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  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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  • A61K51/1203—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules in a form not provided for by groups A61K51/1206 - A61K51/1296, e.g. cells, cell fragments, viruses, virus capsides, ghosts, red blood cells, viral vectors
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  • C07K—PEPTIDES
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  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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  • C12N2710/00011—Details
  • C12N2710/10011—Adenoviridae
  • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
  • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
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  • C12N2830/00—Vector systems having a special element relevant for transcription
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  • C12N2840/00—Vectors comprising a special translation-regulating system
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  • C12N2840/00—Vectors comprising a special translation-regulating system
  • C12N2840/44—Vectors comprising a special translation-regulating system being a specific part of the splice mechanism, e.g. donor, acceptor

Definitions

• the present invention relates generally to the fields of radioimmunobiology, gene therapy and nuclear medicine. More specifically, the present invention relates to a method for monitoring gene transfer and expression into a subject. Description of the Related Ar
• RAIT radioimmunotherapy
• a targeting molecule e. g. a n antibody
• a radionuclide carrying a radionuclide
• This limited efficacy reflects fundamental problems in achieving adequate tumor localization of radiolabeled antibodies, which may be due either to inadequate intratumoral expression of the target antigen or to biodistribution problems associated with the use of intact antibody as the targeting moiety (3-6).
• a second emerging strategy is to use radioactively labeled peptides able to bind to receptor positive tumor cells (e.g. octreotide to somatostatin receptors in malignant carcinoid) (15, 16). Research efforts which provide better radioactive isotope delivery systems and/or targeting strategies will enhance the ability to apply targeted radiation therapy.
• receptor positive tumor cells e.g. octreotide to somatostatin receptors in malignant carcinoid
• Radiolabeled monoclonal antibodies have serious limitations in treating human cancer.
• Successful application of radiolabeled monoclonal antibodies in a single-step protocol for radioimmunodetection and radioimmunotherapy of tumors has been hindered in man by problems related to the low percentage uptake of injected radioactivity in tumors (0.001 to 0.1 % ID/g), the slow penetration of relatively large (160 kD) intact antibodies into tumors and heterogeneous distribution, their long persistence times in normal tissues leading to high background radioactivity and bone marrow suppression, and the development of human anti-mouse antibody (HAMA) responses.
• HAMA human anti-mouse antibody
• the prior art is deficient in the lack of effective means of monitoring gene transfer and expression into a subject to further improve the efficacy of gene therapy.
• the present invention fulfills this long-standing need and desire in the art.
• the present invention is directed to a method for monitoring gene transfer and expression into a subject.
• One method of the present invention includes administering to the subject a genetic vector encoding at least one selected therapeutic gene to be expressed in the subject and a gene for human somatostatin receptor subtype 2 wherein the vector and the genes are capable of being expressed in the subject.
• a somatostatin analogue having a detectable radioisotope label attached thereto is administered to the subject and the subject is imaged to determine if the labeled somatostatin analogue has bound to human somatostatin receptor subtype 2 expressed in the subject. No attempt is being made to distinguish the actual binding to the membrane receptor, and subsequent events that allow for imaging.
• a method for monitoring reporter and/or therapeutic gene transfer and expression into a subject comprising the steps of administering to the subject a vector encoding at least one therapeutic gene and a reporter gene for a membrane expressed targeting molecule, wherein both genes are capable of expressing in the subject; administering a radiolabeled ligand to the subject, wherein the ligand has a high affinity for the membrane expressed targeting molecule; and then imaging the subject by detecting the binding of the radiolabeled ligand with the membrane expressed targeting molecule, wherein the binding is directly proportional to the transfer and expression of the therapeutic gene into the subject.
• the membrane expressed targeting molecule is human somatostatin subtype 2 receptor.
• a recombinant adenoviral vector encoding at least one therapeutic gene and a gene for a membrane expressed targeting molecule.
• membrane expressed targeting molecule is human somatostatin subtype 2 receptor.
• a method of treating an individual having a tumor by administering the recombinant adenoviral vector disclosed herein to the individual.
• the recombinant adenoviral vector may be administered with 5-fluorocytosine (5- FC), ganciclovir (GCV) or a radiolabeled peptide.
• Figure 1 shows recombinant adenovirus vector with expanded tissue tropism expressing HSV-TK and SSTR2.
• Figures 2A and 2B show conversion of tritiated 5-FC to 5-FU and binding of 125 I-somatostatin in SK-OV-3.ipl cell lysates and membrane preparations, respectively.
• SK-OV-3.ipl cells were co-infected at 10, 100, and 300 moi with AdCMVSSTr2, AdCMVCD, or both. Two days later the cells were harvested to prepare lysates for the conversion assay or membrane preparations for the binding assay.
• Figure 2A shows the percentage of tritiated 5-FC converted to 5-FU and Figure 2 B shows the amount of 125 I- somatostatin bound as a percentage of the total radioactivity added. Note that assay for AdCMVSSTr2-infected cells at 300 moi was not performed.
• Figures 3A and 3B show gamma camera images of a 24-well plate containing mixtures of AdSSTr2-infected and uninfected cells during incubation with [' ' 'Inj-DTPA-D-Phe 1 - octreotide ( Figure 3 A ) and after 1 h and an acid wash (Figure 3B). The percentages of SSTr2 positive cells are indicated on the right. The last 3 columns had excess unlabeled octreotide added as an inhibitor.
• Figure 3C shows the percentage dose internally bound per mg protein. After imaging, the cells were harvested, counted in a gamma counter and the percentages of internally bound radioactivity standardized for the total amount of protein.
• Figures 4A-4D show representative gamma camera images of mice injected with [' ' 'Inj-DTPA-D-Phe'-octreotide. Mice bearing s.c. A-427 tumors were administered a single intratumoral injection ( Figures 4 A and 4B ) or two intratumoral injections ( Figures 4C and 4D) of AdSSTr2 (right solid circles) and AdTRHr (left dashed circles).
• [ l ' 'Inj-DTPA-D-Phe 1 -octreotide was administered t.v. 48 h ( Figures 4A and 4C) and 96 h ( Figures 4B and 4 D) after adenoviral injection and the mice imaged 5.5 h later. The squares show the clearance of the radioactivity through the bladder.
• Figure 6 A shows growth of A-427 tumors following intratumoral injection of AdSSTr2 and i. v. injection of [ 90 Y]-SMT 487.
• Groups of mice (n 10) received a total of two AdSSTr2 injections and four injections of either 400 ⁇ Ci or 500 ⁇ Ci [ 90 Y]- SMT 487.
• Untreated controls or no adeno virus plus 500 ⁇ Ci [ 90 Y]- SMT 487 controls are also shown.
• Data represent the median change in tumor surface area for surviving mice in each group (n > 7) relative to the tumor surface area at the time of the first AdSSTr2 injection (Day 0).
• Figure 6B shows proportion of mice with tumors that quadrupled in size from time of first AdSSTr2 injection (Day 0). Solid arrows represent AdSSTr2 injections and dashed arrows represent [ 9 0 Y]-SMT 487 injections.
• the no treatment (_), AdSSTr2 + 400 ⁇ Ci [ 90 Y]-SMT 487 (_), AdSSTr2 + 500 ⁇ Ci [ 90 Y]-SMT 487 (_), and no virus + 500 ⁇ Ci [ 90 Y]-SMT 487 (_) are shown.
