U.S. patent number RE35,491 [Application Number 07/421,096] was granted by the patent office on 1997-04-08 for methods and compositions for detecting human tumors.
This patent grant is currently assigned to The Regents of the University of California. Invention is credited to Martin J. Cline, Dennis J. Slamon.
United States Patent |
RE35,491 |
Cline , et al. |
April 8, 1997 |
Methods and compositions for detecting human tumors
Abstract
Methods and compositions for detecting the presence of tumors
are provided, where a physiological sample is assayed for the
expression product of a c-onc gene as diagnostic for the presence
of the tumor. The method finds use in both pre-and postoperative
situations with a host suspected of having transformed malignant
cells.
Inventors: |
Cline; Martin J. (Pacific
Palisades, CA), Slamon; Dennis J. (Woodland Hills, CA) |
Assignee: |
The Regents of the University of
California (Alameda, CA)
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Family
ID: |
27412019 |
Appl.
No.: |
07/421,096 |
Filed: |
October 12, 1989 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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439252 |
Nov 4, 1982 |
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496027 |
May 19, 1983 |
|
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Reissue of: |
673469 |
Nov 20, 1984 |
04699877 |
Oct 13, 1987 |
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Current U.S.
Class: |
435/6.14;
435/7.23; 436/503; 530/326; 530/327; 530/328; 530/387.7;
530/388.35; 530/388.8; 530/389.7; 530/391.1; 530/391.3; 530/391.5;
530/391.7; 530/391.9 |
Current CPC
Class: |
C07K
14/82 (20130101); C07K 16/32 (20130101); C12Q
1/6886 (20130101); G01N 33/57426 (20130101); G01N
33/57484 (20130101); A61K 39/00 (20130101); G01N
2333/82 (20130101); C12Q 2600/158 (20130101) |
Current International
Class: |
C07K
14/82 (20060101); C07K 16/18 (20060101); C07K
16/32 (20060101); C12Q 1/68 (20060101); G01N
33/574 (20060101); A61K 39/00 (20060101); C12Q
001/68 (); G01N 033/53 (); C07K 007/06 (); C07K
007/08 (); C07K 016/10 (); C07K 017/00 () |
Field of
Search: |
;435/67.23,172.1
;424/1.1,9,85 ;436/503,504,508,813
;530/326,387,403,826,828,327,328,387.7,388.21,388.35,388.8,389.7,390.1,391.1
;935/78,81 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
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(1979). .
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.
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.
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Dhar, Science (1982) 217:934-936..
|
Primary Examiner: Kim; Kay K. A.
Attorney, Agent or Firm: Rowland; Bertram I.
Parent Case Text
RELATED APPLICATIONS
This application is a continuation-in-part application of copending
application Ser. No. 439,252, filed Nov. 4, 1982, .Iadd.now
abandoned, .Iaddend.and copending application Ser. No. 496,027
filed May 19, 1983, now abandoned, incorporating herein by
reference.
Claims
What is claimed is:
1. A method for evaluating the probability of cellular malignancy
in a human host, said method comprising:
bringing into close associate (1) a probe specific for a cellular
product, said cellular product being mRNA or its expression
product, where said mRNA is complementary to a DNA sequence of a
retrovirus capable of transforming a normal cell to malignancy and
said probe is a nucleic acid sequence capable of duplexing with
said mRNA or antibody capable of binding to said expression
product, and (2) a source from said human host suspected of
containing cellular product; and
determining the level of binding of said probe to said cellular
product, wherein an elevated level is indicative of the presence of
cellular malignancy.
2. A method according to claim 1, wherein said source is cells from
said human host.
3. A method according to claim 1, wherein said source is a
physiological fluid from said human host.
4. A method according to claim 1, wherein said DNA sequence is
selected from the group consisting of the oncogenes src, fps, yes,
fos, myc, erb, myb, rel, mos, bas, abl, ras, fes, fms, and sis.
5. A method according to any of claims 1, 2, 3, or 4, wherein said
probe is an antibody.
6. A method according to claim 5, wherein said antibody is labeled
with a label capable of providing a detectible signal.
7. A method according to claims 1, 2, 3, or 4, wherein said probe
is a polynucleotide of at least 14 bases complementary to said
mRNA.
8. A method for evaluating the probability of leukemia in a human
host, said method comprising:
combining antibodies .[.specific.]. .Iadd.diagnostic for the
presence of .Iaddend.for the oncogene myb and blood cells from a
human host suspected of having leukemia; and
determining the level of binding of said antibodies to said host
blood cells as diagnostic of a leukemic host.
