Syngeneic Mouse Models Of Cancer Research Articles

Abstract LB352: Unraveling therapeutic potentials of mRNA-based cancer vaccine concurrently targeting heterogeneous KRAS mutant cancer

Abstract The utilization of messenger RNA (mRNA)-based cancer vaccines represents a compelling therapeutic strategy, providing distinct advantages over alternative modalities such as peptide vaccines or small-molecule-based cancer therapies. This innovative approach enables the concurrent targeting of heterogeneous cancer populations characterized by multiple mutations while preserving the integrity of normal cells. The Kristen rat sarcoma viral oncogene (KRAS) mutation is prevalent in lung, pancreatic, and colon cancer, and patients harboring KRAS mutations face a particularly dismal prognosis. This study focuses on the optimization of an mRNA-based cancer vaccine designed to target various KRAS mutations, demonstrating its efficacy inducing potent anti-tumor immune responses in both ex vivo immune cell and cancer cell co-culture, as well as in vivo syngeneic mouse cancer models. For the ex vivo evaluation of the multi-targeting capabilities of the vaccine, human peripheral blood mononuclear cells (PBMCs) primed with our mRNA cancer vaccine were subsequently co-incubated with human normal and cancer cell lines harboring KRAS mutations to assess antigen-specific immune responses and the normal cell-sparing effect of the cancer vaccine. Four types of KRAS mutant cancer cells co-cultured with the vaccine-activated PBMCs showed substantially decreased cell viability. We found enrichment of cytotoxic and pro-inflammatory cytokines including ganzyme B, IFNγ, IL-6 and IL-10 in co-culture system with KRAS vaccine transfected monocyte and PBMCs. This suggests that multiple KRAS mutations can be targeted with a single type of mRNA vaccine candidate. In the LL/2 syngeneic model (murine Lewis lung carcinoma, KRAS G12C mutant), the in vivo efficacy of KRAS cancer vaccine candidates was evaluated, revealing a notable reduction in tumor growth in response to the vaccine treatment. Also, during the treatment period, there were no significant clinical symptoms in the mouse between groups indicating the vaccine doses were tolerable. These results indicate that the KRAS vaccine effectively inhibits the growth of an LL/2 tumor which has the immunosuppressive tumor microenvironment. The ability to address the challenges posed by diverse KRAS mutations underscores the promising potential of this multiple-KRAS mutation targeting mRNA-based cancer vaccine in the pursuit of enhanced therapeutic outcomes for patients afflicted with KRAS-mutant cancer. Citation Format: Chang Gyu Lim, Seung-Hyun Shin, Youngjin Han, Yong Ho Heo, Innah Kim, Ji Hee Lee, Miyoung Lee, Seongju Jeong, Yunjae Kim, Jooyun Byun, Daejin Kim, In Young Choi. Unraveling therapeutic potentials of mRNA-based cancer vaccine concurrently targeting heterogeneous KRAS mutant cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB352.

PVRL2 Suppresses Antitumor Immunity through PVRIG- and TIGIT-independent Pathways.

Poliovirus receptor-related 2 (PVRL2, also known as nectin-2 or CD112) is believed to act as an immune checkpoint protein in cancer; however, most insight into its role is inferred from studies on its known receptor, poliovirus receptor (PVR)-related immunoglobulin domain protein (PVRIG, also known as CD112R). Here, we study PVRL2 itself. PVRL2 levels were found to be high in tumor cells and tumor-derived exosomes. Deletion of PVRL2 in multiple syngeneic mouse models of cancer showed a dramatic reduction in tumor growth that was immune dependent. This effect was even greater than that seen with deletion of PD-L1. PVRL2 was shown to function by suppressing CD8+ T and natural killer cells in the tumor microenvironment. The loss of PVRL2 suppressed tumor growth even in the absence of PVRIG. In contrast, PVRIG loss showed no additive effect in the absence of PVRL2. T-cell immunoreceptor with Ig and ITIM domains (TIGIT) blockade combined with PVRL2 deletion resulted in a near complete block in tumor growth. This effect was not recapitulated by the combined deletion of PVRL2 with its paralog, PVR, which is the ligand for TIGIT. These data uncover PVRL2 as a distinct inhibitor of the antitumor immune response with functions beyond that of its known receptor PVRIG. Moreover, the data provide a strong rationale for combinatorial targeting of PVRL2 and TIGIT for cancer immunotherapy.

Abstract 684: DT-9045, a novel PAR2 inhibitor with best-in-class properties that reduces resistance to both EGFR-targeting therapies and immunotherapy in oncology models

