WO1989005862A1

WO1989005862A1 - Human cell life extension

Info

Publication number: WO1989005862A1
Application number: PCT/US1988/004415
Authority: WO
Inventors: Charles Hsu; Michael P. Mcgrogan; Christian C. Simonsen, Sr.
Original assignee: Invitron Corporation
Priority date: 1987-12-09
Filing date: 1988-12-09
Publication date: 1989-06-29
Also published as: AU2921289A

Abstract

Human differentiated cells are modified but not transformed using the Ela region of adenovirus or an expression system for SV40 T-antigen. The modified cells have reduced doubling times and are capable of an increased number of passages, while retaining their ability to generate a desired product, such as t-PA. Therefore, the modified cells are useful for the large-scale production of factors natively or recombinantly produced.

Description

HUMAN CELL LIFE EXTENSION

Field of the Invention The invention relates to propagation of^"human cell lines for the purpose of producing their natural or recombinantly generated products. More particularly, it relates to the use of selected viral geno ic DNA to overcome the inherent mortality of differentiated human cells and to permit their establishment as extended life cell lines.

Background of the Invention

Normal or nontransformed di ferentiated mammalian cells, when cultured in vitro, typically have a defined mortality. After a relatively small number of doublings, a stage of senescence is reached in which no further cell division occurs. Only a limited number of passages into fresh medium can, therefore, be made before the cell population is no longer capable of growth. What this number is varies greatly with species origin and with dif¬ ferentiation status, but for each cell type there is nevertheless a finite upper limit. This property, of course, limits the total number of cells which can ever be obtained from an individual parent cell.

Deviations from this "norm" occur spontaneously by virtue of the appearance of an occasional mutation to a tumorigenic or transformed cell in primary and secondary cell cultures. The descendants of the transformed cell are "immortal"— i.e., the culture can be passaged in- -2-

definitely and is established as a permanent cell line. Clearly immortality is advantageous if the cells are to be used as production means for materials of biological interest. It would also be of advantage, if the "life" of the cell could be extended, i.e., although the total number of doublings would be finite, the permissible number would be increased. In addition, an increase in doubling rate would yield useful quantities of cells more efficiently. These transformation phenomena occur with high relative frequency in rodent cell cultures, but almost never in cultures of normal human cells. The transforma¬ tions can often be characterized by a variety of phenotypic changes including a change from a diploid to aneuploid karyotype, cytoskeletal and structural changes which reflect alterations in immunoreaction characteristics, acquisition of the ability to grow in soft agar and to produce tumors in nude mice, and a number of less well defined metabolic changes. Nevertheless, several human-derived spontaneously generated cell lines have been reported, including an epithelial line from a colon mucosal culture (Danes, B.S., et al, J Natl Cancer Inst (1982) 69.:1271-1276) .

Consonant with the more frequent occurrence of spontaneous -transformation in rodent cell cultures is the ease with which they are transformed using oncogenes or viral genes in comparison to human cell cultures. Accord¬ ingly, the transforming DNAs used in the methods of the present invention have been studied most widely in cultures of rodent-derived cells. The results of such transformation may or may not include the phenotypic changes associated with "transformed" cells? the cells may be simply immortalized or have extended lifetimes.

The most frequently means for in vitro transformation of human cells is insertion of SV40 DNA. A review of in -3-

vitro transformation in human epithelial cells by Chang, S.E., appeared in 1986 (Biochem Biophys Acta (1986) 823: 161-194). The ability of the SV40 DNA to extend the life of human or other mammalian cells appears to be due to presence of the early region of the viral genome, and specifically to the production of the large T-antigen (94 kd) and, peripherally, to small t-antigen (17 kd) . In human cells, which are semipermissive for this virus (i.e., only some of the cells are lytic and permit viral replication while many express some viral genes but fail to lyse) SV40 transfection for purposes of immortalization has been made more effective by introducing SV40 DNA which lacks its own origin of replication, thus eliminating the possibility of successful mobilization of the cells' resources to the end of reproducing the virus.

Typically, human cells transfected with SV40 DNA produce dominating subcultures which show production of large amounts of large T-antigen and show extended lifespans, but reach a crisis phase from which only a few ever emerge to form immortal cell lines. In short, the pattern of transformation and reproduction of SV40- infected or transfected cells is highly unpredictable.

