U.S. patent application number 11/841530 was filed with the patent office on 2008-10-09 for immunoglobulin variants outside the fc region.
This patent application is currently assigned to Xencor, Inc.. Invention is credited to Sher Bahadur Karki, Gregory Alan Lazar.
Application Number | 20080248028 11/841530 |
Document ID | / |
Family ID | 34972142 |
Filed Date | 2008-10-09 |
United States Patent
Application |
20080248028 |
Kind Code |
A1 |
Lazar; Gregory Alan ; et
al. |
October 9, 2008 |
Immunoglobulin Variants Outside the Fc Region
Abstract
The present invention relates to antibody variants outside the
Fc region that alter binding affinity to one or more effector
ligands, methods for their generation, and their therapeutic
application.
Inventors: |
Lazar; Gregory Alan; (Los
Angeles, CA) ; Karki; Sher Bahadur; (Pasadena,
CA) |
Correspondence
Address: |
MORGAN, LEWIS & BOCKIUS, LLP
ONE MARKET SPEAR STREET TOWER
SAN FRANCISCO
CA
94105
US
|
Assignee: |
Xencor, Inc.
Monrovia
CA
|
Family ID: |
34972142 |
Appl. No.: |
11/841530 |
Filed: |
August 20, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11090981 |
Mar 24, 2005 |
7276585 |
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11841530 |
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60556353 |
Mar 24, 2004 |
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60573302 |
May 21, 2004 |
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60585328 |
Jul 1, 2004 |
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60586837 |
Jul 9, 2004 |
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60599741 |
Aug 6, 2004 |
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60607398 |
Sep 2, 2004 |
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60614944 |
Sep 29, 2004 |
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60619409 |
Oct 14, 2004 |
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Current U.S.
Class: |
424/133.1 ;
424/130.1; 424/172.1; 530/387.1; 530/387.3 |
Current CPC
Class: |
C07K 2317/92 20130101;
C07K 2317/56 20130101; C07K 16/32 20130101; C07K 2317/567 20130101;
A61P 43/00 20180101; C07K 16/2893 20130101; C07K 2317/24 20130101;
C07K 2317/732 20130101; C07K 16/00 20130101; C07K 2317/52 20130101;
C07K 2317/53 20130101 |
Class at
Publication: |
424/133.1 ;
530/387.1; 530/387.3; 424/130.1; 424/172.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/18 20060101 C07K016/18; A61P 43/00 20060101
A61P043/00 |
Claims
1. An antibody or immunoadhesin comprising at least one amino acid
modification in the hinge region said modification selected from
the group consisting of D221K, D221Y, T223E, T223K, H224E, T225E,
T225K, T225W, P227K, P227G, L234D, L234E, L234Q, L234V, L234A,
L234M, L234G, L235D, L235T, L235Y, G236D, G236E, G236S, G236T, and
G236A, wherein numbering is according to the EU numbering
scheme.
2. An antibody or immunoadhesin according to claim 1, wherein said
antibody or immunoadhesin further comprises an amino acid
modification in the Fc region.
3. An antibody or immunoadhesin according to claim 1, wherein said
antibody or immunoadhesin further comprises an amino acid
substitution at a position selected from the group consisting of
240, 241, 243, 244, 245, 247, 256, 262, 263, 264, 265, 266, 267,
269, 270, 290, 296, 297, 298, 299, 312, 313, 322, 325, 326, 327,
328, 329, 330, 331, 332, 333, 334 and 339, wherein numbering is
according to the EU index.
4. An antibody or immunoadhesin according to claim 1, wherein said
antibody or immunoadhesin further comprises at least one an amino
acid substitution at a position selected from the group consisting
of 239 and 332, wherein numbering is according to the EU index.
5. An antibody or immunoadhesin according to claim 1, wherein said
antibody or immunoadhesin increases binding affinity to an
Fc.gamma.R as compared to said parent polypeptide.
6. An antibody or immunoadhesin according to claim 5, wherein said
Fc.gamma.R is Fc.gamma.RIIIa.
7. An antibody or immunoadhesin according to claim 6, wherein said
Fc.gamma.RIIIa is a V158 or F158 allotype of Fc.gamma.RIIIa.
8. An antibody or immunoadhesin according to claim 1, wherein said
antibody or immunoadhesin decreases binding affinity to an
Fc.gamma.R as compared to said parent polypeptide.
9. An antibody or immunoadhesin according to claim 1, wherein said
antibody or immunoadhesin is an antibody.
10. An antibody or immunoadhesin according to claim 9, wherein said
antibody is selected from the group consisting of a human antibody,
a humanized antibody, a monoclonal antibody and an antibody
fragment.
11. An antibody or immunoadhesin according to claim 1 wherein said
antibody or immunoadhesin further comprises an engineered
glycoform.
12. An antibody or immunoadhesin according to claim 1 wherein said
antibody or immunoadhesin has specificity for a target antigen
selected from the group consisting of CD19, CD20, CD22, CD30, CD33,
CD40, CD40L, CD52, Her2/neu, EGFR, EpCAM, MUC1, GD3, CEA, CA 125,
HLA-DR, TNFalpha, MUC18, prostate specific membrane antigen (PMSA)
and VEGF.
13. A composition comprising the antibody or immunoadhesin
according to claim 1, further comprising a pharmaceutically
acceptable carrier.
14. A method of treating a mammal in need of said treatment,
comprising administering an antibody or immunoadhesin of a parent
Fc polypeptide, said antibody or immunoadhesin comprising an amino
acid substitution selected from the group consisting of D221K,
D221Y, T223E, T223K, H224E, T225E, T225K, T225W, P227K, P227G,
L234D, L234E, L234Q, L234V, L234A, L234M, L234G, L235D, L235T,
L235Y, G236D, G236E, G236S, G236T, and G236A, and wherein numbering
is according to the EU index.
15. A method according to claim 14, wherein said antibody or
immunoadhesin further comprises an amino acid substitution at a
position selected from the group consisting of 240, 241, 243, 244,
245, 247, 256, 262, 263, 264, 265, 266, 267, 269, 270, 290, 296,
297, 298, 299, 312, 313, 322, 325, 326, 327, 328, 329, 330, 331,
332, 333, 334 and 339.
16. A method according to claim 14, wherein said antibody or
immunoadhesin increases binding affinity to an Fc.gamma.R as
compared to said parent polypeptide.
17. A method according to claim 16, wherein said Fc.gamma.R is
Fc.gamma.RIIIa.
18. A method according to claim 17, wherein said Fc.gamma.RIIIa is
a V158 or F158 allotype of Fc.gamma.RIIIa.
19. A method according to claim 14, wherein said antibody or
immunoadhesin decreases binding affinity to an Fc.gamma.R as
compared to said parent polypeptide.
20. A method according to claim 14, wherein said antibody or
immunoadhesin is an antibody.
21. A method according to claim 14, wherein said antibody is
selected from the group consisting of a human antibody, a humanized
antibody, a monoclonal antibody and an antibody fragment.
22. A method according to claim 14, wherein said antibody or
immunoadhesin further comprises an engineered glycoform.
23. A method according to claim 14, wherein said antibody or
immunoadhesin has specificity for a target antigen selected from
the group consisting of CD19, CD20, CD22, CD30, CD33, CD40, CD40L,
CD52, Her2/neu, EGFR, EpCAM, MUC1, GD3, CEA, CA 125, HLA-DR,
TNFalpha, MUC18, prostate specific membrane antigen (PMSA) and
VEGF.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/090,981, filed Mar. 24, 2005, and U.S.
application Ser. No. 11/090,981 claims the benefit of priority
under 35 U.S.C .sctn. 119(e) to the following U.S. Provisional
Application Serial Nos.: U.S. Ser. No. 60/556,353, filed Mar. 24,
2004; U.S. Ser. No. 60/573,302, filed May 21, 2004; U.S. Ser. No.
60/585,328, filed Jul. 1, 2004; U.S. Ser. No. 60/586,837, filed
Jul. 9, 2004; U.S. Ser. No. 60/599,741, filed Aug. 6, 2004; U.S.
Ser. No. 60/607,398, filed Sep. 2, 2004; U.S. Ser. No. 60/614,944,
filed Sep. 29, 2004; and U.S. Ser. No. 60/619,409, filed, Oct. 14,
2004, all hereby incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to novel optimized antibody
variants wherein one or more amino acid modifications are made
outside the Fc region that alter binding of the antibody to one or
more effector ligands, and their application, particularly for
therapeutic purposes.
BACKGROUND OF THE INVENTION
[0003] Antibodies are immunological proteins that bind a specific
antigen. In most mammals, including humans and mice, antibodies are
constructed from paired heavy and light polypeptide chains. Each
chain is made up of individual immunoglobulin (Ig) domains, and
thus the generic term immunoglobulin is used for such proteins.
Each chain is made up of two distinct regions, referred to as the
variable and constant regions. The light and heavy chain variable
regions show significant sequence diversity between antibodies, and
are responsible for binding the target antigen. The constant
regions show less sequence diversity, and are responsible for
binding a number of natural proteins to elicit important
biochemical events. In humans there are five different isotypes or
classes of antibodies including IgA (which includes subclasses IgA1
and IgA2), IgD, IgE, IgG (which includes subclasses IgG1, IgG2,
IgG3, and IgG4), and IgM. The distinguishing features between these
antibody isotypes are their constant regions, although subtler
differences may exist in the V region. FIG. 1 shows an IgG1
antibody, used here as an example to describe the general
structural features of immunoglobulins. IgG antibodies are
tetrameric proteins composed of two heavy chains and two light
chains. The IgG heavy chain is composed of four immunoglobulin
domains linked from N- to C-terminus in the order VH-CH1-CH2-CH3,
referring to the heavy chain variable domain, heavy chain constant
domain 1, heavy chain constant domain 2, and heavy chain constant
domain 3 respectively (also referred to as
VH-C.gamma.1-C.gamma.2-C.gamma.3, referring to the heavy chain
variable domain, constant gamma 1 domain, constant gamma 2 domain,
and constant gamma 3 domain respectively for the IgG class). The
IgG light chain is composed of two immunoglobulin domains linked
from N- to C-terminus in the order VL-CL, referring to the light
chain variable domain and the light chain constant domain
respectively.
[0004] The variable region of an antibody contains the antigen
binding determinants of the molecule, and thus determines the
specificity of an antibody for its target antigen. The variable
region is so named because it is the most distinct in sequence from
other antibodies within the same isotype. The majority of sequence
variability occurs in the complementarity determining regions
(CDRs). There are 6 CDRs total, three each per heavy and light
chain, designated VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and
VL CDR3. The variable region outside of the CDRs is referred to as
the framework (FR) region. Although not as diverse as the CDRs,
sequence variability does occur in the FR region between different
antibodies. Overall, this characteristic architecture of antibodies
provides a stable scaffold (the FR region) upon which substantial
antigen binding diversity (the CDRs) can be explored by the immune
system to obtain specificity for a broad array of antigens. A
number of high resolution structures are available for a variety of
variable region fragments from different organisms, some unbound
and some in complex with antigen. The sequence and structural
features of antibody variable regions are well characterized (Morea
et al., 1997, Biophys Chem 68:9-16; Morea et al., 2000, Methods
20:267-279), and the conserved features of antibodies have enabled
the development of a wealth of antibody engineering techniques
(Maynard et al., 2000, Annu Rev Biomed Eng 2:339-376). For example,
it is possible to graft the CDRs from one antibody, for example a
murine antibody, onto the framework region of another antibody, for
example a human antibody. This process, referred to in the art as
humanization, enables generation of less immunogenic antibody
therapeutics from nonhuman antibodies. Fragments comprising the
variable region can exist in the absence of other regions of the
antibody, including for example the antigen binding fragment (Fab)
comprising VH-CH1 and VL-CL, the variable fragment (Fv) comprising
VH and VL, the single chain variable fragment (scFv) comprising VH
and VL linked together in the same chain, as well as a variety of
other variable region fragments (Little et al., 2000, Immunol Today
21:364-370).
[0005] The Fc region of an antibody interacts with a number of Fc
receptors and ligands, imparting an array of important functional
capabilities referred to as effector functions. For IgG the Fc
region, as shown in FIG. 1, comprises Ig domains CH2 and CH3. An
important family of Fc receptors for the IgG isotype are the Fc
gamma receptors (Fc.gamma.Rs). These receptors mediate
communication between antibodies and the cellular arm of the immune
system (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220;
Ravetch et al., 2001, Annu Rev Immunol 19:275-290). In humans this
protein family includes Fc.gamma.RI (CD64), including isoforms
Fc.gamma.RIa, Fc.gamma.RIb, and Fc.gamma.RIc; Fc.gamma.RII (CD32),
including isoforms Fc.gamma.RIIa (including allotypes H131 and
R131), Fc.gamma.RIIb (including Fc.gamma.RIIb-1 and
Fc.gamma.RIIb-2), and Fc.gamma.RIIc; and Fc.gamma.RIII (CD16),
including isoforms Fc.gamma.RIIIa (including allotypes V158 and
F158) and Fc.gamma.RIIIb (including allotypes Fc.gamma.RIIIb-NA1
and Fc.gamma.RIIIb-NA2) (Jefferis et al., 2002, Immunol Lett
82:57-65). These receptors typically have an extracellular domain
that mediates binding to Fc, a membrane spanning region, and an
intracellular domain that may mediate some signaling event within
the cell. These receptors are expressed in a variety of immune
cells including monocytes, macrophages, neutrophils, dendritic
cells, eosinophils, mast cells, platelets, B cells, large granular
lymphocytes, Langerhans' cells, natural killer (NK) cells, and
.gamma..delta. T cells. Formation of the Fc/Fc.gamma.R complex
recruits these effector cells to sites of bound antigen, typically
resulting in signaling events within the cells and important
subsequent immune responses such as release of inflammation
mediators, B cell activation, endocytosis, phagocytosis, and
cytotoxic attack. The ability to mediate cytotoxic and phagocytic
effector functions is a potential mechanism by which antibodies
destroy targeted cells. The cell-mediated reaction wherein
nonspecific cytotoxic cells that express Fc.gamma.Rs recognize
bound antibody on a target cell and subsequently cause lysis of the
target cell is referred to as antibody dependent cell-mediated
cytotoxicity (ADCC) (Raghavan et al., 1996, Annu Rev Cell Dev Biol
12:181-220; Ghetie et al., 2000, Annu Rev Immunol 18:739-766;
Ravetch et al., 2001, Annu Rev Immunol 19:275-290). The
cell-mediated reaction wherein nonspecific cytotoxic cells that
express Fc.gamma.Rs recognize bound antibody on a target cell and
subsequently cause phagocytosis of the target cell is referred to
as antibody dependent cell-mediated phagocytosis (ADCP). A number
of structures have been solved of the extracellular domains of
human Fc.gamma.Rs, including Fc.gamma.RIIa (pdb accession code
1H9V)(Sondermann et al., 2001, J Mol Biol 309:737-749) (pdb
accession code 1 FCG)(Maxwell et al., 1999, Nat Struct Biol
6:437-442), Fc.gamma.RIIb (pdb accession code 2FCB)(Sondermann et
al., 1999, Embo J 18:1095-1103); and Fc.gamma.RIIIb (pdb accession
code 1 E4J)(Sondermann et al., 2000, Nature 406:267-273.). All
Fc.gamma.Rs bind the same region on Fc, at the N-terminal end of
the C.gamma.2 domain and the preceding hinge, shown in FIG. 2. This
interaction is well characterized structurally (Sondermann et al.,
2001, J Mol Biol 309:737-749), and several structures of the human
Fc bound to the extracellular domain of human Fc.gamma.RIIIb have
been solved (pdb accession code 1 E4K) (Sondermann et al., 2000,
Nature 406:267-273.) (pdb accession codes 1IIS and 1IIX)(Radaev et
al., 2001, J Biol Chem 276:16469-16477), as well as has the
structure of the human IgE Fc/Fc.epsilon.RI.alpha. complex (pdb
accession code 1F6A)(Garman et al., 2000, Nature 406:259-266).
[0006] The different IgG subclasses have different affinities for
the Fc.gamma.Rs, with IgG1 and IgG3 typically binding substantially
better to the receptors than IgG2 and IgG4 (Jefferis et al., 2002,
Immunol Lett 82:57-65). All Fc.gamma.Rs bind the same region on IgG
Fc, yet with different affinities: the high affinity binder
Fc.gamma.RI has a Kd for IgG1 of 10.sup.-8 M.sup.-1, whereas the
low affinity receptors Fc.gamma.RII and Fc.gamma.RIII generally
bind at 10.sup.-6 and 10.sup.-5 respectively. The extracellular
domains of Fc.gamma.RIIIa and Fc.gamma.RIIIb are 96% identical,
however Fc.gamma.RIIIb does not have a intracellular signaling
domain. Furthermore, whereas Fc.gamma.RI, Fc.gamma.RIIa/c, and
Fc.gamma.RIIIa are positive regulators of immune complex-triggered
activation, characterized by having an intracellular domain that
has an immunoreceptor tyrosine-based activation motif (ITAM),
Fc.gamma.RIIb has an immunoreceptor tyrosine-based inhibition motif
(ITIM) and is therefore inhibitory. Thus the former are referred to
as activation receptors, and Fc.gamma.RIIb is referred to as an
inhibitory receptor. The receptors also differ in expression
pattern and levels on different immune cells. Yet another level of
complexity is the existence of a number of Fc.gamma.R polymorphisms
in the human proteome. A particularly relevant polymorphism with
clinical significance is V158/F158 Fc.gamma.RIIIa. Human IgG1 binds
with greater affinity to the V158 allotype than to the F158
allotype. This difference in affinity, and presumably its effect on
ADCC and/or ADCP, has been shown to be a significant determinant of
the efficacy of the anti-CD20 antibody rituximab (Rituxan.RTM., a
registered trademark of IDEC Pharmaceuticals Corporation). Patients
with the V158 allotype respond favorably to Rituxan treatment;
however, patients with the lower affinity F158 allotype respond
poorly (Cartron et al., 2002, Blood 99:754-758). Approximately
10-20% of humans are V158/V158 homozygous, 45% are V158/F158
heterozygous, and 35-45% of humans are F158/F158 homozygous
(Lehrnbecher et al., 1999, Blood 94:4220-4232; Cartron et al.,
2002, Blood 99:754-758). Thus 80-90% of humans are poor responders,
that is they have at least one allele of the F158
Fc.gamma.RIIIa.
[0007] Although IgG is the principal antibody isoform used for
therapeutic applications, other isoforms have therapeutic
potential. For example, a growing body of evidence suggests that
interaction of IgA Fc with its Fc receptor Fc.alpha.RI (CD89)
elicits a plethora of effector functions (Egmond et al., 2001,
Trends in Immunology, 22: 205-210). IgA is the most prominent
isotype of antibodies at mucosal surfaces, and the second most
predominant isotype in human serum. A number of recent studies
using bispecific antibody fragment constructs that simultaneously
target a cancer antigen and Fc.alpha.RI indicate that engagement of
Fc.alpha.RI can result in cell-mediated tumor cell killing
(Stockmeyer et al., 2000, J. Immunol. 165: 5954-5961; Stockmeyer et
al., 2001, J. Immunol. Methods 248: 103-111; Sundarapandiyan et
al., 2001, J. Immunol. Methods 248: 113-123; dDechant et al., 2002,
Blood 100: 4574-80; (van Egmond et al., 2001, Cancer Research 61:
4055-4060). The structure of the extracellular domain of
Fc.alpha.RI has recently been solved (Ding et al., 2003, J. Biol.
Chem. 278: 27966-27970), as has the receptor in complex with IgA Fc
(Herr et al., 2003, Nature 423: 614-620), and the interface has
been characterized with mutagenesis (Wines et al., 1999, J.
Immunol., 162: 2146-2153; Wines et al., 2001, J. Immunol. 166:
1781-1789). Fc.alpha.RI binds to IgA Fc at a site between the CH2
and CH3 domains, and thus despite substantial structural homology
between gamma and alpha Fc and Fc.gamma.Rs, the IgA/Fc.alpha.RI
interaction is structurally distinct on Fc from the IgG/Fc.gamma.R
interaction.
[0008] A site on Fc that is overlapping but separate from the
Fc.gamma.R binding site serves as the interface for the complement
protein C1q (shown in FIG. 1). In the same way that Fc/Fc.gamma.R
binding mediates ADCC, Fc/C1q binding mediates complement dependent
cytotoxicity (CDC). C1q forms a complex with the serine proteases
C1r and C1s to form the C1 complex. C1q is capable of binding six
antibodies, although binding to two IgGs is sufficient to activate
the complement cascade. Similar to Fc interaction with Fc.gamma.Rs,
different IgG subclasses have different affinity for C1q, with IgG1
and IgG3 typically binding substantially better to the Fc.gamma.Rs
than IgG2 and IgG4 (Jefferis et al., 2002, Immunol Lett 82:57-65).
The structure of human C1q has been solved (Gaboriaud et al., 2003,
J Biol Chem 278:46974-46982). There is currently no structure
available for the Fc/C1q complex; however, mutagenesis studies have
mapped the binding site on human IgG for C1q to a region involving
residues D270, K322, K326, P329, and P331, and E333 (Idusogie et
al., 2000, J Immunol 164:4178-4184; Idusogie et al., 2001, J
Immunol 166:2571-2575).
[0009] A site on Fc between the CH2 and CH3 domains, shown in FIG.
1, mediates interaction with the neonatal receptor FcRn, the
binding of which recycles endocytosed antibody from the endosome
back to the bloodstream (Raghavan et al., 1996, Annu Rev Cell Dev
Biol 12:181-220; Ghetie et al., 2000, Annu Rev Immunol 18:739-766).
This process, coupled with preclusion of kidney filtration due to
the large size of the full length molecule, results in favorable
antibody serum half-lives ranging from one to three weeks. Binding
of Fc to FcRn also plays a key role in antibody transport. The
binding site for FcRn on Fc is also the site at which the bacterial
proteins A and G bind. The tight binding by these proteins is
typically exploited as a means to purify antibodies by employing
protein A or protein G affinity chromatography during protein
purification. Thus the fidelity of this region on Fc is important
for both the clinical properties of antibodies and their
purification. Available structures of the rat Fc/FcRn complex
(Martin et al., 2001, Mol Cell 7:867-877), and of the complexes of
Fc with proteins A and G (Deisenhofer, 1981, Biochemistry
20:2361-2370; Sauer-Eriksson et al., 1995, Structure 3:265-278;
Tashiro et al., 1995, Curr Opin Struct Biol 5:471-481) provide
insight into the interaction of Fc with these proteins.
