bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5405: Expression of cathepsin D in early-stage breast cancer and its prognostic and predictive value
Authors: Alhudiri Inas M. , Nolan Christopher C. , Ellis Ian O. , Rakha Emad A. , Elzagheid Adam , Green Andrew R. , Chapman Caroline J.
Published: June 2022

Purpose: Cathepsin D is a proteolytic enzyme that is normally localized in the lysosomes and is involved in the malignant progression of breast cancer. The expression of Cathepsin D is regulated by steroid hormones including oestrogen and growth factors. There are conflicting results between different studies regarding Cathepsin D significance as prognostic and predictor marker in breast cancer. Furthermore, Cathepsin D has not been recommended for clinical use by the American Society of Clinical Oncology because of insufficient evidence. This study was aimed to fill the gap in the lack of recent and large studies on prognostic significance of Cathepsin D expression by studying a large well-characterized series of primary breast cancer for cathepsin D expression using immunohistochemistry.

Methods: Expression of Cathepsin D was assessed by immunohistochemical staining of tissue microarrays, in a large well-characterized series of early-stage operable breast cancer (n=954) from Nottingham Primary Breast Carcinoma Series between the period of 1988 and 1998 who underwent primary surgery. Correlation of cathepsin D expression with clinicopathological parameters and prognosis was evaluated.

Results: Cathepsin D expression was positive in 28.8% (275/954) of breast cancer tumors. Positive expression of cathepsin D was significantly associated with high histological grade (p=0.007), pleomorphism (p=0.002), poor Nottingham Prognostic Index score (NPI) (p<0.002), recurrence (p=0.005) and distant metastasis (p<0.0001). Kaplan-Meier analysis showed that Cathepsin D expression was significantly associated with shorter breast cancer specific survival (BCSS) (p=0.001), higher risk of recurrence (p=0.001) and distant metastasis (p<0.0001). Cathepsin D positive tumors with ER positive staining who were treated with tamoxifen were associated with lower BCSS and higher risk of developing distant metastasis (p=0.001).

Conclusion: This study shows Cathepsin D expression as a promising prognostic biomarker for early operable invasive breast cancer. Our results indicate that Cathepsin D expressing tumors are associated with shorter breast cancer specific survival and higher risk of recurrence and metastasis. Cathepsin D could be clinically useful to predict response to tamoxifen treatment. Targeting Cathepsin D either through inhibition of action or interference with the proteolytic pathway could be used to treat metastatic breast cancers expressing Cathepsin D.

Citation Format: Inas M. Alhudiri, Christopher C. Nolan, Ian O. Ellis, Emad A. Rakha, Adam Elzagheid, Andrew R. Green, Caroline J. Chapman. Expression of cathepsin D in early-stage breast cancer and its prognostic and predictive value [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5405.
Abstract 5425: Quantitative determination of fibroblast growth factor receptor inhibitor-AZD4547 using HPLC analysis in a mouse model of ultraviolet B (UVB)-induced skin carcinogenesis
Authors: Kandula Rema Anisha , Babin Katherine Claire , Latimer Brian , Martin Kenneth Mc , Linquest Lauren , Moore-Medlin Tara , Thakur Megha A. , Khandelwal Alok R. , DiGiovanni John , Nathan Cherie-Ann O.
Published: June 2022

Cutaneous squamous cell carcinoma (cSCC) is the second most common form of skin cancer affecting more than two million Americans annually. Cumulative sun exposure plays a leading role in the pathogenesis of cSCC. In our previous studies, topical application of Fibroblast growth factor receptor (FGFR) inhibitor AZD4547 was associated with a significant reduction in Ultraviolet B-induced (UVB)-induced acute skin changes such as epidermal hyperplasia and hyperproliferation. Further, chronic, topical application of AZD4547 was associated with significant inhibition in UVB-induced tumor incidence and multiplicity. Unfortunately, oral administration of FGFR inhibitors in clinical trials for solid tumors is associated with notable side effects such as hyperphosphatemia, diarrhea, decreased appetite, fatigue, and liver dysfunction. Therefore, our objective was to investigate whether topical administration of AZD4547 could potentially circumvent systemic absorption, leading to a low toxicity profile. Accordingly, mice were topically administered AZD4547 (400ug/200ul acetone) followed by exposure to UVB three times a week for two weeks. Blood was collected at the end of the study. AZD4547 was extracted using methanol extraction and concentrated using a Savant SpeedVac system. Samples were reconstituted in 25% acetone/75% water solution and analyzed using Reverse-phase HPLC (Shimadzu) with a Zorbax Eclipse C18 3.5-µm 4.6x100mm analytical column operated at ambient temperature and gradient elution using trifluoroacetic acid (TFA) (Mobile A) and 0.1% TFA in Acetonitrile. Data from HPLC analysis demonstrated non-appreciable absorption of AZD4547 (blood levels only from 108-358nM) suggesting that the topical application of AZD4547 was not systemically absorbed and the effect of treatment was restricted to the local skin tissue. Based on the efficacy and low systemic absorption profile, AZD4547 could potentially be utilized as a topical agent to both prevent and treat UVB-induced cSCC.

