U.S. patent application number 11/324356 was filed with the patent office on 2009-07-30 for compositions and methods for regulating nk cell activity.
This patent application is currently assigned to Novo Nordisk A/S. Invention is credited to Pascale Andre, Mariella Della Chiesa, Laurent Gauthier, Alessandro Moretta, Soren Berg Padkjaer, Peter Andreas Nicolai Reumert Wagtmann.
Application Number | 20090191213 11/324356 |
Document ID | / |
Family ID | 37524330 |
Filed Date | 2009-07-30 |
United States Patent
Application |
20090191213 |
Kind Code |
A9 |
Padkjaer; Soren Berg ; et
al. |
July 30, 2009 |
Compositions and methods for regulating NK cell activity
Abstract
The present invention relates to novel compositions and methods
for regulating an immune response in a subject. More particularly,
the invention relates to specific antibodies that regulate the
activity of NK cells and allow a potentiation of NK cell
cytotoxicity in mammalian subjects. The invention also relates to
fragments and derivatives of such antibodies, as well as
pharmaceutical compositions comprising the same and their uses,
particularly in therapy, to increase NK cell activity or
cytotoxicity in subjects.
Inventors: |
Padkjaer; Soren Berg;
(Vaerlose, DK) ; Moretta; Alessandro; (Genoa,
IT) ; Della Chiesa; Mariella; (Genoa, IT) ;
Andre; Pascale; (Marseille, FR) ; Gauthier;
Laurent; (Marseille, FR) ; Reumert Wagtmann; Peter
Andreas Nicolai; (Rungsted Kyst, DK) |
Correspondence
Address: |
SALIWANCHIK LLOYD & SALIWANCHIK;A PROFESSIONAL ASSOCIATION
PO Box 142950
GAINESVILLE
FL
32614
US
|
Assignee: |
Novo Nordisk A/S
Novo Alle
Bagsvaerd
DK
DK-2880
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20060280740 A1 |
December 14, 2006 |
|
|
Family ID: |
37524330 |
Appl. No.: |
11/324356 |
Filed: |
January 3, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/DK04/00470 |
Jul 1, 2004 |
|
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11324356 |
Jan 3, 2006 |
|
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60545471 |
Feb 19, 2004 |
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60483894 |
Jul 2, 2003 |
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Current U.S.
Class: |
424/144.1 ;
435/362; 435/70.21; 530/388.22 |
Current CPC
Class: |
A61P 37/00 20180101;
C07K 16/2803 20130101; C07K 2317/21 20130101; G01N 33/5047
20130101 |
Class at
Publication: |
424/144.1 ;
530/388.22; 435/070.21; 435/362 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12P 21/04 20060101 C12P021/04; C12N 5/06 20060101
C12N005/06; C07K 16/28 20060101 C07K016/28 |
Claims
1. A method of producing an antibody which cross-reacts with
multiple KIR2DL gene products (KIRS) and which neutralizes the
inhibitory activity of such KIRs, said method comprising the steps
of: (a) immunizing a non-human mammal with an immunogen comprising
a KIR2DL polypeptide; (b) preparing antibodies from said immunized
mammal, wherein said antibodies bind said KIR2DL polypeptide, (c)
selecting antibodies of (b) that cross-react with at least two
different KIR2DL gene products, (d) selecting antibodies of (c)
that potentiate NK cells, and (e) selecting and isolating an
antibody from the antibodies of (d) that binds a primate NK cell or
KIR polypeptide.
2. The method of claim 1, wherein the primate in step (e) is a
cynomolgus monkey.
3. An isolated antibody, antibody fragment, or an antibody
derivative comprising one or more light variable region CDRs of
Pan2D as set forth in FIG. 12.
4. The antibody, antibody fragment, or an antibody derivative of
claim 3, wherein the antibody, antibody fragment, or antibody
derivative comprises the light chain variable region sequence of
Pan2D as set forth in FIG. 12.
5. An isolated antibody, antibody fragment, or an antibody
derivative comprising one or more of the light variable region CDRs
of DF-200 as set forth in FIG. 12.
6. The antibody, antibody fragment, or antibody derivative of claim
5, wherein the antibody, antibody fragment, or antibody derivative
comprises the light chain variable region sequence of DF-200 as set
forth in FIG. 12.
7. An isolated antibody, antibody fragment, or an antibody
derivative comprising one or more of the heavy variable region CDRs
of DF-200 as set forth in FIG. 13.
8. The antibody, antibody fragment, or an antibody derivative of
claim 10, wherein the antibody, antibody fragment, or antibody
derivative comprises the heavy chain variable region sequence of
DF-200 as set forth in FIG. 13.
9. An isolated antibody, antibody fragment, or antibody derivative
that exclusively binds to KIR2DL1 within a region defined by amino
acid residues 105, 106, 107, 108, 109, 110, 111, 127, 129, 130,
131, 132, 133, 134, 135, 152, 153, 154, 155, 156, 157, 158, 159,
160, 161, 162, 163, 181, 192.
10. An isolated antibody, antibody fragment, or antibody derivative
that binds to KIR2DL1 and KIR 2DL2/3 without interacting with amino
acid residues outside the region defined by residues 105, 106, 107,
108, 109, 110, 111, 127, 129, 130, 131, 132, 133, 134, 135, 152,
153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 181,
192.
11. An isolated antibody, antibody fragment, or antibody derivative
that binds to KIR2DL1 and does not bind to a mutant of KIR2DL1
characterized by one or more of R131 is Ala, R157 is Ala, and R158
is Ala.
12. An isolated antibody, antibody fragment, or antibody derivative
that binds to KIR2DL1 residues 131, 157, 158.
13. An isolated antibody, antibody fragment, or antibody derivative
that binds to KIR2DS3(R131W), but that does not bind to wild type
KIR2DS3.
14. An isolated antibody, antibody fragment, or antibody derivative
that binds KIR2DL1, KIR2DL2/3, and KIR2DS4.
15. An isolated antibody, antibody fragment, or antibody derivative
that binds to both KIR2DL1 and KIR2DL2/3, but not to KIR2DS4.
16. A pharmaceutically acceptable composition comprising an
effective amount of an antibody, antibody fragment, or antibody
derivative according to claim 15.
17. A pharmaceutically acceptable composition comprising an
effective amount of an antibody, antibody fragment, or antibody
derivative according to claim 14.
18. A pharmaceutically acceptable composition comprising an
effective amount of an antibody, antibody fragment, or antibody
derivative according to claim 3.
19. A pharmaceutically acceptable composition comprising an
effective amount of an antibody, antibody fragment, or antibody
derivative according to claim 4.
20. A pharmaceutically acceptable composition comprising an
effective amount of an antibody, antibody fragment, or antibody
derivative according to claim 5.
21. A pharmaceutically acceptable composition comprising an
effective amount of an antibody, antibody fragment, or antibody
derivative according to claim 6.
22. A pharmaceutically acceptable composition comprising an
effective amount of an antibody, antibody fragment, or antibody
derivative according to claim 7.
23. A pharmaceutically acceptable composition comprising an
effective amount of an antibody, antibody fragment, or antibody
derivative according to claim 8.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This patent application is a continuation of co-pending
International Patent Application PCT/DK2004/000470 (which is
published as WO2005003168 and designates the U.S.) and claims the
benefit of U.S. Provisional Patent Applications No. 60/483,984,
filed Jul. 2, 2003, and 60/545,471, filed Feb. 19, 2004, each of
which being hereby entirely incorporated by reference.
FIELD OF INVENTION
[0002] The present invention relates to antibodies, antibody
fragments, and derivatives thereof that cross-react with two or
more inhibitory receptors present on the cell surface of NK cells
and potentiate NK cell cytotoxicity in mammalian subjects or in a
biological sample. The invention also relates to methods of making
such antibodies, fragments, variants, and derivatives;
pharmaceutical compositions comprising the same; and the use of
such molecules and compositions, particularly in therapy, to
increase NK cell activity or cytotoxicity in subjects.
BACKGROUND
[0003] Natural killer (NK) cells are a sub-population of
lymphocytes, involved in non-conventional immunity. NK cells can be
obtained by various techniques known in the art, such as from blood
samples, cytapheresis, collections, etc.
[0004] Characteristics and biological properties of NK cells
include the expression of 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 NK activating receptor-ligand;
the ability to release cytokines that stimulate or inhibit the
immune response; and the ability to undergo multiple rounds of cell
division and produce daughter cells with similar biologic
properties as the parent cell. Within the context of this invention
"active" 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 fail to express "self"
MHC/HLA antigens (KIR-incompatible cells).
[0005] Based on their biological properties, various therapeutic
and vaccine strategies have been proposed in the art that rely on a
modulation of NK cells. However, NK cell activity is regulated by a
complex mechanism that involves both stimulating and inhibitory
signals. Accordingly, effective NK cell-mediated therapy may
require both a stimulation of these cells and a neutralization of
inhibitory signals.
[0006] NK cells are negatively regulated by major
histocompatibility complex (MHC) class I-specific inhibitory
receptors (Karre et al., 1986; (Ohlen et al, 1989). These specific
receptors bind to polymorphic determinants of MHC class I molecules
or HLA present on other cells and inhibit 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.
[0007] KIRs are a large family of receptors present on certain
subsets of lymphocytes, including NK cells. The nomenclature for
KIRs is based upon the number of extracellular domains (KIR2D or
KIR3D) and whether the cytoplasmic tail is either long (KIR2DL or
KIR3DL) or short (KIR2DS or KIR3DS). Within humans, the presence or
absence of a given KIR is variable from one NK cell to another
within the NK population present in a single individual. Within the
human population there is also a relatively high level of
polymorphism of the KIR molecules, with certain KIR molecules being
present in some, but not all individuals. Certain KIR gene products
cause stimulation of lymphocyte activity when bound to an
appropriate ligand. The confirmed stimulatory KIRs all have a short
cytoplasmic tail with a charged transmembrane residue that
associates with an adapter molecule having an immunostimulatory
motif (ITAM). Other KIR gene products are inhibitory in nature. All
confirmed inhibitory KIRs have a long cytoplasmic tail and appear
to interact with different subsets of HLA antigens depending upon
the KIR subtype. Inhibitory KIRs display in their intracytoplasmic
portion one or several inhibitory motifs that recruit phosphatases.
The known inhibitory KIR receptors include members of the KIR2DL
and KIR3DL subfamilies. KIR receptors having two Ig domains (KIR2D)
identify HLA-C allotypes: KIR2DL2 (formerly designated p58.2) or
the closely related gene product KIR2DL3 recognizes an epitope
shared by group 2 HLA-C allotypes (Cw1, 3, 7, and 8), whereas
KIR2DL1 (p58.1) recognizes an epitope shared by the reciprocal
group 1 HLA-C allotypes (Cw2, 4, 5, and 6). The recognition by
KIR2DL1 is dictated by the presence of a Lys residue at position 80
of HLA-C alleles. KIR2DL2 and KIR2DL3 recognition is dictated by
the presence of an Asn residue at position 80. Importantly the
great majority of HLA-C alleles have either an Asn or a Lys residue
at position 80. One KIR with three Ig domains, KIR3DL1 (p70),
recognizes an epitope shared by HLA-Bw4 alleles. Finally, a
homodimer of molecules with three Ig domains KIR3DL2 (p140)
recognizes HLA-A3 and -A11.
[0008] Although inhibitory KIRs and other class-I inhibitory
receptors (Moretta et al, 1997; Valiante et al, 1997a; Lanier,
1998) may be co-expressed by NK cells, in any given individual's NK
repertoire there are cells that express a single KIR and thus, the
corresponding NK cells are blocked only by cells expressing a
specific class I allele group.
[0009] NK cell population or clones that are KIR mismatched, i.e.,
population of NK cells that express KIR that are not compatible
with a HLA molecules of a host, have been shown to be the most
likely mediators of the graft anti-leukemia effect seen in
allogeneic transplantation (Ruggeri et al., 2002). One way of
reproducing this effect in a given individual would be to use
reagents that block the KIR/HLA interaction.
[0010] Monoclonal antibodies specific for KIR2DL1 have been shown
to block the interaction of KIR2DL1 with Cw4 (or the like) alleles
(Moretta et al., 1993). Monoclonal antibodies against KIR2DL2/3
have also been described that block the interaction of KIR2DL2/3
with HLACw3 (or the like) alleles (Moretta et al., 1993). However,
the use of such reagents in clinical situations would require the
development of two therapeutic mAbs to treat all patients,
regardless of whether any given patient was expressing class 1 or
class 2 HLA-C alleles. Moreover, one would have to pre-determine
which HLA type each patient was expressing before deciding which
therapeutic antibody to use, thus resulting in much higher cost of
treatment.
[0011] Watzl et al., Tissue Antigens, 56, p. 240 (2000) produced
cross-reacting antibodies recognizing multiple isotypes of KIRs,
but those antibodies did not exhibit potentiation of NK cell
activity. G. M. Spaggiara et al., Blood, 100, pp. 4098-4107 (2002)
carried out experiments utilizing numerous monoclonal antibodies
against various KIRs. One of those antibodies, NKVSF1, was said to
recognize a common epitope of CD158a KIR2DL1), CD158b (KIR2DL2) and
p50.3 (KIR2DS4). It is not suggested that NKVSF1 can potentiate NK
cell activity and there is no suggestion that it could be used as a
therapeutic. Accordingly, practical and effective approaches in the
modulation of NK cell activity have not been made available so far
in the art and still require HLA allele-specific intervention using
specific reagents.
SUMMARY OF THE INVENTION
[0012] The present invention now provides novel antibodies,
compositions, and methods that overcome current difficulties in NK
cell activation and provide additional advantageous features and
benefits. In one exemplary aspect, the invention provides a single
antibody that facilitates the activation of human NK cells in
virtually all humans. More particularly, the invention provides
novel specific antibodies that cross-react with various inhibitory
KIR groups and neutralize their inhibitory signals, resulting in
potentiation of NK cell cytotoxicity in NK cells expressing such
inhibitory KIR receptors. This ability to cross-react with multiple
KIR gene products allows the antibodies of the invention to be
effectively used to increase NK cell activity in most human
subjects, without the burden or expense of pre-determining the HLA
type of the subject.
[0013] In a first aspect, the invention provides antibodies,
antibody fragments, and derivatives of either thereof, wherein said
antibody, fragment, or derivative cross-reacts with at least two
inhibitory KIR receptors at the surface of NK cells, neutralizes
the inhibitory signals of the NK cells, and potentiates the
activity of the NK cells. More preferably, the antibody binds a
common determinant of human KIR2DL receptors. Even more
specifically, the antibody of this invention binds at least
KIR2DL1, KIR2DL2, and KIR2DL3 receptors. For the purposes of this
invention, the term "KIR2DL2/3" refers to either or both of the
KIR2DL2 and KIR2DL3 receptors. These two receptors have a very high
homology, are presumably allelic forms of the same gene, and are
considered by the art to be interchangeable. Accordingly, KIR2DL2/3
is considered to be a single inhibitory KIR molecule for the
purposes of this invention and therefore an antibody that
cross-reacts with only KIR2DL2 and KIR2DL3 and no other inhibitory
KIR receptors is not within the scope of this invention.
[0014] The antibody of this invention specifically inhibits binding
of MHC or HLA molecules to at least two inhibitory KIR receptors
and facilitates NK cell activity. Both activities are inferred by
the term "neutralize the inhibitory activity of KIR," as used
herein. The ability of the antibodies of this invention to
"facilitate NK cell activity," "facilitate NK cell cytotoxicity,"
"facilitate NK cells," "potentiate NK cell activity, " "potentiate
NK cell cytotoxicity," or "potentiate NK cells" in the context of
this invention means that the antibody permits NK cells expressing
an inhibitory KIR receptor on their surface to be capable of lysing
cells that express on their surface a corresponding ligand for that
particular inhibitory KIR receptor (e.g., a particular HLA
antigen). In a particular aspect, the invention provides an
antibody that specifically inhibits the binding of HLA-C molecules
to KIR2DL1 and KIR2DL2/3 receptors. In another particular aspect,
the invention provides an antibody that facilitates NK cell
activity in vivo.
[0015] Because at least one of KIR2DL1 or KID2DL2/3 is present in
at least about 90% of the human population, the more preferred
antibodies of this invention are capable of facilitating NK cell
activity against most of the HLA-C allotype-associated cells,
respectively group 1 HLA-C allotypes and group 2 HLA-C allotypes.
