Requisitos físicos

Characterization of anti-tumor immune responses and Effects on Survival of Neoadjuvant Oncolytic Virotherapy in spontaneous Osteosarcoma

Shruthi Naik, PhD1, Kelly Makielski, DVM, MS2, Michael Henson, DVM, PhD2_{, Kathleen Stuebner}2_{, Alexander-Flaviu Tabaran, DVM, MSc, PhD}2_,

Ingrid Cornax, PhD2, Gerard O'Sullivan, MVB, PhD2, Andrea Eckert2, Lauren Mills, PhD2_{, Milcah Scott, BS}2_{, Aaron Sarver, PhD}2_{, Michael Farrar}2_,

Stephen Russell, MD, PhD1, Jaime Modiano, VMD, PhD2

1_{Mayo Clinic, Rochester, MN, USA;}2_{University of Minnesota, Minneapolis,}

MN, USA

Correspondence:Jaime Modiano ([email protected])

Journal for ImmunoTherapy of Cancer2018,6(Suppl 1):O43

Background

Development of effective therapies for osteosarcoma, an infrequent dis- ease that primarily affects adolescents and young adults, is a critical un- met medical need. The rarity and heterogeneity of these cancers have hindered research in this field. Comparative oncology studies in naturally occurring osteosarcoma in companion dogs provide opportunities to ad- vance development in a clinically realistic setting where the tumors re- semble their human counterparts but where the barriers of rarity and cost are lowered [1]. Vesicular stomatitis virus (VSV) is a rapidly replicating, robustly immunogenic oncolytic virus platform with demonstrated cyto- toxicity in canine osteosarcoma cell lines. VSV-IFNβ-NIS, a recombinant oncolytic VSV, can be safely administered intravenously (IV) to reach sites of metastatic disease and has been shown selectively replicate in and destroy tumor cells and activate an anti-tumor immune response [2]. The aim of this study is to evaluate neoadjuvant VSV-IFNβ-NIS therapy for osteosarcoma using a comparative oncology approach and characterize anti-tumor immune responses in spontaneous canine osteosarcoma.

Methods

A veterinary clinical study was initiated. Dogs with osteosarcoma are ran- domized to receive neoadjuvant oncolytic VSV-IFNβ-NIS (109 TICD50/kg) or placebo followed by amputation and carboplatin chemotherapy. Pre- and post-treatment tumor biopsies and serial peripheral blood mono- nuclear cells are obtained to assess anti-tumor immunity by histopath- ology, RNA and DNA sequencing, and lymphocyte effector functions.

Results

To date 24 dogs have been enrolled, of 30 dogs planned. The VSV safety profile is excellent, with mild, transient changes in body temperature and evidence of acute cytokine responses. Preliminary analyses show survival outcomes exceed the expectation for standard-of-care alone. Focal tumor necrosis that is potentially treatment-related has been ob- served in osteosarcoma lesions in resected tissue. Assessment of naïve and treatment-associated gene cluster expression summary scores from RNA sequencing is ongoing, as is massive parallel sequencing of lymphocyte antigen receptors to describe clonal expansion and attrition.

Conclusions

Neoadjuvant VSV treatment is well-tolerated, and shows preliminary evidence of biological activity and clinical efficacy. Updated results describing anti-tumor immunity that is attributable to oncolytic VSV, and its effects on patient outcomes will be presented. These data will indicate if intravenous neoadjuvant VSV-IFNβ-NIS therapy improves clinical outcomes in canine osteosarcoma and inform clinical studies to evaluate this therapeutic approach as an addition to current chemotherapy protocols for osteosarcoma patients.

Consent

1. Scott, M.C., et al., Molecular subtypes of osteosarcoma identified by reducing tumor heterogeneity through an interspecies compara- tive approach. Bone, 2011. 49(3): p. 356-67.

