Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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ANTIGEN BINDING FRAGMENTS 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 antibodies specific to an antigen detected on
neoplastic
cells but not on normal cells. This antigen is termed herein the "C-antigen."
The
C-antigen is recognized by the human monoclonal antibody (Mab) termed "H11."
The
invention encompasses a wide variety of antibodies, and functional derivatives
thereof that
retain the immunologic specificity of H 11 and are termed herein "aC." The
exemplary
antibody, H 11, compositions comprising the H 11, and hybridomas producing H
11 are
included herein. The H11 V region polynucleotides 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 aC
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.
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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
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 13% 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 1973 to 41% in 1993, largely due to
improvements in surgical techniques. The 5 year survival rate for all stages
combined is
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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.
Small cell lung cancer is the most malignant and fastest growing fonn 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 5-10%.
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 of North 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 10 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
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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
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 37,100 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.
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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
5 to these antigens have been used in immunotherapy of several types of
cancer.
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 "VLJL") 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 (VHDHJli) 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 (CE1).
The VLJL and
VHDHJH 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 (CH) 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 (C , Cy, C8, CE, and Ca), and some species have multiple CN subtypes
(e.g.,
CyI, Cy2, Cy3, and C74, 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 (k), and only one of these C regions is present in a single L chain
protein (i.e.,
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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 K or X 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).
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 Vii, 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 Jf.I 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
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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
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. Originallv, the human B cells were immortalized using Epstein-Barr
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) Bio/Technology 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.
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(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.
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 bispecific 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.
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. 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 also been found to react specifically with small cell lung
carcinoma. Cheresh et al. (1986) Cancer Res. 46:5112-5118. 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
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Cell. Dev. Biol. 21:593; Schadendorf (1989) J. Immunol. 142:1621-1625;
Yoshikawa et al.
(1986) Jpn_ J. Cancer Res. 77:1122-1133; Pickering and Misra (1984) Clin.
Imnninol.
Immunoparhol. 32:253-260; Hagiwara and Sato (1983) Mol. Biol. Med. 1:245-252;
and
Sclilom et al. (1980) Proc. Natl. Acad. Sci. USA 77:6841-6845. Human anti-
cancer Mabs
and the antigens tliey 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 11 trial results have been reported on
combined
treatment with murine Mab and colony stimulating factor in metastatic
gastrointestinal
cancer. Saleli 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
prospects of cancers in general would improve.
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 V region having the amino acid sequence of SEQ
ID NO:2
and a L chain V region having the amino acid sequence of SEQ ID NO:4.
Preferably, the
antibody is H11. The invention further encompasses antibodies comprising the H
and L
chain V regions of H11 (SEQ ID NOS:2 and 4, respectively). H11 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 H11 or are only poorly recognized by the antibody. The antibodies
are
designated aC and include HI 1 and any antibody with the "immunologic
specificity" of
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H 11, that is, recognizing the antigen recognized by H 11, and that is
specific for at least one
type of cancer cell but does not recognize normal cells. These antigen binding
fragments
include, but are not limited to, whole native antibodies, exemplified by H 11;
bispecific
antibodies; chimeric antibodies; Fab, Fab', single chain V region fragments
(scFv) and
5 fusion polypeptides.
The invention further encompasses H 11 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 VI.F-VL or VL-VH V region, and
polynucleotides
encoding such polypeptides.
10 Also embodied in the invention are polypeptides having the immunologic
specificity of H 11, wherein the polypeptide comprises at least 5 consecutive
amino acids
from a V region of an aC antibody. The V region may be from a L chain or H
chain. The
5 consecutive amino acids preferably play a role in immunologic specificity,
and may be
from a CDR (Complementarity Determining Region of an antibody). Intact H1 l,
functionally active fragments of H 11, fusion proteins, chimeric antibodies,
multiple
antigen proteins, and other polypeptide derivatives of aC antibodies are
included. Of
special interest are single-chain V regions and fusion proteins.
The compounds and compositions of this invention may 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 the aC antigen
binding fragments.
Another embodiment of this invention is a polynucleotide comprising a sequence
encoding a polypeptide with the immunologic specificity of Hl 1, wherein the
encoded
polypeptide comprises at least 5 consecutive amino acids from a V region of
H11. The V
region may be from either the H 11 L chain or H chain. The 5 consecutive amino
acids
preferably play a role in H 11 immunologic reactivity, and may be from a CDR.
The V
region of H11 has been found to have a small region of homology to an antibody
designated A6. Peptides comprised solely of this region of homology and
lacking other
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H11-specific amino acid residues are specif~callv excluded from the claimed
invention.
A6 is described in W095/35374.
The invention also encompasses isolated polynucleotides of at lcast 20
consecutive
nucleotides capable of forming a stable duplex with the H i 1 L or H chain
encoding
sequences. but not with sequences for other previously described
inununoglobulin
molecules. Any of these polynucleotides may be in the form of cloning vectors,
expression vectors, or transfected into host cells.
A further embodiment'of this invention comprises prophylactic treatment of a
cancer patient with at least one aC antigen binding fragment. Preferablv. aC
is fused to a
] 0 thcrapeutic 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 may 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
antigen recognized bv aC (hereinafter, the "C-antigen") in a sample,
comprising H11 or a
polypeptide of this invention in suitable packaging. Also embodied by the
invention are
methods for detecting the C-antigen or cells expressing the C-antigen by
employing a
reagent or kit embodied in this invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts a schematic of the general antibody structure.
Figure 2 depicts flow cytometric analysis of cells recognized by Hi l.
Figure 3 depicts flow cytometric analysis of cells recognized by Hi l.
Figure 4 depicts flow cytometric analysis of cells recognized by H11. A is A-
375
(melanoma), B is SKMG-I (glioma), C is SK-BR-3 (breast adenocarcinoma), D is
HT-29
(colon adenocarcinoma), E is MB-468 (breast carcinoma), and F is T47D (breast
carcinoma).
Figure 5 depicts binding of H11 to tumor cell extracts. The light bars
represent
H 11 and the dark bars represent control antibody.
Figure 6 depicts binding of H11 to tumor cell extracts.
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Figure 7 depicts binding of H11 to human tumor cell lines by cell-fixed ELISA.
The light bars are H11 IgM and the dark bars are control IgM.
Figure 8 depicts a schematic of the expression vector pSJFI .
Figure 9 depicts the determination of the antigenic similarities between Mab H
11
and H11-scFv by cell fixed ELISA. Reactivity was determined by rabbit antibody
to H11
scFv.
Figure 10 depicts the relative fluorescence intensity of biotinylated H 11-
scFv (thick
line) and BGA scFv (thin line) to lymphoma cells.
Figure 11 depicts the titration of the reactivity of biotinylated HI 1-scFv
for the
binding to lymphonia cells. RAMOS, Daudi. CA-46 and CCRF-CEM cells as
determined
by cell fixed ELISA. The antibody concentrations decrease from an initial 10
g/mL (open
bar) to 5 g/mL. then 2.5 gg/mL and finally, 1.25 g/mL (doubly cross-hatched
line).
Figure 12 depicts the relative fluorescence intensity of HI l-seFv and control
scFv
binding to tumor cell lines. A is A-375 (melanoma), B is SK-BR-3 (breast
adenocarcinoma), C is HT-29 (colon adenocarcinoma). D is CA-46 (Burkitt's
lymphoma),
E is RAMOS (Burkitt's lymphoma), F is H9 (T cell lymphoma), and G is CCRF-CEM
(acute lymphoblastoid lymphoma).
Figure 13 depicts the binding of 1251-H11-scFv to LS174T cells.
Figure 14 depicts the binding of 1 1 1I-H 11-scFv to A375.
Figure 15 depicts the mammalian expression vector pNB2 used to transfect and
express recombinant H 11-IgG.
Figure 16 depicts the mammalian expression vector pNB3 used to transfect and
express recombinant H 1 I-IgG.
Figure 17 depicts the purification of 125I-H11-scFv on P-2 minicolumn (A) and
analysis of 125I-H11-scFv by paper chromatography in 85% methanol (B).
TM
Figure 18 depicts the purification of 125I-H11 IgM on a Sephadex G-25
minicolumn
(A) and analysis of 125I-H1 I IgM by paper chromatography in 85% methanol (B).
Figure 19 depicts the purification of 'In-DTPA-H I 1-scFv on Sephadex G-50
minicolumn (A) and analysis of III In-DTPA-H1 I-scFv by ITLC-SG/0.1M citrate
(B).
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Figure 20 depicts in vivo binding of In-H11-scFv to A375 xenograft tumors in
nude mice.
BEST MODE FOR CARRYING OUT THE INVENTION
This invention encompasses antigen binding fragments exemplified by a newly
identified human Mab that recognizes specifically cancerous cells. This
specificity extends
only to cancer cells, and the antibody does not recognize non-cancerous cells.
The
exemplary antibody is designated H I 1 and the variable regions are encoded by
SEQ ID
NOS: I and 4 (SEQ ID NOS:3 and 6 being the complementary strands of 1 and 4,
respectively) and recognizes the antigen designated "C-antigen." The
specificity of H11
includes, but is not limited to, glioblastoma, neuroblastoma, malignant
melanoma, breast
adenocarcinoma, lung adenocarcinoma, small cell lung carcinoma, colon
adenocarcinoma
and prostate adenocarcinoma.
As shown in the examples herein, H 11 and H 11-scFv do not recognize non-
cancerous cells from all normal tissues tested. H11 and aC antigen binding
fragments are
therefore useful in palliating the clinical conditions related to a wide
variety of cancers.
The invention comprises antigen binding fragments recognizing the antigen HI I
is specific
for (designated C antigen). The invention further comprises polypeptide
derivatives of
Hi I and methods for using these compositions in diagnosis. treatment, and
manufacture of
novel reagents. The invention further encompasses polynucleotides encoding aC,
H 11 and
derivatives thereof. Methods of use thereof are also encompassed by the
invention.
The invention further encompasses a,C derivatives with immunologic specificity
for the C-antigen. These derivatives comprise regions of the polypeptide
sequence
comprising part of the H11 VDJ junction. Also encompassed are regions spanning
at least
one, preferably 2, and more preferably 3 or more of the H 11 CDR amino acid
sequences.
The full sequences of the H11 L and H chain C regions have not been
determined,
but are expected to be identical or nearly identical to those of other human
immunoglobulin molecules. Further, knowledge of the V region amino acid
sequences
allows subcloning with any C region. Such subcloning techniques are well known
in the
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art. The chimeric molecules produced by these cloning techniques are also
encompassed
by the invention.
Screening a commercial heptapeptide phage library with H11 IgM and scFv
antibody clones has shown a very strong consensus sequence at the N-terminus
having the
following amino acid sequence: Phe-His-Arg-Tyr-Ser/Thr. The results are shown
in Table
TABLE 1
IgM H 11 pannings Ml Phe-His-Arg-Tyr-Ser-Leu-Pro
M2 Phe-His-Arg-Tyr-Ser-Asp-Tyr
M3 Phe-His-Arg-Tyr-Ser-Leu-Pro
M4 Phe-His-Arg-Tyr-Ser-Pro-Thr
M7 Phe-His-Arg-Tyr-Thr-Pro-Gly
M8 Phe-His-Arg-Tyr-Ser-Leu-Pro
M 10 Phe-His-Arg-Tyr-Ser-Pro-Thr
sfFv H 11 pannings S2 Phe-His-Arg-Tyr-Ser-Leu-Pro
S5 Met-His-Arg-Tyr-Thr-Pro-Leu
The DNA sequences use multiple codons, indicating quite different phage
origins.
For example, the Arg is coded by triplets CGx and AGx families. In addition,
comparison
of the H 11 pentapeptide consensus with sequence databases showed homology to
the S 100
family of Ca2+ binding proteins. The results are shown in Table 2.
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TABLE 2
H11 pentapeptide Phe-His-Arg-Tyr-Ser/Thr
S. griseus protein Phe-His-Arg-Tyr-Ser (amino acids 251-255)
Peanut stunt virus Phe-His-Arg-Tyr-Ser (amino acids 540-544)
Human calcyclin Phe-His-Lys-Tyr-Ser (amino acids 16-20)
Cystic Fibrosis Ag Tyr-His-Lys-Tyr-Ser (amino acids 16-21)
The consensus pentapeptide sequences described herein are encompassed by the
present invention.
Certain compounds, compositions and methods described in this application
relate
5 generally to aC and derivatives thereof which are routinely generated by
classical
techniques of immunochemistry. This includes aC 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 binds to the C antigen
in a cancer
cell-specific manner. Typically, these derivatives include such immunoglobulin
fragments
10 as Fab, F(ab')2, Fab', scFv (both monomers and polymeric forms) and
isolated H and L
chains. An antigen binding fragment retains the specificity of H 11, although
avidity and/or
affinity may be altered. Especialiy preferred is the H11-scFv described
herein.
The antigen binding fragments (also termed "derivatives" herein) are typically
generated by genetic engineering, although they may alternatively be obtained
by other
15 methods and combinations of methods. This classification includes, but is
not limited to,
engineered peptide fragments and fusion peptides. Preferred compounds include
polypeptide fragments of the H11 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 H 11 antibody V
regions and derivatives thereof. These include isolated polynucleotide
fragments,
recombinant polynucleotides, and therapeutic plasmids and vectors, such as
vaccinia
vectors, comprising the polynucleotides. These polynucleotides are exemplified
by SEQ
ID NOS:1, 3, 4, 6, 13, 15, 16, and 18.
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Pharmaceutical compositions and treatment modalities of this invention are
suitable
for eliciting an immune response against neoplasia. Human cancer patients,
including, but
not limited to, glioblastoma, melanoma, neuroblastoma, adenocarcinoma, glioma,
soft
tissue sarcoma, and various carcinomas (including small cell lung cancer) are
especially
appropriate subjects.
As H 11 has been shown to recognize specifically a variety of carcinomas, it
is
particularly useful in diagnosis, imaging and treatment of carcinomas.
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,
aciite and chronic leukemias, 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.
Additionally, the antigen binding fragments of this invention can be used as
diagnostic and imaging reagents. These applications are described in more
detail in the
sections that follow.
"H11" is an antibody obtained from the fusion of peripheral blood lymphocytes
of
a 64 year old male with a low grade glioma and fused to a human myeloma cell
line to
produce a hybridoma designated NBGM1/H1 l. The generation and characterization
of
H11 is described in Example 1. "aC" represents any antibody, or antigen
binding
fragment thereof, either monoclonal, polyclonal or derivative thereof that
recognizes
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specifically the C antigen and distinguishes between cancer and noncancer
cells. aC
includes H 11.
"Immunologic activity" of aC refers to the ability to specifically bind C
antigen.
