bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2447: Identification of tumor marker gangliosides for early cancer detection: A mass spectrometry approach
Authors: Culp-Hill Rachel , Hill Collin , Blackler Adele , Ricketts William
Published: March 2024

Tumor-marker gangliosides (TMGs) have enormous potential for early-stage cancer detection as diagnostic biomarkers despite being relatively understudied in this context. AOA Dx is focused on the development of a mass spectrometry platform for the quantitation of TMGs in human serum, particularly disialogangliosides GD2 and GD3. The development of a mass spectrometry platform for TMG quantitation may allow for the identification of diagnostic signatures for early-stage cancer detection. Briefly, we monitored fragment moieties characteristic of gangliosides in an untargeted fashion by mass spectrometry. We then identified those differentially altered in cancerous groups compared to normal, and subsequently performed targeted MRM analysis. Through these investigations, we have identified multiple TMGs, specifically multiple types of GD2 and GD3 species characterized by unique lipid tails across different cancer types in human serum from confirmed cancer diagnoses. Other ganglioside classes including GMs and relatively understudied GTs were also identified in this study. Notably, the serum of melanoma and ovarian cancer patients exhibited significantly increased levels of several ganglioside species compared to age-matched healthy serum, highlighting the potential of TMGs as a promising avenue for cancer diagnosis. Further experiments will focus on refining this mass spectrometry method and expand demographic representation to confirm our initial findings and identify additional TMGs of interest. In addition, we intend to apply immune-based platforms such as thin-layer chromatography and ELISA, which have already shown promising results in the detection and quantitation of these compounds. Taken together, AOA Dx is advancing the development of a high-throughput mass spectrometry platform designed for the detection of TMGs, with a primary focus on GD2 and GD3. Our objective is the identification of a diagnostic disease signature for early-stage cancer detection. The validation of this type of diagnostic panel would allow for expedited diagnosis and treatment of cancers currently diagnosed at late stages, ultimately reducing healthcare costs and increasing survival rates. Future research will aim to validate these biomarkers in independent prospective studies.

Citation Format: Rachel Culp-Hill, Collin Hill, Adele Blackler, William Ricketts. Identification of tumor marker gangliosides for early cancer detection: A mass spectrometry approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2447.
Abstract 2448: Field cancerization proteomic analysis provides a novel class of functional risk biomarkers for non small cell lung cancer
Authors: Alhammad Rayan Ibrahim , Vuong Ngoc Bao , Zhou Weidong , Johann Donald J , Birkaya Barbara , Liotta Lance , Luchini Alessandra
Published: March 2024

Lung cancer carcinogenesis is the outcome of a field of premalignant changes that occur in the bronchial tree leading to the overt emergence of the malignant lung cancer (field cancerization). A tissue or blood proteomic signature of these premalignant changes represent a novel concept of risk biomarkers that can predict the probability of future emergence of Lung Cancer. In this study, we analyzed two independent cohorts of non-small cell lung cancer (NSCLC) patients. One cohort consisted of 18 patients who donated blood before and after tumor resection surgery and matched tissue samples (UAMS Medical Center). The second set derived from a longitudinal case-control study of plasma sample collected during the physician health study that were donated at different times prior to the diagnosis of Non-Small Cell Lung Cancer (N=272). We used affinity-enriched mass spectrometry analysis and statistical learning techniques to identify a set of common markers that discriminated plasma cases and controls and were conserved in different bronchial tissue compartments including tumor and adjacent tissue. The tumor microenvironment adjacent to and surrounding cancer cells undergoes to progressive stages of carcinogenesis during field cancerization. We identified 22 proteins involved in cell adhesion, protein folding, immune response, angiogenesis, and cancer proliferation. This is a novel class of functional risk biomarkers to determine the probability of NSCLC onset. In conclusion, our findings shed light on a new class of functional biomarkers that underly tumor pathogenesis mechanisms and can provide hints for new strategies of tumor prevention therapy or lifestyle modification.

Citation Format: Rayan Ibrahim Alhammad, Ngoc Bao Vuong, Weidong Zhou, Donald J Johann Jr, Barbara Birkaya, Lance Liotta, Alessandra Luchini. Field cancerization proteomic analysis provides a novel class of functional risk biomarkers for non small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2448.
Abstract 2529: Comparison of small cell lung cancer transcriptional factor expression by immunohistochemistry, multiplex immunofluorescence, and a targeted RNA expression panel
Authors: Ozakinci Hilal , Alontaga Aileen , Nguyen Jonathan V , Segura Carlos M Moran , Saeed-Vafa Daryoush , Gray Jhanelle , Haura Eric , Chiappori Alberto A , Boyle Theresa
Published: March 2024

