Regular and Young Investigator Award Abstracts最新文献

{"title":"1220 Orthogonal CRISPR screens to identify transcriptional and epigenetic regulators of human CD8 T cell function","authors":"Sean McCutcheon, Adam Swartz, Michael Brown, Alejandro Barrera, Christian McRoberts Amador, Keith Siklenka, Lucas Humayun, James Isaacs, Timothy Reddy, Smita Nair, Scott Antonia, Charles Gersbach","doi":"10.1136/jitc-2023-sitc2023.1220","DOIUrl":"https://doi.org/10.1136/jitc-2023-sitc2023.1220","url":null,"abstract":"<h3>Background</h3> The clinical response to adoptive T cell therapies is strongly associated with transcriptional and epigenetic state. Thus, technologies to discover regulators of T cell gene networks and their corresponding phenotypes have great potential to improve the efficacy of T cell therapies. <h3>Methods</h3> We developed pooled CRISPR screening approaches with compact epigenome editors to systematically profile the effects of activation and repression of 120 transcription factors and epigenetic modifiers on human CD8 T cell state. <h3>Results</h3> CRISPR interference and activation screens nominated known and novel regulators of T cell phenotypes with BATF3 emerging as a high confidence gene in both screens. We found that BATF3 overexpression promoted specific features of memory T cells such as increased IL7R expression and glycolytic capacity, while attenuating gene programs associated with cytotoxicity, regulatory T cell function, and T cell exhaustion. In the context of chronic antigen stimulation, BATF3 overexpression countered phenotypic and epigenetic signatures of T cell exhaustion. For example, only 13% of BATF3 engineered T cells co-expressed canonical exhaustion markers (LAG3, TIM3, TIGIT), whereas 65% of wild type T cell co-expressed all three markers after multiple rounds of antigen stimulation. CAR T cells overexpressing BATF3 significantly outperformed control CAR T cells in both in vitro and in vivo tumor models. Moreover, we found that BATF3 programmed a transcriptional profile that correlated with positive clinical response to adoptive T cell therapy. Finally, we performed CRISPR knockout screens with and without BATF3 overexpression to define co-factors and downstream factors of BATF3, as well as other therapeutic targets. <h3>Conclusions</h3> BATF3 overexpression markedly enhanced the therapeutic potential of CD8 T cells in both in vitro and in vivo tumor models. The compact size of BATF3 could seamlessly integrate into current manufacturing processes of FDA-approved adoptive T cell therapies, which all use lentivirus to deliver the CAR construct to donor T cells. To our knowledge, this work is the first example that combines overexpression of a specific transcription factor with a transcription factor wide knockout screen to dissect co-factors and downstream factors and highlights the power of this approach. These screens pointed to a model where BATF3 interacts with JUNB and IRF4 to regulate gene expression and illuminated several other novel targets for further investigation.","PeriodicalId":500964,"journal":{"name":"Regular and Young Investigator Award Abstracts","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135161894","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"1122 Vidutolimod, an immunostimulatory virus-like particle, reduces proliferation but enhances the activation of tumor-specific T cells","authors":"Travis D Fischer, Caitlin D Lemke-Miltner, George J Weiner","doi":"10.1136/jitc-2023-sitc2023.1122","DOIUrl":"https://doi.org/10.1136/jitc-2023-sitc2023.1122","url":null,"abstract":"<h3>Background</h3> One approach to enhancing the anti-tumor T cell response is to alter the tumor microenvironment (TME) through intratumoral injection (IT) of immunostimulatory agents such as Vidutolimod (<b>Vidu</b>). Vidu is a virus-like particle (VLP) composed of a TLR9 agonist (CpG-A, known as G10) encapsulated by the Qβ bacteriophage capsid. IT Vidu shows considerable promise in early phase clinical trials. The immune response to Vidu is initiated by induction of IFNa production by pDCs within the TME. This effect is dependent on coating of Vidu with antibodies against the Qβ capsid. This is followed by a series of changes