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
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."
aC "activity" or "function" refers to any of the immunologic activities of aC,
or to
any other biological activity ascribed to H11 in this disclosure, including
the role of H11 in
the detection, amelioration or palliation of cancer.
The "V region" of H11 refers to the V region of the H11 L chain or the V
region of
the H11 H chain, either alone or in combination. These V regions are depicted
in SEQ ID
NOS: 2 and 5; 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.
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 aC or H11 includes any
polypeptide monomer or polymer with H 11 immunologic specificity, including
the intact
aC antibody, and smaller and larger functionally equivalent polypeptides.
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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 aC 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 aC 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 aC polynucleotide either encodes
a
polypeptide that is functionally equivalent to Hl 1 when produced by an
expression system,
or has similar hybridization specificity as a H 11 polynucleotide when used in
a
hybridization assay. A functionally equivalent fragment of a aC polypeptide
typically has
one or more of the following properties: ability to bind C antigen; ability to
bind 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 H11.
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
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polynucleotide may comprise modified nucleotides, such as methylated
nucleotides and
nucleotide analogs, uracyl, other sugars and linking groups such as
fluororibose and
thioate, and nucleotide branches. The sequence of nucleotides may be
interrupted by non-
nucleotide components. A polynucleotide may 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.
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.
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
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.
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
murine 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.
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"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
5 sequence and operatively linked to a coding sequence other than the native
sequence is a
heterologous promoter.
A "signal peptide" or "leader sequcnce" 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
10 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.
An "isolated" polynucleotide or polypeptide is one that is substantially free
of the
15 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
20 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 "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
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
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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
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,
diminishment of any direct or indirect pathological consequences of the
disease,
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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,
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 manunal,
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 (Sambrook 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
Molecular Biology" (F.M. Ausubel et al., eds., 1987); "PCR: The Polymerase
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 aC conjugated to a chemically functional
moiety.
Typically, the moiety is a label capable of producing a detectable signal.
These conjugated
aC 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
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 may be covalently linked to aC, recombinantly linked, or conjugated
to the aC
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
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 aC 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 11:231-238.
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The aC antibody of this invention can be prepared in several ways. It is most
conveniently obtained from cells engineered to express an antigen binding
fragment
containing SEQ ID NOS:1 and 5 or other polynucleotides encoding aC binding
fragments.
For example, the cells can be cultured in a suitable medium, and spent medium
can be used
5 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
inununologically naive histocompatible or immunotolerant mammal, especially a
mouse.
10 The mammal may be primed for ascites production by prior administration of
a suitable
composition; e.g., Pristane.
Alternatively, aC 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
15 synthesizers are commercially available, such as those manufactured by
Applied
Biosystems, Inc. (Foster City, CA).
aC may also be obtained by employing routine recombinant methods such as
described in Sambrook et al. (1989). For instance, using the amino acid and
polynucleotide (SEQ ID NOS:1-6, and 13-18) sequences and information provided
herein,
20 a polynucleotide encoding either the aC 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 aC may be produced separately, and then combined by
disulfide bond
25 rearrangement. Alternatively, vectors with separate polynucleotides
encoding each chain
of aC, or a vector with a single polynucleotide encoding both chains as
separate
transcripts, may be transfected into a single host cell which may 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 aC
thus
produced can be purified using standard techniques in the art. Polynucleotides
encoding
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aC for use in the production of aC can in turn be obtained from a hybridoma
producing a
aC antibody, or produced synthetically or recombinantly from the DNA sequences
provided herein.
Another method of obtaining aC is to immunize suitable host animals with C
antigen and to follow standard procedures for polyclonal or Mab production.
Mabs thus
produced can be "humanized" by methods known in the art. Examples of humanized
antibodies are provided, for instance, in United States Patent Nos. 5,530,101
and
5,585,089.
"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
immunoglobuiins. 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 HI 1 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 murine 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. (1987). Various computer programs such
as
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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 H 1 I antibody is a human immunoglobulin of the IgM
subclass, and may be isolated by any technique suitable for immunoglobulins of
this
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. H 11 may also be purified on affinity columns comprising the C
antigen;
for example, in the form of a purified Abl or Ab3. Preferably, HI l 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 aC. In one example, each L-H chain pair binds different
epitopes of the C
antigen. Such hybrids may also be formed using humanized H or L chains.
Another aC 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. A recombinant antibody may also be designed to aid the specific
delivery of a
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substance (such as a cytokine) to a tumor cell. 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 aC 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 aC is
purified.
The aC antibodies may be used for a number of purposes. These include
eliciting
an antibody response to produce aaC which can then be used to elicit a T cell
response to
aC or the C antigen and treating various types of cancer. These uses are
elaborated more
fully in a later section.
The invention encompasses polypeptide fragments of aC containing at least a
portion of a V region of aC. Preferred fragments are those with the
immunologic activity
of H 11. Also preferred are fragments which comprise amino acid sequences
substantially
different from other immunoglobulins, and fragments comprising a CDR. In one
embodiment, the invention includes a polypeptide fragment of the aC H chain V
region,
comprising at least 25 consecutive amino acids, more preferably 30 consecutive
aminq
acids of SEQ ID NO:2, or 5 consecutive amino acids of the CDRI 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 aC L
chain V
region, comprising at least 25 consecutive amino acids, more preferably 30
consecutive
amino acids of SEQ ID NO:5, or 7 consecutive amino acids of the CDR2 thereof,
or at
least 8 consecutive amino acids, preferably 10 consecutive amino acids of the
CDRI or
CDR3 thereof.
The size of the aC polypeptides need be only the minimum size required to
provide
a desired function. The polypeptides can optionally comprise additional
sequence, either
native to aC, or from a heterologous source, as desired. aC peptides need
contain only 5
consecutive amino acids from a H11 V region sequence that are not the same as
the
homologous region of A6. Polypeptides comprising 7 amino acids, more
preferably about
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amino acids, more 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 aC L or H chain V region are also included. Even more preferred are
polypeptides
comprising the entire aC L or H chain V region. Preferably the polypeptides
are derived
5 from H11. Preferably, the polypeptides are the scFvs depicted in SEC ID NOS:
14 and 17.
The invention includes modified aC polypeptides which are functionally
equivalent
to H11, or have altered but measurable H11 immunologic activity. Modified
polypeptides
with improved H 11 immunologic activity are preferred. Examples of modified
polypeptides include those with conservative substitutions of amino acid
residues, and one
10 or more deletions or additions of amino acids which do not significantly
deleteriously alter
the immunologic activity.
One example of this is H11 polypeptides comprising one or more amino acid
substitution in comparison with the prototype H11 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
acidlglutamic acid; serine/threonine/methionine; lysine/arginine; and
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).
H11 polypeptide derivatives comprising both a H11 L chain and a H11 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.
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H chain loss mutants can be obtained by treating approximately 2 x 107 H11
producing cells with fluorescein-labeled rabbit anti-mouse IgG (H chain
specific, DAKO
Corporation, Carpinteria, CA) according to the supplier's instruction. The
stained and
unstained cell populations are analyzed in a fluorescence-activated cell
sorter. The
5 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
H 11 (-H) mutants. mRNA from putative H chain loss mutant H 11 (-H) clones can
be isolated
10 and the sequence of the L chain V region cDNA determined. Reverse PCR of
the mRNA
for the HI 1 VH is performed with 2 sets of 5'- and 3'- primers, used for
cloning of Hl 1(-H).
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 aC derivative encompassed by this invention is an antibody in which
the
15 aC 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 C region domain. Changes contemplated
affect
complement fixation, interaction with membrane receptors, and other effector
functions. A
20 recombinant H11 antibody can also be designed to aid the specific delivery
of a substance
(such as a lymphokine) to an effector cell. 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.
The invention also encompasses single chain V region fragments ("scFv") of H1
1.
25 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)3, which bridges approximately 3.5 mn between the carboxy terminus of
one V
30 region and the amino terminus of another V region. Other linker sequences
can also be
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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 H 11, 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 H11 polypeptide and another component is a different polypeptide, such as
a T cell
epitope.
The scFvs can be assembled in any order, for example, Vu-(linker}-VL or
VL-(linker)-VH. For example, SEQ ID NOS:13 and 16 show H11-scFv2 constructs
having the form VL-(linker)-VH. However, the construct shown in SEQ ID NO: 13
forms monomers, while the construct shown in SEQ ID NO: 16 forms dimers. There
may
be a difference in the level of expression of these two configurations in
particular
expression systems, in which case one of these forms may be preferred. Tandem
scFvs can
also be made, such as (X)-{linker)-(X)---(linker)--{X), in which X are aC
polypeptides,
or combinations of aC polypeptides with other polypeptides. In another
embodiment,
single chain antibody polypeptides have no linker polypeptide, or just a
short, inflexible
linker. Exemplary configurations include V[7-VH and VFi-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 Escherichia coli, and the protein
expressed by
the polynucleotide can be isolated using standard protein purification
techniques.
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A particularly useful system for the production of scFvs is plasmid pET-22b(+)
(Novagen, Madison, WI) in E. coli. 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 NO:2
and,
at least 10 consecutive amino acids of SEQ. ID NO:5, especially wherein the
amino acids
of SEQ. ID NO:2 and the amino acids of SEQ. ID NO:5 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
H11.
The invention also encompasses polymeric forms of aC polypeptides, containing
a
plurality of aC polypeptides. One embodiment is a linear polymer of aC
polypeptides,
optionally conjugated to carrier. These linear polymers can comprise multiple
copies of a
single aC polypeptide, or combinations of different aC polypeptides, and can
have tandem
aC polypeptides, or aC polypeptides separated by other amino acid sequences.
Another
embodiment is aC multiple antigen peptides (MAPs). MAPs have a small
immunologically inert core having radially branching lysine dendrites, onto
which a
number of aC 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 aC polypeptides to core. MAPs are
efficient
immunogens and useful antigens for immunoassays. The core for creating an aC
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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When using aC 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.
aC 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. Using
a series of
polypeptides of 20 or 50 amino acids in length, each aC 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 D2, or those involved in idiotype-anti-idiotype contact.
The invention further encompasses various adaptations of aC described in this
section combined in various fashions to yield other ctC polypeptides with
desirable
properties. For instance, aC polypeptides with modified amino acid residues
can be
comprised in a MAP. In another example, a aC 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 aC 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 aC 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.
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aC 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 aC
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 H 11 polypeptide is expressed in a
suitable
storage medium such as a plant seed, the H 11 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.
The invention also encompasses methods of detecting C antigen in a biological
sample. The methods include obtaining a biological sample, contacting the
sample with
aC under conditions that allow antibody antigen binding and detecting binding,
if any, of
the antibody to the antigen.
The invention also encompasses methods of detecting anti-H 11 or anti-aC in a
biological sample. Anti-aC is detectable whenever it cross-reacts with H11.
Anti-aC with
this activity can spontaneously arise during the course of a tumor-associated
disease. Anti-
aC with this activity is especially likely in individuals who have received a
course of
therapy with aC. These methods are applicable in a clinical setting, for
example, for
monitoring antibody levels in an individual, as well as an industrial setting,
as in
commercial production of anti-H 11 or anti-aC.
The assay methods entail contacting any anti-H11 or anti-aC target antibody in
the
sample with a H11 antibody or polypeptide under conditions suitable to allow
the
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formation of a stable complex between the target and H 11, and detecting any
stable
complex formed. The sample is suitably prepared before conducting the assay,
optionally
by enriching for antibody concentration. When using intact murine aC, it is
generally
preferable to deplete the sample of any anti-mouse immunoglobulin activity
that may be
5 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 aC and other reagents such as humanized aC or H 11, with
fewer
10 mouse determinants are appropriate.
After the sample is suitably prepared, it is mixed with a excess aC under
conditions
that permit formation of a complex between aC 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
the range
15 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 125I, enzymes such
as peroxidase
and (3-galactosidase, or fluorochromes such as fluorescein.
The invention provides various polynucleotides encoding the antibody H 11 or
20 fragments of H 11, based on the polynucleotide sequences provided herein
(SEQ ID NOS:1
and 4). Various embodiments are described in this section, comprising a number
of
different combinations of the H11 H or L chain V region sequences. In general,
a HI 1
polynucleotide of this invention encodes at least one feature that is unique
to the H 11
molecule (in comparison with other immunoglobulins and, in particular, the A6
antibody).
25 Preferably, this feature is related in some way to an immunologic
reactivity of H11.
The invention encompasses polynucleotides encoding a portion of the H11 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 NO:4. The invention
also
30 encompasses a polynucleotide encoding a portion of the H 11 L chain V
region, comprising
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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 H 11 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 H11 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 Hl 1 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 H 11
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.
The invention includes isolated HI 1 polynucleotides encoding a polypeptide
having immunologic activity of H 11, wherein the polypeptide encodes at least
5 amino
acids of a V L chain of H 11 as depicted in SEQ. ID NO:5. The invention also
includes
isolated H11 polynucleotides encoding a polypeptide having immunologic
activity of H11,
wherein the polynucleotide encodes at least 5 amino acids of a V H chain of H
I 1 as
depicted in SEQ. ID NO:2. The polynucleotide sequence can be similar to those
depicted
in SEQ. ID NO:1 or SEQ ID NO: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 NO:2 or SEQ ID NO:5, to design such
polynucleotides. Preferred polynucleotides encode at least five amino acids of
a H11
CDR.
The invention also encompasses polynucleotides encoding for functionally
equivalent variants and derivatives of H11 and functionally equivalent
fragments thereof
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which may enhance, decrease or not significantly affect properties of the
polypeptides
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,ityrosine/tryptophan.
The polynucleotides of the invention may 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
desirab-le 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
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
H 11, 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 H11 encoding sequences to which the test polynucleotide binds
are those
shown in SEQ. ID NOS:1 and 4. Since the known immunoglobulin sequences fall
into a
hierarchy of similarity with that of H 1l, the test may be performed by
comparing the
hybridization of the test polynucleotide with the H 11 sequence with the
hybridization with
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the most closely related sequences. Preferred is a panel of about 10 of the
most closely
related sequences to SEQ. ID NO:1, 3, 4 or 5.
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, I 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 5
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, I x SSC, 0.1 x SSC, or deionized water.
Useful H 11 polynucleotides encoding fragments of H 11 may be identified by
generating polynucleotide fragments (based on SEQ ID NO:1 or SEQ ID NO: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 aC
polypeptide with
desirable properties, polynucleotides can be designed that encode the
polypeptide.
Included in all these embodiments are polynucleotides with encoding regions
for
H11 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.
