AU604753B2 - Tissue-specific viral vectors based on LPV, and their use - Google Patents

Description

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FEE STAMP TO YALUII OF A ACHED MAIL OPFlAfi.m I by James Murray Form COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952-69 COMPLETE SPECIFICATION

(ORIGINAL)

Class I t. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority: Related Art: '~'~wdis coriect for Na,.Tie of Applicant: Adfiress of Applicant: Actual inventor: Address for Service BEHRINGWERKE AKTIENGESELLSCHAFT D-3550 Marburg, Federal Republic of Germany MICHAEL PAWLITA and HARALD ZUR HAUSEN EDWD. WATERS SONS, 50 QUEEN STREET, MELBOURNE, AUSTRALIA, 3000.

Complete Specification for the invention entitled: TISSUE-SPECIFIC VIRAL VECTORS BASED ON LPV,

USE

AND THEIR The following statement is a full description of this invention, including the best method of performing it known to us BEHRINGWERKE AKTIENGESELLSCHAFT HOE 86/B 030 Dr.KL/mU Specification S Tissue-specific viral vectors based on LPV, and their use Lymphotropic papovavirus (LPV) is propagated only in cell Lines derived from 3-lymphomas, for example the BJA-B cell line which originates from a human malignant B-Lymphoma (Menezes et al., Biomedicine 22 (1975) 276-284), or, to a very small extent, in B-Lymphoblastoid cell lines (B-lymphocytes which have been immortalized by the Epstein-Barr virus, Pawlita et at., Virology 143 (1985) 196-211, and Literature cited therein).

i 4 SThis tissue-specificity is a property of the virus particle since, after transfection, isolated viral DNA is also expressed in non-permissive cells. Thus, there arises from this the possibility of introducing this Shighly specific virus, or a vector based on this virus, Sinto malignant B cells in order to insert Di'A sequences coding for toxins or other active compounds (Mechanisms of B Cell Neoplasia 1985, Editiones Roche, Basle, pages 274-278).

i The invention is defined in the patent claims. Further i 25 embodiments are described in detail hereinafter.

i LPV is deposited at the American Type Culture Collection under the number ATCC VR 961.

The entire DNA sequence of a LPV clone (LPV K38) is depicted in Pawlita et al., Loc. cit. The plasmid pLPV K38 which is mentioned therein contains the entire genome, which has been opened at the single BamHI cleavage site and ligated into the BamHI site of pBR322. This plasmid is suitable as starting material for the preparation of the vectors and host cells according to the invention.

i i 1 I 2- Of course, it is also possible to synthesize, on the basis of the known LPV DNA sequence and in a manner known per se, the LPV DNA segments which are used according to the invention, where appropriate using homologous segi! 5 ments from other viruses (Pawlita et al., loc. cit.).

The figures show, in the form of a flow diagram, the preparation of vectors and of LPV DNA segments with which eukaryotic cells are transfected. The figures are not drawn true to scale, in particular the scale is stretched in the region of the polylinkers. The LPV DNA is emphasized by thick Lines.

Figure 1, and its continuation Figure la, show the syn- 15 thesis of the plasmid pL30 which contains the early gene i region of LPV under the control of a foreign promoter, together with a neomycin-resistance gene as marker. This plasmid is suitable for the transfection of a permissive cell line, resulting in a high yield of cells which express the LPV T antigen.

Figure 2 shows the synthesis of the plasmid pL9 which, Safter cleavage with Hinc I, provides an approximately 4.9 kb fragment which, by reason of its size, can be packaged into virus particles. When this DNA fragment is introduced into the LPV T cells which have been generated the virus particles which are produced are infectious but have a replication defect.

Figure 3, and its continuation Figure 3a, show the synthesis of vectors which are able to express foreign DNA.

This initially entails synthesis of the vector pL61 which contains the DNA replication origin and the Late gene region of LPV. Replacement of the pUC19 portion by the desired gene results in the vector LPV X which, in the cells according to the invention in which stable expression of the early gene region of LPV takes place, results in replication and packaging of the foreign gene X in w~Lirr-~CC~--I-- tl- 3 defective, infectious virus particles.

