Jonathan Maynard

Conditionally replication-competent adenoviral vectors with enhanced infectivity for use in gene therapy of melanoma.

To generate vector Ad.Tyr-E1A, which is cytolytic for tyrosinase-positive melanoma cells, we replaced the adenoviral E1A promoter with a human tyrosinase enhancer/promoter. To overcome the low transduction efficiency in populations of melanoma cells that exhibit a low level of the coxsackievirus-adenovirus receptor (CAR), we inserted an RGD-4C peptide into the HI loop of the fiber knob domain of the Ad.Tyr-E1A vector. The resulting vector was named Ad.Tyr-E1A(RGD). As a result of these changes, the transduction efficiency of the RGD-modified vector was increased both in vitro and in vivo. Western blot analysis proved that infection of cells with the Ad.Tyr-E1A(RGD) vector led to expression of the E1A gene selectively in tyrosinase-positive melanoma cell lines, but not in tyrosinase-negative cell lines. The Ad.Tyr-E1A(RGD) vector was as potent in its cytotoxic effect as a tumor nonselective vector (Ad.CMV-E1A) in tyrosinase-positive melanoma cell lines. The Ad.Tyr-E1A(RGD) vector produced a higher vector particle yield in tumor cells than did the Ad.Tyr-E1A vector. Intratumoral injection of the Ad.Tyr-E1A(RGD) vector into xenotransplanted human melanoma tumors led to tumor regression in vivo. The combination of tumor-specific replication and enhanced infectivity generates a more potent CRAD vector for gene therapy of melanoma.

Vector Targeting Makes 5-Fluorouracil Chemotherapy Less Toxic and More Effective in Animal Models of Epithelial Neoplasms

Purpose: 5-Fluorouracil (5-FU) has been combined in the past with other drugs for the combination chemotherapy for cancers of the breast, ovary, and colon. These drug regimens were limited by the fact that 5-FU fails to kill nondividing cancer cells at the doses that are safe to deliver. The goal of the present study is to test the feasibility of replacing 5-FU in established 5-FU combination chemotherapy with the Ad-LpCDIRESE1A/5-fluorocytosine (5-FC) system for the purpose of reducing toxicity and increasing efficacy. Experimental Design: We have replaced 5-FU in the weekly combination of CPT-11, folinic acid (FA) and 5-FU chemotherapy by 5-FC and an adenoviral vector that carries the L-plastin (Lp) tumor-specific promoter-driven transcription unit encoding the cytosine deaminase gene linked to the E1A gene by an internal ribosomal entry site element. This combination is called “genetic combination therapy.” The goal of using the vector was to decrease the toxicity to normal tissue and to increase the efficacy of therapy in the cancer cells by increasing the concentration of 5-FU sufficiently high that even nondividing cancer cells would be killed by 5-FU through its incorporation into mRNA and consequent inhibition of synthesis of functional proteins. We compared the in vivo efficacy of the genetic combination therapy with the conventional combination chemotherapy in a mouse colon cancer model. Results: Both replication-competent and -noncompetent adenoviral vectors carrying an L-plastin–driven cytosine deaminase transcription unit when combined with 5-FC, CPT-11, and FA showed increased in vitro therapeutic activity that was significantly higher than that of the conventional chemotherapy combination. Tumor-bearing mice treated with the genetic combination therapy showed a statistically significant advantage in terms of increased response rate, response duration, survival, and reduced toxicity when compared with tumor-bearing mice treated with the conventional combination chemotherapy. Conclusions: Replacement of 5-FU in 5-FU–based combination chemotherapy with the Ad-LpCDIRESE1A vector and 5-FU reduces toxicity and increases efficacy. This is a concept that could be potentially applied widely for many forms of cancer treatment.

