bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 170: Anticancer potential of curcuma and target identification in hematologic malignancies: A focus on mismatch repair
Authors: Bhattacharya Priyanjali , Patel Trupti N.
Published: July 2021

Introduction- Cancer, an heterogeneous condition, arises through mutations in the tumor suppressors and proto oncogenes which are otherwise involved in numerous cellular processes.These mutations rewire the signaling cascades to promote ‘malignant phenotype'. Under such circumstances the DNA repair system aids in rectifying the mutations and establishing normalcy.Plant derived products have shown promising success in treatment of cancer. In the present study, we make an attempt to screen various curcumin compounds to identify putative targets related to hematologic malignancies which in turn modulate the functionally altered mismatch repair system (MMR) using computational tools.

Methods-A total of 536 curcumin compounds, retrieved from peer reviewed literature and black turmeric database, were analyzed for molecular characteristics, biological activities, ADME/Tox and, drug-like properties through Molinspiration and PreADMET. This was followed by prediction of site of metabolism via FAME3SOM tool. Simultaneously, COSMIC database was used for selection of proteins associated to haematologic malignancies. The database helped us to identify 9 proteins viz. abl1, max, myb, myc, pcna, p73, top3a, blm and vbp1; molecular aberrations of which are associated with malignant transformation in blood related cancers. Additionally, STRING identified these proteins having significant interaction with proteins of mismatch repair system. The crystal structures of the proteins were obtained from PDB and their domains and active sites were predicted using scanPROSITE, MOTIF Search, metaPOCKET2 and PrankWeb tools. Finally the proteins and ligands were energy minimized using YASARA and subjected to virtual screening and molecular docking using Autodock Vina and Autodock Tools respectively.

Results-The overall results revealed six of the curcuma compounds (two monocarbonyl derivatives, one non-curcuminoid and three black turmeric natural compounds) may have the potential to modulate the identified targets and thus revive the MMR functionality; rescuing the genome from being unstable.

Conclusion-With these analyses we predict that curcumin compounds can modulate therapeutic targets identified here and in turn rescue the function of MMR in hematologic malignancies.

Citation Format: Priyanjali Bhattacharya, Trupti N. Patel. Anticancer potential of curcuma and target identification in hematologic malignancies: A focus on mismatch repair [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 170.
Abstract 1746: Designing a dendritic cell-targeted immunotherapy for the treatment of pancreatic ductal adenocarcinoma
Authors: Kleiner Ron , Vaskovich Daniella , Pozzi Sabina , Eldar-Boock Anat , Ben-Shushan Dikla , Florindo Helena , Satchi-Fainaro Ronit
Published: July 2021

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death. Low response rate, acquired resistance, severe side effects, highly desmoplastic and immunosuppressive microenvironment have limited the clinical outcomes of immunotherapy in PDAC. To overcome these hurdles, we set to design a novel immunotherapy that would be safe, efficacious, and selective to PDAC. The failure of immunotherapies to yield clinical response in PDAC is partially attributed to the inefficient antigen presentation by dendritic cells (DC) which results in insufficient T cells stimulation despite their expression of tumor-associated antigens (TAA). DC have an important role in the initiation and regulation of the innate and adaptive arms of the immune system. By efficient presentation of antigens, DC elicit an antigen-specific immune response. Trp53R172H is among the most common TP53 mutations in human PDAC, and is known as one of PDAC TAA. Despite the poor clinical outcomes, longer survival time was observed in PDAC patients with tumors that express high number of TAA combined with an abundant presentation of CD8+ T cell. To that end, we designed a DC-targeted nanoparticle (NP) containing PDAC Trp53R172H TAA and immune potentiators, with the DC targeting moiety, mannose, that was covalently conjugated to the NP, to enhance TAA presentation on DC. This approach led to specific activation of T cells against PDAC's TAA. The therapeutic activity of the NP was evaluated in vitro by our newly established 3D PDAC spheroids model, which is composed of freshly isolated murine stromal cells, therefore, better imitates the complex microenvironment found in PDAC. Along with an in vivo PDAC model, we evaluated the safety profile and therapeutic efficacy of our NP. The NP proposed herein successfully activated splenocytes and induced a specific cytotoxic reaction against PDAC cells ex vivo, which may result in improved survival rate.

Citation Format: Ron Kleiner, Daniella Vaskovich, Sabina Pozzi, Anat Eldar-Boock, Dikla Ben-Shushan, Helena Florindo, Ronit Satchi-Fainaro. Designing a dendritic cell-targeted immunotherapy for the treatment of pancreatic ductal adenocarcinoma [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 1746.
Abstract 1749: DRAK1 suppresses inflammation-induced cervical cancer progression by targeting TRAF6 protein
Authors: Pang Kyoungwha , Lee Jihee , Park Jinah , Ooshima Akira , Yang Kyung-Min , Kim Seong-Jin
Published: July 2021

