Abstract While cancer-associated fibroblasts (CAFs) have emerged as a key cell type capable of influencing malignant cell growth, this axis of cancer biology has not yet been exploited for therapeutic benefit. CAFs enable tumor growth through multiple mechanisms including deposition of extracellular matrix, production of soluble factors, and inhibition of the immune response. Recent advancements have revealed how distinct CAF populations correlate with patient prognosis and response to therapies across many solid tumor types. Amongst these CAF subsets, LRRC15 expression has been shown to identify a myofibroblast population with a central functional role in supporting tumor cell growth and inhibiting anti-tumor immune responses. Approaches to reprogram or reduce this LRRC15+ population of CAFs may provide therapeutic benefit in multiple solid tumor indications. IFN-α signaling directly on tumor-supporting CAFs drives reprogramming, countering the TGF-β signal on which the phenotype depends. IFN-α also effectively promotes both innate and adaptive immune responses, including dendritic cell maturation, repolarization of suppressive myeloid cells, and CD8+ T cell activation. However, the use of systemic IFN-α therapy has been limited by significant dose-limiting toxicities. We have generated a conditionally active cLRRC15-IFNα therapeutic that targets IFN-α activity to LRRC15+ cells while remaining largely inactive on other cells. Our approach uses a novel dual-binding antibody (DBA) mechanism that takes advantage of the ability of an antibody to bind specifically and competitively to two distinct antigens. With this technology, IFN-α is bound and inactive in circulation and only becomes active when the therapeutic binds to LRRC15. Once localized to the surface of an LRRC15+ CAF, cLRRC15-IFNα exerts anti-tumor activity both by direct cis-signaling of IFN-α on CAFs and by trans-signaling to adjacent immune cells. In vitro, reporter cell and receptor binding assays demonstrate that cLRRC15-IFNα has >100-fold preferential IFN-α activity in the presence of LRRC15. In primary cells, cLRRC15-IFNα preferentially induces IFN-α signaling in LRRC15-expressing activated human fibroblasts. cLRRC15-IFNα inhibits tumor growth in mouse syngeneic tumor models, avoids clinical signs of IFN-α-mediated toxicity, and demonstrates robust combinatorial activity with anti-PD-1. In the TME, cLRRC15-IFNα drives the activation of CD8+ T cells. Collectively, these results demonstrate the potential of the DBA platform and support the clinical development of cLRRC15-IFNα. Citation Format: Justin Killebrew, Linda Liang, Shannon Okada, Aelish Guinn, Alton Etheridge, Brett Robison, David Colby, David Jurchen, Jacqueline Pham, Jamie Nguyen, John Skonier, Kendyl Daniels, Kerri Thomas, Laura Carlucci, Lynn Amon, Megan Sprague, Meri Galindo, Remington Lance, Sam Wrenn, Sandra Notonier, Shannon Fallen, Shea McClain, Wendy Curtis, Zane Kraft, John Mulligan, Diane Hollenbaugh, . Safety and activity of cLRRC15-IFNa, a conditionally active biologic targeting IFNa specifically to LRRC15+ CAFs [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 2892.

Abstract IL-2 is a powerful cytokine central to the generation of an effective immune response. However, its use in cancer immunotherapy has been limited by toxicities arising from its broad activity as well as the narrow patient population in which efficacy is seen. To address these limitations, many strategies have been pursued including IL-2 attenuation and targeting of IL-2 activity. These approaches, however, retain unacceptable toxicity and/or are not targeted to the appropriate cell populations. Using a novel approach that takes advantage of the ability of an antibody to bind specifically and competitively to two distinct antigens, we have generated a conditionally active cLAG3-IL2 therapeutic that targets IL-2 to LAG3+ cells while remaining inert on IL-2Rβγ+ cells lacking LAG3 expression, even at high treatment doses. As a marker of antigen-activated and tumor-specific T cells, LAG3 is an ideal target toward which to direct IL-2 activity. In vitro, using both reporter cell lines and receptor binding assays, the regulated cLAG3-IL2 demonstrates >100 fold difference in activity in the presence or absence of LAG3. In primary immune cells, regulated cLAG3-IL2 preferentially induces IL-2 cis-signaling in activated human LAG3+ CD8 T cells compared to LAG3- T cells. In mouse syngeneic tumor models, cLAG3-IL2 inhibits tumor growth while avoiding clinical signs of IL-2-mediated toxicity at doses well above the level where the non-regulated IL2 is toxic. Additionally, cLAG3-IL2 does not induce expansion of circulating LAG3- IL-2Rβγ+ cell populations, including NK cells and LAG3- T cells. In the TME, cLAG3-IL2 drives the expansion and activation of tumor-specific CD8+ T cells. Furthermore, cLAG3-IL2 demonstrates robust combinatorial activity with anti-PD1. These results demonstrate the strength of the Bonum platform, currently applied to multiple target/effector pairs including PD1, PDL1, ATP, LRRC15 and effectors IFNα, IL12 and TGFβ inhibition, and support the clinical development of our conditionally active cLAG3-IL2. Citation Format: Justin Killebrew, Shannon Okada, Lynn Amon, David Bienvenue, Laura Carlucci, David Colby, Wendy Curtis, Kendyl Daniels, Alton Etheridge, Zane Kraft, Jamie Nguyen, Sandra Notonier, Jacqueline Pham, Megan Sprague, Kerri Thomas, Diane Hollenbaugh, John Mulligan. A novel method for generating regulated cytokine therapeutics: Safety and activity of a conditionally active cLAG3-IL2 capable of delivering IL2 to LAG3+ cells while remaining inert on LAG3- cells [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 4062.

