Operacionalización de Variables

P306

A protein extract from fermented wheat germ promotes NK cell-mediated lymphoma eradication in mouse xenografts Gustavo Barisone1, Yunpeng Ma1, Mastewal Abuhay1, Robert O'Donnell1, Kathleen Lundeberg1_{, Sonia Gowda}1_{, William Murphy}2_{, Joseph Tuscano}1 1

University of California Davis, Sacramento, CA, USA;2University of California, Sacramento, CA, USA

Correspondence:Joseph Tuscano ([email protected]) Journal for ImmunoTherapy of Cancer2017,5(Suppl 2):P306 Background

Proteomic and genomic data has allowed for the development of promising targeted agents for NHL [1]. Most have acute and chronic toxicities that limit efficacy. The use of complementary and alterna- tive medicines has increased during the last decade. However, scien- tific evidence of their efficacy is scarce. Fermented wheat germ extract (FWGE) has been claimed to have anti-cancer properties in many tumor types. FWGE therapeutic activity has been attributed to its content of benzoquinones [2].

Methods

A protein fraction (FWGP) was isolated by FPLC and proteins identi- fied by mass spectrometry. Direct cytotoxic was studiedin vitrousing NHL cell lines. Immunomodulatory properties were evaluatedex vivo

by measure immune cell activation in human PBMCs and isolated NK cells.In vivoexperiments used nu/nu NHL xenografts with or without NK cell depletion; endpoints were tumor volume and toxicity. In vivo immunomodulatory effects were evaluated by treating tumor-free BALB/c mice with FWGP and measuring NK cell killing activity and degranulation.

Results

FWGP was cytotoxic in 17 cancer cell lines (IC50 = 20-171μg/ml in NHL, 12-27μg/ml in colon and 70-144 μg/ml in lung) and induced apoptosis by increasing levels of caspase-3, PARP, BAK, BAD and p53, while reducing levels of AKT. FWGP increased % NK cells, production of IFg and GrB, and NK-mediated killing. In vivo efficacy was con- firmed, with no toxicity, in pre-emptive and established models. In

vivo treatment with FWGP+rituximab was as effective as R-CHOP, with 90% complete remission. NK depletion resulted in no response to FWGP. These results support the hypotheses that FWGP augments NK-mediated tumor killing. Proteomic profiling identified 844 pro- teins. An active fraction consisted of 169 proteins.

Conclusions

FWGP represents a promising immunomodulatory agent with anti- tumor activity, minimal toxicity and low cost. Our results suggest FWGP has direct lymphomacidal activity by inducing apoptosis and indirect anti-tumor efficacy by enhancing NK-mediated tumor eradi- cation. Further experimental validation will allow translation of al “alternative”product into mainstream medicine.

References

1. Abuhay, M., et al. The HB22.7-vcMMAE antibody-drug conjugate has efficacy against non-Hodgkin lymphoma mouse xenografts with minimal systemic toxicity. Cancer Immunol Immunother. 2016; 65(10):1169-75. 2. Hidvegi, M., et al. MSC, a new benzoquinone-containing natural product

P307

Expression and function of PD-1 and TIM-3 in non-small cell lung cancer (NSCLC)

Jonathan Travers, Krtisten McEachern, Srimoyee Ghosh, Sridhar Ramaswamy, David Jenkins

1_{TESARO Inc., Waltham, MA, USA}

Correspondence:David Jenkins ([email protected]) Journal for ImmunoTherapy of Cancer2017,5(Suppl 2):P307

Background

The use of anti-programmed cell death protein 1 (PD-1) and PD-Ligand 1 agents in the treatment of non-small cell lung cancer (NSCLC) has been well established but many patients are either intrinsically resistant or become refractory during therapy. One potential resistance mechan- ism is the upregulated expression of additional checkpoint receptors such as T cell immunoglobulin and mucin domain 3 (TIM-3), a trans- membrane receptor that binds multiple putative ligands, and that has been shown to negatively regulate the function of T cells that co- express PD-1 [1].

Methods

We examined the immunophenotype and checkpoint receptor ex- pression of over 100 NSCLC samples from primary surgical resections. From a subset of these samples, we evaluated T cell functional status by gene expression analysis on sorted PD-1+ and TIM-3+ CD8+ T cells as well asex vivostimulation assays to evaluate cytokine pro- duction. Furthermore, we usedex vivoandin vivostudies to assess the effect of blockade of PD-1 and TIM-3 alone and in combination on T cell activation and anti-tumor activity.

