A 60 MeV proton beam-line dedicated to research and development programs.

A new proton beam-line dedicated to R&D programs has been developed at CentreAntoine Lacassagne (CAL), in Nice (France), in collaboration with the Centrenational d'études spatiales (CNES). This is the second beam-line of the MEDICYC 65 MeV cyclotron that is currently in operation, the first being the clinical 'eye-line' used for ocular proton therapy. The R&D beam-line is proposed with two configurations, the first producing a Gaussian narrow beam of a few mm width, the second a 100 mm diameter flat beam with a homogeneity better than ±3%. The energy range is (20 - ∼60) MeV, where the exact upper limit depends on the beam configuration being used. The energy spread of the non-degraded beam is (0.3 ± 0.1) MeV. A beam current between 10 pA and 10 µA can be produced with a stability better than 0.2% above 100 pA, and 2% below. The beam can be monitored online at a precision better than 5% in the flux range 1E5 (1E6) - 1E9 (1E10) p/cm2/s for a flat (Gaussian) configuration, although work is in progress to extend this range. Targeted applications for the R&D beam-line are instrumentation research, radiation tolerance tests of components and radiobiology.

Toxicity and exposure of an adenovirus containing human interferon alpha-2b following intracystic administration in cynomolgus monkeys.

The safety and toxicokinetics of SCH 721015, an adenovirus encoding the human interferon alpha-2b gene, and Syn3 (SCH 209702), a novel excipient, were assessed in cynomolgus monkeys administered intravesical doses of 2.5 × 10E11 or 1.25 × 10E13 particles SCH 721015 in 25 mg Syn3 or 25 mg Syn3 alone on study days 1 and 91. There was no systemic toxicity. Monkeys dosed with SCH 721015 in Syn3 were positive for SCH 721015-specific DNA in the urine for 2 to 3 days following each dose and had interferon alpha-2b protein in the urine for 1-3 days after a single dose and in fewer animals after a second dose. Intracystic administration was associated with inflammation and focal/multifocal ulceration in the urinary bladder and irritation in the ureters and urethra at necropsy. The physical trauma from catheterization and filling/emptying of the bladder was likely a contributing factor and Syn3 exacerbated the trauma. There was nearly complete resolution of these findings 2 months after the last dose. The trauma to the bladder likely contributed to low, transient systemic exposure to Syn3, SCH 721015 and human interferon protein. The results of this study support the clinical investigation of SCH 721015 in Syn3.

Simulation of a 6 MV Elekta Precise Linac photon beam using GATE/GEANT4.

The GEANT4-based GATE Monte Carlo (MC) platform was initially focused on PET and SPECT simulations. The new release v6.0 (February 2010) proposes new tools dedicated for radiation therapy simulations. In this work, we investigated some part of this extension and proposed a general methodology for Linac simulations. Details of the modeling of a 6 MV photon beam delivered by an Elekta Precise Linac, with radiation fields ranging from 5 × 5 to 30 × 30 cm(2) at the isocenter are presented. Comparisons were performed with measurements in water. The simulations were performed in two stages: first, the patient-independent part was simulated and a phase space (PhS) was built above the secondary collimator. Then, a multiple source model (MSM) derived from the PhS was proposed to simulate the photon fluence interacting with the patient-dependent part. The selective bremsstrahlung splitting (SBS) variance reduction technique proposed in GATE was used in order to speed up the accelerator head simulation. Further investigations showed that the SBS can be safely used without biasing the simulations. Additional comparisons with full simulations performed on the EGEE grid, in a single stage from the electron source to the water phantom, allowed the evaluation of the MSM. The proposed MSM allowed for calculating depth dose and transverse profiles in 48 hours on a single 2.8 GHz CPU, with a statistical uncertainty of 0.8% for a 10 × 10 cm(2) radiation field, using voxels of 5 × 5 × 5 mm(3). Good agreement between simulations and measurements in water was observed, with dose differences of about 1% and 2% for depth doses and dose profiles, respectively. Additional gamma index comparisons were performed; more than 90% of the points for all simulations passed the 3%/3 mm gamma criterion. To our knowledge, this feasibility study is the first one illustrating the potential of GATE for external radiotherapy applications.

