CA1108988A - Viral related protein assay for detection of cancer - Google Patents

Abstract

Viral Related Protein Assay For Detection of Cancer Abstract The existence and status of cancers in humans can be de-tected by assaying for viral related proteins in plasma samples.
Suitable viral related proteins include enzyme RNA-dependent DNA polymerase (reverse transcriptase) or an extracellular tumor associated protein which is of viral origin. The afore-said enzyme and tumor associated protein are immunologically cross reactive with antibodies to Mason-Pfizer Monkey Virus (MPMV) which thereby provides a convenient source of reagents for the instant method.

Description

Background of the Invention Studies with the murine mammary tumor model have established the ~easibility of using plasma-concentrations of viral protein to assess the presence and status of solid tumor7 The viral protein utilized for such studies was a 52,000 dalton glycopro-tein (gp 52) isolated from the murine mammary tumor virus (MMTV) using affinity chromatography. ~vailability of purified gp 52 by the a~oresaid procedure allowea development of ~ radioimmuno-assay to this protein sensitive to plasma levels down to 0.1 ng/
100 ~1. See in regard to the above Ritzi et al., Virology, 75, 188 (1976) and Ritzi et al., Proc. Natl. Acad. Sci. USA, 73, No. 11,4190 (1976) The relationship between the mammary tumors and the plasma levels of gp 52 were found to be as follows:
(a) tumor-bearing mice, male or female, showed markedly elevated (100-1000 ng/ml) levels of gp 52 as a free soluble protein in the plasma and the mean concentration increased with average tumor siz~;

- ., ... . . . .

.
~. ;~ ' ' (b) the presence of another malignanc~ (leukernia) did not result in any change o~ gp 52 le~els in the plasma;
(cl mammary tumor tissue located ~y transplantation outside the mammary g~ is also detected by highlplasma gp 52 leveIs;
(d) low (2-10 ng/ml) plasma levels of gp 52 are found in tumor free mice whether they orginate from strains characterized by high or low frequencies of spontaneous mammary tumors;
(e) tumor-free lactating females exhibit the normally low level~ of plasma gp 52 despite the fact that their milk contains an average of 20,000 ng/ml of this protein; and (f) the circulatory clearance time of gp 52 in tumorous animals is sufficiently rapid (a half-life of 4-6 hr.) to suggest a requirement for continued replenishment to maintain the high levels observed.

The MMTV model provides the basis for establishing the feasibility of utlizing a viral protein "marker" in plasma for monitoring the presence and status of human breast cancer.
The u~e of a viral protein marker would be distinguishable from assays, such as disclosed in U.S. Patent 3,999,944, which rely on detecting antigen induced leukocyte adherence inhibition caused by tumor specific cell mediated immunity.

Description of the Invention ; The present invention relates to a method ~or detecting the presence and status o~ cancer in humans by assaying ~or certain tumor specific viral related proteins in plasma samples and novel reagents use~ul therein. Such method is thus useful in diagnosis as in initial screening programs ~or early detection of the disease, in therapy as in evaluating the status of the disease after surgical, radiation and/or chemothe~apeutic

- 2 -:

L.~ 8~88 treatment ~nd in pxogno5is such as ~n det~cting the poss~bility of xecurrence or metast~ses, Viral related proteins which can be employed as markers for the detect~on of cancer, particularly breast cancer, include viral enzymes such as RNA-dependent DNA polymerase (reverse transcriptase) or alternatively a tumor associated protein which is of viral oriyin.
A first aspect of the present invention therefore is lQ directed to the detection of human breast cancer by utilizing RNA-dependent DNA polymerase as the plasma marker.

It has been previously known in the art that human breast tumor particles possessmany of the features characteristic of RNA tumor viruses. In addition to the expected size (600S) and density (1.16g/ml), these features include possession of an outer membrane and an inner one surrounding a "core" containing a DNA polymerase and a large molecular weight (70S) RNA possess-ing detectable homology to the RNA's of the mouse marMnary tumor virus (MMTV) and of the Mason-Pfizer Monkey Virus (MPMV).

The purification and characterization of the DNA polymerase from the human breast cancer particles has now been accomplished and forms a part of the present invention. Key properties of this enzyme are very similar to those of the reverse transcriptases found in MMTV and MPMV. Thus, like these viral enzymes, the purified human breast cancer DNA polymerase exhibits the following three features that together di~tinguish the known viral reverse transcriptases from normal cellular DNA
polymerases: a) a strong preference for oligo(dT):poly(rA) over oligo(dT~:poly(dA) as a template for the synthesis of poly(dT);
b) the acceptance of the highly specific ol~go(dG):poly(rCm) as a template for the formation o~ poly~dG); c) the abilîty to , use a viral ~NA (AMV~ as a template to ~ash~on a faith~ul DNA
cornplementary cop~. The re~emblance o~ the human enzyme to the reverse transcriptases of MMTV and MPMV extends further in its possessing a molecular weight of 70,000 daltons and in its pre~erence for Mg~ over Mn~. To date, an enzyme with these properties has not been detected in normal breast tissues or in benign tumors of the breast.

Isolation of human breast cancer DNA polymerase was accomplished by homogenizing breast cancer tissue and layering over discontinuous sucrose gradients~ The density region at 1.16-l.l9g/cc was pooled, diluted and centrifuged. Suspension of the pellets were fractionated by polyacrylamide agarose gel filtration. Fractions found active by DNA polymerase assay were pooled and chromatographed over a phosphocellulose column.
Elution with a linear gradient of a O.lM to 0.5M phosphate buffer pH 7.2. The main peak o~ polymerase activity was pooled and the enzyme concentrated by dialysis.

The purified human breast cancer DNA-polymerase can be utilized to develop a diagnostic assay for human breast cancer in a number of ways. It can be used to elicit human breast can-cer DNA-polmerase specific antibodies by injecting the purified enæyme preferably in an emulsion of complete Freund's adjuvant into a suitable host animal such as a rabbit, guinea pig, goat, horse, etc. over a period of time and then bleeding the host (usually after booster injections have been given) ko yield the desired antisera.

