U.S. patent application number 16/397271 was filed with the patent office on 2019-08-15 for anti-nkg2a antibodies and uses thereof.
The applicant listed for this patent is NOVO NORDISK A/S. Invention is credited to Soeren Berg Padkaer, Petrus Johannes Louis Spee.
Application Number | 20190248896 16/397271 |
Document ID | / |
Family ID | 38626279 |
Filed Date | 2019-08-15 |
View All Diagrams
United States Patent
Application |
20190248896 |
Kind Code |
A1 |
Spee; Petrus Johannes Louis ;
et al. |
August 15, 2019 |
ANTI-NKG2A ANTIBODIES AND USES THEREOF
Abstract
Described herein are anti-NKG2A antibodies suitable for human
therapy, including humanized versions of murine anti-NKG2A antibody
Z270, as well as related methods and materials for producing and
using such antibodies. Exemplary complementarity-determining
regions (CDRs) sequences and sites for optional amino acid
back-substitutions in framework region (FR) and/or CDRs of such
antibodies are also described.
Inventors: |
Spee; Petrus Johannes Louis;
(Alleroed, DK) ; Padkaer; Soeren Berg; (Vaerloese,
DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NOVO NORDISK A/S |
BAGSVAERD |
|
DK |
|
|
Family ID: |
38626279 |
Appl. No.: |
16/397271 |
Filed: |
April 29, 2019 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15624882 |
Jun 16, 2017 |
|
|
|
16397271 |
|
|
|
|
14548327 |
Nov 20, 2014 |
9683041 |
|
|
15624882 |
|
|
|
|
13477888 |
May 22, 2012 |
8901283 |
|
|
14548327 |
|
|
|
|
12305683 |
May 26, 2009 |
8206709 |
|
|
PCT/EP2007/056485 |
Jun 28, 2007 |
|
|
|
13477888 |
|
|
|
|
60818550 |
Jul 5, 2006 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/56 20130101;
A61P 31/12 20180101; C07K 2317/24 20130101; A61K 2039/505 20130101;
C07K 16/2803 20130101; C07K 16/2851 20130101; A61P 35/00 20180101;
C07K 16/468 20130101; C07K 2317/31 20130101; C07K 2317/76 20130101;
A61P 29/00 20180101; A61P 37/00 20180101; C07K 2317/565 20130101;
A61P 37/08 20180101; C07K 2317/92 20130101; A61P 37/06 20180101;
C07K 2317/73 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 16/46 20060101 C07K016/46 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 30, 2006 |
EP |
06116429.9 |
Claims
1-18. (canceled)
19. An isolated antibody that specifically binds NKG2A, comprising
antigen-binding residues from the complementarity-determining
regions (CDRs) of murine antibody Z270 and human acceptor framework
sequences, wherein said antibody comprises a VL domain that is at
least 90% identical to SEQ ID NO: 4 and a VH domain that is at
least 90% identical to SEQ ID NO: 5, and wherein said antibody is
more effective than an antibody comprising murine antibody Z270
variable light (VL) (SEQ ID NO: 1) and VH (SEQ ID NO: 2) sequences
in potentiating the cytotoxic activity of a CD94/NKG2A-expressing
cytotoxic lymphocyte.
20. The antibody of claim 19, comprising a CDR-H1 corresponding to
residues 31-35 of SEQ ID NO:5 and a CDR-H3 corresponding to
residues 95-102 of SEQ ID NO:5, wherein the CDR-H2 comprises
residues 50-59 of SEQ ID NO:5, wherein the residue numbering is
referenced with respect to Kabat numbering.
21. The antibody of claim 19, wherein (a) the amino acid at
position 5 of the VH domain is V or Q; (b) the amino acid at
position 69 of the VH domain is M or L; (c) the amino acid at
position 71 of the VH domain is T or V; (d) the amino acid at
position 73 of the VH domain is T or K; or (e) the amino acid at
position 75 of the VH domain is T or S, wherein the residue
numbering is referenced with respect to Kabat numbering.
22. The antibody of claim 19, wherein the VH domain human acceptor
framework sequences are free of any back-mutations.
23. The antibody of claim 20, wherein the VH domain comprises the
sequence of SEQ ID NO:5.
24. The antibody of claim 23, comprising a CDR-L1 corresponding to
residues 24-34 of SEQ ID NO:4, a CDR-L2 corresponding to residues
50-56 of SEQ ID NO:4, and a CDR-L3 corresponding to residues 89-97
of SEQ ID NO:4, wherein the residue numbering is referenced with
respect to Kabat numbering.
25. The antibody of claim 24, wherein the VL domain comprises SEQ
ID NO:4.
26. The antibody of claim 19, which is an IgG4 antibody.
27. The antibody of claim 19, which is an antibody fragment or a
multispecific antibody.
28. The antibody of claim 19, wherein the antibody VH domain
comprises residues D52, D54, F99, T(100C), and W(100F) from the VH
CDRs of murine antibody Z270, wherein the residue numbering is
referenced with respect to Kabat numbering.
29. The antibody of claim 28, wherein the antibody VH domain
comprises residues N35, Y53, E56, D98, V(100A), and L(100D) from
the VH CDRs of murine antibody Z270, wherein the residue numbering
is referenced with respect to Kabat numbering.
30. The antibody of claim 29, comprising a CDR-H1 corresponding to
residues 31-35 of SEQ ID NO: 5 and a CDR-H3 corresponding to
residues 95-102 of SEQ ID NO: 5, wherein the CDR-H2 comprises
residues 50-59 of SEQ ID NO: 5, wherein the residue numbering is
referenced with respect to Kabat numbering.
31. The antibody of claim 19, comprising a CDR-H1 corresponding to
residues 31-35 of SEQ ID NO: 5 and a CDR-H3 corresponding to
residues 95-102 of SEQ ID NO: 5, wherein the CDR-H2 comprises
residues 50-59 of SEQ ID NO: 5, wherein the residue numbering is
referenced with respect to Kabat numbering.
32. A method for producing an anti-NKG2A antibody, comprising
culturing a host cell comprising a nucleic acid encoding the
anti-NKG2A antibody of claim 19 so that the nucleic acid is
expressed and the antibody produced.
33. A pharmaceutical composition comprising the antibody of claim
19 and a pharmaceutically acceptable carrier.
34. A method of treating a human patient suffering from a disorder
selected from a cancer, a viral disease, an inflammatory disorder,
and an autoimmune disorder, comprising administering the
pharmaceutical composition of claim 33.
35. A diagnostic method for NKG2A protein comprising contacting the
antibody of claim 19 with a sample selected from cells, tissue or
serum and detecting the binding of said antibody to NKG2A in said
sample.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/624,882, filed Jun. 16, 2017, which is a continuation of
U.S. application Ser. No. 14/548,327, filed Nov. 20, 2014, now U.S.
Pat. No. 9,683,041, which is a divisional of U.S. application Ser.
No. 13/477,888, filed May 22, 2012, now U.S. Pat. No. 8,901,283,
which is a continuation of U.S. application Ser. No. 12/305,683,
filed May 26, 2009, now U.S. Pat. No. 8,206,709, which is a 35
U.S.C. .sctn. 371 national stage application of International
Patent Application PCT/EP2007/056485 (published as WO 2008/009545),
filed Jun. 28, 2007, which claimed priority of European Patent
Application 06116429.9, filed Jun. 30, 2006; this application
further claims priority under 35 U.S.C. .sctn. 119 of U.S.
Provisional Application 60/818,550, filed Jul. 5, 2006; the
contents of all above-named applications are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to anti-NKG2A antibodies, in
particular humanized versions of murine anti-NKG2A antibody Z270,
as well as methods of producing and using such antibodies.
BACKGROUND OF THE INVENTION
[0003] CD94/NKG2A is a cytotoxicity inhibitory receptor found on
subsets of NK, NKT and T cells, which restricts their killing of
cells expressing the CD94/NKG2A-ligand HLA-E (see, e.g.,
WO99/28748). Antibodies that inhibit CD94/NKG2A may increase the
cytolytic activity of tumor-specific lymphocytes against tumor
cells. Therefore, therapeutic antibodies that inhibit CD94/NKG2A in
cancer patients without killing CD94/NKG2A-expressing cells may be
able to control tumor-growth. In addition, certain lymphomas such
as, e.g., NK-lymphomas, are characterized by CD94/NKG2A expression.
In such patients, therapeutic antibodies that target and kill
CD94/NKG2A-expressing cells may be able to eradicate tumor cells.
Anti-NKG2A antibodies have also been suggested for use in treating
autoimmune or inflammatory diseases (see, e.g., US20030095965A1,
WO02006070286).
[0004] Various antibodies against CD94/NKG2A have been described in
the art. For example, Sivori et al. (Eur J Immunol 1996;
26:2487-92) refers to the murine anti-NKG2A antibody Z270;
Carretero et al. (Eur J Immunol 1997; 27:563-7) describes murine
anti-NKG2A antibody Z199 (now commercially available via Beckman
Coulter, Inc., Product No. IM2750, USA); Vance et al. (J Exp Med
1999; 190: 1801-12) refers to murine anti-NKG2-antibody 20D5 (now
commercially available via BD Biosciences Pharmingen, Catalog No.
550518, USA); and U.S. patent application publication 20030095965
describes murine antibody 3S9, which purportedly binds to NKG2A,
NKG2C and NKG2E.
[0005] Currently available anti-CD94/NKG2A antibodies are of
non-human origin, which makes them unsuitable for most therapeutic
applications in humans due to their immunogenicity. While simple
humanization approaches such as, e.g., CDR-grafting, are available,
individualized humanization approaches are typically needed to
obtain an optimal humanized variant, minimizing immunogenicity
while sufficiently retaining or improving functional properties.
Accordingly, there is a need for anti-CD94/NKG2A antibodies that
are suitable for treatment of human patients.
SUMMARY OF THE INVENTION
[0006] The present invention provides anti-NKG2A antibodies, as
well as compositions comprising such antibodies, and methods of
producing and using such antibodies. In one embodiment, the
antibody is a humanized version of murine anti-NKG2A antibody Z270,
herein denoted a "humZ270". In another embodiment, the antibody is
a humanized version of an anti-NKG2A antibody having substantially
identical variable heavy-chain (VH) and/or variable light-chain
(VL) domains to those of Z270.
[0007] Exemplary complementarity-determining region (CDR) residues
and/or sites for amino acid substitutions in framework region (FR)
and/or of such antibodies to produce antibodies having improved
properties such as, e.g., lower immunogenicity, improved
antigen-binding or other functional properties, and/or improved
physicochemical properties such as, e.g., better stability, are
provided. In one aspect, the invention provides humanized
antibodies in which at least a portion of a Kabat CDR is identical
to the corresponding portion in the human acceptor sequence. In one
embodiment, the human acceptor framework sequence does not comprise
any amino acid substitutions or back-mutations. In another
embodiment, the human framework sequence comprises at least one
amino acid substitution. Kabat positions for such exemplary amino
acid substitutions include 5, 66, 67, 69, 71, 73, and 75 in the
framework region of the VH domain, 46 and 48 in the framework
region of the VL domain, and 60, 63, 64, and 65 in CDR-H2.
[0008] In other aspects, the invention provides for pharmaceutical
compositions comprising such antibodies and a carrier, and for
immunoconjugates comprising such antibodies conjugated to a
cytotoxic or detectable agent.
[0009] In other aspects, the invention provides for nucleic acids
and vectors encoding such antibodies, and host cells containing
such nucleic acids and/or vectors. Also provided for are
recombinant methods of producing anti-NKG2A antibodies by culturing
such host cells so that the nucleic acids are produced.
[0010] In other aspects, the invention provides for articles of
manufacture comprising a container comprising such anti-NKG2A
antibodies and instructions directing a user to treat a disorder
such as cancer or a viral disease in a patient. Optionally, the
article may comprise another container containing another agent,
wherein the instructions direct the user to treat the disorder with
the antibody in combination with the agent.
[0011] The invention also provides for methods of using such
anti-NKG2A antibodies in the treatment of disorders such as cancer,
a viral disease, an inflammatory disorder or an autoimmune disorder
in a patient, optionally in conjunction with another anti-cancer,
anti-viral disease agent, or anti-inflammatory agent.
DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 shows an alignment of Z270VL and Z270VH with selected
germline V- and J-segments and exemplary humZ270VL1 (SEQ ID NO:4)
and humZ270VH1 (SEQ ID NO:5) sequences, with amino acid residue
numbering according to the Kabat scheme (Kabat et al, 1991,
Sequences of Proteins of Immunological Interest, 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.) Mask
residues are shaded in the Kabat scheme; CDR residues are shown in
bold in the Kabat scheme; mouse/germline differences are shaded in
the VKI_02/JK4 (SEQ ID NO:9) and VH1_18/JH6 (SEQ ID NO:10)
sequences; and potential back-mutation residues are shaded in the
humZ270VHNL sequences. The identified potential back-mutations in
the VL were L46F and 148V. The identified potential back-mutations
in the heavy chain were V5Q, M69L, T71V, T73K, and T75S. Also shown
are the resulting humZ270VL consensus ("humZ270VL1 cons;" SEQ ID
NO:6) and humZ270VH1 ("humZ270VH1 cons;" SEQ ID NO:7) consensus
sequences. In humZ270VL1 cons, the amino acid at position 46 is L
or F, and the amino acid at position 48 is I or V. In humZ270VH1
cons, the amino acid at position 5 is V or Q; the amino acid at
position 69 is M or L; the amino acid at position 71 is T or V; the
amino acid at position 73 is T or K; and/or the amino acid at
position 75 is T or S. In an alternative humZ270VH1,
humZ270VH1cons2 (SEQ ID NO:8), the amino acid at position 5 is V or
Q; the amino acid at position 60 is S or A; the amino acid at
position 63 is L or F; the amino acid at position 64 is Q or K; the
amino acid at position 65 is G or D; the amino acid at position 66
is R or K; the amino acid at position 67 is V or A; the amino acid
at position 69 is M or L; the amino acid at position 71 is T or V;
the amino acid at position 73 is T or K; and/or the amino acid at
position 75 is T or S.
[0013] FIG. 2 shows the CDRs of an exemplary humZ270 antibody,
according to the Kabat definitions. The differences compared to
murine Z270 CDRs are shown in bold. CDR_L1 corresponds to residues
24-34 of SEQ ID NO: 4. CDR_L2 corresponds to residues 50-56 of SEQ
ID NO: 4. CDR_L3 corresponds to residues 89-97 of SEQ ID NO: 4.
CDR_H1 corresponds to residues 31-35 of SEQ ID NO: 5. CDR_H2
corresponds to residues 50-66 of SEQ ID NO: 5. CDR_H3 corresponds
to residues 99-114 of SEQ ID NO: 5. The residue numbering is
referenced with respect to each SEQ ID NO recited.
[0014] FIGS. 3A-3H show plasmids referred to in the Examples. (FIG.
3A) pMD19-T vector map for TA cloning. (FIG. 3B) pJSV002 cloning
vector map for transient expression. (FIG. 3C) Light chain is
inserted into pJSV002 with murine kappa constant region. (FIG. 3D)
pJSV002-mlgG1 variant containing murine IgG1 constant region. (FIG.
3E) pJSV002-mlgG1 Z270 H1 containing Z270 heavy chain variable
region and IgG1 heavy chain constant region. (FIG. 3F)
pJSV002-hKappa Z270 L11 containing light chain variable region and
human kappa chain constant region. (FIG. 3G) pJSV002-lgG4-S241P.
(FIG. 3H) pJSV002-lgG4-S241P Z270H1.
[0015] FIGS. 4A-4J show different sequences derived from or based
on Z270, FIG. 4A to FIG. 4J, SEQ ID NOS:14-23, respectively. See
Examples 2-5 for details.
[0016] FIG. 5 shows an exemplary design of a vector for expression
of humanized Z270 heavy-chains.
[0017] FIG. 6 shows an exemplary design of a vector for expression
of humanized Z270 light-chains.
[0018] FIG. 7 shows an exemplary outline of a procedure for
transient expression of humanized Z270 antibodies in HEK293
cells.
[0019] FIG. 8 shows an exemplary Biacore assay to determine humZ270
binding affinity for antigen.
[0020] FIGS. 9A-9B show affinity determinations of chimeric Z270
(FIG. 9A) and humZ270VL1NH1 (FIG. 9B).
[0021] FIG. 10 shows affinity determination of humZ270VL1NH1,
having different back-mutations in the light chain or heavy chain.
Chimeric Z270 was used as a comparison.
[0022] FIG. 11 shows the strategy for standard CDR-grafting of
murine Z270 Kabat CDRs H1-H3 into a VH1_18/JH6 heavy chain acceptor
framework, without (humZ270H3) or with (humZ270VH4)
back-mutations.
[0023] FIG. 12 shows an alignment between humZ270VH constructs in
different human acceptor sequences, all with a partly human portion
of CDR-H2.
[0024] FIG. 13 shows the results of Biacore affinity evaluation of
humZ270 variantsVL1NH1 and VH3-VH8, all normalized to the KD of
hZ270VL1NH1.
[0025] FIG. 14 shows the selected residues for alanine-scan
mutagenesis to identify critical paratope residues in the Z270 VL
(SEQ ID NO: 1) and VH (SEQ ID NO: 2) segments.
[0026] FIG. 15 shows the results of Biacore affinity evaluation of
Z270VH alanine mutants, normalized to the binding of chimeric
Z270.
[0027] FIG. 16 shows the results of Biacore affinity evaluation of
Z270VL alanine mutants, normalized to the binding of chimeric
Z270.
[0028] FIG. 17 shows the binding of various recombinant Z270
variants to Ba/F3 cells stably over-expressing either CD94/NKG2A or
CD94/NKG2C, using flow-cytometry.
[0029] FIG. 18 shows the results of an assay to evaluate the
ability of recombinant Z270, chimeric Z270, humZ270VL1NH1, and Z199
to induce killing of .sup.51Cr-labeled LCL 721.221-Cw3 cells by
CD94/NKG2A+ NKL cells, showing that humZ270VL1NH1 was more
efficient in inducing killing.
[0030] FIG. 19 shows that humZ270VL1NH1 efficiently binds
Ba/F3-CD94/NKG2A cells in a concentration dependent fashion
(diamonds). However, when cells were pre-incubated with HLA-E
tetramers, humZ270VL1NH1 was prevented from binding to
Ba/F3-CD94/NKG2A cells (squares).
[0031] FIG. 20 shows that two different preparations of
humZ270VL1NH1 and a humZ270 variant with a V5Q mutation in VH bind
to CD94/NKG2A--but not CD94/NKG2C-expressing cells, though the
V5Q-variant slightly less efficiently.
DEFINITIONS
[0032] The term "antibody" herein is used in the broadest sense and
specifically includes full-length monoclonal antibodies, polyclonal
antibodies, multispecific antibodies (e.g., bispecific antibodies),
and antibody fragments, so long as they exhibit the desired
biological activity. Various techniques relevant to the production
of antibodies are provided in, e.g., Harlow, et al., ANTIBODIES: A
LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1988).
[0033] An "antibody fragment" comprises a portion of a full-length
antibody, preferably antigen-binding or variable regions thereof.
Examples of antibody fragments include Fab, Fab', F(ab).sub.2,
F(ab').sub.2, F(ab).sub.3, Fv (typically the VL and VH domains of a
single arm of an antibody), single-chain Fv (scFv), dsFv, Fd
fragments (typically the VH and CH1 domain), and dAb (typically a
VH domain) fragments; VH, VL, VhH, and V-NAR domains; minibodies,
diabodies, triabodies, tetrabodies, and kappa bodies (see, e.g.,
III et al., Protein Eng 1997; 10: 949-57); camel IgG; IgNAR; and
multispecific antibody fragments formed from antibody fragments,
and one or more isolated CDRs or a functional paratope, where
isolated CDRs or antigen-binding residues or polypeptides can be
associated or linked together so as to form a functional antibody
fragment. Various types of antibody fragments have been described
or reviewed in, e.g., Holliger and Hudson, Nat Biotechnol 2005; 23,
1126-1136; WO2005040219, and published U.S. Patent Applications
20050238646 and 20020161201.
[0034] The term "antibody derivative", as used herein, comprises a
full-length antibody or a fragment of an antibody, preferably
comprising at least antigen-binding or variable regions thereof,
wherein one or more of the amino acids are chemically modified,
e.g., by alkylation, PEGylation, acylation, ester formation or
amide formation or the like, e.g., for linking the antibody to a
second molecule. This includes, but is not limited to, PEGylated
antibodies, cysteine-PEGylated antibodies, and variants
thereof.
[0035] An "immunoconjugate" comprises an antibody derivative
associated with or linked to a second agent, such as a cytotoxic
agent, a detectable agent, etc.
[0036] A "humanized" antibody is a human/non-human chimeric
antibody that contains a minimal sequence derived from non-human
immunoglobulin. For the most part, humanized antibodies are human
immunoglobulins (recipient antibody) in which residues from a
hypervariable region of the recipient are replaced by residues from
a hypervariable region of a non-human species (donor antibody) such
as mouse, rat, rabbit, or non-human primate having the desired
specificity, affinity, and capacity. In some instances, framework
region (FR) residues of the human immunoglobulin are replaced by
corresponding non-human residues. Furthermore, humanized antibodies
may comprise residues that are not found in the recipient antibody
or in the donor antibody. These modifications are made to further
refine antibody performance. In general, a humanized antibody will
comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the
hypervariable loops correspond to those of a non-human
immunoglobulin and all or substantially all of the FR residues are
those of a human immunoglobulin sequence. The humanized antibody
can optionally also comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see, e.g., Jones et al.,
Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329
(1988); and Presta, Curr. Op. Struct Biol. 2:593-596 (1992), WO
92/02190, US Patent Application 20060073137, and U.S. Pat. Nos.
6,750,325, 6,632,927, 6,639,055, 6,548,640, 6,407,213, 6,180,370,
6,054,297, 5,929,212, 5,895,205, 5,886,152, 5,877,293, 5,869,619,
5,821,337, 5,821,123, 5,770,196, 5,777,085, 5,766,886, 5,714,350,
5,693,762, 5,693,761, 5,530,101, 5,585,089, and 5,225,539.
[0037] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody that are responsible for
antigen-binding. The hypervariable region generally comprises amino
acid residues from a "complementarity-determining region" or "CDR"
(residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light-chain
variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the
heavy-chain variable domain; Kabat et al. 1991, supra) and/or those
residues from a "hypervariable loop" (residues 26-32 (L1), 50-52
(L2) and 91-96 (L3) in the light-chain variable domain and 26-32
(H1), 53-55 (H2) and 96-101 (H3) in the heavy-chain variable
domain; Chothia and Lesk, J. Mol. Biol 1987; 196:901-917).
Typically, the numbering of amino acid residues in this region is
performed by the method described in Kabat et al., supra. Phrases
such as "Kabat position", "using Kabat numbering", "variable domain
residue numbering as in Kabat" and "according to Kabat" herein
refer to this numbering system for heavy chain variable domains or
light chain variable domains. Using the Kabat numbering system, the
actual linear amino acid sequence of a peptide may contain fewer or
additional amino acids corresponding to a shortening of, or
insertion into, a FR or CDR of the variable domain. For example, a
heavy chain variable domain may include a single amino acid insert
(residue 52a according to Kabat) after residue 52 of CDR H2 and
inserted residues (e.g. residues 82a, 82b, and 82c, etc. according
to Kabat) after heavy chain FR residue 82. The Kabat numbering of
residues may be determined for a given antibody by alignment at
regions of homology of the sequence of the antibody with a
"standard" Kabat numbered sequence. Unless otherwise indicated or
contrary to context, the position of all amino acid residues in a
VL or VH sequence described herein are according to Kabat.
[0038] "Framework region" or "FR" residues are those VH or VL
residues other than the CDRs as herein defined.
[0039] A "variant" of a polypeptide refers to a polypeptide having
an amino acid sequence that is substantially identical to a
reference polypeptide, typically a native or "parent" polypeptide.
The polypeptide variant may possess one or more amino acid
substitutions, deletions, and/or insertions at certain positions
within the native amino acid sequence.
[0040] "Conservative" amino acid substitutions are those in which
an amino acid residue is replaced with an amino acid residue having
a side chain with similar physicochemical properties. Families of
amino acid residues having similar side chains are known in the
art, and include amino acids with basic side chains (e.g., lysine,
arginine, histidine), acidic side chains (e.g., aspartic acid,
glutamic acid), uncharged polar side chains (e.g., glycine,
asparagine, glutamine, serine, threonine, tyrosine, cysteine,
tryptophan), nonpolar side chains (e.g., alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine), beta-branched side
chains (e.g., threonine, valine, isoleucine) and aromatic side
chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
[0041] The term "substantially identical" in the context of two
amino acid sequences means that the sequences, when optimally
aligned, such as by the programs GAP or BESTFIT using default gap
weights, share at least about 50, at least about 60, at least about
70, at least about 80, at least about 90, at least about 95, at
least about 98, or at least about 99 percent sequence identity. In
one embodiment, residue positions that are not identical differ by
conservative amino acid substitutions. Sequence identity is
typically measured using sequence analysis software. Protein
analysis software matches similar sequences using measures of
similarity assigned to various substitutions, deletions and other
modifications, including conservative amino acid substitutions. For
instance, the publicly available GCG software contains programs
such as "Gap" and "BestFit" which can be used with default
parameters to determine sequence homology or sequence identity
between closely related polypeptides, such as homologous
polypeptides from different species of organisms or between a
wild-type protein and a mutein thereof. See, e.g., GCG Version 6.1.
Polypeptide sequences can also be compared using FASTA or ClustalW,
applying default or recommended parameters. A program in GCG
Version 6.1., FASTA (e.g., FASTA2 and FASTA3) provides alignments
and percent sequence identity of the regions of the best overlap
between the query and search sequences (Pearson, Methods Enzymol.
1990; 183:63-98; Pearson, Methods Mol. Biol. 2000; 132:185-219).
Another preferred algorithm when comparing a sequence to a database
containing a large number of sequences from various organisms, or
when deducing the computer program BLAST, especially blastp, using
default parameters. See, e.g., Altschul et al., J. Mol. Biol. 1990;
215:403-410; Altschul et al., Nucleic Acids Res. 1997; 25:3389-402
(1997); each herein incorporated by reference. "Corresponding"
amino acid positions in two substantially identical amino acid
sequences are those aligned by any of the protein analysis software
mentioned herein, typically using default parameters.
[0042] An antibody having a "biological characteristic" of a
reference antibody, (e.g., Z270), is one that possesses one or more
of the biological characteristics of that antibody that distinguish
it from other antibodies that bind to the same antigen (e.g.
NKG2A). For example, an antibody with a biological characteristic
of Z270 may block activation of NKG2A, and/or cross-compete with
Z270 in binding the extracellular domain of NKG2A.
[0043] NKG2A (OMIM 161555, the entire disclosure of which is herein
incorporated by reference) is a member of the NKG2 group of
transcripts (Houchins, et al. (1991) J. Exp. Med. 173:1017-1020).
NKG2A is encoded by 7 exons spanning 25 kb, showing some
differential splicing. NKG2A is an inhibitory receptor found on the
surface of subsets of NK cells, .alpha./.beta. T cells,
.gamma./.delta. cells, and NKT cells. Like inhibitory KIR
receptors, it possesses an ITIM in its cytoplasmic domain. As used
herein, "NKG2A" refers to any variant, derivative, or isoform of
the NKG2A gene or encoded protein. Also encompassed are any nucleic
acid or protein sequences sharing one or more biological properties
or functions with wild type, full length NKG2A, and sharing at
least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or higher nucleotide
or amino acid identity. Human NKG2A comprises 233 amino acids in 3
domains, with a cytoplasmic domain comprising residues 1-70, a
transmembrane region comprising residues 71-93, and an
extracellular region comprising residues 94-233, of the following
sequence:
TABLE-US-00001 (SEQ ID NO: 11)
MDNQGVIYSDLNLPPNPKRQQRKPKGNKSSILATEQEITYAELNLQKASQ
DFQGNDKTYHCKDLPSAPEKLIVGILGIICLILMASVVTIVVIPSTLIQR
HNNSSLNTRTQKARHCGHCPEEWITYSNSCYYIGKERRTWEESLLACTSK
NSSLLSIDNEEEMKFLSIISPSSWIGVFRNSSHHPWVTMNGLAFKHEIKD
SDNAELNCAVLQVNRLKSAQCGSSIIYHCKHKL
[0044] NKG2C (OMIM 602891, the entire disclosure of which is herein
incorporated by reference) and NKG2E (OMIM 602892, the entire
disclosure of which is herein incorporated by reference) are two
other members of the NKG2 group of transcripts (Gilenke, et al.
