Cytokine Release Assays Research Articles

Abstract 188: Autologous organoids and T cell co-cultures as a powerful personalized platform for immunotherapy development

Abstract Recent advances in cancer immunotherapy had a positive impact on the life expectancy of patients for a large range of clinical indications. With new treatment strategies and druggable targets being identified at an increasing pace, the number of patients eligible for cancer immunotherapy is expected to expand steadily. However, promising therapeutic developments face hurdles in translating preclinical findings into therapy since conventional 2D cancer models hold low clinical predictive value. We developed an innovative alternative, built on the discovery that adult stem cells proliferate and organise into three-dimensional organotypic structures when they are embedded into extracellular matrix. Patient specific organoids are generated from healthy and malignant tissues and stored as biobanks with high quality and reproducibility. HUB Organoids recapitulate complex characteristics of the original parental tissue, including molecular heterogeneity, and morphological and functional traits. Since cancer progression and responses to immunotherapy are governed by immune cell interactions in the tumor microenvironment, we developed an assay in which colorectal (CRC) and non-small cell lung cancer (NSCLC) tumor organoids are co-cultured with simultaneously expanded paired tumor infiltrating lymphocytes (TIL) as a source of tumor reactive cells. Our system allows robust expansion of TIL with preserved T-cell receptor repertoires and has an establishment rate of 75%. Paired resources allow for screening of immune-modulatory therapies under physiologic conditions not depending on peptide-pulsing and allo-reactivity. For this purpose, we developed a organoid-TIL co-culture system which quantifies and characterizes tumor organoid apoptosis by combining image-based analysis with flow cytometry and cytokine release assays as measurements of T-cell cytotoxicity against tumor (and normal-matched) organoids. Our system offers a powerful tool for the development and validation of cancer immunotherapy and may help to stratify cancer patients for susceptibility of various types of immunotherapies. Citation Format: Soura Mardpour, Claudia Beaurivage, Jane Sun, Lorenz Jahn, Rene Overmeer, Lars-Eric Fielmich, Pleun Hombrink, Farzin Pourfarzad, Sylvia F. Boj. Autologous organoids and T cell co-cultures as a powerful personalized platform for immunotherapy development [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 188.

Abstract 1880: A novel CD137 agonist exhibits potent antitumor efficacy with a good safety profile

Abstract Background: CD137 (TNFRSF9, 4-1BB) is a member of the tumor necrosis factor receptor superfamily that functions as a costimulatory molecule of immunocytes and is mainly expressed on the surface of immune cells including activated T and natural killer (NK) cells. Agonists against CD137 have shown promising therapeutic activity in mouse tumor models but have suffered setback in the clinic due to notably hepatotoxicity, or suboptimal agonistic potency. Here, we developed a fully human agonistic anti-CD137 monoclonal IgG4 antibody, ZG033, with an exclusive epitope, to achieve a better efficacy and safety profile for immunotherapy. Methods: The antigen binding specificity and affinity of ZG033 were determined by ELISA, surface plasmon resonance (SPR). X-ray crystallography was conducted to resolve the binding epitopes of the respective binding arms. The activities were determined using cell-based and reporter gene assays. In vivo antitumor activities were evaluated in human 4-1BB transgenic mice bearing with syngeneic tumors, and possible toxicity was evaluated in humanized mice and cynomolgus monkeys. Cytokine release assay was conducted by incubating ZG033 with PBMCs from healthy donors. The pharmacokinetic (PK) behavior of ZG033 were characterized in cynomolgus monkeys. A phase 1 study was initiated to evaluate the safety and preliminary clinical activities of ZG033, as well as the immunogenicity and pharmacokinetics. Results: ZG033 showed high specificity for human CD137 without cross-species reactivity across mouse, rat and exhibited potent CD137 agonist activity dependent on FcγR crosslinking. The binding affinity of ZG033 determined by surface plasmon resonance (SPR) was moderate (KD=10 nM). X-ray crystallography revealed that the Fab of ZG033 binds CD137 at an epitope overlap with the ligand binding interface. ZG033 increased CD137-driven NFκB reporter gene activation and the production of IFN-γ by human T cells in a FcγR-dependent manner. In human CD137 transgenic mice, ZG033 showed robust antitumor activity and induced durable antigen-specific immunological memory that prevented the tumor formation in the rechallenged mice. There were no signs of toxicity in human cytokine release assay which confirmed that ZG033 did not induce pro-inflammatory cytokines in the absence of TCR stimulation. Toxicology data in mice showed that ZG033 was well-tolerated with normal ALT and AST levels. ZG033 was further confirmed to well tolerated up to 180 mg/kg (MTD≥180mg/kg) dose without systemic toxicity in cynomolgus monkeys. Patients with advanced tumors, who are refractory after exhausting available therapies, were enrolled for ZG033 treatment. We will report the results from early dose escalation part of the study. Conclusion: These data demonstrate that ZG033 is an FcγR crosslinking-dependent CD137 agonist that displays a favorable safety profile and has potential in either mono- or combinational immunotherapies. Citation Format: Yang Huang, Liangwei Li, Wenting Liu, Dayan Zhang, Pengfei Zhou, Qing Fang, Weiming Zhou, Fengrong Wang, Guodong Shen, Liansheng Cheng. A novel CD137 agonist exhibits potent antitumor efficacy with a good safety profile [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 1880.