Herceptin.

The biology of the human epidermal growth factor (EGF) receptor-2 (HER2) has been reviewed numerous times and provides an excellent example for developing a targeted cancer therapeutic. Herceptin, the FDA-approved therapeutic monoclonal antibody against HER2, has been used to treat over 150,000 women with breast cancer. However, the developmental history of Herceptin, the key events within the program that created pivotal decision points, and the reasons why decisions were made to pursue the monoclonal antibody approach have never been adequately described. The history of Herceptin is reviewed in a way which allows the experience to be shared for the purposes of understanding the drug discovery and development process. It is the objective of this review to describe the pivotal events and explain why critical decisions were made that resulted in the first therapeutic to successfully target tyrosine kinases in cancer. New approaches and future prospects for therapeutics targeting the HER family are also discussed.

Enhancement of intravesical delivery with Syn3 potentiates interferon-alpha2b gene therapy for superficial bladder cancer.

Intravesical administration of interferon alpha-2b protein (IFN) has been successfully used in the treatment of patients with superficial bladder tumors. Local dosing of IFN minimizes well-known systemic side effects of the drug, but exposure to bladder tumors is limited by the duration of instillation and transient concentrations achieved in the urothelium. Intravesical delivery of the gene encoding interferon results in an alternative strategy for IFN-based therapy of the disease, enabling sustained exposure of IFN protein that results from production by tumor and non-tumor cells in the urothelium. Efficient gene delivery and expression of IFN has been achieved using a recombinant adenovirus gene delivery system (rAd-IFN) in conjunction with the novel small molecule excipient Syn3. Studies with rAd-IFN/Syn3 in animal models result in urine concentrations of IFN that persisted for weeks and correlated with potent anti-tumor effects. The objective of this review is to communicate the rationale and preclinical findings that support ongoing clinical investigation of intravesical rAd-IFN/Syn3 in superficial bladder cancer.

SCH 412499: biodistribution and safety of an adenovirus containing P21(WAF-1/CIP-1) following subconjunctival injection in Cynomolgus monkeys.

Monkey studies were conducted for the preclinical safety assessment of SCH 412499, an adenovirus encoding p21, administered by subconjunctival injection prior to trabeculectomy for postoperative maintenance of the surgical opening. Biodistribution of SCH 412499 was minimal and there was no systemic toxicity. Findings included swollen, partially closed or shut eye(s) and transient congestion in the conjunctiva. A mononuclear cell infiltrate was present in the conjunctiva, choroid and other ocular tissues, but completely or partially resolved over time. Electroretinograms and visual evoked potentials revealed no adverse findings. Thus, the findings are not expected to preclude the clinical investigation of SCH 412499.

Adenovirus delivery provides extended interferon-alpha exposure and augments treatment of metastatic carcinoma.

Type I interferons (e.g. IFNalpha2b) have been successfully used to treat a variety of hematological malignancies, but have not been efficacious for treatment of most solid tumors. We tested the hypothesis that delivery of type I interferon utilizing recombinant adenovirus (rAd) vectors may improve treatment efficacy of metastatic carcinomas by providing increased interferon exposure resulting from continuous transgene expression. Treatment of mice with a rAd-vector expressing hybrid-IFN (rAd-IFNalpha2alpha1) inhibited 4T1 mammary carcinoma tumor growth and induced tumor regression in a dose-dependent manner. Moreover, rAd-IFNalpha2alpha1 treatment reduced hepatic and pulmonary metastatic burden. A comparison of local and systemic routes of administration demonstrated that intratumoral delivery of rAd-IFNalpha2alpha1 was sufficient for inhibition of tumor growth. Moreover, it reduced toxicity associated with high-dose systemic IFNalpha2alpha1 exposure. Interestingly, antitumor activity following intratumoral treatment was due, in part, to the immunostimulatory capacity of the rAd vector component. Furthermore, systemic administration of rAd-IFNalpha2alpha1 potentiated the immunotherapeutic effect induced by local intralesional delivery of empty-rAd vector. These results suggest continuous interferon-alpha exposure may provide improved antitumor responses for metastatic carcinomas. Additionally, immunostimulatory responses induced by rAd-IFNalpha2alpha1 may mitigate the immune-evasive tumor microenvironment.

