Cancer Immunotherapy Clinical Trials Research Articles

Clocking Cancer Immunotherapy Responses.

Two recent papers document that responses to immunotherapy are circadian and peak at the end of resting phase (evening) of mice with syngeneic and genetic models of cancers. The circadian effect is attributed to diurnal T-cell trafficking through the endothelium on the one hand, and to the circadian expression of PD-L1 on myeloid suppressors on the other. Overall, it appears that tumor immunity as a system, including dendritic cell function, behaves in a circadian manner that is also observed in patients in cancer immunotherapy clinical trials. Importantly, these observations uncover time-of-day as an unforeseen variable for cancer immunotherapy responses. This insight on the immune circadian clock should be further explored to enhance immunotherapy responses in the clinic.

CD155-TIGIT Axis as a Therapeutic Target for Cancer Immunotherapy.

Immune checkpoint inhibitors (ICIs) have shown unprecedented efficacy in treating many advanced cancers. Although FDA-approved ICIs have shown promising efficacy in treating many advanced cancers, their application is greatly limited by the low response rate, immune-related adverse events (irAE), and drug resistance. Developing novel ICIs holds great promise to improve the survival and prognosis of advanced cancer patients. T-Cell immunoglobulin and ITIM domain (TIGIT) is an inhibitory receptor expressed on T cells, natural killer (NK) cells, and T regulatory cells. Increasing reports have shown that the disrupting CD155-TIGIT axis could activate the immune system and restore antitumor immune response. This review briefly summarized the role of TIGIT in tumor immune escape and targeting CD155-TIGIT axis drugs in preclinical and clinical trials for cancer immunotherapy.

Studentized permutation method for comparing two restricted mean survival times with small sample from randomized trials.

Recent observations, especially in cancer immunotherapy clinical trials with time-to-event outcomes, show that the commonly used proportional hazard assumption is often not justifiable, hampering an appropriate analysis of the data by hazard ratios. An attractive alternative advocated is given by the restricted mean survival time (RMST), which does not rely on any model assumption and can always be interpreted intuitively. Since methods for the RMST based on asymptotic theory suffer from inflated type-I error under small sample sizes, a permutation test was proposed recently leading to more convincing results in simulations. However, classical permutation strategies require an exchangeable data setup between comparison groups which may be limiting in practice. Besides, it is not possible to invert related testing procedures to obtain valid confidence intervals, which can provide more in-depth information. In this paper, we address these limitations by proposing a studentized permutation test as well as respective permutation-based confidence intervals. In an extensive simulation study, we demonstrate the advantage of our new method, especially in situations with relatively small sample sizes and unbalanced groups. Finally, we illustrate the application of the proposed method by re-analyzing data from a recent lung cancer clinical trial.

Abstract 6245: Selective HDAC6 inhibition by ITF3756 modifies the landscape of gene and protein expression in human monocytes: Possible identification of a specific biomarker signature

