Syngeneic Mouse Tumor Models Research Articles

Improving poxvirus-mediated antitumor immune responses by deleting viral cGAMP-specific nuclease

cGAMP-specific nucleases (poxins) are a recently described family of proteins dedicated to obstructing cyclic GMP-AMP synthase signaling (cGAS), an important sensor triggered by cytoplasmic viral replication that activates type I interferon (IFN) production. The B2R gene of vaccinia viruses (VACV) codes for one of these nucleases. Here, we evaluated the effects of inactivating the VACV B2 nuclease in the context of an oncolytic VACV. VACV are widely used as anti-cancer vectors due to their capacity to activate immune responses directed against tumor antigens. We aimed to elicit robust antitumor immunity by preventing viral inactivation of the cGAS/STING/IRF3 pathway after infection of cancer cells. Activation of such a pathway is associated with a dominant T helper 1 (Th1) cell differentiation of the response, which benefits antitumor outcomes. Deletion of the B2R gene resulted in enhanced IRF3 phosphorylation and type I IFN expression after infection of tumor cells, while effective VACV replication remained unimpaired, both in vitro and in vivo. In syngeneic mouse tumor models, the absence of the VACV cGAMP-specific nuclease translated into improved antitumor activity, which was associated with antitumor immunity directed against tumor epitopes.

Abstract 1546: ADCC enhanced anti-CD25 mAb (TST010) demonstrated antitumor activity via depleting Treg cells and increasing CD8+T/Treg ratio in preclinical tumor models

Abstract Regulatory T (Treg) cells are a distinctive lineage of CD4+ T cells as characterized by expression of FoxP3 and CD25 markers. Treg cells are involved in tumor development and progression by inhibiting antitumor immunity. Accumulation of Treg cells in the tumor microenvironment (TME) is often associated with an adverse prognosis in multiple cancers and also has implication for predicting response to immunotherapy. Treg cell depletion strategy has been explored in preclinical and clinical settings to induce effective antitumor immune responses in TME. CD25, a high-affinity binding subunit alpha of the IL-2 receptor, is constitutively and highly expressed on Treg cells but also transiently upregulated on effector T cells. The high level of CD25 expression on Treg cells could deprive of IL-2 for effector T cells and inhibit their proliferation. Thus, an effective approach to selectively suppress Treg cell function is to deplete Treg cells while avoid blocking IL-2 binding to CD25 for allowing T effect cell proliferation and activation. Using our immune tolerance breaking antibody platform, we generated a humanized IgG1 subtype anti-CD25 monoclonal antibody (TST010). TST010 bound to human CD25 with high affinity and selectivity but did not block IL-2 binding to CD25. No blocking activity to IL-2 signaling on human PBMC was further confirmed by the phosphorylation of Stat5 using FACS analysis. By reducing fucosylation during cell culture process, TST010 mAb gained the enhanced ADCC activity. In an ADCC reporter assay, TST010 showed its sub-nanomolar ADCC activity against CD25-overexpression SU-DHL-1 cell line and human induced Treg cells, but much less to human activated CD8+ T effect cells due to lower CD25 expression. Similarly, in a NK cytotoxicity assay, TST010 selectively killed human induced Treg cells in contrast to activated CD8+ T cells. In vivo antitumor activity of TST010 was evaluated in the syngeneic mouse tumor models. The mouse colon cancer MC38 model was developed on the human CD25 knock-in mice. In the MC38 tumor model, TST010 markedly reduced the Treg (FoxP3+CD4+) population in both peripheral blood and TME. Importantly, the CD8+T/Treg ratio in tumor was increased starting at 2 weeks post TST010 treatment, and it resulted in a significant single agent tumor growth inhibition at a 10 mg/kg dose. Furthermore, in the LLC tumor model which does not respond well to checkpoint inhibitors, the anti-CD25 mAb showed a good combination effect with a PD-L1 inhibitor. In summary, we have discovered a novel therapeutic anti-CD25 mAb that can deplete Treg cells without blocking IL-2 signaling. As demonstrated in our preclinical tumor models, it has a good potential to induce effective antitumor immune responses in TME and tumor growth inhibition especially in combination with PD-1/PD-L1 treatment. Citation Format: Di Sun, Huanhuan Guo, Hongjun Li, Xinlai Yao, Yiqing Xu, Shenjie Zhang, Shijie Zhou, Xiaoli Zi, Lisa Zheng, Yi Gu, Xueming Qian. ADCC enhanced anti-CD25 mAb (TST010) demonstrated antitumor activity via depleting Treg cells and increasing CD8+T/Treg ratio in preclinical tumor models [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 1546.

Abstract 5124: The effect of lenvatinib in combination with chemotherapy plus immune checkpoint inhibitors on tumor microenvironments in the mouse syngeneic tumor model