Citation Format: Rema Anisha Kandula, Katherine Claire Babin, Brian Latimer, Kenneth Mc Martin, Lauren Linquest, Tara Moore-Medlin, Megha A. Thakur, Alok R. Khandelwal, John DiGiovanni, Cherie-Ann O. Nathan. Quantitative determination of fibroblast growth factor receptor inhibitor-AZD4547 using HPLC analysis in a mouse model of ultraviolet B (UVB)-induced skin carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5425.
Abstract 5463: BPI-442096: A potent and selective inhibitor of SHP2 for the treatment of multiple cancers
Authors: Li Ling , Fu Bang , Han Han , Sun Zhongxin , Zhao Xiangdong , Jv Xuepeng , Tong Jun , Zhao Jiayu , Zou Zhengyao , Chen Haibo , Liu Xiaoyun , Ren Wei , Li Yinlong , Wu Wenmao , Guo Jing , Yan Dan , Liu Xiangyong , Lan Hong , Wu Hao , Ding Lieming , Wang Jiabing
Published: June 2022

Src homology region 2 domain-containing phosphatase-2 (SHP-2) is a key node in the RAS signaling pathway. Allosteric inhibition of SHP2 phosphatase is a potential therapeutic strategy for cancers harboring oncogenic mutations in the KRAS pathway. SHP2 also participates in the signal transduction downstream of regulatory immunoreceptors, and it has been shown in preclinical models that SHP2 inhibition drives anti-tumor immunity through modulation of both innate and adaptive mechanism. BPI-442096 is a potent, selective, and orally bioavailable small molecule SHP2 inhibitor. It exhibited significant anti-proliferation activities against multiple KRAS mutant cancer cell lines, including those from NSCLC, PDAC, CRPC, etc. BPI-442096 dose-dependently inhibited SHP2 phosphatase and downstream ERK phosphorylation in cancer cells, as well as NFAT reporter gene expression downstream of PD-1/PD-L1 signaling in immune cells. In vivo, BPI-442096 demonstrated strong tumor growth inhibition in KRASG12C, KRASG12D, and KRASG12V mutant xenograft mouse models. BPI-442096 also exhibited anti-tumor immunity in the MC38 syngeneic model, as a single agent or in combination with anti-PD1/PD-L1 drugs. Moreover, BPI-442096 combining with KRASG12C inhibitor may reverse intrinsic and acquired resistance to KRASG12C inhibition. Adequate oral exposure across multiple pre-clinical species and good ADME properties ensured the druggability of BPI-442096. In conclusion, BPI-442096 exhibits a robust anti-tumor effect in multiple KRAS mutant models and enhanced anti-cancer immunity in syngeneic mouse models, and it shows multiple combination potentials to overcome drug resistance. Phase 1 clinical trial is planned in early 2022.

Citation Format: Ling Li, Bang Fu, Han Han, Zhongxin Sun, Xiangdong Zhao, Xuepeng Jv, Jun Tong, Jiayu Zhao, Zhengyao Zou, Haibo Chen, Xiaoyun Liu, Wei Ren, Yinlong Li, Wenmao Wu, Jing Guo, Dan Yan, Xiangyong Liu, Hong Lan, Hao Wu, Lieming Ding, Jiabing Wang. BPI-442096: A potent and selective inhibitor of SHP2 for the treatment of multiple cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5463.
Abstract 5514: Therapeutic potential of activated natural killer cells in nasopharyngeal carcinoma
Authors: Hui Shinyee , Lo Kwok Wai , Xu Mingjing , Cheung Siu Tim
Published: June 2022

One of the key features of nasopharyngeal carcinoma (NPC) is dense lymphocyte infiltration. Despite elevated lymphocyte populations in tumor microenvironment, these lymphocytes have modest killing activity. One of the possible speculations is constitutive STAT3 overexpression within NPC microenvironment which results in immunosuppression of cytolytic immune cells. Natural killer (NK) cells act as first line defense of innate immunity through cytolytic killing of virus-infected cells and/or cancer cells. Unlike T cells, NK killing does not require antigen presentation and therefore would be effective even for MHC defective cancers. This makes NK therapy attractive in form of autologous and allogeneic transfer.

Here we hypothesized that activated NK cells would show enhanced anti-NPC activity. We also sought to investigate the potential of combined NK with STAT3 inhibitor in NPC treatment. In this study, NK cells from peripheral blood mononuclear cells (PBMC) of healthy donors (N=3) were isolated and expanded. Three Epstein Barr Virus (EBV) positive NPC cell lines (NPC43, C666-1, C17) were employed in this study. Naïve and activated NK cells were subjected to NPC coculture at various effector: target (E:T) cell ratios. In combination treatment with NK cells, a small molecule inhibitor Napabucasin was employed for STAT3 treatment. NK killing activity was assessed through flow cytometry analysis of NPC cell death.

The current study demonstrated significant elevation of NK activation marker expressions after 4 weeks of ex vivo expansion (CD69 34.7±2.3 fold, p=0.002; NKG2D 4.2±0.8 fold, p=0.018). Concomitantly, ex vivo activated NK cells demonstrated augmented killing activity against NPC cells at a dose-dependent manner, compared to their naïve status (p<0.001). In NPC43, net cytotoxicity of expanded NK was 35.5±7.2% compared to naïve NK with only 2.5±4.3% (p=0.003). NPC43 was most sensitive towards NK killing, followed by C666-1, then C17. This corresponds to NPC43’s low PD-L1 protein expression, a known downstream STAT3 target and immune checkpoint molecule. In addition, activated NK could kill chemoresistant NPC cells indiscriminately compared to the paralleled parental cells (18.5±2.3% and 16.1±3.8%, p=0.589). Importantly, augmented NPC43 cell death was observed in combined treatment (47.1±2.1%) compared to single treatment (NK alone: 9.2±6.5%; Napabucasin alone: 25.0±4.5%). In summary, this work demonstrated encouraging therapeutic potential of both NK therapy and in combination with STAT3 inhibitor Napabucasin for NPC treatment.

