Exhaustion Markers Research Articles

The predominant Quillaja Saponaria fraction, QS-18, is safe and effective when formulated in a liposomal murine cancer peptide vaccine

Extracts of the Chilean soapbark tree, Quillaja Saponaria (QS) are the source of potent immune-stimulatory saponin compounds. This study compared the adjuvanticity and toxicity of QS-18 and QS-21, assessing the potential to substitute QS-18 in place of QS-21 for vaccine development. QS-18, the most abundant QS saponin fraction, has been largely overlooked due to safety concerns. We found that QS-18 spontaneously inserted into liposomes, thereby neutralizing hemolytic activity, and following administration did not induce local reactogenicity in a footpad swelling test in mice. With high-dose intramuscular administration, transient weight loss was minor, and QS-18 did not induce significantly more weight loss compared to a liposome vaccine adjuvant system lacking it. Two days after administration, no elevation of inflammatory cytokines was detected in murine serum. In a formulation including cobalt-porphyrin-phospholipid (CoPoP) for short peptide sequestration, QS-18 did not impact the formation of peptide nanoparticles. With immunization, QS-18 peptide particles induced higher levels of cancer neoepitope-specific and tumor-associated antigen-specific CD8+ T cells compared to QS-21 particles, without indication of greater toxicity based on mouse body weight. T cell receptor sequencing of antigen-specific CD8+ T cells showed that QS-18 induced significantly more T cell transcripts. In two murine cancer models, vaccination with QS-18 peptide particles induced a similar therapeutic effect as QS-21 particles, without indication of increased toxicity. Antigen-specific CD8+ T cells in the tumor microenvironment were found to express the exhaustion marker PD-1, pointing to the rationale for exploring combination therapy. Taken together, these data demonstrate that QS-18, when formulated in liposomes, can be a safe and effective adjuvant to induce tumor-inhibiting cellular responses in murine models with potential to facilitate or diminish costs of production for vaccine adjuvant systems. Further studies are warranted to assess liposomal QS-18 immunogic, reactogenic and toxicological profiles in mice and other animal species.

Effector Function Characteristics of Exhausted CD8+ T-Cell in Microsatellite Stable and Unstable Gastric Cancer.

Gastric cancer exhibits molecular heterogeneity, with the microsatellite instability-high (MSI-H) subtype drawing attention for its distinct features. Despite a higher survival rate, MSI-H gastric cancer lack significant benefits from conventional chemotherapy. The immune checkpoint inhibitors, presents a potential avenue, but a deeper understanding of the tumor immune microenvironment of MSI-H gastric cancer is essential. We explored the molecular characteristics of CD8+ T-cell subtypes in three MSI-H and three microsatellite stable (MSS) gastric cancer samples using single-cell RNA sequencing and spatial transcriptome analysis. In MSI-H gastric cancer, significantly higher proportions of effector memory T cell (Tem), exhausted T cell (Tex), proliferative exhausted T cell (pTex), and proliferative T cell were observed, while MSS gastric cancer exhibited significantly higher proportions of mucosal-associated invariant T cell and natural killer T cell. In MSI-H gastric cancer, Tex and pTex exhibited a significant upregulation of the exhaustion marker LAG3, as well as elevated expression of effector function markers such as IFNG, GZMB, GZMH, and GZMK, compared to those in MSS gastric cancer. The interferon γ (IFN-γ) signaling pathway of Tex and pTex was retained compared to those of MSS gastric cancer. The spatial transcriptome analysis demonstrates the IFN-γ signaling pathway between neighboring Tex and malignant cell, showcasing a significantly elevated interaction in MSI-H gastric cancer. Our study reveals novel finding indicating that IFN-γ signaling pathway is retained in Tex and pTex of MSI-H gastric cancer, offering a comprehensive perspective for future investigations into immunotherapy for gastric cancer.

A quality-by-design approach to improve process understanding and optimise the production and quality of CAR-T cells in automated stirred-tank bioreactors.

Ex vivo genetically-modified cellular immunotherapies, such as chimeric antigen receptor T cell (CAR-T) therapies, have generated significant clinical and commercial outcomes due to their unparalleled response rates against relapsed and refractory blood cancers. However, the development and scalable manufacture of these novel therapies remains challenging and further process understanding and optimisation is required to improve product quality and yield. In this study, we employ a quality-by-design (QbD) approach to systematically investigate the impact of critical process parameters (CPPs) during the expansion step on the critical quality attributes (CQAs) of CAR-T cells. Utilising the design of experiments (DOE) methodology, we investigated the impact of multiple CPPs, such as number of activations, culture seeding density, seed train time, and IL-2 concentration, on CAR-T CQAs including, cell yield, viability, metabolism, immunophenotype, T cell differentiation, exhaustion and CAR expression. Initial studies undertaken in G-Rex® 24 multi-well plates demonstrated that the combination of a single activation step and a shorter, 3-day, seed train resulted in significant CAR-T yield and quality improvements, specifically a 3-fold increase in cell yield, a 30% reduction in exhaustion marker expression and more efficient metabolism when compared to a process involving 2 activation steps and a 7-day seed train. Similar findings were observed when the CPPs identified in the G-Rex® multi-well plates studies were translated to a larger-scale automated, controlled stirred-tank bioreactor (Ambr® 250 High Throughput) process. The single activation step and reduced seed train time resulted in a similar, significant improvement in CAR-T CQAs including cell yield, quality and metabolism in the Ambr® 250 High Throughput bioreactor, thereby validating the findings of the small-scale studies and resulting in significant process understanding and improvements. This study provides a methodology for the systematic investigation of CAR-T CPPs and the findings demonstrate the scope and impact of enhanced process understanding for improved CAR-T production.