Biological activities of a recombinant adenovirus p53 (SCH 58500) administered by hepatic arterial infusion in a Phase 1 colorectal cancer trial.

The major focus of intrahepatic arterial (IHA) administration of adenoviruses (Ad) has been on safety. Currently, there is little published data on the biological responses to Ad when administered via this route. As part of a Phase I study, we evaluated biological responses to a replication-defective adenovirus encoding the p53 transgene (SCH 58500) when administered by hepatic arterial infusion to patients with primarily colorectal cancer metastatic to the liver. In analyzing biological responses to the Ad vector, we found that both total and neutralizing Ad antibodies increased weeks after SCH 58500 infusion. The fold increase in antibody titers was not dependent on SCH 58500 dosage. The proinflammatory cytokine interleukin-6 (IL-6) transiently peaked within 6 h of dosing. The cytokine sTNF-R2 showed elevation by 24 h post-treatment, and fold increases were directly related to SCH 58500 doses. Cytokines TNF-alpha, IL-1beta, and sTNF-R1 showed no increased levels over 24 h. Predose antibody levels did not appear to predict transduction, nor did serum Ad neutralizing factor (SNF). Delivery of SCH 58500 to tumor tissue occurred, though we found distribution more predominantly in liver tissues, as opposed to tumors. RT-PCR showed significantly higher expression levels (P<0.0001, ANOVA) for adenovirus type 2 and 5 receptor (CAR) in liver tissues, suggesting a correlation with transduction. Evidence of tumor-specific apoptotic activity was provided by laser scanning cytometry, which determined a coincidence of elevated nuclear p53 protein expression with apoptosis in patient tissue. IHA administration of a replication defective adenovirus is a feasible mode of delivery, allowing for exogenous transfer of the p53 gene into target tissues, with evidence of functional p53. Limited and transient inflammatory responses to the drug occurred, but pre-existing immunity to Ad did not preclude SCH 58500 delivery.

Gene therapy using p21WAF-1/Cip-1 to modulate wound healing after glaucoma trabeculectomy surgery in a primate model of ocular hypertension.

Glaucoma is a common eye disease associated with elevated intraocular pressure (IOP). Lowering IOP is the only acceptable therapy for glaucoma and slows progression of the disease. Filtration surgery, which introduces a guarded ostomy through the sclera into the anterior chamber of the eye to allow the escape of aqueous humor, is the most reliable method for effective IOP lowering. Success of this surgery is limited by scarring of the ostomy, so this procedure is often accompanied by the use of antimetabolites, such as mitomycin C (MMC), to block the wound healing response. Although effective in preventing scarring, antimetabolites also yield unwanted side effects, such as hypotony and tissue degeneration due to cellular destruction. This study presents an alternative to antimetabolites by using gene therapy to introduce the human gene for p21(WAF-1/cip-1) (p21) to cause cell cycle arrest of surrounding cells rather than their destruction. In this procedure, p21 was delivered using a recombinant adenovirus to ocular hypertensive monkey eyes. These eyes then underwent filtration surgery. Results show that eyes treated with p21 exhibited open surgical ostomies by both functional and histological criteria, and did not display any side effects seen in control animals that were treated with MMC.

Interferon alpha2b gene delivery using adenoviral vector causes inhibition of tumor growth in xenograft models from a variety of cancers.