Abstract Introduction: Lenvatinib (LEN) is an oral, multiple-receptor, tyrosine kinase inhibitor that mainly targets vascular endothelial growth factor and fibroblast growth factor receptors. Combination therapy with LEN and everolimus is approved for people with advanced renal cell carcinoma (RCC). Combination therapy with LEN and pembrolizumab, an anti human-programmed-cell-death-1 (PD-1) antibody, is also approved for the treatment of people with advanced endometrial cancer and advanced RCC. In this study, we investigated the antitumor activity of LEN in combination with chemotherapy and immune checkpoint inhibitors (ICIs), and explored the mechanism of action underlying these effects, in the mouse lung cancer syngeneic model. Methods: In the LL/2 mouse Lewis lung carcinoma model, LEN was dosed at either 3 or 10 mg/kg (orally; once daily for 2 weeks) with or without cisplatin 4 mg/kg (intravenously; 3 or 4 times, twice weekly) and with or without anti-PD-1 200 ug/head + anti cytotoxic T-lymphocyte antigen 4 (CTLA4) antibodies 10mg/kg (intraperitoneally; twice weekly). Functional blood vessels were evaluated in tumor microenvironments by injections of Hoechst 33342 (Hoechst) into the tail veins of mice. The Hoechst-stained area and expressions of angiogenesis/immune-cell-related molecules were analyzed by immunohistochemistry (IHC) analysis. Results: In this model, the combination treatment of LEN 3 mg/kg + cisplatin showed significant tumor growth inhibitory activity compared with each single agent without any marked body-weight loss. LEN 10 mg/kg + cisplatin did not show enhanced antitumor activity vs LEN 10 mg/kg as a single agent. IHC analysis using anti-CD31 antibody showed that LEN 3 mg/kg daily decreased microvessel density in tumors after 4 days. In addition, LEN 3 mg/kg daily for 4 days showed a trend toward an increase of the number of functional blood vessels stained with Hoechst in the tumor microenvironment compared with untreated controls. The combination of LEN 3 mg/kg + cisplatin + ICIs (anti-PD-1+anti-CTLA4) significantly inhibited tumor growth compared with combinations of cisplatin + ICIs. Conclusion: Our findings show that LEN 3 mg/kg enhances the antitumor activity of cisplatin in the LL/2 mouse Lewis lung carcinoma tumor model; and LEN 3 mg/kg + cisplatin + ICIs shows greater antitumor activity compared with cisplatin + ICIs. The results of the IHC analysis suggest that LEN 3 mg/kg inhibits angiogenesis. Conversely, LEN 3 mg/kg increases functional vessels in tumor microenvironments. This situation could enhance combination antitumor activity of chemotherapy, and also chemotherapy + ICIs combination treatment. Further analysis of the mechanism of action of LEN 3 mg/kg in combination with chemotherapy + ICIs is warranted. Citation Format: Yu Kato, Yoichi Ozawa, Keito Adachi, Masahiko Kume, Yusuke Adachi, Yudai Narita, Megumi Kuronishi, Junji Matsui, Akira Yokoi, Yasuhiro Funahashi. The effect of lenvatinib in combination with chemotherapy plus immune checkpoint inhibitors on tumor microenvironments in the mouse syngeneic tumor model. [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 5124.

Abstract 1824: Discovery and evaluation of an anti-IL18BP antibody to enhance anti-tumor immunity

Abstract Interleukin-18 (IL18) is a proinflammatory cytokine that modulates both innate and adaptive immune responses. Although recombinant mIL18 has demonstrated efficacy in mouse tumor models, recombinant human IL18 has not been efficacious in clinical studies, likely due to upregulation of IL18 binding protein (IL18BP), a negative regulator of the IL18 signaling axis. IL18BP is induced 10 - 100 fold after IL18 administration, binds to IL18 with high affinity, and prevents IL18 binding to the IL18 receptor. Analysis of expression data from the TCGA database as well as archived patient samples from select indications revealed that both IL18 and IL18BP are co-expressed in a number of tumor types, suggesting there may be a preexisting pool of IL18/IL18BP complex at the site of the tumor in some patients. Inhibition of IL18/IL18BP binding using a monoclonal antibody may therefore allow release of IL18 in situ, leading to IL18-mediated proinflammatory effects and enhanced anti-tumor immunity. Although the interaction of IL18 with IL18BP is a high affinity interaction, we have been able to identify very high affinity antibodies which can both inhibit complex formation and disrupt existing mouse IL18/IL18BP complexes. These antibodies are active in a number of cell-based functional assays, resulting in potent stimulation of interferon gamma production from cell lines or splenocytes. In vivo, we observed efficacy in a mouse syngeneic tumor model using an anti-mIL18BP antibody. RNA and protein analysis demonstrated expression of both IL18BP and IL18 within the tumors. Combined, these data suggest that inhibition of IL18/IL18BP binding may prove efficacious in the treatment of cancers where existing pools of IL18/IL18BP complexes prevent an IL18-mediated proinflammatory response. Experiments exploring anti-IL18BP therapy in combination with other checkpoint inhibitors such as anti-PD-1 are under current investigation. Citation Format: H. Toni Jun, Deborah A. Witherden, Eric K. Elliot, Christine M. Chidester, Maggie Willen, Bryan R. Peguero, Merrick Chai, Robert A. Horlick, David J. King. Discovery and evaluation of an anti-IL18BP antibody to enhance anti-tumor immunity [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 1824.

Abstract 4127: ANV600 is a potent, Cis-signaling, non-alpha IL-2 agonist which efficiently expands intratumoral stem-like CD8 T cells

Abstract Non-alpha IL-2-based therapeutic modalities with preferential signaling through the IL-2 beta(CD122)/gamma(CD132) receptor are in clinical development and have the potential to substantially increase the therapeutic index of recombinant IL-2 (aldesleukin) for cancer therapy. ANV600 is a novel bispecific compound, which features an anti-IL-2 antibody/IL-2 fusion protein and a proprietary hPD-1 binding moiety for specific delivery of the non-alpha IL-2 to tumor antigen experienced PD-1+ T cells. Compared to the untargeted control, ANV600 has increased potency to induce STAT5 phosphorylation in human PD-1+CD8 T cells in vitro. At the same time, stimulation with ANV600 results in markedly reduced STAT5 phosphorylation on Treg cells compared to aldesleukin. In activated human PBMCs, ANV600 induces PD-1 co-internalization with the CD122-CD132 complex, reducing detectable PD-1 on CD8 and CD4 T-cells. Surface PD-1 levels decrease is not observed on cells incubated with untargeted bispecific compound. In transgenic human PD-1 mice ANV600 treatment leads to marked tumor growth retardation in the B16F10 and MC38 subcutaneous tumor models compared to untargeted compound and vehicle. Immunophenotyping of the tumor infiltrating lymphocytes revealed a dose-dependent increase of intratumoral stem-like PD-1+T cells and cytotoxic GrzB+PD-1+T cells in mice treated with ANV600. ANV600 has potent and selective effects on antigen-experienced CD8 T cells both in human PBMCs and mouse syngeneic tumor models. This agent may be a promising anti-tumor therapeutic against poorly immunogenic tumors and warrants further pharmaceutical development. Citation Format: Patrizia Murer, Ulisse Salazar, Nicole Egli, Laetitia Petersen, Pia Neubert, Kirsten Richter, Christian Stocker, Alexander Rau, Andreas Katopodis, Christoph Huber. ANV600 is a potent, Cis-signaling, non-alpha IL-2 agonist which efficiently expands intratumoral stem-like CD8 T cells. [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 4127.

