Abstract Background: STK-012 is a first-in-class α/β-IL-2R biased partial agonist that drives antitumor activity by selectively stimulating CD25+ antigen-activated T-cells and avoids hallmark IL-2 toxicities by sparing pleiotropic activation of lymphocytes including NK cells. In this phase 1a/b study, STK-012 is combined with standard of care pembrolizumab + chemotherapy (PCT) in 1L PD-L1 negative NSQ NSCLC where PCT alone has poor outcomes (ORR 32% and median PFS 6.2 months). Methods: 1L NSQ NSCLC subjects received STK-012 SC Q3W + PCT. Serial blood samples were analyzed for changes in cytokines in chemoluminescence assays, T cell proliferation and activation markers were analyzed in spectral flow cytometry, and T cell clonality was analyzed by TCR sequencing. Data are shown for a cohort of 22 efficacy-evaluable 1L NSCLC subjects (N=18 PD-L1<1%, N=4 PD-L1=1%) which was enriched for loss-of-function tumor suppressor gene (LoF TSG) mutations (n=11; STK11, KEAP1, SMARCA4) or mucinous histology (n=5) associated with a “cold” TME and primary immune resistance. Results: STK-012 + PCT demonstrated an ORR of 55% in all efficacy evaluable patients; 50% in patients with PD-L1<1% tumors; 55% in LoF TSG mutations; and 80% in mucinous histology. STK-012 + PCT induced sustained proliferation of CD8+ and CD4+ T cells (13X and 5x increase from baseline, respectively), proliferation of antigen activated T cells including PD-1+ CD8+ (14X) and 4-1BB+ CD8+ (14X) and re-invigoration and proliferation of previously exhausted CD39+ (11x) and TIM3+ (18x) CD8+ T cells. After the first cycle, 3.35% of the total T cell repertoire were T cell clones which were newly detected or expanded >10x (n=12). STK-012 + PCT induced key cytokines IFNγ and IL-18 characterizing an activated CD8+ T cell response and increased IP-10 which is associated with enhanced T cell trafficking into tumor tissues (13x,14x and 23x mean increase from baseline, respectively). Sustained elevation of cytokines across treatment cycles (IFNγ median peak of 71, 95, 96 pg/mL in C1, C2 and C6) was observed, supportive of the emerging durability of the combination. Induction of cytokines TNFα and IL-6 was limited (median peak 4, 6 pg/mL), consistent with limited activation of naïve T cells and NK cells and the lack of capillary leak syndrome with STK-012. Conclusions: STK-012 + PCT led to robust cytokine induction, proliferation and expansion of antigen activated T cells, proliferation of previously exhausted T cells, and remodeling of the T cell repertoire. The addition of STK-012 to PCT has the potential to overcome resistance in immune excluded populations including PD-L1<1% tumors, LoF TSG mutations and mucinous histology. A global, randomized Phase 2 study, SYNERGY-101, of STK-012 + PCT vs. PCT in 1L PD-L1<1% NSQ NSCLC is ongoing (NCT05098132). Citation Format: Salman Punekar, Adam Jacob Schoenfeld, Edward B. Garon, So Yeon Kim, Kai He, Jennifer Marks, Brian S. Henick, Stephen V. Liu, Nagashree Seetharamu, Alexander Spira, Justin Gainor, Tim Larson, Ticiana A. Leal, Cameron Oswalt, Benjamin Izar, Alize Marangoz-Stager, Vic Danh, Grace Lunardi, Krystle Bach, Naiyer A. Rizvi, Alex Azrilevich, Anita Mehta-Damani, Martin Oft. Selective immune activation of antigen activated T cells with STK-012, an a/b IL-2 receptor biased partial agonist, with pembrolizumab and chemotherapy in 1L PD-L1 negative non-squamous NSCLC [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 6740.

