Note: Descriptions are shown in the official language in which they were submitted.
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EXPRESS MAIL NO.: EV 767823612 US PATENT
DATE DEPOSITED: SEPTEMBER 7, 2006
ANTIBODIES AS T CELL RECEPTOR MIMICS, METHODS OF PRODUCTION AND USES
THEREOF
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0001] The government owns certain rights in the present invention pursuant to
a grant from
the Advanced Technology Program of the National Institute of Standards and
Technology
(Grant #70NANB4H3048).
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0002] The present invention relates generally to a methodology of producing
antibodies that
recognize peptides associated with a tumorigenic or disease state, wherein the
peptides are
displayed in the context of HLA molecules. These antibodies will mimic the
specificity of a T
cell receptor (TCR) such that the molecules may be used as therapeutic,
diagnostic and
research reagents.
2. Description of the Background Art
[0003] Class I major histocompatibility complex (MHC) molecules, designated
HLA class I in
humans, bind and display peptide antigen ligands upon the cell surface. The
peptide antigen
ligands presented by the class I MHC molecule are derived from either normal
endogenous
proteins ("self") or foreign proteins ("nonself') introduced into the cell.
Nonself proteins may be
products of malignant transformation or intracellular pathogens such as
viruses. In this manner,
class I MHC molecules convey information regarding the internal milieu of a
cell to immune
effector cells including but not limited to, CD8+ cytotoxic T lymphocytes
(CTLs), which are
activated upon interaction with "nonself' peptides, thereby lysing or killing
the cell presenting
such "nonself' peptides.
[0004] Class II MHC molecules, designated HLA class II in humans, also bind
and display
peptide antigen ligands upon the cell surface. Unlike class I MHC molecules
which are
expressed on virtually all nucleated cells, class II MHC molecules are
normally confined to
specialized cells, such as B lymphocytes, macrophages, dendritic cells, and
other antigen
presenting cells which take up foreign antigens from the extracellular fluid
via an endocytic
pathway. The peptides they bind and present are derived from extracellular
foreign antigens,
such as products of bacteria that multiply outside of cells, wherein such
products include protein
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toxins secreted by the bacteria that often have deleterious and even lethal
effects on the host
(e.g., human). In this manner, class II molecules convey information regarding
the fitness of
the extracellular space in the vicinity of the cell displaying the class II
molecule to immune
effector cells, including but not limited to, CD4+ helperT cells, thereby
helping to eliminate such
pathogens. The extermination of such pathogens is accomplished by both helping
B cells make
antibodies against microbes, as well as toxins produced by such microbes, and
by activating
macrophages to destroy ingested microbes.
[0005] Class I and class II HLA molecules exhibit extensive polymorphism
generated by
systematic recombinatorial and point mutation events during cell
differentiation and maturation
resulting from allelic diversity of the parents; as such, hundreds of
different HLA types exist
throughout the world's population, resulting in a large immunological
diversity. Such extensive
HLA diversity throughout the population is the root cause of tissue or organ
transplant rejection
between individuals as well as of differing individual susceptibility and/or
resistance to infectious
diseases. HLA molecules also contribute significantly to autoimmunity and
cancer.
[0006] Class I MHC molecules alert the immune response to disorders within
host cells.
Peptides which are derived from viral- and tumor-specific proteins within the
cell are loaded into
the class I molecule's antigen binding groove in the endoplasmic reticulum of
the cell and
subsequently carried to the cell surface. Once the class I MHC molecule and
its loaded peptide
ligand are on the cell surface, the class I molecule and its peptide ligand
are accessible to
cytotoxic T lymphocytes (CTL). CTLs survey the peptides presented by the class
I molecule
and destroy those cells harboring ligands derived from infectious or
neoplastic agents within
that cell.
[0007] While specific CTL targets have been identified, little is known about
the breadth and
nature of ligands presented on the surface of a diseased cell. From a basic
scientific
perspective, many outstanding questions remain in the art regarding peptide
presentation. For
instance, it has been demonstrated that a virus can preferentially block
expression of HLA class
I molecules from a given locus while leaving expression at other loci intact.
Similarly, there are
numerous reports of cancerous cells that downregulate the expression of class
I HLA at
particular loci. However, there is no data describing how (or if) the
classical HLA class I loci
differ in the peptides they bind. It is therefore unclear how class I
molecules from the different
loci vary in their interaction with viral- and tumor-derived ligands and the
number of peptides
each will present.
[0008] Discerning virus- and tumor-specific ligands for CTL recognition is an
important
component of vaccine design. Ligands unique to tumorigenic or infected cells
can be tested
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and incorporated into vaccines designed to evoke a protective CTL response.
Several
methodologies are currently employed to identify potentially protective
peptide ligands. One
approach uses T cell lines or clones to screen for biologically active ligands
among
chromatographic fractions of eluted peptides (Cox et al., 1994). This approach
has been
employed to identify peptide ligands specific to cancerous cells. A second
technique utilizes
predictive algorithms to identify peptides capable of binding to a particular
class I molecule
based upon previously determined motif and/or individual ligand sequences (De
Groot et al.,
2001); however, there have been reports describing discrepancies between these
algorithms
and empirical data. Peptides having high predicted probability of binding from
a pathogen of
interest can then be synthesized and tested for T cell reactivity in various
assays, such as but
not limited to, precursor, tetramer and ELISpot assays.
[0009] Many cancer cells display tumor-specific peptide-HLA complexes derived
from
processing of inappropriately expressed or overexpressed proteins, called
tumor associated
antigens (TAAs) (Bernhard et al., 1996; Baxevanis et al., 2006; and Andersen
et al., 2003).
With the discovery of mAb technology, it was believed that "magic bullets"
could be developed
which specifically target malignant cells for destruction. Current strategies
for the development
of tumor specific antibodies rely on creating monoclonal antibodies (mAbs) to
TAAs displayed
as intact proteins on the surface of malignant cells. Though targeting surface
tumor antigens
has resulted in the development of several successful anti-tumor antibodies
(Herceptin and
Rituxan), a significant number of patients (up to 70%) are refractory to
treatment with these
antibody molecules. This has raised several questions regarding the rationale
for targeting
whole molecules displayed on the tumor cell surface for developing cancer
therapeutic
reagents. First, antibody-based therapies directed at surface antigens are
often associated with
lower than expected killing efficiency of tumor cells. Free tumor antigens
shed from the surface
of the tumor occupy the binding sites of the anti-tumor specific antibody,
thereby reducing the
number of active molecules and resulting in decreased tumor cell death.
Second, current mAb
molecules do not recognize many potential cancer antigens because these
antigens are not
expressed as an intact protein on the surface of tumor cells. The tumor
suppressor protein p53
is a good example. p53 and similar intracellular tumor associated proteins are
normally
processed within the cell into peptides which are then presented in the
context of either HLA
class I or class II molecules on the surface of the tumor cell. Native
antibodies are not
generated against peptide-HLA complexes. Third, many of the antigens
recognized by
antibodies are heterogenic by nature, which limits the effectiveness of an
antibody to a single
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tumor histology. For these reasons it is apparent that antibodies generated
against surface
expressed tumor antigens may not be optimal therapeutic targets for cancer
immunotherapy.
[0010] The majority of proteins produced by a cell reside within intracellular
compartments,
thus preventing their direct recognition by antibody molecules. The abundance
of intracellular
proteins that is available for degradation by proteasome-dependent and
independent
mechanisms yields an enormous source of peptides for surface presentation in
the context of
the MHC class I system (Rock et al., 2004). A new class of antibodies that
specifically
recognizes HLA-restricted peptide targets (epitopes) on the surface of cancer
cells would
significantly expand the therapeutic repertoire if it could be shown that they
have anti-tumor
properties which could lead to tumor cell death.
[0011] Many T cell epitopes (specific peptide-HLA complexes) are common to a
broad range
of tumors which have originated from several distinct tissues. The primary
goal of epitope
discovery has been to identify peptide (tumor antigens) for use in the
construction of vaccines
that activate a clinically relevant cellular immune response against the tumor
cells. The goal
of vaccination in cancer immunotherapy is to elicit a cytotoxicT lymphocyte
(CTL) response and
activate T helper responses to eliminate the tumor. Although many of the
epitopes discovered
by current methods are immunogenic, shown by studies that generate peptide-
specific CTL in
vitro and in vivo, the application of vaccination protocols to cancer
treatment has not been
highly successful. This is especially true for cancer vaccines that target
self-antigens ("normal"
proteins that are overexpressed in the malignant cells). Although this class
of antigens may not
be ideal for vaccine formulation due to an individual "tolerance" of self
antigens, they still
represent good targets for eliciting antibodies ex vivo.
[0012] The value of monoclonal antibodies which recognize peptide-MHC
complexes has
been recognized by others (see for example Reiter, US Publication No. US
2004/0191260 Al,
filed March 26, 2003; Andersen et al., US Publication No. US 2002/0150914 Al,
filed
September 19, 2001; Hoogenboom et al., US Publication No. US 2003/0223994 Al,
filed
February 20, 2003; and Reiter et al., PCT Publication No. WO 03/068201 A2,
filed February 11,
2003). However, these processes employ the use of phage display libraries that
do not produce
a whole, ready-to-use antibody product. The majority of these antibodies were
isolated from
bacteriophage libraries as Fab fragments (Cohen et al., 2003; Held et al.,
2004; and Chames
et al., 2000) and have not been examined for anti-tumor activity since they do
not activate
innate immune mechanisms (e.g., complement-dependent cytotoxicity [CDC]) or
antibody-dependent cellular cytotoxicity (ADCC). Demonstration of anti-tumor
activity is critical
as therapeutic mAbs are thought to act through several mechanisms which engage
the innate
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response, including antibody or complement-mediated phagocytosis by
macrophage, CDC and
ADCC (Liu et al., 2004; Prang et al., 2005; Akewaniop et al., 2001; Clynes et
al., 2000; and
Masui et al., 1986). These prior art methods also have not demonstrated
production of
antibodies capable of staining tumor cells in a robust manner, implying that
they are of low
affinity or specificity. The immunogen employed in the prior art methods uses
MHC which has
been "enriched" for one particular peptide, and therefore such immunogen
contains a pool of
peptide-MHC complexes and is not loaded solely with the peptide of interest.
In addition, there
has not been a concerted effort in these prior art methods to maintain the
structure of the three
dimensional epitope formed by the peptide/HLA complex, which is essential for
generation of
the appropriate antibody response. For these reasons, immunization protocols
presented in
these prior art references had to be carried out over long periods of time
(i.e., approximately
5 months or longer).
[0013] Therefore, there exists a need in the art for diagnostic and
therapeutic antibodies with
novel recognition specificity for peptide-HLA domain in complexes present on
the surface of
tumor or diseased/infected cells. The presently claimed and disclosed
invention provides
innovative processes for creating antibody molecules endowed with unique
antigen recognition
specificities for peptide-HLA complexes, and the present invention recognizes
that these
peptide-HLA molecules are unique sources of tumor/disease/infection specific
antigens
available as therapeutic targets. In addition, the development of this
technology will provide
new tools to detect, visualize, quantify, and study antigen (peptide-HLA)
presentation in tumors
or diseased/infected cells. Antibodies with T cell receptor-like specificity
of the present
invention enable the measurement of antigen presentation on tumors or
diseased/infected cells
by direct visualization. Previous studies attempting to visualize peptide-HLA
complexes using
a soluble TCR found that the poor affinity of the TCR made it difficult to
consistently detect low
levels of target on tumor cells (Weidanz, 2000). Therefore, in addition to
being used as
targeting agents, TCRm of the present invention serve as valuable tools to
obtain information
regarding the presence, expression pattern, and distribution of the target
peptide-HLA complex
antigens on the tumor surface and in tumor metastasis.
SUMMARY OF THE INVENTION
[0014] The present invention relates to a methodology of producing antibodies
that recognize
peptides associated with a tumorigenic or disease state, wherein the peptides
are displayed in
the context of HLA molecules. These antibodies will mimic the specificity of a
T cell receptor
(TCR) such that the molecules may be used as therapeutic, diagnostic and
research reagents.
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In one embodiment, the T cell receptor mimics will have higher binding
affinity than a T cell
receptor. In another embodiment, the T cell receptor mimic has a binding
affinity of about 10
nanomolar or greater.
[0015] The present invention is directed to a method of producing a T-cell
receptor mimic.
The method of the presently disclosed and claimed invention includes
identifying a peptide of
interest, wherein the peptide of interest is capable of being presented by an
MHC molecule.
Then, an immunogen comprising at least one peptide/MHC complex is formed,
wherein the
peptide of the peptide/MHC complex is the peptide of interest. An effective
amount of the
immunogen is then administered to a host for eliciting an immune response, and
the
immunogen retains a three-dimensional form thereof for a period of time
sufficient to elicit an
immune response against the three-dimensional presentation of the peptide in
the binding
groove of the MHC molecule. Serum collected from the host is assayed to
determine if desired
antibodies that recognize a three-dimensional presentation of the peptide in
the binding groove
of the MHC molecule are being produced. The desired antibodies can
differentiate the
peptide/MHC complexfrom the MHC molecule alone, the peptide alone, and a
complex of MHC
and irrelevant peptide. Finally, the desired antibodies are isolated.
[0016] The peptide of interest may be associated with at least one of a
tumorigenic state, an
infectious state and a disease state, or the peptide of interest may be
specific to a particular
organ or tissue. The presentation of the peptide in context of an MHC molecule
may be novel
to cancer cells, or it may be greatly increased in cancer cells when compared
to normal cells.
[0017] In one embodiment, the step of forming an immunogen in the method of
the presently
disclosed and claimed invention may include recombinantly expressing the
peptide/MHC
complex in the form of a single chain trimer. In another embodiment, the step
of forming an
immunogen in the method of the presently disclosed and claimed invention may
include
recombinantly expressing the peptide/MHC complex and chemically cross-linking
the
peptide/MHC complex to aid in stabilization of the immunogen. In another
embodiment, the
step of forming the immunogen of the present invention includes recombinantly
expressing the
MHC heavy chain and the MHC light chain separately in E. coli, and then
refolding the MHC
heavy and light chain's with peptide in vitro.
[0018] In addition, the immunogen may be formed by multimerizing two or more
peptide/MHC complexes, such as but not limited to, a dimer, a trimer, a
tetramer, a pentamer,
or a hexamer. The two or more peptide/MHC complexes may be covalently
attached, and they
may be modified to enable covalent attachment of the peptide/MHC complexes to
one another.
Optionally, the two or more peptide/MHC complexes may be non-covalently
attached. The two
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or more peptide/MHC complexes may be attached to a substrate. When the
peptide/MHC
complexes are attached to a substrate, the desired antibodies should not
recognize the
substrate utilized in multimerization of the peptide/MHC complexes. A tail may
be attached to
the two or more peptide/MHC complexes to aid in multimerization, wherein the
tail may be
selected from the group including but not limited to, a biotinylation signal
peptide tail, an
immunoglobulin heavy chain tail, a TNF tail, an IgM tail, a Fos/Jun tail, and
combinations
thereof. In a further alternative, the peptide/MHC complexes may be
multimerized through
liposome encapsulation, through the use of an artificial antigen presenting
cell, or through the
use of polymerized streptavidin.
[0019] In one embodiment, the immunogen may be further modified to aid in
stabilization
thereof. For example but not by way of limitation, the modification may be
selected from the
group consisting of modifying an anchor in the peptide/MHC complex, modifying
amino acids
in the peptide/MHC complex, PEGalation, chemical cross-linking, changes in pH
or salt,
addition of at least one chaperone protein, addition of at least one adjuvant,
and combinations
thereof.
[0020] The host immunized for eliciting an immune response in the presently
disclosed and
claimed method may be, for example but not by way of limitation, a rabbit, a
rat, or a mouse,
such as but not limited to, a Balb/c mouse or a transgenic mouse. The
transgenic mouse may
be transgenic for the MHC molecule of the immunogen, or the transgenic mouse
may be
capable of producing human antibodies.
[0021] The assaying step of the presently disclosed and claimed invention may
further
include preabsorbing the serum to remove antibodies that are not peptide
specific.
[0022] The step of isolating the desired antibodies of the presently disclosed
and claimed
invention may further include a method for isolating at least one of B cells
expressing surface
immunoglobulin, B memory cells, hybridoma cells and plasma cells producing the
desired
antibodies. The step of isolating the B memory cells may include sorting the B
memory cells
using at least one of FACS sorting, beads coated with peptide/MHC complex,
magnetic beads,
and intracellular staining. The method may further include the step of
differentiating and
expanding the B memory cells into plasma cells.
[0023] The method of the presently disclosed and claimed invention may further
include the
step of assaying the isolated desired antibodies to confirm their specificity
and to determine if
the isolated desired antibodies cross-react with other MHC molecules.
[0024] The present invention is also directed to a T cell receptor mimic that
includes an
antibody or antibody fragment reactive against a specific peptide/MHC complex,
wherein the
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antibody or antibody fragment can differentiate the specific peptide/MHC
complex from the
MHC molecule alone, the peptide alone, and a complex of MHC and an irrelevant
peptide. The
T cell receptor mimic is produced by immunizing a host with an effective
amount of an
immunogen comprising a multimer of two or more specific peptide/MHC complexes.
The
immunogen may be in the form of a tetramer. The peptide of the specific
peptide/MHC complex
may be associated with at least one of a tumorigenic state, an infectious
state and a disease
state, or the peptide of the specific peptide/MHC complex may be specific to a
particular organ
or tissue. Alternatively, the presentation of the peptide of the specific
peptide/MHC complex
in the context of an MHC molecule may be novel to cancer cells, or may be
greatly increased
in cancer cells when compared to normal cells. The peptide of the specific
peptide/MHC
complex may comprise any of SEQ ID NOS:1-3 and 6.
[0025] In one embodiment, the T cell receptor mimic may have at least one
functional moiety,
such as but not limited to, a detectable moiety or a therapeutic moiety, bound
thereto. For
example but not by way of limitation, the detectable moiety may be selected
from the group
consisting of a fluorophore, an enzyme, a radioisotope and combinations
thereof, while the
therapeutic moiety may be selected from the group consisting of a cytotoxic
moiety, a toxic
moiety, a cytokine moiety, a bi-specific antibody moiety, and combinations
thereof.
[0026] The present invention is also directed to a hybridoma cell or a B cell
producing a T
cell receptor mimic comprising an antibody or antibody fragment reactive
against a specific
peptide/MHC complex, wherein the antibody or antibody fragment can
differentiate the specific
peptide/MHC complex from the MHC molecule alone, the peptide alone, and a
complex of MHC
and an irrelevant peptide. The peptide of the specific peptide/MHC complex may
be associated
with at least one of a tumorigenic state, an infectious state and a disease
state, or the peptide
of the specific peptide/MHC complex may be specific to a particular organ or
tissue.
Alternatively, the presentation of the peptide of the specific peptide/MHC
complex in the context
of an MHC molecule may be novel to cancer cells, or may be greatly increased
in cancer cells
when compared to normal cells. The peptide of the specific peptide/MHC complex
may
comprise any of SEQ ID NOS:1-3 and 6.
[0027] The present invention is further directed to an isolated nucleic acid
segment encoding
a T cell receptor mimic comprising an antibody or antibody fragment reactive
against a specific
peptide/MHC complex, wherein the antibody or antibody fragment can
differentiate the specific
peptide/MHC complex from the MHC molecule alone, the peptide alone, and a
complex of MHC
and an irrelevant peptide. The peptide of the specific peptide/MHC complex may
be associated
with at least one of a tumorigenic state, an infectious state and a disease
state, or the peptide
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of the specific peptide/MHC complex may be specific to a particular organ or
tissue.
Alternatively, the presentation of the peptide of the specific peptide/MHC
complex in the context
of an MHC molecule may be novel to cancer cells, or may be greatly increased
in cancer cells
when compared to normal cells. The peptide of the specific peptide/MHC complex
may
comprise any of SEQ ID NOS:1-3 and 6.
[0028] The present invention is also related to an immunogen used in
production of a T cell
receptor mimic. The immunogen includes a multimer of two or more identical
peptide/MHC
complexes, such as a tetramer, wherein the peptide/MHC complexes are capable
of retaining
their 3-dimensional form for a period of time sufficient to elicit an immune
response in a host
such that antibodies that recognize a three-dimensional presentation of the
peptide in the
binding groove of the MHC molecule are produced. The antibodies so produced
are capable
of differentiating the peptide/MHC complex from the MHC molecule alone, the
peptide alone,
and a complex of MHC and irrelevant peptide. The peptide of the specific
peptide/MHC
complex may be associated with at least one of a tumorigenic state, an
infectious state and a
disease state, or the peptide of the specific peptide/MHC complex may be
specific to a
particular organ or tissue. Alternatively, the presentation of the peptide of
the specific
peptide/MHC complex in the context of an MHC molecule may be novel to cancer
cells, or may
be greatly increased in cancer cells when compared to normal cells. The
peptide of the specific
peptide/MHC complex may comprise any of SEQ ID NOS:1-3 and 6.
[0029] Other objects, features and advantages of the present invention will
become apparent
from the following detailed description when read in conjunction with the
accompanying figures
and appended claims.
DESCRIPTION OF THE DRAWINGS
[0030] The patent or application file contains at least one drawing executed
in color. Copies
of this patent or patent application publication with color drawing(s) will be
provided by the
Office upon request and payment of the necessary fee.
[0031] Fig. 1 illustrates size exclusion chromatography on a Sephadex S-75
column of a
mixture of refolded heavy and light (R2m) chains of HLA-A2 with synthetic
peptide
(LLGRNSFEV; SEQ ID NO:1). Peptide-HLA-A2 folded monomers were prepared and
purified
using S-75 size exclusion chromatography. Monomers consisting of peptide-HLA-
A2 were
prepared by mixing heavy chain (1 pM) together with beta-2 microglobulin (2
pM) and 10 mg
of the desired peptide in buffer (1 L) optimized to facilitate folding of
conformationally correct
peptide loaded HLA complexes. After 3 days of folding, the sample is
concentrated 100-fold
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to 10 mL using an Amicon concentrator. The concentrated sample was filtered
through a 0.2
pm filter (Millipore) and purified by FPLC (Pharmacia) chromatography using an
S-75 size
exclusion column (Pharmacia). The sample was applied to the column and washed
at 2
mL/min with buffer (PBS pH 7.4). Fig. 1 shows the typical chromatogram profile
for the
purification of refolded peptide-HLA-A2 monomer. In this Fig., 5 peaks are
seen, which are
marked as aggregates, refolded monomer, HLA-A2 heavy chain, beta2-
microglobulin, and
peptide alone. A typical purification will yield 8 to 12 mg of peptide-HLA-A2
monomer. After
collecting the desired fractions (generally in 50 mL) the sample is
concentrated to approximately
5 mL using an Amicon concentrator and biotinylated with biotin ligase
following standard
procedures (Avidity, CO). The biotin labeled monomer was isolated using the
same approach
as described above (data not shown). The biotin labeled material may then be
used for making
tetramers as described in Fig. 2.
[0032] Fig. 2 illustrates preparation and purification of peptide-HLA tetramer
using size
exclusion chromatography on a Sephadex S-200 column of the multimerized
refolded monomer
peak of Fig. 1. To form tetramers of peptide-HLA-A2, biotin labeled monomer
was mixed with
streptavidin at either 4:1 or 8:1 molar ratios. The precise ratio was
determined for each
peptide-HLA preparation and was based on the ratio of the two proteins which
generates the
largest amount of tetramer band as determined by gel shift assays by SDS-PAGE.
Generally,
8 mg of biotin labeled monomer was used, and after mixing with the appropriate
amount of
streptavidin, the sample (usually in 5 to 10 mL) was applied to the S-200
column for purification
by FPLC. Fig. 2 shows the chromatogram profile for a typical tetramer
purification run on an
S-200 column, and as shown, 4 peaks are present which represent tetramer,
trimer, dimer and
monomer forms of the pepticte-HLA-A2 complex. 3 and 4 mg of purified tetramer
was routinely
produced.
[0033] Fig. 3 illustrates the stability of the 264 peptide-HLA-A2 tetramers.
Tetramer stability
was assessed in mouse serum at 4 C and 37 C. 25 pg of 264 peptide-tetramer
complex was
added to 5 mL of 100% mouse serum and incubated at 4 C and 37 C for 75 hr. At
designated
times, 50 pL aliquots of sample were removed and stored at -20 C and remained
frozen until
completion of the experiment. To determine the integrity of the peptide-HLA
tetramer,
samples were evaluated using a sandwich ELISA and two antibodies, BB7.2 and
W6/32 that
bind only conformationally intact peptide-HLA tetramers. An ELISA protocol was
developed
using 96-well plates (Nunc maxisorb plates) that were coated overnight (O/N)
at 4 C with 0.5
pg of BB7.2,washed with buffer (PBS/0.05% Tween-20) and then blocked with 200
pl of 5%
milk for 1 hr at room temperature. Sample (50 pL) from each time point was
assayed in
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duplicate wells, incubated for 1 hr at room temperature and washed; then, 50
pL of a 1:1000
dilution of biotin conjugated W6/32 antibody was added to each well and
incubated for 1 hr at
room temperature. To detect bound antibody, the streptavidin-HRP (horseradish
peroxidase)
conjugate was added to wells at 1:500 dilution, incubated for 15 minutes and
washed; the assay
was then developed using ABTS substrate. All sample signals were plotted as %
of control.
Control tetramerwas added to serum, mixed, and immediately removed for
assaying by ELISA.
The stability half-life for the 264-peptide-HLA-A2 tetramer at 4 C was
greaterthan 72 hrs, while
at 37 C the stability half-life was approximately 10 hrs.
[0034] Fig. 4 illustrates the complete structure of the peptide-HLA-A2
tetramer immunogen,
as obtained from the tetramer peak of Fig. 2, and recognition of the peptide-
HLA epitope by a
TCR mimic.
[0035] Fig. 5 illustrates the development of an ELISA assay to screen mouse
bleeds to
determine if there are antibodies specific to the peptide-of-interest-HLA-
molecule complex
present. The schematic illustrates two newly developed screening assays for
detection of
anti-peptide-HLA specific antibodies from immunized mouse serum. Assay #2
evolved from
Assay #1.
[0036] Fig. 6 illustrates the results from an ELISA of 6 individual bleeds
from Balb/c mice
immunized with tetramers of 264 peptide-HLA-A2, using assay format #2 as
described in Fig.
5. Mice (male and female Balb/c; 13 and 12 groups, respectively) were
immunized 4 times every
2 weeks by subcutaneous injection in the region behind the head or in the side
flanks with 100
pl containing 50 pg of 264 peptide-HLA-A2 tetramer and 25 pg of QuilA
(adjuvant). Bleeds
were taken at 3 weeks, 5 weeks and just prior to sacrificing the mice. Fig. 6
shows screening
results from mice sera after 3 immunizations (week 5). Detection of polyclonal
antibodies
reactive for 264 peptide-HLA-A2 tefiramerwas carried out by ELISA (assay #2
described in Fig.
5). The ELISA results demonstrate that a 264 peptide-HLA-A2 antibody response
can be
elicited in both male (13M1-M3) and female (12M1-M3) mice using the
immunization protocol
and screening assay of the presently disclosed and claimed invention.
[0037] Fig. 7 illustrates development of cell-based direct and competitive
binding assays for
screening mouse bleeds for antibodies specific to the peptide-of-interest-HLA-
molecule
complex. The schematic illustrates two newly developed cell-based screening
assays for
detection of anti-peptide-HLA specific antibodies from immunized mouse serum.
Two cell
based assays were developed: Assay #3 is a Cell-based direct binding approach
and Assay #4
is a Cell-based competitive binding approach which uses soluble monomer or
tetramer
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peptide-HLA-A2 complexes as competitors and non-competitors. The sensitivity
of Assay # 4
is much greater than Assay #3.