Recent studies have identified distinct molecular subtypes of small cell lung cancer (SCLC) (SCLC-A, SCLC-N, SCLC-P, and SCLC-Y) based on ASCL1, NEUROD1, POU2F3, and YAP1 expression. In this study, we investigated the correlation between immunohistochemistry (IHC), multiplex immunofluorescence (mIF), and RNA Salah Targeted Expression Panel (RNA STEP) for ASCL1, NEUROD1, POU2F3, and YAP1 in formalin-fixed-paraffin-embedded (FFPE) samples from 14 SCLC samples collected under an IRB-approved protocol (MCC19163). For the mIF evaluation, FFPE samples were immunostained using the AKOYA Biosciences OPAL TM 7-Color Automation IHC kit on the BOND RX autostainer. For RNA STEP, nanoString nCounter TagSet chemistry was used to measure gene expression levels of test FFPE samples relative to a reference pooled human normal mRNA control. All three methods demonstrated significant correlation for ASCL1 levels (Table 1). IHC and RNA STEP NEUROD1 results exhibited significant correlation. However, mIF NEUROD1 results did not correlate with the results of other methods likely due to non-specific positivity of the NEUROD1 mIF antibody. No strong correlation was observed between any of the methods for POU2F3 and YAP1. Only 2 of the 14 samples displayed positive POU2F3 protein expression by IHC; one of these also had elevated POU2F3 expression by RNA STEP. Importantly, the sample with the highest POU2F3 protein expression by IHC also had the highest values by mIF and RNA STEP. All samples were negative for YAP1 by IHC and RNA STEP. Notably, ASCL1 staining was primarily observed in CD56+ POU2F3- cells by mIF. These findings highlight the reliability and utility of IHC and RNA STEP in assessing transcriptional factors in SCLC. Also, mIF presents an innovative method for the simultaneous analysis of multiple markers using a single tissue section, enabling the concurrent detection of multiple markers within individual cells.

Table 1. Statistical Significance (p-values) and Correlation Analysis (Spearman's Coefficients) IHC vs RNA STEP IHC vs mIF RNA STEP vs mIF p r P r p r ASCL1 0.021 0.62 <0.0001 0.91 0.010 0.67 NEUROD1 0.002 0.78 0.517 -0.14 0.671 0.12 POU2F3 0.201 0.36 0.422 0.18 0.045 0.55 YAP1 0.299 0.30 0.364 0.20 0.295 0.30

Citation Format: Hilal Ozakinci, Aileen Alontaga, Jonathan V Nguyen, Carlos M Moran Segura, Daryoush Saeed-Vafa, Jhanelle Gray, Eric Haura, Alberto A Chiappori, Theresa Boyle. Comparison of small cell lung cancer transcriptional factor expression by immunohistochemistry, multiplex immunofluorescence, and a targeted RNA expression panel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2529.
Abstract 2563: Understanding the new subgroup- Her 2 low breast cancer
Authors: Kulkarni Radhika , Shahid Muhammad Ali , Castro Oscar Hinojosa , Patel Kunj , Lin Chun-Hui , Rose Courtney , Dabak Vrushali
Published: March 2024

Introduction: Outcomes have greatly improved for patients with HER2+ breast cancer. Recently, tumors with IHC 1+ or 2+/negative FISH have been classified as ‘HER2-low’. The development of novel HER2 agents has shown clinical benefit in this subset of patients. Information on the clinical characteristics of these patients which could be prognostic, and which could impact therapeutic options is limited.

Methods: This was an IRB-approved study. Relevant patient demographics, disease characteristics, and treatment details of patients with metastatic breast cancer seen at our center from 2018 to 2021 were collected by chart review. All data analyses were performed using R version 4.3.1, and a p-value ≤ 0.05 was considered statistically significant.

Results: A total of 102 patients with metastatic breast cancer were included. Patients were divided as HER2+, HER-, HER2 low ER+ and HER2 low ER -. 66.6% of patients had HER2 low disease. Patients with HER2 low ER- disease were more likely to have tumors that were of higher grade (grade 3, 71.4%, p=0.003), higher mean Ki67 (60%, p<0.001), and of ductal histology (100%, p=0.006). There was a statistically significant difference in PFS among the four groups (p=0.001). Median survival was longer in patients with HER2 low ER+ compared to the HER2- group. (1.42 vs 0.58, p<0.001, Table 1). Amongst HER2 low ER+ patients, there was a significant PFS difference in those treated with CDK4/6 inhibitors compared to chemotherapy (2.83 vs. 0.67, p=0.009). We found no difference in PFS between patients who were HER2 1+ ER+ and HER2 2+ ER+. In multivariate analysis, there was no significant association of race, grade, menopausal status, Ki67, histologic subtype, and metastatic sites with PFS.

Table 1. PFS Summary Group Total number Progression/Death Median Survival HER2- 13 13 0.58 HER2 low ER- 14 11 0.88 HER2 low ER+ 54 32 2.42 HER2+ 20 12 1.08

Conclusion: In this small single-center retrospective study, patients with metastatic HER2 low ER- breast cancer had higher-grade tumors. HER2 low ER+ patients had a better PFS compared to HER2- patients. This seems to suggest that the HER2 low group may have some inherent biologic differences. Larger studies are needed.

Citation Format: Radhika Kulkarni, Muhammad Ali Shahid, Oscar Hinojosa Castro, Kunj Patel, Chun-Hui Lin, Courtney Rose, Vrushali Dabak. Understanding the new subgroup- Her 2 low breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2563.
Abstract 2618: BCG017, a bispecific ADC targeting PTK7 and EGFR exhibits anti-tumor efficacy in PDX models
Authors: Yao Sufei , Shang Chengzhang , Li Zhuolin , An Gao , Guo Chaoshe , An W. Frank , Yang Yi
Published: March 2024

EGFR is a favored target in various cancer therapies. However, EGFR-based treatments have been challenged by drug resistance and cytotoxicity. Targeting EGFR with an antibody-drug conjugate (ADC) is a promising new therapeutic strategy to address the drug resistance, due to the potent killing effects of the payload. Furthermore, we hypothesized that development of a bispecific ADC (bsADC) targeting EGFR and another tumor-associated antigen (TAA) might improve the tumor selectivity, thereby limiting the on-target off-tumor toxicity. One such TAA candidate is PTK7, which belongs to the same receptor tyrosine kinase family as EGFR, and is co-expressed with EGFR in multiple types of solid tumors, including lung, esophageal, head and neck, and colon cancers.