(1998) Immunogenetics 48:163-173). NKG2C and NKG2E are activating
receptors found on the surface of NK cells.
[0045] HLA-E (OMIM 143010, the entire disclosure of which is herein
incorporated by reference) is a nonclassical MHC molecule that is
expressed on the cell surface and regulated by the binding of
peptides derived from the signal sequence of other MHC class I
molecules. HLA-E binds natural killer (NK) cells and some T cells,
binding specifically to CD94/NKG2A, CD94/NKG2B, and CD94/NKG2C
(see, e.g., Braud et al. (1998) Nature 391:795-799, the entire
disclosure of which is herein incorporated by reference). Surface
expression of HLA-E is sufficient to protect target cells from
lysis by CD94/NKG2A+NK, T, or NKT cell clones. As used herein,
"HLA-E" refers to any variant, derivative, or isoform of the HLA-E
gene or encoded protein. Also encompassed are any nucleic acid or
protein sequences sharing one or more biological properties or
functions with wild type, full length HLA-E, and sharing at least
70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or higher nucleotide or
amino acid identity.
[0046] A nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome-binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice.
[0047] An "isolated" molecule is a molecule that is the predominant
species in the composition wherein it is found with respect to the
class of molecules to which it belongs (i.e., it makes up at least
about 50% of the type of molecule in the composition and typically
will make up at least about 70%, at least about 80%, at least about
85%, at least about 90%, at least about 95%, or more of the species
of molecule, e.g., peptide, in the composition). Commonly, a
composition of an antibody molecule will exhibit 98%, 98%, or 99%
homogeneity for antibody molecules in the context of all present
peptide species in the composition or at least with respect to
substantially active peptide species in the context of proposed
use.
[0048] In the context of the present invention, "treatment" or
"treating" refers to preventing, alleviating, managing, curing or
reducing one or more symptoms or clinically relevant manifestations
of a disease or disorder, unless contradicted by context. For
example, "treatment" of a patient in whom no symptoms or clinically
relevant manifestations of a disease or disorder have been
identified is preventive or prophylactic therapy, whereas
"treatment" of a patient in whom symptoms or clinically relevant
manifestations of a disease or disorder have been identified
generally does not constitute preventive or prophylactic
therapy.
DETAILED DESCRIPTION OF THE INVENTION
[0049] The present invention concerns antibodies binding to NKG2A.
In one aspect, the antibody is a humanized version of antibody
Z270, which is a murine monoclonal antibody that specifically binds
NKG2A, but not to human NKG2C or NKG2E. Z270 can block the function
of human CD94/NKG2A, and specifically induce killing of cells by
CD94/NKG2A-restricted lymphocytes in a concentration-dependent
fashion.
[0050] The invention provides, e.g., humZ270 variants in which at
least a portion of a VH CDR such as the CDR-H2 is identical to the
corresponding portion of the human VH acceptor sequence, thus
reducing the immunogenicity of the humanized antibody.
Surprisingly, such humanized variants can be more effective in
potentiating the cytotoxicity of a CD94/NKG2A-expressing cytotoxic
lymphocyte than the murine or a chimeric form of Z270. In other
aspects, the invention provides antibodies having CDRs comprising
certain antigen-binding residues corresponding to those in murine
antibody Z270, and human framework sequences. These and other
aspects are described in more detail in the following sections and
in the Examples.
Humanized Anti-NKG2A Antibodies
[0051] Methods for humanizing non-human antibodies have been
described in the art. Generally, in a humanization process,
nucleotides encoding the interaction-regions of a murine antibody
can be cloned into a cDNA-vector encoding human IgG, which can be
done such that a chimeric antibody is generated consisting of a
human IgG backbone harbouring the murine CDRs. Such chimeric
antibodies may exhibit a lower affinity, lower stability, or other
undesired features in comparison with the original murine antibody,
and may also be immunogenic. Therefore, individual amino acids in
the chimeric Ab may need to be optimized to obtain a functional mAb
of high quality for therapeutic applications in humans.
[0052] Typically, a humanized antibody has one or more amino acid
residues introduced into it from a source that is non-human. These
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: 522-525
(1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et
al., Science, 239: 1534-1536 (1988)), by substituting hypervariable
region sequences for the corresponding sequences of a human
"acceptor" antibody. Accordingly, such "humanized" antibodies are
chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially
less than an intact human variable domain has been substituted by
the corresponding sequence from a non-human species. In practice,
humanized antibodies are typically human antibodies in which some
hypervariable region residues and possibly some FR residues are
substituted by residues from analogous sites in rodent
antibodies.
[0053] Another method for making humanized antibodies is described
in U.S. patent application publication 2003/0017534, wherein
humanized antibodies and antibody preparations are produced from
transgenic non-human animals. The non-human animals are genetically
engineered to contain one or more humanized immunoglobulin loci
that are capable of undergoing gene rearrangement and gene
conversion in the transgenic non-human animals to produce
diversified humanized immunoglobulins.
[0054] 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 a rodent antibody is
screened against a library of known human variable-domain sequences
or a library of human germline sequences. The human sequence that
is closest to that of the rodent can then be accepted as the human
framework region for the humanized antibody (Sims et al., J.
Immunol. 1993; 151:2296 et seq.; Chothia et al, Chothia and Lesk,
J. Mol. Biol 1987; 196:901-917). Another method uses a particular
framework region derived from the consensus sequence of all human
antibodies of a particular subgroup of light or heavy chains. The
same framework may be used for several different humanized
antibodies (Carter et al., PNAS USA, 1992; 89:4285 et seq.; Presta
et al., J Immunol 1993; 151:2623 et seq.). Other methods designed
to reduce the immunogenicity of the antibody molecule in a human
patient include veneered antibodies (see, e.g., U.S. Pat. No.
6,797,492 and U.S. patent application publications 20020034765 and
20040253645) and antibodies that have been modified by T-cell
epitope analysis and removal (see, e.g., U.S. patent application
publications 20030153043 and U.S. Pat. No. 5,712,120).
[0055] It is further important that antibodies be humanized with
retention of high affinity for the antigen 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 that 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 recipient and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
hypervariable region residues are directly and most substantially
involved in influencing antigen binding.
[0056] Example 1 below describes the design of exemplary humanized
anti-NKG2A antibodies that bind NKG2A, and the analysis is, at
least in part, illustrated in FIG. 1. As shown in FIG. 1, in one
humZ270 antibody of the invention, the C-terminal portion of CDR-H2
(corresponding to Kabat residues 61-65) are identical to the
corresponding portion of the human acceptor sequence. Further, as
described in the figure legend, a humZ270VL sequence (SEQ ID NO:4)
may optionally comprise mutations in one or both of the indicated
FR residues L46 and 148, and a humZ270VH sequence (SEQ ID NO:5) may
optionally comprise mutations in one or more of the indicated FR
residues V5, M69, T71, T73 and T75, with amino acid numbering
according to Kabat. Table 1 describes exemplary humZ270VL and
humZ270VH variants comprising exemplary human-to-murine
back-mutations in the humZ270VH and humZ270VL FR sequences, as well
as exemplary combinations of FR mutations. In Table 1 and elsewhere
herein, the amino acid positions are designated according to Kabat,
in which amino acids V5, M69, T71, T73, and T75 in the humZ270VH
domain correspond to amino acids V5, M70, T72, T74, and T76 in SEQ
ID NO:5, SEQ ID NO:7, SEQ ID NO:8, or other exemplary humZ270VH
sequences described herein.
TABLE-US-00002 TABLE 1 humZ270VL and humZ270VH variants comprising
exemplary FR back-mutations in native or consensus humZ270VL (SEQ
ID NO: 4 or 6) and/or humZ270VH (SEQ ID NO: 5, 7, or 8) sequences.
humZ270VL Variants humZ270VH Variants None None L46F V5Q I48V M69L
L46F and I48V T71V T73K T75S V5Q and M69L V5Q and T71V V5Q and T73K
V5Q and T75S M69L and T71V M69L and T73K M69L and T75S T71V and
T73K T71V and T75S T73K and T75S V5Q, T73K and T75S V5Q, T71V and
T75S V5Q, T71V and T73K V5Q, M69L and T75S V5Q, M69L and T73K V5Q,
M69L and T71V T71V, T73K and T75S M69L, T73K and T75S M69L, T71V
and T75S, M69L, T71V and T73K, V5Q, M69L, T71V and T73K, V5Q, M69L,
T71V and T75S, V5Q, M69L, T73K and T75S, V5Q, T71V, T73K and T75S,
M69L, T71V, T73K and T75S, V5Q, M69L, T71V, T73K and T75S
[0057] Accordingly, the present invention provides for humanized
versions of an anti-NKG2A antibody produced by the Z270 hybridoma,
as well as for humanized versions of non-human antibodies sharing
biological characteristics and/or substantial sequence identity
with Z270. In another embodiment, the monoclonal antibody or a
fragment or derivative thereof is capable of binding to a non-human
primate NKG2A.
[0058] The humanized antibody herein comprises non-human
hypervariable region or CDR residues incorporated into human VH and
VL domains.
[0059] In one aspect, the invention provides a humanized antibody
comprising antigen-binding residues from the CDRs of murine
antibody Z270 in a human acceptor framework, wherein at least the 6
C-terminal amino acid residues of the CDR-H2 are the same as those
in the human acceptor sequence. Such humanized antibodies can be
more effective than the original murine Z270 antibody or a chimeric
version thereof in, e.g., potentiating the cytotoxic activity of a
CD94/NKG2A-expressing cytotoxic lymphocyte, such as, e.g., an
NK-cell, an NKT-cell, an .alpha./.beta. T-cell, and/or a
.gamma./.delta. T-cell, or of a population of CD94/NKG2A-expressing
cytotoxic lymphocytes.
[0060] Typical antibodies of the invention comprise antigen-binding
residues corresponding to, or portions of the Z270 CDR-H2 and
CDR-H3 that comprise, residues D52, D54, R94, F99, T(100C), and
W(100F) of SEQ ID NO:2 or SEQ ID NO:5, which have been shown in the
Examples to be critical for antigen-binding. Optionally, the
antibodies also comprise VH residues N35, Y53, E56, D98, V(100A),
and L(100D).
[0061] In another aspect, a humanized antibody comprises a CDR-H1
sequence corresponding to residues 31-35 of SEQ ID NO:5 and a
CDR-H3 sequence corresponding to residues 95-102 of SEQ ID NO:5,
wherein the CDR-H2 sequence comprises residues 50-59 of SEQ ID
NO:5. In such humanized antibodies, the VH region can have, for
example 50% or more sequence identity to SEQ ID NO:5, such as at
least 60%, at least 70%, or at least 75% sequence identity.
Exemplary humanized VH domains having such sequences are described
in the examples and below.
[0062] In one embodiment, in such humanized antibodies, the amino
acid at position 5 of the VH region can be V or Q; the amino acid
at position 69 of the VH region can be M or L; the amino acid at
position 71 of the VH region can be T or V; the amino acid at
position 73 of the VH region can be T or K; and/or the amino acid
at position 75 of the VH region can be T or S. In separate
embodiments, the VH region comprises V5 or Q5, M69 or L69, T71 or
V71, T73, or K71, and/or T75 or S75 residues. In another
embodiment, the amino acid at position 69 is L. In another
additional or alternative embodiment, the amino acid at position 71
is V.
[0063] The humanized antibody may comprise a VH human acceptor
framework from a human acceptor sequence selected from, e.g.,
VH1_18, VH5_a, VH5_51, VH1_f, and VH1_46, and the J-segment is JH6,
such as, e.g., a VH1_18, VH5_a, VH5_51, or VH1_f, or other human
germline VH framework sequences known in the art. In one
embodiment, the VH segment is VH1_18. In a particular embodiment,
the VH region comprises the sequence of SEQ ID NO:8. In another
particular embodiment, the VH region comprises the sequence of SEQ
ID NO:7. For example, the VH region may comprise the sequence of
SEQ ID NO:5.
[0064] The VL region human acceptor sequence may be, e.g.,
VKI_02/JK4. In a particular embodiment, the VL region comprises SEQ
ID NO:4.
[0065] In one aspect, a humanized antibody of the invention
comprises a CDR-H2 comprising a murine portion consisting of
residues 50-59 of SEQ ID NO:2 linked to a CDR-H3 consisting of
residues 95-102 of SEQ ID NO:2 via suitable FR sequences.
[0066] As described herein, in one embodiment, the humanized
antibody comprises no FR substitution in the VH domain. In another
embodiment, the humanized antibody comprises a VH domain FR
substitution at one or more positions selected from 5, 69, 71, 73
and 75, utilizing the variable domain numbering system according to
Kabat. In another embodiment, the humanized antibody comprises VH
domain FR substitutions at two or more of positions 5, 69, 71, 73
and 75; and in other embodiments, at three, four, or all of such
positions. In separate and independent embodiments, the humanized
antibody comprises one VH domain FR substitution at a position
selected from 5, 69, 71, 73 and 75. In other separate and
independent embodiments, the humanized antibody comprises VH domain
FR substitutions at positions 5 and 69, or 5 and 71, or 5 and 73,
or 5 and 75, or 69 and 71, 69 and 73, or 69 and 75, or 71 and 73,
or 71 and 75, or 73 and 75, or 5, 69 and 71, or 5, 69 and 73, or 5,
69 and 75, or 69, 71 and 73, or 69, 71 and 75, or 71, 73, and 75,
or 5, 69, 71, and 73, or 5, 69, 71, and 75, or 5, 71, 73, and 75,
or 69, 71, 73, and 75, or 5, 69, 71, 73, and 75. Fewer rather than
more framework substitutions can minimize immunogenicity, but
binding efficacy may be an important consideration for some
applications. Thus, preferred substitutions are back-mutations,
i.e., mutations which replace an amino acid at a certain position
in the human FR with the amino acid at the corresponding position
in a non-human donor FR. Thus, in separate and independent
embodiments, the VH domain amino acid substitution at position 5 is
V5Q, the amino acid substitution at position 69 is M69L, the amino
acid substitution at position 71 is T71V, the amino acid
substitution at position 73 is T73K, and the amino acid
substitution at position 75 is T75S. In a particular embodiment,
the humanized antibody comprises a V at position 71 and/or an L at
position 69.
[0067] The humanized antibody herein also comprises non-human
hypervariable region residues incorporated into a human VL domain.
In one embodiment, the humanized antibody comprises no FR
substitution in the VL domain. In another embodiment, the humanized
antibody comprises a VL domain FR substitution at one of positions
46 and 48, utilizing the variable domain numbering system according
to Kabat. In another embodiment, the humanized antibody comprises
VL domain FR substitutions both of positions 46 and 48. Preferred
substitutions are back-mutations, i.e., mutations which replace an
amino acid at a certain position in the human FR with the amino
acid at the corresponding position in a non-human donor FR. Thus,
in separate and independent embodiments, the VL domain amino acid
substitution at position 46 is L46F, and the VL domain amino acid
substitution at position 48 is 148V.
[0068] An exemplary humanized antibody comprises a VH domain
comprising a CDR-H1 sequence corresponding to residues 31-35 of SEQ
ID NOS:5 or 7, a CDR-H2 sequence corresponding to residues 50-66 of
SEQ ID NOS:5 or 7, and a CDR-H3 sequence corresponding to residues
95-102 of SEQ ID NOS:5 or 7. The humanized antibody may further
comprise an amino acid at Kabat position 5 which is V or Q, an
amino acid at Kabat position 69 which is M or L, an amino acid at
Kabat position 71 which is T or V, an amino acid at Kabat position
73 is T or K, and/or an amino acid at Kabat position 75 which is T
or S, in the VH domain. In one embodiment, the VH domain comprises
a framework region substitution in at least one Kabat position
selected from the group consisting of 5, 69, 71, 73, and 75, e.g.,
corresponding to any of the VH FR substitutions, or combinations
thereof, listed in Table 1. In one embodiment, the VH domain
comprises the sequence of SEQ ID NO:7. In another embodiment, the
VH domain comprises the sequence of SEQ ID NO:5.
[0069] An exemplary humanized antibody may also or alternatively
comprise a VL domain comprising a CDR-L1 sequence corresponding to
residues 24-34 of SEQ ID NO:4, a CDR-L2 sequence corresponding to
residues 50-56 of SEQ ID NO:4, and an CDR-L3 sequence corresponding
to residues 89-97 of SEQ ID NO:4. The humanized antibody may
further comprise an amino acid at Kabat position 46 which is L or F
and/or an amino acid at Kabat position 48 which is I or V. In one
embodiment, the VL domain comprises a framework region substitution
in at least one Kabat position selected from 46 and 48, e.g.,
corresponding to any of the VL FR substitutions, or combinations
thereof, listed in Table 1. In one embodiment, the VL domain
comprises the amino acid sequence of SEQ ID NO:4. In another
embodiment, the VL domain comprises the sequence of SEQ ID
NO:6.
[0070] In another aspect, the invention provides a humanized
antibody that binds human NKG2A, the antibody comprising a VH
domain that comprises the amino acid sequence of SEQ ID NO:5,
optionally with one or more FR substitutions at Kabat positions 5,
69, 71, 73, and/or 75. The optional FR substitutions can be
selected from, e.g., V5Q, M69L, T71V, T73K, and/or T75S, as well as
any combination thereof. Such a humanized antibody may also or
alternatively comprise a VL domain that comprises the amino acid
sequence of SEQ ID NO:4, optionally with one or more FR
substitutions at Kabat positions 46 and/or 48. The optional FR
substitutions can be selected from, e.g., L46F and/or 148V.
[0071] Optionally, in a particular aspect, the VH domain comprises
amino acid modifications of one or more CDR residues, e.g. where
the modifications essentially maintain or improve affinity of the
antibody. For example, the antibody variant may have one, two,
three, or from one to about seven amino acid substitutions in the
above VH CDR sequences.
[0072] In a particular aspect, amino acids in the humanized
antibody VH CDRs which are different from the amino acids at the
corresponding positions in the non-human donor VH CDRs can be
substituted to improve the binding properties and/or stability of
the humanized antibody. For example, one or more of these amino
acids can be substituted for the amino acid at the corresponding
position(s) in the non-human donor VH CDR. In one embodiment, the
variant antibody comprises CDRH2 substitutions at one or more
positions selected from 60, 63, 64, and 65, according to Kabat
(corresponding to positions 61, 64, 65, and 66 in SEQ ID NO:5 or
7). In separate and independent embodiments, the antibody variant
comprises a CDRH2 amino acid substitution at one position selected
from 60, 63, 64, and 65. In other separate and independent
embodiments, the antibody variant comprises CDRH2 amino acid
substitution at positions 60 and 63, or 60 and 64, or 60 and 65, or
63 and 64, or 63 and 65, or 64 and 65, or 60, 63, and 64, or 60,
63, and 65, or 60, 64, and 65, or 63, 64, and 65, or 60, 63, 64,
and 65. Preferred substitutions are back-mutations, i.e., mutations
which replace an amino acid at a certain position in the humanized
CDR with the amino acid at the corresponding position in a
non-human donor CDR. Thus, in separate and independent embodiments,
the CDRH2 amino acid substitution at position 60 is A60S, the amino
acid substitution at position 63 is L63F, the amino acid
substitution at position 64 is Q64K, and the amino acid
substitution at position 65 is G65D. In aspects where the antibody
variant comprises one or more of the CDRH2 amino acid substitutions
A60S, L63F, Q64K, and G65D, the antibody variant comprises the VH
FR amino acid substitutions at positions 66 and 67 according to
Kabat, optionally in conjunction with one or more of the VH FR
substitutions previously described. Preferably, the amino acid
substitutions are R66K and V67A. For example, in one embodiment,
the antibody variant comprises a VH domain comprising a CDR-H1
sequence corresponding to residues 31-35 of SEQ ID NO:5, a CDR-H2
sequence corresponding to residues 50-66 of SEQ ID NO:5, and a
CDR-H3 sequence corresponding to residues 95-102 of SEQ ID NO:5,
with A60S, L63F, Q64K, G65D, R66K and V67A "back-mutations,"
optionally in conjunction with additional amino acid
modifications.
[0073] In another particular aspect, the invention provides for a
humanized antibody that binds human NKG2A, the antibody comprising
a VH domain that comprises non-human CDR residues incorporated into
a human VH domain, the VH domain comprising a CDR-H1 sequence
corresponding to residues 31-35 of SEQ ID NO:8, a CDR-H2 sequence
corresponding to residues 50-66 of SEQ ID NO:8, and a CDR-H3
sequence corresponding to residues 95-102 of SEQ ID NO:8, wherein
the amino acids at Kabat positions 63, 64, 65, 66, and 67 are F, K,
D, K, A, respectively. In one embodiment, the VH domain comprises
the amino acid sequence of SEQ ID NO:8. In another embodiment, the
residue at Kabat position 60 is A. In another embodiment, the
residue at Kabat position 60 is S, and the humanized antibody
comprises the CDR-H1, -H2, and H3 sequences of Z270, with FR
back-mutations in the two amino acids adjacent to the C-terminal
end of CDR-H2. The humanized antibodies described in this section
may further comprise FR back-mutations in other residues, e.g., at
Kabat positions 5, 69, 71, 73, and/or 75, as described herein.
[0074] In another aspect, the invention provides for a humanized
antibody wherein the Kabat CDRs in the VH region all derive from
the murine Z270VH (SEQ ID NO:2), further comprising S60A, F63L,
K64Q, and/or D65G mutations. In one embodiment, the humanized
antibody comprises all of the mutations S60A, F63L, K64Q, and/or
D65G.
[0075] The humanized antibody can also comprise a VL domain
comprising a CDR-L1 sequence corresponding to residues 24-34 of SEQ
ID NO:6, a CDR-L2 sequence corresponding to residues 50-56 of SEQ
ID NO:6, and an CDR-L3 sequence corresponding to residues 89-97 of
SEQ ID NO:6, e.g., in addition to the VH domain CDRs described
above. In one embodiment, the VL domain of the humanized antibody
comprises the amino acid sequence of SEQ ID NO:6. In another
embodiment, the VL domain of the humanized antibody comprises the
amino acid sequence of SEQ ID NO:4. Optionally, such a humanized
antibody comprises amino acid modifications of one or more VL CDR
residues, e.g., where the modifications essentially maintain or
improve affinity of the antibody. For example, the antibody variant
may have one, two, three, or from one to about seven amino acid
substitutions in the above VL CDR sequences.
[0076] The present application also contemplates affinity-matured
antibodies that bind anti-NKG2A, containing additional CDR or FR
mutations that improve the affinity of the humanized antibody for
the antigen. Methods for preparing such affinity-matured antibodies
are known in the art. The parent antibody may be a humanized
antibody, e.g., one comprising the VL/VH sequences of SEQ ID NOS:4
and 5 (optionally with one or more of the CDRH2 and/or FR
substitutions described herein), or one comprising the consensus
VL/VH sequences of SEQ ID NOS:6 and 7, respectively. The
affinity-matured antibody preferably binds to NKG2A with an
affinity comparable or superior to that of murine Z270, e.g. from
at least about one-, about two- or about four-fold to about
100-fold or about 1000-fold improved affinity, e.g. as assessed
using a binding assay as described below.
[0077] In another aspect, the invention provides humanized
antibodies that comprise a VH domain having at least about 50%, at
least about 70%, at least about 80% sequence identity (e.g., at
least about 85%, 90%, 95%, 97%, or more identity) to the VH domain
of Z270, humZ270, humZ270cons, and/or humZ270cons2 (SEQ ID NOS:2,
5, 7, and 8, respectively). In another particular aspect, the
invention provides a humanized antibody that binds NKG2A,
comprising a VH domain that comprises non-human CDR residues
incorporated into a human VH domain, wherein the VH domain is at
least about 50% identical to humZ270VH1 (SEQ ID NO:5). In one
embodiment, the VH domain is at least 90% identical to SEQ ID NO:5.
For example, the humanized antibody may comprise a CDR-H1 sequence
corresponding to residues 31-35 of SEQ ID NO:5, a CDR-H2 sequence
corresponding to residues 50-66 of SEQ ID NO:5, and a CDR-H3
sequence corresponding to residues 95-102 of SEQ ID NO:5. The
humanized antibody may also or alternatively comprise a V or Q at
Kabat position 5, an M or L at Kabat position 69, a T or V at Kabat
position 71, a T or K at Kabat position 73, and a T or S at Kabat
position 75, in the VH domain.
[0078] In another aspect, the invention provides an antibody
molecule that also or alternatively comprises a VL domain having at
least about 50%, at least about 70%, or at least about 80% sequence
identity (e.g., at least about 85%, 90%, 95%, 97%, or more
identity) to the VL domain of Z270, humZ270, and/or humZ270 cons
(SEQ ID NOS:1, 4, and 6, respectively). In another particular
aspect, the invention provides a humanized antibody that binds
NKG2A, comprising a VL region that comprises non-human CDR residues
incorporated into a human VL domain, wherein the VL domain is at
least 50% identical to SEQ ID NO:4. In one embodiment, the VL
region is at least 90% identical to SEQ ID NO:4. For example, the
humanized antibody may comprise a CDR-L1 sequence corresponding to
residues 24-34 of SEQ ID NO:4, a CDR-L2 sequence corresponding to
residues 50-56 of SEQ ID NO:4, and an CDR-L3 sequence corresponding
to residues 89-97 of SEQ ID NO:4. The humanized antibody may also
or alternatively comprise a L or F at Kabat position 46 and/or an I
or V at Kabat position 48, in the VL domain.
[0079] In another aspect, the invention provides an isolated
humanized antibody that specifically binds NKG2A and that comprises
either (a) CDR-L1, CDR-L2, and/or CDR-L3 sequences of Z270 VL (SEQ
ID NO:1), humZ270 VL (SEQ ID NO:4), or humZ270 VL cons (SEQ ID
NO:6) or (b) CDR-H1, CDR-H2, and/or CDR-H3 sequences of Z270 VH
(SEQ ID NO:2), humZ270 (SEQ ID NO:5), humZ270 cons (SEQ ID NO:7),
or humZ270 cons2 (SEQ ID NO:8). In another aspect, the invention
provides an antibody molecule that comprises at least a complete
set of VH CDRs from Z270, humZ270, or humZ270 cons, wherein the 6
C-terminal amino acids are identical to those of the human acceptor
sequence. In a particular aspect, the invention provides an
antibody molecule that comprises CDR-H1, CDR-H2 (or an N-terminal
portion thereof), and CDR-H3 of Z270, humZ270, or humZ270 cons and
at least some of CDR-L1, CDR-L2, and CDR-L3 of Z270, humZ270, or
humZ270 cons. In a more particular aspect, the invention provides
an antibody molecule wherein the antibody molecule comprises
CDR-L1, CDR-L2, and CDR-L3, CDR-H1, CDR-H2, and CDR-H3 of Z270,
humZ270, or humZ270 cons, wherein CDR-L1 is linked to CDR-L2,
CDR-L2 is linked to CDR-L3, CDR-H1 is linked to CDR-H2, and CDR-H2
is linked to CDR-H3, via suitable FR sequences.
[0080] In a particular aspect, the invention provides antibody
molecules that comprise essentially all of the VH and/or VL domains
of humZ270, humZ270 cons, or humZ270 cons2.
