Antitumor Efficacy In Preclinical Models Research Articles

Development of NR0B2 as a therapeutic target for the re-education of tumor associated myeloid cells

Immune checkpoint blockade (ICB) has had limited utility in several solid tumors such as breast cancer, a major cause of cancer-related mortality in women. Therefore, there is considerable interest in alternate strategies to promote an anti-cancer immune response. A paper co-published in this issue describes how NR0B2, a protein involved in cholesterol homeostasis, functions within myeloid immune cells to modulate the inflammasome and reduce the expansion of immune-suppressive regulatory T cells (Treg). Here, we develop NR0B2 as a potential therapeutic target. NR0B2 in tumors is associated with improved survival for several cancer types including breast. Importantly, NR0B2 expression is also prognostic of ICB success. Within breast tumors, NR0B2 expression is inversely associated with FOXP3, a marker of Tregs. While a described agonist (DSHN) had some efficacy, it required high doses and long treatment times. Therefore, we designed and screened several derivatives. A methyl ester derivative (DSHN-OMe) emerged as superior in terms of (1) cellular uptake, (2) ability to regulate expected expression of genes, (3) suppression of Treg expansion using in vitro co-culture systems, and (4) efficacy against the growth of primary and metastatic tumors. This work identifies NR0B2 as a target to re-educate myeloid immune cells and a novel ligand with significant anti-tumor efficacy in preclinical models.

A novel SLC3A2-targeting antibody-drug conjugate exerts potent antitumor efficacy in head and neck squamous cell cancer

The development of innovative therapeutic strategies for head and neck squamous cell carcinoma (HNSCC) is a critical medical requirement. Antibody-drug conjugates (ADC) targeting tumor-specific surface antigens have demonstrated clinical effectiveness in treating hematologic and solid malignancies. Our investigation revealed high expression levels of SLC3A2 in HNSCC tissue and cell lines. This study aimed to develop a novel anti-SLC3A2 ADC and assess its antitumor effects on HNSCC both in vitro and in vivo. This study developed a potent anti-SLC3A2 ADC (19G4-MMAE) and systematically investigated its drug delivery potential and antitumor efficacy in preclinical models. This study revealed that 19G4-MMAE exhibited specific binding to SLC3A2 and effectively targeted lysosomes. Moreover, 19G4-MMAE induced a significant accumulation of reactive oxygen species (ROS) and apoptosis in SLC3A2-positive HNSCC cells. The compound demonstrated potent antitumor effects derived from MMAE against SLC3A2-expressing HNSCC in preclinical models, displaying a favorable safety profile. These findings suggest that targeting SLC3A2 with an anti-SLC3A2 ADC could be a promising therapeutic approach for treating HNSCC patients.

Abstract 3131: Novel CDH17-targeting antibody-drug conjugate exhibits anti-tumor efficacy in preclinical models of gastrointestinal cancers

Abstract Antibody-drug conjugates (ADC) for oncology indications have been developed, however, only few of them are approved for the treatment of gastrointestinal (GI) cancers, which represent as a huge unmet medical need. As such, we developed a novel ADC of which monomethyl auristatin E (MMAE), an antimitotic agent, was conjugated to a cadherin-17 (CDH17)-targeting monoclonal antibody via protease-cleavable peptide valine-citrulline linker. CDH17 is highly restricted to the intestinal adherent junctions which is not accessible in healthy individuals. In contrast, it is overexpressed and becomes reachable in over half of the gastric and pancreatic cancer patients and in more than 95% of patients with colorectal cancer, making it a perfect target for ADC. Our ADC is designed to deliver MMAE specifically to CDH17-expressing tumors to exert the cytotoxic effect of MMAE on cancer cell, mitigating the undesired off-target cytotoxicity. LC/MS analysis revealed that MMAE was conjugated in a drug-to-antibody ratio of 4 with a yield of higher than 75%. ELISA showed that the binding of naked antibody and ADC to CDH17 was comparable (EC50, 0.17 nM & 0.3 nM, respectively), indicating the conjugation process did not affect antigen binding. This ADC also bound to different CDH17-expressing cell lines, including pancreatic AsPC1 and gastric AGS (EC50, 2.1 nM & 1.53 nM, respectively). By labelling the ADC with pH-sensitive fluorescent indicator, we illustrated that ADC internalization only occurred in CDH17+ but not CDH17- cells, suggesting the high specificity of the ADC. Similarly, the ADC showed selective killing on CDH17+ cells (EC50, 0.02-0.5 nM), and the data together proposed a positive correlation between ADC internalization level and its cytotoxicity (R2 = 0.4307). This supported the specificity and safety of the ADC in killing CDH17+ cancer cells only without affecting normal cells. Furthermore, a preliminary in-vivo study on subcutaneous AsPC1 xenograft model demonstrated that the ADC significantly suppressed tumor growth without causing any changes in body weight. Our ADC would potentially be a “First-in-class” ADC for the treatment of GI cancers with minimal adverse systemic toxicity. Further pharmacokinetics, safety, and other IND-enabling studies are underway. Citation Format: Po Yee Wong, Lin He, Kronos Chow, Kwan Wa Wong, Chui Yee O, Dennis Wong, John Moon Luk, Pui-Chi Lo, Kwong Fai Wong. Novel CDH17-targeting antibody-drug conjugate exhibits anti-tumor efficacy in preclinical models of gastrointestinal 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 3131.