Thus, compositions of this invention may be used to effectively
activate or potentiate NK cells in most human individuals,
typically in about 90% of human individuals or more. Accordingly, a
single antibody composition according to the invention may be used
to treat most human subjects, and there is seldom need to determine
allelic groups or to use antibody cocktails.
[0016] The invention demonstrates, for the first time, that
cross-reactive and neutralizing antibodies against inhibitory KIRs
may be generated, and that such antibodies allow effective
activation of NK cells in a broad range of human groups.
[0017] A particular object of this invention thus resides in an
antibody, wherein said antibody specifically binds both KIR2DL1 and
KIR2DL2/3 human receptors and reverses inhibition of NK cell
cytotoxicity mediated by these KIRs. In one embodiment, the
antibody competes with monoclonal antibody DF200 produced by
hybridoma DF200. Optionally said antibody which competes with
antibody DF200 is not antibody DF200 itself.
[0018] In another embodiment, the antibody competes with monoclonal
antibody NKVSF1, optionally wherein the antibody which competes
with antibody NKVSF1 is not antibody NKVSF1.
[0019] In another embodiment, the antibody competes with antibody
1-7F9.
[0020] Preferably said antibodies are chimeric antibodies,
humanized antibodies, or human antibodies.
[0021] The term "competes with" when referring to a particular
monoclonal antibody (e.g. DF200, NKVSF1, 1-7F9, EB6, GL183) means
that an antibody competes with the monoclonal antibody (e.g. DF200,
NKVSF1, 1-7F9, EB6, GL183) in a binding assay using either
recombinant KIR molecules or surface expressed KIR molecules. For
example, if an antibody reduces binding of DF200 to a KIR molecule
in a binding assay, the antibody "competes" with DF200. An antibody
that "competes" with DF200 may compete with DF200 for binding to
the KIR2DL1 human receptor, the KIR2DL2/3 human receptor, or both
KIR2DL1 and KIR2DL2/3 human receptors.
[0022] In a preferred embodiment, the invention provides an
antibody that binds both KIR2DL1 and KIR2DL2/3 human receptors,
reverses inhibition of NK cell cytotoxicity mediated by these KIRs,
and competes with DF200, 1-7F9, or NKVSF1 for binding to the
KIR2DL1 human receptor, the KIR2DL2/3 human receptor, or both
KIR2DL1 and KIR2DL2/3 human receptors. Optionally, said antibody is
not NKVSF1. Optionally, said antibody is a chimeric, human, or
humanized antibody.
[0023] In another embodiment, the invention provides an antibody
that binds both KIR2DL1 and KIR2DL2/3 human receptors, reverses
inhibition of NK cell cytotoxicity mediated by these KIRs, and
competes with EB6 for binding to the KIR2DL1 human receptor,
competes with GL183 for binding to the KIR2DL2/3 human receptor, or
competes with both EB6 for binding to the KIR2DL1 human receptor
and GL183 for binding to the KIR2DL2/3 human receptor. Optionally,
said antibody is not NKVSF1; optionally said antibody is not DF200.
Optionally, said antibody is a chimeric, human, or humanized
antibody.
[0024] In an advantageous aspect, the invention provides an
antibody that competes with DF200 and recognizes, binds to, or has
immunospecificity for substantially or essentially the same, or the
same, epitope or "epitopic site" on a KIR molecule as the
monoclonal antibody DF200. Preferably, said KIR molecule is a
KIR2DL1 human receptor or a KIR2DL2/3 human receptor.
[0025] A particular object of this invention resides in an
antibody, wherein said antibody binds a common determinant present
in both KIR2DL1 and KIR2DL2/3 human receptors and reverses
inhibition of NK cell cytotoxicity mediated by these KIRs. The
antibody more specifically binds substantially the same epitope on
KIR as monoclonal antibody DF200 produced by hybridoma DF200 or
antibody NKVSF1 produced by hybridoma NKVSF1, wherein the antibody
is not NKVSF1.
[0026] In a preferred embodiment, the antibody of this invention is
a monoclonal antibody. The most preferred antibody of this
invention is monoclonal antibody DF200 produced by hybridoma
DF200.
[0027] The hybridoma producing antibody DF200 has been deposited at
the CNCM culture collection, as Identification no. "DF200",
registration no. CNCM I-3224, registered 10 Jun. 2004, Collection
Nationale de Cultures de Microorganismes, Institut Pasteur, 25, Rue
du Docteur Roux, F-75724 Paris Cedex 15, France. The antibody
NKVSF1 is available from Serotec (Cergy Sainte-Christophe, France),
Catalog ref no. MCA2243. NKVSF1 is also referred to as pan2D mAb
herein.
[0028] The invention also provides functional fragments and
derivatives of the antibodies described herein, having
substantially similar antigen specificity and activity (e.g., which
can cross-react with the parent antibody and which potentiate the
cytotoxic activity of NK cells expressing inhibitory KIR
receptors), including, without limitation, a Fab fragment, a Fab'2
fragment, an immunoadhesin, a diabody, a CDR, and a ScFv.
Furthermore, the antibodies of this invention may be humanized,
human, or chimeric.
[0029] The invention also provides antibody derivatives comprising
an antibody of the invention conjugated or covalently bound to a
toxin, a radionuclide, a detectable moiety (e.g., a fluor), or a
solid support.
[0030] The invention also provides pharmaceutical compositions
comprising an antibody as disclosed above, a fragment thereof, or a
derivative of either thereof. Accordingly, the invention also
relates to use of an antibody as disclosed herein in a method for
the manufacture of a medicament. In preferred embodiments, said
medicament or pharmaceutical composition is for the treatment of a
cancer or other proliferative disorder, an infection, or for use in
transplantation.
[0031] In another embodiment, the invention provides a composition
comprising an antibody that binds at least two different human
inhibitory KIR receptor gene products, wherein said antibody is
capable of neutralizing KIR-mediated inhibition of NK cell
cytotoxicity on NK cells expressing at least one of said two
different human inhibitory KIR receptors, wherein said antibody is
incorporated into a liposome. Optionally said composition comprises
an additional substance selected from a nucleic acid molecule for
the delivery of genes for gene therapy; a nucleic acid molecule for
the delivery of antisense RNA, RNAi, or siRNA for suppressing a
gene in an NK cell; or a toxin or a drug for the targeted killing
of NK cells additionally incorporated into said liposome.
[0032] The invention also provides methods of regulating human NK
cell activity in vitro, ex vivo, or in vivo, comprising contacting
human NK cells with an effective amount of an antibody of the
invention, a fragment of such an antibody, a derivative of either
thereof, or a pharmaceutical composition comprising at least one of
any thereof. Preferred methods comprise administration of an
effective amount of a pharmaceutical compositions of this invention
and are directed at increasing the cytotoxic activity of human NK
cells, most preferably ex vivo or in vivo, in a subject having a
cancer, an infectious disease, or an immune disease.
[0033] In further aspects, the invention provides a hybridoma
comprising: (a) a B cell from a mammalian host (typically a
non-human mammalian host) that has been immunized with an antigen
that comprises an epitope present on an inhibitory KIR polypeptide,
fused to (b) an immortalized cell (e.g., a myeloma cell), wherein
said hybridoma produces a monoclonal antibody binds at least two
different human inhibitory KIR receptors and is capable of at least
substantially neutralizing KIR-mediated inhibition of NK cell
cytotoxicity in a population of NK cells expressing said at least
two different human inhibitory KIR receptors. Optionally, said
hybridoma does not produce monoclonal antibody NKVSF1. Preferably
said antibody binds KIR2DL1 and KIR2DL2/3 receptors. Preferably
said antibody binds a common determinant present on KIR2DL1 and
KIR2DL2/3. Preferably said hybridoma produces an antibody that
inhibits the binding of a HLA-c allele molecule having a Lys
residue at position 80 to a human KIR2DL1 receptor, and the binding
of a HLA-C allele molecule having an Asn residue at position 80 to
human KIR2DL2/3 receptors. Preferably said hybridoma produces an
antibody that binds to substantially the same epitope as monoclonal
antibody DF200 produced by hybridoma DF200 on either KIR2DL1 or
KIR2DL2/3 or both KIR2DL1 and KIR2DL2/3. An example of such a
hybridoma is DF200.
[0034] The invention also provides methods of producing an antibody
which cross-reacts with multiple KIR2DL gene products and which
neutralizes the inhibitory activity of such KIRs, said method
comprising the steps of: [0035] immunizing a non-human mammal with
an immunogen comprising a KIR2DL polypeptide; [0036] preparing
antibodies from said immunized mammal, wherein said antibodies bind
said KIR2DL polypeptide, [0037] selecting antibodies of (b) that
cross-react with at least two different KIR2DL gene products, and
[0038] (d) selecting antibodies of (c) that potentiate NK cells. In
one embodiment, said non-human mammal is a transgenic animal
engineered to express a human antibody repertoire (e.g., a
non-human mammal comprising human immunoglobulin loci and native
immunoglobulin gene deletions, such as a Xenomousem.TM.
(Abgenix-Fremont, Calif., USA) or non-human mammal comprising a
minilocus of human Ig-encoding genes, such as the HuMab-mouse.TM.
(Medarex-Princeton, N.J., USA)). . Optionally, the method further
comprises selecting an antibody that binds a primate, preferably a
cynomolgus monkey, NK cell or KIR polypeptide. Optionally, the
invention further comprises a method of evaluating an antibody,
wherein an antibody produced according to the above method is
administered to a primate, preferably a cynomolgus monkey,
preferably wherein the monkey is observed for the presence or
absence of an indication of toxicity of the antibody.
[0039] The inventors also provide a method of producing an antibody
that binds at least two different human inhibitory KIR receptor
gene products, wherein said antibody is capable of neutralizing
KIR-mediated inhibition of NK cell cytotoxicity on a population of
NK cells expressing said at least two different human inhibitory
KIR receptor gene products, said method comprising the steps of:
[0040] immunizing a non-human mammal with an immunogen comprising
an inhibitory KIR polypeptide; [0041] preparing antibodies from
said immunized animal, wherein said antibodies bind said KIR
polypeptide, [0042] selecting antibodies of (b) that cross-react
with at least two different human inhibitory KIR receptor gene
products, and [0043] selecting antibodies of (c) that capable of
neutralizing KIR-mediated inhibition of NK cell cytotoxicity on a
population of NK cells expressing said at least two different human
inhibitory KIR receptor gene products, wherein the order of steps
(c) and (d) is optionally reversed and any number of the steps are
optionally repeated 1 or more times. Preferably, the inhibitory KIR
polypeptide used for immunization is a KIR2DL polypeptide and the
antibodies selected in step (c) cross-react with at least KIR2DL1
and KIR2DL2/3. Preferably said antibody recognizes a common
determinant present on at least two different KIR receptor gene
products; most preferably said KIR are KIR2DL1 and KIR2DL2/3.
Optionally, said method further comprises selecting an antibody
that binds a primate, preferably a cynomolgus monkey, NK cell or
KIR polypeptide. Optionally, the invention further comprises a
method of evaluating an antibody, wherein an antibody produced
according to the above method is administered to a primate,
preferably a cynomolgus monkey, preferably wherein the monkey is
observed for the presence or absence of an indication of toxicity
of the antibody.
[0044] Optionally, in the above-described methods, the antibody
selected in step c) or d) is not NKVSF1. Preferably, the antibody
prepared in step (b) in the above methods is a monoclonal antibody.
Preferably the antibody selected in step (c) in the above methods
inhibits the binding of a HLA-C allele molecule having a Lys
residue at position 80 to a human KIR2DL1 receptor, and the binding
of a HLA-C allele molecule having an Asn residue at position 80 to
human KIR2DL2/3 receptors. Preferably, the antibodies selected in
step (d) in the above methods cause a potentiation in NK
cytotoxicity, for example any substantial potentiation, or at least
5%, 10%, 20%, 30% or greater potentiation in NK cytotoxicity, e.g.
at least about 50% potentiation of target NK cytotoxicity (e.g., at
least about 60%, at least about 70%, at least about 80%, at least
about 85%, at least about 90%, or at least about 95% (such as, for
example about 65-100%) potentiation of NK cell cytotoxicity).
Preferably, the antibody binds to substantially the same epitope as
monoclonal antibody DF200 on KIR2DL1 and/or KIR2DL2/3. Optionally
said methods also or alternatively comprise the additional step of
making fragments of the selected monoclonal antibodies, making
derivatives of the selected monoclonal antibodies (e.g., by
conjugation with a radionuclide, cytotoxic agent, reporter
molecule, or the like), or making derivatives of antibody fragments
produced from or that comprise sequences that correspond to the
sequences of such monoclonal antibodies.
[0045] The invention further provides a method of producing an
antibody that binds at least two different human inhibitory KIR
receptor gene products, wherein said antibody is capable of
neutralizing KIR-mediated inhibition of NK cell cytotoxicity on a
population of NK cells expressing said at least two different human
inhibitory KIR receptor gene products, said method comprising the
steps of: [0046] (a) selecting, from a library or repertoire, a
monoclonal antibody or an antibody fragment that cross-reacts with
at least two different human inhibitory KIR2DL receptor gene
products, and [0047] (b) selecting an antibody of (a) that is
capable of neutralizing KIR-mediated inhibition of NK cell
cytotoxicity in a population of NK cells expressing said at least
two different human inhibitory KIR2DL receptor gene products.
Preferably the antibody binds a common determinant present on
KIR2DL1 and KIR2DL2/3. Optionally, said antibody selected in step
(b) is not NKVSF1. Preferably, the antibody selected in step (b)
inhibits the binding of a HLA-c allele molecule having a Lys
residue at position 80 to a human KIR2DL1 receptor, and the binding
of a HLA-C allele molecule having an Asn residue at position 80 to
human KIR2DL2/3 receptors. Preferably, the antibody selected in
step (b) causes a potentiation in NK cytotoxicity, for example any
substantial potentiation, or at least 5%, 10%, 20%, 30% or greater
potentiation in NK cytotoxicity, e.g. at least about 50%
potentiation of target NK cytotoxicity (e.g., at least about 60%,
at least about 70%, at least about 80%, at least about 85%, at
least about 90%, or at least about 95% (such as, for example about
65-100%) potentiation of NK cell cytotoxicity). Preferably, the
antibody binds to substantially the same epitope as monoclonal
antibody DF200 on KIR2DL1 and/or KIR2DL2/3. Optionally the method
comprises the additional step of making fragments of the selected
monoclonal antibodies, making derivatives of the selected
monoclonal antibodies, or making derivatives of selected monoclonal
antibody fragments.
[0048] Additionally, the invention provides a method of producing
an antibody that binds at least two different human inhibitory KIR
receptor gene products, wherein said antibody is capable of
neutralizing KIR-mediated inhibition of NK cell cytotoxicity in a
population of NK cells expressing said at least two different human
inhibitory KIR receptor gene products, said method comprising the
steps of: [0049] culturing a hybridoma of the invention under
conditions permissive for the production of said monoclonal
antibody; and [0050] separating said monoclonal antibody from said
hybridoma. Optionally the method comprises the additional step of
making fragments of the said monoclonal antibody, making
derivatives of the monoclonal antibody, or making derivatives of
such monoclonal antibody fragments . Preferably the antibody binds
a common determinant present on KIR2DL1 and KIR2DL2/3.
[0051] Also provided by the present invention is a method of
producing an antibody that binds at least two different human
inhibitory KIR receptor gene products, wherein said antibody is
capable of neutralizing KIR-mediated inhibition of NK cell
cytotoxicity in a population of NK cells expressing said at least
two different human inhibitory KIR receptor gene products, said
method comprising the steps of: [0052] isolating from a hybridoma
of the invention a nucleic acid encoding said monoclonal antibody;
[0053] optionally modifying said nucleic acid so as to obtain a
modified nucleic acid that comprises a sequence that encodes a
modified or derivatized antibody comprising an amino acid sequence
that corresponds to a functional sequence of the monoclonal
antibody or is substantially similar thereto (e.g., is at least
about 65%, at least about 75%, at least about 85%, at least about
90%, at least about 95% (such as about 70-99%) identical to such a
sequence) selected from a humanized antibody, a chimeric antibody,
a single chain antibody, an immunoreactive fragment of an antibody,
or a fusion protien comprising such an immunoreactive fragment;
inserting said nucleic acid or modified nucleic acid (or related
nucleic acid coding for the same amino acid sequence) into an
expression vector, wherein said encoded antibody or antibody
fragment is capable of being expressed when said expression vector
is present in a host cell grown under appropriate conditions;
[0054] transfecting a host cell with said expression vector,
wherein said host cell does not otherwise produce immunoglobulin
protein; [0055] culturing said transfected host cell under
conditions which cause the expression of said antibody or antibody
fragment; and [0056] isolating the antibody or antibody fragment
produced by said transfected host cell. Preferably the antibody
binds a common determinant present on KIR2DL1 and KIR2DL2/3.