Background: Bispecific antibodies have emerged as a promising class of therapeutics, enabling simultaneous targeting of two distinct antigens. Single-domain antibodies (sdAbs) comprising camelid variable heavy chains (VHHs) provide a compact and adaptable platform for bispecific antibody design due to their small size and ease of linkage. Methods: Here we investigate structure-activity relationship of VHH-based cytokine surrogates by combining cell signaling and functional assays with x-ray crystallography and other biophysical techniques. Results: We describe crystal structures of four unique bispecific VHHs that engage and activate the cytokine receptor pairs IL-18Rα/IL-18Rβ and IL-2Rβ/IL-2Rγ. These bispecific VHH molecules, referred to as surrogate cytokine agonists (SCAs), create unique cytokine signals that can be tuned by linker engineering. Our structural analysis reveals multiple dimeric conformations for these bispecific SCAs, where the two VHH domains can interact to form a compact structure. We demonstrate that the dimeric conformation can be enforced via engineering of a non-native disulfide bond between the VHH subunits, thus enhancing molecular thermostability. Conclusion: Our findings have important implications for the design and engineering of bispecific VHHs or sdAbs, offering a novel strategy for tuning their activity and increasing their stability.

Introduction and Objective: Interleukin-22 (IL-22) is a pleiotropic cytokine that signals through the heterodimeric IL-22Rα:IL-10Rβ receptor complex. IL-22 exerts its activity predominantly on epithelial and epithelial-like cells (e.g. skin keratinocytes, gut/lung epithelium, hepatocytes), where it can promote mucosal barrier integrity, regulate tissue remodeling, and improve metabolic disorders. However, clinical studies with IL-22-based therapeutics have reported dose-limiting dermatological adverse effects. Here we report on novel IL-22 partial agonists (IL-22 PAs) that have attenuated binding to IL-10Rβ and are designed to preferentially signal on colon but not skin tissue, relative to wild-type (WT). Selected IL-22 PAs demonstrated efficacy by improving weight and metabolic outcomes in diet-induced obese (DIO) mice while showing attenuated skin hyperplasia when compared to IL-22 WT treatment. Methods: Murine IL-22 PAs were screened and tested for efficacy in a DIO model. Effects on skin histopathology, metabolic hormones, glucose, insulin, triglycerides, and proinflammatory cytokines/chemokines were also measured. Results: DIO mice treated with IL-22 PAs showed robust weight loss, associated with increased expression of satiety hormones, and improvement of hyperleptinemia, hyperglycemia, and hyperinsulinemia. IL-22 PAs promoted hypophagia and increased energy expenditure as measured by elevated body temperature. IL-22 PA treated animals had improved glucose and insulin tolerance, reduced circulating triglycerides, and reduced liver lipid burden. Importantly, compared to IL-22 WT, treatment with IL-22 PAs avoided induction of systemic and proinflammatory cytokines and epidermal hyperplasia. Furthermore, IL-22 PAs and a GLP-1 agonist combined well to promote increased weight loss in DIO mice. Conclusion: IL-22 PAs improved body weight and metabolic syndrome in mice and avoided inducing signs of systemic and skin inflammation associated with IL-22 WT treatment. L.A. Zuniga: Employee; Synthekine, Inc. M.C. Bauer: None. M. Ali: None. H. Rosas: None. D. Chaturvedi: None. B. Jayaraman: Employee; Synthekine. D. Rokkam: None. S. Singh: None. R.A. Burgess: None. A.J. Gibbs: None. S. Vivona: None. P. Lupardus: None. M. Oft: None.

Interleukin-12 (IL-12) is a proinflammatory T cell- and natural killer (NK) cell-activating cytokine with potent preclinical anti-tumor efficacy, but clinical toxicity has limited its use as an immunotherapy. In a mouse tumor model, wild-type IL-12 induces NK cell hyperactivation and cytokine storm followed by rapid NK cell loss. To avoid NK hyperactivation, we engineered an IL-12-Fc with attenuated IL-12Rβ1 binding (STK-026) to preferentially target activated T cells expressing high levels of IL-12 receptors. STK-026 avoids NK cell hyperactivation but supports sustained T cell activation, interferon γ (IFNγ) production, and intratumoral infiltration of tumor-infiltrating lymphocytes (TILs), macrophages, and cytotoxic NK cells. STK-026 induces tumor control in immune-competent mice, with a substantial therapeutic window between efficacy and toxicity. STK-026 also controls "cold" tumors and synergizes with anti-PD-1 treatment. In non-human primates, STK-026 avoids toxicity associated with IL-12 treatment but sustains effector T cell and NK cell activation. In summary, STK-026 provides anti-tumor efficacy without acute toxicity, expanding the therapeutic index of IL-12 treatment.