Abstract 1865: Development of CTIM-76, a highly specific Claudin 6 bispecific antibody for treatment of ovarian cancer

Abstract Solid tumors lead to 580,000 deaths annually in the US, and safe and effective therapeutics for many late-stage solid tumors are lacking. Ovarian cancer alone kills 14,000 people each year, and many patients do not respond to currently available treatments. Here we introduce CTIM-76, a CLDN6 T-cell engager antibody as a potential treatment of ovarian, endometrial, and other solid tumors. The tight junction protein Claudin 6 (CLDN6) is a validated therapeutic target for many solid tumor types, including ovarian, endometrial, testicular, and gastric. It is differentially expressed on cancer cells with no reported expression in normal, healthy tissue. Despite being an attractive target, therapeutic monoclonal antibodies (MAbs) targeting CLDN6 are difficult to discover due to an abundance of closely related family members and an absolute need for high specificity. The extracellular region of CLDN6 closely resembles the widely expressed family member CLDN9 (3 amino acids different). The few CLDN6 MAbs in clinical development have demonstrated significant binding to other CLDN family members and most have now been halted from further development. Starting with a panel of highly specific CLDN6 antibodies, we engineered a large set (> 50) of CLDN6xCD3 bispecific antibodies (CLDN6 bispecifics) using multiple formats and CD3 arms that encompass different geometries and binding stoichiometries, as these factors are expected to play a critical role in the potency of these molecules both in vitro and in vivo. In vitro T cell cytotoxicity and cytokine release assays as well as developability studies were used to select a small panel of antibodies. These were tested in vivo and demonstrated efficacy in xenograft studies in PBMC-engrafted mice. From this smaller panel, we chose a lead molecule, CTIM-76 for IND-enabling studies. CTIM-76 shows a combination of potent killing of CLDN6-expressing cells, no killing of cells expressing other closely related claudin family members, and excellent productivity and developability. The exquisite specificity of CTIM-76 suggests the potential to address the need for potent therapeutic modalities for ovarian and other cancers without compromising patient safety. Citation Format: Joseph Rucker, Ileine Sanchez, Kyle Doolan, Breanna Tyrell, Anna Lobley, Nicholas Molino, Kristen Shema, Kyle Guldner, Hayley Roth, Alyssa Cunningham, Kate Slovik, Riley Payne, Eric Butz, Evan Dick, Edward Calamai, Ross Chambers. Development of CTIM-76, a highly specific Claudin 6 bispecific antibody for treatment of ovarian cancer [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 1865.

Abstract 6336: Heterodimeric Fc-fused IL12 shows potent antitumor activity and low toxicity

Abstract Background: Interleukin-12 (IL12) is a heterodimeric pro-inflammatory cytokine (70 kDa) composed of two different polypeptide chains: an α-chain (p35 subunit) and a β-chain (p40 subunit). IL12 could induce the proliferation and cytotoxicity of T and NK cell and production of interferon-γ (IFN-γ), promote the differentiation of T helper 1 (TH1) cells and regulate innate resistance and adaptive immunity. However, the clinical efficacy of IL12 has been hindered for its high dose-related toxicities and short serum half-life. Here we have developed a heterodimeric Fc-fused IL12, HK054-V, with reduced potency to improve tolerability and prolong half-life. Methods: We assessed the binding, bioactivity, anti-tumor efficacy and safety of HK054-V. The binding ability of IL12-Fc lead variants to activated PBMCs was analyzed by flow cytometry. The bioactivity of HK054-V was determined by assessing the activated T-cell proliferation and IFN-γ production in human PBMCs, and STAT-4 phosphorylation in a reporter gene assay. A murine surrogate, mHK054-V, was engineered to mimic HK054-V to evaluate the antitumor efficacy in MC38 and CT26 syngeneic tumor mouse models. Cytokine release assay was conducted by incubating HK054-V with peripheral blood mononuclear cells (PBMCs) from healthy donors. The safety profiles and pharmacokinetic (PK) behavior of HK054-V were characterized in non-tumor-bearing Balb/c mice. Results: HK054-V is a heterodimeric Fc-fused IL12, which was designed with reduced potency. HK054-V was observed to stimulate the proliferation of activated T cells and NK cells and induce production of IFN-γ in a dose-dependent manner. HK054-V could activate pSTAT4 signal in luciferase reporter gene assay and showed up to 100-fold reduction of potency compared to wild-type IL12-Fc. Human IL12 is inactive in mice, so in vivo anti-tumor activity was assessed by a murine surrogate, mHK054-V. mHK054-V demonstrated anti-tumor efficacy in syngeneic mouse models and was better tolerated than WT mIL12-Fc. Furthermore, mHK054-V has improved PK and therapeutic index in mice. Conclusion: These data demonstrate that HK054-V, a heterodimeric Fc-fused IL12 with reduced potency, retain strong anti-tumor efficacy with a favorable tolerability profile, which may provide a practical alternative to the systemic administration of IL12 for antitumor therapy. Citation Format: Yang Huang, Wenting Liu, Dayan Zhang, Pengfei Zhou, Weiming Zhou, Xiaoli Zeng, Qun Zhao, Liangwei Li, Guodong Shen, Liansheng Cheng. Heterodimeric Fc-fused IL12 shows potent antitumor activity and low toxicity. [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 6336.

Abstract 5657: HK010, a novel anti-PDL1 × CD137 bispecific antibody, exhibits potent anti-tumor immunity and low toxicity

Abstract Background: Checkpoint inhibitors targeting the PD-1/PD-L1 have changed the treatment and prognosis for patients with advanced solid tumors. There remains a unmet need for additional therapies due to treatment resistance. Tumor-necrosis superfamily members, such as CD137 and CD40, have been shown to synergize with the checkpoint inhibitors in preclinical studies. However, agonists antibodies have exhibited limited clinical activity for severe liver toxicity. Here we have developed a Fc-silenced bispecific antibody targeting PD-L1 and CD137, HK010, which could overcome these limitations by tumor-directed T-cell activation and checkpoint blockade. Methods: The antigen binding specificity and affinity of HK010 were determined by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) and flow cytometry. The functional activity of HK010 was determined using in vitro cell-based assays and reporter gene assay. Transgenic mice expressing human PD-L1 and 4-1BB were transplanted with human PD-L1-expressing murine MC38 cells to assess in vivo antitumor activity. Cytokine release assay was conducted by incubating HK010 with peripheral blood mononuclear cells (PBMCs) from healthy donors (n=6). The pharmacokinetic (PK) behavior and safety profiles of HK010 were characterized in cynomolgus monkeys. Results: HK010 is a IgG4 PD-L1 × 4-1BB bsAb, which was designed to maintain a high affinity for human PD-L1 and a low affinity for human 4-1BB (KD PD-L1: 2.27 nM; KD 4-1BB: 493 nM) to achieve strong blocking of PD-1/PD-L1 and appropriate agonism of 4-1BB. HK010 retained full blockade activity on PD-1/PD-L1 signaling and enhances T-cell proliferation and IFN-γ production in vitro primary cell assays and Mixed lymphoid reaction (MLR). In addition, it led to a dose-dependent increase in the CD137-driven NFκB reporter gene activation through the bridging of PD-L1 on target cells and CD137 on effector cells. In humanized PD-L1/4-1BB transgenic mice bearing with MC38/hPDL1 tumor, HK010 showed robust single agent anti-tumor activity and induced durable antigen-specific immunological memory that prevents the growth of the same tumor cells in the mice re-challenged. There were no signs of toxicity in human cytokine release assay for HK010. Toxicology data in cynomolgus monkeys showed that HK010 was well-tolerated up to 150 mg/kg (MTD≥150mg/kg) dose without systemic toxicity. Furthermore, HK010 was confirmed to be highly safe in the 5-week repeated-dose toxicity study with no observed treatment-related mortality, abnormality in hematological indexes, important organs. Conclusion: These data demonstrate that HK010, an anti-PD-L1 × CD137 bispecific antibody, may exert a strong localized anti-tumor therapeutic efficacy with a low risk of liver toxicity through tumor-directed T-cell activation and checkpoint blockade in tumors. Citation Format: Liangwei Li, Wenting Liu, Guodong Shen, Dayan Zhang, Weiming Zhou, Fengrong Wang, Xiaoli Zeng, Yang Huang, Liansheng Cheng. HK010, a novel anti-PDL1 × CD137 bispecific antibody, exhibits potent anti-tumor immunity and low toxicity. [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 5657.

