Note: Descriptions are shown in the official language in which they were submitted.
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ANTIGEN BINDING FRAGMENTS, DESIGNATED 4B5, THAT SPECIFICALLY
DETECT CANCER CELLS, NUCLEOTIDES ENCODING THE FRAGMENTS,
AND USE THEREOF FOR THE PROPHYLAXIS AND DETECTION OF
CANCERS
TECHNICAL FIELD
This invention relates to polynucleotides that encode anti-idiotypic
antibodies that
mimic the GD2 antigen. The recombinant human monoclonal antibody (Mab) and
antigenic fragments thereof are termed "4B5." The invention encompasses a wide
variety
of antibodies, and functional derivatives thereof that retain the immunologic
specificity of
4B5 and are termed herein "4B5." The polynucleotides encoding 4B5 and
polypeptides
encoded thereby and recombinant molecules containing these polynucleotides are
also
encompassed by the invention. Methods of use including therapeutic and
diagnostic of the
4B5 antibodies are also included in the invention.
BACKGROUND ART
In spite of numerous advances in medical research, cancer remains the second
leading cause of death in the United States. In the industrialized nations,
roughly one in
five persons will die of cancer. Traditional modes of clinical care, such as
surgical
resection, radiotherapy and chemotherapy, have a significant failure rate,
especially for
solid tumors. Failure occurs either because the initial tumor is unresponsive,
or because of
recurrence due to regrowth at the original site and/or metastases. Even in
cancers such as
breast cancer where the mortality rate has decreased, successful intervention
relies on early
detection of the cancerous cells. The etiology, diagnosis and ablation of
cancer remain a
central focus for medical research and development.
Neoplasia resulting in benign tumors can usually be completely cured by
removing
the mass surgically. If a tumor becomes malignant, as manifested by invasion
of
surrounding tissue, it becomes much more difficult to eradicate. Once a
malignant tumor
metastasizes, it is much less likely to be eradicated.
The three major cancers, in terms of morbidity and mortality, are colon,
breast and
lung. New surgical procedures offer an increased survival rate for colon
cancer. Improved
CONFIRMATION COPY
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screening methods increase the detection of breast cancer, allowing earlier,
less aggressive
therapy. Numerous studies have shown that early detection increases survival
and
treatment options. Lung cancer remains largely refractory to treatment.
Excluding basal cell carcinoma, there are over one million new cases of cancer
per
year in the United States alone, and cancer accounts for over one half million
deaths per
year in this country. In the world as a whole, the five most common cancers
are those of
lung, stomach, breast, colon/rectum, and uterine cervix, and the total number
of new cases
per year is over 6 million. Only about half the number of people who develop
cancer die
of it.
Melanoma is one of the human diseases for which there is an acute need of new
therapeutic modalities. It is a particularly aggressive form of skin cancer,
and occurs in
increased frequency in individuals with regular unguarded sun exposure. In the
early
disease phases, melanoma is characterized by proliferation at the dermal-
epidermal
junction, which soon invades adjacent tissue and metastasizes widely. Once it
has
metastasized, it is often impossible to extirpate and is consequently fatal.
Worldwide,
70,000 patients are diagnosed with melanoma and it is responsible for 25,000
reported
deaths each year. The American Cancer Society projects that by the year 2000,
1 out of
every 75 Americans will be diagnosed with melanoma.
Neuroblastoma is a highly malignant tumor occurring during infancy and early
childhood. Except for Wilm's tumor, it is the most common retroperitoneal
tumor in
children. This tumor metastasizes early, with widespread involvement of lymph
nodes,
liver, bone, Lung, and marrow. While the primary tumor is resolvable by
resection, the
recurrence rate is high.
An estimated 178,100 new cases of lung cancer will be diagnosed in 1997,
accounting for I3% of cancer diagnoses. An estimated 160,400 deaths due to
lung cancer
will occur in 1997 accounting for 29% of all cancer deaths. The one year
survival rates for
lung cancer have increased from 32% in I 973 to 41 % in 1993, largely due to
improvements in surgical techniques. The 5 year survival rate for all stages
combined is
only 14%. The survival rate is 48% for cases detected when the disease is
still localized,
but only 15% of lung cancers are discovered that early.
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Small cell lung cancer is the most malignant and fastest growing form of lung
cancer and accounts for 20-25% of new cases of lung cancer. 60,000 cases will
be
diagnosed in the U.S. in 1996. The primary tumor is generally responsive to
chemotherapy, but is followed by wide-spread metastasis. The median survival
time at
diagnosis is approximately 1 year, with a 5 year survival rate of S-10%.
Breast cancer is one of the most common cancers and is the third leading cause
of
death from cancers in the United States with an annual incidence of about
180,200 new
cases among women in the United States during 1997. About 1,400 new cases of
breast
cancer will be diagnosed in men in 1997. In industrialized nations,
approximately one in
eight women can expect to develop breast cancer. The overall mortality rate
for breast
cancer has remained unchanged since 1930. It has increased an average of 0.2%
per year,
but decreased in women under 65 years of age by an average of 0.3% per year.
Preliminary data suggest that breast cancer mortality may be beginning to
decrease,
probably as a result of increased diagnoses of localized cancer and carcinoma
in situ. See
e.g., Marchant (1994) Contemporary Management of Breast Disease II: Breast
Cancer, in:
Obstetrics and Gynecology Clinics ofNorth America 21:555-560; and Colditz
(1993)
Cancer Suppl. 71:1480-1489. An estimated 44,190 deaths (43,900 women, 290 men)
in
1997 will occur due to breast cancer. In women, it is the second major cause
of cancer
death after lung cancer. The five-year survival rate for localized breast
cancer has
increased from 72% in the 1940s to 97% today. If the cancer has spread
regionally,
however, the rate is 76%, and for women with distant metastases the rate is
20%. Survival
after a diagnosis of breast cancer continues to decline beyond five years.
Sixty-five
percent of women diagnosed with breast cancer survive I 0 years and 56%
survive 15
years.
Non-Hodgkin's B cell lymphomas are cancers of the immune system that are
expected to afflict approximately 225,000 patients in the United States in
1996. These
cancers are diverse with respect to prognosis and treatment, and are generally
classified
into one of three grades. The median survival of the lowest grade is 6.6 years
and the
higher grade cancers have much lower life expectancy. Virtually all non-
Hodgkin's B cell
lymphomas are incurable. New diagnoses of non-Hodgkins lymphomas have
increased
approximately 7% annually over the past decade, with 52,700 new diagnoses
estimated for
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1996. The increase is due in part to the increasing prevalence of lymphomas in
the AIDS
patient population.
Colon and rectal cancer will account for an estimated 131,200 cases in 1997,
including 94,100 of colon cancer and 3?,100 of rectal cancer. Colorectal
cancers account
for about 9% of new cancer diagnoses. An estimated 54,900 deaths due to
colorectal
cancer will occur in 1997, accounting for about 10% of cancer deaths.
Mortality rates for
colorectal cancer have fallen 32% for women and 14% for men during the past 20
years,
reflecting decreasing incidence rates and increasing survival rates. However,
the mortality
rate in African American men continues to rise. The 1 and 5 year relative
survival rates for
patients with colon and rectal cancer are 82% and 61 %, respectively. When
colorectal
cancers are detected in an early, localized stage, the 5 year survival rate is
91 %; however,
only 37% of colorectal cancers are discovered at that stage. After the cancer
has spread
regionally to involve adjacent organs or lymph nodes, the rate drops to 63%.
Survival
rates for persons with distant metastases is 7%. Survival continues to decline
beyond 5
years, and 50% survive 10 years.
In spite of the difficulties, effective cures using anticancer drugs (alone or
in
combination with other treatments) have been devised for some formerly highly
lethal
cancers. Most notable among these are Hodgkin's lymphoma, testicular cancer,
choriocarcinoma, and some leukemias and other cancers of childhood. For
several of the
more common cancers, early diagnosis, appropriate surgery or local
radiotherapy enables a
large proportion of patients to recover.
Current methods of cancer treatment are relatively non-selective. Surgery
removes
the diseased tissue, radiotherapy shrinks solid tumors and chemotherapy kills
rapidly
dividing cells. Chemotherapy, in particular, results in numerous side effects,
in some cases
so severe to preclude the use of potentially effective drugs. Moreover,
cancers often
develop resistance to chemotherapeutic drugs.
Numerous efforts are being made to enhance the specificity of cancer therapy.
For
review, see Kohn and Liotta (1995) Cancer Res. 55:1856-1862. In particular,
identification of cell surface antigens expressed exclusively or
preferentially on certain
tumors allows the formulation of more selective treatment strategies.
Antibodies directed
to these antigens have been used in immunotherapy of several types of cancer.
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The basic immunoglobulin (Ig) structural unit in vertebrate systems is
composed of
two identical light ("L") polypeptide chains (approximately 23 kDa), and two
identical
heavy ("H") chains (approximately 53 to 70 kDa). The four chains are joined by
disulfide
bonds in a "Y" configuration. At the base of the Y, the two H chains are bound
by
covalent disulfide linkages.
Figure 1 shows a schematic of an antibody structure. The L and H chains are
each
composed of a variable (V) region at the N-terminus, and a constant (C) region
at the C-
terminus. In the L chain, the V region (termed "VLJ~,") is composed of a V
(VL) region
connected through the joining (JL) region to the C region (CL). In the H
chain, the V region
(VHDHJH) is composed of a variable (VH) region linked through a combination of
the
diversity (DH) region and the joining (JH) region to the C region (CH). The
VLJL and
V,_,DHJH regions of the L and H chains, respectively, are associated at the
tips of the Y to
form the antigen binding portion and determine antigen binding specificity.
The (Cj.,) region defines the isotype, i. e., the class or subclass of
antibody.
Antibodies of different isotypes differ significantly in their effector
functions, such as the
ability to activate complement, bind to specific receptors {e.g., Fc
receptors) present on a
wide variety of cell types, cross mucosal and placental barriers, and form
polymers of the
basic four-chain IgG molecule.
Antibodies are categorized into "classes" according to the CH type utilized in
the
immunoglobulin molecule (IgM, IgG, IgD, IgE, or IgA). There are at least five
types of
CH genes (Cp., Cy, C8, Ce, and Ca), and some species have multiple CH subtypes
(e.g.,
Cy,, Cyz, Cy3, and Cy4, in humans). There are a total of nine CH genes in the
haploid
genome of humans, eight in mouse and rat, and several fewer in many other
species. In
contrast, there are normally only two types of L chain C regions (CL), kappa
(K) and
lambda (~,), and only one of these C regions is present in a single L chain
protein (i.e.,
there is only one possible L chain C region for every VLJL produced). Each H
chain class
can be associated with either of the L chain classes (e.g., a CHy region can
be present in the
same antibody as either a x or 7~ L chain), although the C regions of the H
and L chains
within a particular class do not vary with antigen specificity (e.g., an IgG
antibody always
has a Cy H chain C region regardless of the antigen specificity).
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Each of the V, D, J, and C regions of the H and L chains are encoded by
distinct
genomic sequences. Antibody diversity is generated by recombination between
the
different V,,, DH, and JH, gene segments in the H chain, and VL and JL gene
segments in the
L chain. The recombination of the different VH, DH, and J,., genes is
accomplished by DNA
recombination during B cell differentiation. Briefly, the H chain sequence
recombines first
to generate a DHJH complex, and then a second recombinatorial event produces a
VHDHJH
complex. A functional H chain is produced upon transcription followed by
splicing of the
RNA transcript. Production of a functional H chain triggers recombination in
the L chain
sequences to produce a rearranged VLJL region which in turn forms a functional
VLJLCL
region, i.e., the functional L chain.
The value and potential of antibodies as diagnostic and therapeutic reagents
has
been long-recognized in the art. Unfortunately, the field has been hampered by
the slow.
tedious processes required to produce large quantities of an antibody of a
desired
specificity. The classical cell fusion techniques allowed for efficient
production of Mabs
by fusing the B cell producing the antibody with an immortalized cell line.
The resulting
cell line is a hybridoma cell line.
Antibodies and functional derivatives thereof have been used in a variety of
clinical
settings. For instance, digoxin-specific Fab antibody fragments were used to
treat life-
threatening digitalis intoxication. Antibodies are becoming more routinely
useful in
diagnostic techniques such as radioimmune diagnosis of colon cancers. Koda et
al. (1995)
Am. J. Gastroenterol. 90:1644. A number of uses of Mabs, previously thought to
be
untenable, have recently been put into practice. For instance, see Hall (
1995) Science
279:915-916.
A number of autoantibodies (antibodies that recognize and bind to self
antigens)
are found in humans. Many of these are associated with particular diseases
such as
rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, primary
biliary
cirrhosis, polymyositis, systemic vasculitis, idiopathic necrotizing and
crescentic
glomerulonephritis and amyotrophic lateral sclerosis. For review, see Shattner
(1986/1987) Immunol. Lett. 14:143-153. Other autoantibodies are naturally-
occurring.
Lutz and Wipp (1982) J. Immunol. 128:1965; and Guilbert et al. (1982) J.
Immunol.
128:2779-2787. Recently, human autoantibodies to specific cancer antigens have
been
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detected and, in some cases, are being produced by hybridoma technology. These
antibodies have also been produced by active immunization. United States
Patent No.
5,474,755. Originally, the human B cells were immortalized using Epstein-Barn
Virus or
mouse myelomas. For review, see Buck et al. ( 1984) "Monoclonal Antibodies"
NY,
Plenum Press. More recent techniques have allowed immortalization without the
use of
this potentially harmful virus. See, e.g., U.S. Patent No. 4,618,477; and
Glassy (1987)
Cancer Res. 47:5181-5188. In most instances, the antibodies are specific for
one, or in
some instances, a few, cancer types. For instance, a Mab has been described
that
specifically recognizes glioma cells but no other tumor or normal cells. These
antibodies
were used to image the glioma in the patient's brain. Fischer et al. (1991)
Immunobiol.
Prot. Pep. VI (M. Atassi, ed.) Plenum Press, NY. pp. 263-270. No antibody has
been
described that is capable of recognizing a wide range of tumors while failing
to recognize.
or only poorly recognize, normal, non-cancerous cells.
Recombinant genetic techniques have allowed cloning and expression of
antibodies, functional fragments thereof and the antigens recognized. These
engineered
antibodies provide novel methods of production and treatment modalities. For
instance,
functional immunoglobulin fragments have been expressed in bacteria and
transgenic
tobacco seeds and plants. Skerra (1993) Curr. Opin. Immunol. 5:256-262;
Fiedler and
Conrad (1995) BiolTechnology 13:1090-1093; Zhang et al. (1993) Cancer Res.
55:3384-
3591; Ma et al. (1995) Science 268:916; and, for a review of synthetic
antibodies, see
Barbas (1995) Nature Med 1:836-839.
Several human Mabs against tumor associated antigens have been produced and
characterized. The tumor associated antigens recognized by human Mabs include
cell
surface, cytoplasmic and nuclear antigens. Yoshikawa et al. ( 1989) Jpn. J.
Cancer Res.
(Gann) 80:546-553; Yamaguchi et al. (1987) Proc. Natl. Acad. Sci. USA 84:2416-
2420;
Haspel et al. (1985) Cancer Res. 45:3951-3961; Cote et al. (1986) Proc. Natl.
Acad. Sci.
USA 83:2959-2963; Glassy (1987) Cancer Res. 47:5181-5188; Borup-Christensen et
al.
(1987) Cancer Detect. Prevent. Suppl. 1:207-215; Haspel et al. (1985) Cancer
Res.
45:3951-3961; Kan-Mitchell et al. (1989) Cancer Res. 49:4536-4541; Yoshikawa
et al.
(1986) Jpn. J. Cancer Res. 77:1122-1133; and McKnight et al. {1990) Human
Antibod.
Hybridomas 1:125-129. Dan et al. ( 1992) J. Neurosurg. 76: 660-669.
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Human Mabs have been used in cancer imaging, diagnosis and therapy. Olsson
(1985) J. Nat. Cancer Inst. 75:397-404; Larrick and Bourla (1986) J. Biol.
Resp. Mod.
5:379-393; McCabe et al. (1988) Cancer Res. 48:4348-4353; Research News (1993)
Science 262:841; Ditzel et al. (1994) Cancer 73:858-863; and Alonso (1991) Am.
J. Clin.
Oncol. 4:463-471. A recombinant single chain bispeciflc antibody has been
reported that
has high tumor cell toxicity. This molecule recognizes both the CD3 antigen of
human T
cells and EpCAM, which is associated with disseminated tumor cells in patients
with
minimal residual colorectal cancer. Mack et al. (1995) Proc. Natl. Acad. Sci.
USA
92:7021-7025.
Immunobiologists have learned that a poor antigen (in terms of eliciting an
immune
response) can be turned into a strong antigen by changing the molecular
environment.
Changes of hapten carrier allow T cell helper cells to become active, making
the overall
immune response stronger. Thus, changing the carrier can also turn a
tolerogenic antigen
into an effective antigen. McBridge et al. ( 1986) Br. J. Cancer 53:707. Often
the
immunological status of a cancer patient is suppressed so that the patient is
only able to
respond to certain T-dependent antigens and not to other antigen forms. From
these
considerations, it would make sense to introduce molecular changes into the
tumor
associated antigens before using them as vaccines. Unfortunately, this is
impossible to
accomplish for most tumor antigens, because they are not well defined and are
very hard to
purify.
The network hypothesis of Lindemann ((1973) Ann. Immunol. 124:171-184) and
Jerne ((1974) Ann. Immunol. 125:373-389) offers an elegant approach to
transform epitope
structures into idiotypic determinants expressed on the surface of antibodies.
According to
the network concept, immunization with a given tumor-associated antigen will
generate
production of antibodies against this tumor-associated antigen, termed Abl;
this Abl is
then used to generate a series of anti-idiotype antibodies against the Abl,
termed Ab2.
