bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1777: Examining dendritic cell heterogeneity in the tumor microenvironment
Authors: To Tsun Ki Jerrick , Devalajara Samir , Folket Ian W. , Haldar Malay
Published: July 2021

Dendritic cells (DCs) are key regulators of anti-tumor immunity with critical roles in priming and stimulating anti-tumor T cells. DCs have heterogeneous phenotype, ontogeny, activation status, and function. However, the distribution of DC subsets within solid tumors remains unclear. Moreover, the study of intratumoral DCs is hampered by their rarity and difficulties associated with distinguishing DCs from other similar cell types in the tumor microenvironment. Using genetically engineered DC reporter mice (ZBTB46-GFP), we performed single-cell RNA-sequencing (scRNA-seq) on DCs from tumors and normal tissue as an unbiased approach for examining DC heterogeneity. Within our scRNA-seq analysis, we identified the major ontological DC subsets (cDC1s and cDC2s) along with two activated DC subsets: one that expressed a migratory signature (mDC) and the other that expressed an interferon-signaling signature (IFN-DC). By comparing DCs in different tumor models and healthy lung tissue, we found that mDCs were present in both tumor and normal tissue, while IFN-DCs were specific to tumors. IFN-DCs were Sca-1high and were selectively enriched for key anti-tumor interferon-stimulated genes such as Cxcl9 and Cxcl10. Additionally, IFN-DCs were further separated into a type I IFN-enriched subcluster (IFN-DC1) and a type II IFN-enriched subcluster (IFN-DC2). IFN-DC2s were lost in tumors of cDC1-deficient Batf3-KO mice as revealed by scRNA-seq of tumor DCs in Batf3-KO mice. Further experiments suggested that this loss of IFN-DC2 was a cell-extrinsic effect due to the dramatic decrease in IFNy in the tumor microenvironment. Specifically, the decrease in IFNy was associated with the absence of cDC1-primed IFNy-producing CD8 T cells in Batf3-KO tumors. In summary, our analysis of tumor DCs using scRNA-seq and DC reporter mice reveals heterogeneous activated DC subsets, including DCs enriched for distinct type I and type II IFN signaling. Further elucidation of the functional relevance of each subset to the tumor immune response can enable more specific targeting of DCs for cancer immunotherapy.

Citation Format: Tsun Ki Jerrick To, Samir Devalajara, Ian W. Folket, Malay Haldar. Examining dendritic cell heterogeneity in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1777.
Abstract 1793: Regional administration of class C CpG Oligodeoxynucleotides results in superior intrahepatic TLR9 activation and immunomodulation compared to systemic infusion
Authors: Ghosh Chandra , Heatherton Kara , O'Connell Kyle , Laporte Jason , Guha Prajna , Jaroch David , Cox Bryan , Katz Steven C.
Published: July 2021

CpG oligodeoxynucleotides (ODNs) are TLR9 agonists (TLR9As) that stimulate pDC and B cells. Yet, multiple other cell types in the liver express TLR9. The effect of TLR9A in reshaping the tumor microenvironment (TME), particularly in the myeloid-derived suppressor cells (MDSC) compartment, is yet to be determined. Clinical success of TLR9As has been limited by toxicity when given systemically and delivery limitations when administered by direct needle injection. Here, we hypothesized that regional intra-vascular infusion of TRL9As would reprogram the liver MDSC compartment to enable immune control of liver metastases (LM). In this study, we evaluated the effect of class C ODN-2395 as TLR9A in inhibiting LM progression and its impact on the liver MDSC population. C57/BL6 mice were challenged with 2.5e6 MC38-CEA-Luc cells via intra-splenic route. After a week, mice were treated with 1, 3, 10 or 30 µg ODN2395 via PV or IV (30 µg). Bioluminescence was measured at 24 and 48 hrs post-ODN administration. CD45+ cells were isolated, and FACS analysis was performed to quantify MDSCs, monocytic MDSCs (M-MDSC), and M1-macrophage subsets, along with downstream signaling. We found that 30 µg ODN2395 delivered via PV compared to IV reduced tumor burden at 24 hrs and persisted up to 48 hrs. Also, 30 µg ODN2395 infused via PV was optimal in controlling MDSC, M-MDSC cells and polarized CD11b+F4/80+ monocytic cells towards pro-inflammatory/anti-tumorigenic M1 macrophage. Using an NFκB-dependent soluble alkaline phosphatase assay (SEAP), we determined that ODN2395 dose-dependently enhanced NFκB transcription factor activity (p<0.001). Western Blot data of tumor lysates show that ODN delivery by PV increased NFκB (pP65) activity and production of IL-6, significantly relative to IV. Overall, our data suggest that the regional delivery of a TLR9A has the potential to provide superior modulation of the TME within the liver.

Table:Readout30µg IV30µg PVP valuenBioluminescence-24 h (photon/s)4e71e7<0.0113Bioluminescence-48h (photon/s)7e72e7<0.0513FACSCD11b+Gr1+ cells/CD45+ NPC (MDSC)29.70 %20.75%<0.0113CD11b+LY6C+ (M-MDSC)60.03%38.98%<0.0113CD11b+F4/80+CD38+EGR2-(M1 macrophage)34.82%58.20%<0.0113Western BlottingpNFκB/NFκB (pP65s276/pP65): Fold change0.862.028<0.016IL6/GAPDH Fold change12.387<0.056