Abstract 4552: 3D-EXplore platform of fresh patient tumoroids with intact TME allows assessment of the efficacy of drugs targeting the tumor stroma on ex vivo tumor immunotherapy

Abstract Introduction: Cancer associated fibroblasts (CAFs) are a major component of the tumormicroenvironment (TME) and exhibit diverse tumorigenic functions such as immunosuppressionand extracellular matrix (ECM) remodeling. Treatment of solid tumors is often hindered by acomplex TME, which persists despite effective tumor-cell directed therapies. Therefore,treatment of solid tumors in combination with stromal-targeting therapies is essential toovercome CAF-facilitated tumor growth, and immunosuppression. Herein we interrogate theimpact of tumor stroma targeting therapeutics, in combination with nivolumab, on the efficacy oftumor cell killing (TCK) in a 3D human tumoroid ex vivo culture model (3D-EXplore). Resultingdata suggests that impairing the recruitment and activation of CAFs within the TME leads toenhanced TCK when combined with immune checkpoint blockade therapy. Materials and Methods: All patients tumor samples were collected with patient consent andrelevant IRB approval. 3D tumoroids measuring 150 microns in size were generated from freshpatient tumors including endometrial, ovarian, and colorectal cancer tissues. Tumoroids werethen treated with stromal targeting strategies for TGF-beta (galunisertib), the FGF pathway(Dovitinib), FAK inhibitors (defactinib), and cell adhesion modulators (plerixafor) alone or incombination with nivolumab for 72-hours ex vivo. Tumor responses to ex vivo treatments wereassessed using a proprietary tumor cell killing assay and 21-color flow cytometry analysis. Results and Summary: Here we evaluated the impact of stromal targeting therapeutics onCAFs and associated tumor cell killing as well as overcoming immune evasion mechanismspreserved in the tumoroid models. Furthermore, we used a multicolor flow panel to analyzewhether combination of stromal targeting drugs enhances nivolumab’s effect on the activation oftumor resident CD4, CD8 T-cells, NKT, and NK cell populations as well as on macrophagepolarization. Treatment-mediated changes in the tumor immune microenvironment was furthercorroborated by a multiplex cytokine release assay detecting GM-CSF, sCD137, IFNγ, sFas,sFasL, Granzyme A, Granzyme B, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, MIP-1α, MIP-1β, TNF-α,Perforin in tumoroid culture media and correlated with clinicopathologic findings and PD-L1expression for individual tumnors. Further, this 3D-tumoroid platform provides unique insightinto the microenvironment of both treatment responsive and non-responsive tumors and can aidin the development of patient-centered therapeutic regimens. Citation Format: Seth Currlin, Brittney Ruedlinger, Sharon Camacho, Angie Rivera, Jasmin D'Andrea, Jared Ehrhart, Soner Altiok. 3D-EXplore platform of fresh patient tumoroids with intact TME allows assessment of the efficacy of drugs targeting the tumor stroma on ex vivo tumor immunotherapy. [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 4552.

Abstract 4557: 3D-EXpress platform utilizing tumoroids from patients with MSS and MSI-H tumors allows rapid assessment of anti-tumor activity of immune checkpoint inhibitors and development of clinically relevant biomarkers of treatment response

Abstract Introduction: Recent studies showed that patients with deficient mismatch repair(dMMR)/microsatellite instability-high (MSI-H) tumors, have higher sensitivity to immune checkpointinhibitors (ICIs) compared to patients with microsatellite-stable (MSS)/microsatellite instability-low(MSI-L) tumors. However, the mechanisms of treatment responsiveness and resistance is not wellunderstood. Here, we used a novel 3D-EXpress ex vivo fresh patient tumoroid platform to assess theefficacy of nivolumab/ipilimumab combination therapy in tumors with known MSS/MSI-H status andperformed correlative studies. Materials and Methods: All tumor samples were obtained with patient consent and relevant IRBapproval. Tumoroids measuring 150 µm in size retaining tumor cell heterogeneity, tumor-residentimmune cells, stromal components, and cell-extracellular matrix interaction were prepared frompatients with endometrial and colorectal tumors among others. Tumoroid aliquots were cryopreservedin Nilogen’s tumoroid Biorepository for future studies. For the 3D-EXpress studies cryopreservedtumoroids were selected based on MSS/MSI-H status and treated ex vivo with nivolumab andipilimumab alone and in combinations for 72h. Tumor responses to treatments were evaluated by aproprietary tumor cell killing assay and changes in tumor immune microenvironment. Furthermore,tumor PD-L1 expression levels were analyzed on the associated TMA slides. Results: Treatment-induced tumor cell killing activity in intact tumoroids was assessed by a 3D high-content confocal imaging technique using a proprietary algorithm for data analysis. The impact of exvivo treatment by nivolumab and/or ipilimumab on tumor resident immune cell populations wasmonitored by a multiplex cytokine release assay analyzing release of GM-CSF, sCD137, IFNγ, sFas, sFasL,Granzyme A, Granzyme B, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, MIP-1α, MIP-1β, TNF-α, and Perforin usingculture supernatants isolated from treated tumoroid samples. Based on the ex vivo responses tumorswere assigned to treatment sensitive and resistant groups and correlative analyses were performed withindividual tumors’ innate and adaptive immune cell populations detected by a 21-color flow cytometrypanel, in addition to tumor MSS/MSI-H and p53 status, and detailed clinicopathologic data readilyavailable for the tumors cryopreserved in the Biorepository. Conclusion: The 3D-Express platform, using cryopreserved 3D tumoroids with intact TME is an effectivetool for the assessment of rational combinations which may prove relevant in the treatment of solidtumors. Furthermore, we believe this platform is a useful tool for the pre-clinical assessment of specifictherapeutic regimens designed for individualized patient care. Citation Format: Brittney Ruedlinger, Seth Currlin, Sharon Camacho, Alliyah Humphrey, Jared Ehrhart, Soner Altiok. 3D-EXpress platform utilizing tumoroids from patients with MSS and MSI-H tumors allows rapid assessment of anti-tumor activity of immune checkpoint inhibitors and development of clinically relevant biomarkers of treatment response. [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 4557.