FEATURES FUNCTIONING OF THE DETOXIFICATION SYSTEM AT THE THREAT TERMINATION OF PREGNANCY AS A CONSEQUENCE OF ACCUMULATION OF 137CS

To date, there is no consensus on the pathogenesis of miscarriage. In modern life, human health is signifi cantly infl uenced by environmental factors. In this regard, 37 years after the accident at the Chornobyl nuclear power plant, the question of remote cytogenetic and hereditary eff ects in the off spring of parents aff ected by radiation remains relevant. Ecosystems contaminated with radioactive substances are a source of exposure for humans. Internal irradiation is the cause of reproductive losses. Radiation stress aff ects the course of redox processes in the body. Biochemical indicators are markers of the infl uence of environmental factors on a person. The study is dedicated to determining the state of peroxide hemostasis in the case of miscarriage. Aim: to determine the peculiarities of the functioning of the detoxification system in case of miscarriage due to the accumulation of 137Cs.Material and methods. The fi rst (research) group included women with reproductive losses in anamnesis and signs oftermination of the current pregnancy; the second (control) group included women with an uncomplicated history and course of pregnancy. Additionally, the fi rst group was divided into subgroups according to pregnancy outcomes: А – 38 women who gave birth at 37-40 weeks, despite the complicated course of the current pregnancy, В – 13 women who gave birth at 28-36 weeks + 6 days, С – 9 women who birth at 22-27 weeks + 6 days. The state of peroxide hemostasis in women was studied using biochemical studies. Accumulation of 137Cs with diff erent activity was detected in the placentas of the examined women of both groups using β-spectrometry. Morphological examination of placentas was performed according to the protocol. The severity of placental damage was studied by Olympus BX51 and Axioskop 40 microscopes. Analyzing the results revealed a relationship between biochemical indicators, the activity of 137Cs, and pregnancy scenarios. Statistical data analysis was performed using Microsoft Excel (2016) and Fisher angular transformation. The diff erence between comparative values was considered signifi cant at p < 0.05 (probability index greater than 95 %). Permission to conduct research was obtained from the Medical Ethics Committee of the SI «Institute of Pediatrics, Obstetrics, and Gynecology named academic Elena M. Lukyanova of the National Аcademy of Мedical Sciences of Ukraine» (protocol No 3 of 07.06.2017).Scientifi c research work is to «Develop the latest and improve existing technologies for diagnosis, prevention, and treatmentof premature termination of pregnancy in women with miscarriage taking into account the passport of the placenta» (2018-2020). Code VN.20.00.02.18, state registration number 0118U000039, KPKV 6561040.Results. It has been confi rmed that the state of the environment plays a decisive role in the pathogenesis of miscarriage. Ithas been proven that placental dysfunction caused by abnormal oxidative stress due to the action of incorporated 137Cs leads to pregnancy termination. As a result of the depletion of the antioxidant reserve, the compensatory capabilities of the placenta decrease. It was established that activity in the placenta up to 1.0 Bq/kg of 137Cs does not aff ect the course of gestation. The compensatory capacity of the placenta remains preserved even with the accumulation of 1.1 to 4.4 Bq/kg of 137Cs. At the same time, it is possible to prolong the pregnancy until the term of timely delivery. Internal irradiation with an activity of 4.5-10.4 Bq/kg of 137Cs causes damage to the stroma of the maternal surface of the placenta and premature birth at 28-36 weeks + 6 days. At the same time, newborns are viable due to the preservation of compensatory reactions in the placenta. Accumulation of more than 10.4 Bq/kg of 137Cs causes antenatal death of the fetus and early premature birth due to damage to maternal and fetal structures of the placenta. An increase in the content of malondialdehyde and a decrease in the activity of superoxide dismutase in the blood indicate the severity of radiation damage.Conclusions. Internal exposure to 137Cs disrupts the architecture and functional capacity of the placenta. Extreme eff ectsdepend on the activity of 137Cs, the compensatory capabilities of the placenta, and the pregnant woman’s body. Activation of lipid peroxidation acts as a biochemical amplifi er of radiation exposure. An increase in the blood of malondialdehyde, diene conjugates, and free SH groups is associated with the severity of radiation damage. An early marker of primary placental dysfunction, premature birth, and antenatal fetal loss is an increase in MDA content in the blood by 12.0 % and a decrease in SOD by 6.5 % relative to permissible values. An early marker of exhaustion of the compensatory reserve is an increase in the content of MDA in the blood by 23.3 % and a decrease in SOD by more than 18.2 % relative to the permissible values. Decompensation of adaptation mechanisms in the system «mother- placenta-fetus» leads to antenatal losses.

CD4+ CAR T-cell exhaustion associated with early relapse of multiple myeloma after BCMA CAR T-cell therapy

AbstractMultiple myeloma is characterized by frequent clinical relapses after conventional therapy. Recently, chimeric antigen receptor (CAR) T cells targeting B-cell maturation antigen (BCMA) has been established as a treatment option for patients with relapsed or refractory disease. However, although >70% of patients initially respond to this treatment, clinical relapse and disease progression occur in most cases. Recent studies showed persistent expression of BCMA at the time of relapse, indicating that immune-intrinsic mechanisms may contribute to this resistance. Although there were no preexisting T-cell features associated with clinical outcomes, we found that patients with a durable response to CAR T-cell treatment had greater persistence of their CAR T cells than patients with transient clinical responses. They also possessed a significantly higher proportion of CD8+ T-effector memory cells. In contrast, patients with short-lived responses to treatment have increased frequencies of cytotoxic CD4+ CAR T cells. These cells expand in vivo early after infusion but express exhaustion markers (hepatitis A virus cellular receptor 2 [HAVCR2] and T-cell immunoglobulin and mucin domain-containing-3 [TIGIT]) and remain polyclonal. Finally, we demonstrate that nonclassical monocytes are enriched in the myeloma niche and may induce CAR T-cell dysfunction through mechanisms that include transforming growth factor β. These findings shed new light on the role of cytotoxic CD4+ T cells in disease progression after CAR T-cell therapy.