• Figures 7A and 7B show the imaging of SSTr2 and
• TK gene expression following i. t. injection of 5 x 10 8 pfu AdSSTr2 (left tumor) or AdTKSSTr2 (right tumor) ( Figure 7 A) or 5 x 10 8 pfu AdTK (left tumor) or 5 x 10 8 pfu AdTKSSTr2 (right tumor) ( Figure 7B ) into s.c. A427 tumor xenografts at 5 h after t ' . v. injection of 99m Tc-P2049 and 131 I-FIAU.
• Figures 8A and 8B show immunohistochemistry of adenovirus-injected tumor xenografts.
• Stained frozen sections (x 200) are from Ad5-CMVhSSTr2-injected tumor, which shows cells expressing somatostatin receptors along apparent injection tract ( Figure 8 A , arrows) (necrosis is at apparent end of tract, upper and lower arrows) and Ad5-CMVTRHr-injkected tumor from same mouse as in Figure 7A showing only scattered macrophage staining ( Figure 8B).
• Figure 9 A shows the images in two mice of gastrin releasing peptide receptor (GRPr) expression using a 99m Tc-labeled BBN analogue.
• GRPr gastrin releasing peptide receptor
• male CD1 mice were injected in the rear footpad with either an adenoviral vector encoding GRPr (Ad-CMVGRPr) or a negative control adenoviral vector encoding ⁇ -galactosidase (AdLacZ).
• Figure 9 B shows tissue biodistribution of 99 m Tc-labeled BBN analogue in the two Experiments.
• Figure 1 0 shows binding of 64 Cu-TETA-octreotide to A427 and SKOV3.ipl membrane preparations from cells which were either uninfected or infected with 10 or 100 pfu/cell of AdSSTR2.
• A427 cells were infected with 10 pfu/cell, while SKOV3.ipl cells were infected with 100 pfu/cell. Binding to infected cells was blocked using a 1 , 000-fold molar excess of unlabeled octreotide.
• Each bar represents the mean amount of 64 Cu-TETA-octreotide bound as a percentage of total radioactivity added ⁇ SD (n > 6).
• Figure 1 1 shows biodistribution of 64 Cu-TETA- octreotide in nude mice inoculated with 2 x 10 7 SKOV3.ipl human ovarian cancer cells i.p. Five days after tumor cell inoculation, AdSSTR2 or AdLacZ was injected i.p., followed by i.p. administration of 64 Cu-TETA-octreotide 2 days later. Four hours after radioligand injection, groups of mice were killed and the organs harvested and counted in a gamma counter.
• Each bar represents the median tissue concentration from a group of 5 animals with a range from the 25th percentile to the 75th percentile.
• Figure 12 shows biodistribution of 6 4 Cu-TETA- octreotide in nude mice inoculated with 2 x 10 7 SKOV3.ipl human ovarian cancer cells i.p. Five days after tumor cell inoculation, AdSSTR2 was injected i.p., followed by i.p.
• Figure 1 3 shows biodistribution of 64 Cu-TETA- octreotide in nude mice inoculated with 2 x 10 7 SKOV3.ipl human ovarian cancer cells i.p.
• AdSSTR2 was injected i.p., followed by i.p. administration of 64 Cu- TETA-octreotide 2 days later.
• 4 and 18 hours after radioligand injection groups of mice were killed and the organs harvested and counted in a gamma counter.
• Each bar represents the median tissue concentration from a group of 5 to 15 animals with a range from the 25th percentile to the 75th percentile.
• Abbreviations are defined in Figure 11.
• Figure 1 4 shows surviving proportion of athymic nude mice that were injected i.p. with 3 x 10 7 SKOV3.ipl cells.
• One group of 5 animals received 1 x 10 9 pfu AdSSTr2 intratumorally on Day 5 followed by 1.4 mCi 64 Cu-TETA-octreotide i.p. on Day 7.
• Another group of 5 animals received 1 x 10 9 pfu AdSSTr2 on Days 5 and 19, and received 1.4 and 0.7 mCi 64 Cu- TETA-octreotide i.p. on Days 7 and 21 , respectively.
• Another group of 5 animals received 1 x 10 9 pfu AdSSTr2 on Day 5 and 2.0 mCi 64 Cu-TETA-octreotide on Day 7.
• An unrelated group of 5 animals served as a control.
• Figure 1 5 shows schema for generation of recombinant GRPr encoding adenovirus under the control of the DF3 promoter element.
• the GRPr gene was subcloned into the sites of pACDF3pLpARS (+) to form the plasmid pACDF3GRPrpLpARS(+).
• the GRPr expression shuttle vector and the adenoviral packaging plasmid pJM17 were cotransfected into 293 cells to generate the recombinant GRPr encoding adenovirus under the control of the DF3 promoter element.
• Figures 16A-C show analysis of the GRPr encoding recombinant adenoviral vectors, AdDF3GRPr and AderbGRPr, under the control of the DF3 and erbB-2 promoters, respectively.
• Figure 16 A is a structural map of AdDF3GRPr genomic DNA demonstrating localization of GRPr expression cassette at the site of El deletion. Restriction endonuclease sites are indicated. The genome is subdivided into 0-100 map units (m.u.). ITR indicates adenoviral inverted terminal repeats of genomic termini.
• Figure 16B shows PCR validation of the AdDF3GRPr and AderbGRPr adenoviral vectors.
• Figure 16C shows restriction analysis of the AdDF3GRPr (lanes 1-6) and AderbGRPr (lanes 8-13) adenoviral vector genomic DNA using the following restriction enzymes.
• Products of the digestion were analyzed by electrophoresis on 1 % agarose gel and ethidium bromide staining.
• Figure 1 7 shows binding of 125 I-BBN to breast cancer cells infected with adenoviral vectors encoding the GRPr under the control of the CMV (AdCMVGRPr) or the erbB-2 (AderbGRPr) promoter at an MOI of 50, 200, and 500 pfu/ cell.
• the cells were harvested 48 hours later and assayed for 1 2 5 I-BBN binding employing an in vitro live-cell binding assay. Uninfected and AdCMVlacZ cells served as controls.
• As a positive control the murine fibroblast cell line, BNR-11 , stably transfected to express the GRPr gene was used. The cells were counted in a well-type gamma scintillation counter. A representative experiment for each cell line done in triplicate is shown.
• Figure 1 8 shows binding of 1 25 I-BBN to breast cancer cells infected with adenoviral vectors encoding the GRPr under the control of the CMV (AdCMVGRPr) or the DF3 (AdDF3GRPr) promoter at an MOI of 50, 200, and 500 pfu/cell.
• the cells were harvested 48 hours later and assayed for 1 2 5 I-BBN binding employing an in vitro live-cell binding assay. Uninfected and AdCMVlacZ infected cells served as controls.
• As a positive control the murine fibroblast cell line, BNR-11 , stably transfected to express the GRPr gene was used. The cells were counted in a well- type gamma scintillation counter. A representative experiment for each cell line done in triplicate is shown.
• Figure 1 9 shows binding of 125 I-BBN to pancreatic a n d cholangiocarcinoma cancer cells infected with adenoviral vectors encoding the GRPr under the control of the CMV (AdCMVGRPr) or the DF3 (AdDF3GRPr) promoter at an MOI of 50, 200, and 500 pfu/cell.
• the cells were harvested 48 hours later and assayed for 125 I-BBN binding employing an in vitro live-cell binding assay. Uninfected and AdCMVlacZ infected cells served as controls.
