Syngeneic Mouse Tumor Models Research Articles

Anti-TIGIT antibody tiragolumab improves PD-L1 blockade via myeloid and Treg cells

Abstract Tiragolumab, an anti-TIGIT antibody with an active IgG1/kappa Fc, demonstrated improved outcomes in the phase 2 CITYSCAPE trial when combined with atezolizumab (anti-PD-L1) versus atezolizumab alone. To understand the mechanism(s) of response with this combination, we leveraged tumor pretreatment samples, and peripheral blood mononuclear cells (PBMC) and serum samples (baseline and on-treatment) collected from patients enrolled in the clinical trials, as well as the syngeneic CT26 mouse tumor model by using mouse tiragolumab surrogate antibodies. Bulk RNA-seq, proteomics, scSeq, and flow cytometry were performed. We found that high baseline intratumoral macrophages and Tregs were associated with better outcomes in patients treated with atezolizumab plus tiragolumab but not with atezolizumab alone. Comparing the on-treatment versus baseline serum samples revealed that macrophage activation was associated with clinical benefit in patients receiving the combination treatment. In mouse tumor models, tiragolumab surrogate antibodies inflamed tumor-associated macrophages, monocytes, and dendritic cells through Fc gamma receptors (FcɣR), in turn driving anti-tumor CD8+ T cells from an exhausted effector-like state to a more memory-like one. These results uncover a mechanism of action by which TIGIT checkpoint inhibitors can remodel immunosuppressive tumor microenvironments, and suggest that FcɣR engagement is an important consideration in anti-TIGIT antibody development.

CBX3 antagonizes IFNγ/STAT1/PD-L1 axis to modulate colon inflammation and CRC chemosensitivity

As an important immune stimulator and modulator, IFNγ is crucial for gut homeostasis and its dysregulation links to diverse colon pathologies, such as colitis and colorectal cancer (CRC). Here, we demonstrated that the epigenetic regulator, CBX3 (also known as HP1γ) antagonizes IFNγ signaling in the colon epithelium by transcriptionally repressing two critical IFNγ-responsive genes: STAT1 and CD274 (encoding Programmed death-ligand 1, PD-L1). Accordingly, CBX3 deletion resulted in chronic mouse colon inflammation, accompanied by upregulated STAT1 and CD274 expressions. Chromatin immunoprecipitation indicated that CBX3 tethers to STAT1 and CD274 promoters to inhibit their expression. Reversely, IFNγ significantly reduces CBX3 binding to these promoters and primes gene expression. This antagonist effect between CBX3 and IFNγ on STAT1/PD-L1 expression was also observed in CRC. Strikingly, CBX3 deletion heightened CRC cells sensitivity to IFNγ, which ultimately enhanced their chemosensitivity under IFNγ stimulation in vitro with CRC cells and in vivo with a syngeneic mouse tumor model. Overall, this work reveals that by negatively tuning IFNγ-stimulated immune genes’ transcription, CBX3 participates in modulating colon inflammatory response and CRC chemo-resistance.

A dietary commensal microbe enhances antitumor immunity by activating tumor macrophages to sequester iron.

Innate immune cells generate a multifaceted antitumor immune response, including the conservation of essential nutrients such as iron. These cells can be modulated by commensal bacteria; however, identifying and understanding how this occurs is a challenge. Here we show that the food commensal Lactiplantibacillus plantarum IMB19 augments antitumor immunity in syngeneic and xenograft mouse tumor models. Its capsular heteropolysaccharide is the major effector molecule, functioning as a ligand for TLR2. In a two-pronged manner, it skews tumor-associated macrophages to a classically active phenotype, leading to generation of a sustained CD8+ T cell response, and triggers macrophage 'nutritional immunity' to deploy the high-affinity iron transporter lipocalin-2 for capturing and sequestering iron in the tumor microenvironment. This process induces a cycle of tumor cell death, epitope expansion and subsequent tumor clearance. Together these data indicate that food commensals might be identified and developed into 'oncobiotics' for a multi-layered approach to cancer therapy.

Targeting PD-L1 in solid cancer with myeloid cells expressing a CAR-like immune receptor.

Solid cancers Myeloid cells are prevalent in solid cancers, but they frequently exhibit an anti-inflammatory pro-tumor phenotype that contribute to the immunosuppressive tumor microenvironment (TME), which hinders the effectiveness of cancer immunotherapies. Myeloid cells' natural ability of tumor trafficking makes engineered myeloid cell therapy an intriguing approach to tackle the challenges posed by solid cancers, including tumor infiltration, tumor cell heterogenicity and the immunosuppressive TME. One such engineering approach is to target the checkpoint molecule PD-L1, which is often upregulated by solid cancers to evade immune responses. Here we devised an adoptive cell therapy strategy based on myeloid cells expressing a Chimeric Antigen Receptor (CAR)-like immune receptor (CARIR). The extracellular domain of CARIR is derived from the natural inhibitory receptor PD-1, while the intracellular domain(s) are derived from CD40 and/or CD3ζ. To assess the efficacy of CARIR-engineered myeloid cells, we conducted proof-of-principle experiments using co-culture and flow cytometry-based phagocytosis assays in vitro. Additionally, we employed a fully immune-competent syngeneic tumor mouse model to evaluate the strategy's effectiveness in vivo. Co-culturing CARIR-expressing human monocytic THP-1 cells with PD-L1 expressing target cells lead to upregulation of the costimulatory molecule CD86 along with expression of proinflammatory cytokines TNF-1α and IL-1β. Moreover, CARIR expression significantly enhanced phagocytosis of multiple PD-L1 expressing cancer cell lines in vitro. Similar outcomes were observed with CARIR-expressing human primary macrophages. In experiments conducted in syngeneic BALB/c mice bearing 4T1 mammary tumors, infusing murine myeloid cells that express a murine version of CARIR significantly slowed tumor growth and prolonged survival. Taken together, these results demonstrate that adoptive transfer of PD-1 CARIR-engineered myeloid cells represents a promising strategy for treating PD-L1 positive solid cancers.