Abstract 1829: Generation of IL-21 INDUKINETM molecules for the treatment of cancer

Abstract In the past decade, great strides have been made in the development of novel immunotherapies, such as immune checkpoint inhibitors (ICI) to treat cancer. However, despite these improvements, subsets of patients do not respond to ICI, highlighting an unmet need for alternative immunotherapies. As potent immunomodulators, cytokines have been explored as treatments for cancer but have 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. A member of the common γ-chain family of cytokines, IL-21 acts on a broader range of cells than IL-2 and does not induce vascular leak syndrome. Despite being a potent inducer of immune activation, clinical activity of IL-21 has been hampered by poor PK and adverse events at dose levels associated with efficacy. To explore the potential therapeutic benefits of IL-21 as a treatment for cancer, Werewolf Therapeutics has developed different formats of IL-21 INDUKINETM polypeptides containing native human IL-21, an inactivation domain and a half-life extension domain tethered together by protease sensitive linkers. IL-21 INDUKINETM polypeptides are peripherally inactive, but due to dysregulation of the protease milieu in the tumor microenvironment, upon dosing and distribution to the tumor, the linkers are cleaved and IL-21 is released intratumorally. We will present data demonstrating that IL-21 INDUKINETM polypeptides showed in vitro and in vivo inducibility and activity. In mouse syngeneic tumor models, the IL-21 INDUKINETM polypeptides showed improved tolerability and therapeutic window compared to equal molar dosing of a half-life extended IL-21 protein (IL-21-HLE). Mechanistically, IL-21 triggered a pronounced activation of the adaptive immunity in the tumor. Efficacy in these tumor models was linked to expansion and activation of tumor infiltrating T cells, increased polyfunctionality in CD8+ T cells, as well as signs of increased activation of B cells. Together, these data support continued exploration of an IL-21 INDUKINETM polypeptide as a therapy for cancer. Citation Format: Jenna M. Sullivan, Pamela A. Aderhold, Heather R. Brodkin, Kyriakos Economides, Daniel J. Hicklin, Nesreen Ismail, Yuka Lewis, Cynthia Seidel-Dugan, Cierra Spencer, William M. Winston, Andres Salmeron. Generation of IL-21 INDUKINETM molecules for the treatment 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 1829.

Abstract 2717: A potent and highly tumor-selective KRAS inactivator for tumor targeting

Abstract Cancer driver mutations in the RAS-RAF-MEK-ERK pathway occur in 46% of all human cancers. This has inspired the successful development of many small molecule inhibitors of BRAF and MEK. However, development of RAS small molecule inhibitors has not been successful in large part due to the lack of a sufficiently large and deep hydrophobic pocket in RAS proteins to allow for small molecule binding. Notably, in this regard, many bacterial pathogens have evolved potent protein toxins to disrupt the RAS-RAF-MEK-ERK pathway. Fortunately, these naturally designed potent toxins, such as anthrax toxin, Fortunately, these potent, naturally designed toxins, such as anthrax toxin and DUF5, can be structurally modified to achieve high tumor specificity. DUF5 (amino acids Glu3581-Gln4089) is a toxin effector domain of MARTX (a multifunctional-autoprocessing repeats-in-toxin) from Vibrio vulnificus. DUF5 was recently identified to be a specific and potent endopeptidase that cleaves and inactivates RAS. To address the above critical unmet medical needs, in this work, we have generated an anthrax toxin/DUF5-based, highly tumor-selective RAS inactivator and evaluated its anti-tumor activity in a variety of tumor models. This tumor-selective RAS inactivator can specifically bind to the major anthrax toxin receptor CMG2 on tumor cells and tumor stromal cells, and strictly relies on the simultaneous presence of two distinct tumor-associated proteases, MMPs and urokinase, to gain entry into tumor cells and tumor stromal cells to proteolytically inactivate RAS signaling, achieving potent selective targeting. Our data show that our tumor-selective RAS inactivator displays potent cytotoxicity to cancer cells whose survival depends on oncogenic RAS signaling. Our engineered toxin displayed potent in vivo antitumor activity to a range of tumors with oncogenic RAS mutations in syngeneic and human xenograft mouse tumor models. Therefore, we have developed a tumor-selective, safe, and highly potent RAS inactivator that has high potential for targeting tumors with oncogenic RAS mutations. Citation Format: Shihui Liu, Zehua Zuo, Jie Liu. A potent and highly tumor-selective KRAS inactivator for tumor targeting [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 2717.

Abstract 2799: Pharmacodynamic biomarkers for TGFBR1 inhibition in oncology

Abstract Transforming growth factor-beta (TGF-ß) is a master regulatory cytokine with pleotropic effects important in tumor development. TGF-ß1 binds TGF-ß receptor 2, which recruits and phosphorylates TGF-ß receptor 1 (TGFBR1). TGFBR1 in turn phosphorylates SMAD2 and SMAD3. Phosphorylated SMAD2/3 (pSMAD2/3) complexes with SMAD4 to translocate to the nucleus and enact transcriptional programs. In late-stage tumors, TGF-ß signaling is oncogenic, promoting growth, invasion, immune suppression, tissue remodeling, and epithelial to mesenchymal transition (EMT). In an effort to counter the oncogenic effects of TGF-ß signaling, Sumitomo Pharma Oncology, Inc. is developing TP-6379, an investigational, orally available, small molecule, selective TGFBR1 inhibitor. TP-6379 has a biochemical IC50 of 1.07 nM and has been shown to reduce pSMAD2 in Rhabdomyosarcoma cells with an IC50 of 108 nM. A dose dependent increase of TP-6379 was observed in plasma and tumors of A549 tumor bearing athymic nude mice (10, 50, 75, and 100 mg/kg PO). In another study using the same mouse model, pSMAD2 has shown an inverse correlation with pharmacokinetics showing > 90% inhibition for up to 4 hours (75 mg/kg PO). Pharmacodynamic (PD) biomarkers are critical for determining biological activity, but procuring serial tumor biopsies for PD analysis is challenging for many tumor types. We hypothesize that pSMAD2 can be measured in non-tumor tissues as a surrogate for activity within the tumor. Surrogate tissues, such as peripheral blood mononuclear cells (PBMCs), skin punches, and even circulating tumor cells (CTCs) are much less invasive and can be sampled over multiple timepoints. We observed a measurable decrease of PD biomarkers pSMAD2/3 in PBMCs and skin after treatment with TP-6379 using a bead-based immunoassay (Luminex). Healthy human donor PBMCs treated ex vivo for 2 hours with up to 1 µM TP-6379 showed a 96% reduction in pSMAD2/3 signal. Although TP-6379 was still active in reducing pSMAD2 in PBMCs when treated in whole blood for 2 hours, it required around 30-fold more drug to achieve a similar effect, possibly due to serum protein binding. Skin samples from in vivo dosed tumor bearing mice also show significant inhibition of pSMAD2/3, which correlated with significant pSMAD2/3 inhibition in tumors from both EMT6 and 4T1 syngeneic triple-negative breast cancer mouse tumor models. We confirmed that the Luminex assay is sensitive enough to reliably measure pSMAD2/3 in healthy human doner skin punches down to 3 mm in diameter. Lastly, CTCs from breast cancer patients’ whole blood treated ex vivo for 24 hours with as low as 1 µM TP-6379 showed a decrease in pSMAD2 and EMT marker SNAI1 by as much as 96% and 86%, respectively, when measured by immunofluorescence. In summary, our research has shown that pSMAD2/3 and EMT marker SNAI1 are valid PD biomarkers for TP-6379 therapy. We propose that these biomarkers would be best measured clinically using PBMCs, CTCs and/or skin punches. Citation Format: Richard E. Heinz, Curtis A. Allred, David A. Kircher, Yuta Matsumura, Bettina Franz, Zhizhou Ye, Jinny Lee, Clifford J. Whatcott, Jason M. Foulks, Adam Siddiqui, Steven L. Warner. Pharmacodynamic biomarkers for TGFBR1 inhibition in oncology [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 2799.

