U.S. patent application number 13/479762 was filed with the patent office on 2012-10-18 for engineering fc antibody regions to confer effector function.
This patent application is currently assigned to MacroGenics, Inc.. Invention is credited to Scott Koenig, Jeffery Stavenhagen.
Application Number | 20120263711 13/479762 |
Document ID | / |
Family ID | 37772044 |
Filed Date | 2012-10-18 |
United States Patent
Application |
20120263711 |
Kind Code |
A1 |
Stavenhagen; Jeffery ; et
al. |
October 18, 2012 |
Engineering Fc Antibody Regions to Confer Effector Function
Abstract
The present invention relates to molecules having a variant Fc
region, wherein said variant Fc region comprises at least one amino
acid modification relative to a wild-type Fc region. These modified
molecules confer an effector function to a molecule, where the
parent molecule does not detectably exhibit this effector function.
In one embodiment, the variant Fc region binds Fc.gamma.RIIIA
and/or Fc.gamma.RIIA with a greater affinity, relative to a
comparable molecule comprising the wild-type Fc region. The
molecules of the invention have particular utility in treatment,
prevention or management of a disease or disorder, in a
sub-population of patients, wherein the target antigen is expressed
at low levels in the target cell population.
Inventors: |
Stavenhagen; Jeffery;
(Brookville, MD) ; Koenig; Scott; (Rockville,
MD) |
Assignee: |
MacroGenics, Inc.
Rockville
MD
|
Family ID: |
37772044 |
Appl. No.: |
13/479762 |
Filed: |
May 24, 2012 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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12565911 |
Sep 24, 2009 |
8216574 |
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13479762 |
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11271140 |
Nov 10, 2005 |
7632497 |
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12565911 |
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60636056 |
Dec 13, 2004 |
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60626510 |
Nov 10, 2004 |
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Current U.S.
Class: |
424/133.1 ;
530/387.3 |
Current CPC
Class: |
C07K 2317/732 20130101;
C07K 2317/734 20130101; A61P 35/00 20180101; C07K 16/32 20130101;
C07K 16/00 20130101; C07K 16/2896 20130101; C07K 2317/72 20130101;
C07K 2317/41 20130101; C07K 2319/30 20130101; C07K 2317/52
20130101; C07K 16/283 20130101; C07K 16/2887 20130101; C07K 16/30
20130101; G01N 33/574 20130101 |
Class at
Publication: |
424/133.1 ;
530/387.3 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61P 35/00 20060101 A61P035/00; A61K 39/395 20060101
A61K039/395 |
Claims
1. A modified antibody that binds a CD32B antigen, said modified
antibody comprising a variant human IgG1 Fc region, wherein said
variant human IgG1 Fc region comprises at least one amino acid
modification relative to the human IgG1 Fc region of a parent
antibody that binds said antigen, said amino acid modification(s)
comprising amino acid modification(s) that alter the affinity or
avidity of the variant Fc region for binding to an Fc.gamma.R such
that said modified antibody is therapeutically effective in a
patient refractory to treatment with said parent antibody, said
amino acid modification(s) that alter the affinity or avidity of
the variant Fc region for binding to an Fc.gamma.R consisting of
the modification of 1, 2, 3, 4 or 5 amino acid residues of the IgG1
Fc region of said parent antibody.
2. The modified antibody of claim 1, wherein said modified antibody
exhibits, in an in vitro assay, detectable effector function
activity in cells derived from said patient, which cells are
positive for said antigen, and said parent antibody does not
exhibit detectable function activity in said cells using said in
vitro assay.
3. The modified antibody of claim 1, wherein said amino acid
modification(s) that alter the affinity or avidity of the variant
Fc region for binding to an Fc.gamma.R comprise(s) a substitution:
(i) at position 370 with glutamic acid, at position 396 with
leucine and at position 270 with glutamic acid; (ii) at position
419 with histidine, at position 396 with leucine and at position
270 with glutamic acid; (iii) at position 240 with alanine, at
position 396 with leucine and at position 270 with glutamic acid;
(iv) at position 255 with leucine, at position 396 with leucine and
position 270 with glutamic acid; (v) at position 255 with leucine,
at position 396 with leucine, at position 270 with glutamic acid
and at position 292 with glycine; or (vi) at position 255 with
leucine, at position 396 with leucine, at position 270 with
glutamic acid and at position 300 with leucine.
4. The modified antibody of claim 1, wherein at least one of said
amino acid modification(s) that alter the affinity or avidity of
the variant Fc region for binding to an Fc.gamma.R is in a CH2
domain of said variant human IgG1 Fc region.
5. The modified antibody of claim 4, wherein said amino acid
modification(s) that alter the affinity or avidity of the variant
Fc region for binding to an Fc.gamma.R in said CH2 domain comprises
a substitution at position 240, 243, 247, 255, 270, 292, or 300
with another amino acid at that position.
6. The modified antibody of claim 1, wherein at least one of said
amino acid modification(s) that alter the affinity or avidity of
the variant Fc region for binding to an Fc.gamma.R is in a CH3
domain of said variant human IgG1 Fc region.
7. The modified antibody of claim 6, wherein said amino acid
modification(s) that alter the affinity or avidity of the variant
Fc region for binding to an Fc.gamma.R in said CH3 domain comprises
a substitution at position 370, 392, 396, 419, or 421 with another
amino acid at that position.
8. The modified antibody of claim 1, wherein said amino acid
modification(s) that alter the affinity or avidity of the variant
Fc region for binding to an Fc.gamma.R comprises at least one amino
acid modification in the CH2 domain and at least one amino acid
modification in the CH3 domain of the Fc region.
9. The modified antibody of claim 1, wherein said amino acid
modification(s) that alter the affinity or avidity of the variant
Fc region for binding to an Fc.gamma.R is in the hinge region of
the human IgG1 heavy chain.
10. The modified antibody of claim 8, comprising at least one amino
acid modification that alters the affinity or avidity of the
variant Fc region for binding to an Fc.gamma.R in the hinge region
of the human IgG1 heavy chain.
11. The modified antibody of claim 1 which variant IgG1 Fc region
specifically binds Fc.gamma.RIIIA with a greater affinity than said
parent antibody binds Fc.gamma.RIIIA.
12. The modified antibody of claim 1 which variant IgG1 Fc region
specifically binds Fc.gamma.RIIA with a greater affinity than said
parent antibody binds Fc.gamma.RIIA.
13. The modified antibody of claim 1 which variant IgG1 Fc region
specifically binds Fc.gamma.RIIB with a lower affinity than said
parent antibody binds Fc.gamma.RIIB.
14. The modified antibody of claim 11 which variant IgG1 Fc region
specifically binds Fc.gamma.RIIB with a lower affinity than said
parent antibody binds Fc.gamma.RIIB.
15. The modified antibody of claim 12 which variant IgG1 Fc region
specifically binds Fc.gamma.RIIB with a lower affinity than said
parent antibody binds Fc.gamma.RIIB.
16. The modified antibody of claim 1 which detectably binds cells
positive for said antigen, which antigen is expressed at a density
of 200 to 1,000 molecules/cell on said cells.
17. The modified antibody of claim 2 wherein said effector function
is antibody dependent cell-mediated cell cytotoxicity (ADCC).
18. The modified antibody of claim 2 wherein said effector function
is phagocytosis, opsonization, cell binding, rosetting, complement
dependent cell mediated cytotoxicity (CDC), or antibody dependent
cell-mediated cell cytotoxicity (ADCC).
19. The modified antibody of claim 2 wherein said in vitro assay is
performed using effector cells and target cells, under conditions
in which the effector cell:target cell ratio is 10:1.
20. The modified antibody of claim 1 which is a monoclonal
antibody.
21. The modified antibody of claim 1 which is a chimeric, human or
humanized antibody.
22. The modified antibody of claim 1, in which the parent antibody
has immunomodulatory activity.
23. A pharmaceutical composition comprising the modified antibody
of claim 1 and a pharmaceutically acceptable carrier.
24. The modified antibody of claim 1, wherein said amino acid
modification(s) that alter the affinity or avidity of the variant
Fc region for binding to an Fc.gamma.R consist of the modification
of 1, 2 or 3 amino acid residues of the IgG1 Fc region of said
parent antibody.
25. The modified antibody of claim 1, wherein said amino acid
modification(s) that alter the affinity or avidity of the variant
Fc region for binding to an Fc.gamma.R consist of the modification
of 4 or 5 amino acid residues of the IgG1 Fc region of said parent
antibody.
26. The modified antibody of claim 2, wherein said in vitro assay
is performed using effector cells and target cells, under
conditions in which the effector cell:target cell ratio is
30:1.
27. The modified antibody of claim 2 wherein said in vitro assay is
performed using effector cells and target cells, under conditions
in which the effector cell:target cell ratio is 50:1.
28. The modified antibody of claim 2 wherein said in vitro assay is
performed using effector cells and target cells, under conditions
in which the effector cell:target cell ratio is 75:1.
29. The modified antibody of claim 2 wherein said in vitro assay is
performed using effector cells and target cells, under conditions
in which the effector cell:target cell ratio is 100:1.
30. The modified antibody of claim 1, wherein said modified
antibody exhibits, in an in vitro assay, detectable cell killing in
cells positive for said antigen, and said parent antibody does not
exhibit detectable cell killing in said cells using said in vitro
assay.
31. The modified antibody of claim 30 wherein said in vitro assay
is performed using effector cells and target cells, under
conditions in which the effector cell:target cell ratio is
10:1.
32. The modified antibody of claim 30 wherein said in vitro assay
is performed using effector cells and target cells, under
conditions in which the effector cell:target cell ratio is
30:1.
33. The modified antibody of claim 30 wherein said in vitro assay
is performed using effector cells and target cells, under
conditions in which the effector cell:target cell ratio is
50:1.
34. The modified antibody of claim 30 wherein said in vitro assay
is performed using effector cells and target cells, under
conditions in which the effector cell:target cell ratio is
75:1.
35. The modified antibody of claim 30 wherein said in vitro assay
is performed using effector cells and target cells, under
conditions in which the effector cell:target cell ratio is 100:1.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/565,911, filed Sep. 24, 2011, which
application is a continuation of U.S. patent application Ser. No.
11/271,140, filed Nov. 10, 2005 (now issued as U.S. Pat. No.
7,632,497), which claims priority to U.S. Provisional Application
Nos. 60/626,510 and 60/636,056, filed on Nov. 10, 2004 and Dec. 13,
2004, respectively, all of which applications are incorporated
herein by reference in their entireties and to which priority is
claimed.
1. FIELD OF THE INVENTION
[0002] The present invention relates to molecules having a variant
Fc region, wherein said variant Fc region comprises at least one
amino acid modification relative to a wild-type Fc region. These
modified molecules confer an effector function to a molecule, where
the parent molecule does not detectably exhibit this effector
function. In particular, the molecules of the invention have an
increased effector cell function mediated by a Fc.gamma.R, such as,
but not limited to, ADCC. In one embodiment, the variant Fc region
binds Fc.gamma.RIIIA and/or Fc.gamma.RIIA with a greater affinity,
relative to a comparable molecule comprising the wild-type Fc
region. The molecules of the invention have particular utility in
treatment, prevention or management of a disease or disorder, such
as cancer, in a sub-population of patients, wherein the target
antigen is expressed at low levels in the target cell population,
in particular, in patients refractory to treatment with an existing
therapeutic antibody due to the low level of target antigen
expression on the cancer or associated cells.
2. BACKGROUND OF THE INVENTION
2.1 Fc Receptors and their Roles in the Immune System
[0003] The interaction of antibody-antigen complexes with cells of
the immune system results in a wide array of responses, ranging
from effector functions such as antibody-dependent cytotoxicity,
mast cell degranulation, and phagocytosis to immunomodulatory
signals such as regulating lymphocyte proliferation and antibody
secretion. All these interactions are initiated through the binding
of the Fc domain of antibodies or immune complexes to specialized
cell surface receptors on hematopoietic cells. The diversity of
cellular responses triggered by antibodies and immune complexes
results from the structural heterogeneity of Fc receptors. Fc
receptors share structurally related ligand binding domains which
presumably mediate intracellular signaling.
[0004] The Fc receptors, members of the immunoglobulin gene
superfamily of proteins, are surface glycoproteins that can bind
the Fc portion of immunoglobulin molecules. Each member of the
family recognizes immunoglobulins of one or more isotypes through a
recognition domain on the .alpha. chain of the Fc receptor. Fc
receptors are defined by their specificity for immunoglobulin
subtypes. Fc receptors for IgG are referred to as Fc.gamma.R, for
IgE as F.epsilon.R, and for IgA as Fc.alpha.R. Different accessory
cells bear Fc receptors for antibodies of different isotype, and
the isotype of the antibody determines which accessory cells will
be engaged in a given response (reviewed by Ravetch J. V. et al.
1991, Annu. Rev. Immunol. 9: 457-92; Gerber J. S. et al. 2001
Microbes and Infection, 3: 131-139; Billadeau D. D. et al. 2002,
The Journal of Clinical Investigation, 2(109): 161-1681; Ravetch J.
V. et al. 2000, Science, 290: 84-89; Ravetch J. V. et al., 2001
Annu. Rev. Immunol. 19:275-90; Ravetch J. V. 1994, Cell, 78(4):
553-60). The different Fc receptors, the cells that express them,
and their isotype specificity is summarized in Table 1 (adapted
from Immunobiology: The Immune System in Health and Disease,
4.sup.th ed. 1999, Elsevier Science Ltd/Garland Publishing, New
York).
[0005] Fc.gamma. Receptors
[0006] Each member of this family is an integral membrane
glycoprotein, possessing extracellular domains related to a C2-set
of immunoglobulin-related domains, a single membrane spanning
domain and an intracytoplasmic domain of variable length. There are
three known Fc.gamma.Rs, designated Fc.gamma.RI(CD64),
Fc.gamma.RII(CD32), and Fc.gamma.RIII(CD16). The three receptors
are encoded by distinct genes; however, the extensive homology
between the three family members suggest they arose from a common
progenitor perhaps by gene duplication.
[0007] Fc.gamma.RII (CD32)
[0008] Fc.gamma.RII proteins are 40 KDa integral membrane
glycoproteins which bind only the complexed IgG due to a low
affinity for monomeric Ig (10.sup.6 M.sup.-1). This receptor is the
most widely expressed Fc.gamma.R, present on all hematopoietic
cells, including monocytes, macrophages, B cells, NK cells,
neutrophils, mast cells, and platelets. Fc.gamma.RII has only two
immunoglobulin-like regions in its immunoglobulin binding chain and
hence a much lower affinity for IgG than Fc.gamma.RI. There are
three human Fc.gamma.RII genes (Fc.gamma.RII-A, Fc.gamma.RII-B,
Fc.gamma.RII-C), all of which bind IgG in aggregates or immune
complexes.
[0009] Distinct differences within the cytoplasmic domains of
Fc.gamma.RII-A and Fc.gamma.RII-B create two functionally
heterogenous responses to receptor ligation. The fundamental
difference is that the A isoform initiates intracellular signaling
leading to cell activation such as phagocytosis and respiratory
burst, whereas the .beta. isoform initiates inhibitory signals,
e.g., inhibiting B-cell activation.
[0010] Signaling Through Fc.gamma.Rs
[0011] Both activating and inhibitory signals are transduced
through the Fc.gamma.Rs following ligation. These diametrically
opposing functions result from structural differences among the
different receptor isoforms. Two distinct domains within the
cytoplasmic signaling domains of the receptor called immunoreceptor
tyrosine based activation motifs (ITAMs) or immunoreceptor tyrosine
based inhibitory motifs (ITIMS) account for the different
responses. The recruitment of different cytoplasmic enzymes to
these structures dictates the outcome of the Fc.gamma.R-mediated
cellular responses. ITAM-containing Fc.gamma.R complexes include
Fc.gamma.RI, Fc.gamma.RIIA, Fc.gamma.RIIIA, whereas ITIM-containing
complexes only include Fc.gamma.RIIB.
[0012] Human neutrophils express the Fc.gamma.RIIA gene.
Fc.gamma.RIIA clustering via immune complexes or specific antibody
cross-linking serves to aggregate ITAMs along with
receptor-associated kinases which facilitate ITAM phosphorylation.
ITAM phosphorylation serves as a docking site for Syk kinase,
activation of which results in activation of downstream substrates
(e.g., PI.sub.3K). Cellular activation leads to release of
proinflammatory mediators.
[0013] The Fc.gamma.RIIB gene is expressed on B lymphocytes; its
extracellular domain is 96% identical to Fc.gamma.RIIA and binds
IgG complexes in an indistinguishable manner. The presence of an
ITIM in the cytoplasmic domain of Fc.gamma.RIIB defines this
inhibitory subclass of Fc.gamma.R. Recently the molecular basis of
this inhibition was established. When coligated along with an
activating Fc.gamma.R, the ITIM in Fc.gamma.RIIB becomes
phosphorylated and attracts the SH2 domain of the inosital
polyphosphate 5'-phosphatase (SHIP), which hydrolyzes
phosphoinositol messengers released as a consequence of
ITAM-containing Fc.gamma.R-mediated tyrosine kinase activation,
consequently preventing the influx of intracellular Ca.sup.++. Thus
crosslinking of Fc.gamma.RIIB dampens the activating response to
Fc.gamma.R ligation and inhibits cellular responsiveness. B cell
activation, B cell proliferation and antibody secretion is thus
aborted.
[0014] Current approaches to optimize the Fc region function (e.g.,
antibody-dependent cell mediated cytotoxicity (ADCC), complement
dependent cytotoxicity (CDC) activity) in therapeutic monoclonal
antibodies and soluble polypeptides fused to Fc regions have
focused on a limited number of single amino acid changes based on
structural analysis and/or computer aided designs. Alternative
approaches in engineering Fc regions have focused on the
glycosylation of the Fc region to optimize Fc region function.
TABLE-US-00001 TABLE 1 Receptors for the Fc Regions of
Immunoglobulin Isotype Receptor Binding Cell Type Effect of
Ligation Fc.gamma.RI (CD64) IgG1 10.sup.8 M.sup.-1 Macrophages
Uptake Neutrophils Stimulation Eosinophils Activation of
respiratory Dendritic cells burst Induction of killing
Fc.gamma.RII-A (CD32) IgG1 2 .times. 10.sup.6 M.sup.-1 Macrophages
Uptake Neutrophils Granule release Eosinophils Dendritic cells
Platelets Langerhan cells Fc.gamma.RII-B2 IgG1 2 .times. 10.sup.6
M.sup.-1 Macrophages Uptake (CD32) Neutrophils Inhibition of
Stimulation Eosinophils Fc.gamma.RII-B1 IgG1 2 .times. 10.sup.6
M.sup.-1 B cells No uptake (CD32) Mast cells Inhibition of
Stimulation Fc.gamma.RIII (CD16) IgG1 5 .times. 10.sup.5 M.sup.-1
NK cells Induction of Killing Eosinophil Macrophages Neutrophils
Mast Cells Fc.epsilon.RI IgE 1010 M.sup.-1 Mast cells Secretion of
granules Eosinophil Basophils Fc.alpha.RI (CD89) IgA1, IgA2
10.sup.7 M.sup.-1 Macrophages Uptake Neutrophils Induction of
killing Eosinophils
3. SUMMARY OF THE INVENTION
[0015] The present invention is based, in part, on the inventors'
discovery of methods for engineering the Fc region of an antibody
to confer one or more effector function activities to a parent
antibody, which parent antibody does not exhibit the particular
effector function activity at a detectable level when tested
against a target cell. Such methods of engineering include
introducing one or more amino acid modifications (substitutions,
deletions or insertions) in one or more portions of the Fc region,
which modifications introduce a detectable level of the effector
function activity in the modified antibody. In certain embodiments,
the modifications alter the parent antibody's affinity for certain
Fc.gamma.R receptors (e.g., activating Fc.gamma.Rs, inhibitory
Fc.gamma.Rs) and one or more effector functions, such as
antibody-dependent cell mediated cytotoxicity (ADCC). In other
embodiments, the modifications confer homo-oligomerization activity
to the parent Fc region such that oligomerization of the modified
antibody cross-links cell-surface antigens, resulting in apoptosis,
negative-growth regulation or cell killing.
[0016] The inventors have found that modification of an Fc region
of a chimeric 2B6 antibody (anti-Fc.gamma.RIIB antibody)
surprisingly conferred an effector function activity (particularly,
ADCC) on chimeric 2B6 antibodies, which normally exhibit no
detectable ADCC in routine in vitro ADCC assays. The inventors have
found that modification of an Fc region of a chimeric 4D5 antibody
(anti-Fc.gamma.RIIB antibody) surprisingly improved the effector
function activity (particularly, ADCC) of chimeric 4D5 antibodies
in cells with low levels of antigen expression. The inventors have
further found that modification of an Fc region of rituximab
(anti-CD20 monoclonal antibody) conferred effector function
activity on the rituximab antibody in cells from a patient
population whose cells were otherwise refractory to
rituximab-induced effector function activity.
[0017] In one aspect, the invention encompasses molecules,
preferably polypeptides, and more preferably immunoglobulins (e.g.,
antibodies) comprising a variant Fc region having one or more amino
acid modifications (e.g., substitutions, but also including
deletions or insertions) in one or more Fc regions, relative to a
parent molecule, which modifications confer a particular effector
function activity on the modified molecule, as compared to the
parent molecule which has little or no detectable activity of that
effector function (as measured using standard in vitro methods
known in the art and exemplified herein). The effector function
activities that may be conferred using the methods of the invention
include, but are not limited to, ADCC, antibody-dependent
phagocytosis, phagocytosis, opsonization, opsonophagocytosis, cell
binding, rosetting, complement dependent cell mediated cytotoxicity
(CDC).
[0018] Another aspect of the invention relates to molecules,
preferably polypeptides, and, more preferably, immunoglobulins
(e.g., antibodies) comprising a variant Fc region having one or
more amino acid modifications (e.g., substitutions, deletions,
insertions) in one or more portions, which modifications increase
the affinity and avidity of the variant Fc region for an Fc.gamma.R
(including activating and inhibitory Fc.gamma.Rs). In some
embodiments, said one or more amino acid modifications increase the
affinity of the variant Fc region for Fc.gamma.RIIIA and/or
Fc.gamma.RIIA. In another embodiment, the variant Fc region further
specifically binds Fc.gamma.RIIB with a lower affinity than does
the Fc region of the comparable parent antibody (i.e., an antibody
having the same amino acid sequence as the antibody of the
invention except for the one or more amino acid modifications in
the Fc region). In some embodiments, such modifications increase
the affinity of the variant Fc region for Fc.gamma.RIIIA and/or
Fc.gamma.RIIA and also enhance the affinity of the variant Fc
region for Fc.gamma.RIIB relative to the parent antibody. In other
embodiments, said one or more amino acid modifications increase the
affinity of the variant Fc region for Fc.gamma.RIIIA and/or
Fc.gamma.RIIA but do not alter the affinity of the variant Fc
regions for Fc.gamma.RIIB relative to the Fc region of the parent
antibody. In another embodiment, said one or more amino acid
modifications enhance the affinity of the variant Fc region for
Fc.gamma.RIIIA and Fc.gamma.RIIA but reduce the affinity for
Fc.gamma.RIIB relative to the parent antibody.
[0019] Increased affinity and/or avidity results in detectable
binding to the Fc.gamma.R or detectable Fc.gamma.R-related activity
in cells that express low levels of the Fc.gamma.R when binding
activity of the parent molecule (without the modified Fc region)
cannot be detected on the cells. In certain embodiments the target
antigen of the modified antibody is an Fc.gamma.R, and the modified
antibody exhibits Fc.gamma.R-binding or related activity in cells
which express the target Fc.gamma.R at a density of 10,000
molecules/cell or less, at a density of 5000 molecules/cell or
less, at a density of 1000 molecules/cell or less, at a density of
500 molecules or less, or at a density of 200 molecules or less
(but at least 10, at least 50, at least 100 or at least 150
molecules/cell). In embodiments wherein the target antigen is an
Fc.gamma.R, the increased binding to the on the cell surface may be
mediated by the CDR region of the antibody to an epitope on the
target Fc.gamma.R. Furthermore, this mechanism of increased antigen
binding may occur with antibodies against non-Fc.gamma. receptors
or surface proteins.
[0020] The invention encompasses molecules, e.g., antibodies, with
altered affinities and avidities for one or more target
Fc.gamma.Rs. The antibodies of the invention with enhanced affinity
and avidity for one or more target Fc.gamma.Rs are particularly
useful in cellular systems (for example for research or diagnostic
purposes) where the Fc.gamma.Rs are expressed at low levels, for
example, tumor specific B cells with low levels of Fc.gamma.RIIB
(e.g., non-Hodgkins lymphoma, CLL, and Burkitt's lymphoma).
Although not intending to be bound by a particular mechanism of
action, the molecules of the invention with enhanced affinity and
avidity for a particular target Fc.gamma.R are valuable as research
and diagnostic tools by enhancing the sensitivity of detection of
Fc.gamma.Rs which are normally undetectable due to a low level of
expression. The antibodies of the invention with enhanced affinity
and avidity for Fc.gamma.Rs are particularly useful for the
treatment, prevention or management of a cancer, or another disease
or disorder, in a subject, wherein the Fc.gamma.Rs are expressed at
low levels in the target cell populations. As used herein,
Fc.gamma.R expression in cells is defined in terms of the density
of such molecules per cell as measured using common methods known
to those skilled in the art. The molecules of the invention
comprising variant Fc regions preferably also have an enhanced
avidity and affinity and/or effector function in cells which
express a target antigen to which the modified antibody
immunospecifically binds, e.g., a cancer antigen, at low density,
for example, at a density of 30,000 to 20,000 molecules/cell, at a
density of 20,000 to 10,000 molecules/cell, at a density of 10,000
to 5,000 molecules/cell, at a density of 5,000 to 1,000
molecules/cell, at a density of 1,000 to 200 molecules/cell or at a
density of 200 molecules/cell or less. The molecules of the
invention have particular utility in treatment, prevention or
management of a disease or disorder, such as cancer, in a
sub-population of patients, wherein the target antigen is expressed
at low levels in the target cell population, in particular, in
patients refractory to treatment with an existing therapeutic
antibody due to the low level of target antigen expression on the
cancer or other cells associated with the disease or disorder to be
treated, prevented or managed.
[0021] The invention encompasses engineering human, chimeric or
humanized therapeutic antibodies in the Fc region by modifying one
or more Fc region amino acids, which modifications alter the
detectable affinity and avidity of the antibodies for one or more
target antigens, e.g., Fc.gamma.R receptors or cancer antigens,
and/or the detectable effector function activity or cell killing
activity of the modified antibody. In one embodiment, said one or
more modifications to the amino acids of the Fc region enhance the
affinity and avidity of the antibody for one or more target
antigens, e.g., Fc.gamma.R receptors or cancer antigens. These
therapeutic antibodies, by virtue of the modifications of the
invention, have increased efficacy in patients refractory to
treatment with the parent antibody, due, in certain instances, to
reduced levels of the expression of the target antigen, as well as
in patients who respond to the parent antibody.
[0022] Although not intending to be bound by a particular mechanism
of action, therapeutic antibodies engineered in accordance with the
invention have enhanced therapeutic efficacy, in part, due to the
ability of the Fc portion of the antibody to bind a target cell
which expresses the particular Fc.gamma.Rs at reduced levels, for
example, by virtue of the ability of the antibody to remain on the
target cell longer due to an improved off rate for Fc-Fc.gamma.R
interaction. In another embodiment, said one or more modifications
to the amino acids of the Fc region modifies the affinity and
avidity of the antibody for one or more Fc.gamma.R receptors. In a
specific embodiment, the invention encompasses antibodies
comprising a variant Fc region, wherein said variant Fc region
comprises at least one amino acid modification relative to the
parent Fc region, which variant Fc region only binds one
Fc.gamma.R, wherein said Fc.gamma.R is Fc.gamma.RIIIA. In another
specific embodiment, the invention encompasses antibodies
comprising a variant Fc region, wherein said variant Fc region
comprises at least one amino acid modification relative to the
parent Fc region, which variant Fc region only binds one
Fc.gamma.R, wherein said Fc.gamma.R is Fc.gamma.RIIA. In yet
another embodiment, the invention encompasses antibodies comprising
a variant Fc region, wherein said variant Fc region comprises at
least one amino acid modification relative to the parent Fc region,
which variant Fc region only binds one Fc.gamma.R, wherein said
Fc.gamma.R is Fc.gamma.RIIB.
[0023] The affinities and binding properties of the antibodies of
the invention for the target antigen or an Fc.gamma.R are initially
determined using in vitro assays (biochemical or immunological
based assays) known in the art for determining antigen-antibody or
Fc-Fc.gamma.R interactions (i.e., specific binding of an Fc region
to an Fc.gamma.R), respectively, including but not limited to,
ELISA assay, surface plasmon resonance assay or immunoprecipitation
assay. Preferably, the binding properties of the molecules of the
invention are also characterized by in vitro functional assays for
determining one or more Fc.gamma.R mediated effector cell
functions. In most preferred embodiments, the antibodies of the
invention have similar binding properties in in vivo models (such
as those described and disclosed herein) as those in in vitro based
assays. However, the present invention does not exclude molecules
of the invention that do not exhibit the desired phenotype in in
vitro based assays but do exhibit the desired phenotype in
vivo.
[0024] The invention also encompasses molecules, preferably
polypeptides, and more preferably, immunoglobulins (e.g.,
antibodies) comprising a variant Fc region having one or more amino
acid modifications (e.g., substitutions, deletions, insertions) in
one or more portions, which modifications confer detectable
effector function activity to the molecule not detectable in the
parent molecule. In certain embodiments, the parent molecule is an
antibody. In yet other embodiments, the parent antibody is
rituximab or humanized 2B6 (see U.S. Patent Application Publication
2004/0185045 and U.S. patent application Ser. No. 11/126,978, filed
May 10, 2005, by Johnson et al., which are incorporated herein by
reference in their entireties), and the modified antibodies are
used to treat the indications associated with the parent
antibodies. Although not intending to be bound to a particular
mechanism of action, the molecules of the invention with conferred
effector function activity are particularly useful for the
treatment and/or prevention of a disease or disorder where an
effector cell function (e.g., ADCC) mediated by an Fc.gamma.R is
desired (e.g., cancer, infectious disease). Alternately, the
molecules of the invention with Fc modifications may exhibit
enhanced therapeutic efficacy due to the introduction of
homo-oligomerization activity in the Fc region, resulting in
apoptosis, negative-growth regulation or cell killing associated
with surface antigen cross-linking.
[0025] The invention encompasses methods and compositions for
treatment, prevention or management of a cancer in a subject,
comprising administering to the subject a therapeutically effective
amount of one or more molecules comprising a variant Fc region
engineered in accordance with the invention, which molecule further
binds a cancer antigen. In certain embodiments, the subject is
human. In other embodiments, the molecules of the invention are
modified rituximab, and are preferably used in the treatment of
lymphoma, such as Non-Hodgkins lymphoma, or modified humanized 2B6
antibodies engineered according to the methods of the invention,
which modified antibodies possess the same indications as the
parent antibodies. Molecules of the invention comprising the
variant Fc regions are particularly useful for the prevention,
inhibition, reduction of growth or regression of primary tumors, or
metastasis of cancer cells. Although not intending to be bound by a
particular mechanism of action, molecules of the invention enhance
the efficacy of cancer therapeutics by i) enhancing antibody
mediated effector function or ii) enhancing the apoptosis
signaling, negative-growth regulation or cell killing associated
with surface antigen cross-linking by introducing
homo-oligomerization activity in the modified molecules, resulting
in an enhanced rate of tumor clearance or an enhanced rated of
tumor reduction or a combination thereof.
[0026] According to an aspect of the invention, immunotherapeutics
may be modifying in accordance with the invention to increase the
potency of an antibody effector function activity, e.g., ADCC, CDC,
phagocytosis, opsonization, etc. In a specific embodiment, antibody
dependent cellular toxicity and/or phagocytosis (e.g., of tumor
cells) is enhanced by modifying immunotherapeutics with variant Fc
regions of the invention. Molecules of the invention may render
immunotherapy cancer treatment efficacious in a patient population
by enhancing (or rendering detectable) at least one
antibody-mediated effector function activity. In one particular
embodiment, the efficacy of immunotherapy treatment is enhanced by
rendering the complement dependent cascade activity detectable. In
another embodiment of the invention, the efficacy of immunotherapy
treatment is enhanced by rendering the phagocytosis and/or
opsonization of the targeted cells, e.g., tumor cells, detectable.
In another embodiment of the invention, the efficacy of treatment
is enhanced by enhancing antibody-dependent cell-mediated
cytotoxicity ("ADCC") in destruction of the targeted cells, e.g.,
tumor cells, detectable. Determining whether such activity is
detectable is done using routine assays known in the art and
described herein.
[0027] In a specific embodiment, the invention encompasses a
molecule comprising a variant Fc region, wherein said variant Fc
region comprises at least one amino acid modification relative to
the parent Fc region such that the molecule has an enhanced
effector activity, provided said one or more amino acid
modifications includes substitutions at one or more positions. The
amino acid positions recited herein are numbered according to the
EU index as set forth in Kabat et al., Sequence of Proteins of
Immunological Interest, 5.sup.th Ed. Public Health Service, NH1, MD
(1991), expressly incorporated herein by reference. In a specific
embodiment, the variant Fc region has a leucine at position 247, a
lysine at position 421, or a glutamic acid at position 270. In
other specific embodiments, the variant Fc region has a leucine at
position 247, a lysine at position 421 and a glutamic acid at
position 270 (MgFc31/60); a threonine at position 392, a leucine at
position 396, a glutamic acid at position 270, and a leucine at
position 243 (MgFc38/60/F243L); a histidine at position 419, a
leucine at position 396, and a glutamic acid at position 270
(MGFc51/60); an alanine at position 240, a leucine at position 396,
and a glutamic acid at position 270 (MGFc52/60); a histidine at
position 419, a leucine at position 396, a glutamic acid at
position 270, and a leucine at position 243 (MGFc51/60/F243L); a
lysine at position 255 and a leucine at position 396 (MgFc55); a
lysine at position 255, a leucine at position 396, and a glutamic
acid at position 270 (MGFc55/60); a lysine at position 255, a
leucine at position 396, a glutamic acid at position 270, and a
lysine at position 300 (MGFc55/60/Y300L); a lysine at position 255,
a leucine at position 396, a glutamic acid at position 270, and a
leucine at position 243 (MgFc55/60/F243L); a lysine at position
255, a leucine at position 396, a glutamic acid at position 270,
and a glycine at position 292 (MgFc55/60/R292G); a glutamic acid at
position 370, a leucine at position 396, and a glutamic acid at
position 270 (MGFc59/60); a glutamic acid at position 270, an
aspartic acid at position 316, and a glycine at position 416
(MgFc71); a leucine at position 243, a proline at position 292, an
isoleucine at position 305, and a leucine at position 396
(MGFc74/P396L); a leucine at position 243, a glutamic acid at
position 270, a glycine at position 292, and a leucine at position
396; a leucine at position 243, a lysine at position 255, a
glutamic acid at position 270, and a leucine at position 396; or a
glutamine at position 297.
[0028] The invention also encompasses methods for treating or
preventing an infectious disease in a subject comprising
administering a therapeutically or prophylactically effective
amount of one or more molecules of the invention that bind an
infectious agent or cellular receptor therefore. Infectious
diseases that can be treated or prevented by the molecules of the
invention are caused by infectious agents including but not limited
to viruses, bacteria, fungi, protozae, and viruses. Although not
intending to be bound by a particular mechanism of action, the
methods and/or molecules of the invention confer a therapeutic
effect not detectable in the parent antibody or enhance the
therapeutic effect of the parent antibody by i) enhancing or
rendering detectable the antibody mediated effector function toward
an infectious agent or ii) enhancing or rendering detectable the
apoptosis signaling, negative-growth regulation or cell killing
associated with surface antigen cross-linking by conferring
homo-oligomerization activity in the modified molecules.
[0029] According to one aspect of the invention, molecules of the
invention comprising variant Fc regions have detectable antibody
effector function towards an infectious agent, which was not
detectable in the parent molecule comprising a wild-type Fc region.
In a specific embodiment, molecules of the invention enhance the
efficacy of treatment of an infectious disease by enhancing or
rendering detectable phagocytosis and/or opsonization of the
infectious agent causing the infectious disease. In another
specific embodiment, molecules of the invention enhance the
efficacy of treatment of an infectious disease by enhancing or
rendering detectable ADCC of infected cells causing the infectious
disease.
[0030] The invention encompasses characterization of the molecules
of the invention (e.g., therapeutic monoclonal antibodies
engineered according to the methods of the invention) using assays
known to those skilled in the art for identifying the effector cell
function of the molecules. In particular, the invention encompasses
characterizing the molecules of the invention for
Fc.gamma.R-mediated effector cell function. Examples of effector
cell functions that can be assayed in accordance with the
invention, include but are not limited to, antibody-dependent cell
mediated cytotoxicity (ADCC), phagocytosis, opsonization,
opsonophagocytosis, C1q binding, and complement dependent cell
mediated cytotoxicity (CDC). Cell-based or cell free assays for
determining effector cell function activity are routine and known
to those skilled in the art and described herein.
[0031] In one embodiment, the molecules of the invention can be
assayed for Fc.gamma.R-mediated phagocytosis in human monocytes.
Alternatively, the Fc.gamma.R-mediated phagocytosis of the
molecules of the invention may be assayed in other phagocytes,
e.g., neutrophils (polymorphonuclear leuckocytes; PMN); human
peripheral blood monocytes, monocyte-derived macrophages, which can
be obtained using standard procedures known to those skilled in the
art. In another embodiment, the molecules of the invention may be
assayed using an antibody-dependent opsonophagocytosis assay
(ADCP). In yet another embodiment, the molecules of the invention
can be assayed for Fc.gamma.R-mediated ADCC activity in effector
cells, e.g., natural killer cells, using any of the standard
methods known to those skilled in the art. In yet another
embodiment, the molecules of the invention are characterized for
antibody dependent cellular cytotoxicity (ADCC).
[0032] Preferably, the effector cells used in the ADCC assays of
the invention are peripheral blood mononuclear cells (PBMC) that
are purified from normal human blood, using standard methods known
to one skilled in the art, e.g., using Ficoll-Paque density
gradient centrifugation. Preferred effector cells for use in the
methods of the invention express different Fc.gamma.R activating
receptors. The invention encompasses, effector cells expressing
Fc.gamma.RI, Fc.gamma.RIIA and Fc.gamma.RIIB, and monocyte derived
primary macrophages derived from whole human blood expressing both
Fc.gamma.RIIIA and Fc.gamma.RIIB. Both the ratio of effector
cell:target cell and concentration of antibody to be used in the
functional assays in accordance with the invention will be
appreciated to be dependent of the particular assay and system to
be tested. The invention encompasses use of the effector cells in
the functional assays of effector function activity at an effector
cell:target cell ratio of 1:1, 10:1, 30:1, 60:1, 75:1 or 100:1. The
invention encompasses the use of antibody in the functional assays
of effector function activity at an concentration of 0.2 .mu.g/ml
to 3 .mu.g/ml, 0.5 .mu.g/ml to 2 .mu.g/ml or 0.5 .mu.g/ml to 1
.mu.g/ml.
[0033] In another embodiment, the molecules of the invention may be
assayed for C1q binding, which mediates complement dependent
cytotoxicity (CDC). To determine C1q binding, a C1q binding ELISA
may be performed. To assess complement activation, a complement
dependent cytotoxicity (CDC) assay may be performed using standard
methods known in the art.
[0034] The Fc variants of the present invention may be combined
with other Fc modifications known in the art. The invention
encompasses combining an Fc variant of the invention with other Fc
modifications to provide additive, synergistic, or novel properties
to the modified antibody. Preferably, the Fc variants of the
invention enhance the phenotype of the modification with which they
are combined. For example, if an Fc variant of the invention is
combined with a mutant known to bind Fc.gamma.RIIIA with a higher
affinity than a comparable wild type Fc region; the combination
with a mutant of the invention results in a greater fold
enhancement in Fc.gamma.RIIIA affinity.
[0035] In one embodiment, the Fc variants of the present invention
may be combined with other known Fc variants such as those
disclosed in Duncan et al, 1988, Nature 332:563-564; Lund et al.,
1991, J. Immunol. 147:2657-2662; Lund et al, 1992, Mol Immunol
29:53-59; Alegre et al, 1994, Transplantation 57:1537-1543;
Hutchins et al., 1995, Proc Natl. Acad Sci USA 92:11980-11984;
Jefferis et al, 1995, Immunol Lett. 44:111-117; Lund et al., 1995,
Faseb J 9:115-119; Jefferis et al, 1996, Immunol Lett 54:101-104;
Lund et al, 1996, J Immunol 157:49634969; Armour et al, 1999, Eur J
Immunol 29:2613-2624; Idusogie et al, 2000, J Immunol 164:41784184;
Reddy et al, 2000, J Immunol 164:1925-1933; Xu et al., 2000, Cell
Immunol 200:16-26; Idusogie et al, 2001, J Immunol 166:2571-2575;
Shields et al., 2001, J Biol Chem 276:6591-6604; Jefferis et al,
2002, Immunol Lett 82:57-65; Presta et al., 2002, Biochem Soc Trans
30:487-490); U.S. Pat. No. 5,624,821; U.S. Pat. No. 5,885,573; U.S.
Pat. No. 6,194,551; PCT WO 00/42072; PCT WO 99/58572; each of which
is incorporated herein by reference in its entirety.
[0036] The present invention also encompasses antibodies that are
homodimers or heterodimers of Fc regions. Homodimeric or
heterodimeric antibodies of the invention comprise variant Fc
regions, wherein the two Fc chains have the same or different amino
acid sequences, respectively. In one embodiment, each Fc chain of
the heterodimeric antibody comprises one or more different amino
acid modifications relative to the other chain. In another
embodiment, one Fc chain of the heterodimeric antibody comprises
the wild type Fc chain and the other Fc chain comprises one or more
amino acid modifications relative to the wild type chain.
[0037] The present invention also includes polynucleotides that
encode a molecule of the invention, including polypeptides and
antibodies, identified by the methods of the invention. The
polynucleotides encoding the molecules of the invention may be
obtained, and the nucleotide sequence of the polynucleotides
determined, by any method known in the art. The invention relates
to an isolated nucleic acid encoding a molecule of the invention.
The invention also provides a vector comprising said nucleic acid.
The invention further provides host cells containing the vectors or
polynucleotides of the invention.
[0038] The invention further provides methods for the production of
the molecules of the invention. The molecules of the invention,
including polypeptides and antibodies, can be produced by any
method known to those skilled in the art, in particular, by
recombinant techniques. In certain embodiments, antibodies of the
invention are created by engineering mutations identified as
conferring therapeutically effective, detectable, effector function
activity into the Fc regions of antibodies which do not natively
exhibit such activity. In other embodiments, the invention relates
to a method for recombinantly producing a molecule of the
invention, said method comprising: (i) culturing in a medium a host
cell comprising a nucleic acid encoding said molecule, under
conditions suitable for the expression of said molecule; and (ii)
recovery of said molecule from said medium.
[0039] The invention also encompasses methods for improving the
therapeutic efficacy of molecules, preferably polypeptides, and
more preferably immunoglobulins (e.g., antibodies) comprising a
variant Fc region having, which Fc regions have been engineered
according to the methods of the invention, which engineering
confers detectable effector function activity on the modified
molecule, as compared to the parent molecule which exhibited little
or no detectable activity of that effector function (as measured
using standard in vitro methods known in the art and exemplified
herein).
[0040] The invention provides pharmaceutical compositions
comprising a molecule of the invention, e.g., a polypeptide
comprising a variant Fc region, an immunoglobulin comprising a
variant Fc region, a therapeutic antibody engineered in accordance
with the invention, and a pharmaceutically acceptable carrier. The
invention additionally provides pharmaceutical compositions further
comprising one or more additional therapeutic agents, including but
not limited to anti-cancer agents, anti-inflammatory agents,
immunomodulatory agents.
[0041] In detail, the invention thus pertains to a modified
antibody that binds an antigen, the modified antibody comprising a
variant human IgG Fc region, wherein the variant human IgG Fc
region comprises at least one amino acid modification relative to
the human IgG Fc region of a parent antibody that binds the
antigen, such that the modified antibody exhibits, in an in vitro
assay, detectable effector function activity (especially
phagocytosis, opsonization, cell binding, rosetting, complement
dependent cell mediated cytotoxicity (CDC), or antibody dependent
cell-mediated cell cytotoxicity (ADCC)) in cells positive for the
antigen, wherein the parent antibody does not exhibit detectable
effector function activity in the cells using the in vitro assay
(and especially wherein the in vitro assay is performed at an
effector cell:target cell ratio of 10:1, 30:1, 50:1, 75:1 or
100:1). The invention particularly pertains to all such modified
antibodies, wherein the human IgG Fc region is a human IgG1, IgG2,
IgG3, or IgG4 Fc region.
[0042] The invention further pertains to a modified antibody that
binds an antigen, the modified antibody comprising a variant human
IgG Fc region, wherein the variant human IgG Fc region comprises at
least one amino acid modification relative to the human IgG Fc
region of a parent antibody that binds the antigen, such that the
modified antibody is therapeutically effective in a patient
refractory to treatment with the parent antibody. The invention
particularly pertains to such a modified antibodies, wherein the
modified antibody exhibits, in an in vitro assay, detectable
effector function activity (especially phagocytosis, opsonization,
cell binding, rosetting, complement dependent cell mediated
cytotoxicity (CDC), or antibody dependent cell-mediated cell
cytotoxicity (ADCC)) in cells derived from the patient, which cells
are positive for the antigen, wherein the parent antibody does not
exhibit detectable function activity in the cells using the in
vitro assay (and especially wherein the in vitro assay is performed
at an effector cell:target cell ratio of 10:1, 30:1, 50:1, 75:1 or
100:1). The invention particularly pertains to all such modified
antibodies, wherein the human IgG Fc region is a human IgG1, IgG2,
IgG3, or IgG4 Fc region.
[0043] The invention particularly pertains to all such modified
antibodies, wherein the antigen is a cancer antigen (for example,
MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2,
N-acetylglucosaminyltransferase, p15, beta-catenin, MUM-1, CDK4,
HER-2/neu, human papillomavirus-E6, human papillomavirus-E7, MUC-1,
CD20 or CD32B) or an antigen that is expressed on the surface of a
cell.
[0044] The invention particularly pertains to all such modified
antibodies, wherein the parent antibody is HERCEPTIN.RTM., IC14,
PANOREX.TM., IMC-225, VITAXIN.TM., CAMPATH.TM. 1H/LDP-03,
LYMPHOCIDE.TM., ZEVLIN.TM. or rituximab.
[0045] The invention particularly pertains to all such modified
antibodies, wherein the at least one amino acid modification
comprises substitution at position 370 with glutamic acid, at
position 396 with leucine and at position 270 with glutamic acid;
at position 419 with histidine, at position 396 with leucine and at
position 270 with glutamic acid; at position 240 with alanine, at
position 396 with leucine and at position 270 with glutamic acid;
at position 240 with alanine, at position 396 with leucine and at
position 270 with glutamic acid; at position 255 with leucine, at
position 396 with leucine and position 270 with glutamic acid; at
position 255 with leucine, at position 396 with leucine, at
position 270 with glutamic acid and at position 292 glycine; at
position 255 with leucine, at position 396 with leucine, at
position 270 with glutamic acid and at position 300 leucine; at
position 243 with leucine, at position 270 with glutamic acid, at
position 392 with asparagine and at position 396 with leucine; or
at position 243 with leucine, at position 255 with leucine, at
position 270 with glutamic acid and at position 396 with
leucine.
[0046] The invention particularly pertains to all such modified
antibodies, wherein the amino acid modification comprises at least
one amino acid modification in the CH2 domain (and especially
wherein the amino acid modification in the CH2 domain comprises
substitution at position 240, 243, 247, 255, 270, 292, or 300 with
another amino acid at that position), and/or in wherein the amino
acid modification comprises at least one amino acid modification in
the CH3 domain (and especially wherein the amino acid modification
in the CH3 domain comprises substitution at position 370, 392, 396,
419, or 421 with another amino acid at that position) and/or in the
hinge region of the human IgG heavy chain.
[0047] The invention particularly pertains to all such modified
antibodies, wherein the variant IgG Fc region specifically binds
Fc.gamma.RIIIA with a greater affinity than the parent antibody
binds Fc.gamma.RIIIA and especially wherein the variant IgG Fc
region specifically binds Fc.gamma.RIIB with a lower affinity than
the parent antibody binds Fc.gamma.RIIB
[0048] The invention particularly pertains to all such modified
antibodies, wherein the variant IgG Fc region specifically binds
Fc.gamma.RIIA with a greater affinity than the parent antibody
binds Fc.gamma.RIIA and especially wherein the variant IgG Fc
region specifically binds Fc.gamma.RIIB with a lower affinity than
the parent antibody binds Fc.gamma.RIIB
[0049] The invention particularly pertains to all such modified
antibodies, wherein the variant IgG Fc region specifically binds
Fc.gamma.RIIB with a lower affinity than the parent antibody binds
Fc.gamma.RIIB.
[0050] The invention particularly pertains to all such modified
antibodies, which detectably binds cells positive for the antigen,
which antigen is expressed at a density of 200 to 1,000
molecules/cell on the cells.
[0051] The invention particularly pertains to all such modified
antibodies, wherein the antibody is a monoclonal antibody or a
chimeric, human or humanized antibody.
[0052] The invention particularly pertains to all such modified
antibodies, wherein the antibody is a chimeric 2B6 antibody.
[0053] The invention additionally pertains to such modified
antibodies, wherein the antigen is associated with an infectious
disease (an especially in which the antigen is a viral, bacterial
or fungal antigen).
[0054] The invention additionally pertains to such modified
antibodies, wherein the parent antibody has immunomodulatory
activity.
[0055] The invention additionally pertains to a method of treating
cancer in a patient (especially a human patient) having a cancer
characterized by a cancer antigen (especially MAGE-1, MAGE-3, BAGE,
GAGE-1, GAGE-2, N-acetylglucosaminyltransferase, p15, beta-catenin,
MUM-1, CDK4, HER-2/neu, human papillomavirus-E6, human
papillomavirus-E7, MUC-1, CD20 or CD32B), wherein the method
comprises administering to the patient a therapeutically effective
amount of the above-described modified antibody that binds a cancer
antigen (and especially in the embodiment in which the patient is
refractory to treatment with the parent antibody (especially
wherein the parent antibody is HERCEPTIN.RTM., IC14, PANOREX.TM.,
IMC-225, VITAXIN.TM., Campath 1H/LDP-03, LYMPHOCIDE.TM., ZEVLIN.TM.
or rituximab). The invention additionally pertains to the use of
such a method of treating cancer that further comprises the
administration of one or more additional cancer therapies.
[0056] The invention additionally pertains to such a method of
treating cancer in a patient, wherein the modified antibody binds a
cancer antigen that is a colon, breast, ovarian, prostate,
cervical, pancreatic carcinoma, non-Hodgkins lymphoma or chronic
lymphocytic leukemia antigen.
[0057] The invention particularly pertains to all such methods of
treatment wherein the modified antibody is a monoclonal antibody or
a chimeric, human or humanized antibody.
[0058] The invention particularly pertains to all such methods of
treatment wherein the human IgG Fc region is a human IgG1, IgG2,
IgG3, or IgG4 Fc region.
[0059] The invention further pertains to a pharmaceutical
composition comprising any of the above-described modified
antibodies and a pharmaceutically acceptable carrier.
[0060] The invention further pertains to a nucleotide sequence
encoding a heavy or a light chain of any of the above-described
modified antibodies, and especially a vector containing such a
sequence. The invention further pertains to a host cell comprising
such nucleic acid molecule or such vector. In particular, the
invention concerns a host cell comprising a first nucleotide
sequence encoding a heavy chain of a modified antibody and a second
nucleotide sequence encoding a light chain of a modified antibody,
the modified antibody being any of those described above.
[0061] The invention further pertains to a method for recombinantly
producing a modified antibody, the method comprising: [0062] a.
culturing in a medium the host cell of claim 46 under conditions
suitable for the expression of the modified antibody; and [0063] b.
recovery of the modified antibody from the medium.
[0064] The invention further pertains to a method for improving a
therapeutic antibody that specifically binds to an antigen and that
does not exhibit, in an in vitro assay, detectable effector
function activity, the method comprising [0065] a. introducing at
least one amino acid modification in the Fc region of the
therapeutic antibody to generate a modified therapeutic antibody;
and [0066] b. determining whether the modified therapeutic antibody
exhibits detectable effector function activity using the in vitro
assay.
[0067] The invention particularly pertains to such a method for
improving a therapeutic antibody, wherein the modified antibody
exhibits any set or subset of the following attributes: ability to
bind to cells expressing the antigen at a density of 200 to 1,000
molecules/cell, ability to bind to a Fc.gamma.RIIA or
Fc.gamma.RIIIA target molecule, ability to bind to an antigen
target molecule, ability to bind to a cancer antigen target
molecule, and exhibiting an increase in at least one effector
function.
[0068] The invention further pertains to a modified antibody that
binds an antigen, the modified antibody comprising a variant human
IgG Fc region, wherein the variant human IgG Fc region comprises at
least one amino acid modification relative to the human IgG Fc
region of a parent antibody that binds the antigen, such that the
modified antibody exhibits, in an in vitro assay, detectable cell
killing in cells positive for the antigen, wherein the parent
antibody does not exhibit detectable cell killing in the cells
using the in vitro assay.
[0069] The invention further pertains to a method of treating
cancer in a patient having a cancer characterized by a cancer
antigen, the method comprising administering to the patient a
therapeutically effective amount of the modified antibody of claim
54, which modified antibody binds the cancer antigen.
[0070] The invention further pertains to a modified antibody that
binds an antigen, the modified antibody comprising a variant human
IgG Fc region, wherein the variant human IgG Fc region comprises at
least one amino acid modification relative to the human IgG Fc
region of a parent antibody that binds the antigen, such that the
modified antibody exhibits, in an in vitro assay, detectable
effector function activity in cells positive for the antigen,
wherein the parent antibody does not exhibit detectable effector
function activity in the cells using the in vitro assay, which in
vitro assay is performed at an effector cell:target cell ratio of
75:1, which in vitro assay is performed at an effector cell:target
cell ratio of 30:1 or which in vitro assay is performed at an
effector cell:target cell ratio of 10:1.
[0071] The invention further pertains to a modified antibody that
binds an antigen, the modified antibody comprising a variant human
IgG Fc region, wherein the variant human IgG Fc region comprises at
least one amino acid modification relative to the human IgG Fc
region of a parent antibody that binds the antigen, such that the
modified antibody exhibits, in an in vitro assay, detectable cell
killing in cells positive for the antigen, wherein the parent
antibody does not exhibit detectable cell killing in the cells
using the in vitro assay, which in vitro assay is performed at an
effector cell:target cell ratio of 75:1, which in vitro assay is
performed at an effector cell:target cell ratio of 30:1, or which
in vitro assay is performed at an effector cell:target cell ratio
of 10:1.
3.1 Definitions
[0072] As used herein, the term "Fc region" is used to define a
C-terminal region of an IgG heavy chain. Throughout the present
specification, the numbering of the residues in an IgG heavy chain
is that of the EU index as in Kabat et al., Sequences of Proteins
of Immunological Interest, 5.sup.th Ed. Public Health Service, NH1,
MD (1991), expressly incorporated herein by references. The "EU
index as in Kabat" refers to the numbering of the human IgG1 EU
antibody. An example of the amino acid sequence containing the
human IgG1 Fc region is SEQ ID NO:11 and is shown in FIG. 1. SEQ ID
NO:11 and FIG. 1 set forth the amino acid sequence of the IgG1
hinge-Fc region. Although boundaries may vary slightly, as numbered
according to the Kabat system, the Fc domain extends from amino
acid 231 to amino acid 447 (which corresponds to amino acid 16 to
amino acid 232 as numbered in SEQ ID NO:11 and FIG. 1).
[0073] The Fc region of an IgG comprises two constant domains, CH2
and CH3. The CH2 domain of a human IgG Fc region usually extends
from amino acids 231 to amino acid 341 according to the numbering
system of Kabat (corresponding to amino acids 16 to 126 as numbered
in SEQ ID NO:11 and FIG. 1). The CH3 domain of a human IgG Fc
region usually extends from amino acids 342 to 447 according to the
numbering system of Kabat (corresponding to amino acids 127 to 232
as numbered in SEQ ID NO:11 and FIG. 1). The CH2 domain of a human
IgG Fc region (also referred to as "C.gamma.2" domain) is unique in
that it is not closely paired with another domain. Rather, two
N-linked branched carbohydrate chains are interposed between the
two CH2 domains of an intact native IgG.
[0074] The "hinge region" is generally defined as stretching from
Glu216 to Pro230 of human IgG1 (corresponding to amino acids 1-15
as numbered in SEQ ID NO:11 and FIG. 1). Hinge regions of other IgG
isotypes may be aligned with the IgG1 sequence by placing the first
and last cysteine residues forming inter-heavy chain S--S binds in
the same positions.
[0075] As used herein, the terms "antibody" and "antibodies" refer
to monoclonal antibodies, multispecific antibodies, human
antibodies, humanized antibodies, synthetic antibodies, chimeric
antibodies, polyclonal antibodies, camelized antibodies,
single-chain Fvs (scFv), single chain antibodies, Fab fragments,
F(ab') fragments, disulfide-linked bispecific Fvs (sdFv),
intrabodies, and anti-idiotypic (anti-Id) antibodies (including,
e.g., anti-Id and anti-anti-Id antibodies to antibodies of the
invention), and epitope-binding fragments of any of the above. In
particular, antibodies include immunoglobulin molecules and
immunologically active fragments of immunoglobulin molecules, i.e.,
molecules that contain an antigen binding site. Immunoglobulin
molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and
IgY), class (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4,
IgA.sub.1 and IgA.sub.2) or subclass.
[0076] As used herein, the term "derivative" in the context of
polypeptides or proteins refers to a polypeptide or protein that
comprises an amino acid sequence which has been altered by the
introduction of amino acid residue substitutions, deletions or
additions. The term "derivative" as used herein also refers to a
polypeptide or protein which has been modified, i.e, by the
covalent attachment of any type of molecule to the polypeptide or
protein. For example, but not by way of limitation, an antibody may
be modified, e.g., by glycosylation, acetylation, pegylation,
phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, linkage to a
cellular ligand or other protein, etc. A derivative polypeptide or
protein may be produced by chemical modifications using techniques
known to those of skill in the art, including, but not limited to
specific chemical cleavage, acetylation, formylation, metabolic
synthesis of tunicamycin, etc. Further, a derivative polypeptide or
protein derivative possesses a similar or identical function as the
polypeptide or protein from which it was derived.
[0077] As used herein, the term "derivative" in the context of a
non-proteinaceous derivative refers to a second organic or
inorganic molecule that is formed based upon the structure of a
first organic or inorganic molecule. A derivative of an organic
molecule includes, but is not limited to, a molecule modified,
e.g., by the addition or deletion of a hydroxyl, methyl, ethyl,
carboxyl or amine group. An organic molecule may also be
esterified, alkylated and/or phosphorylated.
[0078] As used herein, the terms "disorder" and "disease" are used
interchangeably to refer to a condition in a subject. In
particular, the term "autoimmune disease" is used interchangeably
with the term "autoimmune disorder" to refer to a condition in a
subject characterized by cellular, tissue and/or organ injury
caused by an immunologic reaction of the subject to its own cells,
tissues and/or organs. The term "inflammatory disease" is used
interchangeably with the term "inflammatory disorder" to refer to a
condition in a subject characterized by inflammation, preferably
chronic inflammation. Autoimmune disorders may or may not be
associated with inflammation. Moreover, inflammation may or may not
be caused by an autoimmune disorder. Thus, certain disorders may be
characterized as both autoimmune and inflammatory disorders.
[0079] As used herein, the term "cancer" refers to a neoplasm or
tumor resulting from abnormal uncontrolled growth of cells. As used
herein, cancer explicitly includes leukemias and lymphomas. In some
embodiments, cancer refers to a benign tumor, which has remained
localized. In other embodiments, cancer refers to a malignant
tumor, which has invaded and destroyed neighboring body structures
and spread to distant sites. In some embodiments, the cancer is
associated with a specific cancer antigen that is expressed on
cancer cells.
[0080] As used herein, the term "immunomodulatory agent" and
variations thereof refer to an agent that modulates a host's immune
system. In certain embodiments, an immunomodulatory agent is an
immunosuppressant agent. In certain other embodiments, an
immunomodulatory agent is an immunostimulatory agent.
Immunomodatory agents include, but are not limited to, small
molecules, peptides, polypeptides, fusion proteins, antibodies,
inorganic molecules, mimetic agents, and organic molecules.
[0081] As used herein, the term "epitope" refers to a fragment of a
polypeptide or protein or a non-protein molecule having antigenic
or immunogenic activity in an animal, preferably in a mammal, and
most preferably in a human. An epitope having immunogenic activity
is a fragment of a polypeptide or protein that elicits an antibody
response in an animal. An epitope having antigenic activity is a
fragment of a polypeptide or protein to which an antibody
immunospecifically binds as determined by any method well-known to
one of skill in the art, for example by immunoassays. Antigenic
epitopes need not necessarily be immunogenic.
[0082] As used herein, the term "fragment" refers to a peptide or
polypeptide comprising an amino acid sequence of at least 5
contiguous amino acid residues, at least 10 contiguous amino acid
residues, at least 15 contiguous amino acid residues, at least 20
contiguous amino acid residues, at least 25 contiguous amino acid
residues, at least 40 contiguous amino acid residues, at least 50
contiguous amino acid residues, at least 60 contiguous amino
residues, at least 70 contiguous amino acid residues, at least
contiguous 80 amino acid residues, at least contiguous 90 amino
acid residues, at least contiguous 100 amino acid residues, at
least contiguous 125 amino acid residues, at least 150 contiguous
amino acid residues, at least contiguous 175 amino acid residues,
at least contiguous 200 amino acid residues, or at least contiguous
250 amino acid residues of the amino acid sequence of another
polypeptide. In a specific embodiment, a fragment of a polypeptide
retains at least one function of the polypeptide.
[0083] As used herein, the terms "nucleic acids" and "nucleotide
sequences" include DNA molecules (e.g., cDNA or genomic DNA), RNA
molecules (e.g., mRNA), combinations of DNA and RNA molecules or
hybrid DNA/RNA molecules, and analogs of DNA or RNA molecules. Such
analogs can be generated using, for example, nucleotide analogs,
which include, but are not limited to, inosine or tritylated bases.
Such analogs can also comprise DNA or RNA molecules comprising
modified backbones that lend beneficial attributes to the molecules
such as, for example, nuclease resistance or an increased ability
to cross cellular membranes. The nucleic acids or nucleotide
sequences can be single-stranded, double-stranded, may contain both
single-stranded and double-stranded portions, and may contain
triple-stranded portions, but preferably is double-stranded
DNA.
[0084] As used herein, a "therapeutically effective amount" refers
to that amount of the therapeutic agent sufficient to treat or
manage a disease or disorder. A therapeutically effective amount
may refer to the amount of therapeutic agent sufficient to delay or
minimize the onset of disease, e.g., delay or minimize the spread
of cancer. A therapeutically effective amount may also refer to the
amount of the therapeutic agent that provides a therapeutic benefit
in the treatment or management of a disease. Further, a
therapeutically effective amount with respect to a therapeutic
agent of the invention means the amount of therapeutic agent alone,
or in combination with other therapies, that provides a therapeutic
benefit in the treatment or management of a disease.
[0085] As used herein, the terms "prophylactic agent" and
"prophylactic agents" refer to any agent(s) which can be used in
the prevention of a disorder, or prevention of recurrence or spread
of a disorder. A prophylactically effective amount may refer to the
amount of prophylactic agent sufficient to prevent the recurrence
or spread of hyperproliferative disease, particularly cancer, or
the occurrence of such in a patient, including but not limited to
those predisposed to hyperproliferative disease, for example those
genetically predisposed to cancer or previously exposed to
carcinogens. A prophylactically effective amount may also refer to
the amount of the prophylactic agent that provides a prophylactic
benefit in the prevention of disease. Further, a prophylactically
effective amount with respect to a prophylactic agent of the
invention means that amount of prophylactic agent alone, or in
combination with other agents, that provides a prophylactic benefit
in the prevention of disease.
[0086] As used herein, the terms "prevent", "preventing" and
"prevention" refer to the prevention of the recurrence or onset of
one or more symptoms of a disorder in a subject as result of the
administration of a prophylactic or therapeutic agent.
[0087] As used herein, the term "in combination" refers to the use
of more than one prophylactic and/or therapeutic agents. The use of
the term "in combination" does not restrict the order in which
prophylactic and/or therapeutic agents are administered to a
subject with a disorder. A first prophylactic or therapeutic agent
can be administered prior to (e.g., 5 minutes, 15 minutes, 30
minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours,
24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4
weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before),
concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes,
30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12
hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3
weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the
administration of a second prophylactic or therapeutic agent to a
subject with a disorder.
[0088] "Effector function" as used herein is meant a biochemical
event that results from the interaction of an antibody Fc region
with an Fc receptor or ligand. Effector functions include but are
not limited to antibody dependent cell mediated cytotoxicity
(ADCC), antibody dependent cell mediated phagocytosis (ADCP), and
complement dependent cytotoxicity (CDC). Effector functions include
both those that operate after the binding of an antigen and those
that operate independent of antigen binding.
[0089] "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 may
be from any organism including but not limited to humans, mice,
rats, rabbits, and monkeys.
[0090] "Fc ligand" as used herein is meant a molecule, preferably a
polypeptide, from any organism that binds to the Fc region of an
antibody to form an Fc-ligand complex. Fc ligands include but are
not limited to Fc.gamma.Rs, Fc.gamma.Rs, Fc.gamma.Rs, FcRn, C1q,
C3, staphylococcal protein A, streptococcal protein G, and viral
Fc.gamma.R. Fc ligands may include undiscovered molecules that bind
Fc.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0091] FIG. 1 Amino Acid Sequence of Human IgG1 Hinge-Fc Region
[0092] FIG. 1 shows the amino acid sequence of the human IgG1
hinge-Fc region (SEQ ID NO:11). The amino acid residues shown in
the figure, 1-232, correspond to amino acid residues 231 to 447 of
the IgG heavy chain according to the numbering system of Kabat.
[0093] FIG. 2 SDS-Page Analysis of Recombinant Soluble
Fc.gamma.R
[0094] The purity of recombinant soluble Fc.gamma.R proteins was
assessed by 10% polyacrylamide gel electrophoresis. The gels were
stained with Coomassie blue. Lane 1: purified recombinant soluble
Fc.gamma.RIIIA; Lane 2: molecular weight marker; Lane 3: molecular
weight marker; Lane 4: purified recombinant soluble Fc.gamma.RIIB.
The dashes refer to the molecular weight of the markers, from top
to bottom, they correspond to a molecular weight of 98, 50, 36, and
22 KDa respectively.
[0095] FIG. 3 ELISA Assay of Recombinant Soluble Fc.gamma.R
[0096] The direct binding of purified recombinant soluble
Fc.gamma.RIIIA to aggregated and monomeric IgG was determined using
an ELISA assay. Binding of (.tangle-solidup.) aggregated IgG with
3G8; (.diamond-solid.) Biotinylated IgG; (.box-solid.) aggregated
IgG; (X) aggregated IgG with mouse IgG1.
[0097] FIGS. 4 A and B Characterization of Fc.gamma.RIIIA
Tetrameric Complex Using an ELISA Assay
[0098] A. Soluble tetrameric Fc.gamma.RIIIA complex binds soluble
monomeric human IgG specifically. Binding of soluble tetrameric
Fc.gamma.RIIIA to human IgG is blocked by 3G8 (.diamond-solid.), a
mouse anti-Fc.gamma.IIIA monoclonal antibody; the 4-4-20 monoclonal
antibody harboring the D265A mutation was not able to block the
binding of soluble tetrameric Fc.gamma.RIIIA to aggregated human
IgG (.DELTA.).
[0099] B. Binding of soluble tetrameric Fc.gamma.RIIIA complex to
soluble monomeric human IgG (.box-solid.) is compared to the
binding of monomeric soluble Fc.gamma.RIIIA to soluble monomeric
human IgG (.diamond-solid.).
[0100] FIGS. 5A-5E Characterization of Fc.gamma.RIIIA Tetrameric
Complex Using a Magnetic Bead Assay
[0101] A. FIG. 5A: Fc.gamma.RIIIA Complex: two
Fc.gamma.RIIIA(filled shape) are joined by a monoclonal antibody
DJ130c (1.sup.st Ab); the anti-mouse F(ab).sub.2 is conjugated to
PE (circle).
[0102] B. FACS analysis of Fc.gamma.RIIIA bound to Fc coated beads:
FIG. 5B: beads alone; FIG. 5C: complex without Fc.gamma.RIIIA; FIG.
5D: complex with Fc.gamma.RIIIA; FIG. 5E: complex with
Fc.gamma.RIIIA and LNK16.
[0103] FIG. 6 Schematic Presentation of Fc Containing
Constructs
[0104] A schematic diagram of the IgG1 Fc domains cloned into pYD1
is presented. The open box represents the hinge-CH2-CH3 domains;
parallel vertical lines represent the CH1 domain. In the case of
the GIF206 and 227 constructs; the N-terminal amino acids (SEQ ID
NO:12 and SEQ ID NO:13, respectively) are shown. The underlined
residues correspond to the hinge region; the * represents the
Xpress epitope tag; hatched boxes represent the Gly4-Ser linker,
and the stippled boxes represent the Aga2p gene.
[0105] FIGS. 7A-H FACS Analysis of the Fc Fusion Proteins on the
Yeast Cell Wall
[0106] Cells were incubated with either a PE-conjugated polyclonal
goat anti-human Fc antibody (FIGS. 6A-D) or with HP6017 (Sigma), a
mouse anti-human IgG1 Fc (CH3) specific monoclonal antibody (FIGS.
6E-H). A and E represent vector alone; Panels B and F represent the
CH1--CH3 construct; Panels C and G represent the GIF227; and Panels
D and H represent the GIF 206 construct.
[0107] FIGS. 8A-C Binding of Soluble Tetrameric Fc.gamma.RIIIA to
the Surface Displayed Fc Fusion Proteins
[0108] Cells containing pYD1-CH1 (A); pYD-CH1-D265A (B); and pYD
vector (C) were grown under conditions to express Aga2p fusion
proteins on the cell surface. Cells were incubated with
Fc.gamma.RIIIA at 0.15 mM, 7.5 mM, and 7.5 mM, respectively, and
analyzed by FACS.
[0109] FIG. 9 Characterization of the Binding of Soluble Tetrameric
Fc.gamma.RIIIA to the Surface Displayed Fc Fusion Proteins
[0110] Binding of Fc.gamma.RIIIA tetrameric complex to Fc fusion
proteins on the yeast cell surface was analyzed. PE-conjugated
Fc.gamma.RIIIA tetrameric complexes were pre-incubated with
different concentrations of 3G8 (.diamond-solid.), LNK
(.tangle-solidup.) or an irrelevant isotype control (.box-solid.),
and subsequently incubated with the yeast cells. Cells were
analyzed by FACS for PE fluorescence. The percent cells that bound
the Fc.gamma.RIIIA tetrameric complex were plotted on the
y-axis.
[0111] FIG. 10 Example of Sort Gate for Selecting Fc Mutants With
Increased Binding to Fc.gamma.RIIIA
[0112] Cells were stained with PE-conjugated Fc.gamma.RIIIA
tetrameric complexes (y-axis) and anti-Fc-FITC conjugated antibody
.alpha.-axis). Boxed area represents sort gate set to select
.about.1.0% of the cell population.
[0113] FIGS. 11A-N FACS Analysis of Some of the Fc Mutants
Identified Having an Increased Affinity for Fc.gamma.RIIIA
Tetrameric Complexes
[0114] Individual clones harboring the pYD-CH1 plasmid containing
independent Fc mutations were amplified in selective media
containing glucose, induced for Fc expression in selective media
containing galactose, and subsequently analyzed by FACs. FIGS. 10A
and B represent cells harboring wild-type Fc; FIGS. 10C and D
represent mutant #5; FIGS. 10E and F represent mutant #20; FIGS.
10G and H represent mutant #21; FIGS. 10 I and J represent mutant
#24; FIGS. 10K and L represent mutant #25; FIGS. 10M and N
represent mutant #27. Cells were stained with Fc.gamma.RIIIA
tetrameric complex (FIGS. 10 A, C, E, G, I, K, and M) or
Fc.gamma.RIIB tetrameric complex (FIGS. 10B, D, F, H, J, L, and
N).
[0115] FIGS. 12 A-B Characterization of Fc Mutants in the 4-4-20
Monoclonal Antibody by ELISA
[0116] Fc domains from the pYD-CH1 plasmids were cloned into the
heavy chain of the chimeric 4-4-20 monoclonal antibody. The 4-4-20
monoclonal antibody was expressed in 293 cells and supernatants
were collected. ELISA plates were coated with fluoresceine
conjugated BSA to capture the chimeric 4-4-20 mutant antibodies.
Fc.gamma.RIIIA (A) and Fc.gamma.RIIB (B) receptors were then coated
onto the ELISA plates to which the 4-4-20 monoclonal antibodies had
been absorbed in order to determine the relative affinities of the
variant receptors to the Fc domains. Mutants #15 and #29 were
non-binding isolates included as controls.
[0117] FIG. 13 ADCC Activity of Mutants in the 4-4-20 Monoclonal
Antibody
[0118] 4-4-20 antibodies containing mutant Fc regions were assessed
for their ADCC activity, and compared to the ADCC activity of a
wild type 4-4-20 antibody. The mutants analyzed are as follows:
MGFc-10 (K288N, A330S, P396L), MGFc-26 (D265A), MGFc-27 (G316D,
A378V, D399E), MGFc28 (N315I, A379M, D399E), MGFc29 (F243I, V379L,
G420V), MGFc30 (F275V), MGFc-31 (P247L, N421K), MGFc-32 (D280E,
S354F, A431D, L441I), MGFc-33 (K317N, F423 deleted), MGFc-34
(F241L, E258G), MGFc-35 (R255Q, K326E), MGFc-36 (K218R, G281D,
G385R)
[0119] FIGS. 14 A and B ADCC Activity of Mutants in the HER2/neu
Humanized Monoclonal Antibody
[0120] A. Humanized HER2/neu monoclonal antibodies containing
mutant Fc regions were assessed for their ADCC activity and
compared to the ADCC activity of a wild type Her2/neu antibody. The
mutants analyzed are as follows: MGFc-5 (V379M), MGFc-9 (F243I,
V379L), MGFc-10 (K288N, A330S, P396L), MGFc-13 (K334E, T359N,
T366S), MGFc-27 (G316D, A378V, D399E).
[0121] B. ADCC activity of additional mutants in the context of the
humanized Her2/neu monoclonal antibody MGFc-37 (K248M), MGFc-39
(E293V Q295E, A327T), MGFc-38 (K392T, P396L), MGFc-41 (H268N,
P396L), MGFc-23 (K334E, R292L), MGFc-44, MGFc-45. Two independent
clones were tested for each mutant.
[0122] FIG. 15 Capture of ch 4-4-20 Antibody on BSA-FITC
Surface
[0123] 6 .mu.L of antibody at a concentration of approximately 20
.mu.g/mL was injected at 5 .mu.L/min over a BSA-fluoroscein
isothiocyanate (FITC) surface. BIAcore sensogram of the binding of
ch 4-4-20 antibodies with mutant Fc regions on the surface of the
BSA-FITC immobilized sensor ship is shown. The marker was set on
wild-type captured antibody response.
[0124] FIG. 16 Sensogram of Real Time Binding of Fc.gamma.RIIIA to
ch 4-4-20 Antibodies Carrying Variant Fc Regions
[0125] Binding of Fc.gamma.RIIIA to ch-4-4-20 antibodies carrying
variant Fc regions was analyzed at 200 nM concentration. Responses
were normalized at the level of ch-4-4-20 antibody obtained for
wild-type.
[0126] Mutants used were as follows: Mut 6 (S219V), Mut 10 (P396L,
A330S, K288N); Mut 18 (K326E); Mut 14 (K334E, K288N); Mut 11
(R255L, F243L); Mut 16 (F372Y); Mut 19 (K334N, K246I).