2. Naik, S., et al., Curative one-shot systemic virotherapy in murine myeloma. Leukemia, 2012. 26(8): p. 1870-8.

T-cell Checkpoints and Checkpoint Inhibitors

FS120 mAb2, a dual agonist bispecific antibody targeting OX40 and CD137, activates T cellsin vitroand induces potent, FcγR- independent anti-tumour activity

Miguel Gaspar, PhD, Cyril Privezentzev, PhD, Katy Everett, PhD, Sandra Uhlenbroich, John Pravin, Leonor Rodrigues, MSc, Delphine Buffet, Marine Houee, Francisca Wollerton, Melanie Medcalf, Edouard Souteyrand, Alexander Koers, PhD, Emma McConnell, Master in Phyhsiology and Pharmacology for Researc, Martyn Rhoades, miat, Mateusz Wydro, PhD, Maud Berthelot, MSc, Sarah Batey, Michael Davies, Jacqueline Doody, PhD, Michelle Morrow, PhD, Mihriban Tuna, PhD, Neil Brewis, PhD

F-star Biotechnology ltd, Cambridge, UK

Correspondence:Miguel Gaspar ([email protected])

Journal for ImmunoTherapy of Cancer2018,6(Suppl 1):O44

Background

Following the success of checkpoint blockade, activation of the co- stimulatory Tumour Necrosis Factor Receptor (TNFR) superfamily re- ceptors represents the next stage of cancer immunotherapy with clinical trials underway for antibodies stimulating T cell co- stimulatory pathways. Targeting OX40 and CD137 has the potential to strongly activate the immune system due to their broad expres- sion across CD4+ and CD8+ T cells and NK cells. However, FcγR- mediated crosslinking is often required for the activity of monoclonal antibodies, and this likely limits clinical activity, due to the inherently low affinity of Fc:FcγR interactions, as well as due to FcγR-mediated depletion of T cells through ADCC. FS120 is a novel, dual agonist bis- pecific antibody that does not bind to FcγR, but instead crosslinks OX40 and CD137 resulting in potent activation of both CD4+ and CD8+ T cells, independent of FcγR binding.

Methods

FS120 was generated by introducing an OX40-binding specificity into the Fc-region of a human IgG1 targeting CD137. FcγR binding was significantly decreased using the LALA mutation. A parallel approach was taken to generate a murine surrogate molecule to testin vivo.

Results

FS120 binds simultaneously to OX40 and CD137 with subnanomolar affinity and has strong activity in PBMC and T cell stimulation assays. Conventional OX40 and CD137 agonist antibodies require crosslink- ing, e.g. via anti-Fc secondary antibodies for their activity. OX40 agonist antibodies activate CD4+ T cells, but not CD8+ T cells and CD137 agonist antibodies activate CD8+ T cells but not CD4+ T cells. FS120 has subnanomolar dual agonist activity on both CD4+ and CD8+ T cells, which is independent of additional crosslinking. This ac- tivity is dependent on concurrent binding to the two receptors. An anti-mouse OX40/CD137 bispecific antibody showed greater anti- tumour activity than a combination of OX40 and CD137 agonists in a CT26 mouse tumour model, which was associated with peripheral T cell activation and proliferation. The anti-tumour activity was inde- pendent of T regulatory cell (Treg) depletion, as evidenced by similar levels of tumour infiltrating Treg cells in mice treated with the anti- mouse OX40/CD137 mAb² and isotype control. Anti-tumour activity was also demonstrated in a B16-F10 syngeneic model.

Conclusions

An OX40/CD137-specific mAb2 can autonomously stimulate both CD4+ and CD8+ T cellsin vitroindependent of FcγR binding and me- diate potent anti-tumour activity in vivo via an FcγR-independent mechanism of action. These data support initiation of clinical devel- opment of FS120, a first-in-class dual agonist bispecific antibody for the treatment of human cancer.

Acknowledgements

We thank the following people for their technical assistance:In vivoteam for animal studies, Protein sciences team for protein production

Ethics Approval

Murine studies were conducted under a U.K. Home Office License in accordance with the U.K. Animal (Scientific Procedures) Act 1986 and EU Directive EU 2010/63.

O45

Refractory renal cell cancer (RCC) exhibits high adenosine A2A receptor (A2AR) expression and prolonged survival following treatment with the A2AR antagonist, CPI-444

Lawrence Fong, MD1_{, Lawrence Fong, MD}1_{, John Powderly, MD, CPI}2_,

Jason Luke, MD, FACP3, Drew Hotson, PhD4, Mario Sznol, MD5, Saby George, MD, FACP6_{, Toni Choueiri}7_{, Brian Rini, MD}8_{, Matthew Hellmann,}

MD9, Shivaani Kummar, MD10, Leonel Hernandez-Aya, MD PhD11, Daruka Mahadevan, MD, PhD12_{, Brett Hughes, MD}13_{, Ben Markman}14_{, Matthew}

Riese, MD, PhD15, Joshua Brody, MD16, Daniel Renouf, MD17, Rebecca Heist, MD18_{, Rachel Goodwin, MD}19_{, Amy Weise, DO}20_{, Leisha Emens,}

