Efficacy In Preclinical Models Research Articles

Local radiotherapy and E7 RNA-LPX vaccination show enhanced therapeutic efficacy in preclinical models of HPV16+ cancer

Human papilloma virus (HPV) infection is a causative agent for several cancers types (genital, anal and head and neck region). The HPV E6 and E7 proteins are oncogenic drivers and thus are ideal candidates for therapeutic vaccination. We recently reported that a novel ribonucleic acid lipoplex (RNA-LPX)-based HPV16 vaccine, E7 RNA-LPX, mediates regression of mouse HPV16+ tumors and establishes protective T cell memory. An HPV16 E6/E7 RNA-LPX vaccine is currently being investigated in two phase I and II clinical trials in various HPV-driven cancer types; however, it remains a high unmet medical need for treatments for patients with radiosensitive HPV16+ tumors. Therefore, we set out to investigate the therapeutic efficacy of E7 RNA-LPX vaccine combined with standard-of-care local radiotherapy (LRT). We demonstrate that E7 RNA-LPX synergizes with LRT in HPV16+ mouse tumors, with potent therapeutic effects exceeding those of either monotherapy. Mode of action studies revealed that the E7 RNA-LPX vaccine induced high numbers of intratumoral-E7-specific CD8+T cells, rendering cold tumors immunologically hot, whereas LRT primarily acted as a cytotoxic therapy, reducing tumor mass and intratumor hypoxia by predisposing tumor cells to antigen-specific T cell-mediated killing. Overall, LRT enhanced the effector function of E7 RNA-LPX-primed T cell responses. The therapeutic synergy was dependent on total radiation dose, rather than radiation dose-fractionation. Together, these results show that LRT synergizes with E7 RNA-LPX and enhances its anti-tumor activity against HPV16+ cancer models. This work paves into a new translational therapy for HPV16+ cancer patients.

Infrared Spectroscopy-Determined Bone Compositional Changes Associated with Anti-Resorptive Treatment of the oim/oim Mouse Model of Osteogenesis Imperfecta.

Applications of vibrational spectroscopy to assess bone disease and therapeutic interventions are continually advancing, with tissue mineral and protein composition frequently investigated. Here, we used two spectroscopic approaches for determining bone composition in a mouse model (oim) of the brittle bone disease osteogenesis imperfecta (OI) with and without antiresorptive agent treatment (alendronate, or ALN, and RANK-Fc). Near-infrared (NIR) spectral analysis using a fiber optic probe and attenuated total reflection Fourier transform infrared spectroscopy (ATR FTIR) mode were applied to investigate bone composition, including water, mineral, and protein content. Spectral parameters revealed differences among the control wildtype (WT) and OIM groups. NIR spectral analysis of protein and water showed that OIM mouse humerii had ∼50% lower protein and ∼50% higher overall water content compared to WT bone. Moreover, some OIM-treated groups showed a reduction in bone water compared to OIM controls, approximating values observed in WT bone. Differences in bone quality based on increased mineral content and reduced carbonate content were also found between some groups of treated OIM and WT bone, but crystallinity did not differ among all groups. The spectroscopically determined parameters were evaluated for correlations with gold-standard mechanical testing values to gain insight into how composition influenced bone strength. As expected, bone mechanical strength parameters were consistently up to threefold greater in WT mice compared to OIM groups, except for stiffness in the ALN-treated OIM groups. Furthermore, bone stiffness, maximum load, and post-yield displacement showed the strongest correlations with NIR-determined protein content (positive correlations) and bound-water content (negative correlations). These results demonstrate that in this study, NIR spectral parameters were more sensitive to bone composition differences than ATR parameters, highlighting the potential of this nondestructive approach for screening of bone diseases and therapeutic efficacy in pre-clinical models.

Pharmacodynamic effects of direct AMP kinase activation in humans with insulin resistance and non-alcoholic fatty liver disease: A phase 1b study

SummaryAMPK is an energy sensor modulating metabolism, inflammation, and a target for metabolic disorders. Metabolic dysfunction results in lower AMPK activity. PXL770 is a direct AMPK activator, inhibiting de novo lipogenesis (DNL) and producing efficacy in preclinical models. We aimed to assess pharmacokinetics, safety, and pharmacodynamics of PXL770 in humans with metabolic syndrome-associated fatty liver disease. In a randomized, double-blind four-week trial, 12 overweight/obese patients with non-alcoholic fatty liver disease (NAFLD) and insulin resistance received PXL770 500 mg QD; 4 subjects received matching placebo. Endpoints included pharmacokinetics, hepatic fractional DNL, oral glucose tolerance testing, additional pharmacodynamic parameters, and safety. PK parameters show adequate plasma exposure in NAFLD patients for daily oral dosing. PXL770 decreases DNL—both peak and AUC are reduced versus baseline—and improves glycemic parameters and indices of insulin sensitivity versus baseline. Assessment of specific lipids reveals decrease in diacyglycerols/triacylglycerols. Safety/tolerability are similar to placebo. These results unveil initial human translation of AMPK activation and support this therapeutic strategy for metabolic disorders.