• As a positive control the murine fibroblast cell line, BNR-11, stably transfected to express the GRPr gene was used.
• Figure 2 0 demonstrates the expression in 4 cell lines of MUC1 (A Panels) and broad spectrum cytokeratins (B Panels). All photographs are at 400 X magnification.
• the cell lines are 1 : SK-ChA-1; 2: MDA-MB-231; 3: SK-BR-3; 4: Oz.
• Figure 2 1 demonstrates the expression in 3 cell lines of MUC1 (A Panels) and broad spectrum cytokeratins (B Panels). All photographs are 400 X magnification.
• the cell lines are 1 : BXPC-3; 2: ZR-75-1 ; 3: ASPC-1.
• Figure 2 2 shows production of adenoviral vectors encoding gene of choice.
• the recombinant shuttle plasmid containing the gene of choice and parts of the adenovirus genome are co-transfected using a transfection reagent into 293 cells with a rescue plasmid containing the majority of the adenovirus genome with deletions in the El and E3 regions.
• the 293 cells contain the El region deleted in the rescue plasmid and thus allow for adenoviral production by homologous recombination between the gene of choice and the rescue plasmid through the Ad5 genome sequences flanking the gene of choice.
• Areas of adenoviral production appear as cytopathic effect plaques in the 293 cell monolayer. These plaques are picked and screened for the gene of choice and the positive plaques are used for large scale production of adenovirus .
• Figure 2 3 shows partial El-defective adenovirus strategy for increasing the expression of hSSTr2 throughout the tumor volume.
• Figures 24A and 24B show initial internalization imaging of different Ad vectors monitored by 99m Tc-P2045 ( Figure 24A) and 125 I-FIAU ( Figure 24B), respectively.
• SKOV3 cells were infected with different vectors (Ad-TK, Ad-TK-hSSTr2, Ad-TK-hSSTr2 (RGD) and Ad-hSSTr2), and then imaged with the injection of the radiotracer 99m Tc-P2045 or 125 I-FIAU.
• Figures 25A and 25B show internalization imaging of different Ad vectors monitored by 99m Tc-P2045 ( Figure 25 A) a n d 125 I-FIAU ( Figure 25B), respectively, wherein the imaging was obtained 20 minutes after the injection of the radiotracer.
• Figures 26 A and 26B show internalization imaging of different Ad vectors monitored by 99m Tc-P2045 ( Figure 26 A) a n d 125 I-FIAU ( Figure 26B), respectively, wherein the imaging was obtained 60 minutes after the injection of the radiotracer.
• Figures 27 A and 27B show internalization imaging of different Ad vectors monitored by 99m Tc-P2045 ( Figure 27 A) an d 125 I-FIAU ( Figure 27B), respectively, wherein the imaging was obtained 90 minutes after the injection of the radiotracer.
• Figures 28A and 28B show internalization imaging of different Ad vectors monitored by 99m Tc-P2045 ( Figure 28 A) a n d 125 I-FIAU ( Figure 28B), respectively, wherein the imaging was obtained 120 minutes after the injection of the radiotracer.
• Figures 29A a n d 29B show the comparison of percentage dose internally bound per ⁇ g protein ( 99m Tc-P2045/ ⁇ g protein) between the cells infected with Ad-TK-SSTr2 ( Figure 29 A) and with Ad-TK-SSTr2-RGD ( Figure 29B) .
• Figure 30 shows the schema of a reporter system to image gene transfer, wherein SSTr2 receptor/ 9 9 m Tc-labeled peptide were used.
• Figure 31 shows imaging of the gene transfer to subcutaneous A427 tumors.
• Nude mice were injected with control Ad on left flank and Ad-hSSTr2 on right flank. 48 hours later, a radiotracer was injected into the mice (' ' l In-octreotide, 99m Tc-P829 and 99m Tc-P2045). Images v ere obtained 5 h after the injection.
• Figures 32 A an d 32B show the imaging of the gene transfer to i.p. ovarian tumors.
• Three mice having SKOV3.ipl tumors were injected with Ad-hSSTr2 vector 48 hours before the injection of a radiotracer, while three others received no Ad vector. Both groups were imaged 5 h after injection of the radiotracer, (e. g. 99 m Tc-P2045).
• Figure 32 A shows the imaging obtained on the non-Ad group, while Figure 32B shows the Ad- treated group.
• Figure 33 A shows the initial images of 1 25 I-FIAU i n both MKN-28 and KATO-3 cells.
• Figure 33B shows the final images of 125 I-FIAU in both MKN-28 and KATO-3 cells, which were taken after 1 hr incubation.
• Figures 34A and 34B show imaging of hSSTr2 reporter gene transfer to A427 cells using the herpes simplex virus.
• Figure 34A shows the initial images of P2045, while Figure 33B shows the final images, which were taken after 1 hr incubation.
• Figures 35A and 35B show imaging of hSSTr2 reporter gene transfer to A427 cells using Ad-hSSTr2 and 188 Re- P2045.
• Figure 35 A shows the initial images of P2045, while Figure 35B shows the final images, which were taken after 1 hr incubation.
• the methods of the present invention induce normal or cancerous cells to synthesize a membrane expressed targeting molecule (an antigen, a receptor or a modification thereof) which will have a high affinity for infused radioisotopic ligands (including antibodies, peptides, drugs, growth factors, hormones, vitamins and proteins).
• a membrane expressed targeting molecule an antigen, a receptor or a modification thereof
• radioisotopic ligands including antibodies, peptides, drugs, growth factors, hormones, vitamins and proteins.
• the use of gene therapy technology to induce expression of high affinity membrane molecules/receptors can be used to image gene transfer of reporter and/or therapeutic genes.
• a bicistronic adenoviral vector encoding both the cytosine deaminase (CD) gene and the human somatostatin subtype 2 receptor gene were produced.
• a bicistronic vector encoding the herpes simplex virus-thymidine kinase (HSV-TK) gene and the human somatostatin receptor subtype 2 gene has also begun to be constructed. These vectors are used to demonstrate imaging of reporter and/or therapeutic gene transfer.
• HSV-TK herpes simplex virus-thymidine kinase
• the present invention provides a method for monitoring gene transfer and expression into a subject by administering to the subject a genetic vector encoding at least one therapeutic gene (e.g. HSV-TK, CD, p53) to be expressed in the subject and a gene for human somatostatin receptor subtype 2 with the genes being capable of expression in the subject.
• a somatostatin analogue or other molecule with affinity for the receptor having a detectable radioisotope attached thereto is administered to the subject and the subject is then imaged in order to determine if the radiolabeled somatostatin analogue has bound to the human somatostatin subtype 2 receptor expressed in the subject.
• the detection of specific binding of the radiolabeled somatostatin analogue with the human somatostatin subtype 2 receptor indicates the transfer and expression of the genes transferred into the subject.
• Such method could be universally used to image gene transfer following the administration of any genetic vector encoding a therapeutic gene and the human somatostatin receptor subtype 2 gene.
• a method for monitoring therapeutic gene transfer and expression into a subject comprising the steps of administering to the subject a vector encoding at least one therapeutic gene and a reporter gene for a membrane expressed targeting molecule, wherein both genes are capable of expressing in the subject; administering a radiolabeled ligand to the subject, wherein the ligand has a high affinity for the membrane expressed targeting molecule; and then imaging the subject by detecting the binding of the radiolabeled ligand with the membrane expressed targeting molecule, wherein the binding is directly proportional to the transfer and expression of the reporter and therapeutic gene into the subject.
