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Purpose: To evaluate AZD4635, an adenosine A2A receptor antagonist, as monotherapy or in combination with durvalumab in patients with advanced solid tumors. Patients and Methods: In phase Ia (dose escalation), patients had relapsed/refractory solid tumors; in phase Ib (dose expansion), patients had checkpoint inhibitor–naïve metastatic castration-resistant prostate cancer (mCRPC) or colorectal carcinoma, non–small cell lung cancer with prior anti–PD-1/PD-L1 exposure, or other solid tumors (checkpoint-naïve or prior anti–PD-1/PD-L1 exposure). Patients received AZD4635 monotherapy (75–200 mg once daily or 125 mg twice daily) or in combination with durvalumab (AZD4635 75 or 100 mg once daily). The primary objective was safety; secondary objectives included antitumor activity and pharmacokinetics; exploratory objectives included evaluation of an adenosine gene signature in patients with mCRPC. Results: As of September 8, 2020, 250 patients were treated (AZD4635, n = 161; AZD4635+durvalumab, n = 89). In phase Ia, DLTs were observed with monotherapy (125 mg twice daily; n = 2) and with combination treatment (75 mg; n = 1) in patients receiving nanosuspension. The most common treatment-related adverse events included nausea, fatigue, vomiting, decreased appetite, dizziness, and diarrhea. The RP2D of the AZD4635 capsule formulation was 75 mg once daily, as monotherapy or in combination with durvalumab. The pharmacokinetic profile was dose-proportional, and exposure was adequate to cover target with 100 mg nanosuspension or 75 mg capsule once daily. In patients with mCRPC receiving monotherapy or combination treatment, tumor responses (2/39 and 6/37, respectively) and prostate-specific antigen responses (3/60 and 10/45, respectively) were observed. High versus low blood-based adenosine signature was associated with median progression-free survival of 21 weeks versus 8.7 weeks. Conclusions: AZD4635 monotherapy or combination therapy was well tolerated. Objective responses support additional phase II combination studies in patients with mCRPC.

Supplementary Table from Phase Ia/b, Open-Label, Multicenter Study of AZD4635 (an Adenosine A2A Receptor Antagonist) as Monotherapy or Combined with Durvalumab, in Patients with Solid Tumors

Purpose:To evaluate AZD4635, an adenosine A2A receptor antagonist, as monotherapy or in combination with durvalumab in patients with advanced solid tumors.Patients and Methods:In phase Ia (dose escalation), patients had relapsed/refractory solid tumors; in phase Ib (dose expansion), patients had checkpoint inhibitor–naïve metastatic castration-resistant prostate cancer (mCRPC) or colorectal carcinoma, non–small cell lung cancer with prior anti–PD-1/PD-L1 exposure, or other solid tumors (checkpoint-naïve or prior anti–PD-1/PD-L1 exposure). Patients received AZD4635 monotherapy (75–200 mg once daily or 125 mg twice daily) or in combination with durvalumab (AZD4635 75 or 100 mg once daily). The primary objective was safety; secondary objectives included antitumor activity and pharmacokinetics; exploratory objectives included evaluation of an adenosine gene signature in patients with mCRPC.Results:As of September 8, 2020, 250 patients were treated (AZD4635, n = 161; AZD4635+durvalumab, n = 89). In phase Ia, DLTs were observed with monotherapy (125 mg twice daily; n = 2) and with combination treatment (75 mg; n = 1) in patients receiving nanosuspension. The most common treatment-related adverse events included nausea, fatigue, vomiting, decreased appetite, dizziness, and diarrhea. The RP2D of the AZD4635 capsule formulation was 75 mg once daily, as monotherapy or in combination with durvalumab. The pharmacokinetic profile was dose-proportional, and exposure was adequate to cover target with 100 mg nanosuspension or 75 mg capsule once daily. In patients with mCRPC receiving monotherapy or combination treatment, tumor responses (2/39 and 6/37, respectively) and prostate-specific antigen responses (3/60 and 10/45, respectively) were observed. High versus low blood-based adenosine signature was associated with median progression-free survival of 21 weeks versus 8.7 weeks.Conclusions:AZD4635 monotherapy or combination therapy was well tolerated. Objective responses support additional phase II combination studies in patients with mCRPC.

