Downregulation of DNA damage repair genes has attracted considerable research attention recently due to the success of poly(ADP-ribose) polymerase inhibitors. Identification of additional targets and therapies that exploit synthetic lethality could greatly benefit cancer patients. Cyclin-dependent kinases 12 and 13 (CDK12 and CDK13), which regulate RNA polymerase II (RNA Pol II) and, therefore, gene transcription, represented promising therapeutic targets. Although several inhibitors for these kinases have been disclosed, few have progressed to the clinic. Most existing inhibitors utilize a covalent warhead to obtain potency and selectivity. In this study, we reported the design and development of a series of highly selective noncovalent inhibitors targeting CDK12 and 13. This campaign led to the identification of a lead compound exhibiting outstanding potency and favorable absorption, distribution, metabolism, and excretion profiles, as well as favorable pharmacokinetic properties, thereby demonstrating significant potential for therapeutic applications.

Host immune responses play an important role in fighting cancers; therefore, improving these responses against tumor cells is of high interest in cancer therapy. Diacylglycerol (DAG) is a key second messenger that transduces T-cell receptor (TCR) activation signal to downstream effectors through DAG-binding proteins. DAG kinase (DGK) isoforms α and ζ are the major enzymes that modulate DAG levels in T cells and serve as intracellular checkpoints to attenuate TCR activation. Here, we describe INCB177054, a novel, potent, selective, and orally bioavailable small molecule DGKα/ζ dual inhibitor that enhances T-cell activation in vitro and in vivo and demonstrates antitumor activity in mouse tumor models. Consistent with the DGKα and DGKζ biology in regulating DAG levels in T cells, our genetic data and pharmacological inhibition studies in Jurkat T cells indicated that optimal T-cell activation requires inhibition of both DGKα and DGKζ activity. In biochemical ADP-Glo assays, INCB177054 demonstrated potent inhibition of DGKα and DGKζ with subnanomolar half-maximal inhibitory concentration and >500-fold selectivity over other DGK isoforms, such as DGKβ, DGKγ, and DGKδ. Mechanism-based biochemical studies suggest a noncompetitive mode of inhibition vs both ATP and DAG substrates. In cellular assays, INCB177054 enhanced both ERK and NFκB phosphorylation, expression of early T-cell activation marker CD69, and production of cytokines, such as IL-2 and IFNγ. In vitro, INCB177054 treatment enhanced T-cell responses to weak antigens and enhanced both allogenic T cell- and NK cell-mediated killing in coculture assays. In vivo, INCB177054 induced T-cell activation upon antigen stimulation in an OT-1 mouse model. INCB177054 monotherapy induced modest tumor growth inhibition in MC38 and CT26 syngeneic mouse models. However, when combined with PD-1 checkpoint inhibition, INCB177054 showed significant tumor growth inhibition in both models, ultimately leading to tumor rejection in most of the mice. In addition, those mice were protected against a rechallenge with the same tumor, suggesting generation of antitumor T-cell memory. Collectively, our data indicate INCB177054 is a potent and selective DGKα/ζ dual inhibitor that enhances T-cell activity and induces significant tumor growth inhibition when combined with anti-PD(L)-1, suggesting this potential therapy might increase responses to checkpoint inhibitor treatment in patients with cancer. Xiaodi Ren, Yvonne Lo, Michelle Pusey, Lisa Truong, Michelle Frascella, Rodrigo Hess, Kelly Federowicz, Joshua Hummel, Bo Wei, Gengjie Yang, Maryanne Covington, Chen Bao, Guofeng Zhang, Melissa Chan, Yu Li, Jennifer Rocha, Rina Pan, Anthony Perry, Sarah Li, Jinyuan (Jay) Zhang, Brad Yuska, David Rodrigues, Xiaozhao Wang, Jeff Jackson, Patrick Mayes, Sunkyu Kim. INCB177054: A novel, potent, orally bioavailable DGKα/ζ dual inhibitor enhances T-cell function and demonstrates potent antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3789.