• the membrane expressed targeting molecule is an antigen or a receptor or a modification thereof, selected from the group consisting of the human somatostatin subtype 2 receptor, the gastrin releasing peptide receptor, the vasoactive intestinal peptide receptor and the dopamine receptor, and the ligand is an antibody, a peptide, a drug, a growth factor, a hormone, a vitamin or a protein for binding the respective receptor.
• Representative therapeutic genes include herpes simplex virus-thymidine kinase, cytosine deaminase, p53, pl6, bax, tumor antigens, antiangiogenesis molecules, pro-angiogenesis molecules, pro-apoptosis molecules, anti-apoptosis molecules, platelet factor-4, soluble Fit- 1 , angiopoietin-2, thrombospondin-1 , angiostatin, endostatin, Fas, FasLigand, cytokines, GM-CSF, IL-2, IL- 4, IL-6, IL-7, IL-12, TNF- ⁇ and interferon.
• Representative vectors include an adenoviral vector, a retroviral vector, an adeno- associated viral vector, herpes simplex viral vector, vaccinia virus vector, a liposome, plasmid DNA and a synthetic vector.
• Representative radioisotopes used for labeling the ligand include 99m Tc, U 1 ln, 124 I, 18 F, 64 Cu, 123 I, n C, 131 I, 67 Cu, 21 1 At, 177 Lu, 186 Re, 212 Bi, 47 Sc, 105 Rh, 109 Pd, 153 Sm, 188 Re, 198 Au, 199 Au, 75 Br, 76 Br, 77 Br, 13 N, 34m Cl,
• a recombinant adenoviral vector encoding at least one therapeutic gene and a gene for a membrane expressed targeting molecule.
• the membrane expressed targeting molecule is human somatostatin subtype 2 receptor, wherein the gene for the receptor is tagged with the HA sequence of 9 amino acids (YPYDVPDYA, SEQ ID No. 1) at the N-terminus.
• the adenoviral vector may be further tropism-modified by incorporating the tumor cell targeting motifs in the HI loop of adenovirus fiber protein.
• Representative therapeutic genes include herpes simplex virus-thymidine kinase, cytosine deaminase, p53, p l 6, bax, tumor antigens, antiangiogenesis molecules, pro-angiogenesis molecules, pro-apoptosis molecules, anti-apoptosis molecules, platelet factor-4, soluble Flt- 1 , angiopoietin-2, thrombospondin- 1 , angiostatin, endostatin, Fas, FasLigand, cytokines, GM-CSF, IL-2, IL-4, IL-6, IL-7, IL-12, TNF- ⁇ and interferon.
• a method of treating an individual having a tumor by administering the above claimed recombinant adenoviral vector to the individual and further administering 5- fluorocytosine (5-FC), ganciclovir (GCV) or a radiolabeled peptide to the individual, wherein the peptide has a high affinity for a membrane expressed targeting molecule.
• the tumor is selected from the group consisting of ovarian tumor, colon tumor, lung tumor, pancreatic tumor, prostate tumor, breast tumor, head and neck tumor, and glioma tumor.
• the vector is administered systemically or locally by direct injection or via intraperitoneal injection.
• the peptide may be labeled by an radioisotope, e.g., 32 P, 90 Y, 125 I, 212 Pb, 213 Bi, l u In, 64 Cu, 123 I, 13 1 I, 67 Cu, 21 1 At, 177 Lu, 186 Re, 212 Bi, 7 Sc, 105 Rh, 109 Pd, 153 Sm, 188 Re, 198 Au, 199 Au, 77 Br and 175 Yb.
• an radioisotope e.g., 32 P, 90 Y, 125 I, 212 Pb, 213 Bi, l u In, 64 Cu, 123 I, 13 1 I, 67 Cu, 21 1 At, 177 Lu, 186 Re, 212 Bi, 7 Sc, 105 Rh, 109 Pd, 153 Sm, 188 Re, 198 Au, 199 Au, 77 Br and 175 Yb.
• tissue specific promoters include the insulin promoter, which is functional only in insulin-producing cells; oligodendrocyte-specific myelin basic protein (MBP) gene promoter, which is functional only in oligodendrocytes, and tumor specific promoters, which are active only in cancerous cells, or at least significantly more active in these cells (for example a prostate specific antigen or a carcinoembryonic antigen).
• insulin promoter which is functional only in insulin-producing cells
• MBP myelin basic protein
• tumor specific promoters which are active only in cancerous cells, or at least significantly more active in these cells (for example a prostate specific antigen or a carcinoembryonic antigen).
• tissue specific promoters are erbB-2 promoter, MUC 1 promoter, alpha fetoprotein promoter, Cox-2 promoter, E2F promoter and myosin light chain 2 promoter.
• a second mechanism for control of gene expression is provided by inducible promoters or fragments thereof, wherein the fragments are cis-acting control sequences. The expression of genes under control of such promoters or fragments is afforded by addition of an exogeneous compound.
• An example of an inducible promoter is the tetracycline-inducible promoter which becomes active in a subject following administration of tetracycline or doxycycline, a tetracycline derivative.
• Another example of an inducible promoter is the multidrug resistance gene (mdrl) promoter, which is induced by mdrl activating drugs.
• This modification at the N-terminus is an "HA" tag of 9 amino acids (YPYDVPDYA, SEQ ID No. 1) derived from the influenza virus hemaglutinin. When expressed as a protein product, this epitope is recognized by a commercially available antibody.
• the HA sequence has been successfully inserted into both the N-terminal and C-terminal ends of recombinant proteins as a means of protein tagging (39).
• HA-SSTR2 adenovirus generation The expression plasmid used to HA-tag hSSTR2 was provided by Kerry J. Koller, Affymax Research Institute, Palo Alto, CA. Briefly, _+12CA5-KH contains the nine amino acid tag sequence YPYDVPDYA (SEQ ID No. 1) immediately following a starting methionine (39). The presence of unique EcoRI and Notl sites after this sequence enables the insertion of hSSTR2, which was released from the plasmid pAChSSTR2 using the same restriction enzymes .
• pAdTrack-CMV AdEasy system of recombinant adenovirus generation, which takes advantage of homologous recombination in bacteria rather than in eukaryotic cells (40). This system was provided by Bert Vogelstein, Johns Hopkins Oncology Center.
• HA-hSSTR2-pAdTrack-CMV was linearized with Pmel and purified using Wizard DNA clean-up purification resin (Promega, Madison, WI).
• 1 _g of linearized HA- hSSTR2-pAdTrack-CMV and 0.1 _g of the supercoiled adenoviral plasmid p AdEasy- 1 were added. Electroporation was performed in 0.1 mm cuvettes at 1800V in an Eppendorf electroporator 2510, with a time constant between 4-5 sec. The cells were immediately resuspended in 1 ml SOC medium and grown at 37°C for 1 hour.
• the bacteria were then pelleted, resuspended in a smaller volume and plated onto kanamycin resistant plates. Smaller colonies, thought to represent the recombinants, were picked and grown overnight in L-Broth containing 50 _g/ml kanamycin.
