Stimulator of IFN genes (STING) is an innate immune pathway that activates a type I IFN response upon detection of intracellular DNA from foreign pathogens as well as tumor cells. STING signaling is critical for antiviral immunity and can be co-opted to drive an antitumor immune response. However, STING activation requires careful and controlled agonism to drive immune activation in the tumor microenvironment (TME) while avoiding toxic systemic immune activation. Indeed, nontargeted small-molecule STING agonist therapeutics have shown limited antitumor activity in the clinic, likely due to their short half-life and poor retention within the TME. We hypothesized that targeted delivery of a potent STING agonist payload directly to the TME via an antibody-drug conjugate (ADC) may overcome some of these limitations. In this study, we report the development of a novel STING agonist ADC with a noncleavable linker-payload (ncSTING). Tumor-targeted ADCs employing this linker-payload (ncSTING ADC) elicited robust antitumor activity in a variety of preclinical murine tumor models. We found that Fcγ receptor binding affected antitumor activity as ADCs with a wild-type Fc drove more antitumor activity than ADCs with an Fcγ receptor-binding mutant Fc in a subset of tumor models. Moreover, tumor-targeted ncSTING ADCs elicited tumor regression with reduced systemic immune activation compared with the systemic administration of the released payload. Altogether, these data provide a therapeutic rationale for the targeted delivery of a potent STING agonist payload via an ADC.

Plasma stability of quaternary ammonium linker in rodent plasma.

Background: BCL-2 inhibition to target intrinsic apoptotic pathways that confer a survival advantage to leukemic blasts has become a key therapeutic strategy for patients (pts) with myeloid malignancies. LP-108 is an oral highly potent and selective inhibitor of BCL-2 with comparable or more potent in vitro inhibitory activity compared to the FDA approved oral BCL-2 inhibitor venetoclax. Aims: We present safety and preliminary efficacy data of pts treated in an ongoing phase 1 study (NCT04139434) evaluating LP-108 as monotherapy (Arm 1) and in combination with azacitidine (Arm 2) in pts with relapsed or refractory (r/r) MDS, CMML or AML. Methods: Pts age ≥18 with r/r MDS with excess blasts (MDS-EB) or high/very-high risk per IPSS-R (HR-MDS), r/r AML, or r/r CMML were enrolled in Arm 1, and pts with r/r MDS-EB/HR-MDS, r/r AML, or r/r CMML were enrolled in Arm 2. Pts with prior hypomethylating agents or venetoclax exposure were allowed to enroll in Arm 2. Blast count was required to be ≤ 30 × 109 cells/L at therapy initiation and ECOG performance status was ≤2. For Arm 1, LP-108 was given in several dose cohorts (100 mg, 200 mg, 400 mg, or 600 mg daily in 28-day (d) cycle). For Arm 2 azacitidine 75 mg/m2 was administered on d1-7 (or d1-5,8-9) of each 28-d cycle, and LP-108 was given in several dose cohorts (100 mg, 200 mg, or 400 mg daily in 28-d cycles). Primary objectives were to determine the safety/tolerability and the RP2D of LP-108 as a single agent and in combination with azacitidine. Key secondary objectives were to evaluate the objective response rate (ORR) of LP-108 (monotherapy and combination therapy) in r/r MDS/CMML/AML as well as progression free survival and overall survival. Results: At data cutoff February 20, 2023, 20 pts had received LP-108 monotherapy (Arm 1) and 12 pts in combination with azacitidine (Arm 2). 3 pts received LP-108 at the RP2D of 400 mg with azacitidine. In Arm 1, median age was 72 years (range 40-84), 55% male; 12 pts (60%) had r/r AML, 7 (35%) r/r MDS, and 1 (5%) r/r CMML. In Arm 2, median age was 61 years (range 46-83), 75% male; 8 pts (66.7%) had r/r AML, 1 (8.3%) r/r MDS, and 3 (25%) r/r CMML. 14 pts (70%) experienced any TRAE in Arm 1 (2 serious TRAEs) and 7 (58.3%) in Arm 2 (2 serious), the most common being neutropenia (4 pts, 20% Arm 1; 3pts, 25% Arm 2), nausea (3pts, 15% Arm 1; 1 pt, 8.3% Arm 2) and vomiting (3pts, 15% Arm 1; 2 pts,16.7% Arm 2). Grade 3/4 neutropenia was seen in 3 pts (15%) in Arm 1 and 3 pts (25%) in Arm 2; febrile neutropenia occurred in 1 pt in Arm 1 only. A grade 5 AE did occur in a pt receiving 200 mg LP-108 with azacitidine due to fusarium bacteremia that was not related to LP-108. Across all dosing cohorts in Arm 1, the ORR was 11.1% (2 pts) and 10 pts (55.5%) had SD as best response (18 evaluable pts). Across all dosing cohorts in Arm 2, the ORR was 54.5% (6 pts) and the remaining 5 pts (45.4%) experienced SD as best response (11 evaluable pts). In Arm 1 an outstanding responder with MDS was seen in the 200 mg LP-108 cohort, and in Arm 2 responses were seen in both CMML and AML pts. In Arm 1, the median PFS ranged from 7.0 to 37.0 weeks and the median OS ranged from 3.6 to 12.1 weeks across dosing cohorts. In Arm 2, the median PFS ranged from 12.3 to 29.1 weeks and the median OS ranged from 5.2 to not reached, across dosing cohorts. PFS/OS data are still maturing. Summary/Conclusion: LP-108 as monotherapy and in combination with azacitidine was overall well tolerated with an acceptable safety profile for r/r MDS, CMML, and AML pts. Notably, the combination was associated with encouraging efficacy including ORR and PFS.Swimmers’ plot of patients in Arm 1 (A) and Arm 2 (B). LP-108 dose level cohort indicated by different colors and disease assessment results are indicated. Keywords: CMML, Targeted therapy, AML, MDS