Transient TCR-based T cell therapy in a patient with advanced treatment-resistant MSI-high colorectal cancer

We previously demonstrated the antitumor effectiveness oftransiently Tcell receptor (TCR)-redirected Tcells recognizing a frameshift mutation in transforming growth factor beta receptor 2. We here describe a clinical protocol using mRNATCR-modified Tcells to treat a patient with progressive, treatment-resistant metastatic microsatellite instability-high (MSI-H) colorectal cancer. Following 12 escalating doses of autologous Tcells electroporated with in-vitro-transcribed Radium-1 TCR mRNA, we assessed Tcell cytotoxicity, phenotype, and cytokine production. Tumor markers and growth on computed tomography scans were evaluated and immune cell tumor infiltrate at diagnosis assessed. At diagnosis, tumor-infiltrating CD8+ Tcells had minimal expression of exhaustion markers, except for PD-1. Injected Radium-1 Tcells were mainly naive and effector memory Tcells with low expression of exhaustion markers, except for TIGIT. We confirmed cytotoxicity of transfected Radium-1 Tcells against target cells and found key cytokines involved in tumor metastasis, growth, and angiogenesis to fluctuate during treatment. The treatment was well tolerated, and despite his advanced cancer, the patient obtained a stable disease with 6months survival post-treatment. We conclude that treatment of metastatic MSI-H colorectal cancer with autologous Tcells electroporated with Radium-1 TCR mRNA is feasible, safe, and well tolerated and that it warrants further investigation in a phase 1/2 study.

Abstract CT065: Modakafusp alfa shows intratumoral immune pharmacodynamics (PD) in a subset of patients (pts) in a phase 1b study in advanced/metastatic solid tumors

Abstract Background: Modakafusp alfa (moda), a novel immunocytokine, is an innate immune enhancer comprising an anti-CD38 IgG4 antibody fused to 2 attenuated interferon (IFN) α2b molecules. Moda is being evaluated in phase 1/2 trials for multiple myeloma and in a phase 1/2 trial in solid tumors. Here, we present PD data in tumor biopsies and peripheral blood (PB) from the phase 1/2 study of moda as monotherapy in pts with advanced/metastatic solid tumors (NCT04157517). Methods: A total of 21 pts were treated with moda 0.1-1.5 mg/kg every 3 weeks. Tumor biopsy samples were collected at screening and on cycle 2, day 2 (C2D2). Biopsies were evaluated by multiplex immunofluorescence (using a Multiomyx platform) and RNASeq. PB samples were collected pre-dose on C1D1 and at timepoints after treatment. PB samples were evaluated by CyTOF to assess PD changes in immune cells. Results: No clinical tumor responses (partial or better) were observed in the 21 pts. Analysis of tumor biopsies revealed two subgroups defined by differences in upregulation of CD38 expression after moda treatment, with differential intratumoral PD changes. The CD38 gene has IFN response elements within its promoter region. Thus, correlation of tumor PD with changes in CD38 expression is of interest because, along with being a target of moda, an increase in CD38 expression may indicate drug activity in the tumor microenvironment. Interestingly, in the subset of pts in whom an increase in the percentage of CD38+ lymphocytes in response to moda was reported, there were also increases in CD8 and CD4 T cell activation, (%CD69) as well as CD8 T cell cytotoxic function (%GzB). Additionally, moda led to increase in CD14+CD16+ intermediate monocytes, indicating an enhanced myeloid response in several pts, and minimal changes in FoxP3+ Tregs regardless of changes in CD38 expression. Moda treatment led to an increase in activated and cytolytic natural killer (NK) and CD8 T cells in all pts. This was accompanied by increased NK and CD8 T cell proliferation (%Ki67) in PB, without sustained increase in exhaustion markers. CD4 T cells were also activated in response to moda and there was no increase in circulating FoxP3+ Tregs. Enhanced myeloid cell response, measured by increased proliferation of monocytes and dendritic cells, along with upregulation of costimulatory molecules, were also seen. Importantly, immune PD changes in the PB were seen in most pts and there were no immune PD differences identified between the two patient subsets defined in the biopsy analysis. Conclusion: PD biomarker data from this phase 1/2 trial in pts with advanced solid tumors demonstrated that moda enhances innate and adaptive immune activation in PB and within tumors. The presence of a differential intratumoral PD response in a subset of pts with elevated CD38+ cells highlights that tumor intrinsic factors may play a role in driving moda’s activity in tumors. Citation Format: Gurpanna Saggu, Janet L. Markman, Min Young Lee, Faith Dunbar, Adarsh Joshi, Shining Wang, Sabrina Collins. Modakafusp alfa shows intratumoral immune pharmacodynamics (PD) in a subset of patients (pts) in a phase 1b study in advanced/metastatic solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT065.

Abstract LB070: In vitro modeling of antigen-driven exhaustion in human CAR T cells

Abstract A major hurdle for chimeric antigen receptor (CAR) T cells to function in solid tumors is chronic antigen exposure leading to terminal lymphocyte exhaustion and impaired anti-tumor activity. Therefore, it is necessary to establish improved in vitro models of antigen-driven CAR T cell exhaustion to evaluate new CAR design candidates prior to validation with costly in vivo studies. Using the human tumor antigen mesothelin (MSLN) and MSLN-targeting CAR T cells as a model, we developed two in vitro assays that drive an exhausted phenotype in human CAR T cells and can be used to predict anti-tumor responses in vivo: 1) a chronic stimulation assay with plate-bound recombinant antigen (MSLN), ICAM-1, and CD58 and 2) a high antigen-expressing 3D tumor spheroid co-culture assay. In the chronic antigen stimulation assay, repetitive exposure to plate-bound MSLN (alongside co-stimulation signals provided by recombinant ICAM-1 and CD58) reprogrammed the transcriptomic profile of MSLN CAR T cells leading to increased surface expression of inhibitory checkpoint molecules and reduced cytokine production. Moreover, chronically stimulated MSLN CAR T cells demonstrated a drastically impaired ability to kill MSLN+ tumor cells compared to their unstimulated or acutely stimulated counterparts. A similar exhausted phenotype was observed in the high-antigen tumor spheroid model: when MSLN CAR T cells were co-cultured with MSLN-high human NCI-H226 spheroids for 7 days in a 96-well plate, expression of inhibitory checkpoint molecules markedly increased on the surface of CAR T cells. This increased expression of exhaustion markers coincided with impairment of NCI-H226 spheroid clearance. By contrast, other tumor spheroid models expressing low to intermediate levels of MSLN (NCI-H1650, NCI-H2052, NCI-H292, and SKOV-3 tumor cells) did not substantially induce expression of inhibitory checkpoint molecules and the spheroids were successfully cleared by MSLN CAR T cells. Importantly, the ability (or inability) of a CAR construct to clear NCI-H226 high-antigen tumor spheroids in vitro accurately predicted CAR T cell performance in in vivo solid tumor studies: the CAR construct that showed effective killing in the in vitro spheroid model was the only condition to provide durable regression of NCI-H1650 tumors in a NSG xenograft mouse model. Additionally, the rank order of CAR efficacy for a series of MSLN CAR binders obtained using the tumor spheroid model mirrored in vivo efficacy. As the CAR T cell field develops novel strategies to combat antigen-driven exhaustion for use in solid tumors, these new antigen-dependent in vitro models of exhaustion can enable more cost-efficient and effective evaluation of those strategies than using in vivo models alone. Citation Format: Maria Steele, Tina Tan, Jason Yokoyama, Allan Wang, Ty Crowl, Jared Hammer, Jerry Chen, Kevin Haworth, Howell Moffett, Aaron Foster, Marc Lajoie, Scott Boyken, Rupesh Amin. In vitro modeling of antigen-driven exhaustion in human CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB070.