Abstract 6432: Glutamine antagonist prodrug JHU083 reprograms immunosuppressive tumor-associated macrophages (TAMs) to drive tumor immunity in urologic cancers

Abstract Tumor metabolism is emerging as a regulator of immune mediated anti-tumor responses. Previously, we reported increased immunosuppressive tumor-associated macrophages (TAMs) infiltration with disease progression in prostate adenocarcinoma (PCa).1 Glutamine metabolism has been implicated in metastatic castration resistant prostate cancer (mCRPC)2. To harness the potent anti-tumor effects of 6-Diazo-5-oxo-L-norleucine (DON), which targets glutamine utilizing enzymes and to mitigate known significant toxicities, we here use a novel pro-drug moiety, i.e., JHU083. We hypothesize that JHU083 will enhance anti-tumor immunity by simultaneously targeting TAMs and cancer cells.Using scRNA-seq dataset from mCRPC patients, we investigated the importance of glutamine metabolism in TAMs in the tumors.3 Enriched expression of glutamine utilizing enzymes was observed in TAMs in the metastasized tumor tissue relative to benign. We used JHU083 to treat two urological syngeneic immunogenic mouse tumor models in vivo; B6CaP (PCa) and MB49 (bladder cancer). JHU083 showed significant tumor regression in both models. Using in vivo depletion of CD4 or CD8 T cells or adoptively transferring in-vivo JHU083 treated TAMs in TME, we established a direct anti-tumor role of TAMs. Moreover, to characterize the effect of JHU083 on TAMs transcriptional & translational signatures and metabolite flux in vivo, we used scRNA-sequencing and bulk RNA sequencing at two different time points, multi-parameter flow cytometry and targeted LC-MS/MS metabolic profiling. Clustering of the transcriptional signatures at both time points using UMAP dimensionality reduction classified 6 discreet differentially expressed clusters of TAMs. It elutes to an overall induction of TNF signaling and increased proliferation in the TAM sub-clusters. Strikingly, this was observed translationally as well. Moreover, translationally in the TME at a later timepoint, an increase of glycolytic transporter and enzyme, i.e., Glut1 and Hexokinase II was also observed. The overall impact of this on the metabolome, specifically glycolytic pathway changes in the sorted TAMs, remains to be elucidated. Lastly, we assessed in-vivo the functional phagocytosis of TAMs with flow cytometry and IF microscopy. JHU083-treated TAMs showed significantly increased phagocytic activity, providing direct evidence of functional reprogramming. Conclusions: We found that JHU083 reprograms TAMs from an immunosuppressive to an inflammatory state which we show has a direct anti-tumor effect. These macrophages convert to a highly proliferative and glycolytic state, have increased TNF production which might be resulting in improved phagocytic activity against tumor cells. As urologic cancers are heavily infiltrated with immunosuppressive TAMs, JHU083 is an excellent preclinical candidate. Citation Format: Monali Praharaj, Fan Shen, Alex J. Lee, Liang Zhao, Thomas Nirschl, Xiaoxu Wang, Debebe Theodros, Alok K. Singh, Raekwon A. Williams, Laura A. Sena, Elizabeth A. Thompson, Ada Tam, Srinivasan Yegnasubramanian, Edward J. Pearce, Robert D. Leone, Jesse Alt, Rana Rais, Barbara S. Slusher, Andrew M. Pardoll, Jonathan D. Powell, Jelani C. Zarif. Glutamine antagonist prodrug JHU083 reprograms immunosuppressive tumor-associated macrophages (TAMs) to drive tumor immunity in urologic cancers. [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 6432.

Abstract 691: Irradiated whole tumor cells pulsed with mannan-BAM, TLR ligands and anti-CD40 antibody serve as a potent tumor cell vaccine against glioblastoma

