Multiple Preclinical Tumor Models Research Articles

YF550-C1, an E3 ligase inhibitor of CBL-B, to demonstrate T cell and NK cell activation and anti-tumor activities in syngeneic and PDX tumor model.

e14570 Background: Casitas B-lineage lymphoma b (CBL-B) is one of ligase in E3 ubiquitin family, which mainly expressed in matured hematopoietic cells, including T and NK cells. Study showed that CBL-B deficiency upregulated interleukin-2 and interferon-gamma secretion during anti-CD3 and anti-CD28 stimulated TCR activation, even without co-stimulator anti-CD28[3]. Except T cells, CBL-B plays important role in NK cells as well. CBL-B was upregulated during NK cell activation. While downregulation of CBL-B enhanced the cytotoxicity of primary human NK cells against hematological cancer cell lines. In vivo studies proved that CBL-B deficiency protected mice from tumor growth by inducing antitumor immunity. Thus, CBL-B shows a promising target for cancer therapy. Methods: We developed a small molecule YF550-C1 that targeted CBL-B, and evaluated the activation on T cell and NK cell function, and the therapeutical effect on syngeneic tumor models. The T cell activation was indicated by levels of IL-2 and IFN-γ from CBL-B knockout or WT CD8+ T cells since stimulated with anti-CD3 or anti-CD3/28 for 48 hours. The resistance of PGE2-induced immunosuppressive was evaluated by measuring IL-2 and IFN-γ since human T cells treated with PGE2 and followed by series diluted YF550-C1 or comparable inhibitor cpd23. OVA specific antigen stimulated CD8+ T activation assay was conducted by coculture OT-1 CD8+ T cells and WT DC cells with 50:1 for 72 hours in the present of 5ug/ml OVA peptide and series diluted YF550-C1. The NK cell cytotoxicity was evaluated by co-culture human NK cells and K562-luc cells with 1.25:1 ratio in the present of 1000nM or 333nM YF550-C1, which indicated by luminance. Syngeneic mouse model that inoculated with different types of murine cancer cell lines was used to evaluate tumor growth. Results: CBL-B deficiency suppressed tumor growth in mouse tumor models and enhanced cytokines release in T cells upon TCR engagement. CBL-B inhibitor, YF550-C1, alleviated PGE2-mediated immunosuppression of human T cell activation. CBL-B inhibitor, YF550-C1, enhanced OVA-specific antigen stimulated CD8+ T cell activation. CBL-B inhibitor, YF550-C1, enhanced cytotoxicity of human NK cell against K562-luc cells. CBL-B inhibitor, YF550-C1, or in combinate with anti-PD-L1 suppressed tumor growth in syngeneic tumor models. Single-agent of CBL-B inhibitor YF550-C1 treatment was sufficient to suppress PD1/PD-L1 resistant NSCLC patient-derived tumor growth. Conclusions: These studies provided insight into the activity of the inhibitor of CBL-B, demonstrating that YF550-C1 displays excellently T cell activation, T cell immunosuppression alleviation, NK cell activation and antitumor activity in multiple preclinical tumor models. Our data showed that YF550-C1 is a promising candidate for cancer treatment as monotherapy or in combination with PD-L1 blockade.

A phase I trial of intratumoral STX-001: A novel self-replicating mRNA expressing IL-12 alone or with pembrolizumab in advanced solid tumors.

TPS2696 Background: STX-001 is a multi-mechanistic lipid nanoparticle encapsulated synthetic self-replicating mRNA, engineered to express the IL-12 cytokine for an extended duration when administered intratumorally. A murine surrogate of STX-001 induces deep durable responses in multiple preclinical solid tumor models. This Phase 1 open-label, multi-center first-in-human dose-escalation trial evaluates the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary antitumor activity of STX-001 alone, or in combination with pembrolizumab in patients with treatment refractory advanced cancers. Methods: Approximately 30 adults ≥18 years of age with histologically confirmed advanced solid tumors that are refractory to standard treatments will be enrolled. The study will consist of two arms, an STX-001 monotherapy arm and an STX-001 + pembrolizumab combination arm, enrolling approximately 15 patients each. Key eligibility criteria include ECOG status 0 or 1, a tumor lesion amendable to injection, and confirmed prior disease progression. STX-001 will be administered every 3 weeks (q3w) for 3 doses and then every 6 weeks (q6w) for a further 3 doses. Tumor biopsies will be obtained at baseline and after the first STX-001 administration. Primary endpoints will include safety and maximum tolerated dose (MTD), with secondary endpoints encompassing pharmacokinetics and preliminary efficacy as measured by RECIST 1.1. To determine the MTD, this study will employ the Bayesian optimal interval (BOIN) design, with the 3 + 3 design run-in and a recommended phase 2 dose (RP2D) will be determined. Tumor response will be assessed using RECIST 1.1 criteria. Exploratory pharmacodynamic analyses will assess tumor-infiltrating lymphocytes and STX-001 mRNA in baseline and on-treatment biopsies, evaluate inflammatory cytokine profiles in plasma, determine immune activation in circulating immune cells, and measure circulating tumor DNA (ctDNA) for molecular response assessment. Clinical trial information: NCT06249048 .