Herein, we developed a fully human anti-human PTK7 EGFR bsAb using our proprietary common light chain RenLite® mouse platform and knobs-into-holes technology. The anti-PTK7 EGFR bsAb demonstrates good purity by SEC-HPLC, and can cross-react with human and cynomolgus monkey antigens with favorable affinity. The PTK7 x EGFR bsAb demonstrated higher binding to various types of cancer cell lines, as well as enhanced internalization in vitro compared with PTK7 parental antibody. Next, the bsAb was conjugated with monomethyl auristatin E (MMAE) to generate an anti-PTK7 x EGFR bsADC, BCG017, with a drug-to-antibody ratio of approximately 4. BCG017 demonstrated superior anti-tumor activity compared to benchmarks and its PTK7 parental ADC in cell-derived non-small-cell lung cancer (NSCLC) xenografts, and showed enhanced anti-tumoractivity when compared to benchmark ADCs and parental ADCs in patient-derived breast and pancreatic ductal adenocarcinoma xenograft models. These results suggest that BCG017 have the potential to be a novel therapeutic alternative for PTK7 and EGFR co-expressing tumors.

Citation Format: Sufei Yao, Chengzhang Shang, Zhuolin Li, Gao An, Chaoshe Guo, W. Frank An, Yi Yang. BCG017, a bispecific ADC targeting PTK7 and EGFR exhibits anti-tumor efficacy in PDX models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2618.
Abstract 2640: HBI-2375, a first-in-class IND ready selective MLL1-WDR5 inhibitor, retains desirable preclinical characteristics in solid tumors and in leukemias to be pursued in clinical studies
Authors: Shojaei Farbod , Fang Che , Ricono Jill M. , Gillings Mireille
Published: March 2024

MLL1 (mixed lineage leukemia1) interaction with WDR5 (WD repeat domain 5) is critical for its histone methyl transferase (HMT) activity leading to transcriptional regulation of gene expression. MLL1 dysregulation such as its translocation at chromosome 11q23 results in generation of MLL1 fusion genes and leukemia development. Additionally, recent studies indicated a role for MLL1-WDR5 interaction in solid tumors progression via directly affecting cancer cells or by suppression of tumor immunity. Therefore, MLL1-WDR5 interaction is an important target in the discovery of anti-cancer drugs for both leukemia and solid tumors. We recently identified HBI-2375 as a selective inhibitor of MLL1-WDR5 interaction. HBI-2375 showed potent inhibition of WDR5 (IC50: 4.48nM) in biochemical assay and inhibited MV4-11 cellular proliferation (average IC50: 3.17µM) in CTG assays. In addition, HBI-2375 was stable in liver S9 and whole blood, with moderate to high plasma protein binding rates in multiple species, and acceptable hERG (IC50: 17µM). Furthermore, HBI-2375 displayed reasonable oral PK properties (Cmax, T1/2, AUC, and F%) in mouse, rat and dog and showed good safety profile in GLP tox studies. In pharmacology studies HBI-2375 showed significant dose dependent anti-tumor activity in MV4-11 AML model along with reduction in methylation of H3K4 in the tumors harvested from the treated animals demonstrating the targeted disruption of HMT activity in cancer cells by HBI-2375. Furthermore HBI-2375 in combination with PD-1 mAb significantly inhibited tumor growth in MC38, colorectal tumor model, and in 3LL lung carcinoma syngeneic tumor models. Flow cytometry and IHC showed significant increase in the infiltration of CD8+ CTLs in the combination group further providing immune-driven mechanisms for greater efficacy of combination therapy. Gene expression analysis also identified genes that are involved in promoting immune system activity in the combination group. Tissue distribution studies also showed greater accumulation of HBI-2375 in brain tissue indicative of a potential to target glioma and glioblastoma in clinical studies. Therefore, HBI-2375 represents a first-in-class MLL1-WDR5 inhibitor with promising characteristics for future testing in the clinic.

Citation Format: Farbod Shojaei, Che Fang, Jill M. Ricono, Mireille Gillings. HBI-2375, a first-in-class IND ready selective MLL1-WDR5 inhibitor, retains desirable preclinical characteristics in solid tumors and in leukemias to be pursued in clinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2640.
Abstract 2654: HPK1 inhibits CD8+ T cell effector gene expression following T cell activation
Authors: Ames Rachel Y. , Zhao Rongqi , Mace Parker , Grant Adam , Xie Guorui , Milestone Heather , Grandcolas Molly , Fang Eva , Kiosea Nicolae , Wong Stephen , Brovarney Martin , Jacobson Scott , Nebalasca Danilo , Arguello Jorge , Gomez Blanca , Kandala Hiranmayee , Ko Michelle Y. , Nguyen Lan , Robles Omar , Shibuya Grant , Shakhmin Anton A. , Tivitmahaisoon Parcharee , Vu Vi-Anh , Younai Ashkaan , Zibinsky Mikhail , Subramanyam Babu , Poon Daniel , Ghomi Mohsen Sabouri , Wustrow David J. , Kassner Paul D. , Katibah George E. , Brockstedt Dirk G.
Published: March 2024