• Clones were first screened by comparing their supercoiled DNA sizes on agarose gels to pAdEasy-1 , and possible recombinants were further subjected to PCR analysis using primers for hSSTR2 (forward: 5' TGG ATC CTT GGC CTC CAG 3' (SEQ ID No. 2), reverse: 5' ATT CTA GAA GCC AGG TGT GAG 3' (SEQ ID No. 3)), and Ad5 (forward: 5' TGT CGT TTC TCA GCA GCT GT 3' (SEQ ID No. 4), reverse: 5' TCT GAA CTC AAA GCG TGG GA 3' (SEQ ID No. 5)).
• ElectroMAX DH10B cells Gibco BRL Life Technologies
• Chosen clones were digested with Pac I, transfected into 293 cells (Microbix Biosy stems Inc., Toronto, Ontario) and plaque purified according to standard protocols. Purified plaques were also validated by PCR as described above, and used in a second round of purification in 293 cells. Plaques were again PCR validated and used to make seed stock of the adenovirus. Results for HA-HSSTR2 adenovirus generation:
• HA sequence has been inserted on the N-terminal end of the hSSTR2, and done so in a manner that the chimeric hSSTR2 was unchanged in function. This is the first to utilize the HA-tagged human somatostatin receptor subtype 2 in an Ad5 vector. Inclusion of the HA-tag allows the human somatostatin receptor subtype 2 protein product to be identified in cells by flow cytometry, or by immunohistochemistry.
• Athymic nude mice were utilized for the animal model.
• Human ovarian cancer cells (SKOV3.ipl) were implanted either subcutaneously or intraperitoneally.
• the Ad5 vector targeting the ovarian tumor cells was administered. This was accomplished by either direct injection or via intraperitoneal injection.
• Human non-small cell lung cancer cells were implanted subcutaneously.
• the Ad5 vector encoding the somatostatin receptor subtype 2 gene (AdhSSTR2) was administered by direct injection.
• Control experiments to verify specificity include dilution of specific activity of the radiolabeled peptide in order to saturate the human somatostatin receptor subtype 2 receptors at the tumor site. Additional controls were used. One was an irrelevant peptide without affinity for the receptor under study, while another was a control adenovirus encoding the thyrotropin
• ⁇ _ releasing hormone receptor which does not bind somatostatin analogues. Histology analysis of the tumors was conducted. These studies determine the level and uniformity of the reporter gene expression in the tumors.
• Herpes simplex virus thymidine kinase (HSV-TK) gene was employed as a therapeutic agent.
• Adenovirus vectors were derived encoding the tumor cell targeting motifs within the HI loop, as well as isogenic controls. These vectors were then used in treatment regimens. Analysis of treated groups included reduction of tumor mass within the peritoneum, as well as survival.
• a double expression cassette which replaces an El region of the viral genome, contains herpes simplex virus- thymidine kinase and human somatostatin receptor subtype 2 genes each driven by an immediate-early cytomegalovirus promoter (CMV PR ) and followed by a polyadenylation signal (pA) ( Figure 1).
• CMV PR immediate-early cytomegalovirus promoter
• pA polyadenylation signal
• herpes simplex virus-thymidine kinase allows for therapeutic application of the vector in combination with ganciclovir, whereas expression of the human somatostatin receptor subtype 2 gene provides an opportunity for the vector biodistribution studies via gamma camera imaging.
• An RGD motif incorporated into the HI loop of the adeonvirus fiber protein results in expanded tropism of the vector by allowing the virus to utilize cellular integrins as primary receptors for virus attachment.
• An adenoviral vector which contains the SSTr2 gene and another therapeutic gene, such as cytosine deaminase (CD) gene, could be used to non-invasively determine the level of in vivo expression of the therapeutic gene indirectly by imaging the SSTr2 expression.
• the CD enzyme converts non-toxic 5- fluorocytosine (5-FC) into the toxic 5-fluorouracil (5-FU).
• An initial step towards validating this system is to ensure that both SSTr2 and CD are expressed simultaneously.
• the cells were split into two flasks and grown for an additional 24 hr after which one flask was harvested for a tritiated 5-FC to 5-FU conversion assay and the other flask used in a 125 I-Tyr '-somatostatin binding assay as described above.
• FIG. 2A The results of the tritiated 5-FC conversion assay are shown in Figure 2A.
• the percentage of tritiated 5-FU produced from tritiated 5-FC was 9%, 45%, and 78% when the cells were infected at 10, 100, and 300 moi, respectively.
• Cells infected with a control adenovirus demonstrated only 3% conversion.
• Figure 2B shows the specific binding of ⁇ I-Tyr'-somatostatin to 50 ⁇ g of membrane preparation was 0.4%, 2%, and 5% of the total radioactivity added when the cells were infected at 10, 100, and 300 moi.
• Uninfected SK-OV-3.ip l membrane preparations demonstrated ⁇ 0.2% binding of 125 I-Tyr '-somatostatin.
• both SSTr2 and CD can be expressed in cells after adenoviral co- infection suggesting that a two-gene vector encoding for SSTr2 and CD would yield similar results.
• A-427 cells co- infected with an adenovirus encoding the gene for TK (AdCMVTK) and AdCMVSSTr2 expressed both gene products 48 hr after infection.
• hSSTR2 Expression of hSSTR2 in Lung Cancer Cells
• the adenovirus vector containing the hu m an somatostatin receptor subtype 2 gene was used to infect human lung cancer A427 cells.
• a cell membrane preparation was prepared from these infected cells and 50 _g was incubated with 1 1 'In-labeled octreotide. There was 80% binding of the l u In- labeled octreotide to the cell membrane preparation, which was specifically blocked by excess unlabeled octreotide.
• SSTr2 SSTr2 on A-427 cells following AdSSTr2 infection and binding of radiolabeled peptide.
• the internalization of SSTr2 is important for the intracellular accumulation and retention of radiolabeled octreotide analogues.
• FIG. 5 A The ROI analysis and tissue counts after the second imaging session for each group of animals are shown in Figures 5 A and 5B.
• the ROI analysis in Figure 5 A shows tumor uptake of [" 'Inl-DTPA-D-Phe ⁇ octreotide of -2.8% ID/g 48 and 96 h after a single intratumoral AdSSTr2 injection compared to -1.5% ID/g 48 and 96 h after a second intratumoral AdSSTr2 injection. Uptake of [ 1 1 1 In]-DTPA-D-Phe 1 -octreotide in AdTRHr-injected tumors was ⁇ 0.3% ID/g at both time points.
• Figure 5B shows the biodistribution of [" 'Inj-DTPA-D-Phe 1 -octreotide.
• the uptake in tumors after a single or double injection of AdSSTr2 was greater than in normal tissues ( ⁇ 0.5% ID/g except kidney) and the AdTRHr-injected tumors (0.08% ID/g) for both groups of animals.
• This uptake of [' ' 'Inj-DTPA-D-Phe 1 -octreotide in AdSSTr2-injected tumors was similar to that previously reported in rats bearing pancreatic tumors that natively express SSTr2.
• the kidneys had -18% ID/g in both groups of animals, which agrees with the results of other studies (44).
• AdSSTr2-injected tumors by tissue counting was similar to the ROI analysis for two injections of AdSSTr2, but not a single injection of AdSSTr2. This is likely due to an overestimate of the ROI after a single AdSSTr2 injection. Since the tumors were smaller for the single injection of AdSSTr2, it is hypothesized that some normal tissue surrounding the tumor was induced to express SSTr2 and included in the tumor ROI analysis. Thus, this could account for the discrepancy between the ROI analysis and the tissue counting. These studies showed a high level of SSTr2 expression in tumors up to 4 days following one or two direct intratumoral AdSSTr2 injections.
