution at residues N99, H100, E130,
H131, F132, V178, P179, H180, S181, P182, Y183 and/or residue Q184
of SEQ ID NO: 1.
[0238] In some embodiments, an anti-KIR3DL2 antibody binds a
wild-type KIR3DL2 polypeptide having a sequence of SEQ ID NO: 1 but
has decreased binding to a mutant KIR3DL2 polypeptide having any
one or more (e.g., 1, 2, 3 or 4) of the following mutations: P179T
and/or S181T (with reference to SEQ ID NO:1). In one embodiment,
binding to the mutant KIR3DL2 is significantly reduced compared to
binding to the wild-type KIR3DL2.
[0239] In some embodiments, anti-KIR3DL2 antibodies exhibit
significantly lower binding for a mutant KIR3DL2 polypeptide in
which a residue in a segment corresponding to residues 1-98,
residues 99-292, or residues 99-192 (or a subsequence thereof) in a
wild-type KIR3DL2 polypeptide (e.g., SEQ ID NO:1) is substituted
with a different amino acid.
[0240] In one aspect, an antibody can compete with monoclonal
antibody 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9
and recognizes bind to, or have immunospecificity for substantially
or essentially the same, or the same, epitope or "epitopic site" on
a KIR3DL2 molecule as monoclonal antibody 10F6, 2B12, 18C6, 9E10,
10G5, 13H1, 5H1, 1E2, 1C3 or 20E9. In other embodiments, the
monoclonal antibody consists of, or is a derivative or fragment of,
antibody 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or
20E9.
[0241] It will be appreciated that, while antibodies may bind to
the same epitope as antibody 10F6, 2B12, 18C6, 9E10, 10G5, 13H1,
5H1, 1E2, 1C3 or 20E9, suitable antibodies can recognize and be
raised against any part of the KIR3DL2 polypeptide so long as the
antibody binds KIR3DL2 and has the desired functionality. For
example, any fragment of KIR3DL2, e.g., human KIR3DL2, or any
combination of KIR3DL2 fragments, can be used as immunogens to
raise antibodies, and the antibodies can recognize epitopes at any
location within the KIR3DL2 polypeptide, so long as they can do so
on KIR3DL2 expressing NK cells as described herein. In an
embodiment, the recognized epitopes are present on the cell
surface, i.e. they are accessible to antibodies present outside of
the cell. Optionally, the epitope is the epitope specifically
recognized by antibody 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1,
1E2, 1C3 or 20E9. Further, antibodies recognizing distinct epitopes
within KIR3DL2 can be used in combination, e.g. to bind to KIR3DL2
polypeptides with maximum efficacy and breadth among different
individuals.
[0242] The antibodies may be produced by a variety of techniques
known in the art. Typically, they are produced by immunization of a
non-human animal, optionally a mouse, with an immunogen comprising
a KIR3DL2 polypeptide, optionally a human KIR3DL2 polypeptide. The
KIR3DL2 polypeptide may comprise the full length sequence of a
human KIR3DL2 polypeptide, or a fragment or derivative thereof,
typically an immunogenic fragment, i.e., a portion of the
polypeptide comprising an epitope exposed on the surface of cells
expressing a KIR3DL2 polypeptide, optionally the epitope recognized
by the 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9
antibody. Such fragments typically contain at least about 7
consecutive amino acids of the mature polypeptide sequence, or at
least about 10 consecutive amino acids thereof. Fragments typically
are essentially derived from the extra-cellular domain of the
receptor. In one embodiment, the immunogen comprises a wild-type
human KIR3DL2 polypeptide in a lipid membrane, typically at the
surface of a cell. In one embodiment, the immunogen comprises
intact cells, particularly intact human cells, optionally treated
or lysed. In another embodiment, the polypeptide is a recombinant
KIR3DL2 polypeptide.
[0243] The step of immunizing a non-human mammal with an antigen
may be carried out in any manner well known in the art for
stimulating the production of antibodies in a mouse (see, for
example, E. Harlow and D. Lane, Antibodies: A Laboratory Manual.,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1988), the entire disclosure of which is herein incorporated by
reference). For exemplary monoclonal antibodies, the next step is
the isolation of splenocytes from the immunized non-human mammal
and the subsequent fusion of those splenocytes with an immortalized
cell in order to form an antibody-producing hybridoma. Once
isolated and present in single cell suspension, lymphocytes can be
fused to an immortal cell line.
[0244] Antibodies may also be produced by selection of
combinatorial libraries of immunoglobulins, as disclosed for
instance in (Ward et al. Nature, 341 (1989) p. 544, the entire
disclosure of which is herein incorporated by reference).
[0245] The identification of one or more antibodies that bind(s) to
KIR3DL2 can be readily determined using any one of a variety of
immunological screening assays in which antibody competition can be
assessed. Many such assays are routinely practiced and are well
known in the art (see, e.g., U.S. Pat. No. 5,660,827, issued Aug.
26, 1997, which is specifically incorporated herein by reference).
It will be understood that actually determining the epitope to
which an antibody described herein binds is not in any way required
to identify an antibody that binds to the same or substantially the
same epitope as the monoclonal antibody described herein.
[0246] For example, where the test antibodies to be examined are
obtained from different source animals, or are even of a different
Ig isotype, a simple competition assay may be employed in which the
control (10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9,
for example) and test antibodies are admixed (or pre-adsorbed) and
applied to a sample containing KIR3DL2 polypeptides. Protocols
based upon western blotting and the use of BIACORE analysis are
suitable for use in such competition studies.
[0247] In certain embodiments, one pre-mixes the control antibodies
(10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9, for
example) with varying amounts of the test antibodies (e.g., about
1:10 or about 1:100) for a period of time prior to applying to the
KIR3DL2 antigen sample. In other embodiments, the control and
varying amounts of test antibodies can simply be admixed during
exposure to the KIR3DL2 antigen sample. As long as one can
distinguish bound from free antibodies (e.g., by using separation
or washing techniques to eliminate unbound antibodies) and 10F6,
2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 from the test
antibodies (e.g., by using species-specific or isotype-specific
secondary antibodies or by specifically labeling 10F6, 2B12, 18C6,
9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 with a detectable label)
one can determine if the test antibodies reduce the binding of
10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 to the
antigens. The binding of the (labeled) control antibodies in the
absence of a completely irrelevant antibody can serve as the
control high value. The control low value can be obtained by
incubating the labeled (10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1,
1E2, 1C3 or 20E9) antibodies with unlabelled antibodies of exactly
the same type (10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or
20E9), where competition would occur and reduce binding of the
labeled antibodies. In a test assay, a significant reduction in
labeled antibody reactivity in the presence of a test antibody is
indicative of a test antibody that may recognize substantially the
same epitope. A test antibody may for example reduce the binding of
10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 to
KIR3DL2 antigens by at least about 50%, such as at least about 60%,
or more preferably at least about 80% or 90% (e.g., about 65-100%),
at any ratio of 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3
or 20E9: test antibody between about 1:10 and about 1:100. For
example such test antibody can reduce the binding of 10F6, 2B12,
18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 to the KIR3DL2
antigen by at least about 90% (e.g., about 95%).
[0248] Competition can also be assessed by, for example, a flow
cytometry test. In such a test, cells bearing a given KIR3DL2
polypeptide can be incubated first with 10F6, 2B12, 18C6, 9E10,
10G5, 13H1, 5H1, 1E2, 1C3 or 20E9, for example, and then with the
test antibody labeled with a fluorochrome or biotin. The antibody
is said to compete with 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1,
1E2, 1C3 or 20E9 if the binding obtained upon preincubation with a
saturating amount of 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2,
1C3 or 20E9 is about 80%, about 50%, about 40% or less (e.g., about
30%, 20% or 10%) of the binding (as measured by mean of
fluorescence) obtained by the antibody without preincubation with
10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9.
Alternatively, an antibody is said to compete with 10F6, 2B12,
18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 if the binding
obtained with a labeled 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1,
1E2, 1C3 or 20E9 antibody (by a fluorochrome or biotin) on cells
preincubated with a saturating amount of test antibody is about
80%, about 50%, about 40%, or less (e.g., about 30%, 20% or 10%) of
the binding obtained without preincubation with the test antibody.
A simple competition assay in which a test antibody is pre-adsorbed
and applied at saturating concentration to a surface onto which a
KIR3DL2 antigen is immobilized may also be employed. The surface in
the simple competition assay is for example a BIACORE chip (or
other media suitable for surface plasmon resonance analysis). The
control antibody (e.g., 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1,
1E2, 1C3 or 20E9) is then brought into contact with the surface at
a KIR3DL2-saturating concentration and the KIR3DL2 and surface
binding of the control antibody is measured. This binding of the
control antibody is compared with the binding of the control
antibody to the KIR3DL2-containing surface in the absence of test
antibody. In a test assay, a significant reduction in binding of
the KIR3DL2-containing surface by the control antibody in the
presence of a test antibody indicates that the test antibody
competes and may recognize substantially the same epitope as the
control antibody. Any test antibody that reduces the binding of
control (such as 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3
or 20E9) antibody to a KIR3DL2 antigen by at least about 30% or
more, or about 40%, can be selected. For example, such a test
antibody will reduce the binding of the control antibody (e.g.,
10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9) to the
KIR3DL2 antigen by at least about 50% (e.g., at least about 60%, at
least about 70%, or more). It will be appreciated that the order of
control and test antibodies can be reversed: that is, the control
antibody can be first bound to the surface and the test antibody is
brought into contact with the surface thereafter in a competition
assay. For example, the antibody having higher affinity for the
KIR3DL2 antigen is bound to the surface first, as it will be
expected that the decrease in binding seen for the second antibody
(assuming the antibodies are cross-reacting) will be of greater
magnitude. Further examples of such assays are provided in, e.g.,
Saunal (1995) J. Immunol. Methods 183: 33-41, the disclosure of
which is incorporated herein by reference.
[0249] Determination of whether an antibody binds within an epitope
region can be carried out in ways known to the person skilled in
the art. As one example of such mapping/characterization methods,
an epitope region for an anti-KIR3DL2 antibody may be determined by
epitope "foot-printing" using chemical modification of the exposed
amines/carboxyls in the KIR3DL2 protein. One specific example of
such a foot-printing technique is the use of HXMS
(hydrogen-deuterium exchange detected by mass spectrometry) wherein
a hydrogen/deuterium exchange of receptor and ligand protein amide
protons, binding, and back exchange occurs, wherein the backbone
amide groups participating in protein binding are protected from
back exchange and therefore will remain deuterated. Relevant
regions can be identified at this point by peptic proteolysis, fast
microbore high-performance liquid chromatography separation, and/or
electrospray ionization mass spectrometry. See, e.g., Ehring H,
Analytical Biochemistry, Vol. 267 (2) pp. 252-259 (1999) Engen, J.
R. and Smith, D. L. (2001) Anal. Chem. 73, 256A-265A. Another
example of a suitable epitope identification technique is nuclear
magnetic resonance epitope mapping (NMR), where typically the
position of the signals in two-dimensional NMR spectra of the free
antigen and the antigen complexed with the antigen binding peptide,
such as an antibody, are compared. The antigen typically is
selectively isotopically labeled with 15N so that only signals
corresponding to the antigen and no signals from the antigen
binding peptide are seen in the NMR-spectrum. Antigen signals
originating from amino acids involved in the interaction with the
antigen binding peptide typically will shift position in the
spectrum of the complex compared to the spectrum of the free
antigen, and the amino acids involved in the binding can be
identified that way. See, e.g., Ernst Schering Res Found Workshop.
2004; (44): 149-67; Huang et Journal of Molecular Biology, Vol. 281
(1) pp. 61-67 (1998); and Saito and Patterson, Methods. 1996 Jun. 9
(3): 516-24.
[0250] Epitope mapping/characterization also can be performed using
mass spectrometry methods. See, e.g., Downward, J Mass Spectrom.
2000 April; 35 (4): 493-503 and Kiselar and Downard, Anal Chem.
1999 May 1; 71 (9): 1792-801. Protease digestion techniques also
can be useful in the context of epitope mapping and identification.
Antigenic determinant-relevant regions/sequences can be determined
by protease digestion, e.g. by using trypsin in a ratio of about
1:50 to KIR3DL2 or o/n digestion at and pH 7-8, followed by mass
spectrometry (MS) analysis for peptide identification. The peptides
protected from trypsin cleavage by the anti-KIR3DL2 binder can
subsequently be identified by comparison of samples subjected to
trypsin digestion and samples incubated with antibody and then
subjected to digestion by e.g. trypsin (thereby revealing a
footprint for the binder). Other enzymes like chymotrypsin, pepsin,
etc., also or alternatively can be used in similar epitope
characterization methods. Moreover, enzymatic digestion can provide
a quick method for analyzing whether a potential antigenic
determinant sequence is within a region of the KIR3DL2 polypeptide
that is not surface exposed and, accordingly, most likely not
relevant in terms of immunogenicity/antigenicity. See, e.g., Manca,
Ann 1st Super Sanita. 1991; 27: 15-9 for a discussion of similar
techniques.
[0251] Site-directed mutagenesis is another technique useful for
elucidation of a binding epitope. For example, in
"alanine-scanning", each residue within a protein segment is
replaced with an alanine residue, and the consequences for binding
affinity measured. If the mutation leads to a significant reduction
in binding affinity, it is most likely involved in binding.
Monoclonal antibodies specific for structural epitopes (i.e.,
antibodies which do not bind the unfolded protein) can be used to
verify that the alanine-replacement does not influence overall fold
of the protein. See, e.g., Clackson and Wells, Science 1995;
267:383-386; and Wells, Proc Natl Acad Sci USA 1996; 93:1-6.
[0252] Electron microscopy can also be used for epitope
"foot-printing". For example, Wang et al., Nature 1992; 355:275-278
used coordinated application of cryoelectron microscopy,
three-dimensional image reconstruction, and X-ray crystallography
to determine the physical footprint of a Fab-fragment on the capsid
surface of native cowpea mosaic virus.
[0253] Other forms of "label-free" assay for epitope evaluation
include surface plasmon resonance (SPR, BIACORE) and reflectometric
interference spectroscopy (RifS). See, e.g., Fagerstam et al.,
Journal Of Molecular Recognition 1990; 3:208-14; Nice et al., J.
Chromatogr. 1993; 646:159-168; Leipert et al., Angew. Chem. Int.
Ed. 1998; 37:3308-3311; Kroger et al., Biosensors and
Bioelectronics 2002; 17:937-944.
[0254] It should also be noted that an antibody binding the same or
substantially the same epitope as an antibody can be identified in
one or more of the exemplary competition assays described
herein.
[0255] Once antibodies are identified that are capable of binding
KIR3DL2 and/or having other desired properties, they will also
typically be assessed, using standard methods including those
described herein, for their ability to bind to other polypeptides,
including unrelated polypeptides. Ideally, the antibodies only bind
with substantial affinity to KIR3DL2, e.g., human KIR3DL2, and do
not bind at a significant level to unrelated polypeptides. However,
it will be appreciated that, as long as the affinity for KIR3DL2 is
substantially greater (e.g., 5.times., 10.times., 50.times.,
100.times., 500.times., 1000.times., 10,000.times., or more) than
it is for other, unrelated polypeptides), then the antibodies are
suitable for use in the present methods.
[0256] In one aspect of any of the embodiments, the antibodies
prepared according to the present methods are monoclonal
antibodies. In another aspect, the non-human animal used iesto
produce antibodies is a mammal, such as a rodent, bovine, porcine,
fowl, horse, rabbit, goat, or sheep.
[0257] According to an alternate embodiment, the DNA encoding an
antibody that binds an epitope present on KIR3DL2 polypeptides is
isolated from the hybridoma and placed in an appropriate expression
vector for transfection into an appropriate host. The host is then
used for the recombinant production of the antibody, or variants
thereof, such as a humanized version of that monoclonal antibody,
active fragments of the antibody, chimeric antibodies comprising
the antigen recognition portion of the antibody, or versions
comprising a detectable moiety.