Vector Prime/Protein Boost Vaccine That Overcomes Defects Acquired during Aging and Cancer

We showed that the Ad-sig-TAA/ecdCD40L vaccine induces a tumor suppressive immune response to the hMUC-1 and rH2N tumor-associated self Ags (TAA) and to the Annexin A1 tumor vascular Ag, even in mice in which anergy exists to these Ags. When the TAA/ecdCD40L protein is given s.c. as a boost following the Ad-sig-TAA/ecdCD40L vector, the levels of the TAA-specific CD8 T cells and Abs increase dramatically over that seen with vector alone, in young (2-mo-old) as well as old (18-mo-old) mice. The Abs induced against hMUC-1 react with human breast cancer. This vaccine also induces a 4-fold decrement of negative regulatory CD4CD25FOXP3-T cells in the tumor tissue of 18-mo-old mice. These results suggest that the Ad-sig-TAA/ecdCD40L vector prime-TAA/ecdCD40L protein boost vaccine platform may be valuable in reducing postsurgery recurrence in a variety of epithelial neoplasms.

Antitumor immune response induced by i.t. injection of vector-activated dendritic cells and chemotherapy suppresses metastatic breast cancer

S.c. injection of the Ad-sig-tumor-associated antigen (TAA)/ecdCD40L vector vaccine has been shown to induce a CD8 immune response against TAA for up to 1 year. The first goal of this article is to test if the injection of autologous dendritic cells infected ex vivo with the Ad-sig-TAA/ecdCD40L can increase the immune response induced against TAA. The second goal is to test the effect of adding local chemotherapy in the form of i.t. injection of the AdCDIRESE1A vector-directed chemotherapy on the immune response induced by i.t. injection of adenoviral vector-activated dendritic cells. The results show that the i.t. injection of the AdCDIRESE1A chemotherapy sensitization vector, which encodes the cytosine deaminase chemotherapy sensitization transcription unit, to the i.t. injection of Ad-sig-ecdCD40L vector-infected dendritic cells increased the level of suppression of the growth of the CCL-51 breast cancer cells. The combination of i.t. injection of the AdCDIRESE1A chemotherapy sensitization vector and Ad-sig-ecdCD40L vector-infected dendritic cells into s.c. CCL-51 breast cancer nodules suppressed the growth of uninjected metastatic tumor nodules in the lung. Finally, adding the i.t. injection of the AdCDIRESE1A chemotherapy sensitization vector to the i.t. administration of dendritic cells infected with a rat HER-2/neu (rH2N)–expressing vector (Ad-sig-rH2N/ecdCD40L) led to the induction of rH2N-specific antitumoral immunity in rH2N transgenic mice (which are anergic to the rH2N antigen). This anti-rH2N immune response suppressed the growth of established H2N-positive NT2 breast cancer more efficiently than did the vector-targeted chemotherapy or Ad-sig-rH2N/ecdCD40L-infected dendritic cell vaccine alone. [Mol Cancer Ther 2006;5(8):1975–85]

Chemotherapy targeted to cancer tissue potentiates antigen-specific immune response induced by vaccine for in vivo antigen loading and activation of dendritic cells.

Our laboratory has created an Ad-sig-TAA/ecdCD40L vaccine platform designed to activate dendritic cells (DCs). Two subcutaneous (s.c.) injections of the TAA/ecdCD40L protein following the s.c. injection of the Ad-sig-TAA/ecdCD40L vector (TAA/ecdCD40L VPP vaccine) further increases the levels of the tumor-associated antigen (TAA)-specific CD8 effector T cells induced by the vector. We tested the combined effect of chemotherapy-induced destruction of tumor cells and TAA/ecdCD40L VPP vaccine which further increases the levels of TAA available to the DCs at the time of vaccination. The chemotherapy was delivered selectively to the tumor cells using intratumoral (i.t.) injection of the AdCDIRESE1A vector followed by intraperitoneal (i.p.) 5-fluorocytosine (5FC). The 5-fluorouracil (5FU) produced in the vector infected the tumor cells, destroys them and releases the TAA for processing and presentation by the DCs. This mode of delivery spares the TAA CD8 effector T cells from the destructive effect of the 5FU when their proliferation is induced by the vaccine. Test mice treated with both the s.c. administered TAA/ecdCD40L VPP vaccine and the AdCDIRESE1A/5FC chemosensitization vector had the smallest tumor volumes and survived longer than mice treated with either of these agents alone (P < 0.001).

