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Purpose: Treatment options are limited beyond JAK inhibitors for patients with primary myelofibrosis (MF) or secondary MF. Preclinical studies have revealed that PI3Kδ inhibition cooperates with ruxolitinib, a JAK1/2 inhibitor, to reduce proliferation and induce apoptosis of JAK2V617F-mutant cell lines. Patients and Methods: In a phase I dose-escalation and -expansion study, we evaluated the safety and efficacy of a selective PI3Kδ inhibitor, umbralisib, in combination with ruxolitinib in patients with MF who had a suboptimal response or lost response to ruxolitinib. Enrolled subjects were required to be on a stable dose of ruxolitinib for ≥8 weeks and continue that MTD at study enrollment. The recommended dose of umbralisib in combination with ruxolitinib was determined using a modified 3+3 dose-escalation design. Safety, pharmacokinetics, and efficacy outcomes were evaluated, and spleen size was measured with a novel automated digital atlas. Results: Thirty-seven patients with MF (median age, 67 years) with prior exposure to ruxolitinib were enrolled. A total of 2 patients treated with 800 mg umbralisib experienced reversible grade 3 asymptomatic pancreatic enzyme elevation, but no dose-limiting toxicities were seen at lower umbralisib doses. Two patients (5%) achieved a durable complete response, and 12 patients (32%) met the International Working Group-Myeloproliferative Neoplasms Research and Treatment response criteria of clinical improvement. With a median follow-up of 50.3 months for censored patients, overall survival was greater than 70% after 3 years of follow-up. Conclusions: Adding umbralisib to ruxolitinib in patients was well tolerated and may resensitize patients with MF to ruxolitinib without unacceptable rates of adverse events seen with earlier generation PI3Kδ inhibitors. Randomized trials testing umbralisib in the treatment of MF should be pursued.

Clinical evidence of immune checkpoint inhibitors combined with antiangiogenic drugs in patients with advanced non-small cell lung cancer (NSCLC) was limited. Recombinant human endostatin (rh-endostatin), an antiangiogenic drug, and camrelizumab, an anti-PD-1 antibody, have been approved for the treatment of advanced NSCLC in China. This study aimed to investigate the efficacy and safety of rh-endostatin plus camrelizumab and chemotherapy in the treatment of advanced NSCLC.Eligible patients were enrolled and received camrelizumab (200 mg, day 1) every 3 weeks and continuous intravenous infusion of rh-endostatin (70 mg/day, days 1-3) and cisplatin combined with pemetrexed (for adenocarcinoma) or paclitaxel (for NSCLC other than adenocarcinoma) every 3 weeks. Primary endpoint was progression-free survival (PFS). Secondary endpoints were objective response rate (ORR), disease control rate (DCR), overall survival (OS), and safety profiles.Overall, 27 patients were included, and 25 patients were eligible for efficacy evaluation. For these 25 patients, ORR was 48.15% (13/27) and DCR was 85.19% (23/27). With a median follow-up of 10.37 months, the median PFS was 8.9 (95% CI: 4.23-13.57) months. Median OS was not reached. Overall, 96.3% of patients experienced at least one treatment-related adverse event, and grade 3 TRAEs occurred in 9 (33.3%) patients. No unexpected AEs were observed.Rh-endostatin plus camrelizumab and chemotherapy showed favorable efficacy and safety profile in patients with advanced NSCLC, representing a promising treatment regimen for these patients.

The combination of cytotoxic treatment modalities, including oncolytic viral gene therapies and immunotherapy, usually yields a synergistic effect. In the current study, a bicistronic adenoviral vector, Ad-CD-GMCSF, carrying the cytosine deaminase (CD) and granulocyte-macrophage colony-stimulating factor (GM-CSF) transcription units driven by a cytomegalovirus promoter was constructed, and the