[0127] FIGS. 17 A-H Analysis of Kinetic Parameters of
Fc.gamma.RIIIA Binding to Antibodies Carrying Variant Fc
Regions
[0128] Kinetic parameters for Fc.gamma.RIIIA binding to antibodies
carrying variant Fc regions were obtained by generating separate
best fit curves for 200 nM and 800 nM. Solid line indicates an
association fit which was obtained based on the k.sub.off values
calculated for the dissociation curves in the 32-34 sec interval.
K.sub.d and k.sub.off values represent the average from two
concentrations.
[0129] FIG. 18 Sensogram of Real Time Binding of Fc.gamma.RIIB-Fc
Fusion Proteins to Antibodies Carrying Variant Fc Regions
[0130] Binding of Fc.gamma.RIIB-Fc fusion proteins to ch-4-4-20
antibodies carrying variant Fc regions was analyzed at 200 nM
concentration. Responses were normalized at the level of ch-4-4-20
antibody obtained for wild type.
[0131] FIGS. 19 A-C Analysis of Kinetic Parameters Fc.gamma.RIIB-Fc
Fusion Proteins to Antibodies Carrying Variant Fc Regions
[0132] Kinetic parameters for Fc.gamma.RIIB-Fc binding to
antibodies carrying variant Fc regions were obtained by generating
separate best fit curves for 200 nM and 800 nM. Solid line
indicates an association fit which was obtained based on the
k.sub.off values calculated for the dissociation curves in the
32-34 sec. interval. K.sub.d and K.sub.off values represent the
average from two concentrations.
[0133] Mutants used were as follows: Mut 6 (S219V), Mut 10 (P396L,
A330S, K288N); Mut 18 (K326E); Mut 14 (K334E, K288N); Mut 11
(R255L, F243L); Mut 16 (F372Y); Mut 19 (K334N, K246I).
[0134] FIG. 20 Ratios of K.sub.off (Wt)/K.sub.off (Mut) for
Fc.gamma.RIIIA-Fc Plotted Against ADCC Data
[0135] Numbers higher than one show a decreased dissociation rate
for Fc.gamma.RIIIA binding and increased dissociation rate for
Fc.gamma.RIIB-Fc binding relative to wild-type. Mutants in the box
have lower off rate for Fc.gamma.RIIIA binding and higher off rate
for Fc.gamma.RIIB-Fc binding.
[0136] FIG. 21 Competition with Unlabeled Fc.gamma.RIIIA
[0137] A kinetic screen was implemented to identify Fc region
mutants with improved K.sub.off rates for binding Fc.gamma.RIIIA. A
library of Fc region variants containing P396L mutation was
incubated with 0.1 .mu.M biotinylated
Fc.gamma.RIIIA-Linker-AVITAG.TM. peptide for one hour and then
washed. Subsequently 0.8 .mu.M unlabeled Fc.gamma.RIIIA was
incubatd with the labeled yeast for different time points. Yeast
was spun down and unlabeled Fc.gamma.RIIIA was removed, Receptor
bound yeast was stained with SA (streptavidin):PE (phycoerythrin)
for FACS analysis.
[0138] FIGS. 22 A-C FACS Analysis Based on the Kinetic Screen
[0139] Based on the calculated K.sub.off from the data presented in
FIG. 20, a one minute time point selection was chosen. A 10-fold
excess of library was incubated with 0.1 .mu.M biotinylated
Fc.gamma.RIIIA-Linker-AVITAG.TM. peptide monomer; cells were washed
and incubated with unlabeled ligand for one minute; then washed and
labeled with SA:PE. The cells were then sorted by FACS, selecting
the top 0.3% binders. The non-selected P396L library was compared
to the yeast cells selected for improved binding by FACS. The
histograms show the percentage of cells that are costained with
both Fc.gamma.RIIIA/PE and goat anti-human Fc/FITC.
[0140] FIGS. 23 A-F and G-L Selection Based on Solid Phase
Depletion of Fc.gamma.RIIB Fc Binders
[0141] FIGS. 23 A-F. The P396L library was screened based on
Fc.gamma.RIIB depletion and Fc.gamma.RIIIA selection using magnetic
beads. The Fc.gamma.RIIB depletion by magnetic beads was repeated 5
times. The resulting yeast population was analyzed and found to
show greater than 50% cell staining with goat anti-human Fc and a
very small percentage of cells stained with Fc.gamma.RIIIA.
Subsequently cells were selected twice by FACS using 0.1 .mu.M
biotinylated Fc.gamma.RIIIA linker-AVITAG.TM. peptide. Yeast cells
were analyzed for both Fc.gamma.RIIIA and Fc.gamma.RIIB binding
after each sort and compared to wild type binding.
[0142] FIGS. 23 G-L. Fc Mutants were selected from the
Fc.gamma.RIIB depleted yeast population using biotinylated
Fc.gamma.RIIIA 158F linker avitag monomer as a ligand. The sort
gate was set to select the top 0.25% Fc.gamma.RIIIA 158F binders.
The resulting enriched population was analyzed by FACS for binding
to the different Fc.gamma.RIIIA (158F and 158V), Fc.gamma.RIIIB and
Fc.gamma.RIIA (131R).
[0143] FIG. 24 Relative Rates of SKBR3Target Cell Lysis Mediated by
Chimeric 4D5 Harboring Fc Mutants
[0144] Relative rates of lysis was calculated for each Fc mutant
tested. Lysis rates for 4D5 antibody with Fc mutants were divided
by the rate of lysis mediated by wild type 4D5 antibody. Data from
at least 2 independent assays were averaged and plotted on the
histogram. For each Fc mutant data from two different antibody
concentrations are shown. The antibody concentrations were chosen
to flank the point along the curve at which lysis was
.about.50%.
[0145] FIG. 25 Relative Rates of Daudi Cell Lysis Mediated by
Chimeric 2H7 Harboring Fc Mutants
[0146] Relative rates of lysis was calculated for each Fc mutant
tested. Lysis rates for 2H7 antibody with Fc mutants were divided
by the rate of lysis mediated by wild type 2H7 antibody. Data from
at least 1-2 independent assays were averaged and plotted on the
histogram. For each Fc mutant, data from two different antibody
concentrations are shown The antibody concentrations were chosen
based on the point along the curve at which lysis was
.about.50%.
[0147] FIG. 26 Scheme for Library Production.
[0148] DNA strands are represented. Forward arrows represent
primers containing mutant codons. Reverse arrow represent reverse
gene specific oligo.
[0149] FIG. 27 Strategy for Production of Libraries by Build a Gene
Protocol.
[0150] The rectangular boxes represent the hinge, CH2, and CH3
domains, respectively. The short black lines represent the double
stranded oligos with 5' overhangs.
[0151] FIG. 28 Novel Fc Mutants Improve PBMC Mediated ADCC in
SKBR3Cells.
[0152] The plot shows linear regression analysis of a standard ADCC
assay. Antibody was titrated over 3 logs using an effector to
target ratio of 75:1. % lysis=(Experimental
release--SR)/(MR-SR)*100.
[0153] FIG. 29 Novel Fc Mutants Improve PBMC Mediated ADCC in Daudi
Cells.
[0154] The plot shows linear regression analysis of a standard ADCC
assay. Antibody was titrated over 3 logs using an effector to
target ratio of 75:1. % lysis=(Experimental
release--SR)/(MR-SR)*100.
[0155] FIGS. 30A-30O Fc Receptor Profiles Via FACS Upon Cytokine
Treatment of Monocytes.
[0156] Cytokine treatment of monocytes increases low affinity Fc
receptor expression Elutriated monocytes were cultured using
specific cytokines in serum free media. Fc receptor profiles were
assayed using FACS.
[0157] FIG. 31 Improved Tumor Cell Killing Using Fc Mutants in
Macrophage-Derived Monocytes Based ADCC.
[0158] Ch4D5 MAb concentration over 2 logs was tested using
effector:target ratio of 35:1. Percent lysis was calculated as in
FIG. 28.
[0159] FIG. 32 Complement Dependent Cytotoxicity Assay Flow
Chart.
[0160] The flow chart summarizes the CDC assays used.
[0161] FIG. 33 Complement Dependent Cytotoxicity Activity
[0162] Fc mutants that show enhanced binding to Fc.gamma.RIIIA also
showed improved complement activity. Anti-CD20 ChMAb over 3 orders
of magnitude was titrated. Percent lysis was calculated as in as in
FIG. 28.
[0163] FIG. 34 Decision Tree for Selection of Fc Mutants
[0164] An exemplary protocol for selecting Fc mutants. In the
Figure, the term "Rituxan" denotes RITUXAN.TM. anti-CD20
antibody.
[0165] FIGS. 35A-35C C1q Binding to 2B6 Antibody
[0166] FIG. 35A. The diagram depicts the BIAcore format for
analysis of 2B6 binding to the first component of the complement
cascade.
[0167] FIGS. 35B-35C. Sensogram of real time binding of 2B6
antibody carrying variant Fc regions to C1q.
[0168] FIGS. 36 A-D C1q Binding to 2B6 Mutant Antibody.
[0169] Sensogram of real time binding of 2B6 mutants to C1q (3.25
nM). Mutants depicted at MgFc51 (Q419H, P396L); MgFc51/60 in Panel
A; MgFc55 and MgFc55/60 (Panel B), MgFc59 and MgFc59/60 (Panel C);
and MgFc31/60 (Panel D).
[0170] FIGS. 37 A-D Fc Variants with Decreased Binding to
Fc.gamma.RIIB
[0171] Binding of FcR to ch4D5 antibodies to compare effect of
D270E (60) on R255L, P396L double mutant (MgFc55). K.sub.D was
analyzed at different concentrations of FcR; 400 nM CD16A 158V; 800
nM CD16A 158F; 200 nM CD32B; 200 nM CD32A 131H. Analysis was
performed using separate K.sub.D using Biacore 3000 software.
[0172] FIGS. 38 A-D Kinetic Characteristics of 4D5 Mutants Selected
from Fc.gamma.RIIB Depletions/Fc.gamma.RIIAH131 Selection
[0173] Binding of FcR to ch4D5 antibodies carrying different Fc
mutations selected by CD32B depletion and CD32A H131 screening
strategy. K.sub.D was analyzed at different concentrations of FcR;
400 nM CD16A 158V; 800 nM CD16A 158F; 200 nM CD32B; 200 nM CD32A
131H. Analysis was performed using separate K.sub.D using Biacore
3000 software.
[0174] FIG. 39 Plot of MDM ADCC Data Against the K.sub.OFF
Determined for CD32A 131H Binding as Determined by Biacore.
[0175] The mutants are as follows: MgFc 25 (E333A, K334A, S298A);
MgFc68 (D270E); MgFc38 (K392T, P396L); MgFc55 (R255L, P396L);
MgFc31 (P247L, N421K); MgFc59(K370E, P396L).
[0176] FIG. 40 A-D Fc.gamma.R Binding to 4D5 Mutant Antibody,
Triple Mutation
[0177] Sensogram of real time binding of 4D5 mutants to
Fc.gamma.RIII3A (CD16Z V.sup.158, panel A, and CD16A F.sup.158,
panel B), Fc.gamma.RIIB (CD32B, panel C) and Fc.gamma.RIIA (CD32A
H.sup.131, panel D). Mutants depicted are MgFc31/60 (P247L; N421K;
D270E) (short dashed line), MgFc71 (D270E; G316D; R416G) (solid
line) and AAA (E333A; K334A; S298A) (long dashed line). The binding
of wild-type 4D5 (dash-dot line) is also provided.
[0178] FIG. 41A-D Fc.gamma.R Binding to 4D5 Mutant Antibody,
Quadruple Mutation
[0179] Sensogram of real time binding of 4D5 mutants to
Fc.gamma.RIII3A (CD16Z V.sup.158, panel A, and CD16A F.sup.158,
panel B), Fc.gamma.RIIB (CD32B, panel C) and Fc.gamma.RIIA (CD32A
H.sup.131, panel D). Mutants depicted are MgFc55/60/F243L (R255L;
P396L; D270E; F243L), MgFc38/60/F243L (K392T; P396L; D270E; F243L)
and AAA (E333A; K334A; S298A). The binding of wild-type 4D5 is also
provided.
[0180] FIG. 42 A-E Binding of 4D5 Variant 31/60 to HT29 Cells
[0181] FACS analysis was used to characterize the binding of
monoclonal anti-HER2/neu antibody ch4D5, variant 31/60 (P247L;
N421K; D270E), to HT29 cells (low expression of HER2/neu).
Incubation with primary antibody was at 10 .mu.g/ml (A), 1 .mu.g/ml
(B), 0.1 .mu.g/ml (C), 0.001 .mu.g/ml (D), or 0.001 .mu.g/ml (E).
Wild-type ch4D5 and SYNAGIS.RTM. (palivizumab) were used as
controls. PE-conjugated polyclonal F(ab).sub.2 goat
anti-humanFC.gamma.R was used as the secondary antibody.
[0182] FIG. 43 A-B ADCC Activity of Mutants in the Anti-HER2/neu
Antibody, ch4D5
[0183] CH4D5 antibodies containing mutant Fc regions were assessed
for their ADCC activity and compared to the ADCC activity of wild
type ch4D5. SKBR3 (high expression of HER2/neu) and HT29 (low
expression of HER2/neu) cells lines were used as targets (panels A
and B, respectively). Effector to target ratio (E:T ratio) was 50:1
with 18 h incubation. Mutants analyzed were MGFc59/60 (K370E;
P396L; D270E), MGFc55/60 (R255L; P396L; D270E), MGFc51/60 (Q419H;
P396L; D270E), MGFc55/60/F243L (R255L; P396L; D270E; F243L);
MGFc74/P396L (F243L; R292P; V3051; P396L).
[0184] FIG. 44 A-B ADCC Activity of Mutants in the Anti-HER2/neu
Antibody, ch4D5
[0185] Ch4D5 antibodies containing mutant Fc regions were assessed
for their ADCC activity and compared to the ADCC activity of wild
type ch4D5. SKBR3 (high expression of HER2/neu) and HT29 (low
expression of HER2/neu) cells lines were used as targets (panels A
and B, respectively). Effector to target ratio (E:T ratio) was 75:1
with 18 h incubation. Mutants analyzed were MgFc31/60 (P247L;
N421K; D270E) and MgFc71 (D270E; G316D; R416G).
[0186] FIGS. 45A-45H Binding of Mutants in the Monoclonal
Anti-CD32B Antibody Ch2B6 to Daudi Cells and Ramos Cells
[0187] FACS analysis was used to characterize the binding of
monoclonal anti-CD32B antibody ch2B6 variant 31/60 (P247L; N421K;
D270E), variant 71 (D270E; G316D; R416G) and variant 59/60 (K370E;
P396L; D270E) to either Daudi cells (high expression of CD32B) or
Ramos cells (low expression of CD32B). Incubation with primary
antibody was at 5 .mu.g/ml, 0.5 .mu.g/ml, 50 ng/ml, or 5 ng/ml.
Wild-type ch2B6 and IgG (SYNAGIS) were used as controls.
PE-conjugated polyclonal F(ab).sub.2 goat anti-humanFC.gamma.R was
used as the secondary antibody.
[0188] FIG. 46 A-B ADCC Activity of Mutants in the Anti-CD32B
Antibody, ch2B6
[0189] Ch2B6 antibodies containing mutant Fc regions were assessed
for their ADCC activity and compared to the ADCC activity of wild
type 2B6. The Ramos cell line (low expression of CD32B) was used as
target. Effector to target ratio (E:T ratio) was 75:1 with 18 h
incubation. Mutants analyzed were variant 31/60 (P247L; N421K;
D270E) and ch2B6 N297Q (aglycoslyated Fc, no FcR binding) (panel
A); and MGFc51/60/F243L (Q419H; P396L; D270E; F243L);
MGFc55/60/F243L (R255L; P396L; D270E; F243L) and MGFc38/60/F243L
(K392T; P396L; D270E; F243L) (panel B). Wild-type ch2B6 or
RITUXAN.TM. (rituximab) were used as controls.
[0190] FIG. 47 A-C CDC Activity of Mutants in the Anti-CD32B
Antibody, ch2B6
[0191] Ch2B6 antibodies containing mutant Fc regions were assessed
for their CDC activity and compared to the CDC activity of wild
type ch2B6. BL41 (a Burkitt's lymphoma cell line) (panel A and B)
and Ramos (low expression of CD32B) (panel C) cells lines were used
as targets. Effector to target ratio (E:T ratio) was 75:1 with 18 h
incubation. Mutants analyzed were MgFc31/60 (P247L; N421K; D270E)
and, MGFc55/60/Y300L (R255L; P396L; D270E; Y300L) (panel A); MgFc71
(D270E; G316D; R416G), MGFc51/60/F243L (Q419H; P396L; D270E;
F243L), and MGFc55/60/F243L (R255L; P396L; D270E; F243L) (panel B);
and MgFc31/60 (P247L; N421K; D270E) (panel C). Wild-type ch2B6,
wild-type humanized ch2B6 (hu2B6 wt) or RITUXAN.TM. (rituximab)
were used as controls.
[0192] FIG. 48 A-B ADCC Activity of Mutants in the Anti-CD32B
Antibody, ch2B6
[0193] Ch2B6 antibodies containing mutant Fc regions were assessed
for their ADCC activity and compared to the ADCC activity of wild
type ch2B6. The Daudi cell line (high expression of CD32B) was used
as target. Effector to target ratio (E:T ratio) was 75:1 with 18 h
incubation. Mutants analyzed were MgFc31/60 (P247L; N421K; D270E),
ch2B6 Ag (N297Q; aglycoslyated Fc, no FcR binding) and MgFc71
(D270E; G316D; R416G) (panel A); and MGFc55/60/F243L (R255L; P396L;
D270E; F243L), MGFc51/60/F243L (Q419H; P396L; D270E; F243L) and
MGFc38/60/F243L (K392T; P396L; D270E; F243L) (panel B). Wild-type
ch2B6 or RITUXAN.TM. (rituximab) were used as controls.
[0194] FIG. 49 A-B FACS Analysis of the Binding of the Anti-CD32B
Antibody, ch2B6, and the Anti-CD20 Antibody, Rituxan.TM., to a
Transgenic Cho Cell Line.
[0195] Cho cells were engineered to express both recombinant CD32B
and recombinant CD20 on the cell surface. Following incubation and
amplification in selective media, cells were analyzed by FACS.
Cells were incubated in either FITC-conjugated wild-type 2B6 (A) or
FITC-conjugated Rituxan.TM. (B).
[0196] FIG. 50 A-B ADCC Activity of Mutants in the Anti-CD20
Antibody, Rituxan.TM.
[0197] RITUXAN.TM. anti-CD20 antibodies containing mutant Fc
regions were assessed for their ADCC activity and compared to the
ADCC activity of wild type RITUXAN.TM. anti-CD20 and ch2B6. A Cho
cell line engineered to express both CD32B and CD20 was used as
target. Effector to target ratio (E:T ratio) was 75:1 with 18 h
incubation. Figure A shows the ADCC activity of wild type ch2B6 and
RITUXAN.TM. anti-CD20. Figure B shows a comparison of the ADCC
activity of wild type RITUXAN.TM. anti-CD20 and RITUXAN.TM.
anti-CD20 comprising mutation variant MGFc55/60 (R255L; P396L;
D270E).
[0198] FIG. 51A-E Comparison of Binding Affinity and Kinetic
Characteristics of ch2B6 Mutants
[0199] FACS analysis was used to characterize the binding of mutant
ch2B6 antibodies to Ramos cells (low expression of CD32B). Data
were compared to a BIAcore analysis of the k.sub.off for the same
variant antibodies. Mutants analyzed were MgFc55 (R255L; P396L),
MgFc55/60 (R255L; P396L; D270E) and MgFc55/60/F243L (R255L; P396L;
D270E; F243L). Wild-type ch2B6 was used as control. Incubation with
primary antibody was at 10 .mu.g/ml (A), 1 .mu.g/ml (B), 0.1 ng/ml
(C), or 0.01 ng/ml (D). PE-conjugated polyclonal F(ab).sub.2 goat
anti-humanFC.gamma.R was used as the secondary antibody.
[0200] FIG. 52 A-C Binding of Activating Receptor CD16A to Ramos
Cells Opsonized with Mutant ch2B6 Antibody
[0201] FACS analysis was used to characterize the binding of
activating receptor CD16A to Ramos cells opsonized with mutant
ch2B631/60 antibody (P247L; N421K; D270E). Opsonization with
wild-type ch2B6, hu2B6YA (humanized 2B6 with YA substitution at
positions 50,51 of antibody light-chain--eliminates glycosylation
at position 50 of the light-chain protein), or antibody-free buffer
was used as a control. PE-conjugated polyclonal F(ab).sub.2 goat
anti-humanFC.gamma.R was used as the secondary antibody.
[0202] FIG. 53 A-J Binding of Mutants in the Monoclonal Anti-CD32B
Antibody ch2B6 to Daudi Cells
[0203] FACS analysis was used to characterize the binding of
monoclonal anti-CD32B antibody ch2B6 variant 31/60 (P247L; N421K;
D270E), hu2B6YA (humanized 2B6 with YA substitution at positions
50,51 of antibody light-chain--eliminates glycosylation at position
50 of the light-chain protein) or hu2B6YA31/60 to Daudi cells (high
expression of CD32B). Wild-type ch2B6, hu2B6, SYNAGIS.RTM.
(palivizumab) and ch2B6 Agly (N297Q; aglycoslyated Fc, no FcR
binding) were used as controls. Incubation with primary antibody
was at either 37.degree. C. (panels A-E) or 4.degree. C. (panels
F-J) for 0.5 h and at a concentration of 10 .mu.g/ml (A, F), 1
.mu.g/ml (B, G), 0.1 ng/ml (C, H), 0.01 .mu.g/ml (D, I), or 0.001
.mu.g/ml (E, J). PE-conjugated polyclonal F(ab).sub.2 goat
anti-humanFC.gamma.R was used as the secondary antibody.
[0204] FIG. 54 A-E Binding of Mutants in the Monoclonal Anti-CD32B
Antibody ch2B6 to EL-4/CD32B Cells
[0205] FACS analysis was used to characterize the binding of
monoclonal anti-CD32B antibody ch2B6 variant 31/60 (P247L; N421K;
D270E), hu2B6YA (humanized 2B6 with YA substitution at positions
50,51 of antibody light-chain--eliminates glycosylation at position
50 of the light-chain protein) or hu2B6YA31/60 to EL-4/CD32B cells.
Wild-type ch2B6, hu2B6, SYNAGIS.RTM. (palivizumab) and ch2B6 Agly
(N297Q; aglycoslyated Fc, no FcR binding) were used as controls.
Incubation with primary antibody was at 37.degree. C. for 0.5 h and
at a concentration of 10 .mu.g/ml (A), 1 .mu.g/ml (B), 0.1 .mu.g/ml
(C), 0.01 .mu.g/ml (D), or 0.001 .mu.g/ml (E). PE-conjugated
polyclonal F(ab).sub.2 goat anti-humanFC.gamma.R was used as the
secondary antibody.
[0206] FIG. 55 A-J Binding of Mutants in the Monoclonal Anti-CD32B
Antibody ch2B6 to Ramos Cells
[0207] FACS analysis was used to characterize the binding of
monoclonal anti-CD32B antibody ch2B6 variant 31/60 (P247L; N421K;
D270E), hu2B6YA (humanized 2B6 with YA substitution at positions
50,51 of antibody light-chain--eliminates glycosylation at position
50 of the light-chain protein) or hu2B6YA31/60 to Ramos cells (low
expression of CD32B). Wild-type ch2B6, hu2B6, SYNAGIS.RTM.
(palivizumab) and ch2B6 Agly (N297Q; aglycoslyated Fc, no FcR
binding) was used as control. Incubation with primary antibody was
at either 37.degree. C. (panels A-E) or 4.degree. C. (panels F-J)
for 0.5 h and at a concentration of 10 .mu.g/ml (A, F), 1 .mu.g/ml
(B, G), 0.1 ng/ml (C, H), 0.01 .mu.g/ml (D, I), or 0.001 .mu.g/ml
(E, J). PE-conjugated polyclonal F(ab).sub.2 goat
anti-humanFC.gamma.R was used as control.
[0208] FIG. 56 ADCC Activity of Mutants in the Anti-CD32B Antibody,
ch2B6
[0209] Ch2B6 antibodies containing mutant Fc regions were assessed
for their ADCC activity and compared to the ADCC activity of wild
type ch2B6. The Ramos cell line (low expression of CD32B) was used
as target. Effector to target ratio (E:T ratio) was 75:1 with 18 h
incubation. Mutants analyzed were MGFc31/60 (P247L; N421K; D270E),
MGFc51/60 (Q419H; P396L; D270E), MGFc55/60 (R255L; P396L; D270E);
hu2B6YA (humanized 2B6 with YA substitution at positions 50,51 of
antibody light-chain--eliminates glycosylation at position 50 of
the light-chain protein), hu2B6YA MGFc51/60; hu2B6YA MGFc31/60 and
hu2B6YA MGFc55/60. Wild-type ch2B6, hu2B6, RITUXAN.TM. (rituximab)
and ch2B6 Agly (N297Q; aglycoslyated Fc, no FcR binding) were used
as controls.
[0210] FIG. 57 CDC Activity of Mutants in the Anti-CD32B Antibody,
ch2B6
[0211] Ch2B6 antibodies containing mutant Fc regions were assessed
for their CDC activity and compared to the CDC activity of wild
type ch2B6. The Ramos cell line (low expression of CD32B) was used
as target. Effector to target ratio (E:T ratio) was 75:1 with 18 h
incubation. Mutants analyzed were MGFc31/60 (P247L; N421K; D270E),
MGFc51/60 (Q419H; P396L; D270E), MGFc55/60 (R255L; P396L; D270E);
hu2B6YA (humanized 2B6 with YA substitution at positions 50,51 of
antibody light-chain--eliminates glycosylation at position 50 of
the light-chain protein), hu2B6YA MGFc51/60; hu2B6YA MGFc31/60 and
hu2B6YA MGFc55/60. Wild-type ch2B6 and RITUXAN.TM. anti-CD20
antibodies were used as controls.
[0212] FIG. 58 A-F ADCC Activity of Modified Rituximab Antibodies
in Human Patients Treated with Rituximab
[0213] Rituximab antibodies containing mutant Fc regions were
assessed for their ADCC activity and compared to the ADCC activity
of wild type rituximab. Patient derived cells were used as target.
Effector to target ratio (E:T ratio) was 30:1 and 10:1. Mutants
analyzed were MGFc55/60/300L (R255L; P396L; D270E; Y300L);
MGFc51/60 (Q419H; P396L; D270E); MGFc52/60 (V240A; P396L; D270E);
MGFc59/60 (K370E; P396L; D270E); MGFc38/60 (K392T; P396L; D270E);
MGFc59 (K370E; P396L); MGFc51 (Q419H; P396L); MGFc31/60 (P247L;
N421K; D270E); MGFc55/292G (R255L; P396L; D270E; R292G).
5. DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0214] The present invention relates to engineering molecules,
preferably polypeptides, and more preferably immunoglobulins (e.g.,
antibodies) to confer one or more effector function activities to
the molecule, which effector functions the parent molecule does not
have or has at low levels (e.g., not detectable in in vitro and/or
in vivo assays known in the art). In particular, the modified
molecules, e.g., antibodies, of the invention comprise a variant Fc
region, having one or more amino acid modifications (e.g.,
substitutions, but also including insertions or deletions) in one
or more regions, which modifications confer at least one effector
function. In particular, the modifications alter the affinity and
avidity of the variant Fc region for an Fc.gamma.R (e.g.,
activating Fc.gamma.Rs or inhibitory Fc.gamma.Rs) and thereby
altering the activity of one or more effector functions. In other
embodiments, the modifications confer homo-oligomerization activity
to the parent Fc region such that oligomerization of the modified
antibody cross-links cell-surface antigens, resulting in apoptosis,
negative-growth regulation or cell killing. Effector function
activities that may be conferred include, but are not limited to,
antibody-dependent cell mediated cytotoxicity (ADCC),
antibody-dependent phagocytosis, phagocytosis, opsonization,
opsonophagocytosis, cell binding, rosetting, and complement
dependent cell mediated cytotoxicity (CDC). In some embodiments of
the invention, the modifications alter the affinity of the variant
Fc region such that the variant Fc regions oligomerize and
homo-oligomers of the modified antibody are formed. In certain
embodiments of the invention, the engineered molecule is not an
anti-CD20 antibody, more particularly, does not compete for CD20
binding with rituximab or is not rituximab.
[0215] The present invention also relates to molecules (e.g.,
antibodies) comprising a variant Fc region having one or more amino
acid modifications (e.g., substitutions, deletions, insertions) in
one or more portions, which modifications increase the affinity and
avidity of the variant Fc region for an Fc.gamma.R (including
activating and inhibitory Fc.gamma.Rs). In some embodiments, said
one or more amino acid modifications increase the affinity of the
variant Fc region for Fc.gamma.RIIIA and/or Fc.gamma.RIIA. In
another embodiment, the variant Fc region further specifically
binds Fc.gamma.RIIB with a lower affinity than does the Fc region
of the comparable parent antibody (i.e., an antibody having the
same amino acid sequence as the antibody of the invention except
for the one or more amino acid modifications in the Fc region). In
some embodiments, such modifications increase the affinity of the
variant Fc region for Fc.gamma.RIIIA and/or Fc.gamma.RIIA and also
enhance the affinity of the variant Fc region for Fc.gamma.RIIB
relative to the parent antibody. In other embodiments, said one or
more amino acid modifications increase the affinity of the variant
Fc region for Fc.gamma.RIIIA and/or Fc.gamma.RIIA but do not alter
the affinity of the variant Fc regions for Fc.gamma.RIIB relative
to the Fc region of the parent antibody. In another embodiment,
said one or more amino acid modifications enhance the affinity of
the variant Fc region for Fc.gamma.RIIIA and Fc.gamma.RIIA but
reduce the affinity for Fc.gamma.RIIB relative to the parent
antibody. Increased affinity and/or avidity results in detectable
binding to the Fc.gamma.R or Fc.gamma.R-related activity in cells
that express low levels of the Fc.gamma.R when binding activity of
the parent molecule (without the modified Fc region) can not be
detected in the cells. In other embodiments, the modified molecule
exhibits detectable binding in cells which express non-Fc.gamma.R
receptor target antigens at a density of 30,000 to 20,000
molecules/cell, at a density of 20,000 to 10,000 molecules/cell, at
a density of 10,000 to 5,000 molecules/cell, at a density of 5,000
to 1,000 molecules/cell, at a density of 1,000 to 200
molecules/cell or at a density of 200 molecules/cell or less (but
at least 10, 50, 100 or 150 molecules/cell).
[0216] In another embodiment, said one or more modifications to the
amino acids of the Fc region reduce the affinity and avidity of the
antibody for one or more Fc.gamma.R receptors. In a specific
embodiment, the invention encompasses antibodies comprising a
variant Fc region, wherein said variant Fc region comprises at
least one amino acid modification relative to a wild type Fc
region, which variant Fc region only binds one Fc.gamma.R, wherein
said Fc.gamma.R is Fc.gamma.RIIIA. In another specific embodiment,
the invention encompasses antibodies comprising a variant Fc
region, wherein said variant Fc region comprises at least one amino
acid modification relative to a wild type Fc region, which variant
Fc region only binds one Fc.gamma.R, wherein said Fc.gamma.R is
Fc.gamma.RIIA.
[0217] Preferably, the binding properties of the molecules of the
invention are characterized by in vitro functional assays for
determining one or more Fc.gamma.R mediator effector cell functions
(See Section 5.2.7). The affinities and binding properties of the
molecules, e.g., antibodies, of the invention for an Fc.gamma.R can
be determined using in vitro assays (biochemical or immunological
based assays) known in the art for determining antibody-antigen or
Fc-Fc.gamma.R interactions, i.e., specific binding of an antigen to
an antibody or specific binding of an Fc region to an Fc.gamma.R,
respectively, including but not limited to ELISA assay, surface
plasmon resonance assay, immunoprecipitation assays (See Section
5.2.1). In most preferred embodiments, the molecules of the
invention have similar binding properties in in vivo models (such
as those described and disclosed herein) as those in in vitro based
assays. However, the present invention does not exclude molecules
of the invention that do not exhibit the desired phenotype in in
vitro based assays but do exhibit the desired phenotype in
vivo.
[0218] In some embodiments, the molecules of the invention
comprising a variant Fc region comprise at least one amino acid
modification in the CH3 domain of the Fc region, which is defined
as extending from amino acids 342-447. In other embodiments, the
molecules of the invention comprising a variant Fc region comprise
at least one amino acid modification in the CH2 domain of the Fc
region, which is defined as extending from amino acids 231-341. In
some embodiments, the molecules of the invention comprise at least
two amino acid modifications, wherein one modification is in the
CH3 region and one modification is in the CH2 region. The invention
further encompasses amino acid modification in the hinge region. In
a particular embodiment, the invention encompasses amino acid
modification in the CH1 domain of the Fc region, which is defined
as extending from amino acids 216-230.
[0219] In particularly preferred embodiments, the invention
encompasses molecules comprising a variant Fc region wherein said
variant confers or has an increased ADCC activity and/or an
increased binding to Fc.gamma.RIIA (CD32A), as measured using
methods known to one skilled in the art and exemplified herein. The
ADCC assays used in accordance with the methods of the invention
may be NK dependent or macrophage dependent.
[0220] The Fc variants of the present invention may be combined
with other Fc modifications known in the art. The invention
encompasses combining an Fc variant of the invention with other Fc
modifications to provide additive, synergistic, or novel properties
to the modified antibody. Preferably, the Fc variants of the
invention enhance the phenotype of the modification with which they
are combined. For example, if an Fc variant of the invention is
combined with a mutant known to bind Fc.gamma.RIIIA with a higher
affinity than a comparable wild type Fc region; the combination
with a mutant of the invention results in a greater fold
enhancement in Fc.gamma.RIIIA affinity.
[0221] The Fc variants of the present invention may be combined
with any modifications in the art such as those disclosed in Table
2 below.
TABLE-US-00002 TABLE 2 Substitution(s) V264A V264I/N297D/I332E
V264L Y296D/N297D/I332E V264I Y296E/N297D/I332E F241W
Y296N/N297D/I332E F241L Y296Q/N297D/I332E F243W Y296H/N297D/I332E
F243L Y296T/N297D/I332E F241L/F243L/V262I/V264I N297D/T299V/I332E
F241W/F243W N297D/T299I/I332E F241W/F243W/V262A/V264A
N297D/T299L/I332E F241L/V262I N297D/T299F/I332E F243L/V2641
N297D/T299H/I332E F243L/V262I/V264W N297D/T299E/I332E
F241Y/F243Y/V262T/V264T N297D/A330Y/I332E F241E/F243R/V262E/V264R
N297D/S298A/A330Y/I332E F241E/F243Q/V262T/V264E S239D/A330Y/I332E
F241R/F243Q/V262T/V264R S239N/A330Y/I332E F241E/F243Y/V262T/V264R
S239D/A330L/I332E L328M S239N/A330L/I332E L328E V264I/S298A/I332E
L328F S239D/S298A/I332E I332E S239N/S298A/I332E L328M/I332E
S239D/V264I/I332E P244H S239D/V264I/S298A/I332E P245A
S239D/V264I/A330L/I332E P247V T256A W313F K290A P244H/P245A/P247V
D312A P247G *K326A V264I/I332E S298A F241E/F243R/V262E/V264R/I332E
E333A F241E/F243Q/V262T/V264E/I332E K334A
F241R/F243Q/V262T/V264R/I332E E430A F241E/F243Y/V262T/V264R/I332E
T359A S298A K360A S298A/I332E E430A S298A/E333A/K334A K320M
S239E/I332E K326S S239Q/I332E K326N S239E K326D D265G K326E D265N
K334Q S239E/D265G K334E S239E/D265N K334M S239E/D265Q K334H Y296E
K334V Y296Q K334L S298T A330K S298N T335K T299I A339T A327S E333A,
K334A A327N T256A, S298A S267Q/A327S T256A, D280A, S298A, T307A
S267L/A327S S298A, E333A, K334A S298A, K334A A327L S298A, E333A
P329F T256A A330L K290A A330Y K326A I332D R255A N297S E258A N297D
S267A N297S/I332E E272A N297D/I332E N276A N297E/I332E D280A
D265Y/N297D/I332E E283A D265Y/N297D/T299L/I332E H285A
D265F/N297E/I332E N286A L328I/I332E P331A L328Q/I332E S337A I332N
H268A I332Q E272A V264T E430A V264F A330K V240I R301M V263I H268N
V266I H268S T299A E272Q T299S N286Q T299V N286S N325Q N286D N325L
K290S N325I K320M S239D K320Q S239N K320E S239F K320R S239D/I332D
K322E S239D/I332E K326S S239D/I332N K326D S239D/I332Q K326E
S239E/I332D A330K S239E/I332N T335E S239E/I332Q S267A, E258A
S239N/I332D S267A, R255A S239N/I332E S267A, D280A S239N/I332N
S267A, E272A S239N/I332Q S267A, E293A S239Q/I332D S267A, E258A,
D280A, R255A S239Q/I332N P238A S239Q/I332Q D265A K326E E269A Y296D
D270A Y296N N297A F241Y/F243Y/V262T/V264T/ P329A N297D/I332E
A330Y/I332E A327Q V264I/A330Y/I332E S239A A330L/I332E E294A
V264I/A330L/I332E Q295A L234D V303A L234E K246A L234N I253A L234Q
T260A L234T K274A L234H V282A L234Y K288A L234I Q311A L234V K317A
L234F E318A L235D K338A L235S K340A L235N Q342A L235Q R344A L235T
E345A L235H Q347A L235Y R355A L235I E356A L235V M358A L235F K360A
S239T N361A S239H Q362A S239Y Y373A V240A S375A V240T D376A V240M
E380A V263A E382A V263T S383A V263M N384A V264M Q386A V264Y E388A
V266A N389A V266T N390A V266M Y391A E269H K392A E269Y L398A E269F
S400A E269R D401A Y296S D413A Y296T K414A Y296L S415A Y296I R416A
A298H Q418A T299H Q419A A330V N421A A330I V422A A330F S424A A330R
E430A A330H H433A N325D N434A N325E H435A N325A Y436A N325T T437A
N325V Q438A N325H K439A L328D/I332E S440A L328E/I332E S442A
L328N/I332E S444A L328Q/I332E K447A L328V/I332E K246M L328T/I332E
K248M L328H/I332E Y300F L328I/I332E A330Q L328A K338M I332T K340M
I332H A378Q I332Y Y391F I332A S239E/V264I/I332E S239Q/V264I/I332E
S239E/V264I/A330Y/I332E S239E/V264I/S298A/A330Y/I332E
S239D/N297D/I332E S239E/N297D/I332E S239D/D265V/N297D/I332E
S239D/D265I/N297D/I332E S239D/D265L/N297D/I332E
S239D/D265F/N297D/I332E S239D/D265Y/N297D/I332E
S239D/D265H/N297D/I332E S239D/D265T/N297D/I332E
[0222] In other embodiments, the Fc variants of the present
invention may be combined with any of the known Fc modifications in
the art such as those disclosed in Tables 3 A and B below.
TABLE-US-00003 TABLE 3A Starting Position Position Position
Position Position Variant 300 298 296 295 294 Y3001 + .fwdarw. --
S298N, S298V, Y296P, Y296F, Q295K, Q295L, E294N, E294A, S298D,
S298P, or N276Q. or Q295A. E294Q, or S298A, S298G, E294D. S298T, or
S298L. Y300L + .fwdarw. -- S298N, S298V, Y296P, Y296F, Q295K,
Q295L, E294N, E294A, S298D, S298P, or N276Q. or Q295A. E294Q, or
S298A, S298G, E294D. S298T, or S298L. S298N + .fwdarw. Y3001,
Y300L, -- Y296P, Y296F, Q295K, Q295L, E294N, E294A, or Y300F. or
N276Q. or Q295A. E294Q, or E294D. S298V + .fwdarw. Y3001, Y300L, --
Y296P, Y296F, Q295K, Q295L, E294N, E294A, or Y300F. or N276Q. or
Q295A. E294Q, or E294D. S298D + .fwdarw. Y3001, Y300L, -- Y296P,
Y296F, Q295K, Q295L, E294N, E294A, or Y300F. or N276Q. or Q295A.
E294Q, or E294D. S298P + .fwdarw. Y3001, Y300L, -- Y296P, Y296F,
Q295K, Q295L, E294N, E294A, or Y300F. or N276Q. or Q295A. E294Q, or
E294D. Y296P + .fwdarw. Y3001, Y300L, S298N, S298V, -- Q295K,
Q295L, E294N, E294A, or Y300F. S298D, S298P, or Q295A. E294Q, or
S298A, S298G, E294D. S298T, or S298L. Q295K + .fwdarw. Y3001,
Y300L, S298N, S298V, Y296P, Y296F, -- E294N, E294A, or Y300F.
S298D, S298P, or N276Q. E294Q, or S298A, S298G, E294D. S298T, or
S298L. Q295L + .fwdarw. Y3001, Y300L, S298N, S298V, Y296P, Y296F,
-- E294N, E294A, or Y300F. S298D, S298P, or N276Q. E294Q, or S298A,
S298G, E294D. S298T, or S298L. E294N + .fwdarw. Y3001, Y300L,
S298N, S298V, Y296P, Y296F, Q295K, Q295L, -- or Y300F. S298D,
S298P, or N276Q. or Q295A. S298A, S298G, S298T, or S298L. ** Note
that table uses EU numbering as in Kabat.
TABLE-US-00004 TABLE 3B Starting Position Position Position
Position Position Variant 334 333 324 286 276 Y3001 + .fwdarw.
K334A, K334R, E33A, E333Q, S324A, S324N, N286Q, N276Q, K334Q,
K334N, E333N, S324Q, S324K, N286S, N276A, or K334S, K334E, E333S,
or S324E. N286A, or N276K. K334D, K334M, E333K, N286D. K334Y,
K334W, E333R, K334H, K334V, or E333D, or K334L. E333G. Y300L +
.fwdarw. K334A, K334R, E33A, E333Q, S324A, S324N, N286Q, N276Q,
K334Q, K334N, E333N, S324Q, S324K, N286S, N276A, or K334S, K334E,
E333S, or S324E. N286A, or N276K. K334D, K334M, E333K, N286D.
K334Y, K334W, E333R, K334H, K334V, or E333D, or K334L. E333G. S298N
+ .fwdarw. K334A, K334R, E33A, E333Q, S324A, S324N, N286Q, N276Q,
K334Q, K334N, E333N, S324Q, S324K, N286S, N276A, or K334S, K334E,
E333S, or S324E. N286A, or N276K. K334D, K334M, E333K, N286D.
K334Y, K334W, E333R, K334H, K334V, or E333D, or K334L. E333G. S298V
+ .fwdarw. K334A, K334R, E33A, E333Q, S324A, S324N, N286Q, N276Q,
K334Q, K334N, E333N, S324Q, S324K, N286S, N276A, or K334S, K334E,
E333S, or S324E. N286A, or N276K. K334D, K334M, E333K, N286D.
K334Y, K334W, E333R, K334H, K334V, or E333D, or K334L. E333G. S298D
+ .fwdarw. K334A, K334R, E33A, E333Q, S324A, S324N, N286Q, N276Q,
K334Q, K334N, E333N, S324Q, S324K, N286S, N276A, or K334S, K334E,
E333S, or S324E. N286A, or N276K. K334D, K334M, E333K, N286D.
K334Y, K334W, E333R, K334H, K334V, or E333D, or K334L. E333G. S298P
+ .fwdarw. K334A, K334R, E33A, E333Q, S324A, S324N, N286Q, N276Q,
K334Q, K334N, E333N, S324Q, S324K, N286S, N276A, or K334S, K334E,
E333S, or S324E. N286A, or N276K. K334D, K334M, E333K, N286D.
K334Y, K334W, E333R, K334H, K334V, or E333D, or K334L. E333G. Y296P
+ .fwdarw. K334A, K334R, E33A, E333Q, S324A, S324N, N286Q, N276Q,
K334Q, K334N, E333N, S324Q, S324K, N286S, N276A, or K334S, K334E,
E333S, or S324E. N286A, or N276K. K334D, K334M, E333K, N286D.
K334Y, K334W, E333R, K334H, K334V, or E333D, or K334L. E333G. Q295K
+ .fwdarw. K334A, K334R, E33A, E333Q, S324A, S324N, N286Q, N276Q,
K334Q, K334N, E333N, S324Q, S324K, N286S, N276A, or K334S, K334E,
E333S, or S324E. N286A, or N276K. K334D, K334M, E333K, N286D.
K334Y, K334W, E333R, K334H, K334V, or E333D, or K334L. E333G. Q295L
+ .fwdarw. K334A, K334R, E33A, E333Q, S324A, S324N, N286Q, N276Q,
K334Q, K334N, E333N, S324Q, S324K, N286S, N276A, or K334S, K334E,
E333S, or S324E. N286A, or N276K. K334D, K334M, E333K, N286D.
K334Y, K334W, E333R, K334H, K334V, or E333D, or K334L. E333G. E294N
+ .fwdarw. K334A, K334R, E33A, E333Q, S324A, S324N, N286Q, N276Q,
K334Q, K334N, E333N, S324Q, S324K, N286S, N276A, or K334S, K334E,
E333S, or S324E. N286A, or N276K. K334D, K334M, E333K, N286D.
K334Y, K334W, E333R, K334H, K334V, or E333D, or K334L. E333G. **
Note that table uses EU numbering as in Kabat.
[0223] In a preferred specific embodiment, the invention
encompasses molecules comprising a variant Fc region, wherein said
variant Fc region comprises at least one amino acid modification
relative to a wild-type Fc region, such that said molecule has a
conferred effector function (i.e., in a particular assay, the
modified molecule has an effector function activity not detectable
in the parent molecule) and/or an altered affinity for an
Fc.gamma.R, provided that said variant Fc region does not have a
substitution at positions that make a direct contact with
Fc.gamma.R based on crystallographic and structural analysis of
Fc-Fc.gamma.R interactions, such as those positions disclosed by
Sondermann et al., 2000 (Nature, 406: 267-273 which is incorporated
herein by reference in its entirety). Examples of positions within
the Fc region that make a direct contact with Fc.gamma.R are amino
acids 234-239 (hinge region), amino acids 265-269 (B/C loop), amino
acids 297-299 (C'/E loop), and amino acids 327-332 (F/G) loop. In
some embodiments, the molecules of the invention comprising variant
Fc regions comprise modification of at least one residue that makes
a direct contact with an Fc.gamma.R based on structural and
crystallographic analysis.
[0224] The Fc.gamma.R interacting domain maps to the lower hinge
region and select sites within the CH2 and CH3 domains of the IgG
heavy chain. Amino acid residues flanking the actual contact
positions and amino acid residues in the CH3 domain play a role in
IgG/Fc.gamma.R interactions as indicated by mutagenesis studies and
studies using small peptide inhibitors, respectively (Sondermann et
al., 2000 Nature, 406: 267-273; Diesenhofer et al., 1981,
Biochemistry, 20: 2361-2370; Shields et al., 2001, J. Biol. Chem.
276: 6591-6604; each of which is incorporated herein by reference
in its entirety). Direct contact as used herein refers to those
amino acids that are within at least 1 .ANG., at least 2 .ANG., or
at least 3 .ANG. of each other or within 1 .ANG., 1.2 .ANG., 1.5
.ANG., 1.7 .ANG. or 2 .ANG. Van Der Waals radius. An exemplary list
of previously identified sites on the Fc that affect binding of Fc
interacting proteins is listed in the Table 4 below. In some
embodiments, the invention encompasses Fc variants that do not have
any modifications at the sites listed below. In other embodiments,
the invention encompasses Fc variants comprising amino acid
modifications at one or more sites listed below in combination with
other modifications disclosed herein such that such modification
has a synergistic or additive effect on the property of the
mutant.
TABLE-US-00005 TABLE 4 PREVIOUSLY IDENTIFIED SITES ON THE Fc THAT
EFFECT BINDING OF Fc INTERACTING PROTEINS. FcR-Fc Domain residue
FcRI FcRII FcRIII C1q FcRn CH2 233 C C C C A, B CH2 234 C C C G C
A, B CH2 235 C C C G C A, B CH2 236 C C C C A, B CH2 237 A, B CH2
238 D A, B CH2 239 C CH2 241 D CH2 243 D CH2 246 D CH2 250 E CH2
254 C CH2 255 C CH2 256 C C CH2 258 C B CH2 265 C C C F C B CH2 267
C CH2 268 C C B CH2 269 C CH2 270 C C F CH2 272 C CH2 276 C CH2 285
C CH2 286 C CH2 288 C CH2 290 C C CH2 292 C CH2 293 C CH2 295 C C
CH2 296 C B CH2 297 X X X X B CH2 298 B CH2 299 CH2 301 D C C CH2
311 C CH2 312 C CH2 315 C CH2 317 C CH2 322 C C F CH2 326 C F A, B
CH2 327 D, C C C A CH2 328 A CH2 329 D, C C C F A CH2 330 CH2 331 C
F A CH2 332 CH2 333 C F CH2 334 C CH2 337 C CH2 338 C CH3 339 C CH3
360 C CH3 362 C CH3 376 C CH3 378 C CH3 380 C CH3 382 C CH3 414 C
CH3 415 C CH3 424 C CH3 428 E CH3 430 C CH3 433 C CH3 434 C CH3 435
C CH3 436 C
[0225] Table 4 lists sites within the Fc region that have
previously been identified to be important for the Fc-FcR
interaction. Columns labeled FcR-Fc identifies the Fc chain
contacted by the FcR. Letters identify the reference in which the
data was cited. C is Shields et al., 2001, J. Biol. Chem. 276:
6591-6604; D is Jefferis et al., 1995, Immunol. Lett. 44: 111-7; E
is Hinton et al; 2004, J. Biol. Chem. 279(8): 6213-6; F is Idusogie
et al., 2000, J. Immunol. 164: 4178-4184; each of which is
incorporated herein by reference in its entirety.
[0226] In another embodiment, the invention encompasses a molecule
comprising a variant Fc region, wherein said variant Fc region
comprises at least one amino acid modification relative to a
wild-type Fc region, such that said molecule has an enhanced
effector function relative to a molecule comprising a wild-type Fc
region, provided that said variant Fc region does not have or is
not solely a substitution at any of positions 243, 255, 256, 258,
267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290,
292, 293, 294, 295, 296, 298, 300, 301, 303, 305, 307, 309, 312,
320, 322, 326, 329, 330, 332, 331, 333, 334, 335, 337, 338, 339,
340, 359, 360, 373, 376, 416, 419, 430, 434, 435, 437, 438, 439. In
a specific embodiment, the invention encompasses a molecule
comprising a variant Fc region, wherein said variant Fc region
comprises at least one amino acid modification relative to a
wild-type Fc region, such that said molecule binds an Fc.gamma.R
with an altered affinity relative to a molecule comprising a
wild-type Fc region, provided that said variant Fc region does not
have or is not solely a substitution at any of positions 243, 255,
258, 267, 269, 270, 276, 278, 280, 283, 285, 289, 292, 293, 294,
295, 296, 300, 303, 305, 307, 309, 320, 322, 329, 332, 331, 337,
338, 340, 373, 376, 416, 419, 434, 435, 437, 438, 439 and does not
have an alanine at any of positions 256, 290, 298, 312, 326, 333,
334, 359, 360, or 430; an asparagine at position 268; a glutamine
at position 272; a glutamine, serine, or aspartic acid at position
286; a serine at position 290; a methionine at position 301; a
methionine, glutamine, glutamic acid, or arginine at position 320;
a glutamic acid at position 322; an asparagine, serine, glutamic
acid, or aspartic acid at position 326; a lysine at position 330; a
glutamine at position 334; a glutamic acid at position 334; a
methionine at position 334; a histidine at position 334; a valine
at position 334; a leucine at position 334; a glutamine at position
335; a lysine at position 335; or a threonine at position 339.
[0227] In a specific embodiment, the invention encompasses a
molecule comprising a variant Fc region, wherein said variant Fc
region does not have or is not solely a substitution at any of
positions 268, 269, 270, 272, 276, 278, 283, 285, 286, 289, 292,
293, 301, 303, 305, 307, 309, 320, 331, 333, 334, 335, 337, 338,
340, 360, 373, 376, 416, 419, 430, 434, 435, 437, 438 or 439 and
does not have a histidine, glutamine, or tyrosine at position 280;
a serine, glycine, threonine or tyrosine at position 290, an
asparagine at position 294, a lysine at position 295; a proline at
position 296; a proline, asparagine, aspartic acid, or valine at
position 298; or a leucine or isoleucine at position 300. In
another embodiment, the invention encompasses a molecule comprising
a variant Fc region, wherein said variant Fc region comprises at
least one amino acid modification relative to a wild-type Fc
region, such that said molecule binds an Fc.gamma.R with a reduced
affinity relative to molecule comprising a wild-type Fc region
provided that said variant Fc region does not have or is not solely
a substitution at any of positions 243, 252, 254, 265, 268, 269,
270, 278, 289, 292, 293, 294, 295, 296, 298, 300, 301, 303, 322,
324, 327, 329, 333, 335, 338, 340, 373, 376, 382, 388, 389, 414,
416, 419, 434, 435, 437, 438, or 439. In yet another embodiment,
the invention encompasses a molecule comprising a variant Fc
region, wherein said variant Fc region comprises at least one amino
acid modification relative to a wild-type Fc region, such that said
molecule binds an Fc.gamma.R with an enhanced affinity relative to
a molecule comprising a wild-type Fc region provided that said
variant Fc region does not have or is not solely a substitution at
any of positions 280, 283, 285, 286, 290, 294, 295, 298, 300, 301,
305, 307, 309, 312, 315, 331, 333, 334, 337, 340, 360, 378, 398, or
430.
[0228] In a specific embodiment, the invention encompasses molecule
comprising a variant Fc region, wherein said variant Fc region does
not include or are not solely a substitution at any of positions
330, 243, 247, 298, 241, 240, 244, 263, 262, 235, 269, or 328 and
does not have a leucine at position 243, an asparagine at position
298, a leucine at position 241, and isoleucine or an alanine at
position 240, a histidine at position 244, a valine at position
330, or an isoleucine at position 328.
[0229] In alternative embodiments, the molecules of the invention,
having variant Fc regions with enhanced effector function and/or
altered affinities for activating and/or inhibitory receptors, have
one or more amino acid modifications, wherein said one or more
amino acid modification is a substitution at position 288 with
asaparagine, at position 330 with serine and at position 396 with
leucine (MgFc10)(See Table 5); or a substitution at position 334
with glutamic acid, at position 359 with asparagine, and at
position 366 with serine (MgFcl3); or a substitution at position
316 with aspartic acid, at position 378 with valine, and at
position 399 with glutamic acid (MgFc27); or a substitution at
position 247 with leucine, and a substitution at position 421 with
lysine (MgFc31); or a substitution at position 392 with threonine,
and at position 396 with leucine (MgFc38); or a substitution at
position 221 with glutamic acid, at position 270 with glutamic
acid, at position 308 with alanine, at position 311 with histidine,
at position 396 with leucine, and at position 402 with aspartic
acid (MgFc42); or a substitution at position 419 with histidine,
and a substitution at position 396 with leucine (MgFc51); or a
substitution at position 240 with alanine, and at position 396 with
leucine (MgFc52); or a substitution at position 410 with histidine,
and at position 396 with leucine (MgFc53); or a substitution at
position 243 with leucine, at position 305 with isoleucine, at
position 378 with aspartic acid, at position 404 with serine, and
at position 396 with leucine (MgFc54); or a substitution at
position 255 with isoleucine, and at position 396 with leucine
(MgFc55); or a substitution at position 370 with glutamic acid and
at position 396 with leucine (MgFc59); or a substitution at
position 270 with glutamic acid; or a combination of the foregoing.
In one specific embodiment, the invention encompasses a molecule
comprising a variant Fc region wherein said variant Fc region
comprises a substitution at position 396 with leucine, at position
270 with glutamic acid and at position 243 with leucine. In another
specific embodiment the molecule further comprises one or more
amino acid modification such as those disclosed herein.
[0230] In some embodiments, the invention encompasses molecules
comprising a variant Fc region having an amino acid modification at
one or more of the following positions: 119, 125, 132, 133, 141,
142, 147, 149, 162, 166, 185, 192, 202, 205, 210, 214, 215, 216,
217, 218, 219, 221, 222, 223, 224, 225, 227, 229, 231, 232, 233,
235, 240, 241, 242, 243, 244, 246, 247, 248, 250, 251, 252, 253,
254, 255, 256, 258, 261, 262, 263, 268, 269, 270, 272, 274, 275,
276, 279, 280, 281, 282, 284, 287, 288, 289, 290, 291, 292, 293,
295, 298, 301, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312,
313, 315, 316, 317, 318, 319, 320, 323, 326, 327, 328, 330, 333,
334, 335, 337, 339, 340, 343, 344, 345, 347, 348, 352, 353, 354,
355, 358, 359, 360, 361, 362, 365, 366, 367, 369, 370, 371, 372,
375, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388,
389, 390, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402,
404, 406, 407, 408, 409, 410, 411, 412, 414, 415, 416, 417, 419,
420, 421, 422, 423, 424, 427, 428, 431, 433, 435, 436, 438, 440,
441, 442, 443, 446, or 447. Preferably such mutations result in
molecules that have been conferred an effector cell mediated
function and, optionally, have an altered affinity for an
Fc.gamma.R as determined using methods disclosed and exemplified
herein and known to one skilled in the art.
[0231] The invention encompasses molecules comprising variant Fc
regions consisting of or comprising any of the mutations listed in
the table below in Table 5.
TABLE-US-00006 TABLE 5 EXEMPLARY MUTATIONS SINGLE SITE MUTANTS
DOUBLE SITE MUTANTS K392R Q347H, A339V N315I S415I, L251F S132I
K290E, L142P P396L G285E, P247H P396H K409R, S166N A162V E334A,
K334A R292L R292L. K334E T359N K288N, A330S T366S R255L, E318K
V379L F243L, E318K K288N V279L, P395S A330S K246T, Y319F F243L
F243I, V379L E318K K288M, K334E V379M K334E, E308D S219Y E233D,
K334E V282M K246T, P396H D401V H268D, E318D K222N K246I, K334N
K334I K320E, K326E K334E S375C, P396L I377F K288N, K326N P247L
P247L, N421K F372Y S298N, W381R K326E R255Q, K326E H224L V284A,
F372L F275Y T394M. V397M L398V P247L, E389G K334N K290T, G371D
S400P P247L, L398Q S407I P247L, I377F F372Y K326E, G385E T366N
S298N, S407R K414N E258D, N384K M352L F241L, E258G T225S K370N,
S440N I377N K317N, F423-DELETED K248M P227S, K290E R292G K334E,
E380D S298N P291S, P353Q D270E V240I, V281M E233G P232S, S304G
P247L, L406F D399E, M428L L251F, F372L D399E, G402D D399E, M428L
K392T, P396L H268N, P396L K326I, P396L H268D, P396L K210M, P396L
L358P, P396L K334N, P396L V379M, P396L P227S, P396L P217S, P396L
Q419H, P396L K370E, P396L L242F, P396L R255L, P396L V240A, P396L
T250A, P396L P247S, P396L L410H, P396L Q419L, P396L V427A, P396L
E258D, P396L N384K, P396L V323I, P396L P244H, P396L V305L, P396L
S400F, P396L V303I, P396L A330V, Q419H V263Q, E272D K326E,
A330T
[0232] In yet other embodiments, the invention encompasses
molecules comprising variant Fc regions having more than two amino
acid modifications. A non-limiting example of such variants is
listed in the table below (Table 6). The invention encompasses
mutations listed in Table 6 which further comprise one or more
amino acid modifications such as those disclosed herein.
TABLE-US-00007 TABLE 6 EXEMPLARY COMBINATION VARIANTS D399E, R292L,
V185M R301C, M252L, S192T P291S, K288E, H268L, A141V S383N, N384K,
T256N, V262L, K218E, R214I, K205E, F149Y, K133M S408I, V215I, V125L
G385E, P247H V348M, K334N, F275I, Y202M, K147T H310Y, T289A, Y407V,
E258D R292L, P396L, T359N F275I, K334N, V348M F243L. R255L, E318K
K334E, T359N, T366S T256S, V305I, K334E, N390S T335N, K370E, A378V,
T394M, S424L K334E, T359N, T366S, Q386R K288N, A330S, P396L P244H,
L358M, V379M, N384K, V397M P217S, A378V, S408R P247L, I253N, K334N
D312E, K327N, I378S D280E, S354F, A431D, L441I K218R, G281D, G385R
P247L, A330T, S440G T355N, P387S, H435Q P247L, A431V, S442F P343S,
P353L, S375I, S383N E216D, E345K, S375I K288N, A330S, P396L K222N,
T335N, K370E, A378V, T394M G316D, A378V, D399E N315I, V379M, T394M
K326Q, K334E, T359N, T366S A378V, N390I, V422I V282E, V369I, L406F
V397M, T411A, S415N T223I, T256S, L406F L235P, V382M, S304G, V305I,
V323I P247L, W313R, E388G D221Y, M252I, A330G, A339T, T359N, V422I,
H433L F243I, V379L, G420V A231V, Q386H, V412M T215P, K274N, A287G,
K334N, L365V, P396L P244A, K326I, C367R, S375I, K447T R301H, K340E,
D399E C229Y, A287T, V379M, P396L, L443V E269K, K290N, Q311R, H433Y
E216D, K334R, S375I T335N, P387S, H435Q K246I, Q362H, K370E K334E,
E380D, G446V V303I, V369F, M428L K246E, V284M, V308A E293V, Q295E,
A327T Y319F, P352L, P396L D221E, D270E, V308A, Q311H, P396L, G402D
K290T, N390I, P396L K288R, T307A, K344E, P396L V273I, K326E, L328I,
P396L K326I, S408N, P396L K261N, K210M, P396L F243L, V305I, A378D,
F404S, P396L K290E, V369A, T393A, P396L K210N, K222I, K320M, P396L
P217S, V305I, I309L, N390H, P396L K246N, Q419R, P396L P217A, T359A,
P396L V215I, K290V, P396L F275L, Q362H, N384K, P396L A330V, H433Q,
V427M V263Q, E272D, Q419H N276Y, T393N, W417R V282L, A330V, H433Y,
T436R V284M, S298N, K334E, R355W A330V, G427M, K438R S219T, T225K,
D270E, K360R K222E, V263Q, S298N E233G, P247S, L306P S219T, T225K,
D270E S254T, A330V, N361D, P243L V284M, S298N, K334E, R355W R416T
D270E, G316D, R416G K392T, P396L, D270E R255L, P396L, D270E V240A,
P396L, D270E Q419H, P396L, D270E K370E, P396L, D270E P247L, N421K,
D270E R292P, V305I R292P, V305I, F243L V284M, R292L, K370N R255L,
P396L, D270E, Y300L R255L, P396L, D270E, R292G F243L, D270E, K392N,
P396L F243L, R255L, D270E, P296L
[0233] In specific embodiments, the variant Fc region has a leucine
at position 247, a lysine at position 421 and a glutamic acid at
position 270 (MgFc31/60); a threonine at position 392, a leucine at
position 396, a glutamic acid at position 270, and a leucine at
position 243 (MgFc38/60/F243L); a histidine at position 419, a
leucine at position 396, and a glutamic acid at position 270
(MGFc51/60); a histidine at position 419, a leucine at position
396, a glutamic acid at position 270, and a leucine at position 243
(MGFc51/60/F243L); an alanine at position 240, a leucine at
position 396, and a glutamic acid at position 270 (MGFc52/60); a
lysine at position 255 and a leucine at position 396 (MgFc55); a
lysine at position 255, a leucine at position 396, and a glutamic
acid at position 270 (MGFc55/60); a lysine at position 255, a
leucine at position 396, a glutamic acid at position 270, and a
lysine at position 300 (MGFc55/60/Y300L); a lysine at position 255,
a leucine at position 396, a glutamic acid at position 270, and a
glycine at position 292 (MGFc55/60/R292G); a lysine at position
255, a leucine at position 396, a glutamic acid at position 270,
and a leucine at position 243 (MgFc55/60/F243L); a glutamic acid at
position 370, a leucine at position 396, and a glutamic acid at
position 270 (MGFc59/60); a glutamic acid at position 270, an
aspartic acid at position 316, and a glycine at position 416
(MgFc71); a leucine at position 243, a proline at position 292, an
isoleucine at position 305, and a leucine at position 396
(MGFc74/P396L); a leucine at position 243, a glutamic acid at
position 270, an asparagine at position 392 and a leucine at
position 396; or a leucine at position 243, a leucine at position
255, a glutamic acid at position 270 and a leucine at position 396;
a glutamine at position 297, or any combination of the individual
substitutions.
[0234] In some embodiments, the molecules of the invention further
comprise one or more glycosylation sites, so that one or more
carbohydrate moieties are covalently attached to the molecule.
Preferably, the molecules of the invention with one or more
glycosylation sites and/or one or more modifications in the Fc
region confer or have an enhanced antibody mediated effector
function, e.g., enhanced ADCC activity, compared to a parent
antibody. In some embodiments, the invention further comprises
molecules comprising one or more modifications of amino acids that
are directly or indirectly known to interact with a carbohydrate
moiety of the antibody, including but not limited to amino acids at
positions 241, 243, 244, 245, 245, 249, 256, 258, 260, 262, 264,
265, 296, 299, and 301. Amino acids that directly or indirectly
interact with a carbohydrate moiety of an antibody are known in the
art, see, e.g., Jefferis et al., 1995 Immunology Letters, 44:
111-7, which is incorporated herein by reference in its
entirety.
[0235] In another embodiment, the invention encompasses molecules
that have been modified by introducing one or more glycosylation
sites into one or more sites of the molecules, preferably without
altering the functionality of the molecules, e.g., binding activity
to target antigen or Fc.gamma.R. Glycosylation sites may be
introduced into the variable and/or constant region of the
molecules of the invention. As used herein, "glycosylation sites"
include any specific amino acid sequence in an antibody to which an
oligosaccharide (i.e., carbohydrates containing two or more simple
sugars linked together) will specifically and covalently attach.
Oligosaccharide side chains are typically linked to the backbone of
an antibody via either N- or O-linkages. N-linked glycosylation
refers to the attachment of an oligosaccharide moiety to the side
chain of an asparagine residue. O-linked glycosylation refers to
the attachment of an oligosaccharide moiety to a hydroxyamino acid,
e.g., serine, threonine. The molecules of the invention may
comprise one or more glycosylation sites, including N-linked and
O-linked glycosylation sites. Any glycosylation site for N-linked
or O-linked glycosylation known in the art may be used in
accordance with the instant invention. An exemplary N-linked
glycosylation site that is useful in accordance with the methods of
the present invention is the amino acid sequence: Asn-X-Thr/Ser,
wherein X may be any amino acid and Thr/Ser indicates a threonine
or a serine. Such a site or sites may be introduced into a molecule
of the invention using methods well known in the art to which this
invention pertains. See, for example, "In vitro Mutagenesis,"
Recombinant DNA: A Short Course, J. D. Watson, et al. W. H. Freeman
and Company, New York, 1983, chapter 8, pp. 106-116, which is
incorporated herein by reference in its entirety. An exemplary
method for introducing a glycosylation site into a molecule of the
invention may comprise: modifying or mutating an amino acid
sequence of the molecule so that the desired Asn-X-Thr/Ser sequence
is obtained.
[0236] In some embodiments, the invention encompasses methods of
modifying the carbohydrate content of a molecule of the invention
by adding or deleting a glycosylation site. Methods for modifying
the carbohydrate content of antibodies are well known in the art
and encompassed within the invention, see, e.g., U.S. Pat. No.
6,218,149; EP 0 359 096 B1; U.S. Publication No. US 2002/0028486;
WO 03/035835; U.S. Publication No. 2003/0115614; U.S. Pat. No.
6,218,149; U.S. Pat. No. 6,472,511; all of which are incorporated
herein by reference in their entirety. In other embodiments, the
invention encompasses methods of modifying the carbohydrate content
of a molecule of the invention by deleting one or more endogenous
carbohydrate moieties of the molecule. In a specific embodiment,
the invention encompasses shifting the glycosylation site of the Fc
region of an antibody, by modifying positions adjacent to 297. In a
specific embodiment, the invention encompasses modifying position
296 so that position 296 and not position 297 is glycosylated.
5.1 Polypeptides and Antibodies with Variant Fc Regions
[0237] The present invention is based, in part, on the inventors'
discovery of methods for engineering the Fc region of an antibody
to confer an effector function activity to the antibody, which the
parent antibody did not exhibit when tested against a target cell.
Such methods of engineering include introducing one or more amino
acid modifications (substitutions, deletions or insertions) in one
or more portions of the Fc region, which modifications introduce a
detectable effector function activity on the parent antibody. In
particular, the modifications alter the parent antibody's affinity
for certain Fc.gamma.R receptors (e.g., activating Fc.gamma.Rs,
inhibitory Fc.gamma.Rs) and one or more effector functions, such as
ADCC. Alternately, the modifications alter the affinity of the
variant Fc region such that the variant Fc regions oligomerize and
homo-oligomers of the modified antibody are formed. The inventors
have found that modification of an Fc region of a chimeric 2B6 or
4D5 antibody (anti-Fc.gamma.RIIB antibody) surprisingly conferred a
particular effector function activity (ADCC) on chimeric 2B6
antibodies, which normally exhibit no detectable ADCC activity, and
improved effector function activity (particularly ADCC) of chimeric
4D5 antibodies in cells with low levels of antigen expression. The
inventors have further found that modification of an Fc region of
rituximab (anti-CD20 monoclonal antibody) conferred effector
function activity on the rituximab antibody in a patient population
whose cells were otherwise refractory to rituximab-induced effector
function activity.
[0238] It will be appreciated by one skilled in the art that aside
from amino acid substitutions, the present invention contemplates
other modifications of the Fc region amino acid sequence in order
to generate an Fc region variant with one or more altered
properties, e.g., enhanced effector function. The invention
contemplates deletion of one or more amino acid residues of the Fc
region in order to, e.g., reduce binding to an Fc.gamma.R.
Preferably, no more than 5, no more than 10, no more than 20, no
more than 30, no more than 50 Fc region residues will be deleted
according to this embodiment of the invention. The Fc region herein
comprising one or more amino acid deletions will preferably retain
at least about 80%, and preferably at least about 90%, and most
preferably at least about 95%, of the wild type Fc region. In some
embodiments, one or more properties of the molecules are maintained
such as for example, non-immunogenicity, Fc.gamma.RIIIA binding,
Fc.gamma.RIIA binding, or a combination of these properties.
[0239] In alternate embodiments, the invention encompasses amino
acid insertion to generate the Fc region variants, which variants
have altered properties including enhanced effector function. In
one specific embodiment, the invention encompasses introducing at
least one amino acid residue, for example, one to two amino acid
residues and preferably no more than 10 amino acid residues
adjacent to one or more of the Fc region positions identified
herein. In alternate embodiments, the invention further encompasses
introducing at least one amino acid residue, for example, one to
two amino acid residues and preferably no more than 10 amino acid
residues adjacent to one or more of the Fc region positions known
in the art as impacting Fc.gamma.R interaction and/or binding.
[0240] The invention further encompasses incorporation of unnatural
amino acids to generate the Fc variants of the invention. Such
methods are known to those skilled in the art such as those using
the natural biosynthetic machinery to allow incorporation of
unnatural amino acids into proteins, see, e.g., Wang et al., 2002
Chem. Comm. 1:1-11; Wang et al., 2001, Science, 292: 498-500; van
Hest et al., 2001. Chem. Comm. 19: 1897-1904, each of which is
incorporated herein by reference in its entirety. Alternative
strategies focus on the enzymes responsible for the biosynthesis of
amino acyl-tRNA, see, e.g., Tang et al., 2001, J. Am. Chem.
123(44): 11089-11090; Kiick et al., 2001, FEBS Lett. 505(3): 465;
each of which is incorporated herein by reference in its
entirety.
[0241] The effector function properties of the molecules of the
invention are determined for one or more Fc.gamma.R mediator
effector cell functions as described in Section 5.2.7. The
affinities and binding properties of the molecules of the invention
for a target antigen or an Fc.gamma.R are initially determined
using in vitro assays (biochemical or immunological based assays)
known in the art for determining antibody-antigen or Fc-Fc.gamma.R
interactions, i.e., specific binding of an antibody to an antigen
or an Fc region to an Fc.gamma.R, respectively, including but not
limited to ELISA assay, surface plasmon resonance assay,
immunoprecipitation assays (See Section 5.2.1). In most preferred
embodiments, the molecules of the invention have similar binding
properties in in vivo models (such as those described and disclosed
herein) as those in in vitro based assays. However, the present
invention does not exclude molecules of the invention that do not
exhibit the desired phenotype in in vitro based assays but do
exhibit the desired phenotype in vivo. A representative flow chart
of the screening and characterization of molecules of the invention
is described in FIG. 34.
[0242] The invention encompasses molecules comprising a variant Fc
region that binds with a greater affinity to one or more
Fc.gamma.Rs. Such molecules preferably mediate effector function
more effectively as discussed infra. In other embodiments, the
invention encompasses molecules comprising a variant Fc region that
bind with a weaker affinity to one or more Fc.gamma.Rs. In general,
increased or added effector function would be directed to tumor and
foreign cells.
[0243] The Fc variants of the present invention may be combined
with other Fc modifications, including but not limited to other
modifications that enhance effector function. The invention
encompasses combining an Fc variant of the invention with other Fc
modifications to provide additive, synergistic, or novel properties
in antibodies or Fc fusions. Preferably the Fc variants of the
invention enhance the phenotype of the modification with which they
are combined. For example, if an Fc variant of the invention is
combined with a mutant known to bind Fc.gamma.RIIIA with a higher
affinity than a comparable molecule comprising a wild type Fc
region; the combination with a mutant of the invention results in a
greater fold enhancement in Fc.gamma.RIIIA affinity.
[0244] In one embodiment, the Fc variants of the present invention
may be combined with other known Fc variants such as those
disclosed in Duncan et al, 1988, Nature 332:563-564; Lund et al.,
1991, J. Immunol. 147:2657-2662; Lund et al, 1992, Mol Immunol
29:53-59; Alegre et al, 1994, Transplantation 57:1537-1543;
Hutchins et al., 1995, Proc Natl. Acad Sci USA 92:11980-11984;
Jefferis et al, 1995, Immunol Lett. 44:111-117; Lund et al., 1995,
Faseb J 9:115-119; Jefferis et al, 1996, Immunol Lett 54:101-104;
Lund et al, 1996, J Immunol 157:49634969; Armour et al., 1999, Eur
J Immunol 29:2613-2624; Idusogie et al, 2000, J Immunol
164:41784184; Reddy et al, 2000, J Immunol 164:1925-1933; Xu et
al., 2000, Cell Immunol 200:16-26; Idusogie et al, 2001, J Immunol
166:2571-2575; Shields et al., 2001, J Biol Chem 276:6591-6604;
Jefferis et al, 2002, Immunol Lett 82:57-65; Presta et al., 2002,
Biochem Soc Trans 30:487-490); U.S. Pat. No. 5,624,821; U.S. Pat.
No. 5,885,573; U.S. Pat. No. 6,194,551; PCT WO 00/42072; PCT WO
99/58572; each of which is incorporated herein by reference in its
entirety.
[0245] In some embodiments, the Fc variants of the present
invention are incorporated into an antibody or Fc fusion that
comprises one or more engineered glycoforms, i.e., a carbohydrate
composition that is covalently attached to an antibody comprising
an Fc region, wherein said carbohydrate composition differs
chemically from that of a parent antibody comprising an Fc region.
Engineered glycoforms may be useful for a variety of purposes,
including, but not limited to, enhancing effector function.