Leishmania infantum infection after visiting southern Spain in patients on biological treatment; an observational, longitudinal, cohort study

BackgroundReports of leishmaniasis in immunosuppressed patients after visiting the Mediterranean Basin are becoming increasingly common. Still, awareness of the risk of infection and its clinical manifestations may be insufficient among healthcare professionals in the travellers’ home countries. MethodsThis observational, longitudinal study included 47 patients from Sweden with rheumatic disease and ongoing immunomodulatory treatment, who visited a rehabilitation centre in southern Spain where leishmaniasis is endemic. Patients were evaluated for clinical signs of leishmaniasis at baseline and after three years. Patients with leishmaniasis were followed for 4–5 years. The treatment outcome was assessed by clinical evaluation and determination of the cell-mediated immunological response to Leishmania by a whole blood cytokine release assay. ResultsSeven patients (15%) were diagnosed with leishmaniasis. The median time from exposure to the onset of symptoms was 3 [1–17] months. The median delay between the onset of symptoms and treatment start was 9 [1–12] months. All patients with leishmaniasis responded well to treatment. Only one patient had a relapse, which occurred within the first year. ConclusionHealthcare professionals need to be aware of the increased risk of leishmaniasis for travellers who are immunosuppressed. Knowledge of the symptoms is crucial for a timely diagnosis and early treatment.

A human Caco-2-based co-culture model of the inflamed intestinal mucosa for particle toxicity studies

The intestinal barrier is a complex interface of the human body, possessing the largest contact surface to nutrients and antigens and containing a major part of the immune system. It has to deal with continuous exposure to a broad mixture of essential, harmful, or useless substances and particles. In the context of plastic pollution and the ubiquitous occurrence of micro- and nanoplastics, oral exposure to such particles is of particular interest. Standard intestinal in vitro models, however, are unable to mimic the role of the immune system in the particle-exposed intestine. To allow for a closer look on the effect of particles on the intestinal immune system, we here developed a co-culture model to enable investigation of the epithelial brush border monolayer in a healthy and inflamed state. The model is based on well-established Caco-2 intestinal epithelial cells cultured in a Transwell™ system. Intraepithelial immune cells were mimicked by THP-1-derived M0-macrophages and MUTZ-3-derived dendritic cells. To fulfill the requirements needed for the investigation of particles, the co-culture system was developed without an additional matrix layer. Cell–cell contacts were established between interstitial and immune cells, and the Caco-2 standard cell culture medium was used, which is well-characterized for its role in defining the identity of particle dispersions. The model was characterized using confocal microscopy, membrane integrity measurements, and cytokine release assays from inflamed and healthy cells. Finally, the new co-culture model was used for investigation on polylactic acid, melamine formaldehyde resin, and polymethylmethacrylate plastic micro- and nanoparticles.

Cell-Derived Vesicles for Antibiotic Delivery-Understanding the Challenges of a Biogenic Carrier System.

Recently, extracellular vesicles (EVs) sparked substantial therapeutic interest, particularly due to their ability to mediate targeted transport between tissues and cells. Yet, EVs' technological translation as therapeutics strongly depends on better biocompatibility assessments in more complex models and elementary in vitro-in vivo correlation, and comparison of mammalian versus bacterial vesicles. With this in mind, two new types of EVs derived from human B-lymphoid cells with low immunogenicity and from non-pathogenic myxobacteria SBSr073areintroduced here. A large-scale isolation protocol to reduce plastic waste and cultivation space toward sustainable EV research is established. The biocompatibility of mammalian and bacterial EVsiscomprehensively evaluated using cytokine release and endotoxin assays in vitro, and an in vivo zebrafish larvae modelisapplied. A complex three-dimensional human cell culture model is used to understand the spatial distribution of vesicles in epithelial and immune cells and again used zebrafish larvae to study the biodistribution in vivo. Finally, vesiclesaresuccessfully loaded with the fluoroquinolone ciprofloxacin (CPX) and showed lower toxicity in zebrafish larvae than free CPX. The loaded vesiclesarethen tested effectively on enteropathogenic Shigella, whose infections are currently showing increasing resistance against available antibiotics.

Glyco-binding domain chimeric antigen receptors as a new option for cancer immunotherapy

In the last decade, treatment using Chimeric Antigen Receptor (CAR) are largely studied and demonstrate the potential of immunotherapeutic strategies, as seen mainly for blood related cancers. Still, efficient CAR-T cell approaches especially for the treatment of solid tumors are needed. Tn- and Sialyl-Tn antigens are tumor associated carbohydrate antigens correlating with poor prognosis and tumor metastasis on a variety of tumor entities. These glycans can be recognized by CD301 (CLEC10A, MGL), which is a surface receptor found primarily on immune cells. In the present study, we hypothesized, that it is possible to use newly generated CD301-bearing CARs, enabling cytotoxic effector cells to recognize and eliminate breast cancer cells. Thus, we genetically modified human NK92 cells with different chimeric receptors based on the carbohydrate recognition domain (CRD) of human CD301. We assessed their cytotoxic activity in vitro demonstrating the specific recognition of CD301 ligand positive cell lines. These results were confirmed by degranulation assays and in cytokine release assays. Overall, this study demonstrates CD301-CARs represent a cost-effective and fast alternative to conventional scFv CARs for cancer immunotherapy.