• the median tumor quadrupling times of the no treatment group and the no virus + 4 doses of 500 ⁇ Ci [ 90 Y]-SMT 487 group were 16 and 25 days, respectively.
• the median tumor areas at the time of treatment were 47-68 mm 2 and the tumor doubling times (7-9 days) were not significantly different among groups.
• the differences in median tumor area between groups began to appear at the beginning of the second week of treatment.
• a second experiment showed differences in the median time to tumor doubling when the initial median tumor areas of the groups were 58-132 mm 2 (data not shown). Possible reasons for the differences between these experiments could be that a smaller fraction of the radiation dose was deposited in the smaller tumors due to the long path length of [ 9 0 Y] emissions or that the smaller tumors were still in an exponential phase of growth that was not inhibited by the first week of treatment. Anderson et al.
• Figures 7A and 7B show the imaging of the expression of two genes (SSTr2 and TK) following Ad transfer to A427 tumors.
• 5 x 10 8 pfu AdSSTr2 (left tumor) or AdTKSSTr2 (right tumor) ( Figure 7 A) or 5 x 10 8 pfu AdTK (left tumor) or 5 x 10 8 pfu AdTKSSTr2 (right tumor) ( Figure 7B) were injected into s.c. A427 tumor xenografts and animals imaged 2 days later at 5 h after i. v. injection of 99m Tc-P2049 and 131 I-FIAU.
• Ad5-CMVTRHr-injected tumors showed minimal background staining, primarily in scattered macrophages.
• Ad5-CMVhSSTr2-injected tumors showed cells expressing somatostatin receptors in 3 of the 4 tumors that were examined. Of interest, the negative tumors had low uptake of the radiolabeled peptide (data not shown). The location of the positive areas appeared to be along the Ad5 needle tract and at the termination of the probable injection site. In addition, areas of necrosis were noted frequently at the terminations of the probable injection tracts for the Ad5-CMVhSSTr2-injected tumors.
• Figure 8A shows cells expressing somatostatin receptor along an apparent injection tract.
• Figure 8B shows no staining in a similarly stained frozen section from the matching control tumor from the same mouse.
• a technique was developed to image gastrin releasing peptide receptor (GRPr) expression using a 99m Tc-labeled bombesin (BBN) analogue.
• BBN 99m Tc-labeled bombesin
• the BBN analogue QWAVGHLM, SEQ ID No. 6
• the HYNIC-peptide was radiolabeled with 99m Tc using tricine as the transchelator and purified again by HPLC. Binding specificity and affinity of the 99m Tc-labeled BBN analogue for GRPr were verified by Scatchard analysis using BNR- 1 1 cells (3T3 cells stably expressing GRPr).
• GRPr expression was induced in the footpad of mice after local injection of a recombinant GRPr-expressing adenoviral vector (Figure 9A).
• Mice were injected IV 48 hours later with 99m Tc-BBN analogue (0.5 ⁇ g, 1.5 MBq), imaged with a gamma camera using a pinhole collimator, and terminated.
• TETA (Aldrich Chemical Co., Milwaukee, WI) was conjugated to octreotide as described elsewhere (43).
• the mixture was added to the SepPak, rinsed with 5 ml of 0.1 M ammonium acetate, pH 5.5 to remove uncomplexed 64 Cu, and rinsed with ethanol to elute the 64 Cu-TETA-octreotide product. Purity was confirmed by reverse phase HPLC and thin layer chromatography.
• the A427 cells were infected at 0 and 10 pfu/cell with AdhSSTR2, while the SKOV3.ipl cells were infected with 0 and 100 pfu/cell of AdhSSTR2 because they are less easily transduced (44).
• the cells were grown for 48 hours and then harvested to form membrane preparations as previously described (44).
• the membrane preparations were then diluted with buffer (10 mM HEPES, 5 mM MgCl 2 , 1 mM EDTA, 0.1% BSA, 10 ⁇ g/ml leupeptin, 10 ⁇ g/ml pepstatin, 0.5 ⁇ g/ml aprotinin, and 200 ⁇ g/ml bacitracin, pH 7.4) to 0.5 mg/ml.
• buffer 10 mM HEPES, 5 mM MgCl 2 , 1 mM EDTA, 0.1% BSA, 10 ⁇ g/ml leupeptin, 10 ⁇ g/ml pepstatin, 0.5 ⁇ g/ml aprotinin, and 200 ⁇ g/ml bacitracin, pH 7.4
• Tumor localization and pharmacokinetics of 64 Cu- TETA-octreotide was investigated in mice bearing i.p. SKOV3.ipl human ovarian tumors induced to express hSSTR2 with AdhSSTR2. Mice bearing i.p. SKOV3.ipl tumors infected with 1 x 1 0 9 pfu AdhSSTR2 or AdLacZ 5 days after tumor cell inoculation followed by i.p.
• mice given AdhSSTR2 had tumor-to-blood, kidney, and liver ratios of 27.3, 2.7, and 2.2 at 4 h after 64 Cu-TETA-octreotide administration respectively.
• mice killed 4 or 18 hours after injection of 6 4 Cu-TETA-octreotide showed a median tumor uptake of 24.3 (17.0-31.1) %ID/g at 4 hours which declined to 4.9 (2.9-8.0) %ID/g at 18 hours (Figure 13).
• Pharmacokinetic analysis demonstrated that clearance of 64 Cu- TETA-octreotide from the tumor, blood, liver, and kidney was best fit using a two compartment model. The radioactivity cleared from tumor with an ⁇ half-life of 1.2 hours and a ⁇ half-life of 27.1 hours. In contrast, blood demonstrated an initial clearance half-life of 0.3 hours followed by accumulation of radioactivity with a doubling time of 46.2 hours.
• One group of 5 animals received 1 x 10 9 pfu AdSSTr2 intratumorally on Day 5 followed by 1.4 mCi 64 Cu-TETA-octreotide i.p. on Day 7.
• Another group of 5 animals received 1 x 10 9 pfu AdSSTr2 on Days 5 and 19, and received 1.4 and 0.7 mCi 64 Cu-TETA-octreotide i.p. on Days 7 and 21 , respectively.
• Another group of 5 animals received 1 x 10' pfu AdSSTr2 on Day 5 and 2.0 mCi 64 Cu-TETA- octreotide on Day 7.
• An unrelated group of 5 animals served as a control.
• Recombinant adenoviral vectors encoding the GRPr gene under the control of the DF3 (AdDF3GRPr) or erbB-2 (AderbGRPr) promoters were constructed.
• the schema used to generate the recombinant adenoviral vectors AdDF3GRPr is shown in Figure 15.
• AderbGRPr was generated using standard methods. The methodology for recombinant adenovirus construction is based upon in vivo homologous recombination between the recombinant adenoviral shuttle vector and the adenoviral packaging plasmid pJM 17.
• the resulting adenoviral vector, AdDF3GRPr would be predicted to contain the GRPr expression cassette inserted in place of deleted adenoviral El sequences ( Figure 16A).
• a similar result would be expected for AderbGRPr generated from pACerbGRPrpLpARS(+) (map not shown) .
• AdDF3GRPr and AderbGRPr genomic DNA yielded the predicted bands for the DF3 and erbB-2- specific primers, respectively (lanes 2 and 6, Figure 16B).
