Society for Immunotherapy of Cancer (SITC) recommendations on intratumoral immunotherapy clinical trials (IICT): from premalignant to metastatic disease.

BACKGROUND: Intratumorally delivered immunotherapies have the potential to favorably alter the local tumor microenvironment and may stimulate systemic host immunity, offering an alternative or adjunct to other local and systemic treatments. Despite their potential, these therapies have had limited success in late-phase trials for advanced cancer resulting in few formal approvals. The Society for Immunotherapy of Cancer (SITC) convened a panel of experts to determine how to design clinical trials with the greatest chance of demonstrating the benefits of intratumoral immunotherapy for patients with cancers across all stages of pathogenesis. METHODS: An Intratumoral Immunotherapy Clinical Trials Expert Panel composed of international key stakeholders from academia and industry was assembled. A multiple choice/free response survey was distributed to the panel, and the results of this survey were discussed during a half-day consensus meeting. Key discussion points are summarized in the following manuscript. RESULTS: The panel determined unique clinical trial designs tailored to different stages of cancer development-from premalignant to unresectable/metastatic-that can maximize the chance of capturing the effect of intratumoral immunotherapies. Design elements discussed included study type, patient stratification and exclusion criteria, indications of randomization, study arm determination, endpoints, biological sample collection, and response assessment with biomarkers and imaging. Populations to prioritize for the study of intratumoral immunotherapy, including stage, type of cancer and line of treatment, were also discussed along with common barriers to the development of these local treatments. CONCLUSIONS: The SITC Intratumoral Immunotherapy Clinical Trials Expert Panel has identified key considerations for the design and implementation of studies that have the greatest potential to capture the effect of intratumorally delivered immunotherapies. With more effective and standardized trial designs, the potential of intratumoral immunotherapy can be realized and lead to regulatory approvals that will extend the benefit of these local treatments to the patients who need them the most.

Protein profiling in potato (Solanum tuberosum L.) leaf tissues by differential centrifugation.

Foliar diseases, such as late blight, result in serious threats to potato production. As such, potato leaf tissue becomes an important substrate to study biological processes, such as plant defense responses to infection. Nonetheless, the potato leaf proteome remains poorly characterized. Here, we report protein profiling of potato leaf tissues using a modified differential centrifugation approach to separate the leaf tissues into cell wall and cytoplasmic fractions. This method helps to increase the number of identified proteins, including targeted putative cell wall proteins. The method allowed for the identification of 1484 nonredundant potato leaf proteins, of which 364 and 447 were reproducibly identified proteins in the cell wall and cytoplasmic fractions, respectively. Reproducibly identified proteins corresponded to over 70% of proteins identified in each replicate. A diverse range of proteins was identified based on their theoretical pI values, molecular masses, functional classification, and biological processes. Such a protein extraction method is effective for the establishment of a highly qualified proteome profile.

A soluble high molecular weight copolymer of benzo[1,2-b:4,5-b']dithiophene and benzoxadiazole for efficient organic photovoltaics.

The synthesis and characterization of a soluble high molecular weight copolymer based on 4,8-bis(1-pentylhexyloxy)benzo[1,2-b:4,5-b']dithiophene and 2,1,3-benzoxadiazole is presented. High efficiency organic photovoltaic (OPV) devices comprised of this polymer and phenyl-C(71) -butyric acid methyl ester (PC(71) BM) were fabricated by additive processing with 1-chloronapthalene (CN). When the active layer is cast from pristine chlorobenzene (CB), power conversion efficiencies (PCEs) average 1.41%. Our best condition-using 2% chloronapthalene as a solvent additive in CB-results in an average PCE of 5.65%, with a champion efficiency of 6.05%.

Effect of processing additive on the nanomorphology of a bulk heterojunction material.

The bulk heterojunction (BHJ) material Si-PDTBT:PC(70)BM is sensitive to the use of a small amount of 1-chloronaphthalene (CN) as a processing additive; CN as a cosolvent (e.g., 4% in chlorobenzene) causes in a factor of 2 increase in the power conversion efficiency of BHJ solar cells. The morphology of the BHJ material, prepared with and without the CN additive is studied with top-down transmission electron microscopy, cross-sectional transmission electron microscopy, and atomic force microscopy. The improved performance is the result of changes in the nanoscale morphology. Field-effect transistor measurements are consistent with the observed changes in morphology.