MD, PhD21, Stephen Willingham, PhD4, Long Kwei, PhD4, Ginna Laport, MD4_{, Richard Miller, MD}4

1

University of California, San Francisco, CA, USA;2Carolina BioOncology Institute, Huntersville, NC, USA;3_{University of Chicago, Chicago, MA,}

USA;4Corvus Pharmaceuticals, Burlingame, CA, USA;5Yale University, New Haven, CT, USA;6_{Roswell Park Cancer Institute, Buffalo, NY, USA;} 7

Dana Farber Cancer Institute, Boston, MA, USA;8Cleveland Clinic Foundation, Cleveland, OH, USA;9_{Memorial Sloan Kettering Cancer}

Center, New York, NY, USA;10Stanford University School of Medicine, Stanford, CA, USA;11_{Washington University, Saint Louis, MO, USA;} 12

University of Arizona, Tucson, AZ, USA;13Royal Brisbane Hospital, Herston, Australia;14_{Monash Health and Monash University, Melbourne,}

VIC, Australia;15Medical College of Wisconsin, Milwaukee, WI, USA;

16_{Icahn School of Medicine at Mt Sinai, New York, NY, USA;}17_British

Columbia Cancer Agency, Vancouver, Canada;18Massachusetts General Hospital, Boston, MA, USA;19_{Ottawa Hospital Cancer Center, Ottawa,}

Canada;20Karmanos Cancer Institute, Detroit, MI, USA;21Johns Hopkins University, Pittsburgh, PA, USA

Correspondence:Richard Miller ([email protected])

Journal for ImmunoTherapy of Cancer2018,6(Suppl 1):O45

Background

Adenosine plays a role in blocking the function of immune cells through binding to the A2AR. CPI-444 is an oral A2AR antagonist that has been evaluated in Phase 1 trials in advanced cancer.

Methods

This Phase 1b trial was conducted at 30 centers. RCC patients (pts) with progressive disease were randomized to receive either CPI-444, 100 mg po bid monotherapy or CPI-444 in combination with atezolizumab 840 mg IV every 2 weeks and treated until progression or unacceptable tox- icity. Endpoints included safety, tumor response (RR), progression free survival and overall survival (OS). Peripheral blood and tumor biopsies were obtained at baseline and on-treatment and evaluated for lympho- cyte subsets and gene expression of A2AR using Nanostring. A2AR sig- naling was assessed by measurement of inhibition of pCREB.

Results

Results: 68 pts enrolled (N=33 monotherapy, N=35 combination); me- dian prior therapies was 3 (range 1-5) including TKI, 84%; anti-PD(L)1 (IO), 72%; median time since last IO was 3.1 and 1.7 months for monotherapy and combination, respectively. Median treatment dur- ation was 4.6 months. CPI-444 was well-tolerated with no Gr3/4 tox- icity in monotherapy; 7 pts had reversible Gr3 toxicity in combination. CPI-444 blocked A2AR signaling based on inhibition of phosphorylation of CREB in blood lymphocytes. A2AR mRNA was measured in 31 pre-treatment RCC tumor biopsies and compared to other cancers (36 lung; 12 bladder; 5 colon; 8 prostate, 9 melanoma and 20 triple negative breast). RCC showed increase in A2AR expres- sion compared to lung (p<0.01) and the other tumors (p<0.001). Ob- jective clinical responses were observed in 8% of pts; another 21% had reduction of tumor not meeting criteria for response. Responses were seen in both monotherapy and combination arms and in pts who failed prior IO agents. 40% and 58% of pts experienced disease control (DC) for > 3 months (confirmed scans) in monotherapy and combination, respectively, including in pts resistant to prior IO. The

OS for both monotherapy and combination exceeded 80% at 16 months. Treatment increased CD8+ T cells in tumors in pts with DC>6mo compared to DC<6mo (p<0.02).

Conclusions

CPI-444 is active in RCC and is associated with prolonged survival and DC in treatment refractory pts, including those who have failed prior IO. Prolonged DC was associated with treatment-induced in- crease in tumor infiltrating CD8+ cells. Our results compare favorably to those reported with atezolizumab demonstrating 1 yr OS of 81% and RR of 15% in IO naïve RCC with median 2 prior therapies[1].

