U.S. patent application number 10/671328 was filed with the patent office on 2004-11-25 for immune reactivity to expressed activated oncogenes for diagnosis and treatment of malignancy.
This patent application is currently assigned to Washington Research Foundation. Invention is credited to Cheever, Martin A., Peace, David J..
Application Number | 20040235070 10/671328 |
Document ID | / |
Family ID | 23868429 |
Filed Date | 2004-11-25 |
United States Patent
Application |
20040235070 |
Kind Code |
A1 |
Cheever, Martin A. ; et
al. |
November 25, 2004 |
Immune reactivity to expressed activated oncogenes for diagnosis
and treatment of malignancy
Abstract
Methods for the detection, monitoring and treatment of
malignancies are disclosed. Detection of the proliferation of T
cells in response to in vitro exposure to a protein expression
product of an activated oncogene or cancer-related gene associated
with a malignancy, or detection of immunocomplexes formed between
the protein expression product and antibodies in body fluid, allows
the diagnosis of the presence of a malignancy. The present
invention also discloses methods and compositions for treating a
malignancy.
Inventors: |
Cheever, Martin A.; (Mercer
Island, WA) ; Peace, David J.; (Chicago, IL) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE
SUITE 6300
SEATTLE
WA
98104-7092
US
|
Assignee: |
Washington Research
Foundation
Seattle
WA
|
Family ID: |
23868429 |
Appl. No.: |
10/671328 |
Filed: |
September 24, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10671328 |
Sep 24, 2003 |
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08473762 |
Jun 7, 1995 |
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08473762 |
Jun 7, 1995 |
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08251590 |
May 31, 1994 |
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5601989 |
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08251590 |
May 31, 1994 |
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07820409 |
Jan 13, 1992 |
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5320947 |
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07820409 |
Jan 13, 1992 |
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07470645 |
Jan 26, 1990 |
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Current U.S.
Class: |
435/7.23 ;
435/6.14 |
Current CPC
Class: |
A61K 39/00 20130101;
A61P 35/00 20180101; C07K 14/82 20130101; G01N 33/5748 20130101;
A61P 31/04 20180101; Y10S 436/813 20130101; A61K 35/17 20130101;
G01N 2800/52 20130101; C07K 14/4746 20130101; G01N 33/5091
20130101 |
Class at
Publication: |
435/007.23 ;
435/006 |
International
Class: |
C12Q 001/68; G01N
033/574 |
Claims
1-23. (Cancelled)
24. A method for treating a malignancy in a warm-blooded animal,
wherein an activated oncogene or cancer-related gene is associated
with the malignancy, comprising the steps of: (a) isolating T cells
from a warm-blooded animal; (b) incubating the T cells in the
presence of at least one protein expression product of an activated
oncogene or cancer-related gene associated with the malignancy,
such that the T cells proliferate; and (c) administering to the
warm-blooded animal an effective amount of the proliferated T
cells.
25. The method of claim 24 wherein the protein expression product
of an activated oncogene is a protein encoded by an oncogene
selected from the group consisting of ras, src, abl, fgr, rel, yes,
fes, net, mos, raf, erb B, erb A, fms, neu, ros, kit, sea, sis,
myc, myb, fos, ski, jun and ets.
26. The method of claim 24 wherein the step of incubating the T
cells is repeated one or more times.
27-33. (Cancelled)
34. A method for eliciting or enhancing in a human a T cell
response to the protein expression product of an activated oncogene
or cancer-related gene associated with a malignancy, comprising
immunizing the human with at least one protein expression product
of an activated oncogene or cancer-related gene associated with a
malignancy wherein the expression product is recognized by T cells,
the cancer-related gene product having an altered primary sequence
relative to the product expressed in non-malignant tissue of the
human, and wherein the expression product of the activated oncogene
or cancer-related gene has an altered primary sequence which occurs
in nature when the oncogene or cancer-related gene is
activated.
35. The method of claim 34 wherein the protein expression product
of an activated oncogene is a protein encoded by an oncogene
selected from the group consisting of ras, src, abl, fgr, rel, yes,
fes, met, mos, raf, erb B, erb A, fms, neu, ros, kit, sis, myc,
myb, fos, jun and ets.
36. The method of claim 34 wherein the step of immunizing comprises
administering the protein expression product repetitively to the
human.
37. A method for prolonging the survival time of a human
subsequently exposed or reexposed to a malignancy, wherein an
activated oncogene or cancer-related gene is associated with the
malignancy, comprising prior to exposure or reexposure to the
malignancy immunizing the human with at least one protein
expression product of an activated oncogene or cancer-related gene
associated with the malignancy wherein the expression product is
recognized by T cells, the cancer-related gene product having an
altered primary sequence relative to the product expressed in
non-malignant tissue of the human, and wherein the expression
product of the activated oncogene or cancer-related gene has an
altered primary sequence which occurs in nature when the oncogene
or cancer-related gene is activated.
38. The method of claim 37 wherein the protein expression product
of an activated oncogene is a protein encoded by an oncogene
selected from the group consisting of ras, src, abl, fgr, rel, yes,
fes, met, mos, raf, erb B, erb A, fms, neu, ros, kit, sis, myc,
myb, fos, jun and ets.
39. The method of claim 37 wherein the step of immunizing comprises
administering the protein expression product repetitively to the
human.
40. The method of claim 34 wherein the protein expression product
of an activated oncogene is a protein encoded by a ras
oncogene.
41. The method of claim 34 wherein the protein expression product
of an activated oncogene is a protein encoded by a bcr-abl
oncogene.
42. The method of claim 37 wherein the protein expression product
of an activated oncogene is a protein encoded by a ras
oncogene.
43. The method of claim 37 wherein the protein expression product
of an activated oncogene is a protein encoded by a bcr-abl
oncogene.
44. The method of claim 34 wherein the protein expression product
is of an activated oncogene.
45. The method of claim 37 wherein the protein expression is of an
activated oncogene.
Description
CROSS REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of pending U.S. patent
application Ser. No. 08/473,762, filed Jun. 07, 1995; which
application is a division of U.S. patent application Ser. No.
08/251,590, filed May 31, 1994, which issued as U.S. Pat. No.
5,601,989; which application is a division of U.S. patent
application Ser. No. 07/820,409, filed Jan. 13, 1992, which issued
as U.S. Pat. No. 5,320,947; which application is a
continuation-in-part of U.S. patent application Ser. No.
07/470,645, filed Jan. 26, 1990 and abandoned; all of which
applications are incorporated by reference herein in their
entirety.
TECHNICAL FIELD
[0002] The present invention is generally directed toward the
detection, monitoring, and treatment of malignancies through the
use of a cancer patient's own to the protein expression product of
an activated oncogene or cancer-related gene associated with
malignancy.
BACKGROUND OF THE INVENTION
[0003] Despite enormous investments of financial and human
resources, cancer remains one of the major causes of death. A
common characteristic of malignancies is uncontrolled cell growth.
Cancer cells appear to have undergone a process of transformation
from the normal phenotype to a malignant phenotype capable of
autonomous growth. Mutation of somatic cell genes is considered to
be a common primary event that results in the transformation of
normal cells to malignant cells. The malignant phenotypic
characteristics encoded by the mutated genes are passed on during
cell division to the progeny of the transformed cells. Various
genes involved with transformation have been designated as
oncogenes. Oncogenes were originally identified as components of
the genetic material of oncogenic viruses. The homologous genes on
human chromosomes are commonly termed oncogenes or
proto-oncogenes.
[0004] Ongoing research involving oncogenes has identified at least
forty oncogenes operative in malignant cells and responsible for,
or associated with, transformation. Oncogenes have been classified
into different groups based on the putative function or location of
their gene products (such as the protein expressed by the
oncogene).
[0005] Proto-oncogenes are believed to be essential for certain
aspects of normal cellular physiology. Certain proto-oncogenes
appear to be activated to a cellular oncogene through quantitative
mechanisms that result from increased or deregulated expression of
an essentially normal gene product. For example, the myc gene
family has been associated with initiation and/or progression of
certain human lymphomas and carcinomas, whose transforming
activation is the result of quantitative mechanisms. Alternatively,
other proto-oncogenes appear to be activated to transforming
cellular oncogenes through qualitative mechanisms, including
mutation in the coding sequence of the gene. This creates a gene
product with an altered primary structure and biochemical
properties as a result of one or more differences in the amino acid
sequence of the protein. For example, the ras gene family, causally
associated with the most common forms of human malignancy (e.g.,
colon cancer) is activated as a result of single codon changes.