[0038] Fig. 8 illustrates peptide loading of T2 cells. T2 cells (HLA-A2+, TAP
deficient) were
stained with BB7 antibody (specific for properly folded HLA-A2, ATCC #HB-82)
to demonstrate
that addition of exogenous peptide increased the surface expression of the HLA-
A2 molecule.
5x105 T2 cells were incubated in 100pI of buffer containing 100 pg of either
264 or eIF4G
peptide for 6 hours at 37 C, washed and stained with 0.5 pg BB7.2 for 20 min.
Negative control
cells were not pulsed with peptide. After staining, the reaction was washed
once with 3-4 ml
wash buffer and resuspended in approximately 100 pl of wash buffer containing
0.5 pg of
FITC-conjugated goat anti-mouse IgG (Caltag, Burlingame, CA). Cells were
washed as above
and resuspended in 0.5 ml wash bufferfor analysis. Samples were collected on a
FACScan (BD
biosciences, San Diego, California) and analyzed using Cell Quest software
(version 3.3, BD
Biosciences). Peptide pulsed T2 cells (open traces) shifted significantly to
the right when
stained, indicating the presence of HLA-A2 molecules on the surface, while
unpulsed cells did
not.
[0039] Fig. 9 illustrates an example of the cell-based direct binding assay of
Fig. 7, and
contains the results of staining of 264 peptide-loaded T2 cells with the 13M2
mouse bleed. T2
cells (HLA-A2+, TAP deficient) were stained with preabsorbed, diluted serum
from mouse 13M2
(immunized with 264 tetramers) to demonstrate that antibodies exist in the
serum which are
specific for the 264p-HLA-A2 complex. 5x105 T2 cells were incubated in 100 pl
of buffer
containing 100 pg of either 264 or eIF4G peptide for 6 hours at 37 C, washed
and stained with
100 pl of a 1:200 dilution of preabsorbed sera for 20 min. After staining, the
reaction was
washed once with 3-4 ml wash buffer and resuspended in approximately 100 pl of
wash buffer
containing 0.5 pg of FITC-conjugated goat anti-mouse IgG (Caltag, Burlingame,
CA). Cells
were washed as above and resuspended in 0.5 ml wash buffer for analysis.
Samples were
collected on a FACScan (BD biosciences, San Diego, California) and analyzed
using Cell Quest
software (version 3.3, BD Biosciences). 264 peptide-pulsed T2 cells (open
trace) shifted
significantly to the right of the eIF4G peptide pulsed T2s when stained,
indicating the presence
of 264p-HLA-A2 specific antibodies from immunized mice.
[0040] Fig. 10 illustrates that pre-bleed samples (mice bleeds taken prior to
immunization)
show no sign of reactivity to T2 cells pulsed with either the 264- or eIF4G
peptides. T2 cells
(HLA-A2+, TAP deficient) were stained with diluted serum from mouse C3M4
(unimmunized)
to demonstrate that antibodies do not preexist in the serum which are specific
for the 264p-
HLA-A2 complex. 5x105 T2 cells were incubated in 100 pl of buffer containing
100 pg of either
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264 or eIF4G peptide for 6 hours at 37 C, washed and stained with 100 pl of a
1:200 dilution
of sera for 20 min. After staining the reaction was washed once with 3-4 ml
wash buffer and
resuspended in approximately 100 lal of wash buffer containing 0.5 pg of FITC-
conjugated goat
anti-mouse IgG (Caltag, Burlingame, CA). Cells were washed as above and
resuspended in
0.5 ml wash buffer for analysis. Samples were collected on a FACScan (BD
biosciences, San
Diego, California) and analyzed using Cell Quest software (version 3.3, BD
Biosciences). 264
peptide-pulsed T2 cells (filled trace) and eIF4G peptide pulsed T2s (open
trace) did not shift
significantly from the origin when stained, indicating the absence of any HLA-
A2 specific
antibodies in the mouse's serum.
[0041] Fig. 11 depicts development of assays to screen hybridomas to determine
if they are
producing anti-HLA-peptide specific antibodies. The schematic illustrates two
ELISA-based
screening assays for detection of anti-peptide-HLA specific monoclonal
antibodies from culture
supernatant. Assay #1 is an ELISA-based direct binding approach that coats
wells of a 96-well
plate with 0.5 pg of either specific or irrelevant tetramer. Hybridoma cell
culture supernatant (50
pL) was assayed in duplicate by addition to an antibody coated plate blocked
with 5% milk for
1 hr at room temperature. Plates were incubated for 1 hr at room temperature,
washed, and
probed with goat anti-mouse-HRP for 30 minutes. The assay was developed by
adding 50 pL
of either TMB or ABTS and read at 450 or 405 nm, respectively. Assay #2 is an
ELISA that
uses a competitive binding approach in which cell culture supernatant is
incubated in the
presence of either 300 ng of competitor or non-competitor (soluble monomer or
tetramer
peptide-HLA-A2 corriplexes) in wells on 96-well plates that have been coated
with 100 ng of
specific peptide-HLA-A2 tetramer and blocked with 5% milk. After 1 hr
incubation, the plate is
washed, probed with goat anti-mouse HRP and developed using TMB or ABTS.
[0042] Fig. 12 illustrates a competitive ELISA assay for evaluation of
individual hybridomas
(13M1) reactive against 264p-HLA-A2 complexes. Light grey bar = addition of
264p-HLA-A2
tetramer (competitor, 0.3 pg); Dark grey bar = addition of eIF4Gp-HLA-A2
tetramer
(non-competitor, 0.3 pg). Hybridoma cell culture supernatant (50 pL) was
incubated in the
presence of 300 ng of competitor (264 peptide-HLA-A2 tetramer) or non-
competitor (eIF4G
peptide-HLA-A2 tetramer) in wells on a 96-well plate coated previously with
100 ng of 264
peptide-HLA-A2 tetramer. After 1 hr incubation, the plate was washed, probed
with goat
anti-mouse HRP, developed using TMB or ABTS and read at 450 or 405 nm,
respectively.
Results were calculated by dividing the absorbance read in the presence of non-
competitor by
the absorbance read in the presence of competitor [eIF4G/264]. Ratios of 2 or
greater were
considered to be positive, and hybridoma clones with this desired ratio were
selected for further
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analysis. Fig. 12 shows 4 different hybridoma supernatants (M1/3-A5, M1/3-F11,
M1/4-G3,
and M1/6-A12) with a specific binding ratio [eIF4G/264] of 2 or greater.
[0043] Fig. 13 illustrates the results of a competitive ELISA assay for
evaluation of individual
hybridomas to determine if the hybridoma produced from mouse bleed 13M1
expresses anti-
264-HLA-A2 antibodies. Hybridoma cell culture supernatant (50 pL) was
incubated without any
tetramer addition or in the presence of 300 ng of competitor (264 peptide-HLA-
A2 tetramer) or
non-competitor (eIF4G peptide-HLA-A2 tetramer) in wells on a 96-well plate
coated previously
with lOOng of 264 peptide-HLA-A2 tetramer. After lhr incubation, the plate was
washed,
probed with goat anti-mouse HRP, developed using TMB orABTS and read at 450 or
405 nm,
respectively. Fig. 13 illustrates three different hybridoma supernatants with
favorable
eIF4G/264 ratios. These include M1-1 F8, M1-2G5, M1-6C7 and M3-2A6, which were
selected
for further analysis.
[0044] Fig. 14 illustrates the characterization of monoclonal antibody 13.M3-
2A6 by the cell-
based competitive binding assay. T2 cells (HLA-A2+, TAP deficient) were
stained with cell
supernatant from hybridoma 13.M3-2A6 (immunogen = 264 tetramers) in the
presence of
(1) tetramer complex that would compete with specific binding to 264p-HLA-A2;
(2) tetramer
complex that would not compete with specific binding (eIF4Gp); or (3) no
tetramer, to
demonstrate that the antibody specifically recognizes the 264p-HLA-A2 complex
on the cell
surface. Cell supernatant was pre-absorbed against 20 pg of soluble Her2/neu-
peptide-HLA-A2
complexes, diluted 1:200 and added (100 pl) to a tube containing 1 pg of
either 264p-HLA-A2
tetramer (competitor) or eIF4Gp-HLA-A2 tetramer (non competitor) for 15
minutes at room
temperature. 5x105 T2 cells were incubated in 100 pl of buffer containing 100
pg of 264 peptide
for 6 hours at 37 C, washed, resuspended in 100 pl, and added to the
preabsorbed/tetramer
treated supernatant for 20 minutes at room temperature. After staining, the
reaction was
washed once with 3-4 ml wash buffer and resuspended in approximately 100 lal
of wash buffer
containing 0.5 pg of FITC-conjugated goat anti-mouse IgG (Caltag, Burlingame,
CA). Cells
were washed as above and resuspended in 0.5 ml wash buffer for analysis.
Samples were
collected on a FACScan (BD biosciences, San Diego, California) and analyzed
using Cell Quest
software (version 3.3, BD Biosciences). 264 peptide-competition resulted in a
significant shift
of the T2 cell trace (thick line, open trace) to the left (towards the origin)
while the eIF4G
peptide competition (thin line, open trace) resulted in a much smaller shift
away from T2s
stained in the absence of tetramer, indicating the presence of a monoclonal
antibody with a high
degree of specificity for the 264p-HLA-A2 complex.
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[0045] Fig. 15 illustrates a broad outline of the epitope discovery technology
described in
detail in Hildebrand et al. (US Patent Application Publication No. US
2002/0197672A1,
published December 26, 2002, previously incorporated herein by reference).
Soluble HLA-
secreting transfectants are created in a cancerous or diseased cell line of
interest. In a
separate experiment, a normal (i.e., noncancerous or non-diseased) cell line
also transfected
with a construct encoding the soluble HLA is grown and cultured. Soluble HLA
molecules are
collected from both cell lines, and the peptides are eluted. Mass
spectrometric maps are
generated comparing cancerous (or diseased) peptides to normal peptides.
Differences in the
maps are sequenced to identify their precise amino acid sequence, and such
sequence is
utilized to determine the protein from which the peptide was derived (i.e.,
its "source protein").
This method was utilized to identify the peptide eIF4G, which has a higher
frequency of peptide
binding to soluble HLA-A2 in HIV infected cells compared to uninfected cells.
This protein is
known to be degraded in HIV infected T cells, and elevated levels of the eIF4G
peptide
presented by HLA-A2 molecules was determined using this technology.
[0046] Fig. 16 illustrates the stability of the eIF4Gp-HLA-A2 tetramers.
Tetramer stability
was assessed in mouse serum at 37 C (=) and at 4 C (=) using the
conformational antibodies
BB7.2 and W6/32. 25 pg of elF4G peptide-tetramer complex was added to 5 mL of
100%
mouse serum and incubated at 4 C and 37 C for 75 hr. At designated times, 50
pL aliquots
of sample were removed and stored at -20 C and remained frozen until
completion of the
experiment. To determine the integrity of the peptide-HLA tetramer, samples
were evaluated
using a sandwich ELISA and two antibodies, BB7.2 and W6/32, that bind only
conformationally
intact peptide-HLA tetramers. An ELISA protocol was developed using 96-well
plates (Nunc
maxisorb plates) that were coated O/N at 4 C with 0.5 pg of BB7.2, washed with
buffer
(PBS/0.05% Tween-20) and then blocked with 200 pl of 5% milk for 1 hr at room
temperature.
Sample (50 IaL) from each time point was added in duplicate wells, incubated
for 1 hr at room
temperature, washed, and then 50 pL of at 1:1000 dilution of biotin conjugated
W6/32 antibody
was added to each well and incubated for 1 hr at room temperature. To detect
bound antibody
the streptavidin-HRP (horseradish peroxidase) conjugate was added to wells at
1:500 dilution,
incubated for 15 minutes, washed, and then the assay was developed using ABTS
substrate.
All sample signals were plotted as % of control. Control tetramer was added to
serum, mixed,
and immediately removed for assaying by ELISA. The half-life of stability for
the
eIF4G-peptide-HLA-A2 tetramer at 4 C was greater than 72 hrs while at 37 C the
half-life was
approximately 40 hrs.
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[0047] Fig. 17 illustrates the results from an ELISA of bleeds from 6
individual Balb/c mice
immunized with tetramers of eIF4Gp-HLA-A2. Mouse samples from left to right
are 18.M1,
18.M2, 18.M3, 18.M4, 18.M5, 18.M6. P53-264 = 264p-HLA-A2 monomer (0.5
pg/well), Eif4G =
eIF4Gp-HLA-A2 monomer (0.5 pg/well), and Her2/neu = Her2/neu peptide-HLA-A2
monomer
(0.5 pg/well). The dilutions of sample bleeds start at 1:200 (blue bar) and
titrate down to 1:3600
(light blue bar). Mice (female Balb/c) were immunized 4 times every 2 weeks by
subcutaneous
injection in the region behind the head or in the side flanks with 100 pl
containing 50 pg of
eIF4G peptide-HLA-A2 tetramer and 25 pg of QuilA (adjuvant). Bleeds were taken
at 3 weeks,
weeks and just prior to sacrificing mice. Fig. 17 shows results from mice sera
after 3
immunizations (week 5). Detection of polyclonal antibodies reactive for eIF4G
peptide-HLA-A2
tetramer was carried out by ELISA (assay #2 described in Fig. 5). The ELISA
results
demonstrate that a 264 peptide-HLA-A2 antibody response can be elicited in
female Balb/c
(18.M1-M6) mice using the immunization protocol and screening assay of the
presently
disclosed and claimed invention.
[0048] Fig. 18 illustrates T2 cell direct binding assay performed according to
the method of
Fig. 7. T2 cells (HLA-A2+, TAP deficient) were stained with BB7.2 antibody
(specific for
HLA-A2) to demonstrate that HLA-A2 was present on the surface on these cells.
T2 cells were
incubated in 100 pl of buffer containing 100 pg of either 264 or eIF4G peptide
for 6 hours at
37 C, washed and stained with 0.5 pg BB7.2 for 20 min. Negative control cells
were not pulsed
with peptide. After staining, the reaction was washed once with 3-4 ml wash
buffer and
resuspended in approximately 100 pl of wash buffer containing 0.5 pg of FITC-
conjugated goat
anti-mouse IgG (Caltag, Burlingame, CA). Cells were washed as above and
resuspended in
0.5 ml wash buffer for analysis. Samples were collected on a FACScan (BD
biosciences, San
Diego, California) and analyzed using Cell Quest software (version 3.3, BD
Biosciences).
BB7.2 binding was slightly stronger with T2 cells loaded with 264 peptide as
indicated by the
slightly greater rightward shift with 264 pulsed-T2 cells compared to eIF4G
pulsed cells.
[0049] Fig. 19 illustrates the results of staining of eIF4Gp-loaded T2 cells
with a bleed from
an eIF4Gp-HLA-A2 immunized mouse. T2 cells (HLA-A2+, TAP deficient) were
stained with
preabsorbed, diluted serum from mouse 18M2 (immunized with eIF4G tetramers) to
demonstrate that antibodies exist in the serum which are specific for the
eIF4Gp-HLA-A2
complex. 5x105 T2 cells were incubated in 100 pl of buffer containing 100 pg
of either eIF4G
or 264 peptide for 6 hours at 37 C, washed and stained with 100 pl of a 1:200
dilution of
preabsorbed sera for 20 min. After staining, the reaction was washed once with
3-4 ml wash
buffer and resuspended in approximately 100 pl of wash buffer containing 0.5
pg of
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FITC-conjugated goat anti-mouse IgG (Caltag, Burlingame, CA). Samples were
collected on
a FACScan (BD biosciences, San Diego, California) and analyzed using Cell
Quest software
(version 3.3, BD Biosciences). eIF4G peptide-pulsed T2 cells (open trace)
shifted significantly
to the right of the 264 peptide pulsed T2s when stained, indicating the
presence of
eIF4Gp-HLA-A2 specific antibodies from immunized mice.
[0050] Fig. 20 illustrates the results of a T2 cell-competitive binding assay,
the method of
which is outlined in Fig. 7. T2 cells (HLA-A2+, TAP deficient) were stained
with pre-absorbed,
diluted serum from mouse 18M2 (immunized with eIF4Gp tetramers) in the
presence of (1)
monomer complex that would compete with specific binding to eIF4Gp-HLA-A2; (2)
monomer
complex that would not compete with specific binding (264p); or (3) no
monomer, to
demonstrate that the antibody specifically recognizes the eIF4Gp-HLA-A2
complex on the cell
surface. Cell supernatantwas pre-absorbed against 20 pg of soluble Her2/neu-
peptide-HLA-A2
complexes, diluted 1:200 and added (100 pl) to tube containing 1 pg of either
eIF4Gp-HLA-A2
monomer (competitor) or 264p-HLA-A2 monomer (non competitor) for 15 minutes at
room
temperature. 5x105 T2 cells were incubated in 100 pl of buffer containing 100
pg of eIF4G
peptide for 6 hours at 37 C, washed, resuspended in 100 pl, and added to the
preabsorbed/monomer treated supernatant for 20 minutes at room temperature.
Afterstaining,
the reaction was washed once with 3-4 ml wash buffer and resuspended in
approximately 100
tal of wash buffer containing 0.5 pg of FITC-conjugated goat anti-mouse IgG
(Caltag,
Burlingame, CA). Cells were washed as above and resuspended in 0.5 ml wash
buffer for
analysis. Samples were collected on a FACScan (BD biosciences, San Diego,
California) and
analyzed using Cell Quest software (version 3.3, BD Biosciences). e1F4G
peptide-competition
resulted in a significant shift of the T2 cell trace (thick line, open trace)
to the left (towards the
origin) while the 264 peptide competition (thin line, open trace) resulted in
a much smaller shift
away from T2s stained in the absence of monomer, indicating the presence of
polyclonal
antibodies with a high degree of specificity for the eIF4Gp-HLA-A2 complex.
[0051] Fig. 21 illustrates the results of another T2 cell-competitive binding
assay similar to the
one described in Fig. 20, except that the competitor mixed with the mouse
bleed prior to
reacting with the T2 cells was in the form of a tetramer rather than a
monomer. T2 cells
(HLA-A2+, TAP deficient) were stained with pre-absorbed, diluted serum from
mouse 18M2
(immunized with eIF4Gp tetramers) in the presence of (1) tetramer complex that
would compete
with specific binding to eIF4Gp-HLA-A2; (2) tetramer complex that would not
compete with
specific binding (264p); or (3) no tetramer, to demonstrate that the antibody
specifically
recognizes the eIF4Gp-HLA-A2 complex on the cell surface. Cell supernatant was
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pre-absorbed against 20 pg of soluble Her2/neu-peptide-HLA-A2 complexes,
diluted 1:200 and
added (100 lal) to tube containing 1pg of either eIF4Gp-HLA-A2 tetramer
(competitor) or
264p-HLA-A2 tetramer (non competitor) for 15 minutes at room temperature.
5x105 T2 cells
were incubated in 100 pl of buffer containing 100 pg of eIF4G peptide for 6
hours at 37 C,
washed, resuspended in 100 pl, and added to the preabsorbed/tetramer treated
supernatant
for 20 minutes at room temperature. After staining, the reaction was washed
once with 3-4 ml
wash buffer and resuspended in approximately 100 pl of wash buffer containing
0.5 pg of
FITC-conjugated goat anti-mouse IgG (Caltag, Burlingame, CA). Cells were
washed as above
and resuspended in 0.5 mi wash bufferfor analysis. Samples were collected on a
FACScan (BD
biosciences, San Diego, California) and analyzed using Cell Quest software
(version 3.3, BD
Biosciences). ei F4G peptide-competition resulted in a significant shift of
the T2 cell trace (thick
line, open trace) to the left (towards the origin), while the 264 peptide
competition (thin line,
open trace) resulted in a much smaller shift away from T2s stained in the
absence of tetramer,
indicating the presence of polyclonal antibodies with a high degree of
specificity for the
eIF4Gp-HLA-A2 complex.
[0052] Fig. 22 illustrates the binding specificity of mAb 4F7, as determined
by ELISA. To
assess the binding specificity of 4F7 TCR mimic, a 96-well plate was coated
with 0.5 pg of
specific monomer (elF4G-peptide-HLA-A2) and non-specific monomers (264, VLQ
and TMT
peptide-HLA-A2 monomers). The VLQ and TMT peptides are derived from the human
beta-chorionic gonadotropin protein, as described in detail herein after.
After blocking wells
with 5% milk, 100 ng of 4F7 antibody was added to each well and incubated for
1 hr at room
temperature. Plates were washed, probed with 500 ng/well of goat anti-mouse
IgG-HRP and
developed using ABTS. These results show specific binding of 4F7 to eIF4G
peptide-HLA-A2
tetramer coated wells but no binding to wells coated with non-relevant peptide-
loaded HLA-A2
complexes.
[0053] Fig. 23 illustrates 4F7 TCR mimic binding affinity and specificity
evaluated by surface
plasmon resonance (BIACore). SPR (BIACore) was used to determine the binding
affinity
constant for 4F7 TCR mimic. Various concentrations of soluble monomer peptide-
HLA-A2 (10,
20, 50, and 100 nM) were run over a 4F7 coated chip (4F7 coupled to a
biosensor chip via
amine chemistry), and then BIACore software was used to best fit the binding
curves
generated. The affinity constant of 4F7 mAb for its specific ligand was
determined at 2 x10-9M.
[0054] Fig. 24 illustrates the specific binding of purified 4F7 mAb to eIF4G
peptide pulsed
cells. T2 cells (HLA-A2+, TAP deficient) were stained with cell supernatant
from hybridoma 4F7
(immunogen = eIF4Gp tetramers) to demonstrate binding specificity for this
monoclonal
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antibody for the eIF4Gp-HLA-A2 complex. 5x105 T2 cells were incubated in 100
NI of buffer
containing 100 pg of eIF4G, 264, or TMT peptide for 6 hours at 37 C, washed
and stained with
100 pl of 4F7 culture supernatant for 20 min. In addition, cells that were not
peptide pulsed were
stained in an identical manner with 4F7 to determine the level of background
or endogenous
eIF4Gp presented by HLA-A2 on T2 cells. After staining, the reactions were
washed once with
3-4 ml wash buffer and resuspended in approximately 100 pl of wash buffer
containing 0.5 pg
of FITC-conjugated goat anti-mouse IgG (Caltag, Burlingame, CA). Cells were
washed as
above and resuspended in 0.5 mi wash buffer for analysis. As shown in Fig. 24-
A, samples
were collected on a FACScan (BD biosciences, San Diego, California) and
analyzed using Cell
Quest software (version 3.3, BD Biosciences). eIF4G peptide-pulsed T2 cells
shifted most
significantly to the right of the IgG1 isotype stain. Both 264 and TMT peptide
pulsed cells
overlaid exactly with the 4F7 monoclonal stain of T2 cells that were not
peptide pulsed,
indicating that 4F7 recognizes a low level of endogenous eIF4G peptide on T2
cells. These
data also demonstrate specific binding of the 4F7 monoclonal antibody for
eIF4G
peptide-pulsed T2 cells. Because peptide pulsed T2 cells showed a greater
staining intensity
with BB7.2 monoclonal antibody compared to cells that were not pulsed (Fig. 24-
B), it is
concluded that the 4F7 monoclonal antibody does not react non-specifically
against HLA-A2.
[0055] Fig. 25 illustrates that 4F7 TCRm detects endogenous eIF4G(720) peptide-
HLA-A2
complexes on an HLA-A2 positive tumor cell line but not on a normal mammary
epithelial cell
line. (A) A human mammary epithelial cell line (NHMEC) and (B) a human breast
carcinoma
cell line (MDA-MB-231) were grown in medium specified by the ATCC and were
detached using
1 X trypsin/EDTA (0.25% trypsin/2.21 mM EDTA in HBSS without sodium
bicarbonate, calcium
and magnesium) (Mediatech, Herndon, VA). Cells were washed and then stained
with 5 pg/mI
of isotype control mAb or 4F7 TCRm-FITC in PBS/0.5% FBS/2mM EDTA
(staining/wash
buffer). FACS analysis was performed on a FACScan (BD Biosciences, San Diego,
CA). The
results from flow cytometric studies are expressed as mean fluorescence
intensity (MFI) in
histogram plots.
[0056] Fig. 26 illustrates that purified 4F7 mAb binds eIF4Gp-HLA-A2 complexes
on human
breast carcinoma cell line MCF-7. MCF-7 cells (HLA-A2+) were stained with cell
supernatant
from hybridoma 4F7 (immunogen = eIF4Gp tetramers) in the presence of (1)
tetramer complex
that would compete with specific binding to eIF4Gp-HLA-A2; (2) tetramer
complex that would
not compete with specific binding (264p); or (3) no tetramer, to demonstrate
that the antibody
specifically recognizes the endogenous eIF4Gp-HLA-A2 complex on the cell
surface. 5x105
MCF-7 cells were incubated in 100 NI of buffer containing 100 pl of 4F7
culture supernatant plus
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1pg of either eIF4Gp-HLA-A2 tetramer (competitor) or264p-HLA-A2 tetramer (non
competitor)
or no addition for 15 minutes at room temperature. After staining, the
reactions were washed
once with 3-4 ml wash buffer and resuspended in approximately 100 pl of wash
buffer
containing 0.5 pg of PE-conjugated goat anti-mouse IgG (Caltag, Burlingame,
CA). Cells were
washed as above and resuspended in 0.5 ml wash buffer for analysis. Samples
were collected
on a FACScan (BD biosciences, San Diego, California) and analyzed using Cell
Quest software
(version 3.3, BD Biosciences). The data shown in Fig. 26-A demonstrate 4F7
binding specificity
for endogenous peptide eIF4Gp-HLA-A2 complexes on MCF-7 tumor cells. In panel
B, it is
shown that 4F7 and BB7.2 do not bind to HLA-A2 negative BT-20 breast cancer
cells, further
supporting the claim for 4F7 monoclonal antibody binding specificity for eIF4G
peptide
presented in the context of HLA-A2.
[0057] Fig. 27 illustrates staining of MDA-MB-231 cells with 4F7 mAb (50 ng)
in the absence
or presence of soluble peptide-HLA-A2 monomers including el F4Gp (competitor;
25 nM), 264p
(non-competitor; 25 nM) or Her2/neu peptide (non-competitor; 25 nM). MDA-MB-
231 cells
(HLA-A2+) were stained with cell supernatant from hybridoma 4F7 (immunogen =
eIF4Gp
tetramers) in the presence of (1) monomer complex that would compete with
specific binding
to eIF4Gp-HLA-A2; (2) monomer complex that would not compete with specific
binding to
eIF4Gp-HLA-A2 (264p and Her-2/neu); or (3) no monomer, to demonstrate that the
antibody
specifically recognizes endogenous eIF4Gp-HLA-A2 complex on the cell surface.
5x105
MDA-MB-231 cells were incubated in 100 pl of buffer containing 100 pl of 4F7
culture
supernatant plus 25 nM of eIF4Gp-HLA-A2 tetramer (competitor), 264p-HLA-A2
tetramer or
Her-2/neu-HLA-A2 (non competitors) or no addition for 15 minutes at room
temperature. After
staining, the reactions were washed once with 3-4 ml wash buffer and
resuspended in
approximately 100 lal of wash buffer containing 0.5 pg of PE-conjugated goat
anti-mouse IgG
(Caltag, Burlingame, CA). Cells were washed as above and resuspended in 0.5 mi
wash buffer
for analysis. Samples were collected on a FACScan (BD biosciences, San Diego,
California)
and analyzed using Cell Quest software (version 3.3, BD Biosciences). Fig. 27-
A demonstrates
4F7 binding specificity for endogenous eIF4Gp-HLA-A2 complexes on MDA-231
tumor cells.
