TAA/ecdCD40L adenoviral prime-protein boost vaccine for cancer and infectious diseases.

A vaccine platform has been created by attaching the target-associated antigen (TAA) for the vaccine to the extracellular domain (ecd) of the potent immunostimulatory signal CD40 ligand (CD40L). Attachment of the TAA to the CD40L promotes uptake of the TAA into dendritic cells (DCs), binding to Class I as well as Class II MHC leading to presentation of the TAA on the DCs, expansion of the TAA-specific B cell and CD8 effector T-cell lymphocytes, and induction of a memory response. In addition, the TAA/ecdCD40L vaccine can overcome anergy, induce regressions of pre-existing subcutaneous (SC) nodules of cancer cells, and induce high titers of neutralizing antibodies against viral antigens. This vaccine, which can be administered SC as a TAA/ecdCD40L fusion protein, or as expression vectors (viral or plasmid) or as a vector prime-protein boost strategy, is applicable to the development of vaccine for a wide range of cancers and infectious agents.

Addition of adenoviral vector targeting of chemotherapy to the MUC-1/ecdCD40L VPPP vector prime protein boost vaccine prolongs survival of mice carrying growing subcutaneous deposits of Lewis lung cancer cells.

We studied the effect of adding chemotherapy or vector targeted chemotherapy to the administration of an Ad-sig-hMUC-1/ecdCD40L adenoviral vector prime-hMUC-1/ecdCD40L protein boost cancer vaccine (designated hMUC-1/ecdCD40L VPPP vaccine), which were administered to test mice 10 days following subcutaneous (s.c.) inoculation of 500,000 Lewis Lung Carcinoma cells, at a time when the average volume of the s.c. tumors was 50 cu mm. The survival of hMUC-1/ecdCD40L VPPP vaccine-treated mice was twice as long as untreated mice. Addition of vector-targeted chemotherapy (AdCMVCDIRESE1A/5FC) to the hMUC-1/ecdCD40L VPPP vaccine 10 days after tumor inoculation significantly (P=0.0062) prolonged the survival of the test mice over administration of the hMUC-1/ecdCD40L VPPP vaccine alone or the control mice (P<0.00001). Interestingly, the combination of the AdCMVCDIRESE1A/5FC vector-targeted chemotherapy to the hMUC-1/ecdCD40L VPPP vaccine decreased the levels of CD44(+)CD24⁻ cells in s.c. deposits of the human MUC-1-positive Lewis Lung Cancer cell line (LL2/LL1hMUC-1) by 20 fold. These results suggest that the addition of vector-targeted chemotherapy to an adenoviral-based cancer vaccine is a strategy that deserves further testing.

Vector prime protein boost vaccination in the setting of myeloablative-induced lymphopenia suppresses growth of leukemia and solid tumors.

We have developed a vaccine, which is designed to induce tumor-associated antigen (TAA)-specific T cells and antibodies in the setting of profound lymphopenia induced by myeloablative therapy and T-cell-depleted bone marrow transplantation. Test mice were injected subcutaneously (sc) with the 32DP210Bcr-Abl cell line, which is positive for the p210Bcr-Abl protein (Group 1). In Group 2, 7 days after injection of the 32DP210Bcr-Abl positive cell line, the mice received 900 cGy total body irradiation (TBI) followed in 1 h by the intravenous infusion of 10 million T-cell-depleted syngeneic bone marrow cells (TCDBMT) (Group 2). The leukemia-bearing group received an intravenous injection of 10 million spleen cells (donor lymphocyte infusions) from unvaccinated (Group 3) and TAA/ecdCD40L-vaccinated (Group 4) syngeneic mice 3 days after completion of the TBI and TCDBMT. Groups 3 and 4 mice received three additional sc vaccinations at 7-day intervals with the TAA/ecdCD40L vaccine, in which the TAA was taken from the junctional peptide of the P210bcr-Abl protein. The survival of Groups 3 and 4 mice was significantly longer than that in Groups 1 and 2 mice. Vaccinated mice from Group 4, which developed complete responses, survived up to 350 days post-injection of the leukemia cells without any evidence of leukemia regrowth.

