DK174458B1 - Detection and treatment of cancer - using antibodies to oncogenic expression prods. - Google Patents

Abstract

The following are claimed: (A) a method for evaluating the possibility of cellular malignancy in a human host, comprising (a) contacting a probe specific for a cellular product (I) with a source (e.g. cells or physiological fluid) from the host suspected of contg. (I), where (I) is mRNA or its expression product, the mRNA being complementary to a DNA sequence of a retrovirus capable of transforming a normal cell to malignancy, and (b) determining the level of binding of the probe to (I) as indicative of the presence of cellular malignancy; and e.g. (C) 41 antigenic oligopeptides (II), antibodies raised to such oligopeptides, and antigenic oligopeptides contg. up to 50 amino acid residues including one or more of the amino acid sequences of (II).

Description

in DK 174458 B1

The application for this patent is filed from Danish patent application no.

PA 1983 04980 filed October 31, 1983.

The present invention relates to a process for producing an antigenic oligopeptide containing up to 50 amino acid residues, preferably up to 25 amino acid residues, and containing one or more specific amino acid sequences which are expression products of retroviral oncogenes.

The mechanism of mammalian cell malignancy has been and remains the subject of intense investigation. One of the areas that is considered promising in the elucidation of the mechanism is the so-called oncogenes. Although the presence of oncogenes 10 was first detected in retroviruses, it now seems reasonably established that the viral oncogenes have cellular counterparts. The significance or effect of these cellular counterparts is unclear. An excellent overview of oncogenes, their properties and especially the src gene can be found in an article by J. Michael Bishop, Scientific American, March 1982: 81-93. The article also lists a variety of 15 viral oncogenes, showing that a number of them are involved in phosphorylation.

It has been found that the src gene is not only active in malignant cells of chickens, but also in normal cells. The difference seems to consist only of a degree and not a type difference, since the enzyme expressed by the src gene occurs at much higher concentration in the malignant cell compared to the normal cell.

In order to detect the presence of tumor cells, it is necessary to distinguish between normal cells and tumor cells. Therefore, the observed property to be used in diagnosing the tumor cell must be differentiable from a normal cell or from a physiological fluid from a normal host if it is the fluid and not the cell being examined. Furthermore, the property must not be specific to the individual but be common to the malignant nature of the cell.

Both in diagnosis and treatment, the possibility of specific detection of malignant cells is very important. Any technique used, when a high degree of malignancy is suspected, must be able to distinguish malignant cells from normal cells. Furthermore, the diagnostic technique should be suitable for a large number of members of the population and not specific to a small population group.

2 DK 174458 B1

Since a cancer cell is derived from a normal cell, most properties and constituents of the malignant cell will be the same as that of the normal cell.

Furthermore, there is an increasingly widespread notion that malignancy is the result of a natural process that in some contexts results in malignancy.

5 Given that malignancy may be based on normal processes that produce a divergent result at that time, it is not surprising that there have been great difficulties in detecting observable differences between normal cells and cancer cells for a wide range of allogenic hosts.

The following dissertations provide a general description of oncogenes and the effect I0 of retroviruses in tumor genesis: Bishop, Scientific American, supra; Bishop,

New England J. of Med. (1980) 303; 675-681; Lancet, July 24, 1982, pages 195-196; Cooper, Science (1982) 218: 801-806; Varmus, Science (1982) 216: 812-820. Articles on specific oncogenes include: Becker et al., PNAS USA (1982) 79: 3315-3319; Tsuchida et al., Science (1982) 217: 937-938 and Dharet al., Ibid., (1982) 217: 934-936.

It has now been found that DNA that can transform cells of lower vertebrates into malignancy is found in human cells and has a much higher level of transcription and expression in malignant cells than in normal cells. Thus, by determining the higher level of mRNA or the expression product of such mRNA, it is possible to diagnose the presence of malignant cells. In addition, the production of the higher level of peptides in malignant cells may form the basis for the treatment of malignancy. If 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 can be screened as a diagnostic for the presence of a particular tumor.

The following describes methods and compositions for evaluating the probability of the presence of malignant cells in a group of cells, especially human cells in vivo or freshly removed from a human host. The method relies on cellular products, such as mRNA or its expression product, as a diagnostic of the possible presence of malignant cells. The mRNA selected for detection is usually selected as the result of the presence of RNA in a retrovirus genome, which retrovirus is capable of transforming mammalian cells into malignancy. Furthermore, the RNA in the retrovirus is selected as

-IN

B1 is given, a sequence that does not encode a significant function of the retrovirus, but in fact must be dormant.

First, a DNA sequence is defined which is capable of causing mammalian cell malignancy. Once this DNA sequence is defined, polynucleotide sequences can be provided which can serve as probes to determine elevated concentrations of mRNA as a measure of cell malignancy. The sequence can also be used to define polypeptide sequences which can define complementary receptors with high specificity for the peptide sequence. The receptors can then be used to determine the presence or concentration of the peptide in the cells or physiological fluids and for treatment if the receptors can be targeted to malignant cells. Knowing the nature and function of the peptide, other means may also be provided to control the increased production of the particular peptide.

The first step of the method is to define the DNA sequence. Various methods can be used to define the DNA sequence of a retroviral oncogene.

Eg. retroviruses have been found capable of transforming cells from lower vertebrates to malignancy. The retroviruses that have been characterized have been found to have DNA sequences comparable to wild-type genes in the host, and such genes are now called oncogenes. In the case of Rous sarcoma virus, the expression product of the gene is isolated and characterized and has been shown to be a kinase. In the case of this kinase, it is also shown that the kinase is normally produced by the cell, but at a much lower amount when the src gene from Rous's sarcoma virus is introduced. A large number of viral oncogenes have already been determined in various vertebrate species, and the following table lists the oncogenes and their origin.