Although immune checkpoint inhibitors (ICIs) targeting pathways involving programmed cell death 1 (PD-1) and its ligand (PD-L1) are promising for some diseases, they have shown modest responses in ovarian cancer patients. Multiple studies have shown that ICI resistance is associated with the prevalence of transforming growth factor (TGF)-β signaling in the tumor microenvironment (TME). In patients with ovarian cancer, TGF-β expression is associated with immunosuppressive features in the TME and poor prognosis. PD-1 and TGF-β inhibitory pathways play independent yet complementary roles in immunosuppression, providing rationale for simultaneous targeting of both pathways. However, agnostic inhibition of the TGF-β pathway has historically led to detrimental side effects. Therefore, we developed a bispecific Biclonics® antibody that conditionally blocks the TGF-β pathway through TGFβR2, only on immunosuppressed T cells that coexpress PD-1. In this study, we tested the efficacy of the dual PD-1- and TGFβR2-binding Biclonics® antibody, INCA33890, in models of ovarian cancer. We used patient ascites (N=6) as a model of ovarian TME. INCA33890 treatment resulted in significantly higher levels of secreted interferon (IFN)-γ compared with control antibodies targeting only PD-1 (pembrolizumab) or TGFβR2 (TGF-1). A significant increase in T-cell activation markers, such as CD137, CD25, and CD69, was also observed in some samples treated with INCA33890 compared with controls. Next, we tested INCA33890 in an autologous coculture of tumor-infiltrating lymphocytes expanded from an enzymatically digested-patient tumor and enriched tumor cells from the same patient. In these cocultures, IFN-γ levels were significantly higher with INCA33890 treatment compared with pembrolizumab; levels were similar compared with TGF-1. For an in vivo assessment, we used double huPD1/huTGFβR2 knock-in mice implanted with ovalbumin (ova) and luciferase-expressing murine ovarian cancer ID8 cells. Mice treated with INCA33890 showed a significant reduction in tumor burden and improved survival compared with controls. A significant increase in tumor-associated ova-specific CD8+ T cells was observed with INCA33890 treatment compared with controls. Phenotypic analysis of CD8+ T cells revealed a skew toward an effector memory subset (CD44high; CD62Llow expression) with INCA33890 treatment. Thus, INCA33890 showed favorable results in a humanized ovarian cancer model and demonstrated a tumor antigen-specific T-cell response resulting in reduced tumor burden. Altogether, we show that INCA33890 presents a promising immunotherapy option for patients with advanced ovarian cancer by targeting 2 prominent immunosuppressive pathways, leading to a coordinated immune response. Clinical assessment of INCA33890 for various advanced malignancies, including ovarian cancer, is ongoing (NCT05836324). Veethika Pandey, Kay M. Foos, Isabella Pargiolas, Mathilde Poussin, Ashwini Kulkarni, Liang-Chuan Wang, Patrick Mayes, Daniel J. Powell Jr. INCA33890, a bispecific antibody targeting PD-1 and TGFβR2, shows enhanced immune responses in models of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6074.

Anti-programmed cell death (PD)-1 and anti PD-ligand1 antibodies have produced durable clinical responses in a wide variety of solid and hematologic malignancies; however, their clinical benefits are often limited to a subset of patients. One of the immune resistance mechanisms that limits the activity of PD-1 axis pathway blockers involves activation of the transforming growth factor beta (TGFβ) pathway within the tumor microenvironment (TME). To overcome this immune suppression, a PD-1 and TGFβ receptor 2 (TGFβR2) dual targeting bispecific Biclonics® antibody, INCA33890, was developed. INCA33890 inhibits TGFβR2 preferentially on PD-1-positive cells (Wang L-C, et al. AACR 2023. Abs 2936). In this study, various in vitro and ex vivo cell-based systems were used to evaluate changes in T-cell effector function induced by INCA33890 compared with the anti-PD-1 antibodies, nivolumab or pembrolizumab. We observed that INCA33890 induced higher interferon gamma (IFNγ) levels than nivolumab with and without exogenous TGFβ in an exhausted T-cell mixed lymphocyte reaction (MLR) assay. Additionally, INCA33890 was superior to nivolumab in enhancing T-cell proliferation in the presence of TGFβ. RNAseq analysis of samples from the exhausted MLR assay revealed that, compared with nivolumab, expression of cytotoxic genes (GZMB, GNLY), proinflammatory cytokines (IFNG), and T-cell activation markers (CD25, 4-1BB) were elevated following INCA33890 treatment in the presence of TGFβ. We next co-cultured T-cell receptor-engineered T cells with non-small cell lung cancer tumor digests to examine the impact of the TME on antigen-specific T-cell responses. INCA33890 increased the percentage of IFNγ-producing T cells compared with pembrolizumab over 72 hours. In summary, our data suggest that co-inhibition of both PD-1 and TGFβR2 by INCA33890 may provide greater therapeutic benefits than an anti-PD-1 antibody alone. Jun Guan, Aidan Gilmartin, Kenji Kimura, Estela Noguera-Ortega, Ashwini Kulkarni, Pat Feldman, Michelle Kinder, Cynthia Timmers, Alejandro Amador Arjona, Patrick Mayes, Steven M. Albelda, Evgeniy Erulasnov, Liang-Chuan Wang. PD-1xTGFβR2 bispecific Biclonics® antibody INCA33890 augments human T-cell effector functions in vitro and ex vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6071.