[0057] In the context of this invention isolated antibodies and
related isolated molecules include antibodies produced by synthetic
means as well as those recovered from biological media, such as
antibodies recovered from recombinant cells.
[0058] It will be appreciated that the invention also provides a
composition comprising an antibody that binds at least two
different human inhibitory KIR receptor gene products, wherein said
antibody is capable of neutralizing KIR-mediated inhibition of NK
cell cytotoxicity in NK cells expressing at least one of said two
different human inhibitory KIR receptors, said antibody being
present in an amount effective to detectably potentiate NK cell
cytotoxicity in a patient or in a biological sample comprising NK
cells; and a pharmaceutically acceptable carrier or excipient.
Preferably the antibody binds a common determinant present on
KIR2DL1 and KIR2DL2/3. Said composition may optionally further
comprise a second therapeutic agent selected from, for example, an
immunomodulatory agent, a hormonal agent, a chemotherapeutic agent,
an anti-angiogenic agent, an apoptotic agent, a second antibody
that binds to and inhibits an inhibitory KIR receptor, an
anti-infective agent, a targeting agent, or an adjunct compound.
Advantageous immunomodulatory agents may be selected from IL-1
alpha, IL-1 beta, 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, or IFN-gamma. Examples of
said chemotherapeutic agents include alkylating agents,
antimetabolites, cytotoxic antibiotics, 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), other vinca
alkyloids and derivatives or prodrugs thereof. Examples of hormonal
agents include leuprorelin, goserelin, triptorelin, buserelin,
tamoxifen, toremifene, flutamide, nilutamide, cyproterone
bicalutamid anastrozole, exemestane, letrozole, fadrozole medroxy,
chlormadinone, megestrol, other LHRH agonists, other
anti-estrogens, other anti-androgens, other aromatase inhibitors,
and other progestagens. Preferably, said second antibody that binds
to and inhibits an inhibitory KIR receptor is an antibody or a
derivative or fragment thereof that binds to an epitope of an
inhibitory KIR receptor that differs from the epitope bound by said
antibody that binds a common determinant present on at least two
different human inhibitory KIR receptor gene products.
[0059] The invention further provides a method of detectably
potentiating NK cell activity in a patient in need thereof,
comprising the step of administering to said patient a composition
according to the invention. A patient in need of NK cell activity
potentiation can be any patient having a disease or disorder
wherein such potentiation may promote, enhance, and/or induce a
therapeutic effect (or promotes, enhances, and/or induces such an
effect in at least a substantial proportion of patients with the
disease or disorder and substantially similar characteristics as
the patient--as may determined by, e.g., clinical trials). A
patient in need of such treatment may be suffering from, e.g.,
cancer, another proliferative disorder, an infectious disease or an
immune disorder. Preferably said method comprises the additional
step of administering to said patient an appropriate additional
therapeutic agent selected from an immunomodulatory agent, a
hormonal agent, a chemotherapeutic agent, an anti-angiogenic agent,
an apoptotic agent, a second antibody that binds to and inhibits an
inhibitory KIR receptor, an anti-infective agent, a targeting agent
or an adjunct compound wherein said additional therapeutic agent is
administered to said patient as a single dosage form together with
said antibody, or as separate dosage form. The dosage of the
antibody (or antibody fragment/derivative) and the dosage of the
additional therapeutic agent collectively are sufficient to
detectably induce, promote, and/or enhance a therapeutic response
in the patient which comprises the potentiation of NK cell
activity. Where administered separately, the antibody, fragment, or
derivative and the additional therapeutic agent are desirably
administered under conditions (e.g., with respect to timing, number
of doses, etc.) that result in a detectable combined therapeutic
benefit to the patient.
[0060] Further encompassed by the present invention are antibodies
of the invention which are capable of specifically binding
non-human primate, preferably monkey, NK cells and/or monkey KIR
receptors. Also encompassed are methods for evaluating the
toxicity, dosage and/or activity or efficacy of antibodies of the
invention which are candidate medicaments. In one aspect, the
invention encompasses a method for determining a dose of an
antibody that is toxic to an animal or target tissue by
administering an antibody of the invention to an non-human primate
recipient animal having NK cells, and assessing any toxic or
deleterious or adverse effects of the agent on the animal, or
preferably on a target tissue. In another aspect, the invention is
a method for identifying an antibody that is toxic to an animal or
target tissue by administering an antibody of the invention to an
non-human primate recipient animal having NK cells, and assessing
any toxic or deleterious or adverse effects of the agent on the
animal, or preferably on a target tissue. In another aspect, the
invention is a method for identifying an antibody that is
efficacious in treatment of an infected, disease or tumor by
administering an antibody of the invention to a non-human primate
model of infection, disease or cancer, and identifying the antibody
that ameliorates the infection, disease or cancer, or a symptom
thereof. Preferably said antibody of the invention is an antibody
which (a) cross reacts with at least two inhibitory human KIR
receptors at the surface of human NK cells, and (b) cross-reacts
with NK cells or a KIR receptor of the non-human primate.
[0061] Further encompassed by the present invention is a method of
detecting the presence of NK cells bearing an inhibitory KIR on
their cell surface in a biological sample or a living organism,
said method comprising the steps of: [0062] contacting said
biological sample or living organism with an antibody of the
invention, wherein said antibody is conjugated or covalently bound
to a detectable moiety; and [0063] detecting the presence of said
antibody in said biological sample or living organism. [0064] The
invention also provides a method of purifying from a sample NK
cells bearing an inhibitory KIR on their cell surface comprising
the steps of: [0065] contacting said sample with an antibody of the
invention under conditions that allow said NK cells bearing an
inhibitory KIR on their cell surface to bind to said antibody,
wherein said antibody is conjugated or covalently bound to a solid
support (e.g., a bead, a matrix, etc.); and [0066] eluting said
bound NK cells from said antibody conjugated or covalently bound to
a solid support.
[0067] In a further aspect, the invention provides an antibody,
antibody fragment, or derivative of either thereof, that comprises
the light variable region or one or more light variable region CDRs
of antibody DF200 or antibody Pan2D as illustrated in FIG. 12. In
still another aspect, the invention provides an antibody, antibody
fragment, or derivative of either thereof that comprises a sequence
that is highly similar to all or essentially all of the light
variable region sequence of DF200 or Pan2D or one or more of the
light variable region CDRs of one or both of these antibodies.
[0068] In a further aspect, the invention provides an antibody,
antibody fragment, or derivative of either thereof, that comprises
the heavy variable region or one or more light variable region CDRs
of antibody DF200 as illustrated in FIG. 13. In still another
aspect, the invention provides an antibody, antibody fragment, or
derivative of either thereof that comprises a sequence that is
highly similar to all or essentially all of the heavy variable
region sequence of DF200.
[0069] These and additional advantageous aspects and features of
the invention may be further described elsewhere herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0070] FIG. 1 depicts monoclonal antibody DF200 binding to a common
determinant of various human KIR2DL receptors.
[0071] FIG. 2 depicts monoclonal antibody DF200 neutralizing the
KIR2DL-mediated inhibition of KIR2DL1 positive NK cell cytotoxicity
on Cw4 positive target cells.
[0072] FIG. 3 depicts monoclonal antibody DF200, a Fab fragment of
DF200 and KIR2DL1 or KIR2DL2/3 specific conventional antibodies
neutralizing the KIR2DL-mediated inhibition of KIR2DL1 positive NK
cell cytotoxicity on Cw4 positive target cells and the
KIR2DL-mediated inhibition of KIR2DL2/3 positive NK cell
cytotoxicity on Cw3 positive target cells.
[0073] FIG. 4 depicts reconstitution of cell lysis by NK clones of
HLA Cw4 positive target cells in the presence of F(ab')2 fragments
of the DF200 and EB6 antibodies.
[0074] FIGS. 5 and 6 depict monoclonal antibodies DF200, NKVSF1
(pan2D), human antibodies 1-7F9, 1-4F1, 1-6F5 and 1-6F 1, and
KIR2DL1 or KIR2DL2/3 specific conventional antibodies neutralizing
the KIR2DL-mediated inhibition of KIR2DL1 positive NK cell
cytotoxicity on Cw4 positive target cells (Cw4 transfected cells in
FIG. 5 and EBV cells in FIG. 6).
[0075] FIG. 7 depicts an epitope map showing results of competitive
binding experiments obtained by surface plasmon resonance
(BIAcore.RTM.) analysis with anti-KIR antibodies to KIR2DL1, where
overlapping circles designate overlap in binding to KIR2DL1.
Results show that 1-7F9 is competitive with EB6 and 1-4F1, but not
with NKVSF1 and DF200, on KIR 2DL1. Antibody 1-4 F1 in turn is
competitive with EB6, DF200, NKVSF1, and 1-7 F9. Antibody NKVSF1
competes with DF200, 1-4F 1, and EB6, but not 1-7F9, on KIR2DL1.
DF200 competes with NKVSF1, 1-4F1, and EB6, but not 1-7F9, on
KIR2DL1.
[0076] FIG. 8 depicts an epitope map showing results of competitive
binding experiments obtained by BIAcore.RTM. analysis with anti-KIR
antibodies to KIR2DL3, where overlapping circles designate overlap
in binding to KIR2DL3. Results show that 1-4F1 is competitive with
NKVSF1, DF200, gl183, and 1-7F9 on KIR2DL3. 1-7F9 is competitive
with DF200, gl183, and 1-4F1, but not with NKVSF1, on KIR2DL3.
NKVSF1 competes with DF200, 1-4F1, and GL183, but not 1-7F9, on
KIR2DL3. DF200 competes with NKVSF1, 1-4F1, and 1-7F9, but not with
GL183, on KIR2DL3.
[0077] FIG. 9 depicts an epitope map showing results of competitive
binding experiments obtained by BIAcore.RTM. analysis with anti-KIR
antibodies to KIR2DS1, where overlapping circles designate overlap
in binding to KIR2DS1. Results show that antibody 1-4F1 is
competitive with NKVSF1, DF200, and 1-7F9 on KIR2DS1. Antibody
1-7F9 is competitive with 1-4F1, but not competitive with DF200 and
NKVSF1 on KIR2DS1. NKVSF1 competes with DF200 and 1-4F1, but not
with 1-7F9, on KIR2DS1. DF200 competes with NKVSF1 and 1-4F1, but
not with 1-7F9, on KIR2DS1.
[0078] FIG. 10 depicts NKVSF1 (pan2D) mAb titration demonstrating
binding of the mAb to cynomolgus NK cells. Cynomolgus NK cells (NK
bulk day 16) were incubated with different amount of Pan2D mAb
followed by PE-conjugated goat F(ab')2 fragments anti-mouse IgG
(H+L) antibodies. The percentage of positive cells was determined
with an isotypic control (purified mouse IgG1).Samples were done in
duplicate. Mean fluorescence intensity =MFI.
[0079] FIG. 12 provides a comparative alignment of the amino acid
sequences of the light variable regions and light variable region
CDRs of antibodies DF200 and Pan2D mAb.
[0080] FIG. 13 provides the heavy variable region of antibody
DF200.
DETAILED DESCRIPTION OF THE INVENTION
Antibodies
[0081] The present invention provides novel antibodies and
fragments or derivatives thereof that bind common determinants of
human inhibitory KIR receptors, preferably a determinant present on
at least two different KIR2DL gene products, and cause potentiation
of NK cells expressing at least one of those KIR receptors. The
invention discloses, for the first time, that such cross-reacting
and neutralizing antibodies can be produced, which represents an
unexpected result and opens an avenue towards novel and effective
NK-based therapies, particularly in human subjects.
[0082] In a preferred embodiment, the antibody is not monoclonal
antibody NKVSF1.
[0083] Within the context of this invention a "common determinant"
designates a determinant or epitope that is shared by several gene
products of the human inhibitory KIR receptors. Preferably, the
common determinant is shared by at least two members of the KIR2DL
receptor group. More preferably, the determinant is shared by at
least KIR2DL1 and KIR2DL2/3. Certain antibodies of this invention
may, in addition to recognizing multiple gene products of KIR2DL,
also recognize determinants present on other inhibitory KIRs, such
as gene product of the KIR3DL receptor group. The determinant or
epitope may represent a peptide fragment or a conformational
epitope shared by said members. In a more specific embodiment, the
antibody of this invention specifically binds to substantially the
same epitope recognized by monoclonal antibody DF200. This
determinant is present on both KIR2DL1 and KIR2DL2/3.
[0084] Within the context of this invention, the term antibody that
"binds" a common determinant designates an antibody that binds said
determinant with specificity and/or affinity.
[0085] The term "antibody," as used herein, refers to polyclonal
and monoclonal antibodies, as well as to fragments and derivatives
of said polyclonal and monoclonal antibodies unless otherwise
stated or clearly contradicted by context. Depending on the type of
constant domain in the heavy chains, full length antibodies
typically 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. The
heavy-chain constant domains that correspond to the difference
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 because they are
the most common antibodies in the physiological situation and
because they are most easily made in a laboratory setting.
Preferably the antibody of this invention is a monoclonal antibody.
Because one of the goals of the invention is to block the
interaction of an inhibitory KIR and its corresponding HLA ligand
in vivo without depleting the NK cells, isotypes corresponding to
Fc receptors that mediate low effector function, such as IgG4,
typically are preferred.
[0086] The antibodies of this invention may be produced by 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 inhibitory KIR polypeptide,
preferably a KIR2DL polypeptide, more preferably a human KIR2DL
polypeptide. The inhibitory KIR polypeptide may comprise the full
length sequence of a human inhibitory KIR polypeptide, or a
fragment or derivative thereof, typically an immunogenic fragment,
i.e., a portion of the polypeptide comprising an epitope exposed on
the surface of the cell expressing an inhibitory KIR receptor. Such
fragments typically contain at least about 7 consecutive amino
acids of the mature polypeptide sequence, even more preferably at
least about 10 consecutive amino acids thereof. Fragments typically
are essentially derived from the extra-cellular domain of the
receptor. Even more preferred is a human KIR2DL polypeptide which
includes at least one, more preferably both, extracellular Ig
domains, of the full length KIRDL polypeptide and is capable of
mimicking at least one conformational epitope present in a KIR2DL
receptor. In other embodiments, said polypeptide comprises at least
about 8 consecutive amino acids of an extracellular Ig domain of
amino acid positions 1-224 of the KIR2DL1 polypeptide (amino acid
numbering of according to PROW web site describing the KIR gene
family, http://www.ncbi.nlm.nih.gov/prow/guide/1326018082. htm)
[0087] In a most preferred embodiment, the immunogen comprises a
wild-type human KIR2DL 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.
[0088] The step of immunizing a non-human mammal with an antigen
may be carried out in any manner well known in the art for
stimulating the production of antibodies in a mouse (see, for
example, E. Harlow and D. Lane, Antibodies: A Laboratory Manual.,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1988)). The immunogen is then 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. These
parameters may be different for different immunogens, but are
easily elucidated.
[0089] 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 I 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.
[0090] For polyclonal antibody preparation, serum is obtained from
an immunized non-human animal and the antibodies present therein
isolated by well-known techniques. The serum may be affinity
purified using any of the immunogens set forth above linked to a
solid support so as to obtain antibodies that react with inhibitory
KIR receptors.
[0091] In an alternate 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.
[0092] For monoclonal antibodies, the next step is the isolation of
splenocytes from the immunized non-human mammal and the subsequent
fusion of those splenocytes with an immortalized cell in order to
form an antibody-producing hybridoma. The isolation of splenocytes
from a non-human mammal is well-known in the art and typically
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.
[0093] Once isolated and present in single cell suspension, the
lymphocytes can be 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.
[0094] Hybridomas are typically 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 in
Goding, "Monoclonal Antibodies: Principles and Practice," pp.
59-103 (Academic Press, 1986), the disclosure of which is herein
incorporated by reference.
[0095] The cells are allowed to grow in the selection media for
sufficient time for colony formation and antibody production. This
is usually between about 7 and about 14 days. The hybridoma
colonies are then assayed for the production of antibodies that
cross-react with multiple inhibitory KIR receptor gene products.