Binding of the 4F7 TCR mimic to MDA-MB-231 cells was significantly reduced
(see leftward
shift with peak) in the presence of 25nM of competitor (eIF4Gp-HLA-A2
monomer). In panels
B and C, it is shown that 4F7 binding was not blocked when non-relevant (264
and Her-2/neu)
peptide-HLA-A2 monomers were used to compete with 4F7 binding to MDA-231
cells. These
findings support previous binding specificity data and indicate eIF4Gp-HLA-A2
as a novel tumor
antigen.
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[0058] Fig. 28 illustrates endogenous eIF4G peptide presented by HLA-A2
molecules on the
surface of HIV-1 infected and non-infected human CD4+ T cells. Mock infected
(A-C; upper
panels) or HIV-1 infected (D-F and G-I) human CD4+ T cells were stained on day
5 post
infection (PI) with IgG, (isotype control), 1 B8 TCRm (anti-Her2(369)-HLA-
A*0201; specificity and
isotype control) or with 4F7 TCRm. HIV-1 exposed CD4+ T cells were gated based
on p24
expression and analyzed separately as (D-F) infected-p24 positive (middle
panels) or (G-I)
non-infected-p24 negative (bottom panels).
[0059] Fig. 29 illustrates time-dependent expression of eIF4G(720) peptide-HLA-
A2
complexes on HIV-infected cells. Human CD4+ T cells were infected with HIV-1
(strain Ba-L)
at an MOI of 1.0 and stained with (A) 4F7 TCRm or (B) isotype control on days
3 thru 9
post-infection. Non-infected cells (p24 negative) are represented by gray
bars. HIV-1 infected
cells (p24 positive) are represented by black bars.
[0060] Fig. 30 illustrates HLA-peptide tetramer inhibition of 4F7 staining of
HIV-1 infected
cells. Human CD4+ T cells were infected with HIV-1 (strain Ba-L) at an MOI of
1.0 and stained
with mAb 4F7 TCRm on (A) day 4 PI and (B) day 5 PI in the presence of
eIF4G(720)-HLA-A*0201-tetramer (competitor), p53(264)-HLA-A*0201-tetramer (non-
competitor)
or VLQ(44)-HLA-A*'0201 tetramer (non-competitor) or without tetramer addition.
Results are
from staining p24 positive CD4+ T cells and are presented as % eIF4G(720)
expression.
[0061] Fig. 31 illustrates the characterization of 1 B8 TCRm binding
specificity. HLA-A2
tetramer complexes were loaded with 0.1 pg of each of the following peptides:
Her2 (369-377;
KIFGSLAFL (SEQ ID NO:3)), VLQ (44-52; VLQGVLPAL (SEQ ID NO:5)), eIF4G (720-
748;
VLMTEDIKL (SEQ ID NO:2)) and TMT (40-48; TMTRVLQGC (SEQ ID NO:4)). Recombinant
proteins were detected by staining with 1 B8 TCR mAb specific for Her-2369-A2
complex (A), 3F9
TCRm mAb specific for TMT40-A2 complex (B) and BB7.2 mAb specific for HLA-A2.1
(C)
followed by ELISA as described herein. Data are representative of three
independent
experiments.
[0062] Fig. 32 illustrates the characterization of I B8 TCRm binding detection
sensitivity. (A)
T2 cells (5 x 105) were incubated in AIM-V medium (Invitrogen, Carlsbad, CA)
and loaded with
mM Her2369, eIF4G720, TMT40 peptide or no peptide. After 4 hr, the cells were
washed to
remove excess peptide and stained with 0.5 tag/mI of 1 B8 TCRm mAb antibody.
Bound mAb
was detected using the PE-conjugated goat anti-mouse IgG heavy chain specific
polyclonal Ab.
Filled area represents T2 cells stained with IgG, isotype control. Data are
representative of
three independent staining procedures. (B) T2 cells were treated with acid to
remove
endogenous peptide bound to HLA-A2, pulsed with 20 irrelevant peptides or 20
irrelevant
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peptides plus the Her2(369) peptide and then stained with 1 B8 TCRm mAb. T2
cells (5 x 106/mL)
were acid stripped (0.131 M citric acid, 0.067M Na2HPO4, pH 3.3) for 45
seconds, washed
twice with 50 ml of RPMI supplemented with 2mM Hepes and resuspended at 3.3 x
106/ml in
30 pg/mL of P2-microglobulin (Fitzgerald Industries, Concord, MA) (23, 24).
Cells were then
incubated for 3.5 hrs in a 20 C water bath with 2 pM of each peptide, washed,
stained with
antibodies and evaluated on a BD FACScan. Subsequent analysis was performed
using
CeIlQuest software version 3.3 (BD Biosciences, San Diego, CA). As a control,
T2 cells pulsed
with 20 peptides plus p369 peptides were stained with IgG1 isotype-control.
(C) HLA-A2+/Her2-
normal human mammary epithelial cells were stained with 0.5 pg of IgG, isotype
control, 1 B8
TCRm or BB7.2 mAb. (D) HLA-A2+/Her2- human PBMCs were stained with 0.5 pg of
anti-Her2
(TA-1) antibody, 3F9 TCRm, 1 B8 TCRm or BB7.2 antibody. (E) T2 cells were
incubated with
decreasing concentrations (2500-0.08 nM as indicated by the arrows) of p369
peptide and
stained with 1 B8 TCRm mAb. In all experiments bound antibody was .detected
using goat
anti-mouse PE conjugate.
[0063] Fig. 33 illustrates that 1 B8 detects endogenous Her2/neu peptide-HLA-
A2 complexes
on HLA-A2 positive tumor cells. All adherent tumor cell lines were grown in
medium specified
by the ATCC and were detached using 1X trypsin/EDTA (0.25% trypsin/2.21 mM
EDTA in
HBSS without sodium bicarbonate, calcium and magnesium (Mediatech, Herndon,
VA). Cells
were washed and then stained with 5mg/mi of 1 B8 TCRm in PBS/0.5% FBS/2mM EDTA
(staining/wash buffer), and the bound TCRm was detected by subsequent
incubation with
PE-labeled goat anti-mouse IgG. FACS analysis was performed on a FACScan (BD
Biosciences, San Diego, CA). The results from flow cytometric studies are
expressed either as
mean fluorescence intensity (MFI) in histogram plots or as the mean
fluorescence intensity ratio
(MFIR), the ratio between the MFI of cells stained with the selected mAb and
the MFI of cells
stained with the isotype-matched mouse Ig. Generation of MFRI values
normalizes background
staining between the cell lines. (A) Human tumor cell lines were stained with
0.5 pg of isotype
control mAb (thin dark gray line), 3F9 TCRm mAb (thick black line) and 1 B8
TCRm mAb (thick
gray line). (B) Human tumor cells pre-treated with IFN-y (20ng/ml) plus TNF-a
(3ng/ml) for 24
hr and then stained, with the same three antibodies. Isotype control mAb (thin
gray line), 3F9
TCRm mAb (thick black line) and 1 B8 TCRm (thick gray line).
[0064] Fig. 34 illustrates HLA-peptide specific inhibition of human tumor cell
staining and CTL
killing. (A) MDA-MB-231 cells (5 x 105) were incubated for 1 h with 0.5 Ng/mi
of 1 B8 TCRm
mAb in the presence of 0.1 or 1.0 pg/mi of Her2/neu peptide-HLA-A2 tetramer,
1.0 Ng/mi TMT
peptide-HLA-A2 tetramer or no tetramer. After staining, the reactions were
washed once and
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resuspended in 100 pl of wash buffer containing 0.5 pg of PE-conjugated goat
anti-mouse IgG.
Cells were washed as described previously and resuspended in 0.5 ml of wash
buffer for
characterization on a FACScan. Following incubation, cells were analyzed by
flow cytometry
as described herein. (B) Confirmation that the CTL line generated in the HLA-
A2-Kb transgenic
mice was specific for the Her2(369)-A2 epitope. The CTL line was generated as
described by
Lustgarten et al (1997). The Her2369 specific CTL line was maintained in vitro
by weekly
restimulation. Briefly, CTLs (1x106) were restimulated in 2 ml cultures with
0.2 x 106 irradiated
Jurkat-A2.1 cells (20,000 rad) that were preincubated with Her-2/neu peptide
(15 pM).
Irradiated (3000 rad) C57BL/6 spleen cells (5 x 105) were added as fillers.
Restimulation
medium was complete RPMI containing 2% (v/v) supernatant from concanavalin-A
stimulated
rat spleen cells. T2 cells pulsed with Her2(369) peptide or not pulsed were
incubated with CTL
in a 6h 5'Cr release assay at an E:T ratio of 10:1. (C) MDA-231 cells were
either not treated
(white bars) or pre-treated for 24 h with rIFN-y (20 ng/ml) and TNF-a (3
ng/ml) (black bars).
Anti-Her2(369)-A2 CTL activity was then evaluated in the absence or presence
of 0.5 pg of 1 B8
TCRm or BB7.2 mAbs in a 6 h 51Cr release assay at an E:T ratio of 10:1. All
CTL assays were
done in triplicate from 3 independent experiments. T2 cells pulsed with
peptides and tumor
cells (MDA-MB-231, Saos-2, MCF-7, SW620 and COL0205) were incubated with 150
pCi of
5'Cr-sodium chromate for 1 hour at 37 C. Cells were washed three times and
resuspended in
complete RPMI medium. For the cytotoxicity assay, 51Cr-labeled target cells
(104) were
incubated at a 10:1 CTL:target ratio in a final volume of 200 NI in U-bottomed
96-well microtiter
plates. Previous studies have shown optimal killing at a 10:1 CTL:tumor cell
ratio (Lustgarten
et al., 1997). Supernatants were recovered after 4-7 hours of incubation. The
percent specific
lysis was determined by the formula: percent specific lysis = 100 x
[(experimental release -
spontaneous release)/ (maximum release - spontaneous release)]. Anti-Her2(369)-
A2 (1 B8) and
anti-A2.1 mAb (BB7.2) were added to the assay to determine that the CTL lysis
was specific
for the Her2/neu/369-peptide-A2.1 complex and A2.1 restricted, respectively.
Prior to the
addition of the effector cells, tumor cells were incubated in the presence or
absence of 0.5
pg/ml of 1 B8, BB7.2, or murine IgG, and IgG2b isotype control antibodies.
[0065] Fig. 35 illustrates that 1 B8 mAb does not bind to soluble Her2/neu
peptide.
MDA-MB-231 cells (HLA-A2+) were stained with cell supernatant from hybridoma 1
B8
(immunogen = Her-2/neu tetramers) in the presence or absence of 100 pM of
exogenously
added Her-2/neu peptide. 5x105 MDA-MB-231 cells were incubated in 100 pl of
buffer
containing 100 pl of 1 B8 culture supernatant for 15 minutes at room
temperature. After staining
the reactions were washed once with 3-4 ml wash buffer and resuspended in
approximately 100
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pI of wash buffer containing 0.5 pg of PE-conjugated goat anti-mouse IgG
(Caltag, Burlingame,
CA). Cells were washed as above and resuspended in 0.5 ml wash buffer for
analysis.
Samples were collected on a FACScan (BD biosciences, San Diego, California)
and analyzed
using Cell Quest software (version 3.3, BD Biosciences). Fig. 35 demonstrates
that 1 B8 TCR
mimic has dual specificity and does not bind to Her-2/neu peptide alone.
[0066] Fig. 36 illustrates the expression of Her2/neu protein in human tumor
cell lines.
Tumor cell lines were evaluated for the expression of Her2/neu protein by
ELISA and flow
cytometry. Cellular levels of Her2/neu were determined by preparing tumor cell
lysates and
quantifying Her2/neu with the c-erbB2/c-neu Rapid Format ELISA (CalBiochem)
according to
the manufacturer's instructions. Her2/neu protein was detected in a sandwich
ELISA using two
mouse monoclonal antibodies. The detector antibody was bound to horseradish
peroxidase-conjugated streptavidin and color was developed by incubation with
TMT substrate
(Pierce). The concentration of Her2/neu in the samples was quantified by
generating a
standard curve using known concentrations of Her2/neu provided in the kit. (A)
Tumor cell
lysate was prepared from each line and analyzed for Her2/neu levels (ng/106
cells) by ELISA.
(B) Surface expression of Her2/neu on tumor cells was determined by staining
cells with 0.5
pg of anti-Her2/neu mAb (TA-1) and bound antibody was detected using Goat anti-
mouse-PE
conjugate. Results are plotted as mean fluorescence intensity ratio (MFIR)
with standard
deviation from three different experiments. Regression analysis was used to
compare the
relationship between measuring total Her2/neu antigen in cell lysates with
Her2/neu expressed
on the cell's surface. (R2 = 0.82; p < 0.05)
[0067] Fig. 37 illustrates expression of HLA-A*0201 and HLA-Her2(369) peptide
complexes on
human tumor cell lines and CTL lysis of human tumor cell lines. Tumor cell
lines were
evaluated forthe expression of HLA-A2 and Her2(369)A2 complex expression.
Tumor cells were
stained with (A) anti-HLA-A2.1 mAb (BB7.2) and (B) 1 B8 TCRm. Results are
plotted as mean
fluorescence intensity ratio (MFIR) with standard deviation from three
different experiments.
(C) The specificity of the Her2(36s)-A2 reactive CTL line was evaluated
against human tumor cell
lines not treated. CTL cytotoxic activity was evaluated in a 6 h 5'Cr release
assay at an E:T ratio
of 10:1 as described herein above. Regression analysis was determined from
flow cytometric
and cytotoxic data for MDA-MB-231, Saos-2, MCF-7, SW620 and Co1o205 tumor cell
lines. The
analyses did not reach significance for peptide-A2 vs. total Her2, tumor lysis
vs. total Her2,
peptide-A2 vs. HLA-A2, tumor lysis vs. HLA-A2 and peptide-A2 vs. tumor lysis.
[0068] Fig. 38 illustrates expression of HLA-A*0201 molecules and HLA-
Her2(369) peptide
complexes after cytokine treatment of human tumor cell lines. Human tumor cell
lines were
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pre-treated for 24 h with rIFN-y (20 ng/ml) and TNF-a (3 ng/ml) and stained
with (A) anti-A2.1
BB7.2 or (B)1 B8 TCRm mAbs. Results are plotted as mean fluorescence intensity
ratio (MFIR)
with standard deviation from three different experiments. (C) The specificity
of the Her2(369)-A2
reactive CTL line was evaluated against human tumor cell lines pre-treated for
24 h with rIFN-y
(20 ng/ml) and TNF-a (3 ng/ml). CTL cytotoxic activity was evaluated in a 6 h
51Cr release
assay at an E:T ratio of 10:1 as described herein above. (D) Data plotted from
regression
analysis reveals a significant (ps0.05) relationship between tumor specific
lysis and only
Her2(369)-A2 complex level (Rz =0.75). The analyses did not reach significance
for peptide-A2
vs. total Her2, tumor lysis vs. total Her2, peptide-A2 vs. HLA-A2, and tumor
lysis vs. HLA-A2.
[0069] Fig. 39 illustrates the characterization of binding specificity for
3.2G1 TCRm. (A)
Supernatant from hybridoma 3.2G1 was used to probe wells coated with HLA-A2
tetramer
refolded with the different peptides indicated. Bound antibody was detected
with a goat
anti-mouse peroxidase conjugate and developed using ABTS. (B) Hybridoma
supernatant was
used to stain 5 x 105 T2 cells pulsed with the peptides indicated or no
peptide. After washing,
cells were probed with a goat anti-mouse secondary antibody, washed and
analyzed by flow
cytometry. (C) T2 cells pulsed with 20 pg/mi of GVL peptide for four hours
were stained with
serially-diluted 3.2G1 TCRm. The net (pulsed - non-pulsed) mean fluorescence
intensity (MFI)
was calculated for each antibody concentration and plotted. ( D) T2 cells were
pulsed with
varying levels of GVL peptide and stained with 1 pg/mI 3.2G1 TCRm or BB7.2 mAb
followed
by a secondary goat anti-mouse antibody. MFI values are shown for the various
peptide
concentrations. (E) T2 cells were pulsed with 20 lag/mI GVL peptide and then
stained with a
preincubated mixture of 1pg/100 pi 3.2G1 TCRm and either GVL tetramer or VLQ
tetramer.
The tetramer and antibody were preincubated for 40 min before addition to the
pulsed cells.
Tetramer concentrations (pg/stain) ranged from 1 to 0.01 for GVL and 1 to 0.1
for VLQ.
[0070] Fig. 40 illustrates CDC of peptide-pulsed T2 cells. T2 cells were
pulsed with the
various peptide mixes for 4 hours, washed and dispensed into wells in 96 well
plates at 3 x 105
cells/well. Antibody and rabbit complement were added and the reactions
allowed to proceed
for 4 hours, and then cytotoxicity was analyzed using the LDH assay from
Promega. (A) T2
cells were pulsed with mixes of GVL:TMT peptide at the concentrations in mg/mi
shown in the
legend at the top of the figure for 4 hours before incubating with 2.5 pg/mI
3.2G1 TCRm or
BB7.2 antibody and rabbit complement. (B) T2 cells were pulsed with varying
levels of peptide
diluted 1:2 from 50 pg/mI to 0.1 Ng/mI before incubating with 10 pg/mI 3.2G1
TCRm or BB7.2
antibody. (C) T2 cells were pulsed with 20 pg/mI peptide before addition of a
mix containing
varying amounts of antibody and either GVL or VLQ tetramer at a final
concentration of 2 pg/mI
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tetramer. Final antibody concentration was varied from 9 to 0.1 pg/mI and
corresponds to color
coding shown in the legend for (C). Bars representing standard error are shown
for (A), (B) and
(C).
[0071] Fig. 41 illustrates that 3.2G1 detects endogenous GVL-HLA-A2 complexes
on human
tumor lines. Immunofluorescent staining was carried out using 3.2G1, BB7.2,
and isotype
control antibodies on four human tumor lines. 3.2G1 detects various levels of
GVL/A2 on the
cells' surface and does not stain the HLA-A2 negative cell line BT20.
[0072] Fig. 42 illustrates CDC and ADCC of MDA-MB-231 cells by 3.2G1 TCRm. (A)
Complement-dependent cytolysis was carried out using 2 x 105 MDA-MB-231 cells
well in a 96
well plate. The final concentration of the antibodies in the wells was varied
from 25 to 1 pg/mI
and corresponds to color coding shown above the figure. Tetramer concentration
in each well
was 6 pg/mI. Reactions were incubated for 4 hours and analyzed using the LDH
assay. (B)
ADCC reactions included 2 x 105 MDA-MB-231 cells/well and IL-2 stimulated
human PBMC
preparations at an E:T ratio of 30:1 with 10 {ag/ml 3.2G1. Lysis was
determined using the LDH
assay. (C) ADCC reactions using IL-2-stimulated human PBMC at an E:T ratio of
20:1 with
either 10 pg/mi 3.2G1 (black bars) or 10 pg/mi W6/32 (grey bars). Bars
indicate standard error
for each reaction. Data from CDC assays are representative of 4 independent
experiments.
[0073] Fig. 43 illustrates that the 3.2G1 TCRm prevents tumor growth in
athymic nude mice.
Female athymic mice were subcutaneously injected between the shoulders with
5x106
MDA-MB-231 cells in 0.2 ml containing 1:1 mixture of medium and Matrigel. Mice
were given
tumor cells and treated i.p. with 100 pg of either murine IgG2a isotype
control antibody or with
GVL/A2 specific 3.2G1 TCRm antibody. After the initial antibody injection,
mice received one
injection a week (50 pg/injection) for three weeks. Tumor growth was initially
seen in mice
treated with IgG21 control antibody at week 6 and by week 10 the tumor volume
had increased
>30-fold (0). In contrast, no tumor growth was seen in mice treated with the
3.2G1 antibody
(M). Tumors were monitored and final scoring was tabulated at 69 days after
implant at which
time all tumors were at least 6 mm in diameter and no new tumors had appeared
for 21 days.
Tumor volumes were calculated by assuming a spherical shape and using the
formula, volume
= 4r3/3, where r=1/2 of the mean tumor diameter measured in two dimensions.
Points, median;
bars, SEM. Significance P = 0.0007, was determined by the Fisher Exact Test.
[0074] Fig. 44 illustrates that the 3.2G1 TCRm can be used therapeutically to
treat athymic
nude mice with established tumors. Female athymic mice were subcutaneously
injected in the
right flank with 1x10' MDA-MB-231 breast cancer cells containing 1:1 mixture
of medium and
Matrigel. After 10 days of growth, tumors were measured using calipers with
the mean tumor
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27of 101
volume (mm3) ranging between 62 and 105 mm3. At day 10, mice were injected
(100
pg/injection) with either the 3.2G1 TCRm antibody or an IgGaa isotype control
antibody. Mice
then received 3 more injections (50 pg/injection) at weekly intervals. 24 days
after initial
injection, tumor growth was measured and plotted as tumor volume. Tumor growth
in the IgG2a
isotype control group increased almost three-fold from an initial pre-
treatment mean of 105 mm3
to a mean of 295 mm3. In contrast, the 3.2G1 treated group had a mean tumor
volume of 62
mm3 that was reduced to a tumor volume of 8 mm3 after treatment. Even more
impressive was
that 3 out of 4 mice in the 3.2G1 treated group had no tumors. Tumor volumes
were calculated
by assuming a spherical shape and using the formula, volume=4r3/3, where r=1/2
of the mean
tumor diameter measured in two dimension.
[0075] Fig. 45 illustrates a protocol for the generation of peptide-MHC Class
I specific TCR
mimics of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0076] Before explaining at least one embodiment of the invention in detail by
way of
exemplary drawings, experimentation, results, and laboratory procedures, it is
to be understood
that the invention is not limited in its application to the details of
construction and the
arrangement of the components set forth in the following description or
illustrated in the
drawings, experimentation and/or results. The invention is capable of other
embodiments or
of being practiced or carried out in various ways. As such, the language used
herein is
intended to be given the broadest possible scope and meaning; and the
embodiments are
meant to be exemplary - not exhaustive. Also, it is to be understood that the
phraseology and
terminology employed herein is for the purpose of description and should not
be regarded as
limiting.
[0077] Unless otherwise defined herein, scientific and technical terms used in
connection with
the present invention shall have the meanings that are commonly understood by
those of
ordinary skill in the art. Further, unless otherwise required by context,
singular terms shall
include pluralities and plural terms shall include the singular. Generally,
nomenciatures utilized
in connection with, and techniques of, cell and tissue culture, molecular
biology, and protein and
oligo- or polynucleotide chemistry and hybridization described herein are
those well known and
commonly used in the art. Standard techniques are used for recombinant DNA,
oligonucleotide
synthesis, and tissue culture and transformation (e.g., electroporation,
lipofection). Enzymatic
reactions and purification techniques are performed according to
manufacturer's specifications
or as commonly accomplished in the art or as described herein. The foregoing
techniques and
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procedures are generally performed according to conventional methods well
known in the art
and as described in various general and more specific references that are
cited and discussed
throughout the present specification. See e.g., Sambrook et al. Molecular
Cloning: A
Laboratory Manual (2"d ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.
(1989) and Coligan et al. Current Protocols in Immunology (Current Protocols,
Wiley
Interscience (1994)), which are incorporated herein by reference. The
nomenclatures utilized
in connection with, and the laboratory procedures and techniques of,
analytical chemistry,
synthetic organic chemistry, and medicinal and pharmaceutical chemistry
described herein are
those well known and commonly used in the art. Standard techniques are used
for chemical
syntheses, chemical analyses, pharmaceutical preparation, formulation, and
delivery, and
treatment of patients.
[0078] As utilized in accordance with the present disclosure, the following
terms, unless
otherwise indicated, shall be understood to have the following meanings:
[0079] The terms "isolated polynucleotide" and "isolated nucleic acid segment"
as used
herein shall mean a polynucleotide of genomic, cDNA, or synthetic origin or
some combination
thereof, which by virtue of its origin the "isolated polynucleotide" or
"isolated nucleic acid
segment" (1) is not associated with all or a portion of a polynucleotide in
which the "isolated
polynucleotide" or "isolated nucleic acid segment" is found in nature, (2) is
operably linked to
a polynucleotide which it is not linked to in nature, or (3) does not occur in
nature as part of a
larger sequence.
[0080] The term "isolated protein" referred to herein means a protein of cDNA,
recombinant
RNA, or synthetic origin or some combination thereof, which by virtue of its
origin, or source of
derivation, the "isolated protein" (1) is not associated with proteins found
in nature, (2) is free
of other proteins from the same source, e.g., free of murine proteins, (3) is
expressed by a cell
from a different species, or, (4) does not occur in nature.
[0081] The term "polypeptide" as used herein is a generic term to refer to
native protein,
fragments, or analogs of a polypeptide sequence. Hence, native protein,
fragments, and
analogs are species of the polypeptide genus.
[0082] The term "naturally-occurring" as used herein as applied to an object
refers to the fact
that an object can be found in nature. For example, a polypeptide or
polynucleotide sequence
that is present in an organism (including viruses) that can be isolated from a
source in nature
and which has not been intentionally modified by man in the laboratory or
otherwise is
naturally-occurring.
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29 of 101
[0083] The te'rm "operably linked" as used herein refers to positions of
components so
described are in a relationship permitting them to function in their intended
manner. A control
sequence "operably linked" to a coding sequence is ligated in such a way that
expression of the
coding sequence is achieved under conditions compatible with the control
sequences.
[0084] The term "control sequence" as used herein refers to polynucleotide
sequences which
are necessary to effect the expression and processing of coding sequences to
which they are
ligated. The nature of such control sequences differs depending upon the host
organism; in
prokaryotes, such control sequences generally include promoter, ribosomal
binding site, and
transcription termination sequence; in eukaryotes, generally, such control
sequences include
promoters and transcription termination sequence. The term "control sequences"
is intended
to include, at a minimum, all components whose presence is essential for
expression and
processing, and can also include additional components whose presence is
advantageous, for
example, leader sequences and fusion partner sequences.
[0085] The term "polynucleotide" as referred to herein means a polymeric form
of
nucleotides of at least 10 bases in length, either ribonucleotides or
deoxynucleotides or a
modified form of either type of nucleotide. The term includes single and
double stranded forms
of DNA.
[0086] The term "oligonucleotide" referred to herein includes naturally
occurring, and modified
nucleotides linked together by naturally occurring, and non-naturally
occurring oligonucleotide
linkages. Oligonucleotides are a polynucleotide subset generally comprising a
length of 200
bases or fewer. In one embodiment, oligonucleotides are 10 to 60 bases in
length, such as but
not limited to, 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length.
Oligonucleotides are
usually single stranded, e.g., for probes; although oligonucleotides may be
double stranded,
e.g., for use in the construction of a gene mutant. Oligonucleotides of the
invention can be
either sense or antisense oligonucleotides.
[0087] The term "naturally occurring nucleotides" referred to herein includes
deoxyribonucleotides and ribonucleotides. The term "modified nucleotides"
referred to herein
includes nucleotides with modified or substituted sugar groups and the like.
The term
"oligonucleotide linkages" referred to herein includes oligonucleotides
linkages such as
phosphorothioate, phosphorodithioate, phosphoroselenoate,
phosphorodiselenoate,
phosphoroanilothioate, phoshoraniladate, phosphoroamidate, and the like. See
e.g., LaPlanche
et al. Nucl. Acids Res. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077
(1984); Stein
et al. Nucl. Acids Res. 16:3209 (1988); Zon et al. Anti-Cancer Drug Design
6:539 (1991); Zon
et al. Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F.
Eckstein, Ed.,
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Oxford University Press, Oxford England (1991)); Stec et al. U.S. Pat. No.