[0010] A key feature of the Fc region is the conserved N-linked
glycosylation that occurs at N297, shown in FIG. 1. This
carbohydrate, or oligosaccharide as it is sometimes referred, plays
a critical structural and functional role for the antibody, and is
one of the principle reasons that antibodies must be produced using
mammalian expression systems. The structural purpose of this
carbohydrate may be to stabilize or solubilize Fc, determine a
specific angle or level of flexibility between the CH3 and CH2
domains, keep the two CH2 domains from aggregating with one another
across the central axis, or a combination of these. Efficient Fc
binding to Fc.gamma.R and C1q require this modification, and
alterations in the composition of the N297 carbohydrate or its
elimination affect binding to these proteins (Umana et al., 1999,
Nat Biotechnol 17:176-180; Davies et al., 2001, Biotechnol Bioeng
74:288-294; Mimura et al., 2001, J Biol Chem 276:45539-45547.;
Radaev et al., 2001, J Biol Chem 276:16478-16483; Shields et al.,
2001, J Biol Chem 276:6591-6604; Shields et al., 2002, J Biol Chem
277:26733-26740; Simmons et al., 2002, J Immunol Methods
263:133-147). Yet the carbohydrate makes little if any specific
contact with Fc.gamma.Rs (Radaev et al., 2001, J Biol Chem
276:16469-16477), indicating that the functional role of the N297
carbohydrate in mediating Fc/Fc R binding is via the structural
role it plays in determining the Fc conformation. This is supported
by a collection of crystal structures of four different Fc
glycoforms, which show that the composition of the oligosaccharide
impacts the conformation of CH2 and as a result the Fc/Fc.gamma.R
interface (Krapp et al., 2003, J Mol Biol 325:979-989).
[0011] The features of antibodies discussed above--specificity for
target, ability to mediate immune effector mechanisms, and long
half-life in serum--make antibodies powerful therapeutics.
Monoclonal antibodies are used therapeutically for the treatment of
a variety of conditions including cancer, inflammation, and
cardiovascular disease. There are currently over ten antibody
products on the market and hundreds in development. Despite such
widespread application, antibodies are not optimized for clinical
use. A significant deficiency of antibodies is their suboptimal
anticancer potency. This and other shortcomings of antibodies are
addressed by the present invention.
[0012] There are a number of possible mechanisms by which
antibodies destroy tumor cells, including anti-proliferation via
blockage of needed growth pathways, intracellular signaling leading
to apoptosis, enhanced down regulation and/or turnover of
receptors, CDC, ADCC, ADCP, and promotion of an adaptive immune
response (Cragg et al., 1999, Curr Opin Immunol 11:541-547; Glennie
et al., 2000, Immunol Today 21:403-410). Anti-tumor efficacy can be
due to a combination of these mechanisms, and their relative
importance in clinical therapy appears to be cancer dependent.
Despite this arsenal of anti-tumor weapons, the potency of
antibodies as anti-cancer agents is unsatisfactory, particularly
given their high cost. Patient tumor response data show that
monoclonal antibodies provide only a small improvement in
therapeutic success over normal single-agent cytotoxic
chemotherapeutics. For example, just half of all relapsed low-grade
non-Hodgkin's lymphoma patients respond to the anti-CD20 antibody
Rituxan (McLaughlin et al., 1998, J Clin Oncol 16:2825-2833). Of
166 clinical patients, 6% showed a complete response and 42% showed
a partial response, with median response duration of approximately
12 months. Trastuzumab (Herceptin.RTM., a registered trademark of
Genentech), an anti-HER2/neu antibody for treatment of metastatic
breast cancer, has less efficacy. The overall response rate using
Herceptin for the 222 patients tested was only 15%, with 8 complete
and 26 partial responses and a median response duration and
survival of 9 to 13 months (Cobleigh et al., 1999, J Clin Oncol
17:2639-2648). Currently for anticancer therapy, any small
improvement in mortality rate defines success. Thus there is a
significant need to enhance the capacity of antibodies to destroy
targeted cancer cells.
[0013] A promising means for enhancing the anti-tumor potency of
antibodies is via enhancement of their ability to mediate cytotoxic
effector functions such as ADCC, ADCP, and CDC. The importance of
Fc.gamma.R-mediated effector functions for the anti-cancer activity
of antibodies has been demonstrated in mice (Clynes et al., 1998,
Proc Natl Acad Sci USA 95:652-656; Clynes et al., 2000, Nat Med
6:443-446), and the affinity of interaction between Fc and certain
Fc.gamma.Rs correlates with targeted cytotoxicity in cell-based
assays (Shields et al., 2001, J Biol Chem 276:6591-6604; Presta et
al., 2002, Biochem Soc Trans 30:487-490; Shields et al., 2002, J
Biol Chem 277:26733-26740). Additionally, a correlation has been
observed between clinical efficacy in humans and their allotype of
high (V158) or low (F158) affinity polymorphic forms of
Fc.gamma.RIIIa (Cartron et al., 2002, Blood 99:754-758). Together
these data suggest that an antibody that is optimized for binding
to certain Fc.gamma.Rs may better mediate effector functions and
thereby destroy cancer cells more effectively in patients. The
balance between activating and inhibiting receptors is an important
consideration, and optimal effector function may result from an
antibody that has enhanced affinity for activation receptors, for
example Fc.gamma.RI, Fc.gamma.RIIa/c, and Fc.gamma.RIIIa, yet
reduced affinity for the inhibitory receptor Fc.gamma.RIIb.
Furthermore, because Fc.gamma.Rs can mediate antigen uptake and
processing by antigen presenting cells, enhanced Fc.gamma.R
affinity may also improve the capacity of antibody therapeutics to
elicit an adaptive immune response. Fc variants have been
successfully engineered with selectively enhanced binding to
Fc.gamma.Rs, and furthermore these Fc variants provide enhanced
potency and efficacy in cell-based effector function assays. See
for example U.S. Ser. No. 10/672,280, U.S. Ser. No. 10/822,231,
entitled "Optimized Fc Variants and Methods for their Generation",
U.S. Ser. No. 60/627,774, entitled "Optimized Fc Variants", and
U.S. Ser. No. 60/642,477, entitled "Improved Fc Variants", and
references cited therein.
[0014] All research on engineering antibodies to enhance effector
function has focused on the Fc region because it comprises the
binding sites for Fc.gamma.Rs and C1q. The present invention
describes the concept that determinants of effector ligand binding
and effector function reside not only in the Fc region, but also in
the Fab and hinge regions of an antibody. The present invention
describes methods by which to generate Fab and hinge variants, and
provides a series of novel engineered immunoglobulin variants in
the VL, VH, JL, JH, CL, CH1, and hinge regions that provide altered
and optimized effector ligand binding properties. Based on the
documented relationship described above between affinity and
specificity of antibodies for effector ligands, their behavior in
cell based effector function assays, and their clinical behavior in
vivo, engineered Fab and hinge variants that modulate binding to
effector ligands may provide optimal clinical properties.
SUMMARY OF THE INVENTION
[0015] The present invention provides antibody variants outside the
Fc region that are optimized for a number of therapeutically
relevant properties.
[0016] It is an object of the present invention to provide an
antibody variant, wherein said antibody variant comprises one or
more amino acid modifications outside the Fc region of the parent
antibody, wherein said modification alters the affinity of said
antibody for one or more effector ligands.
[0017] In one embodiment, the antibody variant of the present
invention comprises one or amino acid modifications at a position
in the VL region. In one embodiment, the antibody variant comprises
one or amino acid modifications at a position in the JL region. In
an alternate embodiment, the antibody variant comprises one or more
amino acids at a position in the VH region. In one embodiment, the
antibody variant comprises one or more amino acids at a position in
the JH region. In an alternate embodiment, the antibody variant
comprises one or amino acid modifications at a position in the CL
region. In an alternate embodiment, the antibody variant comprises
one or more amino acids at a position in the CH1 region. In an
alternate embodiment, the antibody variant comprises one or amino
acid modifications at a position in the hinge region.
[0018] In one embodiment, the antibody variant of the present
invention variant binds with greater affinity to one or more
effector ligands relative to the parent antibody. The increase in
affinity can range from about 1.5, 2 or 3-fold of wild-type (or
parent), or greater. In an alternate embodiment, the antibody
variant binds with reduced affinity to one or more effector ligands
relative to the parent antibody.
[0019] In one embodiment, the antibody variant of the present
invention binds with greater affinity to one or more Fc.gamma.Rs
relative to the parent antibody. In a preferred embodiment, said
Fc.gamma.R is human Fc.gamma.RIIIa. Additional Fc.gamma.Rs which
may have modified (including increases and decreases) binding
affinity are shown in the tables. In an alternate embodiment, the
antibody variant binds with weaker affinity to one or more
Fc.gamma.Rs relative to the parent antibody. In a preferred
embodiment, said Fc.gamma.R is human Fc.gamma.RIIb. In an alternate
embodiment, the antibody variant binds with greater affinity to one
or more Fc.gamma.Rs but reduced affinity to one or more other
Fc.gamma.Rs relative to the parent antibody.
[0020] In one embodiment, the antibody variant of the present
invention mediates effector function in the presence of effector
cells more effectively than the parent antibody. In one embodiment,
the antibody variant mediates ADCC that is greater than that
mediated by the parent antibody. In an alternate embodiment, the
antibody variant mediates ADCP that is greater than that mediated
by the parent antibody. In an alternate embodiment, the antibody
variant mediates CDC that is greater than that mediated by the
parent antibody.
[0021] It is an object of the present invention to provide an
antibody variant, wherein said antibody variant comprises one or
more substitutions outside the Fc region of the parent antibody,
and further comprises one or more substitutions in the Fc region of
the parent antibody, wherein said antibody variant has altered
affinity for one or more effector ligands; the preferred ligands
are outlined above
[0022] It is an object of the present invention to provide one or
more positions in the Fab region of an antibody, wherein mutation
at said position alters binding of said antibody to one or more
effector ligands. In one embodiment, said position is in the VL
region. In one embodiment, said position is in the JL region. In an
alternate embodiment, said position is in the VH region. In one
embodiment, said position is in the JH region. In an alternate
embodiment, said position is in the CL region. In an alternate
embodiment, said position is in the CH1 region. It is an object of
the present invention to provide one or more substitutions in the
Fab region of an antibody, wherein said substitution alters binding
of said antibody to one or more effector ligands. In one
embodiment, said substitution is in the VL region. In one
embodiment, said substitution is in the JL region. In an alternate
embodiment, said substitution is in the VH region. In one
embodiment, said substitution is in the JH region. In an alternate
embodiment, said substitution is in the CL region. In an alternate
embodiment, said substitution is in the CH1 region.
[0023] It is an additional object of the invention to provide
specific positions and specific modifications of non-Fc regions as
follows: (all numbering is according to the Kabat numbering scheme,
and the parent sequence is human):
[0024] JL region modifications: positions 100, 103, 105 and 106,
and modifications: Q100P, Q100G, Q100K, K103R, K103D, K103L, E105D,
E105K, E105K, E105I, E105I, I106L, and K107E. JH region
modifications: positions 110, 112 and 113; modifications: T110I,
S112D, S113D, and S113R.
[0025] CL region modifications: positions 108, 109, 110, 111, 112,
114, 116, 121, 122, 123, 124, 125, 126, 127, 128, 129, 131, 137,
138, 140, 141, 142, 143, 145, 147, 149, 150, 151, 152, 153, 154,
155, 156, 157, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,
169, 170, 171, 172, 173, 174, 176, 180, 181, 182, 183, 184, 185,
187, 188, 189, 190, 191, 193, 195, 197, 199, 200, 202, 203, 204,
205, 206, 207, 208, 210, 211, 212, and 213, modifications: R108Q,
R108D, R108I, T109P, T109R, T109D, V110K, V110E, V110I, A111K,
A111E, A111L, A112R, A112E, A112Y, S114D, S114K, S114I, F116T,
S121D, D122S, D122R, D122Y, E123R, E123L, Q124E, L125E, L125K,
K126Q, K126D, K126L, S127A, S127D, S127K, G128N, T129K, T129E,
T129I, S131T, N137S, N137K, N138D, N138K, N138L, Y140K, Y140E,
Y140H, P141K, P141E, R142G, R142L, R142D, E143A, E143R, E143L,
K145T, K145D, K145Y, Q147A, Q147E, Q147K, K149D, K149Y, V150A,
D151K, D151I, N152S, N152R, N152L, A153S, A153D, A153H, L154V,
L154E, L154R, Q155K, Q155E, Q155I, S156A, S156D, S156R, G157N,
N158R, N158D, N158L, S159K, S159E, S159L, Q160V, Q160K, E161K,
E161L, S162T, V163T, V163K, V163E, T164Q, E165P, E165K, E165Y,
Q166S, Q166E, Q166M, D167K, D167L, S168Q, S168K, S168Y, K169S,
K169H, K169D, D170N, D170R, D170I, S171N, S171A, S171V, T172K,
T172I, T172E, Y173K, Y173Q, Y173L, S174A, S176T, T180S, T180K,
T180E, L181K, S182T, S182E, S182R, K183P, K183D, K183L, A184E,
A184K, A184Y, D185Q, D185R, D185I, E187K, E187Y, K188S, K188E,
K188Y, H189D, H189K, H189Y, K190R, K190E, K190L, V191S, V191E,
V191R, A193S, A193E, A193K, E195Q, E195K, E195I, T197E, T197K,
T197L, Q199E, Q199K, Q199Y, G200S, S202D, S202R, S202Y, S203D,
S203R, S203L, P204T, V205E, V205K, T206E, T206K, T206I, K207E,
K207L, K207A, S208T, S208E, S208K, N210A, N210E, N210K, R211P,
R211E, R211A, G212T, G212K, G212E, E213R, and E213L.
[0026] CH regions: positions 118, 119, 120, 121, 122, 124, 126,
129, 131, 132, 133, 135, 136, 137, 138, 139, 147, 148, 150, 151,
152, 153, 155, 157, 159, 160, 161, 162, 163, 164, 165, 166, 167,
168, 169, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 183,
187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199,
201, 203, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 216,
217, 218, 219, 221, 222, 223, 224, and 225; modifications: A118K,
A118E, A118Y, S119R, S119E, S119Y, T120R, T120E, T120I, K121E,
K121Y, K121H, G122E, G122R, S124K, S124E, S124Y, F126K, F126D,
A129L, A129D, S131G, S131T, S132D, S132R, S132L, K133R, K133E,
K133L, T135I, T135E, T135K, S136E, S136K, S136I, G137E, G138S,
G138R, G138D, T1391, T139E, T139K, K147A, K147E, D148Y, D148K,
F150L, F150K, F150E, P151A, P151D, E152L, E152K, P153L, P153D,
T155E, T155K, T155I, S157E, S157K, S157Y, N159K, N159D, N159L,
S160K, S160E, S160Y, G161D, A162D, A162K, A162Y, L163R, T164R,
T164E, T164Y, S165D, S165R, S165Y, G166D, V167A, H168L, T169E,
P171G, P171H, A172K, A172L, A172E, V173T, V173D, L174E, L174K,
L174Y, Q175D, Q175L, S176D, S176R, S176L, S177R, S177E, S177Y,
G178D, L179K, L179Y, L179E, Y180K, Y180L, Y180E, S183T, T187I,
T187K, T187E, V188I, P189D, P189G, S190I, S190K, S190E, S191D,
S191R, S191Y, S192N, S192R, S192L, L193F, L193E, G194R, G194D,
T195R, T195D, T195Y, Q196K, Q196D, Q196L, T197R, T197E, T197Y,
Y198L, I199T, I199D, I199K, N201E, N201K, N201L, N203D, N203L,
N203K, K205D, K205L, K205AP206A, P206E, S207K, S207D, N208R, N208E,
N208Y, T209E, T209K, T209Y, K210L, K210E, K210Y, K210A, V211R,
V211E, V211Y, D212Q, D212K, D212H, D212L, D212Y, K213N, K213E,
K213H, K213L, K213Y, K213A, K214N, K214E, K214H, K214L, K214Y,
K214A, E216N, E216K, E216H, E216L, E216Y, P217D, P217H, P217A,
P217V, P217G, K218D, K218E, K218Q, K218T, K218H, K218L, K218Y,
K218A, S219D, S219E, S219Q, S219K, S219T, S219H, S219L, S2191,
S219Y, D221K, D221Y, D221E, D221N, D221Q, D221R, D221S, D221T,
D221H, D221A, D221V, D221L, D221I, D221F, D221M, D221W, D221P,
D221G, K222E, K222Y, K222D, K222N, K222Q, K222R, K222S, K222T,
K222H, K222V, K222L, K222I, K222F, K222M, K222W, K222P, K222G,
K222A, T223D, T223N, T223Q, T223R, T223S, T223H, T223A, T223V,
T223L, T223I, T223F, T223M, T223Y, T223W, T223P, T223G, T223E,
T223K, H224D, H224N, H224Q, H224K, H224R, H224S, H224T, H224V,
H224L, H224I, H224F, H224M, H224W, H224P, H224G, H224E, H224Y,
H224A, T225D, T225N, T225Q, T225R, T225S, T225H, T225A, T225V,
T225L, T225I, T225F, T225M, T225Y, T225P, T225G, T225E, T225K, and
T225W.
[0027] CHI regions: positions: 118, 119, 120 121, 122, 201 and 206;
modifications: A118K, A118E, A118Y, S119E, T120E, K121H, G122E,
N201E, and P206E.
[0028] Hinge regions: positions 221, 223, 224, 225, 227, 234, 235
and 236; modifications D221K, D221Y, T223E, T223K, H224E, T225E,
T225K, T225W, P227K, P227G, L234D, L234E, L234Q, L234V, L234A,
L234M, L234G, L235D, L235T, L235Y, G236D, G236E, G236S, G236T, and
G236A.
[0029] Variable light (VL) regions (based on Herceptin.RTM.
variable region): positions 3, 10, 22, 24, 28, 30, 31, 32, 40, 42,
43, 50, 51, 52, 53, 55, 56, 66, 83, 87, 92, 93, 94, 96, 100, 103,
105, 106, 107, and 108; modifications: Q3K, S10F, T22N, R24K, D28N,
N30D, T31K, A32Y, P40L, K42E, A43S, S50N, A51T, S52N, F53N, Y55Q,
S56T, R66G, F83V, Y87F, Y92I, T93S, T94R, P96R, Q100T, K103A,
E105A, I106L, K107A, R108A, Q100P, Q100G, Q100K, K103R, K103D,
K103L, E105D, E105K, E105I, I106L, K107E, and K107L.
[0030] Variable heavy (VH) regions (based on Herceptin.RTM.
variable region): positions 3, 5, 18, 24, 28, 30, 32, 35, 40, 41,
44, 45, 50, 52, 52a, 52b, 52c, 53, 54, 55, 56, 58, 60, 61, 71, 73,
74, 75, 77, 82b, 89, 95, 97, 98, 99, 100, 100a, 100b, 102, 105,
107, 108, 110, 112, and 113; modifications Q3K, V5L, L18M, A24G,
N28T, K30T, T32F, H35N, A40P, P41A, G44A, L45P, R50F, Y52R, P52aD,
-52bK, -52cA, T53K, N54G, G55Y, Y56T, R58E, A60N, D61P, A71R, T73N,
S74T, K75Q, T77M, S82bT, V89T, W95E, G97H, D98T, G99A, F100A,
Y100aP, A100b-, T107V, L108M, Y102L, Q105E, Q105E, Q105E, Q105L,
S107T, L108T, L108E, L108K, T110K, T110E, T110I, S112D, S112K,
S112Y, S113D, S113R, and S113L.
[0031] In an additional aspect, the invention provides novel
variants, with or without altered affinity, within the human CL
region at positions 108, 109, 110, 111, 112, 114, 116, 121, 122,
123, 124, 125, 126, 127, 128, 129, 131, 137, 138, 140, 141, 142,
143, 145, 147, 149, 150, 151, 152, 153, 154, 155, 156, 157, 159,
160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172,
173, 174, 176, 180, 181, 182, 183, 184, 185, 187, 188, 189, 190,
191, 193, 195, 197, 199, 200, 202, 203, 204, 205, 206, 207, 208,
210, 211, 212, and 213, and specific modifications of R108Q, R108D,
R108I, T109P, T109R, T109D, V110K, V110E, V110I, A111K, A111E, A
111L, A112R, A112E, A112Y, S114D, S114K, S114I, F116T, S121D,
D122S, D122R, D122Y, E123R, E123L, Q124E, L125E, L125K, K126Q,
K126D, K126L, S127A, S127D, S127K, G128N, T129K, T129E, T129I,
S131T, N137S, N137K, N138D, N138K, N138L, Y140K, Y140E, Y140H,
P141K, P141E, R142G, R142L, R142D, E143A, E143R, E143L, K145T,
K145D, K145Y, Q147A, Q147E, Q147K, K149D, K149Y, V150A, D151K,
D151I, N152S, N152R, N152L, A153S, A153D, A153H, L154V, L154E,
L154R, Q155K, Q155E, Q155I, S156A, S156D, S156R, G157N, N158R,
N158D, N158L, S159K, S159E, S159L, Q160V, Q160K, E161K, E161L,
S162T, V163T, V163K, V163E, T164Q, E165P, E165K, E165Y, Q166S,
Q166E, Q166M, D167K, D167L, S168Q, S168K, S168Y, K169S, K169H,
K169D, D170N, D170R, D170I, S171N, S171A, S171V, T172K, T172I,
T172E, Y173K, Y173Q, Y173L, S174A, S176T, T180S, T180K, T180E,
L181K, S182T, S182E, S182R, K183P, K183D, K183L, A184E, A184K,
A184Y, D185Q, D185R, D185I, E187K, E187Y, K188S, K188E, K188Y,
H189D, H189K, H189Y, K190R, K190E, K190L, V191S, V191E, V191R,
A193S, A193E, A193K, E195Q, E195K, E195I, T197E, T197K, T197L,
Q199E, Q199K, Q199Y, G200S, S202D, S202R, S202Y, S203D, S203R,
S203L, P204T, V205E, V205K, T206E, T206K, T206I, K207E, K207L,
K207A, S208T, S208E, S208K, N210A, N210E, N210K, R211P, R211E,
R211A, G212T, G212K, G212E, E213R, and E213L. Similarly, CH
positions include positions 118, 119, 120, 121, 122, 124, 126, 129,
131, 132, 133, 135, 136, 137, 138, 139, 147, 148, 150, 151, 152,
153, 155, 157, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,
169, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 183, 187,
188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 201,
203, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 216, 217,
218, 219, 221, 222, 223, 224, and 225, and modifications at A118K,
A118E, A118Y, S119R, S119E, S119Y, T120R, T120E, T120I, K121E,
K121Y, K121H, G122E, G122R, S124K, S124E, S124Y, F126K, F126D,
A129L, A129D, S131G, S131T, S132D, S132R, S132L, K133R, K133E,
K133L, T135I, T135E, T135K, S136E, S136K, S136I, G137E, G138S,
G138R, G138D, T1391, T139E, T139K, K147A, K147E, D148Y, D148K,
F150L, F150K, F150E, P151A, P151D, E152L, E152K, P153L, P153D,
T155E, T155K, T155I, S157E, S157K, S157Y, N159K, N159D, N159L,
S160K, S160E, S160Y, G161D, A162D, A162K, A162Y, L163R, T164R,
T164E, T164Y, S165D, S165R, S165Y, G166D, V167A, H168L, T169E,
P171G, P171H, A172K, A172L, A172E, V173T, V173D, L174E, L174K,
L174Y, Q175D, Q175L, S176D, S176R, S176L, S177R, S177E, S177Y,
G178D, L179K, L179Y, L179E, Y180K, Y180L, Y180E, S183T, T187I,
T187K, T187E, V188I, P189D, P189G, S190I, S190K, S190E, S191D,
S191R, S191Y, S192N, S192R, S192L, L193F, L193E, G194R, G194D,
T195R, T195D, T195Y, Q196K, Q196D, Q196L, T197R, T197E, T197Y,
Y198L, I199T, I199D, I199K, N201E, N201K, N201L, N203D, N203L,
N203K, K205D, K205L, K205AP206A, P206E, S207K, S207D, N208R, N208E,
N208Y, T209E, T209K, T209Y, K210L, K210E, K210Y, K210A, V211R,
V211E, V211Y, D212Q, D212K, D212H, D212L, D212Y, K213N, K213E,
K213H, K213L, K213Y, K213A, K214N, K214E, K214H, K214L, K214Y,
K214A, E216N, E216K, E216H, E216L, E216Y, P217D, P217H, P217A,
P217V, P217G, K218D, K218E, K218Q, K218T, K218H, K218L, K218Y,
K218A, S219D, S219E, S219Q, S219K, S219T, S219H, S219L, S2191,
S219Y, D221K, D221Y, D221E, D221N, D221Q, D221R, D221S, D221T,
D221H, D221A, D221V, D221L, D221I, D221F, D221M, D221W, D221P,
D221G, K222E, K222Y, K222D, K222N, K222Q, K222R, K222S, K222T,
K222H, K222V, K222L, K222I, K222F, K222M, K222W, K222P, K222G,
K222A, T223D, T223N, T223Q, T223R, T223S, T223H, T223A, T223V,
T223L, T223I, T223F, T223M, T223Y, T223W, T223P, T223G, T223E,
T223K, H224D, H224N, H224Q, H224K, H224R, H224S, H224T, H224V,
H224L, H224I, H224F, H224M, H224W, H224P, H224G, H224E, H224Y,
H224A, T225D, T225N, T225Q, T225R, T225S, T225H, T225A, T225V,
T225L, T225I, T225F, T225M, T225Y, T225P, T225G, T225E, T225K.