4 DK 174458 B1 TABLE 1

Oncogen Origin of origin v-src Chicken v-fps 5 v-yes "v-fos" v-myc v-erb v-myb 10 v-rel turkey v-mos mouse v-bass v-abl v-breed rat 15 v-fes cat v-fms v-sis monkey

Other examples of sources of DNA sequences capable of introducing malignant transformations in vertebrate cells may be isolated DNA from a malignant cell or cell line, cloned DNA from a genomic library or cloned DNA from an mRNA library where the total mRNA from the malignant cell is reverse-transcribed to DNA and cloned. Each of these libraries can be examined for their ability to introduce malignancy. A refinement of the study technique can be obtained by taking the total mRNA from a normal cell and producing the cDNA from the mRNA. Then, the single-stranded DNA can be used as a probe to remove mRNA associated with the normal cell from the total mRNA from a malignant cell. The remaining mRNA will then comprise mRNA expressed by genes associated with malignancy. Then, the mRNA can be used to screen a genomic library and use the cloned DNA which hybridizes with mRNA in a bioassay to determine the ability to transform into malignancy. Over time, other methods will also become available to detect and define DNA sequences capable of transforming normal cells.

5 DK 174458 B1

A further analysis can be used to screen cDNA from fetuses with mRNA from malignant cells. In particular, if the oncogene is a quiescent gene or relatively inactive in mature vertebrates but highly active in the fetus, the screening may serve to further narrow the area of the sequence to be screened.

Once isolated from a DNA sequence capable of inducing malignancy, a cloned viral oncogene or short polynucleotide sequences can be used as probes to determine the level of production of mRNA in the 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 10 sequences have been the subject of extensive description and exemplification in the literature. A sequence should normally have at least 14 nucleotides, usually at least 18 nucleotides, and the polynucleotide probes may be one or more kilobytes. Various labels may be used, usually radionuclides, especially 32P.

However, other techniques, such as the use of biotin-modified nucleotides for introduction into a polynucleotide, may also be used. The biotin then serves as a site for binding to avidin or antibodies that can be labeled with various labels, such as radionuclides, fluorescent substances, enzymes or the like. Instead, antibodies that recognize specific duplexes, including DNA-protein duplexes, can be used. The antibodies, in turn, can be labeled and study 20 can be performed to find where the duplex is bound to the surface, so that the presence of the antibody bound to the duplex can be determined on the surface.

By isolating the nucleotide sequence for the entire oncogene, the sequence of bases can be determined in known manner, e.g. according to Maxam and Gilbert, PNAS USA (1977) 25 74: 560. The sequence can be used to determine the amino acid sequence of the protein expressed by the oncogene. By identifying the codons for methionine followed by a sequence that does not have stop codons that prevent expression, one can usually find a single sequence in the frame with a methionine codon for defining the oncogene.

Instead, hybrid DNA technology can be used to obtain expression. The DNA sequence can be restriction mapped and the appropriate sites for cleavage defined. In this way, the sequence can be excised and introduced into a vector having the appropriate control signals. After expression of the DNA sequence has been obtained, antibodies to the polypeptide can be prepared. By using oocytes to express the mRNA which is then translated to produce the peptide expressed by the oncogene, the protein defined by the mRNA can be produced. The identity of the peptide from the oocyte as the peptide produced by expression of the hybrid DNA can then be determined.

Once the protein has been identified and verified, the protein or its peptide subunits can be used as antigen for the production of antibodies for diagnosis and treatment. The antibodies can be prepared in various ways depending on whether monoclonal or polyclonal antibodies are desired. To obtain polyclonal antibodies, a vertebrate, usually a domestic animal, is hyperimmunized with the antigen, and shortly after repeated immunizations, blood is collected and the γ-globulin is isolated. To obtain monoclonal antibodies, a small animal is hyperimmunized, the spleen is extracted, and the lymphocytes are fused with an appropriate fusion partner. The hybridomas formed are then grown under limiting dilution and clones are selected to yield the desired antibodies.

Instead of producing the entire peptide, various regions likely to be determinant sites can be determined and these oligopeptides having at least 8 amino acids, usually with at least 10 and not more than 20, usually not more than 18 amino acids, can be used to define a hapten. which can be used to induce the antibody formation. The oligopeptide binds to a suitable immunogen and is introduced into a vertebrate to produce antibodies, either polyclonal or monoclonal antibodies as described above.

Accordingly, a series of oligopeptides corresponding to antigenic regions of the peptide expression products of RNA present in 25 retrovirus oncogenes have been found. Examples of species of suitable antigenic oligopeptides are shown in the following list, arranged in groups following the retroviral oncogene (expression product), which is recognized by antibodies produced from the oligopeptide.