9. A method according to claim 8, wherein said antibodies are
produced in response to an oligopeptide mimicking a portion of the
conformation of the myb protein. .[.10. A method for substantially
eliminating human malignant cells from a combination of human
malignant and normal cells, which comprises:
combining under cytotoxic conditions said combination of cells with
an antibody specific for an expression product of a DNA sequence
present in a retrovirus genome or substantially complementary to
said DNA sequence, which sequence is expressed in said malignant
cells as a surface protein; and
isolating normal cells, substantially free of malignant
cells..]..[.11. A method according to claim 10, wherein said
separation occurs in the presence of complement as said cytotoxic
condition..]..[.12. A method according to claim 10, wherein said
antibodies are labeled with a radionuclide as said cytotoxic
condition..]..[.13. A method according to claims 10, 11, or 12,
wherein said DNA sequence is the myb gene..]..[.14. A method for
treating a human host suspected of having malignant cells, which
comprises:
administering to said human host under cytotoxic conditions
antibodies to the expression product of a gene, which gene is part
of a retrovirus genome capable of inducing malignancy in a normal
cell or which gene is substantially complementary to said gene of
said retrovirus genome..]..[.15. A method according to claim 14,
wherein said cytotoxic condition is the presence of
complement..]..[.16. Antibodies specific for the expression product
of the human oncogenes c-myc, c-fos, c-ras.sup.Ha, c-ras.sup.Ki,
c-fes, c-myb, and c-src..]..[.17. Antibodies according to claim 16,
labeled with a label capable of providing a detectible
signal..]..[.18. Antibodies according to claim 16, labeled with
a
cytotoxic agent..].19. An antigenic oligopeptide selected from the
class consisting of:
(a) met-ala-phe-ala-his-asn-pro-pro-ala-gly-pro-leu-pro-gly-ala
(b)
pro-phe-his-lys-asp-gln-thr-phe-thr-glu-tyr-arg-lsy-met-his-gly-gly-ala-va
(c) pro-phe-his-lys-asp-gln-thr-phe-thr-glu-tyr-arg-lys-met
(d)
asp-asn-thr-arg-thr-ser-gly-asp-asn-ala-pro-val-ser-cys-leu-gly-glu
(e)
arg-leu-ileu-gly-asp-asn-glu-tyr-thr-ala-arg-gln-gly-ala-lys-phe-pro
(f) trp-arg-arg-asp-pro-glu-glu-arg-pro-thr
(g)
arg-leu-lys-lys-ileu-ser-lys-glu-glu-lys-thr-pro-gly-cys-val-lys-ileu-lys-
lys
(h)
asp-leu-pro-ser-arg-thr-val-asp-thr-lys-gln-ala-gln-glu-leu-ala-arg
(i)
met-thr-glu-tyr-lys-leu-val-val-val-gly-ala-ser-gly-val-gly-lys-ser-ala
(j)
glu-asp-ileu-his-gln-try-arg-glu-gln-ileu-lys-arg-val-lys-asp-ser-asp-asp
(k)
val-arg-glu-ileu-arg-gln-his-lys-leu-arg-lys-leu-asn-pro-pro-asp-glu-ser-g
ly-pro
(l)
met-thr-gly-tyr-lys-leu-val-val-val-gly-ala-gly-gly-val-gly-lys-ser-ala
(m)
val-asp-glu-tyr-asp-pro-thr-ileu-glu-asp-ser-tyr-arg-lys-gln-val
(n) arg-his-ser-thr-ser-ser-ser-glu-gln-glu-arg-glu-gly-gly-arg
(o) asn-gln-gln-thr-arg-glu-phe-val-glu-lys-gly-gly-arg
(p) pro-glu-val-gln-lys-pro-leu-his-glu-gln
(q) ala-ser-pro-tyr-pro-asn-leu-ser-asn-gln-gln-thr-arg
(r)
arg-leu-ileu-ala-glu-lys-glu-gln-leu-arg-arg-arg-arg-glu-gln
(s) asn-asn-glu-lys-ala-pro-lys-val-val. 20. Antibodies raised to
an antigenic polypeptide selected from the class consisting of:
(a)
met-ala-phe-ala-his-asn-pro-pro-ala-gly-pro-leu-pro-gly-ala;
(b)
pro-phe-his-lys-asp-gln-thr-phe-thr-glu-tyr-arg-lys-met-his-gly-gly-ala-va
l;
(c) pro-phe-his-lys-asp-gln-thr-phe-thr-glu-tyr-arg-lys-met;
(d)
asp-asn-thr-arg-thr-ser-gly-asp-asn-ala-pro-val-ser-cys-leu-gly-glu;
(e)
arg-leu-ileu-glu-asp-asn-glu-tyr-thr-ala-arg-gln-gly-ala-lys-phe-pro;
(f) trp-arg-arg-asp-pro-glu-glu-arg-pro-thr;
(g)
arg-leu-lys-lys-ileu-ser-lys-glu-glu-lys-thr-pro-gly-cys-val-lys-ileu-lys-
lys;
(h)
asp-leu-pro-ser-arg-thr-val-asp-thr-lys-gln-ala-gln-glu-leu-ala-arg;
(i)
met-thr-glu-try-lys-leu-val-val-val-gly-ala-ser-gly-val-gly-lys-ser-ala;
(j)
glu-asp-ileu-his-gln-tyr-arg-glu-gln-ileu-lys-arg-val-lys-asp-ser-asp-asp;
(k)
val-arg-glu-ileu-arg-gln-his-lys-leu-arg-lys-leu-asn-pro-pro-asp-glu-ser-g
ly-pro;
(l)
met-thr-glu-tyr-lys-leu-val-val-gly-ala-gly-gly-val-gly-lys-ser-ala;
(m)
val-asp-glu-tyr-asp-pro-thr-ileu-glu-asp-ser-tyr-arg-lys-gln-val;
(n)
arg-his-ser-thr-ser-ser-ser-glu-gln-glu-arg-glu-gly-gly-arg;
(o) asn-gln-gln-thr-arg-glu-phe-val-glu-lys-gly-gly-arg;
(p) pro-glu-val-gln-lys-pro-leu-his-glu-gln;
(q) ala-ser-pro-tyr-pro-asn-leu-ser-asn-gln-gln-thr-arg;
(r) arg-leu-ileu-ala-glu-lys-glu-gln-leu-arg-arg-arg-arg-glu-gln;
and
(s) asn-asn-glu-lys-ala-pro-lys-val-val. 21. Antibodies according
to claim
20, labeled with a label capable of providing a detectible signal.
22. Antibodies according to claim 20, labeled with a cytotoxic
agent.
.Iadd.23. A method for evaluating the probability of cellular
malignancy in a human host, said method comprising:
bringing into close association (1) a probe diagnostic for the
presence of a cellular product, said cellular product being mRNA or
its expression product, where said mRNA is capable of hybridizing
to a DNA sequence of a retrovirus capable of transforming a normal
cell to malignancy and said probe is a nucleic acid sequence
capable of duplexing with said mRNA or antibody capable of binding
to said expression product, and (2) a source from said human host
suspected of containing cellular product; and
determining the level of said binding of said probe to said
cellular product, wherein an elevated level is indicative of the
presence of
cellular malignancy. .Iaddend..Iadd.24. An antibody diagnostic for
the presence of the expression product of a human oncogene selected
from the group consisting of c-myc, c-fos, c-ras.sup.K, c-fes, and
c-myb. .Iaddend..Iadd.25. An antibody according to claim 24,
wherein said human oncogene is c-myc. .Iaddend..Iadd.26. An
antibody according to claim 24, wherein said human oncogene is
c-fos. .Iaddend..Iadd.27. An antibody according to claim 24,
wherein said human oncogene is c-ras.sup.K. .Iaddend..Iadd.28. An
antibody according to claim 24, wherein said human oncogene is
c-fes. .Iaddend..Iadd.29. An antibody according to claim 24,
wherein said human oncogene is c-myb. .Iaddend..Iadd.30. An
antibody according to claim 24, labeled with a label capable of
providing a detectable signal. .Iaddend..Iadd.31. An antibody
according to claim 24, labeled with a cytotoxic agent. .Iaddend.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The mechanism for malignancy of mammalian cells has been and
continues to be the subject matter of intense investigation. One of
the areas which is considered to be promising in the elucidation of
the mechanism is the area of oncogenes. While the occurrence of
oncogenes was first detected with retroviruses, it now seems
reasonably firm that the viral oncogenes have cellular
counterparts. The role of the cellular counterparts is not clear.