Binuclear initiators for the telechelic synthesis of elastomeric polyolefins.

Novel binuclear complexes, 4,4'-bis{[N-(2,6-diisopropylphenyl)-2-(2,6-diisopropylphenylimino)propanamidato-κ(2)-N,O-(trimethylphosphine)nickel(II)]methyl}-1,1'-biphenyl (2a) and 4,4'-bis{[N-(2,6-diisopropylphenyl)-2-(2,6-diisopropylphenylimino)-4-methylpentamidato-κ(2)-N,O-(trimethylphosphine)nickel(II)]methyl}-1,1'-biphenyl (2b), were synthesized by linking two nickel centers through a bis(benzyl) fragment. When activated with nickel bis(1,5-cyclooctadiene) (Ni(COD)(2)), 2a and 2b are capable of polymerizing ethylene in a quasi-living fashion, producing polymers with approximately twice the molecular weights relative to those obtained by using a structurally related mononuclear system. In addition, 2b/Ni(COD)(2) was utilized to synthesize a series of pseudo-triblock polyethylene (PE) macroinitiating copolymers, bearing atom-transfer radical polymerization (ATRP) initiators. Pseudo-pentablock copolymers were also prepared by taking advantage of a pressure-pulsing technique, wherein the ethylene pressure was increased from 100 to 500 psi in order to produce semicrystalline ethylene-rich end-blocks. Copolymers with elastomeric properties were synthesized by grafting n-butyl acrylate from the PE macroinitiators via ATRP. Examination using monotonic and step-cyclic stress-strain tests demonstrates that the materials exhibit large strains at break (1600-2000%) and excellent elastic recoveries at large strains (∼80%). That materials with such desirable properties could not be attained using a mononuclear initiator demonstrates the clear advantage of growing the polymer via a telechelic mechanism.

Expression of RNA interference triggers from an oncolytic herpes simplex virus results in specific silencing in tumour cells in vitro and tumours in vivo.

BACKGROUND: Delivery of small interfering RNA (siRNA) to tumours remains a major obstacle for the development of RNA interference (RNAi)-based therapeutics. Following the promising pre-clinical and clinical results with the oncolytic herpes simplex virus (HSV) OncoVEX GM-CSF, we aimed to express RNAi triggers from oncolytic HSV, which although has the potential to improve treatment by silencing tumour-related genes, was not considered possible due to the highly oncolytic properties of HSV. METHODS: To evaluate RNAi-mediated silencing from an oncolytic HSV backbone, we developed novel replicating HSV vectors expressing short-hairpin RNA (shRNA) or artificial microRNA (miRNA) against the reporter genes green fluorescent protein (eGFP) and ß-galactosidase (lacZ). These vectors were tested in non-tumour cell lines in vitro and tumour cells that are moderately susceptible to HSV infection both in vitro and in mice xenografts in vivo. Silencing was assessed at the protein level by fluorescent microscopy, x-gal staining, enzyme activity assay, and western blotting. RESULTS: Our results demonstrate that it is possible to express shRNA and artificial miRNA from an oncolytic HSV backbone, which had not been previously investigated. Furthermore, oncolytic HSV-mediated delivery of RNAi triggers resulted in effective and specific silencing of targeted genes in tumour cells in vitro and tumours in vivo, with the viruses expressing artificial miRNA being comprehensibly more effective. CONCLUSIONS: This preliminary data provide the first demonstration of oncolytic HSV-mediated expression of shRNA or artificial miRNA and silencing of targeted genes in tumour cells in vitro and in vivo. The vectors developed in this study are being adapted to silence tumour-related genes in an ongoing study that aims to improve the effectiveness of oncolytic HSV treatment in tumours that are moderately susceptible to HSV infection and thus, potentially improve response rates seen in human clinical trials.

A third-generation herpesvirus is effective against gastroesophageal cancer.