Hematopoietic progenitor kinase 1 (HPK1) has been shown to act as a negative regulator of T cell receptor signaling and of subsequent effector T cell function.  Inhibiting HPK1 to enhance T cell activity has emerged as a promising strategy for cancer immunotherapy. Upon T cell receptor engagement, the kinase domain of HPK1 is activated and targets components of the T cell receptor (TCR) signaling pathway for degradation, including SLP76. However, the molecular events connecting HPK1 kinase activity to observed T cell functions downstream of proximal TCR signaling are not well understood. Through transcriptional profiling of HPK1-kinase-dead (HPK1-KD) versus wild-type CD8+ T cells following an acute, attenuated Listeria monocytogenes infection, we observed increased expression of many genes associated with T cell activation and effector function, including changes in expression of several key transcription factors and their targets. Upon activation, HPK1-KD T cells, as well as T cells treated with our small-molecule HPK1 inhibitor, produce higher levels of a wide range of effector cytokines in vitro and in vivo.  Treatment of mice with the HPK1 inhibitor also inhibited tumor growth in several syngeneic tumor models.  These data expand our understanding of the role of HPK1 in inhibiting CD8+ T cell effector programs, and, importantly, the impact of HPK1 inhibitors on T cell function. 

Citation Format: Rachel Y. Ames, Rongqi Zhao, Parker Mace, Adam Grant, Guorui Xie, Heather Milestone, Molly Grandcolas, Eva Fang, Nicolae Kiosea, Stephen Wong, Martin Brovarney, Scott Jacobson, Danilo Nebalasca, Jorge Arguello, Blanca Gomez, Hiranmayee Kandala, Michelle Y. Ko, Lan Nguyen, Omar Robles, Grant Shibuya, Anton A. Shakhmin, Parcharee Tivitmahaisoon, Vi-Anh Vu, Ashkaan Younai, Mikhail Zibinsky, Babu Subramanyam, Daniel Poon, Mohsen Sabouri Ghomi, David J. Wustrow, Paul D. Kassner, George E. Katibah, Dirk G. Brockstedt. HPK1 inhibits CD8+ T cell effector gene expression following T cell activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2654.
Abstract 2689: Mass cytometry analysis of CXCR3 expression in tumor-infiltrated myeloid cells as a predictive biomarker For immunotherapy response
Authors: Valentin-Guillama Gabriel A. , Chatterjee Nandini , Alimadadi Ahmad , Hedrick Catherine C.
Published: March 2024

Monocytes have emerged as important regulators of cancer progression. We have recently found that CXCR3+ circulating monocytes in humans are associated with better response to immunotherapy. Here, we aimed to study the functions and heterogeneity of CXCR3+ monocytes in two different triple negative breast cancer (TNBC) tumors in mice, using either AT3 or EO771 tumor cell lines. AT-3 tumors exhibit higher resistance to anti-PD-1 and anti-CTLA-4 therapy in C57BL/6 breast cancer mouse models compared to EO771 tumors. We evaluated 36 surface protein markers on tumor-infiltrated myeloid cells in tumors isolated from each model using CyTOF mass cytometry. Analysis of monocytes, macrophages and dendritic cells in the tumors using unsupervised clustering identified 13 cell subsets. Significant differences were discovered between the two tumor types in frequencies of nine of 13 identified subsets. However, 3 of these subsets displayed relatively high expression of CXCR3 in EO771 tumors when compared to AT-3 tumors. The significant CXCR3 positive subsets were identified as CXCR3+ early macrophages, CXCR3+ Ly6C+CD11blo classical monocytes and Trem2+ CXCR3+ dendritic cells. We observed higher expression of CXCR4, Trem2, and CCR1 in CXCR3+ myeloid cells when compared to their CXCR3− counterparts in both tumor models. To elucidate the role of these CXCR3+ cells, we performed low-input bulk RNA sequencing of CD115+/CD11b+ cells from blood and spleen of EO771 tumor bearing mice with a wild type or CXCR3KO genotype. Ingenuity Pathway Analysis (IPA) of the differentially expressed genes suggests a significant inhibition of the IL-10 signaling pathway of CXCR3KO cells. These differentially expressed markers and genes suggest a functional importance in this subset in migration to the tumor and antigen presentation, both of which are anti-tumoral functions. Ongoing studies are aimed towards understanding the role of CXCR3+ myeloid cells in regulating response to immunotherapies.