An excellent review of oncogenes, their properties and particularly
the src gene may be found in the article by J. Michael Bishop,
Scientific American, Mar., 1982:81-93. The article also provides a
list of various viral oncogenes, demonstrating that a number of
them are involved with phosphorylation.
The src gene is found to be not only active in the malignant cell
of chickens, but also in the normal cell. The difference appears to
be one of degree, rather than of kind, in that the enzyme expressed
by the src gene would appear to be of much higher concentration in
the malignant cell as compared to the normal cell.
In order to be able to determine the presence of a tumor cell, it
is necessary to be able to distinguish between normal cells and
tumor cells. Therefore, the observed property which is to be
diagnostic of the tumor cell must be capable of differentiation
from a normal cell or from a physiologic fluid of a normal host,
where the fluid rather than cells are assayed. Furthermore, the
property should not be specific for the individual, but be common
to the malignant nature of the cell.
In both diagnosis and treatment, the opportunity or specifically
detecting malignant cells is very important. Any technique, in a
high percentage of situations where malignancy is suspected, should
be able to distinguish malignant cells from normal cells.
Furthermore, the diagnostic technique should be useful for a large
number of members of the population and not specific for one or a
few members of the population.
Because a cancer cell is derived from a normal cell, most of the
properties and components of the malignant cell are the same as the
normal cell. Furthermore, there is an increasing view that
malignancy is a result of a natural process, which in a certain
context results in malignancy. In view of the fact that malignancy
may be based on normal processes, which at the time in question
have an abberrant result, it is not surprising that there has been
substantial difficulty in demonstrating observable differences
between normal cells and cancer cells over a broad spectrum of
allogeneic hosts.
2. Description of the Prior Art
The following papers provide a general description of oncogenes and
the role of retroviruses in tumorigenesis: Bishop, Scientific
American, supra; Bishop, New England J. of Med. (1980) 303:675-681;
Lancet, Jul. 24, 1982, pages 195-196; Cooper, Science (1982)
218:801-806; Vamus, Science (1982) 216:821-820. Papers concerned
with specific oncogenes include Becker et al., PNAS USA (1982)
79:3315-3319; Tsuchida et al., Science (1982) 217:937-938 and Dhar
et al., ibid., (1982) 217:934-936.
SUMMARY OF THE INVENTION
Methods and compositions are provided for identifying and treating
malignant cells of fresh tumors in a human host. From knowledge of
DNA sequences capable of transforming cells of a lower vertebrate
to malignancy, polynucleotide probes can be made for determining
the level of transcription of said DNA in human cells and receptors
produced capable of specifically recognizing determinant sites of
peptide products of said DNA sequence. The probes and receptors may
be labeled with a wide variety of labels for diagnosis and
treatment.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
In accordance with the subject invention, novel methods and
compositions are provided for the diagnosis and treatment of cancer
in humans and other primates. It has now been observed that DNA
which is capable of transforming cells of lower vertebrates to
malignancy is present in human cells and has a much higher level of
transcription and expression in malignant cells than in normal
cells. Thus, by being able to detect the higher level of messenger
RNA or the expression product of such messenger RNA, the presence
of malignant cells in a host may be diagnosed. In addition, the
production of the higher level of peptides in the malignant cells
can be a basis for treatment of the malignancy. Where the
polypeptide expression product can be found in physiological
fluids, such as blood, and the levels of the expression product are
substantially different in the presence and absence of malignancy,
the physiological fluid may be screened as diagnostic for the
presence of a particular tumor.
The subject invention provides methods and compositions for
evaluating the probability or presence of malignant cells in a
group of cells, particularly human cells in vivo or freshly removed
from a human host. The method looks to cellular products such as
mRNA or its expression product as diagnostic of the probable
presence of malignant cells. The mRNA which is selected from
detection will usually be selected as a result of there being RNA
present in a retrovirus genome, which retrovirus is capable of
transforming mammalian cells to malignancy. Furthermore, the RNA in
the retrovirus which is selected to a sequence which does not
encode an essential function of the retrovirus and, in fact, may be
silent.
The method involves as a first step defining a DNA sequence capable
of causing malignancy in a mammalian cell. Once the DNA sequence is
defined, polynucleotide sequences can be provided which may serve
as probes for detection of elevated levels of messenger RNA to
determine whether a cell is malignant. The sequence can also be
used for defining polypeptide sequences which can define
complementary receptors having high specificity for the peptide
sequence. The receptors may then be used for determining the
presence or the concentration of the peptide in cells or
physiological fluids and for treatment where the receptors can be
directed to malignant cells. Also, knowing the nature of the
peptide and its function, other means may be available for
controlling the elevated production of the particular peptide.
The first step in the subject method is to define the DNA sequence.
Various methods can be used for defining the DNA sequence of a
retroviral oncogene. For example, retroviruses have been found
capable of transforming lower vertebrate cells to malignancy. The
retroviruses which have been characterized have been shown to carry
DNA sequences comparable to wild type genes present in the host,
genes which are now referred to as oncogenes. Furthermore, in the
case of Rous sarcoma virus, the expression product of the gene has
been isolated and characterized and shown to be a kinase. In the
case of this kinase, it has also been shown that the kinase is
normally produced by the cell, but at a much lower level than when
the src gene from the Rous sarcoma virus is introduced. A number of
viral oncogenes have already been detected in a variety of
vertebrates, and the following is a list of the oncogenes and their
species of origin.
TABLE 1 ______________________________________ Species of Oncogene
origin ______________________________________ v-src chicken v-fps "
v-yes " v-fos " v-myc " v-erb " v-myb " v-rel turkey v-mos mouse
v-bas " v-abl " v-ras rat v-fes cat v-fms " v-sis monkey
______________________________________
Other sources of DNA sequences capable of including malignant
transformations in vertebrate cells may include isolated DNA from a
malignant cell or cell line, cloned DNA from a genomic library or
cloned DNA from a messenger RNA library, where the total messenger
of the malignant cell is reverse transcribed to DNA and cloned.