Abstract 1356: The VISTA blocking antibody HMBD-002 promotes type-I interferon signaling and drives anti-tumor responses through macrophage reprograming and cytotoxic CD8+ T cell activation

Abstract VISTA, a predominantly myeloid immune checkpoint, promotes an immunosuppressive tumor microenvironment and has been associated with resistance to approved checkpoint therapies. While blockade of VISTA with antibodies has shown anti-tumor efficacy in preclinical models, the biological mechanisms that underlie this effect remain poorly understood. To investigate the mechanistic basis of anti-tumor responses, we performed single-cell RNA sequencing of tumor infiltrating leukocytes from the cell-derived murine colon carcinoma xenograft model, CT26, which is known to be sensitive to VISTA blockade. We used HMBD-002, a uniquely blocking but non-depleting clinical-stage IgG4 anti-VISTA antibody, to identify the key immune cells and pathways impacted by VISTA blockade in the absence of Fc-mediated cell depletion. The translatability of these findings was validated in a human PBMC mixed leukocyte reaction assay. Subsequently, targeted depletion or inhibition of the identified immune populations and pathways was performed to confirm their contribution to the anti-tumor efficacy of HMBD-002.The blockade of VISTA by HMBD-002 significantly increased CD8+ T cells in the CT26 tumors, promoting an activated cytotoxic Tc1 program without inducing exhaustion. Concurrently, a substantial increase in proinflammatory M1 tumor-associated macrophages (TAMs) was noted within treated tumors, while other immune cell populations remained unaffected. Among the signaling pathways, the type-I interferon (IFN) response exhibited a significant upregulation, particularly in TAMs, indicating a type-I IFN-driven M1 reprogramming by HMBD-002. Validation experiments further demonstrated that the depletion of TAMs or CD8+ T cells, but not CD4+ T cells, abrogated the efficacy of HMBD-002. Moreover, blocking the type-I IFN signaling effectively impeded HMBD-002-induced TAM reprogramming and subsequent anti-tumor efficacy, thus confirming the pivotal roles of CD8+ T cells, TAMs, and type-I IFN signaling in driving the anti-tumor response to VISTA blockade. In conclusion, VISTA blockade with HMBD-002 promotes an anti-tumor response by type-I IFN mediated reprogramming of TAMs and activation of cytotoxic CD8+ T cells. Citation Format: Bhushan Dharmadhikari, Olga Zharkova, Dipti Thakkar, Roberto Tirado-Magallanes, Konrad Paszkiewicz, Piers J. Ingram, Jerome D. Boyd-Kirkup. The VISTA blocking antibody HMBD-002 promotes type-I interferon signaling and drives anti-tumor responses through macrophage reprograming and cytotoxic CD8+ T cell activation [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 1356.

Abstract 4073: Decoy-resistant IL-18 enhances checkpoint inhibitor combinations beyond anti-PD-1 in vitro and in vivo

Abstract Background: Interleukin-18 (IL18) is a proinflammatory cytokine that modulates both innate and adaptive immune responses. Historically, wild-type recombinant IL-18 has shown limited anti-tumor efficacy in preclinical models and clinical trials, likely due to upregulation of IL-18 binding protein (IL-18BP), a negative regulator of the IL-18 signaling axis. Accordingly, an engineered IL-18 cytokine capable of interacting with the IL-18 receptor, but resistant to IL-18BP interactions (i.e., “Decoy-Resistant IL-18”, DR-18), has demonstrated enhanced therapeutic potential in mouse tumor models, both as a single agent and in combination with anti-PD-1 [1, 2]. Here we evaluated murine DR-18 in combination with additional checkpoint inhibitors beyond anti-PD-1 in a set of mouse tumor models, and tested human DR-18 activity in vitro/ex vivo for activation of human immune cells and anti-tumor activity. Results: Treatment with mDR-18 elicited strong efficacy as a single agent and combination activity with immune checkpoint inhibitors (ICIs) across diverse syngeneic mouse models of solid tumors. As a single agent, mDR-18 demonstrated robust single agent activity in tumor growth inhibition for MC38 (>80%), CT26 (>60%) and B16-F10 (>55%). Combination treatment of mDR-18 with anti-PD-1, anti-CTLA-4, or anti-LAG-3 increased the efficacy for the MC38 (>85-95%), CT26 models (>80-85%), while the B16-F10 (>60%) showed smaller efficacy enhancement with the combinations. In vitro assays revealed human DR-18 (ST-067) activated immune cells and enhanced A549 tumor cell killing over 72hrs. Furthermore, ex vivo studies of 3D patient-derived tumor spheroids demonstrated that ST-067 impaired growth and increased immune cell infiltration, highlighting the potential of hDR-18 to enhance immune activity within the human tumor microenvironment. Conclusion: These studies expand the breadth of IL-18/checkpoint synergism beyond anti-PD-1 and confirm enhanced human immune response in vitro with the clinical candidate ST-067. Taken together, these findings strengthen the rationale for clinical combination of ST-067 with ICI agents in patients with solid tumors. 1.Zhou T, Damsky W, Weizman OE, et al. IL-18BP is a secreted immune checkpoint and barrier to IL-18 immunotherapy. Nature. 2020;583(7817):609-614. doi:10.1038/s41586-020-2422-62.Minnie SA, Waltner OG, Ensbey KS, et al. Depletion of exhausted alloreactive T cells enables targeting of stem-like memory T cells to generate tumor-specific immunity. Sci Immunol. 2022;7(76):eabo3420. doi:10.1126/sciimmunol.abo3420 Citation Format: Jeffrey N. Lindquist, Kai Qin, Aaron M. Ring, Hirdesh Uppal. Decoy-resistant IL-18 enhances checkpoint inhibitor combinations beyond anti-PD-1 in vitro and in vivo [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 4073.