Cervical cancer is a major cause of mortality in women worldwide and strongly associated with persistent infection with high-risk (HR) human papillomavirus (HPV), mainly HPV16 and HPV18. Most HPV viruses can be cleared by the immune system, and a preventive and effective vaccine against HPV infection decreases the development of cervical cancer. The adaptor protein TNF receptor-associated factor 6 (TRAF6) is a key mediator in inflammation. However, the molecular mechanisms controlling its activity and stability in cancer progression remain unclear. Here we show that death-associated protein kinase-related apoptosis-inducing kinase 1 (DRAK1) specifically decreases the stability of the TRAF6 protein via an autophagy-mediated degradation pathway by interfering with the homo-oligomerization of TRAF6, eventually suppressing tumor growth and metastasis in inflammation-associated advanced cervical cancer cells. DRAK1 bounds directly to the TRAF domain of TRAF6, preventing its autoubiquitination by interfering homo-oligomerization, eventually leading to autophagy-mediated degradation of TRAF6. Depletion of DRAK1 in cervical cancer cells resulted in markedly increased levels of TRAF6 protein, promoting activation of the IL1β signaling-associated pathway and pro-inflammatory cytokine production. DRAK1 knockdown accelerated cervical cancer tumorigenesis and metastasis whereas DRAK1 overexpression suppressed that phenomenon. DRAK1 was specifically underexpressed in metastatic cervical cancers and inversely correlated with TRAF6 expression in mouse xenograft model tumor tissues and human cervical tumor tissues. Collectively, our findings highlight DRAK1 as a novel potential biomarker and novel therapeutic target for treatment of TRAF6-associated advanced cervical cancers. [This work was supported by National R&D Program for Cancer Control, Ministry for Health and Welfare, Republic of Korea (HA17C0037).]

Citation Format: Kyoungwha Pang, Jihee Lee, Jinah Park, Akira Ooshima, Kyung-Min Yang, Seong-Jin Kim. DRAK1 suppresses inflammation-induced cervical cancer progression by targeting TRAF6 protein [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 1749.
Abstract 1750: Human KIT+myeloid cells facilitate visceral organ colonization by melanoma
Authors: Yu Chun I. , Martinek Jan , Wu Te-Chia , Kim Kyung In , George Joshy , Ahmadzadeh Elaheh , Maser Rick , Marches Florentina , Metang Patrick , Authie Pierre , Oliveira Vanessa K. , Wang Victor G. , Chuang Jeffrey H. , Robson Paul , Banchereau Jacques , Palucka Karolina
Published: July 2021

Metastasis is a major risk factor for poor melanoma outcome, but mechanisms supporting distant organ colonization by melanoma are not fully understood. Here, we found that metastatic melanoma tumors from patients are infiltrated by CD33+ myeloid cells. To determine the role of CD33+ cells in melanoma metastasis, we used NSG mice humanized by engraftment of human CD34+ hematopoietic progenitor cells and transgenic expression of human hematopoietic cytokines SCF/GM-CSF/IL-3 (SGM3). Humanized (h)NSG-SGM3 mice enabled development of human CD33+ myeloid cells in the bone marrow and peripheral tissues, and when implanted subcutaneously with human melanoma cell line, supported melanoma colonization of the spleen, liver, lung, and kidneys. Melanoma growth in distant organs was dependent on host SGM3 expression and facilitated by human CD33+ myeloid cells. Deeper characterization attributed this activity to a rare human IL-3- and SCF-dependent CD33+CD11b+CD117+ progenitor cell subset comprising <4% of the total CD45+ leukocyte population. Metastatic tumor-infiltrating CD33+ cells from patients and hNSG-SGM3 mice showed converging transcriptional profiles. Single-cell RNAseq analysis identified a gene signature of a KIT/CD117 expressing CD33+ subset that correlated with decreased overall survival in TCGA melanoma samples. Thus, human CD33+CD11b+CD117+ myeloid cells facilitate metastatic colonization of distant organs by melanoma, representing a novel candidate biomarker as well as a therapeutic opportunity for metastatic melanoma.

Citation Format: Chun I. Yu, Jan Martinek, Te-Chia Wu, Kyung In Kim, Joshy George, Elaheh Ahmadzadeh, Rick Maser, Florentina Marches, Patrick Metang, Pierre Authie, Vanessa K. Oliveira, Victor G. Wang, Jeffrey H. Chuang, Paul Robson, Jacques Banchereau, Karolina Palucka. Human KIT+myeloid cells facilitate visceral organ colonization by melanoma [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 1750.
Abstract 1756: The roles of Regnase-1 in pancreatic cancer development and progression
Authors: Okabe Junya , Kodama Takahiro , Takehara Tetsuo
Published: July 2021

Background: Regnase-1 is a RNA-degrading enzyme that plays an important role in the regulation of inflammation by degrading mRNA of inflammation-related genes. Although inflammation is known to promote pancreatic carcinogenesis, involvement of Regnase-1 in this process has never been studied. In this study, we thus investigated the role of Regnase-1 in pancreatic cancer development and progression.

Methods: We examined the expression levels of Regnase-1 in the pancreas of chronic pancreatitis by repeated administration of caerullein in mice. We sought for a regulator of the Regnase-1 expression in Panc-1 pancreatic cancer cell line. We assessed the biological phenotypes caused by siRNA-mediated Regnase-1 inhibition in multiple pancreatic cancer cell lines, including cell proliferation, migration, apoptosis and cytokine expressions. We generated pancreas-specific Kras-activated and Regnase-1-deficient (PDX1-Cre; Kras G12D KI Regnase-1 fl/fl) mice (PKR mice) and examined these phenotypes. We assessed the relationship between the expression levels of Regnase-1 in pancreatic tumor tissue and prognosis in 39 pancreatic cancer patients who underwent curative surgical resection.