Either of these libraries may be screened for their ability to
induce malignancy. A refinement in the technique of screening may
be achieved by taking the total messenger from a normal cell and
preparing cDNA from the messenger. One can then use the single
stranded DNA as a probe to remove messenger RNA associated with the
normal cell from the total messenger RNA from a malignant cell. The
residual messenger RNA will then include messenger being expressed
by genes associated with the malignancy. One may then use the
messenger to screen a genomic library and use the cloned DNA which
hybridizes with messenger in a bioassay for the determination of
the ability to transform to malignancy. Other ways will also become
available in time for detecting and defining DNA sequences capable
of transforming normal cells.
A further analysis can be employed by screening cDNA from fetuses
with messenger RNA from malignant cells. Particularly, where the
oncogene is a gene which is silent or relatively quiescent in the
mature vertebrate, while highly active in the embryo, the screening
may further serve to narrow the field of sequences to be
screened.
Once haVing identified a DNA sequence capable of inducing
malignancy, a cloned viral oncogene or short polynucleotide
sequences can be employed as probes for detection of the level of
production of messenger RNA in cells suspected of being malignant.
The preparation of both RNA and DNA nucleotide sequences, the
labeling of the sequences, and the preferred size of the sequences
has received ample description and exemplification in the
literature. Normally, a sequence should have at least about 14
nucleotides, usually at least about 18 nucleotides, and the
polynucleotide probes may be one or more kilobases. Various labels
may be employed, most commonly radionuclides, particularly .sup.32
P. However, other techniques may also be employed, such as using
biotin modified nucleotides for introduction into a polynucleotide.
The biotin then serves as the site of binding to avidin or
antibodies, which may be labeled with a wide variety of labels,
such as radionuclides, fluorescers, enzymes, or the like.
Alternatively, antibodies may be employed which can recognize
specific duplexes, including DNA duplexes, RNA duplexes and DNA-RNA
hybrid duplexes or DNA-protein duplexes. The antibodies in turn may
be labeled and the assay may be carried out where the duplex is
bound to a surface, so that upon the formation of duplex on the
surface, the presence of antibody bound to the duplex can be
detected.
By isolating the nucleotide sequence for the whole oncogene, the
sequence of bases may be determined by known means, e.g., Maxam and
Gilbert, PNAS USA (1977) 74:560. The sequence can be used for the
determination of the amino acid sequence of the protein expressed
by the oncogene. By identifying codons for methionine followed by a
sequence which does not have stop codons which prevent expression,
one can usually find a single sequence in frame with a methionine
codon for defining the oncogene.
Alternatively, hybrid DNA technology may be employed for obtaining
expression. The DNA sequence may be restriction mapped and
appropriate sites for cleavage defined. In this way, the sequence
may be excised and introduced into a vector having the appropriate
regulatory signals. After obtaining expression of the DNA sequence,
antibodies can be made to the polypeptide. By employing oocytes for
expression of the messenger RNA which is then translated to produce
the peptide expressed by the oncogene, the protein defined by the
messenger may be produced. The identity of the peptide from the
oocyte which the peptide produced by the expression of the hybrid
DNA may then be determined.
Once the protein has identified and verified, one can then use the
protein or subunit peptides as an antigen for the production of
antibodies for diagnosis and treatment. Antibodies can be prepared
in a variety of ways, depending upon whether monoclonal or
polyclonal antibodies are desired. For polyclonal antibodies, a
vertebrate, normally a domestic animal, is hyperimmunized with the
antigen and blood collected shortly after repeat immunizations and
the gamma globulin isolated. For monoclonal antibodies, a small
animal is hyperimmunized, the spleen removed and the lymphocytes
fused with an appropriate fusing partner. The resulting hybridomas
are then grown under limiting dilution and clones providing the
desired antibodies selected.
Rather than preparing the entire peptide, one can determine various
regions which are likely to be determinant sites and use these
oligopeptides of at least about eight amino acids, usually at least
about 10 and not more than 20, usually not more than 18 amino
acids, to define a hapten which can be used to induce antibody
formation. The oligopeptide is bound to an appropriate immunogen
and introduced into a vertebrate to produce antibodies, either
polyclonal or monoclonal antibodies, as described previously.
Accordingly, the present invention also provides a series of
oligopeptides corresponding to antigenic regions in the peptide
expression products of RNA present in retrovirus oncogenes.
Exemplary species of the antigenic oligopeptides useful in
accordance with the subject invention are listed below in groups
based on the retroviral oncogene (expression product) which is
recognized by antibodies produced from the oligopeptide.
______________________________________ A. Myb
met-ala-phe-ala-his-asn-pro-pro-ala-gly-pro-leu-pro- gly-ala
pro-phe-his-lys-asp-gln-thr-phe-thr-glu-tyr-arg-lys-met-
his-gly-gly-ala-val
pro-phe-his-lys-asp-gln-thr-phe-thr-glu-tyr-arg-lys-met-
asp-asn-thr-arg-thr-ser-gly-asp-asn-ala-pro-val-ser-cys-
leu-gly-glu B. Src
arg-leu-ileu-glu-asp-asn-glu-tyr-thr-ala-arg-gln-gly-ala-
lys-phe-pro trp-arg-arg-asp-pro-glu-glu-arg-pro-thr C. Ras.sup.Ki
arg-leu-lys-lys-ileu-ser-lys-glu-glu-lys-thr-pro-gly-cys-
val-lys-ileu-lys-lys
asp-leu-pro-ser-arg-thr-val-asp-thr-asp-thr-lys-gln-ala-gln-glu-
leu-ala-arg
met-thr-glu-tyr-lys-leu-val-val-val-gly-ala-ser-gly-val-
gly-lys-ser-ala D. Ras.sup.Ha
glu-asp-ileu-his-gln-tyr-arg-glu-gln-ileu-lys-arg-val-lys-
asp-ser-asp-asp
val-arg-glu-ileu-arg-gln-his-lys-ser-arg-lys-leu-asn-pro-
pro-asp-glu-ser-gly-pro
met-thr-glu-tyr-lys-leu-val-val-val-gly-ala-gly-gly-val-
gly-lys-ser-ala
val-asp-glu-tyr-asp-pro-thr-ileu-glu-asp-ser-tyr-arg-lys- gln-val
E. Fes arg-his-ser-thr-ser-ser-ser-glu-gln-glu-arg-glu-gly-gly- arg
asn-gln-gln-thr-arg-glu-phe-val-glu-lys-gly-gly-arg
pro-glu-val-gln-lys-pro-leu-his-glu-gln
ala-ser-pro-tyr-pro-asn-leu-ser-asn-gln-gln-thr-arg F. Myc
arg-leu-ileu-ala-glu-lys-glu-gln-leu-arg-arg-arg-arg-glu- gln
asn-asn-glu-lys-ala-pro-lys-val-val
______________________________________
In those situations where the human gene is different from the
v-onc, e.g. human c-ras, the above described techniques may be used
for isolating the gene, mRNA or pseudo-gene and obtaining
antibodies to the human expression product. The human oncogene
would be expected to have substantial complementarity to the
related, v-onc, normally differing in fewer than about 5% of the
bases, generally differing by fewer than 5% of the amino acids in
the expression product.