Citation Format: Gabriel A. Valentin-Guillama, Nandini Chatterjee, Ahmad Alimadadi, Catherine C. Hedrick. Mass cytometry analysis of CXCR3 expression in tumor-infiltrated myeloid cells as a predictive biomarker For immunotherapy response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2689.
Abstract 2704: cGAS-STING drives myeloid inflammation in chromosomally unstable esophageal cancer
Authors: Beernaert Bruno , Erdal Erkin , Clark Rose , Liu Tong , Hammond Ester M. , Parkes Eileen E.
Published: March 2024

Esophageal adenocarcinoma (EAC) is an aggressive disease characterized by chromosomal instability (CIN), whereby cells continuously acquire chromosomal abnormalities that promote therapeutic resistance, immune evasion and metastasis. CIN in cancers also fuels the misplacement of genomic DNA in the form of structural abnormalities of the nucleus, such as micronuclei (MN), that lead to chronic activation of the cytosolic DNA sensing cGAS-STING pathway. However, how chronic cGAS-STING pathway activation is tolerated and shapes the tumor landscape of CINhigh EAC cancers is poorly understood. Using publicly available RNA-seq and genome sequencing data of EAC tumours and esophageal cancer cell lines as well as a panel of EAC cell lines, we demonstrate that the core cGAS-STING pathway machinery is rarely inactivated in EAC despite high levels of baseline CIN and aneuploidy. Through transcriptomic profiling of an isogenic EAC precursor cell line model of variable CIN and cGASKO/WT EAC cell lines we show that both chronic and transient CIN-driven cGAS-STING activation converge on the expression of CXCR1/2 ligands and other pro-inflammatory cytokines and chemokines rather than typical anti-tumor type I interferon signalling in EAC cells. Indeed, a novel transcriptional signature of cGAS+ micronuclear burden-correlated genes correlates with orthogonal measures of CIN in EAC tumours and esophagogastric cancer cell lines and is associated with an enrichment in myeloid-derived cells as well as poor prognosis. Correspondingly, using multiplexed immunofluorescence (IF) we find that a high preponderance of cGAS+MN and STING expression in human EAC samples is associated with decreased tumor purity, increased myeloid cell and macrophage infiltration, and increased peripheral blood neutrophil counts, indicative of tumor-promoting myeloid inflammation. Ongoing multiplexed IF and single-nuclei RNAseq analysis of cGAS+MNhigh and cGAS+MNlow EAC tumors will further help characterise the components that comprise the CIN EAC tumor microenvironment (TME) and the CIN- and cGAS-STING-dependent cell-cell interactions that govern it. Taken together, our findings provide an explanation for the observed maintenance of cGAS and STING in EAC and identify disruption of cGAS-STING-dependent myeloid cell inflammation and recruitment as a potential therapeutic target for CINhigh EAC.

Citation Format: Bruno Beernaert, Erkin Erdal, Rose Clark, Tong Liu, Ester M. Hammond, Eileen E. Parkes. cGAS-STING drives myeloid inflammation in chromosomally unstable esophageal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2704.
Abstract 2705: Hyaluronic acid impairs NK killing towards breast tumor spheroids despite elevated cytotoxicity
Authors: Cheung Brian C. , Li Alan T. , Segall Jeffrey E. , Wu Mingming
Published: March 2024

The tumor microenvironment (TME) is often regarded as a critical component in tumor progression and metastasis. Among the main structural components of the TME, such as collagen and glycosaminoglycans (GAGs), hyaluronic acid (HA) is highly elevated in the breast TME, and is associated with poor prognosis. In recent years, immunotherapy has gained tremendous success in the battle against hematological cancers. Yet, solid tumors have remained a significant clinical challenge for immunotherapy due to the physical constraints they impose on immune cells. Mechanically, our previous work has shown that the abundance of high molecular weight (HMW) HA significantly reduces matrix pore sizes and softens the collagen and HA cogel, and impairs breast tumor invasion. However, it is still unclear how modulating tumor invasion through the mechanics of the TME may correlate with the efficacy of immunotherapy. In this study, we aim to investigate the effect of HMW HA on natural killer (NK) cell-based immunotherapy using a tumor spheroid model (GFP-labeled MCF-7). We introduced NK-92 MI cells into tumor spheroid embedded collagen gels with or without HA, and followed spheroid-NK cell interactions through live cell imaging. Preliminary data shows that while tumor spheroids were still able to invade in the collagen only gel, they underwent apoptosis in the presence of NK cells. More importantly, the presence of HA within the ECM reduced NK cells’ ability to kill tumor spheroids. Specifically, when surrounded by NK cells, tumor spheroids remained intact and viable in the collagen-HA cogel, while they underwent apoptosis in collagen only gels. In a parallel experiment, multicolor flow cytometry data showed that HA increased NK activation and certain cytotoxic markers (interferon-gamma, perforin, and granzyme-B), despite the fact that spheroids appeared to be more resistant towards NK cells in the presence of HA. Taken together, we speculate that the presence of HMW HA in the TME may protect solid tumors from immune attacks by modulating the mechanical environment.

Citation Format: Brian C. Cheung, Alan T. Li, Jeffrey E. Segall, Mingming Wu. Hyaluronic acid impairs NK killing towards breast tumor spheroids despite elevated cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2705.
Abstract 2706: Identification and characterization of cancer-associated polymorphonuclear cell subsets in mice
Authors: De Velasco Marco A. , Kura Yurie , Sakai Kazuko , Kikuchi Takashi , Mori Yasunori , Minami Takafumi , Yoshimura Kazuhiro , Fujita Kazutoshi , Nishio Kazuto , Uemura Hirotsugu
Published: March 2024

Background: Neutrophils are the main type of polymorphonuclear cells (PMN) and comprise most immune cells in the body. PMN cells are upregulated in cancer and accumulate in the tumors. Tumor-associated PMNs comprise a heterogeneous population consisting of immunologically active mature neutrophils and immature polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). The phenotype of these cells evolves with disease progression and is implicated in modulating immune response. However, there is yet to be a consensus to define PMN populations in tumors and blood.

