U.S. patent application number 16/226742 was filed with the patent office on 2019-05-09 for monoclonal antibodies against nkg2a.
The applicant listed for this patent is INNATE PHARMA S.A., UNIVERSITY OF GENOVA. Invention is credited to Pascale Andre, Emanuela Marcenaro, Alessandro Moretta, Francois Romagne.
Application Number | 20190135938 16/226742 |
Document ID | / |
Family ID | 36350427 |
Filed Date | 2019-05-09 |
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United States Patent
Application |
20190135938 |
Kind Code |
A1 |
Moretta; Alessandro ; et
al. |
May 9, 2019 |
MONOCLONAL ANTIBODIES AGAINST NKG2A
Abstract
The present invention relates to methods of treating immune
disorders, particularly autoimmune or inflammatory disorders, and
methods of producing antibodies and other compounds for use in
therapeutic strategies for treating such disorders. Generally, the
present methods involve the use of antibodies or other compounds
that prevent the stimulation of NKG2A receptors on NK cells,
leading to the lysis of dendritic cells that contribute to the
pathology of the disorders.
Inventors: |
Moretta; Alessandro;
(Genova, IT) ; Marcenaro; Emanuela; (Genova-Pegli,
IT) ; Romagne; Francois; (Marseille, FR) ;
Andre; Pascale; (Marseille, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INNATE PHARMA S.A.
UNIVERSITY OF GENOVA |
Marseille
Genova |
|
FR
IT |
|
|
Family ID: |
36350427 |
Appl. No.: |
16/226742 |
Filed: |
December 20, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14594353 |
Jan 12, 2015 |
10160810 |
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16226742 |
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11720553 |
May 31, 2007 |
8993319 |
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PCT/IB2005/004013 |
Dec 27, 2005 |
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14594353 |
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60639465 |
Dec 28, 2004 |
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60639832 |
Dec 28, 2004 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 25/00 20180101;
A61P 13/12 20180101; A61P 1/16 20180101; A61P 17/14 20180101; A61K
39/3955 20130101; A61K 45/06 20130101; A61P 19/02 20180101; A61P
1/00 20180101; A61P 5/14 20180101; G01N 33/56966 20130101; G01N
2500/04 20130101; A61P 37/00 20180101; A61P 43/00 20180101; A61P
31/12 20180101; A61P 7/00 20180101; A61P 17/06 20180101; C07K
16/2851 20130101; A61K 2039/572 20130101; A61P 21/04 20180101; A61P
27/02 20180101; C07K 2317/21 20130101; A61P 9/10 20180101; A61P
7/06 20180101; G01N 2333/70596 20130101; C07K 16/2896 20130101;
A61P 37/04 20180101; A61P 37/06 20180101; A61P 35/02 20180101; A61K
2039/505 20130101; A61P 21/00 20180101; A61P 1/04 20180101; A61P
3/10 20180101; A61P 31/00 20180101; A61P 37/08 20180101; A61P 29/00
20180101; A61P 9/00 20180101; A61P 17/00 20180101; A61P 37/02
20180101; C07K 2317/76 20130101; A61P 35/00 20180101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 39/395 20060101 A61K039/395; A61K 45/06 20060101
A61K045/06; G01N 33/569 20060101 G01N033/569 |
Claims
1. A monoclonal antibody or a fragment thereof characterized by: a)
specifically binding to NKG2A and not specifically binding to NKG2C
or NKG2E; b) a human constant region that does not substantially
bind human Fc.gamma.IIIa receptor (CD16); and c) when bound to
NKG2A on a human NK cell, causing said NK cell to lyse a target
human cell bearing HLA-E on the target cell surface, when said
target cell comes into contact with said NK cell.
2. A composition comprising: a) an effective amount of a monoclonal
antibody or a fragment thereof according to claim 1; and b) a
pharmaceutically acceptable carrier or excipient.
3. A method of reconstituting NK cell-mediated lysis of a target
cell in a population comprising a NK cell and said target cell,
wherein said NK cell is characterized by NKG2A on its surface, and
said target cell is characterized by the presence of HLA-E on its
surface, said method comprising the step of contacting said NK cell
with a monoclonal antibody or a fragment thereof according to claim
1.
4. The method according to claim 3, wherein said NK cell is a human
cell and said target cell is a human cell selected from a dendritic
cell, a cancer cell, a virally infected cell.
5. A method of treating a cancer in a patient, wherein said cancer
is characterized by the presence of a cancer cell expressing HLA-E
on its cell surface, said method comprising the step of
administering to said patient a composition according to claim
2.
6. The method according to claim 5, comprising the additional step
of administering to said patient a second therapeutic agent
selected from: an anticancer agent or an antiemetic, wherein said
second therapeutic agent is administered either as a separate
dosage form or as part of said composition.
7. The method according to claim 5, wherein the cancer is a cancer
of the bladder, breast, colon, kidney, liver, lung, ovary,
prostate, pancreas, stomach, cervix, thyroid and skin, including
squamous cell carcinoma.
8. The method according to claim 5, wherein the cancer is a
hematopoietic tumor of lymphoid lineage.
9. A method of treating a viral disease in a patient, wherein said
viral disease is characterized by the presence of a
virally-infected cell expressing HLA-E on its cell surface, said
method comprising the step of administering to said patient a
composition according to claim 2.
10. The method according to claim 9, comprising the addition step
of administering to said patient an antiviral agent, wherein said
antiviral agent is administered either as a separate dosage form or
as part of said composition.
11. A method of improving the engraftment of hematopoietic cells in
a patient, said method comprising the step of administering to said
patient a composition according to claim 2.
12. The method according to claim 11, comprising the additional
step of administering to said patient a second therapeutic agent
selected from an anticancer agent, or a hematopoietic growth
factor, wherein said second therapeutic agent is administered
either as a separate dosage form or as part of said
composition.
13. The method according to claim 11, wherein said patient is
suffering from leukemia.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. Ser. No.
14/594,353, filed Jan. 12, 2015, now U.S. Pat. No. 10,160,810,
which is a divisional of U.S. Ser. No. 11/720,553, filed May 31,
2007, now U.S. Pat. No. 8,993,319, which is the U.S. national stage
application of International Patent Application No.
PCT/M2005/004013, filed Dec. 27, 2005, which claims the benefit of
U.S. Provisional Patent Application No. 60/639,465, filed Dec. 28,
2004, and U.S. Provisional Patent Application No. 60/639,832, filed
Dec. 28, 2004, the disclosures of which are hereby incorporated by
reference in their entireties, including all figures, tables and
amino acid or nucleic acid sequences.
FIELD OF THE INVENTION
[0002] The present invention relates to monoclonal antibodies and
fragments thereof directed against the NK cell surface receptor
NKG2A, as well as to methods of producing and evaluating such
antibodies. The monoclonal antibodies and fragments thereof are
useful in treating immune disorders, particularly autoimmune
disorders, as well as other diseases requiring modulated NK cell
function. Generally, the present methods involve the use of the
antibodies and fragments thereof to prevent the stimulation of
NKG2A receptors on NK cells, leading to the lysis of HLA-E or
Qa1.sup.b expressing cells, such as dendritic cells or activated T
cells, that contribute to the pathology of the disorders to be
treated.
BACKGROUND
[0003] Maintaining effective immune surveillance without provoking
autoimmune reactions requires the precise titration of effector T
cell responses. Autoimmune disorders arise when the immune system
mounts an immune response against self-antigens (see, e.g., Ludewig
et al. (1999) Immunol Rev. 169:45-54). While the mechanisms
involved in the triggering and maintenance of autoimmune reactions
is unclear, it is likely that the appearance of previously
immunologically ignored antigens in secondary lymphoid organs is
involved.
[0004] Dendritic cells are bone-marrow derived antigen presenting
cells (APCs) that play a key role in the immune response (see,
e.g., O'Neill et al. (2004) Blood 104:2235-2246). DCs internalize
bacteria, viruses, dying cells, and various complex molecules
through phagocytosis, endocytosis, and pinocytosis. Incorporated
proteins are broken down into peptides, which are then presented on
the DC cell surface along with MHC class I and class II molecules.
Antigens loaded onto MHC class I are typically derived from
endogenous proteins and are recognized by CD8+ T cells, whereas MHC
class II loaded antigens are generally derived from external
proteins and are recognized by CD4+ T cells. Following antigen
capture, immature DC cells mature to form mature DCs which show
reduced phagocytosis, migrate to lymphoid tissues, and have
enhanced T cell stimulation capacity.
[0005] In lymphoid tissues, DCs prime naive T cells, stimulating
their clonal expansion and differentiation, and can also interact
with B cells and cells of the innate immune system, including NK
cells. Activated NK cells can kill immature, but not mature, DC
cells. As antigen transport and primary sensitization of T
lymphocytes is mainly mediated by antigen presenting dendritic
cells, it is likely that the inappropriate presentation of self
antigens by dendritic cells contributes at least in part to
autoimmune disorders.
[0006] Natural killer (NK) cells are a subpopulation of lymphocytes
involved in non-conventional immunity. NK cells provide an
efficient immunosurveillance mechanism by which undesired cells
such as tumor or virally-infected cells can be eliminated. NK cell
activity is regulated by a complex mechanism that involves both
activating and inhibitory signals (see, e.g., Moretta et al. (2001)
Annu Rev Immunol 19:197-223; Moretta et al. (2003) EMBO J EPub
December 18; Ravetch et al. (2000) Science 290:84-89; Zambello et
al. (2003) Blood 102:1797-805; Moretta et al. (1997) Curr Opin
Immunol 9:694-701; the entire disclosures of which are herein
incorporated by reference).
[0007] Several distinct NK-specific receptors have been identified
that play important roles in the NK cell mediated recognition and
killing of HLA Class I deficient target cells. These receptors,
termed NKp30, NKp46 and NKp44, are members of the Ig superfamily.
Their cross-linking, induced by specific mAbs, leads to a strong NK
cell activation resulting in increased intracellular Ca.sup.++
levels, triggering of cytotoxicity, and lymphokine release.
Importantly, mAb-mediated activation of NKp30, NKp46, and/or NKp44
results in an activation of NK cytotoxicity against many types of
target cells. These findings provide evidence for a central role of
these receptors in natural cytotoxicity.
[0008] NK cells are negatively regulated by major
histocompatibility complex (MHC) class I-specific inhibitory
receptors (Karre et al. (1986) Nature 319:675-8; Ohlen et al,
(1989) Science 246:666-8). These specific receptors bind to
polymorphic determinants of major histocompatibility complex (MHC)
class I molecules or HLA and inhibit natural killer (NK) cell
lysis. In humans, certain members of a family of receptors termed
killer Ig-like receptors (KIRs) recognize groups of HLA class I
alleles (see, e.g., Yawata et al. (2002) Crit Rev Immunol
22:463-82; Martin et al. (2000) Immunogenetics. 51:268-80; Lanier
(1998) Annu Rev Immunol. 16:359-93; the entire disclosures of which
are herein incorporated by reference).
[0009] Another important inhibitory receptor on NK cells is
CD94-NKG2A, which interacts with the non-classical MHC class 1
molecule HLA-E (see, e.g., Braud et al. (1998) Nature 391:795-799;
Lee et al. (1998) PNAS 95:5199-5204; Vance et al. (2002) PNAS
99:868-873; Brooks et al. (1999) J Immunol 162:305-313; Miller et
al. J Immunol (2003) 171:1369-75; Brooks et al. (1997) J Exp Med
185:795-800; Van Beneden et al. (2001) 4302-4311; U.S. patent
application no. 20030095965; the entire disclosures of each of
which are herein incorporated by reference). Some of these
receptors have the capacity to modulate thresholds of T cell
antigen receptor-dependent T cell activation. In the rare absence
of inhibitory receptors, the activating isoforms may augment T cell
effector functions and contribute to autoimmune pathology. The
amino acid sequence of NKG2A varies among mammals, including among
primates. For example, the human and rhesus monkey versions of the
NKG2A proteins share less than 90% identity, including
approximately 86% within the ligand binding domain.
[0010] Efforts towards therapeutics for modulating NKG2A,
essentially for the prevention of inflammation, have focused on the
study of the nonclassical MHC class I molecules, HLA-E for the
human receptor and Qa-1b for the mouse receptor. For cell surface
expression, these MHC molecules preferentially bind peptides
derived from the signal peptides of other MHC class I molecules.
The expression of other class I MHC molecules can regulate the
expression of HLA-E, thereby allowing NK cells to monitor the state
of the MHC class I dependent antigen presentation pathway in
potential target cells. The level of cell surface HLA-E is critical
for the NK cell cytotoxicity towards tumor and virally infected
cells. Therapeutic strategies for modulating HLA-E expression or
function have generally been directed towards using HLA-I or HSP60
peptides to induce a protective state for the prevention of
inflammation such that NK cells are not activated.
[0011] United States patent publication 20030095965 discloses an
antibody, 3S9, that binds to NKG2A, NKG2C and NKG2E. 3 S9
purportedly causes cross-linking of those receptors and concomitant
inhibition of NK cell-mediated lysis. Co-owned PCT patent
publication WO 2005/105849 discloses the use of an antibody that
specifically binds to an NK receptor, including NKG2A, to treat a
patient suffering from NK-type lymphoproliferative disease of
granular lymphocytes (NK-LDGL). Such antibodies inhibit NK cell
activity.
[0012] Monoclonal antibodies have proven to be enormously useful
for the diagnosis and treatment of various diseases. Therapeutic
monoclonal antibodies can act through different mechanisms. Some
antibodies, such as Rituxan, recognize antigens (CD20 in the case
of Rituxan) present on the surface of pathological cells, e.g.,
tumor cells, and act by directing the immune system to destroy the
recognized cells. Other antibodies, such as Bexxar, Oncolym, or
Zevalin, are coupled to radioisotopes, chemotherapeutic agents, or
toxins, leading to the direct killing of cells bound by the
antibodies. Still others, such as Basiliximab and Daclizumab (which
block IL-2), the IgE blocking Omalizumab, and efaluzimab, act to
block the activity of specific proteins. Antibody based therapies
are well known in the art and are reviewed, e.g., in Gatto (2004)
Curr Med Chem Anti-Canc Agents 4(5):411-4, Casadevall et al. (2004)
Nat Rev Microbiol. 2(9):695-703, Hinoda et al. (2004) Cancer Sci.
95(8):621-5, Olszewski et al. (2004) Sci STKE. July 06(241):pe30,
Coiffier (2004) Hematol J. Suppl 3:S154-8, Roque et al. (2004)
Biotechnol Prog. 20(3):639-54, the entire disclosures of each of
which is herein incorporated by reference.
[0013] Before antibodies can be used for therapeutic applications
in humans, or enter clinical trials, they must go through
pre-clinical studies in non-human animals to assess various
parameters such as their toxicity, in vivo efficacy,
bioavailability, half-life and various other pharmacokinetic and
pharmacodynamic parameters. Such assays are typically carried out
in mammals, and, preferably, where they have biological activity,
i.e. where the mAb is reacting to the homolog molecule in the
specie, therefore where one can expect the greatest physiological
similarity to humans. However, studies in nonhuman primates can be
impeded if an antibody directed against a human protein does not
bind to the nonhuman animal homolog of the target protein. When
crossreactivity is present, in contrast, not only can the in vivo
efficacy of the antibody be tested in the animal, but other issues
such as side effects, toxicity, or kinetic properties that are
related to the binding of the antibody to the target protein can be
studied as well. Examples of readily available primates include the
New World monkeys and Old World monkeys, such as the cynomolgus
monkey (Macaca mulatta), the rhesus macaque (Macacus mulatta), the
African green monkey (Chlorocebus aethiops), the marmoset
(Callithrix jacchus), the saimiri (Saimiri sciureus), all available
from "Centre de Primatologie" (CDP: ULP, Fort Foch, 67207
Niederhausbergen, France), and the baboon (Papio hamadryas)
available from "Station de Primatologie du CNRS", CD56, 13790
Rousset/Arc, France). Chimpanzees and apes in general may also be
used for testing a candidate medicament, although such instances
are rare and generally only when no other alternative for testing
exists or has been exhausted.
[0014] As antibodies bind to specific 3-dimensional features of
their targets, slight changes in the amino acid sequence of a
target protein can abolish binding altogether, making it
unpredictable whether a given antibody directed against a protein
from one species will also bind to homologous proteins sharing some
but not complete sequence identity. Many instances have been
described in which antibodies directed against a human protein, for
example, do not bind to homologs in even closely related species.
For example, some antibodies against the human CD4 protein do not
bind to monkey homologs, even though the human and rhesus monkey
CD4 proteins share close to 94% percent identity (see, e.g.,
Genbank IDs GI:116013 and 20981680; Sharma et al. (2000) JPET
293:33-41, 2000, the entire disclosures of which are incorporated
herein by reference). Other examples include some antibodies
against human CD3, a widely pursued pharmaceutical target for
antibody development; antibodies, for example UCHT2, otherwise
having properties suitable for development do not crossreact with
the monkey CD3 protein.
[0015] In view of the prominence and severity of many autoimmune
disorders, and the role of mature dendritic cells in coordinating
the immune response against self-antigens, there is a great need in
the art for new and effective therapies that modulate the activity
or level of dendritic cells underlying such disorders. Moreover,
there is a need for therapies against disorders characterized by
aberrant cells (e.g., certain cancer or virally infected cells)
that are able to shield themselves from destruction by the immune
system. Finally, there is also a need to find a valid in vivo test
system for the therapeutic potential in humans of monoclonal
antibodies against NKG2A. The present invention addresses this and
other needs.
SUMMARY OF THE INVENTION
[0016] The present invention provides monoclonal antibodies and
fragments thereof directed against the NKG2A receptor. The
monoclonal antibodies and fragments thereof of this invention may
either inhibit the ability of NK cells to lyse normally susceptible
target cells ("NK cell inhibitory antibodies") or reconstitute the
ability of NK cells to lyse otherwise protected target cells ("NK
cell activating antibodies"). The function of the monoclonal
antibodies and fragments thereof of this invention is dependent
upon their ability to bind to an Fc receptor.
[0017] Fc receptors, such as Fc gamma receptors, are expressed on
the surface of leukocytes. These receptors bind to the Fc portion
of immunoglobulin (Ig), e.g. Fc gamma receptors bind to the Fc
portion of IgG. This binding helps contribute to immune function by
linking the recognition of antigens by antibodies with cell-based
effector mechanisms. Different immunoglobulin classes trigger
different effector mechanisms through the differential interaction
of immunoglobulin Fc regions with specific Fc receptors (FcRs) on
immune cells. Activating Fc gamma receptors include Fc gamma RI, Fc
gamma RITA, Fc gamma RIIC, and Fc gamma RIII A. Fc gamma RIIB is
considered an inhibitory Fc gamma receptor. (For review, see, e.g.,
Woof et al. (2004) Nat Rev Immunol. 4(2):89-99; Baumann et al.
(2003) Arch Immunol Ther Exp (Warsz) 51(6):399-406; Pan et al.
(2003) Chin Med J (Engl) 116(4):487-94; Takai et al. (1994) Cell
76:519-529; Ravetch et al. (2001) Annu Rev Immunol 19:275-290, the
entire disclosures of each of which are herein incorporated by
reference).
[0018] Without being bound by theory, the inventors believe that
the presence of an Fc receptor binding region in the antibodies and
fragments of this invention causes inhibition of NK cell lysis in
the presence of a cell bearing an Fc receptor. Those antibodies and
fragments that lack an Fc receptor binding region are capable of
reconstituting NK cell lysis of target cells bearing HLA-E or
Qa1.sup.b on their cell surface. Such target cells are typically
protected against NK cell lysis through the interaction of HLA-E or
Qa1.sup.b with the NKG2A receptor.
[0019] The invention also provides compositions comprising the
antibodies and fragments of this invention, as well as therapeutic
methods utilizing such compositions for treating different diseases
and disorders. The invention further provides methods for using
non-human primates to evaluate and characterize the activity,
toxicity and proper dosing regimen of an antibody or fragment
thereof against human NKG2A.
[0020] In one aspect, accordingly, the present invention provides
an activating antibody that is a monoclonal antibody or a fragment
thereof characterized by: a) specifically binding to NKG2A; b) not
specifically binding to an Fc receptor; and c) when bound to NKG2A
on a human NK cell, causing said NK cell to lyse a target human
cell bearing HLA-E or Qa1.sup.b on the target cell surface, when
said target cell comes into contact with said NK cell. Preferably,
the monoclonal antibody or fragment does not bind to other human
NKG2 receptors, specifically the activating receptors NKG2C or
NKG2E. Even more preferred is that the antibody or fragment of this
invention completely compete with an anti-NKG2 monoclonal selected
from Z199 or Z270.
[0021] In one preferred embodiment, the monoclonal antibody or a
fragment thereof is capable of binding to a non-human primate
NKG2A. Even more preferred is when upon binding to NKG2A on a
non-human primate NK cell, the monoclonal antibody or a fragment
thereof has the ability to reconstitute lysis of a target non-human
primate cell bearing HLA-E on the target cell surface, when said
target cell comes into contact with said NK cell.
[0022] In another preferred embodiment, the monoclonal antibody or
a fragment thereof comprises the amino acids sequence of the
variable heavy chain region of Z270 or the variable light chain
region of Z270. In an alternate preferred embodiment, the
monoclonal antibody or a fragment thereof comprises the amino acids
sequence of the variable heavy chain region of Z199 or the variable
light chain region of Z199.
[0023] In yet another preferred embodiment, the monoclonal antibody
or a fragment thereof comprises a mouse or human IgG.sub.1 constant
region that has been modified to prevent binding to an Fc receptor,
or a human IgG.sub.4 constant region.
[0024] In another preferred embodiment, the antibody or fragment is
chimeric or humanized. More preferred is an antibody or fragment
thereof that comprises ch270VK or ch270VH.
[0025] In another embodiment, the antibody or fragment thereof is
derivatized to enhance its bioavailability or stability in vivo. In
another embodiment, the antibody is derivatized with PEG.
[0026] The activating antibodies and fragments of this invention
are useful to reconstitute lysis of certain target cells that are
normally resistant to NK cell-mediated lysis. Thus, in another
embodiment the invention provides a method of reconstituting NK
cell-mediated lysis of a target cell in a population comprising a
NK cell and said target cell, wherein said NK cell is characterized
by NKG2A on its surface, and said target cell is characterized by
the presence of HLA-E or Qa1.sup.b on its surface, said method
comprising the step of contacting said NK cell with a monoclonal
antibody or a fragment described above. Preferably, the target cell
is a human cell. More preferably, the target cell is a dendritic
cell ("DC"), a cancer cell or a virally-infected cell. Most
preferably, the target is a mature dendritic cell ("mDC").
[0027] The activating antibodies and fragments thereof may be
formulated into compositions additionally comprising a
pharmaceutically acceptable carrier or excipient. Such composition
may be formulated so as to be suitable for pharmaceutical
administration. The pharmaceutical compositions may optionally
comprise a second therapeutic agent useful for the particular
disease or condition being treated. All such compositions are also
part of the present invention.
[0028] The activating antibody compositions of this invention may
be utilized to treat or prevent in a patient an autoimmune or
inflammatory disorder, or an immune response; or to treat in a
patient a cancer characterized by the presence of a cancer cell
expressing HLA-E or Qa1.sup.b on its surface, or a viral disease
characterized by the presence of a virally infected cell expressing
HLA-E or Qa1.sup.b on its surface. These methods may additionally
comprise the step of administering to the patient a second
therapeutic agent useful for the particular disease or condition
being treated. The second therapeutic agent may be administered
either as a separate dosage form or as part of said
composition.
[0029] In one embodiment, the second therapeutic agent in the
compositions comprising and the methods utilizing an activating
antibody or fragment of the invention is a compound that agonizes
an activating an NK cell receptor, such as NKp30, NKp44, and NKp46.
In another embodiment, the second therapeutic agent is an
antagonist of an inhibitory NK cell receptor, such as an inhibitor
KIR receptor. In another embodiment, the second therapeutic agent
is an antagonist of TGF-beta 1. In another embodiment, the second
therapeutic agent is selected from the group consisting of a
cytokine inhibitor, a hematopoietic growth factor, a pain reliever,
insulin, an anti-inflammatory agent, and an immunosuppressant. In
another embodiment, the second therapeutic agent is an anticancer
compound or an antiemetic. In another embodiment, the second
therapeutic agent is an antiviral compound.
[0030] In another embodiment, the autoimmune or inflammatory
disorder to be prevented or treated is selected from the group
consisting of autoimmune hemolytic anemia, pernicious anemia,
polyarteritis nodosa, systemic lupus erythematosus, Wegener's
granulomatosis, Alzheimer's disease, autoimmune hepatitis, Behcet's
disease, Crohn's disease, primary bilary cirrhosis, scleroderma,
ulcerative colitis, Sjogren's syndrome, Type 1 diabetes mellitus,
uveitis, Graves' disease, thyroiditis, Type 1 diabetes mellitus,
myocarditis, rheumatic fever, ankylosing spondylitis, rheumatoid
arthritis, glomerulonephritis, sarcoidosis, dermatomyositis,
myasthenia gravis, polymyositis, Guillain-Barre syndrome, multiple
sclerosis, alopecia areata, pemphigus/pemphigoid, psoriasis, and
vitiligo.
[0031] In another aspect, the present invention provides an
inhibitory monoclonal antibody or an inhibitory fragment thereof
characterized by: a) specifically binding to NKG2A; b) specifically
binding to an Fc receptor; c) not binding to NKG2C or NKG2E; d)
complete competition with Z270 or Z199; e) being able to inhibit NK
cell lysis of an NK cell-susceptible target cell, wherein said
cross-linking monoclonal antibody is not Z199. In one preferred
embodiment, the inhibitory antibody is further characterized by
binding to a non-human primate NKG2A.
[0032] In a more preferred embodiment, the inhibitory antibody or
fragment thereof comprises an amino acid sequence of the variable
light chain region of Z270 or an amino acid sequence of the
variable heavy chain region of Z270. In one of the most preferred
embodiments, the antibody is Z270.
[0033] In another preferred embodiment, the inhibitory antibody or
fragment is chimeric or humanized. More preferred is an inhibitory
antibody or inhibitory fragment thereof that comprises ch270VK or
ch270VH. In another of the most preferred embodiments, the antibody
is chZ270 or Z270.
[0034] In another embodiment, the invention provides a composition
comprising an effective amount of an inhibitory antibody or
inhibitory fragment thereof described above, or Z199; and a
pharmaceutically acceptable carrier or excipient. These inhibitory
antibody compositions are preferably formulated for pharmaceutical
use.
[0035] The inhibitory antibody compositions of this invention
optionally comprise a second therapeutic agent useful to treat a
disease or condition characterized by undesired NK cell-mediated
lysis of other cells, hyperactive NK cell activity, or unwanted NK
cell proliferation. Such second therapeutic agents may be selected
from, for example, a cytokine, an anticancer compound (such as a
chemotherapeutic compound, an anti-angiogenic compound, an
apoptosis-promoting compound, a hormonal agent, a compound that
interferes with DNA replication, mitosis and/or chromosomal
segregation, or an agent that disrupts the synthesis and fidelity
of polynucleotide precursors), an adjunct compound, a compound
capable of stimulating an inhibitory NK cell receptor (such as
natural ligands, antibodies or small molecules that can stimulate
the activity of CD94/NKG2A receptors, or an inhibitory KIR receptor
such as KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, and KIR3DL2), or an
inhibitor of an activating NK cell receptor (such as NKp30, NKp44,
or NKp46).