The assay is typically a colorimetric ELISA-type assay, although
any assay may be employed that can be adapted to the wells that the
hybridomas are grown in. 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 re-cloned 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 re-cloned and
re-assayed to insure only one monoclonal antibody is being detected
and produced. Antibodies may also be produced by selection of
combinatorial libraries of immunoglobulins, as disclosed for
instance in Ward et al., Nature, 341 (1989) p. 544).
[0096] The antibodies of this invention are able to neutralize the
KIR-mediated inhibition of NK cell cytotoxicity; particularly
inhibition mediated by KIR2DL receptors and more particularly at
least both the KIR2DL1 and KIR2DL2/3 inhibition. These antibodies
are thus "neutralizing" or "inhibitory" antibodies, in the sense
that they block, at least partially and detectably, the inhibitory
signaling pathway mediated by KIR receptors when they interact with
MHC class I molecules. More importantly, this inhibitory activity
is displayed with respect to several types of inhibitory KIR
receptors, preferably several KIR2DL receptor gene products, and
more preferably at least both KIR2DL1 and KIR2DL2/3 so that these
antibodies may be used in various subjects with high efficacy.
Inhibition of KIR-mediated inhibition of NK cell cytotoxicity can
be assessed by various assays or tests, such as binding or cellular
assays.
[0097] Once an antibody that cross-reacts with multiple inhibitor
KIR receptors is identified, it can be tested for its ability to
neutralize the inhibitory effect of those KIR receptors in intact
NK cells. In a specific variant, the neutralizing activity can be
illustrated by the capacity of said antibody to reconstitute lysis
by KIR2DL-positive NK clones of HLA-C positive targets. In another
specific embodiment, the neutralizing activity of the antibody is
defined by the ability of the antibody to inhibit the binding of
HLA-C molecules to KIR2DL1 and KIR2DL3 (or the closely related
KIR2DL2) receptors, further preferably as it is the capacity of the
antibody to alter: [0098] the binding of a HLA-C molecule selected
from Cw1, Cw3, Cw7, and Cw8 (or of a HLA-C molecule having an Asn
residue at position 80) to KIR2DL2/3; and [0099] the binding of a
HLA-C molecule selected from Cw2, Cw4, Cw5 and Cw6 (or of a HLA-C
molecule having a Lys residue at position 80) to KIR2DL1.
[0100] In another variant, the inhibitory activity of an antibody
of this invention can be assessed in a cell based cytotoxicity
assay, as disclosed in the Examples provided herein.
[0101] In another variant, the inhibitory activity of an antibody
of this invention can be assessed in a cytokine-release assay,
wherein NK cells are incubated with the test antibody and a target
cell line expressing one HLA-C allele recognized by a KIR molecule
of the NK population, to stimulate NK cell cytokine production (for
example IFN-.gamma. and/or GM-CSF production). In an exemplary
protocol, IFN-.gamma. production from PBMC is assessed by cell
surface and intracytoplasmic staining and analysis by flow
cytometry after about 4 days in culture. Briefly, Brefeldin A
(Sigma Aldrich) can be added at a final concentration of about 5
.mu.g/ml for the least about 4 hours of culture. The cells can then
incubated with anti-CD3 and anti-CD56 mAb prior to permeabilization
(IntraPrep.TM.; Beckman Coulter) and staining with
PE-anti-IFN-.gamma. or PE-IgG1 (Pharmingen). GM-CSF and IFN-.gamma.
production from polyclonal activated NK cells can be measured in
supernatants using ELISA (GM-CSF: DuoSet Elisa, R&D Systems,
Minneapolis, MN; IFN-.gamma.: OptE1A set, Pharmingen).
[0102] Antibodies of this invention may partially or fully
neutralize the KIR-mediated inhibition of NK cell cytotoxicity. The
term "neutralize KIR-mediated inhibition of NK cell cytotoxicity,"
as used herein means the ability to increase to at least about 20%,
preferably to at least about 30%, at least about 40%, at least
about 50% or more (e.g., about 25-100%) of specific lysis obtained
at the same ratio with NK cells or NK cell lines that are not
blocked by their KIR, as measured by a classical chromium release
test of cytotoxicity, compared with the level of specific lysis
obtained without antibody when an NK cell population expressing a
given KIR is put in contact with a target cell expressing the
cognate MHC class I molecule (recognized by the KIR expressed on NK
cell). For example, preferred antibodies of this invention are able
to induce the lysis of matched or HLA compatible or autologous
target cell populations, i.e., cell populations that would not be
effectively lysed by NK cells in the absence of said antibody.
Accordingly, the antibodies of this invention may also be defined
as facilitating NK cell activity in vivo.
[0103] Alternatively, the term "neutralize KIR mediated inhibition"
means that in a chromium assay using an NK cell clone or
transfectant expressing one or several inhibitory KIRs and a target
cell expressing only one HLA allele that is recognized by one of
the KIRs on the NK cell, the level of cytotoxicity obtained with
the antibody should be at least about 20%, preferably at least
about 30%, at least about 40%, at least about 50% (e.g., about
25-100%), or more of the cytotoxicity obtained with a known
blocking anti MHC class I molecule, such as W6/32 anti MHC class I
antibody.
[0104] In a specific embodiment, the antibody binds substantially
the same epitope as monoclonal antibody DF200 (produced by
hybridoma DF200). Such antibodies are referred to herein as "DF200
like antibodies." In a further preferred embodiment, the antibody
is a monoclonal antibody. More preferred "DF200 like antibodies" of
this invention are antibodies other than the monoclonal antibody
NKVSF1. Most preferred is monoclonal antibody DF200 (produced by
hybridoma DF200).
[0105] The term "binds to substantially the same epitope or
determinant as" an antibody of interest means that an antibody
"competes" with said antibody of interest. The term "binds to
substantially the same epitope or determinant as" the monoclonal
antibody DF200 means that an antibody "competes" with DF200.
Generally, an antibody that "binds to substantially the same
epitope or determinant as" the monoclonal antibody of interest
(e.g. DF200, NKVSF1, 17F9) means that the antibody "competes" with
said antibody of interest for any one of more KIR molecules,
preferably a KIR molecule selected from the group consisting of
KIR2DL1 and KIR2DL2/3. In other examples, an antibody that binds to
substantially the same epitope or determinant on a KIR2DL1 molecule
as the antibody of interest "competes" with the antibody of
interest for binding to KIR2DL1. An antibody that binds to
substantially the same epitope or determinant on a KIR2DL2/3
molecule as the antibody of interest "competes" with antibody of
interest for binding to KIR2DL2/3.
[0106] The term "binds to essentially the same epitope or
determinant as" an antibody of interest means that an antibody
"competes" with said antibody of interest for any and all KIR
molecules to which said antibody of interest specifically binds.
The term "binds to essentially the same epitope or determinant as"
the monoclonal antibody DF200 means that an antibody "competes"
with DF200 for any and all KIR molecules to which DF200
specifically binds. For example, an antibody that binds to
essentially the same epitope or determinant as the monoclonal
antibodies DF200 or NKVSF1 "competes" with said DF200 or NKVSF1
respectively for binding to KIR2DL1, KIR2DL2/3, KIR2DS1 and
KIR2DS2.
[0107] The identification of one or more antibodies that bind(s) to
substantially or essentially the same epitope as the monoclonal
antibodies described herein can be readily determined using any one
of variety of immunological screening assays in which antibody
competition can be assessed. A number of such assays are routinely
practiced and well known in the art (see, e.g., U.S. Pat. No.
5,660,827, issued Aug. 26, 1997, which is specifically incorporated
herein by reference). It will be understood that actually
determining the epitope to which an antibody described herein binds
is not in any way required to identify an antibody that binds to
the same or substantially the same epitope as the monoclonal
antibody described herein.
[0108] For example, where the test antibodies to be examined are
obtained from different source animals, or are even of a different
Ig isotype, a simple competition assay may be employed in which the
control (DF200, for example) and test antibodies are admixed (or
pre-adsorbed) and applied to a sample containing both KIR2DL1 and
KIR2DL2/3, each of which is known to be bound by DF200. Protocols
based upon ELISAs, radioimmunoassays, Western blotting, and the use
of BIACORE analysis (as set forth, for example, in the Examples
section) are suitable for use in such simple competition
studies.
[0109] In certain embodiments, one would pre-mix the control
antibodies (DF200, for example) with varying amounts of the test
antibodies (e.g., about 1:10 or about 1:100) for a period of time
prior to applying to the inhibitory KIR antigen sample. In other
embodiments, the control and varying amounts of test antibodies can
simply be admixed during exposure to the KIR antigen sample. As
long as one can distinguish bound from free antibodies (e.g., by
using separation or washing techniques to eliminate unbound
antibodies) and DF200 from the test antibodies (e.g., by using
species-specific or isotype-specific secondary antibodies or by
specifically labeling DF200 with a detectable label) one will be
able to determine if the test antibodies reduce the binding of
DF200 to the two different KIR2DL antigens, indicating that the
test antibody recognizes substantially the same epitope as DF200.
The binding of the (labeled) control antibodies in the absence of a
completely irrelevant antibody can serve as the control high value.
The control low value can be obtained by incubating the labeled
(DF200) antibodies with unlabelled antibodies of exactly the same
type (DF200), where competition would occur and reduce binding of
the labeled antibodies. In a test assay, a significant reduction in
labeled antibody reactivity in the presence of a test antibody is
indicative of a test antibody that recognizes substantially the
same epitope, i.e., one that "cross-reacts" with the labeled
(DF200) antibody. Any test antibody that reduces the binding of
DF200 to each of KIR2DL1 and KIR2DL2/3 antigens by at least about
50%, such as at least about 60%, or more preferably at least about
70% (e.g., about 65-100%), at any ratio of DF200: test antibody
between about 1:10 and about 1:100 is considered to be an antibody
that binds to substantially the same epitope or determinant as
DF200. Preferably, such test antibody will reduce the binding of
DF200 to each of the KIR2DL antigens by at least about 90% (e.g.,
about 95%).
[0110] Competition can be assessed by, for example, a flow
cytometry test. In such a test, cells bearing a given KIR can be
incubated first with DF200, for example, and then with the test
antibody labeled with a fluorochrome or biotin. The antibody is
said to compete with DF200 if the binding obtained upon
preincubation with saturating amount of DF200 is about 80%,
preferably about 50%, about 40% or less (e.g., about 30%) of the
binding (as measured by mean of fluorescence) obtained by the
antibody without preincubation with DF200. Alternatively, an
antibody is said to compete with DF200 if the binding obtained with
a labeled DF200 (by a fluorochrome or biotin) on cells preincubated
with saturating amount of test antibody is about 80%, preferably
about 50%, about 40%, or less (e.g., about 30%) of the binding
obtained without preincubation with the antibody.
[0111] A simple competition assay in which a test antibody is
pre-adsorbed and applied at saturating concentration to a surface
onto which both KIR2DL1 and KIR2DL2/3 are immobilized also may be
advantageously employed. The surface in the simple competition
assay is preferably a BIACORE chip (or other media suitable for
surface plasmon resonance analysis). The control antibody (e.g.,
DF200) is then brought into contact with the surface at KIR2DL1 and
KIR2DL2/3-saturating concentration and the KIR2DL1 and KIR2DL2/3
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 KIR2DL1 and KIR2DL2/3-containing surface in the
absence of test antibody. In a test assay, a significant reduction
in binding of the KIR2DL1 and KIR2DL2/3-containing surface by the
control antibody in the presence of a test antibody indicates that
the test antibody recognizes substantially the same epitope as the
control antibody such that the test antibody "cross-reacts" with
the control antibody. Any test antibody that reduces the binding of
control (such as DF200) antibody to each of KIR2DL1 and KIR2DL2/3
antigens by at least about 30% or more preferably about 40% can be
considered to be an antibody that binds to substantially the same
epitope or determinant as a control (e.g., DF200). Preferably, such
test antibody will reduce the binding of the control antibody
(e.g., DF200) to each of the KIR2DL antigens by at least about 50%
(e.g., at least about 60%, at least about 70%, or more). It will be
appreciated that the order of control and test antibodies can be
reversed: that is the control antibody can be first bound to the
surface and the test antibody is brought into contact with the
surface thereafter in a competition assay. Preferably, the antibody
having higher affinity for KIR2DL1 and KIR2DL2/3 antigens is bound
to the KIR2DL1 and KIR2DL2/3-containing surface first, as it will
be expected that the decrease in binding seen for the second
antibody (assuming the antibodies are cross-reacting) will be of
greater magnitude. Further examples of such assays are provided in
the Examples and in, e.g., Saunal and Regenmortel, (1995) J.
Immunol. Methods 183: 33-41, the disclosure of which is
incorporated herein by reference.
[0112] While described in the context of DF200 for the purposes of
exemplification, it will be appreciated that the above-described
immunological screening assays can also be used to identify
antibodies that compete with NKVSF1, 1-7F9, EB6, GL183, and other
antibodies according to the invention.
[0113] Upon immunization and production of antibodies in a
vertebrate or cell, particular selection steps may be performed to
isolate antibodies as claimed. In this regard, in a specific
embodiment, the invention also relates to methods of producing such
antibodies, comprising: [0114] immunizing a non-human mammal with
an immunogen comprising an inhibitory KIR polypeptide; [0115]
preparing antibodies from said immunized animal, wherein said
antibodies bind said KIR polypeptide, [0116] selecting antibodies
of (b) that cross-react with at least two different inhibitory KIR
gene products, and [0117] selecting antibodies of (c) that are
capable of neutralizing KIR-mediated inhibition of NK cell
cytotoxicity on a population of NK cells expressing said at least
two different human inhibitory KIR receptor gene products. The
selection of an antibody that cross-reacts with at least two
different inhibitory KIR gene products may be achieved by screening
the antibody against two or more different inhibitory KIR antigens,
for example as described above.
[0118] In a more preferred embodiment, the antibodies prepared in
step (b) are monoclonal antibodies. Thus, the term "preparing
antibodies from said immunized animal," as used herein, includes
obtaining B-cells from an immunized animal and using those B cells
to produce a hybridoma that expresses antibodies, as well as
obtaining antibodies directly from the serum of an immunized
animal. In another preferred embodiment, the antibodies selected in
step (c) are those that cross-react with at least KIR2DL1 and
KIR2DL2/3.
[0119] In yet another preferred embodiment, the antibodies selected
in step (d) cause at least about 10% specific lysis mediated by NK
cells displaying at least one KIR recognized by the antibody, and
preferably at least about 40% specific lysis, at least about 50%
specific lysis, or more preferably at least about 70% specific
lysis (e.g., about 60-100% specific lysis), as measured in a
standard chromium release assay, towards a target cell expressing
cognate HLA class I molecule, compared with the lysis or
cytotoxicity obtained at the same effector/target ratio with NK
cells that are not blocked by their KIR. Alternatively, the
antibodies selected in step (d) when used in a chromium assay
employing an NK cell clone expressing one or several inhibitory
KIRs and a target cell expressing only one HLA allele that is
recognized by one of the KIRs on the NK clone, the level of
cytotoxicity obtained with the antibody should be at least about
20% preferably at least about 30%, or more of the cytotoxicity
obtained with a blocking anti MHC class I mAb such as W6/32 anti
MHC class I antibody.
[0120] The order of steps (c) and (d) of the immediately
above-described method can be changed. Optionally, the method also
or alternatively may further comprise additional steps of making
fragments of the monoclonal antibody or derivatives of the
monoclonal antibody or such fragments, e.g., as described elsewhere
herein.
[0121] In a preferred embodiment, the non-human animal used to
produce antibodies according to applicable methods of the invention
is a mammal, such as a rodent (e.g., mouse, rat, etc.), bovine,
porcine, horse, rabbit, goat, sheep, etc. Also, the non-human
mammal may be genetically modified or engineered to produce "human"
antibodies, such as the Xenomouse.TM. (Abgenix) or HuMAb-Mouse.TM.
(Medarex).
[0122] In another variant, the invention provides a method for
obtaining an antibody that comprises: [0123] selecting, from a
library or repertoire, a monoclonal antibody, a fragment of a
monoclonal antibody, or a derivative of either thereof that
cross-reacts with at least two different human inhibitory KIR2DL
receptor gene products, and [0124] selecting an antibody, fragment,
or derivative of (a) that is capable of neutralizing KIR-mediated
inhibition of NK cell cytotoxicity on a population of NK cells
expressing said at least two different human inhibitory KIR2DL
receptor gene products.
[0125] 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.). Selection of inhibitory antibodies may be performed as
disclosed above and further illustrated in the examples.
