Glucocorticoids are key components of the current standard-of-care regimens (e.g., R-CHOP, EPOCH-R, Hyper-CVAD) for treatment of B-cell malignancy. However, systemic glucocorticoid treatment is associated with several adverse events. CD19 displays restricted expression in normal B-cells and is up-regulated in B-cell malignancies. ABBV-319 is a CD19-targeting antibody-drug conjugate (ADC) engineered to reduce glucocorticoid-associated toxicities while possessing three distinct mechanisms of action (MOA) to increase therapeutic efficacy: (1) antibody-mediated delivery of glucocorticoid receptor modulator (GRM) payload to activate apoptosis, (2) inhibition of CD19 signaling, and (3) enhanced Fc-mediated effector function via afucosylation of the antibody backbone. ABBV-319 elicited potent GRM-driven anti-tumor activity against multiple malignant B-cell lines in vitro as well as in cell line-derived xenografts (CDXs) and patient-derived xenografts (PDXs) in vivo. Remarkably, a single-dose of ABBV-319 induced sustained tumor regression and enhanced anti-tumor activity compared to repeat dosing of systemic prednisolone at the maximum tolerated dose (MTD) in mice. The unconjugated CD19 monoclonal antibody (mAb) also displayed anti-proliferative activity on a subset of B-cell lymphoma cell lines through the inhibition of PI3K signaling. Moreover, afucosylation of the CD19 mAb enhanced Fc-mediated antibody-dependent cellular cytotoxicity (ADCC), and this activity was maintained after conjugation with GRM payloads. Notably, ABBV-319 displayed superior efficacy compared to afucosylated CD19 mAb in human CD34+ PBMC-engrafted NSG-tg(Hu-IL15) transgenic mice, demonstrating enhanced anti-tumor activity when multiple MOAs are enabled. ABBV-319 also showed durable anti-tumor activity across multiple B-cell lymphoma PDX models, including non-germinal center B-cell (GCB) DLBCL and relapsed lymphoma post R-CHOP treatment. Collectively, these data support the ongoing evaluation of ABBV-319 in Phase I clinical trial (NCT05512390).

Table S1: Primers for Methylation Analysis. Figure S1: Sensitivity of erythroid and myeloid progenitors to veliparib. Figure S2: Reproducibility of colony forming assays. Figure S3: Veliparib IC50 as a function of BRCA1 Promotor Methylation.

Supplementary Figure and Table Legends 1-2 from Progressive Chromatin Repression and Promoter Methylation of CTNNA1 Associated with Advanced Myeloid Malignancies

Supplementary Table 1 from Alterations of the HBP1 Transcriptional Repressor Are Associated with Invasive Breast Cancer

Supplementary Figure 1 from Progressive Chromatin Repression and Promoter Methylation of CTNNA1 Associated with Advanced Myeloid Malignancies

PDF file - 646K, Supp Fig 1: Pathways involved by hypermethylated genes in PV / ET. Supp Fig 2: Validation of HELP assay findings by MassArray bisulfite analysis Supp Fig 3: Pathways involved by genes that are overexpressed and hypomethylated at promoters by the Jak2V617F mutant. Supp Fig 4: Jak2V617F mutation does not have significant effects on global DNA methylation profiles

Figure S1 shows the percentage of γH2AX- and RAD51- foci positive cells with and without radiation.

PDF file - 846K, Supplementary Table 1: Genes hypermethylated in PV / ET Supplementary Table 2: Genes hypermethylated in PMF Supplementary Table 3: Genes hypomethylated in PMF Supplementary Table 4: Genes hypermethylated in PMF cases with ASXL1 mutations / deletions Supp Table 5: Biological pathways affected by hypermethylated genes in ASXL1 mutated/deleted cases of PMF Supplementary Table 6: Genes hypermethylated in MPN cases with TET2 mutations Supp Table 7: Biological pathways affected by hypermethylated genes in TET2 mutated cases of MPNs