Engineered glycoforms may be generated by any method known to one
skilled in the art, for example by using engineered or variant
expression strains, by co-expression with one or more enzymes, for
example, DI N-acetylglucosaminyltransferase III (GnTI11), by
expressing an antibody comprising an Fc region in various organisms
or cell lines from various organisms, or by modifying
carbohydrate(s) after the antibody comprising Fc region has been
expressed. Methods for generating engineered glycoforms are known
in the art, and include but are not limited to those described in
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 01/292246A1; PCT WO
02/311140A1; PCT WO 02/30954A1; Potillegent.TM. technology (Biowa,
Inc. Princeton, N.J.); GlycoMAb.TM. glycosylation engineering
technology (GLYCART biotechnology AG, Zurich, Switzerland); each of
which is incorporated herein by reference in its entirety. See,
e.g., WO 00061739; EA01229125; US 20030115614; Okazaki et al.,
2004, JMB, 336: 1239-49 each of which is incorporated herein by
reference in its entirety.
[0246] The Fc variants of the present invention may be optimized
for a variety of properties. Properties that may be optimized
include, but are not limited to, conferred or enhanced effector
function, enhanced or reduced affinity for an Fc.gamma.R, or
conferred oligomerization activity. In a preferred embodiment, the
Fc variants of the present invention are optimized to possess
enhanced affinity for a human activating Fc.gamma.R, preferably
Fc.gamma.R, Fc.gamma.RIIA, Fc.gamma.RIIc, Fc.gamma.RIIIA, and
Fc.gamma.RIIIB, most preferably Fc.gamma.RIIIA. In an alternate
preferred embodiment, the Fc variants are optimized to possess
reduced affinity for the human inhibitory receptor Fc.gamma.RIIB.
These preferred embodiments are anticipated to provide antibodies
and Fc fusions with new or enhanced therapeutic properties in
humans, for example, enhanced effector function and greater
anti-cancer potency as described and exemplified herein. These
preferred embodiments are anticipated to provide antibodies and Fc
fusions with enhanced tumor elimination in mouse xenograft tumor
models.
[0247] In an alternate embodiment the Fc variants of the present
invention are optimized to have reduced affinity for a human
Fc.gamma.R, including but not limited to Fc.gamma.RI,
Fc.gamma.RIIA, Fc.gamma.RIIB, Fc.gamma.RIIc, Fc.gamma.RIIIA, and
Fc.gamma.RIIIB. These embodiments are anticipated to provide
antibodies and Fc fusions with enhanced therapeutic properties in
humans, for example, reduced toxicity.
[0248] In alternate embodiments, the Fc variants of the present
invention possess a conferred effector function and/or enhanced or
reduced affinity for Fc.gamma.Rs from non-human organisms,
including, but not limited to, mice, rats, rabbits, and monkeys. Fc
variants that are optimized for effector function in a non-human or
binding to a non-human Fc.gamma.R may find use in experimentation.
For example, mouse models are available for a variety of diseases
that enable testing of properties such as efficacy, toxicity, and
pharmacokinetics for a given drug candidate. As is known in the
art, cancer cells can be grafted or injected into mice to mimic a
human cancer, a process referred to as xenografting. Testing of
antibodies or Fc fusions that comprise Fc variants that confer an
effector function and/or are optimized for one or more mouse
Fc.gamma.Rs, may provide valuable information with regard to the
efficacy of the antibody or Fc fusion, its mechanism of action, and
the like.
[0249] In certain embodiments, while it is preferred to alter
binding to an Fc.gamma.R, the instant invention further
contemplates Fc variants with altered binding affinity to the
neonatal receptor (FcRn). Although not intending to be bound by a
particular mechanism of action, Fc region variants with improved
affinity for FcRn are anticipated to have longer serum half-lives,
and such antibodies will have useful applications in methods of
treating mammals where long half-life of the administered
polypeptide is desired, e.g., to treat a chronic disease or
disorder. Although not intending to be bound by a particular
mechanism of action, Fc region variants with decreased FcRn binding
affinity, on the contrary, are expected to have shorter half-lives,
and such antibodies may, for example, be administered to a mammal
where a shortened circulation time may be advantageous, e.g., for
in vivo diagnostic imaging or for polypeptides which have toxic
side effects when left circulating in the blood stream for extended
periods. Fc region variants with decreased FcRn binding affinity
are anticipated to be less likely to cross the placenta, and thus
may be utilized in the treatment of diseases or disorders in
pregnant women.
[0250] In other embodiments, these variants may be combined with
other known Fc modifications with altered FcRn affinity such as
those disclosed in International Publication Nos. WO 98/23289; and
WO 97/34631; and U.S. Pat. No. 6,277,375, each of which is
incorporated herein by reference in its entirety.
[0251] The invention encompasses any other method known in the art
for generating molecules, e.g., antibodies, having an increased
half-life in vivo, for example, by introducing one or more amino
acid modifications (i.e., substitutions, insertions or deletions)
into an IgG constant domain, or FcRn binding fragment thereof
(preferably a Fc or hinge-Fc domain fragment). See, e.g.,
International Publication Nos. WO 98/23289; and WO 97/34631; and
U.S. Pat. No. 6,277,375, each of which is incorporated herein by
reference in its entirety to be used in combination with the Fc
variants of the invention. Further, molecules, e.g., antibodies, of
the invention can be conjugated to albumin in order to make the
antibody or antibody fragment more stable in vivo or have a longer
half-life in vivo. The techniques well-known in the art, see, e.g.,
International Publication Nos. WO 93/15199, WO 93/15200, and WO
01/77137, and European Patent No. EP 413,622, all of which are
incorporated herein by reference in their entirety.
[0252] The variant(s) described herein may be subjected to further
modifications, often times depending on the intended use of the
variant. Such modifications may involve further alteration of the
amino acid sequence (substitution, insertion and/or deletion of
amino acid residues), fusion to heterologous polypeptide(s) and/or
covalent modifications. Such further modifications may be made
prior to, simultaneously with, or following, the amino acid
modification(s) disclosed herein which results in altered
properties such as an enhanced binding to target antigen, conferred
oligomerization activity, or enhanced effector function and/or
alteration of Fc receptor binding.
[0253] Alternatively or additionally, the invention encompasses
combining the amino acid modifications disclosed herein with one or
more further amino acid modifications that confer or enhance
additional effector functions, e.g., C1q binding and/or complement
dependent cytoxicity function, of the Fc region as determined in
vitro and/or in vivo. The further amino acid substitutions
described herein will generally serve to confer the activity on a
parent antibody that does not exhibit detectable levels of the
activity or enhance the ability of the starting antibody to bind to
C1q and/or complement dependent cytotoxicity (CDC) function. For
example, the starting antibody may be unable to bind C1q and/or
mediate CDC and may be modified according to the teachings herein
such that it acquires these further effector functions. Moreover,
antibodies with preexisting C1q binding activity, optionally
further having the ability to mediate CDC may be modified such that
one or both of these activities are enhanced. In some embodiments,
the invention encompasses variant Fc regions with altered CDC
activity without any alteration in C1q binding. In yet other
embodiments, the invention encompasses variant Fc regions with
altered CDC activity and altered C1q binding.
[0254] To generate an Fc region with altered C1q binding and/or
complement dependent cytotoxicity (CDC) function, the amino acid
positions to be modified are generally selected from positions 270,
322, 326, 327, 329, 331, 333, and 334, where the numbering of the
residues in an IgG heavy chain is that of the EU index as in Kabat
et al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(199). These amino acid modifications may be combined with one or
more Fc modifications disclosed herein to provide a synergistic or
additive effect on C1q binding and/or CDC activity. In other
embodiments, the invention encompasses Fc variants with altered C1q
binding and/or complement dependent cytotoxicity (CDC) function
comprising an amino acid substitution at position 396 with leucine
and at position 255 with leucine; or an amino acid substitution at
position 396 with leucine and at position 419 with histidine; an
amino acid substitution at position 396 with leucine and at
position 370 with glutamic acid; an amino acid substitution at
position 396 with leucine and at position 240 with alanine; an
amino acid substitution at position 396 with leucine and at
position 392 with threonine; an amino acid substitution at position
247 with leucine and at position 421 with lysine. The invention
encompasses any known modification of the Fc region which alters
C1q binding and/or complement dependent cytotoxicity (CDC) function
such as those disclosed in Idusogie et al., 2001, J. Immunol.
166(4) 2571-5; Idusogie et al., J. Immunol. 2000 164(8): 4178-4184;
each of which is incorporated herein by reference in its
entirety.
[0255] As disclosed above, the invention encompasses an Fc region
with altered effector function, e.g., modified C1q binding and/or
FcR binding and thereby altered CDC activity and/or ADCC activity.
In specific embodiments, the invention encompasses variant Fc
regions with improved C1q binding and improved Fc.gamma.RIII
binding; e.g. having both improved ADCC activity and improved CDC
activity. In alternative embodiments, the invention encompasses a
variant Fc region with reduced CDC activity and/or reduced ADCC
activity. In other embodiments, one may increase only one of these
activities, and optionally also reduce the other activity, e.g. to
generate an Fc region variant with improved ADCC activity, but
reduced CDC activity and vice versa.
[0256] A. Mutants with Enhanced Altered Affinities for
Fc.gamma.RIIIA and/or Fc.gamma.RIIA
[0257] The invention encompasses molecules comprising a variant Fc
region, having one or more amino acid modifications (e.g.,
substitutions) in one or more regions, wherein such modifications
alter the affinity of the variant Fc region for an activating
Fc.gamma.R. In some embodiments, molecules of the invention
comprise a variant Fc region, having one or more amino acid
modifications (e.g., substitutions) in one or more regions, which
modifications increase the affinity of the variant Fc region for
Fc.gamma.RIIIA and/or Fc.gamma.RIIA by at least 2-fold, relative to
a comparable molecule comprising a wild-type Fc region. In another
specific embodiment, molecules of the invention comprise a variant
Fc region, having one or more amino acid modifications (e.g.,
substitutions) in one or more regions, which modifications increase
the affinity of the variant Fc region for Fc.gamma.RIIIA and/or
Fc.gamma.RIIA by greater than 2 fold, relative to a comparable
molecule comprising a wild-type Fc region. In other embodiments of
the invention, the one or more amino acid modifications increase
the affinity of the variant Fc region for Fc.gamma.RIIIA and/or
Fc.gamma.RIIA by at least 3-fold, 4-fold, 5-fold, 6-fold, 8-fold,
or 10-fold relative to a comparable molecule comprising a wild-type
Fc region. In yet other embodiments of the invention the one or
more amino acid modifications decrease the affinity of the variant
Fc region for Fc.gamma.RIIIA and/or Fc.gamma.RIIA by at least
3-fold, 4-fold, 5-fold, 6-fold, 8-fold, or 10-fold relative to a
comparable molecule comprising a wild-type Fc region. Such fold
increases are preferably determined by an ELISA or surface plasmon
resonance assays. In a specific embodiment, the one or more amino
acid modifications do not include or are not solely a substitution
at any one of positions 329, 331, or 322 with any amino acid. In
certain embodiments, the one or more amino acid modifications do
not include or are not solely a substitution with any one of
alanine at positions 243, 256, 290, 298, 312, 333, 334, 359, 360,
or 430; with lysine at position 330; with threonine at position
339; with methionine or arginine at position 320; with serine,
asparagine, aspartic acid, or glutamic acid at position 326 with
glutamine, glutamic acid, methionine, histidine, valine, or leucine
at position 334. In another specific embodiment, the one or more
amino acid modifications do not include or are not solely a
substitution at any of positions 280, 290, 300, 294, or 295. In
another more specific embodiment, the one or more amino acid
modifications do not include or are not solely a substitution at
position 300 with leucine or isoleucine; at position 295 with
lysine; at position 294 with asparagine; at position 298 with
valine; aspartic acid proline, asparagine, or valine; at position
280 with histidine, glutamine or tyrosine; at position 290 with
serine, glycine, threonine or tyrosine.
[0258] In another specific embodiment, the invention encompasses a
molecule comprising a variant Fc region, wherein said variant Fc
region comprises at least one amino acid modification relative to a
wild-type Fc region, such that said polypeptide specifically binds
Fc.gamma.RIIA with a greater affinity than a comparable molecule
comprising the wild-type Fc region binds Fc.gamma.RIIA, provided
that said variant Fc region does not have an alanine at any of
positions 256, 290, 326, 255, 258, 267, 272, 276, 280, 283, 285,
286, 331, 337, 268, 272, or 430; an asparagine at position 268; a
glutamine at position 272; a glutamine, serine, or aspartic acid at
position 286; a serine at position 290; a methionine, glutamine,
glutamic acid, or arginine at position 320; a glutamic acid at
position 322; a serine, glutamic acid, or aspartic acid at position
326; a lysine at position 330; a glutamine at position 335; or a
methionine at position 301. In a specific embodiment, molecules of
the invention comprise a variant Fc region, having one or more
amino acid modifications (e.g., substitutions) in one or more
regions, which modifications increase the affinity of the variant
Fc region for Fc.gamma.RIIA by at least 2-fold, relative to a
comparable molecule comprising a wild-type Fc region. In another
specific embodiment, molecules of the invention comprise a variant
Fc region, having one or more amino acid modifications (e.g.,
substitutions) in one or more regions, which modifications increase
the affinity of the variant Fc region for Fc.gamma.RIIA by greater
than 2 fold, relative to a comparable molecule comprising a
wild-type Fc region. In other embodiments of the invention the one
or more amino acid modifications increase the affinity of the
variant Fc region for Fc.gamma.RIIA by at least 3-fold, 4-fold,
5-fold, 6-fold, 8-fold, or 10-fold relative to a comparable
molecule comprising a wild-type Fc region.
[0259] In a specific embodiment, the invention encompasses
molecules comprising a variant Fc region, having one or more amino
acid modifications (e.g., substitutions but also include insertions
or deletions), which modifications increase the affinity of the
variant Fc region for Fc.gamma.RIIIA and/or Fc.gamma.RIIA by at
least 65%, at least 70%, at least 75%, at least 85%, at least 90%,
at least 95%, at least 99%, at least 100%, at least 150%, and at
least 200%, relative to a comparable molecule comprising a
wild-type Fc region.
[0260] In a specific embodiment, the one or more amino acid
modifications which increase the affinity of the variant Fc region
comprise a substitution at position 347 with histidine, and at
position 339 with valine; or a substitution at position 425 with
isoleucine and at position 215 with phenylalanine; or a
substitution at position 408 with isoleucine, at position 215 with
isoleucine, and at position 125 with leucine; or a substitution at
position 385 with glutamic acid and at position 247 with histidine;
or a substitution at position 348 with methionine, at position 334
with asparagine, at position 275 with isoleucine, at position 202
with methionine, and at position 147 with threonine; or a
substitution at position 275 with isoleucine, at position 334 with
asparagine, and at position 348 with methionine; or a substitution
at position 279 with leucine and at position 395 with serine; or a
substitution at position 246 with threonine and at position 319
with phenylalanine; or a substitution at position 243 with
isoleucine and at position 379 with leucine; or a substitution at
position 243 with leucine, at position 255 with leucine and at
position 318 with lysine; or a substitution at position 334 with
glutamic acid, at position 359 with asparagine, and at position 366
with serine; or a substitution at position 288 with methionine and
at position 334 with glutamic acid; or a substitution at position
334 with glutamic acid and at position 380 with aspartic acid; or a
substitution at position 256 with serine, at position 305 with
isoleucine, at position 334 with glutamic acid and at position 390
with serine; or a substitution at position 335 with asparagine, at
position 370 with glutamic acid, at position 378 with valine, at
position 394 with methionine, and at position 424 with leucine; or
a substitution at position 233 with aspartic acid and at position
334 with glutamic acid; or a substitution at position 334 with
glutamic acid, at position 359 with asparagine, at position 366
with serine, and at position 386 with arginine; or a substitution
at position 246 with threonine and at position 396 with histidine;
or a substitution at position 268 with aspartic acid and at
position 318 with aspartic acid; or a substitution at position 288
with asparagine, at position 330 with serine, and at position 396
with leucine; or a substitution at position 244 with histidine, at
position 358 with methionine, at position 379 with methionine, at
position 384 with lysine and at position 397 with methionine; or a
substitution at position 217 with serine, at position 378 with
valine, and at position 408 with arginine; or a substitution at
position 247 with leucine, at position 253 with asparagine, and at
position 334 with asparagine; or a substitution at position 246
with isoleucine, and at position 334 with asparagine; or a
substitution at position 320 with glutamic acid and at position 326
with glutamic acid; or a substitution at position 375 with cysteine
and at position 396 with leucine; or a substitution at position 243
with leucine, at position 270 with glutamic acid, at position 392
with asparagine and at position 396 with leucine; or a substitution
at position 243 with leucine, at position 255 with leucine, at
position 270 with glutamic acid and at position 396 with leucine;
or a substitution at position 300 with leucine. Examples of other
amino acid substitutions that results in an enhanced affinity for
Fc.gamma.RIIIA in vitro are disclosed below and summarized in Table
5.
[0261] The invention encompasses a molecule comprising a variant Fc
region, wherein said variant Fc region comprises a substitution at
position 243 with isoleucine and at position 379 with leucine, such
that said molecule binds Fc.gamma.RIIIA with about a 1.5 fold
higher affinity than a comparable molecule comprising the wild type
Fc region binds Fc.gamma.RIIIA, as determined by an ELISA assay. In
a specific embodiment, the invention encompasses a molecule
comprising a variant Fc region, wherein said variant Fc region
comprises a substitution at position 288 with asparagine, at
position 330 with serine, and at position 396 with leucine, such
that said molecule binds Fc.gamma.RIIIA with about a 5 fold higher
affinity than a comparable molecule comprising the wild type Fc
region binds Fc.gamma.RIIIA, as determined by an ELISA assay. In a
specific embodiment, the invention encompasses a molecule
comprising a variant Fc region, wherein said variant Fc region
comprises a substitution at position 243 with leucine and at
position 255 with leucine such that said molecule binds
Fc.gamma.RIIIA with about a 1 fold higher affinity than a
comparable molecule comprising the wild type Fc region binds
Fc.gamma.RIIIA, as determined by an ELISA assay. In a specific
embodiment, the invention encompasses a molecule comprising a
variant Fc region, wherein said variant Fc region comprises a
substitution at position 334 with glutamic acid, at position 359
with asparagine, and at position 366 with serine, such that said
molecule binds Fc.gamma.RIIIA with about a 1.5 fold higher affinity
than a comparable molecule comprising the wild type Fc region binds
Fc.gamma.RIIIA, as determined by an ELISA assay. In a specific
embodiment, the invention encompasses a molecule comprising a
variant Fc region, wherein said variant Fc region comprises a
substitution at position 288 with methionine and at position 334
with glutamic acid, such that said molecule binds Fc.gamma.RIIIA
with about a 3 fold higher affinity than a comparable molecule
comprising the wild type Fc region binds Fc.gamma.RIIIA, as
determined by an ELISA assay. In a specific embodiment, the
invention encompasses a molecule comprising a variant Fc region,
wherein said variant Fc region comprises a substitution at position
316 with aspartic acid, at position 378 with valine, and at
position 399 with glutamic acid, such that said molecule binds
Fc.gamma.RIIIA with about a 1.5 fold higher affinity than a
comparable molecule comprising the wild type Fc region binds
Fc.gamma.RIIIA, as determined by an ELISA assay. In a specific
embodiment, the invention encompasses a molecule comprising a
variant Fc region, wherein said variant Fc region comprises a
substitution at position 315 with isoleucine, at position 379 with
methionine, and at position 399 with glutamic acid, such that said
molecule binds Fc.gamma.RIIIA with about a 1 fold higher affinity
than a comparable molecule comprising the wild type Fc region binds
Fc.gamma.RIIIA, as determined by an ELISA assay. In a specific
embodiment, the invention encompasses a molecule comprising a
variant Fc region, wherein said variant Fc region comprises a
substitution at position 243 with isoleucine, at position 379 with
leucine, and at position 420 with valine, such that said molecule
binds Fc.gamma.RIIIA with about a 2.5 fold higher affinity than a
comparable molecule comprising the wild type Fc region binds
Fc.gamma.RIIIA, as determined by an ELISA assay. In a specific
embodiment, the invention encompasses a molecule comprising a
variant Fc region, wherein said variant Fc region comprises a
substitution at position 247 with leucine, and at position 421 with
lysine, such that said molecule binds Fc.gamma.RIIIA with about a 3
fold higher affinity than a comparable molecule comprising the wild
type Fc region binds Fc.gamma.RIIIA, as determined by an ELISA
assay. In a specific embodiment, the invention encompasses a
molecule comprising a variant Fc region, wherein said variant Fc
region comprises a substitution at position 392 with threonine and
at position 396 with leucine such that said molecule binds
Fc.gamma.RIIIA with about a 4.5 fold higher affinity than a
comparable molecule comprising the wild type Fc region binds
Fc.gamma.RIIIA, as determined by an ELISA assay. In a specific
embodiment, the invention encompasses a molecule comprising a
variant Fc region, wherein said variant Fc region comprises a
substitution at position 293 with valine, at position 295 with
glutamic acid, and at position 327 with threonine, such that said
molecule binds Fc.gamma.RIIIA with about a 1.5 fold higher affinity
than a comparable molecule comprising the wild type Fc region binds
Fc.gamma.RIIIA, as determined by an ELISA assay. In a specific
embodiment, the invention encompasses a molecule comprising a
variant Fc region, wherein said variant Fc region comprises a
substitution at position 268 with asparagine and at position 396
with leucine, such that said molecule binds Fc.gamma.RIIIA with
about a 2 fold higher affinity than a comparable molecule
comprising the wild type Fc region binds Fc.gamma.RIIIA, as
determined by an ELISA assay. In a specific embodiment, the
invention encompasses a molecule comprising a variant Fc region,
wherein said variant Fc region comprises a substitution at position
319 with phenylalanine, at position 352 with leucine, and at
position 396 with leucine, such that said molecule binds
Fc.gamma.RIIIA with about a 2 fold higher affinity than a
comparable molecule comprising the wild type Fc region binds
Fc.gamma.RIIIA, as determined by an ELISA assay.
[0262] In a specific embodiment, the invention encompasses a
molecule comprising a variant Fc region, wherein said variant Fc
region comprises at least one amino acid modification relative to a
wild-type Fc region, such that said molecule specifically binds
Fc.gamma.RIIIA with a greater affinity than a comparable molecule
comprising the wild-type Fc region, wherein said at least one amino
acid modification comprises substitution at position 396 with
histidine. In a specific embodiment, the invention encompasses a
molecule comprising a variant Fc region, wherein said variant Fc
region comprises at least one amino acid modification relative to a
wild-type Fc region, such that said molecule specifically binds
Fc.gamma.RIIIA with a greater affinity than a comparable molecule
comprising the wild-type Fc region, wherein said at least one amino
acid modification comprises substitution at position 248 with
methionine. The invention encompasses a molecule comprising a
variant Fc region, wherein said variant Fc region comprises at
least one amino acid modification relative to a wild-type Fc
region, such that said polypeptide specifically binds
Fc.gamma.RIIIA with a similar affinity than a comparable molecule
comprising the wild-type Fc region, wherein said at least one amino
acid modification comprises substitution at position 392 with
arginine. The invention encompasses a molecule comprising a variant
Fc region, wherein said variant Fc region comprises at least one
amino acid modification relative to a wild-type Fc region, such
that said molecule specifically binds Fc.gamma.RIIIA with a similar
affinity than a comparable molecule comprising the wild-type Fc
region, wherein said at least one amino acid modification comprises
substitution at position 315 with isoleucine. The invention
encompasses a molecule comprising a variant Fc region, wherein said
variant Fc region comprises at least one amino acid modification
relative to a wild-type Fc region, such that said molecule
specifically binds Fc.gamma.RIIIA with a similar affinity than a
comparable molecule comprising the wild-type Fc region, wherein
said at least one amino acid modification comprises substitution at
position 132 with isoleucine. The invention encompasses a molecule
comprising a variant Fc region, wherein said variant Fc region
comprises at least one amino acid modification relative to a
wild-type Fc region, such that said molecule specifically binds
Fc.gamma.RIIIA with a similar affinity than a comparable molecule
comprising the wild-type Fc region, wherein said at least one amino
acid modification comprises substitution at position 162 with
valine. The invention encompasses a molecule comprising a variant
Fc region, wherein said variant Fc region comprises at least one
amino acid modification relative to a wild-type Fc region, such
that said molecule specifically binds Fc.gamma.RIIIA with a greater
affinity than a comparable molecule comprising the wild-type Fc
region, wherein said at least one amino acid modification comprises
substitution at position 396 with leucine. The invention
encompasses a molecule comprising a variant Fc region, wherein said
variant Fc region comprises at least one amino acid modification
relative to a wild-type Fc region, such that said molecule
specifically binds Fc.gamma.RIIIA with a greater affinity than a
comparable polypeptide comprising the wild-type Fc region, wherein
said at least one amino acid modification comprises substitution at
position 379 with methionine. The invention encompasses a molecule
comprising a variant Fc region, wherein said variant Fc region
comprises at least one amino acid modification relative to a
wild-type Fc region, such that said molecule specifically binds
Fc.gamma.RIIIA with a greater affinity than a comparable molecule
comprising the wild-type Fc region, wherein said at least one amino
acid modification comprises substitution at position 219 with
tyrosine. The invention encompasses a molecule comprising a variant
Fc region, wherein said variant Fc region comprises at least one
amino acid modification relative to a wild-type Fc region, such
that said molecule specifically binds Fc.gamma.RIIIA with a greater
affinity than a comparable molecule comprising the wild-type Fc
region, wherein said at least one amino acid modification comprises
substitution at position 282 with methionine. The invention
encompasses a molecule comprising a variant Fc region, wherein said
variant Fc region comprises at least one amino acid modification
relative to a wild-type Fc region, such that said molecule
specifically binds Fc.gamma.RIIIA with a greater affinity than a
comparable molecule comprising the wild-type Fc region, wherein
said at least one amino acid modification comprises substitution at
position 401 with valine. The invention encompasses a molecule
comprising a variant Fc region, wherein said variant Fc region
comprises at least one amino acid modification relative to a
wild-type Fc region, such that said molecule specifically binds
Fc.gamma.RIIIA with a greater affinity than a comparable molecule
comprising the wild-type Fc region, wherein said at least one amino
acid modification comprises substitution at position 222 with
asparagine. The invention encompasses a molecule comprising a
variant Fc region, wherein said variant Fc region comprises at
least one amino acid modification relative to a wild-type Fc
region, such that said molecule specifically binds Fc.gamma.RIIIA
with a greater affinity than a comparable molecule comprising the
wild-type Fc region, wherein said at least one amino acid
modification comprises substitution at position 334 with glutamic
acid. The invention encompasses a molecule comprising a variant Fc
region, wherein said variant Fc region comprises at least one amino
acid modification relative to a wild-type Fc region, such that said
molecule specifically binds Fc.gamma.RIIIA with a greater affinity
than a comparable molecule comprising the wild-type Fc region,
wherein said at least one amino acid modification comprises
substitution at position 377 with phenylalanine. The invention
encompasses a molecule comprising a variant Fc region, wherein said
variant Fc region comprises at least one amino acid modification
relative to a wild-type Fc region, such that said molecule
specifically binds Fc.gamma.RIIIA with a greater affinity than a
comparable molecule comprising the wild-type Fc region, wherein
said at least one amino acid modification comprises substitution at
position 334 with isoleucine. The invention encompasses a molecule
comprising a variant Fc region, wherein said variant Fc region
comprises at least one amino acid modification relative to a
wild-type Fc region, such that said molecule specifically binds
Fc.gamma.RIIIA with a greater affinity than a comparable molecule
comprising the wild-type Fc region, wherein said at least one amino
acid modification comprises substitution at position 247 with
leucine. The invention encompasses a molecule comprising a variant
Fc region, wherein said variant Fc region comprises at least one
amino acid modification relative to a wild-type Fc region, such
that said molecule specifically binds Fc.gamma.RIIIA with a greater
affinity than a comparable molecule comprising the wild-type Fc
region, wherein said at least one amino acid modification comprises
substitution at position 326 with glutamic acid. The invention
encompasses a molecule comprising a variant Fc region, wherein said
variant Fc region comprises at least one amino acid modification
relative to a wild-type Fc region, such that said molecule
specifically binds Fc.gamma.RIIIA with a greater affinity than a
comparable molecule comprising the wild-type Fc region, wherein
said at least one amino acid modification comprises substitution at
position 372 with tyrosine. The invention encompasses a molecule
comprising a variant Fc region, wherein said variant Fc region
comprises at least one amino acid modification relative to a
wild-type Fc region, such that said molecule specifically binds
Fc.gamma.RIIIA with a greater affinity than a comparable molecule
comprising the wild-type Fc region, wherein said at least one amino
acid modification comprises substitution at position 224 with
leucine.
[0263] The invention encompasses a molecule comprising a variant Fc
region, wherein said variant Fc region comprises at least one amino
acid modification relative to a wild-type Fc region, such that said
molecule specifically binds Fc.gamma.RIIIA with a greater affinity
than a comparable molecule comprising the wild-type Fc region,
wherein said at least one amino acid modification comprises
substitution at position 275 with tyrosine. The invention
encompasses a molecule comprising a variant Fc region, wherein said
variant Fc region comprises at least one amino acid modification
relative to a wild-type Fc region, such that said molecule
specifically binds Fc.gamma.RIIIA with a greater affinity than a
comparable molecule comprising the wild-type Fc region, wherein
said at least one amino acid modification comprises substitution at
position 398 with valine. The invention encompasses a molecule
comprising a variant Fc region, wherein said variant Fc region
comprises at least one amino acid modification relative to a
wild-type Fc region, such that said molecule specifically binds
Fc.gamma.RIIIA with a greater affinity than a comparable molecule
comprising the wild-type Fc region, wherein said at least one amino
acid modification comprises substitution at position 334 with
asparagine. The invention encompasses a molecule comprising a
variant Fc region, wherein said variant Fc region comprises at
least one amino acid modification relative to a wild-type Fc
region, such that said molecule specifically binds Fc.gamma.RIIIA
with a greater affinity than a comparable molecule comprising the
wild-type Fc region, wherein said at least one amino acid
modification comprises substitution at position 400 with proline.
The invention encompasses a molecule comprising a variant Fc
region, wherein said variant Fc region comprises at least one amino
acid modification relative to a wild-type Fc region, such that said
molecule specifically binds Fc.gamma.RIIIA with a greater affinity
than a comparable molecule comprising the wild-type Fc region,
wherein said at least one amino acid modification comprises
substitution at position 407 with isoleucine. The invention
encompasses a molecule comprising a variant Fc region, wherein said
variant Fc region comprises at least one amino acid modification
relative to a wild-type Fc region, such that said molecule
specifically binds Fc.gamma.RIIIA with a greater affinity than a
comparable molecule comprising the wild-type Fc region, wherein
said at least one amino acid modification comprises substitution at
position 372 with tyrosine. The invention encompasses a molecule
comprising a variant Fc region, wherein said variant Fc region
comprises at least one amino acid modification relative to a
wild-type Fc region, such that said molecule specifically binds
Fc.gamma.RIIIA with a similar affinity than a comparable molecule
comprising the wild-type Fc region, wherein said at least one amino
acid modification comprises substitution at position 366 with
asparagine. The invention encompasses a molecule comprising a
variant Fc region, wherein said variant Fc region comprises at
least one amino acid modification relative to a wild-type Fc
region, such that said molecule specifically binds Fc.gamma.RIIIA
with a reduced affinity than a comparable molecule comprising the
wild-type Fc region, wherein said at least one amino acid
modification comprises substitution at position 414 with
asparagine. The invention encompasses a molecule comprising a
variant Fc region, wherein said variant Fc region comprises at
least one amino acid modification relative to a wild-type Fc
region, such that said molecule specifically binds Fc.gamma.RIIIA
with a reduced affinity than a comparable molecule comprising the
wild-type Fc region, wherein said at least one amino acid
modification comprises substitution at position 225 with serine.
The invention encompasses a molecule comprising a variant Fc
region, wherein said variant Fc region comprises at least one amino
acid modification relative to a wild-type Fc region, such that said
molecule specifically binds Fc.gamma.RIIIA with a reduced affinity
than a comparable molecule comprising the wild-type Fc region,
wherein said at least one amino acid modification comprises
substitution at position 377 with asparagine.
[0264] In a specific embodiment, the invention encompasses a
molecule comprising a variant Fc region, wherein said variant Fc
region comprises at least one amino acid modification relative to a
wild-type Fc region, such that said molecule specifically binds
Fc.gamma.RIIIA with about a 2 fold greater affinity than a
comparable molecule comprising the wild-type Fc region as
determined by an ELISA assay, wherein said at least one amino acid
modification comprises substitution at position 379 with
methionine. In another specific embodiment, the invention
encompasses a molecule comprising a variant Fc region, wherein said
variant Fc region comprises at least one amino acid modification
relative to a wild-type Fc region, such that said molecule
specifically binds Fc.gamma.RIIIA with about a 1.5 fold greater
affinity than a comparable molecule comprising the wild-type Fc
region as determined by an ELISA assay, wherein said at least one
amino acid modification comprises substitution at position 248 with
methionine.
[0265] In some embodiments, the molecules of the invention have an
altered affinity for Fc.gamma.RIIIA and/or Fc.gamma.RIIA as
determined using in vitro assays (biochemical or immunological
based assays) known in the art for determining Fc-Fc.gamma.R
interactions, i.e., specific binding of an Fc region to an
Fc.gamma.R including but not limited to ELISA assay, surface
plasmon resonance assay, immunoprecipitation assays (See Section
5.2.1). Preferably, the binding properties of these molecules with
altered affinities for activating Fc.gamma.R receptors are also
correlated to their activity as determined by in vitro functional
assays for determining one or more Fc.gamma.R mediator effector
cell functions, e.g., molecules with variant Fc regions with
enhanced affinity for Fc.gamma.RIIIA have a conferred or an
enhanced ADCC activity. In most preferred embodiments, the
molecules of the invention that have an altered binding property
for an activating Fc receptor, e.g., Fc.gamma.RIIIA in an in vitro
assay, also have an altered binding property in in vivo models
(such as those described and disclosed herein). However, the
present invention does not exclude molecules of the invention that
do not exhibit an altered Fc.gamma.R binding in in vitro based
assays but do exhibit the desired phenotype in vivo.
[0266] B. Mutants with Enhanced Affinity for Fc.gamma.RIIIA and
Reduced or No Affinity for Fc.gamma.RIIB
[0267] In a specific embodiment, the molecules of the invention
comprise a variant Fc region, having one or more amino acid
modifications (i.e., substitutions) in one or more regions, which
one or more modifications increase the affinity of the variant Fc
region for Fc.gamma.RIIIA and decrease the affinity of the variant
Fc region for Fc.gamma.RIIB, relative to a comparable molecule
comprising a wild-type Fc region which binds Fc.gamma.RIIIA and
Fc.gamma.RIIB with wild-type affinity. In a certain embodiment, the
one or more amino acid modifications do not include or are not
solely a substitution with alanine at any of positions 256, 298,
333, 334, 280, 290, 294, 298, or 296; or a substitution at position
298 with asparagine, valine, aspartic acid, or proline; or a
substitution 290 with serine. In certain amino embodiments, the one
or more amino acid modifications increases the affinity of the
variant Fc region for Fc.gamma.RIIIA by at least 65%, at least 70%,
at least 75%, at least 85%, at least 90%, at least 95%, at least
99%, at least 100%, at least 200%, at least 300%, at least 400% and
decreases the affinity of the variant Fc region for Fc.gamma.RIIB
by at least 65%, at least 70%, at least 75%, at least 85%, at least
90%, at least 95%, at least 99%, at least 100%, at least 200%, at
least 300%, at least 400%.
[0268] In a specific embodiment, the molecule of the invention
comprising a variant Fc region with an enhanced affinity for
Fc.gamma.RIIIA and a lowered affinity or no affinity for
Fc.gamma.RIIB, as determined based on an ELISA assay and/or an ADCC
based assay using ch-4-4-20 antibody carrying the variant Fc
region, comprises a substitution at any of the following: at
position 275 with isoleucine, at position 334 with asparagine, and
at position 348 with methionine; or a substitution at position 279
with leucine and at position 395 with serine; or a substitution at
position 246 with threonine and at position 319 with phenylalanine;
or a substitution at position 243 with leucine, at position 255
with leucine, and at position 318 with lysine; or a substitution at
position 334 with glutamic acid, at position 359 with asparagine
and at position 366 with serine; or a substitution at position 334
with glutamic acid and at position 380 with aspartic acid; or a
substitution at position 256 with serine, at position 305 with
isoleucine, at position 334 with glutamic acid, and at position 390
with serine; or a substitution at position 335 with asparagine, at
position 370 with glutamic acid, at position 378 with valine, at
position 394 with methionine and at position 424 with leucine; or a
substitution at position 233 with aspartic acid and at position 334
with glutamic acid; or a substitution at position 334 with glutamic
acid, at position 359 with asparagine, at position 366 with serine
and at position 386 with arginine; or a substitution at position
312 with glutamic acid, at position 327 with asparagine, and at
position 378 with serine; or a substitution at position 288 with
asparagine and at position 326 with asparagine; or a substitution
at position 247 with leucine and at position 421 with lysine; or a
substitution at position 298 with asparagine and at position 381
with arginine; or a substitution at position 280 with glutamic
acid, at position 354 with phenylalanine, at position 431 with
aspartic acid, and at position 441 with isoleucine; or a
substitution at position 255 with glutamine and at position 326
with glutamic acid; or a substitution at position 218 with
arginine, at position 281 with aspartic acid and at position 385
with arginine; or a substitution at position 247 with leucine, at
position 330 with threonine and at position 440 with glycine; or a
substitution at position 284 with alanine and at position 372 with
leucine; or a substitution at position 335 with asparagine, as
position 387 with serine and at position 435 with glutamine; or a
substitution at position 247 with leucine, at position 431 with
valine and at position 442 with phenylalanine.
[0269] In a specific embodiment, the molecule of the invention
comprising a variant Fc region with an enhanced affinity for
Fc.gamma.RIIIA and a lowered affinity or no affinity for
Fc.gamma.RIIB as determined based on an ELISA assay and/or an ADCC
based assay using ch-4-4-20 antibody carrying the variant Fc region
comprises a substitution at position 379 with methionine; at
position 219 with tyrosine; at position 282 with methionine; at
position 401 with valine; at position 222 with asparagine; at
position 334 with isoleucine; at position 334 with glutamic acid;
at position 275 with tyrosine; at position 398 with valine.
[0270] The invention encompasses a molecule comprising a variant Fc
region, wherein said variant Fc region comprises at least one amino
acid modification relative to a wild-type Fc region, such that said
molecule specifically binds Fc.gamma.RIIB with about a 3 fold lower
affinity than a comparable molecule comprising the wild-type Fc
region as determined by an ELISA assay, wherein said at least one
amino acid modification comprises substitution at position 288 with
asparagine, at position 330 with serine, and at position 396 with
leucine. The invention encompasses a molecule comprising a variant
Fc region, wherein said variant Fc region comprises at least one
amino acid modification relative to a wild-type Fc region, such
that said molecule specifically binds Fc.gamma.RIIB with about a
10-15 fold lower affinity than a comparable molecule comprising the
wild-type Fc region as determined by an ELISA assay, wherein said
at least one amino acid modification comprises substitution at
position 316 with aspartic acid, at position 378 with valine, and
at position 399 with glutamic acid. The invention encompasses a
molecule comprising a variant Fc region, wherein said variant Fc
region comprises at least one amino acid modification relative to a
wild-type Fc region, such that said molecule specifically binds
Fc.gamma.RIIB with about a 10 fold lower affinity than a comparable
molecule comprising the wild-type Fc region as determined by an
ELISA assay, wherein said at least one amino acid modification
comprises substitution at position 315 with isoleucine, at position
379 with methionine, and at position 399 with glutamic acid. The
invention encompasses a molecule comprising a variant Fc region,
wherein said variant Fc region comprises at least one amino acid
modification relative to a wild-type Fc region, such that said
molecule specifically binds Fc.gamma.RIIB with about a 7 fold lower
affinity than a comparable molecule comprising the wild-type Fc
region as determined by an ELISA assay, wherein said at least one
amino acid modification comprises substitution at position 243 with
isoleucine, at position 379 with leucine, and at position 420 with
valine. The invention encompasses a molecule comprising a variant
Fc region, wherein said variant Fc region comprises at least one
amino acid modification relative to a wild-type Fc region, such
that said molecule specifically binds Fc.gamma.RIIB with about a 3
fold lower affinity than a comparable molecule comprising the
wild-type Fc region as determined by an ELISA assay, wherein said
at least one amino acid modification comprises substitution at
position 392 with threonine and at position 396 with leucine. The
invention encompasses a molecule comprising a variant Fc region,
wherein said variant Fc region comprises at least one amino acid
modification relative to a wild-type Fc region, such that said
molecule specifically binds Fc.gamma.RIIB with about a 5 fold lower
affinity than a comparable molecule comprising the wild-type Fc
region as determined by an ELISA assay, wherein said at least one
amino acid modification comprises substitution at position 268 with
asparagine and at position 396 with leucine. The invention also
encompasses a molecule comprising a variant Fc region, wherein said
variant Fc region comprises at least one amino acid modification
relative to a wild-type Fc region, such that said molecule
specifically binds Fc.gamma.RIIB with about a 2 fold lower affinity
than a comparable molecule comprising the wild-type Fc region as
determined by an ELISA assay, wherein said at least one amino acid
modification comprises substitution at position 319 with
phenylalanine, at position 352 with leucine, and at position 396
with leucine.
[0271] C. Mutants with Enhanced Affinity to Fc.gamma.RIIIA and
Fc.gamma.RIIB
[0272] The invention encompasses molecules comprising variant Fc
regions, having one or more amino acid modifications, which
modifications increase the affinity of the variant Fc region for
Fc.gamma.RIIIA and Fc.gamma.RIIB by at least 65%, at least 70%, at
least 75%, at least 85%, at least 90%, at least 95%, at least 99%,
at least 100%, at least 200%, at least 300%, at least 400% and
decreases the affinity of the variant Fc region for Fc.gamma.RIIB
by at least 65%, at least 70%, at least 75%, at least 85%, at least
90%, at least 95%, at least 99%, at least 100%, at least 200%, at
least 300%, at least 400%. In a specific embodiment, the molecule
of the invention comprising a variant Fc region with an enhanced
affinity for Fc.gamma.RIIIA and an enhanced affinity for
Fc.gamma.RIIB (as determined based on an ELISA assay and/or an ADCC
based assay using ch-4-4-20 antibody carrying the variant Fc region
as described herein) comprises a substitution at position 415 with
isoleucine and at position 251 with phenylalanine; or a
substitution at position 399 with glutamic acid, at position 292
with leucine, and at position 185 with methionine; or a
substitution at position 408 with isoleucine, at position 215 with
isoleucine, and at position 125 with leucine; or a substitution at
position 385 with glutamic acid and at position 247 with histidine;
or a substitution at position 348 with methionine, at position 334
with asparagine, at position 275 with isoleucine, at position 202
with methionine and at position 147 with threonine; or a
substitution at position 246 with threonine and at position 396
with histidine; or a substitution at position 268 with aspartic
acid and at position 318 with aspartic acid; or a substitution at
position 288 with asparagine, at position 330 with serine and at
position 396 with leucine; or a substitution at position 244 with
histidine, at position 358 with methionine, at position 379 with
methionine, at position 384 with lysine and at position 397 with
methionine; or a substitution at position 217 with serine, at
position 378 with valine, and at position 408 with arginine; or a
substitution at position 247 with leucine, at position 253 with
asparagine, and at position 334 with asparagine; or a substitution
at position 246 with isoleucine and at position 334 with
asparagine; or a substitution at position 320 with glutamic acid
and at position 326 with glutamic acid; or a substitution at
position 375 with cysteine and at position 396 with leucine; or a
substitution at position 343 with serine, at position 353 with
leucine, at position 375 with isoleucine, at position 383 with
asparagine; or a substitution at position 394 with methionine and
at position 397 with methionine; or a substitution at position 216
with aspartic acid, at position 345 with lysine and at position 375
with isoleucine; or a substitution at position 288 with asparagine,
at position 330 with serine, and at position 396 with leucine; or a
substitution at position 247 with leucine and at position 389 with
glycine; or a substitution at position 222 with asparagine, at
position 335 with asparagine, at position 370 with glutamic acid,
at position 378 with valine and at position 394 with methionine; or
a substitution at position 316 with aspartic acid, at position 378
with valine and at position 399 with glutamic acid; or a
substitution at position 315 with isoleucine, at position 379 with
methionine, and at position 394 with methionine; or a substitution
at position 290 with threonine and at position 371 with aspartic
acid; or a substitution at position 247 with leucine and at
position 398 with glutamine; or a substitution at position 326 with
glutamine; at position 334 with glutamic acid, at position 359 with
asparagine, and at position 366 with serine; or a substitution at
position 247 with leucine and at position 377 with phenylalanine;
or a substitution at position 378 with valine, at position 390 with
isoleucine and at position 422 with isoleucine; or a substitution
at position 326 with glutamic acid and at position 385 with
glutamic acid; or a substitution at position 282 with glutamic
acid, at position 369 with isoleucine and at position 406 with
phenylalanine; or a substitution at position 397 with methionine;
at position 411 with alanine and at position 415 with asparagine;
or a substitution at position 223 with isoleucine, at position 256
with serine and at position 406 with phenylalanine; or a
substitution at position 298 with asparagine and at position 407
with arginine; or a substitution at position 246 with arginine, at
position 298 with asparagine, and at position 377 with
phenylalanine; or a substitution at position 235 with proline, at
position 382 with methionine, at position 304 with glycine, at
position 305 with isoleucine, and at position 323 with isoleucine;
or a substitution at position 247 with leucine, at position 313
with arginine, and at position 388 with glycine; or a substitution
at position 221 with tyrosine, at position 252 with isoleucine, at
position 330 with glycine, at position 339 with threonine, at
position 359 with asparagine, at position 422 with isoleucine, and
at position 433 with leucine; or a substitution at position 258
with aspartic acid, and at position 384 with lysine; or a
substitution at position 241 with leucine and at position 258 with
glycine; or a substitution at position 370 with asparagine and at
position 440 with asparagine; or a substitution at position 317
with asparagine and a deletion at position 423; or a substitution
at position 243 with isoleucine, at position 379 with leucine and
at position 420 with valine; or a substitution at position 227 with
serine and at position 290 with glutamic acid; or a substitution at
position 231 with valine, at position 386 with histidine, and at
position 412 with methionine; or a substitution at position 215
with proline, at position 274 with asparagine, at position 287 with
glycine, at position 334 with asparagine, at position 365 with
valine and at position 396 with leucine; or a substitution at
position 293 with valine, at position 295 with glutamic acid and at
position 327 with threonine; or a substitution at position 319 with
phenylalanine, at position 352 with leucine, and at position 396
with leucine; or a substitution at position 392 with threonine and
at position 396 with leucine; at a substitution at position 268
with asparagine and at position 396 with leucine; or a substitution
at position 290 with threonine, at position 390 with isoleucine,
and at position 396 with leucine; or a substitution at position 326
with isoleucine and at position 396 with leucine; or a substitution
at position 268 with aspartic acid and at position 396 with
leucine; or a substitution at position 210 with methionine and at
position 396 with leucine; or a substitution at position 358 with
proline and at position 396 with leucine; or a substitution at
position 288 with arginine, at position 307 with alanine, at
position 344 with glutamic acid, and at position 396 with leucine;
or a substitution at position 273 with isoleucine, at position 326
with glutamic acid, at position 328 with isoleucine and at position
396 with leucine; or a substitution at position 326 with
isoleucine, at position 408 with asparagine and at position 396
with leucine; or a substitution at position 334 with asparagine and
at position 396 with leucine; or a substitution at position 379
with methionine and at position 396 with leucine; or a substitution
at position 227 with serine and at position 396 with leucine; or a
substitution at position 217 with serine and at position 396 with
leucine; or a substitution at position 261 with asparagine, at
position 210 with methionine and at position 396 with leucine; or a
substitution at position 419 with histidine and at position 396
with leucine; or a substitution at position 370 with glutamic acid
and at position 396 with leucine; or a substitution at position 242
with phenylalanine and at position 396 with leucine; or a
substitution at position 255 with leucine and at position 396 with
leucine; or a substitution at position 240 with alanine and at
position 396 with leucine; or a substitution at position 250 with
serine and at position 396 with leucine; or a substitution at
position 247 with serine and at position 396 with leucine; or a
substitution at position 410 with histidine and at position 396
with leucine; or a substitution at position 419 with leucine and at
position 396 with leucine; or a substitution at position 427 with
alanine and at position 396 with leucine; or a substitution at
position 258 with aspartic acid and at position 396 with leucine;
or a substitution at position 384 with lysine and at position 396
with leucine; or a substitution at position 323 with isoleucine and
at position 396 with leucine; or a substitution at position 244
with histidine and at position 396 with leucine; or a substitution
at position 305 with leucine and at position 396 with leucine; or a
substitution at position 400 with phenylalanine and at position 396
with leucine; or a substitution at position 303 with isoleucine and
at position 396 with leucine; or a substitution at position 243
with leucine, at position 305 with isoleucine, at position 378 with
aspartic acid, at position 404 with serine and at position 396 with
leucine; or a substitution at position 290 with glutamic acid, at
position 369 with alanine, at position 393 with alanine and at
position 396 with leucine; or a substitution at position 210 with
asparagine, at position 222 with isoleucine, at position 320 with
methionine and at position 396 with leucine; or a substitution at
position 217 with serine, at position 305 with isoleucine, at
position 309 with leucine, at position 390 with histidine and at
position 396 with leucine; or a substitution at position 246 with
asparagine; at position 419 with arginine and at position 396 with
leucine; or a substitution at position 217 with alanine, at
position 359 with alanine and at position 396 with leucine; or a
substitution at position 215 with isoleucine, at position 290 with
valine and at position 396 with leucine; or a substitution at
position 275 with leucine, at position 362 with histidine, at
position 384 with lysine and at position 396 with leucine; or a
substitution at position 334 with asparagine; or a substitution at
position 400 with proline; or a substitution at position 407 with
isoleucine; or a substitution at position 372 with tyrosine; or a
substitution at position 366 with asparagine; or a substitution at
position 414 with asparagine; or a substitution at position 352
with leucine; or a substitution at position 225 with serine; or a
substitution at position 377 with asparagine; or a substitution at
position 248 with methionine.
[0273] E. Mutants with Altered Fc.gamma.R-Mediated Effector
Functions
[0274] The invention encompasses molecules, e.g., immunoglobulins
comprising Fc variants with altered effector functions, preferably,
added effector functions, i.e., where the variants exhibit
detectable levels of one or more effector functions that are not
detectable in the parent antibody. In some embodiments,
immunoglobulins comprising Fc variants mediate effector function
more effectively in the presence of effector cells as determined
using assays known in the art and exemplified herein. In specific
embodiments, the Fc variants of the invention may be combined with
other known Fc modifications that enhance effector function, such
that the combination has an additive, synergistic effect. The Fc
variants of the invention have conferred or enhanced effector
function in vitro and/or in vivo.
[0275] In a specific embodiment, the immunoglobulins of the
invention have an enhanced Fc.gamma.R-mediated effector function as
determined using ADCC activity assays disclosed herein. Examples of
effector functions that could be mediated by the molecules of the
invention include, but are not limited to, C1q binding,
complement-dependent cytotoxicity, antibody-dependent cell mediate
cytotoxicity (ADCC), phagocytosis, etc. The effector functions of
the molecules of the invention can be assayed using standard
methods known in the art, examples of which are disclosed in
Section 5.2.7. In a specific embodiment, the immunoglobulins of the
invention comprising a variant Fc region mediate ADCC where the
parent molecule does not exhibit detectable levels of ADCC activity
or induces ADCC 2-fold more effectively, than an immunoglobulin
comprising a wild-type Fc region. In other embodiments, the
immunoglobulins of the invention comprising a variant Fc region
mediate ADCC where the parent molecule does not exhibit detectable
levels of ADCC activity or induces ADCC at least 4-fold, at least
8-fold, at least 10-fold, at least 100-fold, at least 1000-fold, at
least 10.sup.4-fold, at least 10.sup.5-fold more effectively, than
an immunoglobulin comprising a wild-type Fc region. In another
specific embodiment, the immunoglobulins of the invention have
altered C1q binding activity. In some embodiments, the
immunoglobulins of the invention mediate C1q binding activity where
the parent molecule does not exhibit detectable levels of C1q
binding activity or has at least 2-fold, at least 4-fold, at least
8-fold, at least 10-fold, at least 100-fold, at least 1000-fold, at
least 10.sup.4-fold, at least 10.sup.5-fold higher C1q binding
activity than an immunoglobulin comprising a wild-type Fc region.
In yet another specific embodiment, the immunoglobulins of the
invention have altered complement dependent cytotoxicity. In yet
another specific embodiment, the immunoglobulins of the invention
have complement dependent cytotoxicity where the parent molecule
does not exhibit detectable levels of complement dependent
cytotoxicity or enhances complement dependent cytotoxicity to
levels greater than an immunoglobulin comprising a wild-type Fc
region. In some embodiments, the immunoglobulins of the invention
have at least 2-fold, at least 4-fold, at least 8-fold, at least
10-fold, at least 100-fold, at least 1000-fold, at least
10.sup.4-fold, at least 10.sup.5-fold higher complement dependent
cytotoxicity than an immunoglobulin comprising a wild-type Fc
region.
[0276] In other embodiments, immunoglobulins of the invention have
phagocytosis activity where the parent molecule does not exhibit
detectable levels of phagocytosis activity or have enhanced
phagocytosis activity relative to an immunoglobulin comprising a
wild-type Fc region, as determined by standard assays known to one
skilled in the art or disclosed herein. In some embodiments, the
immunoglobulins of the invention have at least 2-fold, at least
4-fold, at least 8-fold, at least 10-fold higher phagocytosis
activity relative to an immunoglobulin comprising a wild-type Fc
region.
[0277] In a specific embodiment, the invention encompasses an
immunoglobulin comprising a variant Fc region with one or more
amino acid modifications, such that the immunoglobulin has an
effector function, e.g., antibody dependent cell mediated
cytotoxicity or phagocytosis, where the parent molecule does not
exhibit detectable levels of the effector function or has an
enhanced effector function. In a specific embodiment, the one or
more amino acid modifications which increase the ADCC activity of
the immunoglobulin comprise a substitution at position 379 with
methionine; or a substitution at position 243 with isoleucine and
at position 379 with leucine; or a substitution at position 288
with asparagine, at position 330 with serine, and at position 396
with leucine; or a substitution at position 243 leucine and at
position 255 with leucine; or a substitution at position 334 with
glutamic acid, at position 359 with asparagine, and at position 366
with serine; or a substitution at position 288 with methionine and
at position 334 with glutamic acid; or a substitution at position
334 with glutamic acid and at position 292 with leucine; or a
substitution at position 316 with aspartic acid, at position 378
with valine, and at position 399 with glutamic acid; or a
substitution at position 315 with isoleucine, at position 379 with
methionine, and at position 399 with glutamic acid; or a
substitution at position 243 with isoleucine, at position 379 with
leucine, and at position 420 with valine; or a substitution at
position 247 with leucine and at position 421 with lysine; or a
substitution at position 248 with methionine; or a substitution at
position 392 with threonine and at position 396 with leucine; or a
substitution at position 293 with valine, at position 295 with
glutamic acid, and at position 327 with threonine; or a
substitution at position 268 with asparagine and at position 396
with leucine; or a substitution at position 319 with phenylalanine,
at position 352 with leucine, and at position 396 with leucine; or
a substitution at position 255 with leucine, at position 396 with
leucine, at position 270 with glutamic acid, and at position 300
with leucine; or a substitution at position 240 with alanine, at
position 396 with leucine, and at position 270 with glutamic acid;
or a substitution at position 370 with glutamic acid, at position
396 with leucine, and at position 270 with glutamic acid; or a
substitution at position 392 with threonine, at position 396 with
leucine, and at position 270 with glutamic acid; or a substitution
at position 370 with glutamic acid and at position 396 with
leucine; or a substitution at position 419 with histidine and at
position 396 with leucine; or a substitution at position 255 with
leucine, at position 396 with leucine, at position 270 with
glutamic acid, and at position 292 with glycine. In other specific
embodiments, the variant Fc region has a leucine at position 247, a
lysine at position 421 and a glutamic acid at position 270
(MgFc31/60); a threonine at position 392, a leucine at position
396, a glutamic acid at position 270, and a leucine at position 243
(MgFc38/60/F243L); a histidine at position 419, a leucine at
position 396, and a glutamic acid at position 270 (MGFc51/60); a
histidine at position 419, a leucine at position 396, a glutamic
acid at position 270, and a leucine at position 243
(MGFc51/60/F243L); an alanine at position 240, a leucine at
position 396, and a glutamic acid at position 270 (MGFc52/60); a
lysine at position 255 and a leucine at position 396 (MgFc55); a
lysine at position 255, a leucine at position 396, and a glutamic
acid at position 270 (MGFc55/60); a lysine at position 255, a
leucine at position 396, a glutamic acid at position 270, and a
lysine at position 300 (MGFc55/60/Y300L); a lysine at position 255,
a leucine at position 396, a glutamic acid at position 270, and a
glycine at position 292 (MGFc55/60/R292G); a lysine at position
255, a leucine at position 396, a glutamic acid at position 270,
and a leucine at position 243 (MgFc55/60/F243L); a glutamic acid at
position 370, a leucine at position 396, and a glutamic acid at
position 270 (MGFc59/60); a glutamic acid at position 270, an
aspartic acid at position 316, and a glycine at position 416
(MgFc71); a leucine at position 243, a proline at position 292, an
isoleucine at position 305, and a leucine at position 396
(MGFc74/P396L); or a leucine at position 243, a glutamic acid at
position 270, an asparagine at position 392 and a leucine at
position 396; or a leucine at position 243, a leucine at position
255, a glutamic acid at position 270 and a leucine at position 396;
or a glutamine at position 297.
[0278] In another specific embodiment, the one or more amino acid
modifications which confers or increases the ADCC activity of the
immunoglobulin is any of the mutations listed below, in table
7.
TABLE-US-00008 TABLE 7 AMINO ACID MODIFICATIONS WHICH CONFER OR
INCREASE ADCC E333A, K334A R292L, K334E V379M S219Y V282M K222N
F243I, V379L F243L, R255L, E318K K334I K334E, T359N, T366S K288M,
K334E K288N, A330S, P396L K326E G316D, A378V, D399E N315I, V379M,
T394M F243I, V379L, G420V E293V, Q295E, A327T Y319F, P352L, P396L
K392T, P396L K248M H268N, P396L K290T, N390I, P396L K326I, P396L
H268D, P396L K210M, P396L L358P, P396L K288R, T307A, K344E, P396L
V273I, K326E, L328I, P396L K326I, S408N, P396L K334N, P396L V379M,
P396L P227S, P396L P217S, P396L K261N, K210M, P396L Q419H, P396L
K370E, P396L L242F, P396L F243L, V305I, A378D, F404S, P396L R255L,
P396L V240A, P396L T250S, P396L P247S, P396L K290E, V369A, T393A,
P396L K210N, K222I, K320M, P396L L410H, P396L Q419L, P396L V427A,
P396L P217S, V305I, I309L, N390H, P396L E258D, P396L N384K, P396L
V323I, P396L K246N, Q419R, P396L P217A, T359A, P396L P244H, P396L
V215I, K290V, P396L F275L, Q362H, N384K, P396L V305L, P396L S400F,
P396L V303I, P396L D270E, G316D, R416G P247L, N421K P247L, N421K,
D270E Q419H, P396L, D270E K370E, P396L, D270E R255L, P396L, D270E
V240A, P396L, D270E K392T, P396L, D270E R255L, P396L, D270E, Y300L
R255L, P396L, D270E, R292G K392T, P396L, D270E, F243L Q419H, P396L,
D270E, F243L R255L, P396L, D270E, F243L F243L, D270E, K392N, P396L
F243L, R255L, D270E, P396L
[0279] Alternatively or additionally, it may be useful to combine
the above amino acid modifications or any other amino acid
modifications disclosed herein with one or more further amino acid
modifications that confer and/or alter C1q binding and/or
complement dependent cytoxicity function of the Fc region. The
further amino acid substitutions described herein will generally
serve to alter the ability of the starting molecule to bind to C1q
and/or modify its complement dependent cytotoxicity function, e.g.,
to reduce and preferably abolish these effector functions.
Molecules comprising substitutions at one or more of the described
positions with conferred or improved C1q binding and/or complement
dependent cytotoxicity (CDC) function are contemplated herein. For
example, the starting molecule may be unable to bind C1q and/or
mediate CDC and may be modified according to the teachings herein
such that it acquires these further effector functions. Moreover,
molecules with preexisting C1q binding activity, optionally further
having the ability to mediate CDC may be modified such that one or
both of these activities are enhanced.
[0280] As disclosed above, one can design an Fc region with altered
effector function, e.g., by modifying or conferring C1q binding
and/or FcR binding and thereby changing CDC activity and/or ADCC
activity. For example, one can generate a variant Fc region with
improved or conferred C1q binding and improved or conferred
Fc.gamma.RIII binding; e.g., having both conferred or improved ADCC
activity and conferred or improved CDC activity. Alternatively,
where one desires that effector function be reduced or ablated, one
may engineer a variant Fc region with reduced CDC activity and/or
reduced ADCC activity. In other embodiments, one may increase only
one of these activities, and optionally also reduce the other
activity, e.g., to generate an Fc region variant with improved ADCC
activity, but reduced CDC activity and vice versa.
[0281] The invention encompasses molecules with specific variants
of the Fc region that have been identified using the methods of the
invention from a yeast library of mutants after 2nd-4th-round of
sorting are listed in Table 8. Table 8 summarizes the various
mutants that were identified using the methods of the invention.
The mutants were assayed using an ELISA assay for determining
binding to Fc.gamma.RIIIA and Fc.gamma.RIIB. The mutants were also
tested in an ADCC assay, by cloning the Fc variants into a ch
4-4-20 antibody using methods disclosed and exemplified herein.
Bolded items refer to experiments, in which the ch4-4-20 were
purified prior the ADCC assay. The antibody concentration used was
standard for ADCC assays, in the range 0.5 .mu.g/mL-1.0
.mu.g/mL.
TABLE-US-00009 TABLE 8 MUTATIONS IDENTIFIED IN THE Fc REGION
Binding Binding to to 4-4-20 ADCC Fc.gamma.RIIIA Fc.gamma.RIIB
(Relative Mutations Domain (ELISA) (ELISA) Lysis (Mut/Wt) pYD-CH1
library FACS screen with 3A tetramer Q347H; A339V CH3
.uparw..quadrature.0.5x NT S415I; L251F CH2, CH3
.uparw..quadrature.0.5x .uparw..75x 0.82 K392R CH3 N/C NT D399E;
R292L; V185M CH1, CH2, CH3 N/C .uparw..quadrature.0.5x 0.65 0.9
K290E; L142P CH1, CH2 N/C NT R301C; M252L; S192T CH1, CH2
.dwnarw..5x NT P291S; K288E; H268L; A141V CH1, CH2 .dwnarw..5x NT
N315I CH2 N/C .uparw..75x S132I CH1 N/C NT S383N; N384K; T256N;
V262L; K218E; R214I; K205E; All .uparw.0.5x NT F149Y; K133M S408I;
V215I; V125L CH1, CH2, CH3 .uparw..quadrature.0.5x .uparw..75x 0.62
P396L CH3 .uparw.1x .uparw.1x 0.55 G385E; P247H; CH2, CH3 .uparw.1x
.uparw..75x 0.44 P396H CH3 .uparw.1x .quadrature..uparw.1x 0.58
A162V CH1 N/C NT V348M; K334N; F275I; Y202M; K147T CH1, CH2, CH3
.uparw..quadrature.0.5x .uparw..75x 0.33 H310Y; T289A; G337E CH2
.uparw..5x NT S119F; G371S; Y407V; E258D CH1, CH2, CH3 N/C N/C 0.29
K409R; S166N CH1, CH3 N/C NT in vitro Site Directed mutants R292L
CH2 NT NT 0.82 T359N CH3 NT NT 1.06 T366S CH3 NT NT 0.93 E333A,
K334A CH2 NT NT 1.41 R292L, K334E CH2 NT NT 1.41; 1.64 R292L,
P396L, T359N CH2, CH3 NT NT 0.89; 1.15 V379L CH3 NT NT 0.83 K288N
CH2 NT NT 0.78 A330S CH2 NT NT 0.52 F243L CH2 NT NT 0.38 E318K CH2
NT NT 0.86 K288N, A330S CH2 NT NT 0.08 R255L, E318K CH2 NT NT 0.82
F243L, E318K CH2 NT NT 0.07 Mutants in 4-4-20 mini-library
Increased Fc.gamma.RIIIA binding, decreased or no change to
Fc.gamma.RIIB binding N/C means no change; N/B means no binding; NT
means not tested V379M CH3 .uparw.2x N/C 1.47 S219Y Hinge .uparw.1x
.dwnarw. or N/B 1.28 V282M CH2 .uparw.1x .dwnarw. or N/B 1.25; 1
F275I, K334N, V348M CH2 .uparw.0.5x N/C D401V CH3 .uparw. 0.5x N/C
V279L, P395S CH2 .uparw.1x N/C K222N Hinge .uparw.1x .dwnarw.or N/B
1.33; 0.63 K246T, Y319F CH2 .uparw.1x N/C F243I, V379L CH2, CH3
.uparw.1.5x .dwnarw. or N/B 1.86; 1.35 F243L, R255L, E318K CH2
.uparw.1x .dwnarw. or N/B 1.81; 1.45 K334I CH2 .uparw.1x N/C 2.1;
1.97 K334E, T359N, T366S CH2, CH3 .uparw.1.5x N/C 1.49; 1.45 K288M,
K334E CH2 .uparw. 3x .dwnarw. or N/B 1.61; 1.69 K334E, E380D CH2,
CH3 .uparw.1.5x N/C T256S, V305I, K334E, N390S CH2, CH3 .uparw.1.5x
N/C K334E CH2 .uparw.2.5x N/C 1.75; 2.18 T335N, K370E, A378V,
T394M, S424L CH2, CH3 .uparw.0.5x N/C E233D, K334E CH2 .uparw.1.5x
N/C 0.94; 1.02 K334E, T359N, T366S, Q386R CH2 .uparw.1x N/C
Increased Binding to Fc.gamma.IIIA and Fc.gamma.RIIB K246T, P396H
CH2, CH3 .uparw.1x .uparw. 2.5x H268D, E318D CH2 .uparw.1.5x
.uparw. 5x K288N, A330S, P396L CH2, CH3 .uparw. 5x .uparw. 3x 2.34;
1.66; 2.54 I377F CH3 .uparw.1.5x .uparw.0.5x P244H, L358M, V379M,
N384K, V397M CH2, CH3 .uparw.1.75x .uparw.1.5x P217S, A378V, S408R
Hinge, CH3 .uparw. 2x .uparw.4.5x P247L, I253N, K334N CH2 .uparw.
3x .uparw. 2.5x P247L CH2 .uparw.0.5x .uparw. 4x 0.91; 0.84 F372Y
CH3 .uparw.0.75x .uparw.5.5x 0.88; 0.59 K326E CH2 .uparw. 2x
.uparw. 3.5x 1.63; 2 K246I, K334N CH2 .uparw.0.5x .uparw. 4x 0.66;
0.6 K320E, K326E CH2 .uparw.1x .uparw.1x H224L Hinge .uparw.0.5x
.uparw. 5x 0.55; 0.53 S375C, P396L CH3 .uparw.1.5x .uparw.4.5x
D312E, K327N, I378S CH2, CH3 .uparw.0.5x N/C K288N, K326N CH2
.uparw.1x N/C F275Y CH2 .uparw. 3x N/C 0.64 P247L, N421K CH2, CH3
.uparw. 3x N/C 2.0 S298N, W381R CH2, CH3 .uparw. 2x N/C D280E,
S354F, A431D, L441I CH2, CH3 .uparw. 3x N/C 0.62 R255Q, K326E CH2
.uparw. 2x N/C 0.79 K218R, G281D, G385R H, CH2, CH3 .uparw.3.5x N/C
0.67 L398V CH3 .uparw.1.5x N/C P247L, A330T, S440G CH2, CH3
.uparw.0.75x .dwnarw. 0.25x V284A, F372L CH2, CH3 1x N/C T335N,
P387S, H435Q CH2, CH3 1.25x N/C P247L, A431V, S442F CH2, CH3 1x N/C
Increased Binding to Fc.gamma.RIIIA and Fc.gamma.RIIB P343S, P353L,
S375I, S383N CH3 .uparw. 0.5x .uparw. 6x T394M, V397M CH3
.uparw.0.5x .uparw. 3x E216D, E345K, S375I H, CH2, CH3 .uparw. 0.5x
.uparw. 4x K334N. CH2 .uparw.0.5x .uparw. 2x K288N, A330S, P396L
CH2, CH3 .uparw.0.5x .uparw. 9x P247L, E389G CH2, CH3 .uparw.1.5x
.uparw. 9x K222N, T335N, K370E, A378V, T394M H, CH2, CH3 .uparw.1x
.uparw. 7x G316D, A378V, D399E CH2, CH3 .uparw.1.5x .uparw.14x 2.24
N315I, V379M, T394M CH2, CH3 .uparw.1x .uparw. 9x 1.37 K290T,
G371D, CH2, CH3 .uparw. 0.25x .uparw. 6x P247L, L398Q CH2, CH3
.uparw.1.25x .uparw.10x K326Q, K334E, T359N, T366S CH2, CH3
.uparw.1.5x .uparw. 5x S400P CH3 .uparw.1x .uparw. 6x P247L, I377F
CH2, CH3 .uparw.1x .uparw. 5x A378V, N390I, V422I CH3 .uparw. 0.5x
.uparw. 5x K326E, G385E CH2, CH3 .uparw.0.5x .uparw.15x V282E,
V369I, L406F CH2, CH3 .uparw. 0.5x .uparw. 7x V397M, T411A, S415N
CH3 .uparw. 0.25x .uparw.5x T223I, T256S, L406F H, CH2, CH3 .uparw.
0.25x .uparw. 6x S298N, S407R CH2, CH3 .uparw.0.5x .uparw. 7x
K246R, S298N, I377F CH2, CH3 .uparw.1x .uparw. 5x S407I CH3 .uparw.
0.5x .uparw.4x F372Y CH3 .uparw.0.5x .uparw.4x L235P, V382M, S304G,
V305I, V323I CH2, CH3 .uparw. 2x .uparw. 2x P247L, W313R, E388G
CH2, CH3 .uparw.1.5x .uparw.1x D221Y, M252I, A330G, A339T, T359N,
V422I, H433L H, CH2, CH3 .uparw.2.5x .uparw. 6x E258D, N384K CH2,
CH3 .uparw.1.25x .uparw.4x F241L, E258G CH2 .uparw. 2x .uparw. 2.5x
-0.08 K370N, S440N CH3 .uparw.1x .uparw. 3.5x K317N, F423-deleted
CH2, CH3 .uparw. 2.5x .uparw. 7x 0.18 F243I, V379L, G420V CH2, CH3
.uparw. 2.5x .uparw.3.5x 1.35 P227S, K290E H, CH2 .uparw.1x .uparw.
0.5x A231V, Q386H, V412M CH2, CH3 .uparw.1.5x .uparw. 6x T215P,
K274N, A287G, K334N, L365V, P396L H, CH2, CH3 .uparw.2x .uparw. 4x
Increased Binding to Fc.gamma.RIIB but not Fc.gamma.RIIIA K334E,
E380D CH2, CH3 N/C .uparw.4.5x T366N CH3 N/C .uparw. 5x P244A,
K326I, C367R, S375I, K447T CH2, CH3 N/C .uparw. 3x C229Y, A287T,
V379M, P396L, L443V H, CH2, CH3 .dwnarw. 0.25x .uparw.10x Decreased
binding to Fc.gamma.RIIIA and Fc.gamma.RIIB R301H, K340E, D399E
CH2, CH3 .dwnarw. 0.50x .dwnarw. 0.25x K414N CH3 .dwnarw. 0.25x N/B
P291S, P353Q CH2, CH3 .dwnarw. 0.50x .dwnarw. 0.25x V240I, V281M
CH2 .dwnarw. 0.25x .dwnarw. 0.25x P232S, S304G CH2 N/B N/B E269K,
K290N, Q311R, H433Y CH2, CH3 N/B N/B M352L CH3 N/B N/B E216D,
K334R, S375I H, CH2, CH3 N/B N/B P247L, L406F CH2, CH3 N/B N/B
T335N, P387S, H435Q CH2, CH3 N/B N/B T225S CH2 .dwnarw. 0.25x
.dwnarw. 0.50x D399E, M428L CH3 .dwnarw. 0.50x .dwnarw. 0.50x
K246I, Q362H, K370E CH2, CH3 N/B .dwnarw. 0.50x K334E, E380D, G446V
CH2, CH3 N/B N/B I377N CH3 .dwnarw. 0.50x N/B V303I, V369F, M428L
CH2, CH3 N/B N/B L251F, F372L CH2, CH3 N/B N/B K246E, V284M, V308A
CH2, CH3 N/B N/B D399E, G402D CH3 N/B N/B D399E, M428L CH3 N/B N/B
Fc.gamma.RIIB depletion/Fc.gamma.RIIIA selection: Naive Fc library.
E293V, Q295E, A327T CH2 .uparw.0.4x .dwnarw. or N/B 4.29 Y319F,
P352L, P396L CH2, CH3 .uparw.3.4x .uparw.2x 1.09 K392T, P396L CH3
.uparw. 4.5x .uparw. 2.5x 3.07 K248M CH2 .uparw.0.4x .dwnarw. or
N/B 4.03 H268N, P396L CH2, CH3 .uparw. 2.2x .uparw. 4.5x 2.24
Solution competition 40X Fc.gamma.RIIB-G2: P396L Library D221E,
D270E, V308A, Q311H, P396L, G402D .uparw.3.6x .uparw.0.1x 3.17
Equilibrium Screen: 0.8 .mu.M Fc.gamma.RIIIA monomer: P396L library
K290T, N390I, P396L CH2, CH3 .quadrature..uparw.2.8x .uparw. 6.1x
1.93 K326I, P396L CH2, CH3 .quadrature..uparw.2.9x .uparw. 5.9x
1.16 H268D, P396L CH2, CH3 .uparw.3.8x .uparw.13.7x 2.15 K210M,
P396L CH1, CH3 .uparw.1.9x .uparw. 4.6x 2.02 L358P, P396L CH3
.uparw.1.9x .uparw. 4.2x 1.58 K288R, T307A, K344E, P396L CH2, CH3
.uparw. 4.1x .uparw. 2.3x 3.3 V273I, K326E, L328I, P396L CH2, CH3
.uparw.1.3x .uparw.10.8x 0.78 K326I, S408N, P396L CH2, CH3
.uparw.4x .uparw. 9.3x 1.65 K334N, P396L CH2, CH3 .uparw.3.1x
.uparw. 3x 2.43 V379M, P396L CH3 .uparw.1.9x .uparw.5.6x 2.01
P227S, P396L CH2, CH3 .uparw.1.5x .uparw. 4x 2.01 P217S, P396L H,
CH3 .uparw.1.6x .uparw.4.5x 2.04 K261N, K210M, P396L CH2, CH3
.uparw. 2x .uparw. 4.2x 2.06 Kinetic Screen: O.8 .mu.M, 1' with
cold 8 .mu.M Fc.gamma.RIIIA: P396L Library term is M, P396L CH3
.uparw.1.9x .uparw. 7.2x 3.09 Q419H, P396L CH3 .uparw. 2x .uparw.
6.9x 2.24 K370E, P396L CH3 .uparw.2x .uparw.6.6x 2.47 L242F, P396L
CH2, CH3 .uparw. 2.5x .uparw. 4.1x 2.4 F243L, V305I, A378D, F404S,
P396L CH2, CH3 .uparw.1.6x .uparw.5.4x 3.59 R255L, P396L CH2, CH3
.uparw.1.8x .uparw. 6x 2.79 V240A, P396L CH2, CH3 .uparw.1.3x
.uparw. 4.2x 2.35 T250S, P396L CH2, CH3 .uparw.1.5x .uparw.6.8x
1.60 P247S, P396L CH2, CH3 .uparw.1.2x .uparw. 4.2x 2.10 K290E,
V369A, T393A, P396L CH2, CH3 .uparw.1.3x .uparw. 6.7x 1.55 K210N,
K222I, K320M, P396L H, CH2, CH3 .uparw. 2.7x .uparw. 8.7x 1.88
L410H, P396L CH3 .uparw.1.7x .uparw. 4.5x 2.00 Q419L, P396L CH3
.uparw. 2.2x .uparw. 6.1x 1.70 V427A, P396L CH3 .uparw.1.9x
.uparw.4.7x 1.67 P217S, V305I, I309L, N390H, P396L H, CH2, CH3
.uparw.2x .uparw. 7x 1.54 E258D, P396L CH2, CH3 .uparw.1.9x .uparw.