Abstract P157: Targeting Trop2 for treatment of prostate and breast cancer

Abstract Background: Prostate cancer and breast cancer are the most commonly diagnosed non-skin cancers and the second leading cause of cancer related mortalities in men and women respectively in the United States. Fifteen percent of breast cancer is TNBC characterized with lack of receptors for estrogen, progesterone, and HER-2/neu making it resistant to hormone therapies and therapies targeting estrogen, progesterone, and HER-2/neu signaling. The first line of treatment for men with advanced prostate cancer is androgen depravation therapy. While initial responses are observed, unfortunately, the disease frequently recurs as a lethal metastatic form referred to as castration-resistant prostate cancer (CRPC). We recently identified that the cell surface receptor, Trop2, is a new regulator of TNBC and CRPC. We demonstrated that Trop2 is highly expressed in CRPC and TNBC. In this study, we set out to test the therapeutic potential of new fully humanized human anti-Trop2 antibody fragments in CRPC and NEPC. Methods: We have generated new human anti-Trop2 antibody fragments. Lentiviral infection was used to generate prostate and breast cancer cell lines with over-expression of Trop2 and knock-down of Trop2. In vivo functional assays were performed including subcutaneous xenograft growth and metastasis. Results: High protein levels of Trop2 are observed in metastatic CRPC and TNBC. Furthermore, loss of Trop2 significantly delays growth, migration and invasion of prostate and breast cancer cells in vitro and delays tumor growth and metastasis of prostate and breast cancer in vivo. Overexpression of Trop2 enhances tumor growth and metastasis of prostate cancer cells and drives CRPC. We generated new fully humanized human anti-Trop2 antibody fragments that have therapeutic efficacy in preclinical models of prostate and breast cancers. Conclusions: In a previous study, we identified cell surface receptor, Trop2, as a novel driver of metastatic CRPC. Herein, our new findings reveal that Trop2 represents a promising therapeutic target for prostate, breast, and potentially other epithelial cancers. We identified lead antibody candidates that bind to human Trop2 protein with high affinity and stability. Furthermore, our lead anti-Trop2 Abs significantly delay the tumors growth in CRPC and TNBC without measurable toxicity and provide new therapeutic strategy for metastatic CRPC and TNBC. Citation Format: Tanya Stoyanova. Targeting Trop2 for treatment of prostate and breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P157.

Abstract P039: A phase 1 dose escalation study of protein arginine methyltransferase 5 (PRMT5) inhibitor PRT543 in patients with advanced solid tumors and lymphoma

Abstract Background: PRMT5 catalyzes symmetric arginine dimethylation of protein substrates with important roles in cancer cell growth/survival. PRT543 is a potent, selective, oral PRMT5 inhibitor with robust preclinical efficacy (Bhagwat AACR 2020). An open-label Phase I study of PRT543 in unselected patients (pts) with advanced solid tumors and hematologic malignancies (NCT03886831) is ongoing. Dose escalation results from pts with solid tumors and lymphoma are presented herein. Materials and Methods: This study assesses safety, pharmacokinetics, pharmacodynamics, and preliminary tumor response (REClST v1.1) of PRT543 administered at varying schedules beginning with twice weekly (BIW) and increasing to once daily (QD) with doses ranging from 5-50 mg in 28-day cycles. Dose escalation followed a 3+3 design. Serum symmetric dimethylarginine (sDMA) and intron retention, a marker of PRMT5-mediated mRNA splicing fidelity, were assessed as measures of PRMT5 target engagement and function, respectively. Results: As of 18 June 2021, 49 unselected pts with measurable disease refractory to established therapies had enrolled (17 pts BIW, 11 pts 5x/week [wk], 21 pts QD). Median number of prior lines of systemic therapies was 3. Six of 21 pts dosed at 25-50 mg QD experienced dose limiting toxicity (DLT) of thrombocytopenia, and 1 pt of 6 dosed at 45 mg 5x/wk had DLT of fatigue. Most frequent treatment-related adverse events (TRAEs), any grade, in all regimens were fatigue (n=20, 41%), nausea (n=14, 29%), thrombocytopenia (n=13, 27%), and anemia (n=12, 24%). Grade ≥3 TRAEs in all regimens occurring in ≥5 pts included thrombocytopenia (n=10, 20%) and anemia (n=6, 12%). Cytopenias were reversible and managed with dose modifications. 44 pts discontinued treatment, mainly due to disease progression, with 2 due to AEs (Grade 3 thrombocytopenia and Grade 4 cholangitis). The 45 mg 5x/wk regimen was selected as the expansion dose. PRT543 demonstrated dose-dependent increases of Cmax (nM) and AUC (nM.hr). At the expansion dose, T½ was 15 hrs, and plasma drug exposures (Cmax, 2142 nM; AUC, 26,293 nM.hr) exceeded those for preclinical efficacy models. Serum sDMA decreased in a dose-dependent manner reaching 77% reduction with 50 mg QD and 69% reduction with the expansion dose. Increased intron retention was seen in peripheral blood mononuclear cells. A complete response (CR) has been maintained for >1 yr in a pt with homologous recombination deficiency-positive (HRD+) ovarian cancer who remains on study therapy. An additional 5 pts exhibited stable disease ≥6 mo. Conclusions: PRT543 was well tolerated with a favorable safety profile. Dose-dependent inhibition of target engagement and functional activity of PRMT5 were observed. PRT543 demonstrated encouraging clinical activity with a CR in HRD+ ovarian cancer and prolonged stable disease in multiple pts. An expansion phase in biomarker-selected solid tumor cohorts is ongoing. Citation Format: Meredith McKean, Manish R. Patel, Robert Wesolowski, Renata Ferrarotto, Eytan M. Stein, Alexander N. Shoushtari, David Mauro, John Viscusi, Peggy Scherle, Neha Bhagwat, William Sun, Rachel Chiaverelli, Eric Mintah, Shekeab Jauhari, Laura Finn, Neil D. Palmisiano, Robert A. Baiocchi. A phase 1 dose escalation study of protein arginine methyltransferase 5 (PRMT5) inhibitor PRT543 in patients with advanced solid tumors and lymphoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P039.