Table S1. Changes in Proportion (%) of γH2AX-Positive Cells in Peripheral Blood upon Veliparib and Veliparib/Temozolomide Treatment Figure S1. FANCD2 monoubiquitination following ex vivo melphalan treatment of patient bone marrow cells Figure S2.Pretreatment levels of PARP1 and MGMT Figure S3.Pretreatment MGMT promoter methylation in patient peripheral blood (PB) or bone marrow (BM) AML cells Figure S4. PARP inhibition in peripheral blood cells

Supplementary Figure Legends 1-3, Methods, Tables 1-4 from Alterations of the HBP1 Transcriptional Repressor Are Associated with Invasive Breast Cancer

Supplementary Figure A from 250K Single Nucleotide Polymorphism Array Karyotyping Identifies Acquired Uniparental Disomy and Homozygous Mutations, Including Novel Missense Substitutions of c-Cbl, in Myeloid Malignancies

Background: CAR T cell therapy has become an important therapeutic tool for the treatment of B-cell neoplasms. However, the development of CAR T cell therapy outside of hematologic malignancies has stalled. As such, there is an unmet need to understand why CAR T cell therapy fails, which would be aided through the creation of tools to track them in vivo. Additionally, endowing CAR T cells with potent orthogonal anti-tumor activity has emerged as a strategy to improve upon CAR T cell efficacy. To accomplish both goals, we engineered a class of theranostic “THOR” CAR T cells that express a membrane-bound scFv, huC825, that binds DOTA-radiohaptens with pM affinity; these hapten chelands can be conjugated with radionuclides with therapeutic potential or diagnostic imaging capabilities. Methods: We engineered 19BBZ anti-CD19 CAR T cells to express huC825 (THOR CAR T cells) and confirmed transduction of these cells with flow cytometry. To assess the cytolytic ability and effector function of THOR CAR T cells, we used the Matador co-culture assay and measured the cytokine secretion of these co-cultures. We tested the potential to track THOR CAR T cells in vivo using PET/CT following intravenous DOTA-based radiohapten tracer administration in immunodeficient (NSG) mice bearing subcutaneous CD19+ Raji tumors. We also tested the ability of THOR CAR T cells to synergize with therapeutic radionuclide therapy. We evaluated the anti-tumor efficacy using whole animal bioluminescence imaging, tumor size measurements, and median animal survival. Results: We successfully transduced THOR CAR T cells, which demonstrated similar in vitro anti-tumor effects and cytokine secretion as parent 19BBZ CAR T cells. In vivo tracking experiments with 86Y-DOTA-Bn radiotracer showed that THOR CAR T cells can be observed at day 7 post T cell administration, peak at day 14, and persist until at least day 28. 86Y-DOTA-Bn uptake was specific to THOR CAR T cells, as minimal uptake was observed in mice transplanted with 19BBZ CAR T cells. Importantly, we noted rapid renal clearance of unbound tracer resulting in high contrast images. In efficacy assays in vivo, we demonstrated the synergy between 225Ac-Proteus-DOTA and THOR CAR T cells, as shown by a significant decrease in tumor burden and increase in median survival (THOR CAR treated = 26 days, treated with THOR CAR plus 225Ac-Proteus-DOTA = 32 days, p = .04, n = 6 per group). In contrast, mice receiving 19BBZ CAR T cells with or without 225Ac-Proteus-DOTA showed no improvements in antitumor efficacy. Conclusions: We show the pharmacokinetics of THOR CAR T cells can be probed in vivo following systemic administration of radiotracer using serial PET/CT imaging. Furthermore, we demonstrate a CAR T cell can be potentiated by systemically administered cytotoxic radionuclide that are then delivered selectively to tumors by the THOR cells. Conflict of interest: Ownership: MSK has filed for patent protection on behalf of M.M.D, S.M.C., D.R.V., B.H.S., N.K.C., D.A.S., S.M.L. and S.K. for inventions related to this work. Advisory Board: N.K.C. is an advisor to, or owns equity in Abpro, Alexs Lemonade Stand Foundation, Biotec Pharmacon, Eureka Therapeutics, Keystone Symposia, Partner Therapeutics, St. Jude-VIVA Forum, and Y-mAbs Therapeutics that may work in areas related to this study. Other Substantive Relationships: S.M.C. has licensed IP to Y-mAbs.