• Analysis for EIA sequences using adenovirus ElA-specific primers indicated the predicted PCR product of 1070 bp from genomic DNA of the Ad2 adenovirus. In contrast, this fragment was absent in amplification of AdDF3GRPr and AderbGRPr DNA, confirming the absence of EIA sequences in these recombinant adeno viruses .
• PCR analysis is consistent with the derivation of EIA(-), GRPr encoding recombinant adenoviruses under the control of either the DF3 or erbB-2 promoter elements and also indicated that no wild-type virus had been rescued by propagation.
• the ability of the AderbGRPr vector to direct specific gene expression was tested in three human breast cancer cell lines, SK-BR-3, ZR-75-1 erbB-2 positive and MDA-MB-231 erbB-2 negative (45,46). Specific binding of 125 I-BBN to the induced GRPr receptor served as the assay to assess gene expression. The amount of binding was quantified as the percent radioactivity bound compared to total radioactivity added.
• the AdCMVGRPr virus showed high levels of GRPr gene expression that increased with increasing MOI at 50, 200 and 500 ( Figure 17) in the three breast cancer cell lines tested.
• the percent 1 25 I-BBN binding for AdCMVGRPr at MOI 50, 200, and 500 were 46%, 54% and 66% for MDA-MB-231 , 54%, 73% and 77% for ZR-75-1 and 68%, 72% and 74% for SK-BR-3, respectively.
• the BNR-11 cells served as a positive control and demonstrated 58 % 125 I-BBN binding.
• only the two erbB-2 positive cell lines showed high level BBN binding following infection with AderbGRPr ( Figure 17) that also increased as the MOI was increased.
• the percent 125 I-BBN binding for AderbGRPr at MOI 50, 200, and 500 were 8%, 7% and 12% for MDA-MD-231 , 29%, 38% and 56% for ZR-75-1 and 15%, 28% and 50% for SK-BR-3, respectively.
• the level of GRPr gene expression was not as great for the erbB-2 promoter as it was for the CMV promoter.
• the percent binding of AderbGRPr versus AdCMVGRPr was 18%, 73% and 68% for MDA- MB-231 , ZR-75-1 and SK-BR-3, respectively. Therefore, specific gene expression employing the erbB-2 promoter was achieved in an adenoviral vector at high levels relative to the CMV promoter.
• the same breast cancer cell lines were tested with the AdDF3GRPr vector employing the same binding assay.
• Specific GRPr gene expression was observed employing the AdDF3GRPr vector ( Figure 18).
• Increased gene expression was observed with increasing MOI (50, 200 and 500) with the AdDF3GRPr vector in the ZR-75-1 and SK-BR-3 cell lines.
• the percent 125 I-BBN binding for AdDF3GRPr at MOI 50, 200, and 500 were 10%, 11 % and 10% for MDA-MB-231, 25%, 51% and 62% for ZR-75-1 and 18%, 41% and 65% for SK-BR-3 cells, respectively.
• GRITS Genetic Radio-Isotope Targeting Strategy
• the percent 125 I-BBN binding for AdCMVGRPr at MOI 50, 200, and 500 were 21%, 31% and 48% for BXPC-3; 36%, 63% and 64% for ASPC-1 ; 21%, 39% and 70% for SK-ChA- 1 and 45%, 58% and 70% for Oz cells, respectively.
• the percent 125 I-BBN binding for AdDF3GRPr at MOI 50, 200, and 500 were 2%, 4%, and 3% for BXPC-3 and 14%, 20% and 18 % for ASPC-1 cells, respectively.
• the DF3 mediated GRPr expression in ASPC-1 was not as great as the other DF3 positive cell lines and did not increase with increasing MOI.
• the SK-ChA-1 cell line gave results similar to the breast cancer cell lines with 17%, 33% and 51% compared to 5%, 5% and 10% for Oz cells at MOIs of 50, 200 and 500 respectively with AdDF3GRPr. Therefore, the DF3 promoter can be used to achieve tumor-specific gene expression in breast, pancreatic and cholangiocarcinoma cells expressing DF3. Weak tumor-specific gene expression was observed in one pancreatic cell line.
• MUCl and cytokeratin expression was analyzed in the panel of human breast, pancreatic, and cholangiocarcinoma cell lines by immunohistochemistry.
• the two breast cancer cell lines with the highest level of MUCl expression, ZR-75-1 and SK-BR-3 had the highest GRPr expression following infection with AdDF3GRPr detected by 125 I-BBN binding (Table 1 , Figures 20 and 21).
• MDA-MB-231 had low MUCl expression and low GRPr expression following infection with AdDF3GRPr (Table 1 , Figure 20).
• a similar result was observed for the cholangiocarcinoma cell lines.
• SKChA-1 cells had a higher level of MUCl expression compared to Oz cells which corresponded to a higher level of I 25 I- BBN binding to AdDF3GRPr infected cells (Table 1 and Figure 20).
• the pancreatic cell lines showed a different relationship. While BXPC-3 cells had an intermediate level of MUCl expression, GRPr expression after infection with AdDF3GRPr was very low (Table 1 and Figure 21). However, GRPr expression after infection with AdCMVGRPr was lower in BXPC-3 cells than for the other cell lines.
• ASPC-1 cells had low level MUCl expression but a higher level of GRPr expression following AdDF3GRPr infection than BXPC-3 cells.
• GRPr expression was higher in AdCMVGRPr infefcted ASPC-1 cells than BXPC-3 cells. All cell lines were positive for control cytokeratin staining. Therefore, the expression of MUC l correlated well with GRPr expression using AdDF3GRPr in breast and cholangiocarcinoma cell lines, but not with the pancreatic cell lines.
• Adenoviral vectors with an El deletion are replication-deficient and thus are capable of infecting a cell only once. Therefore, the cell does not die as a result of infection.
• the desired cDNA is usually cloned into a shuttle (expression) plasmid that contains a portion of the El region of the viral genome. The large size of the adenoviral genome prevents the use of a single plasmid system.
• a system called homologous recombination whereby the cDNA recombinant shuttle plasmid and a rescue plasmid containing the rest of the adenoviral genome are co-transfected into a packaging cell line ( Figure 22).
• the 293 human transformed primary embryonal kidney cell is the standard packaging cell line that has been stably transfected with the El region of the adenoviral genome (50). This allows the El -deleted vector to be made at high titer and remain replication-deficient.
• Another method of adenoviral generation allows for recombination in bacteria. This method, described by He et al. , permits broad screening of recombinants before introduction into 293 cells (51).
• adenoviral vectors The main advantage of using adenoviral vectors is their high in vivo transduction efficiency. Another advantage is their ability to infect non- dividing as well as dividing cells. The capability to produce adenoviral vectors at high titers is also important. Disadvantages of using adenoviral vectors include host immune response against the vector preventing re-administration, transient expression because it is non-integrating, and lack of cell-specific infection resulting in gene delivery to normal cells.
• Ad ⁇ 24SSTr2 Another replication-competent vector that has been constructed and is being evaluated is an adenovirus encoding SSTr2 that contains a 24 base pair deletion ( ⁇ 24) in the EIA region (Ad ⁇ 24SSTr2).
• the ⁇ 24 should allow the adenovirus to replicate only in cancers that have disruption of the Rb protein.
• Ad ⁇ 24SSTr2 was used to infect A-427 cells at 1 and 10 moi. Forty-eight hr after infection, the cells were harvested for a membrane preparation and a binding assay was conducted with 125 I-Tyr '-somatostatin.
