Sistemas de representación

P569

Novel Pharmacobiotic approach to enhance the tamoxifen efficacy using bacterial extracellular vesicles as the immunotherapy in breast cancer

Jeongshin An, MD,PhD1_{, Yeun-yeoul Yang}1_{, Won-Hee Lee}2_{, Jinho Yang}2_,

Jong-kyu Kim1, HyunGoo Kim1, Se Hyun Paek1, Jun Woo Lee1, Joohyun Woo1_{, Jong Bin Kim}1_{, Hyungju Kwon}1_{, Woosung Lim}1_{, Nam Sun Paik}1_,

Yoon-Keun Kim2

1_{Ewha Womans University, Seoul, Korea, Republic of;}2_{MD healthcare}

company, Seoul, Korea, Republic of

Correspondence:Jeongshin An ([email protected])

Journal for ImmunoTherapy of Cancer2018,6(Suppl 1):P569

Background

The anti-cancer effect of bacteria has a long history. According to Bierman et al., spontaneous remission of cancer has been observed in patients with severe bacteremia[1]. The reason was not revealed at that time, but we studied that in breast cancer. There are four main ways in which microbiota affects cancer: probiotics, prebiotics, drugs that target microbial enzymes and microbial products that have anticancer properties[2]. Among them, bacterial extracellular vesicles(EVs) are one of microbial products. In this study, we investi- gated the effects of bacterial EVs on the growth of breast cancer cells and tamoxifen efficacy.

Methods

Here, we analized microbiota of urine samples by NGS to select the target EVs that were expected to affect the growth of breast cancer cells. A total of 347 female urine samples–from 127 breast cancer patients (cancer group) and 220 normal individuals (control group)_– were collected and analyzed by NGS using a universal bacterial pri- mer of 16S rDNA. Human breast cancer cells were cultured, and the cells were treated with EVs of S. aureus and K.pneumoniae for 72 h. Real-time polymerase chain reaction (PCR) and Western blotting for signalling molecule analysis were performed after treatment of EVs in each breast cancer cell.

Results

There was a significant difference in the distribution of bacterial EVs be- tween the urine samples from breast cancer patients and from normal controls. Especially, S.aureus EVs were predominant in the normal group, and K.pneumoniae was abundant in the breast cancer group. Therefore, we selected these two bacterial EVs that may have an effect on breast cancer cell growth. We found that S.aureus and K.pneumoniae EVs down-regulated cell growth in MDA-MB-231 cells. We also found that S.aureus or K.pneumoniae EVs had a synergic effect on growth inhibition of while co-treated with tamoxifen. S.aureus EVs down-regulated mRNA expression of cyclin E2 and up- regulated that of TNF-alpha which was re- lated ERK pathway while co-treated with tamoxifen.

Conclusions

The anti-cancer effect of S.aureus and K.pneumoniae was initiated by its bacterial EVs and consequently inhibited the growth of breast cancer cells in triple negative breast cancer cells and improved the efficacy of tamoxifen in ER-positive cells. In the near future, we plan to conduct animal studies which are expected to further clarify the effect of bacterial EV on breast cancer.

References

1 Bierman, H. R. et al. Remissions in leukemia of childhood following acute infectious disease. Staphylococcus and streptococcus, varicella, and feline panleukopenias. Cancer 6, 591-605 (1953).2 Zitvogel, L., Daillère, R., Roberti, M. P., Routy, B. & Kroemer, G. Anticancer effects of the micro- biome and its products. Nature Reviews Microbiology 15, 465 (2017).

Ethics Approval

The study was approved by Ewha Womans University Medical Center_‘s Ethics Board.

Consent

Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

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P570

Commensal bacteria Bifidobacterium stimulates an anti-tumor response via cross-reactivity

Catherine Bessell, BA1, Catherine Bessell, BA1, Ariel Isser1, Jonathan Havel, PhD2, Sangyun Lee, PhD3, Ruhong Zhou, PhD4, Jonathan Schneck, MD, PhD1_{, David Bell, PhD}3

1_{Johns Hopkins University, Baltimore, MD, USA;}2_{Memorial Sloan}

Kettering Cancer Center, New York, NY, USA;3IBM Thomas J. Watson Research Center, New York, NY, USA;4Columbia University, New York, NY, USA

Correspondence:Jonathan Schneck ([email protected])

Journal for ImmunoTherapy of Cancer2018,6(Suppl 1):P570

Background

While recent studies have shown an important role of the micro- biome in modulating anti-tumor immune responses, its mechanism remains unclear. One proposed mechanism is due to cross-reactivity between antigens expressed in commensal bacteria and neoepitopes found in tumors. We have identified a cross-reactive antigen expressed in commensal bacteria Bifidobacterium (Bifido) “SVYR- YYGL” (henceforth called SVY) and show that it conveys a neoantigen-specific cross-reactivity to the classic neoantigen“SIY.”

