bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5614: Targeting the radiation-inducible neoantigen GRP78 as a novel strategy for non-small cell lung carcinoma treatment
Authors: Singh Abhay , Kapoor Vaishali , Dadey David , Thotala Dinesh , Hallahan Dennis
Published: August 2018

Non-small cell lung cancer (NSCLC) ranks among the highest cancer-related mortalities worldwide. NSCLC immunotherapy has not been successful in the past. Although lung cancer is not typically believed to be an immunogenic malignancy, more and more evidence suggests that immune responses to lung cancer may be important. The objective of this research was to address a critical barrier and develop a new paradigm in the treatment of NSCLC. Development of anticancer antibodies is limited by the scarcity of antigens that are specifically overexpressed in lung cancer, resulting in too few molecular targets and small percentages of patients who can be treated with the therapeutic antibody. We established a platform technology to develop antibodies to radiation-inducible neoantigens. Several radiation-inducible antigens have been identified by our group. Glucose-regulated protein 78 (GRP78), a multifunctional protein folding chaperone and co-receptor that is highly expressed on the surface of NSCLC, holds significant promise as a cancer-specific target. Although the potential of targeting GRP78 has been demonstrated in prostate cancer, melanoma, and breast cancer, there are limited data on the impact of targeting cell surface-associated GRP78 in NSCLC. We show that GRP78 undergoes radiation-induced translocation to the surface of NSCLC cells. We developed several GRP78-targeting monoclonal antibodies. NSCLC-specific antibody binding is achieved for several days by anti-GRP78 monoclonal antibodies after irradiation. Optical imaging and immunohistochemical staining indicated that the GRP78-specific antibodies achieve specific binding to irradiated NSCLC in mouse models. The anti-GRP78 antibodies also have a direct cytotoxic effect on NSCLC cells. The combination of anti-GRP78 antibodies and radiation showed an additive effect. Overall, this study suggests that GRP78 is a promising therapeutic target for the treatment of lung cancer. We propose to harness the physiologic response of cancer to radiotherapy and evolve it for immunotherapy of lung cancer using specific targeting antibodies.

Citation Format: Abhay Singh, Vaishali Kapoor, David Dadey, Dinesh Thotala, Dennis Hallahan. Targeting the radiation-inducible neoantigen GRP78 as a novel strategy for non-small cell lung carcinoma treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5614.
Abstract 5618: Anti-FGFR4 antibody drug conjugate for immune therapy of rhabdomyosarcoma and hepatocellular carcinoma
Authors: Cheuk Adam , Shivaprasad Nitya , Skarzynski Martin , Baskar Sivasubramanian , Azorsa Peter , Khan Javed
Published: August 2018

Rhabdomyosarcoma (RMS), an aggressive soft tissue sarcoma originating from skeletal muscle in children and adolescent young adults. It is divided in two main histological subtypes including embryonal RMS driven by RAS pathway mutations and alveolar RMS driven by a chimeric fusion gene involving PAX3 or PAX7 with FOXO1. FGFR4 is a cell surface tyrosine kinase receptor that is a critical molecule in RMS biology. In RMS it is a direct target and strongly induced by PAX3-FOXO1 as well as PAX3, and PAX7. By immunohistochemistry we found high expression of FGFR4 in RMS cancers but low expression in normal human organs. Of note FGFR4 has been reported overexpressed in hepatocellular carcinoma (HCC). We therefore hypothesized that FGFR4 will provide a rational target for immune therapy in cancers with high expression. We generated fifteen binders against FGFR4 and characterized them further as candidates for immunotherapy. We found that 3A11, a mouse IgG antibody, bound to FGFR4 positive cell lines with an observed avidity of 1.9nM. By ELISA using the extra cellular domain of human FGFR1, FGFR2, FGFR3 or FGFR4, 3A11 showed dose dependent binding to FGFR4 only. 3A11 was also found internalized upon binding to FGFR4 positive cell lines. The VL and VH domain of 3A11 was cloned and chimeric 3A11 scFvFc (mouse Fv and Human IgG1 Fc) construct was made. The chimeric form of 3A11 antibody was successfully produced in vitro and retained its specificity. To investigate FGFR4-mediated cytotoxicity in RMS cell lines, we cultured RH30 (a RMS cell line expresses FGFR4) in the presence of the chimeric 3A11 and polyclonal anti-mouse 2°ADC conjugated to Duocarmycin DM for 72 hours. Dose-dependent cytotoxicity was observed in RH30, suggesting that chimeric form of 3A11 could deliver a cytotoxic payload to FGFR4 positive cells. We are currently developing antibody drug conjugate using the cytotoxic drug, pyrrolobenzodiazepine which will be tested in vitro and in vivo models of RMS. We have thus developed a chimeric antibody that may provide effective immune therapy for cancers with high FGFR4 expression including RMS and HCC

Citation Format: Adam Cheuk, Nitya Shivaprasad, Martin Skarzynski, Sivasubramanian Baskar, Peter Azorsa, Javed Khan. Anti-FGFR4 antibody drug conjugate for immune therapy of rhabdomyosarcoma and hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5618.
Abstract 5620: Multispecific antibodies targeting CD38 and PD-L1 show potent tumor cytotoxicity
Authors: Schooten Wim van
Published: August 2018