• the membrane preparations infected at 1 and 10 moi showed 18% and 16% binding, respectively, which was inhibited to ⁇ 8% with an excess of unlabeled somatostatin.
• the ability of Ad ⁇ 24SSTr2 to replicate was confirmed by seeding uninfected A-427 cells into the original flask of A-427 cells that were harvested for the membrane preparation. The hypothesis is that Ad ⁇ 24SSTr2 left in the flask could infect the newly seeded A- 427 cells. This was confirmed by performing a binding assay on these cells 48 hr after seeding which resulted in 18% binding from both the original 1 and 10 moi flasks. Three subsequent passages in this manner resulted in 15-17% binding.
• Figure 23 shows partial El -defective adenovirus strategy for increasing the expression of hSSTr2 throughout the tumor volume.
• Ad-TK Ad-TK-hSSTr2
• Ad-TK- hSSTr2 RGD
• Ad-hSSTr2 Ad-TK- hSSTr2
• Figures 24-28 show the time course imaging study (initial, 20 minutes, 60 minutes, 90 minutes and 120 minutes, respectively). After imaging, the cells were harvested, counted in a gamma counter and the percentages of internally bound radioactivity were standardized for the total amount of protein.
• Figure 29 shows the comparison of percentage dose internally bound per ⁇ g protein ( 99m Tc-P2045/ ⁇ g protein) between the cells infected with Ad-TK-SSTr2 (A) and with Ad-TK-SSTr2-RGD (B).
• Human lung cancer cells (A427) were injected s. c. in the right and left flanks of 8 nude mice (2 groups of 4/group) (see Figure 30 for the schema of a reporter system to image gene transfer). After 14 days all right tumors were directly injected with Ad-TK-hSSTr2 (5 x 10 8 plaque forming units). The left tumors were injected with the same dose of Ad encoding TK alone (Ad-TK, Group A) or Ad encoding hSSTr2 alone (Ad-hSSTr2, Group B).
• hSSTr2 The expression of hSSTr2 following gene transfer was monitored using a new 9 9 m Tc-labeled somatostatin-analogue, P2045 (Diatide, Inc.), while TK enzymatic activity was monitored w i th l 3 1 I-2'-deoxy-2'-fluro-_-D-arabinofuranosyl-5-iodouracil (FIAU). 99m Tc-P2045 (54 MBq/nmol, 22 MBq) and 13 1 1-FIAU (23 MBq/nmol, 5.6 MBq) were injected i. v. 48 h later.
• Radiotracers in tumors and other tissues were determined by region of interest (ROI) and gamma counter analyses.
• Left Group A tumors accumulated 3.7 ⁇ 1.3% ID/g for 131 I-FIAU, while left Group B tumors accumulated 9.0 ⁇ 2.5% ID/g for 99 m Tc-P2045.
• Gamma counter and ROI analyses were in agreement for the levels of tumor uptake for the radiotracers.
• the 99 m Tc-P2045 showed 7-fold greater accumulation in Ad-TK-hSSTr2 injected tumors as compared with 13 I I-FIAU at 26 h.
• the rapid blood clearance and high retention of 99m Tc .P2045 in Ad-TK-hSSTr2-injected tumors offers great potential for this agent to image expression of the hSSTr2 reporter gene.
• 99 m Tc-P2045 was linearly correlated with 13 1 I-FIAU uptake, the 9 9 m Tc-P2045 imaging provides a convenient assessment of TK gene transfer. Imaging hSSTr2 expression with 99m Tc-P2045 is thus a highly sensitive procedure for detecting Ad- mediated gene transfer of both the reporter and therapeutic genes.
• Ad-Cox2L-TK vector was used in this study.
• MKN-28 cell line permissive for Cox-2 expression
• KATO-3 cell line negative for Cox-2 expression
• Uninfected cells no Ad
• Internal binding of 125 I- FIAU was imaged according to the method described above.
• Figure 33A shows the initial images of 125 I-FIAU in both MKN-28 and KATO-3 cells
• Figure 33B shows the final images of 125 I- FIAU, which was taken after 1 hr incubation.
• MKN-28 cells infected 48 h earlier with Ad-Cox-2-TK showed increased specific internal binding of 125 I-FIAU relative to uninfected cells. Such binding was specific since it was inhibited with excess unlabeled FIAU.
• EXAMPLE 2 2 Imaging hSSTr2 Reporter Gene Transfer Using the HSV Herpes virus encoding hSSTr2 was used to infect A427 cells. 99m Tc-P2045 was used to monitor the expression of hSSTr2 following gene transfer. 48 hours later, the internalization of 99m Tc-P2045 was imaged. Excess P2045 was used to show the specificity. Two sets of triplicates were tested in a 12-well plate. The initial and final images are shown in Figures 34A and 34B.
• A427 cells infected with the Herpes virus encoding hSSTr2 showed increased specific internal binding of 99m Tc-P2045, relative to no internal binding with uninfected cells. Such binding was specific since it was inhibited with excess unlabeled P2045.
• Ad-hSSTr2 vector was used to infect A427 cells.
• the infected cells were undergone 100, 75, 50 and 25% positive cell dilution.
• 188 Re-P2045 was used to monitor the expression of hSSTr2 following gene transfer (_-shield was required on collimator). 48 hours later, the internalization of 188 Re-P2045 was imaged. Excess P2045 was used to show the specificity. The initial and final images are shown in Figures 35 A and 35B.
• 64 Cu is a positron emitter which can be detected by positron emission tomography (PET). Such detection is very sensitive with high resolution.
• PET positron emission tomography
• a mouse is imaged with a subcutaneous SKOV3.ipl ovarian tumor or A427 lung tumor following intratumoral injection of AdCMVhS STR2 and intravenous injection of 64 Cu-TETA-octreotide.
• radioisotopes used for imaging are 99m Tc, m In, 124 I, 1 8 F, 123 I, n C, 131 I 67 Cu, 21 1 At, 177 Lu, 186 Re, 212 Bi, 47 Sc, , 05 Rh, 109 Pd, 153 Sm, 188 Re, 198 Au 1 99 Au, 75 Br, 76 Br, 77 Br, 1 3 N, 34m Cl, 38 C1, 52m Mn, 55 Co, 62 Cu, 68 Ga, 72 As 76 As, 72 Se, 73 Se, 75 Se and 94m Tc.
• radioisotopes 32 P, 90 Y, 125 I, 212 Pb, 213 Bi, U 1 ln, 123 I, 131 I, 67 Cu, 21 1 At, 177 Lu, 186 Re, 175 Yb, 212 Bi 47 Sc, 105 Rh, 109 Pd, 153 Sm, 188 Re, 198 Au, 199 Au and 77 Br may also be used for therapy.
• Some of the radioisotopes can be detected by PET or single photon emission computed tomography (SPECT). Others can be detected by planar imaging.
• SPECT single photon emission computed tomography
• the demonstration of enhanced binding/gene transfer to ovarian and lung cancer cells in vitro would warrant their evaluation in stringent models relevant to human diseases.
• a number of adenoviral-vector based strategies for carcinoma of the ovary were developed and an orthotopic h u m an/murine xenograft model for the analysis of the therapeutic efficacy of these approaches was employed (41-42).
• the present invention provides the herpes simplex virus thymidine kinase (HSV-TK) gene and cytosine deaminase (CD) gene as examples of the therapeutic agents.
• Adenovirus vectors were derived encoding the tumor cell targeting motifs within the HI loop, as well as isogenic controls.

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