[0081] A humanized anti-NKG2A antibody according to the invention
may comprise any full-length or partial heavy-chain (HC) comprising
a humZ270VH described herein and/or any full-length or partial HC
comprising a humZ270VH may be combined with any humZ270VL described
herein in, and the resulting antibody or fragment tested for
antigen binding, functional effects on CD94/NKG2A-expressing cells,
and/or immunogenicity.
[0082] Various forms of the humanized antibody are contemplated.
For example, the humanized antibody may be an antibody fragment,
such as a Fab or other type of fragment described herein.
Alternatively, the humanized antibody may be a full-length or
intact antibody, such as a full-length or intact IgG1 or IgG4
antibody. In one embodiment, the humanized antibody is a
full-length IgG4 antibody or a fragment thereof.
[0083] In one aspect, the present invention provides a humanized
antibody characterized by: a) specifically binding to NKG2A; b) not
specifically binding to an Fc receptor; and c) when bound to NKG2A
on a human NK cell, causing said NK cell to lyse a target human
cell bearing HLA-E on the target cell surface, when said target
cell comes into contact with said NK cell. In one embodiment, the
humanized antibody comprises a mouse or human IgG1 constant region
that has been modified to prevent binding to an Fc receptor, or a
human IgG4 constant region. Such antibodies, as well as antibody
fragments that do not bind an Fc receptor, are particularly useful
in applications where it is desired to activate NK cells (e.g.
cancer, infectious disease), without leading to the depletion of
the NK cell themselves, as might be mediated by antibody dependent
cell cytotoxicity, and can be referred to as "non-depleting"
antibodies.
[0084] In another aspect, the humanized antibody comprises a mouse
or human IgG1 constant region that binds an Fc receptor, or a human
IgG1, 2, 3 or 4 constant region has been modified to bind an Fc
receptor or increase binding to an Fc receptor, or a human
IgG.sub.4 constant region. In another embodiment, the monoclonal
antibody or a fragment thereof is linked to a moiety that is toxic
to a cell to which the antibody is bound. Such antibodies are
particularly useful in applications where it is desired to deplete
an NK cell, useful in certain applications such as NK-LDGL (NK-type
lymphoproliferative disease of granular lymphocytes; alternatively
called NK-LGL), and can be referred to as "depleting"
antibodies.
[0085] For recombinant production of humanized antibodies,
humanized VH and VL regions, or variant versions thereof, can be
cloned into expression vectors encoding full-length or truncated
constant regions from a human antibody according to standard
recombinant methods (see, e.g., Sambrook et al., Molecular Cloning:
A Laboratory Manual, 2.sup.nd Ed., Cold Spring Harbor Laboratory,
Cold Spring Harbor, N.Y., 1989). The result is a transfected cell
line that expresses and secretes the humanized antibody molecule of
interest, comprising the selected VH and VL regions and constant
regions. cDNA sequences encoding the constant regions of human
antibodies are known. Exemplary cDNA sequences available via, e.g.,
GenBank, each of which incorporated by reference in its entirety,
are as follows:
[0086] Human IgG1 constant heavy chain region: GenBank accession
No.: J00228;
[0087] Human IgG2 constant heavy chain region: GenBank accession
No.: J00230;
[0088] Human IgG3 constant heavy chain region: GenBank accession
No.: X04646;
[0089] Human IgG4 constant heavy chain region: GenBank accession
No.: K01316; and
[0090] Human kappa light chain constant region: GenBank accession
No.: J00241.
[0091] If desired, the class of a humanized antibody may also be
"switched" by known methods. For example, an antibody that was
originally produced as an IgM molecule may be class switched to an
IgG antibody. Class switching techniques also may be used to
convert one IgG subclass to another, e.g., from IgG1 to IgG2. Thus,
the effector function of the antibodies of the invention may be
changed by isotype switching to, e.g., an IgG1, IgG2, IgG3, IgG4,
IgD, IgA, IgE, or IgM antibody for various therapeutic uses.
[0092] The constant region may further be modified according to
known methods. For example, in an IgG4 constant region, residue
S241 may be mutated to a proline (P) residue to allow complete
disulphide bridge formation at the hinge (see, e.g., Angal et al.,
Mol Immunol. 1993; 30:105-8).
Antibody Fragments
[0093] The humanized antibodies of the invention may be prepared as
antibody fragments, or antibody fragments may be prepared from
humanized full-length antibodies.
[0094] Various techniques have been developed for the production of
antibody fragments of humanized antibodies. Traditionally, these
fragments were derived via proteolytic digestion of full-length
antibodies (see, e.g., Morimoto et al., Journal of Biochemical and
Biophysical Methods, 24:107-117 (1992); and Brennan et al.,
Science, 229:81 (1985)). However, these fragments can now be
produced directly by recombinant host cells. Alternatively, Fab'-SH
fragments can be directly recovered from E. coli and chemically
coupled to form F(ab')2 fragments (Carter et al., Bio/Technology,
10:163-167 (1992)). According to another approach, F(ab')2
fragments can be isolated directly from recombinant host cell
culture. Other techniques for the production of antibody fragments
will be apparent to the skilled practitioner. In other embodiments,
the antibody of choice is a single-chain Fv fragment (scFv). See WO
1993/16185; U.S. Pat. Nos. 5,571,894; and 5,587,458. The antibody
fragment may also be a "linear antibody", e.g., as described in
U.S. Pat. No. 5,641,870, for example. Such linear antibody
fragments may be monospecific or bispecific.
[0095] Bispecific antibodies are antibodies that have binding
specificities for at least two different epitopes. Methods for
making bispecific antibodies are known in the art, and traditional
production of full-length bispecific antibodies is usually based on
the coexpression of two immunoglobulin heavy-chain-light-chain
pairs, where the two chains have different specificities (Millstein
et al., Nature, 305: 537-539 (1983)). In the bispecific antibodies
according to the present invention, at least one binding epitope is
on the NKG2A protein. The anti-NKG2A-binding "arm" may be combined
with an "arm" that binds to a triggering molecule on a leukocyte
such as a T-cell receptor molecule (e.g. CD2 or CD3), or Fc
receptors for IgG (Fcgamma-R), such as Fo-gamma-RI (CD64),
Fo-gamma-RII (CD32) and Fo-gamma-RIII (CD16), so as to focus
cellular defense mechanisms to the NKG2A-expressing cell.
Bispecific antibodies may also be used to localize cytotoxic agents
to cells that express NKG2A. These antibodies possess a
NKG2A-binding arm and an arm that binds the cytotoxic agent (e.g.
saporin, anti-interferon-alpha, vinca alkaloid, ricin A chain,
methotrexate, or radioactive isotope hapten). Bispecific antibodies
can be prepared as full-length antibodies or antibody fragments
(e.g. F(ab')2 bispecific antibodies). Antibodies with more than two
valencies are contemplated. For example, trispecific antibodies can
be prepared. Tutt et al., J. Immunol, 147: 60 (1991).
[0096] Typical antibodies, antibody fragments and multispecific
antibodies of the invention comprise portions of the CDR-H2 and
CDR-H3 that comprise residues D52, D54, R94, F99, T(100C), and
W(100F) of SEQ ID NO:2 or SEQ ID NO:5, which have been shown to be
critical for antigen-binding (see Examples). Optionally, the
humanized antibody fragments and multispecific antibodies also
comprise Z270 heavy chain residues N35, Y53, E56, D98, V(100A), and
L(100D). In one aspect, a humanized antibody fragment or
multispecific antibody of the invention comprises residues 50-59 of
the CDR-H2 and residues 95-102 of the CDR-H3 of SEQ ID NO:2 or SEQ
ID NO:5. In one embodiment, the humanized antibody fragment or
multispecific antibody does not comprise one, two, or all of Z270
CDR-L1, CDR-L2, and CDR-L3. In an additional or alternative
embodiment, the antibody fragment or multispecific antibody does
not comprise the Z270 CDR-H1.
Antibody Derivatives
[0097] Antibody derivatives within the scope of this invention
include humanized antibodies conjugated or covalently bound to a
second agent.
[0098] For example, in one aspect, the invention provides
immunoconjugates comprising a humanized antibody conjugated or
covalently bonded to a cytotoxic agent. The term "cytotoxic agent"
as used herein is a molecule that is capable of killing a cell
bearing a NKG2A receptor on its cell surface. Any type of moiety
with a cytotoxic or cytoinhibitory effect can be conjugated to the
present antibodies to form a cytotoxic conjugate of the present
invention and to inhibit or kill specific NK receptor expressing
cells, including therapeutic radioisotopes, toxic proteins, toxic
small molecules, such as drugs, toxins, immunomodulators, hormones,
hormone antagonists, enzymes, oligonucleotides, enzyme inhibitors,
therapeutic radionuclides, angiogenesis inhibitors,
chemotherapeutic drugs, vinca alkaloids, anthracyclines,
epidophyllotoxins, taxanes, antimetabolites, alkylating agents,
antibiotics, COX-2 inhibitors, SN-38, antimitotics, antiangiogenic
and apoptotoic agents, particularly doxorubicin, methotrexate,
taxol, CPT-11, camptothecans, nitrogen mustards, gemcitabine, alkyl
sulfonates, nitrosoureas, triazenes, folic acid analogs, pyrimidine
analogs, purine analogs, platinum coordination complexes,
Pseudomonas exotoxin, ricin, abrin, 5-fluorouridine, ribonuclease
(RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral
protein, gelonin, diphtherin toxin, Pseudomonas exotoxin, and
Pseudomonas endotoxin and others (see, e.g., Remington's
Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co. 1995);
Goodman and Gilman's The Pharmacological Basis of Therapeutics
(McGraw Hill, 2001); Pastan et al. (1986) Cell 47:641; Goldenberg
(1994) Cancer Journal for Clinicians 44:43; U.S. Pat. No.
6,077,499; the entire disclosures of which are herein incorporated
by reference). It will be appreciated that a toxin can be of
animal, plant, fungal, or microbial origin, or can be created de
novo by chemical synthesis.
[0099] In another embodiment, the antibody is derivatized with a
radioactive isotope, such as a therapeutic radionuclide or a
radionuclide suitable for detection purposes. Any of a number of
suitable radioactive isotopes can be used, including, but not
limited to, 1-131, Indium-111, Lutetium-171, Bismuth-212,
Bismuth-213, Astatine-211, Copper-62, Copper-64, Copper-67,
Yttrium-90, Iodine-125, Iodine-131, Phosphorus-32, Phosphorus-33,
Scandium-47, Silver-Ill, Gallium-67, Praseodymium-142,
Samarium-153, Terbium-161, Dysprosium-166, Holmium-166,
Rhenium-186, Rhenium-188, Rhenium-189, Lead-212, Radium-223,
Actinium-225, Iron-59, Selenium-75, Arsenic-77, Strontium-89,
Molybdenum-99, Rhodium-105, Palladium-109, Praseodymium-143,
Promethium-149, Erbium-169, Iridium-194, Gold-198, Gold-199, and
Lead-211. In general, the radionuclide preferably has a decay
energy in the range of 20 to 6,000 keV, preferably in the ranges 60
to 200 keV for an Auger emitter, 100-2,500 keV for a beta emitter,
and 4,000-6,000 keV for an alpha emitter. Also preferred are
radionuclides that substantially decay with generation of
alpha-particles.
[0100] In other embodiments, the second agent is a detectable
moiety, which can be any molecule that can be quantitatively or
qualitatively observed or measured. Examples of detectable markers
useful in the conjugated antibodies of this invention are
radioisotopes, fluorescent dyes, or a member of a complementary
binding pair, such as a member of any one of: and antigen/antibody
(other than an antibody to NKG2A), lectin/carbohydrate;
avidin/biotin; receptor/ligand; or molecularly imprinted
polymer/print molecule systems.
[0101] The second agent may also or alternatively be a polymer,
intended to increase the circulating half-life of the humanized
antibody, for example. Exemplary polymers and methods to attach
such polymers to peptides are illustrated in, e.g., U.S. Pat. Nos.
4,766,106; 4,179,337; 4,495,285; and 4,609,546. Additional
illustrative polymers include polyoxyethylated polyols and
polyethylene glycol (PEG) moieties (e.g., a full-length antibody or
antibody fragment can be conjugated to one or more PEG molecules
with a molecular weight of between about 1,000 and about 40,000,
such as between about 2000 and about 20,000, e.g., about
3,000-12,000).
[0102] The cytotoxic agents or other compounds can be linked to the
antibody directly or indirectly, using any of a large number of
available methods. For example, an agent can be attached at the
hinge region of the reduced antibody component via disulfide bond
formation, using cross-linkers such as N-succinyl
3-(2-pyridyldithio)proprionate (SPDP), or via a carbohydrate moiety
in the Fc region of the antibody (see, e.g., Yu et al. (1994) Int.
J. Cancer 56: 244; Wong, Chemistry of Protein Conjugation and
Cross-linking (CRC Press 1991); Upeslacis et al., "Modification of
Antibodies by Chemical Methods," in Monoclonal antibodies:
principles and applications, Birch et al. (eds.), pages 187-230
(Wiley-Liss, Inc. 1995); Price, "Production and Characterization of
Synthetic Peptide-Derived Antibodies," in Monoclonal antibodies:
Production, engineering and clinical application, Ritter et al.
(eds.), pages 60-84 (Cambridge University Press 1995), Cattel et
al. (1989) Chemistry today 7:51-58, Delprino et al. (1993) J.
Pharm. Sci 82:699-704; Arpicco et al. (1997) Bioconjugate Chemistry
8:3; Reisfeld et al. (1989) Antibody, Immunicon. Radiopharm. 2:217;
the entire disclosures of each of which are herein incorporated by
reference).
[0103] Alternatively, a fusion protein comprising the anti-NKG2A
antibody and a second (cytotoxic or other) polypeptide agent may be
made, e.g. by recombinant techniques or peptide synthesis.
Binding Assays
[0104] The present invention provides for antibodies that bind
human NKG2A, in particular humanized versions of an anti-NKG2A
antibody produced by the Z270 hybridoma.
[0105] Any of a wide variety of assays can be used to assess
binding of an antibody to human NKG2A. Protocols based upon ELISAs,
radioimmunoassays, Western blotting, BIACORE, and other competition
assays, inter alia, are suitable for use and are well known in the
art.
[0106] For example, simple binding assays can be used, in which a
test antibody is incubated in the presence of a target protein or
epitope (e.g., NKG2A or a portion thereof), unbound antibodies are
washed off, and the presence of bound antibodies is assessed using,
e.g., radiolabels, physical methods such as mass spectrometry, or
direct or indirect fluorescent labels detected using, e.g.,
cytofluorometric analysis (e.g. FACScan). Such methods are well
known to those of skill in the art. Any amount of binding above the
amount seen with a control, non-specific antibody indicates that
the antibody binds specifically to the target.
[0107] In such assays, the ability of the test antibody to bind to
the target cell or human NKG2A can be compared with the ability of
a (negative) control protein, e.g. an antibody raised against a
structurally unrelated antigen, or a non-Ig peptide or protein, to
bind to the same target. Antibodies or fragments that bind to the
target cells or NKG2A using any suitable assay with 25%, 50%, 100%,
200%, 1000%, or higher increased affinity relative to the control
protein, are said to "specifically bind to" or "specifically
interact with" the target, and are preferred for use in the
therapeutic methods described below. The ability of a test antibody
to affect the binding of a (positive) control antibody against
NKG2A, e.g. Z270, or derivatives thereof, may also be assessed.
[0108] The humanized anti-NKG2A antibodies may or may not bind
human NKG2C, may or may not bind human NKG2E, or may or may not
bind any of human NKG2C and E. In a particular embodiment, the
monoclonal antibody or fragment does not bind to other human NKG2
receptors, specifically the activating receptors NKG2C or NKG2E.
The NKG2C- and NKG2E-binding properties of the antibodies of the
invention can be evaluated in similar assays as those described
above, simply exchanging NKG2A for the molecule of interest.
[0109] In one aspect, the invention provides for humanized versions
of non-human antibodies sharing biological characteristics and/or
substantial sequence identity with Z270. One exemplary biological
characteristic is the binding to the Z270 epitope, i.e., the region
in the extracellular domain of NKG2A to which the Z270 antibody
binds. To screen for antibodies that bind to the Z270 epitope, a
routine cross-blocking assay, such as that described in Antibodies,
A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and
David Lane (1988), can be performed.
[0110] In an exemplary cross-blocking or competition assay, Z270
(control) antibody and a test antibody are admixed (or
pre-adsorbed) and applied to a sample containing NKG2A. In certain
embodiments, one would pre-mix the control antibodies with varying
amounts of the test antibody (e.g., 1:10 or 1:100) for a period of
time prior to applying to the NKG2A-containing sample. In other
embodiments, the control and varying amounts of test antibody can
simply be admixed during exposure to the antigen/target sample. As
long as one can distinguish bound from free antibodies (e.g., by
using separation or washing techniques to eliminate unbound
antibodies) and the control antibody from test antibody (e.g., by
using species- or isotype-specific secondary antibodies, by
specifically labeling the control antibody with a detectable label,
or by using physical methods such as mass spectrometry to
distinguish between different compounds) one will be able to
determine if the test antibody reduces the binding of the control
antibody to the antigen, indicating that the test antibody
recognizes substantially the same epitope as the control. In this
assay, the binding of the (labeled) control antibody in the
presence of a completely irrelevant antibody is the control high
value. The control low value is be obtained by incubating the
labeled (positive) control antibody (Z270) with unlabeled control
antibody, where competition would occur and reduce binding of the
labeled antibody.
[0111] In a test assay, a significant reduction in labeled antibody
reactivity in the presence of a test antibody is indicative of a
test antibody that recognizes the same epitope, i.e., one that
"cross-reacts" with the labeled control antibody. Any test antibody
or compound that reduces the binding of the labeled control to the
antigen/target by at least 50% or more preferably 70%, at any ratio
of control:test antibody or compound between about 1:10 and about
1:100 is considered to be an antibody or compound that binds to
substantially the same epitope or determinant as the control.
Preferably, such test antibody or compound will reduce the binding
of the control to the antigen/target by at least 90%. Nevertheless,
any compound or antibody that reduces the binding of a control
antibody or compound to any measurable extent can be used in the
present invention.
[0112] Similar cross-blocking assays can also be used to evaluate
whether a test (humanized) antibody affects the binding of the
natural ligand for human NKG2A, HLA-E, to NKG2A, by exchanging Z270
for a suitable form of HLA-E. For example, to determine whether a
humanized anti-NKG2A antibody preparation reduces or blocks
CD94/NKG2A interactions with HLA-E, the following test can be
performed: A cell line expressing CD94/NKG2A, such as
Ba/F3-CD94/NKG2A, NKL or NK92, is incubated for 30 min on ice, with
increasing concentrations of a test anti-NKG2A antibody. The cells
are then incubated with PE-labeled HLA-E tetramers for 30 minutes
on ice, washed again, and HLA-E tetramer binding analyzed on a flow
cytometer (FACScalibur, Beckton Dickinson), by standard methods. In
the absence of test antibodies, the HLA-E tetramer binds to the
cells. In the presence of an antibody preparation that blocks
CD94/NKG2A-binding to HLA-E, there is a reduced binding of HLA-E
tetramers to the cells, and such mAbs are designated "blocking
antibodies".
[0113] In some aspects of the invention, e.g., where it is not
desired to kill NKG2A-expressing cells, the humanized antibodies of
this invention preferably do not demonstrate substantial specific
binding to Fc receptors. Such antibodies may comprise constant
regions of various heavy chains that are known not to bind Fc
receptors. One such example is an IgG4 constant region.
Alternatively, antibody fragments that do not comprise constant
regions, such as Fab or F(ab')2 fragments, can be used to avoid Fc
receptor binding. Fc receptor binding can be assessed according to
methods known in the art, including for example testing binding of
an antibody to Fc receptor protein in a BIACORE assay. Also, any
other antibody type can be used in which the Fc portion is modified
to minimize or eliminate binding to Fc receptors (see, e.g.,
WO03101485, the disclosure of which is herein incorporated by
reference). Assays such as, e.g., cell based assays, to assess Fc
receptor binding are well known in the art, and are described in,
e.g., WO03101485.
Cytotoxicity Assays
[0114] If an anti-NKG2A antibody reduces or blocks CD94/NKG2A
interactions with HLA-E, it may increase the cytotoxicity of
CD94/NKG2A-restricted lymphocytes. This can be evaluated by a
typical cytotoxicity assay, examples of which are described
below.
[0115] The ability of an antibody to reduce CD94/NKG2A-mediated
signaling can be tested in a standard 4-hour in vitro cytotoxicity
assay using, e.g., NKL cells that express CD94/NKG2A, and target
cells that express HLA-E. Such NKL cells do not efficiently kill
targets that express HLA-E because CD94/NKG2A recognizes HLA-E,
leading to initiation and propagation of inhibitory signaling that
prevents lymphocyte-mediated cytolysis. Such an in vitro
cytotoxicity assay can be carried out by standard methods that are
well known in the art, as described for example in Coligan et al.,
eds., Current Protocols in Immunology, Greene Publishing Assoc. and
Wiley Interscience, N.Y., (1992, 1993). The target cells are
labeled with .sup.51Cr prior to addition of NKL cells, and then the
killing is estimated as proportional to the release of .sup.51Cr
from the cells to the medium, as a result of killing. The addition
of an antibody that prevents CD94/NKG2A from binding to HLA-E
results in prevention of the initiation and propagation of
inhibitory signaling via CD94/NKG2A. Therefore, addition of such
agents results in increases in lymphocyte-mediated killing of the
target cells. This step thereby identifies agents that prevent
CD94/NKG2A-induced negative signaling by, e.g., blocking ligand
binding. In a particular .sup.51Cr-release cytotoxicity assay,
CD94/NKG2A-expressing NKL effector-cells can kill HLA-E-negative
LCL 721.221 target cells, but less well HLA-E-expressing LCL
721.221-Cw3 control cells. In contrast, YTS effector-cells that
lack CD94/NKG2A kill both cell-lines efficiently. Thus, NKL
effector cells kill less efficiently HLA-E.sup.+ LCL 721.221-Cw3
cells due to HLA-E-induced inhibitory signaling via CD94/NKG2A.
When NKL cells are pre-incubated with blocking anti-CD94/NKG2A
antibodies according to the present invention in such a
.sup.51Cr-release cytotoxicity assay, HLA-E-expressing LCL
721.221-Cw3 cells are more efficiently killed, in an
antibody-concentration-dependent fashion.
[0116] The inhibitory or potentiating activity of an antibody of
this invention can also be assessed in any of a number of other
ways, e.g., by its effect on intracellular free calcium as
described, e.g., in Sivori et al., J. Exp. Med. 1997;
186:1129-1136, the disclosure of which is herein incorporated by
reference. NK, T, or NKT cell activity can also be assessed using a
cell based cytotoxicity assays, e.g., measuring chromium release or
other parameter to assess the ability of the antibody to stimulate
NK cells to kill target cells such as P815, K562 cells, or
appropriate tumor cells as disclosed in Sivori et al., J. Exp. Med.
1997; 186:1129-1136; Vitale et al., J. Exp. Med. 1998;
187:2065-2072; Pessino et al. J. Exp. Med. 1998; 188:953-960; Neri
et al. Clin. Diag. Lab. Immun. 2001; 8:1131-1135; Pende et al. J.
Exp. Med. 1999; 190:1505-1516, the entire disclosures of each of
which are herein incorporated by reference.
[0117] In one embodiment, an antibody preparation causes at least a
10% augmentation in the cytotoxicity of a CD94/NKG2A-restricted
lymphocyte, preferably at least a 40% or 50% augmentation in NK
cytotoxicity, or more preferably at least a 70% augmentation in NK
cytotoxicity.
[0118] The activity of a cytotoxic lymphocyte can also be addressed
using a cytokine-release assay, wherein NK cells are incubated with
the antibody to stimulate the cytokine production of the NK cells
(for example IFN-.gamma. and TNF-.alpha. production). In an
exemplary protocol, IFN-.gamma. production from PBMC is assessed by
cell surface and intracytoplasmic staining and analysis by flow
cytometry after 4 days in culture. Briefly, Brefeldin A (Sigma
Aldrich) is added at a final concentration of 5 .mu.g/ml for the
last 4 hours of culture. The cells are 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 are measured in supematants using ELISA (GM-CSF:
DuoSet Elisa, R&D Systems, Minneapolis, Minn., IFN-: OptEIA
set, Pharmingen).
[0119] In a particular aspect, the invention provides antibodies
that are more capable of, or more effective in, increasing the
cytotoxicity of CD94/NKG2A-restricted lymphocytes, potentiating
cytotoxic activity of a CD94/NKG2A-restricted lymphocyte, or
reducing or inhibiting CD94/NKG2A-mediated signaling, than the
original, non-humanized antibody and/or a chimeric version thereof.
Such antibodies can be, for example, at least 2%, at least 5%, at
least 10%, at least 15%, or at least 20% more capable or effective
an original, non-humanized antibody or chimeric version
thereof.
Recombinant Methods
[0120] The invention also provides isolated nucleic acids encoding
the anti-NKG2A antibodies described herein, as well as vectors and
host cells comprising such nucleic acids. Also provided for are
methods of producing such anti-NKG2A antibodies using recombinant
techniques such as, e.g., culturing suitable host cells comprising
such nucleic acids or vectors so that the nucleic acid is expressed
and the humanized antibody produced. Before culturing, the host
cell may, for example, be co-transfected with a vector comprising
nucleic acids encoding a variable heavy domain and with a vector
comprising nucleic acid encoding a variable light domain.
Additionally, the antibody may be recovered and/or purified from
the host cell culture using known techniques. Useful vectors, host
cells, and techniques are further described below and in the
Examples. Additionally, strategies for expressing humanized
anti-NKG2A antibodies are outlined in FIGS. 4-6.
[0121] Generally, for recombinant production of the antibody, a
nucleic acid encoding it is isolated and inserted into a replicable
vector for further cloning (amplification of the DNA) or for
expression, typically operably linked to one or more expression
control elements. DNA encoding the monoclonal antibody 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 the
antibody). Many vectors are known and available. The vector
components generally include, but are not limited to, one or more
of the following: a signal sequence, an origin of replication, one
or more marker genes, an enhancer element, a promoter, and a
transcription-termination sequence.
Signal Sequence Component
[0122] The anti-NKG2A antibody of this invention may be produced
recombinantly not only directly, but also as a fusion polypeptide
with a heterologous polypeptide, which is preferably a signal
sequence or other polypeptide having a specific cleavage site at
the N-terminus of the mature protein or polypeptide. The
heterologous signal sequence selected preferably is one that is
recognized and processed (i.e., cleaved by a signal peptidase) by
the host cell. See, e.g., Example 2 for exemplary signal sequences
for expression of humanized heavy and/or light chains of anti-NKG2A
antibodies such as humanized Z270 antibodies.
[0123] For prokaryotic host cells that do not recognize and process
the native anti-NKG2A antibody signal sequence, the signal sequence
is substituted by a prokaryotic signal sequence selected, for
example, from the group of the alkaline phosphatase, penicillinase,
Ipp, or heat-stable enterotoxin II leaders. For yeast secretion the
native signal sequence may be substituted by, e.g., the yeast
invertase leader, alpha-factor leader (including Saccharomyces and
Kluyveromyces alpha-factor leaders), acid-phosphatase leader, the
C. albicans glucoamylase leader, or the signal described in WO
1990/13646. In mammalian cell expression, mammalian signal
sequences as well as viral secretory leaders, for example, the
herpes simplex gD signal, are available.
[0124] The DNA for such precursor region is ligated in reading
frame to DNA encoding the anti-NKG2A antibody.
Origin of Replication Component
[0125] Both expression and cloning vectors contain a nucleic acid
sequence that enables the vector to replicate in one or more
selected host cells. Generally, in cloning vectors this sequence is
one that enables the vector to replicate independently of the host
chromosomal DNA, and includes origins of replication or
autonomously replicating sequences. Such sequences are well known
for a variety of bacteria, yeast, and viruses. The origin of
replication from the plasmid pBR322 is suitable for most
Gram-negative bacteria, the 2p plasmid origin is suitable for
yeast, and various viral origins (SV40, polyoma, adenovirus, VSV,
EBV, or BPV) are useful for cloning vectors in mammalian cells.