The principle of the early replacement vector whict ras been described by Gluzman, Cell 23 (1981) 175-182 COS system) was used to construct the LPV vector. The "early" gene region in the genome of LPV codes for a protein (T antigen) which is necessary for the replication of the viral DNA and for the expression of the "late" gene region coding for the virus capsid proteins VP1, VP2 and VP3. The LPV particles which have a replication defect and are used according to the invention as vectors I harbor, in their genome, foreign DNA sequences in place of the early gene region. During the synthesis of these defective particles it is necessary to have the LPV T 15 antigen present in the synthesizing cells, namely the LPVpermissive cells according to the invention, in which stable expression of the early gene region of LPV takes p- place.

The advantageous process, which is described hereinafter, for the preparation of cell lines in which stable expression of LPV T antigen takes place, and for the preparation of infectious LPV particles which have a replication de- 1 fect and are able efficiently and specifically to transport foreign DNA into LPV-susceptible cells and bring about its expression there, can, of course, also be carried out in a modified manner by an expert, for example using other genome fragments, other cell lines and other foreign DNA fragments.

The LPV-susceptible human B-lymphoma cell line which was used was the established cell Line BJA-B (Menezes et al., loc. cit.), since this can be efficiently transfected.

This cell line can be obtained from the European Collection of Animal Cell Cultures (ECACC), Porton Down, Salisbury deposited in accordance with the provisions of the Budapest Treaty, under the number 86081105.

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4 Since the BamHI restriction cleavage site in the starting plasmid pLPV K38 is located in the "early" gene region, it is necessary initially to obtain viral DNA whose early region, which codes for the LPV T antigen, is not interrupted. For this purpose, the plasmid DNA is cleaved out of pLPV K38 with BamHI and is ligated for circularization of the LPV DNA. This ligated DNA is used to transfect BJA-B cells, and the LPV K38 which is synthesized in this culture is used as inoculating virus for the preparation of larger amounts of LPV DNA.

The XmaI restriction fragment, which is 4695 bp in size, from the LPV K38 DNA is cloned into the Xmal restriction cleavage site of the bacterial vector pUC19. The resulting plasmid pL3 contains the entire early gene region and the 3'-terminal part of the late gene region which, however, cannot be expressed because of the loss of the late promoter region and the 5'-terminal sequences. To delete the origin of LPV DNA replication, the remaining, noncoding LPV control region (nucleotides 1 to 528) is replaced by a promoter-enhancer element from the immediate early region of human cytomegalovirus (HCMV) (European i Patent Application with the publication number 0 173 177).

SThis entails the plasmid pL3 being digested with the restriction enzymes NcoI and SaLI, and the ends being filled in by use of the Klenow fragment of DNA polymerase I from E. coli. The AvaII-Hincil restriction I fragment which is 650 bp in size (nucleotides -598 to t is isolated from the HCMV DNA (PstI-m fragment) and, after the 5'-protruding ends have been filled in, is cloned into the prepared fragment of the plasmid pL3, resuiting in the plasmid pL12. The correct orientation of the HCMV promoter-enhancer element with respect to the early LPV gene region is evident, on the one hand, from the reconstitution of the NcoI-AvaII cleavage site and, on the other hand, by digestion with BgLI, by reason of the two cleavage sites for this restriction enzyme in the pUC19 DNA and the BglI cleavage sites in the HCMV fragment.

6 +1 3- 5 The plasmid pSV2Neo (Southern and Berg, J. Mot. Appl.