Vector vaccination and vector targeted chemotherapy in solid tumors.

Gene therapy is one of the promising treatment modalities in cancer therapy. The current gene therapy modalities are mainly focused on the introduction of suppressed tumor suppressor genes into cancer cells, modulation of anti-tumoral immune response, and the suicide gene therapy by introducing pro-drug-activating enzyme genes into the tumor cells. Currently, various gene therapy trials are being conducted in cancer patients. However, the early results of these trials conducted so far are not so encouraging. Combination of gene therapy strategies with conventional treatment modalities such as chemotherapy, immunotherapy or radiotherapy has yielded encouraging results in experimental models and early clinical trials.

Mapping of angiogenic markers for targeting of vectors to tumor vascular endothelial cells.

The vasculature of mouse breast tumor spheroids grown on mammary fat pad tissue in an intravital microscopy (IVM) viewing chamber was shown to derive from infiltrating angiogenic mammary vessels. The receptors tissue factor (TF), alpha V beta 3 integrin and Tie-2 were expressed on the vascular endothelium in the periphery but not in the center of the tumor spheroids nor in the mammary tissue nor in smooth muscle tissue, whereas Tie-1 and PCAM-1 were expressed extensively in the entire tumor and in the vascular endothelium of the entire tumor nodule and in normal mammary tissue. TF is a specific target for adenoviral vector-mediated cancer immunotherapy. Subcutaneous injection of the AdfVII/IgG(1)Fc vector leads to the release into the system circulation of a fVII/IgG(1)Fc immunoconjugate molecule that binds specifically and tightly to TF on vascular endothelial cells and tumor cells, activating a cytolytic immune response against the targeted cells. We show that a single administration of the AdfVII/IgG(1)Fc vector destroys the peripheral but not the central vasculature of a tumor spheroid, causing partial tumor regression; additional administrations prevent regeneration of the peripheral vasculature and regrowth of the tumor. These findings indicate that a critical parameter for optimizing tumor damage is the schedule for successive administrations of the AdfVII/IgG(1)Fc, which should coincide with the regeneration of the peripheral vasculature and continue until the tumor is destroyed.

International Society for Cell and Gene Therapy of Cancer: 2005 meeting in Shenzhen, China.

The 2005 International Society for Cell and Gene Therapy of Cancer (ISCGT) Congress was held in Shenzhen, China (www.iscgtchina2005.com) from December 9th-11th 2005. Here, we describe a representation of the most seminal presentations providing an overview of the progress in the field of cancer gene therapy including the successful introduction of the first approved gene therapy drug.

Oncolytic adenoviral vector carrying the cytosine deaminase gene for melanoma gene therapy.

We constructed an oncolytic adenoviral vector Ad.HE1HCD3, in which the adenoviral E1A promoter was replaced by a human tyrosinase enhancer (HTE)/promoter. The RGD-4C peptide was inserted into the HI loop of the fiber knob domain to increase the transduction efficiency of this vector for tumor cell lines. We also inserted the prodrug activating cytosine deaminase gene driven by the HTE/promoter into the E3 region of the Ad.HE1HCD3 vector. The in vitro cytotoxic effect of the Ad.HE1HCD3 vector with 5-fluorocytosine (5-FC) was greater than that of a wild-type adenovirus or that of the Ad.HE1HCD3 vector alone in tyrosinase-positive melanoma cell lines at low multiplicity of infection. Intratumoral injection of low doses of the Ad.HE1HCD3 vector into xenotransplanted human melanoma cell lines followed by the intraperitoneal injection of 5-FC led to a greater degree of tumor regression in vivo than did the intratumoral injection of the same dose of the Ad.HE1HCD3 vector alone. This oncolytic vector with a melanoma-specific prodrug activation therapeutic transcription unit and a RGD targeted fiber protein offers a potent therapeutic combination for the gene therapy of melanoma.