BACKGROUND: Gastroesophageal cancer remains a leading cause of cancer deaths and is uniformly fatal in patients presenting with metastases and recurrence. This study sets out to determine the effect of a third-generation, replication-competent, oncolytic herpes simplex type 1 virus containing transgenes encoding for a fusogenic membrane glycoprotein and Fcy::Fur, against gastroesophageal cancer. METHODS: The cytotoxic effect of the virus was tested on human gastroesophageal cancer cell lines OCUM-2MD3, MKN-45, AGS, MKN-1, MKN-74, and BE-3 at sequential multiplicities of infection (MOI). Cytotoxicity was measured using a lactate dehydrogenase assay. Viral replication was tested by serially diluting supernatants from viral infections and titering on VERO cells via standard plaque assay. Correlations of cytotoxicity and viral replication were also investigated. RESULTS: All cell lines were susceptible to viral infection and demonstrated a dose-dependent effect, with greater and faster cytotoxicity at higher MOIs. Viral replication was supported in the cell lines tested, with peak titers by d 5, some supporting as high as >40,000× amplification. Cell lines with longer doubling times (>30 h) also achieved higher viral titers at a MOI of 0.1. Cell lines with shorter doubling times achieved 50% cell kill in fewer days, with an average of 2.3 d for cell lines with doubling times under 30 h compared with 4.4 d for cell lines with doubling times over 30 h. CONCLUSION: These results suggest that this third-generation oncolytic herpesvirus can effectively infect and lyse gastroesophageal cancer cells and may provide a novel therapy against gastroesophageal cancer.

Efficient delivery of RNA Interference to peripheral neurons in vivo using herpes simplex virus.

Considerable interest has been focused on inducing RNA interference (RNAi) in neurons to study gene function and identify new targets for disease intervention. Although small interfering RNAs (siRNAs) have been used to silence genes in neurons, in vivo delivery of RNAi remains a major challenge limiting its applications. We have developed a highly efficient method for in vivo gene silencing in dorsal root ganglia (DRG) using replication-defective herpes simplex viral (HSV-1) vectors. HSV-mediated delivery of short-hairpin RNA (shRNA) targeting reporter genes resulted in highly effective and specific silencing in neuronal and non-neuronal cells in culture and in the DRG of mice in vivo including in a transgenic mouse model. We further establish proof of concept by demonstrating in vivo silencing of the endogenous trpv1 gene. These data are the first to show silencing in DRG neurons in vivo by vector-mediated delivery of shRNA. Our results support the utility of HSV vectors for gene silencing in peripheral neurons and the potential application of this technology to the study of nociceptive processes and in pain gene target validation studies.

Combining BRAF inhibition with oncolytic herpes simplex virus enhances the immune-mediated antitumor therapy of BRAF-mutant thyroid cancer.

BACKGROUND: The aggressive clinical behavior of poorly differentiated and anaplastic thyroid cancers (PDTC and ATC) has proven challenging to treat, and survival beyond a few months from diagnosis is rare. Although 30%-60% of these tumors contain mutations in the BRAF gene, inhibitors designed specifically to target oncogenic BRAF have shown limited and only short-lasting therapeutic benefits as single agents, thus highlighting the need for improved treatment strategies, including novel combinations. METHODS: Using a BRAFV600E-driven mouse model of ATC, we investigated the therapeutic efficacy of the combination of BRAF inhibition and oncolytic herpes simplex virus (oHSV). Analyses of samples from tumor-bearing mice were performed to immunologically characterize the effects of different treatments. These immune data were used to inform the incorporation of immune checkpoint inhibitors into triple combination therapies. RESULTS: We characterized the immune landscape in vivo following BRAF inhibitor treatment and detected only modest immune changes. We, therefore, hypothesized that the addition of oncolytic virotherapy to BRAF inhibition in thyroid cancer would create a more favorable tumor immune microenvironment, boost the inflammatory status of tumors and improve BRAF inhibitor therapy. First, we showed that thyroid cancer cells were susceptible to infection with oHSV and that this process was associated with activation of the immune tumor microenvironment in vivo. Next, we showed improved therapeutic responses when combining oHSV and BRAF inhibition in vivo, although no synergistic effects were seen in vitro, further confirming that the dominant effect of oHSV in this context was likely immune-mediated. Importantly, both gene and protein expression data revealed an increase in activation of T cells and natural killer (NK) cells in the tumor in combination-treated samples. The benefit of combination oHSV and BRAF inhibitor therapy was abrogated when T cells or NK cells were depleted in vivo. In addition, we showed upregulation of PD-L1 and CTLA-4 following combined treatment and demonstrated that blockade of the PD-1/PD-L1 axis or CTLA-4 further improved combination therapy. CONCLUSIONS: The combination of oHSV and BRAF inhibition significantly improved survival in a mouse model of ATC by enhancing immune-mediated antitumor effects, and triple combination therapies, including either PD-1 or CTLA-4 blockade, further improved therapy.

