NCI Microbial Based Cancer Therapy Conference

POSTER SESSION

Conference sponsored by the National Cancer Institute

Technologies to Overcome Cancer Challenges

July 11-12, 2017 Natcher Center, NIH campus- Building 45, Bethesda, Maryland

POSTER SESSION

No Name Title

Cancer virotherapy

1 Eric Bartee Tumor localized inhibition of the PD1/PDL1 enhances the efficacy of oncolytic myxoma

2 David A. Ornelles MAP3K7 and CHD1 are novel mediators of resistance to VSV oncolysis in prostate cancer

3 Lauren Oldfield Genome-wide modification of herpesvirus genomes using synthetic genomics methods

4 Bernardo A. Mainou Reoviruses Have Enhanced Oncolytic Properties Against Triple-Negative Breast Cancer

5 Liang Deng Intratumoral delivery of inactivated modified vaccinia virus Ankara (iMVA)

6 Liang Deng Intratumoral Delivery of Modified Vaccinia Virus Ankara Expressing Human Flt3L

7 Kate Chiappinelli Epigenetic control of endogenous retroviruses in cancer: implications for immune therapy

8 Biswajit Biswas Therapeutic and Prophylactic Applications of Bacteriophages in Cancer Therapy

9 Ann B. Hill Cytomegalovirus-based vaccines in breast and melanoma mouse tumor models

Bacterial and eukaryotes based cancer therapy

10 Abel Baerga-Ortiz Direct detection of genotoxic or pro-inflammatory bacterial genes in stool samples

11 Adam Fisher Modulation of the Tumor Microenvironment using Synthetic BioticsTM

12 Hassan Brim Gut microbiome analysis reveals dysbiosis in sickle cell diseases patients with veillonella

13 Weiguo Cui Reenergized Adoptive Cell Transfer -A Multi-Pronged Strategy to Treat Solid Tumors

14 David J. Bzik Uncorking the remarkable anti-cancer biology of Toxoplasma

15 Jill Zeilstra-Ryalls Developing a photosynthetic bacterial vector for intratumorial photodynamic therapy

16 Greg Phillips Use of a gnotobiotic mouse model to characterize bacteria/colorectal tumor interactions

17 Wei Kong TRAIL-armed Self-destructing Salmonella serve as “Time-Bombs” to Combat Cancer

18 Bin Xue Multiple Responding Mechanisms in Intestinal Cancer Tissues Mediated by Microbiome

19 Shiladitya DasSarma An Archaeal Therapeutic Drug and Antigen Delivery Employing Proteinaceous Nanoparticles

20 Qiuhong He Loss of Cancer Immune Privilege in Bacterial-based Therapy

21 Roger A Laine Bacterial Secreted Polysaccharide Toxins Bind to Sialin on Tumor Capillary Endothelium

22 Gilad Bachrach Tumor targeting by Fusobacterium nucleatum

23 Mark Gomelsky Optogenetic and chemogenetic platforms for listeria-mediated intratumoral drug delivery

24 Alejandro Alice Vγ9Vδ2 T cells dominate the response to Listeria monocytogenes-based vaccines

25 Shifeng Wang Regulated invasion, chemotaxis, attenuation and endotoxicity of Salmonella

26 Katherine Broadway Contribution of S. enterica Chemotaxis on Mouse Mammary Carcinoma Progression

27 Bahareh Behkam Nanoscale Bacteria-Enabled Autonomous Drug Delivery Systems for Cancer Therapy

28 Yasser Heakal Autophagy and Apoptosis in Triple-Negative Breast Cancer Cells by Heat-labile Enterotoxin

29 Dudley H. McNitt The Streptococcal Adhesin, Scl1, Recognizes Oncofetal Fibronectin

30 Melissa Garcia Dietary fatty acids modulate fungal-host interactions

31 Arturo Ferreira Could Trypanosoma cruzi Infection Be a Good Thing in the Presence of a Tumor?

32 Jesus Vera Combined Parasite-Derived Peptides for Melanoma Therapy

Technologies to Support Research on Microbial based cancer therapy

33 Tsang, Hsiny Cloud-based platform for analyzing TCGA and microbe sequencing data

34 Wenyun Lu Mass spectrometry based metabolomics platform for cancer research

35 Yanming Li Weak Signal Detection of Lung Cancer Risk

0

Cancer virotherapy

1

Tumor localized inhibition of the PD1/PDL1 checkpoint enhances the efficacy of

oncolytic myxoma virus against both local and metastatic melanoma.

Mee Y Bartee, Katherine M Dunlap, Eric Bartee

Medical University of South Carolina, Charleston

While traditional oncolytic therapy is effective at rapidly debulking directly injected tumor masses,

achieving complete eradication of either established local tumors or disseminated metastatic disease

has proven difficult. One potential method to overcome this is to use oncolytic infections to induce

secondary anti-tumor immunotherapy. Unfortunately, while the initial induction of this

immunotherapy is typically robust, its subsequent clinical efficacy is often inhibited through a variety

of immuno-regulatory mechanisms, such as activation of the PD1/PDL1 T-cell checkpoint. In order

to improve the clinical potential of oncolytic therapy, we therefore examined whether inhibition of

the PD1/PDL1 pathway would improve the efficacy of oncolytic myxoma virus against well-

established B16/F10 melanomas.

Our results indicate that systemic inhibition of the PD1/PDL1 pathway during myxoma virus

treatment significantly enhances the efficacy of viral therapy and allows for the complete eradiation

of established melanomas. This increased efficacy was not due to changes in the direct oncolytic

capacity of myxoma virus and instead correlated with enhanced activation of anti-tumor CD8+ T cell

responses. To advance this finding, we further generated a novel oncolytic myxoma virus (vMYX­

PD1) which secretes a soluble form of PD1 from infected cells within the tumor microenvironment.

Once secreted, this soluble PD1 binds to PDL1 on the surface of neighboring tumor cells thus

preventing activation of the inhibitory PD1/PDL1 checkpoint. Analysis of this virus indicated that

vMYX-PD1 both induced and maintained CD8+ anti-tumor T-cell responses and that monotherapy

with this virus actually outperformed therapy using the combination of unmodified myxoma and

systemic PD1-blockade. Additionally, localized vMYX-PD1 treatment induced a systemic abscopal

effect capable of significantly reducing established metastatic tumor burden in non-injected

secondary solid organs such as the lung. These results demonstrate that tumor localized inhibition of

the PD1 T-cell checkpoint significantly improves outcomes during oncolytic virotherapy and

establishes a feasible path to translate these findings against clinically relevant disease.

2

MAP3K7 and CHD1 are novel mediators of resistance to VSV oncolysis in prostate cancer

ROBERT S. BAYNE1, DAVID A. ORNELLES2 and DOUGLAS S. LYLES3

1Department of Biochemistry, 2Department of Microbiology and Immunology, Wake Forest School

of Medicine, Winston-Salem, NC

Innate immunity is a prominent determinant of viral pathogenicity and viral tropism; viruses

preferentially infect tissues with abrogated immune responses, such as cancers that downregulated

their antiviral responses. However, some cancers constitutively express antiviral genes and display

viral resistance. This phenomenon will significantly impact the efficacy of several dozen oncolytic

viral therapies currently in clinical trials. We tested the effects of two genes, MAP3K7 and CHD1, on

antiviral gene expression in virus-resistant prostate cancer. These two genes are frequently co-deleted

in high-grade prostate cancers. MAP3K7 and/or CHD1 expression was silenced with shRNA in virus-

resistant PC3 human prostate cancer cells. Transcriptome analysis by RNA-Seq showed that silencing

CHD1 alone and silencing both MAP3K7 and CHD1 decreased antiviral gene expression in PC3 cells,

whereas silencing MAP3K7 alone increased antiviral gene expression. Silencing expression of

MAP3K7, CHD1, or both genes increased susceptibility to infection with oncolytic vesicular

stomatitis virus (VSV). These results indicate that CHD1 plays an important role in the constitutive

expression of antiviral genes. However, the increase in antiviral gene expression in MAP3K7-silencd

cells indicated that the increased viral susceptibility is mediated by other mechanisms. These findings

demonstrate that MAP3K7 and CHD1 regulate viral resistance in prostate cancer. Antiviral gene

expression may be a determinant of viral resistance in prostate cancer, but other mechanisms are

involved as well.