[0126] According to another embodiment, the invention provides a
hybridoma comprising a B cell from a non-human host, wherein said B
cell produces an antibody that binds a determinant present on at
least two different human inhibitory KIR receptor gene products and
said antibody is capable of neutralizing the inhibitory activity of
said receptors. More preferably, the hybridoma of this aspect of
the invention is not a hybridoma that produces the monoclonal
antibody NKVSF1. The hybridoma according to this aspect of the
invention can be created as described above by the fusion of
splenocytes from the immunized non-human mammal with an immortal
cell line. Hybridomas produced by this fusion can be screened for
the presence of such a cross-reacting antibody as described
elsewhere herein. Preferably, the hybridoma produces an antibody
the recognizes a determinant present on at least two different
KIR2DL gene products, and cause potentiation of NK cells expressing
at least one of those KIR receptors. Even more preferably, the
hybridoma produces an antibody that binds to substantially the same
epitope or determinant as DF200 and which potentiates NK cell
activity. Most preferably, that hybridoma is hybridoma DF200 which
produces monoclonal antibody DF200.
[0127] Hybridomas that are confirmed to produce a monoclonal
antibody of this invention can be 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.
[0128] 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.
[0129] According to an alternate embodiment, the DNA encoding an
antibody that binds a determinant present on at least two different
human inhibitory KIR receptor gene products, is isolated from the
hybridoma of this invention and placed in an appropriate expression
vector for transfection into an appropriate host. The host is then
used for the recombinant production of the antibody, or variants
thereof, such as a humanized version of that monoclonal antibody,
active fragments of the antibody, 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 at least two different KIR2DL gene products,
and cause potentiation of NK cells expressing at least one of those
KIR receptors. Even more preferably, the DNA encodes an antibody
that binds to substantially the same epitope or determinant as
DF200 and which potentiates NK cell activity. Most preferably, that
DNA encodes monoclonal antibody DF200.
[0130] DNA encoding the monoclonal antibodies of the invention is
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.,
Curr. Opinion in Immunol., 5, pp. 256 (1993); and Pluckthun,
Immunol. Revs., 130, pp. 151 (1992).
Fragments and Derivatives of a Monoclonal Antibody
[0131] Fragments and derivatives of antibodies of this invention
(which are encompassed by the term "antibody" or "antibodies" as
used in this application, unless otherwise stated or clearly
contradicted by context), preferably a DF-200-like antibody, can be
produced by techniques that are known in the art. "Immunoreactive
fragments" comprise a portion of the intact antibody, generally the
antigen binding site or variable region. Examples of antibody
fragments include Fab, Fab', Fab'-SH, F(ab').sub.2, and Fv
fragments; diabodies; any antibody fragment that is a polypeptide
having a primary structure consisting of one uninterrupted sequence
of contiguous amino acid residues (referred to herein as a
"single-chain antibody fragment" or "single chain polypeptide"),
including without limitation (1) single-chain Fv (scFv) molecules
(2) single chain polypeptides containing only one light chain
variable domain, or a fragment thereof that contains the three CDRs
of the light chain variable domain, without an associated heavy
chain moiety and (3) single chain polypeptides containing only one
heavy chain variable region, or a fragment thereof containing the
three CDRs of the heavy chain variable region, without an
associated light chain moiety; and multispecific antibodies formed
from antibody fragments. For instance, Fab or F(ab')2 fragments may
be produced by protease digestion of the isolated antibodies,
according to conventional techniques. It will be appreciated that
immunoreactive fragments can be modified using known methods, for
example to slow clearance in vivo and obtain a more desirable
pharmacokinetic profile the fragment may be modified with
polyethylene glycol (PEG). Methods for coupling and
site-specifically conjugating PEG to a Fab' fragment are described
in, for example, Leong et al, Cytokine 16(3):106-119 (2001) and
Delgado et al, Br. J. Cancer 73(2):175-182 (1996), the disclosures
of which are incorporated herein by reference.
[0132] In a particular aspect, the invention provides antibodies,
antibody fragments, and antibody derivatives comprising the light
chain variable region sequence of DF-200 as set forth in FIG. 12.
In another particular aspect, the invention provides antibodies,
antibody fragments, and antibody derivatives that comprise the
light chain variable region sequence of Pan2D as set forth in FIG.
12. In another aspect, the invention provides antibodies, antibody
fragments, and derivatives thereof that comprise one or more of the
light variable region CDRs of DF-200 as set forth in FIG. 12. In
yet another aspect, the invention provides antibodies, antibody
fragments, and derivatives thereof that comprise one or more light
variable region CDRs of Pan2D as set forth in FIG. 12. Functional
variants/analogs of such sequences can be generated by making
suitable substitutions, additions, and/or deletions in these
disclosed amino acid sequences using standard techniques, which may
be aided by the comparison of the sequences. Thus, for example, CDR
residues that are conserved between Pan2D and DF-200 may be
suitable targets for modification inasmuch as such residues may not
contribute to the different profiles in competition these
antibodies have with respect to other antibodies disclosed herein
(although Pan2D and DF-200 do compete) and thus may not contribute
to the specificity of these antibodies for their particular
respective epitopes. In another aspect, positions where a residue
is present in a sequence of one of these antibodies, but not
another, may be suitable for deletions, substitutions, and/or
insertions.
[0133] In a particular aspect, the invention provides antibodies,
antibody fragments, and antibody derivatives comprising the heavy
chain variable region sequence of DF-200 as set forth in FIG. 13.
In another aspect, the invention provides antibodies, antibody
fragments, and derivatives thereof that comprise one or more of the
heavy variable region CDRs of DF-200 as set forth in FIG. 13.
Functional variants/analogs of such sequences can be generated by
making suitable substitutions, additions, and/or deletions in these
disclosed amino acid sequences using standard techniques, which may
be aided by the comparison of the sequences. In another aspect,
positions where a residue is present in a sequence of one of these
antibodies, but not another, may be suitable for deletions,
substitutions, and/or insertions.
[0134] Alternatively, the DNA of a hybridoma producing an antibody
of this invention, preferably a DF-200-like antibody, may be
modified so as to encode for a fragment of this invention. The
modified DNA is then inserted into an expression vector and used to
transform or transfect an appropriate cell, which then expresses
the desired fragment.
[0135] In an alternate embodiment, the DNA of a hybridoma producing
an antibody of this invention, preferably a DF-200-like antibody,
can be modified prior to insertion into an expression vector, for
example, by substituting the coding sequence for human heavy- and
light-chain constant domains in place of the homologous non-human
sequences (e.g., Morrison et al., Proc. Natl. Acad. Sci. U.S.A.,
81, pp. 6851 (1984)), or by covalently joining to the
immunoglobulin coding sequence all or part of the coding sequence
for a non-immunoglobulin polypeptide. In that manner, "chimeric" or
"hybrid" antibodies are prepared that have the binding specificity
of the original antibody. Typically, such non-immunoglobulin
polypeptides are substituted for the constant domains of an
antibody of the invention.
[0136] Thus, according to another embodiment, the antibody of this
invention, preferably a DF-200-like antibody, 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') .sub.2, or other
antigen-binding subsequences of antibodies) which contain minimal
sequence derived from the murine immunoglobulin. For the most part,
humanized antibodies are human immunoglobulins (recipient antibody)
in which residues from a complementary-determining region (CDR) of
the recipient are replaced by residues from a CDR of the original
antibody (donor antibody) while maintaining the desired
specificity, affinity, and capacity of the original antibody. In
some instances, Fv framework residues of the human immunoglobulin
may be replaced by corresponding non-human residues. Furthermore,
humanized antibodies can comprise residues that are not found in
either the recipient antibody or in the imported CDR or framework
sequences. These modifications are made to further refine and
optimize antibody performance. In general, the humanized antibody
will comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the CDR
regions correspond to those of the original antibody and all or
substantially all of the FR regions are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally
also will comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin. For further
details see Jones et al., Nature, 321, pp. 522 (1986); Reichmann et
al., Nature, 332, pp. 323 (1988); and Presta, Curr. Op. Struct.
Biol., 2, pp. 593 (1992).
[0137] Methods for humanizing the antibodies of this invention are
well known in the art. Generally, a humanized antibody according to
the present invention has one or more amino acid residues
introduced into it from the original antibody. These murine or
other non-human amino acid residues are often referred to as
"import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers (Jones et al.,
Nature, 321, pp. 522 (1986); Riechmann et al., Nature, 332, pp. 323
(1988); Verhoeyen et al., Science, 239, pp. 1534 (1988)).
Accordingly, such "humanized" antibodies are chimeric antibodies
(Cabilly et al., U.S. Pat. No. 4,816,567), wherein substantially
less than an intact human variable domain has been substituted by
the corresponding sequence from the original antibody. In practice,
humanized antibodies according to this invention are typically
human antibodies in which some CDR residues and possibly some FR
residues are substituted by residues from analogous sites in the
original antibody.
[0138] 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., J. Immunol., 151, pp. 2296
(1993); Chothia and Lesk, J. Mol. Biol., 196, pp. 901 (1987)).
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., Proc. Natl. Acad.
Sci. U.S.A., 89, pp. 4285 (1992); Presta et al., J. Immunol., 51,
pp. 1993)).
[0139] It is further important that antibodies be humanized with
retention of high affinity for multiple inhibitory KIR receptors
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.
[0140] Another method of making "humanized" monoclonal antibodies
is to use a XenoMouse(.RTM. (Abgenix, Fremont, Calif.) as the mouse
used for immunization. A XenoMouse is a murine host according to
this invention 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.RTM. (Medarex).
[0141] Human antibodies may also be produced according to various
other techniques, such as by using, for immunization, other
transgenic animals that have been engineered to express a human
antibody repertoire (Jakobovitz et al., Nature 362 (1993) 255), or
by selection of antibody repertoires using phage display methods.
Such techniques are known to the skilled person and can be
implemented starting from monoclonal antibodies as disclosed in the
present application.
[0142] The antibodies of the present invention, preferably a
DF-200-like antibody, 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
(Cabilly et al., supra; Morrison et al., Proc. Natl. Acad. Sci.
U.S.A., 81, pp. 6851 (1984)).
[0143] Other derivatives within the scope of this invention include
functionalized antibodies, i.e., antibodies that are conjugated or
covalently bound to a toxin, such as ricin, diphtheria toxin, abrin
and Pseudomonas exotoxin; to a detectable moiety, such as a
fluorescent moiety, a radioisotope or an imaging agent; or to a
solid support, such as agarose beads or the like. Methods for
conjugation or covalent bonding of these other agents to antibodies
are well known in the art.
[0144] Conjugation to a toxin is useful for targeted killing of NK
cells displaying one of the cross-reacting KIR receptors on its
cell surface. Once the antibody of the invention binds to the cell
surface of such cells, it is internalized and the toxin is released
inside of the cell, selectively killing that cell. Such use is an
alternate embodiment of the present invention.
[0145] Conjugation to a detectable moiety is useful when the
antibody of this invention is used for diagnostic purposes. Such
purposes include, but are not limited to, assaying biological
samples for the presence of the NK cells bearing the cross-reacting
KIR on their cell surface and detecting the presence of NK cells
bearing the cross-reacting KIR in a living organism. Such assay and
detection methods are also alternate embodiments of the present
invention.
[0146] Conjugation of an antibody of this invention to a solid
support is useful as a tool for affinity purification of NK cells
bearing the cross-reacting KIR on their cell surface from a source,
such as a biological fluid. This method of purification is another
alternate embodiment of the present invention, as is the resulting
purified population of NK cells.
[0147] In an alternate embodiment, an antibody that binds a common
determinant present on at least two different human inhibitory KIR
receptor gene products, wherein said antibody is capable of
neutralizing KIR-mediated inhibition of NK cell cytotoxicity on NK
cells expressing at least one of said two different human
inhibitory KIR receptors of this invention, including NKVSF1, may
be incorporated into liposomes ("immunoliposomes"), alone or
together with another substance for targeted delivery to an animal.
Such other substances include nucleic acids for the delivery of
genes for gene therapy or for the delivery of antisense RNA, RNAi
or siRNA for suppressing a gene in an NK cell, or toxins or drugs
for the targeted killing of NK cells.
[0148] Computer modelling of the extra-cellular domains of KIR2DL1,
-2 and -3 (KIR2DL1-3), based on their published crystal-structures
(Maenaka et al. (1999), Fan et al. (2001), Boyington et al.
(2000)), predicted the involvement of certain regions or KIR2DL1,
-2 and -3 in the interaction between KIR2DL1 and the
KIR2DL1-3-cross-reactive mouse monoclonal antibodies DF200 and
NKVSF1. Thus, in one embodiment, the present invention provides
antibodies that exclusively bind to KIR2DL1 within a region defined
by the amino acid residues (105, 106, 107, 108, 109, 110, 111, 127,
129, 130, 131, 132, 133, 134, 135, 152, 153, 154,155,156, 157, 158,
159, 160, 161, 162, 163, 181, 192). In another embodiment the
invention provides antibodies that bind to KIR2DL1 and KIR 2DL2/3
without interacting with amino acid residues outside the region
defined by the residues (105, 106, 107,108, 109, 110, 111, 127,
129, 130, 131, 132, 133, 134, 135, 152, 153, 154, 155, 156, 157,
158, 159, 160, 161, 162, 163, 181, 192).
[0149] In another embodiment, the invention provides antibodies
that bind to KIR2DL1 and which does not bind to a mutant of KIR2DL1
in which R131 is Ala.
[0150] In another embodiment, the invention provides antibodies
that bind to KIR2DL1 and which does not bind to a mutant of KIR2DL1
in which R157 is Ala.
[0151] In another embodiment, the invention provides antibodies
that bind to KIR2DL1 and which does not bind to a mutant of KIR2DL1
in which R158 is Ala.
[0152] In another embodiment, the invention provides antibodies
that bind to KIR2DL1 residues (131, 157, 158).
[0153] In another embodiment, the invention provides antibodies
that bind to KIR2DS3(R131W), but not to wild type KIR2DS3.
[0154] In another embodiment, the invention provides antibodies
that bind to both KIR2DL1 and KIR2DL2/3 as well as KIR2DS4.
[0155] In another embodiment, the invention provides antibodies
that bind to both KIR2DL1 and KIR2DL2/3, but not to KIR2DS4.
[0156] Determination of whether an antibody binds within one of the
epitope regions defined above can be carried out in ways known to
the person skilled in the art. As one example of such
mapping/characterization methods, an epitope region for an anti-KIR
antibody may be determined by epitope "foot-printing" using
chemical modification of the exposed amines/carboxyls in the
KIR2DL1 or KIR2DL2/3 protein. One specific example of such a
foot-printing technique is the use of HXMS (hydrogen-deuterium
exchange detected by mass spectrometry) wherein a
hydrogen/deuterium exchange of receptor and ligand protein amide
protons, binding, and back exchange occurs, wherein the backbone
amide groups participating in protein binding are protected from
back exchange and therefore will remain deuterated. Relevant
regions can be identified at this point by peptic proteolysis, fast
microbore high-performance liquid chromatography separation, and/or
electrospray ionization mass spectrometry. See, e.g., Ehring H,
Analytical Biochemistry, Vol. 267 (2) pp. 252-259 (1999) and/or
Engen, J. R. and Smith, D. L. (2001) Anal. Chem. 73, 256A-265A.
Another example of a suitable epitope identification technique is
nuclear magnetic resonance epitope mapping (NMR), where typically
the position of the signals in two-dimensional NMR spectres of the
free antigen and the antigen complexed with the antigen binding
peptide, such as an antibody, are compared. The antigen typically
is selectively isotopically labeled with .sup.15N so that only
signals corresponding to the antigen and no signals from the
antigen binding peptide are seen in the NMR-spectrum. Antigen
signals originating from amino acids involved in the interaction
with the antigen binding peptide typically will shift position in
the spectres of the complex compared to the spectres of the free
antigen, and the amino acids involved in the binding can be
identified that way. See, e.g., Ernst Schering Res Found Workshop.
2004; (44):149-67; Huang et al, Journal of Molecular Biology, Vol.
281 (1) pp. 61-67 (1998); and Saito and Patterson, Methods. 1996
Jun;9(3):516-24.
[0157] Epitope mapping/characterization also can be performed using
mass spectrometry methods. See, e.g., Downward, J Mass Spectrom.
2000 Apr;35(4):493-503 and Kiselar and Downard, Anal Chem. 1999 May
1;71(9):1792-801.