Pinoresinol stimulates keratinocyte proliferation and downregulates TNF-α secretion in peripheral blood mononuclear cells: An experimental in vitro study.

Natural coniferous resins are used in traditional medicine for the treatment of skin wounds. Coniferous wood resins ("callus" resin) are a mixture of abietic (resin) acids, lignans such as pinoresinol, and p-coumaric acid. The wound-healing properties of resins are thought to be related to their antimicrobial properties, but also to their effects on cell proliferation and inflammation. The purpose of this study was to identify and investigate the effects of novel aqueous dispersions of resin and its main components in the proliferation of human primary keratinocytes in vitroand in the expression of proinflammatory cytokines in human peripheral blood mononuclear cells. The proliferation studies were performed under low and high calcium conditions with or without added growth stimulators at the time points of 2and 6 days using AlamarBlue Cell Viability Reagent. The cytokine release assay was carried out by incubating the cells with the test articles for 18 h, after which the levels of tumor necrosis factor-α(TNF-α),interleukin-1β (IL-1β), IL-6, and IL-8 were measured in the supernatant by enzyme-linked immunosorbent assay. Resin and the purified lignan PINO, but not p-coumaric acid or abietic acid (industrial tall oil rosin), enhanced the proliferation of human keratinocytes in vitroand inhibited the expression of TNF-α, and to a lesser extent the expression of IL-1β in peripheral blood mononuclear cells. In this study, novel aqueous dispersions of spruce resin were used to investigate the effects of main resin components on keratinocyte proliferation and on the expression of key proinflammatory cytokines known to be associated with chronic wounds. The observations suggest that lignans, such as PINO, but not resin acids, are the components of resins that mediate the proliferative and TNF-α-suppressing effects. Lignans including PINO were identified as novel potential compounds in the treatment of chronic skin ulcers.

Correction: Longitudinal T Cell Responses against Ancestral, Delta, and Omicron SARS-CoV-2 Variants Determined by Rapid Cytokine Release Assay in Whole Blood

Correction: Longitudinal T Cell Responses against Ancestral, Delta, and Omicron SARS-CoV-2 Variants Determined by Rapid Cytokine Release Assay in Whole Blood

Single and Combinational Multiplex Base-Edited 'Universal' CAR T Cells in a Humanised Model of Primary CD7+CD33+ AML

Introduction Universal allogeneic donor CAR T cells are being developed as part of a 'deep conditioning' strategy against AML to secure disease clearance ahead of allogeneic stem cell transplantation (Allo-SCT). CD33 is widely expressed on AML, sometimes on the entire leukemia-associated populations, but is also present on healthy precursors; CD7 may be expressed on AML subpopulations and is present on normal T and NK cells, resulting in problematic fratricide effects during CAR T manufacture. Healthy donor derived CAR T cell banks generated using cytidine deamination base-editing and lentiviral transduction with CARs against both CD33 and CD7 circumvent such issues and could offer cell based anti-leukemic therapy alongside preparative effects ahead of allo-SCT. Here we investigated single and combinational BE-CAR T cells against primary human AML in vivo. Methods: Healthy donor lymphocytes were activated by anti-CD3/28 stimulation ahead of electroporation to deliver codon optimised base editor mRNA and guide RNAs against CD7, CD52 and TRBC, and lentiviral transduction to express either an anti-CD7 or anti-CD33 CAR. Magnetic bead depletion removed residual TCRab T cells, and disruption of CD7 ensured cells avoided fratricide. In vitro, antigen specific function was verified in cytotoxicity and cytokine release assays, and in vivo potency was confirmed against luciferase modified Molm14 and Kasumi lines in NSG mice. Responses against primary AML were investigated in a patient derived xenograft model (PDX) from a KIT mutated AML patient, established by serial transplantation, with full expression of CD33 (100% of blasts) and partial expression of CD7 (30% of blasts). Cells were injected into irradiated NSG mice (n=10 per group). On day 24 at a mean leukemic engraftment of 15% human CD45 positive cells in peripheral blood, mice were treated with either 10x106 BE-CAR33, BE-CAR7 or a combination of both or with anti-CD19-CAR-T cells (CAR19) as a control group (in total 20x106 T cells per mouse). AML engraftment, T cell persistence and expression of checkpoint markers were monitored by multicolour flow cytometric analysis. Results: On days 14 and 28 after CAR-T cell infusion, AML cells were readily detected in peripheral blood of control CAR19 animals (42.8% and 90% respectively) and the BE-CAR7 group (27.8% and 67.6%), but were notably reduced in the BE-CAR33 alone group (2.3% and 8.3%) and the BE-CAR33 and BE-CAR7 combination group (2.5% and 3.4%) (Figure 1A). Survival was significantly prolonged for BE-CAR33 and BE-CAR33/BE-CAR7 treated mice compared to the other groups (P<0.001) (Figure 1B). CD7+ AML cells were depleted to a mean of 1% and 4.5% in the BE-CAR33 group, to 0.6% and 0.7% in the BE-CAR33/BE-CAR7 and to 2.6% and 2.9% in the BE-CAR7 group, while CD7+ AML cells increased to 12% and 30% in the CAR19 group at day 14 and 28 after CAR-T cell infusion. Total CD2+CAR-T cell detection in peripheral blood ranged from 1.6 to 9.8% at day 14, 1 to 3.7% at day 28, and 0.8 to 2.7% at day 42, indicating durable persistence over the investigation period. Interestingly, PD-L1 on AML cells was present in all groups while checkpoint markers PD1, GAL9, TIM3, and LAG3 were significantly upregulated in BE-CAR33 cell treated mice, but not BE-CAR7 or CAR19 cell treated mice (P<0.001 for all comparisons). Conclusions: Lentiviral transduction and multiplexed base editing to remove TCR, CD52 and the shared AML/T lineage antigen CD7, enabled the generation of universal donor CAR T cells for combinational use. This data demonstrates robust activity of BE-CAR33 alone and in combination with BE-CAR7 against human CD7+CD33+AML in mice and highlights the importance of strategies that ensure complete targeting of leukemic populations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