Cyclin-dependent kinase 8 (CDK8), a gene encoding the cyclin-dependent kinase (CDK) component of the Mediator complex, is known as a colon cancer oncogene. Our recent study showed that CDK8 plays an important role in the formation of pancreatic cancer, but the CDK8 expression levels were not completely identical in different pancreatic cancer samples. The level of CDK8 expression depended on whether the K-ras gene was mutated; its expression was much higher in samples carrying a K-ras mutation than in wild-type K-ras samples. Moreover, CDK8 expression was reduced following mutated K-ras knockdown in K-ras-mutated pancreatic cancer cells, whereas CDK8 expression was increased following expression of mutated K-ras in wild-type K-ras cells. Our study demonstrates that mutated K-ras stimulates CDK8 expression, possibly by regulating HIF-1α, and both CDK8 and mutated K-ras were confirmed to promote cell proliferation and prevent apoptosis in vitro. Additionally, we found that both CDK8 and mutated K-ras promote the invasion and migration of pancreatic cancer cells via the positive regulation of the Wnt/β-catenin signaling pathway, thereby increasing the expression of Snail1 and ZEB1, which act as important stimulating factors of the epithelial-to-mesenchymal transition (EMT). Finally, knockdown of either CDK8 or mutated K-ras contributed to attenuated pancreatic cancer growth in BALB/c nude mice. In conclusion, these findings demonstrate that mutated K-ras promotes CDK8 expression and that the regulatory effects of CDK8 on the EMT are partially attributed to the Wnt/β-catenin signaling pathway.

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.

Background: Combination of cytotoxic treatments with immunotherapeutic agents seem more efficacious than using either strategy alone. Oncolytic viral vectors carrying immunostimulatory genes like GM-CSF have emerged as treatment options in various tumors. Previously, we have shown that combination of suicide gene therapy either with GM-CSF or vector-induced dendritic cells has significant anti-tumor efficacy. In the current study, we aimed to combine the cytotoxic effect of the suicide gene cytosine deaminase and the immunostimulatory effect of granulocyte macrophage colony stimulating factor in a single vector construct. Methods: We have constructed a bicistronic adenoviral vector carrying cytosine deaminase (CD) and granulocyte macrophage-colony stimulating factor (GM-CSF) genes combined with an IRES element and driven by CMV promoter (Ad-CMV-CDiresGMCSF). We tested the in vitro efficacy of the vector in various tumor cell lines and in a mouse model of colon cancer. Results: Our bicistronic vector Ad-CMV-CDiresGMCSF showed significant in-vitro cytotoxic effects on tumor cell lines similar to the vector carrying CD gene alone when 5-FC added. Likewise, the GM-CSF producing efficacy of the bicistronic vector was comparable to the vector carrying GM-CSF gene alone. In the syngeneic colon cancer model, the bicistronic vector carrying CD and GM-CSF genes yielded significant tumor shrinkage and overall survival when compared to the control suicide vector carrying CD gene alone. Accordingly, anti-tumor immune response parameters including CTL assay and immune cell infiltration of tumor tissue were significantly improved in the Ad-CMV-CDiresGMCSF vector treated group (p Citation Format: Hakan Akbulut, Arzu Coleri, Gunce Sahin, Yucheng Tang, Albert Deisseroth, Fikri Icli. A bicistronic adenoviral vector carrying cytosine deaminase and GM-CSF genes significantly improves antitumor immunity and overall survival in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5911.

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.