TAS‐116 (pimitespib), a heat shock protein 90 inhibitor, shows efficacy in preclinical models of adult T‐cell leukemia

Adult T‐cell leukemia/lymphoma (ATL) is a highly chemoresistant malignancy of peripheral T lymphocytes caused by human T‐cell leukemia virus type 1 infection, for which there is an urgent need for more effective therapeutic options. The molecular chaperone heat shock protein 90 (HSP90) plays a crucial role in nuclear factor‐κB (NF‐κB)‐mediated antiapoptosis in ATL cells, and HSP90 inhibitors are new candidate therapeutics for ATL. Accordingly, we investigated the anti‐ATL effects of a novel oral HSP90 inhibitor, TAS‐116 (pimitespib), and the mechanisms involved in ex vivo and in vivo preclinical models. TAS‐116 achieved IC50 values of less than 0.5 μmol/L in 10 ATL‐related cell lines and less than 1 μmol/L in primary peripheral blood cells of nine ATL patients; no toxicity was observed toward CD4+ lymphocytes from healthy donors, indicating the safety of this agent. Given orally, TAS‐116 also showed significant inhibitory effects against tumor cell growth in ATL cell‐xenografted mice. Furthermore, gene expression profiling of TAS‐116‐treated Tax‐positive or ‐negative cell lines and primary ATL cells using DNA microarray and multiple pathway analysis revealed the significant downregulation of the NF‐κB pathway in Tax‐positive cells and cell‐cycle arrest in Tax‐negative cells and primary ATL cells. TAS‐116 suppressed the activator protein‐1 and tumor necrosis factor pathways in all examined cells. These findings strongly indicate the efficacy of TAS‐116, regardless of the stage of ATL progression, and its potential application as a novel clinical anti‐ATL therapeutic agent.

Safety and efficacy of multipotent adult progenitor cells in acute respiratory distress syndrome (MUST-ARDS): a multicentre, randomised, double-blind, placebo-controlled phase 1/2 trial

PurposeBone marrow-derived, allogeneic, multipotent adult progenitor cells demonstrated safety and efficacy in preclinical models of acute respiratory distress syndrome (ARDS).MethodsThis phase 1/2 trial evaluated the safety and tolerability of intravenous multipotent adult progenitor cells in patients with moderate-to-severe ARDS in 12 UK and USA centres. Cohorts 1 and 2 were open-label, evaluating acute safety in three subjects receiving 300 or 900 million cells, respectively. Cohort 3 was a randomised, double-blind, placebo-controlled parallel trial infusing 900 million cells (n = 20) or placebo (n = 10) within 96 h of ARDS diagnosis. Primary outcomes were safety and tolerability. Secondary endpoints included clinical outcomes, quality of life (QoL) and plasma biomarkers.ResultsNo allergic or serious adverse reactions were associated with cell therapy in any cohort. At baseline, the cohort 3 cell group had less severe hypoxia. For cohort 3, 28-day mortality was 25% for cell vs. 45% for placebo recipients. Median 28-day free from intensive care unit (ICU) and ventilator-free days in the cell vs. placebo group were 12.5 (IQR 0,18.5) vs. 4.5 (IQR 0,16.8) and 18.5 (IQR 0,22) vs. 6.5 (IQR 0,18.3), respectively. A prospectively defined severe ARDS subpopulation (PaO2/FiO2 < 150 mmHg (20 kPa); n = 16) showed similar trends in mortality, ICU-free days and ventilator-free days favouring cell therapy. Cell recipients showed greater recovery of QoL through Day 365.ConclusionsMultipotent adult progenitor cells were safe and well tolerated in ARDS. The clinical outcomes warrant larger trials to evaluate the therapeutic efficacy and optimal patient population.