Eltrombopag has been shown to improve response rates when added to standard therapy in adults with severe aplastic anemia in controlled trial settings. However, outcomes in real-world populations have mostly been examined in small retrospective studies. This robust, multicenter, retrospective cohort study across six academic health systems compared outcomes in patients who received immunosuppressive therapy with or without eltrombopag. The study included 82 patients who received front-line therapy from January 2014 to August 2021. Overall response rates at 6 months did not differ significantly for patients receiving eltrombopag versus immunosuppressive therapy alone (58% v. 65%, p = 0.56). However, complete response rates at 6 and 12 months were over two times higher in the eltrombopag arm (29% v. 12%, p = 0.06 and 48% v. 18%, p = 0.005). Rates of hepatotoxicity were similar across both arms. Eltrombopag addition did not impact overall survival (median not reached in either arm at 2 years, p = 0.86) or disease-free survival (median not reached v. 13.3 months at 2 years, p = 0.20). Eltrombopag may not produce as large of a benefit in real-world settings compared to controlled trial settings but may offer patients deeper responses with similar rates of toxicity to immunosuppressive therapy alone.

Pharmacokinetic profile of clearance for three ADCs.

Experimental details for the chemical synthesis of the drug-linkers.

In vitro dose response curves

Novel payloads with orthogonal mechanisms of action (MOAs) to established ADC technologies are important for expanding the utility of antibody-drug conjugates (ADCs) in cancer treatment. Anthracyclines, a class of cytotoxic small molecules that include important clinical chemotherapeutics such as doxorubicin, have long been of interest to the ADC field due to their high potency and unique MOA. Interest in anthracyclines has increased in recent years due to their status as canonical inducers of immunogenic-cell death (ICD), a feature that may lead to more durable tumor regressions and increased synergy with immune checkpoint blockade. In this work, we investigate several drug-linkers based on oxidized derivatives of the highly potent anthracycline analogue PNU-159682. Resultant ADCs are potent and immunologically specific with linker-dependent bystander activity in co-culture cytotoxicity assays displaying heterogeneous antigen (Ag+/Ag-) expression. Rodent in vivo anti-tumor activity and tolerability are shown with significant differences emerging between the toxicity profile of the cleavable and non-cleavable analogues. In vitro, tumor cells treated with anthracycline ADCs showed key hallmarks of ICD including markers of endoplasmic reticulum (ER) stress and the induction of ICD damage-associated molecular patterns (DAMPs) such as HMGB1 and ATP. In vivo mouse xenograft studies show increased tumoral F4/80+ macrophage infiltration by IHC in anthracycline ADC treated animals relative to controls. ADCs delivering monomethyl auristatin E (MMAE) show comparable activity in ICD assays, confirming MMAE as an ICD inducing ADC payload alongside canonical inducers such as anthracyclines. Our results indicate there is a therapeutic window in rodents alongside ICD induction by anthracycline ADCs, positioning anthracyclines as a potentially impactful class of ADC payloads. Citation Format: Joseph Z. Hamilton, Kerry Klussman, Rebecca Mahzereh, Jessica Simmons, Michelle Ulrich, Shyra J. Gardai, Peter D. Senter, Patrick J. Burke. Oxidized anthracycline payloads induce anti-tumor immunogenic cell-death and show linker-dependent tolerability when delivered as ADCs [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 2013.