in the TME that ultimately result in an enhanced anti-tumor T cell response. Mouse models have shown that the efficacy of IT Vidu depends on the presence of both CD4+ and CD8+ T cells. Most studies to date exploring the impact of Vidu on T cells have focused on the overall T cell population. <h3>Methods</h3> The current studies were designed to further assess the complex mechanisms by which Vidu induces an anti-tumor T cell response through use of the well-established OT-1 mouse model that allows for analysis of the tumor-specific T cell population. OT-1 mice contain CD8+ T cells with a transgenic TCR that recognizes the ovalbumin (OVA) peptide SIINFEKL sequence (OVA257–264) presented on MHC Class I. Prior to culture, OT-1 splenocytes were labeled with CellTrace Violet in order to monitor proliferation over time. OT-1 splenocytes were then cultured with EL4 cells (an OVA-negative T lymphoblast cell line) or E.G7-OVA (OVA-expressing EL4 derivative cells). <h3>Results</h3> Minimal proliferation or evidence of T cell activation was seen when OT-1 CD8+ T cells were cultured with EL4 cells regardless of the addition of Vidu and anti-Qβ antibodies. OT-1 CD8+ T cells cultured with E.G7-OVA cells showed both proliferation and activation as indicated by increased intracellular IFNy and surface PD-1. Addition of Vidu and anti-Qb antibody reduced OT-1 CD8+ proliferation but enhanced production of IFNγ and expression of PD-1. The increase in IFNγ and PD-1 expression was strongest in the dividing OT-1 CD8+ T cell population. Preliminary results of ongoing <i>in vivo</i> studies are consistent with these results. <h3>Conclusions</h3> Vidu reduces proliferation but enhances phenotypic markers of activation expressed by tumor-specific CD8+ T cells (OT-1 cells) when co-cultured with cells expressing OVA, their target antigen. Markers of activation are most notable in dividing OT-1 CD8+ T cells. <h3>Ethics Approval</h3> Mouse studies were approved and performed according to guidelines established by the University of Iowa Institutional Animal Care and Use Committee (IACUC) under the approved Protocol #1011236.","PeriodicalId":500964,"journal":{"name":"Regular and Young Investigator Award Abstracts","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135161895","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"799 Combination intraperitoneal chemoimmunotherapy triggers a T-cell chemotactic locoregional response in patients with recurrent platinum-sensitive ovarian cancer","authors":"Mackenzy Radolec, Brian Orr, Lixin Zhang, Mary Strange, Syed Zaidi, Robert Edwards, Anda Vlad","doi":"10.1136/jitc-2023-sitc2023.0799","DOIUrl":"https://doi.org/10.1136/jitc-2023-sitc2023.0799","url":null,"abstract":"<h3>Background</h3> The increased prevalence of CD8+ tumor-infiltrating lymphocytes (TIL) in the tumor microenvironment (TME) correlates with improved outcomes in patients with epithelial ovarian cancer (EOC). We hypothesize that a combination of intraperitoneal (IP) chemotherapy (via immunogenic cell death-inducing cisplatin) with dual agent immunotherapy using IV pembrolizumab (anti-PD1) and IP rintatolimod (dsRNA and TLR-3 agonist) promotes increased T cell chemotaxis and cytolytic function, for improved clinical outcomes. <h3>Methods</h3> We have performed translational studies focused on the immune TME, using a longitudinal collection of biospecimens, including plasma, PBMC, IP washes and tumor tissue. The samples were obtained from patients treated in a phase II, investigator- initiated trial (NCT03734692) that tests the efficacy/safety of IP cisplatin/IP rintatolimod/IV pembrolizumab administered in 6 cycles, three weeks apart. Serial collection of biologic samples includes aspiration of peritoneal resident cells (IP washes) before and after each treatment, at each of the 6 cycles. RNA sequencing of IP wash cells was performed using the Novogene platform. Additionally, the MesoScale Delivery (MSD) platform was used to profile 20 biomarkers in the peritoneal samples throughout treatment. <h3>Results</h3> Sequential sampling of the intraperitoneal cavity showed an increase in cellularity immediately after treatment consistent with an ‘acute’ pro-inflammatory reaction. RNA