U.S. patent application number 12/184855 was filed with the patent office on 2009-03-05 for enhancement of antibody-mediated immune responses.
This patent application is currently assigned to THE ROCKEFELLER UNIVERSITY. Invention is credited to Jeffrey V. Ravetch.
Application Number | 20090060911 12/184855 |
Document ID | / |
Family ID | 26893900 |
Filed Date | 2009-03-05 |
United States Patent
Application |
20090060911 |
Kind Code |
A1 |
Ravetch; Jeffrey V. |
March 5, 2009 |
ENHANCEMENT OF ANTIBODY-MEDIATED IMMUNE RESPONSES
Abstract
The present invention is related to enhancing the function of
anti-tumor antibodies by regulating Fc.gamma.RIIB-mediated
activity. In particular disrupting SHIP activation by Fc.gamma.RIIB
enhances cytotoxicity elicited by a therapeutic antibody in vivo in
a human. The invention further provides an antibody, e.g., an
anti-tumor antibody, with a variant Fc region that results in
binding of the antibody to Fc.gamma.RIIB with reduced affinity. A
variety of transgenic mouse models demonstrate that the inhibiting
Fc.gamma.RIIB molecule is a potent regulator of cytotoxicity in
vivo.
Inventors: |
Ravetch; Jeffrey V.; (New
York, NY) |
Correspondence
Address: |
Pepper Hamilton LLP
400 Berwyn Park, 899 Cassatt Road
Berwyn
PA
19312-1183
US
|
Assignee: |
THE ROCKEFELLER UNIVERSITY
New York
NY
|
Family ID: |
26893900 |
Appl. No.: |
12/184855 |
Filed: |
August 1, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09834321 |
Apr 13, 2001 |
7416726 |
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12184855 |
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60198550 |
Apr 13, 2000 |
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60204254 |
May 15, 2000 |
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Current U.S.
Class: |
424/133.1 ;
424/174.1; 514/44R; 530/387.3; 530/387.7 |
Current CPC
Class: |
A61P 31/12 20180101;
C07K 16/32 20130101; C07K 2317/52 20130101; A61P 43/00 20180101;
Y02A 50/412 20180101; C07K 2317/732 20130101; A61P 31/04 20180101;
A61P 35/04 20180101; A61K 2039/505 20130101; C07K 2317/73 20130101;
A61P 35/02 20180101; A61P 33/00 20180101; A61P 35/00 20180101; C07K
16/30 20130101; A61K 38/00 20130101; Y02A 50/30 20180101; A61P
31/00 20180101; A61P 37/04 20180101; C07K 2317/72 20130101 |
Class at
Publication: |
424/133.1 ;
514/44; 424/174.1; 530/387.3; 530/387.7 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 31/7088 20060101 A61K031/7088; C07K 16/18
20060101 C07K016/18; A61P 31/00 20060101 A61P031/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] The research leading to the present invention was supported
in part, by National Institutes of Health Grant No. CA 80757.
Accordingly, the U.S. Government may have certain rights in this
invention.
Claims
1. A method for enhancing cytotoxicity elicited by a therapeutic
antibody in vivo in a subject, which method comprises disrupting
activation of SHIP by Fc-gamma-receptor IIB (Fc.gamma.RIIB).
2. The method according to claim 1, wherein the SHIP activation by
Fc.gamma.RIIB results from antibody binding to Fc.gamma.RIIB.
3. The method according to claim 2, wherein antibody binding is
inhibited by a competitive inhibitor.
4. The method according to claim 2, wherein antibody binding is
inhibited by modifying the Fc portion of the antibody to reduce its
affinity for Fc.gamma.RIIB.
5. The method according to claim 1, wherein SHIP activation by
Fc.gamma.RIIB is disrupted by inhibiting the expression of
Fc.gamma.IIB.
6. The method according to claim 5, wherein Fc.gamma.RIIB
expression is disrupted with an antisense nucleic acid specific for
the .gamma.IIB chain mRNA.
7. The method according to claim 5, wherein Fc.gamma.RIIB
expression is disrupted with an intracellular antibody specific for
the .gamma.IIB chain.
8. The method according to claim 1, wherein SHIP activation is
inhibited by an inositol phosphatase inhibitor.
9. The method according to claim 1, wherein SHIP activation is
inhibited by inhibiting SHIP expression.
10. The method according to claim 1, wherein the antibody is an
anti-tumor antibody.
11. The method according to claim 10 wherein the antibody is
specific for a tumor cell growth receptor.
12. The method according to claim 11, wherein the antibody is
specific for a HER12/neu growth factor receptor.
13. The method according to claim 11, wherein the antibody is
specific for a CD20 B cell antigen.
14. The method according to claim 1, wherein the antibody binds to
human activating Fc receptors.
15. The method according to claim 14, wherein the subject expresses
human Fc receptors.
16. An antibody with a variant Fc region, which antibody binds
Fc.gamma.RIIB with reduced affinity.
17. The antibody of claim 16, which binds activating Fc-receptors
with at least the same affinity as wildtype antibody.
18. The antibody of claim 16, which is an anti-tumor antibody.
19. The antibody of claim 18, which is specific for a tumor cell
growth receptor.
20. The antibody of claim 19, which is specific for a HER2/neu
growth factor receptor.
21. The antibody of claim 19, which is specific for a CD20 B cell
antigen.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Applications, Ser. No. 60/198,550, filed Apr. 13, 2000, and Ser.
No. 60/204,254, filed May, 15, 2000, each of which is incorporated
herein by reference.
FIELD OF THE INVENTION
[0003] The present invention relates to enhancing the function of
anti-tumor antibodies by regulating Fc.gamma.R activity.
BACKGROUND OF THE INVENTION
[0004] The interaction of antibodies and antibody-antigen complexes
with cells of the immune system effects a variety of responses,
including antibody-dependent cell-mediated cytotoxicity (ADCC) and
complement-dependent cytotoxicity (CDC), phagocytosis, inflammatory
mediator release, clearance of antigen, and antibody half-life
(reviewed in Daeron, Annu. Rev. Immunol., 1997, 15:203-234; Ward
and Ghetie, Therapeutic Immunol., 1995, 2:77-94; Ravetch and Kinet,
Annu. Rev. Immunol., 1991; 9:457-492, each of which is incorporated
herein by reference).
[0005] Antibody constant domains are not involved directly in
binding an antibody to an antigen, but exhibit various effector
functions. Depending on the amino acid sequence of the constant
region of their heavy chains, antibodies or immunoglobulins can be
assigned to different classes. There are five major classes of
immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these
may be further divided into subclasses (isotypes), e.g., IgG1,
IgG2, IgG3, and IgG4; IgA1 and IgA2. The heavy chain constant
regions that correspond to the different classes of immunoglobulins
are called .alpha., .delta., .epsilon., .gamma., and .mu.,
respectively. Of the various human immunoglobulin classes, human
IgG1 and IgG3 mediate ADCC more effectively than IgG2 and IgG4.
[0006] Papain digestion of antibodies produces two identical
antigen binding fragments, called Fab fragments, each with a single
antigen binding site, and a residual "Fc" fragment, whose name
reflects its ability to crystallize readily. The Fc region is
central to the effector functions of antibodies. The crystal
structure of the human IgG Fc region has been determined
(Deisenhofer, Biochemistry, 20:2361-2370 (1981), which is
incorporated herein by reference). In human IgG molecules, the Fc
region is generated by papain cleavage N-terminal to Cys 226.
[0007] The effector functions mediated by the antibody Fc region
can be divided into two categories: (1) effector functions that
operate after the binding of antibody to an antigen; these
functions involve the participation of the complement cascade or Fc
receptor (FcR)-bearing cells; and (2) effector functions that
operate independently of antigen binding; these functions confer
persistence in the circulation and the ability to be transferred
across cellular barriers by transcytosis (Ward and Ghetie,
Therapeutic Immunology, 1995, 2:77-94, which is incorporated herein
by reference).
[0008] While binding of an antibody to the requisite antigen has a
neutralizing effect that might prevent the binding of a foreign
antigen to its endogenous target (e.g., receptor or ligand),
efficient effector functions are also required for removing and/or
destroying foreign antigens.
[0009] Several antibody effector functions are mediated by Fc
receptors (FcRs), which bind the Fc region of an antibody. FcRs are
defined by their specificity for immunoglobulin isotypes: Fc
receptors for IgG antibodies are referred to as Fc.gamma.R, for IgE
as Fc.epsilon.R, for IgA as Fc.alpha.R and so on. Surface receptors
for immunoglobulin G are present in two distinct classes--those
that activate cells upon their crosslinking ("activation FcRs") and
those that inhibit activation upon co-engagement ("inhibitory
FcRs"). Activation FcRs for IgG require the presence of the Immune
Tyrosine Activation Motif (ITAM) to mediate cellular activation.
This 19 amino acid sequence, found in the cytoplasmic tail of the
receptors or their associated subunits, interacts with src and syk
families of tyrosine kinases sequentially. Upon crosslinking of an
activation Fc.gamma.R by an immune complex, ITAM sequences trigger
the activation of these tyrosine kinases, which in turn activate a
variety of cellular mediators, like PI3K, PLC.gamma. and Tec
kinases. The net result of these activation steps is to increase
intracellular calcium release from the endoplasmic reticulum stores
and opening of the capacitance coupled calcium channel to generate
a sustained calcium response. These calcium fluxes are critical for
the exocytosis of granular contents, stimulation of phagocytosis
and ADCC responses and activation of specific nuclear transcription
factors. Opposing these activation responses is the inhibitory
Fc.gamma.R. Inhibitory signaling is dependent on a 13 amino acid
cytoplasmic sequence called the Immune Tyrosine Inhibitory Motif
(ITIM). Upon co-ligation of an ITAM containing receptor to the
inhibitory Fc.gamma.R, a critical tyrosine residue in the ITIM
becomes phosphorylated, leading to the recruitment of a specific
SH2-containing inositol polyphosphate 5 phosphatase called SHIP.
SHIP catalyzes the hydrolysis of the membrane inositol lipid, PIP3,
thereby preventing activation of PLC.gamma. and Tec kinases and
abrogating the sustained calcium flux mediated by the influx of
calcium through the capacitance coupled channel.
[0010] Three subclasses of Fc.gamma.R have been identified:
Fc.gamma.RI (CD64), Fc.gamma.RII (CD32) and Fc.gamma.RIII (CD16).
Because each Fc.gamma.R subclass is encoded by two or three genes,
and alternative RNA splicing leads to multiple transcripts, a broad
diversity in Fc.gamma.R isoforms exists. The three genes encoding
the Fc.gamma.RI subclass (Fc.gamma.RIA, Fc.gamma.RIB and
Fc.gamma.RIC) are clustered in region 1q21.1 of the long arm of
chromosome 1; the genes encoding Fc.gamma.RII isoforms
(Fc.gamma.RIIA, Fc.gamma.RIIB and Fc.gamma.RIIC) and the two genes
encoding Fc.gamma.RIII (Fc.gamma.RIIIA and Fc.gamma.RIIIB) are all
clustered in region 1q22.
[0011] The mouse expresses two activation Fc.gamma.Rs, FcRI and
FcRIII, oligomeric surface receptors with a ligand binding a
subunit and an ITAM containing y subunit. The inhibitory receptor
is Fc.gamma.RIIB, a single chain receptor with an ITIM sequence
found in the cytoplasmic tail of the ligand binding a chain. FcRIIB
and FcRIII bind monomeric IgG with an affinity constant of
1.times.10.sup.6; hence, under physiological conditions they do not
bind monomeric IgG, but interact with multimeric IgG immune
complexes with low affinity and high avidity. FcRIII is the
physiologically important activation FcR for mediating inflammatory
disease triggered by cytotoxic antibodies or pathogenic immune
complexes. FcRIII is expressed on NK cells, macrophages, mast cells
and neutrophils in the mouse. It is not found on B cells, T cells
or circulating monocytes. FcRIIB is found on B cells, macrophages,
mast cells, neutrophils. It is not found on T cells or NK cells.
FcRII and III have greater than 90% sequence identity in their
extracellular, ligand binding domain.