Generally, the origin of replication component is not needed for
mammalian expression vectors (the SV40 origin may typically be used
only because it contains the early promoter).
Selection Gene Component
[0126] Expression and cloning vectors may contain a selection gene,
also termed a selectable marker. Typical selection genes encode
proteins that (a) confer resistance to antibiotics or other toxins,
e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)
complement auxotrophic deficiencies, or (c) supply critical
nutrients not available from complex media, e.g., the gene encoding
D-alanine racemase for Bacilli.
[0127] One example of a selection scheme utilizes a drug to arrest
growth of a host cell. Those cells that are successfully
transformed with a heterologous gene produce a protein conferring
drug resistance and thus survive the selection regimen. Examples of
such dominant selection use the drugs neomycin, mycophenolic acid
and hygromycin.
[0128] Another example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the anti-NKG2A antibody-encoding nucleic acid, such as
DHFR, thymidine kinase, metallothionein-I and -II, preferably
primate metallothionein genes, aderosine deaminase, omithine
decarboxylase, etc.
[0129] For example, cells transformed with the DHFR selection gene
are first identified by culturing all of the transformants in a
culture medium that contains methotrexate (Mtx), a competitive
antagonist of DHFR. An appropriate host cell when wild-type DHFR is
employed is the Chinese hamster ovary (CHO) cell line deficient in
DHFR activity.
[0130] Alternatively, host cells (particularly wild-type hosts that
contain endogenous DHFR) transformed or co-transformed with DNA
sequences encoding anti-NKG2A antibody, wild-type DHFR protein, and
another selectable marker such as aminoglycoside
3'-phosphotransferase (APH) can be selected by cell growth in
medium containing a selection agent for the selectable marker such
as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or
G418. See U.S. Pat. No. 4,965,199.
[0131] A suitable selection gene for use in yeast is the trp1 gene
present in the yeast plasmid YRp7 (Stinchcomb et al., Nature,
282:39 (1979)). The trp1 gene provides a selection marker for a
mutant strain of yeast lacking the ability to grow in tryptophan,
for example, ATCC No. 44076 or PEP4-1. Jones, Genetics, 85:12
(1977). The presence of the trp1 lesion in the yeast host cell
genome then provides an effective environment for detecting
transformation by growth in the absence of tryptophan. Similarly,
Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are
complemented by known plasmids bearing the Leu2 gene.
[0132] In addition, vectors derived from the 1.6-.mu.m circular
plasmid pKD1 can be used for transformation of Kluyveromyces
yeasts. Alternatively, an expression system for large-scale
production of recombinant calf chymosin was reported for K. lactis
(Van den Berg, Bio/Technology, 8:135 (1990). Stable multi-copy
expression vectors for secretion of mature recombinant human serum
albumin by industrial strains of Kluyveromyces have also been
disclosed. Fleer et al., Bio/Technolog, 9: 968-975 (1991).
Promoter Component
[0133] Expression and cloning vectors usually contain a promoter
that is recognized by the host organism and is operably linked to
the anti-NKG2A antibody-encoding nucleic acid. Promoters suitable
for use with prokaryotic hosts include the phoA promoter,
.beta.-lactamase and lactose promoter systems, alkaline
phosphatase, a tryptophan (trp) promoter system, and hybrid
promoters such as the tac promoter. However, other known bacterial
promoters are suitable. Promoters for use in bacterial systems also
will contain a Shine-Dalgamo (S.D.) sequence operably linked to the
DNA encoding the anti-NKG2A antibody.
[0134] Various promoter sequences are known for eukaryotes.
Virtually all eukaryotic genes have an AT-rich region located
approximately 25 to 30 bases upstream from the site where
transcription is initiated. Another sequence found 70 to 80 bases
upstream from the start of transcription of many genes is a CNCAAT
region where N may be any nucleotide. At the 3' end of most
eukaryotic genes is an AATAAA sequence that may be the signal for
addition of the poly-A tail to the 3' end of the coding sequence.
All of these sequences are suitably inserted into eukaryotic
expression vectors. Examples of suitable promoting sequences for
use with yeast hosts include the promoters for 3-phosphoglycerate
kinase or other glycolytic enzymes, such as enolase,
glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase.
[0135] Other yeast promoters, which are inducible promoters having
the additional advantage of transcription controlled by growth
conditions, are the promoter regions for alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase, degradative enzymes associated
with nitrogen metabolism, metallothionein,
glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible
for maltose and galactose utilization. Suitable vectors and
promoters for use in yeast expression are further described in
EP73657. Yeast enhancers also are advantageously used with yeast
promoters.
[0136] Anti-NKG2A antibody transcription from vectors in mammalian
host cells is controlled, for example, by promoters obtained from
the genomes of viruses such as polyoma virus, fowipox virus,
adenovirus (such as Adenovirus 2), bovine papilloma virus, avian
sarcoma virus, cytomegalovirus (CMV), a retrovirus, hepatitis-B
virus and most preferably Simian Virus 40 (SV40), heterologous
mammalian promoters, e.g., the actin promoter or an immunoglobulin
promoter, and heat-shock promoters, provided such promoters are
compatible with the host cell systems.
[0137] The early and late promoters of the SV40 virus are
conveniently obtained as an SV40 restriction fragment that also
contains the SV40 viral origin of replication. The immediate early
promoter of the human cytomegalovirus is conveniently obtained as a
HindIII E restriction fragment. A system for expressing DNA in
mammalian hosts using the bovine papilloma virus as a vector is
disclosed in U.S. Pat. No. 4,419,446. A modification of this system
is described in U.S. Pat. No. 4,601,978. See also Reyes et al.,
Nature, 297:598-601 (1982) on expression of human .beta.-interferon
cDNA in mouse cells under the control of a thymidine kinase
promoter from herpes simplex virus. Alternatively, the rous sarcoma
virus long-terminal repeat can be used as the promoter.
Enhancer Element Component
[0138] Transcription of a DNA encoding the anti-NKG2A antibody of
this invention by higher eukaryotes is often increased by inserting
an enhancer sequence into the vector. Many enhancer sequences are
now known from mammalian genes (globin, elastase, albumin,
.alpha.-fetoprotein, and insulin). Typically, however, one will use
an enhancer from a eukaryotic cell virus. Examples include the SV40
enhancer on the late side of the replication origin (bp 100-270),
the cytomegalovirus early-promoter enhancer, the polyoma enhancer
on the late side of the replication origin, and adenovirus
enhancers. See also Yaniv, Nature, 297:17-18 (1982) on enhancing
elements for activation of eukaryotic promoters. The enhancer may
be spliced into the vector at a position 5' or 3' to the anti-NKG2A
antibody-encoding sequence, but is preferably located at a site 5'
from the promoter.
Transcription Termination Component
[0139] Expression vectors used in eukaryotic host cells (for
example, yeast, fungi, insect, plant, animal, human, or nucleated
cells from other multicellular organisms) will also contain
sequences necessary for the termination of transcription and for
stabilizing the mRNA. Such sequences are commonly available from
the 5' end, occasionally 3' end, of untranslated regions of
eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide
segments transcribed as polyadenylated fragments in the
untranslated portion of the mRNA encoding anti-NKG2A antibody. One
useful transcription termination component is the bovine growth
hormone polyadenylation region. See WO 1994/11026 and the
expression vector disclosed therein.
Selection and Transformation of Host Cells
[0140] Suitable host cells for cloning or expressing the DNA in the
vectors herein are the prokaryote, yeast, or higher eukaryote cells
described above. Suitable prokaryotes for this purpose include
eubacteria, such as Gram-negative or Gram-positive organisms, for
example, Enterobacteriaceae such as Escherichia, e.g., E. coli,
Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g.,
Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and
Shigella, as well as Bacilli such as B. subtilis and B.
licheniformis (e.g. B. licheniformis 41 P disclosed in DD 266,710
published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and
Streptomyces. One preferred E. coli cloning host is E. coli 294
(ATCC 31,446), although other strains such as E. coli B, E. coli
X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.
These examples are illustrative rather than limiting.
[0141] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for anti-NKG2A antibody-encoding vectors. Saccharomyces cerevisiae,
or common baker's yeast, is the most commonly used among lower
eukaryotic host microorganisms. However, a number of other genera,
species, and strains are commonly available and useful herein, such
as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K.
lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.
wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum
(ATCC 36,906), K. thermotolerans, and K. marxianus; yarrowia (EP
402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia
(EP 244,234); Neurospora crassa; Schwanniomyces such as
Schwanniomyces occidentalis; and filamentous fungi such as, e.g.,
Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such
as A. nidulans and A. niger.
[0142] Suitable host cells for the expression of glycosylated
anti-NKG2A antibody are derived from multicellular organisms.
Examples of invertebrate cells include plant and insect cells.
Numerous baculoviral strains and variants and corresponding
permissive insect host cells from hosts such as Spodoptera
frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes
albopictus (mosquito), Drosophila melanogaster (fruitfly), and
Bombyx mori have been identified. A variety of viral strains for
transfection are publicly available, e.g. the L-1 variant of
Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,
and such viruses may be used as the virus herein according to the
present invention, particularly for transfection of Spodoptera
frugiperda cells.
[0143] Plant cell cultures of cotton, corn, potato, soybean,
petunia, tomato, and tobacco can also be utilized as hosts.
[0144] However, interest has been greatest in vertebrate cells, and
propagation of vertebrate cells in culture (tissue culture) has
become a routine procedure. Examples of useful mammalian host cell
lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC
CRL 1651); human embryonic kidney (HEK) line (293 or 293 cells
subcloned for growth in suspension culture, Graham et al., J. Gen
Virol., 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL
10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc.
Natl. Acad. Sci. USA, 77:4216 (1980), including DG44 (Urlaub et
al., Som. Cell and Mol. Gen., 12: 555-566 (1986)) and DP12 cell
lines); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251
(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green
monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical
carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC
CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human
lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB
8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells
(Mather et al., Annals N.Y. Acad. Sci., 383:44-68 (1982)); MRC 5
cells; FS4 cells; and a human hepatoma line (Hep G2).
[0145] Host cells are transformed with the above-described
expression or cloning vectors for anti-NKG2A antibody production
and cultured in conventional nutrient media modified as appropriate
for inducing promoters, selecting transformants, or amplifying the
genes encoding the desired sequences.
Culturing the Host Cells
[0146] The host cells used to produce the anti-NKG2A antibody of
this invention may be cultured in a variety of media. Commercially
available media such as Ham's F10 (Sigma), Minimal Essential Medium
((MEM), Sigma), RPMI-1640 (Sigma), FreeStyle.TM. (Cibco) and
Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for
culturing the host cells. In addition, any of the media described,
for example, in Ham et al., Meth. Enz. 58:44 (1979); Barnes et al.,
Anal. Biochem., 102:255 (1980); U.S. Pat. Nos. 4,767,704;
4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 1990/03430; WO
1987/00195; or U.S. Pat. Re. 30,985 may be used as culture media
for the host cells. Any of these media may be supplemented as
necessary with hormones and/or other growth factors (such as
insulin, transferrin, or epidermal growth factor), salts (such as
sodium chloride, calcium, magnesium, and phosphate), buffers (such
as HEPES), nucleotides (such as adenosine and thymidine),
antibiotics (such as GENTAMYCIN.TM. drug), trace elements (defined
as inorganic compounds usually present at final concentrations in
the micromolar range), and glucose or an equivalent energy source.
Any other necessary supplements may also be included at appropriate
concentrations that would be known to those skilled in the art. The
culture conditions, such as temperature, pH, and the like, are
those previously used with the host cell selected for expression,
and will be apparent to the ordinarily skilled artisan.
Purification of Anti-NKG2A Antibody
[0147] When using recombinant techniques, the antibody can be
produced intracellularty or in the periplasmic space, or directly
secreted into the medium. If the antibody is produced
intracellularly, as a first step, the particulate debris, either
host cells or lysed fragments, is removed, for example, by
centrifugation or ultrafiltration. Carter et al., Bio/Technology,
10: 163-167 (1992) describes a procedure for isolating antibodies
that are secreted to the periplasmic space of E. coli. Briefly,
cell paste is thawed in the presence of sodium acetate (pH 3.5),
EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.
Cell debris can be removed by centrifugation. Where the antibody is
secreted into the medium, superatants from such expression systems
are generally first concentrated using a commercially available
protein concentration filter, for example, an AMICON.TM. or
MILLIPORE PELLICON.TM. ultrafiltration unit. A protease inhibitor
such as phenylmethylsulphonyl fluoride (PMSF) may be included in
any of the foregoing steps to inhibit proteolysis, and antibiotics
may be included to prevent the growth of adventitious
contaminants.
[0148] The antibody composition prepared from the cells can be
purified using, for example, hydroxylapatite chromatography, gel
electrophoresis, dialysis, and affinity chromatography, with
affinity chromatography being the preferred purification technique.
The suitability of protein A as an affinity ligand depends on the
species and isotype of any immunoglobulin Fc domain that is present
in the antibody. Protein A can be used to purify antibodies that
are based on human gamma1, gamma, or gamma4 heavy chains (iUndmark
et al., J. Immunol. Meth., 62:1-13 (1983)). Protein G is
recommended for all mouse isotypes and for human y3 (Guss et al.,
EMBO J., 5:15671575 (1986)). The matrix to which the affinity
ligand is attached is most often agarose, but other matrices are
available. Mechanically stable matrices such as controlled-pore
glass or poly(styrenedivinyl)benzene allow for faster flow rates
and shorter processing times than can be achieved with agarose.
Where the antibody comprises a CH3 domain, the BAKERBOND ABX.TM.
resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.
Other techniques for protein purification such as fractionation on
an ion-exchange column, ethanol precipitation, reverse-phase HPLC,
chromatography on silica, chromatography on heparin SEPHAROSE.TM.,
chromatography on an anion- or cation-exchange resin (such as a
polyaspartic acid column), chromatofocusing, SDS-PAGE, and
ammonium-sulfate precipitation are also available depending on the
antibody to be recovered.
[0149] An exemplary Protein A-based purification method for Z270 or
humanized versions thereof is described in Example 6.
Pharmaceutical Formulations
[0150] In one embodiment, the present invention provides
pharmaceutical composition comprising antibodies as described
herein together with one or more carriers.
[0151] Accordingly, one object of the invention is to provide a
pharmaceutical formulation comprising such an antibody which is
present in a concentration from 1 mg/ml to 500 mg/ml, and wherein
said formulation has a pH from 2.0 to 10.0. The formulation may
further comprise a buffer system, preservative(s), tonicity
agent(s), chelating agent(s), stabilizers and surfactants. In one
embodiment, the pharmaceutical formulation is an aqueous
formulation, i.e., formulation comprising water. Such formulation
is typically a solution or a suspension. In a further embodiment,
the pharmaceutical formulation is an aqueous solution. The term
"aqueous formulation" is defined as a formulation comprising at
least 50% w/w water. Likewise, the term "aqueous solution" is
defined as a solution comprising at least 50% w/w water, and the
term "aqueous suspension" is defined as a suspension comprising at
least 50% w/w water.
[0152] In another embodiment, the pharmaceutical formulation is a
freeze-dried formulation, whereto the physician or the patient adds
solvents and/or diluents prior to use.
[0153] In another embodiment, the pharmaceutical formulation is a
dried formulation (e.g. freeze-dried or spray-dried) ready for use
without any prior dissolution.
[0154] In a further aspect, the pharmaceutical formulation
comprises an aqueous solution of such an antibody, and a buffer,
wherein the antibody is present in a concentration from 1 mg/ml or
above, and wherein said formulation has a pH from about 2.0 to
about 10.0.
[0155] In another embodiment, the pH of the formulation is in the
range selected from the list consisting of from about 2.0 to about
10.0, about 3.0 to about 9.0, about 4.0 to about 8.5, about 5.0 to
about 8.0, and about 5.5 to about 7.5.
[0156] In a further embodiment, the buffer is selected from the
group consisting of sodium acetate, sodium carbonate, citrate,
glycylglycine, histidine, glycine, lysine, arginine, sodium
dihydrogen phosphate, disodium hydrogen phosphate, sodium
phosphate, and tris(hydroxymethyl)-aminomethan, bicine, tricine,
malic acid, succinate, maleic acid, fumaric acid, tartaric acid,
aspartic acid or mixtures thereof. Each one of these specific
buffers constitutes an alternative embodiment of the invention.
[0157] In a further embodiment, the formulation further comprises a
pharmaceutically acceptable preservative. The preservative may be
selected from, e.g., the group consisting of phenol, o-cresol,
m-cresol, p-cresol, methyl p-hydroxybenzoate, propyl
p-hydroxybenzoate, 2-phenoxyethanol, butyl p-hydroxybenzoate,
2-phenylethanol, benzyl alcohol, chlorobutanol, and thiomerosal,
bronopol, benzoic acid, imidurea, chlorohexidine, sodium
dehydroacetate, chlorocresol, ethyl p-hydroxybenzoate, benzethonium
chloride, chlorphenesine (3p-chlorphenoxypropane-1,2-diol) or
mixtures thereof. The preservative may, e.g., be present in a
concentration from 0.1 mg/ml to 20 mg/ml, from 0.1 mg/ml to 5
mg/ml, from 5 mg/ml to 10 mg/ml, or from 10 mg/ml to 20 mg/ml. Each
one of these specific preservatives constitutes an alternative
embodiment of the invention. The use of a preservative in
pharmaceutical compositions is well-known to the skilled person.
For convenience reference is made to Remington: The Science and
Practice of Pharmacy, 19.sup.th edition, 1995.
[0158] In a further embodiment, the formulation further comprises
an isotonic agent. The isotonic agent may be, e.g., selected from
the group consisting of a salt (e.g. sodium chloride), a sugar or
sugar alcohol, an amino acid (e.g. L-glycine, L-histidine,
arginine, lysine, isoleucine, aspartic acid, tryptophan,
threonine), an alditol (e.g. glycerol (glycerine), 1,2-propanediol
(propyleneglycol), 1,3-propanediol, 1,3-butanediol)
polyethyleneglycol (e.g. PEG400), or mixtures thereof. Any sugar
such as mono-, di-, or polysaccharides, or water-soluble glucans,
including for example fructose, glucose, mannose, sorbose, xylose,
maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin,
cyclodextrin, soluble starch, hydroxyethyl starch and
carboxymethylcellulose-Na may be used. In one embodiment, the sugar
additive is sucrose. Sugar alcohol is defined as a C4-C8
hydrocarbon having at least one --OH group and includes, for
example, mannitol, sorbitol, inositol, galactitol, dulcitol,
xylitol, and arabitol. In one embodiment, the sugar alcohol
additive is mannitol. The sugars or sugar alcohols mentioned above
may be used individually or in combination. There is no fixed limit
to the amount used, as long as the sugar or sugar alcohol is
soluble in the liquid preparation and does not adversely effect the
stabilizing effects achieved using the methods of the invention.
The sugar or sugar alcohol concentration can, e.g., be between
about 1 mg/ml and about 150 mg/ml. The isotonic agent can be
present in a concentration from, e.g., 1 mg/ml to 50 mg/ml, from 1
mg/ml to 7 mg/ml, from 8 mg/ml to 24 mg/ml, or from 25 mg/ml to 50
mg/ml. Each one of these specific isotonic agents constitutes an
alternative embodiment of the invention. The use of an isotonic
agent in pharmaceutical compositions is well-known to the skilled
person. For convenience reference is made to Remington: The Science
and Practice of Pharmacy, 19.sup.th edition, 1995.
[0159] In a further embodiment, the formulation also comprises a
chelating agent. The chelating agent can, for example, be selected
from salts of ethylenediaminetetraacetic acid (EDTA), citric acid,
and aspartic acid, and mixtures thereof. The chelating agent may,
for example, be present in a concentration from 0.1 mg/ml to 5
mg/ml, from 0.1 mg/ml to 2 mg/ml, or from 2 mg/ml to 5 mg/ml. Each
one of these specific chelating agents constitutes an alternative
embodiment of the invention. The use of a chelating agent in
pharmaceutical compositions is well-known to the skilled person.
For convenience reference is made to Remington: The Science and
Practice of Pharmacy, 19.sup.th edition, 1995.
[0160] In a further embodiment of the invention the formulation
further comprises a stabilizer. The use of a stabilizer in
pharmaceutical compositions is well-known to the skilled person.
For convenience reference is made to Remington: The Science and
Practice of Pharmacy, 19.sup.th edition, 1995. More particularly,
compositions of the invention can be stabilized liquid
pharmaceutical compositions whose therapeutically active components
include a polypeptide that possibly exhibits aggregate formation
during storage in liquid pharmaceutical formulations. By "aggregate
formation" is intended a physical interaction between the
polypeptide molecules that results in formation of oligomers, which
may remain soluble, or large visible aggregates that precipitate
from the solution. By "during storage" is intended a liquid
pharmaceutical composition or formulation once prepared, is not
immediately administered to a subject. Rather, following
preparation, it is packaged for storage, either in a liquid form,
in a frozen state, or in a dried form for later reconstitution into
a liquid form or other form suitable for administration to a
subject. By "dried form" is intended the liquid pharmaceutical
composition or formulation is dried either by freeze drying (i.e.,
lyophilization; see, for example, Williams and Polli (1984) J.
Parenteral Sci. Technol. 38:48-59), spray drying (see Masters
(1991) in Spray-Drying Handbook (5th ed; Longman Scientific and
Technical, Essez, U.K.), pp. 491-676; Broadhead et al. (1992) Drug
Devel. Ind. Pharm. 18:1169-1206; and Mumenthaler et al. (1994)
Pharm. Res. 11:12-20), or air drying (Carpenter and Crowe (1988)
Cryobiology 25:459-470; and Roser (1991) Biopharm. 4:47-53).
Aggregate formation by a polypeptide during storage of a liquid
pharmaceutical composition can adversely affect biological activity
of that polypeptide, resulting in loss of therapeutic efficacy of
the pharmaceutical composition. Furthermore, aggregate formation
may cause other problems such as blockage of tubing, membranes, or
pumps when the polypeptide-containing pharmaceutical composition is
administered using an infusion system.
[0161] The pharmaceutical compositions of the invention may further
comprise an amount of an amino acid base sufficient to decrease
aggregate formation by the polypeptide during storage of the
composition. By "amino acid base" is intended an amino acid or a
combination of amino acids, where any given amino acid is present
either in its free base form or in its salt form. Where a
combination of amino acids is used, all of the amino acids may be
present in their free base forms, all may be present in their salt
forms, or some may be present in their free base forms while others
are present in their salt forms. In one embodiment, amino acids to
use in preparing the compositions of the invention are those
carrying a charged side chain, such as arginine, lysine, aspartic
acid, and glutamic acid. Any stereoisomer (i.e., L, D, or a mixture
thereof) of a particular amino acid (e.g. methionine, histidine,
imidazole, arginine, lysine, isoleucine, aspartic acid, tryptophan,
threonine and mixtures thereof) or combinations of these
stereoisomers, may be present in the pharmaceutical compositions of
the invention so long as the particular amino acid is present
either in its free base form or its salt form. In one embodiment
the L-stereoisomer is used. Compositions of the invention may also
be formulated with analogues of these amino acids. By "amino acid
analogue" is intended a derivative of the naturally occurring amino
acid that brings about the desired effect of decreasing aggregate
formation by the polypeptide during storage of the liquid
pharmaceutical compositions of the invention. Suitable arginine
analogues include, for example, aminoguanidine, omithine and
N-monoethyl L-arginine, suitable methionine analogues include
ethionine and buthionine and suitable cysteine analogues include
S-methyl-L cysteine. As with the other amino acids, the amino acid
analogues are incorporated into the compositions in either their
free base form or their salt form. In a further embodiment of the
invention the amino acids or amino acid analogues are used in a
concentration, which is sufficient to prevent or delay aggregation
of the protein.
[0162] In a further embodiment of the invention methionine (or
other sulphuric amino acids or amino acid analogous) may be added
to inhibit oxidation of methionine residues to methionine sulfoxide
when the polypeptide acting as the therapeutic agent is a
polypeptide comprising at least one methionine residue susceptible
to such oxidation. By "inhibit" is intended minimal accumulation of
methionine oxidized species over time. Inhibiting methionine
oxidation results in greater retention of the polypeptide in its
proper molecular form. Any stereoisomer of methionine (L or D) or
combinations thereof can be used. The amount to be added should be
an amount sufficient to inhibit oxidation of the methionine
residues such that the amount of methionine sulfoxide is acceptable
to regulatory agencies. Typically, this means that the composition
contains no more than about 10% to about 30% methionine sulfoxide.
Generally, this can be achieved by adding methionine such that the
ratio of methionine added to methionine residues ranges from about
1:1 to about 1000:1, such as 10:1 to about 100:1.
[0163] In a further embodiment, the formulation further comprises a
stabilizer selected from the group of high molecular weight
polymers or low molecular compounds. In a further embodiment of the
invention the stabilizer is selected from polyethylene glycol (e.g.
PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone,
carboxy-/hydroxycellulose or derivates thereof (e.g. HPC, HPC-SL,
HPC-L and HPMC), cyclodextrins, sulphur-containing substances as
monothioglycerol, thioglycolic acid and 2-methylthioethanol, and
different salts (e.g. sodium chloride). Each one of these specific
stabilizers constitutes an alternative embodiment of the
invention.
[0164] The pharmaceutical compositions may also comprise additional
stabilizing agents, which further enhance stability of a
therapeutically active polypeptide therein. Stabilizing agents of
particular interest to the present invention include, but are not
limited to, methionine and EDTA, which protect the polypeptide
against methionine oxidation, and a nonionic surfactant, which
protects the polypeptide against aggregation associated with
freeze-thawing or mechanical shearing.
[0165] In a further embodiment, the formulation further comprises a
surfactant. The surfactant may, for example, be selected from a
detergent, ethoxylated castor oil, polyglycolyzed glycerides,
acetylated monoglycerides, sorbitan fatty acid esters,
polyoxypropylene-polyoxyethylene block polymers (eg. poloxamers
such as Pluronic F68, poloxamer 188 and 407, Triton X-100),
polyoxyethylene sorbitan fatty acid esters, polyoxyethylene and
polyethylene derivatives such as alkylated and alkoxylated
derivatives (tweens, e.g. Tween-20, Tween-40, Tween-80 and
Brij-35), monoglycerides or ethoxylated derivatives thereof,
diglycerides or polyoxyethylene derivatives thereof, alcohols,
glycerol, lectins and phospholipids (eg. phosphatidyl serine,
phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl
inositol, diphosphatidyl glycerol and sphingomyelin), derivates of
phospholipids (eg. dipalmitoyl phosphatidic acid) and
lysophospholipids (eg. palmitoyl lysophosphatidyl-L-serine and
1-acyl-sn-glycero-3-phosphate esters of ethanolamine, choline,
serine or threonine) and alkyl, alkoxyl (alkyl ester), alkoxy
(alkyl ether)-derivatives of lysophosphatidyl and
phosphatidylcholines, e.g. lauroyl and myristoyl derivatives of
lysophosphatidylcholine, dipalmitoylphosphatidylcholine, and
modifications of the polar head group, that is cholines,
ethanolamines, phosphatidic acid, serines, threonines, glycerol,
inositol, and the positively charged DODAC, DOTMA, DCP, BISHOP,
lysophosphatidylserine and lysophosphatidylthreonine, and
glycerophospholipids (eg. cephalins), glyceroglycolipids (eg.
galactopyransoide), sphingoglycolipids (eg. ceramides,
gangliosides), dodecylphosphocholine, hen egg lysolecithin, fusidic
acid derivatives--(e.g. sodium tauro-dihydrofusidate etc.),
long-chain fatty acids and salts thereof C6-C12 (eg. oleic acid and
caprylic acid), acylcamitines and derivatives,
N.sup..alpha.-acylated derivatives of lysine, arginine or
histidine, or side-chain acylated derivatives of lysine or
arginine, Na-acylated derivatives of dipeptides comprising any
combination of lysine, arginine or histidine and a neutral or
acidic amino acid, Na-acylated derivative of a tripeptide
comprising any combination of a neutral amino acid and two charged
amino acids, DSS (docusate sodium, CAS registry no [577-11-7]),
docusate calcium, CAS registry no [128-49-4]), docusate potassium,
CAS registry no [7491-09-0]), SDS (sodium dodecyl sulphate or
sodium lauryl sulphate), sodium caprylate, cholic acid or
derivatives thereof, bile acids and salts thereof and glycine or
taurine conjugates, ursodeoxycholic acid, sodium cholate, sodium
deoxycholate, sodium taurocholate, sodium glycocholate,
N-Hexadecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, anionic
(alkyl-aryl-sulphonates) monovalent surfactants, zwitterionic
surfactants (e.g. N-alkyl-N,N-dimethylammonio-1-propanesulfonates,
3-cholamido-1-propyldimethylammonio-1-propanesulfonate, cationic
surfactants (quaternary ammonium bases) (e.g.
cetyl-trimethylammonium bromide, cetylpyridinium chloride),
non-ionic surfactants (eg. Dodecyl .beta.-D-glucopyranoside),
poloxamines (eg. Tetronic's), which are tetrafunctional block
copolymers derived from sequential addition of propylene oxide and
ethylene oxide to ethylenediamine, or the surfactant may be
selected from the group of imidazoline derivatives, or mixtures
thereof. Each one of these specific surfactants constitutes an
alternative embodiment of the invention.