Favorable Modulation of Chimeric Antigen Receptor T Cells Safety and Efficacy By the Non-Covalent BTK Inhibitor Vecabrutinib

Favorable Modulation of Chimeric Antigen Receptor T Cells Safety and Efficacy By the Non-Covalent BTK Inhibitor Vecabrutinib

Optimized Inhibition of GM-CSF in Preclinical Models of Anti-CD19 Chimeric Antigen Receptor T Cell Therapy

Anti-CD19 chimeric antigen receptor T (CART19) cell therapy has resulted in unprecedented outcomes in patients with relapsed/refractory B-cell malignancies, which led to the FDA approval for several indications. However, CART19 cell therapy is limited by the development of severe life-threatening toxicities, as well as by the limited rates of durable response. It has become apparent that myeloid cells contribute to the development of both CART cell toxicity and also to the inhibitory tumor microenvironment. We and others have identified that granulocyte-monocyte-colony-stimulating factor (GM-CSF) depletion results in decreased myeloid activation, reduced toxicities, and enhancement of CART19 cell therapy efficacy in pre-clinical models. Furthermore, we observed that GM-CSF knockout (GM-CSF k/o) in CART19 cells resulted in the improvement of their functions (in vitro and in vivo). These findings suggest that there is also a direct effect of GM-CSF on CART19 cells, which is independent of the GM-CSF impact on myeloid cell activation.To further evaluate this, we first examined GM-CSF receptor alpha (GM-CSFRα) expression by flow cytometry on resting and activated CART19 cells (using FMC63-41BBζ). When CART19 cells were stimulated with either anti-CD3/CD28 beads or lethally irradiated (120 Gy) CD19 + Nalm6 cells (B cell acute lymphoblastic leukemia cancer cell line), GM-CSFRα expression was upregulated upon both T cell receptor (TCR) (data not shown) and CAR stimulation (Figure 1A).Having demonstrated that GM-CSFRα is significantly upregulated on stimulated CART19 cells, we aimed to determine the impact of GM-CSF neutralization (clinical-grade anti-GM-CSF antibody, lenzilumab, 10 µg/mL) versus GM-CSFRα blockade (research-grade antibody, 10 µg/mL) on CART19 cell function and CART cell-monocyte interactions. An IgG isotype antibody was used as a control antibody. Neither the GM-CSF neutralizing antibody, nor GM-CSFRα blocking antibody, had any impact on CART19 cell antigen-specific killing against the CD19 + JeKo-1 cells (mantle cell lymphoma cancer cell line), in the presence or absence of CD14 + monocytes (ratio 1:1:1) isolated by magnetic beads from healthy donors (Figures 1B-C).Next, we compared the effects of GM-CSF neutralization versus GM-CSFRα blockade on CART19 cell antigen-specific proliferation. Here, CART19 cells were co-cultured with lethally irradiated CD19 + cell line JeKo-1 at 1:1 ratio in the presence of 10 µg/mL of the GM-CSF neutralizing antibody, increasing doses of the GM-CSFRα blocking antibody (10-100 µg/mL), or an IgG control. The absolute number of CART cells was measured by flow cytometry on day 5. GM-CSF neutralization did not affect CART19 cell proliferation, but GM-CSFRα blocking antibody significantly inhibited CART19 cell proliferation in a dose-dependent manner. Then, we assessed the effects of GM-CSF neutralizing antibody (20 µg/mL) versus GM-CSFRα blocking antibody (20 µg/mL) on CART19 cell antigen-specific proliferation in the presence of healthy donor monocytes (ratio 1:1:0.5) on day 3. Flow cytometric analysis revealed that GM-CSF neutralization, but not GM-CSFRα blockade, mitigated monocyte-suppression of CART19 antigen-specific proliferation (Figure 1E).In summary, our findings indicate significant differences on CART cell functions and CART cell-monocyte interactions when a specific cytokine, GM-CSF, is neutralized compared to blocking its receptor. Further mechanistic studies are ongoing to assess the functions of GM-CSFRα k/o and GM-CSF k/o CART cells. [Display omitted] DisclosuresSakemura: Humanigen: Patents & Royalties. Parikh: Pharmacyclics, MorphoSys, Janssen, AstraZeneca, TG Therapeutics, Bristol Myers Squibb, Merck, AbbVie, and Ascentage Pharma: Research Funding; Pharmacyclics, AstraZeneca, Genentech, Gilead, GlaxoSmithKline, Verastem Oncology, and AbbVie: Membership on an entity's Board of Directors or advisory committees. Kay: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Research Funding; CytomX Therapeutics: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Research Funding; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Research Funding; Acerta Pharma: Research Funding; Genentech: Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees; Behring: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sunesis: Research Funding; Targeted Oncology: Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees. Durrant: Humanigen Inc.: Current Employment. Ahmed: Humanigen Inc.: Current Employment, Current equity holder in publicly-traded company. Chappell: Humanigen Inc.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Cox: Humanigen: Patents & Royalties. Kenderian: Humanigen, Inc.: Consultancy, Honoraria, Research Funding.