Abstract 6594: 4-1BB: A double-edged sword for T cells

Abstract Background: The costimulatory receptor 4-1BB (CD137) is expressed on the surface of activated T cells, playing a pivotal role in enhancing T cell responses and emerging as a promising target for cancer immunotherapy. Its primary ligand, 4-1BBL (CD137L), is found on antigen-presenting cells and various immune cells. The interaction between 4-1BBL and 4-1BB provides crucial costimulatory signals to both CD4+ and CD8+ T cells, amplifying their activation and effector functions. However, recent studies have shed light on a distinct role of 4-1BB in T cells. Prior work has revealed that 4-1BB can distinguish intratumoral regulatory T cells (Tregs) from peripheral Tregs and other CD4 lineage T cells in cancer patients, and the overexpression of 4-1BB on Tregs has been associated with unfavorable survival outcomes. Additionally, targeting 4-1BB with IgG2a isotype antibodies has shown significant anti-tumor efficacy in preclinical models by depleting Tregs. Furthermore, agonist monoclonal antibodies targeting 4-1BB have not only shown promise in inducing tumor regressions but also demonstrated improved outcomes in mouse models of autoimmune diseases induced by autoreactive CD4+ T cells. These findings indicate the existence of specific ligands or factors that modulate 4-1BB function on T cells that can vary depending on the disease state. Methods: To identify potential ligand(s) involved in the modulation of 4-1BB function, we developed an innovative CRISPR/Cas9-based transcriptional activation (CRISPRa) screening system that integrates CRISPR/Cas9-guide mediated gene activation, high avidity bead-based selection, serial enrichment, and flow cytometry-based monitoring. This screening platform provides enhanced sensitivity for identifying extracellular interactions between receptor-ligand partners that have not been fully characterized before, even with low-affinity. Results: Through our unbiased CRISPRa screen, we discovered that Myelin-Associated Glycoprotein (MAG), also known as siglec-4, a member of the siglec (sialic acid-binding immunoglobulin-like lectins) family, interacts with 4-1BB in trans. Our study demonstrated that MAG binds to activated T cells via 4-1BB and inhibits their activation and proliferation, suggesting a potential role of the MAG-4-1BB interaction in modulating T cell function. Notably, our data revealed that trans-binding to MAG-positive cells inhibits c-Jun signaling in activated T cells. Furthermore, the presence of MAG on target cells can hinder Chimeric Antigen Receptor (CAR) T-cell activity. Conclusion: Utilizing a highly efficient cell-based CRISPRa system, we discovered a novel interactor of 4-1BB capable of modulating T cell activation through its interaction with 4-1BB. This interaction suggests an immunosuppressive role of 4-1BB, explaining the paradoxical roles of 4-1BB in T cells. These findings provide crucial insights for the development of precise therapeutics targeting 4-1BB. Citation Format: Liping Yang, Timothy P. Sheets, Yang Feng, Kuo-Sheng Hsu, Guojun Yu, Steven Seaman, Pradip Bajgain, Daeho So, Jaewon Lee, Raj Chari, Brad St. Croix. 4-1BB: A double-edged sword for T 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 6594.

A Phase I Study of the Oral Dual-Acting Pan-PI3K/mTOR Inhibitor Bimiralisib in Patients with Advanced Solid Tumors.

Bimiralisib is a pan-PI3K/mTOR inhibitor demonstrating antitumor efficacy in preclinical models. The objectives of this study were to identify a maximum tolerated dose (MTD), pharmacokinetics (PK), a dosing schedule, and adverse events (AEs) in patients with advanced solid tumors. Patients received oral bimiralisib to determine the MTD of one continuous (once daily) and two intermittent schedules (A: Days 1, 2 weekly; B: Days 1, 4 weekly) until progression or unacceptable AEs occurred. The MTD for the continuous schedule was 80 mg, with grade three fatigue as the dose-limiting toxicity (DLT). No MTD was reached with intermittent schedules, with only one DLT in schedule B. PK analysis suggested that 140 mg (schedule A) was within the biologically active dose range and was selected for further exploration. The most frequent treatment-emergent AEs were hyperglycemia (76.2%) in the continuous schedule, and nausea (56-62.5%) in schedules A and B. The most frequent treatment-emergent > grade three AE for all schedules combined was hyperglycemia (28.6%, continuous schedule; 12.0%, schedule A; 12.5%, schedule B). There was one partial response in a head and neck squamous cancer patient with a NOTCH1T1997M mutation. Bimiralisib demonstrated a manageable AE profile consistent with this compound class. Intermittent schedules had fewer > grade three AEs, while also maintaining favorable PK profiles. Intermittent schedule A is proposed for further development in biomarker-selected patient populations.

The Discovery of Exarafenib (KIN-2787): Overcoming the Challenges of Pan-RAF Kinase Inhibition.

RAF, a core signaling component of the MAPK kinase cascade, is often mutated in various cancers, including melanoma, lung, and colorectal cancers. The approved inhibitors were focused on targeting the BRAFV600E mutation that results in constitutive activation of kinase signaling through the monomeric protein (Class I). However, these inhibitors also paradoxically activate kinase signaling of RAF dimers, resulting in increased MAPK signaling in normal tissues. Recently, significant attention has turned to targeting RAF alterations that activate dimeric signaling (class II and III BRAF and NRAS). However, the discovery of a potent and selective inhibitor with biopharmaceutical properties suitable to sustain robust target inhibition in the clinical setting has proven challenging. Herein, we report the discovery of exarafenib (15), a highly potent and selective inhibitor that intercepts the RAF protein in the dimer compatible αC-helix-IN conformation and demonstrates anti-tumor efficacy in preclinical models with BRAF class I, II, and III and NRAS alterations.