5,151,510; Uhlmann
and Peyman Chemical Reviews 90:543 (1990), the disclosures of which are hereby
incorporated by reference. An oligonucleotide can include a label for
detection, if desired.
[0088] The term "selectively hybridize" referred to herein means to detectably
and
specifically bind. Polynucleotides, oligonucleotides and fragments thereof in
accordance with
the invention selectively hybridize to nucleic acid strands under
hybridization and wash
conditions that minimize appreciable amounts of detectable binding to
nonspecific nucleic acids.
.High stringency conditions can be used to achieve selective hybridization
conditions as known
in the art and discussed herein. Generally, the nucleic acid sequence homology
between the
polynucleotides, oligonucleotides, and fragments of the invention and a
nucleic acid sequence
of interest will be at least 80%, and more typically with increasing
homologies of at least 85%,
90%, 95%, 99%, and 100%. Two amino acid sequences are homologous if there is a
partial
or complete identity between their sequences. For example, 85% homology means
that 85%
of the amino acids are identical when the two sequences are aligned for
maximum matching.
Gaps (in either of the two sequences being matched) are allowed in maximizing
matching; gap
lengths of 5 or less are preferred with 2 or less being more preferred.
Alternatively and
preferably, two protein sequences (or polypeptide sequences derived from them
of at least 30
amino acids in length) are homologous, as this term is used herein, if they
have an alignment
score of at more than 5 (in standard deviation units) using the program ALIGN
with the mutation
data matrix and a gap penalty of 6 or greater. See Dayhoff, M. 0., in Atlas of
Protein Sequence
and Structure, pp. 101-110 (Volume 5, National Biomedical Research Foundation
(1972)) and
Supplement 2 to this volume, pp. 1-10. The two sequences or parts thereof are
more
preferably homologous if their amino acids are greater than or equal to 50%
identical when
optimally aligned using the ALIGN program. The term "corresponds to" is used
herein to mean
that a polynucleotide sequence is homologous (i.e., is identical, not strictly
evolutionarily
related) to all or a portion of a reference polynucleotide sequence, or that a
polypeptide
sequence is identical. to a reference polypeptide sequence. In
contradistinction, the term
"complementary to" is used herein to mean that the complementary sequence is
homologous
to all or a portion of a reference polynucleotide sequence. For illustration,
the nucleotide
sequence "TATAC" corresponds to a reference sequence "TATAC" and is
complementary to
a reference sequence "GTATA".
[0089] The following terms are used to describe the sequence relationships
between two or
more polynucleotide or amino acid sequences: "reference sequence", "comparison
window",
"sequence identity", "percentage of sequence identity", and "substantial
identity". A"reference
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sequence" is a defined sequence used as a basis for a sequence comparison; a
reference
sequence may be a subset of a larger sequence, for example, as a segment of a
full-length
cDNA or gene sequence given in a sequence listing or may comprise a complete
cDNA or gene
sequence. Generally, a reference sequence is at least 18 nucleotides or 6
amino acids in
length, frequently at least 24 nucleotides or 8 amino acids in length, and
often at least 48
nucleotides or 16 amino acids in length. Since two polynucleotides or amino
acid sequences
may each (1) comprise a sequence (i.e., a portion of the complete
polynucleotide or amino acid
sequence) that is similar between the two molecules, and (2) may further
comprise a sequence
that is divergent between the two polynucleotides or amino acid sequences,
sequence
comparisons between two (or more) molecules are typically performed by
comparing
sequences of the two molecules over a "comparison window" to identify and
compare local
regions of sequence similarity. A "comparison window", as used herein, refers
to a conceptual
segment of at least 18 contiguous nucleotide positions or 6 amino acids
wherein a
polynucleotide sequence or amino acid sequence may be compared to a reference
sequence
of at least 18 contiguous nucleotides or 6 amino acid sequences and wherein
the portion of the
polynucleotide sequence in the comparison window may comprise additions,
deletions,
substitutions, and the like (i.e., gaps) of 20 percent or less as compared to
the reference
sequence (which does not comprise additions or deletions) for optimal
alignment of the two
sequences. Optimal alignment of sequences for aligning a comparison window may
be
conducted by the local homology algorithm of Smith and Waterman Adv. Appl.
Math. 2:482
(1981), by the homology alignment algorithm of Needleman and Wunsch J. Mol.
Biol. 48:443
(1970), by the search for similarity method of Pearson and Lipman Proc. Natl.
Acad. Sci.
(U.S.A.) 85:2444 (1988), by computerized implementations of these algorithms
(GAP,
BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release
7.0,
(Genetics Computer Group, 575 Science Dr., Madison, Wis.), Geneworks, or
MacVector
software packages), or by inspection, and the best alignment (i.e., resulting
in the highest
percentage of homology over the comparison window) generated by the various
methods is
selected.
[0090] The term "sequence identity" means that two polynucleotide or amino
acid
sequences are identical (i.e., on a nucleotide-by-nucleotide or residue-by-
residue basis) over
the comparison window. The term "percentage of sequence identity" is
calculated by
comparing two optimally aligned sequences over the window of comparison,
determining the
number of positions at which the identical nucleic acid base (e.g., A, T, C,
G, U, or I) or residue
occurs in both sequences to yield the number of matched positions, dividing
the number of
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matched positions by the total number of positions in the comparison window
(i.e., the window
size), and multiplying the result by 100 to yield the percentage of sequence
identity. The terms
"substantial identity" as used herein denotes a characteristic of a
polynucleotide or amino acid
sequence, wherein the polynucleotide or amino acid comprises a sequence that
has at least
85 percent sequence identity, such as at least 90 to 95 percent sequence
identity, or at least
99 percent sequence identity as compared to a reference sequence over a
comparison window
of at least 18 nucleotide (6 amino acid) positions, frequently over a window
of at least 24-48
nucleotide (8-16 amino acid) positions, wherein the percentage of sequence
identity is
calculated by comparing the reference sequence to the sequence which may
include deletions
or additions which total 20 percent or less of the reference sequence over the
comparison
window. The reference sequence may be a subset of a larger sequence.
[0091] As used herein, the twenty conventional amino acids and their
abbreviations follow
conventional usage. See Immunology--A Synthesis (2"d Edition, E. S. Golub and
D. R. Gren,
Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated
herein by
reference. Stereoisomers (e.g., D-amino acids) of the twenty conventional
amino acids,
unnatural amino acids such as a-,a-disubstituted amino acids, N-alkyl amino
acids, lactic acid,
and other unconventional amino acids may also be suitable components for
polypeptides of the
present invention. Examples of unconventional amino acids include: 4-
hydroxyproline,
y-carboxyglutamate, s-N,N,N-trimethyllysine, s-N-acetyllysine, 0-
phosphoserine, N-acetylserine,
N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, a-N-methylarginine,
and other similar
amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide
notation used herein,
the Iefthand direction is the amino terminal direction and the righthand
direction is the
carboxy-terminal direction, in accordance with standard usage and convention.
[0092] Similarly, unless specified otherwise, the lefthand end of single-
stranded
polynucleotide sequences is the 5' end; the lefthand direction of double-
stranded polynucleotide
sequences is referred to as the 5' direction. The direction of 5' to 3'
addition of nascent RNA
transcripts is referred to as the transcription direction; sequence regions on
the DNA strand
having the same sequence as the RNA and which are 5' to the 5' end of the RNA
transcript are
referred to as "upstream sequences"; sequence regions on the DNA strand having
the same
sequence as the RNA and which are 3' to the 3' end of the RNA transcript are
referred to as
"downstream sequences".
[0093] As applied to polypeptides, the term "substantial identity" means that
two peptide
sequences, when optimally aligned, such as by the programs GAP or BESTFIT
using default
gap weights, share at least 80 percent sequence identity, such as at least 90
percent sequence
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identity, or at least 95 percent sequence identity, or at least 99 percent
sequence identity.
Preferably, residue positions which are not identical differ by conservative
amino acid
substitutions. Conservative amino acid substitutions refer to the
interchangeability of residues
having similar side chains. For example, a group of amino acids having
aliphatic side chains
is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids
having
aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids
having
amide-containing side chains is asparagine and glutamine; a group of amino
acids having
aromatic side chains is phenylaianine, tyrosine, and tryptophan; a group of
amino acids having
basic side chains is lysine, arginine, and histidine; and a group of amino
acids having
sulfur-containing side chains is cysteine and methionine. Preferred
conservative amino acids
substitution groups are: valine-leucine-isoleucine, phenylaianine-tyrosine,
lysine-arginine,
alanine-valine, glutamic-aspartic, and asparagine-glutamine.
[0094] As discussed herein, minor variations in the amino acid sequences of
antibodies or
immunoglobulin molecules are contemplated as being encompassed by the present
invention,
providing that the variations in the amino acid sequence maintain at least
75%, such as at least
80%, 90%, 95%, and 99%. In particular, conservative amino acid replacements
are
contemplated. Conservative replacements are those that take place within a
family of amino
acids that are related in their side chains. Genetically encoded amino acids
are generally
divided into families: (1) acidic=aspartate, glutamate; (2) basic=lysine,
arginine, histidine;
(3) nonpolar=alanine, valine, leucine, isoleucine, proline, phenylalanine,
methionine, tryptophan;
and (4) uncharged polar=glycine, asparagine, glutamine, cysteine, serine,
threonine, tyrosine.
More preferred families are: serine and threonine are aliphatic-hydroxy
family; asparagine and
glutamine are an amide-containing family; alanine, valine, leucine and
isoleucine are an
aliphatic family; and phenylalanine, tryptophan, and tyrosine are an aromatic
family. For
example, it is reasonable to expect that an isolated replacement of a leucine
with an isoleucine
or valine, an aspartate with a glutamate, a threonine with a serine, or a
similar replacement of
an amino acid with a structurally related amino acid will not have a major
effect on the binding
or properties of the resulting molecule, especially if the replacement does
not involve an amino
acid within a framework site. Whether an amino acid change results in a
functional peptide can
readily be determined by assaying the specific activity of the polypeptide
derivative. Fragments
or analogs of antibodies or immunoglobulin molecules can be readily prepared
by those of
ordinary skill in the art. Preferred amino- and carboxy-termini of fragments
or analogs occur
near boundaries of functional domains. Structural and functional domains can
be identified by
comparison of the nucleotide and/or amino acid sequence data to public or
proprietary
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sequence databases. Preferably, computerized comparison methods are used to
identify
sequence motifs or predicted protein conformation domains that occur in other
proteins of
known structure and/or function. Methods to identify protein sequences that
fold into a known
three-dimensional structure are known. Bowie et al. Science 253:164 (1991).
Thus, the
foregoing examples demonstrate that those of skill in the art can recognize
sequence motifs
and structural conformations that may be used to define structural and
functional domains in
accordance with the invention.
[0095] Preferred amino acid substitutions are those which: (1) reduce
susceptibility to
proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding
affinity for forming protein
complexes, (4) alter binding affinities, and (5) confer or modify other
physicochemical or
functional properties of such analogs. Analogs can include various mutations
of a sequence
other than the naturally-occurring peptide sequence. For example, single or
multiple amino acid
substitutions (preferably conservative amino acid substitutions) may be made
in the
naturally-occurring sequence (preferably in the portion of the polypeptide
outside the domain(s)
forming intermolecular contacts. A conservative amino acid substitution should
not substantially
change the structural characteristics of the parent sequence (e.g., a
replacement amino acid
should not tend to break a helix that occurs in the parent sequence, or
disrupt other types of
secondary structure that characterizes the parent sequence). Examples of art-
recognized
polypeptide secondary and tertiary structures are described in Proteins,
Structures and
Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York
(1984));
Introduction to Protein Structure . Branden and J. Tooze, eds., Garland
Publishing, New York,
N.Y. (1991)); and Thornton et at. Nature 354:105 (1991), which are each
incorporated herein
by reference.
[0096] The term "polypeptide fragment" as used herein refers to a polypeptide
that has an
amino-terminal and/or carboxy-terminal deletion, but where the remaining amino
acid sequence
is identical to the corresponding positions in the naturally-occurring
sequence deduced, for
example, from a full-length cDNA sequence. Fragments typically are at least 5,
6, 8 or 10
amino acids long, such as at least 14 amino acids long or at least 20 amino
acids long, usually
at least 50 amino acids.long or at least 70 amino acids long.
[0097] "Antibody" or "antibody peptide(s)" refer to an intact antibody, or a
binding fragment
thereof that competes with the intact antibody for specific binding. Binding
fragments are
produced by recombinant DNA techniques, or by enzymatic or chemical cleavage
of intact
antibodies. Binding fragments include Fab, Fab', F(ab')2, Fv, and single-chain
antibodies. An
antibody other than a "bispecific" or "bifunctional" antibody is understood to
have each of its
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binding sites identical. An antibody substantially inhibits adhesion of a
receptor to a
counterreceptor when an excess of antibody reduces the quantity of receptor
bound to
counterreceptor by at least about 20%, 40%, 60% or 80%, and more usually
greater than about
85% (as measured in an in vitro competitive binding assay).
[0098] The term "MHC" as used herein will be understood to refer to the Major
Histocompability Complex, which is defined as a set of gene loci specifying
major
histocompatibility antigens. The term "HLA" as used herein will be understood
to refer to
Human Leukocyte Antigens, which is defined as the histocompatibility antigens
found in
humans. As used herein, "HLA" is the human form of "MHC".
[0099] The terms "MHC light chain" and "MHC heavy chain" as used herein will
be
understood to refer to portions of the MHC molecule. Structurally, class I
molecules are
heterodimers comprised of two noncovalently bound polypeptide chains, a larger
"heavy" chain
(a) and a smaller "light" chain (R-2-microglobulin or P2m). The polymorphic,
polygenic heavy
chain (45 kDa), encoded within the MHC on chromosome six, is subdivided into
three
extracellular domains (designated 1, 2, and 3), one intracellular domain, and
one
transmembrane domain. The two outermost extracellular domains, 1 and 2,
together form the
groove that binds antigenic peptide. Thus, interaction with the TCR occurs at
this region of the
protein. The 3 domain of the molecule contains the recognition site for the
CD8 protein on the
CTL; this interaction serves to stabilize the contact between the T cell and
the APC. The
invariant light chain (12 kDa), encoded outside the MHC on chromosome 15,
consists of a
single, extracellular polypeptide. The terms "MHC light chain", "(3-2-
microglobulin", and "(32m"
may be used interchangeably herein.
[0100] The term "epitope" includes any protein determinant capable of specific
binding to an
immunoglobulin or T-cell receptor. Epitopic determinants usually consist of
chemically active
surface groupings of molecules such as amino acids or sugar side chains and
usually have
specific three dimensional structural characteristics, as well as specific
charge characteristics.
An antibody is said to specifically bind an antigen when the dissociation
constant is <1 pM, or
<100 nM, or <10 nM.
[0101] 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 (e.g., Fab,
F(ab')2 and Fv) so
long as they exhibit the desired biological activity. Antibodies (Abs) and
immunoglobulins (Igs)
are glycoproteins having the same structural characteristics. While antibodies
exhibit binding
specificityto a specific antigen, immunoglobulins include both antibodies and
other antibody-like
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molecules which lack antigen specificity. Polypeptides of the latter kind are,
for example,
produced at low levels by the lymph system and at increased levels by
myelomas.
[0102] Native antibodies and immunoglobulins are usually heterotetrameric
glycoproteins of
about 150,000 daltons, composed of two identical light (L) chains and two
identical heavy (H)
chains. Each light chain is linked to a heavy chain by one covalent disulfide
bond. While the
number of disulfide linkages varies between the heavy chains of different
immunoglobulin
isotypes. Each heavy and light chain also has regularly spaced intrachain
disulfide bridges.
Each heavy chain has at one end a variable domain (VH) followed by a number of
constant
domains. Each light chain has a variable domain at one end (VL) and a constant
domain at its
other end. The constant domain of the light chain is aligned with the first
constant domain of
the heavy chain, and the light chain variable domain is aligned with the
variable domain of the
heavy chain. Particular amino acid residues are believed to form an interface
between the light
and heavy chain variable domains (Clothia et al., J. Mol. Biol. 186, 651-66,
1985); Novotny and
Haber, Proc. Natl. Acad. Sci. USA 82 4592-4596 (1985).
[0103] An "isolated" antibody is one which has been identified and separated
and/or
recovered from a component of the environment in which is was produced.
Contaminant
components of its production environment are materials which would interfere
with diagnostic
or therapeutic uses for the antibody, and may include enzymes, hormones, and
other
proteinaceous or nonproteinaceous solutes. In certain embodiments, the
antibody will be
purified as measurable by at least three different methods: 1) to greater than
50% by weight
of antibody as determined by the Lowry method, such as more than 75% by
weight, or more
than 85% by weight, or more than 95% by weight, or more than 99% by weight; 2)
to a degree
sufficient to obtain at least 10 residues of N-terminal or internal amino acid
sequence by use
of a spinning cup sequentator, such as at least 15 residues of sequence; or 3)
to homogeneity
by SDS-PAGE under reducing or non-reducing conditions using Coomasie blue or,
preferably,
silver stain. Isolated antibody includes the antibody in situ within
recombinant cells since at
least one component of the antibody's natural environment will not be present.
Ordinarily,
however, isolated antibody will be prepared by at least one purification step.
[0104] The term "antibody mutant" refers to an amino acid sequence variant of
an antibody
wherein one or more of the amino acid residues have been modified. Such
mutants necessarily
have less than 100% sequence identity or similarity with the amino acid
sequence having at
least 75% amino acid sequence identity or similarity with the amino acid
sequence of either the
heavy or light chain variable domain of the antibody, such as at least 80%, or
at least 85%, or
at least 90%, or at least 95%.
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[0105] The term "variable" in the context of variable domain of antibodies,
refers to the fact
that certain portions of the variable domains differ extensively in sequence
among antibodies
and are used in the binding and specificity of each particular antibody for
its particular antigen.
However, the variability is not evenly distributed through the variable
domains of antibodies.
It is concentrated in three segments called complementarity determining
regions (CDRs) also
known as hypervariable regions both in the light chain and the heavy chain
variable domains.
There are at least two techniques for determining CDRs: (1) an approach based
on
cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins
of Immunological
Interest (National Institute of Health, Bethesda, Md. 1987); and (2) an
approach based on
crystallographic studies of antigen-antibody complexes (Chothia, C. et al.
(1989), Nature 342:
877). The more highly conserved portions of variable domains are called the
framework (FR).
The variable domains of native heavy and light chains each comprise four FR
regions, largely
adopting a(3-sheet configuration, connected by three CDRs, which form loops
connecting, and
in some cases forming part of, the R-sheet structure. The CDRs in each chain
are held
together in close proximity by the FR regions and, with the CDRs from the
other chain,
contribute to the formation of the antigen binding site of antibodies (see
Kabat et al.) The
constant domains are not involved directly in binding an antibody to an
antigen, but exhibit
various effector function, such as participation of the antibody in antibody-
dependent cellular
toxicity.
[0106] The term "antibody fragment" refers to a portion of a full-length
antibody, generally
the antigen binding or variable region. Examples of antibody fragments include
Fab, Fab',
F(ab')z and Fvfragments. Papain digestion of antibodies produces two identical
antigen binding
fragments, called the Fab fragment, each with a single antigen binding site,
and a residual "Fc"
fragment, so-called for its ability to crystallize readily. Pepsin treatment
yields an F(ab')2
fragment that has two antigen binding fragments which are capable of cross-
linking antigen,
and a residual other fragment (which is termed pFc'). As used herein,
"functional fragment"
with respect to antibodies, refers to Fv, F(ab) and F(ab')2 fragments.
[0107] An "Fv" fragment is the minimum antibody fragment which contains a
complete
antigen recognition and binding site. This region consists of a dimer of one
heavy and one light
chain variable domain in a tight, non-covalent association (VH -VL dimer). It
is in this
configuration that the three CDRs of each variable domain interact to define
an antigen binding
site on the surface of the VH -VL dimer. Collectively, the six CDRs confer
antigen binding
specificity to the antibody. However, even a single variable domain (or half
of an Fv comprising
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only three CDRs specific for an antigen) has the ability to recognize and bind
antigen, although
at a lower affinity than the entire binding site.
[0108] The Fab fragment [also designated as F(ab)] also contains the constant
domain of
the light chain and the first constant domain (CH1) of the heavy chain. Fab'
fragments differ
from Fab fragments by the addition,of a few residues at the carboxyl terminus
of the heavy
chain CHI domain including one or more cysteines from the antibody hinge
region. Fab'-SH
is the designation herein for Fab' in which the cysteine residue(s) of the
constant domains have
a free thiol group. F(ab') fragments are produced by cleavage of the disulfide
bond at the hinge
cysteines of the F(ab')2 pepsin digestion product. Additional chemical
couplings of antibody
fragments are known to those of ordinary skill in the art.
[0109] The light chains of antibodies (immunoglobulin) from any vertebrate
species can be
assigned to one of two clearly distinct types, called kappa (.kappa.) and
lambda (.lambda.),
based on the amino sequences of their constant domain.
[0110] Depending on the amino acid sequences of the constant domain of their
heavy
chains, "immunoglobulins" can be assigned to different classes. There are at
least five (5) major
classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these
may be further
divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3 and IgG4; IgA-1
and IgA-2. The
heavy chains constant domains that correspond to the different classes of
immunoglobulins are
called a, A, E, y and p, respectively. The subunit structures and three-
dimensional
configurations of different classes of immunoglobulins are well known.
[0111] 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 which typically include different antibodies directed against
different determinants
(epitopes), each monoclonal antibody is directed against a single determinant
on the antigen.
In additional to their specificity, the monoclonal antibodies are advantageous
in that they are
synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
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 256,495 (1975), or may be made by recombinant
methods, e.g.,
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as described in U.S. Pat. No. 4,816,567. The monoclonal antibodies for use
with the present
invention may also be isolated from phage antibody libraries using the
techniques described in
Clackson et al. Nature 352: 624-628 (1991), as well as in Marks et al., J.
Mol. Biol. 222: 581-597
(1991).
[0112] Utilization of the monoclonal antibodies of the present invention may
require
administration of such or similar monoclonal antibody to a subject, such as a
human. However,
when the monoclonal antibodies are produced in a non-human animal, such as a
rodent,
administration of such antibodies to a human patient will normally elicit an
immune response,
wherein the immune response is directed towards the antibodies themselves.
Such reactions limit
the duration and effectiveness of such a therapy. In order to overcome such
problem, the
monoclonal antibodies of the present invention can be "humanized", that is,
the antibodies are
engineered such that antigenic portions thereof are removed and like portions
of a human antibody
are substituted therefor, while the antibodies' affinity for specific
peptide/MHC complexes is
retained. This engineering may only involve a few amino acids, or may include
entire framework
regions of the antibody, leaving only the complementarity determining regions
of the antibody
intact. Several methods of humanizing antibodies are known in the art and are
disclosed in US
Patent Nos. 6,180,370, issued to Queen et al on January 30, 2001; 6,054,927,
issued to Brickell
on April 25, 2000; 5,869,619, issued to Studnicka on February 9, 1999;
5,8~61,155, issued to Lin
on January 19, 1999;, 5,712,120, issued to Rodriquez et al on January 27,
1998; and 4,816,567,
issued to Cabilly et al on March 28, 1989, the Specifications of which are all
hereby expressly
incorporated herein by reference in their entirety.
[0113] Humanized forms of antibodies are chimeric immunoglobulins,
immunoglobulin chains
or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding
subsequences of
antibodies) that are principally comprised of the sequence of a human
immunoglobulin, and contain
minimal sequence derived from a non-human immunoglobulin. Humanization can be
performed
following the method of Winter and co-workers (Jones et al., 1986; Riechmann
et al., 1988;
Verhoeyen et al., 1988), by substituting rodent CDRs or CDR sequences for the
corresponding
sequences of a human antibody. (See also U.S. Patent No.5,225,539.) In some
instances, F,
framework residues of the human immunoglobulin are replaced by corresponding
non-human
residues. Humanized antibodies can also comprise residues which are found
neither in the recipient
antibody nor in the imported CDR or framework sequences. In general, the
humanized antibody
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will comprise substantially all of at least one, and typically two, variable
domains, in which all or
substantially all of the CDR regions correspond to those of a non-human
immunoglobulin and all
or substantially all of the framework regions are those of a human
immunoglobulin consensus
sequence. The humanized antibody optimally also will comprise at least a
portion of an
immunoglobulin constant region (Fc), typically that of a human immunoglobulin
(Jones et al., 1986;
Riechmann et al., 1988; and Presta, 1992).
[0114] 97 published articles relating to the generation or use of humanized
antibodies were
identified by a PubMed search of the database as of April 25, 2002. Many of
these studies teach
useful examples of protocols that can be utilized with the present invention,
such as Sandborn et
al., Gatroenterology, 120:1330 (2001); Mihara et al., Clin. Immunol. 98:319
(2001); Yenari et al.,
Neurol. Res. 23:72 (2001); Morales et al., Nucl. Med. Biol. 27:199 (2000);
Richards et al., Cancer
Res. 59:2096 (1999); Yenari et al., Exp. Neurol. 153:223 (1998); and Shinkura
et al., Anticancer
Res. 18:1217 (1998), all of which are expressly incorporated in their entirety
by reference. For
example, a treatment protocol that can be utilized in such a method includes a
single dose,
generally administered intravenously, of 10-20 mg of humanized mAb per kg
(Sandborn, et al.
2001). In some cases, alternative dosing patterns may be appropriate, such as
the use of three
infusions, administered once every two weeks, of 800 to 1600 mg or even higher
amounts of
humanized mAb (Richards et al., 1999). However, it is to be understood that
the invention is not
limited to the treatment protocols described above, and other treatment
protocols which are known
to a person of ordinary skill in the art may be utilized in the methods of the
present invention.
[0115] The presently disclosed and claimed invention further includes fully
human monoclonal
antibodies against specific peptide/MHC complexes. Fully human antibodies
essentially relate to
antibody molecules in which the entire sequence of both the light chain and
the heavy chain,
including the CDRs, arise from human genes. Such antibodies are termed "human
antibodies", or
"fully human antibodies" herein. Human monoclonal antibodies can be prepared
by the trioma
technique; the human B-cell hybridoma technique (see Kozbor, et al.,
Hybridoma, 2:7 (1983)) and
the EBV hybridoma technique to produce human monoclonal antibodies (see Cole,
et al., PNAS
82:859 (1985)). Human monoclonal antibodies may be utilized in the practice of
the present
invention and may be produced by using human hybridomas (see Cote, et al.,
PNAS 80:2026
(1983)) or by transforming human B-cells with Epstein Barr Virus in vitro (see
Cole, et al., 1985).
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[0116] In addition, human antibodies can also be produced using additional
techniques, including
phage display libraries (Hoogenboom et al., Nucleic Acids Res. 19:4133 (1991);
Marks et al., J Mol
Biol. 222:581 (1991)). Similarly, human antibodies can be made by introducing
human
immunoglobulin loci into transgenic animals, e.g., mice in which the
endogenous immunoglobulin
genes have been partially or completely inactivated. Upon challenge, human
antibody production
is observed, which closely resembles that seen in humans in all respects,
including gene
rearrangement, assembly, and antibody repertoire. This approach is described,
for example, in
U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;
5,661,016, and in Marks
et al., J Biol. Chem. 267:16007 (1992); Lonberg et al., Nature, 368:856
(1994); Morrison, 1994;
Fishwild et al., Nature Biotechnol. 14:845 (1996); Neuberger, Nat. Biotechnol.
14:826 (1996); and
Lonberg and Huszar, Int Rev Immunol. 13:65 (1995).