Methods: Here, we used single-cell RNA-seq, bulk RNA sequencing, flow cytometry, and immunohistochemistry (IHC) analysis to characterize cancer-associated PMN-MDSCs in tumor-bearing mice.

Results: Single-cell RNA-seq data from lung tumors and healthy individuals revealed five metaclusters (MC): MCA, MCB, MCC, MCD, and MCE. MCA and MCB made up 34.4 and 65.1% of PMNs in healthy tissues, respectively. MCA, MCB, MCC, MCD, and MCE comprised 3.8%, 23.5% 3.3% 14.6%, and 54.7% of PMNs in tumors, respectively. Genes enriched in MCA and MCE were associated with MDSCs and myeloid progenitor cells. MCB and MCC were associated with inflammatory responses mediated by TNF-alpha signaling and neutrophil degranulation. MCD was associated with a CD33 myeloid cell phenotype and had high expression levels of CD274, Pdcd1lg2, Havcr2, and Tigit genes. Cells from MCE were associated with protein translation signatures and had elevated levels of Nt5e. Gene expression signatures from single-cell RNA-seq data were compared with bulk transcriptomic data from healthy mouse prostates and Pten-null prostate tumors characterized by high PMN cell infiltration. Gene set enrichment analysis showed enrichments after androgen deprivation therapy and Cluster MCE was associated with castration-resistant tumors. Flow cytometric and IHC analysis confirmed the enrichment of PMN cells expressing elevated levels of proteins identified from gene expression analysis including CD73, PD-L1, and PD-L2.

Conclusion: This study offers further insights into the distinct subsets of PMN-MDSCs present in tumors.

Citation Format: Marco A. De Velasco, Yurie Kura, Kazuko Sakai, Takashi Kikuchi, Yasunori Mori, Takafumi Minami, Kazuhiro Yoshimura, Kazutoshi Fujita, Kazuto Nishio, Hirotsugu Uemura. Identification and characterization of cancer-associated polymorphonuclear cell subsets in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2706.
Abstract 2717: Bifunctional fusion protein PDL1sFv/MICAe for cancer immunotherapy
Authors: Li Junyi , Wei Yanzhang
Published: March 2024

Many cancer cells upregulate the expression of programmed death ligand 1 (PD-L1) to surpass T-cell mediated immune response. Immune checkpoint blockers targeting PD-L1 or its receptor PD1 have been shown to suppress the inhibitory signals and enhance T cell function. As a new immunotherapy, immune checkpoint blockers have been used to treat multiple cancers. However, most clinically available blockers only engage with one type of immune cell: the T cells. Natural killer (NK) cells, on the other hand, play a significant role in anti-cancer immune responses. It will represent an advancement in anti-cancer immunotherapy if NK cells are engaged with immune checkpoint blockers. In this study, we designed a unique bifunctional fusion gene, PDL1sFv/MICAe, consisting of the variable regions of an anti-PD-L1 antibody and the extracellular domain of stress protein MICA and hypothesized that fusion protein PDL1sFv/MICAe will simultaneously bind to PD-L1 on tumor cells to block the inhibitory signal and extend T cells activity, and to NKG2D to activate NK Cells. cDNA sequence of fusion gene PDL1sFv/MICAe was designed based on the variable region of an established clinically used antibody, Atezolizumab, and the extracellular domain of MICA with linkers in between. After a computational confirmation of its structure and binding affinities, the fusion gene was synthesized and cloned into an expressing vector, and the fusion protein was produced for evaluation. Western blotting and fluorescent microscopic analyses confirmed that the protein binds to PD-L1 of human tumor cells, including MCF7 and MDAMB231, and NKG2D of KHYG1 cells. Preliminary results also show that the fusion protein enhances NK cell cytotoxicity against tumor cells. Studies on confirming the fusion protein’s activities on other tumor cells, NK cell IFN-γ production, T cell function, etc., are underway. In summary, fusion protein PDL1sFv/MICAe represents an improved version of the immune checkpoint blockers and could treat cancer patients quickly and effectively.

Citation Format: Junyi Li, Yanzhang Wei. Bifunctional fusion protein PDL1sFv/MICAe for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2717.
Abstract 2722: CTN001, a first Twin Fc-immune cell engager, with a superior potency against Her2+ and Her2-low cancers by engaging multiple Fc-mediated mechanisms
Authors: Lee Deokjae , Yoon Sujin , Bae Jiseon , Kim Minju , Lee Ki-June , Kim Eunmi , Park Hyunkyu , Choi Eun S. , Yang Gi-Hyeok
Published: March 2024