[0036] The inhibitory antibody and fragments of this invention may
be utilized in a method of reducing NK cell-mediated lysis of
cells. Alternatively, the inhibitory antibody and fragments of this
invention may be utilized in a method of reducing the number of NK
cells in a cell population. Both of these methods comprise the step
of contacting said NK cell with the inhibitor monoclonal antibody
or fragment.
[0037] The pharmaceutically suitable compositions of this invention
comprising an inhibitory antibody may be employed in a method of
treating or preventing a patient suffering from a condition or
disorder characterized by undesired NK cell-mediated lysis of other
cells, hyperactive NK cell activity, or unwanted NK cell
proliferation, said method comprising the step of administering to
the patient said composition. One such condition is NK-LDGL.
NK-LDGL (NK-type lymphoproliferative disease of granular
lymphocytes; alternatively called NK-LGL) refers to a class of
proliferative disorders that is caused by the clonal expansion of
NK cells or NK-like cells, i.e., large granular lymphocytes showing
a characteristic combination of surface antigen expression (e.g.,
CD3-, CD56+, CD16+, etc.; see, e.g., Loughran (1993) Blood
82:1).
[0038] In an alternate embodiment, any of the methods utilizing an
inhibitory antibody of this invention may comprise the additional
step of administering to said patient a second therapeutic agent.
The second therapeutic agent is an agent normally used to treat a
disease or condition characterized by undesired NK cell-mediated
lysis of other cells, hyperactive NK cell activity, or unwanted NK
cell proliferation. Examples of such agents are set forth above.
The second therapeutic agent may be administered as a separate
dosage form or as a component of the inhibitory antibody or
fragment composition.
[0039] In another aspect, the present invention provides kits
comprising any one or more of the herein-described antibodies or
fragments thereof. Typically, the kit also comprises instructions
for using the antibodies according to the present methods. In a
related embodiment, the kit additionally comprises, in a separate
vessel, a second therapeutic agent, such as any of those described
above for use in conjunction with either activating or inhibitory
antibodies or fragments in the treatment or prevention of various
diseases or conditions.
[0040] According to another aspect, the invention provides a method
of evaluating an antibody against human NKG2A comprising the steps
of: a) contacting said antibody with a non-human primate cell
characterized by NKG2A on its surface, or a non-human primate NKG2A
polypeptide; and b) assessing the ability of said antibody to bind
to or affect the activity of said cell or polypeptide. In a related
embodiment, the method is used to evaluate an activating antibody;
said antibody is contacted with a cell population comprising a
non-human primate NK cell and a target cell, wherein said NK cell
is characterized by NKG2A on its surface, and said target cell is
characterized by the presence of HLA-E on its surface; and said
assessing step is determining if said target cell is lysed.
[0041] In another embodiment, the invention provides a method of
producing an antibody suitable for use in disease treatment in
humans, said method comprising: a) immunizing a nonhuman mammal
with a composition comprising human NKG2A; b) selecting a
monoclonal antibody that binds NKG2A, but not NKG2C or NKG2E; c)
rendering said antibody suitable for use in humans; d)
administering said antibody to a nonhuman primate; and e)
evaluating the ability of said antibody to bind to NKG2A in vivo in
said primate and the tolerance of said primate to said antibody. If
the antibody binds to and is tolerated by said nonhuman primate, it
indicates that said antibody is suitable for use in disease
treatment in humans. In a preferred embodiment, the method
comprises the additional step of modifying said antibody to not
bind an Fc receptor prior to step d.
[0042] The invention also provides an antibody produced by this
method.
[0043] In yet another embodiment, the invention provides a method
of identifying a suitable administration regimen for a therapeutic
antibody directed against human NKG2A, said method comprising: a)
administering said antibody to a nonhuman primate using a series of
administration regimens in which the dose or frequency of said
antibody is varied; and b) determining the activity of
NKG2A-expressing cells in said non-human primate and the tolerance
of said primate for each of said administration regimens. Once it
is determined that a regimen is tolerated by said primate and leads
to a detectable modulation in said activity of NKG2A-expressing
cells, that administration regimen is considered suitable for use
in humans.
[0044] According to an alternative embodiment, the invention
provides a conjugate comprising: a) an inhibitory or activating
antibody, and b) a cytotoxic agent. The resulting conjugate is used
to kill NK cells. Thus, conjugation of an activating antibody with
a cytotoxic agent will produce a molecule that will kill the NK
cell, as opposed to the activation of that cell achieved by the
activating antibody alone. The cytotoxin/antibody conjugates of
this invention can be formulated into compositions and used in
methods in a manner similar to the inhibitory antibodies of this
invention.
DESCRIPTION OF THE FIGURES
[0045] FIG. 1 depicts the effect of three different concentrations
of Z270 on NK cell lysis of HLA-E expressing PHA blasts at varying
ratios of NK cells to PHA blasts.
[0046] FIG. 2 depicts the effect of three different concentrations
of Z199 on NK cell lysis of HLA-E expressing PHA blasts at varying
ratios of NK cells to PHA blasts.
[0047] FIG. 3 depicts the effect of an F(ab')2 fragment of Z270 on
NK cell lysis of HLA-E expressing PHA blasts.
[0048] FIG. 4 shows binding to cynomolgus monkey NK cells of
antibody Z270 as well as IgG1 and anti-CD16, demonstrating that
Z270 binds to cynomolgus monkey NK cells. Binding was also shown
for macaca mulatta and baboons.
DETAILED DESCRIPTION OF THE INVENTION
Introduction
[0049] The present invention provides novel antibodies against
NKG2A that activate NK cell-mediated lysis of target cells
characterized by the presence of cells expressing HLA-E or
Qa1.sup.b on their cell surface, methods for producing, evaluating
and characterizing those antibodies for therapeutic use, and
compositions comprising and methods of using those antibodies for
the treatment of autoimmune or inflammatory disorders and other
conditions characterized by the presence of cells expressing HLA-E
or Qa1.sup.b on their cell surface, such as dendritic cells. The
present invention is based, in part, on the surprising discovery
that NKG2A has a primary responsibility for inhibiting the lysis of
mature dendritic cells by many NK cells. Mature dendritic cells
express significant levels of HLA-E, which acts through NKG2A
receptors present on NK cells to inhibit the targeting of the
dendritic cells. Accordingly, without being bound by the following
theory, it is believed that blocking the NKG2A-mediated inhibition
of NK cells leads to an increase in dendritic cell targeting by NK
cells, thereby providing an effective treatment for autoimmune or
inflammatory disorders or indeed any condition that could be
alleviated or cured by reducing the activity of dendritic cells,
particularly mature dendritic cells. The present invention thus
also provides methods of, more generally, inhibiting or reducing
the number of dendritic cells, preferably mature dendritic cells,
in a mammal, as well as to generally reduce an immune response,
preferably an autoreactive immune response.
[0050] Conversely, the present invention also provides novel
antibodies against NKG2A that inhibit NK cell-mediated lysis of
target cells, methods of producing, evaluating and characterizing
those antibodies for therapeutic use, and compositions comprising
and methods of using those antibodies for the treatment of
autoimmune disorders or transplant rejection.
Definitions
[0051] As used herein, the following terms have the meanings
ascribed to them unless specified otherwise.
[0052] As used herein, "NK" cells refers to a sub-population of
lymphocytes that is involved in non-conventional immunity. NK cells
can be identified by virtue of certain characteristics and
biological properties, such as the expression of specific surface
antigens including CD16, CD56 and/or CD57, the absence of the
alpha/beta or gamma/delta TCR complex on the cell surface, the
ability to bind to and kill cells that fail to express "self"
MHC/HLA antigens by the activation of specific cytolytic enzymes,
the ability to kill tumor cells or other diseased cells that
express a ligand for NK activating receptors, and the ability to
release protein molecules called cytokines that stimulate or
inhibit the immune response. Any of these characteristics and
activities can be used to identify NK cells, using methods well
known in the art.
[0053] Dendritic cells are a heterogeneous population of immune
cells produced in the bone marrow (see, e.g., O'Neill et al. (2004)
Blood 104:2235-2246, Mohamadzadeh et al. (2004) J Immune Based Ther
Vaccines. 2004; 2: 1; the entire disclosures of which are herein
incorporated by reference). As referred to herein, DCs can include
DC precursors, immature DCs, and mature DCs. DC precursors and
immature DCs are lineage negative (CD3-CD14-CD19-CD56-) HLA-DR+
mononuclear cells. These cells can be further classified into two
populations, myeloid DCs and plasmacytoid DCs. Myeloid DCs are
CD11c+ and CD123 low and have a monocytoid appearance, and
plasmacytoid DCs are CD11c- and CD123 high, with morphological
features similar to plasma cells. Following antigen capture, DCs
undergo a process of maturation in which the captured antigens are
processed into peptides and loaded onto MHC class I or II for
presentation on the cell surface. Mature DCs show lower phagocytic
uptake, have cytoplasmic extensions called veils, migrate to
lymphoid tissues, and express characteristic markers such as CD83
and DC-LAMP. TLRs are also expressed in DCs, with different DC
types expressing different TLR markers (see, e.g., O'Neill et al.
(2004).
[0054] NKG2A (OMIM 161555, the entire disclosure of which is herein
incorporated by reference) is a member of the NKG2 group of
transcripts (Houchins, et al. (1991) J. Exp. Med. 173:1017-1020).
NKG2A is encoded by 7 exons spanning 25 kb, showing some
differential splicing. NKG2A is an inhibitory receptor found on the
surface of NK cells. Like inhibitory KIR receptors, it possesses an
ITIM in its cytoplasmic domain. As used herein, "NKG2A" refers to
any variant, derivative, or isoform of the NKG2A gene or encoded
protein. Also encompassed are any nucleic acid or protein sequences
sharing one or more biological properties or functions with wild
type, full length NKG2A, and sharing at least 70%, 80%, 90%, 95%,
96%, 97%, 98%, 99%, or higher nucleotide or amino acid identity.
NKG2A is also referred to as the "NKG2A receptor" throughout this
disclosure.
[0055] NKG2C (OMIM 602891, the entire disclosure of which is herein
incorporated by reference) and NKG2E (OMIM 602892, the entire
disclosure of which is herein incorporated by reference) are two
other members of the NKG2 group of transcripts (Gilenke, et al.
(1998) Immunogenetics 48:163-173). NKG2C and NKG2E are activating
receptors found on the surface of NK cells. As used herein, "NKG2C"
and "NKG2E" refer to any variant, derivative, or isoform of the
NKG2C or NKG2E gene or encoded protein, respectively. Also
encompassed are any nucleic acid or protein sequences sharing one
or more biological properties or functions with wild type, full
length NKG2C or NKG2E, and sharing at least 70%, 80%, 90%, 95%,
96%, 97%, 98%, 99%, or higher nucleotide or amino acid identity
with the disclosed gene or encoded protein.
[0056] CD94 (OMIM 602894, the entire disclosure of which is herein
incorporated by reference in its entirety) is an antigen
preferentially expressed on NK cells (Chang et al. (1995) Europ. J.
Immun. 25: 2433-2437). CD94 is expressed as 3 major transcripts of
0.8, 1.8, and 3.5 kb and a minor transcript of 5.5 kb in NK cell
lines, and encodes a protein with a 147-amino acid extracellular
domain and several motifs characteristic of C-type lectins. The
amino acid sequence of CD94 is 27 to 32% identical to those of NKG2
family members NKG2A, NKG2C, NKG2D, and NKG2E. Due to the virtual
absence of a cytoplasmic domain, CD94 requires association with
other receptors forming disulfide-bonded heterodimers with NKG2A,
NKG2C, and NKG2E (Lazetic et al. (1996) J. Immun. 157: 4741-4745).
As used herein, "CD94" refers to any variant, derivative, or
isoform of the CD94 gene or encoded protein. Also encompassed are
any nucleic acid or protein sequences sharing one or more
biological properties or functions with wild type, full length
CD94, and sharing at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%,
or higher nucleotide or amino acid identity.
[0057] HLA-E (OMIM 143010, the entire disclosure of which is herein
incorporated by reference) is a nonclassical MHC molecule that is
expressed on the cell surface and regulated by the binding of
peptides derived from the signal sequence of other MHC class I
molecules. HLA-E binds natural killer (NK) cells and some T cells,
binding specifically to CD94/NKG2A, CD94/NKG2B, and CD94/NKG2C, and
not to the inhibitory KIR receptors (see, e.g. OMIM 604936, the
entire disclosure of which is herein incorporated by reference)
(see, e.g., Braud et al. (1998) Nature 391:795-799, the entire
disclosure of which is herein incorporated by reference). Surface
expression of HLA-E is sufficient to protect target cells from
lysis by CD94/NKG2A+NK cell clones. As used herein, "HLA-E" refers
to any variant, derivative, or isoform of the HLA-E gene or encoded
protein. Also encompassed are any nucleic acid or protein sequences
sharing one or more biological properties or functions with wild
type, full length HLA-E, and sharing at least 70%, 80%, 90%, 95%,
96%, 97%, 98%, 99%, or higher nucleotide or amino acid
identity.
[0058] Qa1.sup.b is a mouse cell surface antigen that is the
physiological ligand for NKG2A. As used herein, "Qa1.sup.b" refers
to any variant, derivative, or isoform of the Qa1.sup.b gene or
encoded protein. Also encompassed are any nucleic acid or protein
sequences sharing one or more biological properties or functions
with wild type, full length Qa1.sup.b, and sharing at least 70%,
80%, 90%, 95%, 96%, 97%, 98%, 99%, or higher nucleotide or amino
acid identity.
[0059] "Autoimmune" disorders include any disorder, condition, or
disease in which the immune system mounts a reaction against self
cells or tissues, due to a breakdown in the ability to distinguish
self from non-self or otherwise. Examples of autoimmune disorders
include Hashimoto's thyroiditis, pernicious anemia, Addison's
disease, type I diabetes, rheumatoid arthritis, systemic lupus
erythematosus, dermatomyositis, Sjogren's syndrome, lupus
erythematosus, multiple sclerosis, myasthenia gravis, Reiter's
syndrome, Grave's disease, polymyositis, Guillain Barre, Wegener's
granulomatosis, polyarteritis nodosa, polymyalgia rheumatica,
temporal arteritis, Bechet's disease, Churg-Strauss syndrome,
Takayasu's arteritis, and others. An "inflammatory disorder"
includes any disorder characterized by an unwanted immune response.
Autoimmune and inflammatory disorders can involve any component of
the immune system, and can target any cell or tissue type in the
body.
[0060] The terms "inhibiting," "reducing," "blocking,"
"downmodulating," and "downregulating," with respect to NKG2A
activity refer to any process, method, or compound that can slow
down, reduce, reverse, or in any way negatively affect the
stimulation or expression of NKG2A receptors on cells, preferably
NK cells. These terms can refer to compounds that inhibit the
stimulation of NKG2A by a ligand, that act antagonistically in the
absence of a ligand to decrease the activity of the receptor, that
decrease the expression level of the receptor, that block
NKG2A-triggered signaling or gene expression, or that block any
other activity of the cell that results from NKG2A activation. In a
preferred embodiment, the inhibiting compound or method prevents
the binding of the receptor by a ligand, e.g. HLA-E. The number of
NKG2A receptor molecules or any of the herein-described activities
can be measured in any standard way, e.g. as disclosed elsewhere in
the present application.
[0061] 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 and/or IgM
are the preferred classes of antibodies employed in this invention,
with IgG being particularly preferred, because they are the most
common antibodies in the physiological situation and because they
are most easily made in a laboratory setting.
[0062] Preferably the antibody of this invention is a monoclonal
antibody. Particularly preferred are humanized, chimeric, human, or
otherwise-human-suitable antibodies. The term "antibody" also
includes any fragment or derivative of any of the herein described
antibodies except in those contexts of the present disclosure where
such inclusion causes a redundancy (e.g., a specific reference to
"an antibody or a fragment thereof"). In one preferred embodiment,
the antibodies are non-depleting antibodies, meaning that they bind
to NK cells and inhibit NKG2A stimulation (which leads to the lysis
of cells bearing HLA-E or Qa1.sup.b on their cell surface), but do
not lead to the killing of the NKG2A expressing cell. Non-depleting
antibodies or antibody fragments are those that are not recognized,
or only poorly recognized, by Fc receptors, such as IgG4
antibodies, antibody fragments lacking the Fc portion, or any other
antibody whose Fc tail has been modified to reduce or eliminate
binding by Fc receptors (see, e.g., WO03101485, the entire
disclosure of which is herein incorporated by reference).
[0063] In another preferred embodiment, the antibodies or antibody
fragments bind to an Fc receptor. Such antibodies and fragments
cause cross-linking of NKG2A molecules leading to inhibition of NK
cell activity and, in some cases, to NK cell death.
[0064] The term "specifically binds to" means that an antibody can
bind, preferably in a competitive binding assay, to the binding
partner, e.g. NKG2A, as assessed using either recombinant forms of
the protein, epitopes therein, or native proteins present on the
surface of isolated NK or other cells. Competitive binding assays
and other methods for determining specific binding are further
described below and are well known in the art.
[0065] A "human-suitable" antibody refers to any antibody,
derivatized antibody, or antibody fragment that can be safely used
in humans for, e.g. the therapeutic methods described herein.
Human-suitable antibodies include all types of humanized, chimeric,
or fully human antibodies, or any antibodies in which at least a
portion of the antibodies is derived from humans or otherwise
modified so as to avoid the immune response that is generally
provoked when native non-human antibodies are used.
[0066] For the purposes of the present invention, a "humanized"
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
humanized 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.
[0067] 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.
[0068] A "human" antibody is an antibody 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).
[0069] Within the context of this invention, "active" or
"activated" NK cells designate biologically active NK cells, more
particularly NK cells having the capacity of lysing target cells.
For instance, an "active" NK cell is able to kill cells that
express an NK activating receptor-ligand and fails to express
"self" MHC/HLA antigens (KIR-incompatible cells). Such cells are
also referred to herein as "NK cell-susceptible target cells."
Examples of such target cells, which are suitable for use in
redirected killing assays, are P815 and K562 cells. However, any of
a number of cell types can be used and are well known in the art
(see, e.g., Sivori et al. (1997) J. Exp. Med. 186: 1129-1136;
Vitale et al. (1998) J. Exp. Med. 187: 2065-2072; Pessino et al.
(1998) J. Exp. Med. 188: 953-960; Neri et al. (2001) Clin. Diag.
Lab. Immun. 8:1131-1135). "Active" or "activated" cells can also be
identified by any other property or activity known in the art as
associated with NK activity, such as cytokine (e.g. IFN-.gamma. and
TNF-.alpha.) production of increases in free intracellular calcium
levels. For the purposes of the present invention, activated NK
cells ideally refer to NK cells in which NKG2A receptors are not
stimulated, and in which an NCR, preferably NKp30, is stimulated,
thereby leading to cytotoxicity of the cell against mature
dendritic cells.
[0070] The term "NKG2A stimulation," as used herein refers to the
process that occurs in a cell bearing NKG2A, e.g., a NK cell, when
NKG2A binds to its natural ligand (e.g., HLA-E or Qa1.sup.b) or a
functional fragment thereof. Because NKG2A is an inhibitory
receptor, such binding can cause inhibition of NK cell activity.
Thus, "inhibition of NKG2A stimulation" refers to a process whereby
the binding of NKG2A to its natural ligand or a functional fragment
thereof is either reduced or prevented, where the binding occurs,
but does not cause inhibition of NK cell activity.
[0071] Thus, the term "activating antibody," as used herein in
reference to antibodies against NKG2A, is intended to mean an
antibody which, through binding to NKG2A on a NK cell, prevents
association of NKG2A with its natural ligand (e.g., HLA-E or
Qa1.sup.b) on a target cell, or prevents NKG2A dependent signal
transduction normally mediated by a HLA-E positive target, and thus
reverses the inhibition of lysis of the target cell by the NK cell
caused by the association of NKG2A with the ligand. Thus, an
activating antibody causes inhibition of NKG2A stimulation.
[0072] The term "inhibitory antibody," as used herein in reference
to antibodies against NKG2A, is intended to mean an antibody which,
through binding to NKG2A on a NK cell, causes inhibition of a NK
cell's ability to lyse cells that would otherwise be lysed. The
inhibitory antibodies of this invention typically cause
cross-linking of NKG2A molecules in a NK cell, which leads to
inhibition, and sometimes death, of that NK cell. It should be
noted that an inhibitory antibody against NKG2A of this invention
may prevent the association of NKG2A with its natural ligand or an
active fragment thereof, but will not result in the lysis of a cell
bearing that natural ligand because the NK cell's ability to lyse
cells had been inhibited by the antibody.
[0073] 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.
[0074] The term "biological sample" as used herein includes but is
not limited to a biological fluid (for example serum, lymph,
blood), cell sample or tissue sample (for example bone marrow).
[0075] 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.
[0076] 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
(nonrecombinant) form of the cell or express native genes that are
otherwise abnormally expressed, under expressed or not expressed at
all.
[0077] The term "competes with" when referring to a particular
monoclonal antibody (e.g. Z199 or Z270) means that the antibody or
fragment thereof being tested reduces the binding of that reference
monoclonal antibody (e.g. Z199 or Z270) to NKG2A (as compared to a
control comprising that reference monoclonal antibody and NKG2A,
but lacking the test antibody) in a binding assay using either
recombinant NKG2A molecules or surface expressed NKG2A molecules.
For example, if an antibody reduces binding of Z270 to a human
NKG2A molecule in a binding assay, the antibody "competes" with
Z270 for binding to human NKG2A.
[0078] The term "completely competes with," as used herein means
that the test antibody binds to substantially or essentially the
same epitope as the reference monoclonal.
[0079] As used herein, an "effective amount" refers to any amount
that is necessary or sufficient for achieving or promoting a
desired outcome. In some instances an effective amount is a
therapeutically effective amount. A therapeutically effective
amount is any amount that is necessary or sufficient for promoting
or achieving a desired biological response in a subject. The
effective amount for any particular application can vary depending
on such factors as the disease or condition being treated, the
particular agent being administered, the size of the subject, or
the severity of the disease or condition. One of ordinary skill in
the art can empirically determine the effective amount of a
particular agent without necessitating undue experimentation.
[0080] The term non-human primates include any mammals within the
Order Primates, including apes, New World monkeys, Old World
monkeys, prosimians, Pongo pygmaeus pygmaeus (Borneo orangutan),
Pongo pygmaeus abelii (Sumatran orangutan), Gorilla gorilla
(western lowland gorilla), Pan paniscus (bonobo), Pan troglodytes
(chimpanzee), Pan troglodytes verus (chimpanzee), Lemur fulvus
(brown lemur), Saguinus fuscicollis (white-lipped tamarin),
Saguinus labiatus (red-bellied tamarin), Callicebus molloch
pallescens (Paraguayan titi), Saimiri sciureus (squirrel monkey),
Ateles geoffroyi (black-handed spider monkey), Lagothrix lagotricha
(woolly monkey), Macaca arctoides (stumptail macaque), Macaca
fascicularis (crab-eating macaque), Macaca fuscata (Japanese
macaque), Macaca mulatta (rhesus monkey), Macaca nemestrina
(pigtailed macaque), Macaca nigra (Celebes ape), Erythrocebus patas
(patas monkey), baboons, marmosets, capuchins, cynomolgus, howlers,
spider monkeys, mandrills, guenon, patas monkeys, colobus, gibbons,
lemurs, aye-ayes, loris, bushbabies, and tarsiers. In a preferred
embodiment, the nonhuman primate used in the present invention is
not an ape, e.g. is a nonhuman primate other than a chimpanzee,
gorilla, orangutan, or gibbon. For the purposes of the invention,
assays said to be carried out using nonhuman primates can include
in vivo assays in which antibodies are administered to the
primates, ex vivo assays in which, e.g. cells taken from a primate
are treated with the antibodies and returned to the primate, and in
vitro assays involving cells, proteins, or tissue taken from a
primate.
[0081] If a mammal such as a nonhuman primate is said to "tolerate"
an administration regime of an anti-NKG2A antibody, it means that
the administration is not lethal and does not have any severe side
effects in the animal, although side effects may be still be
present as long as they are not severe, and, generally, that they
are outweighed by the therapeutic benefit provided by the
administration.
Obtaining Compounds that Specifically Bind to NKG2A
[0082] The present invention involves both activating and
inhibitory antibodies that bind to NKG2A on immune cells,
preferably NK cells, as well as their identification, production,
evaluation and use. One way of identifying such antibodies is to
find those that are capable of binding to NKG2A. Once specifically
binding antibodies are identified, they can be tested for their
ability to inhibit or activate NKG2A, e.g. on NK cells. It will be
appreciated, however, that carrying out such binding assays is in
no way necessary for the practice of the present invention.
[0083] Any of a wide variety of assays can be used to assess
binding of an antibody to NKG2A. Protocols based upon ELISAs,
radioimmunoassays, Western blotting, BIACORE, and other competition
assays, inter alia, are suitable for use and are well known in the
art.
[0084] For example, simple binding assays can be used, in which a
test antibody is incubated in the presence of a target protein or
epitope (e.g., NKG2A or a portion thereof), unbound antibodies are
washed off, and the presence of bound antibodies is assessed using,
e.g., radiolabels, physical methods such as mass spectrometry, or
direct or indirect fluorescent labels detected using, e.g.,
cytofluorometric analysis (e.g. FACScan). Such methods are well
known to those of skill in the art. Any amount of binding above the
amount seen with a control, non-specific antibody indicates that
the antibody binds specifically to the target.
[0085] In such assays, the ability of the test antibody to bind to
the target cell or human NKG2A can be compared with the ability of
a (negative) control protein, e.g. an antibody raised against a
structurally unrelated antigen, or a non-Ig peptide or protein, to
bind to the same target. Antibodies or fragments that bind to the
target cells or NKG2A using any suitable assay with 25%, 50%, 100%,
200%, 1000%, or higher increased affinity relative to the control
protein are said to "specifically bind to" or "specifically
interact with" the target, and are preferred for use in the
therapeutic methods described below.
[0086] In one embodiment, the ability of a test antibody to affect
the binding of a (positive) control antibody against NKG2A, e.g.
3S9, 20d5, Z270 or Z199, or derivatives thereof, is assessed. In
another, the ability of a test antibody to affect the binding of a
natural ligand for NKG2A, e.g. HLA-E, is measured. 3S9 is described
in United States patent publication 20030095965, the disclosure of
which is herein incorporated by reference. 3S9 binds to NKG2C and
NKG2E, as well as to NKG2A. 20d5 is a commercially available
antibody (BD Biosciences Pharmingen, Catalog No. 550518, USA). 20d5
binds to mouse NKG2A, NKG2E and NKG2C. Z199 is a commercially
available antibody (Beckman Coulter, Inc., Product No. IM2750,
USA). Z270 is described fully herein. Z270 binds specifically to
human NKG2A, but not to human NKG2C or NKG2E.