Band gap control in conjugated oligomers via Lewis acids.

A simple and effective strategy for optical band gap control is demonstrated through the use of a novel small acceptor/donor/acceptor molecule, 1, and group-13 Lewis acids. Chromophore 1 contains a dithienolesilole donor unit end-capped with benzo-2,1,3-thiadiazole (BT) acceptor units. Addition to 1 of stoichiometeric quantities of Lewis acids of varying strength resulted in the formation of Lewis adducts with progressively red-shifted primary charge-transfer absorption bands. These complexes have absorption spectra that approach those of known conjugated copolymers containing related units. NMR spectroscopy and X-ray diffraction studies revealed that 1 binds 2 equiv of Lewis acid via the nitrogen at the 3-position of BT.

Construction and characterization of an oncolytic HSV vector containing a fusogenic glycoprotein and prodrug activation for enhanced local tumor control.

A large number of oncolytic viral vectors are currently under clinical development for cancer therapy. Herpes simplex virus type 1 (HSV-1) has demonstrated particular promise in this field, showing genetically engineered selective tumor replication and cytotoxicity in a wide variety of tumor types, without damaging healthy tissues. Enhanced activity has been observed when a range of therapeutic genes has been inserted into various oncolytic HSV genomes. Here, we discuss methods used to develop and characterize an oncolytic HSV virus that combines expression of a highly potent prodrug activating gene (yeast cytosine deaminase/uracil phosphoribosyltransferase fusion [Fcy::Fur]) and the fusogenic glycoprotein from gibbon ape leukemia virus (GALV) for enhanced local tumor control.

Development of a new fusion-enhanced oncolytic immunotherapy platform based on herpes simplex virus type 1.

BACKGROUND: Oncolytic viruses preferentially replicate in tumors as compared to normal tissue and promote immunogenic cell death and induction of host systemic anti-tumor immunity. HSV-1 was chosen for further development as an oncolytic immunotherapy in this study as it is highly lytic, infects human tumor cells broadly, kills mainly by necrosis and is a potent activator of both innate and adaptive immunity. HSV-1 also has a large capacity for the insertion of additional, potentially therapeutic, exogenous genes. Finally, HSV-1 has a proven safety and efficacy profile in patients with cancer, talimogene laherparepvec (T-VEC), an oncolytic HSV-1 which expresses GM-CSF, being the only oncolytic immunotherapy approach that has received FDA approval. As the clinical efficacy of oncolytic immunotherapy has been shown to be further enhanced by combination with immune checkpoint inhibitors, developing improved oncolytic platforms which can synergize with other existing immunotherapies is a high priority. In this study we sought to further optimize HSV-1 based oncolytic immunotherapy through multiple approaches to maximize: (i) the extent of tumor cell killing, augmenting the release of tumor antigens and danger-associated molecular pattern (DAMP) factors; (ii) the immunogenicity of tumor cell death; and (iii) the resulting systemic anti-tumor immune response. METHODS: To sample the wide diversity amongst clinical strains of HSV-1, twenty nine new clinical strains isolated from cold sores from otherwise healthy volunteers were screened across a panel of human tumor cell lines to identify the strain with the most potent tumor cell killing ability, which was then used for further development. Following deletion of the genes encoding ICP34.5 and ICP47 to provide tumor selectivity, the extent of cell killing and the immunogenicity of cell death was enhanced through insertion of a gene encoding a truncated, constitutively highly fusogenic form of the envelope glycoprotein of gibbon ape leukemia virus (GALV-GP-R-). A number of further armed derivatives of this virus were then constructed intended to further enhance the anti-tumor immune response which was generated following fusion-enhanced, oncolytic virus replication-mediated cell death. These viruses expressed GMCSF, an anti-CTLA-4 antibody-like molecule, CD40L, OX40L and/or 4-1BB, each of which is expected to act predominantly at the site and time of immune response initiation. Expression of these proteins was confirmed by ELISA and/or western blotting. Immunogenic cell death was assessed by measuring the levels of HMGB1 and ATP from cell free supernatants from treated cells, and by measuring the surface expression of calreticulin. GALV-GP-R- mediated cell to cell fusion and killing was tested in a range of tumor cell lines in vitro. Finally, the in vivo therapeutic potential of these viruses was tested using human A549 (lung cancer) and MDA-MB-231(breast cancer) tumor nude mouse xenograft models and systemic anti-tumor effects tested using dual flank syngeneic 4434 (melanoma), A20 (lymphoma) mouse tumor models alone and in combination with a murine anti-PD1 antibody, and 9 L (gliosarcoma) tumors in rats. RESULTS: The twenty nine clinical strains of HSV-1 isolated and tested demonstrated a broad range of tumor cell killing abilities allowing the most potent strain to be identified which was then used for further development. Oncolytic ability was demonstrated to be further augmented by the expression of GALV-GP-R- in a range of tumor cell lines in vitro and in mouse xenograft models in nude mice. The expression of GALV-GP-R- was also demonstrated to lead to enhanced immunogenic cell death in vitro as confirmed by the increased release of HMGB1 and ATP and increased levels of calreticulin on the cell surface. Experiments using the rat 9 L syngeneic tumor model demonstrated that GALV-GP-R- expression increased abscopal uninjected (anenestic) tumor responses and data using mouse 4434 tumors demonstrated that virus treatment increased CD8+ T cell levels both in the injected and uninjected tumor, and also led to increased expression of PD-L1. A combination study using varying doses of a virus expressing GALV-GP-R- and mGM-CSF and an anti-murine PD1 antibody showed enhanced anti-tumor effects with the combination which was most evident at low virus doses, and also lead to immunological memory. Finally, treatment of mice with derivatives of this virus which additionally expressed anti-mCTLA-4, mCD40L, m4-1BBL, or mOX40L demonstrated enhanced activity, particularly in uninjected tumors. CONCLUSION: The new HSV-1 based platform described provides a potent and versatile approach to developing new oncolytic immunotherapies for clinical use. Each of the modifications employed was demonstrated to aid in optimizing the potential of the virus to both directly kill tumors and to lead to systemic therapeutic benefit. For clinical use, these viruses are expected to be most effective in combination with other anti-cancer agents, in particular PD1/L1-targeted immune checkpoint blockade. The first virus from this program (expressing GALV-GP-R- and hGM-CSF) has entered clinical development alone and in combination with anti-PD1 therapy in a number of tumor types (NCT03767348).