Citation Format: Shinyee Hui, Kwok Wai Lo, Mingjing Xu, Siu Tim Cheung. Therapeutic potential of activated natural killer cells in nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5514.
Abstract 5525: An Fc-competent bispecific antibody targeting PD-L1 and TIGIT induces strong immune responses and potent anti-tumor efficacy
Authors: Dai Shuang , Huang Weifeng , Yuan Zhijun , Peng Shaogang , Si Jiayi , Wang Chao , Yan Yao , Miao Xiaoniu , Xu Yingda , Sun Joanne , Liu Xiaolin , Tsun Andy , Zhai Tianhang
Published: June 2022

Background: TIGIT (T-cell immunoglobulin and ITIM domain), which is primarily expressed on activated and 'exhausted' T and NK cells, is one of the most promising 'next generation' immune checkpoint target. Engagement of TIGIT to its ligands (i.e., PVR and PVRL2) leads to inhibitory signaling in T cells and promotes the functional exhaustion of tumor-infiltrating T lymphocytes. Anti-TIGIT monoclonal antibodies have shown clinical benefit when combined with anti-PD-L1 agents in NSCLC. Here, we describe our novel anti-PD-L1 × TIGIT bispecific antibody (PD-L1 × TIGIT biAb) that blocks both the PD-L1/PD-1 and TIGIT/PVR/PVRL2 pathways and has the potential to exhibit equal clinical benefit compared to current combination therapies.

Methods: PD-L1 × TIGIT biAb was engineered with a fully-human IgG targeting TIGIT in a g1-Fc backbone, fused to a VHH at the C-terminus targeting PD-L1. Binding affinities and specificity testing were studied by flow cytometry and biolayer interferometry. The co-binding of the PD-L1 × TIGIT biAb to TIGIT and PD-L1 was detected by ELISA. The immunomodulatory functions of the PD-L1 × TIGIT biAb were evaluated using luciferase reporter cell assays and mixed lymphocyte reaction (MLR) assays in vitro, and human PBMC models in vivo.

Results: The PD-L1 × TIGIT biAb binds with high affinity to the extracellular domain of human TIGIT and PD-L1 and can bind to TIGIT and PD-L1 simultaneously. In a competition assay, the PD-L1 × TIGIT biAb efficiently blocked the interaction between TIGIT and PVR/PVRRL2, and likewise PD-L1 to PD-1. The PD-L1 × TIGIT biAb induced higher luciferase signals than the anti-TIGIT or anti-PD-L1 mAbs alone in a luciferase reporter-based cell system and enhanced IFN-γ production in an MLR assay. In vivo, the PD-L1 × TIGIT biAb demonstrates similar anti-tumor efficacy to the combination of anti-TIGIT and anti-PD-L1 mAbs, which is stronger than the single-agents alone. We have also completed GLP-toxicity studies that have shown excellent safety.

Conclusion: We have discovered a novel PD-L1 × TIGIT biAb, which induces strong immune responses in vitro and in vivo, supporting its clinical development for the treatment of human cancers. Clinical trials shall be initiating in early 2022.

Citation Format: Shuang Dai, Weifeng Huang, Zhijun Yuan, Shaogang Peng, Jiayi Si, Chao Wang, Yao Yan, Xiaoniu Miao, Yingda Xu, Joanne Sun, Xiaolin Liu, Andy Tsun, Tianhang Zhai. An Fc-competent bispecific antibody targeting PD-L1 and TIGIT induces strong immune responses and potent anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5525.
Abstract 5531: PD-1 blockades plus targeted therapy and cytotoxic drugs as conversion therapy in unresectable metastatic colorectal cancer (mCRC): A real-world experience
Authors: Huang Ling , Zhao Liying , Liu Xinyi , Huang Mengli
Published: June 2022

Background: Resection of metastatic tumor site has been proven beneficial for survival in mCRC, thus the need for conversion therapy of mCRC is strong. Anti-PD-1 antibody has presented durable clinical response in mismatch repair deficient (dMMR)/microsatellite instable (MSI-H) mCRC as first-line treatment while its efficacy in microsatellite stable (MSS) mCRC is still controversial. Combination of anti-PD-1 and targeted therapy or chemotherapies was regarded as potential regimens in MSS mCRC. Moreover, it is fully elucidated that immune checkpoint inhibitor used in initial therapy would have higher probability to achieve treatment response. We retrospectively analyzed the efficacy of anti-PD-1 plus targeted drugs and cytotoxic drugs as conversion/first-line therapy in fifteen untreated mCRC.

Methods: Thirteen MSS and two MSI-H mCRC were included. All patients received at least one cycle of anti-PD-1 (Pembrolizumab, Sintilimab or Toripalimab, q3w) combined with bevacizumab or cetuximab plus chemotherapy (FOLFOX or FOLFOXIRI regimens).

Results: 8 patients were RAS-mutant and 1 patient harbored BRAF-V600E. 13 patients had liver metastasis, 3 had lung metastasis and 3 had retroperitoneal lymph metastasis. 4 patients, including 2 MSI-H patients and 2 MSS patients had complete response (CR), 8 patients had partial response (PR), 2 patients had stable disease (SD) and 1 patient had progression disease (PD). The conversion rate was 80% (12/15), including 10 patients received resection or thermal ablation of metastatic tumor and 2 patients achieved CR. 9 patients were R0 resection and 10 patients achieved NED (no evidence of disease) status. G3-4 treatment-related adverse events (AEs) occurred in 1 patient (neutropenia, n=1). No patient discontinued treatment due to adverse events.

Conclusions: The triplet regimen of anti-PD-1, bevacizumab or cetuximab and chemotherapy was safe and tolerant and showed encouraging anti-tumor activities in MSS mCRC. Our experience suggested the clinical value of PD-1 blockades as initial treatment in MSS mCRC.