[0117] Human antibodies may additionally be produced using transgenic nonhuman
animals
which are modified so as to produce fully human antibodies rather than the
animal's endogenous
antibodies in response to challenge by an antigen. (See PCT publication WO
94/02602). The
endogenous genes encoding the heavy and light immunoglobulin chains in the
nonhuman host
have been incapacitated, and active loci encoding human heavy and light chain
immunoglobulins
are inserted into the host's genome. The human genes are incorporated, for
example, using yeast
artificial chromosomes containing the requisite human DNA segments. An animal
which provides
all the desired modifications is then obtained as progeny by crossbreeding
intermediate transgenic
animals containing fewer than the full complement of the modifications. One
embodiment of such
a nonhuman animal is a mouse, and is termed the XENOMOUSET"' as disclosed in
PCT
publications WO 96/33735 and WO 96/34096. This animal produces B cells which
secrete fully
human immunoglobulins. The antibodies can be obtained directly from the animal
after
immunization with an immunogen of interest, as, for example, a preparation of
a polyclonal
antibody, or alternatively from immortalized B cells derived from the animal,
such as hybridomas
producing monoclonal antibodies. Additionally, the genes encoding the
immunoglobulins with
human variable regions can be recovered and expressed to obtain the antibodies
directly, or can
be further modified to obtain analogs of antibodies such as, for example,
single chain Fv molecules.
[0118] An example of a method of producing a nonhuman host, exemplified as a
mouse, lacking
expression of an endogenous immunoglobulin heavy chain is disclosed in U.S.
Pat. No. 5,939,598,
issued to Kucherlapati et al. on August 17, 1999, and incorporated herein by
reference. It can be
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obtained by a method including deleting the J segment genes from at least one
endogenous heavy
chain locus in an embryonic stem cell to prevent rearrangement of the locus
and to prevent
formation of a transcript of a rearranged immunoglobulin heavy chain locus,
the deletion being
effected by a targeting vector containing a gene encoding a selectable marker;
and producing from
the embryonic stem cell a transgenic mouse whose somatic and germ cells
contain the gene
encoding the selectable marker.
[0119] A method for producing an antibody of interest, such as a human
antibody, is disclosed
in U.S. Pat. No. 5,916,771, issued to Hori et al. on June 29, 1999, and
incorporated herein by
reference. It includes introducing an expression vector that contains a
nucleotide sequence
encoding a heavy chain into one mammalian host cell in culture, introducing an
expression vector
containing a nucleotide sequence encoding a light chain into another mammalian
host cell, and
fusing the two cells to form a hybrid cell. The hybrid cell expresses an
antibody containing the
heavy chain and the light chain.
[0120] The term "agent" is used herein to denote a chemical compound, a
mixture of chemical
compounds, a biological macromolecule, or an extract made from biological
materials.
[0121] As used herein, the terms "label" or "labeled" refers to incorporation
of a detectable
marker, e.g., by incorporation of a radiolabeled amino acid or attachment to a
polypeptide of
biotinyl moieties that can be detected by marked avidin (e.g., streptavidin
containing a fluorescent
marker or enzymatic activity that can be detected by optical or calorimetric
methods). In certain
situations, the label or marker can also be therapeutic. Various methods of
labeling polypeptides
and glycoproteins are known in the art and may be used. Examples of labels for
polypeptides
include, but are not limited to, the following: radioisotopes or radionuclides
(e.g., 3H, 14C, 15N, 35S
90Y, 99Tc, 111In' 1251, 1311), fluorescent labels (e.g., FITC, rhodamine,
lanthanide phosphors),
enzymatic labels (e.g., horseradish peroxidase, R-galactosidase, luciferase,
alkaline phosphatase),
chemiluminescent, biotinyl groups, predetermined polypeptide epitopes
recognized by a secondary
reporter (e.g., leucine zipper pair sequences, binding sites for secondary
antibodies, metal binding
domains, epitope tags). In some embodiments, labels are attached by spacer
arms of various
lengths to reduce potential steric hindrance.
[0122] The term "pharmaceutical agent or drug" as used herein refers to a
chemical compound
or composition capable of inducing a desired therapeutic effect when properly
administered to a
patient. Other chemistry terms herein are used according to conventional usage
in the art, as
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exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed.,
McGraw-Hill, San
Francisco (1985)), incorporated herein by reference).
[0123] The term "antineoplastic agent" is used herein to refer to agents that
have the functional
property of inhibiting a' development or progression of a neoplasm in a human,
particularly a
malignant (cancerous) lesion, such as a carcinoma, sarcoma, lymphoma, or
leukemia. Inhibition
of metastasis is frequently a property of antineoplastic agents.
[0124] As used herein, "substantially pure" means an object species is the
predominant species
present (i.e., on a molar basis it is more abundant than any other individual
species in the
composition), and preferably a substantially purified fraction is a
composition wherein the object
species comprises at least about 50 percent (on a molar basis) of all
macromolecular species
present. Generally, a substantially pure composition will comprise more than
about 80 percent of
all macromolecular species present in the composition, such as more than about
85%, 90%, 95%,
and 99%. In one embodiment, the object species is purified to essential
homogeneity (contaminant
species cannot be detected in the composition by conventional detection
methods) wherein the
composition consists essentially of a single macromolecular species.
[0125] The term patient includes human and veterinary subjects.
[0126] A'9iposome" is a small vesicle composed of various types of lipids,
phospholipids and/or
surfactant. The components of the liposome are commonly arranged in a bilayer
formation, similar
to the lipid arrangement of biological membranes.
[0127] "Treatment" refers to both therapeutic treatment and prophylactic or
preventative
measures. Those in need of treatment include those already with the disorder
as well as those in
which the disorder is to be prevented.
[0128] A "disorder" is any condition that would benefit from treatment with
the polypeptide. This
includes chronic and acute disorders or diseases including those pathological
conditions which
predispose the mammal to the disorder in question.
[0129] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in
mammals that is typically characterized by unregulated cell growth. Examples
of cancer include
but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
More particular
examples of such cancers include squamous cell cancer, small-cell lung cancer,
non-small cell lung
cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical
cancer, ovarian cancer,
liver cancer, bladder cancer, hopatoma, breast cancer, colon cancer,
colorectal cancer, endometrial
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carcinoma, salivary gland carcinoma, kidney cancer, renal cancer, prostate
cancer, vulval cancer,
thyroid cancer, hepatic carcinoma and various types of head and neck cancer.
[0130] "Mammal" for purposes of treatment refers to any animal classified as a
mammal,
including human, domestic and farm animals, nonhuman primates, and zoo,
sports, or pet animals,
such as dogs, horses, cats, cows, etc.
[0131] As mentioned hereinabove, depending on the application and purpose, the
T cell
receptor mimic of the presently disclosed and claimed invention may be
attached to any of various
functional moieties. A T cell receptor mimic of the present invention attached
to a functional moiety
may be referred to herein as an "immunoconjugate". In one embodiment, the
functional moiety
is a detectable moiety or a therapeutic moiety.
[0132] As is described and demonstrated in further detail hereinbelow, a
detectable moiety or
a therapeutic moiety may be particularly employed in applications of the
present invention involving
use of the T cell receptor mimic to detect the specific peptide/MHC complex,
or to kill target cells
and/or damage target tissues.
[0133] The present invention include the T cell receptor mimics described
herein attached to
any of numerous types of detectable moieties, depending on the application and
purpose. For
applications involving detection of the specific peptide/MHC complex, the
detectable moiety
attached to the T cell receptor mimic may be a reporter moiety that enables
specific detection of
the specific peptide/MHC complex bound by the T cell receptor mimic of the
presently disclosed
and claimed invention.
[0134] While various types of reporter moieties may be utilized to detect the
specific
peptide/MHC complex, depending on the application and purpose, the reporter
moiety may be a
fluorophore, an enzyme or a radioisotope. Specific reporter moieties that may
utilized in
accordance with the present invention include, but are not limited to, green
fluorescent protein
(GFP), alkaline phosphatase (AP), peroxidase, orange fluorescent protein
(OFP), R-galactosidase,
fluorescein isothiocyanate (FITC), phycoerythrin, Cy-chrome, rhodamine, blue
fluorescent protein
(BFP), Texas red, horseradish peroxidase (HPR), and the like.
[0135] A fluorophore may be employed as a detection moiety enabling detection
of the specific
peptide/MHC complex via any of numerous fluorescence detection methods.
Depending on the
application and purpose, such fluorescence detection methods include, but are
not limited to,
fluorescence activated flow cytometry (FACS), immunofluorescence confocal
microscopy,
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fluorescence in-situ hybridization (FISH), fluorescence resonance energy
transfer (FRET), and the
like.
[0136] Various types of fluorophores, depending on the application and
purpose, may be
employed to detect the specific peptide/MHC complex. Examples of suitable
fluorophores include,
but are not limited to, phycoerythrin, fluorescein isothiocyanate (FITC), Cy-
chrome, rhodamine,
green fluorescent protein (GFP), blue fluorescent protein (BFP), Texas red,
and the like.
[0137] Ample guidance regarding fluorophore selection, methods of linking
fluorophores to
various types of molecules, such as a T cell receptor mimic of the present
invention, and methods
of using such conjugates to detect molecules which are capable of being
specifically bound by
antibodies or antibody fragments comprised in such immunoconjugates is
available in the literature
of the art [for example, referto: Richard P. Haugland, "Molecular Probes:
Handbook of Fluorescent
Probes and Research Chemicals 1992-1994", 5th ed., Molecular Probes, Inc.
(1994); U.S. Pat. No.
6,037,137 to Oncoimmunin Inc.; Hermanson, "Bioconjugate Techniques", Academic
Press New
York, N.Y. (1995); Kay M. et al., 1995. Biochemistry 34:293; Stubbs et al.,
1996. Biochemistry
35:937; Gakamsky D. et al., "Evaluating Receptor Stoichiometry by Fluorescence
Resonance
Energy Transfer," in "Receptors: A Practical Approach," 2nd ed., Stanford C.
and Horton R. (eds.),
Oxford University Press, UK. (2001); U.S. Pat. No. 6,350,466 to Targesome,
Inc.]. Therefore, no
further description is considered necessary.
[0138] Alternately, an enzyme may be utilized as the detectable moiety to
enable detection of
the specific peptide/MHC complex via any of various enzyme-based detection
methods. Examples
of such methods include, but are not limited to, enzyme linked immunosorbent
assay (ELISA; for
example, to detect the specific peptide/MHC complex in a solution), enzyme-
linked
chemiluminescence assay (for example, to detect the complex on solubilized
cells), and
enzyme-linked immunohistochemical assay (for example, to detect the complex in
a fixed tissue).
[0139] Numerous types of enzymes may be employed to detect the specific
peptide/MHC
complex, depending on the application and purpose. Examples of suitable
enzymes include, but
are not limited to, horseradish peroxidase (HPR), (3-galactosidase, and
alkaline phosphatase (AP).
Ample guidance for practicing such enzyme-based detection methods is provided
in the literature
of the art (for example, refer to: Khatkhatay M I. and Desai M., 1999. J
Immunoassay 20:151-83;
Wisdom G B., 1994. Methods Mol Biol. 32:433-40; Ishikawa E. et al., 1983. J
Immunoassay
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4:209-327; Oellerich M., 1980. J Clin Chem Clin Biochem. 18:197-208; Schuurs A
H. and van
Weemen B K., 1980. J Immunoassay 1:229-49).
[0140] The present invention include the T cell receptor mimics described
herein attached to any
of numerous types of therapeutic moieties, depending on the application and
purpose. Various
types of therapeutic moieties that may be utilized in accordance with the
present invention include,
but are not limited to, a cytotoxic moiety, a toxic moiety, a cytokine moiety,
a bi-specific antibody
moiety, and the like. Specific examples of therapeutic moieties that may be
utilized in accordance
with the present invention include, but are not limited to, Pseudomonas
exotoxin, Diptheria toxin,
interleukin 2, CD3, CD16, interleukin 4, interleukin 10, Ricin A toxin, and
the like.
[0141] The functional moiety may be attached to the T cell receptor mimic of
the present
invention in various ways, depending on the context, application and purpose.
A polypeptidic
functional moiety, in particular a polypeptidic toxin, may be attached to the
antibody or antibody
fragment via standard recombinant techniques broadly practiced in the art (for
Example, refer to
Sambrook et al., infra, and associated references, listed in the Examples
section which follows).
A functional moiety may also be attached to the T cell receptor mimic of the
presently disclosed
and claimed invention using standard chemical synthesis techniques widely
practiced in the art [for
example, refer to the extensive guidelines provided by The American Chemical
Society (for
example at: http://www.chemistry.org/portal/Chemistry)]. One of ordinary skill
in the art, such as
a chemist, will possess the required expertise for suitably practicing such
such chemical synthesis
techniques.
[0142] Alternatively, a functional moiety may be attached to the T cell
receptor mimic by
attaching an affinity tag-coupled T cell receptor mimic of the present
invention to the functional
moiety conjugated to a specific ligand of the affinity tag. Various types of
affinity tags may be
employed to attach the T cell receptor mimic to the functional moiety. In one
embodiment, the
affinity tag is a biotin molecule or a streptavidin molecule. A biotin or
streptavidin affinity tag can
be used to optimally enable attachment of a streptavidin-conjugated or a
biotin-conjugated
functional moiety, respectively, to the T cell receptor mimic due to the
capability of streptavidin and
biotin to bind to each other with the highest non covalent binding affinity
known to man (i.e., with
a Kd of about 10-14 to 10-15)
[0143] A pharmaceutical composition of the present invention includes a T cell
receptor mimic
of the present invention and a therapeutic moiety conjugated thereto. The
pharmaceutical
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composition of the present invention may be an antineoplastic agent. A
diagnostic composition of
the present invention includes a T cell receptor mimic of the present
invention and a detectable
moiety conjugated thereto.
[0144] The present invention relates to methodologies for producing antibodies
that function as
T-cell receptor mimics (TCRms) and recognize peptides displayed in the context
of HLA molecules,
wherein the peptide is associated with a tumorigenic, infectious or disease
state. These antibodies
will mimic the specificity of a T cell receptor (TCR) such that the molecules
may be used as
therapeutic and diagnostic reagents. In one embodiment, the T cell receptor
mimics of the
presently disclosed and claimed invention will have a higher binding affinity
than a T cell receptor.
In one embodiment, the T cell receptor mimic produced by the method of the
presently disclosed
and claimed invention has a binding affinity of about 10 nanomolar or greater.
[0145] The methods of the presently claimed and disclosed invention begin with
the production
of an immunogen. The immunogen comprises a peptide/MHC complex, wherein the 3-
dimensional
presentation of the peptide in the binding groove is the epitope recognized
with high specificity by
the antibody. The immunogen may be any form of a stable peptide/MHC complex
that may be
utilized for immunization of a host capable of producing antibodies to the
immunogen, and the
immunogen may be produced by any methods known to those skilled in the art.
The immunogen
is used in the construction of an agent that will activate a clinically
relevant cellular immune
response against the tumor cell which displays the particular peptide/MHC
complex.
[0146] The peptide epitopes of the peptide/MHC complex of the immunogen are
antigens that
have been discovered as being novel to cancer cells, and such peptide epitopes
are present on the
surface of cells associated with a tumorigenic, infectious or disease state,
such as but not limited
to cancer cells, and displayed in the context of MHC molecules. The peptide
may be a known
tumor antigen, or a peptide identified in U.S. Patent Application Publication
No. US 2002/0197672
Al, filed by Hildebrand et al. on October 10, 2001 and published on December
26, 2002; or U.S.
Patent Application Publication No. US 2005/0003483 Al, filed by Hildebrand et
al. on May 13, 2004
and published on January 6, 2005; the contents of each of which are expressly
incorporated herein
by reference in their entirety, or the peptide may be a previously
unidentified peptide that is
identified by methods such as those described in the two Hildebrand et al.
published applications
incorporated immediately hereinabove by reference.
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[0147] The immunogen may be produced in a manner so that it is stable, or it
may be modified
by various means to make it more stable. Two different methods of producing a
stable form of an
immunogen of the present invention will be described in more detail
hereinbelow. However, it is
to be understood that other methods, or variations of the below described
methods, are within the
ordinary skill of a person in the art and therefore fall within the scope of
the present invention.
[0148] In one embodiment, the immunogen is produced by a cell-based approach
through
genetic engineering and recombinant expression, thus significantly increasing
the half-life of the
complex. The genetically-engineered and recombinantly expressed peptide/MHC
complex may
be chemically cross-linked to aid in stabilization of the complex.
Alternatively or in addition to
chemical cross-linking, the peptide/MHC complex may be genetically engineered
such that the
complex is produced in the form of a single-chain trimer. In this method, the
MHC heavy chain, R-2
microglobulin and peptide are all produced as a single-chain trimer that is
linked together. Methods
of producing single-chain trimers are known in the art and are disclosed
particularly in Yu et al.
(2002). Other methods involve forming a single-chain dimer in which the
peptide-R2m molecules
are linked together, and in the single-chain dimer, the (32m molecule may or
may not be membrane
bound.
[0149] In a second embodiment, the immunogen of the presently claimed and
disclosed
invention is produced by multimerizing two or more peptide/MHC complexes. The
term "multimer"
as used herein will be understood to include two or more copies of the
peptide/MHC complexwhich
are covalently or non-covalently attached together, either directly or
indirectly. The MHC molecules
of the complexes may be produced by any methods known in the art. Examples of
MHC
production include but are not limited to endogenous production and
purification, or recombinant
production and expression in host cells. In one embodiment, the MHC heavy
chain and R2m
molecules are expressed in E. coli and folded together with a synthesized
peptide. In another
embodiment, the peptide/MHC complex may be produced as the genetically-
engineered single-
chain trimer (or the single-chain dimer plus MHC heavy chain) described
hereinabove.
[0150] For multimerizing the two or more copies of the peptide/MHC complex to
form the
immunogen, each of the peptide/MHC complexes may be modified in some manner
known in the
art to enable attachment of the peptide/MHC complexes to each other, or the
multimer may be
formed around a substrate to which each copy of the peptide/MHC complex is
attached. The
multimer can contain any desired number of peptide/MHC complexes and thus form
any multimer
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desired, such as but not limited to, a dimer, a trimer, a tetramer, a
pentamer, a hexamer, and the
like. Specific examples of multimers which may be utilized in accordance with
the present invention
are described hereinbelow; however, these examples are not to be regarded as
limiting, and other
methods of multimerization known to those of skill in the art are also within
the scope of the present
invention. Streptavidin has four binding sites for biotin, so a BSP
(biotinylation signal peptide) tail
may be attached to the MHC molecule during production thereof, and a tetramer
of the desired
peptide/MHC complex could be formed by combining the peptide/MHC complexes
with the BSP
tails with biotin added enzymatically in vitro. An immunoglobulin heavy chain
tail may be utilized
as a substrate for forming a dimer, while a TNF tail may be utilized as a
substrate for forming a
trimer. An IgM tail could be utilized as a substrate for forming various
combinations, such as
tetramers, hexamers and pentamers. In addition, the peptide/MHC complexes may
be
multimerized through liposome encapsulation or artificial antigen presenting
cell technology (see
U.S. Serial No. 10/050,231, filed by Hildebrand et al. on January 16, 2002,
the contents of which
are hereby expressly incorporated herein by reference). Further, the
peptide/MHC complexes may
be multimerized through the use of polymerized streptavidin and would produce
what is termed a
"streptamer" (see http://www.streptamer.com/streptamer/, which is hereby
expressly incorporated
herein by reference in its entirety).
[0151] The immunogen of the present invention may further be modified for
providing better
performance or for aiding in stabilization of the immunogen. Examples of
modifications which may
be utilized in accordance with the present invention include but are not
limited to, modifying
anchor/tail or modifying amino acids in peptide/MHC complex, PEGalation,
chemical cross-linking,
changes in pH or salt depending on the specific peptide of the peptide/MHC
complex, addition of
one or more chaperone proteins that stabilize certain peptide/MHC complexes,
addition of one or
more adjuvants that enhance immunogenicity (such as but not limited to the
addition of a T cell
epitope on a multimer), and the like.
[0152] Once the immunogen is produced and stabilized, it is delivered to a
host for eliciting an
immune response. The host may be any animal known in the art that is useful in
biotechnological
screening assays and is capable of producing recoverable antibodies when
administered an
immunogen, such as but not limited to, rabbits, mice and rats. In one
embodiment, the host is a
mouse, such as a Balb/c mouse or a transgenic mouse. In another embodiment,
the mouse is
transgenic for the particular MHC molecule of the immunogen so as to minimize
the antigenicity
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of the immunogen, thereby ensuring that the 3-dimensional domain of the
peptide sitting in the
binding pocket of the MHC molecule is the focus of the antibodies generated
thereto and thus is
preferentially recognized with high specificity. In yet another embodiment,
the mouse is transgenic
and produces human antibodies, thereby greatly easing the development work for
creating a
human therapeutic.
[0153] After the host is immunized and allowed to elicit an immune response to
the immunogen,
a screening assay is performed to determine if the desired antibodies are
being produced. In one
embodiment, the assay requires four components plus the sera of the mouse to
be screened. The
four components include: (A) a binding/capture material (such as but not
limited to, streptavidin,
avidin, biotin, etc.) coated on wells of a solid support, such as a microtiter
plate; (B) properly folded
HLA trimer (HLA heavy chain plus [i2m plus peptide) molecule containing an
irrelevant peptide; (C)
properly folded HLA tetramer or trimer containing the peptide of interest; and
(D) at least one antibody which recognizes mouse IgG and IgA constant regions
and is covalently
linked to a disclosing agent, such as but not limited to, peroxidase or
alkaline phosphatase.
[0154] The solid support of (A) must be able to bind the HLA molecule of
interest in such a way
as to present the peptide and the HLA to an antibody without stearic or other
hindrance. One
configuration of the properly folded HLA trimers in (B) and (C) above is a
single-site biotinylation.
If single-site biotinylation cannot be achieved, then other methods of
capture, such as antibody may
be used. If antibody is used to capture the HLA molecule onto the solid
support, it cannot cross-
react with the anti-mouse IgG and IgA in (D) above.
[0155] Prior to assaying the serum from immunized mice, it is preferred that
the bleeds from the
immunized mice be preabsorbed to remove antibodies that are not peptide
specific. The
preabsorption step should remove antibodies that are reactive to epitopes
present on any
component of the immunogen other than the peptide, including but not limited
to, R2m, HLA heavy
chain, a substrate utilized for multimerization, an immunogen stabilizer, and
the like.
[0156] One embodiment of methods of assaying serum from immunized mice is
described in
the attached figures (see for example Fig. 5), as well as in the Examples
provided hereinafter.
Once it is determined that the desired antibodies are being produced, a
standard hybridoma fusion
protocol can be employed to generate cells producing monoclonal antibodies.
These cells are
plated such that individual clones can be identified, selected as individuals,
and grown up in
individual wells in plates. The supernatants from these cells can then be
screened for production
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of antibodies of the desired specificity. These hybridoma cells can also be
grown as individual
clones and mixed and sorted or grown in bulk and sorted as described below for
cells expressing
surface immunoglobulin of the desired reactivity.
[0157] In another embodiment of the present invention, cell sorting is
utilized to isolate desired
B cells, such as B memory cells, prior to hybridoma formation. One method of
sorting which may
be utilized in accordance with the present invention is FACS sorting, as B
memory cells have
immunoglobulin on their surface, and this specificity may be utilized to
identify and capture these
cells. FACS sorting is a preferred method as it involves two color staining.
Optionally, beads can
be coated with peptide/HLA complex (with FITC or PE) and attached to a column,
and B cells with
immunoglobulin on their surface can be identified by FACS as well as by
binding to the complex.
In yet another alternative, a sorting method using magnetic beads, such as
those produced by
Dynal or Miltenyi, may be utilized.
[0158] In another embodiment of the present invention, the sorted B cells may
further be
differentiated and expanded into plasma cells, which secrete antibodies,
screened for specificity
and then used to create hybridomas or have their antibody genes cloned for
expression in
recombinant form.
[0159] Once the antibodies are sorted, they are assayed to confirm that they
are specific for one
peptide/MHC complex and to determine if they exhibit any cross reactivity with
other HLA
molecules. One method of conducting such assays is a sera screen assay as
described in U.S.
Patent Application Publication No. US 2004/0126829 Al, filed by Hildebrand et
al. on September
24, 2003 and published on July 1, 2004, the contents of which are hereby
expressly incorporated
herein by reference. However, other methods of assaying for quality control
are within the skill of
a person of ordinary skill in the art and therefore are also within the scope
of the present invention.
[0160] The present invention also includes a predictive screen to determine if
a particular
peptide can be utilized in an immunogen of the present invention for producing
the desired
antibodies which act as T-cell receptor mimics. These screens include but are
not limited to,
stability, refolding, IC50, Kd, and the like. The present invention may
provide a threshold of binding
affinity of peptide so that a predictive threshold can be created for
examining entire proteins of
interest for potential peptides. This threshold can also be used as a
predictor of yield that can be
obtained in the refolding process of producing the peptide/MHC complex. In
addition, if a potential
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peptide is shown to be low to medium in the predictive screens, methods of
modifying the
immunogen can be attempted at the onset of the production of immunogen.
[0161] The TCR mimics of the present invention have a variety of uses. The TCR
mimic
reagents could be utilized in a variety of vaccine-related uses. In one
embodiment, the TCR
mimics could be utilized as direct therapeutic agents, either as an antibody
or bispecific molecule.
In another embodiment, the TCR mimics of the present invention could be
utilized for carcinogenic
profiling, to provide an individualized approach to cancer detection and
treatment. The term
"carcinogenic profiling" as used herein refers to the screening of cancer
cells with TCRm's of
various specificities to define a set of peptide/MHC complexes on the tumor.
In another
embodiment, the TCR mimics of the present invention could be utilized for
vaccine validation, as
a useful tool to determine whether desired T cell epitopes are displayed on
cells such as but not
limited to, tumor cells, viral infected cells, parasite infected cells, and
the like. The TCR mimics of
the present invention could also be used as research reagents to understand
the fate of antigen
processing and presentation in vivo and in vitro, and these processes could be
evaluated between
solid tumor cells, metastatic tumor cells, cells exposed to chemo-agents,
tumor cells after exposure
to a vaccine, and the like. The TCR mimics of the present invention could also
be utilized as
vehicles for drug transport to transport payloads of toxic substances to tumor
cells or viral infected
cells. Further, the TCR mimics of the present invention could also be utilized
as diagnostic
reagents for identifying tumor cells, viral infected cells, and the like. In
addition, the TCR mimic
reagents of the present invention could also be utilized in metabolic typing,
such as but not limited
to, to identify disease-induced modifications to antigen processing and
presentation as well as
peptide-HLA presentation and tumor sensitivity to drugs.
[0162] Examples are provided hereinbelow. However, the present invention is to
be understood
to not be limited in its application to the specific experimentation, results
and laboratory procedures.
Rather, the Examples are simply provided as one of various embodiments and is
meant to be
exemplary, not exhaustive.
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Table I - Peptides Utilized in the Methods of the Present Invention
Name Sequence SEQ Origin Position IC50* Tetramer
ID Yield (mg)
NO:
p53 (264) LLGRNSFEV 1 Tumor suppressor p53 (264-272) 1273 1.99 +/- 0.76
eIF4G VLMTEDIKL 2 eukaryotic transcription (720-728) 690.3 2.77 +/-1.09
initiation factor 4 gamma
Her2/neu KIFGSLAFL 3 tyrosine kinase-type cell (369-377) 881.9 0.89 +/- 0.69
surface receptor Her2
(EC 2.7.1.112) (C-erbB-
2)
TMT TMTRVLQGV 4 human chorionic (40-48) 1862 2-3
gonadotropin-(3
VLQ VLQGVLPAL 5 human chorionic (44-53) 914.1 2-3
gonadotropin-(3
GVL GVLPALPQV 6 human chorionic (47-55) 926.8 2-3
gonadotropin-R
*Peptide IC50 values less than 5000 are considered high affinity binders.