Abstract Protease-activated receptor 2 (PAR2) is a promising therapeutic target in oncology and immuno-oncology. It is upregulated and associated with poor prognosis in several cancer types. A pan-cancer meta-analysis showed that PAR2 is one of the genes most significantly associated with resistance to immune checkpoint blockade (ICB) and T cells dysfunction in cancer patients. Mechanistically, being expressed by various cells of the tumor microenvironment, PAR2 promotes both survival and proliferation of cancer cells and dampens the anti-cancer immune response. Domain Therapeutics has developed a novel PAR2 inhibitor, DT-9045, with best-in-class properties. Indeed, compared to its most advanced competitors, DT-9045 is a small molecule, orally bioavailable, insurmountable, biased, and active in tumor-like conditions (i.e. acidic pH and high level of activating proteases). This compound is also highly potent and selective on PAR2. Pharmacokinetic properties are suitable with a once-a-day oral administration. In vitro, DT-9045 completely prevented PAR2-mediated resistance to EGFR-targeting drugs. In vivo, its combination with anti-PD1 therapy showed a significant increase in efficacy over monotherapies in preclinical syngeneic mouse cancer models. This result was similar to the one observed in PAR2 knockout mice, indicating that oral administration of DT-9045 induces a complete inhibition of the receptor. We have further demonstrated that treatment with a PAR2 inhibitor enhances dendritic cell-mediated T cell activation and intratumoral infiltration of CD4+ and CD8+ T cells as well as a decrease in M2 macrophage infiltration. In conclusion, Domain Therapeutic has identified a novel PAR2 inhibitor, DT-9045, with clear competitive advantages. This preclinical candidate has shown strong potency in alleviating the resistance to both EGFR-targeting therapies and immunotherapy in preclinical cancer models. IND-enabling studies are currently ongoing to bring this new hope for cancer patients to the clinic. Citation Format: Thibaut Brugat, Maleck Kadiri, Samya Aouad, Anne-Laure Blayo, Baptiste Rugeri, Antoine Mousson, Aurélie Janvier, Edith Steinberg, Luc Baron, Mandy Recolet, Xavier Wirth, Maria Jesus Garcia-Leon, Quentin Ruet, Meriem Semache, Laurent Sabbagh, Orphée Blanchard, Christel Franchet, Stanislas Mayer, John Stagg, Nathalie Lenne, Stephan Schann. DT-9045, a novel PAR2 inhibitor with best-in-class properties that reduces resistance to both EGFR-targeting therapies and immunotherapy in oncology models [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 684.

Abstract 5171: TP-6379, an investigational TGFBR1 inhibitor, shown to remodel the tumor microenvironment and enhance anti-tumorigenic immunological responses in syngeneic mouse models of cancer

Abstract Transforming growth factor-β (TGF-β) receptors regulate SMAD signal transduction and a wide-range of biological processes including, wound healing, angiogenesis, immune modulation, epithelial-mesenchymal transition, cell differentiation, apoptosis, growth, and motility. In transformed cells, TGF-β signaling is frequently exploited to reshape the architecture of the tumor microenvironment (TME) and establish a fibrotic barrier that insulates the tumor from surrounding normal tissue. As a result, immunological threats to the tumor are restricted, as is the anti-tumorigenic potential of immunotherapeutic interventions. Clinically, TGF-β hyperactivity is associated with immunotherapy resistance and poor outcome in a variety of malignancies. We hypothesize that TP-6379, an investigational small molecule inhibitor of TGFBR1, may remodel the TME to expand the access of tumor-seeking lymphocytes to tumor tissue. Herein we describe several anti-tumorigenic effects of TP-6379 observed in multiple mouse models of cancer both as a single agent, and in combination with immunotherapy. In an EMT6 syngeneic mouse model of triple-negative breast cancer (TNBC), we observed that tumor phospho-SMAD2/3 (pSMAD2/3) levels quickly declined post-TP-6379-treatment and were suppressed by 74% after 8 hours. Preclinical data showed TP-6379 plasma levels inversely correlated with pSMAD2/3 suppression. TP-6379 alone was observed to inhibit tumor growth in EMT6 and 4T1 TNBC models, an activity that was enhanced when augmented by immunotherapy. In a Cloudman S91 syngeneic melanoma mouse model, there was a clear subset of mice that responded to combination treatment. Histological analyses of TNBCs and responsive melanomas showed that TP-6379 treatment conferred a loss of induration of tumors, increased vascularization, and increased infiltration of CD45+ leukocytes (including CD8+ T cells), particularly when combined with immunotherapy. High-throughput analyses of tumors uncovered an array of MHC class I and II factors that were observed elevated subject to TP-6379 treatment, an activity that was oftentimes enhanced when combined with immunotherapy. Collectively, these data suggest that TP-6379 may improve immune cell access to tumor tissue via TME remodeling and normalization of vascular networks. Thus, TP-6379 treatment may present a unique and multifactorial anti-tumor strategy, 1) as a single agent to improve anti-tumorigenic immunological responses, and 2) as a combination treatment to boost immunotherapeutic activity. Citation Format: David A. Kircher, Tetyana V. Forostyan, Richard E. Heinz, Curtis A. Allred, Sal Sommakia, Yuta Matsumura, Adam Siddiqui, Jason M. Foulks, Steven L. Warner. TP-6379, an investigational TGFBR1 inhibitor, shown to remodel the tumor microenvironment and enhance anti-tumorigenic immunological responses in syngeneic mouse models of 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 5171.

Evaluation of a CD206-Targeted Peptide for PET Imaging of Macrophages in Syngeneic Mouse Models of Cancer.