Abstract Background: STK-012 is a first-in-class α/β-IL-2R biased partial agonist designed to drive antitumor activity by selectively stimulating CD25+ antigen activated T cells, while avoiding hallmark IL-2 toxicities by sparing pleotropic activation of lymphocytes including NK cells. Methods: STK-012-101 is a Phase 1a/b study of STK-012 administered subcutaneously in subjects with advanced, relapsed/refractory (r/r) solid tumors. During Phase 1a, subjects are enrolled in a 3+3 dose escalation to STK-012 as monotherapy (QW or Q3W) or STK-012 + pembrolizumab (Q3W) followed by Phase 1b expansions. Results: Phase 1a and preliminary Phase 1b data are being presented for STK-012 monotherapy. As of Oct 23rd, 2023, 45 subjects were treated at 7 dose levels (DL) across 2 schedules (QW at 0.375 mg and 0.75mg; Q3W at 0.75mg-3mg). The most common tumors were NSCLC (35.6%) and RCC (20%). The most common treatment related AEs (TRAEs) were maculo-papular rash (38%), injection site reactions (28.9%), fatigue (28.9%), and nausea (24.4%). Grade 3 TRAEs occurred in 12 subjects (26.6%) and included maculo-papular rash (4), vomiting (2), nausea (1), diarrhea (1), enterocolitis (1), arthralgia (1), urticaria (1), periorbital edema (1), blood creatinine increase (1) and leukocytosis (1). One subject had a Grade 4 TRAE of anaphylaxis in Cycle 9 after the data extract date. TRAEs were reversible, and no subjects had Grade 5 TRAEs. No subjects had capillary leak syndrome and <5% experienced other IL-2 hallmark TRAEs (hypotension [4.4%], AST/ALT increase [0%], pyrexia [4.4%], peripheral edema [4.4%]). No DLTs were observed. The maximum administered dose of STK-012 was 3mg Q3W. A DL of 2.25mg Q3W was advanced into Phase 1b based on safety, pharmacokinetic (PK) and pharmacodynamic (PD) data. Peripheral PD data showed STAT5 phosphorylation in CD25+ T cells and a dose dependent increase in activated T cells (Ki-67+CD38+CD8+) and serum IFNγ. Expansion of NK cells and Tregs was limited. STK-012 has a half-life of 4 days. Of 38 efficacy evaluable subjects, 3 had a partial response (PR) and 17 had stable disease as best overall response by RECIST V1.1. The PRs included 2 confirmed (anti-PD-1 r/r NSCLC and RCC) and 1 unconfirmed (anti-PD-1 r/r SCCHN) with the RCC subject in response for over 9 months with treatment ongoing. After the data extract date, a fourth subject achieved a PR (confirmed) with 80% reduction in target lesions (anti-PD-1/CTLA-4 and TKI r/r RCC) with treatment ongoing. Monotherapy Phase 1b dose expansions in r/r NSCLC and r/r RCC are ongoing. Conclusions: In our ongoing Phase 1a/b trial, STK-012 demonstrated a favorable safety, PK, and PD profile which is distinct from that of aldesleukin and non-α IL-2 analogues. Peripheral PD and PK data support selectivity for IL-2R α/β. Preliminary monotherapy efficacy in subjects who are r/r to prior immunotherapy warrants further development. Citation Format: Benjamin Izar, Dmitriy Zamarin, David R. Spigel, Christopher J. Hoimes, David F. McDermott, Kartik Sehgal, Yana G. Najjar, Adam J. Schoenfeld, Edward B. Garon, Ryan J. Sullivan, Brian S. Henick, Ticiana A. Leal, Michael E. Hurwitz, Rana R. McKay, Natalie Busby, Anita Mehta-Damani, Alex Azrilevich, Tony Tran, Naiyer Rizvi, Martin Oft, Alexander I. Spira. Initial results from a phase 1a/1b study of STK-012, a first-in-class α/β IL-2 receptor biased partial agonist in advanced solid tumors (NCT05098132) [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 CT183.