Abstract Histone deacetylase 6 (HDAC6) modulates tumor immunogenicity through several pathways, such as the control of PD-L1 expression on tumor and on lymphoid cells. Thus, HDAC6 is a potential pharmacological target for cancer treatment. In humans, HDAC6 inhibition can be monitored by measuring the ratio between tubulin and histone acetylation. However, the identification of biomarkers to be used as a signature for patient stratification that correlates with the treatment efficacy is missing. Since we have previously demonstrated that the activity of the selective HDAC6 inhibitor ITF3756 on myeloid cells plays a role in the antitumor immune response in the TME, we reasoned that an ITF3756-specific signature could be identified in unstimulated monocytes treated with the compound. Monocytes were purified from healthy donors, treated with ITF3756 and RNAseq and proteomic analyses were performed. Gene Ontology analysis shows that ITF3756 has a widespread effect on monocyte transcriptome and proteome. We observed the down-modulation of genes and proteins involved in inflammatory pathways and in transcription regulation. Up-regulation of neutrophil-mediated immunity, metabolic pathways and protein phosphorylation was also identified. Focusing on genes that could be relevant in the tumor context, we selected a panel of genes from the most up- (SCL27A1, ANXA6 and IRF6) and down-modulated (CD84, RANK, CXCL2, CXCL3, STAB-1, CD163, PD-L1, CD206 and ADA). Interestingly, we found among the downmodulated genes markers of M2 macrophages, genes related to the tumor associated macrophages, and STAB-1, whose targeting is currently in clinical trials for cancer immunotherapy. Peripheral Blood Mononuclear Cells (PBMCs) could be a less expensive starting material for biomarker identification to be collected during a clinical trial when compared to purified monocytes. Thus, to ascertain whether the regulation by ITF3756 of our selected genes was detectable also in PBMCs we performed gene expression analysis after ITF3756 treatment and we confirmed that all selected genes were reproducibly modulated. Surface protein expression of M2 macrophages biomarkers in our panel, were determined by flow cytometry and all of them were significantly modulated in PBMCs. These data show how our approach successfully led both to the identification of a biomarker signature that could be used in clinical trials to monitor the pharmacodynamic effect of ITF3756, and to the discovery of previously unknown targets of HDAC6 inhibition. Citation Format: Chiara Ripamonti, Valeria Spadotto, Monica Forino, Christian Steinkuhler, Gianluca Fossati. Selective HDAC6 inhibition by ITF3756 modifies the landscape of gene and protein expression in human monocytes: Possible identification of a specific biomarker signature. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6245.

Abstract 2256: Cancer stem cells and exosomal PD-L1 suppress anti-tumor B cells

Abstract We have conducted clinical trials for cancer immunotherapy using vaccine-primed lymph node (VPLN) T calls based on our animal studies with tumor-draining lymph node (TDLN) T cells. More recently we have identified antitumor effector TDLN B cells which demonstrated significant tumor reactivity in vitro and therapeutic efficacy in vivo upon adoptive transfer which can be enhanced by anti-PD-L1 administration. It is unknown whether tumor cells, particularly cancer stem cells (CSCs) could suppress the antitumor effector B cells. We found that depletion of B cells with anti-CD20 mAb prior to anti-PD-L1 administration resulted in more aggressive tumor growth, and the anti-tumor efficacy of anti-PD-L1 was significantly reduced in both 4T1 and CT26 murine tumor-bearing hosts, suggesting the involvement of host B cells in the anti-tumor effect of anti-PD-L1. In addition, administration of anti-PD-L1 partially recovered the humoral immune response, confirming the PD-L1/PD-1 pathway involvement in B cell suppression. We detected elevated expression of PD-1 on activated B cells, and higher PD-L1 expression on ALDHhigh CSCs than on ALDHlow non-CSCs. Co-culturing ALDHhigh CSCs with purified B cells significantly reduced IgG secretion, and such CSC-mediated B cell suppression was rescued by adding anti-PD-L1 mAb in a dose-dependent manner to the CSC-B cell co-culture. These experiments thus indicate that CSCs suppress the IgG production by B cells via the PD-L1/PD-1 axis. Tumor cells were recently recognized to suppress T cell function via secretion of exosomes that express PD-L1. To investigate if exosomal PD-L1 contributes to B cell immunosuppression, we generated Rab27ako D5 cells to delete exosomes as well as PD-L1ko D5 cells by CRISPR/Cas9 gene editing. D5-derived exosomes suppressed B cell proliferation and IgG production in vitro, and administration of these exosomes promoted tumor growth and reduced animal survival, revealing the role of tumor-derived exosomes in B cell immunosuppression. Tumor-derived exosomes expressed PD-L1, but PD-L1 was absent in the exosomes isolated from the PD-L1ko tumor cells as evident by western blot and flow cytometry. Deletion of Rab27a or PD-L1 did not affect the proliferation of the tumor cells in vitro. However, Rab27ako or PD-L1ko resulted in significantly enhanced host anti-tumor immunity evident by reduced 4T1 tumor growth in vivo compared with the WT tumor. Importantly, anti-PD-L1 therapy significantly reduced Rab27ako or PD-L1ko 4T1 tumor growth and prolonged animal survival vs. the WT tumor control. We conclude that PD-L1-expresing CSCs and PD-L1-expresing exosomes suppress antitumor effector B cells via the PD-L1/PD-1 axis. To characterize the suppression of CSC-derived exosomes on host B cells as well as T cells, isolation of Rab27ako ALDHhigh CSCs warranties further investigation. Citation Format: Ying Xin, Jiayu Liu, You Qin, Kexing Lyu, Sifei Yu, Ming Lin, Max Wicha, Alfred E. Chang, Qiao Li. Cancer stem cells and exosomal PD-L1 suppress anti-tumor B cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2256.