[0032] The present invention also provides methods for engineering
optimized antibody variants.
[0033] The present invention provides isolated nucleic acids
encoding the antibody variants described herein. The present
invention provides vectors comprising said nucleic acids,
optionally, operably linked to control sequences. The present
invention provides host cells containing the vectors, and methods
for producing and optionally recovering the antibody variants.
[0034] The present invention provides novel antibodies that
comprise the antibody variants disclosed herein. Said novel
antibodies may find use in a therapeutic product.
[0035] The present invention provides compositions comprising
antibodies that comprise the antibody variants described herein,
and a physiologically or pharmaceutically acceptable carrier or
diluent.
[0036] The present invention contemplates therapeutic and
diagnostic uses for antibodies that comprise the antibody variants
disclosed herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] FIG. 1. Antibody structure and function. Shown is a model of
a full length human IgG1 antibody, modeled using a humanized Fab
structure from pdb accession code 1CE1 (James et al., 1999, J Mol
Biol 289:293-301) and a human IgG1 Fc structure from pdb accession
code 1 DN2 (DeLano et al., 2000, Science 287:1279-1283). The
flexible hinge that links the heavy chain CH1 and CH2 regions is
not shown. IgG1 is a homodimer of heterodimers, made up of two
light chains and two heavy chains. The Ig domains that comprise the
antibody are labeled, and include VL and CL for the light chain,
and VH, CH1, CH2, and CH3 for the heavy chain. The Fab and Fc
regions are labeled. Binding sites for relevant proteins are
labeled, including the antigen binding site in the variable region,
and the binding sites for Fc.gamma.Rs, FcRn, C1q, and proteins A
and G in the Fc region.
[0038] FIG. 2. The Fc/Fc.gamma.RIIIb complex structure 1IIS (Radaev
et al., 2001, J Biol Chem 276:16469-16477). Fc is shown as a gray
ribbon diagram, and Fc.gamma.RIIIb is shown as a black ribbon. The
N297 carbohydrate is shown as black sticks.
[0039] FIGS. 3a-3f. Aligned sequences for the kappa light chain
variable region V.kappa. (FIG. 3a), heavy chain variable region VH
(FIG. 3b), light chain constant region CL (FIG. 3c), IgG CH1 (FIG.
3d), IgG hinge (FIG. 3e), and IgG CH2 and CH3 regions (FIG. 3f).
For the VL and VH sequences in FIGS. 3a and 3b, the sequences of
antibodies Campath-1G, Herceptin, Rituxan, and Erbitux are
provided, along with the sequences of the human germline J.kappa.
and JH segments. For these sequences, position numbering according
to the Kabat and Chothi numbering schemes are provided, and CDRs as
defined according to the two schemes are bolded. Bolded residues in
the Campath-1G and Herceptin sequences illustrate positions and
residues that are different between the two antibodies, as
described in Example 2. Herceptin residue M100d and Campath-1G
residue F100d were aligned sequentially with the JH region and as a
result are not numbered according to Kabat. FIG. 3c shows an
alignment of the C.kappa. and C.lamda. light chain constant
regions, with numbering of the C.kappa. sequence according to the
EU numbering scheme. Differences between the two aligned sequences
are shown in grey. FIGS. 3d-3f show the heavy chain IgG constant
regions, with numbering of the IgG1 sequence according to the EU
numbering scheme. Differences between IgG1 and IgG2, IgG3, and IgG4
are shown in grey. The boundaries of the VL, VH, JL, JH, and CL
regions are defined genetically, whereas the boundaries of the CH1,
hinge, CH2, and CH3 regions are defined structurally, as described
in Example 4. Polymorphisms have been observed at a number of
immunoglobulin positions (for example see Kim et al., 2001, J Mol
Evol 53:1-9), and thus slight differences between the presented
sequence and sequences in the prior art may exist.
[0040] FIG. 4. Binding to human V158 Fc.gamma.RIIIa by Campath and
Herceptin as determined by the AlphaScreen.TM. assay. In the
presence of competitor Campath or Herceptin antibody, a
characteristic inhibition curve is observed as a decrease in
luminescence signal. Phosphate buffer saline (PBS) alone was used
as the negative control. These data were normalized to the maximum
and minimum luminescence signal provided by the baselines at low
and high concentrations of competitor antibody respectively. The
curves represent the fits of the data to a one site competition
model using nonlinear regression.
[0041] FIGS. 5a-5b. Impact of Fc mutations distal to the
Fc/Fc.gamma.R binding site on Fc/Fc.gamma.R affinity. FIG. 5a
provides AlphaScreen data showing enhanced binding of Herceptin Fc
variants E272Y and K274E to human Fc.gamma.RIIIa. The data were
normalized, and the curves represent the fits of the data to a one
site competition model. PBS was used as a negative control. FIG. 5b
shows the Fc/Fc.gamma.RIIIb complex structure 1IIS showing the
positions of E272 and K274. Fc is shown as a gray ribbon diagram,
and Fc.gamma.RIIIb is shown as a black ribbon. The N297
carbohydrate is shown as black sticks, and E272 and K274 are shown
as black ball and sticks.
[0042] FIG. 6a-6b. Structure of a full length human antibody IgG1
b12 (pdb accession code 1HZH, Saphire et al., 2002, J Mol Biol
319:9-18). FIG. 6a shows the entire structure, with the heavy chain
shown as a gray ribbon, the light chain is shown as a black ribbon,
and the carbohydrate is shown as black sticks. FIG. 6b shows a
closer view of the structure, illustrating interactions at the
Fab/Fc interface. The heavy chain is shown as a gray ribbon, the
light chain is shown as a black ribbon. Fab residues at the
interface are shown as black sticks, and Fc residues at the
interface are shown as grey sticks.
[0043] FIG. 7. The Fab/Fc interface in the 1HZH structure, showing
interaction of positively charged Fab residues R77, R106, K107, and
R108 (Kabat and 1HZH numbering) with negatively charged Fc residues
E269, D270, and E272 (EU numbering scheme numbering, which
correspond to 1HZH residues E282, D283, and E285 respectively).
[0044] FIGS. 8a-8b. Full length antibody complex structures mAb
61.1.3, a murine IgG1 (pdb accession code 1GY, Harris et al., 1995,
Nature 360:369-372) (FIG. 8a) and mAb 231, a murine IgG2a (pdb
accession code 1GT, Harris et al., 1997, Biochemistry 36:1581-1597)
(FIG. 8b). The heavy chains are shown as grey ribbon, the light
chains are shown as black ribbon, and the carbohydrates are shown
as black sticks.
[0045] FIGS. 9a-9b. Models illustrating how residues outside the Fc
region may affect interaction of the antibody with effector
ligands. FIG. 9a illustrates an inhibition, repression, or
competition model, whereby Fab/Fc and/or Fab/Fab interactions may
compete for Fc/Fc.gamma.R interactions. FIG. 9b illustrates an
activation or enhancement model, whereby Fab/Fc.gamma.R and/or
Fab/Fc/Fc.gamma.R interactions enhance affinity of the
Fc/Fc.gamma.R complex. Although the models are shown with an
Fc.gamma.R as the effector ligand, they are meant to generally
apply for any Fc or effector ligands as defined herein.
[0046] FIG. 10a-10c. Binding to human V158 Fc.gamma.RIIa by VH and
VL variants in the context of Herceptin as determined by the
AlphaScreen assay. In the presence of competitor antibody, a
characteristic inhibition curve is observed as a decrease in
luminescence signal. Phosphate buffer saline (PBS) alone was used
as the negative control. These data were normalized to the maximum
and minimum luminescence signal provided by the baselines at low
and high concentrations of competitor antibody respectively. The
curves represent the fits of the data to a one site competition
model using nonlinear regression.
[0047] FIG. 11. SPR kinetic data curves. 5 ul of 200 nM
Fc.gamma.RIIIa(V158)-GST was injected at 1 ul/min over an anti-GST
antibody flowcell, 1 uM antibody was flowed over the bound chip by
KINJECT of 120 ul injection at 40 ul/min, followed by a
dissociation time of 600 seconds. The chip was regenerated each
cycle with 2 injections of 10 mM glycine, pH 1.5. Equilibrium
constants (Kd's) from the fits to the data are provided in Table
3.
[0048] FIG. 12. Correlation between fold IC50's relative to WT
obtained from the AlphaScreen and fold Kd's relative to WT obtained
from SPR for binding of VH and VL Herceptin variants to human V158
Fc.gamma.RIIIa.
[0049] FIG. 13. Structure of Herceptin (pdb accession code 1FVE,
Eigenbrot et al, 1993, J Mol Biol 229:969-995) showing residues
mutated in variants VH2, VH7, VH9, and VH14. The VL and VH chains
are shown as black and grey ribbon respectively.
[0050] FIGS. 14a and 14b. Computational structure-based
calculations on the kappa JL and CL (FIG. 14a) and JH and IgG CH1
(FIG. 14b) domains of Fab structure 1L7I (Vajdos et al., 2002, J
Mol Biol 320(2):415-28) as described in Example 3. Column 1 lists
the positions, and column 2 lists the wild-type amino acid identity
at each position. The J regions are numbered according to the Kabat
numbering scheme, and the constant domains are numbered according
to EU numbering scheme. The remaining columns provide the energy
for each of the natural 20 amino acids, shown in the top row. HID,
HIE, and HSP represent respectively histidine with hydrogen on the
delta nitrogen, histidine with hydrogen on the epsilon nitrogen,
and positively charged histidine with hydrogens on both nitrogens.
All substitutions were normalized with respect to the lowest energy
substitution, which was set to 0 energy and is shown in dark grey.
Light grey indicates substitutions within 2 kcal/mol of the lowest
energy substitution, and white indicates substitutions greater than
2 kcal/mol from the lowest energy substitution. Favorable
substitutions may be considered to be the lowest energy
substitution for each position, and substitutions that have small
energy differences from the lowest energy substitution, for example
substitutions within 1-2, 1-3, 1-5, or 1-10 kcal/mol. nd indicates
that the substitution is not determined, typically due to extremely
high energy.
[0051] FIGS. 15a and 15b. Alignments, structural analysis, and
library design for the J.kappa. and JH segments (FIG. 15a), and
C.kappa. and IgG CH1 chains (FIG. 15b). The sequences for the human
J (heavy, kappa light and lambda light) chains, four human IgG
(IgG1, IgG2, IgG3, and IgG4) CH1, and two human light (kappa and
lambda) first constant regions are aligned. The J regions are
numbered according to the Kabat numbering scheme, and the constant
domains are numbered according to EU numbering scheme of IgG1 for
the heavy chain and C.kappa. for the light chain. For the J region,
Cam indicates the sequence of Campath. Structural analysis, by
visual inspection of the 1 L7I Fab pdb structure, was used to
indicate residues that reside at a critical interface (iface) (as
an example, some residues in CH interact with VH and CL), in the
core (core), or which potentially impact binding to antigen
(antgn). This information and the calculations described in FIG. 14
were used to design the variant library, shown in the Substitutions
column. Substitutions in parentheses indicate that those mutations
are part of a multiple variant; these include C.kappa. K207A/R211A,
CH1 K205A/K210A, and CH1 K213A/K214A/K218A.
[0052] FIG. 16. Binding to human V158 Fc.gamma.RIIIa by select Fab
variant and WT Campath antibodies as determined by the AlphaScreen
assay. Also shown for comparison are two Fc variants, I332E and
S239D/I332E, that have been previously been shown to enhance
binding to Fc.gamma.RIIIa and ADCC in a cell based assay. Numbering
is according to the EU numbering scheme. Phosphate buffer saline
(PBS) alone was used as the negative control. These data were
normalized to the maximum and minimum luminescence signal provided
by the baselines at low and high concentrations of competitor
antibody respectively. The curves represent the fits of the data to
a one site competition model using nonlinear regression.
[0053] FIG. 17. Structure of a human Fab (pdb accession code 1 L7I)
highlighting Fab heavy chain residues at which mutation provides
enhanced Fc.gamma.R binding. The light chain (VL-CL) is shown in
black, and the heavy chain (VH-CH1) is shown in grey. Residues on
the heavy chain at which single substitution results in Fc.gamma.R
binding greater than 3-fold relative to WT are shown as black
sticks. Numbering is according to Kabat or EU numbering schemes as
indicated in Table 4.
[0054] FIG. 18. Structure of a human Fab (pdb accession code 1L7I)
highlighting Fab light chain variants with enhanced Fc.gamma.R
binding. The light chain (VL-CL) is shown in dark grey, and the
heavy chain (VH-CH1) is shown in light grey. Residues on the light
chain at which single substitution results in Fc.gamma.R binding
greater than 3-fold relative to WT are shown as black sticks.
Numbering is according to Kabat and EU numbering schemes as
indicated in Table 4.
[0055] FIG. 19. Binding to human V158 Fc.gamma.RIIIa by select J
and constant region variant/Fc variant combinations, as determined
by the AlphaScreen assay. Phosphate buffer saline (PBS) alone was
used as the negative control. Fc variants I332E and S239D/I332E are
also shown for direct comparison. Numbering is according to Kabat
and EU numbering schemes as indicated in Table 4. The data were
normalized to the maximum and minimum luminescence signal provided
by the baselines at low and high concentrations of competitor
antibody respectively. The curves represent the fits of the data to
a one site competition model using nonlinear regression.
[0056] FIGS. 20a-20b. Binding to human effector ligands by select
hinge variants as determined by the AlphaScreen assay. FIG. 20a
shows binding to human V158 Fc.gamma.RIIIa by select hinge region
variants. FIG. 20b shows binding to human Fc.gamma.RI,
Fc.gamma.RIIa, Fc.gamma.RIIb, Fc.gamma.RIIc, Fc.gamma.RIIIa, C1q,
and FcRn for the D221K hinge variant. The data were normalized to
the maximum and minimum luminescence signal provided by the
baselines at low and high concentrations of competitor antibody
respectively. The curves represent the fits of the data to a one
site competition model using nonlinear regression.
DETAILED DESCRIPTION OF THE INVENTION
[0057] In order that the invention may be more completely
understood, several definitions are set forth below. Such
definitions are meant to encompass grammatical equivalents.
[0058] By "amino acid" and "amino acid identity" as used herein is
meant one of the 20 naturally occurring amino acids or any
non-natural analogues that may be present at a specific, defined
position. By "amino acid modification" herein is meant an amino
acid substitution, insertion, and/or deletion in a polypeptide
sequence. The preferred amino acid modification herein is a
substitution. By "amino acid substitution" or "substitution" herein
is meant the replacement of an amino acid at a particular position
in a parent polypeptide sequence with another amino acid. For
example, the substitution S119E refers to a variant polypeptide in
which serine at position 119 is replaced with glutamic acid. By
"amino acid insertion" or "insertion" as used herein is meant the
addition of an amino acid at a particular position in a parent
polypeptide sequence. For example, -52bK designates an insertion of
lysine at position 52b. By "amino acid deletion" or "deletion" as
used herein is meant the removal of an amino acid at a particular
position in a parent polypeptide sequence. For example,
A100b--designates the deletion of alanine at position 100b.
[0059] By "antibody" herein is meant a protein consisting of one or
more proteins substantially encoded by all or part of the
recognized immunoglobulin genes. The recognized immunoglobulin
genes, for example in humans, include the kappa (.kappa.), lambda
(.lamda.), and heavy chain genetic loci, which together comprise
the myriad variable region genes, and the constant region genes mu
(.mu.), delta (.delta.), gamma (.gamma.), sigma (.sigma.), and
alpha (.alpha.) which encode the IgM, IgD, IgG, IgE, and IgA
isotypes respectively. Antibody herein is meant to include full
length antibodies and antibody fragments. By "full length antibody"
herein is meant the structure that constitutes the natural
biological form of an antibody, including variable and constant
regions. For example, in most mammals, including humans and mice,
the full length antibody of the IgG class is a tetramer and
consists of two identical pairs of two immunoglobulin chains, each
pair having one light and one heavy chain, each light chain
comprising immunoglobulin domains VL and CL, and each heavy chain
comprising immunoglobulin domains VH, C.gamma.1, C.gamma.2, and
C.gamma.3. In some mammals, for example in camels and llamas, IgG
antibodies may consist of only two heavy chains, each heavy chain
comprising a variable domain attached to the Fc region. Antibody
fragments, as are known in the art, include proteins such as Fab,
Fab', F(ab')2, Fv, scFv, or other antigen-binding subsequences of
antibodies, either produced by the modification of whole antibodies
or those synthesized de novo using recombinant DNA technologies.
Antibody may refer to a natural antibody from any organism, an
engineered antibody, or an antibody generated recombinantly for
experimental, therapeutic, or other purposes. The term antibody
comprises monoclonal and polyclonal antibodies. Antibodies can be
antagonists, agonists, neutralizing, inhibitory, or
stimulatory.
[0060] By "antibody dependent cell-mediated cytotoxicity" or "ADCC"
or as used herein is meant the cell-mediated reaction wherein
nonspecific cytotoxic cells that express one or more effector
ligands recognize bound antibody on a target cell and subsequently
cause lysis of the target cell. By "antibody dependent
cell-mediated phagocytosis" or "ADCP" or as used herein is meant
the cell-mediated reaction wherein nonspecific cytotoxic cells that
express one or more effector ligands recognize bound antibody on a
target cell and subsequently cause phagocytosis of the target
cell.
[0061] By "effector function" as used herein is meant a biochemical
event that results from the interaction of an antibody with an
effector ligand. Effector functions include but are not limited to
antibody dependent cell-mediated cytotoxicity (ADCC), antibody
dependent cell-mediated phagocytosis (ADCP), complement dependent
cytotoxicity (CDC), complement dependent cellular cytotxicity
(CDCC), oxidative burst, and release of inflammatory mediators. By
"effector cell" as used herein is meant a cell of the immune system
that expresses one or more Fc receptors and mediates one or more
effector functions. Effector cells include but are not limited to
monocytes, macrophages, neutrophils, dendritic cells, eosinophils,
mast cells, platelets, B cells, large granular lymphocytes,
Langerhans' cells, natural killer (NK) cells, and .gamma..delta. T
cells, and may be from any organism including but not limited to
humans, mice, rats, rabbits, and monkeys. By "effector ligand" as
used herein is meant a molecule, preferably a protein or
polypeptide, from any organism that binds to an antibody to mediate
one or more effector functions (Jefferis et al., 2002, Immunol Lett
82:57-65). Effector ligands include but are not limited to Fc
receptors, Fc.gamma.Rs, Fc.alpha.Rs, Fc.epsilon.Rs, FcRn, C1q, C3,
mannan binding lectin, mannose receptor, staphylococcal protein A,
streptococcal protein G, and viral Fc.gamma.R. Effector ligands
also include Fc receptor homologs (FcRH), which are a family of Fc
receptors that are homologous to the Fc.gamma.Rs (Davis et al.,
2002, Immunological Reviews 190:123-136). Effector ligands may
include undiscovered effector molecules.
[0062] By "Fab" or "Fab region" as used herein is meant the
polypeptides that comprises the VL, VH, CL, and CH1 immunoglobulin
domains or regions. Because VL comprises the JL region and VH
comprises the JH region, JL and JH also compose the Fab region. It
is generally viewed in the art that the Fab region is demarcated
N-terminally by the N-terminus and C-terminally by the disulfide
bond that covalently links the heavy and light chains. Accordingly,
for the purposes of the present invention, "Fab region" as used
herein comprises from the N-terminus to residue 214 of the light
chain and from the N-terminus to residue 220 of the heavy chain,
wherein the numbering of the C-terminal residues is according to
the EU numbering scheme. Fab may refer to this region in isolation,
or this region in the context of a full length antibody or antibody
fragment. Positional definitions of the regions within the Fab,
including the VL, VH, JL, JH, CL, and CH1 regions, are illustrated
in FIG. 3. The VL kappa and VH regions are well defined genetically
and in the art, and accordingly "VL region" as used herein
comprises residues 1-107, and "VH region" as used herein comprises
residues 1-113, wherein numbering is according to the Kabat
numbering scheme. The JL kappa region is made up of 5 germline
sequences of equal length, and accordingly "JL region" as used
herein comprises residues 96-107, wherein numbering is according to
Kabat. There are 6 JH germline sequences of differing length, and
the exact Kabat position at which this segment combines with the VH
germline varies. For the purposes of the present invention, the JH
region is defined to comprise the residues of these sequences that
are clearly defined in a Kabat sequence alignment; based on this
definition, "JH region" as used herein comprises residues 100-113,
wherein numbering is according to the Kabat numbering scheme. The
remaining C-terminal light and heavy chain sequences of the Fab are
made up of the CL and CH1 regions respectively. Thus, "CL region"
as used herein comprises residues 108-214, and "CH1 region" as used
herein comprises residues 118-220, wherein numbering is according
to the EU numbering scheme.