Some of these Ab2 molecules can effectively mimic the three-dimensional
structure of the
tumor-associated antigen identified by the Ab 1. These particular anti-
idiotypes called
Ab2~i fit into the paratopes of Abl, and express the internal image of the
tumor-associated
antigen. The Ab2(3 can induce specific immune responses similar to those
induced by the
original tumor-associated antigen and can, therefore, be used as surrogate
tumor-associated
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antigens. Immunization with Ab2~3 can lead to the generation of anti-anti-
idiotype
antibodies (Ab3) that recognize the corresponding original tumor-associated
antigen
identified by Abl. Because of this Abl-like reactivity, the Ab3 is also called
Abl' to
indicate that it might differ in its other idiotopes from Ab 1.
A potentially promising approach to cancer treatment is immunotherapy
employing
anti-idiotype antibodies. In this form of therapy, an antibody mimicking an
epitope of a
tumor-associated protein is administered in an effort to stimulate the
patient's immune
system against the tumor, via the tumor-associated protein. WO 91/11465
describes
methods of stimulating an immune response in a human against malignant cells
or an
infectious agent using primate anti-idiotype antibodies. However, not all anti-
idiotype
antibodies can be used in therapeutic regimens against tumors. First, only a
fraction of
antibodies raised against an Abl are limited in their reactivity to the
paratope of Abi (i.e.,
are non-reactive against features shared with other potential antibodies in
the host).
Second, anti-idiotype antibodies are not necessarily immunogenic. Third, even
if an anti-
idiotype elicits an immune response, only a fraction of these immunogenic anti-
idiotypes
elicit an immune response against the tumor antigen and not against other
antigens with
less specificity. Moreover, since different cancers have widely varying
molecular and
clinical characteristics, it has been suggested that anti-idiotype therapy
should be evaluated
on a case by case basis, in terms of tumor origin and antigens express.
Anti-Id monoclonal antibodies structurally resembling tumor-associated
antigens
have been used as antigen substitutes in cancer patients. Herlyn et al. (
1987) PNAS
84:8055-8059; Mittleman et al. (1992) PNAS 89:466-470; Chatterjee et al.
(1993) Ann.
N. Y. Acad. Sci. 690:376-377. It has been proposed that the anti-Id provides a
partial
analog of the tumor-associated antigen in an immunogenic context.
Several murine monoclonal anti-GD2 antibodies were reported to suppress the
growth of tumors of neuroectodermal origin in athymic (nu/nu) mice or cause
remission in
patients with metastatic melanoma. Several antibodies specific for GD2 have
been
described and deposited with the ATCC. These are HB-9326, described in U.S.
Pat.
4,849,509: HB-9325, described in U.S. Pat. 4,849,509; and HB-8568, described
in U.S.
Pat. 4,675,287. Anti-idiotypic monoclonal antibodies providing an internal
anti-idiotypic
determinant on an antibody to GD2 have also been described. U.S. Pat.
5,612,030; and
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Saleh et al. (1993) J. Immunol. 151:3390-3398. The anti-idiotypic antibodies
are
described for use in treatment of melanoma and small cell carcinoma. A human-
mouse
chimeric anti-GD2 antibody caused remission in patients with metastatic
neuroblastoma.
The mechanism of action of the antibodies is thought to involve antibody
dependent
cellular cytotoxicity (ADCC) or complement-mediated cytotoxicity (CMC).
Clinical
responses have been obtained by treating melanoma with Mabs against GM2, GD2
and
GD3. Cheresh et al. (1985) Proc. Natl. Acad. Sci. USA 82:5155-5159. Active
immunization with a ganglioside vaccine comprising GM2 produced anti-GM2
antibodies
in 50/58 patients, who survived longer on average than patients without
detectable anti-
GM2 antibody.
Mabs to GD2 have been found to react specifically with small cell lung
carcinoma.
Cheresh et al. ( 1986) Cancer Res. 46:5112-5 I 18. Human Mabs specific for
other cancers
including lung, melanoma, stomach, squamous cell carcinoma, cervical
carcinoma, and
mammary carcinoma have also been produced. Murakami (1985) in Vitro Cell. Dev.
Biol.
21:593; Schadendorf (1989) J. Immunol. 142:1621-1625; Yoshikawa et al. (1986)
.Ipn. J.
Cancer Res. 77:1122-1133; Pickering and Misra (I984) Clin. Immunol.
Immunopathol.
32:253-260; Hagiwara and Sato (1983) Mol. Biol. Med. 1:245-252; and Schlom et
al.
( 1980) Proc. Natl. Acad. Sci. USA 77:6841-6845. Human anti-cancer Mabs and
the
antigens they recognize have also been suggested for use in vaccines. See,
e.g. Finn et al.
(1995) Immunol. Rev. 145:61-89. A human Mab to malignant brain tumors was used
in a
phase I clinical trial without adverse side effects. Matsumoto et al. (1994)
The Clinical
Report 28:118-126. Phase II trial results have been reported on combined
treatment with
murine Mab and colony stimulating factor in metastatic gastrointestinal
cancer. Saleh et
al. (1995) Cancer Res. 55:4339-4346. A single chain immunotoxin has also been
found to
cure carcinomatous meningitis in a rat model. Pastan et al. (1995) Proc. Natl.
Acad. Sci.
USA 92:2765-2769. Human Mabs that specifically recognize ovarian cancer cells
have
been shown to effectively image this cancer. Chaudhuri et al. ( 1994) Cancer
73:1098-
1104.
If there were a simple and reliable strategy for providing immune reactivity
against
an antigen common to these cancers rather than cancer-specific immunity, the
clinical
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prospects of cancers in general would improve. All references cited herein are
hereby
incorporated by reference in their entirety.
DISCLOSURE OF THE INVENTION
This invention encompasses compositions containing antigen binding fragments
of
an antibody where the antibody specifically recognizes the antigen recognized
by an
antibody comprising a H chain having the amino acid sequence of SEQ ID N0:2
and a L
chain having the amino acid sequence of SEQ ID N0:4. Preferably, the antibody
is
recombinant 4B5. The invention further encompasses antibodies comprising the H
and L
chain of 4B5 (SEQ ID NOS:2 and 4, respectively). 4B5 specifically recognizes
cancer
cells from a wide variety of cancers but does not recognize normal, non-
cancerous cells.
By "does not recognize" is meant that noncancer cells are either not
specifically bound to
by 4B5 or are only poorly recognized by the antibody. These antigen binding
fragments
include, but are not limited to, whole native antibodies, exemplified by the
recombinant
4B5 described herein; bispecific antibodies; chimeric antibodies; Fab, Fab',
single chain V
region fragments (scFv) and fusion polypeptides.
The invention further encompasses 4B5 antibody fusion molecules comprising a
polypeptide region with an antigenic, therapeutic, toxic or labeling molecule
attached to
the H chain C region, a single-chain VH VL or VL VH V region, and
polynucleotides
encoding such polypeptides.
Also embodied in the invention is a polynucleotide comprising a sequence
encoding a polypeptide with the immunologic specificity of 4B5, wherein the
encoded
polypeptide comprises at least S consecutive amino acids from a V region of
4B5. The V
region may be from either the 4B5 L chain or H chain. The 5 consecutive amino
acids
preferably play a role in 4B5 immunologic reactivity, and may be from a CDR.
The invention also encompasses isolated polynucleotides of at least 20
consecutive
nucleotides capable of forming a stable duplex with the 4B5 L or H chain
encoding
sequences, but not with sequences for other previously described
immunoglobulin
molecules. Any of these polynucleotides can be in the form of cloning vectors,
expression
vectors, or transfected into host cells.
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Another embodiment of this invention are polypeptides having the immunologic
specif city of 4B5, wherein the polypeptide comprises at least 5 consecutive
amino acids
from a V region of an 4B5 antibody. The V region may be from a L chain or H
chain. The
consecutive amino acids preferably play a role in immunologic specificity, and
may be
5 from a CDR (Complementarity Determining Region of an antibody). Intact
recombinant
4B5, functionally active fragments of 4B5, fusion proteins, chimeric
antibodies, multiple
antigen proteins, and other polypeptide derivatives of 4B5 antibodies are
included. Of
special interest are single-chain V regions and fusion proteins.
The compounds and compositions of this invention can be used inter alia for
detecting or treating a cancer; including therapy of such cancer, and
prophylactic care,
particularly for decreasing the risk of recurrence.
The invention further embodies cells and cell lines producing 4B5.
A further embodiment of this invention comprises prophylactic treatment of a
cancer patient with at least one 4B5 antigen binding fragment. Preferably, 4B5
is fused to
a therapeutic molecule to effect delivery of the therapeutic molecule to the
cancer cell.
The individual may have a clinically detectable tumor, or the tumor may have
been
previously treated and rendered undetectable. The method can be for palliating
the
disease, or for reducing the risk of recurrence.
A further embodiment of the invention is a kit for detection or quantitation
of the
antibodies specific for GD2 in a sample, comprising 4B5 in suitable packaging.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts a schematic of the general antibody structure.
Figure 2 depicts the purification of recombinant 4B5 on protein G Sepharose
column. The
4B5 antibodies produced by the hybridoma cells are shown in lane 1 and 2.
Recombinant
4B5 antibodies are shown in lane 3 and 4. Lane 1 and 3 contain 3~g of protein
and lane 2
and 4 contain 6q.g of protein.
Figure 3 depicts the determination of the antigenic similarity between
recombinant
4B5 and hybridoma 4B5 by ELISA analysis. Dark bars represent binding of the
4B5
antibodies or control human IgG to antigen 14G2A. Light bars represent non-
specific
binding of the 4B5 antibodies or control human IgG to mouse IgG.
12
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Figure 4 depicts the binding specificity of recombinant 4B5 to antigen 14G2A
by
Western blot analysis.
Figure 5 depicts the plasmid expression vector pNB2-4B5.
BEST MODE FOR CARRYING OUT THE INVENTION
This invention encompasses antigen binding fragments exemplified by the
recombinant antibody described herein. The exemplary antibody is designated
4B5 and is
encoded by SEQ ID NOS: 1 and 4 (SEQ ID NOS:2 and S being the complementary
strands
of l and 4, respectively) and recognizes antibodies specific for GD2
antibodies.
Antibodies specific for GD2 recognize various cancers including, but not
limited to,
glioblastoma, neuroblastoma, malignant and/or metastatic melanoma, breast
adenocarcinoma, lung adenocarcinoma, small cell lung carcinoma, colon
adenocarcinoma
and prostate adenocarcinoma.
The invention further encompasses 4B5 derivatives with immunologic specificity
for antibodies specific for GD2. These derivatives comprise regions of the
polypeptide
sequence comprising part of the 4B5 VDJ junction. Also encompassed are regions
spanning at least one, preferably 2, and more preferably 3 or more of the 4B5
CDR amino
acid sequences.
Certain compounds, compositions and methods described in this application
relate
generally to 4B5 and derivatives thereof which are routinely generated by
classical
techniques of immunochemistry. This includes 4B5 which has been coupled to
another
compound by chemical conjugation, or by mixing with an excipient or an
adjuvant. The
term antigen binding fragment includes any peptide that is recognized
specifically by
antibodies specific for GD2. Typically, these derivatives include such
immunoglobulin
fragments as Fab, F(ab')z, Fab', scFv (both monomers and polymeric forms) and
isolated
H and L chains. An antigen binding fragment retains the antigenic specificity
of 4B5,
although avidity and/or affinity may be altered.
The antigen binding fragments (also termed "derivatives" herein) are typically
generated by genetic engineering, although they may alternatively be obtained
by other
methods and combinations of methods. This classification includes, but is not
limited to,
engineered peptide fragments and fusion peptides. Preferred compounds include
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polypeptide fragments of the 4B5 CDRs, antibody fusion proteins comprising
cytokine
effector components, antibody fusion proteins comprising adjuvants or drugs,
and single-
chain V region proteins.
The invention further comprises polynucleotides encoding the 4B5 antibody V
regions and derivatives thereof. These include isolated polynucleotide
fragments both
coding and complimentary strands, recombinant polynucleotides, and therapeutic
plasmids
and vectors, such as vaccinia vectors, comprising the polynucleotides. These
polynucleotides are exemplified by SEQ ID NOS:1, 2, 4 and 5 and plasmid pNB2-
4B5
(Fig. 5).
As 4B5 has been shown to mimic GD2, it is particularly useful in generating a
host
immune response to cancer. Pharmaceutical compositions and treatment
modalities of this
invention are suitable for eliciting an immune response against neoplasia.
Suitable
carcinomas include any known in the field of oncology, including, but not
limited to,
astrocytoma, oligodendroglioma, ependymoma, medulloblastoma, primitive neural
ectodermal tumor (PNET), pancreatic ductal adenocarcinoma, small and large
cell lung
adenocarcinomas, squamous cell carcinoma, bronchoalveolarcarcinoma, epithelial
adenocarcinoma, and liver metastases thereof, hepatoma, cholangiocarcinoma,
breast
tumors such as ductal and lobular adenocarcinoma, squamous and adenocarcinomas
of the
uterine cervix, uterine and ovarian epithelial carcinomas, prostatic
adenocarcinomas,
transitional squamous cell carcinoma of the bladder, B and T cell lymphomas
(nodular and
diffuse) plasmacytoma, acute and chronic Ieukemias, malignant melanoma, soft
tissue
sarcomas and leiomyosarcomas.
The subjects may have an advanced form of disease, in which case the treatment
objective may include mitigation or reversal of disease progression, and
amelioration of
side effects. The subjects may have had a history of the condition, for which
they have
already been treated, in which case the objective will typically include a
decrease or delay
in the risk of recurrence.
Immunologic activity" of 4B5 refers to the ability to specifically bind
antibodies
specific for GD2. Such binding may or may not elicit an immune response. A
specific
immune response may comprise antibody, B cells, T cells, and any combination
thereof,
and effector functions resulting therefrom. Included are the antibody-mediated
functions
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ADCC and complement-mediated cytolysis (CDC). The T cell response includes T
helper
cell function, cytotoxic T cell function, inflammation/inducer T cell
function, and T cell
mediated suppression. A compound able to elicit a specific immune response
according to
any of these criteria is referred to as "immunogenic."
4B5 "activity" or "function" refers to any of the immunologic activities of
4B5, or
to any other biological activity ascribed to 4B5 in this disclosure, including
the role of 4B5
in the detection of antibodies specific for GD2, amelioration or palliation of
cancer.
The "V region" of 4B5 refers to the V region of the 4B5 L chain or the V
region of
the 4B5 H chain, either alone or in combination. These V regions are depicted
in SEQ ID
NOS: 3 and 6; the DNA encoding these regions is depicted in SEQ ID NOS: 1 and
4,
respectively.
GM-CSF, IL-2, and other biologically active molecules referred to herein are
meant
to include fragments and derivatives based on the respective parent molecule
that have the
same biologic or physiologic function.
A "polynucleotide" is a polymeric form of nucleotides of any length, which
contain
deoxyribonucleotides, ribonucleotides, and analogs in any combination analogs.
Polynucleotides may have any three-dimensional structure, and may perform any
function,
known or unknown. The term "polynucleotide" includes double- , single-
stranded, and
triple-helical molecules. Unless otherwise specified or required, any
embodiment of the
invention described herein that is a polynucleotide encompasses both the
double-stranded
form and each of two complementary single-stranded forms known or predicted to
make
up the double stranded form of either the DNA, RNA or hybrid molecules.
The following are non-limiting examples of polynucleotides: a gene or gene
fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant
polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of
any
sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A
polynucleotide can comprise modified nucleotides, such as methylated
nucleotides and
nucleotide analogs, uracyl, other sugars and linking groups such as
fluororibose and
thioate, and nucleotide branches. Analogs of purines and pyrimidines are known
in the art,
and include, but are not limited to, aziridinylcytosine, 4-acetylcytosine, 5-
fluorouracil, 5-
bromouracil, ~-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-
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aminomethyluracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-
methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethyiguanine, 2-
methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine,
pseudoruacil, 5-
pentynyluracil and 2,6-diaminopurine. The use of uracil as a substitute for
thymine in a
deoxyribonucleic acid is also considered an analogous form of pyrimidine.
The sequence of nucleotides can be interrupted by non-nucleotide components. A
polynucleotide can be further modified after polymerization, such as by
conjugation with a
labeling component. Other types of modifications included in this definition
are caps,
substitution of one or more of the naturally occurring nucleotides with an
analog, and
introduction of means for attaching the polynucleotide to proteins, metal
ions, labeling
components, other polynucleotides, or a solid support.
If present, modification to the nucleotide structure can be imparted before or
after
assembly of the polymer. The sequence of nucleotides can be interrupted by non-
nucleotide components. A polynucleotide can be further modified after
polymerization,
such as by conjugation with a labeling component. Other types of modifications
included
in this definition are, for example, "caps", substitution of one or more of
the naturally
occurring nucleotides with an analog, internucleotide modifications such as,
for example,
those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters,
phosphoamidates, carbamates, etc.) and with charged linkages (e.g.,
phosphorothioates,
phosphorodithioates, etc.), those containing pendant moieties, such as, for
example,
proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine,
etc.), those with
intercalators (e.g., acridine, psoralen, etc.), those containing chelators
(e.g., metals,
radioactive metals, boron, oxidative metals, etc.), those containing
alkylators, those with
modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as
unmodified forms of
the polynucleotide(s).
Further. any of the hydroxyl groups ordinarily present in the sugars may be
replaced by phosphonate groups, phosphate groups, protected by standard
protecting
groups, or activated to prepare additional linkages to additional nucleotides,
or may be
conjugated to solid supports. The S' and 3' terminal OH groups can be
phosphorylated or
substituted with amines or organic capping group moieties of from 1 to 20
carbon atoms.
Other hydroxyls can also be derivatized to standard protecting groups.
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Polynucleotides can also contain analogous forms of ribose or deoxyribose
sugars
that are generally known in the art, including, but not limited to, 2'-O-
methyl-, 2'-O-allyl,
2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, oc-anomeric sugars,
epimeric
sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose
sugars,
sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl
riboside.