7 DK 174458 B1 A. Myb pro-gln-glu-ser-ser-lys-ala-gly-pro-pro-ser-gly-thr-thr-gly with ala-phe-ala-his-asn-pro- pro-ala-gly-pro-leu-pro-gly-ala pro-pro-val-asp-his-gly-cys-leu-pro-glu-glu-ser-ala-ser-pro-ala 5 pro-phe -his-lys-asp-gln-thr-phe-thr-glu-tyr-arg-lys-with-his-gly-gly-ala-val pro ^ he-his-lys-asp-gln-thr-phe- thr-glu-tyr-arg-lys-with asp-asn-thr-arg-thr-ser-gly-asp-asn-ala-pro-val-ser-cys-leu-gly-glu pro-ser-pro pro-val-asp-his-gly-cys-leu-pro-glu-glu-ser-ala-ser-pro-ala-arg leu-gln-glu-ser-ser-lys-ala-ser-gln-pro -ala-val-ala-thr-ser 10 asn-pro-glu-val-lys-lys-thr-ser-trp-thr-glu-glu-glu-asp-arg B. Src arg-leu-ileu-glu -asp-asn-glu-tyr-thr-ala-arg-gln-gly-ala-lys-phe-pro asn-arg-glu-val-leu-asp-gln-val-glu-arg-gly-tyr- arg-met-pro bull-arg-arg-asp-pro-glu-glu-arg-pro-thr 15 C. Ras * 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-lys-gln-ala-gln-glu-leu-ala-arg with Thr-glu-Tyr-Lys-leu-val-val-gly-ala-ser-gly-val-gly-light- ser-ala D. RasHa 20 glu-asp-ileu-his-gln-bull-arg-glu-gln-ileu-lys-arg-val-lys-asp-ser-asp-asp val-arg-glu-ileu- arg-gln-his-lys-leu-arg-lys-leu-asn-pro-pro-asp-glu-ser-gly-pro with-thr-glu-tyr-lys-leu-val-val-val-gly -ala-arg-gly-val-gly-lys-ser-ala met-thr-glu-tyr-lys-leu-val-val-val-gly-ala-val-gly-val-gly-lys-ser-ala ala met-thr-glu-tyr-lys-leu-val-val-val-gly-ala-gly-gly-val-gly-lys-ser-ala 25 val-asp-glu-tyr-asp-pro-thr -ileu-glu-asp-ser-tyr-arg-lys-gln-val 8 DK 174458 B1 E. Fes arg-his-ser-thr-ser-ser-ser-glu-gln-glu-arg-glu-gly -gly-arg ser-arg-glu-ala-ala-asp-gly asn-gln-gln-thr-arg-glu-phe-val-glu-lys-gly-gly-arg 5 pro-glu-val-gln -lys-pro-leu-his-glu-gln phe-leu-arg-thr-glu-gly-ala-ala-ala-asp-giu-asp-ala-ala-ala phe-leu-arg-thr-glu -gly-ala-arg-leu-arg-met-lys-thr-leu-leu ser-ala-pro-arg-ser-ser-pro-ser-thr-ser-ser-trp-ser-ser ala-ser -pro-tyr-pro-asn-leu-ser-asn-gln-thr-arg 10 F. Myc asp-gln-gln-arg-leu-gly-arg-arg-thr-arg-arg-arg-arg-val arg-lys glu-arg-gln-glu-ala-glu-arg-his-asn-val-leu -glu-arg-gln-arg-arg-asn-glu arg-leu-ileu-ala-glu-lys-glu-gln-leu-arg-arg-arg-arg-glu-gln arg-lys-lys-asn -lys-lys-lys-ser-glu-glu-ileu-asp] 5-pro-thr-thr-ser-ser-asp-ser-glu-glu-glu-gln-glu arg-thr-leu-asp -ser-glu-glu-asn-asp-lys-arg-arg glu-arg-gln-arg-arg-asn-glu-leu-lys-leu-arg glu-gln-leu-arg-arg-arg-arg -glu-gln-leu-lys asn-asn-glu-lys-ala-pro-lys-val-val 20 arg-arg-glu-gln-leu-lys-his G. Fos lys-glu-lys-glu- lys-leu-glu-phe-ileu-leu pro-glu-glu-glu-glu-lys-arg-arg-ileu-arg-arg-glu-arg

Several amino acid groups can be coupled to both ends of each of the oligomeric peptide sequences mentioned in sections A 25 - F, without changing the antigenic nature of the peptide formed, up to 50, preferably 25, amino acid residues. containing at least one of the amino acid sequences listed in sections A - F.

The antigenic oligopeptides listed above containing one or more of the amino acid sequences set forth in sections A - F may be prepared synthetically or by hybrid DNA technology. For such polypeptides and oligopeptides with up to 50 amino acid residues, conventional solid phase peptide synthesis is particularly suitable.

Accordingly, the present invention provides a process for producing an antigenic oligopeptide containing up to 50 amino acid residues, preferably up to 25 amino acid residues, and containing one or more of the above-mentioned amino acid sequences, which is unique in that the amino acids are sequentially coupled. obtaining the oligopeptide, on a suitable carrier.

In this ordinary synthesis procedure for the preparation of peptides which, e.g. is disclosed in US Patent No. 4,341,761, known side chain protecting groups and conventional carriers, e.g. polystyrene resin, such as chloromethylated resin, hydroxymethyl resin or benzhydrylamine resin, to obtain the amino acid coupling.

In the practice of recombinant or hybrid DNA technology, such oligopeptides containing up to e.g. 20 amino acid residues are used to deduce the co-20 don sequence of the nucleotide (DNA) encoding the subject oligopeptide. Using this codon sequence as a template, double-stranded DNA can be synthesized in known manner and inserted into a vector DNA or cloning medium such as E. coli plasmid. Transforming a suitable host, e.g. a microorganism such as E. coli or e.g. other cell lines, with recombinant vectors, provide a means of obtaining expression of the desired oligopeptide. In those situations where the human gene is different from v-onc, e.g. human c-ras, the technique described above can be used to isolate the gene, mRNA or pseudo-gene and obtain antibodies to the human expression product. The human oncogene is expected to have substantial complementarity to the related v-onc, usually deviating in less than 5% of the bases, and generally deviating by less than 5% of the amino acids in the expression product.