Citation Format: Ling Huang, Liying Zhao, Xinyi Liu, Mengli Huang. PD-1 blockades plus targeted therapy and cytotoxic drugs as conversion therapy in unresectable metastatic colorectal cancer (mCRC): A real-world experience [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5531.
Abstract 5549: First-in-class TNFR2 agonist & antagonist antibodies as Treg-Teff modulator for cancer immunotherapy
Authors: Jiang Xiaoling , Wu Chonging , Yin Liusong
Published: June 2022

Tumor necrosis factor receptor 2 (TNFR2) is expressed on highly suppressive tumor-infiltrating T regulatory cells (Treg), activated T effector cells (Teff), as well as many tumor cells in the tumor microenvironment, which makes TNFR2 a novel and very promising target for cancer immunotherapy. We have identified and characterized a wide panel of first-in-class highly selective TNFR2 agonist and antagonist antibodies, from murine hybridoma and camelid phage display campaigns. Our Treg antagonist lead candidate IAT0981-1A8 is a humanized single domain antibody (VHH), which showed tight TNFR2 binding with low-digit nanomolar affinity. IAT0981-1A8 potently blocked ligand TNFa binding to TNFR2, and elicited strong Treg proliferation inhibition activities. Our Teff agonist lead candidate IAT0981-231 is a humanized monoclonal antibody with sub-nanomolar affinity to TNFR2, which potently stimulated CD8+ T cell activation, proliferation and cytokine secretion, without blocking endogenous TNFa activity. Both IAT0981-1A8 and IAT0981-231 did not cross-react with TNFR1, and exhibited extremely potent tumor growth inhibition activities as single agent in multiple tumor models in TNFR2 transgenic animals, better than tested benchmarks. Our epitope binning studies demonstrated that IAT0981-1A8 and IAT0981-231 bound to totally different epitopes on TNFR2. We also evaluated several bispecific antibodies composing of tumor associated antigens or other immune cell modulators in tumor microenvironment, with either IAT0981-1A8 or IAT0981-231. We have generated high quality commercial stable cell line for the Teff agonist lead candidate IAT0981-231, and completed several batches of 200L non-GMP and GMP production. Our preclinical cynomolgus monkey toxicity studies revealed extremely good safety profile for IAT0981-231, with no significant toxicity observed even with high 200mg/kg repeated dose. We are currently performing IND-enabling studies for the agonist lead candidate IAT0981-231, and aim to enter clinical studies in the first half of 2022, with first-in-class potential as monotherapy and I/O combination therapy for advanced cancers.

Citation Format: Xiaoling Jiang, Chonging Wu, Liusong Yin. First-in-class TNFR2 agonist & antagonist antibodies as Treg-Teff modulator for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5549.
Abstract 5563: Development of an integrated in vitro and in vivo OVA-specific system for cancer vaccine discovery
Authors: Xiang Jian , Huang Jun , Zhou Wenrong , Peng Zhengang , Gu Qingyang , Ji Qunsheng
Published: June 2022

As the development of cancer immunotherapy and urgent demand of precision medicine, cancer vaccines have been envisioned as a novel and effective tool to cure cancer disease or prevent cancer from recurrence or metastasis. In order to support the research and development of new modality in drug discovery, we have established an advanced and integrated in vitro and in vivo platform for various types of cancer vaccine, such as peptide vaccine, mRNA vaccine, DC vaccine and so on. Ovalbimin (OVA) is a key model antigen which has been widely used in vaccination experiments. Here we established an OVA overexpressed system based on different types of tumor cells, such as B16F10-OVA and MC38-OVA. Firstly, we proved that MHC-I:OVA complex were presented on the surface of stable cell lines successfully through IFN stimulation. The results of in vitro CD8+ T cells killing assay showed the B16F10-OVA cells can be largely killed which indicates OVA protein has been presented and can be utilized as a specific target for analysis. In vitro ELISPOT assay is well characterized for efficiency tool for immunogenicity determination. Using ELISPOT assay, we compared the immunogenicity of OVA specific peptide and mRNA administration via different injection manners. Furthermore, in vivo tumor model of B16F10-OVA and MC38-OVA have also been established to evaluate the anti-tumor efficacy of peptide and mRNA vaccine. The mechanism of cancer vaccine has also been investigated via T cell, B cell and other cell populations blocking. Additionally, we explored the target protein biodistribution and in vivo behavior of mRNA vaccine carrier with Lumina LT system. In summary, the integrated OVA-specific system can be used for new adjuvants and peptide/mRNA carrier exploration in cancer vaccine discovery.

Citation Format: Jian Xiang, Jun Huang, Wenrong Zhou, Zhengang Peng, Qingyang Gu, Qunsheng Ji. Development of an integrated in vitro and in vivo OVA-specific system for cancer vaccine discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5563.
Abstract 5569: CAR-T cells with αPDL1/CD28 switch-receptor synergize radiotherapy and anti-PD1 therapy in solid tumors
Authors: Yin Limei , Wang Wenbo , Yao Zhuoran , Xue Jianxin , Tong Ruizhan , Wang Hui , Liao Shuangsi , Wang Laduona , Zheng Yue , Na Feifei , Yu Min , Zhang Xuanwei , Gong Youling , Huang Meijuan , Mo Xianming , Chen Chong , Lu You
Published: June 2022