Multivalent antibodies targeting either CD38 alone or CD38 in conjunction with PD-L1 may yield therapeutics with superior biologic activities and provide benefit for treating malignancies expressing low levels of CD38 (DLCBL, daratumumab refractory MM, and NSCLC). Multivalent, multispecific antibodies kill CD38low cells through a variety of mechanisms including stronger and more specific engagement of CD38. Potent and directed immune checkpoint inhibition is realized by adding an anti-PD-L1 binding domain. Teneobio's discovery platform utilizes VH domains (UniDabs) of fully human heavy chain antibodies (UniAbs) to develop bi-, tri-, and tetravalent antibodies. Individual UniDabs targeting CD38 and PDL1 were identified using our unique sequence-based discovery platform and high-throughput lead evaluation pipeline (TeneoSeek). This robust screening workflow enables evaluation of a large diversity of natural fully human antibodies, targeting multiple epitopes on a single antigen and uncovering important sequence activity relationships. We have identified UniDabs that bind five different functional epitopes on human, cynomolgus and mouse CD38 including potent inhibitors of hydrolase and cyclase enzymatic functions. In addition, we generated a large panel of PD-L1 inhibitors that bind with high affinities human and cynomolgus PD-L1. Using different combinations and arrangements of UniDabs, a variety of multivalent antibodies were constructed and evaluated in in vitro models. Data from a range of assay types show that multivalent UniAbs targeting CD38 can be engineered to display superior tumor cell cytotoxicity through multiple mechanisms of action. Summary: Teneobio's discovery platform utilizes VH domains (UniDabs) of fully human heavy chain antibodies (UniAbs) to develop bi-, tri-, and tetravalent antibodies. Multivalent UniAbs targeting CD38 and PD-L1 can be engineered to display superior tumor cell cytotoxicity through multiple mechanisms of action.

Citation Format: Wim van Schooten. Multispecific antibodies targeting CD38 and PD-L1 show potent tumor cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5620.
Abstract 5623: Development of a CD47-blocking antibody as a cancer therapy
Authors: Oaks Joshua , Wang Ming , Zou Hui
Published: August 2018

Immune system avoidance by cancer cells is controlled through a variety of mechanisms such as CTLA-4 and PD-1/L1, which inhibit the T-cell mediated adaptive immune response to cancer. Similar mechanisms exist for evasion from innate immunity, for example, through CD47 overexpression. On healthy cells, CD47 serves as a “don't eat me” signal by the binding to the transmembrane SIRPα protein on phagocytic cells, and prevents the engulfment of “self” by macrophages. In several cancer types, tumor cells overexpress CD47 to elude the immune system. This property of CD47 makes it an attractive target for cancer treatment. Here we describe a humanized monoclonal antibody that is a potent disruptor of the CD47/SIRPα interaction and therefore presents itself as a potential therapeutic anti-CD47 antibody. This antibody binds robustly to recombinant extracellular domains (ECD) of human and cynomolgus CD47, and to full-length human and cynomolgus CD47 expressed on HEK293 cells. Functionally, it blocks the CD47/SIRPα interaction in an ELISA-based assay with an IC50 of single-digit nM. In a cell-based assay, effect on phagocytosis by this antibody was assessed. In addition, disruption of the TSP-1/CD47 interaction has been postulated to have positive outcomes in cancer therapy and the efficacy of this antibody on inhibiting this interaction is also investigated. Finally, the effect of the antibody on Raji human Burkitt's lymphoma xenograft growth in mice will be presented. A concern with anti-CD47 therapy is the undesired targeting of red blood cells (RBCs). This antibody does not induce marked hemagglutination, suggesting a limited impact on RBCs. Taken together, our data suggest that we have developed a novel, humanized anti-CD47 antibody that can restore immune system targeting of cancer cells without inducing significant RBC agglutination.

Citation Format: Joshua Oaks, Ming Wang, Hui Zou. Development of a CD47-blocking antibody as a cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5623.
Abstract 5624: Development of novel bispecific immune modulating antibodies
Authors: Vitale Laura A. , Thomas Lawrence J. , He Li-Zhen , O'Neill Thomas , Widger Jenifer , Crocker Andrea , Testa James , Sundarapandiyan Karuna , Forsberg Eric , Boyer James , Storey James , Goldstein Joel , Marsh Henry C. , Keler Tibor
Published: August 2018

The initial success with checkpoint blocking antibodies has evolved into a tremendous effort in combination therapy to further improve outcome in responsive cancers and to overcome resistance. There is strong rationale for this approach, as the cancer-immunity cycle has many points for intervention, and the immune system has developed multiple pathways to the same end. The use of bispecific antibodies provides opportunities to engage two pathways simultaneously that can result in greater efficacy, improved safety profile, and simplified development compared to separately administered antibodies. To generate bispecific antibodies that blocked the PD-1 signaling pathway, we developed and characterized fully human PD-L1 specific antibodies generated using human Ig transgenic mice. A lead antibody was chosen for its potent activity in functional assays and good manufacturing properties. Our initial bispecific construct couples our CD27 agonist antibody with the PD-L1 antibody (αCD27xαPD-L1) as we have previously shown that this combination led to improved therapeutic activity in the BCL1 disseminated lymphoma model. We found that the αCD27xαPD-L1 was more potent in CD27 costimulation assays such as NFkB activation, and T cell cytokine production compared to the parental antibodies. Similarly, αCD27xαPD-L1 was more efficacious in vivo as measured by Ag-specific T cell IFN-γ production and antitumor activity. Importantly, the bispecific antibody was more effective than the combination of anti-CD27 and anti-PD-L1 antibodies in the BCL1 lymphoma model. The enhanced efficacy was attributed to more efficient cross-linking of the bispecific antibody/CD27 receptor, resulting in stronger T cell activation. In addition, we observed that CD27 signaling increased PD-L1 expression on both tumor and immune cells, which may further enhance the activity of the bispecific antibody. The use of bispecific antibodies can improve the efficacy and profile of immunotherapeutic monoclonal antibodies by combining complementary functionalities and enhancing target cross-linking in the relevant tissues. We are currently exploring this approach with multiple antibodies from our pipeline.