Additionally, the purified enzyme can be employed as a ~` substrate for radio-iodination to yield the [125~ enzyme. A
suita~le procedure for radioiodination ~nvolves treating the enzyme with [125~ -3(4-hydroxphenyl) propionic acid N-~r , , hydrox~succini~ide ester ~Bolton~Hunter reagentl ~ollowed by puri~ication over a G-100 column.
The a~oresaid ant~body and labelled enzyme can be utilized in a rad~oimmunoassay ~or human breast cancer DNA-polymerase in~human plasma samples. Suitable radioi~nunoassay procedures are known in the art. Thus, for example, an analogous procedure which can be employed is described by Ritzi et al,, Virology 75, 188 (1976). In such a procedure a buffered sample was treated with the antisera, i.e., rabbit anti-DNA
polymerase and then after incubation for about 45 minutes at 37C
the rr25~ -labelled enzyme was added. The sample was incubated for a further two hours at 37C and the bound radioactivity was separated from the free by the addition o normal IgG (rabbit) and a sufficient amount of second antibody (goat anti-rabbit IgG) to yield optimal preciptation of the IgG (rabbit).

The concentration of D~A-polymerase in the sample can be determined by comparing the counts of radioactivity observed in the bound and/or free fractions to a standard curve obtained by utilizing different known amount of DNA-polymerase in the same assay procedure.

In an alte~nate procedure, the enzyme concentration in plasma samples can be determined by isolating the enzyme and measuring for enzymatic activity~ Isolation can be readily accomplished by ammoni.um sulphate treatment o~ the plasma sample to precipitate the enzyme ~ollowed by affinity chromatography ~he reconstituted precipitate through a column of SEPHAROSE bead to which a synthetic template for the enzyme had been covalently bonded. Suitable templates ~or this purpose include polyriboadenylate (poly(rA) or poly (2'-O-methylcytidylate) (poly(rCm)~ Elut~on o~ the enzyme from the column is accomplished using a gradient of 0.01 M KCl to 1.0 M KCl in * Trademark .OlM phosphate bu~fered at pH 7.2. The D~A-polymerase activity of the isolated enzyme can be assayed using the same assay procedures employed in the following the purification of the enzyme from breast cancer tissue discussed previously.

A further embodiment of the method of the present invention relates to the discovery of a human tumor associated protein which is of viral origin. One such protein can be demonstrated in substantial concentration in the intercellular spaces of human breast cancer tissue specimens and also circulating in the plasma of breast cancer patients. Tumor associated protein appears to be excess protein produced either by the virus after it has infected the breast cell or by the cell itself under viral control. It is therefore a further aspect of this invention to assay fox the presence of this tumor associated protein in plasma samples as a diagnostic and prognostic test for cancer, such as, breast cancer.

The isolation of tumor associated protein from homogenized cancer tissue can be carried out by a combination of affinity chromatography and column chromatography. The affinity column comprises Concavalin A* coupled to Sepharose 4B* and is an article of commerce (Con A Sepharose). Elution of the protein from the affinity column is accomplished using buffered O~methyl-D-manno-side solution. Additional purification of the protein is accomplished by DEAE cellulose chromatography. The aforesaid procedures are directly analogous to the procedures employed by Ri~zi et al., Virology 75, 188 (1976) for purification of gp 52 from MMTV and are descr;bed in greater detail therein.

* Trademark --6w The purified tumor associated protein o~ viral vrigin Gan be utilized in the same manner as described prev~ously for DNA-poly-merase for the development of a radioimmunoassay useful in the detection of breast cancer. Thus, the protein can be injected into host animals in a known manner to elicit antibodies specific to the tumor associated protein of viral origin.
Moreover, the purified tumor associated protein can be radio-labelled, preferably radioiodinated with 125I Bol~on-Hunter reagent to yield the labelled protein, i.e., 125I-tumor associated protein r used as the marker in such radioimmunoassay. The radioimmunoassay procedure used in this aspect of the invention is not narrowly critical and any conventional technique can be employed. Preferably, the assay procedure employed will be the blocking double antibody procedure used for the radioimmuno-assay of DNA~polymerase, described previously above.
In a further aspect of the present invention, it has now been discovered that Mason-Pfizer Monkey Virus (MPMV) specific antibodies cross-react at reasonably high levels with both human breast cancer DNA-polymerase and human breast cancer viral origin tumor associated protein. It is thus possible to utilize such antibodies and radiolabelled antigen, preferably 125I-MPMV, in the human breast cancer radioimmunoassays of this invention.
Since MPMV can be grown in tissue culture and thus is readily available in substantial amounts, this provides an especially convenient source of reagents for the wide~scale application of this invention.
Thus the present invention provides a human breast cancer specific viral related protein essentially free ~rom other viral and breast tissue components, said protein selected from the group consisting of human breast cancer RNA-dependent DNA
polymerase and human breast cancer viral origin tumor associated prote~n.

In one aspect the in~ention pro~ides human breast cancer RN~-dependent DNA polymerase essentially ~ree ~r~ o~her viral and breast tissue components. In another embodiment the inven-tion provides 125I~human breast cancer RNA dependent DNA poly-merase~ In another embod~ment the inVention prov;des human breast cancer viral orig;n tumor associated prote~n essentially free from other viral and breast tissue components. In another embodiment the invention provides 125I-human breast cancer vira]
origin tumor associated protein. In another embodiment the invention provides an antibody specific to human breast cancer RNA-dependent DNA polymexase. In anotner embodiment the inven- -tion provides an antibody specific to human breast cancer viral origin tumor associated protein.
In yet another aspect of the invention a suitable en-zyme is covalently bound to the desired antibody. The antibody-enzyme complex is still enzymatically active and when placed in contact with tissue specimens that contain the proper antigen the antihody combines with it. A substrate of the en~yme is then added which results in the release of a colored precipitate at the site of activity. In a specific embodiment peroxidase was coupled to antibodies against Mason-Pfizer viral proteins.
The appearance and distribution of the colored product enable not only the identification of the presence of the antigens, but to actually localize it in the malignant cells in the tissue specimens.