4.9x 1.54 N384K, P396L CH3 .uparw. 2.2x .uparw.5.2x 1.49 V323I,
P396L CH2, CH3 .uparw.1.1x .uparw. 8.2x 1.29 K246N, Q419R, P396L
CH2, CH3 .uparw.1.1x .uparw. 4.8x 1.10 P217A, T359A, P396L H, CH2,
CH3 .uparw.1.5x .uparw. 4.8x 1.17 P244H, P396L CH2, CH3 .uparw.2.5x
.uparw. 4x 1.40 V215I, K290V, P396L H, CH2, CH3 .uparw.2.2x .uparw.
4.6x 1.74 F275L, Q362H, N384K, P396L CH2, CH3 .uparw. 2.2x .uparw.
3.7x 1.51 V305L, P396L CH2, CH3 .uparw.1.3x .uparw. 5.5x 1.50
S400F, P396L CH3 .uparw.1.5x .uparw.4.7x 1.19 V303I, P396L CH3
.uparw.1.1x .uparw. 4x 1.01 Fc.gamma.RIIB depletion Fc.gamma.RIIIA
158V solid phase selection: Naive Library A330V, H433Q, V427M CH2,
CH3 NT NT NT V263Q, E272D, Q419H CH2, CH3 NT NT NT N276Y, T393N,
W417R CH2, CH3 NT NT NT V282L, A330V, H433Y, T436R CH2, CH3 NT NT
NT A330V, Q419H CH2, CH3 NT NT NT V284M, S298N, K334E, R355W CH2,
CH3 NT NT NT A330V, G427M, K438R CH2, CH3 NT NT NT S219T, T225K,
D270E, K360R CH2, CH3 NT NT NT K222E, V263Q, S298N CH2 NT NT NT
V263Q, E272D CH2 NT NT NT R292G CH2 NT NT NT S298N CH2 NT NT NT
E233G, P247S, L306P CH2 NT NT NT D270E CH2 NT NT NT S219T, T225K,
D270E CH2 NT NT NT K326E, A330T CH2 NT NT NT E233G CH2 NT NT NT
S254T, A330V, N361D, P243L CH2, CH3 NT NT NT Fc.gamma.RIIB
depletion Fc.gamma.RIIIA 158F solid phase selection: Naive Library
158F by FACS top 0.2% V284M, S298N, K334E, R355W R416T CH2, CH3 NT
NT Fc.gamma.RIIB depletion FcgRIIA 131H solid phase selection:
Naive Library R292P, V305I CH2, CH2 NT NT D270E, G316D, R416G CH2,
CH3 NT NT V284M, R292L, K370N CH2, CH3 NT NT R292P, V305I, F243L
CH2 NT NT
[0282] In certain embodiments, the invention provides modified
molecules with variant Fc regions, having one or more amino acid
modifications, which one or more amino acid modifications confer an
effector function and/or increase the affinity of the molecule for
Fc.gamma.RIIIA and/or Fc.gamma.RIIA. Such molecules include IgG
molecules that naturally contain Fc.gamma.R binding regions (e.g.,
Fc.gamma.RIIIA and/or Fc.gamma.RIIB binding region), or
immunoglobulin derivatives that have been engineered to contain an
Fc.gamma.R binding region (e.g., Fc.gamma.RIIIA and/or
Fc.gamma.RIIB binding region). The modified molecules of the
invention include any immunoglobulin molecule that binds,
preferably, immunospecifically, i.e., competes off non-specific
binding as determined by immunoassays well known in the art for
assaying specific antigen-antibody binding, an antigen and contains
an Fc.gamma.R binding region (e.g., a Fc.gamma.RIIIA and/or
Fc.gamma.RIIB binding region). Such antibodies include, but are not
limited to, polyclonal, monoclonal, bi-specific, multi-specific,
human, humanized, chimeric antibodies, single chain antibodies, Fab
fragments, F(ab').sub.2 fragments, disulfide-linked Fvs, Fc
fusions, and fragments containing either a VL or VH domain or even
a complementary determining region (CDR) that specifically binds an
antigen, in certain cases, engineered to contain or fused to an
Fc.gamma.R binding region.
[0283] In some embodiments, the molecules of the invention comprise
portions of an Fc region. As used herein the term "portion of an Fc
region" refers to fragments of the Fc region, preferably a portion
with effector activity and/or Fc.gamma.R binding activity (or a
comparable region of a mutant lacking such activity). The fragment
of an Fc region may range in size from 5 amino acids to the entire
Fc region minus one amino acids. The portion of an Fc region may be
missing up to 10, up to 20, up to 30 amino acids from the
N-terminus or C-terminus.
[0284] The IgG molecules of the invention are preferably IgG1
subclass of IgGs, but may also be any other IgG subclasses of given
animals. For example, in humans, the IgG class includes IgG1, IgG2,
IgG3, and IgG4; and mouse IgG includes IgG1, IgG2a, IgG2b, IgG2c
and IgG3.
[0285] The immunoglobulins (and other polypeptides used herein) may
be from any animal origin including birds and mammals. Preferably,
the antibodies are human, rodent (e.g., mouse and rat), donkey,
sheep, rabbit, goat, guinea pig, camel, horse, or chicken. As used
herein, "human" antibodies include antibodies having the amino acid
sequence of a human immunoglobulin and include antibodies isolated
from human immunoglobulin libraries or from animals transgenic for
one or more human immunoglobulin and that do not express endogenous
immunoglobulins, as described infra and, for example, in U.S. Pat.
No. 5,939,598 by Kucherlapati et al.
[0286] The antibodies of the present invention may be monospecific,
bispecific, trispecific or of greater multispecificity.
Multispecific antibodies may be specific for different epitopes of
a polypeptide or may be specific for heterologous epitopes, such as
a heterologous polypeptide or solid support material. See, e.g.,
PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO
92/05793; Tutt, et al., J. Immunol., 147:60-69, 1991; U.S. Pat.
Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819;
Kostelny et al., J. Immunol., 148:1547-1553, 1992.
[0287] Multispecific antibodies have binding specificities for at
least two different antigens. While such molecules normally will
only bind two antigens (i.e. bispecific antibodies, BsAbs),
antibodies with additional specificities such as trispecific
antibodies are encompassed by the instant invention. Examples of
BsAbs include without limitation those with one arm directed
against a tumor cell antigen and the other arm directed against a
cytotoxic molecule.
[0288] Methods for making bispecific antibodies are known in the
art. Traditional production of full length bispecific antibodies is
based on the coexpression of two immunoglobulin heavy chain-light
chain pairs, where the two chains have different specificities
(Millstein et al., Nature, 305:537-539 (1983); which is
incorporated herein by reference in its entirety). Because of the
random assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a potential mixture of 10 different
antibody molecules, of which only one has the correct bispecific
structure. Purification of the correct molecule, which is usually
done by affinity chromatography steps, is rather cumbersome, and
the product yields are low. Similar procedures are disclosed in WO
93/08829, and in Traunecker et al., EMBO J., 10:3655-3659
(1991).
[0289] According to a different approach, antibody variable domains
with the desired binding specificities (antibody-antigen combining
sites) are fused to immunoglobulin constant domain sequences. The
fusion preferably is with an immunoglobulin heavy chain constant
domain, comprising at least part of the hinge, CH2, and CH3
regions. It is preferred to have the first heavy-chain constant
region (CH1) containing the site necessary for light chain binding,
present in at least one of the fusions. DNAs encoding the
immunoglobulin heavy chain fusions and, if desired, the
immunoglobulin light chain, are inserted into separate expression
vectors, and are co-transfected into a suitable host organism. This
provides for great flexibility in adjusting the mutual proportions
of the three polypeptide fragments in embodiments when unequal
ratios of the three polypeptide chains used in the construction
provide the optimum yields. It is, however, possible to insert the
coding sequences for two or all three polypeptide chains in one
expression vector when, the expression of at least two polypeptide
chains in equal ratios results in high yields or when the ratios
are of no particular significance.
[0290] In a preferred embodiment of this approach, the bispecific
antibodies are composed of a hybrid immunoglobulin heavy chain with
a first binding specificity in one arm, and a hybrid immunoglobulin
heavy chain-light chain pair (providing a second binding
specificity) in the other arm. It was found that this asymmetric
structure facilitates the separation of the desired bispecific
compound from unwanted immunoglobulin chain combinations, as the
presence of an immunoglobulin light chain in only one half of the
bispecific molecule provides for a facile way of separation. This
approach is disclosed in WO 94/04690. For further details of
generating bispecific antibodies see, for example, Suresh et al.,
Methods in Enzymology, 121:210 (1986). According to another
approach described in WO96/27011, a pair of antibody molecules can
be engineered to maximize the percentage of heterodimers which are
recovered from recombinant cell culture. The preferred interface
comprises at least a part of the CH3 domain of an antibody constant
domain. In this method, one or more small amino acid side chains
from the interface of the first antibody molecule are replaced with
larger side chains (e.g. tyrosine or tryptophan). Compensatory
"cavities" of identical or similar size to the large side chain(s)
are created on the interface of the second antibody molecule by
replacing large amino acid side chains with smaller ones (e.g.
alanine or threonine). This provides a mechanism for increasing the
yield of the heterodimer over other unwanted end-products such as
homodimers.
[0291] Bispecific antibodies include cross-linked or
"heteroconjugate" antibodies. For example, one of the antibodies in
the heteroconjugate can be coupled to avidin, the other to biotin.
Such antibodies have, for example, been proposed to target immune
system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for
treatment of HIV infection (WO 91/00360, WO 92/200373, and EP
03089). Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents are well known
in the art, and are disclosed in U.S. Pat. No. 4,676,980, along
with a number of cross-linking techniques.
[0292] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. See, e.g.,
Tutt et al., 1991, J. Immunol. 147: 60, which is incorporated
herein by reference.
[0293] The antibodies of the invention include derivatives that are
otherwise modified, i.e., by the covalent attachment of any type of
molecule to the antibody such that covalent attachment does not
prevent the antibody from binding antigen and/or generating an
anti-idiotypic response. For example, but not by way of limitation,
the antibody derivatives include antibodies that have been
modified, e.g., by glycosylation, acetylation, pegylation,
phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, linkage to a
cellular ligand or other protein, etc. Any of numerous chemical
modifications may be carried out by known techniques, including,
but not limited to, specific chemical cleavage, acetylation,
formylation, metabolic synthesis of tunicamycin, etc. Additionally,
the derivative may contain one or more non-classical amino
acids.
[0294] For some uses, including in vivo use of antibodies in humans
and in vitro detection assays, it may be preferable to use
chimeric, humanized, or human antibodies. A chimeric antibody is a
molecule in which different portions of the antibody are derived
from different animal species, such as antibodies having a variable
region derived from a murine monoclonal antibody and a constant
region derived from a human immunoglobulin. Methods for producing
chimeric antibodies are known in the art. See e.g., Morrison,
Science, 229:1202, 1985; Oi et al., BioTechniques, 4:214 1986;
Gillies et al., J. Immunol. Methods, 125:191-202, 1989; U.S. Pat.
Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated
herein by reference in their entireties. Humanized antibodies are
antibody molecules from non-human species that bind the desired
antigen having one or more complementarity determining regions
(CDRs) from the non-human species and framework regions and
constant domains from a human immunoglobulin molecule. Often,
framework residues in the human framework regions will be
substituted with the corresponding residue from the CDR donor
antibody to alter, preferably improve, antigen binding. These
framework substitutions are identified by methods well known in the
art, e.g., by modeling of the interactions of the CDR and framework
residues to identify framework residues important for antigen
binding and sequence comparison to identify unusual framework
residues at particular positions. See, e.g., Queen et al., U.S.
Pat. No. 5,585,089; Riechmann et al., Nature, 332:323, 1988, which
are incorporated herein by reference in their entireties.
Antibodies can be humanized using a variety of techniques known in
the art including, for example, CDR-grafting (EP 239,400; PCT
publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101 and
5,585,089), veneering or resurfacing (EP 592,106; EP 519,596;
Padlan, Molecular Immunology, 28(4/5):489-498, 1991; Studnicka et
al., Protein Engineering, 7(6):805-814, 1994; Roguska et al., Proc
Natl. Acad. Sci. USA, 91:969-973, 1994), and chain shuffling (U.S.
Pat. No. 5,565,332), all of which are hereby incorporated by
reference in their entireties. Humanized antibodies may be
generated using any of the methods disclosed in U.S. Pat. Nos.
5,693,762 (Protein Design Labs), 5,693,761, (Protein Design Labs)
5,585,089 (Protein Design Labs), 6,180,370 (Protein Design Labs),
and U.S. Publication Nos. 20040049014, 200300229208, each of which
is incorporated herein by reference in its entirety.
[0295] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. Human antibodies can be
made by a variety of methods known in the art including phage
display methods described above using antibody libraries derived
from human immunoglobulin sequences. See U.S. Pat. Nos. 4,444,887
and 4,716,111; and PCT publications WO 98/46645; WO 98/50433; WO
98/24893; WO 98/16654; WO 96/34096; WO 96/33735; and WO 91/10741,
each of which is incorporated herein by reference in its
entirety.
[0296] Human antibodies can also be produced using transgenic mice
which are incapable of expressing functional endogenous
immunoglobulins, but which can express human immunoglobulin genes.
For an overview of this technology for producing human antibodies,
see Lonberg and Huszar, Int. Rev. Immunol., 13:65-93, 1995. For a
detailed discussion of this technology for producing human
antibodies and human monoclonal antibodies and protocols for
producing such antibodies, see, e.g., PCT publications WO 98/24893;
WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598
877; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825;
5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and
5,939,598, which are incorporated by reference herein in their
entireties. In addition, companies such as Abgenix, Inc. (Freemont,
Calif.), Medarex (NJ) and Genpharm (San Jose, Calif.) can be
engaged to provide human antibodies directed against a selected
antigen using technology similar to that described above.
[0297] Completely human antibodies which recognize a selected
epitope can be generated using a technique referred to as "guided
selection." In this approach a selected non-human monoclonal
antibody, e.g., a mouse antibody, is used to guide the selection of
a completely human antibody recognizing the same epitope (Jespers
et al., Bio/technology, 12:899-903, 1988).
[0298] The invention encompasses engineering human or humanized
therapeutic antibodies (e.g., tumor specific monoclonal antibodies)
in the Fc region, by modification (e.g., substitution, insertion,
deletion) of at least one amino acid residue, which modification
confers an effector function activity, e.g., enhanced ADCC
activity, phagocytosis activity, etc., as determined by standard
assays known to those skilled in the art. The engineered
therapeutic antibodies may further have increased affinity of the
Fc region for Fc.gamma.RIIIA and/or Fc.gamma.RIIA. In other
embodiments, the engineered therapeutic antibodies may exhibit
oligomerization activity mediated by the variant Fc region. In
another embodiment, the invention relates to engineering human or
humanized therapeutic antibodies (e.g., tumor specific monoclonal
antibodies) in the Fc region, by modification of at least one amino
acid residue, which modification increases the affinity of the Fc
region for Fc.gamma.RIIIA and/or Fc.gamma.RIIA and further
decreases the affinity of the Fc region for Fc.gamma.RIIB.
[0299] In another specific embodiment, the invention encompasses
engineering an monoclonal antibody by modification (e.g.,
substitution, insertion, deletion) of at least one amino acid
residue in the Fc region which modification confers an effector
function as determined by standard assays known in the art and
disclosed and exemplified herein. In another embodiment,
modification of the monoclonal antibody increases the affinity of
the Fc region for Fc.gamma.RIIIA and/or Fc.gamma.RIIA. In another
specific embodiment, modification of the monoclonal antibody may
also further decrease the affinity of the Fc region for
Fc.gamma.RIIB.
[0300] In a specific embodiment, the invention encompasses a
modified molecule comprising an Fc chain with a substitution at
position 255 with leucine, at position 396 with leucine, at
position 270 with glutamic acid, and at position 300 with leucine;
or a substitution at position 419 with histidine, at position 396
with leucine, and at position 270 with glutamic acid; or a
substitution at position 240 with alanine, at position 396 with
leucine, and at position 270 with glutamic acid; or a substitution
at position 370 with glutamic acid, at position 396 with leucine,
and at position 270 with glutamic acid; or a substitution at
position 392 with threonine, at position 396 with leucine, and at
position 270 with glutamic acid; or a substitution at position 370
with glutamic acid and at position 396 with leucine; or a
substitution at position 419 with histidine and at position 396
with leucine; or a substitution at position 247 with leucine, at
position 421 with lysine, and at position 270 with glutamic acid;
or a substitution at position 255 with leucine, at position 396
with leucine, at position 270 with glutamic acid, and at position
292 with glycine. In other specific embodiments, the variant Fc
region has a leucine at position 247, a lysine at position 421 and
a glutamic acid at position 270 (MgFc31/60); a threonine at
position 392, a leucine at position 396, a glutamic acid at
position 270, and a leucine at position 243 (MgFc38/60/F243L); a
histidine at position 419, a leucine at position 396, and a
glutamic acid at position 270 (MGFc51/60); a histidine at position
419, a leucine at position 396, a glutamic acid at position 270,
and a leucine at position 243 (MGFc51/60/F243L); an alanine at
position 240, a leucine at position 396, and a glutamic acid at
position 270 (MGFc52/60); a lysine at position 255 and a leucine at
position 396 (MgFc55); a lysine at position 255, a leucine at
position 396, and a glutamic acid at position 270 (MGFc55/60); a
lysine at position 255, a leucine at position 396, a glutamic acid
at position 270, and a lysine at position 300 (MGFc55/60/Y300L); a
lysine at position 255, a leucine at position 396, a glutamic acid
at position 270, and a glycine at position 292 (MGFc55/60/R292G); a
lysine at position 255, a leucine at position 396, a glutamic acid
at position 270, and a leucine at position 243 (MgFc55/60/F243L); a
glutamic acid at position 370, a leucine at position 396, and a
glutamic acid at position 270 (MGFc59/60); a glutamic acid at
position 270, an aspartic acid at position 316, and a glycine at
position 416 (MgFc71); a leucine at position 243, a proline at
position 292, an isoleucine at position 305, and a leucine at
position 396 (MGFc74/P396L); or a leucine at position 243, a
glutamic acid at position 270, an asparagine at position 392 and a
leucine at position 396; or a leucine at position 243, a leucine at
position 255, a glutamic acid at position 270 and a leucine at
position 396; or a glutamine at position 297.
[0301] 5.1.1 Polypeptide and Antibody Conjugates
[0302] Molecules of the invention comprising variant Fc regions may
be recombinantly fused or chemically conjugated (including both
covalently and non-covalently conjugations) to heterologous
polypeptides (i.e., an unrelated polypeptide; or portion thereof,
preferably at least 10, at least 20, at least 30, at least 40, at
least 50, at least 60, at least 70, at least 80, at least 90 or at
least 100 amino acids of the polypeptide) to generate fusion
proteins. The fusion does not necessarily need to be direct, but
may occur through linker sequences.
[0303] Further, molecules of the invention comprising variant Fc
regions may be conjugated to a therapeutic agent or a drug moiety
that modifies a given biological response. Therapeutic agents or
drug moieties are not to be construed as limited to classical
chemical therapeutic agents. For example, the drug moiety may be a
protein or polypeptide possessing a desired biological activity.
Such proteins may include, for example, a toxin such as abrin,
ricin A, pseudomonas exotoxin (i.e., PE-40), or diphtheria toxin,
ricin, gelonin, and pokeweed antiviral protein, a protein such as
tumor necrosis factor, interferons including, but not limited to,
.alpha.-interferon (IFN-.alpha.), .beta.-interferon (IFN-.beta.),
nerve growth factor (NGF), platelet derived growth factor (PDGF),
tissue plasminogen activator (TPA), an apoptotic agent (e.g.,
TNF-.alpha., TNF-.beta., AIM I as disclosed in PCT Publication No.
WO 97/33899), AIM II (see, PCT Publication No. WO 97/34911), Fas
Ligand (Takahashi et al., J. Immunol., 6:1567-1574, 1994), and VEGI
(PCT Publication No. WO 99/23105), a thrombotic agent or an
anti-angiogenic agent (e.g., angiostatin or endostatin), or a
biological response modifier such as, for example, a lymphokine
(e.g., interleukin-1 ("IL-1"), interleukin-2 ("IL-2"),
interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating
factor ("GM-CSF"), and granulocyte colony stimulating factor
("G-CSF"), macrophage colony stimulating factor, ("M-CSF"), or a
growth factor (e.g., growth hormone ("GH"); proteases, or
ribonucleases.
[0304] Molecules of the invention can be fused to marker sequences,
such as a peptide to facilitate purification. In preferred
embodiments, the marker amino acid sequence is a hexa-histidine
peptide, such as the tag provided in a pQE vector (QIAGEN, Inc.,
9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of
which are commercially available. As described in Gentz et al.,
1989, Proc. Natl. Acad. Sci. USA, 86:821-824, for instance,
hexa-histidine provides for convenient purification of the fusion
protein. Other peptide tags useful for purification include, but
are not limited to, the hemagglutinin "HA" tag, which corresponds
to an epitope derived from the influenza hemagglutinin protein
(Wilson et al., Cell, 37:767 1984) and the "flag" tag (Knappik et
al., Biotechniques, 17(4):754-761, 1994).
[0305] Additional fusion proteins may be generated through the
techniques of gene-shuffling, motif-shuffling, exon-shuffling,
and/or codon-shuffling (collectively referred to as "DNA
shuffling"). DNA shuffling may be employed to alter the activities
of molecules of the invention (e.g., antibodies with higher
affinities and lower dissociation rates). See, generally, U.S. Pat.
Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and
Patten et al., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama,
1998, Trends Biotechnol. 16:76; Hansson, et al., 1999, J. Mol.
Biol. 287:265; and Lorenzo and Blasco, 1998, BioTechniques 24:308
(each of these patents and publications are hereby incorporated by
reference in its entirety). Molecules of the invention comprising
variant Fc regions, or the nucleic acids encoding the molecules of
the invention, may be further altered by being subjected to random
mutagenesis by error-prone PCR, random nucleotide insertion or
other methods prior to recombination. One or more portions of a
polynucleotide encoding a molecule of the invention, may be
recombined with one or more components, motifs, sections, parts,
domains, fragments, etc. of one or more heterologous molecules.
[0306] The present invention also encompasses molecules of the
invention comprising variant Fc regions conjugated to a diagnostic
or therapeutic agent or any other molecule for which serum
half-life is desired to be increased and/or targeted to a
particular subset of cells. The molecules of the invention can be
used diagnostically to, for example, monitor the development or
progression of a disease, disorder or infection as part of a
clinical testing procedure to, e.g., determine the efficacy of a
given treatment regimen. Detection can be facilitated by coupling
the molecules of the invention to a detectable substance. Examples
of detectable substances include various enzymes, prosthetic
groups, fluorescent materials, luminescent materials,
bioluminescent materials, radioactive materials, positron emitting
metals, and nonradioactive paramagnetic metal ions. The detectable
substance may be coupled or conjugated either directly to the
molecules of the invention or indirectly, through an intermediate
(such as, for example, a linker known in the art) using techniques
known in the art. See, for example, U.S. Pat. No. 4,741,900 for
metal ions which can be conjugated to antibodies for use as
diagnostics according to the present invention. Such diagnosis and
detection can be accomplished by coupling the molecules of the
invention to detectable substances including, but not limited to,
various enzymes, enzymes including, but not limited to, horseradish
peroxidase, alkaline phosphatase, beta-galactosidase, or
acetylcholinesterase; prosthetic group complexes such as, but not
limited to, streptavidin/biotin and avidin/biotin; fluorescent
materials such as, but not limited to, umbelliferone, fluorescein,
fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine
fluorescein, dansyl chloride or phycoerythrin; luminescent material
such as, but not limited to, luminol; bioluminescent materials such
as, but not limited to, luciferase, luciferin, and aequorin;
radioactive material such as, but not limited to, bismuth
(.sup.213Bi), carbon (.sup.14C), chromium (.sup.51Cr), cobalt
(.sup.57Co), fluorine (.sup.18F), gadolinium (.sup.153Gd, 159 Gd),
gallium (.sup.68Ga, .sup.67Ga), germanium (.sup.68Ge), holmium
(.sup.166Ho), indium (.sup.115In, .sup.113In, .sup.112In, 111In),
iodine (.sup.131I, .sup.125I, .sup.123I, .sup.121I), lanthanium
(.sup.140La), lutetium (.sup.177Lu), manganese (.sup.54Mn),
molybdenum (.sup.99Mo), palladium (.sup.103Pd), phosphorous
(.sup.32P), praseodymium (.sup.142Pr), promethium (.sup.149Pm),
rhenium (.sup.186Re, .sup.188Re), rhodium (.sup.105Rh), ruthemium
(.sup.97Ru), samarium (.sup.153Sm), scandium (.sup.47Sc), selenium
(.sup.75Se), strontium (.sup.85Sr), sulfur (.sup.35S), technetium
(.sup.99Tc), thallium (.sup.201Ti), tin (.sup.113Sn, .sup.117Sn),
tritium (.sup.3H), xenon (.sup.133Xe), ytterbium (.sup.169Yb,
.sup.175Yb), yttrium (.sup.90Y), zinc (.sup.65Zn); positron
emitting metals using various positron emission tomographies, and
nonradioactive paramagnetic metal ions.
[0307] Molecules of the invention comprising a variant Fc region
may be conjugated to a therapeutic moiety such as a cytotoxin
(e.g., a cytostatic or cytocidal agent), a therapeutic agent or a
radioactive element (e.g., alpha-emitters, gamma-emitters, etc.).
Cytotoxins or cytotoxic agents include any agent that is
detrimental to cells. Examples include paclitaxol, cytochalasin B,
gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,
tenoposide, vincristine, vinblastine, colchicin, doxorubicin,
daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,
actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,
tetracaine, lidocaine, propranolol, and puromycin and analogs or
homologs thereof. Therapeutic agents include, but are not limited
to, antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and anthramycin (AMC), and anti-mitotic agents (e.g.,
vincristine and vinblastine).
[0308] Moreover, a molecule of the invention can be conjugated to
therapeutic moieties such as a radioactive materials or macrocyclic
chelators useful for conjugating radiometal ions (see above for
examples of radioactive materials). In certain embodiments, the
macrocyclic chelator is
1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid
(DOTA) which can be attached to the antibody via a linker molecule.
Such linker molecules are commonly known in the art and described
in Denardo et al., 1998, Clin Cancer Res. 4:2483-90; Peterson et
al., 1999, Bioconjug. Chem. 10:553; and Zimmerman et al., 1999,
Nucl. Med. Biol. 26:943-50 each of which is incorporated herein by
reference in their entireties.
[0309] Techniques for conjugating such therapeutic moieties to
antibodies are well known; see, e.g., Arnon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
1985, pp. 243-56, Alan R. Liss, Inc.); Hellstrom et al.,
"Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd
Ed.), Robinson et al. (eds.), 1987, pp. 623-53, Marcel Dekker,
Inc.); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer
Therapy: A Review", in Monoclonal Antibodies '84: Biological And
Clinical Applications, Pinchera et al. (eds.), 1985, pp. 475-506);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
1985, pp. 303-16, Academic Press; and Thorpe et al., Immunol. Rev.,
62:119-58, 1982.
[0310] In one embodiment, where the molecule of the invention is an
antibody comprising a variant Fc region, it can be administered
with or without a therapeutic moiety conjugated to it, administered
alone, or in combination with cytotoxic factor(s) and/or
cytokine(s) for use as a therapeutic treatment. Alternatively, an
antibody of the invention can be conjugated to a second antibody to
form an antibody heteroconjugate as described by Segal in U.S. Pat.
No. 4,676,980, which is incorporated herein by reference in its
entirety. Antibodies of the invention may also be attached to solid
supports, which are particularly useful for immunoassays or
purification of the target antigen. Such solid supports include,
but are not limited to, glass, cellulose, polyacrylamide, nylon,
polystyrene, polyvinyl chloride or polypropylene.
5.2 Screening of Molecules with Variant Fc Regions for Effector
Function, Enhanced Fc.gamma.RIII Binding and Characterization of
Same
[0311] The affinities and binding properties of the molecules of
the invention for an Fc.gamma.R are initially determined using in
vitro assays (biochemical or immunological based assays) known in
the art for determining Fc-Fc.gamma.R interactions, i.e., specific
binding of an Fc region to an Fc.gamma.R including but not limited
to ELISA assay, surface plasmon resonance assay,
immunoprecipitation assays. Preferably, the binding properties of
the molecules of the invention are also characterized by in vitro
functional assays for determining one or more Fc.gamma.R mediator
effector cell functions. In most preferred embodiments, the
antibodies of the invention have similar binding properties in in
vivo models (such as those described and disclosed herein) as those
in in vitro based assays. However, the present invention does not
exclude molecules of the invention that do not exhibit the desired
phenotype in in vitro based assays but do exhibit the desired
phenotype in vivo.
[0312] In preferred embodiments, screening and identifying
molecules comprising variant Fc regions with altered Fc.gamma.R
affinities (e.g., enhanced Fc.gamma.RIIIA affinity) are done using
the yeast display technology as described herein in combination
with one or more biochemical based assays, preferably in a high
throughput manner. In some embodiments, screening and identifying
molecules comprising variant Fc regions with altered Fc.gamma.R
affinities (e.g., enhanced Fc.gamma.RIIIA affinity) are done using
the yeast display technology as described herein in combination
with one or more functional based assays, preferably in a high
throughput manner. The functional based assays can be any assay
known in the art for characterizing one or more Fc.gamma.R mediated
effector cell functions such as those described herein in Section
5.2.7. Non-limiting examples of effector cell functions that can be
used in accordance with the methods of the invention, include but
are not limited to, antibody-dependent cell mediated cytotoxicity
(ADCC), antibody-dependent phagocytosis, phagocytosis,
opsonization, opsonophagocytosis, cell binding, rosetting, C1q
binding, and complement dependent cell mediated cytotoxicity.
[0313] The term "specific binding" of an Fc region to an Fc.gamma.R
refers to an interaction of the Fc region and a particular
Fc.gamma.R which has an affinity constant of at least about 150 nM,
in the case of monomeric Fc.gamma.RIIIA and at least about 60 nM in
the case of dimeric Fc.gamma.RIIB as determined using, for example,
an ELISA or surface plasmon resonance assay (e.g., a BIAcore.TM.).
The affinity constant of an Fc region for monomeric Fc.gamma.RIIIA
may be 150 nM, 200 nM or 300 nM. The affinity constant of an Fc
region for dimeric Fc.gamma.RIIB may be 60 nM, 80 nM, 90 nM, or 100
nM. Dimeric Fc.gamma.RIIB for use in the methods of the invention
may be generated using methods known to one skilled in the art.
Typically, the extracellular region of Fc.gamma.RIIB is covalently
linked to a heterologous polypeptide which is capable of
dimerization, so that the resulting fusion protein is a dimer,
e.g., see, U.S. Application No. 60/439,709 filed on Jan. 13, 2003
(Attorney Docket No. 11183-005-888), which is incorporated herein
by reference in its entirety. A specific interaction generally is
stable under physiological conditions, including, for example,
conditions that occur in a living individual such as a human or
other vertebrate or invertebrate, as well as conditions that occur
in a cell culture such conditions as used for maintaining and
culturing mammalian cells or cells from another vertebrate organism
or an invertebrate organism.
[0314] In a specific embodiment, screening for and identifying
molecules comprising variant Fc regions and altered Fc.gamma.R
affinities comprise: displaying the molecule comprising a variant
Fc region on the yeast surface; and characterizing the binding of
the molecule comprising the variant Fc region to a Fc.gamma.R (one
or more), using a biochemical assay for determining Fc-Fc.gamma.R
interaction, preferably, an ELISA based assay. Once the molecule
comprising a variant Fc region has been characterized for its
interaction with one or more Fc.gamma.Rs and determined to have an
altered affinity for one or more Fc.gamma.Rs, by at least one
biochemical based assay, e.g., an ELISA assay, the molecule maybe
engineered into a complete immunoglobulin, such as a molecule,
using standard recombinant DNA technology methods known in the art,
and the immunoglobulin comprising the variant Fc region expressed
in mammalian cells for further biochemical characterization. The
immunoglobulin into which a variant Fc region of the invention is
introduced (e.g., replacing the Fc region of the immunoglobulin)
can be any immunoglobulin including, but not limited to, polyclonal
antibodies, monoclonal antibodies, bispecific antibodies,
multi-specific antibodies, humanized antibodies, and chimeric
antibodies. In preferred embodiments, a variant Fc region is
introduced into an immunoglobulin specific for a cell surface
receptor, a tumor antigen, or a cancer antigen. The immunoglobulin
into which a variant Fc region of the invention is introduced may
specifically bind a cancer or tumor antigen for example, including,
but not limited to, KS 1/4 pan-carcinoma antigen (Perez and Walker,
1990, J. Immunol. 142: 3662-3667; Bumal, 1988, Hybridoma 7(4):
407-415), ovarian carcinoma antigen (CA125) (Yu et al., 1991,
Cancer Res. 51(2): 468-475), prostatic acid phosphate (Tailor et
al., 1990, Nucl. Acids Res. 18(16): 4928), prostate specific
antigen (Henttu and Vihko, 1989, Biochem. Biophys. Res. Comm.
160(2): 903-910; Israeli et al., 1993, Cancer Res. 53: 227-230),
melanoma-associated antigen p97 (Estin et al., 1989, J. Natl.
Cancer Instil. 81(6): 445-446), melanoma antigen gp75
(Vijayasardahl et al., 1990, J. Exp. Med. 171(4): 1375-1380), high
molecular weight melanoma antigen (HMW-MAA) (Natali et al., 1987,
Cancer 59: 55-63; Mittelman et al., 1990, J. Clin. Invest. 86:
2136-2144), prostate specific membrane antigen, carcinoembryonic
antigen (CEA) (Foon et al., 1994, Proc. Am. Soc. Clin. Oncol. 13:
294), polymorphic epithelial mucin antigen, human milk fat globule
antigen, colorectal tumor-associated antigens such as: CEA, TAG-72
(Yokata et al., 1992, Cancer Res. 52: 3402-3408), C017-1A
(Ragnhammar et al., 1993, Int. J. Cancer 53: 751-758); GICA 19-9
(Herlyn et al., 1982, J. Clin. Immunol. 2: 135), CTA-1 and LEA,
Burkitt's lymphoma antigen-38.13, CD19 (Ghetie et al., 1994, Blood
83: 1329-1336), human B-lymphoma antigen-CD20 (Reff et al., 1994,
Blood 83:435-445), CD33 (Sgouros et al., 1993, J. Nucl. Med.
34:422-430), melanoma specific antigens such as ganglioside GD2
(Saleh et al., 1993, J. Immunol., 151, 3390-3398), ganglioside GD3
(Shitara et al., 1993, Cancer Immunol. Immunother. 36:373-380),
ganglioside GM2 (Livingston et al., 1994, J. Clin. Oncol. 12:
1036-1044), ganglioside GM3 (Hoon et al., 1993, Cancer Res. 53:
5244-5250), tumor-specific transplantation type of cell-surface
antigen (TSTA) such as virally-induced tumor antigens including
T-antigen DNA tumor viruses and Envelope antigens of RNA tumor
viruses, oncofetal antigen-alpha-fetoprotein such as CEA of colon,
bladder tumor oncofetal antigen (Hellstrom et al., 1985, Cancer.
Res. 45:2210-2188), differentiation antigen such as human lung
carcinoma antigen L6, L20 (Hellstrom et al., 1986, Cancer Res. 46:
3917-3923), antigens of fibrosarcoma, human leukemia T cell
antigen-Gp37 (Bhattacharya-Chatterjee et al., 1988, J. of Immun.
141:1398-1403), neoglycoprotein, sphingolipids, breast cancer
antigen such as EGFR (Epidermal growth factor receptor), HER2
antigen (p185.sup.HER2), polymorphic epithelial mucin (PEM)
(Hilkens et al., 1992, Trends in Bio. Chem. Sci. 17:359), malignant
human lymphocyte antigen-APO-1 (Bernhard et al., 1989, Science 245:
301-304), differentiation antigen (Feizi, 1985, Nature 314: 53-57)
such as I antigen found in fetal erythrocytes, primary endoderm I
antigen found in adult erythrocytes, preimplantation embryos, I(Ma)
found in gastric adenocarcinomas, M18, M39 found in breast
epithelium, SSEA-1 found in myeloid cells, VEP8, VEP9, Myl, VIM-D5,
D.sub.156-22 found in colorectal cancer, TRA-1-85 (blood group H),
C14 found in colonic adenocarcinoma, F3 found in lung
adenocarcinoma, AH6 found in gastric cancer, Y hapten, Le.sup.y
found in embryonal carcinoma cells, TL5 (blood group A), EGF
receptor found in A431 cells, E.sub.1 series (blood group B) found
in pancreatic cancer, FC10.2 found in embryonal carcinoma cells,
gastric adenocarcinoma antigen, CO-514 (blood group Le.sup.a) found
in Adenocarcinoma, NS-10 found in adenocarcinomas, CO-43 (blood
group Le.sup.b), G49 found in EGF receptor of A431 cells, MH2
(blood group ALe.sup.b/Le.sup.y) found in colonic adenocarcinoma,
19.9 found in colon cancer, gastric cancer mucins, T.sub.5A.sub.7
found in myeloid cells, R.sub.24 found in melanoma, 4.2, G.sub.D3,
D1.1, OFA-1, G.sub.M2, OFA-2, G.sub.D2, and M1:22:25:8 found in
embryonal carcinoma cells, and SSEA-3 and SSEA-4 found in 4 to
8-cell stage embryos. In one embodiment, the antigen is a T cell
receptor derived peptide from a Cutaneous T cell Lymphoma (see,
Edelson, 1998, The Cancer Journal 4:62).
[0315] In some embodiments, a variant Fc region of the invention is
introduced into an anti-fluoresceine monoclonal antibody, 4-4-20
(Kranz et al., 1982, J. Biol. Chem. 257(12): 6987-6995; which is
incorporated herein by reference in its entirety). In other
embodiments, a variant Fc region of the invention is introduced
into a mouse-human chimeric anti-CD20 monoclonal antibody, 2H7,
which recognizes the CD20 cell surface phosphoprotein on B cells
(Liu et al., 1987, Journal of Immunology, 139: 3521-6; which is
incorporated herein by reference in its entirety). In other
embodiments, the monoclonal antibody is not an anti-CD20 antibody.
In yet other embodiments, a variant Fc region of the invention is
introduced into a humanized antibody (Ab4D5) against the human
epidermal growth factor receptor 2 (p185 HER2) as described by
Carter et al. (1992, Proc. Natl. Acad. Sci. USA 89: 4285-9; which
is incorporated herein by reference in its entirety). In yet other
embodiments, a variant Fc region of the invention is introduced
into a humanized anti-TAG72 antibody (CC49) (Sha et al., 1994
Cancer Biother. 9(4): 341-9; which is incorporated herein by
reference in its entirety). In other embodiments, a variant Fc
region of the invention is introduced into RITUXAN.TM. (humanized
anti-CD20 antibody; rituximab) (International Patent Publication
No. WO 02/096948; which is incorporated herein by reference in its
entirety) which is used for treating lymphomas.
[0316] In another specific embodiment, the invention encompasses
engineering an anti-Fc.gamma.RIIB antibody including but not
limited to any of the antibodies disclosed in U.S. Provisional
Application No. 60/403,266 filed on Aug. 12, 2002; U.S. application
Ser. No. 10/643,857 filed on Aug. 14, 2003 (having Attorney Docket
No. 011183-010-999); the U.S. Provisional Application No.
60/562,804 (having Attorney Docket No. 011183-014-888) that was
filed on Apr. 16, 2004; U.S. Provisional Application No. 60/569,882
(having Attorney Docket No. 011183-013-888) that was filed on May
10, 2004 and U.S. Provisional Application Nos. 60/582,044,
60/582,045, and 60/582,043, having Attorney Docket Nos.
011183-016-888, 011183-017-888, and 011183-018-888, respectively,
each of which was filed on Jun. 21, 2004, by modification (e.g.,
substitution, insertion, deletion) of at least one amino acid
residue which modification increases the affinity of the Fc region
for Fc.gamma.RIIIA and/or Fc.gamma.RIIA. Examples of
anti-Fc.gamma.RIIB antibodies that may be engineered in accordance
with the methods of the invention are 2B6 monoclonal antibody
having ATCC accession number PTA-4591 and 3H7 having ATCC accession
number PTA-4592, 1D5 monoclonal antibody having ATCC accession
number PTA-5958, 1F2 monoclonal antibody having ATCC accession
number PTA-5959, 2D11 monoclonal antibody having ATCC accession
number PTA-5960, 2E1 monoclonal antibody having ATCC accession
number PTA-5961 and 2H9 monoclonal antibody having ATCC accession
number PTA-5962 (all deposited at 10801 University Boulevard,
Manassas, Va. 02209-2011 under the terms of the Budapest Treaty on
the International Recognition of the Deposit of Microorganisms for
the Purposes of Patent Procedure), which are incorporated herein by
reference. In another specific embodiment, modification of the
anti-Fc.gamma.RIIB antibody may also further decrease the affinity
of the Fc region for Fc.gamma.RIIB. In yet another specific
embodiment, the engineered anti-Fc.gamma.RIIB antibody may further
have an enhanced effector function as determined by standard assays
known in the art and disclosed and exemplified herein. In some
embodiments, a variant Fc region of the invention is introduced
into a therapeutic monoclonal antibody specific for a cancer
antigen or cell surface receptor including but not limited to,
ERBITUX.TM. anti-EGFR antibody (also known as IMC-C225) (ImClone
Systems Inc.), a chimerized monoclonal antibody against EGFR;
HERCEPTIN.RTM. anti-HER2 antibody (Trastuzumab) (Genentech, CA)
which is a humanized anti-HER2 monoclonal antibody for the
treatment of patients with metastatic breast cancer; REOPRO.RTM.
anti-glycoprotein IIb/IIIa receptor antibody (abciximab) (Centocor)
which is an anti-glycoprotein IIb/IIIa receptor on the platelets
for the prevention of clot formation; ZENAPAX.RTM. anti-CD25
antibody (daclizumab) (Roche Pharmaceuticals, Switzerland) which is
an immunosuppressive, humanized anti-CD25 monoclonal antibody for
the prevention of acute renal allograft rejection. Other examples
are a humanized anti-CD18 F(ab').sub.2 (Genentech); CDP860 which is
a humanized anti-CD18 F(ab').sub.2 (Celltech, UK); PRO542 which is
an anti-HIV gp120 antibody fused with CD4 (Progenics/Genzyme
Transgenics); C14 which is an anti-CD14 antibody (ICOS Pharm); a
humanized anti-VEGF IgG1 antibody (Genentech); OVAREX.TM. anti-CA
125 antibody which is a murine anti-CA 125 antibody (Altarex);
PANOREX.TM. anti-17-IA antibody which is a murine anti-17-IA cell
surface antigen IgG2a antibody (Glaxo Wellcome/Centocor); IMC-C225
which is a chimeric anti-EGFR IgG antibody (ImClone System);
VITAXIN.TM. anti-.alpha.V.beta.3 integrin antibody which is a
humanized anti-.alpha.V.beta.3 integrin antibody (Applied Molecular
Evolution/MedImmune); CAMPATH.RTM. anti CD52 antibody 1H/LDP-03
which is a humanized anti CD52 IgG1 antibody (Leukosite); SMART
195.TM. anti-CD33 antibody which is a humanized anti-CD33 IgG
antibody (Protein Design Lab/Kanebo); RITUXAN.TM. (rituximab)
anti-CD20 antibody which is a chimeric anti-CD20 IgG1 antibody
(IDEC Pharm/Genentech, Roche/Zettyaku); LYMPHOCIDE.TM. anti-CD22
antibody which is a humanized anti-CD22 IgG antibody
(Immunomedics); SMART ID10.TM. anti-HLA antibody which is a
humanized anti-HLA antibody (Protein Design Lab); ONCOLYM.TM.
(Lym-1) anti-HLA DR antibody is a radiolabelled murine anti-HLA DR
antibody (Techniclone); anti-CD11a is a humanized IgG1 antibody
(Genetech/Xoma); ICM3.TM. anti-ICAM3 is a humanized anti-ICAM3
antibody (ICOS Pharm); IDEC-114.TM. anti-CD80 antibody is a
primatized anti-CD80 antibody (IDEC Pharm/Mitsubishi); ZEVALIN.TM.
anti-CD20 antibody is a radiolabelled murine anti-CD20 antibody
(IDEC/Schering AG); IDEC-131.TM. anti-CD40L antibody is a humanized
anti-CD40L antibody (IDEC/Eisai); IDEC-151.TM. anti-CD4 antibody is
a primatized anti-CD4 antibody (IDEC); IDEC-152.TM. anti-CD23
antibody is a primatized anti-CD23 antibody (IDEC/Seikagaku); SMART
anti-CD3.TM. anti-CD3 antibody is a humanized anti-CD3 IgG (Protein
Design Lab); 5G1.1 is a humanized anti-complement factor 5 (C5)
antibody (Alexion Pharm); IDEC-151.TM. anti-CD4 antibody is a
primatized anti-CD4 IgG1 antibody (IDEC Pharm/SmithKline Beecham);
MDX-CD4.TM. anti-CD4 antibody is a human anti-CD4 IgG antibody
(Medarex/Eisai/Genmab); CDP571.TM. anti-TNF-.alpha. antibody is a
humanized anti-TNF-.alpha. IgG4 antibody (Celltech); LDP-02.TM.
anti-.alpha.4.beta.7 antibody is a humanized anti-.alpha.4.beta.7
antibody (LeukoSite/Genentech); OrthoClone OKT4A.TM. anti-CD4
antibody is a humanized anti-CD4 IgG antibody (Ortho Biotech);
ANTOVA.TM. anti-CD40 antibody is a humanized anti-CD40L IgG
antibody (Biogen); ANTEGREN.TM. anti-VLA-4 antibody is a humanized
anti-VLA-4 IgG antibody (Elan); MDX-33.TM. anti-CD64 antibody is a
human anti-CD64 (Fc.gamma.R) antibody (Medarex/Centeon);
rhuMab-E25.TM. anti-IgE antibody is a humanized anti-IgE IgG1
antibody (Genentech/Norvartis/Tanox Biosystems); IDEC-152.TM.
anti-CD64 antibody is a primatized anti-CD23 antibody (IDEC Pharm);
ABX-CBL.TM. anti-CD147 antibody is a murine anti CD-147 IgM
antibody (Abgenix); BTI-322.TM. anti-CD2 antibody is a rat anti-CD2
IgG antibody (Medimmune/Bio Transplant); Orthoclone/OKT3.TM.
anti-CD3 antibody is a murine anti-CD3 IgG2a antibody (ortho
Biotech); SIMULECT.TM. anti-CD25 antibody is a chimeric anti-CD25
IgG1 antibody (Novartis Pharm); LDP-01.TM.
anti-.beta..sub.2-integrin antibody is a humanized
anti-.beta..sub.2-integrin IgG antibody (LeukoSite); Anti-LFA-1.TM.
anti-CD18 antibody is a murine anti CD18 F(ab').sub.2
(Pasteur-Merieux/Immunotech); CAT-152.TM. anti-TGF-.beta..sub.2
antibody is a human anti-TGF-.beta..sub.2 antibody (Cambridge Ab
Tech); and Corsevin M.TM. anti-Factor VII antibody is a chimeric
anti-Factor VII antibody (Centocor). In certain embodiments, the
antibody is not RITUXAN.TM. anti-CD20 antibody.
[0317] The variant Fc regions of the invention, preferably in the
context of an immunoglobulin, can be further characterized using
one or more biochemical assays and/or one or more functional
assays, preferably in a high throughput manner. In some alternate
embodiments, the variant Fc regions of the inventions are not
introduced into an immunoglobulin and are further characterized
using one or more biochemical based assays and/or one or more
functional assays, preferably in a high throughput manner. The one
or more biochemical assays can be any assay known in the art for
identifying immunoglobulin-antigen or Fc-Fc.gamma.R interactions,
including, but not limited to, an ELISA assay, and surface plasmon
resonance-based assay, e.g., BIAcore assay, for determining the
kinetic parameters of immunoglobulin-antigen or Fc-Fc.gamma.R
interaction. Characterization of target antigen binding affinity or
assessment of target antigen density on a cell surface may be
assessed by methods well known in the art such as Scatchard
analysis or by the use of kits as per manufacturer's instructions,
such as Quantum.TM. Simply Cellular.RTM. (Bangs Laboratories, Inc.,
Fishers, Ind.). The one or more functional assays can be any assay
known in the art for characterizing one or more Fc.gamma.R mediated
effector cell function as known to one skilled in the art or
described herein. In specific embodiments, the immunoglobulins
comprising the variant Fc regions are assayed in an ELISA assay for
binding to one or more Fc.gamma.Rs, e.g., Fc.gamma.RIIIA,
Fc.gamma.RIIA, Fc.gamma.RIIA; followed by one or more ADCC assays.
In some embodiments, the immunoglobulins comprising the variant Fc
regions are assayed further using a surface plasmon resonance-based
assay, e.g., BIAcore. Surface plasmon resonance-based assays are
well known in the art, and are further discussed in Section 5.2.7,
and exemplified herein in Example 6.8.
[0318] An exemplary high throughput assay for characterizing
immunoglobulins comprising variant Fc regions may comprise:
introducing a variant Fc region of the invention, e.g., by standard
recombinant DNA technology methods, in a 4-4-20 antibody;
characterizing the specific binding of the 4-4-20 antibody
comprising the variant Fc region to an Fc.gamma.R (e.g.,
Fc.gamma.RIIIA, Fc.gamma.RIIB) in an ELISA assay; characterizing
the 4-4-20 antibody comprising the variant Fc region in an ADCC
assay (using methods disclosed herein) wherein the target cells are
opsonized with the 4-4-20 antibody comprising the variant Fc
region; the variant Fc region may then be cloned into a second
immunoglobulin, e.g., 4D5, 2H7, and that second immunoglobulin
characterized in an ADCC assay, wherein the target cells are
opsonized with the second antibody comprising the variant Fc
region. The second antibody comprising the variant Fc region is
then further analyzed using an ELISA-based assay to confirm the
specific binding to an Fc.gamma.R.
[0319] Preferably, a variant Fc region of the invention binds
Fc.gamma.RIIIA and/or Fc.gamma.RIIA with a higher affinity than a
wild type Fc region as determined in an ELISA assay. Most
preferably, a variant Fc region of the invention binds
Fc.gamma.RIIIA and/or Fc.gamma.RIIA with a higher affinity and
binds Fc.gamma.RIIB with a lower affinity than a wild type Fc
region as determined in an ELISA assay. In some embodiments, the
variant Fc region binds Fc.gamma.RIIIA and/or Fc.gamma.RIIA with at
least 2-fold higher, at least 4-fold higher, more preferably at
least 6-fold higher, most preferably at least 8 to 10-fold higher
affinity than a wild type Fc region binds Fc.gamma.RIIIA and/or
Fc.gamma.RIIA and binds Fc.gamma.RIIB with at least 2-fold lower,
at least 4-fold lower, more preferably at least 6-fold lower, most
preferably at least 8 to 10-fold lower affinity than a wild type Fc
region binds Fc.gamma.RIIB as determined in an ELISA assay.
[0320] The immunoglobulin comprising the variant Fc regions may be
analyzed at any point using a surface plasmon based resonance based
assay, e.g., BIAcore, for defining the kinetic parameters of the
Fc-Fc.gamma.R interaction, using methods disclosed herein and known
to those of skill in the art. Preferably, the Kd of a variant Fc
region of the invention for binding to a monomeric Fc.gamma.RIIIA
and/or Fc.gamma.RIIA as determined by BIAcore analysis is about 100
nM, preferably about 70 nM, most preferably about 40 nM.; and the
Kd of the variant Fc region of the invention for binding a dimeric
Fc.gamma.RIIB is about 80 nM, about 100 nM, more preferably about
200 nM.
[0321] In most preferred embodiments, the immunoglobulin comprising
the variant Fc regions is further characterized in an animal model
for interaction with an Fc.gamma.R. Preferred animal models for use
in the methods of the invention are, for example, transgenic mice
expressing human Fc.gamma.Rs, e.g., any mouse model described in
U.S. Pat. Nos. 5,877,397, and 6,676,927 which are incorporated
herein by reference in their entirety. Transgenic mice for use in
the methods of the invention include, but are not limited to, nude
knockout Fc.gamma.RIIIA mice carrying human Fc.gamma.RIIIA; nude
knockout Fc.gamma.RIIIA mice carrying human Fc.gamma.RIIA; nude
knockout Fc.gamma.RIIIA mice carrying human Fc.gamma.RIIB and human
Fc.gamma.RIIIA; nude knockout Fc.gamma.RIIIA mice carrying human
Fc.gamma.RIIB and human Fc.gamma.RIIA; nude knockout Fc.gamma.RIIIA
and Fc.gamma.RIIA mice carrying human Fc.gamma.RIIIA and
Fc.gamma.RIIA and nude knockout Fc.gamma.RIIIA, Fc.gamma.RIIA and
Fc.gamma.RIIB mice carrying human Fc.gamma.RIIIA, Fc.gamma.RIIA and
Fc.gamma.RIIB.
[0322] 5.2.1 Design Strategies
[0323] The present invention encompasses engineering methods to
generate Fc variants including but not limited to computational
design strategies, library generation methods, and experimental
production and screening methods. These strategies may be applied
individually or in various combinations to engineer the Fc variants
of the instant invention.
[0324] In most preferred embodiments, the engineering methods of
the invention comprise methods in which amino acids at the
interface between an Fc region and the Fc ligand are not modified.
Fc ligands include but are not limited to Fc.gamma.Rs, C1q, FcRn,
C3, mannose receptor, protein A, protein G, mannose receptor, and
undiscovered molecules that bind Fc. Amino acids at the interface
between an Fc region and an Fc ligand is defined as those amino
acids that make a direct and/or indirect contact between the Fc
region and the ligand, play a structural role in determining the
conformation of the interface, or are within at least 3 angstroms,
preferably at least 2 angstroms of each other as determined by
structural analysis, such as x-ray crystallography and molecular
modeling The amino acids at the interface between an Fc region and
an Fc ligand include those amino acids that make a direct contact
with an Fc.gamma.R based on crystallographic and structural
analysis of Fc-Fc.gamma.R interactions such as those disclosed by
Sondermann et al., (2000, Nature, 406: 267-273; which is
incorporated herein by reference in its entirety). Examples of
positions within the Fc region that make a direct contact with
Fc.gamma.R are amino acids 234-239 (hinge region), amino acids
265-269 (B/C loop), amino acids 297-299 (C'/E loop), and amino
acids 327-332 (F/G) loop. In some embodiments, the molecules of the
invention comprising variant Fc regions comprise modification of at
least one residue that does not make a direct contact with an
Fc.gamma.R based on structural and crystallographic analysis, e.g.,
is not within the Fc-Fc.gamma.R binding site.
[0325] Preferably, the engineering methods of the invention do not
modify any of the amino acids as identified by Shields et al.,
which are located in the CH2 domain of an Fc region proximal to the
hinge region, e.g., Leu234-Pro238; Ala327, Pro329, and affect
binding of an Fc region to all human Fc.gamma.Rs.
[0326] In other embodiments, the invention encompasses Fc variants
with altered Fc.gamma.R affinities and/or altered effector
functions, such that the Fc variant does not have an amino acid
modification at a position at the interface between an Fc region
and the Fc ligand. Preferably, such Fc variants in combination with
one or more other amino acid modifications which are at the
interface between an Fc region and the Fc ligand have a further
impact on the particular altered property, e.g. altered Fc.gamma.R
affinity. Modifying amino acids at the interface between Fc and an
Fc ligand may be done using methods known in the art, for example
based on structural analysis of Fc-ligand complexes. For example
but not by way of limitation by exploring energetically favorable
substitutions at Fc positions that impact the binding interface,
variants can be engineered that sample new interface conformations,
some of which may improve binding to the Fc ligand, some of which
may reduce Fc ligand binding, and some of which may have other
favorable properties. Such new interface conformations could be the
result of, for example, direct interaction with Fc ligand residues
that form the interface, or indirect effects caused by the amino
acid modifications such as perturbation of side chain or backbone
conformations
[0327] The invention encompasses engineering Fc variants comprising
any of the amino acid modifications disclosed herein in combination
with other modifications in which the conformation of the Fc
carbohydrate at position 297 is altered. The invention encompasses
conformational and compositional changes in the N297 carbohydrate
that result in a desired property, for example increased or reduced
affinity for an Fc.gamma.R. Such modifications may further enhance
the phenotype of the original amino acid modification of the Fc
variants of the invention. Although not intending to be bound by a
particular mechanism of actions such a strategy is supported by the
observation that the carbohydrate structure and conformation
dramatically affect Fc-Fc.gamma.R and Fc/C1q binding (Umaha 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;
Radaev et al., 2001, J Biol Chem 276:16478-16483; Shields et al.
2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol
Chem 278:3466-3473).
[0328] Another design strategy for generating Fc variants in
accordance with the invention is provided in which the Fc region is
reengineered to eliminate the structural and functional dependence
on glycosylation. This design strategy involves the optimization of
Fc structure, stability, solubility, and/or Fc function (for
example affinity of Fc for one or more Fc ligands) in the absence
of the N297 carbohydrate. In one approach, positions that are
exposed to solvent in the absence of glycosylation are engineered
such that they are stable, structurally consistent with Fc
structure, and have no tendency to aggregate. Approaches for
optimizing aglycosylated Fc may involve but are not limited to
designing amino acid modifications that enhance aglycosylated Fc
stability and/or solubility by incorporating polar and/or charged
residues that face inward towards the Cg2-Cg2 dimer axis, and by
designing amino acid modifications that directly enhance the
aglycosylated Fc-Fc.gamma.R interface or the interface of
aglycosylated Fc with some other Fc ligand.
[0329] The Fc variants of the present invention may be combined
with other Fc modifications, including but not limited to
modifications that enhance effector function. The invention
encompasses combining an Fc variant of the invention with other Fc
modifications to provide additive, synergistic, or novel properties
in antibodies or Fc fusions. Such modifications may be in the CH1,
CH2, or CH3 domains or a combination thereof. Preferably the Fc
variants of the invention enhance the property of the modification
with which they are combined. For example, if an Fc variant of the
invention is combined with a mutant known to bind Fc.gamma.RIIIA
with a higher affinity than a comparable molecule comprising a wild
type Fc region; the combination with a mutant of the invention
results in a greater fold enhancement in Fc.gamma.RIIIA
affinity.
[0330] In one embodiment, the Fc variants of the present invention
may be combined with other known Fc variants such as those
disclosed in Duncan et al, 1988, Nature 332:563-564; Lund et al.,
1991, J. Immunol. 147:2657-2662; Lund et al, 1992, Mol Immunol
29:53-59; Alegre et al, 1994, Transplantation 57:1537-1543;
Hutchins et al., 1995, Proc Natl. Acad Sci USA 92:11980-11984;
Jefferis et al, 1995, Immunol Lett. 44:111-117; Lund et al., 1995,
Faseb J 9:115-119; Jefferis et al, 1996, Immunol Lett 54:101-104;
Lund et al, 1996, J Immunol 157:49634969; Armour et al., 1999, Eur
J Immunol 29:2613-2624; Idusogie et al, 2000, J Immunol
164:41784184; Reddy et al, 2000, J Immunol 164:1925-1933; Xu et
al., 2000, Cell Immunol 200:16-26; Idusogie et al, 2001, J Immunol
166:2571-2575; Shields et al., 2001, J Biol Chem 276:6591-6604;
Jefferis et al, 2002, Immunol Lett 82:57-65; Presta et al., 2002,
Biochem Soc Trans 30:487-490); U.S. Pat. No. 5,624,821; U.S. Pat.
No. 5,885,573; U.S. Pat. No. 6,194,551; PCT WO 00/42072; PCT WO
99/58572; each of which is incorporated herein by reference in its
entirety.
[0331] 5.2.2 Fc.gamma.R-Fc Binding Assay
[0332] An Fc.gamma.R-Fc binding assay was developed for determining
the binding of the molecules of the invention comprising variant Fc
regions to Fc.gamma.R, which allowed detection and quantization of
the interaction, despite the inherently weak affinity of the
receptor for its ligand, e.g., in the micro molar range for
Fc.gamma.RIIB and Fc.gamma.RIIIA. The method involves the formation
of an Fc.gamma.R complex that has an improved avidity for an Fc
region, relative to an uncompleted Fc.gamma.R. According to the
invention, the preferred molecular complex is a tetrameric immune
complex, comprising: (a) the soluble region of Fc.gamma.R (e.g.,
the soluble region of Fc.gamma.RIIIA, Fc.gamma.RIIA or
Fc.gamma.RIIB); (b) a biotinylated 15 amino acid AVITAG.TM. peptide
sequence operably linked to the C-terminus of the soluble region of
Fc.gamma.R (e.g., the soluble region of Fc.gamma.RIIIA,
Fc.gamma.RIIA or Fc.gamma.RIIB); and (c) streptavidin-phycoerythrin
(SA-PE); in a molar ratio to form a tetrameric Fc.gamma.R complex
(preferably in a 5:1 molar ratio). According to a preferred
embodiment of the invention, the fusion protein is biotinylated
enzymatically, using for example, the E. coli Bir A enzyme, a
biotin ligase which specifically biotinylates a lysine residue in
the 15 amino acid AVITAG.TM. peptide sequence. In a specific
embodiment of the invention, 85% of the fusion protein is
biotinylated, as determined by standard methods known to those
skilled in the art, including but not limited to streptavidin shift
assay. According to preferred embodiments of the invention, the
biotinylated soluble Fc.gamma.R proteins are mixed with SA-PE in a
1.times. SA-PE:5.times. biotinylated soluble Fc.gamma.R molar ratio
to form a tetrameric Fc.gamma.R complex.
[0333] In a preferred embodiment of the invention, molecules
comprising Fc regions bind the tetrameric Fc.gamma.R complexes,
formed according to the methods of the invention, with at least an
8-fold higher affinity than the monomeric uncomplexed Fc.gamma.R.
The binding of polypeptides comprising Fc regions to the tetrameric
Fc.gamma.R complexes may be determined using standard techniques
known to those skilled in the art, such as for example,
fluorescence activated cell sorting (FACS), radioimmunoassays,
ELISA assays, etc.
[0334] The invention encompasses the use of the immune complexes
formed according to the methods described above, for determining
the functionality of molecules comprising an Fc region in
cell-based or cell-free assays.
[0335] As a matter of convenience, the reagents may be provided in
an assay kit, i.e., a packaged combination of reagents for assaying
the ability of molecules comprising variant Fc regions to bind
Fc.gamma.R tetrameric complexes. Other forms of molecular complexes
for use in determining Fc-Fc.gamma.R interactions are also
contemplated for use in the methods of the invention, e.g., fusion
proteins formed as described in U.S. Provisional Application
60/439,709, filed on Jan. 13, 2003 (Attorney Docket No.
11183-005-888); which is incorporated herein by reference in its
entirety.
[0336] 5.2.3 Mutagenesis and Construction of Yeast Display
Libraries
[0337] Molecular interactions between the IgG Fc and Fc receptors
have been previously studied by both structural and genetic
techniques. These studies identified amino acid residues that are
critical for functional binding of Fc to different Fc.gamma.R. None
of these changes have been shown to improve human Fc.gamma.R
mediated efficacy of therapeutic antibodies in animal models. A
complete analysis of all potential amino acid changes at these
residues or other potentially important residues has not been
reported. The platform described herein has the ability to both
construct mutant libraries with all possible amino acid changes,
screen libraries using multiple functional assays, and finally
analyze libraries in relevant humanized animal models.
[0338] The instant invention encompasses construction of multiple
libraries based on both genetic and structural data known in the
art or being developed. The method described and exemplified herein
incorporates building individual libraries that contain mutants
testing all 20 amino acid changes at between 3-6 residues in the Fc
region. The complete set of mutations will be assembled in all
possible combinations of mutations. The number of independent
mutations generated is based on the number of sites being saturated
during library assembly (Table 9 below). Library size will
determine the choice of primary screen and therefore the choice of
vector for initial cloning steps.
TABLE-US-00010 TABLE 9 Number of Independent mutants based on
number of targeted sites. Library # of residues # independent
mutants Primary screen Small 3 or less 8000 max. ELISA Large 4-6
1.6 .times. 10.sup.5-6.4 .times. 10.sup.7 Surface display
[0339] The instant invention encompasses construction of
combinatorial libraries, focusing on a limited number of critical
residues (e.g., 3-6). Using a library of randomly mutagenized IgG1
Fc and the screening assays described and exemplified herein Fc
variants will be identified. In the initial rounds, the best 5
mutations, based on both FcR binding profile and functional
activity will be selected. It will take 20.sup.5 individual mutants
to cover all possible amino acid changes and their combinations at
five locations. A library with at least 10-fold coverage for each
mutant will be generated. In addition regions will be chosen based
on available information, e.g., crystal structure data, Mouse/Human
isotype Fc.gamma.R binding differences, genetic data, and
additional sites identified by mutagenesis.
[0340] The biggest disadvantage of current site directed mutagenic
protocols is production of bias populations, over-representing
variations in some regions and under-representing or completely
lacking mutations in others. The present invention overcomes this
problem by generating unbiased arrays of desirable Fc mutants using
a well-developed gene building technology to eliminate the bias
introduced in library construction by PCR based approaches such as
overlapping PCR and inverted PCR. The key distinctions of the
approach of the present invention are: 1) Employment of equimolar
mix of 20 individual oligos for every targeted codon instead of
degenerated primers. This way each amino acid is represented by a
single, most used codon, whereas degenerated primers over represent
those amino acids encoded by more codons over those encoded by
fewer codons. 2) Building mutants by a chain replacement approach.
This insures unbiased introduction of all desirable changes into
the final product.
[0341] An exemplary protocol comprises of the following steps: 1)
phosphorylated oligos, representing desirable changes at one or
several locations, all complementary to the same strand, added to
the template along with a thermostable, 5'>3' exonuclease
deficient, DNA polymerase and ligase (FIG. 26 a). 2) assembled mix
undergoes a number of polymerization/ligation cycles, sufficient to
generate desirable amount of product. Use of a 5'>3' exonuclease
deficient DNA polymerase insures integrity of the primer sequence
and its phosphate residue, when a thermostable ligase assembles
individual primer-extended fragments into a contiguous
single-stranded chain. Reaction cycles can continue until complete
exhaustion of the oligos pool without introducing bias into the
final product (FIG. 26 b). 3) generated pool of single-stranded
mutants is converted into double-stranded DNA by adding a reverse
gene-specific primer to the reaction (FIG. 26 1c). 4)
double-stranded product gets digested at the end-designed
restriction sites and cloned into an appropriate expression vector
(FIG. 26 1d)
[0342] To insure quality of the library, PCR amplified fragments
will be analyzed by electrophoresis to determine the length of the
final PCR products. The reaction will be characterized as
successful if >99% of the PCR products are of the expected
length. The final library will be cloned into an expression vector.
A fraction of the mutant library will be sequenced to determine the
rate of mutant codon incorporation. The number of fragments
sequenced will be based on the number of target sites mutated and
library validation will be determined by the observed rate of
mutation at targeted sites (Table 10). The rate of vector without
inserts should be less than 2%. The rate of mutation at
non-targeted sites should be less than 8%. Libraries containing
clones with >90% correct inserts will allow us to maintain
screening timelines.
TABLE-US-00011 TABLE 10 Expected rates of Mutation for Libraries
Approx. rates of mutation for library validation Targeted # of seq.
Sin- Residues reactions gle Double Triple Quad. Pent. Hex. 3 20 42%
43% 15% NA NA NA 4 50 29% 43% 21% 7% NA NA 5 75 18% 35% 32% 11% 4%
NA 6 100 12% 20% 40% 20% 6% 2%
[0343] In other embodiments, the invention the invention
encompasses overlapping or inverted PCR for construction of
libraries. In order to remain unbiased, individual primers for each
codon will be used rather than degenerative primers. A similar
validation scheme as disclosed supra will be employed.
[0344] Most preferably automated protocols will be employed for
high throughput library production. Automation allows for improved
throughput, walk away operation, and an overall reduction in
experimental error for tasks requiring tedious repetitive
operations. Oligo synthesis capabilities is based on 2 Mermade DNA
synthesizers (Bioautomation, Inc.) with a total output capability
of 575 60mer Oligos/12 hrs. Proprietary software handles all
aspects of design, synthesis, and storage of the final
oligonucleotides. Robotic liquid handlers will be employed to set
up oligos for synthesis of full length Fc mutants and ligation
reactions for incorporating the mutant Fcs into antibody heavy
chain expression vectors will be set up. After ligation it is
estimated that it would take 1 FTE .about.10 days to array the
library clones and generate .about.8000 minipreps, equivalent to a
combinatorial library saturated at 3 sites. Subsequent to bacterial
transformation a Qpix-2 clone picker robot will be used for picking
colonies into 96 deep well plates. Culture growth will be done
using a magnetic levitation stirrer, capable of incubating 12
plates and resulting in dense growth in 12-16 hr at 37.degree. C. A
Qiagen miniprep robot will be used to perform DNA preps at the rate
of 4 96 well plates in 2.5 hrs. By overlapping tasks 5 such
libraries could be constructed in 9 months with 1 FTE.
[0345] Affinity maturation requires the assembly of a new set of
combinations of mutations, from a preselected mutant pool or
members of a gene family, which can be enriched by a selection
protocol. The process is repeated several times until the isolation
of a mutant with the desired phenotype is achieved. The
disadvantage of the current enzymatic approach, DNA shuffling, to
accomplish this process is bias which can be introduced due to
specific sites within gene that are hot spots for nucleases,
dominance of specific mutants in the final reassembled pool and
loss of some of the original mutants in the final pool. In order to
overcome this shortcoming a build-a-gene (BAG) technology will be
used to generate a highly complex library of Fc mutants containing
random amino acid changes at all potential locations that may be
important for receptor(s) binding. Sets of degenerated oligos
covering specific regions of the IgG Fc will be used (See FIG.
27).
[0346] Oligos will be .about.30 nt and degenerate oligos
synthesized to change one (4 oligos) or two AAs (8 oligos) will be
constructed. The oligos are designed to be overlapping with no
gaps. It will take .about.200 oligos to accommodate all single AA
changes and .about.2000 to change two AAs per oligonucleotide. All
2000+ oligos will be used individually and in combinations to
generate arrays of Fc mutants using the protocol outlined above
(A.20). We will use a home-written randomizer program and a robotic
liquid handler for pooling selected combinations of mutant and wild
type oligos. Large libraries will be cloned into vectors that will
allow for screening using yeast surface display. This approach
utilizes a magnetic bead selection followed by flow cytometry and
has been successfully applied to libraries with a complexity
>10.sup.9 (Feldhaus et al., 2003, Nat. Biotech. 21(2): 163-170;
which is incorporated herein by reference in its entirety). This
limits the number of sites to test at any one pool to 7, resulting
in .about.1.3.times.10.sup.9 possible mutations/pool.
[0347] To insure quality of the library PCR amplified fragments
will be analyzed by electrophoresis to determine the length of the
final PCR products. The reaction will be characterized as
successful if >99% of the PCR products are of the expected
length. A fraction of the mutant library will be sequenced to
determine the rate of mutant codon incorporation. The number of
fragments sequenced will be based on the number of target sites
mutated and library validation will be determined by the observed
rate of mutation at targeted sites (Table 10). The rate of vectors
without inserts should be less than 2%. The rate of mutation at
non-targeted sites should be less than 8%.
[0348] The ability to generate the desired level of efficiency of
mutagenesis by this approach will be determined by sequencing of a
subset of clones. The alternative to BAG will be using a "DNA
shuffle" protocol. This requires pooling all of the mutants,
single, double, triple, etc. Following DNA preparation, Fc regions
will be amplified by PCR using flanking primers that selectively
amplify the mutated region of the Fc, .about.700 bp. Novel mutants
are constructed by reshuffling of mutations in the Fc via DNAseI
treatment of the amplified DNA and isolation of 150-200 bp
fragments (see, e.g., Stemmer et al., 1994, Proc. Natl. Acad. Sci.
U.S.A. 91: 10747-51). Fragments will be religated, PCR amplified
with nested primers and cloned into the yeast surface display
vector, pYD 1. The recombined library will be reselected in the
yeast Fc display screen as described and exemplified herein.
[0349] BAG libraries will utilize most of the same equipment as the
combinatorial library. However cloning will be in a vector suitable
for yeast surface display and will not require arraying of
individual clones as the yeast surface display will initially be
employed for enrichment of large libraries. Subsequent to the
appropriate level of enrichment individual clones will be
arrayed.
[0350] An initial library of molecules comprising variant Fc
regions is produced using any random based mutagenesis techniques
known in the art. It will be appreciated by one of skill in the art
that amino acid sequence variants of Fc regions may be obtained by
any mutagenesis technique known to those skilled in the art. Some
of these techniques are briefly described herein, however, it will
be recognized that alternative procedures may produce an equivalent
result. In a preferred embodiment molecules of the invention
comprising variant Fc regions are prepared by error-prone PCR as
exemplified in Example 6, infra (See Leung et al., 1989, Technique,
1:11). It is especially preferred to have error rates of 2-3 bp/Kb
for use in the methods of the invention. In one embodiment, using
error prone PCR a mutation frequency of 2-3 mutations/kb is
obtained.
[0351] Mutagenesis may be performed in accordance with any of the
techniques known in the art including, but not limited to,
synthesizing an oligonucleotide having one or more modifications
within the sequence of the Fc region of an antibody or a
polypeptide comprising an Fc region (e.g., the CH2 or CH3 domain)
to be modified. Site-specific mutagenesis allows the production of
mutants through the use of specific oligonucleotide sequences which
encode the DNA sequence of the desired mutation, as well as a
sufficient number of adjacent nucleotides, to provide a primer
sequence of sufficient size and sequence complexity to form a
stable duplex on both sides of the deletion junction being
traversed. Typically, a primer of about 30 to about 45 nucleotides
or more in length is preferred, with about 10 to about 25 or more
residues on both sides of the junction of the sequence being
altered. A number of such primers introducing a variety of
different mutations at one or more positions may be used to
generated a library of mutants.
[0352] The technique of site-specific mutagenesis is well known in
the art, as exemplified by various publications (see, e.g., Kunkel
et al., Methods Enzymol., 154:367-82, 1987, which is hereby
incorporated by reference in its entirety). In general,
site-directed mutagenesis is performed by first obtaining a
single-stranded vector or melting apart of two strands of a double
stranded vector which includes within its sequence a DNA sequence
which encodes the desired peptide. An oligonucleotide primer
bearing the desired mutated sequence is prepared, generally
synthetically. This primer is then annealed with the
single-stranded vector, and subjected to DNA polymerizing enzymes
such as T7 DNA polymerase, in order to complete the synthesis of
the mutation-bearing strand. Thus, a heteroduplex is formed wherein
one strand encodes the original non-mutated sequence and the second
strand bears the desired mutation. This heteroduplex vector is then
used to transform or transfect appropriate cells, such as E. coli
cells, and clones are selected which include recombinant vectors
bearing the mutated sequence arrangement. As will be appreciated,
the technique typically employs a phage vector which exists in both
a single stranded and double stranded form. Typical vectors useful
in site-directed mutagenesis include vectors such as the M13 phage.
These phage are readily commercially available and their use is
generally well known to those skilled in the art. Double stranded
plasmids are also routinely employed in site directed mutagenesis
which eliminates the step of transferring the gene of interest from
a plasmid to a phage.
[0353] Alternatively, the use of PCR.TM. with commercially
available thermostable enzymes such as Taq DNA polymerase may be
used to incorporate a mutagenic oligonucleotide primer into an
amplified DNA fragment that can then be cloned into an appropriate
cloning or expression vector. See, e.g., Tomic et al., Nucleic
Acids Res., 18(6):1656, 1987, and Upender et al., Biotechniques,
18(1):29-30, 32, 1995, for PCR.TM.--mediated mutagenesis
procedures, which are hereby incorporated in their entireties.