Abstract C074: VPS34 inhibition delays activation-induced STING degradation to prolong STING signaling and improve anti-tumor efficacy in preclinical models

Abstract Background: The STING/interferon (IFN) response pathway has been a target of cancer immunotherapy for many years, as a method for reactivating the anti-tumor immune response. Stimulation of the STING pathway leads to activation and infiltration of immune cells into the tumor, and cancer cell death. However, multiple STING agonists have failed in the clinic due to poor efficacy or toxicity1. STING activation leads to its degradation within a few hours through trafficking to the lysosome, as a mechanism to control immune cell hyperactivation2. Although this safety mechanism is important for preventing autoimmunity and chronic inflammation, it diminishes the continuous activation of immune-mediated cancer cell killing, especially in an immunosuppressive tumor microenvironment. VPS34 is a lipid kinase which plays an integral role in endosomal trafficking and autophagy3. Previous publications have demonstrated a role for VPS34 inhibition in increased effector immune cell infiltration, which synergizes with anti-PD1 therapy, demonstrating a link to immune activation4. Herein, we demonstrate that the in vivo combination efficacy with a VPS34 inhibitor and a STING agonist is due to reduced degradation of activated STING, through the inhibition of VPS34-mediated endosomal trafficking. Materials and Methods: Cancer cell lines were cultured using recommended medium. STING activity was measured by Western blot and via a CisBio pTBK1 kit. ADU-S100/MSA-2 were sourced from MedChemExpress. CCL5, CXCL10 and IFNb ELISA’s were purchased from R&D systems. Primers used to measure mRNA expression were obtained from IDT. Results: DP-9244 is a potent and selective VPS34 inhibitor. The combination of DP-9244 with the STING agonist, ADU-S100, enhances IFN-mediated gene expression and cytokine secretion in the murine melanoma cell line, B16F10, and in the human and murine myeloid cell lines THP-1 and DC2.4, demonstrating combination activity in both cancer and immune cells. This combination activity is STING-dependent as demonstrated by the complete loss of IRF and NFκB activity in the THP-1 STING-/- cell line when treated with ADU-S100, DP-9244 or the combination. Mechanistically, VPS34 inhibition delays degradation of activated STING and prolongs downstream signaling of pTBK1 in multiple human and murine cancer cell lines. Importantly, the in vitro effects extend to in vivo efficacy in the B16F10 model, in which the combination of DP-9244 and ADU-S100 results in increased tumor growth control and number of mice responding to treatment. Conclusions: These data provide a strong preclinical rationale to combine a VPS34 inhibitor with a STING agonist as a method of improving STING pathway mediated anti-tumor efficacy.

Abstract C077: STC-15, a small molecule inhibitor of the RNA methyltransferase METTL3, activates anti-tumor immunity and reshapes the tumor microenvironment

Abstract Background METTL3 is the RNA methyltransferase responsible for the deposition of N-6-methyladenosine modification (m6A) on mRNA, to regulate its stability, splicing and protein translation. Small molecule inhibitors of METTL3 catalytic activity have previously demonstrated anti-tumor efficacy in pre-clinical models of acute myeloid leukemia (AML) and solid tumors. Inhibition of METTL3 was shown to induce a cell-intrinsic innate immune response through activation of the double strand RNA (dsRNA) sensing machinery and interferon (IFN) signalling. Here we present new pre-clinical data, investigating the combination of METTL3 inhibitor (METTL3i) with DNA damaging therapies. We show that METTL3 inhibition profoundly changes the tumor microenvironment (TME), and we further demonstrate the generation of durable immune memory in mice treated with METTL3i, with or without anti-PD1 therapy. Materials & Methods The orally bioavailable small molecule clinical-stage METTL3i STC-15, or the equipotent tool compound STM3675 were used in these studies. To characterise transcriptomic changes following METTL3 inhibition, RNA sequencing was performed. Induction of specific genes was validated by Western Blot, and cytokine secretion assessed by ELISA. In-vitro cell viability was assessed by Cell TiterGlo (Promega). In vivo efficacy studies used subcutaneous MC38 and GL261 syngeneic tumor models, and characterisation of the TME was performed by flow cytometry. Results Treatment with METTL3i sensitized cells to DNA damaging agents such as doxorubicin. On the molecular level, further enhancement of the IFN pathway by the combination treatment was observed. Moreover, combination of METTL3i with radiation treatment (RT) in vivo in the MC38 colorectal syngeneic mouse model demonstrated a clear benefit of the combination over each of the single treatments. METTL3i alone or in combination with RT led to a major TME remodulation, reducing the number of Tregs, M2 macrophages and neutrophils, and increasing the number of M1 macrophages and CD8+ T-cells in tumors. The GL261 syngeneic glioma model is exquisitely sensitive to anti-PD1 treatment. Similarly, METTL3i treatment led to multiple complete regressions, and the combination of METTL3i and anti-PD1 further accelerated the occurrence of regressions. Following a 3-weeks’ observation period, tumor free mice were re-inoculated with GL261 cells. All tumor-free mice remained tumor free after the rechallenge. Conclusions In pre-clinical cancer models, treatment with METTL3i such as STC-15 resulted in activation of innate immune pathways and subsequent enhancement of cytotoxic T-cell activity. In-vivo, STC-15 had a profound effect on the TME, shifting it from an immunosuppressive, pro-tumorigenic state to an immunostimulatory, anti-tumor state. In combination with anti-PD1, METTL3 inhibitors generate a durable anti-tumor response and lasting immune memory. A First-in-Human dose escalating study evaluating STC-15 monotherapy in patients with solid tumors is ongoing (NCT05584111). Citation Format: Yaara Ofir-Rosenfeld, Lina Vasiliauskaitė, Joanna Obacz, Georgia Tsagkogeorga, Josefin-Beate Holz, Jerry McMahon, Oliver Rausch. STC-15, a small molecule inhibitor of the RNA methyltransferase METTL3, activates anti-tumor immunity and reshapes the tumor microenvironment [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C077.