VIP943 Is a Novel CD123 Antibody Drug Conjugate with in Vitro and In Vivo Efficacy in Acute Myeloid Leukemia (AML) Models

Background: Current treatment options (cytarabine with anthracyclines) for patients with acute myeloid leukemia (AML) are often associated with severe and barely tolerable toxicities. In patients who tolerate induction therapy, the complete remission rate is 60%-80%; however, 50% of patients relapse due to the presence of leukemic stem cells (LSC). Antibody-drug conjugates (ADCs) are putatively designed to deliver potent drugs directly to cancer cells while sparing normal tissue; however, first-generation ADCs are associated with severe bone marrow and liver toxicity. VIP943 is a CD123-targeting ADC which uses a kinesin spindle protein inhibitor (KSPi) as a new payload class and a unique legumain linker designed to overcome liabilities of currently approved ADC therapies. The payload is delivered with a drug-antibody ratio of 6. It consists of the KSPi warhead which is modified with a CellTrapperTM moiety so it accumulates within the CD123-targeted tumor cell and cannot enter normal cells. Non-human primate toxicology studies demonstrated a highly favorable and differentiated safety profile for VIP943 (Kirchhoff et al, Cancers 2020). Here we characterize VIP943 in various patient-derived AML models. Methods: For co-localization experiments, MOLM-13 AML cell line was stained with labelled anti-CD123 antibody or isotype control and CytoPainter LysoGreen indicator reagent and analyzed by imaging flow cytometry. AML primary patient mononuclear cells from blood and bone marrow were treated with VIP943 for 24, 48, and 72 hours. Flow cytometry immunophenotyping for AML blasts and CD34+/CD38- LSCs was used to analyze these populations. An in vivo patient-derived AML PDX mouse model was treated with VIP943 (5 mg/kg IV every 7 days) or in combination with 5-azacytidine (2.5 mg/kg SC days 1-5 x 3) and venetoclax (50 mg/kg PO days 1-5 x 3). Cytokine release syndrome was analyzed comparing VIP943 to isotype control ADC, anti-CD123 antibody, and positive comparator antibodies by in vitro cytokine release assays (CRA) in two different assay formats using whole blood and isolated mononuclear cells from 10 healthy donors. Flow cytometric lymphocyte and leukemic immunophenotyping was performed from blood of non-human primates. A leukemia cell line panel drug screen with VIP943 was evaluated and baseline genomic features were correlated with response. Results: Co-localization studies confirm specific internalization and efficient intracellular trafficking to the lysosome of VIP943. In vitro treatment with VIP943 (66 nM, continuous exposure) delivers blast reductions of 37% after a 24h incubation with VIP943, 45% after 48h incubation and 83% after an 72h incubation. In the CD34+/CD38- cell population, a reduction of 71% was achieved. In a patient-derived AML model, VIP943 with 5-azacytidine and venetoclax led to complete tumor growth inhibition after 21 days showing superiority of this triplet therapy (complete regression: 8/9 mice) with high tolerability (maximum body weight loss -4.8 %). The immunotoxicological evaluation in both CRA formats and with the tested antibody concentrations (100µg, 10µg, 1µg) exerts no induction of cytokines by VIP943 compared to controls. Immunophenotyping demonstrates no impact of VIP943 treatment on lymphocytic subpopulations, whereas the CD123-positive basophils are reduced after 24h followed by an almost complete recovery. VIP943 IC50 ranges from 0.1nM-300nM in a leukemia cell line panel screen and features associated with sensitivity are investigated. Conclusions: VIP943 is a next-generation ADC with a differentiated safety profile from currently approved ADCs, including lack of in vitro cytokine release. In vitro and in vivo studies using patient-derived AML cells show VIP943 has favorable monotherapy and combination efficacy including targeting of leukemic stem cells, which drive relapse. These findings warrant evaluating VIP943 in clinical trials.

Preclinical Characterization of NGM936, a Novel Bispecific T Cell Engager Targeting ILT3 for the Treatment of Acute Myeloid Leukemia with Monocytic Differentiation

Acute myeloid leukemia (AML) with monocytic differentiation [M4 and M5 AML per the French-American-British (FAB) classification] accounts for 25-30% of all AML cases and has a particularly poor prognosis, with a <15% five-year overall survival rate and an increased risk of bone marrow and extramedullary relapse after stem cell transplant compared with other AML subtypes. The use of T cell engagers in AML has been limited by the lack of suitable target antigens with high specificity for AML cells and minimal expression on healthy bone marrow progenitors. Immunoglobulin-like transcript 3 (ILT3, also known as LILRB4) is a highly specific marker of AML with monocytic differentiation and is not expressed on non-monocytic blood cells or hematopoietic progenitor cells. This highly restricted expression profile makes ILT3 a promising target for monocytic AML, with the potential to enable effective elimination of AML cells with less toxicity than current therapies. Here, we report the pre-clinical characterization of NGM936, an ILT3-based T cell engager for the treatment of monocytic AML and other ILT3+ hematologic malignancies. NGM936 is a monovalent scFv-Fab bispecific on an effectorless human IgG1 backbone (LALA) with a knob-in-hole Fc design for heterodimeric assembly. NGM936 demonstrates sub-nM affinity towards ILT3 of human origin (KD = 0.2 nM at 25°C by surface plasmon resonance) and is not cross-reactive towards other LILRA/B family members. A panel of more than 30 ILT3 x CD3 engagers in various formats was generated and screened in assays to measure both T cell-dependent cytotoxicity and cytokine release. NGM936 was identified for its ability to potently induce T cell-dependent cytotoxicity (TDCC) against ILT3+ AML cells while inducing minimal cytokine release. In both whole blood cytokine release assays and in TDCC assays in which cytokine secretion was measured after target engagement, induction of TNF-α, IL-6, IFN-γ, and IL-2 by NGM936 was considerably lower than that induced by a vibecotamab biosimilar (CD123 x CD3). NGM936 induced potent cytotoxicity when both expanded and naïve T cells were used as effectors. Importantly, NGM936 efficiently ablated tumor cells with a range of ILT3 expression, from ~1500 copies/cell to ~40,000 copies/cell. In addition, NGM936 failed to induce T cell-dependent cytotoxicity against CD34+ hematopoietic stem cells or non-monocytic immune cells, consistent with the lack of ILT3 expression on these cell types. In ex vivo cultures of primary M5 AML bone marrow, NGM936 induced a dose-dependent depletion of AML cells and a concordant increase in T cell proliferation and activation. Finally, NGM936 induced a dose-dependent depletion of circulating tumor cells in a xenograft model in which irradiated, immunodeficient NSG mice were engrafted with human PBMCs and human ILT3+ AML cells. NGM936 thus represents a promising new treatment strategy for monocytic AML, with the potential to eliminate monocytic leukemia cells while minimizing the myelotoxicity associated with ablation of healthy bone marrow.