3

Rapid, genome-wide modification of herpesvirus genomes using synthetic genomics methods

Lauren M. Oldfield, Peter Grzesik, Alexander Voorhies, Sanjana Prasad, Nina Alperovich, Derek

MacMath, Claudia Najera, Alexandra Rocha, Diya Chandra, Vladimir Noskov, Michael Montague,

Prashant Desai, Sanjay Vashee

J. Craig Venter Institute

Herpesviruses are clinically important human pathogens and some members of this family, Epstein-

Barr virus (EBV) and Kaposi’s sarcoma-associated virus (KSHV), are oncogenic. The genomes of

herpesviruses range in size between 125 – 250 kb, contain regions of repeated sequence, and have a

high GC content. Historically, manipulation and sequencing of herpesvirus genomes has been

difficult. The cloning of herpesvirus genomes as bacterial artificial chromosomes has greatly

facilitated the manipulation of these DNA sequences using Escherichia coli recombineering,

however, the long timeframe to generate complex mutants and the instability of some herpesvirus

genomes over repeated rounds of mutagenesis still represent significant drawbacks. To improve our

capacity to engineer herpesvirus genomes, we utilized synthetic genomics methods in yeast to clone

overlapping fragments of the herpes simplex virus type 1 (HSV-1) genome and then to assemble the

fragments to regenerate a full-length genome. This modular DNA assembly method allows

researchers to make changes quickly in the genomic fragments in parallel and assemble genomes

using a mixture of wild-type and mutant fragments, rapidly generating mutant HSV-1 genomes with

multiple mutations. Using a number of different techniques, including in vitro CRISPR-Cas9 editing,

we demonstrated the utility of this method by making modifications to a single gene, two genes at a

time and, finally, combinatorial deletions of five tegument genes. HSV-1 was chosen as a model

because infectious virus can be quickly reconstituted in tissue culture and, because of our success

with HSV-1, we have initiated similar genome assemblies of EBV and KSHV. Rapid manipulation

of HSV-1 could also help to engineer effective delivery platforms for therapeutics, such as oncolytic

viruses, or vaccines.

4

Engineered Reoviruses Have Enhanced Oncolytic Properties Against Triple-Negative Breast

Cancer

Roxana M. Rodriguez Stewart1, Angela K. Berger2,3, Jaime Guberman4, and Bernardo A.

Mainou1,2,3

Microbiology and Molecular Genetics1, Department of Pediatrics2, Children’s Healthcare of

Atlanta3, Emory University4, Atlanta, GA 30322

Triple-negative breast cancer (TNBC) constitutes approximately 15% of all breast cancer, has a

higher rate of relapse, and shorter overall survival after metastasis than other subtypes of breast

cancer. There is a need for targeted therapeutics to treat this type of breast cancer, as current therapies

are largely limited to cytotoxic chemotherapy. Mammalian orthoreovirus (reovirus), a nonenveloped

segmented dsRNA virus in the Reoviridae family causes a mostly asymptomatic infection in humans.

Reovirus has been shown to preferentially kill transformed cells and is currently in Phase I-III clinical

trials to assess its efficacy as an oncolytic against a variety of cancers. To engineer reovirus with

enhanced infective and cytopathic properties against triple-negative breast cancer cells, we coinfected

a TNBC cell line (MDA-MB-231) with prototype reoviruses T1L, T2J, and T3D. Following serial

passage, we isolated two reassortant reoviruses, r1Reovirus and r2Reovirus. r1Reovirus and

r2Reovirus contain gene segments predominately from T1L, with one (r2Reovirus) or three

(r1Reovirus) gene segments from T3D and synonymous and nonsynonymous point mutations.

Infection of two TNBC cell lines, MDA-MB-231 and HCC1937, showed that r1Reovirus and

r2Reovirus infect both cell lines more efficiently than T1L and have enhanced capacity to induce cell

death in MDA-MB-231 cells. Although r2Reovirus infected HCC1937 cells more efficiently, all

reovirus serotypes tested impaired cell growth equally without promoting cell death. These data

suggest that r1Reovirus and r2Reovirus encode genomic changes that enhance their ability to infect

TNBC cells. The different inhibitory effects on cell growth by virus infection also suggests reovirus

can impair cancer cell growth through different mechanisms depending on the genetic composition

of the TNBC cells.

5

Intratumoral Delivery of Modified Vaccinia Virus Ankara Expressing Human Flt3L as Cancer

Immunotherapy

Peihong Dai1,2#, Weiyi Wang1#, Ning Yang1#, Stewart Shuman2, Taha Merghoub3,4,5, Jedd D.

Wolchok3,4,5,6, and Liang Deng1,6*

1 Dermatology Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY 10065., 2Molecular

Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065., 3Parker Institute for Cancer

Immunotherapy, Memorial Sloan Kettering Cancer Center, New York, NY 10065., 4Ludwig Center for Cancer Immunotherapy,

Memorial Sloan Kettering Cancer Center, New York, NY 10065., 5Melanoma and Immunotherapeutics Service, Department

of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY 10065, 6Weill Cornell Medical and Graduate Colleges,

New York, NY 10065

Modified vaccinia virus Ankara (MVA) is a highly attenuated vaccinia strain that is an important vaccine

vector for infectious diseases and cancers. MVA has a 31-kb deletion of the parental vaccinia genome and

was shown to be safe for human use during smallpox vaccination. The investigation of MVA as cancer

therapeutics has so far been limited to its use as a vaccine vector to express tumor antigens. We

hypothesize that intratumoral delivery of recombinant MVA∆E3L (with deletion of vaccinia virulence

factor E3) expressing human flt3L (Fms-like tyrosine kinase 3 ligand) would provide "in situ therapeutic

vaccine effects." Flt3L plays a critical role in the development of DC subsets, including CD103+/CD8α+

DCs, which are critical for cross-presentation of tumor antigens. We found that MVA∆E3L infection of

B16-F10 and MC38 induces higher levels of IFN-ȕ, IL-6, CCL4 and CCL5 than MVA. MVA∆E3L ­

induction of type I IFN in cDCs is mainly dependent on the cGAS/STING pathway. Intratumoral injection

of MVA∆E3L is more efficacious than MVA in tumor eradication and extension of survival in bilateral

tumor implantation models, which correlates with stronger induction of activated CD8+ and CD4+ effector

T cells in both injected and non-injected tumors from MVA∆E3L-treated mice compared with MVA-

treated mice. Furthermore, intratumoral injection of MVA∆E3L-hFlt3L exerts stronger anti-tumor effects

than MVA∆E3L in a murine melanoma bilateral implantation model. Our results show that intratumoral

injection of MVA or MVA∆E3L leads to alteration of tumor immune suppressive microenvironment,

which facilitates tumor antigen presentation, recruitment and activation of anti-tumor CD8+ and CD4+ T

cells. Using immune activating virus to produce hFlt3L within tumor microenvironment further enhances

antitumor immune responses. Lastly intratumoral injection of MVA∆E3L-hFlt3L overcomes resistance to

immune checkpoint blockade therapy in murine tumor models. Taken together, our results indicate that

MVA∆E3L-hFlt3L is a promising cancer immunotherapeutic agent.

6

Intratumoral delivery of inactivated modified vaccinia virus Ankara (iMVA) induces systemic

antitumor immunity via STING and Batf3-dependent dendritic cells

Peihong Dai1,2#, Weiyi Wang1#, Ning Yang1, Cristian Serna-Tamayo1, Jacob M. Ricca3, Dmitriy

Zamarin3,4,5,6, Stewart Shuman2, Taha Merghoub3,4,5, Jedd D. Wolchok3,4,5,6, and Liang Deng1,6*

1 Dermatology Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New

York, NY 10065. 2Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York,

NY 10065. 3Parker Institute for Cancer Immunotherapy, Memorial Sloan Kettering Cancer Center,

New York, NY 10065. 4Ludwig Center for Cancer Immunotherapy, Memorial Sloan Kettering

Cancer Center, New York, NY 10065. 5Melanoma and Immunotherapeutics Service, Department of

Medicine, Memorial Sloan Kettering Cancer Center, New York, NY 10065 6Weill Cornell Medical

and Graduate Colleges, New York, NY 10065

Advanced cancers remain a therapeutic challenge despite recent progress in targeted therapy and

immunotherapy. Novel approaches are needed to alter the tumor immune-suppressive

microenvironment and to facilitate the recognition of tumor antigens that leads to antitumor

immunity. Poxviruses, such as modified vaccinia virus Ankara (MVA), have potential as

immunotherapeutic agents. Here we show that infection of conventional dendritic cells (DCs) with

heat-inactivated or UV-inactivated MVA leads to higher levels of IFN induction than MVA via the

cGAS–STING cytosolic DNA-sensing pathway. Intratumoral injection of inactivated MVA (iMVA)

was effective and generated adaptive antitumor immunity in murine melanoma and colon cancer

models. iMVA-induced antitumor therapy was less effective in STING- or Batf3-deficient mice than

in wild-type mice, indicating that both cytosolic DNA-sensing and Batf3-dependent CD103+/CD8+

DCs are essential for iMVA immunotherapy. The combination of intratumoral delivery of iMVA and

systemic delivery of immune checkpoint blockade generated synergistic antitumor effects in bilateral

tumor implantation models as well as in a unilateral large established tumor model. Our results

suggest that inactivated vaccinia virus could be used as a safe and effective cancer immunotherapeutic

agent for human cancers.