Results: The expression levels of Regnase-1 significantly decreased in the murine pancreas of chronic pancreatitis together with the increase in IL-1β. The expression levels of Regnase-1 was downregulated by IL-1β administration in Panc-1 cells. siRNA-mediated Rengase-1 Inhibition significantly promoted cell proliferation, migration and inhibited apoptosis with Bcl-xL upregulation in Panc-1 cells. Rengase-1 Inhibition also upregulated a variety of chemokines involved in the recruitment and activation of myeloid-derived suppressor cells (MDSCs) including CXCL1, CXCL2, CXCL8, CSF2 and CSF3 in Panc1cells. All PKR mice developed pancreatic tumors and became moribund by 3 months of age. We observed massive infiltration of CD11b+Ly6g+Ly6c- PMN-MDSCs and significant upregulation of Arg1, Nos2, Nox2, and S100a8/9 mRNA levels, which are suppressors of anti-tumor immunity known to be secreted from MDSCs. Surgically-resected pancreatic cancer patients with low levels of intra-tumor Regnase-1 expression had significantly shorter recurrence-free and overall survival than those with high levels of Regnase-1. Intratumor Regnase-1 expression levels were negatively correlated with the number of CD11b+ cell infiltration.

Conclusion: Inflammation-mediated downregulation of Regnase-1 may contribute to pancreatic cancer development and progression in cell-autonomous and -heteronomous manners.

Citation Format: Junya Okabe, Takahiro Kodama, Tetsuo Takehara. The roles of Regnase-1 in pancreatic cancer development and progression [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 1756.
Abstract 1757: IFN-γ signaling in myeloid and fibroblastic cells regulates pancreatic cancer growth and metastasis
Authors: Ivleva Elena , Crisostomo Ashley , Andreeva Natalia , Grivennikov Sergei
Published: July 2021

Pancreatic ductal adenocarcinoma (PDAC) represents a significant healthcare problem which is on the rise, primarily because of the late stage diagnosis, early metastasis, and rapidly emerging resistance to therapy. As cancer cells in a typical pancreatic tumor are outnumbered by stromal and immune cells, the role of tumor microenvironment in cancer growth and progression is essential. One of the key regulators of tumor microenvironment in numerous cancers is IFN-γ signaling pathway, which is implicated into regulation of multiple cell types. Using a genetic murine model with conditional IFN-γR2 deficiency in myeloid cells, we performed an orthotopic injection of cell line derived from Pdx1Cre-Kras-P53 model (“KPC” cells). The absence of IFN-γ signaling in myeloid cells led to an increase of tumor burden and size of tumors in these mice. Further analysis demonstrated that IFN-γR2 deletion in myeloid cells changes the differentiation of cancer cells in primary tumors, resulting in a loss of characteristic histological pattern and shifting it towards more aggressive phenotype. Subsequent intrasplenic injection/liver spread model revealed that loss of IFN-γR2 in myeloid cells led to increased metastatic outgrowth of PDAC KPC cells. This effect was partially dependent on the gut microbiota. Deficiency of IFN-γR2 in myeloid cells increases recruitment and infiltration of monocytes in normal and tumor liver tissue. Meanwhile, deletion of IFN-γR2 in several subsets of cancer-associated fibroblasts demonstrated that IFN-γ signaling in different subsets of fibroblast/myofibroblasts may play opposing role in PDAC tumor growth. Our data identifies cell type specific IFN-γ signaling pathway as an important regulator of pancreatic cancer progression and metastasis, that can be potentially exploited as a novel immunooncology target or a biomarker of the disease progression.

Citation Format: Elena Ivleva, Ashley Crisostomo, Natalia Andreeva, Sergei Grivennikov. IFN-γ signaling in myeloid and fibroblastic cells regulates pancreatic cancer growth and metastasis [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 1757.
Abstract 1794: Human cancer associated fibroblast subsets can differentially regulate immunotherapy response in head and neck cancer patients
Authors: Graves Diana , Korrer Michael J. , Obradovic Aleksandar Z. , Roy Sohini , Califano Andrea , Drake Charles G. , Kim Young J.
Published: July 2021

The stromal cancer associated fibroblasts (CAF) have been postulated to modulate immune responses, but their role in clinical immunotherapy has not been rigorously explored in detail. To determine potential biomarkers of response and resistance for αPD-1 therapy in head & neck cancer patients, we performed high dimensional immunophenotyping to include single cell RNA sequencing (scRNA-SEQ) from a neoadjuvant trial of 50 advanced-stage head and neck squamous cell carcinoma (HNSCC) patients that were treated with nivolumab. Sufficient suspension cells from the tumors harvested pre and post nivolumab treatment allowed single-cell RNA sequencing on 4 patients as well as bulk RNA sequencing on 40 patients. Using the Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNe) and Virtual Inference of Protein-activity by Enriched Regulon (VIPER) bioinformatic analysis platform on the matched scRNA-Seq dataset to capture low expressing genes such as transcription factors, we identified 14 distinct cell types present in HNSCC patients. While CD8 T cells did not show increased number among the responder cohort, T cell cluster analysis showed enhanced IFNγ, granzyme, and perforin. Of these 14 cell types, the CAF cluster showed significant changes among the nivolumab responder cohort. Upon deeper analysis of this fibroblast population, we identified 5 distinct clusters of cancer associated fibroblast subsets (CAF-A, B, C, D, and E) of which, one cluster, CAF-A correlated with patient response to αPD-1 therapy. In order to determine the significance of this CAF subset's function, we isolated CD45-EpCAM-CD31-CD29+FAP+ CAF-A's from primary HNSCC tumor specimens and co-cultured with primary human T cells. Analysis by flow cytometry showed that CAF-A suppresses both CD4+ and CD8+ T cell proliferation as well as activation of these T cells. When we queried the cancer genome atlas, CAF-A gene signature correlated with increased survival, corroborating the important regulatory role of these CAF subsets in the context of endogenous immune responses. Overall, our findings demonstrate the functional importance of CAF subsets in modulating the immunoregulatory milieu of the human HNSCC for the first time, and justifies a more in depth characterization of these stromal cells in human squamous carcinoma in the current era of cancer immunotherapy.