PCR.TM. employing a thermostable ligase in addition to a
thermostable polymerase may also be used to incorporate a
phosphorylated mutagenic oligonucleotide into an amplified DNA
fragment that may then be cloned into an appropriate cloning or
expression vector (see e.g., Michael, Biotechniques, 16(3):410-2,
1994, which is hereby incorporated by reference in its
entirety).
[0354] Another method for preparing variants for use in the
invention, is cassette mutagenesis based on the technique described
by Wells et al. (1985, Gene, 34: 315). The starting material is the
plasmid comprising the desired DNA encoding the protein to be
mutated (e.g., the DNA encoding a polypeptide comprising an Fc
region). The codon(s) in the DNA sequence to be mutated are
identified; there must be a unique restriction endonuclease site on
each side of the identified mutations site(s). If no such
restriction site exits, it may be generated by oligonucleotide
directed mutagenesis. After the restriction sites have been
introduced into the plasmid, the plasmid is cut at these sites and
linearized. A double-stranded oligonucleotide encoding the sequence
of the DNA between the restriction sites but containing the
mutation is synthesized using standard procedures known to those
skilled in the art. The double stranded oligonucleotide is referred
to as the cassette. This cassette is designed to have 3' and 5'
ends that are compatible with the ends of the linearized plasmid,
such that it can be directly ligated to the plasmid.
[0355] Other methods known to those of skill in the art for
producing sequence variants of the Fc region of an antibody or
polypeptides comprising an Fc region can be used. For example,
recombinant vectors encoding the amino acid sequence of the
constant domain of an antibody or a fragment thereof may be treated
with mutagenic agents, such as hydroxylamine, to obtain sequence
variants.
[0356] Once a mutant library is produced according to the methods
described, the mutagenized library is transformed into a yeast
strain, preferably EBY100 (Invitrogen), MAT.alpha. ura3-52 trpl
leu2.DELTA.l his3.DELTA.200 pep4::HIS3 prb1.DELTA.1.6R can1
GAL::GAL-AGA1 using a standard lithium acetate transformation
protocol known to those skilled in the art.
[0357] It will be appreciated by one of skill in the art, that once
molecules of the invention with desired effector function and/or
binding properties (e.g., molecules with variant Fc regions with at
least one amino acid modification, which modification confers ADCC
activity or enhances the affinity of the variant Fc region for
Fc.gamma.RIIIA relative to a comparable molecule, comprising a
wild-type Fc region) have been identified (See Section 5.1 and
Table 2) according to the methods of the invention, other molecules
(i.e, therapeutic antibodies) may be engineered using standard
recombinant DNA techniques and any known mutagenesis techniques, as
described in this section to produce engineered molecules carrying
the identified mutation sites.
[0358] 5.2.4 Yeast Surface Display
[0359] The preferred method for screening and identifying molecules
comprising variant Fc regions with altered Fc.gamma.R affinities
(i.e., enhanced Fc.gamma.RIIIA affinity and/or Fc.gamma.RIIA) is
yeast surface display technology (for review see Boder and Wittrup,
2000, Methods in Enzymology, 328: 430-444, which is incorporated
herein by reference in its entirety) which addresses the deficiency
in the prior art for screening binding interactions of
extracellular post-translationally modified proteins. Specifically,
the yeast surface display is a genetic method whereby polypeptides
comprising Fc mutants are expressed on the yeast cell wall in a
form accessible for interacting with Fc.gamma.R. Yeast surface
display of the mutant Fc containing polypeptides of the invention
may be performed in accordance with any of the techniques known to
those skilled in the art. See U.S. Pat. Nos. 6,423,538; 6,114,147;
and 6,300,065, all of which are incorporated herein by reference in
their entirety. See Boder et al., 1997 Nat. Biotechnol., 15:553-7;
Boder et al., 1998 Biotechnol. Prog., 14:55-62; Boder et al., 2000
Methods Enzymol., 328:430-44; Boder et al., 2000 Proc. Natl. Acad.
Sci. U.S.A., 2000, 97:10701-5; Shusta et al., 1998 Nat.
Biotechnol., 1998, 16:773-7; Shusta et al., 1999 J. Mol. Biol.,
292:949-56; Shusta et al., 1999 Curr. Opin. Biotechnol., 10:117-22;
Shusta et al., 2000 Nat. Biotechnol., 18:754-9; Wittrup et al.,
1994 Ann. N.Y. Acad. Sci., 745:321-30; Wittrup et al., 1994
Cytometry, 16:206-13; Wittrup, 1995 Curr. Opin. Biotechnol.,
6:203-8; Wittrup, 1999 Trends Biotechnol., 17:423-4; Wittrup, 2000
Nat. Biotechnol., 18:1039-40; Wittrup, 2001 Curr. Opin.
Biotechnol., 12:395-9.
[0360] Yeast Surface Display will be used to enrich libraries
containing >10.sup.7 independent clones. This approach will
provide the ability to enrich large libraries >20-fold in single
sort. Fc mutant libraries with >10,000 independent mutants (4 or
more sites) will be cloned into the appropriate vectors for yeast
surface display and enriched by FACS sorting until <8000 mutants
are able to be tested by other biochemical and functional assays as
described below.
[0361] The invention provides methods for constructing an Fc mutant
library in yeast for displaying molecules comprising Fc regions,
which have been mutated as described in Section 5.2.2. Preferably,
the Fc mutant libraries for use in the methods of the invention
contain at least 10.sup.7 cells, up to 10.sup.9 cells. One
exemplary method for constructing a Fc library for use in the
methods of the invention comprises the following: nucleic acids
encoding molecules comprising Fc regions are cloned into the
multiple cloning site of a vector derived from a yeast replicating
vector, e.g., pCT302; such that the Fc encoding nucleic acids are
expressed under the control of the GAL1 galactose-inducible
promoter and in-frame with a nucleotide sequence encoding Aga2p,
the mating agglutinin cell wall protein. In a preferred embodiment,
nucleic acids encoding molecules comprising Fc regions are cloned
C-terminal to the Aga2p coding region, such that a Fc-region Aga2p
fusion protein is encoded. A fusion protein comprising the Aga2p
protein and polypeptides comprising Fc regions will be secreted
extracellularly and displayed on the cell wall via disulfide
linkage to the Aga1p protein, an integral cell wall protein, using
the preferred construct of the invention. In an alternative
embodiment, the constructs may further comprise nucleotide
sequences encoding epitope tags. Any epitope tag nucleotide coding
sequence known to those skilled in the art can be used in
accordance with the invention, including, but not limited to
nucleotide sequences encoding hemagglutinin (HA), c-myc Xpress TAG,
His-TAG, or V5TAG. The presence of the fusion protein on the yeast
cell surface may be detected using FACS analysis, confocal
fluorescence microscopy or standard immunostaining methods, all of
which are known to those skilled in the art. In one embodiment, the
presence of the Fc fusion proteins of the invention on the yeast
cell surface are detected using Fc-specific monoclonal antibodies
(CH3 specific), including but not limited to IgG1 Fc-specific
monoclonal antibody, HP6017 (Sigma), JL512 (Immunotech), and any
antibody disclosed in Partridge et al., 1986, Molecular Immunology,
23 (12): 1365-72, which is incorporated herein by reference in its
entirety. In another embodiment, the presence of the Fc fusion
proteins of the invention are detected by immunofluorescent
labeling of epitope tags using techniques known to those skilled in
the art. It is particularly useful in the methods of the invention,
to use nucleotide sequences encoding epitope tags to flank the
nucleic acids encoding the Fc fusion proteins, as an internal
control, to detect if the fusion proteins are displayed on the cell
wall in a partially proteolyzed form.
[0362] 5.2.5 Screening of Yeast Display Libraries
[0363] The invention encompasses screening the yeast display
libraries using immunological based assays including but not
limited to cell based assays, solution based assays, and solid
phase based assays.
[0364] In some embodiments, the invention encompasses
identification of Fc mutants with altered Fc.gamma.R affinities
using affinity maturation methods which are known to those skilled
in the art and encompassed herein. Briefly, affinity maturation
creates novel alleles by randomly recombining individual mutations
present in a mutant library, see, e.g., Hawkins et al., 1992, J.
Mol. Biol. 226: 889-896; Stemmer et al., 1994 Nature, 370: 389-91;
both of which are incorporated herein by reference in their
entireties. It has been used successfully to increase the affinity
of antibodies, T cell receptors and other proteins.
[0365] The invention encompasses using mutations that show
increased Fc.gamma.R binding as a baseline to construct new mutant
libraries with enhanced phenotypes. Using the methods of the
invention, a population of IgG1 Fc mutants enriched by yeast
surface display for increased binding to an Fc.gamma.R, e.g.,
Fc.gamma.RIIIA, may be selected. Following DNA preparation, Fc
regions can be amplified by PCR using flanking primers that
selectively amplify the mutated region of the Fc, which is about
.about.700 bp using methods known to one skilled in the art and
exemplified or disclosed herein. Novel mutants can thus be
constructed by reshuffling of mutations in the Fc region for
example via DNAseI treatment of the amplified DNA and isolation of
fragments using methods such as those disclosed by Stemmer et al.,
1994 Proc. Natl. Acad. Sci. USA 91: 10747-51, which is incorporated
herein by reference in its entirety. Fragments can then be
religated, PCR amplified with nested primers and cloned into the
yeast display vector, e.g., pYD1 using methods known to one skilled
in the art. The recombined library can then be reselected in the
yeast Fc display screen. As the K.sub.D decreases, below 10 nM,
conditions can be established to allow for further increases in
affinity based on the reduction of the off rate of the
Fc.gamma.RIIIA ligand from the Fc receptor using methods known in
the art such as those disclosed in Boder et al., 1998, Biotechnol.
Prog. 14: 55-62, which is incorporated herein by reference in its
entirety. The invention encompasses a kinetic screen of the yeast
library. A kinetic screen may be established by labeling of the Fc
displaying cells to saturation with a labeled ligand, e.g., a
fluorescent ligand followed by incubation with an excess of
non-labeled ligand for a predetermined period. After termination of
the reaction by the addition of excess buffer (e.g., 1.times.PBS,
0.5 mg/ml BSA) cells will be analyzed by FACS and sort gates set
for selection. After each round of enrichment individual mutants
can be tested for fold increases in affinity and sequenced for
diversity. The in vitro recombination process can be repeated. In
some embodiments, the in vitro recombination process is repeated at
least 3 times.
[0366] Selection of the Fc variants of the invention may be done
using any Fc.gamma.R including but not limited to polymorphic
variants of Fc.gamma.R. In some embodiments, selection of the Fc
variants is done using a polymorphic variant of Fc.gamma.RIIIA
which contains a phenylalanine at position 158. In other
embodiments, selection of the Fc variants is done using a
polymorphic variant of Fc.gamma.RIIIA which contains a valine at
position 158. Fc.gamma.RIIIA 158V displays a higher affinity for
IgG1 than 158F and an increased ADCC activity (see, e.g., Koene et
al., 1997, Blood, 90:1109-14; Wu et al., 1997, J. Clin. Invest.
100: 1059-70, both of which are incorporated herein by reference in
their entireties); this residue in fact directly interacts with the
lower hinge region of IgG1 as recently shown by IgG1-Fc.gamma.RIIIA
co-crystallization studies, see, e.g., Sonderman et al., 2000,
Nature, 100: 1059-70, which is incorporated herein by reference in
its entirety. Studies have shown that in some cases therapeutic
antibodies have improved efficacy in Fc.gamma.RIIIA-158V homozygous
patients. Therapeutic antibodies may also be more effective in
patients heterozygous for Fc.gamma.RIIIA-158V and
Fc.gamma.RIIIA-158F, and in patients with Fc.gamma.RIIA H131.
[0367] The invention encompasses screening yeast libraries based on
Fc.gamma.RIIB depletion and Fc.gamma.RIIIA selection so that Fc
mutants are selected that not only have an enhanced affinity for
Fc.gamma.RIIIIA but also have a reduced affinity for Fc.gamma.RIIB.
Yeast libraries may be enriched for clones that have a reduced
affinity for Fc.gamma.RIIB by sequential depletion methods, for
example, by incubating the yeast library with magnetic beads coated
with Fc.gamma.RIIB. Fc.gamma.RIIB depletion is preferably carried
out sequentially so that the library is enriched in clones that
have a reduced affinity for Fc.gamma.RIIB. In some embodiments, the
Fc.gamma.RIIB depletion step results in a population of cells so
that only 30%, preferably only 10%, more preferably only 5%, most
preferably less than 1% bind Fc.gamma.RIIB. In some embodiments,
Fc.gamma.RIIB depletion is carried out in at least 3 cycles, at
least 4 cycles, at least 6 cycles. The Fc.gamma.RIIB depletion step
is preferably combined with an Fc.gamma.RIIIIA selection step, for
example using FACS sorting so that Fc variants with an enhanced
affinity for Fc.gamma.RIIIIA are selected.
[0368] 5.2.5.1 FACs Assays; Solid Phased Assays and Immunological
Based Assays
[0369] The invention encompasses characterization of the mutant Fc
fusion proteins that are displayed on the yeast surface cell wall,
according to the methods described in Section 5.2.3. One aspect of
the invention provides a method for selecting mutant Fc fusion
proteins with a desirable binding property, specifically, the
ability of the mutant Fc fusion protein to bind Fc.gamma.RIIIA
and/or Fc.gamma.RIIA with a greater affinity than a comparable
polypeptide comprising a wild-type Fc region binds Fc.gamma.RIIIA
and/or Fc.gamma.RIIA. In another embodiment, the invention provides
a method for selecting mutant Fc fusion proteins with a desirable
binding property, specifically, the ability of the mutant Fc fusion
protein to bind Fc.gamma.RIIIA and/or Fc.gamma.RIIA with a greater
affinity than a comparable polypeptide comprising a wild-type Fc
region binds Fc.gamma.RIIIA and/or Fc.gamma.RIIA, and further the
ability of the mutant Fc fusion protein to bind Fc.gamma.RIIB with
a lower affinity than a comparable polypeptide comprising a
wild-type Fc region binds Fc.gamma.RIIB. It will be appreciated by
one skilled in the art, that the methods of the invention can be
used for identifying and screening any mutations in the Fc regions
of molecules, with any desired binding characteristic.
[0370] Yeast cells displaying the mutant Fc fusion proteins can be
screened and characterized by any biochemical or immunological
based assays known to those skilled in the art for assessing
binding interactions.
[0371] Preferably, fluorescence activated cell sorting (FACS),
using any of the techniques known to those skilled in the art, is
used for screening the mutant Fc fusion proteins displayed on the
yeast cell surface for binding Fc.gamma.RIIIA, preferably the
Fc.gamma.RIIIA tetrameric complex, or optionally Fc.gamma.RIIB.
Flow sorters are capable of rapidly examining a large number of
individual cells that contain library inserts (e.g., 10-100 million
cells per hour) (Shapiro et al., Practical Flow Cytometry, 1995).
Additionally, specific parameters used for optimization including,
but not limited to, ligand concentration (i.e., Fc.gamma.RIIIA
tetrameric complex), kinetic competition time, or FACS stringency
may be varied in order to select for the cells which display Fc
fusion proteins with specific binding properties, e.g., higher
affinity for Fc.gamma.RIIIA compared to a comparable polypeptide
comprising a wild-type Fc region. Flow cytometers for sorting and
examining biological cells are well known in the art. Known flow
cytometers are described, for example, in U.S. Pat. Nos. 4,347,935;
5,464,581; 5,483,469; 5,602,039; 5,643,796; and 6,211,477; the
entire contents of which are incorporated by reference herein.
Other known flow cytometers are the FACS Vantage.TM. system
manufactured by Becton Dickinson and Company, and the COPAS.TM.
system manufactured by Union Biometrica.
[0372] According to a preferred embodiment of the invention, yeast
cells are analyzed by fluorescence activated cell sorting (FACS).
In most preferred embodiments, the FACS analysis of the yeast cells
is done in an iterative manner, at least twice, at least three
times, or at least 5 times. Between each round of selection cells
are regrown and induced so the Fc regions are displayed on the
maximum number of yeast cell surfaces. Although not intending to be
bound by a particular mode of action, this iterative process helps
enrich the population of the cells with a particular phenotype,
e.g., high binding to Fc.gamma.RIIIA.
[0373] In preferred embodiments, screening for Fc variants of the
invention comprises a selection process that has multiple rounds of
screening, e.g., at least two rounds of screening. In one
embodiment, screening for Fc variants that have an enhanced
affinity for Fc.gamma.RIIIA may comprise the following steps: in
the first round of screening, a library of yeast cells, e.g., a
naive library of 10.sup.7 cells is enriched by FACS, preferably in
an iterative manner, using for example labeled tetrameric
Fc.gamma.RIIIA to select for Fc variants that have an enhanced
affinity for Fc.gamma.RIIIA; the variant Fc region that is selected
with the desired phenotype, e.g., enhanced binding to
Fc.gamma.RIIIA, is then introduced into an antibody, e.g., a 4-4-20
antibody, and the engineered antibody is assayed using a secondary
screen, e.g., ELISA for binding to an Fc.gamma.R. In the second
round of screening, a single mutation library may be generated
based on the first screen so that the Fc region harbors the variant
displaying the enhanced affinity for Fc.gamma.RIIIA; and enriched
by FACS using for example labeled monomeric Fc.gamma.RIIIA in both
the presence and absence of unlabeled receptor; and the variant Fc
region is then introduced into an antibody, e.g., a 4-4-20
antibody, and the engineered antibody is assayed using a secondary
screen, e.g., ELISA for binding to an Fc.gamma.R. In some
embodiments, the secondary screen may further comprise
characterizing the antibodies comprising Fc variants in an ADCC or
BIAcore based assay using methods disclosed herein
[0374] The invention encompasses FACS screening of the mutant yeast
library under equilibrium or kinetic conditions. When the screening
is performed under equilibrium conditions, an excess of the yeast
library carrying Fc mutants is incubated with Fc.gamma.RIIIA,
preferably labeled Fc.gamma.RIIIA at a concentration 5-10 fold
below the Kd, for at least one hour to allow binding of Fc mutants
to Fc.gamma.RIIIA under equilibrium conditions. When the screening
is performed under kinetic conditions, the mutant yeast library is
incubated with labeled Fc.gamma.RIIIA; the cells are then incubated
with equimolar unlabeled Fc.gamma.RIIIA for a pre-selected time,
bound Fc.gamma.RIIIA is then monitored.
[0375] One exemplary method of analyzing the yeast cells expressing
mutant Fc fusion proteins with FACS is costaining the cells with
Fc.gamma.RIIIA-tetrameric complex which has been labeled with a
fluorescent label such as, PE and an anti-Fc antibody, such as
F(ab).sub.2 anti-Fc which has been fluorescently labeled.
Fluorescence measurements of a yeast library produced according to
the methods of the invention preferably involves comparisons with
controls; for example, yeast cells that lack the insert encoding
molecules comprising an Fc region (negative control). The flow
sorter has the ability not only to measure fluorescence signals in
cells at a rapid rate, but also to collect cells that have
specified fluorescent properties. This feature may be employed in a
preferred embodiment of the invention to enrich the initial library
population for cells expressing Fc fusion proteins with specific
binding characteristics, e.g., higher affinity for Fc.gamma.RIIIA
compared to a comparable polypeptide comprising a wild-type Fc
region. In a preferred embodiment of the invention, yeast cells are
analyzed by FACS and sort gates established to select for cells
that show the highest affinity for Fc.gamma.RIIIA relative to the
amount of Fc expression on the yeast cell surface. According to a
preferred embodiment, four consecutive sorts are established,
wherein the gates for each successive sort is 5.5%, 1%, 0.2%, and
0.1%. It is preferred that the yeast display library formed
according to the methods of the invention be over-sampled by at
least 10-fold to improve the probability of isolating rare clones
(e.g., analyze .about.10.sup.8 cells from a library of 10.sup.7
clones). Alternatively, 2-5 sorts are established to select for
cells of the desired phenotype. Sort gates can be established
empirically by one skilled in the art.
[0376] In other preferred embodiments, mutant Fc fusion proteins
displayed on the yeast cell surface are screened using solid phase
based assays, for example assays using magnetic beads, e.g.,
supplied by Dynal, preferably in a high through put manner for
binding to an Fc.gamma.R, e.g., Fc.gamma.RIIIA. In one embodiment,
magnetic bead assays may be used to identify mutants with enhanced
affinity for Fc.gamma.RIIIA and/or reduced affinity for
Fc.gamma.RIIB. An exemplary assay to identify mutants with enhanced
affinity for Fc.gamma.RIIIA and reduced affinity for Fc.gamma.RIIB
may comprise selecting mutants by a sequential solid phase
depletion using magnetic beads coated with Fc.gamma.RIIB followed
by selection with magnetic beads coated with Fc.gamma.RIIIA. For
example, one assay may comprise the following steps: incubating the
library of yeast cells generated in accordance with the methods of
the invention with magnetic beads coated with Fc.gamma.RIIB;
separating yeast cells bound to beads from the non bound fraction
by placing the mixture in a magnetic field, removing the non-bound
yeast cells and placing them in a fresh media; binding the yeast
cells to beads coated with Fc.gamma.RIIIA, separating yeast cells
bound to beads from the non bound fraction by placing the mixture
in a magnetic field, removing the non-bound yeast cells; removing
the bound cells by rigorous vortexing; growing the recovered cells
in glucose containing media; re-inducing in selective media
containing galactose. The selection process is repeated at least
once. Inserts containing the Fc domain are then amplified using
common methodologies known in the art, e.g., PCR, and introduced
into an antibody by methods already described for further
characterization.
[0377] In an alternative embodiment, a non-yeast based system is
used to characterize the binding properties of the molecules of the
invention. One exemplary system for characterizing the molecules of
the invention comprises a mammalian expression vector containing
the heavy chain of the anti-fluorescein monoclonal antibody 4-4-20,
into which the nucleic acids encoding the molecules of the
invention with variant Fc regions are cloned. The resulting
recombinant clone is expressed in a mammalian host cell line (i.e.,
human kidney cell line 293H), and the resulting recombinant
immunoglobulin is analyzed for binding to Fc.gamma.R using any
standard assay known to those in the art, including but not limited
to ELISA and FACS.
[0378] Molecules of the present invention may be characterized in a
variety of ways. In particular, molecules of the invention
comprising modified Fc regions may be assayed for the ability to
immunospecifically bind to a ligand, e.g., Fc.gamma.RIIIA
tetrameric complex. Such an assay may be performed in solution
(e.g., Houghten, Bio/Techniques, 13:412-421, 1992), on beads (Lam,
Nature, 354:82-84, 1991, on chips (Fodor, Nature, 364:555-556,
1993), on bacteria (U.S. Pat. No. 5,223,409), on spores (U.S. Pat.
Nos. 5,571,698; 5,403,484; and 5,223,409), on plasmids (Cull et
al., Proc. Natl. Acad. Sci. USA, 89:1865-1869, 1992) or on phage
(Scott and Smith, Science, 249:386-390, 1990; Devlin, Science,
249:404-406, 1990; Cwirla et al., Proc. Natl. Acad. Sci. USA,
87:6378-6382, 1990; and Felici, J. Mol. Biol., 222:301-310, 1991)
(each of these references is incorporated by reference herein in
its entirety). Molecules that have been identified to
immunospecifically bind to an ligand, e.g., Fc.gamma.RIIIA can then
be assayed for their specificity and affinity for the ligand.
[0379] Molecules of the invention that have been engineered to
comprise modified Fc regions (e.g., therapeutic antibodies) or have
been identified in the yeast display system to have the desired
phenotype (see Section 5.1) may be assayed for immunospecific
binding to an antigen (e.g., cancer antigen and cross-reactivity
with other antigens (e.g., Fc.gamma.R) by any method known in the
art. Immunoassays which can be used to analyze immunospecific
binding and cross-reactivity include, but are not limited to,
competitive and non-competitive assay systems using techniques such
as western blots, radioimmunoassays, ELISA (enzyme linked
immunosorbent assay), "sandwich" immunoassays, immunoprecipitation
assays, precipitin reactions, gel diffusion precipitin reactions,
immunodiffusion assays, agglutination assays, complement-fixation
assays, immunoradiometric assays, fluorescent immunoassays, protein
A immunoassays, to name but a few. Such assays are routine and well
known in the art (see, e.g., Ausubel et al., eds, 1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,
Inc., New York, which is incorporated by reference herein in its
entirety).
[0380] The binding affinity of the molecules of the present
invention comprising modified Fc regions to a ligand, e.g.,
Fc.gamma.R tetrameric complex and the off-rate of the interaction
can be determined by competitive binding assays. One example of a
competitive binding assay is a radioimmunoassay comprising the
incubation of labeled ligand, such as tetrameric Fc.gamma.R (e.g.,
.sup.3H or .sup.125I) with a molecule of interest (e.g., molecules
of the present invention comprising modified Fc regions) in the
presence of increasing amounts of unlabeled ligand, such as
tetrameric Fc.gamma.R, and the detection of the molecule bound to
the labeled ligand. The affinity of the molecule of the present
invention for the ligand and the binding off-rates can be
determined from the saturation data by scatchard analysis.
[0381] In a preferred embodiment, BIAcore kinetic analysis is used
to determine the binding on and off rates of molecules of the
present invention to a ligand such as Fc.gamma.R. BIAcore kinetic
analysis comprises analyzing the binding and dissociation of a
ligand from chips with immobilized molecules (e.g., molecules
comprising modified Fc regions) on their surface.
[0382] 5.2.6 Sequencing of Mutants
[0383] Any of a variety of sequencing reactions known in the art
can be used to directly sequence the molecules of the invention
comprising variant Fc regions. Examples of sequencing reactions
include those based on techniques developed by Maxim and Gilbert
(Proc. Natl. Acad. Sci. USA, 74:560, 1977) or Sanger (Proc. Natl.
Acad. Sci. USA, 74:5463, 1977). It is also contemplated that any of
a variety of automated sequencing procedures can be utilized
(Bio/Techniques, 19:448, 1995), including sequencing by mass
spectrometry (see, e.g., PCT Publication No. WO 94/16101, Cohen et
al., Adv. Chromatogr., 36:127-162, 1996, and Griffin et al., Appl.
Biochem. Biotechnol., 38:147-159, 1993).
[0384] 5.2.7 Functional Assays of Molecules with Variant Fc
Regions
[0385] The invention encompasses characterization of the molecules
of the invention (e.g., an antibody comprising a variant Fc region
identified by the yeast display technology described supra; or
therapeutic monoclonal antibodies engineered according to the
methods of the invention) using assays known to those skilled in
the art for identifying the effector cell function of the
molecules. In particular, the invention encompasses characterizing
the molecules of the invention for Fc.gamma.R-mediated effector
cell function. Examples of effector cell functions that can be
assayed in accordance with the invention, include but are not
limited to, antibody-dependent cell mediated cytotoxicity,
phagocytosis, opsonization, opsonophagocytosis, C1q binding, and
complement dependent cell mediated cytotoxicity. Any cell-based or
cell free assay known to those skilled in the art for determining
effector cell function activity can be used (For effector cell
assays, see Perussia et al., 2000, Methods Mol. Biol. 121: 179-92;
Baggiolini et al., 1998 Experientia, 44(10): 841-8; Lehmann et al.,
2000 J. Immunol. Methods, 243(1-2): 229-42; Brown E J. 1994,
Methods Cell Biol., 45: 147-64; Munn et al., 1990 J. Exp. Med.,
172: 231-237, Abdul-Majid et al., 2002 Scand. J. Immunol. 55:
70-81; Ding et al., 1998, Immunity 8:403-411, each of which is
incorporated by reference herein in its entirety).
[0386] In one embodiment, the molecules of the invention can be
assayed for Fc.gamma.R-mediated phagocytosis in human monocytes.
Alternatively, the Fc.gamma.R-mediated phagocytosis of the
molecules of the invention may be assayed in other phagocytes,
e.g., neutrophils (polymorphonuclear leuckocytes; PMN); human
peripheral blood monocytes, monocyte-derived macrophages, which can
be obtained using standard procedures known to those skilled in the
art (e.g., see Brown E J. 1994, Methods Cell Biol., 45: 147-164).
In one embodiment, the function of the molecules of the invention
is characterized by measuring the ability of THP-1 cells to
phagocytose fluoresceinated IgG-opsonized sheep red blood cells
(SRBC) by methods previously described (Tridandapani et al., 2000,
J. Biol. Chem. 275:20480-7). For example, an exemplary assay for
measuring phagocytosis of the molecules of the invention comprising
variant Fc regions with enhanced affinities for Fc.gamma.RIIIA,
comprises of: treating THP-1 cells with a molecule of the invention
or with a control antibody that does not bind to Fc.gamma.RIIIA,
comparing the activity levels of said cells, wherein a difference
in the activities of the cells (e.g., rosetting activity (the
number of THP-1 cells binding IgG-coated SRBC), adherence activity
(the total number of SRBC bound to THP-1 cells), and phagocytic
rate) would indicate the functionality of the molecule of the
invention. It can be appreciated by one skilled in the art that
this exemplary assay can be used to assay any of the molecules
identified by the methods of the invention.
[0387] Another exemplary assay for determining the phagocytosis of
the molecules of the invention is an antibody-dependent
opsonophagocytosis assay (ADCP) which can comprise the following:
coating a target bioparticle such as Escherichia coli-labeled FITC
(Molecular Probes) or Staphylococcus aureus-FITC with (i) wild-type
4-4-20 antibody, an antibody to fluorescein (See Bedzyk et al.,
1989, J. Biol. Chem., 264(3): 1565-1569, which is incorporated
herein by reference in its entirety), as the control antibody for
Fc.gamma.R-dependent ADCP; or (ii) 4-4-20 antibody harboring the
D265A mutation that knocks out binding to Fc.gamma.RIII, as a
background control for Fc.gamma.R-dependent ADCP (iii) 4-4-20
antibody carrying variant Fc regions identified by the methods of
the invention and produced as exemplified in Example 6.6; and
forming the opsonized particle; adding any of the opsonized
particles described (i-iii) to THP-1 effector cells (a monocytic
cell line available from ATCC) at a 1:1, 10:1, 30:1, 60:1, 75:1 or
100:1 ratio to allow Fc.gamma.R-mediated phagocytosis to occur;
preferably incubating the cells and E. coli-FITC/antibody at
37.degree. C. for 1.5 hour; adding trypan blue after incubation
(preferably at room temperature for 2-3 min.) to the cells to
quench the fluoroscence of the bacteria that are adhered to the
outside of the cell surface without being internalized;
transferring cells into a FACS buffer (e.g., 0.1%, BSA in PBS,
0.1%, sodium azide), analyzing the fluorescence of the THP1 cells
using FACS (e.g., BD FACS Calibur). Preferably, the THP-1 cells
used in the assay are analyzed by FACS for expression of Fc.gamma.R
on the cell surface. THP-1 cells express both CD32A and CD64. CD64
is a high affinity Fc.gamma.R that is blocked in conducting the
ADCP assay in accordance with the methods of the invention. The
THP-1 cells are preferably blocked with 100 .mu.g/mL soluble IgG1
or 10% human serum. To analyze the extent of ADCP, the gate is
preferably set on THP-1 cells and median fluorescence intensity is
measured. The ADCP activity for individual mutants is calculated
and reported as a normalized value to the wild type chMab 4-4-20
obtained. The opsonized particles are added to THP-1 cells such
that the ratio of the opsonized particles to THP-1 cells is 30:1 or
60:1. In most preferred embodiments, the ADCP assay is conducted
with controls, such as E. coli-FITC in medium, E. coli-FITC and
THP-1 cells (to serve as Fc.gamma.R-independent ADCP activity), E.
coli-FITC, THP-1 cells and wild-type 4-4-20 antibody (to serve as
Fc.gamma.R-dependent ADCP activity), E coli-FITC, THP-1 cells,
4-4-20 D265A (to serve as the background control for
Fc.gamma.R-dependent ADCP activity).
[0388] In another embodiment, the molecules of the invention can be
assayed for Fc.gamma.R-mediated ADCC activity in effector cells,
e.g., natural killer cells, using any of the standard methods known
to those skilled in the art (See e.g., Perussia et al., 2000,
Methods Mol. Biol. 121: 179-92; Weng et al., 2003, J. Clin. Oncol.
21:3940-3947). An exemplary assay for determining ADCC activity of
the molecules of the invention is based on a .sup.51Cr release
assay comprising of: labeling target cells with
[.sup.51Cr]Na.sub.2CrO.sub.4 (this cell-membrane permeable molecule
is commonly used for labeling since it binds cytoplasmic proteins
and although spontaneously released from the cells with slow
kinetics, it is released massively following target cell necrosis);
opsonizing the target cells with the molecules of the invention
comprising variant Fc regions; combining the opsonized radiolabeled
target cells with effector cells in a microtitre plate at an
appropriate ratio of target cells to effector cells; incubating the
mixture of cells for 16-18 hours at 37.degree. C.; collecting
supernatants; and analyzing radioactivity. The cytotoxicity of the
molecules of the invention can then be determined, for example
using the following formula: % lysis=(experimental cpm-target leak
cpm)/(detergent lysis cpm-target leak cpm).times.100%.
Alternatively, % lysis=(ADCC-AICC)/(maximum release-spontaneous
release). Specific lysis can be calculated using the formula:
specific lysis=% lysis with the molecules of the invention--% lysis
in the absence of the molecules of the invention. A graph can be
generated by varying either the target:effector cell ratio or
antibody concentration.
[0389] In yet another embodiment, the molecules of the invention
are characterized for antibody dependent cellular cytotoxicity
(ADCC) see, e.g., Ding et al., Immunity, 1998, 8:403-11; which is
incorporated herein by reference in its entirety.
[0390] Preferably, the effector cells used in the ADCC assays of
the invention are peripheral blood mononuclear cells (PBMC) that
are preferably purified from normal human blood, using standard
methods known to one skilled in the art, e.g., using Ficoll-Paque
density gradient centrifugation. Preferred effector cells for use
in the methods of the invention express different Fc.gamma.R
activating receptors. The invention encompasses, effector cells,
THP-1, expressing Fc.gamma.RI, Fc.gamma.RIIA and Fc.gamma.RIIB, and
monocyte derived primary macrophages derived from whole human blood
expressing both Fc.gamma.RIIIA and Fc.gamma.RIIB, to determine if
Fc antibody mutants show increased ADCC activity and phagocytosis
relative to wild type IgG1 antibodies.
[0391] The human monocyte cell line, THP-1, activates phagocytosis
through expression of the high affinity receptor Fc.gamma.RI and
the low affinity receptor Fc.gamma.RIIA (Fleit et al., 1991, J.
Leuk. Biol. 49: 556). THP-1 cells do not constitutively express
Fc.gamma.RIIA or Fc.gamma.RIIB. Stimulation of these cells with
cytokines effects the FcR expression pattern (Pricop et al., 2000
J. Immunol. 166: 531-7). Growth of THP-1 cells in the presence of
the cytokine IL4 induces Fc.gamma.RIIB expression and causes a
reduction in Fc.gamma.RIIA and Fc.gamma.RI expression.
Fc.gamma.RIIB expression can also be enhanced by increased cell
density (Tridandapani et al., 2002, J. Biol. Chem. 277: 5082-9). In
contrast, it has been reported that IFN.gamma. can lead to
expression of Fc.gamma.RIIIA (Pearse et al., 1993 PNAS USA 90:
4314-8). The presence or absence of receptors on the cell surface
can be determined by FACS using common methods known to one skilled
in the art. Cytokine induced expression of Fc.gamma.R on the cell
surface provides a system to test both activation and inhibition in
the presence of Fc.gamma.RIIB. If THP-1 cells are unable to express
the Fc.gamma.RIIB the invention also encompasses another human
monocyte cell line, U937. These cells have been shown to terminally
differentiate into macrophages in the presence of IFN.gamma. and
TNF (Koren et al., 1979, Nature 279: 328-331).
[0392] Fc.gamma.R dependent tumor cell killing is mediated by
macrophage and NK cells in mouse tumor models (Clynes et al., 1998,
PNAS USA 95: 652-656). The invention encompasses the use of
elutriated monocytes from donors as effector cells to analyze the
efficiency Fc mutants to trigger cell cytotoxicity of target cells
in both phagocytosis and ADCC assays. Expression patterns of
Fc.gamma.RI, Fc.gamma.RIIIA, and Fc.gamma.RIIB are affected by
different growth conditions. Fc.gamma.R expression from frozen
elutriated monocytes, fresh elutriated monocytes, monocytes
maintained in 10% FBS, and monocytes cultured in FBS+GM-CSF and or
in human serum may be determined using common methods known to
those skilled in the art. For example, cells can be stained with
Fc.gamma.R specific antibodies and analyzed by FACS to determine
FcR profiles. Conditions that best mimic macrophage in vivo
Fc.gamma.R expression is then used for the methods of the
invention.
[0393] In some embodiments, the invention encompasses the use of
mouse cells especially when human cells with the right Fc.gamma.R
profiles are unable to be obtained. In some embodiments, the
invention encompasses the mouse macrophage cell line RAW264.7(ATCC)
which can be transfected with human Fc.gamma.RIIIA and stable
transfectants isolated using methods known in the art, see, e.g.,
Ralph et al., J. Immunol. 119: 950-4). Transfectants can be
quantitated for Fc.gamma.RIIIA expression by FACS analysis using
routine experimentation and high expressors can be used in the ADCC
assays of the invention. In other embodiments, the invention
encompasses isolation of spleen peritoneal macrophage expressing
human Fc.gamma.R from knockout transgenic mice such as those
disclosed herein.
[0394] Lymphocytes may be harvested from peripheral blood of donors
(PBM) using a Ficoll-Paque gradient (Pharmacia). Within the
isolated mononuclear population of cells the majority of the ADCC
activity occurs via the natural killer cells (NK) containing
Fc.gamma.RIIIA but not Fc.gamma.RIIB on their surface. Results with
these cells indicate the efficacy of the mutants on triggering NK
cell ADCC and establish the reagents to test with elutriated
monocytes.
[0395] Target cells used in the ADCC assays of the invention
include, but are not limited to, breast cancer cell lines, e.g.,
SK-BR-3 with ATCC accession number HTB-30 (see, e.g., Tremp et al.,
1976, Cancer Res. 33-41); B-lymphocytes; cells derived from
Burkitts lymphoma, e.g., Raji cells with ATCC accession number
CCL-86 (see, e.g., Epstein et al., 1965, J. Natl. Cancer Inst. 34:
231-240), and Daudi cells with ATCC accession number CCL-213 (see,
e.g., Klein et al., 1968, Cancer Res. 28: 1300-10). The target
cells must be recognized by the antigen binding site of the
immunoglobulin to be assayed.
[0396] The ADCC assay is based on the ability of NK cells to
mediate cell death via an apoptotic pathway. NK cells mediate cell
death in part by Fc.gamma.RIIIA's recognition of IgG bound to an
antigen on a cell surface. The ADCC assays used in accordance with
the methods of the invention may be radioactive based assays or
fluorescence based assays. The ADCC assay used to characterize the
molecules of the invention comprising variant Fc regions comprises
labeling target cells, e.g., SK-BR-3, MCF-7, OVCAR3, Raji, Daudi
cells, opsonizing target cells with an antibody that recognizes a
cell surface receptor on the target cell via its antigen binding
site; combining the labeled opsonized target cells and the effector
cells at an appropriate ratio, which can be determined by routine
experimentation; harvesting the cells; detecting the label in the
supernatant of the lysed target cells, using an appropriate
detection scheme based on the label used. The target cells may be
labeled either with a radioactive label or a fluorescent label,
using standard methods known in the art. For example the labels
include, but are not limited to, [.sup.51Cr]Na.sub.2CrO.sub.4; and
the acetoxymethyl ester of the fluorescence enhancing ligand,
2,2':6',2''-terpyridine-6-6''-dicarboxylate (TDA).
[0397] In a specific preferred embodiment, a time resolved
fluorimetric assay is used for measuring ADCC activity against
target cells that have been labeled with the acetoxymethyl ester of
the fluorescence enhancing ligand,
2,2':6',2''-terpyridine-6-6''-dicarboxylate (TDA). Such
fluorimetric assays are known in the art, e.g., see, Blomberg et
al., 1996, Journal of Immunological Methods, 193: 199-206; which is
incorporated herein by reference in its entirety. Briefly, target
cells are labeled with the membrane permeable acetoxymethyl diester
of TDA (bis(acetoxymethyl)
2,2':6',2''-terpyridine-6-6''-dicarboxylate, (BATDA), which rapidly
diffuses across the cell membrane of viable cells. Intracellular
esterases split off the ester groups and the regenerated membrane
impermeable TDA molecule is trapped inside the cell. After
incubation of effector and target cells, e.g., for at least two
hours, up to 3.5 hours, at 37.degree. C., under 5% CO.sub.2, the
TDA released from the lysed target cells is chelated with Eu3+ and
the fluorescence of the Europium-TDA chelates formed is quantitated
in a time-resolved fluorometer (e.g., Victor 1420, Perkin
Elmer/Wallac).
[0398] In another specific embodiment, the ADCC assay used to
characterize the molecules of the invention comprising variant Fc
regions comprises the following steps: Preferably
4-5.times.10.sup.6 target cells (e.g., SK-BR-3, MCF-7, OVCAR3, Raji
cells) are labeled with bis(acetoxymethyl)
2,2':6',2''-terpyridine-t-6''-dicarboxylate (DELFIA BATDA Reagent,
Perkin Elmer/Wallac). For optimal labeling efficiency, the number
of target cells used in the ADCC assay should preferably not exceed
5.times.10.sup.6. BATDA reagent is added to the cells and the
mixture is incubated at 37.degree. C. preferably under 5% CO.sub.2,
for at least 30 minutes. The cells are then washed with a
physiological buffer, e.g., PBS with 0.125 mM sulfinpyrazole, and
media containing 0.125 mM sulfinpyrazole. The labeled target cells
are then opsonized (coated) with a molecule of the invention
comprising a variant Fc region, i.e., an immunoglobulin comprising
a variant Fc region of the invention, including, but not limited
to, a polyclonal antibody, a monoclonal antibody, a bispecific
antibody, a multi-specific antibody, a humanized antibody, or a
chimeric antibody. In preferred embodiments, the immunoglobulin
comprising a variant Fc region used in the ADCC assay is specific
for a cell surface receptor, a tumor antigen, or a cancer antigen.
The immunoglobulin into which a variant Fc region of the invention
is introduced may specifically bind any cancer or tumor antigen,
such as those listed in section 5.4. Additionally, the
immunoglobulin into which a variant Fc region of the invention is
introduced may be any therapeutic antibody specific for a cancer
antigen, such as those listed in section 5.4. In some embodiments,
the immunoglobulin comprising a variant Fc region used in the ADCC
assay is an anti-fluoresceine monoclonal antibody, 4-4-20 (Kranz et
al., 1982 J. Biol. Chem. 257(12): 6987-6995) a mouse-human chimeric
anti-CD20 monoclonal antibody 2H7 (Liu et al., 1987, Journal of
Immunology, 139: 3521-6); or a humanized antibody (Ab4D5) against
the human epidermal growth factor receptor 2 (p185 HER2) (Carter et
al. (1992, Proc. Natl. Acad. Sci. USA 89: 4285-9). The target cells
in the ADCC assay are chosen according to the immunoglobulin into
which a variant Fc region of the invention has been introduced so
that the immunoglobulin binds a cell surface receptor of the target
cell specifically. Preferably, the ADCC assays of the invention are
performed using more than one engineered antibody, e.g., anti
Her2/neu, 4-4-20, 2B6, RITUXAN.TM., and 2H7, harboring the Fc
variants of the invention. In a most preferred embodiment, the Fc
variants of the invention are introduced into at least 3 antibodies
and their ADCC activities are tested. Although not intending to be
bound by a particular mechanism of action, examining at least 3
antibodies in these functional assays will diminish the chance of
eliminating a viable Fc mutation erroneously.
[0399] Target cells are added to effector cells, e.g., PBMC, to
produce effector:target ratios of approximately 1:1, 10:1, 30:1,
50:1, 75:1, or 100:1. In a specific embodiment, when the
immunoglobulin comprising a variant Fc region has the variable
domain of 4-4-20, the effector:target is 75:1. The effector and
target cells are incubated for at least two hours, up to 3.5 hours,
at 37.degree. C., under 5% CO.sub.2. Cell supernatants are
harvested and added to an acidic europium solution (e.g., DELFIA
Europium Solution, Perkin Elmer/Wallac). The fluorescence of the
Europium-TDA chelates formed is quantitated in a time-resolved
fluorometer (e.g., Victor 1420, Perkin Elmer/Wallac). Maximal
release (MR) and spontaneous release (SR) are determined by
incubation of target cells with 1% TX-100 and media alone,
respectively. Antibody independent cellular cytotoxicity (AICC) is
measured by incubation of target and effector cells in the absence
of antibody. Each assay is preferably performed in triplicate. The
mean percentage specific lysis is calculated as: Experimental
release (ADCC)-AICC)/(MR-SR).times.100.
[0400] The invention encompasses characterization of the Fc
variants in both NK-dependent and macrophage dependent ADCC assays.
Fc variants of the invention have altered phenotypes such as an
altered effector function as assayed in an NK dependent or
macrophage dependent assay.
[0401] The invention encompasses assays known in the art and
exemplified herein, to bind C1q and mediate complement dependent
cytotoxicity (CDC). To determine C1q binding, a C1q binding ELISA
may be performed. An exemplary assay may comprise the following:
assay plates may be coated overnight at 4.degree. C. with
polypeptide variant or starting polypeptide (control) in coating
buffer. The plates may then be washed and blocked. Following
washing, an aliquot of human C1q may be added to each well and
incubated for 2 hrs at room temperature. Following a further wash,
100 uL of a sheep anti-complement C1q peroxidase conjugated
antibody may be added to each well and incubated for 1 hour at room
temperature. The plate may again be washed with wash buffer and 100
ul of substrate buffer containing OPD (O-phenylenediamine
dihydrochloride (Sigma)) may be added to each well. The oxidation
reaction, observed by the appearance of a yellow color, may be
allowed to proceed for 30 minutes and stopped by the addition of
100 ul of 4.5 NH.sub.2SO.sub.4. The absorbance may then read at
(492-405) nm.
[0402] A preferred variant in accordance with the invention is one
that displays a significant reduction in C1q binding, as detected
and measured in this assay or a similar assay. Preferably the
molecule comprising an Fc variant displays about 50 fold reduction,
about 60 fold, about 80 fold, or about 90 fold reduction in C1q
binding compared to a control antibody having a nonmutated IgG1 Fc
region. In the most preferred embodiment, the molecule comprising
an Fc variant does not bind C1q, i.e. the variant displays about
100 fold or more reduction in C1q binding compared to the control
antibody.
[0403] Another exemplary variant is one which has a better binding
affinity for human C1q than the molecule comprising wild type Fc
region. Such a molecule may display, for example, about two-fold or
more, and preferably about five-fold or more, improvement in human
C1q binding compared to the parent molecule comprising wild type Fc
region. For example, human C1q binding may be about two-fold to
about 500-fold, and preferably from about two-fold or from about
five-fold to about 1000-fold improved compared to the molecule
comprising wild type Fc region.
[0404] To assess complement activation, a complement dependent
cytotoxicity (CDC) assay may be performed, e.g. as described in
Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), which
is incorporated herein by reference in its entirety. Briefly,
various concentrations of the molecule comprising a variant Fc
region and human complement may be diluted with buffer. Cells which
express the antigen to which the molecule comprising a variant Fc
region binds may be diluted to a density of about 1.times.10.sup.6
cells/ml. Mixtures of the molecule comprising a variant Fc region,
diluted human complement and cells expressing the antigen may be
added to a flat bottom tissue culture 96 well plate and allowed to
incubate for 2 hrs at 37.degree. C. and 5% CO.sub.2 to facilitate
complement mediated cell lysis. 50 uL of alamar blue (Accumed
International) may then be added to each well and incubated
overnight at 37.degree. C. The absorbance is measured using a
96-well fluorometer with excitation at 530 nm and emission at 590
nm. The results may be expressed in relative fluorescence units
(RFU). The sample concentrations may be computed from a standard
curve and the percent activity as compared to nonvariant molecule,
i.e., a molecule comprising wild type Fc region, is reported for
the variant of interest.
[0405] In some embodiments, an Fc variant of the invention does not
activate complement. Preferably the variant does not appear to have
any CDC activity in the above CDC assay. The invention also
pertains to a variant with enhanced CDC compared to a parent
molecule (a molecule comprising wild type Fc region), e.g.,
displaying about two-fold to about 100-fold improvement in CDC
activity in vitro or in vivo (e.g., at the IC50 values for each
molecule being compared). Complement assays may be performed with
guinea pig, rabbit or human serum. Complement lysis of target cells
may be detected by monitoring the release of intracellular enzymes
such as lactate dehydrogenase (LDH), as described in Korzeniewski
et al., 1983 Immunol. Methods 64(3): 313-20; and Decker et al.,
1988, J. Immunol. Methods 115(1): 61-9, each of which is
incorporated herein by reference in its entirety; or the release of
an intracellular lable such as europium, chromium 51 or indium 111
in which target cells are labeled as described herein.
[0406] 5.2.8 Other Assays
[0407] The molecules of the invention comprising variant Fc regions
may also be assayed using any surface plasmon resonance based
assays known in the art for characterizing the kinetic parameters
of Fc-Fc.gamma.R interaction binding. Any SPR instrument
commercially available including, but not limited to, BIAcore
Instruments, available from Biacore AB (Uppsala, Sweden); IAsys
instruments available from Affinity Sensors (Franklin, Mass.); IBIS
system available from Windsor Scientific Limited (Berks, UK),
SPR-CELLIA systems available from Nippon Laser and Electronics Lab
(Hokkaido, Japan), and SPR Detector Spreeta available from Texas
Instruments (Dallas, Tex.) can be used in the instant invention.
For a review of SPR-based technology see Mullet et al., 2000,
Methods 22: 77-91; Dong et al., 2002, Review in Mol. Biotech., 82:
303-23; Fivash et al., 1998, Current Opinion in Biotechnology 9:
97-101; Rich et al., 2000, Current Opinion in Biotechnology 11:
54-61; all of which are incorporated herein by reference in their
entirety. Additionally, any of the SPR instruments and SPR based
methods for measuring protein-protein interactions described in
U.S. Pat. Nos. 6,373,577; 6,289,286; 5,322,798; 5,341,215; and
6,268,125 are contemplated in the methods of the invention, all of
which are incorporated herein by reference in their entirety.
[0408] Briefly, SPR based assays involve immobilizing a member of a
binding pair on a surface, and monitoring its interaction with the
other member of the binding pair in solution in real time. SPR is
based on measuring the change in refractive index of the solvent
near the surface that occurs upon complex formation or
dissociation. The surface onto which the immobilization occur is
the sensor chip, which is at the heart of the SPR technology; it
consists of a glass surface coated with a thin layer of gold and
forms the basis for a range of specialized surfaces designed to
optimize the binding of a molecule to the surface. A variety of
sensor chips are commercially available especially from the
companies listed supra, all of which may be used in the methods of
the invention. Examples of sensor chips include those available
from BIAcore AB, Inc., e.g., Sensor Chip CMS, SA, NTA, and HPA. A
molecule of the invention may be immobilized onto the surface of a
sensor chip using any of the immobilization methods and chemistries
known in the art, including but not limited to, direct covalent
coupling via amine groups, direct covalent coupling via sulfhydryl
groups, biotin attachment to avidin coated surface, aldehyde
coupling to carbohydrate groups, and attachment through the
histidine tag with NTA chips.
[0409] In some embodiments, the kinetic parameters of the binding
of a molecule of the invention comprising variant Fc regions, e.g.,
immunoglobulins comprising variant Fc region, to an Fc.gamma.R may
be determined using a BIAcore instrument (e.g., BIAcore instrument
1000, BIAcore Inc., Piscataway, N.J.). Any Fc.gamma.R can be used
to assess the interaction with the molecules of the invention
comprising variant Fc regions. In a specific embodiment the
Fc.gamma.R is Fc.gamma.RIIIA, preferably a soluble monomeric
Fc.gamma.RIIIA. For example, in one embodiment, the soluble
monomeric Fc.gamma.RIIIA is the extracellular region of
Fc.gamma.RIIIA joined to the linker-AVITAG sequence (see, U.S.
Provisional Application No. 60/439,498, filed on Jan. 9, 2003
(Attorney Docket No. 11183-004-888) and U.S. Provisional
Application No. 60/456,041 filed on Mar. 19, 2003, which are
incorporated herein by reference in their entireties). In another
specific embodiment, the Fc.gamma.R is Fc.gamma.RIIB, preferably a
soluble dimeric Fc.gamma.RIIB. For example, in one embodiment, the
soluble dimeric Fc.gamma.RIIB protein is prepared in accordance
with the methodology described in U.S. Provisional application No.
60/439,709 filed on Jan. 13, 2003, which is incorporated herein by
reference in its entirety.
[0410] An exemplary assay for determining the kinetic parameters of
a molecule comprising a variant Fc region, wherein the molecule is
the 4-4-20 antibody, to an Fc.gamma.R using a BIAcore instrument
comprises the following: BSA-FITC is immobilized on one of the four
flow cells of a sensor chip surface, preferably through amine
coupling chemistry such that about 5000 response units (RU) of
BSA-FITC is immobilized on the surface. Once a suitable surface is
prepared, 4-4-20 antibodies carrying the Fc mutations are passed
over the surface, preferably by one minute injections of a 20
.mu.g/mL solution at a 5 .mu.L/mL flow rate. The level of 4-4-20
antibodies bound to the surface ranges between 400 and 700 RU.
Next, dilution series of the receptor (Fc.gamma.RIIA and
Fc.gamma.RIIB-Fc fusion protein) in HBS-P buffer (20 mM HEPES, 150
mM NaCl, 3 mM EDTA, pH 7.5) are injected onto the surface at 100
.mu.L/min Antibody regeneration between different receptor
dilutions is carried out preferably by single 5 second injections
of 100 mM NaHCO.sub.3 pH 9.4; 3M NaCl. Any regeneration technique
known in the art is contemplated in the method of the
invention.
[0411] Once an entire data set is collected, the resulting binding
curves are globally fitted using computer algorithms supplied by
the SPR instrument manufacturer, e.g., BIAcore, Inc. (Piscataway,
N.J.). These algorithms calculate both the K.sub.on and K.sub.off,
from which the apparent equilibrium binding constant, K.sub.d is
deduced as the ratio of the two rate constants (i.e.,
K.sub.off/K.sub.on). More detailed treatments of how the individual
rate constants are derived can be found in the BlAevaluaion
Software Handbook (BIAcore, Inc., Piscataway, N.J.). The analysis
of the generated data may be done using any method known in the
art. For a review of the various methods of interpretation of the
kinetic data generated see Myszka, 1997, Current Opinion in
Biotechnology 8: 50-7; Fisher et al., 1994, Current Opinion in
Biotechnology 5: 389-95; O'Shannessy, 1994, Current Opinion in
Biotechnology, 5:65-71; Chaiken et al., 1992, Analytical
Biochemistry, 201: 197-210; Morton et al., 1995, Analytical
Biochemistry 227: 176-85; O'Shannessy et al., 1996, Analytical
Biochemistry 236: 275-83; all of which are incorporated herein by
reference in their entirety.
[0412] In preferred embodiments, the kinetic parameters determined
using an SPR analysis, e.g., BIAcore, may be used as a predictive
measure of how a molecule of the invention will function in a
functional assay, e.g., ADCC. An exemplary method for predicting
the efficacy of a molecule of the invention based on kinetic
parameters obtained from an SPR analysis may comprise the
following: determining the K.sub.off values for binding of a
molecule of the invention to Fc.gamma.RIIIA and Fc.gamma.RIIB;
plotting (1) K.sub.off (wt)/K.sub.off (mut) for Fc.gamma.RIIIA; (2)
K.sub.off (mut)/K.sub.off (wt) for Fc.gamma.RIIB against the ADCC
data. Numbers higher than one show a decreased dissociation rate
for Fc.gamma.RIIIA and an increased dissociation rate for
Fc.gamma.RIIB relative to wild type; and possess and enhanced ADCC
function.
5.3 Methods of Recombinantly Producing Molecules of the
Invention
[0413] 5.3.1 Polynucleotides Encoding Molecules of the
Invention
[0414] The present invention also includes polynucleotides that
encode the molecules, including the polypeptides and antibodies, of
the invention identified by the methods of the invention. The
polynucleotides encoding the molecules of the invention may be
obtained, and the nucleotide sequence of the polynucleotides
determined, by any method known in the art.
[0415] Once the nucleotide sequence of the molecules (e.g.,
antibodies) that are identified by the methods of the invention is
determined, the nucleotide sequence may be manipulated using
methods well known in the art, e.g., recombinant DNA techniques,
site directed mutagenesis, PCR, etc. (see, for example, the
techniques described in Sambrook et al., 2001, Molecular Cloning, A
Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y.; and Ausubel et al., eds., 1998, Current
Protocols in Molecular Biology, John Wiley & Sons, NY, which
are both incorporated by reference herein in their entireties), to
generate, for example, antibodies having a different amino acid
sequence, for example by generating amino acid substitutions,
deletions, and/or insertions.
[0416] In a specific embodiment, when the nucleic acids encode
antibodies, one or more of the CDRs are inserted within framework
regions using routine recombinant DNA techniques. The framework
regions may be naturally occurring or consensus framework regions,
and preferably human framework regions (see, e.g., Chothia et al.,
1998, J. Mol. Biol. 278: 457-479 for a listing of human framework
regions).
[0417] In another embodiment, human libraries or any other
libraries available in the art, can be screened by standard
techniques known in the art, to clone the nucleic acids encoding
the molecules of the invention.
[0418] 5.3.2 Recombinant Expression of Molecules of the
Invention
[0419] Once a nucleic acid sequence encoding molecules of the
invention (i.e., antibodies) has been obtained, the vector for the
production of the molecules may be produced by recombinant DNA
technology using techniques well known in the art. Methods which
are well known to those skilled in the art can be used to construct
expression vectors containing the coding sequences for the
molecules of the invention and appropriate transcriptional and
translational control signals. These methods include, for example,
in vitro recombinant DNA techniques, synthetic techniques, and in
vivo genetic recombination. (See, for example, the techniques
described in Sambrook et al., 1990, Molecular Cloning, A Laboratory
Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y. and Ausubel et al. eds., 1998, Current Protocols in Molecular
Biology, John Wiley & Sons, NY).
[0420] An expression vector comprising the nucleotide sequence of a
molecule identified by the methods of the invention (i.e., an
antibody) can be transferred to a host cell by conventional
techniques (e.g., electroporation, liposomal transfection, and
calcium phosphate precipitation) and the transfected cells are then
cultured by conventional techniques to produce the molecules of the
invention. In specific embodiments, the expression of the molecules
of the invention is regulated by a constitutive, an inducible or a
tissue, specific promoter.
[0421] The host cells used to express the molecules identified by
the methods of the invention may be either bacterial cells such as
Escherichia coli, or, preferably, eukaryotic cells, especially for
the expression of whole recombinant immunoglobulin molecule. In
particular, mammalian cells such as Chinese hamster ovary cells
(CHO), in conjunction with a vector such as the major intermediate
early gene promoter element from human cytomegalovirus is an
effective expression system for immunoglobulins (Foecking et al.,
1998, Gene 45:101; Cockett et al., 1990, Bio/Technology 8:2).
[0422] A variety of host-expression vector systems may be utilized
to express the molecules identified by the methods of the
invention. Such host-expression systems represent vehicles by which
the coding sequences of the molecules of the invention may be
produced and subsequently purified, but also represent cells which
may, when transformed or transfected with the appropriate
nucleotide coding sequences, express the molecules of the invention
in situ. These include, but are not limited to, microorganisms such
as bacteria (e.g., E. coli and B. subtilis) transformed with
recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression
vectors containing coding sequences for the molecules identified by
the methods of the invention; yeast (e.g., Saccharomyces Pichia)
transformed with recombinant yeast expression vectors containing
sequences encoding the molecules identified by the methods of the
invention; insect cell systems infected with recombinant virus
expression vectors (e.g., baculovirus) containing the sequences
encoding the molecules identified by the methods of the invention;
plant cell systems infected with recombinant virus expression
vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic
virus (TMV)) or transformed with recombinant plasmid expression
vectors (e.g., Ti plasmid) containing sequences encoding the
molecules identified by the methods of the invention; or mammalian
cell systems (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells), lymphotic
cells (see U.S. Pat. No. 5,807,715), Per C.6 cells (human retinal
cells developed by Crucell) harboring recombinant expression
constructs containing promoters derived from the genome of
mammalian cells (e.g., metallothionein promoter) or from mammalian
viruses (e.g., the adenovirus late promoter; the vaccinia virus
7.5K promoter).
[0423] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
molecule being expressed. For example, when a large quantity of
such a protein is to be produced, for the generation of
pharmaceutical compositions of an antibody, vectors which direct
the expression of high levels of fusion protein products that are
readily purified may be desirable. Such vectors include, but are
not limited, to the E. coli expression vector pUR278 (Ruther et
al., 1983, EMBO J. 2:1791), in which the antibody coding sequence
may be ligated individually into the vector in frame with the lac Z
coding region so that a fusion protein is produced; pIN vectors
(Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van
Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the
like. pGEX vectors may also be used to express foreign polypeptides
as fusion proteins with glutathione S-transferase (GST). In
general, such fusion proteins are soluble and can easily be
purified from lysed cells by adsorption and binding to a matrix
glutathione-agarose beads followed by elution in the presence of
free glutathione. The pGEX vectors are designed to include thrombin
or factor Xa protease cleavage sites so that the cloned target gene
product can be released from the GST moiety.
[0424] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. The antibody
coding sequence may be cloned individually into non-essential
regions (e.g., the polyhedrin gene) of the virus and placed under
control of an AcNPV promoter (e.g., the polyhedrin promoter).
[0425] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, the antibody coding sequence of interest may be
ligated to an adenovirus transcription/translation control complex,
e.g., the late promoter and tripartite leader sequence. This
chimeric gene may then be inserted in the adenovirus genome by in
vitro or in vivo recombination. Insertion in a non-essential region
of the viral genome (e.g., region E1 or E3) will result in a
recombinant virus that is viable and capable of expressing the
immunoglobulin molecule in infected hosts (e.g., see Logan &
Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specific
initiation signals may also be required for efficient translation
of inserted antibody coding sequences. These signals include the
ATG initiation codon and adjacent sequences. Furthermore, the
initiation codon must be in phase with the reading frame of the
desired coding sequence to ensure translation of the entire insert.
These exogenous translational control signals and initiation codons
can be of a variety of origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (see Bittner et al., 1987, Methods in Enzymol.
153:51-544).
[0426] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the
protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins and gene products. Appropriate cell lines or host
systems can be chosen to ensure the correct modification and
processing of the foreign protein expressed. To this end,
eukaryotic host cells which possess the cellular machinery for
proper processing of the primary transcript, glycosylation, and
phosphorylation of the gene product may be used. Such mammalian
host cells include but are not limited to CHO, VERY, BHK, Hela,
COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and
T47D, CRL7030 and Hs578Bst.
[0427] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express an antibody of the invention may be
engineered. Rather than using expression vectors which contain
viral origins of replication, host cells can be transformed with
DNA controlled by appropriate expression control elements (e.g.,
promoter, enhancer, sequences, transcription terminators,
polyadenylation sites, etc.), and a selectable marker. Following
the introduction of the foreign DNA, engineered cells may be
allowed to grow for 1-2 days in an enriched media, and then are
switched to a selective media. The selectable marker in the
recombinant plasmid confers resistance to the selection and allows
cells to stably integrate the plasmid into their chromosomes and
grow to form foci which in turn can be cloned and expanded into
cell lines. This method may advantageously be used to engineer cell
lines which express the antibodies of the invention. Such
engineered cell lines may be particularly useful in screening and
evaluation of compounds that interact directly or indirectly with
the antibodies of the invention.
[0428] A number of selection systems may be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler et
al., 1977, Cell 11: 223), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, 1992, Proc.
Natl. Acad. Sci. USA 48: 202), and adenine
phosphoribosyltransferase (Lowy et al., 1980, Cell 22: 817) genes
can be employed in tk-, hgprt- or aprt-cells, respectively. Also,
antimetabolite resistance can be used as the basis of selection for
the following genes: dhfr, which confers resistance to methotrexate
(Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et
al., 1981, Proc. Natl. Acad. Sci. USA 78: 1527); gpt, which confers
resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc.
Natl. Acad. Sci. USA 78: 2072); neo, which confers resistance to
the aminoglycoside G-418 Clinical Pharmacy 12: 488-505; Wu and Wu,
1991, 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol.
32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and
Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH
11(5):155-215). Methods commonly known in the art of recombinant
DNA technology which can be used are described in Ausubel et al.
(eds.), 1993, Current Protocols in Molecular Biology, John Wiley
& Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A
Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13,
Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics,
John Wiley & Sons, NY.; Colberre-Garapin et al., 1981, J. Mol.
Biol. 150:1; and hygro, which confers resistance to hygromycin
(Santerre et al., 1984, Gene 30:147).
[0429] The expression levels of an antibody of the invention can be
increased by vector amplification (for a review, see Bebbington and
Hentschel, The use of vectors based on gene amplification for the
expression of cloned genes in mammalian cells in DNA cloning, Vol.
3 (Academic Press, New York, 1987). When a marker in the vector
system expressing an antibody is amplifiable, increase in the level
of inhibitor present in culture of host cell will increase the
number of copies of the marker gene. Since the amplified region is
associated with the nucleotide sequence of the antibody, production
of the antibody will also increase (Crouse et al., 1983, Mol. Cell.
Biol. 3:257).
[0430] The host cell may be co-transfected with two expression
vectors of the invention, the first vector encoding a heavy chain
derived polypeptide and the second vector encoding a light chain
derived polypeptide. The two vectors may contain identical
selectable markers which enable equal expression of heavy and light
chain polypeptides. Alternatively, a single vector may be used
which encodes both heavy and light chain polypeptides. In such
situations, the light chain should be placed before the heavy chain
to avoid an excess of toxic free heavy chain (Proudfoot, 1986,
Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197).
The coding sequences for the heavy and light chains may comprise
cDNA or genomic DNA.
[0431] Once a molecule of the invention has been recombinantly
expressed, it may be purified by any method known in the art for
purification of polypeptides or antibodies, for example, by
chromatography (e.g., ion exchange, affinity, particularly by
affinity for the specific antigen after Protein A, and sizing
column chromatography), centrifugation, differential solubility, or
by any other standard technique for the purification of
polypeptides or antibodies.
5.4 Prophylactic and Therapeutic Methods
[0432] The molecules of the invention with conferred effector
function activity are particularly useful for the treatment and/or
prevention of a disease, disorder or infection where an enhanced
efficacy of effector cell function (e.g., ADCC) mediated by
Fc.gamma.R is desired (e.g., cancer, infectious disease), and in
enhancing the therapeutic efficacy of therapeutic antibodies, the
effect of which is mediated by an effector function activity, e.g.,
ADCC.
[0433] The invention encompasses methods and compositions for
treatment, prevention or management of a cancer in a subject,
comprising administering to the subject a therapeutically effective
amount of one or more molecules comprising a variant Fc region
engineered in accordance with the invention, which molecule further
binds a cancer antigen. Molecules of the invention comprising the
variant Fc regions are particularly useful for the prevention,
inhibition, reduction of growth or regression of primary tumors,
metastasis of cancer cells, and infectious diseases. Although not
intending to be bound by a particular mechanism of action,
molecules of the invention enhance the efficacy of cancer
therapeutics by enhancing antibody mediated effector function
resulting in an enhanced rate of tumor clearance or an enhanced
rated of tumor reduction or a combination thereof. In alternate
embodiments, the modified antibodies of the invention enhance the
efficacy of cancer therapeutics by conferring oligomerization
activity to variant Fc region, resulting in cross-linking of cell
surface antigens and/or receptors and enhanced apoptosis or
negative growth regulatory signaling.
[0434] According to an aspect of the invention, immunotherapeutics
may be enhanced by modifying the Fc region in accordance with the
invention to confer or increase the potency of an antibody effector
function activity, e.g., ADCC, CDC, phagocytosis, opsonization,
etc., of the immunotherapeutic. In a specific embodiment, antibody
dependent cellular toxicity and/or phagocytosis of tumor cells or
infected cells is enhanced by modifying immunotherapeutics with
variant Fc regions of the invention. Molecules of the invention may
enhance the efficacy of immunotherapy treatment by enhancing at
least one antibody-mediated effector function activity. In one
particular embodiment, the efficacy of immunotherapy treatment is
enhanced by enhancing the complement dependent cascade. In another
embodiment of the invention, the efficacy of immunotherapy
treatment is enhanced by enhancing the phagocytosis and/or
opsonization of the targeted cells, e.g., tumor cells. In another
embodiment of the invention, the efficacy of treatment is enhanced
by enhancing antibody-dependent cell-mediated cytotoxicity ("ADCC")
in destruction of the targeted cells, e.g., tumor cells. The
molecules of the invention may make an antibody that does not have
a therapeutic effect in patients or in a subpopulation of patients
have a therapeutic effect.
[0435] Although not intending to be bound by a particular mechanism
of action, therapeutic antibodies engineered in accordance with the
invention have enhanced therapeutic efficacy, in part, due to the
ability of the Fc portion of the antibody to bind a target cell
which expresses the particular Fc.gamma.Rs at reduced levels, for
example, by virtue of the ability of the antibody to remain on the
target cell longer due to an improved off rate for Fc.gamma.R
interaction.
[0436] The antibodies of the invention with enhanced affinity and
avidity for Fc.gamma.Rs are particularly useful for the treatment,
prevention or management of a cancer, or another disease or
disorder, in a subject, wherein the Fc.gamma.Rs are expressed at
low levels in the target cell populations. As used herein,
Fc.gamma.R expression in cells is defined in terms of the density
of such molecules per cell as measured using common methods known
to those skilled in the art. The molecules of the invention
comprising variant Fc regions preferably also have a conferred or
an enhanced avidity and affinity and/or effector function in cells
which express a target antigen, e.g., a cancer antigen, at a
density of 30,000 to 20,000 molecules/cell, at a density of 20,000
to 10,000 molecules/cell, at a density of 10,000 molecules/cell or
less, at a density of 5000 molecules/cell or less, or at a density
of 1000 molecules/cell or less. The molecules of the invention have
particular utility in treatment, prevention or management of a
disease or disorder, such as cancer, in a sub-population, wherein
the target antigen is expressed at low levels in the target cell
population.
[0437] The molecules of the invention may also be advantageously
utilized in combination with other therapeutic agents known in the
art for the treatment or prevention of diseases, such as cancer,
autoimmune disease, inflammatory disorders, and infectious
diseases. In a specific embodiment, molecules of the invention may
be used in combination with monoclonal or chimeric antibodies,
lymphokines, or hematopoietic growth factors (such as, e.g., IL-2,
IL-3 and IL-7), which, for example, serve to increase the number or
activity of effector cells which interact with the molecules and,
increase immune response. The molecules of the invention may also
be advantageously utilized in combination with one or more drugs
used to treat a disease, disorder, or infection such as, for
example anti-cancer agents, anti-inflammatory agents or anti-viral
agents, e.g., as detailed in sections 5.4.1.2 and 5.4.2.1
below.
[0438] 5.4.1 Cancers
[0439] The invention encompasses methods and compositions for
treatment or prevention of cancer in a subject comprising
administering to the subject a therapeutically effective amount of
one or more molecules comprising a variant Fc region. In some
embodiments, the invention encompasses methods and compositions for
the treatment or prevention of cancer in a subject with Fc.gamma.R
polymorphisms such as those homozygous for the FyRIIIA-158V or
Fc.gamma.RIIIA-158F alleles. In some embodiments, the invention
encompasses engineering therapeutic antibodies, e.g., tumor
specific monoclonal antibodies in accordance with the methods of
the invention such that the engineered antibodies have enhanced
efficacy in patients homozygous for the low affinity allele of
Fc.gamma.RIIIA (158F). In other embodiments, the invention
encompasses engineering therapeutic antibodies, e.g., tumor
specific monoclonal antibodies in accordance with the methods of
the invention such that the engineered antibodies have enhanced
efficacy in patients homozygous for the high affinity allele of
Fc.gamma.RIIIA (158V).
[0440] The efficacy of monoclonal antibodies may depend on the
Fc.gamma.R polymorphism of the subject (Carton et al., 2002 Blood,
99: 754-8; Weng et al., 2003 J Clin Oncol. 21(21):3940-7 both of
which are incorporated herein by reference in their entireties).
These receptors are expressed on the surface of the effector cells
and mediate ADCC. High affinity alleles, of the low affinity
activating receptors, improve the effector cells' ability to
mediate ADCC. The methods of the invention allow engineering
molecules harboring Fc mutations to enhance their affinity to
Fc.gamma.R on effector cells via their altered Fc domains. The
engineered antibodies of the invention provide better immunotherapy
reagents for patients regardless of their Fc.gamma.R
polymorphism.
[0441] Molecules harboring the Fc variants are tested by ADCC using
either a cultured cell line or patient derived PMBC cells to
determine the ability of the Fc mutations to enhance ADCC. Standard
ADCC is performed using methods disclosed herein. Lymphocytes are
harvested from peripheral blood using a Ficoll-Paque gradient
(Pharmacia). Target cells, i.e., cultured cell lines or patient
derived cells, are loaded with Europium (PerkinElmer) and incubated
with effectors for 4 hrs at 37.degree. C. Released Europium is
detected using a fluorescent plate reader (Wallac). The resulting
ADCC data indicates the efficacy of the Fc variants to trigger NK
cell mediated cytotoxicity and establish which Fc variants can be
tested with both patient samples and elutriated monocytes. Fc
variants showing the greatest potential for enhancing the efficacy
of the molecule are then tested in an ADCC assay using PBMCs from
patients. PBMC from healthy donors are used as effector cells.
[0442] According to an aspect of the invention, molecules of the
invention comprising variant Fc regions enhance the efficacy of
immunotherapy by conferring or increasing the potency of an
antibody effector function relative to a molecule containing the
wild-type Fc region, e.g., ADCC, CDC, phagocytosis, opsonization,
etc. In a specific embodiment, antibody dependent cellular toxicity
and/or phagocytosis of tumor cells is conferred or enhanced using
the molecules of the invention with variant Fc regions. Molecules
of the invention may enhance the efficacy of immunotherapy cancer
treatment by conferring or enhancing at least one antibody-mediated
effector function. In one particular embodiment, a molecule of the
invention comprising a variant Fc region confers or enhances the
efficacy of immunotherapy treatment by enhancing the complement
dependent cascade. In another embodiment of the invention, the
molecule of the invention comprising a variant Fc region enhances
the efficacy of immunotherapy treatment by conferring or enhancing
the phagocytosis and/or opsonization of the targeted tumor cells.
In another embodiment of the invention, the molecule of the
invention comprising a variant Fc region enhances the efficacy of
treatment by conferring or enhancing antibody-dependent
cell-mediated cytotoxicity ("ADCC") in destruction of the targeted
tumor cells.
[0443] The invention further contemplates engineering therapeutic
antibodies (e.g., tumor specific monoclonal antibodies) for
enhancing the therapeutic efficacy of the therapeutic antibody, for
example, by enhancing the effector function of the therapeutic
antibody (e.g., ADCC), or conferring effector function to a
therapeutic antibody which doesn't have that effector function (at
least detectable in an in vitro or in vivo assay). Preferably the
therapeutic antibody is a cytotoxic and/or opsonizing antibody. It
will be appreciated by one of skill in the art, that once molecules
of the invention with desired binding properties (e.g., molecules
with variant Fc regions with at least one amino acid modification,
which modification enhances the affinity of the variant Fc region
for Fc.gamma.RIIIA and/or Fc.gamma.RIIA relative to a comparable
molecule, comprising a wild-type Fc region) have been identified
(See Section 5.2 and Table 8) according to the methods of the
invention, therapeutic antibodies may be engineered using standard
recombinant DNA techniques and any known mutagenesis techniques, as
described in Section 5.2.2 to produce engineered therapeutic
carrying the identified mutation sites with the desired binding
properties. Any of the therapeutic antibodies listed in Table 11
that have demonstrated therapeutic utility in cancer treatment, may
be engineered according to the methods of the invention, for
example, by modifying the Fc region to confer an effector function
or have an enhanced affinity for Fc.gamma.RIIIA and/or
Fc.gamma.RIIA compared to a therapeutic antibody having a wild-type
Fc region.