Abstract 4690: SC-2882, a novel QPCTL inhibitor, with anti-tumor activity in solid tumor models

Abstract Background: SC-2882 is an orally administered first-in-class small molecule inhibitor of glutaminyl-peptide cyclotransferase like (QPCTL). QPCTL is an intracellular enzyme that modifies several proteins containing an N-terminal glutamine or glutamate, resulting in pyroglutamylated amino acids. Pyroglutamylation can alter protein function in different ways and dependent on context. Key substrate proteins include the “don’t eat me” signal protein CD47 and several chemokines (CCL2 and CCL7) (Nat Med 2019, Nat Imm. 2022). Previously, we have shown activity of SC-2882 in a murine model of diffuse large B-cell lymphoma (Di Siervi, 2023). Here we report for the first time, the evaluation of SC-2882 in several preclinical mouse models for solid tumors. Methods: Potency of SC-2882 was evaluated in a cell-based assay in several human and mouse tumor cell lines: SIRPα binding to CD47 in response to increasing concentrations of SC-2882 was measured in a flowcytometry-based assay. Several syngeneic mouse tumor models were evaluated for their response towards SC-2882 as a single agent and in combination with either a murine anti-PD-L1 antibody or cisplatin. Animals were dosed orally with SC-2882 once daily for the duration of the experiment. Results: In all human and mouse cancer cell lines tested, SC-2882 was able to reduce SIRPα binding to CD47 significantly. This reduction was equivalent to the reduction observed in QPCTL genetic knockouts created in the same cell line. In the same experiments, no differences in CD47 expression were observed. From anti-tumor efficacy studies, we learned that SC-2882 was well tolerated in mice as a single agent and in combination with anti-PD-L1 or cisplatin for at least 4 weeks (this was the maximum period tested). No clinical signs or changes in body weight were observed, even over prolonged periods of treatment. Tumor growth inhibition and tumor shrinkage by SC-2882 was observed in 5 different syngeneic mouse tumor models. In two out of these five models, SC-2882 was able to reduce tumor growth as a single agent. In the other three models, tumor growth inhibition was observed in combination with either anti-PD-L1 or cisplatin. Conclusions: SC-2882 is an orally administered small molecule inhibitor with a favorable PK profile. Preclinical studies show that SC-2882 was well tolerated in mice. In several mouse tumor models, SC-2882 reduced tumor growth in either single agent treated animals or in combination with anti-PD-L1 or cisplatin. SC-2882 is currently in IND enabling studies and scheduled to enter the clinic in 2024. Citation Format: Jasper Mullenders, Marcel Scheepstra, Michel R. Faas, Annette B. Galler, Bastiaan Evers, Jens U. Wuerthner. SC-2882, a novel QPCTL inhibitor, with anti-tumor activity in solid tumor 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 4690.

Abstract 4078: WTX-712, a conditionally active IL-21 INDUKINETM molecule, induces a strong anti-tumor phenotype through a differentiated mechanism

Abstract Despite great strides seen recently in the development of immunotherapies such as immune checkpoint inhibitors (ICI), many patients do not respond or acquire resistance, highlighting a need for alternative immunotherapies. As potent immunomodulators, cytokines have been explored as treatments for cancer, but their use has been limited due to toxicity and poor pharmacokinetics (PK). One of these key cytokines, interleukin 21 (IL-21), is a pluripotent cytokine that activates anti-tumor T cell responses, induces B cell activation, and promotes generation and maintenance of germinal centers and tertiary lymphoid structures. IL-21 acts on a broader range of cells and does not induce vascular leak syndrome compared to IL-2, another common-gamma chain family member. Half-life extended IL-21 (IL-21-HLE) drives robust anti-tumor activity in several syngeneic tumor mouse models. Of particular interest, IL-21-HLE showed superior anti-tumor activity compared to IL-2-HLE in the EMT-6 and Renca models, both of which are highly resistant to anti-PD-1/PD-L1 treatment. Although IL-21-HLE and IL-2-HLE induced a similar frequency of tumor infiltrating CD8+ T cells, IL-21-HLE treatment led to higher polyfunctionality in CD8+ T cells than IL-2-HLE treatment. Of note, IL-21-HLE induced greater amounts of effector cytokines, Granzyme B and perforin, than IL-2-HLE. Transcriptomic analysis revealed that IL-21 drives upregulation of a type I IFN signature in the tumor promoting an anti-tumorigenic microenvironment. Clinical activity of IL-21 has been hampered by poor PK and adverse events at dose levels associated with signs of efficacy. To overcome these limitations Werewolf Therapeutics has developed WTX-712, an IL-21 INDUKINETM molecule, which contains wild-type human IL-21, an inactivation domain, and a half-life extension domain tethered together by protease sensitive linkers. In preclinical studies with mouse syngeneic tumor models, WTX-712 is inactive in the periphery, and wild-type IL-21 is selectively released within the tumor, resulting in anti-tumor efficacy with an expanded therapeutic window compared to IL-21-HLE. Anti-tumor efficacy was linked to expansion and activation of tumor infiltrating CD8 T cells with increased polyfunctionality. We will show additional mechanistic data evaluating the effect of WTX-712 dosing on immune populations, including tumor spatial profiling using different models. Citation Format: Jenna M. Sullivan, Pamela A. Aderhold, Heather R. Brodkin, Celesztina Nagy-Domonkos, Kyriakos Economides, Daniel J. Hicklin, Nesreen Ismail, Yuka Lewis, Cynthia Seidel-Dugan, William M. Winston, Andres Salmeron. WTX-712, a conditionally active IL-21 INDUKINETM molecule, induces a strong anti-tumor phenotype through a differentiated mechanism [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 4078.