Abstract Despite numerous therapeutic advances in cancers, the treatment of glioblastoma multiforme (GBM) remains a challenge. Boosting T-lymphocyte mediated responses against GBM offers a promising approach towards solving this problem. Herein, we present a therapeutic vaccination strategy that promotes the phagocytosis of tumor cells, enhances tumor antigen presentation, and induces a tumor-specific adaptive immune response with subsequent tumor eradication. This strategy consists of subcutaneous injection of irradiated whole tumor cells (rWTC) pulsed with phagocytic agonists (Mannan-BAM), TLR ligands (LTA, Poly (I:C), and R-848), and anti-CD40 agonistic antibody (collectively abbreviated as rWTC-MBTA). We evaluated the therapeutic efficacy of rWTC-MBTA strategy in mouse syngeneic GBM tumor models with GL261 and SB28 cells. In GL261 GBM model, complete regression (CR) of intracranial tumors was achieved in 70% (7/10) of rWTC-MBTA treated animals while none survived in the control group. Of note, the therapeutic efficacy of rWTC-MBTA was abolished in CD4-T and/or CD8-T lymphocyte depleted mice. Immunophenotyping analyses of peripheral lymph nodes and brain tumors of rWTC-MBTA treated mice demonstrated increased antigen presenting cells (dendritic cells and MHC II+ monocytes) and increased cytotoxic IFNγ, TNFα, and granzyme B-secreting CD4-T and CD8-T cells. All three CR mice that were rechallenged with GL261 cells intracranially 14 months after their last rWTC-MBTA treatment resisted tumor development, confirming the establishment of long-term immunological memory. In SB28 GBM model, 80% (8/10) rWTC-MBTA treated mice survived past 95 days after tumor cell implantation without any GBM-related symptoms, with median survival being only 35 days in control groups. In summary, our study demonstrated that rWTC-MBTA strategy can induce potent adaptive immune response against GBM in pre-clinical models. Citation Format: Herui Wang, Rogelio Medina, Juan Ye, Samik Chakraborty, Ondrej Uher, Mitchell Sun, Jan Zenka, Mark R. Gilbert, Karel Pacak, Zhengping Zhuang. Irradiated whole tumor cells pulsed with mannan-BAM, TLR ligands and anti-CD40 antibody serve as a potent tumor cell vaccine against glioblastoma [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 691.

Abstract 6336: Heterodimeric Fc-fused IL12 shows potent antitumor activity and low toxicity

Abstract Background: Interleukin-12 (IL12) is a heterodimeric pro-inflammatory cytokine (70 kDa) composed of two different polypeptide chains: an α-chain (p35 subunit) and a β-chain (p40 subunit). IL12 could induce the proliferation and cytotoxicity of T and NK cell and production of interferon-γ (IFN-γ), promote the differentiation of T helper 1 (TH1) cells and regulate innate resistance and adaptive immunity. However, the clinical efficacy of IL12 has been hindered for its high dose-related toxicities and short serum half-life. Here we have developed a heterodimeric Fc-fused IL12, HK054-V, with reduced potency to improve tolerability and prolong half-life. Methods: We assessed the binding, bioactivity, anti-tumor efficacy and safety of HK054-V. The binding ability of IL12-Fc lead variants to activated PBMCs was analyzed by flow cytometry. The bioactivity of HK054-V was determined by assessing the activated T-cell proliferation and IFN-γ production in human PBMCs, and STAT-4 phosphorylation in a reporter gene assay. A murine surrogate, mHK054-V, was engineered to mimic HK054-V to evaluate the antitumor efficacy in MC38 and CT26 syngeneic tumor mouse models. Cytokine release assay was conducted by incubating HK054-V with peripheral blood mononuclear cells (PBMCs) from healthy donors. The safety profiles and pharmacokinetic (PK) behavior of HK054-V were characterized in non-tumor-bearing Balb/c mice. Results: HK054-V is a heterodimeric Fc-fused IL12, which was designed with reduced potency. HK054-V was observed to stimulate the proliferation of activated T cells and NK cells and induce production of IFN-γ in a dose-dependent manner. HK054-V could activate pSTAT4 signal in luciferase reporter gene assay and showed up to 100-fold reduction of potency compared to wild-type IL12-Fc. Human IL12 is inactive in mice, so in vivo anti-tumor activity was assessed by a murine surrogate, mHK054-V. mHK054-V demonstrated anti-tumor efficacy in syngeneic mouse models and was better tolerated than WT mIL12-Fc. Furthermore, mHK054-V has improved PK and therapeutic index in mice. Conclusion: These data demonstrate that HK054-V, a heterodimeric Fc-fused IL12 with reduced potency, retain strong anti-tumor efficacy with a favorable tolerability profile, which may provide a practical alternative to the systemic administration of IL12 for antitumor therapy. Citation Format: Yang Huang, Wenting Liu, Dayan Zhang, Pengfei Zhou, Weiming Zhou, Xiaoli Zeng, Qun Zhao, Liangwei Li, Guodong Shen, Liansheng Cheng. Heterodimeric Fc-fused IL12 shows potent antitumor activity and low toxicity. [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 6336.

Abstract 4630: Deep Immuno-profiling of syngeneic tumor mouse models for preclinical studies

Abstract Identification of major cellular players involved in progression of cancer is key to success of new immunotherapies. It is however a daunting challenge due to the complex interplay between tumor cells and the immune system. Component of innate and adaptive immunity may be directed to a pro- or anti-tumor function. In order to cope with the complexity of the tumor microenvironment (TME), it is necessary to use an experimental approach characterizing the heterogeneity of cell types, the evolution of their relative proportion and their fine-tuned functional specificity. This approach should lead to the identification of new cellular biomarkers characteristics of different stages of tumor cancer progression. To decipher the impact of immunotherapy treatments, cellular phenotyping of leukocytes infiltrating TME but also those present in peripheral organs is necessary. In order to increase our understanding in the precise mode of action of anti-PD1 treatment at the cellular level in sensitive and unsensitive syngeneic models, we investigated immunophenotypes and responses to immune checkpoint inhibitor (ICI) of several hallmark 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 arewere 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 well-characterized syngeneic tumor model and highlight the importance of tumor immune landscape changes across models that will drive selection of 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, Lillia Hadjem, Magali Grange, Olivier Deas, Ana Zarubica, Bernard Malissen, Hervé Luche. Deep Immuno-profiling of syngeneic tumor mouse models for preclinical studies. [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 4630.