The antibodies can be used in various ways. In particular, they can be used for diagnosis. In cases where the antigen can only appear in a physiological fluid 5 at a higher concentration when malignancy occurs, the physiological fluid such as serum, plasma, whole blood or cerebrospinal fluid may be subjected to a study. The antibodies used in the study may be labeled or unlabeled. Non-labeled antibodies can be used for agglutination, the labeled antibodies can be used in a large number of studies, using widely different labels such as radionuclides, enzymes, fluorescent substances, enzyme substrates or cofactors. These methods are well defined in the literature and examples of test procedures can be found in U.S. Patent Nos. 3,817,834, 3,935,074, 4,233,402, and 4,318,980.

In some techniques, it will be useful to label the antigen or fragment thereof instead of the antibody and have a competition between labeled antigen and antigen in the antibody sample. In this situation, it is common to provide kits which comprise a combination of the labeled antigen or labeled fragment and the antibody in amounts that provide optimum sensitivity and accuracy.

In other situations, it may be desirable to have a solid carrier which is either bound to an antigen or an antibody. A polyepitopic antigen can serve as a bridge between antibody bound to a carrier and labeled antibody in the test medium. Instead, one can compete between labeled antigen and any antigen in the sample for a limited amount of antibody.

If the antigen is not present in a physiological fluid or if it does not detect malignancy, the cells must be isolated and the cells examined for the presence of mRNA or antigen. Methods for determining mRNA have already been described. For detecting the antigen, the tissue sample may be lysed in conventional manner, e.g. base, detergents or the like, celiac residues are separated by filtration or centrifugation, and the filtrate or supernatant is isolated and examined.

For therapy purposes, either xenogenic or allogenic antibodies may be used depending on the nature of the treatment and whether the foreign antibodies induce an immune response. The literature describes many methods for producing human antibodies insofar as antibodies from mice or other mammals are not satisfactory. The antibodies can be used in many ways. By using the appropriate IgG (different from IgGi), light can be induced via the natural complement process. Alternatively, the lysing portion of a toxin may be attached to the antibody, in particular a Fab fragment. The antibodies may be bound to liposomes to direct the liposomes to the malignant cells to be taken up into the cells by fusion of the membranes. Other labels can also be bound to the antibodies, such as radionuclides, fluorescent substances or enzymes.

10 Upon introduction of the antibodies in vivo, the antibodies will direct the label to the malignant cell where the presence of malignancy can be diagnosed or treated.

The composition 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 directed, and the like. The antibodies are usually introduced into a physiologically acceptable carrier, e.g. saline or a phosphate buffered saline solution, and injected into the host at the desired location, if possible, and otherwise into a circulating system such as blood.

The described antibodies may 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). The same technique can be used to identify and isolate cells expressing the oncogene. To remove cells expressing the oncogene from a mixture of cells, the above antibodies can be combined with complement, joined to the lysing fragment (A-25 fragment) of a toxin (see EP Patent Application No. 17,507 and GB Patent Application No. 2,034,324 ), or the cells are agglutinated and separated by physical methods there.

The following examples serve to illustrate the invention.

Tumors were obtained from fresh surgical specimens at the time of resection and 30 were untreated with chemotherapy or radiotherapy. Only viable tumors were obtained and the tissue treated as quickly as possible to avoid mRNA degradation. The samples were quickly frozen and stored in liquid nitrogen until the processing for RNA was performed. If surgical specimens included larger amounts of normal tissue, some of this was taken for analysis as an internal control for the amount of c-onc gene expression. Thus, the C-onc gene expression could be compared for normal and malignant tissue from the same patient. As little as 20 µg of Maloney murine sarcoma virus, equivalent to 1 with approximately one RNA transcript of 3 kilobases (kb) per cell or approximately 2 micrograms of poly-A RNA applied to the filter could be determined by this method (Kafatos et al., Nucleic Acids Res. (1979) 7: 1541). By using appropriate controls, including unrelated RNAs, poly-A-negative fraction-10 RNA, plasmid DNA and mouse and human DNA, false-negative as well as false-positive results could be reasonably avoided. The deposited spots were quantitatively evaluated using a soft laser scanning densitometer. If sufficient material was available, mRNA was further characterized by Northern analysis to confirm the presence and extent of specific transcripts (Thomas, PNAS USA (1980) 77: 5201).

Expression of 13 cellular oncogenes in 14 tumors was investigated by DNA-RNA hybridization technique. These data are summarized in Table 2.

DK 174458 B1 6 g n E + Ο-Q .- ,,, + ,,,,,,,, E l +>, ro _l (Λ 0 © F CVI + '>' * '+' + + + · '

"ISLAND ISLAND

* P C

© ofs -ί '' '·' + 'ίί + ·' I n + ..... + 1 + + '1 1 o + c 0 8 ™ ΐ ..... t> xt' '' 0 ^ o * o * o -t ...... ΐ ί · i i-? LU> H + rs Z (0 ^ + ..... + '+ +' '' <- E + 2 o j- ^ <£ + J, ^ R CO + ..... + 'ί t · · 1 5> IS + ii = + + 2 XO (N + '' '' i + '+ +' i 'g <5 + c ©. & X i +. .S

'C

£ t 3 + * 4- i i © t5'r- + 11 ,,, + -1 + ,,, «© © ++ c

H— »> v O

· = Π3 LL on * - t_ · O) H ~ c ^ © g I g

c £ iS ++. . H

£ 0-πτ - + ...... - t I I '' · 2 © C c + +++ ^ J5 u u 7

.Ξ © H S

ti v, (Λ o 3 © R ++. o.

™ + <> << I + <X X <>> -

e <o I

ω ° ϋ © ^ ++. . e e <= O ^ + ..... + '+ +' Jp. £ 'g <Ν c / 5 <2 _1 ULI (j © * * <o.