sequencing data showed a significant upregulation acutely in genes associated with anti-tumor immunity (STAT1/STAT2 and downstream targets), T lymphotactic chemokines (CXCL9, 10, 11), and TH1 type response (IFNgamma, Tbet) (p<0.05) all of which are important for T lymphotaxis and function via TCR engagement with cognate tumor antigens. Gene Set Enrichment Analysis demonstrates an acute enrichment in Interferon α response as well as the Interferon γ response (figure 1). MSD measurements in IP washes demonstrated an acute increase in granzyme B, perforin, TNF alpha, CXCL9, 10 and 11, IFN gamma, and IL-15 after treatment (p<0.05) (figure 2). Longitudinal data revealed a progressive increase in CXCL9, 10 and 11, as well as perforin, IFN gamma and TNF alpha from baseline levels at cycle 1 to cycle 6, suggestive of a sustained, ‘chronic’ response during treatment. <h3>Conclusions</h3> Analysis of the locoregional immune environment taken from patients receiving this novel, triple drug combination has demonstrated an acute and persistent increase in biomarkers associated with T cell chemotaxis and T cell function. Ongoing chip cytometry profiling of both IP wash cells and tumor samples will further elucidate the treatment induced changes in various innate and adaptive immune cell types in the TME. <h3>Acknowledgements</h3> Acknowledgments: Pembrolizumab was provided by MERCK and Rintatolimod was provided by AIM Immunotech. This study was funded in part by the National Institutes o","PeriodicalId":500964,"journal":{"name":"Regular and Young Investigator Award Abstracts","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135161903","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"48 Blood-based peripheral T cell cytotoxicity assay in predicting response to immune checkpoint inhibitors: a US pilot study","authors":"Rajesh Nair, Jonathon Woo, Suzanna Lee, Shumei Kato, Michele Baltay, Yali Li, Kota Iwahori, Junichi Akatsuka, Srinath Sampath, Christian Schmedt, Srihari Sampath","doi":"10.1136/jitc-2023-sitc2023.0048","DOIUrl":"https://doi.org/10.1136/jitc-2023-sitc2023.0048","url":null,"abstract":"<h3>Background</h3> Immunotherapy with Immune checkpoint inhibitors (ICIs) has revolutionized cancer treatment over the last decade. Despite the phenomenal success of ICIs, the clinical response is confined to a small subset of patients. Conventional biomarkers such as PDL1 expression and Tumor Mutational Burden (TMB) rely on invasive biopsies and remain inadequate in predicting clinical benefits for patients. Therefore, there is an urgent need to develop better noninvasive biomarkers to predict the response to ICIs and to identify patients for whom these therapies are both safe and effective. A novel blood-based functional assay, peripheral T cell cytotoxicity (PeriCyto), has previously been shown to accurately predict the clinical response for advanced non-small cell lung cancer (NSCLC) in Japanese patients.^{1 2} The present study aimed to assess clinical feasibility of PeriCyto in predicting the response to ICIs in a US patient cohort. <h3>Methods</h3> Prospective samples (n=13) were obtained from patients with diverse solid tumors prior to treatment with ICIs either as monotherapy or in combination with chemotherapy/targeted therapy. Peripheral Blood Mononuclear Cells (PBMCs) isolated from whole blood were cocultured with U251 cells in the presence of an EphA2/CD3 Bispecific T cell Engager antibody (BiTE). After 48hrs, peripheral T cell cytotoxicity was measured using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay (MTS) assay. PBMCs from a healthy donor with an established cytotoxicity score served as an internal positive control for the assay. <h3>Results</h3> The study evaluated the positive predictive value (PPV), negative predictive value (NPV), sensitivity and specificity of PeriCyto in 13 patients with diverse set of cancers. As previously published^{1 2} the assay was able to accurately predict the clinical response in the subgroup of patients with advanced NSCLC (4/4). Combining data from patients with all cancer types, the PPV was ~70% and NPV was determined to be 100% (n=13). The assay identified all patients who subsequently responded to ICIs (sensitivity 100%), whereas specificity was 50%. The latter was driven by incorrect prediction of positive treatment response in tumor types traditionally known to be immunologically ‘cold’, raising the possibility that these non-responding histologies may reflect tumor types wherein peripheral T cells are activatable but remain absent or suppressed in the TME. <h3>Conclusions</h3> Overall, these early pilot findings indicate that PeriCyto can help to identify patients who may benefit from ICIs. Limitations include small sample size and single site design. Future efforts will focus on expansion of this patient cohort and extending followup to further assess PeriCyto predictive value. <h3>Acknowledgements</h3> We would like to thank the patients and healthy donors who participated in the study. <h3>References</h3> Iwahori K, S","PeriodicalId":500964,"journal":{"name":"Regular and Young Investigator Award Abstracts","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135161917","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"1465 Multiplex imaging identifies unique immunophenotypic and spatial characteristics associated with response to immune checkpoint inhibitors (ICIs) in metastatic urothelial cancer (mUC)","authors":"Jonathan Anker, John-William Sidhom, Guray Akturk, Sudeh Izadmehr, Justin David, Saurabh Gupta, Seunghee Kim-Schulze, Padmanee Sharma, Sacha Gnjatic, Matthew Galsky","doi":"10.1136/jitc-2023-sitc2023.1465","DOIUrl":"https://doi.org/10.1136/jitc-2023-sitc2023.1465","url":null,"abstract":"<h3>Background</h3> ICIs increase survival in mUC¹ but only a subset (~15–25%) of patients experience durable disease control.² Differences in the tumor microenvironment (TME) might underlie such differential responses. However, the complex network of cellular interactions within the TME that associate with response and resistance to ICIs remains underexplored. <h3>Methods</h3> Multiplex Immunohistochemical Consecutive Staining on a Single Slide (MICSSS)³ was performed on UC specimens (N=40) from CheckMate 275⁴ prior to treatment with nivolumab. 9 immunohistochemical stains (PD-L1, CD8, CD3, pan-cytokeratin, fibronectin, CD68, FAP, DC-LAMP,⁵ CD11b) were sequentially performed on a single slide per patient. Image processing, whole slide annotation (median 464,554 cells/slide), and intra- and extra-tumoral compartment training, were performed using QuPath (figure 1).⁶ Responders (CR, PR) and non-responders (SD, PD) were defined per RECIST v1.1. Immunophenotypic designations of ‘inflamed’, ‘excluded’, and ‘desert’ were defined via tumor margin CD8 analysis.⁷ Lymphoid aggregates were identified morphologically with dense CD3 positivity. Single-cell spatial analysis was performed defining neighborhoods as the 25 nearest neighboring cells. <h3>Results</h3> TME characterization demonstrated inter-tumoral heterogeneity, both in the intra- and extra-tumoral compartments (figure 2). Responders contained &gt;2-fold increased intra-tumoral CD8 cells, though no cell types were significantly altered in comparison to non-responders. In contrast, extra-tumoral CD3, CD8, CD3CD8-, DC-LAMP (PD-L1- and PD-L1+), and PD-L1+ CD11b cells were significantly enriched in responders (figure 3). Inflamed tumors were more prevalent and excluded/desert tumors less prevalent in responders, with inflamed tumors containing increased intra-tumoral T cell and DC-LAMP infiltration. There were no significant differences in infiltrate composition between inflamed responders and inflamed non-responders, while excluded/desert responders demonstrated enrichment for extra-tumoral DC-LAMP cells (PD-L1- and PD-L1+) and intra-tumoral PD-L1- DC-LAMP cells as compared to excluded/desert non-responders (figure 4). Responder tumors also contained an increased density of lymphoid aggregates, which were found in closer proximity to tumor regions, were associated with increased survival, and were comprised of a greater degree of DC-LAMP cells (figure 5). Spatial analysis of extra-tumoral cells identified a unique immune and tumor-enriched PD-L1+ neighborhood (cluster 0) predominant in responders, and a distinct CD11b and tumor-based neighborhood devoid of PD-L1 and other immune cells (cluster 2) predominant