Alternatively, aC polynucleotide sequences can be obtained from a aC antibody
producing cell line, aC cloning vector, or aC expression vector. RNA or DNA
encoding
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the desired sequence can be isolated, amplified, and processed by standard
recombinant
techniques. Such techniques include digestion with restriction nucleases, and
amplification by polymerase chain reaction (PCR), or a suitable combination
thereof. PCR
technology is described in U.S. Patent Nos. 4,683,195, 4,800,159, 4,754,065
and
4,683,202, as well as PCR: The Polymerase Chain Reaction, Mullis et al. eds.,
Birkauswer Press, Boston (1994).
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 H
11
polynucleotide. These vectors can be used for expression of recombinant
polypeptides are
also a source of H11 polynucleotides. Cloning vectors can be used to obtain
replicate
copies of the H11 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 H11
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
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
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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,
5 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
10 selected from a large number of cloning vectors availabie in the art. While
the cloning
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., pUC18, mp18, mp19, pBR322, pMB9, ColEl,
pCRI,
15 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 aC polypeptide of interest. The polynucleotide
encoding aC
polypeptide is operatively linked to suitable transcriptional controlling
elements, such as
20 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 H11 gene, or heterologous (i.e.,
derived from
other genes or other organisms). A polynucleotide sequence encoding a signal
peptide can
25 also be included to allow a aC 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
example of an expression vector is pcDNA3 (Invitrogen, San Diego, CA), in
which
transcription is driven by the cytomegalovirus (CMV) early promoter/enhancer.
This
30 vector also contains recognition sites for muitiple restriction enzymes for
insertion of an
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aC 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 aC polynucleotide. Suitable
systems are
described for instance by Brown et al. (1994) Virol. 198:477-488; 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 aC polynucleotides will often depend on features of
the host cell.
Once introduced into a suitable host cell, expression of a aC polypeptide can
be
determined using any assay known in the art. For example, presence of aC
polypeptide
can be detected by RIA or ELISA of the culture supernatant (if the H11
polypeptide is
secreted) or cell lysates.
A particularly useful expression vector for Hl 1 polynucleotides is a vaccinia
virus
comprised of a H11 polynucleotide sequence, which can also be used in vaccine
preparations. Moss (1991) Science 252:1662-1667. To introduce polynucleotide
sequences encoding H11 polypeptide, including H11 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 Hi I polypeptide-encoding polvnucleotide sequence
into the
vaccinia virus genome. Typically, the H1 I polynucleotide is inserted into the
viral TK
(thymidine kinase) gene. Insertion into the TK site attenuates the virus more
than 10,000
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 H11 polynucleotide is under the control of the
vaccinia
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early/late promoter (7.5 K), whereby the resultant Hl 1 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 H
11
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
aC
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 5' untranslated
region that
are capable of forming a hairpin loop. Also embodied in this invention are
vaccinia
vectors encoding for recombinant aC variants, such as scFvs, chimeras, and
polymers.
Other embodiments of this invention are host cells transformed with aC
polynucleotides and vectors comprising aC polynucleotide sequences, as
described above.
Both prokaryotic and eukaryotic host cells may be used. Prokaryotic hosts
include
bacterial cells, for example E. colf 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
aC
polynucleotides, or as vehicles for production of aC polynucleotides and
polypeptides.
They may also be used as vehicles for in vivo expression of aC polypeptides.
The H11
polynucleotides of this invention can be used in expression systems to produce
H11
polypeptides, intact H 11, or recombinant forms of H 11, such as are described
below.
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The polynucleotides of this invention have several uses. They are useful, for
example, in expression systems for the production of aC. They are also useful
as
hybridization probes to assay for the presence of aC 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
aC
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
polynucleotide is then contacted with the HI I polynucleotide probe under
conditions that
permit a stable duplex to form if the sample contains a matching aC sequence.
Any stable
duplexes formed are detected by any suitable means. For example, the aC
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 aC encoding sequences.
A short aC 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 aC
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 aC polynucleotides to an individual. In
these
methods, a aC polynucleotide encoding a ctC polypeptide is administered to an
individual,
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either directly or via cells transfected with the aC polynucleotide.
Preferably, the aC
polynucleotide is in the form of a circular plasmid, preferably in a
supercoiled
configuration. Preferably, the aC polynucleotide is replicated inside a cell.
Thus, the aC
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 affinity and/or avidity.
To determine whether plasmids containing aC polynucleotides are capable of aC
expression in eukaryotic cells, cells such as COS-7, CHO, or HeLa can be
transfected with
the plasmids. Expression of aC is then determined by immunoassay; for example,
by
Western blot. Smaller aC polypeptides can be detected, for example, by
constructing the
plasmid so that the resultant aC polypeptide is fused with a tag, such as a
target epitope or
enzyme label. Further characterization of the expressed aC 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 aC
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.
aC polypeptides can be characterized in several ways. For instance, a aC
polypeptide may be tested for its ability to bind specifically to cancer
cells, for its ability to
specifically inhibit the binding between cancer cells and intact H11. A aC
polypeptide can
also react with anti-CDR3 polypeptides. aC 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
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the scope of this invention, although preferably more than one of these
properties is
present.
The ability of a aC polypeptide to bind cancer cells or antigenic fractions
thereof
can be tested by immunoassay. Any form of direct binding assay is suitable. In
one such
5 assay, the cancer cell or the putative aC polypeptide is labeled. Suitable
labels include
radioisotopes such as 125I, 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 component. the solid
phase is
10 washed and the amount of bound label is determined. Another such assay is a
sandwich
assay, in which the putative aC polypeptide is captured by a first anti-
immunoglobulin on
a solid phase and developed with aC antibody. In either of these examples, the
extent of
binding of aC is directly related to the amount of label bound to the solid
phase.
To conduct the inhibition assays, the putative aC polypeptide is titered for
its
15 ability to decrease the binding of H 11 to cancer cells. Either of the
binding pairs in the
reaction to be inhibited is labeled, while the other is typically
insolubilized in order to
facilitate washing. The putative aC polypeptide is typically mixed with the
labeled
component, and then the mixture is combined with the solid phase. Polypeptides
with the
characteristics of H11 will proportionatelv decrease the amount of label
attached to the
20 solid phase, compared with control polypeptides. This test may be more
sensitive than
measuring direct binding, because lower affinity interaction between aC and C
antigen
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 present invention encompasses pharmaceutical compositions and immunogenic
25 compositions containing aC 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 aC antibody, or a
30 polynucleotide or a polypeptide derivative thereof as an active ingredient
is conducted in
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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.
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
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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.
Also included in this invention are methods for treating cancer. The methods
comprise administering an amount of a pharmaceutical composition containing aC
effective to achieve the desired effect, be it palliation of an existing tumor
mass or
prevention of recurrence. For treatment of cancer, the amount of a
pharmaceutical
composition administered is an amount effective in producing the desired
effect. An
effective amount can be provided in one or a series of administrations.
The effective amount of aC 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
gg to 20 mg,
preferably 250 gg to 10 mg, more preferably 300 gg to 5 mg, even more
preferably 500 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.
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
therapy. Suitable active agents include the anti-neoplastic drugs and
bioresponse modifiers
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described above and effector cells such as those described by Douillard et al.
(1986)
Hybridomas (Supp. 1:5139).
When used for immunotherapy, aC can be unlabeled or labeled with a therapeutic
agent as described above. These agents can be coupled either directly or
indirectly to the
polypeptides of the invention. One example of indirect coupling is by use of a
spacer
moiety. These spacer moieties, in turn, can be either insoluble or soluble
(Diener et al.
(1986) Science 231:148) and can be selected to enable drug release from aC at
the target
site. Alternatively, an aC and a therapeutic agent can be translated,
synthesized, ligated or
otherwise produced as a single molecule which has both aC and therapeutic
agent
functions. Examples of therapeutic agents which can be coupled to aC for
immunotherapy
include, but are not limited to, bioresponse modifiers, drugs, radioisotopes,
lectins, and
toxins. Bioresponse modifiers include lymphokines which include, but are not
limited to,
tumor necrosis factor, interleukins 1, 2, and 3, lymphotoxin, macrophage
activating factor,
migration inhibition factor, colony stimulating factor, and interferon.
Interferons with
which aC can be labeled include a-interferon, (3-interferon, and y-interferon
(IFN-y) and
their subtypes.
In using radioisotopically conjugated aC for immunotherapy, certain isotypes
may
be more preferable than others depending on such factors as leukocyte
distribution as well.
as isotype stability and emission. If desired, the maiignant cell distribution
can be
evaluated by the in vivo diagnostic techniques described below. Depending on
the
malignancy, some emitters may be preferable to others. In general, alpha and
beta particle-
emitting radioisotopes are preferred in immunotherapy. For example, if an
animal has
solid tumor foci, as in a carcinoma, a high energy beta emitter capable of
penetrating
several millimeters of tissue, such as 90Y, may be preferable. On the other
hand, if the
malignancy consists of simple target cells, as in the case of leukemia, a
short range, high
energy alpha emitter, such as 212Bi, may be preferable. Radioisotopes which
can be bound
to the antigen binding fragments of the invention for therapeutic purposes
include, but are
not limited to, 125I1131 I, 90 Y, 67 Cu, 212Bi, 211 At, 212Pb, 47Sc, 109Pd,
and ' ggRe.
Lectins are proteins, usually isolated from plant material, which bind to
specific
sugar moieties. Many lectins are also able to agglutinate cells and stimulate
lymphocytes.
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However, ricin is a toxic lectin which has been used immunotherapeutically.
This is
preferably accomplished by binding the alpha-peptide chain of ricin, which is
responsible
for toxicity, to the antibody molecule to enable site specific delivery of the
toxic effect.
Toxins are poisonous substances produced by plants, animals, or microorganisms
that, in sufficient dose, are often lethal. Diphtheria toxin is a substance
produced by
Corynebacterium diphtheria which can be used therapeutically. This toxin
consists of an
alpha and beta subunit which under proper conditions can be separated. The
toxic A chain
component can be bound to an antibody and used for site specific delivery to a
neoplastic
cell.
Thus, for example, aC can be used in combination with alpha-interferon. This
treatment modality enhances Mab targeting of melanomas by increasing the
expression of
Mab reactive antigen by the melanoma cells. Greiner et al. (1987) Science
235:895.
Alternatively, aC could be used, for example, in combination with IFN-y to
thereby
activate and increase the expression of Fc receptors by effector cells which,
in turn, results
in an enhanced binding of the antigen binding fragments to the effector cell
and killing of
target inalignant cells. Those of skill in the art will be able to select from
the various
biological response modifiers to create a desired effector function which
enhances the
efficacy of K.
When aC is used in combination with various therapeutic agents. such as those
described herein, the administration of both usually occurs substantially
contemporaneously. The term "substantially contemporaneously" means that they
are
administered reasonably close together with respect to time. Usually, it is
preferred to
administer the therapeutic agent before K. For example, the therapeutic agent
can be
administered I to 6 days before aC. The administration of the therapeutic
agent can be
daily, or at any other suitable interval, depending upon such factors, for
example, as the
nature of the malignancy, the condition of the patient and half-life of the
agent.
Using aC, it is possible to design combination therapies. It may be desirable
to
administer a therapeutic agent, or agents, prior to the administration of aC
in combination
with effector cells and the same, or different, therapeutic agent or agents.
For example,
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patients can be treated by first administering IFN-y and interleukin-2 (I1-2)
daily for 3 to 5
days, and on day 5 administering aC in combination with effector cells, IFN-y,
and 11-2.
The present invention also encompasses the use of liposomes with membrane
bound aC to specifically deliver the liposome to the area of the tumor or
neoplastic cells
5 expressing C antigen. These liposomes can be produced such that they
contain, in addition
to aC, such immunotherapeutic agents as those described above which would then
be
released at the site of malignancy. Wolff et al. (1984) Biochem. Biophys. Acta
802:259.
Another such delivery system described by Brown et al. (1994) Virology 198:477-
488; and
Miyamura et al. (1994) Proc. Natl. Acad. Sci. USA 91:8507-8511 utilizes
chimeric
10 parvovirus B 19 capsids for presentation of the antigen binding fragments.
Such chimeric
systems are encompassed for use in the claimed methods.
The dosage ranges for the administration of aC 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
15 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 500 mg/kg,
in one or
more dose administrations daily, for one or several days. Generally, when aC
are
20 administered conjugated with therapeutic agents, lower dosages, comparable
to those used
for in vivo immunodiagnostic imaging, can be used.
Therapeutic compositions of aC can be administered by injection or by gradual
perfusion. The aC antigen binding fragments can be administered intravenously,
intraperitoneally, intramuscularly, subcutaneously, intracavity, intrathecally
or
25 transdermally, alone or in combination with effector cells.
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. (1987) Lymphokine Res. 6:45; Rabinowich et
al.
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(1987) Cancer Res. 47:173; Rosenberg et al. (1989) Science 233:1318; and Pizz
et al.
(1984) Int. J. Cancer 34:359.
aC is particularly suitable for use in treating and imaging brain cancer. When
the
site of delivery is the brain, the therapeutic agent must be capable of being
delivered to the
brain. The blood-brain barrier limits the uptake of many therapeutic agents
into the brain
and spinal cord from the general circulation. Molecules which cross the blood-
brain
barrier use two main mechanisms: free diffusion; and facilitated transport.
Because of the
presence of the blood-brain barrier, attaining beneficial concentrations of a
given
therapeutic agent in the CNS may require the use of specific drug delivery
strategies.
Delivery of therapeutic agents to the CNS can be achieved by several methods.
One method relies on neurosurgical techniques. In the case of gravely ill
patients,
surgical intervention is warranted despite its attendant risks. For instance,
therapeutic
agents can be delivered by direct physical introduction into the CNS, such as
intraventricular, intralesional, or intrathecal injection. Intraventricular
injection can be
facilitated by an intraventricular catheter, for example, attached to a
reservoir, such as an
Ommaya reservoir. Methods of introduction are also provided by rechargeable or
biodegradable devices. Another approach is the disruption of the blood-brain
barrier by
substances which increase the permeability of the blood-brain barrier.
Examples include
intra-arterial infusion of poorly diffusible agents such as mannitol,
pharmaceuticals which
increase cerebrovascular permeability such as etoposide, or vasoactive agents
such as
leukotrienes. Neuwelt and Rappoport (1984) Fed. Proc. 43:214-219; Baba et al.
(1991) J.
Cereb. Blood Flow Metab. 11:638-643; and Gennuso et al. (1993) Cancer Invest.
11:638-
643.
Further, it may be desirable to administer the compositions locally to the
area in
need of treatment; this can be achieved by, for example, local infusion during
surgery, by
injection, by means of a catheter, or by means of an implant, said implant
being of a
porous, non-porous, or gelatinous material, including membranes, such as
silastic
membranes, or fibers. A suitable such membrane is Gliadel provided by
Guilford
Pharmaceuticals Inc.