[0444] The Fc variants of the invention may be incorporated into
therapeutic antibodies such as those disclosed herein or other Fc
fusion clinical candidates, i.e., a molecule comprising an Fc
regions which has been approved for us in clinical trials or any
other molecule that may benefit from the Fc variants of the instant
invention, humanized, affinity matured, modified or engineered
versions thereof.
[0445] The invention also encompasses engineering any other
polypeptide comprising an Fc region which has therapeutic utility,
including but not limited to ENBREL, according to the methods of
the invention, in order to enhance the therapeutic efficacy of such
polypeptides, for example, by enhancing the effector function of
the polypeptide comprising an Fc region.
TABLE-US-00012 TABLE 11 THERAPEUTIC ANTIBODIES THAT CAN BE
ENGINEERED ACCORDING TO THE METHODS OF THE INVENTION Company
Product Disease Target Abgenix ABX-EGF .TM. anti-EGF Cancer EGF
receptor Receptor Antibody AltaRex OvaRex .TM. anti-CA125 ovarian
cancer tumor antigen CA125 Antibody BravaRex .TM. anti-MUC1
metastatic tumor antigen MUC1 Antibody cancers Antisoma Theragyn
.TM. ovarian cancer PEM antigen (pemtumomabytrrium- 90) Therex .TM.
breast cancer PEM antigen Boehringer Blvatuzumab head & neck
CD44 Ingelheim cancer Centocor/J&J Panorex .TM. anti-17-1A
Colorectal 17-1A Antibody cancer ReoPro .TM. PTCA gp IIIb/IIIa
ReoPro .TM. Acute MI gp IIIb/IIIa ReoPro .TM. Ischemic stroke gp
IIIb/IIIa Corixa Bexocar .TM. NHL CD20 CRC MAb, idiotypic 105AD7
colorectal cancer gp72 Technology vaccine Crucell Anti-EpCAM .TM.
cancer Ep-CAM Cytoclonal MAb, lung cancer non-small cell NA lung
cancer Genentech Herceptin .TM. anti-Her-2 metastatic breast HER-2
Antibody cancer Herceptin .TM. anti-Her-2 early stage HER-2
Antibody breast cancer Rituxan .TM. anti-CD20 Relapsed/refractory
CD20 Antibody low-grade or follicular NHL Rituxan .TM. anti-CD20
intermediate & CD20 Antibody high-grade NHL MAb-VEGF NSCLC,
VEGF metastatic MAb-VEGF Colorectal VEGF cancer, metastatic AMD Fab
age-related CD18 macular degeneration E-26 (2.sup.nd gen. IgE)
allergic asthma IgE & rhinitis IDEC Zevalin .TM. (Rituxan .TM.
low grade of CD20 anti-CD20 Antibody + follicular, yttrium-90)
relapsed or refractory, CD20-positive, B-cell NHL and Rituximab-
refractory NHL ImClone Cetuximab + innotecan refractory EGF
receptor colorectal carcinoma Cetuximab + cisplatin & newly
diagnosed EGF receptor radiation or recurrent head & neck
cancer Cetuximab + newly diagnosed EGF receptor gemcitabine
metastatic pancreatic carcinoma Cetuximab + cisplatin + recurrent
or EGF receptor 5FU or Taxol metastatic head & neck cancer
Cetuximab + newly diagnosed EGF receptor carboplatin + paclitaxel
non-small cell lung carcinoma Cetuximab + cisplatin head & neck
EGF receptor cancer (extensive incurable local- regional disease
& distant metasteses) Cetuximab + radiation locally advanced
EGF receptor head & neck carcinoma BEC2 + Bacillus small cell
lung mimics ganglioside Calmette Guerin carcinoma GD3 BEC2 +
Bacillus melanoma mimics ganglioside Calmette Guerin GD3 IMC-1C11
colorectal cancer VEGF-receptor with liver metasteses ImmonoGen
nuC242-DM1 Colorectal, nuC242 gastric, and pancreatic cancer
ImmunoMedics LymphoCide .TM. anti- Non-Hodgkins CD22 CD22 Antibody
lymphoma LymphoCide Y-90 .TM. Non-Hodgkins CD22 anti-CD22 Antibody
lymphoma CEA-Cide .TM. metastatic solid CEA tumors CEA-Cide .TM.
Y-90 metastatic solid CEA tumors CEA-Scan .TM. (Tc-99m- colorectal
cancer CEA labeled arcitumomab) (radioimaging) CEA-Scan .TM.
(Tc-99m- Breast cancer CEA labeled arcitumomab) (radioimaging)
CEA-Scan .TM. (Tc-99m- lung cancer CEA labeled arcitumomab)
(radioimaging) CEA-Scan .TM. (Tc-99m- intraoperative CEA labeled
arcitumomab) tumors (radio imaging) LeukoScan .TM. (Tc-99m- soft
tissue CEA labeled sulesomab) infection (radioimaging) LymphoScan
.TM. (Tc- lymphomas CD22 99m-labeled) (radioimaging) AFP-Scan .TM.
(Tc-99m- liver 7 gem-cell AFP labeled) cancers (radioimaging)
Intracel HumaRAD-HN .TM. (+ head & neck NA yttrium-90) cancer
HumaSPECT .TM. colorectal NA imaging Medarex MDX-101 .TM. (CTLA-4)
Prostate and CTLA-4 other cancers MDX-210 .TM. (her-2 Prostate
cancer HER-2 overexpression) MDX-210 .TM./MAK Cancer HER-2
MedImmune Vitaxin .TM. Cancer .alpha.v.beta..sub.3 Merck KGaA MAb
425 Various cancers EGF receptor IS-IL-2 Various cancers Ep-CAM
Millennium Campath .RTM. anti-CD52 chronic CD52 Antibody
lymphocytic (alemtuzumab) leukemia NeoRx CD20-streptavidin (+
Non-Hodgkins CD20 biotin-yttrium 90) lymphoma Avidicin .TM.
(albumin + metastatic NA NRLU13) cancer Peregrine Oncolym .TM. (+
iodine- Non-Hodgkins HLA-DR 10 beta 131) lymphoma Cotara .TM. (+
iodine-131) unresectable DNA-associated malignant proteins glioma
Pharmacia C215 .TM. (+ pancreatic NA Corporation staphylococcal
cancer enterotoxin) MAb, lung/kidney lung & kidney NA cancer
cancer nacolomab tafenatox colon & NA (C242 + staphylococcal
pancreatic enterotoxin) cancer Protein Design Nuvion .TM. T cell
CD3 Labs malignancies SMART M195 .TM. anti- AML CD33 CD33 Antibody
SMART 1D10 .TM. anti- NHL HLA-DR antigen HLA-DR Antibody Titan
CEAVac .TM. colorectal CEA cancer, advanced TriGem .TM. metastatic
GD2-ganglioside melanoma & small cell lung cancer TriAb .TM.
metastatic breast MUC-1 cancer Trilex CEAVa .TM. c colorectal CEA
cancer, advanced TriGem .TM. metastatic GD2-ganglioside melanoma
& small cell lung cancer TriAb .TM. metastatic breast MUC-1
cancer Viventia NovoMAb-G2 .TM. Non-Hodgkins NA Biotech
radiolabeled lymphoma Monopharm C .TM. colorectal & SK-1
antigen pancreatic carcinoma GlioMAb-H .TM. (+ gliorna, NA gelonin
toxin) melanoma & neuroblastoma Xoma Rituxan .TM. anti-CD20
Relapsed/refractory CD20 Antibody low-grade or follicular NHL
Rituxan .TM. anti-CD20 intermediate & CD20 Antibody high-grade
NHL ING-1 .TM. adenomcarcinoma Ep-CAM
[0446] Accordingly, the invention provides methods of preventing or
treating cancer characterized by a cancer antigen, using a
therapeutic antibody that binds a cancer antigen and is cytotoxic
and has been modified at one or more sites in the Fc region,
according to the invention, to bind Fc.gamma.RIIIA and/or
Fc.gamma.RIIA with a higher affinity than the parent therapeutic
antibody, and/or mediates one or more effector functions (e.g.,
ADCC, phagocytosis) either not detectably mediated by the parent
antibody or more effectively than the parent antibody. In another
embodiment, the invention provides methods of preventing or
treating cancer characterized by a cancer antigen, using a
therapeutic antibody that binds a cancer antigen and is cytotoxic,
and has been engineered according to the invention to bind
Fc.gamma.RIIIA and/or Fc.gamma.RIIA with a higher affinity and bind
Fc.gamma.RIIB with a lower affinity than the parent therapeutic
antibody, and/or mediates one or more effector functions (e.g.,
ADCC, phagocytosis) either not detectably mediated by the parent
antibody or more effectively than the parent antibody. The
therapeutic antibodies that have been engineered according to the
invention are useful for prevention or treatment of cancer, since
they have an enhanced cytotoxic activity (e.g., enhanced tumor cell
killing and/or enhanced for example, ADCC activity or CDC
activity).
[0447] Cancers associated with a cancer antigen may be treated or
prevented by administration of a therapeutic antibody that binds a
cancer antigen and is cytotoxic, and has been engineered according
to the methods of the invention to have, for example, a conferred
or an enhanced effector function. In one particular embodiment, the
therapeutic antibodies engineered according to the methods of the
invention enhance the antibody-mediated cytotoxic effect of the
antibody directed at the particular cancer antigen. For example,
but not by way of limitation, cancers associated with the following
cancer antigens may be treated or prevented by the methods and
compositions of the invention: KS 1/4 pan-carcinoma antigen (Perez
and Walker, 1990, J. Immunol. 142:32-37; Bumal, 1988, Hybridoma
7(4):407-415), ovarian carcinoma antigen (CA125) (Yu et al., 1991,
Cancer Res. 51(2):48-475), prostatic acid phosphate (Tailor et al.,
1990, Nucl. Acids Res. 18(1):4928), prostate specific antigen
(Henttu and Vihko, 1989, Biochem. Biophys. Res. Comm.
10(2):903-910; Israeli et al., 1993, Cancer Res. 53:227-230),
melanoma-associated antigen p97 (Estin et al., 1989, J. Natl.
Cancer Instil. 81(6):445-44), melanoma antigen gp75 (Vijayasardahl
et al., 1990, J. Exp. Med. 171(4):1375-1380), high molecular weight
melanoma antigen (HMW-MAA) (Natali et al., 1987, Cancer 59:55-3;
Mittelman et al., 1990, J. Clin. Invest. 86:2136-2144)), prostate
specific membrane antigen, carcinoembryonic antigen (CEA) (Foon et
al., 1994, Proc. Am. Soc. Clin. Oncol. 13:294), polymorphic
epithelial mucin antigen, human milk fat globule antigen,
Colorectal tumor-associated antigens such as: CEA, TAG-72 (Yokata
et al., 1992, Cancer Res. 52:3402-3408), C017-1A (Ragnhammar et
al., 1993, Int. J. Cancer 53:751-758); GICA 19-9 (Herlyn et al.,
1982, J. Clin. Immunol. 2:135), CTA-1 and LEA, Burkitt's lymphoma
antigen-38.13, CD19 (Ghetie et al., 1994, Blood 83:1329-1336),
human B-lymphoma antigen-CD20 (Reff et al., 1994, Blood
83:435-445), CD33 (Sgouros et al., 1993, J. Nucl. Med. 34:422-430),
melanoma specific antigens such as ganglioside GD2 (Saleh et al.,
1993, J. Immunol., 151, 3390-3398), ganglioside GD3 (Shitara et
al., 1993, Cancer Immunol. Immunother. 36:373-380), ganglioside GM2
(Livingston et al., 1994, J. Clin. Oncol. 12:1036-1044),
ganglioside GM3 (Hoon et al., 1993, Cancer Res. 53:5244-5250),
tumor-specific transplantation type of cell-surface antigen (TSTA)
such as virally-induced tumor antigens including T-antigen DNA
tumor viruses and envelope antigens of RNA tumor viruses, oncofetal
antigen-alpha-fetoprotein such as CEA of colon, bladder tumor
oncofetal antigen (Hellstrom et al., 1985, Cancer. Res.
45:2210-2188), differentiation antigen such as human lung carcinoma
antigen L6, L20 (Hellstrom et al., 1986, Cancer Res. 46:3917-3923),
antigens of fibrosarcoma, human leukemia T cell antigen-Gp37
(Bhattacharya-Chatterjee et al., 1988, J. of Immun. 141:1398-1403),
neoglycoprotein, sphingolipids, breast cancer antigen such as EGFR
(Epidermal growth factor receptor), HER2 antigen (p185.sup.HER2),
polymorphic epithelial mucin (PEM) (Hilkens et al., 1992, Trends in
Bio. Chem. Sci. 17:359), malignant human lymphocyte antigen-APO-1
(Bernhard et al., 1989, Science 245:301-304), differentiation
antigen (Feizi, 1985, Nature 314:53-57) such as I antigen found in
fetal erthrocytes and primary endoderm, I(Ma) found in gastric
adencarcinomas, M18 and M39 found in breast epithelium, SSEA-1
found in myeloid cells, VEP8, VEP9, Myl, VIM-D5, and D.sub.156-22
found in colorectal cancer, TRA-1-85 (blood group H), C14 found in
colonic adenocarcinoma, F3 found in lung adenocarcinoma, AH6 found
in gastric cancer, Y hapten, Le found in embryonal carcinoma cells,
TL5 (blood group A), EGF receptor found in A431 cells, E.sub.1
series (blood group B) found in pancreatic cancer, FC10.2 found in
embryonal carcinoma cells, gastric adenocarcinoma, CO-514 (blood
group Le.sup.a) found in adenocarcinoma, NS-10 found in
adenocarcinomas, CO-43 (blood group Le.sup.b), G49, EGF receptor,
(blood group ALe.sup.b/LeY) found in colonic adenocarcinoma, 19.9
found in colon cancer, gastric cancer mucins, T.sub.5A.sub.7 found
in myeloid cells, R.sub.24 found in melanoma, 4.2, G.sub.D3, D1.1,
OFA-1, G.sub.M2, OFA-2, G.sub.D2, M1:22:25:8 found in embryonal
carcinoma cells and SSEA-3, SSEA-4 found in 4-8-cell stage embryos.
In another embodiment, the antigen is a T cell receptor derived
peptide from a cutaneous T cell lymphoma (see Edelson, 1998, The
Cancer Journal 4:62).
[0448] Cancers and related disorders that can be treated or
prevented by methods and compositions of the present invention
include, but are not limited to, the following: Leukemias
including, but not limited to, acute leukemia, acute lymphocytic
leukemia, acute myelocytic leukemias such as myeloblastic,
promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias
and myelodysplastic syndrome, chronic leukemias such as but not
limited to, chronic myelocytic (granulocytic) leukemia, chronic
lymphocytic leukemia, hairy cell leukemia; polycythemia vera;
lymphomas such as but not limited to Hodgkin's disease,
non-Hodgkin's disease; multiple myelomas such as but not limited to
smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic
myeloma, plasma cell leukemia, solitary plasmacytoma and
extramedullary plasmacytoma; Waldenstrom's macroglobulinemia;
monoclonal gammopathy of undetermined significance; benign
monoclonal gammopathy; heavy chain disease; bone and connective
tissue sarcomas such as but not limited to bone sarcoma,
osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell
tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma,
soft-tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma,
Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma,
neurilemmoma, rhabdomyosarcoma, synovial sarcoma; brain tumors
including but not limited to, glioma, astrocytoma, brain stem
glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic
neurinoma, craniopharyngioma, medulloblastoma, meningioma,
pineocytoma, pineoblastoma, primary brain lymphoma; breast cancer
including, but not limited to, adenocarcinoma, lobular (small cell)
carcinoma, intraductal carcinoma, medullary breast cancer, mucinous
breast cancer, tubular breast cancer, papillary breast cancer,
Paget's disease, and inflammatory breast cancer; adrenal cancer,
including but not limited to, pheochromocytom and adrenocortical
carcinoma; thyroid cancer such as but not limited to papillary or
follicular thyroid cancer, medullary thyroid cancer and anaplastic
thyroid cancer; pancreatic cancer, including but not limited to,
insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting
tumor, and carcinoid or islet cell tumor; pituitary cancers
including but not limited to, Cushing's disease,
prolactin-secreting tumor, acromegaly, and diabetes insipius; eye
cancers including but not limited to, ocular melanoma such as iris
melanoma, choroidal melanoma, and cilliary body melanoma, and
retinoblastoma; vaginal cancers, including but not limited to,
squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar
cancer, including but not limited to, squamous cell carcinoma,
melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and
Paget's disease; cervical cancers including but not limited to,
squamous cell carcinoma, and adenocarcinoma; uterine cancers
including but not limited to, endometrial carcinoma and uterine
sarcoma; ovarian cancers including but not limited to, ovarian
epithelial carcinoma, borderline tumor, germ cell tumor, and
stromal tumor; esophageal cancers including but not limited to,
squamous cancer, adenocarcinoma, adenoid cyctic carcinoma,
mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma,
melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small
cell) carcinoma; stomach cancers including but not limited to,
adenocarcinoma, fungating (polypoid), ulcerating, superficial
spreading, diffusely spreading, malignant lymphoma, liposarcoma,
fibrosarcoma, and carcinosarcoma; colon cancers; rectal cancers;
liver cancers including but not limited to hepatocellular carcinoma
and hepatoblastoma, gallbladder cancers including but not limited
to, adenocarcinoma; cholangiocarcinomas including but not limited
to, pappillary, nodular, and diffuse; lung cancers including but
not limited to, non-small cell lung cancer, squamous cell carcinoma
(epidermoid carcinoma), adenocarcinoma, large-cell carcinoma and
small-cell lung cancer; testicular cancers including but not
limited to, germinal tumor, seminoma, anaplastic, classic
(typical), spermatocytic, nonseminoma, embryonal carcinoma,
teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate
cancers including but not limited to, adenocarcinoma,
leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers
including but not limited to, squamous cell carcinoma; basal
cancers; salivary gland cancers including but not limited to,
adenocarcinoma, mucoepidermoid carcinoma, and adenoidcystic
carcinoma; pharynx cancers including but not limited to, squamous
cell cancer, and verrucous; skin cancers including but not limited
to, basal cell carcinoma, squamous cell carcinoma and melanoma,
superficial spreading melanoma, nodular melanoma, lentigo malignant
melanoma, acral lentiginous melanoma; kidney cancers including but
not limited to, renal cell cancer, adenocarcinoma, hypernephroma,
fibrosarcoma, transitional cell cancer (renal pelvis and/or
uterer); Wilms' tumor; bladder cancers including but not limited
to, transitional cell carcinoma, squamous cell cancer,
adenocarcinoma, carcinosarcoma. In addition, cancers include
myxosarcoma, osteogenic sarcoma, endotheliosarcoma,
lymphangioendotheliosarcoma, mesothelioma, synovioma,
hemangioblastoma, epithelial carcinoma, cystadenocarcinoma,
bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland
carcinoma, papillary carcinoma and papillary adenocarcinomas (for a
review of such disorders, see Fishman et al., 1985, Medicine, 2d
Ed., J.B. Lippincott Co., Philadelphia and Murphy et al., 1997,
Informed Decisions: The Complete Book of Cancer Diagnosis,
Treatment, and Recovery, Viking Penguin, Penguin Books U.S.A.,
Inc., United States of America).
[0449] Accordingly, the methods and compositions of the invention
are also useful in the treatment or prevention of a variety of
cancers or other abnormal proliferative diseases, including (but
not limited to) the following: carcinoma, including that of the
bladder, breast, colon, kidney, liver, lung, ovary, pancreas,
stomach, prostate, cervix, thyroid and skin; including squamous
cell carcinoma; hematopoietic tumors of lymphoid lineage, including
leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia,
B-cell lymphoma, T-cell lymphoma, Burketts lymphoma; hematopoietic
tumors of myeloid lineage, including acute and chronic myelogenous
leukemias and promyelocytic leukemia; tumors of mesenchymal origin,
including fibrosarcoma and rhabdomyoscarcoma; other tumors,
including melanoma, seminoma, tetratocarcinoma, neuroblastoma and
glioma; tumors of the central and peripheral nervous system,
including astrocytoma, neuroblastoma, glioma, and schwannomas;
tumors of mesenchymal origin, including fibrosafcoma,
rhabdomyoscarama, and osteosarcoma; and other tumors, including
melanoma, xenoderma pegmentosum, keratoactanthoma, seminoma,
thyroid follicular cancer and teratocarcinoma. It is also
contemplated that cancers caused by aberrations in apoptosis would
also be treated by the methods and compositions of the invention.
Such cancers may include but not be limited to follicular
lymphomas, carcinomas with p53 mutations, hormone dependent tumors
of the breast, prostate and ovary, and precancerous lesions such as
familial adenomatous polyposis, and myelodysplastic syndromes. In
specific embodiments, malignancy or dysproliferative changes (such
as metaplasias and dysplasias), or hyperproliferative disorders,
are treated or prevented by the methods and compositions of the
invention in the ovary, bladder, breast, colon, lung, skin,
pancreas, or uterus. In other specific embodiments, sarcoma,
melanoma, or leukemia is treated or prevented by the methods and
compositions of the invention.
[0450] In a specific embodiment, a molecule of the invention (e.g.,
an antibody comprising a variant Fc region, or a therapeutic
monoclonal antibody engineered according to the methods of the
invention) inhibits or reduces the growth of cancer cells by at
least 99%, at least 95%, at least 90%, at least 85%, at least 80%,
at least 75%, at least 70%, at least 60%, at least 50%, at least
45%, at least 40%, at least 45%, at least 35%, at least 30%, at
least 25%, at least 20%, or at least 10% relative to the growth of
cancer cells in the absence of said molecule of the invention.
[0451] In a specific embodiment, a molecule of the invention (e.g.,
an antibody comprising a variant Fc region, or a therapeutic
monoclonal antibody engineered according to the methods of the
invention) kills cells or inhibits or reduces the growth of cancer
cells at least 5%, at least 10%, at least 20%, at least 25%, at
least 30%, at least 35%, at least 40%, at least 45%, at least 50%,
at least 60%, at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, at least 95%, or at least 100% better than the
parent molecule.
[0452] 5.4.1.1 Combination Therapy
[0453] The invention further encompasses administering the
molecules of the invention in combination with other therapies
known to those skilled in the art for the treatment or prevention
of cancer or infectious disease, including but not limited to,
current standard and experimental chemotherapies, hormonal
therapies, biological therapies, immunotherapies, radiation
therapies, or surgery. In some embodiments, the molecules of the
invention may be administered in combination with a therapeutically
or prophylactically effective amount of one or more anti-cancer
agents, therapeutic antibodies (e.g., antibodies listed in Table
11), or other agents known to those skilled in the art for the
treatment and/or prevention of cancer (See Section 5.4.1.2).
[0454] In certain embodiments, one or more molecule of the
invention is administered to a mammal, preferably a human,
concurrently with one or more other therapeutic agents useful for
the treatment of cancer. The term "concurrently" is not limited to
the administration of prophylactic or therapeutic agents at exactly
the same time, but rather it is meant that a molecule of the
invention and the other agent are administered to a mammal in a
sequence and within a time interval such that the molecule of the
invention can act together with the other agent to provide an
increased benefit than if they were administered otherwise. For
example, each prophylactic or therapeutic agent (e.g.,
chemotherapy, radiation therapy, hormonal therapy or biological
therapy) may be administered at the same time or sequentially in
any order at different points in time; however, if not administered
at the same time, they should be administered sufficiently close in
time so as to provide the desired therapeutic or prophylactic
effect. Each therapeutic agent can be administered separately, in
any appropriate form and by any suitable route. In various
embodiments, the prophylactic or therapeutic agents are
administered less than 1 hour apart, at about 1 hour apart, at
about 1 hour to about 2 hours apart, at about 2 hours to about 3
hours apart, at about 3 hours to about 4 hours apart, at about 4
hours to about 5 hours apart, at about 5 hours to about 6 hours
apart, at about 6 hours to about 7 hours apart, at about 7 hours to
about 8 hours apart, at about 8 hours to about 9 hours apart, at
about 9 hours to about 10 hours apart, at about 10 hours to about
11 hours apart, at about 11 hours to about 12 hours apart, no more
than 24 hours apart or no more than 48 hours apart. In preferred
embodiments, two or more components are administered within the
same patient visit.
[0455] In other embodiments, the prophylactic or therapeutic agents
are administered at about 2 to 4 days apart, at about 4 to 6 days
apart, at about 1 week part, at about 1 to 2 weeks apart, or more
than 2 weeks apart. In preferred embodiments, the prophylactic or
therapeutic agents are administered in a time frame where both
agents are still active. One skilled in the art would be able to
determine such a time frame by determining the half life of the
administered agents.
[0456] In certain embodiments, the prophylactic or therapeutic
agents of the invention are cyclically administered to a subject.
Cycling therapy involves the administration of a first agent for a
period of time, followed by the administration of a second agent
and/or third agent for a period of time and repeating this
sequential administration. Cycling therapy can reduce the
development of resistance to one or more of the therapies, avoid or
reduce the side effects of one of the therapies, and/or improves
the efficacy of the treatment.
[0457] In certain embodiments, prophylactic or therapeutic agents
are administered in a cycle of less than about 3 weeks, about once
every two weeks, about once every 10 days or about once every week.
One cycle can comprise the administration of a therapeutic or
prophylactic agent by infusion over about 90 minutes every cycle,
about 1 hour every cycle, about 45 minutes every cycle. Each cycle
can comprise at least 1 week of rest, at least 2 weeks of rest, at
least 3 weeks of rest. The number of cycles administered is from
about 1 to about 12 cycles, more typically from about 2 to about 10
cycles, and more typically from about 2 to about 8 cycles.
[0458] In yet other embodiments, the therapeutic and prophylactic
agents of the invention are administered in metronomic dosing
regimens, either by continuous infusion or frequent administration
without extended rest periods. Such metronomic administration can
involve dosing at constant intervals without rest periods.
Typically the therapeutic agents, in particular cytotoxic agents,
are used at lower doses. Such dosing regimens encompass the chronic
daily administration of relatively low doses for extended periods
of time. In preferred embodiments, the use of lower doses can
minimize toxic side effects and eliminate rest periods. In certain
embodiments, the therapeutic and prophylactic agents are delivered
by chronic low-dose or continuous infusion ranging from about 24
hours to about 2 days, to about 1 week, to about 2 weeks, to about
3 weeks to about 1 month to about 2 months, to about 3 months, to
about 4 months, to about 5 months, to about 6 months. The
scheduling of such dose regimens can be optimized by the skilled
oncologist.
[0459] In other embodiments, courses of treatment are administered
concurrently to a mammal, i.e., individual doses of the
therapeutics are administered separately yet within a time interval
such that molecules of the invention can work together with the
other agent or agents. For example, one component may be
administered one time per week in combination with the other
components that may be administered one time every two weeks or one
time every three weeks. In other words, the dosing regimens for the
therapeutics are carried out concurrently even if the therapeutics
are not administered simultaneously or within the same patient
visit.
[0460] When used in combination with other prophylactic and/or
therapeutic agents, the molecules of the invention and the
prophylactic and/or therapeutic agent can act additively or, more
preferably, synergistically. In one embodiment, a molecule of the
invention is administered concurrently with one or more therapeutic
agents in the same pharmaceutical composition. In another
embodiment, a molecule of the invention is administered
concurrently with one or more other therapeutic agents in separate
pharmaceutical compositions. In still another embodiment, a
molecule of the invention is administered prior to or subsequent to
administration of another prophylactic or therapeutic agent. The
invention contemplates administration of a molecule of the
invention in combination with other prophylactic or therapeutic
agents by the same or different routes of administration, e.g.,
oral and parenteral. In certain embodiments, when a molecule of the
invention is administered concurrently with another prophylactic or
therapeutic agent that potentially produces adverse side effects
including, but not limited to, toxicity, the prophylactic or
therapeutic agent can advantageously be administered at a dose that
falls below the threshold that the adverse side effect is
elicited.
[0461] The dosage amounts and frequencies of administration
provided herein are encompassed by the terms therapeutically
effective and prophylactically effective. The dosage and frequency
further will typically vary according to factors specific for each
patient depending on the specific therapeutic or prophylactic
agents administered, the severity and type of cancer, the route of
administration, as well as age, body weight, response, and the past
medical history of the patient. Suitable regimens can be selected
by one skilled in the art by considering such factors and by
following, for example, dosages reported in the literature and
recommended in the Physician's Desk Reference (56.sup.th ed.,
2002).
[0462] 5.4.1.2 Other Therapeutic/Prophylactic Agents
[0463] In a specific embodiment, the methods of the invention
encompass the administration of one or more molecules of the
invention with one or more therapeutic agents used for the
treatment and/or prevention of cancer. In one embodiment,
angiogenesis inhibitors may be administered in combination with the
molecules of the invention. Angiogenesis inhibitors that can be
used in the methods and compositions of the invention include but
are not limited to: Angiostatin (plasminogen fragment);
antiangiogenic antithrombin III; Angiozyme; ABT-627; Bay 12-9566;
Benefin; Bevacizumab; BMS-275291; cartilage-derived inhibitor
(CDI); CAI; CD59 complement fragment; CEP-7055; Col 3;
Combretastatin A-4; Endostatin (collagen XVIII fragment);
Fibronectin fragment; Gro-beta; Halofuginone; Heparinases; Heparin
hexasaccharide fragment; HMV833; Human chorionic gonadotropin
(hCG); IM-862; Interferon alpha/beta/gamma; Interferon inducible
protein (IP-10); Interleukin-12; Kringle 5 (plasminogen fragment);
Marimastat; Metalloproteinase inhibitors (TIMPs);
2-Methoxyestradiol; MMI 270 (CGS 27023A); MoAb IMC-1C11; Neovastat;
NM-3; Panzem; PI-88; Placental ribonuclease inhibitor; Plasminogen
activator inhibitor; Platelet factor-4 (PF4); Prinomastat;
Prolactin 16 kD fragment; Proliferin-related protein (PRP); PTK
787/ZK 222594; Retinoids; Solimastat; Squalamine; SS 3304; SU 5416;
SU6668; SU11248; Tetrahydrocortisol-S; tetrathiomolybdate;
thalidomide; Thrombospondin-1 (TSP-1); TNP-470; Transforming growth
factor-beta (TGF-b); Vasculostatin; Vasostatin (calreticulin
fragment); ZD6126; ZD 6474; farnesyl transferase inhibitors (FTI);
and bisphosphonates.
[0464] Anti-cancer agents that can be used in combination with the
molecules of the invention in the various embodiments of the
invention, including pharmaceutical compositions and dosage forms
and kits of the invention, include, but are not limited to:
acivicin; aclarubicin; acodazole hydrochloride; acronine;
adozelesin; aldesleukin; altretamine; ambomycin; ametantrone
acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin;
asparaginase; asperlin; azacitidine; azetepa; azotomycin;
batimastat; benzodepa; bicalutamide; bisantrene hydrochloride;
bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar
sodium; bropirimine; busulfan; cactinomycin; calusterone;
caracemide; carbetimer; carboplatin; carmustine; carubicin
hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin;
cisplatin; cladribine; crisnatol mesylate; cyclophosphamide;
cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride;
decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate;
diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride;
droloxifene; droloxifene citrate; dromostanolone propionate;
duazomycin; edatrexate; eflornithine hydrochloride; elsamitrucin;
enloplatin; enpromate; epipropidine; epirubicin hydrochloride;
erbulozole; esorubicin hydrochloride; estramustine; estramustine
phosphate sodium; etanidazole; etoposide; etoposide phosphate;
etoprine; fadrozole hydrochloride; fazarabine; fenretinide;
floxuridine; fludarabine phosphate; fluorouracil; fluorocitabine;
fosquidone; fostriecin sodium; gemcitabine; gemcitabine
hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide;
ilmofosine; interleukin II (including recombinant interleukin II,
or rIL2), interferon alfa-2a; interferon alfa-2b; interferon
alfa-n1; interferon alfa-n3; interferon beta-I a; interferon
gamma-I b; iproplatin; irinotecan hydrochloride; lanreotide
acetate; letrozole; leuprolide acetate; liarozole hydrochloride;
lometrexol sodium; lomustine; losoxantrone hydrochloride;
masoprocol; maytansine; mechlorethamine hydrochloride; megestrol
acetate; melengestrol acetate; melphalan; menogaril;
mercaptopurine; methotrexate; methotrexate sodium; metoprine;
meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin;
mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone
hydrochloride; mycophenolic acid; nocodazole; nogalamycin;
ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin;
pentamustine; peplomycin sulfate; perfosfamide; pipobroman;
piposulfan; piroxantrone hydrochloride; plicamycin; plomestane;
porfimer sodium; porfiromycin; prednimustine; procarbazine
hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin;
riboprine; rogletimide; safingol; safingol hydrochloride;
semustine; simtrazene; sparfosate sodium; sparsomycin;
spirogermanium hydrochloride; spiromustine; spiroplatin;
streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan
sodium; tegafur; teloxantrone hydrochloride; temoporfin;
teniposide; teroxirone; testolactone; thiamiprine; thioguanine;
thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone
acetate; triciribine phosphate; trimetrexate; trimetrexate
glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard;
uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine
sulfate; vindesine; vindesine sulfate; vinepidine sulfate;
vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;
vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;
zinostatin; zorubicin hydrochloride. Other anti-cancer drugs
include, but are not limited to: 20-epi-1,25 dihydroxyvitamin D3;
5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol;
adozelesin; aldesleukin; ALL-TK antagonists; altretamine;
ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin;
amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis
inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing
morphogenetic protein-1; antiandrogen, prostatic carcinoma;
antiestrogen; antineoplaston; antisense oligonucleotides;
aphidicolin glycinate; apoptosis gene modulators; apoptosis
regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase;
asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2;
axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III
derivatives; balanol; batimastat; BCR/ABL antagonists;
benzochlorins; benzoylstaurosporine; beta lactam derivatives;
beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor;
bicalutamide; bisantrene; bisaziridinylspermine; bisnafide;
bistratene A; bizelesin; breflate; bropirimine; budotitane;
buthionine sulfoximine; calcipotriol; calphostin C; camptothecin
derivatives; canarypox IL-2; capecitabine;
carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN
700; cartilage derived inhibitor; carzelesin; casein kinase
inhibitors (ICOS); castanospermine; cecropin B; cetrorelix;
chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin;
cladribine; clomifene analogues; clotrimazole; collismycin A;
collismycin B; combretastatin A4; combretastatin analogue;
conagenin; crambescidin 816; crisnatol; cryptophycin 8;
cryptophycin A derivatives; curacin A; cyclopentanthraquinones;
cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;
cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;
dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;
diaziquone; didemnin B; didox; diethylnorspermine;
dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl
spiromustine; docetaxel; docosanol; dolasetron; doxifluridine;
droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine;
edelfosine; edrecolomab; eflornithine; elemene; emitefur;
epirubicin; epristeride; estramustine analogue; estrogen agonists;
estrogen antagonists; etanidazole; etoposide phosphate; exemestane;
fadrozole; fazarabine; fenretinide; filgrastim; finasteride;
flavopiridol; flezelastine; fluasterone; fludarabine;
fluorodaunorunicin hydrochloride; forfenimex; formestane;
fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate;
galocitabine; ganirelix; gelatinase inhibitors; gemcitabine;
glutathione inhibitors; hepsulfam; heregulin; hexamethylene
bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene;
idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod;
immunostimulant peptides; insulin-like growth factor-1 receptor
inhibitor; interferon agonists; interferons; interleukins;
iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine;
isobengazole; isohomohalicondrin B; itasetron; jasplakinolide;
kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin;
lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia
inhibiting factor; leukocyte alpha interferon;
leuprolide+estrogen+progesterone; leuprorelin; levamisole;
liarozole; linear polyamine analogue; lipophilic disaccharide
peptide; lipophilic platinum compounds; lissoclinamide 7;
lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone;
lovastatin; loxoribine; lurtotecan; lutetium texaphyrin;
lysofylline; lytic peptides; maitansine; mannostatin A; marimastat;
masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase
inhibitors; menogaril; merbarone; meterelin; methioninase;
metoclopramide; MIF inhibitor; mifepristone; miltefosine;
mirimostim; mismatched double stranded RNA; mitoguazone;
mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast
growth factor-saporin; mitoxantrone; mofarotene; molgramostim;
monoclonal antibody, human chorionic gonadotrophin; monophosphoryl
lipid A+myobacterium cell wall sk; mopidamol; multiple drug
resistance gene inhibitor; multiple tumor suppressor 1-based
therapy; mustard anticancer agent; mycaperoxide B; mycobacterial
cell wall extract; myriaporone; N-acetyldinaline; N-substituted
benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin;
naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid;
neutral endopeptidase; nilutamide; nisamycin; nitric oxide
modulators; nitroxide antioxidant; nitrullyn; O6-benzylguanine;
octreotide; okicenone; oligonucleotides; onapristone; ondansetron;
ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone;
oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;
paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic
acid; panaxytriol; panomifene; parabactin; pazelliptine;
pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin;
pentrozole; perflubron; perfosfamide; perillyl alcohol;
phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil;
pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A;
placetin B; plasminogen activator inhibitor; platinum complex;
platinum compounds; platinum-triamine complex; porfimer sodium;
porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2;
proteasome inhibitors; protein A-based immune modulator; protein
kinase C inhibitor; protein kinase C inhibitors, microalgal;
protein tyrosine phosphatase inhibitors; purine nucleoside
phosphorylase inhibitors; purpurins; pyrazoloacridine;
pyridoxylated hemoglobin polyoxyethylene conjugate; raf
antagonists; raltitrexed; ramosetron; ras farnesyl protein
transferase inhibitors; ras inhibitors; ras-GAP inhibitor;
retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin;
ribozymes; RII retinamide; rogletimide; rohitukine; romurtide;
roquinimex; rubiginone B1; ruboxyl; safingol; saintopin; SarCNU;
sarcophytol A; sargramostim; Sd.+-.1 mimetics; semustine;
senescence derived inhibitor 1; sense oligonucleotides; signal
transduction inhibitors; signal transduction modulators; single
chain antigen binding protein; sizofuran; sobuzoxane; sodium
borocaptate; sodium phenylacetate; solverol; somatomedin binding
protein; sonermin; sparfosic acid; spicamycin D; spiromustine;
splenopentin; spongistatin 1; squalamine; stem cell inhibitor;
stem-cell division inhibitors; stipiamide; stromelysin inhibitors;
sulfinosine; superactive vasoactive intestinal peptide antagonist;
suradista; suramin; swainsonine; synthetic glycosaminoglycans;
tallimustine; tamoxifen methiodide; tauromustine; tazarotene;
tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors;
temoporfin; temozolomide; teniposide; tetrachlorodecaoxide;
tetrazomine; thaliblastine; thiocoraline; thrombopoietin;
thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist;
thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin;
tirapazamine; titanocene bichloride; topsentin; toremifene;
totipotent stem cell factor; translation inhibitors; tretinoin;
triacetyluridine; triciribine; trimetrexate; triptorelin;
tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins;
UBC inhibitors; ubenimex; urogenital sinus-derived growth
inhibitory factor; urokinase receptor antagonists; vapreotide;
variolin B; vector system, erythrocyte gene therapy; velaresol;
veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin;
vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin
stimalamer. Preferred additional anti-cancer drugs are
5-fluorouracil and leucovorin.
[0465] Examples of therapeutic antibodies that can be used in
methods of the invention include but are not limited to
ZENAPAX.RTM. (daclizumab) anti-CD25 antibody (Roche
Pharmaceuticals, Switzerland) which is an immunosuppressive,
humanized anti-CD25 monoclonal antibody for the prevention of acute
renal allograft rejection; PANOREX.TM. anti-17-IA antibody which is
a murine anti-17-IA cell surface antigen IgG2a antibody (Glaxo
Wellcome/Centocor); BEC2.TM. anti-idiotype (GD3 epitope) antibody
which is a murine anti-idiotype (GD3 epitope) IgG antibody (ImClone
System); IMC-C225.TM. anti-EGFR antibody which is a chimeric
anti-EGFR IgG antibody (ImClone System); VITAXIN.TM.
anti-.alpha.V.beta.3 antibody which is a humanized
anti-.alpha.V.beta.3 integrin antibody (Applied Molecular
Evolution/MedImmune); Smart M195.TM. anti-CD33 antibody which is a
humanized anti-CD33 IgG antibody (Protein Design Lab/Kanebo);
LYMPHOCIDE.TM. .TM. anti-CD22 antibody which is a humanized
anti-CD22 IgG antibody (Immunomedics); ICM3.TM. anti-ICAM3 antibody
is a humanized anti-ICAM3 antibody (ICOS Pharm); IDEC-114.TM.
anti-CD80 antibody is a primatied primatized anti-CD80 antibody
(IDEC Pharm/Mitsubishi); IDEC-131.TM. anti-CD40L antibody is a
humanized anti-CD40L antibody (IDEC/Eisai); IDEC-151.TM. anti-CD4
antibody is a primatized anti-CD4 antibody (IDEC); IDEC-152.TM.
anti-CD23 antibody is a primatized anti-CD23 antibody
(IDEC/Seikagaku); SMART anti-CD3.TM. anti-CD3 antibody is a
humanized anti-CD3 IgG (Protein Design Lab); 5G1.1.TM. anti-CS
antibody is a humanized anti-complement factor 5 (C5) antibody
(Alexion Pharm); D2E7.TM. anti-TNF-.alpha. antibody is a humanized
anti-TNF-.alpha. antibody (CAT/BASF); CDP870.TM. anti-TNF.alpha.
antibody is a humanized anti-TNF-.alpha. Fab fragment (Celltech);
IDEC-151.TM. anti-CD4 antibody is a primatized anti-CD4 IgG1
antibody (IDEC Pharm/SmithKline Beecham); MDX-CD4.TM. anti-CD4
antibody is a human anti-CD4 IgG antibody (Medarex/Eisai/Genmab);
CDP571.TM. anti-TNF-.alpha. antibody is a humanized
anti-TNF-.alpha. IgG4 antibody (Celltech); LDP-02.TM.
anti-.alpha.4.beta.7 antibody is a humanized anti-.alpha.4.beta.7
antibody (LeukoSite/Genentech); OrthoClone OKT4A.RTM. anti-CD4
antibody is a humanized anti-CD4 IgG antibody (Ortho Biotech);
ANTOVA.TM. anti-CD40L antibody is a humanized anti-CD40L IgG
antibody (Biogen); ANTEGREN.TM. anti-VLA-4 antibody is a humanized
anti-VLA-4 IgG antibody (Elan); and CAT-152.TM.
anti-TGF-.beta..sub.2 antibody is a human anti-TGF-.beta..sub.2
antibody (Cambridge Ab Tech). Other examples of therapeutic
antibodies that can be used in accordance with the invention are
presented in Table 11.
[0466] 5.4.2 Autoimmune Disease and Inflammatory Diseases
[0467] In some embodiments, molecules of the invention comprise a
variant Fc region, having one or more amino acid modifications in
one or more regions, which modification increases the affinity of
the variant Fc region for Fc.gamma.RIIB but decreases the affinity
of the variant Fc region for Fc.gamma.RIIIA and/or Fc.gamma.RIIA.
Molecules of the invention with such binding characteristics are
useful in regulating the immune response, e.g., in inhibiting the
immune response in connection with autoimmune diseases or
inflammatory diseases. Although not intending to be bound by any
mechanism of action, molecules of the invention with an enhanced
affinity for Fc.gamma.RIIB and a decreased affinity for
Fc.gamma.RIIIA and/or Fc.gamma.RIIA may lead to dampening of the
activating response to Fc.gamma.R and inhibition of cellular
responsiveness.
[0468] In some embodiments, a molecule of the invention comprising
a variant Fc region is not an immunoglobulin, and comprises at
least one amino acid modification which modification increases the
affinity of the variant Fc region for Fc.gamma.RIIB relative to a
molecule comprising a wild-type Fc region. In other embodiments,
said molecule further comprises one or more amino acid
modifications, which modifications decreases the affinity of the
molecule for an activating Fc.gamma.R. In some embodiments, the
molecule is a soluble (i.e., not membrane bound) Fc region. The
invention contemplates other amino acid modifications within the
soluble Fc region which modulate its affinity for various Fc
receptors, including those known to one skilled in the art as
described herein. In other embodiments, the molecule (e.g., the Fc
region comprising at least one or more amino acid modification) is
modified using techniques known to one skilled in the art and as
described herein to increase the in vivo half life of the Fc
region. Such molecules have therapeutic utility in treating and/or
preventing an autoimmune disorder. Although not intending to be
bound by any mechanism of actions, such molecules with enhanced
affinity for Fc.gamma.RIIB will lead to a dampening of the
activating receptors and thus a dampening of the immune response
and have therapeutic efficacy for treating and/or preventing an
autoimmune disorder.
[0469] In certain embodiments, the one or more amino acid
modifications, which increase the affinity of the variant Fc region
for Fc.gamma.RIIB but decrease the affinity of the variant Fc
region for Fc.gamma.RIIIA comprise a substitution at position 246
with threonine and at position 396 with histidine; or a
substitution at position 268 with aspartic acid and at position 318
with aspartic acid; or a substitution at position 217 with serine,
at position 378 with valine, and at position 408 with arginine; or
a substitution at position 375 with cysteine and at position 396
with leucine; or a substitution at position 246 with isoleucine and
at position 334 with asparagine. In one embodiment, the one or more
amino acid modifications, which increase the affinity of the
variant Fc region for Fc.gamma.RIIB but decrease the affinity of
the variant Fc region for Fc.gamma.RIIIA comprise a substitution at
position 247 with leucine. In another embodiment, the one or more
amino acid modification, which increases the affinity of the
variant Fc region for Fc.gamma.RIIB but decreases the affinity of
the variant Fc region for Fc.gamma.RIIIA comprise a substitution at
position 372 with tyrosine. In yet another embodiment, the one or
more amino acid modification, which increases the affinity of the
variant Fc region for Fc.gamma.RIIB but decreases the affinity of
the variant Fc region for Fc.gamma.RIIIA comprise a substitution at
position 326 with glutamic acid. In one embodiment, the one or more
amino acid modification, which increases the affinity of the
variant Fc region for Fc.gamma.RIIB but decreases the affinity of
the variant Fc region for Fc.gamma.RIIIA comprise a substitution at
position 224 with leucine.
[0470] The variant Fc regions that have an enhanced affinity for
Fc.gamma.RIIB and a decreased affinity for Fc.gamma.RIIIA and/or
Fc.gamma.RIIA relative to a comparable molecule comprising a
wild-type Fc region, may be used to treat or prevent autoimmune
diseases or inflammatory diseases. The present invention provides
methods of preventing, treating, or managing one or more symptoms
associated with an autoimmune or inflammatory disorder in a
subject, comprising administering to said subject a therapeutically
or prophylactically effective amount of one or more molecules of
the invention with variant Fc regions that have an enhanced
affinity for Fc.gamma.RIIB and a decreased affinity for
Fc.gamma.RIIIA and or Fc.gamma.RIIA relative to a comparable
molecule comprising a wild type Fc region.
[0471] The invention also provides methods for preventing,
treating, or managing one or more symptoms associated with an
inflammatory disorder in a subject further comprising,
administering to said subject a therapeutically or prophylactically
effective amount of one or more anti-inflammatory agents. The
invention also provides methods for preventing, treating, or
managing one or more symptoms associated with an autoimmune disease
further comprising, administering to said subject a therapeutically
or prophylactically effective amount of one or more
immunomodulatory agents. Section 5.4.2.1 provides non-limiting
examples of anti-inflammatory agents and immunomodulatory
agents.
[0472] Examples of autoimmune disorders that may be treated by
administering the molecules of the present invention include, but
are not limited to, alopecia greata, ankylosing spondylitis,
antiphospholipid syndrome, autoimmune Addison's disease, autoimmune
diseases of the adrenal gland, autoimmune hemolytic anemia,
autoimmune hepatitis, autoimmune oophoritis and orchitis,
autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid,
cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune
dysfunction syndrome (CFIDS), chronic inflammatory demyelinating
polyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid,
CREST syndrome, cold agglutinin disease, Crohn's disease, discoid
lupus, essential mixed cryoglobulinemia,
fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease,
Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary
fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA
neuropathy, juvenile arthritis, lichen planus, lupus erthematosus,
Meniere's disease, mixed connective tissue disease, multiple
sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia
gravis, pemphigus vulgaris, pernicious anemia, polyarteritis
nodosa, polychrondritis, polyglandular syndromes, polymyalgia
rheumatica, polymyositis and dermatomyositis, primary
agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic
arthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoid
arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man
syndrome, systemic lupus erythematosus, lupus erythematosus,
takayasu arteritis, temporal arteristis/giant cell arteritis,
ulcerative colitis, uveitis, vasculitides such as dermatitis
herpetiformis vasculitis, vitiligo, and Wegener's granulomatosis.
Examples of inflammatory disorders include, but are not limited to,
asthma, encephilitis, inflammatory bowel disease, chronic
obstructive pulmonary disease (COPD), allergic disorders, septic
shock, pulmonary fibrosis, undifferentitated spondyloarthropathy,
undifferentiated arthropathy, arthritis, inflammatory osteolysis,
and chronic inflammation resulting from chronic viral or bacteria
infections. Examples of inflammatory disorders which can be
prevented, treated or managed in accordance with the methods of the
invention include, but are not limited to, asthma, encephilitis,
inflammatory bowel disease, chronic obstructive pulmonary disease
(COPD), allergic disorders, septic shock, pulmonary fibrosis,
undifferentitated spondyloarthropathy, undifferentiated
arthropathy, arthritis, inflammatory osteolysis, and chronic
inflammation resulting from chronic viral or bacteria
infections.
[0473] Molecules of the invention with variant Fc regions that have
an enhanced affinity for Fc.gamma.RIIB and a decreased affinity for
Fc.gamma.RIIIA relative to a comparable molecule comprising a
wild-type Fc region can also be used to reduce the inflammation
experienced by animals, particularly mammals, with inflammatory
disorders. In a specific embodiment, a molecule of the invention
reduces the inflammation in an animal by at least 99%, at least
95%, at least 90%, at least 85%, at least 80%, at least 75%, at
least 70%, at least 60%, at least 50%, at least 45%, at least 40%,
at least 45%, at least 35%, at least 30%, at least 25%, at least
20%, or at least 10% relative to the inflammation in an animal,
which is not administered the said molecule or which is
administered the parent molecule.
[0474] Molecules of the invention with variant Fc regions that have
an enhanced affinity for Fc.gamma.RIIB and a decreased affinity for
Fc.gamma.RIIIA relative to a comparable molecule comprising a
wild-type Fc region can also be used to prevent the rejection of
transplants.
[0475] The invention further contemplates engineering any of the
antibodies known in the art for the treatment and/or prevention of
autoimmune disease or inflammatory disease, so that the antibodies
comprise a variant Fc region comprising one or more amino acid
modifications, which have been identified by the methods of the
invention to have a conferred effector function and/or enhanced
affinity for Fc.gamma.RIIB and a decreased affinity for
Fc.gamma.RIIIA relative to a comparable molecule comprising a wild
type Fc region. A non-limiting example of the antibodies that are
used for the treatment or prevention of inflammatory disorders
which can be engineered according to the invention is presented in
Table 12A, and a non-limiting example of the antibodies that are
used for the treatment or prevention of autoimmune disorder is
presented in Table 12B.
TABLE-US-00013 TABLE 12A ANTIBODIES FOR INFLAMMATORY DISEASES AND
AUTOIMMUNE DISEASES THAT CAN ENGINEERED IN ACCORDANCE WITH THE
INVENTION. Antibody Target Product Name Antigen Type Isotype
Sponsors Indication 5G1.1 Complement Humanized IgG Alexion
Rheumatoid (C5) Pharm Inc Arthritis 5G1.1 Complement Humanized IgG
Alexion SLE (C5) Pharm Inc 5G1.1 Complement Humanized IgG Alexion
Nephritis (C5) Pharm Inc 5G1.1-SC Complement Humanized ScFv Alexion
Cardiopulmonary (C5) Pharm Inc Bypass 5G1.1-SC Complement Humanized
ScFv Alexion Myocardial (C5) Pharm Inc Infarction 5G1.1-SC
Complement Humanized ScFv Alexion Angioplasty (C5) Pharm Inc
ABX-CBL CBL Human Abgenix Inc GvHD ABX-CBL CD147 Murine IgG Abgenix
Inc Allograft rejection ABX-IL8 IL-8 Human IgG2 Abgenix Inc
Psoriasis Antegren VLA-4 Humanized IgG Athena/Elan Multiple
Sclerosis Anti- CD11a Humanized IgG1 Genentech Psoriasis CD11a
Inc/Xoma Anti- CD18 Humanized Fab'2 Genentech Inc Myocardial CD18
infarction Anti- CD18 Murine Fab'2 Pasteur- Allograft rejection
LFA1 Merieux/ Immunotech Antova CD40L Humanized IgG Biogen
Allograft rejection Antova CD40L Humanized IgG Biogen SLE BTI-322
CD2 Rat IgG Medimmune GvHD, Psoriasis Inc CDP571 TNF-alpha
Humanized IgG4 Celltech Crohn's CDP571 TNF-alpha Humanized IgG4
Celltech Rheumatoid Arthritis CDP850 E-selectin Humanized Celltech
Psoriasis Corsevin M Fact VII Chimeric Centocor Anticoagulant D2E7
TNF-alpha Human CAT/BASF Rheumatoid Arthritis Hu23F2G CD11/18
Humanized ICOS Pharm Multiple Sclerosis Inc Hu23F2G CD11/18
Humanized IgG ICOS Pharm Stroke Inc IC14 CD14 ICOS Pharm Toxic
shock Inc ICM3 ICAM-3 Humanized ICOS Pharm Psoriasis Inc IDEC-114
CD80 Primatised IDEC Psoriasis Pharm/Mitsubishi IDEC-131 CD40L
Humanized IDEC SLE Pharm/Eisai IDEC-131 CD40L Humanized IDEC
Multiple Sclerosis Pharm/Eisai IDEC-151 CD4 Primatised IgG1 IDEC
Rheumatoid Pharm/Glaxo Arthritis SmithKline IDEC-152 CD23
Primatised IDEC Pharm Asthma/Allergy Infliximab TNF-alpha Chimeric
IgG1 Centocor Rheumatoid Arthritis Infliximab TNF-alpha Chimeric
IgG1 Centocor Crohn's LDP-01 beta2- Humanized IgG Millennium Stroke
integrin Inc (LeukoSite Inc.) LDP-01 beta2- Humanized IgG
Millennium Allograft rejection integrin Inc (LeukoSite Inc.) LDP-02
alpha4beta7 Humanized Millennium Ulcerative Colitis Inc (LeukoSite
Inc.) MAK- TNF alpha Murine Fab'2 Knoll Pharm, Toxic shock 195F
BASF MDX-33 CD64 (FcR) Human Medarex/Centeon Autoimmune
haematogical disorders MDX- CD4 Human IgG Medarex/Eisai/ Rheumatoid
CD4 Genmab Arthritis MEDI-507 CD2 Humanized Medimmune Psoriasis Inc
MEDI-507 CD2 Humanized Medimmune GvHD Inc OKT4A CD4 Humanized IgG
Ortho Biotech Allograft rejection OrthoClone CD4 Humanized IgG
Ortho Biotech Autoimmune OKT4A disease Orthoclone/ CD3 Murine
mIgG2a Ortho Biotech Allograft rejection anti-CD3 OKT3 RepPro/
gpIIbIIIa Chimeric Fab Centocor/Lilly Complications of Abciximab
coronary angioplasty rhuMab- IgE Humanized IgG1 Genentech/Novartis/
Asthma/Allergy E25 Tanox Biosystems SB-240563 IL5 Humanized
GlaxoSmithKline Asthma/Allergy SB-240683 IL-4 Humanized
GlaxoSmithKline Asthma/Allergy SCH55700 IL-5 Humanized
Celltech/Schering Asthma/Allergy Simulect CD25 Chimeric IgG1
Novartis Allograft rejection Pharm SMART CD3 Humanized Protein
Autoimmune a-CD3 Design Lab disease SMART CD3 Humanized Protein
Allograft rejection a-CD3 Design Lab SMART CD3 Humanized IgG
Protein Psoriasis a-CD3 Design Lab Zenapax CD25 Humanized IgG1
Protein Allograft rejection Design Lab/Hoffman- La Roche
TABLE-US-00014 TABLE 12B ANTIBODIES FOR AUTOIMMUNE DISORDERS THAT
CAN BE ENGINEERED IN ACCORDANCE WITH THE INVENTION Antibody
Indication Target Antigen ABX-RB2 antibody to CBL antigen on T
cells, B cells and NK cells fully human antibody from the Xenomouse
5c8 (Anti CD-40 Phase II trials were halted in October CD-40 ligand
antibody) 1999 examine "adverse events" IDEC 131 systemic lupus
erythyematous anti CD40 (SLE) humanized IDEC 151 rheumatoid
arthritis primatized; anti-CD4 IDEC 152 Asthma primatized;
anti-CD23 IDEC 114 Psoriasis primatized anti-CD80 MEDI-507
rheumatoid arthritis; multiple anti-CD2 sclerosis Crohn's disease
Psoriasis LDP-02 (anti-b7 inflammatory bowel disease a4b7 integrin
receptor on white mAb) Chron's disease blood cells (leukocytes)
ulcerative colitis SMART Anti- autoimmune disorders Anti-Gamma
Interferon Gamma Interferon antibody Verteportin rheumatoid
arthritis MDX-33 blood disorders caused by monoclonal antibody
against FcRI autoimmune reactions receptors Idiopathic
Thrombocytopenia Purpurea (ITP) autoimmune hemolytic anemia MDX-CD4
treat rheumatoid arthritis and other monoclonal antibody against
CD4 autoimmunity receptor molecule VX-497 autoimmune disorders
inhibitor of inosine monophosphate multiple sclerosis dehydrogenase
rheumatoid arthritis (enzyme needed to make new RNA inflammatory
bowel disease and DNA lupus used in production of nucleotides
psoriasis needed for lymphocyte proliferation) VX-740 rheumatoid
arthritis inhibitor of ICE interleukin-1 beta (converting enzyme
controls pathways leading to aggressive immune response) VX-745
specific to inflammation inhibitor of P38MAP kinase involved in
chemical signalling of mitogen activated protein kinase immune
response onset and progression of inflammation Enbrel (etanercept)
targets TNF (tumor necrosis factor) IL-8 fully human monoclonal
antibody against IL-8 (interleukin 8) Apogen MP4 recombinant
antigen selectively destroys disease associated T-cells induces
apoptosis T-cells eliminated by programmed cell death no longer
attack body's own cells specific apogens target specific T-
cells
[0476] 5.4.2.1 Immunomodulatory Agents and Anti-Inflammatory
Agents
[0477] The present invention provides methods of treatment for
autoimmune diseases and inflammatory diseases comprising
administration of the molecules with variant Fc regions having an
enhanced affinity for Fc.gamma.RIIB and a decreased affinity for
Fc.gamma.RIIIA and/or Fc.gamma.RIIA in conjunction with other
treatment agents. Examples of immunomodulatory agents include, but
are not limited to, methothrexate, ENBREL, REMICADE.TM.,
leflunomide, cyclophosphamide, cyclosporine A, and macrolide
antibiotics (e.g., FK506 (tacrolimus)), methylprednisolone (MP),
corticosteroids, steroids, mycophenolate mofetil, rapamycin
(sirolimus), mizoribine, deoxyspergualin, brequinar,
malononitriloamindes (e.g., leflunamide), T cell receptor
modulators, and cytokine receptor modulators.
[0478] Anti-inflammatory agents have exhibited success in treatment
of inflammatory and autoimmune disorders and are now a common and a
standard treatment for such disorders. Any anti-inflammatory agent
well-known to one of skill in the art can be used in the methods of
the invention. Non-limiting examples of anti-inflammatory agents
include non-steroidal anti-inflammatory drugs (NSAIDs), steroidal
anti-inflammatory drugs, beta-agonists, anticholingeric agents, and
methyl xanthines. Examples of NSAIDs include, but are not limited
to, aspirin, ibuprofen, celecoxib (CELEBREX.TM.), diclofenac
(VOLTAREN.TM.), etodolac (LODINE.TM.), fenoprofen (NALFON.TM.),
indomethacin (INDOCIN.TM.), ketoralac (TORADOL.TM.), oxaprozin
(DAYPRO.TM.), nabumentone (RELAFEN.TM.), sulindac (CLINORIL.TM.),
tolmentin (TOLECTIN.TM.), rofecoxib (VIOXX.TM.), naproxen
(ALEVE.TM., NAPROSYN.TM.), ketoprofen (ACTRON.TM.) and nabumetone
(RELAFEN.TM.). Such NSAIDs function by inhibiting a cyclooxygenase
enzyme (e.g., COX-1 and/or COX-2). Examples of steroidal
anti-inflammatory drugs include, but are not limited to,
glucocorticoids, dexamethasone (DECADRON.TM.), cortisone,
hydrocortisone, prednisone (DELTASONE.TM.), prednisolone,
triamcinolone, azulfidine, and eicosanoids such as prostaglandins,
thromboxanes, and leukotrienes.
[0479] 5.4.3 Infectious Disease
[0480] The invention also encompasses methods for treating or
preventing an infectious disease in a subject comprising
administering a therapeutically or prophylatically effective amount
of one or more molecules of the invention. Infectious diseases that
can be treated or prevented by the molecules of the invention are
caused by infectious agents including but not limited to viruses,
bacteria, fungi, protozae, and viruses.
[0481] Viral diseases that can be treated or prevented using the
molecules of the invention in conjunction with the methods of the
present invention include, but are not limited to, those caused by
hepatitis type A, hepatitis type B, hepatitis type C, influenza,
varicella, adenovirus, herpes simplex type I (HSV-I), herpes
simplex type II (HSV-II), rinderpest, rhinovirus, echovirus,
rotavirus, respiratory syncytial virus, papilloma virus, papova
virus, cytomegalovirus, echinovirus, arbovirus, huntavirus,
coxsackie virus, mumps virus, measles virus, rubella virus, polio
virus, small pox, Epstein Barr virus, human immunodeficiency virus
type I (HIV-I), human immunodeficiency virus type II (HIV-II), and
agents of viral diseases such as viral miningitis, encephalitis,
dengue or small pox.
[0482] Bacterial diseases that can be treated or prevented using
the molecules of the invention in conjunction with the methods of
the present invention, that are caused by bacteria include, but are
not limited to, mycobacteria rickettsia, mycoplasma, neisseria, S.
pneumonia, Borrelia burgdorferi (Lyme disease), Bacillus antracis
(anthrax), tetanus, streptococcus, staphylococcus, mycobacterium,
tetanus, pertissus, cholera, plague, diptheria, chlamydia, S.
aureus and legionella.
[0483] Protozoal diseases that can be treated or prevented using
the molecules of the invention in conjunction with the methods of
the present invention, that are caused by protozoa include, but are
not limited to, leishmania, kokzidioa, trypanosoma or malaria.
[0484] Parasitic diseases that can be treated or prevented using
the molecules of the invention in conjunction with the methods of
the present invention, that are caused by parasites include, but
are not limited to, chlamydia and rickettsia.
[0485] According to one aspect of the invention, molecules of the
invention comprising variant Fc regions have an enhanced antibody
effector function towards an infectious agent, e.g., a pathogenic
protein, relative to a comparable molecule comprising a wild-type
Fc region. Examples of infectious agents include but are not
limited to bacteria (e.g., Escherichia coli, Klebsiella pneumoniae,
Staphylococcus aureus, Enterococcus faecials, Candida albicans,
Proteus vulgaris, Staphylococcus viridans, and Pseudomonas
aeruginosa), a pathogen (e.g., B-lymphotropic papovavirus (LPV);
Bordatella pertussis; Borna Disease virus (BDV); Bovine
coronavirus; Choriomeningitis virus; Dengue virus; a virus, E.
coli; Ebola; Echovirus 1; Echovirus-11 (EV); Endotoxin (LPS);
Enteric bacteria; Enteric Orphan virus; Enteroviruses; Feline
leukemia virus; Foot and mouth disease virus; Gibbon ape leukemia
virus (GALV); Gram-negative bacteria; Heliobacter pylori; Hepatitis
B virus (HBV); Herpes Simplex Virus; HIV-1; Human cytomegalovirus;
Human coronovirus; Influenza A, B & C; Legionella; Leishmania
mexicana; Listeria monocytogenes; Measles virus; Meningococcus;
Morbilliviruses; Mouse hepatitis virus; Murine leukemia virus;
Murine gamma herpes virus; Murine retrovirus; Murine coronavirus
mouse hepatitis virus; Mycobacterium avium-M; Neisseria
gonorrhoeae; Newcastle disease virus; Parvovirus B19; Plasmodium
falciparum; Pox Virus; Pseudomonas; Rotavirus; Samonella
typhiurium; Shigella; Streptococci; T-cell lymphotropic virus 1;
Vaccinia virus).
[0486] In a specific embodiment, molecules of the invention enhance
the efficacy of treatment of an infectious disease by enhancing
phagocytosis and/or opsonization of the infectious agent causing
the infectious disease. In another specific embodiment, molecules
of the invention enhance the efficacy of treatment of an infectious
disease by enhancing ADCC of infected cells causing the infectious
disease.
[0487] In some embodiments, the molecules of the invention may be
administered in combination with a therapeutically or
prophylactically effective amount of one or additional therapeutic
agents known to those skilled in the art for the treatment and/or
prevention of an infectious disease. The invention contemplates the
use of the molecules of the invention in combination with
antibiotics known to those skilled in the art for the treatment and
or prevention of an infectious disease. Antibiotics that can be
used in combination with the molecules of the invention include,
but are not limited to, macrolide (e.g., tobramycin (Tobi.RTM.)), a
cephalosporin (e.g., cephalexin (Keflex.RTM.), cephradine
(Velosef.RTM.), cefuroxime (Ceftin.RTM.), cefprozil (Cefzil.RTM.),
cefaclor (Ceclor.RTM.), cefixime (Suprax.RTM.) or cefadroxil
(Duricef.RTM.)), a clarithromycin (e.g., clarithromycin
(Biaxin.RTM.)), an erythromycin (e.g., erythromycin (EMycin.RTM.)),
a penicillin (e.g., penicillin V (V-Cillin K.RTM. or Pen Vee
K.RTM.)) or a quinolone (e.g., ofloxacin (Floxin.RTM.),
ciprofloxacin (Cipro.RTM.) or norfloxacin (Noroxin.RTM.)),
aminoglycoside antibiotics (e.g., apramycin, arbekacin,
bambermycins, butirosin, dibekacin, neomycin, neomycin,
undecylenate, netilmicin, paromomycin, ribostamycin, sisomicin, and
spectinomycin), amphenicol antibiotics (e.g., azidamfenicol,
chloramphenicol, florfenicol, and thiamphenicol), ansamycin
antibiotics (e.g., rifamide and rifampin), carbacephems (e.g.,
loracarbef), carbapenems (e.g., biapenem and imipenem),
cephalosporins (e.g., cefaclor, cefadroxil, cefamandole,
cefatrizine, cefazedone, cefozopran, cefpimizole, cefpiramide, and
cefpirome), cephamycins (e.g., cefbuperazone, cefmetazole, and
cefminox), monobactams (e.g., aztreonam, carumonam, and tigemonam),
oxacephems (e.g., flomoxef, and moxalactam), penicillins (e.g.,
amdinocillin, amdinocillin pivoxil, amoxicillin, bacampicillin,
benzylpenicillinic acid, benzylpenicillin sodium, epicillin,
fenbenicillin, floxacillin, penamccillin, penethamate hydriodide,
penicillin o-benethamine, penicillin 0, penicillin V, penicillin V
benzathine, penicillin V hydrabamine, penimepicycline, and
phencihicillin potassium), lincosamides (e.g., clindamycin, and
lincomycin), amphomycin, bacitracin, capreomycin, colistin,
enduracidin, enviomycin, tetracyclines (e.g., apicycline,
chlortetracycline, clomocycline, and demeclocycline),
2,4-diaminopyrimidines (e.g., brodimoprim), nitrofurans (e.g.,
furaltadone, and furazolium chloride), quinolones and analogs
thereof (e.g., cinoxacin, clinafloxacin, flumequine, and
grepagloxacin), sulfonamides (e.g., acetyl sulfamethoxypyrazine,
benzylsulfamide, noprylsulfamide, phthalylsulfacetamide,
sulfachrysoidine, and sulfacytine), sulfones (e.g.,
diathymosulfone, glucosulfone sodium, and solasulfone),
cycloserine, mupirocin and tuberin.
[0488] In certain embodiments, the molecules of the invention can
be administered in combination with a therapeutically or
prophylactically effective amount of one or more antifungal agents.
Antifungal agents that can be used in combination with the
molecules of the invention include but are not limited to
amphotericin B, itraconazole, ketoconazole, fluconazole,
intrathecal, flucytosine, miconazole, butoconazole, clotrimazole,
nystatin, terconazole, tioconazole, ciclopirox, econazole,
haloprogrin, naftifine, terbinafine, undecylenate, and
griseofuldin.
[0489] In some embodiments, the molecules of the invention can be
administered in combination with a therapeutically or
prophylactically effective amount of one or more anti-viral agent.
Useful anti-viral agents that can be used in combination with the
molecules of the invention include, but are not limited to,
protease inhibitors, nucleoside reverse transcriptase inhibitors,
non-nucleoside reverse transcriptase inhibitors and nucleoside
analogs. Examples of antiviral agents include but are not limited
to zidovudine, acyclovir, gangcyclovir, vidarabine, idoxuridine,
trifluridine, and ribavirin, as well as foscarnet, amantadine,
rimantadine, saquinavir, indinavir, amprenavir, lopinavir,
ritonavir, the alpha-interferons; adefovir, clevadine, entecavir,
pleconaril.
5.5 Vaccine Therapy
[0490] The invention further encompasses using a composition of the
invention to induce an immune response against an antigenic or
immunogenic agent, including but not limited to cancer antigens and
infectious disease antigens (examples of which are disclosed
infra). The vaccine compositions of the invention comprise one or
more antigenic or immunogenic agents to which an immune response is
desired, wherein the one or more antigenic or immunogenic agents is
coated with a variant antibody of the invention that has a
conferred effector function and/or an enhanced affinity to
Fc.gamma.RIIIA. Although not intending to be bound by a particular
mechanism of action, coating an antigenic or immunogenic agent with
a variant antibody of the invention that has an enhanced affinity
to Fc.gamma.RIIIA, enhances the immune response to the desired
antigenic or immunogenic agent by inducing humoral and
cell-mediated responses. The vaccine compositions of the invention
are particularly effective in eliciting an immune response,
preferably a protective immune response against the antigenic or
immunogenic agent.
[0491] In some embodiments, the antigenic or immunogenic agent in
the vaccine compositions of the invention comprise a virus against
which an immune response is desired. The viruses may be recombinant
or chimeric, and are preferably attenuated. Production of
recombinant, chimeric, and attenuated viruses may be performed
using standard methods known to one skilled in the art. The
invention encompasses a live recombinant viral vaccine or an
inactivated recombinant viral vaccine to be formulated in
accordance with the invention. A live vaccine may be preferred
because multiplication in the host leads to a prolonged stimulus of
similar kind and magnitude to that occurring in natural infections,
and therefore, confers substantial, long-lasting immunity.
Production of such live recombinant virus vaccine formulations may
be accomplished using conventional methods involving propagation of
the virus in cell culture or in the allantois of the chick embryo
followed by purification.
[0492] In a specific embodiment, the recombinant virus is
non-pathogenic to the subject to which it is administered. In this
regard, the use of genetically engineered viruses for vaccine
purposes may require the presence of attenuation characteristics in
these strains. The introduction of appropriate mutations (e.g.,
deletions) into the templates used for transfection may provide the
novel viruses with attenuation characteristics. For example,
specific missense mutations which are associated with temperature
sensitivity or cold adaption can be made into deletion mutations.
These mutations should be more stable than the point mutations
associated with cold or temperature sensitive mutants and reversion
frequencies should be extremely low. Recombinant DNA technologies
for engineering recombinant viruses are known in the art and
encompassed in the invention. For example, techniques for modifying
negative strand RNA viruses are known in the art, see, e.g., U.S.
Pat. No. 5,166,057, which is incorporated herein by reference in
its entirety.
[0493] Alternatively, chimeric viruses with "suicide"
characteristics may be constructed for use in the intradermal
vaccine formulations of the invention. Such viruses would go
through only one or a few rounds of replication within the host.
When used as a vaccine, the recombinant virus would go through
limited replication cycle(s) and induce a sufficient level of
immune response but it would not go further in the human host and
cause disease. Alternatively, inactivated (killed) virus may be
formulated in accordance with the invention. Inactivated vaccine
formulations may be prepared using conventional techniques to
"kill" the chimeric viruses. Inactivated vaccines are "dead" in the
sense that their infectivity has been destroyed. Ideally, the
infectivity of the virus is destroyed without affecting its
immunogenicity. In order to prepare inactivated vaccines, the
chimeric virus may be grown in cell culture or in the allantois of
the chick embryo, purified by zonal ultracentrifugation,
inactivated by formaldehyde or .beta.-propiolactone, and
pooled.
[0494] In certain embodiments, completely foreign epitopes,
including antigens derived from other viral or non-viral pathogens
can be engineered into the virus for use in the intradermal vaccine
formulations of the invention. For example, antigens of non-related
viruses such as HIV (gp160, gp120, gp41) parasite antigens (e.g.,
malaria), bacterial or fungal antigens or tumor antigens can be
engineered into the attenuated strain.
[0495] Virtually any heterologous gene sequence may be constructed
into the chimeric viruses of the invention for use in the
intradermal vaccine formulations. Preferably, heterologous gene
sequences are moieties and peptides that act as biological response
modifiers. Preferably, epitopes that induce a protective immune
response to any of a variety of pathogens, or antigens that bind
neutralizing antibodies may be expressed by or as part of the
chimeric viruses. For example, heterologous gene sequences that can
be constructed into the chimeric viruses of the invention include,
but are not limited to, influenza and parainfluenza hemagglutinin
neuraminidase and fusion glycoproteins such as the FIN and F genes
of human PIV3. In yet another embodiment, heterologous gene
sequences that can be engineered into the chimeric viruses include
those that encode proteins with immuno-modulating activities.
Examples of immuno-modulating proteins include, but are not limited
to, cytokines, interferon type 1, gamma interferon, colony
stimulating factors, interleukin-1, -2, -4, -5, -6, -12, and
antagonists of these agents.
[0496] In yet other embodiments, the invention encompasses
pathogenic cells or viruses, preferably attenuated viruses, which
express the variant antibody on their surface.
[0497] In alternative embodiments, the vaccine compositions of the
invention comprise a fusion polypeptide wherein an antigenic or
immunogenic agent is operatively linked to a variant antibody of
the invention that has an enhanced affinity for Fc.gamma.RIIIA.
Engineering fusion polypeptides for use in the vaccine compositions
of the invention is performed using routine recombinant DNA
technology methods and is within the level of ordinary skill.
[0498] The invention further encompasses methods to induce
tolerance in a subject by administering a composition of the
invention. Preferably a composition suitable for inducing tolerance
in a subject, comprises an antigenic or immunogenic agent coated
with a variant antibody of the invention, wherein the variant
antibody has a higher affinity to Fc.gamma.RIIB. Although not
intending to be bound by a particular mechanism of action, such
compositions are effective in inducing tolerance by activating the
Fc.gamma.RIIB mediated inhibitory pathway.
5.6 Compositions and Methods of Administering
[0499] The invention provides methods and pharmaceutical
compositions comprising molecules of the invention (i.e.,
antibodies, polypeptides) comprising variant Fc regions. The
invention also provides methods of treatment, prophylaxis, and
amelioration of one or more symptoms associated with a disease,
disorder or infection by administering to a subject an effective
amount of a fusion protein or a conjugated molecule of the
invention, or a pharmaceutical composition comprising a fusion
protein or a conjugated molecule of the invention. In a preferred
aspect, an antibody, a fusion protein, or a conjugated molecule, is
substantially purified (i.e., substantially free from substances
that limit its effect or produce undesired side-effects). In a
specific embodiment, the subject is an animal, preferably a mammal
such as non-primate (e.g., cows, pigs, horses, cats, dogs, rats
etc.) and a primate (e.g., monkey such as, a cynomolgous monkey and
a human). In a preferred embodiment, the subject is a human. In yet
another preferred embodiment, the antibody of the invention is from
the same species as the subject.