[0158] Protease digestion techniques also can be useful in the
context of epitope mapping and identification. Antigenic
determinant-relevant regions/sequences can be determined by
protease digestion, e.g. by using trypsin in a ratio of about 1:50
to KIR2DL1 or KIR2DL2/3 o/n digestion at 37.degree. C. and pH 7-8,
followed by mass spectrometry (MS) analysis for peptide
identification. The peptides protected from trypsin cleavage by the
anti-KIR binder can subsequently be identified by comparison of
samples subjected to trypsin digestion and samples incubated with
antibody and then subjected to digestion by e.g. trypsin (thereby
revealing a foot print for the binder). Other enzymes like
chymotrypsin, pepsin, etc., also or alternatively can be used in a
similar epitope characterization methods. Moreover, enzymatic
digestion can provide a quick method for analyzing whether a
potential antigenic determinant sequence is within a region of the
KIR2DL1 in the context of a Anti-KIR polypeptide that is not
surface exposed and, accordingly, most likely not relevant in terms
of immunogenicity/antigenicity. See, e.g., Manca, Ann Ist Super
Sanita. 1991;27(l):15-9 for a discussion of similar techniques.
Crossreactivity with Cynomolgus Monkeys
[0159] It has been found that antibody NKVSF1 also binds to NK
cells from cynomolgus monkeys, see example 7. The invention
therefore provides an an antibody, as well as fragments and
derivatives thereof, wherein said antibody, fragment or derivative
cross-reacts with at least two inhibitory human KIR receptors at
the surface of human NK cells, and which furthermore binds to NK
cells from cynomolgus monkeys. In one embodiment hereof, the
antibody is not antibody NKVSF1. The invetion also provdes a method
of testing the toxicity of an antibody, as well as fragments and
derivatives thereof, wherein said antibody, fragment or derivative
cross-reacts with at least two inhibitory human KIR receptors at
the surface of human NK cells, wherein the method comprises testing
the antibody in a cynomolgus monkey.
Compositions and Administration
[0160] The invention also provides pharmaceutical compositions that
comprise an antibody, as well as fragments and derivatives thereof,
wherein said antibody, fragment or derivative cross-reacts with at
least two inhibitory KIR receptors at the surface of NK cells,
neutralizes their inhibitory signals and potentiates the activity
of those cells, in any suitable vehicle in an amount effective to
detectably potentiate NK cell cytotoxicity in a patient or in a
biological sample comprising NK cells. The composition further
comprises a pharmaceutically acceptable carrier. Such compositions
are also referred to as "antibody compositions of this invention."
In one embodiment, antibody compositions of this invention comprise
an antibody disclosed in the antibody embodiments above. The
antibody NKVSF1 is included within the scope of antibodies that may
be present in the antibody compositions of this invention.
[0161] 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).
[0162] Pharmaceutically acceptable carriers that may be used in
these compositions include, but are not limited to, ion exchangers,
alumina, aluminum stearate, lecithin, serum proteins, such as human
serum albumin, buffer substances such as phosphates, glycine,
sorbic acid, potassium sorbate, partial glyceride mixtures of
saturated vegetable fatty acids, water, salts or electrolytes, such
as protamine sulfate, disodium hydrogen phosphate, potassium
hydrogen phosphate, sodium chloride, zinc salts, colloidal silica,
magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based
substances, polyethylene glycol, sodium carboxymethylcellulose,
polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,
polyethylene glycol and wool fat.
[0163] The compositions of this invention may be employed in a
method of potentiating the activity of NK cells in a patient or a
biological sample. This method comprises the step of contacting
said composition with said patient or biological sample. Such
method will be useful for both diagnostic and therapeutic
purposes.
[0164] For use in conjunction with a biological sample, the
antibody composition can be administered by simply mixing with or
applying directly to the sample, depending upon the nature of the
sample (fluid or solid). The biological sample may be contacted
directly with the antibody in any suitable device (plate, pouch,
flask, etc.). For use in conjunction with a patient, the
composition must be formulated for administration to the
patient.
[0165] 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, intrastemal, intrathecal, intrahepatic,
intralesional and intracranial injection or infusion techniques.
Preferably, the compositions are administered orally,
intraperitoneally or intravenously.
[0166] 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.
[0167] 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.
[0168] 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.
[0169] The compositions of this invention may also 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, or the lower intestinal tract.
Suitable topical formulations are readily prepared for each of
these areas or organs.
[0170] 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.
[0171] 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.
[0172] 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.
[0173] 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.
[0174] Several monoclonal antibodies have been shown to be
efficient in clinical situations, such as Rituxan (Rituximab),
Herceptin (Trastuzumab) or Xolair (Omalizumab), 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 of the antibody
in the pharmaceutical compositions of the present invention can be
determined in accordance with known methods for these products, for
example using the manufacturers' instructions. For example, an
antibody present in a pharmaceutical composition of this invention
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 in a pharmaceutical
composition of this invention may between about 10 mg/m.sup.2 and
500 mg/m.sup.2. However, it will be appreciated that these
schedules are exemplary and that an optimal schedule and regimen
can be adapted taking into account the affinity and tolerability of
the particular antibody in the pharmaceutical composition that must
be determined in clinical trials. Quantities and schedule of
injection of an antibody in a pharmaceutical composition of this
invention that saturate NK 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.
[0175] According to another embodiment, the antibody compositions
of this invention may further comprise another therapeutic agent,
including agents normally utilized for the particular therapeutic
purpose for which the antibody is being administered. The
additional therapeutic agent will normally be present in the
composition in amounts 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, therapeutic
agents used to treat infectious disease, therapeutic agents used in
other immunotherapies, cytokines (such as IL-2 or IL-15), other
antibodies and fragments of other antibodies.
[0176] For example, a number of therapeutic agents are available
for the treatment of cancers. The antibody compositions and methods
of the present invention may be combined with any other methods
generally employed in the treatment of the particular disease,
particularly a tumor, cancer disease, or other disease or disorder
that the patient exhibits. 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 activity of
the antibody in a pharmaceutical composition of this invention, its
combination with the present invention is contemplated.
[0177] In connection with solid tumor treatment, the pharmaceutical
compositions of the present invention may be used in combination
with classical approaches, such as surgery, radiotherapy,
chemotherapy, and the like. The invention therefore provides
combined therapies in which a pharmaceutical composition of this
invention is used simultaneously with, before, or after surgery or
radiation treatment; or are administered to patients with, before,
or after conventional chemotherapeutic, radiotherapeutic or
anti-angiogenic agents, or targeted immunotoxins or
coaguligands.
[0178] When one or more agents are used in combination with an
antibody-containing composition of this invention in a therapeutic
regimen, 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, any increased anti-cancer effect above one of the single
therapies would be of benefit. Also, there is no particular
requirement for the combined treatment to exhibit synergistic
effects, although this is certainly possible and advantageous.
[0179] To practice combined anti-cancer therapy, one would simply
administer to an animal an antibody composition of this invention
in combination with another anti-cancer agent in a manner effective
to result in their combined anti-cancer actions within the animal.
The agents would therefore be provided in amounts effective and for
periods of time effective to result in their combined presence
within the tumor vasculature and their combined actions in the
tumor environment. To achieve this goal, an antibody composition of
this invention and anti-cancer agents may be administered to the
animal simultaneously, either in a single combined composition, or
as two distinct compositions using different administration
routes.
[0180] Alternatively, the administration of an antibody composition
of this invention may precede, or follow, the anti-cancer agent
treatment by, e.g., intervals ranging from minutes to weeks and
months. One would ensure that the anti-cancer agent and an antibody
in the antibody composition of this invention exert an
advantageously combined effect on the cancer.
[0181] Most anti-cancer agents would be given prior to an
inhibitory KIR antibody composition of this invention in an
anti-angiogenic therapy. However, when immunoconjugates of an
antibody are used in the antibody composition of this invention,
various anti-cancer agents may be simultaneously or subsequently
administered.
[0182] In some situations, it may even be desirable to extend the
time period for treatment significantly, where several days (2, 3,
4, 5, 6 or 7), several weeks (1, 2, 3, 4, 5, 6, 7 or 8) or even
several months (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the
respective administration of the anti-cancer agent or anti-cancer
treatment and the administration of an antibody composition of this
invention. This would be advantageous in circumstances where the
anti-cancer treatment was intended to substantially destroy the
tumor, such as surgery or chemotherapy, and administration of an
antibody composition of this invention was intended to prevent
micrometastasis or tumor re-growth.
[0183] It also is envisioned that more than one administration of
either an inhibitory KIR antibody-based composition of this
invention or the anti-cancer agent will be utilized. These agents
may be administered interchangeably, on alternate days or weeks; or
a cycle of treatment with an inhibitory KIR antibody composition of
this invention, followed by a cycle of anti-cancer agent therapy.
In any event, to achieve tumor regression using a combined therapy,
all that is required is to deliver both agents in a combined amount
effective to exert an anti-tumor effect, irrespective of the times
for administration.
[0184] In terms of surgery, any surgical intervention may be
practiced in combination with the present invention. In connection
with radiotherapy, any mechanism for inducing DNA damage locally
within cancer cells is contemplated, such as gamma-irradiation,
X-rays, UV-irradiation, microwaves and even electronic emissions
and the like. The directed delivery of radioisotopes to cancer
cells is also contemplated, and this may be used in connection with
a targeting antibody or other targeting means.
[0185] In other aspects, immunomodulatory compounds or regimens may
be administered in combination with or as part of the antibody
compositions of the present invention. Preferred examples of
immunomodulatory compounds include cytokines. Various cytokines may
be employed in such combined approaches. Examples of cytokines
useful in the combinations contemplated by this invention include
IL-1 alpha IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,
IL-9, IL-10, IL-I11, 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.
[0186] In certain embodiments, the cross-reacting inhibitory KIR
antibody-comprising therapeutic compositions of the present
invention may be administered in combination with or may further
comprise a chemotherapeutic or hormonal therapy agent. A variety of
hormonal therapy and chemotherapeutic agents may be used in the
combined treatment methods disclosed herein. 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.
[0187] 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.
[0188] As will be understood by those of ordinary skill in the art,
the appropriate doses of chemotherapeutic agents will approximate
those already employed in clinical therapies wherein the
chemotherapeutics are administered alone or in combination with
other chemotherapeutics. By way of example only, agents such as
cisplatin, and other DNA alkylating may be used. Cisplatin has been
widely used to treat cancer, with efficacious doses used in
clinical applications of 20 mg/m.sup.2 for 5 days every three weeks
for a total of three courses. Cisplatin is not absorbed orally and
must therefore be delivered via injection intravenously,
subcutaneously, intratumorally or intraperitoneally.
[0189] Further useful chemotherapeutic agents include compounds
that interfere with DNA replication, mitosis and chromosomal
segregation, and agents that disrupt the synthesis and fidelity of
polynucleotide precursors. 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.
[0190] The present cross-reacting inhibitory KIR antibody
compositions of this invention may be used in combination with any
one or more other anti-angiogenic therapies or may further comprise
anti-angiogenic agents. Examples of such 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). 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.
[0191] The inhibitory KIR antibody compositions of this invention
may also be advantageously used in combination with methods to
induce apoptosis or may comprise apoptotic agents. For example, a
number of oncogenes have been identified that inhibit apoptosis, or
programmed cell death. Exemplary oncogenes in this category
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).
[0192] The inhibitory KIR antibody compositions of this invention
may also comprise or be used in combination with molecules that
comprise a targeting portion, e.g., antibody, ligand, or conjugate
thereof, directed to a specific marker of a target cell ("targeting
agent"), for example a target tumor cell. Generally speaking,
targeting agents for use in these additional aspects of the
invention will preferably recognize accessible tumor antigens that
are preferentially, or specifically, expressed in the tumor site.
The targeting agents will generally bind to a surface-expressed,
surface-accessible or surface-localized component of a tumor cell.
The targeting agents will also preferably exhibit properties of
high affinity; and 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 "not exert significant side
effects," as used herein, refers to the fact that a targeting
agent, when administered in vivo, will produce only negligible or
clinically manageable side effects, such as those normally
encountered during chemotherapy.
[0193] In the treatment of tumors, an antibody composition of this
invention may additionally comprise or may be used in combination
with adjunct compounds. 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.
[0194] In another embodiment, two or more antibodies of this
invention having different cross-reactivities, including NKVSF1,
may be combined in a single composition so as to neutralize the
inhibitory effects of as many inhibitory KIR gene products as
possible. Compositions comprising combinations of cross-reactive
inhibitory KIR antibodies of this invention, or fragments or
derivatives thereof, will allow even wider utility because there
likely exists a small percentage of the human population that may
lack each of the inhibitory KIR gene products recognized by a
single cross-reacting antibody. Similarly, an antibody composition
of this invention may further comprise one or more antibodies that
recognize single inhibitory KIR subtypes. Such combinations would
again provide wider utility in a therapeutic setting.
[0195] The invention also provides a method of potentiating NK cell
activity in a patient in need thereof, comprising the step of
administering a composition according to this invention to said
patient. The method is more specifically directed at increasing NK
cell activity in patients having a disease in which increased NK
cell activity is beneficial, which involves, affects or is caused
by cells susceptible to lysis by NK cells, or which is caused or
characterized by insufficient NK cell activity, such as a cancer,
another proliferative disorder, an infectious disease or an immune
disorder. More specifically, the methods of the present invention
are utilized for the treatment of a variety of cancers and other
proliferative diseases including, but 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.
[0196] Preferred disorders that can be treated according to the
invention include hematopoietic tumors of lymphoid lineage, for
example T-cell and B-cell tumors, including but not limited to
T-cell disorders such as T-prolymphocytic leukemia (T-PLL),
including of the small cell and cerebriform cell type; large
granular lymphocyte leukemia (LGL) preferably of the T-cell type;
Sezary syndrome (SS); Adult T-cell leukemia lymphoma (ATLL); a/d
T-NHL hepatosplenic lymphoma; peripheral/post-thymic T cell
lymphoma (pleomorphic and immunoblastic subtypes); angio
immunoblastic T-cell lymphoma; angiocentric (nasal) T-cell
lymphoma; anaplastic (Ki 1+) large cell lymphoma; intestinal T-cell
lymphoma; T-lymphoblastic; and lymphoma/leukaemia
(T-Lbly/T-ALL).
[0197] Other proliferative disorders can also be treated according
to the invention, including for example hyperplasias, fibrosis
(especially pulmonary, but also other types of fibrosis, such as
renal fibrosis), angiogenesis, psoriasis, atherosclerosis and
smooth muscle proliferation in the blood vessels, such as stenosis
or restenosis following angioplasty. The cross-reacting inhibitory
KIR antibody of this invention can be used to treat or prevent
infectious diseases, including preferably any infections caused by
viruses, bacteria, protozoa, molds or fungi. Such viral infectious
organisms include, but are not limited to, hepatitis type A,
hepatitis type B, hepatitis type C, influenza, varicella,
adenovirus, herpes simplex type I (HSV-1), herpes simplex type 2
(HSV-2), rinderpest, rhinovirus, echovirus, rotavirus, respiratory
syncytial virus, papilloma virus, papilloma virus, cytomegalovirus,
echinovirus, arbovirus, huntavirus, coxsackie virus, mumps virus,
measles virus, rubella virus, polio virus and human
immunodeficiency virus type I or type 2 (HIV-1, HIV-2).
[0198] Bacterial infections that can be treated according to this
invention include, but are not limited to, infections caused by the
following: Staphylococcus; Streptococcus, including S. pyogenes;
Enterococcl; Bacillus, including Bacillus anthracis, and
Lactobacillus; Listeria; Corynebacterium diphtheriae; Gardnerella
including G. vaginalis; Nocardia; Streptomyces; Thermoactinomyces
vulgaris; Treponerna; Camplyobacter, Pseudomonas including
Raeruginosa; Legionella; Neisseria including N. gonorrhoeae and N.
meningitides; Flavobacterium including F. meningosepticum and F.
odoratum; Brucella; Bordetella including B. pertussis and B.
bronchiseptica; Escherichia including E. coli, Klebsiella;
Enterobacter, Serratia including S. marcescens and S. liquefaciens;
Edwardsiella; Proteus including P. mirabilis and P. vulgaris;
Streptobacillus; Rickettsiaceae including R. fickettsfi, Chlamydia
including C. psittaci and C. trachomatis; Mycobacterium including
M. tuberculosis, M. intracellulare, M. folluitum, M. lapraern, M.
avium, M. bovis, M. africanum, M. kansasii, M. intracellulare, and
M. lepraemurium; and Nocardia. Protozoa infections that may be
treated according to this invention include, but are not limited
to, infections caused by leishmania, kokzidioa, and trypanosoma. A
complete list of infectious diseases can be found on the website of
the National Center for Infectious Disease (NCID) at the Center for
Disease Control (CDC) (http://www.cdc.gov/ncidod/diseases/), which
list is incorporated herein by reference. All of said diseases are
candidates for treatment using the cross-reacting inhibitory KIR
antibodies of the invention.