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Another method involves pharmacological techniques such as modification or
selection of the aC to provide an analog which will cross the blood-brain
barrier.
Exampies include increasing the hydrophobicity of the molecule, decreasing net
charge or
molecular weight of the molecule, or modifying the molecule, such as to
resemble one
normally transported across the blood-brain barrier. Levin (1980) J. Med.
Chem. 23:682-
684; Pardridge (1991) in: Peptide Drug Delivery to the Brain; and Kostis et
al. (1994) J.
Clin. Pharmacol. 34:989-996.
Encapsulation of aC in a hydrophobic environment such as liposomes is also
effective in delivering drugs to the CNS. For example, WO 91/04014 describes a
liposomal delivery system in which the drug is encapsulated within liposomes
to which
molecules have been added that are normally transported across the blood-brain
barrier.
Another method of formulating aC to pass through the blood-brain barrier is
encapsulation in cyclodextrin. Any suitable cyclodextrin which passes through
the blood-
brain barrier can be employed, including, but not limited to, 0-cyclodextrin,
y-cyclodextrin
and derivatives thereof. See generally, U.S. Patent Nos. 5,017,566, 5,002,935
and
4,983,586. Such compositions can also include a glycerol derivative as
described by U.S.
Patent No. 5,153,179.
Yet another method takes advantage of physiological techniques such as
conjugation of aC to a transportable agent to yield a new chimeric
transportable aC. For
example, vasoactive intestinal peptide analog (VIPa) exerts its vasoactive
effects only after
conjugation to a Mab to the specific carrier molecule transferrin receptor,
which facilitates
the uptake of the VIPa-Mab conjugate through the blood-brain barrier.
Pardridge (1991);
and Bickel et al. (1993) Proc. Natl. Acad. Sci. USA 90:2618-2622. Several
other specific
transport systems have been identified, these include, but are not limited to,
those for
transferring insulin, or insulin-like growth factors I and II. Other suitable,
non-specific
carriers include, but are not limited to, pyridinium, fatty acids, inositol,
cholesterol, and
glucose derivatives. Certain prodrugs have been described whereby, upon
entering the
central nervous system, the drug is cleaved from the carrier to release the
active drug. U.S.
Patent No. 5,017,566.
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Suitable subjects 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
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 aC treatment prophylactically to prevent the occurrence of
cancer
until it is suitable to perform preventive surgery.
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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, aC therapy can be conducted before or during
traditional
therapy of a patient with high tumor burden, and continued after the tumor
becomes
clinically undetectable. aC 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.
Various compounds and compositions of this invention have other clinical
indications, of which the following section provides only a survey.
One indication is the treatment of cells ex vivo. This may be desirable for
experimental purposes, or for treatment of an individual with a neoplastic
disease. In one
example, aC is administered to a culture of cells, such as peripheral blood
cells obtained
from a donor, or a suitable cell line. About 0.5 to 2 g/mL of H11 is an
effective dose for
this purpose. In a second example, donor cells are genetically altered with an
expression
vector of this invention, to provide for ongoing secretion of aC after
administration of the
cells to the recipient.
The present invention further encompasses methods for in vivo detection of
cancer
cells. A diagnostically effective amount of detectably labeled aC is given to
the subject in
need of tumor imaging. The term "diagnostically effective" means that the
amount of
detectably labeled aC is administered in sufficient quantity to enable
detection of the
neoplasia.
The concentration of detectably labeled aC which is administered should be
sufficient such that the binding to those cells having C antigen is detectable
compared to
the background. Further, it is desirable that the detectably labeled aC be
rapidly cleared
from the circulatory system in order to give the best target-to-background
signal ratio.
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As a rule, the dosage of detectably labeled aC for in vivo diagnosis is
somewhat
patient-specific and depends on such factors as age, sex, and extent of
disease. The dosage
of aC can vary from about 0.01 mg/m2 to about 500 mg/mZ, preferably 0.1 mg/m2
to about
200 mg/mz, most preferably about 0.1 mg/m2 to about 10 mg/m2. Such dosages may
vary,
5 for example, depending on number of injections given, tumor burden, and
other factors
known to those of skill in the art. For instance, tumors have been labeled in
vivo using
cyanine-conjugated Mabs. Ballou et al. (1995) Cancer Immunol. Immunother.
41:257-263.
For in vivo diagnostic imaging, the type of detection instrument available is
a major
factor in selecting a given radioisotope. The radioisotope chosen must have a
type of
10 decay which is detectable for a given type of instrument. Still another
important factor in
selecting a radioisotope for in vivo diagnosis is that the half-life of the
radioisotope be long
enough so that it is still detectable at the time of maximum uptake by the
target, but short
enough so that deleterious radiation with respect to the individual is
minimized. Ideally, a
radioisotope used for in vivo imaging lacks a particle emission, but produces
a large
15 number of photons in the 140-250 keV range, to be readily detected by
conventional
gamma cameras. For imaging, doses of III In-H1 l-scFv (for instance, 2 mg of
scFv labeled
with 5 mCi of ". Indium) the range administered is about 0.01 mg to 20 mg,
more
preferably about 0.1-10 mg and even more preferably about 1-5 mg per patient.
For in vivo diagnosis, radioisotopes can be bound to aC either directly or
indirectly
20 by using an intermediate functional group. Intermediate functional groups
which are often
used to bind metallic ion radioisotopes to immunoglobulins are the
bifunctional chelating
agents such as diethylene triaminepentacetic acid (DTPA) and
ethylenediaminetetraacetic
acid (EDTA) and similar molecules. Typical examples of metallic ions which can
be
bound to aC are I I I In, 97Ru, 67 Ga, 68Ga, 72As, 89Zr, 9 Y, and 201T1.
25 aC can also be labeled with a paramagnetic isotope for purposes of in vivo
diagnosis, as in magnetic resonance imaging (MRI) or electron spin resonance
(ESR). In
general, any conventional method for visualizing diagnostic imaging can be
utilized.
Usually, gamma and positron emitting radioisotopes are used for camera imaging
and
paramagnetic isotopes for MRI. Elements which are particularly useful in such
techniques
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include 157Gd, 55Mn, 162DY, 52Cr, and 56Fe. aC can also be labeled with a
fluorescent dye
for the purpose of in vivo diagnosis.
aC can also be used to detect neoplasias using in vitro assays. Samples are
taken
from the patient and subject to any suitable immunoassay with aC to detect the
presence of
C antigen. This is particularly useful in detecting lymphomas and leukemias
where the
tumor cells bearing C antigen are circulating in the patient's bloodstream.
aC can also be used to monitor the course of amelioration of malignancy in an
individual. Thus, by measuring the increase or decrease in the number of cells
expressing
C antigen or changes in the concentration of C antigen present in various body
fluids, it is
possible to determine whether a particular therapeutic regimen aimed at
ameliorating the
malignancy is effective.
The present invention encompasses kits containing aC. Diagnostic procedures
using aC 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.
Each kit comprises aC used for detecting C antigen in the sample. Optionally,
the
reagent 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 aC. Labeled 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 aC
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,
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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
H1 l,
along with H11 encoding polynucleotides, H11 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 H11, which is provided herein.
The
following examples are provided to illustrate but not limit the claimed
invention.
EXAMPLE I
Method of obtaining Mab H I l
Mab NBGM 1/H 11 ("H I 1"), is a human monoclonal IgM antibody reactive against
the following human tumor tissues and corresponding tumor cell lines: glioma,
malignant
melanoma, colon adenocarcinoma and breast adenocarcinoma. In vitro
characterization of
Mab NBGM 1/H 11 is shown in Example 2.
Fusion of H11 was accomplished by fusing 8 x 106 peripheral blood lymphocytes
obtained from a 64 year old male with a low grade glioma with the TM-H2-SP2
human
myeloma cell line. The TM-H2-SP2 cell line is the immunoglobulin nonsecreting
subline
of the IgG(K) parental cell line TM-H2, a hypoxanthine guanine
phosphoribosyltransferase
(EC 2.4.2.8)-deficient derivative of an unknown human myeloma-like line
selected in
0.8% methylcellulose for its resistance to 6-thioguanine (6 g/mL) and failure
to grow in
hypoxanthine-aminopterin-thymidine medium. The karyotype of TM-H2-SP2 is 46 2,
XX.
The resultant viable hybridoma cells were split among 40 microwells at a
density of
2 x 10 5 cells/mL and 0.2 mL/well. The frequency of outgrowth from fusion H 11
was 12 of
40 (30%) potential hybridoma-containing wells. Outgrowth resulting from
sustained
growth is defined as prolonged growth with culture expansion for periods
longer than 3
months; instances of hybridoma growth failure occurring later than 3 months
post-fusion
were not observed.
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Screening of hybridoma clones was performed by antigen-capture enzyme-linked
immunosorbent assay (ELISA) in microtiter plates using polyclonal anti-human
IgM or
IgG as coating antigen. A hybridoma culture supernatant was positive if the
measured
optical density (O.D.) value exceeded the mean background level of a control
culture
supernatant by greater than two standard deviations.
Selection of hybridoma clone NBGM1/H11 was performed by cell-fixed ELISA.
Culture supernatants from 6 microtiter wells, which tested high for IgM or IgG
secretion,
were screened against previously attached and fixed human tumor cell lines:
Glioblastoma
(SKMG-1 and D-54MG); melanoma (A-375); and colon adenocarcinoma (SK-CO-1). A
hybridoma supernatant was considered to be positive if the measured O.D. value
exceeded
the mean background level of control culture supernatants by greater than two
standard
deviations. Mab produced by hybridoma NBGM1/H1 l continues to be reactive
against
these tumor cell lines. The "H 11 " antibodies are IgM(k).
Characterization of the hybridoma NBGM I/H I I seed bank was performed by
Microbiological Associates (Rockville, MD). The cells tested negative for (1)
bacterial
and fungal contamination, (2) mycoplasma contamination, (3) HIV-1 and HIV-2
antigens
and (4) HTLV-1 and HTLV-2 antigens.
The methods used for the characterization of Mab NBGM1/H11 include: antigen-
capture ELISA, antigen ELISA, cell-fixed ELISA, flow cytometry,
immunoperoxidase
staining of human tumor cell lines and immunohistochemistry of human tumor and
normal
tissues (see following examples).
Binding characteristics of this human Mab to human tumor cell lines as
determined
by flow cytometry, immunoperoxidase staining, cell-fixed ELISA and antigen
ELISA (i.e.,
tumor cell freeze-thaw extracts) are presented below.
MP
Binding of Mab H11 to Human Glioblastoma (SKMG-1) and Melanoma (A375)
Cell Lines by Flow vtometric Analy,i
In order to determine the binding of Mab H11 to tumor cells, tumor cells
growing
in T-flasks were detached by incubation with PBS-EDTA. Cells were collected by
low
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speed centrifugation, washed with ice-cold PBS-1 % FBS, centrifuged and the
supernatant
aspirated. The cell pellet was resuspended in culture medium spiked with one
of the
following: a control human melanoma IgM; hybridoma NBGM 1/H 11 culture
supernatant;
or PBS containing purified Mab H11; and incubated on ice for 30 minutes. After
incubation, the cells were collected by centrifugation, washed by resuspension
in PBS-FBS
and centrifuged. The cell pellet was then incubated for 30 min. with FITC-
conjugated goat
anti-human IgM. After incubation, the cells were washed with PBS-FBS. Finally,
the
cells were resuspended in PBS-FBS propidium iodide (PI) was added and the
cells washed.
PI-positive and FITC-positive cells were analyzed by flow cytometry.
The results of the flow cytometric analyses are shown in Figs. 2, 3 and 4.
These
results indicate that crude and purified forms of Mab H 11 bind to a cell
surface-associated
antigen(s) expressed on live human tumor cell lines, including glioblastoma,
melanoma,
breast adenocarcinoma and colon adenocarcinoma.
EXAMPLE 3
Binding of Mab HI I to Freeze-Thaw Extracts of Human Tumor Cell ines
by ELISA Analysis
In order to determine the ability of H11 to bind specifically to human tumor
antigen(s), ELISA plates were coated with human tumor cell extracts prepared
by repeated
freezing and thawing of glioblastoma (SKMG-1), breast adenocarcinoma (BT-20,
MB-468
and MB-453), colon adenocarcinoma (SK-CO-1 and HT-29) cells.
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 Mab HI I in PBS or control IgM in PBS or culture medium for 2 hrs
at room
temperature. The plates were washed and incubated with biotinylated anti-human
IgM
followed by incubation with biotinylated anti-human IgM followed by incubation
with
streptavidin-conjugated alkaline phosphatase for I 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.
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The binding of Mab H 11 to the tumor cell extracts is shown in Figs. 5 and 6.
These
results indicate that Mab H 11 binds to tumor cell extracts prepared from
glioblastoma,
breast adenocarcinoma and colon adenocarcinoma cells in a dose-dependent
manner.
EXAMPLE 4
5 Binding of Mab HI I to Human Tumor Cells Determined by
Im unoperoxidase Staining
In order to determine immunoreactivity of H 11, the following experiment was
performed. Tumor cells were grown in 24-well plates on coverslips for 48-96
hrs. The
cells were washed with PBS, fixed with formaldehyde and incubated with 5%
normal goat
10 serum on PBS for 30 min. After washing, the cells were incubated for 2 hrs
with either
hybridoma NBGM I/H 11 culture supernatants or purified Mab H 11 (10 gg/mL) in
PBS or
culture medium spiked with control human myeloma IgM (10 gg/mL) for 2 hrs. The
cells
were then washed and incubated with anti-human IgM conjugated to HRP. Finally,
the
cells were washed, incubated with DAB substrate to visualize Mab H I 1
binding, counter-
15 stained with hematoxylin and mounted in GVA.
The results of the immunoreactivity of Mab H 11 are shown in Table 3 where
reactivity is indicated as negative (--), weak positive (+), positive (++),
strong positive
(+++). These results indicate that, as determined by immunoperoxidase
staining, the
epitope recognized by Mab H 11 is expressed by a number of different types of
human
20 tumor cells and cell lines.