7

Epigenetic control of endogenous retroviruses in cancer: implications for immune therapy

Katherine B. Chiappinelli, PhD, George Washington University Cancer Center

Therapies that activate the host immune system have shown tremendous promise for a wide variety

of solid tumors, with patients exhibiting vigorous and durable responses. However, in most cancer

types, fewer than half of patients respond to these immune therapies. We propose epigenetic therapy

as a mechanism to sensitize these patients. DNA methyltransferase inhibitors (DNMTis) upregulate

immune attraction, including the interferon response, in solid tumors. We have shown that in human

epithelial ovarian cancer cells, DNMTis upregulate viral defense by cytosolic sensing of double-

stranded RNA (dsRNA), triggering a Type I Interferon response and apoptosis. Demethylation and

expression of bidirectionally transcribed endogenous retroviruses (ERVs) is a major component of

the dsRNA that activates the response. Our work shows that treatment with the DNMTi 5-azacytidine

(Aza) sensitizes mouse melanoma cells to subsequent anti-CTLA4 therapy, likely through activation

of the interferon response and subsequent signaling to host immune cells. Our current work aims to

verify this hypothesis. In addition, we observe that adding histone deacetylase inhibitors (HDACis)

to DNMTis can augment the upregulation of specific ERVs and the resulting downstream interferon

response in human cancer cell lines. Specifically, the ERV-K family as well as the Fc2 and ERV-9

families are increased by DNMTi treatment but further augmented by HDACi treatment, while

HDACis alone have minimal effects on the ERVs and the downstream interferon response. We tested

the hypothesis that epigenetic activation of ERVs sensitize tumors to immune therapy by recruiting

host immune cells in an immunocompetent mouse model of serous ovarian cancer. Treatment of this

model with DNMTi and HDACi results in a Type I Interferon response and increased recruitment of

(CD3+) T cells, including tumor-killing T Effector cells, to the tumor. This epigenetic therapy causes

increased activation of CD8 T cells and natural killer cells, an increase in helper T cells, and a

reduction in myeloid derived suppressor cells and macrophages. Both the immune cell activation and

the tumor burden decrease by DNMTi are dependent on activation of the Type I Interferon response

and associated with an increase in transcription of mouse ERVs. Treatment of this mouse model with

the above drug combination plus an antibody to the immune checkpoint PD-1 significantly reduces

tumor burden and increases survival. We thus define a major mechanism for how DNMTis and

HDACis may induce cancer cells to increase attraction and activation of immune cells and sensitize

patients to immunotherapy.

8

Therapeutic and Prophylactic Applications of Bacteriophages in Cancer Therapy

Biswajit Biswas, Matthew Henry, Javier Quinones, David Wolfe, and Theron Hamilton Naval Medical Research Center

Recent global surveillance indicates that bacterial pathogens are gaining Antibiotics resistances at

an alarming rate. This raises a concern about the potential of saving cancer patients from various

multidrug resistant (MDR) bacterial infections due to their reduced immune status during and after

cancer therapy. Recently, scientists have been evaluating the possibility of using bacteriophages

(phages) as therapeutic and vaccine delivery agents, but Phage therapy is also being considered

for treating cancer patients if they are infected with MDR bacteria during or after cancer therapy.

Additionally, phage can be used to develop vaccines due to the inherent properties of phages as

potent immune-stimulators. Currently, we are modifying phage heads in order to produce complex

nanoparticles which can provoke the immune system in different ways depending on the nature of

the display antigens. Importantly, these phage-based nanoparticles are non-pathogenic to humans

and the resultant vaccines do not require additional adjuvants. Phage based vaccines (PBV) can

deliver all relevant information to the immune system in order to provoke a rapid response. It is

the particulate nature of PBVs that attracts the antigen presenting cells (APC) that engulf, process,

and present phage mediated antigens through MHCs (class I and Class II pathways) and evoke

both cell mediated and humoral immunities. In addition, the immunostimulatory unmethylated

CpG motifs of the phage genome are recognized by the innate immune cells through Toll-like

receptors (TLR), which further enhance immunity. Finally, isolation of natural phages from

environmental sources is rapid, facile, and inexpensive. Therefore, developing targeted phage

cocktails for MDR-infected cancer patients is rapid. These facts encouraged us to consider phage

as prophylactic and therapeutic measures against cancer and some complications associated with

its treatment. As a preliminary attempt we have designed, developed, and produced a nanoparticle­

based anticancer vaccine targeting human aspartyl (asparaginyl) b-hydroxylase (HAAH). This and

several other phage based therapeutics and prophylactic experimental results related to these

applications are presented here.

9

Cytomegalovirus-based vaccines in breast and melanoma mouse tumor models

Ann B. Hill, Michael W. Munks, Guangxu Xu, Tameka Smith, Conrad Barry, Xiaoyan Wang, Savannah Smart, Shawn Jensen, Bernard Fox, Christopher Snyder and Rosalie Sears.

Oregon Health and Science University, Portland, OR, Thomas Jefferson University, Pennsylvania, PA, and Earle A Chiles Cancer Research Center, Portland, OR.

The common herpesvirus, cytomegalovirus (CMV), is of interest as a vaccine vector because it

elicits very large, sustained T cells and antibody responses, and can be used as a vector even in

CMV seropositive individuals. In different mouse cancer models CMV vaccines have shown

therapeutic efficacy by eliciting tumor-specific CD8 T cell or antibody responses, or by an

antigen-independent impact on the tumor microenvironment when injected intratumorally. We

generated a murine (m)CMV based vaccine expressing rat HER-2/neu (CMV-neu). The vaccine

was effective prophylactically and therapeutically in the transplanted TUBO mammary tumor

model in BALB/c mice. We next tested it in a genetically engineered mouse model of mammary

cancer, in which beta lactoglobin-Cre-driven expression of neuNT and c-myc is induced in

mammary epithelium by lactation. Blg-cre/neuNT/myc mice develop mostly single mammary

tumors within several months of lactation; the tumors phenocopy human HER2 tumors, and

about 50% metastasize. A single i.p. injection of CMV-neu caused complete regression of the

majority of palpable tumors in Blg-cre/neuNT/myc mice; the CMV vector alone had no effect.

The tumors begin to regress around five days after vaccination. Immune depletion studies are

underway, but preliminary results suggest that antibody and CD4 T cells are needed for vaccine

efficacy.

0

Bacterial and eukaryotes based cancer therapy

10

Direct detection of genotoxic or pro-inflammatory bacterial genes in stool

samples: implications for colorectal cancer and inflammatory diseases

Abel Baerga-Ortiz

University of Puerto Rico Medical School

The risk of developing colorectal cancer (CRC) and inflammatory bowel diseases (IBDs) is

dependent upon a number of factors which include the gut microbiota. In recent year, advances in

automated and parallel DNA sequencing technologies have enabled a more complete

understanding of how the gut microbiota modulates the risk for intestinal disorders, resulting in

the identification of bacterial species and bacterial genes whose presence correlates with disease

state. Our group has developed a quick PCR test for the detection of some of these pro-

inflammatory or genotoxic bacterial genes directly in a stool sample. Interestingly, some of the

genes in this panel can be detected in a large percentage (>20%) of the population experimentally,

yet they only appear in a small set (1%-2%) of patient-derived sequences in the shotgun

metagenomic databases such as the Human Microbiome Project (HMP) or the European

Nucleotide Archive. Despite their low abundance in the metagenomic databases, these bacterial

pro-inflammatory and gentoxic have been shown to correlate with CRC in a case-control study

that will also be presented

11

Modulation of the Tumor Microenvironment using Synthetic BioticsTM

Adam Fisher

Synlogictx

The immunosuppressive milieu found within the tumor microenvironment (TME) has long been

understood to be a key driver of tumor initiation and progression. More recently it has been

appreciated that metabolites derived from the tryptophan and ATP/adenosine pathways are major

drivers in forming this immunosuppressive environment within the tumor. We are using

synthetic biology in combination with natural probiotics to develop engineered bacteria or

“Synthetic BioticsTM”, which are programmed with precision to correct disease-causing

metabolic dysregulation. Here we present the development of two engineered bacterial strains

that have been designed to consume either kynurenine or adenosine, two molecules known to

play central roles in promoting tumor immune tolerance. In in vitro biochemical assays, the

adenosine-consuming strain or the kynurenine-consuming strain were able to consume 180 and

80 mM adenosine or kynurenine, respectively, within 2 hours. These levels of adenosine and

kynurenine are ~100-fold and 20-fold higher respectively than found in cancer patient tumors.

For the kynurenine-consuming strain, this in vitro kynurenine consumption translated to in vivo

activity where in tumor-bearing mice, the administration of this strain led to significant decreases

in tumor kynurenine levels. Taken together these results demonstrate that engineered bacteria

can be designed to modulate the tumor microenvironmen

12

Gut microbiome analysis reveals major dysbiosis in Sickle Cell Disease patients with a prevalence of Veillonella strains

Hassan Brim, Kimberly Vilmenay, Nazli Atefi, Mohamad Daremipouran, Edward L. Lee, Sudhir

Varma, Patricia O'Neal, Hassan Ashktorab

Howard University Pathology Department & College of Medicine, Washington D.C.

Background: Sickle cell disease (SCD) is an inherited blood disorder that occurs primarily in

patients of African descent and generally associates with frequent pain crises. It has been

suggested that the gut microbiome structure and function may have a major impact on host health.

Here we used high throughput sequencing technologies to explore the gut microbiome and

specifics in SCD patients.

Aim: To characterize the gut microbiome in patients with sickle cell disease.

Materials & Methods: Stool samples from 14 controls and 14 SCD patients were used for DNA

extraction. Among the SCD patients, 7 had mild pain crises (< 3 hospitalizations/year) while 7 had

severe pain crises (≥ 3 hospitalizations/year). The 16S rRNA gene V4 variable region was PCR

amplified, purified using calibrated Ampure XP beads and used to prepare illumina DNA library.