Abstract 514: Salt-inducible kinase inhibitor OMX-0407 drives cell-cycle arrest in vitro and in vivo: An in-depth MoA analysis by phospho-proteomics

Abstract Based on iOmx’ proprietary iOTargTM genetic screening platform, salt-inducible kinase (SIK) 3 was identified as a novel cell signaling modulator in cancer biology. OMX-0407, an orally available, spectrum-selective kinase inhibitor targets several members of the SIK family and is known to prevent tumor-promoting function of the SIK-family through repolarization of the tumor microenvironment by enhancing caspase-mediated apoptosis upon death-receptor signaling. In addition, OMX-0407 inhibits individual key members of the tyrosine and tyrosine-like kinase family that are involved in cancer cell proliferation and cell cycle regulation. Via this dual mode of action, OMX-0407 has the potential to effectively fight solid tumors as exemplified by its striking single-agent efficacy in multiple pre-clinical tumor models. A comprehensive anti-tumor viability screen of >200 human cancer cell lines revealed striking effects of OMX-0407 on cancer cell viability across various cancer indications, resulting in a distinct sensitivity profile across various tumor indications with particularly strong effects on renal cell carcinoma (RCC) and squamous non-small cell lung cancer (sqNSCLC). Phospho-proteomics screening demonstrated pharmacodynamic activity of OMX-0407 in sensitive tumor cell lines via inhibition of key cellular processes such as cell motility, proliferation, and cell cycle regulation across different indications. Orthogonal functional in vitro assays confirmed the association of the OMX-0407-mediated anti-tumor efficacy with the arrest of G1/S transition and corresponding downregulation of cell cycle associated proteins PAK1/2 and ARHGAP35. The profound impact of OMX-0407 on fundamental regulatory mechanisms of cell division and cancer cell apoptosis translates into dose-dependent single-agent, anti-tumor efficacy in various pre-clinical tumor models of selected indications. Combination with Axitinib, a selective orally available inhibitor of vascular endothelial growth factor receptor 2, significantly enhanced anti-tumor efficacy and pharmacodynamic inhibition of cell growth as well as angiogenesis. These data further strengthen the potential of OMX-0407 in the treatment of RCC as well as other indications. In summary, OMX-0407 is a novel spectrum selective kinase inhibitor, currently under evaluation in a clinical Phase I trial (NCT05826600). OMX-0407 demonstrates strong anti-tumor efficacy in monotherapy in selected sensitive indications through a dual-pronged MoA, by modulating the tumor microenvironment and exerting direct anti-tumor activity in solid tumor indications with high unmet medical need. Citation Format: Ilona-Petra Maser, Marisa Stebegg-Wagner, Bettina Bauer, Filippos Konstantinidis, Andreas Schirmer, Moritz Zulley, Sonja Lacher, Barbara Kracher, Parastou Kohvaei, Murray Yule, Hannes Loferer, Stefan Bissinger. Salt-inducible kinase inhibitor OMX-0407 drives cell-cycle arrest in vitro and in vivo: An in-depth MoA analysis by phospho-proteomics [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 514.

The Human Soluble NKG2D Ligand Differentially Impacts Tumorigenicity and Progression in Temporal and Model-Dependent Modes.

NKG2D is an activating receptor expressed by all human NK cells and CD8 T cells. Harnessing the NKG2D/NKG2D ligand axis has emerged as a viable avenue for cancer immunotherapy. However, there is a long-standing controversy over whether soluble NKG2D ligands are immunosuppressive or immunostimulatory, originating from conflicting data generated from different scopes of pre-clinical investigations. Using multiple pre-clinical tumor models, we demonstrated that the impact of the most characterized human solid tumor-associated soluble NKG2D ligand, the soluble MHC I chain-related molecule (sMIC), on tumorigenesis depended on the tumor model being studied and whether the tumor cells possessed stemness-like properties. We demonstrated that the potential of tumor formation or establishment depended upon tumor cell stem-like properties irrespective of tumor cells secreting the soluble NKG2D ligand sMIC. Specifically, tumor formation was delayed or failed if sMIC-expressing tumor cells expressed low stem-cell markers; tumor formation was rapid if sMIC-expressing tumor cells expressed high stem-like cell markers. However, once tumors were formed, overexpression of sMIC unequivocally suppressed tumoral NK and CD8 T cell immunity and facilitated tumor growth. Our study distinguished the differential impacts of soluble NKG2D ligands in tumor formation and tumor progression, cleared the outstanding controversy over soluble NKG2D ligands in modulating tumor immunity, and re-enforced the viability of targeting soluble NKG2D ligands for cancer immunotherapy for established tumors.

Intralesional administration of VAX014 facilitates in situ immunization and potentiates immune checkpoint blockade in immunologically cold tumors

BackgroundImmunologically cold tumors with an ‘immune desert’ phenotype lack tumor-infiltrating lymphocytes (TILs) and are typically impervious to systemic immune checkpoint blockade (ICB). Intratumoral treatment of tumors with immunomodulatory agents can...