Results

We showed that primary NSCLC samples display heterogeneity in both their baseline immune infiltrate and also PD-1 and TIM-3 check- point receptor expression. We examined mRNA expression of mul- tiple immune genes on sorted PD-1+ and TIM-3+ CD8+ T cells, and found that PD-1/TIM-3 double positive cells express reduced interleukin-2 (IL-2) and tumor necrosis factor alpha (TNFα), but similar mRNA levels of interferon gamma (IFNγ) when compared to double negative cells. This phenotype is recapitulated in CD8+ T cells de- rived from patient samples stimulated with PMA and ionomycin, where we found PD-1 and TIM-3 double positive cells to be signifi- cantly deficient in IL-2, but not IFNγ production. Importantly, in addition to their expression being associated with T-cell dysfunction, we also found that blockade of PD-1 and TIM-3 was associated with increased T cell activation and anti-tumor activity in ex vivo and

in vivomodels, suggesting a potential functional role for the inhib- ition of TIM-3, in addition to PD-1, in the enhancement of anti-tumor immunity.

Conclusions

Taken together, these data provide further evidence that TIM-3 may play a role in intrinsic resistance to single agent anti-PD1 therapy in NSCLC and support evaluating the combination of anti-PD-1 and anti-TIM-3 agents in the clinic.

References

1. Koyama, et al. Adaptive resistance to therapeutic PD-1 blockade is associated with upregulation of alternative immune checkpoints. Nat Commun. 2016;7:10501.

P308

A PD-1 x CTLA-4 bispecific DART® protein with optimal dual checkpoint blockade and favorable tolerability in non-human primates

Alexey Berezhnoy1_{, Kurt Stahl}1_{, Kalpana Shah}1_{, Tim Gaynutdinov}1_{, Gurunadh}

Chichili1, Daorong Liu1, Rebecca Johnson1, Ross La Motte-Mohs1, Jessica Hill1, Jonathan Li2_{, Sergey Gorlatov}1_{, Valentina Ciccarone}1_{, Ralf Alderson}1_{, Hua Li}1_,

James Tamura1, Jennifer Brown1, Jon Wigginton1, Ezio Bonvini1, Paul Moore1, Syd Johnson1

1

Macrogenics, INC, Rockville, MD, USA;2Macrogenics, INC, South San Fancisco, CA, USA

Correspondence:Alexey Berezhnoy ([email protected]) Journal for ImmunoTherapy of Cancer2017,5(Suppl 2):P308

Background

Immunotherapy with the combination of monoclonal antibodies that block PD-1 and CTLA-4 has shown clinical benefit beyond that ob- served with either mAb alone. A PD-1xCTLA-4 bispecific DART protein was designed to induce antitumor immunity through simultaneous targeting of both checkpoint pathways via administration of a single molecule. The DART protein increases checkpoint blocking activity on PD-1/CTLA-4 dually expressing cells, while displaying distinct immuno- logical effects of CTLA-4 blockadein vivoabsent evidence of toxicity. Methods

A PD-1xCTLA-4 DART protein was engineered as a tetravalent bispecific molecule from humanized anti-PD-1 and anti-CTLA-4 mAb sequences in a human hinge-stabilized IgG4 backbone. PK, PD and toxicology studies were performed in cynomolgus monkeys.

Results

The PD-1xCTLA-4 DART molecule demonstrated binding to immobi- lized PD-1 protein and PD-1-expressing cells lines, inhibition of PD-1 interaction with PD-L1 or PD-L2, as well as reversal of PD-1-mediated T-cell signal inhibition in gene-reporter assays comparable to that supported by a replica of nivolumab. Similarly, binding, ligand blocking and rescue of CTLA-4-mediated T-cell suppression was comparable to that supported by a replica of ipilimumab. The DART molecule demonstrated activation properties comparable to the combination of ipilimumab and nivolumab replicas in a variety of hu- man primary T-cell assays and showed enhanced B7-ligand binding blockade over that mediated by the ipilimumab replica on PD-1/CTLA- 4 double-positive cells. In the cynomolgus monkey, the PD-1xCTLA-4 DART molecule exhibited a PK profile consistent with that of an IgG4 and was well tolerated, with no mortality or significant adverse findings up to 75 mg/kg QWx3, the highest dose tested. T-cell expansion in the peripheral blood and lymphoid organs was observed, which was attrib- utable to the CTLA-4 blocking arm, since no such finding was observed with similar or higher doses of the anti-PD-1 constituent of the bispeci- fic molecule.