[0006] Studies to develop cancer therapies have, in general,
focused on the use of characteristic differences between normal and
malignant cells. Mutated, translocated or otherwise overexpressed
proto-oncogenes and the products of such genes represent potential
identifiable characteristic differences between normal and
malignant cells. The identified differences have been utilized in
attempts to develop diagnostic assays or therapeutic regimens.
[0007] An approach to developing a diagnostic assay has been to
attempt to quantify the expressed product of an oncogene in tissue
or body fluids, utilizing antibodies directed toward the unique or
abnormal oncogene product. In general, xenogeneic antibodies have
been raised against the abnormally expressed proto-oncogene
product. Problems in the development of diagnostic assays based on
detecting abnormal oncogene products include the following factors:
(1) a lack of antibodies with high specificity, affinity and
selectivity for the abnormal product; (2) only small amounts of
abnormal oncogene product may be released by tumor cells; (3)
oncogenic products may be released only intermittently by tumor
cells; (4) the oncogene product may be absorbed out of the body
fluid by antibody or may be formed into immune complexes; and (5)
the free antigen may be rapidly cleared or degraded.
[0008] Due to the difficulties in the current approaches to cancer
diagnosis and therapy, there is a need in the art for improved
methods and compositions. The present invention fills this need,
and further provides other related advantages.
SUMMARY OF THE INVENTION
[0009] Briefly stated, the present invention provides a variety of
methods for the detection of a malignancy in a warm-blooded animal,
wherein an activated oncogene or cancer-related gene is associated
with the malignancy. The methods may be used on a one time basis
when a malignancy is suspected or on a periodic basis to monitor an
individual with an elevated risk of acquiring a malignancy. In one
embodiment, the method comprises the steps of: (a) isolating T
cells from a warm-blooded animal; (b) incubating the T cells with
at least one protein expression product of an activated oncogene or
cancer-related gene associated with the malignancy; and (c)
detecting the presence or absence of proliferation of the T cells,
thereby determining the presence or absence of the malignancy. In
another embodiment, the method comprises the steps of: (a)
contacting a body fluid, suspected of containing antibodies
specific for a protein expression product of an activated oncogene
or cancer-related gene associated with the malignancy, with at
least one protein expression product of an activated oncogene or
cancer-related gene associated with the malignancy; (b) incubating
the body fluid under conditions and for a time sufficient to allow
immunocomplexes to form; and (c) detecting the presence or absence
of one or more immunocomplexes formed between the protein
expression product and antibodies in the body fluid specific for
the protein expression product, thereby determining the presence or
absence of the malignancy.
[0010] In another aspect, the present invention provides methods
for monitoring the effectiveness of cancer therapy in a
warm-blooded animal with a malignancy, wherein an activated
oncogene or cancer-related gene is associated with the malignancy.
In one embodiment, the method comprises the steps of: (a)
contacting a first body fluid sample, taken from the warm-blooded
animal prior to initiation of therapy, with at least one protein
expression product of an activated oncogene or cancer-related gene
associated with the malignancy; (b) incubating the body fluid under
conditions and for a time sufficient to allow immunocomplexes to
form; (c) detecting immunocomplexes formed between the protein
expression product and antibodies in the body fluid specific for
the protein expression product; (d) repeating steps (a), (b), and
(c) on a second body fluid sample taken from the animal subsequent
to the initiation of therapy; and (e) comparing the number of
immunocomplexes detected in the first and second body fluid
samples, thereby monitoring the effectiveness of the therapy in the
animal.
[0011] The present invention is also directed toward methods for
treating a malignancy in a warm-blooded animal, wherein an
activated oncogene or cancer-related gene is associated with the
malignancy. In one embodiment, the method comprises the steps of:
(a) isolating T cells from a warm-blooded animal; (b) incubating
the T cells in the presence of at least one protein expression
product of an activated oncogene or cancer-related gene associated
with the malignancy, such that the T cells proliferate; and (c)
administering to the warm-blooded animal an effective amount of the
proliferated T cells. In another embodiment, the method comprises
the steps of: (a) isolating T cells from a warm-blooded animal; (b)
incubating the T cells in the presence of at least one protein
expression product of an activated oncogene or cancer-related gene
associated with the malignancy, such that the T cells proliferate;
(c) cloning one or more cells that proliferated in the (d)
administering to the warm-blooded animal an effective amount of the
cloned T cells. In a third embodiment, the method comprises
immunizing the animal with at least one protein expression product
of an activated oncogene or cancer-related gene associated with the
malignancy.
[0012] Within a related aspect, the present invention provides
anti-cancer therapeutic compositions comprising T cells
proliferated in the presence of at least one protein expression
product of an activated oncogene or cancer-related gene associated
with a malignancy, in combination with a physiologically acceptable
carrier or diluent.
[0013] These and other aspects of the present invention will become
evident upon reference to the following detailed description and
attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 graphically illustrates that specific T cell
responses to point-mutated ras peptide can be generated by in vivo
priming on day 0 with 50 .mu.g of a peptide of 12 amino acid length
constructed identically to murine ras-p21 (amino acids 5-16) but
with the substitution of arginine for the glycine normally found at
position 12 (i.e., position 12 of ras-p21 but position 8 of the
peptide). This peptide is denoted as ras p5-16[R12] or as ras-R12.
Mice were immunized with ras-R12 peptide plus adjuvant or with
adjuvant alone. After 10 days, the draining lymph nodes were
harvested and lymphocytes stimulated in vitro with ras-R12 peptide
or as specificity controls with similar peptides substituted with
serine, cysteine or glycine at position 12 as opposed to arginine,
denoted as ras-S12, ras-C12 and ras-G12, respectively. Four days
later, cultures were pulsed with [.sup.3H]-thymidine (.sup.3HTdR)
for 8 hours, as described in Example 1 below. Results are
represented as .DELTA.CPM.
[0015] FIG. 2 shows that ras-peptide specific T cells grown
long-term in vitro in response to intermittent stimulation by
ras-peptide retain specific function. T cells derived from
mesenteric lymph nodes of C57BL/6 mice primed to ras-R12 (as
defined for FIG. 1 above) were cultured long-term in vitro in
response to intermittent stimulation with ras-R12 peptide (5
.mu.g/ml) on irradiated B6 spleen cells (3000 rad) as antigen
presenting cells. The specificity of the T cell line was tested on
day 85 by stimulating with graded concentrations of various ras
peptides (as defined for FIG. 1 above) or with trypsinized OVA
(TOVA) containing several potential immunogenic peptides. Data is
represented as .sup.3HTdR uptake in counts per minute (c.p.m.).
[0016] FIG. 3 graphically illustrates that T cells specific for a
ras peptide respond specifically to intact ras p21 protein
containing the same residue substitution. This peptide, denoted as
p5-17[R12], consists of 13 amino acid residues and was constructed
identically to murine ras-p21 (amino acids 5-17) but with the
substitution of arginine (R) for the glycine (G) normally found at
position 12 of ras-p21. Cloned B6 T cells specific for the Arg-12
ras peptide were cultured with irradiated B6 spleen cells and
either p5-17[R12] or intact ras p21 proteins bearing the designated
amino acid at position-12 (1 .mu.g/ml). Proliferative responses
were measured after four days (as described in FIG. 1). The data
represent the mean of triplicate determinations of the c.p.m. of
incorporated .sup.3HTdR. The standard deviations were <10% of
the c.p.m.
[0017] FIG. 4 shows that specific T cell responses can be generated
by in vivo priming with a peptide of 13 amino acid length
constructed identically to murine ras-p21 (amino acids 54-66) but
with the substitution of leucine (L) for the glutamine (Q) normally
found at position 61 of ras-p21. This peptide is denoted as
p54-66[L61] or [L61]. C3H/HeN mice were immunized twice
subcutaneously at two week intervals with Ribi adjuvant alone or
adjuvant emulsified with p54-66[L61] ras peptide (100 .mu.g). Two
weeks after the final immunization, spleen cells from mice injected
with adjuvant alone (open bar) or adjuvant plus p54-66[L61] ras
peptide (black bar) were harvested and tested in vitro for
proliferative response to the indicated p54-66 ras peptide (100
.mu.g/ml). The peptide containing the substitution of lysine (K)
for glutamine at position 61 is denoted as [K61]. The data
represent the mean of triplicate determinations of the c.p.m. of
incorporated .sup.3HTdR.+-.S.D.