The therapeutic efficacy of CAR-T cells in solid tumors, unlike in hematologic tumor, is greatly limited by the insufficient infiltration and persistence of CAR-T cells as well as the immunosuppressive tumor microenvironment. The aim of this study was to overcome these obstacles by introducing an αPDL1/CD28 switch-receptor construct and by seeking combinatorial strategies for CAR-T cells in solid tumors. We found that in non-transduction T cells, the cytokine release and T cell proliferation were repressed in response to PD-L1 protein, while T cells that express αPDL1/CD28 switch-receptor showed enhanced functions, indicating that αPDL1/CD28 could revert PD-L1 inhibition into CD28 costimulation. CAR-T cells with αPDL1/CD28 switch-receptor showed better effector function than that of unitary CAR-T in vitro studies and significant responses in tumor-bearing mice, with more effector memory T cells infiltrated in the tumor. On this basis, PD-1 inhibitor can further enhance the efficacy and persistence of αPDL1/CD28 CAR-T cells, especially in PDL1+ tumor models. We found in vitro studies that radiotherapy increased the expression of T-cell recruiting chemokines and promoted CAR-T cell transmigration. In tumor-bearing mice, synergistic anti-tumor efficacy of mice treated with radiotherapy and αPDL1/CD28 CAR-T cells was further observed. Finally, triple therapy with radiotherapy and anti-PD1 plus αPDL1/CD28 CAR-T cells maximized antitumor responses and induced complete cures in the tumor-bearing mice. Our study suggests that αPDL1/CD28 augments the function of CAR-T cells, and the combinatorial regime of αPDL1/CD28 CAR-T cells, radiotherapy and anti-PD1 in solid tumors could be further investigated.

Citation Format: Limei Yin, Wenbo Wang, Zhuoran Yao, Jianxin Xue, Ruizhan Tong, Hui Wang, Shuangsi Liao, Laduona Wang, Yue Zheng, Feifei Na, Min Yu, Xuanwei Zhang, Youling Gong, Meijuan Huang, Xianming Mo, Chong Chen, You Lu. CAR-T cells with αPDL1/CD28 switch-receptor synergize radiotherapy and anti-PD1 therapy in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5569.
Abstract 557: Engineering off-the-shelf anti mesothelin t-cell receptor fusion construct (TRuC™) t-cells
Authors: Donaghey Julie , Kwong Cecilia , Powell Allison , Patterson Troy , Liberzon Ella , Decker Richard , Rodriguez Julio Gomez , Kreienberg Darby , Bian Jennifer , Patankar Urmi , Horton Holly , Ding Jian , Hofmeister Robert , Gutierrez Dario , Tighe Robert
Published: June 2022

We have previously described the generation of autologous T Cell Receptor Fusion Construct (TRuC™) T cells which are engineered to express a fusion protein comprised of an antibody-derived binder tethered to the extracellular domain of the CD3ε signaling subunit. Upon integration of the TRuC into the T cell receptor (TCR), it targets specific tumor surface antigens independent of HLA and uses the complete receptor complex to trigger a comprehensive T cell response. Here, we report about engineering of off-the-shelf TRuC-T cells directed against mesothelin (MSLN). To eliminate the alloreactivity of α/β T cells and reduce the risk of graft-versus-host-disease (GvHD), the TRAC locus is knocked-out. To enable the re-assembly of the TCR, the endogenous TCRα and β subunits are replaced with fusion proteins comprised of antibody-derived binders fused to the TCRγ and δ constant domains. Allogeneic anti-MSLN TRuC-T cells upregulate activation markers, secrete robust cytokines, and lyse tumor cells in an antigen-specific manner without allo-reactivity. In comparison with control autologous TRuC-T cells targeting mesothelin (TC-210), allogeneic TRuC-Ts demonstrate extended tumor clearance in NSG xenograft models due to their enhanced expansion and persistence. To reduce host rejection and further boost the persistence of the allogeneic TRuC-T cells, we eliminated MHC class I surface expression by B2M gene knockout and over-expressed membrane-bound IL15 tethered to its receptor alpha. In summary we have engineered persistence enhanced, allogeneic TRuC-T cells that maintain the signaling properties of the TCR complex with improved efficacy compared to donor-matched autologous TRuC-T cells.

Citation Format: Julie Donaghey, Cecilia Kwong, Allison Powell, Troy Patterson, Ella Liberzon, Richard Decker, Julio Gomez Rodriguez, Darby Kreienberg, Jennifer Bian, Urmi Patankar, Holly Horton, Jian Ding, Robert Hofmeister, Dario Gutierrez, Robert Tighe. Engineering off-the-shelf anti mesothelin t-cell receptor fusion construct (TRuC™) t-cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 557.
Abstract 5582: Modulation of intracellular kinase signaling to improve TIL manufacturing for adoptive cell therapy
Authors: Shi Zixiao , Yao Sheng , Zhao Peipei , Li Fei , Zhou Di , Cui Jun , Sun Jingwei , Liu Yarong
Published: June 2022

Tumor-infiltrating lymphocytes (TILs) has demonstrated significant clinical benefits for patients with solid tumor. A robust expansion process is needed in order to produce a large number of cells for clinical usage. We modulated the PI3K-AKT signaling pathway in TILs isolated from cervical and ovarian cancer patients and found the inhibition of either PI3K or AKT can promote TIL proliferation during the REP stage. Inhibiting PI3K-AKT led to an increase of the effector CD8+T cell population with higher CD25+ and CD28+ in the CD8+ population, more TCF-1+ and CD39-CD69- T cells in both CD4+ and CD8+ T cell population, and downregulated exhaustion markers CD39+ and TIM-3+ in CD8+ T cell population. This inhibition significantly increased cytotoxicity when cocultured with tumor cell lines and patient-derived tumor samples. Moreover, dual knockout of ATK1 and ATK2 promoted TIL proliferation, long-term survival and decreased apoptosis of TIL, which were further confirmed by the RNA-seq results. Thus, inhibition of PI3K-AKT signaling represents a promising strategy to generate large numbers of TIL with enhanced stemness, activation and cytotoxicity, and could be used to support adoptive cell therapy to treat solid tumors.