Citation Format: Chandra Ghosh, Kara Heatherton, Kyle O'Connell, Jason Laporte, Prajna Guha, David Jaroch, Bryan Cox, Steven C. Katz. Regional administration of class C CpG Oligodeoxynucleotides results in superior intrahepatic TLR9 activation and immunomodulation compared to systemic infusion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1793.
Abstract 180: Identifying signatures of vulnerability through machine learning in an umbrella trial for glioblastoma
Authors: Lee Matthew Eric , Tang Nanyun , Ahluwalia Manmeet , Fonkem Ekokobe , Fink Karen , Dhurv Harshil , Dhurv Harshil , Berens Michael E. , Peng Sen
Published: July 2021

Glioblastoma is characterized by intra- and inter-tumoral heterogeneity. An umbrella trial tests multiple investigational treatment arms depending on corresponding biomarker signatures. A contingency of an efficient umbrella trial is a suite of preferably orthogonal molecular biomarkers to classify patients into the likely-most-beneficial arm. Assigning thresholds of molecular signatures to classify a patient as a “most-likely responder” for one specific treatment arm is a crucial task. Gene Set Variation Analysis (GSVA) of specimens from a GBM clinical trial of methoxyamine associated differential enrichment in DNA repair pathways activities with patient response. However, the large number of DNA-repair related pathways confound confident “high” enrichment of responder, as well as obscuring to what degree each feature contributes to the likelihood of a patient's response. Here, we utilized semi-supervised machine learning, Entropy-Regularized Logistic Regression (ERLR) to predict vulnerability classification. By first training all available data using semi-supervised algorithms we transformed unclassified TCGA GBM samples with highest certainty of predicted response into a self-labeled dataset. In this case, we developed a predictive model which has a larger sample size and potential better performance. Our umbrella trial design currently includes three treatment arms for GBM patients: arsenic trioxide, methoxyamine, and pevonedistat. Each treatment arm manifests its own signature developed by the above (or similar) machine learning pipeline based on selected gene mutation status and whole transcriptome data. In order to increase the robustness and scalability (with future more treatment arms), we also developed a multi-label classification ensemble model that's capable of predicting a probability of “fitness” of each novel therapeutic agent for each patient. By expansion to three, independent treatment arms within a single umbrella trial, a “mock” stratification of TCGA GBM patients labeled 56% of all cases into at least one “high likelihood of response” arm. Predicted vulnerability using genomic data from preclinical PDX models placed 4 out of 6 models into a “high likelihood of response” regimen. Our utilization of multiple vulnerability signatures in an umbrella trial demonstrates how a precision medicine model can support an efficient clinical trial for heterogeneous diseases such as GBM.

Citation Format: Matthew Eric Lee, Nanyun Tang, Manmeet Ahluwalia, Ekokobe Fonkem, Karen Fink, Harshil Dhurv, Harshil Dhurv, Michael E. Berens, Sen Peng. Identifying signatures of vulnerability through machine learning in an umbrella trial for glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 180.
Abstract 1801: Development of a comprehensive biomarker strategy to support phase 1 clinical trial of SRK-181 the latent TGFβ1 inhibitor
Authors: Brueckner Christopher , Faucette Ryan , Caldwell Charles , Reitsma Sofia , Wawersik Stefan , Kalra Ashish , Gan Lu , Lee-Hoeflich Si Tuen
Published: July 2021

TGFβ signaling appears to be a key mediator of primary resistance to programmed cell death protein (PD-1) pathway blockade. SRK-181 is an investigational stage, high-affinity, fully human antibody that selectively binds to latent TGFβ1 and inhibits its activation. Preclinical data demonstrate that combining SRK-181 with a PD-1 inhibitor modulates tumor microenvironment (TME), including an influx of CD8 positive T cells that correlates with anti-tumor responses. The ongoing DRAGON trial is a multi-center, open-label, Phase 1, first in-human (FIH), dose-escalation, and dose-expansion study. The goal of the trial is to evaluate the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and efficacy of SRK-181 administered alone and in combination with an anti-PD-(L)-1 in adult patients with locally advanced or metastatic solid tumors. To support this ongoing DRAGON clinical trial and further explore the mechanism of action of SRK-181, a comprehensive biomarker strategy is being developed to assess the alternation of immune profile in TME and potential predictors of therapeutic response to SRK-181. Here we describe the development and refinement of several biomarker assays. First, an image analysis-based algorithm for CD8 immunohistochemistry (IHC) is established utilizing human cancer tissue in a pre-clinical study. This novel digital pathology analysis enables identification of CD8 positive T cells in discrete compartments, including the tumor nests, stroma and tumor/stromal margins, to better capture the heterogeneity of the CD8 signal within tissues. Second, we describe methods to evaluate the TGFβ pathway including quantitative analysis of tumor tissue phospho-Smad2 and circulatory levels of TGFβ1 ligand. A companion assay to exclude blood samples with nonspecific background signals has been characterized and will be performed in parallel when evaluating circulatory TGFβ1 in clinic. In summary, we present several novel, tailored biomarker readouts that are part of a broader biomarker strategy aimed at maximizing detection of relevant clinical data to both support the ongoing clinical trial and provide further insight into the mechanism of action of SRK-181.