Abstract 736: ACTM-838 safely and comprehensively re-activates the immunosuppressive TME by exploiting myeloid biology intrinsic in many cancers

Abstract ACTM-838 is a genetically modified bacterial vehicle encoding a DNA plasmid with two engineered payloads, IL-15plex and constitutively active eSTING. The bacterial vehicle is a highly attenuated S. typhimurium, engineered to facilitate safe IV administration. ACTM-838 is designed to specifically enrich in the TME via auxotrophic dependency on extracellular adenosine and purine metabolites. ACTM-838 is then selectively internalized by tumor-resident myeloid cells achieving tumor-specific payload delivery. Previously, we showed ACTM-838 exhibited significant and durable anti-tumor efficacy across syngeneic tumor mouse models (EMT6 breast, MC38 colon) and MMTV-PyMT GEMM. To elucidate TME changes induced by ACTM-838, EMT6 tumors were analyzed post-treatment by scRNA-seq, which demonstrated that ACTM-838 induced significant transcriptional changes in several cell populations in the TME. Cytolytic pathways were upregulated in T and NK cells, while type I IFN and PDL1 were upregulated in myeloid populations consistent with previous bulk RNAseq analyses. To understand the tolerability and safety of ACTM-838, a GLP toxicology study was performed on naïve cynomolgus monkeys (NHP). No ACTM-838-related clinical or veterinary observations were reported at any dose level. Acute dose-dependent increases in serum cytokines were observed at 4 hours which reverted to baseline by 24 hours. ACTM-838 was cleared from blood by 24 hours post-dose with no significant shedding in urine or feces. ACTM-838 was only detected in the spleen in 2 and 1 animals at 22- and 49-days post treatment, respectively. Pre-existing ADA was observed in some animals with subsequent increases in ADA upon dosing in most animals. However, no correlation of serum ADA and blood PK levels suggested that the presence of ADA does not impact drug stability. The ACTM-838 mechanism of action requires high levels of adenosine or purine metabolites to enrich and colonize the TME as well as phagocytic internalization by tumor-intrinsic myeloid cells to achieve tumor-specific payload delivery. Myeloid cells comprise roughly half of all cells in the TME in human tumors. Bioinformatics analyses of the public patient datasets identified key tumor indications enriched in both adenosine and myeloid pathways. To further understand the spatial heterogeneity in the TME across tumors, we performed multiplex immunofluorescence on patient tumor microarrays to validate tumor types with a high prevalence of myeloid populations coupled with an active adenosine pathway, which will help guide future clinical study design. In summary, we demonstrate that ACTM-838 modulates the TME and is stable and safe in animal models. Additionally, our studies recommend the prioritization of certain tumor indications for treatment with ACTM-838 in the clinic. Citation Format: William Lu, Kyle Cron, Julie Janes, Leigh Berryman, Gurvinder Singh, Christopher Thanos, Akshata Udyavar. ACTM-838 safely and comprehensively re-activates the immunosuppressive TME by exploiting myeloid biology intrinsic in many 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 736.

Abstract 4060: Characterization of MDNA113, a tumor-targeting anti-PD1-IL-2SK immunocytokine with conditional activation to increase tolerability and maximize efficacy

Abstract Background-MDNA113 consists of an IL-13 decoy receptor-binding domain (MDNA213) fused to anti-PD1-IL-2SK (MDNA223) immunocytokine using a matrix metalloprotease (MMP) sensitive linker (PSL). MDNA213 is an IL-13 superkine with high selectivity towards IL-13Rα2, a tumor associated antigen overexpressed in numerous aggressive solid tumors with limited expression in normal tissues. IL-13Rα2 overexpression is associated with MMPs abundancy in tumor microenvironment (TME) and poor clinical outcomes. MDNA223, a fusion of ‘not-alpha, beta enhanced’ IL-2 superkine with anti-PD1 is designed to facilitate cis-binding to PD1 and IL-2Rβγ on CD8+ T cells. MDNA223 is superior to co-administration of anti-PD1 and IL-2 agonist at inhibiting both immunologically ‘hot’ and ‘cold’ tumors in mice. MDNA113 is designed to target and accumulate in tumors via the MDNA213 domain, which also attenuates immune stimulation by hindering IL-2R activation to reduce peripheral activity. The dual targeting/masking MDNA213 domain is released within tumor microenvironment following MMP cleavage of PSL to fully restore IL-2R agonism while maintaining PD1/PDL-1 blockade. Methods-In vitro signaling studies were performed in reporter cell assays and human PBMCs. Tolerability and pharmacodynamic studies were conducted in mice. Tumor growth inhibition was assessed in syngeneic mouse tumor models in monotherapy and combination settings. Results-MDNA113 showed reduced IL-2R agonism (~38-fold) while maintaining PD1/PD-L1 blockade compared to MDNA223 (non-masked) in cell-based reporter assays. Capacity to stimulate human PBMC proliferation was reduced while potency in CD8+ T cell p-STAT5 signaling also decreased. MMP cleavage of MDNA113 to release MDNA213 domain fully restored IL-2R signaling. Systemic delivery of MDNA113 to mice showed reduction, but not complete blockade, of lymphocyte expansion compared to equimolar dose of MDNA223. A 3x increase in MDNA113 restored lymphocyte expansion to a similar level achieved with MDNA223, indicating a ‘partial masking’ effect of MDNA213 domain. Accordingly, MDNA113 was better tolerated than MDNA223 in viability studies in mice. In syngeneic tumor models, equimolar treatment with MDNA113 and MDNA223 (non-mask version) resulted in similar extent of tumor growth inhibition. In contrast, in vivo activity was partially compromised when mice were treated with a non-cleavable version of MDNA113, consistent with a role for MMP-cleavage to restore full anti-tumor effect within TME. Conclusion-MDNA113 is a tumor-targeting and conditional-activatable anti-PD1/IL-2SK fusion characterized with potent PD1/PDL1 blockade and ‘not-alpha, beta enhanced’ IL-2R agonism. MDNA113 is better tolerated than the non-mask version while achieving similar effect on tumor control, therefore offering a much broader therapeutic index. Citation Format: Aanchal Sharma, Minh D. To, Qian Liu, Fahar Merchant. Characterization of MDNA113, a tumor-targeting anti-PD1-IL-2SK immunocytokine with conditional activation to increase tolerability and maximize efficacy [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 4060.