[0258] DNA encoding a monoclonal antibody, e.g., antibody 10F6,
2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9, can be readily
isolated and sequenced using conventional procedures (e.g., by
using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies). Once isolated, the DNA can be placed into expression
vectors, which are then transfected into host cells such as E. coli
cells, simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of monoclonal antibodies in the recombinant
host cells. As described elsewhere in the present specification,
such DNA sequences can be modified for any of a large number of
purposes, e.g., for humanizing antibodies, producing fragments or
derivatives, or for modifying the sequence of the antibody, e.g.,
in the antigen binding site in order to optimize the binding
specificity of the antibody.
[0259] Recombinant expression in bacteria of DNA encoding the
antibody is well known in the art (see, for example, Skerra et al.,
Curr. Opinion in Immunol., 5, pp. 256 (1993); and Pluckthun,
Immunol. 130, p. 151 (1992).
[0260] In one embodiment, an antibody is capable of mediating the
depletion of pathogenic KIR3DL2-expressing cells (e.g. tumor cells)
via ADCC (and optionally further via ADCP). Once an antigen-binding
compound is obtained it may be assessed for its ability to induce
ADCC towards, inhibit the activity and/or proliferation of and/or
cause the elimination of KIR3DL2-expressing target cells. Assessing
the antigen-binding compound's ability to induce ADCC or generally
lead to the elimination or inhibition of activity of
KIR3DL2-expressing target cells, can be carried out at any suitable
stage of the method. This assessment can be useful at one or more
of the various steps involved in the identification, production
and/or development of an antibody (or other compound) destined for
therapeutic use. For example, activity may be assessed in the
context of a screening method to identify candidate antigen-binding
compounds, or in methods where an antigen-binding compound is
selected and made human suitable (e.g. made chimeric or humanized
in the case of an antibody), where a cell expressing the
antigen-binding compound (e.g. a host cell expressing a recombinant
antigen-binding compound) has been obtained and is assessed for its
ability to produce functional antibodies (or other compounds),
and/or where a quantity of antigen-binding compound has been
produced and is to be assessed for activity (e.g. to test batches
or lots of product). Generally the antigen-binding compound will be
known to specifically bind to a KIR3DL2 polypeptide. The step may
involve testing a plurality (e.g., a very large number using high
throughput screening methods or a smaller number) of
antigen-binding compounds.
[0261] Testing ADCC can be carried out can be determined by various
assays including those known in the art and those described in the
experimental examples herein. Testing ADCC typically involves
assessing cell-mediated cytotoxicity in which a KIR3DL2-expressing
target cell (e.g. a celiac disease cell or other KIR3DL2-expressing
cell) with bound anti-KIR3DL2 antibody is recognized by an effector
cell bearing Fc receptors, without the involvement of complement. A
cell which does not express a KIR3DL2 antigen can optionally be
used as a control. Activation of NK cell cytotoxicity is assessed
by measuring an increase in cytokine production (e.g. IFN-.gamma.
production) or cytotoxicity markers (e.g. CD107 mobilization). In
one embodiment, the antibody will induce an increase in cytokine
production, expression of cytotoxicity markers, or target cell
lysis of at least 20%, 50%, 80%, 100%, 200% or 500% in the presence
of target cells, compared to a control antibody (e.g. an antibody
not binding to KIR3DL2, a KIR3DL2 antibody having murine constant
regions). In another example, lysis of target cells is detected,
e.g. in a chromium release assay, for example the antibody will
induce lysis of at least 10%, 20%, 30%, 40% or 50% of target
cells.
[0262] In one embodiment, an anti-KIR3DL2 antibody does not
substantially increase or induce intracellular internalization of
KIR3DL2 expressed at the surface of a cell. As used herein, an
anti-KIR3DL2 antibody that is not "internalized" or that does not
"internalize" is one that is not substantially taken up by (i.e.,
enters) the cell upon binding to KIR3DL2 on a mammalian cell (i.e.
cell surface KIR3DL2).
[0263] In one embodiment, an anti-KIR3DL2 antibody is capable of
causing an increase of cell surface KIR3DL2 polypeptide available
for binding by an anti-KIR3DL2 antibody, notably on malignant
cells. The antibodies may, in one embodiment, increase the level of
expression of KIR3DL2 polypeptides on the cell surface (e.g. of
malignant cells). The antibodies may, in one embodiment, increase
the amount or number of KIR3DL2 polypeptides on the cell surface
available for binding by an anti-KIR3DL antibody. The antibodies
may, in one embodiment, stabilize and/or cause accumulation of
KIR3DL2 polypeptides present on the cell surface, e.g., they may
decrease receptor cycling or internalization of KIR3DL2
polypeptides. Antibodies that increase cell surface KIR3DL2, e.g.
on pathogenic CD4+ T cells, have increased potency because they
permit a greater number of antibodies to be bound to a
KIR3DL2-expressing cell (e.g. target cell, malignant cell). In one
embodiment, provided is an isolated monoclonal antibody that binds
a KIR3DL2 polypeptide on the surface of a KIR3DL2-expressing cell,
wherein the antibody causes an increase of the amount or numbers of
KIR3DL2 polypeptides detectable at the cell surface after being in
contact with cells (in vivo or in vitro) for at least 1 hour, 3
hours, 6 hours, 12 hours or 24 hours. The increase can be in
comparison to a control antibody, e.g. an isotype control, or
another antibody that binds KIR3DL2 (e.g. an antibody that has a
different heavy and/or light chain variable region amino acid
sequence).
[0264] Whether an anti-KIR3DL2 antibody internalizes upon binding
KIR3DL2 on a mammalian cell, or whether a KIR3DL2 polypeptide
undergoes intracellular internalization (e.g. upon being bound by
an antibody) can be determined by various assays including those
described in the experimental examples PCT/EP2013/069302 and
PCT/EP2013/069293, both filed 17 Sep. 2013. For examples cells can
be incubated in tissue culture dishes in the presence or absence of
the relevant antibodies added to the culture media and processed
for microscopic analysis at desired time points. The presence of an
internalized, labelled antibody in the cells can be directly
visualized by microscopy or by autoradiography if radiolabelled
antibody is used. Optionally, in microscopy, co-localization with a
known polypeptide or other cellular component can be assessed; for
example co-localization with endosomal/lysosomal marker LAMP-1
(CD107a) can provide information about the subcellular localization
of the internalized antibody.
[0265] Testing whether an antibody is capable of increasing the
number of KIR3DL2 polypeptides at the surface of a cell can be
carried out by incubating the test antibody with a
KIR3DL2-expressing cell (e.g. a T cell lymphoma) and detecting
KIR3DL2 polypeptides at the surface of the cell after the
incubation period. KIR3DL2 polypeptides can be carried out using a
suitable affinity regent, e.g. one or more antibodies. Exemplary
assays are shown in PCT/EP2013/069302 and PCT/EP2013/069293. For
example, an antibody may induce an increase of at least 20%, 50%,
75% or 100% of the number of KIR3DL2 polypeptides detectable at the
surface of cells after incubation (e.g. for at least 1, 3, 6, 12,
24 or 48 hours) in the presence of test antibody, compared to a
control antibody (e.g. an antibody not binding to KIR3DL2, a
different anti-KIR3DL2 antibody). Optionally, the number of KIR3DL2
polypeptides detectable at the surface of cells after incubation is
the number detectable using the test antibody. Optionally, the
number of KIR3DL2 polypeptides detectable at the surface of cells
after incubation is the number detectable using a second
anti-KIR3DL2 antibody that does not compete with the test antibody
for binding to KIR3DL2.
[0266] In one embodiment, an anti-KIR3DL2 antibody can be tested
for its ability to detectably reduce (or eliminate) binding between
the KIR3DL2 and an HLA natural ligand of KIR3DL2. Exemplary assays
are shown in PCT/EP2013/069302 and PCT/EP2013/069293. In one
embodiment, provided is an antibody that binds a KIR3DL2
polypeptide, wherein said antibody detectably reduces (or
eliminates) binding between the KIR3DL2 and a first HLA natural
ligand of KIR3DL2 but does not detectably reduce (or eliminate)
binding between the KIR3DL2 and a second HLA natural ligand of
KIR3DL2.
[0267] In one embodiment, the antibody optionally detectably
reduces binding between the KIR3DL2 and an HLA class 1-ligand of
KIR3DL2 (e.g. HLA-B27). In one embodiment, the antibody optionally
detectably reduces binding between the KIR3DL2 and HLA-B27 but does
not detectably reduce binding between KIR3DL2 and HLA-A3.
[0268] When an agent that binds a KIR3DL2 polypeptide has been
identified, it can be tested to determine the concentration that
provide a specified "NK % lytic capacity" by testing the ability of
NK cells to lyse tumor cells (e.g. HUT78 cells) in an in vitro
cytotoxicity assay, as measured in a .sup.51Cr release assay, by
the percentage of maximal tumor cell lysis obtained (=Tumor cell
lysis/Max tumor cell lysis at saturation.times.100). Examples of
suitable assays employing PBMC and HUT78 cells as effector and
target cells are described in the Examples herein. The anti-KIR3DL2
agent can be tested to determine the EC.sub.10, the EC.sub.60,
EC.sub.80, EC.sub.90, or EC.sub.100 of its maximum response or
effect with respect to such NK lytic capacity. Typically, the NK
cells are NK cells from a healthy human donor, e.g. within PBMC. A
suitable number of experiments using samples from different donors
can be carried out, e.g. 10, 20 or more different donor
samples.
[0269] In certain embodiments, the DNA of a hybridoma producing an
antibody can be modified prior to insertion into an expression
vector, for example, by substituting the coding sequence for human
heavy- and light-chain constant domains in place of the homologous
non-human sequences (e.g., Morrison et al., PNAS pp. 6851 (1984)),
or by covalently joining to the immunoglobulin coding sequence all
or part of the coding sequence for a non-immunoglobulin
polypeptide. In that manner, "chimeric" or "hybrid" antibodies are
prepared that have the binding specificity of the original
antibody. Typically, such non-immunoglobulin polypeptides are
substituted for the constant domains of an antibody.
[0270] Thus, according to another embodiment, the antibody is
humanized. "Humanized" forms of antibodies according are specific
chimeric immunoglobulins, immunoglobulin chains or fragments
thereof (such as Fv, Fab, Fab', F (ab') 2, or other antigen-binding
subsequences of antibodies) which contain minimal sequence derived
from the murine immunoglobulin. For the most part, humanized
antibodies are human immunoglobulins (recipient antibody) in which
residues from a complementary-determining region (CDR) of the
recipient are replaced by residues from a CDR of the original
antibody (donor antibody) while maintaining the desired
specificity, affinity, and capacity of the original antibody.
[0271] In some instances, Fv framework residues of the human
immunoglobulin may be replaced by corresponding non-human residues.
Furthermore, humanized antibodies can comprise residues that are
not found in either the recipient antibody or in the imported CDR
or framework sequences. These modifications are made to further
refine and optimize antibody performance. In general, the humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the CDR regions correspond to those of the original antibody and
all or substantially all of the FR regions are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally
also will comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin. For further
details see Jones et al., Nature, 321, pp. 522 (1986); Reichmann et
al, Nature, 332, pp. 323 (1988); Presta, Curr. Op. Struct. Biol.,
2, pp. 593 (1992); Verhoeyen et Science, 239, pp. 1534; and U.S.
Pat. No. 4,816,567, the entire disclosures of which are herein
incorporated by reference.) Methods for humanizing the antibodies
are well known in the art.
[0272] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity. According to the so-called "best-fit" method,
the sequence of the variable domain of an antibody is screened
against the entire library of known human variable-domain
sequences. The human sequence which is closest to that of the mouse
is then accepted as the human framework (FR) for the humanized
antibody (Sims et al., J. Immunol. 151, pp. 2296 (1993); Chothia
and Lesk, J. Mol. 196, 1987, pp. 901). Another method uses a
particular framework from the consensus sequence of all human
antibodies of a particular subgroup of light or heavy chains. The
same framework can be used for several different humanized
antibodies (Carter et al., PNAS 89, pp. 4285 (1992); Presta et al.,
J. Immunol., 151, p. 2623 (1993)).
[0273] It is further important that antibodies be humanized with
retention of high affinity for KIR3DL2 receptors and other
favorable biological properties. To achieve this goal, according to
one method, humanized antibodies are prepared by a process of
analysis of the parental sequences and various conceptual humanized
products using three-dimensional models of the parental and
humanized sequences. Three-dimensional immunoglobulin models are
commonly available and are familiar to those skilled in the art.
Computer programs are available which illustrate and display
probable three-dimensional structures of selected candidate
immunoglobulin sequences. Inspection of these displays permits
analysis of the likely role of the residues in the functioning of
the candidate immunoglobulin sequence, i.e., the analysis of
residues that influence the ability of the candidate immunoglobulin
to bind its antigen. In this way, FR residues can be selected and
combined from the consensus and import sequences so that the
desired antibody characteristic, such as increased affinity for the
target antigen (s), is achieved. In general, the CDR residues are
directly and most substantially involved in influencing antigen
binding.
[0274] Another method of making "humanized" monoclonal antibodies
is to use a XenoMouse (Abgenix, Fremont, Calif.) as the mouse used
for immunization. A XenoMouse is a murine host that has had its
immunoglobulin genes replaced by functional human immunoglobulin
genes. Thus, antibodies produced by this mouse or in hybridomas
made from the B cells of this mouse, are already humanized. The
XenoMouse is described in U.S. Pat. No. 6,162,963, which is herein
incorporated in its entirety by reference.
[0275] Human antibodies may also be produced according to various
other techniques, such as by using, for immunization, other
transgenic animals that have been engineered to express a human
antibody repertoire (Jakobovitz et al., Nature 362 (1993) 255), or
by selection of antibody repertoires using phage display methods.
Such techniques are known to the skilled person and can be
implemented starting from monoclonal antibodies as disclosed in the
present application.
[0276] In view of the ability of the anti-KIR3DL2 antibodies to
induce ADCC, the antibodies can be made with modifications that
increase their ability to bind Fc receptors which can affect
effector functions such as antibody-dependent cytotoxicity, mast
cell degranulation, and phagocytosis, as well as immunomodulatory
signals such as regulation of lymphocyte proliferation and antibody
secretion. Typical modifications include modified human IgG1
constant regions comprising at least one amino acid modification
(e.g. substitution, deletions, insertions), and/or altered types of
glycosylation, e.g., hypofucosylation. Such modifications can
affect interaction with Fc receptors: Fc.gamma.RI (CD64),
Fc.gamma.RII (CD32), and Fc.gamma.RIII (CD 16). Fc.gamma.RI (CD64),
Fc.gamma.RIIA (CD32A) and Fc.gamma.RIII (CD 16) are activating
(i.e., immune system enhancing) receptors while Fc.gamma.RIIB
(CD32B) is an inhibiting (i.e., immune system dampening) receptor.
A modification may, for example, increase binding of the Fc domain
to Fc.gamma.RIIIa on effector (e.g. NK) cells.
[0277] Anti-KIR3DL2 antibodies may comprise an Fc domain (or
portion thereof) of human IgG1 or IgG3 isotype, optionally
modified. Residues 230-341 (Kabat EU) are the Fc CH2 region.
Residues 342-447 (Kabat EU) are the Fc CH3 region. Anti-KIR3DL2
antibodies may comprise a variant Fc region having one or more
amino acid modifications (e.g., substitutions, deletions,
insertions) in one or more portions, which modifications increase
the affinity and avidity of the variant Fc region for an Fc.gamma.R
(including activating and inhibitory Fc.gamma.Rs). In some
embodiments, said one or more amino acid modifications increase the
affinity of the variant Fc region for Fc.gamma.RIIIA and/or
Fc.gamma.RIIA. In another embodiment, the variant Fc region further
specifically binds Fc.gamma.RIIB with a lower affinity than does
the Fc region of the comparable parent antibody (i.e., an antibody
having the same amino acid sequence as the antibody except for the
one or more amino acid modifications in the Fc region). For
example, the one or both of the histidine residues at amino acid
positions 310 and 435 may be substituted, for example by lysine,
alanine, glycine, valine, leucine, isoleucine, proline, methionine,
tryptophan, phenylalanine, serine or threonine (see, e.g. PCT
publication no. WO 2007/080277); such substituted constant regions
provide decreased binding to the inhibitory Fc.gamma.RIIB without
decreasing binding to the activatory Fc.gamma.RIIIA. In some
embodiments, such modifications increase the affinity of the
variant Fc region for Fc.gamma.RIIIA and/or Fc.gamma.RIIA and also
enhance the affinity of the variant Fc region for Fc.gamma.yRIIB
relative to the parent antibody. In other embodiments, said one or
more amino acid modifications increase the affinity of the variant
Fc region for Fc.gamma.RIIIA and/or Fc.gamma.RIIA but do not alter
the affinity of the variant Fc regions for Fc.gamma.RIIB relative
to the Fc region of the parent antibody. In another embodiment,
said one or more amino acid modifications enhance the affinity of
the variant Fc region for Fc.gamma.RIIIA and Fc.gamma.RIIA but
reduce the affinity for Fc.gamma.RIIB relative to the parent
antibody. Increased affinity and/or avidity results in detectable
binding to the Fc.gamma.R or Fc.gamma.R-related activity in cells
that express low levels of the Fc.gamma.R when binding activity of
the parent molecule (without the modified Fc region) cannot be
detected in the cells.