Supplementary Figure from Phase Ia/b, Open-Label, Multicenter Study of AZD4635 (an Adenosine A2A Receptor Antagonist) as Monotherapy or Combined with Durvalumab, in Patients with Solid Tumors

Purpose: Danvatirsen is a therapeutic antisense oligonucleotide (ASO) that selectively targets STAT3 and has shown clinical activity in two phase I clinical studies. We interrogated the clinical mechanism of action using danvatirsen-treated patient samples and conducted back-translational studies to further elucidate its immunomodulatory mechanism of action. Experimental Design: Paired biopsies and blood samples from danvatirsen-treated patients were evaluated using immunohistochemistry and gene-expression analysis. To gain mechanistic insight, we used mass cytometry, flow cytometry, and immunofluorescence analysis of CT26 tumors treated with a mouse surrogate STAT3 ASO, and human immune cells were treated in vitro with danvatirsen. Results: Within the tumors of treated patients, danvatirsen uptake was observed mainly in cells of the tumor microenvironment (TME). Gene expression analysis comparing baseline and on-treatment tumor samples showed increased expression of proinflammatory genes. In mouse models, STAT3 ASO demonstrated partial tumor growth inhibition and enhanced the antitumor activity when combined with anti–PD-L1. Immune profiling revealed reduced STAT3 protein in immune and stromal cells, and decreased suppressive cytokines correlating with increased proinflammatory macrophages and cytokine production. These changes led to enhanced T-cell abundance and function in combination with anti–PD-L1. Conclusions: STAT3 ASO treatment reverses a suppressive TME and promotes proinflammatory gene expression changes in patients' tumors and mouse models. Preclinical data provide evidence that ASO-mediated inhibition of STAT3 in the immune compartment is sufficient to remodel the TME and enhance the activity of checkpoint blockade without direct STAT3 inhibition in tumor cells. Collectively, these data provide a rationale for testing this combination in the clinic.

Purpose:There are several agents in early clinical trials targeting components of the adenosine pathway including A2AR and CD73. The identification of cancers with a significant adenosine drive is critical to understand the potential for these molecules. However, it is challenging to measure tumor adenosine levels at scale, thus novel, clinically tractable biomarkers are needed.Experimental Design:We generated a gene expression signature for the adenosine signaling using regulatory networks derived from the literature and validated this in patients. We applied the signature to large cohorts of disease from The Cancer Genome Atlas (TCGA) and cohorts of immune checkpoint inhibitor–treated patients.Results:The signature captures baseline adenosine levels in vivo (r2 = 0.92, P = 0.018), is reduced after small-molecule inhibition of A2AR in mice (r2 = −0.62, P = 0.001) and humans (reduction in 5 of 7 patients, 70%), and is abrogated after A2AR knockout. Analysis of TCGA confirms a negative association between adenosine and overall survival (OS, HR = 0.6, P < 2.2e–16) as well as progression-free survival (PFS, HR = 0.77, P = 0.0000006). Further, adenosine signaling is associated with reduced OS (HR = 0.47, P < 2.2e–16) and PFS (HR = 0.65, P = 0.0000002) in CD8+ T-cell–infiltrated tumors. Mutation of TGFβ superfamily members is associated with enhanced adenosine signaling and worse OS (HR = 0.43, P < 2.2e–16). Finally, adenosine signaling is associated with reduced efficacy of anti-PD1 therapy in published cohorts (HR = 0.29, P = 0.00012).Conclusions:These data support the adenosine pathway as a mediator of a successful antitumor immune response, demonstrate the prognostic potential of the signature for immunotherapy, and inform patient selection strategies for adenosine pathway modulators currently in development.