Background Systemic glucocorticosteroids (ie, glucocorticoid receptor modulators [GRMs]) show robust monotherapy activity against B-cell malignancies at high doses; however, they may be accompanied by steroid-associated toxicities that limit dosing. CD19 is a marker essential for B-cell proliferation and has high expression across B-cell malignancies including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and chronic lymphocytic leukemia (CLL). ABBV-319 is a CD19-GRM antibody-drug conjugate (ADC) composed of an optimized high-affinity immunoglobulin G1 antibody conjugated to a potent proprietary glucocorticosteroid payload. ABBV-319 takes advantage of both enhanced antibody-dependent cellular cytotoxicity (via afucosylation) and targeted GRM payload delivery to maximize anticancer activity while lowering the risk of systemic steroid toxicities. Preclinically, ABBV-319 demonstrates sustained antitumor efficacy in models of human B-cell malignancies that compares favorably with approved therapeutics. This phase 1 study evaluates safety and clinical activity of ABBV-319 monotherapy in patients with relapsed or refractory (R/R) B-cell malignancies. Methods This is a phase 1, first-in-human, open-label, dose-escalation, dose-expansion, biomarker/pharmacodynamic (PD) study in patients aged ≥18 years with R/R B-cell malignancies. Key eligibility criteria include measurable disease and Eastern Cooperative Oncology Group performance status of 0 or 1. In addition, patients must meet predefined criteria for adrenal, bone marrow, kidney, and liver function, coagulation parameter levels, and hemoglobin A1c levels (dose escalation only) during screening. The primary objectives are to evaluate safety, tolerability, pharmacokinetics (PK), and immunogenicity of ABBV-319 and identify a recommended phase 2 dose (RP2D). The secondary objective is to evaluate the efficacy of ABBV-319 in patients with R/R DLBCL, FL, and CLL. Exploratory objectives include evaluating the effect of ABBV-319 on QT prolongation and evaluating PD and predictive biomarkers. This study will be conducted in 2 parts - dose escalation (Part 1) and dose expansion (Part 2). Part 1 aims to determine the RP2D for ABBV-319 following a Bayesian optimal interval design, with a maximum of 2-fold increments that reduce as the dose increases. RP2D will be determined on the basis of all the data collected in Part 1 including safety, tolerability, PK, PD, and efficacy, if available. Part 2 will further evaluate ABBV-319 at the RP2D in 3 separate subtypes of B-cell malignancies - DLBCL, FL, and CLL. A total of 114 patients are planned to be enrolled - 54 patients with various R/R B-cell malignancies in Part 1, and 60 patients in Part 2 (20 patients included for each R/R B-cell malignancy subtype: DLBCL, FL, and CLL). ABBV-319 will be administered intravenously until disease progression, intolerable toxicity, or other study discontinuation criteria are met, for a maximum of ~24 months from the last patient enrolled. Safety assessments include adverse event monitoring (per National Cancer Institute Common Terminology Criteria for Adverse Events v5.0), physical examinations, vital sign measurements, and clinical laboratory testing. Dose-limiting toxicities will be assessed. PK parameters including maximum observed serum/plasma concentration (Cmax), time to Cmax, terminal plasma elimination half-life, and area under the serum/plasma concentration-time curve will be analyzed using noncompartmental methods for ABBV-319 total antibody, ADC, and unconjugated GRM payload. Antidrug antibodies (ADAs) and neutralizing ADAs may also be determined, as appropriate. Efficacy will be evaluated in terms of response per disease-specific criteria (including International Workshop on Chronic Lymphocytic Leukemia, International Workshop on Waldenstrom's Macroglobulinemia, and Lugano classification). Duration of response, time to response, progression-free survival, and overall survival will be evaluated per Kaplan-Meier analysis. QT prolongation, PD, and biomarker data will be assessed as changes from baseline and may be summarized for each scheduled postbaseline visit.