Another method employing this principle involves the quantitative estimate Oæ the same antigens in the body Eluids (e.g., plasma), The procedure is as Eollows: 1) coat the antibody on a sol~d sur;Eace ~e.y., polystyrene~; 2) add a known volume o~ plasma fxom a patient to the tube and allow the antibody coated on the surface to pick up any of the relevant antigens present in the sample; 3~ the sample is then removed and the ~! .

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tube washed; 4) add the antibody~enz~me complex and incubate for attachment; any unabsorbed enzyme-l~nked antihody i5 then washed out; and 5) add the chromogenic su~strate which gives either ~he color or the fluorescence which can be measured to estimate the amount of tumor antigen present. Preferred enzymes which can be employed lnclude alkal~ne-phosphatase and ~-galactos~dase, both of which have excellent chromogenic substrates Further details relating to procedures useful in the practice of this aspect of the invention are available in the prior art. See for example U.S. Patent Nos. 4,002,532 and

4,016,0~3.

The several assays which form the method aspects of this invention may be utilized to detect the presence of breast cancer in humans. To effectuate such use a statistically significant number of blood plasma samples from clinically established breast cancer patients, from normal patients, and~
from patients with benign or non-breast cancer tumors are assayed by either the direct DNA-polymerase activity method, the DNA-polymerase immunoassay, or the tumor associated protein immunoassay. The concentration of marker protein found in these assays is markedly elevated in the case of the breast cancer plasma samples when compared to the levels found for the normal and non-breast cancer tumor samples. It is thus possible to draw an arbitrary control level line between concentration levels of marker protein in each assay method which corresponds to the presence of breast aancer and levels which correspond to normal or non-breast cancer skates. An unknown plasma sample can then be evaluated for the possibility of breast cancer in the subject by as~aying the sample in accord-~ccordance with one of ~ methods of the pres~lt invention and deten~ng whether the marker protein is present in a concentrat~on in .
~.
_ g _ excess of the control level.

Alternatively, a patient's own levels o~ marker protein can serve as an internal control~ Thus a patient with a confirmed breast cancer can be assayed before and after the initiat~on of therapy. A marked drop in the level of the marker protein would be ;ndicative o~ a ~avorable prognosis of the kreat-ment. A subsequent substantial ~ncrease in the marker protein concentration levels would be ~ndicative of a possible recurrence or metastases of the disease and would allow the attending physician to initiate therapy at an early time.
Moreover, the effectiveness of such therapy could be monitored by the instant method.

The present invention is furthex illustrated by reference to the Examples which follow.

Example 1 Subcellular Fractionation of Breast Tumor Tissue Depending of the amounts of material available, between 9 and 30 g of tumor were thawed, minced, suspended in four volumes of cold 5% sucrose (w/v)-TNE (O.OlM Tris-HCl, pH 8.0, 0.15 M
NaCl, 3mM EDTA) and blended in a Silverson homogenizer. The homogenate was centrifuged at 4000 X g and then 10,000 X g to remove nuclei and mitochondria, respectively. Trypsin was added to the post-mitoahondrial supernatent to a final concentration of 0.5 mg/ml 7 AEter incubation at 20 for 10 min, proteolytic activity was inhibited by the addition o~ two poly-peptides, lima bean trypsin inhibitor (one-fold excess) and Trasylol*(100 KIU/ml), The sample was layered over discontinuous sucrose gradients composed o 6 ml of 50~ sUcrose -TNE and 8 ml of 25% sucrose-TNE. Following centrifugat~on at 25,000 rpm for 90 min at 4~ in a Spinco S~-27* rotor, material ~t the 25/50 *Trademark - 10 ~

interface was collected, diluted with TNE, and la~ered over line~r 20-50% sucrose-TNE ~rad~ents~ The samples were centr~
fuged as above for 16 hr and the di~ferent density regions collected. The density region ~1.16-1.19 g~cc) in which RNA tumor viruses localtze was pooled, diluted,and centrifuged as above for 90 min. The result~ng pellets were resuspended in approximately 0.6 ml of 0Ol M Tris-HCl, pH 8Ø

Six lots of tumors were processed for enzyme in the manner described. Four of these ~A, B, C, and D) yielded enough enzyme to characterize. One preparation (A), a metastatic liver tumor,c~me from a single patient, all the others being pooled material from a number of different individuals.

Pol~acrylamide Agarose Gel Filtration _ The resuspended pellet was solubilized and disrupted at 0 for 15 min by the addition of KCl (to 0.4M), DTT ~dithiothreitol, to 0.0I M), and a non-ionic detergent such as Triton X-100 ~to 0.6%~. The sample, approximately 0.9 ml, was applied to a 0.9 X
50 cm column of polyacrylamide agarose gel (Ultrogel*AcA44) equilibrated with 0.3 M potassium phosphate, pH 8.0, in buffer A
(2 mM DTT, 1 mM EDTA, 0.02% Triton X-100, and 10% glycerol).
Elution was with 0.3 M phosphate~ufer A at a flow rate of about 2 ml/hr. Fractions (0~5 ml) were assayed for DNA polymerase and terminal transferase activity as described below.

Phosphocellulose Chromatography The peak fractions from the Ultrogeloolumn were pooled (3 ml) and Trasylol was added to a concentration of 100 KIU/ml.
The sample was dialyzed against 0.01 M potassium phosphate, . pH 7.2, in bu~er A until the phosphate concentration was less than 0.02 M and then was loaded onto a 0.9 X 10 cm phosphocel-lulose column ~Whatman* P-ll) e~uilibrated with the same buffer.
.^0 *Tra~rk q~he colum, was washed with 30 ml o~ the O.Ol M phosphate buffer and the enzyme activi~y was eluted with a 120 ml linear gradient of O~Ol M to U.5 M pot~ssium phosphate buf~er A, pH 7.2, at a flow rate of l4~ml/hr. Fractions (1.2ml) ~ere assayed for both DN~ pol~merase and terminal transferase activities, The ma~n peak of polymerase activity was pooled and Trasylol was added to lO0 KIU~ml, This enzyme ~raction (called PC enzyme) was concentrated by dialysis at 0~ ag~inst an osmotically active, high molecular weight synthetic polymer such as ~quacide*ll-A, Glycerol Gradient Centrifugation For estimation of molecular weight, the concentrated PC enzyme was diluted three-fold with O.l M potassium phosphate, pH 8.0, and layered on a linear 10-30% glycerol gradient containing Ool M potassium phosphate, pH 8.0, 2 mM DTT, and 0.02% Triton X-lO0. Centrifugation was at 48,000 rpm fox 12 hr at 1 in a Spinco SW-50.l rotor. Fractions were collected from the bottom and assayed for reverse transcriptase activity with oligo(dG)~poly(,rC) as template. Bovine serum albumin served as a sedimentation marker in a parallel gradient.