Nanoparticle-Mediated STING Activation for Cancer Immunotherapy.

As the first line of host defense against pathogenic infections, innate immunity plays a key role in antitumor immunotherapy. The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) (cGAS-STING) pathway attracts numerous attentions because of the secretion of various proinflammatory cytokines and chemokines. Many STING agonists have been identified and applied into preclinical or clinical trials for cancer immunotherapy. However, the fast excretion, low bioavailability, non-specificity and adverse effects of the small molecule STING agonists limit their therapeutic efficacy and in vivo application. Nano-delivery systems with appropriate size, charge and surface modification are capable of addressing these dilemmas. In this review, we discuss the mechanism of cGAS-STING pathway and summarize the STING agonists, focusing on nanoparticle-mediated STING therapy and combined therapy for cancers. Finally, the future direction and challenges of nano-STING therapy are expounded, emphasizing the pivotal scientific problems and technical bottlenecks and hoping to provide general guidance for its clinical application. This article is protected by copyright. All rights reserved.

Abstract 1970: Single cell proteomic quantification of T cell states using mass cytometry for applications in monitoring immune responses in cancer immunotherapy clinical trials

Abstract Although immunotherapies have been established as a successful treatment option for several types of cancers, additional treatment strategies are necessary to broaden the cohorts of patients who may receive clinical benefit. Common immunological profiling for immunotherapy response depends on assessment of the composition of immune cell types in tissue samples and the periphery. Still, the function and state of each of these cells further contributes to the ultimate clinical benefit of immunotherapeutic response. Additionally, our ability to comprehensively study treatment strategies depends on elucidating the temporal effects different immunotherapy regimens have on the immune system of patients. Single-cell proteomics profiling with CyTOF mass cytometry enables robust protein-level profiling of cell types and is also high-dimensional enough to characterize functional states of immune cells. To construct an immunological framework that may feasibly serve to empower functional assessment of immune cell states for clinical trial monitoring strategies using CyTOF, we are employing single cell trajectory inference methods and non-negative matrix factorization (NMF). We demonstrate this approach using peripheral blood derived CyTOF mass cytometry data to model T cell states at the proteomic level. First, we extract T helper (Th) and T cytotoxic (Tc) cell integrated metrics from CyTOF mass cytometry data obtained from diverse cell marker panel designs, immunological states, and diseases to benchmark that these computational pipelines can accurately reflect Th and Tc activation and exhaustion. Next, we develop a new pipeline that enables the use of continuous functional cell-state metrics from continuous pseudotime and NMF pattern weights as immunological profiling markers from CyTOF that can be applied to cancer immunotherapy clinical trials. For instance, we find CyTOF Tc pseudotime and memory pattern weights are higher in PDAC patients with stable disease treated with ipilimumab and a pancreatic cancer-specific vaccine. Thus, our pipeline has the ability to integrate metrics from mass cytometry data to empower translational analyses for determining patient responses in cancer immunotherapy, such as adaptive immune responses to checkpoint inhibitors and cancer vaccines. Moreover, applying this pipeline to peripheral blood samples obtained before and after treatment also enables us to map further temporal cell state transitions in Tc and Th cells resulting from that treatment. Future work will include leveraging CyTOF T cell integrated metrics in larger scale survival analyses to test the predictive potential in a clinical proof of concept. Citation Format: Dimitrios N. Sidiropoulos, Zaw Phyo, Nicole Gross, Soren Charmsaz, Stephanie Xavier, James Leatherman, Mark Yarchoan, Elizabeth M. Jaffee, Elana J. Fertig, Won Jin Ho. Single cell proteomic quantification of T cell states using mass cytometry for applications in monitoring immune responses in cancer immunotherapy clinical trials [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 1970.