Tumor-associated macrophages (TAMs) are large phagocytic cells that play numerous roles in cancer biology and are an important component of the relationship between immune system response and tumor progression. The peptide, RP832c, targets the Mannose Receptor (CD206) expressed on M2-like macrophages and is cross-reactive to both human and murine CD206. Additionally, it exhibits therapeutic properties through its ability to shift the population of TAMs from an M2-like (protumor) toward an M1-like phenotype (antitumor) and has demonstrated promise in inhibiting tumor resistance in PD-L1 unresponsive melanoma murine models. In addition, it has shown inhibition in bleomycin-induced pulmonary fibrosis through interactions with CD206 macrophages.1,2 Our work aims to develop a novel CD206 positron emission tomography (PET) imaging probe based on RP832c (Kd = 5.64 μM) as a direct, noninvasive method for the assessment of TAMs in mouse models of cancer. We adapted RP832c to incorporate the chelator DOTA to allow for radiolabeling with the PET isotope 68Ga (t1/2 = 68 min; ß+ = 89%). In vitro stability studies were conducted in mouse serum up to 3 h. The in vitro binding characteristics of [68Ga]RP832c to CD206 were determined by a protein plate binding assay and Surface Plasmon Resonance (SPR). PET imaging and biodistribution studies were conducted in syngeneic tumor models. Stability studies in mouse serum demonstrated that 68Ga remained complexed up to 3 h (less than 1% free 68Ga). Binding affinity studies demonstrated high binding of [68Ga]RP832c to mouse CD206 protein and that the binding of the tracer was able to be blocked significantly when incubated with a blocking solution of native RP832c. PET imaging and biodistribution studies in syngeneic tumor models demonstrated uptake in tumor and CD206 expressing organs of [68Ga]RP832c. A significant correlation was found between the percentage of CD206 present in each tumor imaged with [68Ga]RP832c and PET imaging mean standardized uptake values in a CT26 mouse model of cancer. The data shows that [68Ga]RP832c represents a promising candidate for macrophage imaging in cancer and other diseases.

Polymeric microneedles enable simultaneous delivery of cancer immunomodulatory drugs and detection of skin biomarkers.

Background: Immune-modulating therapies impart positive outcomes in a subpopulation of cancer patients. Improved delivery strategies and non-invasive monitoring of anti-tumor effects can help enhance those outcomes and understand the mechanisms associated with the generation of anti-tumor immune responses following immunotherapy. Methods: We report on the design of a microneedle (MN) platform capable of simultaneous delivery of immune activators and collection of interstitial skin fluid (ISF) to monitor therapeutic responses. While either approach has shown promise, the integration of the therapy and diagnostic arms into one MN platform has hardly been explored before. MNs were synthesized out of crosslinked hyaluronic acid (HA) and loaded with a model immunomodulatory nanoparticle-containing drug, CpG oligodinucleotides (TLR9 agonist), for cancer therapy in melanoma and colon cancer models. The therapeutic response was monitored by longitudinal analysis of entrapped immune cells in the MNs following patch retrieval and digestion. Results: Transdermal delivery of CpG-containing NPs with MNs induced anti-tumor immune responses in multiple syngeneic mouse cancer models. CpG-loaded MNs stimulated innate immune cells and reduced tumor growth. Intravital microscopy showed deposition and spatiotemporal co-localization of CpG-NPs within the tumor microenvironment when delivered with MNs. Analysis of MN-sampled ISF revealed similar immune signatures to those seen in the bulk tumor homogenate, such as increased populations of macrophages and effector T cells following treatment. Conclusions: Our hydrogel-based MNs enable effective transdermal drug delivery into immune cells in the tumor microenvironment, and upon retrieval, enable studying the immune response to the therapy over time. This platform has the theranostic potential to deliver a range of combination therapies while detecting biomarkers.

Optimization of the Solvent and In Vivo Administration Route of Auranofin in a Syngeneic Non-Small Cell Lung Cancer and Glioblastoma Mouse Model.

The antineoplastic activity of the thioredoxin reductase 1 (TrxR) inhibitor, auranofin (AF), has already been investigated in various cancer mouse models as a single drug, or in combination with other molecules. However, there are inconsistencies in the literature on the solvent, dose and administration route of AF treatment in vivo. Therefore, we investigated the solvent and administration route of AF in a syngeneic SB28 glioblastoma (GBM) C57BL/6J and a 344SQ non-small cell lung cancer 129S2/SvPasCrl (129) mouse model. Compared to daily intraperitoneal injections and subcutaneous delivery of AF via osmotic minipumps, oral gavage for 14 days was the most suitable administration route for high doses of AF (10-15 mg/kg) in both mouse models, showing no measurable weight loss or signs of toxicity. A solvent comprising 50% DMSO, 40% PEG300 and 10% ethanol improved the solubility of AF for oral administration in mice. In addition, we confirmed that AF was a potent TrxR inhibitor in SB28 GBM tumors at high doses. Taken together, our results and results in the literature indicate the therapeutic value of AF in several in vivo cancer models, and provide relevant information about AF's optimal administration route and solvent in two syngeneic cancer mouse models.

Systemic TLR7/8 micelles trigger a novel and potent anti-tumor response by strong recruitment of neutrophils leading to massive tumor cell killing.