[0087] In addition, simple competition assays may be employed in
which a control antibody (e.g. 3S9, Z270 or Z199) and a test
antibody are admixed (or pre-adsorbed) and applied to a sample
containing NKG2A. In certain embodiments, one would pre-mix the
control antibodies with varying amounts of the test antibody (e.g.,
1:10 or 1:100) for a period of time prior to applying to the
NKG2A-containing sample. In other embodiments, the control and
varying amounts of test antibody can simply be admixed during
exposure to the antigen/target sample. As long as one can
distinguish bound from free antibodies (e.g., by using separation
or washing techniques to eliminate unbound antibodies) and the
control antibody from test antibody (e.g., by using species- or
isotype-specific secondary antibodies, by specifically labeling the
control antibody with a detectable label, or by using physical
methods such as mass spectrometry to distinguish between different
compounds) one will be able to determine if the test antibody
reduces the binding of the control antibody to the antigen,
indicating that the test antibody recognizes substantially the same
epitope as the control.
[0088] In the above-described competition assays, the binding of
the (labeled) control antibody in the presence of a completely
irrelevant antibody is the control high value. The control low
value is obtained by incubating the labeled (positive) control
antibody (e.g. 3S9, Z270 or Z199) with unlabeled antibody of
exactly the same type (e.g. 3S9, Z270 or Z199), where competition
would occur and reduce binding of the labeled antibody.
[0089] 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 recognizes the same epitope, i.e., one that
"cross-reacts" with the labeled control antibody. Any test antibody
or compound that reduces the binding of the labeled control to the
antigen/target by at least 50% or more preferably 70%, at any ratio
of control:test antibody or compound between about 1:10 and about
1:100 is considered to be an antibody or compound that binds to
substantially the same epitope or determinant as the control.
Preferably, such test antibody or compound will reduce the binding
of the control to the antigen/target by at least 90%. Nevertheless,
any compound or antibody that reduces the binding of a control
antibody or compound to any measurable extent can be used in the
present invention.
[0090] The identification of one or more antibodies that bind(s) to
substantially the same epitope as the monoclonal antibody in
question can be readily determined using any one of a variety of
immunological screening assays in which antibody competition can be
assessed. Such assays are routine in the art (see, e.g., U.S. Pat.
No. 5,660,827, which is herein incorporated by reference). It will
be understood that actually determining the epitope to which the
antibody 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 in question.
[0091] In one embodiment, competition can be assessed by a flow
cytometry test. For example, cells bearing an NKG2A/CD94 receptor
are incubated first with a control antibody that is known to
specifically bind to the receptor (e.g., 3S9, Z270 or Z199), and
then with the test antibody that has been labeled with, e.g., a
fluorochrome or biotin. The test antibody is said to compete with
the control if the binding obtained with preincubation with
saturating amounts of control antibody is 80%, preferably, 50%, 40%
or less of the binding (mean of fluorescence) obtained by the
antibody without preincubation with the control. Alternatively, a
test antibody is said to compete with the control if the binding
obtained with a labeled control (by a fluorochrome or biotin) on
cells preincubated with saturating amount of antibody to test is
80%, preferably 50%, 40%, or less of the binding obtained without
preincubation with the antibody.
[0092] In one preferred example, a simple competition assay may be
employed in which a test antibody is pre-adsorbed and applied at
saturating concentration to a surface onto which is immobilized the
substrate for the antibody binding, e.g. NKG2A/CD94 receptor, or
epitope-containing portion thereof, which is known to be bound by,
e.g., 3S9. The surface is preferably a BIACORE chip. The control
antibody (e.g. 3S9, Z270 or Z199) is then brought into contact with
the surface at a substrate-saturating concentration and the
substrate 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 substrate-containing surface in the absence
of a test antibody. In a test assay, a significant reduction in
binding of the substrate-containing surface by the control antibody
in the presence of a test antibody is indicative of a test antibody
that recognizes the same epitope, i.e., one that "cross-reacts"
with the control antibody. Any test antibody that reduces the
binding of the control antibody to the antigen-containing substrate
by at least 30% or more preferably 40% is considered to be an
antibody that binds to substantially the same epitope or
determinant as the control antibody. Preferably, such test antibody
will reduce the binding of the control antibody to the substrate by
at least 50%. It will be appreciated that the order of control and
test antibodies can be reversed, that is the control antibody is
first bound to the surface and the test antibody is brought into
contact with the surface thereafter. Preferably, the antibody
having higher affinity for the substrate antigens is bound to the
substrate-containing surface first since 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 Saunal et al.
(1995) J. Immunol. Meth 183: 33-41, the entire disclosure of which
is herein incorporated by reference.
[0093] Preferably, monoclonal antibodies according to this
invention that recognize an NKG2A will react with an epitope that
is present on a substantial percentage of NK cells in patients with
an autoimmune or inflammatory disorder, but will not significantly
react with other cells, i.e., immune or non-immune cells that do
not express NKG2A. Accordingly, once an antibody that specifically
recognizes NKG2A on cells such as NK, preferably human NK cells, is
identified, it can be tested for its ability to bind to NK cells
taken from patients with autoimmune or inflammatory disorders.
Similarly, it will be appreciated that the present methods can be
practiced using multiple antibodies, e.g. directed against
different epitopes or isoforms of NKG2A in a way that is designed
to maximally inhibit the stimulation of NKG2A. In one embodiment,
NK cells and dendritic cells are taken from a patient prior to the
administration of the antibodies or compounds, and the ability of
test antibodies to overcome NKG2A-mediated inhibition of lysis of
the dendritic cells is assessed.
[0094] In those embodiments of the invention where specific binding
or lack of specific binding to other antigens (e.g., NKG2A from
other species, NKG2C, NKG2E, Fc receptor) must be measured, assays
similar to those set forth above may be employed substituting the
appropriate antigen for NKG2A and employing control antibodies that
are specific for the antigen to which binding is being assayed.
Such antigens and control antibodies are well-known in the art and
many are commercially available.
Assessing the Ability of Antibodies to Inhibit NKG2A
Stimulation
[0095] The identification of activating antibodies of this
invention that are capable of inhibiting the stimulation of
NKG2A/CD94 by HLA-E or Qa1.sup.b will generally involve cell-based
assays to assess NKG2A activity in the presence of test antibody.
In some embodiments, candidate antibodies will be first identified
based on their ability to bind to NKG2A, as described supra. In
other embodiments, cell-based screening will be performed to
directly identify antibodies capable of inhibiting NKG2A
stimulation, regardless of their binding affinity.
[0096] In one embodiment, modulators of NKG2A will be identified
using methods or assays described in U.S. patent application no.
20030171280, Braud et al. (1998) Nature 391:795-799; Lee et al.
(1998) PNAS 95:5199-5204; Vance et al. (2002) PNAS 99:868-873;
Brooks et al. (1999) J Immunol 162:305-313; Miller et al. J Immunol
(2003) 171:1369-75; Brooks et al. (1997) J Exp Med 185:795-800; Van
Beneden et al. (2001) 4302-4311; U.S. patent application no.
20030095965; the entire disclosures of which are herein
incorporated by reference.
[0097] In one embodiment, the activating antibodies of this
invention are assessed for their ability to inhibit the stimulation
of the NKG2A receptor by ligands. Any of a large number of assays,
including molecular cell-based, and animal-based models can be
used. In typical embodiments, cell-based assays will be used in
which cells, e.g. NK cells expressing NKG2A, are exposed to an
NKG2A ligand (or cells expressing the ligand), preferably HLA-E,
and the ability of the antibody to disrupt the stimulation of the
receptor is assessed.
[0098] Any of a number of cell-based assays can be used to assess
NKG2A activity, including gene expression-based activities,
cytotoxicity-based assays, and proliferation assays. In certain
embodiments, in vitro assays will use cells, e.g. NK cells, taken
from patients with an autoimmune or inflammatory disorder, but in
general any NKG2A-expressing cell can be used, including NK cell
lines such as YTS or NK-92 (available from the ATCC). For example,
cell lines can be transfected with an NKG2A-encoding transgene and
used in the present assays, so long as the stimulation of the
expressed receptor alters the activity or properties of the cells
in a detectable way, e.g., activates signal transduction pathways,
affects proliferation, or alters the cytotoxicity of the cells. It
will be appreciated that, for such assays, any isoform of NKG2A,
CD94, or HLA-E (see, e.g. OMIM refs. 161555, 602894, and 143010,
the entire disclosures of which are herein incorporated by
reference) can be used in such assays (or any other assay or method
involving NKG2A described herein).
[0099] In one preferred embodiment, a cellular assay is used in
which NKG2A-expressing cells, e.g., NK cells, are incubated with an
NKG2A ligand such as HLA-E, or a cell expressing an NKG2A ligand,
preferably a dendritic cell, and the ability of a test compound to
block the inhibition of the NK cell is assessed. In such assays,
the lysis of the dendritic cells can itself be measured as a
reflection of NK cell activity.
[0100] In one embodiment, cell lines will be established using NK
cells from patients with an autoimmune or inflammatory disorder. In
numerous embodiments, assays will be used using non-human cells or
non-human NKG2A/CD94, e.g. non-human primate cells expressing
NKG2A/CD94, or mouse cells expressing either mouse or human
NKG2A/CD94, with the inclusion of the appropriate ligand (e.g., in
the case of mouse, Qa-1).
[0101] The binding of NKG2A to the appropriate ligand causes a
number of physiological changes in the cell bearing NKG2A. These
include changes in gene expression, cell growth, cell
proliferation, pH, intracellular second messengers, e.g.,
Ca.sup.2+, IP3, cGMP, or cAMP, cytokine production, or activity
such as cytotoxic activity. Such changes are referred to herein as
"NKG2A activity". Any reversal of these changes in the presence of
a NKG2A ligand can be used to assess the utility of a test
antibody. Such reversal is referred to herein as "inhibition of
NKG2A activity." In one embodiment, NKG2A activity is assessed by
detecting the expression or activity of NKG2A-responsive genes or
proteins, e.g., SHP-1 or SHP-2 or their targets (see, e.g., Le
Drean et al. (1998) Eur J Immunol 28:264-276, Augugliaro et al.
(2003) Eur J Immunol 33:1235-141; the entire disclosures of which
are herein incorporated by reference).
[0102] In any of the herein-described assays, a decrease of 5%,
10%, 20%, preferably 30%, 40%, 50%, most preferably 60%, 70%, 80%,
90%, 95%, or greater reduction in any detectable measure of NKG2A
activity in the cells indicates that the test antibody is a
suitable candidate for use in the present methods.
[0103] In addition to binding, the ability of antibodies or
compounds to cause NK cells to inhibit the proliferation or
activation of, or, preferably, kill NKG2A ligand-bearing target
cells, e.g. dendritic cells, certain cancer cells, or certain
virally-infected cells, can be assessed. In one embodiment, human
NK cells expressing the NKG2A receptor are introduced along with
NKG2A ligand-bearing target cells into plates, e.g., 96-well
plates, and exposed to various amounts of test antibody. By adding
a vital dye, i.e. one taken up by intact cells, such as AlamarBlue
(BioSource International, Camarillo, Calif.), and washing to remove
excess dye, the number of viable cells can be measured by virtue of
the optical density (the more cells killed by the antibody, the
lower the optical density). (See, e.g., Connolly et al. (2001) J
Pharm Exp Ther 298:25-33, the disclosure of which is herein
incorporated by reference in its entirety).
[0104] Most preferably, the activating antibodies of this invention
do not demonstrate substantial specific binding to Fc receptors.
Such antibodies may comprise constant regions of various heavy
chains that are known not to bind Fc receptors. One such example is
an IgG4 constant region. Alternatively, antibody fragments that do
not comprise constant regions, such as Fab or F(ab')2 fragments,
can be used to avoid Fc receptor binding. FC receptor binding can
be assessed according to methods known in the art, including for
example testing binding of an antibody to Fc receptor protein in a
BIACORE assay. Also, any other antibody type can be used in which
the Fc portion is modified to minimize or eliminate binding to Fc
receptors (see, e.g., WO03101485, the disclosure of which is herein
incorporated by reference). Assays, e.g., cell based assays, to
assess Fc receptor binding are well known in the art, and are
described, e.g., in WO03101485.
[0105] Preferably, the activating monoclonal antibody of this
invention comprises an Fc region, preferably an Fc region of the
IgG4 or G2 subtype, or an Fc region of the IgG1 or G3 subtype that
has been modified to reduce binding to Fc receptors. Most
preferably the G4 or G2 Fc region is modified to further minimize
or completely abolish binding to Fc receptors (see, e.g., Angal et
al. (1993) Molecular Immunology 30:105-108, the entire disclosure
of which is herein incorporated by reference.)
[0106] IgG4 isotypes are not totally devoid of Fc binding activity,
showing some binding to Fc gamma ("Fcg") receptors (Newman et al.
(2001) Clin. Immunol. (98(2):164-174). An unmodified IgG4
monoclonal antibody can cause cell depletion in vivo (Isaacs et al,
(1996) Clin. Exp. Immunol. 106, 427). The sequence reported to be
primarily responsible for the binding to Fcg receptors has been
defined as LLGGPS (Burton et al, (1992) Adv. Immunol. 51:1). This
sequence, located at the N terminal end (EU numbering 234-239) of
the heavy chain CH2 region, is conserved in human IgG1, IgG3, and
murine IgG2a isotypes, all of which bind Fcg receptors strongly.
The wild-type sequence for the IgG4 isotype contains a
phenylalanine at position 234, giving the motif FLGGPS. The murine
IgG2b isotype, also a poor binder of Fcg receptors, contains the
sequence LEGGPS. Newman et al. (2001) incorporated the glutamic
acid residue associated with murine IgG2b into the human wildtype
IgG4 CH2 domain to give the sequence FEGGPS which reduced even
further CDC and ADCC activities and virtually eliminated binding to
FcgRI and FcgRII in vitro. In addition to the introduction of
glutamic acid, the replacement of serine 228 by a proline resulted
in a molecule that was more stable than the wild-type IgG4. The
IgG4 molecule tends to show inefficient formation of interchain
disulfide bonds in the hinge region. The introduction of a proline
was said to provide rigidity to the hinge and promote more
efficient interchain bonding, and that the presence of a serine at
position 228 might promote preferential linkage of intrachain
rather than inter-chain disulfide bonds by neighboring cysteine
molecules. Any such modification and others can readily be made to
the antibodies of the invention.
[0107] In many instances, an inhibitory antibody of this invention
can be converted to an activating antibody of this invention by
abolishing most or all of the former's ability to bind an Fc
receptor.
Assessing the Ability of Antibodies Against NKG2A to Inhibit NK
Cell Activity
[0108] The identification of inhibitory antibodies of this
invention that are capable of binding NKG2A and inhibiting NK cell
activity, particularly NK cell lysis of cells is assayed using
cell-based assays. Typically, a NKG2A-bearing cell, such as an NK
cell, will be contacted with a NK-susceptible cell, such as RMA, a
TAP-2 derivative of RMA, P815 and K562 in the presence of varying
amounts of test antibody. The percentage of NK-susceptible cells
killed in the presence of test antibody is compared with killing in
the absence of antibody.
[0109] In another assay for an inhibitory antibody of this
invention, NK cells are incubated in the presence of varying
amounts of test antibody to determine that antibody's direct
killing affect on NK cells as compared to NK cell death in the
absence of antibody. NK cell killing may also be determined in an
assay including the presence of NK-susceptible cells.
[0110] In any of the herein-described assays, a decrease of 5%,
10%, 20%, preferably 30%, 40%, 50%, most preferably 60%, 70%, 80%,
90%, or 95% of NK-susceptible cell killing and/or an increase of
5%, 10%, 20%, preferably 30%, 40%, 50%, most preferably 60%, 70%,
80%, 90%, or 95% of NK cell death indicates that the test antibody
is an inhibitory antibody of this invention.
Cross-Reactivity of NKG2A Between Primate Species
[0111] It has been discovered that there is crossreactivity between
human and nonhuman primate NKG2A. Thus, assays to assess the effect
of an anti-NKG2A antibody on receptor activity can be carried out
using an NKG2A polypeptide from any primate. For example, such
assays can be performed using nonhuman primate NK cells in vitro,
or the antibodies can be administered to nonhuman primates and
their ability to modulate NKG2A activity, e.g. as reflected in
alterations in NK cell activity, can be measured.
Producing Antibodies
[0112] The antibodies of this invention may be produced by any of a
variety of techniques known in the art. Typically, they are
produced by immunization of a non-human animal, preferably a mouse,
with an immunogen comprising an NKG2A (or, for all embodiments
described herein, for CD94, or HLA-E) receptor on the surface of
cells such as T cells or NK cells or dendritic cells. The receptor
may comprise entire cells or cell membranes, the full length
sequence of an NKG2A (or CD94, etc.), or a fragment or derivative
of any NKG2A, typically an immunogenic fragment, i.e., a portion of
the polypeptide comprising an epitope exposed on the surface of
cells expressing the receptor. Any isoform or splicing fragment of
NKG2A can be used (see, e.g., OMIM 161555; the disclosure of which
is herein incorporated by reference). Such fragments typically
contain at least 7 consecutive amino acids of the mature
polypeptide sequence, even more preferably at least 10 consecutive
amino acids thereof. They are essentially derived from the
extracellular domain of the receptor. In preferred embodiments, the
NKG2A receptor used to generate antibodies is a human receptor. In
certain embodiments, NKG2A present in a heterodimer, e.g. in
association with CD94, can be used to generate antibodies.
[0113] In a most preferred embodiment, the immunogen comprises a
wild-type human NKG2A receptor polypeptide in a lipid membrane,
typically at the surface of a cell. In a specific embodiment, the
immunogen comprises intact NK cells, particularly intact human NK
cells, optionally treated or lysed. In a preferred embodiment, the
immunogen is an NK cell taken from a patient with an autoimmune or
inflammatory disorder.
[0114] In one embodiment, the antibodies are derived from one or
more already-existing monoclonal antibodies that recognize NKG2A,
e.g. Z199 (Della Chiesa et al, (2003) Eur. J. Immunol.
33:1657-1666), Z270, 3S9 (see, e.g., U.S. patent application no.
20030095965), or 20D5 (Vance et al, (1990) J. Exp. Med.
190(12):1801-1812), the entire disclosures of which are herein
incorporated by reference). For certain applications, such
antibodies can be directly or indirectly labeled (i.e., used with a
labeled secondary antibody) for use as diagnostic antibodies to
determine the presence of NKG2A on the presence of cells,
preferably NK cells from patients with autoimmune or inflammatory
disorders. In addition, the antibodies can be made suitable for
human administration as described herein for use in the present
therapeutic methods.
[0115] The present antibodies can be full length antibodies or
antibody fragments or derivatives. Examples of antibody fragments
include Fab, Fab', Fab'-SH, F(ab').sub.2, and Fv fragments;
diabodies; single-chain Fv (scFv) molecules; 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; 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 antibodies formed from antibody fragments. Such
fragments and derivatives and methods of preparing them are well
known in the art. For example, pepsin can be used to digest an
antibody below the disulfide linkages in the hinge region to
produce F(ab)'.sub.2, a dimer of Fab which itself is a light chain
joined to V.sub.H-C.sub.H1 by a disulfide bond. The F(ab)'.sub.2
may be reduced under mild conditions to break the disulfide linkage
in the hinge region, thereby converting the F(ab)'.sub.2 dimer into
an Fab' monomer. The Fab' monomer is essentially Fab with part of
the hinge region (see Fundamental Immunology (Paul ed., 3d ed.
1993)). While various antibody fragments are defined in terms of
the digestion of an intact antibody, one of skill will appreciate
that such fragments may be synthesized de novo either chemically or
by using recombinant DNA methodology.
[0116] In a preferred embodiment, the activating antibodies are
non-depleting antibodies, meaning that they bind to NK cells and
inhibit NKG2A stimulation, but do not lead to the killing of the
NKG2A expressing cell. The ability to kill NKG2A expressing cells
can be assessed using standard methods, including in vitro assays
to ensure that the antibodies are not cytotoxic, directly killing
bound cells, as well as in vivo assays in which the antibodies are
administered and the level and activity of NKG2A expressing cells
are assessed. In a particularly preferred embodiment, as described
supra, antibodies will be used that are not recognized (or only
poorly recognized) by Fc receptors. Accordingly, preferred
antibodies include IgG4, fragments such as Fab or F(ab')2, or any
other IgG, IgE, IgM, etc. of which the Fc portion has been modified
to reduce or eliminate binding by Fc receptors (see, e.g.,
WO03101485, the entire disclosure of which is herein incorporated
by reference).
[0117] The preparation of monoclonal or polyclonal antibodies is
well known in the art, and any of a large number of available
techniques can be used (see, e.g., Kohler & Milstein, Nature
256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983);
Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy
(1985)). Techniques for the production of single chain antibodies
(U.S. Pat. No. 4,946,778) can be adapted to produce antibodies to
desired polypeptides, e.g., NKG2A. Also, transgenic mice, or other
organisms such as other mammals, may be used to express humanized,
chimeric, or similarly-modified antibodies. Alternatively, phage
display technology can be used to identify antibodies and
heteromeric Fab fragments that specifically bind to selected
antigens (see, e.g., McCafferty et al., Nature 348:552-554 (1990);
Marks et al., Biotechnology 10:779-783 (1992)). In one embodiment,
the method comprises selecting, from a library or repertoire, a
monoclonal antibody or a fragment or derivative thereof that cross
reacts with an NKG2A receptor polypeptide. For example, the
repertoire may be any (recombinant) repertoire of antibodies or
fragments thereof, optionally displayed by any suitable structure
(e.g., phage, bacteria, synthetic complex, etc.).
[0118] The step of immunizing a non-human mammal with an antigen
may be carried out in any manner well known in the art for (see,
for example, E. Harlow and D. Lane, Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. (1988)). Generally, the immunogen is suspended or dissolved in
a buffer, optionally with an adjuvant, such as complete Freund's
adjuvant. Methods for determining the amount of immunogen, types of
buffers and amounts of adjuvant are well known to those of skill in
the art and are not limiting in any way on the present
invention.
[0119] Similarly, the location and frequency of immunization
sufficient to stimulate the production of antibodies is also well
known in the art. In a typical immunization protocol, the non-human
animals are injected intraperitoneally with antigen on day 1 and
again about a week later. This is followed by recall injections of
the antigen around day 20, optionally with adjuvant such as
incomplete Freund's adjuvant. The recall injections are performed
intravenously and may be repeated for several consecutive days.
This is followed by a booster injection at day 40, either
intravenously or intraperitoneally, typically without adjuvant.
This protocol results in the production of antigen-specific
antibody-producing B cells after about 40 days. Other protocols may
also be utilized as long as they result in the production of B
cells expressing an antibody directed to the antigen used in
immunization.
[0120] In another embodiment, lymphocytes from an unimmunized
non-human mammal are isolated, grown in vitro, and then exposed to
the immunogen in cell culture. The lymphocytes are then harvested
and the fusion step described below is carried out.
[0121] For monoclonal antibodies, which are preferred for the
purposes of the present invention, the next step is the isolation
of cells, e.g., lymphocytes, splenocytes, or B cells, from the
immunized non-human mammal and the subsequent fusion of those
splenocytes, B cells, or lymphocytes with an immortalized cell in
order to form an antibody-producing hybridoma. Accordingly, the
term "preparing antibodies from an immunized animal," as used
herein, includes obtaining B-cells/splenocytes/lymphocytes from an
immunized animal and using those cells to produce a hybridoma that
expresses antibodies, as well as obtaining antibodies directly from
the serum of an immunized animal. The isolation of splenocytes,
e.g., from a non-human mammal is well-known in the art and, e.g.,
involves removing the spleen from an anesthetized non-human mammal,
cutting it into small pieces and squeezing the splenocytes from the
splenic capsule and through a nylon mesh of a cell strainer into an
appropriate buffer so as to produce a single cell suspension. The
cells are washed, centrifuged and resuspended in a buffer that
lyses any red blood cells. The solution is again centrifuged and
remaining lymphocytes in the pellet are finally resuspended in
fresh buffer.
[0122] Once isolated and present in single cell suspension, the
antibody-producing cells are fused to an immortal cell line. This
is typically a mouse myeloma cell line, although many other
immortal cell lines useful for creating hybridomas are known in the
art. Preferred murine myeloma lines include, but are not limited
to, those derived from MOPC-21 and MPC-11 mouse tumors available
from the Salk Institute Cell Distribution Center, San Diego, Calif.
U.S.A., X63 Ag8653 and SP-2 cells available from the American Type
Culture Collection, Rockville, Md. U.S.A. The fusion is effected
using polyethylene glycol or the like. The resulting hybridomas are
then grown in selective media that contains one or more substances
that inhibit the growth or survival of the unfused, parental
myeloma cells. For example, if the parental myeloma cells lack the
enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or
HPRT), the culture medium for the hybridomas typically will include
hypoxanthine, aminopterin, and thymidine (HAT medium), which
substances prevent the growth of HGPRT-deficient cells.
[0123] The hybridomas can be grown on a feeder layer of
macrophages. The macrophages are preferably from littermates of the
non-human mammal used to isolate splenocytes and are typically
primed with incomplete Freund's adjuvant or the like several days
before plating the hybridomas. Fusion methods are described (e.g.,
in Goding, "Monoclonal Antibodies: Principles and Practice," pp.
59-103 (Academic Press, 1986), the disclosure of which is herein
incorporated by reference).
[0124] The cells are allowed to grow in the selection media for
sufficient time for colony formation and antibody production. This
is usually between 7 and 14 days. The hybridoma colonies are then
assayed for the production of antibodies that specifically
recognize the desired substrate, e.g. NKG2A. The assay is typically
a colorimetric ELISA-type assay, although any assay may be employed
that can be adapted to the wells in which the hybridomas are grown.
Other assays include immunoprecipitation and radioimmunoassay. The
wells positive for the desired antibody production are examined to
determine if one or more distinct colonies are present. If more
than one colony is present, the cells may be recloned and grown to
ensure that only a single cell has given rise to the colony
producing the desired antibody. Positive wells with a single
apparent colony are typically recloned and re-assayed to ensure
that only one monoclonal antibody is being detected and
produced.
[0125] Hybridomas that are confirmed to be producing a monoclonal
antibody of this invention are then grown up in larger amounts in
an appropriate medium, such as DMEM or RPMI-1640. Alternatively,
the hybridoma cells can be grown in vivo as ascites tumors in an
animal.
[0126] After sufficient growth to produce the desired monoclonal
antibody, the growth media containing monoclonal antibody (or the
ascites fluid) is separated away from the cells and the monoclonal
antibody present therein is purified. Purification is typically
achieved by gel electrophoresis, dialysis, chromatography using
protein A or protein G-Sepharose, or an anti-mouse Ig linked to a
solid support such as agarose or Sepharose beads (all described,
for example, in the Antibody Purification Handbook, Amersham
Biosciences, publication No. 18-1037-46, Edition AC, the disclosure
of which is hereby incorporated by reference). The bound antibody
is typically eluted from protein A/protein G columns by using low
pH buffers (glycine or acetate buffers of pH 3.0 or less) with
immediate neutralization of antibody-containing fractions. These
fractions are pooled, dialyzed, and concentrated as needed.