Citation Format: Zixiao Shi, Sheng Yao, Peipei Zhao, Fei Li, Di Zhou, Jun Cui, Jingwei Sun, Yarong Liu. Modulation of intracellular kinase signaling to improve TIL manufacturing for adoptive cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5582.
Abstract 564: Targeting axonal guidance with anti-ROBO1 CAR T cells: A new therapeutic strategy for malignant brain cancer
Authors: Singh Sheila Kumari , Chokshi Chirayu R. , Brakel Benjamin , Rossotti Martin A. , Venugopal Chitra , Salim Sabra , Henry Kevin
Published: June 2022

No standardized treatment exists for patients with recurrent glioblastoma (GBM). Given the aggressive nature of the disease and difficulty in modeling tumor recurrence, minimal efforts have been made to design rational therapies against it. The roundabout guidance receptor 1 (ROBO1) protein is involved in axonal guidance during neurodevelopment and is aberrantly upregulated in glioma where it mediates glioma cell migration. Here, we present that ROBO1 is highly expressed on the surface of malignant and treatment-refractory brain tumor initiating cells (BTICs), prompting the development of an anti-ROBO1 CAR-T cell therapy. Using the binding region of a single-domain antibody targeting ROBO1, we developed second-generation anti-ROBO1 CAR-T cells specific and effective against ROBO1-expressing BTICs. Upon antigen exposure, anti-ROBO1 CAR-T cells upregulated markers of activation and degranulation. Additionally, treatment of orthotopic and patient-derived brain tumor xenograft models with anti-ROBO1 CAR-T cells resulted in reduced tumor burden and prolonged survival, demonstrating the therapy’s therapeutic potential for treating neoplastic brain malignancies.

Citation Format: Sheila Kumari Singh, Chirayu R. Chokshi, Benjamin Brakel, Martin A. Rossotti, Chitra Venugopal, Sabra Salim, Kevin Henry. Targeting axonal guidance with anti-ROBO1 CAR T cells: A new therapeutic strategy for malignant brain cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 564.
Abstract 5656: Design and development of novel anticancer dimethoxyquinazoline-4-amine analog, DW-8, against colorectal cancer
Authors: Neupane Rabin , Malla Saloni , Abou-Dahech Mariam Sami , Balaji Swapnaa , Kumari Shikha , Tang Yuan , Karthikeyan Chandrabose , Tiwari Amit K.
Published: June 2022

Patients with advanced or metastatic colorectal cancer (CRC) often have poor prognosis and survival. There are ongoing efforts to identify novel molecules to manage advanced and metastatic CRC. Modifications in the quinazoline ring has resulted in effective and clinically approved drugs such as gefitinib, erlotinib, vandetanib among others. We rationalized and synthesized a series of 4-anilinoquinazoline analogues, DW (1-10) and evaluated them for anticancer efficacy in human CRC cell lines (HCT116, HT29, SW620). We identified a lead molecule DW-8, with sub-micromolar potency against CRC lines with selectivity index of DW-8 being >2 folds in CRC cells compared to non-cancerous line, CRL1459. Mechanistically, DW-8 produced significant morphological changes in SW620 cells. This included nuclear condensation, cell shrinkage, blebbing and apoptotic bodies formation which are the hallmarks of apoptosis. We found that the DW-8 induced apoptosis by i) producing cell cycle arrest at G2 phase; ii) activated intrinsic apoptosis as shown by cleaved caspases such as caspase-9, the executioner caspases 3 and 7; iii) produced nuclear fragmentation; and iv) increased the level of reactive oxygen species. We are now synthesizing the analogs of DW-8 and are optimizing the formulations for efficient delivery to CRC tumors in vivo. Overall, our result suggests DW-8 may represent a suitable lead for developing novel compound to treat CRC.

Citation Format: Rabin Neupane, Saloni Malla, Mariam Sami Abou-Dahech, Swapnaa Balaji, Shikha Kumari, Yuan Tang, Chandrabose Karthikeyan, Amit K. Tiwari. Design and development of novel anticancer dimethoxyquinazoline-4-amine analog, DW-8, against colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5656.
Abstract 567: CRISPR/Cas9-mediated CD39 disruption can be combined with TCR editing in T cells to improve the adoptive T cell therapy of colorectal cancer
Authors: Potenza Alessia , Balestrieri Chiara , Albarello Luca , Pedica Federica , Stasi Lorena , Manfredi Francesco , Spiga Martina , Tassi Elena , Cianciotti Beatrice Claudia , Abbati Danilo , Elmore Ugo , Biondi Andrea , Aldrighetti Luca , De Lalla Claudia , Lullo Giulia Di , Dellabona Paolo , Ruggiero Eliana , Rosati Riccardo , Bonini Chiara
Published: June 2022

Colorectal cancer (CRC) is the 2nd cause of cancer-related death. Despite standard therapies, more than 50% of patients experience relapse, eventually with metastatic disease. The CRC microenvironment is densely infiltrated by T-cells, which presence correlates with improved overall survival, thus sustaining the rational for immunotherapy. Here, we paired high-dimensional flow cytometry, bulk RNA sequencing and immunohistochemistry to describe the phenotype and the exhaustion status of T-cells infiltrating primary and metastatic CRC. Analysis of the healthy, peritumoral and neoplastic tissues of treatment-naïve primary CRCs and of the peritumoral and tumoral tissues of CRC patients undergoing surgery for liver metastasis, revealed extensive transcriptional and spatial remodeling across tumors. Unsupervised analysis of flow cytometry data performed by an advanced pipeline of data handling by dimensionality reduction and clustering algorithms allowed the definition of a peculiar inhibitory receptors signature on TILs enriched both in primary CRCs and liver metastases. Of note, CD39 was upregulated in both the signatures retrieved from primary and metastatic CRC, thus suggesting its relevance as molecular target for T-cells engineering. By CRISPR/Cas9 we disrupted the CD39 gene in T cells with >80% efficiency. We combined CD39 knock-out with the genetic disruption of alpha and beta chains of the endogenous TCR, observing >90% efficiency for both genes, thus generating triple-knockout T-cells. By repetitively stimulating healthy donors’ peripheral blood mononuclear cells with autologous antigen-presenting cells loaded with a pool of peptides selected to be immunogenic and expressed by CRC, we obtained a library of anti-tumor TCRs to redirected the specificity of triple knock-out lymphocytes. Our preliminary experiments showed a functional advantage for TCR-redirected, CD39 disrupted T-cells in recognizing and killing CRC target cells.