2576 Background: Clinical use of TLR7/8 agonists is currently restricted to topical application of Imiquimod for treatment of superficial basal cell carcinoma since systemic administration is prevented due to dose-limiting toxicity. Here, we present a novel micelle-based drug delivery technology containing a lipid-anchored TLR7/8 agonist for intravenous administration. The MBS8 formulation shows good efficacy in mouse cancer models, is well tolerated in rodents and non-human primates and is currently being tested in clinical studies (NCT04855435). Methods: MBS8 was tested in 12 syngeneic mouse cancer models as monotherapy or in combination anti-PD-1 and anti-PD-L1 antibodies. MBS8 was administered IV as slow bolus. Tumors and spleens were analysed using histology, immunohistochemistry, ELISPOT and flow cytometry. Phenotyping of tumor microenvironment was done using flow cytometry and Nanostring analyses. Safety and tolerability were tested in mice, rats and monkeys. Safety-related and PD biomarkers were identified in monkeys using a multiplex MSD technology and validated in whole human blood stimulated with MBS8 ex-vivo. Results: In several syngeneic mouse tumor models, MBS8 led to complete eradication of established tumors. The complete responders showed tumor rejection upon re-challenge. In these mice, a CD8+-dependent tumor-specific immune memory response was evident. Tumors demonstrated a massive tissue necrosis ( > 80%) within 24-48h after the first drug administration and showed massive neutrophil infiltration 6h after dosing. Antigen specific CD8+ T-cells were detected in tumors 24 hours after treatment with increased CD8+:Treg cell ratio and enhanced antigen presentation in tdLN. MBS8 showed both additive and synergistic effect when combined with anti-PD-1 or anti-PD-L1 antibody treatment. Moreover, in tumors being resistant to anti-PD-1 treatment, co-administration of MBS8 reverted sensitivity to anti-PD-1 treatment in a synergistic manner. A panel of cytokines induced by MBS8 upon repeat dose administration in monkeys was identified in plasma as potential biomarkers. Ten of them were further evaluated in whole human blood treated with MBS8 ex vivo. Currently, these cytokines are being used in Phase I clinical studies. Conclusions: MBS8 showed significant anti-cancer activity in multiple in vivo solid tumor models when administered either as monotherapy or in combination with ICIs. MBS8 demonstrated a novel mode of action with neutrophils playing a central role as primary effector cells causing a rapid killing of tumors. Further, adaptive immune response was initiated including generation of tumor specific CD8+ cells and establishment of the immune memory. MBS8 was well tolerated in rodents and cynomolgus monkeys at the dose levels above therapeutic effective doses identified in mice, thus providing a good therapeutic window.

Chitooligosaccharides Improve the Efficacy of Checkpoint Inhibitors in a Mouse Model of Lung Cancer.

YKL-40 (also named chitinase 3 like-1 protein [CHI3L1]) is a secreted chitinase-like protein which is upregulated in cancers and suggested to have pro-tumorigenic activity. YKL-40 lacks enzymatic function, but it can bind carbohydrates such as chitin. Chitooligosaccharides (COS) derived from deacetylation and hydrolysis of chitin might be used for the blockade of YKL-40 function. Here, public single-cell RNA sequencing datasets were used to elucidate the cellular source of YKL-40 gene expression in human tumors. Fibroblasts and myeloid cells were the primary sources of YKL-40. Screening of YKL-40 gene expression in syngeneic mouse cancer models showed the highest expression in the Lewis lung carcinoma (LL2) model. LL2 was used to investigate COS monotherapy and combinations with immune checkpoint inhibitors (anti-PD-L1 and anti-CTLA-4) (ICIs) and radiotherapy (8 Gy × 3) (RT). COS tended to reduce plasma YKL-40 levels, but it did not affect tumor growth. LL2 showed minimal responses to ICIs, or to RT alone. Interestingly, ICIs combined with COS led to delayed tumor growth. RT also enhanced the efficacy of ICIs; however, the addition of COS did not further delay the tumor growth. COS may exert their anti-tumorigenic effects through the inhibition of YKL-40, but additional functions of COS should be investigated.

In Vivo Measurement of Granzyme Proteolysis from Activated Immune Cells with PET.

The biology of human granzymes remains enigmatic in part due to our inability to probe their functions outside of in vitro assays or animal models with divergent granzyme species. We hypothesize that the biology of human granzymes could be better elaborated with a translational imaging technology to reveal the contexts in which granzymes are secreted and biochemically active in vivo. Here, we advance toward this goal by engineering a Granzyme targeting Restricted Interaction Peptide specific to family member B (GRIP B) to measure secreted granzyme B (GZMB) biochemistry with positron emission tomography. A proteolytic cleavage of 64Cu-labeled GRIP B liberates a radiolabeled form of Temporin L, which sequesters the radioisotope by binding to adjacent phospholipid bilayers. Thus, at extended time points postinjection (i.e., hours, not seconds), tissue biodistribution of the radioisotope in vivo reflects relative units of the GZMB activity. As a proof of concept, we show in three syngeneic mouse cancer models that 64Cu-GRIP B detects GZMB from T cells activated with immune checkpoint inhibitors (CPI). Remarkably, the radiotracer detects the proteolysis within tumors but also in lymphoid tissue, where immune cells are activated by a systemic CPI. Control experiments with an uncleavable analogue of 64Cu-GRIP B and tumor imaging studies in germline GZMB knockout mice were applied to show that 64Cu-GRIP B is specific for GZMB proteolysis. Furthermore, we explored a potential noncytotoxic function for GZMB by applying 64Cu-GRIP B to a model of pulmonary inflammation. In summary, we demonstrate that granzyme biochemistry can be assessed in vivo using an imaging modality that can be scaled vertically into human subjects.