Citation Format: Laura A. Vitale, Lawrence J. Thomas, Li-Zhen He, Thomas O'Neill, Jenifer Widger, Andrea Crocker, James Testa, Karuna Sundarapandiyan, Eric Forsberg, James Boyer, James Storey, Joel Goldstein, Henry C. Marsh, Tibor Keler. Development of novel bispecific immune modulating antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5624.
Abstract 5628: Measurement of Fc-mediated ADCC and CDC activity of anti-TNFA and anti-VEGF therapeutic antibodies using reporter-based bioassays and engineered TNFA+ and VEGF+ target cells
Authors: Garvin Denise , Grailer Jamison , Moravec Rich , Hartnett Jim , Ott Vanessa , Fan Frank , Cong Mei , Cheng Zhi-jie Jey
Published: August 2018

Fc-mediated effector functions are critical to the efficacy and safety of therapeutic antibodies. Measurement of Fc-mediated antibody-mediated cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) during antibody drug discovery and development is not only important for antibodies that harness ADCC and/or CDC as their primary mechanism of action (e.g. rituximab, trastuzumab), but also for antibodies designed to target and block soluble ligands such as TNFA and VEGF. We previously reported the development of a cell-based reporter bioassay platform which has been used to measure ADCC and ADCP mediated through FcΓRI, FcΓRIIa and FcΓRIIIa. These reporter bioassays exhibit the specificity, accuracy, precision, and robustness necessary for qualification according to ICH guidelines and have been used extensively to characterize and measure the potency of antibody-based biologics drugs that target cell surface immune receptors. In the current study, we sought to evaluate Fc-mediated ADCC and CDC activities of therapeutic antibodies designed to target and block soluble ligands including TNFA and VEGF. To measure ADCC activity of anti-TNFA and anti-VEGF blocking antibodies, we developed engineered target cells that express either membrane-bound TNFA or VEGF. When used as target cells with reporter-based effector cells expressing a relevant FcΓR, ADCC activity of adalimumab (anti-TNFA) and bevacizumab (anti-VEGF) was detected in a specific and dose-dependent manner. Similarly, when used in a luminescence-based CDC assay, the engineered target cells elicited an appropriate FcΓR-mediated response. The assay signals demonstrated IgG isotype specificity as IgG4 variants showed minimal activity in both ADCC and CDC assays. Our results demonstrate that the combined use of cell-based reporter bioassays with target cells engineered to express membrane-bound soluble ligands can provide a simple, specific, and quantitative platform to measure Fc-mediated effector functions of therapeutic antibodies targeting soluble ligands.

Citation Format: Denise Garvin, Jamison Grailer, Rich Moravec, Jim Hartnett, Vanessa Ott, Frank Fan, Mei Cong, Zhi-jie Jey Cheng. Measurement of Fc-mediated ADCC and CDC activity of anti-TNFA and anti-VEGF therapeutic antibodies using reporter-based bioassays and engineered TNFA+ and VEGF+ target cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5628.
Abstract 563: Identification of a novel biomarker to predict pathologic complete response for neoadjuvant chemoradiotherapy in locally advanced rectal cancer
Authors: Kim Jong Gwang , Lee In Hee , Lee Soo Jung , Chae Yee Soo , Kang Byung Woog
Published: August 2018

Neoadjuvant chemoradiotherapy (CRT) followed by total mesorectal excision (TME) is the standard treatment for locally advanced rectal cancer (LARC). Good pathologic response to CRT is associated with better outcome. However, there is a lack of effective methods to select rectal cancer patients who would or would not have a benefit from CRT. Therefore, we aimed to perform whole-exome sequencing (WES) for rectal cancer to identify the novel genetic variations predicting CRT benefit in LARC. Two independent sets were used to evaluate the genetic differences between the good response group and poor response group to CRT. Fifteen patients who achieved pathologic complete response were divided into the good response group and 15 patients with pathologic stage III were classified as poor response group. Among these 30 patients, WES was examined. To validate discovery set, additional samples (n=67) were genotyped for candidate variants using TaqMan SNP genotyping assays or Sanger sequencing. Overall, the current study included a total of 97 LARC patients who were treated with neoadjuvant CRT followed by curative surgery at Kyungpook National University Medical Center. We found 5 candidate variants significantly associated with pathologic complete response (BCL2L10 rs2231292, DLC1 rs3816748, DNAH14 rs3105571, ITIH5 rs3824658, RAET1L rs912565) using WES. In the dominant model, patients with GC/CC genotype of DLC1 rs3816748 (p=0.032), AC/CC genotype of DNAH14 rs3105571 (p=0.009) and TT genotype of RAET1 rs912565 (p<0.0001) showed higher rate of pCR compared to those of other genotype patients. In the recessive model, the BCL2L10 rs2231292 (p=0.036) and ITIH5 rs3824658 (p=0.003) were significantly associated with pathologic CR. Four SNPs (DLC1 rs3816748, DNAH14 rs3105571, ITIH5 rs3824658, RAET1L rs912565) found to be significantly correlated with pCR in codominant model. However, none of the SNPs was associated with the relapse-free survival or overall survival. The present results suggest that the genetic variation of the BCL2L10 rs2231292, DLC1 rs3816748, DNAH14 rs3105571, ITIH5 rs3824658, and RAET1L rs912565 genes can be used as biomarkers predicting CRT response for patients with LARC.