Gen. 1 (1982), 327-341) contains the prokaryotic Neo gene together with three eukaryotic regulation elements, namely the early enhancer-promoter, a RNA splice sequence and a polyadenylation signal of the SV40 genome. Eukaryotic cells in which this gene is expressed are resistant to Sthe toxic, neomycin-related aminoglycoside antibiotic G 418. Cleavage of pSV2Neo with AccI, filling-in of the protruding 5'-ends, and subsequent cleavage with EcoRI results in isolation of the fragment which is 2867 bp in size, and which is cloned into the plasmid pL12 which has been digested with EcoRI and SmaI. This results in the plasmid The plasmid pL30 is Linearized with EcoRI, and this DNA is used to transfect the BJA-B cell line. Selection for resistance to G 418 is used to identify Neor cell clones, which are isolated by subcultivation. It is possible by Ij indirect immunofluorescence using an anti-LPV-T serum to identify ceLl lines expressing LPV T antigen. The BJA-B (Neor LPV-T lines obtained in this way are cultivated, and one of these cell clones (31B3-K6) is used for the subsei quent experiments.

j 25 To prepare defective virus particles, the LPV K38 genome i is cloned via the BclI restriction cleavage site (nucleotide 3328) into the BamHI restriction cleavage site of the plasmid pSP64, resulting in the plasmid pL9. This pSP64 insertion ensures, via the inactivation of the early 30 LPV region, that pL9 does not synthesize any functional T antigen. Transfection of the 31B3-K6 cells with the plasmid pL9, which is 8.27 kb in size, in the closed, circular form results in 0.1% LPV-VP cells 48 hours after transfection, and this proportion subsequently decreases again. There is no detectable LPV-VP synthesis in BJA-B cells transfected for control purposes. This proves that the LPV T antigen present in the 31B3-K6 cells is functional.

i LYI 3 -rvy 6- Since, by reason of its size, the plasmid pL9 cannot be packaged into LPV particles, the restriction endonuclease HincII is used to cleave it into a fragment which is 3415 bp in size and contains 370 bp of the early LPV gene region (nucleotides 3328 to 3698) in addition to the pSP64 sequence, together with a fragment which is 4912 bp in size and comprises 4900 bp of the LPV DNA sequence (nucleotides 3698 to 3327) plus 12 bp of the pSP64 DNA sequence. In addition to the non-coding LPV control region with the origin of LPV DNA replication, the fragment which is 4912 bp in size contains the complete late LPV region, and parts of the early '.PV region which, however, are no longer able to code for LPV T antigen.

pL9 plasmid DNA is digested with HincII, and the product is used to transfect 31B3-K6 cells. Once again, 0.1% LPV- VP cells are obtained 48 hours after transfection. In contrast to the cells obtained with undigested pL9 plasmid, the proportion of LPV-VP cells rose to more than 50% during six subsequent days of incubation, which is evidence of the production of infectious LPV particles.

Treatment of BJA-B cells and 31B3-K6 cells with culture supernatants which have been sterilized by filtration resuLts, 48 hours after inoculation, in 10% LPV-VP cells in the 31B3-K6 cells, whereas the inoculated BJA-B cells remain negative for at least eight days. This proves the existence of LPV particles which are infectious but have a replication defect.

S, 30 The LPV capsid is able to harbor a genome which iq up to 5.3 kb in size. The non-coding control region and the late gene region, both of which are required in the genome for the production of defective particles, together comprise 2605 bp. Thus, if all the sequences of the early gene region are deleted, there remain a maximum of about 2.7 kb for cloning in foreign DNA.

After introduction of an EcoRI restriction cleavage site 7 at position 2843 by targeted in vitro mutagenesis (Grundstrom et al., Nucleic Acids Res. 13 (1985) 3305- 3316) it is possible to cut out the entire early LPV gene region by double digestion with the restriction endonucleases NcoI (position 5268) and EcoRI.

This entails the vector pUC19 initially being opened with BamHI and then ligated with the LPV-K38 genome which has likewise been opened with BamHI, resulting in the plasmid pLPV K38-1. The latter is cut with Apal to e.iminate the fragment comprising 240 bp, and the remaining plasmid is religated, resulting in the plasmid pL24. pL24 is cut with BamHI to isoLate the viraL DNA, which is cLoned via Ii the BamHI restriction cleavage site into the vector M13mp18 15 (New England Biolabs), resulting in the vector mL58.