Many therapeutic monoclonal antibodies mediate the immune effector functions through the engagement of their fragment crystallizable (Fc) domain and Fcγ receptors on the immune cells. However, these functions are limited due to the low affinity of their interactions and the requirement of abundancy of antigens for the avidity effects. Recently, mutation-based Fc engineered antibodies showed their enhanced immune effector functions mostly through the NK cell-mediated ADCC. To overcome the limited improvement of current Fc engineering, we developed an innovated platform, Twin Fc-Immune Cell Engager (Twin Fc-ICE) composed of a single Fab domain and two Fc domain with drug-like properties. Here we demonstrated the superior efficacy of CTN001, a first Twin Fc-ICE, in in vitro and in vivo Her2+ tumor models. CTN001 bound to various types of Fcγ receptor-expressing immune cells with stronger affinity, supported by its better in vitro affinity to all types of Fcγ receptors than wild type and Fc-engineered antibodies. To evaluate the pharmacological efficacies, CTN001 was tested for its functions of in vitro antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) and in vivo efficacy with Her2+ xenograft models. In Her2+ models, both CTN001 and trastuzumab induced ADCC-mediated effects, but CTN001 showed better anti-tumor cytotoxicity through ADCP and CDC than trastuzumab. Importantly, CTN001 also showed cytotoxic effects on Her2-low tumor cells. Consistently, CTN001 showed better anti-tumor efficacy than trastuzumab or its combination with pertuzumab in Her2+ tumor xenograft model and, surprisingly, even in Her2-low or resistant models. These results suggest that CTN001 can provide new therapeutic options for treating Her2-low patients differentiated from antibody-drug conjugates (ADCs), and that the Twin Fc-ICE can be an innovative platform to overcome limitations of existing therapeutics.

Citation Format: Deokjae Lee, Sujin Yoon, Jiseon Bae, Minju Kim, Ki-June Lee, Eunmi Kim, Hyunkyu Park, Eun S. Choi, Gi-Hyeok Yang. CTN001, a first Twin Fc-immune cell engager, with a superior potency against Her2+ and Her2-low cancers by engaging multiple Fc-mediated mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2722.
Abstract 2729: Discovery and characterization of LILRB2XPD-L1 bispecific antibody SPX-303
Authors: Haight Anthony , Chen Qian , William Brandon , Nguyen Dinh-Duc , Cai Robert , Ma Jichun , White Michael , Jin Jindong , Shen Jadon , Siekierzycki Martin , Hall Victoria , Jin Hua , Meier Roland , Moser Justin
Published: March 2024

The advent of immunotherapy has led to deep and sometimes durable remissions in certain cancers. However, the variable response rates and development of resistance mechanisms pose significant challenges to the current therapies. The immunosuppressive nature in the tumor microenvironment (TME) can drive many of these observations. Reversing this immunosuppression is a promising avenue for enhancing and broadening the applicability of IO therapies. Macrophages are critically modulated by LILRB2, a pivotal receptor in their maturation and polarization to the M2 immunosupressive phenotype. We report the discovery and development of a novel anti-LILRB2/PD-L1 bispecific antibody, SPX-303, designed using an anti-LILRB2 IgG4 derived from a mouse hybridoma screen and linked with an anti-PD-L1 epitope. SPX-303 demonstrates sub-nanomolar binding affinity to both LILRB2 and PD-L1 targets, effectively blocking the interaction between LILRB2 and its ligand, as well as inhibiting PD-1 binding to PD-L1. In in vitro functional assays, SPX-303 exhibits effects comparable to the parental monomeric anti-LILRB2 (SPX-104) in redirecting macrophage polarization from M2 to M1 stage, as observed in both cultured PBMC and human monocyte-derived macrophage (HMDM) models. Furthermore, SPX-303 demonstrates a synergistic effect in inducing T cell activation in the autologous mixed lymphocyte reaction assay (auto-MLR), surpassing the effects of the anti-LILRB2 epitope (SPX-104) and the anti-PDL1 fragment (single-domain anti-PDL1) alone or in combination. In vivo experiments reveal that SPX-303 significantly inhibits tumor growth. Collectively, these results support SPX-303 as a novel therapeutic antibody that effectively enhances anti-cancer immunity in the treatment of advanced solid tumors. The pre-clinical data as well as the clinical plan of SPX-303 will be presented.

Citation Format: Anthony Haight, Qian Chen, Brandon William, Dinh-Duc Nguyen, Robert Cai, Jichun Ma, Michael White, Jindong Jin, Jadon Shen, Martin Siekierzycki, Victoria Hall, Hua Jin, Roland Meier, Justin Moser. Discovery and characterization of LILRB2XPD-L1 bispecific antibody SPX-303 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2729.
Abstract 2735: High affinity and potent anti-tumor efficacy of Nivo813, an anti-PD-1/ALK-1 bispecific antibody, in pre-clinical studies for solid tumors
Authors: Zhou Qingqing , Tang Rong , Zhuang Lanfang , Huang Jianyu , Qian Jiang , Ren Zhihua , Tong Youzhi
Published: March 2024

Background: The activin-like kinase receptor 1 (ALK-1, encoded by ACVRL1) is a member of the transforming growth factor β (TGF-β) superfamily. Combination treatment using anti-PD-1/PD-L1 agents with anti-angiogenetic reagents might bring additional benefits, such as establishing a favorable TME leading to better responses towards immunotherapy. In the phase II clinical trial (NCT03893695) of combination therapy of ALK-1 (GT90001) and Nivolumab for the second line therapy in patients with metastatic hepatocellular carcinoma (“HCC”) in Taiwan, among the 20 evaluable patients, the overall response rate is 40%, and the side effects were well tolerated and manageable. Nowadays, the combination of GT90001 and nivolumab shows a remarkable long-tail effect, since 7 patients are still alive as of March 30, 2023, 6 of them had received no further systematic anti-tumor therapy after trial completion. Among them, 4 patients are still on progression free status. Our pre-clinical data demonstrated that Nivo813 has improved cell-binding potency and potent anti-tumor effects, lighting up its potential capacity as a therapeutic drug in the treatment of human cancers.