Abstract 5301: Tumor-targeted activation of CD137 using Bicycles: New insights into mechanism of action and discovery of BT7455, a clinical candidate for the treatment of EphA2-expressing cancers

Abstract Introduction: Clinical studies in cancer patients have validated CD137 agonism as an activator of the immune system to enable tumor rejection. We have demonstrated that small, chemically synthetic bicyclic peptides can drive tumor-localized agonism of CD137 and anti-tumor immunity in mouse models. Here, we report the next stage of our work - delving into the mechanism of action of these novel agents and extending our program to serve patients whose tumors express EphA2. Experimental Procedures: MultiOmyx™ hyperplexed immunofluorescence assay was used to evaluate the expression of CD137 and EphA2 in head and neck squamous cell carcinoma samples. Human PBMC/tumor cell co-culture assays were used to assess CD137 Bicycle TICA™ in vitro bioactivity and syngeneic mouse tumor models were used to evaluate CD137 Bicycle TICA anti-tumor activity as mono- or combination therapies. Pharmacodynamic activity was evaluated by transcriptional profiling using NanoString assays or single cell RNA sequencing. Summary of the Data: Studies utilizing a syngeneic mouse model and deep RNA sequencing of tumors from CD137 Bicycle TICA-treated mice implicated intratumoral dendritic cells in addition to T cells as early responders to localized CD137 agonism and potential contributors to the anti-tumor response. Furthermore, a mouse efficacy model demonstrated synergy with checkpoint inhibitor therapy. Multiplex imaging revealed that EphA2 and CD137 are co-expressed in human tumors of high unmet medical need and therefore, using our highly modular Bicycle platform, we discovered BT7455, a Bicycle TICA that engages EphA2 and CD137 with high affinity, resulting in potent EphA2-dependent activity in vitro and robust anti-tumor activity in vivo with intermittent dosing in mouse models. Gene expression profiling of tumors revealed that BT7455 led to increased production of cytokines and chemokines known to drive T cell infiltration, the extent of which differentiated it from both a checkpoint inhibitor and an anti-CD137 monoclonal antibody agonist. BT7455 was well tolerated in preclinical species and liver function tests indicated no evidence of hepatic toxicity. Statement of Conclusions: In summary, we have identified EphA2 as a promising target to pair with a CD137 agonist. In addition to CD8+ T cells, tumor-resident dendritic cells are a likely contributor to anti-tumor immunity following treatment with Bicycle® TICAs, supporting utility for Bicycle® tumor-targeted CD137 agonists in solid tumors beyond those that are highly T cell-infiltrated. We have advanced a clinical development candidate, BT7455, to realize this potential for patients with EphA2-expressing cancers. Citation Format: Johanna Lahdenranta, Kristen Hurov, Heather Cohen, Lia Luus, Cara Bray, Peter Brown, Anna Devlen, Carly Campbell, Matthew Gray, Mike Kelly, Gemma Mudd, Punit Upadhyaya, Sailaja Battula, Kelvin Zhang, Anne-Sophie Dugast, Kevin McDonnell, Phil Brandish, Nicholas Keen. Tumor-targeted activation of CD137 using Bicycles: New insights into mechanism of action and discovery of BT7455, a clinical candidate for the treatment of EphA2-expressing 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 5301.

An IRF2-Expressing Oncolytic Virus Changes the Susceptibility of Tumor Cells to Antitumor T Cells and Promotes Tumor Clearance.

IFN regulatory factor 1 (IRF1) can promote antitumor immunity. However, we have shown previously that in the tumor cell, IRF1 can promote tumor growth, and IRF1-deficient tumor cells exhibit severely restricted tumor growth in several syngeneic mouse tumor models. Here, we investigate the potential of functionally modulating IRF1 to reduce tumor progression and prolong survival. Using inducible IRF1 expression, we established that it is possible to regulate IRF1 expression to modulate tumor progression in established B16-F10 tumors. Expression of IRF2, which is a functional antagonist of IRF1, downregulated IFNγ-induced expression of inhibitory ligands, upregulated MHC-related molecules, and slowed tumor growth and extended survival. We characterized the functional domain(s) of IRF2 needed for this antitumor activity, showing that a full-length IRF2 was required for its antitumor functions. Finally, using an oncolytic vaccinia virus as a delivery platform, we showed that IRF2-expressing vaccinia virus suppressed tumor progression and prolonged survival in multiple tumor models. These results suggest the potency of targeting IRF1 and using IRF2 to modulate immunotherapy.

Abstract 6535: Targeting estrogen receptor signaling in tumor associated myeloid cells to enhance anti-tumor immunity

Abstract Immune checkpoint inhibitors (ICB) have significantly improved outcomes across the spectrum of cancers, especially in patients with metastatic melanoma or non-small cell lung cancer (NSCLC). Despite encouraging efficacy in some patients, the development of primary and acquired resistance and severe toxicity associated with these treatment regimens, together with the overall low response rates, have limited their widespread use. Mining publicly available transcriptomic datasets, we have demonstrated that the response to ICB in patients with melanoma is influenced by the functionality of tumor associated macrophages (TAMs). Thus, it is of significance that we have demonstrated that 17-β estradiol (E2), working through myeloid cell intrinsic estrogen receptor alpha (ERα), promotes tumor growth in preclinical mouse models by polarizing tumor associated macrophages towards an immune-suppressive state in a manner that suppresses CD8+T cell functionality and limits ICB efficacy independent of gender. Transcriptomic analysis of tumor infiltrating myeloid cells isolated from E2-treated syngeneic tumors revealed that the type I interferon (IFN) signaling pathway was suppressed and that this activity could be reversed by the addition of fulvestrant a Selective Estrogen Receptor Downregulator. The importance of type I IFN signaling in E2-regulated tumor growth was confirmed in syngeneic mouse tumor models (B16F10-melanoma and LLC1-NSCLC cells) by showing that inhibiting type I interferon receptor (IFNAR) activity using blocking antibodies reversed the protective effects of E2 deprivation (ovariectomy) on tumor growth. Further, using in vitro cultures of macrophages from mouse bone marrow or peripheral blood mononuclear cells from deidentified human donors, we demonstrated that E2-dependent inhibition of type I IFN signaling could be attributed to increased efferocytosis by myeloid cells. These findings were recapitulated in vivo using an IFN reporter mouse (Mx1-egfp) implanted with labelled tumor cells (mCherry-Spectrin labelled LLC1), where we demonstrated that E2 mediated-suppression of myeloid cell intrinsic interferon signaling within the tumor microenvironment is contingent upon their engulfment of apoptotic cancer cells. Mechanistically, this enhanced efferocytosis was attributed to increased expression of the fractalkine receptor (CX3CR1) in E2-treated myeloid cells- as either genetic or pharmacological depletion of CX3CR1 in mouse reversed E2-mediated tumor growth (B16F10 and LLC1), myeloid cell polarization (from immunosuppressive to proinflammatory), myeloid cell intrinsic type I IFN signaling and reinvigorated CD8+T cell function. Taken together, our results highlight the potential clinical utility of targeting the E2/ER-CX3CR1 pathway as a means to enhance response to ICBs. Citation Format: Binita Chakraborty, Michael Brown, Prabuddha Chakraborty, Aditi Goyal, Ching Yi Chang, Donald McDonnell. Targeting estrogen receptor signaling in tumor associated myeloid cells to enhance anti-tumor immunity [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 6535.