A modified IL-18 drug in combination with CTLA-4 blockade enhances anti-tumor efficacy in preclinical models of renal cell carcinoma

Abstract Background Cytokine-based drugs are currently being explored as alternative cancer immunotherapies. While the cytokine interleukin-18 (IL-18) has immunostimulatory effects, it is negatively regulated by a secreted high-affinity binding protein, IL-18BP, that functions as an immune checkpoint that limits IL-18’s efficacy as a cancer therapeutic. A modified version of IL-18, termed “decoy-resistant” or DR-18, that can avoid trapping by IL-18BP while still maintaining its immune signaling potential, has recently been developed. DR-18 has shown promising preclinical activity in melanoma and colorectal murine models, including potential synergy with anti-PD-1 therapy, and is currently in Phase I trials. In this study, we aim to test the efficacy and determine the cellular mechanism of action of DR-18 in combination with immune checkpoint inhibitors (ICIs) in immunocompetent preclinical models of renal cell carcinoma (RCC), with the goal of establishing the basis for testing these combinations in early phase clinical trials. Methods We engrafted tumors subcutaneously using two different syngeneic, immunocompetent murine RCC models: Renca and RAG. Mice were treated with single-agent DR-18 and combinations of DR-18 with single- and dual-agent anti-PD-1 and anti-CTLA-4. Tumor growth and survival were monitored. In the Renca model, plasma was collected at early time-points and cytokine/chemokine levels were profiled using a 31-plex discovery assay. Single-cell RNA and TCR sequencing was also performed on Renca tumors. Additionally, immune cell depletion studies were conducted in the Renca model with antibodies targeting CD8, CD4, NK cells, and interferon-gamma. Results In the Renca model, DR-18 monotherapy modestly inhibited tumor growth and prolonged survival. The effects were comparable to single- and dual-agent ICIs. Adding PD-1 blockade to DR-18 did not enhance efficacy whereas the addition of anti-CTLA-4 to DR-18 significantly increased anti-tumor effects. Triple-therapy (DR-18 plus anti-PD-1 plus anti-CTLA-4) did not further inhibit tumor growth or prolong survival compared to the doublet (DR-18 plus anti-CTLA-4). The RAG model was more sensitive to ICIs but produced similar results, again showing modest anti-tumor activity of single-agent DR-18 and enhanced benefit of combining with anti-CTLA-4 but not anti-PD-1. Cytokine/chemokine profiling revealed significantly elevated levels of IP-10 (CXCL10) and MIG (CXCL9) after one cycle of DR-18 plus anti-CTLA-4 compared to control and single-agent treatments, suggesting that these chemokines may be key early mediators of the anti-tumor immune response. Single-cell transcriptomic analysis demonstrated changes in intra-tumoral T cell, macrophage, and granulocyte populations with DR-18 plus anti-CTLA-4 relative to other regimens, including enrichment of CD8+ precursor and terminally exhausted T cells and a neutrophil population associated with interferon signaling. Additionally, single-cell TCR analysis showed a reduction in intra-tumoral clonotype diversity with DR-18 plus anti-CTLA-4 compared to other treatments. Immune cell depletion studies identified CD8+ T cells, NK cells, and interferon-gamma, but not CD4+ T cells, as equally required for efficacy of DR-18 plus anti-CTLA-4. Conclusions In this study, we identify DR-18, an IL-18-based drug engineered with resistance to a secreted decoy-receptor protein, in combination with anti-CTLA-4 as having enhanced anti-tumor activity in preclinical models of RCC. This regimen was associated with a more pro-inflammatory immune microenvironment. Further investigation is ongoing to elucidate the cellular mechanism of action of this regimen more fully and lay the groundwork for clinical testing of DR-18-based combination therapy in RCC. In the future, testing novel partner agents outside of anti-PD-1/CTLA-4 and using RCC models of ICI-resistance could be particularly informative and clinically relevant.

Abstract 4478: Up284, A small molecule inhibitor of ADRM1 demonstrates significant anti-tumor efficacy in preclinical models of aggressive breast cancer

Abstract Quadruple-negative breast cancer (QNBCs) is a highly aggressive and metastatic disease and remains clinically challenging breast cancer subtype with worst prognosis. Due to its exceeding heterogeneity, absence of the AR, ER, PR, Her2 receptors and lack of established therapeutic targets, QNBC is hard to treat cancer and recurs rapidly with standard chemo regimen. QNBC demonstrated vulnerability to proteasome inhibition due to their higher metabolic needs. But FDA approved 20S proteasome inhibitors failed to treat solid tumors including QNBC. Hence, developing an effective strategy to overcome the limitations associated with current 20S proteasome inhibitors is mandated to provide alternate treatment options for QNBC patients. Preclinical, Up Therapeutics lead candidate small molecule inhibitor Up284 has shown significant tumor growth inhibition as a single agent in animal models. To explore potential synergy of Up284 with other therapeutic agents, we conducted the in vivo combination experiments. In this work, we present ADRM1 amplification in QNBC tumors, identification of a small molecule selective inhibitor of ADRM1, Up284 (Up Therapeutics), Up284 demonstrated efficacy against panel of BC cell lines and patient derived organoids. In vivo combination results for Up284 with widely used BC standard-of care chemotherapy regimen demonstrated strong in vivo synergy in tumor mouse syngeneic models. Taken together, these data demonstrated combination synergy of a small molecule ADRM1 inhibitor with other agents. These results pave the road for potential clinical evaluation of combination treatment of QNBC in patients. Citation Format: Balasubramanyam Karanam, Ravi Anchoori, Yung-Nien Chang, Rachel Martini, Florencia Madorsky Rowdo, Melissa B. Davis. Up284, A small molecule inhibitor of ADRM1 demonstrates significant anti-tumor efficacy in preclinical models of aggressive breast cancer. [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 4478.

Withaferin A: A Pleiotropic Anticancer Agent from the Indian Medicinal Plant Withania somnifera (L.) Dunal.