Description of stability experiment, rat tolerability, and chemistry experimentals.

Topic: 4. Acute myeloid leukemia - Clinical Background: Despite improved outcomes with azacitidine (AZA) and venetoclax (VEN) in frontline unfit AML (CR 37% and CR/CRi 66%), long-term survival for these patients remains inadequate (mOS <15m, DiNardo NEJM 2020). CD123 is expressed on the majority of AML blasts and leukemic stem cells while minimally expressed on normal hematopoietic stem cells (Kovtun Blood Adv 2018). Pivekimab sunirine (PVEK, IMGN632) is an antibody-drug conjugate (ADC) comprising a high-affinity CD123 antibody, cleavable linker, and an indolinobenzodiazepine pseudodimer (IGN) payload. The IGN payload alkylates DNA and causes single strand breaks without crosslinking. IGNs are designed to have high potency against tumor cells, while demonstrating less toxicity to normal marrow progenitors than other DNA-targeting payloads. Clinical data in relapsed/refractory (R/R) AML (Daver ASH 2022) supported the continued clinical exploration of the PVEK+AZA+VEN triplet. Aims: The current enrolling dose expansion cohorts (Regimen C; Cohorts 1 and 2) in frontline AML will evaluate antileukemia activity and MRD levels in the combination of PVEK+AZA+VEN. Regimen E will enroll patients with R/R AML to evaluate the safety and antileukemia activity of PVEK in combination with magrolimab. Methods: This is an open-label, multicenter, Phase 1b/2 study of PVEK administered in combination regimens in patients with frontline and R/R CD123-positive AML. Patients will receive the established recommended phase 2 dose (RP2D) of PVEK 0.045 mg/kg IV, as a < 30-minute outpatient infusion. In Regimen C, patients will receive PVEK 0.045 mg/kg IV on D7 + azacitidine 75 mg/m2 SC or IV daily on days 1 to 7, and venetoclax up to 400 mg PO daily for at least 14 days (Cohort 1) or for up to 28 days (Cohort 2) in a 28-day cycle. In Regimen E, R/R AML patients will receive PVEK 0.045 mg/kg IV on D1 of the 28-day cycle in combination with magrolimab at 30 mg/kg Q2W, after its standard ramp up schedule. Regimen C will enroll adults with untreated AML, CD123+ by flow cytometry or IHC and no prior HMA treatments. The primary endpoints for Regimen C are composite CR rate, overall response rate (ORR), duration of remission (DOR), MRD levels and key secondary endpoints are safety, PK and immunogenicity. Regimen E will enroll adult patients with relapsed or refractory CD123+ AML with up to 2 prior lines of therapy. The primary endpoint for Regimen E is dose limiting toxicities (DLTs) and key secondary endpoints are safety, antileukemic activity, PK and immunogenicity. Results: N/A Summary/Conclusion: The PVEK+AZA+VEN triplet (Regimen C) is currently enrolling frontline unfit patients across sites in France, Germany, Italy, Spain, UK and USA. The PVEK+Magro doublet (Regimen E) is planned to be open for enrollment mid-2023 at sites in the USA. Clinical trial information: NCT04086264. Keywords: Acute myeloid leukemia, Clinical trial