Of particular interest in connection with the present invention are attempts to immortalize colon epithelial cells. Moyer, M.P., et al, Science (1984) 224:7445-7447, showed that treatment of human mucosal epithelial cells with SV40 virus (or with a chemical carcinogen) produced cells of altered phenotypic characteristics, including the synthesis of intranuclear T-antigen and additional characteristics associated with transformation into malignancy. The study was undertaken in an attempt to understand the mechanisms of carcinogenesis, and the extended lifespan exhibited by the cells was associated with additional properties characteristic of conversion to malignancy. More recently, Moyer, M.P., et al (In Vitro -4-

Cellular and Developmental Biology (1987) 23:141-147) used a carrier plasmid (pBR322) into which a portion of the SV40 genome containing the early large T-antigen coding region was inserted. This resulted in cells with increased longevity, but also with variable phenotypes having characteristics of malignant cells. These characteristics include decreased growth factor require¬ ments, altered cell surface features, and capacity to grow in soft agar. As this paper clearly indicates, trans- formation with SV40 DNA capable of generating T-antigen inside the cells results in a range of phenotypic and growth pattern modifications which covers many pos¬ sibilities and is unpredictable with respect to various parameters such as capacity to produce cellular products not included in the study.

In addition to immortalization using SV40 DNA, adenovirus has also been employed, though less frequently, to extend the lifetime of mammalian cells. It has been known for some time that the early region of adenovirus is capable of effecting the transformation of rodent cell cultures and the function of the various portions of the early region genes have been reasonably well character¬ ized. It has been shown that the early region "1", which is demonstrably capable of effecting transformation to tumorigenicity in rodents, consists of two transcriptional subunits, regions Ela and Elb, each encoding several early viral proteins. The presence of both the Ela region and the Elb region is necessary for the morphological and tumorigenic characteristics of fully transformed cells to be exhibited_? however, transfeetion with Ela alone appears to immortalize rodent cells without rendering them tumorigenic or noticeably morphologically different. (Transfection with Elb alone appears not to have any ef¬ fect. ) -5-

It has also been shown using embryonic rat kidney cells that the Ela and Elb regions can be transfected into the recipient cell on separate vectors and that the Ela regions of both of two adenovirus serotypes, Ad5 and Adl2, are capable of interacting with the Elb region of Adl2 to effect full transformation (Van den Elsen, P., et al, Gene (1982) 18_^:175-185) .

There are also several reports of treating cultured human epithelial cells with adenovirus or adenovirus- derived DNA. Rhim, J.S. et al. Science (1985) 227: 1250- 1252 reported the infection of keratinocytes with a hybrid Adl2-SV40 virus followed by later infection with Kirsten sarcoma virus. One of the by-products of this double infection was a immortalized line, not capable of inducing tumors or producing virus, which was aneuploid, expressed keratins, failed to grow in soft agar, and was not tumorigenic in mice. This line produced no transcripts of Ela or Elb of the adenovirus, although transcripts of the transforming region of SV40 were detected, and the cells appeared to show the flattened morphology of an SV40 transformant. It was concluded by Rhim that the effective transformation of this cell line was as if by SV40 alone.

Graham, F.L, et al (1977) J Gen Virol 36:59-72, transformed human epithelial cells from embryonic kidneys by transfection with sheared fragments of adenovirus-5

DNA. Embryonic cells are not fully differentiated and are generally much more easily transformed and immortalized than the differentiated cells derived from adult tissues. A single cell line now in common use among practitioners was immortalized from this transformation and has been subcultured over one hundred times, having passed a crisis phase which endured through passages 13-16- These cells, designated 293 cells, were confirmed as epithelial cells by virtue of their ability to express keratin antigens, and are characterized by Chang (supra) as the first human -6-

cells to be transformed by a human adenovirus. They are mildly tumorigenic. Chang attributes the success in transformation to the use of sheared DNA rather than virus infection, since infection generally leads to lysis. It can not be known for certain whether infection might also have occurred. Subsequent studies have shown that the portion of the Ad5 genome which was integrated into the chromosome of this cell line encompasses all the El region. Whittaker, J.L., et al, Mol Cell Biol (1984) 4_:110- 116, established four transformed lines from cultures of human embryo kidney (HEK) cells by microinjection or transfeetion with a fragment of adenovirus-12 DNA representing map positions 0-16.5 on the left hand side of the viral genome. This fragment was cloned and used to transform the cells. Subsequent studies showed that all four transformed and immortalized cell lines which resulted expressed El proteins, although Elb was more dramatically expressed than Ela- All of the resulting cell lines were tumorigenic and are confirmed to be epithelial since they express keratin antigens.