Use of Adenoviral Vectors to Target Chemotherapy to Tumor Vascular Endothelial Cells Suppresses Growth of Breast Cancer and Melanoma

To target chemotherapy to tumor vascular endothelial cells (TVECs), we created the AdTie2RprCDFib(knob-RGD+) vector by inserting into an AdEasy adenoviral vector (Ad) backbone: (i) the cytosine deaminase (CD) gene driven by the Tie2 receptor promoter (Tie2Rpr) into the E1 region of Ad; (ii) mutations that reduce binding of the fiber knob to the Coxsackie adenovirus receptor (CAR); and (iii) the RGD peptide into the H1 loop of fiber for binding to the alpha(V)beta(3) integrin receptors on TVECs. To reduce uptake of the AdTie2RprCDFib(knob-RGD+) by reticuloendothelial (RE) and liver cells, we intravenously (i.v.) injected Hetastarch and low-dose Ad (one million vector particles (VPs)) prior to i.v. injection of a therapeutic dose (one billion VPs) of the AdTie2RprCDFib(knob-RGD+) vector. This treatment induced regressions of N202 breast cancer and B16 melanoma without toxicity to normal tissues. We showed that the tumor regression was induced by infection of the TVECs and not by the infection of tumor cells by the AdTie2RprCDFib(knob-RGD+) vector.

Targeting of chemotherapy to Tie-2-receptor positive angiogenic endothelial cells of tumor vasculature.

Abstract #902 Background: There are at least three types of cells in tumor tissue which express the Tie2 receptor (Tie2R): dividing vascular endothelial cells (TVECs), Tie2R-expressing monocytes (TEMs) and Tie2R-expressing mesenchymal progenitor cells (TMPCs).
 We tested if the targeting of chemotherapy to these classes of Tie2R positive cells could suppress tumor cell growth.
 Material and Methods: We constructed replication incompetent adenoviral vectors carrying the cytosine deaminase (CD) suicide gene driven by the mouse Tie2R transcriptional promoter and enhancer sequences. In these viruses, we inserted an RGD-4C peptide into the HI loop of the fiber knob domain of the vector to increase its transduction efficiency in TVECs. At the same time, we added two mutations (S408E and P409A) in the AB loop of the fiber, which ablated the CAR-binding ability of the vector. The resulting vector was named AdTie2RCD(MRGD).
 Results: In vitro viral infection assays involving the human umbilical vein endothelial cells (HUVECs) showed that the RGD-modified vectors have a higher transduction efficiency as compared with their adenoviral vector counterparts which have a wild type fiber protein. Mice carrying the rat Her-2-Neu positive N202 mouse breast cancer cells and the mouse B16 melanoma cells were treated with intravenous injections of Hetastarch and the AdTie2RCD(MRGD) vector followed by intraperitoneal injections of 5-Fluorocytosine (5-FC). The Hetastarch was given before the intraveinous infusion of the adenoviral vector in order to reduce the uptake of the adenoviral vector by the reticuloendothelial cell system. The Tie2R targeted chemotherapy sensitization vectors induced greater degrees of suppression of tumor cell growth than did the control group, achieved the similar treatment effect to its CMV promoter counterpart, but with less side effect.
 Histological analysis showed that these vectors specifically targeted the TVECs, TEMs and TMPCs through which they may have exerted cytotoxic effects in the presence of 5-FC on the tumor cells.
 Discussion: Our results showed that N202 breast cancer cell line is more responsive to the Tie2R-targeting therapy compared to B16 mouse melanoma cell line and the Tie2R-directed anti-angiogenic therapy deserves further study, in combination with other types of therapy to pave the way for future clinic trails. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 902.

1128. Ad-sig-TAA/ecdCD40L Vector Prime, TAA/CD40L Protein Boost Vaccine Breaks Tolerance for Tumor Assoicated Antigens (TAA) and Suppresses Growth and Metastasis of Epithelial Neoplastic Cells