153 Spatial Transcriptomic Profiling of T Cell Exhaustion Markers in Atypical Teratoid Rhabdoid Tumor Subtypes

INTRODUCTION: Atypical teratoid/rhabdoid tumor (ATRT) is one of the most common brain tumors in pediatric populations. Despite advances in treatment, overall survival remains poor and novel effective therapies are needed. METHODS: Tumor purities of 49 atypical ATRT tumors, encompassing 3 epigenetic subtypes (MYC, SHH, TYR) were estimated using known mRNA expression signatures of stromal and immune cells. Using the purity estimates and bulk tumor expression values, non-negative linear regression was implemented to estimate the average expression of each gene in the stromal and tumor compartments for each subtype. RESULTS: Compared to other subtypes, SHH-ATRTs expressed relatively higher levels of T-cell immunoglobulin domain and mucin domain protein 3 (TIM-3) and its ligand GAL9. MYC-ATRTs expressed higher levels of numerous T cell inhibitory receptors including lymphocyte activation gene 3 protein (LAG-3), band T lymphocyte attenuator (BTLA), cytotoxic T lymphocyte antigen-4 (CTLA-4), and programmed cell death protein 1 (PD-1). CONCLUSIONS: The predicted stromal and tumor compartment deconvolution yielded previously unrecognized expression patterns of T cell exhaustion markers which should be evaluated further to develop effective targeted therapies for ATRT.

Spectral Flow Cytometry Methods and Pipelines for Comprehensive Immunoprofiling of Human Peripheral Blood and Bone Marrow.

This study introduces optimized methods and analysis algorithms that enhance capabilities in comprehensive immunophenotyping of human blood and bone marrow using spectral flow cytometry. This approach facilitates detection of rare cell types, enables measurement of cell variations across donors, and provides proof-of-concept in identifying known hematologic malignancies. By unlocking complexities of hematopoietic and immune landscapes at the single-cell level, this advancement holds potential for understanding disease states and therapeutic responses.

Abstract 1365: INB03: a new immune checkpoint inhibitor that reprograms macrophage polarization, boosts ADCP and reverts T-cell exhaustion markers

Abstract Trastuzumab resistance is an important clinical issue in HER2+ breast cancer (BC), and few actionable targets are available. We demonstrated that soluble TNF (sTNF) upregulates mucin 4 (MUC4) expression, which shields trastuzumab epitope on HER2 hindering its therapeutic effect, and that sTNF blockade with INB03 decreases MUC4 expression which, together with trastuzumab, triggers an effective antitumor immune response based on antitumoral macrophage-NK cell collaboration. Because immune checkpoint molecules (ICP) and T cell exhaustion foster tumor immune escape, we addressed whether INB03 could modulate macrophage polarization, T cell exhaustion, and ICP expression in both populations to boost an antitumor immune response. For in vitro assays, human monocytes and T cells were isolated from buffy coats and enriched with specific RosetteSep. Macrophages (Mϕ) were differentiated for 6 days with M-CSF (10 ng/ml) to M0, and polarized for 48h to the M1-like subtype with LPS (50 ng/ml)+IFNγ (20 ng/ml), or to the M2-like subtype with IL-4 (20 ng/ml)+IL-10 (20 ng/ml). Then, INB03 was added (10 μg/ml) for 96h with corresponding cytokines to already-polarized Mϕ to test its ability to revert polarization. Also, Mϕ antibody-dependent cellular phagocytosis (ADCP) against human HER2+ breast cancer cell line JIMT-1 was assessed. T cells were activated with CD3/CD28 beads and cultured for 7 or 14 days. INB03 was added at those times for 48h to test its ability to modulate ICP and exhaustion markers. For in vivo assays, tumor-free mice or bearing the HER2+ C4HD tumor were treated with vehicle or INB03 (10 mg/kg, i.p.) for 21 days. Polarization, exhaustion and ICP markers were studied in Mϕ and T cells from spleen and tumor. Adding INB03 to already-polarized human Mϕ enhanced polarization towards M1-like (CD86+CD206−). Polarized Mϕ exposed to INB03 showed decreased PD1 and PDL-1 expression. Also, the expression of ADCP-inhibitory markers B7H4 and SIRPα was diminished when INB03 was present. These Mϕ exhibited increased ADCP against JIMT-1 cells treated with INB03, which showed downregulation of the inhibitory signal CD47. Murine Mϕ from tumor-bearing mice treated with INB03 showed increased polarization to the M1-like phenotype. Human CD8+ T cells showed decreased expression of PD1, PD-L1, LAG3, TIM3 and TIGIT. However, only TIM3 and PD1 were downregulated in murine CD8+ T cells of tumor-bearing mice treated with INB03. In all, sTNF blockade skews Mϕ to the M1-like phenotype and reprograms already-polarized pro-tumoral M2-like Mϕ to antitumoral ones. INB03 can tailor the tumor microenvironment by promoting Mϕ ADCP against tumor cells and by acting as an ICP inhibitor for CD8+ T cells, possibly relieving their exhaustion. HER2+MUC4+ BC patients could benefit from the administration of INB03 to boost targeted therapy efficacy and overcome tumor-induced macrophage and T-cell immune escape. Citation Format: Sofia Bruni, Maria F. Mercogliano, Roxana Schillaci. INB03: a new immune checkpoint inhibitor that reprograms macrophage polarization, boosts ADCP and reverts T-cell exhaustion markers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1365.