[0278] The affinities and binding properties of the molecules for
an Fc.gamma.R can be determined using in vitro assays (biochemical
or immunological based assays) known in the art for determining
antibody-antigen or Fc-Fc.gamma.R interactions, i.e., specific
binding of an antigen to an antibody or specific binding of an Fc
region to an Fc.gamma.R, respectively, including but not limited to
ELISA assay, surface plasmon resonance assay, immunoprecipitation
assays.
[0279] Specific mutations (in IgG1 Fc domains) which affect
(enhance) Fc.gamma.RIIIa or FcRn binding are also set forth
below.
TABLE-US-00007 Isotype Species Modification Effector Function
Effect of Modification IgG1 Human T250Q/M428L Increased Increased
half-life binding to FcRn IgG1 Human 1M252Y/S254T/T256E + Increased
Increased half-life H433K/N434F binding to FcRn IgG1 Human E333A
Increased Increased ADCC and binding to CDC Fc.gamma.RIIIa IgG1
Human S239D/I332E or Increased Increased ADCC S239D/A330L/I332E
binding to Fc.gamma.RIIIa IgG1 Human P257I/Q311 Increased Unchanged
half-life binding to FcRn IgG1 Human S239D/I332E/G236A Increased
Increased macrophage Fc.gamma.RIIa/Fc.gamma.RIIb phagocytosis
ratio
[0280] In some embodiments, the molecules comprising a variant Fc
region comprise at least one amino acid modification (for example,
possessing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid
modifications) in the CH3 domain of the Fc region. In other
embodiments, the molecules comprising a variant Fc region comprise
at least one amino acid modification (for example, possessing 1, 2,
3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) in the CH2
domain of the Fc region, which is defined as extending from amino
acids 231-341. In some embodiments, the molecules comprise at least
two amino acid modifications (for example, possessing 2, 3, 4, 5,
6, 7, 8, 9, or more amino acid modifications), wherein at least one
such modification is in the CH3 region and at least one such
modification is in the CH2 region. Amino acid modifications may be
made for example in the hinge region. In a particular embodiment,
the invention encompasses amino acid modification in the CH1 domain
of the Fc region, which is defined as extending from amino acids
216-230.
[0281] Any combination of Fc modifications can be made, for example
any combination of different modifications disclosed in U.S. Pat.
Nos. 7,632,497; 7,521,542; 7,425,619; 7,416,727; 7,371,826;
7,355,008; 7,335,742; 7,332,581; 7,183,387; 7,122,637; 6,821,505
and 6,737,056; in PCT Publications Nos. WO2011/109400; WO
2008/105886; WO 2008/002933; WO 2007/021841; WO 2007/106707; WO
06/088494; WO 05/115452; WO 05/110474; WO 04/1032269; WO 00/42072;
WO 06/088494; WO 07/024249; WO 05/047327; WO 04/099249 and WO
04/063351; and in Presta, L. G. et al. (2002) Biochem. Soc. Trans.
30(4):487-490; Shields, R. L. et al. (2002) J. Biol. Chem. 26;
277(30):26733-26740 and Shields, R. L. et al. (2001) J. Biol. Chem.
276(9):6591-6604).
[0282] Anti-KIR3DL2 antibodies may comprise a variant Fc region,
wherein the variant Fc region comprises at least one amino acid
modification (for example, possessing 1, 2, 3, 4, 5, 6, 7, 8, 9, or
more amino acid modifications) relative to a wild-type Fc region,
such that the molecule has an enhanced effector function relative
to a molecule comprising a wild-type Fc region, optionally wherein
the variant Fc region comprises a substitution at any one or more
of positions 221, 239, 243, 247, 255, 256, 258, 267, 268, 269, 270,
272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295,
296, 298, 300, 301, 303, 305, 307, 308, 309, 310, 311, 312, 316,
320, 322, 326, 329, 330, 332, 331, 332, 333, 334, 335, 337, 338,
339, 340, 359, 360, 370, 373, 376, 378, 392, 396, 399, 402, 404,
416, 419, 421, 430, 434, 435, 437, 438 and/or 439.
[0283] Anti-KIR3DL2 antibodies may comprise a variant Fc region,
wherein the variant Fc region comprises at least one amino acid
modification (for example, possessing 1, 2, 3, 4, 5, 6, 7, 8, 9, or
more amino acid modifications) relative to a wild-type Fc region,
such that the molecule has an enhanced effector function relative
to a molecule comprising a wild-type Fc region, optionally wherein
the variant Fc region comprises a substitution at any one or more
of positions 329, 298, 330, 332, 333 and/or 334 (e.g. S239D, S298A,
A330L, 1332E, E333A and/or K334A substitutions).
[0284] In one embodiment, antibodies having variant or wild-type Fc
regions may have altered glycosylation patterns that increase Fc
receptor binding ability of antibodies. Such carbohydrate
modifications can be accomplished by, for example, expressing the
antibody in a host cell with altered glycosylation machinery. Cells
with altered glycosylation machinery have been described in the art
and can be used as host cells in which to express recombinant
antibodies to thereby produce an antibody with altered
glycosylation. See, for example, Shields, R. L. et al. (2002) J.
Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech.
17:176-1, as well as, European Patent No: EP 1,176,195; PCT
Publications WO 06/133148; WO 03/035835; WO 99/54342, each of which
is incorporated herein by reference in its entirety.
[0285] Generally, such antibodies with altered glycosylation are
"glyco-optimized" such that the antibody has a particular N-glycan
structure that produces certain desirable properties, including but
not limited to, enhanced ADCC and effector cell receptor binding
activity when compared to non-modified antibodies or antibodies
having a naturally occurring constant region and produced by murine
myeloma NSO and Chinese Hamster Ovary (CHO) cells (Chu and
Robinson, Current Opinion Biotechnol. 2001, 12: 180-7),
HEK293T-expressed antibodies as produced herein in the Examples
section, or other mammalian host cell lines commonly used to
produce recombinant therapeutic antibodies.
[0286] Monoclonal antibodies produced in mammalian host cells
contain an N-linked glycosylation site at Asn297 of each heavy
chain. Glycans on antibodies are typically complex biatennary
structures with very low or no bisecting N-acetylglucosamine
(bisecting GlcNAc) and high levels of core fucosylation. Glycan
temini contain very low or no terminal sialic acid and variable
amounts of galactose. For a review of effects of glycosylation on
antibody function, see, e.g., Wright & Morrison, Trend
Biotechnol. 15:26-31(1997). Considerable work shows that changes to
the sugar composition of the antibody glycan structure can alter Fc
effector functions. The important carbohydrate structures
contributing to antibody activity are believed to be the fucose
residues attached via alpha-1,6 linkage to the innermost
N-acetylglucosamine (GlacNAc) residues of the Fc region N-linked
oligosaccharides (Shields et al., 2002).
[0287] Fc.gamma.R binding requires the presence of oligosaccharides
covalently attached at the conserved Asn297 in the Fc region of
human IgGI, IgG2 or IgG3 type. Non-fucosylated oligosaccharides
structures have recently been associated with dramatically
increased in vitro ADCC activity. "Asn 297" means amino acid
asparagine located at about position 297 in the Fc region; based on
minor sequence variations of antibodies, Asn297 can also be located
some amino acids (usually not more than + 3 amino acids) upstream
or downstream.
[0288] Historically, antibodies produced in CHO cells contain about
2 to 6% in the population that are nonfucosylated. YB2/0 (rat
myeloma) and Lecl3 cell line (a lectin mutant of CHO line which has
a deficient GDP-mannose 4,6-dehydratase leading to the deficiency
of GDP-fucose or GDP sugar intermediates that are the substrate of
alpha6-fucosyltransferase have been reported to produce antibodies
with 78 to 98% non-fucosylated species. In other examples, RNA
interference (RNAi) or knock-out techniques can be employed to
engineer cells to either decrease the FUT8 mRNA transcript levels
or knock out gene expression entirely, and such antibodies have
been reported to contain up to 70% non-fucosylated glycan.
[0289] An antibody that binds to KIR3DL2 may be glycosylated with a
sugar chain at Asn297. In one embodiment, an antibody will comprise
a constant region comprising at least one amino acid alteration in
the Fc region that improves antibody binding to FcyRIIIa and/or
ADCC.
[0290] In one aspect, the antibodies are hypofucosylated in their
constant region. Such antibodies may comprise an amino acid
alteration or may not comprise an amino acid alteration but be
produced or treated under conditions so as to yield such
hypofucosylation. In one aspect, an antibody composition comprises
a chimeric, human or humanized antibody described herein, wherein
at least 20, 30, 40, 50, 60, 75, 85, 90, 95% or substantially all
of the antibody species in the composition have a constant region
comprising a core carbohydrate structure (e.g. complex, hybrid and
high mannose structures) which lacks fucose. In one embodiment,
provided is an antibody composition which is free of antibodies
comprising a core carbohydrate structure having fucose. The core
carbohydrate will preferably be a sugar chain at Asn297.
[0291] In one embodiment, an antibody composition, e.g. a
composition comprising antibodies which bind to KIR3DL2, are
glycosylated with a sugar chain at Asn297, wherein the antibodies
are partially fucosylated. Partially fucosylated antibodies are
characterized in that the proportion of anti-KIR3DL2 antibodies in
the composition that lack fucose within the sugar chain at Asn297
is between 20% and 90%, between 20% and 80%, between 20% and 50%,
55%, 60%, 70% or 75%, between 35% and 50%, 55%, 60%, 70% or 75%, or
between 45% and 50%, 55%, 60%, 70% or 75%. Optionally the antibody
is of human IgGI or IgG3 type.
[0292] The sugar chain show can further show any characteristics
(e.g. presence and proportion of complex, hybrid and high mannose
structures), including the characteristics of N-linked glycans
attached to Asn297 of an antibody from a human cell, or of an
antibody recombinantly expressed in a rodent cell, murine cell
(e.g. CHO cell) or in an avian cell.
[0293] In one embodiment, the antibody is expressed in a cell that
is lacking in a fucosyltransferase enzyme such that the cell line
produces proteins lacking fucose in their core carbohydrates. For
example, the cell lines Ms704, Ms705, and Ms709 lack the
fucosyltransferase gene, FUT8 (alpha (1,6) fucosyltransferase),
such that antibodies expressed in the Ms704, Ms705, and Ms709 cell
lines lack fucose on their core carbohydrates. These cell lines
were created by the targeted disruption of the FUT8 gene in
CHO/DG44 cells using two replacement vectors (see U.S. Patent
Publication No. 20040110704 by Yamane et al.; and Yamane-Ohnuki et
al. (2004) Biotechnol Bioeng 87:614-22, the disclosures of which
are incorporated herein by reference). Other examples have included
use of antisense suppression, double-stranded RNA (dsRNA)
interference, hairpin RNA (hpRNA) interference or intron-containing
hairpin RNA (ihpRNA) interference to functionally disrupt the FUT8
gene. In one embodiment, the antibody is expressed in a cell line
with a functionally disrupted FUT8 gene, which encodes a fucosyl
transferase, such that antibodies expressed in such a cell line
exhibit hypofucosylation by reducing or eliminating the alpha 1,6
bond-related enzyme.
[0294] In one embodiment, the antibody is expressed in cell lines
engineered to express glycoprotem-modifying glycosyl transferases
(e.g., beta(1,4)-N-acetylglucosaminyl-transferase III (GnTHI)) such
that antibodies expressed in the engineered cell lines exhibit
increased bisecting GlcNac structures which results in increased
ADCC activity of the antibodies (PCT Publication WO 99/54342 by
Umana et al.; and Umana et al. (1999) Nat. Biotech. 17:176-180, the
disclosures of which are incorporated herein by reference).
[0295] In another embodiment, the antibody is expressed and the
fucosyl residue(s) is cleaved using a fucosidase enzyme. For
example, the fucosidase alpha-L-fucosidase removes fucosyl residues
from antibodies (Tarentino, et al. (1975) Biochem. 14:5516-5523).
In other examples, a cell line producing an antibody can be treated
with a glycosylation inhibitor; Zhou et al. Biotech. and Bioengin.
99: 652-665 (2008) described treatment of CHO cells with the
alpha-mannosidase 1 inhibitor, kifunensine, resulting in the
production of antibodies with non-fucosylated oligomannose-type
N-glucans.
[0296] In one embodiment, the antibody is expressed in a cell line
which naturally has a low enzyme activity for adding fucosyl to the
N-acetylglucosamine that binds to the Fc region of the antibody or
does not have the enzyme activity, for example the rat myeloma cell
line YB2/0 (ATCC CRL 1662). Other example of cell lines include a
variant CHO cell line, Led 3 cells, with reduced ability to attach
fucosyl to Asn(297)-linked carbohydrates, also resulting in
hypofucosylation of antibodies expressed in that host cell (WO
03/035835 (Presta et al); and Shields, R X. et al. (2002) J. Biol.
Chem. 277:26733-26740, the disclosures of which are incorporated
herein by reference). In another embodiment, the antibody is
expressed in an avian cell, e.g., a EBx.RTM. cell (Vivalis, France)
which naturally yields antibodies with low fucose content e.g.
WO2008/142124. Hypofucosylated glycans can also be produced in cell
lines of plant origin, e.g. WO 07/084926A2 (Biolex Inc.), WO
08/006554 (Greenovation Biotech GMBH), the disclosures of which are
incorporated herein by reference.
Antibody Formulations
[0297] Pharmaceutically acceptable carriers that may be used in
these compositions include, but are not limited to, ion exchangers,
alumina, aluminum stearate, lecithin, serum proteins, such as human
serum albumin, buffer substances such as phosphates, glycine,
sorbic acid, potassium sorbate, partial glyceride mixtures of
saturated vegetable fatty acids, water, salts or electrolytes, such
as protamine sulfate, disodium hydrogen phosphate, potassium
hydrogen phosphate, sodium chloride, zinc salts, colloidal silica,
magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based
substances, polyethylene glycol, sodium carboxymethylcellulose,
polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,
polyethylene glycol and wool fat. This method comprises the step of
contacting said composition with said patient. Such method will be
useful for both prophylaxis and therapeutic purposes.
[0298] For use in administration to a patient, the composition will
be formulated for administration to the patient. The compositions
may be administered parenterally, in particular by intravenous
injection or infusion techniques.
[0299] Sterile injectable forms of the compositions may be aqueous
or an oleaginous suspension. These suspensions may be formulated
according to techniques known in the art using suitable dispersing
or wetting agents and suspending agents. The sterile injectable
preparation may also be a sterile injectable solution or suspension
in a non-toxic parenterally acceptable diluent or solvent, for
example as a solution in 1,3-butanediol. Among the acceptable
vehicles and solvents that may be employed are water, Ringer's
solution and isotonic sodium chloride solution. In addition,
sterile, fixed oils are conventionally employed as a solvent or
suspending medium. For this purpose, any bland fixed oil may be
employed including synthetic mono- or diglycerides. Fatty acids,
such as oleic acid and its glyceride derivatives are useful in the
preparation of injectables, as are natural
pharmaceutically-acceptable oils, such as olive oil or castor oil,
especially in their polyoxyethylated versions. These oil solutions
or suspensions may also contain a long-chain alcohol diluent or
dispersant, such as carboxymethyl cellulose or similar dispersing
agents that are commonly used in the formulation of
pharmaceutically acceptable dosage forms including emulsions and
suspensions. Other commonly used surfactants, such as Tweens, Spans
and other emulsifying agents or bioavailability enhancers which are
commonly used in the manufacture of pharmaceutically acceptable
solid, liquid, or other dosage forms may also be used for the
purposes of formulation.