Citation Format: Christopher Brueckner, Ryan Faucette, Charles Caldwell, Jr., Sofia Reitsma, Stefan Wawersik, Ashish Kalra, Lu Gan, Si Tuen Lee-Hoeflich. Development of a comprehensive biomarker strategy to support phase 1 clinical trial of SRK-181 the latent TGFβ1 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1801.
Abstract 1803: Newly emerged immunogenic neoantigens enable to break the resistance of immune checkpoint inhibitors
Authors: Sugiyama Daisuke , Muramatsu Tomoaki , Noguchi Takuro , Akatsuka Yoshiki , Nishikawa Hiroyoshi
Published: July 2021

Immune checkpoint inhibitors (ICIs) and other cancer immunotherapies resurge T cell responses that target tumor antigens expressed by cancer cells, particularly tumor neoantigens derived from genetic alterations. However, tumors establish immune escape mechanisms during tumor development and progression, including the loss of pre-existing tumor antigens and impaired T cell responses. In the last decade, various strategies to overcome the immune escape mechanisms have been investigated in the clinic to achieve a cure for cancer. Here, we show that a newly emerged immunogenic neoantigen changes the tumor environment and inhibits tumor growth. To develop animal models, we employed a Tet-on gene expression system, in which immunogenic neoantigens including chicken ovalbumin (OVA) and human cancer-testis antigen NY-ESO-1 were newly emerged upon doxycycline (Dox) treatment. MC-38 (colorectal cancer) inducing OVA, CT26 (colorectal cancer) inducing NY-ESO-1 and B16 (melanoma) inducing NY-ESO-1 were developed, and these cell lines were administered into mice. Induction of these model neoantigens by Dox treatment after tumor establishment (approximately 8~10 mm) significantly inhibited tumor growth. The antitumor effects were not observed in nude mice lacking T cells or CD8+ T-cell depleted mice, indicating an important role of CD8+ T cells. Furthermore, PD-1/PD-L1 blockade further enhanced CD8+ T cell responses against the newly emerged immunogenic neoantigens, resulting in a far stronger tumor growth inhibition. We then propose that newly-emerged immunogenic neoantigens are sufficient to induce antitumor effects that can overcome immune escape mechanisms in the tumor microenvironment, thereby inhibiting tumor growth and that induction of immunogenic tumor neoantigens is a novel strategy to overcome the resistance to ICI therapy.

Citation Format: Daisuke Sugiyama, Tomoaki Muramatsu, Takuro Noguchi, Yoshiki Akatsuka, Hiroyoshi Nishikawa. Newly emerged immunogenic neoantigens enable to break the resistance of immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1803.
Abstract 1804: Modulation of Nr2f2 reprograms tumor blood vessels to enhance anti-tumor immunity and immunotherapy
Authors: Zhu Yu , Lazarus Nicole , Brulois Kevin , Salazar Nicole , Dinh Theresa , Pan Junliang , Butcher Eugene
Published: July 2021

A critical limitation to cancer immunotherapy is posed by the paucity of anti-tumor T cells in tumor tissues, even after therapeutic intervention. Development of effective strategies to enhance T cell recruitment remains challenging. As a critical regulator of immune cell trafficking, the tumor vasculature is a promising target that can be utilized to enhance anti-tumor immunity. Functional vasculature requires specialization of endothelial cells (ECs): capillary ECs transport oxygen and nutrients to promote tumor growth, while venular ECs regulate the recruitment and extravasation of immune cells. The majority of the current vessel-targeting therapies are designed to deplete endothelial cells indiscriminately with the goal of starving tumor cells. Loss of venules as a consequence could further block sustained recruitment of anti-tumor T cells. An alternative strategy that instead promotes capillary-to-venule “reprogramming” could simultaneously inhibit the tumor-promoting activities of capillaries and preserve the T cell-recruiting functions of tumor vessels, and thus better augment anti-tumor immunity and benefit immunotherapies. Through single cell transcriptomic profiling of tumor ECs, we found the nuclear receptor Nr2f2 to be specifically expressed in tumor-associated venules but not in capillaries. Upon ectopic expression of Nr2f2 in tumor ECs, capillary vessels undergo fate switch and adopt the venular phenotype and functions, as confirmed by immune profiling and single cell transcriptomic analyses. Nr2f2-driven capillary-to-venule reprogramming enhanced the recruitment of cytotoxic T cells into the tumor tissue, and led to a significant reduction of tumor burden in mouse breast and pancreatic tumor models. Interestingly, Nr2f2-driven EC fate switch enhanced the anti-tumor efficacy of immunotherapies, including immune checkpoint blockade and adoptive cell therapy by transferring T cells specific for a surrogate tumor cell antigen. Collectively, these data suggest that strategies that reprogram tumor vessels from capillaries into venules can be exploited to enhance anti-tumor immune responses and sensitize tumors to immunotherapy, especially in immunologically cold tumors.

Citation Format: Yu Zhu, Nicole Lazarus, Kevin Brulois, Nicole Salazar, Theresa Dinh, Junliang Pan, Eugene Butcher. Modulation of Nr2f2 reprograms tumor blood vessels to enhance anti-tumor immunity and immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1804.
Abstract 1811: A novel OX40 humanized mouse model for efficacy assessment of OX40-targeting therapies
Authors: Sônego Fabiane , Martin Gaëlle , Beringer Audrey , Beuraud Chloé , Cherifi Yacine , Fraichard Alexandre , Petit Patricia Isnard , Thiam Kader
Published: July 2021