Methods

The SVY-specific response was analyzed through biophysical experi- ments and molecular dynamics simulations to determine antigen processing and MHC binding. T cell expansion studies from SIY and SVY T cell populations along with cross specificity studies reveals the cross-reactive T cell populations. B6 mice housed from Jackson, Bifido colonized mice, and Taconic, Bifido lacking, mice were used for examine Bifido colonization on T cell expansion. Sorting cross- reactive T cell populations from Bifido positive or negative mice based on antigen specificity and T cell receptor (TCR) beta sequen- cing allows to examine the effect of colonization on TCR repertoire composition. Finally the anti-tumor activity of the commensal bac- teria population against the cross- reactive tumor antigen was tested by adoptive transfer studies with B16-SIY melanoma model.

Results

The SVY-specific response results from SVY peptide binding the H2- Kb MHC and can be processed from whole bacteria. The commensal bacteria SVY-specific T cells population has a cross-reactive SIY- specific T cell response and can recognize tumors expressing the

“SIY”antigen. Mice lacking Bifido have a decreased SVY-specific T cell response and an altered (TCR) repertoire compared to Bifido. colo- nized animals. Bifido. colonization not only shapes the SVY-specific TCR repertoire but selects for clones that are represented in the SIY TCR repertoire. Cross- reactive SVY-specific T cells recognize tumors bearing SIY in vivo in an adoptive T cell transfer model of murine melanoma and leads to decreased tumor growth and extended survival.

Conclusions

Our work demonstrates that commensal bacteria can directly stimu- late anti-tumor immune responses via T cell cross-reactivity and pro- vides a proof of principle for how bacterial antigens can shape the T- cell landscape.

P571

Targeted sequencing of 16s rRNA Gene to understand the diversity and composition of the gut microbiome

Rajesh Gottimukkala, MS1, Jianping Zheng2, Karen Clyde, PhD2, Fiona Hyland2_{, Janice Au-Young, PhD}2

1

ThermoFisher Scientific, Fremont, CA, USA;2Thermo Fisher Scientific, south san francisco, CA, USA

Correspondence:Rajesh Gottimukkala ([email protected])

Journal for ImmunoTherapy of Cancer2018,6(Suppl 1):P571

Background

Recent studies in humans and experiments in mouse models demon- strated the key role of the gut microbiota in modulating the tumor response to check point blockade immunotherapy. One study

showed an association between negative outcome using CTLA-4 blockade therapy and the absence of a specific gut microbiome. So, the gut microbiota has emerged as a promising biomarker to assess the efficacy of immune-modulatory drugs. Next generation sequen- cing of the 16S rRNA Gene is widely used as standard for under- standing the composition of the gut microbiome.

Methods

The AmpliSeq pan-Bacterial Research panel that contains 24 primer pairs targeting the 16S rRNA gene provides a cost-effective approach to identify the bacterial species present in the sample. Due to highly homologous nature of 16S sequences, it is challenging to correctly identify organisms at the Genus/Species level using short reads. We have developed a new algorithm that can identify all the organisms in the 16S database at Genus level and a majority at Species level. For every sequence in the database, we construct a coverage pattern using the aligned reads across the multiple amplicons. By matching the observed pattern per sequence with an expected pattern that is pre-computed we can identify the organisms present in the sample. The algorithm reports the identified microbes with Genus/Species level taxonomic classifications and the relative abundance of the or- ganisms in the sample.