Abstract 1866: D02, a novel, non-blocking antibody targeting IL2RA, exhibits significant anti-tumor activity in IL2RA-humanized mice

Abstract Regulatory T cells, or Tregs, are a subset of CD4+ T cells that negatively regulate immune responses to promote their resolution and protect against autoimmunity. Infiltration of Tregs in the tumor microenvironment (TME), and a low ratio of effector T cells to Tregs, is frequently associated with tumor progression and poor prognosis. The alpha subunit of the IL-2 cytokine receptor (IL2RA), a component of the high-affinity IL-2 receptor along with the β and γ subunits (IL2Rβγ), is constitutively expressed on Tregs and transiently expressed on effector T cells. IL-2 is a key cytokine for effector T cell survival and cytolytic responses. We aimed to discover an IL2RA-targeting antibody capable of depleting Tregs but permitting IL-2 stimulation of effector T cells in the TME. We developed a fully human IgG1 antibody specific to IL2RA, D02, from our proprietary RenMab™ mice, which contain the entire human immunoglobulin variable domain. D02 binds both human and cynomolgus monkey IL2RA with high affinity without hindering IL2RA-IL-2 binding or inhibiting IL-2 signaling. To evaluate the safety and efficacy of D02 in vivo, we established a syngeneic mouse tumor model in IL2RA-humanized mice. We observed that D02 potently inhibited MC38 (colon adenocarcinoma) tumor growth at 10 mg/kg. Further studies will aim to characterize the effect of D02 on the ratio of effector T cells to Tregs in the TME. These data indicate that D02 is a novel, IL2RA-specific monoclonal antibody that uniquely exhibits significant anti-tumor activity in vivo while preserving IL-2 signaling to effector T cells. Citation Format: Shuzhen Cao, Wenjiao Zhang, Qingcong Lin. D02, a novel, non-blocking antibody targeting IL2RA, exhibits significant anti-tumor activity in IL2RA-humanized mice [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 1866.

Abstract 1850: A first-in-class PD1-IL18 immunocytokine (BPT567) targets PD-1+ IL18R+ CD8+ T effector cells enriched in the tumor microenvironment and exhibits potent antitumor efficacy with excellent tolerability

Abstract Immunocytokines (IC) leverage orthogonal mechanisms of action in one molecule to induce potent antitumor immune responses. PD-1-targeting ICs are of particular interest since they harbor the multifunctional ability to selectively target antigen-experienced PD-1+ CD8+ T cells enriched in the tumor microenvironment (TME), release them from PD-(L)1 pathway inhibition, be retained within the TME, and simultaneously deliver potent cytokine receptor stimulation to the same T cell in cis (cis-signaling). Interleukin (IL-18) is a proinflammatory cytokine that stimulates both innate and adaptive immunity and generates potent antitumor activity mediated by both T effector and NK cells. Recent evidence indicates that a subset of tumor-infiltrated PD-1+ CD8+ T effector cells hallmarked by high expression of IL-18 receptor exhibits a superior cytotoxic and proliferative phenotype. Hence, we developed a PD1-IL18 IC to specifically target and activate intratumoral IL-18R-expressing PD-1+ CD8+ T cells. We engineered a conjugatable variant of human IL-18 with enhanced potency and significant resistance to IL-18 binding protein (IL-18BP) and utilized this enhanced IL-18 payload to create a PD1-IL18 IC (BPT567) via site-specific chemical conjugation to a defined lysine residue within the heavy chain of an anti-human PD-1 antibody (Ab). Conjugation did not affect the basic properties of the Ab as neither binding to PD-1 nor the interaction with the neonatal Fc receptor (FcRn) or Fcγ receptors were significantly impacted. Of note, the conjugation handle in our enhanced IL-18 variant was inserted at a site distinct from its N- or C-terminus to preserve the full potency and selectivity of the IL-18 payload. As a result, BPT567 exhibits increased potency and marked resistance to IL-18BP inhibition compared to wild-type IL-18. In vivo, BPT567 shows striking antitumor efficacy in multiple syngeneic mouse tumor models that is superior to responses induced by an anti-PD-1 Ab alone, a non-targeted IL-18 IC or the combination of both agents. Our evidence suggests that the strong in vivo efficacy observed is attributed to a preferential activation of PD-1+ tumor-infiltrated immune cells. The excellent tolerability and large therapeutic window observed in mice may be driven by less potent immune cell activation in the periphery. Citation Format: Jean-Philippe Carralot, Kea Martin, Rubén Alvarez Sanchez, Roy Meoded, Caoimhe Herr, Philipp Moosman, Arnaud Goepfert, Andrew Chi, Vijaya J. Pattabiraman, Bertolt Kreft. A first-in-class PD1-IL18 immunocytokine (BPT567) targets PD-1+ IL18R+ CD8+ T effector cells enriched in the tumor microenvironment and exhibits potent antitumor efficacy with excellent tolerability [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 1850.

Abstract 4436: A novel antibody-enabled dual precision targeted protein stabilization (TPS2) that augments anti-tumor immune response by targeting CBL-B inhibitor to exhausted T cells while blocking checkpoint molecule, PD-1

Abstract It is well-established that T cells play a key role in the success of cancer immunotherapies. The goals of T cell-targeting therapies have been to restore the effector function of tumor-specific T cells that are either dysfunctional due to the immunosuppressive mechanisms in the tumor microenvironment, the lack of co-stimulatory signals, or negative regulation by expression of checkpoint molecules such as PD-1. E3 ligase Casitas B-Lineage Lymphoma Proto-Oncogene B (CBL-B) has recently gained attention as a master regulator of multiple immune-activation mechanisms. Inhibition of CBL-B enables the activation of antigen-specific T cells, and has been shown in syngeneic mouse tumor models to be efficacious as a cancer immunotherapy agent. CBL-B inhibition has also been shown to enhance and prolong the effects of anti-PD-1 antibody (Ab) therapy. However, genetic knockout of CBL-B in mice led to spontaneous autoimmunity characterized by auto-antibody production and infiltration of activated T cells and B cells into multiple organs that could result in tissue damage. In order to mitigate safety issues that could result from systemic CBL-B inhibition, we developed novel antibody-drug conjugates (ADCs) that target CBL-B inhibitors (CBL-Bi) to T cells via binding to PD-1. Using PD-1-expressing Jurkat NFAT reporter cells and mixed lymphocyte reaction assay, we demonstrated that our TPS2 approach enhanced the activation of T cells in vitro compared to treatment with anti-PD-1 Ab alone. Evaluation of pathways downstream of TCR activation in primary exhausted T cells showed increased Notch1, phospho-PLCγ2, and ZAP-70 accumulation following anti-PD-1/CBL-Bi treatment and is correlated with IFNγ induction. When autologous mature dendritic cells were co-cultured with dissociated tumor cells from melanoma patients, the highest magnitude of tumor-infiltrating lymphocyte activation was observed using delivery of CBL-Bi by anti-PD-1 ADC, compared to CBL-Bi alone, anti-PD-1 Ab alone, or combination treatment. The potential of anti-PD-1/CBL-Bi ADC for activation effector T cell function is also evident by increased intratumoral transcript levels of Granzyme B and Perforin following treatment in a humanized mouse model. In conclusion, we validated that the biological activity of CBL-Bi is retained after conjugation with an anti-PD-1 antibody. The use of anti-PD-1 Ab for developing the ADC not only targets the CBL-Bi payload to exhausted T cells but also blocks negative regulation of T cells via PD-1. Together, anti-PD-1/CBL-Bi ADC enhanced T cell activation in vitro as well as in vivo. Evaluation of safety and efficacy in animal tumor models is ongoing, to support further development of the TPS2 approach. Citation Format: Joanne Lim, Anna Skaletskaya, Uttapol Permpoon, Yeonjoon Kim, Shikha Saini, Khuloud Takrouri, Zinaida Ribkovskaia, Palash Bhar, Nathan Fishkin, Dong-Ki Choi, Ji Hyun Park, James Palacino, Peter U. Park. A novel antibody-enabled dual precision targeted protein stabilization (TPS2) that augments anti-tumor immune response by targeting CBL-B inhibitor to exhausted T cells while blocking checkpoint molecule, PD-1. [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 4436.

