Amissah, Obed Boadi

Background: The dysregulation of the CDK1/Cyclin B1 complex drives tumor progression in breast cancer (BC). The natural flavonoid luteolin (LT) shows anti-cancer potential, but its mechanism targeting CDK1/CCNB1 remains unclear. Methods: CDK1, CCNB1, and CCNB2 expression were profiled in normal and BC cell lines. An engineered HEK293T GST-CDK1/CCNB1 cell model was used to evaluate LT’s effects on proliferation, ROS levels, and target gene transcription. Computational approaches (molecular docking, dynamics simulations, pharmacophore modeling, MM/GBSA, ADMET, and network pharmacology) assessed LT and its analogues. Results: CDK1/CCNB1 expression was lower in MCF7 BC cells than in normal cells, suggesting the loss of a growth barrier. In engineered HEK293T cells, LT suppressed CCNB1 transcription with minimal effect on CDK1 levels, correlating with anti-proliferative and ROS-modulating effects. Computational analyses confirmed stable LT binding to the CDK1/CCNB1 complex. Designed LT analogues showed improved binding and favorable ADMET profiles. Network pharmacology identified cell cycle regulation, particularly in BC stem cells, as the primary pathway targeted. Conclusions: LT and its analogues inhibit the CDK1/Cyclin B1 complex, revealing a dual mechanism that suppresses both tumor growth and BC stemness.

Abstract Background: Colorectal cancer (CRC) remains a leading cause of cancer-associated mortality and is difficult to treat due to therapy resistance and limited durable responses. Esophageal cancer (EC) is similarly challenging, exhibiting highly aggressive and metastatic behavior that contribute to its poor prognosis. Adoptive TCR-engineered T cell (TCR-T) therapies show promise in these settings, but their efficacy is limited by suboptimal antigen specificity and an immunosuppressive tumor microenvironment (TME). To address these barriers, we isolated high-affinity TCRs from a phage-displayed TCR library targeting KRASG12V neoantigen in CRC and CTAG1B cancer/testis antigen (CTA) in EC. Methods: KRASG12V(8-16)/HLA-A11:01-restricted and CTAG1B(157-165)/HLA-A02:01-restricted TCRs were isolated from an optimized phage-displayed TCR library generated from healthy donor CD8+ T cells through high-throughput biopanning. Binders were screened against antigen-family peptides and irrelevant controls by phage ELISA to eliminate cross-reactivity. Binding affinities were measured by SPR. Antigen-specific TCRs (asTCRs) were cloned into lentiviral vectors and transduced into primary human T cells. TCR-T function was assessed using peptide-pulsed T2 cells and KRASG12V+ or CTAG1B+ tumor cells through LDH-release cytotoxicity and cytokine secretion assays. Normal human cells were used to evaluate off-target toxicity. In vivo antitumor activity was tested in NOD/SCID-IL2rγ-/- xenografts with histological confirmation of TCR-T infiltration. Results: Next-generation sequencing confirmed that the 109-scale library contained >90% functional genes and >80% sequence diversity. Three high-affinity TCRs were identified for each antigen (KD as low as 1.07 μM). These TCRs exhibited strict antigen specificity with no detectable cross-reactivity. TCR-T cells expressing asTCRs showed potent antigen-dependent cytotoxicity against KRASG12V+/HLA-A11:01+ colorectal and CTAG1B+/HLA-A02:01+ esophageal tumor cells (up to 80% lysis, p<0.01), accompanied by robust IFN-γ and TNF-α secretion (>1000 pg/mL, p<0.01). No killing was observed against any of seven healthy cell types. TCR-T cells remained effective at low effector-to-target ratios and maintained stable proliferation and effector-memory phenotypes. In xenograft models, KRAS- and CTAG1B-targeted TCR-T cells induced significant tumor regression compared to unmodified T cell controls (p<0.05), with increased effector TCR-T infiltration, and reduced antigen escape. Conclusions: This work establishes a rapid and robust platform for isolating potent, safe, antigen-specific TCRs targeting both oncogenic driver mutation (KRASG12V) and an immunogenic CTA (CTAG1B). TCR-T cells demonstrated strong antitumor activity in vitro and in vivo, supporting the translational potential of this approach for solid tumor treatment. Citation Format: Obed Boadi Amissah, Wenfang Chen, Zhiyuan Li, Gloria Bora Kim. KRASG12V- and CTAG1B-targeted TCR-T cells from phage TCR library suppress colorectal and esophageal tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5618.