[0500] Various delivery systems are known and can be used to
administer a composition comprising molecules of the invention
(i.e., antibodies, polypeptides), comprising variant Fc regions,
e.g., encapsulation in liposomes, microparticles, microcapsules,
recombinant cells capable of expressing the antibody or fusion
protein, receptor-mediated endocytosis (See, e.g., Wu and Wu, 1987,
J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as
part of a retroviral or other vector, etc. Methods of administering
a molecule of the invention include, but are not limited to,
parenteral administration (e.g., intradermal, intramuscular,
intraperitoneal, intravenous and subcutaneous), epidural, and
mucosal (e.g., intranasal and oral routes). In a specific
embodiment, the molecules of the invention are administered
intramuscularly, intravenously, or subcutaneously. The compositions
may be administered by any convenient route, for example, by
infusion or bolus injection, by absorption through epithelial or
mucocutaneous linings (e.g., oral mucosa, rectal and intestinal
mucosa, etc.) and may be administered together with other
biologically active agents. Administration can be systemic or
local. In addition, pulmonary administration can also be employed,
e.g., by use of an inhaler or nebulizer, and formulation with an
aerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968; 5,985,320;
5,985,309; 5,934,272; 5,874,064; 5,855,913; 5,290,540; and
4,880,078; and PCT Publication Nos. WO 92/19244; WO 97/32572; WO
97/44013; WO 98/31346; and WO 99/66903, each of which is
incorporated herein by reference in its entirety.
[0501] The invention also provides that the molecules of the
invention (i.e., antibodies, polypeptides) comprising variant Fc
regions, are packaged in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of antibody. In one
embodiment, the molecules of the invention are supplied as a dry
sterilized lyophilized powder or water free concentrate in a
hermetically sealed container and can be reconstituted, e.g., with
water or saline to the appropriate concentration for administration
to a subject. Preferably, the molecules of the invention are
supplied as a dry sterile lyophilized powder in a hermetically
sealed container at a unit dosage of at least 5 mg, more preferably
at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at
least 45 mg, at least 50 mg, or at least 75 mg. The lyophilized
molecules of the invention should be stored at between 2 and
8.degree. C. in their original container and the molecules should
be administered within 12 hours, preferably within 6 hours, within
5 hours, within 3 hours, or within 1 hour after being
reconstituted. In an alternative embodiment, molecules of the
invention are supplied in liquid form in a hermetically sealed
container indicating the quantity and concentration of the
molecule, fusion protein, or conjugated molecule. Preferably, the
liquid form of the molecules of the invention are supplied in a
hermetically sealed container at least 1 mg/ml, more preferably at
least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least 10
mg/ml, at least 15 mg/kg, at least 25 mg/ml, at least 50 mg/ml, at
least 100 mg/ml, at least 150 mg/ml, at least 200 mg/ml of the
molecules.
[0502] The amount of the composition of the invention which will be
effective in the treatment, prevention or amelioration of one or
more symptoms associated with a disorder can be determined by
standard clinical techniques. The precise dose to be employed in
the formulation will also depend on the route of administration,
and the seriousness of the condition, and should be decided
according to the judgment of the practitioner and each patient's
circumstances. Effective doses may be extrapolated from
dose-response curves derived from in vitro or animal model test
systems.
[0503] For antibodies encompassed by the invention, the dosage
administered to a patient is typically 0.0001 mg/kg to 100 mg/kg of
the patient's body weight. Preferably, the dosage administered to a
patient is between 0.0001 mg/kg and 20 mg/kg, 0.0001 mg/kg and 10
mg/kg, 0.0001 mg/kg and 5 mg/kg, 0.0001 and 2 mg/kg, 0.0001 and 1
mg/kg, 0.0001 mg/kg and 0.75 mg/kg, 0.0001 mg/kg and 0.5 mg/kg,
0.0001 mg/kg to 0.25 mg/kg, 0.0001 to 0.15 mg/kg, 0.0001 to 0.10
mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kg or 0.01 to 0.10 mg/kg
of the patient's body weight. Generally, human antibodies have a
longer half-life within the human body than antibodies from other
species due to the immune response to the foreign polypeptides.
Thus, lower dosages of human antibodies and less frequent
administration is often possible. Further, the dosage and frequency
of administration of antibodies of the invention or fragments
thereof may be reduced by enhancing uptake and tissue penetration
of the antibodies by modifications such as, for example,
lipidation.
[0504] In one embodiment, the dosage of the molecules of the
invention administered to a patient are 0.01 mg to 1000 mg/day,
when used as single agent therapy. In another embodiment the
molecules of the invention are used in combination with other
therapeutic compositions and the dosage administered to a patient
are lower than when said molecules are used as a single agent
therapy.
[0505] In a specific embodiment, it may be desirable to administer
the pharmaceutical compositions of the invention locally to the
area in need of treatment; this may be achieved by, for example,
and not by way of limitation, local infusion, by injection, or by
means of an implant, said implant being of a porous, non-porous, or
gelatinous material, including membranes, such as sialastic
membranes, or fibers. Preferably, when administering a molecule of
the invention, care must be taken to use materials to which the
molecule does not absorb.
[0506] In another embodiment, the compositions can be delivered in
a vesicle, in particular a liposome (See Langer, Science
249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of
Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.),
Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 3
17-327; see generally ibid.).
[0507] In yet another embodiment, the compositions can be delivered
in a controlled release or sustained release system. Any technique
known to one of skill in the art can be used to produce sustained
release formulations comprising one or more molecules of the
invention. See, e.g., U.S. Pat. No. 4,526,938; PCT publication WO
91/05548; PCT publication WO 96/20698; Ning et al., 1996,
"Intratumoral Radioimmunotheraphy of a Human Colon Cancer Xenograft
Using a Sustained-Release Gel," Radiotherapy & Oncology
39:179-189, Song et al., 1995, "Antibody Mediated Lung Targeting of
Long-Circulating Emulsions," PDA Journal of Pharmaceutical Science
& Technology 50:372-397; Cleek et al., 1997, "Biodegradable
Polymeric Carriers for a bFGF Antibody for Cardiovascular
Application," Pro. Int'l. Symp. Control. Rel. Bioact. Mater.
24:853-854; and Lam et al., 1997, "Microencapsulation of
Recombinant Humanized Monoclonal Antibody for Local Delivery,"
Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, each of
which is incorporated herein by reference in its entirety. In one
embodiment, a pump may be used in a controlled release system (See
Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:20;
Buchwald et al., 1980, Surgery 88:507; and Saudek et al., 1989, N.
Engl. J. Med. 321:574). In another embodiment, polymeric materials
can be used to achieve controlled release of antibodies (see e.g.,
Medical Applications of Controlled Release, Langer and Wise (eds.),
CRC Pres., Boca Raton, Fla. (1974); Controlled Drug
Bioavailability, Drug Product Design and Performance, Smolen and
Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J.,
Macromol. Sci. Rev. Macromol. Chem. 23:61; See also Levy et al.,
1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351;
Howard et al., 1989, J. Neurosurg. 7 1:105); U.S. Pat. No.
5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S.
Pat. No. 5,989,463; U.S. Pat. No. 5,128,326; PCT Publication No. WO
99/15154; and PCT Publication No. WO 99/20253). Examples of
polymers used in sustained release formulations include, but are
not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl
methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate),
poly(methacrylic acid), polyglycolides (PLG), polyanhydrides,
poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide,
poly(ethylene glycol), polylactides (PLA),
poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In yet
another embodiment, a controlled release system can be placed in
proximity of the therapeutic target (e.g., the lungs), thus
requiring only a fraction of the systemic dose (see, e.g., Goodson,
in Medical Applications of Controlled Release, supra, vol. 2, pp.
115-138 (1984)). In another embodiment, polymeric compositions
useful as controlled release implants are used according to Dunn et
al. (See U.S. Pat. No. 5,945,155). This particular method is based
upon the therapeutic effect of the in situ controlled release of
the bioactive material from the polymer system. The implantation
can generally occur anywhere within the body of the patient in need
of therapeutic treatment. In another embodiment, a non-polymeric
sustained delivery system is used, whereby a non-polymeric implant
in the body of the subject is used as a drug delivery system. Upon
implantation in the body, the organic solvent of the implant will
dissipate, disperse, or leach from the composition into surrounding
tissue fluid, and the non-polymeric material will gradually
coagulate or precipitate to form a solid, microporous matrix (See
U.S. Pat. No. 5,888,533).
[0508] Controlled release systems are discussed in the review by
Langer (1990, Science 249:1527-1533). Any technique known to one of
skill in the art can be used to produce sustained release
formulations comprising one or more therapeutic agents of the
invention. See, e.g., U.S. Pat. No. 4,526,938; International
Publication Nos. WO 91/05548 and WO 96/20698; Ning et al., 1996,
Radiotherapy & Oncology 39:179-189; Song et al., 1995, PDA
Journal of Pharmaceutical Science & Technology 50:372-397;
Cleek et al., 1997, Pro. Int'l. Symp. Control. Rel. Bioact. Mater.
24:853-854; and Lam et al., 1997, Proc. Int'l. Symp. Control Rel.
Bioact. Mater. 24:759-760, each of which is incorporated herein by
reference in its entirety.
[0509] In a specific embodiment where the composition of the
invention is a nucleic acid encoding an antibody, the nucleic acid
can be administered in vivo to promote expression of its encoded
antibody, by constructing it as part of an appropriate nucleic acid
expression vector and administering it so that it becomes
intracellular, e.g., by use of a retroviral vector (See U.S. Pat.
No. 4,980,286), or by direct injection, or by use of microparticle
bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with
lipids or cell-surface receptors or transfecting agents, or by
administering it in linkage to a homeobox-like peptide which is
known to enter the nucleus (See e.g., Joliot et al., 1991, Proc.
Natl. Acad. Sci. USA 88:1864-1868), etc. Alternatively, a nucleic
acid can be introduced intracellularly and incorporated within host
cell DNA for expression by homologous recombination.
[0510] For antibodies, the therapeutically or prophylactically
effective dosage administered to a subject is typically 0.1 mg/kg
to 200 mg/kg of the subject's body weight. Preferably, the dosage
administered to a subject is between 0.1 mg/kg and 20 mg/kg of the
subject's body weight and more preferably the dosage administered
to a subject is between 1 mg/kg to 10 mg/kg of the subject's body
weight. The dosage and frequency of administration of antibodies of
the invention may be reduced also by enhancing uptake and tissue
penetration (e.g., into the lung) of the antibodies or fusion
proteins by modifications such as, for example, lipidation.
[0511] Treatment of a subject with a therapeutically or
prophylactically effective amount of molecules of the invention can
include a single treatment or, preferably, can include a series of
treatments. In a preferred example, a subject is treated with
molecules of the invention in the range of between about 0.1 to 30
mg/kg body weight, one time per week for between about 1 to 10
weeks, preferably between 2 to 8 weeks, more preferably between
about 3 to 7 weeks, and even more preferably for about 4, 5, or 6
weeks. In other embodiments, the pharmaceutical compositions of the
invention are administered once a day, twice a day, or three times
a day. In other embodiments, the pharmaceutical compositions are
administered once a week, twice a week, once every two weeks, once
a month, once every six weeks, once every two months, twice a year
or once per year. It will also be appreciated that the effective
dosage of the molecules used for treatment may increase or decrease
over the course of a particular treatment.
[0512] 5.6.1 Pharmaceutical Compositions
[0513] The compositions of the invention include bulk drug
compositions useful in the manufacture of pharmaceutical
compositions (e.g., impure or non-sterile compositions) and
pharmaceutical compositions (i.e., compositions that are suitable
for administration to a subject or patient) which can be used in
the preparation of unit dosage forms. Such compositions comprise a
prophylactically or therapeutically effective amount of a
prophylactic and/or therapeutic agent disclosed herein or a
combination of those agents and a pharmaceutically acceptable
carrier. Preferably, compositions of the invention comprise a
prophylactically or therapeutically effective amount of one or more
molecules of the invention and a pharmaceutically acceptable
carrier.
[0514] In one particular embodiment, the pharmaceutical composition
comprises a therapeutically effective amount of one or more
molecules of the invention comprising a variant Fc region, wherein
said variant Fc region binds Fc.gamma.RIIIA and/or Fc.gamma.RIIA
with a greater affinity than a comparable molecule comprising a
wild-type Fc region binds Fc.gamma.RIIIA and/or Fc.gamma.RIIA
and/or said variant Fc region confers an effector function or
mediates an effector function at least 2-fold more effectively than
a comparable molecule comprising a wild-type Fc region, and a
pharmaceutically acceptable carrier. In another embodiment, the
pharmaceutical composition comprises a therapeutically effective
amount of one or more molecules of the invention comprising a
variant Fc region, wherein said variant Fc region binds
Fc.gamma.RIIIA with a greater affinity than a comparable molecule
comprising a wild-type Fc region binds Fc.gamma.RIIIA, and said
variant Fc region binds Fc.gamma.RIIB with a lower affinity than a
comparable molecule comprising a wild-type Fc region binds
Fc.gamma.RIIB, and/or said variant Fc region confers and effector
function or mediates an effector function at least 2-fold more
effectively than a comparable molecule comprising a wild-type Fc
region, and a pharmaceutically acceptable carrier. In another
embodiment, said pharmaceutical compositions further comprise one
or more anti-cancer agents.
[0515] The invention also encompasses pharmaceutical compositions
comprising a therapeutic antibody (e.g., tumor specific monoclonal
antibody) that is specific for a particular cancer antigen,
comprising one or more amino acid modifications in the Fc region as
determined in accordance with the instant invention, and a
pharmaceutically acceptable carrier.
[0516] In a specific embodiment, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more
particularly in humans. The term "carrier" refers to a diluent,
adjuvant (e.g., Freund's adjuvant (complete and incomplete),
excipient, or vehicle with which the therapeutic is administered.
Such pharmaceutical carriers can be sterile liquids, such as water
and oils, including those of petroleum, animal, vegetable or
synthetic origin, such as peanut oil, soybean oil, mineral oil,
sesame oil and the like. Water is a preferred carrier when the
pharmaceutical composition is administered intravenously. Saline
solutions and aqueous dextrose and glycerol solutions can also be
employed as liquid carriers, particularly for injectable solutions.
Suitable pharmaceutical excipients include starch, glucose,
lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel,
sodium stearate, glycerol monostearate, talc, sodium chloride,
dried skim milk, glycerol, propylene, glycol, water, ethanol and
the like. The composition, if desired, can also contain minor
amounts of wetting or emulsifying agents, or pH buffering agents.
These compositions can take the form of solutions, suspensions,
emulsion, tablets, pills, capsules, powders, sustained-release
formulations and the like.
[0517] Generally, the ingredients of compositions of the invention
are supplied either separately or mixed together in unit dosage
form, for example, as a dry lyophilized powder or water free
concentrate in a hermetically sealed container such as an ampoule
or sachette indicating the quantity of active agent. Where the
composition is to be administered by infusion, it can be dispensed
with an infusion bottle containing sterile pharmaceutical grade
water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0518] The compositions of the invention can be formulated as
neutral or salt forms. Pharmaceutically acceptable salts include,
but are not limited to those formed with anions such as those
derived from hydrochloric, phosphoric, acetic, oxalic, tartaric
acids, etc., and those formed with cations such as those derived
from sodium, potassium, ammonium, calcium, ferric hydroxides,
isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, etc.
[0519] 5.6.2 Gene Therapy
[0520] In a specific embodiment, nucleic acids comprising sequences
encoding molecules of the invention, are administered to treat,
prevent or ameliorate one or more symptoms associated with a
disease, disorder, or infection, by way of gene therapy. Gene
therapy refers to therapy performed by the administration to a
subject of an expressed or expressible nucleic acid. In this
embodiment of the invention, the nucleic acids produce their
encoded antibody or fusion protein that mediates a therapeutic or
prophylactic effect.
[0521] Any of the methods for gene therapy available in the art can
be used according to the present invention. Exemplary methods are
described below.
[0522] For general reviews of the methods of gene therapy, see
Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu,
1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol.
Toxicol. 32:573-596; Mulligan, Science 260:926-932 (1993); and
Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May,
1993, TIBTECH 11(5):155-215. Methods commonly known in the art of
recombinant DNA technology which can be used are described in
Ausubel et al. (eds.), Current Protocols in Molecular Biology, John
Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and
Expression, A Laboratory Manual, Stockton Press, NY (1990).
[0523] In a preferred aspect, a composition of the invention
comprises nucleic acids encoding an antibody, said nucleic acids
being part of an expression vector that expresses the antibody in a
suitable host. In particular, such nucleic acids have promoters,
preferably heterologous promoters, operably linked to the antibody
coding region, said promoter being inducible or constitutive, and,
optionally, tissue-specific. In another particular embodiment,
nucleic acid molecules are used in which the antibody coding
sequences and any other desired sequences are flanked by regions
that promote homologous recombination at a desired site in the
genome, thus providing for intrachromosomal expression of the
antibody encoding nucleic acids (Koller and Smithies, 1989, Proc.
Natl. Acad. Sci. USA 86:8932-8935; and Zijlstra et al., 1989,
Nature 342:435-438).
[0524] In another preferred aspect, a composition of the invention
comprises nucleic acids encoding a fusion protein, said nucleic
acids being a part of an expression vector that expresses the
fusion protein in a suitable host. In particular, such nucleic
acids have promoters, preferably heterologous promoters, operably
linked to the coding region of a fusion protein, said promoter
being inducible or constitutive, and optionally, tissue-specific.
In another particular embodiment, nucleic acid molecules are used
in which the coding sequence of the fusion protein and any other
desired sequences are flanked by regions that promote homologous
recombination at a desired site in the genome, thus providing for
intrachromosomal expression of the fusion protein.
[0525] Delivery of the nucleic acids into a subject may be either
direct, in which case the subject is directly exposed to the
nucleic acid or nucleic acid-carrying vectors, or indirect, in
which case, cells are first transformed with the nucleic acids in
vitro, then transplanted into the subject. These two approaches are
known, respectively, as in vivo or ex vivo gene therapy.
[0526] In a specific embodiment, the nucleic acid sequences are
directly administered in vivo, where it is expressed to produce the
encoded product. This can be accomplished by any of numerous
methods known in the art, e.g., by constructing them as part of an
appropriate nucleic acid expression vector and administering it so
that they become intracellular, e.g., by infection using defective
or attenuated retroviral or other viral vectors (see U.S. Pat. No.
4,980,286), or by direct injection of naked DNA, or by use of
microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or
coating with lipids or cell-surface receptors or transfecting
agents, encapsulation in liposomes, microparticles, or
microcapsules, or by administering them in linkage to a peptide
which is known to enter the nucleus, by administering it in linkage
to a ligand subject to receptor-mediated endocytosis (See, e.g., Wu
and Wu, 1987, J. Biol. Chem. 262:4429-4432) (which can be used to
target cell types specifically expressing the receptors), etc. In
another embodiment, nucleic acid-ligand complexes can be formed in
which the ligand comprises a fusogenic viral peptide to disrupt
endosomes, allowing the nucleic acid to avoid lysosomal
degradation. In yet another embodiment, the nucleic acid can be
targeted in vivo for cell specific uptake and expression, by
targeting a specific receptor (See, e.g., PCT Publications WO
92/06180; WO 92/22635; WO92/20316; WO93/14188; WO 93/20221).
Alternatively, the nucleic acid can be introduced intracellularly
and incorporated within host cell DNA for expression, by homologous
recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci.
USA 86:8932-8935; and Zijlstra et al., 1989, Nature
342:435-438).
[0527] In a specific embodiment, viral vectors that contain nucleic
acid sequences encoding a molecule of the invention (e.g., an
antibody or a fusion protein) are used. For example, a retroviral
vector can be used (See Miller et al., 1993, Meth. Enzymol.
217:581-599). These retroviral vectors contain the components
necessary for the correct packaging of the viral genome and
integration into the host cell DNA. The nucleic acid sequences
encoding the antibody or a fusion protein to be used in gene
therapy are cloned into one or more vectors, which facilitates
delivery of the nucleotide sequence into a subject. More detail
about retroviral vectors can be found in Boesen et al., (1994,
Biotherapy 6:291-302), which describes the use of a retroviral
vector to deliver the mdr 1 gene to hematopoietic stem cells in
order to make the stem cells more resistant to chemotherapy. Other
references illustrating the use of retroviral vectors in gene
therapy are: Clowes et al., 1994, J. Clin. Invest. 93:644-651;
Klein et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993,
Human Gene Therapy 4:129-141; and Grossman and Wilson, 1993, Curr.
Opin. in Genetics and Devel. 3:110-114.
[0528] Adenoviruses are other viral vectors that can be used in
gene therapy. Adenoviruses are especially attractive vehicles for
delivering genes to respiratory epithelia. Adenoviruses naturally
infect respiratory epithelia where they cause a mild disease. Other
targets for adenovirus-based delivery systems are liver, the
central nervous system, endothelial cells, and muscle. Adenoviruses
have the advantage of being capable of infecting non-dividing
cells. Kozarsky and Wilson (Current Opinion in Genetics and
Development 3:499-503, 1993, present a review of adenovirus-based
gene therapy. Bout et al., (Human Gene Therapy, 5:3-10, 1994)
demonstrated the use of adenovirus vectors to transfer genes to the
respiratory epithelia of rhesus monkeys. Other instances of the use
of adenoviruses in gene therapy can be found in Rosenfeld et al.,
1991, Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155;
Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234; PCT
Publication WO94/12649; and Wang et al., 1995, Gene Therapy
2:775-783. In a preferred embodiment, adenovirus vectors are
used.
[0529] Adeno-associated virus (AAV) has also been proposed for use
in gene therapy (see, e.g., Walsh et al., 1993, Proc. Soc. Exp.
Biol. Med. 204:289-300 and U.S. Pat. No. 5,436,146).
[0530] Another approach to gene therapy involves transferring a
gene to cells in tissue culture by such methods as electroporation,
lipofection, calcium phosphate mediated transfection, or viral
infection. Usually, the method of transfer includes the transfer of
a selectable marker to the cells. The cells are then placed under
selection to isolate those cells that have taken up and are
expressing the transferred gene. Those cells are then delivered to
a subject.
[0531] In this embodiment, the nucleic acid is introduced into a
cell prior to administration in vivo of the resulting recombinant
cell. Such introduction can be carried out by any method known in
the art, including but not limited to, transfection,
electroporation, microinjection, infection with a viral or
bacteriophage vector, containing the nucleic acid sequences, cell
fusion, chromosome-mediated gene transfer, microcellmediated gene
transfer, spheroplast fusion, etc. Numerous techniques are known in
the art for the introduction of foreign genes into cells (See,
e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618, Cohen et
al., 1993, Meth. Enzymol. 217:618-644; and Clin. Pharma. Ther.
29:69-92, 1985) and may be used in accordance with the present
invention, provided that the necessary developmental and
physiological functions of the recipient cells are not disrupted.
The technique should provide for the stable transfer of the nucleic
acid to the cell, so that the nucleic acid is expressible by the
cell and preferably heritable and expressible by its cell
progeny.
[0532] The resulting recombinant cells can be delivered to a
subject by various methods known in the art. Recombinant blood
cells (e.g., hematopoietic stem or progenitor cells) are preferably
administered intravenously. The amount of cells envisioned for use
depends on the desired effect, patient state, etc., and can be
determined by one skilled in the art.
[0533] Cells into which a nucleic acid can be introduced for
purposes of gene therapy encompass any desired, available cell
type, and include but are not limited to epithelial cells,
endothelial cells, keratinocytes, fibroblasts, muscle cells,
hepatocytes; blood cells such as T lymphocytes, B lymphocytes,
monocytes, macrophages, neutrophils, eosinophils, megakaryocytes,
granulocytes; various stem or progenitor cells, in particular
hematopoietic stem or progenitor cells, e.g., as obtained from bone
marrow, umbilical cord blood, peripheral blood, fetal liver,
etc.
[0534] In a preferred embodiment, the cell used for gene therapy is
autologous to the subject.
[0535] In an embodiment in which recombinant cells are used in gene
therapy, nucleic acid sequences encoding an antibody or a fusion
protein are introduced into the cells such that they are
expressible by the cells or their progeny, and the recombinant
cells are then administered in vivo for therapeutic effect. In a
specific embodiment, stem or progenitor cells are used. Any stem
and/or progenitor cells which can be isolated and maintained in
vitro can potentially be used in accordance with this embodiment of
the present invention (See e.g., PCT Publication WO 94/08598;
Stemple and Anderson, 1992, Cell 7 1:973-985; Rheinwald, 1980,
Meth. Cell Bio. 21A:229; and Pittelkow and Scott, 1986, Mayo Clinic
Proc. 61:771).
[0536] In a specific embodiment, the nucleic acid to be introduced
for purposes of gene therapy comprises an inducible promoter
operably linked to the coding region, such that expression of the
nucleic acid is controllable by controlling the presence or absence
of the appropriate inducer of transcription.
[0537] 5.6.3 Kits
[0538] The invention provides a pharmaceutical pack or kit
comprising one or more containers filled with the molecules of the
invention (i.e., antibodies, polypeptides comprising variant Fc
regions). Additionally, one or more other prophylactic or
therapeutic agents useful for the treatment of a disease can also
be included in the pharmaceutical pack or kit. The invention also
provides a pharmaceutical pack or kit comprising one or more
containers filled with one or more of the ingredients of the
pharmaceutical compositions of the invention. Optionally associated
with such container(s) can be a notice in the form prescribed by a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals or biological products, which notice reflects
approval by the agency of manufacture, use or sale for human
administration.
[0539] The present invention provides kits that can be used in the
above methods. In one embodiment, a kit comprises one or more
molecules of the invention. In another embodiment, a kit further
comprises one or more other prophylactic or therapeutic agents
useful for the treatment of cancer or infectious disease, in one or
more containers. In another embodiment, a kit further comprises one
or more cytotoxic antibodies that bind one or more antigens
associated with cancer or infectious disease. In certain
embodiments, the other prophylactic or therapeutic agent is a
chemotherapeutic. In other embodiments, the prophylactic or
therapeutic agent is a biological or hormonal therapeutic.
5.7 Characterization and Demonstration of Therapeutic Utility
[0540] Several aspects of the pharmaceutical compositions,
prophylactic, or therapeutic agents of the invention are preferably
tested in vitro, in a cell culture system, and in an animal model
organism, such as a rodent animal model system, for the desired
therapeutic activity prior to use in humans. For example, assays
which can be used to determine whether administration of a specific
pharmaceutical composition is desired, include cell culture assays
in which a patient tissue sample is grown in culture, and exposed
to or otherwise contacted with a pharmaceutical composition of the
invention, and the effect of such composition upon the tissue
sample is observed. The tissue sample can be obtained by biopsy
from the patient. This test allows the identification of the
therapeutically most effective prophylactic or therapeutic
molecule(s) for each individual patient. In various specific
embodiments, in vitro assays can be carried out with representative
cells of cell types involved in an autoimmune or inflammatory
disorder (e.g., T cells), to determine if a pharmaceutical
composition of the invention has a desired effect upon such cell
types.
[0541] Combinations of prophylactic and/or therapeutic agents can
be tested in suitable animal model systems prior to use in humans.
Such animal model systems include, but are not limited to, rats,
mice, chicken, cows, monkeys, pigs, dogs, rabbits, etc. Any animal
system well-known in the art may be used. In a specific embodiment
of the invention, combinations of prophylactic and/or therapeutic
agents are tested in a mouse model system. Such model systems are
widely used and well-known to the skilled artisan. Prophylactic
and/or therapeutic agents can be administered repeatedly. Several
aspects of the procedure may vary. Said aspects include the
temporal regime of administering the prophylactic and/or
therapeutic agents, and whether such agents are administered
separately or as an admixture.
[0542] Preferred animal models for use in the methods of the
invention are, for example, transgenic mice expressing human
Fc.gamma.Rs on mouse effector cells, e.g., any mouse model
described in U.S. Pat. No. 5,877,396 (which is incorporated herein
by reference in its entirety) can be used in the present invention.
Transgenic mice for use in the methods of the invention include,
but are not limited to, mice carrying human Fc.gamma.RIIIA; mice
carrying human Fc.gamma.RIIA; mice carrying human Fc.gamma.RIIB and
human Fc.gamma.RIIIA; mice carrying human Fc.gamma.RIIB and human
Fc.gamma.RIIA.
[0543] Preferably, mutations showing the highest levels of activity
in the functional assays described above will be tested for use in
animal model studies prior to use in humans. Sufficient quantities
of antibodies may be prepared for use in animal models using
methods described supra, for example, using mammalian expression
systems and IgG purification methods disclosed and exemplified
herein.
[0544] Mouse xenograft models may be used for examining efficacy of
mouse antibodies generated against a tumor specific target based on
the affinity and specificity of the CDR regions of the antibody
molecule and the ability of the Fc region of the antibody to elicit
an immune response (Wu et al., 2001, Trends Cell Biol. 11: S2-9).
Transgenic mice expressing human Fc.gamma.Rs on mouse effector
cells are unique and are tailor-made animal models to test the
efficacy of human Fc-Fc.gamma.R interactions. Pairs of
Fc.gamma.RIIIA, Fc.gamma.RIIIB and Fc.gamma.RIIA transgenic mouse
lines generated in the lab of Dr. Jeffrey Ravetch (Through a
licensing agreement with Rockefeller U. and Sloan Kettering Cancer
center) can be used such as those listed in the Table 13 below.
TABLE-US-00015 TABLE 13 Mice Strains Strain Background Human FcR
Nude/CD16A KO none Nude/CD16A KO Fc.gamma.RIIIA Nude/CD16A KO
Fc.gamma.R IIA Nude/CD16A KO Fc.gamma.R IIA and IIIA Nude/CD32B KO
none Nude/CD32B KO Fc.gamma.R IIB
[0545] Preferably Fc mutants showing both enhanced binding to
Fc.gamma.RIIIA and reduced binding to Fc.gamma.RIIB, increased
activity in ADCC and phagocytosis assays are tested in animal model
experiments. The animal model experiments examine the increase in
efficacy of Fc mutant bearing antibodies in Fc.gamma.RIIIA
transgenic, nude mCD16A knockout mice compared to a control which
has been administered native antibody. Preferably, groups of 8-10
mice are examined using a standard protocol. An exemplary animal
model experiment may comprise the following steps: in a breast
cancer model, .about.2.times.10.sup.6 SK-BR-3 cells are injected
subcutaneously on day 1 with 0.1 mL PBS mixed with Matrigel (Becton
Dickinson). Initially a wild type chimeric antibody and isotype
control are administered to establish a curve for the predetermined
therapeutic dose, intravenous injection of 4D5 on day 1 with an
initial dose of 4 .mu.g/g followed by weekly injections of 2
.mu.g/g. Tumor volume is monitored for 6-8 weeks to measure
progress of the disease. Tumor volume should increase linearly with
time in animals injected with the isotype control. In contrast very
little tumor growth should occur in the group injected with 4D5.
Results from the standard dose study are used to set an upper limit
for experiments testing the Fc mutants. These studies are done
using subtherapeutic doses of the Fc mutant containing antibodies.
A one tenth dose was used on xenograft models in experiments done
in Fc.gamma.RIIB knockout mice, see, Clynes et al., 2000, Nat. Med.
6: 443-6, with a resultant block in tumor cell growth. Since the
mutants of the invention preferrably show an increase in
Fc.gamma.RIIIA activation and reduction in Fc.gamma.RIIB binding
the mutants are examined at one tenth therapeutic dose. Examination
of tumor size at different intervals indicates the efficacy of the
antibodies at the lower dose. Statistical analysis of the data
using t test provides a way of determining if the data is
significant. Fc mutants that show increased efficacy are tested at
incrementally lower doses to determine the smallest possible dose
as a measure of their efficacy.
[0546] The anti-inflammatory activity of the combination therapies
of invention can be determined by using various experimental animal
models of inflammatory arthritis known in the art and described in
Crofford L. J. and Wilder R. L., "Arthritis and Autoimmunity in
Animals", in Arthritis and Allied Conditions: A Textbook of
Rheumatology, McCarty et al. (eds.), Chapter 30 (Lee and Febiger,
1993). Experimental and spontaneous animal models of inflammatory
arthritis and autoimmune rheumatic diseases can also be used to
assess the anti-inflammatory activity of the combination therapies
of invention. The following are some assays provided as examples,
and not by limitation.
[0547] The principle animal models for arthritis or inflammatory
disease known in the art and widely used include: adjuvant-induced
arthritis rat models, collagen-induced arthritis rat and mouse
models and antigen-induced arthritis rat, rabbit and hamster
models, all described in Crofford L. J. and Wilder R. L.,
"Arthritis and Autoimmunity in Animals", in Arthritis and Allied
Conditions: A Textbook of Rheumatology, McCarty et al.(eds.),
Chapter 30 (Lee and Febiger, 1993), incorporated herein by
reference in its entirety.
[0548] The anti-inflammatory activity of the combination therapies
of invention can be assessed using a carrageenan-induced arthritis
rat model. Carrageenan-induced arthritis has also been used in
rabbit, dog and pig in studies of chronic arthritis or
inflammation. Quantitative histomorphometric assessment is used to
determine therapeutic efficacy. The methods for using such a
carrageenan-induced arthritis model is described in Hansra P. et
al., "Carrageenan-Induced Arthritis in the Rat," Inflammation,
24(2): 141-155, (2000). Also commonly used are zymosan-induced
inflammation animal models as known and described in the art.
[0549] The anti-inflammatory activity of the combination therapies
of invention can also be assessed by measuring the inhibition of
carrageenan-induced paw edema in the rat, using a modification of
the method described in Winter C. A. et al., "Carrageenan-Induced
Edema in Hind Paw of the Rat as an Assay for Anti-inflammatory
Drugs" Proc. Soc. Exp. Biol Med. 111, 544-547, (1962). This assay
has been used as a primary in vivo screen for the anti-inflammatory
activity of most NSAIDs, and is considered predictive of human
efficacy. The anti-inflammatory activity of the test prophylactic
or therapeutic agents is expressed as the percent inhibition of the
increase in hind paw weight of the test group relative to the
vehicle dosed control group.
[0550] Animal models for autoimmune disorders can also be used to
assess the efficacy of the combination therapies of invention.
Animal models for autoimmune disorders such as type 1 diabetes,
thyroid autoimmunity, systemic lupus eruthematosus, and
glomerulonephritis have been developed (Flanders et al., 1999,
Autoimmunity 29:235-246; Krogh et al., 1999, Biochimie 81:511-515;
Foster, 1999, Semin. Nephrol. 19:12-24).
[0551] Further, any assays known to those skilled in the art can be
used to evaluate the prophylactic and/or therapeutic utility of the
combinatorial therapies disclosed herein for autoimmune and/or
inflammatory diseases.
[0552] Toxicity and efficacy of the prophylactic and/or therapeutic
protocols of the instant invention can be determined by standard
pharmaceutical procedures in cell cultures or experimental animals,
e.g., for determining the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index and it can be
expressed as the ratio LD.sub.50/ED.sub.50. Prophylactic and/or
therapeutic agents that exhibit large therapeutic indices are
preferred. While prophylactic and/or therapeutic agents that
exhibit toxic side effects may be used, care should be taken to
design a delivery system that targets such agents to the site of
affected tissue in order to minimize potential damage to uninfected
cells and, thereby, reduce side effects.
[0553] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage of the
prophylactic and/or therapeutic agents for use in humans. The
dosage of such agents lies preferably within a range of circulating
concentrations that include the ED.sub.50 with little or no
toxicity. The dosage may vary within this range depending upon the
dosage form employed and the route of administration utilized. For
any agent used in the method of the invention, the therapeutically
effective dose can be estimated initially from cell culture assays.
A dose may be formulated in animal models to achieve a circulating
plasma concentration range that includes the IC.sub.50 (i.e., the
concentration of the test compound that achieves a half-maximal
inhibition of symptoms) as determined in cell culture. Such
information can be used to more accurately determine useful doses
in humans. Levels in plasma may be measured, for example, by high
performance liquid chromatography.
[0554] The anti-cancer activity of the therapies used in accordance
with the present invention also can be determined by using various
experimental animal models for the study of cancer such as the SCID
mouse model or transgenic mice or nude mice with human xenografts,
animal models, such as hamsters, rabbits, etc. known in the art and
described in Relevance of Tumor Models for Anticancer Drug
Development (1999, eds. Fiebig and Burger); Contributions to
Oncology (1999, Karger); The Nude Mouse in Oncology Research (1991,
eds. Boven and Winograd); and Anticancer Drug Development Guide
(1997 ed. Teicher), herein incorporated by reference in their
entireties.
[0555] Preferred animal models for determining the therapeutic
efficacy of the molecules of the invention are mouse xenograft
models. Tumor cell lines that can be used as a source for xenograft
tumors include but are not limited to, SKBR3 and MCF7 cells, which
can be derived from patients with breast adenocarcinoma. These
cells have both erbB2 and prolactin receptors. SKBR3 cells have
been used routinely in the art as ADCC and xenograft tumor models.
Alternatively, OVCAR3 cells derived from a human ovarian
adenocarcinoma can be used as a source for xenograft tumors.
[0556] The protocols and compositions of the invention are
preferably tested in vitro, and then in vivo, for the desired
therapeutic or prophylactic activity, prior to use in humans.
Therapeutic agents and methods may be screened using cells of a
tumor or malignant cell line. Many assays standard in the art can
be used to assess such survival and/or growth; for example, cell
proliferation can be assayed by measuring .sup.3H-thymidine
incorporation, by direct cell count, by detecting changes in
transcriptional activity of known genes such as proto-oncogenes
(e.g., fos, myc) or cell cycle markers; cell viability can be
assessed by trypan blue staining, differentiation can be assessed
visually based on changes in morphology, decreased growth and/or
colony formation in soft agar or tubular network formation in
three-dimensional basement membrane or extracellular matrix
preparation, etc.
[0557] Compounds for use in therapy can be tested in suitable
animal model systems prior to testing in humans, including but not
limited to in rats, mice, chicken, cows, monkeys, rabbits,
hamsters, etc., for example, the animal models described above. The
compounds can then be used in the appropriate clinical trials.
[0558] Further, any assays known to those skilled in the art can be
used to evaluate the prophylactic and/or therapeutic utility of the
combinatorial therapies disclosed herein for treatment or
prevention of cancer, inflammatory disorder, or autoimmune
disease.
5.8 Diagnostic Assays
[0559] The invention encompasses molecules, e.g., antibodies, with
altered affinities and avidities for one or more Fc.gamma.Rs. The
antibodies of the invention with enhanced affinity and avidity for
one or more Fc.gamma.Rs are particularly useful in cellular systems
(for example for research or diagnostic purposes) where the
Fc.gamma.Rs are expressed at low levels. Although not intending to
be bound by a particular mechanism of action, the molecules of the
invention with enhanced affinity and avidity for a particular
Fc.gamma.R are valuable as research and diagnostic tools by
enhancing the sensitivity of detection of Fc.gamma.Rs which are
normally undetectable due to a low level of expression.
6. EXAMPLES
[0560] Using a yeast display system, mutant human IgG1 heavy chain
Fc regions were screened for modified affinity to different Fc
receptors. In particular, a mutant Fc library was generated by
error prone PCR (Genemorph, Stratagene), and then the mutant Fc
proteins were fused to the Aga2p cell wall protein, which allowed
the fusion protein to be secreted extracellularly and displayed on
the yeast cell wall.
[0561] Soluble forms of the human receptors (Fc.gamma.RIIIA and
Fc.gamma.RIIB) were cloned. Detection of the IgG1 Fc domains on the
yeast cell surface, however, is hindered due to the low affinity of
Fc.gamma.R for its ligand. In order to circumvent this limitation,
soluble Fc.gamma.R tetrameric complexes were formed using an AVITAG
sequence which could be enzymatically biotinylated and subsequently
reacted with streptavidin conjugated to phycoerythrin (SA-PE;
Molecular Probes) to form soluble tetrameric Fc.gamma.R complexes.
ELISA assays confirmed that the soluble Fc.gamma.R tetrameric
complexes had a higher avidity for human IgG1 relative to the
monomeric Fc.gamma.R. Fc fusion proteins on the yeast cell surface
also bound the soluble Fc.gamma.R tetrameric complexes as assessed
by FACS analysis.
[0562] The differential binding of the Fc fusion proteins expressed
on the yeast cell surface to soluble tetrameric Fc.gamma.R
complexes was monitored by a FACS analysis. Fc fusion proteins with
altered affinities for one or more soluble tetrameric Fc.gamma.R
complexes were thus identified and were then incorporated into a
complete immunoglobulin and expressed in mammalian cells. The
mammalian expressed product was used in ELISA assays to confirm the
results obtained in the yeast surface display system. Finally, the
mutant Fc regions were sequenced to confirm the altered
residue(s).
6.1 Cloning, Expression and Purification of Fc.gamma.RIIIA
[0563] Materials and Methods
[0564] Soluble Fc.gamma.RIIB and Fc.gamma.RIIIA were cloned as
follows. The cDNA clones for the human Fc.gamma.R genes
(Fc.gamma.RIIB and Fc.gamma.RIIIA) were obtained (gift from Ravetch
lab). Soluble region of the Fc.gamma.RIIIA gene (amino acids 7-203)
was amplified by PCR (Table 14), digested with BamHI/HindIII and
ligated into the pET25vector (Novagen). This vector was digested
with Sall/Notl and a 370 by fragment was gel isolated. The vector
hu3A, (gift from J. Ravetch) was digested with BamHI/Sall and a 270
by fragment containing the N-terminus of Fc.gamma.RIIIA was
isolated. Both fragments were coligated into pcDNA3.1 cut with
BamH/NotI to create pcDNA3-Fc.gamma.RIIIA (amino acids 1-203). The
soluble region of Fc.gamma.RIIB (amino acids 33-180) was amplified
by PCR (Table 14), digested with BglII/HindIII and ligated into
pET25b(+) (Novagen). This vector was digested with BamHI/NotI and a
140 bp fragment was gel isolated. The vector huRIIb1 (gift from J.
Ravetch) was digested with BamHI/EcoRI and a 440 bp N-terminal
Fc.gamma.RIIB fragment was isolated. Both of these fragments were
coligated into pcDNA3.1 cut with BamHI/Notl to create
pcDNA3-Fc.gamma.RIIB (amino acids 1-180). Recombinant clones were
transfected into 293H cells, supernatants were collected from cell
cultures, and soluble recombinant Fc.gamma.R (rFc.gamma.R) proteins
were purified on an IgG sepharose column.
[0565] Results
[0566] Recombinant Soluble Fc.gamma.RIIIA (rFc.gamma.RIIIA) and
Recombinant Soluble Fc.gamma.RIIB (rFc.gamma.RIIB) were Purified to
Homogeneity
[0567] Subsequent to expression and purification of the recombinant
soluble Fc.gamma.R proteins on an IgG sepharose column, the purity
and apparent molecular weight of the recombinant purified soluble
receptor proteins were determined by SDS-PAGE. As shown in FIG. 1,
soluble rFc.gamma.RIIIA (FIG. 2, lane 1) had the expected apparent
molecular weight of .about.35 KDa and soluble rFc.gamma.RIIB (FIG.
2, lane 4) had the expected apparent molecular weight of .about.20
KDa. As shown in FIG. 2, soluble rFc.gamma.RIIIA migrates as a
diffuse "fuzzy" band which has been attributed to the high degree
of glycosylation normally found on Fc.gamma.RIIIA (Jefferis, et
al., 1995 Immunol Lett. 44, 111-117).
[0568] 6.1.1 Characterization of Purified Recombinant Soluble
Fc.gamma.RIIIA
[0569] Materials and Methods
[0570] Purified soluble rFc.gamma.RIIIA, which was obtained as
described above, was analyzed for direct binding against human
monomeric or aggregated IgG using an ELISA assay. The plate is
coated with 10 ng of soluble rFc.gamma.RIIIA overnight in
1.times.PBS. Subsequent to coating, the plate is washed three times
in 1.times. PBS/0.1% Tween 20. Human IgG, either biotinylated
monomeric IgG or biotinylated aggregated IgG, is added to the wells
at a concentration ranging from 0.03 mg/mL to 2 mg/mL, and allowed
to bind to the soluble rFc.gamma.RIIIA. The reaction is carried out
for one hour at 37.degree. C. The plate is washed again three times
with 1.times. PBS/0.1% Tween 20. The binding of human IgG to
soluble rFc.gamma.RIIIA is detected with streptavidin horseradish
peroxidase conjugate by monitoring the absorbance at 650 nm. The
absorbance at 650 nm is proportional to the bound aggregated
IgG.
[0571] In a blocking ELISA experiment, the ability of an
Fc.gamma.RIIIA monoclonal antibody, 3G8, a mouse
anti-Fc.gamma.RIIIA antibody (Pharmingen), to block the binding of
the receptor to aggregated IgG is monitored. The washing and
incubation conditions were the same as described above, except that
prior to IgG addition, a 5-fold molar excess of 3G8 was added and
allowed to incubate for 30 minutes at 37.degree. C.
[0572] Results
[0573] Purified, recombinant soluble Fc.gamma.RIIIA binds
aggregated IgG specifically
[0574] The direct binding of purified recombinant soluble
Fc.gamma.RIIIA to aggregated and monomeric IgG was tested using an
ELISA assay (FIG. 3). At an IgG concentration of 2 .mu.g/ml, strong
binding to the aggregated IgG was observed. However, at a similar
concentration, no binding was detected to the monomeric IgG. The
binding to aggregated IgG was blocked by 3G8, a mouse
anti-Fc.gamma.RIIIA monoclonal antibody that blocks the ligand
binding site, indicating that the aggregated IgG binding is via
that of the normal Fc.gamma.RIIIA ligand binding site (FIG. 3).
Soluble rFc.gamma.RIIB was also characterized and shown to bind to
IgG with similar characteristics as the soluble rFc.gamma.RIIIA
(data not shown).
6.2 Formation of Soluble Fc.gamma.R Tetrameric Complexes
[0575] Materials and Methods
[0576] Construction of plasmids for expression of soluble FcRyIIIA
and FcRyIIB fused to the AVITAG peptide.
[0577] To generate soluble Fc.gamma.R tetrameric complexes, the
soluble region of the human FcRgIIIA gene (amino acids 7-203) was
amplified by PCR (Table 14), digested with BamHI/HindIII and
ligated into the pET25b(+) (Novagen). This vector was digested with
SalI/Notl, and a 370 bp fragment was isolated by agarose gel
electrophoresis. The vector hu3A, (gift from J. Ravetch) was
digested with BamHI/SalI, and a 270 bp fragment containing the
N-terminus of FcRyIIIA was isolated. Both fragments were coligated
into pcDNA3.1 (Invitrogen), which had been digested with BamH/NotI
to create pcDNA3-FcRgIIIA (amino acids 1-203).
[0578] The soluble region of FcRyIIB (amino acids 33-180) was
amplified by PCR (Table 14), digested with BglII/HindIII and
ligated into pET25b(+) (Novagen). This vector was digested with
BamHI/NotI, and a 140 bp fragment was isolated by agarose gel
electrophoresis. The vector huRIIb.sub.1 (gift from J. Ravetch) was
digested with BamHI/EcoRI, and a 440 by FcRyIIB N-terminal fragment
was isolated. Both of these fragments were co-ligated into
pcDNA3.1, which had been digested with BamHI/Notl to create
pcDNA3-FcRyIIB (amino acids 1-180). Subsequently, the
linker-AVITAG.TM. peptide sequence was fused to the C-terminus of
both Fc.gamma.RIIIA and Fc.gamma.RIIB. To generate the
Fc.gamma.RIIIA-linker-avitag and Fc.gamma.RIIB-linker-AVITAG.TM.
peptide constructs, the pcDNA3.1 Fc.gamma.RIIIA and Fc.gamma.RIIB
constructs were digested with Not I and XbaI (both cut in the
vector sequence) and a 86 base pair double stranded oligonucleotide
consisting of NotI site at the 5' end and XbaI at the 3' end was
ligated into the vector. This 86 bp fragment was generated by
annealing two 5' phosphorylated reverse complement oligonucleotides
(shown in Table 12 as 5' and 3' linker.avitag primers) with the
restrictions sites for NotI and XbaI already pre-designed. Equal
volumes of each primer at 100 ng per ul were mixed and the DNA
heated to 90.degree. C. for 15 minutes and cooled at room
temperature for an hour to anneal. This created a double-stranded
DNA fragment ready to be ligated to the pcDNA3.1-Fc.gamma.RIIIA and
Fc.gamma.RIIB constructs digested with the respective enzymes.
Therefore, the pcDNA3.1-FcRyIIIA-linker-AVITAG and
pcDNA3.1-FcRyIIB-linker-AVITAG.TM. peptide, were constructed.
TABLE-US-00016 TABLE 14 PRIMERS USED FOR CONSTRUCTION OF Fc.gamma.R
AND IgG VECTORS Oligomer Sequence 5' linker.avitag
GGCCGCAGGTGGTGGTGGTTCTGGTGGTGGTGGT (SEQ. ID NO. 1)
TCTGGTCTGAACGACATCTTCGAGGCTCAGAAAA TCGAATGGCACGAATGAT 3'
linker.avitag CTAGATCATTCGTGCCATTCGATTTTCTGAGCCT (SEQ. ID NO. 2)
CGAAGATGTCGTTCAGACCAGAACCACCACCACC AGAACCACCACCACCTGC FcRIIIA left
G TTG GAT CCT CCA ACT GCT CTG CTA (SEQ. ID NO. 3) CTT CTA GTT T
FcRIIIA right GAA AAG CTT AAA GAA TGA TGA GAT (SEQ. ID NO. 4) GGT
TGA CAC T FcRIIBright GAA GTC GAC AAT GAT CCC CAT TGG (SEQ. ID NO.
5) TGA AGA G FcRIIBleft G TTA GAT CTT GCT GTG CTA TTC (SEQ. ID NO.
6) CTG GCT CC IgG1 right ATA GTC GAC CAC TGA TTT ACC CGG (SEQ. ID
NO. 7) AGA IgG1left GGAA TTC AAC ACC AAG GTG GAC AAG (SEQ. ID NO.
8) AAA GTT mcr025; ch1 (f') AAA GGATCC GCG AGC TCA GCC TCC (SEQ. ID
NO. 9) ACC AAG G H021 GTCTGCTCGAAGCATTAACC (SEQ. ID NO. 10)
[0579] Biotinylation by BirA
[0580] Soluble Fc receptors (Fc.gamma.R) fused to the 15 amino acid
AVITAG.TM. peptide sequence (Avidity, CO) (Schatz P. J., 1993,
Biotechology, 11:1138-1143) at the C-terminus of the protein cloned
into pcDNA3.1 were generated by transiently transfecting 293H cells
using Lipofectamine 2000 reagent (Invitrogen, CA). Supernatants
were collected from the cultures and soluble FcR proteins were
purified by passing the supernatants over an IgG sepharose column.
Concentration of the soluble FcR-AVITAG.TM. peptide fusion protein
was quantitated by absorbance at 280 nm. The AVITAG.TM. peptide
present on the soluble FcR proteins was biotinylated according to
the manufacturer's protocol (Avidity, CO) with the E. coli BirA
enzyme, a biotin ligase. A 1:100 final dilution of a cocktail of
protease inhibitors (Sigma catalog #P8849) and 1 mg/ml final
concentration of Leupeptin (Sigma L-8511) were added to the mixture
to prevent degradation of the proteins. The BirA reaction was
incubated at room temperature overnight, following which the
solution was concentrated using a Biomax 10K-ultrafiltration device
(Millipore) by centrifugation at 3500 rpm at 4.degree. C. The
protein was loaded onto an FPLC Superdex 200 HR 10/30 column
(Pharmacia Biotech) in Tris-HCl (20 mM, pH 8.0), 50 mM NaCl to
separate the labeled soluble Fc.gamma.R from free biotin.
[0581] Determination of the Extent of Biotinylation by Streptavidin
Shift Assay
[0582] Approximately 80-85% of the protein was biotinylated by the
BirA enzyme (Avidity, CO). The streptavidin-shift assay was used to
determine the extent of biotinylation of the protein. Biotinylated
protein was incubated with streptavidin (MW 60,000 Daltons) in
different ratios. Unbiotinylated protein alone and streptavidin
alone are included as controls to determine the extent of
biotinylation. The incubation is carried out either on ice for 2
hours or overnight at 4.degree. C. Samples are analyzed on a 4-12%
SDS-PAGE Bis-Tris (Invitrogen, CA) with reducing agent and without
boiling of the samples. Streptavidin bound biotinylated protein
migrates as a high molecular weight band. The extent of
biotinylation is estimated by the amount of monomeric protein left
in the sample. Absence of monomeric low molecular weight species
and presence of a complex with molecular weight greater than
streptavidin alone indicates a high degree of biotinylation.
[0583] Formation of Fc.gamma.R Tetrameric Complexes
[0584] Formation of Fc.gamma.R tetrameric complexes was performed
according to previously established methodologies for MHC class I
tetramers (See Busch, D. H. et al., 1998 Immunity 8:353-362;
Altman, J. D. et al., 1996, Science 274: 94-96). The concentration
of the biotinylated monomeric Fc.gamma.R was calculated based on
absorbance at 280 nm. One molecule of streptavidin-phycoerythrin
(SA-PE) (Molecular Probes, OR) has the capacity to bind 4 molecules
of biotin. A 5:1 molar ratio of monomeric biotinylated Fc.gamma.R
to SA-PE (5.times. monomeric biotinylated Fc.gamma.R: 1.times.
SA-PE) was used to ensure an excess of biotinylated protein. The
calculated molecular weight of SA-PE is 300,000 Daltons, therefore
303 mL of a 1 mg/mL solution of streptavidin-PE has 1 nmole of
SA-PE, which was added to 5 nmole of protein. Efficient formation
of tetrameric protein requires SA-PE to be added in step-wise
increments. Half the amount of SA-PE was added upfront, and the
remaining SA-PE was added in small aliquots every 20-30 minutes at
4.degree. C. in the dark. The intervals for the addition of the
remaining SA-PE is flexible. After the addition of SA-PE was
complete, the solution was concentrated and loaded over an FPLC
size exclusion column as above in phosphate buffered saline, at pH
7.4. The fraction that eluted in the void volume with a molecular
weight greater than SA-PE alone was collected. Protease inhibitors
were replenished to prevent protein degradation. The solution was
concentrated and additional protease inhibitors were added to the
final complex for storage. The final concentration of the soluble
Fc.gamma.R tetrameric complex was calculated based on the starting
concentration of the biotinylated monomeric protein. For example,
if 500 .mu.g of biotinylated protein was used to make the
tetrameric complex and the final concentrated tetramers were in a
volume of 500 .mu.L, the concentration is estimated to be
approximately 1 mg/mL (The losses incurred during concentration are
not taken into account as it is difficult to accurately determine
how much is lost during each step of the formation of the
tetramers. It is also not possible to take an absorbance at 280 nm
to measure the concentration due to interference from the PE).
Soluble Fc.gamma.R tetrameric complexes were dispensed in small
aliquots at -80.degree. C. for long term storage with protease
inhibitors. Sodium azide was not added to these preparations as the
tetramers were used for screening a yeast display library. On
thawing an aliquot, the tetramers were stored at 4.degree. C. for
up to 1 week.
[0585] ELISA Assay for Characterizing the Tetrameric Fc.gamma.R
Complexes
[0586] An ELISA was used to characterize the tetrameric Fc.gamma.R
complexes. Maxisorb F96 well plate (Nunc) was coated with 25 ng of
human IgG in PBS buffer, and incubated overnight at 4.degree. C.
The plates were washed with PBS/0.5% BSA/0.1% Tween 20 (wash and
diluent buffer) before adding the combination of Fc.gamma.RIIIA
tetramers and test antibodies to determine blocking with 3G8, a
mouse anti-human Fc.gamma.RIIIA antibody as described below: The
blocking step was performed as follows: soluble Fc.gamma.RIIIA
tetramers at a fixed 0.5 mg/ml final concentration were
pre-incubated with antibodies for 1 h at room temperature in
buffer, PBS/0.5% BSA/0.1% Tween 20. The final concentrations of the
antibodies ranged from 60 mg/mL to 0.25 mg/mL. 3G8 is a mouse
anti-human Fc.gamma.RIIIA antibody, and for the purpose of this
experiment, a chimeric version was used, i.e., the variable region
of the antibody is a mouse anti-human Fc.gamma.RIIIA and the
constant region of the heavy and light chains is from the IgG1
human region. A chimeric 4.4.20. D265A was also used in this
experiment, which is an anti-fluorescein antibody, such that the Fc
region contains a mutation at position 265, where an aspartic acid
is substituted with alanine in the human IgG1, which results in a
reduced binding to Fc.gamma.R. This antibody has been characterized
previously (See Clynes et al., 2000, Nat. Med. 6: 443-446; Shields
et al., 2001, J. Biol. Chem., 276: 6591-6604). This antibody was
used as negative isotype control.
[0587] The antibodies were allowed to bind to Fc.gamma.RIIIA
tetramers, by pre-incubation for 1 hour at room temperature. The
mixture was then added to the IgG on the washed plate and incubated
for and additional hour at room temperature. The plate was washed
with buffer and DJ130c (a mouse anti-human Fc.gamma.RIIIA antibody
available from DAKO, Denmark; its epitope is distinct from that of
the 3G8 antibody) at 1:5000 dilution was added and allowed to
incubate for 1 hr. at room temperature in order to detect the bound
Fc.gamma.RIIIA tetramers. Unbound antibodies were washed out with
buffer and the bound DJ130c was detected with goat anti-mouse
peroxidase (Jackson laboratories). This reagent will not detect the
human Fc. After washing out the unbound peroxidase-conjugated
antibody, the substrate, TMB reagent (BioFx), was added to detect
the extent of blocking with 3G8 versus the isotype control and the
developed color was read at 650 nm.
[0588] For direct binding of soluble tetrameric Fc.gamma.RIIIA to
IgG by ELISA, maxisorb plates were coated with 25 ng IgG as
described above. The soluble tetrameric Fc.gamma.RIIIA were added
from 20 mg/mL to 0.1 mg/mL and the biotinylated monomeric soluble
tetrameric Fc.gamma.RIIIA were added at concentrations ranging from
20 mg/mL to 0.16 mg/mL. Detection was the same as above with
DJ130c, followed by goat anti-mouse-peroxidase antibody. Color
developed with the TMB reagent and the plate was read at 650
nm.
[0589] Results
[0590] Soluble Fc.gamma.RIIIA Tetrameric Complex Binds Monomeric
Human IgG Via its Normal Ligand Binding Site
[0591] Soluble Fc.gamma.RIIIA-AVITAG.TM. peptide fusion proteins
were generated, isolated, and analyzed as described in the Material
and Methods section using an ELISA assay and were shown to have
similar properties as the non-AVITAG.TM. peptide soluble
Fc.gamma.RIIIA protein (data not shown). The fusion proteins were
biotinylated, and the tetrameric complexes were generated as
described above.
[0592] The soluble Fc.gamma.R tetrameric complex was then assessed
for binding its ligand, monomeric human IgG, using an ELISA assay.
Analysis by ELISA showed the soluble tetrameric Fc.gamma.R
complexes bind monomeric human IgG specifically. As shown in FIG.
3A, binding of soluble tetrameric Fc.gamma.RIIIA to monomeric human
IgG is blocked by 3G8, a mouse anti-human Fc.gamma.IIIA monoclonal
antibody, as monitored by the absorbance at 650 nm. On the other
hand, the 4-4-20 monoclonal antibody harboring the D265A mutation
was not able to block the binding of soluble tetrameric
Fc.gamma.RIIIA to monomeric human IgG (FIG. 4A). This experiment
thus confirms that binding of the soluble tetrameric Fc.gamma.RIIIA
complex occurs through the native ligand binding site.
[0593] Soluble Fc.gamma.RIIIA Tetrameric Complex Binds Monomeric
Human IgG with a Greater Avidity than Monomeric Soluble
Fc.gamma.RIIIA
[0594] The direct binding of soluble tetrameric Fc.gamma.RIIIA to
aggregated human IgG was assessed using an ELISA assay and compared
to the direct binding of soluble monomeric Fc.gamma.RIIIA to
monometic human IgG. As shown in FIG. 4B, soluble tetrameric
Fc.gamma.RIIIA binds human IgG with a higher avidity (8-10 fold)
than the soluble monomeric receptor, as monitored by the absorbance
at 450 nm.
[0595] The binding of soluble Fc.gamma.RIIIA tetrameric complex was
also assayed using magnetic beads coated with Fc Fragment purified
from IgG1 (FIG. 5). Soluble Fc.gamma.RIIIA tetrameric complex binds
to the IgG1 Fc-coated beads, under conditions in which monomer
binding is not detected. Specificity of binding was shown by
pre-incubating the receptor complex, with an anti-Fc.gamma.RIIIA
monoclonal antibody, LNK16, which blocks Fc binding. This assay
further confirms that soluble Fc.gamma.RIIIA tetrameric complex
binds monomeric IgG through its normal ligand binding site, and the
avidity of the receptor is increased due to multiple binding sites
within the complex.
6.3 Construction of Yeast Strain for Display of Mutant IgG1 Fc
Domains
[0596] Materials and Methods
[0597] The pYD1 vector (Invitrogen) is derived directly from a
yeast replicating vector, pCT302 (Shusta, et al., 2000 Nat.
Biotechnol. 18: 754-759, that has been successfully used to display
T-cell receptors and a number of scFVs. This plasmid is centromeric
and harbors the TRP1 gene enabling a relatively constant copy
number of 1-2 plasmids per cell in a trpl yeast strain. Directional
cloning into the polylinker places the gene of interest under the
control of the GAL1 promoter and in-frame with AGA2. Fusion of the
IgG Fc domain to the yeast Aga2p results in the extracellular
secretion of the Aga2-Fc fusion protein and subsequent display of
the Fc protein on the cell wall via disulfide bonding to the yeast
Aga1p protein, which is an integral cell wall protein.
[0598] In order to optimize the display levels, different fragments
from the IgG1 heavy chain were amplified by PCR and cloned into
pYD1. Specifically, the Fc region of the IgG1 heavy chain (allotype
IGlm(a); amino acids 206-447) was amplified by PCR (Table 14) from
the IMAGE clone 182740, digested with EcoRI/SaII and ligated into
the pYD1 vector (Invitrogen). The initial clone from IMAGE
contained a deletion of a single nucleotide at position 319 which
was corrected by in vitro site directed mutagenesis to construct
pYD-GIF206 (Quickchange, Stratagene).
[0599] The CH1--CH3 fragment (amino acids 118-447) was amplified
from the heavy chain clone of the MAb B6.2 in the pCINEO vector
using a 5' oligo (mcr025;chl(f)) and a 3' oligo (H021) (See Table
14). The fragment was digested with BamHI/NotI and ligated into the
pYD1 vector to construct pYD-CH1.
[0600] FIG. 6 shows a schematic presentation of the constructs. The
CH1--CH3 construct contains the CH1 domain in addition to the
hinge-CH2--CH3 domains of the heavy chain, GIF206 contains 6 amino
acid residues upstream of the hinge and GIF227 starts within the
hinge region at an endogenous proteolytic cleavage site (Jendeberg
et al., 1997J. Immunol. Meth. 201: 25-34).
6.4 Immunolocalization and Characterization of Fc Domains on the
Yeast Cell Wall
[0601] Materials and Methods
[0602] Constructs containing the Aga2p-Fc fusion proteins and a
control vector, pYD1, lacking any insert, were transformed into the
yeast strain EBY100 (Invitrogen), MAT.alpha. ura3-52 trpl
leu2.DELTA.l his3.DELTA.200 pep4::HIS3 prb1.DELTA.1.6R can1
GAL::GAL-AGA1, using a standard lithium acetate yeast
transformation protocol (Gietz et al., 1992 Nucleic Acids Res. 20:
1425) Subsequently, tryptophan prototrophs were selected on defined
media. Amplification of independent cell populations and induction
of Aga1p and the Aga2p-Fc fusion proteins were accomplished by
growth in glucose, followed by growth in media containing galactose
as the primary carbon source for 24-48 hrs at 20.degree. C. Growth
in galactose induces expression of the Aga2-Fc fusion proteins via
the GAL1 promoter, which subsequently leads to the display of the
Fc fusion proteins on the yeast cell surface.
[0603] Results
[0604] FACS Analysis of Fc Fusion Proteins
[0605] Expression of Fc fusion proteins on the yeast cell surface
was analyzed by immunostaining using a PE-conjugated polyclonal
F(ab).sub.2 goat anti-human Fc.gamma.R and HP6017 (Sigma) antibody
(Jackson Immununoresearch Laboratories, Inc.). Fluorescence
microscopy shows peripheral staining for the three Fc fusion
proteins. The control strain, harboring vector alone, shows little
or no staining (data not shown). FACS analysis was used to
quantitate the staining (FIG. 7). The yeast strain containing the
CH1--CH3 fusion demonstrated the highest percentage of cells
stained with both antibodies (FIGS. 7B and F). The GIF227 construct
showed the greatest mean fluorescence intensity (FIG. 7, panels C
and G).
[0606] Characterization of the Binding of Fc Fusion Proteins
Expressed on the Yeast cell Surface
[0607] The natural context of the Fc and Fc.gamma.R proteins places
the receptor on the cell surface and the Fc as the soluble ligand;
however, the yeast Fc surface display reverses the geometry of the
natural interaction. Detection of the IgG1 Fc proteins on the
surface of the yeast cell wall is complicated by both the low
affinity of the Fc.gamma.R for its ligand and the reverse geometry
inherent in the display system. Although the latter point cannot be
altered, the avidity of the ligand was improved as explained above
by forming soluble Fc.gamma.R tetrameric complexes, which allows
detection of Fc.gamma.R binding to the Fc fusion proteins expressed
on the surface yeast cell wall.
[0608] To characterize the binding of soluble tetrameric Fc.gamma.R
complexes to the surface displayed Fc fusion proteins, yeast cells
expressing different Fc constructs were incubated with the soluble
rFc.gamma.RIIIA tetrameric complex and analyzed by FACS. Yeast
cells harboring pYD-CH1, displaying the wild type CH1--CH3
construct were bound by the soluble rFc.gamma.RIIIA tetrameric
complex as shown by FACS analysis. The GIF206 and GIF227 strains,
however, showed little or no binding to the soluble rFc.gamma.RIIIA
tetrameric complex as shown by FACS analysis (data not shown).
[0609] Mutations in the Fc region that block binding to the
Fc.gamma.Rs have been identified (Shields et al., 2001; J Biol.
Chem. 276: 6591-6604). One of these mutations, D265A, was
incorporated into pYD-CH1 and this mutant was expressed on the
yeast cell surface. These cells were incubated with the soluble
Fc.gamma.RIIIA tetrameric complex using a high concentration of
ligand (0.15 mM of Fc; 7.5 mM of D265A) FACS analysis indicated
that soluble Fc.gamma.RIIIA tetrameric complex bound to wild type
Fc (FIG. 8A) but soluble Fc.gamma.RIIIA tetrameric complex did not
bind to the D265A-Fc mutant indicating that Fc.gamma.R is
interacting with the normal FcR binding site in the lower hinge-CH2
region (FIG. 8B).
[0610] Antibodies against the Fc.gamma.RIIIA ligand binding site
blocked binding of the soluble Fc.gamma.RIIIA tetrameric complex to
the wild type Fc protein displayed on the yeast cell surface wall,
as analyzed by FACS (FIG. 9). The binding of soluble Fc.gamma.RIIIA
tetrameric complex was blocked by the 3G8 antibody, as well as the
LNK16 antibody, another anti-Fc.gamma.RIIIA monoclonal antibody
(Advanced Immunological) (Tam et al., 1996 J. Immunol. 157:,
1576-1581) and was not blocked by an irrelevant isotype control.
Therefore, binding of soluble Fc.gamma.RIIIA tetrameric complex to
the Fc proteins displayed on the yeast cell surface occurs through
the normal ligand binding site. The limited binding of the
Fc.gamma.RIIIA tetrameric complex indicates that a subpopulation of
cells have a correctly folded Fc that is accessible to Fc.gamma.R.
There are numerous reasons why only a subpopulation of cells may be
able to bind the ligand, for example, they may be at different
stages of cell cycle or the fusion proteins may not have been
exported.
[0611] In order to determine the dissociation constant of the
Fc.gamma.RIIIA-tetramer binding to the Fc fusion proteins on the
yeast cell surface, the binding of a range of Fc.gamma.RIIIA
tetrameric complex was analyzed using FACS. Fc.gamma.RIIIA
tetrameric complex was titrated at concentrations of 1.4 .mu.M to
0.0006 .mu.M. Using the mean fluorescence intensity as a measure of
binding affinity and nonlinear regression analysis, the K.sub.D was
determined to be 0.006 .mu.M (+/-0.001) (data not shown).
6.5 Construction of Fc Mutant Library
[0612] A mutant Fc library was constructed using primers flanking
the Fc fragment in the Fc-CH1 construct and error-prone PCR
(Genemorph, Stratagene). The CH1--CH3 insert in vector pYD-CHI was
amplified using a mutagenic PCR (Genemorph, Stratagene). Five
reactions were carried out using the pYD-upstream and
pYD-downstream primers (Invitrogen). The resultant amplified
fragment was digested with XHOI/BamHI and ligated into pYD1. The
ligation reaction was then transformed into XL10 ultracompetent
cells (Stratagene), which resulted in .about.1.times.10.sup.6
transformants, with 80% of the transformants containing
inserts.
[0613] Sequence analysis of 28 random plasmids from the library
indicated a mutation frequency .about.2-3 mutations/kb with a
breakdown of 40% conserved nucleotide changes and 60% of the
mutations resulting in amino acid changes.
[0614] The library was transformed into the yeast strain EBY100,
MAT.alpha. ura3-52 trp 1 leu2.DELTA.1 his3.DELTA.200 pep4::HIS3
prb1.DELTA.1.6R can 1 GAL GAL-AGA 1::URA3 to a high efficiency,
.about.3.3.times.10.sup.5 transformants/ug, in 30 independent
transformation reactions to create a total of .about.10.sup.7 yeast
transformants (Gietz, et al., 1992, Nucleic Acids Res. 20: 1425).
The library was pooled and amplified by growth in glucose.
6.6 Selection and Analysis of Fc Mutants
[0615] Materials and Methods
[0616] Elisa Assay for Screening Fc Mutants
[0617] ELISA plates (Nunc F96 MaxiSorp Immunoplate) were coated
with 50 ml/well of 0.5 mg/ml BSA-FITC in carbonate buffer at
4.degree. C., and allowed to incubate overnight. Plates were washed
with 1.times.PBS/0.1% Tween 20 (PBST) 3 times. 200 ml/well of
PBST/0.5% BSA was added and the plates were incubated for 30 mins
at room temperature. Plates were washed three additional times with
PBST. 50 ml/well of 1:4 diluted 4-4-20 antibody (approximately 3
mg/mL which would lead to a final concentration of 0.7-0.8 mg/well)
either wild type or containing an Fc mutant, was added from
conditional medium in PBST/0.5% BSA and allowed to incubate for 2
hrs at room temperature. Plates were washed with PBST three times.
Purified, biotinylated monomeric Fc.gamma.RIIIA at 3 mg/ml (in
PBST/0.5% BSA) was added (50 .mu.l/well) to the plates and allowed
to incubate for 1.5 hours at room temperature. Plates were washed
with PBST three times. 50 ml/well of a 1:5000 dilution of
Streptavidin-HRP(Pharmacia, RPN 123v) in PBST/0.5% BSA was added
and the plates were incubated for 30 minutes at room temperature.
Plates were washed with PBST three times. 80 ml/well of TMB reagent
(BioFX) was then added to the plates, and allowed to incubate for
10-15 minutes at room temperature in a dark place. The reactions
were finally stopped by adding 40 ml/well of stop solution (0.18 M
sulfuric acid). Plates were then monitored for absorbance at 450
nm. After the first screen, the interesting candidates were further
confirmed by serial titration of 4-4-20-Fc mutants in the
immuno-complex based binding ELISA. A few modifications were made
in this ELISA. For coating the plates, 2 mg/ml BSA-FITC was used.
Based on IgG quantitation results, diluted 4-4-20Fc (wild type or
mutants) from conditional medium was added to a final concentration
of 1, 0.5, 0.25, 0.125, 0.063, and 0 mg/ml in PBST-/0.5% BSA.
[0618] FACS Screen for the Cell Surface Displayed Fc Proteins
[0619] Cells were grown in at least 10 mls of HSM-Trp-Ura pH 5.5
with glucose for 16-24 hrs or until OD.sub.600 was greater than
2.0. Cells were spun down at .about.2000 rpm for 5 minutes. Cells
were resuspended in an equal volume of HSM-Trp-Ura, pH 7.0 with
galactose. In a 125 ml flask, 36 mls of galactose media was added,
and inoculated with 9 mls of culture, which was incubated at
20.degree. C. with shaking for 24-48 hrs. Growth was monitored by
measuring OD.sub.600 at 8-16 hr intervals. Cells were harvested at
2K rpm for 5 minutes, and resuspended in an equal volume of
1.times.PBS, pH 7.4.
[0620] Equilibrium Screen:
[0621] An appropriate amount of cells was incubated while
maintaining an excess of ligand. For example, it is preferred to
start with a number of cells needed to ensure 10-fold coverage of
the library. For the first sort with a library containing 10.sup.7
transformants, 10.sup.8 cells should be used. In fact it is best to
start with 10.sup.9 cells to compensate for loss during the
staining protocol.
[0622] Incubation was typically done in a 1.5 mL tube in volumes of
20-100 mls for 1 hour at 4.degree. C. in the dark on a rotator
(incubation buffer: 1.times.PBS pH7.4; 1 mg/ml BSA). Cells were
washed once in 500 ml of incubation buffer and spun down at 4K rpm
for 2.5 minutes. Cells were resuspended in 100 ml incubation buffer
and incubated with the second staining reagent. For Fc-CH1, a
F(ab).sub.2 goat anti-hFc F(ab).sub.2--FITC antibody (Jackson
Immunoresearch Laboratories, Inc.) can be used to stain for CH1
expression. Staining was done with 1 mL for 30 minutes. Cells were
washed additionally in 500 mL of incubation buffer and spun down at
4K rpm for 2.5 minutes, resuspended in 1 mL 1.times.PBS1 mg/mL BSA
and analyzed by FACS.
[0623] Typical equilibrium screen sort gates and number of cells
collected are shown in Table 15.
TABLE-US-00017 TABLE 15 SORT GATES AND NUMBER OF CELLS SORTED Sort
Gate total cells screened cells collected 1.sup.st 5% 10.sup.8 5
.times. 10.sup.6 2.sup.nd 1% 10.sup.7 1 .times. 10.sup.5 3.sup.rd
0.2% 10.sup.7 2 .times. 10.sup.4 4.sup.th 0.2% 10.sup.7 2 .times.
10.sup.4
[0624] After the 3rd and 4th sorts, cells were plated directly on
-trp-ura plates to identify individual mutants. This typically
recovered .about.200-400 colonies per plate. After collection the
cells were placed in 10 mLs of glucose media in a 50 mL conical
tube and grown at 30.degree. C. The whole procedure was repeated
iteratively.
[0625] Results
[0626] FACS Analysis of Fc Mutants
[0627] After induction in galactose media, cells were harvested and
co-stained with soluble Fc.gamma.RIIIA tetrameric complex-PE
labeled and F(ab.sub.2) of mouse anti-human Fc-FITC labeled
(Jackson Immunoresearch Laboratories, Inc.). Cells were analyzed by
FACS and sort gates were used to select the cells that showed the
highest affinity for the soluble Fc.gamma.RIIIA tetrameric complex
relative to the amount of Fc expression on the cell surface (FIG.
10). For example, a cell containing a mutant Fc that binds better
to the soluble Fc.gamma.RIIIA tetrameric complex may express fewer
Fc fusion proteins on the yeast cell surface, and this cell will be
in the lower left hand corner of the sort gate.
[0628] Four consecutive sorts were done to enrich for those mutants
that showed the highest affinity for the soluble Fc.gamma.RIIIA
tetrameric complex. The gates for each successive sort were 5.5%,
1%, 0.2% and 0.1%. After the last sort, cells were plated onto
selective media and individual colonies were isolated. Each
individual colony represented a clonal population of cells
harboring a single Fc mutant within the Aga2-Fc fusion protein.