Results

We sequenced DNA from 12 fecal samples with the assay using Ion GeneStudio S5 System and detected the 25 frequently observed Genera across all the samples including Bifidobacterium, Lactobacil- lus, Clostridium, Ruminococcus and Bacteroides etc. We sequenced a metagenomics mock community sample comprising of 20 different strains and identified all the 20 species including few organisms rele- vant to cancer microbiome studies like H.pylori, E.Faecalis, B.vulgatus etc. We did an in-silico analysis using the primers in the assay and demonstrated that using the assay we can identify the frequent bac- terial microbes in Gut microbiome resolved to Genus and/or Species level.

Conclusions

The AmpliSeq Pan-Bacterial Research panel with the described Bio- informatics pipeline will enable usage of 16s rRNA sequencing to as- sess the Gut microbiome as a biomarker for immunotherapy.

P572

Variation of the gut microbiome of complete responders to immune checkpoint blockade and healthy individuals– implications for clinical trial design

Beth Helmink, MD PhD1, Vancheswaran Gopalakrishnan, MPH, PhD1, Abdul Wadud Khan, MD1_{, Pierre-Olivier Gaudreau}1_{, Elizabeth Sirmans}1_,

Elizabeth Burton1, Vanessa Jensen, DVM1, Adrienne Duran, BAS1, Linsey Martin1_{, Angela Harris}1_{, Miles Andrews, MD, PhD}1_{, Jennifer McQuade,}

MD1, Alexandria Cogdill, MEng1, Christine Spencer, PhD1, Reetakshi Arora1_{, Nadim Ajami, PhD}1_{, Joseph Petrosino, PhD}2_{, Jamal Mohamed}1_,

Sapna Patel, MD1, Michael Wong, MD PhD FRCPC1, Rodabe Amaria, MD1, Jeffrey Gershenwald, MD1_{, Patrick Hwu, MD}1_{, Wen-Jen Hwu, MD, PhD}1_,

Michael Davies, MD, PhD1, Isabella Glitza, MD, PhD1, Hussein Tawbi, MD, PhD1_{, George Marnellos}3_{, Jaclyn Sceneay}3_{, Jennifer Wortman}3_{, Lata}

Jayaraman3, David Cook3, Theresa LaVallee4, Robert Jenq, MD1, Timothy Heffernan, PhD1_{, Jennifer Wargo, MD, MMSc}1

1

MD Anderson Cancer Center, Houston, TX, USA;2Baylor College of Medicine, Houston, TX, USA;3_{Seres Therapeutics, Cambridge, MA, USA;} 4

Parker Institute Cancer Immunotherapy, San Francisco, CA, USA

Correspondence:Jennifer Wargo ([email protected])

Journal for ImmunoTherapy of Cancer2018,6(Suppl 1):P572

Background

The gut microbiome has been shown to have profound influences on host and anti-tumor immunity, and pre-clinical studies suggest that gut microbiota may be modulated to enhance responses to immune check- point blockade [1-4]. Recent studies demonstrate differences in the gut microbiome of responders (Rs) versus non-responders (NRs) to anti-PD- 1 therapy in patients [5-8], with identification of a microbiome signa- ture associated with a 100% response rate (Type-1 signature) [5]. Several clinical trials are in development/underway that aim to modu- late the microbiome to augment responses to immune checkpoint

blockade. These are, in part, based on foundational evidence that treat- ment with fecal microbiota transplant (FMT) from healthy donors is as- sociated with clinical responses in other diseases (C. difficileinfection and inflammatory bowel disease, CDI and IBD)[9]; however, the optimal donors for FMT to enhance responses to immune checkpoint blockade remain incompletely understood.

Methods

To address this critical question, we performed profiling of the gut microbiota (via 16s and metagenomic sequencing) in a cohort of pa- tients with complete responses (CRs) to anti-PD-1 therapy (n=11) versus healthy controls10_{(n=116). Importantly, immune profiling was also per-}

formed in available baseline tumor biopsies from CRs. Diversity (inverse Simpson) and composition of the gut microbiota was assessed in each of these cohorts, and FMT of selected CR donors versus a known NR (n=3 and 1, respectively) was then performed into gnotobiotic mice and melanoma tumors were implanted. Mice were then treated with immune checkpoint blockade. Tumor outgrowth was assessed and lon- gitudinal microbiome analyses and immune profiling of tumor and the periphery in FMT- treated mice were also performed.

