WO1998018489A1 - Augmentation de la chimiosensibilite et de la radiosensibilite de cellules tumorales au moyen d'anticorps intracellulaires a une seule chaine - Google Patents

Augmentation de la chimiosensibilite et de la radiosensibilite de cellules tumorales au moyen d'anticorps intracellulaires a une seule chaine Download PDF

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WO1998018489A1
WO1998018489A1 PCT/US1997/019911 US9719911W WO9818489A1 WO 1998018489 A1 WO1998018489 A1 WO 1998018489A1 US 9719911 W US9719911 W US 9719911W WO 9818489 A1 WO9818489 A1 WO 9818489A1
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sfv
cell
cells
erbb
bcl
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PCT/US1997/019911
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Donald J. Buchsbaum
David T. Curiel
Murray Stackhouse
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The Uab Research Foundation
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Priority to AU52431/98A priority Critical patent/AU5243198A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA

Definitions

  • the present invention relates generally to the fields of immunology and protein chemistry. More specifically, the present invention relates to a enhancement of tumor cell chemosensitivity and radiosensitivity using single chain intracellular antibodies.
  • Ovarian carcinoma is the leading cause of death from gynecologic cancer in the United States. Approximately 26,600 new cases were estimated to occur in 1995, resulting in 14,500 deaths from this disease. This figure exceeds the number of deaths from all other gynecologic malignancies combined. Over 70% of the patients present with late stage disease, the majority of which cannot be completely resected at the time of initial surgery. Chemotherapy has become the primary adjunct to surgery in obtaining a clinical remission or enhanced disease free survival in ovarian cancer patients . Although response to initial chemotherapy in ovarian cancer patients approaches 70%, most are transient and approximately 80% of patients (particularly those with advanced stage disease) will recur and eventually die of disease. Although a variety of salvage agents and strategies have been investigated, few have demonstrated long term effectiveness. In this regard, the five-year survival of patients with stage III disease remains, 15% to 30%.
  • cytotoxic prodrugs may be adjunctive to conventional chemotherapeutic modalities.
  • methods to enhance tumor cell conversion of cytotoxic prodrugs to their active forms have been developed. These include methods to enhance tumor cell metabolism of standard anti-tumor agents , such as oxazaphosphorines, by tumor cell transduction with cytochrome P- 450.
  • transfer of viral or prokaryotic genes such as the herpes simplex virus thymidine kinase (HSVTK), and E.
  • HVTK herpes simplex virus thymidine kinase
  • coli cytosine deaminase are employed to sensitize tumor cells to the prodrugs ganciclovir or 5-fluorucytosine (5-FC), respectively, by conversion to toxic metabolites.
  • methods have been proposed based upon specifically reverting the molecular basis of the drug-resistant phenotype. This approach is based upon the concept that tumor cell drug resistance may be the result of diverse genetic alterations. These include mutational changes that lead to modifications in the structure of level of topoisomerase, to increased detoxification reactions, or to interference with the delivery of cytotoxic drug to intracellular targets.
  • alterations affecting the regulation of the cell cycle and apoptosis are highly associated with drug resistance.
  • Gene transfer approaches may be adjunctive to conventional radiation therapy.
  • methods to enhance tumor cell conversion of non-cytotoxic prodrugs to their active forms have been developed.
  • the active forms of these drugs are potential or known radiosensitizers.
  • transfer of viral or prokaryotic genes such as herpes simplex thymidine kinase (HSVTK), and E. coli cytosine deaminase are employed to sensitize tumor cells to the prodrugs ganciclovir or 5- fluorocytosine (5-FC), respectively, by conversion to toxic metabolites. Both of these systems have also been employed to demonstrate enhanced radiation sensitivity.
  • An alternative employed to enhance radiosensitivity in tumors involves the use of radiation inducible promoters to control gene expression.
  • TNF ⁇ tumor necrosis factor- ⁇
  • methods have been proposed based upon specifically reverting the molecular basis of the radiation resistant phenotype. Alterations affecting the regulation of the cell cycle and apoptosis are highly associated with radiation resistance or sensitization. These alterations include inactivation of tumor suppressor genes, in particular p53 and Rb, and overexpression of proto-oncogenes such as those belonging to the ras and myc families, although this is not universal.
  • Inactivating DNA DSB repair genes could be an effective method to dramatically increase the radiosensitivity of human tumor cell lines.
  • gene therapy strategies may provide novel mechanisms to enhance radiation efficacy.
  • the erbB-2 oncogene is important to the malignant transformation of selected neoplasms including ovarian carcinomas .
  • ErbB-2 is a 185 kDa transmembrane protein kinase receptor with homology to the family of epithelial growth factor receptors.
  • Aberrant expression of the erbB-2 gene may play a role in neoplastic transformation and progression.
  • ectopic expression of erbB-2 is capable of transforming rodent fibroblasts in vitro.
  • transgenic mice carrying either normal or mutant erbB-2 develop a variety of tumors, including neoplasms of mammary origin.
  • amplification and/or overexpression of the erbB-2 gene occurs in a variety of human epithelial carcinomas, including malignancies of the ovary, breast, gastrointestinal tract, salivary gland, and lung.
  • ovarian carcinoma a direct correlation has been noted between overexpression of erbB-2 and aggressive tumor growth with reduced overall patient survival.
  • erbB-2 overexpression may be a key event in malignant transformation and progression, strategies to ablate its expression would be therapeutic.
  • erbB-2 Overexpression of erbB-2 is associated with tumor cell chemoresistance. In addition to its direct role in neoplastic conversion, erbB-2 overexpression is associated with tumor cell resistance to chemotherapeutic agents . In this regard, heterologous overexpression of human erbB-2 accomplished by genetic transduction has been shown to increase the chemoresistance of murine fibroblasts and human lung carcinoma cells to a variety of chemotherapeutic agents. These findings are corroborated by the clinical observation that erbB -2 overexpressing tumors possess a higher intrinsic chemoresistance and thus are associated with a shorter relapse-free interval.
  • Another line of evidence supporting the role of erbB-2 in modulating tumor cell chemoresistance is the observed therapeutic synergy between cisplatin and anti-erbB-2 monoclonal antibodies. These studies have documented that anti-erbB-2 antibodies capable of down-regulating the erbB-2 oncoprotein achieve enhanced tumor cell sensitivity to this chemotherapeutic agent. Thus, the erbB-2 oncoprotein plays a key role in determining tumor cell chemoresistance. Therapeutic strategies for cancer have been developed which target the erbB-2 gene product. The association of overexpression of the erbB-2 gene product with neoplastic transformation and chemoresistance has led to the development of therapeutic strategies to down modulate erbB-2 levels in target tumor cells.
  • mAbs monoclonal antibodies
  • mAbs monoclonal antibodies
  • Gene therapy methods have been proposed to target erbB-2 overexpressing tumor cells to achieve down modulation of the oncoprotein. These approaches have included antisense strategies targeted to the transcriptional and post-transcriptional levels of gene expression. In the former instance, triplex-forming oligonucleotides binding the erbB-2 promoter region inhibit transcription of the erbB-2 gene. In addition, antisense oligonucleotides targeted to the erbB-2 transcript have accomplished phenotypic alterations in erbB-2 overexpressing tumor cells including down-regulation of cell surface expression and inhibition of proliferation. Alternative methods to achieve targeted knockout of erbB-2 have been developed.
  • single-chain immunoglobulin (sFv) molecules retain the antigen-binding specificity of the immunoglobulin from which they are derived, however, they lack other functional domains characterizing the parent molecule.
  • the Bcl-2-related protein family is an important regulator of programmed cell death or apoptosis.
  • Members of this family with death antagonist properties include Bcl-2, Bcl-X, Bcl- w, Bfl-1 , Brag- 1 , Mcl- 1 and Al .
  • Most of these proteins have to localize to the mitochondria to regulate apoptosis.
  • overexpression of death antagonist genes from the Bcl-2 family have been shown to protect a variety of cell lines from apoptosis induced by anti-cancer drugs.
  • the Bcl-2 gene encodes a 26 kD protein that regulates apoptosis, at least in part, via its interaction with other members of the Bcl-2 family.
  • Bcl-2 is mainly localized as an integral mitochondrial membrane protein, although Bcl-2 is also found to be associated with other membranes, including those of the endoplasmic reticulum (ER) and the nucleus.
  • ER endoplasmic reticulum
  • Bcl-2 promotes cell survival by preventing the onset of apoptosis induced by a wide variety of stimuli, including essentially all classes of anticancer drugs and x- irradiation.
  • a role for Bcl-2 in cancer was initially identified in follicular lymphoma bearing the chromosomal translocation t(14; 18) that juxtaposes the Bcl-2 gene with the immunoglobulin heavy chain locus, thereby up-regulating its expression.
  • Bcl-2 may play an important role in protecting cancer cells from death induced by anti-cancer drugs.
  • Estrogen-induced increases in Bcl-2 in the context of an estrogen-responsive human breast cancer cell line significantly enhanced their resistance to apoptosis, whereas antisense mediated reduction in Bcl-2 increased their sensitivity to anticancer drugs.
  • Taxol-mediated inactivation of Bcl-2 by phosphorylation in prostate cancer cell lines renders them susceptible to apoptosis.
  • Bcl-2 expression in ovarian cancer cells affects the cellular response to apoptosis and modulates their resistance to anti-cancer drugs.
  • many non-Hodgkin lymphomas (NHL) and some acute myeloid leukemias (AMLs) often overexpress Bcl- 2.
  • Clinical studies of these hematological malignancies suggest an association between Bcl-2 expression, resistance to apoptosis, poor response to chemotherapy and shorter patient survival. Taken together, these results suggest a central role for Bcl-2 in the promotion of cell survival in solid and hematopoietic tumors.
  • antisense (AS) oligonucleotides targeted against Bcl-2 mRNA sequences and plasmid derived Bcl-2 AS transcripts have been shown to alter the growth and survival of lymphoid cells overexpressing Bcl-2 in vitro .
  • AS antisense
  • several independent Bcl-2 AS studies have demonstrated a significant increase in apoptosis in treated cells, as well as more effective tumor cell killing following exposure to chemotherapeutic drugs.
  • BAG- 1 a newly described Bcl-2 binding protein, functions in concert with Bcl-2 to prolong cell survival.
  • Bcl-2 functions in concert with Bcl-2 to prolong cell survival.
  • gene transfer experiments have shown that coexpression of BAG- 1 and Bcl-2 markedly enhanced protection from apoptosis induced by a variety of stimuli compared to cells transduced with either BAG-1 or Bcl-2 alone.
  • overexpression of BAG- 1 in liver progenitor cells increased hepatocyte growth factor (HGF)- and platelet-derived growth factor (PDGF)-induced protection from apoptosis.
  • HGF hepatocyte growth factor
  • PDGF platelet-derived growth factor
  • BAG-1 Although the predicted amino-acid sequence of BAG- 1 shares no homology with other proteins of the Bcl-2 family, it specifically interacts with Bcl-2 and can activate Raf-1 kinase. Of note, BAG-1 lacks the Bcl-2 family transmembrane domain and thereby localizes to the cytoplasm where it can interact with the cytoplasmic domain of the HGF and PDGF receptors. Despite these findings, the precise role of BAG-1 remains unclear, but the fact that it is expressed ubiquitously, and that it acts in conjunction with different growth factor receptors in preventing apoptosis, suggest that BAG- 1 can function as a common adaptor protein between tyrosine kinase receptors and the anti-apoptotic machinery of the cell.