Immunotherapies through targeting of activating and inhibitory immune checkpoints (ICP) has revolutionized the treatment of cancers. To facilitate the translation of basic research into clinical application of these immunotherapies, we developed a pipeline of immunocompetent mouse models expressing humanized ICP instead of their mouse counterparts, thus compounds can be tested in the absence of endogenous cross-reacting mouse targets. The humanization strategy is target-dependent but ensures that the biology of the target, its physiological regulation and interacting partners are preserved. Herein, we describe a novel OX40 humanized mouse model (hOX40 model), designed to assess the efficacy and mechanism of action of compounds targeting human OX40 (hOX40). The expression of the fully human OX40 protein (human extracellular, transmembrane and intracellular domains) did not alter the distribution of immune cells in spleen, blood and bone marrow. hOX40 was expressed on CD4 and CD8 T cells upon activation and the kinetic of expression mirrored the expression of mouse OX40 in WT cells activated under the same conditions. Treg cells isolated from hOX40 mice expressed hOX40 and this expression was upregulated upon activation, also recapitulating the expression of mouse OX40 in WT cells, and suggesting that the regulation of OX40 expression was kept in the hOX40 mice. hOX40 was also found to be functional on CD4 T cells, as evidenced by the increased proliferation and cytokine production upon stimulation with human OX40 ligand. Importantly, hOX40 mice bearing MC38 tumors showed a tumor growth inhibition upon treatment with an anti-human OX40 therapy with agonist activity. This model should also enable the investigation of mechanism of action as well as tumor immunological memory, as a similar design of the hOX40 model was used for the assessment of a bispecific therapy targeting OX40 and CTLA-4 (Kvarnhammar et.al, 2019). The hOX40 mouse model is currently being crossed with other models expressing humanized immune checkpoint to enable the assessment of bispecifics and combo therapies.

Citation Format: Fabiane Sônego, Gaëlle Martin, Audrey Beringer, Chloé Beuraud, Yacine Cherifi, Alexandre Fraichard, Patricia Isnard Petit, Kader Thiam. A novel OX40 humanized mouse model for efficacy assessment of OX40-targeting therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1811.
Abstract 1815: Defining cell population diversity and T cell dysfunction in a mouse model of obesity
Authors: Baracho Gisele V. , Widmann Stephanie , Tyznik Aaron
Published: July 2021

Obesity creates a chronic inflammatory environment that can lead to an exhausted phenotype among T cells. Since exhausted T cells gradually lose their effector functions, obese patients may be at higher risk of developing different disorders, including infectious diseases and cancer. Using the BD FACSymphonyTM A5 Cell Analyzer, we applied a 21-color high-parameter flow cytometry panel to assess various immune cell types in a mouse model of obesity. Also, a flow-based functional assay provided insights into obesity-associated T cell dysfunction. To address progressive changes that might have been caused by a high-fat diet over time, C57BL/6 mice were fed with a high-fat diet for a period of 4 or 16 weeks. Cell populations were examined in blood, spleen and epididymal fat tissue with the latter being known as a location that harbors local inflammation in obese mice. Computational tools for high-dimensional data analysis allowed a thorough characterization of tissue-specific cell phenotypes and their relationship with obesity. Notably, the frequency of effector/memory CD8 T cells and TCRγδ T cells were increased in the fat of obese mice. However, assessment of T cell effector functions in vitro revealed an impairment in cell proliferation, suggesting that fat CD8 T cells from obese mice may not effectively respond to stimulation. Though further studies are required, these results support previous published evidence that low-grade chronic inflammation associated with obesity may impact T cell responses. The data also represent an efficient stepwise approach for staining of surface and intracellular markers and for evaluation of T cell function in vitro and demonstrates the utility of computational tools available in FlowJo™ v10 Software.

For Research Use Only. Not for use in diagnostic or therapeutic procedures. Class 1 Laser Product.BD-23275 (v1.0) 1120BD, the BD Logo, FACSymphony and FlowJo are trademarks of Becton, Dickinson and Company or its affiliates. © 2020 BD. All rights reserved.

Citation Format: Gisele V. Baracho, Stephanie Widmann, Aaron Tyznik. Defining cell population diversity and T cell dysfunction in a mouse model of obesity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1815.
Abstract 1822: Novel DNA encoded tumor attacking bridges (dTABs) targeting IL13Rα2 display potent tumor control in an animal model of glioblastoma multiforme
Authors: Bhojnagarwala Pratik Sanjivkumar , O'Connell Ryan , Park Daniel , Gary Ebony , Zhu Xizhou , Weiner David
Published: July 2021

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor with poor clinical prognosis and there is significant need for new, improved therapies. Immunotherapy is a growing therapeutic research area for patients with advanced brain cancers. DNA encoded Tumor Attacking Bridges (dTABs) are bispecific antibodies that redirect T cell activity against Tumor Associated Antigens (TAAs) of interest. Here, we describe the design and development of in vivo generated dTABs targeting IL13Rα2, which is expressed in tumors of approximately 75% of GBM patients. Four dTABs targeting 2 different epitopes of IL13Rα2 were designed to compare alternate orientations of heavy and light chains in the dTABs. All dTABs bound to U87 cells expressing target as well as CD3 on T cells. The dTABs induced T cell activation, cytokine production, and tumor killing in the presence of target expressing tumor cells in a dose dependent manner. An important issue in the field of antibody guided therapies is on target - off tumor T cell activity which can cause serious side effects in patients. In order to minimize such toxicities, we identified one arrangement of dTAB which induced high T cell activation with potent tumor killing in the presence of target expressing tumor cells, but not when co-cultured with tumors that do not express IL13Rα2. We were able to detect expression of this lead dTAB for up to 19 days in the serum of NSG mice after a single DNA injection followed by electroporation. Serum from mice injected with this dTAB activated T cells which potently killed target expressing U87 GBM cells. In a subcutaneous xenograft mouse model of human GBM, this in vivo produced dTAB demonstrated significant tumor control and improved survival of mice. This study illustrates the potential of dTABs targeting IL13Rα2 as a novel approach for immunotherapy of this difficult to treat brain cancer.