Multiple investigators have reported the presence of defects in the immune response of the elderly [Castle In: Clin Infect Dis 31:578, 2000; Ortqvist et al. In: Eur Respir J 30:414-422, 2007; Saurwein-Teissl et al. In: J Immunol 168:5893, 2002; Haynes et al. In: Proc Natl Acad Sci USA 100:15053-15058, 2003]. These defects reduce the magnitude of the immune response to infection and to vaccination. In individuals greater than 55 years of age, the probability of developing a fully protective neutralizing antibody response to the yearly multivalent particle inactivated influenza vaccine is less than 20% [Jefferson et al. In: Lancet 264:1165-1174, 2005; Goodwin et al. In: Vaccine 24:1159-1169, 2006; Jackson et al. In: Lancet 372:398-405, 2008; Simonsen and Taylor In: Lancet 7:658-666, 2007]. The defects in the aged immune system that are responsible for this limited response to vaccination in the older age groups include functional defects of the antigen presenting cells, functional defects in CD4 helper CD4 T cells and monocytes, and an altered microenvironment [Eaton et al. In: J Exp Med 200:1613-1622, 2004; Dong et al. In: J Gen Virol 84:1623-1628, 2003; Deng et al. In: Immunology 172:3437-3446, 2004; Cella et al. In: J Exp Med 184:747-752, 1996]. Starting at puberty, the involution of the thymus and the consequent reduction of the export of naïve T cells specific to neo-antigens leads to the reduction of the ratio of antigen naïve to memory cells as chronological age advances [Prelog In: Autoimmun Rev 5:136-139, 2006; McElhaney et al. In: J Immunology 176:6333-6339, 2006]. Changes in glycosylation of T cells and target antigens acquired during the aging process and the antibodies to these new glycopeptides and glycoproteins may also contribute to a reduction in the functioning of the adaptive immune response [Ishii et al. In: J Clin Neurosci 14:110-115, 2007; Shirai et al. In: Clin Exp Immunol 12:455-464, 1972; Adkins and Riley In: Mech Ageing Dev 103:147-164, 1998; Ben-Yehuda and Weksler In: Cancer Investigation 10:525-531, 1992]. One of the more interesting examples of the functional defects in the cells of the adaptive immune response is a reduced level of expression in the surface cytoadhesion and activation receptor molecules on CD4 helper T cells undergoing activation during vaccination. Upon infection or vaccination, CD40L is typically increased on the surface of CD4 helper T cells during activation, and this increased expression is absolutely essential to the CD40L promotion of expansion of antigen-specific B cells and CD 8 effector T cells in response to infection or vaccination [Singh et al. In: Protein Sci 7:1124-1135, 1998; Grewal and Flavell In: Immunol Res 16: 59-70, 1997; Kornbluth In: J Hematother Stem Cell Res 11:787-801, 2002; Garcia de Vinuesa et al. In: Eur J Immunol 29:3216-3224, 1999]. In aged human beings and mice, the reduced levels of expression of CD40 ligand (CD40L) in activated CD4 helper T cells is dramatically reduced [Eaton et al. In: J Exp Med 200:1613-1622, 2004; Dong et al. In: J Gen Virol 84:1623-1628, 2003]. To circumvent the reduction in CD40L expression and the subsequent reduction in immune response in the elderly, we have developed a chimeric vaccine comprised of the CD40L linked to the target antigen, in a replication incompetent adenoviral vector and in booster protein. This review will discuss the implementation the potential use of this approach for the vaccination of the older populations for cancer and infection.

To develop a method to overcome the anergy that exists in tumor hosts to cancer, we have designed an adenoviral vector for thein vivoactivation and tumor antigen loading of dendritic cells. This adenoviral vector encodes a fusion protein composed of an amino-terminal tumor-associated antigen fragment fused to the CD40 ligand (CD40L). Subcutaneous injection of an adenoviral vector encoding a fusion protein of the human papillomavirus E7 foreign antigen linked to the CD40L generates CD8+T cell-dependent immunoresistance to the growth of the E7-positive syngeneic TC-1 cancer cells in C57BL/6 mice for up to 1 year. We also studied the s.c. injection of a vector carrying the gene for the human MUC-1 (hMUC-1) self-antigen fused to the CD40L. When this vector was injected into hMUC-1.Tg mice, which are transgenic for the hMUC-1 antigen, the growth of syngeneic hMUC-1-positive LL1/LL2hMUC-1 mouse cancer cells was suppressed in 100% of the injected animals. The hMUC-1.Tg mice are anergic to the hMUC-1 antigen before the injection of the vector. These experimental results show that it is possible to use vector injection to activate a long-lasting cellular immune response against self-antigens in anergic animals. The vector-mediatedin vivoactivation, and tumor-associated antigen loading of dendritic cells does not require additional cytokine boosting to induce the immune response against the tumor cells. This vector strategy may therefore be of use in the development of immunotherapy for the many carcinomas in which the hMUC-1 antigen is overexpressed.