Background: STK-012 is a first-in-class α/β-IL-2R biased partial agonist designed to drive antitumor activity by selectively stimulating CD25+ antigen activated T cells, while avoiding hallmark IL-2 toxicities by sparing pleotropic activation of lymphocytes including NK cells. Methods: STK-012-101 is a Phase 1a/b study of STK-012 administered subcutaneously in subjects with advanced, relapsed/refractory (r/r) solid tumors. During Phase 1a, subjects are enrolled in a 3+3 dose escalation to STK-012 as monotherapy (QW or Q3W) or STK-012 + pembrolizumab (Q3W) followed by Phase 1b expansions. Results: Phase 1a and preliminary Phase 1b data are being presented for STK-012 monotherapy. As of Oct 23rd, 2023, 45 subjects were treated at 7 dose levels (DL) across 2 schedules (QW at 0.375 mg and 0.75mg; Q3W at 0.75mg-3mg). The most common tumors were NSCLC (35.6%) and RCC (20%). The most common treatment related AEs (TRAEs) were maculo-papular rash (38%), injection site reactions (28.9%), fatigue (28.9%), and nausea (24.4%). Grade 3 TRAEs occurred in 12 subjects (26.6%) and included maculo-papular rash (4), vomiting (2), nausea (1), diarrhea (1), enterocolitis (1), arthralgia (1), urticaria (1), periorbital edema (1), blood creatinine increase (1) and leukocytosis (1). One subject had a Grade 4 TRAE of anaphylaxis in Cycle 9 after the data extract date. TRAEs were reversible, and no subjects had Grade 5 TRAEs. No subjects had capillary leak syndrome and <5% experienced other IL-2 hallmark TRAEs (hypotension [4.4%], AST/ALT increase [0%], pyrexia [4.4%], peripheral edema [4.4%]). No DLTs were observed. The maximum administered dose of STK-012 was 3mg Q3W. A DL of 2.25mg Q3W was advanced into Phase 1b based on safety, pharmacokinetic (PK) and pharmacodynamic (PD) data. Peripheral PD data showed STAT5 phosphorylation in CD25+ T cells and a dose dependent increase in activated T cells (Ki-67+CD38+CD8+) and serum IFNγ. Expansion of NK cells and Tregs was limited. STK-012 has a half-life of 4 days. Of 38 efficacy evaluable subjects, 3 had a partial response (PR) and 17 had stable disease as best overall response by RECIST V1.1. The PRs included 2 confirmed (anti-PD-1 r/r NSCLC and RCC) and 1 unconfirmed (anti-PD-1 r/r SCCHN) with the RCC subject in response for over 9 months with treatment ongoing. After the data extract date, a fourth subject achieved a PR (confirmed) with 80% reduction in target lesions (anti-PD-1/CTLA-4 and TKI r/r RCC) with treatment ongoing. Monotherapy Phase 1b dose expansions in r/r NSCLC and r/r RCC are ongoing. Conclusions: In our ongoing Phase 1a/b trial, STK-012 demonstrated a favorable safety, PK, and PD profile which is distinct from that of aldesleukin and non-α IL-2 analogues. Peripheral PD and PK data support selectivity for IL-2R α/β. Preliminary monotherapy efficacy in subjects who are r/r to prior immunotherapy warrants further development. Citation Format: Benjamin Izar, Dmitriy Zamarin, David R. Spigel, Christopher J. Hoimes, David F. McDermott, Kartik Sehgal, Yana G. Najjar, Adam J. Schoenfeld, Edward B. Garon, Ryan J. Sullivan, Brian S. Henick, Ticiana A. Leal, Michael E. Hurwitz, Rana R. McKay, Natalie Busby, Anita Mehta-Damani, Alex Azrilevich, Tony Tran, Naiyer Rizvi, Martin Oft, Alexander I. Spira. Initial results from a phase 1a/1b study of STK-012, a first-in-class α/β IL-2 receptor biased partial agonist in advanced solid tumors (NCT05098132) [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 CT183.

Discovered over 4 decades ago in the supernatants of activated T cells, interleukin-2 (IL-2) is a potent pleiotropic cytokine involved in the regulation of immune responses. It is required for effector T cell expansion and differentiation as well as for peripheral tolerance induced by regulatory T cells. High-dose IL-2 treatment was the first FDA-approved immunotherapy for renal cell carcinoma and melanoma, achieving single agent complete and durable responses, albeit only in a small proportion of patients. The therapeutic potential of wild type IL-2 is clinically limited by its short half-life and severe vascular toxicity. Moreover, the activation of regulatory T cells and the terminal differentiation of effector T cells on IL-2 pose additional restrictions. To overcome the toxicity of IL-2 in order to realize its full potential for patients, several novel engineering strategies are being developed and IL-2 based immunotherapy for cancer has emerged as a burgeoning field of clinical and experimental research. In addition, combination of IL-2 with PD-1/L1 pathway blockade shows vastly improved anti-tumor efficacy over either monotherapy in preclinical tumor models. In this review we discuss the biological characteristics of IL-2 and its receptors, as well as its efficacy and treatment limiting toxicities in cancer patients. We also explore the efforts aimed at developing novel and safer IL-2 therapies to harness the full therapeutic potential of this cytokine.