Abstract 1888: IAE0972, A novel EGFR/IL10 immunocytokine of monovalent anti-EGFR antibody fused with IL10 homodimer

Abstract Exhaustion of immune cells in the tumor microenvironment has been found and confirmed in many tumor cases, accounting for the major problems of current PD1/L1-oriented immunotherapy. There are two types of exhausted T cells present in the tumor microenvironment: progenitor exhausted T cells and terminally exhausted T cells. Data from various mouse syngeneic tumor models suggest that the dominance of progenitor exhausted T cells will be replaced by terminally exhausted T cells as the tumor progresses. The PD1/PDL1 antibodies mainly act on progenitor exhausted T cells and can develop acquired resistance as terminally exhausted T cells become the dominant cells in the tumor microenvironment. Recently, there are studies demonstrating that IL10 can restore the oxidative phosphorylation metabolism of terminally exhausted T cells and restore their potent killing activities of tumor cells. We therefore designed IAE0972, a novel EGFR/IL10 immunocytokine to solve the problems of immune cell exhaustion in current immunotherapy. It is a monovalent anti-EGFR antibody fused with IL10 homodimer. The anti-EGFR antibody can specifically enrich IAE0972 in tumor microenvironment, so that IL10 can reinvigorate antigen specific CD8+ T cells and facilitate its proliferation, while preserving the tumor cell proliferation inhibition activities of EGFR antibody. The design of monovalent form anti-EGFR antibody can also avoid the skin toxicity problem. In this study, we studied the target binding activity, CD8+ T cell activation activity and cancer cell proliferation inhibition activity of IAE0972. We also observed a much higher anti-tumor efficacy of IAE0972 in a MC38 syngeneic tumor model compared with that of Cetuximab. In addition, the molecule has a very good safety profile with MTD over 6 mg/kg and no skin toxicities, which is about 300-fold higher than that of cytokine drugs. In summary, IAE0972 is an innovative EGFR/IL10 immunocytokine with robust preclinical anti-tumor efficacy and favorable safety profile, which has the potential to address acquired resistance of current immunotherapy. IAE0972 is the first EGFR/IL10 immunocytokine with IND approval from both FDA and NMPA. The indications of IAE0972 include colorectal cancer, head and neck squamous cell carcinoma, squamous non-small cell lung cancer, and other EGFR positive tumors. A Phase I/IIa clinical trial to evaluate the safety, tolerability and preliminary efficacy of IAE0972 in patients with locally advanced or metastatic malignant tumors is on-going (NCT05396339). Citation Format: Liusong Yin, Xiaoling Jiang, Chongbing Wu, Zi Chen. IAE0972, A novel EGFR/IL10 immunocytokine of monovalent anti-EGFR antibody fused with IL10 homodimer [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 1888.

Abstract 1807: CB307: a novel targeted CD137 agonist for enhancement of immune cell responses to PSMA+ tumors

Abstract Introduction: CD137 (HGNC:TNFRSF9, 4-1BB) is a TNF receptor superfamily costimulatory receptor expressed by T cells and NK cells. Upon engagement, CD137 enhances immune cell survival, proliferation, cytokine production and memory formation. CD137 agonists in the clinic have shown tantalizing anti-tumor effects, however, hepatic toxicity has hampered clinical utility. To circumvent this limitation, we developed CB307, a half-life extended heavy-chain only Humabody® that selectively agonizes CD137 only in the presence of prostate specific membrane antigen (PSMA, HGNC:FOLH1). PSMA is highly upregulated on tumor cells in prostate cancer and tumor neovasculature in other solid tumors including lung (NSCLC), kidney (clear cell RCC), bladder and colorectal cancers. CB307 enhances immune cell activation in a PSMA-dependent manner. We present here preclinical mechanistic differentiators of CB307 with respect to non-targeted CD137 agonists. Methods: CD137 agonism, immune cell activation and cytokine secretion were evaluated in vitro using coculture assays in the presence/absence of PSMA-expressing tumor cells and in vivo with a transgenic mouse syngeneic tumor model. The ability of CB307 or non-targeted CD137 agonists to enhance tumor cell killing was additionally characterized in 3D spheroid cocultures. In vitro cocultures were used to determine the impact of PSMA expression level on CB307 activity. CB307 was evaluated as a single agent and in combination with PD-1 or PD-L1 inhibitors; tumoral checkpoint contribution was investigated using isogenic PD-L1 positive vs. PD-L1 CRISPR-deleted tumor cells. Results: CB307 or PD-1/PD-L1 inhibition alone induced IL-2 secretion in PBMC/PSMA-expressing tumor cell cocultures; this effect was synergistically enhanced when CB307 was combined with either PD-1 or PD-L1 inhibition. CB307 augmented tumor cell killing in 3D spheroids containing PSMA-expressing cells. Enhanced tumor cell killing was observed in spheroid cultures containing CB307 in combination with PD-1/PD-L1 inhibition. In in vivo pharmacology studies in a syngeneic mouse model, CB307 demonstrated statistically significant inhibition of tumor growth versus control without peripheral T cell activation - consistent with pharmacology restricted to the PSMA+ tumor microenvironment, whereas an anti-CD137 monoclonal antibody induced tumor growth inhibition accompanied by peripheral immune cell proliferation and cytokine production. Summary: CB307 is a novel CD137 agonist that selectively enhances immune cell activity only in the presence of PSMA-positive cells. Our data demonstrate PSMA-targeted CB307 immunological pharmacology both in vitro and in vivo which has supported the ongoing Phase 1 ‘POTENTIA’ clinical trial (NCT04839991). Citation Format: Andrew J. Pierce, Phillip M. Brailey, Sophie Archer, Clare Song, Anca-Giuilia Cionca, Joanne Fernando, Phillip D. Bartlett, Philip Bland-Ward. CB307: a novel targeted CD137 agonist for enhancement of immune cell responses to PSMA+ tumors [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 1807.