As noted above, one or more phosphodiester linkages can be replaced by
alternative linking groups. These alternative linking groups include, but are
not limited to,
embodiments wherein phosphate is replaced by P{O)S ("thioate"), P(S)S
("dithioate"),
"(O)NRZ ("amidate"), P(O)R, P(O)OR', CO or CHZ ("formacetal"), in which each R
or R'
is independently H or substituted or unsubstituted alkyl (1-20 C) optionally
containing an
ether (-O-) linkage, aryl, alkenyl, cycloalky, cycloalkenyl or araldyl. Not
all linkages in a
polynucleotide need be identical.
Although conventional sugars and bases will be used in applying the method of
the
invention, substitution of analogous forms of sugars, purines and pyrimidines
can be
advantageous in designing a final product, as can alternative backbone
structures like a
polyamide backbone.
The term "recombinant" polynucleotide means a polynucleotide of genomic,
cDNA, semisynthetic, or synthetic origin which either does not occur in nature
or is linked
to another polynucleotide in a nonnatural arrangement.
An "isolated" polynucleotide or polypeptide is one that is substantially free
of the
materials with which it is associated in its native environment. By
substantially free is
meant at least 50%, preferably at least 70%, more preferably at least 80%, and
even more
preferably at least 90% free of these materials.
A "stable duplex" of polynucleotides, or a "stable complex" formed between any
two or more components in a biochemical reaction, refers to a duplex or
complex that is
sufficiently long-lasting to persist between the formation of the duplex or
complex and
subsequent detection, including any optional washing steps or other
manipulation that may
take place in the interim.
A "vector" refers to a recombinant DNA or RNA plasmid or virus that comprises
a
heterologous polynucleotide to be delivered into a target cell, either in
vitro or in vivo.
The heterologous polynucleotide may comprise a sequence of interest for
purposes of
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therapy, and may optionally be in the form of an expression cassette. As used
herein, a
vector need not be capable of replication in the ultimate target cell or
subject. The term
includes cloning vectors for the replication of a polynucleotide, and
expression vectors for
translation of a polynucleotide encoding sequence. Also included are viral
vectors, which
comprise a polynucleotide encapsidated or enveloped in a viral particle.
The terms "polypeptide", "peptide" and "protein" are used interchangeably
herein
to refer to polymers of amino acid residues of any length. The polymer may be
linear or
branched, it may comprise modified amino acids or amino acid analogs, and it
may be
interrupted by chemical moieties other than amino acids. The terms also
encompass an
amino acid polymer that has been modified naturally or by intervention; for
example,
disulfide bond formation, glycosylation, lipidation, acetylation,
phosphorylation, or any
other manipulation or modification, such as conjugation with a labeling or
bioactive
component. Unless stated or implied otherwise, the term 4B5 includes any
polypeptide
monomer or polymer with 4B5 immunologic specificity, including the intact
recombinant
1 S antibody, and smaller and larger functionally equivalent polypeptides.
A "fusion polypeptide" is a polypeptide comprising regions in a different
position
in the sequence than occurs in nature. The regions may normally exist in
separate proteins
and are brought together in the fusion polypeptide; they may normally exist in
the same
protein but are placed in a new arrangement in the fusion polypeptide; or they
may be
synthetically arranged. For instance, as described below, the invention
encompasses
recombinant proteins (and the polynucleotides encoding the proteins) that are
comprised of
a functional portion of 4B5 and a toxin. Methods of making these fusion
proteins are
known in the art and are described for instance in W093/07286.
A "functionally equivalent fragment" of a 4B5 polypeptide varies from the
native
sequence by any combination of additions, deletions, or substitutions while
preserving at
least one functional property of the fragment relevant to the context in which
it is being
used. A functionally equivalent fragment of a 4B5 polynucleotide either
encodes a
polypeptide that is functionally equivalent to 4B5 when produced by an
expression system,
or has similar hybridization specificity as a 4B5 polynucleotide when used in
a
hybridization assay. A functionally equivalent fragment of a 4B5 polypeptide
typically has
one or more of the following properties: ability to bind antibodies specific
for GD2;
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ability to generate antibodies specific at least one type of cancer cell in a
specific manner;
and an ability to elicit an immune response with a similar antigen specificity
as that
elicited by 4B5 or GD2.
A "cell Line" or "cell culture" denotes bacterial, plant, insect or higher
eukaryotic
cells grown or maintained in vitro. The descendants of a cell may not be
completely
identical (either morphologically, genotypically, or phenotypically) to the
parent cell. A
Mab may be produced by a hybridoma or other cell. Methods of making
hybridomas, both
marine and human, are known in the art. Particular methods of producing human
hybridomas are described and referenced throughout the specification.
A "host cell" denotes a prokaryotic or eukaryotic cell that has been
genetically
altered, or is capable of being genetically altered by administration of an
exogenous
polynucleotide, such as a recombinant plasmid or vector. When referring to
genetically
altered cells, the term refers both to the originally altered cell, and to the
progeny thereof.
"Heterologous" means derived from a genotypically distinct entity from the
rest of
the entity to which it is being compared. For example, a polynucleotide may be
placed by
genetic engineering techniques into a plasmid or vector derived from a
different source,
and is a heterologous polynucleotide. A promoter removed from its native
coding
sequence and operatively linked to a coding sequence other than the native
sequence is a
heterologous promoter.
A "signal peptide" or "leader sequence" is a short amino acid sequence that
directs
a newly synthesized protein through a cellular membrane, usually the
endoplasmic
reticulum in eukaryotic cells, and either the inner membrane or both inner and
outer
membranes of bacteria. Signal peptides are typically at the N terminal portion
of a
polypeptide and are typically removed enzymatically between biosynthesis and
secretion
of the polypeptide from the cell. The signal peptide is not present in the
secreted protein,
only during protein production.
A "biological sample" encompasses a variety of sample types, including blood
and
other liquid samples of biological origin, solid tissue samples such as a
biopsy specimens
or tissue cultures, or cells derived therefrom and the progeny thereof. The
definition also
includes samples that have been manipulated in any way after their
procurement, such as
by treatment with reagents, solubilization, or enrichment for certain
components, such as
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proteins or polynucleotides. The term encompasses various kinds of clinical
samples
obtained from any species, and also includes cells in culture, cell
supernatants, and cell
lysates. Particularly, for the purposes described herein, biological samples
comprise tumor
tissue or tissue thought to be tumorous and are obtained for instance by
surgical resection,
biopsy, aspiration or any method known in the art.
An "immunogen" refers to composition for human or animal use, which is
administered with the intention of conferring to the recipient a degree of
specific
immunologic reactivity against a particular antigen. The immunologic
reactivity may be
carried out by antibodies or cells (particularly B cells, plasma cells, T
helper cells, and
cytotoxic T lymphocytes, and their precursors) that are immunologically
reactive against
the target, or any combination thereof. For purposes of this invention, the
target is
primarily tumor-associated C antigen or a tumor-specific portion thereof. The
immunologic reactivity may be desired for experimental purposes, for treatment
of a
particular condition, for the elimination of a particular substance, or for
prophylaxis. An
1 S active immunogen is intended to elicit an immune response that persists in
the absence of
the vaccine components.
"Adjuvant" as used herein has several meanings, all of which will be clear
depending on the context in which the term is used. In the context of a
pharmaceutical
preparation, an adjuvant is a chemical or biological agent given in
combination with or
recombinantly fused to an antigen to enhance immunogenicity of the antigen. In
the
context of cancer diagnosis or management, adjuvant refers to a class of
cancer patients
with no clinically detectable tumor mass, but who are suspected of being at
risk of
recurrence.
When referring to a type of cancer that normally manifests as a solid tumor, a
"clinically detectable" tumor is one that is detectable on the basis of tumor
mass; i.e., by
such procedures as CAT scan, X-Ray, or palpation. Biochemical, histological or
immunologic findings alone may be insufficient to meet this definition.
As used herein, "treatment" refers to clinical intervention in an attempt to
alter the
natural course of the individual or cell being treated, and may be performed
either for
prophylaxis or during the course of clinical pathology. Desirable effects of
the treatment
include preventing occurrence or recurrence of disease, alleviation of
symptoms,
_. _ ___._____~. _ _ -__ _..____ _._.._._~__
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diminishment of any direct or indirect pathological consequences of the
disease,
preventing metastasis, decreasing the rate of disease progression,
amelioration or palliation
of the disease state, and remission or improved prognosis.
The "pathology" associated with a disease condition is any condition that
compromises the well-being, normal physiology, or quality of life of the
affected
individual. This may involve, but is not limited to, destructive invasion of
affected tissues
into previously unaffected areas, growth at the expense of normal tissue
function, irregular
or suppressed biological activity, aggravation or suppression of an
inflammatory or
immunologic response, increased susceptibility to other pathogenic organisms
or agents,
and undesirable clinical symptoms such as pain, fever, nausea, fatigue, mood
alterations,
and such other features as may be determined by an attending physician.
An "effective amount" is an amount sufficient to effect a beneficial or
desired
clinical result. An effective amount can be administered in one or more doses.
In terms of
treatment, an effective amount is amount that is sufficient to palliate,
ameliorate, stabilize,
i 5 reverse or slow the progression of the disease, or otherwise reduce the
pathological
consequences of the disease. In terms of an adjuvant, an effective amount is
one sufficient
to enhance the immune response to the immunogen. The effective amount is
generally
determined by the physician on a case-by-case basis and is within the skill of
one in the art.
Several factors are typically taken into account when determining an
appropriate dosage.
These factors include age, sex and weight of the patient, the condition being
treated, the
severity of the condition and the form of the antibody being administered. For
instance,
the concentration of scFv need not be as high as that of native antibodies in
order to be
therapeutically effective.
An "individual", "patient" or "subject" is a vertebrate, preferably a mammal,
more
preferably a human. Mammals include, but are not limited to, humans, farm
animals, sport
animals, and pets.
The practice of the present invention employs, unless otherwise indicated,
conventional techniques of molecular biology (including recombinant
techniques),
microbiology, cell biology, biochemistry and immunology, which are within the
skill of
the art. Such techniques are explained fully in the literature, such as,
"Molecular Cloning:
A Laboratory Manual", second edition (Sarnbrook et al., 1989);
"Oligonucleotide
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Synthesis" (M.J. Gait, ed., 1984); "Animal Cell Culture" (R.I. Freshney, ed.,
1987);
"Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental
Immunology" (D.M. Wei & C.C. Blackwell, eds.); "Gene Transfer Vectors for
Mammalian Cells" (J.M. Miller & M.P. Calos, eds., 1987); "Current Protocols in
S Molecular Biology" (F.M. Ausubel et al., eds., 1987); "PCR: The Poiymerase
Chain
Reaction", (Mullis et al., eds., 1994); "Current Protocols in Immunology"
(J.E. Coligan et
al., eds., 1991 ). These techniques are applicable to the production of the
polynucleotides
and polypeptides of the invention, and, as such, may be considered in making
and
practicing the invention. Particularly useful techniques for particular
embodiments will be
discussed in the sections that follow.
The invention also encompasses 4BS conjugated to a chemically functional
moiety.
Typically, the moiety is a label capable of producing a detectable signal.
These conjugated
4BS are useful, for example, in detection systems such as quantitation of
tumor burden,
and imaging of metastatic foci and tumor imaging. Such labels are known in the
art and
1 S include, but are not limited to, radioisotopes, enzymes, fluorescent
compounds,
chemiluminescent compounds, bioluminescent compounds substrate cofactors and
inhibitors. See, for examples of patents teaching the use of such labels, U.S.
Patent Nos.
3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and
4,366,241. The
moieties can be covalently linked to 4BS, recombinantly linked, or conjugated
to 4BS
through a secondary reagent, such as a second antibody, protein A, or a biotin-
avidin
complex.
Other functional moieties include signal peptides, agents that enhance
immunologic
reactivity, agents that facilitate coupling to a solid support, vaccine
carriers, bioresponse
modifiers, paramagnetic labels and drugs. Signal peptides are described above
and include
2S prokaryotic and eukaryotic forms. Agents that enhance immunologic
reactivity include,
but are not limited to, bacterial superantigens. Agents that facilitate
coupling to a solid
support include, but are not limited to, biotin or avidin. Immunogen carriers
include, but
are not limited to, any physiologically acceptable buffers. Bioresponse
modifiers include
cytokines, particularly tumor necrosis factor (TNF), interleukin-2,
interleukin-4,
granulocyte macrophage colony stimulating factor and y interferons.
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Suitable drug moieties include antineoplastic agents. These include, but are
not
limited to, radioisotopes, vinca alkaloids such as the vinblastine,
vincristine and vindesine
sulfates, adriamycin, bleomycin sulfate, carboplatin, cisplatin,
cyclophosphamide,
cytarabine, dacarbazine, dactinomycin, duanorubicin hydrochloride, doxorubicin
hydrochloride, etoposide, fluorouracil, lomustine, mechlororethamine
hydrochloride,
melphalan, mercaptopurine, methotrexate, mitomycin, mitotane, pentostatin,
pipobroman,
procarbaze hydrochloride, streptozotocin, taxol, thioguanine, and uracil
mustard.
Immunotoxins, including single chain molecules, can be produced by recombinant
means. Production of various immunotoxins is well-known in the art, and
methods can be
found, for example, in "Monoclonal Antibody-toxin Conjugates: Aiming the Magic
Bullet," Thorpe et al. (1982) Monoclonal Antibodies in Clinical Medicine,
Academic
Press, pp. 168-190; Vitatta (1987) Science 238:1098-1104; and Winter and
Milstein
( 1991 ) Nature 349:293-299. Suitable toxins include, but are not limited to,
ricin,
radionuclides, pokeweed antiviral protein, Pseudomonas exotoxin A, diphtheria
toxin, ricin
A chain, fungal toxins such as restrictocin and phospholipase enzymes. See,
generally,
"Chimeric Toxins," Olsnes and Pihl, Pharmac. Ther. 15:355-381 (1981); and
"Monoclonal Antibodies for Cancer Detection and Therapy," eds. Baldwin and
Byers, pp.
159-179, 224-266, Academic Press (1985).
The chemically functional moieties can be made recombinantly for instance by
creating a fusion gene encoding the antigen binding fragment and functional
regions from
other genes (e.g. enzymes). In the case of gene fusions, the two components
are present
within the same polypeptide gene. Alternatively, the 4B5 antigen binding
fragments can
be chemically bonded to the moiety by any of a variety of well known chemical
procedures. For example, when the moiety is a protein, the linkage may be by
way of
heterobifunctional cross linkers, e.g., SPDP, carbodiimide glutaraldehyde, or
the like. The
moieties may be covalently linked, or conjugated, through a secondary reagent,
such as a
second antibody, protein A, or a biotin-avidin complex. Paramagnetic moieties
and the
conjugation thereof to antibodies are well-known in the art. See, e.g.,
Miltenyi et al.
( 1990) Cytometry 11:231-23 8.
The 4B5 antibody of this invention can be prepared in several ways. It is most
conveniently obtained from cells engineered to express the GD2 mimic fragment
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containing SEQ ID NOS:1 and 4 or other polynucleotides encoding 4B5 binding
fragments. For example, the cells can be cultured in a suitable medium, and
spent medium
can be used as an antibody source. Optionally, matrix-coated channels or beads
and cell
co-cultures may be included to enhance growth of antibody-producing cells. For
the
production of large amounts of antibody, it is generally more convenient to
obtain an
ascites fluid. The method of raising ascites generally comprises injecting
hybridoma cells
into an immunologicaily naive histocompatible or immunotolerant mammal,
especially a
mouse. The mammal may be primed for ascites production by prior administration
of a
suitable composition; e.g., Pristane.
IO Alternatively, 4B5 can be chemically synthesized using sequence data and
other
information provided in this disclosure, in conjunction with standard methods
of protein
synthesis. A suitable method is the solid-phase Merrifield technique.
Automated peptide
synthesizers are commercially available, such as those manufactured by Applied
Biosystems, Inc. (Foster City, CA).
4B5 can also be obtained by employing routine recombinant methods such as
described in Sambrook et al. (1989). For instance, using the amino acid and
polynucleotide sequences provided herein and information provided herein, a
polynucleotide encoding either the 4B5 H or L chain can be cloned into a
suitable
expression vector (which contains control sequences for transcription, such as
a promoter).
The expression vector is in turn introduced into a host cell. The host cell is
grown under
suitable conditions such that the polynucleotide is transcribed and translated
into a protein.
H and L chains of 4B5 can be produced separately, and then combined by
disulfide bond
rearrangement. Alternatively, vectors with separate polynucleotides encoding
each chain
of 4B5, or a vector with a single polynucleotide encoding both chains as
separate
transcripts, can be transfected into a single host cell which can then produce
and assemble
the entire molecule. Preferably, the host cell is derived from a higher
eukaryote that can
provide the normal carbohydrate complement of the molecule. The 4B5 thus
produced can
be purified using standard techniques in the art. Polynucleotides encoding 4B5
for use in
the production of 4B5 can be produced synthetically or recombinantly from the
DNA
sequences provided herein.
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"Humanized" antibodies are antibodies in which at least part of the sequence
has
been altered from its initial form to render it more like human
immunoglobulins. In one
version, the H chain and L chain C regions are replaced with human sequence.
This is a
fusion polypeptide comprising a H11 V region and a heterologous immunoglobulin
C
region. In another version, the CDR regions comprise H 11 amino acid
sequences, while
the V framework regions have also been converted human sequences. See, for
example,
EP 0329400. In a third version, V regions are humanized by designing consensus
sequences of human and mouse V regions, and converting residues outside the
CDRs that
are different between the consensus sequences. The invention encompasses
humanized
Mabs.