Results

Characterization of gut microbiota revealed wide variation in the diver- sity and composition of the gut microbiota, with preliminary work dem- onstrating a trend towards higher diversity in CR donors versus healthy controls (p=0.2); validation in a larger cohort of CRs is ongoing. Interest- ingly, not all CRs demonstrated a Type-1-like signature (with higher rela- tive abundance of Clostridiales versus Bacteroidales) (27%, n=3/11) nor did healthy controls 28% (n=33/116). This has critical implications for FMT donor selection in immune checkpoint blockade trials (versus those for CDI or IBD). Murine studies demonstrated reduced tumor growth in CR-FMT mice vs. NR-FMT mice, with variability noted between donors. Immune profiling in available patient tumor samples and in murine stud- ies and comparisons to gut microbiota are currently being performed.

Conclusions

Together, these studies provide important information about poten- tial donor selection in FMT trials in immunotherapy, warranting add- itional studies and translational research.

References

1. Borody TJ, Khoruts A. Fecal microbiota transplantation and emerging applications. Nat Rev Gastroenterol Hepatol. 2011;9:88-96.

2. Frankel AE, Coughlin LA, Kim J, Froehlich TW, Xie Y, Frenkel EP, Koh AY. Metagenomic shotgun sequencing and unbiased metabolomic profiling identify specific human gut microbiota and metabolites associated with immune checkpoint therapy efficacy in melanoma patients(). Neoplasia (New York, NY). 2017;19,848-855.

3. Garrett WS. Cancer and the microbiota. Science. 2015; 348, 80-86. 4. Gopalakrishnan V, Spencer CN, Nezi L, Reuben A, Andrews MC,

Karpinets TV, Prieto PA, Vicente D, Hoffman K, Wei SC, et al. Gut microbiome modulates response to anti-PD-1 immunotherapy in mel- anoma patients. Science. 2017.

5. Matson V, Fessler J, Bao R, Chongsuwat T, Zha Y, Alegre ML, Luke JJ, and Gajewski TF. The commensal microbiome is associated with anti-PD-1 ef- ficacy in metastatic melanoma patients. Science. 2018; 359, 104-108. 6. McDonald D, Hyde E, Debelius JW, Morton JT, Gonzalez A, Ackermann G,

Aksenov AA, Behsaz B, Brennan C, Chen Y, et al. American gut: an open platform for citizen science microbiome research. mSystems 3. 2018. 7. Routy B, Le Chatelier E, Derosa L, Duong CPM, Alou MT, Daillere R,

Fluckiger A, Messaoudene M, Rauber C, Roberti MP, et al. Gut microbiome influences efficacy of PD-1-based immunotherapy against epithelial tumors. Science. 2017.

8. Segre JA. Microbiome. Microbial growth dynamics and human disease. Science. 2015; 349, 1058-1059.

9. Sivan A, Corrales L, Hubert N, Williams JB, Aquino-Michaels K, Earley ZM, Benyamin FW, Lei YM, Jabri B, Alegre ML, et al. Commensal

Bifidobacterium promotes antitumor immunity and facilitates anti-PD- L1 efficacy. Science. 2015; 350, 1084-1089.

10. Vetizou M, Pitt JM, Daillere R, Lepage P, Waldschmitt N, Flament C, Rusakiewicz S, Routy B, Roberti MP, Duong CP, et al. Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota. Sci- ence. 2015; 350,1079-1084.

Ethics Approval

The study was approved by MD Anderson Cancer Center Institutution_‘s Ethics Board, approval numbers LAB00-063, PA15-0232, and RN00001344-RN01.

P573

Antibiotic use and clinical outcomes of PD-1 antagonists in advanced non-small cell lung cancers

Amit Kulkarni, MD, Manish Patel, DO, Ying Wang, MD, PhD, Todd Defor, MS University of Minnesota, Minneapolis, MN, USA

Correspondence:Manish Patel ([email protected])

Journal for ImmunoTherapy of Cancer2018,6(Suppl 1):P573

Background

Pre-clinical evidence in mice suggests that antibiotics induced dys- biosis can negatively influence efficacy of immune check-point inhib- itors (ICI). The effects of antibiotic use on clinical outcomes of ICI are limited and has yielded inconsistent results. We evaluated whether antibiotic use impacts efficacy of PD-1 inhibitors in patients with ad- vanced non-small-cell lung cancer (NSCLC)