Abstract 6808: Investigating the role of CXCR3+ monocytes on CAR T cell function

Abstract Chimeric antigen receptor T cells (CAR-Ts) are an emerging immunotherapy that have remarkable efficacy against leukemias and lymphomas but limited success against solid tumors. We conducted a phase I clinical trial (NCT 02107963) of GD2 CAR-T (GD2-CAR.OX40.28.z.ICD9) administration to children and young adults with neuroblastoma and, for the first time, osteosarcoma. While all patients progressed and were switched to alternative therapy, we observed a significant association between good CAR-T expanders and expression of C-X-C Motif Chemokine receptor 3 (CXCR3) on monocytes detected in pretreatment apheresis. This study seeks to uncover the possible interactions between CXCR3 and CAR-Ts that may alter CAR-T function and ultimately lead to better CAR-T expansion. We hypothesize that CXCR3+ monocytes induce functional change in CAR-Ts resulting in improved expansion. To recapitulate the interaction between tumor, CAR-Ts and monocytes, we took 143Bs (osteosarcoma cell line) and GD2 CAR-Ts and co cultured them with THP-1s (a monocytic leukemia cell line) that were either untransduced (UTD) or transduced via lentivirus to express CXCR3 (CXCR3+). The ratio of UTD or CXCR3+ THP-1s to CAR-Ts and 143Bs was varied between co cultures. Enzyme-linked immunoassay (ELISA) for interferon gamma (IFNy) expression was run as a proxy measurement for CAR-T cell activity. Flow cytometry was used to assess for expression of activation and exhaustion markers. Differences between UTD and CXCR3+ co cultures were evaluated for statistical significance via unpaired t-test. We observed that CXCR3 was elevated on GD2 CAR-Ts co cultured with CXCR3+ monocytes. This pattern was seen most prominently in CD4+ CAR-Ts, but also subtly in CD8+ T-cells. Exhaustion markers CD39 and LAG3 were elevated in CAR-Ts co cultured with CXCR3+ monocytes. Additionally, CAR-Ts co cultured with CXCR3+ monocytes displayed greater activity via elevated IFNy expression. We conclude that GD-2 CAR-T cells co cultured with CXCR3+ monocytes express elevated exhaustion markers due to possible increased activity when in the presence of CXCR3+ monocytes. Additionally, we suspect that elevated CXCR3 expression on CAR-Ts co cultured with CXCR3+ monocytes may be helping CAR-Ts better target tumor cells, thus leading to improved expansion. If CXCR3 can improve CAR-T cell function, this could lead to better optimization of CAR-Ts against solid tumors and create a viable treatment option for neuroblastoma and osteosarcoma patients. Citation Format: Claire Victoria Ong, Sabina Kaczanowska, Wei Ju, James Cronk, Sneha Ramakrishna, Rosandra Kaplan. Investigating the role of CXCR3+ monocytes on CAR T cell function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6808.

Abstract 4273: Genetically engineered mesenchymal stromal cells degrade hyaluronic acid to disrupt the ECM and enhance cancer therapy outcomes

Abstract The extracellular matrix (ECM) is a vital component of cancer pathophysiology. However, little is known about how to modulate the ECM for therapeutic benefit effectively. Hyaluronic acid (HA) is a polysaccharide component of the ECM with increased deposition during tissue injury or inflammation. HA in the tumor microenvironment (TME) has been linked to aggressive cancer progression. We have found enhanced ECM remodeling behavior linked to pre-metastatic niche formation and metastatic progression. To remodel this ECM-enriched microenvironment, we developed HA-targeting genetically engineered mesenchymal stromal cells (GEMesys) expressing hyaluronidase (Hyal), the enzyme responsible for HA degradation. We examined the impact of Hyal-GEMesys in osteosarcoma (F42010), rhabdomyosarcoma (M-3-9M), and pancreatic cancer (Panc02) syngeneic murine models. Hyal-GEMesys delivered to tumor-bearing mice homed to primary tumor sites and successfully degraded HA in the TME. The degradation of HA also resulted in a significant remodeling of collagen. T cells in Hyal-GEMesy treated tumors display an activated CD4+CD44+ phenotype with a reduction of LAG3+PD1+ exhaustion markers. Additionally, Hyal-GEMesy treatment results in a significant decrease in hypoxic regions within tumors and reorganization of the tumor vasculature to a more normalized phenotype, marked by diminished CD31 expression. Hyal-GEMesy treatment reduces tumor volumes in osteosarcoma, rhabdomyosarcoma, and pancreatic cancer models and enhances chemotherapeutic efficacy. These findings suggest that reorganizing the tumor ECM with Hyal-GEMesys holds the potential to dismantle the protective stroma of the tumor, which often shields malignant cells from therapeutic reach, and combining anti-cancer and anti-stroma agents can enhance anti-tumor efficacy and patient outcomes. Citation Format: Alice T. Browne, Miranda Clements, Wei Ju, Rosandra Kaplan. Genetically engineered mesenchymal stromal cells degrade hyaluronic acid to disrupt the ECM and enhance cancer therapy outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4273.