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 679: In vivo synergy of 90Y-NM600 and Bempegaldesleukin improves anti-tumor efficacy of immune checkpoint inhibitors in syngeneic murine cancer models

Abstract We have observed in preclinical studies that the delivery of low dose targeted radionuclide therapy (TRT) therapy to sites of primary and metastatic cancer can improve the anti-tumor immune response to immune checkpoint inhibition (ICI) with anti-CTLA4 or anti-PDL1. NM600, an alkylphosphocholine that can be radiolabeled with 90Y, is taken up and retained in most cancer cells following intravenous injection. Bempegaldesleukin (BEMPEG) is a first in class, CD122-preferential IL2 pathway agonist that can selectively stimulate an immune response. We hypothesized that combining low dose TRT with BEMPEG would increase immune activation to enhance the response to ICIs in immunologically “cold” murine cancer models. C57Bl/6 female mice were subcutaneously engrafted in the flank with the murine head and neck squamous cell carcinoma (HNSCC) cell line, MOC2. In vivo dosimetry performed using the Monte Carlo based RAPID platform following serial 86Y-NM600 PET/CT imaging demonstrated that 100 µCi of 90Y-NM600 would deliver ~8 Gy to the MOC2 tumor. Mice bearing MOC2 tumors (mean volume ~100 mm3) received combinations of 90Y-NM600 (100 µCi, day 1 IV), BEMPEG (16 µg, days 6, 15, and 24 IV), and anti-CTLA4 (200 µg, days 4, 7, and 10 IP) using a 2x2x2 study design. Tumor growth and survival were monitored. Blood was collected weekly and analyzed on the Abaxis VetScan HM5 to evaluate for toxicity. In a parallel 2x2x2 survival study, an orthotopic model generated by engrafting MOC2 tumors in the cheek was used, and mice were treated with combinations of BEMPEG, 90Y-NM600, and anti-PDL1 (200 µg, days 4, 7, and 10, IP). Mice were euthanized when >25% weight loss was observed. Similar experiments using 90Y-NM600 and BEMPEG were performed in additional syngeneic mouse cancer models including SCC7 (HNSCC), 4T1 (breast), LLC (lung), and Panc02 (pancreatic). In the immunologically “cold” MOC2 HNSCC model, 62.5% of mice treated with 90Y-NM600 (TRT), BEMPEG, and anti-CTLA4; and 44.4% of mice treated with 90Y-NM600, BEMPEG, and anti-PDL1 experienced complete tumor response with no observable primary or metastatic disease at day 60. In mice treated with 90Y-NM600 and BEMPEG, tumor regression followed by escape was seen without ICI. By day 60, no mice receiving single or dual treatment combinations exhibited a complete tumor response. Comprehensive whole blood analysis did not show any major hematologic toxicities. This treatment was explored in mice bearing SCC7, 4T1, Panc02, and LLC tumors, and similar trends were seen. Combination of 90Y-NM600, BEMPEG, and ICI displays robust anti-tumor activity that prevents metastatic disease progression and prolongs survival in spontaneously metastatic, immunologically “cold” tumor models. Clinical studies are warranted to test the safety and efficacy of this promising combined modality treatment regimen. Citation Format: Elizabeth G. Sumiec, Amber M. Bates, Reinier Hernandez, Joseph J. Grudzinski, Ian R. Marsh, Sarah E. Emma, Erin J. Nystuen, Justin C. Jagodinsky, Alexander A. Pieper, Gustavo A. Sosa, Bryan P. Bednarz, Ravi B. Patel, Jamey Weichert, Zachary S. Morris. In vivo synergy of 90Y-NM600 and Bempegaldesleukin improves anti-tumor efficacy of immune checkpoint inhibitors in syngeneic murine cancer 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 679.

Targeting the Extracellular HSP90 Co-Chaperone Morgana Inhibits Cancer Cell Migration and Promotes Anticancer Immunity.

HSP90 is secreted by cancer cells into the extracellular milieu, where it exerts protumoral activities by activating extracellular substrate proteins and triggering autocrine signals through cancer cell surface receptors. Emerging evidence indicates that HSP90 co-chaperones are also secreted and may direct HSP90 extracellular activities. In this study, we found that the HSP90 co-chaperone Morgana is released by cancer cells and, in association with HSP90, induces cancer cell migration through TLR2, TLR4, and LRP1. In syngeneic cancer mouse models, a mAb targeting Morgana extracellular activity reduced primary tumor growth via macrophage-dependent recruitment of CD8+ T lymphocytes, blocked cancer cell migration, and inhibited metastatic spreading. Overall, these data define Morgana as a new player in the HSP90 extracellular interactome and suggest that Morgana may regulate HSP90 activity to promote cancer cell migration and suppress antitumor immunity. SIGNIFICANCE: This work suggests the potential therapeutic value of targeting the extracellular HSP90 co-chaperone Morgana to inhibit metastasis formation and enhance the CD8+ T-cell-mediated antitumor immune response.