[0300] Several monoclonal antibodies have been shown to be
efficient in clinical situations, such as Rituxan.TM. (rituximab),
Herceptin.TM. (Trastuzumab) or Xolair.TM. (Omalizumab), and similar
administration regimens (i.e., formulations and/or doses and/or
administration protocols) may be used with the antibodies. For
example, an antibody present in a pharmaceutical composition can be
supplied at a concentration of 10 mg/mL in either 100 mg (10 mL) or
500 mg (50 mL) single-use vials.
[0301] Further aspects and advantages will be disclosed in the
following experimental section, which should be regarded as
illustrative and not limiting the scope of this application.
EXAMPLES
Example 1--Generation of KIR3DL2-Selective Antibodies
[0302] Immunization and Screening
[0303] Antibodies which bind KIR3DL2 but not closely related
KIR3DL1 were generated by immunizing mice with recombinant
KIR3DL2-Fc fusion protein, described in US patent publication
number US-2015-0232556-A1. Supernatant (SN) of the growing
hybridomas were tested by flow cytometry on Sezary Syndrome cell
lines (HUT78, COU-L) and HEK-293T/KIR3DL2 Domain 0-eGFP.
Potentially interesting hybridomas selected from the initial
screening were cloned by limiting dilution techniques in 96-wells
plates. The secondary screen involved selection of hybridomas of
interest by testing supernatants of the subclones by flow cytometry
on HUT78, COU-L, HEK-293T/KIR3DL1 Domain 0-eGFP and
HEK-293T/KIR3DL2 Domain 0-eGFP. Positive subclones were injected
into mice to produce ascitis and antibodies of interest were
purified before being tested in a Biacore assay using rec KIR3DL2
chips, followed by various assays formats based on binding to human
KIR3DL2-expressing cells.
[0304] Sequences of the variable domains of heavy (VH) and light
(VL) chain of antibodies were amplified by PCR from the cDNA of
each antibody. Sequences amplified were run on agarose gel then
purified using the Qiagen Gel Extraction kit. VH and VL sequences
were then sub-cloned into the Lonza expression vectors (Double-Gene
Vectors) using the InFusion system (Clontech) according to the
manufacturer's instructions. After sequencing, vectors containing
the VH and VL sequences were prepared as Maxiprep using the Promega
PureYield.TM. Plasmid Maxiprep System. Vectors were then used for
HEK-293T cell transfection using Invitrogen's Lipofectamine 2000
according to the manufacturer instructions. Antibodies generated
included, inter alia, 10G5, 2B12, 19H12 and 12B11.
[0305] Epitope Mapping
[0306] Antibodies were further tested for binding to a series of
KIR3DL2 mutants. Antibodies 19H12 and 12B11 did not show any loss
of binding to un-mutated wild type KIR3DL2 (WTaKIR3DL2), but lost
binding to mutant 11 having P179T and S181T substitutions as well
as to mutant 11A1 having V178A and H1805 substitutions. The
principal epitope of these antibodies 19H12, 18B10 and 12B11
therefore includes residues P179, S181, V178 and/or H180. These
residues at positions 179 and 181 in mutant 11 correspond to the
residues present at in KIR3DL1 (KIR3DL1 has T179 and T181).
Residues P179 and S181 in particular are within the D1 domain of
KIR3DL2 and on the opposite face on the KIR3DL2 protein of the
HLA-binding regions (i.e. the HLA binding pocket). Each of
antibodies 15C11, 19H12, 18B10 and 12B11 had reduced binding (full
loss of binding for 15C11 and 19H12) to mutant M11A4 having
substitutions E1305, H131S and R145S. These residues at positions
179 and 181 in mutant 11 correspond to the residues present at in
KIR3DL1 (KIR3DL1 has T179 and T181). Residues P179 and S181 in
particular are within the D1 domain of KIR3DL2 and on the opposite
face on the KIR3DL2 protein of the HLA-binding regions (i.e. the
HLA binding pocket). Surface-exposed residues adjacent to these
mutated residues can also contribute to the epitopes of the
antibodies, including for example residues N99, H100, E130, H131,
F132, V178, H180, P182, Y183, and Q184 (reference to SEQ ID NO: 1)
located at the surface of KIR3DL2 in the region of the P179/5181
epitope but outside of the region of the KIR3DL2 mutations which
did not result in loss of binding of the antibodies (e.g., mutant 5
(residue P66) and mutant 8 (residues V127)). Antibody 2B12 had loss
of binding to mutants having 160N and G62S substitutions and
decrease in binding to mutants having P14S, S15A and H23S
substitutions, but did not lose binding to any other mutants. The
principal epitope of these antibodies therefore includes residues
160 and/or G62 (and the epitope optionally further includes one or
more of P14, S15, and H23). Residues 60 and 62 are within the D0
domain of KIR3DL2. Residues 14, 15, 23, 60 and 61 are within the D0
domain of KIR3DL2.
[0307] Antibodies Increase the Number of Available Cell Surface
KIR3DL2 Receptors
[0308] Part 1:
[0309] Impact of Staining Conditions on 2B12 Labelling of
KIR3DL2-Expressing Cells
[0310] This study aimed to evaluate the impact of staining
conditions on 2B12 labelling of KIR3DL2-expressing cells, gated on
total cells, at 4.degree. C. or 37.degree. C., and with incubation
times of 2 hours, 4 hours or 24 hours. Briefly, 100,000 HUT78 cells
per well were incubated with a dose range of 2B12 antibody starting
from 0.0005 .mu.g/ml to 30 .mu.g/ml (serial dilution 1/3 in
complete medium). The protocol used was as follows: incubation 2H;
4H and overnight at 4.degree. C. and 37.degree.; staining in RPMI
10% with or without PFA fixation; 2 washes in SB (150 .mu.l/w);
addition of anti-human-Fc PE for 30 min at 4.degree. C.; 2 washes
in SB (100 .mu.l/w); and detection using FACS CANTO II.
[0311] Results are shown in FIG. 1A. While incubation at 4.degree.
C. which inhibits receptor internalization/cycling was expected to
result in an at least equal level of available cell-surface
KIR3DL2, staining with antibody 2B12 (human IgG1) was higher at
37.degree. C. than at 4.degree. C. Furthermore, higher median
fluorescence was observed with increasing duration of incubation,
the greatest KIR3DL2 expression was observed after 24 hours of
incubation.
[0312] Part 2:
[0313] Total, Free and 2B12-Bound KIR3DL2 Detection on HUT78 Tumor
Cells after Overnight Incubation
[0314] This study aimed to evaluate the impact of a 20 hour
incubation with antibody 2B12 on cell surface KIR3DL2 level by
observing the amount of bound 2B12 (human IgG1), free (non-antibody
bound) cell surface KIR3DL2 polypeptide, and total cell surface
KIR3DL2 polypeptide. Briefly, HUT78 (100,000 cells/well) were
incubated for 20 h at 37.degree. C. with a dose range of 2B12
antibody starting from (decreasing) 8.88 .mu.g/ml, 1/3 serial
dilution, 11 concentrations. Dose ranges were made in duplicate in
order to perform the following 2 staining conditions: [0315] Total
KIR3DL2+bound KIR3DL2 (GaH IgG Fc-PE+mAb2-APC (a non-competing
anti-KIR3LD2 mAb) (10 .mu.g/ml) [0316] Free KIR3DL2: 2B12-PE (10
.mu.g/ml)
[0317] Staining was performed at 4.degree. C. in staining buffer
for 1 h and analyzed with a FACS Canto II HTS. Results are shown in
FIG. 1B. The dark line/squares represents antibody 2B12 while the
light line/circles represents isotype control. It can be seen that
free KIR3DL2 receptors can be detected when incubating cells with
10 .mu.g/ml of 2B12-PE and detecting free receptors with
non-competing anti-KIR3DL2 antibody. It can be seen that 2B12-bound
KIR3DL2 receptors can be detected by incubating cells with goat
anti-human IgG Fc-PE secondary Ab. Both read-outs were correlated,
with similar EC.sub.50. The rightmost panel shows that a 20 hour
incubation with 2B12 increases KIR3DL2 receptor level at cell
surface as detected by the non-competing anti-KIR3DL2 antibody
mAb2-APC. Antibody 2B12 may be causing conformational change upon
binding, receptor stabilization/accumulation at cell surface and/or
internalization/recycling blockade.
[0318] Part 3:
[0319] Total, Free and 2B12-Bound KIR3DL2 Detection on HUT78 Tumor
Cells after 1, 24 or 48 Hours
[0320] This study aimed to evaluate the dynamics of KIR3DL2
receptor expression by observing by observing the amount of total,
free and 2B12-bound KIR3DL2 after different periods of incubation
with antibody 2B12. Briefly, HUT78 cells (50,000 cells/well) were
incubated for 1 h, 24 h or 48 h at 37.degree. C. in complete medium
with 2B12 (human IgG1), dose range starting from 10 .mu.g/ml
(decreasing), 1/3 serial dilution, 11 concentrations, or with
isotype control (IC), dose range starting (decreasing) from 10
.mu.g/ml, 1/3 serial dilution, 11 concentrations. Dose ranges were
made in triplicate in order to perform 3 staining conditions:
[0321] Bound KIR3DL2 (30 min at 4.degree. C.): GaH IgG Fc-PE,
[0322] Free KIR3DL2+ total KIR3DL2 (1 h at 4.degree. C.): 2B12-PE
(10 .mu.g/ml)+ mAb2-APC (non-competing anti-KIR3DL2) (10 .mu.g/ml),
[0323] Total KIR3DL2 (1 h+ 30 min at 4.degree. C.): 2B12 (10
.mu.g/ml)+ GaH IgG Fc-PE.
[0324] Staining was performed at 4.degree. C. in staining buffer,
and analysis conducted with HTFC Intellicyt.
[0325] Results are shown in FIG. 1C. In these culture conditions
(96 well-plates, 50,000 HUT78/well at TO), KIR3DL2 detection at
cell surface decreases in the absence of any Ab (as detected by
mAb2-APC, 2B12-PE or 2B12+ GaH-PE, points on the Y-axis).
[0326] Incubation with 2B12 at 37.degree. C. increases surface
expression of KIR3DL2 (as detected by non-competing mAb2 or by 2B12
itself+ secondary Ab), in a dose-dependent manner. Isotype control
did not give rise to any change in KIR3DL2. This increase is
already observed after 1 h at 37.degree. C., and seems to reach its
maximum after 24 h. Staining is optimal after 24 h (in terms of
total staining and of detected Ab-bound receptors).
[0327] Antibodies do not Internalize into Sezary Syndrome Cell
Line
[0328] Internalization of antibodies 10F6, 2B12, 18C6, 9E10, 10G5,
13H1, 4B5, 5H1, 1E2, 1C3 and 20E9, as well as antibody AZ158 (an
anti-domain 0 mAb as comparator) and other anti-D1 antibodies as
disclosed in PCT applications WO2014/044686 and WO2014/044681 were
assessed by fluoro-microscopy using the HUT78 SS cell line.
[0329] Materials and Methods:
[0330] Hut-78 cells were incubated during 1H at 4.degree. C. with
10 .mu.g/ml of the different antibodies. After this incubation
cells were either fixed (t=OH) or incubated for 2H at 37.degree. C.
Cells incubated for 2H were then fixed and stained. Antibodies were
stained using goat anti-mouse antibodies coupled to Alexa594
(Invitrogen, A11032). LAMP-1 compartments were stained using rabbit
anti-LAMP-1 antibodies (Abcam, ab24170) revealed by goat
anti-rabbit polyclonal antibodies coupled to FITC (Abcam ab6717).
Pictures were acquired using an Apotome device (Zeiss) and analyzed
using the Axiovision software.
[0331] Results:
[0332] Anti-KIR3DL2 mAbs were visible in red while LAMP-1
compartments were visible in green. At the time of addition of
antibodies, KIR3DL2 staining in red was visible at the cell surface
while green LAMP-1 were visible intracellularly in green. However,
at 2 hours following the addition of antibodies, each of antibodies
AZ158, 13H1 and 4B5, and anti-D1 antibodies caused red staining to
be colocalized with green staining, along with a decrease in red
staining at the cell surface, indicating that AZ158, 13H1 and 4B5,
and anti-D1 antibodies were rapidly internalized. Antibodies 10F6,
2B12, 18C6, 9E10, 10G5, 5H1, 1E2, 1C3 and 20E9, however was not
internalized, and at 2 hours following the addition of antibody,
red staining remained entirely on the cell surface.
[0333] Antibodies are Able to Kill KIR3DL2 Expressing Targets Via
Antibody Dependent Cellular Cytotoxicity (ADCC)
[0334] Cell lysis through an ADCC mechanism was monitored in a
radioactivity-based .sup.51Cr release experiment (the level of
radioactivity released from the preloaded target cells being
proportional to their death). One million target cells were loaded
with .sup.51Cr for 1 hour at 37.degree. C. and washed 3 times.
3,000 cells were seeded per well (U-shaped bottom 96-well plates)
and test mAbs are added at 10 or 20 .mu.g/ml final concentration
(or increasing concentrations if dose-response relationship is
studied). Effector cells were added at a defined effector:target
ratio (in general 10:1) and the mixture was incubated at 37.degree.
C. for 4 h. Supernatant is analyzed on a Lumaplate apparatus.
[0335] Anti-KIR3DL2 mAbs selected in Example 1 were tested at the
same final concentration (10 .mu.g/ml), to kill KIR3DL2-transfected
B221 target cells. The mAbs were effective in mediating ADCC
against KIR3DL2-expressing B221 targets.
Example 2--Activity in Mouse Xenograft Models of KIR3DL2 Expressing
Human Tumors
[0336] Tumor cells lines B221 and RAJI were made to express human
KIR3DL2. Immune compromised mice used for B221-KIR3DL2 and
RAJI-KIR3DL2 models were NOD-SCID purchased from Charles River
Laboratories. In the following models, 5 million human B221-KIR3DL2
or RAJI-KIR3DL2 tumor cells (in 100 .mu.l PBS as vehicle) were
engrafted IV on Day 0 (D0), i.e. 1 day before treatment initiation
(D1). From D1, mice were treated IV with different doses of
anti-KIR3DL2 mAbs (doses were adapted to mouse body weight) diluted
in PBS, 2 injections per week for the duration of the whole
experiment.
[0337] Control Groups Included, Depending on the Experiment: [0338]
PBS/placebo-treated mice as a control of normal/unaffected tumor
growth; [0339] mice injected with the same dose of isotype
control-matched mAbs directed against an irrelevant antigen.
[0340] Mice were weighed and observed for clinical signs every 2 to
5 days depending on the model. Percent of body weight changes were
calculated as compared to body weight at D0 before tumor
engraftment or to the highest body weight reached during the
experiment. Mouse deaths or important weight losses were recorded
and used to draw survival Kaplan-Meier curves and calculate
improvement in survival as compared to control groups of mice. The
efficacy of IgG2b isotype murine anti-KIR3DL2 19H12 antibodies
(given at 300 .mu.g/mouse, twice a week) was separately tested
against SC B221-KIR3DL2 xenografts or RAJI-KIR3DL2 xenografts (n=6
NOD-SCID mice per group). Animals treated with anti-KIR3DL2
antibodies showed an increase in survival in comparison to mice
treated with isotype control-matched mAbs.
Example 3--Improved Detection Methods Reveal KIR3DL2 Positive
Tumors
[0341] Tumor biopsies from RAJI-KIR3DL2 models and RAJI-KIR3DL2
cell lines were obtained and staining was performed on frozen
sample using AZ158 antibody (see WO2010/081890) or antibodies 12B11
(see Example 1). KIR3DL2 was stained with anti-KIR3DL2 antibody by
DAB chromogenic detection according to standard protocols, adapted
for immunostaining with BenchMark XT Ventana Roche. For all
staining control isotype (mIgG1) and control DAB were performed.