Citation Format: Jong Gwang Kim, In Hee Lee, Soo Jung Lee, Yee Soo Chae, Byung Woog Kang. Identification of a novel biomarker to predict pathologic complete response for neoadjuvant chemoradiotherapy in locally advanced rectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 563.
Abstract 5638: A tetanus-way of improving synthetic long peptide tumor vaccination
Authors: Drijfhout Jan Wouter , Fletcher Erika , Leja-Jarblad Justyna , Kerzeli Iliana , Cordfunke Robert , Tornqvist Gunilla , Lindqvist Frida , Hultqvist Greta , Valentijn Rob , Ladjervardi Sam , Haggman Michael , Ullenhag Gustav , Mangsbo Sara
Published: August 2018

We have previously identified a natural B cell epitope derived from tetanus toxin named Minimal Tetanus Toxin Epitope (MTTE)[1]. Most healthy individuals have IgG1 antibodies to this peptide sequence but no IgM antibodies. We have assessed the possibility to apply this MTTE sequence in drug development with the intention to create immune complexes and use them as vehicles to carry antigen-material into dendritic cells. Thru a conjugation method three MTTE sequences and a longer synthetic peptide stretch harboring T cell epitopes are linked together. As immune complexes are efficient carriers of antigen material and can promote cross-presentation the aim is to use these conjugates for therapeutic purposes in diseases where a strong cellular immune response should be induced.

To further evaluate the MTTE peptide conjugates in drug development we have investigated how a diphtheria, tetanus, pertussis (DTP) vaccination induces antibody titers against the linear tetanus sequence. Herein we have studied the MTTE location in the tertiary structure of the protein, how cancer patients respond with antibody titers against MTTE along with a subsequent T cell response and in vivo anti-tumor responses (mice). In addition we have studied safety in terms of cytokine release assessment pre and post a DTP vaccination. We found that the MTTE sequence is part of an alpha-helix that is externally oriented and thereby accessible for peptide binding. We found that not helper epitope is part of the MTTE sequence and that healthy individuals as well as cancer patients can mount IgG titers against the epitope, this also result in more efficient induction of recall responses post a DTP booster than pre boost. Mice seropositive to the MTTE sequence can mount anti-tumor responses, while this is not seen in seronegative animals. In addition, a cytokine release assessment using a modified chandler loop model with intact complement does not display a broad immediate cytokine release pre or post a DTP booster. We conclude that it is efficacious and safe to make use of a peptide conjugate in a drug development project with the aim to use it as a vaccine strategy.

[1] Mangsbo et al. In press. Molecular Immunology 2017

Citation Format: Jan Wouter Drijfhout, Erika Fletcher, Justyna Leja-Jarblad, Iliana Kerzeli, Robert Cordfunke, Gunilla Tornqvist, Frida Lindqvist, Greta Hultqvist, Rob Valentijn, Sam Ladjervardi, Michael Haggman, Gustav Ullenhag, Sara Mangsbo. A tetanus-way of improving synthetic long peptide tumor vaccination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5638.
Abstract 5649: Multi-omics data indicate that primary and lymph node oral cancer cells-derived extracellular vesicles carry cargo molecules with a specific aggressive pattern
Authors: Busso-Lopes Ariane F. , Carnielli Carolina M. , Winck Flávia , Riaño-Pachón Diego M. , Oliveira Ana K. , Costa Rute A. , Rivera César , Caldana Camila , Fox Jay W. , Leme Adriana F. Paes
Published: August 2018

Oral squamous cell carcinoma (OSCC) exhibits high incidence and morbidity, representing 300,000 new cases and 145,000 deaths per year. Moreover, the 5-year survival rate is 50% and this is directly associated with regional lymph node metastases. To explore how the extracellular vesicles (EVs) play a significant role on OSCC lymph node metastasis, we characterized the EVs released by tumor primary site cells and their paired lymph node metastasis cells using nanoparticle tracking analysis, high-resolution microscopy (Cryo-EM), EVs uptake by HUVEC, BJ-5ta and gingival primary fibroblasts recipient cells and the proteins, lipids, metabolites and miRNAs contents. Both cells-derived EVs showed a similar size, morphology, and the presence of annexin-2 and flotillin-1 surface markers. Interesting, only gingival primary recipient cells were more likely to uptake primary OSCC-derived EVs compared with lymph node-derived EVs. This effect is probably associated with the differential abundance of 490 proteins, 316 miRNAs, 63 lipids and 17 metabolites between the oral cancer cells-derived EVs. Markedly, multilevel omic data integration indicates that metabolic processes were over-represented mainly in primary OSCC-derived EVs, suggesting that these EVs carry specific cargo molecules associated with a more aggressive pattern compared with lymph node OSCC-derived EVs.