  • the prior art is deficient in the lack of effective means of enhancing tumor cell chemosensitivity to cancer drugs and enhancing sensitivity to radiation.
  • the present invention fulfills this longstanding need and desire in the art.
  • a method of enhancing the chemosensitivity and radiosensitivity of a neoplastic cell comprising the step of: introducing into the cell an antibody homologue, wherein the antibody homologue is expressed intracellularly and binds to a target protein intracellularly.
  • a method of enhancing the radiosensitivity of a neoplastic cell comprising introducing into the cell a nucleic acid molecule encoding an antibody homologue, wherein the antibody homologue is expressed intracellularly and binds to the oncoprotein intracellularly in the endoplasmic reticulum of the cell.
  • a method for enhancing the inhibition of proliferation of a neoplastic cell expressing an oncoprotein that stimulates proliferation of the cell comprising the steps of: introducing into the cell a nucleic acid molecule encoding an antibody homologue, wherein the antibody homologue is expressed intracellularly and binds to the protein intracellularly; and contacting said cell with an anti-neoplastic agent.
  • a method of enhancing the chemosensitivity and radiosensitivity of a neoplastic cell comprising the step of: introducing into the cell an antibody homologue, wherein the antibody homologue is expressed intracellularly and binds to a target protein intracellularly; and contacting said cell with an anti- neoplastic agent, radiation or a combination thereof.
  • Figure 1 shows the effect of intracellular anti-erbB-2 sFv on cell surface expression of erbB-2 protein.
  • the human ovarian carcinoma cell line SKOV3 was transfected by the AdpL method with the described plasmid constructs and analyzed for cell surface erbB-2 at 96 hours post-transfection using an anti- human erbB-2 polyclonal antibody.
  • Original magnification 400X shows the transfection with control plasmid pcDNA3.
  • Figure IB shows the transfection with non-ER form of anti-erbB- 2 sFv plasmid, pGT20.
  • Figure IC shows the transfection with ER form of anti-erbB-2 sFv plasmid, pGT21.
  • Figure 2 shows the effect of expression of intracellular anti-erbB-2 sFv genes on tumor cell viability in the erbB-2 overexpressing human ovarian carcinoma cell line SKOV3 ( Figure 2A) and the non-erbB-2 expressing cervical carcinoma cell line HeLa ( Figure 2B).
  • Tumor cell targets were transfected with the plasmids ⁇ cDNA3, pGT20 and pGT21. At indicated times post- transfection, cell viability was determined employing the XTT assay. Assays were performed xl2 at each time point.
  • Figure 3 shows the determination of apoptotic DNA fragmentation induced by ER anti-erbB-2 sFv.
  • Tumor cells were transfected with the plasmids pcDNA3, pGT20 and pGT21. At indicated time points post-transfection, cells were harvested and chromosomal DNA analyzed by gel electrophoresis.
  • Figure 3A shows transfection of the non-erbB-2 expressing human cervical cell line HeLa.
  • Figure 3B shows transfection of the erbB-2 overexpressing human ovarian carcinoma cell line SKOV3.
  • Figure 4 shows the effect of coexpression of erbB-2 and the anti-erbB-2 sFv on HeLa cell viability.
  • the non-erbB-2 expressing human tumor cell line HeLa was transfected with plasmids encoding the ER form of the anti-erbB-2 sFv (pGT21 ) and/or the human erbB-2 expression vector LTR-2/erbB-2.
  • pGT21 the anti-erbB-2 sFv
  • LTR-2/erbB-2 human erbB-2 expression vector
  • Figure 5 shows the effect of expression of the anti- erbB-2 sFv gene on human ovarian tumor cell viability.
  • ErbB-2 expressing human primary ovarian carcinoma cells isolated from malignant ascites were transfected with pcDNA3, pGT20 or pGT21.
  • ErbB-2 overexpressing ovarian carcinoma cells (SKOV3) were used as additional controls. Cells were assayed for viability by the XTT assay at 96 hours post-transfection. This experiment was replicated lOx. Data represents mean ⁇ ' SEM.
  • Figure 6 shows the efficacy of various vectors in accompli shing in vivo gene delivery.
  • Intraperitoneally transplanted SKOV3.ipl cells were challenged with different vector systems delivering the lacZ reporter gene.
  • Peritoneal lavage contents were subjected to FACS analysis for lacZ expression in erbB-2 overexpressing tumor cells.
  • Figure 7 shows the effect of expression of a recombinant adenovirus encoding anti-erbB-2 sFv gene on viability of erbB-2 overexpressing human ovarian tumor cells.
  • SKOV3.i ⁇ l cells were infected with recombinant adenovirus encoding the cytosolic or ER directed anti-erbB-2 sFvs.
  • the XTT assay was employed to determine cell viability 96 hours post infection. This experiment was replicated lOx. Data represents mean ⁇ SEM.
  • Figure 8 shows the in vivo efficacy of the recombinant adenovirus encoding the ER form of the anti-erbB-2 sFv in prolongation of survival.
  • Figure 9 shows the cytotoxic effect of an anti-erbB-2 sFv in combination with CDDP.
  • the erbB-2 overexpressing ovarian carcinoma cell line SKOV3 was transfected with a plasmid construct encoding an ER form of an anti-erbB-2 sFv (pGT21 ) or a control plasmid construct (pcDNA3) and treated with CDDP (2 ⁇ g/ml). The cells were incubated for 72 hours and the number of viable cells determined by an MTS assay. Experiments were performed in triplicate and the results represent the mean ⁇ SEM.
  • Figure 10 shows the characterization of anti-erbB-2 sFv expressing SKOV3.ip l clones.
  • Figure 10A shows the determination of cell surface erbB-2 protein expression in stable clones as determined by an ELISA assay. Relative erbB-2 levels were calculated from a standard curve. SKOV3.ipl cells are a positive control while HeLa, an erbB-2 negative human cervical cell line, served as a negative control. Results are expressed a mean ⁇ SEM.
  • Figure 10B shows the determination of the' presence of anti-erbB-2 sFv in stable clones by Western blot. Cell were solubilized and the samples electrophoresed with probe analysis using a polyclonal rabbit anti-erbB-2 sFv antibody.
  • Figure 11 shows the sensitivity of anti-erbB-2 sFv expressing SKOV3 clones to CDDP.
  • SKOV3/pGT21 clones expressing the ER form of the anti-erbB-2 sFv demonstrate enhanced chemosensitivity to CDDP.
  • SKOV3/pGT21 clones were treated with CDDP (2 ⁇ g/ml) and incubated for 72 hours. Cell viability was then measured using an MTS assay.
  • SKOV3 cells and SKOV3/pGT20 clones served as controls. Experiments were performed in triplicate and the results are reported as mean ⁇ SEM.
  • Figure 12 shows the effect of single fraction cobalt-60 external beam irradiation on the growth of established subcutaneous human ovarian cancer xenografts of either untransfected SKOV3 or SKOV3/pGT21 cells expressing the ER form of the anti-erbB-2 sFv.
  • Figure 13 shows a different experiment depicting the effect of single fraction cobalt-60 external beam irradiation on the growth of established subcutaneous human ovarian cancer xenografts of either untransfected SKOV3 or SKOV3/pGT21 cells expressing the ER form of the anti-erbB-2 sFv.
  • Figure 14 shows ( Figure 14A) the schema of the pCANTAB5 vector showing control regions.
  • the sFv cDNA (750 bp) is introduced between the Sfil and No tl sites.
  • the g3p leader sequence directs transport of the protein to the inner membrane/periplasm of E. coli whereas the functional domain of g3p (fd3) attaches the fusion protein to the tip of the assembling phage.
  • the sFv is expressed as a fusion protein with the E-tag peptide to allow easy detection.
  • Figure 14B shows the schema of the pSTCF.KDEL eukaryotic vector expressing sFv genes. Expression of the sFv protein is driven by the CMV promoter.
  • the sFv cDNA is introduced between Sfil and Notl.
  • the IgK leader sequence directs the sFv protein to the ER, and the KDEL signal at the c-terminus leads to retention in this cellular compartment.
  • the sFv open reading frame is also fused with a c-myc epitope to allow easy detection by Western blot.
  • Figure 15A shows the screening of positive clones for sFv inserts obtained after the colony lift selection.
  • Figure 16 shows the binding affinity of the anti-Bcl-2 sFvs to the Bcl-2 protein as measured by ELISA.
  • Various concentrations of periplasmic extracts anti-Bcl-2 sFv 1 and 4 were added onto a 96-well plate coated with recombinant Bcl-2 protein.
  • a periplasmic extract containing no sFv protein was used as a negative control.
  • Figure 17A shows the expression of the anti-Bcl-2 sFvs 1 and 4 in HeLa cells as determined by Western blot.
  • Cells were transduced with either the anti-Bcl-2 sFvs 1 and 4 alone (sFv 1 , sFv 4) or cotransfected with the sFv constructs and the pRC/CMV/hBcl-2 plasmid (sFv 1 + Bcl-2, sFv 4 + Bcl-2).
  • the sFv protein migrates around 34 kDa
  • Figure 17B shows the modulation of Bcl-2 expression in HeLa cells as determined by Western blot.
  • Mock indicates that cells were treated with AdpL only; sFv 1 , sFv 1 vector only; sFvl + Bcl- 2, sFv 1 vector and pRC/CMV/hBcl-2; sFv 4, sFv 4 vector only; sFv
  • Figure 18 shows the western blot analysis of different ratios of Bcl-2 versus anti-Bcl-2 sFv in HeLa cells. The ratios used in each experiments is indicated on the left. Equal amounts (30 ⁇ g) of total protein were loaded in each lane and separated by SDS-PAGE. The position of the Bcl-2 protein is indicated.
  • Figure 19A s h ow s a western blot analysis showing down-regulation of Bcl-2 expression in DU145 and MCF-7 cells.
  • DU145 were transfected with either pRC/CMV/hBcl-2 alone (Bcl- 2) or pRC/CMV/hBcl-2 plus sFv 4 vector (sFv 4 + Bcl-2) at a DNA ratio of 1 : 10.
  • MCF-7 cells were treated with AdpL alone (mock) or the sFv 4 vector (sFv 4). Equal amounts (25 ⁇ g) of total protein were loaded in each lane and separated by SDS-PAGE; and Figure
  • 19B shows the expression of the sFv 4 protein in DU145 and MCF- 7 as determined by Western blot. Equal amounts (25 ⁇ g) of protein were separated by SDS-PAGE.
  • Figure 20 shows that expression of the anti-Bcl-2 sFv 4 does not affect the growth rate of cells overexpressing or not
  • DU145 ( Figure 20A) and MCF-7 ( Figure 20B) were mock-transfected (square), transfected with the pSTCF.KDEL (diamond) or the anti-Bel— 2 sFv (circle) and followed over time. The growth rate was determined by MTT assay.