Abstract 5075: Transcriptomic landscape of tumor microenvironment in gastric cancer in treatment with different types of cancer drugs

Abstract Gastric cancer (GC) in the fifth-leading types of cancer and the third-leading cause of death from cancer. Characterization of tumor microenvironment (TME) in GC is important not only to comprehensively understand tumor biology in GC but also to develop a novel cancer therapeutics targeting TME. In this study, we conducted multi-omics analyses of single-cell RNA sequencing (scRNA-seq), bulk RNA sequencing (bulk RNA-seq), and whole exome sequencing (WES) using biopsied tumor samples through endoscopy from 23 GC patients: 3 treatment-naïve patients, 7 patients in treatment with chemotherapy (Chemo), 5 patients in combination treatment with chemotherapy and immune checkpoint blockade (Chemo+CPI), 3 patients in combination treatment with chemotherapy and Her2-antibody targeted therapy (Chemo+Her2i), and 5 patients in third-line or later treatment. We initially classified the GC samples into 4 molecular subtypes (CIN, GS, MSI, and EBV) using datasets of WES, bulk RNA-seq and scRNA-seq, showing that our samples were mainly composed of CIN (n=7) and GS (n=16) types. In scRNA-seq, we revealed that drug treatments profoundly modulated not only proportions of TME components but also their molecular characteristics. For instance, Chemo+CPI treated patients showed higher proportion of CD8+ cytotoxic T cells with activation and/or exhaustion markers. Combination treatments with Chemo+Her2i, in addition, resulted in higher Trail and Estrogen signaling pathways in epithelial cells of TME components. These findings suggest that interventions with certain cancer therapeutic drugs could substantially convert transcriptome landscape and reconstitute GC TME components. High-resolution of GC TME analyses should provide a clue for developing a next generation of cancer therapeutic drugs. Citation Format: Hiroki Yamashita, Jason Guoqiang Zhang, Chiaki Mashima, Ryo Kamata, Tomoko Yamamori, Izuma Nakayama, Akihito Kawazoe, Kohei Shitara, Tomonori Yano, Qunli Xu, Akihiro Ohashi, Takeshi Saito. Transcriptomic landscape of tumor microenvironment in gastric cancer in treatment with different types of cancer drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5075.

Abstract 62: Intra-tumor persistence of activated anti-mesothelin hYP218 CAR T cells is associated with increased efficacy in gastric and colorectal cancers

Abstract Purpose: Patients with gastric and colorectal cancers that have progressed after standard therapies have poor prognosis. Given the high expression of mesothelin in these tumor types, we examined the potential utility of the anti-mesothelin hYP218 CAR T cells to treat these cancers. Experimental Design: Cell surface mesothelin expression in a panel of gastric and colorectal cancer cell-lines was analyzed using flow cytometry. In vitro efficacy of hYP218 CAR T cells against these cell lines was evaluated using cytotoxicity and cytokine release assays. In vivo efficacy of hYP218 CAR T cells was studied using gastric (HGC27) and colorectal (SW48) tumor xenografts in NSG mice. NSG mice were injected with 1 × 106 cancer cells and the tumors were allowed to grow until they reached a volume of 80-100mm³, after which the mice were treated with either 5 × 106 hYP218 CAR T cells, 5 × 106 untransduced T cells, or saline and monitored over time. Persistence of CAR T cells in the tumor was evaluated over time and expression of activation and exhaustion markers were studied. Results: hYP218 CAR T cells demonstrated strong cytotoxic activity against mesothelin positive gastric and colorectal cancer cell lines and exhibited increased pro-inflammatory cytokine production. In both the HGC27 and SW48 tumor models hYP218 CAR T cells demonstrated significant reduction in tumor volume and overall survival compared to mice treated with untransduced T cells or saline. CAR T cells isolated from tumors of both the xenograft models, at day 40 after treatment, displayed higher expression levels of activation markers CD39 and CD69 when compared to the infused product. Furthermore, a concomitant elevation in the levels of effector cytokines, TNF-α and IFN-γ, was detected in the blood of treated mice demonstrating the functional capacity of the CAR T cells to not only persist but also to maintain an activated state. Conclusions: hYP218 CAR T cells show anti-tumor efficacy against gastric and colorectal tumor xenografts due to increased accumulation of activated CAR T cells in the tumor and warrants further investigation for treatment of patients with these cancers. Citation Format: Sameer A. Mir, Abhilash Venugopalan, Jingli Zhang, Mana Kamana Khanal, Chaido Stathopoulou, Manjistha Sengupta, Qun Jiang, Raffit Hassan. Intra-tumor persistence of activated anti-mesothelin hYP218 CAR T cells is associated with increased efficacy in gastric and colorectal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 62.

Abstract 6877: Progesterone promotes immunosuppression in the murine mammary gland by causing T cell death

Abstract Breast Cancer is the most common cancer diagnosed in women in the United States. Second only to lung cancer, breast cancer results in the greatest number of cancer deaths for women. Hormone receptor positive (HR+) breast cancers - cancers expressing the nuclear receptors for steroid hormones estrogen and progesterone, termed the estrogen receptor (ER) and progesterone receptor (PR) - make up the largest proportion of diagnosed breast cancers by a significant margin. So, although the HR+ subtype is less aggressive compared to others, it continues to be the largest contributor to breast cancer mortality. Treatment consists of anti-endocrine therapies, largely targeting ER/estrogen, the established drivers of breast cancer growth. Most women respond well to these therapies, but for women who do not, there are few alternative options. With the advent of immunotherapy and its successes in treating and controlling certain tumor types, many were hopeful that it could be employed for breast cancer as well. However, breast cancers, especially HR+ breast cancers, are considered “immune cold.” They lack high proportions of tumor-infiltrating lymphocytes (TILs), have low neoantigen production, and high percentages of immunosuppressive cells. A contributing factor could be the hormones present in the tumor microenvironment. The accepted paradigm is that lifetime exposure to estrogens drives the development of HR+ breast cancer, but both estrogen and progesterone are part of the normal menstrual cycle. Prior work from our lab has uncovered a novel immune-modulatory role for progesterone, pivotal to promoting the growth of PR-positive mammary tumors. Recent preliminary data from our lab show that progesterone treatment conveys immune protection against T cell killing in a co-culture with E0771 mammary gland tumor cells. However, when activated in monoculture, T cells exposed to increasing concentrations of progesterone have no differing expression of cell surface activation or exhaustion markers, implying that progesterone is not eliciting this effect by interfering with the T cell activation cascade nor is it inducing premature exhaustion. Interestingly, progesterone treatment instead leads to a concentration-dependent increase in T cell death compared to the untreated control. In vivo, these results are supported as progesterone treatment decreases the amount of total CD3+ T cells, and specifically the cytotoxic CD8+ subset, in the mammary glands of mice. These results suggest that progesterone could modulate the breast and tumor microenvironment, providing tumor cells with protection from immunosurveillance and contributing to HR+ breast cancer development. These data also argue for the use of anti-progestins to be added to the HR+ breast cancer treatment regimen to boost anti-tumor immune activity. Citation Format: Amanda Heard, Lauryn R. Werner, Eilidh I. Chowanec, Harmony Saunders, Julio Tinoco, Zachary C. Hartman, Christy R. Hagan. Progesterone promotes immunosuppression in the murine mammary gland by causing T cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6877.