Mouse Lymphoma Model-Based Senescence Exploitation to Predict Treatment Outcome of DLBCL Patients

Introduction: Molecularly informed treatment decisions are the basis of personalized cancer precision medicine. While gene expression profiling has been used to map the molecular landscape of tumors and to study the impact of molecularly defined subtypes on treatment outcome, biological effector principles, specifically cellular senescence, remain largely understudied. Syngeneic mouse models of cancer that recapitulate core molecular features of human malignancies could serve as useful models to explore mechanisms of drug sensitivity, and, likewise, to dissect molecular mechanisms of treatment failure. In particular, we focus here on the role of therapy-induced senescence, a histone H3 lysine 9 trimethylation (H3K9me3)-governed terminal cell-cycle arrest program.Methods: The Eµ- myc transgenic lymphoma mouse was used as a cross-species model of chemoresistance for patients diagnosed with diffuse large B-cell lymphoma (DLBCL). Gene expression profiles (GEP) of primary Eµ- myc lymphomas were obtained at diagnosis and after exposure to cyclophosphamide chemotherapy in vivo, with long-term outcome monitored in a clinical trial-like fashion. Lymphoma senescence capacity, a pivotal drug effector principle, was interrogated in mice by unbiased approaches as well as genetic perturbations.Results: DLBCL-established gene expression-based classifiers related to cell-of-origin (COO - i.e. GCB/ABC) subtypes and distinct DLBCL biologies (e.g. comprehensive consensus clusters [CCC]) were instrumental to identify comparable lymphoma subsets in the murine system. In turn, mice carrying senescence-defective lymphomas due to engineered loss of the H3K9me3 methyltransferase Suv39h1 or overexpression of the H3K9-active demethylase LSD1 succumbed faster to chemotherapy, as DLBCL patients with high LSD1 or low H3K9me3 expression in their lymphomas presented with significantly inferior outcome. Likewise, cross-species application of senescence-associated characteristics identified in unengineered primary mouse lymphomas unveiled molecular features important for treatment outcome in human DLBCL. Ongoing mouse model-based work aims at specific targeting of aberrant H3K9 demethylase activity to assess therapeutic long-term effects and to determine the conditions for future early-phase clinical testing in DLBCL patients.Conclusions: Primary Eµ- myc transgenic lymphomas serve as a faithful model for human DLBCL, since they share key pathologic and genetic features, including CCC/COO-based clustering. In turn, mouse lymphoma-based molecular exploration, genetic dissection and functional interrogation of treatment effector mechanisms, which is difficult to conduct in patient material, can be re-applied to molecularly and clinically co-annotated datasets from DLBCL patients to inform novel, lesion- and state-driven personalized treatment approaches and the design of future clinical trials. DisclosuresNo relevant conflicts of interest to declare.

Single-Domain Antibody Nuclear Imaging Allows Noninvasive Quantification of LAG-3 Expression by Tumor-Infiltrating Leukocytes and Predicts Response of Immune Checkpoint Blockade

Recent advances in the field of immune-oncology led to the discovery of next-generation immune checkpoints (ICPs). Lymphocyte activation gene-3 (LAG-3), being the most widely studied among them, is being explored as a target for the treatment of cancer patients. Several antagonistic anti-LAG-3 antibodies are being developed and are prime candidates for clinical application. Furthermore, validated therapies targeting cytotoxic T-lymphocyte–associated protein-4, programmed cell-death protein-1, or programmed cell-death ligand-1 showed that only subsets of patients respond. This finding highlights the need for better tools for patient selection and monitoring. The potential of molecular imaging to detect ICPs noninvasively in cancer is supported by several preclinical and clinical studies. Here, we report on a single-domain antibody to evaluate whole-body LAG-3 expression in various syngeneic mouse cancer models using nuclear imaging. <b>Methods:</b> SPECT/CT scans of tumor-bearing mice were performed 1 h after injection with radiolabeled single-domain antibody. Organs and tumors of mice were isolated and evaluated for the presence of the radiolabeled tracer and LAG-3–expressing immune cells using a γ-counter and flow cytometry respectively. PD-1/LAG-3–blocking antibodies were injected in MC38-bearing mice. <b>Results:</b> The radiolabeled single-domain antibody detected LAG-3 expression on tumor-infiltrating lymphocytes (TILs) as soon as 1 h after injection in MC38, MO4, and TC-1 cancer models. The single-domain antibody tracer visualized a compensatory upregulation of LAG-3 on TILs in MC38 tumors of mice treated with PD-1–blocking antibodies. When PD-1 blockade was combined with LAG-3 blockade, a synergistic effect on tumor growth delay was observed. <b>Conclusion:</b> These findings consolidate LAG-3 as a next-generation ICP and support the use of single-domain antibodies as tools to noninvasively monitor the dynamic evolution of LAG-3 expression by TILs, which could be exploited to predict therapy outcome.