A recombinant adenovirus expressing human interferon alpha2b driven by the cytomegalovirus promoter, IACB, was shown to produce and secrete biologically active protein in vitro and in vivo. Intravenous administration of IACB in Buffalo rats resulted in circulating levels of biologically active human interferon at 70,000 international units/mL for up to 15 days. Distribution of interferon protein after IACB administration was different from that seen with the subcutaneous delivery of interferon protein. Higher levels of interferon protein were observed in liver and spleen after IACB delivery compared to protein delivery. The antitumor efficacy of IACB, as measured by suppression of tumor growth, was tested in athymic nude mice bearing established human tumor xenografts from different types of human cancer. Subcutaneous tumors most responsive to the intratumoral administration of IACB ranked as U87MG (glioblastoma) and K562 (chronic myelogenous leukemia), followed by Hep 3B (hepatocellular carcinoma) and LN229 cells (glioblastoma). Intravenous administration of IACB in animals bearing U87MG or Hep 3B xenografts was also effective in suppressing tumor growth, although to a lesser extent than the intratumoral administration. IACB was also tested in a metastatic model in beige/SCID mice generated with H69 (small cell lung carcinoma) cells and was found to prolong survival in tumor-bearing animals. This suggested that interferon gene delivery can be effective in suppressing tumor growth in a wide variety of cells.

Identification of polyamides that enhance adenovirus-mediated gene expression in the urothelium.

Adenovirus-mediated gene therapy of bladder diseases has been limited by the inability to transduce the urothelium successfully using adenoviral vectors. We have sought to identify agents that would increase adenovirus-mediated transgene expression in the bladder. We have utilized a rat model to screen compounds for their ability to enhance viral transgene expression in the rat bladder. Rats received intravesical administration of replication-deficient adenovirus (rAd) formulated in various agents, and transgene expression was evaluated after 48 h by determining the amount of lacZ expression in the luminal epithelium of the bladder. We report the identification of two different polyamides, each capable of dramatically increasing viral transgene expression in the bladder without causing detectable alteration of the umbrella cell layer of the urothelium. We have utilized a carcinogen-induced rat bladder tumor model to demonstrate that these polyamides are also capable of enhancing viral transgene expression in tumor tissue. The identification of these polyamides potentiates the use of adenovirus-mediated gene therapy for the treatment of superficial bladder cancer or other bladder diseases.

Specific depletion of human anti-adenovirus antibodies facilitates transduction in an in vivo model for systemic gene therapy.

Recombinant adenoviral (rAd) vectors are capable of mediating high-efficiency gene transfer in vivo. Under conditions requiring systemic administration, however, the use of rAd vectors can be problematic due to the presence of circulating anti-adenovirus antibodies developed either through natural infection or during the course of treatment. We developed a passive immunization model in SCID/Beige mice to assess the effect of human and mouse anti-adenovirus antibodies on systemic administration of a rAd vector expressing beta-galactosidase (rAd-betagal). In this model, the in vitro neutralizing activity of human or mouse antibodies used for passive immunization correlated well with inhibition of transduction of the liver following i.v. administration of rAd-betagal. Depletion of antibodies to individual adenovirus structural proteins (hexon, penton, fiber) by affinity chromatography demonstrated that antibodies to each of the three virion components contributed to neutralization of infectivity in vitro and to inhibition of transduction in vivo. Depletion of antibodies against all three structural proteins from human or mouse immune serum prior to passive immunization restored in vivo transduction activity to levels comparable to those obtained with nonimmune serum. Our data suggest that depletion of both murine and human anti-adenoviral antibodies can restore transduction in vivo during systemic rAd gene therapy in hosts previously exposed to adenovirus.

Ex vivo purging by adenoviral p53 gene therapy does not affect NOD-SCID repopulating activity of human CD34+ cells.