While early studies with regard to viral gene trans¬ formation were pursued using direct virus infection, transformation with isolated DNA, as in the foregoing Van den Elsen paper, permits more defined characterization of the source of the observed effects. The entire Ela region is found on the Hpal-E fragment, which contains the region representing map positions 0-4.3 of the Ad5 genome (Van Ormondt, H. , et al, Gene (1980) ] :299-309). the Elb region is contained on the Ad5 Smal-F DNA representing map positions 2.8-10.7 of the genome. Preparation of these fragments is described in detail by Van den Elsen et al. (supra) .

Partial transformation of rodent cells with the El region alone has been achieved (Houweling, A. et al,

^■7 -

Virology (1980) 10_5_:537-550; Ruley, H.E., Nature (1983) 3_0_4:602-606) . In both cases, primary rat cell cultures were used as substrates-

Roberts, B.E., et al, J Virol (1985) 56.404-413 reviews the detailed structure of the Ela region. It is known that the Ela transcription unit encodes three over¬ lapping mRNAs which share common sequences at their 5' and 3' termini. These three mRNAs encode different proteins because they differ in length, and, in one case, in read- ing frame. The 13S and 12S mRNAs are the first transcripts expressed after adenovirus infection and contain identical amino and carboxy termini, but differ in length corresponding to the deletion of an internal frag¬ ment from the protein encoded by the 12S mRNA. The resulting proteins have molecular weights of 32 kd and 26 kd. The remaining mRNA encodes a 6.1 kd protein which is identical in its amino terminus to the others, but is spliced in a different reading frame to alter the remainder of the peptide. The precise functions of these proteins are not known, but it appears that the 13S encoded protein regulates the accumulation of mRNA transcripts in the infected cell. Furthermore, it is not known at present whether the Ela and/or Elb regions alter the cells into which they are incorporated by virtue of the proteins, they encode, or whether such alteration is effected at the DNA level, or both.

Moran, E. et al, Mol Cell Biol (1986) 6_:3470-3480 have further studied the Ela region and find that certain modifications are more significant to the effect of this viral region than others. Deletions of amino acids from positions 86-120 had little effect on lytic or transform¬ ing functions, while modifications in positions 121-150 were destructive.

Although transfection of human cell cultures is inherently difficult, a good deal of work has been done in -8-

attempting to form human permanent cell lines. Much of the work has, however, been done on fibroblast cells, rather than epithelial cells, since fibroblast cells are inherently easier to culture in the first place (Moyer, M.P-, et al, Science (1984) 22^:1445-1448 (supra)).

It should be noted parenthetically that there are fundamental distinctions between SV40 transformed and adenoviral transformed cells. The distinction is in part morphological, in that most adenoviral transformed cells are highly rounded and resemble the spherical cells found in the midst of mitosis, with no intracellular muscle-like fibers. SV40 transformed cells are less similar to mitotic cells and contain many more organized muscle fibers throughout the cell cycle. These morphological differences appear to have behavioral consequences in that the adenoviral transformed cells do not readily attach to solid surfaces. This distinction is sufficiently well recognized and known in the art that it is referred to in standard beginning Molecular Biology textbooks. See, for example, Watson, J.D. "Molecular Biology of the Gene" 3d ed. (1977) W.A. Benjamin and Company, p. 667).

Applicants are not aware of any reports of trans¬ formation of human differentiated cells yielding altered cell lines without substantial modification of most phenotypic characteristics, wherein these lines have extended lifespans and increased doubling rates and wherein the cells continue to secrete, at enhanced levels, products normally produced by these cells. Previous stud¬ ies have focused on the mechanism of conversion of cells from normal, differentiated, limited-lifetime cultures to malignant, immortalized cell lines, and have not explored the use of these techniques for providing an extended source for normally secreted products. Transfection of human cell lines which secrete valuable products such as human tissue plasminogen activator (t-PA) results in a -9-

reliable, convenient, and quantitatively significant source of these products.