The antibodies may be used in a variety of ways. Particularly, they
may be used for diagnosis. In instances where the antigen may be
found in a physiological fluid at an elevated concentration only
when malignancy exists, the physiological fluid, such as serum,
plasma, whole blood or cerebrospinal fluid may be assayed.
Antibodies employed in assays may be labeled or unlabeled.
Unlabeled antibodies may be employed in agglutination; labeled
antibodies may be employed in a wide variety of assays, employing a
wide variety of labels, such as radionuclides, enzymes,
fluorescers, enzyme substrates or cofactors, or the like. These
techniques are amply defined in the literature and exemplary assays
may be found in U.S. Pat. Nos. 3,817,834, 3,935,074, 4,233,402 and
4,318,980, as illustrative.
In some techniques, it will be useful to label the antigen or
fragment thereof, rather than the antibody and have a competition
between labeled antigen and antigen in the sample for antibody. In
this situation, it is common to provide kits which have the
combination of the labeled antigen or labeled fragment and the
antibody in amounts which provide for optimum sensitivity and
accuracy.
In other situations, it is desirable to have a solid support, where
either antigen or antibody is bound. A polyepitopic antigen can
serve as a bridge between antibody bound to a support and labeled
antibody in the assay medium. Alternatively, one may have a
competition between labeled antigen and any antigen in the sample
for a limited amount of antibody.
Where the antigen may not be found in a physiological fluid or if
found there is not diagnostic of malignancy, then cells will have
to be isolated and the cells assayed for the presence of messenger
RNA or the antigen. Methods of detecting messenger RNA have already
been described. For detecting the antigen, the tissue sample may be
lysed by conventional methods, e.g. base, detergents, or the like,
cellular debris separated by filtration or centrifugation and the
filtrate or supernatant isolated and assayed.
For purposes of therapy, either xenogeneic or allogeneic antibodies
may be employed, depending upon the nature of the treatment, and
whether the foreign antibodies will induce an immune response. The
literature has described a number of ways of making human
antibodies, where it is found that mouse or other mammalian
antibodies are not satisfactory. The antibodies may be used in a
wide variety of ways. By employing the appropriate IgG (other than
IgG.sub.1), one may induce lysis through the natural complement
process. Alternatively, the lysing portion of a toxin may be joined
to the antibodies, particularly a Fab fragment. The antibodies may
be bound to liposomes for directing the liposomes to the malignant
cells to become ingested by the cells by merging of the membranes.
Other labels may also be bound to the antibodies, such as
radionuclides, fluorescers, enzymes, and the like. By introducing
the antibodies in vivo, the antibodies will direct the label to the
malignant cells, where the presence of malignancy may be diagnosed
or treated.
The formation of the antibodies will vary widely, depending on the
nature of the label, the purpose of the antibodies, the site to
which the antibodies are to be directed, and the like. Usually, the
antibodies will be formulated in a physiologically acceptable
carrier, e.g. saline or phosphate buffered saline, and injected
into the host, when possible at the desired site, and when this is
not possible, into a circulating system, such as blood.
The antibodies obtained in accordance with this invention can also
be used to isolate cells expressing the oncogene and to remove
cells in vitro from a heterogeneous cell population containing
cells expressing the oncogene. Separation can be achieved with a
fluorescence activated cell sorter (FACS). This same technique can
be used for identifying and isolating cells expressing the
oncogene. For removing cells expressing the oncogene from a mixture
of cells, the subject antibodies may be combined with complement,
joined to the lysing fragment (A fragment) of a toxin (see E.P.O.
application no. 17,507 and U.K. Patent Application No. 2,034,324)
or the cells agglutinated and separated by physical means.
The following examples are offered by way of illustration and not
by way of limitation.
Tumors were obtained from fresh surgical specimens at the time of
resection and were untreated by chemotherapy or radiotherapy. An
effort was made to obtain only viable tumor and to process the
tissues as rapidly as possible to avoid messenger RNA (mRNA)
degradation. Specimens were quickly frozen and stored in liquid
nitrogen until processed for RNA. When the surgical specimens
included wide margins of normal tissue, some of this was taken for
analysis as an internal control of the level of c-onc gene
expression. C-onc gene expression could then be compared in normal
and malignant tissue from the same patient. As little as 20 pg of
Maloney murine sarcoma virus equivalent to approximately one RNA
transcript of 3 kilobases (kb) per cell or approximately 2
micrograms of poly A RNA applied to the filter could be detected by
this method (Kafatos et al., Nucleic Acids Res. (1979)7:1541). By
use of appropriate controls including unrelated RNA's, poly
A-negative fraction RNA, plasmid DNA, and mouse and human DNA,
false-negative as well as false-positive results could reasonably
be excluded. The dot blots were quantitatively evaluated by means
of a soft laser scanning densitometer. Where sufficient material
was available, mRNA was further characterized by Northern analysis
to confirm the presence of, and to size, specific transcripts
(Thomas, PNAS USA (1980)77:5201).
Expression of 13 cellular oncogenes in 14 tumors was examined by
DNA-RNA hybridization techniques. These data are summarized in
Table 2.
TABLE 2
__________________________________________________________________________
HUMAN MALIGNANCY* GASTROINESTINAL MALIGNANCIES LUNG Adenocarcinoma
of ADENO- LYMPHO- Small Rec- RENAL CELL OVARAIN CARCI- SAR- V-ONC
Colon Bowel tum CARCINOMA CARCINOMA NOMA COMA PROBE 1 2 1 1 1 2 3 4
1 2 3 1 2 1
__________________________________________________________________________
Myc** +++ +++ +++ +++ ++++ ++++ ++ ++++ ++ ++ ++ ++ ++ - Myb - - -
- - - - - - - - - ++ - Erb - - - - - - - - - - - - - - Src - - - -
- - - - - - - - - +++ Yes - - - - - - - - - - - - - - Abi*** - - -
- - - - - - - - - - - Fos +++ +++ +++ +++ ++ +++ +++ ++ + ++ ++++ +
++ - Mos - - - - - - - - - - - - - - Ras.sup.Ha ++ ++ ++ ++ ++ ++
++ ++ ++ ++ ++ ++ ++ - Ras.sup.Ki ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
++ ++ - Fes - - - - - - - - - - - + ++ - Fms - - - - - - - - - - -
- - - Sis - - - - - - - - - - - - - -
__________________________________________________________________________
*Increasing numbers of pluses indicates increasing intensity of
hybridization of tissue mRNA to vonc probes. **Avian ***Murine
Three patterns were observed: (1) expression of specific c-onc mRNA
sequences in all or nearly all tumor samples (e.g., c-myc); (2)
detection of c-onc expression in sporadic tumors(e.g., c-fes); and
(3) no detectable expression (e.g., c-mos).