Citation Format: Diana Graves, Michael J. Korrer, Aleksandar Z. Obradovic, Sohini Roy, Andrea Califano, Charles G. Drake, Young J. Kim. Human cancer associated fibroblast subsets can differentially regulate immunotherapy response in head and neck cancer patients [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 1794.
Abstract 1806: Novel preclinical orthotopic and bone metastasis TNBC models for assessment of immunotherapy and chemotherapy responses
Authors: Zdrojewska Justyna , Gladwyn-Ng Ivan , Mäki-Jouppila Jenni H. , Fagerlund Katja M. , Suominen Mari I. , Horn Ross , Rissanen Jukka
Published: July 2021

Triple-negative breast cancer (TNBC) is one of the most complicated types of breast cancer to treat. However, clinical trials continue to demonstrate that combining immunotherapy with chemotherapy can improve survival in patients with metastatic TNBC. In preclinical immunotherapy development, one of the challenges and limitations is the lack of representative TNBC models for advanced metastatic disease that could be used to study survival benefit of these compounds. The aim of this study was to develop local and bone metastatic TNBC models where survival could be followed as primary endpoint, and to assess effects of standard-of-care (Nab-paclitaxel, Abraxane) and anti-PD-1 (pembrolizumab, Keytruda).

Age-matched immunodeficient female CIEA NOG® mice (NOG) and NOG mice engrafted with human CD34+ hematopoietic stem cells (huNOG) were used in the study. In the orthotopic model, the luciferase labelled human TNBC cells, MDA-MB-231(SA)-luc, were inoculated into mammary fat pad of the mice. In the intratibial model, MDA-MB-231(SA)-luc cells were inoculated into bone marrow cavity of tibia to model the growth of primary and bone metastatic TNBC. The treatment with vehicle or paclitaxel (10 mg/kg) in NOG mice or isotype control or pembrolizumab (5 mg/kg) in huNOG mice was started 3 days after cancer cell inoculation and continued twice a week until termination. The mice were sacrificed individually according to predefined sacrifice criteria or latest at study week 8.

Nab-paclitaxel prolonged survival of immunodeficient mice with both primary and metastatic TNBC, but the effect was more profound in the orthotopic model. Pembrolizumab treatment in humanized mice with either tumor site did not result in survival benefit. In an earlier study, we have observed modest tumor growth inhibition with pembrolizumab in the orthotopic model suggesting donor variation effect. The findings are consistent with the clinical studies where pembrolizumab as monotherapy has demonstrated benefit for only some of the TNBC patients. In summary, novel preclinical models mimicking primary and bone metastatic TNBC were established that could be used to study the survival benefit of developing new treatments, including immunotherapies, against local and advanced TNBC.

Citation Format: Justyna Zdrojewska, Ivan Gladwyn-Ng, Jenni H. Mäki-Jouppila, Katja M. Fagerlund, Mari I. Suominen, Ross Horn, Jukka Rissanen. Novel preclinical orthotopic and bone metastasis TNBC models for assessment of immunotherapy and chemotherapy responses [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 1806.
Abstract 1819: The immunosuppressive role of YAP1 on the tumor immune microenvironment
Authors: Zhu Ling-Ling , Liu Jiewei , Chen Jiang
Published: July 2021

Background and aims: Lung adenocarcinoma (LUAD), the most prevalent histologic subtype of non-small cell lung cancer, is one of the most fatal malignancies. Despite the reported direct link between Hippo pathway and suppression of CD8+ and CD4+ T cell function in the context of cancer, its dynamic modulation and tumor microenvironment (TME) in LUAD remain unclear. We aim to investigate the correlation between YAP1 (a major component of Hippo signalling pathway), TME, and the overall survival of patients with LUAD.

Methods: The SsGSEA, CIBERSORT, and ESTIMATE algorithms were applied, and gene set enrichment analysis (GSEA) was conducted to screen Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways between patients with LUAD expressing low and high levels of YAP1 utilizing data downloaded from The Cancer Genome Atlas database. Flow cytometry was performed to detect the immune cell infiltration in LUAD mice model and patients. Western blot and qRT-PCR were used to investigate the mRNA and protein expression of YAP1 from mice model and patients.