Citation Format: Alessia Potenza, Chiara Balestrieri, Luca Albarello, Federica Pedica, Lorena Stasi, Francesco Manfredi, Martina Spiga, Elena Tassi, Beatrice Claudia Cianciotti, Danilo Abbati, Ugo Elmore, Andrea Biondi, Luca Aldrighetti, Claudia De Lalla, Giulia Di Lullo, Paolo Dellabona, Eliana Ruggiero, Riccardo Rosati, Chiara Bonini. CRISPR/Cas9-mediated CD39 disruption can be combined with TCR editing in T cells to improve the adoptive T cell therapy of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 567.
Abstract 571: Inclusion of a Dap10 costimulatory domain decreases cytokine release syndrome in chimeric antigen-expressing gamma delta T cells
Authors: Barber Amorette E.
Published: June 2022

Adoptive transfer of T cells is a promising anti-tumor therapy for many cancers. To enhance tumor recognition by T cells, chimeric antigen receptors (CAR) consisting of signaling domains fused to receptors that recognize tumor antigens can be expressed in T cells. While CAR T cells can successfully treat hematopoietic malignancies, CAR T cells have not shown success in treating solid tumors. There are many factors that may enhance CAR T cell efficacy and safety for solid tumor treatment, including addition of costimulatory domains to increase T cell functions and switching the T cell type used from alpha beta to gamma delta T cells. We developed a novel CAR (chimeric PD-1, chPD1) that targets the programmed death 1 receptor (PD-1) ligands expressed on many types of solid tumors. The aim of this study was to compare anti-tumor efficacy and safety of chPD1-expressing gamma delta and alpha beta T cells in murine breast and ovarian cancer models. Inclusion of different costimulatory domains, CD28, 4-1BB, and Dap-10, in the chPD1 receptor was also compared. There were no differences in T cell viability and proliferation when comparing costimulatory domains or T cell type. Additionally, there were no significant differences in killing of murine breast and ovarian cancer cell lines. ChPD1-CD28 T cells had increased secretion of proinflammatory and cytokine release syndrome (CRS)-inducing cytokines, including IFN-gamma, IL-2, IL-6, GM-CSF, and nitric oxide, compared to chPD1-41BB and chPD1-Dap10 receptors. Additionally, alpha beta chPD1 T cells had increased cytokine secretion of inflammatory cytokines compared to chPD1 gamma delta T cells. Furthermore, while gamma delta and alpha beta T cells expressing chPD1 receptors with a Dap10 domain had similar anti-tumor efficacy in vivo, chPD1-Dap10 gamma delta T cells had the lowest serum levels of inflammatory cytokines IFN-gamma, IL-6, and GM-CSF. Therefore, inclusion of Dap10 domains in gamma delta T cells may be a safer and more efficacious option for CAR T cell therapy of solid tumors.

Citation Format: Amorette E. Barber. Inclusion of a Dap10 costimulatory domain decreases cytokine release syndrome in chimeric antigen-expressing gamma delta T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 571.
Abstract 5722: Targeting the mRNA endonuclease CPSF73 inhibits breast cancer cell migration and invasion
Authors: Liu Huiyun , Heller-Trulli Daniel , Moore Claire
Published: June 2022

Cleavage by the endonuclease CPSF73 and polyadenylation of nascent RNA is an essential step in co-transcriptional mRNA maturation, and abnormal poly(A) site choices have been associated with human diseases, including cancer. Recent work has identified CPSF73 as a promising drug target for inhibiting the growth of specific cancers, and higher level of CPSF73 correlates to poor survival in breast cancer patients. However, our understanding of the regulation of CPSF73 is still at an early stage. Here, we report that a HECT-like E3 ligase, UBE3D, participates in stabilizing CPFS73 protein by preventing its ubiquitin-mediated degradation by the proteasome. Depletion of UBE3D in both non-cancer cells and cancer cells leads to CPSF73 downregulation, a pre-mRNA cleavage defect and dysregulated gene expression. UBE3D dysfunction or chemical inactivation of CPFS73 inhibited migration and invasion as well as stem cell renewal phenotypes in vitro in triple negative breast cancer cells. In addition, genetic overexpression of CPSF73 promoted breast cancer stemness and knocking down of CPSF73 inhibited stem cell renewal property. Together, our findings indicate that targeting the pre-mRNA processing nuclease CPSF73 has potential for breast cancer therapy.

This work was supported by the NSF grant NS0224 to C. Moore and the NIH grant R01 GM101010-01A1 to C. Moore.

Citation Format: Huiyun Liu, Daniel Heller-Trulli, Claire Moore. Targeting the mRNA endonuclease CPSF73 inhibits breast cancer cell migration and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5722.
Abstract 5765: Genomic characterization of Chinese biliary tract cancers identifies differences between subtypes and therapeutic biomarkers
Authors: Chen Xiaofeng , Pang Jiaohui , Liu Cuicui , Wang Sha , Shao Yang
Published: June 2022

Background: Biliary tract carcinoma (BTC) is a relatively rare cancer worldwide and accounts for 3% gastrointestinal cancers in China. BTCs are mainly classified as gallbladder cancer (GBC) and cholangiocarcinomas (CCA) with dismal prognosis. Genetic landscape of BTC can offer a comprehensive understanding of its carcinogenesis and clinical associations. The present study aims to compare the molecular features between BTC subtypes and explore clues for treatment therapies.