Co-incubation of a replication-deficient, recombinant adenovirus carrying the wild-type p53 gene (rAd-p53) and hematopoietic stem cell (HSC) products from patients with breast cancer can significantly reduce tumor cell contamination. Whereas this approach provides a powerful tumor cell purging strategy, potential detrimental effects on the HSC population have not been investigated. The ability of human HSC to reconstitute hematopoiesis in severe combined immunodeficient (SCID) mice and to undergo secondary transplantation provides the only nonclinical measure of self-renewing, stem cell function. The objective of this study was to investigate whether co-incubation with rAd-p53 compromised the SCID repopulating activity (SRA) of HSC. Granulocyte colony-stimulating factor-mobilized human CD34+ cells were co-cultured with rAd-p53 at our targeted clinical dose, and the ability of these cells to establish multilineage hematopoiesis in sublethally irradiated, nonobese diabetic (NOD)-SCID mice was investigated. The persistence of human cells in the mice was investigated by flow cytometry, granulocyte-macrophage colony-forming unit assay, and polymerase chain reaction of human Alu sequences. Further, limiting dilution analysis provided a quantitative comparison between the SRA of CD34+ cells co-incubated with rAd-p53 and control CD34+ cells (no rAd-p53 co-incubation). We conclude that co-incubation with rAd-p53 has little effect on the SRA of HSC.

Purging of human breast cancer cells from stem cell products with an adenovirus containing p53.

Tumor cell contamination of stem cell products can contribute to tumor relapse following high-dose chemotherapy and stem cell rescue. Numerous techniques have been used to remove the tumor cells from stem cell products with the objective of prolonging relapse-free survival. However, to date these techniques have been relatively ineffectual and/or toxic to hematopoietic stem and progenitor cells. The differential infectivity of adenovirus (Adv) vectors for breast cancer cells, compared with hematopoietic cells, has suggested that Adv-p53 might provide an effective purging strategy. To facilitate the use of Adv-p53 as a clinical strategy, we undertook studies to determine the parameters necessary for optimal stem cell product purging. The parameters studied were the particle number to nucleated cell ratio, the duration of coincubation, the incubation volume, and the presence or absence of hematopoietic progenitor cells. We have found that these parameters are interdependent and conclude that a 4-hour coincubation with an Adv-p53 particle to nucleated cell ratio of 2000:1 with 2 x 10(8) nucleated cells/mL is optimal for tumor cell purging. Furthermore, this appeared to be a safe procedure, with total loss of clonogenic growth of breast cancer cells as well as no significant effect on progenitor cell function as determined by granulocyte-macrophage colony-forming unit assays.

A bacteriophage encoding a pathogenicity island, a type-IV pilus and a phage receptor in cholera bacteria.

The virulence properties of many pathogenic bacteria are due to proteins encoded by large gene clusters called pathogenicity islands, which are found in a variety of human pathogens including Escherichia coli, Salmonella, Shigella, Yersinia, Helicobacter pylori, Vibrio cholerae, and animal and plant pathogens such as Dichelobacter nodosus and Pseudomonas syringae. Although the presence of pathogenicity islands is a prerequisite for many bacterial diseases, little is known about their origins or mechanism of transfer into the bacterium. The bacterial agent of epidemic cholera, Vibrio cholerae, contains a bacteriophage known as cholera-toxin phage (CTXphi), which encodes the cholera toxin, and a large pathogenicity island called the VPI (for V. cholerae pathogenicity island) which itself encodes a toxin-coregulated pilus that functions as a colonization factor and as a CTXphi receptor. We have now identified the VPI pathogenicity island as the genome of another filamentous bacteriophage, VPIphi. We show that VPIphi is transferred between V. cholerae strains and provide evidence that the TcpA subunit of the toxin-coregulated type IV pilus is in fact a coat protein of VPIphi. Our results are the first description of a phage that encodes a receptor for another phage and of a virus-virus interaction that is necessary for bacterial pathogenicity.

Ethanol improves adenovirus-mediated gene transfer and expression to the bladder epithelium of rodents.