[0012] The situation in the human is more complex. There are two
low-affinity activation FcRs for IgG-Fc.gamma.RIIA and
Fc.gamma.RIIIA. Fc.gamma.RIIA is a single-chain low affinity
receptor for IgG, with an ITAM sequence located in its cytoplasmic
tail. It is expressed on macrophages, mast cells, monocytes,
neutrophils and some B cells. It is 90% homologous in its
extracellular domain to the human inhibitory FcRIIB molecule, which
has an ITIM sequence in its cytoplasmic domain, expressed on B
cells, macrophages, mast cells, neutrophils, monocytes but not NK
cells or T cells. FcRIIIA is an oligomeric activation receptor
consisting of a ligand binding a subunit and an ITAM containing
.gamma. or .xi. subunit. It is expressed on NK cells, macrophages
and mast cells. It is not expressed on neutrophils, B cells or T
cells. In addition, a receptor with greater than 95% sequence
identity in its extracellular domain called FcRIIIB is found on
human neutrophils as a GPI-anchored protein. It is capable of
binding immune complexes but not activating cells in the absence of
association with an ITAM containing receptor like FcRIIA. FcRII and
FcRIII are about 70% identical in their ligand binding
extracellular domains.
[0013] Thus, in the human, IgG cytotoxic antibodies interact with
four distinct low-affinity receptors--two of which are capable of
activating cellular responses, FcRIIA and FcRIIIA, one of which is
inhibitory, FcRIIB and one of which will bind IgG complexes but not
trigger cellular responses, FcRIIIB. Macrophages express FcRIIA,
FcRIIB and FcRIIIA, neutrophils express FcRIIA, FcRIIB and FcRIIIB,
while NK cells express only FcRIIIA. The efficacy of a therapeutic
anti-tumor antibody will thus depend on the specific interactions
with activation, inhibition and inert low-affinity FcRs,
differentially expressed on distinct cell types.
[0014] Well-defined tumor models for which therapeutic anti-tumor
antibodies have been developed are known. For example, antibodies
directed against the HER2/neu growth factor receptor prevent the
growth of breast carcinoma cells in vitro and in vivo. Similarly,
antibodies directed to the CD20 antigen on B cells arrests the
growth of non-Hodgkin's lymphoma (Taji, H. et al., Jpn. J. Cancer
Res., 1998, 89:748, which is incorporated herein by reference).
These antibodies were developed based on their ability to interfere
with tumor cell growth in vitro and are representative of a class
which include those with specificities for the EGF receptor (Masul,
H. et al., J. Cancer Res., 1986, 46:5592, which is incorporated
herein by reference), IL-2R (Waldmann, T. A., Ann. Oncol., 1994, 5
Supp. 1:13-7, which is incorporated herein by reference) and others
(Tutt, A. L. et al., J. Immunol., 1998, 161:3176, which is
incorporated herein by reference). HERCEPTIN.RTM., a humanized
antibody specific for the cellular proto-oncogene p185HER-2/neu
(Pegram, M. D. et al., J. Clin. Oncol. 1998, 16:2659; Carter, P. et
al., Proc. Natl. Acad. Sci. USA, 1992, 89:4285-4289, each of which
is incorporated herein by reference), and RITUXAN.RTM., a chimeric
antibody specific for the B cell marker CD20 (Leget, G. A. and
Czuczman, M. S., Curr. Opin. Oncol., 1998, 10:548-51, which is
incorporated herein by reference), are approved for the treatment
of HER-2 positive breast cancer and B cell lymphoma, respectively.
A number of in vitro studies indicated that the critical mechanism
responsible for the anti-tumor activities of HERCEPTIN.RTM. and its
mouse parent molecule 4D5 are due to receptor-ligand blockade
(Kopreski, M. et al., Anticancer Res., 1996, 16:433-6; Lewis, G. D.
et al., Cancer Immunol. Immunother., 1993, 37:255-63, each of which
is incorporated herein by reference), while other in vitro studies
have suggested that activities such as antibody dependent cellular
cytotoxicity (ADCC) may be of importance (Carter, 1992, supra;
Lewis, G. D. et al., Cancer Immunol. Immunother., 1993, 37:255-63,
which is incorporated herein by reference). In vitro studies with
RITUXAN.RTM. and its murine parent 2B8 have suggested a direct
pro-apoptotic activity may be associated with this antibody (Shan,
D. et al., Blood, 1998, 91:1644-52, which is incorporated herein by
reference).
[0015] Thus, multiple mechanisms have been proposed for the ability
of anti-tumor antibodies to mediate their effects in vivo,
including extended half-life, blockade of signaling pathways,
activation of apoptosis and effector cell mediated cytotoxicity.
The elucidation of a mechanism that enhances the ability of
anti-tumor antibodies to effectively treat tumors is highly
desirable.
SUMMARY OF THE INVENTION
[0016] The present invention represents an important improvement
over prior art efforts to regulate antibody-mediated immune
responses by recognizing the key role played by Fc.gamma.RIIB in
modulating antibody-mediated cytotoxicity. Thus the invention
advantageously provides a method for enhancing cytotoxicity
elicited by a therapeutic antibody in vivo in a human. The method
of the invention comprises disrupting activation of SHIP by
Fc-gamma-receptor IIB (Fc.gamma.RIIB). Preferably, antibody binding
is inhibited by modifying the Fc portion of the antibody to reduce
its affinity for Fc.gamma.RIIB. The invention is particularly
useful to enhance the activity and thus effectiveness of anti-tumor
antibodies.
[0017] The invention also provides an antibody with a variant Fc
region, which antibody binds Fc.gamma.RIIB with reduced affinity.
Preferably, the antibody binds activating Fc-receptors with at
least the same affinity as wildtype antibody. As noted above, these
characteristics are particularly useful for an anti-tumor
antibody.
[0018] These and other aspect of the invention will be better
understood by reference to the Drawings, Detailed Description, and
Examples.
DESCRIPTION OF THE DRAWINGS
[0019] FIGS. 1A, 1B, 1C, 1D, 1E, and 1F show that anti-tumor
activity of 4D5, HERCEPTIN.RTM., and RITUXAN.RTM. require
Fc.gamma.R activating receptors. Nude mice (6-10 per group) were
injected subcutaneously with 5.times.10.sup.6 BT474M1 cells
followed by weekly injections of mAb4D5 (FIG. 1A and FIG. 1B) or
HERCEPTIN.RTM. (FIGS. 1C and 1D) or RITUXAN.RTM. (FIGS. 1E and 1F).
Antibody-dependent tumor protection observed in BALB/c nude mice
(FIGS. 1A, 1C, and 1E) is absent in Fc.gamma.R.gamma..sup.-/- nude
mice (FIGS. 1B, 1D, and 1F). All experiments were repeated three
times with similar results.
[0020] FIGS. 2A and 2B show that anti-breast tumor activity of 4D5
and HERCEPTIN.RTM. is enhanced in Fc.gamma.RIIB deficient mice.
Nude mice (8 per group) were injected with BT474M1 cells, as in
FIG. 1, and treated with 0.4 .mu.g/gm loading dose and 0.2 .mu.g/gm
weekly, a sub-therapeutic dose for wild-type mice. Complete
inhibition is observed in RIIB deficient and partial inhibition in
RIIB heterozygous mice.
[0021] FIGS. 3A, 3B, 3C, and 3D show in vitro and in vivo
properties of D265A mutant antibody. FIG. 3A--Fc.gamma.RIII
binding. Both wildtype and mutant Fc fragments were grafted onto an
antihuman IgE Fab fragment. Solid phase binding assays were
performed with human IgE/antihuman IgE hexamenic complexes and
recombinant Fc.gamma.RIII coated plates. FIG. 3B--Growth inhibition
of BT474MI cells. FIG. 3C-NK cell ADCC of chromium-labeled tumor
targets. Chromium labeled SKBR-3 cells were incubated with NK
effector cells at varying ratios and release of label quantitated.
FIG. 3D--In vivo growth of breast carcinoma cells: Athymic BALB/c
nu/nu animals were implanted with BT474M1 xenografts and their
growth measured as described in FIG. 1 in response to treatment
with 4D5, D265A or PBS.
DETAILED DESCRIPTION
[0022] The present invention provides an advantageous strategy for
enhancing effector function of therapeutic antibodies, particular
anti-tumor, anti-viral, and anti-microbial (bacteria and
unicellular parasites) antibodies, in humans. Enhancing
cytotoxicity elicited by a therapeutic antibody in vivo in a human
comprises disrupting activation of SHIP by Fc-gamma-receptor IIB
(Fc.gamma.RIIB or FcRIIB). In particular, by disrupting therapeutic
antibody binding to the inhibitory Fc receptor FcRIIB while
retaining or enhancing binding to FcRIIA and FcRIIIA, or by
preventing FcRIIB from activating SHIP, the invention significantly
improves antibody efficacy.
[0023] The present invention is based, in part, on recognition that
inhibitory receptors modulate the in vivo cytotoxic response
against tumor targets. Experiments using a variety of syngenic and
xenograft models demonstrated that the inhibitory Fc.gamma.RIIB
molecule is a potent regulator of cytotoxicity in vivo, modulating
the activity of Fc.gamma.RIII on effector cells. While multiple
mechanisms have been proposed to account for the anti-tumor
activities of therapeutic antibodies, engagement of Fc.gamma.Rs on
effector cells is now demonstrated to be a significant component of
the in vivo activity of anti-tumor antibodies.
[0024] Murine monoclonal antibodies as well as the humanized,
clinically effective therapeutics HERCEPTIN.RTM. and RITUXAN.RTM.
engage both activation and inhibitory antibody receptors on myeloid
cells, thus modulating their cytotoxic potential. Mice deficient in
Fc.gamma.RIIB display greatly enhanced antibody-mediated
cytotoxicity; conversely, mice deficient in activating Fc receptors
as well as antibodies engineered to disrupt Fc binding to those
receptors are unable to arrest tumor growth in vivo. These results
demonstrate that FcR-dependent mechanisms significantly contribute
to the action of cytotoxic anti-tumor antibodies and suggest that
an optimal anti-tumor antibody for human therapy binds
preferentially to activation FcRs and minimally to the inhibitory
partner Fc.gamma.RIIB.
[0025] These data substantiate the importance of inhibiting FcRIIB
function to a greater degree than earlier work has done, because it
demonstrates the effects in vivo by measuring the direct effects of
antibody-mediated cytotoxicity on a valid therapeutic model (tumor
cells in transgenic mice). The in vivo results reflect the effects
of multiple receptor interactions of both the activation and
inhibitory classes, i.e., FcRI, FcRIIB, and FcRIII, under
physiological conditions. These results stand in contrast,
therefore, to ADCC data, which measure in vitro antibody activity
mediated by FcRIII engagement. Indeed, the in vivo data were
critical to the discovery that FcRIIB makes a dominant contribution
to antibody-mediated cytotoxicity, and that disrupting FcRIIB
greatly improves cytotoxicity.
DEFINITIONS
[0026] Throughout the present specification and claims, the
numbering of the residues in an immunoglobulin 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. (1991), which is expressly
incorporated herein by reference. The "EU index as in Kabat" refers
to the residue numbering of the human IgG1 EU antibody.
[0027] A "parent polypeptide" is a polypeptide comprising an amino
acid sequence which lacks one or more of the Fc region
modifications disclosed herein and which differs in effector
function compared to a polypeptide variant as herein disclosed. The
parent polypeptide may comprise a native sequence Fc region or an
Fc region with pre-existing amino acid sequence modifications (such
as additions, deletions and/or substitutions).
[0028] The term "Fc region" is used to define a C-terminal region
of an immunoglobulin heavy chain. The "Fc region" may be a native
sequence Fc region or a variant Fc region. Although the boundaries
of the Fc region of an immunoglobulin heavy chain might vary, the
human IgG heavy chain Fc region is usually defined to stretch from
an amino acid residue at position Cys226, or from Pro230, to the
carboxyl-terminus thereof.
[0029] The "CH2 domain" of a human IgG Fc region (also referred to
as "C.gamma.2" domain) usually extends from about amino acid 231 to
about amino acid 340. The CH2 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 molecule. It has been speculated that the
carbohydrate may provide a substitute for the domain-domain pairing
and help stabilize the CH2 domain (Burton, Mol. Immunol., 1985,
22:161-206, which is incorporated herein by reference).