  • Figure 21 shows that expression of the anti-Bcl-2 sFv 4 increases cell death in tumor cells overexpressing Bcl-2 in the presence of drugs.
  • Figure 21A DU 145 cells were mock- transduced (square), transduced with pRC/CMV/hBcl-2 (diamond), or transfected with pRC/CMV/hBcl-2 plus the anti-Bcl-2 sFv 4 (circle) at a DNA ratio of 1 : 10 and treated with various concentrations of CDDP.
  • Figure 22 shows the expression of BAG-1 and Bcl-2 in DU145 and MCF-7 cell lines. Equal amount of protein cell lysates (30 ⁇ g) were subjected to SDS-PAGE/immunoblot analysis.
  • Figure 22A Lysates from DU145 cells probed with an anti- BAG-1 antibody. BAG- 1 protein migrates as a ⁇ 29 kDa protein. The anti-BAG-1 antibody also detects a non-specific cellular band at -47 kDa.
  • Figure 22B Lysates from MCF-7 cells probed with the anti-BAG-1 antibody.
  • Figure 22C Cell extracts from DU145 probed with an anti-BCl-2 antibody.
  • FIG. 22D MCF-7 cell lysates probed with an anti-Bcl-2 antibody. The positions of BAG- 1 and Bcl-2 protein are indicated by an arrow.
  • Figure 23A MCF-7 cells survival.
  • Figure 23B DU145 cells survival.
  • Figure 24 shows the expression and binding affinity of anti-BAG-1 sFvs produced by differents E. coli HB2151 clones after induction with IPTG 1 mM.
  • Figure 24A Representative Western blot analysis of 9 different clones (out of 20) selected after colony lift assay screening. The periplasmic extracts prepared form IPTG-induced clones were run on SDS-PAGE gel ( 12%). After transfer, the membrane was probed with an horseradish peroxidase labeled anti-E-tag antibody. The anti-BAG- 1 sFv migrates as a -34-36 kDa protein.
  • FIG. 24B Binding affinity of the anti-BAG- 1 sFvs to BAG- 1 protein as measured by ELISA.
  • Various concentrations of periplasmic extracts from anti- BAG-1 sFv clones 11 , 15 and 20 were added onto a 96 well plate coated with recombinant BAG- 1 protein.
  • a periplasmic extract containing no sFv protein was used as a negative control.
  • Figure 25 s h o w s Figure 25A the modulation of BAG-1 expression in HeLa cells as determined by Western blot. Equal amount of protein cell lysates (30 ⁇ g) were loaded in each lane. Mock indicates that cells were treated with AdpL only; pCDNA3, the empty vector only; ⁇ CDNA3/BAG- l , the BAG- 1 eucaryotic vector; anti-BAG-1 sFv, sFv expression vector; anti- BAG- 1 sFv + BAG-1 , sFv expression vector + BAG- 1 eucaryotic vector (pCDNA3/BAG- l).
  • the membrane was probed with an anti-BAG- 1 antibody; and (Figure 25B) the expression of the anti-BAG-1 sFvs in HeLa cells. Equal amount of cell lysates (30 ⁇ g) in each lane and protein expression was analysed by immunoblot assay using an anti-c-myc monoclonal antibody.
  • Figure 26 shows that the anti-BAG- 1 sFv down- regulates the expression of BAG- 1 in DU145 and MCF-7 cells.
  • DU145 Figure 26A
  • MCF-7 Figure 26B
  • Thirty ⁇ g of total protein lysates were loaded per lane and
  • Figure 27 shows that the anti-BAG- 1 sFv abolishes BAG- 1 -mediated resistance to cell killing in MCF-7 but not in DU145.
  • MCF-7 Figure 27A-B or DU145 ( Figure 27C-D) cells were transfected with BAG-1 , BAG-1 and anti-BAG-1 sFv 20 (BAG- 1 + sFv) or BAG-1 and pSTCF.KDEL (BAG-1 + pSTCF) and then treated with either staurosporine or CDDP. The percentage of surviving cells was determined by MTS assay 4 days later. Samples were done in quadruplicate. Data are presented as mean ⁇ standard deviation.
  • Figure 28 shows that BAG- 1 expression does not affect the growth rate of DU145 and MCF-7 under normal growth conditions.
  • Figure 28B similar experiments done in DU145 cells.
  • Figure 29A Molecular cloning of anti-cyclin-Dl sFv from RNA derived from the DCS-6 hybridoma cell line. The V L and V H domains of the RNA were amplified separately by RT-PCR, ligated together and reamplified with outstream primers. These products were visualized on an agarose gel (1 %).
  • Figure 29B Expression of anti-cyclin-Dl sFv in E. coli. SDS-PAGE (12%) of IPTG-induced and uninduced periplasmic protein.
  • Figure 29C Enzyme-linked ELISA was used to measure the binding activity of the periplasmic expressed anti-cyclin-D l sFv clones 3 and 34. Cyclin-D l protein was coated on 96-well plates at the final concentration of 80 ng/well. After blocking with 3% milk, the periplasmic preparations were added to the plates. HRP-labeled mouse anti-E tag was used. Bound antibodies were detected by the addition of HRP substrate and determining the O.D. at 405 nm.
  • Figure 30 A intracellular localization of GFP fusion protein in HeLa cells 48 hr after transfection with the ER and nuclear localizing vectors.
  • Figure 30B Expression of intracellular anti-cyclin-Dl sFv in HeLa cells.
  • Figure 30C Expression of cyclin- Dl protein five days post-transfection of anti-cyclin-Dl sFv. Fifty mg of total cellular protein from each of the indicated cell lines were subjected to 12% SDS-PAGE, transferred to nitrocellulose and immunoblotted.
  • Figure 31 shows the cell cycle analysis by FACS following staining with PI. 48 hr after transfection with the anti- cyclin-Dl sFv using AdpL nonsynchronized, log phase cells (10 4 ) were harvested and analyzed by FACS for cellular DNA content. Regions were set over the cell cycle phases and the percentage of cells within each region were determined using the CellQuest program.
  • Figure 32 shows the morphological appearance of MDA-MD-453 cells 5 days after transfection.
  • Figure 32A Control vector pcDNA3
  • Figure 32B erCDl scFv34.1
  • Figure 32C nCDl scFv34.1
  • Figure 33 shows the percentage of cell viability after anti-cyclin-D l scFv treatment.
  • Plasmid DNAs pcDNA3, erCDl scFv34.1 and nCDl scFv34.1 were transfected into HBL- 100, MCF-7 and MDA-MB-453. At day 5 post-transfection, the number of viable cells were counted in the Coulter Counter Model ZF.
  • the present invention is directed to a method of enhancing the chemosensitivity and radiosensitivity of a neoplastic cell expressing an oncoprotein that stimulates proliferation of the cell, comprising introducing into the cell a nucleic acid molecule encoding an antibody homologue, wherein the antibody homologue is expressed intracellularly and binds to the oncoprotein intracellularly in the endoplasmic reticulum (ER) of the cell.
  • the oncoprotein is erbB2 although others can be targeted by those having ordinary skill in this art.
  • the antibody homologue is selected from the group consisting of a single chain Fv fragment and a Fab fragment.
  • the nucleic acid molecule is, for example, a recombinant expression vector selected from the group consisting of a viral vector and a plasmid vector.
  • the neoplastic cell is from a tissue or organ selected from the group consisting of breast, gastrointestinal tract, lung, ovarian and salivary gland.
  • the present invention is also directed to a method of enhancing the chemosensitivity and radiosensitivity of a neoplastic cell, comprising the step of: introducing into the cell an antibody homologue, wherein the antibody homologue is expressed intracellularly and binds to a target protein intracellularly.
  • the antibody homologue binds in the endoplasmic reticulum of the cell.
  • the antibody homologue binds to a target protein in the nucleus.
  • the neoplastic cell expresses an oncoprotein that stimulates proliferation of the cell.
  • this method further comprises the step of contacting the cell with an anti- neoplastic agent, radiation or a combination thereof.
  • an anti-neoplastic agent is selected from the group consisting of cisplatin, a halogenated pyrimidine, fluoropyrimidines, taxol, BCNU, 5-fluorouracil, bleomycin, mitomycin, hydroxyurea, fludarabine, nucleoside analogues, topoisomerase I inhibitors, hypoxic cell sensitizers and etoposide.
  • neoplastic cell include ovarian cancer, bladder cancer, lung cancer, cervical cancer, breast cancer, prostate cancer, gliomas, fibrosarcomas, retinoblastomas, melanomas, soft tissue sarcomas, ostersarcomas, leukemias, colon cancer, carcinoma of the kidney, gastrointestinal cancer, salivary gland cancer and pancreatic cancer.
  • the target protein bound by the antibody homologue is a growth factor receptor protein, cell cycle control protein and anti-apoptotic protein.
  • growth factor receptor proteins are erbB2 and epidermal growth factor receptor.
  • anti-apoptotic proteins are Bcl-2 and BAG-1.
  • cell cycle control proteins are cyclin DI and cyclin B.
  • the antibody homologue is selected from the group consisting of a single chain Fv fragment and a Fab fragment.
  • the antibody homologue is introduced to the cell via a nucleic acid molecule encoding said antibody homologue.
  • the method nucleic acid molecule is preferably a recombinant expression vector such as a viral vector and a plasmid vector.
  • the present invention is also directed to a method of enhancing the chemosensitivity and radiosensitivity of a neoplastic cell, comprising the step of: introducing into the cell an antibody homologue, wherein the antibody homologue is expressed intracellularly and binds to a target protein intracellularly; and contacting said cell with an anti-neoplastic agent, radiation or a combination thereof.
  • the present invention is also directed to a method of inhibiting proliferation of erbB2-overexpressing tumor cells in a mammal, comprising the steps of: introducing into the cell an antibody homologue, wherein the antibody homologue is expressed intracellularly and binds to erbB2 intracellularly in the endoplasmic reticulum of the tumor cells; and contacting said cell with an anti-neoplastic agent, radiation or a combination thereof.
  • Anti-erbB-2 single-chain intracellular antibody fsFv is specifically cytotoxic in erbB-2 overexpressing tumor cells
  • a strategy was developed involving construction of a gene encoding a single-chain immunoglobulin (sFv) directed against erbB-2. If an anti-erbB-2 sFv were localized to the endoplasmic reticulum of SKOV3 cells (an ovarian carcinoma cell line which overexpresses erbB-2), the nascent erbB-2 protein would be entrapped within the ER of the cells, and unable to achieve its normal cell surface localization.
  • SKOV3 cells an ovarian carcinoma cell line which overexpresses erbB-2
  • sFv constructs were cloned into the eucaryotic expression vector pcDNA3 (Invitrogen), which directs high level gene expression from the cytomegalovirus (CMV) early intermediate promoter/enhancer.
  • pcDNA3 eucaryotic expression vector
  • CMV cytomegalovirus
  • plasmid DNAs pcDNA3, pGT20, and ⁇ GT21 were transfected into the erbB-2 overexpressing ovarian carcinoma cell line SKOV3 using the adenovirus-polylysine (AdpL) method.