Impaired axonal regeneration by isolectin B4-binding dorsal root ganglion neurons in vitro.

The subpopulation of dorsal root ganglion (DRG) neurons recognized by Griffonia simplicifolia isolectin B4 (IB4) differ from other neurons by expressing receptors for glial cell line-derived neurotrophic factor (GDNF) rather than neurotrophins. Additionally, IB4-labeled neurons do not express the laminin receptor, alpha7-integrin (Gardiner et al., 2005), necessary for optimal axonal regeneration in the peripheral nervous system. In cultures of dissociated DRG neurons of adult mice on laminin, robust spontaneous neurite outgrowth from IB4-negative neurons occurs and is strongly enhanced by previous axotomy. In contrast, IB4-labeled neurons show little neurite outgrowth and do not express GAP 43, even after axotomy or culture with GDNF. Moreover, growth of their axons through collagen gels is impaired compared with other DRG neurons. To determine whether the sparse neurite outgrowth of IB4-labeled neurons is attributable to lack of integrin expression, DRG cultures were infected with a herpes simplex 1 vector encoding alpha7-integrin, but its forced expression failed to promote neurite outgrowth in either IB4-labeled or other DRG neurons or in cultured adult retinal ganglion cells. Forced coexpression of both alpha7-integrin and GAP 43 also failed to promote neurite outgrowth in IB4-labeled neurons. In addition, cultured sciatic nerve segments were found to release much lower levels of GDNF, demonstrated by ELISA, than nerve growth factor. These findings together with their impaired intrinsic axonal regeneration capacity may contribute to the known vulnerability of the IB4-labeled population of DRG neurons to peripheral nerve injury.

New polyethylene macroinitiators and their subsequent grafting by atom transfer radical polymerization.