In making humanized antibodies, the choice of framework residues can be
critical
in retaining high binding affinity. In principle, a framework sequence from
any HuAb can
serve as the template for CDR grafting; however, it has been demonstrated that
straight
CDR replacement into such a framework can lead to significant loss of binding
affinity to
the antigen. Glaser et al. ( 1992) J. Immunol. 149:2606; Tempest et al. (
1992)
Biotechnology 9:266; and Shalaby et al. (1992) J. Exp. Med. 17:217. The more
homologous a HuAb is to the original muAb, the less likely that the human
framework will
introduce distortions into the marine CDRs that could reduce affinity. Based
on a
sequence homology search against an antibody sequence database, the HuAb IC4
provides
good framework homology to muM4TS.22, although other highly homologous HuAbs
would be suitable as well, especially kappa L chains from human subgroup I or
H chains
from human subgroup III. Kabat et al. (I987). Various computer programs such
as
ENCAD (Levitt et al. (1983) J. Mol. Biol. 168:595) are available to predict
the ideal
sequence for the V region. The invention thus encompasses HuAbs with different
V
regions. It is within the skill of one in the art to determine suitable V
region sequences and
to optimize these sequences. Methods for obtaining antibodies with reduced
immunogenicity are also described in U.S. Patent No. 5,270,202 and EP 699,755.
Methods of antibody production and isolation are well known in the art. See,
for
example, Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring
Harbor
Laboratory, New York. The 4B5 antibody is a human immunoglobulin of the IgG
subclass, and may be isolated by any technique suitable for immunoglobulins of
this
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isotype. Purification methods may include salt precipitation (for example,
with
ammonium sulfate), ion exchange chromatography (for example, on a cationic or
anionic
exchange column run at neutral pH and eluted with step gradients of increasing
ionic
strength), gel filtration chromatography (including gel filtration HPLC), and
chromatography on affinity resins such as protein A, protein G,
hydroxyapatite, and anti-
immunoglobulin. 4B5 can also be purified on affinity columns comprising the
anti-GD2
antibodies. Preferably, 4B5 is purified using Protein-A-CL-SepharoseTM 4B
chromatography followed by chromatography on a DEAE-SepharoseTM 4B ion
exchange
column.
The invention also encompasses hybrid antibodies, in which one pair of H and L
chains is obtained from a first antibody, while the other pair of H and L
chains is obtained
from a different second antibody. For purposes of this invention, one pair of
L and H
chains is from 4B5. In one example, each L-H chain pair binds different
epitopes of the
4B5 antigen. Such hybrids can also be formed using humanized H or L chains.
Another 4B5 contemplated by this invention is an antibody in which the H or L
chain has been modified to provide additional properties. For instance, a
change in amino
acid sequence can result in reduced immunogenicity of the resultant
polypeptide. The
changes range from changing of one or more amino acids to the complete
redesign of a
region such as a C region domain. Typical changes include, but are not limited
to, those
related to complement fixation, interaction with membrane receptors, and other
effector
functions. Also encompassed by the invention are peptides in which various
immunoglobulin domains have been placed in an order other than that which
occurs in
nature.
If 4B5 is to be administered to an individual, it is preferably at least 80%
pure,
more preferably it is at least 90% pure, even more preferably it is at least
95% pure and
free of pyrogens and other contaminants. In this context, the percent purity
is calculated as
a weight percent of the total protein content of the preparation, and does not
include
constituents which are deliberately added to the composition after the 4B5 is
purified.
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Polynucleotides of the invention
The invention provides various polynucleotides encoding the antibody 4B5 or
fragments of 4B5, based on the polynucleotide sequences provided herein (SEQ
ID NOS:1
and 4) and the complimentary polynucleotides (SEQ ID NOS:2 and 6). Various
embodiments are described in this section, comprising a number of different
combinations
of the 4B5 H or L chain V region sequences. In general, a 4B5 polynucleotide
of this
invention encodes at least one feature that is unique to the 4B5 molecule (in
comparison
with other immunoglobulins). Preferably, this feature is related in some way
to an
immunologic reactivity of 4B5.
The invention encompasses polynucleotides encoding a portion of the 4B5 L
chain
V region, comprising at least about 70 consecutive nucleotides, preferably at
least about 80
consecutive nucleotides, more preferably at least about 100 consecutive
nucleotides, even
more preferably at least about 150 nucleotides of SEQ ID N0:4. The invention
also
encompasses a polynucleotide encoding a portion of the 4B5 L chain V region,
comprising
at least about 25 consecutive nucleotides, preferably at least about 30
consecutive
nucleotides, and even more preferably at least about 35 consecutive
nucleotides of the
CDR1 encoding sequence thereof. The invention also encompasses a
polynucleotide
encoding a portion of the 4B5 L chain V region, comprising at least about 20
consecutive
nucleotides, preferably at least about 25 consecutive nucleotides, and even
more preferably
at least about 35 consecutive nucleotides of the CDR2 or CDR3 encoding
sequence
thereof.
The invention also encompasses polynucleotides encoding a portion of the 4B5 H
chain V region, comprising at least about 70 consecutive nucleotides,
preferably at least
about 80 consecutive nucleotides, more preferably at least about 100
consecutive
nucleotides, even more preferably at least about 150 nucleotides of SEQ ID
NO:1. The
invention also encompasses a polynucleotide encoding a portion of the 4B5 L
chain V
region, comprising 15 consecutive nucleotides of the CDR1 encoding sequence
thereof.
The invention also encompasses a polynucleotide encoding a portion of the 4B5
L chain V
region, comprising at least about 20 consecutive nucleotides, preferably at
least about 25
consecutive nucleotides, and even more preferably at least about 35
consecutive
nucleotides of the CDR2 or CDR3 coding sequence thereof.
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The invention includes isolated 4B5 polynucleotides encoding a polypeptide
having immunalogic activity of 4B5, wherein the polypeptide encodes at least 5
amino
acids of a V L chain of 4B5 as depicted in SEQ ID NO:S. The invention also
includes
isolated 4B5 polynucleotides encoding a polypeptide having immunologic
activity of 4B5,
wherein the polynucleotide encodes at least 5 amino acids of a V H chain of
4B5 as
depicted in SEQ ID N0:3. The polynucleotide sequence can be similar to those
depicted
in SEQ ID NO:1 or SEQ ID N0:4 with changes designed to optimize codon usage,
stability, facilitate cloning, or any other purpose. It is within the skill of
one in the art,
given the amino acid sequence in SEQ ID N0:3 or SEQ ID N0:6, to design such
polynucleotides. Preferred polynucleotides encode at least five amino acids of
a 4B5
CDR.
The invention also encompasses polynucleotides encoding for functionally
equivalent variants and derivatives of 4B5 and functionally equivalent
fragments thereof
which may enhance, decrease or not significantly affect properties of the
polypeptides
1 S encoded thereby. These functionally equivalent variants, derivatives, and
fragments
display the ability to specifically recognize C antigen. For instance, changes
in a DNA
sequence that do not change the encoded amino acid sequence, as well as those
that result
in conservative substitutions of amino acid residues, one or a few amino acid
deletions or
additions, and substitution of amino acid residues by amino acid analogs are
those which
will not significantly affect properties of the encoded polypeptide.
Conservative amino
acid substitutions are glycine/alanine; valine/isoleucine/leucine;
asparagine/glutamine;
aspartic acid/glutamic acid; serine/threonine/methionine; lysine/arginine; and
phenylalanine/tyrosine/tryptophan.
The polynucleotides of the invention can comprise additional sequences, such
as
additional encoding sequences within the same transcription unit, controlling
elements
such as promoters, ribosome binding sites, and polyadenylation sites,
additional
transcription units under control of the same or a different promoter,
sequences that permit
cloning, expression, and transformation of a host cell, and any such construct
as may be
desirable to provide embodiments of this invention.
The invention encompasses a polynucleotide of at least about 15 consecutive
nucleotides, preferably at least about 20 nucleotides, more preferably at
least about 25
28
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consecutive nucleotides, more preferably at least about 35 consecutive
nucleotides, more
preferably at least about 50 consecutive nucleotides, even more preferably at
least about 75
nucleotides, still more preferably at least about 100 nucleotides, still more
preferably at
least about 200 nucleotides, and even more preferably at least about 300
nucleotides that
forms a stable hybrid with a polynucleotide encoding the L chain or H chain V
region of
4B5, but not with other immunoglobulin encoding regions known at the time of
filing of
this application. Any set of conditions may be used for this test, as long as
at least one set
of conditions exist wherein the test polynucleotide demonstrates the required
specificity.
Preferably, the 4B5 encoding sequences to which the test polynucleotide binds
are those
shown in SEQ ID NOS:2 and 5. Since the known immunoglobulin sequences fall
into a
hierarchy of similarity with that of 4B5, the test may be performed by
comparing the
hybridization of the test polynucleotide with the 4B5 sequence with the
hybridization with
the most closely related sequences. Preferred is a panel of about 10 of the
sequences most
closely related to SEQ ID NO: l or 4.
Hybridization reactions can be performed under conditions of different
"stringency". Conditions that increase stringency of a hybridization reaction
are well
known. See, for example, Sambrook and Maniatis. Examples of relevant
conditions
include (in order of increasing stringency): incubation temperatures of
25°C, 37°C, 50°C
and 68°C; buffer concentrations of 10 x SSC, 6 x SSC, 1 x SSC, 0.1 x
SSC (where SSC is
0.15 M NaCI and 15 mM citrate buffer) and their equivalent using other buffer
systems;
formamide concentrations of 0%, 25%, 50%, and 75%; incubation times from S
minutes to
24 hours; 1, 2, or more washing steps; wash incubation times of 1, 2, or 15
minutes; and
wash solutions of 6 x SSC, 1 x SSC, 0.1 x SSC, or deionized water.
Useful 4B5 polynucleotides encoding fragments of 4B5 can be identified by
generating polynucleotide fragments (based on SEQ ID NO:1 or SEQ ID N0:4, for
example) and testing the polypeptides encoded thereby for a function of
interest.
Alternatively, the polypeptide fragment encoded by a particular polynucleotide
can be
prepared and tested for a function of interest. Alternatively, given a 4B5
polypeptide with
desirable properties, polynucleotides can be designed that encode the
polypeptide.
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Included in all these embodiments are polynucleotides with encoding regions
for
4B5 polymers, fusion proteins, humanized immunoglobulins, single-chain V
regions, and
other particular polypeptides of interest. These polypeptides are described
above.
The invention also provides polynucleotides covalently linked with a
detectable
label. Such polynucleotides are useful, for example, as probes for detection
of related
nucleotide sequences.
The polynucleotides of this invention can be obtained using chemical
synthesis,
recombinant cloning methods, PCR, or any combination thereof. Methods of
chemical
polynucleotide synthesis are well known in the art and need not be described
in detail
herein. One of skill in the art can use the sequence data provided herein to
obtain a desired
polynucleotide by employing a DNA synthesizer or ordering from a commercial
service.
Polynucleotides comprising a desired sequence can be inserted into a suitable
vector, and the vector in turn can be introduced into a suitable host cell for
replication and
amplification. Polynucleotides can be introduced into host cells by any means
known in
the art. Cells are transformed by introducing an exogenous polynucleotide by
direct
uptake, endocytosis, transfection, f mating or electroporation. Once
introduced, the
exogenous polynucleotide can be maintained within the cell as a non-integrated
vector
(such as a plasmid) or integrated into the host cell genome. Amplified DNA can
be
isolated from the host cell by standard methods. See, e.g., Sambrook et al.
(1989). RNA
can also be obtained from transformed host cell, or it can be obtained
directly from the
DNA by using a DNA-dependent RNA polymerase.
The present invention further encompasses a variety of vectors comprising a
4B5
polynucleotide. These vectors can be used for expression of recombinant
polypeptides are
also a source of 4B5 polynucleotides. Cloning vectors can be used to obtain
replicate
copies of the 4B5 polynucleotides they contain, or as a means of storing the
polynucleotides in a depository for future recovery. Expression vectors (and
host cells
containing these expression vectors) can be used to obtain polypeptides
produced from the
polynucleotides they contain. They can also be used where it is desirable to
express 4B5 in
an individual and thus have intact cells capable of synthesizing the
polypeptide, such as in
gene therapy. Suitable cloning and expression vectors include any known in the
art, e.g.,
those for use in bacterial, mammalian, yeast and insect expression systems.
Specific
CA 02295375 1999-12-24
WO 99/02545 PCT/IB98/01046
vectors and suitable host cells are known in the art and are not described in
detail herein.
See e.g. Gacesa and Ramji, Vectors, John Wiley & Sons (1994).
Cloning and expression vectors typically contain a selectable.marker (for
example,
a gene encoding a protein necessary for the survival or growth of a host cell
transformed
with the vector), although such a marker gene can be carried on another
polynucleotide
sequence co-introduced into the host cell. Only those host cells into which a
selectable
gene has been introduced will grow under selective conditions. Typical
selection genes
either: (a) confer resistance to antibiotics or other toxins, e.g.,
ampicillin, neomycin,
methotrexate; (b) complement auxotrophic deficiencies; or (c) supply critical
nutrients not
available from complex media. The choice of the proper marker gene will depend
on the
host cell, and appropriate genes for different hosts are known in the art.
Vectors also
typically contain a replication system recognized by the host.
Suitable cloning vectors can be constructed according to standard techniques,
or
selected from a large number of cloning vectors available in the art. While
the cloning
I S vector selected may vary according to the host cell intended to be used,
useful cloning
vectors will generally have the ability to self replicate, may possess a
single target for a
particular restriction endonuclease, or may carry marker genes. Suitable
examples include
plasmids and bacterial viruses, e.g., pUClB, mpl8, mpl9, pBR322, pMB9, ColEl,
pCRl,
RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28. These and other
cloning
vectors are available from commercial vendors such as BioRad, Stratagene, and
Invitrogen.
Expression vectors generally are replicable polynucleotide constructs that
contain a
polynucleotide encoding a 4B5 polypeptide of interest. The polynucleotide
encoding 4B5
polypeptide is operatively linked to suitable transcriptional controlling
elements, such as
promoters, enhancers and terminators. For expression (i.e., translation), one
or more
translational controlling elements are also usually required, such as ribosome
binding sites,
translation initiation sites, and stop codons. These controlling elements
(transcriptional
and translational) can be derived from the 4B5 gene, or heterologous (i.e.,
derived from
other genes or other organisms). A polynucleotide sequence encoding a signal
peptide can
also be included to allow a 4B5 polypeptide to cross or lodge in cell
membranes or be
secreted from the cell. A number of expression vectors suitable for expression
in
eukaryotic cells including yeast, avian, and mammalian cells are known in the
art. One
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example of an expression vector is pcDNA3 (Invitrogen, San Diego, CA), in
which
transcription is driven by the cytomegalovirus (CMV) early promoter/enhancer.
This
vector also contains recognition sites for multiple restriction enzymes for
insertion of an
4B5 polynucleotide of interest. Another example of an expression vector
(system) is the
baculovirus/insect system.
Also encompassed by the invention are expression systems suitable for use in
antibody-targeted gene therapy comprising a 4B5 polynucleotide. Suitable
systems are
described for instance by Brown et al. (1994) Virol. 198:47788; and Miyamura
et al.
(1994) Proc. Natl. Acad. Sci. USA 91:8507-8511.
The vectors containing the polynucleotides of interest can be introduced into
the
host cell by any of a number of appropriate means, including electroporation,
transfection
employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-
dextran, or
other substances; microprojectile bombardment; lipofection; and infection
(where the
vector is an infectious agent, such as vaccinia virus, which is discussed
below). The choice
of introducing vectors or 4B5 polynucleotides will often depend on features of
the host
cell.
Once introduced into a suitable host cell, expression of a 4B5 polypeptide can
be
determined using any assay known in the art. For example, presence of 4B5
polypeptide
can be detected by RIA or ELISA of the culture supernatant (if the 4B5
polypeptide is
secreted) or cell lysates.
A particularly useful expression vector for 4B5 polynucleotides is a vaccinia
virus
comprised of a 4B5 polynucleotide sequence, which can also be used in vaccine
'
preparations. Moss (1991) Science 252:1662-1667. To introduce polynucleotide
sequences encoding 4B5 polypeptide, including 4B5 polypeptide fragments, into
vaccinia,
the polynucleotide sequence of interest is first inserted into a plasmid
containing a vaccinia
virus promoter with flanking sequences homologous to vaccinia DNA not required
for
replication. Plasmid-containing cells are then infected with vaccinia, which
leads to a low
level of homologous recombination between plasmid and virus, with resultant
transfer of
the vaccinia promoter and 4B5 polypeptide-encoding polynucleotide sequence
into the
vaccinia virus genome. Typically, the 4B5 polynucleotide is inserted into the
viral TK
(thymidine kinase) gene. Insertion into the TK site attenuates the virus more
than 10,000
32
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fold compared to wild type. Flexner et al. (1980) Vaccine 88 (Cold Spring
Harbor
Laboratory), pp. 179-184. Recombinant virus is identified by the TK-
phenotype.
Preferably, expression of the 4B5 polynucleotide is under the control of the
vaccinia
early/late promoter (7.5 K), whereby the resultant 4B5 polypeptides can be
expressed in
infected cells throughout the life cycle of the virus. However, other
promoters known in
the art can be used, such as pH6, or synthetic promoters. Expression of the
4B5
polypeptide occurs in cells infected with the recombinant vaccinia or
individuals
immunized with the live recombinant vaccinia virus. Any one of several strains
of
vaccinia can be used, including, but not limited to, WR, ALVAC, and NYVAC.
A vector of this invention can contain one or more polynucleotides encoding a
4B5
polypeptide. It can also contain polynucleotide sequences encoding other
polypeptides
that enhance, facilitate, or modulate the desired result, such as lymphokines,
including, but
not limited to, IL-2, IL-4, GM-CSF, TNF-a, and IFN-y. A preferred lymphokine
is GM-
CSF. Preferred GM-CSF constructs are those which have been deleted for the AU-
rich
elements from the 3' untranslated regions and sequences in the S' untranslated
region that
are capable of forming a hairpin loop. Also embodied in this invention are
vaccinia
vectors encoding for recombinant 4B5 variants, such as scFvs, chimeras, and
polymers.
Other embodiments of this invention are host cells transformed with 4B5
polynucleotides and vectors comprising 4B5 polynucleotide sequences, as
described
above. Both prokaryotic and eukaryotic host cells may be used. Prokaryotic
hosts include
bacterial cells, for example E. coli and Mycobacteria. Among eukaryotic hosts
are yeast,
insect, avian, plant and mammalian cells. Host systems are known in the art
and need not
be described in detail herein. Examples of mammalian host cells include CHO
and NSO,
obtainable from the European Collection of Cell Cultures (England).