Results: In this study, we demonstrated that YAP1 was negatively related to activated CD4/CD8 T cells and positively linked to immature dendritic cell, Tregs, and MDSC in 497 LUAD patients from TCGA, while it was not associated with signatures of 37 immunoregulatory and effector molecules, 37 cytokines. GSEA revealed that the high YAP1 group was not enriched in GO terms and KEGG pathways related to the immune response. However, high level of YAP1 positively correlated to immune score. In LUAD mice model, YAP1 knockout was associated with a reduced Treg cell intratumoural representation, and with higher production of interferon-γ and tumour necrosis factor by CD8+ and CD4+ effector cells (markers of increased cytotoxic activity). Moreover, YAP1 level was negatively related to overall survival in patients.

Conclusion: YAP1 expression within the TME might be useful as a predictive biomarker for immune response and a novel immunotherapeutic prognostic biomarker for LUAD.

Citation Format: Ling-Ling Zhu, Jiewei Liu, Jiang Chen. The immunosuppressive role of YAP1 on the tumor immune 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 1819.
Abstract 1831: Affinity tuned XmAb®2+1 GPC3 x CD3 bispecific antibodies demonstrate selective activity in liver cancer models
Authors: Nisthal Alex , Hassanzadeh-Kiabi Nargess , Avery Kendra N. , Rashid Rumana , Diaz Juan E. , Muchhal Umesh S. , Chu Seung Y. , Moore Gregory L. , Bykova Katrina , Desjarlais John R.
Published: July 2021

Bispecific T cell engagers simultaneously bind CD3 on T cells and tumor-associated antigens to promote T cell-mediated killing of tumor cells. These agents provide synthetic immunity by expanding, activating, and redirecting T cells against a target of interest. While targeting lineage-restricted antigens such as CD19 or CD20 have found clinical success in hematopoietic cancers, targeting solid tumors requires high expressing tumor associated antigens (TAAs) with minimal normal tissue expression.

Glypican 3 (GPC3), a lipid-anchored cell surface protein, is over-expressed in the majority of hepatocellular carcinoma (HCC) and a smaller subset of lung squamous cell carcinoma. It is considered an ideal target because its expression is not detected in adult tissues, and its function can promote tumor growth through the Wnt/beta-catenin pathway. Although GPC3 demonstrates good differential expression, bispecific T cell engagers are powerful immunomodulatory agents and careful tuning can improve the therapeutic window on all targets.

Building upon the XmAb® heterodimeric Fc platform, we generated bispecific antibodies in an XmAb 2+1 Fab2-scFv-Fc format that are bivalent for GPC3 and monovalent for CD3. Reducing the affinity of GPC3 encouraged avid binding and strong killing activity only on high GPC3 expressing cell lines while minimizing reactivity on low expressing cell lines. We found modulating the CD3 affinity, either directly through mutation or indirectly by the molecular format, contributes to the selectivity of the bispecific antibodies. To ensure biologically valid antigen densities were being considered, IHC was conducted on paraffin embedded arrays of tumor tissue, normal tissue, and cell lines. Upon matching the staining intensity between the three sample types, we identified cell lines with corresponding GPC3 antigen density that could serve as proxies of tumor and normal tissue. In vitro T cell-dependent cellular cytotoxicity (TDCC) assays on the relevant cell lines confirmed selective potency on high versus low expressing cells.

Citation Format: Alex Nisthal, Nargess Hassanzadeh-Kiabi, Kendra N. Avery, Rumana Rashid, Juan E. Diaz, Umesh S. Muchhal, Seung Y. Chu, Gregory L. Moore, Katrina Bykova, John R. Desjarlais. Affinity tuned XmAb®2+1 GPC3 x CD3 bispecific antibodies demonstrate selective activity in liver cancer 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 1831.
Abstract 1832: Development of a splicing modulator-based ADC payload class with immune stimulatory properties for cancer therapy
Authors: Mitra Satyajit K. , Jammalamadaka Vasu , Kang Jeffrey , Losic Teodora , Tuffy Greg , Liang Tony W. , Tipton Kim , Lopez Adriana , Savage Scott , Monteith William , Haskins William E. , Jurica Melissa S. , Satyal Sanjeev , Do Mary
Published: July 2021

Background: Thailanstatins are naturally occurring anti-proliferative compounds that target spliceosomes and modulate pre-mRNA splicing. Alterations in splicing machinery and mRNA splicing is common in cancer and represents a potential susceptibility that can be exploited by targeted delivery of a splicing modulator to tumors with antibody drug conjugates (ADCs).

Results: PH1 payload is a novel derivative of Thailanstatin optimized for metabolic stability and anti-tumor activity. PH1 is an efficient modulator of splicing in vitro and in vivo and a poor substrate for the MDR1 transporter. Transcriptome analysis of PH1- treated NCI-N87 cells revealed multiple classes of mis-splicing events including elevated expression of transcripts with skipped exons (3364 genes/4x change) and retained introns (332 genes/2.5x change). Translation of transcripts with retained introns and novel exon-exon junctions results in generation of neoepitopes.

Upon conjugation to Trastuzumab, Tras PH1 ADC exhibited nanomolar potency specific to HER2-expressing cancer cells, with durable anti-tumor response in vivo and favorable pharmacokinetic exposure in mice. Due to the potential for generation of neoepitopes, combination studies of Tras PH1 with a checkpoint inhibitor antibody was performed in a syngeneic MC38-HuHer2 model. Single agent and combination treatments induced tumor recruitment of CD11b+ positive myeloid cell subsets. While the combination treatment eradicated tumors, the treated animals also developed tumor-specific immunity.