Prodrug activating transcription unit gene therapy is one of several promising approaches to cancer gene therapy. Combining that approach with conditionally replication-competent viral vectors that are truly tumor specific has been an important objective of recent work. In this study, we report the construction of a new conditionally replication-competent bicistronic adenoviral vector in which the cytosine deaminase (CD) gene and the E1a gene are driven by the L-plastin tumor-specific promoter (AdLpCDIRESE1a). A similar vector driven by the CMV promoter has also been constructed (AdCMVCDIRESE1a) as a control. We have carried out in vitro cytotoxicity in carcinomas of the breast, ovary and colon, and in vivo efficacy studies with these vectors in an animal model of colon cancer. While the addition of the AdLpCDIRESE1a vector to established cancer cell lines showed significant cytotoxicity in tumor cells derived from carcinomas of the breast (MCF-7), colon (HTB-38) and ovary (Ovcar 5), no significant toxicity was seen in explant cultures of normal human mammary epithelial cells (HMEC) exposed to this vector. The addition of 5-fluorocytosine (5FC) significantly increased the cytotoxicity in an additive fashion of both the AdLpCDIRESE1a and AdCMVCDIRESE1a vectors as well as that of the AdLpCD replication incompetent vector to established tumor cell lines. However, no significant cytotoxicity was observed with the addition of 5FC to explant cultures of normal human mammary epithelial cells that had been exposed to the L-plastin-driven vectors. Studies with mixtures of infected and uninfected tumor cell lines showed that the established cancer cell lines infected with the AdLpCDIRESE1a vector generated significant toxicity to surrounding uninfected cells (the "bystander effect") even at a ratio of 0.25 of infected cells to infected + uninfected cells in the presence of 5FC. The injection of the AdLpCDIRESE1a vector into subcutaneous deposits of human tumor nodules in the nude mice was potentiated by administering 5-FC by intraperitoneal injection. This treatment resulted in a decreased tumor size and a decreased tumor cell growth rate. The mice treated with a combination of the AdLpCDIRESE1a vector intratumoral injection and intraperitoneal 5FC injections lived much longer than the other experimental groups exposed to the viral vector alone or to the combination of the intratumoral AdLpCD replication incompetent vector injections plus intraperitoneal 5-FC injections. These encouraging results with our newly constructed AdLpCDIRESE1a vector suggest a need for further study of its utility in a preclinical model of intracavitary therapy of pleural or peritoneal carcinomatosis.

High mobility group protein A2 (HMGA2) and octamer-binding transcription factor 4 (Oct4) are transcription factors that play major roles in the acquisition of cancer stemness phenotypes and tumorigenicity of malignant neoplasms. The aim of this study was to analyze the association between HMGA2 and Oct4 expression and various clinicopathologic features in gastric cancer patients including invasion, metastasis, and clinical prognosis, in addition to overall survival. Immunohistochemistry was performed to explore the expression of HMGA2 and Oct4 in 158 gastric cancer and surrounding non-tumor tissues. Moreover, HMGA2 and Oct4 mRNA and protein levels were also detected by qRT-PCR and Western blotting, respectively, in 86 clinical tissue specimens and various gastric epithelial cell lines (GES-1, SGC7901, MKN45, and MKN27). Finally, associations between HMGA2 and Oct4 expression and clinicopathological features were analyzed by Pearson correlation coefficient. Survival analysis was performed by univariate and multivariate analyses. Taken together, we found that HMGA2 and Oct4 expression was significantly higher in gastric cancer tissues compared with non-cancerous tissues (P < 0.01), and HMGA2 and Oct4 protein levels were significantly higher in poorly differentiated gastric cancer cell lines (MKN45), moderately differentiated cell lines (SGC7901), and well-differentiated cell lines (MKN28) compared with human immortalized gastric epithelial cell lines (GES-1) (P < 0.01). Elevated HMGA2 and Oct4 levels were significantly associated with poor clinical prognosis (P < 0.05). Further conclusion showed that coexpression of HMGA2 and Oct4 in gastric cancer correlated with tumor invasion, metastasis, and clinical prognosis and predicted an unfavorable clinical outcome. These transcription factors may represent useful biomarkers to identify patients at high risk of postoperative recurrence.

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.

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]

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.