[0063] By "Fc" or "Fc region" as used herein is meant the
polypeptides comprising the last two constant region immunoglobulin
domains of IgA, IgD, and IgG, and the last three constant region
immunoglobulin domains of IgE and IgM, and part of the flexible
hinge N-terminal to these domains. For IgA and IgM Fc may include
the J chain. For IgG, Fc comprises immunoglobulin domains CH2 and
CH3, also referred to as Cgamma2 and Cgamma3 (C.gamma.2 and
C.gamma.3). For IgA, Fc comprises immunoglobulin domains CH2 and
CH3, also referred to as Calpha2 and Calpha3 (C.alpha.2 and
C.alpha.3). Although the boundaries of the Fc region may vary, the
human IgG heavy chain Fc region is usually defined to comprise
residues C226 or P230 to its carboxyl-terminus, wherein the
numbering is according to the EU numbering scheme. Fc may refer to
this region in isolation, or this region in the context of a full
length antibody or antibody fragment. Ergo, by "outside the Fc
region" as used herein is meant the region of an antibody that does
not comprise the Fc region of the antibody. In accordance with the
aforementioned definition of Fc region, "outside the Fc region" for
an IgG1 antibody is herein defined to be from the N-terminus up to
and including residue T225 or C229, wherein the numbering is
according to the EU numbering scheme. Thus the Fab region and part
of the hinge region of an antibody are outside the Fc region.
[0064] By "Fc gamma receptor" or "Fc.gamma.R" as used herein is
meant any member of the family of proteins that bind the IgG
antibody Fc region and are substantially encoded by the Fc.gamma.R
genes. In humans this family includes but is not limited to
Fc.gamma.RI (CD64), including isoforms Fc.gamma.RIa, Fc.gamma.RIb,
and Fc.gamma.RIc; Fc.gamma.RII (CD32), including isoforms
Fc.gamma.RIIa (including allotypes H131 and R131), Fc.gamma.RIIb
(including Fc.gamma.RIIb-1 and Fc.gamma.RIIb-2), and Fc.gamma.RIIc;
and Fc.gamma.RIII (CD16), including isoforms Fc.gamma.RIIIa
(including allotypes V158 and F158) and Fc.gamma.RIIIb (including
allotypes Fc.gamma.RIIIb-NA1 and Fc.gamma.RIIIb-NA2) (Jefferis et
al., 2002, Immunol Lett 82:57-65), as well as any undiscovered
human Fc.gamma.Rs or Fc.gamma.R isoforms or allotypes. An
Fc.gamma.R may be from any organism, including but not limited to
humans, mice, rats, rabbits, and monkeys. Mouse Fc.gamma.Rs include
but are not limited to Fc.gamma.RI (CD64), Fc.gamma.RII (CD32),
Fc.gamma.RIII (CD16), and Fc.gamma.RIII-2 (CD16-2), as well as any
undiscovered mouse Fc.gamma.Rs or Fc.gamma.R isoforms or
allotypes.
[0065] By "germline" as used herein is meant the set of sequences
that compose the natural genetic repertoire of a protein, and its
associated alleles.
[0066] By "hinge" or "hinge region" herein is meant the flexible
polypeptide comprising the amino acids between the first and second
constant domains of an antibody. The hinge is defined structurally
for the purposes of the present invention, and "hinge region" as
used herein for IgG comprises residues 221-236, wherein numbering
is according to the EU numbering scheme.
[0067] By "immunoglobulin (Ig)" herein is meant a protein
consisting of one or more polypeptides substantially encoded by
immunoglobulin genes. Immunoglobulins include but are not limited
to antibodies. Immunoglobulins may have a number of structural
forms, including but not limited to full length antibodies,
antibody fragments, and individual immunoglobulin domains. By
"immunoglobulin (Ig) domain" herein is meant a region of an
immunoglobulin that exists as a distinct structural entity as
ascertained by one skilled in the art of protein structure. Ig
domains typically have a characteristic .beta.-sandwich folding
topology. The known Ig domains in the IgG isotype of antibodies are
VH, CH1, CH2, CH3, VL, and CL.
[0068] By "IgG" as used herein is meant a protein belonging to the
class of antibodies that are substantially encoded by a recognized
immunoglobulin gamma gene. In humans this class comprises IgG1,
IgG2, IgG3, and IgG4.
[0069] By "parent" or "parent protein" as used herein is meant a
protein that is subsequently modified to generate a variant. The
parent protein may be a naturally occurring protein, or a variant
or engineered version of a naturally occurring protein. Parent
protein may refer to the protein itself, compositions that comprise
the parent protein, or the amino acid sequence that encodes it.
Accordingly, by "Parent antibody" as used herein is meant an
antibody that is subsequently modified to generate a variant
antibody. Accordingly, by "Parent sequence" as used herein is meant
the sequence that encodes the parent protein or parent
antibody.
[0070] By "position" as used herein is meant a location in the
sequence of a protein. Positions may be numbered sequentially, or
according to an established format, for example the Kabat or EU
numbering schemes. For example, position 297 is a position in the
human antibody IgG1.
[0071] By "protein" herein is meant at least two covalently
attached amino acids, which includes proteins, polypeptides,
oligopeptides and peptides.
[0072] By "residue" as used herein is meant a position in a protein
and its associated amino acid identity. For example, Asparagine 297
(also referred to as Asn297, also referred to as N297) is a residue
in the human antibody IgG1.
[0073] By "target antigen" as used herein is meant the molecule
that is bound specifically by the variable region of a given
antibody. A target antigen may be a protein, carbohydrate, lipid,
or other chemical compound. By "target cell" as used herein is
meant a cell that expresses a target antigen.
[0074] By "variable region" as used herein is meant the region of
an immunoglobulin that comprises one or more Ig domains
substantially encoded by any of the VL (including V.kappa. and
V.lamda.), VH, JL (including J.kappa. and J.lamda.), and JH genes
that make up the light chain (including .kappa. and .lamda.) and
heavy chain immunoglobulin genetic loci respectively. A light or
heavy chain variable region (VL and VH) consists of a "framework"
or "FR" region interrupted by three hypervariable regions referred
to as "complementarity determining regions" or "CDRs". The extent
of the framework region and CDRs have been precisely defined, for
example as in Kabat (see "Sequences of Proteins of Immunological
Interest," E. Kabat et al., U.S. Department of Health and Human
Services, (1983)), and as in Chothia (Chothia & Lesk, 1987, J.
Mol. Biol. 196: 901-917; Chothia et al., 1989, Nature 342: 877-883;
AI-Lazikani et al., 1997, J. Mol. Biol. 273: 927-948). The
framework regions of an antibody, that is the combined framework
regions of the constitutent light and heavy chains, serves to
position and align the CDRs, which are primarily responsible for
binding to an antigen.
[0075] By "variant protein" or "protein variant", or "variant" as
used herein is meant a protein that differs from that of a parent
protein by virtue of at least one amino acid modification. Protein
variant may refer to the protein itself, a composition comprising
the protein, or the amino sequence that encodes it. Preferably, the
protein variant has at least one amino acid modification compared
to the parent protein, e.g. from about one to about ten amino acid
modifications, and preferably from about one to about five amino
acid modifications compared to the parent. The protein variant
sequence herein will preferably possess at least about 80% homology
with a parent protein sequence, and most preferably at least about
90% homology, more preferably at least about 95% homology.
Accordingly, by "antibody variant" or "variant antibody" as used
herein is meant an antibody that differs from a parent antibody by
virtue of at least one amino acid modification.
[0076] By "wild type or WT" herein is meant an amino acid sequence
or a nucleotide sequence that is found in nature, including allelic
variations. A WT protein has an amino acid sequence or a nucleotide
sequence that has not been intentionally modified.
[0077] Antibody variants of the present invention may be
substantially encoded by genes from any organism, preferably
mammals, including but not limited to humans, rodents including but
not limited to mice and rats, lagomorpha including but not limited
to rabbits and hares, camelidae including but not limited to
camels, llamas, and dromedaries, and non-human primates, including
but not limited to Prosimians, Platyrrhini (New World monkeys),
Cercopithecoidea (Old World monkeys), and Hominoidea including the
Gibbons and Lesser and Great Apes. In a most preferred embodiment,
the antibody variants of the present invention are substantially
human. The antibody variants of the present invention may be
substantially encoded by immunoglobulin genes belonging to any of
the antibody classes. In a most preferred embodiment, the antibody
variants of the present invention comprise sequences belonging to
the IgG class of antibodies, including human subclasses IgG1, IgG2,
IgG3, and IgG4. In an alternate embodiment, the antibody variants
of the present invention comprise sequences belonging to the IgA
(including human subclasses IgA1 and IgA2), IgD, IgE, IgG, or IgM
classes of antibodies. The antibody variants of the present
invention may comprise more than one protein chain. That is, the
present invention may find use in an antibody variant that is a
monomer or an oligomer, including a homo- or hetero-oligomer.
[0078] In the most preferred embodiment, the antibody variants of
the invention are based on human IgG sequences, and thus human IgG
sequences are used as the "base" sequences against which other
sequences are compared, including but not limited to sequences from
other organisms, for example rodent and primate sequences, as well
as sequences from other immunoglobulin classes such as IgA, IgE,
IgGD, IgGM, and the like. It is contemplated that, although the
antibody variants of the present invention are engineered in the
context of one parent antibody variant, the variants may be
engineered in or "transferred" to the context of another, second
parent antibody variant. This is done by determining the
"equivalent" or "corresponding" residues and substitutions between
the first and second antibody variants, typically based on sequence
or structural homology between the sequences of the two antibody
variants. In order to establish homology, the amino acid sequence
of a first antibody variant outlined herein is directly compared to
the sequence of a second antibody variant. After aligning the
sequences, using one or more of the homology alignment programs
known in the art (for example using conserved residues as between
species), allowing for necessary insertions and deletions in order
to maintain alignment (i.e., avoiding the elimination of conserved
residues through arbitrary deletion and insertion), the residues
equivalent to particular amino acids in the primary sequence of the
first antibody variant are defined. Alignment of conserved residues
preferably should conserve 100% of such residues. However,
alignment of greater than 75% or as little as 50% of conserved
residues is also adequate to define equivalent residues. Equivalent
residues may also be defined by determining structural homology
between a first and second Antibody variant that is at the level of
tertiary structure for antibody variants whose structures have been
determined. In this case, equivalent residues are defined as those
for which the atomic coordinates of two or more of the main chain
atoms of a particular amino acid residue of the parent or precursor
(N on N, CA on CA, C on C and O on O) are within 0.13 nm and
preferably 0.1 nm after alignment. Alignment is achieved after the
best model has been oriented and positioned to give the maximum
overlap of atomic coordinates of non-hydrogen protein atoms of the
proteins. Regardless of how equivalent or corresponding residues
are determined, and regardless of the identity of the parent
antibody variant in which the antibody variants are made, what is
meant to be conveyed is that the antibody variants discovered by
the present invention may be engineered into any second parent
Antibody variant that has significant sequence or structural
homology with said antibody variant. Thus for example, if a variant
antibody is generated wherein the parent antibody is human IgG1, by
using the methods described above or other methods for determining
equivalent residues, said variant antibody may be engineered in
another IgG1 parent antibody that binds a different antigen, a
human IgG2 parent antibody, a human IgA parent antibody, a mouse
IgG2a or IgG2b parent antibody, and the like. Again, as described
above, the context of the parent antibody variant does not affect
the ability to transfer the antibody variants of the present
invention to other parent antibody variants.
[0079] Virtually any binding partner or antigen may be targeted by
the antibody variants of the present invention, including but are
not limited to proteins, subunits, domains, motifs, and epitopes
belonging to the following list of proteins: CD2; CD3, CD3E, CD4,
CD11, CD11a, CD14, CD16, CD18, CD19, CD20, CD22, CD23, CD25, CD28,
CD29, CD30, CD32, CD33 (p67 protein), CD38, CD40, CD40L, CD52,
CD54, CD56, CD80, CD147, GD3, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-5,
IL-6, IL-6R, IL-8, IL-12, IL-15, IL-18, IL-23, interferon alpha,
interferon beta, interferon gamma; TNF-alpha, TNFbeta2, TNFc,
TNFalphabeta, TNF-RI, TNF-RII, FasL, CD27L, CD30L, 4-1BBL, TRAIL,
RANKL, TWEAK, APRIL, BAFF, LIGHT, VEGI, OX40L, TRAIL Receptor-1, A1
Adenosine Receptor, Lymphotoxin Beta Receptor, TACI, BAFF-R, EPO;
LFA-3, ICAM-1, ICAM-3, EpCAM, integrin beta1, integrin beta2,
integrin alpha4/beta7, integrin alpha2, integrin alpha3, integrin
alpha4, integrin alpha5, integrin alpha6, integrin alphav,
alphaVbeta3 integrin, FGFR-3, Keratinocyte Growth Factor, VLA-1,
VLA-4, L-selectin, anti-Id, E-selectin, HLA, HLA-DR, CTLA-4, T cell
receptor, B7-1, B7-2, VNRintegrin, TGFbeta1, TGFbeta2, eotaxin1,
BLyS (B-lymphocyte Stimulator), complement C5, IgE, factor VII,
CD64, CBL, NCA 90, EGFR (ErbB-1), Her2/neu (ErbB-2), Her3 (ErbB-3),
Her4 (ErbB-4), Tissue Factor, VEGF, VEGFR, endothelin receptor,
VLA-4, Hapten NP-cap or NIP-cap, T cell receptor alpha/beta,
E-selectin, digoxin, placental alkaline phosphatase (PLAP) and
testicular PLAP-like alkaline phosphatase, transferrin receptor,
Carcinoembryonic antigen (CEA), CEACAM5, HMFG PEM, mucin MUC1,
MUC18, Heparanase I, human cardiac myosin, tumor-associated
glycoprotein-72 (TAG-72), tumor-associated antigen CA 125, Prostate
specific membrane antigen (PSMA), High molecular weight
melanoma-associated antigen (HMW-MAA), carcinoma-associated
antigen, Gcoprotein IIb/IIIa (GPIIb/IIIa), tumor-associated antigen
expressing Lewis Y related carbohydrate, human cytomegalovirus
(HCMV) gH envelope glycoprotein, HIV gp120, HCMV, respiratory
syncitial virus RSV F, RSVF Fgp, VNRintegrin, IL-8, cytokeratin
tumor-associated antigen, Hep B gp120, CMV, gpIIbIIIa, HIV IIIB
gp120 V3 loop, respiratory syncytial virus (RSV) Fgp, Herpes
simplex virus (HSV) gD glycoprotein, HSV gB glycoprotein, HCMV gB
envelope glycoprotein, and Clostridium perfringens toxin. A number
biotherapeutic antibodies that are approved for use, in clinical
trials, or in development may thus benefit from antibody variants
of the present invention. Thus in a preferred embodiment, the
antibody variants of the present invention may find use in a range
of clinical products and candidates. Other targets and clinical
products and candidates that may benefit from the antibody variants
of the present invention include but are not limited to those
described in U.S. Ser. No. 10/672,280 and U.S. Ser. No.
60/627,774.
[0080] The antibody variants of the present invention may comprise
one or more additional modifications that provide optimized
properties. Said modifications may be amino acid modifications, or
may be modifications that are made enzymatically or chemically.
Such modification(s) likely provide some improvement in the
antibody, for example an enhancement in its stability, solubility,
function, or clinical use. The present invention contemplates a
variety of improvements that made be made by coupling the antibody
variants of the present invention with additional
modifications.
[0081] In a preferred embodiment, the antibody variants of the
present invention may comprise modifications to reduce
immunogenicity in humans. In a most preferred embodiment, the
immunogenicity of an antibody variant of the present invention is
reduced using a method described in U.S. Ser. No. 11/004,590, filed
Dec. 3, 2004, entitled "Methods of Generating Variant Proteins with
Increased Host String Content and Compositions Thereof". In
alternate embodiments, the antibody variants of the present
invention are humanized (Clark, 2000, Immunol Today 21:397-402). By
"humanized" antibody as used herein is meant an antibody comprising
a human framework region (FR) and one or more complementarity
determining regions (CDR's) from a non-human (usually mouse or rat)
antibody. The non-human antibody providing the CDR's is called the
"donor" and the human immunoglobulin providing the framework is
called the "acceptor". Humanization relies principally on the
grafting of donor CDRs onto acceptor (human) VL and VH frameworks
(Winter U.S. Pat. No. 5,225,539). This strategy is referred to as
"CDR grafting". "Backmutation" of selected acceptor framework
residues to the corresponding donor residues is often required to
regain affinity that is lost in the initial grafted construct (U.S.
Pat. No. 5,530,101; U.S. Pat. No. 5,585,089; U.S. Pat. No.
5,693,761; U.S. Pat. No. 5,693,762; U.S. Pat. No. 6,180,370; U.S.
Pat. No. 5,859,205; U.S. Pat. No. 5,821,337; U.S. Pat. No.
6,054,297; U.S. Pat. No. 6,407,213). The humanized antibody
optimally also will comprise at least a portion of an
immunoglobulin constant region, typically that of a human
immunoglobulin, and thus will typically comprise a human Fc region.
A variety of techniques and methods for humanizing and reshaping
non-human antibodies are well known in the art (See Tsurushita
& Vasquez, 2004, Humanization of Monoclonal Antibodies,
Molecular Biology of B Cells, 533-545, Elsevier Science (USA), and
references cited therein). Humanization methods include but are not
limited to methods described in Jones et al., 1986, Nature
321:522-525; Riechmann et al., 1988; Nature 332:323-329; Verhoeyen
et al., 1988, Science, 239:1534-1536; Queen et al., 1989, Proc Natl
Acad Sci, USA 86:10029-33; He et al., 1998, J. Immunol. 160:
1029-1035; Carter et al., 1992, Proc Natl Acad Sci USA 89:4285-9,
Presta et al., 1997, Cancer Res. 57(20):4593-9; Gorman et al.,
1991, Proc. Natl. Acad. Sci. USA 88:4181-4185; O'Connor et al.,
1998, Protein Eng 11:321-8. Humanization or other methods of
reducing the immunogenicity of nonhuman antibody variable regions
may include resurfacing methods, as described for example in
Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969-973. In one
embodiment, the parent antibody has been affinity matured, as is
known in the art. Structure-based methods may be employed for
humanization and affinity maturation, for example as described in
U.S. Ser. No. 11/004,590. Selection based methods may be employed
to humanize and/or affinity mature antibody variable regions,
including but not limited to methods described in Wu et al., 1999,
J. Mol. Biol. 294:151-162; Baca et al., 1997, J. Biol. Chem.
272(16):10678-10684; Rosok et al., 1996, J. Biol. Chem. 271(37):
22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci. USA 95:
8910-8915; Krauss et al., 2003, Protein Engineering 16(10):753-759.
Other humanization methods may involve the grafting of only parts
of the CDRs, including but not limited to methods described in U.S.
Ser. No. 09/810,502; Tan et al., 2002, J. Immunol. 169:1119-1125;
De Pascalis et al., 2002, J. Immunol. 169:3076-3084.
[0082] Modifications to reduce immunogenicity may include
modifications that reduce binding of processed peptides derived
from the parent sequence to MHC proteins. For example, amino acid
modifications would be engineered such that there are no or a
minimal number of immune epitopes that are predicted to bind, with
high affinity, to any prevalent MHC alleles. Several methods of
identifying MHC-binding epitopes in protein sequences are known in
the art and may be used to score epitopes in an Antibody variant of
the present invention. See for example WO 98/52976; WO 02/079232;
WO 00/3317; U.S. Ser. No. 09/903,378; U.S. Ser. No. 10/039, 170;
U.S. Ser. No. 60/222,697; U.S. Ser. No. 10/754,296; PCT WO
01/21823; and PCT WO 02/00165; Mallios, 1999, Bioinformatics 15:
432-439; Mallios, 2001, Bioinformatics 17: 942-948; Sturniolo et
al., 1999, Nature Biotech. 17: 555-561; WO 98/59244; WO 02/069232;
WO 02/77187; Marshall et al., 1995, J. Immunol. 154: 5927-5933; and
Hammer et al., 1994, J. Exp. Med. 180: 2353-2358. Sequence-based
information can be used to determine a binding score for a given
peptide--MHC interaction (see for example Mallios, 1999,
Bioinformatics 15: 432-439; Mallios, 2001, Bioinformatics 17:
p942-948; Sturniolo et. al., 1999, Nature Biotech. 17:
555-561).
[0083] The Fc region of the antibody may be modified in some way to
make it more effective therapeutically. For example, the Fc region
may comprise substitutions that enhance therapeutic properties.
Most preferred substitutions and optimized effector function
properties are described in U.S. Ser. No. 10/672,280, U.S. Ser. No.
10/822,231, entitled "Optimized Fc Variants and Methods for their
Generation", U.S. Ser. No. 60/627,774, entitled "Optimized Fc
Variants", and U.S. Ser. No. 60/642,477, entitled "Improved Fc
Variants". Other known Fc variants that may find use in the present
invention include but are not limited to those described in U.S.
Pat. No. 6,737,056; PCT US2004/000643; U.S. Ser. No. 10/370,749;
PCT/US2004/005112; US 2004/0132101; U.S. Ser. No. 10/672,280;
PCT/US03/30249; U.S. Pat. No. 6,737,056, US 2004/0002587; WO
2004/063351; Idusogie et al., 2001, J. Immunology 166:2571-2572;
Hinton et al., 2004, J. Biol. Chem. 279(8): 6213-6216. In alternate
embodiments, the constant region may comprise one or more
engineered glycoforms, as is known in the art (Umana et al., 1999,
Nat Biotechnol 17:176-180; Davies et al., 2001, Biotechnol Bioeng
74:288-294; Shields et al., 2002, J Biol Chem 277:26733-26740;
Shinkawa et al., 2003, J Biol Chem 278:3466-3473); (U.S. Pat. No.
6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No. 10/113,929; PCT
WO 00/61739A1; PCT WO 01/29246A1; PCT WO 02/31140A1; PCT WO
02/30954A1; Potelligent.TM. technology [Biowa, Inc., Princeton,
N.J.]; GlycoMAb.TM. glycosylation engineering technology [GLYCART
biotechnology AG, Zurich, Switzerland]).
[0084] In an alternate embodiment, the antibody variant of the
present invention is conjugated or operably linked to another
therapeutic compound. The therapeutic compound may be a cytotoxic
agent, a chemotherapeutic agent, a toxin, a radioisotope, a
cytokine, or other therapeutically active agent. The antibody may
be linked to one of a variety of nonproteinaceous polymers, e.g.,
polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or
copolymers of polyethylene glycol and polypropylene glycol.
[0085] The present invention provides methods for engineering,
producing, and screening antibody variants. The described methods
are not meant to constrain the present invention to any particular
application or theory of operation. Rather, the provided methods
are meant to illustrate generally that one or more antibody
variants may be engineered, produced, and screened experimentally
to obtain antibodies with optimized effector function. A variety of
methods are described for designing, producing, and testing
antibody and protein variants in U.S. Ser. No. 10/754,296, and U.S.
Ser. No. 10/672,280, which are herein expressly incorporated by
reference.
[0086] A variety of protein engineering methods may be used to
design Fab and/or hinge variants with altered effector function. In
one embodiment, a structure-based engineering method may be used,
wherein available structural information is used to guide
substitutions. In a preferred embodiment, a computational screening
method may be used, wherein substitutions are designed based on
their energetic fitness in computational calculations. See for
example U.S. Ser. No. 10/754,296 and U.S. Ser. No. 10/672,280, and
references cited therein.