Structure activity relationship of 3‐methylcytidine‐5’‐α,β‐methylenediphosphates as CD73 inhibitors

Adenosine in the tumor microenvironment acts on A2A and A2B adenosine receptors (ARs) on immune cells (T cells, dendritic cells, NK cells, macrophages and neutrophils) to prevent their activation. Therefore, a means of lowering extracellular adenosine would be beneficial in cancer immunotherapy either as a co‐therapy or monotherapy. Inhibitors of CD73 (ecto‐5’‐nucleotidase, the main extracellular adenosine‐forming enzyme) are already in clinical trials for cancer immunotherapy. Many purine (ADP) analogues have been shown to inhibit this enzyme, but we have explored pyrimidine (UDP/CDP) analogues. We recently reported N4‐substituted 3‐methylcytidine‐5’‐α,β‐methylenediphosphates as potent CD73 inhibitors. We now expand the structure activity relationship (SAR) of pyrimidine nucleotides as human CD73 inhibitors, focusing mainly on cytidine functionalization at the N4 position. 4‐Chloro (MRS4598 16, Ki 0.673 nM) and 4‐iodo (MRS4620 18, Ki 0.436 nM) substitution of the N4‐benzyloxy group increased inhibitory potency Ki by ~20‐fold. Primary alkylamine derivatives coupled through a p‐amido group and varying methylene chain length (24and 25) were functionalized congeners, for subsequent conjugation to carrier or reporter moieties. hCD73 X‐ray structures with two of the newly synthesized inhibitors indicated a ribose ring conformational adaptation, and the benzyloxyimino group (E configuration) binds to the same region (between C‐terminal and N‐terminal domains) as N4‐benzyl groups in potent adenine‐derived inhibitors. Molecular dynamics simulation identified stabilizing interactions and predicted conformational diversity around the N4‐benzyloxy moiety. Thus, by structure‐guided substitution of the N4‐benzyloxy moiety, we have greatly enhanced their inhibitory potency and added functionality enabling molecular probes. Their potential as anticancer drugs was confirmed by blocking CD73 activity in human head and neck squamous cell carcinoma (HNSCC) and palatine tonsils in situ.

Cytokine Based Immunotherapy for Cancer and Lymphoma: Biology, Challenges and Future Perspectives.

Cytokines regulate both the innate and adaptive immune responses to cancer. Although antitumor activity has been seen for several cytokines in preclinical models, they have had limited success as single therapeutic agents in clinical trials of cancer immunotherapy. However, the possible combinations of cytokines with other immune therapeutics and the advancement in genetic engineering, synthetic biology and cellular and immune therapy has led to the revival of interest in cytokines as anticancer agents. This article will review several immunostimulatory cytokines with anticancer activity, focusing on the those that have been studied in treatment of lymphoma and highlighting recent advances of potential clinical relevance.

Neoadjuvant immunotherapy prior to surgery for mucosal head and neck squamous cell carcinoma: Systematic review.

Given the recent successes of anti-PD-1 immunotherapy, many clinical trials have sought to assess the safety and efficacy of this treatment modality in the neoadjuvant setting. This systematic review provides a comprehensive summary of findings from neoadjuvant head and neck cancer immunotherapy clinical trials with a focus on PD-1/PD-L1 axis blockade. Pubmed, Embase, Cochrane Library, Web of Science, Google Scholar, and clinicaltrials.gov were systematically searched for all eligible neoadjuvant head and neck cancer immunotherapy clinical trials. Eight clinical trials met the inclusion criteria comprising a total of 260 patients. Study drugs included nivolumab, pembrolizumab, ipilimumab, durvalumab, and tremelimumab. The overall mean objective response rate (ORR) was 45.9 ± 5.7% with a 41.5 ± 5.6% single agent mean ORR. There were no deaths due to immune-related toxicities. Neoadjuvant immunotherapy for mucosal head and neck squamous cell cancer has demonstrated favorable response rates with no unexpected immune-related toxicities in phase I/II clinical trials.