in non-responders (figure 6). <h3>Conclusions</h3> Multiplex immunohistochemistry identified unique immunophenotypic and spatial TME features specific to mUC responders to ICI. Both increased infiltration and the geogra","PeriodicalId":500964,"journal":{"name":"Regular and Young Investigator Award Abstracts","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135162078","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"1164 BSA01, a bispecific antibody-drug conjugate targeting EGFR and membrane-bound MUC-1-C, exhibits anti-tumor efficacy<i>in vivo</i>","authors":"Yifu Zhang, Chengzhang Shang, Anqi Wang, Jia Zhang, Yuji Liu, Hao Li, Xiaopeng Li, Gao An, W Frank An, Chaoshe Guo, Yi Yang","doi":"10.1136/jitc-2023-sitc2023.1164","DOIUrl":"https://doi.org/10.1136/jitc-2023-sitc2023.1164","url":null,"abstract":"<h3>Background</h3> EGFR and the surface glycoprotein MUC-1 are commonly co-expressed in several malignancies, including esophageal squamous cell carcinomas, non-small cell lung cancers (NSCLC), and triple-negative breast cancers. To overcome limitations of current EGFR- and MUC-1-targeting therapies, we generated a novel bispecific antibody-drug conjugate conjugated with monomethyl auristatin E, BSA01, which targets both antigens. EGFR and MUC-1 antibodies were identified using RenLite® fully human common light chain mice and further evaluated for efficacy and specificity <i>in vitro</i> and <i>in vivo.</i> <h3>Methods</h3> Parental MUC1 antibody was tested in a binding competition assay alongside a benchmark identifying cleaved MUC-1.¹ Surface plasmon resonance and flow cytometry was employed to determine the affinity and species cross-reactivity of anti-MUC1. Binding avidity was assessed by flow cytometry. Internalization and cytotoxicity were assessed by Incucyte® live cell imaging. The specificity of BSA01 was assessed by comparing the cytotoxicity of tumor cells and neonatal Human Epidermal Keratinocytes (HEKn). The efficacy of BSA01 in preventing growth of cell line-derived (CDX) and patient-derived xenograft (PDX) tumors <i>in vivo</i> was subsequently evaluated. <h3>Results</h3> Binding competition assays indicate that the parental MUC1 antibody of BSA01 binds to MUC1-C*, which remains membrane-bound after cleavage. The affinity of the anti-MUC1 antibody was similar to human and cynomolgus monkey antigens. BSA01 BsAbs bound EGFR+MUC-1+ cell lines (HCC827 and HCC70) with stronger avidity than a single-positive cell line (A431). The internalization activity of BSA01 BsAbs was superior to its monovalent parental antibodies. BSA01 was able to effectively induce cytotoxicity <i>in vitro</i>, while only marginally affecting human normal cells that express low levels of MUC1 and EGFR. Notably, BSA01 showed superior anti-tumor efficacy when compared with benchmark ADCs in CDX and pancreatic PDX models <i>in vivo</i>, In NSCLC PDX tumors, BSA01 performed similar to MUC-1 benchmark. <h3>Conclusions</h3> We generated a novel bispecific ADC targeting EGFR and MUC-1. The MUC-1 arm of BSA01 binds to the cleaved MUC1-C*protein, which remains membrane bound on tumor cells. BSA01 exhibits strong affinity and internalization activity <i>in vitro</i>, while also demonstrating a good safety profile. Moreover, BSA01 shows superior anti-tumor efficacy to benchmarks in certain <i>in vivo</i> PDX models evaluated. <h3>Reference</h3> Wu G, Kim D, Kim JN, Park S, Maharjan S, Koh H, Moon K, Lee Y, Kwon HJ. A Mucin1 C-terminal Subunit-directed Monoclonal Antibody Targets Overexpressed Mucin1 in Breast Cancer. <i>Theranostics</i>, 2018;<b>8</b>(1):78–91. https://doi.org/10.7150/thno.21278 <h3>Ethics Approval</h3> All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Biocytogen Beijing Co., Ltd.","PeriodicalId":500964,"journal":{"name":"Regular and Young Investigator Award Abstracts","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135162090","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"5 Severely immunodeficient NOG-EXL mice allow for humanization and development of a human glioblastoma-derived tumor microenvironment","authors":"Zev Binder, Lamia Lamrani, Anthony Secreto, Nicholas Skuli, Moriah Jacobson, Charles-Antoine Assenmacher, Elinor Willis, Enrico