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TABLE 3
CELL LINES/TYPE OF TUMOR REACTIVITY
Control IgM Mab H11
HUMAN GLIOBLASTOMA
SKMG 1 -- +++
U-118 MG -- ++
U-87 MG -- ++
HUMAN MALIGNANT MELANOMA
A-375 -- +++
SK-MEL-5 -- ++
HUMAN COLON ADENOCARCINOMA
SK-CO- l -- ++
HUMAN BREAST ADENOCARCINOMA
MG-468 -- ++
MB-453 -- +
BT-20 -- ++
BT-474 -- ++
HUMAN KIDNEY ADENOCARCINOMA
S W-83 9 -- ++
HUMAN OSTEOGENIC SARCOMA
SAOS-2 -- ++
HUMAN OVARY ADENOCARCINOMA
SK-OV-3 -- ++
EXAMPLE 5
Binding of Mab H 11 to Human Tumor Cell Lines Determined by Cell-Fixed ELISA
The binding of H 11 to human tumor cells and cell lines was also determined by
cell-
fixed ELISA. Growing tumor cells were detached from the T-flask surface by
incubating with
EDTA-PBS. Cells were collected by centrifugation, washed with PBS, resuspended
in culture
medium, counted, and 50 l of cell suspension containing 5,000-10,000 cells
placed in each
well of 96-well ELISA plates. After allowing the cells to attach to the
plates, the culture
supernatants were removed and the plates were blocked with PBS-BSA. The cells
were then
incubated with different concentrations (1-20 g/mL) of either Mab H 11 or
control human
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myeloma IgM for 2 hrs. After incubation, the plates were washed, incubated
with biotin-
conjugated goat anti-human IgM, washed again and incubated with streptavidin-
conjugated
alkaline phosphatase. Finally, the plates were washed, incubated with p-
nitrophenyl phosphate
substrate and read at 405 nm in an ELISA plate reader.
Results of the reactivity of Mab H 11 to human tumor cell lines by cell-fixed
ELISA are
shown in Table 4 and Figure 7. In Table 4, Control IgM 10 gg/mL and HI 1 10
gg/mL were
used for testing the reactivity, and values are given as absorbance at 405 nm
standard
deviation. These results indicate that: 1) Mab H 11 reacts strongly with
glioblastoma cells
(SKMG-1), even at a low concentration of 1 gg/mL, whereas control IgM at 20
gg/mL does
not react with SKMG-1 cells; and 2) Mab H I 1 recognizes the tumor antigen(s)
present on
numerous tumor cell lines (breast adenocarcinoma, colon adenocarcinoma,
malignant
melanoma, neuroblastoma, glioblastoma, lung adenocarcinoma, small cell lung
carcinoma and
prostate adenocarcinoma). The degree for Mab reactivity varies both with the
type of cancer
and the tumor cell lines. The reactivity of Mab H11 for cancer and tumor cells
was between
three and ten times greater than that of the control IgM.
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TABLE 4
Cell lines/Tumor Type Reactivity (O.D. at 405 nm)
Control 1gM Mab H 11
Human Glioblastoma
SKMG-1 0.21 0.01 0.95 0.06
D-54-MG 0.13 0.02 0.43 t 0.07
U-87MG 0.13 t 0.02 0.60 t 0.01
Neuroblastoma
SK-N-SH 0.14 0.02 0.96 0.06
SK-N-MC 0.17 f 0.03 1.00 t 0.05
Malignant Melanoma
SK-MEL-5 0.18 f 0.03 1.42 t 0.04
SK-MEL-28 0.19 0.03 1.79 0.05
Breast adenocarcinoma
MB-453 0.68 f 0.18 2.85 f 0.14
MB-468 0.60 t 0.03 2.39 t 0.10
SK-BR-3 0.60 t 0.03 2.14 t 0.13
T47D 0.58f0.01 2.13t0.04
BT-20 0.57 t 0.02 2.07 t 0.13
BT-474 0.61 t0.03 2.20t0.17
Lung adenocarcinoma
SW-900 0.20 t 0.02 0.68 0.10
SK-LU-1 0.19 f 0.02 0.57 t 0.07
A-427 0.22 0.01 0.88 0.07
Small cell lung carcinoma
NCI-H69 0.25 0.04 1.42 0.20
NCI-H82 0.20 f 0.09 1.16 t 0.13
Colon adenocarcinoma
SK-Co-1 0.27 f 0.03 0.98 0.11
HT-29 0.37 t 0.02 1.78 t 0.20
Prostate adenocarcinoma
PC-3 0.17t0.01 0.60t0.01
DU-145 0.15 0.01 0.52 0.01
Kidney adenocarcinoma
SW-839 0.2 t 0.01 1.43 0.01
Osteogenic sarcoma
SAOS-2 0.24 t 0.02 1.22 0.07
U-2 0S 0.13 t 0.04 1.93 t 0.05
Bladder cell carcinoma
T-24 0.13t0.01 1.25 0.03
Ovarian adenocarcinoma
SKOOV-3 0.12f0.01 1.14t0.02
Larynx carcinoma
HEP-2 0.25 t 0.01 1.25 f 0.01
Normal human fibroblast
GM-8333 0.13 f 0.01 0.39 t 0.01
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EXAMPLE 6
Immunoanatomic Distribution and ImmunQpathologic Analysis of H11
Immunohistochemistry was used to determine Hl I specificity for micro-
anatomical
detail and heterogeneity in tissues and tumors. Limitations of this technique
include
possible false negative results due to low levels of expression of the
molecule under study,
as well as false positive results (cross-reactivity) due to antibody-binding
to similar
epitopes or epitopes shared by other antigens. To address these limitations,
this study was
carried out at the highest concentration of antibody that did not show non-
specific binding.
This allowed for detection of all levels of cross-reactivity in different
tissues. Also,
fixation analysis established the best combination of antigenic staining
intensity and
morphological preservation. The present example presents results obtained from
IMPATH
Inc., New York, retained to study the cellular specificity and antigen
expression of H11, on
a selected panel of cryostat-cut frozen sections of normal and tumor tissues.
The study
used an indirect immunoperoxidase technique.
Histologically normal human tissues were obtained from surgical and autopsy
specimens. These fresh tissues were embedded in OCT (Miles Laboratories, Inc.,
NaperviIle, IL) in cryomolds, snap-frozen in isopentane, cooled by liquid
nitrogen. The
tissues from IMPATH's frozen tissue bank were cut at 5 microns, placed on poly-
L-lysine
coated slides, air-dried, and stored at -70 C.
H11, received on wet ice and stored at 2-8 C, was supplied non-biotinylated at
a
concentration of 200 gg/mL, total volume of 3.0 mL. A human myeloma IgM
(Pierce Cat.
#31146), also supplied by Novopharm, was used as the negative control. Both
antibodies
were diluted in phosphate buffered saline to the same working concentrations
dictated by
titration analysis of antibody H 11. The peroxidase-labeled secondary antibody
was a goat
anti-human IgM (American Qualex, San Clemente, CA, lot #AI 12PN) diluted in
PBS to
1:500.
Immunoperoxidase Techniques: Immunohistochemical studies were performed
using an indirect immunoperoxidase method. The cryostat cut sections were
removed
from the -70 C freezer, air-dried and fixed according to the fixation protocol
(fixation
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details, provided below). Tissue sections were blocked for 10 minutes with 5%
normal
goat seruni diluted in PBS, then incubated with the primary antibody overnight
at 4 C.
tM
Slides were washed in PBS, followed bv a wash with 0.5% Tween/PBS solution,
then PBS
again. Endogenous peroxidase activity was blocked with a 30 minute 3% hydrogen
~ peroxide/methanol incubation, followed bv 3 washes of PBS. The sections were
then
incubated with goat anti-human 1gM (peroxidase-labeled) secondary antibody for
15
minutes, at room temperature, and washed in PBS as described above. .
The peroxidase reaction was visualized by incubating tissue sections for 2-5
minutes kvith 3,3-diaminobenzidine-tetrahydrochloride (DAB) (Sigma Chemical
Co., St.
10 Louis. MO). Tissue sections were thoroughly washed. counterstained with a
modified
Harris liematoYviin (Fisher Scientific. Fairlawn, NJ) dehydrated through
graded alcohols,
cleared in xylene, and coverslipped. Tissues that demonstrated high levels of
background
staining \vith the negative control antibody were repeated with more extensive
Nvashing.
Human breast carcinoma (F95-036), supplied by IMPATI-i, was the positive
control for
15 H 11. Negative controls substituted the primarv test antibody with purified
human
myeloma IgM.
The purpose of the fixation analysis was to establish the conditions which
provide
the optimal combination of antigenic staining intensity and morphological
preservation.
The positive control tissue was tested with five fixation protocols. including
no fixation.
20 The fixation protocols tested were 10% neutral buffered formalin (23-25 C),
acetone (2-
8 C), methyl/acetone (1:1 VN, 2-8 C) and 95% ethanol (23-25 C). For this
study, 10%
neutral buffered formalin (NBF) gave optimal results for HI 1.
Using 10% NBF as the fixative, serial antibody dilutions (20.0 g/mL to 0.1
g/mL) were tested on the positive control, human breast carcinoma. A
concentration of
25 10.0 g/mL of antibody H 11 gave optimal results-maximum staining intensity
without
significant background staining of the negative control.
The results obtained are depicted in Tables 5 and 6. Table 5 depicts H11
reactivity
on normal tissues and Table 6 depicts H 11 reactivity on human tumors.
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TABLE 5
Tested Range of
(0-3+)
Tissue Positive/Total Reactivity
Adrenal 0/3 0
Bladder 0/3 0
Bone Marrow 1/3 1+
Brain 0/3 0
Breast 0/3 0
Cervix 0/3 0
Esophagus 0/3 0
Eye 0/3 0
Heart 0/3 0
Kidney 0/3 0
Large Intestine 0/3 0
Liver 0/3 0
Lung 0/3 0
Lymph Node 0/3 0
Muscle 0/3 0
Ovary 0/2 0
Pancreas 0/3 0
Parotid 0/3 0
Pituitary 0/1 0
Prostate 0/3 0
Skin 0/3 0
Small intestine 0/3 0
Spinal cord 0/3 0
Spleen 0/3 0
Stomach 0/3 0
Testis 0/3 0
Thymus 0/3 0
Thyroid 0/3 0
Tonsil 1 /3 1 +
Uterus 0/3 0
WBC 0/3 0
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TABLE 6
Tested % of Tumor Range of
Tumor Positive/Total Cells Staining Reactivity (0-3+)
Breast carcinoma 2/3 30-90 1-3+
Colon carcinoma 3/3 40-70 1-2+
Glioma 4/6 30-90 1-2+
Gastric carcinoma 3/3 30-50 1-2+
Lung adenocarcinoma 3/4 10-70 1-2+
Lung squamous carcinoma 3/3 10-95 1-3+
Lung small cell carcinoma 1/2 30 1+
Lymphoma 8/8 10-95 1-3+
Melanoma 3/3 20-95 1-2+
Ovarian carcinoma 3/3 20-30 1-3+
Prostate carcinoma 3/3 20-95 1-2+
Sarcoma 0/3 0 0
The results obtained indicate that weak (1+) to strong (3+) reactivity was
observed
in over 70% of the positive control sample. The antigen recognized by H11 has
a
restricted pattern of distribution. H11 was largely unreactive with normal
human tissues
tested in the IMPATH system. All simple epithelial cells, as well as the
stratified epithelia
and squamous epithelia of different organs were found to be unreactive.
Reactivity was
also not seen in neuroectodermal cells, including those in the brain, spinal
cord and
peripheral nerves. Mesenchymal elements such as skeletal and smooth muscle
cells,
fibroblasts, and endothelial cells were negative. Tissues of lymphoid origin
including bone
marrow, lymph node, spleen, and thymus were largely unreactive with antibody H
11.
Weak (1+) reactivity was observed in rare cells in one specimen of bone marrow
and in the
germinal centers of one of three specimens of tonsil tested.
Positive immunoreactivity was observed in almost all specimens of tumor tested
including breast, colon, glioma, gastric, lung (adeno, squamous, and small
cell),
lymphoma, melanoma, ovarian, and prostate. Reactivity was seen in 10% to
greater than
95% of the tumor cells present in these specimens; staining intensity ranged
from weak
(1 +) to strong (3+). Antibody H 11 was, however, unreactive with all three
specimens of
sarcoma tested. Some but not all normal counterparts of the tumor cells, when
present in
the specimens, were reactive with H11. A few normal cells present in breast,
gastric and
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prostate carcinoma were reactive with antibody H 11. The large granular cells
that were
reactive with antibody H 11 are believed to be inflammatory cells of the
eosinophile-mast
cell lineage.
In summary, antibody Hi 1 is largely unreactive with normal human tissues with
the exception of some normal tissues present in tumors. The H 11 antibody
detects an
antigen that is expressed in almost all of the tumors tested.
EXAMPLE 7
H 11 Cloninc_ Expression and immunologic reactivity
In order to determine the ability of H11-scFv antibody fragments to bind
specifically to cancer cells, the following experiments were performed.
The single chain antibody constructs were made by the following procedure.
Primers specific to the 5' and 3' ends of the H 11 kappa and mu V regions were
synthesized
on an Applied Biosystems DNA synthesizer. All the primers contained a
restriction
endonuclease site for cloning. Primers 5 and 6 also contained additional
nucleotides that
encode a (SGGGG)3 linker. The primers used are listed in Table 7 where the
restriction
endonuclease site introduced is underlined.
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TABLE 7
Pt'imer Sequence (5'--+3') SjLe
Introduced
1. TATGAAGACACCAGGCCGATATTGTGTTGACGCAG Bbsl
(SEQ ID NO:7)
2. TATCCGGATGCAGCCACAGTTCGTTT (SEQ ID NO:8) BspEl
3. TATTCGGACAGGTGCAGCTGGTGGAG (SEQ ID NO:9) BspEl
4. TATGGATCCTGAGGAGACGGTGACCGT (SEQ ID NO:10) BamHl
5. TATATAT G AGGTGGTGGATCAGGTGGAGGTGGCTC BspEl
CCAGGTGCAGCTGGTGGAGTCT(SEQ ID NO:11)
6. ACCTCCGGAACCGCCACCGCCAGAGACAGATGGTGCA BspEl
GCCACATTC (SEQ ID NO:12)
PCR reactions were carried out using primers I and 2 for the kappa dimer,
primers
3 and 4 for the mu dimer, primers I and 6 for the kappa monomer and primers 4
and 5 for
the mu monomer. The PCR fragments were then purified and digested with their
respective restriction endonucleases. The coding nucleotides are depicted in
SEQ ID
NOS:13 and 16 and the complementary nucleotides are depicted in SEQ ID NOS:15
and
18, respectively.
The expression vector pSJF I containing a ribosome binding site, OmpA signal
peptide sequence, c-myc (9E 10) detection tag and histidine tail (See Figure
8) was
prepared by cutting with Bbsl and BamHl. The monomer and dimer constructs were
assembled by ligating the respective kappa and mu fragments into pSJF 1 and
transforming
them into competent TG1 E. coli. Resulting colonies were screened by colony
PCR and
restriction endonuclease digests to confirm the correct size inserts and the
sequences were
verified by dideoxy fluorescent sequencing.