Conclusions

PD-1xCTLA-4 DART protein binds and blocks its targets, with increased activity on dual PD-1/CTLA-4-expressing cells.

The DART molecule enhances T-cell responses in vitro to the

level achieved by a combination of nivolumab and ipilimumab replicas.

PD-1xCTLA-4 DART protein was well tolerated in cynomolgus monkeys, with a safety profile similar to that observed with PD-1 blockade alone, while demonstrating biological effects of CTLA-4 antagonism.

The favorable safety and tolerability profile of the PD-1xCTLA-4 DART molecule combined with its enhanced activity on PD-1/CTLA-4 double- positive cells suggest a potential for an improved therapeutic window for PD-1/CTLA-4 co-blockade strategies, with the administration of a single molecule providing dosing convenience and ease of incorpor- ation into additional therapeutic regimens.

P309

Treatment with heterodimeric IL-15 promotes effector T cell infiltration into tumors

Cristina Bergamaschi1, Konstantinos Dimas1, Dimitrios Stellas1, Bethany Nagy1, Shawn Jensen2_{, Bernard Fox}2_{, Barbara Felber}1_{, George Pavlakis}1

1

National Cancer Institute at Frederick, Frederick, MD, USA;2Earle A Chiles Research Institute, Portland Providence Cancer Center, Portland, OR, USA

Correspondence:Cristina Bergamaschi ([email protected]) Journal for ImmunoTherapy of Cancer2017,5(Suppl 2):P309

Background

The presence of tumor-infiltrating effector T cells is considered the most predictive biomarker for clinical benefit in response to immuno- therapies. IL-15 is a cytokine important for the proliferation, activa- tion and mobilization of lymphocytes, including natural killer and

CD8+T cells. We have previously shown that bioactive IL-15 in vivo comprises a complex of the IL-15 chain with the IL-15 receptor alpha chain that are together termed heterodimeric IL-15 (hetIL-15). Several preclinical models have indicated the ability of IL-15 to enhance the response of the immune system against cancer, and based on these results hetIL-15 has advanced to clinical trials.

Methods

We have produced hetIL-15 and tested its anti-tumor activity in several murine cancer models. Analysis of lymphocytes in lymphoid organs and in tumors was performed by flow cytometry and multi- color immunohistochemistry. Chemokine and cytokine levels were determined using electrochemiluminescence (MSD) and ELISA assays. Results

Repeated injections of hetIL-15 in mice were effective in delaying tumor growth in the MC38 colon carcinoma, TC-1 cervical carcinoma and B16 melanoma models. The combination of hetIL-15 and adop- tive cell transfer of melanoma specific Pmel-1 cells showed anti- tumor efficacy in B16-bearing mice in absence of lymphodepletion. The E0771 orthotopic breast cancer model showed delay in tumor progression and significantly reduced lung metastasis upon hetIL-15 treatment. A significantly reduced onset of lung metastasis was also observed in 4T1 breast cancer-bearing mice. Flow cytometry and multi-color immunohistochemistry assays showed increased traffick- ing and persistence of CD8+T cells, including tumor specific T cells, into the tumors and an increased CD8+/Treg ratio, upon hetIL-15 ad- ministration. Importantly, hetIL-15 treatment led to preferential en- richment of adoptively transferred tumor-specific CD8+_{T cells in the} B16 tumor in an antigen-dependent manner. Tumor infiltration by CD8+T cells was accompanied by increased plasma levels of CXCL10. Tumor-resident CD8+ _{T cells showed features of activated effector} cells with enhanced proliferation (Ki67+_{) and high cytotoxic potential} (Granzyme B+). Upon ex-vivo stimulation, an increased frequency of both CD8+ and CD4+ T cells producing IFNg was observed in the tumors of mice treated with hetIL-15.

Conclusions

Our results show that hetIL-15 administration may be a general method to enhance T cell entry in non-inflamed tumors, increasing the success rate of immunotherapy interventions. Preclinical cancer studies support the use of hetIL-15 in tumor immunotherapy approaches to promote the development of anti-tumor responses by favoring effector over regulatory cells. The effect of hetIL-15 on me- tastasis establishment in orthotopic models may provide synergies against metastatic disease.