CQ C "5 U jQ * o) * rn W [fl 0) (ft ^ <? §> '> L ^ iDXioO®ro (i) E.! I! # * I-> W 55oJW> - <U. 2irQ; LLLlM * ϊ Ϊ 14 DK 174458 B1

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 was measured. This was not the result of a lack of homology between the viral gene probe and the human mRNA, as it was possible to detect homologs of all these probes in human genomic DNA. RNA from microscopically normal tissue did not contain any detectable 10 transcripts in this assay.

Four cellular oncogenes exhibited a consistent expression pattern in various human tumors. These were c-myc, c-fos, c-rasHa, and c-rasK1. A comparison was made of the intensity of hybridization, which was possible since all probes were labeled for approximately the same specific activity, v-myc 15 and v-fos showed the highest intensity of hybridization to human tumor RNAs, suggesting the presence of a large number of copies of mRNA per cell. Expression of both of these genes was observed in all malignancies examined. c-rasHa and c-rasK 'sequences were also detected in most of the human tumors, but with less intense hybridization.

20 mRNA sequences related to c-fes were detected only in 2 of 14 tumors examined; both of these were lung cancers.

C myb expression was detected in only one of 14 tumors; this one was also a lung cancer.

C-src mRNA sequences were only observed in circulating tumor cells in a patient with lymphosarcoma.

To investigate whether expression of cellular oncogene sequences was related to neoplasia, an effort was made to obtain both substantially normal-looking tissue and obviously malignant tissue from the same area of the patient at the same time. Hybridization studies were then performed on RNA samples from the tumor and from closely located uninvolved tissues. In 6 of the 14 patients, this analysis was possible. In one of these cases, it was later shown by histological analysis that the suspected normal tissue was infiltrated with tumor. In 4 of the remaining 5 cases, there was differential expression between the tumor and the normal tissue, observing low or undetectable levels in the normal tissues and elevating nine levels in the malignancy. Three of the renal cell carcinomas and one colon carcinoma showed this phenomenon.

Depletion analysis of RNA from cells in the regions of the tumor sample and the control sample showed a correlation between the presence or absence of tumor and c-onc gene expression. In one tumor, an adenocarcinoma of the small intestine, c-onc-related sequences were found in histologically normal adjacent tissues. Analyzes of poly-A RNA from tumors and control tissues were performed by the Northern technique. Two c-mys-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 mRNA in these hybridization assays, most likely resulting from degradation during tissue anoxia in the period following surgical removal of the tissue. Using the above procedures, several other tumor types, obtained from fresh surgical specimens, were examined for c-onc gene expression. In this series of assays, DNA-RNA hybridization was used to search for expression of 10 different 20 cellular oncogenes in 9 tumors. The data obtained are summarized in Table 3 below.

DK 174458 B1 + ^ CM ..... + + + + 1 o E +

£ (B

ε -¾ å -2 + + i + + c - i + i '' J i + i '£ (Λ

C

ro cs e Ό o 0? ^ + '+' '+ +' + '

é S

o z u <u

*ISLAND

C

(Λ O

~ V) C υ _ +. +,, + + .., 9 O) -T- + + + '' + + + + 'l

* TJ

l_ O S

w X <t § 2: <9 s s 2 I s

-—J C T— + I I I I ++! IN W

<>, i.

S .c «> z H δ

<'S

2 a? g § x ώ E S? 3 o c J- C T- I 1 I I <I 1 I I & 5 e is ro _ £ o>.

α o c +, + + + -3 co + '· ·' T + T + 1 «ro + + + + + jj P L * o 8 C T3 'o +,, +. p

S ^ ϊ ++ +, + ί ++ + I

JL, js ¢ /) * 3.

^ s CQ 4. _i_ "f · π TT + + ,, j ++ + I λ + + + ω α scc as · = r> Co <É _i lu r λ ro * + s 3 oC 0X10 - Lw« (nw H-> «5lwct: <uK [tiL (o * 5 i 17 DK 174458 B1

The results reported in Table 3 correlate fairly well with the results previously reported in Table 2 in that the cellular oncogenes c-myc, c-fos, c-rasHa and o-rasK 'show a consistent expression pattern in the further investigated tumor. types. Furthermore, c-myb, c-src and c-fes were also detected in several additional tumor types, while c-rel, c-abl and c-sis expression were not observed in any of the further examined tumor types. . It is interesting that none of the cellular oncogenes searched for were found to be expressed at any significant level in the only uterine carcinoma evaluated.

To determine if the mRNA shown to be present in malignant cells in elevated amounts was linked to genes involved in embryogenesis, experiments were generally performed as set forth below. Total RNA was isolated from embryos / fetuses of randomly bred Swiss mice at daily intervals beginning with the 6th day of pregnancy (the day of gestation was designated as day 0 for prenatal development). Beginning at day 10 of prenatal development, the embryo itself was separated from the extraembryonic membranes and the placenta. The small size prior to day 10 prevented separation and therefore the embryos from days 6-9 represent the entire concept as dissected from the uterine wall.

Twenty aliquots of poly (A) -containing RNA (poly (A *) - RNA) were isolated by affinity chromatography on oligo (dT) cellulose columns and placed as spots on nitrocellulose paper (dot blots) (Kafatos et al., Nucl. Acids Res. (1979) 7: 1541-; 1552). The samples were hybridized to [32P] labeled molecular cloned oncogene specific probes. Dot blots were continuously evaluated using a soft-laser scanning densitometer. Transcriptional activity of c-oncs was further studied in more detail in different tissues of newborn and 10-day-old mice. Agarose gel electrophoresis followed by dipping on nitrocellulose paper (Northern blotting) (Thomas, PNAS USA (1980) 77: 5201-5205) was used to confirm the results obtained from dot blot analysis and further to determine the size of the various c-onc-related transcripts.