' DNA PoIymerase Assays Assay mixtures for polymerase activity with synthetic polymer templates contained in ~lO0 ~ 5 ~ mol Tr~s-HCl, p~ 8.0, O.S ~mol MgCl2l O~l ~mol DTT, and the following combinations of polymer and dNTP~-04. ~g oligo(dG):
poly~rC) or oligo(dG):poly(rCm), 0.02~mol dCTP and l.0 nmol [ H] dGTP (4000 cpm/pmoll; 0.4~g oligo(dT):po1y(rA) or oligo(dT~:pol~(,dAl~ 0.02~ mol dATP and l.0 mmol [3H~ dTTP
(,4000 cpm~pmoll. In reactions w~th oligo(dG):poly(rCm), MnC12 (0'~02 ~moll replaced MgC12.

*Trademark - l2 A5sa~s emplo~in~ AMY RNA cont~ined (.in 100 ~ 5 pmol Tris-HClr pH 8~0~ 0~8 ~ol MgC12, U,l ~mol DTT~ lQ/~y actinomycin D (Sigma co~p.), 5 ~g distamycin ~ ~C~lb~ochem) 2 ~g AMV 70S' RN~, 0.1 ~g oligo (dT~ 12 1~' O.li~mol each o~ dATP, dGTP and dTTP, and 5 nmol [3H] d~TP ~1.5 X 1~4 cpm~pmol).

All reactions were incubated at 36v for 15-30 min and were terminated by the addition of 0~5 ml cold 0.067 M sodium pyrophosphate - 1 M sodium phosphate, pH 7.2, followed by 0.5 ml cold 80~ TCA. Acid-insoluble radio activity was collected on membrane filters and measured in a scintillation counter.

Terminal deoxynucleotidyl transferase activity was measured by the polymerization of [3H] dGTP in the absence of a complemen-tary polymer template. Reactions were carried out as described above except that polymer dNTP combination was replaced with 0.4 ~g oligo(dG)10 18 plus 0.02 ~mol dC~P and 1.0 nmol [3H] dGTP.

, All synthetic oligo- and polynucleotides tritiated dGTP, dTTP, and dCTP were articles of commerce. AMV 70S RNA was isolated from the purified virus as described by Marcus et al.
Virology 71, 242 (1976).

Hybridization Re_ctions Procedures for the hybridization reac~ion~ and their analysis with S nuclease have been reported by Weiss et al. supra.
Conditions for Cs2SO4 equilibrium density centrifugation as disclosed by Axel et al., Natuxe 235, 32 (1972) were modified by the addition of 0.02~ sodium N-lauroyl sarcosinate and ~0 ~g each of E. coli DNA and RNA to the gradients.

esults The data described below are based on independent enzyme . ~ .

isol~tions ~Xom ~ouX dif~e~en~ tu,m,or collecti.on~, ~abeled A
through D~.~ The beh.~ior du~ng ~xactionati.on and the properties of the breast tumor pol~m,e~se di.d not vary sign~ficantly from one prepaxation to another~
Polymerase preparation A was ~solated ~rom a metastatic les~on in the liver of a patient with breast cancer. Nine grams of tumox were homogenized and the particulate material, bending at a density of 1.16-1.19 g/ml, was collected as describe~above. The recovered pellet was solubilized by the addition of Triton X-100 and then fractionated through a polyacrylamide~agarose gel ~Ultrogen ACA-443 column. Each column ~raction was assayed for 1) DNA polyermase with oligo (dG)12 18poly(rC) as a template and 2) for terminal transferase, using oligo(.dG)12-18 as a primer. Three peaks (two major and one minor) of DNA polymerase activity were observed and it was evident that the first major peak also contained terminal transferase. The two major peaks were found (Table 1) to contain 90~ of the applied polymerase activity and 3~ of the protein as measured by the fluorescamine procedure of Udenfriend et al., Science 178, 871 (1972).

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To examine the reality of the separation of the pol~m~se activities observed abwe, the two major pe~ks were pooled, dialy ~ to reduce the phosphate buffer concentration, and then chromatographed through a phosphocellulose column(PC) with a linear (0.01 M to 0.5 M) phosphate gradient. me polymerase activities were seen to again resolve into two major peaks one eluting at 0.08 M phosphate and the other at 0.18 M. It will be noted that again the second major polymerase peak i5 devoid of terminal transferase activity. The latter splits into two peaks, one associated with the first DNA polymer~se activity at 0.08 M phosphate, and another eluting by itself at 0.23 M
phosphate.
The second (O.l~M phosphate~ peak of polymerase activity observed on the PC column is not found in normal tissues (3 samples of breast tissue and 3 samples from spleens) or in `' benign ~ibroadenomas of the breast (3 pools o~ 3-4 fibn~e~
each). and is thus the DNA polymerase unique to breast cancer tissueO The fractions composing peak ~ of the PC column are found to contain 65% of the applied DNA polymerase activity and 8% of the protein. These fractions are pooled and concentrated as described above to yield the breast tumor polymerase. Table 1 summarizes the yields of activity and protein at each of the three steps in a typical purification.