Abstract 3862: Identification, profiling and clonotypic tracking of tumor-reactive T cells derived from bronchoalveolar lavage in lung cancer patients

Abstract Immunotherapeutic strategies for disinhibiting T cell responses have revolutionized lung cancer treatment. However, the identification of tumor-reactive T cells and the acquisition of adequate tissue for molecular and immune profiling remains a challenge that hinders our ability to adequately characterize the mechanisms of immunotherapy response versus resistance. To address this gap, we are investigating the use of bronchoalveolar lavage (BAL) as an alternative to blood or tumor biopsy samples to identify tumor reactive T cells. We collected cells from a prospective bronchoscopic biorepository of patients undergoing lung cancer evaluation that were subjected to bilateral BAL, phlebotomy, and tissue sampling. We then employed flow cytometry and single-cell multi-omic analyses to identify, characterize and clonotypically track tumor-reactive T cells obtained from BAL, blood and tumor samples from lung cancer patients. Analysis of BAL samples revealed a significant enrichment of terminally exhausted CD8+CD39+CD103+ T cells in BAL from tumor-bearing lungs compared to non-tumor bearing lung BAL and blood PBMCs in 14 patients. We then used single-cell sequencing-based multi-omics to deeply profile bilateral BAL cells, blood PBMCs and tumor tissues from 5 of these patients. By tracking T cell receptor (TCR) sequences across patient-matched samples, we found BAL derived T cell clonotypes that expressed markers of terminal exhaustion, such as CD39 and CXCL13 in the tumor, which strongly implicates them as being tumor reactive and relatively abundant in BAL. Further validation through functional assays is being used to confirm the tumor reactivity of these clones. These data demonstrate the feasibility of capturing tumor-specific T cells during bronchoscopy, providing promising opportunities for patient outcome characterization and exploration of novel therapies. This relatively non-invasive and practical test during routine bronchoscopy offers a cost-effective and safe alternative to identifying tumor-reactive T cells from surgical specimens or biopsies. Citation Format: Heeju Ryu, Wei Sun, Michelle A. Wurscher, Viswam S. Nair, Evan W. Newell. Identification, profiling and clonotypic tracking of tumor-reactive T cells derived from bronchoalveolar lavage in lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3862.

Abstract 9: AFNT-212: A TRAC-knocked-in KRASG12D-specific TCR-T cell product enhanced with CD8αβ and a chimeric cytokine receptor for treatment of solid cancers

Abstract The KRASG12D mutation is an ideal target for anti-cancer therapies as its expression is typically clonal, restricted to cancer tissue, and is among the most common oncogenic drivers in solid tumors. TCR-T cell therapies have demonstrated clinical activity in some solid cancers but have been limited by heterogeneous antigen expression and unfavorable tumor microenvironments. By targeting the KRASG12D mutation for which the cancer has established genetic dependency, AFNT-212 is designed to selectively target all cancer cells while avoiding on-target/off-tumor toxicities. AFNT-212 is non-virally engineered to knock-in a 5-transgene cassette expressing a high-avidity TCR specific for the KRASG12D mutation, a CD8α/β coreceptor, and a chimeric cytokine receptor. Transgene insertion at the TRAC locus disrupts expression of the endogenous TCRα, further enhancing the expression/activity of the transgenic KRASG12D TCR. Primary human CD8+ and CD4+ T cells were genetically engineered by a novel CRISPR-Cas nuclease system to integrate AFNT-212 transgenes within the TRAC locus. A cGMP compatible scale-up process for non-viral knock-in was established to support AFNT-212 clinical manufacturing. The activity of AFNT-212 was assessed against a panel of human KRASG12D tumor cell lines in vitro and established mouse xenograft models in vivo. The preclinical safety profile of AFNT-212 was evaluated by X-scan and crossreactivity assessment, alloreactivity studies, and cytokine independent growth studies. The specificity of gene-editing (GE) was assessed by an unbiased oligo-capture method followed by targeted sequencing. AFNT-212 TCR-T cells demonstrated potent in vitro anti-tumor activity against endogenously expressing HLA-A*11:01 KRASG12D tumor cells, including during chronic exposure to viable tumor cells. AFNT-212 TCR-T cells showed robust antitumor activity in established xenograft mouse models in vivo. No cross-reactivity was identified for the KRASG12D TCR against potential self-peptides even at supraphysiological levels, demonstrating high specificity of the TCR. No alloreactivity or cytokine-independent proliferation was observed. GE safety evaluations did not reveal any off-target activity using high sensitivity (~0.1%) NGS-based analyses or any GE-associated chromosomal rearrangements. The manufacturing of AFNT-212 consistently delivered >50-fold expansion of engineered TCR-T cells to meet expected clinical dose levels and exhibit memory/stemness phenotypes and negligible markers of immunologic exhaustion. AFNT-212, a novel TCR T cell therapy targeting KRASG12D mutant tumors, demonstrates robust activity against KRASG12D mutant tumors in vitro and in vivo. The robust manufacturing process developed using non-viral gene editing in the TRAC locus will support future clinical development of AFNT-212. Citation Format: Allison Drain, Nicholas Rouillard, Nathaniel Swanson, Martina Canestraro, Santosh Narayan, Tyler Warner, Nicole Danek, Ken Gareau, Jinsheng Liang, Luhua Shen, Tanya Tetrault, Iqraa Priyata, Sarah Vidyasagar, Taylor Riggins-Walker, Hui-Wen Liu, Klaus Pechhold, Lauren Brown, Joshua Francis, Xingyue He, Patrick Browne, Rebecca Lamothe, Meghan Storlie, Gregory Cost, Thomas M. Schmitt, Philip D. Greenberg, Smita S. Chandran, Christopher A. Klebanoff, Hubert Lam, Ankit Gupta, Damien Hallet, Gary Shapiro, Kim Nguyen, Loïc Vincent. AFNT-212: A TRAC-knocked-in KRASG12D-specific TCR-T cell product enhanced with CD8αβ and a chimeric cytokine receptor for treatment of solid cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 9.