Disclosure of the Invention The invention provides a method for extending the life of and accelerating the doubling time of cultures of human cells, especially differentiated cells, which are useful in the in vitro production of the products gener¬ ated naturally by such cells or for the production of products which they are capable of synthesizing as a result of manipulation by recombinant techniques. The invention method results in the ability to proliferate these cells to a quantity sufficient to produce desired amounts of the product without the undesired further ef- feet of rendering them tumorigenic in mammals, and without accompanying undesirable changes in cellular morphology. The altered cells are therefore capable of being cultured under conditions standard for the culture of mammalian cells in general and have the ability to sustain an increased number of passages without exhibiting senescence. The cell line thus provides an amplified resource for whatever materials these cells are naturally capable of producing or are capable of producing through genetic manipulation. Therefore, in one aspect, the invention relates to a method of extending the life and increasing the doubling rate of human cells, especially differentiated cells, which comprises transfecting said cells with a DNA sequence consisting essentially either of the Ela region of the adenovirus genome or of an SV40 T-antigen expres¬ sion system, followed by selection of normal phenotypes capable of producing the desired product. In other aspects, the invention relates to the rapidly proliferat¬ ing, extended-life human cells so produced, to methods of -10-

producing desired proteins by culturing these cells, and to the products so produced.

Brief Description of the Drawings Figure 1 shows a diagram of the Ela region of the adenovirus genome.

Figure 2 is a diagram of the pAd2-BalI-10 plasmid containing and Ela region.

Figure 3 is a diagram of the pSV40 and pSV VP. plasmids containing an SV40 T-antigen expression system.

Modes of Carrying Out the Invention

A. Definitions As used herein,

"Differentiated" cells are cells which are in their

"final" form and not converted to modified cell types.

Embryonic cells are, of course, not fully differentiated; neither, however, are certain other cells found in mature individuals. Colon cells of the intestinal mucosa are considered differentiated.

"Human differentiated epithelial cell" refers to a cell or cell culture or group of cells of human origin which is defined as epithelial by standards commonly recognized in the art, and which is fully differentiated.

At present, epithelial cells are characterized by their ability to produce keratin antigens. They are derived from a variety of tissues, and while skin and the intestinal mucosa are the most fertile sources, cells of this type exist in a variety of tissues such as kidney, placenta, mammary glands, and so forth. However, only a subset of such cells is differentiated.

"Consisting essentially of the Ela region of adenovirus genome or its equivalent" refers to a DNA frag- ment whose functional portion in conferring altered -li¬

metabolie and reproductive characteristics (as opposed, for example, to conferring the ability to produce a re- combinant protein) on a cell line into which it is transfeeted is that conferred by DNA residing in the Ela region of the adenovirus genome or its equivalent.

A diagram of the Ela gene of Ad2 and the RNA spliced transcripts is shown in Figure 1. It is recognized that the Ela region in a variety of serotypes is at the extreme left hand portion of the genome and is responsible for encoding a number of early proteins. The DNA sequence of the Ela region is known, and it is clear that while the Ela region per se could be derived, and is indeed most simply derived, by isolation from the adenovirus genome, it could also be produced synthetically. The region defines an overlapping region of three mRNA transcripts as set forth in the Background section above, and equivalents could thus be prepared in a variety of forms. The upstream sequence, at least, where all transcripts are in the same reading frame may permit considerable variation due to the redundancy of the genetic code? where reading frames differ, the options are less numerous. All modifications of Ela DNA which encode mRNA transcripts that translate into the three early proteins encoded by this region are, of course, included within the definition of Ela and its equivalents.

There are a number of known serotypes of adenovirus, for example Ad2, Ad5 and Adl2, but it is understood that the functionality of the Ela regions of all members of this family is interchangeable among the group, and regardless of the serotypes used for illustration, the Ela region could be isolated from any adenovirus using analogous methods, or could be chemically synthesized using standard techniques for DNA synthesis, or could be prepared by some combination of these methods. -12-

"Expression system for SV40 T-antigen" refers to a DNA fragment containing the coding sequence for the protein represented by the large T-antigen from the Simian virus (SV40) operably linked to control sequences capable of effecting its expression in mammalian hosts. These control systems may include the SV40 early promoter normally associated with the T-antigen-encoding gene but can also include substitute promoters such as the murine leukemia virus (MuLV) promoter. The T-antigen itself may be of the usual amino acid sequence, or may have an altered amino acid sequence which is. sensitive to temperature—i.e., which is denatured at moderately elevated temperatures. Temperature-specific T-antigens have been disclosed. The T-antigen, in affecting the life of the cell in which is resides, is known to operate at the protein level—i.e., the T-antigen protein in its ac¬ tive form binds to nuclear or other DNA and alters the pattern of expression otherwise found. There is no known direct effect of the DNA encoding the T-antigen protein, although this possibility is not excluded. Nevertheless, the invention requires an expression system assuring the production of T-antigen protein.