Transfection of NSO
cells with a plasmid, for example, which is driven by a CMV promoter, followed
by
amplification of this plasmid in using glutamine synthetase provides a useful
system for
protein production. Cockett et al. (1990) Bio. Technology 8:662-667.
The host cells of this invention can be used, inter alia, as repositories of
4B5
polynucleotides, or as vehicles for production of 4B5 polynucleotides and
polypeptides.
They may also be used as vehicles for in vivo expression of 4B5 polypeptides.
The 4B5
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polynucleotides of this invention can be used in expression systems to produce
4B5
polypeptides, intact 4B5, or recombinant forms of 4B5, such as are described
below.
The polynucleotides of this invention have several uses. They are useful, for
example, in expression systems for the production of 4B5. They are also useful
as
hybridization probes to assay for the presence of 4B5 polynucleotide or
related sequences
in a sample using methods well known to those in the art. Further, the
polynucleotides are
also useful as primers to effect amplification of desired polynucleotides. The
polynucleotides of this invention are also useful in pharmaceutical
compositions including
vaccines and for gene therapy.
The polynucleotides can also be used as hybridization probes for detection of
4B5
encoding sequences. Suitable samples include cells transformed ex vivo for use
in gene
therapy. In one illustration, DNA or RNA is extracted from a sample, and
optionally run
on a gel and/or digested with restriction endonucleases. The processed sample
polynucleotide is typically transferred to a medium suitable for washing. The
sample
1 S polynucleotide is then contacted with the 4B5 polynucleotide probe under
conditions that
permit a stable duplex to form if the sample contains a matching 4B5 sequence.
Any
stable duplexes formed are detected by any suitable means. For example, the
4B5
polynucleotide probe can be supplied in labeled form, and label remaining with
the sample
after washing will directly reflect the amount of stable duplex formed. In a
second
illustration, hybridization is performed in situ. A suitably prepared tissue
sample is
overlaid with a labeled probe to indicate the location 4B5 encoding sequences.
A short 4B5 polynucleotide can also be used as a primer for a PCR reaction,
particularly to amplify a longer sequence comprising a region hybridizing with
the primer.
This can be conducted preparatively, in order to produce polynucleotide for
further genetic
manipulation. It can also be conducted analytically, to determine whether a
4B5 encoding
polynucleotide is present, for example, in a sample of diagnostic interest.
Another use of the polynucleotides is in vaccines and gene therapy. The
general
principle is to administer the polynucleotide so that it either promotes or
attenuates the
expression of the polypeptide encoded therein. Thus, the present invention
includes
methods of inducing an immune response and methods of treatment comprising
administration of an effective amount 4B5 polynucleotides to an individual. In
these
34
_.. ........_._.. T_---__-..__._.~ .__....____..... e.. ........
__..........__._.._..__........_..... ._.-...._.._...
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methods, a 4B5 polynucleotide encoding a 4B5 polypeptide is administered to an
individual, either directly or via cells transfected with the 4B5
polynucleotide. Preferably,
the 4B5 polynucleotide is in the form of a circular plasmid, preferably in a
supercoiled
configuration. Preferably, the 4B5 polynucleotide is replicated inside a cell.
Thus, the
4B5 polynucleotide is operatively linked to a suitable promoter, such as a
heterologous
promoter that is intrinsically active in cells of the target tissue type.
Preferably, once in
cell nuclei, plasmids persist as circular non-replicating episomal molecules.
In vitro
mutation can be carried out with plasmid constructs to encode, for example,
molecules
with greater immunogenicity.
To determine whether plasmids containing 4B5 polynucleotides are capable of
4B5
expression in eukaryotic cells, cells such as COS-7, CHO, or HeLa can be
transfected with
the plasmids. Expression of 4B5 is then determined by immunoassay; for
example, by
Western blot. Smaller 4B5 polypeptides can be detected, for example, by
constructing the
plasmid so that the resultant 4B5 polypeptide is fused with a tag, such as a
target epitope or
enzyme label. Further characterization of the expressed 4B5 polypeptide can be
achieved
by purifying the peptide and then conducting one of the functional assays
described herein.
In one mode of gene therapy, the polynucleotides of this invention are used
for
genetically altering cells ex vivo. In this strategy, cells removed from a
donor or obtained
from a cell line are transfected or transduced with vectors encoding a 4B5
polypeptide, and
then administered to a recipient. Suitable cells for transfection include
peripheral blood
mononuclear cells.
In another mode of gene therapy, the polynucleotides of this invention are
used for
genetically altering cells in vivo. The purpose includes, but is not limited
to, treating
various types of cancer.
Polypeptides of tl:e invention
The invention also encompasses poiypeptide fragments of 4B5 containing at
least a
portion of a V region of 4B5. Preferred fragments are those with the
immunogenic activity
of 4B5. Also preferred are fragments which comprise amino acid sequences
substantially
different from other immunoglobuiins, and fragments comprising a CDR. In one
embodiment, the invention includes a polypeptide fragment of the 4B5 H chain V
region,
CA 02295375 1999-12-24
WO 99/02545 PCT/IB98/01046
comprising at least 25 consecutive amino acids, more preferably 30 consecutive
amino
acids of SEQ ID N0:3, or 5 consecutive amino acids of the CDR1 thereof, or at
least 7
consecutive amino acids, preferably at least 9 consecutive amino acids of the
CDR2 or
CDR3 thereof. The invention also includes a polypeptide fragment of the 4B5 L
chain V
region, comprising at least 25 consecutive amino acids, more preferably 30
consecutive
amino acids of SEQ ID N0:6, or 7 consecutive amino acids of the CDR2 thereof,
or at
least 8 consecutive amino acids, preferably 10 consecutive amino acids of the
CDR1 or
CDR3 thereof.
The size of the 4B5 polypeptides can be only the minimum size required to
provide
a desired function. The polypeptides can optionally comprise additional
sequence, either
native to 4B5, or from a heterologous source, as desired. 4B5 peptides can
contain only 5
consecutive amino acids from a 4B5 V region. Polypeptides comprising 7 amino
acids,
more preferably about 10 amino acids, mare preferably about 15 amino acids,
more
preferably about 25 amino acids, more preferably about 50 amino acids, more
preferably
about 75 amino acids from the 4B5 L or H chain V region are also included.
Even more
preferred are polypeptides comprising the entire 4B5 L or H chain V region.
Preferably
the polypeptides are derived from 4B5.
The invention includes modified 4B5 polypeptides which are functionally
equivalent to 4B5, or have altered but measurable 4B5 immunogenic activity.
Modified
polypeptides with improved 4B5 immunogenic activity are preferred. Examples of
modified polypeptides include those with conservative substitutions of amino
acid
residues, and one or more deletions or additions of amino acids which do not
significantly
deleteriously alter the immunologic activity.
One example of this is 4B5 polypeptides comprising one or more amino acid
substitution in comparison with the prototype 4B5 sequence. Substitutions can
range from
changing or modifying one or more amino acid residues to complete redesign of
a region.
such as the V region. Amino acid substitutions, if present, are preferably
conservative
substitutions that do not deleteriously affect folding or functional
properties of the peptide.
Groups of functionally related amino acids within which conservative
substitutions can be
made are glycine/alanine; valine/isoleucine/leucine; asparagine/glutamine;
aspartic
acid/glutamic acid; serine/threonine/methionine; lysine/arginine; and
36
-__ ~_____..._ __ _.___.. _ __. _._.
CA 02295375 1999-12-24
WO 99102545 PCT/IB98/01046
phenylalanine/tryosine/tryptophan. Polypeptides of this invention can be in
glycosylated
or unglycosylated form, can be modified post-translationally (e.g.,
acetylation, and
phosphorylation) or can be modified synthetically (e.g., the attachment of a
labeling
group).
4B5 polypeptide derivatives comprising both a 4B5 L chain and a 4B5 H chain
can
be formed as separate L and H chains and then assembled, or assembled in situ
by an
expression system for both chains. Such expression systems can be created by
transfecting
a suitable cell with a plasmid comprising separate transcribable regions for
the L and H
chain, or by co-transfecting the same cell with plasmids for each chain. In a
third method,
a suitable plasmid with a H chain encoding region is transfected into a H
chain loss mutant.
H chain loss mutants can be obtained by treating approximately 2 x 10~ 4B5
producing cells with fluorescein-labeled rabbit anti-mouse IgG (H chain
specific, DAKO
Corporation, Carpinteria, CA) according to the supplier's instructions. The
stained and
unstained cell populations are analyzed in a fluorescence-activated cell
sorter. The
1 S unstained cells are collected in a sterilized tube and placed in 96-well
plates with 1
cell/well by limiting dilution. The culture supernatants are then assayed by
ELISA using
goat anti-mouse IgG (H chain specific) and goat anti-mouse kappa. The clones
with
kappa-positive and IgG-negative phenotype are subcloned at least 3 times to
obtain stable
4B5~-"~ mutants. mRNA from putative H chain loss mutant 4B5~-H' clones can be
isolated
and the sequence of the L chain V region cDNA determined. Reverse PCR of the
mRNA
for the 4B5 VH is performed with 2 sets of 5'- and 3'- primers, used for
cloning of 4B5~-"~
cDNA (Example 7). A H chain loss mutant yields no detectable DNA band.
Transfection
of the cells proceeds with a suitable H chain plasmid.
Another 4B5 derivative encompassed by this invention is an antibody in which
the
4B5 H or L chain has been modified to provide additional properties. For
instance, a
change in amino acid sequence can result in greater immunogenicity of the
resultant
polypeptide. The changes range from changing of one or more amino acids to the
complete redesign of a region such as a 4B5 region domain. Changes
contemplated affect
complement fixation, interaction with membrane receptors, and other effector
functions.
Also encompassed by the invention are proteins in which various immunoglobulin
domains have been placed in an order other than that which occurs in nature.
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WO 99/02545 PCT/IB98/01046
The invention also encompasses single chain V region fragments ("scFv") of
4B5.
Single chain V region fragments are made by linking L and/or H chain V regions
by using
a short linking peptide. Bird et al. ( 1988) Science 242:423-426. Any peptide
having
sufficient flexibility and length can be used as a linker in a scFv. Usually
the linker is
selected to have little to no immunogenicity. An example of a linking peptide
is
(GGGGS)~, which bridges approximately 3.5 nm between the carboxy terminus of
one V
region and the amino terminus of another V region. Other linker sequences can
also be
used, and can provide additional functions, such as a means for attaching a
drug or a solid
support.
All or any portion of the H or L chain can be used in any combination.
Typically,
the entire V regions are included in the scFv. For instance, the L chain V
region can be
linked to the H chain V region. Alternatively, a portion of the L chain V
region can be
linked to the H chain V region, or a portion thereof. Also contemplated are
scFvs in which
the H chain V region is from 4B5, and the L chain V region is from another
immunoglobulin. It is also possible to construct a biphasic, scFv in which one
component
is a 4B5 polypeptide and another component is a different polypeptide, such as
a T cell
epitope.
The scFvs can be assembled in any order, for example, V,,-{linker--VL or
VL-(linker)-VH. Tandem scFvs can also be made, such as
(X}-(linker--(X}--(linker-(X), in which X are 4B5 polypeptides, or
combinations of
4B5 polypeptides with other polypeptides. In another embodiment, single chain
antibody
polypeptides have no linker polypeptide, or just a short, inflexible linker.
Exemplary
configurations include VL V,., and V,~VL. The linkage is too short to permit
interaction
between VL and VH within the chain, and the chains form homodimers with a
VL/VH
antigen binding site at each end. Such molecules are referred to in the art as
"diabodies".
ScFvs can be produced either recombinantly or synthetically. For synthetic
production of scFv, an automated synthesizer can be used. For recombinant
production of
scFv, a suitable plasmid containing a polynucleotide that encodes the scFv can
be
introduced into a suitable host cell, either eukaryotic, such as yeast, plant,
insect or
mammalian cells, or prokaryotic, such as E. coli, and the protein expressed by
the
polynucleotide can be isolated using standard protein purification techniques.
38
_ __. _ __._.__.~..~._~___.. ._ . ._ __ _ ___ ~__.. T
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WO 99/02545 PCT/IB98/01046
A particularly useful system for the production of scFvs is plasmid pET-22b(+)
(Novagen, Madison, WI) in E. toll. pET-22b(+) contains a nickel ion binding
domain
consisting of 6 sequential histidine residues, which allows the expressed
protein to be
purified on a suitable affinity resin. Another example of a suitable vector is
pcDNA3
(Invitrogen, San Diego, CA), described above.
Expression conditions should ensure that the scFv assumes functional and,
preferably, optimal tertiary structure. Depending on the plasmid used
(especially the
activity of the promoter) and the host cell, it may be necessary to modulate
the rate of
production. For instance, use of a weaker promoter, or expression at lower
temperatures,
may be necessary to optimize production of properly folded scFv in prokaryotic
systems;
or, it may be preferably to express scFv in eukaryotic cells.
Preferred scFv comprise at least 10 consecutive amino acids of SEQ ID N0:3 and
at least 10 consecutive amino acids of SEQ ID N0:6, especially wherein the
amino acids
of SEQ ID N0:3 and the amino acids of SEQ ID N0:6 are joined by a linker
polypeptide
of 5 to 20 amino acids, or comprising the L chain V region and the H chain V
region of
4B5.
The invention also encompasses polymeric forms of 4B5 polypeptides, containing
a plurality of 4B5 polypeptides. One embodiment is a linear polymer of 4B5
polypeptides,
optionally conjugated to carrier. These linear polymers can comprise multiple
copies of a
single 4B5 polypeptide, or combinations of different 4B5 polypeptides, and can
have
tandem 4B~ polypeptides, or 4B5 polypeptides separated by other amino acid
sequences.
Another embodiment is 4B5 multiple antigen peptides (MAPS). MAPS have a small
immunologically inert core having radially branching lysine dendrites, onto
which a
number of 4B5 polypeptides are covalently attached. See for instance, Posnett
et al. ( 1988)
J. Biol. Chem. 263:1719-1725; and Tam (1989) Meth. Enz. 168:7-15. The result
is a large
macromolecule having a high molar ratio of 4B5 polypeptides to core. MAPs are
efficient
immunogens and useful antigens for immunoassays. The core for creating an 4B5
MAP
can be made by standard peptide synthesis techniques, or obtained
commercially, e.g.,
from Quality Controlled Biochemicals, Inc., Hopkinton, MA. A typical core
matrix is
made up of three levels of lysine and eight amino acids.
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WO 99/02545 PCT/IB98/01046
When using 4B5 polypeptides as immunogens, preferably the polypeptides are
delivered in conjunction with a carrier. Any carrier can be used which is not
harmful to the
host. Suitable carriers are typically large, slowly metabolized macromolecules
such as
proteins; polysaccharides (such as latex functionalized Sepharose, agarose,
cellulose,
cellulose beads and the like); polymeric amino acids (such as polyglutamic
acid,
polylysine, and the like); amino acid copolymers; and inactive virus particles
or attenuated
bacteria, such as Salmonella. Especially useful carrier proteins are serum
albumins,
keyhole limpet hemacyanin (KLH), certain Ig molecules, thyroglobulin,
ovalbumin, and
tetanus toxoid. KLH is especially preferred.
4B5 polypeptides of the invention can be identified in a number of ways. For
example, the V regions of the L and H chains can be screened by preparing a
series of short
polypeptides that together span the entire V region amino acid sequence and
determining
whether they are recognized specifically by antibodies specific for GD2. Using
a series of
polypeptides of 20 or 50 amino acids in length, each 4B5 V region can be
surveyed for
useful functional properties. It is also possible to carry out a computer
analysis of a protein
sequence to identify potentially interesting. polypeptides, such as those that
bear the shape
of GD2, or those involved in idiotype-anti-idiotype contact.
The invention further encompasses various adaptations of 4B5 described in this
section combined in various fashions to yield other 4B5 polypeptides with
desirable
properties. For instance, 4B5 polypeptides with modified amino acid residues
can be
comprised in a MAP. In another example, a 4B5 scFv is fused to a cytokine,
such as IL-2.
All such combinations are contemplated in this invention.
The polypeptides of this invention can be made by any suitable procedure,
including proteolysis of the 4B5 antibody, by recombinant methods or by
chemical
synthesis. These methods are known in the art and need not be described in
detail herein.
Examples of proteolytic enzymes include, but are not limited to, trypsin,
chymotrypsin,
pepsin, papain, V8 protease, subtilisin, plasmin, and thrombin. Intact 4B5 can
be
incubated with one or more proteinases simultaneously or sequentially.
Alternatively, or
in addition, intact antibody can be treated with disulfide reducing agents.
Peptides can
then be separated from each other by techniques known in the art including,
but not limited
to, gel filtration chromatography, gel electrophoresis, and reverse-phase
HPLC.
_ _._. _._.____T_ ___.___.. _.__.. .__._... _..._._.___._-~__-..-...___ _____
_~....
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WO 99/02545 PCT/IB98/01046
4B5 polypeptides can also be made by expression from a polynucleotide encoding
the peptide according to the information provided elsewhere in this
application, in a
suitable expression system. Typically, polynucleotides encoding a 4B5
polypeptide are
ligated into an expression vector under control of a suitable promoter and
used to
genetically alter the intended host cell. Both eukaryotic and prokaryotic host
systems can
be used. The polypeptide is then isolated from lysed cells or from the culture
medium and
purified to the extent needed for its intended use. Examples of prokaryotic
host cells
appropriate for use with this invention include E. coli. Examples of
eukaryotic host cells
include avian, insect, plant, and animal cells including, but not limited to,
COS7, HeLa,
and CHO cells.
In certain applications, such as when a 4B5 polypeptide is expressed in a
suitable
storage medium such as a plant seed, the 4B5 polypeptide can be used without
purification.
Fiedler et al. (1995) Biotechnology 13:1090-1093. For most applications, it is
generally
preferable that the polypeptide is at least partially purified from other
cellular constituents.
Preferably, the polypeptide is at least about 50% pure as a weight percent of
total protein.