Sequencing was performed on a MiSeq following the manufacturer’s guidelines. Sequences were

joined, and depleted of barcodes. Sequences less than 150 bp , or ambiguous base calls were then

removed. OTUs clustering was performed after the sequences were denoised, and chimeras

removed. Operational taxonomic units (OTUs) were defined by clustering at 3% divergence (97%

similarity). The final OTUs were taxonomically classified using BLASTn against a curated

database derived from RDPII and NCBI. A LeFSe analysis was used to determine differential

bacteria.

Results: A major dysbiosis was noticed in the SCD gut microbiome. Several bacterial groups have

been depleted from the SCD patients when compared with controls, including Clostridiales, a

protector against pathogens. The SCD gut microbiome has been defined by the prevalence of

Bifidobacteria, Campylobacter, Veillonella, Actinomyces, Scardovia and Atopobium. The analysis

among the two SCD groups revealed a much higher microbiota symbiosis within the SCD patients

with severe pain crises with a higher prevalence of Campylobacter strains in this group.

Conclusion: We report a major dysbiosis in SCD patients’ gut microbiota. This symbiosis is more

pronounced in patients with severe pain crises that displayed a higher prevalence of

Campylobacter. Veillonella, a normal oral and colon inhabitant, is known for its ability to form

biofilms and as a facilitator of Streptococcus strains pathogenesis. Its high prevalence in SCD

patients might exacerbate pain crises primarily due to blood vessels occlusion as a consequence of

sickle shaped blood cells. Indeed, Veillonella biofilms might block blood vessels as well and

increase Streptococcus strains virulence.

13

Reenergized Adoptive Cell Transfer (ReACT)- A Multi-pronged Strategy to Treat Solid

Tumors

Weiguo Cui PhD

Blood Research Institute, Blood Center of Wisconsin, 8727 West Watertown Plank Road, Milwaukee, WI 53213

Adoptive cell transfer (ACT) of genetically engineered T cells provides unprecedented

opportunities to treat hematological malignancies. However, due to insufficient migration and

antitumor function of transferred T cells, especially inside the immunosuppressive tumor

microenvironment (TME), the efficacy of ACT is much curtailed in treating solid tumors.

Pathogen-based cancer vaccines can break immunosuppression in TME, but are less efficient at

mobilizing tumor specific T cell response with ample magnitude to eradicate established tumors.

To overcome these challenges, we sought to combine the strengths of ACT and pathogen-based

cancer vaccines with a new strategy named Reenergized ACT (ReACT). To bridge ACT with a

pathogen, we genetically engineered tumor-specific CD8 T cells in vitro with a second T cell

receptor (TCR) that recognizes a bacterial antigen. We then transfer these dual-specific T cells in

combination with intratumoral bacteria injection to treat solid tumors in mice. The dual-specific

CD8 T cells expand robustly in response to bacteria, migrate to the very site of tumor, and confer

tumor eradication in the majority of tumor-bearing mice. ReACT demonstrates greater efficacy in

tumor control than either ACT or pathogen-based vaccine alone. The mice cured from ReACT

also develop immunological memory against subsequent tumor rechallenge. Mechanistically, we

have found that this combined approach reverts the immunosuppressive TME and recruits CD8 T

cells with enhanced killing ability to the tumors. Overall, we provide the proof-of-principle of a

promising strategy to treat various malignancies.

14

Cancer therapy in a microbial bottle: Uncorking the remarkable anti-cancer biology of

Toxoplasma gondii

Barbara A. Fox, Kiah L. Butler, Rebekah B. Guevara, and David J. Bzik

Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth,

Lebanon, New Hampshire, 03756, United States of America

Cancers emerge after the immune system fails to control and contain tumors. Multiple tumor-

specific mechanisms create tumor environments where the immune system is forced to tolerate

tumors and their cells instead of eliminating them. The goal of cancer immunotherapy is to

rescue the immune systems natural ability to eliminate tumors. Acute inflammation associated

with microbial infection has been observationally linked with the spontaneous elimination of

tumors for centuries. Here, we uncork the unique biology of the bottle-shaped protozoan parasite

Toxoplasma gondii and describe how this microbe when engineered into a safe nonreplicating

vaccine unleashes mechanisms that effectively breaks tumor control over the immune system to

stimulate potent immunity against already established cancer thereby promoting survival and

preventing cancer recurrence.

15

Developing a photosynthetic bacterial vector for intratumorial photodynamic therapy

Jill Zeilstra-Ryalls

Bowling Green State University

Many bacterial species injected into the bloodstream of mice or even administered orally

preferentially accumulate in tumors and propagate in the immunoprotective tumor

environment. In murine models, intratumorally grown bacteria inhibit tumor growth and in some

cases eradicate tumors. However, limited efficacy and concerns over safety of introducing live

bacteria into cancer patients have hindered translational applications of bacterial anticancer

therapies; at the same time, the hypoxic environment of the tumor restricts the choice of bacteria

to those species that do not require oxygen for energy production. We are investigating the

potential to use highly penetrant (but safe) near-infrared light to support intratumoral anaerobic­

phototrophic growth of the nonpathogenic bacterium Rhodobacter sphaeroides. The bacteria have

been engineered to overproduce the pro-drug 5-aminolevulinic acid, which is widely used in

photodynamic therapy, and so will serve as a vehicle for targeted therapy.

16

Use of a gnotobiotic mouse model to characterize bacteria/colorectal

tumor interactions

Alexandra Proctor and Gregory J. Phillips

Department of Veterinary Microbiology, Iowa State University, Ames, IA 50010

Individuals with inflammatory bowel disease (IBD) are at a greater risk of developing colorectal

cancer (CRC). It is now evident that both IBD and CRC are associated with dysbiosis of the

gastrointestinal (GI) microbiota. Use of rodent models to study these links are complicated by the

complexity of the mammalian GI tract, however, as mice harbor hundreds of species of bacteria,

many of which are poorly characterized. Consequently, we have used altered Schaedler flora

(ASF) mice, which are colonized with only 8 bacterial species, in combination with a model of

chemically induced inflammation and mutagenesis, to better understand how members of the GI

microbiota interact with the host. Use of RNA-seq and RNA-scope technologies reveal new details

of how the microbiota responds to GI inflammation and cancer development and to identify

specific bacterial species that interact with diseased tissue. These results have implications for

developing new strategies for microbial-based CRC therapy.

17

TRAIL-armed Self-destructing Salmonella serve as “Time-Bombs” to Combat Cancer

Wei Kong Biodesign Center for Immunotherapy, Vaccines, and Virotherapy. Tempe, AZ 85287-7501

Cancer is one of the leading causes of death worldwide. In 2016, an estimated 1,685,210 new cases

of cancer will be diagnosed in the United States and 595,690 people will die from the disease due

to lack of efficient therapeutic method (NCI statistics). Bacterial cancer therapy was invented more

than 100 years ago. It is known that many passive therapeutics induce variety of side effects, but

bacteria offer unique features that can avoid these limitations. Salmonella Typhimurium (STM) is

one of the most promising bacteria using in cancer therapy. In this study, we constructed self-

destructing S. Typhimurium stains with tumor navigating feature to express TNF-related

apoptosis-inducing ligand (TRAIL) and release them inside of tumor by programmed Salmonella

cell lysis. These genetically engineered STM stains (GMS) could dramatically induce variety types

of cancer cell death in vitro. In addition, GMS stains, administrated by intra-tumor injection or

orally, induced significant tumor regression and extend survival rate of tumor bearing mice in

multiple colon cancer mouse models. Our results indicate that these TRAIL-armed Self-

destructing Salmonella can serve as an alternative novel approach for cancer therapy.

18

Presence of Multiple Responding Mechanisms in Intestinal Cancer Tissues Mediated by Microbiome Revealed by Secondary Analysis of Existing Gene Expression Data

Bi Zhao and Bin Xue

The Department of Cell Biology, Microbiology and Molecular Biology, College of Arts and Sciences, University of South Florida, Tampa, FL. USA 33620

Gene expression analysis provides valuable information on the molecular mechanisms through

which the samples undergo genetic and phenotypic changes as a respond to various internal and

external factors. By using multiple sets of existing gene expression data of intestinal cancer tissues

mediated by microbiome, differentially expressed genes (DEGs) were analyzed to: (1) characterize

the heterogeneity of tissue samples in the same control or treatment group; (2) evaluate the

influence of heterogeneity on the identification of DEGs; (3) determine different responses of

samples in the same treatment group under the same treatment; and (4) discover various

mechanisms associated with different responses. It has been found that the heterogeneity of

samples is a critical factor for tissue-level gene expression analysis.

19

Loss of Cancer Immune Privilege in Bacterial-based Therapy

*Qiuhong He, Vitaly Chibisov, Gabrielle Ramus, Huiwen Liu, Kaung-Ti

Yung, Peter Wu, Weike Lai, Xiang-jin Song, and Han H. Liu

Departments of Radiology and Bioengineering, University of Pittsburgh Cancer

Institute, University of Pittsburgh, 200 Lothrop Street, Pittsburgh, PA 15213.