Abstract 5534: Needle biopsy induces pro-metastatic progression and systemic dissemination of estrogen receptor-positive breast cancer cells through the COX-2/PGE2 cascade

Abstract Breast cancer is the most commonly diagnosed malignancy in the U.S. and over two-thirds of invasive breast cancer cases are diagnosed early stage. However, 15-year survival rates for early-stage breast cancer remain in the range of 70-77%. Mounting evidence showed an increased risk of disease progression and mortality among early-stage breast cancer patients whose surgery was delayed over 60 days after the diagnostic biopsy. Yet, the mechanism(s) underlying the rapidly increased mortality due to delay of surgery after diagnosis remains unknown. Our cohort analyses of early-stage breast cancer patients reveal the emergence of a significantly rising mortality risk when the biopsy-to-surgery interval was extended beyond 53 days. Additionally, histology of post-biopsy tumors shows prolonged retention of a metastasis-permissive wound stroma dominated by M2-like macrophages capable of promoting cancer cell epithelial-to-mesenchymal transition and angiogenesis by secretion of transforming growth factor beta 1 (TGF-β1) and vascular endothelial growth factor (VEGF). We show that needle biopsy promotes systemic dissemination of cancer cells through a mechanism of sustained activation of the cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) cascade, which favors M2-polarization and its associated pro-metastatic changes, but are abrogated by oral treatment with COX-2 inhibitors in estrogen receptor-positive (ER+) syngeneic mouse tumor models. Therefore, we conclude that needle biopsy of ER+ breast cancer provokes progressive pro-metastatic changes, which may explain the mortality risk posed by surgery delay after diagnosis. Also, our data may provide a rationale for a pharmacologic strategy to inhibit the COX-2/PGE2 cascade in cases when delay of surgery is unavoidable. Citation Format: Hiroyasu Kameyama, Macall Leslie, Reese Simmons, Yunguang Sun, Misung Yi, Priya Dondapati, John F. Langenheim, Kar-Ming Fung, Inna Chervoneva, Hallgeir Rui, Takemi Tanaka. Needle biopsy induces pro-metastatic progression and systemic dissemination of estrogen receptor-positive breast cancer cells through the COX-2/PGE2 cascade [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 5534.

Abstract 1321: In vivo immune cell programming using mRNA-LNP chimeric antigen receptors

Abstract Introduction: The tumor microenvironment (TME) is a dynamic and multifaceted system that comprises largely myeloid lineage immune cells including monocytes, neutrophils, macrophages, and dendritic cells. Reprogramming the myeloid compartment within the context of cancer therapy presents an attractive strategy to overcome current limitations in solid tumor treatment. We have developed a novel approach that harnesses the natural ability of Myeloid cells to take up mRNA loaded lipid nanoparticles, to reprogram these cells with chimeric antigen receptors (CAR’s) that arm these cells to recognize and kill tumor cells. Methods: Current LNPs technology is sufficient to deliver significant mRNA cargo to numerous cell types, including B cells, T cells and myeloid cells. Leveraging this phenomenon we have developed a new class of chimeric antigen receptors (CARs), using protein biology that imparts targeted immune cell expression and functionality, specifically in Myeloid cells. These CARs harness Immunoreceptor Tyrosine-based Activation Motif (ITAM) signaling adaptors expressed exclusively in immune cells, achieving immune cell-type specific CAR-directed tumor lysis and cytokine responses. With a FcaR I (CD89) based CAR design, we have developed a CAR that is dependent on the common Fc receptor gamma chain, primarily restricted to myeloid cells, for stable expression and function. Results: We have demonstrated specific expression of CD89-based CAR in myeloid cells in mouse and monkey following systemic mRNA/LNP delivery, and in human myeloid cells following in vitro treatment with mRNA/LNPs. Systemic mRNA/LNP delivery of CD89-based CAR has demonstrated robust anti-tumor efficacy against multiple tumor antigens in various xenograft mouse models of human breast cancer, hepatocellular carcinoma, and ovarian cancer. Furthermore, in syngeneic mouse tumor models, robust anti-tumor immune responses, characterized by tumoral infiltration of activated CD8+ T cells, diminished tumor-associated Tregs, systemic enhancement of dendritic cells and anti-tumor IgG production, were observed for treated animals. Conclusions: Taken together, we present CAR designs that achieve expression and function in targeted immune cell populations, capable of eliciting anti-tumor efficacy against multiple target antigens by leveraging innate and adaptive immune responses, and demonstrating a class of novel CAR designs to overcome limitations faced by current in vivo mRNA/LNP delivery strategy. A phase I clinical trial is currently on-going to assess the safety and preliminary efficacy of CD89-based CAR against TROP2+ epithelial malignancies. Citation Format: Shannon Argueta, Franciele K. Melber, Michael Gorgievski, Neha Divangi, Edward Cochran, Thach Chu, Yuxiao Wang, Jian Ding, Daniel Getts. In vivo immune cell programming using mRNA-LNP chimeric antigen receptors [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 1321.