Financial support: FAPESP (Process number 2016/50005-7).

Citation Format: Ariane F. Busso-Lopes, Carolina M. Carnielli, Flávia Winck, Diego M. Riaño-Pachón, Ana K. Oliveira, Rute A. Costa, César Rivera, Camila Caldana, Jay W. Fox, Adriana F. Paes Leme. Multi-omics data indicate that primary and lymph node oral cancer cells-derived extracellular vesicles carry cargo molecules with a specific aggressive pattern [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5649.
Abstract 5654: Fully automated tissue classification by imaging mass spectrometry, moving towards a hundred samples a day
Authors: Jones Emrys A. , Chapman Richard , Hoyes Emmy , Migas Lukasz , Henderson Fiona , Pringle Steven , Langridge James , McMahon Adam
Published: August 2018

Desorption electrospray ionisation mass spectrometry is a surface analysis technique that can rapidly analyse the lipid and metabolite composition of a tissue section. By moving the tissue under the analysis probe, a chemical map of the sample can be generated in a matter of minutes. As has been shown in the literature 1,2, each tissue type or disease state has its own characteristic molecular fingerprint, therefore, using histologically annotated databases of these molecular profiles these maps can be classified using machine learning approaches. With the increased rate of analysis made possible by recent technological advances, the timeframes of a single sample makes automation an attractive proposition. With a slide loading system and a range of software solutions, up to two hundred slides can be queued for analysis. Each will be loaded in turn onto the analysis stage, the location of the tissue section determined with optically directed object detection algorithms, and the analysis started. During the analysis the information from each pixel is projected into the classification model and colour coded by designated tissue type. Applications of this approach that have been demonstrated from a research perspective are the analysis of serial sections of drug dosed spheroids and tumour xenografts for studying the chemical heterogeneity of these systems in three dimensions, in the latter case allowing comparison with other imaging modalities such as MRI and PET. Initial studies will also be presented which will investigate the use of the system for the classification of large cohorts of unknown tissues with subsequent comparison to histological classification by conventional methodologies.l 1. Calligaris, D.Caragacianu, D.Liu, et al; PNAS, 2014, 111, 15185-15189 2. Guenther, S., Muirhead, L.J., et al, Cancer Res, 2015, 75, 1828-1837

Citation Format: Emrys A. Jones, Richard Chapman, Emmy Hoyes, Lukasz Migas, Fiona Henderson, Steven Pringle, James Langridge, Adam McMahon. Fully automated tissue classification by imaging mass spectrometry, moving towards a hundred samples a day [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5654.
Abstract 566: High dimensional single cell analysis predicts response to anti-PD-1 immunotherapy
Authors: Krieg Carsten , Nowicka Malgorzata , Guglietta Silvia , Schindler Sabrina , Hartmann Felix J. , Weber Lukas M. , Dummer Reinhard , Robinson Mark D. , Levesque Mitchell P. , Becher Burkhard
Published: August 2018

Checkpoint inhibition of programmed cell death protein 1 (PD-1) has revolutionized cancer therapy. As more and more patients with metastatic melanoma but also other cancers show a dramatic increase in progression-free survival, still a large proportion of patients do not show durable response. As options for monotherapies, as well as combination immunotherapies, increase in the clinics, predictive biomarkers of clinical response are urgently needed. We employed high-dimensional single cell mass cytometry and a machine-learning bioinformatics pipeline for the in-depth characterization of all immune cells in the peripheral blood of stage IV melanoma patients before and at 12 weeks of anti-PD-1 immunotherapy. We observed a clear treatment response of patients under immunotherapy. However, a strong predictor of progression free and overall survival in response to anti-PD-1 immunotherapy before therapy was the frequency of CD14+CD16-HLA-DRhi monocytes. We could confirm this by conventional flow cytometry in an independent, blinded validation cohort and propose this as a novel predictive biomarker for therapy decisions in the clinic. We further present a high dimensional analysis workflow to analyze predictive and prognostic immune biomarkers during future clinical trials.

Citation Format: Carsten Krieg, Malgorzata Nowicka, Silvia Guglietta, Sabrina Schindler, Felix J. Hartmann, Lukas M. Weber, Reinhard Dummer, Mark D. Robinson, Mitchell P. Levesque, Burkhard Becher. High dimensional single cell analysis predicts response to anti-PD-1 immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 566.
Abstract 5664: Chemical proteomic platforms to probe metabolic heterogeneity in the tumor microenvironment
Authors: Moellering Raymond E.
Published: August 2018

Proteomic platforms provide quantitative snapshots of the proteins present in a biological sample. Despite widespread use, these methods typically require homogenization of samples, signal-averaging over thousands-to-millions of cells and provide no information on protein function. This talk will focus on several novel technologies to circumvent these limitations and to interrogate protein activity within the complex architecture of biological samples, and on spatial scales ranging from single cells to whole organisms. At the single cell level we have developed a chemical proteomic technology, activity-dependent proximity ligation, which enables multiplexed quantification of enzyme activity with high spatial resolution in cellular co-culture and in patient tissue samples. Application of this platform has enabled detection of several metastasis-associated biomarker activities in primary patient samples, as well as simultaneous detection of activity and mRNA expression profiles. These data provide insight into the progression lineage of metastatic ovarian cancers. On the other end of the spectrum, we have recently developed a first-in-class activity-dependent PET imaging probe that specifically and irreversibly labels one of these enzyme biomarkers in vivo. This provides an opportunity to detect aggressive tumor populations, both at sites of primary occurrence and metastasis, with this novel PET radiotracer. Together, these approaches offer new paradigms for studying protein function in basic and translational biology and medicine, ideally providing new tools and biological hypotheses to understand and target tumor malignancy.