We have genetically engineered the Attenuated Salmonella Typhimurium VNP20009

strain to express anti-cancer proteins TNFα and/or methioninase (METase), with or without an

imaging marker myoglobin (Mb). The therapeutic VNP20009-METase-TNFα -Mb, VNP20009­

METase, VNP20009-TNFα, and the control VNP20009 and VNP20009-Mb strains were

administered i.t. or i.v. into the immunocompetent C57BL/6 mice carrying Lewis lung carcinoma

(LLC). The Salmonella Typhimurium strains carrying anti-cancer proteins have selectively

amplified in the tumor tissues, including the secondary tumor site. Rapid tumor tissue destruction

was observed by non-invasive MRI and histochemical tissue staining, accompanied with

neovascular damage and tumor cell apoptosis. Large number of macrophages was observed in

the treated LLC tumors by immunochemical staining of the tumor tissues. For tumors treated

with the control VNP20009 or VNP20009-Mb strains, the confocal microscopic images displayed

macrophage engulfment of the bacteria in the tumor tissues. This explained why the control

bacteria did not amplify in the tumor tissues and consequently caused no tumor tissue damage.

In contrast, macrophage no longer engulfed the Salmonella strains in the tumors treated with

the therapeutic VNP20009-METase-TNFα-Mb strains. The anti-cancer proteins methioninase

and/or TNFα enabled the VNP20009 strains to overcome the host immune barrier in C57BL/6J

mice for selective tumor infiltration. In addition, the LLC tumors lost immune privilege and got

destroyed upon amplification of the therapeutic VNP20009-METase-TNFα- Mb, VNP20009­

METase, VNP20009-TNFα strains in the tumor tissues. The macrophages may have elicited both

innate and adaptive immune responses for tumor destruction and bacterial clearance,

respectively, in the therapeutic and control cases. Detailed molecular mechanisms merit further

investigation to induce similar anticancer effect in human cancer treatment.

20

An Archaeal Therapeutic Drug and Antigen Delivery System Employing Buoyant,

Bioengineerable Proteinaceous Nanoparticles

Shiladitya DasSarma and Priya DasSarma

University of Maryland School of Medicine, Department of Microbiology and Immunology, Institute of Marine and Environmental Technology, Baltimore, Maryland 21202

Some aquatic microbes produce large quantities of buoyant nanoparticles called gas vesicles

(or GVNPs) to promote flotation and photosynthesis. These hollow, gas-filled organelles are

bounded by an extremely stable yet biocompatible protein membrane. In the salt-loving

Archaea Halobacterium sp. NRC-1, GVNPs have been shown to be genetically engineerable

and capable of displaying foreign proteins (1). These nanoparticles are also easily purified after

hypotonic cell lysis and may serve as an ideal vehicle for therapeutic drug and antigen delivery

due to their non-toxicity and immunogenicity (1,2). Genetic analysis showed that GVNPs are

encoded by a gene cluster on Halobacterium megaplasmids, with gvpA and gvpC encoding the

major protein components. GvpC protein is bound to the exterior surface of nanoparticles and

promotes their growth and stability. We have developed a genetic system for bioengineering

the nanoparticles by insertion of foreign sequences near the C-terminal coding region of gvpC.

This expression system has permitted the display of diverse peptides and proteins on GVNPs,

including the easily detectable Gaussia princeps luciferase reporter (3). In another recent study,

GVNP display and delivery of bactericidal permeability-increasing protein (BPI) resulted in

protection of mice in a sepsis model (4). Other proteins successfully displayed by GVNPs

include antigens from the simian immunodeficiency virus (SIV), the facultative intracellular

pathogen Salmonella enterica, and parasitic protozoa Plasmodium species (1). All together, our

results have shown that the Halobacterium GVNP display and delivery system represents a

unique platform for the development of potential diagnostic assays and therapeutic

interventions.

1. DasSarma, S., and DasSarma, P. 2015. Gas Vesicle Nanoparticles for Antigen Display.

Vaccines 3:686-702.

2. Andar, A.U., Karan, R., Pecher, W.T., DasSarma, P., Hedrich, W.D., Stinchcomb, A.L.,

and DasSarma, S. 2017. Microneedle-Assisted Skin Permeation by Nontoxic Bioengineerable

Gas Vesicle Nanoparticles. Mol. Pharm. 14:953-958.

3. DasSarma, S., Karan, R., DasSarma, P., Barnes, S., Ekulona, F., and Smith, B. 2013. An

improved genetic system for bioengineering buoyant gas vesicle nanoparticles from

Haloarchaea. BMC Biotechnol. 13:112.

4. Balakrishnan, A., DasSarma, P., Bhattacharjee, O., Kim, J.M., DasSarma, S., and

Chakravortty, D. 2016. Halobacterial nano vesicles displaying murine bactericidal

permeability-increasing protein rescue mice from lethal endotoxic shock. Scientific Rep.

6:33679.

21

Bacterial Secreted Polysaccharide Toxins Bind to HP59 (Sialin) on Tumor Capillary Endothelium, Activate Complement and Recruit CD69+ Granulocytes, Disrupt Capillaries.

Roger A Laine, Department of Biological Sciences

Louisiana State University and A&M College, Baton Rouge, LA 70803. TumorEnd, LLC, Louisiana

In 1867, Busch in Germany1 observed that a sarcoma patient who developed nosocomial erysipelas

had spontaneous tumor regression. He followed up by wounding sarcoma patients, putting them in

the same hospital bed as the erysipelas patient and observed further sarcoma patients to have tumor

regression if they contracted erysipelas. Fehleisen, in 18812, identified the organism that caused

erysipelas as Streptococcus erysipelas, now called Streptococcus pyogenes, and, following Busch’s

work, injected bacterial cultures into sarcoma patients to initiate erysipelas, and infected patients had

tumor regression. Independently, William B Coley, MD, at Cornell, in 18913 discovered in hospital

records an erysipelas infected sarcoma patient who had spontaneous tumor regression, and followed

up by injecting S. erysipelas into sarcoma patients, with tumor regression in successfully infected

patients. Hemorrhage in the tumor was universal, followed by inflammation and regression. Coley

tried heat-killed bacterial cultures which had the same effect. No other organ or tissue was affected

except the tumor, and the tumors were affected whether the heat-killed cultures were injected directly

into the tumor or at distant sites. Coley began using mixed cultures of S. erysipelas and S. marscesens,

heat killed, to treat his patients. This combination became known as “Coley’s Toxin”, and was used

by a number of other physicians to significant success against sarcomas and other tumors. Beebe and

Tracy4, in New York, used Coleys toxin to treat transplantable sarcomas in dogs, also experimenting

with other bacterial extracts, and found universal tumor regression in the majority of dogs.

Hellerqvist, et al. in the 1990’s5 independently found a Group B Streptococcus (agalactica)

polysaccharide exotoxin that was associated with destruction of neocapillaries in infant human lungs

“Early Onset Disease”. Hellerqvist’s group found that tumor also attract hypoxically driven

neovasculature susceptible to GBS Toxin, which they called CM101. They showed effective tumor

therapy with CM101 in preclinical models.6 A published Phase I clinical safety trial, under IND 4578

showed 33% effectivity in Stage 4 cancer patients, including one cure7 . The receptor was later

identified by Fu, et al.8 as Sialin SLC17A5. CM101 showed safety at 10X the effective dose in a

Phase I trial in dogs, and canine cancer patients are being recruited for a Phase II trial.

1(Busch W. Aus der Sitzung der medicinischen Section vom 13 November 1867. Berl Klin Wochenschr. 1868;5:137). 2(Fehleisen F. Ueber die Zuchtung der Erysipelkokken auf kunstlichem Nahrboden und ihre Ubertragbarkeit auf den Menschen. Dtsch Med Wochenschr. 1882;8:553–554. doi: 10.1055/s¬0029¬1196806.) 3(Coley, WB, Ann. of Surg. St. Louis, 1891, xiv, p. 199; , Coley, WB, Amer. Journ. Med. Sci., Philad.1893, cv, p. 487.) 4Beebe SP, Tracy M, 1907, “The Treatment of Experimental Tumors with Bacterial Toxins”, J. Am. Med. Assoc. 18: 1493 -1498 5Sundell HW, Yan H, Carter CE, Wamil BD, Wu K, Gaddipati R, Li D, Hellerqvist CG (2000). "Isolation and identification of group B ß-hemolytic streptococcal (GBS) toxin from septic newborn infants". The Journal of Pediatrics. 137: 338–344. doi:10.1067/mpd.2000.107839. PMID 10969257 6(Thurman, GB; Russell, BA; York, GE; Wang, Y-F; Page, DL; Sundell, HW; Hellerqvist, CG (1994). "Effects of GBS toxin on long-term survival of mice bearing transplanted Madison lung tumors". J. Can. Res. Clin. Oncol. 120 (8): 479–484. doi:10.1007/BF01191801). 7(DeVore RF, Hellerqvist CG, Wakefield GB, Wamil BD, Thurman GB, Minton PA, Sundell HW, Yan HP, Carter CE, Wang YF, York GE, Zhang MH, Johnson DH (1997). "A phase I study of the antineovascularization drug CM101". J. Clin. Can. Res. 3 (3): 365–372. PMID 9815693..

22

Tumor targeting by Fusobacterium nucleatum

Jawad Abed1, Naseem Maalouf1, Lishay Parhi1, Ofer Mandelboim3 and Gilad Bachrach1

1The Institute of Dental Sciences, The Hebrew University-Hadassah School of Dental

Medicine, Jerusalem, 91120, Israel

3The Lautenberg Center of General and Tumor Immunology, The Hebrew University

Hadassah Medical School, IMRIC Jerusalem, Israel.