[0087] An alignment of sequences may be used to guide substitutions
at the identified positions. One skilled in the art will appreciate
that the use of sequence information may curb the introduction of
substitutions that are potentially deleterious to antibody
structure. The source of the sequences may vary widely, and include
one or more of the known databases, including but not limited to
the Kabat database (.immuno.bme.nwu.edu; Johnson & Wu, 2001,
Nucleic Acids Res. 29:205-206; Johnson & Wu, 2000, Nucleic
Acids Res. 28:214-218), the IMGT database (IMGT, the international
ImMunoGeneTics Information System.RTM.; imgt.cines.fr; Lefranc et
al., 1999, Nucleic Acids Res. 27:209-212; Ruiz et al., 2000 Nucleic
Acids Res. 28:219-221; Lefranc et al., 2001, Nucleic Acids Res.
29:207-209; Lefranc et al., 2003, Nucleic Acids Res. 31:307-310),
and VBASE (.mrc-cpe.cam.ac.uk/vbase-ok.php?menu=901). Antibody
sequence information can be obtained, compiled, and/or generated
from sequence alignments of germline sequences or sequences of
naturally occurring antibodies from any organism, including but not
limited to mammals. One skilled in the art will appreciate that the
use of sequences that are human or substantially human may further
have the advantage of being less immunogenic when administered to a
human. Other databases which are more general nucleic acid or
protein databases, i.e. not particular to antibodies, include but
are not limited to SwissProt (expasy.ch/sprot/), GenBank
(ncbi.nlm.nih.gov/Genbank) and Entrez (ncbi.nlm.nih.gov/Entrez/),
and EMBL Nucleotide Sequence Database (ebi.ac.uk/embl/). Aligned
sequences may comprise VH, VL, CH1, and/or CL sequences. There are
numerous sequence-based alignment programs and methods known in the
art, and all of these find use in the present invention for
generation of sequence alignments.
[0088] Alternatively, random or semi-random mutagenesis methods may
be used to make amino acid modifications at the desired positions.
In these cases positions are chosen randomly, or amino acid changes
are made using simplistic rules. For example all residues may be
mutated to alanine, referred to as alanine scanning. Such methods
may be coupled with more sophisticated engineering approaches that
employ selection methods to screen higher levels of sequence
diversity. As is well known in the art, there are a variety of
selection technologies that may be used for such approaches,
including, for example, display technologies such as phage display,
ribosome display, cell surface display, and the like, as described
below.
[0089] Methods for production and screening of antibody variants
are well known in the art. General methods for antibody molecular
biology, expression, purification, and screening are described in
Antibody Engineering, edited by Duebel & Kontermann,
Springer-Verlag, Heidelberg, 2001; and Hayhurst & Georgiou,
2001, Curr Opin Chem Biol 5:683-689; Maynard & Georgiou, 2000,
Annu Rev Biomed Eng 2:339-76. Also see the methods described in
U.S. Ser. No. 10/754,296, filed on Mar. 3, 2003, U.S. Ser. No.
10/672,280, filed Sep. 29, 2003, and U.S. Ser. No. 10/822,231,
filed Mar. 26, 2004.
[0090] In one embodiment of the present invention, the antibody
variant sequences are used to create nucleic acids that encode the
member sequences, and that may then be cloned into host cells,
expressed and assayed, if desired. These practices are carried out
using well-known procedures, and a variety of methods that may find
use in the present invention are described in Molecular Cloning--A
Laboratory Manual, 3.sup.rd Ed. (Maniatis, Cold Spring Harbor
Laboratory Press, New York, 2001), and Current Protocols in
Molecular Biology (John Wiley & Sons). The nucleic acids that
encode the antibody variants of the present invention may be
incorporated into an expression vector in order to express the
antibody. Expression vectors typically comprise a antibody operably
linked, that is placed in a functional relationship, with control
or regulatory sequences, selectable markers, any fusion partners,
and/or additional elements. The antibody variants of the present
invention may be produced by culturing a host cell transformed with
nucleic acid, preferably an expression vector, containing nucleic
acid encoding the antibody variants, under the appropriate
conditions to induce or cause expression of the antibody. A wide
variety of appropriate host cells may be used, including but not
limited to mammalian cells, bacteria, insect cells, and yeast. For
example, a variety of cell lines that may find use in the present
invention are described in the ATCC cell line catalog, available
from the American Type Culture Collection. The methods of
introducing exogenous nucleic acid into host cells are well known
in the art, and will vary with the host cell used.
[0091] In a preferred embodiment, antibody variants are purified or
isolated after expression. Antibodies may be isolated or purified
in a variety of ways known to those skilled in the art. Standard
purification methods include chromatographic techniques,
electrophoretic, immunological, precipitation, dialysis,
filtration, concentration, and chromatofocusing techniques. As is
well known in the art, a variety of natural antibodys bind
antibodies, for example bacterial antibodys A, G, and L, and these
antibodys may find use in the present invention for purification.
Purification can often be enabled by a particular fusion partner.
For example, antibodys may be purified using glutathione resin if a
GST fusion is employed, Ni.sup.+2 affinity chromatography if a
His-tag is employed, or immobilized anti-flag antibody if a
flag-tag is used. For general guidance in suitable purification
techniques, see Antibody Purification: Principles and Practice,
3.sup.rd Ed., Scopes, Springer-Verlag, N.Y., 1994.
[0092] Antibody variants may be screened using a variety of
methods, including but not limited to those that use in vitro
assays, in vivo and cell-based assays, and selection technologies.
Automation and high-throughput screening technologies may be
utilized in the screening procedures. Screening may employ the use
of a fusion partner or label, for example an immune label, isotopic
label, or small molecule label such as a fluorescent or
calorimetric dye.
[0093] In a preferred embodiment, the functional and/or biophysical
properties of antibody variants are screened in an in vitro assay.
In a preferred embodiment, the antibody is screened for
functionality, for example its ability to catalyze a reaction or
its binding affinity to its target. Binding assays can be carried
out using a variety of methods known in the art, including but not
limited to FRET (Fluorescence Resonance Energy Transfer) and BRET
(Bioluminescence Resonance Energy Transfer)-based assays,
AlphaScreen.TM. (Amplified Luminescent Proximity Homogeneous
Assay), Scintillation Proximity Assay, ELISA (Enzyme-Linked
Immunosorbent Assay), SPR (Surface Plasmon Resonance, also known as
BIACORE.RTM.), isothermal titration calorimetry, differential
scanning calorimetry, gel electrophoresis, and chromatography
including gel filtration. These and other methods may take
advantage of some fusion partner or label. Assays may employ a
variety of detection methods including but not limited to
chromogenic, fluorescent, luminescent, or isotopic labels. The
biophysical properties of antibodys, for example stability and
solubility, may be screened using a variety of methods known in the
art. Antibody stability may be determined by measuring the
thermodynamic equilibrium between folded and unfolded states. For
example, antibody variants of the present invention may be unfolded
using chemical denaturant, heat, or pH, and this transition may be
monitored using methods including but not limited to circular
dichroism spectroscopy, fluorescence spectroscopy, absorbance
spectroscopy, NMR spectroscopy, calorimetry, and proteolysis. As
will be appreciated by those skilled in the art, the kinetic
parameters of the folding and unfolding transitions may also be
monitored using these and other techniques. The solubility and
overall structural integrity of a antibody variant may be
quantitatively or qualitatively determined using a wide range of
methods that are known in the art. Methods which may find use in
the present invention for characterizing the biophysical properties
of antibody variants include gel electrophoresis, chromatography
such as size exclusion chromatography and reversed-phase high
performance liquid chromatography, mass spectrometry, ultraviolet
absorbance spectroscopy, fluorescence spectroscopy, circular
dichroism spectroscopy, isothermal titration calorimetry,
differential scanning calorimetry, analytical ultra-centrifugation,
dynamic light scattering, proteolysis, and cross-linking, turbidity
measurement, filter retardation assays, immunological assays,
fluorescent dye binding assays, antibody-staining assays,
microscopy, and detection of aggregates via ELISA or other binding
assay. Structural analysis employing X-ray crystallographic
techniques and NMR spectroscopy may also find use.
[0094] As is known in the art, a subset of screening methods are
those that select for favorable members of a library. The methods
are herein referred to as "selection methods", and these methods
find use in the present invention for screening antibody variants.
When antibody libraries are screened using a selection method, only
those members of a library that are favorable, that is which meet
some selection criteria, are propagated, isolated, and/or observed.
As will be appreciated, because only the most fit variants are
observed, such methods enable the screening of libraries that are
larger than those screenable by methods that assay the fitness of
library members individually. Selection is enabled by any method,
technique, or fusion partner that links, covalently or
noncovalently, the phenotype of a antibody with its genotype, that
is the function of a antibody with the nucleic acid that encodes
it. For example the use of phage display as a selection method is
enabled by the fusion of library members to the gene III antibody.
In this way, selection or isolation of antibody variants that meet
some criteria, for example binding affinity to the antibody's
target, also selects for or isolates the nucleic acid that encodes
it. Once isolated, the gene or genes encoding Fc variants may then
be amplified. This process of isolation and amplification, referred
to as panning, may be repeated, allowing favorable antibody
variants in the library to be enriched. Nucleic acid sequencing of
the attached nucleic acid ultimately allows for gene
identification.
[0095] A variety of selection methods are known in the art that may
find use in the present invention for screening antibody libraries.
These include but are not limited to phage display (Phage display
of peptides and antibodys: a laboratory manual, Kay et al., 1996,
Academic Press, San Diego, Calif., 1996; Lowman et al., 1991,
Biochemistry 30:10832-10838; Smith, 1985, Science 228:1315-1317)
and its derivatives such as selective phage infection (Malmborg et
al., 1997, J Mol Biol 273:544-551), selectively infective phage
(Krebber et al., 1997, J Mol Biol 268:619-630), and delayed
infectivity panning (Benhar et al., 2000, J Mol Biol 301:893-904),
cell surface display (Witrrup, 2001, Curr Opin Biotechnol,
12:395-399) such as display on bacteria (Georgiou et al., 1997, Nat
Biotechnol 15:29-34; Georgiou et al., 1993, Trends Biotechnol
11:6-10; Lee et al., 2000, Nat Biotechnol 18:645-648; Jun et al.,
1998, Nat Biotechnol 16:576-80), yeast (Boder & Wittrup, 2000,
Methods Enzymol 328:430-44; Boder & Wittrup, 1997, Nat
Biotechnol 15:553-557), and mammalian cells (Whitehorn et al.,
1995, Bio/technology 13:1215-1219), as well as in vitro display
technologies (Amstutz et al., 2001, Curr Opin Biotechnol
12:400-405) such as polysome display (Mattheakis et al., 1994, Proc
Natl Acad Sci USA 91:9022-9026), ribosome display (Hanes et al.,
1997, Proc Natl Acad Sci USA 94:4937-4942), mRNA display (Roberts
& Szostak, 1997, Proc Natl Acad Sci USA 94:12297-12302; Nemoto
et al., 1997, FEBS Lett 414:405-408), and ribosome-inactivation
display system (Zhou et al., 2002, J Am Chem Soc 124, 538-543).
[0096] Other selection methods that may find use in the present
invention include methods that do not rely on display, such as in
vivo methods including but not limited to periplasmic expression
and cytometric screening (Chen et al., 2001, Nat Biotechnol
19:537-542), the antibody fragment complementation assay (Johnson
& Varshavsky, 1994, Proc Natl Acad Sci USA 91:10340-10344;
Pelletier et al., 1998, Proc Natl Acad Sci USA 95:12141-12146), and
the yeast two hybrid screen (Fields & Song, 1989, Nature
340:245-246) used in selection mode (Visintin et al., 1999, Proc
Natl Acad Sci USA 96:11723-11728). In an alternate embodiment,
selection is enabled by a fusion partner that binds to a specific
sequence on the expression vector, thus linking covalently or
noncovalently the fusion partner and associated Fc variant library
member with the nucleic acid that encodes them. For example, U.S.
Ser. No. 09/642,574; U.S. Ser. No. 10/080,376; U.S. Ser. No.
09/792,630; U.S. Ser. No. 10/023,208; U.S. Ser. No. 09/792,626;
U.S. Ser. No. 10/082,671; U.S. Ser. No. 09/953,351; U.S. Ser. No.
10/097, 100; U.S. Ser. No. 60/366,658; PCT WO 00/22906; PCT WO
01/49058; PCT WO 02/04852; PCT WO 02/04853; PCT WO 02/08023; PCT WO
01/28702; and PCT WO 02/07466 describe such a fusion partner and
technique that may find use in the present invention. In an
alternative embodiment, in vivo selection can occur if expression
of the antibody imparts some growth, reproduction, or survival
advantage to the cell.
[0097] A subset of selection methods referred to as "directed
evolution" methods are those that include the mating or breading of
favorable sequences during selection, sometimes with the
incorporation of new mutations. As will be appreciated by those
skilled in the art, directed evolution methods can facilitate
identification of the most favorable sequences in a library, and
can increase the diversity of sequences that are screened. A
variety of directed evolution methods are known in the art that may
find use in the present invention for screening antibody variants,
including but not limited to DNA shuffling (PCT WO 00/42561 A3; PCT
WO 01/70947 A3), exon shuffling (U.S. Pat. No. 6,365,377; Kolkman
& Stemmer, 2001, Nat Biotechnol 19:423-428), family shuffling
(Crameri et al., 1998, Nature 391:288-291; U.S. Pat. No.
6,376,246), RACHITT.TM. (Coco et al., 2001, Nat Biotechnol
19:354-359; PCT WO 02/06469), STEP and random priming of in vitro
recombination (Zhao et al., 1998, Nat Biotechnol 16:258-261; Shao
et al., 1998, Nucleic Acids Res 26:681-683), exonuclease mediated
gene assembly (U.S. Pat. No. 6,352,842; U.S. Pat. No. 6,361,974),
Gene Site Saturation Mutagenesis.TM. (U.S. Pat. No. 6,358,709),
Gene Reassembly.TM. (U.S. Pat. No. 6,358,709), SCRATCHY (Lutz et
al., 2001, Proc Natl Acad Sci USA 98:11248-11253), DNA
fragmentation methods (Kikuchi et al., Gene 236:159-167),
single-stranded DNA shuffling (Kikuchi et al., 2000, Gene
243:133-137), and AMEsystem.TM. directed evolution antibody
engineering technology (Applied Molecular Evolution) (U.S. Pat. No.
5,824,514; U.S. Pat. No. 5,817,483; U.S. Pat. No. 5,814,476; U.S.
Pat. No. 5,763,192; U.S. Pat. No. 5,723,323).
[0098] In a preferred embodiment, antibody variants are screened
using one or more cell-based or in vivo assays. For such assays,
purified or unpurified antibodys are typically added exogenously
such that cells are exposed to individual variants or pools of
variants belonging to a library. These assays are typically, but
not always, based on the function of the antibody; that is, the
ability of the antibody to bind to its target and mediate some
biochemical event, for example effector function, ligand/receptor
binding inhibition, apoptosis, and the like. Such assays often
involve monitoring the response of cells to the antibody, for
example cell survival, cell death, change in cellular morphology,
or transcriptional activation such as cellular expression of a
natural gene or reporter gene. For example, such assays may measure
the ability of antibody variants to elicit ADCC, ADCP, or CDC. For
some assays additional cells or components, that is in addition to
the target cells, may need to be added, for example serum
complement, or effector cells such as peripheral blood monocytes
(PBMCs), NK cells, macrophages, and the like. Such additional cells
may be from any organism, preferably humans, mice, rat, rabbit, and
monkey. Antibodys may cause apoptosis of certain cell lines
expressing the target, or they may mediate attack on target cells
by immune cells which have been added to the assay. Methods for
monitoring cell death or viability are known in the art, and
include the use of dyes, immunochemical, cytochemical, and
radioactive reagents. For example, caspase staining assays may
enable apoptosis to be measured, and uptake or release of
radioactive substrates or fluorescent dyes such as alamar blue may
enable cell growth or activation to be monitored. In a preferred
embodiment, the DELFIA.RTM. EuTDA-based cytotoxicity assay (Perkin
Elmer, Mass.) is used. Alternatively, dead or damaged target cells
may be monitored by measuring the release of one or more natural
intracellular antibodys, for example lactate dehydrogenase.
Transcriptional activation may also serve as a method for assaying
function in cell-based assays. In this case, response may be
monitored by assaying for natural genes or antibodys which may be
upregulated, for example the release of certain interleukins may be
measured, or alternatively readout may be via a reporter construct.
Cell-based assays may also involve the measure of morphological
changes of cells as a response to the presence of a antibody. Cell
types for such assays may be prokaryotic or eukaryotic, and a
variety of cell lines that are known in the art may be employed.
Alternatively, cell-based screens are performed using cells that
have been transformed or transfected with nucleic acids encoding
the variants. That is, antibody variants are not added exogenously
to the cells. For example, in one embodiment, the cell-based screen
utilizes cell surface display. A fusion partner can be employed
that enables display of antibody variants on the surface of cells
(Witrrup, 2001, Curr Opin Biotechnol, 12:395-399).
[0099] In a preferred embodiment, the immunogenicity of the
antibody variants is determined experimentally using one or more
cell-based assays. Several methods can be used for experimental
confirmation of epitopes. In a preferred embodiment, ex vivo T-cell
activation assays are used to experimentally quantitate
immunogenicity. In this method, antigen presenting cells and naive
T cells from matched donors are challenged with a peptide or whole
antibody of interest one or more times. Then, T cell activation can
be detected using a number of methods, for example by monitoring
production of cytokines or measuring uptake of tritiated thymidine.
In the most preferred embodiment, interferon gamma production is
monitored using Elispot assays (Schmittel et. al., 2000, J.
Immunol. Meth., 24: 17-24).
[0100] The biological properties of the antibody variants of the
present invention may be characterized in cell, tissue, and whole
organism experiments. As is known in the art, drugs are often
tested in animals, including but not limited to mice, rats,
rabbits, dogs, cats, pigs, and monkeys, in order to measure a
drug's efficacy for treatment against a disease or disease model,
or to measure a drug's pharmacokinetics, toxicity, and other
properties. The animals may be referred to as disease models.
Therapeutics are often tested in mice, including but not limited to
nude mice, SCID mice, xenograft mice, and transgenic mice
(including knockins and knockouts). Such experimentation may
provide meaningful data for determination of the potential of the
antibody to be used as a therapeutic. Any organism, preferably
mammals, may be used for testing. For example because of their
genetic similarity to humans, monkeys can be suitable therapeutic
models, and thus may be used to test the efficacy, toxicity,
pharmacokinetics, or other property of the antibodys of the present
invention. Tests of the in humans are ultimately required for
approval as drugs, and thus of course these experiments are
contemplated. Thus the antibodys of the present invention may be
tested in humans to determine their therapeutic efficacy, toxicity,
immunogenicity, pharmacokinetics, and/or other clinical
properties.
[0101] The antibody variants of the present invention may find use
in a wide range of antibody products. In one embodiment the
antibody variant of the present invention is a therapeutic, a
diagnostic, or a research reagent, preferably a therapeutic. The
antibody variant may find use in an antibody composition that is
monoclonal or polyclonal. In a preferred embodiment, the antibody
variants of the present invention are used to kill target cells
that bear the target antigen, for example cancer cells. In an
alternate embodiment, the antibody variants of the present
invention are used to block, antagonize, or agonize the target
antigen, for example for antagonizing a cytokine or cytokine
receptor. In an alternately preferred embodiment, the antibody
variants of the present invention are used to block, antagonize, or
agonize the target antigen and kill the target cells that bear the
target antigen.
[0102] The antibody variants of the present invention may be used
for various therapeutic purposes. In a preferred embodiment, an
antibody comprising the antibody variant is administered to a
patient to treat an antibody-related disorder. A "patient" for the
purposes of the present invention includes both humans and other
animals, preferably mammals and most preferably humans. By
"antibody related disorder" or "antibody responsive disorder" or
"condition" or "disease" herein are meant a disorder that may be
ameliorated by the administration of a pharmaceutical composition
comprising an antibody variant of the present invention. Antibody
related disorders include but are not limited to autoimmune
diseases, immunological diseases, infectious diseases, inflammatory
diseases, neurological diseases, and oncological and neoplastic
diseases including cancer. By "cancer" and "cancerous" herein refer
to or describe the physiological condition in mammals that is
typically characterized by unregulated cell growth. Examples of
cancer include but are not limited to carcinoma, lymphoma,
blastoma, sarcoma (including liposarcoma), neuroendocrine tumors,
mesothelioma, schwanoma, meningioma, adenocarcinoma, melanoma, and
leukemia and lymphoid malignancies.
[0103] In one embodiment, an antibody variant of the present
invention is the only therapeutically active agent administered to
a patient. Alternatively, the antibody variant of the present
invention is administered in combination with one or more other
therapeutic agents, including but not limited to cytotoxic agents,
chemotherapeutic agents, cytokines, growth inhibitory agents,
anti-hormonal agents, kinase inhibitors, anti-angiogenic agents,
cardioprotectants, or other therapeutic agents. The antibody
variants may be administered concomitantly with one or more other
therapeutic regimens. For example, an antibody variant of the
present invention may be administered to the patient along with
chemotherapy, radiation therapy, or both chemotherapy and radiation
therapy. In one embodiment, the antibody variant of the present
invention may be administered in conjunction with one or more
antibodies, which may or may not comprise a antibody variant of the
present invention. In accordance with another embodiment of the
invention, the antibody variant of the present invention and one or
more other anti-cancer therapies are employed to treat cancer cells
ex vivo. It is contemplated that such ex vivo treatment may be
useful in bone marrow transplantation and particularly, autologous
bone marrow transplantation. It is of course contemplated that the
antibodies of the invention can be employed in combination with
still other therapeutic techniques such as surgery.
[0104] A variety of other therapeutic agents may find use for
administration with the antibody variants of the present invention.
In one embodiment, the antibody is administered with an
anti-angiogenic agent. By "anti-angiogenic agent" as used herein is
meant a compound that blocks, or interferes to some degree, the
development of blood vessels. The anti-angiogenic factor may, for
instance, be a small molecule or a protein, for example an
antibody, Fc fusion, or cytokine, that binds to a growth factor or
growth factor receptor involved in promoting angiogenesis. The
preferred anti-angiogenic factor herein is an antibody that binds
to Vascular Endothelial Growth Factor (VEGF). In an alternate
embodiment, the antibody is administered with a therapeutic agent
that induces or enhances adaptive immune response, for example an
antibody that targets CTLA-4. In an alternate embodiment, the
antibody is administered with a tyrosine kinase inhibitor. By
"tyrosine kinase inhibitor" as used herein is meant a molecule that
inhibits to some extent tyrosine kinase activity of a tyrosine
kinase. In an alternate embodiment, the antibody variants of the
present invention are administered with a cytokine. By "cytokine"
as used herein is meant a generic term for proteins released by one
cell population that act on another cell as intercellular
mediators.
[0105] Pharmaceutical compositions are contemplated wherein an
antibody variant of the present invention and one or more
therapeutically active agents are formulated. Formulations of the
antibody variants of the present invention are prepared for storage
by mixing said antibody having the desired degree of purity with
optional pharmaceutically acceptable carriers, excipients or
stabilizers (Remington's Pharmaceutical Sciences 16th edition,
Osol, A. Ed., 1980), in the form of lyophilized formulations or
aqueous solutions. The formulations to be used for in vivo
administration are preferrably sterile. This is readily
accomplished by filtration through sterile filtration membranes or
other methods. The antibodies and other therapeutically active
agents disclosed herein may also be formulated as immunoliposomes,
and/or entrapped in microcapsules
[0106] The concentration of the therapeutically active antibody
variant in the formulation may vary from about 0.1 to 100 weight %.