Our goal in the present work was to characterize the multiple steps involved in overcoming the anergy that exists in tumor hosts to tumor-associated antigen (TAA). Our studies showed that the subcutaneous injection of the Ad-sig-TAA/ecdCD40L vector resulted in secretion of the TAA/ecdCD40L protein for at least 10 days from infected cells. Binding of the TAA/ecdCD40L protein to dendritic cells (DCs) resulted in the induction of CCR-7 chemokine receptor expression and cytokine release. This was followed by migration of the DCs to regional lymph nodes. Tetramer staining, enzyme-linked immunospot (ELISPOT) assay, and cytotoxicity assay all showed that the Ad-sig-TAA/ecdCD40L vector increased the levels of splenic CD8+ T cells specific for the 2 TAAs (human MUC1 [hMUC1] and HPV E7) tested. Vaccination with the Ad-sighMUC1/ecdCD40L vector suppressed the growth of hMUC1 antigen-positive tumor cells in 100% of the test mice that were previously anergic to the hMUC1 antigen. These data suggest that Ad-sig-TAA-ecd/ecdCD40L vector injections may be of value in treating the many epithelial malignancies in which TAA-like hMUC1 is overexpressed. (Blood. 2004;104:2704-2713)

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.

Since a significant portion (30%) of individuals diagnosed with epithelial neoplasms have highly chemotherapy resistant CD44+CD24−/low cancer stem cells in the peripheral blood as well as in solid tumor nodules at diagnosis, we have been developing methods of cancer treatment which depend on methods of killing the cancer stem cells which depend on mechanisms other than chemotherapy for treatment. Vaccination has been one of these approaches. Unfortunately, it has been known for a long time that the immune response is diminished in older individuals due to decreased numbers of CD4 and CD8 T cells and due to acquisition of functional defects in CD4 cells. An example is the diminished expression of the CD40L in activated CD4 T cells in older individuals which limits the cellular and humoral response to vaccines in the older age groups. The presence of the CD40L on the CD4 helper T cells is important for vaccine induced expansion of antigen specific T cells and B cells. In order to overcome these problems, we have developed an Ad-sig-TAA/ecdCD40L vector prime-TAA/ecdCD40L protein boost vaccine strategy. The Ad-sig-TAA/ecdCD40L carries a transcription unit encoding the extracellular domain (ecd) of the CD40 ligand (CD40L) linked to tumor associated antigen (TAA). The TAA are in turn linked to a secretory signal peptide (sig) so that the TAA/ecdCD40L protein will be secreted from the vector infected cells at the injection site of the vector continuously over a 10–14 day period. The CD40L targets the TAA to the DCs, activates the DCs, and carries the TAA into the DC so that the TAA fragments are presented on Class I MHC. The sc injection of the TAA/ecdCD40L protein booster following the sc administration of the Ad-sig-TAA/ecdCD40L (we call this the TAA/ecdCD40L VPP vaccine) induces complete regressions of pre-existing tumor even in aged mice (18 months old). We then chose human MUC-1 as the TAA for testing the efficacy of our vaccine platform for cancer stem cells. Over 90% of the late stage patients with cancers of the breast, ovary, prostate, and lung have been reported to overexpress a hypoglycosylated form of MUC-1 which correlates with decreased time to progression. The hypoglycosylation on MUC-1 in cancer cells creates a tumor specific target for vaccines. Kufe’s lab has shown that overexpression of MUC-1 in the cancer cell promotes resistance to therapy by reducing p53 (Cancer Cell7: 167, 2005) and induces proliferation by activation of the NFkappaB (Nature Cell Biology9: 1419, 2007). Finn’s lab has shown that MUC-1 is a marker present on >90% of the “tumor stem cell” population as well as the more mature cells in breast cancer (Ca Res68: 2419, 2008). In order to test the efficacy of the hMUC-1/ecdCD40L VPP vaccine on tumor stem cells, we first isolated hMUC-1 positive Lewis Lung carcinoma cells by stably transfecting the hMUC-1 cDNA into these cells. We showed that the Ad-sig-hMUC-1/ecdCD40L vector prime-hMUC-1/ecdCD40L protein boost vaccine overcomes anergy to hMUC-1 antigen (hMUC-1) in hMUC-1.Tg mice. We then injected 100,000 human MUC- 1 positive Lewis Lung mouse carcinoma cells (LL2/LL1hMUC-1) mouse cancer cells into the syngeneic C57BL/6J mice. ELISPOT assay showed that the hMUC-1/ecdCD40L VPP vaccine increased the level of antigen specific CD8 effector cells in the lymph node draining the site of injection of the tumor to 900 hMUC-1 specific CD8 T cells/100,000 total cells and to 325 hMUC-1 specific CD8 T cells/100,000 cells in the spleen. The level of cytotoxicity of spleen cells increased 6 fold following vaccination (spleen cells from control and vaccinated mice were exposed in vitro to mitomycin C treated hMUC positive LL2/LL1hMUC-1 cells for 5 days before testing). Measurement of the growth of the sc tumor nodule at the sc injection site showed a 20 fold decrease in the total size of the tumor in the vaccinated vs the control mice and the size of the tumor was decreasing in the vaccinated mice vs the control group at the time of sacrifice at 24 days. Finally, the tumor nodule from the vaccinated and unvaccinated mice were excised post mortem, minced, treated with collagenase and DNAse I and strained through gauze. FACS analysis showed that in the vaccinated mice, the number of the MUC-1 positive tumor stem cells with the CD44+CD24− immunophenotype decreased over 20 fold during the treatment period. These results suggest that the hMUC-1/ecdCD40L VPP vaccine could suppress the levels of hMUC-1 cancer stem cells in pre-existing tumor nodules. This vaccine platform has entered clinical phase I testing.