Acknowledgements

not applicable

Trial Registration

NCT02655822

References

1. McDermott D, Sosman J, Sznol M, Massard C, Gordon M, Hamid O, Powderly J, Infante J, Fasso M, Wang Y, Zou W, Hegde P, Fine G, Powles T. Atezolizumab, an anti-programmed death- ligand 1 antibody, in meta- static renal cell carcinoma: long-term safety, clinical activity, and immune correlates from a Phase 1a study. J Clin Oncol. 2016;34:1-10.

Ethics Approval

The protocol was approved by the institutional review board or ethics committee at each participating center.

O46

Increased tumor-resident memory T cells in breast cancer is associated with improved prognosis

Peter Savas1_{, Balaji Virassamy}1_{, Chengzhong Ye}2_{, Agus Salim}3_{, Chris}

Mintoff1, Franco Caramia1, Roberto Salgado4, Zhi Ling Teo1, Sathana Dushyanthen1_{, Ann Byrne}1_{, Stephen Luen}1_{, Paul Beavis, PhD}1_{, Stephen}

Fox1, Phillip Darcy1, Terence Speed2, Laura Mackay5, Paul Neeson, PhD6, Sherene Loi, MD, PhD6

1

Peter MacCallum Cancer Center, Melbourne, Australia;2WEHI, Melbourne, Australia;3_{La Trobe University, Melbourne, Australia;}4_GZA

Ziekenhuizen, Antwerp, Belgium;5University of Melbourne, Melbourne, Australia;6_{Peter MacCallum Cancer Centre, Melbourne, Australia;}7_Peter

Mac Callum Cancer Center, Victoria, Australia

Correspondence:Paul Neeson ([email protected])

Journal for ImmunoTherapy of Cancer2018,6(Suppl 1):O46

Background

The level of tumor infiltrating lymphocytes (TIL) is a prognostic factor for improved patient survival in triple negative and HER2-overexpressing breast cancer (BC) subtypes. T cells are the main immune subset in BC tu- mors with a high TIL content, TIL(hi); however the influence of the T cell qualitative response on patient prognosis is unknown.

Methods

To address this issue, TILs were isolated from 129 primary and metastatic BC samples, T cells were sorted and single-cell RNA sequencing (scRNA- seq) performed to reveal the BC TIL T cell clusters present. BC TIL scRNA- seq data was confirmed by multi-parameter flow cytometry (FACS). We also performed bulk RNA seq on FACS-sorted T cell populations, and in- terrogated BC TIL T cell receptor (TCR) sequences to compare the differ- ence in TCR usage between clusters. We explored BC TIL functional responses (cytokines, effector proteins) following co-culture with autolo- gous BC cells. Finally we compared the clinical outcome data for triple negative BC patients who expressed high vs low levels of the signature clusters in BC TILs.

Results

Our study showed BC T cells were heterogeneous in sub-type and func- tional polarization. In particular, BC TIL(hi) cases contained increased CD8+ tumor-resident memory T (TRM) cells. These cells expressed CD103 and high levels of immune checkpoint molecules (PD1, CTLA-4, TIM-3, Lag-3) and T cell effector proteins (perforin and granzyme B). Further analysis of BC TILs by multi-parameter FACS confirmed the presence of increased CD8+ TRM in TIL(hi) BC tumors, these CD8+ TRM also released granzyme B on co-culture with autologous BC cells. In two primary tumors, the BC TRM had different TCR usage to TEM

suggesting these are clonally distinct populations. Using the scRNAseq data, we developed a CD8+ TRM gene signature that was associated with improved patient disease free survival (DFS), (n=329, log-rank p=0.003) in early-stage triple negative breast cancers (TNBC) from the METABRIC data set. The CD8+ TRM gene signature also stratified pa- tients with high vs low CD8A expression for DFS (log-rank p = 0.03).

Conclusions

In conclusion, patients with BC TIL(hi) tumours have a qualitatively different T cell response which includes CD8+ TRM differentiation. These cells express high levels of immune checkpoint molecules, plus T cell effector proteins suggesting they could be the key responders to immune checkpoint inhibitor therapy. Further studies exploring BC-associated CD8+ TRM cell development and homeostasis will be critical for creating new opportunities for BC immunotherapy.