Colorectal adenocarcinoma (CRC) is a common tumor with high mortality rates. Recently, CRC

was found to be colonized by the oral anaerobic bacteria Fusobacterium nucleatum. F.

nucleatum accelerates tumor progression and protects the tumor from immune cells. CRC-

specific colonization by fusobacteria is mediated through the recognition of tumor displayed

Gal-GalNAc moieties by the fusobacterial Fap2 Gal-GalNAc lectin. Here, we show high Gal-

GalNAc levels in additional adenocarcinomas including those found in the stomach, prostate,

ovary, colon, uterus, pancreas, breast, lung and esophagus. This observation coincides with

recent reports that found fusobacterial DNA in some of these tumors. Given the tumorigenic

role of fusobacteria and its immune evasion properties, we suggest that fusobacterial

elimination might improve treatment outcome of the above tumors. Furthermore, if F.

nucleatum specifically homes-in to Gal-GalNAc – displaying tumors, it might be engineered as

a platform for treating CRC and the above common, lethal, adenocarcinomas.

23

Optogenetic and chemogenetic platforms for listeria-mediated intratumoral drug

delivery

R. Abrar1, C. Gravekamp2 and M. Gomelsky1

1Department of Molecular Biology, University of Wyoming, Laramie, WY

2Department of Microbiology, Albert Einstein College of Medicine, Bronx, NY

Attenuated strains of Listeria monocytogenes (Lm) accumulate and propagate in primary tumors

and metastases while being quickly cleared from healthy tissues. These strains expressing

specific tumor antigens are currently in advanced clinical trials as anticancer vaccines; however,

their curative power remains limited. We hypothesize that efficacy of the Lm-vaccines can be

significantly increased by expanding the potential of Lm as a tumor-specific payload delivery

vehicle. The payload may involve activators of innate immunity as well as tumor toxins. Since

uncontrolled production of these agents in healthy tissues infected with Lm would be

deleterious, expression needs to be inducible. We will present our efforts on engineering protein

and gene (bactofection) delivery platforms that are regulated by light or benign chemicals. Light

within the near-infrared therapeutic window penetrates deeply through mammalian tissues and

is harmless. Photoreceptors of the bacteriophytochrome class that respond to such light have

been engineered. One of the systems to be discussed involves inducible intratumoral synthesis

of c-di-GMP, the bacterial cyclic dinucleotide activator of the STING innate immunity pathway,

which results in the production of type 1 interferons. Injections of high doses of c-di-GMP in

tumor-carrying mice have been shown to induce immunogenic tumor cell death resulting in

release of tumor-associated antigens, while low c-di-GMP doses activate tumor antigen-specific

T cells. We intend to replace systemic c-di-GMP injections with localized, intratumoral c-di-

GMP production via chemically and light-controlled systems.

24

Vγ9Vδ2 T cells dominate the response to Listeria monocytogenes-based vaccines in

patients

Alejandro F. Alice1, Gwen Kramer1, Yoshinobu Koguchi1, Chris Fountain1, Rom Leidner1, Michael J. Gough1, Keith Bahjat2, and Marka R. Crittenden1,3.

1 Earle A. Chiles Research Institute, Robert W. Franz Cancer Center, Providence Portland Medical Center, 4805 NE Glisan St, Portland, OR, 97213, USA.

2 Bristol-Myers Squibb, Biologics Discovery California, Redwood City, CA, 94063, USA. 3 The Oregon Clinic, Portland, OR, 97213, USA Correspondence: alejandro.alice@providence.org

Vector-neutralizing immunity represents a major impediment to the induction of cellular

immune responses using infectious agents as vaccine platforms. An ongoing trial

(NCT01967758) of a novel recombinant Listeria monocytogenes (Lm) –based cancer vaccine

at our institution has yielded poor tumor antigen-specific T cell immune responses. This result

is consistent with other clinical trials with Lm-based vaccines. Importantly, we observed the

marked expansion of VȖ9Vβ2 T cells in every subject (3-10 fold, 10/10 subjects). This finding

is in direct contrast to murine models, where Lm-based vaccines generate a strong αȕ T cell

response to both endogenous Lm antigens and the tumor antigen. VȖ9Vβ2 T cells recognize (E) ­

4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), an intermediate of the non­

mevalonate pathway of isoprenoid biosynthesis. Recognition of HMBPP complexed with

butyrophilin 3A1 (BTN3A1) is a property of human and non-human primate VȖ9Vβ2 T cells

that is not shared with rodent species. Therefore, traditional murine models do not take into

account the effects that these VȖ9Vβ2 vector-specific cytolytic effector cells have during Lm

immunization in humans. Following clearance of the vaccine (>5 days), VȖ9Vβ2 T cells

persisted in a constitutively activated effector-memory state (30+ days). Therefore, at the time

of secondary immunization, ~25% of the peripheral T cell repertoire is comprised of activated

Lm-specific cytolytic effector T cells. We propose that VȖ9Vβ2 T cells facilitate the rapid

clearance of Lm during secondary immunization and repeated boosts, minimizing the quantity

and quality of antigen presentation to αȕ T cells. We have designed a novel HMBPP-deficient

Lm strain that we hypothesize does not activate the dominant human Ȗβ T cell response and

may be a superior vaccine platform to generate αȕ T cell responses for human immunotherapy

approaches. These results have implications for human application of alternative Eubacteria

platforms, which similarly use the non-mevalonate isoprenoid synthesis pathway

.

25

Regulated invasion, chemotaxis, attenuation and endotoxicity of Salmonella to eliminate

tumors in tumor-bearing syngeneic mice

Shifeng Wang

University of Florida

Salmonella is a facultative anaerobe that thrives in solid tumors. We have therefore endeavored

to modify Salmonella to enhance this activity while retaining safety for healthy tissues. We first

genetically manipulated S. Typhimurium to exhibit regulated delayed in vivo attenuation and

lysis in vivo, to constitutively express invasiveness, display chemotaxis to aspartate and serine

that are produced by tumor cells and deliver TRAIL. Each of these mutational alterations

enhanced S. Typhimurium destruction of subcutaneous breast cancer tumors in syngeneic

BALB/c mice. In other studies, it was demonstrated that mutations that caused synthesis of a

truncated LPS core made Salmonella hyper invasive to colorectal tumor cells but very

susceptible to being killed by macrophages. Such mutations also caused enhanced susceptibility

of the Salmonella to being killed due to complement-mediated cytotoxicity. However, this

sensitivity to killing by host defense mechanisms also reduces the ability of the Salmonella cells

to reach and invade tumor cells. We therefore used a regulated delayed cessation in synthesis

of the LPS core by fusing the gene imparting the defect in LPS core synthesis to the arabinose­

activated araC PBAD cassette. Growth of the strain in the presence of arabinose results in

complete LPS synthesis and ability after inoculation into the tumor-bearing mouse to efficiently

gain access to the tumor. LPS core synthesis ceases in vivo and the LPS Outer core and O-

antigen decreases in amount at each in vivo cell division since arabinose is not present in animal

tissues. During the course of these studies we learned that the virulence attributes of the

Salmonella parental strain very much enhanced efficacy in destroying tumors. It was also

demonstrated that inclusion of an aroA mutation also enhanced tumor cell killing. We believe

that liberation of peptidoglycan constituents during Salmonella cell lysis and which are also

elevated in aroA mutants contributes to inflammasomes to augment tumor cell killing. We are

now constructing new Salmonella combining these and additional traits to enhance tumor cell

killing and inability to be harmful for normal tissues external to the tumor

26

Contribution of Salmonella enterica serovar Typhimurium VNP20009 Chemotaxis on 4T1

Mouse Mammary Carcinoma Progression

Katherine M. Broadway, Sheryl L. Coutermarsh-Ott, Seungbeum Suh, Bahareh Behkam, Irving C. Allen, Birgit E. Scharf

Virginia Tech, 970 Washington St. SW Blacksburg, VA 24060

Attenuated bacterial strains have been investigated on the premise of selective tumor colonization

and drug delivery potential for decades. Salmonella Typhimurium VNP20009 was derived from

the parental strain 14028 through genetic modification and tumor targeting ability, being well

studied for anticancer effects in mice. In 2001 Phase 1 Clinical Trials, patients diagnosed with

melanoma were introduced with VNP20009, resulting in safe delivery of the strain and targeting

to the tumor, however no anticancer effects were observed. Recently, it was discovered that

VNP20009 contains a SNP in cheY, which encodes the chemotaxis response regulator of flagellar

motor function, rendering the strain deficient in chemotaxis. Replacement of cheY with the 14028

wild-type copy resulted in a 70% restoration of phenotype in traditional capillary assays compared

to the parental strain. We attempted to optimize the chemotactic potential of VNP20009 but were

unable without reversing the attenuated state of VNP20009.