Background: The 5-year survival rate for metastatic pancreatic cancer is ~3%, indicating an urgent need for novel therapies. Combination therapy with modified FOLFIRINOX (leucovorin, irinotecan, 5-fluorouracil, and oxaliplatin) and immunotherapy has been proposed for first-line metastatic pancreatic cancer to improve tolerability and clinical efficacy, respectively (NCCN, Pancreatic. 2021; Vonderheide, Annu. Rev. Med. 2020). The present study evaluates the safety, pharmacokinetics, and preliminary antitumor activity of modified FOLFIRINOX + giloralimab (CD40 agonist) with or without budigalimab (anti-PD-1) in patients with untreated metastatic pancreatic cancer. Methods: Multicenter, randomized phase 1b/2 study (NCT04807972) in patients (18-75 years) with untreated metastatic pancreatic cancer. The phase 1b (dose escalation) examines the safety dose level of giloralimab in a triplet of modified FOLFIRINOX + giloralimab + budigalimab using a Bayesian optimal interval [BOIN] design. BOIN design is utilized to guide giloralimab escalation decisions. In phase 2 (dose expansion), patients are randomized 1:1:1 to receive treatment with modified FOLFIRINOX (cohort A), modified FOLFIRINOX + giloralimab (cohort B), or modified FOLFIRINOX + giloralimab + budigalimab (cohort C). Randomization is stratified according to Eastern Cooperative Oncology Group performance status. Primary objectives are to assess the safety and tolerability of modified FOLFIRINOX + giloralimab + budigalimab (phase 1b) and to evaluate overall survival in patients treated with modified FOLFIRINOX + giloralimab with or without budigalimab (versus those receiving modified FOLFIRINOX alone; phase 2). Secondary objectives include characterizing the pharmacokinetics of giloralimab and budigalimab in combination with modified FOLFIRINOX, assessing the efficacy of modified FOLFIRINOX + giloralimab with or without budigalimab, and evaluating the safety/tolerability of modified FOLFIRINOX + giloralimab with or without budigalimab. Patients will receive giloralimab and budigalimab intravenously in combination with modified FOLFIRINOX in a 28-day cycle. Dose-limiting toxicities are assessed during the first cycle of dosing. Adverse events are evaluated according to the National Cancer Institute Common Terminology Criteria for Adverse Events. Blood samples for pharmacokinetic analysis are collected at designated time points throughout the study. Responses are assessed by Response Evaluation Criteria in Solid Tumors version 1.1. Survival outcomes are described using the Kaplan-Meier method. Approximately 129 patients are planned to be included. Enrollment started in June 2021, with 7 patients enrolled as of November 2021. Citation Format: Dung T. Le, Marcia Cruz-Correa, David L. Bajor, Rocio Garcia-Carbonero, Marion Harris, Roberto Pazo-Cid, Hedy Kindler, Nelson Yee, Suneel Kamath, Maulik Patel, Hua Fang, William Henner, Patrick Hardesty, Martha Blaney, Michael McDevitt, Talia Golan. Phase 1b/2 study of giloralimab in combination with modified FOLFIRINOX with or without budigalimab in patients with untreated metastatic pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT246.