Radaelli, Donald O’Rourke, Cecile Alanio, Maclean Nasrallah","doi":"10.1136/jitc-2023-sitc2023.0005","DOIUrl":"https://doi.org/10.1136/jitc-2023-sitc2023.0005","url":null,"abstract":"<h3>Background</h3> Cellular immunotherapy has resulted in tremendous therapeutic advances across liquid tumors. However, for solid tumors, including glioblastoma (GBM), significant limitations still exist in successfully translating immune-based adoptive cell therapeutics from the bench to the clinic. The majority of preclinical <i>in vivo</i> GBM models either use human cell lines grown in immunodeficient animals or syngeneic tumors grown in immunocompetent animals. Both approaches lack critical immune components of the tumor microenvironment (TME), thus inherently limiting the ability to adequately model immunotherapies. <h3>Methods</h3> Here, we demonstrate the ability of hematopoietic stem cells (HSCs) from a GBM patient to successfully engraft in the severely immunodeficient NOG-EXL mouse strain and generate a patient-derived immune system. Mouse brains, with and without tumors, were evaluated by immunohistochemistry and flow cytometry. <h3>Results</h3> Intraosseous injections of a low number (10,000) of CD34+ HSCs, obtained from a bone marrow draw from the patient at the time of tumor resection, resulted in an average of 20% human CD45+ cells in peripheral blood 17 weeks post-transplant. GBM organoids, established from the same patient, were implanted intracranially at Week 18 and allowed to grow for 5 weeks without significant clinical evidence of graft-vs-host disease or macrophage activation syndrome. Immunohistochemical and flow cytometry analyses of mouse brains, with and without tumors, revealed infiltration of human-derived, CD45+/CD33+ cells, in proximity to the tumor engraftment site. <h3>Conclusions</h3> These results demonstrate the ability of patient-obtained CD34+ HSCs to engraft and form a TME in severely immunodeficient mice. Subsequent work will focus on the differences between autologous and allogeneic humanized tumor-bearing mice in the context of the originating tumor. Additional future work will focus on testing cellular therapies directed towards the relevant areas of the autologous model.","PeriodicalId":500964,"journal":{"name":"Regular and Young Investigator Award Abstracts","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135162099","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"1106 Molecular mechanisms of immunogenic cell death driven by PT-112","authors":"Emma Guilbaud, Takahiro Yamazaki, Maria Congenie, Christina Yim, Tyler Ames, Lorenzo Galluzzi","doi":"10.1136/jitc-2023-sitc2023.1106","DOIUrl":"https://doi.org/10.1136/jitc-2023-sitc2023.1106","url":null,"abstract":"<h3>Background</h3> PT-112 is a novel immunogenic small molecule¹ under Phase II clinical development for cancer therapy.^2–8 Besides mediating cytostatic and cytotoxic effects in numerous human and mouse cancer cells, PT-112 elicits various danger signals that are linked to immunogenic cell death (ICD) such as calreticulin exposure, as well as ATP and HMGB1 secretion.^{1 9–11} Accordingly, mouse cancer cells succumbing to PT-112 <i>in vitro</i> efficiently protect immunocompetent, tumor-naïve mice from challenge with living cancer cells of the same type.^{1 9} Moreover, PT-112 synergizes with PD-1 or PD-L1 blockade to control mouse tumors developing in immunologically competent hosts.^{1 9} This work focuses on elucidating the underlying mechanisms of PT-112-induced ICD. <h3>Methods</h3> We harnessed a panel of human and mouse cell lines optionally engineered to lack specific genes involved in mitochondrial apoptosis (namely, <i>Bcl2, Bax</i> and <i>Bak1</i>) coupled with flow cytometry, immunoblotting, RT-PCR, immunofluorescence microscopy and clonogenic assays to determine the impact of reticular and mitochondrial events on the established ability of PT-112 to kill malignant cells in an immunogenic manner.