More preferably, the protein is at least about 50-75% pure. For clinical use,
the
polypeptide is preferably at least about 80% pure.
4B5 polypeptides can be characterized in several ways. For instance, a 4B5
polypeptide may be tested for its ability to bind specifically to antibodies
specific for GD2,
or for its ability to specifically inhibit the binding between cancer cells
and antibodies
specific for GD2. A 4B5 polypeptide can also react with anti-CDR3
polypeptides. 4B5
polypeptides can also be tested for their ability to palliate or ameliorate
neoplastic disease,
such as carcinomas. It is understood that only one of these properties need be
present in
order for a polypeptide to come within the scope of this invention, although
preferably
more than one of these properties is present.
The ability of a 4B5 polypeptide to bind specific antibodies can be tested by
immunoassay. Any form of direct binding assay is suitable. In one such assay,
the
antibody is labeled. Suitable labels include radioisotopes such as'ZSI,
enzymes such as
peroxidase, fluorescent labels such as fluorescein, and chemiluminescent
labels. Typically,
the other binding partner is insolubilized (for example, by coating onto a
microtiter plate)
to facilitate washing. After combining the labeled component with the
insolubilized
41
CA 02295375 1999-12-24
WO 99/02545 PCT/IB98/01046
component, the solid phase is washed and the amount of bound label is
determined.
Another such assay is a sandwich assay, in which the putative 4B5 polypeptide
is captured
by a first anti-immunoglobulin on a solid phase and developed with 4B5
antibody. In
either of these examples, the extent of binding of 4B5 is directly related to
the amount of
label bound to the solid phase.
To conduct the inhibition assays, the putative 4B5 polypeptide is titered for
its
ability to decrease the binding of antibodies specific for GD2 to GD2.
Typically, the anti-
GD2 antibodies are labeled, while the GD2 is typically insolubilized in order
to facilitate
washing. The putative 4B5 polypeptide is typically mixed with the anti-GD2
antibodies
and the mixture is combined with the solid phase. Polypeptides with the
characteristics of
4B5 will proportionately decrease the amount of label attached to the solid
phase,
compared with control polypeptides. This test may be more sensitive than
measuring
direct binding. because lower affinity interaction between 4B5 and anti-GD2
antibodies
may be too weak to form a stable bond, but adequate to interfere with the
binding of
another ligand-receptor pair when present at sufficient concentration.
The invention also encompasses methods of detecting antibodies specific for
GD2
in a biological sample. The methods include obtaining a biological sample,
contacting the
sample with 4B5 under conditions that allow antibody antigen binding and
detecting
binding, if any, of 4B5 with the anti-GD2 antibodies.
When using intact murine 4B5, it is generally preferable to deplete the sample
of
any anti-mouse immunoglobulin activity that may be present. Anti-mouse
immunoglobulin antibody can be removed from a sample, for example, by
precipitation
with normal mouse IgG or adsorption with a mouse Ig adsorbent. Binding of anti-
mouse
immunoglobulin antibody, particularly that specific for the Fc region, can be
minimized by
judicious choice of the reagents of the assay. F(ab')2 or Fab fragments of
murine 4B5 and
other reagents such as humanized 4B~ or 4B5, with fewer mouse determinants are
appropriate.
After the sample is suitably prepared, it is mixed with a excess 4B5 under
conditions that permit formation of a complex between 4B5 and any target
antibody that
may be present. The amount of complex is then determined, and compared with
complexes formed with standard samples containing known amounts of target
antibody in
42
... _...__..__.__._____T ._._. -y. ....... _.
CA 02295375 1999-12-24
WO 99/02545 PCT/IB98/01046
the range expected. Complex formation can be observed by any method known in
the art
such as immunoprecipitation or nephelometry, but it is generally more
sensitive to employ
a reagent labeled with such labels as radioisotopes such as'ZSI, enzymes such
as peroxidase
and [3-galactosidase, or fluorochromes such as fluorescein.
Pharmaceutical compositions of the invention
The present invention encompasses pharmaceutical compositions and immunogenic
compositions containing 4B5 either alone or in combination. Such
pharmaceutical
compositions and vaccines are useful for eliciting an immune response and
treating
neoplastic diseases, either alone or in conjunction with other forms of
therapy, such as
chemotherapy or radiotherapy.
The preparation of pharmaceutical compositions that contain 4B5 antibody, or a
polynucleotide or a polypeptide derivative thereof as an active ingredient is
conducted in
accordance with generally accepted procedures for the preparation of
pharmaceutical
preparations. See, for example, Remington's Pharmaceutical Sciences 18th
Edition (1990),
E.W. Martin ed., Mack Publishing Co., PA. Depending on the intended use and
mode of
administration, it may be desirable to process the active ingredient further
in the
preparation of pharmaceutical compositions. Appropriate processing may include
sterilizing, mixing with appropriate non-toxic and non-interfering components,
dividing
into dose units, and enclosing in a delivery device.
Liquid pharmaceutically acceptable compositions can. for example, be prepared
by
dissolving or dispersing a polypeptide embodied herein in a liquid excipient,
such as water,
saline, aqueous dextrose, glycerol, or ethanol. The composition can also
contain other
medicinal agents, pharmaceutical agents, adjuvants, carriers, and auxiliary
substances such
as wetting or emulsifying agents, and pH buffering agents.
Pharmaceutical compositions of the present invention are administered by a
mode
appropriate for the form of composition. Typical routes include subcutaneous,
intramuscular, intraperitoneal, intradermal, oral, intranasal, and
intrapulmonary (i.e., by
aerosol). Pharmaceutical compositions of this invention for human use are
typically
administered by a parenteral route, most typically intracutaneous,
subcutaneous, or
intramuscular.
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WO 99/02545 PCT/IB98/01046
Pharmaceutical compositions for oral, intranasal, or topical administration
can be
supplied in solid, semi-solid or liquid forms, including tablets, capsules,
powders, liquids,
and suspensions. Compositions for injection can be supplied as liquid
solutions or
suspensions, as emulsions, or as solid forms suitable for dissolution or
suspension in liquid
prior to injection. For administration via the respiratory tract, a preferred
composition is
one that provides a solid, powder, or liquid aerosol when used with an
appropriate
aerosolizer device. Although not required, pharmaceutical compositions are
preferably
supplied in unit dosage form suitable for administration of a precise amount.
Also
contemplated by this invention are slow release or sustained release forms,
whereby a
relatively consistent level of the active compound are provided over an
extended period.
Compositions embodied in this invention can be assessed for their ability to
recognize specifically a neoplasia. Accordingly, test compounds are prepared
as a suitable
pharmaceutical composition and administered to test subjects. Initial studies
are preferably
done in small animals such as mice or rabbits, optionally next in non-human
primates and
then ultimately in humans. Immunogenicity is preferably tested in individuals
without a
previous antibody response. A test composition in an appropriate dose is
administered on
an appropriate treatment schedule. It may be appropriate to compare different
doses and
schedules within the predicted range. Such testing is within the skill of one
in the art.
Compositions of this invention are particularly suitable for administration to
humans with a neoplastic disease. Especially relevant are melanoma,
neuroblastoma,
glioma, sarcoma, lymphoma, and small cell lung cancer.
The present invention includes methods of eliciting an immune response in a
cancer patient that entail administering an effective amount of 4B5 to the
individual. In
this context, an "effective amount" is an amount sufficient to elicit an
immune response,
whether humoral and/or cellular. Preferably, the immune response includes the
production
of anti-GD2.
In a preferred embodiment, 4B5 is used to elicit an immune response and/or for
treatment of andlor for palliating advanced cancer. A suitable cancer is one
that expresses
GD2, expressed on the surface of tumor cells, such as lung cancers and
melanoma. As
used herein, "advanced" cancers means that there is detectable metastasis,
that is,
detectable tumor masses at sites other than the primary site of the tumor.
Masses are
44
CA 02295375 1999-12-24
WO 99/02545 PCT/IB98/0104b
preferably detected by imaging techniques known in the art such as X-ray or CT
scan. For
eliciting an immune response, palliation, or treatment, an effective amount of
4B5 is
administered to an individual with advanced tumor(s). Administration of an
effective
amount of 4B5 to individuals with advanced cancer may delay or slow the rate
of
progression of the disease or ameliorate disease, in comparison with other
individuals who
are not so treated.
It is understood that for some situations involving advanced cancers, the
individual
receiving 4B5 may be moderately to severely immunocompromised, either due to
the
nature of previous treatment, the disease itself, or both. Thus, the time
required to mount
an immune response and/or the number of injections of 4B5 and/or the amount of
4B5 per
administration may vary. For example, an individual may require a longer time
to elicit an
immune response once 4B5 has been administered. In this case, it is
recommended that the
individual continue to be monitored for an immune response, even if no initial
(i.e., within
the first month) no immune response has been detected. As another example, an
individual
1 S may require more than the average number of injections to elicit an immune
response.
The effective amount of 4B5 antigen binding fragments to be administered will
depend upon several factors, such as the route of administration, the
condition of the
individual, and the desired objective. The term "therapeutically effective"
means that the
amount of antigen binding fragment used is of sufficient quantity to
ameliorate the cancer.
"Ameliorate" denotes a lessening of the detrimental effect of the cancer on
the individual.
Typically, if administered directly, the amount per administration is about 10
~g to 20 mg,
preferably 250 p,g to 10 mg, more preferably 300 ug to 5 mg, even more
preferably 500 p.g
to 2.5 mg. Administrations are typically conducted on a weekly or biweekly
basis until a
desired, measurable parameter is detected, such as diminution of disease
symptoms.
Administration can then be continued on a less frequent basis, such as
biweekly or
monthly, as appropriate.
4B5 is typically administered bi-weekly for four injections, followed by
monthly
injections as required. Timing of subsequent injections (i.e., a maintenance
dose) will
depend, inter alia, upon the condition and response of the individual being
treated. Ab3
levels can be monitored, for example, preferably by the diagnostic methods
described
CA 02295375 1999-12-24
WO 99/02545 PCT/IB98/01046
herein, to determine when maintenance (booster) administrations should be
given, which
would typically be about every three months.
The dosage ranges for the administration of 4B5 are those large enough to
produce
the desired effect in which the symptoms of the malignant disease are
ameliorated without
causing undue side effects such as unwanted cross-reactions, anaphylactic
reactions, and
the like. Generally, the dosage will vary with the patient's age, condition,
sex and extent
of the disease and can be determined by one of skill in the art. The dosage
can be adjusted
by the individual physician in the event of any complication. Dosage can vary
from about
0.1 mg/kg to about 2000 mg/kg, preferably about 0.1 mg/kg to about S00 mg/kg,
in one or
more dose administrations daily, for one or several days. Generally, when 4B5
are
administered conjugated with therapeutic agents, lower dosages, comparable to
those used
for in vivo immunodiagnostic imaging, can be used.
Another method of administration is intralesionally, for instance by direct
injection
directly into the tumor. Intralesional administration of various forms of
immunotherapy to
cancer patients does not cause the toxicity seen with systemic administration
of
immunologic agents. Fletcher et al. ( I 987) Lymphokine Res. 6:45; Rabinowich
et al.
(1987) Cancer Res. 47:173; Rosenberg et al. (1989) Science 233:1318; and Pizz
et al.
( 1984) Int. J. Cancer 34:359.
One possible indication of effectiveness of administration of 4B5, whether for
eliciting an immune response and/or treatment, or whether administration of
4B5 is
indicated, is the density of GD2 on the tumor cells. This density can vary
widely from
individual to individual, and may vary over the course of administration of
4B5 and/or
over the course of the disease. As used herein, "density" of GD2 can refer to
either or both
of the following: (a) the number of cells per total cells in a given
biological sample that
have GD2 on their surface; (b) the amount of GD2 on the surface of each cell.
Density (a)
is calculated by noting the number of cells in a sample that are stained or
otherwise
indicate that GD2 is present divided by the total number of cells. This
density would be
preferably greater than about 20%, more preferably greater than about 30%,
more
preferably greater than about 50%, even more preferably greater than about
70%, even
more preferably greater than about 80%, most preferably greater than about
90%. Thus,
the invention includes administration of 4B5 to an individual having density
of GD2
46 .
CA 02295375 1999-12-24
WO 99/02545 PCT/IB98/01046
greater than about 20%, preferably greater than 30%, more preferably greater
than 70%,
even more preferably greater than about 80%, most preferably greater than
about 90%.
Density (b) is indicated by the relative intensity of staining (or intensity
of any
measurement indicating the presence of GD2) of cells in a sample from one
individual
relative to, for example, a sample from another individual. For this density,
one skilled in
the art can make an empirical determination of density. Density (b) is
relative to normal
tissues (i.e., cells lacking GD2, or unaffected cells), so preferred ranges
may be about 1.5
fold, preferably about 3 fold, more preferably about 10 fold higher expression
over
unaffected cells, as detected by immunohistochemical staining density.
Unaffected cells
could also be from the same individual.
This is not to say that individuals with lower densities, for example, less
than about
50% are not indicated for administration of 4B5. While not wishing to be bound
by a
single theory, it is possible that administration of 4B5 could elicit a series
of immuno-
reactions that result in a more general response that is effective against a
GD2-associated
tumor, such as a cytotoxic T cell response. A lower density, however, may
indicate that
additional therapies are desirable.
It is understood that density can also be used as an indicator of extent of
disease
and response to administration of 4B5. For example, a sample taken from an
individual at
the onset of 4B5 administration may exhibit about 80% density (i.e., about 80%
of the cells
exhibit GD2). After receiving 4B5, a sample taken from the same location may
exhibit
only about 50% density, indicating that GD2-expressing cells are being
destroyed.
Similarly, if the intensity of staining of a sample from an individual
receiving 4B5
diminishes upon receiving 4B5, this indicates that GD2-bearing tumor cells are
being
destroyed.
For purposes of raising an immune response or providing treatment to
individuals
with advanced GD2-associated tumors, 4B5 is administered parenterally,
preferably
intracutaneously. Other routes of administration include, but are not limited
to,
intramuscular, subcutaneous and intradermal. 4B5 can also be administered
indirectly, by
treatment of cultured cells followed by introduction of these cultured cells
into an
individual.
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WO 99/02545 PCT/IB98/01046
Preferably, 4B5 is administered with a pharmaceutically acceptable excipient.
A
pharmaceutically acceptable excipient is a relatively inert substance that
facilitates
administration of a pharmacologically effective substance. For example, an
excipient can
give form or consistency to the vaccine composition, or act as a diluent.
Suitable
excipients include but are not limited to stabilizing agents, wetting and
emulsifying agents,
salts for varying osmolarity, encapsulating agents, buffers, and skin
penetration enhancers.
Examples of pharmaceutically acceptable excipients are described in
Remington's
Pharmaceutical Sciences (Alfonso R. Gennaro, ed., 18th edition, 1990).
Preferably, 4B5 is used with an adjuvant that enhances presentation of 4B5 or
otherwise enhances the immune response against 4B5. Suitable adjuvants include
aluminum hydroxide, alum, QS-21 (U.S. Pat. No. 5,057,540), DHEA (U.S. Pat.
Nos. 5,407,684 and 5,077,284) including its precursors and modified forms,
(e.g., DHEA-
S, the sulfonated form of DHEA), (3-2 microglobulin (WO 91/16924), muramyl
dipeptides,
muramyl tripeptides (U.S. Pat. No. 5,171,568) and monophosphoryl lipid A (U.S.
Pat. No.
4,436,728; WO 92/16231) and its derivatives, e.g., DetoxTM, and BCG (U.S. Pat.
No. 4,726,947). Other suitable adjuvants include, but are not limited to,
aluminum salts,
squalene mixtures (SAF-1 ), muramyl peptide, saponin derivatives,
mycobacterium wall
preparations, mycolic acid derivatives, nonionic block copolymer surfactants,
Quil A,
cholera toxin B subunit, polyphosphazene and derivatives, and
immunostimulating
complexes (ISCOMs) such as those described by Takahashi et al. {1990) Nature
344:873-
875. For veterinary use and for production of antibodies in animals, mitogenic
components of Freund's adjuvant can be used.
The choice of an adjuvant will depend in part on the stability of the vaccine
in the
presence of the adjuvant, the route of administration, and the regulatory
acceptability of the
adjuvant, particularly when intended for human use. For instance, alum is
approved by the
United States Food and Drug Administration (FDA) for use as an adjuvant in
humans. The
4B5 can be administered in a precipitated form; for example, alum-precipitated
4B5 can be
used. If QS-21 is used, preferably 100 pg is used for each dose which
preferably is
administered subcutaneously within about 30 minutes of mixing with 4B5 (with
care taken
to mix gently). If DetoxTM is used, preferably 0.12 ml is used for each dose,
which is
preferably administered subcutaneously within about 30 minutes of mixing with
4B5.
48
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Manufacturers of adjuvants generally provide recommendations regarding
amounts,
volume, preparation, and routes) of administration.
Alternatively, 4B5 can be encapsulated, for example, in liposomes. Liposomes
suitable for packaging polypeptides for delivery to cells are known in the
art.
4B5 can be heat treated before administration, and the heat treatment can be
in the
presence of adjuvant, for example, alum. For instance, 4B5 can be heated at
about 40 to
60°C, preferably 45°C to 55°C, for a period of about 5
minutes to 2 hours, preferably I 5
minutes to 1 hour. Heat treatment is more preferably at 45°C for 30
minutes in a sterile
vial, in a water bath. The heat treatment can occur anytime before
administration.
Preferably, heat treatment is within 7 days of administration. Other heat
treatment
procedures can be used, as long as the desired activity of 4B5 is not
significantly
compromised.
For the purpose of raising an immune response, 4B5 can be administered in an
unmodified form. 4B5 can be modified to improve its immunogenicity. Methods of
improving immunogenicity include, inter alia, crosslinking with agents such as
gluteraldehyde or bifunctional couplers, or attachment to a polyvalent
platform molecule.
Immunogenicity may also be improved by coupling to a protein carrier,
particularly one
that comprises T cell epitopes.
4B5 can be used alone or in conjunction with other agents which promote the
desired activity/objective. In this context, an "agent" can be any of a
variety of substances.