Abstract 6349: 2MW4991, a novel ADCC-enhanced integrin αvβ8 blocker, exhibits high anti-tumor potency and was well tolerated in cynomolgus monkeys

Abstract Background: The three highly structurally related TGF-β isoforms (TGF-β1, TGF-β2, and TGF-β3) exert greatly different effects on cancer and immune cells under various physiological conditions. Efforts to target the TGF-β pathway have been hampered by systemic toxicity or limited efficacy due to challenges associated with the global inhibition of TGF-βs. Recently, TGF-β1 was proven to be a critical player in generating an immunosuppressive tumor microenvironment (TME). TGF-β1 is expressed as an inactive latent form (L-TGF-β1) and activated upon binding to integrins, and integrin αvβ8 is a key activator of TGF-β1 in TME. Here, an ADCC-enhanced αvβ8 blocking antibody, 2MW4991, was developed to specifically inhibit TGF-β1-mediated signaling for the treatment of αvβ8-expressing cancers. Methods: Binding ability, specificity, and affinity of antibodies were measured by Flow cytometry and Biacore T200. A blocking assay was performed using U251MG cells and TGF-β signaling reporter cells. Jurkat/h FcγRIIIa reporter system was used to assess the ADCC activity of 2MW4991. Mouse syngeneic tumor models were used to characterize the in vivo anti-tumor efficacy. A repeat dose administration study was performed in cynomolgus monkeys to evaluate the pharmacokinetic and toxicity profiles of 2MW4991. Results: ScRNA-seq analysis of kidney renal clear cell carcinoma (KIRC) and head and neck squamous cell carcinoma (HNSCC) clinical samples revealed that αvβ8 highly expresses in tumor and some myeloid-derived cells, suggesting the development of ADCC-enhanced αvβ8 blocking antibodies for the treatment of αvβ8-expressing cancers. 2MW4991 showed sub-nanomolar binding affinity and high specificity to αvβ8, with no cross-reactivity to other integrins. 2MW4991 could effectively block TGF-β1 release in a GARP/L-TGF-β1/αvβ8 reporter system. In an immune-excluded EMT6 syngeneic model, 2MW4991 promoted substantial tumor regression and reversed immune suppressive TME by significantly increasing tumor infiltration of CD8+ T and innate immune cells. As expected, TGF-β1 blockade significantly increased the sensitivity of anti-PD-1 therapy in a combination study. Furthermore, 2MW4991 displayed potent anti-tumor efficacy in the human αvβ8 knock-in syngeneic model mimicking αvβ8 expression in human cancers. In a repeat dose administration NHP study, 2MW4991 showed a good pharmacokinetic profile and was well tolerated at a high dose of 100 mg/kg, accompanied by significantly declined pharmacodynamic biomarker TGF-β1 in peripheral blood. Conclusion: Integrin αvβ8 is a main activator of TGF-β1 in the TME and overexpressed in some cancer types. 2MW4991 demonstrated potent anti-tumor activity by unleashing the immune cells suppressed by TGF-β1 in TME and Fc-mediated immune functions. These compelling preclinical results support further evaluation of 2MW4991 in clinical trials. Citation Format: Cuicui Guo, Zhen Han, Xiaowei Cen, Hai Wu, Jinchao Zhang, Xun Gui. 2MW4991, a novel ADCC-enhanced integrin αvβ8 blocker, exhibits high anti-tumor potency and was well tolerated in cynomolgus monkeys [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 6349.

Abstract 1183: A first-in-class and SHP1 allosteric inhibitor, SB8091, shows a good efficacy and safety profile in preclinical models

Abstract Introduction: Src homology 2 domain-containing phosphatase 1 (SHP1) is a cytoplasmic protein tyrosine phosphatase (PTP) that is primarily expressed in hematopoietic cells and is commonly recognized as a negative regulator of multiple signaling pathways1. Previously, we showed that inhibition of the phosphatase activity of SHP1 by a novel SHP1 allosteric inhibitor, SB6299, results in the activation of innate and adaptive immune cells and enhanced anti-tumor immune responses2. Here, we report SB8091, an advanced SHP1 allosteric inhibitor with a promising initial safety profile and improved pharmacokinetic properties. Methods: SHP1 inhibitors were developed through a structure-based drug design approach, and optimized for cellular activity on target, in vitro ADME, and in vivo pharmacokinetic properties. Pharmacokinetic (PK) and preliminary toxicity studies were performed with mice. The lead series was assessed in in-vitro studies with mouse or human primary immune cells. In-vivo efficacy and pharmacodynamic data were generated using mouse syngeneic tumor models. Results: Structurally, SB8091 seemed to stabilize the autoinhibitory form of SHP1 as an allosteric inhibitor, in a nanomolar range and which can explain the outstanding selectivity compared to other PTP enzymes, including SHP2. In several human primary immune cell types, SB8091 significantly increased cytokine responses and demonstrated significantly enhanced activity in NK cells and macrophages against various cancer target cells. As a single agent, this compound exhibited inhibitory effects on tumor growth when administered orally, and the efficacy was further enhanced when co-administered with anti-PD1 antibodies in various syngeneic tumor models. Notably, this effect was associated with a pharmacodynamic response marked by increased levels of phosphorylated substrates regulated by SHP1. SB8091 exhibited favorable pharmacokinetic properties for oral administration, and SafetyScreen data showed a very low potential for off-target activity and drug-drug interactions. Additionally, during a 2-week repeat pilot toxicity study in mice, SB8091 demonstrated excellent tolerability even at high doses, indicating a high therapeutic index (TI > 100). Conclusion: Our advanced compound, SB8091, is a potent, highly selective, and orally bioavailable allosteric inhibitor of SHP1. It has demonstrated excellent efficacy as a monotherapy or in combination with immune checkpoint blockers, with favorable drug properties and safety profiles. Research to advance to the clinical stage is being conducted.