EXAMPLE 1
[0163] The human p53 protein is an intracellular tumor suppressor protein.
Point mutations in
the p53 gene inactivate or reduce the effectiveness of the p53 protein and
leave cells vulnerable
to transformation during progression towards malignancy. As cells attempt to
compensate for a
lack of active p53, over production of the p53 protein is common to many human
cancers including
breast cancer, resulting in cytoplasmic increases in p53 peptide fragments
such as the peptide
264-272. There are many reports demonstrating that surface HLA-A2 presents the
264-peptide
epitope from wild-type p53 (Theobald et al., 1995; and Theobald et al., 1998).
Cytotoxic T
lymphocytes have been generated against the 264-peptide-HLA-A2 complexes
(referred to herein
as 264p-HLA-A2) on breast cancer cells from peripheral blood monolayer cells
(PBMC) of healthy
donors and individuals with breast cancer (Nikitina et al., 2001; Barfoed et
al., 2000; and Gnjatic
et al., 1998). Further, several studies have reported successful immunization
with the 264 peptide
in HLA-A2 transgenic mice (Yu et al., 1997; and Hoffmann et al., 2005). The
studies were
successful in generated murine CTL lines reactive against the 264p-HLA-A2
complex and showed
that these murine CTL lines could detect and destroy human breast cancer
cells. Because the
264-peptide presented by HLA-A2 on the surface of malignant cells is
recognized by the immune
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system and it has relatively high affinity (IC50 <1 nM) (Chikamatsu et al.,
1999), the 264 peptide was
utilized in Example 1 to construct 264p-HLA-A2 tetramers for use in immunizing
mice for production
of T cell receptor mimics in accordance with the present invention.
[0164] Preparation of 264p-HLA-A2 peptide tetramers: The heavy and light (R2m)
chains of the
HLA-A2 Class I molecule were expressed and prepared separately in E. coli as
insoluble inclusion
bodies according to established protocols. The inclusion bodies were dissolved
in 10 M urea, and
the heavy and light chains were mixed at a molar ratio of 1:2 at a
concentration of 1 and 2 mM
respectively with 10 mg of a synthetic peptide (LLGRNSFEV; SEQ ID NO:1) from
the human p53
tumor suppressor protein (amino acids 264-272) in a protein refolding buffer
and were allowed to
refold over 60 hr at 4 C with stirring. The filtrate of this mix was
concentrated, and the buffer was
exchanged with 10 mM Tris pH 8Ø The mix was biotinylated using a recombinant
birA ligase for
two hours at room temperature and then subjected to size exclusion
chromatography on a
Sephadex S-75 column (Superdex S-75, Amersham GE Health Sciences) (Fig. 1).
Alternatively,
a monomer HLA-A2-peptide can be purified from a Sephadex S-75 column,
concentrated and then
biotinylated using birA ligase for 2 hours at room temperature. The refolded
biotinylated monomer
peak was reisolated on the S-75 column and then multimerized with streptavidin
(SA) at a 4:1 molar
ratio. The multimerized sample was subjected to size exclusion chromatography
on a Sephadex
S-200 column (Fig. 2).
[0165] The stability of the 264p-HLA-A2 tetramers was assessed in mouse serum
at different
temperatures using the conformational antibodies BB7.2 and W6/32 (Fig. 3). The
results suggest
that 50% of the 264p-HLA-A2 tetramers maintain a conformational integrity
after 10 h incubation
at 37 C. Only 10% of tetramers remain stable after 40 h incubation. However,
the multimerization
of 264p-HLA-A2 greatly increased the half life of the molecules; normally
monomers only have a
few hours half life in mouse serum. It was not clear a priori that these
tetramers would be stable
long enough to elicit a robust immune response in mouse, but recent results
indicated that at least
a fraction of the injected tetramers were stable long enough in mice to elicit
a specific antibody
response.
[0166] Immunization of Baib/c mice (female and male) with peptide-HLA-A2: The
complete
structure of the peptide-HLA-A2 tetramer immunogen is shown in Fig. 4. Balb/c
mice (female and
male) were immunized with the 264p-HLA A2 tetramers. Each mouse was injected
subcutaneously
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every 2 weeks (up to 5 times) with immunogen (50 pg) in PBS which also
contained 25 pg of Quil
A (adjuvant) in 100 NI.
[0167] Blood samples from mice were collected into 1.5 ml eppendorf
microcentrifuge tubes
containing heparin, and plasma was clarified by centrifugation at 6,000 x g
for 10 minutes. The
recovered plasma samples were then frozen at -20 C and later used in screening
assays.
Samples were diluted 1:200 into 0.5% milk in Phosphate Buffered Saline
solution (PBS) and
pre-absorbed with refolded monomer HLA-A2 containing an irrelevant peptide
(Her2/neu) before
screening.
[0168] Effective assays were needed to analyze anti-peptide-HLA antibodies in
the serum of
immunized mice, and several factors complicate this analysis. One of these
factors is predicated
on the fact that a specific antibody response against a complex epitope
represented by both the
peptide and the binding site of the HLA molecule is being sought, and this
epitope may represent
only a minor target to B cells. A significant portion of the antibodies raised
against peptide-HLA
tetramers are generated against HLA as well as streptavidin (SA) utilized to
tetramerize the
peptide-HLA complexes; consequently, an assay protocol had to be developed
that allowed for
detection of a low concentration of specific antibodies in a milieu of non-
specific ones. To resolve
this problem, a pre-absorption step was incorporated into an ELISA assay
format. This step was
designed to remove antibodies against HLA and (32-microglobulin from the
reaction. In a variation
of this assay, biotinylated non-relevant monomers were used to pre-absorb and
then remove the
formed complexes from the reaction on a sold surface-bound SA. In the ELISA
format, sera from
immunized mice are first reacted with HLA-A2 monomers containing another
irrelevant peptide
before reacting them with HLA-A2 complexes of the relevant peptide. The
specifics of these
assays are described in more detail herein below.
[0169] Pre-Absorption assay: Serum from the immunized mice was used in an
ELISA format
to identify "peptide-specific" antibody responses. Remember that TCR mimics
are antibodies
having dual specificity for both peptide and HLA. In addition, the immunized
mice will produce
antibody specificities against HLA epitopes. It is these antibodies that the
pre-absorption protocol
substantially removes from the serum samples. In order to substantially remove
antibodies that
were not peptide specific, a pre-absorption step was included in the protocol.
It was assumed that
12 pg of IgG is present in 1 ml of mouse serum, and that 10% of the IgG in
immunized mouse
serum is specific for an epitope on the peptide-HLA-A2 immunogen. Based on
these assumptions,
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1.2 pg of IgG in 1 ml of serum from an immunized mouse is potentially specific
for some position
on the peptide-A2 molecules and is not "peptide specific". In order to remove
these non-specific
antibodies, 20 pg of biotinylated Her2/neu-peptide-HLA-A2 (which differs from
264p-HLA-A2 only
in the peptide) was added to 1 mi of a 1:200 dilution of each mouse bleed.
Samples were
incubated overnight at 4 C with agitation. The next morning 0.5 ml of sample
was added to a well
in a 12 well plate (which had been coated the previous night with 10 mg of
streptavidin and blocked
in 5% milk protein) and incubated for 1 hour. The pre-absorbed samples were
then transferred to
a second streptavidin coated well on the plate. This process was repeated one
more time (a total
of 3) to ensure efficient removal of antibody-HLA complexes and antibodies
reactive to streptavidin
and/or biotin. After completing the pre-absorption steps, samples were ready
for use in the
screening ELISA.
[0170] Screening ELISA: Fig. 5 demonstrates the development of an ELISA assay
for screening
mouse bleeds to determine if there are antibodies specific to the peptide-of-
interest-HLA-molecule
complex present. Pre-absorbed serum samples from six Balb/c mice were
individually tested in
the ELISA screening assay of Fig. 5 (see Fig. 6). Briefly, 96 well plates
(maxisorb; Nunc) were
coated the night before with 0.5 pg of either biotinylated 264p-HLA-A2 monomer
or biotinylated
eIF4Gp-HLA-A2 monomer at 4 C. (Subsequence interactions used non-biotinylated
forms of the
relevant and irrelevant HLAs.) The following day, wells were blocked with 1%
milk for 1 h at room
temperature and rinsed lx in PBS. The pre-absorbed serum samples (50 pl/well)
were then added
to wells starting at 1:200 dilution and titrating down to a final dilution
equivalent to either 1:1600 or
1:3200. After 2 hr incubation at room temperature, the plate was washed 2x in
PBS followed by
the addition of antibody conjugate (goat anti-mouse-HRP, 1:500 dilution) and
incubated for 1 h at
room temperature. The plate was then washed 3x in PBS and developed after
addition of 50 ial of
tetramethylbenzidine (TMB) substrate. Development time was 5 to 10 minutes,
and the reaction
was stopped with the addition of 50 pl quench buffer (2 M sulfuric acid). The
results were read at
450 nm absorbance (Fig. 6).
[0171] For a positive control in the assay, BB7.2 mAb was used at 50 to 200
ng/well. This mAb
recognizes only conformationally correct forms of the refolded peptide-HLA-A2
molecule. For a
negative control in the assay, a peptide-HLA-A2 complex containing an
irrelevant peptide was
coated on the plate. In this particular assay, the negative control was eIF4G
peptide-loaded
HLA-A2 monomer.
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[0172] In addition, the mice used for the production of the antibodies were
pre-bled in order to
ensure that Balb/c mice do not harbor antibodies specific for the desired
antigens before
immunization. Assay background was determined using pre-bleed samples at 1:200
and 1:400
dilution. The highest absorbance reading recorded for pre-bleeds was less than
OD 0.06 at 450
nm.
[0173] Fig. 6 shows the results from an ELISA of six individual bleeds from
Balb/c mice
immunized with tetramers of 264p-HLA-A2. The data shown in Fig. 6 demonstrates
that both male
and female mice immunized with 264p-HLA-A2 tetramers make specific antibody to
264p-HLA-A2
monomers. Bleeds incubated in wells containing eIF4Gp-HLA-A2 monomers
(irrelevant peptide)
were used to evaluate non-specific reactivity of bleeds. The findings shown in
Fig. 6 demonstrate
minimal reactivity to eIF4Gp/A2 with signal to noise ratios ranging from 3 to
6 fold, indicating that
immunization of mice with peptide-A2 tetramers leads to the generation of
specific antibody
responses to the immunogen.
[0174] The results presented in Fig. 6 demonstrate that antibodies in the
serum reacted twice
as strongly or stronger with 264p-HLA-A2 as compared to eIF4Gp-HLA-A2,
suggesting that some
specific antibodies against the p53-264p epitope are present. The larger the
difference in the
response between reactivity with HLA-A2 complexes with a relevant or
irrelevant peptide, the
higher the titer for specific antibodies in the sera. The results in Fig. 6
clearly demonstrate that
serially diluted sera from all six mice generated a signal with 264p-HLA-A2
monomers that was 2-5
times stronger than the signal with eIF4Gp-HLA-A2 monomers, clearly
demonstrating the
effectiveness of the methods of the present invention.
[0175] T2 binding assay: To confirm the ELISA findings, the binding of the
different mouse
bleed samples to T2 cells pulsed with either the 264 peptide (peptide of
interest) or the eIF4G
peptide (irrelevant peptide) was investigated, as shown in Fig. 7. T2 cells
are a human B
lymphoblastoid cell line (ATCC CRL-1 999) that has been well characterized by
Peter Creswell (Wei
et al., 1992). T2 cells are useful for studying recognition of HLA-A2 antigens
because they are
deficient in peptide loading. These cells have been found to be deficient in
TAP1/2 proteins, which
are necessary proteins for transporting peptides from the cytosol into the
endoplasmic reticulum
for loading HLA class I molecules. Because of the TAP1/2 deficiency, these
cells express a low
level of empty HLA-A2 molecules on the surface. Thus, these cells can be
primed (loaded) with
peptides of choice, and the cells will display them appropriately in the
context of HLA-A2 molecules
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on their surface. Addition of peptide to these cells leads to peptide binding
to the HLA-A2
molecules which are constantly cycling to the surface and stabilization of the
HLA-A2 structure.
The more stable structure increases the density of surface displayed HLA-A2
molecules that are
loaded with the particular peptide of interest. T2 cells can be loaded with
relevant or irrelevant
peptide, and the reactivity of immune sera from immunized mice against them
can be measured.
The larger the difference in the response between T2 cells loaded with
relevant or irrelevant
peptide, the higher the titer for specific antibodies in the sera.
[0176] T2 cells were loaded with either the 264 or the eIF4G peptide, and then
the cells were
stained with the BB7.2 antibody to detect the level of HLA-A2 molecules
present on the surface of
T2 cells. Fig. 8 shows that both 264 and eIF4G peptides have been successfully
loaded by
comparing the BB7.2 staining profile of cells that received peptide versus the
cells that did not
receive peptide (negative controls). These findings demonstrate that eIF4G
peptide may be more
efficient at loading and stabilizing HLA-A2 on T2 cells than the 264 peptide.
[0177] Fig. 9 illustrates the results of staining of 264 peptide-loaded T2
cells with the 13M2
mouse bleed. The pre-absorbed mouse sample preferentially binds cells pulsed
with 264 peptide.
In contrast, Fig. 10 demonstrates that the pre-bleed samples (mice bleeds
taken prior to
immunization) show no sign of reactivity to T2 cells pulsed with either the
264- or eIF4G peptide.
In combination, these results clearly demonstrate that a polyclonal peptide-
HLA specific antibody
response can be generated to the specific three-dimensional, and that these
antibodies are specific
for the immunogen that was used. They confirm that the antibodies produced
also recognize a
"native" or natural form of the peptide-HLA-A2 complex and are not restricted
in reactivity to the
refolded form used to prepare the immunogen.
[0178] Hybridomas were generated by submitting 12 mice immunized with 264p-HLA-
A2 to the
Hybridoma Center, Oklahoma State University, Stillwater, Oklahoma, for
hybridoma generation
using standard technology. In total, the center returned 1440 supernatants
from p53-264
hybridoma isolates for screening. Fig. 11 depicts development of assays to
screen hybridomas to
determine if they are producing anti-peptide-HLA specific antibodies. In a
primary ELISA screen,
40 positives were identified, and in a secondary screen, 7 positives against
264p-HLA-A2 were
identified. The results from screening hybridoma supernatants by a competitive
binding ELISA are
shown in Fig. 12. Supernatants that had ratios of eIF4G/264 greater than 1.7
were considered
positive, and after expanding hybridoma numbers, the supernatant was re-
screened.
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Approximately 1500 wells were screened, and approximately 50 positives were
identified after the
primary screen.
[0179] Hybridomas determined positive after a first screening were expanded,
and the
supernatant was diluted and rescreened by competitive ELISA two weeks after
cell growth. Fig.
13 represents data obtained from a competitive ELISA of these positive
hybridoma clones.
TCRm's specific for 264p-HLA-A2 were determined by showing a reduction in
absorbance (read
at 450 nm) after addition of competitor (no tetramer versus 264p tetramer),
while no change in
absorbance was observed after addition of non-competitor (no tetramer versus
eIF4Gp tetramer).
These findings confirm anti-264p-HLA-A2 specificity of TCRm's and validate the
protocols of the
presently disclosed and claimed invention for generating monoclonal antibodies
specific for
peptide-HLA complexes.
[0180] Supernatant from 13.M3-2A6 was characterized further by a cell-based
competitive
binding assay, as shown in Fig. 14. These findings demonstrate that 13.M3.2A6
TCRm has
specificity for the authentic 264p-HLA-A2 epitope. This is illustrated by the
significant reduction of
TCRm binding to 264p pulsed T2 cells in the presence of the competitor versus
the non-competitor.
The competitor reduces binding by greater than 3.5 fold (as measured by mean
channel
fluorescence) compared to the effect of an equivalent amount of non-
competitor.
[0181] Therefore, the results presented herein in Example 1 clearly
demonstrate that the
immunogen of the present invention is capable of eliciting an immune response
in a host that is
specific for an epitope formed by a desired peptide presented in the context
of an HLA molecule.
[0182] These results also indicate there is a significant component of the
antibody reactivity in
most of the immunized mice that recognizes epitopes that are not specific to
the peptide in the
context of the HLA binding groove. Rather, these antibodies probably recognize
other epitopes
common to properly folded HLA-A2 molecules (independent of the peptide region)
or epitopes
which form as the immunogen is processed, unfolded and denatured in the body.
[0183] Appropriate measures must be taken to remove these "non-peptide-
specific" antibodies
from the serum prior to evaluating it for the presence of a true TCR mimic
antibody. The ability to
discover an antibody which recognizes the peptide of interest in its authentic
three-dimensional
configuration when the HLA-binding groove is dependent upon (1) the creation
of an immunogen
capable of presenting the peptide in this context, and (2) the ability to
prepare the serum from the
immunized animal in such a way that the peptide specific reactivity is
revealed.
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EXAMPLE 2
[0184] The eukaryotic translation initiation factor 4 gamma (eIF4G) is a
protein which is part of
a complex of molecules that are critical in regulating translation. When
breast carcinoma cell lines
(MCF-7 and MDA-MB-231) were stressed with serum starvation, the eIF4G protein
degrades ihto
smaller peptide fragments (Morley et al., 2000; Morley et al., 2005; Bushell
et al., 2000; and
Clemens, 2004). A peptide of eIF4G has been identified as being presented by
HLA molecules on
HIV infected cells at a higher frequency than in uninfected cells by the
epitope discovery method
of Hildebrand et al. (US Patent Application Publication No. US 2002/0197672
Al, which has
previously been incorporated herein by reference). The epitope discovery
methodology is shown
in Fig. 15. Briefly, an expression construct encoding a secreted HLA molecule
is transfected into
a normal cell line and an infected, diseased or cancerous cell line (in this
case, an HIV infected
cell line), and the cell lines are cultured at high density in hollow-fiber
bioreactors. Then, the
secreted HLA molecules are harvested and affinity purified, and the peptides
bound therein are
eluted. The peptides from the uninfected cell line and the HIV infected cell
line are then
comparatively mapped using mass spectroscopy to identify peptides that are
presented by HLA
at a higher frequency in the HIV infected than in the uninfected cells. Using
this method, the
peptide VLMTEDIKL (SEQ ID NO:2), was identified, and determined to be a
peptide fragment of
eukaryotic translation initiation factor 4 gamma (eIF4G). The peptide of SEQ
ID NO:2 is referred
to herein as the "eIF4G peptide", or "eIF4Gp".
[0185] Monomers and tetramers of eIF4Gp-HLA-A2 complexes were produced in a
similar
manner as described in Example I for the 264p-HLA-A2 complexes. Briefly, 10 mg
(10 pM) of
peptide were refolded with 46 mg (1 pM) of HLA-A2 heavy chain and 28 mg (2 pM)
of HLA light
chain under appropriate redox conditions over approximately 60 hours at 4 C.
The monomers
were biotinylated and multimerized with streptavidin to form tetramers, and
the tetramers were
purified on a Superdex S200 column. Under the abovementioned conditions,
typically 10-20 mg
properly folded monomer, 8-12 mg of biotinylated monomer, and 2-3 mg of
tetramers were
produced.
[0186] Tetramer stability was assessed as described in Example 1 for the 264p-
HLA-A2
tetramers. In contrast to the 264p-HLA-A2 tetramers, which have a half life of
10 hours at 37 C,
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eIF4Gp-HLA-A2 tetramers have a half life of 20 hours, and 40% of tetramers
remain stable after
40 hours of incubation.
[0187] The eIF4Gp-HLA-A2 tetramers were utilized to immunize Balb/c mice as
described in
Example 1, and the mice were bled and sera assayed using the ELISA method
described above
in Example 1 and in Fig. 5. Sera from a mouse immunized with eIF4Gp-HLA-A2
tetramers was
pre-absorbed with biotinylated 264p-HLA-A2 monomers. The serum was reacted
with SA on a
solid surFace and then used in an ELISA format. Serum was reacted with solid
surface bound (1)
264p-HLA-A2 monomers; (2) eIF4Gp-HLA-A2 monomers; or (3) Her2/neu-peptide-HLA-
A2
monomers, and the bound antibody was detected with a goat anti mouse (GAM)-HRP
conjugate
antibody. The ELISA reactions were then developed with TMB (an HRP chromogenic
substrate),
and the absorbance read at 450 nm. The results shown in Fig. 17 illustrate
that antibodies in the
serum generated a signal thatwas twice as strong or strongerwith eIF4Gp-HLA-A2
than with either
264p-HLA-A2 or Her2/neu-peptide-HLA-A2, suggesting that some specific
antibodies against the
eIF4Gp epitope are present.
[0188] To confirm the ELISA findings, cell based assays were performed. T2
cell direct binding
assays, as described in Example 1 and in Fig. 7, were performed, and the
results shown in Fig.s
18 and 19. In these assays, T2 cells were loaded with a relevant (eIF4Gp) or
irrelevant (264p)
peptide, and the reactivity of immune sera from immunized mice against them
were measured.
Fig. 18 demonstrates the detection of HLA-A2 levels on peptide-pulsed T2 cells
using BB7.2 mAb.
This figure demonstrates the successful and relatively equivalent loading of
both the 264 and
eIF4G peptides on the surface of HLA-A2 T2 cells.
[0189] Fig. 19 demonstrates the results of staining eIF4G and 264 peptide-
loaded T2 cells with
a bleed from a mouse immunized with eIF4Gp-HLA-A2. 264 peptide loaded cells
are shown in the
solid peak. The pre-absorbed serum sample was used at a dilution of 1:400 for
staining and
preferentially binds cells pulsed with the eIF4G peptide (as shown by the
rightward shift). The pre-
bleed sample shows no sign of reactivity to T2 cells pulsed with either
peptide (not shown).
[0190] Next, T2 cell-based competitive assays, as described in Example 1 and
in Fig. 7, were
used to further evaluate the specificity of the polyclonal antibody to eI F4Gp-
HLA-A2, and the results
are shown in Figs. 20 and 21. In these assays, sera from mice immunized with
eIF4Gp-HLA-A2
tetramers were diluted 1:200 in PBS and pre-absorbed against Her2/neu-peptide-
HLA-A2. The
sera was then mixed with eIF4Gp-HLA-A2 or with 264p-HLA-A2, either in the form
of monomers
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(Fig. 20) or tetramers (Fig. 21) and before being reacted with T2 cells loaded
with eIF4G peptide
(100 iag/mI).
[0191] In the Figures, the maximum staining signal (filled peak) is shown for
the anti-serum.
To assess the specificity of antibody binding, a competitor (eIF4Gp-HLA-A2) or
a non-competitor
(264p-HLA-A2) was included in the cell staining reaction mix at three
different concentrations (0.1,
1.0 and 10 pg). The results shown in Figs. 20 and 21 reveal that the addition
of the 264p-HLA-A2
monomer ortetramer had little inhibitory activity on anti-serum binding to
eIF4G peptide-loaded T2
cells. In contrast, a dose-response effect of specific binding to T2 cells was
observed in the
presence of the competitor eIF4Gp-HLA-A2 monomer or tetramer. These findings
provide
additional evidence that the immunization strategy of the presently disclosed
and claimed invention
can elicit a specific anti-peptide-HLA-A2 IgG antibody response.
[0192] Mouse hybridomas were generated as described in Example 1 using
standard
technology, and immunogen specific monoclonals were identified using a
competitive binding
ELISA (as described herein before). From over 800 clones, 27 mAb candidates
were identified,
and 4F7 mAb (IgG1 isotype) was selected for further characterization. After
expanding the 4F7
hybridoma cell line by known methods in the art, the mAb was purified from 300
ml of culture
supernatant on a Protein-A column that yielded 30 mg of 4F7 mAb. The
specificity of antibody
binding to relevant peptide-HLA-A2 tetramers and 3 irrelevant peptide-HLA-A2
tetramers was
determined by ELISA, as shown in Fig. 22. The 4F7 mAb showed specific binding
only to eIF4Gp-
HLA-A2 tetramers; no signal was detected using irrelevant peptide-A2 controls,
which included
peptide VLQ and TMT, both derived from the human beta chorionic gonadotropin
protein, and 264
peptide derived from the human p53 tumor suppressor protein.
[0193] Next, the binding affinity and specificity of the 4F7 mAb was
determined by plasmon
surface resonance (BIACore). 4F7 mAb was coupled to a biosensor chip via amine
chemistry, and
soluble monomers of HLA-A2 loaded with 264 or eIF4G peptide were passed over
the antibody
coated chip. In Fig. 23, specific binding of soluble eIF4Gp-HLA-A2 monomer to
4F7 mAb was
observed, while no binding to 264p-HLA-A2 complexes containing the irrelevant
peptide p53-264
was observed. The affinity constant of 4F7 mAb for its specific ligand was
determined at 2
x10-9M.
[0194] In Figs. 22 and 23, 4F7 binding to recombinant eIF4Gp-HLA-A2 molecules
was
demonstrated. In Fig. 24, 4F7 binding to eIF4Gp-HLA-A2 complexes on the
surface of T2 cells
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was demonstrated. In this experiment cells were pulsed at 10 pg/ml with the
following peptides:
eIF4G, 264, and TMT. Unpulsed T2 cells were also used as a control. In Fig.
24A, T2 cells
pulsed with irrelevant peptides or no peptide and stained with 4F7 (50 ng)
displayed minimal signal.
In contrast, 4F7 staining of eIF4G peptide loaded T2 cells resulted in a
significant rightward shift,
indicating specific binding of 4F7. In Panel B, T2 cells were stained with
BB7.2 mAb (specific for
HLA-A2). T2 cells loaded with any of the peptides resulted in a rightward
shift of the peak,
indicating that each of the peptides efficiently loads the HLA on the cell
surface. These data also
indicate that the 4F7 binding to T2 cells is dependent on the antibody
recognizing both peptide and
HLA-A2.
[0195] Characterization of 4F7 TCRm binding specificity using human epithelial
cell lines. It was
observed that the 4F7 TCRm mAb recognizes recombinant HLA-A2 protein or T2
cells pulsed with
eIF4G(720) peptide. Next, it was evaluated whether this antibody would
recognize the eIF4G(720)
peptide-A2 complex on a tumor cell line expressing HLA-A2. Several groups have
reported on the
overexpression of eIF4G protein in malignant cells (Bauer et al., 2001 and
2002; and Fukuchi-
Shimogori et al., 1997). However, there are no reports describing the
presentation of the
eIF4G(720) peptide by MHC class I molecules on cancer cells. To address
whether the self peptide
was presented on cancer cells, the 4F7 TCRm mAb was used to stain a normal
human mammary
epithelial cell line and a human breast carcinoma cell line (MDA-MB-231).
Although both cell lines
expressed similar levels of HLA-A2 on their surface, the 4F7 TCRm mAb stained
only the breast
carcinoma cell line (Fig. 25), indicating that cancer cells express this
peptide-HLA-A2 epitope. In
addition, these results support the binding specificity of 4F7 TCRm mAb for
the eIF4G(720)
peptide-HLA-A2 complex.
[0196] In Fig. 26A, MCF-7 cells were stained with 100 ng of 4F7 mAb and showed
a significant
rightward shift compared to the isotype control. To determine if binding was
indeed specific for the
eIF4G peptide, soluble tetramers (competitor and non-competitor) were used to
block 4F7 binding.
As expected, eIF4Gp-HLA-A2 tetramer completely blocked 4F7 staining, while the
non-competitor,
264p-HLA-A2, failed to block 4F7 mAb from binding to cells. In Fig. 26B, the
HLA-A2 negative
breast carcinoma cell line BT-20 was not stained with 4F7 mAb. These findings
support the
specific binding of 4F7 antibody to eIF4Gp-HLA-A2 complex.