Example 2 Evidence that the Breast Cancer Polymeras~ is a Reverse : Transcri~tase There are several useul criteria which distinguish the reverse transcriptases of the RNA tumor viruses from normal `. mammalian DNA polymerases. The viral reverse transcriptases show a pre~erence for oligo~dT):poly~rA) over oligo(d~
` poly(dA) and they al~o accept oligo~dG):poly(rC) and oliyo~dG):
poly(rCm) as excellent templates or the synthesis of poly(dG~.
Another, and more diagno~tic characterlstic, is the ability , ..

'il~8~38 o~ a reVerse t~nscriptase to use ~ heteropolymeriC RNA to direct the synthesis of a faithful co~ple~entary DNA as demonstrated by proper back-hybridization of the cDNA to the template used in the synthesis.

The responses of four of the breast cancer polymerases to the synthetic polyribonucleoti~es are summariæed in Table 2.
The results show a pattern of activities completely consistent with that obtained with reverse transcriptases isolated from authentic animal RNA tumor viruses. Thus, ln all cases, oligo (dT~:poly(rA~ is superior to oliyo(dT~:poly~dA) for the synthesis of poly(dT). Further, both oligo~dG):poly~rC~ and oligotdG):
poly(rCn) were excellent templates for the formation of poly~dG). It should be noted that in addition to the four breast tumor enzyme preparations descri~ed here, many others : (more than 50) obtained from additional patients in an ongoing effort have been examined in the same way at different stages of purity using varlous methods of fractionation and they all exhibited the response pattern described in Table 2, ,,~
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The operational definition of a reverse t~anscriptase requIres the demonstxation that it can use a heteropolymeric RNA to make a DN~ transcript. lhe response ~Table 3) o~ the breast cancer polymerase to the RNA of the avian myelo-blastosis virus (AMV) is that expected ~rom the synthesis of a heteropolymer~c DNA. Leaving out any one, or all of the required three unlabeled deoxyribosidetrisphosphates leads to the same virtual disappearance of synthetic activity as occurs on omission of the RNA template.

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The most telling test of a putatiVe reVerse transcriptase reaction comes from an exam~nat~on of the f~delity of the DNA
transcript, This requires ~solation of the I 3H3 DNA product and challenging it in anneal;ng react~on with the ~NA template used ~n the synthesis. To thiS end, a 2-ml reaction was run for 15 min with the DNA polymerase preparation A and AMV-RNA
as the template, leading to the synthesis of approx;matelY
3 ng ~3~ DNA at ~ X 107 cpm~g. The [3~] DNA product was purified and recovered by the usual procedures and then annealed with AMV and RLV 70S RNAs. The outcome was examined ~y separation in Cs2SO4 gradients and by resistance to Sl nuclease.
Both methods yielded less than 5~ annealing to the unrelaked RLV-RNA and between 80 and 85% hydribidzation to AMV-RNA, the template used to direct the synthesis. These results suggest therefore that the [ ~] DNA is a singLe-stranded complement of the AMV-RNA. A more informative examination of the [3H]
DNA product is provided by a kinetic examination of the annealing reaction. A comparison was made of the annealing kinetics to AMV-RNA of two [3H] DNA products, one synthesized under the 2~ direction of AMV-RNA by AMV reverse transcriptase and the other synthesized by the human breast cancer polymerase instructed by the same template. It was found that the kinetics of annealing to AMV-RNA of the two DNA products are indistinguish-able. Thus, the human enzyme is every bit as efficient in reverse transcribing AMV-RNA as is the homologous reverse transcriptase purified rom the avian virus.

O ~39~93~ the Bxeast Cancer Pol~merase ~ariations in t~rature and pH were ~ned for their effects on ~he acti~ities of several preparations o~ the breast cancer polymerase using oligo~dG):poly(rCJ and oligo~dT):poly(rA) as templates.
The max~mal rate of polymerization occurred at 37 ;n 5 mM
~gCl2 over a pH range of 7.~ to 8.5.
X

The divalent ion requirements ~ reverse tr~nscriptases are of some interest since they ser~e,~o ~ de the,v~ruses o~
origin ~nto different groups. Thus~ all six stra~ns of MMTV
from a variety of sources contain a reverse transcriptase show-ing a strong preference for Mg~+ as compared with Mn . The same holds true ~or the Mason~P~izer monkey virus, the bromo-deoxyurid~ne-induced guinea pig v~rus and the bovine leukemia virus, In contrast, the reverse transcriptases of the murine leukem~a and sarcoma viruses function much more effectively in the presence of Mn~. For the human breast cancer enzyme, Mn at its optimum yields only about one seventh of the activity attainable with Mg~+. It is clear that as between the murine mammary tumor and leukemia viruses, the human breast cancer enzyme shows a divalent ion requirement most closely resembling the mammary tumor virus reverse transcriptases.
The molecular size of the breast cancer enzyme was estimated by sedimentation through a linear (10-30%) glycerol gradient. Ths enzyme was located by assaying fractions with ~MV-RNA as template. ~l~he enzyme activity sediments between 5S and 6S, slightly faster than the bovine serum albumin marker, placing the molecular weight at around 70,000 daltons. This result is also consistent with the relative elut~on positions of these same proteins on ultrogel. A
number of en~yme preparations from different breast tumor sources yielded identical sedimentation values.
'~

Materials and Methods Viruses Mason-Pfizer monkey virus was propagated in a __ suspension culture of the noxmal human lymphocytic cell line NC-37 and concentrated as previously descrlbed by Schlom and Spiegelm~nf Proc. Nat. Acad. Sci. U.S.A. 68, 1613 (1971). The ; virus was further puri~ied by centri~ugation through an 8-ml column of 20% glyercol in TN~ buf~er (0.01 M Tris-HCl, pH 8.3, , ~2 -0.15 M N~Cl, 0~002 EDTA~ onto ,a, pad o~ 100~ ylyercol at 98,000 X ~ for 60 min at 4, The viral pellet was taken up ~n TN~
buffer and spun to equil~brium ~n a continuous 20~50~ sucrose gradient ~n TNE at 98,000 X g for 16 hr. The particles banding between densIt~es of 1.14-1.19 g/ml were collected, diluted, and centr~fuged at 98,000 X g ~or 45 min at 4. The pellet was used ~mmediately for DNA polymerase purification.
Avian myeloblastos~s virus ~AMV, BAI straln-A), simian sarcoma virus stra~n-l (SSV-l), Friend leukemia virus (FLV), feline leukemia virus (FeLV), and Rauscher leukemia virus ~RLV) were also used in this Example. All viral concentrates were purified as described a~ove.