[0030] The "CH3 domain" comprises the stretch of residues
C-terminal to a CH2 domain in an Fc region (i.e., from about amino
acid residue 341 to about amino acid residue 447 of an IgG).
[0031] "Hinge region" is generally defined as stretching from
Glu216 to Pro230 of human IgG1 (Burton, Mol. Immunol., 1985,
supra). 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 bonds in the same positions.
[0032] The "lower hinge region" of an Fc region is normally defined
as the stretch of residues immediately C-terminal to the hinge
region, i.e., residues 233 to 239 of the Fc region. Prior to the
present invention, Fc.gamma.R binding was generally attributed to
amino acid residues in the lower hinge region of an IgG Fc
region.
[0033] The term "binding domain" refers to the region of a
polypeptide that binds to another molecule. In the case of an FcR,
the binding domain can comprise a portion of a polypeptide chain
thereof (e.g., the a chain thereof) which is responsible for
binding an Fc region. One useful binding domain is the
extracellular domain of an FcR chain.
[0034] A "functional Fc region" possesses an "effector function" of
a native sequence Fc region. Exemplary "effector functions" include
C1q binding; complement dependent cytotoxicity; Fc receptor
binding; antibody-dependent cell-mediated cytotoxicity (ADCC);
phagocytosis; down regulation of cell surface receptors (e.g., B
cell receptor; BCR), etc. Such effector functions generally require
the Fc region to be combined with a binding domain (e.g., an
antibody variable domain) and can be assessed using various assays
as herein disclosed, for example.
[0035] A "native sequence Fc region" comprises an amino acid
sequence identical to the amino acid sequence of an Fc region found
in nature. A "variant Fc region" comprises an amino acid sequence
which differs from that of a native sequence Fc region by virtue of
at least one "amino acid modification" as herein defined.
Preferably, the variant Fc region has at least one amino acid
substitution compared to a native sequence Fc region or to the Fc
region of a parent polypeptide, e.g., from about one to about ten
amino acid substitutions, and preferably from about one to about
five amino acid substitutions in a native sequence Fc region or in
the Fc region of the parent polypeptide. The variant Fc region
herein will preferably possess at least about 80% homology with a
native sequence Fc region and/or with an Fc region of a parent
polypeptide, and most preferably at least about 90% homology
therewith, more preferably at least about 95% homology
therewith.
[0036] The term "Fc region-containing polypeptide" refers to a
polypeptide, such as an antibody or immunoadhesin (see definitions
below), which comprises an Fc region.
[0037] The terms "Fc receptor" or "FcR" are used to describe a
receptor that binds to the Fc region of an antibody. The preferred
FcR is a native sequence human FcR. Moreover, a preferred FcR is
one which binds an IgG antibody (a gamma receptor) and includes
receptors of the Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII
subclasses, including allelic variants and alternatively spliced
forms of these receptors. Fc.gamma.RII receptors include
Fc.gamma.RIIA (an "activating receptor") and Fc.gamma.RIIB (an
"inhibiting receptor"), which have similar amino acid sequences
that differ primarily in the cytoplasmic domains thereof.
Activating receptor Fc.gamma.RIIA contains an immunoreceptor
tyrosine-based activation motif (FAM) in its cytoplasmic domain.
Inhibiting receptor Fc.gamma.RIIB contains an immunoreceptor
tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain
(see review in Daeron, Annu. Rev. Immunol., 1997, 15:203-234; FcRs
are reviewed in Ravetch and Kinet, Annu. Rev. Immunol., 1991,
9:457-92; Capel et al., Immunomethods, 1994, 4:25-34; and de Haas
et al., J. Lab. Clin. Med., 1995, 126:330-41, each of which is
incorporated herein by reference).
[0038] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC"
refer to an in vitro cell-mediated reaction in which nonspecific
cytotoxic cells that express FcRs (e.g., monocytic cells such as
Natural Killer (NK) cells and macrophages) recognize bound antibody
on a target cell and subsequently cause lysis of the target cell.
In principle, any effector cell with an activating Fc.gamma.R can
be triggered to mediate ADCC. The primary cells for mediating ADCC,
NK cells, express Fc.gamma.RIII only, whereas monocytes, depending
on their state of activation, localization, or differentation, can
express Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII. FcR
expression on hematopoietic cells is summarized in Ravetch and
Kinet, Annu. Rev. Immunol., 1991, 9:457-92, each of which is
incorporated herein by reference.
[0039] "Human effector cells" are leukocytes which express one or
more FcRs and perform effector functions. Preferably, the cells
express at least Fc.gamma.RIII and perform ADCC effector function.
Examples of human leukocytes which mediate ADCC include peripheral
blood mononuclear cells (PBMC), natural killer (NK) cells,
monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK
cells being preferred. The effector cells may be isolated from a
native source thereof, e.g., from blood or PBMCs as described
herein.
Antibodies
[0040] The term "antibody" is used in the broadest sense and
specifically covers monoclonal antibodies (including full length
monoclonal antibodies), polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies), and antibody fragments so
long as they exhibit the desired biological activity.
[0041] "Antibody fragments", as defined for the purpose of the
present invention, comprise a portion of an intact antibody,
generally including the antigen binding or variable region of the
intact antibody or the Fc region of an antibody which retains FcR
binding capability. Examples of antibody fragments include linear
antibodies; single-chain antibody molecules; and multispecific
antibodies formed from antibody fragments. The antibody fragments
preferably retain at least part of the hinge and optionally the CHI
region of an IgG heavy chain. More preferably, the antibody
fragments retain the entire constant region of an IgG heavy chain,
and include an IgG light chain.
[0042] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to conventional
(polyclonal) antibody preparations that typically include different
antibodies directed against different determinants (epitopes), each
monoclonal antibody is directed against a single determinant on the
antigen. The modifier "monoclonal" indicates the character of the
antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by the hybridoma method first described by
Kohler and Milstein, Nature, 1975, 256:495-497, which is
incorporated herein by reference, or may be made by recombinant DNA
methods (see, e.g., U.S. Pat. No. No. 30 4,816,567, which is
incorporated herein by reference). The "monoclonal antibodies" may
also be isolated from phage antibody libraries using the techniques
described in Clackson et al., Nature, 1991, 352:624-628 and Marks
et al., J. Mol. Biol, 1991, 222:581-597, for example, each of which
is incorporated herein by reference.
[0043] The monoclonal antibodies herein specifically include
"chimeric" antibodies (immunoglobulins) in which a portion of the
heavy and/or light chain is identical with or homologous to
corresponding sequences in antibodies derived from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the chain(s) is identical with or homologous
to corresponding sequences in antibodies derived from another
species or belonging to another antibody class or subclass, as well
as fragments of such antibodies, so long as they exhibit the
desired biological activity (see U.S. Pat. No. 4,816,567; Morrison
et al, Proc. Natl. Acad. Sci. USA, 1984, 81:6851-6855; Neuberger et
al., Nature, 1984, 312:604-608; Takeda et al., Nature, 1985,
314:452-454; International Patent Application No. PCT/GB85/00392,
each of which is incorporated herein by reference).
[0044] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a hypervariable region of the recipient are replaced by
residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity, and capacity. In some instances, Fv
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FR
residues are those of a human immunoglobulin sequence. The
humanized antibody optionally also will comprise at least a portion
of an immunoglobulin constant region (Fc), typically that of a
human immunoglobulin. For further details, see Jones et al.,
Nature, 1986, 321:522-525; Riechmann et al., Nature, 1988,
332:323-329; Presta, Curr. Op. Struct. Biol., 1992, 2:593-596; U.S.
Pat. No. 5,225,539, each of which is incorporated herein by
reference.
Inhibition by a Competitive Inhibitor
[0045] The present invention contemplates administering an
effective amount of a competitive inhibitor that binds specifically
to FcRIIB, without activating it, and thus prevents binding by a
tumor specific antibody. For example, binding of monomeric
molecules to FcRIIB prevents crosslinking of the receptor, which is
required for activation.
[0046] Various competitive inhibitors can be used in the practice
of this invention, including but by no means limited to anti-FcRIIB
antibodies (preferably Fv antibodies to preclude development of a
cytotoxic response) and peptides corresponding to the
FcRIIB-binding sequence of immunoglobulins.
[0047] Small molecular weight competitive inhibitors of the FcRIIB
binding site are effective at preventing the binding of cytotoxic
antibodies to the inhibitory Fc receptor. Other targets for
preventing activation of the inhibitory receptor include the
dominant signaling molecule, SHIP. SHIP, an inositol polyphosphate
5-phosphatase, is essential for the biological activity of FcRIIB
(Ono et al, Nature, 1996, 383:263; Ono et al., Cell, 1997, 90:293;
Bolland et al., Immunity, 1998, 8:509, each of which is
incorporated herein by reference). Competitive inhibitors of the
inositol phosphatase activity of SHIP will abrogate the inhibitory
activity of FcRIIB and thereby amplify the effective cytotoxic
activity of IgG antibodies. The competitive inhibitors can include
antibodies as well as small molecular weight antagonists.
Antibodies with Modified FcRIIB Binding Site
[0048] In a preferred embodiment, antibody binding is inhibited by
modifying the Fc portion of the antibody to reduce its affinity for
Fc.gamma.RIIB, thus creating an antibody variant. A number of
references describe techniques for modifying Fc portions to
modulate binding affinity for FcRs (see PCT Publication Nos. WO
99/58572, WO 99/51642, WO 98/23289, WO 89/07142, WO 88/07089; U.S.
Pat. Nos. 5,834,597 and 5,624,821, each of which is incorporated
herein by reference).
[0049] An antibody variant with "altered" FcR binding affinity is
one which has diminished Fc.gamma.RIIB binding activity and
enhanced cytotoxicity compared to a parent polypeptide or to a
polypeptide comprising a native sequence Fc region.
[0050] The antibody variant which "mediates antibody-mediated
cytotoxicity in the presence of human effector cells more
effectively" than a parent antibody is one which in vitro or in
vivo is substantially more effective at mediating cytotoxicity,
when the amounts of antibody preferred variant is from about
1.5-fold to about 100-fold, e.g., from about two-fold to about
fifty-fold, more effective at mediating cytotoxicity than the
parent, e.g., in one or more of the in vivo assays disclosed
herein.
[0051] An "amino acid modification" refers to a change in the amino
acid sequence of a predetermined amino acid sequence. Exemplary
modifications include an amino acid substitution, insertion and/or
deletion. The preferred amino acid modification herein is a
substitution.
[0052] An "amino acid modification at" a specified position, e.g.,
of the Fc region, refers to the substitution or deletion of the
specified residue, or the insertion of at least one amino acid
residue adjacent the specified residue. By insertion "adjacent" a
specified residue is meant insertion within one to two residues
thereof. The insertion may be N-terminal or C-terminal to the
specified residue.
[0053] An "amino acid substitution" refers to the replacement of at
least one existing amino acid residue in a predetermined amino acid
sequence with another different "replacement" amino acid residue.
The replacement residue or residues may be "naturally occurring
amino acid residues" (i.e., encoded by the genetic code) and
selected from the group consisting of: alanine (Ala); arginine
(Arg); asparagine (Asn); aspartic acid (Asp); cysteine (Cys);
glutamine (Gln); glutamic acid (Glu); glycine (Gly); histidine
(His); isoleucine (Ile); leucine (Leu); lysine (Lys); methionine
(Met); phenylalanine (Phe); proline (Pro); serine (Ser); threonine
(Thr); tryptophan (Trp); tyrosine (Tyr); and valine (Val).