P310

CSF1/CSF1R signaling blockade triggers release of matrix-degrading proteases in mouse models

Stefan Bissinger1_{, Martina Schmittnaegel}2_{, Ioanna Keklikoglou}2_,

Michele De Palma2, Sabine Hoves1, Carola Ries1

1_{Roche Innovation Center Munich, 82377 Penzberg, Germany;}2_The

Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland

Correspondence:Stefan Bissinger ([email protected]) Journal for ImmunoTherapy of Cancer2017,5(Suppl 2):P310 Background

The heterotypic interplay between cancer cells and their microenviron- ment provides an opportunity for therapeutic targeting. The abundant tumor-associated macrophage (TAM) infiltrate can be substantially reduced in mouse tumor models and cancer patients by colony- stimulating factor 1 receptor (CSF1R) signaling blockade [1]. Notably, TAM depletion provides marked clinical benefits in diffuse-type tenosy- novial giant cell tumors [2,3]. However, facial edema is reported as the most common adverse event of TAM elimination in patients [2,4]. We here sought to gain insight into the molecular mechanisms mediating edema formation. To this aim, we characterized antibody exposure, CSF1 levels and an array of extracellular matrix-degrading and restruc- turing metalloproteinases (MMPs) in tumor-bearing and tumor-free mice treated with an anti-CSF1R antibody.

Methods

Western Blotting and multiplex assays of tumors and sera of anti-mouse CSF1R mAb (clone 2G2)-treated mice showed an association of TAM elimination with an early intratumoral and systemic release of a specific set of MMPs, including MMP-2, -3 and -8, in multiple transplant (MC38, E0771, KPL-4 and PyMT) and de novo (MMTV-PyMT) tumor models. Results

In addition, we found that the early increase of MMPs in the face of CSF1/CSF1R pathway blockade was independent of tumor burden and was accompanied by a significant increase of body weight in tumor-free mice following long-term exposure to the antibody. The body weight gain may therefore indicate the enhanced retention of body fluids. Discontinuation of the CSF1R antibody reinstated un- altered body weight and MMP levels. We excluded platelets and neu- trophils as sources of MMP release, even if they accumulated in tumor-bearing mice during CSF1R inhibition. We also examined the effects of blocking the CSF1R ligand CSF1, which is one of the two known ligands of the CSF1R [2,4]. CSF1 antibodies had no impact on the binding of IL-34 (the second CSF1R ligand) to CSF1R. Similar to CSF1R blockade, an anti-mouse CSF1 antibody (clone 5A1) provoked an early systemic surge of a subset of MMPs.

Conclusions

Collectively, our data suggest that CSF1 and CSF1R blocking antibodies induce the release of a distinct set of matrix-degrading proteases (MMP-2,-3 and -8), but not metastasis-promoting proteases (MMP-9 and -12), which may be potentially causative of edema formation. Fur- ther studies may inform optimized dosing schedules of CSF1/CSF1R tar- geting regimens for cancer patients.

References

1. Ries CH, et al. Cancer Cell 2014;25(6):846-59. 2. Cassier PA, et al. Lancet Oncol 2015;16(8):949-56. 3. Tap WD, et al. N Engl J Med 2015;373(5):428-37. 4. Papadopoulos KP, et al. Clin Cancer Res. 2017.

P311

Clinical outcomes of PD-1 inhibition by PD-L1 expression level across malignancies in 204 consecutive patients in a real world oncology setting

Kenneth Byrd1_{, Tristan Hayes}1_{, Mike Martin}2_{, Lee Schwartzberg}2_,

Ari Vanderwalde2

1_{University of Tennessee Health Sciences Center, Memphis, TN, USA;} 2

West Cancer Center, Germantown, TN, USA

Correspondence:Kenneth Byrd ([email protected]) Journal for ImmunoTherapy of Cancer2017,5(Suppl 2):P311 Background

The utility of the PD-L1 biomarker in predicting response to anti-PD- 1 agents has been inconsistent across malignancies. In this study, we describe outcomes of patients treated with anti-PD-1 agents by PD- L1 expression level.