Citation Format: Pratik Sanjivkumar Bhojnagarwala, Ryan O'Connell, Daniel Park, Ebony Gary, Xizhou Zhu, David Weiner. Novel DNA encoded tumor attacking bridges (dTABs) targeting IL13Rα2 display potent tumor control in an animal model of glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1822.
Abstract 1833: Development of anti-CLDN18.2-anti-CD3 bispecific antibody as cancer therapeutics
Authors: Lin Haishan , Li Huey , Drobenaire Hunter , Pizzarella Veronica , Zhang Richard
Published: July 2021

Claudin-18 (CLDN18) is a member of the large Claudin family of four-span transmembrane proteins, which are essential components of the mammalian tight junctions (TJs) in the epithelial cells. Claudin-18 has two splice variants, CLDN18.1 and CLDN18.2. While CLDN18.1 is specifically expressed in the lung tissue, CLDN18.2 expression in normal tissue is more restricted and is only detected in small patches of stomach mucosal. CLDN18.2 expression is elevated in many types of epithelial cancers including stomach, esophagus, pancreatic and ovarian cancers. Anti-CLDN18.2 monoclonal antibody Claudiximab (IMAB362) demonstrated promising results in clinical trials for gastric cancers. Using a combination of DNA, protein, and transfected cells as immunogens, we have generated a panel of mouse monoclonal antibodies which are highly specific for CLDN18.2 but do not cross-react to CLDN18.1. The lead antibody E9 had higher binding affinity for CLDN18.2, compared to the IMAB362. The humanized anti-CLDN18.2 antibody showed a strong antibody dependent cytotoxicity (ADCC) activity against Mia PaCa-2 cells overexpressing CLDN18.2. To redirect T-cell-mediated lysis toward CLDN18.2-overexpressed tumor cells, we generated an anti-CLDN18.2/anti-CD3 bispecific antibody. We demonstrated that the bispecific antibody binds to both human T cells and CLDN18.2-expressing tumor cells. Furthermore, the anti-CLDN18.2/anti-CD3 bispecific antibody had robust activity in killing Mia PaCa-2 cells overexpressing CLDN18.2 in the in vitro T cell-dependent cellular cytotoxicity (TDCC) assay. In addition, the bispecific antibody retained the in vitro ADCC activity against Mia PaCa-2 cells overexpressing CLDN18.2. Taken together, our data suggest that the anti-CLDN18.2/anti-CD3 bispecific antibody is a promising cancer therapeutic candidate utilizing multiple anti-tumor mechanisms of action, including ADCC and TDCC.

Citation Format: Haishan Lin, Huey Li, Hunter Drobenaire, Veronica Pizzarella, Richard Zhang. Development of anti-CLDN18.2-anti-CD3 bispecific antibody as cancer therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1833.
Abstract 1854: A Fc-competent anti-human TIGIT blocking antibody BGB-A1217 elicits strong immune responses and potent anti-tumor efficacy in pre-clinical models
Authors: Chen Xin , Xue Liu , Ding Xiao , Liu Qi , Zhang Jing , Jiang Lei , Liu Sha , Hou Hongjia , Zhu Qing , Jiang Bin , Zhang Lijie , Zhou Xiaosui , Ma Jie , Li Yucheng , Jin Wei , Wei Min , Shen Zhirong , Liu Mike , Li Kang , Zhang Tong
Published: July 2021

Background: TIGIT (T-cell immunoglobulin and ITIM domain) is a “checkpoint” inhibitory receptor, which is primarily expressed on activated and “exhausted” T and NK cells. Engagement of TIGIT to its ligands (i.e., PVR and PVR-L2) leads to inhibitory signaling in T cells, promoting functional exhaustion of tumor-infiltrating T lymphocytes. BGB-A1217 is a novel humanized IgG1 anti-TIGIT antibody under clinical development. The immunomodulatory activity of BGB-A1217 was evaluated both in vitro and in vivo.

Materials and methods: BGB-A1217 was generated through hybridoma fusion, humanized by CDR grafting and structural simulation. The binding affinity and specificity were studied by FACS and SPR. The immunomodulatory functions of BGB-A1217 were evaluated using primary immune cells as well as using animal models.

Results: BGB-A1217 binds to the extracellular domain of human TIGIT with high affinity (KD = 0.135 nM) and specificity. In a competition assay, BGB-A1217 efficiently blocks the interaction between TIGIT and PVR. In vitro, BGB-A1217 significantly enhances T-cell functions and induces potential ADCC against TIGITHi targets. In a human T-cell assay, BGB-A1217 enhances IFN-γ production of CMV-specific T cells. In a PBMC assay, BGB-A1217 augments T cell response, either alone or in combination with an anti-PD-1 antibody BGB-A317. Besides blocking TIGIT signaling, BGB-A1217 can also remove TIGIT from cell surface through trogocytosis. This activity is Fc-dependent. In vivo, the Fc effector function is critical for the activity of BGB-A1217 against CT26WT tumor implanted in humanized TIGIT knock-in mice. The observed anti-tumor efficacy is associated with pharmacodynamic change of TIGIT down-regulation, CD226 upregulation and Treg depletion at 48hrs after first dosing.

Conclusions: BGB-A1217, either alone or in combination with anti-PD-1 mAb promotes immune cell activation both in vitro and in vivo, supporting its clinical development for the treatment of human cancers.