In order to develop a method to overcome the immune tolerance of cancer, we have designed an Ad-sig-TAA/ecdCD40L adenoviral vector vaccine for the in vivo activation and tumor antigen loading of dendritic cells (DCs). Subcutaneous (sc) injection of the Ad-sig-TAA/ecdCD40L adenoviral vector results in the secretion for 10 days from the vector infected cells of a fusion protein composed of a fragment of a tumor associated antigen (TAA) fused to the extracellular domain (ecd) of the CD40 ligand (CD40L). CD40L is a homo-trimeric protein normally found on B cells and helper CD4+ T cell lymphocytes. All of the sequences necessary to stabilize this trimeric structure of the protein are contained within the ecd of the CD40L protein. The binding of the TAA/ecdCD40L protein to DCs induces migration of these DCs to the regional lymph nodes. These DCs carry fragments of TAA bound to surface MHC Class I molecules. We have shown that the Ad-sig-TAA/ecdCD40L vector strategy can induce a cellular and humoral immune that persists for over a year indicating that a durable memory response is generated. We showed that sc injections of the Ad-sig-rH2N/ecdCD40L vector in rH2N.Tg mice induces a cellular and humoral immune response against the rat Her-2-Neu (rH2N) antigen which is associated with breast cancer. We showed that the sc injection of the Ad-sig-rH2N/ecdCD40L adenoviral vector in a rH2N.Tg transgenic mouse induced resistance to the growth of rH2N positive cancer cells in mice previously anergic to the rH2N antigen. We demonstrated that the sc injection of the Ad-sig-hMUC-1/ecdCD40L vector suppressed the growth of tumor cells positive for the human MUC-1 (hMUC-1) antigen in hMUC-1.Tg mice which were previously anergic to the hMUC-1 antigen. The sc injection of the Ad-sig-hMUC-1/ecdCD40L vector followed by sc injection of two booster injections of the hMUC-1/ecdCD40L protein induced high levels of hMUC-1 specific tumor infiltrating effector CD8 positive T cells and hMUC-1 antibodies which bound to human breast and prostate cancer cells. In addition, we recently showed that the Ad-sig-TAA/ecdCD40L strategy could be used to activate a cellular and humoral immune response against Annexin A1 (AnxA1), which is a marker uniquely displayed on the luminal membrane of tumor vascular endothelial cells but not on the luminal membrane of vascular endothelial cells of normal tissue. The subcutaneous injection of the Ad-sig-AnxA1/ecdCD40L vector suppressed the growth of AnxA1 negative tumor cells in a syngeneic mouse tumor model. This vector prime/protein boost vaccination was found to induce increased levels of effector CD8 positive T cells in the target tumor. These effector T cells were shown express increased levels of the genes encoding the CCR5 chemokine receptor and the CCL3 chemokine ligand which promote the infiltration of antigen specific effector T cells in the target tumor tissues. The response to cancer vaccines is often reduced in older individuals in part due to an intrinsic functional defect in CD4 cells. The Ad-sig-TAA/ecdCD40L vaccine may circumvent this defect because we have shown that the induction of the immune response is CD4 independent. These data suggest that this vector prime-protein boost vaccination strategy will be useful in the reduction of the frequency of recurrence following initial therapy for a wide variety of neoplastic diseases.