  • AdpL adenovirus-polylysine
  • the adenovirus- polylysine-DNA complexes containing a ⁇ -galactosidase reporter gene (pCMV ⁇ ) produced detectable levels of reporter gene expression in >99% of targeted cells.
  • erbB-2 At various times after transfection, the cells were evaluated for cell surface expression of erbB-2 using an anti-human erbB-2 polyclonal antibody.
  • SKOV3 cells transfected with the non-ER (cytosolic) form of the anti-erbB-2 sFv (pGT20) exhibited levels of cell surface erbB-2 similar to the control.
  • SKOV3 cells transfected with pGT21 which encodes an ER form of the anti- erbB-2 sFv, demonstrated marked down-regulation of cell surface erbB-2 expression.
  • the ER anti-erbB-2 sFv exhibited a prominent anti-proliferative effect, it was hypothesized that it might also exhibit an anti-tumor effect in cells stably modified to express this gene construct.
  • the plasmids pcDNA3, pGT20 and pGT21 contained neomycin selectable markers, they were used to derive stable clones.
  • the various plasmid constructs were used to derive G418 resistant clones in HeLa cells, a human cervical carcinoma cell line not characterized by overexpression of erbB-2. After selection, the number of clones derived from transfection with pGT20 and pGT21 was not significantly different (Table 1 ).
  • the number of clones did not differ after transfection with the control plasmid pcDNA3.
  • a similar analysis was then carried out with the erbB-2 overexpressing tumor line SKOV3 as the target.
  • the number of clones derived with pGT20 did not differ from the number derived with the control plasmid pcDNA3 (Table 1).
  • the ER form of the anti-erbB-2 sFv again had no observable effect on the non-erbB-2 expressing human cervical carcinoma line HeLa.
  • Non-erbB-2 expressing human cell lines from a variety of different tissues were also transduced and not significant cytotoxicity was noted. A subset of the cells was not eradicated by this intervention. Despite that >99% of the cells were transfected to transiently express the anti-erbB-2 sFv, in a subset of these transfected cells the anti- erbB-2 sFv did not appear to effectively induce cytotoxicity. This agrees with the derivation of anti-erbB-2 sFv-expressing SKOV3.ipl stable clones noted in Table 1.
  • SKOV3 cells transfected with the plasmid DNA pGT21 showed intense nuclear staining characteristic of cellular apoptosis. These alterations were not seen in cells transfected with the control plasmids pcDNA3 and pGT20.
  • ErbB-2 down-regulation mediated by antisense oligonucleotides induces proliferative arrest, but not apoptosis in erbB-2 overexpressing tumor targets. In contrast, apoptosis was induced by virtue of an alternate mechanism of erbB-2 down- regulation. This suggests that erbB-2 down-regulation, per se, was not inductive of apoptosis. To determine the basis whereby the anti-erbB-2 sFv induced apoptosis, this phenomenon in a different system was reproduced.
  • Ectopic localization of erbB-2, in non- erbB -2 transformed tumor cells was accomplished by cotransfection of HeLa cells with wild-type human erbB-2 cDNA and the cDNA for the ER form of the anti-erbB-2 sFv.
  • Transfection of the non-erbB-2 expressing HeLa cell line with the erbB-2 cDNA did not result in any change in cell viability, identical to that observed employing the control plasmid DNA ⁇ cDNA3.
  • cotransfection of the erbB-2 cDNA with the anti-erbB-2 sFv construct caused a marked cytocidal effect ( Figure 4).
  • This cytotoxicity was also shown to be the result of induction of apoptosis as was observed in SKOV3 cells transfected with the anti-erbB-2 sFv.
  • erbB-2 does not contribute to the transformed phenotype
  • coexpression of the anti-erbB-2 sFv and heterologous erbB-2 still induced apoptosis.
  • the effects of the anti-erbB-2 sFv in human tumor material isolated from a patient with primary ovarian carcinoma was shown. Methods were developed to isolate primary ovarian tumor cells which maintain their viability and proliferative capacity in vitro for approximately 7- 10 days.
  • the amount of cell surface erbB-2 in these tumor explants was estimated employing an immunohistochemistry assay.
  • the various anti- erbB-2 sFv constructs were delivered to cells employing the AdpL vector followed by the XTT assay for determination of cell viability.
  • Control experiments employing a LacZ reporter gene demonstrated that >99% of the isolated human primary ovarian carcinoma cells could be transduced.
  • the human ovarian carcinoma cell line SKOV3 was employed as a control.
  • the ER form of the anti-erbB-2 sFv exhibited a specific cytotoxic effect in the human primary tumor cells at 96 hours post transfection.
  • the magnitude of the sFv-mediated cell killing observed in the primary tumor material was as great as that observed in the erbB-2 overexpressing cell line SKOV3.
  • Oncoprotein ablation mediated by the anti-erbB-2 sFv accomplishes a therapeutic effect in a murine model of human ovarian carcinoma
  • human ovarian cancer cells could be selectively killed in a murine model of malignant ascites was determined.
  • Athymic nude mice with the erbB-2 overexpressing human ovarian carcinoma line SKOV3.ipl were engrafted. This model allows for the development of malignant ascites and peritoneal implants of neoplastic cells in a manner which parallels the human disease.
  • vector strategies must be capable of accomplishing direct, in situ delivery of heterologous genes to tumor in vivo.
  • a vector system that accomplishes efficient in situ transduction of the tumor cells found in ovarian carcinoma malignant ascites fluid was determined.
  • candidate vector systems capable of achieving therapeutic levels of in vivo gene transfer were evaluated.
  • Athymic nude mice (Balb/c) were transplanted intraperitoneally with 1 x 10? SKOV3.ip l cells. After 48 hours, vectors were administered intraperitoneally to deliver an E. coli ⁇ -galactosidase reporter gene construct (lacZ) to the mobile neoplastic cells.
  • Evaluated vector systems included adenovirus-polylysine-DNA-complexes (AdpL), liposomes (DOTAP), and a recombinant adenovirus encoding lacZ (AdCMVLacZ). Forty- eight hours after vector administration, mobile tumor cells were harvested by peritoneal lavage and analyzed for expression of the lacZ reporter gene. This was accomplished by a fluorescent activated cell sorting (FACS) double-sorting procedure ( Figure 6). The highest level of gene transfer was accomplished with the recombinant adenovirus, the transduction frequency achieved with this vector was >80%.
  • FACS fluorescent activated cell sorting
  • a recombinant adenovirus proved useful for i n situ transduction of mobile neoplastic cells in vivo, whether the anti-erbB-2 sFv-mediated selective toxicity in this setting was determined.
  • a recombinant adenovirus was, therefore, constructed encoding the ER form of the anti-erbB-2 sFv (Ad21) using methods of homologous recombination.
  • the resultant recombinant virus is E 1A/B deleted and, thus, replication- incompetent. Studies confirmed the structural integrity of the recombinant adenovirus genome.
  • a replication-defective adenovirus encoding the anti-erbB-2 sFv has been constructed which retains the capacity to express an ER-anti- erbB-2 sFv.
  • This vector can achieve selective cytotoxicity based on the encoded sFv in human ovarian carcinoma cell lines.
  • SKOV3.ipl cells were xenotransplanted into athymic nude mice. Forty-eight hours after engraftment with SKOV3.ipl cells, the SCID mice were challenged intraperitoneally with the ElA/B-deleted recombinant adenovirus encoding the anti-erbB-2 sFv (Ad21 ) or an El A/B-deleted recombinant adenovirus encoding the reporter gene lacZ (AdCMVLacZ).
  • the animals underwent peritoneal lavage for analysis of harvested mobile tumor cells.
  • Cells were analyzed for cell viability employing the XTT assay.
  • the number of viable cells was dramatically decreased in the Ad21 group compared to the AdCMVLacZ group.
  • This cytotoxicity appeared to be specifically associated with the anti- erbB-2 sFv encoding adenovirus.
  • Analysis of the mechanism of cell death demonstrated that the Ad21 virus induced cellular apoptosis.
  • the recombinant adenovirus encoding the anti- erbB-2 sFv was specifically cytotoxic in mobile neoplastic cells in an orthotopic murine model of human ovarian cancer.
  • the efficacy of the anti-erbB-2 sFv approach was established employing a murine model of human ovarian carcinoma.
  • SCID mice were xenografted i.p. with 2 x 10 ⁇ SKOV3.ipl cells.
  • animals were challenged by the same route with either the control adenovirus, AdCMVLacZ, or the anti-erbB-2 encoding adenovirus, Ad21 , and animals monitored for survival.
  • the anti-erbB-2 sFv encoding adenovirus, Ad21 accomplished a statistically significant survival prolongation (Figure 8).
  • direct in situ tumor transduction to accomplish selective tumor cell cytotoxicity via the anti-erbB-2 sFv has therapeutic utility.
  • the expression of the erbB-2 oncoprotein is a parameter which may affect tumor cell chemosensitivity.
  • An inverse relationship between erbB-2 levels and chemosensitivity was noted in the studies of Gazdar et al.
  • a strategy to down- regulate the erbB -2 oncoprotein enhances tumor cell chemosensitivity.
  • the anti-erbB-2 sFv was capable of inducing apoptotic cell death in a tumor cell subset, in those tumor cells in which sFv expression was achieved but cytotoxicity not induced, sensitivity to a second apoptotic stimulus might occur. That the anti-erbB-2 sFv directly affects tumor cell sensitivity to chemotherapeutic agents was demonstrated.
  • Plasmids encoding a cytosolic form of the anti-erbB-2 sFv (pGT20) as well as an endoplasmic reticulum directed form of the anti-erbB-2 sFv (pGT21) have been described.
  • the plasmid pcDNA3 (Invitrogen, San Diego, CA.) served as a control.
  • the phagemid pCANTAB5/sFv contains the anti-Bcl-2 sFv DNA under the control of the inducible lac promoter.
  • This vector also encodes a peptide tag (E-tag) located at the 3' end of the sFv to allow easy immunological detection of sFv protein expression.
  • E-tag peptide tag
  • the Bcl-2 expression plasmid pRc-CMV/hBcl-2 contains wild type human Bcl-2 under the control of the CMV promoter.
  • the pGEX-hBcl-2 encodes the human Bcl-2.
  • the ER-targeted vector is a derivative of the pSecTag C vector (Invitrogen, Carlsbad, CA). A DNA sequence encoding the c-myc peptide tag and KDEL signal was inserted between Notl and Apal sites of pSecTag C to generate pSTCF.KDEL.
  • the anti-Bcl-2 sFv fragments generated by Sfil/ No tl digest of pCANTAB5/sFv were ligated into the S fill Notl sites in pSTCF.KDEL just upstream from and in-frame with the added c-myc/KDEL sequence.
  • the prokaryotic BAG- 1 expression vector, pGEX 3X- hBAG-1 was obtained from JC Reed (Burnham Institute, La Jolla, CA).
  • pGEX 3X-hBAG-l was digested with Ba mHI and EcoRl to release BAG- 1 open reading frame (ORF), and this fragment was subcloned into BamHl/EcoR l sites of pcDNA3 (Invitrogen, Carlsbad, CA).
  • the BAG-1 expression cassette is under the control of the CMV promoter in this vector.