[0018] FIG. 5 graphically illustrates that intact ras p21[L61]
protein is specifically recognized by a ras peptide-induced T cell
line. Specific T cell lines and clones were generated and
maintained as described in Example 2 below. A T cell line which
exhibits specific reactivity to the ras peptide p54-66[L61] (panel
A) and a T cell clone which exhibits specific reactivity to the ras
peptide p5-17[R12] (panel B) were stimulated with (a) no antigen,
(b) sensitizing ras peptide, or (c) intact ras p21[L61] protein.
The ras p54-66[L61] specific T cell line was of C3H origin and the
ras p5-17[R12] specific T cell clone was of B6 origin.
Proliferative assays were performed with syngeneic irradiated
spleen cells as antigen presenting cells (as described in FIG. 1).
Stimulating peptides and proteins were used at a concentration of 5
.mu.g/ml. The ras p21[L61] protein was produced in E. coli HB101
using the prokaryotic expression plasmid pHR-L9 and purified by
sequential DEAE-Sephacel and Sephadex column chromatography. The
data represent the mean c.p.m. of incorporated
.sup.3HTdR.+-.S.D.
[0019] FIG. 6 graphically illustrates chat lymphocytes from spleens
and lymph nodes of BALB/c mice primed in vivo with bcr3-abl2
12-amino acid peptide corresponding to the joining region of
p210.sup.bcr-abl chimeric protein proliferate specifically in
response to the sensitizing peptide in vitro. Ten days following
immunization with peptide, draining lymph node cells (Panel A) and
spleen cells (Panel B) were cultured alone or with bcr3-abl2 12 mer
peptide, or bcr2-abl2 12 mer peptide, or ras-c12 12 mer peptide at
25 .mu.g/ml, and assayed after 96 hours for proliferation by
[.sup.3H] thymidine uptake. The data represent the mean of
triplicate determinations of the c.p.m.+S.D.
[0020] FIG. 7 graphically illustrates that spleen cells sensitized
with FBL ras [Val-12] tumor cells in vivo respond specifically to
ras [Val-12] peptide in vitro. C57BL/6 mice were immunized with
irradiated FBL tumor cells (a Friend virus induced leukemia cell
line) that had been transfected with the ras [Val-12] oncogene.
Mice were injected twice with 5.times.10.sup.6 irradiated tumor
cells at an interval of two weeks. Approximately four weeks after
the second inoculation, spleen cells were harvested and tested for
the ability to respond to p5-17 ras [Val-12] peptide at the
indicated concentrations. Spleen cells from mice immunized with FBL
tumor that had been transfected with a normal ras [Gly-12]
proto-oncogene evidenced no increased proliferative response to the
same concentrations of the ras [Val-12] peptide.
[0021] FIG. 8 graphically illustrates that T cells specific for ras
p21 [Val-12] protein protect mice from challenge with a tumor that
expresses the corresponding p21.sup.ras protein. BALB/c.times.B6 F1
mice (6 per group) were injected i.p. with RAS4 tumor cells
(3.times.10.sup.5 cells per mouse) alone or in conjunction with B6
anti-p5-17 ras [Val-12] T cells (2.times.10.sup.7 cells per mouse).
RAS4 is a tumorigenic line that was derived by transfection of the
NIH3T3 cell line with the T24 oncogene encoding ras p21 [Val-12]
protein. The anti-ras [Val-12] T cell line was generated by in vivo
priming of B6 mice and subsequent periodic in vitro stimulation of
spleen cells with the p5-17 [Val-12] ras peptide. The T cell line
proliferates specifically to the sensitizing peptide and to intact
ras p21 [Val-12] protein.
DETAILED DESCRIPTION OF THE INVENTION
[0022] Prior to setting forth the invention, it may be helpful to
an understanding thereof to set forth definitions of certain terms
to be used hereinafter.
[0023] Activated oncogene--as used herein, refers to
proto-oncogenes that have become activated, leading to the
expression of protein products with amino acid sequences other than
those of the protein products expressed by the normal
proto-oncogenes.
[0024] Cancer-related gene--as used herein, refers to any altered
gene (other than an oncogene) associated with the development or
maintenance of the malignant-phenotype. Examples of such altered
genes include mutants of the p53 tumor suppressor gene.
[0025] Protein expression product--as used herein, includes
proteins, polypeptides, and peptides; and may be an intact
molecule, a fragment thereof, or a functional equivalent thereof;
and may be genetically engineered.
[0026] Proliferation of T cells--as used herein, includes the
multiplication of T cells as well as the stimulation of T cells
leading to multiplication, i.e., the initiation of events leading
to mitosis and mitosis itself. Methods for detecting proliferation
of T cells are discussed below.
[0027] As noted above, the present invention is directed toward
methods and compositions for the diagnosis, monitoring and
treatment of malignancies in a warm-blooded animal, wherein
activated oncogenes or cancer-related genes are associated with the
malignancies. The disclosure of the present invention shows that
the protein expression products of activated oncogenes and
cancer-related genes can be recognized by thymus-dependent
lymphocytes (hereinafter "T cells") and, therefore, the
autochthonous immune response can be utilized to diagnose and treat
malignancies expressing such protein expression products.
[0028] Activation of proto-oncogenes is associated with or leads to
transformation and expression of the malignant phenotype.
Activation by mechanisms such as mutation or chromosomal
translocation (gene rearrangement) creates a gene product with
altered primary structure, i.e., a protein expression product with
one or more amino acid differences relative to the normal protein
expressed by the proto-oncogene. Mutation mechanisms include point
mutation of nucleotides, recombination, deletion and insertion.
Examples of activation of proto-oncogenes through mutation include
activation of ras and neu proto-oncogenes. Chromosomal
translocation results in a fused protein, such as that associated
with chronic myeologenous leukemia. Similarly, cancer-related genes
express abnormal protein products with altered amino acid
sequences. For example, mutation of the p53 tumor suppressor gene
results in amino acid substitutions.
[0029] As disclosed within the present invention, protein products
with altered primary structure expressed by activated oncogenes and
cancer-related genes are recognized by T cells. Such abnormal
protein expression products "turn over" within cells, i.e., undergo
a cycle wherein a protein is synthesized, functions and then is
degraded and replaced by a newly synthesized molecule. The ensuing
peptide fragments from the degraded protein bind to major
histocompatibility complex (MHC) antigens. By display of an
abnormal peptide bound to MHC antigen on the cell surface and
recognition by host T cells of the combination of abnormal peptide
plus self MHC antigen, a malignant cell will be immunogenic to T
cells. The exquisite specificity of the T cell receptor enables
individual T cells to discriminate between fragments of proteins
which differ by a single amino acid residue.
[0030] During the immune response to an abnormal peptide, T cells
expressing a T cell receptor with high affinity binding of the
peptide-MHC complex will bind to the peptide-MHC complex and
thereby become activated and induced to proliferate. In the first
encounter with an abnormal peptide, small numbers of immune T cells
will proliferate and differentiate into effector and memory T
cells. The primary immune response will occur in vivo but is not
detected in vitro. Subsequent encounter with the same antigen by
the memory T cell will lead to a faster and more intense immune
response. The secondary response will occur either in vivo or in
vitro. The in vitro response is easily gauged by measuring the
degree of proliferation of the T cell population re-exposed in the
antigen. Proliferation of the T cell population in response to a
particular antigen is considered to be indicative of prior exposure
or priming to the antigen.
[0031] Within one aspect of the present invention, a malignancy in
which an activated oncogene or cancer-related gene is associated
with the malignancy may be detected. An immune response to an
abnormal protein expressed by an activated oncogene or
cancer-related gene, once generated, can be long-lived and can be
detected long after immunization, regardless of whether the protein
is present or absent in the body at the time of testing. In one
embodiment, prior exposure of a warm-blooded animal, such as
humans, to the protein expression product of an activated oncogene
or cancer-related gene can be detected by examining for the
presence or absence of T cell proliferative responses. More
specifically, T cells isolated from an individual by routine
techniques are incubated with a protein expression product of an
activated oncogene or cancer-related gene. Examples of oncogenes
include ras, src, abl, fgr, rel, yes, fes, net, mos, raf, erb B,
erb A, fms, neu, ros, kit, sea, sis myc, myb, fos, ski, jun and
ets. Alternatively, more than one protein expression product can be
examined with the T cell sample. It may be desirable to incubate
individual aliquots of a T cell sample with only one protein
expression product if such a protein interferes with another
protein expression product.