Abstract 4514: Discovery of potent and orally bioavailable degraders of HPK1 based on a novel HPK1 binder

Abstract Background: Proteolysis targeting chimeras (PROTACs) are novel structures designed to target a protein of interest (POI) for ubiquitination and degradation, leading to the selective reduction in the expression levels of the POI. Hematopoietic progenitor kinase 1 (HPK1) is considered a promising target for tumor immunotherapy. PROTAC-involved disruption of HPK1 protein activity enhanced the treatment efficacy of CAR-T-cell-based immunotherapies in different solid tumors and mouse models. Thus, targeting HPK1 by PROTAC to restore T-cell activity is an attractive approach to induce a greater immune responses in cancer. Results: We designed and synthesized a series of HPK1 degraders by tethering our previously discovered novel HPK1 inhibitor with CRBN binder through various linkage. The most potent PROTAC effectively degrades HPK1 proteins in the Ramous cell in a dose-dependent manner and achieves DC50 value <50 nM. The maximum degradation (D max ) values achieved are >90%. Our PROTAC showed EC50 value <100 nM for IL-2 and IFNγ production. PK studies revealed a low clearance, high plasma exposure and oral bioavailability in mice and rats. Furthermore, our PROTAC is shown to induce robust and statistically significant tumor growth inhibition in the MC38 and CT26 syngeneic model with increased T cell signatures observed within the tumor. The PROTAC in combination with anti-PD1 also resulted in robust anti-tumor activity with TGI > 90%. The 14 days DRF experiment shows that the compound has a great safety window(>200 folds). Conclusion: We show here that a potent oral HPK1 degrader demonstrates strong immune cell activation and robust anti-tumor activity in mouse syngeneic tumor models, as a single agent and in combination with anti-PD1. Further evaluation of these potent degraders in additional in vivo studies will continue to build upon our mechanistic understanding of HPK1 degradation as a novel immunomodulatory approach for anti-tumor immunity. Citation Format: Zhimin Zhang, Mengqiang Wu, Ling Wang, Xi Yang, Zhiping Zhang, Liubin Guo, Hao Pan, Mengting Zhao, Linli Wang, Sirui Liu, Zhao Dong, Chunhua Jiang, Haowen Zheng, Dongzhou Liu. Discovery of potent and orally bioavailable degraders of HPK1 based on a novel HPK1 binder [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 4514.

Abstract 4062: A novel method for generating regulated cytokine therapeutics: Safety and activity of a conditionally active cLAG3-IL2 capable of delivering IL2 to LAG3+ cells while remaining inert on LAG3- cells

Abstract IL-2 is a powerful cytokine central to the generation of an effective immune response. However, its use in cancer immunotherapy has been limited by toxicities arising from its broad activity as well as the narrow patient population in which efficacy is seen. To address these limitations, many strategies have been pursued including IL-2 attenuation and targeting of IL-2 activity. These approaches, however, retain unacceptable toxicity and/or are not targeted to the appropriate cell populations. Using a novel approach that takes advantage of the ability of an antibody to bind specifically and competitively to two distinct antigens, we have generated a conditionally active cLAG3-IL2 therapeutic that targets IL-2 to LAG3+ cells while remaining inert on IL-2Rβγ+ cells lacking LAG3 expression, even at high treatment doses. As a marker of antigen-activated and tumor-specific T cells, LAG3 is an ideal target toward which to direct IL-2 activity. In vitro, using both reporter cell lines and receptor binding assays, the regulated cLAG3-IL2 demonstrates >100 fold difference in activity in the presence or absence of LAG3. In primary immune cells, regulated cLAG3-IL2 preferentially induces IL-2 cis-signaling in activated human LAG3+ CD8 T cells compared to LAG3- T cells. In mouse syngeneic tumor models, cLAG3-IL2 inhibits tumor growth while avoiding clinical signs of IL-2-mediated toxicity at doses well above the level where the non-regulated IL2 is toxic. Additionally, cLAG3-IL2 does not induce expansion of circulating LAG3- IL-2Rβγ+ cell populations, including NK cells and LAG3- T cells. In the TME, cLAG3-IL2 drives the expansion and activation of tumor-specific CD8+ T cells. Furthermore, cLAG3-IL2 demonstrates robust combinatorial activity with anti-PD1. These results demonstrate the strength of the Bonum platform, currently applied to multiple target/effector pairs including PD1, PDL1, ATP, LRRC15 and effectors IFNα, IL12 and TGFβ inhibition, and support the clinical development of our conditionally active cLAG3-IL2. Citation Format: Justin Killebrew, Shannon Okada, Lynn Amon, David Bienvenue, Laura Carlucci, David Colby, Wendy Curtis, Kendyl Daniels, Alton Etheridge, Zane Kraft, Jamie Nguyen, Sandra Notonier, Jacqueline Pham, Megan Sprague, Kerri Thomas, Diane Hollenbaugh, John Mulligan. A novel method for generating regulated cytokine therapeutics: Safety and activity of a conditionally active cLAG3-IL2 capable of delivering IL2 to LAG3+ cells while remaining inert on LAG3- cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4062.

Abstract 4081: rhIL-7-hyFc (efineptakin-alfa, NT-I7) increases tumor-specific CD8+ T cells despite FOLFOX cytotoxicity effect

Abstract rhIL-7-hyFc (efineptakin-alfa; NT-I7) is a potent T cell amplifier, comprised of two molecules of interleukin-7 (IL-7) fused to the hybrid Fc domain of IgD/IgG4 immunoglobulin. NT-I7 safely, significantly, and persistently increases the absolute lymphocyte counts (ALC) in humans in a dose-proportional manner. Importantly, this increase is primarily due to an increase in T cells (1). Previous studies have shown that NT-I7 not only significantly increases the number of CD8+ T cells in peripheral blood but also dramatically boosts the presence of tumor-infiltrating CD8+ T cells, including both tumor-specific (p15E+PD-1+) and tumor non-specific (PD-1-) CD8+ T cells in mice (2). FOLFOX is a chemotherapy regimen composed of 5-fluorouracil, leucovorin, and oxaliplatin that inhibits DNA duplication and eradicates rapidly dividing cells (3). Although the mechanism of FOLFOX results in direct tumor killing, it also has the potential to kill newly dividing lymphocytes during NT-I7 treatment (4-6). Therefore, in this study, we aimed to evaluate the feasibility of combining FOLFOX with NT-I7 in a syngeneic MC38 mouse tumor model. Our results show that NT-I7 improves the efficacy of FOLFOX, irrespective of the timing of NT-I7 administration: simultaneous administration or delayed 3 hours, or 2 days after FOLFOX administration. This improvement persists despite a significant decrease in ALC for the first 6 days, which was significantly lower than the ALC observed with FOLFOX alone. FOLFOX decreases the absolute numbers of tumor non-specific CD8+ T cells. However, adding FOLFOX does not affect the NT-I7-driven increase of tumor-specific CD8+ T cells within the tumor. Taken together, our data demonstrate that FOLFOX does not compromise NT-I7’s ability to increase tumor-specific T cells within the tumor and the combination enhances the anti-tumor response irrespective of the timing of NT-I7 administration. Citation Format: Soung-Min Lee, Miyoung Kim, Sara Ferrando-Martínez, Sun-Kyoung Im, Mankyu Ji, Donghoon Choi. rhIL-7-hyFc (efineptakin-alfa, NT-I7) increases tumor-specific CD8+ T cells despite FOLFOX cytotoxicity effect [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 4081.