¹ <h3>Results</h3> In line with previous findings,^10–13 PT-112 elicited eukaryotic translation initiation factor 2 subunit alpha (EIF2S1, best known as eIF2α) phosphorylation and mitochondrial dysfunction in malignant cells, a process that was accompanied by the release of interferogenic mitochondrial DNA (mtDNA)¹⁴ in the cytoplasm of PT-112 treated cells, and was differentially affected by the deletion of <i>Bcl2</i>, <i>Bax</i>, <i>Bak1</i>, or <i>Bax</i> plus <i>Bak1</i>. Similarly, the lack of <i>Bcl2</i>, <i>Bax</i>, <i>Bak1</i>, or <i>Bax</i> plus <i>Bak1</i> had a differential impact on the ability of PT-112 to elicit early signs of mitochondrial apoptosis including reactive oxygen species (ROS) generation, mitochondrial transmembrane potential loss and ultimately plasma membrane permeabilization. <h3>Conclusions</h3> ER stress and mitochondrial dysfunction appear to underlie the ability of PT-112 to drive ICD, the integrated stress response, and viral mimicry. This is in line with the well-established connections between ER stress and cytoplasmic nucleic acid sensing, which are pristine mechanisms of antiviral defense in mammalian cells, with the capacity of dying cells to emit immunostimulatory signals.¹⁵ Whether PT-112-driven stress also shifts the antigenic properties of cancer cells as a consequence of the accumulation of non-mutational neoantigens¹⁶ remains to be determined. Despite these and other open questions, PT-112 stands out as a powerful immunotherapeutic agent with promising clinical activity in patients with a variety of tumors¹ under Phase II clinical development for cancer therapy.^2–8 <h3>Reference","PeriodicalId":500964,"journal":{"name":"Regular and Young Investigator Award Abstracts","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135162314","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"359 Developing human placental CD34-derived natural killer cells with high affinity and cleavage resistant CD16 and secreted IL-15 (CYNK-201) for cancer immunotherapy","authors":"Marina Gergues, Yuechao Zhao, Irene Raitman, Shengchen Lin, Valentina Rousseva, Siyara Kilcoyne, Adrian Kilcoyne, Robert Hariri, Shuyang He, Lin Kang","doi":"10.1136/jitc-2023-sitc2023.0359","DOIUrl":"https://doi.org/10.1136/jitc-2023-sitc2023.0359","url":null,"abstract":"<h3>Background</h3> Natural killer (NK) cells can display antibody dependent cellular cytotoxicity (ADCC) activity against tumor cells via the CD16 Fc receptor in combination with tumor specific antibodies. IL-15 is important for NK cell survival, proliferation and function. Celularity is developing human placental CD34⁺-derived, cryopreserved, off-the-shelf, allogenic NK cells (CYNK-201) with high IgG binding affinity, protease cleavage resistant CD16 and secreted IL-15 for cancer treatment. Here we report the preclinical efficacy results of CYNK-201 against tumor cells. <h3>Methods</h3> CYNK-201 cells were generated by transduction of human placental CD34 cells with lentivirus vector expressing CD16 and IL-15, followed by culture expansion in the presence of cytokines. Transgene expression and phenotype of CYNK-201 cells were characterized by flow cytometry. The concentration of secreted IL-15 by CYNK-201 was measured by a human IL-15 ELISA kit. ADCC activity of CYNK-201 against HER2⁺ gastric cancer cell line NCI-N87 and CD20⁺ Burkitts lymphoma cell line Daudi was assessed in combination with trastuzumab and rituximab, respectively. <i>In vivo</i> persistence and efficacy of CYNK-201 were assessed using NSG mice and NCI-N87 xenografted NSG mice, respectively. <h3>Results</h3> CYNK-201 was generated from multiple placental CD34⁺ donors (n=5) with 91.8±1.1% CD56⁺CD3^- , 78.9± 9.77% CD16 expression, 3274± 1605 fold expansion and the average of 203±1.1 pg/mL secreted IL-15. While 2h PMAi treatment resulted in 61.6±11.3% CD16 cleavage on non-transduced (NT)-CYNK cells, no cleavage was observed from CYNK-201 demonstrating CD16 shedding resistance. CYNK-201 showed increased expression of activation markers including CD11a, NKG2D, CD226 and NKp30 as compared to the NT-CYNK cells. CYNK-201 displayed enhanced <i>in vitro</i> ADCC against NCI-N87 and Daudi as compared to that of NT-CYNK cells. At an E:T ratio of 1:1, CYNK-201 elicited increased ADCC against NCI-N87 with trastuzumab compared to that of NT-CYNK cells, with 65.6±15.1% vs. 52.0±9.1% at 4 hours. At an E:T ratio of 2:1, CYNK-201 showed higher cytotoxicity against Daudi in combination with