No significant expression of mRNA sequences homologous to c-erb,
c-yes, c-abl, c-mos, c-fms, or c-sis could be detected. This was
not the result of lack of homology between the viral gene probe and
the human messenger RNA, since it was possible to detect homologues
of all these probes in human genomic DNA. RNA from microscopically
normal tissue did not contain any detectable transcripts by this
analysis.
Four cellular oncogenes showed a consistent pattern of expression
in a variety of human tumors. These were c-myc, c-fos,
c-ras.sup.Ha, and c-ras.sup.Ki. A comparison was made of the
intensity of hybridization, which was possible since all probes
were labeled to approximately the same specific activity. V-myc and
v-fos demonstrated the highest intensity of hybridization to human
tumor RNA's suggesting a large number of copies of mRNA per cell.
Expression of both these genes was observed in all malignancies
examined. C-ras.sup.Ha and c-ras.sup.Ki sequences were also
detected in most of the human tumors but with less intense
hybridization.
Messengers RNA sequences related to c-fes were detected in only 2
of 14 tumors examined; both of these were lung cancers.
C-myb expression was detected in only one of 14 tumors; this, too,
was a lung cancer.
C-src messenger RNA sequences were observed only in circulating
tumor cells of a patient with lymphosarcoma.
In order to test whether expression of cellular oncogene sequences
was related to neoplasia, an effort was made to obtain both grossly
normal-appearing tissue and obviously malignant tissue from the
same site in the same patient at the same time. Hybridization
studies were then performed on RNA samples from the tumor and from
adjacent noninvolved tissues. In 6 of the 14 patients it was
possible to perform this analysis. In 1 of these 6 cases the
presumed normal tissue was subsequently shown by histologic
analysis to be infiltrated by tumor. In 4 of the remaining 5 cases
there was differential expression between the tumor and normal
tissue, with low or undetectable levels observed in the normal
tissues and elevated levels observed in the malignancy. Three of
the renal cell carcinomas and one colon carcinoma demonstrated this
phenomenon.
Blot analysis of RNA from cells in the areas of the tumor sample
and the control sample shows a correlation between the presence or
absence of tumor and c-onc gene expression. In one tumor, an
adenocarcinoma of the small bowel, c-onc-related sequences were
found in histologically normal adjacent tissue.
Analyses of poly A RNA from tumors and control tissues were
performed by the Northern technique. Two c-myc-related transcripts
of 4.0 and 2.0 kb were found in all tumors examined. In addition to
these transcripts, there was obvious degradation of some of the
messenger RNA in these hybridization analyses, most likely
resulting from degradation occurring during tissue anoxia in the
period after surgical removal of the tissue.
Using the procedures given above, several other tumor types
obtained from fresh surgical specimens were examined for c-onc gene
expression. In this series of tests, DNA-RNA hybridization was used
to look for expression of 10 different cellular oncogenes in 9
tumors. The data obtained are summarized in Table 3 below.
TABLE 3
__________________________________________________________________________
HUMAN MALIGNANCY* CHRONIC BREAST UTERINE NON-HODGKIN'S MYELOCYTIC
V-ONC CARCINOMA CARCINOMA THYMOMA HODGKIN'S LYMPHOMA LEUKEMIA PROBE
1 2 3 1 1 1 1 1 2
__________________________________________________________________________
Myc** ++ +++ +++ - + ++ + ++ - Myb + ++ - - - + - + - Src + + - - -
++ + +++ - Rel - - - - - - - - - Abi*** - - - - - - - - - Fos ++ ++
++ - + ++ + ++++ ++++ Ras.sup.Ha +++ +++ +++ - + ++ + ++ +
Ras.sup.Ki + + ++ - - + - + + Fes + ++ +++ - - + + +++ ++ Sis - - -
- - - - - -
__________________________________________________________________________
*Increasing numbers of pulses indicates increasing intensity of
hybridization of tissue mRNA to vonc probes. **Avian ***Murine
The results given in Table 3 correlate fairly well with the results
previously reported in Table 2 in that the cellular oncogenes
c-myc, c-fos, c-ras.sup.Ha, and c-ras.sup.Ki show a consistent
pattern of expression in the additional tumor types examined.
Further, c-myb, c-src and c-fes were also detected in several
additional tumor types whereas c-rel, c-abl and c-sis expression
was not observed in any of the additional tumor types examined.
Interestingly, none of the cellular oncogenes looked for were found
to be expressed at any significant level in the single uterine
carcinoma evaluated.
To determine whether the messenger RNA shown to be present in
malignant cells in elevated amounts were related to genes involved
in embryogenesis, experiments were carried out generally as
follows. Total RNA was isolated from embryo/fetuses of random-bred
Swiss mice at daily intervals starting on the 6th day of gestation
(day of coital plug was taken as day 0 of prenatal development).
Beginning at day 10 of prenatal development, the embryo proper was
separated from the extraembryonal membranes and placenta. The small
size prior to day 10 prevented separation and therefore the embryos
of days 6-9 represent the entire conceptus as dissected from the
uterine wall.
Aliquots of poly(A)-containing RNA (poly(A+) RNA), were isolated by
affinity chromatography on oligo(dT)-cellulose columns and spotted
on nitrocellulose paper (dot blots) (Kafatos et al., Nucl. Acids
Res. (1979) 7:1541-1552). The samples were hybridized to [.sup.32
P]-labeled molecularly cloned oncogene-specific probes. Dot blots
were quantitatively evaluated by means of a soft laser scanning
densitometer. Transcriptional activity of c-onc's was additionally
studied in more detail in various tissues of newborn and 10 day old
mice. Agarose gel electrophoresis followed by blotting on
nitrocellulose paper (Northern blotting), (Thomas, PNAS USA (1980)
77:5201-5205) was used to confirm the results obtained by dot blot
analysis and additionally to determine the sizes of the different
c-onc-related transcripts.