Cell-type abundance GE signatures are useful for informing drug mechanism of action and may be useful in-patient selection for cancer immunotherapies. Recently, we validated natural killer cell- and dendritic cell-type specific expression signatures using a tiered strategy that included both computational and ex vivo validation. Here, we built upon this strategy to validate expression signatures for immunosuppressive myeloid cells (IMC). IMC play a critical role in impairing anti-tumor immunity and increased levels of peripheral IMC have been associated with advanced tumor progression and poor prognosis in various cancers. However, their low abundance in normal tissues and heterogeneous surface expression render their profiling difficult. To address this, we focused on validating GE signatures for IMC abundance using mRNA expression methods which are more clinically tractable than established flow cytometry methods that require intact cells. We implemented a three-stage GE signature validation strategy. First, we generated GE data from human ex vivo differentiated IMC (LIN- CD11B+ CD33+ HLADR-). Then, we evaluated the concordance of 16 previously published myeloid signatures with cell type abundance in a series of spike-ins with varying but known quantities of ex vivo differentiated IMC in a background of undifferentiated peripheral blood mononuclear cells (PBMCs). Gene-set variation analysis (GSVA) was used to score the signatures and Spearman's rank (rS) correlation coefficient was calculated to assess the significance of correlations between GSVA scores and IMC abundance. With this method, we validated 3 of the 16 published myeloid signatures (1. MacB3/PMID:26873985 (rS = 0.87; p = 8.8e-07); 2. MacB2_3w/PMID:26873985 (rS = 0.83; p = 6.7e-06); 3. TAM/PMID:27424807 (rS = 0.80; p= 2.6e-05)). Next, we generated a ‘de novo' signature by performing differential GE analysis and identified up-regulated genes in IMC in comparison to PBMCs with log2 fold change &gt 2, p-adj &lt 0.05). We further refined this list by selecting genes with significant concordance to IMC abundance (rS &gt 0.80, p-adj &lt 0.05) and confirmed their specificity to IMC in published scRNAseq studies [PMID: 30979687; 31033233; 32302573]. This led to a 6-gene ‘de novo' signature (MRC1, APOE, C1QA, C1QB, MMP9, SPP1) associated with IMC function with highly significant concordance (rS = 0.96; p = 1.2e-11) to IMC abundance. Lastly, we showed that in whole blood baseline samples from HNSCC patients (n=53) treated with danvatirsen + durvalumab [NCT02499328], our ‘de novo' signature was significantly elevated (p=0.033) in patients with progressive disease compared to complete responders. Application to additional studies will be required to more fully determine its clinical utility. Citation Format: Srimathi S. Srinivasan, Gozde Kar, Deanna L. Russell, Peter Gathungu, Minoo Rafati, David Whitston, Grant Duclos, Ben Sidders, J Carl Barrett, Tim Slidel, Patricia McCoon. Identification of a novel immunosuppressive myeloid gene expression signature for clinical biomarker development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 390.