Due to the role of chemotaxis in bacterial tumor colonization and eradication remaining unclear,

we aimed to compare VNP20009 and VNP20009 cheY+ primary tumor colonization and impact

on metastasis in an aggressive 4T1 mouse mammary carcinoma model. Based on our pilot study,

the presence of VNP20009 cheY+ significantly increased the number of metastatic foci in the lungs

and cytokine expression of cancer cells derived from the primary tumor, as compared to

VNP20009. To confirm this preliminary finding a full study was implemented, where both

intravenous and direct tumor injection routes were tested. In contrast to our pilot study, tumor

colonization and metastatic potential caused by the bacteria appear chemotaxis independent.

Moreover, mice bearing tumors exposed to Salmonella exhibited increased morbidity that was

associated with significant liver disease. Our results suggest that VNP20009 may not be safe or

efficacious when used in the context of immunocompetent animals with aggressive, metastatic

breast cancer.

27

Nanoscale Bacteria-Enabled Autonomous Drug Delivery Systems (NanoBEADS) for

Cancer Therapy

Bahareh Behkam, Associate Professor of Health Sciences Department of Mechanical

Engineering

School of Biomedical Engineering and Sciences Virginia Tech

Systemic chemotherapy is a major therapeutic approach for nearly all types and stages of cancer.

Despite significant recent progress in nanomedicine, drug delivery to solid tumors remains a

formidable challenge often associated with limited penetration of the drug in poorly vascularized

regions. In contrast, attenuated strains of tumor-targeting bacteria have been demonstrated to

have exceptionally high selectivity to cancerous tissue, a good safety profile, and effective

tumor penetration capability. However, therapeutic efficacy in immunocompetent host can be

difficult to achieve. We hypothesize that a combinatorial therapy approach based on

integrating bacteria with chemotherapeutics-loaded nanoparticles will amplify the therapeutic

potential of both modalities. Thus, we have developed a Nanoscale Bacteria-Enabled Drug

Delivery System (NanoBEADS) in which the functional capabilities of the engineered tumor-

targeting bacteria are interfaced with and augmented by chemotherapeutic-loaded

nanoparticles. We report (1) our in vitro results elucidating the mechanism of bacteria and

NanoBEADS intratumoral transport and (2) our in vivo results in mouse 4T1 breast tumor model

characterizing the intratumoral transport efficacy of NanoBEADS compared to passively

diffusing nanoparticles.

28

Induction of Autophagy and Apoptosis in Triple-Negative Breast Cancer Cells by LT-IIc, a

Type II Heat-labile Enterotoxin

Yasser Heakala, Sofia A Girald Berlingeria, c, John Hub, Matthew Federowicza, Terry D.

Connellb, Patricia Masso-Welchc

aDepartment of Pharmaceutical Sciences, D’Youville College School of Pharmacy, 320 Porter

Avenue, Buffalo, NY 14201. bDepartment of Microbiology and Immunology and the Witebsky

Center for Microbial Pathogenesis and Immunology, Jacobs School of Medicine, The University

at Buffalo, 3435 Main Street, Buffalo, NY 14214 cDepartment of Biotechnical and Clinical

Laboratory Sciences, Jacobs School of Medicine The University at Buffalo, 3435 Main Street,

Buffalo, NY 14214

Despite the recent advances [1]in breast cancer treatment, triple negative breast cancer (TNBC)

remains a serious health problem with poor prognosis and limited therapeutic options. Although

hampered by development of resistance, chemotherapy-based regimens remain the first-line

adjuvant choice for the treatment of TNBC. To combat development of resistance, it is essential

to identify agents that induce cell death by independent induction of multiple cell death pathways.

In this report, we screened cholera toxin (CT), a type I heat-labile enterotoxin (HLT) of Vibrio

cholerae, and LT-IIa and LT-IIb, type II HLT of Escherichia coli, for capacity to induce cell death

specifically in triple-negative MDA-MB-231 breast cancer cells. While CT, LT-IIa and LT-IIb had

little effect on the cells, LT-IIc reduced cell viability by ~50%, concomitant with development of

extensive intracellular vacuolation. LT-IIc-induced cytotoxicity was not observed in MCF10A, a

non-tumorigenic breast epithelial cell line, or in MCF7, an estrogen receptor-positive breast cancer

cell line. LT-IIc independently and simultaneously stimulated autophagy and apoptosis in MDA­

MB-231 cells. Neither knockdown of ATG5, a key mediator of cellular autophagy, nor activation

of caspase 3/7 disrupted LT-IIc-mediated cell death. Similarly, pharmacological disruption of

autophagy using bafilomycin A1, did not interfere with LT-IIc-mediated cell death. In contrast,

treatment of MDA-MB-231 cells with rapamycin, a known inducer of autophagy, enhanced LT-

IIc mediated cell death. Collectively, these findings indicate that LT-IIc uniquely induces cell

death through the divergent induction of autophagy and autophagy-independent apoptotic

pathways.

1. Nascimento, A.V., et al., Overcoming cisplatin resistance in non-small cell lung cancer with Mad2

silencing siRNA delivered systemically using EGFR-targeted chitosan nanoparticles. Acta

Biomater, 2017. 47: p. 71-80.

29

The Streptococcal Adhesin, Scl1, Recognizes Oncofetal Fibronectin

Dudley H. McNitt1, Soo Choi1 , Flavia Squeglia2, Rita Berisio2, Slawomir Lukomski1

1Department of Microbiology, Immunology, and Cell Biology, West Virginia University School of Medicine, Morgantown, WV, USA

2Institute of Biostructures and Bioimaging, National Research Council, Naples, Italy

Group A Streptococcus (GAS) gains access to host tissue by binding cellular fibronectin isoforms

containing extra domain A (EDA/cFn). EDA/cFn is expressed during embryogenesis but not in

normal human tissue, except during wound healing and within the tumor microenvironment. The

pathogen adherence to EDA/cFn is mediated by the surface adhesin, the streptococcal collagen-

like protein 1 (Scl1). Scl1 is a homotrimeric protein that binds EDA via its N-terminal globular

sequence-variable (V) domain. The Scl1-V domain has predicted conserved structure composed

of three pairs of anti-parallel α-helices connected by flexible loops. We hypothesized that the

surface-exposed loops in the Scl1 globular domain mediate EDA recognition and binding. To test

our hypothesis, we designed, cloned, expressed in E. coli, and affinity-purified a series of the

chimeric recombinant Scl constructs by replacing the loop sequences. Circular dichroism and

rotary shadowing analyses demonstrated that chimeric proteins were not altered structurally. We

identified that the C-terminal 11 amino acids of the Scl1-V-loop segment was sufficient for rEDA

binding. Surface potential maps of the homology models of Scl1-V domains revealed conserved

negatively-charged pockets within the V-domain loop segment. Current work is focused on

immobilizing rScl1 to the surface of nanoparticles, and assessing the capacity of these

functionalized nanoparticles to adhere to rEDA-coated surfaces and to cancer-cell deposited

matrices. Our work aims to demonstrate the potential of rScl-functionalized nanoparticles as

vehicles targeting cancer tissue for diagnostic and drug delivery applications.

30

Dietary fatty acids modulate fungal-host interactions

Safanah T. Siddiqui and Melissa C. Garcia-Sherman

Department of Biology, City University of New York Brooklyn College, Brooklyn, NY 11210

Candida albicans is a major species of the fungal component of the human microbiome. It

commonly colonizes the oral, gastrointestinal and genital regions of the human body. C. albicans

becomes pathogenic when the host microbiota and immune defenses are altered causing superficial

and systemic candidiasis. The human diet can modulate immune response, inflammation levels

and the human microbiome. The average American diet is high in omega-6 fatty acids but low in

omega-3 fatty acids. Here we demonstrate that an omega-3 fatty acid, docosahexaenoic acid

(DHA), inhibits switching from yeast to the pathogenic-associated hyphal morphology. Treatment

of C. albicans with DHA mitigates biofilm formation on abiotic surfaces. Furthermore DHA also

prevents binding of C. albicans to human oral epithelial cell monolayers. Our results suggest that

DHA interferes with C. albicans morphogenesis, adhesion to biotic and abiotic surfaces. These

findings serve as a basis for future studies to determine whether DHA moderates pathogenic

interactions between C. albicans and its host.

31

Could Trypanosoma cruzi Infection Be a Good Thing in the Presence of a Tumor?

Arturo Ferreira1, Paula Abello1 , Viviana Ferreira2

1Immunology Program, Faculty of Medicine, University of Chile, Santiago, Chile; 2Department of Medical Microbiology and Immunology, College of Medical and Life

Sciences, The University of Toledo, Ohio, USA

Worldwide, 10 million people are infected with Trypanosoma cruzi, an intracellular

protozoan, but only 30% will present symptoms of Chagas’ disease. Several observations

associate T. cruzi with resistance to solid tumors: i) cancer is rare in endemic areas; ii) infected

rats resist carcinomas; iii) this parasite has tropism for tumors; iv) used as a vector for a testis

tumor antigen, it inhibits tumor growth and; v) rats treated with parasite extracts show CD4+

and CD8+ anti-tumor responses. However, no responsible parasite molecule was identified in

these studies.