OBJECTIVES: Replication deficient adenoviral vectors (rAds) are used as gene delivery systems that can efficiently transduce a variety of tissues and may be appropriate vectors to develop gene therapeutics for many urologic applications. However, the bladder epithelium has been shown to be highly resistant to transgene expression after intracystic administration. A potential explanation for this low gene transfer efficiency may be the protective structure of the urothelium. Since this protective barrier can be disrupted by organic solvents, we assessed whether ethanol co-administration can enhance adenovirus-mediated transgene expression. METHODS: Normal and bladder tumor-bearing rats received a single intracystic administration of rAd encoding beta-galactosidase (rAd-beta gal) or p53 (rAd-p53). rAd was administered in a saline solution or in solutions with increasing concentrations of ethanol. Transgene expression was evaluated in the bladder tissues. RESULTS: A dramatic increase in urothelial beta-galactosidase transgene expression was achieved by rAd-beta gal administered in a 22% ethanol solution. Transgene expression was enhanced in normal urothelium and in superficial bladder tumors. p53 transgene expression was similarly enhanced. CONCLUSIONS: Co-administration of 22% ethanol enhanced local rAd-mediated transgene expression in the normal and neoplastic bladder epithelium in rodents. Improvement of rAd-mediated transgene expression is progress toward local gene delivery to the urothelium and may enable local gene therapy for superficial bladder cancer or other bladder diseases.

Adenovirus p53 purging for human breast cancer stem cell products.

Tumor cell (TC) contamination of stem cell products can contribute to relapse after high dose chemotherapy and stem cell rescue. A new purging technology using replication-deficient recombinant adenovirus (Adv) containing the p53 tumor suppressor gene (Adv-p53) has been suggested to reduce tumor contamination of autologous stem cell product. We demonstrate herein a safe and effective Adv-p53 purging procedure using four human breast cancer TC lines. Multiple parameters need to be achieved to successfully purge stem cell products, including a high cell:virus ratio, a small incubation volume, a long incubation time and 37 degrees C rather than room temperature. These parameters are all interrelated and equally important for the inhibition of TC clonogenic growth. In our studies, we also observed that Adv could nonspecifically inhibit TC clonogenic growth, although Adv-p53 treatment led to a significantly greater inhibition of clonogenic growth by cells expressing mutated p53. The presence of peripheral stem cell (PSC) products was found to decrease the effect of Adv-p53 on TC clonogenic growth, suggesting that PSC products could compete with TC for infection by recombinant Adv. However, X-Gal staining after incubation with Adv containing-galactosidase demonstrated that PSC products were 2, 000-fold more resistant to Adv infection than TC. We conclude that a 4-hour incubation of stem cell products (2 x 10(8)/ml) with 4 x 10(11) Adv-p53 particles is sufficient to completely purge TC with no effect on hematopoietic cell function.

Strategy for cross-protection among Shigella flexneri serotypes.

Based upon the lipopolysaccharide (LPS) structure and antigenicity of Shigella group B, a strategy for broad cross-protection against 14 Shigella flexneri serotypes was designed. This strategy involves the use of two S. flexneri serotypes (2a and 3a), which together bear the all of the major antigenic group factors of this group. The novel attenuated strains used in these studies were S. flexneri 2a strain CVD 1207 (DeltaguaB-A DeltavirG Deltaset1 Deltasen) and S. flexneri 3a strain CVD 1211 (DeltaguaB-A DeltavirG Deltasen). Guinea pigs were immunized with an equal mixture of these strains and later challenged (Sereny test) with a wild-type S. flexneri serotype 1a, 1b, 2b, 4b, 5b, Y, or 6 strain of demonstrated virulence in the same model. Guinea pigs that were immunized with these two vaccine strains produced serum and mucosal antibodies that cross-reacted with all the S. flexneri serotypes tested (except of S. flexneri serotype 6) as assessed by enzyme-linked immunosorbent assay, immunoblotting, and slide agglutination. Furthermore, the combination vaccine conferred significant protection against challenge with S. flexneri serotypes 1b, 2b, 5b, and Y but not with serotypes 1a, 4b, or (as predicted) 6.

Remission of human breast cancer xenografts on therapy with humanized monoclonal antibody to HER-2 receptor and DNA-reactive drugs.