More specifically, RNA was isolated from Swiss-Webster mouse embryo
fetuses at various stages of development using the guanidine
thiocyanate method. (Cox, Methods Enzymol. (1967) 12:120-129; Adams
et al., PNAS USA (1980) 74:3399-3043).
As indicated above, days 6-9 Swiss-Webster mouse embryos represent
the entire conceptuses including all extraembryonal tissues, such
as membranes and those cells giving rise to the placenta at later
developmental stages. At all later stages, the embryo proper was
dissected free of extraembryonal tissues. RNA was selected for
poly(A.sup.+) -RNA by one cycle of chromatography on
oligo(dT)-cellulose columns (Aviv and Leder, PNAS USA (1972)
69:1408-1412). Poly(A.sup.+)-RNA was dissolved in water, boiled,
quick-cooled on ice and 3 .mu.g-(1.5 .mu.l) were applied to sheets
of nitrocellulose paper which had previously been equilibrated with
20.times.SSC (1.times.SSC is 0.15 NaCl, 0.015M sodium citrate) and
air dried. After baking overnight at 80.degree. C., the blots were
prehybridized for at least 4 h at 45.degree. C. in a buffer
containing 0.75M NaCl, 0.05M sodium phosphate (pH7.5), 0.005M EDTA,
0.2% SDS, 10 mg of glycine/ml, 5.times.Denhardt's reagent
(1.times.Denhardt's reagent is 0.02% each of ficoll, bovine serum
albumin and polyvinylpyrrolidone), 0.25 mg of denatured herring
DNA/ml and 50% formamide.
The blots were hybridized for about 20 h at 45.degree. C. with
1.times.10.sup.6 cpm of nick-translated probe/ml of hybridization
buffer (prehybridization buffer with Denhardt's reagent at
1.times.). The cloned oncogene fragments purified from vector
sequences by preparative agarose gel electrophoresis were
nick-translated (Rigby et al., J. Mol. Biol. (1977) 113.237-251) in
the presence of [.sup.32 P]-dCTP(3200 Ci/mmol) to specific
radioactivities of about 1-2.times.10.sup.9 cpm/.mu.g of DNA. After
hybridization, the blots were washed three times in 1.times.SSC at
50.degree. C. for a total of about 2 h and exposed to preflashed
X-ray films with intensifying screens at -70.degree. C. for 72
h.
Employing the above procedure, a number of known oncogenes were
screened to determine whether they were expressed in the embryos.
The following table indicates the individual oncogene and the
observations concerning their expression in embryonic cells.
TABLE 4
__________________________________________________________________________
mRNA production embryos days Oncogene Virus Disease 6-9 10-18 fetus
__________________________________________________________________________
fos.sup.1. FBJ-osterosarcoma ostersarcoma + - + abl.sup.2. Abelson
leukemia lymphoma + + + ras.sup.Ha3. Harvey sarcoma
erythroleukemia, + + + sarcoma mos.sup.4. Maloney sarcoma sarcoma -
- - myc.sup.5. Avian carcinoma, sarcoma + + myelocytomatosis
leukemia erb.sup.6. Avian leukemia, sarcoma - + erythroblastosis
sarcoma src.sup.7. Rous sarcoma virus sarcoma + + myb.sup.8. Avian
leukemia - - myeloblastosis fes.sup.9. Synder-Theilin sarcoma - -
feline sarcoma sis.sup.10. Simian sarcoma sarcoma + +
__________________________________________________________________________
.sup.1. Curran et al., J. Virol, (1982) .sup.2. Goff et al., Cell
(1980) 22:777785 .sup.3. Ellis et al., J. Virol. (1980) 36:408420
.sup.4. Oskarmon et al., Science (1980) 207:12221227; Jones et al.,
PNAS USA (9180) 77:26512655 .sup.5. Eva et al., Nature (1982)
295:116 .sup.6. Gonda et al., Mol. Cell. Biol. (1982) 2:617 .sup.7.
Wang et al., (1977) J. Virol. 24:64 .sup.8. Vister et al., PNAS USA
(1982) 79:36773681 .sup.9. Fedele et al., PNAS (1982), in press
.sup.10. Devare et al., PNAS (1982) 79:31793182
Relatively high levels of c-fos related sequences were detected in
poly(A.sup.+)-RNA prepared from 6, 7, 8 and 9 day conceptuses
containing the embryo proper and extraembryonal tissues. More than
10-fold lower fos expression was observed in embryos of later
development stages dissected free of extraembryonal tissues. Data
from the placenta and extraembryonal membranes of fetuses from days
10 to 18, showed that expression was primarily in those tissues. In
postnatal tissue, c-fos expression could be observed in all tissues
investigated with stronger hybridization to the fos-specific probe
from bones.
Hybridization showed that for c-abl about three-fold higher levels
in the embryo proper than in extraembryonal membranes and placenta
is observed at the 10th day of gestation, as compared to the
concentration observed in the 6, 7, and 9 day conceptuses.
Expression of c-abl in the fetus appears to decrease after the 11th
day of prenatal development. The oncogene c-abl is
transcriptionally active in all postnatal mouse tissue examined
with spleen and thymus poly(A).sup.+ RNA exhibiting a slightly
stronger hybridization than from other tissues.
The oncogene c-ras.sup.Ha was found to be expressed in
considerable, but similar levels at all stages of prenatal
development both in the embryo proper as well as in extraembryonal
tissues. High levels of c-ras.sup.Ha expression were also observed
in various tissues of newborn or 10 day old mice, particularly in
bone, brain, kidney, skin and spleen.
The oncogene c-myc was detectable at days 7 and 8, but much higher
levels were observed in late embryonic development (days 17 and
18).
The oncogene c-erb had maximum hybridization at 13 days, while no
hybridization was observed at day 6.
The oncogene c-src was detected at its highest levels in the latter
half of mouse embryonic development with an increase beginning at
day 14, peaking at day 15 and gradually decreasing thereafter. For
the oncogene c-sis, peak expressions were observed at days 7 and
16, the day-7 peak was 1.5 to 3 times higher than all other days
and the day-16 peak was 1.5 to 2 times higher than days 9 to 13
days 17 and 18.