In a preferred embodiment, the concentration of the antibody is in
the range of 0.003 to 1.0 molar. In order to treat a patient, a
therapeutically effective dose of the antibody variant of the
present invention may be administered. By "therapeutically
effective dose" herein is meant a dose that produces the effects
for which it is administered. The exact dose will depend on the
purpose of the treatment, and will be ascertainable by one skilled
in the art using known techniques. Dosages may range from 0.01 to
100 mg/kg of body weight or greater, for example 0.1, 1, 10, or 50
mg/kg of body weight, with 1 to 10 mg/kg being preferred. As is
known in the art, adjustments for antibody degradation, systemic
versus localized delivery, and rate of new protease synthesis, as
well as the age, body weight, general health, sex, diet, time of
administration, drug interaction and the severity of the condition
may be necessary, and will be ascertainable with routine
experimentation by those skilled in the art.
[0107] Administration of the pharmaceutical composition comprising
an antibody variant of the present invention, preferably in the
form of a sterile aqueous solution, may be done in a variety of
ways, including, but not limited to, orally, subcutaneously,
intravenously, intranasally, intraotically, transdermally,
topically (e.g., gels, salves, lotions, creams, etc.),
intraperitoneally, intramuscularly, intrapulmonary (e.g., AERx.RTM.
inhalable technology commercially available from Aradigm, or
Inhance.TM. pulmonary delivery system commercially available from
Inhale Therapeutics), vaginally, parenterally, rectally, or
intraocularly.
EXAMPLES
[0108] Examples are provided below to illustrate the present
invention. These examples are not meant to constrain the present
invention to any particular application or theory of operation.
[0109] For all constant region (CL, CH1, hinge, CH2, and CH3)
positions discussed in the present invention, numbering is
according to the EU numbering scheme (Kabat et al., 1991, Sequences
of Proteins of Immunological Interest, 5th Ed., United States
Public Health Service, National Institutes of Health, Bethesda),
which refers to the numbering of the EU antibody (Edelman et al.,
1969, Proc Natl Acad Sci USA 63:78-85). For all variable region (VL
and VH) and J segment (JH and JL) positions discussed in the
present invention, numbering is according to the Kabat numbering
scheme (Kabat et al., 1991, Sequences of Proteins of Immunological
Interest, 5th Ed., United States Public Health Service, National
Institutes of Health, Bethesda). Exceptions to these numbering
schemes are noted where they occur. Those skilled in the art of
antibodies will appreciate that these conventions consist of
nonsequential numbering in specific regions of an immunoglobulin
sequence, enabling a normalized reference to conserved positions in
immunoglobulin families. Accordingly, the positions of any given
immunoglobulin as defined by EU numbering or Kabat numbering will
not necessarily correspond to its sequential sequence.
[0110] FIGS. 3a and 3b provide the VL (.kappa.) and VH sequences
for four commercial antibodies, along with the sequences of human
germline J.kappa. and JH segments, with positions numbered
according to the Kabat numbering scheme. Position numbering
according to another common numbering scheme, that of Chothia, is
also provided. FIGS. 3c-3f provide the CL (.kappa. and .lamda.) and
IgG constant region sequences, with numbering according to the EU
numbering scheme. The boundaries of the regions in FIG. 3 are
designated based on both genetic (variable, J segment, and constant
region boundaries) and structural (CH1, hinge, CH2, and CH3
boundaries). It should be noted that polymorphisms have been
observed at a number of immunoglobulin positions (for example see
Kim et al., 2001, J Mol Evol 53:1-9), and thus slight differences
between the presented sequence and sequences in the scientific
literature may exist.
Example 1
Regions Outside Fc can Impact Interaction of an Antibody with
Effector Ligands
[0111] Essentially all research aimed at modifying antibody
effector function has focused on the Fc region, intuitively because
it comprises the binding sites for the Fc.gamma.Rs and C1q. The
present invention provides support for a role of antibody regions
outside of the Fc region that affect Fc/effector ligand
interaction. An initial basis of support for a role of the variable
region in antibody effector function is the observation that
antibodies that differ solely in their variable region sequences
mediate differing levels of effector function. There are a number
of potential explanations for this result, the most reasonable of
which have concerned the properties of the target antigen,
including expression level, structural accessibility, and so forth.
Data set forth in the present invention suggest, however, that the
variable region can affect antibody/effector ligand affinity.
[0112] Binding affinity to human Fc.gamma.RIIIa was measured for
two antibodies with identical Fc regions yet different variable
regions--Campath.RTM. and Herceptin.RTM.. Campath (alemtuzumab,
Campath-1H, a registered trademark of Ilex Pharmaceuticals LP) is a
humanized anti-CD52 antibody currently approved for treatment of
B-cell chronic lymphocytic leukemia. Herceptin (trastuzumab, a
registered trademark of Genentech) is a humanized anti-Her2/neu
antibody currently approved for treatment of breast cancer. The
genes for the variable regions of Campath and Herceptin were
constructed using recursive PCR, and subcloned into the mammalian
expression vector pcDNA3.1Zeo (Invitrogen) comprising the full
length light kappa (C.kappa.) and heavy chain IgG1 constant
regions. DNA was sequenced to confirm the fidelity of the
sequences. Plasmids containing heavy chain gene (VH-CH1-CH2-CH3)
(wild-type or variants) were co-transfected with plasmid containing
light chain gene (VL-C.kappa.) into 293T cells. Media were
harvested 5 days after transfection, and antibodies were purified
from the supernatant using protein A affinity chromatography
(Pierce).
[0113] In order to screen for Fc.gamma.R binding, the extracellular
region of human V158 Fc.gamma.RIIIa was expressed and purified. The
extracellular region of this receptor was obtained by PCR from a
clone obtained from the Mammalian Gene Collection (MGC:22630). The
receptor was fused with glutathione S-Transferase (GST) to enable
screening. Tagged Fc.gamma.RIIIa was transfected in 293T cells, and
media containing secreted Fc.gamma.RIIIa were harvested 3 days
later and purified. Binding affinity to human Fc.gamma.RIIIa by the
antibodies was measured using a quantitative and extremely
sensitive method, AlphaScreen.TM. assay. The AlphaScreen is a
bead-based luminescent proximity assay. Laser excitation of a donor
bead excites oxygen, which if sufficiently close to the acceptor
bead will generate a cascade of chemiluminescent events, ultimately
leading to fluorescence emission at 520-620 nm. The AlphaScreen was
applied as a competition assay for screening the antibodies.
Wild-type Campath and Herceptin antibodies were biotinylated by
standard methods for attachment to streptavidin donor beads, and
tagged human Fc.gamma.RIIIa (Val158 isoform) was bound to
glutathione chelate acceptor beads. In the absence of competing
antibody, antibody and Fc.gamma.R interact and produce a signal at
520-620 nm. Addition of untagged antibody competes with the
Fc/Fc.gamma.R interaction, reducing fluorescence quantitatively to
enable determination of relative binding affinities. FIG. 4
presents the AlphaScreen binding data for the binding of Campath
and Herceptin to Fc.gamma.RIIIa. The binding data were normalized
to the maximum and minimum luminescence signal provided by the
baselines at low and high concentrations of competitor antibody
respectively. The data were fit to a one site competition model
using nonlinear regression, and these fits are represented by the
curves in the figure. These fits provide the inhibitory
concentration 50% (IC50) (i.e. the concentration required for 50%
inhibition) for each antibody, thus enabling the relative binding
affinities of Fc variants to be quantitatively determined. The
results show that Campath has significantly greater affinity for
the receptor than Herceptin.
[0114] The difference in Fc.gamma.R binding affinities between
Campath and Herceptin was further corroborated using Surface
Plasmon Resonance (SPR) (Biacore, Uppsala, Sweden). SPR is a
sensitive and quantitative method that allows for the measurement
of binding affinities of protein-protein interactions, and has been
used effectively to measure Fc/Fc.gamma.R binding (Radaev et al.,
2001, J Biol Chem 276:16478-16483). GST-tagged V158 or F158
Fc.gamma.RIIIa was immobilized to an SPR chip, and Campath and
Herceptin antibodies were flowed over the chip at a range of
concentrations. Binding constants were obtained from fitting the
data using standard curve-fitting methods. Table 1 presents
dissociation constants (Kd) for binding of Campath and Herceptin to
V158 Fc.gamma.RIIIa and F158 Fc.gamma.RIIIa obtained using SPR.
TABLE-US-00001 TABLE 1 V158 Fc.gamma.RIIIa F158 Fc.gamma.RIIIa Kd
(nM) Kd (nM) Campath 68 730 Herceptin 364 503
[0115] The SPR results confirm the AlphaScreen data, further
indicating that there is a difference in the binding affinities of
the two antibodies for Fc.gamma.RIIa. Furthermore, there is a
substantial difference in binding specificity for the different
Fc.gamma.RIIIa isoforms. Because the constant region sequences of
the Campath and Herceptin antibodies used in these experiments,
including CL, CH1, CH2, and CH3, are identical, there exists some
difference or differences between these antibodies in their
variable regions that impacts the Fc/Fc.gamma.R interaction.
[0116] Another set of data that suggests that there is more to the
antibody/effector ligand interaction than the residues at the
Fc/Fc.gamma.R interface is the observation that a number of
mutations at Fc residues distal to the Fc/Fc.gamma.R interface
affect affinity of the interaction (U.S. Ser. No. 10/672,280, U.S.
Ser. No. 10/822,231, U.S. Ser. No. 60/627,774, and U.S. Ser. No.
60/642,477). FIG. 5a shows AlphaScreen data for binding to human
V158 Fc.gamma.RIIIa by Fc variants E272Y and K274E, carried out as
described above. These substitutions significantly enhance the
affinity of Fc for Fc.gamma.RIIIa, in the case of E272Y by an order
of magnitude. As shown in FIG. 5b, however, these residues, are
distal to the Fc/Fc.gamma.R interface. Thus it appears that these
residues are playing some role in the Fc/Fc.gamma.R binding event,
but one which does not involve direct interaction at the interface.
One possible explanation for the effect of these mutations on
Fc.gamma.R binding is suggested by visual inspection of the
structure of a full length human antibody (pdb accession code 1HZH,
Saphire et al., 2002, J Mol Biol 319:9-18). This structure,
illustrated in FIG. 6a, shows that a full length antibody is not an
extended structure as suggested by the model in FIG. 1, but rather
has intra-molecular interactions between the Fab and Fc regions,
shown in a closer view in FIG. 6b. Comparing the human complex
structure in FIG. 6a with the Fc/Fc.gamma.R complex structure in
FIG. 2, it seems that the Fab/Fc interaction would sterically
occlude binding of Fc to Fc.gamma.Rs, as well as potentially C1q
based on the putative Fc/C1q binding site. E272 (EU numbering) in
the 1IIS structure (Radaev et al, 2001, J Biol Chem
276:16469-16477) corresponds to position E285 in the 1HZH structure
(the Fc sequences in the two structures must be been aligned to
identify the corresponding residues). FIG. 7 provides a closeup
view of the Fab/Fc binding site, showing that E272 in fact resides
at the Fab/Fc interface and makes potential interactions with
residues in the Fab region. Overall there is a substantial amount
of charge complementarity at the interface, with a number of
positively charged Fab residues (R77, R106, K107, and R108; Kabat
and 1HZH numbering) facing a number of negatively charged Fc
residues (E282, D283, and E285 in 1HZH, corresponding to EU
numbering 269, 270, and 272 respectively). Notably, Fc.gamma.RIIIa
also has a number of positively charged residues that mediate
interaction with Fc, further supporting the idea that the
positively charged Fab competes with the positively charged
Fc.gamma.R for the negatively charged Fc.
[0117] Given the high degree of flexibility between the different
antibody domains, the conformation of the binding interface may not
be well-ordered, or may be one of an ensemble of Fab/Fc interaction
conformations. Indeed there are a lower number of specific
interactions in the 1HZH structure relative to other
protein-protein interfaces, and two other structures of full length
murine antibodies, shown in FIGS. 8a and 8b, also indicate
intra-molecular interaction between Fab and Fc, but in different
conformations (pdb accession code 1GY, Harris et al., 1995, Nature
360:369-372; pdb accession code 1GT, Harris et al., 1997,
Biochemistry 36:1581-1597). Accordingly, discussion of E272 and
K274 is not meant to imply that they are necessarily specific
binding determinants of the Fab/Fc interaction, nor that
electrostatics necessarily play the dominant role. Rather, these
residues are provided to illustrate that a collection of loosely
defined interactions may characterize the Fab/Fc binding event.
Because Fab/Fc interaction is intramolecular, a substantial amount
of the entropy cost binding has already been paid. Thus it is
reasonable to suspect that a loose set of interactions, for example
one or more clusters of electrostatic interactions, may provide
sufficient energy to promote binding.
[0118] It follows that a possible explanation for the enhanced
Fc.gamma.R binding properties of E272Y and other Fc variants distal
to the Fc/Fc.gamma.R interface that enhance binding, is that by
disrupting the favorable interaction between Fab and Fc, the
equilibrium between them is disfavored, and thus Fc resides in an
unbound state that is free to interact with Fc.gamma.R. In effect,
the Fab region competes with Fc.gamma.R for binding to Fc. A model
describing this inhibition hypothesis is illustrated in FIG. 9a.
The unbound form of the antibody is in equilibrium with a set of
mutually exclusive bound states--one wherein Fc is bound to
effector ligand, and one wherein Fc is bound to Fab. The model
shows additionally that perhaps interactions between the Fab
regions may also exclude or compete for binding to effector ligand.
The equilibrium constants that govern this system are K.sub.Fc/Fab,
which defines the equilibrium between Fc and Fab, K.sub.Fab/Fab,
which defines the equilibrium between Fab and Fab, and
K.sub.Fc/Fc.gamma.R, which defines the equilibrium between Fc and
Fc.gamma.R. Thus different Fab and Fc sequences, although having
limited impact on K.sub.Fc/Fc.gamma.R directly, may affect the
observed affinity of Fc/Fc.gamma.R by altering K.sub.Fc/Fab or
K.sub.Fab/Fab. An alternative to the inhibition model is one in
which, rather than competing with effector ligand binding, the Fab
region positively impacts effector ligand binding. This activation
model is illustrated in FIG. 9b. In this model, the Fab region
makes favorable interactions with Fc.gamma.R, the Fc/Fc.gamma.R
complex, or both. Thus the Fab/Fc.gamma.R+Fc/Fc.gamma.R state and
the Fab/Fc/Fc.gamma.R state are more stable complexes than the
Fc/Fc.gamma.R state. These two models are meant to provide a
hypothetical account for differences in Fc/Fc.gamma.R binding
observed between antibody variable regions and by Fc mutations
distal to the Fc/Fc.gamma.R interface. Although the models are
shown with an Fc.gamma.R as the effector ligand, they are meant to
generally apply for any effector ligands as defined herein. For
example it follows that, since the C1q site is proximal and
overlapping with the Fc.gamma.R binding site on Fc, the same models
may apply to binding of Fc to C1q.
[0119] An implication of either of the proposed models is that they
suggest that engineering substitutions in regions outside of the
antibody Fc region may be a means to optimize effector ligand
binding and effector function. As discussed, there are many
possible reasons for the differing levels of effector function
observed for therapeutic antibody, including but not limited
expression level, availability, and accessibility of target
antigen. The present invention suggests that another possible
parameter determining the effector function of antibodies, and
potentially in turn their clinical behavior, is the impact of the
Fab and hinge region on interaction of the Fc region with effector
ligands. It is contemplated that substitutions can be engineered
into the Fab and hinge regions that modulate this effect, and thus
favor or disfavor interaction of the antibody with effector ligands
so that antibodies can be tuned for a desired clinical outcome.
Example 2
Engineered Variable Region Variants
[0120] As discussed, the flexibility between the Fab and Fc
regions, as well as the differences between different antibody
variable regions, may dictate that different antibodies have
different Fab/Fc interactions. Thus although the available
structures (1HZH, 1GY, and 1GT as described above) may provide
information on the Fab/Fc interface, it may be imprudent to rely on
this information as a definitive structural picture. Another
important source of information is the structure activity
relationship (SAR) data provided by different antibodies with
different effector ligand affinities. For example, as described
above, there exists some difference or differences between Campath
and Herceptin in their variable regions that impact interaction of
the antibody with Fc.gamma.R. FIGS. 3a and 3b show alignments of
the Campath and Herceptin VL and VH sequences respectively. These
alignments highlight the differences between the two antibodies
(shown in bold) that are putatively involved in determining their
Fc.gamma.R affinity differences. Thus one strategy for
characterizing Fab/Fc interaction, and for designing variable
region variants with altered effector ligand affinity and effector
function, is to replace some or all of the residues in Herceptin
with the corresponding residues in Campath. Substitutions may be
engineered at FR positions, CDR positions, or both. Here, because
Herceptin is the weaker Fc.gamma.R binder, replacement of one or
more residues with the corresponding Campath amino acid is expected
to enhance Fc.gamma.R.
[0121] Based on this strategy, a variant library was designed
wherein for residues at which the two antibody variable regions
differ, the Herceptin residue was replaced with the corresponding
Campath-1G residue. Only surface exposed differences (determined by
visual inspection of a modeled Herceptin structure) were considered
in order not to perturb the stability of the variable region.
Residues were grouped according to structural proximity to each
other; CDR residues were grouped together accordingly (VL CDR1, VL
CDR2, etc.), and were defined according to the structural
definition of Chothia (Chothia & Lesk, 1987, J. Mol. Biol. 196:
901-917; Chothia et al., 1989, Nature 342: 877-883; AI-Lazikani et
al., 1997, J. Mol. Biol. 273: 927-948). Table 2 provides a list of
the designed VL and VH variants. In this library, VL1, VL2, and VL3
represent the light chain CDR differences between Herceptin and
Campath-1G, VH1, VH2, and VH3 represent the heavy chain CDR
differences, VL4 and VH4 provide the combined CDR differences for
VL and VH respectively, and VL4-VH4 provides all mutated CDRs.
Residues are numbered according to Kabat. Dashes denote deletions
or insertions; for example, -52bK indicates an insertion of lysine
at Kabat position 52b, and A100b--indicates a deletion of alanine
at Kabat position 100b.
TABLE-US-00002 TABLE 2 Variable Region Variants Variant
Substitution(s) (Kabat numbering) VL1 D28N/N30D/T31K/A32Y VL2
S50N/A51T/S52N/F53N/Y55Q/S56T VL3 Y92I/T93S/T94R/P96R VL4
D28N/N30D/T31K/A32Y/S50N/A51T/S52N/F53N/
Y55Q/S56T/Y92I/T93S/T94R/P96R VL5 Q3K/Q100T VL6 S10F VL7 T22N/R24K
VL8 P40L/K42E/A43S VL9 R66G VL10 F83V/I106L VL11 Y87F VL12
K103A/E105A.sup.a VL13 K107A/R108A.sup.a b VH1 N28T/K30T/T32F VH2
R50F/Y52R/P52aD/-52bK/-52cA/T53K/N54G/G55Y/Y56T VH3
W95E/G97H/D98T/G99A/F100A/Y100aP/A100b-.sup.c VH4
N28T/K30T/T32F/R50F/Y52R/P52aD/-52bK/-52cA/
T53K/N54G/G55Y/Y56T/W95E/G97H/D98T/G99A/F100A/ Y100aP/A100b-.sup.c
VH5 Q3K/V5L VH6 L18M/A24G VH7 H35N VH8 A40P/P41A VH9 G44A/L45P VH10
R58E/A60N/D61P VH11 A71R VH12 T73N/S74T/K75Q/T77M VH13 S82bT VH14
V89T/T107V/L108M VL4-VH4 VL4 + VH4 .sup.aVL12 and VL13 are double
alanine mutations at Herceptin K103 and E105, and K107 and R108
respectively, and are not differences between Herceptin and
Campath-1G. .sup.bResidue R108 is in the C.kappa. region, and
numbering is according to the EU numbering scheme .sup.cVH residues
Herceptin M100d and Campath-1G F100d were aligned with the
sequential JH region (see FIG. 3b), and as a result are not
numbered according to Kabat.
[0122] These variants were constructed in the variable region of
the antibody Herceptin in the pcDNA3.1Zeo vector using quick-change
mutagenesis techniques (Stratagene), expressed in 293T cells, and
purified as described above. Binding affinity to human
Fc.gamma.RIIIa was measured for the variants using the AlphaScreen
assay as described above. The results, provided in FIG. 10, show
that a number of the variants significantly enhance the binding of
Herceptin to Fc.gamma.RIIIa. The binding data were normalized to
the maximum and minimum luminescence signal for each particular
curve, provided by the baselines at low and high antibody
concentrations respectively. The data were fit to a one site
competition model using nonlinear regression, and these fits are
represented by the curves in the figure. These fits provide the
inhibitory concentration 50% (IC50) (i.e. the concentration
required for 50% inhibition) for each antibody, enabling the
relative binding affinities of Fc variants to be quantitatively
determined. By dividing the IC50 for each variant by that of WT
Herceptin, the fold-enhancement or reduction relative to WT
Herceptin (Fold WT) were obtained. These values are provided in
Table 3. Here a fold above 1 indicates an enhancement in binding
affinity, and a fold below 1 indicates a reduction in binding
affinity relative to WT Herceptin. The Fc.gamma.R binding
affinities of the variants were further investigated using Surface
Plasmon Resonance (SPR) (Biacore, Uppsala, Sweden).
Fc.gamma.RIIIa(V158)-GST was immobilized to an SPR chip, and WT and
variant Herceptin antibodies were flowed over the chip at a range
of concentrations. FIG. 11 shows the raw data obtained from this
set of binding experiments. Binding constants were obtained from
fitting the data using standard curve-fitting methods. Table 3
presents dissociation constants (Kd) and Fold Kd relative to WT
(Fold WT) for binding of select Fc variants to human V158
Fc.gamma.RIIIa obtained using SPR, and compares these with IC50s
obtained from the AlphaScreen assay. The correlation between Fold
WT's obtained from the SPR and AlphaScreen binding measurements is
shown in FIG. 12; the good fit of these data to a straight line
(R.sup.2=0.86) supports the accuracy of the data. The SPR data
corroborate the improvements to Fc.gamma.RIIIa affinity observed by
AlphaScreen assay.