Background STING is an innate immune pathway that detects intracellular DNA from foreign pathogens as well as stressed or tumor cells and activates a proinflammatory, type I interferon (IFN) response.1 STING activation is critical for mounting anti-viral immunity, and recent advances suggest this pathway can be co-opted to drive an anti-tumor immune response.2 However, STING activation requires careful and controlled agonism as dysregulated STING signaling is associated with autoinflammatory diseases including systemic lupus erythematosus (SLE), Aicardi-Goutières syndrome (AGS) and STING-associated vasculopathy with onset of infancy (SAVI).3 Balancing anti-tumor immunity against the risk of systemic immune activation has likely hindered the clinical success of systemic STING agonist small molecules.4 To date, STING agonist therapeutics have shown limited anti-tumor activity potentially due to their short half-life and poor retention within the tumor microenvironment (TME).5 We hypothesized that targeted delivery of a potent STING agonist to the tumor microenvironment via an antibody drug conjugate (ADC) may overcome these limitations. Methods STING agonist ADCs were generated by conjugating novel, potent cleavable and non-cleavable drug-linkers to a tumor-targeting antibody. The potency of these STING agonist ADCs and released free drugs were evaluated on human myeloid cells in vitro. Finally, the antitumor activity of STING ADCs were evaluated in vitro in a tumor and immune cell co-culture system as well as in vivo in syngeneic murine tumor models. Results STING agonist ADCs with both non-cleavable and cleavable drug-linkers demonstrated comparable potency in vitro – with a similar capacity to induce type I IFN signaling and elicit immune-mediated tumor cell killing. However, in vivo, STING agonist ADCs with the non-cleavable drug-linker led to superior antitumor activity compared to ADCs with cleavable drug-linkers. STING agonist ADCs with this novel, potent non-cleavable drug-linker demonstrated robust, durable antitumor activity across multiple tumor models as well as reduced systemic immune activation compared to a small molecule agonist. Conclusions Here, we report the generation of a STING agonist ADC that elicits robust antitumor activity across multiple preclinical murine tumor models. Drug-linker design was critical for maximizing antitumor activity, as ADCs with the non-cleavable drug-linker drove enhanced antitumor activity compared to cleavable drug-linkers. This targeted ADC delivery led to antitumor activity with reduced systemic immune activation compared to a small molecule STING agonist. Altogether, these data demonstrate the potential of STING agonists as ADC payloads to drive tolerated, efficacious anti-cancer responses. Ethics Approval All animal studies were conducted in accordance with protocols reviewed and approved by the Institutional Animal Care and Use Committee at Seagen or the external testing facility that conducted the studies. References Ablasser A, Zhijian CJ, cGAS in action: Expanding roles in immunity and inflammation. Science. 2019;363(6431):eaat8657. Flood BA, Higgs EF, Li S, Luke JJ, Gajewski TF. STING pathway agonism as a cancer therapeutic. Rev. 2019;290(1):24–38. Ahn J, Barber GN. Self-DNA, STING-dependent signaling and the origins of autoinflammatory disease. Opin. Immunol. 2014;31:121–126. Aval LM, Pease JE, Sharma R, Pinato DJ. Challenges and opportunities in the clinical development of STING agonists for cancer immunotherapy. Clin. Med. 2022;9:3323. Marc-Bernstam F, et al. Phase I dose-escalation trial of MIW815 (ASU-S100), an intratumoral STING agonist, in patients with advanced/metastatic solid tumors or lymphomas. Cancer Res. 2022;28:677–688.