Immune checkpoint inhibition in syngeneic mouse cancer models by a silicasome nanocarrier delivering a GSK3 inhibitor

Checkpoint blocking antibodies that interfere in the PD-1/PD-L1 axis provide effective cancer immunotherapy for tumors that are immune inflamed or induced to become “hot”. It has also been demonstrated that a small molecule inhibitor of the signaling hub kinase GSK3 can interfere in the PD-1/PD-L1 axis in T-cells by suppressing PD-1 expression. This provides an alternative approach to intervening in the PD-1/PD-L1 axis to provide cancer immunotherapy. In this communication, we demonstrate the remote loading of GSK3 inhibitor AZD1080 into the porous interior of mesoporous silica nanoparticles coated with a lipid bilayer (a.k.a. silicasomes). In a MC38 colon cancer model, intravenous injection (IV) of silicasome-encapsulated AZD1080 significantly improved biodistribution and drug delivery to the tumor site. The improved drug delivery was accompanied by cytotoxic MC38 tumor cell killing by perforin-releasing CD8+ T-cells, exhibiting reduced PD-1 expression. IV injection of encapsulated AZD1080 also resulted in significant tumor shrinkage in other syngeneic mouse tumor models, including another colorectal tumor (CT26), as well as pancreas (KPC) and lung (LLC) cancer models. Not only was the therapeutic efficacy of encapsulated AZD1080 similar or better than anti-PD-1 antibody, but the treatment was devoid of treatment toxicity. These results provide proof-of-principal demonstration of the feasibility of using encapsulated delivery of a GSK3 inhibitor to provide cancer immunotherapy, with the possibility to be used as a monotherapy or in combination with chemotherapy or other immunomodulatory agents.

Near-Infrared Photoimmunotherapy Combined with CTLA4 Checkpoint Blockade in Syngeneic Mouse Cancer Models.

Near infrared photoimmunotherapy (NIR-PIT) is a newly developed and highly selective cancer treatment that induces necrotic/immunogenic cell death. It employs a monoclonal antibody (mAb) conjugated to a photo-absorber dye, IRDye700DX, which is activated by NIR light. Tumor-targeting NIR-PIT is also at least partly mediated by a profound immune response against the tumor. Cytotoxic T-lymphocyte antigen-4 (CTLA4) is widely recognized as a major immune checkpoint protein, which inhibits the immune response against tumors and is therefore, a target for systemic blockade. We investigated the effect of combining tumor-targeted NIR-PIT against the cell-surface antigen, CD44, which is known as a cancer stem cell marker, with a systemic CTLA4 immune checkpoint inhibitor in three syngeneic tumor models (MC38-luc, LL/2, and MOC1). CD44-targeted NIR-PIT combined with CTLA4 blockade showed greater tumor growth inhibition with longer survival compared with CTLA4 blockade alone in all tumor models. NIR-PIT and CTLA4 blockade produced more complete remission in MOC1 tumors (44%) than NIR-PIT and programmed cell death protein 1 (PD-1) blockade (8%), which was reported in our previous paper. However, the combination of NIR-PIT and CTLA4 blockade was less effective in MC38-luc tumors (11%) than the combination of NIR-PIT and PD-1 blockade (70%). Nonetheless, in many cases ineffective results with NIR-PIT and PD-1 blockade were reversed with NIR-PIT and CTLA4 blockade.

Abstract 2781: Diffusion weighted MRI evaluation of response to immunotherapy and radiotherapy in CT26 and 4T1 syngeneic mouse models of cancer

Abstract Immunotherapy has potential to improve outcome for cancer patients. The MRI biomarker apparent diffusion co-efficient (ADC) can map response to radiotherapy (RT) through its sensitivity to changes in tumor fluid and cellularity. The role of ADC in evaluating immunotherapy agents is unknown, despite investigators using ADC change as an exploratory endpoint in early phase clinical trials. Our study sought to evaluate and validate ADC changes induced by both RT and immunotherapy agents in syngeneic mouse tumor models. Three experiments were performed in BALB/c mice bearing CT26 colorectal cancers: (1) single 10Gy fraction RT versus control (sham); (2) TLR 7/8 agonist R848 versus control (saline); (3) anti-PD-L1 antibody versus control (saline). We acquired MRI data at 7T Bruker system at days 0, 3, 7, +/- 10 after therapy start, with tumors measuring between 250-300 mm3 at day 0. Median ADC and the inter-quartile range (IQR; a measure of tumor heterogeneity) were derived. Three further equivalent experiments were performed in BALB/c mice bearing triple negative 4T1 breast tumors. All CT26 and 4T1 tumors were bisected at cull for immunohistochemistry and FACS analysis. For CT26 model, RT-induced tumor growth inhibition (p&amp;lt;0.001) and increased median ADC and IQR at days 7-10 (p&amp;lt;0.05) were accompanied by increased numbers of CD8 cells at days 7-10 on both immunohistochemistry and FACS (p&amp;lt;0.01) and increased necrosis (P&amp;lt;0.05), relative to control. Neither R848 nor anti-PD-L1 modified tumor growth, CD8 cells or macrophages on FACS. R848 induced marked increase in median ADC and IQR at day 3 (p&amp;lt;0.01) accompanied by decrease in CD4 cells at day 3. In distinction, the increased ADC in 4T1 tumors treated with RT (p&amp;lt;0.05 at d7 and d10) and R848 (p&amp;lt;0.05 at d3) were not associated with any change of immune cell populations in tumors as determined by FACS. Anti-PD-L1 therapy did not alter median ADC in either CT26 or 4T1 models, although diffusion heterogeneity, measured by the IQR of ADC was increased markedly in 3/7 CT26 tumors. In conclusion, our data are the first to evaluate ADC changes induced by RT and immunotherapy agents in syngeneic mouse models of cancer. RT, R848 and anti-PD-L1 induced different ADC responses, each with varied relationships to immune cells. This highlights the need for extensive validation before diffusion weighted MRI biomarkers can be used in clinical trials to monitor response to immunotherapies alone or in combination with RT. Citation Format: Grazyna Lipowska-Bhalla, Damien J. McHugh, Muhammad Babur, Isabel Peset Martin, Michael Berks, Ross A. Little, Susan Cheung, Yvonne Watson, Denis G. Alferez, Kaye J. Williams, Jamie Honeychurch, James P. O'Connor. Diffusion weighted MRI evaluation of response to immunotherapy and radiotherapy in CT26 and 4T1 syngeneic mouse models of cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2781.