Tumor-specific therapeutic effect induced by an oncolytic adenoviral vector containing heat shock protein 70 and prodrug activation genes.

We constructed a melanoma-specific oncolytic adenoviral vector Ad.MCDIRESE1.71Hsp3, in which the cytosine deaminase and adenoviral E1A genes linked by the IRES sequence were under the control of a mouse tyrosinase enhancer/promoter transcriptional element in the E1 region of the vector. We also inserted the human heat shock protein 70 (Hsp70) gene driven by the cytomegalovirus promoter into the E3 region of this vector. The RGD-4C peptide was inserted into the HI loop of the fiber knob domain of the Ad.MCDIRESE1.71Hsp3 vector to increase the transduction efficiency of this vector to tumor cells. The Ad.MCDIRESE1.71Hsp3 vector replicates specifically in melanoma cells, and it has a melanoma-specific cytotoxic effect in the presence of 5-fluorocytosine in vitro and in vivo. Moreover, the in vivo killing of tumor cells associated with the overexpression of Hsp70 generated by the intratumoral injection of the Ad.MCDIRESE1.71Hsp3 vector into established subcutaneous tumors can lead to the suppression of tumor growth and potent melanoma-specific systemic immune responses.

Engineering conditionally replication-competent adenoviral vectors carrying the cytosine deaminase gene increases the infectivity and therapeutic effect for breast cancer gene therapy.

We constructed a conditionally replication-competent adenoviral vector Ad.Lp-CD-IRES-E1A(control) in which the expression of both the prodrug-activating cytosine deaminase gene and the viral replication E1A gene were driven by the L-plastin tumor-specific promoter. In order to overcome the low infectivity of the adenoviral vectors for breast cancer cells, and to increase the safety and efficacy for cancer gene therapy, this vector was further modified on a transductional level by simultaneously ablating the native tropism of the vector to the primary CAR receptor and inserting a RGD-4C peptide into the HI loop of the fiber, which allows the vector to use the alphavbeta3 and alphavbeta5 receptors as alternative receptors. The resulting vector was named Ad.Lp-CD-IRES-E1A(MRGD). The transduction efficiency of the vector for breast cancer cell lines which have low expression level of CAR was increased both in vitro and in vivo. The Ad.Lp-CD-IRES-E1A(MRGD) vector produces a higher vector particle yield and a greater cytotoxic effect in tumor cells which have a low expression level of CAR, than did the Ad.Lp-CD-IRES-E1A(control) vector. Intratumoral injection of the Ad.Lp-CD-IRES-E1A(MRGD) vector following the intraperitoneal injection of 5FC into xenotransplanted human breast cancer cell lines which have low expression level of CAR led to greater degree of tumor regression in vivo than did the intratumoral injection of control adenoviral vectors not so modified.

The use of the L-plastin promoter for adenoviral-mediated, tumor-specific gene expression in ovarian and bladder cancer cell lines.