A toxicology study performed in cynomolgus monkeys confirmed favorable PK profile and a wide safety margin for Tras PH1 ADC.

Conclusions: We present the development of a splicing modulator-based payload class with the ability to target tumor mRNA splicing, induce tumor neoepitopes, recruit myeloid cells and generate anti-tumor immunity. These findings support the development of ADCs using this novel class of immunostimulatory payload.

Citation Format: Satyajit K. Mitra, Vasu Jammalamadaka, Jeffrey Kang, Teodora Losic, Greg Tuffy, Tony W. Liang, Kim Tipton, Adriana Lopez, Scott Savage, William Monteith, William E. Haskins, Melissa S. Jurica, Sanjeev Satyal, Mary Do. Development of a splicing modulator-based ADC payload class with immune stimulatory properties for cancer therapy [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 1832.
Abstract 1843: Dicovery and characterization of a novel anti-human CXCR5 antibody for the treatment of B cell lymphomas
Authors: Kok Ayrin , Margallducos Germain , Beq Stephanie , Monchecourt Clarisse , Poullain Hombline , David Muriel , George Carine , Ellouze Sami , Chen Mingjie , Lu Yun-Yueh , Li Juying , Zhang Qian , Wang Jean , Adrian Francisco , Schweizer Liang , Beltraminelli Nicola
Published: July 2021

CXCR5 is a seven transmembrane protein coupled to protein G(i) expressed mainly on mature, resting B cells, follicular helper T cells (TFH), tonsillar B cells, and B cell lymphoma cells. CXCR5 is activated by its ligand CXCL13 and involved in cell migration. Dysregulated CXCL13 and/or CXCR5 expression and signaling have been associated with cancer promotion, as well as autoimmune and chronic inflammatory disorders. Overexpression of CXCL13 in autoimmune and lymphoproliferative disorders has been shown to correlate with disease progression. We therefore hypothesized that an antibody that selectively depletes CXCR5+ malignant B cells through antibody dependent cell-mediated cytotoxicity (ADCC) could be developed to treat B cell lymphomas. Here, we present preclinical data supporting the rationale for indication selection, biomarker identification, dose selection, and phase I clinical trial design. Using our single-cell screening platform technology, CelliGo™, we identified a panel of monoclonal antibodies against human CXCR5. We selected HFB2-4, which binds to CXCR5 expressing cells, blocks CXCL13-induced intracellular signaling and B cell migration, and induces ADCC of Raji B cell lymphoma cells at sub-nM concentrations. HFB2-4 demonstrates good PK behavior in mice and exhibits potent anti-tumor activity in a Raji xenograft murine model. HFB2-4 efficacy in this model is comparable to that of rituximab, despite a lower number of huCXCR5 receptors compared to CD20 on the surface of Raji cells. Our data shows that HFB2-4 holds promise as a therapeutic agent for B cell lymphoproliferative diseases by depleting CXCR5+ tumor B cells in refractory B cell lymphoma and lymphomas derived from severe Sjogren syndrome. Compared to the emerging treatments for refractory B cell lymphomas such as Antibody-Drug-Conjugates, bi-specific antibodies or cell therapies, HFB2-4 may represent a well-tolerated, efficacious, cost-efficient and easy to manufacture alternative.

Citation Format: Ayrin Kok, Germain Margallducos, Stephanie Beq, Clarisse Monchecourt, Hombline Poullain, Muriel David, Carine George, Sami Ellouze, Mingjie Chen, Yun-Yueh Lu, Juying Li, Qian Zhang, Jean Wang, Francisco Adrian, Liang Schweizer, Nicola Beltraminelli. Dicovery and characterization of a novel anti-human CXCR5 antibody for the treatment of B cell lymphomas [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 1843.
Abstract 1845: Enhancing the efficacy of antitumor antibodies with immune checkpoint inhibitors and targeted therapy in melanoma
Authors: Pérez-Lorenzo Rolando , Erjavec Stephanie O. , Clynes Raphael , Christiano Angela M.
Published: July 2021

The therapeutic outcomes of current strategies in melanoma treatment have greatly improved within the last decade. However, the persistent relatively low success rate of current standard-of-care strategies underscores the urgent need for improvement of these therapeutic approaches. The use of specific antitumor antibodies recently transformed the cancer therapeutics landscape with successes such as anti-HER2 in breast cancer, and anti-EGFR in HNSCC and colorectal cancer. However, antitumor antibodies have not been developed for the treatment of melanoma. Here, using mouse preclinical models of melanoma (both solid tumor and lung metastasis), we found that the therapeutic efficacy of the specific antitumor antibody TA99 (anti-TYRP1) is significantly enhanced by immune checkpoint blockade. This was associated with a greater CD8+, CD8+/Foxp3+, NK1.1+ and dendritic cell infiltrate, suggestive of an increased anti-tumor immune response. Further, MEK inhibition (MEKi) increased the expression of melanosomal antigens in B16 and YUMM mouse melanoma cell lines in vitro. We next combined TA99 and MEKi to treat B16 melanoma in vivo and observed a synergistic reduction in tumor growth. Treatment of mice bearing YUMM melanoma (BRAF mutant) with TA99/MEKi resulted in the complete elimination of tumors in the majority of animals, and produced durable responses. Moreover, we found a synergistic effect when B16 tumor-bearing mice were treated with TA99 combined with MEKi and immune checkpoint blockade (anti-PD1 and anti-CTLA4). Our findings suggest that MEKi induced an increased expression of tumor-associated antigens, which, in combination with antitumor antibodies, generated a robust adaptive antitumor response that was sustained by immune checkpoint inhibition therapy. These improved therapeutic effects of specific antitumor antibodies by current standard-of-care therapeutics have potential near-term translation to the clinic, in light of fully humanized anti-TYRP1 antibodies currently in clinical development.