Background CD40 agonist immunotherapy can potentially license antigen-presenting cells to promote antitumor T-cell activation and re-educate macrophages to destroy tumor stroma. Systemic administration of CD40 agonists has historically been associated with considerable toxicity, providing the rationale for development of tumor-targeted immunomodulators to improve clinical safety and efficacy. This phase I study assessed the safety, tolerability, preliminary antitumor activity, and preliminary biomarkers of ABBV-428, a first-in-class, mesothelin-targeted, bispecific antibody designed for tumor microenvironment-dependent CD40 activation with limited systemic toxicity. Methods ABBV-428 was administered intravenously every 2 weeks to patients with advanced solid tumors. An accelerated titration (starting at a 0.01 mg/kg dose) and a 3+3 dose escalation scheme were used, followed by recommended phase II dose cohort expansions in ovarian cancer and mesothelioma, tumor types associated with high mesothelin expression. Results Fifty-nine patients were treated at doses between 0.01 and 3.6 mg/kg. The maximum tolerated dose was not reached, and 3.6 mg/kg was selected as the recommended phase II dose. Seven patients (12%) reported infusion-related reactions. Treatment-related grade ≥3 treatment-emergent adverse events were pericardial effusion, colitis, infusion-related reaction, and pleural effusion (n=1 each, 2%), with no cytokine release syndrome reported. The pharmacokinetic profile demonstrated roughly dose-proportional increases in exposure from 0.4 to 3.6 mg/kg. Best response was stable disease in 9/25 patients (36%) treated at the recommended phase II dose. CD40 receptor occupancy >90% was observed on peripheral B-cells starting from 0.8 mg/kg; however, no consistent changes from baseline in intratumoral CD8+ T-cells, programmed death ligand-1 (PD-L1+) cells, or immune-related gene expression were detected post-ABBV-428 treatment (cycle 2, day 1). Mesothelin membrane staining showed greater correlation with progression-free survival in ovarian cancer and mesothelioma than in the broader dose escalation population. Conclusions ABBV-428 monotherapy exhibited dose-proportional pharmacokinetics and an acceptable safety profile, particularly for toxicities characteristic of CD40 agonism, illustrating that utilization of a tumor-targeted, bispecific antibody can improve the safety of CD40 agonism as a therapeutic approach. ABBV-428 monotherapy had minimal clinical activity in dose escalation and in a small expansion cohort of patients with advanced mesothelioma or ovarian cancer. Trial registration number NCT02955251.

Abstract Background: ABBV-428 is a first-in-class bispecific antibody against CD40 and MSLN designed to stimulate CD40 for tumor-specific immune activation with limited systemic toxicity (Cancer Immunol Res 2019;7:1864). In phase I findings, ABBV-428 was well tolerated up to 3.6mg/kg; 3/50 patients had long-term stable disease (SD; Luke et al. ESMO 2019). Here, pharmacodynamic and potential predictive biomarkers were assessed in the blood and tumor tissues in this phase I open-label, multicenter study (NCT02955251). Methods: Patients ≥18 y had advanced solid tumors and progression/intolerance to standard therapies. ABBV-428 (≤3.6 mg/kg) was administered biweekly. CD40 receptor occupancy, absolute B cell counts, immune costimulatory activation markers, and serum cytokines were assessed at baseline and after treatment. MSLN expression (H-score) by IHC was reported at baseline. CD40/MSLN colocalization was assessed with a fluorescent multiplex assay. Paired tumor biopsy samples were collected at baseline and after treatment (C1D1, C2D1) to measure CD8+ T cells and PD-L1+ cells (IHC) and gene expression (NanoString Pancancer immune profiling panel). Immunologic constant of rejection (ICR) gene signature scores (J Immunother Cancer 2018;6:50) and IFNγ signatures (J Clin Invest 2017;127:2930) were used to characterize the tumor immune inflammatory microenvironment. BAP1 gene was sequenced (Sanger) in mesothelioma samples in 3 patients with long-term SD. Results: In peripheral blood, there was >90% CD40 receptor occupancy starting at 0.8 mg/kg. There was a transient decrease in circulating B cells 2-24 hrs after administration of ABBV-428, followed by an increase in B cell costimulatory activation markers (CD80, CD86, CD69) across dose groups with no evidence of cytokine response (IFNγ, IL-10, IL-12p70, IL-6, IL-8, TNFα, IL-1β, IL-2). There was no consistent change in tumor-infiltrating CD8+ T cells or PD-L1+ cells after ABBV-428 treatment in paired tumor biopsy samples from 13 patients. Patients with SD had higher ICR scores and a higher IFNγ gene signature at baseline than those with progressive disease. There was no correlation between baseline MSLN expression level and clinical response. Wide intertumor variability of MSLN/CD40 colocalization was observed from 4 tumor biopsy samples. No well-established recurrent mutations were seen in BAP1, which are correlated with better prognosis in mesothelioma patients. Conclusions: These data show that ABBV-428 binds and activates CD40 in the blood leading to B cell activation with no evidence of systemic cytokine production or significant change in the tumor immune microenvironment. Evidence of inflamed immune tumor microenvironment was observed at baseline from long-term SD patients. These results may explain the clinical observations from the current ABBV-428 clinical trial. Citation Format: Hua Fang, Shiming Ye, Anita Reddy, Tolga Turan, Andrew Woolley, Debra T. Chao, Cyril Ramathal, William R. Henner, Kinjal Hew, Michael McDevitt, Joel Hayflick, Frances Fan, Kathryn Allaire, Lawrence Fong. Pharmacodynamics and potential predictive biomarkers of ABBV-428, a first-in-class mesothelin (MSLN)-CD40 bispecific, 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 5672.