[0032] Detection of the proliferation of T cells may be
accomplished by a variety of known techniques. For example, T cell
proliferation can be detected by measuring the rate of DNA
synthesis. T cells which have been stimulated to proliferate
exhibit an increased rate of DNA synthesis. A typical way to
measure the rate of DNA synthesis is, for example, by
pulse-labeling cultures of T cells with tritiated thymidine, a
nucleoside precursor which is incorporated into newly synthesized
DNA. The amount of tritiated thymidine incorporated can be
determined using a liquid scintillation spectrophotometer. Other
ways to detect T cell proliferation include measuring increases in
interleukin-2 (IL-2) production, Ca.sup.2+flux, or dye uptake, such
as 3-(4,5-dimethylthiazol-- 2-yl)-2,5-diphenyl-tetrazolium.
[0033] A representative example of an activated oncogene that
expresses a protein product capable of immunizing T cells is the
ras oncogene. Ras proto-oncogenes encode a highly conserved family
of 21Kd proteins (189 amino acids in length) collectively
designated as "p21". p21 proteins bind to the inner aspect of the
cell membrane, associate with guanosine nucleotides and have
intrinsic GTPase activity. p21 proteins have been implicated as
important intermediary signalling proteins which regulate cell
growth and differentiation. Ras oncogenes were first detected as
genetic material in the transforming Harvey and Kirsten murine
sarcoma retroviruses. In animals, carcinogens induce very specific
and predictable mutations, usually involving codons 12, 13, 59
and/or 61, which impair the intrinsic GTPase activity of the p21
molecule and confer transforming activity. The human genome
contains at least three ras proto-oncogene homologs of the viral
ras oncogene denoted as K-ras, H-ras and N-ras which are located on
three separate human chromosomes. Each of the three ras genes can
become activated through specific mutation. Mutations of one of the
three ras proto-oncogenes occurs commonly in a variety of human
malignancies including pancreas adenocarcinoma, thyroid follicular
carcinoma, colon adenocarcinoma, seminoma, lung adenocarcinoma,
liver adenocarcinoma, melanoma, myeloid leukemia, and myeloma. The
disclosure of the present invention shows that once mutated, the
expressed protein product of any of the three ras genes by virtue
of a single amino acid substitution may be recognized by
autochthonous T cells.
[0034] For example, within the present invention, peptides
consisting of 12 or 13 amino acid residues which corresponded to
the amino acid sequence from residues 5-16, 5-17, or 4-16 of p21
were constructed to contain either the normal glycine (termed
Gly-12 or G-12), or arginine (termed Arg-12 or R-12), or serine
(termed Ser-12 or S-12), or cysteine (termed Cys-12, or C-12).
C57BL/6 mice were immunized with a single dose of the Arg-12
peptide. Lymphocytes from immunized mice were subsequently tested
for proliferative response to the above peptides in vitro and, as
shown in FIG. 1, were found to proliferate specifically in response
to the Arg-12 peptide, but not the Gly-12, Cys-12, or Ser-12
peptides. In addition, the lymphocytes from mice immunized with the
Arg-12 peptide were shown (FIG. 3) to proliferate specifically in
response to stimulation by the intact p21 protein containing this
same amino acid substitution (Oncogene Science, Inc., Manhasset,
N.Y.).
[0035] Another example of a peptide suitable within the present
invention is ras p54-66[L61]. This peptide consists of 13 amino
acid residues which correspond to the amino acid sequence from
residues 54-66 of p21, except leucine (L) has been substituted for
the glutamine (Q) normally found at position 61. Lymphocytes from
mice immunized with p54-66[L61] were found to proliferate in vitro,
as shown in FIG. 4, specifically in response to the Leu-61 peptide,
but not to Gln-61 or Lys-61 peptides. In addition, the lymphocytes
from mice immunized with the Leu-61 peptide were shown (FIG. 5) to
proliferate specifically in response to stimulation by the intact
p21 protein containing the same amino acid substitution.
[0036] Another representative example of an activated oncogene that
expresses a protein product capable of immunizing T cells is the
fusion bcr-abl oncogene. The hallmark of chronic myelogenous
leukemia (CML) is the translocation of the c-abl oncogene from
chromosome 9 to the specific breakpoint cluster region (bcr) of the
BCR gene on chromosome 22. The t(9;22) (q34; q11) translocation is
exceedingly consistent and present in more than 95% of patients
with CML. The translocation of the c-abl proto-oncogene on
chromosome 9 to the breakpoint cluster region (bcr) on chromosome
22 forms a new fusion gene, termed bcr-abl, which encodes a 210 kD
chimeric protein with abnormal tyrosine kinase activity. In humans,
the bcr-abl protein is expressed only by leukemia cells.
p210.sup.bcr-abl can stimulate the growth of hematopoietic
progenitor cells and is essential for the pathogenesis of CML. In
CML, the bcr breakpoint is generally between exons 2 and 3 or exons
3 and 4. Depending on the location of the breakpoint within the bcr
gene, the mRNA either includes or excludes bcr exon 3. Regardless
of the location of the breakpoint, the bcr-abl reading frames are
fused in frame and the translocated mRNA encodes a functional 210
kD chimeric protein consisting of 1,004 c-abl encoded amino acids
plus either 902 or 927 bcr encoded amino acids--both of which are
enzymatically active as protein kinases. In acute lymphoblastic
leukemia (ALL), c-abl is translocated to chromosome 22 but to a
different region of the bcr gene, denoted BCRI, which results in
the expression of a p185-190.sup.bcr-abl chimeric protein kinase.
p185-190.sup.bcr-abl is expressed in approximately 10% of children
and 25% of adults with ALL.
[0037] The disclosure of the present invention shows that the
joining region of the bcr-abl protein is immunogenic to T cells.
Immunization of mice with synthetic peptides corresponding to the
joining region elicited peptide-specific CD4.sup.+ class
II-MHC-restricted T cells. For example, an immunizing peptide was
composed of 6 bcr, 1 fusion, and 5 c-abl amino acids. The bcr-abl
peptide-specific T cells recognized only the combined sequence of
bcr-abl amino acids and not bcr or c-abl amino acid sequences
alone. Thus, immunity was directed specifically against the 12
amino acid residues of the joining region. Importantly,
p210.sup.bcr-abl protein effectively stimulated the bcr-abl
peptide-specific T cells. Therefore, p210.sup.bcr-abl protein can
be processed by antigen-presenting cells so that the joining region
segment is bound to class II MHC molecules in a configuration
similar to that of the immunizing peptide and in a concentration
high enough to stimulate the antigen-specific T cell receptor.
[0038] Similarly, proteins (or peptides based upon or derived
therefrom) other than p21 and bcr-abl may be isolated or
constructed by a variety of known techniques. It will be
appreciated by those skilled in the art that it may be desirable to
increase the length of an overall peptide or of the native flanking
regions to facilitate the induction of T cell responses. As
discussed above, protein expression products of activated oncogenes
other than ras, or cancer-related genes (i.e., with an amino acid
sequence different from that of the proteins expressed by normal
proto-oncogenes or normal genes) are suitable for use within the
methods described herein.
[0039] For therapeutic purposes, T cells that proliferate in the
presence of one or more protein expression products of activated
oncogenes or cancer-related genes can be expanded in number either
in vitro or in vivo. Proliferation of such T cells ii vitro may be
accomplished in a variety of ways. For example, the T cells can be
re-exposed to one or more protein expression products. It may be
desirable to repeat the exposure of T cells to the protein to
induce proliferation. As shown in FIG. 2, protein expression
product-specific T cells can be grown to large numbers in vitro
with retention of specificity in response to intermittent
restimulation with the immunizing peptide.
[0040] Alternatively, one or more T cells that proliferate in the
presence of a protein expression can be expanded in number by
cloning. Methods for cloning cells are well known in the art. For
example, T cell lines may be established in vitro and cloned by
limiting dilution. Responder T cells are purified from the
peripheral blood of sensitized patients by density gradient
centrifugation and sheep red cell rosetting and established in
culture by stimulating with the nominal antigen in the presence of
irradiated autologous filler cells. In order to generate CD4+T cell
lines, intact ras p21 protein bearing the relevant amino acid
substitution is used as the antigenic stimulus and autologous
peripheral blood lymphocytes (PBL) or lymphoblastoid cell lines
(LCL) immortalized by infection with Epstein Barr virus are used as
antigen presenting cells. In order to generate CD8+T cell lines,
autologous antigen-presenting cells transfected with an expression
vector which produces relevant mutated ras p21 protein are used as
stimulator cells. Established T cell lines are cloned 2-4 days
following antigen stimulation by plating stimulated T cells at a
frequency of 0.5 cells per well in 96-well flat-bottom plates with
1.times.10.sup.6 irradiated PBL or LCL cells and recombinant
interleukin-2 (rIL2) (50 U/ml). Wells with established clonal
growth are identified at approximately 2-3 weeks after initial
plating and restimulated with appropriate antigen in the presence
of autologous antigen-presenting cells, then subsequently expanded
by the addition of low doses of rIL2(10 U/ml) 2-3 days following
antigen stimulation. T cell clones are maintained in 24-well plates
by periodic restimulation with antigen and rIL2 approximately every
two weeks.