The primordial differentiation of tumor-specific memory CD8+ T cells as bona fide responders to PD-1/PD-L1 blockade in draining lymph nodes

Blocking PD-1/PD-L1 signaling transforms cancer therapy and is assumed to unleash exhausted tumor-reactive CD8+ Tcells in the tumor microenvironment (TME). However, recent studies have also indicated that the systemic tumor-reactive CD8+ Tcells may respond to PD-1/PD-L1 immunotherapy. These discrepancies highlight the importance of further defining tumor-specific CD8+ Tcell responders to PD-1/PD-L1 blockade. Here, using multiple preclinical tumor models, we revealed that a subset of tumor-specific CD8+ cells in the tumor draining lymph nodes (TdLNs) was not functionally exhausted but exhibited canonical memory characteristics. TdLN-derived tumor-specific memory (TTSM) cells established memory-associated epigenetic program early during tumorigenesis. More importantly, TdLN-TTSM cells exhibited superior anti-tumor therapeutic efficacy after adoptive transfer and were characterized as bona fide responders to PD-1/PD-L1 blockade. These findings highlight that TdLN-TTSM cells could be harnessed to potentiate anti-tumor immunotherapy.

Targeting the Mitochondria with Pseudo-Stealthy Nanotaxanes to Impair Mitochondrial Biogenesis for Effective Cancer Treatment.

The clinical success of anticancer therapy is usually limited by drug resistance and the metastatic dissemination of cancer cells. Mitochondria are essential generators of cellular energy and play a crucial role in sustaining cell survival and metastatic escape. Selective drug strategies targeting mitochondria are able to rewire mitochondrial metabolism and may provide an alternative paradigm to treat many aggressive cancers with high efficiency and low toxicity. Here, we present a pseudo-stealthy mitochondria-targeted pro-nanotaxane and test it against recurrent and metastatic tumor xenografts. The nanoparticle encapsulates a mitochondria-targetable pro-taxane agent, which can be converted into the chemically unmodified cabazitaxel drug, with further surface cloaking with a low-density lipophilic triphenylphosphonium cation. The resultant nanotaxane could be effectively taken up by cells and consequently specifically localized to the mitochondria. The in situ activated cabazitaxel causes mitochondrial dysfunction and ultimately results in potent cell apoptosis. After intravenous administration to animals, pro-nanotaxane mimics the stealthy behavior of polyethylene glycol-cloaked nanoparticles to provide a long circulation time. The antitumor efficacy of this mitochondria-targeted system was validated in multiple preclinical drug-resistant tumor models. Notably, in a patient-derived metastatic melanoma model that was initially pretreated with cabazitaxel, nanotaxane administration not only produced durable tumor reduction but also substantially suppressed metastatic recurrence. Taken together, these results demonstrate that this combination of a pseudo-stealthy platform with a rationally designed pro-drug is an attractive approach to target mitochondria and enhance drug efficacy.

Abstract 593: Anti-tumor immunity and efficacy of combination treatment of M6223 and bintrafusp alfa versus the combination of M6223 and anti-PD-L1 in preclinical tumor models

Abstract Background: T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT) is an inhibitory receptor expressed on lymphocytes and has recently emerged as a target in cancer immunotherapy. M6223 is a fully human antagonistic anti-TIGIT antibody in immunoglobulin (Ig) G1 format with Fc-mediated effector function. Preclinical studies demonstrated that M6223 could induce an anti-tumor immune response by complementary mechanisms including but not limited to direct blockade of the TIGIT pathway, stimulation of CD226 dimerization/activation, and depletion of TIGIT+ immune subsets by Fc-mediated effector function. M6223 showed dose-dependent anti-tumor efficacy in multiple preclinical tumor models. Bintrafusp alfa, a first-in-class bifunctional fusion protein composed of the extracellular domain of the human transforming growth factor β receptor II (TGF-βRII or TGF-β "trap") fused via a flexible linker to the C-terminus of each heavy chain of an IgG1 antibody blocking programmed death ligand 1 (anti-PD-L1), was designed to target two key immunosuppressive pathways in the tumor microenvironment. Methods: To attenuate the tumor immunosuppressive microenvironment, the anti-tumor immunity and efficacy of combining M6223 with bintrafusp alfa was investigated in syngeneic models in humanized mice with huTIGIT knock-in sequences. Results: M6223 combined with bintrafusp alfa significantly enhanced anti-tumor efficacy and extended survival compared with either monotherapies or the combination of M6223 with anti-PD-L1. M6223 combined with bintrafusp alfa stimulated higher CD8+ T cells and natural killer cells infiltration, proliferation and cytotoxicity in tumor microenvironment in comparison to the combination of M6223 with anti-PD-L1. The ratio of CD8+ T cells to regulatory T cells and the ratio of CD226 to TIGIT were significantly increased, indicating the conversion of the tumor microenvironment from an immuno-suppressive phenotype to a more immune-permissive phenotype. With the combination therapy, bintrafusp alfa was the main driver promoting CD8+ T cell proliferation, infiltration, and cytotoxicity. Alternatively, adding M6223 to bintrafusp alfa may decrease the risk of immune resistance caused by elevated TIGIT expression triggered by bintrafusp alfa treatment. Conclusion: These complementary mechanisms of M6223 and bintrafusp alfa orchestrate antitumor activity in preclinical tumor models. Currently, M6223 and bintrafusp alfa combination is being investigated in a Phase I clinical trial (NCT04457778) in patients with metastatic or locally advanced solid unresectable tumors. Citation Format: Chunxiao Xu, Sireesha Yalavarthi, Clotilde Bourin, Christie Kelton, Joern-Peter Halle, Jacques Moisan. Anti-tumor immunity and efficacy of combination treatment of M6223 and bintrafusp alfa versus the combination of M6223 and anti-PD-L1 in preclinical tumor models [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 593.