Methods

Molecular profiling of tumors in patients with advanced cancer was performed at West Cancer Center using Caris Molecular Intelligence Profile testing, which includes PD-L1 percentage assessed by immu- nohistochemical staining. Patients were included in this retrospect- ive analysis if they were treated with a PD-1 inhibitor and had available PD-L1 results between November 2014 and May 2017. Pa- tients were assessed by PD-L1 expression level, defined as negative (0% expression) or positive (>1% expression), regardless of staining intensity. PD-L1 positive samples were further subclassified into PD- L1 low (1-4%), intermediate (5-49%) and high (_≥50%). Best overall response using RECIST 1.1 criteria was retrospectively assessed using 2-physician review of radiologic data. Progression free sur- vival (PFS) and overall survival (OS) were assessed using the Kaplan-Meier method.

Results

204 patients with quantifiable PD-L1 expression were treated with PD-1 inhibitors. Primary tumors included 125 non-small cell lung can- cers, 31 melanomas, 12 renal cell carcinomas, and 36 others. 110

(54%) tumors were PD-L1 negative and 94 (46%) were PD-L1 positive (22 [11%] low, 37 [18%] intermediate, and 35 [17%] high). ORR was 39% for PD-L1 positive versus 17% for PD-L1 negative (p=<0.001). Best response for each PD-L1 level is shown in Table 1. The esti- mated median PFS was 6.4 months for PD-L1 positive versus 3.0 months for PD-L1 negative (HR 0.59;p=0.001; 95% CI, 0.43 to 0.81). The estimated median OS was 17.3 months for PD-L1 positive versus 6.9 months for PD-L1 negative (Fig. 1; HR 0.64;p=0.021, 95% CI, 0.44 to 0.94). Increasing PD-L1 expression was associated with a statisti- cally significant improvement in PFS and OS. There was a statistically significant difference among PFS and OS with increasing PD-L1 levels (PFSp= 0.002; OSp= 0.026). Multivariate analysis did not identify tumor type as a predictor of response.

Conclusions

PD-L1 staining of any level predicted for improvements in ORR, PFS, and OS with PD-1 inhibitors across multiple malignancies. The higher the PD-L1 staining, the greater the likelihood of benefit. These data provide important real world confirmation for the potential utility of global PD-L1 testing in clinical practice, regardless of malignancy.

P312

AGEN2034, a novel anti-PD-1 antibody that combines effectively with CTLA-4 pathway blockade to enhance T cell activity Dhan Chand1_{, David Savitsky}1_{, Ana Gonzalez}1_{, Christopher Clarke}1_,

Andrea Schuster2, Elise E. Drouin1, Jeremy D. Waight1, Cornelia Mundt2, Gerd Ritter1_{, Taha Merghoub}4_{, David Schaer}4_{, Rikke B Homlgaard}4_,

Roberta Zappasodi4, Marc van Dijk5, Jennifer S. Buell1, Jean-Marie Cuillerot1, Robert Stein1_{, Nicholas S Wilson}1

1

Agenus Inc., Lexington, MA, USA;2Former employee of Agenus Switzerland Inc., Basel, Switzerland;4_{Memorial Sloan Kettering Cancer}

Center, New York, NY, USA;5Agenus Switzerland Inc., Basel, Switzerland

Correspondence:Dhan Chand ([email protected]) Journal for ImmunoTherapy of Cancer2017,5(Suppl 2):P312 Background

PD-1 (or CD279) is a co-inhibitory receptor that suppresses T cell function upon binding to its ligands, PD-L1 or PD-L2. PD-1 signal- ing functions cooperatively with CTLA-4 to limit T cell activation during priming by antigen presenting cells, leading to reduced proliferation, cytokine and chemokine production and cell sur- vival. Anti-PD-1 antibody therapies that block the interaction be- tween PD-1 and its ligands have shown durable clinical benefit both as single agents, but particularly in combination with anti- bodies that antagonize CTLA-4.

Methods

AGEN2034 (anti-PD-1; IgG4) was discovered using a proprietary mam- malian display technology, Retrocyte Display™. Binding kinetics and affinity to PD-1 were characterized by surface plasmon resonance. Cell-based potency was determined in a Jurkat PD-1+NFAT reporter- based assay where luciferase activity was measured as an endpoint of PD-1/PD-L1 antagonism. The pharmacological effects of AGEN2034 alone or in combination with CTLA-4 pathway blockade using a novel high affinity anti-CTLA-4 IgG1 antibody, AGEN1884, was assessed in vitro using peripheral blood mononuclear cells (PBMC) from healthy donors. Prior to human clinical trials, pharmacokinetic

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