Abstract CAR T cell therapy has demonstrated remarkable clinical efficacy against blood malignancies. However, prominent barriers, including limited proliferation, CAR T cell persistence, and poor T cell effector function, hinder its potential, especially in solid tumors. Various engineering approaches attempt to address these issues by armoring CAR T cells with factors that enhance the function and maintenance of T cells in the anti-tumor response. We previously described a human orthogonal IL-2/IL-2R system consisting of a pegylated, IL-2 mutein (STK- 009) that does not significantly activate the wild type receptor and a mutated IL-2 Receptor Beta (oRβ) that does not significantly respond to its native ligand, wild type IL-2. This system provides a “private IL-2 signal” in engineered oRβ-expressing cells while avoiding signaling in bystander immune cells, reducing toxicities. We have demonstrated STK-009/oRβ’s ability to selectively expand oRβ-expressing CAR T cells (SYNCAR T cells) targeting heme (SYNCAR-001, CD19) and solid tumor (SYNCAR-002, GPC3) targets at will. Importantly, the system enhanced anti-tumor control driving deep and durable responses in both bulky lymphoma and hepatocellular carcinoma (HCC) tumor models. Additionally, in a non-human primate model, STK-009 did not potentiate wild type T or NK cells and exhibited no evidence of toxicity. Currently, the combination of STK-009 + SYNCAR-001 is undergoing Phase I clinical trials (NCT05665062). Here, we conducted a direct comparison of several armoring approaches including the SYNCAR platform, the overexpression of c-Jun, an IL-15/IL-15Rα fusion, and IL-18 in human CAR T cells targeting GPC3. To assess their effects in overcoming CAR T cell dysfunction, cells were subjected to continuous antigen exposure in vitro. STK-009 treated SYNCAR-002 T cells displayed an enhanced proliferative capacity, sustained effector cytokine secretion, and an increased activation state when compared to other armoring technologies and PBS treatment. In a HCC tumor model, treatment with STK-009/SYNCAR-002 demonstrated superior tumor control with minimal toxicities. Interestingly, IL-18 overexpression also controlled tumors, but was accompanied by uncontrolled CAR T cell expansion and toxicity resembling severe GVHD. scRNAseq analyses revealed STK-009-treated SYNCAR-002 T cells simultaneously activated effector genes and repressed an exhaustion gene signature. This analysis also identified a novel mechanism by which STK-009/oRβ signaling induces a superior synthetic effector state via transcriptional repression of the stress response/proteosome. These findings support the advantages of an orthogonal platform that selectively drives and sustains potent T cell effector function without safety concerns associated with constitutively expressed armored approaches. Citation Format: Ethan Jung, Helena Silva, Marie Semana, Ivan Cheng, Somya Singh, Michele Bauer, Mohammed Ali, Henry Rosas, Ryan Burgess, George Zeng, Woei Chang, Navneet Ratti, Deepti Rokkam, Patrick J. Lupardus, Martin Oft, Paul-Joseph Aspuria. Orthogonal IL-2/IL-2Rβ signaling selectively enhances and sustains a synthetic effector state via a novel mechanism and outperforms constitutive armoring approaches [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 6312.

Supplementary Table 2 from Dinaciclib (SCH 727965), a Novel and Potent Cyclin-Dependent Kinase Inhibitor

PDF file - 206K, Suppl. Figure 1. PEG-IL-10 increases intratumoral CD8 T cells and induces CD8 T cell dependent tumor rejection. Suppl. Figure 2. Anti-tumor efficacy is abolished in IL-10R-/- mice. Suppl Figure 3. FTY720 treatment neither impairs IL-10 induced activation of tumor-resident CD8+ T cells nor rejection of CT26 tumors