[0127] In preferred embodiments, the DNA encoding an antibody that
binds a determinant present on the NKG2A immunogen 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, variants thereof,
active fragments thereof, or humanized or chimeric antibodies
comprising the antigen recognition portion of the antibody.
Preferably, the DNA used in this embodiment encodes an antibody
that recognizes a determinant present on NKG2A receptors on NK
cells, such as NK cells taken from a patient with an autoimmune or
inflammatory disorder.
[0128] DNA encoding the monoclonal antibodies of the invention 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. Recombinant expression in bacteria of DNA encoding the
antibody is well known in the art (see, for example, Skerra et al.
(1993) Curr. Op. Immunol. 5:256; and Pluckthun (1992) Immunol.
Revs. 130:151). Antibodies may also be produced by selection of
combinatorial libraries of immunoglobulins, as disclosed for
instance in Ward et al. (1989) Nature 341:544.
[0129] In a specific embodiment, the antibody binds essentially the
same epitope or determinant as one of monoclonal antibodies Z199 or
Z270. In one preferred embodiment, the monoclonal antibody
comprises the Fab or F(ab).sub.2 portion of Z270. According to
another preferred embodiment, the monoclonal antibody comprises the
three CDRs of the variable heavy chain region of Z270 (CDR1=amino
acids 31 to 35 of SEQ ID NO:2; CDR2=amino acids 50 to 66 of SEQ ID
NO:2; CDR3=amino acids 99-108 of SEQ ID NO:2). More preferred is a
monoclonal antibody that comprises the variable heavy chain region
of Z270 (Z270VH; SEQ ID NO:2). Even more preferred is a monoclonal
antibody that comprises the variable heavy chain region of Z270 and
is transcribed and translated from a nucleotide sequence comprising
chZ270VH (SEQ ID NO:3). According to another preferred embodiment,
the monoclonal antibody comprises the three CDRs of the variable
light chain region of Z270 (CDR1=amino acids 24 to 34 of SEQ ID
NO:6; CDR2=amino acids 50 to 56 of SEQ ID NO:6; CDR3=amino acids
89-95 of SEQ ID NO:6). More preferred is a monoclonal antibody that
comprises the variable light chain region of Z270 (SEQ ID NO:6).
Even more preferred is a monoclonal antibody that comprises the
variable light chain region of Z270 and is transcribed and
translated from a nucleotide sequence comprising chZ270VK (SEQ ID
NO:7). In yet another preferred embodiment the antibody is Z270.
Z270 was deposited on Dec. 22, 2005 at the Collection Nationale de
Culture de Microorganismes, Institute Pasteur, 25, Rue du Docteur
Roux, F-75725 Paris, France, under accession number I-3549.
[0130] Both activating and inhibitory monoclonal antibodies against
NKG2A will generally be modified so as to make them suitable for
therapeutic use in humans. For example, they may be humanized,
chimerized, or selected from a library of human antibodies using
methods well known in the art. Such human-suitable antibodies can
be used directly in the present therapeutic methods, or can be
further derivatized into cytotoxic antibodies, as described infra,
for use in the methods.
[0131] In one preferred embodiment, the DNA of a hybridoma
producing an antibody of this invention, e.g. an antibody that
binds to substantially the same epitope as Z199 or Z270, 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. (1984) PNAS 81:6851), 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 of the invention.
[0132] In a preferred embodiment, the antibody comprises the
variable heavy chain region of Z270 fused (SEQ ID NO:2) to a human
heavy chain constant region. In one preferred embodiment, the human
heavy chain constant region is a IgG4 constant region. In another
preferred embodiment, the human heavy chain constant region is a
IgG1 constant region, preferably a human IgG1m(-1, -2, -3) constant
region. Preferably, such a human heavy chain constant
region-containing antibody is transcribed and translated from a
nucleotide sequence comprising chZ270VH (SEQ ID NO:3).
[0133] In another preferred embodiment, the antibody comprises the
variable light chain region of Z270 fused (SEQ ID NO:6) to a human
light chain constant region. More preferred is an antibody that
comprises the variable light chain region of Z270 fused to the
human kappa (k3) light chain constant region. Preferably, such a
human light chain constant region-containing antibody is
transcribed and translated from a nucleotide sequence comprising
chZ270VK (SEQ ID NO:7).
[0134] Even more preferred is an antibody comprising both 270VK
fused to a human light chain constant region and 270VK fused to a
human heavy chain constant region. Preferably, the light chain
constant region is a kappa (k3) constant region and the heavy chain
constant region is selected from IgG4 or IgG1m(-1, -2, -3). Also,
preferably, each of the heavy and light chains of the antibody are
transcribed from a nucleotide sequence comprising chZ270VH (SEQ ID
NO:3) and a nucleotide sequence comprising chZ270VK (SEQ ID NO:7),
respectively.
In one particularly preferred embodiment, the antibody of this
invention is humanized. "Humanized" forms of antibodies according
to this invention 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 or other
non-human 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. 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. For further details see Jones et
al. (1986) Nature 321: 522; Reichmann et al. (1988) Nature 332:
323; Verhoeyen et al. (1988) Science 239:1534 (1988); Presta (1992)
Curr. Op. Struct. Biol. 2:593; each of which is herein incorporated
by reference in its entirety.
[0135] 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 of this
invention 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. (1993) J. Immun., 151:2296;
Chothia and Lesk (1987) J. Mol. Biol. 196:901). 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. (1992) PNAS 89:4285; Presta et al. (1993)
J. Immunol. 51:1993)).
[0136] It is further important that antibodies be humanized while
retaining their high affinity for NKG2A, preferably human and
non-human primate NKG2A, and other favorable biological properties.
To achieve this goal, according to a preferred 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
conformational 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.
[0137] In preferred examples, the invention provides human or
humanized activating anti-NKG2A antibodies having a half-life of at
least 5, 6, 8, 9, 10, 15 or 20 days, which do not substantially
bind human FcgammaRIIIa (CD16). More preferably, the activating
anti-NKG2A antibody is a humanized antibody and completely competes
with a Z199 or Z270 antibody for binding to human NKG2A. For the
purpose of illustration with preferred antibodies suitable for use
according to the methods herein, a Z199 or Z270 antibody can be
used to prepare a humanized antibody. Preferred humanized
antibodies according to the invention comprise a human framework,
at least one CDR from a non-human antibody, and in which any
constant region present is substantially identical to a human
immunoglobulin constant region, e.g., at least about 60-90%,
preferably at least 95% identical. Hence, all parts of a humanized
antibody, except possibly the CDR's, are substantially identical to
corresponding parts of one or more native human antibody sequences.
In some instances, the humanized antibody, in addition to CDRs from
a non-human antibody, would include additional non-human residues
in the human framework region.
[0138] The design of humanized antibodies can be carried out as
follows. When an amino acid falls under the following categories,
the framework amino acid of a human antibody to be used (acceptor
antibody) is replaced by a framework amino acid from a
CDR-providing non-human antibody (donor antibody): (a) the amino
acid in the human framework region of the acceptor antibody is
unusual for human antibody at that position, whereas the
corresponding amino acid in the donor antibody is typical for human
antibody in that position; (b) the position of the amino acid is
immediately adjacent to one of the CDR's; or (c) the amino acid is
capable of interacting with the CDR's in a tertiary structure
antibody model (see, C. Queen et al. Proc. Natl. Acad. Sci. USA 86,
10029 (1989), and Co et al., Proc. Natl. Acad. Sci. USA 88, 2869
(1991) the disclosures of which are incorporated herein by
reference).
[0139] For further detailed description of the production of
humanized antibody, See Queen et al., op. cit. and Co et al, op.
cit. and U.S. Pat. Nos. 5,585,089; 5,693,762, 5,693,761, and
5,530,101, the disclosures of which are incorporated herein by
reference. Usually, the CDR regions in humanized antibodies are
substantially identical, and more usually, identical to the
corresponding CDR regions in the mouse antibody from which they
were derived. Although not usually desirable, it is sometimes
possible to make one or more conservative amino acid substitutions
of CDR residues without appreciably affecting the binding affinity
of the resulting humanized antibody. Occasionally, substitutions of
CDR regions can enhance binding affinity. Other than for the
specific amino acid substitutions discussed above, the framework
regions of humanized antibodies are usually substantially
identical, and more usually, identical to the framework regions of
the human antibodies from which they were derived. Of course, many
of the amino acids in the framework region make little or no direct
contribution to the specificity or affinity of an antibody. Thus,
many individual conservative substitutions of framework residues
can be tolerated without appreciable change of the specificity or
affinity of the resulting humanized antibody. The antigen binding
region of the humanized antibody (the non-human portion) can be
derived from an antibody of nonhuman origin, referred to as a donor
antibody, having specificity for NKG2A. For example, a suitable
antigen binding region can be derived from Z199 or Z270 monoclonal
antibodies. Other sources include NKG2A-specific (blocking)
antibodies obtained from nonhuman sources, such as rodent (e.g.,
mouse and rat), rabbit, pig, goat or non-human primate (e.g.,
monkey) or camelid animals (e.g., camels and llamas). Additionally,
other polyclonal or monoclonal antibodies, such as antibodies which
bind to the same or similar epitope as Z199 or Z270 antibodies, can
be made (e.g., Kohler et al., Nature, 256:495-497 (1975); Harlow et
al., 1988, Antibodies: A Laboratory Manual (Cold Spring Harbor,
N.Y.); and Current Protocols in Molecular Biology, Vol. 2
(Supplement 27, Summer '94), Ausubel et al., Eds. (John Wiley &
Sons: New York, N.Y.), Chapter 11 (1991)).
[0140] In one embodiment, the humanized antibody having binding
specificity for human and non-human primate NKG2A comprises at
least one CDR of nonhuman origin. For example, a humanized antibody
having a binding specificity for human and non-human primate NKG2A
comprises a heavy chain and a light chain. The light chain can
comprise a CDR derived from an antibody of nonhuman origin which
binds NKG2A and a FR derived from a light chain of human origin.
For example, the light chain can comprise CDR1, CDR2 and/or CDR3
which have the amino acid sequence similar or substantially the
same as that of the respective CDR of any one of the Z199 or Z270
antibodies such that the antibody specifically binds to the human
and non-human primate NKG2A. The heavy chain can comprise a CDR
derived from an antibody of nonhuman origin which binds NKG2A and a
FR derived from a heavy chain of human origin. For example, the
heavy chain can comprise CDR1, CDR2 and CDR3 which have the amino
acid sequence set forth below or an amino acid similar or
substantially the same as that of the respective CDR of the Z199 or
Z270 antibodies such that the antibody specifically binds to the
human and non-human primate NKG2A.
[0141] An embodiment of the invention is a humanized antibody which
specifically binds to human and non-human primate NKG2A and
comprises a humanized light chain comprising three light chain CDRs
from a Z199 or Z270 antibody and a light chain variable region
framework sequence from a human antibody light chain. The invention
further comprises a humanized heavy chain that comprises three
heavy chain CDRs from a Z199 or Z270 antibody and a heavy chain
variable region framework sequence from a human antibody heavy
chain.
[0142] The portion of the humanized antibody or antibody chain
which is of human origin (the human portion) can be derived from
any suitable human antibody or antibody chain. For example, a human
constant region or portion thereof, if present, can be derived from
the kappa or lambda light chains, and/or the gamma (e.g., gamma1,
gamma2, gamma3, gamma4), .mu., alpha (e.g., alpha1, alpha2), delta
or epsilon heavy chains of human antibodies, including allelic
variants. A particular constant region, such as IgG2b or IgG4,
variants or portions thereof can be selected to tailor effector
function. The latter constant regions, or portions thereof are
particularly preferred in that they do not substantially bind
FcgammaIIIa receptor on NK cells (CD16) and therefore do not
substantially induce ADCC mediated lysis of NK effectors to which
the anti-NKG2A antibodies of the invention are bound. For example,
a mutated constant region, also referred to as a "variant," can be
incorporated into a fusion protein to minimize binding to Fc
receptors and/or ability to fix complement (see e.g., Winter et
al., U.S. Pat. No. 5,648,260; Morrison et al., WO 89/07142; Morgan
et al., WO 94/29351). In addition, a mutated IgG2 Fc domain can be
created that reduces the mitogenic response, as compared to natural
Fc regions (see e.g., Tso et al., U.S. Pat. No. 5,834,597, the
teachings of which are incorporated by reference herein in their
entirety). If present, human FRs are preferably derived from a
human antibody variable region having sequence similarity to the
analogous or equivalent region of the antigen binding region donor.
Other sources of FRs for portions of human origin of a humanized
antibody include human variable consensus sequences (see,
Kettleborough, C. A. et al., Protein Engineering 4:773-783 (1991);
Queen et al., U.S. Pat. Nos. 5,585,089, 5,693,762 and 5,693,761,
the teachings of all of which are incorporated by reference herein
in their entirety). For example, the sequence of the antibody or
variable region used to obtain the nonhuman portion can be compared
to human sequences as described in Kabat, E. A., et al., Sequences
of Proteins of Immunological Interest, Fifth Edition, U.S.
Department of Health and Human Services, U.S. Government Printing
Office (1991). In a preferred embodiment, the FRs of a humanized
antibody chain are derived from a human variable region having at
least about 60% overall sequence identity, and preferably at least
about 80% overall sequence identity, with the variable region of
the nonhuman donor (e.g., Z199 or Z270 antibody).
[0143] The phrase "substantially identical," in context of two
nucleic acids or polypeptides (e.g., DNAs encoding a humanized
antibody or the amino acid sequence of the humanized antibody)
refers to two or more sequences or subsequences that have at least
about 80%, most preferably 90-95% or higher nucleotide or amino
acid residue identity, when compared and aligned for maximum
correspondence, as measured using the following sequence comparison
method and/or by visual inspection. Such "substantially identical"
sequences are typically considered to be homologous. Preferably,
the "substantial identity" exists over a region of the sequences
that is at least about 50 residues in length, more preferably over
a region of at least about 100 residues, and most preferably the
sequences are substantially identical over at least about 150
residues, or over the full length of the two sequences to be
compared. As described below, any two antibody sequences can only
be aligned in one way, by using the numbering scheme in Kabat.
Therefore, for antibodies, percent identity has a unique and
well-defined meaning.
[0144] Amino acids from the variable regions of the mature heavy
and light chains of antibodies are designated Hx and Lx
respectively, where x is a number designating the position of an
amino acid according to the scheme of Kabat, Sequences of Proteins
of Immunological Interest (National Institutes of Health, Bethesda,
Md., 1987 and 1991). Kabat lists many amino acid sequences for
antibodies for each subgroup, and lists the most commonly occurring
amino acid for each residue position in that subgroup. Kabat uses a
method for assigning a residue number to each amino acid in a
listed sequence, and this method for assigning residue numbers has
become standard in the field. Kabat's scheme is extendible to other
antibodies not included in his compendium by aligning the antibody
in question with one of the consensus sequences in Kabat. The use
of the Kabat numbering system readily identifies amino acids at
equivalent positions in different antibodies. For example, an amino
acid at the L50 position of a human antibody occupies the
equivalent position to an amino acid position L50 of a mouse
antibody. From N-terminal to C-terminal, both light and heavy chain
variable regions comprise alternating framework and (CDRs): FR1,
CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids
to each region is in accordance with the definitions of Kabat
(1987) and (1991), supra and/or Chothia & Lesk, J. Mol. Biol.
196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989).
[0145] Binding and/or adhesion assays or other suitable methods can
also be used in procedures for the identification and/or isolation
of humanized antibodies (e.g., from a library) with the requisite
specificity (competition assays for example).
[0146] The antibody portions of nonhuman and human origin for use
in the invention include light chains, heavy chains and portions of
light and heavy chains. These antibody portions can be obtained or
derived from antibodies (e.g., by de novo synthesis of a portion),
or nucleic acids encoding an antibody or chain thereof having the
desired property (e.g., binds NKG2A, sequence similarity, for
example with the Z199 or Z270 antibody) can be produced and
expressed. Humanized antibodies comprising the desired portions
(e.g., antigen binding region, CDR, FR, C region) of human and
nonhuman origin can be produced using synthetic and/or recombinant
nucleic acids to prepare genes (e.g., cDNA) encoding the desired
humanized chain. To prepare a portion of a chain, one or more stop
codons can be introduced at the desired position. For example,
nucleic acid sequences coding for newly designed humanized variable
regions can be constructed using PCR mutagenesis methods to alter
existing DNA sequences (see e.g., Kamman, M., et al., Nucl. Acids
Res. 17:5404 (1989)). PCR primers coding for the new CDRs can be
hybridized to a DNA template of a previously humanized variable
region which is based on the same, or a very similar, human
variable region (Sato, K., et al., Cancer Research 53:851-856
(1993)). If a similar DNA sequence is not available for use as a
template, a nucleic acid comprising a sequence encoding a variable
region sequence can be constructed from synthetic oligonucleotides
(see e.g., Kolbinger, F., Protein Engineering 8:971-980 (1993)). A
sequence encoding a signal peptide can also be incorporated into
the nucleic acid (e.g., on synthesis, upon insertion into a
vector). If the natural signal peptide sequence is unavailable, a
signal peptide sequence from another antibody can be used (see,
e.g., Kettleborough, C. A., Protein Engineering 4:773-783 (1991)).
Using these methods, methods described herein or other suitable
methods, variants can be readily produced. In one embodiment,
cloned variable regions can be mutagenized, and sequences encoding
variants with the desired specificity can be selected (e.g., from a
phage library; see e.g., Krebber et al., U.S. Pat. No. 5,514,548;
Hoogengoom et al., WO 93/06213, published Apr. 1, 1993)).
[0147] The invention also relates to isolated and/or recombinant
(including, e.g., essentially pure) nucleic acids comprising
sequences which encode a humanized antibody or humanized antibody
light or heavy chain of the present invention.
[0148] 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. In this technique, elements of the human heavy
and light chain loci are introduced into mice or other animals with
targeted disruptions of the endogenous heavy chain and light chain
loci (see, e.g., Jakobovitz et al. (1993) Nature 362:255; 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
disclosures of which are herein incorporated by reference).
Alternatively, human antibodies can be constructed by genetic or
chromosomal transfection methods, or through the selection of
antibody repertoires using phage display methods. In this
technique, antibody variable domain genes are cloned in-frame into
either a major or minor coat protein gene of a filamentous
bacteriophage, and displayed as functional antibody fragments on
the surface of the phage particle. Because the filamentous particle
contains a single-stranded DNA copy of the phage genome, selections
based on the functional properties of the antibody also result in
selection of the gene encoding the antibody exhibiting those
properties. In this way, the phage mimics some of the properties of
the B cell (see, e.g., Johnson et al. (1993) Curr Op Struct Biol
3:5564-571; McCafferty et al. (1990) Nature 348:552-553, the entire
disclosures of which are herein incorporated by reference). 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, the disclosures
of which are incorporated in their entirety by reference).
[0149] In one embodiment, "humanized" monoclonal antibodies are
made using an animal such as a XenoMouse.RTM. (Abgenix, Fremont,
Calif.) 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. An
analogous method can be achieved using a HuMAb-Mouse.TM.
(Medarex).
[0150] The antibodies of the present invention may also be
derivatized to "chimeric" antibodies (immunoglobulins) in which a
portion of the heavy and/or light chain is identical with or
homologous to corresponding sequences in the original antibody,
while the remainder of the chain(s) is identical with or homologous
to corresponding sequences in antibodies derived from another
species or belonging to another antibody class or subclass, as well
as fragments of such antibodies, so long as they exhibit the
desired biological activity (see, e.g., Morrison et al. (1984) PNAS
81:6851; U.S. Pat. No. 4,816,567).
[0151] In another embodiment the invention provides any of the
antibodies or fragments thereof described above (whether activating
or inhibitory) conjugated to a cytotoxic agent. The term "cytotoxic
agent" as used herein is a molecule that is capable of killing a
cell bearing a NKG2A receptor on its cell surface. The term
"conjugated" as used herein means that the two agents are either
bound to each other through a covalent and/or non-covalent bond, or
tethered or otherwise connected to one another directly or through
a linking moiety.
[0152] Any of a large number of toxic moieties or strategies can be
used to produce such cytotoxic antibody conjugates. In certain
preferred embodiments, the antibodies will be directly derivatized
with radioisotopes or other toxic compounds. In such cases, the
labeled monospecific anti-NKG2A antibody can be injected into the
patient, where it can then bind to and kill cells expressing that
target antigen, particularly NK cells, with unbound antibody simply
clearing the body. Indirect strategies can also be used, such as
the "Affinity Enhancement System" (AES) (see, e.g., U.S. Pat. No.
5,256,395; Barbet et al. (1999) Cancer Biother Radiopharm
14:153-166; the entire disclosures of which are herein incorporated
by reference). This particular approach involves the use of a
radiolabeled hapten and an antibody that recognizes both the NK
cell receptor and the radioactive hapten. In this case, the
antibody is first injected into the patient and allowed to bind to
target cells, and then, once unbound antibody is allowed to clear
from the blood stream, the radiolabeled hapten is administered. The
hapten binds to the antibody-antigen complex on the
overproliferating LGL (e.g. NK or T) cells, thereby killing them,
with the unbound hapten clearing the body.
[0153] Any type of moiety with a cytotoxic or cytoinhibitory effect
can be conjugated to the present antibodies to form a cytotoxic
conjugate of the present invention and to inhibit or kill specific
NK receptor expressing cells, including radioisotopes, toxic
proteins, toxic small molecules, such as drugs, toxins,
immunomodulators, hormones, hormone antagonists, enzymes,
oligonucleotides, enzyme inhibitors, therapeutic radionuclides,
angiogenesis inhibitors, chemotherapeutic drugs, vinca alkaloids,
anthracyclines, epidophyllotoxins, taxanes, antimetabolites,
alkylating agents, antibiotics, COX-2 inhibitors, SN-38,
antimitotics, antiangiogenic and apoptotoic agents, particularly
doxorubicin, methotrexate, taxol, CPT-11, camptothecans, nitrogen
mustards, gemcitabine, alkyl sulfonates, nitrosoureas, triazenes,
folic acid analogs, pyrimidine analogs, purine analogs, platinum
coordination complexes, Pseudomonas exotoxin, ricin, abrin,
5-fluorouridine, ribonuclease (RNase), DNase I, Staphylococcal
enterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin
toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin and others
(see, e.g., Remington's Pharmaceutical Sciences, 19th Ed. (Mack
Publishing Co. 1995); Goodman and Gilman's The Pharmacological
Basis of Therapeutics (McGraw Hill, 2001); Pastan et al. (1986)
Cell 47:641; Goldenberg (1994) Cancer Journal for Clinicians 44:43;
U.S. Pat. No. 6,077,499; the entire disclosures of which are herein
incorporated by reference). It will be appreciated that a toxin can
be of animal, plant, fungal, or microbial origin, or can be created
de novo by chemical synthesis.
[0154] The toxins or other compounds can be linked to the antibody
directly or indirectly, using any of a large number of available
methods. For example, an agent can be attached at the hinge region
of the reduced antibody component via disulfide bond formation,
using cross-linkers such as N-succinyl
3-(2-pyridyldithio)proprionate (SPDP), or via a carbohydrate moiety
in the Fc region of the antibody (see, e.g., Yu et al. (1994) Int.
J. Cancer 56: 244; Wong, Chemistry of Protein Conjugation and
Cross-linking (CRC Press 1991); Upeslacis et al., "Modification of
Antibodies by Chemical Methods," in Monoclonal antibodies:
principles and applications, Birch et al. (eds.), pages 187-230
(Wiley-Liss, Inc. 1995); Price, "Production and Characterization of
Synthetic Peptide-Derived Antibodies," in Monoclonal antibodies:
Production, engineering and clinical application, Ritter et al.
(eds.), pages 60-84 (Cambridge University Press 1995), Cattel et
al. (1989) Chemistry today 7:51-58, Delprino et al. (1993) J.
Pharm. Sci 82:699-704; Arpicco et al. (1997) Bioconjugate Chemistry
8:3; Reisfeld et al. (1989) Antibody, Immunicon. Radiopharrn.
2:217; the entire disclosures of each of which are herein
incorporated by reference).
[0155] In one preferred embodiment, the antibody will be
derivatized with a radioactive isotope, such as 1-131. Any of a
number of suitable radioactive isotopes can be used, including, but
not limited to, Indium-111, Lutetium-171, Bismuth-212, Bismuth-213,
Astatine-211, Copper-62, Copper-64, Copper-67, Yttrium-90,
Iodine-125, Iodine-131, Phosphorus-32, Phosphorus-33, Scandium-47,
Silver-111, Gallium-67, Praseodymium-142, Samarium-153,
Terbium-161, Dysprosium-166, Holmium-166, Rhenium-186, Rhenium-188,
Rhenium-189, Lead-212, Radium-223, Actinium-225, Iron-59,
Selenium-75, Arsenic-77, Strontium-89, Molybdenum-99, Rhodium-105,
Palladium-109, Praseodymium-143, Promethium-149, Erbium-169,
Iridium-194, Gold-198, Gold-199, and Lead-211. In general, the
radionuclide preferably has a decay energy in the range of 20 to
6,000 keV, preferably in the ranges 60 to 200 keV for an Auger
emitter, 100-2,500 keV for a beta emitter, and 4,000-6,000 keV for
an alpha emitter. Also preferred are radionuclides that
substantially decay with generation of alpha-particles.
[0156] In selecting a cytotoxic moiety for conjugation to the
anti-NKG2A antibody in the present cytotoxic compositions, it is
desirable to ensure that the moiety will not exert significant in
vivo side effects against life-sustaining normal tissues, such as
one or more tissues selected from heart, kidney, brain, liver, bone
marrow, colon, breast, prostate, thyroid, gall bladder, lung,
adrenals, muscle, nerve fibers, pancreas, skin, or other
life-sustaining organ or tissue in the human body. The term
"significant side effects", as used herein, refers to an antibody,
ligand or antibody conjugate, that, when administered in vivo, will
produce only negligible or clinically manageable side effects, such
as those normally encountered during chemotherapy.
[0157] In a somewhat related embodiment, the invention also
provides an antibody of this invention conjugated to a detectable
marker. The term "detectable marker" as used herein refers to any
molecule that can be quantitatively or qualitatively observed or
measured. Examples of detectable markers useful in the conjugated
antibodies of this invention are radioisotopes, fluorescent dyes,
or a member of a complementary binding pair, such as a member of
any one of: an antigen/antibody (other than an antibody to NKG2A);
lectin/carbohydrate; avidin/biotin; receptor/ligand; or molecularly
imprinted polymer/print molecule systems.