Acknowledgements

We wish to thank H Thorne, E Niedermayr, all the kConFab research nurses and staff, the heads and staff of the Family Cancer Clinics, and the Clinical Follow Up Study (which has received funding from the NHMRC, NBCF, Cancer Australia, and the National Institute of Health (USA)) for their contributions to this resource, and the many families who contribute to kConFab. kConFab is supported by a grant from NBCF, and previously NHMRC, the Queensland Cancer Fund, the Cancer Councils of New South Wales, Victoria, Tasmania and South Australia, and the Cancer Foundation of Western Australia. We wish to thank the FACS core facility staff R Rossi, V Milovac and S Curcio, and T Tan and P Petrone for additional FACS assistance. We also thank S Ellis for assistance with confocal imaging, G Mir Arnau for facilitating RNASeq, and the Anatomical Pathology staff at the Peter MacCallum Cancer Centre. Special thanks also to J Jabbari and the Australian Genome Research Facility for making the single cell sequencing possible.

Ethics Approval

This project was approved by the Human Research Ethics Committee of the Peter MacCallum Cancer Centre (project approval number_“SEGMENT_”13/ 123, Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab) project approval numbers #129 and #150).

Consent

All participating patients provided written informed consent.There is no sensitive or identifiable information in the abstract.

O47

Peripheral T cell dynamics in resectable NSCLC patients treated with neoadjuvant PD-1 blockade

Jiajia Zhang, MD, MPH1_{, Zhicheng Ji}1_{, Margueritta El Asmar, MD}1_{, Justina}

Caushi1, Valsamo Anagnostou, MD PhD1, Hok Yee Chan, MS1, Prerna Suri, MS1_{, Haidan Guo, BS, and BA}1_{, Kristen Marrone, MD}1_{, Jarushka}

Naidoo, MD1, Taha Merghoub, PhD2, Jamie Chaft, MD2, Matthew Hellmann, MD2_{, Janis Taube, MD, MSC}1_{, Julie Brahmer, MD}1_{, Victor}

Velculescu, MD, PhD1, Ni Zhao1, Patrick Forde, MD1, Drew Pardoll, MD, PhD1_{, Hongkai Ji}1_{, Kellie Smith, PhD}1

1

Johns Hopkins University, Baltimore, MD, USA;2Memorial Sloan Kettering Cancer Center, New York, NY, USA

Correspondence:Kellie Smith ([email protected])

Journal for ImmunoTherapy of Cancer2018,6(Suppl 1):O47

Background

Neoadjuvant PD-1 blockade has recently been shown to induce major pathologic responses (MPRs) and delay time to relapse in pa- tients with resectable non-small cell lung cancer (NSCLC)[1]. While the role of tumor-specific T cells in facilitating tumor regression has been demonstrated in advanced NSCLC, it is unknown how neoadju- vant PD-1 blockade affects the anti-tumor T cell repertoire and how these factors correlate with clinical outcome. The neoadjuvant setting provides a unique opportunity to evaluate T cell mobility into the tumor and other tissues after checkpoint blockade, which is challen- ging in studies of patients with metastatic disease.

Methods

Patients with resectable NSCLC were treated with neoadjuvant PD-1 blockade (NCT02259621). T cell receptor (TCR) sequencing was per- formed on pre- and post-treatment tissues, bulk serial peripheral

blood samples, and pre-treatment blood sorted for PD-1 expression. Whole exome sequencing and neoantigen prediction was performed on pre-treatment tumor biopsies and matched normal lung tissue. Peripheral dynamics of intratumoral T cell clonotypes, as well as en- richment of TCR motifs were evaluated. The associations of T cell dy- namics and neoantigen recognition with MPRs and recurrence-free survival were assessed.

Results

Substantial alterations in the T cell repertoire and influx of peripheral T cell clonotypes into tumor tissue, normal lung, and lymph nodes were observed following PD-1 treatment. Peripheral TCR responses were in- dependent of MPR status. T cell clonality in the resected tumorpost- treatment tissue positively correlated with pre-treatment tumor muta- tional burden and negatively correlated with percent residual tumor. Tumor infiltrating clonotypes underwent dynamic peripheral remodel- ing on-treatment; the magnitude of clonal reshaping was significantly higher than clonotypes not found in the tumor. Tumor-infiltrating clo- notypes that were also detected in the peripheral blood were of signifi- cantly higher frequency than those only present in the tumor. Notably, an anergic/monoclonal anti-tumor TCR repertoire was observed in a pa- tient with KRAS/STK11 co-mutations and an early relapse after PD-1 blockade. Analyses of the dynamics of T cell clonotypes with differential PD-1 expression is ongoing.

Conclusions

Significant and systemic alterations exist in the peripheral anti-tumor T cell repertoire in NSCLC patients treated with neoadjuvant PD-1