Abstract 1821: Discovery of XNW21015, a novel, potent HPK1 inhibitor with excellent immune modulatory activity

Abstract Hematopoietic progenitor kinase 1 (HPK1), also known as MAP4K1, is a member of the STE20 family of serine/threonine kinases, and restrictedly expressed in hematopoietic cells. In T cells, HPK1 negatively regulated TCR signaling by phosphorylating the adaptor protein SLP76 on Ser376, which leads to the dissociation of the SLP-76/LAT signalosomes and downregulation of T cell priming and activation. HPK1 knockout (KO) or kinase-inactive (KI) mice spontaneously reject tumors with an enhanced immune response compared to wild-type ones. Thus, HPK1 is a novel immuno-oncology (I/O) target. In this work, we described the discovery of a potent HPK1 inhibitor starting from comprehensive SAR-based design with guidance from X-ray crystallography and computational modeling. These efforts led to identification of the potent azaindole inhibitor XNW21015, with excellent kinase selectivity and promising PK profile of low clearance and sufficient exposure for in vivo animal studies. XNW21015 dose-dependently inhibited phosphorylation of SLP76(Ser376) in vitro and in vivo in response to anti-CD3 agonist antibody, with concomitant enhancement in T cell activation. Oral administration of XNW21015 in mouse syngeneic tumor models showed favorable PK profile and robust anti-tumor activity as both single agent and in combination with immune checkpoint inhibitors. In a humanized PDX (patient-derived xenograft) model, XNW21015 enhanced tumor-infiltrated lymphocytes (TIL). In summary, we have discovered a novel, highly potent and selective HPK1 inhibitor XNW21015 as promising small molecule I/O agent, as monotherapy or in combination with immune checkpoint inhibitors. Citation Format: Yuchuan Wu, Rui Hao, Xi Chen, Xiao Liu, Yonghua Xie, Shihua Wang, Bingquan Gao, Jichun Wen, Zhenwei Wu, Wengui Wang, Haiyang Wei, Yonghan Hu, Xiaojun Liu, Meijie Le, Jing Qiang. Discovery of XNW21015, a novel, potent HPK1 inhibitor with excellent immune modulatory activity [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 1821.

Сравнение онколитической активности рекомбинантных штаммов вируса осповакцины LIVP-RFP и MVA-RFP в отношении солидных опухолей

Among oncolytic viruses, modified vaccinia virus Ankara (MVA), a highly attenuated vaccinia virus (VV) is a well-studied variant with promising results in preclinical and clinical trials. The Lister VV strain from the Moscow Institute of Viral Preparations (LIVP) has been studied to a lesser extent than MVA and has a different oncolytic property from MVA. The aim of this work was to compare the oncolytic efficacy of LIVP and MVA strains against solid tumors. We developed recombinant variants LIVP-RFP and MVA-RFP; to enhance onco-selectivity thymidine kinase (TK) gene was inactivated by insertion of red fluorescent protein (RFP) gene to the TK locus. The replication kinetics and oncolytic activity of the obtained recombinant strains were evaluated in vitro and in vivo on tumor cell lines and mouse syngeneic tumor models of metastatic mouse 4T1 mammary adenocarcinoma, CT26 colon adenocarcinoma, and B16 melanoma. Both MVA-RFP and LIVP-RFP showed high replication efficiency in tumor cells and pronounced oncolytic activity against B16 melanoma and 4T1 breast adenocarcinoma allografts. In relation to 4T1, which is a model of triple negative human breast cancer, LIVP-RFP showed more than 50% increased cytotoxicity in in vitro tests compared to MVA-RFP, as well as a significant slowdown in the progression of 4T1 allografts and an increase in animal survival in experiments in vivo. Thus, the LIVP strain may be more promising than MVA as a platform for the development of recombinant oncolytic viruses for the breast cancer treatment.

Antimicrobial peptides CS-piscidin-induced cell death involves activation of RIPK1/PARP, and modification with myristic acid enhances its stability and tumor-targeting capability

Ovarian cancer (OC) is a highly lethal gynecological malignancy, often diagnosed at advanced stages with limited treatment options. Here, we demonstrate that the antimicrobial peptide CS-piscidin significantly inhibits OC cell proliferation, colony formation, and induces cell death. Mechanistically, CS-piscidin causes cell necrosis by compromising the cell membrane. Furthermore, CS-piscidin can activate Receptor-interacting protein kinase 1 (RIPK1) and induce cell apoptosis by cleavage of PARP. To improve tumor targeting ability, we modified CS-piscidin by adding a short cyclic peptide, cyclo-RGDfk, to the C-terminus (CS-RGD) and a myristate to the N-terminus (Myr-CS-RGD). Our results show that while CS-RGD exhibits stronger anti-cancer activity than CS-piscidin, it also causes increased cytotoxicity. In contrast, Myr-CS-RGD significantly improves drug specificity by reducing CS-RGD toxicity in normal cells while retaining comparable antitumor activity by increasing peptide stability. In a syngeneic mouse tumor model, Myr-CS-RGD demonstrated superior anti-tumor activity compared to CS-piscidin and CS-RGD. Our findings suggest that CS-piscidin can suppress ovarian cancer via multiple cell death forms and that myristoylation modification is a promising strategy to enhance anti-cancer peptide performance.Graphical