[0158] The detectable marker conjugated antibodies of this
invention may be used to detect the binding of the antibody to
NKG2A, either in vitro or in vivo. Such conjugates may also be
utilized to detect the binding of another molecule to NKG2A in a
competition-type experiment. In an in vivo setting, the detectable
marker-antibody conjugate of this invention may be used to monitor
the efficacy of treatment of a patient with a NKG2A antibody
composition of this invention, by ex vivo detection of the
detectable marker (e.g., via whole body scans or the like) or by
detection in a biological material (e.g., blood, biopsied tissue,
other bodily fluids, skin scrapings, etc.) obtained from the
patient. The detection of the marker in various biological material
will be correlated with the presence of the therapeutic antibody in
said material.
[0159] In a related embodiment the invention provides a kit
comprising, in separate vessels: a detectable marker-anti-NKG2A
antibody conjugate; and an NKG2A-containing material. An
NKG2A-containing material may be isolated NKG2A, a fragment of
NKG2A comprising an epitope to which an anti-NKG2A antibody of this
invention binds, or a cell that expresses NKG2A on its cell
surface.
Evaluation of Anti-Human NKG2A Antibodies in Nonhuman Primates
[0160] In a preferred series of embodiments, the activity of an
anti-NKG2A antibody of this invention will be assessed in vivo in a
nonhuman primate. Such embodiments can be carried out for any of a
wide variety of reasons. In view of the crossreactivity between
human NKG2A and NKG2A from nonhuman primates, and in view of the
physiological similarities among primates, administering antibodies
that recognize human NKG2A to nonhuman primates allows the
antibodies to be assessed in vivo for many aspects including, but
not limited to, ability to modulate the activity of cells
expressing NKG2A (e.g. NK cells), side effects produced, toxicity,
pharmacodynamics, pharmacokinetics, bioavailability, half-life,
optimal dose or frequency of administration, optimal formulations
including combinations with other therapeutic agents, or any other
property that may be measured to determine the efficacy, safety, or
optimal administration of the antibodies. Methods of assessing
candidate therapeutic compounds in vivo are well known in the art,
and are described, e.g., in The Merck Manual of Diagnosis and
Therapy, 17.sup.th edition, Remington's Pharmaceutical Sciences,
20.sup.th edition, the entire disclosures of which are herein
incorporated by reference.
[0161] Any nonhuman primate can be used for the herein-described
methods, including apes, monkeys, and prosimians. Preferred
primates include the Rhesus monkey (Macacus mulatta), African green
monkey (Chlorocebus aethiops), Marmoset (Callithrix jacchus),
Saimiri (Saimiri sciureus), cynomolgus, and Baboon (Papio
hamadryas). In another preferred embodiment, the primate is not an
ape, e.g. is a primate other than a chimpanzee. Non-human primates
are commonly used in safety and efficacy assays for candidate human
therapeutic agents, and their care, administration, biology, and
other relevant features are well known to those in the art. In one
embodiment, prior to the administration of any antibody to any
nonhuman primate (or the use of tissue, cells, or proteins from a
nonhuman primate in any assay), the crossreactivity of the
candidate anti-human NKG2A antibodies with NKG2A from the nonhuman
primate will be confirmed.
[0162] In certain embodiments, the nonhuman primates will serve as
a model for a disease or condition that could be treated by an
NKG2A-modulating compound. For example, models of autoimmune
disorders, allergies, cancers, or infectious diseases can be used,
e.g. to assess the ability of the antibodies to treat or alleviate
the symptoms of the diseases or conditions. While in no way
limiting for the practice of the present invention, certain
nonhuman primates are particularly useful for studying particular
types of diseases or conditions. For example, marmosets have served
as model animals for the study of immunity and of cardiovascular
diseases, saimiri for the study of infectious diseases, macaques
(including rhesus monkeys) for the study of pharmacology and
toxicology of specific compounds, and baboons as a model for
surgical studies, transplants, and biomaterials.
[0163] In one embodiment, anti-NKG2A antibodies are administered to
a nonhuman primate to assess the efficacy of the antibodies in
binding to and/or modulating NKG2A activity. In such embodiments,
the antibodies can be administered in any dose, frequency, or
formulation, and indeed such factors can be varied to assess their
relative influence over the efficacy. Efficacy of the antibodies
can be assessed in any of a large variety of ways. For example, one
can assess the in vivo binding of the antibodies to NKG2A or to
NKG2A-expressing cells, the in vivo effect of the antibodies on the
expression of NKG2A on cells, e.g. NK cells, or the in vivo
influence of the antibodies on the activity of NKG2A, e.g. as
measured using any of the herein-described assays for NK cell
activity. In such embodiments, an antibody is typically
administered to a nonhuman primate and its effects detected, e.g.,
on biological samples obtained from the nonhuman primate.
Alternatively, certain methods can be carried out in vitro, where
the effects of the antibodies on, e.g., NKG2A-expressing cells
obtained from a nonhuman primate are examined.
[0164] To assess the binding of the anti-human NKG2A antibodies,
the antibodies can be directly or indirectly labeled. For example,
the antibody can be labeled with a radioisotope prior to
administration, and its localization within the animal assessed by
examining various biological samples (e.g., blood, various tissues
or organs, immune-related tissues such as bone marrow, spleen,
lymphatic system components, or others) obtained at different times
after administration. In one preferred embodiment, PBLs are
obtained, and the binding of the antibodies to NK cells is
determined using, e.g., fluorescently labeled secondary antibodies,
with bound antibodies detected, e.g., by FACS analysis.
[0165] Similarly, antibodies can be administered to a nonhuman
primate and their effect on NKG2A activity assessed. For example,
NK cells can be obtained prior and subsequent to administration of
an anti-NKG2A antibody, and the activity, expression of NKG2A,
and/or number of the two (or more) sets of cells assessed using any
standard method. Activating antibodies of this invention that block
NKG2A stimulation (and thereby block inhibition of NK cells through
the receptor) would be expected to increase NK cell activity.
Inhibitory antibodies of this invention that cross-link NKG2A
receptors would be expected to decrease NK cell activity and
decrease the number of viable NK cells. Both types of antibodies
that cause altered NK cell activity in the nonhuman primate would
be considered suitable for use in treating disorders in humans
where an increase or a decrease in NK cell activity is
desirable.
[0166] In another set of embodiments, anti-NKG2A antibodies are
administered to a nonhuman primate in order to assess the safety of
the antibodies, as well as their various pharmacokinetic and
pharmacodynamic properties. Safety can be assessed in any of a
large variety of ways. For example, the overall toxicity of the
antibodies can be assessed, by determining the median lethal dose
(LD50), typically expressed as milligram per kilogram (mg/kg), in
which the value 50 refers to the percentage death among the animals
under study. In addition to determining the LD50, safety can also
be assessed by monitoring the animals for any detectable responses
to the administration, including behavioral, physical, or
physiological changes as evidenced by heart rate, blood pressure,
etc. Responses can also involve blood and other laboratory based
tests to examine markers indicative of organ function, such as
creatine or BUN for renal function, prothrombin, bilirubin,
albumin, or various enzymes to determine hepatic function, or
others (see, e.g., The Merck Manual of Diagnosis and Therapy,
17.sup.th edition, herein incorporated by reference).
[0167] Methods for in vivo pharmacokinetic and pharmacodynamic
assessment of the antibodies are standard and well known in the art
(see, e.g., He et al. (1998) J. Immunol. 160:1029-1035; Alyanakian
et al. (2003) Vox Sanguinis 84:188-192, Sharma et al. (2000) WET
293:33-41, the entire disclosures of which are herein incorporated
by reference). Such assays would typically involve administering
anti-NKG2A antibodies to a nonhuman primate and, at various times
after administration, examining the level (in plasma and other
tissues), distribution, binding, stability, and other properties of
the antibodies. Such assays are critical components of pre-clinical
studies and, by determining the in vivo half life, distribution,
bioavailability, etc. of the antibodies, help determine the
therapeutic window and thus proper administration regimens (e.g.
frequency and dose of administration) that will allow optimal
targeting of NK expressing cells by the administered
antibodies.
[0168] In conjunction with studies of the efficacy, safety,
pharmacodynamics and pharmacokinetics of anti-NKG2A antibodies, a
variety of formulations and administration regimens can also be
systematically tested to obtain optimal efficacy and safety for
anti-human NKG2A antibodies. For example, the therapeutic window
(the range of plasma concentrations of the antibodies that have a
high probability of therapeutic success) can be determined, as well
as those regimens and formulations that are optimally safe and
effective in targeting NKG2A and modulating NK cell activity in
vivo. For example, a given antibody can be administered every 1, 2,
3, 4, 5, or 6 days, or every 1, 2, 3, or 4 weeks, etc., and the
safety, efficacy, kinetic, etc. parameters examined. Similarly, the
dose of the antibody administered at any one time can be varied and
the same parameters examined, or any combination of dose and
frequency of administration can be tested. Further, different
formulations, e.g., compositions including different excipients,
different combinations of anti-NKG2A antibodies, or different
combinations of NKG2A antibodies with other therapeutic agents
(depending on the condition that would be treated, e.g. a
chemotherapeutic agent to treat cancer) can be tested in nonhuman
primates. Also, different routes of administration, e.g.
intravenous, pulmonary, topical, etc., can be compared. Such
methods of varying administration parameters are well known to
those of skill in the art.
Pharmaceutical Compositions
[0169] The invention also provides compositions, e.g.,
pharmaceutical compositions, that comprise any of the present
antibodies, including fragments and derivatives thereof, in any
suitable vehicle in an effective amount and a pharmaceutically
acceptable carrier.
[0170] 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
and wool fat.
[0171] The compositions of the present invention may be
administered orally, parenterally, by inhalation spray, topically,
rectally, nasally, buccally, vaginally or via an implanted
reservoir. The term "parenteral" as used herein includes
subcutaneous, intravenous, intramuscular, intra-articular,
intra-synovial, intrasternal, intrathecal, intrahepatic,
intralesional and intracranial injection or infusion techniques.
For localized disorders such as RA, the compositions will often be
administered topically, e.g., in inflamed joints.
[0172] Sterile injectable forms of the compositions of this
invention 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.
[0173] The compositions of this invention may be orally
administered in any orally acceptable dosage form including, but
not limited to, capsules, tablets, aqueous suspensions or
solutions. In the case of tablets for oral use, carriers commonly
used include lactose and corn starch. Lubricating agents, such as
magnesium stearate, are also typically added. For oral
administration in a capsule form, useful diluents include lactose
and dried cornstarch. When aqueous suspensions are required for
oral use, the active ingredient is combined with emulsifying and
suspending agents. If desired, certain sweetening, flavoring or
coloring agents may also be added.
[0174] Alternatively, the compositions of this invention may be
administered in the form of suppositories for rectal
administration. These can be prepared by mixing the agent with a
suitable non-irritating excipient that is solid at room temperature
but liquid at rectal temperature and therefore will melt in the
rectum to release the drug. Such materials include cocoa butter,
beeswax and polyethylene glycols. Such compositions are prepared
according to techniques well-known in the art of pharmaceutical
formulation.
[0175] The compositions of this invention may be administered
topically, especially when the target of treatment includes areas
or organs readily accessible by topical application, including
diseases of the eye, the skin, the joints, or the lower intestinal
tract. Suitable topical formulations are readily prepared for each
of these areas or organs. Topical application for the lower
intestinal tract can be effected in a rectal suppository
formulation (see above) or in a suitable enema formulation.
Topically-transdermal patches may also be used.
[0176] For topical applications, the compositions may be formulated
in a suitable ointment containing the active component suspended or
dissolved in one or more carriers. Carriers for topical
administration of the compounds of this invention include, but are
not limited to, mineral oil, liquid petrolatum, white petrolatum,
propylene glycol, polyoxyethylene, polyoxypropylene compound,
emulsifying wax and water. Alternatively, the compositions can be
formulated in a suitable lotion or cream containing the active
components suspended or dissolved in one or more pharmaceutically
acceptable carriers. Suitable carriers include, but are not limited
to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl
esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and
water.
[0177] For ophthalmic use, the compositions may be formulated as
micronized suspensions in isotonic, pH adjusted sterile saline, or,
preferably, as solutions in isotonic, pH adjusted sterile saline,
either with or without a preservative such as benzylalkonium
chloride. Alternatively, for ophthalmic uses, the compositions may
be formulated in an ointment such as petrolatum.
[0178] The compositions of this invention may also be administered
by nasal aerosol or inhalation. Such compositions are prepared
according to techniques well-known in the art of pharmaceutical
formulation and may be prepared as solutions in saline, employing
benzyl alcohol or other suitable preservatives, absorption
promoters to enhance bioavailability, fluorocarbons, and/or other
conventional solubilizing or dispersing agents.
[0179] In one embodiment, the antibodies or therapeutic compounds
of this invention may be incorporated into liposomes
("immunoliposomes" in the case of antibodies), alone or together
with another substance for targeted delivery to a patient or an
animal. Such other substances can include nucleic acids for the
delivery of genes for gene therapy or for the delivery of antisense
RNA, RNAi or siRNA for activating NK cells or inhibiting mature
dendritic cells, or toxins or drugs for the activation of NK cells
(or inhibition of dendritic cells) through other means, or any
other agent described herein that may be useful for the purposes of
the present invention.
[0180] In another embodiment, the antibodies or other compounds of
the invention can be modified to improve its bioavailability, half
life in vivo, etc. For example, antibodies and other compounds can
be pegylated, using any of the number of forms of polyethylene
glycol and methods of attachment known in the art (see, e.g., Lee
et al. (2003) Bioconjug Chem. 14(3):546-53; Harris et al. (2003)
Nat Rev Drug Discov. 2(3):214-21; Deckert et al. (2000) Int J
Cancer. 87(3):382-90).
Determining Dosage and Frequency of Administration
[0181] As described above, an important part of the present
invention is testing anti-NKG2A antibodies in nonhuman primates to
determine safe and effective doses and frequencies of
administration. Suitable starting administration regimens can be
determined by examining experience with other already developed
therapeutic monoclonal antibodies. Several monoclonal antibodies
have been shown to be efficient in clinical situations, such as
Rituxan (Rituximab), Herceptin (Trastuzumab) Xolair (Omalizumab),
Bexxar (Tositumomab), Campath (Alemtuzumab), Zevalin, Oncolym and
similar administration regimens (i.e., formulations and/or doses
and/or administration protocols) may be used with the antibodies of
this invention. Schedules and dosages for administration can be
determined in accordance with known methods for these products, for
example using the manufacturers' instructions. For example, a
monoclonal antibody can be supplied at a concentration of 10 mg/mL
in either 100 mg (10 mL) or 500 mg (50 mL) single-use vials. The
product is formulated for IV administration in 9.0 mg/mL sodium
chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7 mg/mL
polysorbate 80, and Sterile Water for Injection. The pH is adjusted
to 6.5. An exemplary suitable dosage range for an antibody of the
invention may be between about 10 mg/m2 and 500 mg/m2. However, it
will be appreciated that these schedules are exemplary and that
optimal schedule and regimen can be adapted taking into account the
affinity and anti-NKG2A activity of the antibody and the
tolerability of the antibodies that must be determined in clinical
trials. Quantities and schedule of injection of antibodies to
NKG2As that saturate cells for 24 hours, 48 hours, 72 hours or a
week or a month will be determined considering the affinity of the
antibody and its pharmacokinetic parameters.
[0182] However, it will be appreciated that these schedules are
exemplary and that optimal schedule and regimen can be adapted
taking into account the affinity and anti-NKG2A activity of the
antibody and the tolerability of the antibodies that must be
determined in clinical or preclinical trials. Quantities and
schedule of injection of antibodies to NKG2As that saturate cells
for 24 hours, 48 hours 72 hours or a week or a month will be
determined considering the affinity of the antibody and the its
pharmacokinetic parameters.
[0183] The dose administered to a patient or nonhuman primate in
the present methods should be sufficient to effect a beneficial
response in the subject over time. The dose will be determined by
the efficacy of the particular modulators employed and the
condition of the subject, as well as the body weight or surface
area of the area to be treated. The size of the dose also will be
determined by the existence, nature, and extent of any adverse
side-effects that may accompany administration in a particular
subject. In determining the effective amount of the compound to be
administered in a particular patient, a physician may evaluate
circulating plasma levels of the compound, compound toxicities, and
the production of anti-compound antibodies. In general, the dose
equivalent of a compound is from about 1 ng/kg to 10 mg/kg for a
typical subject. Administration can be accomplished via single or
divided doses.
[0184] The antibodies of the invention that bind both human and
non-human primate NKG2A receptors can be advantageously used in
determining dosage and frequency of administration. The selection
of an optimal therapeutic window for therapy with an anti-NKG2A
antibody can be carried out based on administration of the antibody
to a non-human primate. While NK cell activation in the short term
(24 hour co-culture) has been suggested to avoid bone marrow cell
(BMC) toxicity, it has been shown that longer (48 hour co-culture
of bone marrow cells with activated NK cells) adversely affects
hematopoietic reconstitution (Koh et al. (2002) Biol. Blood Marrow
Transplant. 8:17-25). However, it would be valuable to employ
administration regimens that permit exposure of NK cells in an
individual to an NK cell activation anti-NKG2A antibody for a
longer period, e.g. longer than 24 hours or even 48 hours. While
not wishing to be bound by theory such a regimen where an
anti-NKG2A antibody is present for greater than 24 hours or 48
hours would enable the anti-NKG2A antibody to come into contact
with and activate a sufficient number of NK cells in the individual
for a therapeutic effect against target (e.g. cancer, infected,
inflammatory) cells. The inventors therefore provide a method of
treating an individual with an anti-NKG2A antibody comprising
exposing said individual to an anti-NKG2A antibody for a period for
a period greater than 24 hours, more preferably 48 hours. Most
preferably the invention comprises administering to said individual
an anti-NKG2A antibody having a plasma half-life greater than 24
hours, or 48 hours, or more preferably of at least 5, 6, 7, 10, 14
or 20 days. Most preferably the invention comprises administering
to said individual an anti-NKG2A antibody comprising an Fc portion,
preferably an Fc portion of the G2 or G4 type. As further discussed
herein, any suitable antibody that blocks NKG2A function can be
used, for example an antibody having the binding specificity of
Z199 or Z270. In preferred embodiments the antibody is administered
in a second or further dose and the antibody will be a chimeric,
CDR grafted, human or humanized antibody.
[0185] The present invention provides a method of identifying a
suitable administration regimen for a therapeutic antibody directed
against human NKG2A, the method comprising administering the
antibody to a nonhuman primate using an administration regimen,
preferably a series of regimens in which the dose or frequency of
the antibody is varied, and determining the activity of
NKG2A-expressing cells in the non-human primate and the effect of
therapy on bone marrow cells (BMC) and/or hematopoietic cells,
particularly myeloid cell reconstitution, of the primate for the
particular administration regimen(s). Preferably the method further
comprises assessing myeloid reconstitution following anti-NKG2A
antibody administration, generally involving determining the number
of days required for myeloid reconstitution to normalize, e.g. to
levels approaching that observed prior to anti-NKG2A therapy or to
a predetermined minimum level. It is then possible to select or
identify an administration regimen that allows myeloid
reconstitution to normalize.
[0186] The method can further comprise determining the activity of
NKG2A-expressing cells in the non-human primate and/or identifying
or selecting an administration regimen that leads to a detectable
modulation in the activity of NKG2A-expressing cells.
[0187] Said administration regimen(s) can be expressed for example
in terms of period of exposure of an individual to an anti-NKG2A
antibody that activates an NK cell, and frequency of antibody
administration. Based on such parameters, administration frequency
and dosage can be adapted depending on the particular antibody
used, e.g. taking account of the antibody's plasma half-life,
affinity, bioavailability (or time to peak serum concentration),
etc.
[0188] A determination that a regimen permits partial or complete
recovery or normalization of myeloid reconstitution by the primate
and leads to a detectable modulation in the activity of
NKG2A-expressing cells indicates that the administration regimen is
suitable for use in humans.
[0189] The catabolic rates of the endogenous human immunoglobulins
have been well characterized. The half-life of IgG varies according
to isotype, up to 3 weeks for IgG1, IgG2, and IgG4 and
approximately 1 week for IgG3. Unless pharmacokinetics are altered
by antigen binding or immunogenicity, intact human IgG monoclonal
antibodies will exhibit pharmacokinetics comparable to endogenous
IgG. As discussed previously, the extraordinarily long half-life of
the human IgG1, IgG2, and IgG4 isotypes is due to catabolic
protection by FcRn. FcRn is expressed on hepatocytes, endothelial
cells, and phagocytic cells of the reticuloendothelial system
(RES). When IgG undergoes endocytosis, the low pH of the endosome
promotes binding of the IgG Fc domain to FcRn, which recycles IgG
to the cell surface and salvages IgG from lysosomal degradation.
The short half life of IgG3 compared to the other IgG isotypes is
due to a single amino acid difference (an arginine instead of a
histidine at position 435) in the FcRn binding domain.
[0190] The elimination of intact murine IgG1 and IgG2 antibodies is
much faster than the corresponding human isotypes. Half-lives for
murine antibodies are in the range of 12 to 48 hours in humans. The
short half-life of murine antibodies in humans is due to
low-affinity binding of the murine Fc domain to human FcRn. Human
FcRn binds to human, rabbit, and guinea pig IgG, but not
significantly to rat, bovine, sheep, or mouse IgG; mouse FcRn binds
to IgG from all of these species. Antibody fragments, including
F(a').sub.2, Fab, and scFV, lack the Fc domain and do not bind to
FcRn. Therefore, the half-lives of these fragments are
substantially shorter than intact IgG, with half-life determined
predominantly by their molecular weights. Lower molecular weight
Fab and scFv fragments are subject to renal clearance, which
accelerates elimination. Reported half-lives have ranged from 11 to
27 h for F(ab')2 fragments and 0.5 to 21 h for Fab fragments. The
half-life of monovalent and multivalent scFv constructs may range
from minutes to several hours.
[0191] Antigen binding can significantly affect the
pharmacokinetics of antibodies. If the antibody binds to an
internalized cell membrane antigen or an immune complex formed with
a secreted antigen is efficiently eliminated from circulation, the
antigen may act as a "sink" for antibody clearance. An antigen sink
will produce dose-dependent pharmacokinetics. If the dose level is
insufficient to saturate the antigen pool, antigen-mediated
clearance will predominate and the antibody half-life will be
shorter than the half-life of endogenous IgG; at dose levels that
saturate the antigen, RES-mediated clearance will predominate and
half-life will be similar to endogenous IgG.
[0192] A preferred embodiment of the present invention describes a
dosing regimen wherein anti-NKG2A antibody is administered in a
first administration. The first dose of anti-NKG2A antibody
activates NK cells and may indirectly by activating NK cells
inhibit myeloid cell reconstitution in the individual. The second
dose of anti-NKG2A antibody is administered to coincide with the
pharmacodynamic profile of myeloid cell reconstitution recovery,
e.g. to be administered at a time when an individual's rate of
myeloid cell reconstitution is expected to have at least partially
recovered. Thus, by using an anti-NKG2A antibody which cross-reacts
with the receptor in humans and non-human primates, the inventors
provide a method in which NK cells are brought into contact with an
anti-NKG2A antibody for a period greater than 24 hours during which
myeloid reconstitution has been reported to not be affected.
[0193] In preferred embodiments, the second dose of anti-NKG2A
antibody will be administered at least 6, 7, 8, 9 or 10 days
following the initial dose, and preferably at least 14, 15, 16, or
20 days following the initial dose. Most preferably the second dose
of anti-NKG2A antibody will be administered at least 2, 3, 4, 5, 6,
7, 8, 9 or 10 days or at least 14, 15, 16, or 20 days following the
time (day) at which anti-NKG2A antibody plasma concentration in a
subject is estimated to reach half of the initial (at
administration) concentration, preferably at least 6-10 days or at
least 15-20 days following the duration of at least one plasma
half-life of the anti-NKG2A antibody. Alternatively, the method can
be expressed in terms of peak serum concentration of the anti-NKG2A
antibody, where the second dose of anti-NKG2A antibody will be
administered at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 days or at least
14, 15, 16, or 20 days following the time (day) at which anti-NKG2A
antibody plasma concentration in a subject is estimated to reach
half of the peak serum concentration in the individual.
[0194] In a further embodiment, the second dose of anti-NKG2A
antibody will be administered at least 2, 3, 4, 5, 6, 7, 8, 9 or 10
days or at least 14, 15, 16, or 20 days following the time (day) at
which anti-NKG2A antibody plasma concentration in a subject is
estimated to reach a non-detectable concentration, preferably at
least 2, 3, 4, 5, 6, 7, 8, 9 or 10 days or at least 14, 15, 16, or
20 days following the duration of at least 2, 3, 4 or more plasma
half-lives of the anti-NKG2A antibody.
[0195] In a preferred embodiment, an administration regimen is
described for an antibody comprising an Fc region of the G2b or
preferably G4 subtype (IgG2b or IgG4 respectively). Preferably said
antibody has a plasma half-life of about 5, 6, 7, 8, 9, 10, 12, 15,
18, 20, or 21 days, or preferably of about 10 to 15 days, 15-21
days. Preferably the antibody comprises an Fc region substantially
free of binding to Fc receptors on NK cells (CD16). Said antibody
is preferably administered in a first dose, and a second and/or
subsequent dose, wherein the second and/or subsequent dose is
administered at least 6, 7, 8, 9, 10, 14, 15, 16, or 20 days after
the antibody is estimated to reach half its initial concentration.
Said second and/or subsequent dose can also be expressed in
absolute number of days following administration, e.g. preferably
at least 6, 7, 8, 9 or 10 days following the initial dose, and
preferably at least 14, 15, 16, 20, 21, 24, 28, 30 or 35 days
following the first administration. Said antibody may be an
antibody comprising a naturally occurring Fc portion, preferably a
naturally occurring human Fc portion, or more preferably may
contain modifications such as one or more amino acid substitutions
that increase the plasma half-life of the antibody and/or that
modify binding to Fc receptors, for example increase binding to Fcn
receptors to increase plasma half-life or decrease binding to
FcgammaIIIa to decrease unwanted toxicity (ADCC) towards the NK
cell. Such modifications can be carried out according to methods
well known in the art, several of which modifications are further
described herein.
[0196] In yet another preferred embodiment, an administration
regimen is described for an antibody fragment, preferably a F(ab')2
fragment modified, for example with polyethylene glycol as
described herein, to have a plasma half-life of about 5, 6, 7, 8,
9, 10, 12, 15, 18, 20, or 21 days. Said antibody is preferably
administered in a first dose, and a second and/or subsequent dose,
wherein the second and/or subsequent dose is administered at least
6, 7, 8, 9, 10, 14, 15, 16, or 20 days after the antibody is
estimated to reach half its initial concentration. Said second
and/or subsequent dose can also be expressed in absolute number of
days following administration, e.g. preferably at least 6, 7, 8, 9
or 10 days following the initial dose, and preferably at least 14,
15, 16, 20, 21, 24, 28, 30 or 35 days following the first
administration.
Pharmaceutical Combinations
[0197] According to another important embodiment of the present
invention, the anti-NKG2A antibodies and/or other compounds may be
formulated together with one or more additional therapeutic agents,
including agents normally utilized for the particular therapeutic
purpose for which the antibody or compound is being administered.