In the next study, the nucleotide sequence of the presumed oncogene
region of Avian myeloblastosis virus myb was employed (Vister et
al. (1982) supra). Using the published nucleotide sequence, a
number of antigenic oligopeptide sequences were derived and seven
of the polypeptides so derived were synthesized and evaluated as
being potentially antigenic. These seven oligopeptides, which are
representative antigenic oligopeptides according to the invention,
have the following formulas:
(1) pro-phe-his-lys-asp-gln-thr-phe-glu-tyr-arg-lys-met
(2)
pro-ser-pro-pro-val-asp-his-gly-cys-leu-pro-glu-glu-ser-ala-ser-pro-ala-ar
(3)
asp-asn-thr-arg-thr-ser-gly-asp-asn-ala-pro-val-ser-cys-leu-gly-glu
(4) pro-gln-glu-ser-ser-lys-ala-gly-pro-pro-ser-gly-thr-thr-gly
(5) met-ala-phe-ala-his-asn-pro-pro-ala-gly-pro-leu-pro-gly-ala
(6)
pro-pro-val-asp-his-gly-cys-leu-pro-glu-glu-ser-ala-ser-pro-ala
(7)
pro-phe-his-lys-asp-gln-thr-phe-thr-gly-tyr-arg-lys-met-his-gly-gly-ala-va
l
The polypeptides were linked to keyhole limpet hemocyanin in
accordance with conventional techniques (Dockray, Regulatory
Peptides (1980)1:169) and the resulting immunogen was used to
immunize rabbits in a first injection with complete Freund's
adjuvant, followed by injections with incomplete Freund's adjuvant
over periods of three to four weeks to hyperimmunize the rabbits.
The rabbits were bled repeatedly over a period of six months. Of
the seven oligopeptides which resulted in the production of
antibodies, antibodies to two peptides (5 and 7) were selected for
detailed analysis. The antibodies were reacted with radioactively
labeled cell lysates from a cell line containing multiple copies of
the Avian myeloblastosis virus and with lysates from appropriate
non-infected cell lines. Antibody No. 5 identifies a specific
protein of approximately 58,000 daltons, which is present in the
virus-infected cell line but not in controls.
Antibody against polypeptide 5 was also reacted with the plasma of
chickens bearing tumors induced by amv. A band similar to that
observed with the above lysates of approximately 48,000 daltons was
identified.
Antisera to polypeptide No. 5 was also reacted with lysates of a
myeloid human leukemia cell line (HL-60) which is known to express
messenger RNA transcripts of the c-myb gene (Gallo and Wong-Staal,
Blood (1982) 60:545). This antibody reacted with a protein of about
90,000 daltons. In freshly isolated myeloid leukemia cells, the
antibody identifies a series of proteins, 14 kd to 70 kd, not
present in normal white blood cells.
The polyconal antibodies to the fragment no. 5 of the myb protein
was tested for its ability to kill normal and leukemic cells. The
procedure employed is described in Terasaki and McClelland, infra.
The data are set forth in the following table.
TABLE 5
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CYTOTOXICITY* OF ANTI-Myb2 ANTIBODY AGAINST HUMAN CELLS
Cells.sup.++ Common Normal Normal Molt 4 HL-60 ALL AML T-ALL
Antiserum Dilution B T (T) (AML) 1 2 (5)** 1 2
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Medium Alone 1 1 1 1 1 1 1 1 1 (-control) ALS.sup.+ -- 8 8 8 8 8 8
8 8 8 (+control) Anti-myb -- 1 1 8 6 6 1 6 6 6 1:2 1 1 6 1 1 1 6 6
2 1:4 1 1 6 1 1 1 4 4 1 Pre-immune -- 1 1 4 1 1 1 1 4 2 1:2 1 1 2 1
1 1 1 2 2 1:4 1 1 2 1 1 1 1 2 2
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*8 = 80-100% killing 6 = 60-80% killing 1 = 0-10% killing The
method od Terasaki and McClelland, Nature (1964) 204-998, was
employe for complement lysis. **3 of 5 are killed; a representative
one is shown .sup.+ ALS antileukocyte serum .sup.++ Molt 4 is a
nonmalignant human T lymphoid cell line that is known to express
myb nRNA. HL60 human myeloid leukemia cell line AML acute myeloid
leukemia ALL acute lymphocyte leukemia TALL Tcell active
lymphoblastic leukemia
The above results demonstrate that the expression product of the
myb gene can react with antibodies to produce lysis with
complement. Thus, the myb protein appears to be a surface membrane
protein which is available for binding to antibodies. By
identifying proteins to which specific antibodies will bind, which
proteins have diagnostic value as indicative of malignance, the
malignant cells can be identified and treated. In the subject work,
no determination has been made as to the specificity or
cross-reactivity of the subject antibodies. Since only a fragment
was used to prepare the antibodies, it would be expected that
antibodies of greater binding specificity and avidity could be
prepared with the whole protein, particularly with the whole
protein in a membrane. The subject antibodies can be used to select
for antibodies binding to the same or other determinant site.
The subject data demonstrate that antibodies can be prepared which
do not affect normal B- and T-cells, but are cytotoxic in
combination with complement for a variety of malignant cells.
Therefore, the antibodies can be used for cancer therapy without
the hazard of substantially inactivating the immune system.
It is evident from the above results, that one can detect the
presence of malignancy in a human host by determining the
transcription and/or expression products of the oncogene. One can
screen retroviruses or other source of nucleic acids to
.Iadd.demonstrate the ability of such nucleic acids to
.Iaddend.transform vertebrates to malignancy. One may then use
these nucleic acids to deduce peptide composition and screen
malignant cells for transcripts or peptides, by hybridization in
the former case and with appropriate receptors in the latter case,
employing any of a wide variety of diagnostic assays. Antibodies
can be produced to the peptides, which antibodies may be labeled
and may then be used for diagnosing the presence of a peptide
diagnostic of malignancy. The oncogernic proteins are found to be
available for binding to antibodies as surface membrane proteins.
The antibodies may serve as diagnostic reagents for determining the
presence of malignancy and determining the location of malignant
cells. The antibodies may also serve in treating tumors in vivo by
using radionuclides, toxins, in combination with the host
complement system or opsonins, or other antibody dependent lytic
system or the like. The antibodies find use in pre- and
postoperative systems, in the later determining whether complete
removal has occurred, whether metastases exist. The antibodies can
be used postoperatively to destroy any remnants of the tumor which
may not have been excised.
Although the foregoing invention has been described in some detail
by way of illustration and example for purposes of clarity of
understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
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