[0199] Such methods of treating various infectious diseases may
employ the antibody composition of this invention, either alone or
in combination with other treatments and/or therapeutic agents
known for treating such diseases, including anti-viral agents,
anti-fungal agents, antibacterial agents, antibiotics,
anti-parasitic agents and anti-protozoal agents. When these methods
involve additional treatments with additional therapeutic agents,
those agents may be administered together with the antibodies of
this invention as either a single dosage form or as separate,
multiple dosage forms. When administered as a separate dosage form,
the additional agent may be administered prior to, simultaneously
with, of following administration of the antibody of this
invention.
[0200] Further aspects and advantages of this invention will be
disclosed in the following experimental section, which should be
regarded as illustrative and not limiting the scope of this
application.
EXAMPLE 1
Purification of PBLs and Generation of Polyclonal or Clonal NK Cell
Lines
[0201] PBLs were derived from healthy donors by Ficoll Hypaque
gradients and depletion of plastic adherent cells. To obtain
enriched NK cells, PBLs were 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-cells were 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 were cloned by
limiting dilution and clones of NK cells were characterized by flow
cytometry for expression of cell surface receptors.
[0202] The mAbs used were JT3A (IgG2a, anti CD3), EB6 and GL1 83
(IgG1 anti KIR2DL1 and KIR2DL3 respectively), XA-141 IgM (anti
KIR2DL1 with the same specificity as EB6), anti CD4 (HP2.6), and
anti DR (D1.12, IgG2a). Instead of JT3A, HP2.6, and DR1.12, which
were produced by applicants, commercially available mAbs of the
same specificities can be used (Beckman Coulter Inc., Fullerton,
Calif.). EB6 and GL183 are commercially available (Beckman Coulter
Inc., Fullerton, Calif. XA-141 is not commercially available, but
EB6 can be used for control reconstitution of lysis as described in
(Moretta et al., 1993).
[0203] Cells were stained with the appropriate antibodies (30 mns
at 4.degree. C.) followed by PE or FITC conjugated polyclonal anti
mouse antibodies (Southern Biotechnology Associates Inc). Samples
were analyzed by cytofluorometric analysis on a FACSAN apparatus
(Becton Dickinson, Mountain View, Calif.).
[0204] The following clones were used in this study. CP11, CN5 and
CN505 are KIR2DL1 positive clones and are stained by EB6 ((IgG1
anti KIR2DL1) or XA-141 (IgM anti KIR2DL1 with same specificity as
compared to EB6 antibodies). CN12 and CP502 are KIR2DL3 positive
clones and are stained by GL183 antibody (IgG1 anti KIR2DL3).
[0205] The cytolytic activity of NK clones was assessed by a
standard 4 hour .sup.51Cr release assay in which effector NK cells
were tested on Cw3 or Cw4 positive cell lines known for their
sensitivity to NK cell lysis. All the targets were used at 5000
cells per well in microtitration plate and the effector:target
ratio is indicated in the Figures (usually 4 effectors per target
cells). The cytolytic assay was performed with or without
supematant of the indicated monoclonal antibodies at a 1/2
dilution. The procedure was essentially the same as described in
(Moretta et al., 1993).
EXAMPLE 2
Generation of New mAbs
[0206] mAbs were generated by immunizing 5 week old Balb C mice
with activated polyclonal or monoclonal NK cell lines as described
in (Moretta et al., 1990). After different cell fusions, the mAbs
were first selected for their ability to cross-react with EB6 and
GL183 positive NK cell lines and clones. Positive monoclonal
antibodies were further screened for their ability to reconstitute
lysis by EB6 positive or GL183 positive NK clones of Cw4 or Cw3
positive targets respectively.
[0207] Cell staining was carried out as follows. Cells were stained
with a panel of antibodies (1 .mu.g/ml or 50 .mu.l supernatant, 30
mns at 4.degree. C.) followed by PE-conjugated goat F(ab')2
fragments anti-mouse IgG (H+L) or PE-conjugated goat F(ab')2
fragment anti-human IgG (Fc gamma) antibodies (Beckman Coulter).
Cytofluorometric analysis was performed on an Epics XL.MCL
apparatus (Beckman Coulter).
[0208] One of the monoclonal antibodies, the DF200 mAb, was found
to react with various members of the KIR family including KIR2DL1,
KIR2DL2/3. Both KIR2DL1+ and KIR2DL2/3+ NK cells were stained
brightly with DF200 mAb (FIG. 1).
[0209] NK clones expressing one or another (or even both) of these
HLA class I-specific inhibitory receptors were used as effectors
cells against target cells expressing one or more HLA-C alleles.
Cytotoxicity assays were carried out as follows. The cytolytic
activity of YTS-KIR2DL1 or YTS-Eco cell lines was assessed by a
standard 4 hours 51 Cr release assay. The effector cells were
tested on HLA-Cw4 positive or negative EBV cell lines and HLA-Cw4
transfected 721.221 cells. All targets were used at 3000 cells per
well in microtitration plate. The effector/target ratio is
indicated in the figures. The cytolytic assay was performed with or
without the indicated full length or F(ab')2 fragments of
monoclonal mouse or human antibodies. As expected, KIR2DL1.sup.+ NK
clones displayed little if any cytolytic activity against target
cells expressing HLA-Cw4 and KIR2DL3.sup.+ NK clones displayed
little or no activity on Cw3 positive targets. However, in the
presence of DF200 mnAb (used to mask their KIR2DL receptors) NK
clones became unable to recognize their HLA-C ligands and displayed
strong cytolytic activity on Cw3 or Cw4 targets.
[0210] For example, the C1R cell line (CW4.sup.+ EBV cell line,
ATCC n.degree. CRL 1993) was not killed by KIR2DL1.sup.+ NK clones
(CN5CN505), but the inhibition could be efficiently reversed by the
use of either DF200 or a conventional anti KIR2DL1 mAb. On the
other hand NK clones expressing the KIR2DL2/3.sup.+ KIR2DL1-
phenotype (CN12) efficiently killed C1R cells and this killing was
unaffected by the DF200 mAb (FIG. 2). Similar results are obtained
with KIR2DL2- or KIR2DL3-positive NK clones on Cw3 positive
targets.
[0211] Similarly, the Cw4+221 EBV cell line was not killed by
KIR2DL1.sup.+ transfected NK cells, but the inhibition could be
efficiently reversed by the use of either DF200, a DF200 Fab
fragment, or a conventional anti KIR2DL1 mAb EB6 or XA141. Also, a
Cw3+221 EBV cell line was not killed by KIR2DL2.sup.+ NK cells, but
this inhibition could be reversed by the use of either DF200 or a
DF200 Fab fragment. Finally, the latter Cw3+221 EBV cell line was
not killed by KIR2DL3.sup.+ NK cells, but this inhibition could be
reversed by the use of either a DF200 Fab fragment or a
conventional anti KIR2DL3 mAb GL183 or Y249. The results are shown
in FIG. 3.
[0212] F(ab')2 fragments were also tested for their ability to
reconstite lysis of Cw4 positive targets. F(ab')2 fragments of the
DF200 and EB6 Abs were both able to reverse inhibition of lysis by
KIR2DL1-transfected NK cells of the Cw4 transfected 221 cell line
and the Cw4+TUBO EBV cell line. Results are shown in FIG. 4.
EXAMPLE 4
Generation of New Human mAbs
[0213] Human monoclonal anti-KIR Abs were generated by immunizing
transgenic mice engineered to express a human antibody repertoire
with recombinant KIR protein. After different cell fusions, the
mAbs were first selected for their ability to cross-react with
immobilized KIR2DL1 and KIR2DL2 protein. Several monoclonal
antibodies, including 1-7F9, 1-4F1, 1-6F5 and 1-6F1, were found to
react with KIR2DL1 and KIR2DL2/3.
[0214] Positive monoclonal antibodies were further screened for
their ability to reconstitute lysis by EB6 positive NK
transfectants expressing KIR2DL1 of Cw4-positive target cells. The
NK cells expressing the HLA class I-specific inhibitory receptors
were used as effectors cells against target cells expressing one or
more HLA-C alleles (FIGS. 5 and 6). Cytotoxicity assays were
carried out as described above. The effector/target ratio is
indicated in the Figures, and antibodies were used at either 10
ug/ml or 30 ug/ml.
[0215] As expected, KIR2DL1.sup.+ NK cells displayed little if any
cytolytic activity against target cells expressing HLA-Cw4.
However, in the presence of 1-7F9 mAb, NK cells became unable to
recognize their HLA-C ligands and displayed strong cytolytic
activity on the Cw4 targets. For example, the two cell lines tested
(the HLA-Cw4 transfected 721.221 and the CW4.sup.+ EBV cell lines)
were not killed by KIR2DL1.sup.+ NK cells, but the inhibition could
be efficiently reversed by the use of either Mab 1-7F9 or a
conventional anti KIR2DL1 mAb EB6. Abs DF200 and panKIR (also
referred to as NKVSF1) were compared to 1-7F9. Antibodies 1-4F1,
1-6F5 and 1-6F1 on the other hand were not able to reconstitute
cell lysis by NK cells on Cw4 positive targets.
EXAMPLE 5
Biacore Analysis of DF200 mAb/KIR2DL1 and DF200 mAb/KIR2DL3
Interactions
Production and Purification of Recombinant Proteins
[0216] The KIR2DL1 and KIR2DL3 recombinant proteins were produced
in E. coli. cDNA encoding the entire extracellular domain of
KIR2DL1 and KIR2DL3 were amplified by PCR from pCDM8 clone 47.11
vector (Biassoni et al, 1993) and RSVS(gpt)183 clone 6 vector
(Wagtman et al, 1995) respectively, using the following primers:
TABLE-US-00001 Sense:
5'-GGAATTCCAGGAGGAATTTAAAATGCATGAGGGAGTCCACAG-3' Anti-sense:
5'-CGGGATCCCAGGTGTCTGGGGTTACC-3'
They were cloned into the pML1 expression vector in frame with a
sequence encoding a biotinylation signal (Saulquin et al,
2003).
[0217] Protein expression was performed in the BL21(DE3) bacterial
strain (Invitrogen). Transfected bacteria were grown to
OD.sub.600=0.6 at 37.degree. C. in medium supplemented with
ampicillin (100 .mu.g/ml) and expression was induced with 1 mM
IPTG.
[0218] Proteins were recovered from inclusion bodies under
denaturing conditions (8 M urea). Refolding of the recombinant
proteins was performed in 20 mM Tris, pH 7.8, NaCl 150 mM buffer
containing L-arginine (400 mM, Sigma) and .beta.-mercaptoethanol (1
mM), at room temperature, by decreasing the urea concentration in a
six step dialysis (4, 3, 2, 1 0.5 and 0 M urea, respectively).
Reduced and oxidized glutathione (5 mM and 0.5 mM respectively,
Sigma) were added during the 0.5 and 0 M urea dialysis steps.
Finally, the proteins were dialyzed extensively against 10 mM Tris,
pH 7.5, NaCl 150 mM buffer. Soluble, refolded proteins were
concentrated and then purified on a Superdex 200 size-exclusion
column (Pharmacia; AKTA system).
[0219] Surface plasmon resonance measurements were performed on a
Biacore apparatus (Biacore). In all Biacore experiments HBS buffer
supplemented with 0.05% surfactant P20 served as running
buffer.
Protein immobilisation.
[0220] Recombinant KIR2DL1 and KIR2DL3 proteins produced as
described above were immobilized covalently to carboxyl groups in
the dextran layer on a Sensor Chip CM5 (Biacore). The sensor chip
surface was activated with EDC/NHS
(N-ethyl-N'-(3-dimethylaminopropyl)carbodiimidehydrochloride and
N-hydroxysuccinimide, Biacore). Proteins, in coupling buffer (10 mM
acetate, pH 4.5) were injected. Deactivation of the remaining
activated groups was performed using 100 mM ethanolamine pH 8
(Biacore).
Affinity Measurements.
[0221] For kinetic measurements, various concentrations of the
soluble antibody (1.times.10.sup.-7 to 4.times.10.sup.-10 M) were
applied onto the inmobilized sample. Measurements were performed at
a 20 .mu.l/min continuous flow rate. For each cycle, the surface of
the sensor chip was regenerated by 5 .mu.l injection of 10 mM NaOH
pH 11. The BIAlogue Kinetics Evaluation program (BIAevaluation 3.1,
Biacore) was used for data analysis. The soluble analyte (40 .mu.l
at various concentrations) was injected at a flow rate of 20
.mu.l/min in HBS buffer, on dextran layers containing 500 or 540
reflectance units (RU), and 1000 or 700 RU of KIR2DL1 and KIR2DL3,
respectively. Data are representative of 6 independent experiments.
The results are shown in Table 1, below. TABLE-US-00002 TABLE 1
BIAcore analysis of DF200 mAb binding to immobilized KIR2DL1 and
KIR2DL3. Protein K.sub.D (10.sup.-9 M) KIR2DL1 10.9 +/- 3.8 KIR2DL3
2.0 +/- 1.9 K.sub.D: Dissociation constant.
EXAMPLE 6
Biacore Competitive Binding Analysis of Murine and Human anti-KIR
Antibodies
[0222] Epitope mapping analysis was performed on immobilized KIR
2DL1 (900 RU), KIR 2DL3 (2000 RU) and KIR 2DS1 (1000 RU) with mouse
anti-KIR 2D antibodies DF200, Pan2D, gl 183 and EB6, and human
anti-KIR 2D antibodies 1-4F1, 1-6F1, 1-6F5 and 1-7F9 as described
previously (Gauthier et al 1999, Saunal and van Regenmortel
1995).
[0223] All experiments were done at a flow rate of 5 .mu.l/min in
HBS buffer with 2 min injection of the different antibodies at 15
.mu.g/ml. For each couple of antibodies competitive binding
analysis was performed in two steps. In the first step the first
monoclonal antibody (mAb) was injected on KIR 2D target protein
followed by the second mAb (without removing the first mAb) and
second mAb RU value (RU2) was monitored. In the second step the
second mAb was injected first, directly on nude KIR 2D protein, and
mAb RU value (RU1) was monitored. Percent inhibition of second mAb
binding to KIR 2D protein by first mab was calculated by:
100*(1-RU2/RU1).
[0224] Results are shown in Tables 2,3 and 4, where the antibodies
designated `first antibody` are listed on vertical column and the
`second antibody` are listed on the horizontal column. For each
antibody combination tested, the values for direct binding level
(RU) of the antibodies to the chip are listed in the table, where
direct binding of the second antibody to the KIR2D chip is listed
in the upper portion of the field and the value for binding of the
second antibody to the KIR2D chip when the first antibody is
present is listed in the lower portion of the field. Listed in the
right of each field is the percentage inhibition of second antibody
binding. Table 2 shows binding on a KIR2DL1 chip, Table 3 shows
binding of antibodies to a KIR2DL3 chip, and Table 4 shows binding
of antibodies to a KIR2DS1 chip.
[0225] Competitive binding of murine antibodies DF200, NKVSF1 and
EB6, and human antibodies 1-4F1, 1-7F9 and 1-6F1 to immobilized
KIR2DL1, KIR2DL2/3 and KIR2DS1 was assessed. Epitope mapping (FIG.
7) from experiments with anti-KIR antibodies' binding to KIR2DL1
showed that (a) antibody 1-7F9 is competitive with EB6 and 1-4F1,
but not with NKVSF1 and DF200; (b) antibody 1-4 F1 in turn is
competitive with EB6, DF200, NKVSF1 and 1-7 F9; (c) NKVSF1 competes
with DF200,1-4F1 and EB6, but not 1-7F9; and (d) DF200 competes
with NKVSF1, 1-4F1 and EB6, but not 1-7F9. Epitope mapping (FIG. 8)
from experiments with anti-KIR antibodies' binding to KIR2DL3
showed that (a) 1-4F 1 is competitive with NKVSF1, DF200, gl183 and
1-7F9; (b) 1-7F9 is competitive with DF200, gl183 and 1-4F 1, but
not with NKVSF1; (c) NKVSF1 competes with DF200, 1-4F1 and GL183,
but not 1-7F9; and (d) DF200 competes with NKVSF1,1-4F1 and 1-7F9,
but not with GL183. Epitope mapping (FIG. 9) from experiments with
anti-KIR antibodies' binding to KIR2DS I showed that (a) 1-4F1 is
competitive with NKVSF1, DF200 and 1-7F9; (b) 1-7F9 is competitive
with 1-4F1 but not competitive with DF200 and NKVSF1; (c) NKVSF1
competes with DF200 and 1-4F1, but not 1-7F9; and (d) DF200
competes with NKVSF1 and 1-4F1, but not with 1-7F9.