The additional therapeutic agent will generally be administered at
a dose typically used for that agent in a monotherapy for the
particular disease or condition being treated. Such therapeutic
agents include, but are not limited to, therapeutic agents used in
the treatment of cancers ("anticancer compounds"; including
chemotherapeutic compounds, hormones, angiogenesis inhibitors,
apoptotic agents, etc.); therapeutic agents used to treat
infectious disease (including antiviral compounds); therapeutic
agents used in other immunotherapies, such as the treatment of
autoimmune disease, inflammatory disorders, and transplant
rejection; cytokines; immunomodulatory agents; adjunct compounds;
or other antibodies and fragments of other antibodies against both
activating and inhibitory NK cell receptors. Unless otherwise
specifically stated, the combination compositions set forth below
can comprise either an activating antibody, an inhibitory antibody
or a cytotoxin-antibody conjugate of this invention.
[0198] Therapeutic agents for the treatment of cancer include
chemotherapeutic agents (including agents that interfere with DNA
replication, mitosis and chromosomal segregation, and agents that
disrupt the synthesis and fidelity of polynucleotide precursors),
hormonal therapy agents, anti-angiogenic agents, and agents that
induce apoptosis.
[0199] Chemotherapeutic agents contemplated as exemplary include,
but are not limited to, alkylating agents, antimetabolites,
cytotoxic antibiotics, vinca alkaloids, for example adriamycin,
dactinomycin, mitomycin, carminomycin, daunomycin, doxorubicin,
tamoxifen, taxol, taxotere, vincristine, vinblastine, vinorelbine,
etoposide (VP-16), 5-fluorouracil (5FU), cytosine arabinoside,
cyclophosphamide, thiotepa, methotrexate, camptothecin,
actinomycin-D, mitomycin C, cisplatin (CDDP), aminopterin,
combretastatin(s) and derivatives and prodrugs thereof.
[0200] Hormonal agents include, but are not limited to, for example
LHRH agonists such as leuprorelin, goserelin, triptorelin, and
buserelin; anti-estrogens such as tamoxifen and toremifene;
anti-androgens such as flutamide, nilutamide, cyproterone and
bicalutamide; aromatase inhibitors such as anastrozole, exemestane,
letrozole and fadrozole; and progestagens such as medroxy,
chlormadinone and megestrol.
[0201] A number of exemplary chemotherapeutic agents for combined
therapy are listed in Table C of U.S. Pat. No. 6,524,583, the
disclosure of which agents and indications are specifically
incorporated herein by reference. Each of the agents listed are
exemplary and not limiting. The skilled artisan is directed to
"Remington's Pharmaceutical Sciences" 15th Edition, chapter 33, in
particular pages 624-652. Variation in dosage will likely occur
depending on the condition being treated. The physician
administering treatment will be able to determine the appropriate
dose for the individual subject.
[0202] Examples of anti-angiogenic agents include neutralizing
antibodies, antisense RNA, siRNA, RNAi, RNA aptamers and ribozymes
each directed against VEGF or VEGF receptors (U.S. Pat. No.
6,524,583, the disclosure of which is incorporated herein by
reference).
[0203] Variants of VEGF with antagonistic properties may also be
employed, as described in WO 98/16551, specifically incorporated
herein by reference. Further exemplary anti-angiogenic agents that
are useful in connection with combined therapy are listed in Table
D of U.S. Pat. No. 6,524,583, the disclosure of which agents and
indications are specifically incorporated herein by reference.
[0204] Exemplary apoptotic agents include, but are not limited to,
bcr-abl, bcl-2 (distinct from bcl-1, cyclin D1; GenBank accession
numbers M14745, X06487; U.S. Pat. Nos. 5,650,491 and 5,539,094;
each incorporated herein by reference) and family members including
Bcl-x1, Mcl-1, Bak, A1, and A20. Overexpression of bcl-2 was first
discovered in T cell lymphomas. The oncogene bcl-2 functions by
binding and inactivating Bax, a protein in the apoptotic pathway.
Inhibition of bcl-2 function prevents inactivation of Bax, and
allows the apoptotic pathway to proceed. Inhibition of this class
of oncogenes, e.g., using antisense nucleotide sequences, RNAi,
siRNA or small molecule chemical compounds, is contemplated for use
in the present invention to give enhancement of apoptosis (U.S.
Pat. Nos. 5,650,491; 5,539,094; and 5,583,034; each incorporated
herein by reference).
[0205] Useful anti-viral agents that can be used in combination
with the molecules of the invention include, but are not limited
to, protease inhibitors, nucleoside reverse transcriptase
inhibitors, non-nucleoside reverse transcriptase inhibitors and
nucleoside analogs. Examples of antiviral agents include but are
not limited to zidovudine, acyclovir, gangcyclovir, vidarabine,
idoxuridine, trifluridine, and ribavirin, as well as foscarnet,
amantadine, rimantadine, saquinavir, indinavir, amprenavir,
lopinavir, ritonavir, the alpha-interferons, adefovir, clevadine,
entecavir, and pleconaril.
[0206] For autoimmune or inflammatory disorders, any other compound
known to be effective for one or more types of autoimmune or
inflammatory disorders, or any symptom or feature of autoimmune or
inflammatory disorders, including inter alia, immunosuppressants,
e.g., azathioprine (e.g., Imuran), chlorambucil (e.g., Leukeran),
cyclophosphamide (e.g., Cytoxan), cyclosporine (e.g., Sandimmune,
Neoral), methotrexate (e.g., Rheumatrex), corticosteroids,
prednisone (e.g., Deltasone, Meticorten), Etanercept (e.g.,
Enbrel), infliximab (e.g., Remicade), inhibitors of TNF, FK-506,
rapamycin, mycophenolate mofetil, leflunomide, anti-lymphocyte
globulin, deoxyspergualin or OKT.
[0207] Preferred examples of immunomodulatory compounds include
cytokines. Other examples include compounds that have an effect,
preferably an effect of activation or potentiation of NK cell
activity, or of inducing or supporting the proliferation of NK
cells. Examples of immunomodulating compounds include but are not
limited to ligands of NOD and PKR receptors, agonists of TLRs
(Toll-like receptors), such as agonists of TLR3 (dsRNA, poly I:C
and poly A:U), TLR4 (ANA380, isatoribine, LPS and mimetics such as
MPL), TLR7 (oligonucleotides, ssRNA), TLR9 (oligonucleotides such
as CpGs), a number of examples of which are described in Akira and
Takeda ((2004) Nature Reviews 4: 499), and antibodies that block
inhibitory receptors on NK cells (for example that inhibit KIR2DL1
and KIR2DL2/3 activity) or act as agonists at NK cell activatory
receptors (for example antibodies that crosslink NCR receptors
NKp30, NKp44 or NKp46). Various cytokines may be employed in
combined approaches according to the invention. Examples of
cytokines useful in the combinations contemplated by this invention
include IL-1alpha IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,
IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-21, TGF-beta,
GM-CSF, M-CSF, G-CSF, TNF-alpha, TNF-beta, LAF, TCGF, BCGF, TRF,
BAF, BDG, MP, LIF, OSM, TMF, PDGF, IFN-alpha, IFN-beta, IFN-gamma.
Cytokines used in the combination treatment or compositions of this
invention are administered according to standard regimens,
consistent with clinical indications such as the condition of the
patient and relative toxicity of the cytokine.
[0208] Adjunct compounds may include by way of example anti-emetics
such as serotonin antagonists and therapies such as phenothiazines,
substituted benzamides, antihistamines, butyrophenones,
corticosteroids, benzodiazepines and cannabinoids; bisphosphonates
such as zoledronic acid and pamidronic acid; and hematopoietic
growth factors such as erythropoietin and G-CSF, for example
filgrastim, lenograstim and darbepoietin.
[0209] Other therapeutic agents that can be formulated with the
activating anti-NKG2A antibodies of this invention include other
compounds that can activate NK cells. For example, compounds that
stimulate NCRs, e.g. NKp30, NKp44, and NKp46, can be used (see,
e.g., PCT WO 01/36630, Vitale et al. (1998) J. Exp. Med.
187:2065-2072, Sivori et al. (1997) J. Exp. Med. 186:1129-1136;
Pessino et al. (1998) J. Exp Med. 188:953-960; the entire
disclosures of which are herein incorporated by reference), as can
inhibitors of the KIR inhibitory receptors (see, e.g., Yawata et
al. (2002) Crit Rev Immunol 22:463-82; Martin et al. (2000)
Immunogenetics. 51:268-80; Lanier (1998) Annu Rev Immunol.
16:359-93; the entire disclosures of which are herein incorporated
by reference). Preferably, an activator, e.g. natural ligand or
activating antibody, of NKp30 is used. In one embodiment, an
inhibitor of TGF-beta 1 is used, as TGF-beta 1 can downregulate
NKp30 (see, e.g., Castriconi et al. (2004) C.R. Biologies
327:533-537, the entire disclosure of which is herein incorporated
in its entirety).
[0210] Therapeutic compounds that can be formulated with the
inhibitory anti-NKG2A antibodies of this invention are compounds
that can inhibit NK cells. Such compounds include inhibitors of
NCRs, e.g. NKp30, NKp44, and NKp46, inhibitors of activating NKG2
receptors (e.g., NKG2C), activators of inhibitory KIR receptors, or
activators of an inhibitory Ly49 receptor.
[0211] The activating antibodies of this invention may also be
formulated together with an antigen to which tolerance is desired.
It is believed that the enhanced killing of dendritic cells caused
by the activating antibodies of this invention will cause
tolerization of antigens presented to the immune system at that
time. Such compositions are useful in treating autoimmune disease,
as well as allergies. Examples of antigens that may be formulated
with the activating antibodies of this invention include myelin
basic protein, ragweed and other pollen and plant allergens,
allergens responsible for pet allergies, allergens responsible for
food allergies (such as peanut and other nut allergens, dairy
product allergens, sesame and other seed allergens) or insect
allergens.
[0212] The interrelationship of dosages for animals and humans
(based on milligrams per meter squared of body surface) is
described in Freireich et al., (1966) Cancer Chemother Rep 50: 219.
Body surface area may be approximately determined from height and
weight of the patient. See, e.g., Scientific Tables, Geigy
Pharmaceuticals, Ardley, N.Y., 1970, 537. An effective amount of a
compound of this invention can range from about 0.001 mg/kg to
about 1000 mg/kg, more preferably 0.01 mg/kg to about 100 mg/kg,
more preferably 0.1 mg/kg to about 10 mg/kg; or any range in which
the low end of the range is any amount between 0.001 mg/kg and 900
mg/kg and the upper end of the range is any amount between 0.1
mg/kg and 1000 mg/kg (e.g., 0.005 mg/kg and 200 mg/kg, 0.5 mg/kg
and 20 mg/kg). Effective doses will also vary, as recognized by
those skilled in the art, depending on the diseases treated, route
of administration, excipient usage, and the possibility of co-usage
with other therapeutic treatments such as use of other agents.
[0213] For pharmaceutical compositions that comprise additional
therapeutic agents, an effective amount of the additional
therapeutic agent is between about 20% and 100% of the dosage
normally utilized in a monotherapy regime using just that
additional agent. Preferably, an effective amount is between about
70% and 100% of the normal monotherapeutic dose. The normal
monotherapeutic dosages of these additional therapeutic agents are
well known in the art. See, e.g., Wells et al., eds.,
Pharmacotherapy Handbook, 2nd supp. Edition, Appleton and Lange,
Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket
Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma
Linda, Calif. (2000), each of which are entirely incorporated
herein by reference.
[0214] It is expected that some of the additional therapeutic
agents listed above will act synergistically with the compounds of
this invention. When this occurs, its will allow the effective
dosage of the additional therapeutic agent and/or the compound of
this invention to be reduced from that required in a monotherapy.
This has the advantage of minimizing toxic side effects of either
the additional therapeutic agent of a compound of this invention,
synergistic improvements in efficacy, improved ease of
administration or use and/or reduced overall expense of compound
preparation or formulation.
[0215] It will be recognized by those of skill in the art that
certain therapeutic agents set forth above fall into two or more of
the categories disclosed above. For the purpose of this invention,
such therapeutic agents are to be considered members of each of
those categories of therapeutics and the characterization of any
therapeutic agent as being in a certain specified category does not
preclude it from also being considered to be within another
specified category.
[0216] In yet another embodiment, the invention provides a
composition of matter comprising an antibody of this invention and
a second therapeutic agent or an allergen, selected from any of the
agents or allergens set forth above, wherein the antibody and the
second agent are in separate dosage forms, but associated with one
another. The term "associated with one another" as used herein
means that the separate dosage forms are packaged together or
otherwise attached to one another such that it is readily apparent
that the separate dosage forms are intended to be sold and
administered as part of the same regimen. The agent and the
antibody are preferably packaged together in a blister pack or
other multi-chamber package, or as connected, separately sealed
containers (such as foil pouches or the like) that can be separated
by the user (e.g., by tearing on score lines between the two
containers).
In still another embodiment, the invention provides a kit
comprising in separate vessels, a) an antibody of this invention;
and b) a second therapeutic agent or an allergen. Again, any of the
therapeutic agents or allergens set forth above may be present in
such a kit.
Therapeutic Use of Anti-NKG2A Antibodies and Compositions
[0217] The activating antibodies of the present invention render NK
cells capable of lysing target cells bearing HLA-E or Qa1.sup.b on
their cell surfaces when the NK cell comes into contact with the
target cell. Thus, according to one embodiment, the invention
provides a method of reconstituting NK cell-mediated lysis of a
target cell in a population comprising a NK cell and said target
cell, wherein said NK cell is characterized by NKG2A on its
surface, and said target cell is characterized by the presence of
HLA-E or Qa1.sup.b on its surface, said method comprising the step
of contacting said NK cell with an above-described activating
monoclonal antibody or a fragment thereof.
[0218] This activity is particularly useful in the treatment of
conditions and disorders characterized by deleterious cells
expressing HLA-E or Qa1.sup.b on their cell surface. One such cell
type is a dendritic cell, preferably a mature dendritic cell. Thus,
the invention provides a method of treating an autoimmune or
inflammatory disorder or any other disorder caused at least in part
by an excess of dendritic cells, or hyperactive dendritic cell
activity. The method of treating such disorders comprises the step
of administering to a patient a non-cytotoxic composition of the
present invention that comprises an activating antibody.
[0219] Exemplary autoimmune disorders treatable using the present
methods include, inter alia, hemolytic anemia, pernicious anemia,
polyarteritis nodosa, systemic lupus erythematosus, Wegener's
granulomatosis, autoimmune hepatitis, Behcet's disease, Crohn's
disease, primary bilary cirrhosis, scleroderma, ulcerative colitis,
Sjogren's syndrome, Type 1 diabetes mellitus, uveitis, Graves'
disease, Alzheimer's disease, thyroiditis, myocarditis, rheumatic
fever, ankylosing spondylitis, rheumatoid arthritis,
glomerulonephritis, sarcoidosis, dermatomyositis, myasthenia
gravis, polymyositis, Guillain-Barre syndrome, multiple sclerosis,
alopecia areata, pemphigus/pemphigoid, Bullous pemphigoid,
Hashimoto's thyroiditis, psoriasis, and vitiligo.
[0220] Examples of inflammatory disorders that can be treated by
these methods include, but are not limited to, adrenalitis,
alveolitis, angiocholecystitis, appendicitis, balanitis,
blepharitis, bronchitis, bursitis, carditis, cellulitis,
cervicitis, cholecystitis, chorditis, cochlitis, colitis,
conjunctivitis, cystitis, dermatitis, diverticulitis, encephalitis,
endocarditis, esophagitis, eustachitis, fibrositis, folliculitis,
gastritis, gastroenteritis, gingivitis, glossitis, hepatosplenitis,
keratitis, labyrinthitis, laryngitis, lymphangitis, mastitis, media
otitis, meningitis, metritis, mucitis, myocarditis, myosititis,
myringitis, nephritis, neuritis, orchitis, osteochondritis, otitis,
pericarditis, peritendonitis, peritonitis, pharyngitis, phlebitis,
poliomyelitis, prostatitis, pulpitis, retinitis, rhinitis,
salpingitis, scleritis, selerochoroiditis, scrotitis, sinusitis,
spondylitis, steatitis, stomatitis, synovitis, syringitis,
tendonitis, tonsillitis, urethritis, and vaginitis.
[0221] It has also been shown that alloreactive NK cell killing of
dendritic cells improved engraftment of hematopoietic cells in a
bone marrow transplant (L. Ruggeri et al., Science, 2002,
295:2097-2100). Thus, in another embodiment, the invention provides
a method of improving the engraftment of hematopoietic cells in a
patient comprising the step of administering to said patient a
composition of this invention comprising an activating antibody.
Improvement in grafting is manifested by any one of reduced
incidence or severity of graft versus host disease, prolonged
survival of the graft, or a reduction in or elimination of the
symptoms of the disease being treated by the graft (e.g., a
hematopoietic cancer). This method is preferably used in the
treatment of leukemia.
[0222] Cancer cells have also been shown to evade killing through
the presence of HLA-E on their surface. HLA-E has been detected on
surgically removed glioblastoma specimens, in glioma cell lines and
glioblastoma cell cultures (J. Wischhusen et al., J Neuropathol Exp
Neurol. 2005; 64(6):523-8); and in leukemia-derived cell lines,
melanomas, melanoma-derived cell lines and cervical tumors (R Marin
et al., Immunogenetics. 2003; 54(11):767-75). Thus, in another
embodiment, the invention provides a method of treating a patient
suffering from cancer, wherein said cancer is characterized by a
cell expressing HLA-E, said method comprising the step of
administering to said patient a composition of the present
invention comprising an activating antibody.
[0223] Examples of cancers that may be treated according to this
method include, but are not limited to, carcinoma, including that
of the bladder, breast, colon, kidney, liver, lung, ovary,
prostate, pancreas, stomach, cervix, thyroid and skin, including
squamous cell carcinoma; hematopoietic tumors of lymphoid lineage,
including leukemia, acute lymphocytic leukemia, acute lymphoblastic
leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkins lymphoma,
non-Hodgkins lymphoma, hairy cell lymphoma and Burketts lymphoma;
hematopoietic tumors of myeloid lineage, including acute and
chronic myelogenous leukemias and promyelocytic leukemia; tumors of
mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma;
other tumors, including melanoma, seminoma, teratocarcinoma,
neuroblastoma and glioma; tumors of the central and peripheral
nervous system, including astrocytoma, neuroblastoma, glioma, and
schwannomas; tumors of mesenchymal origin, including fibrosarcoma,
rhabdomyoscaroma, and osteosarcoma; and other tumors, including
melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid
follicular cancer and teratocarcinoma.
[0224] Preferred cancers that can be treated according to the
invention include gliomas, glioblastomas, leukemias, melanomas, and
cervical tumors.
[0225] Virally infected cells also use HLA-E expression as a
mechanism of avoiding NK cell killing. HLA-E expression has been
associated with hepatitis C virus infected cells (J. Mattermann et
al, American Journal of Pathology. 2005; 166:443-453); and
cytomegalovirus infected cells (C. Cerboni et al., Eur J Immunol.
2001; 31(10):2926-35). Thus, in another embodiment, the invention
provides a method of treating a patient suffering from a viral
infection, wherein said viral infection is characterized by a
virally-infected cell expressing HLA-E, said method comprising the
step of administering to said patient a composition of the present
invention comprising an activating antibody.
Examples of viral infections that may be treated by this method
include, but are not limited to, infections caused by viruses of
the family Retroviridae (e.g., human immunodeficiency viruses, such
as HIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV, or
HIV-III; and other isolates, such as HIV-LP)); Picornaviridae
(e.g., polio viruses, hepatitis A virus, enteroviruses, human
Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g.,
strains that cause gastroenteritis); Togaviridae (e.g., equine
encephalitis viruses, rubella viruses); Flaviviridae (e.g., dengue
viruses, encephalitis viruses, yellow fever viruses); Coronaviridae
(e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis
viruses, rabies viruses); Filoviridae (e.g., ebola viruses);
Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measles
virus, respiratory syncytial virus); Orthomyxoviridae (e.g.,
influenza viruses) or avian influenza viruses (e.g. H5N1 or related
viruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses,
phleboviruses and Nairo viruses); Arenaviridae (hemorrhagic fever
viruses); Reoviridae (e.g., reoviruses, orbiviruses and
rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus);
Parvoviridae (parvoviruses); Papovaviridae (papillomaviruses,
polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae
(herpes simplex virus (HSV) 1 and 2, varicella zoster virus,
cytomegalovirus (CMV)); Poxviridae (variola viruses, vaccinia
viruses, pox viruses); Iridoviridae (e.g., African swine fever
virus); and unclassified viruses (e.g., the etiological agents of
spongiform encephalopathies, the agent of delta hepatitis (thought
to be a defective satellite of hepatitis B virus), the agents of
non-A, non-B hepatitis (class 1=internally transmitted; class
2=parenterally transmitted (i.e., Hepatitis C); Norwalk and related
viruses, and astroviruses).
[0226] Most preferably, the viral infection to be treated is
selected from a hepatitis C virus infection or a cytomegalovirus
infection.
[0227] The activating antibodies of this invention can also be used
to induce tolerance to an antigen. Thus, according to another
embodiment, the invention provides a method of inducing tolerance
to an antigen in a patient comprising the steps of administering to
said patient a composition of this invention comprising an
activating antibody; and administering to said patient an antigen
to which tolerance is desired. The method is preferably used to
treat an allergy, wherein the antigen is an allergen. The choice of
antigen can be made from those set forth above for combination
compositions comprising an activating antibody of this invention
and an antigen.
[0228] Compositions comprising the inhibitory antibodies of this
invention or cytotoxin-antibody conjugates are useful for killing
NK cells, reducing the activity of NK cells, reducing proliferation
of NK cells, preventing the lysis of cells susceptible to NK cell
lysis, or reducing the number of NK cells in a population.
According to one embodiment, the invention provides a method of
reducing the activity of NK cells, reducing proliferation of NK
cells, preventing the lysis of cells susceptible to NK cell lysis,
or reducing the number of NK cells in a population comprising the
step of contacting a NK cell with a composition of this invention
comprising an inhibitory antibody or a cytotoxin-antibody
conjugate. These methods are particularly useful in diseases
characterized by NK hyperactivity and/or hyperproliferation.
[0229] For example, co-owned PCT publication WO2005/105849
generally describes the use of antibodies against various NK cell
receptors for the treatment of NK-Type LDGL. PCT publication WO
2005/115517 discloses that NK cell hyperactivity is associated with
the presence, progression, stage and/or aggressiveness of
pancreatic islet autoimmunity and thus plays a role in Type-I
diabetes. Thus, according to one embodiment, the invention provides
a method of treating a patient suffering from a condition
characterized by NK cell hyperactivity or NK cell
hyperproliferation comprising the step of administering to said
patient a composition according to this invention comprising an
inhibitory antibody or a cytotoxin-antibody conjugate. In a
preferred embodiment, the condition is selected from NK-Type LDGL
or Type I diabetes.
[0230] Any of the therapeutic methods described above may comprise
the additional step of administering to the patient a second
therapeutic agent suitable for the condition being treated.
Examples of the types of second therapeutic agents that may be
administered to the patient include a cytokine, a cytokine
inhibitor, a hematopoietic growth factor, insulin, an
anti-inflammatory agent, an immunosuppressant, an anticancer
compound (such as a chemotherapeutic compound, an anti-angiogenic
compound, an apoptosis-promoting compound, a hormonal agent, a
compound that interferes with DNA replication, mitosis and/or
chromosomal segregation, or an agent that disrupts the synthesis
and fidelity of polynucleotide precursors), an adjunct compound
(such as a pain reliever or an antiemetic), a compound that
agonizes an activating NK cell receptor (such as NKp30, NKp44, and
NKp46), an antagonist of an inhibitory NK cell receptor (such as an
inhibitory KIR receptor), an antagonist of TGF-beta 1, a compound
capable of stimulating an inhibitory NK cell receptor (such as
natural ligands, antibodies or small molecules that can stimulate
the activity of CD94/NKG2A receptors, or an inhibitory KIR receptor
such as KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, and KIR3DL2), or an
inhibitor of an activating NK cell receptor (such as NKp30, NKp44,
or NKp46).
[0231] Specific examples of the above-described classes of
compounds are set forth in the section on pharmaceutical
combinations and any of such specific compounds, as well as other
members of any of these classes of therapeutic agents may be
administered to a patient in the methods of this invention. The
choice of therapeutic agent to use is easily made by those of skill
in the medical arts and is dependent upon the nature of the
condition being treated or prevented, the severity of the
condition, the general overall health of the patient being treated,
and the judgment of the treating physician.
[0232] The second therapeutic agent may be administered
simultaneously with, prior to, or following the anti-NKG2A
composition of this invention. When administered simultaneously,
the second therapeutic agent may be administered as either a
separately formulated composition (i.e., as a multiple dosage
form), or as part of the antibody-containing composition.
[0233] In some embodiments, prior to the administration of a NKG2A
antibody composition of this invention, the expression of NKG2A,
and possibly other proteins, on NK cells will be assessed, and/or
the activity or number of dendritic cells (preferably mature
dendritic cells) and/or the presence of a NKG2A ligand (e.g., HLA-E
or Qa1.sup.b) on other cells, will be measured. This can be
accomplished by obtaining a sample of NK or dendritic cells from
the patient, and, for NK cells, testing e.g., using immunoassays,
to determine the relative prominence of markers such as KIR
receptors, other NKG2A receptors, or NCRs (e.g., NKp30, NKp44,
NKp46), on the cells. Other methods can also be used to detect
expression of these proteins, such as RNA-based methods, e.g.,
RT-PCR or Northern blotting. The detection of NK cells expressing
NKG2A in the patient indicates that the present methods are well
suited for use in treating the patient.
[0234] The treatment may involve multiple rounds of antibody. For
example, following an initial round of administration, the level
and/or activity of NKG2A-expressing NK cells, and/or dendritic
cells or other cells expressing NKG2A or HLA-E, or Qa1.sup.b on
their surface, can be re-measured, and, if appropriate, an
additional round of administration can be performed. In this way,
multiple rounds of receptor/cell/ligand detection and antibody
composition administration can be performed, e.g., until the
disorder is brought under control.
[0235] It will also be appreciated that more than one antibody can
be produced and/or used using the present methods. For example,
combinations of antibodies directed against different epitopes of
NKG2A, against different combinations of NKG2A, CD94, or HLA-E, or
against different isoforms of any of the three proteins that may
exist in any individual may be used, as appropriate to obtain the
ideal level of inhibition of NKG2A stimulation or inhibition of NK
cell activity, either generally or in any individual patient (e.g.,
following an analysis of the NKG2A-expressing cells in the patient
to determine an appropriate treatment regimen).
[0236] So long as a particular therapeutic approach is not known to
be detrimental to the patient's condition in itself, and does not
significantly counteract the NKG2A antibody-treatment, its
combination with the present invention is contemplated.