The terms "cells", "cell lines", and "cell strains" are sometimes used interchangeably herein although they may have distinct meanings in a technical sense. In general, the term "cell lines" is reserved for clonal, immortalized, or transformed cultures. Since the inven¬ tion is directed to methods of converting nonimmortalized cells into extended life cells, or if possible to i - mortalized lines, at some point the initial "cells" become "cell lines". However, most often the distinction is not important to the sense of the text. When it is, this will be evident from the context. The foregoing terms as ap¬ plied to particular cells, cell lines, etc. refer also to the progeny of the original characterized cell.

-13-

"Immortalized" as used herein refers to the ability of cells derived from a single parent cell to continue growth through an indefinite number of passages without exhibiting senescence. A crisis phase may be present as it is in "normal" growth, but the ability of the cells to multiply continues essentially undiminished indefinitely. "Immortalized" is to be carefully distinguished from "transformed" . Transformation includes immortality as a feature, but is also characterized by morphological change, and in many cases the cells are also tumorigenic. The cells of the invention are not "transformed" .

"Extended life" refers to a condition of the cells of the invention which is characterized by their ability to divide through an increased number of doublings over and above the number associated with the starting cells.

Typically, in addition to the approximately 50 doublings expected of the differentiated human epithelial cells used in the invention, a further 20 doublings can be obtained. While this additional 20 doublings does not constitute immortality, the purpose of the invention is served by virtue of the availability of 2 20 times as many cells as previously obtainable, and hence 2 20 times the quantity of the secreted product produced.

The extended life of the cells of the invention is also associated with an increased rate of doubling. This characteristic makes it possible to accumulate a desired number of cells in a shorter time frame and thus to render more efficient the process of obtaining a culture for use in product production. "Doubling" and "passaging" have their usual meanings herein. An indication of the increased growth rate of the cells of the invention is the greater increase in cell density when normal time-frame passaging is conducted.

It is, of course, recognized that the cells present after a given number of passages may not be precisely

-14-

genetically identical to those at other stages; however, these progeny cells are included within the scope of the invention so long as they exhibit the essential characteristics referred to herein, namely retain their capacity for extended life, their enhanced doubling rate, and their ability to produce a particular product for which the parent cells are of interest. For example, the cells used for illustration herein are capable of generat¬ ing useful amounts of tissue plasminogen activator (t-PA) , a protein of considerable pharmaceutical utility in dis¬ solving blood clots. Progeny of the original life- extended or immortalized cells are included, within the invention so long as their capability to produce t-PA is maintained or enhanced and so long as they also retain their ability to survive and grow after repeated passag¬ ing, without having become tumorigenic.

"Product generation" refers to the ability of the cells to produce the usual or greater amounts of a produc for which they are cultured. Various human differentiate cells are capable, depending on their origins, of secret¬ ing particular proteins which are useful generally. It i for the production of such products that it is desirable to extend the number of doublings and to shorten the doubling time of the parent cell. "Product generation" thus refers to this general capacity to secrete (or other wise produce) the desired product. Besides the exempli¬ fied t-PA, other useful products generated by human dif¬ ferentiated cells include hormones such as insulin, human growth hormone, and LHRH, antibodies, and various enzymes

B. General Description

Certain human cell lines have been found to secrete, as a product of their "normal" metabolism products that are useful, for example, as pharmaceuticals. Examples of such human cells/cell lines include B lymphocytes which -15-

secrete antibodies and Islet cells which secrete insulin and the various differentiated human cells which secrete t-PA. Illustrated below is a human differentiated epithelial cell line derived from colon mucosa which secretes t-PA in concentrations severalfold higher than those secreted by most corresponding human colon cell lines, and which can be used, if subjected to the method of the invention, for large-scale production of t-PA.

Cell lines which produce useful products natively are thus suitable candidates for application ^"of the process of the invention. Examples include vascular endothelial cells, which produce blood homeostatis and clotting fac¬ tors; adrenal cells; pituitary cells; and lymphoid and myeloid cells, which produce growth, chemostatic, and homeostatic factors. Of course, cells which have been altered to obtain protein production using recombinant techniques are also suitable. Since transfection of expression systems for a wide variety of genes into mam¬ malian cells has become commonplace, this offers a means to produce a large range of products.