[0041] Regardless of how an individual's T cells are proliferated
in vitro, the T cells may be administered to the individual for
therapeutic attack against a tumor. Thus, a patient's own T cells
(autochthonous T cells) can be used as reagents to mediate specific
tumor therapy. Typically, about 1.times.10.sup.9 to
1.times.10.sup.11 T cells/M.sup.2 will be administered
intravenously or intracavitary, e.g., in pleural or peritoneal
cavities, or in the bed of a resected tumor. It will be evident to
those skilled in the art that the number and frequency of
administration will be dependent upon the response of the patient.
Suitable carriers or diluents for T cells include physiological
saline or sera. It will be recognized by one skilled in the art
that the composition should be prepared in sterile form.
[0042] T cells may also be proliferated in vivo. For example,
immunization of an individual with one or more protein expression
products of activated oncogenes or cancer-related genes can induce
continued expansion in the number of T cells necessary for
therapeutic attack against a tumor. It may be desirable to
administer the protein expression product repetitively.
[0043] The present invention also discloses that the protein
expression products of activated oncogenes or cancer-related genes,
in addition to being immunogenic to T cells, appear to stimulate
B-cells to produce antibodies capable of recognizing these
proteins. Detection of such antibodies provides another way to
diagnose a malignancy in which an activated oncogene or
cancer-related gene is associated with the malignancy. Antibodies
specific (i.e., which exhibit a binding affinity of about 10.sup.7
liters/mole or better) for one or more protein expression products
may be found in a variety of body fluids including sera and
ascites. Detection of one or more immunocomplexes formed between a
protein expression product and antibodies specific for the protein
may be accomplished by a variety of known techniques, such as
radioimmunoassays (RIA) and enzyme linked immunosorbent assays
(ELISA).
[0044] Suitable immunoassays include the double monoclonal antibody
sandwich immunoassay technique of David et al. (U.S. Pat. No.
4,376,110); monoclonal-polyclonal antibody sandwich assays (Wide et
al., in Kirkham and Hunter, eds., Radioimmunoassay Methods, E. and
S. Livingstone, Edinburgh, 1970); the "western blot" method of
Gordon et al. (U.S. Pat. No. 4,452,901); immunoprecipitation of
labeled ligand (Brown et al., J. Biol. Chem. 255:4980-4983, 1980);
enzyme-linked immunosorbent assays as described by, for example,
Raines and Ross (J. Biol. Chem. 257:5154-5160, 1982);
immunocytochemical techniques, including the use of fluorochromes
(Brooks et al., Clin. Exp. Immunol. 39: 477, 1980); and
neutralization of activity [Bowen-Pope et al., Proc. Natl. Acad.
Sci. USA 81:2396-2400 (1984)], all of which are hereby incorporated
by reference. In addition to the immunoassays described above, a
number of other immunoassays are available, including those
described in U.S. Pat. Nos.: 3,817,827; 3,850,752; 3,901,654;
3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876, all of
which are herein incorporated by reference.
[0045] For detection purposes, the protein expression products
("antigens") may either be labeled or unlabeled. When unlabeled,
the antigens find use in agglutination assays. In addition,
unlabeled antigens can be used in combination with labeled
molecules that are reactive with immunocomplexes, or in combination
with labeled antibodies (second antibodies) that are reactive with
the antibody directed against the protein expression product, such
as antibodies specific for immunoglobulin. Alternatively, the
antigens can be directly labeled. Where they are labeled, the
reporter group can include radioisotopes, fluorophores, enzymes,
luminescers, or dye particles. These and other labels are well
known in the art and are described, for example, in the following
U.S. Pat. Nos.: 3,766,162; 3,791,932; 3,817,837; 3,996,345; and
4,233,402.
[0046] Typically in an ELISA assay, antigen is adsorbed to the
surface of a microtiter well. Residual protein-binding sites on the
surface are then blocked with an appropriate agent, such as bovine
serum albumin (BSA), heat-inactivated normal goat serum (NGS), or
BLOTTO (buffered solution of nonfat dry milk which also contains a
preservative, salts, and an antifoaming agent). The well is then
incubated with a sample suspected of containing specific antibody.
The sample can be applied neat, or, more often, it can be diluted,
usually in a buffered solution which contains a small amount
(0.1-5.0% by weight) of protein, such as BSA, NGS, or BLOTTO. After
incubating for a sufficient length of time to allow specific
binding to occur, the well is washed to remove unbound protein and
then incubated with an anti-species specific immunoglobulin
antibody labeled with a reporter group. The reporter group can be
chosen from a variety of enzymes, including horseradish peroxidase,
beta-galactosidase, alkaline phosphatase, and glucose oxidase.
Sufficient time is allowed for specific binding to occur, then the
well is again washed to remove unbound conjugate, and the substrate
for the enzyme is added. Color is allowed to develop and the
optical density of the contents of the well is determined visually
or instrumentally.
[0047] In one preferred embodiment of this aspect of the present
invention, a reporter group is bound to the protein expression
product. The step of detecting immunocomplexes involves removing
substantially any unbound protein expression product and then
detecting the presence or absence of the reporter group.
[0048] In another preferred embodiment, a reporter group is bound
to a second antibody capable of binding to the antibodies specific
for a protein expression product. The step of detecting
immunocomplexes involves (a) removing substantially any unbound
antibody, (b) adding the second antibody, (c) removing
substantially any unbound second antibody and then (d) detecting
the presence or absence of the reporter group. Where the antibody
specific for the protein expression product is derived from a
human, the second antibody is an anti-human antibody.
[0049] In a third preferred embodiment for detecting
immunocomplexes, a reporter group is bound to a molecule capable of
binding to the immunocomplexes. The step of detecting involves (a)
adding the molecule, (b) removing substantially any unbound
molecule, and then (c) detecting the presence or absence of the
reporter group. An example of a molecule capable of binding to the
immunocomplexes is protein A.
[0050] It will be evident to one skilled in the art that a variety
of methods for detecting the immunocomplexes may be employed within
the present invention. Reporter groups suitable for use in any of
the methods include radioisotopes, fluorophores, enzymes,
luminescers, and dye particles.
[0051] In a related aspect of the present invention, detection of
immunocomplexes formed between a protein expression product of an
activated oncogene, or cancer-related gene, and antibodies in body
fluid which are specific for the protein may be used to monitor the
effectiveness of cancer therapy. Samples of body fluid taken from
an individual prior to and subsequent to initiation of therapy may
be analyzed for the immunocomplexes by the methodologies described
above. Briefly, the number of immunocomplexes detected in both
samples are compared. A substantial change in the number of
immunocomplexes in the second sample (post-therapy initiation)
relative to the first sample (pre-therapy) reflects successful
therapy.
[0052] The following examples are offered by way of illustration
and not by way of limitation.
EXAMPLES
Example 1
Elicitation of Specific Class-II Restricted T-Cell Response to
Mutated Ras Oncogene Products
[0053] A. Immunization
[0054] C57BL/6 and B6.C--H-2.sup.bm12 mice (Jackson Laboratories,
Bar Harbor, Me.) were inoculated with a synthetic peptide (Amino
acid sequence: KLVVVGARGVGK; Microbiological Associates, Bethesda,
Md.) which corresponds to residue 5-16 of the p21 protein product
of mutated ras proto-oncogene encoding a residue 12 substitution of
Arginine (R) for Glycine (G). The peptide was solubilized in
distilled water at 1 mg/ml, emulsified in complete Freund's
adjuvant (Sigma Co., St. Louis, Mo.) at a ration of 1:1, then
injected subcutaneously into the base of the tail using a 25 gauge
needle. Alternatively peptide was emulsified with Ribi MPL+TDM+CWS
adjuvant (Ribi Immunochem. Res., Hamilton, Mont.) and injected
subcutaneously into both hindquarters. Total amount of peptide
injected was 50 .mu.g per mouse. Seven days later animals were
sacrificed and draining periaortic and inguinal lymph nodes were
removed. Lymph nodes, suspended in buffered saline in petri dishes,
were teased apart with 18 gauge needles. Dislodged lymphocytes were
collected and washed with buffered saline then suspended at
1.times.10.sup.6 cells per ml in culture medium for use in
proliferation assays.