The inhibitory receptor TIM-3 limits activation of the cGAS-STING pathway in intra-tumoral dendritic cells by suppressing extracellular DNA uptake

Blockade of the inhibitory receptor TIM-3 shows efficacy in cancer immunotherapy clinical trials. TIM-3 inhibits production of the chemokine CXCL9 by XCR1+ classical dendritic cells (cDC1), thereby limiting antitumor immunity in mammary carcinomas. We found that increased CXCL9 expression by splenic cDC1s upon TIM-3 blockade required type I interferons and extracellular DNA. Chemokine expression as well as combinatorial efficacy of TIM-3 blockade and paclitaxel chemotherapy were impaired by deletion of Cgas and Sting. TIM-3 blockade increased uptake of extracellular DNA by cDC1 through an endocytic process that resulted in cytoplasmic localization. DNA uptake and efficacy of TIM-3 blockade required DNA binding by HMGB1, while galectin-9-induced cell surface clustering of TIM-3 was necessary for its suppressive function. Human peripheral blood cDC1s also took up extracellular DNA upon TIM-3 blockade. Thus, TIM-3 regulates endocytosis of extracellular DNA and activation of the cytoplasmic DNA sensing cGAS-STING pathway in cDC1s, with implications for understanding the mechanisms underlying TIM-3 immunotherapy.

Current status of cancer immunotherapy for gynecologic malignancies.

Recent cancer immunotherapy development with immune checkpoint inhibitors has shown durable clinical responses in a wide variety of tumor types. These drugs targeting programmed cell death 1, its ligand programmed death ligand 1 and cytotoxic T cell lymphocyte-associated antigen 4 have revolutionized the field of cancer treatment. It is of significant interest in optimizing the immunotherapy for cancer patients beyond the conventional treatments such as surgery, chemotherapy and radiation. Many clinical trials evaluating the safety and efficacy of various combined regimens with immune checkpoint inhibitors have been reported and are in progress. Among gynecologic malignancy, endometrial cancers have distinct subtypes with microsatellite instability-high status and polymerase ɛ mutation. These types have been shown to immunogenic tumors and appropriated candidate for immune checkpoint inhibitors. Also, recurrent cervical cancer showed a promising objective response with single anti-PD1 Ab treatment. Despite their definite outcome and considerable potential of immunotherapy, not all patients received a survival benefit and further understanding of human tumor immunology is essential to improve this type of therapy. In this review, we have summarized the updated results of clinical trials of cancer immunotherapy for gynecologic malignancies and discussed the future perspectives.

Can Molecular Imaging Measure T-cell Activation?

Successful immunotherapy usually depends on activation of T cells in the tumor microenvironment. However, ascertaining whether T-cell activation has occurred in vivo is difficult without invasive tissue sampling. Inducible T-cell costimulator (ICOS) is a specific marker of T-cell activation that can be imaged by radiolabeling an anti-ICOS antibody and performing PET scanning. Hopefully, this agent will be the first of many molecular imaging constructs that can determine whether T-cell activation has occurred and could be used in drug development and clinical trials of cancer immunotherapy.See related article by Xiao et al., p. 3023.