EXAMPLE 7
Anti-KIR mAb titration with cvnomolgus NK cells
[0226] Anti-KIR antibody NKVSF1 was tested for its ability to bind
to NK cells from cynomolgus monkeys. Binding of the antibody to
monkey NK cells is shown in FIG. 10.
Purification of monkey PBMC and generation of polyclonal NK cell
bulk.
[0227] Cynomolgus Macaque PBMC were prepared from Sodium citrate
CPT tube (Becton Dickinson). NK cells purification was performed by
negative depletion (Macaque NK cell enrichment kit, Stem Cell
Technology). NK cells were cultivated on irradiated human feeder
cells, 300 U/ml Interleukin 2 (Proleukin, Chiron Corporation) and
1ng/ml Phytohemagglutinin A (Invitrogen, Gibco) to obtain
polyclonal NK cell populations.
Pan2D mAb titration with cynomolgus NK cells.
[0228] Cynomolgus NK cells (NK bulk day 16) were incubated with
different amount of Pan2D mAb followed by PE-conjugated goat
F(ab')2 fragments anti-mouse IgG (H+L) antibodies. The percentage
of positive cells was determined with an isotypic control (purified
mouse IgG1).Samples were done in duplicate. Mean fluorescence
intensity =MFI. TABLE-US-00003 TABLE 2 KIR2DL1 epitope mapping
First Ab Second Ab .fwdarw. (below) DF200 Pan2D EB6 1-4 F1 1-7 F9
1-6 F1 1-6 F5 DF200 80% 90% 490 92% 480 27% 540 15% 400 15% 40 350
460 340 Pan2D 90% 90% 900 95% 860 2% 750 12% 600 13% 50 840 660 520
EB6 60% 40% 460 57% 370 48% 490 65% 260 23% nd 200 190 170 200 1-4
F1 1-7 F9 600 10% 545 2% 460 60% 360 95% 330 9% nd 545 534 180 16
300 1-6 F1 350 11% 475 7% 260 18% 360 23% 490 10% nd 310 440 320
275 440 1-6 F5 350 17% 475 7% nd 360 17% nd 290 40% 290 440 300
170
[0229] TABLE-US-00004 TABLE 3 KIR2DL3 epitope mapping First Ab
Second Ab .fwdarw. (below) DF200 Pan2D gl183 1-4 F1 1-7 F9 1-6 F1
1-6 F5 DF200 75% 20% 1270 75% 520 62% 550 16% 440 4% 320 200 460
420 Pan2D 95% 85% 2250 68% 880 15% 840 8% 560 18% 730 750 770 460
gl183 8% 40% 1300 75% 670 76% 530 18% nd 330 160 430 1-4 F1 1140
82% 2400 63% 1240 73% 1050 87% 210 890 330 140 1-7 F9 770 42% 870
5% 800 75% 1000 63% 450 830 200 270 1-6 F1 790 4% 990 0% 620 8% 760
1090 570 1-6 F5 800 5% 990 4% nd 760 950
[0230] TABLE-US-00005 TABLE 4 KIR2DS1 epitope mapping First Ab
Second Ab .fwdarw. (below) DF200 Pan2D 1-4 F1 1-7 F9 DF200 70% 660
87% 975 15%.sup. 80 825 Pan2D 100% 650 100% 920 45% * -8 500 1-7 F9
900 17% 1350 11% 660 96% 1090 1200 23
EXAMPLE 8
Epitope-mapping of DF200- and pan2D-binding to KIR2DL1
[0231] Computer modelling of the extra-cellular domains of KIR2DL1,
-2 and -3 (KIR2DL1-3), based on their published crystal-structures
(Maenaka et al. (1999), Fan et al. (2001), Boyington et al.
(2000)), predicted the involvement of amino acids R131.sup.1 in the
interaction between KIR2DL1 and the KIR2DL1-3-cross-reactive mouse
monoclonal antibodies (mAb's) DF200 and pan2D. To verify this,
fusion-proteins were prepared consisting of the complete
extra-cellular domain of KIR2DL1 (amino acids H1-H224), either
wild-type or point-mutated (e.g. R131W.sup.2), fused to human Fc
(hFc). The material and methods used to produce and evaluate the
various KIR2DL1-hFc fusion-proteins have been described (Winter and
Long (2000)). In short, KIR2DL1(R131W)-hFc encoding cDNA-vectors
were generated, by PCR-based mutagenesis (Quickchange II, Promega)
of CL42-Ig, a published cDNA-vector for the production of wild-type
KIR2DLI-hFc (Wagtmann et al. (1995)). KIR2DL1-hFc and
KIR2DL1(R131W)-hFc were produced in COS7 cells and isolated from
tissue-culture media, essentially as described (Wagtmann et al.
(1995)). To test their correct folding, KIR2DL1-hFc and
KIR2DL1(R131W)-hFc were incubated with LCL721.221 cells that
express either HLA-Cw3 (no KIR2DL1 ligand) or HLA-Cw4 (KIR2DL1
ligand), and the interaction between KIR-Fc fusion proteins and
cells analysed by FACS, a standard technique for the study of
protein-interactions at the cell-surface. An example of independent
experiments is given in FIG. 11, panel A. As predicted from the
literature, none of the KIR2DL1-hFc fusion proteins bound HLA-Cw3
expressing LCL721.221 cells. In contrast, both KIR2DL1-hFc and
KIR2DL1(R131W)-hFc bound to HLA-Cw4 expressing LCL721.221 cells,
thereby confirming their correct folding. The binding of
KIR2DL1(RI31W)-hFc and KIR2DL1-hFc to KIR-specific mAb's (DF200,
pan2D, EB6 and GL183) was studied using ELISA, a standard technique
to study protein-interactions. In short, KIR2DL1(R131W)-hFc and
KIR2DL1-hFc were linked to 96-wells plates via goat anti-human
antibodies, after which KIR-specific mAb's were added in various
concentrations (0-1 .mu.g/ml in PBS). The interactions between
KIR2DL1-hFc variants and mAb's were visualised by spectrophotometry
(450 nm), using peroxidase-coupled secondary antibodies specific
for mouse antibodies to convert TMB substrate. An examples of
independent experiments is given in FIG. 11, panel B. Whereas the
KIR2DL2-3-specific mAb GL83 was not able to bind any of the
KIR2DL1-hFc fusion proteins, the KIR2DL1-specific mAb EB6, DF200
and pan2D bound KIR2DL1-hFc variants in a dose-dependent fashion.
The single point-mutation (R131W) affected the binding of DF200 and
pan2D with a reduction in binding compared to wild type of
.about.10% at highest concentrations of mab (1 .mu.g/ml),
confirming that R131 is part of the binding-site of DF200 and pan2D
in extra-cellular domain 2 of KIR2DL1.
REFERENCES
[0232] Moretta, A., Bottino, C., Pende, D., Tripodi, G., Tambussi,
G., Viale, O., Orengo, A., Barbaresi, M., Merli, A., Ciccone, E.,
and et al. (1990). Identification of four subsets of human
CD3-CD16+ natural killer (NK) cells by the expression of clonally
distributed functional surface molecules: correlation between
subset assignment of NK clones and ability to mediate specific
alloantigen recognition. J Exp Med 172, 1589-1598.
[0233] Moretta, A., Vitale, M., Bottino, C., Orengo, A. M.,
Morelli, L., Augugliaro, R., Barbaresi, M., Ciccone, E., and
Moretta, L. (1993). P58 molecules as putative receptors for major
histocompatibility complex (MHC) class I molecules in human natural
killer (NK) cells. Anti-p58 antibodies reconstitute lysis of MHC
class I-protected cells in NK clones displaying different
specificities. J Exp Med 178, 597-604.
[0234] Pende, D., Parolini, S., Pessino, A., Sivori, S.,
Augugliaro, R., Morelli, L., Marcenaro, E., Accame, L., Malaspina,
A., Biassoni, R., et al (1999). Identification and molecular
characterization of NKp30, a novel triggering receptor involved in
natural cytotoxicity mediated by human natural killer cells. J Exp
Med 190, 1505-1516.
[0235] Ruggeri, L., Capanni, M., Urbani, E., Perruccio, K.,
Shlomchik, W. D., Tosti, A., Posati, S., Rogaia, D., Frassoni, F.,
Aversa, F., et al. (2002). Effectiveness of donor natural killer
cell alloreactivity in mismatched hematopoietic transplants.
Science 295, 2097-2100.
[0236] Wagtmann N, Biassoni R, Cantoni C, Verdiani S, Malnati MS,
Vitale M, Bottino C, Moretta L, Moretta A, Long EO.Molecular clones
of the p58 NK cell receptor reveal immunoglobulin-related molecules
with diversity in both the extra- and intracellular
domains.Immunity. 1995 May;2(5):439-49.
[0237] Biassoni R, Verdiani S, Cambiaggi A, Romeo PH, Ferrini S,
Moretta L. Human CD3-CD16+natural killer cells express the hGATA-3
T cell transcription factor and an unrearranged 2.3-kb TcR delta
transcript. Eur J Immunol. 1993 May;23(5):1083-7. Saulquin X,
Gastinel L N, Vivier E.Crystal structure of the human natural
killer cell activating receptor KIR2DS2 (CD158j) J Exp Med. 2003
Apr 7;197(7):933-8.
[0238] Gauthier, L., Lemmers, B., Guelpa-Fonlupt, V., Fougereau,
M., and Schiff, C. .mu.-SLC physico-chemical interactions of the
human preB cell receptor: implications for VH repertoire selection
and cell signaling at the preB cell stage. Journal of Immunology,
162., 41-50. (1999).
Saunal, H. and Van Regenmortel, M. H. V., Mapping of viral
conformation epitopes using biosensor measurements. Journal of
Immunology, 183: 33-41 (1995).
Boyington J C; Motyka S A; Schuck P; Brooks A G; Sun P D. Nature,
Vol. 405 (6786) pp. 537-543 (2000)
Fan Q R; Long E O; Wiley D C. Nature immunology, Vol. 2 (5) pp.
452-460 (2001) Maenaka K; Juji T; Stuart D I; Jones E Y. Structure
with Folding and design, Vol. 7 (4) pp. 391-398 (1999)
Wagtmann N; Rajagopalan S; Winter C C; Peruzzi M; Long E O.
Immunity, Vol. 3 (6) pp. 801-809 (1995)
Winter C C; Long E O. Natural Killer Cells Protocols (edited by
Campbell K S and Colonna M). Human Press. pp. 219-238 (2000)
[0239] All references, including publications, patent applications
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
All headings and sub-headings are used herein for convenience only
and should not be construed as limiting the invention in any
way,
Any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
The terms "a" and "an" and "the" and similar referents as used in
the context of describing the invention are to be construed to
cover both the singular and the plural, unless otherwise indicated
herein or clearly contradicted by context.
[0240] Recitation of ranges of values herein are merely intended to
serve as a shorthand method of referring individually to each
separate value falling within the range, unless otherwise indicated
herein, and each separate value is incorporated into the
specification as if it were individually recited herein. Unless
otherwise stated, all exact values provided herein are
representative of corresponding approximate values (e.g., all exact
exemplary values provided with respect to a particular factor or
measurement can be considered to also provide a corresponding
approximate measurement, modified by "about," where
appropriate).
[0241] All methods described herein can be performed in any
suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context.
[0242] The use of any and all examples, or exemplary language
(e.g., "such as") provided herein, is intended merely to better
illuminate the invention and does not pose a limitation on the
scope of the invention unless otherwise indicated. No language in
the specification should be construed as indicating any element is
essential to the practice of the invention unless as much is
explicitly stated.
[0243] The citation and incorporation of patent documents herein is
done for convenience only and does not reflect any view of the
validity, patentability and/or enforceability of such patent
documents,
[0244] The description herein of any aspect or embodiment of the
invention using terms such as "comprising", "having", "including"
or "containing" with reference to an element or elements is
intended to provide support for a similar aspect or embodiment of
the invention that "consists of", "consists essentially of", or
"substantially comprises" that particular element or elements,
unless otherwise stated or clearly contradicted by context (e.g., a
composition described herein as comprising a particular element
should be understood as also describing a composition consisting of
that element, unless otherwise stated or clearly contradicted by
context).
[0245] This invention includes all modifications and equivalents of
the subject matter recited in the aspects or claims presented
herein to the maximum extent permitted by applicable law.
Sequence CWU 1
1
12 1 128 PRT Mus musculus 1 Met Glu Ser Gln Thr Leu Val Phe Ile Ser
Ile Leu Leu Trp Leu Tyr 1 5 10 15 Gly Ala Asp Gly Asn Ile Val Met
Thr Gln Ser Pro Lys Ser Met Ser 20 25 30 Met Ser Val Gly Glu Arg
Val Thr Leu Thr Cys Lys Ala Ser Glu Asn 35 40 45 Val Val Thr Tyr
Val Ser Trp Tyr Gln Gln Lys Pro Glu Gln Ser Pro 50 55 60 Lys Leu
Leu Ile Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp 65 70 75 80
Arg Phe Thr Gly Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser 85
90 95 Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr His Cys Gly Gln Gly
Tyr 100 105 110 Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys Arg 115 120 125 2 131 PRT mus musculus 2 Met Asp Phe Gln
Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile
Met Ser Arg Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Ser 20 25 30
Met Ser Ala Ser Leu Gly Glu Arg Val Thr Met Thr Cys Thr Ala Ser 35
40 45 Ser Ser Val Ser Ser Ser Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro
Gly 50 55 60 Ser Ser Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Leu
Ala Ser Gly 65 70 75 80 Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly
Thr Ser Tyr Ser Leu 85 90 95 Thr Ile Ser Ser Met Glu Ala Glu Asp
Ala Ala Thr Tyr Tyr Cys His 100 105 110 Gln Tyr His Arg Ser Pro Pro
Thr Phe Gly Gly Gly Thr Lys Leu Glu 115 120 125 Ile Lys Arg 130 3
11 PRT mus musculus 3 Lys Ala Ser Glu Asn Val Val Thr Tyr Val Ser 1
5 10 4 12 PRT mus musculus 4 Thr Ala Ser Ser Ser Val Ser Ser Ser
Tyr Leu Tyr 1 5 10 5 7 PRT mus musculus 5 Gly Ala Ser Asn Arg Tyr
Thr 1 5 6 7 PRT mus musculus 6 Ser Thr Ser Asn Leu Ala Ser 1 5 7 9
PRT mus musculus 7 Gly Gln Gly Tyr Ser Tyr Pro Tyr Thr 1 5 8 9 PRT
mus musculus 8 His Gln Tyr His Arg Ser Pro Pro Thr 1 5 9 140 PRT
mus musculus 9 Met Ala Val Leu Gly Leu Leu Phe Cys Leu Val Thr Phe
Pro Ser Cys 1 5 10 15 Val Leu Ser Gln Val Gln Leu Glu Gln Ser Gly
Pro Gly Leu Val Gln 20 25 30 Pro Ser Gln Ser Leu Ser Ile Thr Cys
Thr Val Ser Gly Phe Ser Phe 35 40 45 Thr Pro Tyr Gly Val His Trp
Val Arg Gln Ser Pro Gly Lys Gly Leu 50 55 60 Glu Trp Leu Gly Val
Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Ala 65 70 75 80 Ala Phe Ile
Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln 85 90 95 Val
Phe Phe Lys Met Asn Ser Leu Gln Val Asn Asp Thr Ala Ile Tyr 100 105
110 Tyr Cys Ala Arg Asn Pro Arg Pro Gly Asn Tyr Pro Tyr Gly Met Asp
115 120 125 Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 130 135
140 10 10 PRT mus musculus 10 Gly Phe Ser Phe Thr Pro Tyr Gly Val
His 1 5 10 11 16 PRT mus musculus 11 Val Ile Trp Ser Gly Gly Asn
Thr Asp Tyr Asn Ala Ala Phe Ile Ser 1 5 10 15 12 13 PRT mus
musculus 12 Asn Pro Arg Pro Gly Asn Tyr Pro Tyr Gly Met Asp Tyr 1 5
10
* * * * *
References