  • the phagemid pCANTAB5 (Pharmacia Biotech, Piscataway, NJ) was used to clone the anti-BAG-1 sFv DNA into the Sfil/ No tl sites, under the control of the IPTG-inducible lac promoter.
  • This vector also encodes a peptide tag (E-tag) located at the carboxy terminus of the sFv to allow easy immunological detection of sFv protein expressed in bacteria.
  • the pCANTAB5 plasmid containing the sFv DNA cassette was digested with Sfil/ Notl, and the sFv fragment was subcloned into Sfil/ Notl sites of pSTCF.KDEL, an eukaryotic expression vector.
  • pSTCF.KDEL an eukaryotic expression vector.
  • This vector targets the expression of an sFv to the ER. Expression of the sFv ORF is driven by the CMV promoter.
  • the human ovarian carcinoma cell line SKOV3 was obtained from the American Type Culture Collection (Rockville, MD) and maintained in Dulbecco's modified Eagle's medium (DMEM, Mediatech, Herndon, VA.) supplemented with glutamine (30 mg/ml), penicillin (10 mg/ml), streptomycin (25 mg/ml), and 10% fetal calf serum (PA A Laboratories Inc. Newport Beach, CA.) at 37°C in a humidified 5% C ⁇ 2 atmosphere.
  • DMEM Dulbecco's modified Eagle's medium
  • Peptomycin 25 mg/ml
  • PA A Laboratories Inc. Newport Beach, CA. 10% fetal calf serum
  • the human breast cancer cell line MCF-7, the human prostate cancer cell line DU145, and the human cervical cancer cell line HeLa were obtained from ATCC (Rockville, MD).
  • DU145 and MCF-7 cells were grown in RPMI 1640 (Cellgro Mediatech, Washington, DC) supplemented with 10% fetal bovine serum (FBS) (Hyclone Laboratories), L-glutamine (300 ⁇ g/ml), penicillin (100 i.u./ml) and streptomycin (25 ⁇ g/ml).
  • FBS fetal bovine serum
  • penicillin 100 i.u./ml
  • streptomycin 25 ⁇ g/ml
  • the 6C8 hybridoma cell line (obtained from JC Reed, The Burnham Institute, La Jolla, CA) was grown in RPMI 1640 supplemented with 10% FB S , oxalate/pyruvate/insulin mix (Sigma), 30 ⁇ g/ml of carboxyethyl gamma-butryic acid (Sigma), 13.6 ⁇ g/ml of hypoxanthine (Sigma), 300 ⁇ g/ml of L-glutamine and penicllin ( 100 i.u./ml) and streptomycin (25 ⁇ g/ml).
  • HeLa cells were maintained and propagated in DMEM/F12 (Cellgro Mediatech) supplemented with 10% FBS, L-glutamine and penicillin/streptomycin. All cell lines were incubated at 37°C in 5% C0 2 . HeLa, MCF-7 and DU145 cells were transfected in 6 well plates using the adenovirus-polylysine- DNA (AdpL) complex method.
  • AdpL adenovirus-polylysine- DNA
  • Target SKOV3 cells were transiently transfected with constructs employing the adenovirus-poly-L-lysine (AdpL) utilizing known techniques.
  • cisplatin treated cell lines 4 mg/ml cis-diamminedichloroplatinum (Bristol-Myers Squibb, Princeton, NJ) was diluted in 100 ml of complete media and added to 100 ml 2% FCS for a final concentration of CDDP at 2 mg/ml.
  • Plasmid DNAs were stably transfected into target SKOV3 cells by the lipofectAMINE (GIBCO BRL, Gaithersberg, MD.) method using conditions described by the manufacturer.
  • both pGT20 and pGT21 contained a neomycin resistance expression cassette to allow for selection of stable transfectants.
  • lipid/DNA complexes consisting of 40 mg plasmid DNA were delivered to cells at -50% confluency in 6.0 cm tissue culture dishes in a volume of 1.0 ml of OptiMEM medium (GIBCO BRL). After 18 hours, the transfection medium was removed and replaced with complete medium and incubation continued for an additional 48 hours. Cells were split into selective medium containing Geneticin (GIBCO BRL) at 1 mg/ml. Colonies were then isolated and expanded in selective medium.
  • GIBCO BRL Geneticin
  • erbB2 Expression in Stable Clones To determine the levels of erbB-2 protein present in the aforementioned stable clones, total clonal cell extracts were obtained. Briefly, total cellular protein was isolated from cells in a cell lysis buffer solution containing lx physiologic buffered saline (pH-7.4), 1.5 mM EDTA, 100 mM PMSF and 1 mg/ml aprotinin. The cell lysate was then plated at 1 mg/ml in a 96 well plate pre- coated with human erbB-2 antibody and assayed according to manufacturer's instructions using a quantitative Her2/neu (erbB- 2) ELISA kit (Oncogene Science, Uniondale, NY).
  • HNU Human Neu Unit
  • Softmax Program BioTek Instruments, Winooski, VT.
  • the neu assay will detect 10 HNU (0.5 femtomoles) per ml of cell lysate.
  • the signal of 10 HNU standard is approximately twice the background signal.
  • the human neu values obtained from the assay were then converted to mole/mg protein.
  • sample buffer 0.175 M Tris HCl pH 6.8, 20% ( v/v) glycerol, 4.1 % (w/v) SDS , 10% (v/v) ⁇ -mercaptoethanol, 0.002% (w/v) bromophenol blue, 6M Urea).
  • sample buffer 0.175 M Tris HCl pH 6.8, 20% ( v/v) glycerol, 4.1 % (w/v) SDS , 10% (v/v) ⁇ -mercaptoethanol, 0.002% (w/v) bromophenol blue, 6M Urea.
  • Alkaline phosphatase conjugated goat anti-rabbit IgG antibody (Jackson ImmunoResearch Labs, Inc., West Grove, PA) was used as secondary antibody at a 1 : 1000 dilution and the blot was developed in carbonate buffer pH 9.8 (0.1 M NaHC03, 1 mM
  • MgCl2 MgCl2 with nitro blue tetrazolium (NBT) and bromochloroindolyl phosphate (BCIP) (Bio-Rad Laboratories).
  • SKOV3 cells were seeded at a density of 5000 cells/well in a 96-well plate and incubated for 24 hr.
  • the AdpL method was employed for transient transfection of target cells with the plasmid constructs pGT21 or pcDNA3.
  • CDDP was added at a concentration of 2 mg/ml.
  • SKOV3/pGT21 clonal cells or SKOV3/pGT20 clonal cells were seeded at a density of 5000 cells/well in a 96-well plate and incubated for 24 hr.
  • CDDP was then added at a concentration of 2 mg/ml.
  • MTS/PMS solution (20 ⁇ l) was then added into each well maintaining a ratio of 20 ml MTS/PMS to 100 ml of medium. After 30 minutes, the reduction product was measured at an absorbance of 490 nm and compared to a standardized curve.
  • the mechanism of cell death induced by expression of the anti-erbB-2 sFv and co-administration of CDDP was determined by evaluating target cells for evidence of apoptosis.
  • Fluorescent DNA-binding dyes combined with fluorescence microscopy were employed to visualize cells demonstrating aberrant chromatin organization.
  • the cell suspension was prepared at - lx l O 5 cells/ml complete medium.
  • the suspended cells 25 ml were combined with 1 ml of dye containing 100 mg/ml acridine orange + 100 mg/ml ethidium bromide and examined by fluorescent microscopy for evidence of apoptosis.
  • Statistical analysis of calculated means was performed using the Student t-test with pooled variances. A p-value of ⁇ 0.01 was considered significant.
  • the erbB-2-overexpressing tumor cells, SKOV3, were treated with either the anti-erbB-2 sFv (via transient transfection), the chemotherapeutic agent cisplatin (CDDP), or a combination of these agents.
  • Figure 9 shows that intracellular expression of the anti-erbB-2 sFv or CDDP induced cytotoxicity, but a synergistic effect was noted when the two agents were employed in combination.
  • the anti-erbB-2 sFv was capable of enhancing tumor cell sensitivity to a chemotherapeutic agent.
  • An experimental model which would allow more direct analysis of the anti-erbB-2 sFv mediated chemosensitization was developed.
  • the sFv-expressing SKOV3 clones derived in Table 1 were expanded and characterized. Clonal cell populations were thus characterized for confirmation of expression of the anti-erbB-2 sFv.
  • clonal populations of the parent cell line (SKOV3) were chosen, as well as stable lines expressing either the cytosolic anti-erbB-2 sFv (SKOV3/GT20) or the ER anti-erbB-2 sFv (SKOV3/GT21) which exhibited comparable growth kinetics.
  • the parental clone and the cytosolic sFv clone would likely have comparable levels of cellular erbB-2.
  • the ER sFv clone would likely reduce cellular erbB-2, based upon a level of sFv-mediated erbB-2 down- regulation. These clones were thus evaluated for cellular erbB-2 by direct ELISA analysis (Figure 10). It could be seen that the ER anti-erbB-2 sFv clone, SKOV3/GT21 , was uniquely characterized by reduced erbB-2 levels.
  • clonal cell populations were further evaluated for their sensitivity to the chemotherapeutic agent CDDP.
  • the cytosolic sFv expressing clone, SKOV3/GT20 did not differ in CDDP sensitivity when compared to the parental clone SKOV3.
  • intracellular expression of the anti-erbB-2 sFv in the cellular cytosol has no effect on either erbB-2 levels ( Figure 10) or sensitivity to CDDP ( Figures 10 and 11).
  • the clonal population expressing the ER form of the anti-erbB-2 sFv exhibited significantly greater sensitivity to CDDP treatment than the parental clone.
  • the ER-sFv-mediated erbB-2 down-regulation was associated with enhanced chemosensitivity.
  • erbB-2 down- regulation is a means to achieve enhanced chemosensitivity in erbB-2-overexpressing tumor cells.
  • these findings address the issue of intracellular antibody-expressing tumor cells not directly killed by the anti-erbB-2 sFv. These cells are phenotypically altered by the sFv-mediated down-regulation. In this instance, they are thus rendered susceptible to a second apoptosis-inducing insult.
  • the mechanism of cell death could be shown to be on the basis of induced apoptosis.
  • Experiments in heterologous cells demonstrated that this phenomenon was not on the basis of "shut-off" of transforming signals, but rather was linked to the co-expression of erbB-2 and the anti-erbB-2 sFv in target tumor cells.
  • the sFv-mediated erbB- 2 down-regulation enhanced chemosensitivity in erbB-2 tumors. From the standpoint of a gene therapy strategy, this was a very desirable result, in that tumor cells were selectively killed on the basis of a targeted tumor marker.
  • this cytotoxicity could be accomplished in a targeted manner whereby non-erbB-2 positive cells were not induced to undergo apoptosis. In these instances where direct cytotoxicity could not be fully accomplished, the sFv rendered tumor cells more sensitive to a second apoptotic insult.
  • AdpL complexes are constituted with various ratios of anti-erbB-2 sFv plasmid and erbB-2 plasmid (sFv:erbB-2 gene copy number: 10: 1 , 8: 1 , 4: 1 , 2: 1 , 1 : 1 , 1 :2, 1 :4, 1 :8, 1 : 10).