Monocarboxylate transporter 1 is a novel target for breast cancer stem like-cell inhibition by diallyl trisulfide.

Diallyl trisulfide (DATS) is a promising small molecule phytochemical that exhibits in vitro and in vivo activity in multiple preclinical solid tumor models including breast cancer, but the underlying mechanism is not fully understood. We have shown previously that forkhead box Q1 (FoxQ1) transcription factor is a novel target for breast cancer stem-like cells (bCSC) inhibition by DATS. Analysis of the breast TCGA (The Cancer Genome Atlas) data revealed that FoxQ1 expression was positively associated with that of SLC16A1/monocarboxylate transporter 1 (MCT1). Western blot analysis confirmed increased expression of MCT1 protein in SUM159 (basal-like) and MCF-7 cells (luminal-type) stably transfected to overexpress FoxQ1. Furthermore, FoxQ1 was recruited to the promoter of SLC16A1/MCT1. Treatment of SUM159 and MCF-7 cell lines with DATS resulted in suppression of MCT1 protein level that was accompanied by a decrease in intracellular and secreted levels of lactate. Overexpression or knockdown of MCT1 protein failed to alter DATS-mediated inhibition of colony formation or cell migration when compared to corresponding control cells. On the other hand, overexpression of MCT1 protein conferred partial but statistically significant protection against DATS-mediated inhibition of bCSC fraction (CD49fhigh /CD44high and aldehyde dehydrogenase 1 activity). The size of the mammospheres was relatively smaller in the DATS-treated group compared to control group. Inhibition of bCSC upon DATS treatment was augmented by knockdown of the MCT1 protein. In conclusion, the present study reveals that MCT1 is a novel target for bCSC inhibition by DATS treatment.

Tie2 Receptor in Tumor-Infiltrating Macrophages Is Dispensable for Tumor Angiogenesis and Tumor Relapse after Chemotherapy.

Multiple preclinical tumor models, cell stimulation experiments, and meta-analysis of published tumor single cell RNA sequencing data challenge the reported role of Tie2-positive macrophages for tumor angiogenesis, metastasis, and relapse after chemotherapy. See related commentary by Zhang and Brekken, p. 1172.

Development of a Stable Peptide-Based PET Tracer for Detecting CD133-Expressing Cancer Cells.

CD133 has been recognized as a prominent biomarker for cancer stem cells (CSCs), which promote tumor relapse and metastasis. Here, we developed a clinically relevant, stable, and peptide-based positron emission tomography (PET) tracer, [64Cu]CM-2, for mapping CD133 protein in several kinds of cancers. Through the incorporation of a 6-aminohexanoic acid (Ahx) into the N terminus of a CM peptide, we constructed a stable peptide tracer [64Cu]CM-2, which exhibited specific binding to CD133-positive CSCs in multiple preclinical tumor models. Both PET imaging and ex vivo biodistribution verified the superb performance of [64Cu]CM-2. Furthermore, the matched physical and biological half-life of [64Cu]CM-2 makes it a state-of-the-art PET tracer for CD133. Therefore, [64Cu]CM-2 PET may not only enable the longitudinal tracking of CD133 dynamics in the cancer stem cell niche but also provide a powerful and noninvasive imaging tool to track down CSCs in refractory cancers.

Abstract P252: IMM-1-104: a novel, oral, selective dual-MEK inhibitor that displays broad antitumor activity and high tolerability across RAS and RAF mutant tumors in vivo