A 2.4-kb truncated L-plastin promoter was inserted either 5' to the LacZ gene (Ad-Lp-LacZ) or 5' to the cytosine deaminase (CD) gene (Ad-Lp-CD) in a replication-incompetent adenoviral vector backbone. Infectivity and cytotoxicity experiments with the LacZ and CD vectors suggested that the L-plastin promoter-driven transcriptional units were expressed at much higher levels in explants of ovarian cancer cells from patients and in established ovarian or bladder cancer cell lines than they were in normal peritoneal mesothelial cells from surgical specimens, in organ cultures of normal ovarian cells, or in the established CCD minimal deviation fibroblast cell line. Control experiments showed that this difference was not attributable to the lack of infectivity of the normal peritoneal cells, the normal ovarian cells, or the minimal deviation CCD fibroblast cell line, because these cells showed expression of the LacZ reporter gene when exposed to the replication-incompetent adenoviral vector carrying the cytomegalovirus (CMV)-driven LacZ gene (Ad-CMV-LacZ). The Ovcar-5 and Skov-3 ovarian cancer cell lines exposed to the Ad-Lp-CD adenoviral vector were much more sensitive to the prodrug 5-fluorocytosine (5FC), which is converted from the 5FC prodrug into the toxic chemical 5-fluorouracil, than was the CCD minimal deviation fibroblast cell line after exposure to the same vector. A mouse xenograft model was used to show that the Ad-Lp-CD vector/5FC system could prevent engraftment of ovarian cancer cells in nude mice. Finally, injection of the Ad-Lp-CD vector into s.c. tumor nodules generated a greater reduction of the size of the tumor nodules than did injection of the Ad-CMV-LacZ vectors into tumor nodules. The Ad-Lp-CD vectors were as suppressive to tumor growth as the Ad-CMV-CD vectors. These results suggest that an adenoviral vector carrying the CD gene controlled by the L-plastin promoter (Ad-Lp-CD) may be of potential value for the i.p. therapy of ovarian cancer.

Combinatorial chemistry in cancer drug development.

The field of combinatorial chemistry has grown at an enormous rate in recent years, both in response to high-throughput capabilities and the discovery of a plethora of novel therapeutic targets. This review attempts to outlinethe recent developments of combinatorial chemistry in the search for novel cancer-related therapeutic agents.

High and low levels of cottontail rabbit papillomavirus E2 protein generate opposite effects on gene expression.

The papillomavirus E2 protein plays an important role in viral transcriptional regulation and replication. We chose to study the cottontail rabbit papillomavirus (CRPV) E2 protein as a transcriptional regulator because of the availability of an animal model for papilloma formation, which may be relevant for human papillomavirus (HPV) infection and replication. We studied the effect of expression levels of E2 on the long control region, which contains transcriptional promoter and enhancer elements, and synthetic E2-dependent artificial promoters in which the E2 was the dominant factor in the transcriptional activation. These experiments indicated that high levels of E2 were inhibitory and low levels were stimulatory for transactivation. In addition, we showed that the complex formed between CRPV E2 and the cognate binding site was less stable than the complex formed between HPV E2 and the same cognate binding site. Furthermore, we showed that CRPV E2 binding to its transcriptional regulatory sequence was stabilized by other proteins such as E1, which produced increments in transcriptional activation of E2-dependent genes. The data may be used to define conditions in which the rabbit model can be used for the screening of drugs which are inhibitory to the HPV and CRPV replication and gene expression.

[Correlation of cell apoptosis induction with expression of human beta5 integrin on hematopoietic cells].

OBJECTIVE: To investigate the function of alpha(v)beta5 integrin in hematopoietic cells. METHODS: Beta5 integrin cDNA was expressed in K562 cells through a retroviral vector system. The changes of the alpha(v)beta5 and alpha(v)beta3 integrins expression in apoptosis and differentiation induced by serum depletion were observed. RESULT: The beta5 integrin cDNA failed to express in K562 cells after the introduction of the beta5 integrin retrovirus vector pG beta5CHT. Imbalanced expression of alpha(v)beta3 and alpha(v)beta5 integrins occurred during apoptosis and differentiation induced by serum depletion. Treatment of hematopoietic cells with anti-alpha(v)beta5 monoclonal antibody inhibited the cells apoptosis. CONCLUSION: Overexpression of the alpha(v)beta5 integrin cDNA in hematopoietic cells was associated with the inhibition of cell proliferation and apoptosis.

Sensitization of prostate cancer cell lines to 5-fluorocytosine induced by adenoviral vector carrying a CD transcription unit.