Surprisingly while AZ158 was negative, tumors were positive when
using 12B11 antibody at the same concentration (5 .mu.g/ml) of
antibody (see FIG. 1). Raising concentrations of antibody AZ158 (to
50 .mu.g/ml) generated extensive background staining that did not
allow tumor samples to be differentiated from healthy tissue. Next,
tumor biopsies from cancer patients previously stained with AZ158
were re--examined using antibody 12B11. Biopsies that had been
KIR3DL2-negative with AZ158 were stained with 12B11 (i.e. becoming
KIR3DL2-positive).
Example 4: NK Lytic Capacity Assay
[0342] Cell lysis through an ADCC mechanism was monitored in a
radioactivity-based .sup.51Cr release experiment (the level of
radioactivity released from the preloaded target cells HUT78 (ATCC
reference TIB-161.TM., available from LGC Standards Corp.) being
proportional to their death). Briefly, human peripheral blood
mononuclear cells (PBMCs) from healthy donors were incubated with
HUT78 target cell line (KIR3DL2.sup.+) in the presence of a dose
range of IPH4102 mAb (humanized 2B12 mAb). HUT78 cell lysis by
PBMCs was monitored in a 4-hour chromium release assay, using an
E:T ratio of 100.
[0343] Effector Cell Preparation
[0344] Human blood was withdrawn on CPT tubes (n=6 to 8 tubes per
donor containing 7-8 ml of blood). Within 30 minutes after
collection, CPT tubes were centrifuged for 30 minutes at 1500 g
with low acceleration and low brake, at room temperature (RT).
After centrifugation, the mononuclear cells, in the supernatant
above the separation gel, were transferred into 50 ml conical tubes
(contents of 2 to 3 CPT tubes were pooled into one 50 ml tube),
completed to 50 ml with RPMI-1640 and centrifuged for 10 minutes at
600 g at RT. All cell pellets were pooled into one 50 ml conical
tube and washed with 50 ml of RPMI-1640 (centrifugation 10 min, 130
g, RT). Remaining red blood cell (RBC) lysis could be performed at
this step, by adding 1 ml of cold NH.sub.4Cl on cell pellet and
incubating 5-10 min at RT. When RBC lysis was necessary, an
additional washing step was performed by filling the tube to 50 ml
with RPMI-1640 (centrifuged 10 min, 130 g, RT). Cell pellet was
resuspended in 20 ml of CCM, and PBMCs were counted by excluding
dead cells with Trypan blue stain, using Cellometer cell
counter.
[0345] PBMC concentration was adjusted with CCM to 6.times.10.sup.6
cells/ml for target cell lysis assay (.sup.51Cr release, 50
.mu.l/w=3.times.10.sup.5 cells), and to 2.5.times.10.sup.6 cells/ml
for NK cell activation assay (CD137 expression, 50
.mu.l/w=1.25.times.10.sup.5 cells).
[0346] Target cell preparation HUT78 target cells were counted by
excluding dead cells with Trypan blue stain, using Cellometer cell
counter. 2.10.sup.6 cells were labelled with .sup.51Cr, by adding
50 .mu.Ci of 51Cr per 10.sup.6 cells on cell pellet in round-bottom
14 ml polypropylene tube, and incubated 1 h at 37.degree. C. After
chromium labeling, cells were washed 3 times with 10 ml of CCM
(centrifugation 5 min, 500 g, RT). Cells were counted by excluding
dead cells with Trypan blue stain, using Kovaslide. Cell
concentration was adjusted to 3.times.10.sup.4 cells/ml (100
.mu.l/w=3.times.10.sup.3 cells).
[0347] mAb Solution Preparation
[0348] 4.times. solutions (50 .mu.l/w in 200 .mu.l/w final) of
anti-KIR3DL2 antibody (1.6 ml), negative isotypic control, 1.6 ml)
and alemtuzumab (anti-CD52, positive control, 1.2 ml) were prepared
in CCM and centrifuged for 10 min at maximal speed (16100 g) at
4.degree. C. in a benchtop centrifuge (to eliminate potential
aggregates).
[0349] The highest tested concentration was 10 .mu.g/ml (i.e. 40
.mu.g/ml as a 4.times. solution) for isotype control and
alemtuzumab and 8.88 .mu.g/ml (i.e. 35.5 .mu.g/ml as a 4.times.
solution) for anti-KIR3DL2 antibody. 1/4 serial dilutions were
performed in 96-deepwell plate for isotype control and anti-KIR3DL2
antibody, by transferring 400 .mu.l of mAb solution into 1.2 ml of
CCM. Eleven concentrations were tested for both Abs, whereas
alemtuzumab was only tested at 10 .mu.g/ml.
[0350] Assay Procedures
[0351] mAb solutions (50 .mu.l/w) were transferred from the 96
deepwell plate into U-bottom plate, in triplicate. Effector cells
(PBMCs, 50 .mu.l/w) and target cells loaded with .sup.51Cr (HUT78,
100 .mu.l/w) were added to the wells. Final E/T ratio is 100/1.
Spontaneous and maximal chromium release from target cells were
measured in dedicated wells (n=8 per plate) containing respectively
target cells in medium and target cells in medium+ 2% Triton X-100.
The plates were centrifuged for 1 minute at 300 g before incubation
at 37.degree. C. for 4 hours. After the 4 h-incubation, plates were
centrifuged for 3 minutes at 300 g, and 50 .mu.l of supernatants
were transferred into Lumaplate containing scintillator.
Supernatants were allowed to dry at 56.degree. C., and chromium
released into culture supernatants was quantified using TopCount
NXT.TM. microplate scintillation counter (Perkin Elmer).
[0352] Specific lysis of target cells was calculated using the
following formula:
specific lysis ( % ) = ( experimental release - spontaneous release
) ( maximal release - spontaneous release ) .times. 100
##EQU00001##
Example 5--Development of a Model Based on NK Cell % Lytic Activity
to Determine Dosing of Anti-KIR3DL2 Antibodies
[0353] Pharmacokinetic of therapeutic mAbs is usually modelled
using a two-compartment model (Dirks and Meibohm, 2010; Lobo et
al., 2004; Morell et al., 1970; Roskos et al., 2004). Anti-KIR3DL2
antibody 2B12 obtained according to Example 1 was humanized (VH and
VL amino acid sequences shown in Table D; see also WO2015/136052,
the disclosure of which is incorporated herein by reference); the
antibody (referred to as IPH4102) was produced as a full-length
human IgG1 isotype antibody and evaluated in cynomolgus monkeys and
mice. Based on preclinical PK results in both cynomolgus monkeys
and mice, IPH4102 is expected to display PK properties similar to
other therapeutic mAbs in humans, except for compound-specific
target-mediated effects. In SS and MF patients, IPH4102 will bind
KIR3DL2.sup.+ tumor cells in blood and in tissues, in addition to
KIR3DL2.sup.+ normal lymphocytes. It is anticipated that
target-mediated drug disposition (TMDD) may influence the PK of
IPH4102 in humans. Hence, parameters for describing TMDD were
included in the PK model.
[0354] The final PK simulation model was a two-compartment model
with parallel first order and saturable elimination pathways, as
illustrated in FIG. 2. This TMDD model can be used to describe the
anticipated human PK for IPH4102, and includes: [0355]
Two-compartment distribution (from blood to periphery),
characterized by an inter-compartmental clearance (Q) and
distribution volumes for the central and peripheral compartments
(respectively Vc and Vp). [0356] First order elimination from the
central compartment, characterized by a single clearance parameter,
CL. [0357] A central and a peripheral maximum Target Binding
capacity (TB.sub.maxc and TB.sub.maxp, respectively), describing
the amount of IPH4102 which may be bound by the available KIR3DL2
antigen at full saturation in the central and peripheral
compartment, respectively. The dynamics in the system are
characterized by a rate constant for association, K.sub.on, a rate
constant for dissociation, K.sub.off, and a turnover rate for the
KIR3DL2-positive cells, K.sub.cell. In practice the rate constant
for association, K.sub.on, was determined as
K.sub.on=K.sub.off/K.sub.D, where K.sub.D is the affinity of
IPH4102 for binding to KIR3DL2.
[0358] The PK model was extended to include a link between the
predicted serum concentration of IPH4102 and NK cell lytic capacity
in humans, as well as KIR3DL2 saturation prediction.
[0359] A standard Emax-type relationship with a single potency
parameter, EC.sub.50, was used to describe the link between IPH4102
concentration (Conc) and NK lytic capacity, as measured for
instance in .sup.51Cr release assay, by the percentage of maximal
tumor cell lysis obtained (=Tumor cell lysis/Max tumor cell lysis
at saturation.times.100):
% NK lytic capacity=100.times.Conc/(Conc+EC.sub.50)
[0360] The maximal Target Binding (TB.sub.max) in a compartment for
a therapeutic mAb can be calculated as follows:
TB.sub.max=Rec.times.C.sub.cell.times.V.times.A.sub.N.times.MW.sub.mAb.t-
imes.10.sup.9 [0361] where: [0362] Rec is the receptor
density=number of Target Receptors/cell, [0363] C.sub.cell is the
concentration of target positive cells in the compartment
(number/mL), [0364] V is the volume of the compartment, [0365]
A.sub.N is Avogadro's number for converting number of entities to
moles=6.023.times.10.sup.23/mol, [0366] MW.sub.mAb is the molecular
weight of IPH4102=150,000 g/mol, and [0367] 10.sup.9 converts from
g to ng.
[0368] The structure of PK/PD model is described in FIG. 1.
Parameters for each compartment of the model were derived based on
in vitro data and literature information, as further described
below.
[0369] Values for the parameters (CL, Vc, Q, Vp) were identified
based on preclinical PK results in both cynomolgus monkeys and
mice, showing that IPH4102 is expected to display PK properties
similar to other therapeutic mAbs in humans, defining a standard
two-compartment model for an IgG in humans.
[0370] Target binding capacity to normal immune cells in blood was
determined. Briefly, the total number of KIR3DL2 expressing
lymphocytes was determined using results from a non-interventional,
mono-centric descriptive and prospective open study in healthy
volunteers. A total of 40 volunteers were enrolled divided in two
cohorts, cohort 1 with 20 volunteers under the age of 60 years old
and cohort 2 with 20 volunteers over the age of 61. The number of
white blood cells (WBC) given by the blood formula of each donor
was used to normalize flow cytometry data. Fresh whole blood
samples were processed and analyzed with a panel of
fluorochrome-conjugated mAbs (8-colors combinations) to define
blood cell subsets. The gating strategy in flow cytometry aimed to
express each cell subset as a percentage of WBC. By using these
percentages and the WBC number from the blood formula, the
different blood cell subsets were defined in cells per .mu.L of
blood. The absolute number of KIR3DL2.sup.+ immune cells
populations among lymphocytes (mean value of 1 to 10 tubes) and the
MESF at saturation, using PE-labelled anti-KIR3DL2 mAb (mean value
of 1 to 10 tubes) were used to calculate the total number of
KIR3DL2 receptors on lymphocytes in humans.
[0371] No specific information about the tissue-to-blood ratio for
KIR3DL2.sup.+ lymphocytes was available initially. Consequently,
target binding capacity to normal immune cells in tissues was
estimated based on the number of blood lymphocytes that express
KIR3DL2, and the assumption that KIR3DL2.sup.+ cells had a
distribution similar to the general distribution of other
lymphocytes, where the number of cells in the tissue is
approximately 50 times higher than in the blood. KIR3DL2 receptor
density was assumed to be comparable between blood and tissue.
[0372] Target binding capacity to leukemic tumor cells in blood,
for SS patients was assessed. For evaluation of IPH4102 target
binding to blood tumor cells in SS patients, the total number of
KIR3DL2.sup.+ tumor cells was determined in 9 SS patients based on
blood formula counts and % of CD3.sup.+ CD4.sup.+ KIR3DL2.sup.+
cells among whole blood cells, using PE-labelled anti-KIR3DL2 mAb.
The number of KIR3DL2 molecules expressed at the cell surface of
primary Sezary tumor cells was determined for each patient. Mean of
all measurements of absolute number of KIR3DL2.sup.+ tumor cells
(CD4.sup.+ CD3.sup.+ KIR3DL2.sup.+) was calculated. The KIR3DL2
density at the cell surface of SS tumor cells was evaluated.
[0373] For target binding capacity to tumor cells in tissues, no
specific information about the total number of tumor T cells in
skin was initially available for CTCL patients. As total skin
resident T cells were evaluated to 20 billion cells, it was
postulate that total tumor KIR3DL2.sup.+ T cells would not largely
exceed this level, and that median KIR3DL2 density on tumor cells,
assumed to be similar to circulating tumor cells in SS patients.
The result was that TBmax resulting for tumor cells in tissues was
found in same range as target binding capacity of IPH4102 to
KIR3DL2 on circulating tumor cells as observed in SS patients.
[0374] Mechanisms for target-mediated disposition were limited to
regular turnover of NK and of tumor cells, assumed to be
independent of IPH4102 concentration.
[0375] In vitro affinity (K.sub.D) and off-rate (K.sub.off).
IPH4102 in vitro binding affinity for KIR3DL2 was evaluated with
PE-labelled IPH4102 in concentration-response flow cytometry
experiments performed on KIR3DL2-transfected cell lines, on KIR3DL2
expressing Sezary Syndrome (SS) tumor cell lines and on SS primary
tumors collected from patient blood samples. The
concentration-response for IPH4102 binding to KIR3DL2 was confirmed
in flow cytometry experiments on whole blood from healthy
volunteers, gated on NK cells and in surface plasmon resonance
(SPR) experiments using recombinant human KIR3DL2 protein (average
bivalent binding affinity on Biacore.
[0376] In vitro concentration-response binding experiments with
IPH4102 on KIR3DL2.sup.+ Sezary cell lines (such as HuT78 or COU-L)
and on primary Sezary tumor cells revealed that, regardless of
KIR3DL2 expression level, the EC.sub.50 for IPH4102 binding on cell
lines and primary Sezary cells are similar (0.06 .mu.g/mL with
HuT78, 0.087 .mu.g/ml for COU-L, 0.07 .mu.g/mL on patients' primary
tumor cells). In conclusion, the binding affinity of IPH4102 to
tumor and immune cells in blood was set to 70 ng/ml in the PK/PD
model. PE-labelling had only small impact on IPH4102 affinity for
KIR3DL2, in overnight staining conditions. Importantly, a similar
affinity was found in SPR (IPH4102 average bivalent binding
affinity to recombinant KIR3DL2 on Biacore, 0.146 nM, corresponding
to 21.9 ng/mL, shown in the Table below. The off-rate for
dissociation of IPH4102 from recombinant KIR3DL2 was obtained from
SPR experiments. The KIR3DL2 antigen binding activity was
determined using a two-step experimental set-up. Firstly, IPH4102
samples were injected at a constant concentration over the
Protein-A chip (antibody capture step). Secondly, KIR3DL2-His
antigen samples were injected at a constant concentration over the
captured antibodies (antigen binding step) and allowed to
dissociate before injection of a regeneration buffer for baseline
correction (blank subtraction). For batch to batch comparison the
mean (n=3) reflectance unit (RU) ratio between bound antigen and
captured antibody was used as a comparative index (0.4).
[0377] The mean of three determinations of the off-rate for
bivalent binding was used (1.4.times.10.sup.-4 s.sup.-1,
corresponding to 0.504 h.sup.-1).