Abstract Background: Clear cell renal cell carcinoma (ccRCC), metastatic castration-resistant prostate cancer (mCRPC), and glioblastoma (GBM) are treatment-refractory tumors marked by high TGF-β1, a master suppressor of antitumor immunity and antigen presentation. Pharmacologic methylseleninic acid (MSA) potently inhibits TGF-β1, PD-L1, and VEGF without significant off-target toxicity. FN14, the TWEAK receptor and a metastasis driver, is selectively overexpressed in these malignancies, making it a high-specificity CAR-T target. Methods: We developed second-generation anti-FN14 CARs with CD28 or 4-1BB costimulation and CD3ζ signaling and lentivirally transduced them into human CD4+/CD8+ T cells. Cytotoxicity against FN14+ ccRCC, mCRPC, GBM lines and patient-derived tumors was measured using LDH release and xCELLigence assays. Effector function was assessed by intracellular cytokines, phenotyping, and multiplex analysis. MSA was tested at physiologic concentrations to evaluate effects on CAR-T viability, metabolism, function, and tumor susceptibility. Results: Anti-FN14 CAR-T cells mediated rapid, antigen-specific lysis across all tumor types, achieving >50% specific lysis at an E:T ratio of 1:1. MSA (2.5-5 µM) reduced tumor PD-L1 and TGF-β by >40% and improved CAR-T metabolic activity, enhancing IL-2 and IFN-γ secretion. Manufacturing CAR-T cells with MSA (2.5 µM) increased cytotoxicity and persistence versus controls (p < 0.01). MSA’s effects were dose- and schedule-dependent at clinically compatible concentrations without detectable off-target toxicity. Conclusion: MSA augments anti-FN14 CAR-T cytotoxicity, metabolic fitness, and sustained effector function by suppressing tumor-derived TGF-β1 and PD-L1 and directly enhancing CAR-T activity. As a clinically relevant TGF-β1 inhibitor and active seleno-L-methionine metabolite, MSA represents a readily translatable strategy to reduce CAR-T exhaustion and improve efficacy in TGF-β-rich solid tumors. Citation Format: Gloria B. Kim, Obed B. Amissah, Casey R. Ager, Yousef Zakharia, Youcef M. Rustum. Methylseleninic acid enhances anti-FN14 CAR-T cell effector function and redirects cytotoxicity against TGF-β-rich kidney, prostate, and brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6496.

Abstract Background: Clear cell renal cell carcinoma (ccRCC), metastatic castration-resistant prostate cancer (mCRPC), and glioblastoma (GBM) are highly treatment-refractory tumors characterized by elevated TGF-β1, a key suppressor of antitumor immunity and antigen presentation. Because CAR-T cells infiltrating these tumors experience persistent physiologic TGF-β exposure, assessing CAR-T functional resilience across increasing TGF-β concentrations is critical for determining therapeutic feasibility. Methylseleninic acid (MSA), a pharmacologic selenium compound, potently inhibits TGF-β1, PD-L1, and VEGF with minimal off-target toxicity. FN14, the TWEAK receptor and a metastasis driver, is selectively overexpressed in these malignancies, making it a high-specificity CAR target. Methods: We developed second-generation anti-FN14 CARs containing CD28 or 4-1BB costimulatory domains and a CD3ζ signaling motif, then lentivirally transduced into primary human CD4+ and CD8+ T cells. Cytotoxicity against FN14+ ccRCC, mCRPC, GBM lines and patient-derived cells was assessed using LDH release and xCELLigence assays. Effector function was evaluated through intracellular cytokine profiling, multiplex analysis, and phenotypic characterization by flow cytometry. CAR-T persistence in TGF-β-rich conditions was measured using CCK-8 proliferation assays, and Ki-67 and activation/exhaustion markers (PD-1, CXCR3) in the presence of 0-10 ng/mL TGF-β. MSA’s immunomodulatory and synergistic effects were tested at physiologically concentrations to measure impacts on CAR-T viability, metabolic activity, cytokine secretion, and tumor cell susceptibility. Results: Anti-FN14 CAR-T cells mediated rapid, antigen-specific lysis across all tumor types, achieving >50% specific killing at a 1:1 E:T ratio. MSA (2.5-5 µM) reduced tumor PD-L1 and TGF-β expression by >40% and enhanced CAR-T metabolic activity, IL-2, and IFN-γ secretion. CAR-T cells maintained comparable proliferation and activation in 0-5 ng/mL TGF-β, indicating that antigen-driven signaling counteracted low-to-moderate TGF-β-mediated suppression. Ex vivo CAR-T manufacturing with MSA (2.5 µM) improved cytotoxicity and persistence versus untreated CAR-T cells (p < 0.01). MSA’s effects were dose- and schedule-dependent and remained within human-relevant exposure ranges without detectable toxicity. Conclusion: MSA enhances anti-FN14 CAR-T cytotoxicity, metabolic fitness, and sustained effector activity by suppressing tumor-derived TGF-β1 and PD-L1 and by directly improving CAR-T functional capacity. Consistent with this mechanism, physiological TGF-β levels (0-5 ng/mL) did not significantly impair CAR-T proliferation or activation, demonstrating intrinsic resilience of FN14 CAR-T cells in TGF-β-rich environments. As a clinically relevant TGF-β1 inhibitor and active metabolite of seleno-L-methionine, MSA represents a readily translatable strategy to reduce CAR-T exhaustion and enhance efficacy in TGF-β-rich solid tumors including ccRCC, mCRPC, and GBM. Citation Format: Obed B. Amissah, Casey Ager, Yousef Zakharia, Youcef M. Rustum, Gloria B. Kim. Anti-FN14 CAR-T cells overcome TGF-β-mediated suppression, and their synergy with methylseleninic acid enhances cytotoxicity and effector function in kidney, prostate, and brain tumors [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr C010.