Shared Clavulanate and Tazobactam Antigenic Determinants Activate T-Cells from Hypersensitive Patients.

β-Lactamase inhibitors such as clavulanic acid and tazobactam were developed to overcome β-lactam antibiotic resistance. Hypersensitivity reactions to these drugs have not been studied in detail, and the antigenic determinants that activate T-cells have not been defined. The objectives of this study were to (i) characterize clavulanate- and tazobactam-responsive T-cells from hypersensitive patients, (ii) explore clavulanate and tazobactam T-cell crossreactivity, and (iii) define the antigenic determinants that contribute to T-cell reactivity. Antigen specificity, pathways of T-cell activation, and crossreactivity with clavulanate- and tazobactam-specific T-cell clones were assessed by proliferation and cytokine release assays. Antigenic determinants were analyzed by mass spectrometry-based proteomics methods. Clavulanate- and tazobactam-responsive CD4+ T-cell clones were stimulated to proliferate and secrete IFN-γ in an MHC class II-restricted and dose-dependent manner. T-cell activation with clavulanate- and tazobactam was dependent on antigen presenting cells because their fixation prevented the T-cell response. Strong crossreactivity was observed between clavulanate- and tazobactam-T-cells; however, neither drug activated β-lactam antibiotic-responsive T-cells. Mass spectrometric analysis revealed that both compounds form multiple antigenic determinants with lysine residues on proteins, including an overlapping aldehyde and hydrated aldehyde adduct with mass additions of 70 and 88 Da, respectively. Collectively, these data show that although clavulanate and tazobactam are structurally distinct, the antigenic determinants formed by both drugs overlap, which explains the observed T-cell cross-reactivity.

Abstract 2906: AGEN1571 is a novel high-affinity ILT2 antagonist antibody that promotes adaptive and innate immune responses

Abstract Background: Ig-Like Transcript 2 (ILT2, LILRB1) is an inhibitory receptor prominently expressed on resting macrophages, monocytes, and dendritic cells (DCs), with variable expression on CD4+ T cells, CD8+ T cells, B cells, NK cells, and NKT cells in the tumor microenvironment. Interaction of ILT2 with its ligands, HLA-G and classical MHC class I molecules, is reported to impair cytotoxic activity of NK and effector T cells, attenuate B cell function, inhibit antigen-presentation by dendritic cells, and promote immunosuppressive activity of myeloid cells. Further, high ILT2 expression has been associated with poor prognosis in several human malignancies. As a result, ILT2 has been proposed as a therapeutic target to promote antitumor immunity and to overcome resistance to checkpoint blockade. Here, we report that AGEN1571, a fully human anti-ILT2 monoclonal IgG4κ antibody, reversed ILT2-mediated immunosuppression and promoted activation of NK, NKT, T cells, and myeloid cells in a series of preclinical studies. Methods: AGEN1571 binding to ILT2 was analyzed by SPR and flow cytometry. HDX-MS was used to identify the AGEN1571 binding epitope on ILT2. Mechanisms of AGEN1571-mediated modulation of immune cell function were evaluated using reporter cell lines and primary cell-based in vitro and ex vivo assays. Results: AGEN1571 bound to recombinant ILT2 with an approximate monovalent binding affinity of 0.38 nM and to cell-expressed ILT2 with an EC50 of 0.07 - 0.55 nM, depending on cell type and donor. AGEN1571 did not cross-react with related LILRA and LILRB family members except with weak reactivity to ILT4 (~370 nM). Consistent with its binding epitope, AGEN1571 potently blocked ILT2 binding to HLA-A, -B, -C, and -G and restored FcγR signaling inhibited by ILT2-HLA-G engagement in dose-dependent manner. AGEN1571 also augmented NFκB and ISRE activation induced by TLR, FcγR, and STING agonists in myeloid cells, and promoted polarization of primary human macrophages towards a pro-inflammatory phenotype. ILT2 blockade with AGEN1571 alone and in combination with a PD-1 antagonist enhanced secretion of pro-inflammatory cytokines and activation of NK, NKT and CD8+ T cells in a PBMC tumor cell co-culture assay. AGEN1571 was designed to have minimal binding to FcγR and complement which has been confirmed by lack of FcγR signaling and complement cascade induction in vitro. An in silico T cell epitope screen predicted AGEN1571 to be non-immunogenic, while AGEN1571 showed no evidence of inducing systemic cytokine release in a cytokine release assay using whole blood and PBMC cultures. Conclusion: AGEN1571 is a novel high-affinity ILT2 antagonist antibody that effectively antagonizes ILT2 to enhance T cell, NK cell, NKT cell, and myeloid activity. These data support clinical development of AGEN1571 as a therapeutic agent for patients with solid tumors. Citation Format: Olga Udartseva, Malgorzata Pupecka-Swider, Pilar Garcia-Broncano, Naxin Guo, Spencer Campbell, Elizabeth Klakus, Rebecca Ward, Priyadarshini Iyer, Christine Brittsan, Beth Wensley, Benjamin Morin, Andrew Slee, Dhan Chand, Claire Galand, Olga Ignatovich, Emmanuel Briend, David Savitsky. AGEN1571 is a novel high-affinity ILT2 antagonist antibody that promotes adaptive and innate immune responses [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 2906.