[0166] The use of a surfactant in pharmaceutical compositions is
well-known to the skilled person. For convenience reference is made
to Remington: The Science and Practice of Pharmacy, 19.sup.th
edition, 1995.
[0167] In a further embodiment, the formulation further comprises
protease inhibitors such as EDTA (ethylenediamine tetraacetic acid)
and benzamidineHCl, but other commercially available protease
inhibitors may also be used. The use of a protease inhibitor is
particular useful in pharmaceutical compositions comprising
zymogens of proteases in order to inhibit autocatalysis.
[0168] It is possible that other ingredients may be present in the
peptide pharmaceutical formulation of the present invention. Such
additional ingredients may include wetting agents, emulsifiers,
antioxidants, bulking agents, tonicity modifiers, chelating agents,
metal ions, oleaginous vehicles, proteins (e.g., human serum
albumin, gelatine or proteins) and a zwitterion (e.g., an amino
acid such as betaine, taurine, arginine, glycine, lysine and
histidine). Such additional ingredients, of course, should not
adversely affect the overall stability of the pharmaceutical
formulation of the present invention.
[0169] Pharmaceutical compositions containing an antibody according
to the present invention may be administered to a patient in need
of such treatment at several sites, for example, at topical sites,
for example, skin and mucosal sites, at sites which bypass
absorption, for example, administration in an artery, in a vein, in
the heart, and at sites which involve absorption, for example,
administration in the skin, under the skin, in a muscle or in the
abdomen.
[0170] Administration of pharmaceutical compositions according to
the invention may be through several routes of administration, for
example, subcutaneous, intramuscular, intraperitoneal, intravenous,
lingual, sublingual, buccal, in the mouth, oral, in the stomach and
intestine, nasal, pulmonary, for example, through the bronchioles
and alveoli or a combination thereof, epidermal, dermal,
transdermal, vaginal, rectal, ocular, for examples through the
conjunctiva, uretal, and parenteral to patients in need of such a
treatment.
[0171] Compositions of the current invention may be administered in
several dosage forms, for example, as solutions, suspensions,
emulsions, microemulsions, multiple emulsion, foams, salves,
pastes, plasters, ointments, tablets, coated tablets, rinses,
capsules, for example, hard gelatine capsules and soft gelatine
capsules, suppositories, rectal capsules, drops, gels, sprays,
powder, aerosols, inhalants, eye drops, ophthalmic ointments,
ophthalmic rinses, vaginal pessaries, vaginal rings, vaginal
ointments, injection solution, in situ transforming solutions, for
example in situ gelling, in situ setting, in situ precipitating, in
situ crystallization, infusion solution, and implants.
[0172] Compositions of the invention may further be compounded in,
or attached to, for example through covalent, hydrophobic and
electrostatic interactions, a drug carrier, drug delivery system
and advanced drug delivery system in order to further enhance
stability of the antibody, increase bioavailability, increase
solubility, decrease adverse effects, achieve chronotherapy well
known to those skilled in the art, and increase patient compliance
or any combination thereof. Examples of carriers, drug delivery
systems and advanced drug delivery systems include, but are not
limited to, polymers, for example cellulose and derivatives,
polysaccharides, for example dextran and derivatives, starch and
derivatives, poly(vinyl alcohol), acrylate and methacrylate
polymers, polylactic and polyglycolic acid and block co-polymers
thereof, polyethylene glycols, carrier proteins, for example
albumin, gels, for example, thermogelling systems, for example
block co-polymeric systems well known to those skilled in the art,
micelles, liposomes, microspheres, nanoparticulates, liquid
crystals and dispersions thereof, L2 phase and dispersions there
of, well known to those skilled in the art of phase behaviour in
lipid-water systems, polymeric micelles, multiple emulsions,
self-emulsifying, self-microemulsifying, cyclodextrins and
derivatives thereof, and dendrimers.
[0173] Compositions of the current invention are useful in the
formulation of solids, semisolids, powder and solutions for
pulmonary administration of an antibody, using, for example a
metered dose inhaler, dry powder inhaler and a nebulizer, all being
devices well known to those skilled in the art.
[0174] Compositions of the current invention are also useful in the
formulation of controlled, sustained, protracting, retarded, and
slow release drug delivery systems. More specifically, but not
limited to, compositions are useful in formulation of parenteral
controlled release and sustained release systems (both systems
leading to a many-fold reduction in number of administrations),
well known to those skilled in the art. Even more preferably, are
controlled release and sustained release systems administered
subcutaneous. Without limiting the scope of the invention, examples
of useful controlled release system and compositions are hydrogels,
oleaginous gels, liquid crystals, polymeric micelles, microspheres,
nanoparticles,
[0175] Methods to produce controlled release systems useful for
compositions of the current invention include, but are not limited
to, crystallization, condensation, co-crystallization,
precipitation, co-precipitation, emulsification, dispersion, high
pressure homogenisation, encapsulation, spray drying,
microencapsulating, coacervation, phase separation, solvent
evaporation to produce microspheres, extrusion and supercritical
fluid processes. General reference is made to Handbook of
Pharmaceutical Controlled Release (Wise, D. L., ed. Marcel Dekker,
New York, 2000) and Drug and the Pharmaceutical Sciences vol. 99:
Protein Formulation and Delivery (MacNally, E. J., ed. Marcel
Dekker, New York, 2000).
[0176] Parenteral administration may be performed by subcutaneous,
intramuscular, intraperitoneal or intravenous injection by means of
a syringe, optionally a pen-like syringe. Alternatively, parenteral
administration can be performed by means of an infusion pump. A
further option is a composition which may be a solution or
suspension for the administration of the antibody compound in the
form of a nasal or pulmonal spray. As a still further option, the
pharmaceutical compositions containing an antibody of the invention
can also be adapted to transdermal administration, e.g. by
needle-free injection or from a patch, optionally an iontophoretic
patch, or transmucosal, e.g. buccal, administration.
[0177] The antibody can be administered via the pulmonary route in
a vehicle, as a solution, suspension or dry powder using any of
known types of devices suitable for pulmonary drug delivery.
Examples of these comprise of, but are not limited to, the three
general types of aerosol-generating for pulmonary drug delivery,
and may include jet or ultrasonic nebulizers, metered-dose
inhalers, or dry powder inhalers (Cf. Yu J, Chien Y W. Pulmonary
drug delivery: Physiologic and mechanistic aspects. Crit Rev Ther
Drug Carr Sys 14(4) (1997) 395-453).
[0178] Based on standardized testing methodology, the aerodynamic
diameter (da) of a particle is defined as the geometric equivalent
diameter of a reference standard spherical particle of unit density
(1 g/cm.sup.3). In the simplest case, for spherical particles, da
is related to a reference diameter (d) as a function of the square
root of the density ratio as described by:
d a = .rho. .rho. a d ##EQU00001##
[0179] Modifications to this relationship occur for non-spherical
particles (cf. Edwards D A, Ben-Jebria A, Langer R. Recent advances
in pulmonary drug delivery using large, porous inhaled particles. J
Appl Physiol 84(2) (1998) 379-385). The terms "MMAD" and "MMEAD"
are well-described and known to the art (cf. Edwards D A,
Ben-Jebria A, Langer R and represent a measure of the median value
of an aerodynamic particle size distribution. Recent advances in
pulmonary drug delivery using large, porous inhaled particles. J
Appl Physiol 84(2) (1998) 379-385). Mass median aerodynamic
diameter (MMAD) and mass median effective aerodynamic diameter
(MMEAD) are used inter-changeably, are statistical parameters, and
empirically describe the size of aerosol particles in relation to
their potential to deposit in the lungs, independent of actual
shape, size, or density (cf. Edwards D A, Ben-Jebria A, Langer R.
Recent advances in pulmonary drug delivery using large, porous
inhaled particles. J Appl Physiol 84(2) (1998) 379-385). MMAD is
normally calculated from the measurement made with impactors, an
instrument that measures the particle inertial behaviour in
air.
[0180] In a further embodiment, the formulation could be
aerosolized by any known aerosolisation technology, such as
nebulisation, to achieve a MMAD of aerosol particles less than 10
.mu.m, more preferably between 1-5 .mu.m, and most preferably
between 1-3 .mu.m. The preferred particle size is based on the most
effective size for delivery of drug to the deep lung, where protein
is optimally absorbed (cf. Edwards D A, Ben-Jebria A, Langer A,
Recent advances in pulmonary drug delivery using large, porous
inhaled particles. J Appl Physiol 84(2) (1998) 379-385).
[0181] Deep lung deposition of the pulmonal formulations comprising
the antibody may optional be further optimized by using
modifications of the inhalation techniques, for example, but not
limited to: slow inhalation flow (eg. 30 L/min), breath holding and
timing of actuation.
[0182] The term "stabilized formulation" refers to a formulation
with increased physical stability, increased chemical stability or
increased physical and chemical stability.
[0183] The term "physical stability" of the protein formulation as
used herein refers to the tendency of the protein to form
biologically inactive and/or insoluble aggregates of the protein as
a result of exposure of the protein to thermo-mechanical stresses
and/or interaction with interfaces and surfaces that are
destabilizing, such as hydrophobic surfaces and interfaces.
Physical stability of the aqueous protein formulations is evaluated
by means of visual inspection and/or turbidity measurements after
exposing the formulation filled in suitable containers (e.g.
cartridges or vials) to mechanical/physical stress (e.g. agitation)
at different temperatures for various time periods. Visual
inspection of the formulations is performed in a sharp focused
light with a dark background. The turbidity of the formulation is
characterized by a visual score ranking the degree of turbidity for
instance on a scale from 0 to 3 (a formulation showing no turbidity
corresponds to a visual score 0, and a formulation showing visual
turbidity in daylight corresponds to visual score 3). A formulation
is classified physically unstable with respect to protein
aggregation, when it shows visual turbidity in daylight.
Alternatively, the turbidity of the formulation can be evaluated by
simple turbidity measurements well-known to the skilled person.
Physical stability of the aqueous protein formulations can also be
evaluated by using a spectroscopic agent or probe of the
conformational status of the protein. The probe is preferably a
small molecule that preferentially binds to a non-native conformer
of the protein. One example of a small molecular spectroscopic
probe of protein structure is Thioflavin T. Thioflavin T is a
fluorescent dye that has been widely used for the detection of
amyloid fibrils. In the presence of fibrils, and perhaps other
protein configurations as well, Thioflavin T gives rise to a new
excitation maximum at about 450 nm and enhanced emission at about
482 nm when bound to a fibril protein form. Unbound Thioflavin T is
essentially non-fluorescent at the wavelengths.
[0184] Other small molecules can be used as probes of the changes
in protein structure from native to non-native states. For instance
the "hydrophobic patch" probes that bind preferentially to exposed
hydrophobic patches of a protein. The hydrophobic patches are
generally buried within the tertiary structure of a protein in its
native state, but become exposed as a protein begins to unfold or
denature. Examples of these small molecular, spectroscopic probes
are aromatic, hydrophobic dyes, such as antrhacene, acridine,
phenanthroline or the like. Other spectroscopic probes are
metal-amino acid complexes, such as cobalt metal complexes of
hydrophobic amino acids, such as phenylalanine, leucine,
isoleucine, methionine, and valine, or the like.
[0185] The term "chemical stability" of the protein formulation as
used herein refers to chemical covalent changes in the protein
structure leading to formation of chemical degradation products
with potential less biological potency and/or potential increased
immunogenic properties compared to the native protein structure.
Various chemical degradation products can be formed depending on
the type and nature of the native protein and the environment to
which the protein is exposed. Elimination of chemical degradation
can most probably not be completely avoided and increasing amounts
of chemical degradation products is often seen during storage and
use of the protein formulation as well-known by the person skilled
in the art. Most proteins are prone to deamidation, a process in
which the side chain amide group in glutaminyl or asparaginyl
residues is hydrolysed to form a free carboxylic acid. Other
degradation pathways involve formation of high molecular weight
transformation products where two or more protein molecules are
covalently bound to each other through transamidation and/or
disulfide interactions leading to formation of covalently bound
dimer, oligomer and polymer degradation products (Stability of
Protein Pharmaceuticals, Ahem. T. J. & Manning M. C., Plenum
Press, New York 1992). Oxidation (of for instance methionine
residues) can be mentioned as another variant of chemical
degradation. The chemical stability of the protein formulation can
be evaluated by measuring the amount of the chemical degradation
products at various time-points after exposure to different
environmental conditions (the formation of degradation products can
often be accelerated by for instance increasing temperature). The
amount of each individual degradation product is often determined
by separation of the degradation products depending on molecule
size and/or charge using various chromatography techniques (e.g.
SEC-HPLC and/or RP-HPLC).
[0186] Hence, as outlined above, a "stabilized formulation" refers
to a formulation with increased physical stability, increased
chemical stability or increased physical and chemical stability. In
general, a formulation must be stable during use and storage (in
compliance with recommended use and storage conditions) until the
expiration date is reached.
[0187] In one embodiment of the invention the pharmaceutical
formulation comprising the antibody is stable for more than 6 weeks
of usage and for more than 3 years of storage.
[0188] In another embodiment of the invention the pharmaceutical
formulation comprising the antibody is stable for more than 4 weeks
of usage and for more than 3 years of storage.
[0189] In a further embodiment of the invention the pharmaceutical
formulation comprising the antibody is stable for more than 4 weeks
of usage and for more than two years of storage.
[0190] In an even further embodiment of the invention the
pharmaceutical formulation comprising the antibody is stable for
more than 2 weeks of usage and for more than two years of
storage.
[0191] Suitable antibody formulations can also be determined by
examining experiences with other already developed therapeutic
monoclonal antibodies. Several monoclonal antibodies have been
shown to be efficient in clinical situations, such as Rituxan
(Rituximab), Herceptin (Trastuzumab) Xolair (Omalizumab), Bexxar
(Tositumomab), Campath (Alemtuzumab), Zevalin, Oncolym and similar
formulations may be used with the antibodies of this invention. For
example, a monoclonal antibody can be supplied at a concentration
of 10 mg/mL in either 100 mg (10 mL) or 500 mg (50 mL) single-use
vials, 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. In another embodiment, the antibody is supplied in a
formulation comprising about 20 mM Na-Citrate, about 150 mM NaCl,
at pH of about 6.0.
Therapeutic Applications
[0192] Methods of treating a patient using an anti-NKG2A antibody
as described herein are also provided for. In one embodiment, the
invention provides for the use of an antibody as described herein
in the preparation of a pharmaceutical composition for
administration to a human patient. Typically, the patient suffers
from, or is at risk for, cancer, a viral disease, an inflammatory
disorder, or an autoimmune disorder. Alternatively, the antibody of
the invention is used to improve bone marrow transplantation in a
patient.
[0193] For example, in one aspect, the invention provides a method
of potentiating the activity of CD94/NKG2A-restricted lymphocytes
in a patient in need thereof, comprising the step of administering
a human or humanized anti-NKG2A antibody to said patient, which
antibody reduces or prevents HLA-E-mediated activation of the
CD94/NKG2A receptor. In one embodiment, the method directed at
increasing the activity of such lymphocytes in patients having a
disease in which increased NK, T, and/or NKT cell activity is
beneficial, which involves, affects or is caused by cells
susceptible to lysis by NK, T, or NKT cells, or which is caused or
characterized by insufficient NK, T, or NKT cell activity, such as
a cancer, an infectious disease or an immune disorder.
[0194] More specifically, the methods and compositions 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, chronic lymphocytic leukemia, acute lymphoblastic
leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma,
non-Hodgkin's lymphoma, hairy cell lymphoma and Burkitt's lymphoma,
and multiple myeloma; hematopoietic tumors of myeloid lineage,
including acute and chronic myelogenous leukemias, promyelocytic
leukemia, and myelodysplastic syndrome; tumors of mesenchymal
origin, including fibrosarcoma and rhabdomyosarcoma; other tumors,
including melanoma, seminoma, terato-carcinoma, neuroblastoma and
glioma; tumors of the central and peripheral nervous system,
including astrocytoma, neuroblastoma, glioma, and schwannomas;
tumors of mesenchymal origin, including fibrosarcoma,
rhabdomyosarcoma, and osteosarcoma; and other tumors, including
melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid
follicular cancer and teratocarcinoma.
[0195] Particular 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); 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; lym-phoma/leukaemia (T-Lbly/T-ALL), multiple
myeloma.
[0196] 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.
[0197] In a particular aspect, antibodies of the invention are used
to treat NK-type lymphoproliferative disease of granular
lymphocytes; alternatively called NK-LGL), referring to a class of
proliferative disorders that is caused by the clonal expansion of
NK cells or NK-like cells, i.e., large granular lymphocytes showing
a characteristic combination of surface antigen expression (e.g.,
CD3-, CD56+, CD16+, etc.; see, e.g., Loughran (1993) Blood 82:1).
The cell proliferation underlying these disorders can have variable
effects, ranging from the mild symptoms seen in some patients to
the aggressive, often-fatal form of the disease called NK-LDGL
leukemia. Symptoms of this class of disorders can include fever,
mild neutropenia, thrombocytopenia, anemia, lymphocytosis,
splenomegaly, hepatomegaly, lymphadenopathy, marrow infiltration,
and others (see, e.g., Zambello et al. (2003) Blood 102:1797;
Loughran (1993) Blood 82:1; Epling-Bumette et al. (2004)
Blood-2003-02-400).
[0198] The CD94/NKG2A antibody based treatment can also be used to
treat or prevent infectious diseases, including preferably any
infections caused by infection 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,
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).
Bacteria constitute another preferred class of infectious organisms
including but are not limited to 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; Treponema;
Camplyobacter, Pseudomonas including P. aeruginosa; 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. laprae, M. avium,
M. bovis, M. africanum, M. kansasii, M. intracellulare, and M.
lepraemurium; and Nocardia. Protozoa may include but are not
limited to, leishmania, kokzidioa, and trypanosoma. Parasites
include but are not limited to, chlamydia and rickettsia. 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) (World-Wide Web (www) address
cdc.gov/ncidod/diseases/), which list is incorporated herein by
reference. All of these diseases are candidates for treatment using
the inhibitory anti-CD94/NKG2A antibodies of the invention.
[0199] In an alternative aspect, the anti-NKG2A antibodies are used
to target and kill NKG2A-expressing cells in, e.g., a patient
suffering from a cancer characterized by CD94/NKG2A expression on
cancerous cells, for example an NK-lymphoma. In one embodiment, the
humanized antibody is administered in the form of an
immunoconjugate comprising the humanized antibody and a cytotoxic
agent.
[0200] In alternative aspect, the anti-NKG2A antibodies are used to
treat or prevent an autoimmune or inflammatory disorder. Exemplary
autoimmune disorders treatable using the present methods include,
inter alia, hemolytic anemia, pemicious anemia, polyarteritis
nodosa, systemic lupus erythematosus, Wegener's granulomatosis,
autoimmune hepatitis, Behcet's disease, Crohn's disease, primary
bilary cirrhosis, scleroderma, ulcerative colitis, Sjogren's
syndrome, Type 1 diabetes mellitus, uveitis, Graves' disease,
Alzheimer's disease, thyroiditis, myocarditis, rheumatic fever,
scleroderma, ankylosing spondylitis, rheumatoid arthritis,
glomerulonephritis, sarcoidosis, dermatomyositis, myasthenia
gravis, polymyositis, Guillain-Barre syndrome, multiple sclerosis,
alopecia areata, pemphigus/pemphigoid, Bullous pemphigoid,
Hashimoto's thyroiditis, psoriasis, and vitiligo.
[0201] Examples of inflammatory disorders that can be treated by
these methods include, but not limited to, adrenalitis, alveolitis,
angiocholecystitis, appendicitis, balanitis, blepharitis,
bronchitis, bursitis, carditis, cellulitis, cervicitis,
cholecystitis, chorditis, cochlitis, colitis, conjunctivitis,
cystitis, dermatitis, diverticulitis, encephalitis, endocarditis,
esophagitis, eustachitis, fibrositis, folliculitis, gastritis,
gastroenteritis, gingivitis, glossitis, hepatosplenitis, keratitis,
labyrinthitis, laryngitis, lymphangitis, mastitis, media otitis,
meningitis, metritis, mucitis, myocarditis, myosititis, myringitis,
nephritis, neuritis, orchitis, osteochondritis, otitis,
pericarditis, peritendonitis, peritonitis, pharyngitis, phlebitis,
poliomyelitis, prostatitis, pulpitis, retinitis, rhinitis,
salpingitis, scleritis, selerochoroiditis, scrotitis, sinusitis,
spondylitis, steatitis, stornatitis, synovitis, syringitis,
tendonitis, tonsillitis, urethritis, and vaginitis.
[0202] It has also been shown that alloreactive NK cell killing of
dendritic cells improved engraftment of hematopoietic cells in a
bone marrow transplant (L. Rugged et al., Science, 2002,
295:2097-2100). Thus, in another embodiment, the invention provides
a method of improving the engraftment of hematopoietic cells in a
patient comprising the step administering to said patient a
composition of this invention comprising an activating antibody.
Improvement in grafting is manifest by any one of reduced
incidience or severity of graft versus host disease, prolonged
survival of the graft, or a reduction in or elimination of the
symptoms of the disease being treated by the graft (e.g., a
hematopoietic cancer). This method is preferably used in the
treatment of leukemia.
Combinations
[0203] A number of therapeutic agents are available for the
treatment of cancers. The antibody compositions and methods of the
present invention may thus also 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
anti-CD94/NKG2A antibody-based treatment, its combination with the
present invention is contemplated.
[0204] In connection with solid tumor treatment, 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
anti-CD94/NKG2A antibodies according to the invention are used
simultaneously with, before, or after surgery or radiation
treatment; or are administered to patients with, before, or after
administration of another anti-cancer agent. One would ensure that
the surgery, radiotherapy, or anti-cancer agent in combination with
the active agent in the composition of this invention exert an
advantageously combined effect on the cancer.
[0205] Exemplary anti-cancer agents include chemotherapeutic
agents, hormonal agents, anti-angiogenic agents, anti-metastatic
agents, anti-cancer antibodies (e.g., Rituximab), antibodies
against inhibitory KIR-molecules, growth-factor inhibitors,
apoptosis-promoting compounds, cytokines and other immunomodulatory
agents, tumor-targeting agents conjugated to toxins or
radionuclides, compounds that interfere with DNA replication,
mitosis and chromosomal segregation, and agents that disrupt the
synthesis and fidelity of polynucleotide precursors.
[0206] For autoimmune or inflammatory disorders, any other compound
known to be effective for one or more types of autoimmune or
inflammatory disorders, or any symptom or feature of autoimmune or
inflammatory disorders, including inter alia, immunosuppressants,
e.g., azathioprine (e.g., Imuran), chiorambucil (e.g., Leukeran),
cyclophosphamide (e.g., Cytoxan), cyclosporine (e.g., Sandimmune,
Neoral), methotrexate (e.g., Rheumatrex), corticosteroids,
prednisone (e.g., Deltasone, Meticorten), Etanercept (e.g.,
Enbrel), infliximab (e.g., Remicade), inhibitors of TNF, FK-506,
rapamycin, mycophenolate mofetil, leflunomide, anti-lymphocyte
globulin, deoxyspergualin or OKT.
[0207] Preferred examples of immunomodulatory compounds include
cytokines. Other examples include compounds that have an effect,
preferably an effect of activation or potentiation NK cell
activity, or of inducing or supporting the proliferation of NK
cells. Other compounds for administration before, simultaneously
with, or after compositions comprising the agents of the invention
are adjunct compounds (e.g., anti-emetics and analgesic agents) and
anti-viral agents.
[0208] As will be understood by those of ordinary skill in the art,
the appropriate doses of anti-cancer agents will approximate those
already employed in clinical therapies wherein the anti-cancer
agents are administered alone or in combination with other agents.
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.
Articles of Manufacture
[0209] In another embodiment of the invention, an article of
manufacture containing materials useful for the treatment of the
disorders described above is provided. For example, the article of
manufacture can comprise a container containing an antibody as
described herein together with instructions directing a user to
treat a disorder such as a cancer or a viral disease in a mammal
with the antibody in an effective amount. In a preferred
embodiment, the mammal is a human. The article of manufacture
typically comprises a container and a label or package insert on or
associated with the container. Suitable containers include, for
example, bottles, vials, syringes, etc. The containers may be
formed from a variety of materials such as glass or plastic. The
container holds a composition that is effective for treating the
condition and may have a sterile access port (for example, the
container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). At least one
active agent in the composition is the humanized anti-NKG2A
antibody herein, or an antibody derivative (e.g., an
immunoconjugate) comprising such a humanized antibody. The label or
package insert indicates that the composition is used for treating
the condition of choice, such as cancer or a viral disease.
[0210] Moreover, the article of manufacture may comprise (a) a
first container with a composition contained therein, wherein the
composition comprises the antibody described herein, and (b) a
second container with a composition contained therein, wherein the
composition comprises a therapeutic agent other than the first
antibody. The article of manufacture in this embodiment of the
invention may further comprise a package insert indicating that the
first and second compositions can be used in combination to treat a
cancer or viral disease. Such therapeutic agent may be any of the
adjunct therapies described in the preceding section (e.g., a
chemotherapeutic agent, an anti-angiogenic agent, an anti-hormonal
compound, a cardioprotectant, and/or a regulator of immune function
in a mammal, including a cytokine). Alternatively, or additionally,
the article of manufacture may further comprise a second (or third)
container comprising a pharmaceutically acceptable buffer, such as
bacteriostatic water for injection (BWFI), phosphate-buffered
saline, Ringer's solution and dextrose solution. It may further
include other materials desirable from a commercial and user
standpoint, including other buffers, diluents, filters, needles,
and syringes.
Administration
[0211] As described above, several monoclonal antibodies have been
shown to be efficient in clinical situations (such as, e.g.,
Rituxan (Rituximab) and others), and similar administration
regimens (i.e., doses and/or administration protocols) may be used
with the antibodies of this invention. Schedules and dosages for
administration can be determined in accordance with known methods
for these products, for example using the manufacturers'
instructions. For example, an antibody preparation can be supplied
at a concentration of 10 mg/mL in either 100 mg (10 mL) or 500 mg
(50 mL) single-use vials. An exemplary suitable dosage range for an
antibody of the invention may between about 10 mg/m.sup.2 and 500
mg/m.sup.2. Quantities and schedule of injection of anti-NKG2A
antibodies that, e.g., saturate cells for 24 hours, 48 hours, 72
hours or a week or a month can be determined considering the
affinity of the antibody and its pharmacokinetic parameters.