HER-2 oncogene encodes a transmembrane growth factor receptor that is overexpressed in 25-30% of patients with primary breast and ovarian cancer. A murine monoclonal antibody, 4D5, to the extracellular domain of HER-2 receptor elicits cytostatic growth inhibition of tumor cells overexpressing HER-2 protein, but clinical use of this antibody is limited by genesis of human anti-mouse antibodies. To avoid this problem, a recombinant humanized 4D5 monoclonal antibody (rhuMAb HER-2) was developed and tested using a human tumor xenograft model. Human breast and ovarian cancer cells which overexpress HER-2 were inhibited in vivo by the rhuMAb HER-2 antibody. Tumor growth relative to control was reduced at all doses of antibody tested, and the magnitude of growth inhibition was directly related to dose of rhuMAb HER-2. Tumor growth resumed on termination of antibody therapy, indicating a cytostatic effect. To elicit a cytotoxic response, human breast tumor xenografts were treated with a combination of antibody and antitumor drugs, cisplatin or doxorubicin. The combination of antibody with either cisplatin or doxorubicin resulted in significantly greater growth inhibition, with the cisplatin combination demonstrating a greater response. In addition, therapy with cisplatin and antireceptor antibody elicited complete tumor remissions after 2-3 cycles of therapy. The schedule of administration of anti-receptor antibody and cisplatin was critical for occurrence of antibody-induced potentiation in cisplatin cytotoxicity. Enhanced killing of tumor cells was found only if antibody and drug were given in close temporal proximity. Since interference with DNA repair pathways may contribute to this receptor-enhanced chemosensitivity, repair of cisplatin-damaged reporter DNA (pCMV-beta) was determined in human breast cells. As in studies of antibody-enhanced cisplatin cytotoxicity in vivo, treatment with rhuMAb HER-2 blocked the repair of cisplatin-damaged DNA only if the antibody was administered in close temporal proximity to transfection of the drug-exposed reporter DNA. An alternative measure of DNA repair, unscheduled DNA synthesis, was also assessed. Treatment with either cisplatin or doxorubicin led to an increase in unscheduled DNA synthesis that was reduced by combined therapy with antireceptor antibody specific to HER-2-overexpressing breast cancer cells. Using a direct measure of DNA repair, therapy of HER-2-overexpressing cells with rhuMAb HER-2 also blocked the removal of cisplatin-induced DNA adducts. Expression of p21/WAF1, an important mediator of DNA repair, was disrupted in breast cancer cells with HER-2 overexpression, but not in control cells, after treatment with HER-2 antibody, thus suggesting cross-communication between the HER-2 signaling and DNA repair pathways. These data demonstrate an in vivo antiproliferative effect of rhuMAb HER-2 on tumors that overexpress HER-2 receptor and a therapeutic advantage in the administration of the antireceptor antibody in combination with chemotherapeutic agents.

A recombinant adenoviral vector expressing full-length human retinoblastoma susceptibility gene inhibits human tumor cell growth.

As a prelude to considering retinoblastoma (RB) gene therapy for cancer, a series of human tumor cell lines with either full-length, mutated, or undetectable RB protein were treated with recombinant adenovirus encoding RB (ACNRB). Both RB protein expression and the cytotoxic and antiproliferative effects of ACNRB treatment were evaluated. While the transgene expression of a reporter virus encoding the beta-galactosidase enzyme (rAd-beta-gal) varied among cell lines, the reintroduction and expression of the RB gene resulted in a pronounced inhibition of cellular proliferation in RB-altered cell lines. An antiproliferative response was observed with control adenovirus treatment in some cell lines. ACNRB treatment did not cause detectable cytotoxicity in either RB+ or RB-altered cells. Dose-dependent cytostasis was observed in RB- cell lines. In vivo tumor suppression was observed in a breast xenograft model subsequent to the treatment of established tumors with ACNRB. These data support a role for RB gene therapy of tumors with RB mutations and provide a basis for the further evaluation of ACNRB gene therapy of human cancer.