Background: Danvatirsen (Da) is a generation 2.5 antisense oligonucleotide targeting STAT3 mRNA under clinical development in combination with Durvalumab (NCT03421353). The recommended Phase II dose is intravenous (IV) 200 mg weekly (QW, A). However, alternative 400 mg IV every two weeks (Q2W, B) and subcutaneous 200 mg QW (C) are also under investigation. The JAK-STAT signaling pathway is involved in immunity, cell division, cell death, and tumor formation. Additionally, activation of hepatic STAT3 results in increased secretion of C-reactive protein (CRP) and fibrinogen into blood. Therefore, target engagement in patients may be better understood by analyzing Da exposure and related biomarkers in the blood/plasma. We investigated Da exposure and subsequent impact on target engagement biomarkers such as CRP, platelets, fibrinogen, neutrophils and STAT3 mRNA levels. Methods: Extensive pharmacokinetics (PK) samples were collected at lead-in monotherapy and at steady state (SS) in combination. Plasma biomarker samples were collected at baseline and every two weeks thereafter. Whole blood samples for the analysis of STAT3 mRNA were collected at baseline, after lead-in monotherapy, and every four weeks thereafter. The PK parameters were estimated by non-compartmental analysis by WinNonlin. The biomarkers were analyzed by standard clinical methods and STAT3 mRNA level was measured by NanoString technology. Results: The area under plasma concentration-time curve of Da at SS within dosing interval (AUC0-τ, ng.h/ml) for A, B, and C were 69110 (24.64%), 101600 (22.11%), and 44600 (30.25%), respectively. Additionally, SS Ctrough (ng/ml) levels for A, B, and C were 28.53 (48.86%), 14.56 (36.14%), and 27.66 (49.22%), respectively. Maximum median % decrease from baseline in CRP levels was observed at week 4; A (-91%), B (-94%), C (-82%). Additionally, maximum median % changes from baseline for fibrinogen were -60%, -40%, -53%, in A, B, and C at week 4, respectively. Maximum % decrease from baseline in platelets was observed at week 8 in A (-42%), B (-41%), and C (-69%). In contrast, maximum % change from baseline in neutrophil count was observed at week 6; -37%, -31%, and -32% in A, B, and C, respectively. Additionally, the median maximum change from baseline for STAT3 mRNA was -42.4% and -33.6% for A and C, respectively. Conclusion: Cumulative AUC in A was very similar to B, whereas, AUC in C was 30% lower as compared to A or B. The SS Ctrough values in A and C were similar, although Ctrough value in B was 50% lower. Maximum % decreases in CRP levels were similar across all the doses. Moreover, route of administration did not appear to impact level of STAT3 knockdown. In summary, a similar degree of changes in pharmacodynamic biomarkers were achieved at all three dose levels regardless of route of administration, despite differences in plasma exposure. Citation Format: Kowser Miah, Patricia McCoon, Deanna Russell, Patrick Mitchell, Martin Scott, Amy Rosenfeld, Li Zhang, Pablo Martinez Rodriguez, Alicia Savage, Ganesh Mugundu. Plasma exposure-target engagement biomarker analysis for danvatirsen after multiple dosing schedules and route of administrations in patients with advanced solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-105.

FLT3 is a member of the class III receptor tyrosine kinase family that includes C-KIT, C-FMS and platelet derived growth factor receptor (PDGFR). FLT3 is primarily expressed in early myeloid and lymphoid progenitors and plays an important role in their proliferation and differentiation. In human leukemia, FLT3 is expressed on 70-90% acute myeloid leukemia (AML) and most B-acute lymphoblastic leukemia (B-ALL). FLT3 genetic aberrations are commonly detected in patients with AML. The most common aberration is internal tandem duplication (ITD), which occurs in 25-30% of AML patients and causes constitutive activation of FLT3. Point mutation in codon D835 of the FLT3 tyrosine kinase domain is reported in 7-10% of AML patients and also causes constitutive activation of the receptor. FLT3 small molecule inhibitors targeting the kinase domain are predominantly active against FLT3 activated AML. The restricted normal tissue expression profile and higher differential in leukemic specimens makes FLT3 amenable to antibody-based therapeutics, requiring only target expression independent of kinase activation status. Therefore, development of an antibody-drug conjugate (ADC) may provide a therapeutic alternative for AML patients. Here, we report the development of the first FLT3specific ADC, AGS62P1, employing site-specific conjugation using the non-natural amino acid, p-acetyl phenylalanine (pAF). AGS62P1 comprises a human gamma one antibody including an inserted pAF residue in each of the heavy chains. The antibody was conjugated to a potent cytotoxic payload via an oxime bond at the pAF sites, thus creating a nearly homogeneous drug distribution, with approximately 2 drug molecules per antibody. Strong binding affinity (0.1-0.9 nM) and potent in vitro cytotoxic activity (IC50 = 0.2-12 nM) was achieved in AML cell lines. The anti-FLT3 ADC was highly efficacious in AML tumor xenografts, leading to statistically significant tumor growth inhibition of both FLT3 ITD and non-ITD models. Additional characterization of both the antibody and ADC was performed, including ligand receptor interaction, degradation, internalization, and apoptosis. In summary, we have developed a site-specific ADC targeting FLT3 that exhibits potent anti-tumor activity in xenograft models regardless of FLT3 activation status. This drug can potentially offer a new and more versatile approach in targeting FLT3-expressing leukemia through a mechanism independent of FLT3 genetic aberration. Citation Format: Nandini Rudra-Ganguly, Pia M. Challita-Eid, Christine Lowe, Mike Mattie, Sung-Ju Moon, Brian A. Mendelsohn, Monica Leavitt, Cyrus Virata, Alla Verlinsky, Linnette Capo, Mi Sook Chang, Deanna L. Russell, Baljinder Randhawa, Gao Liu, René Hubert, Mary Brodey, Hector Aviña, Chunying Zhang, Joseph D. Abad, Banmeet Anand, Sher Karki, Zili An, Roland Luethy, Fernando Doñate, Daniel S. Pereira, Kendall Morrison, Ingrid B.J. Joseph, David R. Stover. AGS62P1, a novel site-specific antibody drug conjugate targeting FLT3 exhibits potent anti-tumor activity regardless of FLT3 kinase activation status. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 574.