The cells, if modified so that sufficient numbers can be provided, can be used for obtaining the desired product during substantial time periods by maintenance of an already grown culture in a "static maintenance reactor" (SMR). This- permits the cells to be maintained at a high cell density for extended periods without necessarily be¬ ing proliferated, but continuously secreting the desired product. Designs for such static maintenance reactors have been disclosed in US Patent 4,537,860- For a 16 liter version of this reactor, approximately 3 x 10 cells can be accommodated and approximately 6-8 months is required using normal passaging techniques in order to obtain sufficient cell densities to supply this reactor for many cell types. In addition, it can be estimated that starting from the primary culture without the -16-

techniques for cell modification of the invention, suf¬ ficient cells could eventually be obtained (by freezing samples and repassaging after thawing) to obtain only a limited, finite supply for the use of this reactor. That is, since the defined lifetime of untransformed or unmodified cells is only 50 doublings or 20-25 passages, the multiple obtainable from the original cell is inher¬ ently limited.

In order to assure a greatly increased total supply of cells and a more rapid accumulation of sufficient numbers of cells to obtain practical production levels, the present invention confers on the modified cells the ability to survive increased numbers of passages, and thus to provide dramatically increased numbers of cells. In general, an additional 15 passages, each representing a fivefold increase in cell count is achievable. In addi¬ tion, senescence does not occur, and the effective doubling time is dramatically reduced, thus resulting in the aforementioned fivefold, rather than twofold, increase per passage. This enhances the rate at which the required large numbers of cells can be produced.

In general, the method of the invention involves using a viral transfection genomic alteration technique, followed by screening of cells on the basis of several criteria. These criteria include formation of tightly packed foci, and retained high level of product generation. The combination of these two screening criteria results in cells which are effectively capable of rapid accumulation and accumulation in increased numbers. The initial transfection for genomic alteration is selected from one of two general approaches: Immortaliza¬ tion by insertion of the Ela genome of adenovirus or of an expression system for the SV40 large T-antigen. Regard¬ less of the initial technique, the cells treated with -17-

either of these viral DNA fragments are screened accordin to the criteria just mentioned.

Thus, one transfection method employed in the presen invention is the insertion of the Ela genome of adenoviru 5 or its equivalent, typically using a standard bacterial cloning vector in order to increase the copy number. Of course, other means of amplification are usable, however, the use of procaryotic cloning vectors such as the pUC series, the pBR322 series, the pSYClOl series and so 10 forth, all of them well known in the art, is^"convenient and effective.

The Ela region of the adenovirus genome is conveniently obtained by isolating the DNA from adenoviru and treating with Hpal restriction enzyme. The relevant

15 fragment is isolated by size fraetionation on SDS gel, if desired, and inserted into a host plasmid using standard procedures. For example, Van den Elsen, P., et al, Gene (1982) 1^:175-185, cited above, describe the preparation of a workable plasmid by treating purified* d5 DNA with

20 exonuclease-III, followed by heat denaturation and rapid chilling in ice. The resulting single stranded termini were then removed by incubation with SI nuclease and the DNA digested with Hpal. The Hpal-E fragment was isolated from the gel, and the leftmost 4.3 percent DNA fragment

25. containing the Ela region was inserted into the Sail site of plasmid pAT153 by the G/C tailing procedure described by Roychoudhury, et al, Nucleic Acids Research (1976) 2:101-116. In the alternative, Roberts, B.E., et al, J Virol (1985) 56_:404-413, also cited above, describe diges-

30 tion of total Ad5 DNA with Hpal, blunting with Klenow, addition of BamHI linkers, and ligation of the BamHI digested fragments into the plasmid pSVX which had been treated with BamHI and calf alkaline phosphatase. The pSVX plasmid is also capable of conferring G418 resistance and

35 this provides a selection marker. -18-

In any event, the Ela region can be provided as an Hpal digest from any of the adenovirus serotypes or may be synthesized using chemical means, or may be obtained using combinations of these approaches as is described above. The cells are transfected with the Ela region or T- antigen expression system by any standard means, most com¬ monly by the standard calcium phosphate precipitation technique of Graham and Van der Eb, Virology (1973)

5_4:536-539. Suitable initial cell densities of 10~ per cm 2 are desirable, and transfection efficiency using this

_3 method can be expected to be on the order of 10 and

10^~ - Modifications of this technique are preferred, including treatment with DMSO as described by Roberts, et al, (supra) .^' In this modification, the host cells are transfected in logarithmic phase with 2 mg genomic equivalents of DNA using the modified calcium phosphate technique of Van der Eb and Graham (Methods in Enzymology (1980) Academic Press New York, pp 826-839). Cells are then washed once with HEBS buffer 4-5 hours post transfection and treated with 1 ml of 25% DMSO and HEBS buffer for two minutes at room temperature, followed by extensive washing with buffer.