Methods

We retrospectively reviewed clinical outcomes of advanced NSCLC patients treated with nivolumab or pembrolizumab at our institution between 5/2015 to 5/2018. Patients who received antibiotics 3 months prior to initiating ICI were considered to be in the Antibiotic exposure (ATB+) group. The remainder of patients were included in antibiotic naïve (ATB-) group. The primary outcome was clinical bene- fit rate (CBR) defined as proportion of patients with complete re- sponse (CR), partial response (PR) or stable disease (SD) per RECIST 1.1 among patients eligible for response assessment. Secondary out- comes of interest included progression-free survival (PFS) and overall survival (OS) across the entire patient population. Logistic regression and Cox proportional hazards model were used to compare out- comes between ATB+ and ATB- groups.

Results

111 patients were included in the analysis. The median age was 66 years (range 36-87 years). The majority of patients were female (55%), Caucasian (94%) and had adenocarcinoma (61%) histology. Most patients received Nivolumab (91%) in the second or subse- quent line. 30% patients had brain metastasis prior to receiving ICI. 44 (39%) patients received antibiotics up to 3 months prior to ICI ini- tiation, most received fluoroquinolones for respiratory infections. 96 patients were eligible for response assessment (>3 doses before re- staging). Antibiotics exposure did not impact CBR, 63% in ATB+ and 53% in ATB- group (OR=1.5; p=0.36). Similarly, no statistically signifi- cant difference was seen in median PFS and OS between the two groups (PFS 4.1 months vs 2.8 months, p=0.66 and OS of 12 months vs 10 months, p=0.4 in ATB+ and ATB- group respectively). HR for as- sociation of antibiotic use with PFS and OS was 0.8 (p=0.36) and 0.8 (p=0.25) respectively. No significant difference was noted when con- trolling for age, sex, ECOG status, prior lines of therapy, brain metas- tasis and steroid use.

Conclusions

To our knowledge, this is the largest study showing clinical outcomes are not affected by prior antibiotic use in NSCLC patients receiving ICI. While our study has limitations, more studies are needed to es- tablish an association. Data analysis of more patients is currently un- derway that will be reported in the final analysis prior to meeting.

P574

A rationally-designed consortium of human gut commensals induces CD8 T cells and modulates host anti- cancer immunity

Bruce Roberts, PhD6, Takeshi Tanoue1, Satoru Morita1, Koji Atarashi1, Wataru Suda2, Damian Plichta3, Seiko Narushima4, Ashwin Skelly1, Atsushi Shiota5_{, Jason Norman}6_{, Vanni Bucci}7_{, Yutaka Kawakami, MD}

PhD1_{, Masahira Hattori}2_{, Ramnik Xavier}3_{, Bernat Olle}6_{, Bruce Roberts,}

PhD6, Kenya Honda, MD, PhD8

1

Keio University School of Medicine, Tokyo, Japan;2Waseda University, Tokyo, Japan;3_{Broad Institute of MIT and Harvard, Cambridge, MA, USA;} 4_{Riken Center for Integrative Medical Science, Kanagawa, Japan;}5_JSR-

Keio University Innovation Center, Tokyo, Japan;6Vedanta Biosciences, Cambridge, MA, USA;7University of Massachusetts, North Dartmouth, MA, USA;8_{Keio University School of Medicine and JSR-Keio University}

Innovation Center, Tokyo, Japan

Correspondence:Bruce Roberts ([email protected])

Journal for ImmunoTherapy of Cancer2018,6(Suppl 1):P574

Background

Clinical data suggests the gut microbiome influences response to checkpoint inhibitor therapy however the precise identity and mode of action of commensals associated with clinical response has not been elucidated. We report the generation of a consor- tium of human gut derived commensals capable of inducing CD8 T cells and augmenting anti- cancer immunity.

Methods

The microbiota of healthy humans was used to inoculate germ-free mice and assess the level of CD8 T cell induction. Human derived com- mensals were isolated from inoculated mice exhibiting high levels of CD8 T cell induction and sequenced. Consortia consisting of isolated human commensals were tested for the ability to induce CD8 T cells in germ-free and SPF mice. A minimal consortium capable of inducing CD8 T cells was administered with checkpoint inhibitor antibodies to tumor-bearing mice to assess anti-cancer activity and the level of accu-