Transformed TG1 containing either the H11 monomer or dimer expression plasmid
were shaken at 26 C for 24 hours followed by the addition IPTG to a final
concentration of
0.1 gM. The cells were incubated for a further 16 hours and then harvested by
centrifugation. The periplasmic proteins containing the H11 antibody were
released by
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treatment with sucrose buffer (25% sucrose, 1 mM EDTA, 10 mM Tris pH 8.0)
followed
by ice cold shock buffer (10 mM Tris 8.0, 0.5 mM MgC12). Expression was
verified by
polyacrylamide gel electrophoresis and Western Blotting. The antibody was
purified using
a nickel-charged column (Pharmacia HiTrap chelating column) and the bound
antibody
5 was eluted with an increasing gradient of imidazole. The purified antibody
was dialyzed
against PBS/0.02% sodium azide and concentrated to 0.5 mg/mL.
The antigenic similarities between Mab H 11 and H 11-scFv were also determined
by cell fixed ELISA. ELISA plates coated with A375 cells were incubated with
Mab H11,
control IgM, H 11-scFv or control BGA-scFv followed by incubation with rabbit
anti-
10 human IgM antibody or rabbit anti-scFv antibody as appropriate. The
detection was by
goat anti-rabbit IgG-horse radish peroxidase followed by substrate. The
results, shown in
Figure 9, demonstrate a high affinity of both H 11 IgM and H 11-scFv, and a
low affinity of
both the control IgM and BGA-SL-6.
In order to determine the specificity of biotinylated H11-scFv relative to a
15 biotinylated control scFv, the following experiment was performed. Human
tumor cells
were fixed to ELISA plates and incubated with either biotinylated H 11-scFv or
biotinylated BGA scFv (control) as described above.
Biotinylated H 11-scFv also demonstrated a much greater affinity (between 8-
and
50-fold) for tumor cell lines than the control in cell fixed ELISA. Data
corresponding to a
20 concentration of 2.5 g/mL of H11-scFv or BGA scFv is shown in Table 8 and
Figure 10.
Figure 11 illustrates the portion of Table 8 related to the titration of
reactivity of
biotinylated H 11-scFv for the binding to lymphoma cells Daudi, Ramos, CA-46
and
CCRF-CEM cells. At every concentration tested (1.25 to 10 gg/mL), H 11-scFv
demonstrated a high affinity for lymphoma cells, but BGA scFv did not.
30
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TABLE 8
Tumor Cell Lines Reactivity (O.D. at 450 nm SD)
Biotinylated BGA scFv Biotin-Hll-scFv
Human Glioblastoma
SKMG-1 0.01 0.01 0.56 0.04
U-118MG 0.01 0.02 0.47 0.03
D-54MG 0.01 0.00 0.50 0.02
Neuroblastoma
SK-N-MC 0.01 0.00 0.50 0.02
Malignant Melanoma
SK-MEL-5 0.02 0.01 0.61 0.04
A-375 0.12 t 0.03 0.97 0.03
SK-MEL-28 0.02 0.00 0.71 0.04
Breast adenocarcinoma
T47D 0.02 0.00 0.64 0.03
MB-468 0.01 0.00 0.65 0.01
SK-BR-3 0.01 0.00 0.58 0.02
BT-20 0.01 0.00 0.54 0.06
BT-474 0.01 0.00 0.60 0.01
Lung adenocarcinoma
SW-9000 0.01 0.00 0.41 0.02
SK-LU-1 0.01 0.00 0.45 0.03
A-427 0.01 0.00 0.40 0.05
Colon adenocarcinoma
SK-Co-1 0.01 0.00 0.56 t 0.01
HT-29 0.01 0.00 0.53 0.06
LS17T 0.01 0.00 0.57 0.02
Osteogenic sarcoma
SAOS-2 0.02 0.00 0.88 0.06
U-2 OS 0.02 0.00 0.93 0.01
Bladder cell carcinoma
T-24 0.02 0.01 0.97 0.05
Ovarian
adenocarcinoma
SK-OV-3 0.01 0.00 0.77 0.02
Larynx carcinoma
HEP-2 0.02 0.00 0.08 0.04
Prostate carcinoma
DU-145 0.01 0.00 0.42 0.02
PC-3 0.01 0.00 0.36 0.01
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Tumor Cell Lines Reactivity (O.D. at 450 nm SD)
Biotinylated BGA scFv Biotin-Hll-scFv
Small cell lung
carcinoma
NCI-H82 0.01 0.00 0.44 0.02
NCI-69 0.01 0.00 0.44 0.01
Lymphoma cell lines
Chronic myelogenous
leukemia
K-562 0.02 0.00 0.65 0.00
Acute lymphoblastic
leukemia
CEM 0.04 0.00 1.4 0.03
Burkitt Lymphoma
CA-46 0.02 0.00 1.2 0.02
RAMOS 0.04 0.00 1.3 t 0.02
DAUDI 0.02 0.00 1.38 0.01
In order to verify the specificity of biotinylated H I 1-scFv for cancerous
cells, the
following experiment was performed. Malignant and normal tissue specimens were
prepared and incubated with biotinylated H 11-scFv as described above.
The H11-scFv was used to stain sections of tumor and normal tissues. The
results
are depicted in Table 8 for normal tissues and Table 9 for tumor tissues.
Figure 12 depicts the relative fluorescence intensity of H 11-scFv and control
scFv
to tumor cell lines.
The data in Table 9 demonstrate that biotinylated H11-scFv generally does not
react to normal tissues. Almost all of the normal tissues tested demonstrated
no
measurable reactivity, with only a weakly positive signal generated by normal
pancreas
and peripheral nerve tissues. In Table 9,.-ve indicates no measurable activity
and +/-
indicates weakly positive activity.
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TABLE 9
Normal Tissues Reactivity of Biotinylated H11-scFv
(50 g/mL)
Cortex -ve
Breast -ve
Colon -ve
Heart -ve
Liver -ve
Lymph node -ve
Prostate -ve
Thyroid -ve
Adrenal -ve
Cerebellum -ve
Lung -ve
Pancreas Peripheral Nerve +/-
Skin -ve
Spleen -ve
Smooth muscle -ve
Stomach -ve
Thymus -ve
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TABLE 10
Tissue Type Number of Percentage of Range of
positives/ Total Tumor Cells Reactivity (0 - +3)
samples tested staining
Breast carcinoma 27/31 40/60 1-3+
Colon carcinoma 23/26 80/100 1-3+
Melanoma 13/14 50/70 1-3+
Prostate carcinoma 17/20 20/70 1-2+
Cervix squamous 22/24 nd 1-2+
cell carcinoma
Cervix 9/9 nd 1-2+
adenocarcinoma
Kaposi Sarcoma 7/8 nd 1-2+
Benign Colon 0/2 0 0
nd: not determined
The results presented in Table 10 indicate that positive staining was found in
most
breast (27/31) and colon (23/26) and prostate (17/20) carcinoma samples
tested. Positive
staining was found at 25 g/mL concentration of H11-scFv. Although the
staining was
predominantly detected in tumor cells, various degrees of reactivity were also
found on
stroma and adjacent tissue. The Hi l-scFv was also tested for its specificity
for normal
tissue. The results obtained are presented in Table 11 which summarizes the
immunohistochemistry staining of H11-scFv with normal human tissue sections.
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TABLE 11
Tissue H 11-scFv (25 g/mL) 3B I scFv (25 g/mL)
Adrenal t ~. f
Cerebellum - -
Cortex - -
Colon t -
Breast -~ -
Kidney f - ~
Aorta -~ -
Heart
Liver f- + t
Lung - -
Lymph node - -
Pancreas ~ -
Pituitary
Prostate -{focal -(focal f)
Peripheral nerve -~ -
Skin -(sweat gland f) -(sweat giand f)
Spleen -~ -
Small intestine -~ -
Stomach -~ -
St. muscle -~ ~
Thymus ---~ -
Thyroid -~ -
S94-7474-2
(Colon Car. control) ++ -
EXAMPLE 8
13eactivitv of H11-scFv to Live Tumor Cells as Determined by Flow Cytometry
5 In order to test the reactivity of H11-scFv to live tumor cells, cells from
tumor cell
lines were prepared for flow cytometry as described above in Example 2. Tumor
cells
were incubated with either biotinylated H 11-scFv or control biotinylated scFv
as described
above at a protein concentration of 100 g/mL or 200 g/mL. The reactivity was
determined as the mean fluorescence and % positive cells. A biotinylated H11-
scFv was
10 prepared as described above and at a protein concentration of either 100
g/mL or
200 gg/mL. The mean fluorescence and % positive cells are shown in Table 12
where # is
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biotinylated 3B1 as control scFv; * is biotinylated BGA SL-6 as control scFv;
** is PBS
5% FCS as control; and *** is Biotin-5B1 as control scFv.
Table 12
Mean Fluorescence % Positive Cells
Cell Line Protein Biotinylated Biotinylated Biotinylated Biotinylated
conc'n 3B1 scFv # H11-scFv 3B1 ScFv # H11-scFv
( g/mL)
SK-BR-3 200 149 233 11 36
(Breast adeno-
carcinoma)
MB-468 200 144 156 9 11
(Breast adeno-
carcinoma)
A-375 200 111 207 10 80
(Melanoma)
A-375 200 161* 235 28 76
(Melanoma)
LS-174T
(Colon adeno 200 182 233 24 37
carcinoma)
HT-29
(Colon adeno- 200 141 179 12 18
carcinoma)
SKMG-1 100 148** 206 9 31
(Glioma) 100 189** 224 14 27
H9 100 185*** 145 20 13
(T cell
Lymphoma)
WI-38
(Human 100 293*** 255 26 15
diploid lung
cells)
125 1 labeled Hl 1-scFv demonstrated an affinity of binding (Ka) of 3 x 10 8
L/mol for
LS174T cells (specific binding shown in Figure 13). 111 In-Hl 1-scFv
demonstrated an
affinity of binding (Ka) of 3.6 x 10 8 L/mol for A-375 cells and 1.4 x 109
L/mol for
SKMG1 cells. The results are depicted in Figure 14. There were approximately
24,000
binding sites/cell for A-375 cells and approximately 5000 binding sites/cell
for SKMG1.
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These results indicate that purified forms of Mab H 11 bind to a cell surface-
associated antigen(s) expressed on breast adenocarcinoma (SK-BR-3),
glioblastoma
(SKMG-1), and melanoma (A-375) lymphoma cell lines.
In order to further test the reactivity of biotinylated H 11-scFv to live
lymphoma
cells, cells from tumor cell lines were prepared and incubated with
biotinylated H 1 I -scFv
at a protein concentration of either 100 g/mL or 200 g/mL and analyzed by
flow
cytometry as described above in Example 2. The mean fluorescence and %
positive cells
were measured by flow cytometry. The control for scFv binding was biotinylated
BGA
scFv. Results are shown in Table 13.
Table 13
% %
CELL SAMPLES CONC. MEAN FLUORESCENCE POSITIVE
LINE ( g/mL) FLUORESCENCE INCREASE CELLS
Burkitt's PBS 123 10
Lymphoma BIOTIN- 200 126 9
BGA scFv 100 133 14
CA-46 BIOTIN- 200 262 108 76
H11-scFv 100 221 66 58
T cell PBS 150 6
lymphoma BIOTIN- 200 155 8
BGA scFv 100 149 7
H9 BIOTIN- 200 186 20 13
H 11-scFv 100 171 15 9
Acute PBS 151 8
lymphoblast BIOTIN- 200 171 14
oid leukemia BGA scFv 100 159 10
BIOTIN- 200 231 35 34
CCRF-CEM H11-scFv 100 242 52 38
Burkitt's PBS 151 10
Lymphoma BIOTIN- 200 174 15
BGA scFv 100 169 13
RAMOS BIOTIN- 200 423 143 95
H11-scFv 100 316 87 67
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EXAMPLE 9
Binding of Biotinylated HI I-scFv to Human Tumor Cells Determined bX
Immunoperoxidase Staining
In order to determine immunoreactivity of H I 1-scFv, the following experiment
was
performed. Tumor cells were grown in T-flasks and cytospins were prepared and
incubated with biotinylated H 11-scFv or PBS to determine binding.
The results of the immunoreactivity of H I I-scFv are shown in Table 14
where reactivity is indicated as negative (--), weak positive ( ), positive (+
or ++). These
results indicate that, as determined by immunoperoxidase staining, the epitope
recognized
by Mab H11 is expressed by a number of different types of human tumor cells
and cell
lines.
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Table 14
CELL LINES/TYPE OF TUMOR REACTIVITY
PBS H11-scFv (50 g/mL)
HUMAN GLIOBLASTOMA
SKMG 1 -- +
U-87 MG -- +
HUMAN MALIGNANT MELANOMA
A-375 -- +
SK-MEL-5 -- ++
HUMAN COLON ADENOCARCINOMA
SK-CO-1 -- +
HT-29 -- +
174T +
HUMAN BREAST ADENOCARCINOMA
SK-BR-3 -- +
BT-20 -- +
HUMAN LYMPHOMA CELL LINES
U-937 Histocytic Lymphoma -- t
H9 T Cell Lymphoma -- +
CEM Acute Lymphoblastoid leukemia -- -
MOLT-3 Acute Lymphoblastoid leukemia -- t
HL-60 Promyelocytic leukemia -- +
KG-i Acute myelogenous leukemia -- +
K-562 Chronic myelogenous leukemia -- +
GASTRIC CARCINOMA
KATO III -- ++
HUMAN OSTEOGENIC SARCOMA
SAOS-2 -- f
HUMAN OVARY ADENOCARCINOMA
SK-OV-3 -- t
BLADDER CELL CARCINOMA
T-24 +
LARYNX CARCINOMA
Hep-2 -- +
EXAMPLE 10
Reactivity of Recombinantly Produced H1 I IgGI
H11 IgGI was produced in Chinese Hamster Ovary (CHO) cells as follows.
Several vectors containing cDNAs encoding light and heavy chain sequences of
Hi I were
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prepared. The orientation, DNA inserts and antibiotic selection criteria of
these constructs
are shown in Table 15 where CMV is cytomegalovirus; DHFR is dihydrofolate
reductase;
HC is heavy chain and LC is light chain.