The synthetic oligonucleotide

TCTTCAATTAGAATTCCATGC,

Swhich is 21 bp in size and contains the LPV sequence nucLeotides 2832 to 2852, with a guanosine in place of a i 20 cytosine residue in position 11 (LPV 2842), is hybridized onto the single-stranded phage DNA and is used as primer for the synthesis of the complementary DNA strand with I the Klenow fragment. Mutated M13 clones with EcoRI restriction cleavage sites (mL60) are identified by restriction analysis of the replicative form. The latter I is cut with BamHI and EcoRI to isolate the fragment which I is 2826 bp in size, and which is cloned, via the corresponding cleavage sites, into pUC19, resulting in the plasmid pL61. In addition to the complete late LPV gene region and the origin of LPV-DNA replication, the latter also contains the promoter-enhancer element and the polyadenylation signal sequences of the early LPV gene region, so that it is merely necessary to clone the coding sequences of a foreign gene X into the plasmid which has been opened by double digestion with the restriction endonucleases NcoI and BamHI in order to obtain a functional eukaryotic gene which can be expressed (plasmid pLX).

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1 I1;LU131 8 STest genes suitable for introduction into this are, in particular, those sequences which code for an easily measurable and quantifiable enzyme, such as parts of B- Sgalactosidase or chloramphenicol acetyltransferase.

Double digestion of the plasmid pLX with the restriction endonucleases EcoRI and BamHI, followed by transfection into 31B3-K6 cells, results in the production of defective virus particles LPV X, which are used to infect LPV-susceptible cell lines such as BJA-B or Namalwa. Expression of the foreign DNA can then be demonstrated in an enzymatic reaction in protein extracts of the infected cells.

Other test genes which are suitable are DNA sequences which code for well-characterized antigens, such as hepaj titis B surface antigen (HBsAg) and whose expression is 15 easily detectable by antibody reactions.

The high tissue-specificity can be used according to the invention in such a way that the foreign gene which is introduced codes for a polypeptide which is toxic for the host cell or has an antitumor action. Thus, for example, it is possible to use the A peptide of diphtheria toxin for the specific killing of LPV-permissive lymphoma cells.

i Another possibility is the expression of antisense RNA against activated oncogenes. Antitumor effects can also be elicited by the expression of membrane-bound antigens against which there is good immunity.

Another conceivable use is the immunization with the vector system according to the invention of non-tumor patients 30 since, presumably, certain normal stages of B cell differentiation are also susceptible to LPV infections. The expression, as membrane proteins, of new antigens which are coded for by the foreign DNA, for example HBsAg, can be used to induce antigen-specific, especially cellmediated, immunity. Another possibility for the use of the invention relates to the elimination of healthy tissue, for example in the sense of immunosuppression.

t -UC .lni1 LI__ U _L nu i i^n;aW i.t)nn-rr L4 ir t-iniu-rwi iwW B.=tM fw r~ TT -w .«li.).rrri) .nn; ;i irirr-l 9 The invention has been explained hereinbefore on the basis of LPV and its pronounced tissue-specificity. However, it is known that other human viruses also exhibit tissuespecificity, for example the two human retroviruses HTLV I and LAV/HTLV III, which have a high preference for attacking cells of the T-lymphocyte series. Human papillomaviruses (HPV) have hitherto been isolated only from epithelial cells of the epidermis or the mucosa. The Epstein-Barr virus (EBV), a human herpes virus, has to date been detected only in B-lymphocytes and epithelial cells of the nasopharyngeal space. Thus, the invention also provides a route to construct further tissue-specific vector systems by use of other tissue-specific virus partides.

SThe processes described in the examples which follow are illustrated by the figures, with the numbering of the figures and the examples being identical.