TABLE-US-00008 TABLE KIR3DL2 binding affinity by SPR K.sub.D (nM) N
= 1 N = 2 N = 3 Mean K.sub.D K.sub.D (nM) 0.1616 0.1314 0.1436
1.46E-01 K.sub.on (1/Ms) 9.68E+05 9.53E+05 9.65E+05 9.62E+05
K.sub.off (1/s) 1.56E-04 1.25E-04 1.39E-04 1.40E-04
[0378] In order to evaluate biological and potential toxic activity
of IPH4102 in a physiological setting, the in vitro
concentration-response assay of IPH4102 was determined in vitro on
15 human healthy donor PBMCs co-incubated with HuT78 cells and
increasing doses of IPH4102 mAb. Three read-outs were studied in
parallel: the activation of NK cells through CD137 expression (flow
cytometry), the lysis of target cells by PBMCs (classical
.sup.51Cr-release assay) and the secretion of 5 cytokines and
chemokines: IFN-.gamma., TNF-.alpha., IL-6, IL-8, MCP-1. Briefly,
PBMCs from healthy donors were incubated with HuT78 target cell
line (KIR3DL2.sup.+) in the presence of a dose range of IPH4102
mAb. The activation of NK cells among PBMCs after 20 hours of
incubation was monitored using the activation marker CD137, using
an E:T (Effector:Target) ratio of 2.5:1. Cytokines produced by
PBMCs in culture supernatants during the 20 h-incubation (CD137
assay) were quantified with AlphaLISA technology (Perkin Elmer). In
parallel, HuT78 cell lysis by PBMCs was monitored in a 4-hour
.sup.51Cr-release assay, using an E:T ratio of 100:1, as described
in Example 4.
[0379] The parameter the most relevant to IPH4102 safety and
pharmacological activity for determination of dosing was selected
as HuT78 tumor cell lysis by healthy donors' PBMC in the .sup.51Cr
release assay (NK cell lytic capacity). The median EC.sub.10 and
EC.sub.50 (.+-.SD) in the .sup.51Cr release assay were respectively
2 (.+-.2.8) ng/mL and 45 (.+-.40) ng/mL.
[0380] Hence, a standard Emax-type relationship with a single
potency parameter, the EC.sub.50 of IPH4102 in the .sup.51Cr
release assay, i.e. =45 ng/mL, was used to describe the link
between IPH4102 concentration (Conc) and % of maximal ability of NK
cells to mediate tumor cells lysis, as measured by % of NK lytic
capacity:
% of NK lytic capacity=100.times.Conc/(Conc+EC.sub.50)
[0381] The final parameters are summarized in the table below.
TABLE-US-00009 TABLE Summary of IPH4102 PK/PD model parameters
Parameter Description Value CL Clearance 0.12 mL/h/kg Vc Central
volume of distribution 40 mL/kg Q Inter compartmental clearance 1
mL/h/kg Vp Peripheral Volume of distribution 40 mL/kg Kcellc
Turnover rate of KIR3DL2-positive cells 0.003/h for HD (lymphocytes
and tumor cells) in blood and MF; 0.02/h for SS Kcellp Turnover
rate of KIR3DL2-positive cells 0.003/h for HD; (lymphocytes and
tumor cells) in periphery 0.02/h for MF and SS K.sub.off
Dissociation rate constant 0.504/h K.sub.D Binding affinity 70
ng/mL TB.sub.maxc Target binding capacity in blood 5 ng/kg for HD
and MF; 198 ng/kg for SS TB.sub.maxp Target binding capacity in
periphery 257 for HD; 450 ng/kg for MF and SS EC.sub.50 EC.sub.50
in .sup.51Cr-release assay HuT 78 tumor 45 ng/mL lysis by PBMC from
healthy volunteers.
[0382] PD/PK simulations were then performed using the software
Phoenix WinNonLin version 6.4 and plotting of the results was done
in GraphPad Prism 5 version 5.04. The model was implemented in
WinNonLin and used to simulate the PK over time following 1 hour
i.v. infusion of IPH4102 to humans for a range of dose levels.
Based on this, doses for the first-in-human (FIH) trial were
identified. The selected pharmacological parameter for MABEL
calculation was Hut 78 tumor cell lysis by healthy donor PBMC in a
.sup.51Cr release assay, which was a conservative evaluation of the
biological IPH4102-mediated response in SS patients. We determined
doses that would result in a low, but discernable effect in the in
vitro assay of HuT 78 tumor lysis (see Example 4). A 10% response
in this assay was adopted as a low MABEL response (EC.sub.10=2
ng/mL). Hence, of particular interest was the dose resulting in the
pre-defined 10% .sup.51Cr-release at C.sub.max. Based on the PK
simulations, C.sub.max, % of NK lytic capacity at C. and max
KIR3DL2-occupancy achieved at t=3-6 h, were predicted for different
doses in Healthy Donors, MF (no circulating tumor cells) and SS
(circulating tumor cells) patients, and helped in identifying the
FHD as 0.1 pg/kg.
[0383] Simulated AUC.sub.0-7days after 1.sup.st and 4.sup.th doses,
C.sub.max and accumulation index for multiple dose phase I clinical
study are presented in Table below, for MF and SS patients.
TABLE-US-00010 TABLE Cmax and accumulation index for multiple dose
phase I C.sub.max Cycle 1 C.sub.trough Cycle 1 C.sub.max Cycle 4
AUC.sub.7 days AUC.sub.7 days Dose Level Dose fold (6 h) (168 h)
(510 h) Cycle 1 Cycle 4 Accumulation .mu.g/kg increase ng/ml ng/ml
ng/ml ng * h/mL ng * h/mL Index MF 0.1 -- 2 1 4 176 373 2.67 1 x10
22 7 37 1770 3923 2.67 10 x10 216 82 407 18476 47084 2.68 50 x5
1081 444 2138 95579 254699 2.69 200 x4 4326 1824 8663 386736
1039066 2.70 750 x3.75 16226 6890 32597 1455032 3916529 2.72 1500
x2 32454 13799 65235 2911884 7840478 2.74 3000 x2 64909 27617
130511 5825602 15688410 2.82 6000 x2 129820 55253 261062 11653058
31384281 3.01 10000 x1.6 216368 92101 435131 19423001 52312143 3.29
SS 0.1 -- 2 1 3 161 318 2.35 1 x10 21 7 34 1639 3412 2.35 10 x10
213 77 394 17818 44704 2.36 50 x5 1077 436 2121 94584 251534 2.36
200 x4 4322 1815 8645 385644 1035723 2.37 750 x3.75 16222 6881
32579 1453914 3913141 2.38 1500 x2 32449 13790 65216 2910759
7837082 2.39 3000 x2 64905 27607 130492 5824478 15685020 2.43 6000
x2 129815 55243 261043 11651930 31380900 2.57 10000 x1.6 216363
92091 435112 19421868 52308744 2.81
[0384] At a dose of 0.1 .mu.g/kg, in MF and SS patients,
KIR3DL2-occupancy would remain below 3% and the % of NK lytic
capacity mediated by IPH4102-stimulated NK cells will remain below
6%. The tables below summarize, for MF and SS patients,
respectively, the simulations in terms of the expected values of
the % of NK lytic capacity in circulation at C.sub.max and
C.sub.trough, for cycle 1 and cycle 4 of repeated weekly
administrations in MF patients, for the dose levels up to 1500
.mu.g/kg.
TABLE-US-00011 TABLE MF patients % of NK lytic % of NK lytic % of
NK lytic % of NK lytic Dose capacity at capacity at capacity at
capacity at Level C.sub.max cycle 1 C.sub.trough cycle1 C.sub.max
cycle 4 C.sub.trough cycle 4 .mu.g/kg (6 h) (168 h) (510 h) (672 h)
0.1 5 2 7 4 1 32 14 45 28 10 83 65 90 84 50 96 91 98 97 200 99 98
99 99 750 100 99 100 100 1500 100 100 100 100
TABLE-US-00012 TABLE SS patients % of NK lytic % of NK lytic % of
NK lytic % of NK lytic Dose capacity at capacity at capacity at
capacity at Level C.sub.max cycle 1 C.sub.trough cycle 1 C.sub.max
cycle 4 C.sub.trough cycle 4 .mu.g/kg (6 h) (168 h) (510 h) (672 h)
0.1 4 1 7 3 1 31 13 43 25 10 83 63 90 83 50 96 91 98 97 200 99 98
99 99 750 100 99 100 100 1500 100 100 100 100
Example 6--a Human Phase I Clinical Trial in Relapsed/Refractory
CTCL
[0385] IPH4102 (humanized IgG1 anti-KIR3DL2 antibody 2B12) is
currently being investigated in a first-in-human dose-finding phase
1 study (NCT02593045) evaluating repeated administrations of
single-agent IPH4102 in relapsed/refractory CTCL patients.
[0386] The primary objective is to assess the safety and
tolerability of increasing doses of IPH4102 by characterizing
dose-limiting toxicity and adverse events. Secondary objectives
include PK, immunogenicity and signals of anti-neoplastic clinical
activity. Exploratory biomarkers aim to characterize
KIR3DL2-expressing and non-expressing cells in involved
tissue/compartments and to monitor their changes with IPH4102
treatment. Measurement of molecular residual disease is performed
in the skin, blood and/or lymph nodes. Assessment of ex vivo NK
cell-mediated ADCC against autologous tumor cells is also performed
pre-dose on SS patients.
[0387] The study has two sequential portions, a dose-escalation
followed by a cohort expansion portion. The dose-escalation portion
has a 3+3 design with accelerated titration and aims to determine
the maximal tolerated dose (MTD) or recommended phase 2 dose
(RP2D). Doses tested included: 0.0001 mg/kg, 0.001 mg/kg, 0.01
mg/kg, 0.05 mg/kg, 0.2 mg/kg, 0.75 mg/kg, 1.5 mg/kg, 3 mg/kg, 6
mg/kg and 10 mg/kg body weight. In the expansion portion, two CTCL
subtype-specific cohorts will be studied, each cohort to include 10
additional patients to further explore the MTD or RP2D. Eligible
CTCL patients must have received at least 2 prior lines of
anti-neoplastic systemic therapy. Centrally assessed KIR3DL2
expression on malignant cells in skin or blood is required for
inclusion.
[0388] Patients received weekly IPH4102 administrations until
progression or unacceptable toxicity. Intra-patient dose-escalation
is allowed, only past the first complete clinical assessment at
week 5 and provided the upper next dose-level is declared safe by
the safety committee.
[0389] Among the 14 patients who were (or are still being) treated,
11 have SS, 2 have MF and 1 has CD4+ CTCL, Not Otherwise Specified
(NOS). Clinical assessment was performed according to the published
recommended standardized scoring system for assessing tumor burden
and defining response in skin, lymph nodes, blood, and viscera, by
using a composite global response score and a common definition of
clinical end points described in Olsen et al. (2011) American
Society of Clinical Oncology 29, 2598-2607. In this system, global
complete response (CR) is defined as the complete disappearance of
all clinical evidence of disease and can only be achieved if CR is
documented in all involved organs, i.e. all TNMB categories. In
contrast, any progressive disease (PD) in any TNMB category
qualifies for global PD. In intermediate situations, the global
scores of partial response (PR) or stable disease (SD) are achieved
according to TNMB categories (described in Olsen et al. (2011),
supra. Clinical assessment performed included: [0390] full TNMB
scoring (may require imaging) performed pre-dose and then at week 5
(W5), W14, W26 and then every 4 weeks until treatment
discontinuation; [0391] skin-specific mSWAT measurement is
performed pre-dose, at week 5, then every 2 weeks until W26, then
every 4 weeks; and [0392] assessment of blood involvement (through
Sezary cell count or immuno-phenotyping or cytomorphology) is also
performed pre-dose, at W5, then every 2 weeks until W26, then every
4 weeks.
[0393] The clinical trial is still ongoing. Clinical assessments
for the patients remaining in the study are detailed in the table
below:
TABLE-US-00013 Initial Number Treatment dose doses CTCL Stage at
Objective best duration Patient (mg/kg) admin subtype study entry
response (days) 1 0.0001 15 SS T4N0M0B2 PR (week 22; >200 0.05
mg/kg) 2 0.001 12 SS T4NxM0B2 SD 133 3 0.01 12 MF T2N0M0B0a PR
(week 10; 161 0.01 mg kg) 4 0.05 11 Transformed T4NxM0B2 PR (week
10; 133 SS 0.05 mg/kg) 5 0.05 7 MF T2NxM0B0a SD 62 6 0.05 9 SS
T2N0M0B2 SD 118 7 0.2 9 SS T4N2aM0B2 SD 91 8 0.2 7 SS T4N2M0B2 SD
64 9 0.2 7 CD4 Tcell TxN0M0 SD 63 (NOS) 10 0.75 5 SS T1N0M0B2 SD 36
11 0.75 4 SS T4N0M0B2 SD 27
[0394] The table above also displays the dose-level at which each
patient entered the trial, the number of IPH4102 administrations
they received, the CTCL subtypes and the TNMB stage at study entry.
Three patients experienced global PR, which have lasted
respectively for 28, 74 and 70 days and are still ongoing. Timing
of occurrence of these responses as well as the dose-level that was
received at occurrence are shown in the same column.
[0395] Specifically for Sezary Syndrome patients, particular
attention was given to clinical response in blood. Among the five
SS patients enrolled in the study, two have achieved PR and one has
achieved CR in blood, as shown in the table below.
TABLE-US-00014 Sezary Sezary Best Initial Number count count
response dose doses CTCL Stage at baseline nadir in blood (1.sup.st
Patient (mg/kg) admin subtype study entry (cells/.mu.L)
(cells/.mu.L) observation) 1 0.0001 13 SS T4N0M0B2 5273 507 PR
(week 5) 2 0.001 11 SS T4NxM0B2 19219 3407 PR (week 14) 4 0.05 7
Transformed T4NxM0B2 4644 76 CR (week 10) SS 6 0.05 7 SS T2N0M0B2
128 108 SD 7 0.2 5 SS T4N2aM0B2 9197 8636 SD
[0396] Overall, only grade 1 or 2 related adverse events (AEs) were
reported. No patient enrolled in the trial experienced a DLT or any
grade 3-5 related AEs. No IPH4102-related skin rashes or infections
have been observed up to the dose-level tested.
[0397] Ex vivo functional assay results confirmed that SS patients'
NK cells are functional and able to kill autologous tumor cells
through ADCC.
[0398] IPH4102 did not result in depletion of NK cells. FIG. 3
shows the % change from baseline (day 1 of week 1) in patients' NK
cells over a period of up to 50 weeks. FIG. 4 shows the number of
patients' nk cells (nk cells per .mu.l) over a period of up to 50
weeks.
[0399] Preliminary IHC results were obtained in skin biopsies taken
before and after IPH4102 repeated administrations. Signals of
IPH4102 pharmacological activity in skin lesions were observed in
patients, both with SS and MF, with evidence of marked decrease in
KIR3DL2.sup.+ cells in some cases. Representative examples
include:
[0400] Patient 3 has MF and started the trial at the 0.01 mg/kg
dose-level. He had 2 biopsies (B1 and B2) taken at screening with
respectively 54% and 26% of KIR3DL2+ cells.
[0401] At week 5, a decrease in KIR3DL2 staining was observed in B1
(0.5%) but not B2 (32%), and at W14, both lesions showed declined
KIR3DL2+ cells (1% and 16% respectively). The patient is in global
PR since week 10.
[0402] Patient 4 has Sezary Syndrome and started the trial at the
0.05 mg/kg dose-level, with 52% of KIR3DL2+ cells in the skin
biopsy taken at screening. At week 5, a marked decrease in KIR3DL2
staining was observed, with only 4.4% of cells being KIR3DL2+. The
patient is in global PR, with CR in blood, since week 10 in the
study.
[0403] Patient 6 has SS and started the trial with 0.05 mg/kg. The
screening biopsy presented 17.5% KIR3DL2+ cells that decreased to
3% at week 5. Also, histology of this lesion improved from plaque
at screening to patch at week 5. However, this patient is still in
global SD (with SD in skin and SD in blood).
[0404] Patient 7 has SS and started the trial with 0.2 mg/kg. The
screening biopsy presented 76% KIR3DL2+ cells that remained stable
at week 5 (62%). Histology of the lesion also improved from plaque
to patch but this patient remains in global SD (SD in skin and in
blood).