Citation Format: Raymond E. Moellering. Chemical proteomic platforms to probe metabolic heterogeneity in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5664.
Abstract 5671: Humanized mouse models for evaluation of cancer therapies
Authors: Mirjolet Jean-Francois , Caradec Josselin , Duchamp Olivier , Bichat Francis , Mignard Caroline
Published: August 2018

Mice with a humanized immune system, so called “humanized” mouse models, can be used to study the complex interactions between the human immune system and tumor cells. In order to assess compounds efficiency in immune-oncology, the in vivo model should recapitulate the biological characteristics of the human tumor and the related immune microenvironment. The choice of mouse strain is the first critical factor as genetically engineered mouse strains differentially express cytokines and growth factors. Then the choice of human antigen/target to be assessed drives the choice of the tumor models and the immune population to be transferred into mice. We developed on different immunodeficient mouse strains multiple humanization strategies using either human PBMCs, Hematopoietic Stem Cells (HSCs), or specific human immune cells such as Dendritic Cells (DCs), T cells, subset of T cells (e.g. gamma9 delta2 T cells) and NK cells. We also developed mouse humanization models using combinations of immune subpopulations such as co-transfer of autologous T cells and DCs.Humanized models were then used for in vivo proof of concept studies with mice xenografted with cell lines (human disseminated lymphoma, orthotopic/subcutaneous solid tumors) or patient-derived xenografts. Tumor-bearing humanized mice were treated with cancer therapeutics such as bispecific antibodies, ADCC-inducing antibodies, Treg targeting antibodies, TLR agonists, vaccines and adoptive T cells transfer therapeutic antibodies. Therapeutics efficiency was assessed by following up mice survival and tumor growth. The impact of therapeutics on tumors and immune cells was also assessed by flow cytometry and immunohistochemistry analyses. In case of disseminated lymphoma and ovarian human tumor in mice reconstituted with human PBMCs, significant antitumor activity of bispecific antibodies were evidenced by an increase in survival, decrease in specific biomarkers (CA125 for ovary) and decrease of residual disease.

Citation Format: Jean-Francois Mirjolet, Josselin Caradec, Olivier Duchamp, Francis Bichat, Caroline Mignard. Humanized mouse models for evaluation of cancer therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5671.
Abstract 5683: Value of new spontaneous and carcinogen-induced mouse-derived isograft (MDI) tumor models for drug development of novel immune therapeutic approaches
Authors: Beshay Janette , Jantscheff Peter , Obodozie Cynthia , Schaechtele Christoph , Weber Holger
Published: August 2018

Checkpoint inhibitor treatment has already become a common therapy of various cancer types. However, there is still a growing need for well-characterized preclinical mouse models, as clinical data indicate that patients only partially respond to this regiment. Currently, cell lines cultured from the 1970s are used frequently to evaluate novel therapies. In a research development program, new mouse-derived isografts (MDI) were established from spontaneously occurring and carcinogen-induced tumors. These tumors are transplanted as tissue pieces in a PDX-like manner and tested for their solid growth. Furthermore, the efficacy of immune checkpoint inhibitor treatment is evaluated and the presence of different immune cell populations in the tumor is characterized by FACS analysis. In addition, RNA-seq data complete the first characterization of these models and will give insights into the expression level and genetic modification of genes in these models. Here, the spontaneous MDI model JA-0009 and two carcinogen-induced MDI models JA-2042 and JA-2011 are presented. Whereas JA-0009 and JA-2011 tumors are only partially affected by immune checkpoint inhibitor treatment, JA-2042 shows a significant response in a combined treatment of anti-PD-1/anti-CTLA-4. Characterization of the infiltrated immune populations revealed a high number of T cells, especially Treg cells. In contrast, the JA-0009 tumor is infiltrated mainly by macrophages of M2 type with a low number of CD8+ T cells and the JA-2011 tumor by neutrophils. Taken together, the new MDI models complement the existing GEMs and cell-line derived syngeneic tumor models by their new properties (low passage number, no tissue culture, conserved original tumor properties and intra-tumoral immune cell populations) to characterize novel immune-modulatory agents in drug development.