Preparation of RNA-lnstructed DNA Polymerase from Human Mali~nant Breast' Tumor , Reverse transcriptase from human malignant breast tumor was prepared as previously described by Ohno et al., Proc.
Natl. Acad. Sci. U.S.A. 74, 764 (1977) from particles purified by isopycnic separation. After disruption by incubation in 0.2% Triton X~100 for 15 min at 0, some of the samples were analyzed for endogenous polymerase activity as well as oligo(dG):
poly(rC) and oligo(dT):poly(rA) directed synthesis of poly(dG) and poly (rA), respectively. The disrupted virus density regions chromatographed on a polyacrylamide agarose column (Ultrogel AcA44, LKB,Co.) and the eluted enzyme peak loaded on a phos-phocellulose (Whatman pll) column. The enzyme was eluted with 0,01 to 0.5 M potassium phosphate gradient and concentrated as described by Ohno et al. supra.
' Preparatio'n o~ ~iral Re'gions ~om Human Leukem~a and Hodgkin's ''S'pl'e'en Spleens from patients with human chronic lymphocytic leukemia (CLL~ r chron~c myelogenous leukemia (CML), and Hodgkin~s lymphoma were use~ as the sources o~ viral density region preparations, and the polymerase act~ties were analyzed by endogenous k~netics as described by Witkins et al., - 23 ~

.
, :

Proc. Natl. Acad. Sci. U~s.A~ 72, 4133 ~lg75).

Preparation of MPMV Polymerase The MPMV pellet prepared as described above ~viruses) was resuspended onto 0.05 M Tris-HCl, pH 9.2, 0.001 M EDTA and Z
M KCl, son~cated, and then centrifuged at Y8,000 X g for l20 min. The pellet was used to prepare purified DN~ polymerase by column chromatography as described previous1y by Witkins et al~, supra.

Preparation of Antiserum Antiserum against MPMV-DNA was induced in New Zealand white rabbits. Three cycles of immunization were required to achieve the desired titer of anti-polymerase IgG. In each cycle the enæyme ~l X 103 pmoles of TMP incorporated per min) was emulsified with an equal ~ol of Freund's adjuvant and injected into ~he two hind footpads. This was followed by two additional similar inoculations given at two-week intervals in the same sites.

Sera were fractionated by chromatography on a Sephadex*
G-200, O.l M Tris-HCl, pH 8.0~ Rabbit gamma glohulins were identified serologically by immuno-diffusion with goat anti-rabblt IgG antiserum. The relevant fractions were concentrated by ammonium sulfate precipitation (50~ saturation) and dialyzed against O.l M Tris-HCl, pH 8O0~ The protein concentration of the ~gG fraction was measured by the Lowry procedure.

* Trademark .
. .

f~

DN~ Polymerase Assays Assay mixtures for polymerase act~ity ~7ith synthet~c polymer templates contained (in 100 ~1~: 5 ~mol Tris-HCl, pH 8.0, 0,5 ~mol MgC12, 0.1 ~mol DTT, and the ~ollowing com~lnations of polymer and dNTPs: 0.4 yg oligo~dGl:poly(rC~ or ol~go(dG~: poly(rCm), 0702 ~mol dCTP and 1.0 nmol [3H] dGTP ~4000 cpm/pmol), V.4 ,ug oligo(dT~:poly(rA] or ol~go(dTI:poly(dA), 0.02 ,umol dATP and 1.0 nmol [ H] dTTP (4000 cpm~pmol), In reaction with oligo(dG):poly (rCm), MnC12 (0.02 ~mol) replaced MgC12.
Assays using endogenous RNA contained (in 100 jul): 5 ~mol Tris-HCl, pH 8.0, O.8 ~mol MgCl~, 0.1 ,umol DTT, 10 ,ug actinomycin D 5 ug distamycin A, 0.1 ~g oligo (dT)12-18, 0.1 ,umol dATP, dGTP, and dTTP, and 5 nmol [3H] dCTP ~1.5 X 104 cpm/pmol).
All reactions were incubated at 36 for 15-30 min as indi-cated, and were terminated by the addition of 0.5 ml cold 0.067 M sodium pyrophosphate, 1 M sodium phosphate, pH 7.2, follow~d by 0.5 m. cold 80~ TCA. Acid-insoluble radioactivity was collected on membrane filters and measured in a scintillation counter.
Terminal deoxynucleotide transferase activity was measured by the polymerization o~ 13H] dGTP in the absence of a complemen-tary polymer template, the latter being replaced by 0.4 ~g oligo ( G)10-18- ....

The Effect of'Antibody on DNA Polymerase'Activities Reaction mixtures ~or the neutralization of DNA polymerase activity (total vol 55 ~1) contained in addition to 25 ~g of bovine serum albumin (BSA) and DNA polymerase, the indicated amount 125-150 ,ug) of purified IgG fraction. The buffer used was 0.01 M Tris-HCl, pH 8.0, 0.15 M of potassium chloride.
After 15 min incubation at 4, a polymerase assay was carried out uslng oligo(dG):poly(rC) as described above. In certain instances, the e~fect of antibody on the activlty by the endogenous RNA was examined.

- 2s ,:: .

Detection of ~nti~ody-enz e ~omple~es; ~n Glycerol Gradients The concentrated enzyme (reverse transcriptase ~r~m MP~V
or from human malignant breast tumors~ fractions were diluted ~hree-flow with 0~1 M potassium phosphate, pH 8.0~ contain~ng 0.5 mg~ml of bov~ne serum album~n, and ~he indica~ed amounts of puri~ied Ig~ fractions were added. After incubation for 15 min at 4, the samples were layered over a 1~-30% glyercol gradi-ent adjusted to 0.1 M potassium phosphate, pH 8.0, .OOZ M
d~thiothreitol and 0.02% Triton X-lO0. ~l~he samples were sedimented at 48,000 rpm for lZ hr in a Spinco S~50-l rotor at 1. Fractions were collected dropwise from the bottom of the tubes and the enzyme act~vity assayed as described above.