[0055] B. Proliferation Assay
[0056] Lymphocytes obtained from immunized mice were suspended in
culture medium (consisting of RPMI 1640 Gibco supplemented with 10%
fetal calf serum, 2 mM L-glutamine, 100 U/ml streptomycin, 100 U/ml
penicillin and 2.5.times.10.sup.-5 M 2-mercaptoethanol) and were
plated in 96-well flat bottom microtiter plates (Costar Co.,
Cambridge, Mass.) at 1.times.10.sup.5 cells per well. Syngeneic
spleen cells which had been irradiated to 3000 rads were added at
2.times.10.sup.5 cells per well to serve as antigen presenting
cells. The immunizing peptide and indicated control peptides (50
.mu.g/ml) were added to triplicate wells (final volume of 200
.mu.l/well). The plates were incubated at 37.degree. C. in
humidified air containing 5% CO.sub.2 for 72 hours then pulsed for
6 hr with 1 .mu.Ci per well of tritiated thymidine (.sup.3H-TdR; 20
Ci/mmol from NEN Products, Boston, Mass.). Lymphocytes from
individual wells were collected onto filter paper disks using a
multi-channel harvester then transferred to scintillation fluid in
individual counting tubes. .beta. emission was measured using a
Beckmann liquid scintillation spectrophotometer. The data presented
in FIG. 1 represents the mean of triplicate determinations.
Example 2
Generation of T Cell Lines and Clones with Specificity for the
Protein Product of Mutated Ras Proto-Oncogene
[0057] Lymphocytes obtained from draining lymph nodes of immunized
mice as described above were plated (4.times.10.sup.6 cells per
well) in 24-well culture plates (Costar Co.) with irradiated
syngeneic spleen cells (10,000 rads; 2.times.10.sup.6 cells per
well) and immunizing peptide (5 .mu.g/well) to give a final volume
of 2 ml (culture medium). Plates were cultured for 5 days
(37.degree. C., 5% Co.sub.2), then split 1:2 onto replicate plates.
After ten days lymphocytes were restimulated by plating
5.times.10.sup.5 cultured lymphocytes with 4.times.10.sup.6
irradiated syngeneic spleen cells and peptide at 5 .mu.g/ml. After
three days, individual wells were redistributed into 2-4 wells of
culture medium containing 10 units of Interleukin-2 (human
recombinant from Hoffmann-LaRoche) to facilitate cell expansion.
Lines were restimulated with antigen and subsequently IL-2 as
described above, every 3-4 weeks.
[0058] Immune lymphocytes which had been twice stimulated with
antigen in vitro were cloned at day 13 following initiation of
culture (3 days following Ag restimulation) by plating immune T
cells at a frequency of 0.5 cells per well in 96-well flat-bottom
plates with irradiated syngeneic spleen cells (1.times.10.sup.6
cells per well) and Interleukin-2 at 50 U/ml. Wells with clonal
growth were expanded and tested for immune specificity as shown in
FIG. 2. Specific T cell clones were maintained as described for T
cell lines.
Example 3
T Cell Immunity to the Joining Region of p210.sup.bcr-abl
Protein
[0059] A. Tumor Cell Lines, Peptides and Proteins
[0060] K562 is a Ph-chromosome-positive human CML cell line
(Konopka & Witte, Cell 37:1035-1042, 1984) 8B1 and 106-5 are
BALBIC murine leukemia cell lines derived by retroviral
transformation of BALB/c bone marrow cells with p210 or p185
bcr-abl fusion genes, respectively (McLaughlin et al., Proc. Natl.
Acad. Sci. USA 84:6558-6562, 1987; McLaughlin et al., Mol. and
Cell. Biol. 9:1866-1674, 1989), and kindly provided by Dr. Owen
Witte. K-BALB is a Kirsten murine sarcoma virus-transformed
BALB/3T3 fibroblast tumor cell line. The amino acid sequences of
synthetic peptides used (Table 1) were synthesized by M. Dojeski
(FHCRC) using a peptide synthesizer (430A; Applied Biosystems,
Foster City, Calif.) and purified by HPLC. To prepare bcr-abl
protein extracts, K562, 8B1, 106-5 and K-BALB cells were harvested
and resuspended in 25 mM potassium phosphate, pH 7.0, plus 2 mM
EDTA, 100 mM NaCl, 1 mM PMSF, 50 .mu.g/ml leupeptin, 100 units/ml
aprotinin and 20 mM benzamidine (Pendergast et al., Mol. and Cell.
Biol. 6:759-766, 1989). The cells were homogenized by use of
ultrasonic cell disrupter Model W-380 (Heat Systems Ultrasonics,
New York), centrifuged for 10 minutes at 3000 g and the supernates
resedimented at 100,000 g for 90 minutes. The high molecular weight
(>100,000 kD) supernatant proteins in the cell extracts were
isolated and concentrated by using DIAFLO ultrafilter PM100
(Amicon, Mass.). The partially purified bcr-abl proteins were
immunoprecipitated from the lysate with monoclonal antibody to bcr
(Oncogene Science, New York), electrophoresed by SDS-PAGE and
identified by Western immunoblotting with antibody to c-abl
(Oncogene Science, New York). The bcr-abl proteins were aliquoted
and stored at -70.degree. C.
1TABLE 1 Bcr-abl Peptide Amino Acid Sequences joining point
.vertline. bcr3-abl2 22 mer* I V H S A T G F K Q S S K A L Q R P V
A S D bcr3-abl2 18 mer H S A T G F K Q S S K A L Q R P V A
bcr3-abl2 16 mer G F K Q S S K A L Q R P V A S D bcr3-abl2 14 mer G
F K Q S S K A L Q R P V A bcr3-abl2 12 mer G F K Q S S K A L Q R P
bcr3 12 mer I V H S A T G F K Q S S bcr2-abl2 12 mer L T I N K E E
A L Q R P BCRI-abl2 14 mer A F H G D A Q A L Q R P V A *Amino acids
are represented by single letter code; bcr3-abl2 denotes sequences
from the p210.sup.bcr-abl chimeric protein composed of bcr exon 3
fused to c-abl exon 2; bcr2-abl2 denotes sequences from the
p210.sup.bcr-abl chimeric protein composed of bcr exon 2 fused to
c-abl exon 2; BCRI-abl2 denotes sequences of p185.sup.bcr-abl
chimeric protein composed of the BCRI region of the bcr gene fused
to c-abl exon 2 (Gehly et al., Blood 78:458-465, 1991).
[0061] B. Immunization Protocols and Proliferative Assays
[0062] Mice (female BALB/c and C57BL/6 (B6), 6 to 8 weeks old;
Jackson Laboratories, Bar Harbor, Me.) were inoculated twice at
2-week intervals by subcutaneous injection with Complete Freund's
Adjuvant (Sigma Chemical Co., St. Louis, Mo.) alone or emulsified
with bcr3-abl2 peptide (100 .mu.g). Ten days after the final
immunization, single cell suspensions of draining lymph nodes and
spleens were prepared in medium consisting of 1:1 mixture of RPMI
1640 (Gibco Laboratories, Inc., Grand Island, N.Y.) and EHAA
(Biofluids, Inc., Rockville, Md.) with 2.5.times.10.sup.-5 M 2-ME,
200 U/ml penicillin, 200 U/ml streptomycin, 10 mM L-glutamine and
10% fetal calf serum. For proliferative assays, lymphocytes were
cultured in 96-well plates at 2.times.10.sup.5 cells per well with
4.times.10.sup.5 irradiated syngeneic spleen cells (3,000 rad) and
designated (total volume of 200 .mu.l per well). Plates were
incubated in a humidified atmosphere under 5% CO.sub.2 tension at
37.degree. C. for 72 hours, and then pulsed for 18 hours with 1 mCi
of [.sup.3H]-thumidine per well. Proliferative responses of cloned
T cells utilized 2.times.10.sup.4 cells per well with
5.times.10.sup.5 irradiated syngeneic spleen cells (3,000 rad) and
designated peptide.