Pirtobrutinib Overcomes Ibrutinib and Venetoclax Resistance in Mantle Cell Lymphoma

BackgroundMantle cell lymphoma (MCL) is a rare and aggressive B-cell lymphoma characterized by poor prognosis. Although remarkable therapeutic advances have been made by covalent Bruton's tyrosine kinase (BTK) inhibition and CAR T cell therapy, therapeutic resistance inevitably occurs and leads to dismal clinical outcome. Pirtobrutinib (LOXO-305) is a next-generation, highly selective and non-covalent BTK inhibitor. A phase 1/2 BRUIN study showed that pirtobrutinib demonstrated promising efficacy in heavily pretreated MCL patients with or without prior covalent BTK inhibition. Here, we investigated the mechanism of action of pirtobrutinib in MCL cells in vitro and proposed the potential combination therapy in a venetoclax-resistant xenograft model.MethodsMCL cell proliferation was monitored by trypan blue exclusion assay after 24-, 48- and 72-hour treatment with pirtobrutinib and ibrutinib. We performed Annexin V/PI staining to measure the apoptosis inductive effects. Cell cycle analysis using propidium iodide (PI) DNA staining was conducted to compare cell cycle progression kinetics between pirtobrutinib and ibrutinib. We performed RNAseq analysis in Z138 cells to compare differentially expressed genes (DEGs) between pirtobrutinib and ibrutinib treatment. Western blotting was utilized to detect specific signaling proteins. Mino-venetoclax-R cells were inoculated subcutaneously into NSG mice and used for in vivo drug efficacy determination.ResultsCompared to covalent BTK inhibitor ibrutinib, the novel non-covalent BTK inhibitor pirtobrutinib was more potent in inhibiting MCL cell proliferation in a panel of MCL cell lines, especially in ibrutinib/venetoclax resistant cell lines (pirtobrutinib vs. ibrutinib, p<0.01). Treatment with pirtobrutinib (10μM) for 24 hours induced higher levels of apoptosis than that by ibrutinib in all the MCL cell lines tested (p<0.05), which was also confirmed at the molecular level by stronger caspase-3 activation and PARP cleavage. To understand the mechanism of action, we performed whole transcriptomic profiling by RNAseq analysis using Z138 cells treated with/without pirtobrutinib or ibrutinib. Pirtobrutinib treatment resulted in upregulation of 137 genes and downregulation of 97 genes compared to the ibrutinib treatment (adjusted p<0.05). In addition to the downregulated MYC targets and PI3K/Akt pathway, gene set enrichment analysis (GSEA) revealed a significant enrichment for G2/M checkpoints and E2F targets signatures (key genes: PLK1, CDKN1A and CCNB1) in pirtobrutinib treated cells. Consistently, follow-up studies showed that γH2AX level was highly increased upon pirtobrutinib treatment. Pirtobrutinib treatment but not ibrutinib treatment resulted in G2/M cell cycle arrest. The blockade of cell cycle progression is positively correlated with decreased protein levels of critical regulators of S and G2/M phase transition such as cyclin B and CDC25C. BTK inhibitor (ibrutinib) in combination with venetoclax has shown great efficacy in preclinical models and in MCL patients. Therefore, here we assessed the in vivo efficacy of pirtobrutinib in combination with venetoclax with side-by-side comparison to ibrutinib & venetoclax in the Mino-venetoclax-R mouse model. Pirtobrutinib & venetoclax combination enhanced the efficacy of pirtobrutinib in restraining the tumor size (p<0.001) in the xenograft model. Notably, this novel combinatorial treatment exerted much higher potency than ibrutinib and venetoclax combination therapy (p<0.001). In addition, the pirtobrutinib & venetoclax combination was well tolerated and did not reduce overall mouse body weights compared with the vehicle treated mice.ConclusionsPirtobrutinib overcame both ibrutinib and venetoclax resistance in MCL cells in vitro and in vivo. G2/M checkpoints and E2F targets pathways were significantly enriched in both cases. Pirtobrutinib & venetoclax showed better in vivo efficacy in MCL models than combination of ibrutinib & venetoclax. [Display omitted] DisclosuresWang: Genentech: Consultancy; Juno: Consultancy, Research Funding; Kite Pharma: Consultancy, Honoraria, Research Funding; Clinical Care Options: Honoraria; CAHON: Honoraria; InnoCare: Consultancy, Research Funding; Moffit Cancer Center: Honoraria; Molecular Templates: Research Funding; Oncternal: Consultancy, Research Funding; DTRM Biopharma (Cayman) Limited: Consultancy; Hebei Cancer Prevention Federation: Honoraria; Lilly: Research Funding; Loxo Oncology: Consultancy, Research Funding; BioInvent: Research Funding; OMI: Honoraria; Miltenyi Biomedicine GmbH: Consultancy, Honoraria; Imedex: Honoraria; Physicians Education Resources (PER): Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Bayer Healthcare: Consultancy; Chinese Medical Association: Honoraria; Dava Oncology: Honoraria; Celgene: Research Funding; Mumbai Hematology Group: Honoraria; Acerta Pharma: Consultancy, Honoraria, Research Funding; BeiGene: Consultancy, Honoraria, Research Funding; Newbridge Pharmaceuticals: Honoraria; CStone: Consultancy; BGICS: Honoraria; The First Afflicted Hospital of Zhejiang University: Honoraria; Scripps: Honoraria; Epizyme: Consultancy, Honoraria; Pharmacyclics: Consultancy, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; VelosBio: Consultancy, Research Funding; Anticancer Association: Honoraria.