Citation Format: Janette Beshay, Peter Jantscheff, Cynthia Obodozie, Christoph Schaechtele, Holger Weber. Value of new spontaneous and carcinogen-induced mouse-derived isograft (MDI) tumor models for drug development of novel immune therapeutic approaches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5683.
Abstract 5686: Utilization of fluorescent multiplex IHC and digital image analysis for studying LAG-3, CD3 and CD8 positive TIL subsets in NSCLC tissue
Authors: Khenkhar Malik , Biljes Daniel , Winkler Nickels , Uhlig Philipp C. , Juhl Hartmut , Bigley Alison L. , Sherry Lorcan
Published: August 2018

Targeting checkpoint molecules expressed on immune cells has shown promising results in the treatment of non-small cell lung cancer (NSCLC). One interesting molecule that is frequently expressed on exhausted tumor-infiltrating lymphocytes (TIL) is lymphocyte activation gene-3 (LAG-3). We recently implemented chromogenic anti-LAG-3/CD3 dual immunohistochemistry (IHC) and digital image analysis to quantify LAG-3 positive T cells in NSCLC tissue. Here, we extended this work by establishing an anti-LAG-3/CD3/CD8/pan-Cytokeratin (pan-CK) fluorescent multiplex IHC (mIHC) assay followed by digital image analysis to examine the composition of T cells infiltrating NSCLC tissue in more detail. Tyramide signal amplification (TSA) based fluorescent 5-color mIHC (LAG-3/CD3/CD8/pan-CK + DAPI) was developed by Indivumed on the Leica BOND RX automated staining platform and applied to formalin-fixed paraffin-embedded (FFPE) NSCLC tissue samples. Image analysis was performed by OracleBio. Tumor and stroma regions of interest (ROI) were classified according to the pan-CK and DAPI signals. LAG-3, CD3 and CD8 single positive cells, as well as dual and triple positive cells, were then quantified in the tumor and stroma ROIs. Fluorescent mIHC allowed for a specific quantification of LAG-3, CD3 and CD8 single, LAG-3/CD3, LAG-3/CD8, CD3/CD8 dual and LAG-3/CD3/CD8 triple labeled cells in the tumor and stroma ROIs of the analyzed NSCLC samples. As part of the assay validation, similar ratios of the differently labeled cells, in particular LAG-3 positive T cells (LAG-3/CD3 dual positive), were observed with fluorescent mIHC and chromogenic IHC, demonstrating a good concordance between the two approaches. Among the analyzed NSCLC samples, different ratios of LAG-3 positive TILs in adeno- and non-adenocarcinoma samples were detected. Determining the ratios of dual or triple positive TIL subsets by mIHC in combination with digital image analysis allows for a clearer understanding of the lymphocyte composition within NSCLC tissue than a simple quantification of LAG-3, CD3 and CD8 single positive cells. Furthermore, such information will contribute to a deeper understanding of the role of LAG-3 positive immune cell sub-populations in the progression and treatment of NSCLC.

Citation Format: Malik Khenkhar, Daniel Biljes, Nickels Winkler, Philipp C. Uhlig, Hartmut Juhl, Alison L. Bigley, Lorcan Sherry. Utilization of fluorescent multiplex IHC and digital image analysis for studying LAG-3, CD3 and CD8 positive TIL subsets in NSCLC tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5686.
Abstract 5705: The key role of kynurenine in anti-PD-1 failure
Authors: Botticelli Andrea , Cerbelli Bruna , Lionetto Luana , Zizzari Ilaria , Pisano Annalina , Roberto Michela , Onesti Elisa , Pietro Francesca Romana DI , Napoletano Chiara , Pizzuti Laura , Vici Patrizia , D'Amati Giulia , Mazzuca Federica , Simmaco Maurizio , Nuti Marianna , Marchetti Paolo
Published: August 2018

Background: Immune checkpoint inhibitors have revolutionized treatment and outcome of severe and often fatal disease, as metastatic lung cancer, demonstrating long-term tumor control and extended patient survival. Unfortunately, only 25-30% of patients have a long-term benefit from immunotherapy, while the remaining 70-75% demonstrate primary or acquired resistance. Recently, indoleamine 2,3-dioxygenase (IDO) has been proposed as a possible mechanism of resistance to anti-PD1 treatment. Indeed, IDO catalyzes the degradation of tryptophan (Trp) into kynurenine (Kyn), which seems to enhance the activity of Treg, leading to an immunosuppressive microenvironment.

Methods: The serum concentrations of Trp and Kyn were measured by high-performance liquid chromatography tandem mass spectrometry in 26 patients affected by non-small cell lung cancer (NCLSC) before the start of the second-line therapy with nivolumab. The IDO activity was expressed with Kyn/Trp ratio. The associations between Kyn/Trip ratio and early progression, PS, age, sex, brain metastases and pleural effusion were analyzed using Spearman test and Mann Whitney test.

Results: 14 out of 26 patients (54%) presented early progression (defined as progression of the disease within 6 months from the beginning of nivolumab treatment). The median value of Kyn/Trp ratio was 0.073 (0.024-0.18). We found a significant association between Kyn/Trp ratio and early progression (p= 0.009), while no statistical associations were found between Kyn/Trp ratio and PS, age, sex, brain metastases and pleural effusion. Indeed, patients with early progressive disease presented a median value of Kyn/Trp ratio significantly higher than other patients (0.094 vs 0.052; p= 0.01).

Conclusion: The pretreatment evaluation of IDO activity, expressed as Kyn/Trp ratio, seems to be associated with response to immunotherapy. In particular, higher Kyn/Trp ratio could predict resistance to anti-PD-1 treatment. These preliminary results suggest the possibility of using anti-PD-1 plus IDO inhibitor in patients with high level of Kyn/Trp ratio.