Example 1 The plasmid pLPV K38 is cleaved with BamHI, and the LPV DNA is Ligated for circularization to LPV K38 I After purification, the ligated DNA is mixed, by the method of McCutchan and Pagano, J. Natl. Cancer Inst. 41 (1968), 351-357, for transfection with 2 x 106 washed BJA-B cells in 600 pl of dextran transfection buffer (RPMI 1640 medium containing 500 pi/ml DEAE-dextran of molecular weight 500,000), and the mixture is incubated at room temperature for 30 minutes. After one wash, the transfected cells are incubated in 10 ml of growth medium (RPMI 1640, 10% fetal calf serum) at 37 0 C and 5% by volume

CO

2 The LPV K38 synthesized in this culture is used as inoculating virus for the production of larger amounts of LPV DNA by the method of Hirt, J. Mol. Biol. 26 (1967), 365-369.

Standard methods are used for the subsequent clonings f 10 into E. coLi (strain 490 A) and preparations of plasmid DNA (Maniatis et at., MoLecuLar Cloning, Cold Spring Harbor Laboratory, 1982).

LPV K38 is digested with XmaI, and the fragment which is 4695 bp in size (nucleotides 1104 to 529) is isolated. The vector pUC19 is opened with XmaI, and the linearized plasmid is Ligated with the fragment The plasmid is digested with NcoI and Sail, and the protruding ends are filled in with KLenow polymerase, resulting in the blunt-ended fragment The HCMV AD169 Pstl-m fragment (EP A1-0 173 177) is digested with Avail and HincII, and the fragment which is 650 bp -n size (nucleotides -598 to +52) is isolated.

The blunt-ended fragment is obtained by filling in with KLenow polymerase.

The two fragments and are Ligated under blunt end conditions, resulting in the plasmid pL12 The orientation of the HCMV promoter element with respect to the early LPV gene region is checked for correctness by restriction analysis: the desired plasmid contains a NcoI cleavage site, and BglI results in the expected cuts in the pUC19 portion and in the HCMV fragment.

The plasmid is opened with EcoRI and SmaI, and the large fragment is isolated.

The vector pSV2Neo is digested with AccI, the protruding ends are filled with Klenow polymerase, and then cutting with EcoRI is carried out, resulting in the fragment Ligation of the fragments and (10) results in the plasmid pL30 (11).

The plasmid pL30 (11) is linearized with EcoRI and used iii ram~ 11 7 to transfect 10 cells of the BJA-B cell Line in 3 ml of dextran transfection buffer as described above. After incubation for 48 hours, the cells are diluted to a concentration of 2 x 105 cells per ml and, after addition of G 418 to a final concentration of 1.2 mg/ml, are inoculated into 150 pl cultures in 96-well tissue culture plates.

After 10 days, 50% of the culture supernatant is replaced by fresh medium. 25 days after transfection it is possible to identify Neor cell clones and to isolate them by subcultivation. After a further 10 days, cell lines expressing LPV T antigen can be identified by indirect immunofluorescence using an anti-LPV-T serum. The BJA-B- (Neor LPV-T lines obtained in this way are cloned by plating out. Single-cell BJA-B(Neor LPV-T clones which are obtained after culture for 20 days are processed further.

One of these BJA-B(Neor LPV-T cell clones, the cell line 31B3-K6, is used for the subsequent experiments.

I

Example 2: The commercially available plasmid pSP64 is opened with BamHI, resulting in the linearized plasmid (12).

LPV-K38 is opened with BclI, and the linearized genome (13) is ligated with This results in the plasmid pL9 The latter is digested with HinclI, resulting i in two fragments, (15) of 4912 bp and (16) of 3415 bp.

The cell line 3183-K6 (Example 1) is transfected with the pL9 plasmid DNA which has been digested with HincII j (transfection conditions as in Example resulting in 1 0.1% LPV-VP cells 48 hours after transfection. The proportion of LPV-VP cells increases to abovj 50% in six subsequent days of incubation. It is possible, by transferring culture supernatants which have been sterilized by filtration to 31B3-K6 cells to detect 10% LPV-VP+ cells 48 hours after inoculation. Inoculation of BJA-B cells with the culture supernatant results in no detectable LPV-VP+ cells li-- i /2 12 even after eight days.