More specifically, RNA was isolated from Swiss-Webster mouse embryos at various developmental stages using the guanidine thiocyanot method.

(Cox, Methods Enzymol. (1967) 12: 120-129; Adams et al., PNAS USA

(1980) 74: 3399-3043).

As noted above, Swiss-Webster mouse embryos from days 6 - 9 represent the entire concept including all extraembryonic tissues, such as 5 membranes and the cells that give rise to the placenta at later developmental stages. At all later stages, the embryo itself was dissected freely from extraembryonic tissues. RNA was selected for poly (A +) RNA by a cycle of chromatography on oligo (dT) cellulose column (Aviv and Leder, PNAS USA (1972) 69: 1408-1412). Poly (A *> RNA was dissolved in water, boiled, rapidly cooled on ice, 10 and 3 µg (1.5 μΙ) applied to sheets of nitrocellulose paper previously equilibrated with 20 x SSC (1 x SSC is 0.15 NaCl, 0.015 M sodium citrate) and air dried After baking for 18 hours at 80 ° C, the blots were pre-hybridized for at least 4 hours at 45 ° C in a buffer containing 0.75 M NaCl, 0.05 M sodium phosphate ( pH 7.5), 0.005 M EDTA, 0.2% SDS, 10 mg glycine / ml, 5 x Denhardt's reagent (1 x Denhardt's reagent is 0.02% each of fibrill, bovine serum and polyvinylpyrrolidone), 0.25 mg of denatured herring DNA / ml and 50% formamide.

The blots were hybridized for ca. 20 hours at 45 ° C with 1 x 10 6 counts / min nick-translated probe / ml hybridization buffer (prehybridization buffer with Denhardt's reagent at 1x). The cloning 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 [32P] dCTP. (3200 Ci / mmol) for specific radioactivity of approx. 1 - 2 x 10 9 counts / min / µg DNA. After hybridization, the clumps were washed 3 times in 1 x SSC at 50 ° C for a total of approx. 2 hours and exposed to pre-exposed x-ray films with intensification screens at -70 ° C for 72 hours.

Using the above procedure, a number of known oncogenes were screened to determine whether they were expressed in the embryos. The following Table 4 lists the individual oncogenes and the observations regarding their expression in embryonic cells.

DK 174458 B1 (Λ i «3 +++ '0 B 8 s ^ 2 s c x- n. Ri ω ® ii n c oo N Λ W (j) 17 ++ ++ + + · + (ogSRfft

1 S 72s'sr? S

about ^ 77 co - f "fe ® 9- n .3n ti.cn cn <σ> ίΤΓ® m, ι- S m E ro +++ +, + l + l, + I70S ^^^ w S§Ssi§ oa §S ^^ CD Uj rr 75 -fe "ro ro CD <ΐ !! ΐΐ _ E c ^ tu ® ^ Ϊ g 1 o ® ^ fca> ®

S -g g S S Æ * i S

E ω S LU O S> ll Q

ro ro ro ro ro 'o o ίο co PT ro cn o 2 fe o o ^ ro ti i ~ v_ c (H CD ro

C yyy W CO

9 C mc TO "05 c feEDJroE-FCOcroro o ™ ooEo | e | EE c- tj o- £ feo. £ roorooo £ 7 a 2 = € yy-§H ^ yy S« <a § i I: s · s I ss vs

sweep CN

ro cn, I 25

. «Y ^ £ 5 S

<ϋ> ro ro LO 'f n s B ro „» »S ro 2 ro ro E I) 2 o Ji ^ ° roo

! E ·? I S> S 4, - fee® S

SajEoS.Sm.ScE g i i i i i 111! 18 isos- * g? I§ „? sr § is: 14 .i 1 ^ .¾ o —5 ro 2 .roro o .ro>, ^ - · 5 • fe OD J3! 5 J>> o> c ί S ^ S ^ n-> h <i2 << q: <ww - ro f-0- - -J · ^ £ ro ro ro ro ° "fe § * ® § $ ^ ro d O ife ro q rrf S f?„ Λ ~ OOtuO ° DO o - ^ xroo ^^ Xl®2 <f C wp: WO>, - Pp> .roro ____ h- O £ ro2EErowEÆ'ro cm co ^ 20 DK 174458 B1

Relatively high levels of c-fbs-related sequences were detected in poly (A *) RNA prepared from 6, 7, 8, and 9 days of concepti containing the embryo itself and extraembryonic tissues. More than 10-fold lower fos expression was observed in embryos at later developmental stages dissected 5 free from extraembryonic tissues. Data from the placenta and extraembryonic diaphragm membranes from days 10-18 showed that expression was primarily in these tissues. In postnatal tissue, c-fos expression could be observed in all tissues examined with more potent hybridization to fos-specific bone probes.

Hybridization showed that for c-abl, approx. three times higher levels in the embryo itself than in extraembryonic membranes and placenta after the tenth day of fertilization compared to the concentration observed in the 6, 7 and 9 day old concepti. Expression of c-abl in the fetus is found to decrease after the 11th day of prenatal development. The oncogene c-abl is transcriptionally active in all postnatal mouse tissues examined, with spleen and thy-15 mouse poly (A +) RNA exhibiting slightly stronger hybridization than from other tissues.

The oncogene c-rasHa was found to be expressed at significant but similar levels at all stages of prenatal development, both in the embryo itself and in extraembryonic tissues. High levels of c-rasHa expression were also observed in 20 different tissues of newborn or 10-day-old mice, especially 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).

Oncogenic c-erb had maximum hybridization at day 13, 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, maximum at day 15, and gradually decreasing thereafter. For oncogenic c-sis, maximum expression was observed at days 7 and 16, with day 7 peak being 30 1.5 to 3 times higher than all other days, and day 16 peak being 1.5 to 2 times higher than at days 9-13 and days 17 and 18.