Table 15
vector DNA insert HC + LC promoter orientation antibiotic amplif.
promoter selection
ppNB 1 cDNA
heavy and CMV* HC- clockwise neomycin DHFR
light chains LC- anticlockwise
pNB2 cDNA
heavy and CMV HC - clockwise zeocin DHFR
light chains LC- clockwise
pNB3 cDNA
heavy and CMV HC - clockwise zeocin DHFR
light chains LC - anticlockwise
5
These expression vectors have separate insertional sites for the sequences
encoding
the light and heavy antibody chains. A high level of constitutive expression
of both the
heavy and light chains is directed by the cytomegalovirus immediate - early
(CMV)
enhancer/promoter. A chimeric intron comprising the 5' donor site of the first
intron of the
10 human (3-globin and the 3' acceptor site from the intron of an
immunoglobulin gene (heavy
chain variable region) is located downstream from the promoter which has
frequently been
shown to enhance gene expression levels. Polyadenylation of mRNAs are provided
by the
poladenylation signal from the simian virus 40 (SV40).
The plasmids also contain the gene encoding dihydrofolate reductase (DHFR) and
15 can thus be grown in Chinese hamster ovary (CHO) DHFR deficient cells.
Amplification
using methotrexate, a folate analogue and potent DHFR inhibitor, results in
amplification
of the DHFR gene and its flanking sequences (namely, the light and heavy
chains of the
antibody in the construct). A stepwise increase in methotrexate (from about
0.01 nM to
about 800 nM) concentration can produce very high levels of protein from the
target
20 gene(s). The constructs also contain a gene which confers antibiotic
resistance was a
selectable marker, either neomycin or zeomycin is used. The vectors are shown
in Figs. 15
and 16.
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Results of flow-cytometry analysis of recombinantly produced H11 IgGl are
shown in Table 16 and illustrate that H11 IgGI which binds to an antigen on SK-
BR-3
breast carcinoma cells can be produced in CHO cells.
Table 16
Cell Lines/I.D. Conc. of IgGI in Mean % Increase of MF
samples (mg/L) Fluorescence (MF) above IgGi control
PBS 156
Control IgGI 5 169
1129 / pNB l 2 172 2
1233 / pNB 1 2 184 9
KL-13 / pNB2 3.3 192 14
KL-14 / pNB2 3.3 186 10
Sb2 / pNB3 4.0 224 33
3sB3 / pNB3 3.9 200 18
EXAMPLE 11
H11 Binding to Cancer Cell Lines
The binding affinities of H11 IgM and HI 1-scFv for various human cancer cell
lines were determined by labeling H 11 antibodies with either radioactive
iodine or
radioactive indium. 1251-H11-scFv was prepared with specific activities of 7,
20 or 150
Ci/ g and 125I-Hl 1 IgM with 0.6 Ci/ g were obtained. In addition, I I 'In-Hl
1-scFv
having a specific activity of 13 and 38 Ci/ g was prepared as described in
Example 12.
The scFv 3B 1, which does not recognize the C-antigen, was used as a control
to indicate
non-specific binding and was labeled with 150 Ci/ g.
125I-H11-scFv was purified using a P-2 minicolumn and analyzed by paper
chromatography in 85% methanol as shown in Figure 17. 125I-H11 IgM was
purified using
a Sephadex G-50 mini-column (Pharmacia) and analyzed by paper chromotography
in
85% methanol as shown in Figure 18. A Sephadex G-50 column was used to purify
111 In-
HI 11 scFv which was then analyzed by ITLC-SG in 0.1 M Citrate as shown in
Figure 19.
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Results of H 11 binding are shown in Figures 13 and 14. Figure 13 shows the
specific binding of 125I-H11-scFv to LS174T human colon cancer cells. Figure
14 shows
the total binding of III In-HI l-scFv to A375 cells.
The results obtained indicate that H11 binds specifically to both LT174T and
human melanoma cells. H11 also binds, but with lower affinity, to the breast
cancer cell
line.
EXAMPLE 1 ?
Tumor Imaging with M Indium-DTPA-H 11-scFv
H11-scFv was conjugated with the bvcyclic anhydride of
diethylenetriaminepentaacetic acid (DTPA) at a molar ratio of 10:1 (DTPA:H11-
scFv)
resulting in a substitution level of 2 moles of DTPA per mole of H 11-scFv.
DTPA-H 11-
scFv was purified from excess DTPA on a Sephadex G-25 (Pharmacia) mini-column
and
reconcentrated to 10 mg/mL using a Centricon-30 microconcentrator (Amicon).
The
DTPA-H 11-scFv was radiolabeled to a specific activity of 25 mCi/mg with 111
Indium
acetate. Unincorporated 11 'In was removed using a Sephadex G-25 minicolumn.
The
III Indium acetate was prepared from III Indium chloride (Nordion) and 1 M
acetate buffer
at pH 6Ø The radiochemical purity of the final 111 In-DTAP-H 11-scFv was
greater than
99% as measured by thin layer silica gel chromatography in 100 mM sodium
citrate pH
5Ø Figure 19 shows the purification and TLC.
A female nude mouse with an existing subcutaneous A375 melanoma xenograft on
the right lateral side and a subcutaneous HT-29 human colon cancer xenograft
in the mid-
abdominal region was injected intravenously in the tail vein with 100 Ci of
"" In-DTAP-
H11-scFv. The mouse was immediately placed under the gamma camera (Siemans
ZL3700) interfaced with a GE Star 4000i computer and a dynamic acquisition was
obtained for 120 minutes, for a total of 480 frames of 15 seconds each. The
frames were
then combined into 12 x 10 minute images. The A375 tumor was visible on the
right
lateral side of the mouse as early as 30 minutes post-injection.
Region-of-interest analysis of the two tumors showed that the A375 tumor
accumulated radioactivity throughout the 120 minute study, whereas the HT-29
tumor
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accumulated radioactivity for the first hour and then the radioactivity
concentration
remained relatively constant. Figure 20 shows 12 frames and the two arrows on
the
bottom right hand frame, taken at 120 minutes, show the accumulation of
radioactivity in
the two tumors. The narrow arrow points to the A375 tumor, and the broad arrow
points to
the HT-29 tumor. Nomial tissues visible on the images include the heart,
liver, kidneys
and bladder. The heart is visible due to circulating amounts of radioactivity,
and the
kidneys and bladder are visible due to renal elimination of "'In-DTAP-H11-
scFv. The
small amount of liver uptake may be due to blood flow to the liver or to
partial binding of
~In-DTAP-HI 1-scFv to the liver.
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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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Dan, Michael D.
Maiti, Pradip K.
Kaplan, Howard A.
(ii) TITLE OF INVENTION: ANTIGEN BINDING FRAGMENTS Hil, THAT
SPECIFICALLY DETECT CANCER CELLS, NUCLEOTIDES ENCODING THE
FRAGMENTS, AND USE THEREOF FOR THE PROPHYLAXIS AND
DETECTION OF CANCERS
(iii) NUMBER OF SEQUENCES: 18
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Morrison & Foerster
(B) STREET: 755 Page Mill Road
(C) CITY: Palo Alto
(D) STATE: CA
(E) COUNTRY: USA
(F) ZIP: 94304-1018
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Lehnhardt, Susan K.
(B) REGISTRATION NUMBER: 33,943
(C) REFERENCE/DOCKET NUMBER: 31608-20001.20
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (415) 813-5600
(B) TELEFAX: (415) 494-0792
(C) TELEX: 706141
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 543 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..543
SUBSTITUTE SHEET (RULE 26)
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
CAAGCTATTT AGGTGACACT ATAGAATACT CAAGCTATGC ATCCAACGCG TTGGGAGCTC 60
TCCCATATGG TCGACCTGCA GGCGGCCGCA CTAGTGATTT CAAGCTTCAT CACTGAACAC 120
AGAGGACTCA CCATGGAGTT TGGGCTGAGC TGGGTTTTCC TCGTTGCTCT TTTAAGAGGT 180
ATCCAGTGTC AGGTGCAGCT GGTGGAGTCT GGGGGAGGCG TGGTCCAGCC TGGGAGGTCC 240
CTGAGACTCT CCTGTGCAGC CTCTGGATTC CCCTTCAGAA GCTTTGCTAT GCACTGGGTC 300
CGCCAGGCTC TAGGCAAGGG GCTGGAGTGG GTGGCAGTTA TATCATATGA TGGAAGCACT 360
AAATACTACG CAGACTCCGT GAAGGGGCGA TTCACCATCT CCAGAGACAC TTCCAAGAAC 420
ACGGTGTATC TAAAAATGAA CAGGCTGAGA ACTGAGGACA CGGCTGTCTT TTACTTGTGC 480
GAAAGACAGA GCCTGCTGGG TGACTATGAC CACTACTACG GNTTGGACGC TTGGGGAAAG 540
GGA 543
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 179 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Gln Ala Ile Val Thr Leu Asn Thr Gln Ala Met His Pro Thr Arg Trp
1 5 10 15
Glu Leu Ser His Met Val Asp Leu Gln Ala Ala Ala Leu Val Ile Ser
20 25 30
Ser Phe Ile Thr Glu His Arg Gly Leu Thr Met Glu Phe Gly Leu Ser
35 40 45
Trp Val Phe Leu Val Ala Leu Leu Arg Gly Ile Gln Cys Gln Val Gln
50 55 60
Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg
65 70 75 80
Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Arg Ser Phe Ala Met His
85 90 95
Trp Val Arg Gln Ala Leu Gly Lys Gly Leu Glu Trp Val Ala Val Ile
100 105 110
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Ser Tyr Asp Gly Ser Thr Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg
115 120 125
Phe Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Val Tyr Leu Lys Met
130 135 140
Asn Arg Leu Arg Thr Glu Asp Thr Ala Val Phe Tyr Leu Cys Glu Arg
145 150 155 160
Gln Ser Leu Leu Gly Asp Tyr Asp His Tyr Tyr Gly Leu Asp Ala Trp
165 170 175
Gly Lys Gly
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 543 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
TCCCTTTCCC CAAGCGTCCA ANCCGTAGTA GTGGTCATAG TCACCCAGCA GGCTCTGTCT 60
TTCGCACAAG TAAAAGACAG CCGTGTCCTC AGTTCTCAGC CTGTTCATTT TTAGATACAC 120
CGTGTTCTTG GAAGTGTCTC TGGAGATGGT GAATCGCCCC TTCACGGAGT CTGCGTAGTA 180
TTTAGTGCTT CCATCATATG ATATAACTGC CACCCACTCC AGCCCCTTGC CTAGAGCCTG 240
GCGGACCCAG TGCATAGCAA AGCTTCTGAA GGGGAATCCA GAGGCTGCAC AGGAGAGTCT 300
CAGGGACCTC CCAGGCTGGA CCACGCCTCC CCCAGACTCC ACCAGCTGCA CCTGACACTG 360
GATACCTCTT AAAAGAGCAA CGAGGAAAAC CCAGCTCAGC CCAAACTCCA TGGTGAGTCC 420
TCTGTGTTCA GTGATGAAGC TTGAAATCAC TAGTGCGGCC GCCTGCAGGT CGACCATATG 480
GGAGAGCTCC CAACGCGTTG GATGCATAGC TTGAGTATTC TATAGTGTCA CCTAAATAGC 540
TTG 543
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 450 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
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(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..450
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
CTCGAGATGG ACATGGAGTT CCAGGCGCAG CTTCTCTTCC TCCTGCTACT CTGGCTCCCA 60
GATATCACCG GAGATATTGT GTTGACGCAG TCTCCAGGCA CCCTGTCTTT GTCTCCAGGG 120
GAAAGAGCCA CCCTCTCCTG CAGGGCCAGT CAGAGTGTTA GTAGCAGCTA CTTAGCCTGG 180
TACCAGCAGA AACCTGGCCA GGCTCCCAGG CTCCTCATCT ATGGTGCATC CACCAGGGCC 240
ACTGGCATGC CAGACAGGTC CAGTGGCAGT GGGTCCGGGA CAGACTTCAC TCTCACCATC 300
AGTAGACTGG AGCCTGAAGA TTTTGCAGTG TATTACTGTC AGCAGTATGG TAGCTCACCT 360
CAGACACCTC AGATCACTTT CGGCGGAGGG ACCAAGGTGG AGATCAAACG AACTGTGGCT 420
GCACCATCTG TCTTCATCTT CCCGCCATCT 450
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 150 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Leu Glu Met Asp Met Glu Phe Gln Ala Gln Leu Leu Phe Leu Leu Leu
1 5 10 15
Leu Trp Leu Pro Asp Ile Thr Gly Asp Ile Val Leu Thr Gln Ser Pro
20 25 30
Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg
35 40 45
Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys
50 55 60
Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala
65 70 75 80
Thr Gly Met Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
85 90 95
Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr
100 105 110
Cys Gln Gln Tyr Gly Ser Ser Pro Gln Thr Pro Gln Ile Thr Phe Gly
115 120 125
SUBSTITUTE SHEET (RULE 26)
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Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val
130 135 140
Phe Ile Phe Pro Pro Ser
145 150
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 450 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
AGATGGCGGG AAGATGAAGA CAGATGGTGC AGCCACAGTT CGTTTGATCT CCACCTTGGT 60
CCCTCCGCCG AAAGTGATCT GAGGTGTCTG AGGTGAGCTA CCATACTGCT GACAGTAATA 120
CACTGCAAAA TCTTCAGGCT CCAGTCTACT GATGGTGAGA GTGAAGTCTG TCCCGGACCC 180
ACTGCCACTG AACCTGTCTG GCATGCCAGT GGCCCTGGTG GATGCACCAT AGATGAGGAG 240
CCTGGGAGCC TGGCCAGGTT TCTGCTGGTA CCAGGCTAAG TAGCTGCTAC TAACACTCTG 300
ACTGGCCCTG CAGGAGAGGG TGGCTCTTTC CCCTGGAGAC AAAGACAGGG TGCCTGGAGA 360
CTGCGTCAAC ACAATATCTC CGGTGATATC TGGGAGCCAG AGTAGCAGGA GGAAGAGAAG 420
CTGCGCCTGG AACTCCATGT CCATCTCGAG 450
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
TATGAAGACA CCAGGCCGAT ATTGTGTTGA CGCA 34
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
SUBSTITUTE SHEET (RULE 26)
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TATCCGGATG CAGCCACAGT TCGTTT 26