Cancer represents the second most deadly disease and one of the most important public health concerns worldwide. Surgery, chemotherapy, radiation therapy, and immune therapy are the major types of treatment strategies that have been implemented in cancer treatment. Unfortunately, these treatment options suffer from major limitations, such as drug-resistance and adverse effects, which may eventually result in disease recurrence. Many phytochemicals have been investigated for their antitumor efficacy in preclinical models and clinical studies to discover newer therapeutic agents with fewer adverse effects. Withaferin A, a natural bioactive molecule isolated from the Indian medicinal plant Withania somnifera (L.) Dunal, has been reported to impart anticancer activities against various cancer cell lines and preclinical cancer models by modulating the expression and activity of different oncogenic proteins. In this article, we have comprehensively discussed the biosynthesis of withaferin A as well as its antineoplastic activities and mode-of-action in in vitro and in vivo settings. We have also reviewed the effect of withaferin A on the expression of miRNAs, its combinational effect with other cytotoxic agents, withaferin A-based formulations, safety and toxicity profiles, and its clinical potential.

Abstract CT147: Phase 1 study of recombinant interleukin 15 (rhIL-15) in combination with checkpoint inhibitors nivolumab and ipilimumab in subjects with refractory cancers

Abstract Introduction: Recombinant human interleukin 15 (rhIL-15) has been shown to stimulate the activation and expansion in the number of effector T lymphocytes and natural killer cells in patients. The addition of the immune checkpoint inhibitors (ICI) CTLA-4 and PD-1 has shown increased antitumor efficacy in preclinical models, and the triplet is hypothesized to enhance anti-tumor immune response by augmenting effector cell expansion, differentiation, cytotoxic activity, and immune checkpoint inhibition. Results of the lead-in safety arms of rhIL-15/ipilimumab (Ipi) or rhIL-15/nivolumab (N) doublets were previously reported (O’Sullivan et al. AACR. 2019) and we now report results for the dose escalation phase of the triplet combination (NCT03388632). Methods: This phase 1, open-label, 3+3 dose escalation trial enrolled adult patients (pts) with measurable metastatic or refractory solid tumors and ECOG performance status ≤2. Prior therapy with 2 of the 3 study agents was permitted. Pts were treated on three dose levels (DL 1-3) of rhIL-15 (0.5, 1, or 2 mcg/kg/day) administered subcutaneously on days (D)1-8 and D22-29 of a 6-week cycle for the first 4 cycles only. Nivolumab (240mg IV) was administered on D8, 22, 36 and ipilimumab (1mg/kg IV) on D8 of every cycle. Response was assessed by RECIST 1.1. Upon progression, pts could continue for response assessment utilizing iRECIST if clinically stable and without toxicity. Treatment-induced changes in circulating and tumor T cell activation, signaling, and the PD-L1 checkpoint will be assessed with validated pharmacodynamic biomarker assays. Results: A total of 17 pts were enrolled on the triplet dose escalation phase; 15 pts were evaluable for response and 6 pts had received prior treatment with a PD-1/PD-L1 inhibitor. Median time on treatment (MTT) was 12 weeks (range 3-101). A confirmed partial response (PR) was observed in a pt with intrahepatic cholangiocarcinoma (MSI-H) after 3 cycles (total time on treatment 101 weeks). Across all dose levels, 7 pts (46.7%) had stable disease (SD) that prolonged their time on treatment with MTT of 20.4 weeks; 7 pts (46.7%) experienced progressive disease (PD) with MTT of 6.4 weeks. To better profile toxicity, 5 pts were replaced due to incomplete dosing of C1. Dose limiting toxicities at DL 3 (rhIL-15 at 2 mcg/kg/day) were photosensitivity and rash. Main drug-related adverse events included grade 4 lymphopenia (n=1) and grade 3 colitis, hyperthyroidism, hyponatremia, myocarditis, neutropenia, and photosensitivity (n=1, each). No pts discontinued therapy due to toxicity events. No deaths occurred on study. Conclusions: The recommended phase 2 dose (RP2D) of rhIL-15 is 1 mcg/kg/day when administered in combination with N+Ipi. The triplet dose expansion phase with the RP2D is currently accruing and includes biomarker studies in blood and tumor biopsies to assess the tumor microenvironment. This study was funded in part by NCI Contract HHSN261200800001E. *In memorium: Dr. Thomas Waldmann7. Citation Format: Sarah J. Shin, Geraldine O'Sullivan Coyne, Howard Streicher, Naoko Takebe, Ashley Bruns, Elad Sharon, Richard Piekarz, Lamin Juwara, Larry Rubinstein, Ralph Parchment, Kristin Fino, King L. Fung, Katherine Ferry-Galow, Arjun Mittra, Abdul Rafeh Naqash, Kevin Conlon, James H. Doroshow, Alice P. Chen. Phase 1 study of recombinant interleukin 15 (rhIL-15) in combination with checkpoint inhibitors nivolumab and ipilimumab in subjects with refractory cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT147.

IBI379, a novel B cell maturation antigen/CD3 bispecific T-cell engager, displays high antitumor efficacy in preclinical models of multiple myeloma

Multiple myeloma (MM) is a hematological malignancy that results from the malignant proliferation of plasma cells in the bone marrow. B cell maturation antigen (BCMA) is highly selectively expressed in malignant plasma cells and is a novel therapeutic target for MM. Here, we developed a bispecific T cell engager, IBI379, that targets BCMA and CD3, and investigated its antitumor efficacy against MM. IBI379 showed strong binding affinity with both BCMA and CD3, which triggered T cell activation, proliferation, and cytokine release. An in vitro study demonstrated that IBI379 induced the lysis of MM cells expressing differing levels of BCMA on the cell surface. Administration of IBI379 in H929 or Daudi-BCMA cell xenograft mouse models significantly inhibited tumor growth without inducing body weight loss. The mechanism of action study revealed the accumulation of CD4+CD8+ T cells and granzyme B-positive T cells in tumors that were treated with IBI379. Moreover, administration of low dose of IBI379 in cynomolgus monkeys was well-tolerated and induced the depletion of BCMA+ B cells and a mild transient increase of cytokines. Collectively, these results demonstrate that IBI379 is a highly potent therapeutic strategy for depleting BCMA-positive B cells and is a promising approach for the treatment of MM.