For the SV40 T-antigen expression system, the coding DNA for the protein can be obtained from SV40 viral genomes using standard techniques for DNA excision with restriction enzymes. In one very simple approach, BamHI linearized SV40 DNA is inserted into BamHI-digested pBR322 to obtain the transfection plasmid pSV40. The insert then contains the complete genome of the virus, including regions under control of both early and late promoters. The pertinent T-antigen is under control of the early promoter and is satisfactorily expressed in cells transfected with the vector. The late promoter is in¬ active in human cells and the extra DNA is therefore harm- less. -19-

In the alternative, the coding sequence for T-antigen per se is excised by digestion and ligated under control of a suitable promoter, such as alternative viral promot¬ ers, including adenoviral, murine leukemia virus, and bovine papilloma viral promoters. Other promoters active in human cells can also be used, such as the metallothionein promoter.

In addition to wild-type T-antigen, strains of SV40 are available which provide a temperature-sensitive form of T-antigen which is active at 35°C but not active at

39 C (reference). Thus, the activity of the T-antigen in modifying the cell can be controlled by regulating the temperature at which the cells are maintained. As was the case with insertion of the Ela region above, the expres- sion system for T-antigen is conveniently transfected into the host cells by means of a host plasmid replicable and selectable in bacterial culture, such as those derived from pUC, pSYClOl, and the pBR series set forth above. The cells which have been successfully transfected are screened, and their ability to form tightly packed colonies against a background of nonproliferating cells indicates their increased rate of reproduction and doubling. Typically, the cells are capable of multiplying tenfold over a period of about a week in contrast to the normal rate of increase by a factor of 3 over a 5-7 day period. These cells can also be grown in approximately 1% serum, but do not exhibit other properties presumed to be characteristic of transformed cells. The resulting cell lines, which are modified, but not "transformed", are established from separated single cell foci.

An additional important component of the screen is to establish the product-generation capability of the established cell lines. This is accomplished by applica¬ tion of assay methods appropriate to the product to be analyzed, and verification that the property of secreting -20-

the required amounts of the desired product is thus verified.

In addition, of course, other transfection techniques may be used, such as DEA/dextran, electroporation, microinjection and infection with recombinant viruses bearing the Ela region, or T-antigen expression system as the case may be, but altered so that only the Ela region or SV40 T-antigen system is effective, so that im¬ mortalization or life extension/doubling rate increase, but not transformation results. With respect to the particular factors used in the illustration, satisfactory results are obtained using calcium phosphate precipita¬ tion. Following transfection, and culturing cells from tightly packed foci, the cell lines are assessed for product generation and those with satisfactory levels are cultured separately through repeated passages.

Examples

The following examples are intended to illustrate but not to limit the invention.

Example 1 Preparation of Cell Line CCD 18 Co The cell strain CCD 18 Co was isolated from the colon mucosa of a neonatal human female and was deposited with the ATCC with acquisition number CRL1459. This strain produces tissue plasminogen activator. The primary cell culture has been propagated from a bank of frozen samples prepared at early passages (2,5,8). The strain karyotype is near-normal, with a consistent monosomy-22 and low oc¬ currences of other monosomies or trisomies for specific chromosomes. The normal lifespan of the culture is ap¬ proximately 50 cell doublings, which is equivalent to 20 passagings according to the standard procedure worked out for these cells. -21-

Example 2 Preparation of an Ela Gene Bearing Vector The plasmid pAd2-BalI-10, which permits bacterial propagation of the Ela gene, was prepared as follows: Th Ball "I" fragment of Adenovirus 2 was purified from a complete digest of subcloned Adeno 2 DNA. This fragment was ligated into the Smal site of ρUC13, yielding subclones of the I fragment in two orientations. Colonies 10 (used here) and 17 were chosen as the prototypes and grown up for DNA stocks. Since the entire Ela gene, including promoter and translation signals, is encoded on the Ball I fragment, the two expression plasmids differ only in the orientation of the gene with respect to the bacterial sequences. Thus, the resulting plasmid contains the adenoviral Ela gene inserted as a Ball fragment in the host vector. The vector is amplified in E coli HBlOl and used for the transformation of the CCD 18 Co cells.

Example 3

Transfection of CCD 18 Co Cells The method of Van den Elsen, referenced above was employed. Five micrograms plasmid per cell plate contain¬ ing 1-2 x 10 cells per 10 cm diameter dish was used, fol- lowed by a two minute DMSO shock after 24 hours.