TABLE-US-00003 TABLE 3 AlphaScreen SPR Antibody IC50 (uM) Fold WT
Kd (uM) Fold WT WT Herceptin 0.502 1.00 1.44 1.00 VL1 1.22 1.18 VL2
0.346 1.45 2.58 0.56 VL3 0.290 1.73 0.42 3.46 VL4 0.352 1.43 0.28
5.15 VL5 0.972 0.52 1.49 0.97 VL6 0.524 0.96 1.88 0.77 VL7 1.002
0.50 2.18 0.66 VL8 0.727 0.69 2.54 0.57 VL9 0.672 0.75 2.92 0.49
VL10 0.392 1.28 2.04 0.71 VL11 0.336 1.49 1.55 0.93 VL12 2.14 0.68
VL13 0.249 2.02 1.35 1.07 VH1 4.98 0.29 VH2 0.004 125.48 0.12 12.50
VH3 0.158 3.17 1.06 1.36 VH4 0.098 5.11 0.79 1.82 VH5 0.111 4.51
0.76 1.90 VH6 0.342 1.47 2.17 0.66 VH7 0.039 12.98 0.42 3.46 VH8
3.367 0.15 3.64 0.40 VH9 0.005 96.53 0.16 8.90 VH10 0.083 6.04 0.65
2.24 VH11 0.446 1.12 1.51 0.95 VH12 0.065 7.72 0.61 2.37 VH13 0.308
1.63 1.67 0.87 VH14 0.013 40.14 0.25 5.77 VL4-VH4 0.106 4.74 0.75
1.92
[0123] In one embodiment, variants that bind Fc.gamma.R with
greater than 1-fold affinity relative to WT may be considered as
providing improved or enhanced binding to an effector ligand. In a
preferred embodiment, variants that bind with greater than 2-fold
affinity may be considered. In a particularly preferred embodiment,
variants that bind Fc.gamma.R with greater than 3-fold affinity
relative to WT may be considered as providing enhanced effector
ligand binding. FIG. 13 shows 4 variants, VH2, VH7, VH9, and VH14,
that provide greater than 10-fold Fc.gamma.R affinity relative to
WT, mapped onto the Herceptin Fab structure (pdb accession code
1FVE, Eigenbrot et al, 1993, J Mol Biol 229:969-995). The structure
shows that the residues reside in VH CDR2 (variant VH2) and at or
proximal to the interface between VL and VH (variants VH7, VH9, and
VH14). The use of Campath and Herceptin sequences here is only an
example, and is not meant to constrain the concept that variable
region engineering can be used to modulate binding to effector
ligands and effector function to these particular antibodies. The
variable regions of other antibodies, particularly ones that show
differential binding to effector ligands, may be used. For example,
also included in FIGS. 3a and 3b are alignments of the VH and VL
sequences of Rituxan and Erbitux, although any two or more
antibodies can be used to generate modifications and tested as
outlined here.
[0124] Indeed it may be that differences in effector ligand binding
and effector function for different antibodies are not determined
by simplistic rules, for example by specific variable region
residues that consistently effector ligand binding for all
antibodies. The implication here is that no set of mutations can be
made in a Fab that are generally applicable for controlling the
effector ligand affinity/specifity and effector function of all
antibodies. A reasonable approach to this problem, and an
embodiment of the present invention, is to characterize the
effector ligand binding and effector function properties of groups
of similar variable regions. In a most preferred embodiment, this
concept is applied to the human variable region and J segment
germline sequences. There are approximately 40 functional V.kappa.
genes located on chromosome 2, about 30 functional V.lamda. genes
on chromosome 22, and approximately 50 functional VH germline genes
located on chromosome 14, along with 5 J.kappa. sequences, 7
J.lamda.sequences, and 6 JH sequences (Cox et al., 1994, Eur J
Immunol 24:827-836; Barbie & Lefranc, 1998, Exp Clin
Immunogenet 15:171-183; Williams et al., 1996, J Mol Biol
264:220-232; Kawasaki et al., 1997, Genome Res 7: 250-261; Pallares
et al., 1998, Exp Clin Immunogenet 15:8-18; Tomlinson et al., 1992,
J Mol Biol 227:776-798; Matsuda & Honjo, 1996, Advan Immunol
62:1-29; Matsuda et al., 1998, J Exp Med 188:2151-62; Scaviner et
al., 1999, Exp Clin Immunogenet 16:234-40). The V.kappa., V.lamda.,
and VH germline sequences vary in size, and can be grouped into
subfamilies based on sequence homology. There are approximately 7
V.kappa. subfamilies, 8-10 V.lamda. subfamilies, and 6-7 VH
subfamilies; the binning into subfamilies is somewhat arbitrary,
depending on how the homology cutoff between families is defined.
The idea is to characterize the impact on effector ligand binding
and/or effector function of each germline sequence, or given that
the sequences within a given subfamily may behave similarly, each
germline subfamily. Antibodies comprising the sequences, or one or
more representative sequences from each subfamily, may be produced
and screened experimentally to determine their binding affinity to
Fc.gamma.R, C1q, or other effector ligands, and/or their ability to
mediate effector function in one or more cell-based ADCC, ADCP,
and/or CDC assays. Ideally the constant regions in such an
experiment would be the same between the different antibodies. The
variable regions for these antibodies may comprise the germline
sequences themselves, or may be naturally occurring antibodies that
were derived from these sequences, determined for example by
sequence homology. The end result would be that for each antibody
germine sequence or each germline subfamily, the relative affinity
for each effector ligand and the relative capacity to mediate each
effector function would be known.
[0125] An important application of such information would be its
use in immunogenicity reduction methods that are employed to
replace nonhuman sequences (typically mouse and rat) in an antibody
with sequences that are more common in humans. The dominant method
in use for antibody immunogenicity reduction, referred to as
"humanization", relies principally on the grafting of "donor"
(typically mouse or rat) complementarity determining regions (CDRs)
onto "acceptor" (human) VL and VH frameworks (FRs) (Tsurushita
& Vasquez, 2004, Humanization of Monoclonal Antibodies,
Molecular Biology of B Cells, 533-545, Elsevier Science (USA)).
This strategy is referred to as "CDR grafting" (Winter U.S. Pat.
No. 5,225,539). "Backmutation" of selected acceptor framework
residues to the corresponding donor residues is often required to
regain affinity that is lost in the initial grafted construct (U.S.
Pat. No. 5,530,101; U.S. Pat. No. 5,585,089; U.S. Pat. No.
5,693,761; U.S. Pat. No. 5,693,762; U.S. Pat. No. 6,180,370; U.S.
Pat. No. 5,859,205; U.S. Pat. No. 5,821,337; U.S. Pat. No.
6,054,297; U.S. Pat. No. 6,407,213). Knowledge of the effector
ligand affinity and effector function of individual germline
variable region sequences and/or subfamilies would allow acceptor
frameworks to be chosen based on the desired effector ligand
affinity and specificity, and/or the desired degree of effector
function.
[0126] A method that is particularly well suited for engineering
variable regions with altered effector ligand properties is
described in U.S. Ser. No. 11/004,590, filed Dec. 3, 2004, entitled
"Methods of Generating Variant Proteins with Increased Host String
Content and Compositions Thereof". This method reduces antibody
immunogenicity by maximizing the content of human linear sequence
strings, typically by utilizing the local sequence information
contained in an alignment of human sequences. In this way
immunogenicity is addressed at the local sequence level. This
strategy provides a more accurate measure of the immunogenicity,
and employs an optimal balance between binding determinants and
humaness. With respect to effector ligand properties, the advantage
of this method is that it samples a large diversity of local
sequence and structure space, which is both quality in structure
and stability and low in immunogenicity. Indeed, the effector
ligand determinants in the variable region need not reside over the
entire sequence, but rather may occur at the local level. By
sampling this diversity, a library of stable, soluble, and low
immunogenic variable region variants can be sampled, some of which
may possess different effector ligand and effector function
properties than others. Thus this method may be employed to
optimize effector function during the immunogenicity reduction
process, or to reengineer a variable region with suboptimal
effector function such that it possesses more favorable effector
function properties. The engineering and screening of multiple
variable region sequences for their effector ligand and effector
function properties, and further the use of this information as a
criteria for evaluating clinical candidates, are embodiments of the
present invention.
Example 3
Engineered JL, JH, CL, and CH1 Variants
[0127] In contrast to VH and VL sequences, the J segments encoding
the C-termini of the variable regions and constant light and first
constant heavy regions are more conserved among antibodies of the
same class and among therapeutically useful antibodies. Thus
residues within these regions that play a role in determining
effector ligand affinity/specificity and effector function
properties may be more consistent from antibody to antibody, and
accordingly variants that alter these properties may be more
generally applicable to therapeutically useful antibodies. In order
to characterize the effector determinants in these regions of the
Fab, and to generate variants that modulate effector ligand binding
and effector function, substitutions were engineered using a
computational, structure-based and sequence-based approach.
[0128] A set of computational structure-based design calculations
were carried out to design point mutations that retain a stable,
well-folded, structure using a design algorithm described generally
as Protein Design Automation.RTM. (PDA.RTM.) technology, as
described in U.S. Pat. No. 6,188,965; U.S. Pat. No. 6,269,312; U.S.
Pat. No. 6,403,312; U.S. Ser. No. 09/782,004; U.S. Ser. No.
09/927,790; U.S. Ser. No. 10/218, 102; PCT WO 98/07254; PCT WO
01/40091; and PCT WO 02/25588. These algorithms use an energy
function with terms that include van der Waals forces,
electrostatic forces, hydrogen bonding, desolvation interactions,
and entropy. Other statistical energy terms include those based on
known structures and those that compensate for effects on the
unfolded state. Calculations were run using the 1.8 .ANG.
resolution structure of the anti-ErbB2 antibody Fab2C4 (pdb
accession code 1L7I, Vajdos et al., 2002, J Mol Biol 320(2):415-28)
as a structural template. The energies of all 20 natural amino
acids were evaluated for all J.kappa., JH, C.kappa., and CH
positions. The lowest energy rotamer conformation for all 20 amino
acids was determined, and this energy was defined as the energy of
substitution for that amino acid at that variable position. These
calculations thus provided an energy of substitution for each of
the 20 amino acids at each variable position. FIG. 14 shows the
energy predictions from these calculations. In addition to
computational screening calculations, sequence information was also
used to guide variant design. Sequences encoding the human
immunoglobulin kappa light J region (J.kappa.), lambda light J
region (J.lamda.), heavy J region (JH), kappa light constant region
(C.kappa.), lambda light constant region (C.lamda.), and IgG1-IgG4
gamma heavy constant regions (CH1, CH2, CH3, and CH4) were aligned,
shown in FIG. 15. Additionally, visual inspection of the 1L7I Fab
structure was used to identify residues that are surface exposed,
boundary residues, core residues, and residues that reside at or
may impact important binding interfaces, such as interfaces with
other Ig domains in the Fab and the antigen binding interface.
These positions are listed in FIG. 15. Together, these
calculations, alignments, and structural analyses were used to
design a set of variants, listed in FIG. 15.
[0129] Designed variants were constructed in the light and heavy
chains of Campath in the pcDNA3.1Zeo vector using quick-change
mutagenesis, expressed in 293T cells, and purified as described
above. Binding affinity to human V158 Fc.gamma.RIIIa, human F158
Fc.gamma.RIIIa, and bacterial protein A were measured for the
variants using the AlphaScreen assay as described above. FIG. 16
provides a representative data set from this series of binding
assays, showing select Fab variants that enhance Fc.gamma.R
binding. Also shown for comparison are two Fc variants, I332E and
S239D/I332E, that have been previously been shown to enhance
binding to Fc.gamma.RIIIa and ADCC in a cell based assay. Fold WT
values for these data were generated as described above, and these
are provided in Table 4. In this table, variants in the JL and JH
regions are numbered according to the Kabat numbering scheme, and
variants in the CL and CH1 regions are numbered according to the EU
numbering scheme. Of particular importance are modifications that
result in greater than 1-fold increase in affinity as compared to
the wild-type (e.g. parent antibody) for any particular receptor.
Thus, for example, in the JL and CL regions, all mutants exhibited
better affinity to the RIIIa receptor than either of the
"wild-types" tested. It is an aspect of the invention to provide
modifications that provide higher affinity for at least one
receptor.
TABLE-US-00004 TABLE 4 Fc.gamma.R Binding Data for JL, JH, CL, and
CH1 Variants Fold WT Fc.gamma.RIIIa Fc.gamma.RIIIa Substitution(s)
V158 F158 ProtA JL Region Kabat Q100P 2.48 4.46 Q100G 2.48 3.47
Q100K 3.91 5.73 K103R 3.54 5.62 K103D 2.98 4.00 K103L 4.28 6.90
E105D 2.90 3.84 E105K 12.36 21.88 E105I 7.68 12.47 I106L 6.05 8.94
K107E 2.75 4.15 K107L 2.22 3.00 CL Region EU R108Q 3.64 5.47 R108D
5.65 9.28 R108I 8.97 16.24 T109P 4.08 5.86 T109R 3.98 5.59 T109D
3.28 4.62 V110K 5.31 8.95 V110E 2.46 3.43 V110I 2.06 2.90 A111K
5.64 8.18 A111E 5.57 9.45 A111L 2.90 4.11 A112R 4.81 8.72 A112E
5.34 8.70 A112Y 6.96 11.48 S114D 1.64 2.32 S114K 3.35 5.06 S114I
3.57 4.67 F116T 2.25 3.30 S121D 1.33 1.30 D122S 2.67 3.09 D122R
D122Y 2.98 3.11 E123R 2.32 3.17 E123L 2.76 3.46 Q124E 3.86 6.46
L125E 4.18 5.79 L125K 0.23 0.17 K126Q 0.39 0.20 K126D 0.44 0.29
K126L 0.48 0.35 S127A 0.70 0.55 S127D S127K 0.42 0.27 G128N 0.70
0.48 T129K 0.76 0.51 T129E 0.83 0.62 T129I 1.28 0.78 S131T 0.45
0.31 N137S N137K 0.88 0.67 N138D 0.84 0.55 N138K 0.29 0.18 N138L
0.53 0.48 Y140K 0.74 0.56 Y140E 1.40 0.95 Y140H 1.91 1.47 P141K
1.89 1.91 P141E 1.26 0.98 R142G 0.49 0.34 R142L 0.39 R142D 0.72
0.52 E143A 1.18 0.90 E143R 1.48 0.94 E143L 1.22 0.80 K145T 1.07
0.79 K145D 0.65 0.53 K145Y 1.64 1.43 Q147A 3.35 2.71 1.41 Q147E
3.21 3.12 1.55 Q147K 3.83 3.53 1.26 K149D 5.19 K149Y 2.57 2.42 1.19
V150A 3.17 2.70 1.05 D151K 10.51 9.83 1.39 D151I 1.71 1.36 1.27
N152S 4.09 3.26 1.07 N152R 2.61 2.27 1.01 N152L 3.52 3.31 1.29
A153S 1.62 1.92 0.96 A153D 2.55 2.12 1.24 A153H 3.34 2.45 1.11
L154V 2.33 1.68 1.21 L154E 3.14 2.33 1.10 L154R 2.66 2.33 1.11
Q155K 4.27 3.47 1.35 Q155E 3.19 2.90 1.61 Q155I 2.49 2.21 1.39
S156A 4.01 3.75 1.42 S156D 1.96 1.63 1.44 S156R 3.61 2.51 1.53
G157N 1.24 0.95 2.32 N158R 4.05 2.83 1.79 N158D 5.00 4.44 1.82
N158L S159K 11.29 9.34 2.40 S159E 3.88 3.91 1.20 S159L 5.49 5.21
1.23 Q160V 0.87 0.92 1.25 Q160K E161K 1.19 1.45 0.93 E161L 0.76
0.80 0.88 S162T 0.95 0.94 1.27 V163T V163K 0.98 1.02 0.79 V163E
0.93 1.19 1.02 T164Q 2.40 2.60 1.41 E165P 2.40 2.51 1.17 E165K 1.42
1.53 1.07 E165Y Q166S 0.94 0.39 1.04 Q166E 0.67 0.90 1.01 Q166M
0.52 0.49 0.99 D167K 0.53 0.58 1.62 D167L 0.44 0.52 1.04 S168Q 0.48
0.43 1.11 S168K 0.41 0.34 2.48 S168Y K169S K169H K169D D170N 0.80
0.71 1.26 D170R 0.45 0.45 0.94 D170I 0.74 0.82 0.73 S171N 1.21 0.65
1.20 S171A 0.83 0.91 1.22 S171V 0.95 1.03 1.15 T172K 0.62 1.84 1.24
T172I 1.20 1.40 1.01 T172E 1.12 1.41 1.01 Y173K 1.04 2.54 1.30
Y173Q 1.35 1.52 1.00 Y173L 1.08 1.18 0.73 S174A 0.63 0.81 1.30
S176T 0.45 0.39 1.11 T180S 0.25 0.87 0.99 T180K 0.84 1.00 0.81
T180E 0.71 0.55 1.09 L181K 1.09 S182T 0.76 0.82 S182E 0.83 0.86
S182R 0.55 1.17 0.94 K183P 0.74 0.46 1.04 K183D 0.52 0.65 1.00
K183L 0.60 0.45 1.03 A184E 0.61 0.64 0.82 A184K 0.43 0.72 1.21
A184Y 0.76 0.87 D185Q D185R D185I 0.58 4.86 1.08 E187K 0.46 0.40
E187Y 0.52 0.49 1.10 K188S 1.05 0.69 1.11 K188E 0.84 1.14 0.97
K188Y 1.89 0.98 1.30 H189D 0.63 1.33 1.22 H189K 0.27 0.02 0.87
H189Y 0.16 0.01 0.89 K190R 0.15 0.02 0.97 K190E 0.07 0.03 1.00
K190L 0.31 0.03 1.03 V191S 0.35 0.03 1.32 V191E 0.52 0.03 1.24
V191R 2.30 4.00 1.20 A193S 0.30 0.02 1.06 A193E A193K 0.21 0.03
1.10 E195Q 0.19 0.03 1.01 E195K 17.77 7.43 4.93 E195I 0.60 0.81
1.09 T197E T197K 0.28 0.02 1.06 T197L Q199E 0.26 0.01 1.08 Q199K
0.45 0.03 1.23 Q199Y 0.52 1.04 1.01 G200S 0.16 0.01 1.00 S202D 0.21
0.01 0.98 S202R 0.18 0.02 1.13 S202Y 0.17 0.02 1.12 S203D 0.06 0.01
1.02 S203R 0.33 0.01 1.05 S203L 5.58 P204T 0.31 0.03 1.10 V205E
0.46 0.03 1.35 V205K 0.16 0.01 1.03 T206E 0.31 0.02 T206K 0.18 0.02
0.92 T206I 0.23 0.05 1.05 K207E 0.06 0.02 1.00 K207L 4.10 5.90 1.10
S208T 4.40 9.60 1.10 S208E S208K N210A N210E 3.10 3.10 0.90 N210K
R211P R211E G212T G212K G212E 8.30 5.30 1.40 E213R 5.30 4.00 0.80
E213L K207A/ 0.32 R211A* JH Region Kabat Y102L 1.58 2.34 Q105E
Q105E 0.99 1.21 Q105E 0.90 0.85 Q105L 1.48 1.69 S107T 1.24 0.99
L108T 1.39 1.21 L108E 1.60 2.41 L108K 1.10 1.15 T110K 1.70 1.63
T110E 2.03 2.95 T110I 2.88 3.37 S112D 3.77 6.18 S112K 2.00 2.03
S112Y 1.41 1.64 S113D 4.11 6.10 S113R 2.86 4.73 S113L CH1 Region EU
A118K 2.80 4.52 A118E 2.67 3.61 A118Y 3.00 3.68 S119R 2.22 2.54
S119E 11.81 15.89 3.39 S119Y 1.67 2.22 T120R 2.41 2.93 T120E 3.62
5.55
T120I 1.50 1.67 K121E 2.06 2.83 K121Y 1.62 1.62 K121H 2.89 3.95
G122E 4.17 5.31 G122R 1.41 S124K 1.33 1.66 S124E 1.43 1.46 S124Y
0.62 0.55 F126K 0.79 0.62 F126D A129L 0.72 0.74 A129D 0.78 0.67
S131G 0.60 0.51 S131T 0.38 0.38 S132D 1.12 1.20 S132R 0.45 S132L
0.72 0.53 K133R 0.84 0.70 K133E 0.83 0.62 K133L 0.38 0.33 T135I
0.61 0.48 T135E 0.76 0.64 T135K 0.63 0.37 S136E 0.56 0.46 S136K
0.43 0.30 S136I 0.76 0.59 G137E 0.64 0.50 G138S 1.04 1.08 G138R
0.69 0.65 G138D 0.90 0.80 T139I 0.65 0.53 T139E 0.92 0.76 T139K
0.66 0.52 K147A 0.68 0.59 K147E 0.65 0.41 D148Y 0.73 0.83 D148K
0.90 0.80 F150L 0.57 0.44 F150K 0.70 0.53 F150E 0.54 0.52 P151A
0.47 0.41 P151D E152L 0.44 0.31 E152K 0.50 0.50 P153L 0.54 0.69
P153D 1.03 1.34 T155E 0.61 0.81 T155K 0.72 0.65 T155I 0.66 0.70
S157E S157K 0.30 0.32 S157Y 0.53 0.60 N159K 0.45 0.41 N159D 0.52
0.49 N159L 0.39 0.44 S160K 0.66 0.55 S160E 0.76 0.45 S160Y 0.59
0.44 G161D 0.66 0.41 A162D 0.79 0.47 A162K A162Y 0.65 0.36 L163R
0.87 0.48 T164R 0.70 0.32 T164E 0.53 0.30 T164Y 0.70 0.41 S165D
0.67 0.37 S165R 0.67 0.45 S165Y G166D 0.76 0.47 V167A 0.46 0.39
H168L 0.98 1.05 T169E P171G 0.51 0.34 P171H 2.14 1.52 A172K 0.42
0.37 A172L 0.54 0.37 A172E 0.58 0.41 V173T 0.63 0.65 V173D 0.47
0.37 L174E 0.93 0.89 L174K L174Y 1.73 1.71 Q175D 0.67 0.52 Q175L
0.58 0.47 S176D 0.66 0.54 S176R 0.74 0.70 S176L 0.67 0.73 S177R
0.67 0.51 S177E 1.00 0.87 S177Y 0.81 0.62 G178D 0.73 0.57 L179K
0.66 0.67 L179Y L179E 0.70 0.48 Y180K 0.58 0.46 Y180L 0.61 0.59
Y180E 0.47 0.37 S183T 2.11 1.02 T187I 0.60 0.47 T187K 0.42 0.35
T187E 0.50 0.41 V188I 0.40 0.33 P189D 0.69 0.65 P189G 0.24 0.16
S190I 0.50 0.53 S190K 0.32 0.34 S190E 0.55 0.44 S191D 0.52 0.43
S191R 0.54 0.56 S191Y 0.51 0.44 S192N 0.67 0.62 S192R 0.69 0.69
S192L 0.32 0.29 L193F 0.57 0.51 L193E 0.72 0.58 G194R 0.63 0.54
G194D 0.79 0.70 T195R 0.29 0.29 T195D 0.53 0.41 T195Y 0.38 0.31
Q196K 0.26 0.29 Q196D 0.40 0.45 Q196L 0.42 0.29 T197R 0.40 0.34
T197E 0.44 0.41 T197Y 0.47 0.51 Y198L 1.04 1.11 I199T 0.37 0.26
I199D 0.32 0.34 I199K 0.31 0.30 N201E 2.23 3.47 N201K 0.87 0.94
N201L 0.77 1.06 N203D 0.87 0.87 N203L N203K 0.93 1.10 K205D 1.84
2.35 K205L 1.18 1.43 P206A 1.44 1.71 P206E 7.35 9.55 2.55 S207K
1.02 1.05 S207D 1.15 1.43 N208R 0.62 1.01 N208E 1.74 N208Y 0.93
T209E 0.86 0.78 T209K 0.90 1.04 T209Y 0.87 1.21 K210L 0.83 0.86
K210E 0.40 K210Y 1.45 V211R 1.23 1.49 V211E 0.97 1.05 V211Y 1.21
1.70 D212Q 0.40 D212K 0.73 D212H 0.30 D212L 0.53 D212Y 0.29 K213N
0.84 K213E 0.79 K213H 1.13 K213L 0.66 K213Y 0.34 K214N 1.20 K214E
1.43 K214H 0.84 K214L K214Y 1.53 E216N 0.30 E216K 2.04 E216H 0.68
E216L E216Y 1.12 P217D 1.22 P217H 0.19 P217A 0.94 P217V 1.56 P217G
1.21 K218D 1.17 K218E 0.66 K218Q 1.19 K218T K218H K218L K218Y S219D
S219E S219Q S219K S219T S219H S219L S219I S219Y 0.27 K205A/ 1.97
K210A* K213A/ 3.70 K214A/ K218A* *CL double variant K207A/R211A,
and CH1 double and triple variants K205A/K210A and
K213A/K214A/K218A were designed in the context of Herceptin, not
Campath
[0130] On the heavy chain, a number of variants show significant
improvements in Fc.gamma.RIIIa binding. In one embodiment, variants
that bind Fc.gamma.R with greater than 1-fold affinity relative to
WT may be considered as providing improved or enhanced binding to
an effector ligand. In a preferred embodiment, variants that bind
with greater than 2-fold affinity may be considered. In a
particularly preferred embodiment, variants that bind Fc.gamma.R
with greater than 3-fold affinity relative to WT may be considered
as providing enhanced effector ligand binding. For example,
mutation variants T110I, S112D, S113D, S113R (Kabat numbering) in
the JH region of the VH domain, and A118K, A118E, A118Y, S119E,
T120E, K121H, G122E, N201E, and P206E (EU numbering) in the first
heavy constant domain (CH1) all show greater than 3-fold Fc.gamma.R
binding relative to WT. FIG. 17 shows these residues mapped onto
the heavy chain of the 1L7I Fab structure. As can be seen, these
residues residue in and around the interface between the VH and
C.gamma.1 domain. Particularly striking is that P206E, which shows
a substantial enhancement in binding to Fc.gamma.RIIIa, is distal
in sequence to the other residues yet very close structurally. On
the light chain, a number of variants show significant improvements
in Fc.gamma.RIIIa binding. For example, a number of single mutation
variants show greater than 3-fold Fc.gamma.R binding relative to
WT, including Q100P, Q100G, Q100K, K103R, K103D, K103L, E105D,
E105K, E105K, E105I, E1051, 1106L, and K107E (Kabat numbering) in
the JL region of VL domain, and R108Q, R108D, R108I, T109P, T109R,
T109D, V110K, V110E, A111K, A111E, A111L, A112R, A112E, A112Y,
S114K, S114I, F116T, D122S, D122Y, E123R, E123L, Q124E, L125E,
Q147A, Q147E, Q147K, K149D, V150A, D151K, N152S, N152L, A153H,
L154E, Q155K, Q155E, S156A, S156R, N158R, N158D, S159K, S159E,
S159L, D185I, V191R, E195K, E195K, S203L, K207L, S208T, N210E,
G212E, and E213R (EU numbering) in light constant domain CL. FIG.