Citation Format: Xin Chen, Liu Xue, Xiao Ding, Qi Liu, Jing Zhang, Lei Jiang, Sha Liu, Hongjia Hou, Qing Zhu, Bin Jiang, Lijie Zhang, Xiaosui Zhou, Jie Ma, Yucheng Li, Wei Jin, Min Wei, Zhirong Shen, Mike Liu, Kang Li, Tong Zhang. A Fc-competent anti-human TIGIT blocking antibody BGB-A1217 elicits strong immune responses and potent anti-tumor efficacy in pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1854.
Abstract 1856: Donor and antibody diversity in NK cell-mediated antibody dependent cellular cytotoxicity (ADCC) detected using an optimized multiplexed assay and advanced flow cytometry
Authors: Lovchik Julie , Carter Mark
Published: July 2021

Tumor-specific monoclonal antibodies (mAb) which induce NK cell-mediated antibody dependent cellular cytotoxicity (ADCC) have proven to be successful against several types of cancer. Evaluating the potency of a new, tumor-specific mAb requires testing with multiple sources of donor cells since the degree of NK cell-mediated ADCC is known to be variable between patients. Thus, in addition to evaluating whether a new tumor-specific mAb can effectively induce NK cell-mediated ADCC, it is also important to characterize the donor NK cells utilized in order to provide insight into potential treatment efficacy. In the current study, we investigated the application of a novel, high throughput assay in assessing the ADCC potency of different anti-hCD20 mAbs against a Burkitt's lymphoma cell line using effector cells obtained from multiple donors. This multiplexed assay incorporates a cells and beads format to enable the simultaneous measurement of target cell killing, along with enumeration and phenotyping of the NK cells, and quantification of up to 8 secreted effector proteins and cytokines. The assay requires a sample volume of just 10 µl and can be performed in a single well of a 96 or 384 well plate using the iQue® platform for advanced flow cytometry. Data analysis and visualization was done using the integrated iQue Forecyt® software package. The results showed a mAb concentration-dependent increase in tumor cell killing, along with corresponding increases in secretion of Granzyme B and CD178 (Fas Ligand), and production of IFNγ, TNF, and RANTES. The degree of NK cell-mediated ADCC and effector protein secretion levels were dependent on both the anti-hCD20 mAb and donor cells used, with higher levels of ADCC and effector protein secretion observed using a non-fucosylated anti-hCD20 mAb as compared to a conventional, fucosylated anti-hCD20 mAb. This method enabled the rapid assessment of the potency of different mAbs in ADCC mediated by NK cells, and also effectively distinguished potential differences in mAb efficacy between donors.

Citation Format: Julie Lovchik, Mark Carter. Donor and antibody diversity in NK cell-mediated antibody dependent cellular cytotoxicity (ADCC) detected using an optimized multiplexed assay and advanced flow cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1856.
Abstract 1867: Characterization in patient derived tumor organoids of novel oncolytic adenoviruses expressing enhanced cross-hybrid IgGA Fc PD-L1 inhibitors
Authors: Hamdan Firas , Ylösmäki Erkko , Chiaro Jacopo , Giannoula Yvonne , Long Maeve , Fusciello Manlio , Feola Sara , Martins Beatriz , Feodoroff Michaela , Antignani Gabriella , Kari Otto , Lee Moon Hee , Järvinen Petrus , Nisen Harry , Kreutzman Anna , Mustjoki Satu , McWilliams Thomas G , Grönholm Mikaela , Cerullo Vincenzo
Published: July 2021

Despite the success of immune checkpoint inhibitors in the clinic, they only benefit a fraction of patients. A theoretical strategy to increase efficacy would be to enhance such antibodies with Fc-mediated effector mechanisms. Current IgG1 antibodies are excellent activators of natural killer cells yet neglect a crucial effector population, neutrophils. Hence, we designed a cross-hybrid Fc-fusion peptide against PD-L1 able to elicit simultaneously effector mechanisms of an IgG1 but also IgA1, consequently activating neutrophils, in order to combine multiple effector mechanisms. Moreover, to prevent toxicities, these Fc-fusion peptides were cloned in oncolytic adenoviruses whose replication is restricted to the tumor. Our oncolytic adenoviruses were able to selectively infect tumor cells, secrete the cross-hybrid Fc-fusion peptides able to bind to PD-L1 and activate multiple immune components enhancing tumor cytotoxicity compared to FDA-approved immune checkpoint inhibitors. We validated this in various human and murine cancer cell lines and also renal cell carcinoma patient derived organoids from four patients. In conclusion, our cross-hybrid Fc-fusion peptides demonstrate that activating multiple immune effector populations increases tumor cytotoxicity potentially leading to improved clinical outcomes.

Citation Format: Firas Hamdan, Erkko Ylösmäki, Jacopo Chiaro, Yvonne Giannoula, Maeve Long, Manlio Fusciello, Sara Feola, Beatriz Martins, Michaela Feodoroff, Gabriella Antignani, Otto Kari, Moon Hee Lee, Petrus Järvinen, Harry Nisen, Anna Kreutzman, Satu Mustjoki, Thomas G McWilliams, Mikaela Grönholm, Vincenzo Cerullo. Characterization in patient derived tumor organoids of novel oncolytic adenoviruses expressing enhanced cross-hybrid IgGA Fc PD-L1 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1867.
Abstract 1868: A long acting bi-specific T cell engager differentially targeting CD47 positive malignant cells but not CD47 expressing healthy cells
Authors: Liu Shumin , Lyu Weidong , Lei Yang , Tan Shuangyu , Wu Zibin , Yin Shuqiang , Zheng Liling , Zhuo Qiudong , Wen Yu (Yvonne) , Wu David
Published: July 2021