[0405] In conclusion, intermediate analysis of preliminary signs of
clinical activity shows that IPH4102 is able to provide meaningful
clinical benefit to advanced CTCL patients at repeated doses;
patients with response received doses as low as 0.0001 mg/kg
(response on blood involvement) or 0.01 mg/kg (skin disease
response). Clinical responses in blood (in SS patients) were
observed even in patients with very high blood involvement (such as
patient 2, who had more than 19,000 blood Sezary cells/.mu.L blood
at study entry. Interestingly, anti-tumor effect were observed at
considerable less than full NK lytic activity during the duration
of treatment. Furthermore, at the 0.01 mg/kg dose level, IPH4102 is
expected to reach at most a very small number of malignant cells in
skin.
[0406] Also, interestingly, anti-tumor response (in skin) was
observed in a patient (patient 3, 0.01 mg/kg) having no blood
involvement. This suggests that IPH4102 will be useful for
treatment of individuals having indolent or early stage CTCL
without significant blood involvement.
[0407] Furthermore, the ability to treat skin disease by
administering IPH4102 intravenously, moreover at low amounts and
furthermore without depletion of NK cells (a significant portion of
NK cells express KIR3DL2) whether at lower and higher doses, is
advantageous, because a single administration regimen can be used
for patients with or without blood involvement, and/or with
different tumor burden. Despite a wide range of blood and skin
tumor burden in CTCL patient, IPH4102 is promising for use even in
high tumor burden at doses below that which would be needed to
occupy KIR3DL2 on tumor cells in these high-burden patients,
suggesting that high dose treatment need not be used in these
patients in order to maintain saturation of KIR3DL2 on malignant
cells (e.g. in skin), and additionally that a single non-NK
depleting treatment regimen can be used independent of levels of
blood or skin tumor burden (achieving full receptor occupancy in
tissues is generally believed to require a blood concentration of
antibody least 10-fold that required to achieve full occupancy in
circulation). Final results from the trial confirmed the good
safety profile and promising activity of IPH4102 in this elderly
and heavily pre-treated patients population (n=25). At or above the
1.5 mg/kg dose level (1.5, 3, 6 and 10 mg/kg), saturation on blood
tumor cells was achieved, whatever injection schedule and in all
patients irrespective of their blood tumor burden. The objective
response rate in the 20 patients with Sezary syndrome was 50%; the
ORR4 (rate of response lasting for at least more than 4 months) was
40%, the disease control rate (DCR), 90%, the median duration of
response (DOR), 9.9 months and the median progression free survival
(PFS), 10.8 months, respectively. Data showed substantial
improvement in pruritis in patients having a global clinical
response but also in patients with stable disease.
[0408] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference in
their entirety and to the same extent as if each reference were
individually and specifically indicated to be incorporated by
reference and were set forth in its entirety herein (to the maximum
extent permitted by law), regardless of any separately provided
incorporation of particular documents made elsewhere herein.
[0409] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention are to be
construed to cover both the singular and the plural, unless
otherwise indicated herein or clearly contradicted by context.
[0410] Unless otherwise stated, all exact values provided herein
are representative of corresponding approximate values (e.g., all
exact exemplary values provided with respect to a particular factor
or measurement can be considered to also provide a corresponding
approximate measurement, modified by "about," where
appropriate).
[0411] The description herein of any aspect or embodiment of the
invention using terms such as "comprising", "having," "including,"
or "containing" with reference to an element or elements is
intended to provide support for a similar aspect or embodiment of
the invention that "consists of", "consists essentially of", or
"substantially comprises" that particular element or elements,
unless otherwise stated or clearly contradicted by context (e.g., a
composition described herein as comprising a particular element
should be understood as also describing a composition consisting of
that element, unless otherwise stated or clearly contradicted by
context).
[0412] The use of any and all examples, or exemplary language
(e.g., "such as") provided herein, is intended merely to better
illuminate the invention and does not pose a limitation on the
scope of the invention unless otherwise claimed. No language in the
specification should be construed as indicating any non-claimed
element as essential to the practice of the invention.
Sequence CWU 1
1
1091434PRThomo sapiens 1Leu Met Gly Gly Gln Asp Lys Pro Phe Leu Ser
Ala Arg Pro Ser Thr1 5 10 15Val Val Pro Arg Gly Gly His Val Ala Leu
Gln Cys His Tyr Arg Arg 20 25 30Gly Phe Asn Asn Phe Met Leu Tyr Lys
Glu Asp Arg Ser His Val Pro 35 40 45Ile Phe His Gly Arg Ile Phe Gln
Glu Ser Phe Ile Met Gly Pro Val 50 55 60Thr Pro Ala His Ala Gly Thr
Tyr Arg Cys Arg Gly Ser Arg Pro His65 70 75 80Ser Leu Thr Gly Trp
Ser Ala Pro Ser Asn Pro Leu Val Ile Met Val 85 90 95Thr Gly Asn His
Arg Lys Pro Ser Leu Leu Ala His Pro Gly Pro Leu 100 105 110Leu Lys
Ser Gly Glu Thr Val Ile Leu Gln Cys Trp Ser Asp Val Met 115 120
125Phe Glu His Phe Phe Leu His Arg Asp Gly Ile Ser Glu Asp Pro Ser
130 135 140Arg Leu Val Gly Gln Ile His Asp Gly Val Ser Lys Ala Asn
Phe Ser145 150 155 160Ile Gly Pro Leu Met Pro Val Leu Ala Gly Thr
Tyr Arg Cys Tyr Gly 165 170 175Ser Val Pro His Ser Pro Tyr Gln Leu
Ser Ala Pro Ser Asp Pro Leu 180 185 190Asp Ile Val Ile Thr Gly Leu
Tyr Glu Lys Pro Ser Leu Ser Ala Gln 195 200 205Pro Gly Pro Thr Val
Gln Ala Gly Glu Asn Val Thr Leu Ser Cys Ser 210 215 220Ser Trp Ser
Ser Tyr Asp Ile Tyr His Leu Ser Arg Glu Gly Glu Ala225 230 235
240His Glu Arg Arg Leu Arg Ala Val Pro Lys Val Asn Arg Thr Phe Gln
245 250 255Ala Asp Phe Pro Leu Gly Pro Ala Thr His Gly Gly Thr Tyr
Arg Cys 260 265 270Phe Gly Ser Phe Arg Ala Leu Pro Cys Val Trp Ser
Asn Ser Ser Asp 275 280 285Pro Leu Leu Val Ser Val Thr Gly Asn Pro
Ser Ser Ser Trp Pro Ser 290 295 300Pro Thr Glu Pro Ser Ser Lys Ser
Gly Ile Cys Arg His Leu His Val305 310 315 320Leu Ile Gly Thr Ser
Val Val Ile Phe Leu Phe Ile Leu Leu Leu Phe 325 330 335Phe Leu Leu
Tyr Arg Trp Cys Ser Asn Lys Lys Asn Ala Ala Val Met 340 345 350Asp
Gln Glu Pro Ala Gly Asp Arg Thr Val Asn Arg Gln Asp Ser Asp 355 360
365Glu Gln Asp Pro Gln Glu Val Thr Tyr Ala Gln Leu Asp His Cys Val
370 375 380Phe Ile Gln Arg Lys Ile Ser Arg Pro Ser Gln Arg Pro Lys
Thr Pro385 390 395 400Leu Thr Asp Thr Ser Val Tyr Thr Glu Leu Pro
Asn Ala Glu Pro Arg 405 410 415Ser Lys Val Val Ser Cys Pro Arg Ala
Pro Gln Ser Gly Leu Glu Gly 420 425 430Val Phe25PRTMus musculus
2Ser Tyr Thr Met His1 5315PRTMus musculus 3Tyr Ile Asn Pro Ser Ser
Gly Tyr Thr Glu Asn Asn Arg Lys Phe1 5 10 15412PRTMus musculus 4Arg
Leu Gly Lys Gly Leu Leu Pro Pro Phe Asp Tyr1 5 10511PRTMus musculus
5Arg Ala Ser Glu Asn Ile Tyr Ser Asn Leu Ala1 5 1067PRTMus musculus
6Ala Ala Thr Asn Leu Ala Asp1 579PRTMus musculus 7Gln His Phe Trp
Gly Thr Pro Tyr Thr1 58107PRTartificialChimeric 8Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn 20 25 30Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu 35 40 45Tyr
Ala Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro
Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
1059107PRTartificialChimeric 9Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Glu Asn Ile Tyr Ser Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Gln Leu Leu Val 35 40 45Tyr Ala Ala Thr Asn Leu
Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Tyr 85 90 95Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys 100
10510107PRTartificialchimeric 10Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Glu Asn Ile Tyr Ser Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Gln Leu Leu Val 35 40 45Tyr Ala Ala Thr Asn Leu
Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Gln Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Tyr 85 90 95Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys 100
10511107PRTArtificialChimeric 11Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Glu Asn Ile Tyr Ser Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys
Gln Gly Lys Ala Pro Gln Leu Leu Val 35 40 45Tyr Ala Ala Thr Asn Leu
Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Gln Tyr Thr Leu Thr Ile Asn Ser Leu Gln Pro65 70 75 80Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Tyr 85 90 95Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys 100
10512107PRTartificialchimeric 12Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Glu Thr Val Thr Ile Thr Cys Arg
Ala Ser Glu Asn Ile Tyr Ser Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys
Gln Gly Lys Ala Pro Gln Leu Leu Val 35 40 45Tyr Ala Ala Thr Asn Leu
Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Gln Tyr Thr Leu Thr Ile Asn Ser Leu Gln Pro65 70 75 80Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Tyr 85 90 95Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys 100
10513120PRTartificialchimeric 13Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Thr Met His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Tyr Ile Asn Pro Ser
Ser Gly Tyr Thr Glu Asn Asn Arg Lys Phe 50 55 60Lys Asp Arg Val Thr
Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Leu Gly Lys Gly Leu Leu Pro Pro Phe Asp Tyr Trp Gly Gln 100 105
110Gly Thr Thr Val Thr Val Ser Ser 115
12014120PRTartificialchimeric 14Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Thr Met His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asn Pro Ser
Ser Gly Tyr Thr Glu Asn Asn Arg Lys Phe 50 55 60Lys Asp Lys Thr Thr
Met Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Leu Gly Lys Gly Leu Leu Pro Pro Phe Asp Tyr Trp Gly Gln 100 105
110Gly Thr Thr Val Thr Val Ser Ser 115
12015120PRTartificialchimeric 15Gln Val Gln Leu Gln Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Thr Met His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asn Pro Ser
Ser Gly Tyr Thr Glu Asn Asn Arg Lys Phe 50 55 60Lys Asp Lys Thr Thr
Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Leu Gly Lys Gly Leu Leu Pro Pro Phe Asp Tyr Trp Gly Gln 100 105
110Gly Thr Thr Leu Thr Val Ser Ser 115
12016120PRTartificialchimeric 16Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Leu Ala Arg Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Thr Met His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asn Pro Ser
Ser Gly Tyr Thr Glu Asn Asn Arg Lys Phe 50 55 60Lys Asp Lys Thr Thr
Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Leu Gly Lys Gly Leu Leu Pro Pro Phe Asp Tyr Trp Gly Gln 100 105
110Gly Thr Thr Val Thr Val Ser Ser 115
12017120PRTartificialchimeric 17Gln Val Gln Leu Gln Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Thr Met His Trp Val Lys Gln
Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asn Pro Ser
Ser Gly Tyr Thr Glu Asn Asn Arg Lys Phe 50 55 60Lys Asp Lys Thr Thr
Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Leu Gly Lys Gly Leu Leu Pro Pro Phe Asp Tyr Trp Gly Gln 100 105
110Gly Thr Thr Leu Thr Val Ser Ser 115 120185PRTMus musculus 18Thr
Ala Gly Met Gln1 51916PRTMus musculus 19Trp Ile Asn Ser His Ser Gly
Val Pro Lys Tyr Ala Glu Asp Phe Lys1 5 10 15209PRTMus musculus
20Gly Gly Asp Glu Gly Val Met Asp Tyr1 52111PRTMus musculus 21Lys
Ala Ser Gln Asp Val Ser Thr Ala Val Ala1 5 10227PRTMus musculus
22Trp Thr Ser Thr Arg His Thr1 5239PRTMus musculus 23Gln Gln His
Tyr Ser Thr Pro Trp Thr1 524107PRTArtificialchimeric 24Asp Ile Gln
Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg
Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30Val
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40
45Tyr Trp Thr Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Ala65 70 75 80Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser
Thr Pro Trp 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
10525107PRTArtificialchimeric 25Asp Ile Gln Met Thr Gln Ser Pro Ser
Phe Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys
Ala Ser Gln Asp Val Ser Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45Tyr Trp Thr Ser Thr Arg
His Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Ala65 70 75 80Glu Asp Val
Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Trp 85 90 95Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys 100
10526107PRTArtificialchimeric 26Asp Ile Val Met Thr Gln Ser Pro Ser
Phe Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys
Ala Ser Gln Asp Val Ser Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45Tyr Trp Thr Ser Thr Arg
His Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Asp Tyr Thr Leu Thr Ile Ser Ser Val Gln Ala65 70 75 80Glu Asp Val
Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Trp 85 90 95Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys 100
10527107PRTArtificialchimeric 27Asp Ile Val Met Thr Gln Ser Pro Ser
Phe Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Phe Thr Cys Lys
Ala Ser Gln Asp Val Ser Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Trp Thr Ser Thr Arg
His Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Asp Tyr Thr Leu Thr Ile Ser Ser Val Gln Ala65 70 75 80Glu Asp Val
Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Trp 85 90 95Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys 100
10528107PRTArtificialChimeric 28Asp Ile Val Met Thr Gln Ser His Lys
Phe Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Phe Thr Cys Lys
Ala Ser Gln Asp Val Ser Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Trp Thr Ser Thr Arg
His Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Asp Tyr Thr Leu Thr Ile Ser Ser Val Gln Ala65 70 75 80Glu Asp Val
Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Trp 85 90 95Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys 100
10529118PRTArtificialChimeric 29Gln Val Gln Leu Val Gln Ser Gly Ser
Glu Leu Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Thr Ala 20 25 30Gly Met Gln Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Ser His
Ser Gly Val Pro Lys Tyr Ala Glu Asp Phe 50 55 60Lys Gly Arg Phe Val
Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr65 70 75 80Leu Gln Ile
Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Gly Gly Asp Glu Gly Val Met Asp Tyr Trp Gly Gln Gly Thr 100 105
110Thr Val Thr Val Ser Ser 11530118PRTArtificialchimeric 30Gln Val
Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala1 5 10 15Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Ala 20 25
30Gly Met Gln Trp Val Gln Lys Ser Pro Gly Gln Gly Leu Glu Trp Met
35
40 45Gly Trp Ile Asn Ser His Ser Gly Val Pro Lys Tyr Ala Glu Asp
Phe 50 55 60Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr
Ala Tyr65 70 75 80Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala
Val Tyr Phe Cys 85 90 95Ala Arg Gly Gly Asp Glu Gly Val Met Asp Tyr
Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser
11531118PRTArtificialchimeric 31Gln Ile Gln Leu Val Gln Ser Gly Ser
Glu Leu Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Thr Ala 20 25 30Gly Met Gln Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Trp Ile Asn Ser His
Ser Gly Val Pro Lys Tyr Ala Glu Asp Phe 50 55 60Lys Gly Arg Phe Val
Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr65 70 75 80Leu Gln Ile
Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95Ala Arg
Gly Gly Asp Glu Gly Val Met Asp Tyr Trp Gly Gln Gly Thr 100 105
110Thr Val Thr Val Ser Ser 11532118PRTArtificialChimeric 32Gln Ile
Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala1 5 10 15Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Ala 20 25
30Gly Met Gln Trp Val Gln Lys Ser Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45Gly Trp Ile Asn Ser His Ser Gly Val Pro Lys Tyr Ala Glu Asp
Phe 50 55 60Lys Gly Arg Phe Ala Phe Ser Leu Asp Thr Ser Val Ser Thr
Ala Tyr65 70 75 80Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala
Val Tyr Phe Cys 85 90 95Ala Arg Gly Gly Asp Glu Gly Val Met Asp Tyr
Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser
11533118PRTArtificialChimeric 33Gln Ile Gln Leu Val Gln Ser Gly Ser
Glu Leu Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Thr Ala 20 25 30Gly Met Gln Trp Val Gln Lys
Thr Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Trp Ile Asn Ser His
Ser Gly Val Pro Lys Tyr Ala Glu Asp Phe 50 55 60Lys Gly Arg Phe Ala
Phe Ser Leu Asp Thr Ser Ala Ser Thr Ala Tyr65 70 75 80Leu Gln Ile
Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95Ala Arg
Gly Gly Asp Glu Gly Val Met Asp Tyr Trp Gly Gln Gly Thr 100 105
110Ser Val Thr Val Ser Ser 11534117PRTMus musculus 34Gln Ile Gln
Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu1 5 10 15Thr Val
Arg Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ile Ala 20 25 30Gly
Met Gln Trp Val Gln Lys Met Pro Gly Lys Gly Leu Lys Trp Ile 35 40
45Gly Trp Ile Asn Thr His Ser Gly Val Pro Lys Tyr Ala Glu Asp Phe
50 55 60Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Asn Ile Ala
Tyr65 70 75 80Leu Gln Ile Ser Asn Leu Lys Asn Glu Asp Thr Ala Thr
Tyr Phe Cys 85 90 95Ala Arg Gly Gly Asp Glu Gly Val Met Asp Tyr Trp
Gly Gln Gly Thr 100 105 110Ser Val Thr Val Ser 11535107PRTMus
musculus 35Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser
Val Gly1 5 10 15Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val
Ser Thr Ala 20 25 30Val Ala Trp Tyr His Gln Lys Pro Gly Gln Ser Pro
Lys Leu Leu Ile 35 40 45Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro
Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr
Ile Ser Ala Leu Gln Ala65 70 75 80Glu Asp Leu Ala Leu Tyr Tyr Cys
Gln Gln His Tyr Asn Thr Pro Trp 85 90 95Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys 100 10536117PRTMus musculus 36Gln Ile Gln Leu Val
Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu1 5 10 15Thr Val Arg Ile
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Ala 20 25 30Gly Met Gln
Trp Val Gln Lys Thr Pro Gly Lys Gly Leu Lys Trp Ile 35 40 45Gly Trp
Ile Asn Ser His Ser Gly Val Pro Lys Tyr Ala Glu Asp Phe 50 55 60Lys
Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr65 70 75
80Leu Gln Ile Ser Thr Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95Ala Arg Gly Gly Asp Glu Gly Val Met Asp Tyr Trp Gly Gln Gly
Thr 100 105 110Ser Val Thr Val Ser 11537107PRTmus musculus 37Asp
Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Leu Gly1 5 10
15Asp Arg Val Ser Phe Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala
20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu
Ile 35 40 45Tyr Trp Thr Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe
Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser
Val Gln Ala65 70 75 80Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His
Tyr Ser Thr Pro Trp 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys 100 10538139PRTMus musculus 38Gln Val Gln Leu Gln Gln Ser Ala
Ala Glu Leu Ala Arg Pro Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Thr Met His Trp Val Lys
Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asn Pro
Ser Ser Gly Tyr Thr Glu Asn Asn Arg Lys Phe 50 55 60Lys Asp Lys Thr
Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln
Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Leu Gly Lys Gly Leu Leu Pro Pro Phe Asp Tyr Trp Gly Gln 100 105
110Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val
115 120 125Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr 130
13539107PRTMus musculus 39Asp Ile Gln Met Thr Gln Ser Pro Ala Ser
Leu Ser Val Ser Val Gly1 5 10 15Glu Thr Val Thr Ile Thr Cys Arg Ala
Ser Glu Asn Ile Tyr Ser Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Gln
Gly Lys Ser Pro Gln Leu Leu Val 35 40 45Tyr Ala Ala Thr Asn Leu Ala
Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Gln
Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser65 70 75 80Glu Asp Phe Gly
Ser Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Tyr 85 90 95Thr Phe Gly
Gly Gly Thr Lys Leu Glu Ile Lys 100 10540119PRTMus musculus 40Glu
Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala1 5 10
15Ser Met Lys Ile Ser Cys Lys Ala Ser His Tyr Ser Phe Ile Gly Tyr
20 25 30Thr Met Asn Trp Val Lys Gln Arg His Gly Lys Asn Leu Glu Trp
Ile 35 40 45Gly Leu Ile Asn Pro Tyr Asn Gly Asp Thr Thr Tyr Asn Gln
Lys Phe 50 55 60Lys Gly Lys Ala Ser Leu Thr Val Asp Lys Ser Ser Ser
Thr Ala Tyr65 70 75 80Met Glu Ile Leu Ser Leu Thr Ser Glu Asp Ser
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Asn Trp Gly Tyr Pro Tyr Ala
Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Ser Val Thr Val Ser
11541111PRTMus musculus 41Asp Ile Val Leu Thr Gln Ser Pro Ala Ser
Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala
Ser Glu Ser Val Asp Asn Phe 20 25 30Gly Ile Ser Phe Met Asn Trp Phe
Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr Ala Ala
Ser Asn Gln Gly Ser Gly Val Pro Ala 50 55 60Arg Phe Ser Gly Ser Arg
Ser Gly Thr Asp Phe Ser Leu Asn Ile His65 70 75 80Pro Met Glu Glu
Asp Asp Thr Ala Met Tyr Phe Cys Gln Gln Ser Lys 85 90 95Glu Val Pro
Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105
11042117PRTMus musculus 42Gln Val Gln Leu Gln Gln Ser Gly Ala Glu
Leu Val Arg Pro Gly Val1 5 10 15Ser Val Lys Ile Ser Cys Lys Gly Ser
Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Ala Met Asn Trp Val Lys Gln Ser
His Ala Lys Ser Leu Glu Trp Ile 35 40 45Gly Val Ile Ser Thr Tyr Tyr
Gly Asp Ala Asn Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Ala Thr Met
Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ala
Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95Ala Leu Ile
Tyr Tyr Asp Tyr Asp Gly Ser Tyr Trp Gly Gln Gly Thr 100 105 110Thr
Leu Thr Val Ser 11543113PRTMus musculus 43Asp Val Val Met Thr Gln
Thr Pro Leu Ser Leu Pro Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile
Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn Gly Asn Thr
Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Lys Leu
Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75
80Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser
85 90 95Thr His Val Pro Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile 100 105 110Lys44120PRTMus musculus 44Gln Val Gln Leu Gln Gln
Ser Ala Ala Glu Leu Ala Arg Pro Gly Ala1 5 10 15Ser Val Lys Met Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Thr Met His Trp
Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile
Asn Pro Ser Ser Gly Tyr Thr Asp Tyr Asn Gln Lys Phe 50 55 60Lys Asp
Lys Thr Thr Leu Thr Ala Asp Arg Ser Ser Ser Thr Ala Tyr65 70 75
80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95Ala Arg Leu Gly Lys Gly Leu Leu Pro Pro Phe Asp Tyr Trp Gly
Gln 100 105 110Gly Ser Thr Leu Thr Val Ser Ser 115 12045113PRTMus
musculus 45Glu Ile Val Leu Thr Gln Ser Ile Pro Ser Leu Thr Val Ser
Ala Gly1 5 10 15Glu Arg Val Thr Ile Ser Cys Lys Ser Asn Gln Asn Leu
Leu Trp Ser 20 25 30Gly Asn Gln Arg Tyr Cys Leu Val Trp His Gln Trp
Lys Pro Gly Gln 35 40 45Thr Pro Thr Pro Leu Ile Thr Trp Thr Ser Asp
Arg Tyr Ser Gly Val 50 55 60Pro Asp Arg Phe Ile Gly Ser Gly Ser Val
Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Val Gln Ala Glu Asp
Val Ala Val Tyr Phe Cys Gln Gln 85 90 95His Leu His Ile Pro Tyr Thr
Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110Lys46107PRTMus
musculus 46Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser
Val Gly1 5 10 15Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile
Tyr Ser Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro
Gln Leu Leu Val 35 40 45Tyr Ala Ala Thr Asn Leu Ala Asp Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys
Ile Asn Ser Leu Gln Ser65 70 75 80Glu Asp Phe Gly Ser Tyr Tyr Cys
Gln His Phe Trp Gly Thr Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys 100 10547118PRTMus musculus 47Gln Val Gln Leu Gln
Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala1 5 10 15Ser Val Lys Leu
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met Gln
Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Ala
Ile Tyr Pro Gly Asp Gly Asp Thr Arg Tyr Thr Gln Lys Phe 50 55 60Lys
Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75
80Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95Ala Arg Arg Tyr Asp Gly Tyr Tyr His Phe Asp Tyr Trp Gly Gln
Gly 100 105 110Thr Thr Leu Thr Val Ser 11548115PRTMus musculus
48Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Val Thr Ala Gly1
5 10 15Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Trp
Ser 20 25 30Val Asn Gln Lys Asn Tyr Leu Ser Trp Tyr Gln Gln Lys Gln
Arg Gln 35 40 45Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Ile Arg Glu
Ser Trp Val 50 55 60Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr65 70 75 80Ile Ser Asn Val His Ala Glu Asp Leu Ala
Val Tyr Tyr Cys Gln His 85 90 95Asn His Gly Ser Phe Leu Pro Leu Thr
Phe Gly Ser Gly Thr Lys Leu 100 105 110Glu Ile Lys 11549120PRTMus
musculus 49Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Ala Arg Pro
Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Lys Ser Ser Gly Phe Thr Phe
Thr Thr Tyr 20 25 30Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly
Leu Glu Trp Ile 35 40 45Gly Ala Ile Tyr Pro Gly Asp Gly Asp Thr Arg
Tyr Thr Gln Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys
Ser Ser Ile Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Leu Ala Ser
Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Gly Asp Tyr Gly
Asn Tyr Gly Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Ser Val Thr
Val Ser Ser 115 12050112PRTMus musculus 50Asp Val Leu Met Thr Gln
Thr Pro Leu Ser Leu Pro Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile
Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30Asn Gly Asn Thr
Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Lys Leu
Leu Ile Tyr Lys Val Ser Asn His Phe Ser Gly Val Pro 50 55 60Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75
80Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly
85 90 95Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys 100 105 110515PRTMus musculus 51Ile Ala Gly Met Gln1 5526PRTMus
musculus 52Gly Tyr Thr Phe Thr Ile1 55310PRTMus musculus 53Gly Tyr
Thr Phe Thr Ile Ala Gly Met Gln1 5 105417PRTMus musculus 54Trp Ile
Asn Thr His Ser Gly Val Pro Lys Tyr Ala Glu Asp Phe Lys1 5 10
15Gly5510PRTMus musculus 55Trp Ile Asn Thr His Ser Gly Val Pro Lys1
5 10566PRTMus musculus 56Gly Tyr Thr Phe Thr Thr1 55710PRTMus
musculus 57Gly Tyr Thr Phe Thr Thr Ala Gly Met Gln1 5 10589PRTMus
musculus 58Trp Ile Asn Ser His Ser Gly Val Pro1 55910PRTMus
musculus 59Gly Gly Asp Glu Gly Val Met Asp Tyr Trp1 5 10606PRTMus
musculus 60Gly Tyr Thr Phe Thr Ser1 56110PRTMus musculus 61Gly Tyr
Thr Phe Thr Ser Tyr Thr Met His1 5 10628PRTMus musculus 62Tyr Ile
Asn Pro Ser Ser Gly Tyr1 56312PRTMus musculus 63Arg Leu Gly Lys Gly
Leu Leu Pro Pro Phe Asp Tyr1 5 10645PRTMus musculus 64Gly Tyr Thr
Met Asn1 5656PRTMus musculus 65His Tyr Ser Phe Ile Gly1 56610PRTMus
musculus 66His Tyr Ser Phe Ile Gly Tyr Thr Met Asn1 5 106717PRTMus
musculus 67Leu Ile Asn Pro Tyr Asn Gly Asp Thr Thr Tyr Asn Gln Lys
Phe Lys1 5 10 15Gly6810PRTMus musculus 68Leu Ile Asn Pro Tyr Asn
Gly Asp Thr Thr1 5 106911PRTMus musculus 69Glu Asn Trp Gly Tyr Pro
Tyr Ala Met Asp Tyr1 5 10705PRTMus musculus 70Asp Tyr Ala Met Asn1
5716PRTMus musculus 71Gly Tyr Thr Phe Thr Asp1 57210PRTMus musculus
72Gly Tyr Thr Phe Thr Asp Tyr Ala Met Asn1 5 107317PRTMus musculus
73Val Ile Ser Thr Tyr Tyr Gly Asp Ala Asn Tyr Asn Gln Lys Phe Lys1
5 10 15Gly7410PRTMus musculus 74Val Ile Ser Thr Tyr Tyr Gly Asp Ala
Asn1 5 10759PRTMus musculus 75Ile Tyr Tyr Asp Tyr Asp Gly Ser Tyr1
5765PRTMus musculus 76Ser Tyr Thr Met His1 5776PRTMus musculus
77Gly Tyr Thr Phe Thr Ser1 57810PRTMus musculus 78Gly Tyr Thr Phe
Thr Ser Tyr Thr Met His1 5 107917PRTMus musculus 79Tyr Ile Asn Pro
Ser Ser Gly Tyr Thr Asp Tyr Asn Gln Lys Phe Lys1 5 10
15Asp8010PRTMus musculus 80Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Asp1
5 108111PRTMus musculus 81Leu Gly Lys Gly Leu Leu Pro Pro Phe Asp
Tyr1 5 10825PRTMus musculus 82Ser Tyr Trp Met Gln1 5836PRTMus
musculus 83Gly Tyr Thr Phe Thr Ser1 58410PRTMus musculus 84Gly Tyr
Thr Phe Thr Ser Tyr Trp Met Gln1 5 108517PRTMus musculus 85Ala Ile
Tyr Pro Gly Asp Gly Asp Thr Arg Tyr Thr Gln Lys Phe Lys1 5 10
15Gly8610PRTMus musculus 86Ala Ile Tyr Pro Gly Asp Gly Asp Thr Arg1
5 108710PRTMus musculus 87Arg Tyr Asp Gly Tyr Tyr His Phe Asp Tyr1
5 10885PRTMus musculus 88Thr Tyr Trp Met Gln1 5896PRTMus musculus
89Gly Phe Thr Phe Thr Thr1 59010PRTMus musculus 90Gly Phe Thr Phe
Thr Thr Tyr Trp Met Gln1 5 109117PRTMus musculus 91Ala Ile Tyr Pro
Gly Asp Gly Asp Thr Arg Tyr Thr Gln Lys Phe Lys1 5 10
15Gly9210PRTMus musculus 92Ala Ile Tyr Pro Gly Asp Gly Asp Thr Arg1
5 109311PRTMus musculus 93Arg Gly Asp Tyr Gly Asn Tyr Gly Met Asp
Tyr1 5 10949PRTMus musculus 94Gln Gln His Tyr Asn Thr Pro Trp Thr1
59515PRTMus musculus 95Arg Ala Ser Glu Ser Val Asp Asn Phe Gly Ile
Ser Phe Met Asn1 5 10 15967PRTMus musculus 96Ala Ala Ser Asn Gln
Gly Ser1 5979PRTMus musculus 97Gln Gln Ser Lys Glu Val Pro Tyr Thr1
59816PRTMus musculus 98Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly
Asn Thr Tyr Leu His1 5 10 15997PRTMus musculus 99Lys Val Ser Asn
Arg Phe Ser1 510010PRTMus musculus 100Ser Gln Ser Thr His Val Pro
Pro Tyr Thr1 5 1010117PRTMus musculus 101Lys Ser Asn Gln Asn Leu
Leu Trp Ser Gly Asn Gln Arg Tyr Cys Leu1 5 10 15Val1027PRTMus
musculus 102Trp Thr Ser Asp Arg Tyr Ser1 51039PRTMus musculus
103Gln Gln His Leu His Ile Pro Tyr Thr1 510417PRTMus musculus
104Lys Ser Ser Gln Ser Leu Leu Trp Ser Val Asn Gln Lys Asn Tyr Leu1
5 10 15Ser1057PRTMus musculus 105Gly Ala Ser Ile Arg Glu Ser1
510611PRTMus musculus 106Gln His Asn His Gly Ser Phe Leu Pro Leu
Thr1 5 1010716PRTMus musculus 107Arg Ser Ser Gln Ser Ile Val His
Ser Asn Gly Asn Thr Tyr Leu Glu1 5 10 151087PRTMus musculus 108Lys
Val Ser Asn His Phe Ser1 51099PRTMus musculus 109Phe Gln Gly Ser
His Val Pro Pro Thr1 5
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References