BACKGROUND:We previously reported the identification of ONC201/TIC10, a novel small molecule inducer of the human TRAIL gene that improves efficacy-limiting properties of recombinant TRAIL and is in clinical trials in advanced cancers based on its promising safety and antitumor efficacy in several preclinical models. METHODS:We performed a high throughput luciferase reporter screen using the NCI Diversity Set II to identify TRAIL-inducing compounds. RESULTS:Small molecule-mediated induction of TRAIL reporter activity was relatively modest and the majority of the hit compounds induced low levels of TRAIL upregulation. Among the candidate TRAIL-inducing compounds, TIC9 and ONC201/TIC10 induced sustained TRAIL upregulation and apoptosis in tumor cells in vitro and in vivo. However, ONC201/TIC10 potentiated tumor cell death while sparing normal cells, unlike TIC9, and lacked genotoxicity in normal fibroblasts. Investigating the effects of TRAIL-inducing compounds on cell signaling pathways revealed that TIC9 and ONC201/TIC10, which are the most potent inducers of cell death, exclusively activate Foxo3a through inactivation of Akt/ERK to upregulate TRAIL and its pro-apoptotic death receptor DR5. CONCLUSION:These studies reveal the selective activity of ONC201/TIC10 that led to its selection as a lead compound for this novel class of antitumor agents and suggest that ONC201/TIC10 is a unique inducer of the TRAIL pathway through its concomitant regulation of the TRAIL ligand and its death receptor DR5.

Supplementary Table 1, Figures 1-6 from Combinatorial Regulation of Neuroblastoma Tumor Progression by N-Myc and Hypoxia Inducible Factor HIF-1α

In recent years, there has been considerable interest in mAb-based induction of costimulatory receptor signaling as an approach to combat cancer. However, promising nonclinical data have yet to translate to a meaningful clinical benefit. Inducible T-cell costimulator (ICOS) is a costimulatory receptor important for immune responses. Using a novel clinical-stage anti-ICOS immunoglobulin G4 mAb (feladilimab), which induces but does not deplete ICOS+ T cells and their rodent analogs, we provide an end-to-end evaluation of the antitumor potential of antibody-mediated ICOS costimulation alone and in combination with programmed cell death protein 1 (PD-1) blockade. We demonstrate, consistently, that ICOS is expressed in a range of cancers, and its induction can stimulate growth of antitumor reactive T cells. Furthermore, feladilimab, alone and with a PD-1 inhibitor, induced antitumor activity in mouse and humanized tumor models. In addition to nonclinical evaluation, we present three patient case studies from a first-time-in-human, phase I, open-label, dose-escalation and dose-expansion clinical trial (INDUCE-1; ClinicalTrials.gov: NCT02723955), evaluating feladilimab alone and in combination with pembrolizumab in patients with advanced solid tumors. Preliminary data showing clinical benefit in patients with cancer treated with feladilimab alone or in combination with pembrolizumab was reported previously; with example cases described here. Additional work is needed to further validate the translation to the clinic, which includes identifying select patient populations that will benefit from this therapeutic approach, and randomized data with survival endpoints to illustrate its potential, similar to that shown with CTLA-4 and PD-1 blocking antibodies.Significance:Stimulation of the T-cell activation marker ICOS with the anti-ICOS agonist mAb feladilimab, alone and in combination with PD-1 inhibition, induces antitumor activity across nonclinical models as well as select patients with advanced solid tumors.

Supplementary figure 1. Tumor growth kinetics in individual mice treated with trametinib (T) in combination with the immunomodulator-targeting PD1 in the CT26 murine syngeneic model.

Antibodies used for in vitro and in vivo assays, and experimental model

Expression dynamics and cellular localization of ICOS.