Transfection of 31B3-K6 cells with undigested plasmid pL9 (14) results in 0.1% LPV-VP cells 48 hours after transfection, but this proportion subsequently decreases again.

Transfection of BJA-B cells under the same conditions, as a check, results in no detectable LPV-VP synthesis.

Example 3: For the subsequent operations, the LPV genome is initially cloned into the vector pUC19. This entails the latter 15 being opened with BamHI, resulting in the linearized oo. plasmid (17).

o LPV-K38 is likewise opened with BamHI, and the linearized LPV genome (18) is ligated with resulting in the plasmid pLPV K38-1 (19).

o The Apal fragment which is 240 bp in size and contains o the duplication of the origin of DNA replication in LPV- K38 is deleted by cutting (19) with Apal, and the Large 25 fragment is ligated to give the plasmid pL24 Cutting of (20) with BanHI results in the shortened LPV genome which is ligated into the phage vector M13mp18 (22) which has been cut with BamHI, resulting in the vector mL58 (23).

The synthetic oligonucleotide (24) is hybridized onto the single-stranded DNA from (23) and is used as the primer for the synthesis of the complementary DNA strand with Klenow polymerase. Mutated M13 clones having the introduced EcoRI restriction cleavage site are identified by restriction analysis of the replicative form. They are called mL60 c i i i' i -a;us= 13 (25) is digested with BamHI and EcoRI, and the fragment (26) which is 2826 bp in size is isolated. pUC19 is likewise dige~,td with BamHI and EcoRI, and the large fragment (27) is ligated with resulting in the pLasmid pL61 (28).

The plasmid pL61 (28) is cut with BamHI and Ncol, and the large fragment (29) is isolated. The latter is suitable for the incorporation of a DNA fragment (30) which is up to 2.7 kb in size, resulting in a plasmid pLX (31).

Cutting of (31) with EcoRI and BamHI, and transfection into 31B3-K6 cells results in the formation of defective LPV X virus particles (32) which contain the foreign gene X.

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Claims (2)

1. A host-vector system comprising a tissue- specific viral vector in which viral DNA has been replaced by foreign DNA, and a permissive host cell with constitutive expression of said viral DNA, wherein the tissue-specific viral vector embraces the origin of DNA replication and the late gene region of lymphotropic papovavirus (LPV) as herein 0*0° defined, and LPV-permissive cells in which stable expression oo a 00 of the early gene region of LPV as herein defined takes 0 0 o 0 place act as host. S* 2. A process for the expression of foreign DNA, which i comprises replacement of HPV DNA coding for early genes as herein defined in a host-vector system as claimed in claim 1 by said foreign DNA. S3. The process as claimed in claim 2, wherein the foreign DNA codes for a protein which is toxic for the host oao 0 cell. o o0 i 4. A process for the preparation of a vector for a host-vector system as claimed in claim 1, which comprises introduction of a single cleavage site into the genome of LPV between the early and late gene region, as herein defined characterised by incorporation of the late gene region, which embraces about 3 kb, from a NcoI cleavage site up to the cleavage site which has been introduced. A process for the preparation of host cells for a host-vector system as claimed in claim 1, which comprises transfection of a LPV-permissive cell line with a LPV DNA fragment which is capable of supplying the early gene as herein defined functions. L r 33 t U .z. LS I o J

6. A medicament comprising a vector as claimed in claim 1, which codes for a protein which is toxic for the host cell. DATED this 17th day of September, 1990. BEHRINGWERKE AKTIENGESELLSCHAFT o 0 D 00n 00 0"00 0 00 00 0 00 WATERMARK PATENT TRADE MARK ATTORNEYS 'THE ATRIUM', 2ND FLOOR 290 BURWOOD ROAD HAWTHORN VIC. 3122. P I i