An Fc-Competent Anti-Human TIGIT Blocking Antibody Ociperlimab (BGB-A1217) Elicits Strong Immune Responses and Potent Anti-Tumor Efficacy in Pre-Clinical Models.

TIGIT (T-cell immunoglobulin and ITIM domain) has emerged as a promising target in cancer immunotherapy. It is an immune “checkpoint” inhibitor primarily expressed on activated T cells, NK cells and Tregs. Engagement of TIGIT to its ligands PVR and PVR-L2 leads to inhibitory signaling in T cells, promoting functional exhaustion of tumor-infiltrating T lymphocytes. Here, we described the pre-clinical characterization of Ociperlimab (BGB-A1217), a novel humanized IgG1 anti-TIGIT antibody (mAb), and systemically evaluated the contribution of Fc functions in the TIGIT mAb-mediated anti-tumor activities. BGB-A1217 binds to the extracellular domain of human TIGIT with high affinity (KD = 0.135 nM) and specificity, and efficiently blocks the interaction between TIGIT and its ligands PVR or PVR-L2. Cell-based assays show that BGB-A1217 significantly enhances T-cell functions. In addition, BGB-A1217 induces antibody dependent cellular cytotoxicity (ADCC) against Treg cells, activates NK cells and monocytes, and removes TIGIT from T cell surfaces in an Fc-dependent manner, In vivo, BGB-A1217, either alone or in combination with an anti-PD-1 mAb elicits strong immune responses and potent anti-tumor efficacy in pre-clinical models. Moreover, the Fc effector function is critical for the anti-tumor activity of BGB-A1217 in a syngeneic human TIGIT-knock-in mouse model. The observed anti-tumor efficacy is associated with a pharmacodynamic change of TIGIT down-regulation and Treg reduction. These data support the selection of BGB-A1217 with an effector function competent Fc region for clinical development for the treatment of human cancers.

A CLDN18.2-Targeting Bispecific T Cell Co-Stimulatory Activator for Cancer Immunotherapy.

BackgroundCo-stimulatory receptor agonist antibodies have shown promising antitumor efficacy in preclinical models. However, their clinical development lags due to systemic or local adverse effects of non-specific T cell activation. Utilization of a bispecific antibody format to reduce off-tumor immune activation is a focus of co-stimulatory receptor agonist antibody design.MethodsIn this study, a bispecific antibody with anti-CLDN18.2 and anti-CD28 moieties was produced. Its T cell costimulation ability was evaluated in T cell coculture assay in vitro. Its safety and anti-tumor efficacy were explored in mouse tumor models.ResultsAnti-CLDN18.2-anti-CD28 bispecific antibody could co-stimulate T cells and increase the expression of effector cytokines in a CLDN18.2-dependent manner. Treatment of anti-CLDN18.2-anti-CD28 could reduce tumor burden and increase tumor-infiltrated T cells. Immunosuppressive cells including tumor-associated macrophages and myeloid-derived suppressor cells were also reduced without systemic adverse effects.ConclusionThis work provided proof-of-concept evidence for a new strategy to develop a bispecific co-stimulatory activator for treating CLDN18.2+ tumors.

Abstract 1854: A Fc-competent anti-human TIGIT blocking antibody BGB-A1217 elicits strong immune responses and potent anti-tumor efficacy in pre-clinical models

Abstract Background: TIGIT (T-cell immunoglobulin and ITIM domain) is a “checkpoint” inhibitory receptor, which is primarily expressed on activated and “exhausted” T and NK cells. Engagement of TIGIT to its ligands (i.e., PVR and PVR-L2) leads to inhibitory signaling in T cells, promoting functional exhaustion of tumor-infiltrating T lymphocytes. BGB-A1217 is a novel humanized IgG1 anti-TIGIT antibody under clinical development. The immunomodulatory activity of BGB-A1217 was evaluated both in vitro and in vivo. Materials and methods: BGB-A1217 was generated through hybridoma fusion, humanized by CDR grafting and structural simulation. The binding affinity and specificity were studied by FACS and SPR. The immunomodulatory functions of BGB-A1217 were evaluated using primary immune cells as well as using animal models. Results: BGB-A1217 binds to the extracellular domain of human TIGIT with high affinity (KD = 0.135 nM) and specificity. In a competition assay, BGB-A1217 efficiently blocks the interaction between TIGIT and PVR. In vitro, BGB-A1217 significantly enhances T-cell functions and induces potential ADCC against TIGITHi targets. In a human T-cell assay, BGB-A1217 enhances IFN-γ production of CMV-specific T cells. In a PBMC assay, BGB-A1217 augments T cell response, either alone or in combination with an anti-PD-1 antibody BGB-A317. Besides blocking TIGIT signaling, BGB-A1217 can also remove TIGIT from cell surface through trogocytosis. This activity is Fc-dependent. In vivo, the Fc effector function is critical for the activity of BGB-A1217 against CT26WT tumor implanted in humanized TIGIT knock-in mice. The observed anti-tumor efficacy is associated with pharmacodynamic change of TIGIT down-regulation, CD226 upregulation and Treg depletion at 48hrs after first dosing. Conclusions: BGB-A1217, either alone or in combination with anti-PD-1 mAb promotes immune cell activation both in vitro and in vivo, supporting its clinical development for the treatment of human cancers. Citation Format: Xin Chen, Liu Xue, Xiao Ding, Qi Liu, Jing Zhang, Lei Jiang, Sha Liu, Hongjia Hou, Qing Zhu, Bin Jiang, Lijie Zhang, Xiaosui Zhou, Jie Ma, Yucheng Li, Wei Jin, Min Wei, Zhirong Shen, Mike Liu, Kang Li, Tong Zhang. A Fc-competent anti-human TIGIT blocking antibody BGB-A1217 elicits strong immune responses and potent anti-tumor efficacy in pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1854.