[0063] C. Generation and Characterization of p210 bcr-abl-Specific
T Cell Clones
[0064] Lymphocytes from the draining lymph nodes of mice immunized
with bcr3-abl2 peptide were cultured in 24-well culture plates at
4.times.10.sup.6 cells per well with 2.times.10.sup.6 irradiated
syngeneic spleen cells and bcr3-abl2 peptide at 25 .mu.g/ml (total
volume 2 ml). The plates were cultured in a humidified atmosphere
under 5% CO.sub.2 tension at 37.degree. C. for 5 days then split
1:2. After 10 days, lymphocytes were restimulated at
1.times.10.sup.6 lymphocytes per well in 24-well culture plates
with 5.times.10.sup.6 irradiated syngeneic spleen cells and peptide
(25 .mu.g/ml). Two days after the second in vitro stimulation, T
cells were-plated at 0.5 cells per well in 96-well flat-bottom
plates with 1.times.10.sup.6 irradiated syngeneic spleen cells and
rIL-2 (50 U/ml). Derived T cell clones were maintained by periodic
stimulation with peptide (1 .mu.g/ml) plus irradiated syngeneic
spleen cells followed by expansion with rIL-2 (10 U/ml) every 2
weeks. T cell clones were stained with FITC-conjugated anti-Thy 1.2
(1%), or FITC-conjugated anti-Lyt2 (2%), or PE-conjugated anti-L3T4
(3%) mAbs (Becton Dickinson Immunocytochemistry Systems, Mountain
View, Calif.) at 4.degree. C. for 45 minutes.
[0065] D. Synthetic Peptides Corresponding to the Joining Region of
p210.sup.bcr-abl Protein Elicit T Cell Responses
[0066] BALB/c mice were immunized with a synthetic 12 amino acid
peptide identical to the joining region of the p210 protein in K562
CML cells, termed bcr3-abl2 peptide (Table 1). Immunity was
validated by the demonstration that host lymph node and splenic
lymphocytes from immunized mice proliferated in vitro in response
to the immunizing peptide but not control peptides (FIG. 1).
Similar data was generated in the B6 mouse strain. The immunizing
peptide, G.cndot.F.cndot.K.cndot.Q.cndot.S.cndot.S-
.cndot.K.cndot.A.cndot.L.cndot.Q.cndot.R.cndot.P contained 6 amino
acids from bcr (G.cndot.F.cndot.K.cndot.Q.cndot.S.cndot.S), 1
fusion amino acid (K) and 5 amino acids from c-abl
(A.cndot.L.cndot.Q.cndot.A.cndot.P) (Table 1). To examine for fine
specificity, the T cell lines were cloned by limiting dilution and
maintained by episodic restimulation with the bcr3-abl2 peptide.
All clones were Thy-1.2.sup.+ CD4.sup.+ CD8.sup.- by analysis with
fluoresceinated antibodies. Depending upon which bcr domains are
included, the bcr-abl chimeric protein takes two major forms in CML
(bcr2-abl2 and bcr3-abl2) and one major form in ALL (BCRI-abl2)
(Canaani et al., in A. Deisseroth and R. B. Arlinghaus, eds.,
Chronic Myelogenous Leukemia: Molecular Approaches to Research and
Therapy, Marcel Dekker, New York, Chap. 8, pp. 217-240, 1991; Gehly
et al., Blood 78:458-465, 1991; Clark et al., Science 235:85-88,
1987; Kurzrock et al., Nature 325:631-635, 1987; Chan et al.,
Nature 325:635-637, 1987). In each case, the c-abl sequence of
amino acids is identical but is joined with distinct bcr amino acid
sequences. Data from 9 representative clones (Table 2) demonstrated
that the immune T cells responded specifically to bcr3-abl2 but not
to the alternate bcr-abl joining regions of bcr2-abl2 or BCRI-abl2.
The immune T cells likewise failed to respond to peptides identical
to the isolated amino acid sequence of bcr3 alone. Thus, the
immunogenic determinant was specifically associated with the unique
12 amino acid residues of the joining region of the
p210.sup.bcr3-abl2 protein.
2TABLE 2 Specificity of CD4.sup.+ T Cell Clones to a 12-Amino-Acid
Peptide Corresponding to the Joining Region of the p210.sup.bcr-abl
Chimeric Protein* T cell [.sup.3H] Thymidine Incorporation
(c.p.m.).sup..dagger. clones Media bcr3-abl2 bcr2-abl2 BCRI-abl2
bcr3 Ras-c12 2E10 243 242,633 287 137 667 258 1D2 267 274,701 274
315 294 357 3B7 230 64,935 349 277 462 497 2E3 535 293,219 402 461
193 372 3E11 269 263,425 299 398 305 579 3G2 618 30,846 478 583 461
504 3G3 394 61,521 217 351 190 273 2B12 424 109,319 341 385 461 307
2D4 532 262,799 143 147 133 259 *Resting CD4.sup.+ T cell clones of
BABL/c origin were cultured with irradiated syngeneic spleen cells
as APC plus the indicated peptides at 5 .mu.g/ml. .sup..dagger.The
data represent the mean of triplicate determinations of [.sup.3H]
thymidine incorporated for the final 18 hours of a 96-hour culture.
The standard deviations were <10% of the c.p.m.
[0067] The concentration of bcr3-abl2 peptide necessary to induce
proliferation varied. In general, optimal stimulation occurred in
the range of 1 to 5 .mu.g/ml, but some clones responded vigorously
to as low as 0.04 .mu.g/ml, the lowest concentration tested (Table
3).
3TABLE 3 Proliferative Response of CD4.sup.+ T Cell Clones to
Varying Concentrations of a 12-Amino Acid Peptide Corresponding to
the Joining Region of the p210.sup.bcr-abl Chimeric Protein*
[.sup.3H] Thymidine Incorporation (c.p.m.) after Stimulation with
bcr3-abl2 12 mer peptide (.mu.g/ml).sup..dagger. Responding T cell
clones Media 25 5 1 0.2 0.04 2E10 471 131,643 312,783 333,056
235,103 26,195 1D2 496 66,329 259,018 183,229 91,251 16,423 3B7 421
53,441 149,640 111,212 56,967 60,957 2E3 399 358,253 407,729
273,273 93,921 1,475 3E11 556 40,755 130,633 189,843 135,985 91,240
3G2 561 132,024 168,387 150,799 49,602 9,440 3G3 455 101,280
143,670 93,219 18,403 11,510 2B12 364 132,620 229,010 213,089
71,651 4,132 2D4 387 42,623 182,266 256,606 150,711 12,599 *Resting
T cell clones were cultured with irradiated syngeneic spleen cells
as APC plus bcr3-abl2 12 mer peptide at the indicated
concentration. .sup..dagger.The data represent the mean of
triplicate determinations of [.sup.3H] thymidine incorporated for
the final 18 hours of a 96-hour culture. The standard deviations
were <10% of the c.p.m.
[0068] Antigen-specific responses of CD4.sup.+ T cells require not
only binding to class II MHC molecules on APC but also display of
the antigenic epitope in the proper orientation in the MHC cleft.
Two bcr3-abl2-specific T cell clones derived by immunization with a
12 mer bcr3-abl2 peptide were tested for response to 14-, 16-, 18-,
and 22-mer peptides synthesized to be identical to the naturally
occurring amino acid sequences of the p210 joining region. The 12
mer bcr3-abl2 peptide-specific T cells responded to each of the
longer peptides (Table 4), implying that a broad spectrum of
joining region peptides could be appropriately presented by class
II MHC molecules.
4TABLE 4 Proliferative Response of CD4.sup.+ T Cell Clones to
Bcr3-abl2 Peptides of Varying Lengths* [.sup.3H] Thymidine
Incorporation (c.p.m.) after Stimulation with bcr3-abl2 peptide (5
.mu.g/ml).sup..dagger. Responding T cell clones Media 12-mer 14-mer
16-mer 18-mer 22-mer 2E10 589 96,525 170,085 139,083 224,674 29,509
1D2 159 120,049 238,297 108,309 150,309 21,208 *Resting T cell
clones were cultured with irradiated syngeneic spleen cells as APC
plus bcr3-abl2 peptides of the above-varied lengths (see Table 1
for structure of peptides). .sup..dagger.The data represent the
mean of triplicate determinations of [.sup.3H] thymidine
incorporated for the final 18 hours of a 96 hour culture. The
standard deviations were <10% of the c.p.m.