Substitution with one or more non-naturally occurring amino acid
residues is also encompassed by the definition of an amino acid
substitution herein. A "non-naturally occurring amino acid residue"
refers to a residue, other than those naturally occurring amino
acid residues listed above, which is able to covalently bind
adjacent amino acid residues(s) in a polypeptide chain. Examples of
non-naturally occurring amino acid residues include norleucine,
ornithine, norvaline, homoserine and other amino acid residue
analogues such as those described in Ellman et al., Meth. Enzym.,
1991, 202:301-336, which is incorporated herein by reference. To
generate such non-naturally occurring amino acid residues, the
procedures of Noren et al., Science, 1989, 244:182, which is
incorporated herein by reference, and Ellman et al., supra, can be
used. Briefly, these procedures involve chemically activating a
suppressor tRNA with a non-naturally occurring amino acid residue
followed by in vitro transcription and translation of the RNA.
[0054] An "amino acid insertion" refers to the incorporation of at
least one amino acid into a predetermined amino acid sequence.
While the insertion will usually consist of the insertion of one or
two amino acid residues, the present application contemplates
larger "peptide insertions", e.g., insertion of about three to
about five or even up to about ten amino acid residues. The
inserted residue(s) may be naturally occurring or non-naturally
occurring as disclosed above.
[0055] An "amino acid deletion" refers to the removal of at least
one amino acid residue from a predetermined amino acid
sequence.
[0056] In a specific embodiment, a modified antibody variant of the
invention has reduced affinity for FcRIIB, but unchanged, or even
enhanced, affinity for the stimulatory FcRs, FcRI and FcRIII.
[0057] In general, generation of these modified Fc domains involves
the expression of a library of mutagenized dimeric IgG Fc domains
in a compatible host, such as a yeast or a mammalian cell, and the
screening of these surface expressed Fc domains with specific Fc
receptors by either solid-phase or solution binding. Modified Fc
domains with reduced binding to FcRIIB are identified in this
manner.
Inhibiting Expression of Fc.gamma.RIIB or SHIP
[0058] As discussed above, one method for enhancing tumor-specific
(or any) antibody-mediated cytotoxicity involves inhibiting the
expression of either the inhibitory Fc receptor, Fc.gamma.RIIB, or
the molecule that mediates signal transduction by this receptor,
SHIP. There are numerous techniques for inhibiting expression of a
target protein, including antisense and intracellular antibodies.
The nucleic acids encoding these targets, and the proteins
themselves, are well known (Brooks et al, J. Exp. Med., 1989,
170:1369; Damein, Proc. Natl. Acad. Sci. USA 1996, 93:1689;
Kavanaugh et al, Curr. Biol. 1996, 6:438, each of which is
incorporated herein by reference).
[0059] An "antisense nucleic acid" is a single stranded nucleic
acid molecule which, on hybridizing under cytoplasmic conditions
with complementary bases in an RNA or DNA molecule, inhibits the
latter's role. If the RNA is a messenger RNA transcript, the
antisense nucleic acid is a countertranscript or mRNA-interfering
complementary nucleic acid. As presently used, "antisense" broadly
includes RNA-RNA interactions, RNA-DNA interactions, ribozymes and
RNase-H mediated arrest. Antisense nucleic acid molecules can be
encoded by a recombinant gene for expression in a cell (e.g., U.S.
Pat. No. 5,814,500; U.S. Pat. No. 5,811,234, each of which is
incorporated herein by reference), or alternatively they can be
prepared synthetically (e.g., U.S. Pat. No. 5,780,607, which is
incorporated herein by reference). There are numerous examples of
the use of antisense nucleic acids to suppress gene expression (see
U.S. Pat. Nos. 5,773,231 and 5,576,208; Hanna et al, J. Vasc. Surg,
2000, 31:770-780; Han et al, Am. J. Physiol. Renal Physiol., 2000,
278:F628-F634; Prati et al, Biotechnol. Bioeng., 2000, 68:239-244;
Yang et al, Clin. Cancer Res., 2000, 6:1024-30; Yang et al., Clin.
Cancer Res., 2000, 6:773-81; Mack and Robitzki, Prog. Neurobiol.,
2000, 60:602-28, each of which is incorporated herein by
reference).
[0060] Specific non-limiting examples of synthetic oligonucleotides
envisioned for this invention include oligonucleotides that contain
phosphorothioates, phosphotriesters, methyl phosphonates, short
chain alkyl, or cycloalkl intersugar linkages or short chain
heteroatomic or heterocyclic intersugar linkages. Most preferred
are those with CH.sub.2--NH--O--CH.sub.2,
CH.sub.2--N(CH.sub.3)--O--CH.sub.2,
CH.sub.2--O--N(CH.sub.3)--CH.sub.2,
CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2 and
O--N(CH.sub.3)--CH.sub.2--CH.sub.2 backbones (where phosphodiester
is O--PO.sub.2--O--CH.sub.2). U.S. Pat. No. 5,677,437, which is
incorporated herein by reference, describes heteroaromatic
olignucleoside linkages. Nitrogen linkers or groups containing
nitrogen can also be used to prepare oligonucleotide mimics (U.S.
Pat. No. 5,792,844 and No. 5,783,682, each of which is incorporated
herein by reference). U.S. Pat. No. 5,637,684, which is
incorporated herein by reference, describes phosphoramidate and
phosphorothioamidate oligomeric compounds. Also envisioned are
oligonucleotides having morpholino backbone structures (U.S. Pat.
No. 5,034,506, which is incorporated herein by reference). In other
embodiments, such as the peptide-nucleic acid (PNA) backbone, the
phosphodiester backbone of the oligonucleotide may be replaced with
a polyamide backbone, the bases being bound directly or indirectly
to the aza nitrogen atoms of the polyamide backbone (Nielsen et ah,
Science 254:1497, 1991, which is incorporated herein by reference).
Other synthetic oligonucleotides may contain substituted sugar
moieties comprising one of the following at the 2' position: OH,
SH, SCH.sub.3, F, OCN, O(CH.sub.2).sub.nNH.sub.2 or
O(CH.sub.2).sub.nCH.sub.3 where n is from 1 to about 10; C.sub.1 to
C.sub.10 lower alkyl, substituted lower alkyl, alkaryl or aralkyl;
Cl; Br; CN; CF.sub.3; OCF.sub.3; O--; S--, or N-alkyl; O-, S-, or
N-alkenyl; SOCH.sub.3; SO.sub.2CH.sub.3; ONO.sub.2; NO.sub.2;
N.sub.3; NH.sub.2; heterocycloalkyl; heterocycloalkaryl;
aminoalkylamino; polyalkylamino; substituted silyl; a fluorescein
moiety; an RNA cleaving group; a reporter group; an intercalator; a
group for improving the pharmacokinetic properties of an
oligonucleotide; or a group for improving the pharmacodynamic
properties of an oligonucleotide, and other substituents having
similar properties. Oligonucleotides may also have sugar mimetics
such as cyclobutyls or other carbocyclics in place of the
pentofuranosyl group. Nucleotide units having nucleosides other
than adenosine, cytidine, guanosine, thymidine and uridine, such as
inosine, may be used in an oligonucleotide molecule.
[0061] Intracellular antibodies are also effective at inhibiting
protein expression or function. Intracellular antibodies are
typically single chain Fv constructs (Bird, Science, 1988,
242:423-426; Huston et al., Proc. Natl. Acad. Sci. USA, 1988,
85:5879-5883; Ward et al., Nature, 1989, 334:544-546; U.S. Pat.
Nos. 5,476,786, 5,132,405, and 4,946,778; Huse et al., Science
246:1275-1281, 1989, each of which is incorporated herein by
reference). A number of studies report on their effectiveness at
inhibiting intracellular protein function (Richardson et al., Gene
Ther., 1998, 5:635-44; Marasco et al., Hum. Gene Ther., 1998,
9:1627-42; Cochet et al., Cancer Res., 1998, 58:1170-6; Curiel,
Adv. Pharmacol., 1997, 40:51-84, each of which is incorporated
herein by reference).
Inhibiting Signal Transduction
[0062] In another embodiment, SHIP activation is inhibited by an
inositol phosphatase inhibitor. The inositol polyphosphate
5-phosphatase activity of SHIP is both necessary and sufficient for
transducing the inhibitory signal of FcRIIB (Ono et al., 1997,
supra; Bolland et al., 1998, supra). It is uniquely associated with
FcRIIB upon its phosphorylation by crosslinking to an
ITAM-containing receptor, such as FcRIIA or FcRIIIA, as occurs in
vivo when a cytotoxic antibody engages surface FcRs on macrophages,
mast cells, neutrophils, or monocytes. Mutation of the phosphatase
activity of SHIP inactivates FcRIIB, preventing inhibitory
signaling and thereby acting to amplify the in vivo effect of
cytotoxic antibody engagement of activation FcRs. While numerous
classes of inositol phosphatases are known to exist, the
5-phosphatase activity of SHIP is distinctive, permitting the
generation of SHIP-specific inhibitors. SHIP is expressed widely in
hematopoeitic cells and is implicated in signaling from a variety
of growth factor receptors, although the contribution of the
phosphatase activity to those activities is not yet established. In
any event, an inhibitor of SHIP phosphatase activity or recruitment
to the phosphorylated FcRIIB ITIM motif will abrogate inhibitory
signaling in the effected cell.
Recombinant Technology
[0063] In accordance with the present invention there may be
employed conventional molecular biology, microbiology, and
recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. See, e.g.,
Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory
Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (herein "Sambrook et al, 1989"); DNA
Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed.
1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic
Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1985);
Transcription And Translation (B. D. Hames & S. J. Higgins,
eds. 1984); Animal Cell Culture (R. I. Freshney, ed. 1986);
Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A
Practical Guide To Molecular Cloning (1984); F. M. Ausubel et al.
(eds.), Current Protocols in Molecular Biology, John Wiley &
Sons, Inc. (1994), each of which is incorporated herein by
reference.
Molecular Biology--Definitions
[0064] The term "host cell" means any cell of any organism that is
selected, modified, transformed, grown, or used or manipulated in
any way, for the production of a substance by the cell, for example
the expression by the cell of a gene, a DNA or RNA sequence, a
protein or an enzyme. Host cells can further be used for screening
or other assays, as described infra.
[0065] Proteins and enzymes are made in the host cell using
instructions in DNA and RNA, according to the genetic code.
Generally, a DNA sequence having instructions for a particular
protein or enzyme is "transcribed" into a corresponding sequence of
RNA. The RNA sequence in turn is "translated" into the sequence of
amino acids which form the protein or enzyme. An "amino acid
sequence" is any chain of two or more amino acids. Each amino acid
is represented in DNA or RNA by one or more triplets of
nucleotides. Each triplet forms a codon, corresponding to an amino
acid. For example, the amino acid lysine (Lys) can be coded by the
nucleotide triplet or codon AAA or by the codon AAG. (The genetic
code has some redundancy, also called degeneracy, meaning that most
amino acids have more than one corresponding codon.) Because the
nucleotides in DNA and RNA sequences are read in groups of three
for protein production, it is important to begin reading the
sequence at the correct amino acid, so that the correct triplets
are read. The way that a nucleotide sequence is grouped into codons
is called the "reading frame."
[0066] A "coding sequence" or a sequence "encoding" an expression
product, such as a RNA, polypeptide, protein, or enzyme, is a
nucleotide sequence that, when expressed, results in the production
of that RNA, polypeptide, protein, or enzyme, i.e., the nucleotide
sequence encodes an amino acid sequence for that polypeptide,
protein or enzyme. A coding sequence for a protein may include a
start codon (usually ATG) and a stop codon.
[0067] The term "gene", also called a "structural gene" means a DNA
sequence that codes for or corresponds to a particular sequence of
amino acids which comprise all or part of one or more proteins or
enzymes, and may or may not include regulatory DNA sequences, such
as promoter sequences, which determine for example the conditions
under which the gene is expressed. Some genes, which are not
structural genes, may be transcribed from DNA to RNA, but are not
translated into an amino acid sequence. Other genes may function as
regulators of structural genes or as regulators of DNA
transcription.
[0068] A "promoter sequence" is a DNA regulatory region capable of
binding RNA polymerase in a cell and initiating transcription of a
downstream (3' direction) coding sequence. For purposes of defining
the present invention, the promoter sequence is bounded at its 3'
terminus by the transcription initiation site and extends upstream
(5' direction) to include the minimum number of bases or elements
necessary to initiate transcription at levels detectable above
background. Within the promoter sequence will be found a
transcription initiation site (conveniently defined for example, by
mapping with nuclease SI), as well as protein binding domains
(consensus sequences) responsible for the binding of RNA
polymerase.