[0197] In Fig. 27, three panels are shown in which MDA-231 cells were stained
with 4F7 mAb
(50 ng) in the absence or presence of soluble peptide-HLA-A2 monomers. The
three peptide-HLA-
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A2 monomers selected were eIF4Gp (competitor) and 264p and Her2/neu peptide
(non-competitors). As shown in Fig. 27A, 4F7 binds to MDA-231 cells, and its
binding is
significantly inhibited using competitor. In contrast, no reduction in binding
signal strength was
seen with either non-competitor, indicating that 4F7 binds to tumor cells in a
specific manner.
[0198] These data confirm the isolation of a novel TCRm monoclonal antibody
with specificity
for a peptide derived from the eIF4G protein that is presented by HLA-A2 on
the surface of breast
cancer cells.
[0199] Direct detection of endogenously presented eIF4G(720)-HLA-A*0201
complexes on
HIV-1 infected CD4+ T cells. Elevated eIF4G(720) peptide bound to soluble HLA-
A*0201 molecules
as well as eIF4G peptide presented by HLA-B "0702, was revealed using HIV-1
infected Sup-T1
cells. Development of the 4F7 TCRm mAb facilitated a more physiologically
relevant analysis of
the eIF4G(720)-HLA-A*0201 epitope through characterization of these complexes
on HIV-1 infected
and non-infected CD4+ T cells. The staining profiles for 4F7 TCRm, I B8 TCRm,
and IgG, isotype
control using mock infected HLA-A*0201 positive PBMCs are shown in Fig. 28A.
The 4F7 TCRm
mAb showed modest staining of mock infected PBMCs, thus validating our Sup-T1
cell findings in
which eIF4G(720) peptide is constitutively expressed at low levels. In
contrast, no cell staining was
observed with the two control mAbs. Moreover, no cell staining with 4F7 TCRm
mAb was detected
in HIV-1 infected, HLA-A*0201 negative CD4+ T cells (data not shown),
indicating that eIF4G(720)
must be presented in the context of HLA-A*0201.
[0200] Next, eIF4G(720) expression was examined in HLA-A*0201 positive CD4+ T
cells infected
with the HIV-1 strain Illb and stained with the 4F7 TCRm five days post-
infection (PI). HIV-1
infected CD4+ T cell$ were identified by HIV-1 p24 expression (Figs. 28D-F and
28G-1) by staining
with the anti-p24-PE conjugate. On day 5 PI, 30.1 % of the cells were p24
positive. At this time the
population of cells was stained with the 4F7 TCRm, 1 B8 TCRm or IgG1 isotype
control mAbs. As
shown in Figs. 28A-C and 28G-I, in both mock infected cells and in p24
negative cells, little if any
difference was observed between 4F7 TCRm and control antibody staining. In
contrast, 4F7 TCRm
staining of the infected cell population (Fig. 28F; p24 positive cells)
revealed a marked rightward
shift in mean fluorescence intensity (MFI = 30.1) compared to the p24 negative
cell population (Fig.
281; MFI = 8.2). Interestingly, the identical 4F7 TCRm staining profile was
observed using HIV-1
strains Ba-L and NL-4.3 (data not shown). This same 4F7 TCRm staining pattern
was not
observed on HIV-1 infected HLA-A*0201 negative CD4+ T cells, supporting MHC-
restriction forthe
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TCRm (data not shown). To determine whether the increase in eIF4G(720)-A2
complexes was
specific for HIV-1 infected cells, the effect of influenza virus infection on
eIF4G(720) -A2 expression
was examined. After staining cells with the 4F7 TCRm mAb, no increase was
detected suggesting
that the elevated levels observed for eIF4G(720)peptide expression may be
specific for HIV-1
infected cells (data not shown). These findings validate the presence of
elevated eIF4G(720) peptide
in HIV-1 infected cells, and demonstrate that a TCRm to eIF4G(720)-HLA-A*0201
can discriminate
HIV-1 infected cells from non-infected cells.
[0201] Next, the 4F7 TCRm mAb was used to directly examine the kinetics of
eIF4G(720)
peptide-HLA-A*0201 complex presentation on HIV-1 infected CD4+ T cellsfor9
days post-infection
(PI). As shown in Fig. 29A, the p24 positive CD4+ T cells had a two-fold
increase in 4F7 TCRm
staining signal compared to the p24 negative cells by the third day PI. By
days 7 and 8 PI, the 4F7
TCRm staining differential had increased by almost 4-fold between the p24
negative and positive
groups (Fig. 29A). In contrast, there were no significant changes in cell
staining using the isotype
control Ab (Fig. 29B). This finding directly validates the expression of the
eIF4G(720)-HLA-A*0201
epitope and reveals the dynamic nature of host-peptide epitope presentation on
HIV infected cells.
[0202] To firmly establish that the 4F7 TCRm specifically recognized the
eIF4G(720) peptide in
the context of HLA-A*0201, CD4+ T cells were infected with HIV-1 strain Ba-L
and evaluated 4F7
TCRm staining on days 3 through 5 Pi in a tetramer competition assay. HLA-
A*0201 tetramer
complexes loaded with eI F4G(720) peptide or irrelevant p5364) and VLQ(44)
peptides were included
in the staining reactions. The infected CD4+ T cells were stained with 0.5 pg
of 4F7 TCRm in the
presence of either (1) eIF4G(720)-HLA-A*0201 tetramer complexthat would
compete with specific
binding to eIF4G(720)-HLA-A*0201; (2) p53(264)-HLA-A*0201 tetramer complexes;
or (3)
VLQ(44)-HLA-A*0201 tetramer complexes, wherein (2) and (3) would not compete
with specific
binding to eIF4G(720)-HLA-A*0201. The results shown in Fig. 30 reveal that 4F7
TCRm mAb
binding to the p24 positive cell population was significantly reduced in the
presence of 0.5 pg of
eIF4G(720)-HLA-A*0201-tetramer at days 4 and 5 (Figs. 30A & B). In contrast,
when tetramers
p53(264)-HLA-A*0201 and VLQ(44)-HLA-A*0201 were added (0.5 pg), there was
little to no inhibition
of 4F7 TCRm mAb staining. The 1 B8 TCRm mAb did not stain the infected or non-
infected CD4+
T cells (data not shown), further supporting the claim that the 4F7 TCRm
specifically recognizes
the eIF4G(720)-HLA-A*0201 complex. To conclude, these findings indicate that
HIV-1 infection of
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primary cells leads to the enhancement of host peptide eIF4G(720) through
which immune receptors
(TCRm here) can distinguish the virally infected from non-infected cells.
EXAMPLE 3
[0203] Her-2(93ss) represents a common epitope expressed by various tumor
types including
breast carcinomas (Brossart et al., 1999). Approximately 20-30% of primary
breast cancers
express Her-2. The Her-2/neu receptor protein is a member of the tyrosine
kinase family of growth
factor receptors (Coussens et al., 1985) that is frequently amplified and
overexpressed in breast
cancer (Slamon et al., 2001). The Her-2/neu protein is generally displayed on
the surface of cells
and, during malignancy, is detected at high levels on tumor cells. Although
its precise anti-tumor
mechanism(s) remain unknown, Herceptin, an anti-Her-2/neu antibody, is used in
breast cancer
treatment to target the receptor on the surface of tumor cells. In addition to
using antibodies to
attack tumors expressing Her-2/neu receptor on their surface, Her-2/neu
oncoprotein contains
several HLA-A2-restricted epitopes that are recognized by CTL on autologous
tumors. The most
extensively studied Her-2 epitope (and the one utilized herein in Example 3)
spans amino acids
369-377 (Her-2(9369)) (KIFGSLAFL; SEQ ID NO:3) (Fisk et al., 1995) and is
recognized by tumor
associated lymphocytes as well as reactive T cell clones as an immunodominant
HLA-A2-restricted
epitope. The peptide has been shown to bind with high affinity to HLA-A2
alleles (Fisk et al., 1995;
and Seliger et al., 2000). The Her-2(9369) epitope binds to HLA-A2 with
intermediate affinity (IC50
- 50 nM) (Rongcun et al., 1999), and because it is grossly overexpressed on
malignant cells, it has
been used as a vaccine candidate in several clinical trials. For instance,
Knutson et al. (2002)
demonstrated that patients immunized with Her-2(9369) could develop interferon-
gamma (IFN-y)
responses to the peptide and exhibited increased Her-2(93ss)-specific
precursor frequencies.
[0204] Her2/neu-peptide-HLA-A2 monomers and tetramers were generated as
described above
in Example 1. However, Her2/neu-peptide-HLA-A2 tetramers were generated at a
lower efficiency
than for either 264p-HLA-A2 tetramers (Example 1) or eIF4Gp-HLA-A2 tetramers
(Example 2), as
shown in Table I. The relatively low tetramer yields with Her2/neu peptide do
not correlate well with
the high affinity of this peptide to HLA-A2. The IC50 of Her2/neu peptide is
lower than p53-264, yet
tetramer yield with Her2/neu peptide is two to three fold less than tetramer
yield with p53-264.
[0205] To solve this yield problem, it was determined that the peptide needed
to be solubilized
in a solvent, such as but not limited to, DMSO or DMF, prior to refolding with
the heavy and light
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chains. Once the Her2/neu peptide was solubilized in DMSO, sufficient amounts
of Her2/neu
peptide monomer and tetramer were produced.
[0206] The Her2/neu-peptide-HLA-A2 tetramers were utilized for immunization of
Balb/c mice
and generation of monoclonal antibodies as described in detail in Examples 1
and 2. Briefly, the
1 B8 TCRm mAb was generated by immunizing mice with soluble recombinant HLA-
A*0201 loaded
with the Her2/neu369 peptide epitope. The soluble heavy chains of HLA-A*0201
(hereafter
designated A2+) and the R2-microglobulin (p2m) were produced in the form of
inclusion bodies in
E. coli, purified and then refolded in the presence of the Her2 KIFGSLAFL
peptide. The
conformation of the refolded protein was assessed using anti-HLA Class I
antibody (W6/32) and
the anti-HLA-A2 specific mAb BB7.2 (data not shown). The refolded protein
served as the
immunogen and as the positive control in screening assays of hybridoma
supernatants. The
eIF4G72D, TMT40 and VLQ44 peptide loaded A2+ molecules served as negative
controls. Over 2000
hybridomas were screened and the 1 B8 TCRm hybridoma was selected because it
specifically
recognized the recombinant HLA-A2 protein loaded with the p369 peptide but did
not bind
recombinant HLA-A2 proteins loaded with irrelevant peptides (Fig. 31A). As a
control forspecificity,
the 3F9 TCRm mAb was used, which is specific for the TMT40 peptide-HLA-A2
complex. As shown
in Fig. 31 B, the 3F9 TCRm mAb binds specifically to the TMT(40)-A2 complex
without binding to the
Her2(3r,9)-A2 complex. To demonstrate that recombinant HLA-A2 proteins were
properlyfolded after
being loaded with the peptide, they were stained with the BB7.2 anti-A2.1 mAb
(Fig. 31 C). These
data demonstrate that the TCRm antibodies recognize a specific MHC-peptide
complex and they
do not have detectable 'cross-reactivity with either A2+ molecules or HLA
complexes loaded with
irrelevant peptides.
[0207] Although 1138 TCRm recognizes the recombinant Her2(,69)-A2 complex
target in coated
wells, it was unclear whether this mAb would recognize the specific peptide
when loaded into
HLA-A*0201 complexes expressed on a cell surface. In order to ensure that 1 B8
recognized the
Her2369 peptide in the context of the native HLA-A2, its binding to T2 cells
pulsed with 10 pM of
p369 peptide, irrelevant peptides (TMT and eIF4G) or no peptide was analyzed.
As shown in Fig.
32A, 1 B8 TCRm only stains T2 cells pulsed with the Her2/neu peptide but does
not bind T2 cells
not pulsed or pulsed with irrelevant peptides. These results confirm the fine
and unique specificity
of the I B8 TCRm for the Her2/neu369 peptide present in the binding pocket of
the HLA-A2 complex.
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[0208] The specificity and sensitivity of the 1 B8 TCRm mAb for the Her2(369)-
A2 complex was
further evaluated using three different methods. In the first series of
experiments, T2 cells were
pulsed with a cocktail consisting of 20 different irrelevant peptides in the
presence or absence of
the p369 peptide. The results indicate that 1B8 TCRm mAb was able to bind to
cells only when
the specific Her2/neu peptide was included in the peptide cocktail (Fig. 32B).
I n these experiments,
Her2/neu peptide represented less than 5% of the total peptide sample in the
pulsing cocktail. In
the second series of experiments, HLA-A2+/neu- human PBMCs were stained with
the 1 B8 TCRm
mAb. As shown in Fig., 32C, the 1138 TCRm failed to stain HLA-A2 positive
cells that lacked
Her2/neu expression (TA-1 mAb). These findings further support the fine
binding specificity of 1 B8
for the Her2(369)-A2 complex. In the third series of experiments, T2 cells
were pulsed with
decreasing concentrations of the p369 peptide (2500-0.08 nM). As shown in Fig.
32D, the 1138
TCRm mAb was able to recognize T2 cells pulsed with the peptide at
concentrations at least as
low as 0.08 nM. Taken together, these results indicate that 1 B8 TCRm mAb is
capable of detecting
low concentrations of MHC-peptide complexes.
[0209] It was observed that the 1 B8 TCRm mAb recognizes recombinant HLA-A2
protein or T2
cells pulsed with the p369 peptide. Next, it was evaluated whether this
antibody would recognize
the Her2(36s)-A2 complex presented by tumor cells using five HLA-A2+/neu+ cell
lines,
MDA-MB-231, Saos-2, MCF-7, SW620 and COL0205. It has previously been
demonstrated herein
that the p369 epitope is processed and presented in MDA-MB-231 and MCF-7
breast carcinoma
cells. HLA-A2-/neu+ cell lines, BT-20 and SKOV3 were used as negative
controls. In the first
series of experiments, cells were stained with 0.5 pg of IgG1 isotype control
mAb, 3F9 or 1B8
TCRm mAbs, and all tumor cells except the BT-20 and SKOV3 cells (Fig. 33A)
were stained with
the 1 B8 TCRm mAb (thick gray line). In contrast, only human chorionic
gonadotropin expressing
cells, COL0205, were weakly positive when stained with 3F9 TCRm mAb (solid
black line). In the
second series of experiments, the cell lines were pre-treated overnightwith
interferon-y and TNF-a
and then stained with the same panel of antibodies used in Fig. 33A. As shown
in Fig. 33B, the
same five cell lines were stained with 1 B8 TCR mAb. In addition, with the
exception of Saos-2, four
cell lines showed enhanced staining with 1138, suggesting an increase in
levels of Her2(36s)-A2
complex. No staining was detected on SKOV3 cells, and low background signal
was detected on
BT-20 cells (Fig. 33B). These results indicate that TCRm mAb can be used in
the validation of
epitopes which are endogenously processed and presented on the surface of
tumor cells.
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[0210] To further demonstrate that the 1 B8 TCRm mAb binds specifically to
endogenously
processed Her2(369)-A2 complex on human tumor cells, the antibody was
evaluated in two different
competition assays. In the first system, HLA-A2 tetramer complexes were loaded
with either (1)
Her-2/neu peptide that would compete with specific binding to Her2 (369)-A2;
or (2) irrelevant TMT
peptide that would not compete for binding sites, and then added to the
staining reactions.
MDA-MB-231 tumor cells were stained with 0.5 pg of 1 B8 in the presence of
Her2(369)-A2 tetramer
or TMT(40)-A2 tetramer complex. The results, shown in Fig. 34A, reveal that
1138 TCRm mAb
binding was reduced by more than 50% in the presence of 0.1 pg of the
Her2(369)-A2-tetramer and
was completely blocked by 1.0 pg of the Her2(369)-A2-tetramer. In contrast,
when TMT(40)-A2
tetramer was added (1.0 pg), there was no inhibition of 1 B8 TCRm mAb
staining.
[0211] In the second system, the target specificity of the CTL line generated
in the HLA-A2-K'
transgenic mice for the Her2 (369)-A2 epitope was first confirmed by showing
lysis of p369 pulsed
T2 cells but not with unpulsed cells (Fig. 34B). CTL activity against
untreated MDA-MB-231 cells
or cells pretreated with interferon-y (IFN-y, 20 ng/ml) plus tumor necrosis
factor-a (TNF-a, 3 ng/ml)
was then blocked by adding 1B8 TCRm (anti-Her2(369)-A2) or BB7.2 (anti-HLA
2.1) mAb (Fig.
34C). In contrast, isotype control antibodies (IgG1 and IgG2b), did not
inhibit the CTL activity (Fig.
34C). Collectively, these data illustrate that the 1 B8 TCRm mAb can
specifically recognize the
Her2(369)-A2 immunodominant epitope on the surface of tumor cells.
[0212] Fig. 35 illustrates that 1 B8 mAb does not bind to soluble Her2/neu
peptide. MDA-MB-231
cells were stained with 1 B8 in the presence or absence of exogenously added
Her-2/neu peptide.
Fig. 35 demonstrates that 1 B8 TCR mimic has dual specificity and does not
bind to Her-2/neu
peptide alone.
[0213] Expression of peptide-HLA class I on the cell surface depends on
multiple parameters
including the quantity and quality of the peptide supplied. The supply of
peptide is also dependent
on the availability of protein and the rate at which the protein is processed.
It is not clear, however,
whether tumor antigen expression and MHC expression are directly linked with
the level of
expression of MHC-peptide complexes. The expression of Her-2/neu molecules,
HLA-A2.1
molecules and Her2(369)-A2 complexes on the surface of different tumor cell
lines was assessed.
Tumor cell lines were stained for Her-2/neu and the expression of this antigen
was variable among
the cell lines (Fig. 36). For example, the COL0205 cell line revealed
noticeably higher levels of
Her2/neu protein than MDA-MB-231, Saos-2, MCF-7 and SW620 tumor cell lines.
The BT-20
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(HLA-A2 negative) cell line had an intermediate level of Her2/neu protein
expression. Detection of
Her2/neu protein expression by two different methods revealed that the level
of cell surface
expression directly correlates (p<0.05) with the cellular level of Her2
protein expression (RZ=0.82)
as evaluated by ELISA (Figs. 36A & B).
[0214] Next, different tumor cell lines were evaluated for cell surface
expression of HLA-A2
molecules. As expected, the cell lines displayed different levels of HLA-A2
molecules (Fig. 37A),
showing only modest changes in levels at different stages of the growth cycle,
thus suggesting that
HLA-A2 and TAA expression is stable (data not shown). To evaluate whether
there was a
correlation between HLA-A2 and Her-2/neu expression with the levels of
Her2(369)-A2 complexes
present on the cell surface, tumor cell lines were stained with the 1 B8 TCRm
mAb. It was observed
that Her2(369)-A2 expression levels (MFIR) of COL0205 were similar to those of
Saos-2, SW620
and MCF-7 cell lines and roughly 3-fold lower than MDA-MB-231 cells, even
though COL0205
demonstrated significantly higher expression of the Her2/neu antigen (Fig.
36). Taken together,
these results indicate the absence of a direct correlation (p>0.05) between
the level of Her-2/neu
or HLA-A2.1 molecules and the number of Her2(369)-A2 complexes on the surface
of these tumor
cell lines.
[0215] To determine whether there is a relationship between CTL recognition
and the level of
expression of MHC-peptide complexes, we took advantage of the Her-2/neu/A2-
p369 specific CTL
line. The p369-CTLs were evaluated for cytotoxic activity against untreated
human tumor cell lines
(Fig. 37C). The level of Her2(389)-A2 complex was found to be a better
indicator of cell lysis by the
CTL line than was cell surface expression of either Her2/neu antigen or HLA-A2
molecule
expression. In fact, poor or no lysis of the cell lines expressing low levels
of Her2(369)-A2 complex
was observed, as identified using the 1 B8 TCRm mAb (e.g., SW620 and COL0205)
(Fig. 37C).
Also noted was the minimal lysis of BT-20 cells observed. The fact that these
cells are HLA-A2-
is something at this time that can not be explained.
[0216] To further examine the relationship between levels of MHC-peptide
complexes present
on the cell surface and the levels of antigen and MHC molecules expressed, the
cell lines were
pretreated with interferon-y (IFN-y, 20 ng/ml) plus tumor necrosis factor-a
(TNF-a, 3 ng/ml).
Treating tumor cells in this way is known to increase the expression of
adhesion molecules (e.g.,
ICAM) and MHC class I heavy chain. These cytokines also enhance protein
processing and
peptide presentation by HLA class I through the activation of the
immunoproteasome, which has
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been hypothesized to cause an increase in the expression of specific MHC-
peptide complexes,
especially in cells with greater availability of antigen. This hypothesis was
tested by treating the
tumor cell lines for 24 hrs with cytokines and then staining with the BB7.2
mAb (Fig. 38A) and the
1 B8 TCR mimic (Fig. 38B). It was observed that, after cytokine treatment, all
tumor cell lines,
except Saos-2, displayed greater 1 B8 TCRm staining intensity (see also Fig.
33B), indicating that
more of the specific complex was expressed on the cell surface. When comparing
cell surface
levels of the Her2(369)-A2 complexes between the different treated cell lines,
it was found that the
1 B8 staining intensity for COL0205 (MFIR = 9.5) was markedly lower than that
of MDA-MB-231
(MFIR=38) and MCF-7 (MFIR=27). This observation suggests that stimulation of
cellular
machinery for antigen processing and presentation did not favor higher levels
of specific
HLA-peptide complex in cells that, as demonstrated previously (Fig. 36A),
expressed significantly
more of the tumor antigen. Validation of cytokine-induced effects on the MHC
class I system was
demonstrated by the increase observed in HLA-A2 expression (Fig. 38A).
Interestingly, in this
group of cell lines, surface levels of HLA-A2 were equivalent in all but MCF-7
cells, which had
noticeably lower HLA-A2 expression. It was thus concluded from these data that
TAA expression
does not correlate with levels of specific MHC-peptide complexes.
[0217] Following treatment with cytokines, which increases the levels of
Her2(369)-A2 complexes,
it was found that lysis was augmented in all HLA-A2 positive cell lines (Fig.
38C). The
enhancement of cytotoxic activity for the cytokine treated tumor cells
significantly (p=0.05)
correlated with an increase in specific HLA-peptide levels on the surface of
the cells (R2=0.75)
suggesting that the susceptibility of tumor cells to lysis is largely linked
to the density of specific
Her2(369)-A2 complexes present (Fig. 38D). Taken together, these data indicate
that protein
antigen expression, which can be high or low on different tumor cells, does
not predict the level of
CTL epitope presentation nor tumor susceptibility to CTL killing.
[0218] Thus, a new angle of attack on a proven anti-cancer target has been
reported herein.
The reported levels of Her2/neu peptide on the surface of MDA cells, which are
reported as being
low or non-existent, contrasts sharply to the staining reaction seen with the
antibody of the present
invention, which recognizes peptides from the protein. This may indicate that
a much higher
percentage of cancer cells express the receptor, but that the receptor does
not traffic effectively
to the surface of the cell; however, it is still a good target based on the
expression level of the
Her2/neu peptide associated with HLA-A2.
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EXAMPLE 4
[0219] Human chorionic gonadotropin (hCG) is a member of the glycoprotein
hormone family that
shares homology with luteinizing hormone, follicle stimulating hormone and
thyroid stimulating
hormone. Each of these is a heterodimer with a variable R chain and a common a
chain. hCG is
most commonly associated with pregnancy assessment but is also a marker for
tumors resulting
from tissues associated with placenta or germ cells. In a comprehensive review
of hCG in cancer,
Stenman et al. (2004) reported that 0 chain (hCGP) is found in the serum of 45-
60% of patients
with biliary and pancreatic cancers, and 10-30% of other cancers.
Immunohistochemical analysis
and urinalysis have been used to demonstrate the presence of hCGP in lung,
gynecological and
head and neck cancers. The aggressiveness and resistance to therapy of bladder
cell carcinoma
expressing hCGP has been associated with an autocrine anti-apoptotic effect
elicited by the free
R chain (Butler et al., 2000). A series of antibodies which bind hCG were
developed for use as
diagnostic reagents, and hCG(3-specific antibodies which have application in
pregnancy testing as
well as monitoring for hCG positive tumors continue to be developed (Charrel-
Dennis et al., 2004).
An anti-hCG[i vaccine (for use in treatment of human cancer) that targets hCGP
to dendritic cells
has been shown to elicit both cytotoxic and helper T cell responses to peptide
pulsed target cells
and tumor cell lines (He et al., 2004). Recently, several MHC class I epitopes
from hCGP have
been identified which bind with high affinity to HLA-A*0201 molecules (Dangles
et al., 2002).
[0220] A first step in evaluating the efficacy of therapeutic antibodies is in
vitro assessment of
their specificity and ability to induce tumor cell lysis via the activation of
complement and ADCC.
The therapeutic successes of the monoclonal antibodies trastuzumab and
rituxamab are thought
to be due, at least in part, to their ability to promote ADCC and CDC (Clynes
et al., 2000; Spiridon
et al., 2004; Harjunpaa et al., 2000; and Golay et al., 2000). In the present
invention, the antigen
binding specificity, in vitro lytic abilities and in vivo tumor growth
inhibition of a TCRm mAb, 3.2G1,
which is specific for the GVL peptide (residues 47-55 from hCGR) presented in
the context of
HLA-A2, are demonstrated.
[0221] Generation of monoclonal antibodies and experimental methods were
performed as
described in detail in Examples 1 and 2, except as described herein below.
[0222] Cell Culture: Cell culture medium included IMDM and RPMI from Cambrex
(Walkerville,
MD), L-15 from Mediatech (Herndon, VA), and Hybridoma SFM and AIM-V from
Invitrogen
(Carlsbad, CA). Media supplements included heat-inactivated fetal bovine serum
(FBS) and
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penicillin/streptomycin from Sigma (St. Louis, MO) and L-glutamine from
HyClone (Logan, UT).
Recombinant human IL-2 was obtained from Peprotech (Rockyhill, NJ). All tumor
lines were
maintained in culture medium containing glutamine, pen/strep and 10% FBS. Cell
cultures were
maintained at 37 C in 5% CO2 atmosphere with the exception of MDA and SW620
which were
cultured without COz. MDA and SW620 cells were cultured in L-1 5, SKOV3.A2 and
T2 in IMDM,
and BT20 in RPMI. When necessary, attached cells were released from flasks
using TrypLE
Express (Invitr,ogen, Carlsbad, CA ).
[0223] Human peripheral blood mononuclear cells (PBMC) from anonymous donors
were
obtained from separation cones of discarded apheresis units from the Coffee
Memorial Blood Bank,
Amarillo, TX, after platelet harvest. Cells were separated on a ficoll
gradient, 'then washed, counted
and resuspended in AIM-V medium containing 200 units of IL-2 per ml at a
concentration of 2-2.5
x 106 cells/mi. PBMC were maintained at this concentration with media changes
and addition of
IL-2 every 2 to 3 days for a maximum of seven days. These conditions have been
shown to
maintain and activate resident NK cells within the PBMC population (Liu et
al., 2002).
[0224] Murine hybridoma cells were initially grown in RPMI supplemented with
10% FBS,
glutamine and pen/strep (RPMI/10) as described below. After selection for
binding specificity,
clones were grown in RPMI/10 to provide supernatant containing the antibodies
of interest or in
SFM to provide supernatant for isolation of purified antibodies from protein G
columns (GE
Healthcare BioSciences, Piscataway, NJ ).