18 shows these residues mapped onto the light chain of the 1L7I Fab
structure. Notably, the J segments and interfaces between both the
VL/CL and VH/CH1 domains are involved in determining effector
ligand binding. Equally important to residues at which mutation
enhances binding are those at which mutation reduces or ablates
binding; these results provide structure activity relationship
(SAR) data that may be used to better understand the effector
ligand binding determinants of the Fab region, and indicate that
additional mutation at these positions may generate variants that
provide enhanced effector ligand binding.
[0131] In an embodiment of the present invention, any of these Fab
variants may be combined to potentially obtain further optimized
effector ligand properties. In a preferred embodiment, any of the
aforedescribed Fab variants is combined with one or variants in the
Fc region, particularly Fc variants that provide optimized or
altered effector function, with Fc variants described in U.S. Ser.
No. 10/672,280, U.S. Ser. No. 10/822,231, U.S. Ser. No. 60/627,774,
and U.S. Ser. No. 60/642,477 being most preferred. Select enhanced
binding variants were combined with a previously described Fc
variant, I332E, that also provides enhanced Fc.gamma.R binding and
effector function (U.S. Ser. No. 10/672,280). These Fab/Fc
combination variants were constructed in the light and heavy chains
of Campath in the pcDNA3.1Zeo vector using quick-change
mutagenesis, expressed in 293T cells, and purified as described
above. Binding affinity to human V158 Fc.gamma.RIIIa, human F158
Fc.gamma.RIIIa, and bacterial protein A were measured for the
variants using the AlphaScreen assay as described above. FIG. 19
provides a representative data set from this series of binding
assays, showing select Fab/Fc variant combinations that enhance
Fc.gamma.R binding. The Fold WT values provided by the fits to
these data, generated as described above, are provided in Table 5.
As can be seen, combination of some Fab variants with the Fc
variant provides additive or synergistic effects. Again, as
described above in Table 4, of particular importance are
modifications that result in greater than 1-fold increase in
affinity as compared to the wild-type (e.g. parent antibody) for
any particular receptor. It is an aspect of the invention to
provide modifications that provide higher affinity for at least one
receptor.
TABLE-US-00005 TABLE 5 Fc.gamma.R Binding Data for Fab/Fc
Combination Variants Fold WT Fc.gamma.RIIIa Fc.gamma.RIIIa
Substitution(s) V158 F158 ProtA Fc Region EU I332E 8.07 0.71
S239D/I332E 57.87 0.81 JL Region/Fc Region Kabat Q100P/I332E 11.36
17.33 0.84 Q100G/I332E 9.50 9.49 1.37 Q100K/I332E 18.18 29.55 0.65
K103R/I332E 13.78 17.82 0.91 K103D/I332E 47.23 56.91 2.01
K103L/I332E 9.95 14.12 1.43 E105D/I332E 24.68 31.61 1.20
E105K/I332E 59.29 93.12 4.39 E105I/I332E 171.02 326.98 4.36
I106L/I332E 23.61 15.94 0.97 CL Region/Fc Region EU R108Q/I332E
57.83 94.56 1.55 R108D/I332E 56.03 75.48 2.64 R108I/I332E 51.43
73.60 3.42 T109P/I332E 13.88 18.63 1.61 T109R/I332E 16.60 18.87
1.72 T109D/I332E 20.39 32.21 2.35 V110K/I332E 70.72 86.60 2.20
A111K/I332E 13.42 15.59 0.83 A112R/I332E 6.44 7.83 0.70 A112E/I332E
8.35 10.16 0.74 A112Y/I332E 16.55 30.45 1.27 S114K/I332E 7.14 12.54
0.88 S114I/I332E 5.99 6.04 0.81 Q124E/I332E 7.20 7.38 0.84
L125E/I332E 20.34 27.70 1.13 Q147K/I332E 8.47 4.80 1.11 D151K/I332E
40.43 82.93 4.73 N152S/I332E 19.17 14.51 1.95 Q155K/I332E 8.00 8.23
0.87 S156A/I332E 4.72 4.62 0.82 N158R/I332E 2.67 N158D/I332E 7.03
8.97 0.89 S159K/I332E 8.30 11.80 21.25 S159E/I332E 13.71 13.32 1.69
D185I/I332E 10.36 13.51 0.79 V191R/I332E 1.97 K207L/I332E 3.22
S208T/I332E 4.19 N210E/I332E 6.35 G212E/I332E 4.86 E213R/I332E
3.96
[0132] Together the results for variants comprising mutations in
the JL, JH, CL, and CH1 regions serve as a set of structure
activity relationship (SAR) data with which to better understand
the impact of these regions on effector ligand binding. These data
may be used to guide additional experiments for further investigate
the impact of these regions on effector properties, as well as for
further engineering to obtain optimal variants. The present
invention contemplates additional substitutions at these positions,
and at other positions that are proximal to these positions in
three-dimensional space. For example, all residues with one or more
atoms that are within 3 .ANG., 5 .ANG., or 10 .ANG. of one or more
atoms belonging to heavy chain residues T110I, S112D, S113D, S113R
(Kabat numbering), or A118K, A118E, A118Y, S119E, T120E, K121H,
G122E, N201E, and P206E (EUnumbering) may be substituted with any
or all of the 19 remaining amino acids. Additionally, Table 4 also
shows a number of positions at which mutation causes a significant
loss in binding affinity to Fc.gamma.RIIa, suggesting that said
position may play a role in determining binding affinity or
specifity between an antibody and Fc.gamma.R. Accordingly, further
substitutions at these positions are contemplated to obtain
variants with enhanced effector ligand properties.
Example 4
Engineered Hinge Variants
[0133] The IgG hinge region is the flexible linker that genetically
connects the IgG CH1 domain to the CH2 domain. For the purposes of
the present invention, the hinge is defined according to a
structural definition. The C-terminus of the CH2 domain is defined
structurally by C220, which forms a disulfide bond with the
C-terminal disulfide of the CL domain. The N-terminus of the CH2
domain is defined herein according to the last heavy chain residue
before the ordered region of CH2. Available structures of the Fc
region show electron density for residues G237 (pdb 1DN2, DeLano et
al., 2000, Science 287(5456):1279-83) and P237 (pdb 1FC2,
Deisenhofer, 1981, Biochemistry 20(9):2361-70). Therefore for the
purposes of the present invention, the IgG1 hinge is defined as
heavy chain constant region residues D221-G236, with numbering
acccording to the EU numbering scheme. It is noted that according
to this definition, the hinge comprises part of the Fc region
(typically defined from 226 or 230 to the C-terminus of the heavy
chain constant region). Similar to the JL, JH, CL, and CH1 regions,
the hinge region is typically conserved among antibodies of the
same subclass. Thus residues within this region that play a role in
determining effector ligand affinity/specificity and effector
function properties may be more consistent from antibody to
antibody, and accordingly variants that alter these properties may
be more generally applicable to therapeutically useful antibodies.
In order to characterize the effector determinants in the hinge
region, and to generate variants that modulate effector ligand
binding and effector function, a series of variants were designed
to explore a large variety of substitutions at positions within the
hinge. These variants are provided in Table 6. Positions are
numbered according to the EU numbering scheme.
TABLE-US-00006 TABLE 6 Designed Hinge Variants Position WT
Substitution(s) (EU numbering) 221 D K Y E N Q R S T H A V L I F M
W P G 222 K E Y D N Q R S T H V L I F M W P G .sup. A)* 223 T D N Q
R S H A V L I F M Y W P G E K 224 H D N Q K R S T V L I F M W P G E
Y (A)* 225 T D N Q R S H A V L I F M Y P G E K W 226 C S 227 P E K
Y G D N Q R S T H A V L I F M W 228 P K Y G D N Q R T H A V L I F M
W 229 C S 230 P A E Y G D N Q K R S T H V L I F M W 231 A K P D N Q
R S T H V L I F M W 232 P E K Y G D N Q R S T H A V L I F M W 233 E
D N Q R S T H A V L I F M Y W G 234 L D E N Q T H Y I V F K R S A M
G 235 L D S N Q T H Y I V F E K R A M W P G 236 G D E N Q K R S T H
A V L I F M Y W P *K222A and H224A were designed as a double mutant
K222A/H224A in the context of Herceptin, not Campath
[0134] These hinge variants were constructed in the light and heavy
chains of Campath in the pcDNA3.1Zeo vector using quick-change
mutagenesis, expressed in 293T cells, and purified as described
above. Binding affinity to human V158 Fc.gamma.RIIIa was measured
using the AlphaScreen assay as described above. Some variants were
also screened for binding to other effector ligands, including
human Fc.gamma.RI, Fc.gamma.RIIa, Fc.gamma.RIIb, Fc.gamma.RIIc,
FcRn, and C1q using the AlphaScreen; binding assays for these
effector ligands were carried out similar to as described above for
Fc.gamma.RIIa. FIG. 20a provides a representative set of raw data
from this series of binding assays for select hinge variants that
enhance Fc.gamma.R binding. FIG. 20b shows the raw data from
binding of the hinge variant D221K to full the spectrum of effector
ligands tested. The Fold WT values provided by the fits to these
data, generated as described above, are provided in Table 6. Again,
of particular importance are modifications that result in greater
than 1-fold increase in affinity as compared to the wild-type (e.g.
parent antibody) for any particular receptor. In some cases, as is
true for the variants outlined in Tables 4 and 5 as well as Table
7, variants that provide higher affinity for at least one receptor,
even if showing decreased affinity for a different receptor (all as
compared to the parent antibody) are preferred embodiments.
Similarly, variants that provide for higher affinity for more than
one receptor are also desirable. It is an aspect of the invention
to provide modifications that provide higher affinity for at least
one receptor.
TABLE-US-00007 TABLE 7 Effector Ligand Binding Data for Hinge
Variants Fold WT Substitution(s) Fc.gamma.RIIa EU numbering
Fc.gamma.RIIIa V158 Fc.gamma.RI H131 Fc.gamma.RIIb Fc.gamma.RIIc
C1q FcRn D221K 65.60 4.53 4.32 3.50 17.75 0.00 5.66 D221Y 0.56 2.43
2.62 2.41 10.01 2.74 D221E 0.13 D221N D221Q 0.16 D221R D221S D221T
D221H D221A D221V D221L D221I 0.24 D221F D221M D221W 0.84 D221P
D221G K222E 1.51 1.77 1.26 1.04 1.22 1.23 0.64 K222Y 0.34 0.77 0.48
0.50 0.34 1.06 0.66 K222D K222N K222Q K222R 0.22 K222S 0.06 K222T
0.12 K222H K222V K222L K222I 0.87 K222F 0.32 K222M 0.44 K222W 0.12
K222P 0.14 K222G 0.12 T223D T223N 0.24 T223Q T223R 0.10 T223S 0.13
T223H T223A T223V T223L 0.17 T223I 0.23 T223F T223M 0.46 T223Y 0.19
T223W T223P 0.82 T223G T223E 0.75 2.12 2.37 1.74 3.70 2.04 T223K
9.37 1.61 2.18 1.31 2.97 H224D 0.13 H224N 0.16 H224Q 0.32 H224K
0.17 H224R H224S 0.39 H224T 0.50 H224V 0.87 H224L 0.95 H224I 0.48
H224F 0.69 H224M 0.39 H224W 0.91 H224P 0.57 H224G 1.27 H224E 14.28
1.91 3.67 2.80 5.11 H224Y 0.86 1.74 1.17 2.01 1.68 T225D 0.95 T225N
1.56 T225Q 0.77 T225R 1.93 T225S 0.60 T225H 1.19 T225A 0.96 T225V
2.09 T225L T225I T225F 1.10 T225M 0.29 T225Y 0.32 T225P 0.27 T225G
T225E 9.84 2.28 2.75 1.96 4.89 T225K 0.22 0.30 0.37 1.12 5.68 T225W
0.59 2.30 1.04 1.92 6.89 C226S 0.37 P227E 2.10 1.47 1.55 1.55 1.67
1.49 P227K 0.38 0.44 0.64 3.04 0.92 P227Y 0.44 1.06 0.89 0.79 0.73
0.80 P227G 15.77 0.70 1.39 6.56 0.59 P227D P227N P227Q 0.81 P227R
0.60 P227S 0.47 P227T 0.30 P227H 0.33 P227A 0.31 P227V P227L 0.60
P227I 0.39 P227F 0.70 P227M 0.83 P227W P228K 1.28 1.91 1.31 1.14
1.13 1.62 1.55 P228Y 0.75 1.15 1.68 1.59 1.61 2.25 1.45 P228G 0.98
1.00 2.60 0.96 0.54 1.89 1.77 P228D P228N P228Q P228R P228T P228H
P228A P228V P228L P228I P228F P228M P228W C229S P230A 0.55 1.28
0.99 2.53 0.77 P230E 1.40 2.07 1.25 2.34 1.47 2.70 1.01 P230Y 0.56
0.44 0.40 0.85 1.22 2.00 0.65 P230G 0.48 1.04 0.99 1.13 0.75 1.27
1.16 P230D P230N P230Q P230K P230R P230S P230T P230H P230V P230L
P230I P230F P230M P230W A231K 0.58 0.50 0.62 0.41 0.58 1.40 0.52
A231P 0.31 1.17 0.81 A231D A231N A231Q A231R A231S A231T A231H
A231V A231L A231I A231F A231M A231W P232E 1.51 2.97 0.80 1.59 0.99
1.18 0.76 P232K 0.77 0.70 0.87 0.85 0.61 0.78 1.56 P232Y 0.99 1.91
1.51 1.49 0.96 0.87 0.68 P232G 0.04 0.10 0.05 0.11 0.14 0.70 P232D
P232N P232Q P232R P232S P232T P232H P232A P232V P232L P232I P232F
P232M P232W E233D 0.64 0.85 0.81 2.66 0.76 E233N 0.48 0.16 0.50
0.34 0.46 1.28 0.74 E233Q 0.51 0.19 0.52 0.27 0.39 1.17 0.61 E233R
0.35 0.14 0.75 0.36 0.60 1.13 1.05 E233S 0.36 0.17 0.62 0.31 0.37
1.18 1.10 E233T 0.42 0.15 1.28 0.78 0.83 0.99 1.19 E233H 0.32 0.17
0.74 0.58 0.78 1.05 0.99 E233A 0.46 0.10 1.23 0.60 0.71 1.02 0.93
E233V 0.50 0.25 0.71 0.37 0.61 0.70 0.91 E233L 0.52 0.53 0.55 0.26
0.54 2.24 0.60 E233I 0.88 0.30 1.09 1.69 1.80 2.30 0.95 E233F 0.58
0.23 0.64 0.73 0.84 1.27 0.90 E233M 0.70 0.29 0.67 0.49 0.85 1.56
1.13 E233Y 0.37 0.31 0.96 0.97 0.55 1.86 E233W 0.35 0.28 0.86 0.82
0.70 0.91 1.64 E233G 1.21 0.36 1.21 0.94 0.74 1.03 2.09 L234D 3.89
0.36 0.40 4.95 0.96 1.54 L234E 1.86 0.42 0.24 4.78 1.19 1.25 L234N
0.49 0.10 0.19 2.05 1.18 1.06 L234Q 0.52 0.28 0.28 3.53 0.94 0.97
L234T 0.26 0.49 0.20 1.79 0.56 0.99 L234H 0.27 0.11 0.29 1.56 0.65
1.48 L234Y 0.80 1.45 0.51 1.93 0.99 1.90 L234I 1.30 1.20 0.78 2.57
1.28 1.26 L234V 1.61 1.66 0.78 3.94 0.64 1.45 L234F 0.37 0.74 0.47
2.36 0.72 1.46 L234K 0.53 0.43 0.65 1.42 1.09 2.02 0.62 L234R 0.73
0.38 0.87 1.49 1.52 1.72 1.19 L234S 0.69 0.49 1.01 1.40 1.30 0.93
L234A 0.35 0.44 0.80 0.85 0.62 0.88 0.58 L234M 0.49 0.64 0.89 0.90
0.65 0.88 0.55 L234G 2.17 0.70 3.26 3.62 3.48 1.91 2.54 L235D 1.61
0.76 5.48 1.05 0.90 L235S 0.95 0.27 2.99 0.66 1.51 L235N 0.37 0.06
0.21 1.59 0.70 1.32 L235Q 1.02 0.09 0.30 1.40 0.85 1.67 L235T 2.15
0.13 0.53 3.55 1.06 1.65 L235H 0.30 0.06 0.51 1.77 0.54 0.96 L235Y
1.74 0.24 3.32 4.44 0.86 1.02 L235I 1.47 0.16 0.67 1.24 0.68 0.81
L235V 0.58 0.16 0.43 0.94 1.31 L235F 0.56 1.25 1.62 0.71 0.80 L235E
1.06 0.34 0.63 0.83 0.80 0.93 0.78 L235K 0.63 0.42 0.56 1.28 1.34
1.55 0.96 L235R 0.62 0.35 0.71 1.93 1.15 1.73 0.53 L235A 0.41 0.34
0.62 0.84 0.78 1.00 0.97 L235M 0.46 0.38 0.79 0.89 0.64 1.05 0.69
L235W 0.32 0.11 0.90 0.77 0.50 0.83 0.46 L235P 0.78 0.13 1.16 1.16
0.89 1.31 0.86 L235G 0.43 0.99 1.02 0.74 1.12 0.68 G236D 0.11 0.15
5.01 3.77 4.74 1.64 1.53 G236E 1.41 0.06 4.88 3.39 0.48 G236N 0.07
0.07 0.26 0.69 0.79 1.89 G236Q 0.17 0.12 0.60 0.26 1.89 1.46 G236K
0.46 0.11 0.55 2.18 1.38 G236R 0.10 0.10 0.31 0.19 2.03 1.99 G236S
5.77 0.55 22.71 2.77 3.68 1.82 1.77 G236T 0.11 0.11 1.89 0.95 0.48
3.25 1.56 G236H 0.08 0.03 0.37 0.31 0.21 0.52 G236A 0.65 0.48 44.99
1.05 1.45 1.64 1.75 G236V 0.27 0.14 1.52 0.82 0.47 1.59 1.35 G236L
0.18 0.04 1.07 G236I 0.02 0.11 1.33 0.12 1.96 1.33 G236F 0.18 0.06
0.43 0.88 1.19 G236M 0.19 0.14 0.34 0.79 0.40 1.40 1.60 G236Y 0.14
0.17 0.78 1.34 1.52 G236W 0.29 0.78 2.13 0.70 0.29 1.40 1.52
G236P 0.10 0.15 0.11 0.24 0.15 1.38 1.41 K222A/H224A* 0.67* *The
K222A/H224A double variant was constructed in the context of
Herceptin, and its Fold WT Fc.gamma.RIIIa binding was determined by
SPR, not AlphaScreen.
[0135] The data show that some hinge variants provide selective
binding of the antibody to the different effector ligands, in some
cases with substantial enhancements in affinity. In one embodiment,
variants that bind an effector ligand with greater than 1-fold
affinity relative to WT may be considered as providing improved or
enhanced binding. In a preferred embodiment, variants that bind
with greater than 2-fold affinity may be considered. In a
particularly preferred embodiment, variants that bind an effector
ligand with greater than 3-fold affinity relative to WT may be
considered as providing enhanced binding. In an embodiment of the
present invention, any of these hinge variants may be combined to
potentially obtain further optimized effector ligand properties.
Likewise, similar to as shown above with the Fab variants, in a
preferred embodiment any of the aforedescribed hinge variants may
be combined with one or variants in the Fc region, particularly Fc
variants that provide optimized or altered effector function, with
Fc variants described in U.S. Ser. No. 10/672,280, U.S. Ser. No.
10/822,231, U.S. Ser. No. 60/627,774, and U.S. Ser. No. 60/642,477
being most preferred.
[0136] Whereas particular embodiments of the invention have been
described above for purposes of illustration, it will be
appreciated by those skilled in the art that numerous variations of
the details may be made without departing from the invention as
described in the appended claims. All references are herein
expressly incorporated by reference.
* * * * *