Abstract 2651: Combination of an anti-angiogenic antibody with PD1/PDL1 blockade agents produces synergistic anti-tumor efficacy in preclinical models

Abstract Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) play a fundamental role in tumor growth and angiogenesis. Signalling through VEGF/VEGFR not only induces cancer angiogenesis but also affects immune cells, making VEGF/VEGFR a pair of well-documented cancer therapeutic targets. Recently more and more studies have focused on combination of anti-VEGF/VEGFR agents and immune checkpoint inhibitors in preclinical and clinical settings. BD0801 is a humanized rabbit anti-VEGF monoclonal antibody currently in clinical development stage. In this study, we have revealed that BD0801 presents more potent activity than Bevacizumab in blockade of VEGF/VEGFR2 binding (IC50=275 ng/ml for BD0801; IC50=1451 ng/ml for Bevacizumab) and VEGFR2 activation in HUVEC assays. BD0801 also showed enhanced inhibitory effects on the proliferation (IC50=87 ng/ml for BD0801; IC50=476 ng/ml for Bevacizumab) and migration of the HUVECs, compared to Bevacizumab. We found that BD0801 exhibits dose-dependent tumor growth inhibitory activities in both lung cancer PC9 xenograft and lung cancer 3LL murine syngeneic tumor models. In addition, we obtained PK parameters of BD0801 using lung cancer PC9 tumor bearing mice and successfully made correlations between the in vitro function, PK profile and in vivo efficacy of BD0801. Notably, combination of BD0801 with either anti-PD-1, or anti-PD-L1 antibodies showed synergistic anti-tumor efficacy in both lung and colorectal cancer mouse models. Mechanistic studies were conducted to reveal possible mechanism of actions (MOA) underlying the modification in tumor microenvironment. Our data demonstrated that the MOA of the anti-tumor synergy may involve enhanced T-cell mediated immunity, including increased tumor infiltration of CD8+ and CD4+ T cells and reduced double positive CD8+PD-1+ T cells, as well as improved vasculature normalization. Taken together, these data provide a foundation for combining BD0801 with immunotherapy for cancer treatment and support further clinical development plans for BD0801 toward this direction. Citation Format: Liting Xue, Haoyu Zhang, Qian Wang, Feng Li, Xinxin Li, Xiaohong Yu, Zhihong Lu, Yue Huang, Wenqing Yang. Combination of an anti-angiogenic antibody with PD1/PDL1 blockade agents produces synergistic anti-tumor efficacy in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2651.

Preliminary clinical and biologic results of GB1275, a first-in-class oral CD11b modulator, alone and with pembrolizumab, in advanced solid tumors (KEYNOTE A36).

2505 Background: GB1275 is a first-in-class, oral CD11b modulator that reduced myeloid-derived suppressor cells (MDSCs) and tumor associated macrophages (TAMs), repolarized M2 immunosuppressive TAMs to an M1 phenotype, resulting in increased tumor infiltration of activated CD8+ T cells and antitumor efficacy in preclinical models. Here, we report preliminary results from an ongoing, first-in-human dose-escalation study in specific advanced tumors using GB1275 alone or with pembrolizumab. (NCT04060342) Methods: Phase 1 comprises dose escalation and expansion. During dose escalation, cohorts of 3 to 6 subjects were sequentially assigned to ascending dose levels of GB1275 from 100 mg to 1200 mg BID in one of two dosing regimens: Regimen A [GB1275 monotherapy orally (PO) twice a day (BID)] and Regimen B [GB1275 PO BID plus pembrolizumab 200 mg IV every 3 weeks (q3wks)]. Dose escalation was based on safety including dose-limiting toxicities (DLTs). Following dose escalation, up to 40 subjects with specific tumor types are to be treated in expansion with the selected GB1275 dose plus pembrolizumab to assess safety, pharmacokinetics, and preliminary clinical and biomarker activity. Results: As of January 8, 2021, 45 subjects were treated [44 in dose escalation: 23, Regimen A; 21, Regimen B. 1 in expansion, Regimen B], with median (range) GB1275 exposure of 42.0 days (4-263). No DLTs were reported. GB1275-related adverse events occurred in 24/45 (53.3%) subjects; photosensitivity reaction (20.0%), dysesthesia (13.3%) and pruritus (13.3%) were most frequent (≥10%). Stable disease was reported in 6/19 (31.6%) response-evaluable subjects in Regimen A and 9/16 (56.3%) in Regimen B. In Regimen B (800 mg), one partial response was reported in a subject with MSS-CRC treated for 263 days, and one prolonged stable disease (227 days) was reported in a gastric cancer (GC) subject previously treated with pembrolizumab plus bavituximab for less than 3 months due to progression; both subjects are continuing study treatment. A dose-dependent increase in GB1275 systemic exposure was observed up to 800 mg BID. Down-regulation of peripheral MDSCs was seen with both regimens. Regimen-dependent gene clusters in whole blood were noted. An increase in tumor infiltrating lymphocyte (TIL) counts was noted in both Regimens A and B. Conclusions: Dose escalation of GB1275, up to 1200 mg in Regimens A and B, demonstrated tolerability as monotherapy and combined with pembrolizumab in subjects with advanced cancers. Encouraging antitumor activity in Regimen B (800 mg) was observed in subjects with MSS-CRC and GC. Biological activity reflected by MDSC modulations in blood and TIL Increases in tumor biopsies with GB1275 alone and with pembrolizumab supports the mechanism of GB1275. GB1275 800 mg BID plus pembrolizumab 200 mg IV q3wks was selected for evaluation in the expansion phase. Clinical trial information: NCT04060342.