Methods: The antigen binding activity of Nivo813 with PD-1 and ALK-1 were assessed by ELISA, Fortebio and flow cytometry. Endothelial cell tube formation assay was performed to identify the anti-angiogenesis function of Nivo813. The anti-tumor activity of Nivo813 was investigated in PBMC implanted with MDA-MB-231 breast cancer cells.

Results:Nivo813 could specifically bind to human PD-1 and ALK-1 with high affinity. Intriguingly, Nivo813 was found to have much higher affinity (40-fold, nM) to cell surface PD-1 than that of nivolumab. Meanwhile, Nivo813 exhibited dose-dependent anti-angiogenesis activity in vitro. Nivo813 demonstrated greater anti-tumor efficacy compared to nivolumab in mice model.

Conclusions: Nivo813, a dual-blocking anti-PD-1/ALK-1 bispecific antibody, shows improved binding potency to cell surface PD-1 and ALK-1 simultaneously, and displays dose-dependent anti-angiogenesis in vitro and great anti-tumor efficacy in vivo, supporting its clinical potentials for the treatment of human cancers.

Citation Format: Qingqing Zhou, Rong Tang, Lanfang Zhuang, Jianyu Huang, Jiang Qian, Zhihua Ren, Youzhi Tong. High affinity and potent anti-tumor efficacy of Nivo813, an anti-PD-1/ALK-1 bispecific antibody, in pre-clinical studies for solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2735.
Abstract 274: Cancer-associated fibroblasts exhibit increased motility under hypoxic conditions via interaction with cancer cells
Authors: Park Jihye , Lee Sieun , Kurie Jonathan M. , Ahn Young-Ho
Published: March 2024

Cancer-associated fibroblasts (CAFs) are key components of the tumor microenvironment, playing various roles through interactions with cancer cells and other stromal cell types. In a 3D co-culture system of CAFs and lung cancer cells, a specific subgroup of CAFs exhibited increased motility, while another subgroup of CAFs remained stationary. Using the photoconvertible fluorescent protein Dendra2, we isolated these distinct CAF subgroups and conducted RNA sequencing. Gene set enrichment and gene ontology analysis revealed that upregulated genes in motile CAFs are associated with hypoxia and glycolysis. Conditioned medium from mesenchymal-like cancer cells enhanced the expression of genes related to hypoxia and glycolysis in CAFs. Among these genes, Aldoa and Ldha were chosen for knockdown in CAFs, resulting in a reduction in CAF motility. We confirmed that mesenchymal-like cancer cells promoted CAF motility and intensified hypoxic conditions within the co-culture system, which was monitored using a hypoxia reporter. Ceruloplasmin (CP) was identified as a secretory protein from mesenchymal-like cancer cells through mass spectrometry. Knockdown of CP led to the inhibition of CAF motility. In conclusion, the motility of CAFs is enhanced under hypoxic conditions facilitated by the secretion of CP from mesenchymal-like cancer cells in the lung tumor microenvironment.

Citation Format: Jihye Park, Sieun Lee, Jonathan M. Kurie, Young-Ho Ahn. Cancer-associated fibroblasts exhibit increased motility under hypoxic conditions via interaction with cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 274.
Abstract 2746: MAGE-A3 co-expression with IDH1-R132H and IL-1β in colon cancer
Authors: Han Andy/Xi , Gonzalez Rachel , Yom Jina , Guo Zhaoying , Gilmore Bailey , Zewdu Eden , Qu Tianli , Guo Hailey/YiChen , Ren Qi , Hu Xiaomin , Zhang Ranran , Wu Zhaohui , Liu Xuan , Fu Wei
Published: March 2024

Colorectal cancer (CRC) remains a significant cause of cancer death worldwide with an estimated 52,000 deaths in 2023. Over the past few decades, the incidence rate of CRC in individuals younger than 50 has been steadily increasing. Currently, diagnostic biomarkers play an important role in the detection and treatment of CRC. MAGE-A3 has been shown to be expressed in many tumor types, including colon cancer by RNA expression. Due to their high sequence homology, members of the MAGEA family have been difficult to evaluate and screen for their protein expression. However, in a previous study, highly specific antibodies were identified using CytoSections for MAGE-A3 immunohistochemistry. IL-1β is a cytokine that induces inflammatory responses and when localized to the nucleus in colon cancer cells it can promote stemness. IL-1β expression has been shown to be increased in wildtype IDH1 positive glioblastoma. However, the relationship between IL-1β and IDH1 has not been studied in colon cancer. In this study, the difference in the expression of MAGE-A3, IL-1β, and wildtype IDH1 compared to mutant IDH1-R132H in colon cancer tissues was evaluated. The results show that IL-1β, wildtype IDH1, and MAGE-A3 were co-expressed in 27 of the 38 colon cancer tumors. However, IDH1-R132H expression was only seen in 20% of the tumors. This study suggests that MAGE-A3, IL-1β, and IDH1 are candidate biomarkers for immunotherapy due to their high expression in colon cancer tissues.

Citation Format: Andy/Xi Han, Rachel Gonzalez, Jina Yom, Zhaoying Guo, Bailey Gilmore, Eden Zewdu, Tianli Qu, Hailey/YiChen Guo, Qi Ren, Xiaomin Hu, Ranran Zhang, Zhaohui Wu, Xuan Liu, Wei Fu. MAGE-A3 co-expression with IDH1-R132H and IL-1β in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2746.