Methods: We analyzed 258 BTC tissue samples including 70 GBCs and 188 CCAs with targeted sequencing (NGS) using a 425 caner-relevant genes panel. We identified the somatic alterations and MSI status of all samples. TMB was counted by summing all base substitutions and indels in the coding region of targeted genes. TMB-high (TMB-H) was defined as mutation number ≥ 10mut/Mb.

Results: The most frequently mutated genes in 258 BTCs were TP53 (56.6%), KRAS (27.9%), CDKN2A (21.3%), TERT (17.8%) and MCL1 (14.3%). Four gene mutations and 2 gene amplifications were significantly enriched in GBC compared to CCA (mutations P<0.05, amplifications P<0.01): TP53 (78.8% vs 47.9%), ARID2 (15.7% vs 3.7%), STK11 (12.9% vs 4.3%), TERT (24.3% vs 11.2%), CCNE1 and SOX2 amplifications (21.4% vs 2.7%, 4.3% vs 0%). However, ARID1A mutation (15.4% vs 5.7%), PBRM1 mutation (10.1% vs 0%), and MCL1 amplification (17.0% vs 5.7%) were more frequently existed in CAA (all P<0.05). Seven actionable alterations, including IDH1/2 (5.4%), FGFR1-4 (4.7%), ERBB2 (6.2%), PIK3CA (5.4%), MET (3.5%), and BRCA1/2 (5.0%), were detected with different occurrence frequencies in two subtypes. ERBB2 was predominant in GBC (15.7% vs 7.4%, p<0.001), while IDH1/2 and KRAS alteration rates were much higher in CCA (8% vs 0%, 31.4% vs 18.6%, both P<0.05). FGFR3 alterations were also tended to be enriched in CCA (7.1% vs 2.7%), although no significant difference was found. In general, there were 25.7% of GBC patients harboring actionable alterations versus 29.3% of CCA patients. In addition, 40 (15.5%, 13 GBCs and 27 CCAs) and 11 (4.3%, 10 GBCs and 1 CCA) patients were TMB-H and MSI-H, respectively, suggesting the possibility of benefit from immunotherapy.

Conclusions: There were distinct molecular features in somatic variations and actionable alterations between GBSs and CCAs. In general, 44.3% of GBC and 46.3% CCA may benefit from targeted therapy and immunotherapy. This study could be helpful to establish treatment therapies for BTCs according to genetic information.

Citation Format: Xiaofeng Chen, Jiaohui Pang, Cuicui Liu, Sha Wang, Yang Shao. Genomic characterization of Chinese biliary tract cancers identifies differences between subtypes and therapeutic biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5765.
Abstract 5777: Germline variants of cancer predisposition genes in a large cohort of Chinese sarcoma patients
Authors: Shen Jingnan , Li Xiaomo , Xie Xianbiao , Liu Si , Ma Tonghui
Published: June 2022

Background: Sarcomas represent a rare and heterogeneous group of soft tissue and bone cancers, including more than 100 histological subtypes. Although genomic profiling has identified oncogenic fusion genes as the major molecular drivers of many sarcoma subtypes, direct targeting of these fusion genes is clinically challenging and novel treatment approaches are needed.

Methods: To explore new therapeutic targets in sarcoma, we performed germline testing of 148-cancer predisposition genes (OncoPanscan࣪, Genetronhealth) on genomic DNA from a cohort of 822 unselected Chinese sarcoma patients.

Results: The median age of this cohort was 45 (1-88) years, and 84% were soft tissue sarcoma (STS), 16% were osteosarcoma. 9.3% (n =82) of patients harbored at least one predicted pathogenic germline variant in 37 cancer disposition genes including APC, BRCA1, BRCA2, CDKN2A, CHEK2, DICER1, MSH6, PMS2, SBDS, SMARCA4, and TP53. Twenty-four (2.9%) patients had a variant that mapped to a known sarcoma-associated cancer predisposition syndrome gene (CHEK2, DICER1, NF1, RECQL4, SMARCA4, TP53, WRN). The most frequently mutated genes are involved in the DNA damage repair pathway, with a prevalence of 6.3% (n =52). Twelve patients carried pathogenic mutations in TP53, which was associated with cancer-predisposing Li-Fraumeni syndrome (LFS). Interestingly, we also observed 12 patients harboring the same SBDS c.258+2T>C variant. Biallelic SBDS mutations were linked to Shwachman-Diamond Syndrome (SDS) associated with leukemia predisposition. Additionally, four patients carried pathogenic mutations in SMARCA4 (n =2) and SMARCE1 (n =2), two genes encoding key components of the BAF chromatin-remodeling complex. Patients with pathogenic SMARCA4 germline variants could participate in clinical trials of EZH2 inhibitor tazemetostat. Four patients harbored pathogenic germline variants in core homologous recombination genes (BRCA1/2 and PALB2), which may be targeted with platinum-based chemotherapy or PARP inhibitor olaparib.

Conclusions: In summary, our genomic profiling approach revealed novel predisposition variants associated with sarcoma, and some of them can serve as therapeutic targets for future clinical investigation. Our work provided proof of principle that sarcoma patients may benefit from genetic counseling and participate in genomically-matched clinical trials.

Citation Format: Jingnan Shen, Xiaomo Li, Xianbiao Xie, Si Liu, Tonghui Ma. Germline variants of cancer predisposition genes in a large cohort of Chinese sarcoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5777.