[0069] A coding sequence is "under the control" or "operatively
associated with" of transcriptional and translational control
sequences in a cell when RNA polymerase transcribes the coding
sequence into mRNA, which is then trans-RNA spliced (if it contains
introns) and translated into the protein encoded by the coding
sequence.
[0070] The terms "express" and "expression" mean allowing or
causing the information in a gene or DNA sequence to become
manifest, for example producing a protein by activating the
cellular functions involved in transcription and translation of a
corresponding gene or DNA sequence. A DNA sequence is expressed in
or by a cell to form an "expression product" such as a protein. The
expression product itself, e.g., the resulting protein, may also be
said to be "expressed" by the cell. An expression product can be
characterized as intracellular, extracellular or secreted. The term
"intracellular" means something that is inside a cell. The term
"extracellular" means something that is outside a cell. A substance
is "secreted" by a cell if it appears in significant measure
outside the cell, from somewhere on or inside the cell.
[0071] The term "transfection" means the introduction of a foreign
nucleic acid into a cell. The term "transformation" means the
introduction of a "foreign" (i.e., extrinsic or extracellular)
gene, DNA or RNA sequence to a host cell, so that the host cell
will express the introduced gene or sequence to produce a desired
substance, typically a protein or enzyme coded by the introduced
gene or sequence. The introduced gene or sequence may also be
called a "cloned" or "foreign" gene or sequence, may include
regulatory or control sequences, such as start, stop, promoter,
signal, secretion, or other sequences used by a cell's genetic
machinery. The gene or sequence may include nonfunctional sequences
or sequences with no known function. A host cell that receives and
expresses introduced DNA or RNA has been "transformed" and is a
"transformant" or a "clone." The DNA or RNA introduced to a host
cell can come from any source, including cells of the same genus or
species as the host cell, or cells of a different genus or
species.
[0072] The terms "vector", "cloning vector" and "expression vector"
mean the vehicle by which a DNA or RNA sequence (e.g., a foreign
gene) can be introduced into a host cell, so as to transform the
host and promote expression (e.g., transcription and translation)
of the introduced sequence. Vectors include plasmids, phages,
viruses, etc.; they are discussed in greater detail below.
[0073] Vectors typically comprise the DNA of a transmissible agent,
into which foreign DNA is inserted. A common way to insert one
segment of DNA into another segment of DNA involves the use of
enzymes called restriction enzymes that cleave DNA at specific
sites (specific groups of nucleotides) called restriction sites. A
"cassette" refers to a DNA coding sequence or segment of DNA that
codes for an expression product that can be inserted into a vector
at defined restriction sites. The cassette restriction sites are
designed to ensure insertion of the cassette in the proper reading
frame. Generally, foreign DNA is inserted at one or more
restriction sites of the vector DNA, and then is carried by the
vector into a host cell along with the transmissible vector DNA. A
segment or sequence of DNA having inserted or added DNA, such as an
expression vector, can also be called a "DNA construct." A common
type of vector is a "plasmid", which generally is a self-contained
molecule of double-stranded DNA, usually of bacterial origin, that
can readily accept additional (foreign) DNA and which can readily
be introduced into a suitable host cell. A plasmid vector often
contains coding DNA and promoter DNA and has one or more
restriction sites suitable for inserting foreign DNA. Coding DNA is
a DNA sequence that encodes a particular amino acid sequence for a
particular protein or enzyme. Promoter DNA is a DNA sequence which
initiates, regulates, or otherwise mediates or controls the
expression of the coding DNA. Promoter DNA and coding DNA may be
from the same gene or from different genes, and may be from the
same or different organisms. A large number of vectors, including
plasmid and fungal vectors, have been described for replication
and/or expression in a variety of eukaryotic and prokaryotic hosts.
Non-limiting examples include pKK plasmids (CLONTECH, Palo Alto,
Calif.), pUC plasmids, pET plasmids (Novagen, Inc., Madison, Wis.),
pRSET or pREP plasmids (Invitrogen, San Diego, Calif.), or pMAL
plasmids (New England BioLabs, Beverly, Mass.), and many
appropriate host cells, using methods disclosed or cited herein or
otherwise known to those skilled in the relevant art. Recombinant
cloning vectors will often include one or more replication systems
for cloning or expression, one or more markers for selection in the
host, e.g., antibiotic resistance, and one or more expression
cassettes.
[0074] Preferred vectors, particularly for cellular assays in vitro
and in vivo, are viral vectors, such as lentiviruses, retroviruses,
herpes viruses, adenoviruses, adeno-associated viruses, vaccinia
virus, baculovirus, and other recombinant viruses with desirable
cellular tropism. Thus, a gene encoding a functional or mutant
protein or polypeptide domain fragment thereof can be introduced in
vivo, ex vivo, or in vitro using a viral vector or through direct
introduction of DNA. Expression in targeted tissues can be effected
by targeting the transgenic vector to specific cells, such as with
a viral vector or a receptor ligand, or by using a tissue-specific
promoter, or both. Targeted gene delivery is described in
International Patent Publication WO 95/28494, published October
1995, which is incorporated herein by reference.
[0075] Viral vectors commonly used for in vivo or ex vivo targeting
and therapy procedures are DNA-based vectors and retroviral
vectors. Methods for constructing and using viral vectors are known
in the art (see, e.g., Miller and Rosman, BioTechniques, 1992,
7:980-990, which is incorporated herein by reference). Preferably,
the viral vectors are replication defective, that is, they are
unable to replicate autonomously in the target cell. Preferably,
the replication defective virus is a minimal virus, i.e., it
retains only the sequences of its genome which are necessary for
encapsidating the genome to produce viral particles.
[0076] DNA viral vectors include an attenuated or defective DNA
virus, such as, but not limited to, herpes simplex virus (HSV),
papillomavirus, Epstein Barr virus (EBV), adenovirus,
adeno-associated virus (AAV), and the like. Defective viruses,
which entirely or almost entirely lack viral genes, are preferred.
Defective virus is not infective after introduction into a cell.
Use of defective viral vectors allows for administration to cells
in a specific, localized area, without concern that the vector can
infect other cells. Thus, a specific tissue can be specifically
targeted. Examples of particular vectors include, but are not
limited to, a defective herpes virus 1 (HSV1) vector (Kaplitt et
al, Mol. Cell. Neurosci., 1991, 2:320-330, which is incorporated
herein by reference), defective herpes virus vector lacking a
glyco-protein L gene (Patent Publication RD 371005 A, which is
incorporated herein by reference), or other defective herpes virus
vectors (International Patent Publication No. WO 94/21807,
published Sep. 29, 1994; International Patent Publication No. WO
92/05263, published Apr. 2, 1994, each of which is incorporated
herein by reference); an attenuated adenovirus vector, such as the
vector described by Stratford-Perricaudet et al., J. Clin. Invest.,
1992, 90:626-630, which is incorporated herein by reference; see
also La Salle et al., Science, 1993, 259:988-990, which is
incorporated herein by reference); and a defective adeno-associated
virus vector (Samulski et al., J. Virol., 1987, 61:3096-3101;
Samulski et al., J. Virol., 1989, 63:3822-3828; Lebkowski et al.,
Mol. Cell. Biol., 1988, 8:3988-3996, each of which is incorporated
herein by reference).
[0077] Various companies produce viral vectors commercially,
including, but by no means limited to, Avigen, Inc. (Alameda,
Calif.; AAV vectors), Cell Genesys (Foster City, Calif.;
retroviral, adenoviral, AAV vectors, and lentiviral vectors),
CLONTECH (Palo Alto, Calif.; retroviral and baculoviral vectors),
Genovo, Inc. (Sharon Hill, Pa.; adenoviral and AAV vectors), Genvec
(Gaithersburg, Md.; adenoviral vectors), IntroGene (Leiden,
Netherlands; adenoviral vectors), Molecular Medicine (retroviral,
adenoviral, AAV, and herpes viral vectors), Norgen (Ontario,
Canada; adenoviral vectors), Oxford BioMedica (Oxford, United
Kingdom; lentiviral vectors), and Transgene (Strasbourg, France;
adenoviral, vaccinia, retroviral, and lentiviral vectors).
[0078] Non-viral vectors can be introduced by lipofection, as naked
DNA, or with other transfection facilitating agents (peptides,
polymers, etc.). Synthetic cationic lipids can be used to prepare
liposomes for transfection of a gene encoding a marker (Felgner, et
al., Proc. Natl. Acad. Sci. U.S.A., 1987, 84:7413-7417; Felgner and
Ringold, Science, 1989, 337:387-388; Mackey et al, Proc. Natl.
Acad. Sci. U.S.A., 1988, 85:8027-8031; Ulmer et al., Science, 1993,
259:1745-1748, each of which is incorporated herein by reference).
Useful lipid compounds and almost entirely lack viral genes, are
preferred. Defective virus is not infective after introduction into
a cell. Use of defective viral vectors allows for administration to
cells in a specific, localized area, without concern that the
vector can infect other cells. Thus, a specific tissue can be
specifically targeted. Examples of particular vectors include, but
are not limited to, a defective herpes virus 1 (HSV1) vector
(Kaplitt et al, Mol. Cell. Neurosci., 1991, 2:320-330, which is
incorporated herein by reference), defective herpes virus vector
lacking a glyco-protein L gene (Patent Publication RD 3 71005 A,
which is incorporated herein by reference), or other defective
herpes virus vectors (International Patent Publication No. WO
94/21807, published Sep. 29, 1994; International Patent Publication
No. WO 92/05263, published Apr. 2, 1994, each of which is
incorporated herein by reference); an attenuated adenovirus vector,
such as the vector described by Stratford-Perricaudet et al, J.
Clin. Invest., 1992, 90:626-630, which is incorporated herein by
reference; see also La Salle et al., Science, 1993, 259:988-990,
which is incorporated herein by reference); and a defective
adeno-associated virus vector (Samulski et al., J. Virol., 1987,
61:3096-3101; Samulski et al, J. Virol., 1989, 63:3822-3828;
Lebkowski et al., Mol. Cell. Biol., 1988, 8:3988-3996, each of
which is incorporated herein by reference).
[0079] Various companies produce viral vectors commercially,
including, but by no means limited to, Avigen, Inc. (Alameda, C A;
AAV vectors), Cell Genesys (Foster City, Calif.; retroviral,
adenoviral, AAV vectors, and lentiviral vectors), CLONTECH (Palo
Alto, Calif.; retroviral and baculoviral vectors), Genovo, Inc.
(Sharon Hill, Pa.; adenoviral and AAV vectors), Genvec
(Gaithersburg, Md.; adenoviral vectors), IntroGene (Leiden,
Netherlands; adenoviral vectors), Molecular Medicine (retroviral,
adenoviral, AAV, and herpes viral vectors), Norgen (Ontario,
Canada; adenoviral vectors), Oxford BioMedica (Oxford, United
Kingdom; lentiviral vectors), and Transgene (Strasbourg, France;
adenoviral, vaccinia, retroviral, and lentiviral vectors).
[0080] Non-viral vectors can be introduced by lipofection, as naked
DNA, or with other transfection facilitating agents (peptides,
polymers, etc.). Synthetic cationic lipids can be used to prepare
liposomes for transfection of a gene encoding a marker (Felgner, et
al, Proc. Natl. Acad. Sci. U.S.A., 1987, 84:7413-7417; Felgner and
Ringold, Science, 1989, 337:387-388; Mackey et al, Proc. Natl.
Acad. Sci. U.S.A., 1988, 85:8027-8031; Ulmer et al, Science, 1993,
259:1745-1748, each of which is incorporated herein by reference).