Further, "in conjunction with" means that the agent can be used concomitantly,
before, or
after 4B5. A desired activity is any activity that facilitates, enhances,
promotes, or
modulates the desired objective in using 4B5. Agents which can be used
include, but are
not limited to, cytokines, lymphokines, adjuvants, and drugs. Agents also
include
substances which facilitate delivery of 4B5, such as liposomes, or substances
which
promote delivery of 4B5 to a particular target, for example, a cellular
receptor. For
example, 4B5 can be administered with a cytokine such as GM-CSF.
In order to determine the effect of administration with 4B5, an individual can
be
monitored for either an antibody (humoral) or cellular immune response against
GD2, or a
combination thereof.
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To determine the level of GD2 antibody (Ab3) in a biological sample, for
example,
serum or plasma is obtained from the individual. Optionally, the sample can be
enriched
for immunoglobulin before the assay is conducted, although this is not usually
required. If
a mouse immunoglobulin is to be used as an assay reagent, the sample is
preferably
pretreated to remove anti-mouse immunoglobulin activity. This can be
performed, for
example, by depletion on a mouse immunoglobulin column, or by mixing non-
specific
mouse immunoglobulin into the sample and removing any immunoprecipitate
formed.
To conduct the assay, anti-GD2 that may be in the sample is contacted with a
non-
limiting amount of an antigenic equivalent of GD2. This may be isolated GD2,
nitrocellulose with GD2 affixed by direct blotting or by transfer from a
polyacrylamide
geI, cells expressing GD2, membrane preparations from such cells, or fixed
tissue sections
containing GD2. Alternatively, an anti-idiotype, particularly 4B5 may be used.
Once the immune complex has formed, it is generally separated from uncomplexed
GD2 analog, and the amount of complex present is determined. The complex can
be
separated, for example, by centrifugation to collect cells or an
immunoprecipitate, or
capture by a solid phase. The amount of complex present can be measured by
providing
the GD2 analog with a label either directly, or by incubating with a secondary
reagent.
Alternatively, a competition assay may be performed, in which the sample is
first
incubated with the GD2 analog, and then a non-limiting amount of a labeled
anti-GD2
reagent is added which competes with the anti-GD2 which may be present in the
sample.
Suitable labels include radiolabels, enzyme labels, fluorescent labels, and
chemiluminescent labels. A standard curve is constructed using solutions known
to
contain no anti-GD2, and solutions with various relative concentrations of
anti-GD2, in
place of the sample. The sample containing the unknown amount of anti-GD2 is
generally
assayed in parallel, and the relative amount of anti-GD2 contained therein is
determined by
comparison with the standard curve. Preferred assays for determining anti-GD2
levels
using 4B5 antibody are described in more detail in a following section.
The isotype of the anti-GD2 antibody can be determined by including in the
immunoassay an isotype-specific reagent, either at the separation or the
labeling stage. For
example, anti-human IgG may be used to separate or detect antibody of the IgG
class
present in a clinical sample of human origin. Presence of specific anti-GD2 of
the IgG
_ ___..__. _T..__._. _. - _.._... . _.._.~_._.__
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class generally indicates a memory response. Presence of anti-GD2 of the IgM
class
generally indicates ongoing immunostimulation, such as may be due to the
presence of an
GD2 expressing tumor, or ongoing treatment with 4B5.
If desired, anti-GD2 antibody detected in a biological sample can be further
characterized; for example, by competition with anti-GD2 (Ab I ) to determine
whether they
are specific for related epitopes on GD2. Competition assays between Abl and
Ab3 are
known in the art and not described in detail herein.
Anti-GD2 antibody can also be tested to determine whether it is cytotoxic.
Complement mediated cytotoxicity {CMC) is determined, for example, by using
GD2-
expressing target cells labeled with 5 I Cr. Labeling can be accomplished by
incubating
about 106 cells with 200 p.Ci Na251 Cr04 for 60 minutes at 37°C,
followed by washing.
The assay is conducted by incubating the antibody (or clinical sample
containing the
antibody) with the target cells. The opsonized cells are then washed and
incubated with a
source of complement; for example, guinea pig serum pre-adsorbed to remove
intrinsic
antibody activity. After a suitable incubation period at 37°C, release
of 51 Cr into the
medium is determined and compared with that from unopsonized control cells.
Release of
51 Cr correlates with CMC activity.
Another way of characterizing the anti-GD2 antibody is by testing its ability
to
participate in an ADCC response (Cheresh et al. ( 1986), Cancer Res. 46:5112).
Radiolabeled GD2-expressing target cells are incubated with the anti-GD2 (in
the form of
heat-inactivated serum), and effector cells. Normal human peripheral blood
mononuclear
cells (PBMC) are suitable effector cells, and preferably are used at an
effectoraarget ratio
of about 100. After approximately 4 hours at 37°C, the proportion of
released 51 Cr is
determined as a measure of ADCC activity.
The cellular immune response in a subject being administered 4B5 may be
quantified by conducting standard functional assays for specific T cell
activity.
One type of assay measures T cell proliferation. In this test, peripheral
blood
mononuclear cells (PBMC) are obtained from a whole blood sample collected from
the
treated subject. For experimental animals, spleen cells can also be used. T
cells can be
enriched, for example, by centrifugation on a gradient such as Ficoll(TM). The
cells are
s~
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then cultured in the presence of GD2 or (more usually) irradiated GD2
expressing cells at
various concentrations. Preferably, the stimulator cells are autologous with
the responder
cells, particularly in terms of histocompatibility Class II antigens.
Another type of assay measures T cell cytotoxicity. In this test, an enriched
T-cell
population is used to effect lysis of 51 Cr-labeled GD2 expression target
cells, prepared as
described above . Preferably, the effector cells are autologous with the
target cells,
particularly in terms of histocompatibility Class I antigens. The T cell
population can
optionally be pre-stimulated with GD2 or a relevant cell line. The T cells are
then
combined at various ratios with about 104 labeled target cells; for example,
in wells of a
microtiter plate. The plate is optionally centrifuged to initiate cell
contact, and the cells are
cultured together for 4-16 hours at 37°C. The percent specific release
of 5lCr into the
medium is measured in comparison with labeled targets cultured alone (negative
control)
and targets lysed with a detergent such as 0.1% Triton (TM) X-100 (positive
control).
Other relevant measurements to determine the effect of 4B5 administration
include
clinical tests as may be appropriate in determining the progression of cancer
of the
suspected type. Such tests include but are not limited to, inflammatory
indicators,
mammography, and radioscintigraphy.
Another way that 4B5 can be used is to assay for the presence of an antibody
or
other immune component that binds to 4B5, or to GD2. Such components may be
present
following therapeutic administration of 4B5, or may spontaneously arise due to
the
presence of an GD2-expressing tumor in an immunocompetent host. Assays can be
conducted on biological samples, usually clinical samples.
In one embodiment of this invention, 4B5 is used to detect the presence of an
anti-
GD2, particularly anti-4B5 idiotype, that may be present in a biological
sample. The
sample is suitably prepared before conducting the assay, optionally by
enriching for
antibody activity. If the biological sample is suspected of containing
antibody activity
against non-idiotypic regions of 4B5 (particularly anti-mouse immunoglobulin),
it is
preferable to remove them or conduct the assay so as to avoid their detection.
Anti-mouse
immunoglobulin antibody can be removed from a sample, for example, by
precipitation
with normal mouse IgG or adsorption with a mouse Ig adsorbant. Binding of anti-
mouse
immunoglobulin antibody, particularly that specific for the Fc region, can be
minimized by
s2
-___~..T-._____ _ ....___.-..___.~_.....
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WO 99/02545 PCT/IB98/01046
judicious choice of the reagents of the assay. F(ab')z or Fab fragments of 4B5
and other
mouse immunoglobulin reagents are especially appropriate.
After the sample is suitably prepared, it is mixed with a excess functional
equivalent of 4B5 under conditions that permit formation of a complex between
4B5 and
any anti-GD2 that may be present. The amount of complex is then determined,
and
compared with complexes formed with standard samples containing known amounts
of
anti-GD2 in the range expected. Complex formation can be observed by
immunoprecipitation or nephelometry, but it is generally more sensitive to
employ a
reagent labeled with such labels as radioisotopes like ~'-SI, enzymes like
peroxidase and 13-
galactosidase, or fluorochromes like fluorescein.
Antibody assays can be conducted in fluid phase. For example, anti-GD2 may be
mixed with labeled 4B5. Alternatively, the anti-GD2 in the sample can be used
to compete
with a labeled anti-GD2 for binding sites on 4B5. Generally, bound and unbound
label is
separated to quantitate the percent bound. Suitable separation methods include
gel
filtration chromatography, and precipitation with antibody against
immunoglobulin of the
species from which the sample is obtained, optionally in the presence of
polyethylene
glycol. Alternatively, the proportion of bound and unbound label may be
determined in
situ, for example, using fluorescence/quench labeling pairs or
enzyme/inhibitor labeling
pairs. See, e.g., U.S. Patent 3,996,345 (Unman et al.).
As described above, it is generally more convenient to conduct a capture assay
using a reagent linked to a solid phase, such as a polyethylene test tube,
microtiter plate
well, or magnetic bead. In a competition-type capture assay, unlabeled anti-
GD2 in the
sample competes with a labeled anti-GD2 reagent for binding to 4B5. The 4B5
may be
attached directly to the solid support, or captured later, for example, using
an anti-4B5. In
this assay, the amount of label associated with the solid phase is inversely
related to the
amount of anti-GD2 in the sample.
In the sandwich-type capture assay, anti-GD2 is captured by 4B5 attached
directly
or through a secondary reagent to a solid phase. After washing, the anti-GD2
is detected
using anti-immunoglobulin of the appropriate species, or a second 4B5
antibody, to which
a label is directly or indirectly attached. Alternatively, the anti-
immunoglobulin may be
attached to the solid phase and labeled 4B5 is used to complete the sandwich.
If the anti-
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immunoglobulin used is isotype-specific, then the class of the antibody can
also be
determined. In this type of assay, the amount of label associated with the
solid phase
correlates positively with the amount of anti-GD2 in the sample.
Other methods of measuring specific antibody are known in the art, and can be
adapted to measure anti-GD2 by using 4B5 as the target antigen. All such
adapted
methods are embodied in this invention.
4B5 can also be used to measure the level of cellular anti-GD2 activity,
particularly
anti-4B5 idiotype. In a preferred example, 4B5 is used to identify anti-GD2 T
cells,
defined for this purpose as lymphocytes expressing a T cell receptor that
binds the 4B5
IO idiotype. 4B5 may be labeled and contacted with a population of cells
suspected of
containing anti-GD2 T cells. Alternatively, unlabeled 4B5 may be mixed with
the cells,
and followed with a labeled secondary reagent such as labeled anti-mouse
immunoglobulin
or protein A. Suitable labels for this purpose include radiolabels and
fluorescent labels.
The use of fluorescent labels would also allow anti-GD2 cells to be separated
from non-
15 specific cells in a fluorescence-activated cell sorter.
The invention also encompasses methods using 4B5 to remove a label, for
example
radioactivity, from an individual who has received a labeled anti-GD2 antibody
(Abl), for
example, for radioscintilligraphy or radiotherapy. One problem common to use
of
antibody targeted radionuclides (i.e., radioimmunotherapy) has been the
presence of excess
20 Ab I in the system which limits the dosage of radiolabeled antibody for
treatment. Further,
effective imaging using radiolabeled antibodies is hampered due to excess
circulating
radiolabeled antibody, which often takes several days to clear circulation and
tissues. In
these methods of the present invention, 4B5 is administered to the individual
at a specified
time after administration of the labeled anti-GD2. The intention is for the
4B5 to complex
25 with anti-GD2 at sites other than the tumor, such as in the circulation and
interstitial
spaces, and thereby promote its clearance. As a result, the level of labeled
moiety (such as
radioisotope) in unaffected tissues is reduced, and the image of the tumor (in
comparison
to neighboring tissues) is enhanced. Similarly, when radionuclides are given
to subjects
for irradiation of a tumor site, it is desirable to reduce collateral exposure
of unaffected
30 tissue. This invention thus includes methods of treatment in which a
radiolabeled anti-
54
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GD2 antibody is administered in a therapeutic dose, and followed by a molar
excess of
4B5.
In either of these applications, an amount of 4B5 is chosen that is in
sufficient
molar excess over the labeled anti-GD2 to locate and bind any anti-GD2 that is
not
localized at the tumor site. The timing of administration and amount of 4B5
will depend
upon the nature of the radiolabeled antibody, the type of radioisotope used
and the
condition of the individual. Preferably, the molar ratio of 4B5 to the anti-
GD2 antibody is
at least about 5:1, more preferably about 25:1 to 200:1. Preferably, 4B5 is
administered 5
to 24 hours after the individual has received the anti-GD2 antibody.
The invention also includes methods of detecting the presence of an anti-GD2
antibody bound to a tumor cell comprising the steps of treating the tumor cell
with 4B5 for
a sufficient time to allow binding to the anti-GD2 antibody, and detecting the
presence of
any complex formed. The intention is for the 4B5 to detect anti-GD2 that has
pre-attached
to the tumor cell; or alternatively, to promote the binding of anti-GD2 to the
tumor cell by
forming a polyvalent anti-GD2/4B5 immune complex. In the former instance, the
4B5 is
provided with a detectable label or a means by which a label can be attached.
In the latter
instance, either the anti-GD2 or the 4B5 is provided with a label.
This strategy can be used, for example, to identify an GD2 antigen-bearing
cell in a
isolated cell suspension. The cells are incubated sequentially or
simultaneously with anti-
GD2 and 4B5, washed, and then the labeled cells are detected. Preferred labels
for this
embodiment include fluorescent labels, such as fluorescein, rhodamine, and
Texas red.
Optionally, labeled cells can be separated from unlabeled cells; for example,
by sorting in a
fluorescence-activated cell sorter or by affinity separation, using any of the
solid phase
positive or negative immunoselection techniques known in the art.
The strategy can also be used, for example, to detect or image tumors in an
affected
subject. The anti-GD2 and 4B5 are administered {usually sequentially) into the
subject
and allowed to accumulate at the tumor site. Suitable labels include
radiolabels such as
I 1 l In~ 131I ~d 99mTc. The tumor is then detected or visualized using
standard
techniques of radioscintigraphy.
The various compositions of this invention can be used alone, or in
conjunction
with other active agents that promote the desired objective, or provide a
desirable adjunct
CA 02295375 1999-12-24
WO 99/02545 PCT/IB98/01046
therapy. Suitable active agents include the anti-neoplastic drugs and
bioresponse modifiers
described above and effector cells such as those described by Douillard et al.
(1986)
Hybridomas (Supp. 1:5139).
Using 4B5, it is possible to design combination therapies. It may be desirable
to
administer a therapeutic agent, or agents, prior to the administration of 4B5
in combination
with effector cells and the same, or different, therapeutic agent or agents.
For example,
patients can be treated by first administering IFN-y and interleukin-2 (Il-2)
daily for 3 to 5
days, and on day 5 administering 4B5 in combination with effector cells, IFN-
y, and Il-2.
Suitable subjects for treatment with 4B5 or combination therapies including
4B5,
include those who are suspected of being at risk of a pathological effect of
any neoplasia,
particularly carcinoma, are suitable for treatment with the pharmaceutical
compositions of
this invention. Those with a history of cancer are especially suitable.
Suitable human
subjects for therapy comprise two groups, which can be distinguished by
clinical criteria.
Patients with "advanced disease" or "high tumor burden" are those who bear a
clinically
measurable tumor. A clinically measurable tumor is one that can be detected on
the basis
of tumor mass (e.g., by palpation, CAT scan, or X-Ray; positive biochemical or
histopathological markers on their own may be insufficient to identify this
population). A
pharmaceutical composition embodied in this invention is administered to these
patients to
elicit an anti-tumor response, with the objective of palliating their
condition. Ideally,
reduction in tumor mass occurs as a result, but any clinical improvement
constitutes a
benefit. Clinical improvement includes decreased risk or rate of progression
or reduction
in pathological consequences of the tumor.
A second group of suitable subjects is known in the art as the "adjuvant
group".
These are individuals who have had a history of cancer, but have been
responsive to
another mode of therapy. The prior therapy may have included, but is not
restricted to,
surgical resection, radiotherapy, and traditional chemotherapy. As a result,
these
individuals have no clinically measurable tumor. However, they are suspected
of being at
risk for progression of the disease, either near the original tumor site, or
by metastases.
This group can be further subdivided into high-risk and low-risk individuals.
The
subdivision is made on the basis of features observed before or after the
initial treatment.
These features are known in the clinical arts, and are suitably defined for
each different
56
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cancer. Features typical of high risk subgroups are those in which the tumor
has invaded
neighboring tissues, or who show involvement of lymph nodes.
Another suitable group of subjects is those with a genetic predisposition to
cancer
but who have not yet evidenced clinical signs of cancer. For instance, women
testing
positive for a genetic mutation associated with breast cancer, but still of
childbearing age,
may wish to receive 4B5 treatment prophylactically to prevent the occurrence
of cancer
until it is suitable to perform preventive surgery.
A pharmaceutical composition embodied in this invention is administered to
patients in the adjuvant group, or in either of these subgroups, in order to
elicit an anti-
cancer response. Ideally, the composition delays recurrence of the cancer, or
even better,
reduces the risk of recurrence (i.e., improves the cure rate). Such parameters
may be
determined in comparison with other patient populations and other modes of
therapy.
Of course, crossovers between these two patient groups occur, and the
pharmaceutical compositions of this invention can be administered at any time
that is
appropriate. For example, 4B5 therapy can be conducted before or during
traditional
therapy of a patient with high tumor burden, and continued after the tumor
becomes
clinically undetectable. 4B5 therapy can be continued in a patient who
initially fell in the
adjuvant group, but is showing signs of recurrence. The attending physician
has the
discretion to determine how or when the compositions of this invention are to
be used.