Initially 32 independent colonies were picked and tested by FACS
for binding to soluble Fc.gamma.RIIIA tetrameric complex (FIG. 11).
Eighteen mutants showed an increase in binding intensity as
measured by the percentage of cells bound by soluble Fc.gamma.RIIIA
tetrameric complex and the mean fluorescence intensity of the bound
cells.
[0629] Mutations showing an increase in binding to Fc.gamma.RIIIA
were also tested for binding to soluble Fc.gamma.RIIB tetrameric
complex (FIG. 11). Most mutations that lead to an increase in
binding to the soluble Fc.gamma.RIIIA tetrameric complex also
resulted in detection of Fc.gamma.RIIB tetrameric complex staining
(FIG. 11). Based on both previous physical and genetic data, some
mutations that increase binding to Fc.gamma.RIIIA, are expected to
also increase binding to Fc.gamma.RIIB (Shields et al., 2001, J
Biol. Chem. 276: 6591-6604; Sondermann et al., 2000, Nature 406:
267-273).
[0630] Analysis of Mutants in a 4-4-20 MAb Produced in a Human Cell
Line.
[0631] Isolation and analysis of mutations in the yeast system
allows for fast identification of novel mutant alleles. The use of
a heterologous system to isolate mutations could result in the
identification of mutations that enhance binding through an
alteration that results in misfolding or alteration in
glycosylation that is specific to yeast. To analyze the Fc
mutations in an immunoglobulin molecule that is produced in human
cells, the mutants were subcloned into a mammalian expression
vector, containing the heavy chain of the anti-fluorescein
monoclonal antibody, 4-4-20 (Kranz et al., 1982 J. Biol. Chem.,
257(12): 6987-6995). The mutant 4-4-20 heavy chains were
transiently coexpressed with the light chain clones in the human
kidney cell line (293H). Supernatants were collected and analyzed
by ELISA (FIG. 12).
[0632] According to the ELISA assay, the majority of the mutants
that were identified as having an enhanced affinity for the soluble
monomeric Fc.gamma.RIIIA complex, in the secondary FACS analysis,
also showed an increase in binding to the soluble Fc.gamma.RIIIA
tetrameric complex when present in the Fc region of the 4-4-20
monoclonal antibody produced in the human cell line (FIG. 12A). Two
mutants, number 16 and number 19, however, showed a decrease in
binding to the soluble Fc.gamma.RIIIA monomeric complex.
[0633] Table 16, summarizes the mutations that have been identified
and their corresponding binding characteristics to Fc.gamma.RIIIA
and Fc.gamma.RIIB, as determined by both yeast display based assays
and ELISA. In Table 16, the symbols represent the following:
.cndot. corresponds to a 1-fold increase in affinity; + corresponds
to a 50% increase in affinity; - corresponds to a 1-fold decrease
in affinity; .fwdarw. corresponds to no change in affinity compared
to a comparable molecule comprising a wild-type Fc region.
TABLE-US-00018 TABLE 16 MUTATIONS IDENTIFIED AND BINDING
CHARACTERISTICS Clone IIIA IIB # Mutation sites Domain binding
binding 4 A339V, Q347H CH2, CH3 + + 5 L251P, S415I CH2, CH3 + + 7
Aga2p-T43I Note: This is a Aga2p- mutation in T43I Aga2P that
enhances display. 8 V185M, K218N, R292L, CH1, hinge, CH2, no -
D399E CH3 change 12 K290E, L142P CH1, CH2 + not tested 16 A141V,
H268L, K288E, CH1, CH2 - not tested P291S 19 L133M, P150Y, K205E,
CH1, CH2, CH3 - not tested S383N, N384K 21 P396L CH3 .cndot.
.cndot.+ 25 P396H CH3 .cndot..cndot..cndot. .cndot..cndot. 6 K392R
CH3 no no change change 15 R301C, M252L, S192T CH1, CH2 - not
tested 17 N315I CH2 no not tested change 18 S132I CH1 no not tested
change 26 A162V CH1 no not tested change 27 V348M, K334N, F275I,
CH1, Ch2 + + Y202M, K147T 29 H310Y, T289A, G337E CH2 - not tested
30 S119F, G371S, Y407N, CH1, CH2, CH3 + no E258D change 31 K409R,
S166N CH1, CH3 no not tested change 20 S408I, V215I, V125I CH1,
hinge, CH3 + no change 24 G385E, P247H CH2, CH3
.cndot..cndot..cndot. + 16 V379M CH3 .cndot..cndot. no change 17
S219Y Hinge .cndot. - 18 V282M CH2 .cndot. - 31 F275I, K334N, V348M
CH2 + no change 35 D401V CH3 + no change 37 V280L, P395S CH2 + - 40
K222N Hinge .cndot. no change 41 K246T, Y319F CH2 .cndot. no change
42 F243I, V379L CH2, CH3 .cndot.+ - 43 K334E CH2 .cndot.+ - 44
K246T, P396H CH2, CH3 .cndot. .cndot..cndot.+ 45 H268D, E318D CH2
.cndot.+ .cndot..cndot..cndot..cndot..cndot. 49 K288N, A330S, P396L
CH2, CH3 .cndot..cndot..cndot..cndot..cndot. .cndot..cndot..cndot.
50 F243L, R255L, E318K CH2 .cndot. - 53 K334E, T359N, T366S CH2,
CH3 .cndot. no change 54 I377F CH3 .cndot.+ + 57 K334I CH2 .cndot.
no change 58 P244H, L358M, V379M, CH2, CH3 .cndot.+ .cndot.+ N384K,
V397M 59 K334E, T359N, T366S CH2, CH3 .cndot.+ no (independent
isolate) change 61 I377F (independent CH3 .cndot..cndot..cndot.
.cndot..cndot.+ isolate) 62 P247L CH2 .cndot..cndot.
.cndot..cndot.+ 64 P217S, A378V, S408R Hinge, CH3 .cndot..cndot.
.cndot..cndot..cndot..cndot.+ 65 P247L, I253N, K334N CH2
.cndot..cndot..cndot. .cndot..cndot.+ 66 K288M, K334E CH2
.cndot..cndot..cndot. - 67 K334E, E380D CH2, CH3 .cndot.+ - 68
P247L (independent CH2 + .cndot..cndot..cndot..cndot. isolate) 69
T256S, V305I, K334E, CH2, CH3 .cndot.+ no N390S change 70 K326E CH2
.cndot.+ .cndot..cndot.+ 71 F372Y CH3 +
.cndot..cndot..cndot..cndot..cndot.+ 72 K326E (independent CH2 +
.cndot..cndot. isolate) 74 K334E, T359N, T366S CH2, CH3
.cndot..cndot. no (independent isolate) change 75 K334E
(independent CH2 .cndot..cndot.+ no isolate) change 76 P396L
(independent CH3 .cndot.+ no isolate) change 78 K326E (independent
CH2 .cndot..cndot. .cndot..cndot..cndot.+ isolate) 79 K246I, K334N
CH2 .cndot. .cndot..cndot..cndot..cndot. 80 K334E (independent CH2
.cndot. no isolate) change 81 T335N, K370E, A378, CH2, CH3 .cndot.
no T394M, S424L change 82 K320E, K326E CH2 .cndot. .cndot. 84 H224L
Hinge .cndot. .cndot..cndot..cndot..cndot..cndot. 87 S375C, P396L
CH3 .cndot.+ .cndot..cndot..cndot..cndot.+ 89 E233D, K334E CH2
.cndot.+ no change 91 K334E (independent CH2 .cndot. no isolate)
change 92 K334E (independent CH2 .cndot. no isolate) change 94
K334E, T359N, T366S, CH2 .cndot. no Q386R change
[0634] Analysis of soluble Fc.gamma.RIIB tetrameric complex binding
shows that 7 out of the 8 mutants that showed an increase in
binding to the soluble Fc.gamma.RIIIA tetrameric complex also had
an increased binding to the soluble Fc.gamma.RIIB tetrameric
complex (FIG. 12B). One mutant, number 8, showed a decrease in
binding to the soluble Fc.gamma.RIIB tetrameric complex. Three of
the mutants show no difference in binding to either the soluble
Fc.gamma.RIIIA tetrameric complex or the soluble Fc.gamma.RIIB
tetrameric complex, possibly due to mutations that result in yeast
specific alterations.
6.7 ADCC Assay of Fc Mutants
[0635] Effector cell preparation: Peripheral blood mononuclear
cells (PBMC) were purified by Ficoll-Paque (Pharmacia, 17-1440-02)
Ficoll-Paque density gradient centrifugation from normal peripheral
human blood (Biowhittaker/Poietics, 1 W-406). Blood was shipped the
same day at ambient temperature, and diluted 1:1 in PBS and glucose
(1 g/1 L) and layered onto Ficoll in 15 mL conical tubes (3 mL
Ficoll; 4 mL PBS/blood) or 50 mL conical tubes (15 mL: Ficoll; 20
mL PBS/blood). Centrifugation was done at 1500 rpm (400 rcf) for 40
minutes at room temperature. The PBMC layer was removed
(approximately 4-6 mL from 50 mL conical tube) and diluted 1:10 in
PBS (which contains no Ca.sup.2+ or Mg.sup.2+) in a 50 mL conical
tube, and spun for an additional ten minutes at 1200 rpm (250 rcf)
at room temperature. The supernatant was removed and the pellets
were resuspended in 10-12 mL PBS (which contains no Ca.sup.2+ or
Mg.sup.2+), transferred to 15 mL conical tubes, and spun for
another 10 minutes at 1200 rpm at room temperature. The supernatant
was removed and the pellets were resuspended in a minimum volume
(1-2 mL) of media (Isocove's media (IMDM)+10% fetal bovine serum
(FBS), 4 mM Gln, Penicillin/Streptomycin (P/S)). The resuspended
PBMC were diluted to the appropriate volume for the ADCC assay; two
fold dilutions were done in an ELISA 96 well plate (Nunc F96
MaxiSorp Immunoplate). The yield of PBMC was approximately
3-5.times.10.sup.7 cells per 40-50 mL of whole blood.
[0636] Target cell preparation: Target cells used in the assay were
SK-BR-3 (ATCC Accession number HTB-30; Trempe et al., 1976, Cancer
Res. 33-41), Raji (ATCC Accession number CCL-86; Epstein et al.,
1965, J. Natl. Cancer Inst. 34: 231-40), or Daudi cells (ATCC
Accession number CCL-213; Klein et al., 1968, Cancer Res. 28:
1300-10) (resuspended in 0.5 mL IMDM media) and they were labeled
with europium chelate bis(acetoxymethyl) 2,2'':6',2'' terpyridine
6,6' dicarboxylate (BATDA reagent; Perkin Elmer DELFIA reagent;
C136-100). K562 cells (ATCC Accession number CCL-243) were used as
control cells for NK activity. The Daudi and Raji cells were spun
down; the SK-BR-3 cells were trypsinized for 2-5 minutes at
37.degree. C., 5% CO.sub.2 and the media was neutralized prior to
being spun down at 200-350 G. The number of target cells used in
the assays was about 4-5.times.10.sup.6 cells and it did not exceed
5.times.10.sup.6 since labeling efficiency was best with as few as
2.times.10.sup.6 cells. Once the cells were spun down, the media
was aspirated to 0.5 mL in 15 mL Falcon tubes. 2.5 .mu.l of BATDA
reagent was added and the mixture was incubated at 37.degree. C.,
5% CO.sub.2 for 30 minutes. Cells were washed twice in 10 mL PBS
and 0.125 mM sulfinpyrazole ("SP"; SIGMA S-9509); and twice in 10
mL assay media (cell media +0.125 mM sulfinpyrazole). Cells were
resuspended in 1 mL assay media, counted and diluted.
[0637] When SK-BR-3 cells were used as target cells after the first
PBS/SP wash, the PBS/SP was aspirated and 500 .mu.g/mL of FITC was
added (PIERCE 461110) in IMDM media containing SP, Gln, and P/S and
incubated for 30 minutes at 37.degree. C., 5% CO.sub.2. Cells were
washed twice with assay media; resuspended in 1 mL assay media,
counted and diluted.
[0638] Antibody Opsonization: Once target cells were prepared as
described supra, they were opsonized with the appropriate
antibodies. In the case of Fc variants, 50 .mu.L of
1.times.10.sup.5 cells/mL were added to 2.times. concentration of
the antibody harboring the Fc variant. Final concentrations were as
follows: Ch-4-4-20 final concentration was 0.5-1 .mu.g/mL; and
Ch4D5 final concentration was 30 ng/mL-1 ng/mL.
[0639] Opsonized target cells were added to effector cells to
produce an effector:target ratio of 75:1 in the case of the 4-4-20
antibodies with Fc variants. In the case of the Ch4D5 antibodies
with Fc variants, effector:target ratio of 50:1 or 75:1 were
achieved. Effective PBMC gradient for the assay ranges from 100:1
to 1:1. Spontaneous release (SR) was measured by adding 100 .mu.L
of assay media to the cells; maximal release (MR) was measured by
adding 4% TX-100. Cells were spun down at 200 rpm in a Beckman
centrifuge for 1 minute at room temperature at 57 G. Cells were
incubated for 3-3.5 hours at 37.degree. C., 5% CO.sub.2. After
incubation, the cells were spun at 1000 rpm in a Beckman centrifuge
(about 220.times.g) for five minutes at 10.degree. C. 20 .mu.l of
supernatant was collected; 2004 of Eu solution was added and the
mixture was shaken for 15 minutes at room temperature at 120 rpm on
a rotary shaker. The fluorescence was quantitated in a time
resolved fluorometer (Victor 1420, Perkin Elmer)
[0640] Results
[0641] As described above, the variant Fc regions were subcloned
into a mammalian expression vector, containing the heavy chain of
the anti-fluoresceine monoclonal antibody, 4-4-20 (Kranz et al.,
1982 J. Biol. Chem., 257(12): 6987-6995). The variant 4-4-20 heavy
chains were transiently coexpressed with the light chain clones in
the human kidney cell line (293H). Supernatants were collected and
analyzed using the ADCC assay. FIG. 13 shows that ADCC activity of
the mutants is concentration-dependent. As summarized in Table 8,
five immunoglobulins with variant Fc regions had an enhanced ADCC
activity relative to wild type ch 4-4-20. The five mutants were as
follows: MGFc-27 (G316D, A378V, D399E); MGFc-31 (P247L, N421K);
MGFc-10 (K288N, A330S, P396L); MGFc-28 (N315I, V379M, T394M);
MGFc-29 (F243I, V379L, G420V).
[0642] Additional 4-4-20 immunoglobulins with variant Fc regions
were assayed for their ADCC activity relative to a 4-4-20
immunoglobulin with a wild-type Fc region. These results are
summarized in Table 17.
[0643] ADCC assays were also carried out using the same protocol as
previously described for the 4-4-20 antibody, however, the variant
Fc regions were cloned into a humanized antibody (Ab4D5) which is
specific for the human epidermal growth factor receptor 2
(HER2/neu). In this case, SK-BR-3 cells were used as the target
cells that were opsonized with a HER2/neu antibody carrying a
variant Fc region. HER2/neu is endogenously expressed by the
SK-BR-3 cells and therefore present on the surface these cells.
FIG. 14 shows the ADCC activity of HER2/neu antibodies carrying
variant Fc regions. Table 18 summarizes the results of ADCC
activity of the mutants in the context of the HER2/neu antibody.
Normalization was carried out by comparing the concentration of the
mutant to the wild type antibody required for a specific value of
percent cell lysis.
[0644] As shown in FIG. 14A, MGFc-5 (V379M), MGFc-9 (P243I, V379L),
MGFc-10 (K288N, A330S, P396L), MGFc-13 (K334E, T359N, T366S), and
MGFc-27 (G316D, A378V, D399E) mutants that were cloned in to the
humanized anti-HER2/neu antibody exhibited a higher % specific
lysis of SK-BR-3 cells relative to the wild antibody.
TABLE-US-00019 TABLE 17 SUMMARY OF ADCC ACTIVITY OF MUTANTS ADCC Fc
Variant 1 ug/ml 0.5 ug/ml Label Ref Amino Acid Variation % specific
lysis Normalized % specific lysis Normalized MGFc-27 2C4 G316D,
A378V, D399E 33% 2.24 22% 3.60 MGFc-31 3B9 P247L, N421K 30% 2.05
17% 2.90 MGFc-10 1E1 K288N, A330S, P396L 24% 1.66 10% 1.67 MGFc-28
2C5 N315I, V379M, T394M 20% 1.37 10% 1.69 MGFc-29 3D11 F243I,
V379L, G420V 20% 1.35 7% 1.17 ch4-4-20 (P54008) 15% 1.00 6% 1.00
MGFc-35 3D2 R255Q, K326E 11% 0.79 3% 0.53 MGFc-36 3D3 K218R, G281D,
G385R 10% 0.67 5% 0.78 MGFc-30 3A8 F275Y 9% 0.64 2% 0.37 MGFc-32
3C8 D280E, S354F, A431D, L441I 9% 0.62 4% 0.75 MGFc-33 3C9 K317N,
F423deleted 3% 0.18 -1% -0.22 MGFc-34 3B10 F241L, E258G -1% -0.08
-4% -0.71 MGFc-26 D265A 1% 0.08 -3% -0.45
TABLE-US-00020 TABLE 18 SUMMARY OF MUTANTS ELISA ELISA 4-4-20
Anti-HER2 Fc FcR3A, FcR2B, IIIA IIB Phagocytosis ADCC ADCC Variant
Amino Acid changes K.sub.D/Koff K.sub.D/K.sub.off binding binding
(mutant/WT) (mutant/wt) (mutant/wt) Wt none 198/0.170 94/.094 1 1 1
1 1 MGFc 5 V379M 160/0.167 70/0.10 2X.sup. N/C 0.86 2.09 1.77 MGFc
9 P243I, V379L 99.7/0.105 120/0.113 1.5X reduced ? 2.25 2.04 MGFc
10 K288N, A330S, P396L 128/0.115 33.4/0.050 5X.sup. 3X 1.2 2.96
2.50 MGFc 11 F243L, R255L 90/0.075 74.7/0.09.sup. 1x reduced 0.8
2.38 1.00 MGFc 13 K334E, T359N, T366S 55.20.128 72/0.11 1.5X N/C [
1.57 3.67 MGFc 14 K288M, K334E 75.4/0.1 95.6/0.089 3X.sup. reduced
[ 1.74 MGFc 23 K334E, R292L 70.2/0.105 108/0.107 [ 2.09 1.6 MGFc 27
G316D, A378V, D399E 72/0.117 46/0.06 1.5X 14X 1.4 3.60 6.88 MGFc 28
N315I, A379M, D399E 1X.sup. 9X 1.37 1.69 1.00 MGFc 29 P243I, V379L,
G420V 108/0.082 93.4/.101.sup. 2.5X 7X 0.93 1.17 1.00 MGFc 31
P247L, N421K 62/0.108 66/0.065 3X.sup. N/C 1.35 2.90 1.00 MGFc 37
K248M 154/0.175 100/0.091 1.4X reduced 0.98 3.83 0.67 MGFc 38
K392T, P396L 84/0.104 50/0.041 4.5X .sup. 2.5X 1.4 3.07 2.50 MGFc
39 E293V, Q295E, A327T 195/0.198 86/0.074 1.4X reduced 1.5 4.29
0.50 MGFc 40 K248M 180/0.186 110/0.09 1.4X reduced 1.14 4.03 MGFc
41 H268N, P396L 178/0.159 46.6/0.036 2.2X .sup. 4.5X 1.96 2.24 0.67
MGFc 43 Y319F, P352L, P396L 125/0.139 55.7/0.041 3.5X 2X 1.58
1.09
6.8 Analysis of Kinetic Parameters of Fc Mutants
[0645] Kinetic parameters of the binding of ch4-4-20 antibodies
harboring Fc mutants to Fc.gamma.RIIIA and Fc.gamma.RIIB were
analyzed using a BIAcore assay (BIAcore instrument 1000, BIAcore
Inc., Piscataway, N.J.). The Fc.gamma.RIIIA used in this assay was
a soluble monomeric protein, the extracellular region of
Fc.gamma.RIIIA joined to the linker-AVITAG sequence as described in
Section 6.2 supra. The Fc.gamma.RIIB used in this assay was a
soluble dimeric protein prepared in accordance with the methodology
described in U.S. Provisional Application No. 60/439,709 filed on
Jan. 13, 2003, which is incorporated herein by reference. Briefly,
the Fc.gamma.RIIB used was the extracellular domain of
Fc.gamma.RIIB fused to the hinge-CH2--CH3 domain of human IgG2.
[0646] BSA-FITC (36 .mu.g/mL in 10 mM Acetate Buffer at pH 5.0) was
immobilized on one of the four flow cells (flow cell 2) of a sensor
chip surface through amine coupling chemistry (by modification of
carboxymethyl groups with mixture of NHS/EDC) such that about 5000
response units (RU) of BSA-FITC was immobilized on the surface.
Following this, the unreacted active esters were "capped off" with
an injection of 1M Et-NH.sub.2. Once a suitable surface was
prepared, ch 4-4-20 antibodies carrying the Fc mutations were
passed over the surface by one minute injections of a 20 .mu.g/mL
solution at a 5 .mu.L/mL flow rate. The level of ch-4-4-20
antibodies bound to the surface ranged between 400 and 700 RU.
Next, dilution series of the receptor (Fc.gamma.RIIIA and
Fc.gamma.RIIB-Fc fusion protein) in HBS-P buffer (10 mM HEPES, 150
mM NaCl, 0.005% Surfactant P20, 3 mM EDTA, pH 7.4) were injected
onto the surface at 100 .mu.L/min Antibody regeneration between
different receptor dilutions was carried out by single 5 second
injections of 100 mM NaHCO.sub.3 pH 9.4; 3M NaCl.
[0647] The same dilutions of the receptor were also injected over a
BSA-FITC surface without any ch-4-4-20 antibody at the beginning
and at the end of the assay as reference injections.
[0648] Once an entire data set was collected, the resulting binding
curves were globally fitted using computer algorithms supplied by
the manufacturer, BIAcore, Inc. (Piscataway, N.J.). These
algorithms calculate both the K. and K.sub.off, from which the
apparent equilibrium binding constant, K.sub.D is deduced as the
ratio of the two rate constants (i.e., K.sub.off/K.sub.on). More
detailed treatments of how the individual rate constants are
derived can be found in the BlAevaluaion Software Handbook
(BIAcore, Inc., Piscataway, N.J.).
[0649] Binding curves for two different concentrations (200 nM and
800 nM for Fc.gamma.RIIIA and 200 nM and 400 nM for Fc.gamma.RIIB
fusion protein) were aligned and responses adjusted to the same
level of captured antibodies, and the reference curves were
subtracted from the experimental curves. Association and
dissociation phases were fitted separately. Dissociation rate
constant was obtained for interval 32-34 sec of the dissociation
phase; association phase fit was obtained by a 1:1 Langmuir model
and base fit was selected on the basis R.sub.max and chi.sup.2
criteria.
[0650] Results
[0651] FIG. 15 shows the capture of ch 4-4-20 antibodies with
mutant Fc regions on the BSA-FITC-immobilized sensor chip. 6 .mu.L
of antibodies at a concentration of about 20 .mu.g/mL were injected
at 5 .mu.L/min over the BSA-FITC surface. FIG. 16 is a sensogram of
real time binding of Fc.gamma.RIIIA to ch-4-4-20 antibodies
carrying variant Fc regions. Binding of Fc.gamma.RIIIA was analyzed
at 200 nM concentration and resonance signal responses were
normalized at the level of the response obtained for the wild type
ch-4-4-20 antibody. Kinetic parameters for the binding of
Fc.gamma.RIIIA to ch-4-4-20 antibodies were obtained by fitting the
data obtained at two different Fc.gamma.RIIIA concentrations, 200
and 800 nM (FIG. 17). The solid line represents the association fit
which was obtained based on the K.sub.off values calculated for the
dissociation curves in interval 32-34 seconds. K.sub.D and
K.sub.off represent the average calculated from the two different
Fc.gamma.RIIIA concentrations used. FIG. 18 is a sensogram of real
time binding of Fc.gamma.RIIB-Fc fusion protein to ch-4-4-20
antibodies carrying variant Fc regions. Binding of Fc.gamma.RIIB-Fc
fusion protein was analyzed at 200 nM concentration and resonance
signal responses were normalized at the level of the response
obtained for the wild type ch-4-4-20 antibody. Kinetic parameters
for the binding of Fc.gamma.RIIB-Fc fusion protein to ch-4-4-20
antibodies were obtained by fitting the data obtained at two
different Fc.gamma.RIIB-Fc fusion protein concentrations, 200 and
800 nM (FIG. 19). The solid line represents the association fit
which was obtained based on the K.sub.off calculated for the
dissociation curves in interval 32-34 seconds. K.sub.D and
K.sub.off represent the average from the two different
Fc.gamma.RIIB-Fc fusion protein concentrations used.
[0652] The kinetic parameters (K.sub.on and K.sub.off) that were
determined from the BIAcore analysis correlated with the binding
characteristic of the mutants as determined by an ELISA assay and
the functional activity of the mutants as determined in an ADCC
assay. Specifically, as seen in Table 19, mutants that had an
enhanced ADCC activity relative to the wild-type protein, and had
an enhanced binding to Fc.gamma.RIIIA as determined by an ELISA
assay had an improved K.sub.off for Fc.gamma.RIIIA (i.e., a lower
K.sub.off). Therefore, a lower K.sub.off value for Fc.gamma.RIIIA
for a mutant Fc protein relative to a wild type protein may be
likely to have an enhanced ADCC function. On the other hand, as
seen in Table 20, mutants that had an enhanced ADCC activity
relative to the wild-type protein, and had a reduced binding for
Fc.gamma.RIIB-Fc fusion protein as determined by an ELISA assay had
a higher K.sub.off for Fc.gamma.RIIB-Fc fusion protein.
[0653] Thus, the K.sub.off values for Fc.gamma.RIIIA and
Fc.gamma.RIIB can be used as predictive measures of how a mutant
will behave in a functional assay such as an ADCC assay. In fact,
ratios of K.sub.off values for Fc.gamma.RIIIA and Fc.gamma.RIIB-Fc
fusion protein of the mutants to the wild type protein were plotted
against ADCC data (FIG. 20). Specifically, in the case of K.sub.off
values for Fc.gamma.RIIIA, the ratio of K.sub.off (wt) K.sub.off
(mutant) was plotted against the ADCC data; and in the case of
K.sub.off values for Fc.gamma.RIIB, the ratio of K.sub.off
(mut)/K.sub.off (wt) was plotted against the ADCC data. Numbers
higher than one (1) show a decreased dissociation rate for
Fc.gamma.RIIIA and an increased dissociation rate for
Fc.gamma.RIIB-Fc relative to wild type. Mutants that fall within
the indicated box have a lower off rate for Fc.gamma.RIIIA binding
and a higher off-rate for Fc.gamma.RIIB-Fc binding, and possess an
enhanced ADCC function.
TABLE-US-00021 TABLE 19 Kinetic parameters of FcRIIIa binding to
ch4-4-20Ab obtained by "separate fit" of 200 nM and 800 nM binding
curves ##STR00001## Highlighted mutants do not fit to the group by
ELISA or ADCC data.
TABLE-US-00022 TABLE 20 Kinetic parameters of FcRIIB-Fc binding to
wild type and mutant ch4-4-20Ab obtained by "separate fit" of 200
nM and 800 nM binding curves. ##STR00002##
6.9 Screening for Fc Mutants Using Multiple Rounds of Enrichment
Using a Solid Phase Assay
[0654] The following mutant screens were aimed at identifying
additional sets of mutants that show improved binding to
Fc.gamma.RIIIA and reduced binding to Fc.gamma.RIIB. Secondary
screening of selected Fc variants was performed by ELISA followed
by testing for ADCC in the 4-4-20 system. Mutants were than
selected primarily based on their ability to mediate ADCC via
4-4-20 using Fluorescein coated SK-BR3 cells as targets and
isolated PBMC from human donors as the effector cell population. Fc
mutants that showed a relative increase in ADCC, e.g., an
enhancement by a factor of 2 were than cloned into anti-HER2/neu or
anti-CD20 chAbs and tested in an ADCC assay using the appropriate
tumor cells as targets. The mutants were also analyzed by BIAcore
and their relative K.sub.off were determined.
[0655] Screen 1: Sequential Solid Phase Depletion and Selection
Using Magnetic Beads Coated with Fc.gamma.RIIB Followed by
Selection with Magnetic Beads Coated with Fc.gamma.RIIIA.
[0656] The aim of this screen was identification of Fc mutants that
either no longer bind Fc.gamma.RIIB or show reduced binding to
Fc.gamma.RIIB. A 10-fold excess of the naive library
(.about.10.sup.7 cells) was incubated with magnetic beads ("My
One", Dynal) coated with Fc.gamma.RIIB. Yeast bound to beads were
separated from the non-bound fraction by placing the tube
containing the mixture in a magnetic field. Those yeast cells that
were not bound to the beads were removed and placed in fresh media.
They were next bound to beads that were coated with Fc.gamma.RIIIA.
Yeast bound to beads were separated from the nonbound fraction by
placing the tube containing the mixture in a magnetic field.
Nonbound yeast were removed and the bound cells were removed by
vigorous vortexing. The recovered cells were regrown in glucose
containing media and reinduced in selective media containing
galactose. The selection process was repeated. The final culture
was than used to harvest DNA. Inserts containing the Fc domain were
amplified by PCR and cloned into 4-4-20. Approximately 90 Fc
mutants were screened by 4-4-20 ELISA and ADCC assays and the
resultant positive mutants are shown in Table 21.
TABLE-US-00023 TABLE 21 Mutants selected by sequential solid phase
depletion and selection using Magnetic beads coated with
Fc.gamma.RIIB followed by selection with magnetic beads coated with
Fc.gamma.RIIIA. Mutant Amino Acid changes MgFc37 K248M MgFc38
K392T, P396L MgFc39 E293V, Q295E, A327T MgFc41 H268N, P396LN MgFc43
Y319F, P352L, P396L MgFc42 D221E, D270E, V308A, Q311H, P396L,
G402D
[0657] Screens 2&3: Mutants Selected by FACS, Equilibrium and
Kinetic Screening:
[0658] The first library screen identified a mutation at position
396, changing the amino acid from Proline to Leucine (P396L). This
Fc variant showed increased binding to both Fc.gamma.RIIIA and
Fc.gamma.RIIB. A second library was constructed using P396L as a
base line. PCR mutagenesis was used to generate .about.10.sup.7
mutants each of which contained the P396L mutation and contained
additional nucleotide changes. The P396L library was screened using
two sets of conditions.
[0659] An equilibrium screen was performed using biotinylated
Fc.gamma.RIIIA-linker-avitag as a monomer, using methods already
described. Approximately 10-fold excess of library (10.sup.8 cells)
was incubated in a 0.5 mL of approximately 7 nM Fc.gamma.RIIIA for
1 hr. The mixture was sorted by FACS, selecting top 1.2% of
binders. Selected yeast cells were grown in selective media
containing glucose and reinduced in selective media containing
galactose. The equilibrium screen was repeated a second time and
the sort gate was set to collect the top 0.2% of binders. The
selected yeast cells were then grown under selective conditions in
glucose. This culture was than used to harvest DNA. Inserts
containing the Fc domain were amplified by PCR and cloned into the
nucleotide sequence encoding 4-4-20 variable domain using methods
already described. Approximately 90 Fc mutants were screened by
4-4-20 ELISA and ADCC and the resultant positive mutants are shown
in Table 22.
TABLE-US-00024 TABLE 22 Mutants selected by FACS using an
Equilibrium screen with concentrations of FcRIIIA of approximately
7 nM. Mutant Amino Acid changes MgFc43b K288R, T307A, K344E, P396L
MgFc44 K334N, P396L MgFc46 P217S, P396L MgFc47 K210M, P396L MgFc48
V379M, P396L MgFc49 K261N, K210M, P396L MgFc60 P217S, P396L
[0660] A kinetic screen was also implemented to identify mutants
with improved K.sub.off in binding Fc.gamma.RIIIA Conditions were
established for screening the P396L library using a strain with the
P396L Fc variant displayed on the yeast surface. Briefly cells
grown under inducing conditions were incubated with 0.1 .mu.M
biotinylated Fc.gamma.RIIIA-linker-avitag monomer for 1 hr. The
cells were washed to remove the labeled ligand. Labeled cells were
then incubated for different times with 0.1 .mu.M unlabeled
Fc.gamma.RIIIA-linker-avitag monomer, washed and then stained with
SA:PE for FACS analysis (FIG. 21). Cells were also stained with
goat anti-human Fc to show that the Fc display was maintained
during the experiment.
[0661] Based on the competition study it was determined that a 1
minute incubation resulted in approximately 50% loss of cell
staining. This time point was chosen for the kinetic screen using
the P396L library. Approximately 10-fold excess of library
(10.sup.8 cells) was incubated with 0.1 nM biotinylated
Fc.gamma.RIIIA-linker-avitag monomer in a 0.5 mL volume. Cells were
washed and then incubated for 1 minute with unlabeled ligand.
Subsequently the cells were washed and labeled with SA:PE. The
mixture was sorted by FACS, selecting the top 0.3% of binders.
Selected yeast cells were grown in selective media containing
glucose and reinduced in selective media containing galactose. The
kinetic screen was repeated a second time and the sort gate was set
to collect the top 0.2% of binders. The nonselcted P396L library
was compared to the yeast cells selected for improved binding by
FACS (FIG. 22). The histograms show the percentage of cells that
are costained with both Fc.gamma.RIIIA/PE and goat anti-human
Fc/FITC (upper right).
[0662] The selected yeast cells from the second sort were then
grown under selective conditions in glucose. This culture was than
used to harvest DNA. Inserts containing the Fc domain were
amplified by PCR and cloned into the nucleotide sequence encoding
4-4-20 variable domain using methods described above. Approximately
90 Fc mutants were screened by 4-4-20 ELISA and ADCC and the
resultant positive mutants are shown in Table 23.
TABLE-US-00025 TABLE 23 Mutants selected by FACS using a Kinetic
screen using equimolar amounts of unlabeled CD16A for 1 minute.
Mutants Amino Acid changes MgFc50 P247S, P396L MgFc51 Q419H, P396L
MgFc52 V240A, P396L MgFc53 L410H, P396L MgFc54 F243L, V305I, A378D,
F404S, P396L MgFc55 R255l, P396L MgFc57 L242F, P396L MgFc59 K370E,
P396L
[0663] Screens 4 and 5: Combining the Solid Phase Fc.gamma.RIIB
Depletion Step with Fc.gamma.RIIIA Selection by FACs Sort, Using
the Fc.gamma.RIIIA 158V Allele
[0664] Analysis of Fc variants from Screen 1 showed that the
mutations that were selected from the secondary screen had improved
binding to both Fc.gamma.RIIIA and Fc.gamma.RIIB. Therefore, the
data suggested that sequential depletion and selection using
magnetic beads (solid phase) under the established conditions did
not efficiently select for differential binding of Fc.gamma.RIIIA
and Fc.gamma.RIIB. Therefore, in order to screen more effectively
for mutants that bind Fc.gamma.RIIIA, while having reduced or no
binding to Fc.gamma.RIIB, the solid phase Fc.gamma.RIIB depletion
step was combined with Fc.gamma.RIIIA selection by FACs sort. This
combination identified Fc variants that bind Fc.gamma.RIIIA with
greater or equal affinity than wild-type Fc.
[0665] A 10-fold excess of the naive library (.about.10.sup.7) was
incubated with magnetic beads coated with Fc.gamma.RIIB. Yeast
bound to beads were separated from the non-bound fraction by
placing the tube containing the mixture in a magnetic field. Those
yeast cells that were not bound to the beads were removed and
placed in fresh media and subsequently reinduced in media
containing galactose. The Fc.gamma.RIIB depletion by magnetic beads
was repeated 5 times. The resulting yeast population was analyzed
and found to show greater than 50% cell staining with goat
anti-human Fc and a very small percentage of cells were stained
with Fc.gamma.RIIIA. These cells were then selected twice by a FACS
sort using 0.1 .mu.M biotinylated Fc.gamma.RIIIA linker-avitag
(data not shown). The Fc.gamma.RIIIA was the 158V allotype. Yeast
cells were analyzed for both Fc.gamma.RIIIA and Fc.gamma.RIIB
binding after each sort and compared to binding by wild-type Fc
domain (FIGS. 23 A-L).
[0666] The selected yeast cells from the second sort were then
grown under selective conditions in glucose. This culture was then
used to harvest DNA. Inserts containing the Fc domain were
amplified by PCR and cloned into the nucleotide sequence encoding
4-4-20 variable domain. Approximately 90 Fc mutants were screened
by 4-4-20 ELISA and ADCC and the resultant positive mutants are
shown in Table 24 (mutants 61-66).
TABLE-US-00026 TABLE 24 Mutants selected by magnetic bead depletion
using beads coated with CD32B and final selection by FACS using
Fc.gamma.RIIIA 158Valine or 158Phenylalanine Mutants Amino Acid
Changes MgFc61 A330V MgFc62 R292G MgFc63 S298N, K360R, N361D MgFc64
E233G MgFc65 N276Y MgFc66 A330V, V427M MgFc67 V284M, S298N, K334E,
R355W, R416T
[0667] Screening of Fc Mutants Using the 158F allele of
Fc.gamma.RIIIA:
[0668] Two different alleles of Fc.gamma.RIIIA receptor exist that
have different binding affinities for the IgG1 Fc domain (Koene et
al., 1997, Blood 90: 1109-1114; Wu et al., 1997, J. Clin. Invest.
100: 1059-70). The 158F allele binds to the Fc domain with a
binding constant 5-10 fold lower than the 158V allele. Previously
all of the Fc screens using yeast display were done using the high
binding 158V allele as a ligand. In this experiment, Fc mutants
were selected from the Fc.gamma.RIIB depleted yeast population
using biotinylated Fc.gamma.RIIIA158F-linker-avitag monomer as a
ligand. The sort gate was set to select the top 0.25 percent
Fc.gamma.RIIIA 158F binders. The resulting enriched population was
analyzed by FACS (FIG. 23B). Individual clones were then isolated
and their binding to different Fc.gamma.Rs were analyzed by FACS
(FIG. 23B). Analysis of individual clones from the population
resulted in the identification of a single mutant harboring 5
mutations MgFc67 (V284M, S298N, K334E, R355W, R416S), which had an
enhanced binding to Fc.gamma.RIIIA and a reduced binding to
Fc.gamma.RIIB.
[0669] Secondary Screen of Mutants by an ADCC Assay for Screens 1,
2, and 3:
[0670] Mutants that were selected in the above screens were then
analyzed using a standard ADCC assay to determine the relative
rates of lysis mediated by ch4-4-20 harboring the Fc mutants.
ch4-4-20 antibodies carrying the Fc variants were constructed using
methods already described above. SK-BR3 cells were used as targets
and effector cells were PBMC that were isolated from donors using a
Ficoll gradient, as described supra (Section 6.7). The ADCC
activity results for the mutants are summarized in Table 25.
[0671] As seen in Table 25, mutants isolated using the above
primary and secondary screens based on Fc.gamma.RIIB depletion and
Fc.gamma.RIIIA selection showed enhanced ADCC activity relative to
wild-type.
TABLE-US-00027 TABLE 25 Analysis of ADCC mediated by 4-4-20
anti-Fluorescein antibody on SKBR3 cells coated with fluorescein.
Relative rate of Mutant Amino Acid Change lysis MgFc37 K248M 3.83
MgFc38 K392T, P396L 3.07 MgFc39 E293V, Q295E, A327T 4.29 MgFc41
H268N, P396LN 2.24 MgFc43 Y319F, P352L, P396L 1.09 MgFc42 D221E,
D270E, V308A, Q311H, P396L, 3.17 G402D MgFc43b K288R, T307A, K344E,
P396L 3.3 MgFc44 K334N, P396L 2.43 MgFc46 P217S, P396L 2.04 MgFc47
K210M, P396L 2.02 MgFc48 V379M, P396L 2.01 MgFc49 K261N, K210M,
P396L 2.06 MgFc50 P247S, P396L 2.1 MgFc51 Q419H, P396L 2.24 MgFc52
V240A, P396L 2.35 MgFc53 L410H, P396L 2 MgFc54 F243L, V305I, A378D,
F404S, P396L 3.59 MgFc55 R255l, P396L 2.79 MgFc57 L242F, P396L 2.4
MgFc59 K370E, P396L 2.47 MgFc60 P217S, P396L 1.44
[0672] Mutants 37, 38, 39, 41, 43 were analyzed using 0.5 .mu.g/mL
ch4-4-20. All other antibodies were tested at 1 .mu.g/mL. All rates
were normalized to wild type ch4-4-20 (IgG1).
[0673] Mutants were additionally cloned into the heavy chain of
antitumor monoclonal antibody 4D5 (anti-HER2/neu) and anti-CD20
monoclonal antibody 2H7 by replacing the Fc domain of these
monoclonal antibodies. These chimeric monoclonal antibodies were
expressed and purified and tested in an ADCC assay using standard
methods by transient transfection into 293H cells and purification
over protein G column. The chimeric 4D5 antibodies were tested in
an ADCC assay using SK-BR3 cells as targets (FIG. 24), whereas the
chimeric 2H7 antibodies were tested in an ADCC assay using Daudi
cells as targets (FIG. 25).
[0674] Secondary Screen of Mutants Via BIAcore:
[0675] Mutants that were selected in the above screens were then
analyzed by BIAcore to determine the kinetic parameters for binding
Fc.gamma.RIIIA(158V) and Fc.gamma.RIIB. The method used was similar
to that disclosed in Section 6.8, supra.
[0676] The data displayed are K.sub.off values relative to wild
type off rates as determined from experiments using the Fc mutants
in the ch4-4-20 monoclonal antibody. Relative numbers greater than
one indicate a decrease in K.sub.off rate. Numbers less than one
indicate an increase in off rate.
[0677] Mutants that showed a decrease in off rates for
Fc.gamma.RIIIA were MgFc38 (K392, P396L), MgFc43(Y319F, P352L,
P396L), MgFc42(D221E, D270E, V308A, Q311H, P396L, G402D), MgFc43b
(K288R, T307A, K344E, P396L), MgFc44 (K334N, P396L), MgFc46 (P217S,
P396L), MgFc49 (K261N, K210M, P396L). Mutants that showed a
decrease in off rate for Fc.gamma.RIIB were, MgFc38(K392, P396L),
MgFc39 (E293V, Q295E, A327T), MgFc43 (K288R, T307A, K344E, P396L),
MgFc44 (K334N, P396L). The Biacore data is summarized in Table
26.
TABLE-US-00028 TABLE 26 BIAcore data. Fc.gamma.RIIIA Fc.gamma.RIIB
Fc 158V (Koff WT/ mutant AA residues (Koff WT/Mut) Mut) MgFc37
K248M 0.977 1.03 MgFc38 K392T, P396L 1.64 2.3 MgFc39 E293V, Q295E,
A327T 0.86 1.3 MgFc41 H268N, P396LN 0.92 1.04 MgFc43 Y319F, P352L,
P396L 1.23 2.29 MgFc42 D221E, D270E, V308A, 1.38 Q311H, P396L,
G402D MgFc43b K288R, T307A, K344E, P396L 1.27 0.89 MgFc44 K334N,
P396L 1.27 1.33 MgFc46 P217S, P396L 1.17 0.95 MgFc47 K210M, P396L
MgFc48 V379M, P396L MgFc49 K261N, K210M, P396L 1.29 0.85 MgFc50
P247S, P396L MgFc51 Q419H, P396L MgFc52 V240A, P396L MgFc53 L410H,
P396L MgFc54 F243L, V305I, A378D, F404S, P396L MgFc55 R255l, P396L
MgFc57 L242F, P396L MgFc59 K370E, P396L MgFc60 P217S, P396L MgFc61
A330V 1 0.61 MgFc62 R292G 1 0.67 MgFc63 S298N, K360R, N361D 1 0.67
MgFc64 E233G 1 0.54 MgFc65 N276Y 1 0.64 MgFc66 A330V, G427M, 1 0.62
MgFc67 V284M, S298N, K334E, R355W, R416T
6.10 PBMC Mediated ADCC Assays
[0678] Materials and Methods
[0679] Fc variants that show improved binding to Fc.gamma.RIIIA
were tested by PBMC based ADCC using 60:1 effector:target ratio.
Two different tumor model systems were used as targets, SK-BR3
(anti-HER2/neu) and Daudi (anti-CD20). Percent specific Lysis was
quantitated for each mutant. Linear regression analysis was used to
plot the data setting the maximal percent lysis at 100%.
[0680] ADCC is activated on immune system effector cells via a
signal transduction pathway that is triggered by an interaction
between low affinity Fc.gamma.R and an immune complex. Effector
cell populations were derived from either primary blood or
activated monocyte derived macrophages (MDM). Target cells were
loaded with europium and incubated with chimeric MAb and
subsequently incubated with effector cell populations. Europium
works the same way as .sup.51Cr, but it is non-radioactive and the
released europium is detected in a fluorescent plate reader.
Lymphocytes harvested from peripheral blood of donors (PBM) using a
Ficoll-Paque gradient (Pharmacia) contain primarily natural killer
cells (NK). The majority of the ADCC activity will occur via the NK
containing Fc.gamma.RIIIA but not Fc.gamma.RIIB on their
surface.
[0681] Experiments were performed using two different target cell
populations, SK-BR-3 and Daudi, expressing HER2/neu and CD20,
respectively. ADCC assays were set up using Ch4-4-20/FITC coated
SK-BR-3, Ch4D5/SKBR3, and Rituxan/Daudi (data not shown). Chimeric
MAbs were modified using Fc mutations identified. Fc mutants were
cloned into Ch4D5. Purified Ab was used to opsonize SK-BR-3 cells
or Daudi cells. Fc mutants were cloned into Ch4D5.
[0682] Results.
[0683] Fc mutants showed improved PBMC mediated ADCC activity in SK
BR3 cells (FIG. 28). The plot shows linear regression analysis of a
standard ADCC assay. Antibody was titrated over 3 logs using an
effector to target ratio of 75:1. % lysis=(Experimental
release--SR)/(MR-SR)*100.
[0684] Fc mutants showed improved PBMC mediated ADCC activity in
Daudi cells (FIG. 29).
6.11 Monocyte Derived Macrophage (MDM) Based ADCC Assays
[0685] Fc.gamma.R dependent tumor cell killing is mediated by
macrophage and NK cells in mouse tumor models (Clynes et al., 1998,
PNAS USA, 95: 652-6). Elutriated monocytes from donors were used as
effector cells to analyze the efficiency Fc mutants to trigger cell
cytotoxicity of target cells in ADCC assays. Expression patterns of
Fc.gamma.RI, Fc.gamma.R3A, and Fc.gamma.R2B are affected by
different growth conditions. Fc.gamma.R expression from frozen
monocytes cultured in media containing different combinations of
cytokines and human serum were examined by FACS using FcR specific
MAbs. (FIGS. 30A-30O). Cultured cells were stained with Fc.gamma.R
specific antibodies and analyzed by FACS to determine MDM
Fc.gamma.R profiles. Conditions that best mimic macrophage in vivo
Fc.gamma.R expression, i.e., showed the greatest fraction of cells
expressing CD16 and CD32B were used in a monocyte derived
macrophage (MDM) based ADCC assay. For the experiment in FIGS.
30A-30O, frozen elutriated monocytes were grown for 8 days in DMEM
and 20% FBS containing either M-CSF (condition 1) or GM-CSF
(condition 2). For the experiment in FIG. 31, frozen elutriated
monocytes were cultured for 2 days in DMEM and 20% FBS containing
GM-CSF, IL-2 and IFN.gamma. prior to ADCC assay. Serum free
conditions have also been developed which allow for high levels of
CD16 and CD32B expression (data not shown). Briefly, purified
monocytes were grown for 6-8 days in Macrophage-SFM (Invitrogen)
containing GM-CSF, M-CSF, IL-6, IL-10, and IL-1.beta.. While the
incidence of CD32B+/CD16+ cells in these cultures is highest using
a mixture of cytokines, combinations of two of more cytokines will
also enhance Fc.gamma.R expression (M-CSF/IL-6, M-CSF/IL-10; or
M-CSF/IL-1.beta.). For ADCC assays, IFN.gamma. is added for the
final 24-48 hours.
[0686] MDM based ADCC required incubation times of >16 hrs to
observe target cell killing. Target cells were loaded with
Indium-111 which is retained for long incubations within the target
cells. Indium release was quantitated using a gamma counter. All
other reagents, Abs and target cells, were similar to the PBMC
based ADCC assay. ADCC activity due to Fc.gamma.RI can be
efficiently blocked using the anti-FcRI blocking antibody (M21,
Ancell). The assay conditions differ slightly from the PBMC based
assay. 20:1 target to effector; 18-14 hr incubation at 37C.
[0687] Fc mutants that show improved PBMC ADCC, increased binding
to Fc.gamma.RIIIA, or decreased binding to Fc.gamma.RIIB were
tested (FIG. 31).
6.12 Effect of Fc Mutants on Complement Activity
[0688] Fc mutants were originally identified based on their
increased binding to Fc.gamma.RIIIA. These mutants were
subsequently validated for their improved affinity for all low
affinity receptors and in many cases improved activity in ADCC
mediated by PBMC. In vivo antibody mediated cytotoxicity can occur
through multiple mechanisms. In addition to ADCC other possible
mechanisms include complement dependent cytotoxicity (CDC) and
apoptosis. The binding of C1q to the Fc region of an immunoglobulin
initiates as cascade resulting in cell lysis by CDC. The
interaction between C1q and the Fc has been studies in a series of
Fc mutants. The results of these experiments indicate that C1q and
the low affinity FcR bind to overlapping regions of the Fc, however
the exact contact residues within the Fc vary.
[0689] Mutants that showed improved ADCC in the PBMC based assay
were examined for their effect in CDC. Antibodies were analyzed in
the anti CD20 Ch-mAb, 2H7. We detected improved CDC for each mutant
ch-mAb tested. Interestingly even though these mutants were
selected for their improved ADCC they also show enhanced CDC
[0690] Materials and Methods.
[0691] CDC assay was used to test the Fc mutants using anti-CD20
and Daudi cells as targets. Guinea Pig Serum was used as the source
for complement (US Biological). The CDC assay was similar to PBMC
based ADCC. Target cells were loaded with europium and opsonized
with ChMAb. However complement, guinea pig serum, was added instead
of effector cells. FIG. 32 shows a flow chart of the assay.
Anti-CD20 ChMab over 3 orders of magnitude was titrated. % lysis
was calculated. Daudi cells, (3.times.10.sup.6) were labeled with
BADTA reagent. 1.times.10.sup.4 cells were aliquoted into wells in
a 96 well plate. Antibodies were titrated into the wells using 3
fold dilutions. The opsonization reaction was allowed to proceed
for 30-40 minutes at 37.degree. C. in 5% CO.sub.2. Guinea pig serum
was added to a final conc. of 20%. The reaction was allowed to
proceed for 3.5 hrs at 37.degree. C. in 5% CO.sub.2. Subsequently,
100 uls of cell media was added to the reaction and cells were spun
down. For detection 20 uls of the supernatant was added to 200 uls
of the Europium solution and the plates were read in the
Victor2(Wallac).
[0692] Results:
[0693] All mutants that show improved binding for either activating
FcR or C1q were placed in the CDC assay (FIG. 33). Fc mutants that
showed enhanced binding to Fc.gamma.RIIIA also showed improved
complement activity Each of the mutants show enhanced complement
activity compared to wild type. The mutants tested are double
mutants. In each case one of the mutations present is P396L.
[0694] To determine whether the increase in CDC correlated with
increased binding of C1q to IgG1 Fc binding between the two
proteins was measured in realtime using surface plasmon resonance.
In order to examine the binding between C1q and an IgG1 Fc the Fc
variants were cloned into an anti-CD32B ch-mAb, 2B6. This allowed
us to capture the wt and mutant antibodies to the glass slide via
soluble CD32B protein (FIG. 35A). Three of the four mutants tested
in CDC were also examined in the Biacore. All 3 showed greatly
enhanced K.sub.off compare to wild type Fc (FIG. 35B). Biacore
format for C1q binding to 2B6 mutants demonstrate enhanced binding
of mutants with P396L mutation (FIG. 36). Mutation D270E can reduce
C1q binding at different extent. A summary of the kinetic analysis
of Fc.gamma.R and C1q binding is depicted in the Table 27
below.
TABLE-US-00029 TABLE 27 KINETIC ANALYSIS OF FcgR and C1q binding to
mutant 2B6 2B6Mutants 3aV158 3aF158 2bfcagl 2aR131Fcagl 2aH131Fcagl
C1q WT 0.192 0.434 0.056 0.070 0.053 0.124 MgFc38 0.114 0.243 0.024
0.028 0.024 0.096 (K392T, P396L) MgFc51 0.142 0.310 0.030 0.036
0.028 0.074 (Q419H, P396L) MgFc55 0.146 0.330 0.030 0.034 0.028
0.080 (R255I, P396L) MgFc59 0.149 0.338 0.028 0.033 0.028 0.078
(K370E, P396L) MgFc31/60 0.084 0.238 0.094 0.127 0.034 0.210
MgFc51/60 0.112 0.293 0.077 0.089 0.028 0.079 MgFc55/60 0.113 0.288
0.078 0.099 0.025 0.108 MgFc59/60 0.105 0.296 0.078 0.095 0.024
0.107
6.13 Designing Fc Variants with Decreased Binding to
Fc.gamma.RIIB
[0695] Based on a selection for Fc mutants that reduce binding to
Fc.gamma.RIIB and increase binding to Fc.gamma.RIIA 131H a number
of mutations including D270E was identified. Each mutation was
tested individually for binding to the low affinity Fc receptors
and their allelic variants by Biacore performed as described above
in the context of the 4D5 ChAb (anti-HER2/neu).
[0696] D270E had the binding characteristics that suggested it
would specifically reduce Fc.gamma.RIIB binding. D270E was tested
in combination with mutations that were previously identified based
on their improved binding to all FcR.
[0697] Results.
[0698] As shown in Tables 28 and 29 and FIGS. 37 and 38 addition of
D270E mutation enhances Fc.gamma.RIIIA and Fc.gamma.RIIA H131
binding and reduces binding to Fc.gamma.RIIB. FIG. 39 shows the
plot of MDM ADCC data against the Koff as determined for CD32A
H131H binding for select mutants.
TABLE-US-00030 TABLE 28 ADDITION OF D270E MUTATION ENHANCES
Fc.gamma.RIIIA AND Fc.gamma.RIIA H131 BINDING AND REDUCES
Fc.gamma.RIIB BINDING 4D5Mutants 3aV158 3aF158 2bfcagl 2aR131Fcagl
2aH131Fcagl Wt pure 0.175 0.408 0.078 0.067 0.046 MgFc55 0.148
0.381 0.036 0.033 0.029 MgFc55/60 0.120 0.320 0.092 0.087 0.013
MgFc55/60 + 0.116 0.405 0.124 0.112 0.037 R292G MgFc55/60 + 0.106
0.304 0.092 0.087 0.015 Y300L MgFc52 0.140 0.359 0.038 0.040 0.026
MgFc52/60 0.122 0.315 0.094 0.087 0.013 MgFc59 0.145 0.378 0.039
0.047 0.033 MgFc59/60 0.117 0.273 0.088 0.082 0.012 MgFc31 0.125
0.305 0.040 0.043 0.030 MgFc31/60 0.085 0.215 0.139 0.132 0.020
MgFc51 0.135 0.442 0.060 0.047 0.062 MgFc51/60 0.098 0.264 0.118
0.106 0.023 MgFc38 0.108 0.292 0.034 0.025 0.032 MgFc38/60 0.089
0.232 0.101 0.093 0.021 D265A n.b. n.b. n.b. 0.223 0.117
TABLE-US-00031 TABLE 29 KINETIC CHARACTERISTICS OF 4D5 MUTANTS
4D5Mutants 3aV158 3aF158 2bfcagl 2aR131Fcagl 2aH131Fcagl MgFc70
0.101 0.250 0.030 0.025 0.025 MgFc71 0.074 0.212 0.102 0.094 0.020
MgFc73 0.132 0.306 0.190 -- 0.024 MgFc74 0.063 0.370 n.b. 0.311
0.166 WT023stable 0.150 0.419 0.071 0.068 0.043
6.14 Ability to Mediate Cell Lysis by ADCC and CDC Correlates with
Enhancement of Functional Range of the Antibody
[0699] Fc mutations which enhance Fc.gamma.RIIIA and Fc.gamma.RIIA
binding and reduce binding to Fc.gamma.RIIB have been suggested to
positively correlate with the appearance or improvement of both
ADCC and complement function (Section 6.8). This hypothesis was
tested by cloning promising mutations into the heavy chain of the
chimeric antitumor monoclonal antibody 4D5 (anti-HER2/neu),
chimeric anti-CD32B monoclonal antibody ch2B6 and the anti-CD20
antibody RITUXIN.TM. anti-CD-20 antibody. Mutations were cloned
into the heavy chains of the antibodies using standard techniques.
These chimeric antibodies were expressed by transient transfection
into 293H cells and purified over a protein G column. Variant 4D5
antibodies were analyzed for alterations in kinetic parameters
using a BIAcore assay (BIAcore instrument 1000, BIAcore Inc.,
Piscataway, N.J.) and associated software as described supra
(Section 6.8). Binding ability of 4D5 and ch2B6 antibodies was
characterized by immunostaining cells with either FITC conjugated
variant antibody or the variant antibodies and a PE-conjugated
polyclonal F(ab).sub.2 goat anti-human Fc antibody (Jackson
Immunoresearch Laboratories, Inc.). FACS analysis was used to
quantitate the staining.
[0700] The chimeric variant antibodies were tested in an ADCC or
CDC assay as described supra (Section 6.10 and 6.11, respectively).
For both 4D5 and ch2B6 antibodies, the effects of antigen density
on binding or on cell lysis by ADCC/CDC were tested by using cells
with high or low expression of antigen. Antigen density was
determined using QUANTUM.TM. SIMPLY CELLULAR.RTM. kit from Bangs
Laboratories, Inc. (Fishers, Ind.) according to the manufacturer's
instructions. Target cells for 4D5 antibodies were SK-BR3 cells
(high Her2/neu expression) and HT29 cells (low Her2/neu
expression). Target cells for ch2B6 antibodies were Daudi cells or
BL41 cells (high CD32B expression) and Ramos cells (low CD32B
expression). Target cells for RITUXIN.TM. anti-CD-20 antibody were
CHO cells, which were engineered to express both CD32B and CD20
using standard techniques.
[0701] Results
[0702] FIGS. 40 and 41 show the capture of 4D5 antibodies with
mutant Fc regions on the BSA-FITC-immobilized sensor chip. BIAcore
data was analyzed as described in Section 6.8. Either triple
mutants (FIG. 40) or quadruple mutants (FIG. 41) showed reduced
K.sub.d to the activating Fc receptors and increased K.sub.d to the
inhibitory Fc receptor.
[0703] Although the Fc mutant 31/60 (P247L; N421K; D270E) did not
enhance 4D5 binding to cells expressing low levels of Her2/neu
(FIG. 42), this modification, as well as variants 71 (D270E; G316D;
R416G), 59/60 (K370E; P396L; D270E), 55/60 (R255L; P396L; D270E),
51/60 (Q419H; P396L; D270E), 55/60/F243L (R255L; P396L; D270E;
F243L), and 74/P396L (F243L; R292P; V3051; P396L) improved the
wild-type ADCC mediated lysis of cells expressing low levels of
antigen (FIGS. 43 and 44).
[0704] When similar Fc mutations, variants 31/60, 59/60, and 71,
are introduced into an antibody with only limited binding to cells
expressing low levels of antigen and no native effector function on
the same cells, the results are more dramatic. FIGS. 45A-45H
demonstrate that wild-type ch2B6 binding to Ramos cells can be
substantially improved by the introduction the Fc mutations of
variant 31/60 and 59/60. Similarly, effector function can be
introduced by Fc mutations. Where the wild-type antibody has no
detectable effector function, Fc mutations can result in a
gain-of-function phenotype. Mutations which improved the binding of
ch2B6 to Ramos cells also enabled the mutated antibody to mediate
ADCC, variant 31/60, or CDC, variants 31/60 and 71 (FIGS. 46 and
47, respectively). FIGS. 48 and 49 also show the spectrum of
response available, dependent on the specific mutation. Where the
wild type ch2B6 antibody is capable of mediating at least some
effector function, e.g. in cells with high expression of CD32B,
Daudi cells, the same Fc mutations, variant 31/60 and 71, improve
the effect (FIG. 48).
[0705] The increase in ADCC activity was shown to be a function of
the Fc modification and not the target antigen. The mutation
variant 55/60, previously identified as improving ADCC activity in
4D5 antibody, conferred effector function to the anti-CD20
antibody, RITUXIN.TM. anti-CD20 antibody. FIGS. 49 A and B show
that the engineered CHO cell line expressed similar levels of CD32B
and CD20 when tested with FITC-conjugated 2B6 or RITUXIN.TM.
anti-CD20 antibody, respectively. Although the cells were sensitive
to ADCC mediated by wild-type 2B6, ADCC activity was completely
undetectable using wild-type RITUXIN.TM. anti-CD20 antibody (FIG.
50 A). The introduction of the mutation variant 55/60 into
RITUXIN.TM. anti-CD20 antibody, as in 4D5, was, however, able to
confer effector function to the modified antibody (FIG. 50 B).
[0706] Possible mechanisms by which the mutated antibodies were
able to improve both binding and effector function were observed
when the binding affinities of variant ch2B6 antibodies to
Fc.gamma.RIIB were correlated with their ability to bind Ramos
cells (FIG. 51A-B). For example, variant 55/60 had both the highest
k.sub.off and binding affinity to Ramos cells. It is theorized the
limited ability of the wild-type antibody to bind Fc.gamma.RIIB is
due to Fc-Fc.gamma.RIIB interaction, effectively withdrawing the
additional cell surface receptors from further antibody binding.
The theory was investigated by challenging opsonized Ramos cells
with CD16A, an activating Fc.gamma.R. In accord with the theory, at
low antigen density, Fc-engineered ch2B6, but not wild type Fc, was
able bind the activating receptor (FIG. 52).
6.15 Effect of Light-Chain Glcosylation on Fc Binding to Fc.gamma.R
and Mediation of ADCC and Compliment Activity
[0707] As demonstrated in Sections 6.8 and 6.14, Fc mutations can
introduce or improve both the binding and effector function of
antibodies. The effect of light-chain glycosylation on these
abilities was investigated by combining a mutation which eliminated
glycosylation of the light-chain region (YA substitution at
positions 50 and 51 of the light-chain amino acid sequence) with
the previously identified Fc mutations which induced or improved
ADCC or complement function as listed in Table 30.
[0708] Mutations were cloned into both the heavy and light chains
of anti-Fc.gamma.R monoclonal antibody ch2B6. These chimeric
antibodies were expresses and purified using standard methods by
transient transfection into 293H cells and purification over a
protein G column. Variant antibodies were analyzed for alterations
in kinetic parameters using a BIAcore assay (BIAcore instrument
1000, BIAcore Inc., Piscataway, N.J.) and associated software as
described supra (Section 6.8). Binding ability was characterized by
immunostaining cells with the variant antibodies and a
PE-conjugated polyclonal F(ab).sub.2 goat anti-human Fc.gamma.R
antibody (Jackson Immunoresearch Laboratories, Inc.). FACS analysis
was used to quantitate the staining The effects of antigen density
were investigated by immunostaining high (Daudi cells and
EL-4/CD32B cells) and low (Ramos) antigen expressing cells.
[0709] The chimeric variant antibodies were tested in an ADCC or
CDC assay using Ramos cells as targets as described supra (Section
6.10 and 6.11, respectively).
[0710] Results
[0711] BIAcore analysis revealed that previously identified
mutations which enhanced Fc.gamma.RIIIA binding and reduced binding
to Fc.gamma.RIIB were unaffected by the glycosylation state of the
light chain (Table 30).
TABLE-US-00032 TABLE 30 ADDITION OF MUTATION ELIMINATING
LIGHT-CHAIN GLYCOSYLATION AT POSITION 50 DOES NOT AFFECT IMPROVED
BINDING TO Fc.gamma.RIIIA AND DECREASED BINDING TO Fc.gamma.RIIB 3A
3A 2B 2A 2A Antibody VL Fc Date V158 F158 Fcagly R131 H131 Ch2B6
Mouse-Wt- WT 0.150 0.427 0.074 0.071 0.043 gly. position 50 Ch2B6
Mouse-Wt- agly nb nb nb nb nb agly gly. position 50 Ch2B6 Mouse-Wt-
Q419H; Mar. 15, 2004 0.098 0.264 0.118 0.106 0.023 51/60 gly.
position P396L; 50 D270E Ch2B6 Mouse-Wt- P247L; Mar. 15, 2004 0.084
0.245 0.156 0.141 0.027 31/60 gly. position N421K; 50 D270E Ch2b6
Mouse-Wt- R255L; Mar. 15, 2004 0.120 0.320 0.092 0.087 0.013 55/60
gly. position P396L; 50 D270E hu2B6YA Human -agly Q419H; Mar. 15,
2004 0.098 0.264 0.118 0.106 0.023 51/60 YA P396L; substitution
D270E at pos. 50, 51 hu2B6YA Human - agly P247L; Mar. 15, 2004
0.084 0.245 0.156 0.141 0.027 31/60 YA N421K; substitution D270E at
pos. 50, 51 hu2B6YA Human - agly R255L; Mar. 15, 2004 0.120 0.320
0.092 0.087 0.013 55/60 YA P396L; substitution D270E at pos. 50,
51s hu2B6 human-Wt- WT 0.150 0.427 0.074 0.071 0.043 WT gly.
position 50
[0712] The improvements in mutant antibody binding noted in the
BIAcore analysis were not observed when cells with high antigen
expression were immunostained and analyzed by FACS (FIGS. 53 and
54). Variant antibodies failed to improve binding, exhibiting
characteristics similar to the wild type antibody. However, when
the Ramos cell line was tested, wild-type ch2B6 and ch2B6 variant
31/60 bound at levels significantly above that of the other
variants tested (FIG. 55). As the immunostaining protocol more
closely approximates the in vivo conditions of antigen-antibody
interaction, the differences in the two analyses may suggest that
light-chain glycosylation is necessary for biologically relevant
activity of the antibody.
[0713] This suggestion was furthered by the ADCC and CDC assays.
Although these Fc mutations resulted in a gain-of-function for 4D5
antibodies as outlined in Section 6.14, for ch2B6, the ability to
effect lysis was dependent on the glycosylation state of the
light-chain of the antibody (FIGS. 56 and 57).
6.16 Effect of Mutations Identified as Enhancing ADCC Function in
ADCC Assays Using Tumor Cells Isolated from Rituxan.TM. Anti-CD-20
Antibody Treated Patients
[0714] Fc mutations which enhance Fc.gamma.RIIIA and Fc.gamma.RIIA
binding, reduce binding to Fc.gamma.RIIB and enhance ADCC and/or
complement function (Section 6.8) were cloned into the anti-CD20
antibody RITUXIN.TM. anti-CD20 antibody using standard techniques.
These chimeric antibodies were expressed by transient transfection
into 293H cells and purified over a protein G column. The variant
antibodies were tested in an ADCC or CDC assay as described supra
(Section 6.10 and 6.11, respectively) in cells isolated from
RITUXIN.TM. anti-CD20 antibody treated patients.
[0715] During the course of phase I and phase II clinical trials of
Rituximab, lymphoma cells from biopsy specimens obtained from
patients with B cell lymphoma prior to receiving the antibody were
collected. Participating patients underwent surgical removal of a
lymph node near the surface of the body. This was done using a
local anesthetic. A portion of the tissue was analyzed by routine
histopathology in the pathology lab. A portion of the lymph node
was used to make a cell suspension for the in vitro studies.
[0716] Additionally, pre- and post-treatment PBMC via leukapheresis
in some of the patients were collected to study the effector cells
and T cell immune response after Rituximab treatment. Peripheral
blood T cells and effector cells were collected via leukapheresis
from patients treated with Rituximab. Participating patients
underwent leukapheresis before the Rituximab treatment and one
month after completion of the treatment to collect the T
lymphocytes and effector cells. The collected blood components were
mixed with an anti-coagulant (ACD-A) as it was drawn to prevent
clotting. The effector cells collected via leukapheresis were used
to determine if effector cells of different Fc.gamma.R genotypes
mediate ADCC differently.
[0717] Results
[0718] The results of the ADCC assays for the different Fc
Engineered rituximab antibodies in six of the patients are shown in
FIGS. 58A-F. Tables 31 and 32 provide a ranking of the
effectiveness of the antibodies in six patients with 1 being the
most effective for that patient and 11 being the least effective
for that patient. A normal donor provided PBMC for this experiment.
The genotype of the normal donor was heterozygous for the FcRIIIA
158V and FcRIIA 131R alleles. In most patients, the Fc engineered
rituximab antibodies showed an improvement over rituximab in ADCC
activity.
TABLE-US-00033 TABLE 31 (10:1 Effector:Target Ratio) Fc Mutant IgG1
Rituximab 55/60/300L 51/60 52/60 59/60 38/60 59 51 31/60 55/60/292G
Patient 1 11 10 5 3 4 1 9 8 7 6 2 Patient 2 11 9 2 10 4 1 7 3 6 5 8
Patient 3 11 10 3 4 8 2 9 5 7 6 1 Patient 4 11 9 1 6 8 5 10 7 3 4 2
Patient 5 11 7 8 10 2 1 9 3 6 5 4 Patient 6 11 10 8 4 1 2 6 5 9 7
2
TABLE-US-00034 TABLE 32 (30:1 Effector:Target Ratio) Fc Mutant IgGl
Rituximab 55/60/300L 51/60 52/60 59/60 38/60 59 51 31/60 55/60/292G
Patient 1 11 10 6 7 8 2 9 4 5 3 1 Patient 2 11 8 1 4 5 2 6 3 10 7 9
Patient 3 11 8 2 1 3 6 7 5 10 4 10 Patient 4 11 5 1 2 9 3 8 6 10 4
7 Patient 5 11 9 2 5 6 1 10 4 8 3 7 Patient 6 11 10 6 8 4 1 2 3 9 5
7
[0719] As shown in FIG. 58 A, rituximab has minimal ADCC killing
activity as compared to the other engineered rituximab antibodies
tested. Patient 1 fits our definition of a non-responder (i.e., is
refractory) to rituximab treatment (FIG. 58 A). In contrast, in
patient 2, wild-type rituximab shows some ADCC activity; however
all tested variants except 59/60 and 52/60 exhibited improved ADCC
activity.
6.17 Efficacy in a Mouse Model
[0720] Efficacy of Fc engineered rituximab antibodies may be
investigated in a mouse xenographic model (nu/nu mice, female,
approximately 10 weeks old) utilizing a B cell lymphoblastic tumor,
e.g., Ramos cell tumors. Ramos cells (ATCC, CRL 1596) are
maintained in culture using RPMI-1640 supplemented with 10% fetal
calf serum and glutamine at 37.degree. C. and 5% CO.sub.2. To
increase tumorigenicity of the cell line, Tumors are first
initiated in female nude mice approximately 7-10 weeks old by
subcutaneous injection of 1.7.times.10.sup.6 Ramos cells in a
volume of 0.10 ml (HBSS) using a 1 cc syringe fitted with 25 g
needle. All animals are manipulated in a laminar flow hood and all
cages, bedding, food and water are autoclaved. Tumor cells are
passaged by excising tumors and passing these through a 40 mesh
screen; cells are washed twice with 1.times.HBSS (50 ml) by
centrifugation (1300 RPM), resuspended in 1.times.HBSS to
10.times.10.sup.6 cells/ml, and frozen at -70.degree. C. until
used.
[0721] The one-passaged tumor cells from several frozen lots are
thawed, pelleted by centrifugation (1300 RPM), washed twice with
1.times.HBSS, and resuspended to approximately 2.0.times.10.sup.6
cells/ml in HBSS. Mice in the study groups are injected with 0.10
ml of the cell suspension (s.c.) using a 1 cc syringe fitted with a
25 g needle; injections are made on the animal's left side,
approximately mid-region. Tumors will develop in approximately two
weeks.
[0722] Study groups are assigned to separate study groups based on
the creation of a comparable tumor size distribution in each group
(average tumor size, expressed as a product of length.times.width
of the tumor, was approximately 80 mm.sup.2). 200 mg of the Fc
engineered rituximab antibodies is used for treatment. The groups
are treated via tail-vein injections using a 100 .mu.l Hamilton
syringe fitted with a 25 g needle.
[0723] Tumor measurements are made every two or three days using a
caliper.
6.18 Clinical Studies of Fc Engineered Rituximab
[0724] Fifteen patients having histologically documented relapsed
non-Hodgkins B cell lymphoma will be treated with Fc engineered
rituximab in a clinical trial. Each patient will receive a single
dose of antibody in a dose-escalating study; there will be three
patients per dose: 10 mg/m.sup.2; 50 mg/m.sup.2; 100 mg/m.sup.2;
250 mg/m.sup.2 and 500 mg/m.sup.2. Treatment will be by i.v.
infusion through an 0.22 micron in-line filter with the antibody
being diluted in a final volume of 250 cc or a maximal
concentration of 1 mg/ml of normal saline. Initial rate will be 50
cc/hr for the first hour; if no toxicity was seen, dose rate will
be escalated to a maximum of 200 cc/hr.
[0725] Toxicity (as indicated by the clinician) will be judged on a
range from "none", to "fever" to "moderate" (two patients) to
"severe" (one patient). Peripheral Blood Lymphocytes will be
analyzed to determine, inter alia, the impact of the antibodies on
T-cells and B-cells.
[0726] The invention described and claimed herein is not to be
limited in scope by the specific embodiments herein disclosed since
these embodiments are intended as illustration of several aspects
of the invention. Any equivalent embodiments are intended to be
within the scope of this invention. Indeed, various modifications
of the invention in addition to those shown and described herein
will become apparent to those skilled in the art from the foregoing
description. Such modifications are also intended to fall within
the scope of the appended claims.
[0727] Throughout this application various publications are cited.
Their contents are hereby incorporated by reference into the
present application in their entireties for all purposes.
Sequence CWU 1
1
13186DNAArtificial SequencePrimer 5' linker.avitag 1ggccgcaggt
ggtggtggtt ctggtggtgg tggttctggt ctgaacgaca tcttcgaggc 60tcagaaaatc
gaatggcacg aatgat 86286DNAArtificial SequencePrimer 3'
linker.avitag 2ctagatcatt cgtgccattc gattttctga gcctcgaaga
tgtcgttcag accagaacca 60ccaccaccag aaccaccacc acctgc
86335DNAArtificial SequencePrimer FcR3A left 3gttggatcct ccaactgctc
tgctacttct agttt 35434DNAArtificial SequencePrimer FcR3A Right
4gaaaagctta aagaatgatg agatggttga cact 34531DNAArtificial
SequencePrimer FcR2B right 5gaagtcgaca atgatcccca ttggtgaaga g
31630DNAArtificial SequencePrimer FcR2B left 6gttagatctt gctgtgctat
tcctggctcc 30727DNAArtificial SequencePrimer IgG1 right 7atagtcgacc
actgatttac ccggaga 27831DNAArtificial SequencePrimer IgG1 left
8ggaattcaac accaaggtgg acaagaaagt t 31931DNAArtificial
SequencePrimer mcr025;chl (f') 9aaaggatccg cgagctcagc ctccaccaag g
311020DNAArtificial SequencePrimer H021 10gtctgctcga agcattaacc
2011232PRTHomo sapiens 11Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala1 5 10 15Pro Glu Leu Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro 20 25 30Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val 35 40 45Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln65 70 75 80Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120
125Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
130 135 140Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser145 150 155 160Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr 165 170 175Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr 180 185 190Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220Ser Leu Ser
Leu Ser Pro Gly Lys225 2301219PRTArtificial SequenceN-Terminal
Sequence of GIF206 12Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys Thr1 5 10 15His Thr Cys137PRTArtificial
SequenceN-Terminal Sequence of GIF227 13Thr His Thr Cys Pro Pro
Cys1 5
* * * * *