A semicrystalline polyethylene (PE) macroinitiator was prepared by copolymerizing ethylene and an inititating monomer (5-norbornen-2-yl-2'-bromo-2'-methyl propanoate) (3) using [N-(2,6-diisopropylphenyl)-2-(2,6-diisopropylphenylimino)isobutanamidato]-Ni(eta1-CH2Ph)(PMe3) (1) and Ni(COD)2 (bis(1,5-cyclooctadiene)-nickel) (2). Although 3 decomposes Ni(COD)2, if the initiating species (1/2) are exposed to ethylene for a period of time, t1, and then 3 is introduced for another period of time, t2, the polymerization takes place in a controlled manner. Variations in temperature, pressure, and [3] afford a great deal of control over the composition and architecture of the PE macroinitiator. Subsequent grafting with methyl methacrylate (MMA) yields PE-graft-PMMA with narrow polydispersities and increasing PMMA content at longer reaction times. Because the products are semicrystalline materials with high melting points, they are anticipated to function as blend compatibilizers for linear polyethylene.

Genetic diversity of Streptomyces spp. causing common scab of potato in eastern Canada.

Common scab is an important disease of potato caused by Streptomyces scabies and other closely related species. In this study, the genetic diversity of Streptomyces spp. causing common scab of potato in eastern Canada was for the first time investigated. Forty-one Streptomyces spp. isolates were retrieved from necrotic lesions of potato tubers harvested from different regions of the Canadian provinces New-Brunswick, Nova Scotia and Prince-Edward-Island. Most isolates were closely related to known pathogenic S. scabies strains on the basis of partial 16S ribosomal (r) RNA and rpoB gene sequence analyses. Two isolates were identified as pathogenic species of Streptomyces acidiscabies. To our knowledge, this species has never been previously isolated in these areas. Genome fingerprinting studies using repetitive elements (rep) polymerase chain reactions (PCR) revealed 10 distinct genetic groups in eastern Canada. The geographical distribution of the genetic groups was region-dependant. Pathogenicity- and virulence-related genes (txtA, txtC, and tomA) were PCR-amplified from each isolate, and nucleotide sequence analysis of partial gene fragments revealed slight polymorphisms in both txtA and txtC genes. No genetic variation was noted in the partial tomA gene sequences.

Combination of a fusogenic glycoprotein, prodrug activation, and oncolytic herpes simplex virus for enhanced local tumor control.

We have previously developed an oncolytic herpes simplex virus-1 based on a clinical virus isolate, which was deleted for ICP34.5 to provide tumor selected replication and ICP47 to increase antigen presentation as well as tumor selective virus replication. A phase I/II clinical trial using a version of this virus expressing granulocyte macrophage colony-stimulating factor has shown promising results. The work reported here aimed to develop a version of this virus in which local tumor control was further increased through the combined expression of a highly potent prodrug activating gene [yeast cytosine deaminase/uracil phospho-ribosyltransferase fusion (Fcy::Fur)] and the fusogenic glycoprotein from gibbon ape leukemia virus (GALV), which it was hoped would aid the spread of the activated prodrug through the tumor. Viruses expressing the two genes individually or in combination were constructed and tested, showing (a) GALV and/or Fcy::Fur expression did not affect virus growth; (b) GALV expression causes cell fusion and increases the tumor cell killing at least 30-fold in vitro and tumor shrinkage 5- to 10-fold in vivo; (c) additional expression of Fcy::Fur combined with 5-fluorocytosine administration improves tumor shrinkage further. These results indicate, therefore, that the combined expression of the GALV protein and Fcy::Fur provides a highly potent oncolytic virus with improved capabilities for local tumor control. It is intended to enter the GALV/Fcy::Fur expressing virus into clinical development for the treatment of tumor types, such as pancreatic or lung cancer, where local control would be anticipated to be clinically advantageous.

White paper on microbial anti-cancer therapy and prevention.

In this White Paper, we discuss the current state of microbial cancer therapy. This paper resulted from a meeting ('Microbial Based Cancer Therapy') at the US National Cancer Institute in the summer of 2017. Here, we define 'Microbial Therapy' to include both oncolytic viral therapy and bacterial anticancer therapy. Both of these fields exploit tumor-specific infectious microbes to treat cancer, have similar mechanisms of action, and are facing similar challenges to commercialization. We designed this paper to nucleate this growing field of microbial therapeutics and increase interactions between researchers in it and related fields. The authors of this paper include many primary researchers in this field. In this paper, we discuss the potential, status and opportunities for microbial therapy as well as strategies attempted to date and important questions that need to be addressed. The main areas that we think will have the greatest impact are immune stimulation, control of efficacy, control of delivery, and safety. There is much excitement about the potential of this field to treat currently intractable cancer. Much of the potential exists because these therapies utilize unique mechanisms of action, difficult to achieve with other biological or small molecule drugs. By better understanding and controlling these mechanisms, we will create new therapies that will become integral components of cancer care.