Abstract 3984: Full-length TCR and BCR repertoire analysis, integrated with mRNA and surface proteins characterization, in a tumor mouse model

Abstract Full-length TCR and BCR analysis with single-cell multiomic assays provides an understanding of immune responses, offering insights into the complete receptor structure and functionality that is crucial for accurately assessing the diversity and specificity of immune repertoires. Studying mouse immune cell heterogeneity can be achieved by simultaneous mRNA and surface protein profiling. However, when coupled with full-length TCR and BCR analysis, this integrated approach provides a more comprehensive understanding of immune responses and cellular function in healthy and diseased conditions. In this study, we used the MC38 colon carcinoma model to study T and B cell heterogeneity and clonotype diversity. We stained single-cell suspensions prepared from bone marrow, thymus, spleen and tumor tissues with 20 AbSeq (CITE-Seq) antibody oligos and sample-multiplexing tags. We then loaded 50,000 isolated T and B cells into two lanes of an 8-lane microwell cartridge for single-cell capture by a single-cell analysis system. To aid in the simultaneous analysis of clonality and gene expression, we carried out library preparation using a full-length TCR/BCR multiomic assay and a mouse immune response panel. The combination of mRNA and protein expression confirmed the elevated expression of exhaustion and activation markers in tumor-infiltrating lymphocytes (TILs). We identified high-frequency clonotypes within TILs and tracked their frequency across other tissues. Tumor-burdened mice splenic T cells showed shared TCR combinations with TILs, which were absent in healthy tissue. The results show the ability to successfully profile T and B cell receptors using a full-length TCR/BCR multiomic assay in a mouse tumor model and the use of transcriptome, protein and TCR/BCR clonal information to investigate immune cell characteristics across multiple samples. For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, the BD Logo and BD Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2023 BD. All rights reserved. NPM-2536 (v1.0) 1023 Citation Format: Ali Rezvan, Xiaoshan Shi, Moen Sen, Stephanie Widmann, Aaron Tyznik. Full-length TCR and BCR repertoire analysis, integrated with mRNA and surface proteins characterization, in a tumor mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3984.

Abstract 6705: A novel tri-specific T cell engager targeting the intracellular oncoprotein WT1

Abstract Background: Wilms' tumor gene 1 (WT1) is considered an oncogenic factor, with high expression observed in several hematological malignancies and solid tumors. However, its expression is rarely found in normal adult tissue. WT1 can be degraded by the proteasome, processed and presented on the cell surface as major histocompatibility complex (MHC) I epitopes and recognized by T cell receptors (TCR). Here, we described the development of WT1-TOPAbody, a novel tri-specific T cell engager (TCE), that activated T cells by both CD3 and 4-1BB signaling upon recognition of WT1 in the context of HLA-A02, leading to superior anti-tumor efficacy. Materials and Methods: WT1-TOPAbody was evaluated for its antigen binding activity and target specificity by a series protein/peptide and cell-based assays. In vitro cytotoxicity and T cell activation mediated by WT1-TOPAbody was further assessed in a panel of WT1+ tumor cell lines in coculture with effector cells. Anti-tumor activity was investigated in a humanized syngeneic mice model bearing WT1/HLA-A02 expressing tumors. Safety assessment including cytokine release assay (CRA) and preliminary CMC developability was also performed. Results: WT1-TOPAbody exhibited superior binding affinity to the WT1/HLA-A02 complex while displaying lower binding to off-target peptides and CD34+ HSC from healthy donor than clinical benchmark WT1-CD3 bispecific antibody, RG-6007. WT1-TOPAbody effectively induced tumor lysis in multiple WT1+ tumors including leukemia, ovarian, breast, and colon cancer cells. Moreover, serial killing of tumor cells were observed in WT1-TOPAbody treated T cells, accompanied by lower exhaustion markers expressed, suggesting a prolonged anti-tumor inhibition effect of WT1-TOPAbody. In a humanized CD3e/4-1BB/HLA-A2.1 mouse model, WT1-TOPAbody significantly inhibited tumor growth in a dose-dependent manner and the efficacy was superior than that of the benchmark. Additionally, IL-6 release was minimal in PBMC upon treatment of the antibody, indicating a reduced risk of inducing cytokine release syndrome (CRS). Drug developability assessment indicated that the leading WT1-TOPAbody was a reasonably developable molecule to be taken forward. Conclusion: WT1-TOPAbody is a differentiated WT1/HLA-A02 targeted T cell engagers that displayed potent and durable anti-tumor activity. These results collectively support the potential of WT1-TOPAbody as a novel therapeutic agent against WT1/HLA-A02+ cancers and warrant its further development. Citation Format: Shan Gao, Liu Yang, Jun Du, Wenqing Jiang, Lei Fang. A novel tri-specific T cell engager targeting the intracellular oncoprotein WT1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6705.