[0237] The present invention may also be used in combination with
classical approaches, such as surgery, and the like. When one or
more second therapeutic agents or approaches are used in
combination with the present therapy, there is no requirement for
the combined results to be additive of the effects observed when
each treatment is conducted separately. Although at least additive
effects are generally desirable, as long as the antibody
compositions of this invention remain effective to inhibit or
activate NK cells, the methods of this invention may additionally
comprise the use of a second therapeutic agent or other approach.
Also, there is no particular requirement for the combined treatment
to exhibit synergistic effects, although this is certainly possible
and advantageous. The NKG2A antibody-based treatment may precede,
or follow, the other treatment by, e.g., intervals ranging from
minutes to weeks and months. It also is envisioned that more than
one administration of an anti-NKG2A composition of the invention
will be utilized. The second therapeutic agent or other approach
may be administered interchangeably with the NKG2A antibody
composition of this invention, on alternate days or weeks; or a
cycle of anti-NKG2A treatment may be given, followed by a cycle of
the other agent therapy or approach. In any event, for methods that
comprise the additional step of administering a second therapeutic
agent to a patient, all that is required is to deliver both the
second therapeutic agent and the antibody of this invention in a
combined amount effective to exert a therapeutically beneficial
effect, irrespective of the times for administration.
[0238] It will be appreciated that the present methods of
administering antibodies and compositions to patients can also be
used to treat animals, or to test the efficacy of any of the
herein-described methods or compositions in animal models for human
diseases. Thus, the term "patient" as used herein means any
warm-blooded animal, preferably a mammal, more preferably a primate
and most preferably a human.
[0239] Further aspects and advantages of this invention are
disclosed in the following experimental section, which should be
regarded as illustrative and not limiting the scope of this
application.
EXAMPLES
[0240] Example 1--Killing of Autologous IDC is Mediated by a Subset
of CD94/NKG2A+KIR-NK Cells
[0241] Polyclonal NK cells cultured in the presence of exogenous
IL-2 were previously shown to display strong cytolytic activity
against iDC. Accordingly, in the present study, polyclonal NK cell
populations isolated from donors AM, AC and DB efficiently killed
both autologous and allogeneic iDC. However, the cytolytic activity
against autologous iDC could be incremented in the presence of
appropriate anti-HLA class I mAb.
[0242] These data could be the consequence of the disruption of
inhibitory interactions occurring between self HLA class I on DC
and inhibitory receptors on NK cells. On the basis of these
results, we formulated the hypothesis that only a fraction of the
total NK cell pool displays spontaneous cytotoxicity against iDC
whereas the other NK cells do not because of effective inhibitory
interactions between their receptors and HLA class I molecules. To
analyze this possibility, a panel of NK cell clones isolated from
donors AM, AC and DB were assessed for cytolytic activity against
autologous (and allogeneic) iDC. Consistent with our hypothesis,
only a fraction of NK cell clones lysed autologous iDC. The other
clones displayed either little or no cytotoxicity. Moreover, the
percentage of cytolytic clones was slightly increased when target
cells were represented by allogeneic iDC (see below).
[0243] To verify whether the inability of certain NK cell clones to
lyse iDC reflected the interaction of their inhibitory NKR with HLA
class I molecules, these clones were analyzed for the ability to
lyse autologous iDC either in the absence or in the presence of
anti-HLA class I mAb (i.e. under conditions that disrupt the
inhibitory interactions). On the basis of the results of these
experiments, NK cell clones were grouped into three different
functional categories and further analyzed for the expression of
HLA class I-specific inhibitory receptors including killer Ig-like
receptor (KIR)2DL, KIR3DL1 and CD94/NKG2A (i.e. the main MHC class
I-specific inhibitory receptors in humans).
[0244] The first group (group A) of NK clones was characterized by
high spontaneous cytolytic activity against iDC. The magnitude of
their cytolytic activity could not, or could only minimally, be
increased in the presence of anti-HLA class I mAb. These clones
were rather homogeneous in terms of expression of inhibitory
receptors as they expressed CD94/NKG2A but lacked KIR2DL and
KIR3DL1, which react with self-HLA class I alleles. The second
group of NK cell clones (group B) was also characterized by the
capability of spontaneously killing iDC. However, at variance with
group A clones, their cytotoxicity increased in the presence of
anti-HLA class I mAb. This suggested the occurrence of inhibitory
interactions that limited, but did not abrogate, the NK-cell
mediated cytolysis. This group was also composed of CD94/NKG2A+
clones and lacked KIR reactive with self-HLA class I alleles.
Remarkably, the cytolytic activity of group B NK clones could also
be incremented in the presence of anti-CD94 mAb thus indicating
that the (partial) inhibition of cytotoxicity was indeed mediated
by CD94/NKG2A.
[0245] NK clones belonging to the third group (group C) did not
display cytotoxicity against autologous iDC. However, in the
presence of anti-HLA class I mAb, iDC were efficiently lysed,
suggesting the occurrence of potent inhibitory interactions. These
NK clones were more heterogeneous regarding the expression of
inhibitory receptors. Remarkably, virtually all NK clones
expressing KIR2DL or KIR3DL1 specific for self-HLA class I alleles
were included in this group. Moreover, some of these clones were
characterized by the expression of a single KIR whereas others
expressed multiple KIRs with different specificities. The
reconstitution of cytolytic activity against iDC could be obtained
not only with anti-HLA class I mAb but also with anti-KIR mAb (see
below).
[0246] Finally a minor fraction of group C NK cell clones was
KIR-CD94/NKG2A+. Their cytotoxicity could be reconstituted by
mAb-mediated blocking of CD94 or by anti-HLA class I mAb. These
data indicate that: (a) Not all NK cells are capable of killing
autologous iDC (although all NK cells could lyse iDC in the
presence of anti-HLA class I mAb); (b) clones displaying
spontaneous cytolytic activity against iDC are restricted to an NK
subset characterized by the CD94/NKG2A+KIR-surface phenotype
(groups A and B); (c) clones expressing KIR2DL or KIR3DL1, which
are specific for self-HLA class I alleles, do not kill autologous
iDC (group C).
[0247] Some NK clones expressed both self-reactive KIR and
CD94/NKG2A. In all instances, they were confined to group C and
their cytolytic activity could be reconstituted both by anti-HLA
class I and anti-KIR mAb, whereas anti-CD94 mAb had little or no
effect. Finally it is worth mentioning that KIR+NKG2A-clones were
found to display cytolytic activity against iDC only in experiments
in which iDC were derived from allogeneic (KIR mismatched)
individuals. In this case, KIR+NKG2A-cells display alloreactivity
because the expressed KIRs fail to recognize HLA class I alleles on
allogeneic DC. The representative NK clone AM4 (KIR3DL1+) was
unable to kill autologous iDC (BW4+BW6-) whereas it lysed
allogeneic, KIR mismatched (BW4-BW6+) iDC. Killing of autologous
iDC could be reconstituted in the presence of anti-HLA class I mAb
whereas killing of allogeneic iDC was not significantly
modified.
[0248] Another example indicating the ability of KIR to distinguish
between autologous and allogeneic, KIR-mismatched, iDC is provided
by clone DB3, which co-expresses KIR2DL1 and KIR2DL2. This clone
can be defined as "non-alloreactive" because, on the basis of its
KIR phenotype, it should recognize all different HLA-C alleles
(both group 1 and group 2). Indeed this clone did not kill
autologous or allogeneic iDC whereas lysis of both targets could be
efficiently reconstituted by anti-HLA class I mAb. Moreover,
reconstitution of lysis was obtained by anti-KIR2DL2 mAb against
autologous (CW1/CW3) iDC and by anti-KIR2DL1 mAb against allogeneic
(CW2/CW4) iDC. Finally, as expected, in the case of
NKG2A+KIR-clones no substantial difference existed in the ability
to kill autologous or allogeneic iDC.
Example 2--the Susceptibility of IDC to NK-Mediated Cytotoxicity
Reflects the Down-Modulation of HLA-E Class I Molecules
[0249] Previous studies demonstrated that iDC and mDC display
remarkable differences in terms of HLA class I surface expression.
Thus, by the use of mAb specific for a monomorphic determinant of
HLA-A, B, C and E molecules, it has been shown that DC undergoing
maturation greatly up-regulate their HLA class I expression at the
cell surface. Moreover, the up-regulation of HLA class I
represented a crucial mechanism by which mDC become resistant to
NK-cell-mediated lysis.
[0250] To directly assess the expression of various HLA class I
molecules on cells representative of different stages of DC
maturation we comparatively analyzed the expression of HLA-A, B, C
and E on monocytes, iDC and mDC derived from the same individual.
All HLA class I molecules were highly up-regulated in mDC as
compared with iDC. Remarkably, they were clearly down-regulated in
iDC as compared with monocytes (i.e. the precursors of iDC). Thus,
it appears that the generation of iDC from monocytes results not
only in the acquisition (or up-regulation) of novel surface
molecules (for example CD1a) and functional properties but also in
the loss (or down-regulation) of the expression of various
molecules including CD14, and HLA-A, B, C and E molecules. This
would suggest that the degree of HLA class I down-regulation is
tuned to levels that allow iDC to become sensitive to lysis
mediated by a particular subset of NK cells (CD94/NKG2A+KIR-).
[0251] Along this line, because KIR+NK cells are unable to kill
iDC, it is conceivable that the amount of HLA-B or HLAC molecules
expressed by iDC is sufficient to generate KIR cross-linking and
delivery of inhibitory signals. On the other hand, the
down-regulation of HLA-E would be sufficient to enable a fraction
of KIR-NKG2A+NK cells to kill iDC. Indeed it can be seen that HLA-E
(as detected by the HLA-E-specific 3D12 mAb) was almost
undetectable in iDC whereas it was only partially re-expressed in
mDC. However, in all instances, the HLA-E expression in mDC was
lower as compared with monocytes or PBL derived from the same
individual. Surprisingly, although HLA-A, B and C molecules were
expressed by mDC at levels higher than by PHA blasts, the surface
expression of HLA-E was consistently lower in mDC than in PHA
blasts. In this context, previous studies provided clear evidence
that autologous PHA blasts are highly resistant to NK lysis
independently of the KIR/NKG2A phenotype of the effector NK
cells.
Example 3--A Small Fraction of NK Clones can Mediate Killing of
MDC
[0252] Consistent with previous reports that polyclonal NK cells do
not efficiently kill mDC, we show that most NK cell clones that
lysed iDC did not to kill mDC. Interestingly, however, mDC were
lysed by a minor fraction of NK clones belonging to group A (i.e.
those displaying spontaneous anti-iDC cytolytic activity that could
not be increased by anti-HLA class I mAb). Lysis of autologous mDC
was lower as compared with that of iDC and could be increased in
the presence of anti-HLA class I mAb. This suggests that the higher
expression of HLA-E in mDC as compared with iDC results in a more
effective signaling via CD94/NKG2A (this is also suggested by the
ability of anti-CD94 mAb to increase their lysis). Concerning group
B NK clones (i.e. capable of killing iDC and whose lysis was
incremented by anti-HLA class I mAb), they displayed no cytolytic
activity again mDC; however, cytolytic activity could be revealed
in the presence of anti-HLA class I or anti-CD94 mAb. Finally
clones belonging to group C (in most instances KIR+), which are
unable to kill iDC, also failed to kill mDC. Cytotoxicity against
mDC could only be detected upon mAb-mediated disruption of the
interaction between HLA class I and KIR.
Example 4--Heterogeneity of KIR-NKG2A+NK Cells in the Ability to
Kill DC
[0253] As illustrated above, NK cell clones belonging to group A
and B are characterized by a homogeneous KIR-NKG2A+ surface
phenotype whereas group C includes either KIR+NKG2A- or KIR-NKG2A+
clones, (or, less frequently, KIR+NKG2A+ clones). Assuming that the
negative signaling via KIR is more effective than that via NKG2A
(either because of an intrinsic difference in their signaling
capability or because of the different availability of the specific
HLA class I ligands on DC) it should be clarified why KIR-NKG2A+
cells are detectable in all three groups of NK clones. Since the
cytolytic activity of a given NK cell clone is the result of a
balance between inhibitory (KIR, NKG2A) and triggering (NCR, NKG2D)
receptors, we analyzed the levels of expression of these molecules
in the different groups of NK clones. In particular, we focused our
attention on the expression of NKG2A and of NKp30 (i.e. the
triggering NCR that plays a predominant role in the induction of
NK-cell-mediated lysis of iDC and mDC).
[0254] First, the NKG2A+KIR-clones belonging to group A, B and C
were evaluated for the level of NKG2A surface expression. NK clones
belonging to group C expressed very high levels of NKG2A as
compared with groups A and B. Moreover, group A clones were
characterized by a lower expression of NKG2A as compared with group
B clones. These data suggest the existence of an inverse
correlation between the levels of NKG2A expression and the ability
to kill iDC (and mDC). The low amounts of HLA-E molecules expressed
in iDC may be differentially sensed by NK cells expressing high or
low levels of NKG2A whereas mDC (expressing higher levels of HLA-E)
are susceptible to lysis only by NK clones characterized by very
low NKG2A surface density. Regarding the expression of NKp30, this
was comparable in most NKG2A+ clones analyzed. Consistent with
these data, their ability to kill iDC in the presence of anti-HLA
class I mAb (i.e. in the absence of inhibitory interactions) did
not show significant differences.
[0255] Discussion.
[0256] Heterogeneity exists even among NKG2A+KIR-cells in the
magnitude of cytolytic responses. This appears to inversely
correlate with the surface density of NKG2A. Accordingly, NK clones
expressing low levels of NKG2A (group A) lysed both iDC and mDC
whereas those expressing higher levels of NKG2A killed only iDC or,
in a few cases, (NKG2Abright) failed to kill both iDC and mDC.
[0257] Notably, we also show that the surface expression of HLA-E
is sharply reduced in iDC as compared with monocytes whereas it is
partially recovered in mDC. On the contrary, the reduced cell
surface levels of HLA-B and HLA-C in iDC are still sufficient to
effectively engage KIR3DL1 or KIR2DL.
[0258] An unexpected finding was the identification of a small
subset of NK cell clones belonging to group A (5-10%) that were
capable of killing autologous mDC. These NK clones do not express
self-reactive KIR and are characterized by low levels of NKG2A.
This allows these NK cells to readily sense the down-regulation of
HLA-E on target cells as compared with NK cells expressing higher
levels of NKG2A. Accordingly no increases of the cytolytic activity
of NKG2A low NK cells against iDC occurred in the presence of
anti-HLA class I mAb. On the other hand, in the case of mDC
(expressing higher levels of HLA-E), addition of anti-HLA class I
mAb resulted in an increase of cytolytic activity, indicating that,
provided a sufficient level of receptor-ligand interaction, NKG2A
molecules expressed by group A clones can inhibit lysis. It is
conceivable that in mDC some degree of heterogeneity might exist in
the expression of HLA-E and, possibly, of ligand(s) of NKp30. Given
the ability of a fraction of NK cells to discriminate between cells
that express different amounts of HLA-E, it is possible that among
mDC only some may express a surface density of HLA-E sufficient to
confer resistance to this particular subset of NK cells.
Example 5--Z270 Anti-NKG2A mAb Increases Lytic Activity of NK Cell
Lines Towards Immature Dendritic Cells
[0259] Z270 is a mouse IgG1 monoclonal antibody against NKG2A.
Because Z270 is a mouse antibody, it does not bind to human Fc
receptors and thus acts as an activating antibody of this invention
in human cell systems or in any system that lacks cells bearing
mouse Fc receptors. In contrast, in a system comprising cells
bearing a mouse Fc receptor, Z270 is an inhibitory antibody of this
invention, due to the fact that its IgG1 constant region binds to
such Fc receptors.
[0260] Human NK cell clones expressing NKG2A and immature dendritic
cells (plasmacytoid dendritic cells or myeloid dendritic cells)
were generated using standard methods. The lytic activity of the
resulting human NK cell clones BH3, BH18 and BH34 was tested on
autologous immature dendritic cells. Lytic activity of each of
these clones against the iDC was tested in parallel in the absence
or presence of monoclonal antibodies to CD94 (IgM) and to NKG2A
(Z270, IgG1). For comparison, lytic activity in the presence of an
anti-HLA class I antibody and a control IgG1 (anti-2B4 antibody)
was also tested.
[0261] As shown in Table 1 below, NK clones showed little lysis of
iDC in the absence of antibody or in the presence of control
antibody anti-2B4 mAb. However, killing of the autologous iDC could
be reconstituted in the presence of either anti-CD94, anti-NKG2A
mAb Z270 or anti-HLA class I mAb. This result demonstrates that
interference with NKG2A function reconstitutes NK cell lysis of
iDC. It also demonstrates that the NKG2A binding region of
monoclonal Z270 is capable of blocking NKG2A's inhibitory
function.
TABLE-US-00001 TABLE 1 lysis of autologous iDC NK Clone BH3 BH18
BH34 control lysis 257 382 318 anti CD94 1341 2455 2376 anti-NKG2A
(Z270) 984 1977 2108 anti-HLA class I 1397 2603 2498 anti-2B4
(control IgG1) 236 353 292
Example 6--Reconstitution of Autologous Target Cell Lysis Using
Anti-NKG2A Antibodies
[0262] The cytolytic activity of human NK bulk cells against
autologous PHA blast target cells expressing HLA-E in the absence
of antibody or the presence of mAbZ199, or mAbZ270, was tested.
Cytolytic activity was assessed by a standard 4 hour .sup.51Cr
release assay. All target cells were used at 3000 cells per well in
microtitration plate. The number of NK cells was varied to produce
effector/target ratios of between 0.01-100, as indicated in FIG.
1.
[0263] In the absence of antibody, NK cells displayed little if any
cytolytic activity against target cells expressing HLA-E. However,
in the presence of the anti-NKG2A antibody Z270 (having a mIgG1
constant region) or Z199 (having a mIgG2b constant region) NK
clones became unable to recognize their HLA-E ligands and displayed
strong cytolytic activity against the PHA blast targets. Z270 has a
murine IgG1 constant region and Z199 has a murine IgG2b constant
region. Neither of those antibodies can significantly bind to human
Fc receptors.
[0264] Similarly, inhibition of NK bulk cell killing of HLA-E
positive autologous PHA blast cells could be efficiently reversed
by the use of a Z270 F(ab')2 fragment (FIG. 2), an anti-KIR mAb
DF200 or pan2D which block signaling through KIR2DL1 and KIR2DL2,3,
or by antibody W6/32. Also, under the conditions tested (E/T
ratio=1, 50 .mu.g/ml mAb) PHA blast cells were not killed by NK
bulk cells, but this inhibition could be reversed by the use of
either Z270 mAb or Z270 Fab fragment.
Example 7--Materials and Methods
[0265] mAb.
[0266] The following mAb, produced in our laboratory, were used in
this study: JT3A (IgG2a, anti-CD3), AZ20 and F252 (IgG1 and IgM,
respectively, anti-NKp30), c127 (IgG1, anti-CD16), c218 (IgG1,
anti-CD56), EB6b (IgG1, anti-KIR2DL1 and KIR2D S1), GL183 (IgG1,
anti-KIR2DL2 KIR2DL3 and KIR2DS2), FES172 (IgG2a, anti-KIR2DS4),
Z27 (IgG1, anti-KIR3DL1), XA185 (IgG1, anti-CD94), Z199, Z270
(IgG2b, anti-NKG2A), A6-136 (IgM, anti-HLA class I), 131 (IgG1,
anti-HLA-A alleles including A3, All and A24) and E59/53 (IgG2a,
anti-HLA-A) [Ciccone et al, (1990) PNAS USA 87:9794-9797; Pende et
al, (1998) J Immunol. 28:2384-2394]. The mAb F4/326 (IgG,
anti-HLA-C) [Marsh et al, (1990) Tissue Antigens 36: 180-186],
116-5-28 (IgG2a, anti-HLA-Bw4 alleles) and 126-39 (IgG3,
anti-HLA-Bw6 alleles) were kindly provided by Dr K. Gelsthorpe
(Sheffield, GB) (XII International HLA Workshop) and 3D12 (IgG1,
anti-HLA-E) [Lee et al. (1998) J. Immunol. 160:4951-4960] was
kindly provided by Dr. Daniel Geraghty (Fred Hutchinson Cancer
Research Center, Seattle, Wash.).
[0267] Anti-CD1a (IgG1-PE), anti-CD14 (IgG2a), anti-CD83 (IgG2b)
and anti-CD86 (IgG2b-PE) were purchased from Immunotech (Marseille,
France). D1.12 (IgG2a, anti-HLA-DR) mAb was provided by Dr R. S.
Accolla (Pavia, Italy). HP2.6 (IgG2a, anti-CD4) mAb was provided by
Dr P. Sanchez-Madrid (Madrid, Spain).
[0268] Generation of polyclonal or clonal NK cell populations. To
obtain PBL, PBMC were isolated on Ficoll-Hypaque gradients and
depleted of plastic-adherent cells. Enriched NK cells were isolated
by incubating PBL with anti-CD3 (JT3A), anti-CD4 (HP2.6) and
anti-HLA-DR (D1.12) mAb (30 min at 4.degree. C.) followed by goat
anti-mouse coated Dynabeads (Dynal, Oslo, Norway) (30 min at
4.degree. C.) and immunomagnetic depletion. CD3-CD4-HLA-DR-cells
were cultured on irradiated feeder cells in the presence of 100
U/ml rIL-2 (Proleukin, Chiron Corp., Emeryville, Calif.) and 1.5
ng/ml PHA (Gibco Ltd, Paisley, GB) to obtain polyclonal NK cell
populations or, after limiting dilution, NK cell clones as
previously described.
[0269] Generation of DC. PBMC were derived from healthy donors and
plastic adherent cells were cultured in the presence of IL-4 and
GMCSF (Peprotech, London, GB) at a final concentration of 20 ng/ml
and 50 ng/ml, respectively. After 6 days of culture, cells were
characterized by the CD14-CD1a+CD83-phenotype corresponding to iDC.
To generate CD14-CD1a+CD83+CD86+ mDC, iDC were stimulated for 2
days with LPS (Sigma-Aldrich, St. Louis, Mich.) at a final
concentration of 1 ug/ml.
[0270] Flow cytofluorimetric analysis and cytolytic activity. For
one- or two-color cytofluorimetric analysis (FACSCalibur, Becton
Dickinson and Co., Mountain View, Calif.), cells were stained with
the appropriate mAb followed by PE- or FITC-conjugated
isotype-specific goat anti-mouse second reagent (Southern
Biotechnology Associated, Birmingham). Polyclonal and clonal NK
cell populations were tested for cytolytic activity in a 4-h
[510]-release assay against either autologous or heterologous DC.
The concentrations of the various mAb added were 10 ug/ml for
masking experiments. The E:T ratios are indicated in the text.
Example 8--Chimerization of Z270 Heavy and Light Chain Variable
Regions
[0271] Frozen cell pellets of mouse hybridoma line, Z270, were
thawed and processed using the RNeasy Midi Kit (Qiagen cat. No.
75142) to isolate 71 .mu.g of total RNA. About 5 micrograms of Z270
RNA was subjected to reverse transcription to produce Z270 cDNA
using the Amersham Biosciences 1st strand synthesis kit (Amersham
Biosciences, Cat. No. 27-9261-01). Immunoglobulin heavy chain
variable region (VH) cDNA was amplified by PCR using a number of
different IgH primers in combination with a constant region primer
in order to determine which primer pair was the most suitable for
PCR. Similarly, immunoglobulin kappa chain variable region (VK) was
amplified using multiple IgK primers in combination with a kappa
constant region primer.
[0272] Suitable primers for each of the heavy and light chain
variable regions were identified and ligated separately into
pCR2.1.RTM.-TOPO Vectors.RTM. for transformation into E. coli TPO10
bacteria, amplification and sequencing (using the BigDye.RTM.
Terminator v3.0 Cycle Sequencing Ready Reaction Kit (ABI). The DNA
sequence of the heavy chain variable region (Z270 VH) and the
corresponding amino acid sequence are set forth in SEQ ID NO:1 and
SEQ ID NO:2, respectively. The DNA sequence of the light chain
variable region (Z270 VK) and the corresponding amino acid sequence
are set forth in SEQ ID NO:3 and SEQ ID NO:4, respectively.
[0273] Chimerization of Z270 VK involved introducing via the
appropriate primers and PCR, a Hind III restriction site, a Kozak
translation initiation site and the K2A/RFT2 kappa leader sequence
at the 5' end and a splice donor site and Bam HI restriction site
at the 3' end of the Z270 VK DNA sequence. The resulting PCR
product was cloned into a vector encoding the constant region of
the human kappa light chain so as to encode a full-length chimeric
light chain containing the variable region of the Z270 light chain.
The DNA sequence of the resulting chZ270VK and the corresponding
amino acid sequence are set forth in SEQ ID NO:5 and SEQ ID NO:6,
respectively.
[0274] Chimerization of Z270 VH involved introducing via the
appropriate primers and PCR, a Hind III restriction site, a Kozak
translation initiation site and the A003 leader sequence at the 5'
end and the 5' end of the gamma1 C region including a natural Apa I
restriction site at the 3' end of the Z270 VH DNA sequence. The
resulting PCR product was cloned into a vector encoding the
constant region of the human IgG1 heavy chain so as to encode a
full-length chimeric IgG1 heavy chain containing the variable
region of the Z270 heavy chain. The DNA sequence of the resulting
chZ270VH and the corresponding amino acid sequence are set forth in
SEQ ID NO:7 and SEQ ID NO:8, respectively.
[0275] The resulting heavy and light chain containing plasmids were
simultaneously electroporated into COS 7 cells which expressed the
resulting human IgG1-kappa chimersation construct of Z270.
Example 9--Generation of New mAbs
[0276] mAbs were generated by immunizing 5 week old Balb C mice NK
clone SA260 (CD94bright). After different cell fusions, the mAbs
Z199 and Z270 were first selected as described in Moretta et al.,
(1994) J. Exp. Med. 180:545. Analysis of resting or activated NK
cell populations for the distribution of the CD94 molecules was
performed using one or two-color fluorescence cytofluorometric
analysis as described in Moretta et al. (1994).
[0277] Positive monoclonal antibodies were further screened for
their ability to reconstitute lysis by NK clones. The cytolytic
activity of NK clones was assessed by a standard 4 hour .sup.51Cr
release assay in which effector NK cells were tested against the
P815 mouse cell line or the C1R human cell line transfected or not
with various HLA class I genes. Other target cells used in these
studies were represented by the human HLA-class I-LCL 721.221 cell
line either untransfected or transfected with various HLA classes
as described in Sivori et al. (1996) Eur. J. Immunol. 26:
2487-2492.
Example 10--Purification of PBLs and Generation of Polyclonal or
Clonal NK Cell Lines
[0278] PBLs are obtained from healthy donors by Ficoll Hypaque
gradients and depletion of plastic adherent cells. To obtain
enriched NK cells, PBLs are incubated with anti CD3, anti CD4 and
anti HLA-DR mAbs (30 minutes at 4.degree. C.), followed by goat
anti mouse magnetic beads (Dynal) (30 minutes at 4.degree. C.) and
immunomagnetic selection by methods known in the art (Pende et al.,
1999). CD3.sup.-, CD4.sup.-, DR.sup.- cells are cultivated on
irradiated feeder cells and 100 U/ml Interleukin 2 (Proleukin,
Chiron Corporation) and 1.5 ng/ml Phytohemagglutinin A (Gibco BRL)
to obtain polyclonal NK cell populations. NK cells are cloned by
limiting dilution and clones of NK cells are characterized by flow
cytometry for expression of cell surface receptors.