Iadademstat in Combination with Azacitidine Generates Robust and Long Lasting Responses in AML Patients (ALICE Trial)

Iadademstat in Combination with Azacitidine Generates Robust and Long Lasting Responses in AML Patients (ALICE Trial)

Potent and Orally Bioavailable BCL6 PROTAC TM Degraders Demonstrate Efficacy in Pre-Clinical Models of Diffuse Large B-Cell Lymphoma (DLBCL)

The BCL6 transcription repressor (B-cell lymphoma 6, BCL6) protein has been shown to be a key molecular driver of diffuse large B-cell lymphoma (DLBCL). Somatic mutations of the BCL6 gene include gross- or cryptic-chromosome translocations and point mutations that have been shown to result in the deregulation of BCL6 expression. These BCL6 abnormalities also contribute to a subgroup of high-risk (HR) aggressive double- and triple-hit (DH/TH) lymphomas with very poor outcomes. We have developed highly specific, potent and orally bioavailable BCL6 PROteolysis TArgeting Chimera (PROTAC TM) degraders that demonstrate potent in-vitro and in-vivo efficacy in multiple pre-clinical DLBCL models.Ten of 12 germinal center B-cell (GCB) and two of four activated B-cell (ABC) DLBCL cell lines show significant growth inhibition in-vitro with BCL6 PROTAC TM treatment, demonstrating a critical dependence on BCL6. This array of sensitivity across genetically variable cell lines suggests that BCL6-dependence is not just associated with BCL6-mutated DLBCLs. A more advanced BCL6 PROTAC TM, ARVN-71228, achieves >95% BCL6 D max in-vitro at a DC 50 of <1 nM in the OCI-Ly1 model following 24 hr treatment, degrading BCL6 equally well in the nuclear, chromatin-bound and cytosolic cell fractions. BCL6 degradation is associated with dose-dependent G1 cell cycle arrest and elevated apoptosis that increases over time (24 vs 72 hours). In head-to-head BCL6 degradation and growth inhibition studies using OCI-Ly1, the ARVN-71228 BCL6 PROTAC TM demonstrates superior activity compared to recently published BCL6-targeted degraders/inhibitors and heterobifunctional molecules.Importantly, medicinal chemistry efforts have resulted in the successful development of orally bioavailable BCL6 PROTAC TM degraders for in-vivo dosing. Time-course studies show >95% BCL6 loss within four hours which is maintained at 8-, 16- and 24-hours. Genes repressed by BCL6 such as BLIMP1 and PTPN6 are derepressed and show increased protein levels 24 hours post-dose. ARVN-71228 achieves regressions in the GCB OCI-Ly1 CDX model. Future studies plan to look at rational drug combinations with BCL6 PROTAC TM degraders to find collaborative or synergistic pathways to target, especially in the HR-DLBCL subtypes where there is a high unmet medical need. DisclosuresGough: Arvinas: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Sherman: Arvinas: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. DeCarr: Arvinas: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Eaton: Arvinas: Current Employment, Current equity holder in publicly-traded company. Milanovic: Arvinas: Current Employment, Current equity holder in publicly-traded company. Bookbinder: Arvinas: Current Employment, Current equity holder in publicly-traded company. Pizzano: Arvinas: Current Employment, Current equity holder in publicly-traded company. Altieri: Arvinas: Current Employment, Current equity holder in publicly-traded company. Corradi: Arvinas: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Xiao: Arvinas: Current Employment, Current equity holder in publicly-traded company. Gallego: Arvinas: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Soto: Arvinas: Current Employment, Current equity holder in publicly-traded company. Lingamaneni: Arvinas: Current Employment, Current equity holder in publicly-traded company. Chen: Arvinas: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Zhang: Arvinas: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Wang: Arvinas: Current Employment, Current equity holder in publicly-traded company. Dong: Arvinas: Current Employment, Current equity holder in publicly-traded company. Chirnomas: Arvinas: Current Employment, Current equity holder in publicly-traded company. Berlin: Arvinas: Current Employment, Current equity holder in publicly-traded company. Hornberger: Arvinas: Current Employment, Current equity holder in publicly-traded company. Snyder: Arvinas: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Taylor: Arvinas: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months.