  • the complexes are then used to cotransduce the non-erbB-2 expressing cell line HeLa with analysis carried out to determine the magnitude of the induced chemosensitivity in these cells with the various chemotherapeutic agents.
  • a relationship can thus be defined between the input anti- erbB -2 sFv and erbB -2 plasmids and the observed chemosensitivity.
  • the present invention demonstrated that sFv-mediated knockout of the overexpressed erbB-2 growth factor receptor enhanced tumor cell chemosensitivity.
  • a person having ordinary skill in this art would readily recognize that other overexpressed cell surface markers are associated with the progression of human ovarian carcinoma. Overexpression of these markers, such as EGFR, have been associated with enhanced chemoresistance, in a manner analogous to erbB-2.
  • erbB-2 positive ovarian tumors represent a minority of the overall ovarian carcinoma population.
  • the development of sFvs targeting other ovarian carcinoma related targets is within the skill of those having ordinary skill in this art given the teachings of the present invention.
  • its precise association with the chemoresistant phenotype has not been clearly established.
  • the achievement of functional knockout of these targets would establish a phenotypic link.
  • EXAMPLE 15 Down-regulation of analogous transforming transmembrane growth factors via intracellular sFvs and induction of chemosensitivity
  • EGFR is overexpressed in a variety of epithelial tumors, including those originating in lung and breast. Its overexpression has been shown to be a key event in neoplastic transformation and progression. In addition, overexpression of this analogous tyrosine kinase receptor has been linked to tumor cell chemoresistance. sFv mediated down-regulation of the EGFR can be accomplished to elicit cellular chemosensitivity. An anti-EGFR sFv is modified to achieve ER localization after expression in an eucaryotic vector. Cotransduction of heterologous non-EGFR expressing cells with the human EGFR cDNA and the anti-EGFR sFv is then performed. Cells are evaluated for chemosensitization employing the XTT assay. In addition, stable clones expressing the anti-EGFR sFvs (cytosolic and
  • ER ovarian carcinoma cell lines
  • Adenoviral vectors can be used for in situ g e n e transfer to cancer cells.
  • a number of replication-defective recombinant adenoviral vectors have been approved for human use in the context of RAC-approved protocols relating to human ovarian carcinoma.
  • the various sFvs directed against ovarian cancer markers can be configured into adenoviral vectors employed.
  • Liposome vectors can be used to accomplish in situ gene transfer to ovarian cancer cells. Liposome gene transfer vectors offer a number of potential advantages required for the sFv-knockout gene therapy strategy, including low immunogenicity and repetitive in vivo gene delivery. These vectors can achieve direct in vivo gene delivery in target organs and tissues such as the lung, liver and vasculature.
  • this gene therapy strategy was shown to induce enhanced tumor cell chemosensitivity.
  • the present invention further demonstrates improved tumor therapy as a result of the combination of this gene therapy approach and radiation therapy treatment.
  • This combination of gene therapy knockout of an oncoprotein and radiation therapy treatment possesses the advantage of targeted gene therapy mediated radiation sensitization of tumors resulting in improved tumor cures following treatment with radiation therapy.
  • Figure 12 shows the effect of single fraction cobalt-60 external beam irradiation on the growth of established human ovarian cancer xenografts.
  • a group of 5 athymic nude mice were injected subcutaneously with lxlO 7 SKOV3.ipl human ovarian cancer cells, and 10 athymic nude mice were injected subcutaneously with lxl 0 7 SKOV3-KO (SKOV3.ipl cells transduced with a gene encoding single chain anti-erbB-2 antibody designated
  • SKOV3.ip l On day -8, a group of 10 athymic nude mice were injected subcutaneously with 1.5x 10 ' SKOV3.ip l or 2.0x 10 7 SKOV3-KO human ovarian cancer cells SKOV3.ipl cells transduced with a gene encoding single chain anti-erbB-2 antibody designated SKOV3/pGT21.
  • the tumors On day 0, when the tumors measured 3-7.5 mm in diameter, 5 animals from each group received 10 Gy cobalt-60 irradiation. The other tumors were not irradiated. The change in tumor size (bidimensional product) was then assessed at varying times after injection. Data are expressed as the average of 5 animals/group.
  • KO cells Some of the tumors were irradiated with 10 Gy cobalt-60 irradiation on day 0. To confirm the finding that radiation interacts with the anti-erbB-2 sFv to radiosensitize SKOV3/pGT21 tumors ( Figure 13 and Table I), the above experiment were repeated with additional controls. Previously, tumors established with SKOV3.ipl parental cells received radiation; there was no group of the parental cells that did not receive radiation. SKOV3.ipl parental cells without irradiation continued to grow without the delay observed with SKOV3/pGT21 tumors ( Figure 13 and Table II). The results of the repeat experiment were very similar to the first experiment out to
  • the murine hybridoma cell line 4D7 which expresses a monoclonal antibody against the human Bcl-2 protein was described.
  • the murine hybridoma cell line 6C8 expresses a monoclonal antibody against the human BAG- 1 protein (from John C Reed, Burnham Institute, La Jolla, CA). This hybridoma was used to generate cDNA from purified mRNA. sFv constructs were generated with the recombinant phage antibody system (Pharmacia Biotech, Piscataway, NJ) according to the manufacturer's instructions.
  • variable heavy (VH ) and variable light (VL ) chains were amplified from the cDNA by polymerase chain reaction (PCR) using mouse variable region primers (Pharmacia Biotech, Piscataway, NJ).
  • the VH and the VL DNA fragments were linked together by overlap extension PCR using a (Gly4Ser)3 linker to generate a 750 bp sFv construct with flanking Sfil and Notl restriction sites.
  • the sFv D ⁇ A fragments were cloned into Sf ill No tl sites of the prokaryotic expression vector pCA ⁇ TAB5 (Pharmacia Biotech, Piscataway, NJ).
  • BAG-1 ORF was cloned into BamHl/EcoRl of the pGEX 3X (Pharmacia Biotech) vector encoding the Glutathion-S-transferase (GST) protein.
  • GST-BAG- 1 fusion protein was purified using the GST purification module kit from Pharmacia Biotech.
  • periplasmic extracts were prepared as follows: bacterial clones containing pCANTAB5/sFvs were induced with 1 mM IPTG for 4 hours, centrifuged and resuspended in ice-cold phosphate buffered saline (PBS)- l mM EDTA, followed by incubation on ice for 30 minutes and centrifugation at 1500g for 10 minutes at 4°C. The supernatant, which contains the soluble sFvs, was stored at 4°C until needed.
  • PBS phosphate buffered saline
  • the supernatant which contains the soluble sFvs, was stored at 4°C until needed.
  • Ninety-six well plates were coated with 10 ⁇ g/ml of purified BAG- 1 protein (200 ⁇ l/well) in
  • Cells were transfected in 6 well plates using the AdpL method and replated the next day into 96 well plates ( 10 4 cells/well). Twenty-four hours later, the medium was changed and fresh medium containing various concentrations of staurosporine (Sigma) or cis-diamminedichloroplatinum (CDDP) was added. The relative percentage of viable cells was determined 4 days later by (3-(4,5-dimethylthiazol-2yl)-5-(3- carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H tetrazolium (MTS) reduction assay using the Celltiter 96 kit (Promega, Madison, WI).
  • MTS cis-diamminedichloroplatinum
  • the hybridoma cell line 4D7 (from John C Reed) produces a monoclonal antibody against the human Bcl-2 protein.
  • the cDNA encoding the VH and the V chains of this antibody were linked together as described and the full length sFv construct was cloned into the bacterial expression vector pCANTAB5 ( Figure 14A).
  • Positive clones were analyzed by PCR for the presence of an sFv insert ( Figure 15 A) and for their ability to generate an -34 kDa sFv protein by Western blot ( Figure 15B).
  • periplasmic extracts produced from IPTG- induced E. coli were prepared. These extracts, which contain soluble sFv proteins, were then used in an enzyme-linked immunosorbent assay (ELISA) to determine their ability to bind to Bcl-2 protein. The untransduced strain and an irrelevant protein (BAG- 1 ) were used as negative controls to confirm the binding specificity.
  • ELISA enzyme-linked immunosorbent assay
  • an anti-Bcl-2 sFv has been derived, that, when expressed in a prokaryotic system, binds specifically to the human Bcl-2 protein.
  • sFvs The intracellular expression of sFvs is a potent way to achieve selective knock-out of cellular proteins.
  • Bcl-2 is a membrane associated protein
  • pSecTag C vector Invitrogen
  • Ig kappa leader sequence directing proteins to the secretory pathway was modified to include the KDEL signal at the carboxy terminal of the sFv in order to localize the anti-Bcl-2 sFv to the ER. The integrity of this vector was verified by DNA sequencing.
  • GFP green fluorescent protein
  • Bcl-2 expression in HeLa cells was assayed for a dose-dependent modulation.
  • a series of different DNA ratios (1 : 1 , 1 :5, 1 : 10) of pRC/CMV/hBcl-2 and anti-Bcl-2 sFvs plasmids were cotransfected into HeLa cells.
  • Bcl-2 expression was determined 48 hours post- transfection by Western blot.
  • a more pronounced down-modulation of Bcl-2 expression was observed with increasing amounts of anti-Bcl-2 sFv DNA, thereby suggesting that, in this system, a relative excess of sFv protein is required for optimal antigen interaction and ablation.
  • the dose-response observed also supports the hypothesis that a specific interaction occurs between the anti-Bcl-2 sFv and Bcl-2 protein. As previously observed, the anti-Bcl-2 #4 was the most efficent to down-regulate Bcl-2.
  • Bcl-2 expressing MCF-7 cells were transfected with the anti-Bcl-2 sFv plasmid or pSTCF.KDEL, as a control, and cellular proliferation was measured at several time points post- transfection by MTT assay.
  • Bcl-2 negative DU145 cells were also transfected with the same plasmids to serve as a control. The growth curves of these cells are shown in Figure 20A and B. No growth inhibition was observed in cells transduced with the anti-Bcl-2 sFv compared to controls.
  • Bcl-2 expression can modulate the sensitivity of cancer cells to drug-induced apoptosis
  • Staurosporine is known to inhibit protein kinase activity and can therefore induce apoptosis in Bcl-2 overexpressing cells.
  • MCF-7 cells transduced with the anti-Bcl-2 sFv were more susceptible to cell killing induced by staurosporine. A similar effect was observed when these cells were treated with CDDP.
  • Bcl-2 negative DU145 cells transfected with the anti-Bcl- 2 sFv did not show any increase in cell death.
  • BAG- 1 is a 29 kDa molecule that does not show homology to the Bcl-2 family, but the N-terminal shares homology with ubiquitin.
  • the cDNA encoding BAG-1 was cloned in the mammalian expression vector pcDNA3 in which the expression of BAG-1 is driven by the CMV promoter.
  • the resulting plasmid (pcDNA3/BAG-l ) or control DNA (pcDNA3) was transfected into the DU145 cell line, that does not endogenously express Bcl-2, and in the MCF-7 cell line which overexpresses Bcl-2.