Example 11--Staining of Whole Blood from Monkeys to Identify
Individual Expression of Receptors Binding Anti-NKG2A mAb
Materials
[0279] Monkey blood: blood for rhesus and cynomolgus monkeys was
purchased at Centre de Primatologie, ULP, Strasbourg. Monkey blood
for Baboons was purchased at Centre de Primatologie, CNRS, Station
Rousset. Monkey blood was collected in "vacutainer" tube containing
EDTA or sodium citrate. Blood was processed within the 24 hours
following collection and kept at room temperature.
[0280] Antibodies: FITC-CD3, -CD4, -CD14, -CD20, and CyCr-CD45 are
from BD Pharmingen, PC7-CD16 was obtained from Beckman Coulter; all
these clones are cross-reacting with monkey PBMCs. PE-GaM (Goat
F(ab')2 fragment anti-Mouse IgG (H+L)-PE), and OptiLyse.RTM. C.
were purchased from Beckman Coulter. Anti-NKG2a mAb (clone Z270,
mouse IgG1) used at 1 .mu.g/ml.
[0281] Other reagents: PBS (1.times.) obtained from Gibco
Invitrogen; mouse serum from NMRI mouse from Janvier; Formaldehyde
37% from Sigma.
Methods:
[0282] Cell staining was carried out according to the following
protocol: [0283] 100 .mu.l of blood+10 .mu.l of 10.times. purified
mAb [0284] Incubate with agitation 30 min at RT [0285] Wash with 3
ml PBS (1400 RPM 10 min RT) [0286] Add 100 .mu.l PE-GaM or PE-GaH,
1:200 final, vortex [0287] Incubate with agitation 30 min at RT
[0288] Wash with 3 ml PBS (1400 RPM 10 min RT) [0289] Add 50 .mu.l
of 20% mouse serum, vortex and incubate 10 min [0290] Add 30 .mu.l
to 60 .mu.l of FITC-CD3,(-CD4,-CD14,-CD20), PC7-CD16, CyCr-CD45
mixture or 10 .mu.l of each corresponding isotypic control [0291]
Incubate with agitation 30 min at RT [0292] Add 500 .mu.l
OptiLyse.RTM. C., vortex and incubate 10 min [0293] Add 500 .mu.l
PBS, vortex and incubate 10 min [0294] Wash with 3 ml PBS (1400 RPM
10 min RT) [0295] Resuspend cell pellet in 300 .mu.l PBS+0.2%
Formaldehyde. Flow cytometry was carried out according to the
following protocol: [0296] Samples are run on a XL/MCL cytometer
(Beckman Coulter). Acquisition and analysis are performed with
EXPO.TM. 32 v1.2 software (Beckman Coulter). [0297] Analysis is
focused on lymphocytes identified by their FSC and SSC features.
[0298] Analysis of the T cell or NK cell compartments:
[0299] T cells=CD3.sup.+ lymphocytes are defined as the positive
cells of the anti-CD3 staining histogram gated on Ly.
[0300] NK cells=CD3.sup.-CD56.sup.+ lymphocytes corresponds to the
CD3.sup.-CD56.sup.+ gate in the CD3/CD56 dot plot (upper left part
of the quadrant).
Results
[0301] Binding of NKG2A monoclonal antibody Z270 to rhesus monkeys,
cynomolgus monkeys and baboons was assessed. Cynomolgus monkey bulk
NK cells (day 16, 300 uml were incubated 30 min at 4.degree. C.
with mAb (1 .mu.g/ml), washed and labelled 20 min at 4.degree. C.
with PE-GaM. FIG. 1 shows binding to cynomolgus monkey NK cells, as
well as IgG1 and anti-CD16 binding demonstrating that Z270 binds to
cynomolgus monkey NK cells. Macaca mulatta (rhesus monkey) NK cells
(from whole blood) were incubated with mAb, washed and labelled
with PE-GaM. Results, shown in Table 2, demonstrate binding of
clone Z270 to the rhesus monkey NK cells. Finally, baboon NK cells
(from whole blood) were incubated with mAb, washed and labelled
with PE-GaM. Results, shown in Table 3, demonstrate binding of
clone Z270 to the baboon NK cells.
Example 12--Staining of Whole Blood from Monkeys to Identify
Individual Expression of Receptors Binding Anti-NKG2A mAb
Materials
[0302] Monkey blood from rhesus and cynomolgus monkeys was
collected in a tube containing EDTA or sodium citrate. Antibodies:
FITC-CD3, -CD4, -CD14,-CD20, and CyCr-CD45 are from BD Pharmingen,
PC7-CD16 was obtained from Beckman Coulter; all these clones are
cross-reacting with monkey PBMCs. PE-GaM (Goat F(ab')2 fragment
anti-Mouse IgG (H+L)-PE), and OptiLyse.RTM. C. were purchased from
Beckman Coulter. Other reagents: PBS (1.times.) obtained from Gibco
Invitrogen; Formaldehyde 37% from Sigma.
Methods:
[0303] Cell staining was carried out according to the following
protocol:
[0304] 100 .mu.l whole blood (EDTA)+11 .mu.l mAb solution, Z270 or
Z199 (10 .mu.g/ml) or isotype control, incubated for 30 min at
RT
[0305] Wash with PBS, add 100 .mu.l PE- or FITC GaM (1/200 final)
and leave for 30 min at RT
[0306] Wash with PBS, add 50 .mu.l mouse serum 20%, add 60 .mu.l
containing FITC-anti-CD3,-CD4, -CD14, -CD20, CyCr-CD45, PC7-CD16
and leave for 30 min at RT
[0307] Add 500 .mu.l of optilyseC, leave for 10 min at RT
[0308] Add 500 .mu.l of PBS and leave for 10 min at RT
[0309] Wash with PBS and with 0.2% Formaldehyde. [0310] Analysis
focus on CD45.sup.bright small cells (CD45/SSC) then on
CD16.sup.+CD3.sup.-CD4.sup.-CD14.sup.-CD20.sup.- cells.
Results
[0311] Binding of NKG2A monoclonal antibodies Z270 and Z199 to
rhesus monkey NK cells and cynomolgus monkey NK cells was assessed
and compared. Cynomolgus monkey bulk NK cells (day 16, 300 .mu.l
were incubated 30 min at 4.degree. C. with mAb (1 .mu.g/ml), washed
and labelled 20 min at 4.degree. C. with PE-GaM. Table 4 shows
binding of both Z199 and Z270 to cynomolgus monkey NK cells, as
well as IgG1 and anti-CD16 binding demonstrating that both Z199 and
Z270 bind to cynomolgus monkey NK cells. Macaca mulatta (rhesus
monkey) NK cells (from whole blood) were incubated with mAb, washed
and labelled with PE-GaM. Results, shown in Table 5, demonstrate
binding of both clones Z199 and Z270 to the rhesus monkey NK
cells.
[0312] It has further been observed (Biassoni et al, (2005) J.
Immunol. 174: 5695-5705, see FIGS. 5 and 6) that Z199 binds
cynomolgus monkey NKG2C in addition to NKG2A, and moreover that
this mAb results in increase in lysis of P815 target cells in a
redirected killing assay. The latter increase in lysis is the
opposite observed with human NK cells and is opposite that which
would be expected for an inhibitor receptor NKG2A. Thus, while not
wishing to be bound by theory present inventors propose that Z199
acts through the activatory receptor NKG2C in cynomolgus monkeys.
Z270 also binds cynomolgus monkey cells and results in an increase
in lysis of P815 target cells in a redirected killing assay
suggesting that Z270 also recognizes NKG2C in the cynomolgus
monkey.
[0313] The level of binding however of the two mAbs on the same
species (cynomolgus for example) is very different both in terms of
percentage of cells stained and intensity of fluorescence. This
means that the two antibodies bind differently to NKG2A
epitopes.
TABLE-US-00002 TABLE 2 weight m IgG1 Z270 mulatta sex (kg) % N % %
MFI+ CH256 F 8.4 3.5 0.8 78.1 6.5 *8703 F 7.1 2.4 0.4 56.1 5.3
P9215 F 5.85 4.4 1.4 89.7 12.9 RU925 F 1 5.9 0.4 95.2 15 201 M 14.6
14.4 1.3 95.7 8.1 PM021 M 3.7 5 0.8 61.7 5.7 MM031 M 2.25 1.8 0.4
88.1 10 N0401 M 1.75 2.6 0.5 87.1 8.99 N0404 M 1.25 1.7 0.6 86.3 9
Mean 0.7 83 9.1 SD 0.4 12.3 3.2 n 9 9 9 Range 61.7-95.7
5.3-12.9
TABLE-US-00003 TABLE 3 m IgG1 Z270 % tot. tot. Baboon sex birth NK
% MFI % MFI K05 F Jan. 1, 1994 6.7 34 0.9 K938A F Dec. 29, 1998 1.3
0.3 0.3 8.7 0.9 O22V F Jul. 7, 1998 2.9 0.5 0.2 0.4 0.2 V992 F Jan.
31, 1999 5.1 0.6 0.3 41.2 1 V997 F Mar. 29, 1999 5.5 0.7 0.2 32.3
0.8 V999 F Apr. 4, 1999 5.2 0.3 0.2 2.3 0.3 V9912 F May 7, 1999 4.7
0.2 0.2 12 1.4 V9914 F May 17, 1999 3.8 0.1 0.3 10.9 1 V9926 F Jun.
13, 1999 5.4 0.5 0.2 2.7 0.3 V9929 F Nov. 7, 1999 7.9 0.3 0.2 0.9
0.2 PA977 M Dec. 3, 1997 2.8 0.9 1.1 11.4 1.6 PA983 M Aug. 13, 1998
4.5 0.1 0.7 24.6 2 V942A M Jun. 10, 1999 0.8 1.7 1 14.3 1.6 V857C M
Oct. 29, 2000 7.2 0.8 1.4 79.7 9 V861B M Jan. 7, 2000 0.8 0.2 0.6
57.1 2.5 V914B M Feb. 19, 2000 2 0.4 1.1 0.8 1.3 V918C M Feb. 19,
2000 4.2 0.3 1.1 79.7 9 V9812 M Jul. 6, 1998 5.6 1.3 1.2 78.1 6.2
V989 M Jun. 1, 1998 1.5 1.2 1.2 12.4 2 V9920 M Aug. 26, 1999 3.7
0.7 0.8 14.2 1.6 Mean 4.1 30.3 SD 2.1 27.35 Range 0.8 to 7.9 2.3 to
79.7 n 20 17
TABLE-US-00004 TABLE 4 Analysis of NK cell subsets from peripheral
rhesus monkey whole blood IgG1 Z270 IgG2b Z199 Weight % NK % % MFI
% % MFI Name (kg) Mean MFI NK.sup.+ MFI NK.sup.+ NK.sup.+ MFI
NK.sup.+ MFI NK.sup.+ NK.sup.+ 34459 9.6 5.81 5.88 6.07 6.06 6.44
6.1 0.76 1.4 2.7 40.8 5.6 0.7 1.2 63.8 96.2 66.3 31828 7.9 9.9 9.95
9.09 9.54 9.44 9.6 0.65 0.4 4.3 88.9 4.6 0.7 0.5 101.0 99.8 101.0
O967 10.4 13.2 14.1 14 13.5 13.5 13.7 0.67 0.5 2.7 40.8 5.6 0.7 0.5
62.3 84.8 73.4 R00093 6.2 4.93 5.22 5.25 5.38 5.09 5.2 0.68 1.0 2.5
40.9 4.5 0.6 0.4 81.9 97.9 83.6 R00013 7.6 6.2 7.36 7.34 7.28 6.21
6.9 0.66 0.3 1.4 12.4 3.8 0.6 0.2 38.8 97.8 39.7 R00085 7.6 7.63
7.59 7.87 7.13 7.88 7.6 0.7 0.9 3.0 49.3 4.7 0.7 0.3 68.4 91.9 74.4
R00055 6.8 8.54 8.04 8.01 8.2 0.7 0.6 4.0 65.8 5.3 0.6 0.9 R99273
9.4 10.1 9.85 8.8 9.8 9.27 9.6 0.54 0.5 2.0 49.5 2.9 0.4 0.6 32.7
98.0 33.4 R00073 5.6 8.43 7.8 7.31 7.29 7.72 7.7 0.4 0.5 2.2 84.5
2.4 0.4 0.39 31.6 99.1 31.9 R00073 6.1 5.65 5.56 5.45 5.69 4.28 5.3
0.59 0.7 3.3 84.8 3.7 0.3 0.3 31.9 98.0 32.6 R00077 7.2 11.2 10.9
11.9 10 7.9 10.4 0.41 0.4 5.0 80.2 6.1 0.4 0.5 30.4 97.1 31.3
R00025 6.3 6.91 6.57 10.3 9.87 9.27 8.6 0.45 0.7 2.2 79.2 2.5 0.4
0.4 30.0 97.3 30.9 R00041 7.7 9.72 9.71 9.16 9.25 8.49 9.3 0.48 0.9
2.9 77.4 3.5 0.4 0.3 33.1 89.6 36.9 R00099 5.8 6.14 5.8 6.12 5.45
5.39 5.8 0.56 1.2 2.5 58.5 3.6 0.5 0.3 31.1 97.6 31.8 R00037 5.1
4.75 5.01 5.01 4.7 3.88 4.7 0.5 0.6 1.5 50.1 1.9 0.5 0.3 26.7 96.9
27.5 R00023 5.8 11.8 11 10.7 8.46 11.3 10.6 0.5 0.3 4.4 90.9 4.7
0.3 0.6 34.0 96.4 35.3 R00101 6.1 12.1 11.6 13 12.1 11.5 12.1 0.58
0.5 6.0 82.5 7.1 0.5 0.4 37.1 98.7 37.6 R00061 5.7 5.96 5.26 5.62
5.6 5.59 5.6 0.5 0.5 5.4 59.4 8.5 0.6 0.2 1.6 40.7 2.4 R00007 6.5
18.4 17.5 16.6 18 18.8 17.9 0.52 1.5 3.8 67.9 4.9 0.4 0.7 34.9 99.6
35.0 Mean 8.7 0.6 0.7 3.4 63.4 4.3 0.5 0.5 29.6 93.2 30.6 SD 3.3
0.1 0.4 1.5 21.3 2.0 0.1 0.3 9.2 13.6 9.3
TABLE-US-00005 TABLE 5 Analysis of NK cell subsets from peripheral
cynomolgus monkey whole blood IgG1 Z270 IgG2b Z199 % NK % % MFI % %
MFI Kg Mean MFI NK.sup.+ MFI NK.sup.+ NK.sup.+ MFI NK.sup.+ MFI
NK.sup.+ NK.sup.+ 1221 9.1 14.9 13.7 15 14.4 14.3 14.4 0.53 0.6 4.6
57.1 7.1 0.8 1.3 105.0 96.7 109.0 M859 9 7.76 8.8 8.18 8.83 8.13
8.3 0.51 0.2 4.6 74.7 5.7 0.6 0.2 144.0 95.1 152.0 R390 3.6 4.11
3.77 3.61 2.84 3.73 3.6 0.39 0.2 3.6 43.9 6.9 0.5 0.2 32.3 99.0
32.6 T270 3.6 16.4 16.1 17.8 18.8 17.3 0.54 0.2 1.1 4.5 3.4 0.6 0.2
113.0 97.7 116.0 T788 3.3 5.55 5.22 5.41 4.95 5.63 5.4 0.5 0.3 1.8
18.8 4.6 0.6 0.4 123.0 97.0 126.0 AK565 3.9 10.7 10.4 9.82 9.98
9.59 10.1 0.66 0.6 3.4 41.7 6.7 0.6 0.3 28.2 90.7 31.0 AK729 3 5.61
5.78 5.75 5.75 5.87 5.8 0.6 0.3 1.3 9.7 4.7 0.6 0.3 37.4 99.4 37.7
AL210 3.3 4.68 4.63 4.85 5 4.8 0.65 0.4 3.3 54.4 5.0 0.7 0.3 126.0
92.7 136.0 AL303 2.8 19.9 19.1 19.5 18.9 18.2 19.1 0.63 0.13 1.9
24.2 3.7 0.7 0.14 129.0 99.1 130.0 AL389 5.2 7.53 7.96 8.23 7.75
6.49 7.6 0.63 0.3 5.3 78.7 6.3 0.8 0.4 223.0 95.5 234.0 Mean 9.6
0.6 0.3 3.1 40.8 5.4 0.7 0.4 106.1 96.3 110.4 SD 5.5 0.1 0.2 1.5
26.0 1.3 0.1 0.3 60.2 2.9 63.2 N 10
[0314] All publications and patent applications cited in this
specification are herein incorporated by reference in their
entireties as if each individual publication or patent application
were specifically and individually indicated to be incorporated by
reference.
[0315] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to one of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
Sequence CWU 1
1
81396DNAMus musculusCDS(1)..(372) 1cag gtc caa ctg cag cag cct ggg
gct gag ctg gtg agg cct ggg gct 48Gln Val Gln Leu Gln Gln Pro Gly
Ala Glu Leu Val Arg Pro Gly Ala1 5 10 15tca gtg aag ctg tcc tgc aag
gct tct ggc tac acg ttc acc agc tac 96Ser Val Lys Leu Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Ser Tyr20 25 30tgg atg aac tgg gtt aag
cag agg cct gag caa ggc ctt cag tgg att 144Trp Met Asn Trp Val Lys
Gln Arg Pro Glu Gln Gly Leu Gln Trp Ile35 40 45gga agg att gat cct
tac gat agt gaa act cac tac agt caa aag ttc 192Gly Arg Ile Asp Pro
Tyr Asp Ser Glu Thr His Tyr Ser Gln Lys Phe50 55 60aag gac aag gcc
ata ttg act gta gac aaa tcc tcc agc aca gcc tac 240Lys Asp Lys Ala
Ile Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80atg cga
ctc agc agc ctg aca tct gag gac tct gcg gtc tat tac tgt 288Met Arg
Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys85 90 95gca
aga ggg ggc tat gat ttc gac gta gga act ctc tac tgg ttc ttc 336Ala
Arg Gly Gly Tyr Asp Phe Asp Val Gly Thr Leu Tyr Trp Phe Phe100 105
110gat gtc tgg ggc gca ggg acc acg gtc acc gtc tcc tcagcctcca
382Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser115 120ccaagggccc
atcg 3962124PRTMus musculus 2Gln Val Gln Leu Gln Gln Pro Gly Ala
Glu Leu Val 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 Asn Trp Val Lys Gln
Arg Pro Glu Gln Gly Leu Gln Trp Ile 35 40 45Gly Arg Ile Asp Pro Tyr
Asp Ser Glu Thr His Tyr Ser Gln Lys Phe 50 55 60Lys Asp Lys Ala Ile
Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Arg Leu
Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Gly Gly Tyr Asp Phe Asp Val Gly Thr Leu Tyr Trp Phe Phe 100 105
110Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser 115
1203472DNAArtificial sequencehuman heavy chain constant
regionCDS(2)..(448) 3c gcc aag ctt gcc gcc acc atg gga tgg aac tat
atc atc ctc ttc ttg 49Ala Lys Leu Ala Ala Thr Met Gly Trp Asn Tyr
Ile Ile Leu Phe Leu1 5 10 15tta gca aca gct aca tgt gtc cac tcc cag
gtc caa ctg cag cag cct 97Leu Ala Thr Ala Thr Cys Val His Ser Gln
Val Gln Leu Gln Gln Pro20 25 30ggg gct gag ctg gtg agg cct ggg gct
tca gtg aag ctg tcc tgc aag 145Gly Ala Glu Leu Val Arg Pro Gly Ala
Ser Val Lys Leu Ser Cys Lys35 40 45gct tct ggc tac acg ttc acc agc
tac tgg atg aac tgg gtt aag cag 193Ala Ser Gly Tyr Thr Phe Thr Ser
Tyr Trp Met Asn Trp Val Lys Gln50 55 60agg cct gag caa ggc ctt cag
tgg att gga agg att gat cct tac gat 241Arg Pro Glu Gln Gly Leu Gln
Trp Ile Gly Arg Ile Asp Pro Tyr Asp65 70 75 80agt gaa act cac tac
agt caa aag ttc aag gac aag gcc ata ttg act 289Ser Glu Thr His Tyr
Ser Gln Lys Phe Lys Asp Lys Ala Ile Leu Thr85 90 95gta gac aaa tcc
tcc agc aca gcc tac atg cga ctc agc agc ctg aca 337Val Asp Lys Ser
Ser Ser Thr Ala Tyr Met Arg Leu Ser Ser Leu Thr100 105 110tct gag
gac tct gcg gtc tat tac tgt gca aga ggg ggc tat gat ttc 385Ser Glu
Asp Ser Ala Val Tyr Tyr Cys Ala Arg Gly Gly Tyr Asp Phe115 120
125gac gta gga act ctc tac tgg ttc ttc gat gtc tgg ggc gca ggg acc
433Asp Val Gly Thr Leu Tyr Trp Phe Phe Asp Val Trp Gly Ala Gly
Thr130 135 140acg gtc acc gtc tcc tcagcctcca ccaagggccc atcg 472Thr
Val Thr Val Ser1454149PRTArtificial sequencehuman heavy chain
constant region 4Ala Lys Leu Ala Ala Thr Met Gly Trp Asn Tyr Ile
Ile Leu Phe Leu1 5 10 15Leu Ala Thr Ala Thr Cys Val His Ser Gln Val
Gln Leu Gln Gln Pro 20 25 30Gly Ala Glu Leu Val Arg Pro Gly Ala Ser
Val Lys Leu Ser Cys Lys 35 40 45Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
Trp Met Asn Trp Val Lys Gln 50 55 60Arg Pro Glu Gln Gly Leu Gln Trp
Ile Gly Arg Ile Asp Pro Tyr Asp65 70 75 80Ser Glu Thr His Tyr Ser
Gln Lys Phe Lys Asp Lys Ala Ile Leu Thr 85 90 95Val Asp Lys Ser Ser
Ser Thr Ala Tyr Met Arg Leu Ser Ser Leu Thr 100 105 110Ser Glu Asp
Ser Ala Val Tyr Tyr Cys Ala Arg Gly Gly Tyr Asp Phe 115 120 125Asp
Val Gly Thr Leu Tyr Trp Phe Phe Asp Val Trp Gly Ala Gly Thr 130 135
140Thr Val Thr Val Ser1455336DNAMus musculusCDS(1)..(321) 5gac atc
cag atg act cag tct cca gcc tcc cta tct gca tct gtg gga 48Asp Ile
Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly1 5 10 15gaa
act gtc acc atc aca tgt cga gca agt gag aat att tac agt tat 96Glu
Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr 20 25
30tta gca tgg tat cag cag aaa cag gga aaa tct cct cag ttc ttg gtc
144Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Phe Leu Val
35 40 45tat aat gca aaa acc tta gca gaa ggt gtg cca tca agg ttc agt
ggc 192Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60agt gga tca ggc aca cag ttt tct ctg aag atc aac agc ctg
cag cct 240Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu
Gln Pro65 70 75 80gaa gat ttt ggg agt tat tac tgt caa cat cac tat
ggt act cct cgg 288Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His Tyr
Gly Thr Pro Arg 85 90 95acg ttc ggt gga ggc acc aag ctg gaa atc aaa
cgtgagtgga tcccg 336Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100
1056107PRTMus musculus 6Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu
Ser Ala Ser Val Gly1 5 10 15Glu Thr Val Thr Ile Thr Cys Arg Ala Ser
Glu Asn Ile Tyr Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Gln Gly
Lys Ser Pro Gln Phe Leu Val 35 40 45Tyr Asn Ala Lys Thr Leu Ala Glu
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Gln Phe
Ser Leu Lys Ile Asn Ser Leu Gln Pro65 70 75 80Glu Asp Phe Gly Ser
Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Arg 85 90 95Thr Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys 100 1057415DNAArtificial sequencehuman
light chain constant regionCDS(2)..(400) 7c gcc aag ctt gcc gcc acc
atg agt gtg ctc act cag gtc ctg gcg ttg 49Ala Lys Leu Ala Ala Thr
Met Ser Val Leu Thr Gln Val Leu Ala Leu1 5 10 15ctg ctg ctg tgg ctt
aca ggt gcc aga tgt gac atc cag atg tct cag 97Leu Leu Leu Trp Leu
Thr Gly Ala Arg Cys Asp Ile Gln Met Ser Gln20 25 30act cca gcc tcc
cta tct gca tct gtg gga gaa act gtc acc atc aca 145Thr Pro Ala Ser
Leu Ser Ala Ser Val Gly Glu Thr Val Thr Ile Thr35 40 45tgt cga gca
agt gag aat att tac agt tat tta gca tgg tat cag cag 193Cys Arg Ala
Ser Glu Asn Ile Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln50 55 60aaa cag
gga aaa tct cct cag ttc ttg gtc tat aat gca aaa acc tta 241Lys Gln
Gly Lys Ser Pro Gln Phe Leu Val Tyr Asn Ala Lys Thr Leu65 70 75
80gca gaa ggt gtg cca tca agg ttc agt ggc agt gga tca ggc aca cag
289Ala Glu Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
Gln85 90 95ttt tct ctg aag atc aac agc ctg cag cct gaa gat ttt ggg
agt tat 337Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro Glu Asp Phe Gly
Ser Tyr100 105 110tac tgt caa cat cac tat ggt act cct cgg acg ttc
ggt gga ggc acc 385Tyr Cys Gln His His Tyr Gly Thr Pro Arg Thr Phe
Gly Gly Gly Thr115 120 125aag ctg gaa atc aaa cgtgagtgga tcccg
415Lys Leu Glu Ile Lys1308133PRTArtificial sequencehuman light
chain constant region 8Ala Lys Leu Ala Ala Thr Met Ser Val Leu Thr
Gln Val Leu Ala Leu1 5 10 15Leu Leu Leu Trp Leu Thr Gly Ala Arg Cys
Asp Ile Gln Met Ser Gln 20 25 30Thr Pro Ala Ser Leu Ser Ala Ser Val
Gly Glu Thr Val Thr Ile Thr 35 40 45Cys Arg Ala Ser Glu Asn Ile Tyr
Ser Tyr Leu Ala Trp Tyr Gln Gln 50 55 60Lys Gln Gly Lys Ser Pro Gln
Phe Leu Val Tyr Asn Ala Lys Thr Leu65 70 75 80Ala Glu Gly Val Pro
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln 85 90 95Phe Ser Leu Lys
Ile Asn Ser Leu Gln Pro Glu Asp Phe Gly Ser Tyr 100 105 110Tyr Cys
Gln His His Tyr Gly Thr Pro Arg Thr Phe Gly Gly Gly Thr 115 120
125Lys Leu Glu Ile Lys 130
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