21 DK 174458 B1

In the next study, the nucleotide sequence from the putative oncogene region of the Avian myeloblastosis virus myb was used (Vister et al. (1982) above). Using the published nucleotide sequence, a number of antigenic oligopeptide sequences were derived and 7 of the polypeptides thus derived were synthesized and evaluated as potentially antigenic.

These 7 oligopeptides, also listed above as antigenic oligopeptides, have the following formulas: (1) pro-phe-his-lys-asp-gln-thr-phe-glu-tyr-arg-lys (2) pro-ser-pro-pro-val-asp-his-gly-cys-leu-pro-glu-glu-ser-ala-ser-pro-ala-arg (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) with-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-glu-tyr-arg- light-met-his-gly-gly-ala-val

The polypeptides were attached to elbow peel hemocyanin according to conventional technique (Dockray, Regulatory Peptides (1980) 1: 169), and the immunogen formed was used to immunize rabbits by a first injection with Freund's complete adjuvant, followed by 20 Freund's injections. incomplete adjuvant for periods of 3 - 4 weeks to hyper-immunize the rabbits. The rabbits were bloodstained repeatedly for a period of 6 months. Of the 7 oligopeptides that resulted in antibody production, antibodies to two peptides (5 and 7) were selected for detailed analysis. The antibodies were reacted with radiolabeled cell lysates from a cell line 25 containing multiple copies of the Avian myeloblastosis virus and with lysates from appropriately uninfected cell lines. Antibody # 5 identifies a specific protein of approximately 58,000 daltons present in the virus-infected cell line, but not in the controls.

Antibody to polypeptide 5 was also reacted with plasma from chickens which had tumors induced by amv. A band similar to that observed with the above lysates of approximately 48,000 daltons was identified.

22 DK 174458 B1

Antisera to polypeptide # 5 were also reacted with lysates of a myeloid human leukemia cell line (HL-60) known to express mRNA transcripts of the c-myb gene (Gallo and Wong-Staal, Blood (1982) 60): 545). This antibody reacted with a protein of approx. 90,000 daltons. In freshly isolated myeloid century chemistry cells, the antibodies identify a series of proteins, 14 kd to 70 kd, that are not found in normal white blood cells.

The polyclonal antibodies to fragment # 5 of the myb protein were tested for their ability to kill normal and leukemic cells. The procedure used is described in Tarasaki and McClelland, below. The data are listed in the following Table 5.

DK 174458 B1

CM T- 00 CO CM CM CM CM

_J _l <

T- T- 00 (0) O M- t (N CM

ω _I * g>

4 S.J

5 r7 ^ T- COCOOtT-rr- 7 <'dl -t; Q)

ΰ E

zi ®

LU CL

O CM T- ΟΟτ-τ-τ— τ-τ- · <- E <

LU g, S O Z

Z U ® = ¥ <8 <ε

2 U- * "S JQ

51 I

£ | o2 f ω ° 2 S - CO (D T- T- T- T- T- - ° §> = P <CO ^ O x ^ $ * I. T? (Λ UL Tf Φ

O O XJ

rc I— · Λ · ^> co in Z is L- fr Q Η Ί- CO 00 CD CD ^ CM CM g Φ z 5 σ>.

<C · .9

CM _ Φ .C

m jd "oj> - - o) -5 - 2 ® S ^ _ ro 5 t ° g -σ ί?« «<co ro ® -C -E u ll ό φ ^ c Φ - <t = ^ * ro 11 o υ 5 S £ LU £ cd * - (χ) τ-ν-τ-Γ-τ-χ-Φ5.ί _ E ro 52

t i «8« ES

o z E ^ ESO

οσ> | S E ^ -S ^ f P s _ φ ex φ. <? o j - 21., **, «>! *» - £? δ f ^^^^ s®s | g S! || SSSigii-? σΉΏ = φΓ3ομ c ^ '[= <5 ro ro i ro ro 1 ro c o »w i ίο i i j a s li. i f f s | «<sx << k 3 = i! 9« ° -i $ ii g h- <2 i <0. S ° Li_ Ϊ + t 24 DK 174458 B1

The above results show that the expression product of the myib gene can react with antibodies to produce lysis with complement. Thus, it is believed that the myb protein is a surface membrane protein available for binding to antibodies. By identifying proteins to which specific antibodies will bind, which proteins have diagnostic value as indicative of malignancy, the malignant cells can be identified and treated. In the work described herein, no determination has been made as to the specificity or cross-reactivity of antibodies. Since only one fragment was used to produce the antibodies, one would expect that antibodies with greater binding specificity and greater binding ability could be produced with the whole protein, especially with the whole protein in a membrane. The antibodies can be used to select for antibodies that bind to the same or different determinant site.

The data presented demonstrate that antibodies that do not affect normal B and T cells can be produced but are cytotoxic in combination with a complement of a number of different malignant cells. Therefore, the antibodies can be used for cancer therapy without the risk of substantially inactivating the immune system.

It is obvious from the above results that the presence of malignancy in human hosts can be detected by determining the transcription and / or expression products of the oncogene. One can screen retroviruses or another source of nucleic acids for transforming vertebrates into malignancy. These nucleic acids can then be used to derive the peptide composition and screen malignant cells for transcripts or peptides by hybridization in the former case and with appropriate receptors in the latter case, using any of a wide variety of diagnostic assays. Antibodies to the peptides can be prepared, which antibodies can be labeled and then used to diagnose the presence of a peptide diagnostic for malignancy. The oncogenic proteins have been found to be available for binding to antibodies such as surface membrane proteins. The antibodies can serve as diagnostic reagents to determine the presence of malignancy and determine the position of malignant cells. The antibodies may also serve to treat tumors in vivo using radionuclides, toxins, in combination with the host's complement system or opsonins, or other antibody-dependent lytic system or the like. The antibodies are used in pre- and post-operative systems, in the latter to determine whether complete removal has occurred or whether metastases are present. The antibodies can be used post-operatively to destroy any remnants of the tumor that may not have been excised.