In a phase-2 study, the telomerase inhibitor imetelstat induced rapid hematologic responses in all patients with essential thrombocythemia who were refractory or intolerant to prior therapies. Significant molecular responses were achieved within 3-6 months in 81% of patients with phenotypic driver mutations in JAK2, CALR and MPL. Here, we investigated the dynamics of additional somatic mutations in response to imetelstat. At study entry, 50% of patients carried 1-5 additional mutations in the genes ASXL1, CBL, DNMT3A, EZH2, IDH1, SF3B1, TET2, TP53 and U2AF1. Three patients with baseline mutations also had late-emerging mutations in TP53, IDH1 and TET2. Most clones with additional mutations were responsive to imetelstat and decreased with the driver mutation, including the poor prognostic ASXL1, EZH2 and U2AF1 mutations while SF3B1 and TP53 mutations were associated with poorer molecular response. Overall, phenotypic driver mutation response was significantly deeper in patients without additional mutations (P = 0.04) and correlated with longer duration of response. In conclusion, this detailed molecular analysis of highly pretreated and partly resistant patients with essential thrombocythemia reveals a high individual patient complexity. Moreover, imetelstat demonstrates potential to inhibit efficiently co-incident mutations occurring in neoplastic clones in patients with essential thrombocythemia. (ClinicalTrials.gov number, NCT01243073. N Engl J Med 2015; 373:920-928, DOI: 10.1056/NEJMoa1503479.)

Stimulation of the immune system to generate antitumor response represents a compelling therapeutic strategy, especially in settings where only some patients (pts) achieve response. ABBV-368 is a novel, immunoglobulin G1 anti-OX40 monoclonal antibody agonist that elicits tumor responses by stimulating effector T cells and inhibiting regulatory T cells (Le Tourneau et al. SITC. 2019;432). Tilsotolimod is a synthetic TLR9 agonist with immunostimulatory activity (Babiker et al. AACR. 2020;134). Budigalimab (ABBV-181) is a humanized, recombinant immunoglobulin G1 monoclonal antibody targeting PD-1 modified to reduce Fc receptor interactions and limit effector function. This phase Ib, multicenter, open-label study (NCT04196283) is designed to evaluate the safety, tolerability, pharmacokinetics, and preliminary efficacy of ABBV-368 in combination with tilsotolimod and other agents (± nab-paclitaxel and budigalimab) in pts with recurrent/metastatic (R/M) head and neck squamous cell carcinoma (SCC). Pts ≥18 years with histologically confirmed SCC (of the oral cavity, oropharynx, larynx, or hypopharynx) with ≥1 lesion accessible for intratumoral (IT) injection, are eligible. Pts must have an ECOG performance status of 0 or 1, a life expectancy of ≥3 months, received 1 prior immunotherapy regimen (including a PD-(L)1 inhibitor), and have ≤3 prior treatments in the R/M setting. Pts with uncontrolled CNS metastases and those who have received prior OX40 or TLR agonist treatment (excluding topical agents) are ineligible. Pts in Arm 1 will receive intravenous (IV) ABBV-368 plus tilsotolimod (IT); pts in Arm 2, ABBV-368 + tilsotolimod + IV nab-paclitaxel; and pts in Arm 3, ABBV-368 + tilsotolimod + nab-paclitaxel and IV budigalimab. Primary objectives are to assess safety, tolerability, and pharmacokinetics of these combinations. Secondary objectives are to assess objective response rate, clinical benefit rate, time to response, progression-free survival, and duration of response. Paired biopsy samples will be taken for exploratory analysis. Planned enrollment is 69 pts. NCT04196283. Medical writing support was provided by Devon Roll, PhD, of Bio Connections, LLC. AbbVie, Inc. AbbVie, Inc. funded this study.