''Results A ComParison of the Effects o~ Anti-MPMV DNA Polymerase IgG on a Var'iety o'f~~Pol'ymer'ases e effects of the anti-MPMV ~NA polymerase on a nu~r of viral DNA
poi~nerases and on the corresponding enzyme of the human breast cancer particles were compared. In these experiments, isopycnical-"
ly banded particles, purified as described in Materials andMethods, were employed as a source of the DNA polymerase and oligo(dG):poly(rC) was used as the template. lhe antibody inhi~its the MPMV-DNA polymerase more than ~0% and ach~eves a 26% inhibition of the DNA polymerase associated with the human breast cancer particles. In contrast, no detectable effect is observed on the DNA polymeraces of any of the other animal oncornaviruses, including avian myeloblastosis virus (AMV~, ~auscher and Friend murine leukemia viruses ~RLV and FLV), feline leukemia virus (FeLV), a simlan sarcoma virus ~SSV-l), or murine mammary tumor virus ~MTV).

':

'Speci~i_it~ o~:the'Tnh~ on by ~the'Anti-MPMV Pclymerase IgG
~ he next issue exam~ned centered on whethex the lnhibition obser-ved w~th the breast cancer particle enzyme was confined to this malignancy. As already known in the art, spleens from pat~ents with mesenchymal cancers constitute a convenient source of particle enzyme, and these were chosen for immunologic comparison. The particle fractions were prepared and the endogenous polymerase activities were assayed as previously described for human breast tumors and for spleens involved in mesenchymal neoplasias. At least five instances of each kind of neoplastic tissue were examined and typical results are sho~m in Table 1.

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. : : , ':' . ~ . ., $ignificant ~nhibitions are not seen with the particle en-z~mes derived from the leukem~c and the lymphoma spleens.
Howe~er, the breast cancer particulate enzymes were inhibited from 59~ to over 95%. Note that these endogenous reac~ions are more severely affected by the anti-MPMV polymerase IgG than the synthesis directed by synthetic templates. As expected, all the DNA polymerase activities described in Table 1 are sensitive to ~Nase and resistant to the presence of actinornycin D (100 ~g/ml~ and distamycin t50 ~y/ml), features characteristic of RNA-directed DNA polymerase, The data shown in Table 1 were obtained with the endogenous -reactions of detergent-disrupted particles isolated from the indicated neoplastic tissues. For completeness a similar comparison was carried out using the corresponding purified enzymes directed by oligo(dG):poly(rC). ~xperiments using ; this approach provided the responses to anti-MPMV polymerase IgG of the reverse transcriptases purified from the particles prepared from breast cancers and ~rom a chronic myelogenous leukemic spleen. Over the whole concentration range of IgG
examined, the leukemic reverse transcriptase is not significantly affected. In contrast, the anti-MPMV polymerase IgG does suppress the activity of the hreast cancer reverse transcriptase at all concentrations tested, achieving a 37~ inhibition at 150 ~g per reaction mixture.
,~ .
Table 2 examines another aspect of the speci~icity of the interaction by testing the responses o~ normal cellular DNA
polymerases to the anti-MPM~ polymerase IgG, ~ - 29 -. a u~ N 0 ~r ~ ~1 ~ ~ ' S~ O ~ ~o IJ I` ~ a) o ~
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~I t~) U D ~1 00 D a) ta -O ~ ~ --~ N _I _1 3 ~I f:C o ~ u~ 0 ~ m ~,1 , ~1 ~ .

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a) 4~ ~ ~ ~) O ~
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.`, . . `

Neither preparation o~ DNA polymerase y, whether isolated from a breast tumor or from the HeLa cell strain, was detectably ~nhibited, and the same was true for DNA polymerase ~. At the same level the anti-MPMV polymerase IgG suppressed the breast cancer reverse transcriptase by 40%. Similar : experiments were carried out with the normal DNA polymerase a and again have found no evidence of inhibition by the anti-MPMV polymerase IgG.

The Demonstration by Sedimentation of Complexes between the Breast Cancer DNA Polymerase and the Anti-Polymerase IgG
It was desirable to see whether ~urther evidence could be provided for the existence of physical complexes between the breast cancer reverse transcriptase and the anti-MPMV
polymerase IgG. Fixst, such evidence would add direct support to the conclusions derived from simple suppression of enzyme activity. Second, experiments along these lines could identi~y the basis underlying the apparent inability of the anti-MPMV DNA polymerase IgG to achieve total neutralization of the breast cancer reverse transcriptase activity. Basically, two mechanisms can be offered to explain the incompleteness o~ the inhibition. One would suggest that the enzyme prepara-tion is heterogeneous and that only a sub-population forms inactive complexes with the added IgG. The other would assume that the population of enzyme molecules is homogenous in this respect and that all form complexes, which can, -; howevex, express a fraction of the original activity. ~l~hese two possibilities are readily distinguishable by a sedimentation analysis of the enzyme activity before and after reaction with the relevant IgG.
To monitor the e~fectiveness o~ this approach, a positive control experiment was carried out with the homologous system consisting of MPMV-DNA polymerase and its antibody. A negative X