However, it will be appreciated that these schedules are exemplary
and that optimal schedule and regimen and the tolerability of the
antibodies must be determined in clinical trials.
Non-Therapeutic Applications
[0212] The antibodies (e.g. the humanized anti-NKG2A antibodies) of
the invention also have non-therapeutic applications.
[0213] For example, the antibodies may be used as
affinity-purification agents. In this process, the antibodies are
immobilized on a solid phase such as a SEPHADEX.TM. resin or filter
paper, using methods well known in the art. The immobilized
antibody is contacted with a sample containing the NKG2A protein
(or fragment thereof) to be purified, and thereafter the support is
washed with a suitable solvent that will remove substantially all
the material in the sample except the NKG2A protein, which is bound
to the immobilized antibody. Finally, the support is washed with
another suitable solvent, such as glycine buffer, pH 5.0, that will
release the NKG2A protein from the antibody.
[0214] Anti-NKG2A antibodies may also be useful in diagnostic
assays for NKG2A protein, e.g. detecting its expression in specific
cells, tissues, or serum.
[0215] For diagnostic applications, the antibody typically will be
labeled with a detectable moiety. Numerous labels are available
that can be generally grouped into the following categories:
[0216] (a) Radioisotopes, such as .sup.35S, .sup.14C, .sup.125I,
.sup.3H, and .sup.131I. The antibody can be labeled with the
radioisotope using the techniques described in Current Protocols in
Immunology, Volumes 1 and 2, Coligen et al., Ed.
Wiley-Interscience, New York, N.Y., Pubs. (1991), for example, and
radioactivity can be measured using scintillation counting.
[0217] (b) Fluorescent labels such as rare-earth chelates (europium
chelates) or fluorescein and its derivatives, rhodamine and its
derivatives, dansyl, Lissamine, phycoerythrin and Texas Red are
available. The fluorescent labels can be conjugated to the antibody
using the techniques disclosed in Current Protocols in Immunology,
supra, for example. Fluorescence can be quantified using a
fluorimeter.
[0218] (c) Various enzyme-substrate labels are available and U.S.
Pat. No. 4,275,149 provides a review of some of these. The enzyme
generally catalyzes a chemical alteration of the chromogenic
substrate that can be measured using various techniques. For
example, the enzyme may catalyze a color change in a substrate,
which can be measured spectrophotometrically. Alternatively, the
enzyme may alter the fluorescence or chemiluminescence of the
substrate. Techniques for quantifying a change in fluorescence are
described above. The chemiluminescent substrate becomes
electronically excited by a chemical reaction and may then emit
light that can be measured (using a chemiluminometer, for example)
or donates energy to a fluorescent acceptor. Examples of enzymatic
labels include luciferases (e.g., firefly luciferase and bacterial
luciferase; U.S. Pat. No. 4,737,456), luciferin,
2,3-dihydrophthalazinediones, malate dehydrogenase, urease,
peroxidase such as horseradish peroxidase (HRPO), alkaline
phosphatase, .beta.-galactosidase, glucoamylase, lysozyme,
saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and
glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as
uricase and xanthine oxidase), lactoperoxidase, microperoxidase,
and the like. Techniques for conjugating enzymes to antibodies are
described in O'Sullivan et al, "Methods for the Preparation of
Enzyme-Antibody Conjugates for use in Enzyme Immunoassay," in
Methods in Enzym. (Ed., J. Langone & H. Van Vunakis), Academic
Press, New York, 73:147-166 (1981).
[0219] Examples of enzyme-substrate combinations include, for
example:
[0220] (i) Horseradish peroxidase (HRPO) with hydrogen peroxidase
as a substrate, wherein the hydrogen peroxidase oxidizes a dye
precursor (e.g., orthophenylene diamine (OPD) or
3,3',5,5'-tetramethyl benzidine hydrochloride (TMB));
[0221] (ii) alkaline phosphatase (AP) with para-nitrophenyl
phosphate as chromogenic substrate; and
[0222] (iii) beta-D-galactosidase (beta-D-Gal) with a chromogenic
substrate (e.g., p-nitrophenyl-beta-D-galactosidase) or fluorogenic
substrate 4-methylumbelliferyl-p-beta-galactosidase.
[0223] Numerous other enzyme-substrate combinations are available
to those skilled in the art. For a general review of these, see
U.S. Pat. Nos. 4,275,149 and 4,318,980.
[0224] Sometimes, the label is indirectly conjugated with the
antibody. The skilled artisan will be aware of various techniques
for achieving this. For example, the antibody can be conjugated
with biotin, and any of the three broad categories of labels
mentioned above can be conjugated with avidin, or vice versa.
Biotin binds selectively to avidin, and thus, the label can be
conjugated with the antibody in this indirect manner.
Alternatively, to achieve indirect conjugation of the label with
the antibody, the antibody is conjugated with a small hapten (e.g.,
digoxin) and one of the different types of labels mentioned above
is conjugated with an anti-hapten antibody (e.g., anti-digoxin
antibody). Thus, indirect conjugation of the label with the
antibody can be achieved.
[0225] In another embodiment of the invention, the anti-NKG2A
antibody need not be labeled, and the presence thereof can be
detected using a labeled secondary antibody that binds to the NKG2A
antibody.
[0226] The antibodies of the present invention may be employed in
any known assay method, such as competitive-binding assays, direct
and indirect sandwich assays, and immunoprecipitation assays. Zola,
Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC
Press, Inc. 1987).
[0227] For immunohistochemistry, the tumor sample may be fresh or
frozen or may be embedded in paraffin and fixed with a preservative
such as formalin, for example.
[0228] The antibodies may also be used for in vivo diagnostic
assays. Generally, the antibody is labeled with a radionuclide or a
non-radioactive indicator detectable by, e.g., nuclear magnetic
resonance, or other means known in the art. Preferably, the label
is a radiolabel, such as, e.g., .sup.125I, .sup.131I, .sup.67Cu,
.sup.99mTc, or .sup.111In. The labeled antibody is administered to
a host, preferably via the bloodstream, and the presence and
location of the labeled antibody in the host is assayed. This
imaging technique is suitably used in the detection, staging and
treatment of neoplasms. The radioisotope is conjugated to the
protein by any means, including metal-chelating compounds or
lactoperoxidase, or iodogen techniques for iodination.
[0229] As a matter of convenience, the antibodies of the present
invention can be provided in a kit, i.e., a packaged combination of
reagents in predetermined amounts with instructions for performing
the diagnostic assay. Where the antibody is labeled with an enzyme,
the kit will include substrates and cofactors required by the
enzyme (e.g., a substrate precursor that provides the detectable
chromophore or fluorophore). In addition, other additives may be
included such as stabilizers, buffers (e.g., a block buffer or
lysis buffer) and the like. The relative amounts of the various
reagents may be varied widely to provide for concentrations in
solution of the reagents that substantially optimize the
sensitivity of the assay. Particularly, the reagents may be
provided as dry powders, usually lyophilized, including excipients
that on dissolution will provide a reagent solution having the
appropriate concentration.
Deposits
[0230] The Z270 hybridoma was deposited on Dec. 22, 2005 at the
Collection Nationale de Culture de Microorganismes, Institute
Pasteur, 25, Rue du Docteur Roux, F-75725 Paris, France, under
accession number 1-3549.
EXAMPLES
[0231] Further details of the invention are illustrated by the
following non-limiting Examples.
Example I--Selection of Parent humZ270VL and humZ270VH
Sequences
[0232] This Example describes the selection of parent humZ270VL and
humZ270VH sequences as well as optional back mutations for variant
h270VL and humZ270VH sequences.
[0233] As described in Example 2, the Z270 hybridoma was cloned,
and the Z270 VH and VL chain sequences of the corresponding
antibody from were determined to be:
[0234] Z270VL:
DIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQFLVYNAKTLAEGVPSRF
SGSGSGTQFSLKINSLQPEDFGSYYCQHHYGTPRTFGGGTKLEIK (SEQ ID NO:1), with
an optional arginine (R) residue at Kabat position 108.
[0235] Z270VH:
QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWMNWVKQRPEQGLQWIGRIDPYDSETH
YSQKFKDKAILTVDKSSSTAYMRLSSLTSEDSAVYYCARGGYDFDVGTLYWFFDVWGAGTT VTVS
(SEQ ID NO:2), with an optional C-terminal serine (S) residue.
[0236] A second light chain, Z270VL-NB, was also identified.
However, this was a common myeloma light chain.
[0237] Z270VL-NB:
NIVMTQSPKSMSMSVGERVTLTCKASENVVTYVSWYQQKPEQSPKLLIYGASNRY
TGVPDRFTGSGSATDFTLTISSVQAEDLADYHCGQGYSYPYTFGGGTKLEIKRA (SEQ ID
NO:3), with an optional arginine (R) residue at Kabat position 108,
and an optional alanine (A) residue at Kabat position 109.
[0238] From an analysis of the murine Z270 sequences, the CDRs
according to Kabats definitions were determined as:
TABLE-US-00003 CDR-L1: RASENIYSYLA (residues 24-34 of SEQ ID NO: 1)
CDR-L2: NAKTLAE (residues 50-56 of SEQ ID NO: 1) CDR-L3: QHHYGTPRT
(residues 89-97 of SEQ ID NO: 1) CDR-H1: SYWMN (residues 31-35 of
SEQ ID NO: 2) CDR-H2: RIDPYDSETHYSQKFKD (residues 50-66 of SEQ ID
NO: 2) CDR-H3: GGYDFDVGTLYWFFDV (residues 95-102 of SEQ ID NO:
2).
[0239] A 3D protein structure model was built using MOE (molecular
Operating Environment; available at www.chemcomp.com) with
structural templates from the Protein Database Bank (PDB): 1OPG and
1XF4. The PDB is described in Berman et al. (Nuc Acids Res 2000;
28:235-242), and is available at www.rcsb.org/pdb. Based on a
statistical analysis of 201 antibody-antigen complexes in the PDB
database the most probable residues in the paratope were determined
to be:
[0240] Z270VL: residues 24-34, 49-56, 89-97 of SEQ ID NO:1
[0241] Z270VH: residues 23-35, 49-58, 93-102 of SEQ ID NO:2.
[0242] Using MOE, residues interacting (hydrophobic, hydrogen
binding, or charge) with the paratope were identified and the
combined set of residues (paratope+interacting residues) were taken
as the mask of Z270.
[0243] Searching the germline V databases (V-base; available at
vbase.mrc-cpe.cam.ac.uk/) with the Z270VL and Z270VH returned the
following potential framework templates (E-value given in
parenthesis):
[0244] Heavy chain: VH1_46 (2e-036), VH1_f (2e-035), VH1_02
(3e-035), VH1_18 (4e-035), VH1_03 (6e-035); and Light chain: VKI_O2
(1e-039), VKI_O12 (1e-039), VKI_L12 (9e-038), VKI_L8 (9e-038),
VKI_A20 (9e-038).
[0245] Searching the germline databases with the mask returned the
following potential framework templates (E-value given in
parenthesis):
[0246] Heavy chain: VH5_a (4e-013), VH5_51 (3e-011), VH1_f
(1e-010), VH1_18 (2e-010), VH1_46 (4e-010): and
[0247] Light chain: VKI_L9 (2e-012), VKI_O2 (3e-012), VKI_O12
(3e-012), VKI_L24 (2e-011), VKI_A20 (2e-011)
[0248] After manual inspections of the alignments and the hits,
VH1_18 and VKI_O2 were selected as the human scaffolds. JH6 and JK4
were chosen as germline J-segments.
[0249] Humanization was now designed with the following rules:
[0250] Residues outside the mask are taken as human. [0251]
Residues inside the mask and inside the Kabat CDR are taken as
murine. [0252] Residues inside the mask and outside the Kabat CDR
with mouse/germline consensus are taken as the consensus sequence.
[0253] Residues inside the mask and outside the Kabat CDR with
mouse/germline difference are subject to potential back
mutations.
[0254] The analysis is illustrated in FIG. 1 for Z270VL and Z270VH,
where mask residues are those shaded in the Kabat scheme; CDR
residues are shown in bold in the Kabat scheme; and mouse/germline
differences are shaded in the VKI_02/JK4 and VH1_18/JH6 sequences.
Z270VL and humZ270VL1, and humZ270VL1 cons may optionally comprise
an arginine (R) residue at Kabat position 108.
[0255] The resulting sequences, humZ270VL1 and humZ270VH1, are
given with the potential back mutation residues as human.
[0256] The CDRs of a humanized Z270 antibody according to the Kabat
definitions are shown in FIG. 2. Of the humZ270 CDRs, only the
CDR-H2 sequence was different than that of the corresponding murine
CDR, differing in 4 positions. However, in effect, this meant that
Kabat CDR-H2 residues 60-65 were identical to the human acceptor
sequence, providing for a more human molecule and a lesser risk for
immunogenicity. In FIG. 2, the differences are indicated in bold
text.
Example 2--Cloning of Z270 IgG1 VH and VL Regions
[0257] This Example describes the cloning and sequencing of murine
Z270 VH and VL regions.
[0258] Z270 Hybridoma Cell Culture for Total RNA Extraction.
[0259] Z270 hybridoma was cultured in RPMI 1640 (Hyclone Cat#
SH30011.04) plus 10% FCS (Biochrom Cat#S0115). 5.times.10.sup.8 to
1.times.10.sup.7 cells were harvested for total RNA extraction.
[0260] Z270 Hybridoma Cell Culture of Antibody Production.
[0261] One week batch culture strategy was adopted for the
production of the Z270 mAb. The culture medium was RPMI 1640 with
10% FCS. The supernatant was harvested every 7 days. The starting
cell density in the culture chamber of CL-1000 flask (INTEGRA
Biosciences, Item No. 90005, Lot No. 08541150) was be
2.times.10.sup.8 cells/ml. During the culture, the cell density and
viability was checked regularly. After 3 days culture in CL-1000
flask, the density was above 1.times.10.sup.7 and the viability was
about 85%. In the next 4 days the cell density varied between
1.5-2.5.times.10.sup.7 cells/ml and the viability decreased
gradually to 60.about.70%. At the 7.sup.th day the supernatant was
collected and reducing SDS-PAGE was used to estimate the antibody
concentration with a quantification control (A-TNP 20050118 2
mg/ml).
[0262] Z270 Total RNA Extraction.
[0263] This was done using TRIZOL reagent Invitrogen, Cat. No.
15596-026, according to the manufacturer's instructions.
[0264] 5'-RACE (Rapid Amplification of cDNA Ends).
[0265] A protocol was adapted from the manufacturer's instructions
for use of SMART.TM. RACE cDNA Amplification Kit product by
Clontech, Catalog no. 634914, with the following design of
Gene-Specific Primers (GSPs):
TABLE-US-00004 GSP1 for amplification of IgG1 heavy chain variable
region (SEQ ID NO: 12) RacePrimerheavy: 5'-GCCAGTGGATAGACAGATGG-3'
GSP2: for amplification of IgG1 Kappa chain variable region (SEQ ID
NO: 13) RacePrimerkappa: 5'-GATGGATACAGTTGGTGCAGC-3'.
[0266] After first-strand cDNA synthesis, RACE, and analysis of the
resulting samples on a 1.5% agarose gel, the RACE DNA band was cut
out and purified with Gel purification kit (QIAGEN QIAquick
Cat#28706) then cloned into pMD-19 using DNA Ligation Kit
(Cat#D6022 from TAKARA) Ver. 2.0 by TA cloning (FIG. 3A). The
positive clones were sent out for DNA sequencing.
[0267] Several clones of light chain and heavy chain were sequenced
with identical sequence as shown in FIG. 4A-D (with bold text
indicating signal peptide sequences):
[0268] Z270 VL cDNA: (SEQ ID NO:14)
[0269] Z270 VL protein (SEQ ID NO:15)
[0270] Z270 VH cDNA (SEQ ID NO:16)
[0271] Z270 VH protein (SEQ ID NO:17).
Example 3--Cloning of Murine IgG1 Light Chain and Heavy Chain into
pJSV002
[0272] In order to test antibody affinity and to use it for
positive control, Z270 antibody light chain and heavy chain is
inserted into pJSV002 as murine antibody IgG1. pJSV002 is a
transient expression vector that can be used in combination with
HEK293 6E cells for Z270 transient expression (FIG. 3B).
[0273] Z270 VL and IgG1 constant region are cloned into EcoRI and
Nhe I sites of pJSV002. The resulted plasmid is as shown in FIG.
3C. The inserted sequence with EcoRI and Nhe I (uppercase letters)
at both ends is shown in FIG. 4E (SEQ ID NO:18).
[0274] H1 variable region and IgG1 constant region is cloned into
EcoRI and Nhe I sites of pJSV002-mlgGI-variant (FIG. 3D). The
pJSV002-mlgGI-variant contains murine IgG1 heavy chain constant
region (FIG. 3E). The inserted sequence is between EcoRI and Nhe I
(gctagc) sites, with restriction sites and murine IgG1 constant
region indicated in lowercase letters, is shown in FIG. 4F (SEQ ID
NO:19).
Example 4--Plasmid Constructs for Chimeric Z270 Antibody
[0275] In order to test antibody affinity and to use it as positive
control, murine Z270 antibody light chain and heavy chain are
inserted into pJSV002 as human chimeric antibody IgG4 S241P. The
antibody contains murine variable region and human IgG4 constant
region with an S241P mutation in the heavy chain.
[0276] For expression of chimeric Z270 antibody (chimZ270), Z270VL
sequence and human light chain (kappa) constant region is inserted
into EcoR I and BamH I sites of pJSV002 (FIG. 3F). The sequence
shown in FIG. 4G is used, where uppercase letters indicate
restriction sites (SEQ ID NO:20).
[0277] Z270 VH and human heavy chain constant region is inserted
into EcoR I and Nhe I sites of pJSV002-IgG4-S241P (FIGS. 3G and
3H). The sequence shown in FIG. 4H is used, with the actual
inserted sequence being between Eco RI and Nhe I (gctagc) sites,
where upper-case letters indicate restriction sites (SEQ ID
NO:21).
Example 5--Expression of Humanized Z270
[0278] According to the Z270 humanization strategy described in
Example 1, Z270 light chain CDRs are grafted into the VKI_02/JK4
template to prepare humZ270VL1. The sequence shown in FIG. 4I
results from synthesizing the sequence between EcoRI and KasI sites
and inserting the it into pJSV002-hKappaC (with human Kappa
constant region in pJSV002), with CDR-encoding sequences in
uppercase letters and restriction sites in bold text (SEQ ID
NO:22).
[0279] For humanization of the heavy chain, Z270 heavy chain CDRs
(optionally with optimized CDR-H2 are grafted into the VH1_18/JK6
template to prepare humZ270VH1. The sequence between EcoRI and NheI
sites is synthesized and cloned into pJSV002-hlgG4 S241P (human
IgG4 S241P constant region in pJSV002, FIG. 3G), with CDR-encoding
sequences in uppercase letters and restriction sites in bold text
(FIG. 4J, SEQ ID NO:23).
[0280] Mutated constructs are likewise cloned into pJSV002 with the
corresponding IgG4 S241P constant region and human Kappa chain
constant region, with the following combinations of framework
back-mutations in each of humZ270VL and humZ270VH:
[0281] humZ270VL: MWt (i.e., no back-mutation), L46F, 148V,
L46F_148V
[0282] humZ270VH: MWt (i.e., no back-mutation), V5Q, M69L, T71V,
T73K, T75S, V5Q_M69L, V5Q_T71V, V5Q_T73K, V5Q_T75S, M69L_T71V,
M69L_T73K, M69L_T75S, T71V_T73K, T71V_T75S, T73K_T75S,
V5Q_M69L_T71V_T73K_T75S, M69L_T71V_T73K_T75S, V5Q_T71V_T73K_T75S,
V5Q_M69L_T73K_T75S, V5Q_M69L_T71V_T75S, V5Q_M69L_T71V_T73K,
T71V_T73K_T75S, M69L_T73K_T75S, M69L_T71V_T75S, M69L_T71V_T73K,
V5Q_T73K_T75S, V5Q_T71V_T75S, V5Q_T71V_T73K, V5Q_M69L_T75S,
V5Q_M69L_T73K, V5Q_M69L_T71V. An exemplary vector design for the
heavy chain (HC) is illustrated in FIG. 5, and an exemplary vector
design for the light chain (LC) is provided in FIG. 6.
[0283] The plasmids are transfected into HEK293 6E for transient
expression, using 293fectin. Materials: Cells: HEK 293 6E (293-6E)
cells are grown in exponential growth phase (0.8 to 1.2.times.106
cells/ml). Culture Medium: FreeStyle.TM. (Cat. No. 12338-018 from
Gibco); 25 .mu.g/ml Geneticin 418 (Cat. No. 10131-019 from Gibco);
0.1% pluronic F-68 (Cat. No. 24040-032 from Gibco). Transfection
Medium: Opti-MEM (Cat. No. 51985-026 from Gibco); 293fectin (Cat.
No. 12347019 from Invitrogen). Plasmid DNA: Purified plasmid DNA of
interest (see above).
[0284] Cell Count and Inoculation.
[0285] Two days before transfection, the necessary volume to get
7.5.times.10.sup.6 cells is transferred into a 125 ml flask, and
fresh Freestyle medium is added to complete to 30 ml (final cell
density should be 0.25.times.10.sup.6 cells/ml). Two days later
(the day of transfection), cell density should be between 1 and
1.2.times.10.sup.6 cells/ml. Alternatively, one day before
transfection, the necessary volume to get 1.5.times.10.sup.7 cells
is transferred into a 125 ml flask, and fresh Freestyle medium is
added to complete to 30 ml (final cell density should be of
0.50.times.10.sup.6 cells/ml). 24h later (the day of transfection),
cell density should be between 0.9 and 1.2.times.10.sup.6
cells/ml.
[0286] 293Fectin-DNA Complexes Preparation.
[0287] To prepare DNA solution, 30 .mu.g DNA is diluted in a total
volume of 1 ml Opti-MEM. To prepare 293fectin solution, 40 .mu.l is
diluted in 960 .mu.l Opti-MEM. After 5 min. incubation at room
temperature, the 293fectin solution and the DNA solution are mixed,
and then incubated 25 minutes at room temperature. The cells are
then transfected with 2 ml 293fectin-DNA mixture, and incubate at
37.degree. C. in a humidified incubator (orbital shaker) containing
5% CO2 for 4 to 6 days. FIG. 7 outlines a general procedure for
transient expression in HEK293 cells, such as e.g., HEK693 6E
cells.
Example 6--Purification of IgG1 from Z270 Hybridoma Culture
[0288] IgG1 Z270 antibody is purified from Z270 hybridoma cell
culture fluid by adding the sample onto a HiTrap Protein A HP (1
ml) column equilibrated in 3M NaCl 50 mM Tris pH8.5, at a flow
rate: 1.0 ml/min, and eluting antibody using 25 mM citric acid, 4.5
mM sodium citric acid pH3.0.
Example 7--Antibody Quantification and Affinity Determination
[0289] Plasmids containing light chain and equivalent heavy chain
expression constructs are mixed in pairs, and the plasmids are used
to transfect HEK6E cells. The culture medium supernatant is then
collected.
[0290] To quantify mouse IgG1 (or IgG4) antibody quantification, an
ELISA plate is coated with Goat poly anti mouse IgG1 (or IgG4) Fc
specific capture antibody. Antibody expression supernatant is
applied, followed by HRP-Goat poly anti mouse kappa or Fab
secondary antibody. HRP substrate is applied, and conversion
detected at OD450.
[0291] To analyse antigen-binding of humanized Z270 antibodies, the
following Biacore assay, illustrated in FIG. 8, is used.
Antigen-capture antibody is immobilized on a Biacore chip. The
antigen and culture supernatant are applied. The on- and off-rates
are analyzed to calculate the affinity.
Example 8--Biacore Analysis of Chimeric, Humanized, and
Back-Mutation Variants of Z270
Materials and Methods
[0292] Chimeric Z270 and humZ270 in VKI_O2/JK4 light chain and
VH1_18/JH6 heavy chain acceptor frameworks were produced according
to the methods described in Examples 4 and 5. The antigen-binding
properties of chimeric Z270, humZ270 and back-mutation variants
were analyzed on a Biacore T100 (Biacore AB, Uppsala, Sweden). The
antigen was in the form of a single-chain NKG2A-CD94-mFc construct
was covalently immobilized on the sensor CM5 chip (Biacore AB,
Uppsala, Sweden) via amine groups using
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS). The immobilization level was
targeted at 300 RU. Z270 antibody variants were diluted to a
concentration series (0.157, 0.313, 0.625, 1.25, 2.5 nM) in the
running buffer HBS-EP (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA,
0.005% (v/v) Tween-20). All the samples were then injected over the
immobilized antigen for 2 min at the flow rate of 40 ul/min.
Subsequently, the running buffer was injected for 3 min at 40
ul/min for antibody dissociation analysis. After each run, the
regeneration buffer (10 mM NaOH, 500 mM NaCl) was injected (30
seconds, 10 ul/min) to completely strip the remaining antibodies
off the antigen. Data were evaluated with Biacore T100 evaluation
software.
Results
[0293] The affinity of humZ270 was determined as 67 pM. This KD
value was higher than that of chimeric Z270 (50 pM) (FIG. 9 and
Table 2). Introduction of back mutations to humZ270 in VKI_02/JK4
light chain and VH1_18/JH6 heavy chain acceptor frameworks did not
substantially improve its affinity (FIG. 10).
TABLE-US-00005 TABLE 2 Chimeric Z270 humZ270 ka (1/Ms) kd (1/s) KD
(M) Chi.sup.2 (RU.sup.2) ka (1/Ms) Kd (1/s) KD (M) Chi.sup.2
(RU.sup.2) 7.970E+6 3.972E-4 4.983E-11 0.0968 7.492E+6 4.982E-4
6.650E-11 0.044
Example 9--Generation of humZ270 with Full-Length CDR-H2
[0294] In an alternative strategy, shown in FIG. 11, it was
investigated whether full-length Kabat CDRs (including a
full-length CDR-H2) would result in an improved affinity of humZ270
in VKI_O2/JK4 light chain and VH1_18/JH6 heavy chain acceptor
frameworks. humZ270VH3 (SEQ ID NO:24) follows directly from the
difference in CDR definitions, resulting basically in a Kabat
CDR-grafted humanized Z270 antibody. humZ270VH4 (SEQ ID NO:25)
follows from the observation that K38, Q46, W47, 148, G49, Y59,
Q61, K62, K66, and A67 are in proximity to S60, F63, K64, D65
side-chains, resulting in a CDR-grafted humanized Z270 antibody
with R38K, E46Q, M481, R66K, and V67A back-mutations.
Example 10--Generation of humZ270 in Other Human Acceptor
Frameworks
[0295] A number of different humanized constructs with different
human heavy chain acceptor framework sequences were made to explore
the framework choice.
[0296] FIG. 12 shows an alignment between the different humanized
Z270VH constructs prepared. humZ270VH5 (SEQ ID NO:26) is based on
VH5_a, humZ270VH6 (SEQ ID NO:27) is based on VH5_51, humZ270VH7
(SEQ ID NO:28) is based on VH1_f, and humZ270VH8 (SEQ ID NO:29) is
based on VH1_46, all with a JH6 J-segment. The 6 C-terminal amino
acid residues of the Kabat CDR-H2 of all humanized constructs were
identical to the human acceptor framework.
[0297] Using the alignment program Vector NTI, the following
sequence identities between humZ270VH1 and humZ270VH5, -6, -7, and
-8 were obtained: 78.2% (VH1 vs. VH5), 79.0% (VH1 vs. VH6), 88.7%
(VH1 vs. VH7), and 96.0% (VH1 vs. VH8).
Example 11--Biacore Analysis of Different humZ270 Variants
[0298] The affinities of huZ270 variants, all comprising a
humZ270VL1 sequence but with different strategies employed for VH
sequence humanization, were analyzed.
Materials and Methods
[0299] A Biacore T100 (Biacore AB, Uppsala, Sweden) was used.