Clinical patient case studies of feladilimab monotherapy and in combination with pembrolizumab. A, Clinical history and treatment details for a patient with melanoma who received feladilimab monotherapy and a patient with oropharyngeal squamous cell carcinoma who received feladilimab in combination with pembrolizumab. Tumor sample immunohistochemistry and expression of markers for TIL activation, cytotoxic function and proliferation for the monotherapy case study (B) and the combination therapy case study (C). See Supplementary Fig. S11B for CT imaging of tumor lesions for the patient with oropharyngeal squamous cell carcinoma. CPS, combined positive score; CT, computed tomography; HLA-DR, human leukocyte antigen-DR; HPV, human papillomavirus; PD-L1, programmed death ligand-1; TIL, tumor-infiltrating lymphocyte.

Exemplar flow cytometry gating strategy.

Supplementary Figures 1-7, Table 1 from Overcoming Hypoxia-Induced Apoptotic Resistance through Combinatorial Inhibition of GSK-3β and CDK1

Affinity and kinetics

Transforming growth factor-β (TGFβ) signaling is common in many solid tumors and is initiated by binding of the high affinity canonical ligands TGFβ1, 2, οr 3 to TGFβR2, which forms a heteromeric receptor complex with TGFβR1 (Derynck et al, Nature Review Clinical Oncology 2020). Activation of the pathway results in potent suppression of immune cell-mediated anti-tumor immunity and has been reported to predict poor response to PD-(L)1 targeted therapy in patients (Mariathasan et al, Nature 2018; Kieffer et al, Cancer Discovery 2020). However, TGFβ drug development has been hampered by the occurrence of adverse events. INCA33890 is a dual PD-1 and TGFβR2 binding bispecific Biclonics® antibody, developed to antagonize the TGFβ signaling pathway specifically in cells co-expressing PD-1 and TGFBR2. Additionally, it potently antagonizes the PD-1 axis independently of TGFβR2 co-expression. The cell-selective action of INCA33890 was designed to mitigate risks of the known adverse effects associated with TGFβ-pathway inhibition in tissues requiring active TGFβ signaling. ΙΝCΑ33890 mediates its specificity through a PD-1 binding arm with a >10-fold higher affinity relative to the TGFβR2 binding arm. Consistent with this profile, in isogenic Jurkat cells expressing TGFβR2 ± PD-1, INCA33890 potently inhibits TGFβ1-induced pSMAD activation in a PD-1-correlated manner. Additionally, in two independent PD-1 reporter assays, INCA33890 inhibited SHP recruitment and enhanced NFAT activation with a potency within an order of magnitude to that of pembrolizumab. In mixed lymphocyte reaction assays with exhausted primary human T-cells, INCA33890 was found to induce a similar level of anti-tumor cytokine production as the combination of pembrolizumab and an anti-TGFβR2 antagonist mAb. Treatment of primary ovarian ascites with INCA33890 ex vivo induced IFNγ production in all donors tested, while pembrolizumab had no activity. Similarly, in human CD34+ cell-engrafted NSG mice, INCA33890 significantly inhibited the growth of human MDA-MB-231 and A375 subcutaneous xenograft tumors, whereas pembrolizumab or an anti-TGFβR2 antibody had little or no monotherapy activity. INCA33890 had a balanced pharmacokinetic and potency profile and was well tolerated in NHPs at exposures required for pharmacodynamic and tumor growth inhibiting activity in rodents. Encouragingly, there was no evidence of adverse effects in NHPs due to TGFβ-pathway blockade. Collectively, these results provide compelling data for an effective and specific approach to simultaneously antagonizing TGFβ and PD-1 signaling in tumors. Clinical development of INCA33890 in checkpoint inhibitor-resistant and other cancers has been initiated. Citation Format: Liang-Chuan S. Wang, Rinse Klooster, Ashwini Kulkarni, Amaya Garcia de Vinuesa, Maxim Soloviev, Linda JA Hendriks, Lu Huo, Michael Weber, Arpita Mondal, Yonghong Zhao, Shane Harvey, Xin He, Hong Chang, April Horsey, Alla Volgina, Yue Zhang, Veethika Pandey, Yan-Ou Yang, Jonathan Rios-Doria, Evgeniy Eruslanov, Daniel J. Powell, Steven M. Albelda, John de Kruif, Horacio Nastri, Cecile Geuijen, Patrick A. Mayes. INCA33890, a novel PD-1×TGFꞵR2 bispecific antibody conditionally antagonizes TGFꞵ signaling in primary immune cells co-expressing PD-1 [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 2936.

Gene expression in human T-cell

Flow cytometry gating strategy for ICOS on individual T cell populations.

Supplementary figure 2. Tumor gene expression changes measured by RT-PCR from mice treated with trametinib (T) in combination with the immunomodulator-targeting PD1 in the CT26 murine syngeneic model.

Supplementary Materials and Methods