[0069] E. Bcr3-abl2 Pentide-Specific T Cells Recognize p210 bcr-abl
Protein
[0070] Partially purified p210.sup.bcr-abl protein from K562 and
8B1 tumor cells, p185.sup.bcr-abl protein from 106-5 tumor cells,
and irrelevant proteins from K-BALB tumor cells were prepared.
Several bcr3-abl2 peptide-specific CD4.sup.+ T cell clones were
tested in a proliferative assay with irradiated syngeneic APC plus
either bcr3-abl2 peptide or partially purified bcr-abl proteins.
Bcr3-abl2 peptide-specific T cells responded to p210.sup.bcr-abl
protein, but not to p185.sup.bcr-abl protein or irrelevant K-BALB
proteins (Table 5). The proliferative response to p210.sup.bcr-abl
protein was less than the response to peptide, presumably due to
either less joining region segments being present in the protein
preparation on a per weight basis or to competition for binding to
MHC molecules by alternative segments of the protein.
5TABLE 5 Proliferative Response of bcr3-abl2 peptide-specific T
Cell Clone to p210.sup.bcr-abl Chimeric Proteins* [.sup.3H]
Thymidine Incorporation (c.p.m.).sup..dagger. after Stimulation
with bcr-abl 12 peptide/proteins (.mu.g/ml) Peptide and Proteins
Media 1 0.2 0.04 bcr3-abl2 14-mer peptide 214 92,970 54,896 6,303
p210.sup.bcr-abl from K562 207 6,679 2,270 338 p210.sup.bcr-abl
from 8B1 521 2,498 1,483 236 p185.sup.bcr-abl from 106-5 231 157
198 312 Proteins from K-BALB 164 197 276 284 *Bcr3-abl2
peptide-specific CD4.sup.+ T cell clone 2E10 (see Table 2 for
specificity) was cultured with irradiated syngeneic spleen cells as
APC plus the indicated concentration of bcr3-abl2 14-mer peptide or
with partially purified protein isolated from K562 and 8B1
containing p210.sup.bcr-abl; or from 106-5 containing
p185.sup.bcr-abl; or from irrelevant K-BALB tumor cells.
.sup..dagger.The data represent the mean of triplicate
determinations of [.sup.3H] thymidine incorporated for the final 18
hours of a 96-hour culture. The standard deviations were <10% of
the c.p.m.
Example 4
Tumors Expressing Aberrant ras Protein in vivo Elicit T Cells
Specific for Corresponding ras Peptide
[0071] The following experiments show that aberrant ras p21 protein
produced by tumor cells in vivo elicits T cells which proliferate
specifically in response to the altered region of the ras p21
protein. A Friend virus induced leukemia cell line, designated as
FBL, was transfected with the T24 Ha-ras-1 oncogene carried by the
pH06T1 plasmid under control of a Moloney virus LTR and an SV40
enhancer to enable eukaryotic expression (Spandidos and Wilkie,
Nature 310:469-475, 1984). The T24 oncogene encodes aberrant ras
p21 protein with valine substituted for the normal glycine at
residue-12 (Roddy et al., Nature300:149-152, 1982). Expression of
ras p21 [Val-12] by the transfected FBL cells was confirmed by
western analysis using a monoclonal antibody which specifically
binds to ras p21 [Val-12] proteins. Syngeneic B6 mice were
immunized twice at two-week intervals with FBL cells transfected
with the pH06T1 plasmid or a corresponding plasmid pH06N1 which
carries the normal Ha-ras-1 gene. Spleen cells from mice immunized
with FBL transfected with the T24 oncogene proliferated in response
to the ras [Val-12] peptide (FIG. 7), whereas spleen cells from
mice immunized with FBL transfected with normal Ha-ras-1 did not
respond.
Example 5
CD4.sup.+ Ras-Specific T Cells can Mediate Tumor Therapy in
vivo
[0072] The finding (Example 4) that tumors which express aberrant
ras p21 in vivo can elicit ras-specific T cells in vivo strongly
implies that ras p21 is available to APC for processing and
presentation to class II-restricted T cells in vivo. Presentation
of the activated ras p21 in proximity to tumor cells may elicit
class II-restricted T cells which can mediate substantial
therapeutic effects in vivo against tumors even if the latter do
not express class II MHC molecules and cannot be directly
recognized. The ras 4 tumor, which expresses a transfected T24
oncogene and is tumorigenic in BALB/c.times.C57BL/6 F1 mice, was
used in therapy. Small doses of tumor cells (3.times.10.sup.6),
injected i.p., kill animals within 2 weeks. A T cell line specific
for the altered region of ras p21 [Val-12] was tested for the
ability to protect BALB/c.times.C57BL/6 F1 mice against challenge
with ras 4 tumor cells (3.times.10.sup.6 i.p.). In repeated
experiments, mice injected with tumor alone all died by day 19,
whereas mice injected concurrently with tumor cells plus ras
[Val-12] specific T cells (2.times.10.sup.7 cells i.p.) all
survived without evidence of tumor for greater than 8 weeks of
observation (FIG. 8).
[0073] A second tumor therapy model was established with K-BALB
tumor cells which express ras p21 [Ser-12] and syngeneic T cells
which specifically recognize a corresponding synthetic ras [Ser-12]
peptide. BALB/c mice injected with tumor cells (2.times.10.sup.5)
received either no additional therapy, or ras [Ser-12] specific T
cells (1.times.10.sup.7 cells per mouse) plus low doses of
exogenous rIL-2 (5,000 U i.p. per day on days 0-4). Mice treated
with ras specific T cells evidenced substantial extension of median
survival. In related experiments, cohorts of BALB/c mice were
sensitized with the ras [Ser-12] peptide or with adjuvant alone
then challenged with a lethal dose of K-BALB tumor cells. Mice
sensitized with ras peptide survived longer than non-immunized
mice. Therefore, ras-specific class II-restricted T cells can
mediate anti-tumor efficacy in vivo.
[0074] From the foregoing, it will be evident that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Sequence CWU 1
1
11 1 12 PRT Artificial Sequence Synthetic peptide corresponding to
the p21 protein product of mutated ras proto-oncogene 1 Lys Leu Val
Val Val Gly Ala Arg Gly Val Gly Lys 1 5 10 2 22 PRT Artificial
Sequence Synthetic peptide corresponding to bcr3-abl2 fusion
protein 2 Ile Val His Ser Ala Thr Gly Phe Lys Gln Ser Ser Lys Ala
Leu Gln 1 5 10 15 Arg Pro Val Ala Ser Asp 20 3 18 PRT Artificial
Sequence Synthetic peptide corresponding to bcr3-abl2 fusion
protein 3 His Ser Ala Thr Gly Phe Lys Gln Ser Ser Lys Ala Leu Gln
Arg Pro 1 5 10 15 Val Ala 4 16 PRT Artificial Sequence Synthetic
peptide corresponding to bcr3-abl2 fusion protein 4 Gly Phe Lys Gln
Ser Ser Lys Ala Leu Gln Arg Pro Val Ala Ser Asp 1 5 10 15 5 14 PRT
Artificial Sequence Synthetic peptide corresponding to bcr3-abl2
fusion protein 5 Gly Phe Lys Gln Ser Ser Lys Ala Leu Gln Arg Pro
Val Ala 1 5 10 6 12 PRT Artificial Sequence Synthetic peptide
corresponding to bcr3-abl2 fusion protein 6 Gly Phe Lys Gln Ser Ser
Lys Ala Leu Gln Arg Pro 1 5 10 7 12 PRT Artificial Sequence
Synthetic peptide corresponding to bcr3 protein 7 Ile Val His Ser
Ala Thr Gly Phe Lys Gln Ser Ser 1 5 10 8 12 PRT Artificial Sequence
Synthetic peptide corresponding to bcr2-abl2 fusion protein 8 Leu
Thr Ile Asn Lys Glu Glu Ala Leu Gln Arg Pro 1 5 10 9 14 PRT
Artificial Sequence Synthetic peptide corresponding to BCRI-abl2
fusion protein 9 Ala Phe His Gly Asp Ala Gln Ala Leu Gln Arg Pro
Val Ala 1 5 10 10 6 PRT Artificial Sequence Synthetic peptide
corresponding to bcr3 protein 10 Gly Phe Lys Gln Ser Ser 1 5 11 5
PRT Artificial Sequence Synthetic peptide corresponding to c-abl
protein 11 Ala Leu Gln Ala Pro 1 5
* * * * *