Systematic Review of Systemic and Neuraxial Effects of Acetaminophen in Preclinical Models of Nociceptive Processing.

Acetaminophen (APAP) in humans has robust effects with a high therapeutic index in altering postoperative and inflammatory pain states in clinical and experimental pain paradigms with no known abuse potential. This review considers the literature reflecting the preclinical actions of acetaminophen in a variety of pain models. Significant observations arising from this review are as follows: 1) acetaminophen has little effect upon acute nociceptive thresholds; 2) acetaminophen robustly reduces facilitated states as generated by mechanical and thermal hyperalgesic end points in mouse and rat models of carrageenan and complete Freund’s adjuvant evoked inflammation; 3) an antihyperalgesic effect is observed in models of facilitated processing with minimal inflammation (eg, phase II intraplantar formalin); and 4) potent anti-hyperpathic effects on the thermal hyperalgesia, mechanical and cold allodynia, allodynic thresholds in rat and mouse models of polyneuropathy and mononeuropathies and bone cancer pain. These results reflect a surprisingly robust drug effect upon a variety of facilitated states that clearly translate into a wide range of efficacy in preclinical models and to important end points in human therapy. The specific systems upon which acetaminophen may act based on targeted delivery suggest both a spinal and a supraspinal action. Review of current targets for this molecule excludes a role of cyclooxygenase inhibitor but includes effects that may be mediated through metabolites acting on the TRPV1 channel, or by effect upon cannabinoid and serotonin signaling. These findings suggest that the mode of action of acetaminophen, a drug with a long therapeutic history of utilization, has surprisingly robust effects on a variety of pain states in clinical patients and in preclinical models with a good therapeutic index, but in spite of its extensive use, its mechanisms of action are yet poorly understood.

713 Novel protease activatable linker with tumor targeting motifs enhances the retention of cytokine prodrug and active cytokine at disease site and demonstrates improved efficacy in preclinical model

BackgroundAn emerging class of new protease-activatable prodrugs designed to enhance on-target activity and reduce off-target toxicity are being actively developed. Cytokines are complex immune mediators which display potent anti-tumor activity in preclinical models and have delivered clinical responses in several advanced tumor types. However, clinical development of cytokine therapies has been hampered by high systemic toxicity, a narrow therapeutic index and short circulatory half-life. To address these shortcomings, we have developed next-generation cytokine therapies, On Demand Cytokines or ODCs.MethodsODCs are protease-activatable cytokine prodrugs in which the cytokine is linked to an inhibitory moiety via a short proprietary peptide motif. These recombinant proteins are designed to exploit the protease activity present within the tumor microenvironment (TME) and enable the local release of active cytokine to trigger an anti-tumor immune response. ODCs contain tumor stroma targeting elements to further enhance their retention and activation within the malignant tissue. We have developed an array of stromaphilic ODCs, including a panel of IL-2 prodrugs containing single or dual tumor stroma binding motifs and report their preclinical in vitro and in vivo characterization.ResultsAll IL-2 prodrugs were successfully manufactured and activated in vitro by Matrix Metalloprotease cleavage which triggered the release of functional cytokine. Binding of prodrugs to tumor stroma components was confirmed in vitro. The ODC-IL2 panel was tested in vivo as single agent in the subcutaneous syngeneic B16F10 melanoma model. The uncleaved drugs were retained in the tumor at 5 to 20-fold higher levels than a control cytokine prodrug lacking any tumor targeting elements. Furthermore, intratumoral levels of IL-2 and IFNg were increased 8 to 80-fold and 10 to 40-fold respectively compared to cytokine levels measured in the control non-targeted ODC treated arm. Finally, stromaphilic ODCs displayed substantially enhanced levels in circulation over non-targeted ODC. Superior anti-tumor efficacy was observed for all stroma targeting pro-cytokines with near complete tumor growth inhibition achieved with the dual targeting site construct.ConclusionsWe have demonstrated that the On Demand Cytokine platform can generate protease-activatable cytokine prodrugs with enhanced tumor retention and on-target activity, to ultimately deliver safer and more effective immunotherapies.

Cyclophosphamide enhances the antitumor potency of GITR engagement by increasing oligoclonal cytotoxic T cell fitness

Only a subset of cancer patients responds to checkpoint blockade inhibition in the clinic. Strategies to overcome resistance are promising areas of investigation. Targeting glucocorticoid-induced tumor necrosis factor receptor–related protein (GITR) has shown efficacy in preclinical models, but GITR engagement is ineffective in controlling advanced, poorly immunogenic tumors, such as B16 melanoma, and has not yielded benefit in clinical trials. The alkylating agent cyclophosphamide (CTX) depletes regulatory T cells (Tregs), expands tumor-specific effector T cells (Teffs) via homeostatic proliferation, and induces immunogenic cell death. GITR agonism has an inhibitory effect on Tregs and activates Teffs. We therefore hypothesized that CTX and GITR agonism would promote effective antitumor immunity. Here we show that the combination of CTX and GITR agonism controlled tumor growth in clinically relevant mouse models. Mechanistically, we show that the combination therapy caused tumor cell death, clonal expansion of highly active CD8+ T cells, and depletion of Tregs by activation-induced cell death. Control of tumor growth was associated with the presence of an expanded population of highly activated, tumor-infiltrating, oligoclonal CD8+ T cells that led to a diminished TCR repertoire. Our studies show that the combination of CTX and GITR agonism is a rational chemoimmunotherapeutic approach that warrants further clinical investigation.