Abstract 4059: Vitokine-2: A TIL-targeting tumor activable engineered IL-2 for cancer treatment

Abstract Background: Interleukin-2 is the first approved immune-oncology (IO) drug to treat multiple metastatic cancers with durable and curative antitumor effects. Yet, its application is restricted due to severe toxicity, short half-life, and a small fraction of response rate. To reduce toxicity and improve efficacy, we developed Vitokine-2, a tumor infiltrating lymphocyte (TIL)-targeting, tumor activable, and precision engineered IL-2 that remains inert systemically but activated in the tumor microenvironment (TME). Methods: Through protein and structure-guided precision engineering, the biological activity of IL-2 was optimized, concealed, guided to exhausted TILs, and activated at TME. The biological activity was determined by measuring proliferation (Ki67) of CD8+ T and NK cells using human peripheral blood mononuclear cells (PBMCs). Pharmacokinetics (PK), pharmacodynamics (PD), and anti-tumor efficacy were evaluated in mice, encompassing healthy and multiple syngeneic tumor mouse models. Results: Vitokine-2 was inactive in stimulating CD8+T and NK cell proliferation in vitro. Yet, its biological activity was fully restored following the cleavage and release of the concealing domain. Vitokine-2 displayed a significantly prolonged in vivo pharmacokinetic profile due to half-life extension, concealing and reduction of target sink. Administration of Vitokine-2 in mice resulted in minimal or no detectable activation, proliferation, and expansion of cytotoxic CD8+T and NK cells in the systemic circulation. However, marked CD8+ T cell proliferation and expansion with enhanced expression of granzyme B, a cytotoxic functional biomarker, was observed within the TME. The effect was specific to cytotoxic lymphocytes but not regulatory T cells. Corroborating with the expansion and enhanced functionality of CD8+ T cells in TME, robust antitumor efficacy was observed with Vitokine-2 but not with the non-activable IL-2 counterpart. Furthermore, dose-dependent tumor killing efficacy and complete eradication of tumors were demonstrated in multiple syngeneic tumor models. Vitokine-2 was found to be safe and well tolerated with minimal interferon gamma release and body weight loss even at much higher doses. Conclusion: Vitokine-2 is a TIL-targeting, tumor activable and precision engineered IL-2. It demonstrated robust anti-tumor efficacy with minimal systemic toxicity. It is safe, well tolerated, and permits high dose administration, yielding a widened therapeutic window. Vitokine-2 presents a paradigm-shifting, potentially a first-in-class, highly safe and efficacious IO therapy with an immense potential to address both IO-responsive and unresponsive patient population. Citation Format: Sreerupa V. Challa, Yao-Te Hsieh, Karen Gelinas, Andrea Umana, Wei-chun Weng, Toska Berisha, Cameron Stephens, Yuesheng Li, Jing Xu. Vitokine-2: A TIL-targeting tumor activable engineered IL-2 for cancer treatment [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 4059.

Abstract 86: Deep immuno-profiling of syngeneic tumor mouse models for preclinical studies

Abstract The identification of the major cellular players involved in the progression of a type of cancer is a key step for the success of new immunotherapies for personalized medicine. Immune cells play critical functions in cancer, and mice with intact immune systems are vital to understanding tumor immunology. It is however a daunting challenge as complex relationships interplay between tumor cells and the immune system. Each component of innate immunity or adaptive immunity, such as T lymphocytes, macrophages or neutrophils, may be directed to a pro- or anti-tumor function. In order to cope with the complexity of the tumor microenvironment, it is necessary to use an experimental approach capable of characterizing the heterogeneity of the cell types present in the tumor, the evolution of their relative proportion and their fine-tuned functional specificity. This approach leads to the identification of new cellular biomarkers of different stages of tumor progression in order to identify new targets for therapeutic time-window of and improve cancer diagnosis To decipher the impact of immunotherapy treatments involved in the anti-tumor response, cellular phenotyping of leukocytes infiltrating a tumor but also those present in peripheral organs is necessary. Essentially based on extracellular labeling, this primary screen aims to quantify the different cell populations present in a syngenic tumor models on B6/N or BalbC genetical backgrounds. In order to increase our understanding in the precise mode of action of anti-PD1 treatment at the cellular level in sensitive and unsensitive models, we investigated immunophenotypes and responses to immune checkpoint inhibitor (ICI) of several hallmark syngeneic tumor models (MC38, CT26, B16F10, B16-OVA, RENCA, EMT6) in immunocompetent mouse models by flow and mass cytometry. We compared growth kinetics and profiled the immune cell composition of tumor microenvironment (TME), draining lymph node (dLN) and blood in order to establish immune-phenotypic cell signatures that correlates with treatment efficacy. Supervised, unsupervised and integrative data analysis as well as visualization tools are used to identify significant changes across different experimental settings. Our results indicate that each model possesses a unique tumor-immune infiltrate profile that can be modulated with immunotherapies. Overall, these studies provide an important resource of highly-characterized syngeneic tumor model and highlight the importance of tumor immune landscape variance across models that will drive selecting the most appropriate model to test novel immunotherapeutic agents and enhance our translation of knowledge from syngeneic models to human tumors. Citation Format: Anais Joachim, Emilie Maturin, Marielle Mello, Lilia Hadjem, Magali Grange, Olivier Deas, Ana Zarubica, Bernard Malissen, Hervé Luche, François Romagne, Stéphanie Blanchin. Deep immuno-profiling of syngeneic tumor mouse models for preclinical studies [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 86.