Abstract Background: Inappropriate activation of the MAPK pathway, often stemming from mutations in RAS or RAF, represents one of the most common oncogenic events in human cancer. Therefore, MEK1 and MEK2 (MEK), which lie downstream of RAS and RAF but upstream of ERK, have represented an attractive drug target for over two decades. Unfortunately, first generation MEK inhibitors have been limited by pathway reactivation events and serious on-target drug toxicities that restrain clinical utility, especially in patients with RAS mutant tumors. We sought to develop a new approach to MEK inhibition that would be more effective in RAS mutant tumors and with improved tolerability. IMM-1-104 is a novel dual-MEK inhibitor that is designed to disrupt phosphorylation of MEK and subsequently prevent activation of ERK1 and ERK2 (ERK). IMM-1-104’s dual-MEK mechanism resists RAF activation of MEK, and its short drug half-life allows for chronic dosing while maintaining a near-zero drug trough for improved tolerability. Materials & Methods: IMM-1-104 was profiled across a series of preclinical experiments to assess its physicochemical and drug-like properties. Cell-free and cell-based in vitro as well as in vivo characteristics were evaluated, including four independent in vivo pharmacology rodent studies in lung, colon and skin tumor models. Results: IMM-1-104 is a highly selective, orally bioavailable, non-ATP competitive, allosteric dual-MEK inhibitor. At drug exposures up to 1 micromolar (uM) in cell-free and in situ kinome screens, thermodynamic interactions and altered activity levels were observed for MEK1 and MEK2. At higher drug exposures up to 10 uM, IMM-1-104 also thermodynamically interacted with RAF1 (CRAF) and prompted reduction of KSR1 and KSR2 activation markers. IMM-1-104 displayed dual-MEK activity (i.e., RAS mutant tumor cell reductions in both ERK and MEK phosphorylation) across multiple human tumor cell-based models including A549 (KRAS-G12S) lung, A375 (BRAF-V600E) and SK-MEL-2 (NRAS-Q61R) melanoma. Pharmacokinetic studies in rodent models revealed a drug plasma half-life for IMM-1-104 of approximately 1.3 hours. In vivo tumor pharmacology studies in A549 and A375 achieved tumor stasis (i.e., 85% to 95% Tumor Growth Inhibition) with regressions observed in Colon-26 and SK-MEL-2. Conclusions: The dual-MEK mechanism and short half-life of IMM-1-104 has proven to be broadly active and well-tolerated in multiple RAS and RAF mutant preclinical tumor models in rodents. IMM-1-104 drives deep, cyclic inhibition of the MAPK pathway (improving tolerability) while resisting pathway bypass mechanisms (improving activity). Our collective data suggest that RAS and RAF mutant tumor cells are not able to tolerate periods of deep but cyclic MAPK pathway inhibition. Overall, these results are consistent with on and off signaling events that can significantly impact cell fate decisions (i.e. signaling dynamics). Citation Format: Peter J. King, Kevin D. Fowler, Sarah E. Kolitz, Scott Barrett, Benjamin J. Zeskind, Brett M. Hall. IMM-1-104: a novel, oral, selective dual-MEK inhibitor that displays broad antitumor activity and high tolerability across RAS and RAF mutant tumors in vivo [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P252.

Abstract P240: Benchmarking the novel dual-MEK inhibitor, IMM-1-104, head-to-head and in combination with sotorasib (AMG-510) in the MIA PaCa-2 (KRAS-G12C) pancreatic cancer xenograft model

Abstract Background: Approximately 9 out of 10 patients diagnosed with pancreatic cancer have tumors driven by a single activating mutation in KRAS. These tumors remain unaddressable by current therapies and collectively represent a high unmet clinical need. KRAS is the most commonly mutated form of RAS and represents approximately 85% of all RAS mutant human tumors with HRAS and NRAS accounting for the remaining 15%. Sotorasib (AMG-510), a covalent KRAS-G12C inhibitor, recently received accelerated regulatory approval for KRAS-G12C lung cancer in the United States. While the G12C mutation has been documented in up to 15% of all KRAS mutant tumors, it is rarely observed (<1%) in human pancreatic cancer, which could severely limit this type of new inhibitor in certain histologies. IMM-1-104 is a novel dual-MEK inhibitor that resists CRAF-bypass (goal: improve activity against RAS mutant tumors), and is designed to have a short plasma drug half-life, which leads to deep, cyclic inhibition of the MAPK pathway (goal: improved tolerability). Materials & Methods: The novel, orally bioavailable dual-MEK inhibitor, IMM-1-104, was evaluated for antitumor activity, as a single agent (50, 100, 150 mg/kg BID p.o.) in a head-to-head format against sotorasib (10, 30, 100 mg/kg QD p.o.) or in combination (50, 100, 150 mg/kg BID p.o. IMM-1-104 with 30 mg/kg QD p.o. sotorasib) in the MIA PaCa-2 KRAS-G12C mutant pancreatic tumor xenograft model. Tumor-bearing animals were treated for 21 days of oral dosing, before drug treatments were terminated and animals monitored for additional 3 weeks. Results: IMM-1-104 and sotorasib were well-tolerated as single agents and in combination with each other across all dose levels tested. Both drugs demonstrated dose-dependent antitumor activity with tumor regressions noted at higher dose levels. IMM-1-104 in combination with sotorasib produced deep regressions that were sustained longer than either drug alone. Conclusions: The dual-MEK mechanism and short half-life of IMM-1-104 has demonstrated broad activity with low toxicity across multiple RAS and RAF mutant preclinical tumor models. Tumor regressions were observed in NRAS mutant melanoma (SK-MEL-2) and KRAS mutant colorectal (Colon-26) rodent tumor models, and tumor stasis was observed in KRAS mutant lung (A549) and BRAF mutant melanoma (A375) tumor xenograft models. Here, we report that IMM-1-104 treatment resulted in tumor regressions similar to that observed for sotorasib in the recently benchmarked KRAS-G12C mutant pancreatic cancer xenograft model (MIA PaCa-2). IMM-1-104 in combination with sotorasib promoted deep, durable tumor regressions, when compared to either drug alone. Therefore, future drug-drug combinations with upstream inhibitors such as sotorasib may afford greater durability in combination for patients with KRAS-G12C and other select tumor types. In totality, these data suggest the potential for broad, single agent activity of IMM-1-104 in tumors with inappropriately elevated MAPK signaling, including a large percentage of KRAS mutant pancreatic cancer. Citation Format: Peter J. King, Amy E. Axel, Kevin D. Fowler, Sarah E. Kolitz, Scott Barrett, Benjamin J. Zeskind, Brett M. Hall. Benchmarking the novel dual-MEK inhibitor, IMM-1-104, head-to-head and in combination with sotorasib (AMG-510) in the MIA PaCa-2 (KRAS-G12C) pancreatic cancer xenograft model [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P240.