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
TATTCGGACA GGTGCAGCTG GTGGAG 26
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
TATGGATCCT GAGGAGACGG TGACCGT 27
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
TATATATCCG GAGGTGGTGG ATCAGGTGGA GGTGGCTCCC AGGTGCAGCT GGTGGAGTCT 60
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 46 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
ACCTCCGGAA CCGCCACCGC CAGAGACAGA TGGTGCAGCC ACATTC 46
SUBSTITUTE SHEET (RULE 26)
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(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 918 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: join(1..906, 913..918)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
GAA TTC ATG AAA AAA ACC GCT ATC GCG ATC GCA GTT GCA CTG GCT GGT 48
Glu Phe Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly
1 5 10 15
TTC GCT ACC GTT GCG CAG GCC GAT ATT GTG TTG ACG CAG TCT CCA GGC 96
Phe Ala Thr Val Ala Gln Ala Asp Ile Val Leu Thr Gln Ser Pro Gly
20 25 30
ACC CTG TCT TTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC AGG GCC 144
Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala
35 40 45
AGT CAG AGT GTT AGT AGC AGC TAC TTA GCC TGG TAC CAG CAG AAA CCT 192
Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro
50 55 60
GGC CAG GCT CCC AGG CTC CTC ATC TAT GGT GCA TCC ACC AGG GCC ACT 240
Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr
65 70 75 80
GGC ATG CCA GAC AGG TTC AGT GGC AGT GGG TCC GGG ACA GAC TTC ACT 288
Gly Met Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
85 90 95
CTC ACC ATC AGT AGA CTG GAG CCT GAA GAT TTT GCA GTG TAT TAC TGT 336
Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys
100 105 110
CAG CAG TAT GGT AGC TCA CCT CAG ACA CCT CAG ATC ACT TTC GGC GGA 384
Gln Gin Tyr Gly Ser Ser Pro Gln Thr Pro Gln Ile Thr Phe Gly Gly
115 120 125
GGG ACC AAG GTG GAG ATC AAA CGA ACT GTG GCT GCA CCA TCT GTC TCT 432
Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Ser
130 135 140
SUBSTITUTE SHEET (RULE 26)
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GGC GGT GGC GGT TCC GGA GGT GGT GGA TCA GGT GGA GGT GGC TCC CAG 480
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
145 150 155 160
GTG CAG CTG GTG GAG TCT GGG GGA GGC GTG GTC CAG CCT GGG AGG TCC 528
Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser
165 170 175
CTG AGA CTC TCC TGT GCA GCC TCT GGA TTC CCC TTC AGA AGC TTT GCT 576
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Arg Ser Phe Ala
180 185 190
ATG CAC TGG GTC CGC CAG GCT CTA GGC AAG GGG CTG GAG TGG GTG GCA 624
Met His Trp Val Arg Gln Ala Leu Gly Lys Gly Leu Glu Trp Val Ala
195 200 205
GTT ATA TCA TAT GAT GGA AGC ACT AAA TAC TAC GCA GAC TCC GTG AAG 672
Val Ile Ser Tyr Asp Gly Ser Thr Lys Tyr Tyr Ala Asp Ser Val Lys
210 215 220
GGC CGA TTC ACC ATC TCC AGA GAC ACT TCC AAG AAC ACG GTG TAT CTA 720
Gly Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Val Tyr Leu
225 230 235 240
AAA ATG AAC AGC CTG AGA ACT GAG GAC ACG GCT GTC TAT TAC TGT GCG 768
Lys Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Val Tyr Tyr Cys Ala
245 250 255
AGA GAT CAG AGC CTG TTG GGT GAC TAT GAC CAC TAC TAC GGT TTG GAC 816
Arg Asp Gln Ser Leu Leu Gly Asp Tyr Asp His Tyr Tyr Gly Leu Asp
260 265 270
GTC TGG GGC AAA GGG ACC ACG GTC ACC GTC TCC TCA GGA TCC GAA CAA 864
Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser Gly Ser Glu Gln
275 280 285
AAA CTG ATC AGC GAA GAA GAT CTG AAC CAT CAC CAT CAC CAT 906
Lys Leu Ile Ser Glu Glu Asp Leu Asn His His His His His
290 295 300
TAGTGA AAG CTT 918
Lys Leu
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 304 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
SUBSTITUTE SHEET (RULE 26)
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Glu Phe Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly
1 5 10 15
Phe Ala Thr Val Ala Gln Ala Asp Ile Val Leu Thr Gln Ser Pro Gly
20 25 30
Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala
35 40 45
Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro
50 55 60
Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr
65 70 75 80
Gly Met Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
85 90 95
Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys
100 105 110
Gln Gln Tyr Gly Ser Ser Pro Gln Thr Pro Gln Ile Thr Phe Gly Gly
115 120 125
Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Ser
130 135 140
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
145 150 155 160
Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gin Pro Gly Arg Ser
165 170 175
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Arg Ser Phe Ala
180 185 190
Met His Trp Val Arg Gln Ala Leu Gly Lys Gly Leu Glu Trp Val Ala
195 200 205
Val Ile Ser Tyr Asp Gly Ser Thr Lys Tyr Tyr Ala Asp Ser Val Lys
210 215 220
Gly Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Val Tyr Leu
225 230 235 240
Lys Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Val Tyr Tyr Cys Ala
245 250 255
Arg Asp Gln Ser Leu Leu Gly Asp Tyr Asp His Tyr Tyr Gly Leu Asp
260 265 270
Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser Gly Ser Glu Gln
275 280 285
SUBSTITUTE SHEET (RULE 26)
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Lys Leu Ile Ser Glu Glu Asp Leu Asn His His His His His Lys Leu
290 295 300
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 918 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
AAGCTTTCAC TAATGGTGAT GGTGATGGTT CAGATCTTCT TCGCTGATCA GTTTTTGTTC 60
GGATCCTGAG GAGACGGTGA CCGTGGTCCC TTTGCCCCAG ACGTCCAAAC CGTAGTAGTG 120
GTCATAGTCA CCCAACAGGC TCTGATCTCT CGCACAGTAA TAGACAGCCG TGTCCTCAGT 180
TCTCAGGCTG TTCATTTTTA GATACACCGT GTTCTTGGAA GTGTCTCTGG AGATGGTGAA 240
TCGGCCCTTC ACGGAGTCTG CGTAGTATTT AGTGCTTCCA TCATATGATA TAACTGCCAC 300
CCACTCCAGC CCCTTGCCTA GAGCCTGGCG GACCCAGTGC ATAGCAAAGC TTCTGAAGGG 360
GAATCCAGAG GCTGCACAGG AGAGTCTCAG GGACCTCCCA GGCTGGACCA CGCCTCCCCC 420
AGACTCCACC AGCTGCACCT GGGAGCCACC TCCACCTGAT CCACCACCTC CGGAACCGCC 480
ACCGCCAGAG ACAGATGGTG CAGCCACAGT TCGTTTGATC TCCACCTTGG TCCCTCCGCC 540
GAAAGTGATC TGAGGTGTCT GAGGTGAGCT ACCATACTGC TGACAGTAAT ACACTGCAAA 600
ATCTTCAGGC TCCAGTCTAC TGATGGTGAG AGTGAAGTCT GTCCCGGACC CACTGCCACT 660
GAACCTGTCT GGCATGCCAG TGGCCCTGGT GGATGCACCA TAGATGAGGA GCCTGGGAGC 720
CTGGCCAGGT TTCTGCTGGT ACCAGGCTAA GTAGCTGCTA CTAACACTCT GACTGGCCCT 780
GCAGGAGAGG GTGCCTCTTT CCCCTGGAGA CAAAGACAGG GTGCCTGGAG ACTGCGTCAA 840
CACAATATCG GCCTGCGCAA CGGTAGCGAA ACCAGCCAGT GCAACTGCGA TCGCGATAGC 900
GGTTTTTTTC ATGAATTC 918
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 867 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
SUBSTITUTE SHEET (RULE 26)
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(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: join(1..855, 862..867)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
GAA TTC ATG AAA AAA ACC GCT ATC GCG ATC GCA GTT GCA CTG GCT GGT 48
Glu Phe Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly
1 5 10 15
TTC GCT ACC GTT GCG CAG GCC GAT ATT GTG TTG ACG CAG TCT CCA GGC 96
Phe Ala Thr Val Ala Gln Ala Asp Ile Val Leu Thr Gln Ser Pro Gly
20 25 30
ACC CTG TCT TTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC AGG GCC 144
Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala
35 40 45
AGT CAG AGT GTT AGT AGC AGC TAC TTA GCC TGG TAC CAG CAG AAA CCT 192
Ser Gln Ser Val Ser Ser Ser Tyr Leu'Ala Trp Tyr Gln Gln Lys Pro
50 55 60
GGC CAG GCT CCC AGG CTC CTC ATC TAT GGT GCA TCC ACC AGG GCC ACT 240
Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr
65 70 75 80
GGC ATG CCA GAC AGG TTC AGT GGC AGT GGG TCC GGG ACA GAC TTC ACT 2BB
Gly Met Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
85 90 95
CTC ACC ATC AGT AGA CTG GAG CCT GAA GAT TTT GCA GTG TAT TAC TGT 336
Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys
100 105 110
CAG CAG TAT GGT AGC TCA CCT CAG ACA CCT CAG ATC ACT TTC GGC GGA 384
Gln Gln Tyr Gly Ser Ser Pro Gln Thr Pro Gln Ile Thr Phe Gly Gly
115 120 125
GGG ACC AAG GTG GAG ATC AAA CGA ACT GTG GCT GCA TCC GGA CAG GTG 432
Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Ser Gly Gln Val
130 135 140
CAG CTG GTG GAG TCT GGG GGA GGC GTG GTC CAG CCT GGG AGG TCC CTG 480
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu
145 150 155 160
AGA CTC TCC TGT GCA GCC TCT GGA TTC CCC TTC AGA AGC TTT GCT ATG 528
Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Arg Ser Phe Ala Met
165 170 175
CAC TGG GTC CGC CAG GCT CTA GGC AAG GGG CTG GAG TGG GTG GCA GTT 576
His Trp Val Arg Gln Ala Leu Gly Lys Gly Leu Glu Trp Val Ala Val
180 185 190
SUBSTITUTE SHEET (RULE 26)
CA 02255540 1998-11-20
WO 97/44461 PCTIUS97/08962
ATA TCA TAT GAT GGA AGC ACT AAA TAC TAC GCA GAC TCC GTG AAG GGC 624
Ile Ser Tyr Asp Gly Ser Thr Lys Tyr Tyr Ala Asp Ser Val Lys Gly
195 200 205
CGA TTC ACC ATC TCC AGA GAC ACT TCC AAG AAC ACG GTG TAT CTA AAA 672
Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Val Tyr Leu Lys
210 215 220
ATG AAC AGC CTG AGA ACT GAG GAC ACG GCT GTC TAT TAC TGT GCG AGA 720
Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
225 230 235 240
GAT CAG AGC CTG TTG GGT GAC TAT GAC CAC TAC TAC GGT TTG GAC GTC 768
Asp Gln Ser Leu Leu Gly Asp Tyr Asp His Tyr Tyr Gly Leu Asp Val
245 250 255
TGG GGC AAA GGG ACC ACG GTC ACC GTC TCC TCA GGA TCC GAA CAA AAA 816
Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser Gly Ser Glu Gln Lys
260 265 270
CTG ATC AGC GAA GAA GAT CTG AAC CAT CAC CAT CAC CAT TAGTGA AAG 864
Leu Ile Ser Glu Glu Asp Leu Asn His His His His His Lys
275 280 285
CTT 867
Leu
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 287 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Glu Phe Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly
1 5 10 15
Phe Ala Thr Val Ala Gln Ala Asp Ile Val Leu Thr Gln Ser Pro Gly
20 25 30
Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala
35 40 45
Ser Gin Ser Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gin Gln Lys Pro
50 55 60
Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr
65 70 75 80
SUBSTITUTE SHEET (RULE 26)
CA 02255540 1998-11-20
WO 97/44461 PCTIUS97/08962
96
Gly Met Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
85 90 95
Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys
100 105 110
Gln Gln Tyr Gly Ser Ser Pro Gln Thr Pro Gln Ile Thr Phe Gly Gly
115 120 125
Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Ser Gly Gln Val
130 135 140
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu
145 150 155 160
Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Arg Ser Phe Ala Met
165 170 175
His Trp Val Arg Gln Ala Leu Gly Lys Gly Leu Glu Trp Val Ala Val
180 185 190
Ile Ser Tyr Asp Gly Ser Thr Lys Tyr Tyr Ala Asp Ser Val Lys Gly
195 200 205
Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Val Tyr Leu Lys
210 215 220
Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
225 230 235 240
Asp Gln Ser Leu Leu Gly Asp Tyr Asp His Tyr Tyr Gly Leu Asp Val
245 250 255
Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser Gly Ser Glu Gln Lys
260 265 270
Leu Ile Ser Glu Glu Asp Leu Asn His His His His His Lys Leu
275 280 285
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 867 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
AAGCTTTCAC TAATGGTGAT GGTGATGGTT CAGATCTTCT TCGCTGATCA GTTTTTGTTC 60
GGATCCTGAG GAGACGGTGA CCGTGGTCCC TTTGCCCCAG ACGACCAAAC CGTAGTAGTG 120
GTCATAGTCA CCCAACAGGC TCTGATCTCT CGCACAGTAA TAGACAGCCG TGTCCTCAGT 180
SUBSTITUTE SHEET (RULE 26)
CA 02255540 1998-11-20
WO 97/44461 PCT/US97/08962
97
TCTCAGGCTG TTCATTTTTA GATACACCGT GTTCTTGGAA GTGTCTCTGG AGATGGTGAA 240
TCGGCCCTTC ACGGAGTCTG CGTAGTATTT AGTGCTTCCA TCATATGATA TAACTGCCAC 300
CCACTCCAGC CCCTTGCCTA GAGCCTGGCG GACCCAGTGC ATAGCAAAGC TTCTGAAGGG 360
GAATCCAGAG GCTGCACAGG AGAGTCTCAG GGACCTCCCA GGCTGGACCA CGCCTCCCCC 420
AGACTCCACC AGCTGCACCT GTCCGGATGC AGCCACAGTT CGTTTGATCT CCACCTTGGT 480
CCCTCCGCCG AAAGTGATCT GAGGTGTCTG AGGTGAGCTA CCATACTGCT GACAGTAATA 540
CACTGCAAAA TCTTCAGGCT CCAGTCTACT GATGGTGAGA GTGAAGTCTG TCCCGGACCC 600
ACTGCCACTG AACCTGTCTG GCATGCCAGT GGCCCTGGTG GATGCACCAT AGATGAGGAG 660
CCTGGGAGCC TGGCCAGGTT TCTGCTGGTA CCAGGCTAAG TAGCTGCTAC TAACACTCTG 720
ACTGGCCCTG CAGGAGAGGG TGGCTCTTTC CCCTGGAGAC AAAGACAGGG TGCCTGGAGA 780
CTGCGTCAAC ACAATATCGG CCTGCGCAAC GGTAGCGAAA CCAGCCAGTG CAACTGCGAT 840
CGCGATAGCG GTTTTTTTCA TGAATTC 867
SUBSTITUTE SHEET (RULE 26)