CD94/NKG2A antigen was used in the form of a single-chain
NKG2A-CD94-mFc construct, covalently immobilized on the sensor CM5
chip (Biacore AB, Uppsala, Sweden) via amine groups using
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS). The immobilization level was
targeted at 300 RU. humZ270 antibody variants were diluted to a
concentration series (0.157, 0.313, 0.625, 1.25, 2.5 nM) in the
running buffer HBS-EP. All the samples were then injected over the
immobilized antigen for 2 min at the flow rate of 40 ul/min.
Subsequently, the running buffer was injected for 3 min at 40
ul/min for antibody dissociation analysis. After each run, the
regeneration buffer (10 mM NaOH, 500 mM NaCl) was injected (40
seconds, 10 ul/min) to completely strip the remaining antibodies
off the antigen. Data were evaluated with Biacore T100 evaluation
software. The KD value of each variant was divided by that of
huZ270 with a humZ270VH1 heavy chain to obtain the relative KD fold
change.
Results and Conclusions
[0300] The results are shown in FIG. 13, where the KD value of each
variant was normalized to that of huZ270 with a humZ270VH1 heavy
chain to obtain the relative change in KD. As shown in FIG. 13,
there were no significant differences between the CDR-grafted
variants (VH3, VH4) and the humZ270 variant comprising fewer murine
residues in the CDR-H2 segment, (humZ270VL1NH1). Nor were there
substantial differences between the "humanized-CDR-H2" variants in
different human acceptor frameworks. Since a more human humZ270
antibody has the benefit of a lower risk for an immunogenic
response in human patients, the humanized-CDR-H2 variants such as
VH1, VH5, VH6, and VH7 can be chosen for therapeutic applications
without the compromise of a significantly lower affinity as
compared to a standard CDR-grafted humZ270 antibody, and with a
lower likelihood of a host immune response.
Example 12--Identification of Critical Residues in Z270VL and
VH
[0301] In order to identify the paratope of Z270, alanine scan
mutagenesis was conducted on the CDRs of the murine antibody. The
following amino acids were selected for alanine mutagenesis (FIG.
14):
[0302] Z270VL: R24A, S26A, E27A, N28A, Y30A, S31A, N50A, K52A,
T53A, E56A, Y92A, T94A
[0303] Z270VH: K23A, S25A, T28A, T30A, S31A, N35A, D52A, Y53A,
D54A, S55A, E56A, R94A, D98A, F99A, D100A, V(100A)A, T(100C)A,
L(100D)A, W(100F)A, D101A.
[0304] Materials and Methods
[0305] The antigen-binding properties of the alanine mutants were
analyzed on a Biacore T100 (Biacore AB, Uppsala, Sweden). Antigen
in the form of sc-NKG2A-CD94-mFc was covalently immobilized on the
sensor CM5 chip (Biacore AB, Uppsala, Sweden) via amine groups
using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDC) and N-hydroxysuccinimide (NHS). The immobilization level was
targeted at 300 RU. Purified Z270 alanine mutants were diluted to
0.75 nM or 1.5 nM in the running buffer HBS-EP. All the samples
were then injected over the immobilized antigen for 3 min at the
flow rate of 10 ul/min. Subsequently, the running buffer was
injected for 1 min at 10 ul/min for antibody binding stability
analysis. After each run, the regeneration buffer (10 mM NaOH, 500
mM NaCl) was injected (35 seconds, 10 ul/min) to completely strip
the remaining antibodies off the antigen. Data were evaluated with
Biacore T100 evaluation software. The relative binding of each
mutant was calculated through dividing its binding level (RU)
obtained from Biacore by that of chimZ270.
Results and Conclusions
[0306] As shown in FIG. 15, Z270VH alanine mutants D52A, D54A,
F99A, T(100C)A, and W(100F)A completely lost their antigen-binding
properties. The heavy-chain mutant R94 remained around 20%
antigen-binding ability. The relative binding levels of heavy-chain
mutants N35A, Y53A, E56A, D98A, V(100A)A, and L(100D)A were between
40-70% (FIG. 1). Accordingly, the amino acids D52, D54, R94, F99,
T(100C), and W(100F) in the Z270 heavy chain CDR-H2 and CDR-H3 are
the critical residues to recognize the antigen. Meanwhile, the
amino acids N35, Y53, E56, D98, V(100A), and L(100D) in the heavy
chain moderately affect the antigen-binding. Interestingly, all
Z270 light-chain alanine mutants retain comparable antigen-binding
properties to that of chimeric Z270 (FIG. 16). Therefore, no amino
acid in the Z270 light chain significantly contributes to antigen
recognition.
Example 13--HumZ270 Specifically Binds Cells Expressing
CD94/NKG2A
[0307] The strength and specificity of humZ270 binding to
CD94/NKG2A was tested in flow-cytometry, by analyzing the binding
of in HEK293 cells produced wild-type Z270 (recZ270), chimeric Z270
with human IgG4 (chimZ270) or humanized Z270 (humZ270VL1NH1) to
Ba/F3 cells stably over-expressing either CD94/NKG2A or CD94/NKG2C.
For this purpose, Ba/F3-CD94/NKG2A and -C cells were incubated with
various concentrations of Z270 variants in tissue-culture medium
containing 2% FCS, for at least 30 minutes on ice. Subsequently
cells were washed, and the cells incubated in similar medium with
APC conjugated secondary Ab's, again for at least 30 minutes on
ice. After two times washing with ice-cold PBS, the binding of
mAb's to cells was visualized using a BD Biosciences FACSarray.
[0308] As shown in FIG. 17, all Z270 variants bind in a
dose-dependent fashion to Ba/F3-CD94/NKG2A cells, but not to
Ba/F3-CD94/NKG2C cells. Thus all variants specifically bind NKG2A,
with humZ270 binding with similar efficacy to NKG2A as chimZ270,
whereas recZ270 binds slightly more efficiently.
Example 14--humZ270 Induces Killing of HLA-E+ Target Cells by
CD94/NKG2A-Expressing NKL Cells
[0309] The ability of recZ270, chimZ270, humZ270VL1NH1 and Z199 to
induce killing of .sup.51Cr-labeled LCL 721.221-Cw3 cells by
CD94/NKG2A+ NKL cells was investigated. In this assay,
.sup.51Cr-labeled LCL 721.221-Cw3 target-cells (HLA-E+) were
incubated with NKL cells in a humidified incubator containing 5%
CO.sub.2, for 4 hours at 37.degree. C. (E:T ratio=6:1), in the
presence or absence of various concentrations of anti-NKG2A mAb's.
The killing of target-cells was analyzed by measuring the amount of
.sup.51Cr in the tissue-culture medium, which was released by
target cells upon killing.
[0310] In FIG. 18, it shown that increasing concentrations of
anti-NKG2A antibody induced the killing of LCL 721.221-Cw3 cells by
NKL cells. Z199, chimZ270 and recZ270 were all equally efficient,
whereas humZ270 induced higher killing of LCL 721.221-Cw3 cells by
NKL cells. Thus, humZ270 can efficiently block the inhibitory
function of CD94/NKG2A on CD94/NKG2A-expressing cytotoxic
lymphocytes, such as subsets of NK-cells, NKT-cells, .alpha./.beta.
T-cells and .gamma./.delta. T-cells, and was more efficient than
the other recombinant variants tested.
Example 15--humZ270 is a Competitive CD94/NKG2A Antagonist
[0311] To test whether humZ270VL1NH1 prevents ligand (i.e. HLA-E)
binding to CD94/NKG2A, we analyzed whether humZ270 could prevent
the binding of HLA-E tetramers to CD94/NKG2A over-expressing Ba/F3
cells (Ba/F3-CD94/NKG2A). For this, Ba/F3-CD94/NKG2A were incubated
with 1) various concentrations of humZ270 or 2) first incubated
with a saturing concentration of HLA-E tetramers (4.7 .mu.g/ml) and
then incubated with various concentrations of humZ270. All
incubations were performed in tissue-culture medium containing 2%
FCS, on ice. Subsequently, cells were incubated with APC-conjugated
secondary antibodies specific for mouse Ab's, and analyzed by
flowcytometry using a BD Biosciences FACSarray.
[0312] As shown in FIG. 19, humZ270 efficiently binds
Ba/F3-CD94/NKG2A cells in a concentration dependent fashion
(diamonds). However, when cells were pre-incubated with HLA-E
tetramers, humZ270 was prevented from binding to Ba/F3-CD94/NKG2A
cells. Thus HumZ270 and HLA-E bind overlapping epitopes on
CD94/NKG2A. Therefore, the CD94/NKG2A-inhibitory effect of humZ270
in NK-cytotoxicity assays is likely a consequence of preventing the
ability of HLA-E inducing negative signals to cytotoxic lymphocytes
via CD94/NKG2A. As such, humZ270 can be considered a competitive
CD94/NKG2A antagonist.
Example 16--humZ270 Specifically Binds to CD94/NKG2A
[0313] The specificity and efficacy of humZ270VL1NH1, and various
humZ270VL1NH1 variants with back-mutations in the variable light
(VL) or variable heavy (VH) regions were tested for binding to
CD94/NKG2A in flow-cytometry. For this, Ba/F3-CD94/NKG2A cells were
incubated with humZ270-L46F (VL), humZ270-148V (VL),
humZ270-L46F/148V (VL), humZ270-V5Q (VH), humZ270-M69L (VH),
humZ270-T71V (VH), humZ270-T73K (VH), humZ270-T75S (VH),
humZ270-V5Q/M69LJT71V/T73K/T75S (VH), humZ270-M69L/T71V/T73KIT75S
(VH) or two different batches of humZ270VL1NH1 without
back-mutations ("DK" and "CHN"). For this purpose, Ba/F3-CD94/NKG2A
or -C cells were incubated with various concentrations of the
humZ270 variants in tissue-culture medium containing 2% FCS, for at
least 30 minutes on ice. The cells were then washed, and incubated
in similar medium with APC conjugated secondary antibodies specific
for human antibodies, again for at least 30 minutes on ice. After
two times washing with ice-cold PBS, the binding of secondary
antibodies to cells was visualized using a BD Biosciences
FACSarray.
[0314] All variants bound specifically to CD94/NKG2A, and not to
CD94/NKG2C. All variants bound with similar efficacy to CD94/NKG2A,
with the exception of variants containing the V5Q (VL) mutation,
which bound slightly less efficiently (see FIG. 20).
Exemplary Embodiments
[0315] The following paragraphs describe exemplary embodiments of
the invention.
[0316] 1. An antibody that specifically binds NKG2A, comprising
antigen-binding residues from the complementarity-determining
regions (CDRs) of murine antibody Z270 and human acceptor framework
sequences, wherein at least the 6 C-terminal amino acid residues of
the CDR-H2 are the same as those in the variable heavy (VH) human
acceptor sequence.
[0317] 2. The antibody of embodiment 1, which is more effective
than an antibody comprising murine antibody Z270 variable light
(VL) and VH sequences in potentiating the cytotoxic activity of a
CD94/NKG2A-expressing cytotoxic lymphocyte.
[0318] 3. The antibody of embodiment 1, which is more effective
than an antibody comprising murine antibody Z270 variable light
(VL) and VH sequences in neutralizing the inhibitory activity of a
CD94/NKG2A receptor expressed on the surface of a cytotoxic
lymphocyte.
[0319] 4. The antibody of embodiment 1, which is more effective
than an antibody comprising murine antibody Z270 variable light
(VL) and VH sequences in reducing CD94/NKG2A-mediated inhibition of
the cytotoxic activity of a CD94/NKG2A-expressing cytotoxic
lymphocyte.
[0320] 5. The antibody of embodiment 1, which is more effective
than an antibody comprising murine antibody Z270 variable light
(VL) and VH sequences in inducing the killing of a Cw3-expressing
target cell by a CD94/NKG2A-expressing cytotoxic lymphocyte.
[0321] 6. The antibody of any of embodiments 2-5, wherein the
CD94/NKG2A-expressing cytotoxic lymphocyte is an NK cell, an NKT
cell, an .alpha./.beta. T-cell, or a .gamma./.delta. T-cell.
[0322] 7. The antibody of embodiment 6, wherein the
CD94/NKG2A-expressing cytotoxic lymphocyte is an NK cell.
[0323] 8. The antibody of any of embodiments 1-7, wherein the
antibody VH domain comprises residues D52, D54, F99, T(100C), and
W(100F) from the VH CDRs of murine antibody Z270.
[0324] 9. The antibody of embodiment 8, wherein the antibody VH
domain further comprises residues N35, Y53, E56, D98, V(100A), and
L(100D) from the VH CDRs of murine antibody Z270.
[0325] 10. The antibody of any of embodiments 1-9, comprising a
CDR-H1 corresponding to residues 31-35 of SEQ ID NO:5 and a CDR-H3
corresponding to residues 95-102 of SEQ ID NO:5, wherein the CDR-H2
comprises residues 50-59 of SEQ ID NO:5.
[0326] 11. The antibody of any of embodiments 1-10, wherein the VH
domain human acceptor framework has 70% or more sequence identity
to SEQ ID NO:5.
[0327] 12. The antibody of any of embodiments 1-11, wherein the VH
segment of the VH human acceptor framework is VH1_18, VH5_a,
VH5_51, VH1_f, or VH1_46, and the J-segment is JH6.
[0328] 13. The antibody of embodiment 12, wherein the VH segment is
VH1_18, VH5_a, VH5_51, or VH1_f.
[0329] 14. The antibody of embodiment 13, wherein the VH segment is
VH1_18.
[0330] 15. The antibody of any of embodiments 1-14, wherein the VH
domain human acceptor sequence is free of any back-mutations.
[0331] 16. The antibody of any of embodiments 1-14, wherein
[0332] (a) the amino acid at position 5 of the VH domain is V or
Q;
[0333] (b) the amino acid at position 69 of the VH domain is M or
L;
[0334] (c) the amino acid at position 71 of the VH domain is T or
V;
[0335] (d) the amino acid at position 73 of the VH domain is T or
K; or
[0336] (e) the amino acid at position 75 of the VH domain is T or
S.
[0337] 17. The antibody of embodiment 16, wherein the amino acid at
position 69 is L.
[0338] 18. The antibody of embodiment 16, wherein the amino acid at
position 71 is V.
[0339] 19. The antibody of any of embodiments 1-15, wherein the VH
domain comprises the sequence of SEQ ID NO:5.
[0340] 20. The antibody of any of embodiments 1-19, comprising a
CDR-L1 corresponding to residues 24-34 of SEQ ID NO:4, a CDR-L2
corresponding to residues 50-56 of SEQ ID NO:4, and a CDR-L3
corresponding to residues 89-97 of SEQ ID NO:4.
[0341] 21. The antibody of embodiment 20, wherein the VL domain
human acceptor sequence is free of any back-mutations.
[0342] 22. The antibody of any of embodiments 20-21, wherein the VL
domain human acceptor framework is from VKI_02/JK4.
[0343] 23. The antibody of any of embodiments 20-22, wherein the VL
domain human acceptor framework comprises SEQ ID NO:4.
[0344] 24. A humanized antibody or antibody fragment that
specifically binds NKG2A, comprising
[0345] (a) a CDR-L1 corresponding to residues 24-34 of SEQ ID
NO:4;
[0346] (b) a CDR-L2 corresponding to residues 50-56 of SEQ ID
NO:4;
[0347] (c) a CDR-L3 corresponding to residues 89-97 of SEQ ID
NO:4;
[0348] (d) a CDR-H1 corresponding to residues 31-35 of SEQ ID
NO:5;
[0349] (e) a CDR-H3 corresponding to residues 95-102 of SEQ ID
NO:5; and
[0350] (f) a CDR-H2 comprising residues 50-59 of SEQ ID NO:5;
and
[0351] (g) human acceptor framework sequences;
[0352] wherein residues 60-65 in the CDR-H2 are from the VH human
acceptor sequence, and
[0353] wherein the humanized antibody is more effective than an
antibody comprising a variable light (VL) sequence corresponding to
SEQ ID NO:1 and a VH sequences corresponding to SEQ ID NO:2 in
potentiating the cytotoxic activity of a CD94/NKG2A-expressing NK
cell.
[0354] 25. A humanized antibody that binds human NKG2A, the
antibody comprising a VH domain that comprises non-human CDR
residues and a human VH acceptor framework, the VH domain
comprising a CDR-H1 corresponding to residues 31-35 of SEQ ID NO:5,
a CDR-H2 corresponding to residues 50-65 of SEQ ID NO:5, and a
CDR-H3 corresponding to residues 95-102 of SEQ ID NO:5.
[0355] 26. The humanized antibody of embodiment 25, wherein the
human VH acceptor framework does not comprise any
back-mutation.
[0356] 27. The humanized antibody of embodiment 25, wherein the
amino acid at Kabat position 5 of the VH domain is V or Q.
[0357] 28. The humanized antibody of embodiment 25, wherein the
amino acid at Kabat position of the VH domain 69 is M or L.
[0358] 29. The humanized antibody of embodiment 25, wherein the
amino acid at Kabat position 71 of the VH domain is T or V.
[0359] 30. The humanized antibody of embodiment 25, wherein the
amino acid at Kabat position 73 of the VH domain is T or K.
[0360] 31. The humanized antibody of embodiment 25, wherein the
amino acid at Kabat position 75 of the VH domain is T or S.
[0361] 32. The humanized antibody of embodiment 25, wherein the VH
domain comprises a framework region substitution in at least one
Kabat position selected from the group consisting of 5, 69, 71, 73,
and 75.
[0362] 33. The humanized antibody of embodiment 32, wherein the VH
domain comprises the amino acid sequence of SEQ ID NO:5, with
framework substitutions according to any one of the following
options:
[0363] (a) none
[0364] (b) V5Q
[0365] (c) M69L
[0366] (d) T71V
[0367] (e) T73K
[0368] (f) T75S
[0369] (g) V5Q and M69L
[0370] (h) V5Q and T71V
[0371] (i) V5Q and T73K
[0372] (j) V5Q and T75S
[0373] (k) M69L and T71V
[0374] (l) M69L and T73K
[0375] (m) M69L and T75S
[0376] (n) T71V and T73K
[0377] (o) T71V and T75S
[0378] (p) T73K and T75S
[0379] (q) V5Q, T73K and T75S
[0380] (r) V5Q, T71V and T75S
[0381] (s) V5Q, T71V and T73K
[0382] (t) V5Q, M69L and T75S
[0383] (u) V5Q, M69L and T73K
[0384] (v) V5Q, M69L and T71V
[0385] (w) T71V, T73K and T75S
[0386] (x) M69L, T73K and T75S
[0387] (y) M69L, T71V and T75S,
[0388] (z) M69L, T71V and T73K,
[0389] (aa) V5Q, M69L, T71V and T73K,
[0390] (bb) V5Q, M69L, T71V and T75S,
[0391] (cc) V5Q, M69L, T73K and T75S,
[0392] (dd) V5Q, T71V, T73K and T75S,
[0393] (ee) M69L, T71V, T73K and T75S, and
[0394] (ff) V5Q, M69L, T71V, T73K and T75S.
[0395] 34. The humanized antibody of any of embodiments 25-32,
comprising a VL domain that comprises non-human CDR residues
incorporated into a human VL acceptor framework, the VL domain
comprising a CDR-L1 corresponding to residues 24-34 of SEQ ID NO:4,
a CDR-L2 corresponding to residues 50-56 of SEQ ID NO:4, and an
CDR-L3 corresponding to residues 89-97 of SEQ ID NO:4.
[0396] 35. The humanized antibody of embodiment 34, wherein the
human VL acceptor framework does not comprise any
back-mutation.
[0397] 36. The humanized antibody of embodiment 34, wherein the
amino acid at Kabat position 46 of the VL domain is L or F.
[0398] 37. The humanized antibody of embodiment 34, wherein the
amino acid at Kabat position 48 of the VL domain is I or V.
[0399] 38. The humanized antibody of embodiment 34, wherein the VL
domain comprises a framework region substitution in at least one
Kabat position selected from 46 and 48.
[0400] 39. The humanized antibody of embodiment 38, wherein the VL
domain comprises the amino acid sequence of SEQ ID NO:4, with
framework substitutions according to any one of the following
options:
[0401] (a) None
[0402] (b) L46F
[0403] (c) 148V
[0404] (d) L46 and 148V.
[0405] 40. A humanized antibody that binds human NKG2A, the
antibody comprising a VH domain that comprises non-human CDR
residues incorporated into a human VH domain, the VH domain
comprising a framework region substitution in at least one Kabat
position in SEQ ID NO:7 selected from the group consisting of 5,
69, 71, 73, and 75.
[0406] 41. The humanized antibody of embodiment 40, comprising a
V5Q substitution.
[0407] 42. The humanized antibody of embodiment 40, comprising a
M69L substitution.
[0408] 43. The humanized antibody of embodiment 40, comprising a
T71V substitution.
[0409] 44. The humanized antibody of embodiment 40, comprising a
T73K substitution.
[0410] 45. The humanized antibody of embodiment 40, comprising a
T75S substitution.
[0411] 46. The humanized antibody of embodiment 40, comprising a VL
domain that comprises non-human CDR residues incorporated into a
human VL domain, the VL domain comprising a framework region
substitution in at least one Kabat position in SEQ ID NO:6 selected
from 46 and 48.
[0412] 47. The humanized antibody of embodiment 40, comprising a
L46F substitution.
[0413] 48. The humanized antibody of embodiment 40, comprising a
148V substitution.
[0414] 49. The humanized antibody of any of embodiments 40-48,
comprising a CDR-H1 corresponding to residues 31-35 of SEQ ID NO:5,
a CDR-H2 corresponding to residues 50-66 of SEQ ID NO:5, and a
CDR-H3 corresponding to residues 95-102 of SEQ ID NO:5.
[0415] 50. The humanized antibody of any of embodiments 40-49,
comprising a VH domain comprising the sequence of SEQ ID NO:7.
[0416] 51. The humanized antibody of any of embodiments 40-50,
comprising a CDR-L1 corresponding to residues 24-34 of SEQ ID NO:6,
a CDR-L2 corresponding to residues 50-56 of SEQ ID NO:6, and an
CDR-L3 corresponding to residues 89-97 of SEQ ID NO:6.
[0417] 52. The humanized antibody of any of embodiments 40-52,
comprising a VL domain comprising the sequence of SEQ ID NO:6.
[0418] 53. A humanized antibody that binds human NKG2A, the
antibody comprising a VH domain that comprises the amino acid
sequence of SEQ ID NO:5, optionally with one or more FR
substitutions at Kabat positions 5, 69, 71, 73, and/or 75.
[0419] 54. The humanized antibody of embodiment 53, wherein the
optional FR substitutions are V5Q, M69L, T71V, T73K, and/or
T75S.
[0420] 55. The humanized antibody of embodiment 52, further
comprising a VL domain that comprises the amino acid sequence of
SEQ ID NO:4, optionally with one or more FR substitutions at Kabat
positions 46 and/or 48.
[0421] 56. The humanized antibody of embodiment 55, wherein the
optional FR substitutions are L46F and/or 148V.
[0422] 57. A humanized antibody that binds NKG2A, comprising a VH
domain that comprises non-human CDR residues incorporated into a
human VH domain, wherein the VH domain is at least 50% identical to
SEQ ID NO:5.
[0423] 58. The humanized antibody of embodiment 46, wherein the VH
domain is at least 90% identical to SEQ ID NO:5.
[0424] 59. The humanized antibody of any of embodiments 57 and 58,
comprising a CDR-H1 corresponding to residues 31-35 of SEQ ID NO:5,
a CDR-H2 corresponding to residues 50-66 of SEQ ID NO:5, and a
CDR-H3 corresponding to residues 95-102 of SEQ ID NO:5.
[0425] 60. The humanized antibody of any of embodiments 57-59,
comprising a V or Q at Kabat position 5, an M or L at Kabat
position 69, a T or V at Kabat position 71, a T or K at Kabat
position 73, and a T or S at Kabat position 75, in the VH
domain.
[0426] 61. The humanized antibody of any of embodiments 57-60,
comprising a VL domain that comprises non-human CDR residues
incorporated into a human VL domain, wherein the VL domain is at
least 50% identical to SEQ ID NO:4.
[0427] 62. The humanized antibody of any of embodiments 57-61,
comprising a VL domain at least 90% identical to SEQ ID NO:4.
[0428] 63. The humanized antibody of any of embodiments 57-62,
comprising a CDR-L1 sequence corresponding to residues 24-34 of SEQ
ID NO:4, a CDR-L2 sequence corresponding to residues 50-56 of SEQ
ID NO:4, and an CDR-L3 sequence corresponding to residues 89-97 of
SEQ ID NO:4.
[0429] 64. The humanized antibody of any of embodiments 57-63,
comprising a L or F at Kabat position 46 and/or an I or V at Kabat
position 48.
[0430] 65. A humanized antibody that binds human NKG2A, the
antibody comprising a VH domain that comprises non-human CDR
residues incorporated into a human VH domain, the VH domain
comprising a CDR-H1 corresponding to residues 31-35 of SEQ ID NO:8,
a CDR-H2 corresponding to residues 50-66 of SEQ ID NO:8, and a
CDR-H3 corresponding to residues 95-102 of SEQ ID NO:8, wherein the
amino acids at Kabat positions 63, 64, 65, 66, and 67 are F, K, D,
K, A, respectively.
[0431] 66. The humanized antibody of embodiment 65, wherein the
amino acid at Kabat position 60 is A.
[0432] 67. The humanized antibody of any of embodiments 65 and 66,
wherein the VH domain comprises the amino acid sequence of SEQ ID
NO:8.
[0433] 68. The humanized antibody of any of embodiments 65-67,
further comprising a VL domain comprising a CDR-L1 corresponding to
residues 24-34 of SEQ ID NO:6, a CDR-L2 corresponding to residues
50-56 of SEQ ID NO:6, and an CDR-L3 corresponding to residues 89-97
of SEQ ID NO:6.
[0434] 69. The humanized antibody of any of embodiments 65-68,
wherein the VL domain comprises the amino acid sequence of SEQ ID
NO:6.
[0435] 70. A humanized version of an anti-NKG2A antibody produced
by the Z270 hybridoma.
[0436] 71. The antibody of any of embodiments 1-70, which is a
multispecific antibody or an antibody fragment.
[0437] 72. The antibody of embodiment 71, which is an antibody
fragment selected from a Fab, a Fab', a F(ab).sub.2, a
F(ab').sub.2, a F(ab).sub.3, an Fv, a single-chain Fv, a dsFv, an
Fd fragment, a dAb fragment, a minibody, a diabody, a triabody, a
tetrabody, a kappa body; a camel IgG; an IgNAR; and a multispecific
antibody fragment.
[0438] 73. The antibody of embodiment 72, which is a bispecific
antibody.
[0439] 74. The antibody of any of embodiments 1-70, which is a
full-length IgG4 antibody or fragment thereof.
[0440] 75. The antibody of embodiment 74, wherein the heavy-chain
constant domain comprises an S241P mutation.
[0441] 76. An isolated nucleic acid encoding the antibody of any of
the preceding embodiments.
[0442] 77. A vector comprising the nucleic acid of embodiment
76.
[0443] 78. A host cell comprising the vector of embodiment 76.
[0444] 79. A method of producing an antibody comprising culturing
the host cell of embodiment 78 so that the nucleic acid is
expressed and the antibody produced.
[0445] 80. The method of embodiment 79, further comprising
recovering the antibody from the host cell culture.
[0446] 81. The method of embodiment 79 wherein, before culturing,
the host cell is co-transfected with a vector comprising nucleic
acid encoding a variable heavy domain and with a vector comprising
nucleic acid encoding a variable light domain.
[0447] 82. An immunoconjugate comprising an antibody according to
any of embodiments 1-75 and a second agent.
[0448] 83. The immunoconjugate of embodiment 82, wherein the second
agent is a cytotoxic agent.
[0449] 84. The immunoconjugate of embodiment 82, wherein the second
agent is a PEG-molecule.
[0450] 85. A pharmaceutical composition comprising the antibody of
any of embodiments 1-75 or the immunoconjugate of any of
embodiments 82-84, and a carrier.