rituximab compared to that of NT-CYNK with 49.0±18.8% vs. 31.8±9.9%. Post 14 days of infusion, CYNK-201 cells were detectable in NSG mice, with significantly higher abundance than that of NT-CYNK cells plus recombinant IL-15. This enhanced persistence of CYNK-201 led to significant tumor reduction in NCI-N87 tumor model in combination with trastuzumab. <h3>Conclusions</h3> Our results demonstrated enhanced <i>in vitro</i> ADCC, <i>in vivo</i> persistence and anti-tumor activity of CYNK-201. Further development of CYNK-201 is warranted.","PeriodicalId":500964,"journal":{"name":"Regular and Young Investigator Award Abstracts","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135162326","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"501 AZD2796, a myeloid checkpoint inhibitor targeting LILRB2 that promotes pro-inflammatory responses by macrophages and enhances T cell anti-tumor activity","authors":"Des C Jones, Lorraine Irving, Becki Dudley, Marcin Wolny, Seraina Blümli, Ellie Chatzopoulou, Alan Sandercock, Georgina Bowyer, Stacy Pryts, Kathy Mulgrew, Simon Dovedi, Fernanda Arnaldez, Mark Cobbold","doi":"10.1136/jitc-2023-sitc2023.0501","DOIUrl":"https://doi.org/10.1136/jitc-2023-sitc2023.0501","url":null,"abstract":"<h3>Background</h3> Inhibition of T cell immune checkpoints has revolutionized cancer therapy. However, responses are not uniformly positive, and many patients develop resistance. Myeloid cells are an abundant component of the tumor microenvironment able to promote cancer progression and suppress immune responses. Consequently, targeting these cells may be an avenue to optimize immune-based cancer therapies. LILRB2 is an inhibitory member of the leukocyte immunoglobulin-like receptor (LILR) family that is expressed on the surface of myeloid cells, including key suppressive myeloid subsets such as macrophages and myeloid-derived suppressor cells (MDSCs) found in tumors. LILRB2 signalling contributes to the suppressive phenotype of myeloid cells by inhibiting the activity of pro-inflammatory signalling pathways. <h3>Methods</h3> AZD2796 is a fully humanised monoclonal antibody that binds LILRB2. AZD2796 was assessed for target specificity, binding affinity and antagonism of ligand binding. The ability of AZD2796 to enhance proinflammatory responses of macrophages was determined by the release of pro inflammatory cytokines from monocyte derived macrophages in vitro, and enhancement of tumor cell lysis by T cells when co-cultured in the presence of macrophages. The anti-tumor effect of AZD2796 was explored <i>in vivo</i> using two xenograft models of human cancer in humanised mice. <h3>Results</h3> AZD2796 is highly specific to LILRB2 and does not bind other members of the LILR family. It binds LILRB2 with high affinity and blocks binding of LILRB2 to its major histocompatibility complex (MHC) class I ligands. In functional assays, AZD2796 enhanced the production of the proinflammatory cytokines TNF-α and GM-CSF from human macrophages stimulated with CD40L, while reducing the production of VEGF, an important driver of angiogenesis. In addition, AZD2796 increased tumor cell killing by T cells when co-cultured with macrophages. <i>In vivo</i>, AZD2796 significantly reduced tumor growth rate of NCI-H358 lung cancer cells and SK-MEL-5 melanoma cancer cells in humanised mice. <h3>Conclusions</h3> AZD2796 is a high affinity anti-LILRB2 monoclonal antibody that promotes pro-inflammatory responses by macrophages and enhances anti-tumor activity of T cells. Our pre-clinical data support the potential of AZD2796 as an anti-cancer therapy with opportunities to combine with T-cell-based therapeutics. <h3>Ethics Approval</h3> All animal studies are run under the Institutional Animal Care and Use Committee (IACUC) approved in vivo protocol number AUP-22–17. AstraZeneca’s US site IACUC committee oversees the specific use of animals by conducting a formal review of the animal use, ethics and protocols and grants approval prior to the work commencing.","PeriodicalId":500964,"journal":{"name":"Regular and Young Investigator Award Abstracts","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135162545","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}