Electrochemical sensors for oxidative stress monitoring

Electrochemical sensors are ideally suited for the detection of reactive oxygen and nitrogen species (ROS and RNS) generated during biological processes. This review discusses the latest work in the development of electrochemical microsensors for ROS/RNS and their applications for monitoring oxidative stress in biological systems. The performance of recent designs of microelectrodes and electrode materials is discussed along with their functionality in preclinical models of drug efficacy, mitochondrial distress, and endothelial dysfunction. Challenges and opportunities in translating this methodology to study the pathophysiology associated with various diseases are discussed.

Therapeutic Potential of Annexins in Sepsis and COVID-19.

Sepsis is a continuing problem in modern healthcare, with a relatively high prevalence, and a significant mortality rate worldwide. Currently, no specific anti-sepsis treatment exists despite decades of research on developing potential therapies. Annexins are molecules that show efficacy in preclinical models of sepsis but have not been investigated as a potential therapy in patients with sepsis. Human annexins play important roles in cell membrane dynamics, as well as mediation of systemic effects. Most notably, annexins are highly involved in anti-inflammatory processes, adaptive immunity, modulation of coagulation and fibrinolysis, as well as protective shielding of cells from phagocytosis. These discoveries led to the development of analogous peptides which mimic their physiological function, and investigation into the potential of using the annexins and their analogous peptides as therapeutic agents in conditions where inflammation and coagulation play a large role in the pathophysiology. In numerous studies, treatment with recombinant human annexins and annexin analogue peptides have consistently found positive outcomes in animal models of sepsis, myocardial infarction, and ischemia reperfusion injury. Annexins A1 and A5 improve organ function and reduce mortality in animal sepsis models, inhibit inflammatory processes, reduce inflammatory mediator release, and protect against ischemic injury. The mechanisms of action and demonstrated efficacy of annexins in animal models support development of annexins and their analogues for the treatment of sepsis. The effects of annexin A5 on inflammation and platelet activation may be particularly beneficial in disease caused by SARS-CoV-2 infection. Safety and efficacy of recombinant human annexin A5 are currently being studied in clinical trials in sepsis and severe COVID-19 patients.

Sulfarotene, a synthetic retinoid, overcomes stemness and sorafenib resistance of hepatocellular carcinoma via suppressing SOS2-RAS pathway

BackgroundRecurrent hepatocellular carcinoma (HCC) shows strong resistance to sorafenib, and the tumor-repopulating cells (TRCs) with cancer stem cell-like properties are considered a driver for its high recurrent rate and drug resistance.MethodsSuppression of TRCs may thus be an effective therapeutic strategy for treating this fatal disease. We evaluated the pharmacology and mechanism of sulfarotene, a new type of synthetic retinoid, on the cancer stem cell-like properties of HCC TRCs, and assessed its preclinical efficacy in models of HCC patient-derived xenografts (PDXs).ResultsSulfarotene selectively inhibited the growth of HCC TRCs in vitro and significantly deterred TRC-mediated tumor formation and lung metastasis in vivo without apparent toxicity, with an IC50 superior to that of acyclic retinoid and sorafenib, to which the recurrent HCC exhibits significant resistance at advanced stage. Sulfarotene promoted the expression and activation of RARα, which down-regulated SOS2, a key signal mediator associated with RAS activation and signal transduction involved in multiple downstream pathways. Moreover, sulfarotene selectively inhibited tumorigenesis of HCC PDXs with high expression for SOS2.ConclusionsOur study identified sulfarotene as a selective inhibitor for the TRCs of HCC, which targets a novel RARα-SOS2-RAS signal nexus, shedding light on a new, promising strategy of target therapy for advanced liver cancer.

Bladder Cancer-related microRNAs With In Vivo Efficacy in Preclinical Models

Progressive and metastatic bladder cancer remain difficult to treat. In this review, we critique seven up-regulated and 25 down-regulated microRNAs in order to identify new therapeutic entities and corresponding targets. These microRNAs were selected with respect to their efficacy in bladder cancer-related preclinical in vivo models. MicroRNAs and related targets interfering with chemoresistance, cell-cycle, signaling, apoptosis, autophagy, transcription factor modulation, epigenetic modification and metabolism are described. In addition, we highlight microRNAs targeting transmembrane receptors and secreted factors. We discuss druggability issues for the identified targets.

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

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