Effect of LB-1 in tumor model without MSLN expression

Simultaneous binding shown in Biacore sensorgrams

Figures S1-S7 show LRRC15 RNA and protein expression in cancer, and demonstrate the anti-tumor efficacy of the LRRC15-targeted ADC ABBV-085 in vitro and in vivo as a monotherapy and in combination with standard-of-care therapies.

Supplementary Figure Legends

Serum concentration of mAbs after the last dose

Liver inflammation and macrophage activation by LB-1

Liver toxicity measured by serum ALT level

Human T cell costimulation activity of anti-CD137

Efficacy and pharmacodynamics analysis of anti-CD137 in vivo

Binding and ADCC function of ABBV-428

Soluble and cell surface expressed MSLN in 4T1-MSLN model and combination treatment with anti-CD40 and anti-MSLN

Combination study of anti-CD137 with anti PD-1 or anti-PD-L1

PC3 cells stably expressing different levels of MSLN

Cross species binding and tolerability of ABBV-085.

Binding kinetics of anti-CD137 measured by Biacore

Costimulatory receptors such as glucocorticoid-induced tumor necrosis factor receptor–related protein (GITR) play key roles in regulating the effector functions of T cells. In human clinical trials, however, GITR agonist antibodies have shown limited therapeutic effect, which may be due to suboptimal receptor clustering-mediated signaling. To overcome this potential limitation, a rational protein engineering approach is needed to optimize GITR agonist-based immunotherapies. Here we show a bispecific molecule consisting of an anti-PD-1 antibody fused with a multimeric GITR ligand (GITR-L) that induces PD-1-dependent and FcγR-independent GITR clustering, resulting in enhanced activation, proliferation and memory differentiation of primed antigen-specific GITR + PD-1 + T cells. The anti-PD-1–GITR-L bispecific is a PD-1-directed GITR-L construct that demonstrated dose-dependent, immunologically driven tumor growth inhibition in syngeneic, genetically engineered and xenograft humanized mouse tumor models, with a dose-dependent correlation between target saturation and Ki67 and TIGIT upregulation on memory T cells. Anti-PD-1–GITR-L thus represents a bispecific approach to directing GITR agonism for cancer immunotherapy.

Abstract CD137 (TNFRSF9, 4-1BB) agonist antibodies (mAb) have demonstrated potent antitumor activity with memory response while causing hepatotoxicity in mouse models. In clinical trials, the degrees of liver toxicity of anti-CD137 vary from grade 4 transaminitis (urelumab) to nonexistent (utomilumab). To exploit the antitumor potential of CD137 signaling, we identified a new class of CD137 agonist mAbs with strong antitumor potency without significant transaminitis in vivo compared with CD137 agonists previously reported. These mAbs are cross-reactive to mouse and cynomolgus monkey and showed cross-linking–dependent T-cell costimulation activity in vitro. Antitumor efficacy was maintained in Fc gamma receptor (FcγR) III–deficient mice but diminished in FcγRIIB-deficient mice, suggesting the critical role for FcγRIIB to provide cross-linking in vivo. Interestingly, a single dose of an affinity-reduced variant was sufficient to control tumor growth, but a higher affinity variant did not improve efficacy. These observations suggest that binding epitope and FcγR interaction, but not necessarily high affinity, are important for antitumor efficacy and reduced liver toxicity of CD137 mAb. Our study suggests the possibility of CD137 agonist therapy with improved safety profile in humans.