Useful lipid compounds and compositions for transfer of nucleic
acids are described in International Patent Publications WO95/18863
and WO96/17823, and in U.S. Pat. No. 5,459,127, each of which is
incorporated herein by reference. Lipids may be chemically coupled
to other molecules for the purpose of targeting (see Mackey et al.,
supra). Targeted peptides, e.g., hormones or neurotransmitters, and
proteins such as antibodies, or non-peptide molecules could be
coupled to liposomes chemically. Other molecules are also useful
for facilitating transfection of a nucleic acid in vivo, such as a
cationic oligopeptide (e.g., International Patent Publication
WO95/21931, which is incorporated herein by reference), peptides
derived from DNA binding proteins (e.g., International Patent
Publication WO96/25508, which is incorporated herein by reference),
or a cationic polymer (e.g., International Patent Publication
WO95/21931, which is incorporated herein by reference). It is also
possible to introduce the vector in vivo as a naked DNA plasmid.
Naked DNA vectors for gene therapy can be introduced into the
desired host cells by methods known in the art, e.g.,
electroporation, microinjection, cell fusion, DEAE dextran, calcium
phosphate precipitation, use of a gene gun, or use of a DNA vector
transporter (see, e.g., Wu et al., J. Biol. Chem., 1992,
267:963-967; Wu and Wu, J. Biol. Chem., 1988, 263:14621-14624;
Hartmut et al., Canadian Patent Application No. 2,012,311, filed
Mar. 15, 1990; Williams et al., Proc. Natl. Acad. Sci. USA, 1991,
88:2726-2730, each of which is incorporated herein by reference).
Receptor-mediated DNA delivery approaches can also be used (Curiel
et al., Hum. Gene Ther., 1992, 3:147-154; Wu and Wu, J. Biol.
Chem., 1987, 262:4429-4432, each of which is incorporated herein by
reference). U.S. Pat. Nos. 5,580,859 and 5,589,466, each of which
is incorporated herein by reference, disclose delivery of exogenous
DNA sequences, free of transfection facilitating agents, in a
mammal. Recently, a relatively low voltage, high efficiency in vivo
DNA transfer technique, termed electrotransfer, has been described
(Mir et al., C.P. Acad. Sci., 1998, 321:893; WO 99/01157; WO
99/01158; WO 99/01175, each of which is incorporated herein by
reference).
[0081] The term "expression system" means a host cell and
compatible vector under suitable conditions, e.g., for the
expression of a protein coded for by foreign DNA carried by the
vector and introduced to the host cell. Common expression systems
include E. coli host cells and plasmid vectors, insect host cells
and Baculovirus vectors, and mammalian host cells and vectors. In a
specific embodiment, the protein of interest is expressed in COS-1
or C.sub.2C.sub.12 cells. Other suitable cells include CHO cells,
HeLa cells, 293T (human kidney cells), mouse primary myoblasts, and
NIH 3T3 cells.
[0082] The term "heterologous" refers to a combination of elements
not naturally occurring. For example, heterologous DNA refers to
DNA not naturally located in the cell, or in a chromosomal site of
the cell. Preferably, the heterologous DNA includes a gene foreign
to the cell. A heterologous expression regulatory element is a such
an element operatively associated with a different gene than the
one it is operatively associated with in nature. In the context of
the present invention, a gene encoding a protein of interest is
heterologous to the vector DNA in which it is inserted for cloning
or expression, and it is heterologous to a host cell containing
such a vector, in which it is expressed, e.g., a CHO cell.
[0083] A nucleic acid molecule is "hybridizable" to another nucleic
acid molecule, such as a cDNA, genomic DNA, or RNA, when a single
stranded form of the nucleic acid molecule can anneal to the other
nucleic acid molecule under the appropriate conditions of
temperature and solution ionic strength (see Sambrook et al.,
supra). The conditions of temperature and ionic strength determine
the "stringency" of the hybridization. For preliminary screening
for homologous nucleic acids, low stringency hybridization
conditions, corresponding to a T.sub.m (melting temperature) of
55.degree. C., can be used, e.g., 5.times.SSC, 0.1% SDS, 0.25%
milk, and no formamide; or 30% formamide, 5.times.SSC, 0.5% SDS).
Moderate stringency hybridization conditions correspond to a higher
T.sub.m, e.g., 40% formamide, with 5.times. or 6.times.SCC. High
stringency hybridization conditions correspond to the highest
T.sub.m, e.g., 50% formamide, 5.times. or 6.times.SCC. SCC is a
0.15M NaCl, 0.015M Na-citrate. Hybridization requires that the two
nucleic acids contain complementary sequences, although depending
on the stringency of the hybridization, mismatches between bases
are possible. The appropriate stringency for hybridizing nucleic
acids depends on the length of the nucleic acids and the degree of
complementation, variables well known in the art. The greater the
degree of similarity or homology between two nucleotide sequences,
the greater the value of T.sub.m for hybrids of nucleic acids
having those sequences. The relative stability (corresponding to
higher T.sub.m) of nucleic acid hybridizations decreases in the
following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater
than 100 nucleotides in length, equations for calculating T.sub.m
have been derived (see Sambrook et al., supra, 9.50-9.51). For
hybridization with shorter nucleic acids, i.e., oligonucleotides,
the position of mismatches becomes more important, and the length
of the oligonucleotide determines its specificity (see Sambrook et
al., supra, 11.7-11.8). A minimum length for a hybridizable nucleic
acid is at least about 10 nucleotides; preferably at least about 15
nucleotides; and more preferably the length is at least about 20
nucleotides.
[0084] In a specific embodiment, the term "standard hybridization
conditions" refers to a T.sub.m of 55.degree. C., and utilizes
conditions as set forth above. In a preferred embodiment, the
T.sub.m is 60.degree. C.; in a more preferred embodiment, the
T.sub.m is 65.degree. C. In a specific embodiment, "high
stringency" refers to hybridization and/or washing conditions at
68.degree. C. in 0.2.times.SSC, at 42.degree. C. in 50% formamide,
4.times.SSC, or under conditions that afford levels of
hybridization equivalent to those observed under either of these
two conditions.
[0085] As used herein, the term "oligonucleotide" refers to a
nucleic acid, generally of at least 10, preferably at least 15, and
more preferably at least 20 nucleotides, preferably no more than
100 nucleotides, that is hybridizable to a genomic DNA molecule, a
cDNA molecule, or an mRNA molecule encoding a gene, mRNA, cDNA, or
other nucleic acid of interest. Oligonucleotides can be labeled,
e.g., with .sup.32P-nucleotides or nucleotides to which a label,
such as biotin, has been covalently conjugated. In one embodiment,
a labeled oligonucleotide can be used as a probe to detect the
presence of a nucleic acid. In another embodiment, oligonucleotides
(one or both of which may be labeled) can be used as PCR primers,
either for cloning full length or a fragment of the gene, or to
detect the presence of nucleic acids encoding the protein. In a
further embodiment, an oligonucleotide of the invention can form a
triple helix with a DNA molecule. Generally, oligonucleotides are
prepared synthetically, preferably on a nucleic acid synthesizer.
Accordingly, oligonucleotides can be prepared with non-naturally
occurring phosphoester analog bonds, such as thioester bonds,
etc.
Therapeutic Modulation of SHIP Activation by FcRIIB
[0086] The present invention provides strategies for enhancing
antibody-based treatments (passive immunotherapy) of tumors,
viruses, and microorganisms, i.e., conditions in which enhancement
of immune response provides a therapeutic benefit.
[0087] The phrase "therapeutically effective" or "therapeutic" is
used herein to mean to reduce by at least about 15 percent,
preferably by at least 50 percent, more preferably by at least 90
percent, and most preferably eliminate, a clinically significant
deficit in the activity, function and response of the subject.
Alternatively, a therapeutically effective amount is sufficient to
cause an improvement in a clinically significant condition in the
subject. In accordance with the present invention, a therapeutic
effect is achieved by inhibiting FcRIIB activity when a therapeutic
antibody achieves greater effect than in the absence of FcRIIB
inhibition. Such effects include improving cancer (by reducing
tumor size, eliminating metastasises, increasing time to
recurrence, or increasing survival); clearing an infection;
quieting an acute infection; or eliminating parasites.
[0088] Therapeutic antibodies, and inhibitors of FcRIIB
(collectively, "therapeutic agents"), can be provided to subjects
in pharmaceutically acceptable formulations. The phrase
"pharmaceutically acceptable" refers to molecular entities and
compositions that are physiologically tolerable and do not
typically produce an allergic or similar untoward reaction when
administered to a human. Preferably, as used herein, 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, excipient, or vehicle with which the
compound 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 or
aqueous solution saline solutions and aqueous dextrose and glycerol
solutions are preferably employed as carriers, particularly for
injectable solutions. Suitable pharmaceutical carriers are
described in "Remington's Pharmaceutical Sciences" by E. W. Martin,
which is incorporated herein by reference.
[0089] According to the invention, the therapeutic agents can be
formulated together or separately in a pharmaceutical composition
of the invention to be introduced parenterally, transmucosally,
e.g., orally, nasally, or rectally, or transdermally. Preferably,
administration is parenteral, e.g., via intravenous injection, and
also including, but is not limited to, intra-arteriole,
intramuscular, intradermal, subcutaneous, intraperitoneal,
intraventricular, and intracranial administration.
[0090] In another embodiment, the therapeutic agents can be
delivered together or separately in a vesicle, in particular a
liposome (see Langer, Science, 1990, 249:1527-1533; 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. 317-327; see generally ibid,
each of which is incorporated herein by reference). To reduce its
systemic side effects, this may be a preferred method for
introducing the agents.
[0091] In yet another embodiment, the therapeutic agents can be
delivered together or separately in a controlled release system.
For example, a polypeptide may be administered using intravenous
infusion with a continuous pump, in a polymer matrix such as
poly-lactic/glutamic acid (PLGA), a pellet containing a mixture of
cholesterol and the estrogen compound (SILASTICR.TM.; Dow Corning,
Midland, Mich.; see U.S. Pat. No. 5,554,601, which is incorporated
herein by reference) implanted subcutaneously, an implantable
osmotic pump, a transdermal patch, liposomes, or other modes of
administration. In one embodiment, a pump may be used (see Langer,
supra; Sefton, CRC Crit. Ref. Biomed. Eng., 1987, 14:201; Buchwald
et al, Surgery, 1980, 88:507; Saudek et al, N. Engl. J. Med., 1989,
321:574, each of which is incorporated herein by reference). In
another embodiment, polymeric materials can be used (see Medical
Applications of Controlled Release, Langer and Wise (eds.), CRC
Press: Boca Raton, Fla. (1974); Controlled Drug Bioavailability,
Drug Product Design and Performance, Smolen and Ball (eds.), Wiley:
New York (1984); Ranger and Peppas, J., Macromol. Sci. Rev.
Macromol. Chem., 1983, 23:61; Levy et al, Science, 1985, 228:190;
During et al, Ann. Neurol., 1989, 25:351; Howard et al., J.
Neurosurg., 1989, 71:105, each of which is incorporated herein by
reference). In yet another embodiment, a controlled release system
can be placed in proximity of the therapeutic target, e.g., a tumor
or site of infection, 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), which is
incorporated herein by reference). Other controlled release systems
are discussed in the review by Langer (Science, 1990,
249:1527-1533), which is incorporated herein by reference.
[0092] A subject in whom administration of the antibody and
FcRIIB-inhibitory agent provides an effective therapeutic regimen
for a disease or disorder that benefits from enhanced immune
activity, such as tumor therapy or treatment of an infectious
microorganism or parasite, is preferably a human, but can be any
animal, including a laboratory animal in the context of a clinical
trial or screening or activity experiment. Thus, as can be readily
appreciated by one of ordinary skill in the art, the methods and
compositions of the present invention are particularly suited to
administration to any animal, particularly a mammal, and including,
but by no means limited to, domestic animals, such as feline or
canine subjects, farm animals, such as but not limited to bovine,
equine, caprine, ovine, and porcine subjects, wild animals (whether
in the wild or in a zoological garden), research animals, such as
mice, rats, rabbits, goats, sheep, pigs, dogs, cats, etc., avian
species, such as chickens, turkeys, songbirds, etc., i.e., for
veterinary medical use. In a specific embodiment, the animal is a
transgenic mouse that expresses human FcR chains.