In recent years, an explosion of novel agents has shifted the treatment paradigm for patients with acute myeloid leukemia. The optimal place in therapy for many of these novel agents remains unknown due to limited guidance from national guidelines and the way these agents were studied prior to entering the market. A critical evaluation of the literature and incorporation of oncology stewardship principles can be helpful in determining an optimal place for these agents while being mindful of the overall cost that is associated with therapies. The purpose of this review is to critically evaluate the efficacy and safety data for five controversial agents and provide examples of the use of stewardship practices in determining their place in the treatment of acute myeloid leukemia.

Patients with relapsed/refractory (R/R) acute lymphoblastic leukemia (ALL) represent a heterogeneous population and therefore there is no standard of care first salvage regimen. We conducted a multicenter, retrospective analysis to compare chemotherapy (e.g. HyperCVAD, MOAD, Larson/CALGB-9511, etc.) to novel agents (blinatumomab or inotuzumab) in first salvage. The primary endpoint, overall survival (OS), was not significantly different among treatment arms, with a median OS of 10.6 months with chemotherapy and 10.1 months with novel therapy (p = .799). Similarly, there was no difference in the CR/CRi rate, with a CR/CRi in 18 patients (41.9%) versus 16 patients (47.1%) treated with salvage chemotherapy and novel therapy, respectively (p = .817). Age significantly impacted the probability of achieving CR/CRi with novel therapy versus chemotherapy. This analysis suggests the use of chemotherapy in first salvage still represents an appropriate treatment option, particularly for young fit patients, as the median OS was roughly 10 months regardless of whether patients received novel therapy or chemotherapy in first salvage. For the reported outcomes, 100% of patients in the novel therapy arm received a novel therapy (per design), whereas only 60.5% of patients in the chemotherapy arm required a novel therapy. Thus, 40% of patients did not require a novel therapy for similar OS. This analysis demonstrates that first-line chemotherapy can achieve similar results to novel therapies, especially now that novel therapies are available for subsequent relapses. However, this study has several limitations including younger age, increased CNS involvement, and higher blast percentage in the chemotherapy arm and potential confounders, including selection of treatment sequence as 43 patients (55.8%) ultimately received both chemotherapy and novel therapy. Therefore, a larger, prospective, randomized study with adequate chemotherapy comparators and availability of novel agents upon relapse is warranted to confirm these results.

The Front Cover shows the antimitotic tubulysin M bound to bovine brain tubulin. Highlighted in yellow is the labile C11 acetate, the loss of which leads to greatly reduced biochemical activity. The corresponding article interrogates the effect of drug-linker chemistry and conjugation site on the stability of this acetate and the in vivo activity of antibody–drug conjugates (ADCs) delivering tubulysin M. The results led the team from a protease cleavable 4-load ADC (top) to an optimized β-glucuronidase cleavable 2-load ADC (bottom). More information can be found in the Communication by Joseph Z. Hamilton et al.

Tubulysins have emerged in recent years as a compelling drug class for delivery to tumor cells via antibodies. The ability of this drug class to exert bystander activity while retaining potency against multidrug-resistant cell lines differentiates them from other microtubule-disrupting agents. Tubulysin M, a synthetic analogue, has proven to be active and well tolerated as an antibody-drug conjugate (ADC) payload, but has the liability of being susceptible to acetate hydrolysis at the C11 position, leading to attenuated potency. In this work, we examine the ability of the drug-linker and conjugation site to preserve acetate stability. Our findings show that, in contrast to a more conventional protease-cleavable dipeptide linker, the beta-glucuronidase-cleavable glucuronide linker protects against acetate hydrolysis and improves ADC activity in vivo. In addition, site-specific conjugation can positively impact both acetate stability and in vivo activity. Together, these findings provide the basis for a highly optimized delivery strategy for tubulysin M.