Currently both bispecific T cell engagers (BiTE) and innate immune checkpoint blockades (eg. Anti-CD47/SIRPα) have been under development. In this study, we developed a novel compound pegylated anti-CD3 x anti-CD47 which could act with dual mechanisms of both a BiTE and an innate immune checkpoint blockade. The compound did not induce hemagglutination of RBC cells at the extremely high concentration of 2250 ug/ml, while the corresponding monoclonal antibody Hu5F9-G4 did at the concentration as low as 16.67 ug/ml. Meanwhile, in a T cell apoptosis assay, the apoptotic T cells were 16.13%, only 5.9% higher than that of the control T cell sample at the concentration of 100ug/ml of the compound and were about the same as the control sample (10.23%) at concentration of 10 ug/ml of the compound. For comparison, the reported T cell apoptosis induced by 10ug/ml Hu5F9-G4 was as high as 70%. Furthermore, the target affinity by flow cytometry demonstrated that at the concentration 10ug/ml of the compound, stained RBCs were 0.551%, while the compound bound BxPC3 cells were 22.1%, a 40-fold difference in binding. At the concentration 100ug/ml of the compound, only 7.24% RBCs were stained positively, but stained BxPC3 cells were as high as 89.3%. Although not completely understood yet, unique structure of the compound (PEGylated scFv antibody v.s. Fc-bearing antibodies) may have contributed to the differential effect of the compound to healthy cells and tumor cells, resulting in high safety performance of the compound. Furthermore, pharmacokinetics study demonstrated that in vivo elimination half-life of the compound in wild type C57BL/6 mice was 18.4 hours for the single dose of 1mg/kg. Further preclinical study about the compound is still ongoing.

Citation Format: Shumin Liu, Weidong Lyu, Yang Lei, Shuangyu Tan, Zibin Wu, Shuqiang Yin, Liling Zheng, Qiudong Zhuo, Yu (Yvonne) Wen, David Wu. A long acting bi-specific T cell engager differentially targeting CD47 positive malignant cells but not CD47 expressing healthy cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1868.
Abstract 1872: Generation of bispecific and trispecific T cell engagers (TCEs) and NK cell engagers (NKE)s without prior IgG reengineering
Authors: van Geel Remon , van Delft Floris
Published: July 2021

T cell and NK cell-redirecting antibodies are emerging as promising modalities in targeted immunotherapy. New technologies are overcoming the limitations of 1st generation immune cell-redirecting bispecifics, especially extending half-life, reducing immunogenicity and improving safety. For example, in the field of T cell engagers (TCEs), a plethora of molecular architectures has entered the clinic, the vast majority generated by genetic engineering of an IgG to bind both a tumor-associated antigen (TAA) and CD3 or 4-1BB. Similarly, recombinant DNA technology enables the C-terminal fusion of a cytokine (e.g. IL-2 or IL-15) to an IgG to obtain NK cell engagers (NKEs).

We have shown earlier that the native glycan of a monoclonal antibody provides a privileged site for attachment of cytotoxic payloads,1 providing antibody-drug conjugates (ADCs) with significantly enhanced therapeutic index. This technology, known as GlycoConnect™, has been adapted by various drug developers (e.g. ADC Therapeutics, Mersana Therapeutics and others) and is now applied in two (2) clinical programs as well as multiple late-stage preclinical programs.

Here we demonstrate how GlycoConnect™ technology can be applied to generate TCEs or NKEs by selective attachment of scFv (anti-CD3, anti-CD28) or cytokines (IL-15), with concomitant Fc-silencing. Moreover, an existing bispecific TCE (TAA/CD3) can be further boosted by attachment of anti-CD28 scFv for further T cell activation. Molecular architectures can be readily tailored to 2:2 or 2:1 ratio (TAA:scFv or cytokine) to mitigate adverse events like cytokine release syndrome, and even 2:1:1 formats. Structural and functional studies will be presented to corroborate the architecture and biological activity of these GlycoConnect™ bispecifics and trispecifics, in terms of binding, immune cell engagement and cell-killing potential.

1van Geel et al. Chemoenzymatic Conjugation of Toxic Payloads to the Globally Conserved N-Glycan of Native mAbs Provides Homogeneous and Highly Efficacious Antibody−Drug Conjugates. Bioconj. Chem. 2015, 26, 2233-2242.

Citation Format: Remon van Geel, Floris van Delft. Generation of bispecific and trispecific T cell engagers (TCEs) and NK cell engagers (NKE)s without prior IgG reengineering [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1872.
Abstract 1879: CIT-801: a bifunctional anti-PD1xIL15 agent demonstrated synergy and enhanced in vivo anti-tumor efficacy
Authors: Li Yiwen , Hu Yuxiang , Wang Welson , Wu Wei
Published: July 2021

PD1/PD-L1 antibody has revolutionized cancer therapy. However, only a subset of cancer patients are responsive to anti-PD1 antibody treatment. Lack of immune effector cells in the tumor microenvironment (cold tumor) is considered a major reason for such a resistance. Interleukin 15 (IL15) is a cytokine critical for the survival and proliferation of T-cells and NK cells. We hypothesized that a combination of IL15 with anti-PD1 antibody might turn a cold tumor into a hot tumor and overcome tumor resistance to PD1 therapy, and therefore benefit a larger cancer patient population. We have constructed a homodimeric bifunctional agent (CIT-801) that is composed of an anti-PD1 antibody (CIT-100) and IL15 plus the sushi domain of IL15Rα. In vitro, this bifunctional anti-PD1xIL15 agent retained the ability of its anti-PD1 component to enhance T-cell activation in both mixed leukocyte reaction (MLR) and PD1 luciferase reporter assays. It also retained the ability of its IL15 component to stimulate the proliferation of T-cells. Moreover, the bifunctional agent was able to enhance the activation, proliferation and cytotoxicity of NK cells in vitro. In a syngeneic MC38 tumor model with human-PD1 knock-in mice, the bifunctional anti-PD1xIL15 agent strongly inhibited tumor growth, superior to anti-PD1 antibody treatment alone. In summary, anti-PD1xIL15 bifunctional compound CIT-801 has shown a great therapeutic potential and is worth further development.