Anti-Tumor Therapy
[0093] The present invention is directed the treatment of tumors,
particularly solid tumors. Examples of solid tumors that can be
treated according to the invention include sarcomas and carcinomas
such as, but not limited to: fibrosarcoma, myxosarcoma,
liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,
angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,
squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,
sweat gland carcinoma, sebaceous gland carcinoma, papillary
carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary
carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma,
bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, meningioma, melanoma, neuroblastoma, and
retinoblastoma. Hematologic malignancies include leukemias,
lymphomas, and multiple myelomas. The following are non-limiting
preferred examples of the cancers treatable with the composition
and methods of the present invention: melanoma, including stage-4
melanoma; ovarian, including advanced ovarian; leukemia, including
but not limited to acute myelogenous leukemia; colon, including
colon metastasized to liver; rectal, colorectal, breast, lung,
kidney, and prostate cancers.
[0094] Anti-tumor antibodies can be generated against the tumor
cells themselves, or against specific tumor cell antigens. There is
substantial evidence that the same tumor antigens are expressed by
different human melanoma tumors, suggesting that
transformation-associated events may give rise to recurrent
expression of the same tumor antigen in tumors of related tissue
and/or cellular origin (Sahasrabudhe et al, J. Immunol., 1993,
151:6302-6310; Shamamian et al, Cancer Immunol. Immunother., 1994,
39:73-83; Cox et al, Science, 1994, 264:716; Peoples et al, J.
Immunol., 1993, 151:5481-5491; Jerome et al, Cancer Res., 1991,
51:2908-2916; Morioke et al., J. Immunol., 1994, 153:5650-5658,
each of which is incorporated herein by reference). Examples of
such antigens include, but are not limited to, MART 1/Melan A,
gp-100, and tyrosinase (melanoma); MAGE-1 and MAGE-3 (bladder, head
and neck, non-small cell carcinoma); HPV E6 and E7 proteins
(cervical cancer); HER2/neu/c-erbB-2 (breast cancer); HER3, HER4,
Mucin (MUC-1) (breast, pancreas, colon, prostate); prostate
specific antigen (PSA) (prostate); CD20 (B cell lymphoma); and CEA
(colon, breast, GI).
Anti-Fungal, Anti-Viral, Anti-Bacterial, and Anti-Parasite
Therapy
[0095] Anti-viral, anti-bacterial, and anti-parasite antibodies,
with enhanced cytotoxic activity as a result of inhibition of
FcRIIB, can be used to treat or clear infections by these
microorganisms. Such viral infections include, but are by no means
limited to, human immunodeficiency virus (HIV); hepatitis A virus,
hepatitis B virus, hepatitis C virus, hepatitis D virus, and other
hepatitis viruses; cytomagalovirus; herpes simplex virus; human
papilloma viruses; Epstein-Barr virus; and other viral infections.
Anti-viral antibodies are well known in the art, and supply a
readily available reservoir of reagents for use with
FcRIIB-inhibitory (including SHIP inhibitory) agents as set forth
above, or can be modified as set forth above to have reduced FcRIIB
binding affinity.
[0096] Examples of infectious bacteria that can be treated in
accordance with the invention include, but are by no means limited
to, S. pneumoniae, S. aureus, E. faecalis, E. coli, Salmonella, M.
leprae, M. tuberculosis, N. gonorrhoeae, etc. Indeed, the present
invention provides an avenue for enhancing the activity of
antibodies generated against bacterial exported (surface) proteins,
located in the bacterial coat or cell wall, e.g., as have been
described for S. pneumoniae (U.S. Pat. No. 5,981,229, which is
incorporated herein by reference).
[0097] The present invention also provides for enhancing the
activity of cytotoxic antibodies generated to pathogenic fungi,
such as C. neoformans and C. albicans (Yuan et al., J. Exp. Med.,
1998, 187:641, which is incorporated herein by reference).
[0098] Examples of parasites that can be treated in accordance with
the invention include, but are not limited to, trypanosomes,
plasmodia (malaria microbe), shistosomes, etc. An important
advantage of the present invention lies in the ability of the
enhanced antibody-mediated cytotoxicity to clear the parasitic
infection early, before the parasite can transform into a different
stage or develop a new antigenic coat.
Pharmaceutical Kits
[0099] Another aspect of the present invention relates to
pharmaceutical kits directed to enhancing the cytotoxicity of a
therapeutic antibody by disrupting SHIP activation by
Fc.gamma.RIIB. In one embodiment a kit according to this aspect of
the invention comprises a therapeutic antibody having reduced
affinity for Fc.gamma.RIIB, such as an antibody having a modified
Fc domain. In another embodiment, the kit comprises a therapeutic
antibody and a competitive inhibitor of Fc.gamma.RIIB binding. In a
further embodiment of the invention, the kit comprises a
therapeutic antibody and an inhibitor of expression of
Fc.gamma.RIIB or SHIP, including, but is not limited to, antisense
nucleic acid molecules and intracellular antibodies. Optionally,
the kit also includes instructions for use of the component
antibodies and inhibitors, controls, and photos or figures
depicting data.
[0100] The invention can be better understood by reference to the
following Examples, which are provided by way of illustration and
not by way of limitation.
EXAMPLES
Example 1
Inhibitory Fc Receptor Modulates In Vivo Cytotoxicity Against Tumor
Targets
Materials and Methods
[0101] Melanoma metastasis model. Mice were injected intravenously
with 1.times.10.sup.6 B16 melanoma cells on day 0 and with either
PBS or 20 .mu.g of purified TA99 i.p. on days 0, 2, 4, 7, 9 and 11.
A dose of 200 .mu.g of mAb TA99 induced greater than 90% reduction
in tumor metastasis in wild-type but not FcR.gamma..sup.-/- mice.
However, at this lowered 20 .mu.g dose TA99 only limited protection
was provided against tumor metastasis in WT mice. Mice were
sacrificed on day 14 and surface lung metastasis counted under a
dissecting microscope.
[0102] Tumor xenograft models. For breast carcinoma xenograft
experiments, 5.times.10.sup.6 BT474M1 cells (BT474 subclone derived
at Genentech, South San Francisco, Calif.) were injected
subcutaneously on day 1 in 0.1 ml PBS mixed with 0.1 ml MATRIGEL
(Collaborative Research, Bedford, Mass.). 2-4 month old BALB/c nude
mice, .gamma..sup.-/- BALB/c nude mice or RII.sup.-/- BALB/c nude
mice were injected subcutaneously with 170-estradiol 60 day release
pellets (0.75 mg/pellet) (Innovative Research of America, Sarasota,
Fla.) 24 hrs prior to tumor cell injection. Therapeutic antibodies
(obtained from vialed, clinical material, Genentech, Inc., South
San Francisco, Calif.) were intravenously injected beginning on day
1 at 4 .mu.g/mg loading dose, with weekly injections of 2 .mu.g/mg
for BALB/c nude and .gamma..sup.-/- BALB/c nude. A ten fold lower
dose was used for the experiments shown in FIG. 3. For B cell
lymphoma xenograft experiments, 2-4 month old BALB/c nude mice or
.gamma..sup.-/- BALB/c nude mice were irradiated with 3.0 cGy prior
to subcutaneous injection of 5.times.10.sup.6 Raji B lymphoma
cells. RITUXAN.RTM. was obtained from IDEC Pharmaceuticals, Inc.
and given at a dose of 10 .mu.g/gm weekly. Tumor measurements were
obtained weekly.
[0103] Engineering of D265A mutant antibody and binding assays.
Site-directed mutagenesis was performed using QUIKCHANGE
Mutagenesis Kit (Stratagene, La Jolla, Calif.). Mutant antibody was
transiently expressed in A293 cells in the pRK expression vector
and conditioned supernatants were harvested and purified by protein
G affinity column chromatography. The ability of various mutants to
bind recombinant Fc.gamma.Rs was evaluated using an in vitro
binding assay. Microtiter plates were coated with recombinant
Fc.gamma.RIII GST fusion protein at a concentration of 100 ng/well
in PBS. Plates were washed with PBS, supplemented with 0.05%
Tween-20 (wash buffer) then blocked for 1 hour at room temperature
with 0.5% BSA, 50 mM TBS, 0.05% Tween-20, 2 mM EDTA pH 8.0 (ELISA
buffer). The IgG1 Fc fragment of murine 4D5 as well as D265 A was
grafted onto the Fab of anti-human IgE (mAb E27) and recombinant
antibody was produced as mentioned above. Addition of human IgE to
antihuman E27 with wild type or mutant Fc domains in a 1:1 molar
ratio in ELISA buffer led to the formation of homogeneous hexameric
complexes. Complexes were added to the plates, washed five times in
wash buffer and were detected by the addition of goat F(ab').sub.2
anti-mouse IgG, and subsequent colorimetric development.
[0104] Growth inhibition assays. BT474M1 cells were plated at
1.times.10.sup.4, and allowed to adhere for 24 hours. Antibody was
added at the indicated concentrations for 48 hours, followed by a
14 hour pulse with [H.sup.3]thymidine. Cells were harvested,
collected on filter mats, and counted in a WALLAC MICROBETA
scintillation counter. BT474M1 cells were incubated with 4D5 or
D265A antibody, and stained with FITC-conjugated goat anti-mouse
IgG. Fluorescence intensity was measured on a FACSCAN flow
cytometer.
In vitro ADCC assay. Adherent NK effector cells were obtained from
IL-2 stimulated (250 U/ml, Sigma, St. Louis, Mo.) 14-day culture of
nylon wool non-adherent splenocytes. 4-hour ADCC reactions were
performed with 5.times.10.sup.4 chromium-labeled HER2
overexpressing SK-BR3 breast carcinoma (ATCC, Manassas, Va.) target
cells in 96-well plates in the presence or absence of antibody (10
.mu.g/ml). Percentage (%) cytotoxicity was expressed as: counts in
supernatant-spontaneous release (without effectors)/total counts
incorporated-spontaneous release. Data are expressed as the mean of
three replicate wells.
Results
[0105] Passive and active protection against pulmonary metastasis
in the syngenic B16 melanoma model has recently been demonstrated
to require the presence of activation FcRs (2) on effector cells,
such as NK cells. To determine whether the inhibitory Fc.gamma.RIIB
was a factor in determining the in vivo anti-tumor activity of mAb
TA99 2, a protective IgG2a antibody specific for the melanoma
differentiation antigen gp75 (TRP-1), C57B1/6 mice were crossed to
an Fc.gamma.RIIB deficient strain and then backcrossed to establish
a syngenic strain. Metastasis of B16 melanoma cells in the RIIB
deficient background were identical to wild-type (almost total
blackening of the lungs with metastatic melanoma tumor cells),
demonstrating that the inhibitory receptor was not involved in
tumor growth or spread. In contrast, when RIIB deficient animals
were given the protective IgG2a antibody a profound enhancement of
the activity of this antibody was observed, as compared to mice
wild-type for Fc.gamma.RIIB. Quantitation of the tumor nodules in
excised lungs revealed that wild-type, treated mice reduced tumor
load by a factor of 3 (300+/-30 compared to 100+/-10) while
antibody treatment of RIIB-/- animals resulted in a 100-fold
reduction (300 compared to 3). As shown previously, deletion of the
activation .gamma. subunit eliminates the in vivo protective effect
of this antibody.
[0106] NK cells, a principal cell type involved in ADCC express the
activation Fc.gamma.R, RIII, but do not express the inhibitory
counterpart, RIIB. Thus, the enhancement observed in RIIB deficient
mice cannot be attributed to NK cell hyperresponsiveness. Rather,
monocytes and macrophages, which express both RIII and RIIB, are
therefore implicated as the dominant effector cell involved in this
antibody-dependent protection in vivo. Thus the activity attributed
to the protective IgG2a antibody in a wild-type animal represents
the sum of the opposing activation and inhibitory pathways
contributed by NK cells, monocytes and macrophages.
Anti-Tumor Activity of 4D5, HERCEPTIN.RTM., and RITUXAN.RTM.
Required Fc.gamma.R Activating Receptors.