Pseudo-tetrablock copolymers with ethylene and a functionalized comonomer.

Pseudo-tetrablock copolymers comprised of ethylene and 5-norbomen-2-yl acetate (1), were synthesized using the initiator system (L(i)Pr2)Ni(eta1-CH2Ph)(PMe3)(2)[(L(i)Pr2) = N-(2,6-diisopropylphenyl)-2-(2,6-diisopropylphenylimino)propanamide] and 2.5 equivalents of Ni(COD)2 [bis(1,5-cyclooctadiene) nickel.

A novel HSV-1 virus, JS1/34.5-/47-, purges contaminating breast cancer cells from bone marrow.

PURPOSE: Oncolytic herpes simplex virus type 1 (HSV-1) vectors show considerable promise as agents for cancer therapy. We have developed a novel recombinant HSV-1 virus (JS1/34.5-/47-) for purging of occult breast cancer cells from bone marrow of patients. Here, we evaluate the therapeutic efficacy of this oncolytic virus. EXPERIMENTAL DESIGN: Electron microscopy was used to determine whether human breast cancer and bone marrow cells are permissive for JS1/34.5-/47- infection. Subsequently, the biological effects of JS1/34.5-/47- infection on human breast cancer cells and bone marrow were established using cell proliferation and colony formation assays, and the efficiency of cell kill was evaluated. Finally, the efficiency of JS1/34.5-/47- purging of breast cancer cells was examined in cocultures of breast cancer cells with bone marrow as well as bone marrow samples from high-risk breast cancer patients. RESULTS: We show effective killing of human breast cancer cell lines with the JS1/34.5-/47- virus. Furthermore, we show that treatment with JS1/34.5-/47- can significantly inhibit the growth of breast cancer cell lines without affecting cocultured mononuclear hematopoietic cells. Finally, we have found that the virus is effective in destroying disseminated tumors cells in bone marrow taken from breast cancer patients, without affecting the hematopoietic contents in these samples. CONCLUSION: Collectively, our data show that the JS1/34.5-/47- virus can selectively target breast cancer cells while sparing hematopoietic cells, suggesting that JS1/34.5-/47- can be used to purge contaminating breast cancer cells from human bone marrow in the setting of autologous hematopoietic cell transplantation.

A phase I study of OncoVEXGM-CSF, a second-generation oncolytic herpes simplex virus expressing granulocyte macrophage colony-stimulating factor.

PURPOSE: To conduct a phase I clinical trial with a second-generation oncolytic herpes simplex virus (HSV) expressing granulocyte macrophage colony-stimulating factor (Onco VEXGM-CSF) to determine the safety profile of the virus, look for evidence of biological activity, and identify a dosing schedule for later studies. EXPERIMENTAL DESIGN: The virus was administered by intratumoral injection in patients with cutaneous or s.c. deposits of breast, head and neck and gastrointestinal cancers, and malignant melanoma who had failed prior therapy. Thirteen patients were in a single-dose group, where doses of 10(6), 10(7), and 10(8) plaque-forming units (pfu)/mL were tested, and 17 patients were in a multidose group testing a number of dose regimens. RESULTS: The virus was generally well tolerated with local inflammation, erythema, and febrile responses being the main side effects. The local reaction to injection was dose limiting in HSV-seronegative patients at 10(7) pfu/mL. The multidosing phase thus tested seroconverting HSV-seronegative patients with 10(6) pfu/mL followed by multiple higher doses (up to 10(8) pfu/mL), which was well tolerated by all patients. Biological activity (virus replication, local reactions, granulocyte macrophage colony-stimulating factor expression, and HSV antigen-associated tumor necrosis), was observed. The duration of local reactions and virus replication suggested that dosing every 2 to 3 weeks was appropriate. Nineteen of 26 patient posttreatment biopsies contained residual tumor of which 14 showed tumor necrosis, which in some cases was extensive, or apoptosis. In all cases, areas of necrosis also strongly stained for HSV. The overall responses to treatment were that three patients had stable disease, six patients had tumors flattened (injected and/or uninjected lesions), and four patients showed inflammation of uninjected as well as the injected tumor, which, in nearly all cases, became inflamed. CONCLUSIONS: Onco VEXGM-CSF is well tolerated and can be safely administered using the multidosing protocol described. Evidence of an antitumor effect was seen.