There is no single standard-of-care salvage regimen for relapsed/refractory acute myeloid leukemia (AML). This retrospective analysis of first relapse/primary refractory patients with AML compares FLAG (fludarabine, cytarabine, and granulocyte colony-stimulating factor) chemotherapy and non-FLAG salvage regimens. Patients treated with FLAG had improved overall response rates, event-free survival, and overall survival, supporting the use of FLAG as an effective salvage regimen in relapsed/refractory AML. Background: Relapsed/refractory acute myeloid leukemia (AML) confers a poor prognosis, and there is no single standard of care first-line salvage regimen. FLAG (fludarabine, cytarabine, and granulocyte colony-stimulating factor) is a common salvage regimen with a favorable toxicity and efficacy profile in poor-risk AML. Materials and Methods: We conducted a single-center, retrospective analysis of first relapse/primary refractory patients with AML that received salvage chemotherapy from January 2009 to July 2019. We propensity-score matched patients 1:1 (based on age at diagnosis, cytogenetic risk group, Charlson comorbidity index, de novo vs. secondary AML, and whether or not they received an allogeneic stem cell transplant in first complete remission) into 2 groups, FLAG (Group 1) or non-FLAG (Group 2) as first-line salvage regimen, with 66 patients in each group. The primary endpoint was overall response rate (complete response and complete response with incomplete hematologic recovery). Results: The median patient age was 59 years (range, 19-80 years). Patients treated with FLAG had a higher overall response rate (complete response/complete response with incomplete hematologic recovery) (71.2% vs. 50.0%; odds ratio, 2.47; 95% confidence interval [CI], 1.21-5.08; P=.013), longer event-free survival (8.9 vs. 2.1 months; hazard ratio [HR], 0.58; 95% CI, 0.39-0.86; P=.005), and longer overall survival (14.2 vs. 5.9 months; HR, 0.62; 95% CI, 0.41-0.93; P=.019). Patients who received FLAG had a shorter median duration of neutropenia (22 vs. 34 days; HR, 0.43; 95% CI, 0.29-0.64; P<.001). Conclusion: This analysis supports the FLAG regimen as an effective and well-tolerated salvage therapy for patients with relapsed/refractory AML. (C) 2021 Elsevier Inc. All rights reserved.

BACKGROUND:Patients diagnosed with acute myeloid leukemia with a FLT3 mutation (FLT3+ AML) have historically had poor outcomes. While the addition of the FLT3 inhibitors to induction therapy has been shown to improve survival outcomes in FLT3+ AML, interactions and overlapping toxicities between FLT3 inhibitors and standard of care medications used during induction therapy (e.g. azole antifungals, anthracyclines) and logistical barriers have complicated their use. To avoid these concerns, our institution has opted to defer initiation of midostaurin until after completion of induction therapy. However, to our knowledge no study confirming the effectiveness of this strategy for real world FLT3 inhibitor use has been published. METHODS:We performed a single center, propensity-score matched, retrospective cohort study characterizing efficacy and safety of our strategy for use of FLT3 inhibitors in the treatment of FLT3+ AML. The primary outcome was median event-free survival (EFS), while secondary endpoints included median overall survival (OS), overall response rate (ORR), 30-day mortality, duration of neutropenia, duration of thrombocytopenia, consolidation cycle delays, documented infections, and all-cause hospital readmission. RESULTS:A total of 83 FLT3+ AML patients treated with intensive induction therapy were included in the study, of whom 48 were propensity-score matched and analyzed. Baseline characteristics were similar between the patients who received a FLT3 inhibitor after induction therapy and the historical control arm. Median EFS was not significantly different but compared favorably between the FLT3 inhibitor cohort and historical controls (not reached vs 8 months, p = 0.343) with 18-month EFS of 54% and 43% for the two cohorts, respectively. Similarly, no significant differences were noted with regard to median OS (not reached vs 28.7 months, p = 0.752), ORR (79.2% vs 79.2%), or safety outcomes between groups. CONCLUSION:Compared to historical controls, addition of a FLT3 inhibitor to intensive chemotherapy post-induction may improve EFS or OS in a real world patient cohort with longer follow-up and a larger sample size. The omission of midostaurin in induction allowed for the use of an azole antifungal and the intensification of anthracycline dose may have contributed to high remission rates in both groups.