[0107] To determine the contribution of interactions between the Fc
domain and effector cell Fc.gamma.Rs to the in vivo activity of
HERCEPTIN.RTM. and RITUXAN.RTM., the orthotopic athymic nude mouse
tumor model was modified to generate a suitable model to address
the role of Fc.gamma.RII and RIII in the anti-tumor response. The
common y chain deficient mouse (FcR.gamma..sup.-/-) (Takai, T. et
al., Cell, 1994, 76:519-29, which is incorporated herein by
reference), lacking the activation Fc.gamma.Rs, I and III or the
Fc.gamma.RIIB deficient mouse (Takai, T. et al, Nature, 1996,
379:346-9, which is incorporated herein by reference) were each
mated with athymic nude mice to generate FcR.gamma..sup.-/- /nu/nu
and Fc.gamma.RIIB.sup.-/- /nu/nu mice for use in xenograft human
tumor models. The anti-tumor activity of the anti-p185HER-2/neu
antibody HERCEPTIN.RTM. (humanized IgG 1) (Carter et al, 1992,
supra) and its mouse parent antibody 4D5 (mouse IgG I) in
preventing the growth of the human breast carcinoma BT474M1, which
over-expresses p185/HER-2/neu, was addressed in FcR.gamma..sup.-/-
and +/+ athymic nude mice (FIGS. 1A-ID). Tumor growth, measured as
volume, was identical in homozygous .gamma..sup.-/- and +/+ nu/nu
mice injected subcutaneously with 5.times.10.sup.6 BT474M1 cells.
In y.sup.+/+ mice, a single 4 .mu.g/gm intravenous dose, followed
by weekly 2 .mu.g/gm i.v. injections, resulted in near complete
inhibition of tumor growth (tumor mass reductions of 90 and 96% in
4D5 and HERCEPTIN.RTM. treated mice) with only 4 of 17 mice
developing palpable tumors. However, this protective effect of
HERCEPTIN.RTM. and 4D5 was reduced in .gamma..sup.-/- mice. Tumor
mass in antibody treated .gamma..sup.-/- mice were reduced by 29
and 44%, respectively and 14 of 15 mice developed palpable
tumors.
[0108] Similar results were obtained in the .gamma..sup.-/- nu/nu
xenograft model on the mechanism by which the chimeric monoclonal
IgG1 anti-CD20 antibody RITUXAN.RTM. inhibits B cell lymphoma
growth in vivo. Tumor growth of the human B cell lymphoma cell line
Raji is indistinguishable in .gamma..sup.-/- and +/+ nu/nu mice
(FIGS. 1E and 1F). However, the protective effect of weekly i.v.
doses of RITUXAN.RTM. (10 .mu.g/gm) seen in .gamma..sup.-/- is
reduced in .gamma..sup.-/- nu/nu mice. RITUXAN.RTM. treatment of
wild-type athymic mice resulted in reductions of tumor mass by more
than 99% and no wild type mice developed palpable tumors. In
contrast, in .gamma..sup.-/- mice little protection was afforded by
RITUXAN.RTM.; 6 of 7 mice developed palpable tumors and tumor mass
reductions averaged just 23%.
Anti-Breast Tumor Activity of 4D5 and HERCEPTIN.RTM. is Enhanced in
Fc.gamma.RIIB Deficient Mice.
[0109] In contrast, Fc.gamma.RHB.sup.-/- mice were more effective
at arresting BT474 growth in this nude mouse model (FIG. 2). At a
sub-therapeutic dose of antibody (0.4 .mu.g/gm loading, 0.2
.mu.g/gm weekly) tumor growth in RIIB deficient mice was arrested,
demonstrating the involvement of the inhibitory RIIB pathway in
this model as well. Nude mice are known to display elevated NK cell
numbers, leading to the presumption that antibody protection in
those mice are not representative of the protection seen in
syngenic systems, as in human disease. The observation that RIIB
deletion enhances protection in nude mice indicates the involvement
of effector cells other than NK cells, such as monocytes and
macrophages in the protective response and further indicates that
the FcR-dependent pathways are not restricted to an NK cell biased
system but, as in the syngenic melanoma system, is likely to be
relevant in other syngenic systems as well.
In Vitro and In Vivo Properties of D265A Mutant Antibody.
[0110] To further demonstrate the involvement of Fc-Fc.gamma.R
interactions in the protective response, a modification of the
mouse IgG1 anti-HER2 antibody 4D5 was engineered to disrupt the
ability of the antibody to engage cellular Fc.gamma.R receptors
while retaining its affinity for its cognate antigen p185
HER-2/neu. Based on alanine-scanning mutagenesis mapping of the
murine IgG1 Fc domain binding for Fc.gamma.R, a single amino acid
replacement at residue 265 in the C.sub.H2 domain of the mouse IgG1
heavy chain was found to reduce binding of IgG1-containing immune
complexes to both Fc.gamma.RII and III in a receptor coated plate
assay (FIG. 3 A). This residue is located at a site within the Fc
portion of the IgG molecule thought to interact directly with
surfaces of FcRs. The 265 (asp-ala) mutation was placed in the 4D5
IgG1 heavy chain gene and transfected in parallel with the
wild-type 4D5 IgG1 heavy chain into A293 cells along with the 4D5
kappa chain to produce 4D5 and mutant (D265A) antibodies. Since the
mutation would not be expected to disrupt antibody-antigen
interactions, as predicted, both 4D5 and D265A antibodies purified
from transfected cell supernatants bound cellular p185HER-2/neu
with equivalent avidity and had comparable in vitro growth
inhibitory activity when added to BT474M1 expressing breast
carcinoma cells in tissue culture (FIG. 3B). However while D265A
retained the wild-type characteristics of in vivo half-life,
antigenic targeting and functional p185HER-2/neu receptor blockade,
the in vitro ADCC capacity of the mutant was lost as a consequence
of its reduced affinity for Fc.gamma.RIII on effector cells (FIG.
3C). In vivo, the anti-tumor activity of D265A, when tested in the
breast carcinoma BT474M1 xenograft model, displayed reduced
anti-tumor activity as compared to 4D5 (FIG. 3D). Palpable tumors
developed in all wild-type athymic mice treated with D265 A while
only in 2 of 5 mice treated with 4D5. D265A treatment reduced tumor
volumes by 30% as compared to the 85% reduction seen with 4D5. The
attenuated anti-tumor responses of D265A correlates with its
impaired ability to activate FcR bearing effector cells despite its
ability to inhibit tumor growth in vitro, supporting the conclusion
that FcR engagement is a significant contributing component of
anti-tumor activity in vivo.
Discussion
[0111] The data presented here suggest that Fc.gamma.R binding
contributes significantly to in vivo activity. This Fc.gamma.R
dependence appears to apply to more than a single antibody since it
has been observed for both syngenic and xenograft models for the
three unrelated tumors and target antigens presented here.
Fc.gamma.R engagement involves both activation and inhibitory
receptors and thus implicates monocytes and macrophages in the
effector cell component of the protective response. Supportive
evidence for this interpretation is found in the ability of
HERCEPTIN.RTM. to mediate ADCC in vitro and the ability of anti-FcR
antibodies to inhibit some of the in vivo activity of anti-CD20
antibodies (Funakoshi, S. et al., J. Immunother., 1996, 19:93-101,
which is incorporated herein by reference). While the studies
presented here demonstrate a significant role for Fc-Fc.gamma.R
interactions, triggering the growth and apoptotic regulatory
pathways by antibody engagement of p185HER2/neu and CD20 may still
contribute to the total in vivo efficacy of anti-tumor antibodies.
Support for this interpretation can be seen in the partial
protection observed in FcR.gamma..sup.-/- mice treated with
anti-HER2/neu antibodies (FIG. 1), where the anti-tumor activity of
these antibodies against the BT474M1 breast carcinoma cells is
reduced but not ablated. Blocking signaling on tumor cells by
antibodies may also act synergistically with immune effector
responses by rendering the tumor cells more susceptible to immune
effector cell triggered apoptotic or lytic cell death (Baselga, J.
et al., Cancer Res., 1998, 58:2825-31, which is incorporated herein
by reference). These studies highlight the fundamental importance
of the inhibitory pathways in vivo and suggest that individual
responses to anti-tumor antibodies may be dependent on expression
of these inhibitory pathways.
Example 2
Generation of Variant IgG1 Fc Domains with Reduced Binding to
FcRIIB
[0112] The underlying principle of Fc domain mutagenesis requires
the expression of the dimeric Fc domain of human IgG1, for example,
in a cellular system which glycosylates the molecule and displays
it on the surface for binding studies. Compatible expression
systems include eukaryotic cells such as yeast or mammalian
cells.
[0113] In this example, yeast cells are employed as the host
system. Error-prone PCR was employed to generate a library of
sequences of the human IgG1 CH2-CH3 domain, according to procedures
described previously (Saviranta et al., Prot. Eng., 1998,
11:143-152; Leung et al., Technique, 1992, 1:11) to generate an
average of 2-4 amino acid changes per molecule. The primers
employed spanned the hinge region (amino acids 218-229) and vector
sequences flanking the 3' integration site in the expression vector
pCT302. Libraries of greater than 10.sup.7 recombinants were
obtained. Expression was performed in the yeast AG1:AG2 surface
display systems, as described (Boder and Wittrup, Nat. Biotechnol.,
1997, 15:553-557, which is incorporated herein by reference) to
generate an Fc fusion protein with the yeast parent Aga 2p, which
is anchored to the yeast cell wall by disulfide interactions with
the surface expressed yeast protein AG1. The PCR mutagenized IgG1
Fc fragment was cloned into the Aga2p-linker-fusion vector pCT302
and transformed into yeast strain EBY100. Transformants were
selected on SD+CAA plates. Induction of expression of Aga2p Fc
fusion was achieved by induction in galactose containing
medium.
[0114] Screening of the mutant libraries was accomplished by
panning or flow cytometry. For example, flow-cytometry screening
was accomplished by FITC-labelled recombinant FcRIIB expressed as a
hexameric complex, using a 1:1 molar ratio of FcRIIA/IgE Fc fusion
and anti-IgE mAb E25 (Liu et al., Biochem., 1995, 34:10474, which
is incorporated herein by reference) in the presence of an
unlabelled 10-fold molar excess of FcRIIB/IgE anti-IgE complex.
FITC positive yeast cells were enriched by multiple rounds of flow
cytometry sorting and the resulting yeast cells plated and pCT302
fusion plasmid isolated and sequenced to determine the mutations
generating reduced RIIB binding with unchanged or enhanced RIIA
binding. Sites of differential interaction of IgG1 Fc with RIIA,
RIIIA and RIIB were further defined by targeted PCR mutagenesis of
those regions and repeating the yeast surface display screening to
fully optimize binding differences. Similar experiments were
performed to identify polypeptide variants with enhanced RIIB
binding, RIIIA binding or RIIIB binding.
[0115] Alternatively, screening was also performed by panning of
the mutagenized pCT302 fusion library on plates on to which
recombinant RIIA, RIIB, RIIIA or RIIIB were immobolized. Multiple
rounds of panning on RIIA were performed and the positive binders
then panned on RIIB plates to remove any mutants which retain RIIB
binding, thus identifying Fc mutants with retained or enhanced RIIA
binding and reduced or eliminated RIIB binding.
[0116] Similar experiments can be performed using a mammalian
expression vector for surface display on such cells and generating
a mutagenized Fc library in an analogous manner.
Example 3
Isolation of a Monoclonal Antibody with Specificity for FcRIIB
[0117] A murine monoclonal antibody is obtained by immunizing mice
with the recombinant human RIIB protein and spleen cells fused to
obtain hybridomas, as described above. The resulting hybridomas are
screening for selective binding to RIIB, while not binding to RIIA
or RIIIA or RIIIB. Antibodies with the desired properties are then
cloned and the mRNA isolated and converted into cDNA for the heavy
and light chains. A single chain Fv is then constructed as
described above and expressed as a gene III fusion protein for
phage display or Aga2p fusion for yeast display. A randomly
mutagenized library is constructed for the single chain Fv binding
and screening by panning for specificity for RIIB over RIIA. The
resulting phage or yeast cells are characterized by isolating the
fusion phage genome or plasmid, respectively, DNA sequenced and
then expressed as a recombinant antibody.
[0118] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and the accompanying figures. Such
modifications are intended to fall within the scope of the appended
claims. Various patents, patent applications, and publications are
cited herein, the disclosures of which are incorporated herein by
reference in their entireties.
* * * * *