[0225] Peptides and HLA-A2 complexes: The following peptides were synthesized
at the
Molecular Biology Resource Facility, University of Oklahoma (Oklahoma City,
OK.): KIFGSLAFL
(residues 369-377, designated Her-2; SEQ ID NO:3), eukaryotic initiation
translation factor 4
gamma VLMTEDIKL (residues 720-728, designated eIF4G; SEQ ID NO:2), human
chorionic
gonadotropin-(3 TMTRVLQGV (residues 40-48, designated TMT; SEQ ID NO:4),
VLQGVLPAL
(residues 44-53, designated VLQ; SEQ ID NO:5), and GVLPALPQV (residues 47-55,
designated
GVL; SEQ ID NO:6). HLA-A2 extracelluar domain and R2 microglobulin were
produced as inclusion
bodies in E. coli and refolded essentially as described previously. After
refolding, the
peptide-HLA-A2 mixture was concentrated, and properly folded complex was
isolated from
contaminants on a Superdex 75 sizing column (GE Healthcare Bio-Sciences AB).
This complex,
designated the monomer, was biotinylated using the BirA biotin ligase enzyme
(Avidity, Denver,
CO) and purified on the S75 column. Purified, biotinylated monomer was mixed
with streptavidin
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at an empirically determined ratio to yield higher order complexes. Complexes
were then
separated on a Superdex 200 column, and the peak corresponding to a
streptavidin plus four
monomers (the tetramer) was isolated. Tetramer concentration was determined by
BCA protein
assay (Pierce, Rockford, IL).
[0226] ELISA assays were performed using Maxisorb 96-well plates (Nunc,
Rochester, N.Y.).
Assays to evaluate binding specificity of the TCRm antibodies were done on
plates coated with
either 500 ng/well HLA monomer or 100 ng/well HLA tetramer. Bound antibodies
were detected
with peroxidase-labeled goat anti-mouse IgG (Jackson lmmunoResearch) followed
by ABTS
(Pierce). Reactions were quenched with 1% SDS. Absorbance was measured at 405
nm on a
Victor I I plate reader (PerkinElmer, Wellesley, MA). The SBA Clonotyping
System/HRP and mouse
immunoglobulin panel from Southern Biotech were used to estimate the
concentration of 3.2G1
(isotype IgGza) in the supernatant of FBS-containing medium. The assay was run
according to
manufacturer's directions, and 3.2G1 signal was compared with that of an IgG2a
standard supplied
by the manufacturer. Development, quenching and analysis of the plate were
performed as
described above for the other TCRms.
[0227] Cell staining: T2 is a mutant cell line that lacks transporter-
associated proteins (TAP) 1
and 2 which allows for efficient loading of exogenous peptides (Wei et al.,
1992). The T2 cells
were pulsed with the peptides at 20 pg/mI for 4 hours in growth medium, with
the exception of the
peptide-titration experiments, in which the peptide concentration was varied
as indicated. Cells
were washed and resuspended in staining buffer (SB; PBS+0.5% BSA+2 mM EDTA)
and then
stained with 1 pg of 3.2G1, BB7.2 or isotype control antibody for 15 to 30
minutes in 100 pl SB.
Cells were then washed with 3 ml SB, and the pellet was resuspended in 100 lal
of SB containing
2pl of either of two goat anti-mouse secondary antibodies (FITC or PE
labeled). After incubating
for 15-30 minutes at room temperature, the wash was repeated, and cells were
resuspended in 0.5
ml SB, analyzed on a FACScan instrument and evaluated using Cell Quest
Software (BD
Biosciences, Franklin Lakes, NJ).
[0228] In Fig. 41, tumor cell lines were stained and evaluated in the same
manner as the T2 cells,
after being released from plates and washed in SB. Tetramer competition stains
were carried out
in the same order described above except that tetramer at the appropriate
concentration was mixed
with the antibody and allowed to stand for 40 minutes before the mix was added
to the cells.
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[0229] Cytotoxicity Analysis: Specific cell lysis in the complement dependent
cytotoxicity (CDC),
natural killer cell (NK) and antibody dependent cellular cytotoxicity (ADCC)
assays was evaluated
using the CytoTox 96 non-radioactive cytotoxicity Lactate DehydrogenaseAssay
(LDH assay) from
Promega (Madison, WI), following the instructions provided by the
manufacturer. This assay
measures the release of cellular LDH into the culture supernatant after cell
lysis. All cells were
grown or pulsed with peptide in their appropriate growth medium, but final
incubations of cells in
the presence of complement (CDC) or human PBMCs (NK and ADCC) was carried out
in AIM-V
medium for 4 hours at 37 C. CDC analysis of T2 cells took place under three
different conditions:
(1) the antibody concentration was varied and competing or non-competing
tetramer added, (2)
peptide mixes were used to pulse cells, or (3) GVL peptide was titrated for
use in cell pulsing. CDC
analysis of MDA-MB-231 cells using antibody dilutions and tetramer competition
was carried out
on adherent cells. Exact conditions are described in the figure legends and/or
results section.
LoTox complement was obtained from Cedarlane (Burlington, N.C.) All cells used
as targets for
cytotoxicity assays were pulsed for 4 hrs with peptide. Specific lysis in the
CDC assays was
calculated as follows: ([experimental release-spontaneous release]/[maximum
release-spontaneous
release]) x 100 = specific release. ADCC reactions using human PBMC effector
cells (E) were
carried out on MDA-MB-231 target cells (T) using 3.2G1 or W6/32 antibodies at
a final
concentration of 10 pg/mI. Effector:target ratios (E:T) were varied as
indicated in the figures. NK
analysis was performed by mixing human effector cells with K562 cells and
incubating as above.
Specific lysis in ADCC analysis was calculated as follows: ([E+T+Ab release -
E+T-Ab
release]/[maximum release - spontaneous release]) x 100 = specific release.
Specific Iysis in NK
analysis was calculated: ([E+T release -spontaneous release]/[maximum release-
spontaneous
release]) x 100 = specific release. Spontaneous and maximum release was
measured before and
after, respectively, lysis of target cells with 0.9% Triton X 100.
[0230] In vivo studies: Six week-old female athymic nude mice (CByJ.Cg-
Foxn1{nu}/j) were
obtained from Jackson Laboratories and housed under sterile conditions in
barrier cages. Each
of nineteen mice was implanted with 5x106 freshly harvested (97% viable) MDA-
MB-231 cells in
0.2 ml containing 1:1 mixture of medium and Matrigel (Sigma, St. Louis, MO)
(Ferguson et al.,
2005; and Hermann et al., 2005). Mice received an i.p. injection of either 100
pg of an isotype
IgGZa control antibody (n=10) or 100 pg of 3.2G1 (n=9) at the same time that
the tumor cells were
implanted s.c. between the shoulders. Either 3.2G1 or isotype control antibody
(50 pg) was
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administered (i.p.) weekly for the following 3 weeks. Animals were held for at
least one week after
the appearance of the last tumor in the isotype control group (a total of 70
days) before totaling
frequency of occurrence. All tumors reached at least 6 mm in diameter before
being scored as
positive. Tumor volumes were measured once a week using a slide caliper. Tumor
volumes were
calculated by assuming a spherical shape and using the formula: volume =
4r3/3, where r=%Z of
the mean tumor diameter measured in two dimensions.
[0231] Statistics: Significance values for GVL peptide concentration and the
amount of CDC lysis
were calculated using one-way analysis of variance (ANOVA) and the
significance value for the
tumor implantation studies was calculated using the Fisher Exact Test in the
program Sigma Stat
(SSPS Inc, Chicago, IL).
RESULTS
[0232] Characterization of the TCRm antibody 3.2G1: To establish that the
3.2G1 TCRm mAb
isolated in the initial screening was HLA-A2 restricted and peptide-specific,
a series of assays to
characterize its binding specificity were performed. The first assessment
utilized refolded
peptide/HLA-A2 molecules as targets for testing the 3.2G1 TCRm in an ELISA.
Fig. 39A shows
the results of ELISA analysis of supernatant from hybridoma 3.2G1 versus HLA-
A2/(32m complex
refolded with its cognate peptide GVL or with one of three other irrelevant
peptides. Significant
reactivity was seen only in wells containing the GVL tetramer, indicating the
TCR-like specificity of
the antibody. Coating of each well was confirmed by ELISA using the HLA-A2
conformation
specific antibody BB7.2 (data not shown).
[0233] To confirm the specificity of 3.2G1 TCRm for the GVL/A2 complex on the
surface of T2
cells, the cells were pulsed with the specific peptide GVL, with irrelevant
peptides VLQ or TMT,
or with no peptide, and then stained with 3.2G1 (Fig. 39B). The concentration
of 3.2G1 in
supernatant was determined by isotype-specific ELISA, and the antibody was
used at 1pg per
stain. Binding to the surface of the cells was detected with goat anti-mouse
FITC labeled
secondary antibody and the cells were analyzed by flow cytometry. The GVL
pulsed cells shifted
significantly (mean fluorescence intensity [MFI] of 141) compared to cells
pulsed with the irrelevant
peptides containing closely related sequences VLQ and TMT or no peptide (MFI
of 7.3, 7.5 and 9.0
respectively).
[0234] A correlation between antibody concentration and level of staining of
peptide-pulsed cells
was established by titration of the antibody (Fig. 39C). 3.2G1 antibody was
diluted over a range
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of 0.01 to 3 pg and then used to stain T2 cells that had been either pulsed
with 20 pg/mi of GVL
or not pulsed with peptide. Staining was carried out and the net MFI was
determined by subtracting
the no peptide MFI value from the MFI of GVL pulsed cells. The staining
reactions appeared to
saturate with 3.2G1 at approximately 1 pg/100 pl and retained the ability to
differentiate
GVL-pulsed cells from those that were not pulsed down to 0.01 pg. The MFI at
0.01 pg of antibody
was 14.3 as compared to 388 for 1pg of antibody. There is a clear relationship
between antibody
concentration and staining intensity of the pulsed cells.
[0235] To assess the effect of peptide-HLA density on the cell surface on
3.2G1 TCRm staining,
T2 cells were next pulsed with varying levels of GVL peptide. The peptide was
serially diluted and
added to cells at concentrations ranging from 50 Iag/mI to 0.1 pg/mi. The net
MFI was determined
by subtracting the VLQ peptide pulsed T2 cell MFI value from the MFI of GVL
pulsed cells. After
pulsing and addition of antibody, cells were stained and analyzed. MFI of
cells stained with the
3.2G1 antibody titrated over a range of 10-150 MFI; there was much less
variation with BB7.2
staining, which ranged from 250-350 MFI (Fig. 39D). It was concluded from
these findings that
3.2G 1 staining intensity is dependent on the density of the specific epitope
on the surface of cells.
[0236] Competition studies using tetramer constructs containing either the GVL
or VLQ peptide
were conducted to evaluate the fine specificity of binding of antibody 3.2G1
(Fig. 39E).
Preincubation of 3.2G1 with the GVL tetramer inhibited the final staining of
GVL pulsed T2 cells
in a concentration-dependent manner with 50% inhibition occurring at roughly
0.07 mg tetramer/pg
of antibody. There was essentially no inhibition of staining by the VLQ
tetramer at any of the
concentrations tested which were up to 40-fold higher than the concentration
of GVL tetramer
required for 50% inhibition, suggesting that the 3.2G1 TCRm mAb specifically
binds to its cognate
epitope GVL/A2 on the surface of T2 cells.
[0237] Complement-Dependent Cytolysis using 3.2G1 antibody: Murine IgG2a
antibodies have
been found to efficiently direct complement dependent cytolysis (CDC) while
the IgG 1 isotype does
not (Dangl et al., 1988). This fact and the corresponding ability of the IgG2a
isotope to bind human
Fc receptors (see below) led to selection of the 3.2G1 TCRm mAb. T2 cells
pulsed with various
peptides were used as targets forthe initial 3.2G 1-directed CDC analysis
because they could easily
be loaded to a high density with any of a number of peptides. The effect of
the relative density of
the appropriate peptide/A2 complex on the surface of T2 cells was probed by
pulsing with GVL,
TMT, a mixture of the two or no peptide while holding the antibody
concentration constant at 2.5
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pg/mi. Fig. 40A illustrates the CDC results of cells pulsed with various
ratios of peptide (GVC:TMT)
for both the HLA-A2 specific BB7.2 antibody and 3.2G1. BB7.2 is a murine IgG2b
antibody, and
this isotype also efficiently fixes complement. BB7.2-driven lysis
demonstrates that there is little
difference between cells pulsed with peptides at the various concentrations.
The addition of 3.2G1
antibody to the cells resulted in CDC which titrated with the ratio of
GVL:TMT. Lysis was not seen
for non-pulsed cells (the value was below the spontaneous release in the
absence of antibody) or
those pulsed with TMT (CDC = 2%). This experiment implies that the degree of
lysis reflects the
antigen density on the cell.
[0238] In a second experiment, an examination of the relationship between
target density and cell
lysis was carried out using T2 cells that were pulsed with varying levels of
GVL peptide alone (Fig.
40B). The peptide was serially diluted and added to cells at concentrations
ranging from 50 pg/mI
to 0.1 pg/mI. VLQ peptide and non-pulsed cells were used as a zero-point
control. After pulsing
and addition of antibody at 10 pg/mI, cells were subjected to CDC analysis.
The HLA-A2 specific
lysis in the presence of BB7.2 varied from 53 to 70% while that driven by
3.2G1 varied from 6 to
73% (Fig. 40B), titrating with the dose of peptide used to pulse cells. While
there was no indication
of any decrease in cell lysis for BB7.2 (p = 0.29), the 3.2G1 TCRm revealed a
clear relationship
between target density and cell Iysis, with half-maximal lysis occurring at a
peptide concentration
around 6 pg/mi as determined by one-way ANOVA (p<0.001).
[0239] In the final CDC experiment involving T2 cells, the specificity of
lysis by the antibody using
HLA-A2-peptide tetramers to compete for 3.2G 1 binding was examined. 3.2G1
TCRm was serially
diluted and preincubated with tetramer such that the final concentrations of
TCRm varied from 9
to 0.1 pg/mI and the tetramer concentration was 2 Ng/mI after addition to the
CDC reaction.
Tetramers refolded with the GVL peptide (competitor) substantially inhibited
CDC while those
refolded in the presence of VLQ peptide (non-competitor) resulted in an
antibody lysis profile
almost identical to that seen with no tetramer (Fig. 40C). Taken together,
these findings support
the fine recognition specificity of the 3.2G1 TCRm mAb for targeting the GVL-
A2 epitope on T2
cells for cell lysis by CDC.
[0240] 3.2G1 detects endogenous GVL peptide-HLA-A2 presented on human tumor
cell lines:
The ability of the 3.2G1'antibody to detect endogenously processed peptide in
the context of the
HLA-A2 molecule was evaluated by immunofluorescent staining of a series of
tumor cell lines (Fig.
41). BB7.2 mAb indicated the level of HLA-A2 expression on cells. SKOV3.A2 and
SW620 are
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ovarian and colon cancer cell lines, respectively, while MDA-MB-231 and BT20
are breast cancer
cell lines. Additional ahalysis of the SKOV3.A2, SW620 and MDA-MB-231 cell
lines by ELISA
indicated that hCGR was present in these lines (data not shown). BT20 cells
were not evaluated
for the presence of hCG(3 but were included as an HLA-A2 negative control. The
three HLA-A2
positive tumor cell lines displayed different levels of GVL/A2 when stained
with the 3.2G1 TCRm
and, as might be anticipated, the staining intensity varied in accordance with
the level of HLA-A2
on the surface. The HLA-A2 negative cell line, BT-20 was not stained with
either 3.2G1 or BB7.2.
Because of its consistently high level of expression of GVL/A2 and in order to
maximize the target
density, the MDA-MB-231 cell line was selected as the target for the following
in vitro and in vivo
assays.
[0241] The 3.2G1 TCRm mAb directs killing of a human tumor cell line in vitro:
The breast
cancer cell line MDA-MB-231 was subjected to competition analysis via tetramer
blockade of CDC
in the same manner in which the T2 cells were evaluated (described above).
Cells were plated and
allowed to adhere overnight before antibody or antibody plus tetramer was
applied. Antibody
concentration was varied from 25 to 1 iag/mI, and tetramer concentration was
held constant at 6
pg/mI. CDC of cells incubated with antibody in the absence of tetramer showed
an antibody
concentration-dependent lysis which was paralleled by cells incubated with
antibody in the
presence of VLQ tetramer. This indicated that there was essentially no
competition provided by
the tetramer (Fig. 42A). In contrast, cells incubated in the presence of
antibody plus GVL tetramer
were almost completely protected from lysis even at the highest concentration
of antibody used.
These findings further demonstrate the specificity of the 3.2G 1 TCRm and
indicate that use of this
class of antibody as a full length molecule offers a novel approach for
targeting and killing tumor
cells.
[0242] A second mechanism which plays an important role in the ability of a
therapeutic antibody
to control or eliminate tumors is antibody-dependent cell-mediated
cytotoxicity (ADCC) (Liu et al.,
2004; Prang et al., 2005; and Clynes et al., 2000). In order to investigate
the ability of the 3.2G1
TCRm mAb to direct'ADCC, peripheral blood mononuclear cells were isolated from
the platelet
chambers of aphaeresis collection devices from anonymous donors. The cells
were held in
serum-free medium (AIM-V) containing 200 units/mI rhlL-2 for 2 to 7 days.with
media changes
every 2 to 3 days in order to maintain and activate the NK population (Liu et
al., 2002). To
determine the level of NK activity present in the different donor samples,
each preparation was
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evaluated using the NK-sensitive cell line K562 at the same time the ADCC
assays were carried
out. All PBMC isolates were shown to exhibit lysis levels of 60% or more with
one exception (35%)
(data not shown).
[0243] MDA-MB-231 cells were first evaluated for sensitivity to ADCC as
adherent cultures using
five different human PBMC preparations to control for variation among the
individual donors. Fig.
42B shows the results of these assays, which contained 10 pg/mI of 3.2G1 TCRm
and were run
at an E:T ratio of 30:1. The PBMC preparations varied in their ability to lyse
MDA cells as might
be anticipated due to differences in receptor expression by NK cells. The
overall ADCC ranged
from 6.8 to 9.6% with an average value of 8.7%.
[0244] To determine the effect epitope density had on overall lysis, 3.2G1
TCRm or the pan-HLA
antibody W6/32, which is also a murine isotype IgGza, were used as targeting
agents. Fig. 42C
shows the results from an ADCC analysis of MDA-231 cells using two different
human donor
preparations at an E:T ratio of 20:1 with 3.2G1 and W6/32. The lysis values
achieved for W6/32
(14.6-22.6%) were greater than those of 3.2G1 (6.4-13.4%) suggesting that
lysis was at least in
part dependent on epitope density. Overall, these results show a modest but
consistent level of
tumor-specific ADCC mediated by the 3.2G1 TCRm.
[0245] In vivo Analysis of 3.2G1 TCRm in Nude Mice Implanted with MDA-MB-231:
To establish
the ability of the 3.2G1 TCRm to inhibit tumor growth in vivo, nude mice were
implanted with
MDA-MB-231 tumor cells. Antibody treatment was initiated at the time of
implantation with an i.p.
injection of either 3.2G1 TCRm or an isotype control antibody. Tumors began to
appear in the
isotype control-treated mice between 36 and 43 days (week 6) after
implantation while none were
evident in any of the mice treated with 3.2G1. Tumors continued to appear and
expand in the
control mice until day 69 (week 6 tumor volume = 4.5 mm3; week 10, tumor
volume = 156 mm3).
Final scoring was tabulated on day 69, 21 days after the appearance of the
last tumor in the control
mice. At day 69, eight of ten mice in the isotype treated group had developed
tumors that were 6
mm in diameter or larger while none of the nine mice in the group treated with
the 3.2G1 TCRm
showed evidence of tumor growth (Fig. 43). The experiment was terminated at 71
days.
[0246] Fig. 44 illustrates that the 3.2G1 TCRm can be used therapeutically to
treat athymic nude
mice with established tumors. Female athymic mice were subcutaneously injected
with
MDA-MB-231 breast cancer cells and after 10 days of growth, the mice were
injected with either
the 3.2G1 TCRm antibody or an IgG2a isotype control antibody. Mice then
received 3 more
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injections at weekly intervals. 24 days after initial injection, tumor growth
was measured and
plotted as tumor volLime. Tumor growth in the IgG2a isotype control group
increased almost
three-fold from an initial pre-treatment mean of 105 mm3 to a mean of 295 mm3.
In contrast, the
3.2G1 treated group had a mean tumor volume of 62 mm3 that was reduced to a
tumor volume of
8 mm3 after treatment. Even more impressive was that 3 out of 4 mice in the
3.2G1 treated group
had no tumors.
[0247] These findings demonstrate that TCRm mAbs can be used therapeutically
to eradicate
established tumors in mice, thus demonstrating the therapeutic effectiveness
of using TCRm to kill
tumors via binding to a specific peptide-MHC complex on the surface of cancer
cells.
[0248] The current study characterizes the functional properties of an
antibody with the type of
HLA-restricted peptide specificity associated with T cell receptors. The
similarity in epitope
recognition to a TCR has led us to designate this antibody a TCR mimic (TCRm)
and to investigate
its potential as a therapeutic agent. The 3.2G1 TCRm is a murine IgGaa
monoclonal antibody that
(1) binds to and mediates both CDC and ADCC lysis of cells bearing the GVL
peptide-HLA complex
on their surface and (2) inhibits the growth of a human breast cancer cell
line when it is implanted
into mice. 3.2G1 TCRm immunofluorescent staining intensity was proportional to
the antibody
concentration and to the amount of peptide present on the surface of the T2
cells. Staining was
also blocked in a dose-dependent manner by GVL/A2 tetramers added to the
staining buffer.
Titration of the peptide used to pulse T2 cells resulted in demonstration of a
direct correlation
between the staining intensity and the extent of specific cell lysis by CDC.
[0249] In the present invention, the potential efficacy of the 3.2G1 TCRm as a
therapeutic agent
has been demonstrated by examining its ability to trigger CDC and ADCC of
tumor cells in vitro and
to prevent tumor growth in vivo as well as to eradicate tumors in vivo.
Elimination of tumors in vivo
by antibody therapy is thought to be the result of any or all of a number of
mechanisms including
but not limited to blockade of growth factor receptors, induction of
apoptosis, CDC and ADCC.
[0250] The results obtained with our novel TCRm indicate that (1) the
peptide/MHC complex is
a legitimate target for cancer therapy by a naked antibody, (2) the level of
expression of specific
complex is high enough on at least one tumor line to lead to efficient lysis,
and (3) there appears
to be a threshold level of expression of the complex above which the antibody
is effective. A large
number of peptide antigens from tumors that are recognized by T cells have
been previously
characterized (Novellino et al., 2005) and now offer new targets available on
the tumor surface for
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antibody therapy. These antibodies open access to a new range of targets
available on the cell
surface which are independent of the ultimate location of the original protein
to which they are
directed. The ability to create effective TCRm recognizing such peptides in
the context of MHC
antigens presents the opportunity to significantly expand the current
repertoire of therapeutic
antibodies.
SUMMARY
[0251] Shown in Fig. 45 is a timeline of the protocol of generating peptide-
MHC specific
monoclonal antibodies of the presently disclosed and claimed invention. As
evidenced by the figure
and the examples provided herein above, a rapid method of generating peptide-
MHC specific
monoclonal antibodies has been demonstrated, wherein the peptide-MHC specific
monoclonal
antibodies can be generated in 8-12 weeks.
[0252] The value of monoclonal antibodies which recognized peptide-MHC
complexes has been
recognized for some time, as described in the Background of the Prior Art
section, and several
groups have generated antibodies of this type for use in investigating the
characteristics of the
complexes (Murphy et al., 1992; Eastman et al., 1996; Dadaglio et al., 1997;
Messaoudi et al.,
1999; Porgador et al., 1997; Rognan et al., 2000; Polakova et al., 2000;
Denkberg et al., 2003;
Denkberg et al., 2002; Biddison et al., 2003; Cohen et al., 2003; and
Steenbakkers et al., 2003).
There are several aspects of the presently disclosed and claimed invention
that are novel over the
prior art methods, and which overcome the disadvantages and defects of the
prior art. First, the
method of the presently disclosed and claimed invention results in hybridoma
cells producing high
affinity, full-length antibodies to specific peptide-HLA complexes. An example
of the affinity range
achieved is shown by the 4F7 monoclonal antibody (see for example, Fig. 23 and
Example 2),
which has a KD of approximately 1 nM. Affinity measurements for the 1 B8
monoclonal antibody
indicate that it is in the same affinity range. The affinity of these two
antibodies is high enough that
they can distinctly stain breast cancer cell lines, and this aspect of the
presently disclosed and
claimed invention contrasts sharply with the weak staining reported for
antibodies from a phage
display library (Denkberg et al., 2003).
[0253] Second, in contrast to the prior art methods that utilize phage display
libraries, the
product produced by the method of the presently disclosed and claimed
invention is "ready to use";
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it is a whole antibody which is easy to purify and characterize, and does not
require any further
manipulation to achieve expression of significant quantities of material.
[0254] Third, the method of the presently disclosed and claimed invention
requires significantly
less time to product when compared to the prior art methods. The method of the
presently
disclosed and claimed invention can complete the cycle from immunization to
identification of
candidate hybridomas in as few as eight weeks, as shown in Fig. 45 and as
achieved as described
herein for monoclonal antibody I B8. The method of the presently disclosed and
claimed invention
is both rapid and reproducible.
[0255] Fourth, the immunogen employed in the method of the presently disclosed
and claimed
invention is novel. The immunogen consists of peptide-HLA complexes that are
loaded solely with
the peptide of interest. The immunogens are made in a form which allows
production and
characterization of milligram quantities of highly purified material which
correctly presents the three
dimensional structure of the peptide-HLA complex. This complex can be easily
manipulated to
form higher order multimers. Preliminary data indicates that the use of
tetrameric forms of the
peptide-HLA immunogen is more efficient at generating a specific response than
are monomeric
or mixed multimeric forms of the immunogen.
[0256] Fifth, the screening processes described in the presently claimed and
disclosed invention
are unique and completely describe methods to discern the presence of anti-
peptide/HLA
antibodies in the serum of immunized mice, even in the presence of antibodies
which react with
other epitopes present on the complex. The screening processes also produce
methods to identify
and characterize monoclonal antibodies produced after hybridoma fusion.
[0257] The presently disclosed and claimed invention overcomes obstacles
encountered in prior
art methods, which reported low yields of specific monoclonal responses
(Eastman et al., 1996;
Dadaglio et al., 1997; and Andersen et al., 1996). The antibodies generated by
the method of the
presently disclosed and claimed invention are also clearly distinct from those
reported from phage
libraries. As an example, a phage-derived Fab which recognized hTERT-HLA-A2
complex would
stain hTERT-peptide pulsed HLA-A2 positive cells (Lev et al., 2002), but would
not stain tumor cells
(Parkhurst et al., 2004), indicating that this prior art antibody had either
low specificity, or low
affinity, or both. Such an antibody would not be useful in applications
described herein for the
presently disclosed and claimed invention, such as but not limited to, epitope
validation in vaccine
development and other clinical applications.
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[0258] Thus, in accordance with the present invention, there has been provided
a method of
producing antibodies that recognize peptides associated with a tumorigenic or
disease state,
wherein the antibodies will mimic fihe specificity of a T cell receptor, that
fully satisfies the objectives
and advantages set forth hereinabove. Although the invention has been
described in conjunction
with the specific drawings, experimentation, results and language set forth
hereinabove, it is
evident that many alternatives, modifications, and variations will be apparent
to those skilled in the
art. Accordingly, it is intended to embrace all such alternatives,
modifications and variations that
fall within the spirit and broad scope of the invention.
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