[0451] 86. The pharmaceutical composition of embodiment 74,
comprising a buffer selected from citrate, phosphate, and a
combination thereof, having a pH from about 6.0 to about 7.5.
[0452] 87. An article of manufacture comprising a container
containing the antibody of any of embodiments 1-75 and instructions
directing a user to treat a disorder selected from a cancer, a
viral disease, an inflammatory disorder, and an autoimmune
disorder, in a mammal with the antibody in an effective amount.
[0453] 88. The article of embodiment 87, wherein the mammal is a
human.
[0454] 89. The antibody of any of embodiments 1-75 for use in
neutralizing the inhibitory activity of a CD94/NKG2A receptor
expressed on the surface of a cytotoxic lymphocyte in a human
patient.
[0455] 90. The antibody of any of embodiments 1-75 for use in
reducing CD94/NKG2A-mediated inhibition of the cytotoxicity
activity of a CD94/NKG2A-expressing cytotoxic lymphocyte in a human
patient.
[0456] 91. The antibody of any of embodiments 1-75 for use in
potentiating the cytotoxic activity of a CD94/NKG2A-expressing
cytotoxic lymphocyte in a human patient.
[0457] 92. The antibody of any of embodiments 1-75 for use in
inducing the killing of a Cw3-expressing target cell by a
CD94/NKG2A-expressing cytotoxic lymphocyte in a human patient.
[0458] 93. The antibody of any embodiments 89-92, wherein the
CD94/NKG2A-expressing cytotoxic lymphocyte is an NK cell, an NKT
cell, an .alpha./.beta. T-cell, or a .gamma./.delta. T-cell.
[0459] 94. A method of treating a human patient suffering from a
disorder selected from a cancer, a viral disease, an inflammatory
disorder, and an autoimmune disorder, comprising administering the
pharmaceutical composition of any of embodiments 85-86.
[0460] 95. The use of the antibody of any of embodiments 1-75 in
the preparation of a medicament for administration to a human
patient suffering from a disorder selected from a cancer, a viral
disease, an inflammatory disorder, and an autoimmune disorder.
[0461] 96. The antibody of any of embodiments 1-75 for use in the
treatment of a human patient suffering from a disorder selected
from a cancer, a viral disease, an inflammatory disorder, and an
autoimmune disorder.
[0462] 97. The method, use, or antibody of any of embodiments
89-96, wherein the patient suffers from squamous cell carcinoma,
leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia,
B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's
lymphoma, hairy cell lymphoma, Burkitt's lymphoma, multiple
myeloma, acute or chronic myelogenous leukemias, promyelocytic
leukemia, fibrosarcoma, rhabdomyosarcoma; melanoma, seminoma,
teratocarcinoma, neuroblastoma, astrocytoma, neuroblastoma, glioma,
schwannomas; fibrosarcoma, rhabdomyosarcoma, osteosarcoma,
melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid
follicular cancer, teratocarcinoma, other carcinoma of the bladder,
breast, colon, kidney, liver, lung, ovary, prostate, pancreas,
stomach, cervix, thyroid or skin, other hematopoietic tumors of
lymphoid lineage, other hematopoietic tumors of myeloid lineage,
other tumors of mesenchymal origin, other tumors of the central or
peripheral nervous system, or other tumors of mesenchymal
origin.
[0463] 98. The use according to embodiment 97, wherein the patient
suffers from multiple myeloma, Non-Hodgkin's lymphoma, or acute
myelogeous lymphoma.
[0464] All references, including publications, patent applications
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference was individually and
specifically indicated to be incorporated by reference and was set
forth in its entirety herein.
[0465] All headings and sub-headings are used herein for
convenience only and should not be construed as limiting the
invention in any way,
[0466] 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.
[0467] Unless otherwise expressly indicated or clearly contradicted
by context, the term "or" herein is used in the inclusive sense of
"and/or", and, accordingly, as implicitly providing support for an
embodiment or aspect in which the term is to be interpreted in the
exclusive sense of "either this or that".
[0468] 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.
[0469] 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).
[0470] All methods described herein can be performed in any
suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context.
[0471] 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.
[0472] 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,
[0473] 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).
[0474] 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
291107PRTMus musculus 1Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu
Ser Ala Ser Val Gly1 5 10 15Glu Thr Val Thr Ile Thr Cys Arg Ala Ser
Glu Asn Ile Tyr Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Gln Gly
Lys Ser Pro Gln Phe Leu Val 35 40 45Tyr Asn Ala Lys Thr Leu Ala Glu
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Gln Phe
Ser Leu Lys Ile Asn Ser Leu Gln Pro65 70 75 80Glu Asp Phe Gly Ser
Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Arg 85 90 95Thr Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys 100 1052124PRTMus musculus 2Gln Val Gln
Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala1 5 10 15Ser Val
Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp
Met Asn Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Gln Trp Ile 35 40
45Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Ser Gln Lys Phe
50 55 60Lys Asp Lys Ala Ile Leu Thr Val Asp Lys Ser Ser Ser Thr Ala
Tyr65 70 75 80Met Arg Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Gly Gly Tyr Asp Phe Asp Val Gly Thr Leu
Tyr Trp Phe Phe 100 105 110Asp Val Trp Gly Ala Gly Thr Thr Val Thr
Val Ser 115 1203107PRTMus musculus 3Asn Ile Val Met Thr Gln Ser Pro
Lys Ser Met Ser Met Ser Val Gly1 5 10 15Glu Arg Val Thr Leu Thr Cys
Lys Ala Ser Glu Asn Val Val Thr Tyr 20 25 30Val Ser Trp Tyr Gln Gln
Lys Pro Glu Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Gly Ala Ser Asn
Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly Ser Ala
Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala65 70 75 80Glu Asp
Leu Ala Asp Tyr His Cys Gly Gln Gly Tyr Ser Tyr Pro Tyr 85 90 95Thr
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100
1054107PRTArtificialHumanized sequence 4Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr 20 25 30Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asn Ala Lys
Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Arg 85 90
95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
1055124PRTArtificialHumanized sequence 5Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met Asn Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile Asp
Pro Tyr Asp Ser Glu Thr His Tyr Ala Gln Lys Leu 50 55 60Gln Gly Arg
Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met
Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Gly Gly Tyr Asp Phe Asp Val Gly Thr Leu Tyr Trp Phe Phe
100 105 110Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115
1206107PRTArtificialHumanized sequenceUNSURE(46)..(46)Xaa is L or
FUNSURE(48)..(48)Xaa is I or V 6Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Glu Asn Ile Tyr Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Xaa Leu Xaa 35 40 45Tyr Asn Ala Lys Thr Leu
Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Arg 85 90 95Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys 100
1057124PRTArtificialHumanized sequenceUNSURE(5)..(5)Xaa is V or
QUNSURE(70)..(70)Kabat position 69; Xaa is M or
LUNSURE(72)..(72)Kabat position 71; Xaa is T or
VUNSURE(74)..(74)Kabat position 73; Xaa is T or
KUNSURE(76)..(76)Kabat position 75; Xaa is T or S 7Gln Val Gln Leu
Xaa Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met
Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Ala Gln Lys Leu 50 55
60Gln Gly Arg Val Thr Xaa Thr Xaa Asp Xaa Ser Xaa Ser Thr Ala Tyr65
70 75 80Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Gly Gly Tyr Asp Phe Asp Val Gly Thr Leu Tyr Trp
Phe Phe 100 105 110Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser
115 1208124PRTArtificialHumanized sequenceUNSURE(5)..(5)Xaa is V or
QUNSURE(61)..(61)Kabat position 60; Xaa is S or
AUNSURE(64)..(64)Kabat position 63; Xaa is L or
FUNSURE(65)..(65)Kabat position 64; Xaa is Q or
KUNSURE(66)..(66)Kabat position 65; Xaa is G or
DUNSURE(67)..(67)Kabat position 66; Xaa is R or
KUNSURE(68)..(68)Kabat position 67; Xaa is V or
AUNSURE(70)..(70)Kabat position 69; Xaa is M or
LUNSURE(72)..(72)Kabat position 71; Xaa is T or
VUNSURE(74)..(74)Kabat position 73; Xaa is T or
KUNSURE(76)..(76)Kabat position 75; Xaa is T or S 8Gln Val Gln Leu
Xaa Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met
Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Xaa Gln Lys Xaa 50 55
60Xaa Xaa Xaa Xaa Thr Xaa Thr Xaa Asp Xaa Ser Xaa Ser Thr Ala Tyr65
70 75 80Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Gly Gly Tyr Asp Phe Asp Val Gly Thr Leu Tyr Trp
Phe Phe 100 105 110Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser
115 1209107PRTHomo sapiens 9Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95Thr Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys 100 10510117PRTHomo sapiens 10Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln
Lys Leu 50 55 60Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Tyr Tyr Tyr Tyr Tyr Gly Met Asp
Val Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser
11511233PRTHomo sapiens 11Met Asp Asn Gln Gly Val Ile Tyr Ser Asp
Leu Asn Leu Pro Pro Asn1 5 10 15Pro Lys Arg Gln Gln Arg Lys Pro Lys
Gly Asn Lys Ser Ser Ile Leu 20 25 30Ala Thr Glu Gln Glu Ile Thr Tyr
Ala Glu Leu Asn Leu Gln Lys Ala 35 40 45Ser Gln Asp Phe Gln Gly Asn
Asp Lys Thr Tyr His Cys Lys Asp Leu 50 55 60Pro Ser Ala Pro Glu Lys
Leu Ile Val Gly Ile Leu Gly Ile Ile Cys65 70 75 80Leu Ile Leu Met
Ala Ser Val Val Thr Ile Val Val Ile Pro Ser Thr 85 90 95Leu Ile Gln
Arg His Asn Asn Ser Ser Leu Asn Thr Arg Thr Gln Lys 100 105 110Ala
Arg His Cys Gly His Cys Pro Glu Glu Trp Ile Thr Tyr Ser Asn 115 120
125Ser Cys Tyr Tyr Ile Gly Lys Glu Arg Arg Thr Trp Glu Glu Ser Leu
130 135 140Leu Ala Cys Thr Ser Lys Asn Ser Ser Leu Leu Ser Ile Asp
Asn Glu145 150 155 160Glu Glu Met Lys Phe Leu Ser Ile Ile Ser Pro
Ser Ser Trp Ile Gly 165 170 175Val Phe Arg Asn Ser Ser His His Pro
Trp Val Thr Met Asn Gly Leu 180 185 190Ala Phe Lys His Glu Ile Lys
Asp Ser Asp Asn Ala Glu Leu Asn Cys 195 200 205Ala Val Leu Gln Val
Asn Arg Leu Lys Ser Ala Gln Cys Gly Ser Ser 210 215 220Ile Ile Tyr
His Cys Lys His Lys Leu225 2301220DNAArtificialPrimer 12gccagtggat
agacagatgg 201321DNAArtificialPrimer 13gatggataca gttggtgcag c
2114381DNAMus musculus 14atgagtgtgc ccactcaggt cctggggttg
ctgctgctgt ggcttacagg tgccagatgt 60gacatccaga tgactcagtc tccagcctcc
ctatctgcat ctgtgggaga aactgtcacc 120atcacatgtc gagcaagtga
gaatatttac agttatttag catggtatca gcagaaacag 180ggaaaatctc
ctcagttctt ggtctataat gcaaaaacct tagcagaagg tgtgccatca
240aggttcagtg gcagtggatc aggcacacag ttttctctga agatcaacag
cctgcagcct 300gaagattttg ggagttatta ctgtcaacat cactatggta
ctcctcggac gttcggtgga 360ggcaccaagc tggaaatcaa a 38115127PRTMus
musculus 15Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp
Leu Thr1 5 10 15Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ala
Ser Leu Ser 20 25 30Ala Ser Val Gly Glu Thr Val Thr Ile Thr Cys Arg
Ala Ser Glu Asn 35 40 45Ile Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln Lys
Gln Gly Lys Ser Pro 50 55 60Gln Phe Leu Val Tyr Asn Ala Lys Thr Leu
Ala Glu Gly Val Pro Ser65 70 75 80Arg Phe Ser Gly Ser Gly Ser Gly
Thr Gln Phe Ser Leu Lys Ile Asn 85 90 95Ser Leu Gln Pro Glu Asp Phe
Gly Ser Tyr Tyr Cys Gln His His Tyr 100 105 110Gly Thr Pro Arg Thr
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 115 120 12516432DNAMus
musculus 16atgggatgga gctatatcat cctcttcttg ttagcaacag ctacatgtgt
ccactcccag 60gtccaactgc agcagcctgg ggctgagctg gtgaggcctg gggcttcagt
gaagctgtcc 120tgcaaggctt ctggctacac gttcaccagc tactggatga
actgggttaa gcagaggcct 180gagcaaggcc ttcagtggat tggaaggatt
gatccttacg atagtgaaac tcactacagt 240caaaagttca aggacaaggc
catattgact gtagacaaat cctccagcac agcctacatg 300cgactcagca
gcctgacatc tgaggactct gcggtctatt actgtgcaag agggggctat
360gatttcgacg taggaactct ctactggttc ttcgatgtct ggggcgcagg
gaccacggtc 420accgtctcct ca 43217144PRTMus musculus 17Met Gly Trp
Ser Tyr Ile Ile Leu Phe Leu Leu Ala Thr Ala Thr Cys1 5 10 15Val His
Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg 20 25 30Pro
Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40
45Thr Ser Tyr Trp Met Asn Trp Val Lys Gln Arg Pro Glu Gln Gly Leu
50 55 60Gln Trp Ile Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr
Ser65 70 75 80Gln Lys Phe Lys Asp Lys Ala Ile Leu Thr Val Asp Lys
Ser Ser Ser 85 90 95Thr Ala Tyr Met Arg Leu Ser Ser Leu Thr Ser Glu
Asp Ser Ala Val 100 105 110Tyr Tyr Cys Ala Arg Gly Gly Tyr Asp Phe
Asp Val Gly Thr Leu Tyr 115 120 125Trp Phe Phe Asp Val Trp Gly Ala
Gly Thr Thr Val Thr Val Ser Ser 130 135
14018723DNAArtificialChimeric sequence 18gaattcgcca ccatgagtgt
gcccactcag gtcctggggt tgctgctgct gtggcttaca 60ggtgccagat gtgacatcca
gatgactcag tctccagcct ccctatctgc atctgtggga 120gaaactgtca
ccatcacatg tcgagcaagt gagaatattt acagttattt agcatggtat
180cagcagaaac agggaaaatc tcctcagttc ttggtctata atgcaaaaac
cttagcagaa 240ggtgtgccat caaggttcag tggcagtgga tcaggcacac
agttttctct gaagatcaac 300agcctgcagc ctgaagattt tgggagttat
tactgtcaac atcactatgg tactcctcgg 360acgttcggtg gaggcaccaa
gctggaaatc aaacgggctg atgctgcacc aactgtatcc 420atcttcccac
catccagtga gcagttaaca tctggaggtg cctcagtcgt gtgcttcttg
480aacaacttct accccaaaga catcaatgtc aagtggaaga ttgatggcag
tgaacgacaa 540aatggcgtcc tgaacagttg gactgatcag gacagcaaag
acagcaccta cagcatgagc 600agcaccctca cgttgaccaa ggacgagtat
gaacgacata acagctatac ctgtgaggcc 660actcacaaga catcaacttc
acccattgtc aagagcttca acaggaatga gtgttaggct 720agc
723191419DNAArtificialChimeric sequence 19gaattcgcca ccatgggatg
gagctatatc atcctcttct tgttagcaac agctacatgt 60gtccactccc aggtccaact
gcagcagcct ggggctgagc tggtgaggcc tggggcttca 120gtgaagctgt
cctgcaaggc ttctggctac acgttcacca gctactggat gaactgggtt
180aagcagaggc ctgagcaagg ccttcagtgg attggaagga ttgatcctta
cgatagtgaa 240actcactaca gtcaaaagtt caaggacaag gccatattga
ctgtagacaa atcctccagc 300acagcctaca tgcgactcag cagcctgaca
tctgaggact ctgcggtcta ttactgtgca 360agagggggct atgatttcga
cgtaggaact ctctactggt tcttcgatgt ctggggcgca 420gggaccacgg
tcaccgtctc ctcagccaaa acgacacccc catctgtcta tccgctagcc
480cctggatctg ctgcccaaac taactccatg gtgaccctgg gatgcctggt
caagggctat 540ttccctgagc cagtgacagt gacctggaac tctggatccc
tgtccagcgg tgtgcacacc 600ttcccagctg tcctgcagtc tgacctctac
actctgagca gctcagtgac tgtcccctcc 660agcacctggc ccagcgagac
cgtcacctgc aacgttgccc acccggccag cagcaccaag 720gtggacaaga
aaattgtgcc cagggattgt ggttgtaagc cttgcatatg tacagtccca
780gaagtatcat ctgtcttcat cttcccccca aagcccaagg atgtgctcac
cattactctg 840actcctaagg tcacgtgtgt tgtggtagac atcagcaagg
atgatcccga ggtccagttc 900agctggtttg tagatgatgt ggaggtgcac
acagctcaga cgcaaccccg ggaggagcag 960ttcaacagca ctttccgctc
agtcagtgaa cttcccatca tgcaccagga ctggctcaat 1020ggcaaggagt
tcaaatgcag ggtcaacagt gcagctttcc ctgcccccat cgagaaaacc
1080atctccaaaa ccaaaggcag accgaaggct ccacaggtgt acaccattcc
acctcccaag 1140gagcagatgg ccaaggataa agtcagtctg acctgcatga
taacagactt cttccctgaa 1200gacattactg tggagtggca gtggaatggg
cagccagcgg agaactacaa gaacactcag 1260cccatcatgg acacagatgg
ctcttacttc gtctacagca agctcaatgt gcagaagagc 1320aactgggagg
caggaaatac tttcacctgc tctgtgttac atgagggcct gcacaaccac
1380catactgaga agagcctctc ccactctcct ggtaaatga
141920723DNAArtificialChimeric sequence 20gaattcgcca ccatgagtgt
gcccactcag gtcctggggt tgctgctgct gtggcttaca 60ggtgccagat gtgacatcca
gatgactcag tctccagcct ccctatctgc atctgtggga 120gaaactgtca
ccatcacatg tcgagcaagt gagaatattt acagttattt agcatggtat
180cagcagaaac agggaaaatc tcctcagttc ttggtctata atgcaaaaac
cttagcagaa 240ggtgtgccat caaggttcag tggcagtgga tcaggcacac
agttttctct gaagatcaac 300agcctgcagc ctgaagattt tgggagttat
tactgtcaac atcactatgg tactcctcgg 360acgttcggtg gaggcaccaa
gctggaaatc aaacggactg tggcggcgcc atctgtcttc 420atcttcccgc
catctgatga gcagttgaaa tctggtaccg ctagcgttgt gtgcctgctg
480aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc
cctccaatcg 540ggtaactccc aggagagtgt cacagagcag gacagcaagg
acagcaccta cagcctcagc 600agcaccctga cgctgagcaa agcagactac
gagaaacaca aagtctacgc ctgcgaagtc 660acccatcagg gcctgagctc
gcccgtcaca aagagcttca acaggggaga gtgttaggga 720tcc
723211434DNAArtificialChimeric sequence 21gaattcgcca ccatgggatg
gagctatatc atcctcttct tgttagcaac agctacatgt 60gtccactccc aggtccaact
gcagcagcct ggggctgagc tggtgaggcc tggggcttca 120gtgaagctgt
cctgcaaggc ttctggctac acgttcacca gctactggat gaactgggtt
180aagcagaggc ctgagcaagg ccttcagtgg attggaagga ttgatcctta
cgatagtgaa 240actcactaca gtcaaaagtt caaggacaag gccatattga
ctgtagacaa atcctccagc 300acagcctaca tgcgactcag cagcctgaca
tctgaggact ctgcggtcta ttactgtgca 360agagggggct atgatttcga
cgtaggaact ctctactggt tcttcgatgt ctggggcgca 420gggaccacgg
tcaccgtctc ctcagctagc accaagggcc catccgtctt ccccctggcg
480ccctgctcca ggagcacctc cgagagcaca gccgccctgg gctgcctggt
caaggactac 540ttccccgaac cggtgacggt gtcgtggaac tcaggcgccc
tgaccagcgg cgtgcacacc 600ttcccggctg tcctacagtc ctcaggactc
tactccctca gcagcgtggt gaccgtgccc 660tccagcagct tgggcacgaa
gacctacacc tgcaacgtag atcacaagcc cagcaacacc 720aaggtggaca
agagagttga gtccaaatat ggtcccccat gcccaccatg cccagcacct
780gagttcctgg ggggaccatc agtcttcctg ttccccccaa aacccaagga
cactctcatg 840atctcccgga cccctgaggt cacgtgcgtg gtggtggacg
tgagccagga agaccccgag 900gtccagttca actggtacgt ggatggcgtg
gaggtgcata atgccaagac aaagccgcgg 960gaggagcagt tcaacagcac
gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 1020tggctgaacg
gcaaggagta caagtgcaag gtctccaaca aaggcctccc gtcctccatc
1080gagaaaacca tctccaaagc caaagggcag ccccgagagc cacaggtgta
caccctgccc 1140ccatcccagg aggagatgac caagaaccag gtcagcctga
cctgcctggt caaaggcttc 1200taccccagcg acatcgccgt ggagtgggag
agcaatgggc agccggagaa caactacaag 1260accacgcctc ccgtgctgga
ctccgacggc tccttcttcc tctacagcag gctaaccgtg 1320gacaagagca
ggtggcagga ggggaatgtc ttctcatgct ccgtgatgca tgaggctctg
1380cacaaccact acacacagaa gagcctctcc ctgtctctgg gtaaatgagg atcc
143422723DNAArtificialHumanized sequence 22gaattcgcca ccatggacat
gagggtcccc gctcagctcc tggggctcct gctactctgg 60ctccgaggtg ccagatgtga
catccagatg acccagtctc catcctccct gtctgcatct 120gtaggagaca
gagtcaccat cacttgccga gcaagtgaga atatttacag ttatttagca
180tggtatcagc agaaaccagg gaaagcccct aagctcctga tctataatgc
aaaaacctta 240gcagaagggg tcccatcaag gttcagtggc agtggatctg
ggacagattt cactctcacc 300atcagcagtc tgcaacctga agattttgca
acttactact gtcaacatca ctatggtact 360cctcggacgt tcggcggagg
gaccaaggtg gagatcaaac ggactgtggc ggcgccatct 420gtcttcatct
tcccgccatc tgatgagcag ttgaaatctg gtaccgctag cgttgtgtgc
480ctgctgaata acttctatcc cagagaggcc aaagtacagt ggaaggtgga
taacgccctc 540caatcgggta actcccagga gagtgtcaca gagcaggaca
gcaaggacag cacctacagc 600ctcagcagca ccctgacgct gagcaaagca
gactacgaga aacacaaagt ctacgcctgc 660gaagtcaccc atcagggcct
gagctcgccc gtcacaaaga gcttcaacag gggagagtgt 720tag
723231428DNAArtificialHumanized sequence 23gaattcgcca ccatggactg
gacctggagc atccttttct tggtggcagc agcaacaggt 60gcccactccc aggttcagct
ggtgcagtct ggagctgagg tgaagaagcc tggggcctca 120gtgaaggtct
cctgcaaggc ttctggttac acctttacca gctactggat gaactgggtg
180cgacaggccc ctggacaagg gcttgagtgg atgggaagga ttgatcctta
cgatagtgaa 240actcactatg cacagaagct ccagggcaga gtcaccatga
ccacagacac atccacgagc 300acagcctaca tggagctgag gagcctgaga
tctgacgaca cggccgtgta ttactgtgcg 360agagggggct atgatttcga
cgtaggaact ctctactggt tcttcgatgt ctggggccaa 420gggacaacgg
tcaccgtctc ttcagctagc accaagggcc catccgtctt ccccctggcg
480ccctgctcca ggagcacctc cgagagcaca gccgccctgg gctgcctggt
caaggactac 540ttccccgaac cggtgacggt gtcgtggaac tcaggcgccc
tgaccagcgg cgtgcacacc 600ttcccggctg tcctacagtc ctcaggactc
tactccctca gcagcgtggt gaccgtgccc 660tccagcagct tgggcacgaa
gacctacacc tgcaacgtag atcacaagcc cagcaacacc 720aaggtggaca
agagagttga gtccaaatat ggtcccccat gcccaccatg cccagcacct
780gagttcctgg ggggaccatc agtcttcctg ttccccccaa aacccaagga
cactctcatg 840atctcccgga cccctgaggt cacgtgcgtg gtggtggacg
tgagccagga agaccccgag 900gtccagttca actggtacgt ggatggcgtg
gaggtgcata atgccaagac aaagccgcgg 960gaggagcagt tcaacagcac
gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 1020tggctgaacg
gcaaggagta caagtgcaag gtctccaaca aaggcctccc gtcctccatc
1080gagaaaacca tctccaaagc caaagggcag ccccgagagc cacaggtgta
caccctgccc 1140ccatcccagg aggagatgac caagaaccag gtcagcctga
cctgcctggt caaaggcttc 1200taccccagcg acatcgccgt ggagtgggag
agcaatgggc agccggagaa caactacaag 1260accacgcctc ccgtgctgga
ctccgacggc tccttcttcc tctacagcag gctaaccgtg 1320gacaagagca
ggtggcagga ggggaatgtc ttctcatgct ccgtgatgca tgaggctctg
1380cacaaccact acacacagaa gagcctctcc ctgtctctgg gtaaatga
142824124PRTArtificialHumanized sequence 24Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met Asn Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile
Asp Pro Tyr Asp Ser Glu Thr His Tyr Ser Gln Lys Phe 50 55 60Lys Asp
Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Gly Tyr Asp Phe Asp Val Gly Thr Leu Tyr Trp Phe
Phe 100 105 110Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115
12025124PRTArtificialHumanized sequence 25Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met Asn Trp
Val Lys Gln Ala Pro Gly Gln Gly Leu Gln Trp Ile 35 40 45Gly Arg Ile
Asp Pro Tyr Asp Ser Glu Thr His Tyr Ser Gln Lys Phe 50 55 60Lys Asp
Lys Ala Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Gly Tyr Asp Phe Asp Val Gly Thr Leu Tyr Trp Phe
Phe 100 105 110Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115
12026124PRTArtificialHumanized sequence 26Glu Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Glu1 5 10 15Ser Leu Arg Ile Ser
Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30Trp Met Asn Trp
Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Arg Ile
Asp Pro Tyr Asp Ser Glu Thr His Tyr Ser Pro Ser Phe 50 55 60Gln Gly
His Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr65 70 75
80Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
85 90 95Ala Arg Gly Gly Tyr Asp Phe Asp Val Gly Thr Leu Tyr Trp Phe
Phe 100 105 110Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115
12027124PRTArtificialHumanized sequence 27Glu Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Glu1 5 10 15Ser Leu Lys Ile Ser
Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30Trp Met Asn Trp
Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Arg Ile
Asp Pro Tyr Asp Ser Glu Thr His Tyr Ser Pro Ser Phe 50 55 60Gln Gly
Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr65 70 75
80Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
85 90 95Ala Arg Gly Gly Tyr Asp Phe Asp Val Gly Thr Leu Tyr Trp Phe
Phe 100 105 110Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115
12028124PRTArtificialHumanized sequence 28Glu Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Thr Val Lys Ile Ser
Cys Lys Val Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met Asn Trp
Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Arg Ile
Asp Pro Tyr Asp Ser Glu Thr His Tyr Ala Glu Lys Phe 50 55 60Gln Gly
Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Thr Gly Gly Tyr Asp Phe Asp Val Gly Thr Leu Tyr Trp Phe
Phe 100 105 110Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115
12029124PRTArtificialHumanized sequence 29Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met Asn Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile
Asp Pro Tyr Asp Ser Glu Thr His Tyr Ala Gln Lys Phe 50 55 60Gln Gly
Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Gly Tyr Asp Phe Asp Val Gly Thr Leu Tyr Trp Phe
Phe 100 105 110Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115
120
* * * * *
References