Citation Format: Andrea Botticelli, Bruna Cerbelli, Luana Lionetto, Ilaria Zizzari, Annalina Pisano, Michela Roberto, Elisa Onesti, Francesca Romana DI Pietro, Chiara Napoletano, Laura Pizzuti, Patrizia Vici, Giulia D'Amati, Federica Mazzuca, Maurizio Simmaco, Marianna Nuti, Paolo Marchetti. The key role of kynurenine in anti-PD-1 failure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5705.
Abstract 5707: On-treatment changes in CRP as a surrogate for overall survival for atezolizumab benefit in 2nd line NSCLC
Authors: Patil Namrata , Zou Wei , Mocci Simonetta , Gupta Vinita , Molinero Luciana , Sandler Alan , Ballinger Marcus , Flynn Susan , Kowanetz Marcin , Hegde Priti
Published: August 2018

Background: Overall survival (OS) is an optimal endpoint for evaluation of checkpoint inhibitors (CPI). Identification of early surrogates of OS is important to inform early clinical signals and treatment decisions. We evaluated C-reactive protein (CRP) in clinical trials with atezolizumab (anti-PD-L1 antibody). CRP is elevated in different inflammatory and malignant conditions and is often considered to be a marker of poor prognosis.

Materials and Methods: Serum from patients enrolled in the 2L+ NSCLC studies POPLAR and OAK (randomized phase 2 and phase 3 controlled trials of patients with non-small cell lung cancer (NSCLC) who progressed on post-platinum chemotherapy) was analyzed for CRP levels over time. Patients were binned by changes in CRP at 6 weeks post-treatment (C3D1; Cycle 3 Day 1) as compared to pre-treatment levels, by either greater than 1.5 fold, less than 1.5 fold or unchanged (within +1.5 fold).

Results: C3D1 decreases in serum CRP were associated with RECIST1.1 based responses to atezolizumab but not with docetaxel in the POPLAR study, and these findings were further validated in the OAK study. Patients with RECIST based disease progression (PD) demonstrated an increase in serum CRP as early as Cycle 2 (3 weeks post atezolizumab therapy). Notably, decrease in CRP levels was strongly associated with improved PFS and OS. Results from OAK are shown below (table). Furthermore, in patients with RECIST PD or stable disease (SD), decrease in on-treatment CRP also predicted long overall survival.

Conclusion: CRP is a soluble biomarker that is modulated post-atezolizumab treatment. This simple lab test may provide utility in informing OS benefit for CPIs and combinations.

C3D1 CRP fold change relative to pre-treatmentHRCIp valueHRCIp valueITT0.750.62-0.910.0040.740.6-0.910.0053CRP <1.5 FC0.480.33-0.690.0000950.490.32-0.750.0011CRP +1.5 FC0.850.62-1.150.280.610.43-0.860.0049CRP>=1.5 FC0.960.69-1.340.811.250.86-1.810.25

Citation Format: Namrata Patil, Wei Zou, Simonetta Mocci, Vinita Gupta, Luciana Molinero, Alan Sandler, Marcus Ballinger, Susan Flynn, Marcin Kowanetz, Priti Hegde. On-treatment changes in CRP as a surrogate for overall survival for atezolizumab benefit in 2nd line NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5707.
Abstract 5709: Effect of IL-18 on the expansion and phenotype of human natural killer cells
Authors: Senju Hiroaki , Kumagai Asuka , Nakamura Yoichi , Takemoto Shinnosuke , Fukuda Masaaki , Gyotoku Hiroshi , Nakatomi Katsumi , Fukuda Minoru , Mukae Hiroshi , Tanaka Yoshimasa
Published: August 2018

When pathogenic stresses are recognized by innate immune cells, inflammasomes are assembled and caspase-1 is activated, resulting in the conversion of pro-IL-18 into mature IL-18. Because natural killer (NK) cells express IL-18 receptors, IL-18 may play roles in the immune functions of NK cells. In the present study, we examined the effect of IL-18 on NK cells derived from lung cancer patients and healthy adult volunteers. When peripheral blood NK cells were stimulated with IL-2, the cells formed clusters beginning on day 5-6 and proliferated thereafter, in which the number of NK cells increased by 10-fold in 10 days. When IL-18 was added, cell clusters were observed as early as on day 4 and NK cells proliferated vigorously. On day 10, the expansion rate was 56-fold on average, showing that IL-18 promoted the expansion of NK cells. It was also notable that IL-18 enhanced the expression of CD80, CD86, HLA-DR and HLA-DQ on NK cells, suggesting that IL-18 conferred NK cells an APC-like phenotype. When cellular cytotoxicity was determined, APC-like NK cells efficiently killed tumor cells and the anti-tumor activity was augmented by the addition of tumor antigen-specific mAbs. In addition, IFN-γ was produced by APC-like NK cells in response to tumor cells, and the cytokine production was further enhanced by mAbs. Taken together, IL-18 not only promoted the expansion of NK cells, but also changed the phenotype of NK cells, and the IL-2/IL-18-induced NK cells might serve as a bridge between innate immunity and adaptive immunity and be useful for cancer immunotherapy.

Citation Format: Hiroaki Senju, Asuka Kumagai, Yoichi Nakamura, Shinnosuke Takemoto, Masaaki Fukuda, Hiroshi Gyotoku, Katsumi Nakatomi, Minoru Fukuda, Hiroshi Mukae, Yoshimasa Tanaka. Effect of IL-18 on the expansion and phenotype of human natural killer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5709.