FLT3 (FMS-like tyrosine kinase 3) is a member of the class III receptor tyrosine kinase family, which is highly expressed in the blasts of both AML and ALL patients. In addition to FL ligand stimulation, FLT3 can also be activated by two distinct clusters of mutations: internal tandem duplications (FLT3/ITDs) in 20% to 25% patients and point mutations at position D835 in the tyrosine-kinase domain (FLT3/TKD) in 7% to 10% patients. FLT3 tyrosine kinase inhibitors (TKI) are mainly active against FLT3 mutant AML. An antibody drug conjugate (ADC), directed against the extracellular domain of FLT3 may only require FLT3 cell surface expression independent of mutation status. The restricted cellular distribution of FLT3 receptor and a higher expression in AML than in normal bone marrow makes FLT3 a favorable ADC target. Therefore, this ADC based strategy may offer a therapeutic alternative for AML patients independent of FLT3 status. Here, we report the preclinical assessment of a novel FLT3 targeting ADC, AGS62P1. AGS62P1 consists of a human anti-FLT3 monoclonal antibody, site specifically conjugated to a potent cytotoxic payload. FLT3 expression is confirmed in a large panel of AML and ALL tumor cells as well as in AML patient specimens via flow cytometry. The anti-leukemic activity of AGS62P1 was evaluated against AML and ALL tumor cell lines, in vitro and in vivo. AGS62P1 demonstrated strong binding affinity (0.1-0.5 nM) and potent cytotoxic activity in FLT3/ITD and Non-ITD tumor models, in vitro. Cytotoxic IC50 potency for AGS62P1 was 0.5-13 nM in FLT3/ITD and 0.2-12 nM in FLT3 non-ITD models. A fluorescence based assay confirmed that AGS62P1 is rapidly internalized in AML tumor cell lines. AGS62P1 is highly efficacious in FLT3/ITD and non-ITD tumor xenografts, leading to significant tumor growth inhibition or complete tumor regression. In primary AML patient xenograft drug treatment studies, the engraftment and outgrowth of 5/6 samples were significantly reduced when treated with AGS62P1. Taken together our data demonstrate that AGS62P1 exhibits potent antitumor activity against a broad panel of AML tumor models and primary AML samples, regardless of FLT3 status. We believe AGS62P1 may be an effective and alternative therapeutic for AML patients, which can bypass the TKI mediated resistance and deliver target specific effect through a different mode of action. Disclosures Jin: Agensys: Research Funding. Anand:Agensys: Employment. Dick:Agensys: Research Funding.