Exclusion rates of patients living with HIV from cancer immunotherapy clinical trials.

e19035 Background: Although patients living with HIV are at increased risk for developing several cancers, they have often been excluded from practice-changing immunotherapy clinical trials across many tumor types. Emerging retrospective and prospective studies indicate that immunotherapies can be safe and effective in patients with HIV. ASCO and Friends of Cancer Research have supported the inclusion of patients with HIV in cancer clinical trials. We determined the rate of HIV exclusion from recent cancer immunotherapy clinical trials according to tumor type, immunotherapy treatment, and phase of trial. Methods: We compiled trials on the ClinicalTrials.gov database with study start dates from January 1, 2019 to October 10, 2020, using the search term “cancer” for disease, and the following intervention terms: “immunotherapy, PD-1, PD-L1, CTLA-4, pembrolizumab, nivolumab, atezolizumab, durvalumab, avelumab, cemiplimab, ipilimumab, tremelimumab, sintilimab, spartalizumab.” Observational studies and trials not offering immune checkpoint inhibitors (ICIs) or cellular immunotherapy were excluded. Eligibility criteria for each trial were reviewed to determine rate of exclusion for HIV, human immunodeficiency virus, acquired immunodeficiency, or immune deficiency. Cellular immunotherapies were defined as modified T cell, dendritic cell, NK cell, or other peripheral blood leukocyte treatments. Results: Of 1090 analyzed trials, 795 (72.9%) specifically excluded patients with HIV, and another 80 (7.3%) trials included patients with HIV only if certain criteria were met (e.g. undetectable viral load, minimum CD4+ T cell count). By tumor type, the rate of HIV exclusion ranged from 60.9% (renal cancer) to 85.7% (cervical cancer), with hematologic malignancies having a significantly higher rate of HIV exclusion than solid tumor trials (85.2% vs. 73.3%, p = 0.015). Trials combining ICIs with cellular immunotherapies were more likely to exclude HIV (N = 22, 90.9% exclusion) than trials with ICIs alone (N = 1003, 71.4% exclusion, p = 0.044). Phase I (N = 271, 73.1% exclusion, p = 0.0012), I/II (N = 165, 70.9% exclusion, p = 0.010), and II trials (N = 519, 77.5% exclusion, p < 0.0001) had higher rates of exclusion than phase III trials (N = 101, 55.4% exclusion). Conclusions: Despite ASCO and Friends of Cancer Research recommendations to include patients with HIV in cancer clinical trials, the vast majority of immunotherapy clinical trials in 2019 exclude patients with cancer and HIV.

CIMAC-CIDC CyTOF harmonization.

e15242 Background: The Cancer Immune Monitoring and Analysis Centers – Cancer Immunology Data Commons (CIMAC-CIDC) network is a National Cancer Institute-funded initiative to identify biomarkers of mechanisms and response to cancer immunotherapy clinical trials, using state-of-the-art assay technologies. A primary platform for CIMAC-CIDC biomarker studies is CyTOF mass cytometry, which is performed at all four CIMAC laboratories. Methods: To test the ability to generate comparable data across labs, a cross-site harmonization effort was undertaken. We first harmonized SOPs between centers. Because of a new acquisition protocol introduced by the vendor (Fluidigm), we also tested this protocol across sites before finalizing the harmonized SOP. We then performed a cross-site assay harmonization experiment, using 5 shared cryopreserved PBMC samples and one lyophilized control cell preparation, along with a shared lyophilized antibody cocktail consisting of 14 markers, as validated in the HIPC consortium, plus CD45. These reagents and samples were distributed to the four sites, and FCS files were centrally analyzed by both manual gating and automated methods (Astrolabe). Results: Average CVs across sites for each cell population were reported and compared to a previous multisite CyTOF study. Once a cell recovery issue at two sites was resolved, this experiment resulted in inter-site reproducibility of under 20% CV for most cell subsets, very similar to the previous study. Conclusions: These results emphasize the ability to reproduce CyTOF across sites, and also highlights procedures, such as use of spike-in control samples, useful for tracking variability in this assay.