OBJECTIVE: To investigate the efficiency of the cytosine deaminase adenoviral/5-fluorocytosine system on prostate cancer cell lines. METHODS: We used cell culture, infectivity and sensitivity tests, to observe bystander effect by animal tests. RESULTS: Established prostate cancer cell lines are eventually infectible by adenoviral vector. The ratio of vector/cell at which infection occurs depends on the specific cell line. The peak of expression of the transferred cytosine deaminase gene occurred in cells at different time, but persisted beyond 11 days. These prostate cell lines are sensitized to 5-fluorocytosine by infection with adenoviral vector carrying the cytosine deaminase gene. Only 5% of the LNCap and 10% of the RM-1 cells were infected and produced 100% cell death. In the animal test, there was significant inhibition of tumor growth at a ratio of 400 vector particles/cell with the systematic treatment of 5-fluorocytosine. CONCLUSIONS: Adenoviral vector carrying a cytosine deaminase transcription unit can sensitize prostate cancer cell lines to 5-fluorocytosine. The system can significantly inhibit the growth of prostatic tumors in mice.

Systemic treatment of malignant melanoma.

Once there is evidence of systemic involvement in malignant melanoma, treatment options become severely limited and the disease is virtually incurable. There are, however, options available to treat patients, including single-agent chemotherapy, single-agent biologic response modifier (BRM), combination chemotherapy, and the combination of chemotherapeutic agents and BRMs. These treatment modalities and their indications for use are discussed.

Randomized trial of high-dose chemotherapy and blood cell autografts for high-risk primary breast carcinoma.

BACKGROUND: Uncontrolled studies have reported encouraging outcomes for patients with high-risk primary breast cancer treated with high-dose chemotherapy and autologous hematopoietic stem cell support. We conducted a prospective randomized trial to compare standard-dose chemotherapy with the same therapy followed by high-dose chemotherapy. PATIENTS AND METHODS: Patients with 10 or more positive axillary lymph nodes after primary breast surgery or patients with four or more positive lymph nodes after four cycles of primary (neoadjuvant) chemotherapy were eligible. All patients were to receive eight cycles of 5-fluorouracil, doxorubicin (Adriamycin), and cyclophosphamide (FAC). Patients were stratified by stage and randomly assigned to receive two cycles of high-dose cyclophosphamide, etoposide, and cisplatin with autologous hematopoietic stem cell support or no additional chemotherapy. Tamoxifen was planned for postmenopausal patients with estrogen receptor-positive tumors and chest wall radiotherapy was planned for all. All P values are from two-sided tests. RESULTS: Seventy-eight patients (48 after primary surgery and 30 after primary chemotherapy) were registered. Thirty-nine patients were randomly assigned to FAC and 39 to FAC followed by high-dose chemotherapy. After a median follow-up of 6.5 years, there have been 41 relapses. In intention-to-treat analyses, estimated 3-year relapse-free survival rates were 62% and 48% for FAC and FAC/high-dose chemotherapy, respectively (P =.35), and 3-year survival rates were 77% and 58%, respectively (P =.23). Overall, there was greater and more frequent morbidity associated with high-dose chemotherapy than with FAC; there was one septic death associated with high-dose chemotherapy. CONCLUSIONS: No relapse-free or overall survival advantage was associated with the use of high-dose chemotherapy, and morbidity was increased with its use. Thus, high-dose chemotherapy is not indicated outside a clinical trial.

Methods for Chemoprotection and Chemosensitization : MDR-1 For Chemoprotection Using Retroviruses to Modify Hematopoietic Cells and Cytosine Deaminase for Chemosensitization Using Adenoviral Vectors to Modify Epithelian Neoplastic Cells.

Surgery, radiation therapy, and chemotherapy have been applied to the curative therapy of 50% of cancer patients in the United States during the past 100 years. It is clear that the chemotherapeutic agents used to develop curative therapy for leukemias, lymphomas, gestational malignancy, and testicular cancer are not as active in the more numerous epithelial neoplasms, perhaps because of the complexity of genetic change in these latter neoplasms.