  • BAG- 1 alone has little effect on drug- mediated cell killing (at least in the prostate cancer cell line DU145) whereas in endogenously Bcl-2 overexpressing MCF-7 cells, BAG-1 can potentiate the anti-apoptotic effect of Bcl-2.
  • Single-chain antibody as a means to abrogate the expression of BAG-1 in human tumor cell lines
  • Intracellular sFvs represent a class of therapeutic agents that can selectively abrogate the expression of an oncogene wihthin a tumor cell.
  • an anti-BAG-1 sFv has similar properties in BAG- 1 overexpressing tumor cells
  • an anti-BAG- 1 sFv was constructed and evaluated for its binding affinity to BAG-1 by ELISA.
  • the VH and V chains were amplified by PCR from cDNA derived from the hybridoma cell line 6C8 (obtained from J. Reed, Burham Institute,
  • BAG-1 The binding affinity of the 20 clones to the BAG-1 protein was determined by ELISA.
  • anti-BAG-1 sFv clones displayed good binding affinity to BAG-1.
  • no binding was observed with the bacterial extract alone or with purified Bcl- 2 protein demonstrating the specificity of these sFvs for BAG- 1.
  • Figure 24B shows the binding affinity data for three anti-BAG-1 clones that demonstrated the highest affinity.
  • BAG- 1 is a cytosolic protein, as shown by immunofluorescence studies, and it lacks a transmembrane signal domain.
  • the anti-BAG- 1 sFv was targeted to the ER because 1 ) most of the previous experiments performed with intracellular sFvs have been carried out in the ER; 2) antibodies are normally assembled and folded in the ER; 3) sFvs expressed in the cytosol are unstable due to an unfavorable redox environment. In addition, ER-targeted sFv have functionally inhibited proteins that were localized into other cellular compartments.
  • the anti-BAG- 1 sFv ORFs from clones 1 1 , 15 and 20 were subcloned into the pSTCF.KDEL eukaryotic vector. These clones were selected based upon their binding affinity on ELISA. The ability of the pSTCF.KDEL plasmid to localize an sFv to the endoplasmic reticulum (ER) has been described.
  • HeLa cells were transfected using the AdpL method with pcDNA3/BAG- l alone, the anti-BAG- 1 sFvs 11 , 15, 20 alone or cotransfected with pcDNA3/BAG-l and the anti-BAG- 1 sFvs 1 1 , 15, 20 respectively.
  • the HeLa cell line was chosen because of its high transducibility by AdpL and the fact that this system was previously validated with another sFv. Forty-eight hours post-transfection, the expression of BAG- 1 was evaluated by Western blot analysis.
  • BAG- 1 protein or the anti-BAG-1 sFv could influence the proliferation of DU145 and MCF-7 under normal growth conditions
  • these cells were transfected by the AdpL method with the BAG- 1 expression vector, the anti-BAG-1 sFv, both or BAG-1 cDNA and pSTCF.KDEL.
  • overexpression of BAG- 1 per se did not significantly influence the growth rate of DU145 and MCF-7.
  • Identical results were obtained with the anti-BAG-1 sFv suggesting that by itself this sFv had no negative effect on cell proliferation.
  • the anti-BAG- 1 sFv-induced modulation of the BAG- 1 protein can abolish the relative resistance to cytotoxicity conferred by this protein in MCF-7 cell
  • the anti-BAG-1 sFv 20, Bag-1 or both plasmids were transfected by the AdpL method and 24 hours later the transfected MCF-7 cells were treated with staurosporine or CDDP.
  • the cell survival was assessed 4 days after the addition of the drugs. Similar experiments were conducted in DU145 as these cells do not express Bcl-2. As expected, the down-regulation of BAG- 1 expression in MCF-7 cells completely abrogated the protective effect of BAG- 1 (Figure 28A-B).
  • BAG- 1 down- regulation in DU145 cells had no impact on their survival following exposure to drugs (Figure 28C-D).
  • Transfection of the anti-BAG- 1 sFv alone had no effect on the survival of both cell lines compared to the control (mock-transfected cells).
  • Cotransfection of BAG-1 cDNA and pSTCF.KDEL in MCF-7 resulted in increased cell survival, demonstrating that pSTCF.KDEL by itself does not affect BAG- 1 -mediated protection from cytotoxicity. Similar results were obtained with the two cytotoxic drugs.
  • the present invention demonstrates selective down- regulation of Bcl-2 protein expression in different epithelial tumor cell lines. This effect was dependent upon the ratio of anti-Bcl-2 sFv/Bcl-2 protein. A relative excess of anti-Bcl-2 sFv protein was required to achieve efficient down-regulation of the Bcl-2 protein in a situation where both cDNAs were driven by the CMV promoter. However, in tumor cells endogenously overexpressing Bcl-2, the level of Bcl-2 expression was significantly lower than that obtained via heterologous gene transfer. Therefore, an excess of anti-Bcl-2 sFv protein can easily be achieved in these cells with a CMV driven vector. In fact, significant down-regulation of the Bcl-2 protein has been achieved in endogenously Bcl-2 expressing MCF-7 cells.
  • the present invention demonstrates that BAG- 1 has anti-apoptotic activity in a breast cancer cell line that overexpresses Bcl-2.
  • Expression of an intracellular ER-targeted anti-BAG-1 sFv was able to abrogate the expression of BAG-1 and thereby abolish the anti-apoptic activity of BAG-1 in these cells.
  • This is the first report that demonstrates the biological importance on down-modulation of BAG-1.
  • Intracellular sFv anti-cyclin-D l can knockout cyclin D I protein expression and alter tumor cell growth
  • Cyclins and cyclin-dependent kinases are central to the regulation of the eukaryotic cell cycle.
  • the role of cell cycle in modifying radiation sensitivity has been well established. Abrogating genes involved in cell cycle regulation can enhance radiosensitization.
  • a sFv to cyclin-Dl was developed from the hybridoma cell line DCS-6.
  • the DCS-6 hybridoma produces an antibody specific for the cyclin-Dl oncoprotein.
  • the corresponding V L and V H chains of the DCS-6 RNA were amplified using RT-PCR and successfully assembled into an anti-cyclin-D l sFv ( Figure 29A).
  • the sFv library was subcloned into the prokaryotic expression vector pCANTAB5E in frame with a C-terminal E-tag for subsequent detection.
  • E. coli were used to obtain periplasmic extract.
  • Western blot analysis demonstrated sFv protein expression in the prokaryotic system only under induced conditions ( Figure 29B).
  • Figure 29B Western blot analysis demonstrated sFv protein expression in the prokaryotic system only under induced conditions.
  • the anti-cyclin-Dl sFv protein of 27 kDa was produced in the eukaryotic system.
  • periplasmic fractions were obtained and an ELISA was used to determine specific binding to 40 mg of purified cyclin-Dl protein.
  • the localization of the green fluorescent protein (GFP) reporter to the different intracellular compartments was evaluated.
  • the ER (KDEL retention signal) and nuclear GFP-fusion protein vectors were transfected in HeLa cells using the AdpL method.
  • Transduced cells were evaluated under a fluorescent microscope ( Figure 30A).
  • the GFP reporter was localized to the targeted subcellular compartment by the appropriate plasmid vector.
  • the expression of anti-cyclin-Dl sFv was assayed for the nuclear and ER forms of the sFv in transfected HeLa cells. Western blot analysis were performed as shown in Figure 30B.
  • a band at 27 kDa corresponding to the expected molecular weight of the anti- cyclin-Dl sFv protein was detected for both expression vectors.
  • the anti-cyclin-Dl sFv can be expressed in both the nuclear and ER of eukaryotic cells.
  • Cyclin-Dl is essential for progression through the Gl phase of the cell cycle.
  • cellular DNA FACS analysis of nonsynchronized MDA-MB-453 cells when treated with the nuclear form of the anti-cyclin-D l sFv showed a 22% increase in the proportion of cells in Gl and a concomitant reduction of actively dividing cells compared to only 8% with the ER form of the sFv.
  • Another cell line that overexpresses cyclin-Dl (MCF-7) was used to confirm the previous results.
  • MCF-7 cells showed a delayed S phase entry with a 37% increase in cells in Gl transfected with the nuclear form of the anti-cyclin-Dl sFv.
  • the anti-cyclin-Dl sFv was able to achieve selective blockage of cell cycle progression by accumulating cells in the Gl phase of the cell cycle.
  • the cell cycle arrest was additionally manifested as a reduction of viable cells at various time points.
  • Five days post transfection MDA-MB-453 cells expressing the nuclear-localized anti-cyclin-Dl sFv showed extensive cell death as assessed by trypan blue exclusion. This cell death was not apparent in MDA- MB-453 cells transfected with the control plasmid pcDNA3 ( Figure
  • the expression of the nuclear form and ER form of the anti-cyclin-D l sFv alters the cell cycle kinetics of cyclin-D l overexpressing breast cancer cells but not in cells expressing normal levels of cyclin-Dl protein. Therefore, the expression of an sFv that alters the cell cycle kinetics of cells overexpressing cyclin-
  • Dl may have an important role in sensitizing tumor cells to ionizing radiation.
  • Figure 33 shows that in a direct analysis of viable cells, the nuclear form of the anti-cyclin-Dl scFv induced 60% reduction in the number of viable cells in MCF-7 and MDA-MB-453. In contrast, the ER form of the scFv only achieved close to 40% reduction in cell viability in those same cells. Thus, the expression of the nuclear and ER form of this scFv alters the cells cycle kinetics and exhibits a selective anti-proliferative effect in overexpressing cyclin DI breast cancer cells.

Abstract

La présente invention concerne un procédé permettant d'augmenter la chimiosensibilité et la radiosensibilité d'une cellule néoplasique exprimant une oncoprotéine qui stimule la prolifération de cette cellule, consistant à introduire dans la cellule une molécule d'acide nucléique codant un homologue d'anticorps, l'homologue d'anticorps étant exprimé de façon intracellulaire et se liant à l'oncoprotéine également de façon intracellulaire dans le réticulum endoplasmique de la cellule. La présente invention concerne également un procédé permettant d'augmenter l'inhibition de la prolifération d'une cellule néoplasique exprimant une encoprotéine qui stimule la prolifération de cette cellule, comprenant les étapes suivantes: introduction, dans la cellule, d'une molécule d'acide nucléique codant un homologue d'anticorps, cet homologue d'anticorps étant exprimé de façon intracellulaire et se liant à la protéine également de façon intracellulaire, et mise en contact de ladie cellule avec un agent antinéoplasique.