Passage 17 cells were used for transfection during logarithmic phase, and the transfection was repeated at passages 18 and 19. The transfection efficiency was ap¬ proximately 10^" , as defined by colony outgrowth at pas- sages 20-21. The tightly packed foci obtained were isolated and were again passaged through passages 20, 21, and 22. Eight candidate cell lines which appeared to continue above-normal rate doubling and retain their vi¬ ability were further explored. -22-

One cell line, 654, was capable of growing through passage 27, but then exhibited no further growth. On the other hand, line 655, retained normal morphology, is not tumorigenic, did not exhibit senescence, was still at growing in continuous culture to passage 35.

Example 4 Use of Modified Cells to Produce Tissue Plasminogen Activator Passage 20 cells of the 655 cell line were multiplie to an appropriate total cell count passages and supplied to the 16 liter static maintenance reactor corresponding to that described in US 4,537,860. The disclosure of thi patent is incorporated herein by reference. After severa days maintenance in the reactor, these cells showed a t-P production at high levels.

Example 5 Construction of SV40 T-Antigen-Containing Expression Vectors

Expression vectors containing the T-antigen coding sequence were produced as follows:

Plasmid pSV40 was obtained by digesting SV40 DNA wit BamHI and inserting the linearized viral genome into BamHI-cleaved pBR322 to give the desired vector.

An additional vector, designated pSV vp , was prepared by digesting pSV40 with BamHI and EcoRI and isolating and relegating the two large fragments. This reorients the insert and deletes unneeded DNA. The resulting vector, designated pSV VP , was then amplified in E. coli. -23-

Example 6 Transfection with T-Antigen Vector The method of Example 3 was applied to the CCD 18 Co cells described above using the vectors prepared in Example 5.

Following transfection, cells are passaged one time and then maintained for an indefinite period of time. Two selections can be used. In the first, cells are fed with standard DMEM containing 5% FBS. The cells become confluent, and transformants appear as colonies of cells which overgrow the main lawn of cells (i.e., are not contact-inhibited) . In the second, cells are fed with DMEM containing 1% FBS. The primary (nontransformed) cells do not grow at all, but the transformed cells do. The latter then form foci of local growth.

In either case, colonies are then isolated using cloning cylinders, and expanded by the usual procedures (serial passaging into successively larger culture ves¬ sels) . Candidate cell lines appeared to continue through increased numbers above-normal doubling rates. Colonies isolated were assayed for t-PA production, and cell lines HCF-2a and HCF-2b, which exhibit high secretion levels of t-PA, were further cultured and then used in the procedure of Example to obtain t-PA production.

Claims

-24-CLAIMS

1. A method to modify differentiated human cells which generate product to extend their life in culture, without rendering them tumorigenic, which method comprises: transfecting said culture of said cells with a DNA selected from the Ela gene of adenovirus or its equivalent, and an SV40 T-antigen expression system or its equivalent? culturing the transfected cell culture and selecting tightly packed foci; cloning cells from said foci? and screening the resulting colonies for product generation.

2. The method of claim 1 wherein the cells are epithelial.

3. The method of claim 2 wherein the cells are colon mucosal cells, and the product generated is tissue plasminogen activator (t-PA) .

4. The method of claim 1 wherein the DNA is the SV40 T-antigen expression system or its equivalent, and wherein the SV40 T-antigen expression system contains a coding sequence selected from that for the na- tive SV40 T-antigen and that for a temperature-sensitive T-antigen, under the control of a viral promoter selected from the SV40 early promoter and the murine leukemia virus promoter.

5. The method of claim 1 wherein the transfection is effected during logarithmic phase of the culture cells, and the modified cells show at least normal doubling rates, and/or multiply at least tenfold in a pas- sage time of 7-10 days, and/or remain viable after at least 25 passages.

6. Modified nontumorigenic human differentiated cells which generate product, have increased doubling rate, and have extended life, obtained by the method of claim 1.

7. A method for producing a protein native to a human cell line which comprises culturing the cells of claim 6.

8. Protein produced by the method of claim 7 which has the activity of tissue plasminogen activator.

9. Modified nontumorigenic human differentiated cells which generate product, have increased doubling rate, and extended life, which contain xenogeneic DNA selected from the Ela gene of adenovirus or its equivalent, and an SV40 T-antigen expression system or its equivalent.

10. The cells of claim 9 wherein the modified cells multiply at least tenfold in a passage time of 7-10 days and/or remain viable after at least 25 passages.

11. Product immunoglobulin or derivative thereof purified by the method of claim 1.