control w~s incLuded uslny normal (pre-immunized) IgG from the same rabbit~ A 250~ reaction containing enz,yme and 150 ,uy of the indicated IgG were incubated at 4 for 15 min as described in Materials and Methods. The mixture is then layered on a 10 to 30 96 gradient and centrifuged at 48,000 rpm for 12 hr.
-at 1. Fractions are then collected from the bottom and assayed for reverse transcriptase and ~or the presence of IgG
by immunodiffusion. Incubation with normal IgG was found not to change the position (tube 131 of the peak o~ MPMV reverse transcriptase activity with respect to the external marker bovine serum albumin (BSA). ~l~he enzyme still sediments at a velocity corresponding to a molecular weight of 70,000 daLtons.
In this same gradient, the IgG was located in tubes 10, 11, and 12. Incubation of the polymerase with 150 jug of anti-MPMV
polymerase results in an 80% loss of enzyme activity and a markedly different sedimentation pattern of the residual activity~ No enzyme is detectable at the original position close to BSA, all of it appearing as complexes sedimenting faster than ~ree enzyme or IgG. The IgG is now detected by immunodiffusion in fractions 13, 14, and 15 as well as in fractions 6 through 10, which encompass the peak of polymexase activity.
A very similar situation is obtained in the experiments with the reverse transcriptase purified from human breast cancer particles. Incubation with normal IgG leaves the human enzyme in its usual position (tube 15) within one tube of the BSA marker, and the IgG is found by i~unod,iffusion in tubes 12 to 14. However, reaction wi-th anti-MP~V
polymerase IgG results in a 45% loss of ackivity an~ shifks the' residue down the tube as ~'ast-moving complexes found in fractions 6 through 10 r in which IgG can also be detected by immunodifusion. IgG is also found in its original posi-tion (fractions 12 through 14).

- 3~ -I

It is evident ~rom the results described above that neither the MPMV reverse transcriptase nor the one isolated ~rom human breast cancer particles conta;ns a significant proportion of molecules unable to complex with ant~-MPMV polymerase IgG.
The fact that the enzyme IgG complexes can express some activity is not a new phenomenon. Indeed, in some reporte~ instances, such complexes are fully active.

Discussion The experiments described here show that anti-MP~ DNA
IgG, when present in excess, can completely complex with and partially inhibit the reverse transcriptase isolated from human breast cancer particles. The specificity of the inhibition is supported by the inability of this same antibody to affect the activities of normal cellular DNA polymerases or of a variety of reverse transcriptases from animal oncornaviruses (e.g., AMV, RLV, FLV, FeLV, SSV-l, and MM~rV~.
Further, normal IgG obtained from the same rabbit prior to immunization does not complex with, or inhibit, either the MPMV or the human breast cancer reverse transcriptase.

Aside from its etiologic interest, the immunologic cross-reactivity between the reverse transcriptases of MPMV and of the human breast cancer partlcles has an implication of more immediate import. It provides the basis for examining human breast cancer by procedures of clinical usefulness. MPMV can ~e produced in tissue culture in yields adequate for purification of its enzyme and other protein components. These can in turn be used to generate antisera for use as specific detecting reayents in immunofluorescent and ~mmunoperoxidase staining of frozen sections as diagnostic aids for surgical pathologists. Of further interest is the use of such development in immunoassays for the system;~c detect~on o~ immunologXcall~ relate~ pxotein in the plasma and other body flu;ds o~ pat~ents with cancers, okher than breast cancer such as lung cancer, stomach, rectal and colon cancers, ovarian cancer, brain cancer, bone cancer, cancer of the lymph glands, skin cancer (squamous cell and basal cell carcinomas and melanoma) and the ltke~ Each of these cancers has its own distinctive particle associated protein.

Claims (21)

I CLAIM:

1. A method for the detection of breast cancer in a human subject, comprising assaying a blood plasma sample from said subject for a breast cancer specific viral related protein selected from the group consisting of RNA-dependent DNA polymerase and viral origin tumor associated protein.

2. The method of claim 1 wherein the presence of said breast cancer specific viral related protein in concentrations greater than control levels is a diagnostic indication of a breast cancer in said subject.

3. The method of claim 1 wherein plasma samples are taken from said subject before and after initiation of therapy and said assay serves to monitor the effectiveness of said therapy.

4. The method of claim 1 wherein said breast cancer specific viral related protein is human breast cancer RNA-dependent DNA polymerase.

5. The method of claim 4 wherein said RNA-dependent DNA polymerase is assayed by isolating the enzyme from the plasma sample and measuring for enzymatic activity.

6. The method of claim 5 wherein said RNA-dependent DNA polymerase is isolated from said plasma sample by ammonium sulphate precipitation, followed by affinity chromatography of the reconstituted precipitate through a column comprising SEPHAROSE (trademark) beads to which a synthetic template for the enzyme had been covalently bonded.

7. The method of claim 6 wherein said synthetic template is selected from polyriboadenylate and poly (2'-0-methylcytidylate).

8. The method of claim 4 wherein said RNA-dependent DNA polymerase is assayed by radioimmunoassay.

9. The method of claim 8 wherein said radioimmunoassay utilizes human breast cancer RNA-dependent DNA polymerase specific antibody and 125I-human breast cancer RNA-dependent DNA polymerase.

10. The method of claim 8 wherein said radio-immunoassay utilizes Mason-Pfizer Monkey Virus specific antibody, which is cross-reactive with human breast cancer RNA-dependent DNA polymerase, and 125I-Mason-Pfizer Monkey Virus.

11. The method of claim 1 wherein said breast cancer specific viral related protein is viral origin tumor associated protein.

12. The method of claim 11 wherein said viral origin tumor associated protein is assayed by radioimmunoassay.

13. The method of claim 12 wherein said radio immunoassay utilizes viral origin tumor associated protein specific antibody and 125I-viral origin tumor associated protein.

14. The method of claim 12 wherein said radio-immunoassay utilizes Mason-Pfizer Monkey Virus specific antibody, which is cross-reactive with said tumor associated protein, and 125I-Mason-Pfizer Monkey Virus.

15. A human breast cancer specific viral related protein essentially free from other viral and breast tissue components, said protein selected from the group consisting of human breast cancer RNA-dependent DNA polymerase and human breast cancer viral origin tumor associated protein.

16. Human breast cancer RNA-dependent DNA polymerase essentially free from other viral and breast tissue components.

17. 125I-human breast cancer RNA-dependent DNA
polymerase.

18. Human breast cancer viral origin tumor associate protein essentially free from other viral and breast tissue components.

19. 125-I-human breast cancer viral origin tumor associated protein.

20. An antibody specific to human breast cancer RNA-dependent DNA polymerase.

21. An antibody specific to human breast cancer viral origin tumor associated protein.