Abstract 3543: Altering tumor immunophenotypes with PD-L1 engineered toxin bodies

Abstract Targeting of PD-L1 checkpoint has shown clinical efficacy in multiple solid tumor indications. Currently approved PD-L1 targeted approaches rely on the blocking activity of monoclonal antibodies (mAbs) which sterically inhibit PD-L1 thus preventing PD-1 mediated immune checkpoint activity. Although these mAbs have shown activity in the clinic, the need for pre-existing tumor specific immunity and tumor immune infiltration precludes responses in some patients and leads to resistance in others. Therefore, there remains a need for new modalities and treatment paradigms. Molecular Templates has developed MT-6402, an engineered toxin body (ETB) targeting PD-L1 designed to overcome the challenges of current PD-L1 targeting approaches by 1) directly depleting PD-L1 positive tumor or immunosuppressive immune cells displaying PD-L1 and 2) alteration of the tumor immunophenotype through the cytoplasmic delivery of an HLA:A*02 restricted cytomegalovirus (CMV) antigen. Delivery of this antigen is meant to leverage recruitment of existing CMV-restricted cytotoxic T lymphocytes (CTLs) for cell-mediated cytotoxic depletion (antigen seeding technology, AST) of CMV antigen presenting tumor cells. MT-6402 is currently in a phase I open-label, dose escalation and expansion study in subjects with advanced solid cancers that express PD-L1 (NCT04795713). Initial assessment of pharmacodynamic markers for PD-L1 and CMV-mediated ETB activity in a subject with confirmed PD-L1+ tumor cells, CMV positivity, and HLA:A*02 restriction has shown serum phenotypes associated with robust checkpoint inhibitor activity and full extravasation of circulating CMV-specific T cells. In addition to assessing early clinical data from the HLA: A*02 restricted MT-6402 trials, expansion of ETB AST to a broader patient population requires testing of ETBs engineered with the ability to deliver CMV antigens across a broad range of HLA restriction, including HLA:A*01, HLA:A*03, and HLA:A*24. To this end, ETBs were benchmarked against MT-6402 to identify candidates that retain comparable specificity, selectivity, and activity. Substitution of peptide antigens did not alter specificity or selectivity of ETBs compared to MT-6402. Candidate ETB binding profiles and potency were comparable to MT-6402. ETBs delivered an antigen seeding response in a PD-L1 dependent and HLA specific manner. In vivo efficacy of candidate ETBs was comparable in a murine efficacy xenograft model with MT-6402. An ex vivo cytokine release assay in a co-culture setting using HLA matched CTLs and PD-L1+ target cells, treatment with AST capable ETBs resulted in secretion of antigen specific T cell mediated immune cytokines compared to AST null controls. These cytokines overlap with cytokine signatures observed after dosing MT-6402 in HLA:A*02 patients. Preclinical assessment of the safety profile of candidates is ongoing and further development is slated for 2021. Citation Format: Swati Khanna, Elizabeth M. Kapeel, Lauren R. Byrne, Elizabeth Saputra, Steven Rivera, Lindsey Aschenbach, Lilia A. Rabia, Garrett L. Cornelison, Rachael M. Orlandella, Brigitte Brieschke, Michaela Sousares, Jay Zhao, Garrett L. Robinson, Chris Moore, Joseph D. Dekker. Altering tumor immunophenotypes with PD-L1 engineered toxin bodies [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 3543.

Abstract 246: Application of the 3d fresh patient tumoroid platform 3d-explore to assess the immunomodulatory and phagocytic activity of the receptor tyrosine kinase inhibitor, Sunitinib

Abstract Background: The receptor tyrosine kinase inhibitor (TKI) Sunitinib has proven useful in the treatment of a variety of tumors. Sunitinib has shown therapeutic activity in patients with metastatic renal cell carcinoma and currently represents a frontline therapy for this disease. The immune modulatory effect of Sunitinib on immune cells within the tumor microenvironment has been well documented. Here, we developed a novel 3D ex-vivo platform (3D-EXplore) using fresh patient tumor samples with intact stromal components and tumor immune microenvironment to assess the therapeutic efficacy of Sunitinib. Methods: All tumor samples were obtained with patient consent and relevant IRB approval. Unpropagated 3D tumoroids with intact TME measuring 150 µm in size were prepared from fresh tumor samples of renal cell carcinoma using a proprietary technology developed at Nilogen Oncosystems. Tumoroids prepared from each patient’s tumor sample were pooled to represent the tumor heterogeneity and treated ex vivo with Sunitinib for 48h to detect treatment-mediated changes in tumor immune cell composition including CD4 and CD8 T-cells, NK cells, and macrophages. Additionally, we analyzed treatment-mediated changes in T-cell activation and phagocytic activity of myeloid cells. Results: Multiparameter flow analysis revealed that Sunitinib treatment led to an increase in CD8+CD25+ central memory-like phenotype and increased granzyme B expression in CD4 and CD8+ T cells indicating T cell activation. Furthermore, multiparameter flow cytometry analysis showed increased EdU uptake by CD8 and NK cells with sunitinib treatment, while cytokine release assays demonstrated increased IFN-γ and IL-2 production accompanied by downregulation of IL-10 and IL-6 cytokines. We observed Sunitinib mediated phagocytotic activity by tumor resident myeloid macrophages, monocytes, and dendritic cells using a quantitative phagocytosis assay. Conclusions: These results demonstrate that 3D-EXplore using fresh tumor samples provides a clinically relevant platform to assess drug response and serves as a critical tool for reflecting the true intact tumor microenvironment for novel immune-oncology drug development. Citation Format: Brittany Bunch, Krithika N. Kodumudi, Jared Ehrhart, Matt Weitzman, Olivia MacIntosh, Kelly Sussman, Soner Altiok. Application of the 3d fresh patient tumoroid platform 3d-explore to assess the immunomodulatory and phagocytic activity of the receptor tyrosine kinase inhibitor, Sunitinib [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 246.