We propose T. cruzi calreticulin (TcCalr, previously known as TcCRT), an ER-resident

chaperone, as a mediator of these effects, because: i) infective trypomastigotes translocate

TcCalr from the ER to the exterior, ii) non-infective T. cruzi epimastigotes fail to translocate

TcCalr; iii) T. cruzi carrying one, two or three TcCalr alleles, increasingly: express TcCalr,

resist human complement and display infectivity; iv) recombinant TcCalr (rTcCalr) inhibits:

angiogenesis in vitro, ex vivo and in ovum, proliferation and chemotaxis of human endothelial

cells in vitro; v) in molar terms, TcCalr is more anti-angiogenic than human calreticulin, vi) the

anti-angiogenic TcCalr effect is reversed by anti-rTcCalr antibodies; vii) the in vivo anti-tumor

effects of T. cruzi infection, or parasite extracts, are fully reproduced by exogenously

administrated rTcCalr and, viii). the in vivo anti-tumor effects of the infection are neutralized

by anti - rTcCalr antibodies.

Perhaps, the relevant uniqueness of TcCalr resides in its capacity to bind tumor cells and

in its amino acidic sequence differences (about 50%) from the mammal counterparts. Upon

TcCalr binding to tumor cells, infiltrating dendritic cells may present TcCalr-derived peptides

to T cells in the regional lymph nodes thus forcing an adaptive anti-tumor immune response.

Acknowledgements: A.F. laboratory is supported by grants from the CONICYT­

CHILE/FONDECYT 1130099

32

Novel Treatment of Melanoma: Combined Parasite-Derived Peptide GK-1 and Anti-

Programmed Death Ligand 1 Therapy

Jesus Vera MD.

Hematology and Oncology, Mayo Clinic

background: Several studies in advanced melanoma patients suggest that combining therapies

that target tumor mechanisms of immune evasion with activation of normal immune cell

functionality, may provide optimal benefits for patients. The synthetic parasite derived GK1

peptide in combination with anti-PD-L1 showed significant longer survival (34 days) compared

to GK1 or Anti-PD-L1 alone (23-27 days) in a murine melanoma model (p<0.05). This means

an increase survival increased in 47.82% in the mice treated with GK-1 + anti-PD-L1, 21.7%

in mice treated with GK-1 alone, and 6.08% in those mice treated with anti-PD-L1 only.

Methods: To elucidate the potential mechanism by which this combination treatment exerts its

anti-melanoma effects, C57BL/6 mice were injected with B16-F10-luc2 cells and separated

according to treatments in four groups: control, GK-1, anti-PD-L1 and GK-1/anti-PDL-1.Blood

samples were collected at day 0, 14, and at euthanization or end of the experiment and monitored

for serum cytokines using mice-specific V-PLEX Pro-inflammatory Panel.

Results: On day 14, TNF-α levels in the Anti-PD-L1 and GK-1 therapy group was significantly

lower compared to control mice. At sacrifice, the combined treatment group demonstrated

significant decrease cytokine production in IL-6 and IL-10.

Conclusions: The decreased cytokine levels observed in the GK-1/anti-PD-L1 group may

partially explain the significant improved survival observed in this group. GK-1 alone is a Th1

response inductor both in vitro and in vivo as it increases IFN-Ȗ, IL-2 but not IL-4 and IL-10. It

is noteworthy that when PD-L1 signaling is reduced in T cells these cells proliferate extensively

in vitroand produce increased levels of IFN-Ȗ and IL-17, suggesting an enhanced pro-

inflammatory phenotype. It has been established that cytokines of Th2 response such as IL-4

and IL-5, IL-10 and IL-6, have tumor-promoting activity. The anti-melanoma effect of the GK­

1/anti-PD-L1 combination observed in the present study could be mediated by decreasing the

pro-tumor Th2 response. These results open a new field for alteratives to potentiate the effect

of the PD-1/PD-L1 blockade pathway. Further studies to evaluate the direct translational bench-

to bed-side translational potential are being performed by our group.

32

Technologies to Support Research on Microbial based cancer therapy

33

Cloud-based Microbe Identification and Characterization Pipeline

Hsinyi Tsang

National Cancer Institute, Rockville, Maryland, U.S.A.

Recent advances in genomics have allowed researchers to sequence microbes in great depths.

As a result, biological sequence repositories, both public and private, are experiencing an

exponential growth of sequence data. This poses a major challenge in microbial analysis,

especially for bacterial and viral pathogens. In this study, we present a flexible, reproducible,

highly-scalable, and user-friendly platform for analyzing microbial sequence data on the

National Cancer Institute’s Cancer Genomics Cloud (CGC) pilots. The cloud pilots were

conceptualized in 2013 and awarded to the Broad Institute, Institute for Systems Biology and

Seven Bridges Genomics, with the goal of enhancing the utility of cancer genomics data and

facilitating analysis. Implemented in the cloud, these cloud-based platforms realize the co­

localization of compute and data in a secure environment that can be easily shared with

collaborators. The tools on the CGC are run in containers and pipelines are described using

Common Workflow Language (CWL) and Workflow Description Language (WDL), which

maximizes portability and interoperability. On these three cloud platforms, we implemented

four microbe analysis pipelines, including mothur, Qiime, bioBakery and a self-contained

sequence analysis pipeline using Basic Local Alignment Search Tool (BLAST) with various

built-in graphical visualization for PCoA analysis, alpha, beta diversity and microbe abundance.

The cloud environment has proven to be a cost-effective, reproducible, and user-friendly

alternative to high-performance computing cluster, with minimal overhead and setup

requirements. These pipelines represents a necessary step in creating a publicly available and

scalable platform for microbe discovery and microbe-host interaction analysis.

34

Mass spectrometry based metabolomics platform for cancer research

Wenyun Lu, Lin Wang, Xiaoyang Su, Eileen White, Joshua D. Rabinowitz

Lewis-Sigler Institute for Integrative Genomics, Department of Chemistry, Princeton University,

NJ 08544 and Rutgers Cancer Institute of New Jersey, New Brunswick, NJ 08903

The development of metabolomics technology has led to new interests regarding metabolism in

cancer development and progression. A number of metabolite cancer biomarkers have been

discovered, as well as the identification of some novel cancer causing metabolites. We present

here a LC-MS and GC-MS based analytical platform for the comprehensive analysis of cellular

metabolites, including water-soluble metabolites, and water insoluble fatty acids, and

phospholipids. The entire workflow consists of metabolic extraction, LC-MS and GC-MS runs,

data analysis and interpretation. Standard operation procedures have been developed for the

metabolite extraction from cell culture, tissue and serum/plasma, which involve liquid extraction

using appropriate extraction solvents. Metabolites were analyzed using multiple analytical

methods on multiple dedicated instruments. Cationic water-soluble metabolites were analyzed on

a Q Exactive Plus Orbitrap mass spectrometer using hydrophilic interaction chromatography.

Anionic water-soluble metabolites were analyzed using Exactive Orbitrap mass spectrometer

coupled to reversed phase ion pairing chromatography. Fatty acids and phospholipids were

analyzed using Agilent Q-TOF instrument coupled to reversed phase chromatography. Data

analyses were performed using MAVEN program which converts the raw data into a validated

table of metabolite-specific signals. Examples will be provided to demonstrate our capability to

analyze a broad range of metabolites from real biological samples, as well as to probe the metabolic

fluxes using stable isotope tracers, all relevant to cancer metabolism and potentially microbial-

based cancer therapy.

35

Weak Signal Detection of Lung Cancer Risk

Yanming Li1, Hyokyong Grace Hong2, Yi Li1

1. Department of Biostatistics, University of Michigan, Ann Arbor, MI 48109, USA.

2. Department of Statistics and Probability, Michigan State University, East Lansing, MI

48824, USA.

Lung cancer risk is strongly associated with exposure to environmental carcinogens, in particular

to cigarette smoking. However, not all heavy smokers develop lung cancer, and lung cancer also

develops in non-smokers. Therefore, other risk factors, including genetic ones, may play a

significant role in lung cancer. A genome-wide association study (GWAS) approach has led to

valuable insights into lung cancer susceptibility genes; however, the identified loci account for an

extremely small proportion of the familial risk. Lung cancer pedigrees are rare, implying the

possibly polygenic nature of the inherited predisposition to lung cancer. Polygene refers to a gene

whose individual effect on a phenotype is too small to be observed, but which can act together

with other genes to produce observable variation. In this project, we use the term ``Marginally

Unimportant (weak) but Jointly Informative" (MUJI) variants to consider such polygenic variants.

Currently, population-based GWAS is a widely used approach in identifying genetic factors that

affect lung cancer. However, those studies (e.g. the analysis of candidate genes/polymorphisms

and genome-wide screenings) point to genetic markers individually associated with lung cancer

risk; while most population-based studies are premised on the assumption of a polygenic control

of disease risk. In this proposal, we hypothesize that lung cancers follow a polygenic model and

propose methods to identify MUJI signals searching for polygenic susceptibility to lung cancer,

which has not yet been studied formally. Given our successful preliminary experiments of the

proposed MUJI variant detection model, we hope to explain the missing heritability of lung cancer,

improving early detection thorough discovery of disease-susceptible polygenic variants.

1. Nascimento, A.V.S., A.; Bousbaa, H.; Ferreira, D.; Sarmento, B.; Amiji, M. M., Overcoming cisplatin resistance in non-small cell lung cancer with Mad2 silencing siRNA delivered systemically using EGFR-targeted chitosan nanoparticles. Acta Biomater, 2017. 47: p. 71-80.