Citation Format: Yiwen Li, Yuxiang Hu, Welson Wang, Wei Wu. CIT-801: a bifunctional anti-PD1xIL15 agent demonstrated synergy and enhanced in vivo anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1879.
Abstract 1885: Potent tumoricidal activity of a FGFR4 CART in rhabdomyosarcoma
Authors: Cheuk Adam , Tian Meijie , Kumar Jeetendra , Azorsa Peter , Shivaprasad Nityashree , Schneider Dina , Gryder Berkley , Wei Jun , Song Young , Wen Xinyu , Sindiri Sivasish , Chung Joon-Yong , Zhu Zhongyu , Dimitrov Dimiter , Hewitt Stephen , Dropulic Boro , Orentas Rimas , Khan Javed
Published: July 2021

Despite decades of multi-module therapies, RMS remains incurable once it has metastasized, thus new therapeutic strategies are warranted. FGFR4 is a developmentally regulated cell surface receptor tyrosine kinase, overexpressed in virtually all, mutationally activated in about 10% of RMS, and directly activated by PAX3-FOXO1 fusion protein which makes it a tractable target for immunotherapy. We generated fifteen binders against FGFR4 and characterized them further as candidate molecules for immune therapy. We found that 3A11, a mouse IgG antibody, bound to FGFR4 positive cell lines. The VL and VH domain of 3A11 was cloned and made into scFvFc format (mouse Fv and Human IgG1 Fc). The chimeric form of 3A11 antibody was successfully produced in vitro and retained its FGFR4 specificity with an observed binding affinity at nanomolar range. By ELISA using the extra cellular domain of human FGFR1, FGFR2, FGFR3 or FGFR4, 3A11 scFvFc showed dose dependent binding to FGFR4 only. We then made 3A11 into a second generation CAR. Human T cells transduced with 3A11 CAR construct were found to be highly potent at inducing IFN-γ, TNF-α, IL-2 and cytotoxicity when the FGFR4-CART was co-cultured with RMS cells, but not with RMS cells with FGFR4 knocked out and FGFR4 negative cells. Our in vivo testing also found that 3A11 CART was able to eliminate RMS cells in murine xenograft metastatic models. Here we report the successful generation of binders specific to human FGFR4. FGFR4 CAR developed from 3A11 was able to kill FGFR4 positive target cells both in-vivo and in-vitro. Thus, 3A11 CAR T cells targeting FGFR4 may provide effective immune therapies for rhabdomyosarcoma and other FGFR4 expressing cancers and clinical trials are planned.

Citation Format: Adam Cheuk, Meijie Tian, Jeetendra Kumar, Peter Azorsa, Nityashree Shivaprasad, Dina Schneider, Berkley Gryder, Jun Wei, Young Song, Xinyu Wen, Sivasish Sindiri, Joon-Yong Chung, Zhongyu Zhu, Dimiter Dimitrov, Stephen Hewitt, Boro Dropulic, Rimas Orentas, Javed Khan. Potent tumoricidal activity of a FGFR4 CART in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1885.
Abstract 1887: Anti-CD38/CD28xCD3 trispecific T cell engager induces proliferation of primary T cells and mediates potent killing of primary malignant plasma cells isolated from Multiple Myeloma bone marrow aspirates
Authors: El-Murr Nizar , Kassem Sahar , Carrié Nadège , Henry Christophe , Virone-Oddos Angela , Yang Zhi-yong , Wiederschain Dmitri , de Velde Helgi Van , Chiron Marielle , Martinet Ludovic
Published: July 2021

Despite recent breakthrough in the treatment of multiple myeloma (MM) relapses are frequent, highlighting the need for novel approaches. Here we describe a novel anti-CD38/CD28xCD3 trispecific T cell engager (TCE) that targets CD38 expressed by malignant plasma cells and activates T cells by engaging CD3 and CD28 (1). Using bone marrow (BM) aspirates from MM patients, we confirmed that binding to malignant plasmocytes is primarily driven by the expression of CD38 and showed ex vivo that CD38/CD28xCD3 TCE induces a dose-dependent proliferation of CD4+ and CD8+ effector T cells. We also showed that MM patients' primary T cells were stimulated by CD38/CD28xCD3 TCE resulting in RPMI 8226 MM cell line lysis in co-culture assays. Killing of tumor cells was accompanied by MM effector CD8+ T cell degranulation and immunostimulatory cytokine production (IFN-γ and TNF-α). Finally, using MM patients' total BM cells, we showed that CD38/CD28xCD3 TCE induced the activation and cytokine production by cytotoxic CD8+ T cells and the CD38-dependent depletion of autologous primary malignant plasmocytes. Importantly, treatment of total BM cells with CD38/CD28xCD3 TCE also led to a CD38-dependent depletion of immunosuppressive myeloid derived suppressor cells (MDSC) that express CD38. Overall, these data show that CD38/CD28xCD3 trispecific TCE is active against primary myeloma cells and support the current evaluation of this next generation anti-CD38 multi-specific antibody in phase I clinical trial in patients with relapsed/refractory MM.

(1) Wu, L., Seung, E., Xu, L. et al. Trispecific antibodies enhance the therapeutic efficacy of tumor-directed T cells through T cell receptor co-stimulation. Nat Cancer 1, 86-98 (2020).

Citation Format: Nizar El-Murr, Sahar Kassem, Nadège Carrié, Christophe Henry, Angela Virone-Oddos, Zhi-yong Yang, Dmitri Wiederschain, Helgi Van de Velde, Marielle Chiron, Ludovic Martinet. Anti-CD38/CD28xCD3 trispecific T cell engager induces proliferation of primary T cells and mediates potent killing of primary malignant plasma cells isolated from Multiple Myeloma bone marrow aspirates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1887.