Abstract 683: Identification of a macrocyclic peptide that blocks the CD47-SIRPα interaction and its antitumor effect

Abstract Much recent attention has focused on the modification of immune responses as a basis for new cancer treatments. Immune checkpoint inhibitors, such as monoclonal antibodies (mAbs) to CTLA-4, PD-1, and PD-L1, have provided clinical benefit in patients with a variety of types of cancer, and a growing number of agents targeting immune checkpoints are in preclinical and clinical development. SIRPα is a transmembrane protein that is abundant in myeloid cells, such as macrophages and dendritic cells. SIRPα interacts with another transmembrane protein CD47 through their ectodomains, and they constitute the cell-cell communication system (the CD47-SIRPα system). We previously demonstrated that the interaction of SIRPα on macrophages with CD47 on tumor cells prevented the phagocytosis of tumor cells by macrophages. In contrast, blockage by anti-SIRPα antibodies of the CD47-SIRPα interaction enhanced the phagocytic activity of macrophages against antibody-opsonized tumor cells, suggesting that the CD47-SIRPα axis acts as an immune checkpoint pathway involved in tumor escape from immunosurveillance. Here, by the use of random nonstandard peptide integrated discovery (RaPID) system, we have identified novel macrocyclic peptides that bind to the extracellular domain of SIRPα with high affinity. These SIRPα-binding macrocyclic peptides inhibited the CD47-SIRPα interaction. Moreover, one of the SIRPα-binding macrocycles promoted the phagocytosis of antibody-opsonized tumor cells by macrophages in vitro and enhanced the antitumor effect of mAbs against tumor antigens in xenograft or syngeneic mouse models of cancer. Our results thus suggest that macrocyclic peptides against SIRPα hold promise as a novel tool for cancer immunotherapy. Citation Format: Daisuke Hazama, Yoji Murata, Takeshi Okamoto, Daisuke Tanaka, Mariko Sakamoto, Yuka Kakuchi, Yasuyuki Saito, Takenori Kotani, Yoshihiro Nishimura, Takashi Matozaki. Identification of a macrocyclic peptide that blocks the CD47-SIRPα interaction and its antitumor effect [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 683.

The Mincle/Syk/NF-κB Signaling Circuit Is Essential for Maintaining the Protumoral Activities of Tumor-Associated Macrophages.

Tumor-associated macrophages (TAM) have important roles in cancer progression, but the signaling behind the formation of protumoral TAM remains understudied. Here, by single-cell RNA sequencing, we revealed that the pattern recognition receptor Mincle was highly expressed in TAM and significantly associated with mortality in patients with non-small cell lung cancer. Cancer cells markedly induced Mincle expression in bone marrow-derived macrophages (BMDM), thus promoting cancer progression in invasive lung carcinoma LLC and melanoma B16F10 in vivo and in vitro Mincle was predominately expressed in the M2-like TAM in non-small cell lung carcinoma and LLC tumors, and silencing of Mincle unexpectedly promoted M1-like phenotypes in vitro Mechanistically, we discovered a novel Mincle/Syk/NF-κB signaling pathway in TAM needed for executing their TLR4-independent protumoral activities. Adoptive transfer of Mincle-silenced BMDM significantly suppressed TAM-driven cancer progression in the LLC-bearing NOD/SCID mice. By modifying our well-established ultrasound microbubble-mediated gene transfer protocol, we demonstrated that tumor-specific silencing of Mincle effectively blocked Mincle/Syk/NF-κB signaling, therefore inhibiting the TAM-driven cancer progression in the syngeneic mouse cancer models. Thus, our findings highlight the function of Mincle as a novel immunotherapeutic target for cancer via blocking the Mincle/Syk/NF-κB circuit in TAM.