Safety and Efficacy of Vibostolimab and Pembrolizumab in Patients with Relapsed or Refractory Hematologic Malignancies: A Multicohort, Open-Label, Phase 2 Study

Background: The T-cell immunoglobulin and ITIM domain (TIGIT) is highly coexpressed with programmed cell death 1 (PD-1) on both CD4 and CD8 T cells in cancers. Blockade of TIGIT with vibostolimab (MK-7684) has demonstrated antitumor activity in multiple preclinical tumor models. Inhibition of the PD-1 pathway with pembrolizumab has demonstrated efficacy and safety in several hematologic malignancies. Enhanced antitumor activity with anti-TIGIT and anti-PD-L1 combination therapy has been seen in a preclinical model. A multicohort, open-label, phase 2 study will evaluate the safety and efficacy of MK-7684A, a coformulation of vibostolimab and pembrolizumab, in patients with relapsed or refractory (r/r) hematologic malignancies.Study Design and Methods: This two-part phase 2 study includes a signal-finding phase (part 1) with the option for a dose-expansion phase (part 2), depending on the observed risk-benefit profile from part 1. Patients must have confirmed diagnosis of r/r disease, including classical Hodgkin lymphoma (cHL; cohort A or B), primary mediastinal B-cell lymphoma (PMBCL; cohort A or B), follicular lymphoma (FL; cohort C), diffuse large B-cell lymphoma (DLBCL; cohort D), multiple myeloma (MM; cohort E), or non-Hodgkin lymphoma (NHL; cohort F) (Table 1). Three hundred thirty participants are expected to enroll in part 1 (signal finding) and part 2 (cohort expansion). In part 1, patients in cohorts A-F will receive MK-7684A (vibostolimab 200 mg + pembrolizumab 200 mg) by IV infusion every 3 weeks (Q3W) for up to 35 cycles. Patients will continue treatment until investigator-determined PD, start of new anticancer treatment, documented complete response, or completion of maximum 35 cycles of treatment. Safety data from the first 12 treated patients from any cohort will be collected. Dose-limiting toxicities (DLTs) will be monitored for the first 6 weeks of continuous treatment. If ≥5 of the 12 patients experience DLTs during this period, enrollment may be discontinued. After the DLT review period, safety data will be collected every 6 months for all enrolled patients. Efficacy assessments will be performed Q3W, with the initial tumor imaging performed ≤28 days after enrollment and repeated at 12 weeks (cohorts A-D, F) or 4 weeks (cohort E) after enrollment. Adverse events (AEs) will be monitored throughout the study, and severity will be graded according to the guidelines outlined in NCI CTCAE version 5.0. Each patient will be monitored for AEs and serious AEs for 30 day and 90 days, respectively, after discontinuation of study intervention. The primary end point for part 1 is the number and proportion of patients with DLTs, AEs, and discontinuations from study treatment due to AEs. Safety and tolerability will be assessed by clinical review. Secondary end points include investigator-assessed objective response rate, investigator-assessed duration of response, disease control rate, and pharmacokinetic end points. Overall survival, progress-free survival, health-related quality of life assessments, and molecular assessments will be exploratory. In the part 2 dose-expansion phase, the plan is to enroll approximately 120 patients from cohorts B-E to receive up to 35 cycles of MK-7684A Q3W; 30 additional patients may be enrolled in a cohort expansion (cohort G) to receive vibostolimab monotherapy Q3W for up to 35 cycles. Cohort G will include patients with cHL or PMBCL with PD after ≥2 or ≥3 prior therapies, FL after ≥2 prior therapies, DLBCL after ≥2 prior therapies, and/or MM after ≥3 prior therapies. [Display omitted] DisclosuresYusuf: Merck & Co., Inc.: Current Employment. Jemielita: Merck & Co., Inc.: Current Employment, Other: Current Stockholder. Marinello: Merck & Co., Inc.: Current Employment, Other: Current Stockholder.

Local delivery of mRNA-encoded cytokines promotes antitumor immunity and tumor eradication across multiple preclinical tumor models.

Local immunotherapy ideally stimulates immune responses against tumors while avoiding toxicities associated with systemic administration. Current strategies for tumor-targeted, gene-based delivery, however, are limited by adverse effects such as off-targeting or antivector immunity. We investigated the intratumoral administration of saline-formulated messenger (m)RNA encoding four cytokines that were identified as mediators of tumor regression across different tumor models: interleukin-12 (IL-12) single chain, interferon-α (IFN-α), granulocyte-macrophage colony-stimulating factor, and IL-15 sushi. Effective antitumor activity of these cytokines relied on multiple immune cell populations and was accompanied by intratumoral IFN-γ induction, systemic antigen-specific T cell expansion, increased granzyme B+ T cell infiltration, and formation of immune memory. Antitumor activity extended beyond the treated lesions and inhibited growth of distant tumors and disseminated tumors. Combining the mRNAs with immunomodulatory antibodies enhanced antitumor responses in both injected and uninjected tumors, thus improving survival and tumor regression. Consequently, clinical testing of this cytokine-encoding mRNA mixture is now underway.