Citation Format: Rolando Pérez-Lorenzo, Stephanie O. Erjavec, Raphael Clynes, Angela M. Christiano. Enhancing the efficacy of antitumor antibodies with immune checkpoint inhibitors and targeted therapy in melanoma [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 1845.
Abstract 1852: A novel PD1-CD40 bispecific antibody YH008 induces potent anti-tumor activity in vivo by PD1 dependent activation of CD40 signaling
Authors: Liu Baihong , Hui Li , Liu Chonghui , Li Zhihong , Pan Fangxia , Yang Benny , Lin Qingcong
Published: July 2021

Immunotherapies that target PD1/PDL1 pathway have marked a new era of cancer therapy. Combinatorial treatment of inhibiting PD1/PDL1 signaling and other immunomodulating agents has been widely tested and validated as a better strategy compared to single agent treatment. CD40 (TNFRSF5) is a member of the TNFR super family, a key T cell co-stimulatory signal critical for B-cell maturation and immunoglobulin class switching. Biocytogen developed a novel first in class PD1-CD40 bispecific antibody (YH008) that agonizes CD40 conditionally upon PD1 binding. In vitro study data shows YH008 can block the interaction of PD1 to PDL1 and induced dose dependent NFAT activation. YH008 was able to activate human CD40 on reporter cells in the presence of PD1-expressing jurkat cells in a dose dependent manner without relying on FcγR expressing cells. This feature is beneficial for conditionally activation of CD40+ cells in tumor microenvironment and tumor-draining lymph nodes, where tumor specific PD1+ T cells are enriched. In vivo study result shows that YH008 demonstrated robust anti-tumor efficacy against MC38 and B16F10 tumors in PD1/CD40 double humanized mice (B-hPD1/hCD40). In hCD40 mice which has no human PD1 engagement, YH008 did not show anti-tumor activity against MC38 tumor. Furthermore, YH008 did not elevate liver enzymes of MC38 bearingB-hPD1/hCD40 mice at dose as high as 26 mg/kg, demonstrating improved liver safety profile than selicrelumab. Taken together, these data demonstrated that YH008 is a promising new immunotherapeutic candidate in cancer treatment.

Citation Format: Baihong Liu, Li Hui, Chonghui Liu, Zhihong Li, Fangxia Pan, Benny Yang, Qingcong Lin. A novel PD1-CD40 bispecific antibody YH008 induces potent anti-tumor activity in vivo by PD1 dependent activation of CD40 signaling [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 1852.
Abstract 1865: Simultaneous de-repression of innate and adaptive immune responses through dual targeting of ILT4 and PD(L)-1 with bispecific antibodies
Authors: Vitale Laura , Murphy Mike , Weidlick Jeff , Wasiuk Anna , O'Neill Thomas , Widger Jenifer , Mills-Chen Laura , Crocker Andrea , Patterson Colleen , Boyer James , Crew Linda , Natoli Edward J. , Lillquist Jay S. , Goldstein Joel , Thomas Lawrence J. , Marsh Henry C. , Alvarado Diego , Keler Tibor
Published: July 2021

Activation of innate immunity using targeted therapies has become an attractive approach to stimulate antitumor responses. Dual inhibition of receptors that suppress myeloid and T cell compartments through the generation of bispecific antibodies (bsAbs) is a promising strategy to overcome resistance to checkpoint inhibition. Activation of the ITIM-bearing ILT4/LILRB2 receptor by its cognate ligands (HLA-G and HLA Class I) in myeloid cells induces a strong immunosuppressive response that may be leveraged by tumors to evade immune surveillance. Monoclonal antibodies (mAbs) that inhibit ILT4 activity have demonstrated promising preclinical and early clinical data in combination with PD-1 inhibitors, including in the checkpoint refractory setting. These findings provide a rationale to co-target ILT4 with PD(L)-1 using a single molecule. We describe the discovery and characterization of ILT4 inhibitory mAbs for engineering bsAbs that revert myeloid cell suppression by antagonizing ILT4 and activate T cell responses through PD(L)-1 inhibition. Humanized anti-ILT4 mAbs were generated that specifically bind human and macaque ILT4. These mAbs exhibit sub-nanomolar HLA-G binding to ILT4 expressing cells. Macrophages treated with ILT4 mAbs release inflammatory cytokines/chemokines and upregulate activation markers resulting in polarization to an M1 inflammatory phenotype. These activities are further potentiated in the presence of toll-like receptor activation with lipopolysaccharide (LPS). We engineered tetravalent bsAbs using an Fc-silenced IgG1 linked to scFv format from our anti-ILT4 and anti-PD-L1 mAbs in both orientations. The bsAbs have good biophysical characteristics and retain functional properties similar to the parental mAbs. Further in vitro and in vivo characterization efforts are ongoing to select the bsAb candidate to enter development activities and IND-enabling studies.