Abstract A34: Cancer immunotherapy clinical trials in K-MASTER project

Abstract The K-MASTER cancer precision medicine diagnosis and treatment enterprise (K-MASTER project) is a precision medicine cancer treatment clinical trial platform, which uses next-generation sequencing (NGS) panel to screen the actionable mutations in 10,000 patients with refractory solid tumors and assigns the patients to matched clinical trials. K-MASTER project was initiated in Korea in June 2017. From October 2017, we have analyzed NGS -based cancer panel to detect genomic variations in Korean cancer patients. Tumor DNA was analyzed by K-MASTER panel, which allows to detect variations of 375 genes. As of September 3, 2019, total 4,240 cancer patients were registered via KM-00 screening protocol. The mean depth of K-MASTER panel was 701x. The patient whose cancer has been confirmed of certain genomic variants can participate in matched clinical studies. As of September 2019, 11 clinical trials are successfully in process and another 6 are awaiting kickoff under the name of K-MASTER project. There are 3 ongoing immunotherapy clinical trials: avelumab for metastatic colon cancer with microsatellite instability-high or POLE mutation (KM-01), nivolumab for advanced solid tumor with DNA repair pathway aberration (KM-06), and spartalizumab combined with lacnotuzumab for esophageal squamous cell carcinoma (KM-12). Among them, the KM-01 study closed its registration and is waiting for data analysis. Other studies are actively enrolling patients along with KM-00 screening protocol. In addition, various new immunotherapy studies are preparing for initiation: basket trial of nivolumab plus ipilimumab for high tumor mutational burden solid tumors (KM-09), phase II study of CAST cell therapy (KM-15), alternative scheduling trial for pembrolizumab (KM-19), and phase II/III trial of novel PD-1 inhibitor INCMGA00012 (KM-20) are on their way to start. In conclusion, K-MASTER project is efficient to detect actionable variations and function as a reliable path between patient and new immunotherapeutic agent. Nationwide NGS-based screening is not only feasible but also effective in recruiting participants in molecular basket trials as well as immunotherapy trials. This novel knowledge provides an intriguing background to investigate new target approaches and to progress precision medicine. (This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute [KHIDI], funded by the Ministry of Health &Welfare, Republic of Korea [Grant number: HI17C2206].) Citation Format: Ju Won Kim, Jae Sook Sung, Hee Jun Jung, Jung Yoon Choi, Soo Hyeon Lee, Won Jin Chang, Yoon Ji Choi, Kyong Hwa Park, Yeul Hong Kim. Cancer immunotherapy clinical trials in K-MASTER project [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A34.

IMMU-41. RNA-MODIFIED T CELLS AS PROFESSIONAL ANTIGEN PRESENTING CELLS

Abstract INTRODUCTION With the lack of antigen presenting cells (APCs) in the central nervous system (CNS), anti-tumor immune responses from intracranial tumors are believed to be initiated in the peripheral lymph nodes. Recently, studies have shown that intratumoral dendritic cells (DCs), a well-known professional APC, play a crucial role at inducing anti-tumor immunity. Although DCs have been tested in cancer immunotherapy clinical trials for two decades, DC delivery strategies have limited access to the CNS, mainly due to the blood brain barrier (BBB). To bypass the limitations of the lack of intracranial APCs, we investigate the HYPOTHESIS that RNA-modified T cells can express and cross-present single tumor antigens locally within the CNS tumor microenvironment. Since activated T cells have an inherent ability to cross the BBB and can be genetically modified, this strategy makes them an attractive professional antigen presenting cells for cancer cell therapy. METHODS Using electroporation to deliver messenger RNA (mRNA) encoding a model tumor antigen to T cells, we evaluated the expression and cross-presentation capability of RNA-modified T cells. RESULTS We demonstrated that activated T cells can be genetically modified to express tumor antigens in vitro while retaining their inherent effector functions. Compared to unmodified T cells, T cells modified with tumor antigen encoding RNA showed high expression of inflammatory cytokines, including IFN gamma and TNF alpha and enhanced cellular proliferation and IL-2 secretion. Tumor antigen RNA-modified T cells demonstrated potent antigen presentation in vitro and importantly were significantly superior to RNA-loaded DCs in antigen presenting capacity. CONCLUSIONS This study strongly supports the implementation of a non-invasive and efficacious strategy to deliver “APC-like” T cell locally to brain tumors, thereby bypassing the lack of APCs within CNS malignancies.