In another embodiment, 4B5 polypeptides can be conjugated with carrier. In
instances where the 4B5 polypeptide is correctly configured so as to provide a
binding site,
but is too small to be immunogenic, the polypeptide may be linked to a
suitable carrier. A
number of techniques for obtaining such linkage are known in the art and need
not be
described in detail herein. Any carrier can be used which does not itself
induce the
production of antibodies harmful to the host. Suitable carriers are typically
large, slowly
metabolized macromolecules such as proteins; polysaccharides, such as latex
functionalized Sepharose, agarose, cellulose, cellulose beads and the like;
polymeric amino
acids, such as polyglutamic acid, polylysine, and the like; amino acid
copolymers; and
inactive virus particles or attenuated bacteria, such as Salmonella.
Especially useful
protein substrates are serum albumins, keyhole limpet hemacyanin,
immunoglobulin
molecules, thyroglobulin, ovalbumin, tetanus toxoid, and other proteins well
known to
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those of skill in the art. As is evident to one skilled in the art, the above-
described
recombinant forms of 4B5 polypeptides and 4B5, such as fusion proteins, can in
turn be
fused with other amino acid sequences. For instance, a 4B5 scFv can be fused
to a
cytokine, such as IL-2.
The ability of a 4B5 polypeptide to bind Abl and/or Ab3 can be assessed
several
ways. In one test, binding of the 4B5 polypeptide(s) to Abl can be tested
directly, for
example, by radioimmunoassay (RIA), for example, by reacting radiolabeled 4B5
polypeptide with Ab I or Ab3 coated on microtiter plates.
In another procedure, binding to Abl or Ab3 is determined by competitive
immunoassay, In one variation of this procedure, binding of labeled 4B5
polypeptide(s) or
functional equivalent fragments to Abl is measured in the presence of
different Abl, other
Ab2s, 4B5 or analogs thereof, other 4B5 polypeptide(s), GD2 or extracts
containing GD2,
or other proteins. Percent inhibition is calculated according to the following
formula:
inhibition = [ 1 - ( RT R~' >, x 100%
R"~ - R~,
In another variation, the test fragment with putative 4B5 activity is tested
for its
ability to interfere with the binding between Abl and Ab2, or Abl and GD2.
This test may
be more sensitive in some applications, because lower affinity interaction
between 4B5 and
Abl may be too weak to form a stable bond, but be adequate to interfere with
the binding
of another ligand-receptor pair when present at sufficient concentration. The
GD2 can be
provided as purified antigen or GD2-expressing cells. The assay can be
conducted by
labeling either the Ab 1 or the GD2 or Ab2, and optionally immobilizing the
other member
of the ligand-receptor pair on a solid support for ease of separation. The
test fragment is
incubated with the labeled reagent, and then the mixture is presented to the
immobilized
target or test cell to determine if the test fragment is able to inhibit
binding. Degree of
inhibition correlates with 4B5 activity.
Various examples of competition assays are known in the art and include the
following. One test that indicates 4B5 polypeptide activity is to measure the
binding of
radiolabeled Abl to semipurified or purified GD2 in the presence of varying
amounts of
58
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4B5 poIypeptide(s). The Abl-GD2 mixture is then added to plates coated with
4B5
polypeptide(s) and binding is compared with binding of labeled Ab 1 alone.
Preferably,
this test is performed with nonsaturating amounts of labeled Ab 1 to detect
changes in
binding with small amounts of competitive GD2. In another competition assay,
GD2
positive target cells are grown in 96-well tissue culture plates as a
confluent monolayer.
Binding of radiolabeled Abl in the absence and presence of 4B5 polypeptides is
determined. The degree of inhibition can be compared with that of intact 4B5
or other 4B5
polypeptides.
A 4B5 polypeptide is considered to bind Abl if there is inhibition when
compared
to a negative control, such as an unrelated anti-idiotype antibody which does
not bind to
Ab 1.
With all of the above-described assays, it is clear to one of skill in the art
that the
labeled molecule can be labeled in various ways, such as with radioisotopes
(i.e., ''-SI) and
non-radioactive labels, such as biotinylated molecules, and molecules for
enzymatic
detection, fluorescent labels and chemiluminescent labels.
The above discussed tests can also be used to compare characteristics of
various
4B5 polypeptide fragments. For example, competitive assays can be conducted in
which a
first 4B5 polypeptide competes for binding to Abl in the presence of varying
amounts of a
second 4B5 polypeptide. Such tests can indicate relative degrees of binding
affinities or
other characteristics.
Another way of characterizing 4B5 polypeptides is testing their ability to
generate
an immune response. As used herein, "immune response" indicates either a
humoral
response, a cellular response, or both. As used herein, the "ability to elicit
an immune
response" pertains to any individual, including human.
The ability of a 4B5 polypeptide to generate a humoral response can be
determined
by testing for the presence of an antibody that binds to the 4B5
polypeptide(s) after
administration of the 4B5 polypeptide(s). It is understood that this antibody
(Ab3) was not
present, or was present in lower amounts, before administration of the 4B5
polypeptide(s).
Immunogenicity is preferably tested in individuals without a previous anti-4B5
response.
Examples of suitable individuals include, but are not limited to, mice,
rabbits, monkeys
and humans. For this test, an individual is administered a 4B5 polypeptide(s).
The amount
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per administration and number of administrations will vary, depending on the
individual.
The requisite amount can be determined readily by empirical observations.
Presence of an Ab3 can be determined by first pre-incubating sera with
autologous
immunoglobulin to block antibodies against isotypic and allotypic determinants
and then
testing sera for binding to GD2 and/or the 4B5 polypeptide(s), for example,
using ELISA
or RIA. For instance, different dilutions of pre-reacted sera are reacted with
4B5 coated on
microtiter plates. An unrelated Ab2 serves as a control. After washing, the
Ab3-4B5
complex is labeled using, for example,'ZSI-labeled 4B5 in a homogeneous
sandwich assay.
Results from this assay are compared to those obtained before administration
of the 4B5
polypeptide. Alternatively, binding to GD2 positive cells, can be tested using
immune
flow cytometry.
Binding of Ab3 to GD2 can also be determined by immunoprecipitation or
immunoreactivity with GD2-positive tissue samples, or dot blot analysis. In
one method
of dot blot analysis, a semi-purified extract of GD2 is directly blotted to a
nitrocellulose
filter. The filter is then incubated with sera containing Ab3, and the
reaction developed by
enzyme-conjugated anti-immunoglobulin. If the Ab3 binds to GD2, a positive
blot should
appear. For testing with tissue samples, an immunoperoxidase assay can be
used.
If desired, Ab3 elicited by 4B5 polypeptide(s) can be further characterized.
For
example, competition assays can be performed to determine whether Ab3 share Ab
1
idiotopes. In this test, serum from an individual immunized with a 4B5
polypeptide is
tested for inhibition of binding of labeled 4B5 polypeptide (or intact 4B5) to
AbI .
Inhibition indicates that Ab3 and Abl contain at least similar binding
determinants.
Similarly, competition of Ab3 with Abl for binding to GD2 (whether partially
purified,
purified, or on the surface of a GD2-positive cell) can be tested by
coincubating a fixed
amount of labeled Abl with different dilutions of Ab3 containing sera or Abl
preparation
and GD2
As is evident to one of skill in the art, the Ab3 can be used in turn to
characterize
4B5 polypeptides, using the assays described above.
Another way of characterizing a 4B5 polypeptide is by testing its ability to
elicit an
antibody that is cytoxic. Methods for this determination are described above.
For
determination of complement mediated cytotoxicity (CMC), SKBR3 (target) cells
(i.e.,
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cells that express GD2) are labeled with 5'Cr. Labeling can be accomplished by
incubating
about 106 cells with approximately 200 pCi NaZS04 for 60 minutes at
37°C, followed by
washing. The assay is conducted by adding and incubating serum suspected of
containing
antibody. Guinea pig serum pre-adsorbed with LS174-T cells (or other source of
complement) is then added. After a suitable incubation period at 37°C,
extent of 5'Cr
release is then measured and compared with that of unopsonized control cells.
Release of
5'Cr correlates with CMC activity. Herlyn et al. (1981) Int. J. Cancer 27:769.
Another way of characterizing a 4B5 polypeptide is by testing its ability to
elicit an
anti-GD2 antibody that participates in an ADCC response. Cheresh et al. {
1986) Cancer
Research 46:5112-51 I 8. In this assay, cultured or GD2-expressing cells are
labeled with
5' Cr and are used as target cells. Normal human peripheral blood mononuclear
cells
(PBMC) are used as effector cells. Preferably, the ADCC assay is conducted in
the
presence of heat-inactivated serum with an effector to target cell ratio of I
00:1 for 4 hours,
although other suitable conditions may be used. The amount of 5' Cr released
is then
measured.
The 4B5 polypeptides of this invention can also be characterized by their
ability to
elicit a cellular response. As used herein, a "cellular response" is a
response that involves
T cells, and can be observed in vitro or in vivo.
One way of detecting a cellular immune response is by assaying for T cell
proliferative activity. In this test, cellular immune response is measured by
proliferation of
peripheral blood mononuclear cells (PBMs) incubated with 4B5 polypeptide(s).
Peripheral
blood mononuclear cells are isolated from blood after a requisite number of
administrations of 4B5 polypeptide(s) and are incubated with varying
concentrations of
4B5 polypeptide(s). If mice are used, T cells are obtained from spleen. T
cells can be
enriched, for example, by centrifugation on a gradient such as FicollTM. A non-
specific
mitogen such as PHA serves as a positive control; incubation with an unrelated
anti-
idiotype antibody serves as a negative control. Preferably, the stimulator
cells are
autologous with the responder cells, particularly in terms of
histoeompatibility Class II
antigens. After incubation of the PBMs for an appropriate number of days to
allow
proliferation, [3H]thymidine incorporation is measured. In many instances a
suitable time
is five days. If desired, determination of which subset of T cells are
proliferating can be
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performed using flow cytometry. Optionally, splenic T cells can be pre-
depleted of either
CD4+ or CD8+ cells before the proliferation assay by incubation with
monoclonal
antibodies such as RL.172 (anti-CD4+) or mAb.168 (anti-CD8+) and complement.
Another way of detecting a cellular immune response is to test for T cell
cytotoxicity (CTL) activity. In this test, T lymphocytes (i.e., an enriched T
cell
population) are isolated (typically from spleen cells) for use as targets in a
standard 5'Cr
release assay. Kantor et al. (1992) J. Natl. Cancer Inst. 84:1084-1091. An
example of a
5'Cr release assay is described above.
Another way of characterizing 4B5 polypeptides is testing their ability to
ameliorate, delay the progression of and/or reduce the extent of GD2-
associated tumors.
Such tests may include inflammatory indicators, radioscintigraphy, or
measurement of
circulating GD2 levels (such assays are available commercially).
In summary, 4B5 polypeptides have a number of uses. 4B5 polypeptides can be
used to induce an immune response in an individual, preferably an anti-GD2
response.
They can also be used to detect and monitor levels of Ab3, or to purify Ab3.
4B5
polypeptides are also useful for treatment of GD2-associated disease, for
example, certain
lung cancers and melanoma.
Thus, the present invention includes methods of inducing an immune response in
an individual comprising administering a 4B5 polypeptide in an amount
effective to induce
an immune response. Preferably, the individual has GD2-associated tumors. In
this
context, an "effective amount" is an amount sufficient to elicit a measurable
immune
response, whether humoral and/or cellular. An effective amount can be
administered in
one or more administrations.
The invention also encompasses methods of detecting an antibody that binds to
4B5 (i.e., Ab3 and/or Abl) in a biological sample. These methods are
applicable in the
clinical setting. for example, for monitoring Abl or Ab3 levels in an
individual, as well as
an industrial setting, in which commercial production of Ab3 is desired. These
methods
entail contacting the Ab3 and/or Abl in the sample with a 4B5 polypeptide
under
conditions suitable to allow the formation of a stable complex between Ab3
and/or Ab 1
and the 4B5 polypeptide, and detecting a stable complex formed, if any. A
number of
immunoassay methods are known in the art and have been described herein. For
further
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illustration, a test sample potentially containing Ab3 and/or Abl can be mixed
with a pre-
determined non-limiting amount of the 4B5 polypeptide which is typically
detectably
labeled (such as with a radioisotope or enzyme). In a liquid phase assay,
unreacted
reagents are removed by a separation technique, such as filtration or
chromatography. In
these immunoassay techniques, the amount of label associated with the complex
positively
correlates with the amount of Ab3 and/or Abl present in the sample. Similar
assays can be
designed in which Ab3 and/or Abl in the test sample competes with labeled
antibody for
binding to a limiting amount of the 4B5 polypeptide. Here, the amount of label
negatively
correlates with the amount of Ab3 and/or Ab 1 in the sample. Suitable samples
in which to
measure Ab3 and/or Abl levels are biological samples, including serum or
plasma,
preferably serum. Other samples include tissue samples.
Further, the invention also includes methods of purifying Ab3 (or Ab 1 ),
comprising
contacting a biological sample containing Ab3 (and/or Ab 1 ) with a 4B5
polypeptide, and
obtaining a complex formed thereby, if any. Typically, the 4B5 polypeptide(s)
is coupled
to an affinity matrix for affinity column purification. Such methods are
routine in the art
and need not be described in detail herein.
Kits comprising 4B5 polynucleotides and/or encoded 4B5
The present invention encompasses kits containing 4B5 polynucleotides and 4B5,
preferably diagnostic kits. Diagnostic procedures using 4B5 polynucleotides
and 4B5 can
be performed by diagnostic laboratories, experimental laboratories,
practitioners, or private
individuals. The clinical sample is optionally pre-treated for enrichment of
the target being
tested for. The user then applies a reagent contained in the kit in order to
detect the
changed level or alteration in the diagnostic component.
Kits embodied by this invention include those that allow someone to detect the
presence of 4B5 polynucleotides and/or anti-GD2 antibodies (both Abl and Ab3).
Optionally, reagents such as 4B5 contained in the kits may be conjugated with
a label to
permit detection of any complex formed with the target in the sample. In
another option, a
second reagent is provided that is capable of combining with the first reagent
after it has
found its target and thereby supplying the detectable label. For example,
labeled anti-
mouse IgG may be provided as a secondary reagent for use with intact 4B5.
Labeled
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avidin may be provided as a secondary reagent when the primary reagent has
been
conjugated with biotin.
The kits can be employed to test a variety of biological samples, including
both
liquid samples, cell suspensions and tissue samples. Suitable assays using 4B5
that can be
supplied in kit form include those described herein. Each reagent is supplied
in a solid
form or dissolved/suspended in a liquid buffer suitable for inventory storage,
and later for
exchange or addition into the reaction medium when the test is performed.
Suitable
packaging is provided. The kit can optionally provide additional components
that are
useful in the procedure. These optional components include, but are not
limited to,
buffers, capture reagents, developing reagents, labels, reacting surfaces,
means for
detection, control samples, instructions, and interpretive information.
The foregoing description provides, inter alia, detailed methods for preparing
4B5,
along with 4B5 encoding poIynucleotides, 4B5 polypeptide fragments, and other
derivatives. A practitioner of ordinary skill in the art can practice
embodiments of this
invention by referring to the sequence data for 4B5, which is provided herein.
The
following examples are provided to illustrate but not limit the claimed
invention.
EXAMPLE 1
Purification of Recombinant 4B5
4B5 antibodies contained in the hybridoma cell-culture medium or in the
lysates of
4B5-expressing cells were purified on a protein G Sepharose column according
to standard
procedures known in the art ( for a reference, see "Methods in Enzymology"
(Academic
Press, Inc.)). Fractions concentrated with purified antibodies were collected,
analyzed by
SDS-polyacrylamide gel electrophoresis, and visualized by staining. The
results are shown .
in Fig. 2. A 55-kDa and a 23-kDa protein, consistent with the molecular weight
of the
heavy chain (55-kDa) and the light chain (23-kDa) of immunoglobulin were
detected in
samples containing recombinant or hybridoma 4B5. An additional band of
approximately
110 kDa, consistent with the molecular weight of a heavy chain/iight chain
dimer, was also
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detected in the sample containing recombinant 4B5. This was most likely due to
incomplete reduction of the disulfide bonds linking the two immunoglobulin
chains.
EXAMPLE 2
Antigenic Similarity between Recombinant and Hybridoma 4B5
Determined by ELISA
In order to determine the ability of recombinant 4B5 to bind to antigen 14G2A,
ELISA plates were coated with 1 p,g/ml of 14G2A or mouse IgG as a negative
control.
The coated ELISA plates were incubated for 16-18 hours at 2-8°C. The
plates were
blocked with PBS-3% BSA for 1 hr at room temperature. Then the plates were
incubated
with either recombinant 4B5 or hybridoma 4B5 in PBS or control human IgG in
PBS or
culture medium for 2 hrs at room temperature. The plates were washed and
incubated with
biotinylated anti-human IgG followed by incubation with streptavidin-
conjugated alkaline
phosphatase for 1 hr. After washing, p-nitrophenyl phosphate substrate was
added to each
plate and, after incubation, the plates were read at 405 nm in an ELISA plate
reader.
Specific binding of recombinant 4B5 to antigen 14G2A is shown in Figs. 3.
These
results indicate that recombinant 4B5 has antigenic binding specificity
indistinguishable
from that of the hybridoma 4B5 antibodies.
EXAMPLE 3
Antigenic Specificity of Recombinant and Hybridoma 4B5
Determined by Western Blot Analysis
Equal quantities of antigen 14G2A and the control sample, mouse monoclonal
antibodies K914 and 9227, were first analyzed by SDS-polyacrylamide gel
electrophoresis,
and then immunoblotted with either recombinant 4B5 or hybridoma 4B5 under
standard
conditions ( for a reference, see "Methods in Enzymology" (Academic Press,
Inc.)). Both
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recombinant 4B5 and hybridoma 4B5 reacted with antigen 14G2A and not with the
mouse
monoclonal antibodies. The results are shown in Fig. 4.
Although the foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity and understanding. it will be
apparent to
those skilled in the art that certain changes and modifications may be
practiced. Therefore.
the description and examples should not be construed as limiting the scope of
the
invention. which is delineated by the appended claims.
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