Citation Format: Laura Vitale, Mike Murphy, Jeff Weidlick, Anna Wasiuk, Thomas O'Neill, Jenifer Widger, Laura Mills-Chen, Andrea Crocker, Colleen Patterson, James Boyer, Linda Crew, Edward J. Natoli, Jay S. Lillquist, Joel Goldstein, Lawrence J. Thomas, Henry C. Marsh, Diego Alvarado, Tibor Keler. Simultaneous de-repression of innate and adaptive immune responses through dual targeting of ILT4 and PD(L)-1 with bispecific antibodies [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 1865.
Abstract 1870: Novel conditionally active bispecific T cell engagers targeting solid tumors
Authors: Liu Haizhen , Wang Jing , Cugnetti Ana Paula G. , Xing Charles , Wheeler Christina , Lucas Matthew , Chang Cathy , Frey Gerhard , Boyle William , Short Jay
Published: July 2021

The use of CD3/target bispecific molecules has great therapeutic potential in oncology. This potential, however, is diminished due to severe cytokine release effects as well as serious on-target off-tumor toxicities. Conditionally Active Biologic (CAB) technology is a proprietary platform that generates bispecific antibodies which have no or very little binding to CD3 and to the tumor target antigen (TAA) in healthy tissue (normal physiological conditions), but have strong binding in the context of diseased tissues (tumor microenvironment) based on the glycolytic tumor metabolism (including Warburg effect). Using our CAB technology, we have developed dual-CAB (CAB TAA x CAB CD3) bispecific antibodies targeting two well-established tumor associated antigens, B7H3 and Nectin4. These dual-CAB bispecific antibodies were developed so that they bind to recombinant CD3 and TAA as well as to CD3 and TAA expressing cells under in vitro tumor microenvironment conditions, but not in physiologic conditions. In vitro characterization and in vivo efficacy data of the dual-CAB TAA x CD3 bispecific antibodies targeting B7H3 and Nectin4 will be presented. The CAB technology allows the generation of a new class of potent T-cell engagers with increased safety margin and therapeutic index in the clinic.

Citation Format: Haizhen Liu, Jing Wang, Ana Paula G. Cugnetti, Charles Xing, Christina Wheeler, Matthew Lucas, Cathy Chang, Gerhard Frey, William Boyle, Jay Short. Novel conditionally active bispecific T cell engagers targeting solid tumors [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 1870.
Abstract 1874: MDX-124, a novel annexin-A1 antibody, induces an anti-tumor immune response and wide-ranging anti-cancer activity in multiple preclinical models
Authors: Dempsey Fiona C. , Al-Ali Hussein , Crichton Scott J. , Pepper Chris , Parris Christopher N.
Published: July 2021

Major recent advances have increased the understanding of both the molecular mechanisms and the immune response in cancer. This has led to the discovery of new agents that regulate the tumor immune microenvironment. Annexin-A1 (ANXA1) is a phospholipid binding protein that has been shown to have immunomodulatory effects on T-cells, macrophages and dendritic cells. Although normally localized in the cytoplasm, ANXA1 is secreted in response to a number of different stimuli and can modulate cellular functions through interactions with formyl peptide receptors (FPR1/2). Overexpression of ANXA1 has been observed in several cancer types including triple-negative breast (TNBC), lung, pancreatic and ovarian, and correlates with poor prognosis and decreased overall survival. Furthermore, ANXA1 has also been shown to influence cancer cell proliferation, angiogenesis, migration and drug resistance. Hence, we explored the role of MDX-124, a novel humanized antibody targeting ANXA1, and here present data from several pre-clinical cancer models.

Results: Incubation of a panel of cell lines representing several histological cancer subtypes with MDX-124 for 72h resulted in a statistically significant dose-dependent reduction in cell viability compared to control. ANXA1 expression in the subcellular compartments (nucleus, cytoplasm and membrane) was quantified in a panel of cancer cell lines via imaging flow cytometry and the anti-proliferative effect of MDX-124 was shown to correlate with membrane ANXA1 expression. Using a transwell assay, TNBC and acute myeloid leukemia cell lines exposed to MDX-124 exhibited a statistically significant reduction in migration compared to untreated controls. We also observed that MDX-124 induced antibody dependent cellular cytotoxicity (ADCC) activity in a dose-dependent manner using a reporter bioassay (EC50 = 0.38 nM). MDX-124 was further evaluated in an additional panel of in-vitro assays and shown to exert consistent anti-cancer activity.

In conclusion, our data indicate that targeting ANXA1 with MDX-124 inhibits tumorigenic processes and induces an immune response in tumors overexpressing ANXA1. MDX-124 therefore provides an innovative approach to cancer therapy. Investigations are continuing to explore the efficacy of MDX-124 in syngeneic mouse models as both a single agent and in combination with standard of care agents.

Citation Format: Fiona C. Dempsey, Hussein Al-Ali, Scott J. Crichton, Chris Pepper, Christopher N. Parris. MDX-124, a novel annexin-A1 antibody, induces an anti-tumor immune response and wide-ranging anti-cancer activity in multiple preclinical 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 1874.