Abstract 1720: Ax-101, an immunostimulatory small molecule, markedly enhances the antitumor activity of PD1 immune checkpoint blockade in multiple preclinical solid tumor models

Abstract Ax-101, a small-molecule immunomodulator with a yet-to-be elucidated MoA, enhances lymphocyte proliferation, induces a Th1 profile, increases T-cell cytotoxicity, and improves the M1/M2 ratio. Specific Ax-101 effects include upregulated cell-surface TNFalpha (sTNF) on human T cells and monocytes, increased (>7.5x) T-cell cytotoxicity, upregulation by human PBMC of antitumor cytokines (e.g., IL-2, IFN-gamma, TNFalpha) and downregulation of cytokines that mediate immune evasion (e.g., IL-10), and decreased immune-suppressive myeloid cells. Syngeneic mouse cancer models including bladder (MBT-2), colorectal (CT-26), melanoma (Cloudman), and lymphoma (A20) show the addition of Ax-101 to anti-PD1 to improve outcomes. For example, neither Ax-101 nor anti-PD1 monotherapy had significant effects on mean tumor volume (MTV) in MBT-2 mice, while an Ax-101/anti-PD1 (same doses/regimen) combo resulted in up to 44% reduction in MTV c/w anti-PD1 alone (p < 0.05). Further, 30% (3/10) of MBT-2 mice achieved CR with the combo, whereas no CRs were seen with anti-PD1 alone. In CT-26 mice, 25% (2/8) achieved CR, whereas no CRs occurred with anti-PD1 only; subset analysis of mice exhibiting at least some antitumor response to anti-PD1 alone (6/8) or the Ax-101/anti-PD1 combo (5/8) revealed a 40% CR rate and MTV reduction of up to 75% in combo-treated animals. In Cloudman melanomas, all (5/5) mice receiving Ax-101/anti-PD1 throughout their treatment achieved CR. Importantly, in mice with rapidly growing melanomas refractory to anti-PD1 for 12 d, Ax-101 addition reversed growth, with 4/5 mice achieving CR. Of note, 9 Cloudman mice achieving CR were re-injected with 3x the melanoma cells they initially received and followed with no therapy. Only 1/9 mice developed tumor over 73 d; thus, Ax-101/anti-PD1 may elicit a vaccine-like effect. Anti-PD1 alone and the Ax-101/antiPD1 combo in A20 mice resulted in 23% and 40% lower MTVs c/w control. Ax-101 was previously in Ph 1/2 monotherapy studies in follicular lymphoma (FL) and myeloma (M) (CT.gov IDs: NCT00006466, NCT00007839). Thirty-two patients received 2 µg SQ Ax-101 q2 wkly or wkly for up to 18 months. No Grade 3 or higher toxicities were observed. Five of 14 FL patients showed tumor reduction from 16-64%, while 1/17 M patients showed 50% reduced M-protein; maximum responses occurred between 43 days and over 1 year. Increased PBMC sTNF was seen in all patients tested (8 with FL, 6 with M) (p = 0.0006). Consistent with Ax-101 activity depending on a functional immune system, delayed type hypersensitivity (DTH) testing showed all responders to be DTH+ (5 tumor reduction, 4 SD); 5 anergic (DTH-) patients had SD at best (2 SD, 3 worse). Current Ax-101 clinical development will leverage its beneficial immunostimulatory properties to augment the efficacy of anti-PD1 inhibitors such as pembrolizumab, nivolumab and cemiplimab. Citation Format: Stephan W. Morris, Betsy Fisher, Kent Murphy. Ax-101, an immunostimulatory small molecule, markedly enhances the antitumor activity of PD1 immune checkpoint blockade in multiple preclinical solid tumor 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 1720.

Abstract 443: Bintrafusp alfa enhances the antitumor efficacy of anti-VEGF and VEGF receptor kinase inhibitors in preclinical solid tumor models

Abstract The targeting of pathways that regulate tumor angiogenesis is an effective strategy for the treatment of solid tumors. Transforming growth factor (TGF)-β and vascular endothelial growth factor (VEGF) are master regulators of tumor angiogenesis that limit the efficacy of cancer therapies and suppress the anti-tumor immune response. Combination therapies consisting of immune checkpoint inhibitors and tumor angiogenesis inhibitors are emerging as effective treatments for solid cancers. Bintrafusp alfa (M7824) is a bifunctional fusion protein composed of the extracellular domain of the TGF-βRII receptor (a TGF-β “trap”) fused to a human IgG1 antibody blocking PD-L1. Here, we tested the antitumor efficacy of a combination therapy consisting of bintrafusp alfa plus a VEGF receptor kinase inhibitor (pazopanib, lenvatinib, regorafenib, axitinib, or nintedanib), or a VEGF neutralizing antibody (B20) in multiple preclinical tumor models. The combination of bintrafusp alfa with lenvatinib, regorafenib, axitinib, or nintedanib showed significantly greater antitumor efficacy than the respective monotherapies in the CT26 mouse syngeneic colon carcinoma model. In the 4T1 mouse breast carcinoma model, the combination of bintrafusp alfa with anti-VEGF, lenvatinib, or regorafenib was significantly more efficacious than the respective monotherapies. Additionally, the combination of bintrafusp alfa with pazopanib also showed superior antitumor efficacy in an orthotopic Renca mouse tumor model. These data indicate that bintrafusp alfa may be a viable combination partner with multiple angiogenesis inhibitors and provide a rational for the continued development of these combination therapies to treat solid cancers. Citation Format: Tsz-Lun Yeung, Adam Lazorchak, Huakui Yu, Jin Qi, Guozhong Qin, Bo Marelli, Kai Fu, Yan Lan. Bintrafusp alfa enhances the antitumor efficacy of anti-VEGF and VEGF receptor kinase inhibitors in preclinical solid tumor 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 443.