In order to develop a method to overcome the immune tolerance of cancer, we have designed an adenoviral vector, designated Ad-sig-TAA/ecdCD40L, which carries a transcription unit encoding the extracellular domain (ecd) of the CD40 ligand (CD40L) linked to TAA. The TAA are in turn linked to a secretory signal peptide (sig). This work was based on earlier DNA vaccines reported by Ralph Reisfeld. The binding of the CD40L to the CD40 receptor on DCs activates them. The binding of the TAA/CD40L chimeric protein leads to the internalization of the TAA and its ultimate presentation of Class I MHC on the DCs. In vivo and in vitro analysis showed that the subcutaneous injection of the Ad-sig-TAA/ecdCD40L adenoviral vector led to the development of a CD8+ T cell lymphocyte systemic immune response against TAA for up to a year. One of the TAA tested is MUC-1, which is overexpressed in epithelial cancer cells. We have shown that the Ad-sig-ecdhMUC-1/ecdCD40L vector makes the hMUC-1.Tg mice (which otherwise are killed by the growth of hMUC-1 positive cancer cell lines) resistant to the engraftment and growth of a hMUC-1 positive cancer cell line. In contrast, the cancer cell line which lacks the hMUC-1 antigen engrafts and grows well in these transgenic mice. We have also shown that subcutaneous injections of the hMUC-1/ecdCD40L protein following the vector injection increase the level of the hMUC-1 specific T cells, and generates high levels of serum antibodies against the hMUC-1 antigen. Importantly, these antibodies bind to breast and prostate cancer cancer cells in biopsy specimens from human subjects. The target of these antibodies is a 20 amino acid repeat in the MUC-1 extracellular domain which can completely block the binding of the antibodies to the human breast cancer epithelial cells in biopsy specimens. The schedule of one subcutaneous (sc) injection of the Ad-sig-MUC-1/ecdCD40L vector followed in 7 and 14 days by a sc injection of the MUC-1/ecdCD40L protein is the best schedule among many other combinations and schedules tested. In addition to the MUC-1 self antigen, we have tested the Ad-sig-rH2N/ecdCD40L vector which contains the rat her-2-neu (rH2N) antigen. We tested this vaccine in rH2N.Tg mice which are anergic to the rH2Nantigen. We found that one sc injection of the Ad-sig-rH2N/ecdCD40L vector followed by 2 sc injections of the rH2N/ecdCD40L protein breaks tolerance which exists in the rH2N.Tg mice which are otherwise tolerant of the rH2N TAA. This Ad-sig-TAA/ecdCD40L vector strategy outperforms both corresponding DNA vaccines as well as cellular based cytokine modified tumor cell vaccines. We plan to complete the preclinical testing necessary to bring this new strategy into phase I toxicity testing in the near future.

444. A New Vector Which Targets Breast Cancer and Its Vasculature

20% of women diagnosed with breast cancer will succumb to their disease due to resistance of their cancer cells to chemotherapy and hormonal therapy. One of the reasons for resistance to existing chemotherapy is that 70% of breast cancer cells are non-dividing and are therefore relatively resistant to the best chemotherapy for breast cancer. In order to overcome this problem of resistance of non-dividing breast cancer cells to chemotherapy, our laboratory designed the AdCDIRESE1A vector, which carries a chimeric transcription unit composed of the cytosine deaminase gene (CD) linked to the viral E1A proliferation gene. 5-Fluorocytosine (5FC) is converted into 5-Fluorouracil (5FU) by the bacterial gene cytosine deaminase (CD), which is produced in the vector infected cells. The levels of 5FU generated in vector infected cells are so high that even non dividing cells will die due to disruption of protein synthesis. Previous data from our laboratory had shown that the intratumoral injection of this AdCDIRESE1A vector followed by 10 days of ip 5FC induced regressions of existing sc breast cancer nodules. The E1A is an adenoviral gene necessary for replication. We then 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. As reported in this abstract this vector has been further modified 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 protein, which allows the vector to use the |[alpha]|v|[beta]|3 and |[alpha]|v|[beta]|5 integrins as alternative receptors. The |[alpha]|v|[beta]|3 integrin receptor is increased in breast cancer cells and in dividing endothelial cells. Dividing endothelial cells are increased (15%) in tumor vasculature as opposed to vasculature in normal tissues (0.5%). 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 led to tumor regression in vivo. The inactivation of the CAR receptor and the addition of the RGD will increase the safety of this vector in vivo, and will decrease the wasteful binding of the vector to non-neoplastic cells. In addition, the altered tropism will circumvent neutralizing antibodies, and will target this vector directly to luminal endothelial cells. Thus, since this vector does not need to cross over from the intravascular to the extravascular space, it is possible that it will be useful clinically.