5

Claims (3)

1, A process for preparing an antigenic oligopeptide containing up to 50 amino acid residues, preferably up to 25 amino acid residues, and containing one or more of the following amino acid sequences: 5 (a) pro-gln-glucose-ser-lys-ala-gly -pro-pro-ser-gly-thr-thr-gly, (b) with-ala-phe-ala-his-asn-pro-pro-ala-gly-pro-leu-pro-gly-ala, (c ) pro-pro-val-asp-his-gly-cys-leu-pro-glu-glu-ser-aia-ser-pro-ala, (d) pro-phe-his-lys-asp-gln-thr- phe-thr-glu-tyr-arg-lys-with-his-gly-gly-ala-val, (e) pro-phe-his-lys-asp-gln-thr-phe-thr-glu-tyr-arg -lys-with, (f) asp-asn-thr-arg-thr-ser-gly-asp-asn-ala-pro-val-ser-cys-leu-gly-glu, (g) pro-ser- pro-pro-val-asp-his-gly-cys-leu-pro-glu-glu-ser-ala-ser-pro-ala-arg, (h) arg-leu-ileu-glu-asp-asn-glu -tyr-thr-ala-arg-gln-gly-ala-lys-phe-pro, (i) asn-arg-glu-val-leu-asp-gln-val-glu-arg-gly-tyr-arg- met-pro, (j) arg-leu-lys-lys-ileu-ser-lys-glu-glu-lys-thr-pro-gly-cys-val-lys-ileu-lys-lys, 15 (k) asp -leu pro-ser-arg-thr-val-asp-thr-lys-gln-ala-gln -glu-leu-ala-arg, (1) with-thr-glu-tyr-lys-leu-val-val-gly-ala-ser-gly-val-gly-lys-ser-ala, (m) glu -asp-ileu-his-gln-tyr-arg-glu-gln-ileu-lys-arg-val-lys-asp-ser-asp-asp, (n) val-arg-glu-ileu-arg-gln- his-lys-leu-arg-lys-leu-asn-pro-pro-asp-glu-ser-gly-pro, (o) with-thr-glu-tyr-lys-leu-val-val-val-val gly-ala-arg-gly-val-gly-lys-ser-ala, (p) with-thr-glu-tyr-Jys-leu-vaJ-val-val-gly-ala-val-gJy'val-g ) y-lys * ser-a) a, (q) met-thr-glu-tyr-lys-leu-val-val-val-gly-ala-gly-gly-val-gly-lys-ser-ala, (r) val-asp-glu-tyr-asp-pro-thr-ileu-glu-asp-ser-tyr-arg-lys-gln-val, (s) arg-his-ser-thr-ser-ser ser-glu-gln-glu-arg-glu-gly-gly-arg, (t) ser-arg-glu-ala-ala-asp-gly, (u) asn-gln-thr-arg-glu-phe -val-glu-lys-gly-gly-arg, (v) pro-glu-val-gln-lys-pro-leu-his-glu-gln, (w) phe-leu-arg-thr-glu-gly -ala-ala-ala-aspen-glu-aspen-ala-ala-ala, DK 174458 B1 (x) phe-leu-arg-thr-glu-gly-ala-arg-leu-arg-with-lys-thr -leu-leu, (y) ser-ala-pro-arg-ser-ser-pro-ser-thr-ser-ser-trp-ser-ser, (z) asp-gln-gln-arg -leu-gly-arg-arg-thr-arg-arg-arg-val-arg-lys, (aa) glu-arg-gln-glu-ala-glu-arg-his-asn-val-leu-glu- arg-gln-arg-arg-asn-glu, (bb) arg-leu-ileu-ala-glu-lys-glu-gln-leu-arg-arg-arg-arg-glu-gln, (cc) arg -lys-lys-asn-lys-lys-lys-ser-glu-glu-ileu asp, (dd) pro-pro-thr-thr-ser-ser-asp-ser-glu-glu-glu-gln- glu, (ee) arg-thr-leu-asp-ser-glu-glu-asn-asp-lys-arg-arg, (ff) glu-arg-gln-arg-arg-asn-glu-leu-lys- leu-arg, 10 (99) glu-gln-leu-arg-arg-arg-arg-glu-gln-leu-lys, (hh) asn-asn-glu-lys-ala-pro-lys-val-val-val , (ii) arg-arg-glu-gln-leu-lys-his, (jj) lys-glu-lys-glu-lys-leu-glu-phe-ileu-leu, (kk) pro-glu-glu- glu-lys-arg-arg-ileu-arg-arg-glu-arg, (II) leu-gln-glu-ser-ser-lys-ala-ser-gln-pro-ala-val-ala-thr. ser, (mm) asn-pro-glu-val-lys-lys-thr-ser-trp-thr-glu-glu-glu-asp-arg, (nn) try-arg-arg-asp ~ pro-glu- glu-arg-pro-thr, and (oo) ala-ser-pro-tyr-pro-asn-leu-ser-asn-gln-thr-arg, characterized in that the amino acids are sequentially coupled to obtain oligo-20 the peptide, on a suitable berry rer. Process according to claim 1, characterized in that the carrier is a polystyrene resin. Process according to claim 2, characterized in that the polystyrene resin support is selected from chloromethylated resins, hydroxymethyl resins and benzhydrylamine resins.