Creatine in T Cell Antitumor Immunity and Cancer Immunotherapy.

Creatine is a broadly used dietary supplement that has been extensively studied for its benefit on the musculoskeletal system. Yet, there is limited knowledge regarding the metabolic regulation of creatine in cells beyond the muscle. New insights concerning various regulatory functions for creatine in other physiological systems are developing. Here, we highlight the latest advances in understanding creatine regulation of T cell antitumor immunity, a topic that has previously gained little attention in the creatine research field. Creatine has been identified as an important metabolic regulator conserving bioenergy to power CD8 T cell antitumor reactivity in a tumor microenvironment; creatine supplementation has been shown to enhance antitumor T cell immunity in multiple preclinical mouse tumor models and, importantly, to synergize with other cancer immunotherapy modalities, such as the PD-1/PD-L1 blockade therapy, to improve antitumor efficacy. The potential application of creatine supplementation for cancer immunotherapy and the relevant considerations are discussed.

Tumor Oxygenation by Myo-Inositol Trispyrophosphate Enhances Radiation Response.

Tumor hypoxia is a major limiting factor for successful radiation therapy outcomes, with hypoxic cells being up to 3-fold more radiation resistant than normoxic cells; tumor hypoxia creates a tumor microenvironment that is hostile to immune response. Thus, pharmaceutical-induced tumor oxygenation before radiation therapy represents an interesting method to enhance the efficacy of radiation therapy. Myo-inositol trispyrophosphate (ITPP) triggers a decrease in the affinity of oxygen to hemoglobin, which leads to an increased release of oxygen upon tissue demand, including in hypoxic tumors. The combined treatment modality of high-dose bolus ITPP with a single high-dose fraction of ionizing radiation (IR) was investigated for its mechanics and efficacy in multiple preclinical animal tumor models in immunocompromised and immunocompetent mice. The dynamics of tumor oxygenation were determined by serial hypoxia-oriented bioimaging. Initial and residual DNA damage and the integrity of the tumor vasculature were quantified on the immunohistochemical level in response to the different treatment combinations. ITPP application did not affect tumor growth as a single treatment modality, but it rapidly induced tumor oxygenation, as demonstrated by invivo imaging, and significantly reduced tumor growth when combined with IR. An immunohistochemical analysis of γH2AX foci demonstrated increased initial and residual IR-induced DNA damage as the primary mechanism for radiosensitization within initially hypoxic but ITPP-oxygenated tumor regions. Scheduling experiments revealed that ITPP increases the efficacy of ionizing radiation only when applied before radiation therapy. Irradiation alone damaged the tumor vasculature and increased tumor hypoxia, which were both prevented by combined treatment with ITPP. Interestingly, the combined treatment modality also promoted increased immune cell infiltration. ITPP-mediated tumor oxygenation and vascular protection triggers immediate and delayed processes to enhance the efficacy of ionizing radiation for successful radiation therapy.

Stimulation of an anti-tumor immune response with "chromatin-damaging" therapy.

Curaxins are small molecules that bind genomic DNA and interfere with DNA-histone interactions leading to the loss of histones and decondensation of chromatin. We named this phenomenon 'chromatin damage'. Curaxins demonstrated anti-cancer activity in multiple pre-clinical tumor models. Here, we present data which reveals, for the first time, a role for the immune system in the anti-cancer effects of curaxins. Using the lead curaxin, CBL0137, we observed elevated expression of several group of genes in CBL0137-treated tumor cells including interferon sensitive genes, MHC molecules, some embryo-specific antigens suggesting that CBL0137 increases tumor cell immunogenicity and improves recognition of tumor cells by the immune system. In support of this, we found that the anti-tumor activity of CBL0137 was reduced in immune deficient SCID mice when compared to immune competent mice. Anti-tumor activity of CBL0137 was abrogated in CD8+ T cell depleted mice but only partially lost when natural killer or CD4+ T cells were depleted. Further support for a key role for the immune system in the anti-tumor activity of CBL0137 is evidenced by an increased antigen-specific effector CD8+ T cell and NK cell response, and an increased ratio of effector T cells to Tregs in the tumor and spleen. CBL0137 also elevated the number of CXCR3-expressing CTLs in the tumor and the level of interferon-γ-inducible protein 10 (IP-10) in serum, suggesting IP-10/CXCR3 controls CBL0137-elicited recruitment of effector CTLs to tumors. Our collective data underscores a previously unrecognized role for both innate and adaptive immunity in the anti-tumor activity of curaxins.