PCT/US1997/019911 1996-10-30 1997-10-30 Augmentation de la chimiosensibilite et de la radiosensibilite de cellules tumorales au moyen d'anticorps intracellulaires a une seule chaine WO1998018489A1 (fr)

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Cited By (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001049839A2 (fr) * 2000-01-07 2001-07-12 Umeda Jimusho Ltd. Procede d'expression genique et son utilisation
EP1165842A1 (fr) * 1999-03-16 2002-01-02 Dana-Farber Cancer Institute, Inc. Systeme vectoriel lentiviral pour le criblage de grande quantite
WO2001040276A3 (fr) * 1999-12-06 2002-01-10 Urogenesys Inc Nouveaux antigenes transmembranaires du type serpentin exprimes dans des cancers humains et utilisation de ces antigenes
US6833438B1 (en) 1999-06-01 2004-12-21 Agensys, Inc. Serpentine transmembrane antigens expressed in human cancers and uses thereof
US6887975B2 (en) 1998-06-01 2005-05-03 Agensys, Inc. Peptides derived from STEAP1
US7262173B2 (en) 1997-03-21 2007-08-28 Georgetown University Chemosensitizing with liposomes containing oligonucleotides
US7494646B2 (en) 2001-09-06 2009-02-24 Agensys, Inc. Antibodies and molecules derived therefrom that bind to STEAP-1 proteins
US7622569B2 (en) 1998-06-01 2009-11-24 Agensys, Inc. Nucleic acid and corresponding protein entitled 98P4B6 useful in treatment and detection of cancer
US7625558B2 (en) 1998-03-04 2009-12-01 The Trustees Of The University Of Pennsylvania Compositions and methods of treating tumors
WO2010108127A1 (fr) 2009-03-20 2010-09-23 Genentech, Inc. Anticorps anti-her di-spécifiques
WO2010136569A1 (fr) 2009-05-29 2010-12-02 F. Hoffmann-La Roche Ag Modulateurs de la signalisation her2 chez des patients exprimant her2 souffrant d'un cancer de l'estomac
US7884179B2 (en) 2001-09-06 2011-02-08 Agensys, Inc. Nucleic acid and corresponding protein entitled STEAP-1 useful in treatment and detection of cancer
US7947459B2 (en) 1998-06-01 2011-05-24 Agensys, Inc. Serpentine transmembrane antigens expressed in human cancers and uses thereof
US7981418B2 (en) 2007-03-02 2011-07-19 Genentech, Inc. Predicting response to a HER inhibitor
US7993834B2 (en) 2000-05-19 2011-08-09 Genentech, Inc. Detection of ErbB2 gene amplification to increase the likelihood of the effectiveness of ErbB2 antibody breast cancer therapy
WO2011103242A1 (fr) 2010-02-18 2011-08-25 Genentech, Inc. Antagonistes de la neuréguline et leur utilisation dans le cadre du traitement du cancer
US8008442B2 (en) 2004-04-22 2011-08-30 Agensys, Inc. Antibodies and molecules derived therefrom that bind to STEAP-1 proteins
US8012937B2 (en) 1998-06-01 2011-09-06 Agensys, Inc. Nucleic acid and corresponding protein entitled 98P4B6 useful in treatment and detection of cancer
WO2011146568A1 (fr) 2010-05-19 2011-11-24 Genentech, Inc. Prédiction de réponses à un inhibiteur de her
US8075892B2 (en) 1997-12-12 2011-12-13 Genentech, Inc. Treatment with anti-ErbB2 antibodies
EP2399605A1 (fr) 2005-02-23 2011-12-28 Genentech, Inc. Extension du laps de temps avant progression de la maladie ou de la survie chez les patients atteints de cancer
WO2012069466A1 (fr) 2010-11-24 2012-05-31 Novartis Ag Molécules multi-spécifiques
WO2012085111A1 (fr) 2010-12-23 2012-06-28 F. Hoffmann-La Roche Ag Complexe polypeptide-polynucléotide et son utilisation dans l'administration d'une fraction effectrice ciblée
WO2013025853A1 (fr) 2011-08-17 2013-02-21 Genentech, Inc. Anticorps anti-neuréguline et utilisations associées
US8404234B2 (en) 2005-01-21 2013-03-26 Genentech, Inc. Fixed dosing of HER antibodies
US8436147B2 (en) 2006-10-27 2013-05-07 Genentech, Inc. Antibodies and immunoconjugates and uses therefor
EP2592156A2 (fr) 2007-06-08 2013-05-15 Genentech, Inc. Marqueurs d'expression de gène de résistance tumorale à un traitement par inhibiteur HER2
WO2013081645A2 (fr) 2011-11-30 2013-06-06 Genentech, Inc. Mutations dans erbb3 dans des cancers
WO2013083810A1 (fr) 2011-12-09 2013-06-13 F. Hoffmann-La Roche Ag Identification de non-répondeurs aux inhibiteurs de her2
WO2013148315A1 (fr) 2012-03-27 2013-10-03 Genentech, Inc. Diagnostic et traitements concernant des inhibiteurs de her3
WO2014083178A1 (fr) 2012-11-30 2014-06-05 F. Hoffmann-La Roche Ag Identification de patients ayant besoin d'une cothérapie par un inhibiteur de pd-l1
EP1745799B1 (fr) 1998-03-04 2015-09-02 The Trustees of The University of Pennsylvania Compositions et méthodes pour le traitement de tumeurs
US9327023B2 (en) 2011-10-25 2016-05-03 The Regents Of The University Of Michigan HER2 targeting agent treatment in non-HER2-amplified cancers having HER2 expressing cancer stem cells
US9551033B2 (en) 2007-06-08 2017-01-24 Genentech, Inc. Gene expression markers of tumor resistance to HER2 inhibitor treatment
WO2017194554A1 (fr) 2016-05-10 2017-11-16 Inserm (Institut National De La Sante Et De La Recherche Medicale) Polythérapies pour le traitement du cancer
US10689457B2 (en) 2008-06-16 2020-06-23 Genentech, Inc. Treatment of metastatic breast cancer
US11180571B2 (en) 2017-04-03 2021-11-23 Hoffmann-La Roche Inc. Antibodies binding to STEAP-1

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ANTISENSE RESEARCH AND DEVELOPMENT, 1994, Vol. 4, No. 2, KITADA S., "Reversal of Chemoresistance of Lymphoma Cells by Antisense-Mediated Reduction of bcl-2 Gene Expression", pages 71-79. *
CLINICAL CANCER RESEARCH, July 1996, Vol. 2, No. 7, BARNES M.N., "Novel Gene Therapy Strategy to Accomplish Growth Factor Modulation Induces Enhanced Tumor Cell Chemosensitivity", pages 1089-1095. *
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY, September 1996, Vol. 107, No. 3, JANSEN B., "Downregulation of Bcl-2 by Antisense Oligonucleotides Reduces Tumor Size and Improves Chemosensitivity of Human Melanoma in SCID Mice", page 469, Abstract Number 132. *

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US7262173B2 (en) 1997-03-21 2007-08-28 Georgetown University Chemosensitizing with liposomes containing oligonucleotides
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US8425908B2 (en) 1997-12-12 2013-04-23 Genentech, Inc. Treatment with anti-ErbB2 antibodies
US8309087B2 (en) 1997-12-12 2012-11-13 Genentech, Inc. Treatment with anti-ErbB2 antibodies
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US8053551B2 (en) 1999-06-01 2011-11-08 Agensys, Inc. Serpentine transmembrane antigens expressed in human cancers and uses therefor
US6833438B1 (en) 1999-06-01 2004-12-21 Agensys, Inc. Serpentine transmembrane antigens expressed in human cancers and uses thereof
US7455991B2 (en) 1999-06-01 2008-11-25 Agensys, Inc. Serpentine transmembrane antigens expressed in human cancers and uses thereof
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US9593167B2 (en) 2006-10-27 2017-03-14 Genentech, Inc. Antibodies and immunoconjugates and uses therefor
US8889847B2 (en) 2006-10-27 2014-11-18 Genentech, Inc. Antibodies and immunoconjugates and uses therefor
EP2899541A1 (fr) 2007-03-02 2015-07-29 Genentech, Inc. Elément de prévision de la réponse à un inhibiteur de HER
US7981418B2 (en) 2007-03-02 2011-07-19 Genentech, Inc. Predicting response to a HER inhibitor
US8940302B2 (en) 2007-03-02 2015-01-27 Genentech, Inc. Predicting response to a HER inhibitor
EP2592156A2 (fr) 2007-06-08 2013-05-15 Genentech, Inc. Marqueurs d'expression de gène de résistance tumorale à un traitement par inhibiteur HER2
US10385405B2 (en) 2007-06-08 2019-08-20 Genentech, Inc. Gene expression markers of tumor resistance to HER2 inhibitor treatment
US9551033B2 (en) 2007-06-08 2017-01-24 Genentech, Inc. Gene expression markers of tumor resistance to HER2 inhibitor treatment
US10689457B2 (en) 2008-06-16 2020-06-23 Genentech, Inc. Treatment of metastatic breast cancer
US11655305B2 (en) 2008-06-16 2023-05-23 Genentech, Inc. Treatment of metastatic breast cancer
WO2010108127A1 (fr) 2009-03-20 2010-09-23 Genentech, Inc. Anticorps anti-her di-spécifiques
EP3088420A1 (fr) 2009-03-20 2016-11-02 F. Hoffmann-La Roche AG Anticorps anti-her bispécifiques
WO2010136569A1 (fr) 2009-05-29 2010-12-02 F. Hoffmann-La Roche Ag Modulateurs de la signalisation her2 chez des patients exprimant her2 souffrant d'un cancer de l'estomac
WO2011103242A1 (fr) 2010-02-18 2011-08-25 Genentech, Inc. Antagonistes de la neuréguline et leur utilisation dans le cadre du traitement du cancer
WO2011146568A1 (fr) 2010-05-19 2011-11-24 Genentech, Inc. Prédiction de réponses à un inhibiteur de her
WO2012069466A1 (fr) 2010-11-24 2012-05-31 Novartis Ag Molécules multi-spécifiques
WO2012085111A1 (fr) 2010-12-23 2012-06-28 F. Hoffmann-La Roche Ag Complexe polypeptide-polynucléotide et son utilisation dans l'administration d'une fraction effectrice ciblée
WO2013025853A1 (fr) 2011-08-17 2013-02-21 Genentech, Inc. Anticorps anti-neuréguline et utilisations associées
US9327023B2 (en) 2011-10-25 2016-05-03 The Regents Of The University Of Michigan HER2 targeting agent treatment in non-HER2-amplified cancers having HER2 expressing cancer stem cells
WO2013081645A2 (fr) 2011-11-30 2013-06-06 Genentech, Inc. Mutations dans erbb3 dans des cancers
WO2013083810A1 (fr) 2011-12-09 2013-06-13 F. Hoffmann-La Roche Ag Identification de non-répondeurs aux inhibiteurs de her2
WO2013148315A1 (fr) 2012-03-27 2013-10-03 Genentech, Inc. Diagnostic et traitements concernant des inhibiteurs de her3
EP3511718A1 (fr) 2012-11-30 2019-07-17 F. Hoffmann-La Roche AG Inhibiteur de pd-l1
WO2014083178A1 (fr) 2012-11-30 2014-06-05 F. Hoffmann-La Roche Ag Identification de patients ayant besoin d'une cothérapie par un inhibiteur de pd-l1
WO2017194554A1 (fr) 2016-05-10 2017-11-16 Inserm (Institut National De La Sante Et De La Recherche Medicale) Polythérapies pour le traitement du cancer
US11180571B2 (en) 2017-04-03 2021-11-23 Hoffmann-La Roche Inc. Antibodies binding to STEAP-1
US11685790B2 (en) 2017-04-03 2023-06-27 Hoffmann-La Roche Inc. Antibodies binding to STEAP-1

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