Replication-Competent Lentivirus Analysis of Vector-Transduced T Cell Products Used in Cancer Immunotherapy Clinical Trials.

Lentiviral vectors are being used in a growing number of clinical applications, including T cell immunotherapy for cancer. As this new technology moves forward, a safety concern is the inadvertent recombination and subsequent development of a replication-competent lentivirus (RCL) during the manufacture of the vector material. To assess this risk, regulators have required screening of T cell products infused into patients for RCL. Since vector particles have many of the proteins and nucleotide sequences found in RCL, a biologic assay has proven the most sensitive method for RCL detection. As regulators have required screening of up to 108 cells per T cell product, this method described a procedure for assessing RCL contamination of large-volume T cell products.

Methods to assess DC-dependent priming of T cell responses by dying cells.

Dendritic cells have been widely investigated in cancer immunotherapy clinical trials for the last two decades mainly due to their robust ability to elicit an adaptive anticancer immune response of the cellular and humoral types. Immature DCs can be easily loaded with desired antigens. However, to become efficient antigen-presenting cells, DCs must first undergo a process of maturation. Protocols for the generation of DCs for use in cancer immunotherapy, including the generation of a large number of immature DCs for antigen pulsing and the selection of a well-defined immunostimulatory agent to achieve complete and reproducible maturation, which is a crucial step for further stimulation of T cell activation, must carefully consider the characteristics of DC physiology. In this report, we provided a detailed protocol for DC generation, pulsation and activation with the subsequent induction of T cell-specific immune responses.

Abstract 690: Optimization of biopsy scheduling in clinical studies of T cell bispecifics through an integrated modeling and simulation, digital pathology and machine learning approach

Abstract In the context of cancer immunotherapy clinical trials, baseline and on-treatment tumor biopsies may provide important insight into whether a treatment is working as expected, and furthermore whether efficacy is anticipated. For tumor-retained antibodies that perturb the behaviour of immune cells, such as T cell bispecific antibodies (TCBs), spatial information derived from biopsy images may be particularly insightful. On-treatment biopsies in clinical trials are usually scheduled at a time point that is considered convenient for the study design and when therapeutic effects, such as T cell infiltration, are expected to be distinguishable in tumor tissue. To our knowledge, there have been no attempts to investigate optimal scheduling of on-treatment biopsy sample collection using quantitative approaches due to lack of clinical data at a sufficiently diverse range of time points. Our integrated tissue pathology, disease modeling and machine learning workflow is designed to select the time point at which on-treatment biopsies could be most informative for making reliable predictions of response to treatment. Leveraging around 20 baseline and on-treatment digitized biopsy images from patients undergoing treatment with immune stimulating TCBs, we train a mathematical model to simulate tumor cell/T cell interactions in the tumor microenvironment. The mathematical model produces an enriched dataset of “virtual” biopsy images corresponding to predictions at intermediate time points. The virtual biopsies are evaluated based on their ability to predict treatment response. Specific mechanisms of action of bispecific antibodies, such as upregulation of T cell activation and/or proliferation, are taken into account in the structure of the mathematical model. The model is tuned and validated using machine learning techniques, and a reserved “test” dataset comprising images that were not used to estimate model parameters is used to evaluate model performance. Our workflow has the potential to inform clinical study design by promoting a scientific basis for the selection of an on-treatment biopsy schedule. Future applications of this workflow include identification of tissue properties that may contribute to inter-individual variability, and simulations of novel doses and schedules for combinations of immune-modulating cancer therapies. Citation Format: Lucy G. Hutchinson, Antoine Soubret, Benjamin Ribba, Jean-Eric Charoin, Alex Phipps, Richard Peck, Oliver Grimm. Optimization of biopsy scheduling in clinical studies of T cell bispecifics through an integrated modeling and simulation, digital pathology and machine learning approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 690.