Chanan‐Khan, Asher

Abstract Purpose: This global phase I trial investigated the safety, efficacy, pharmacokinetics, and pharmacodynamics of lisaftoclax (APG-2575), a novel, orally active, potent selective B-cell lymphoma 2 (BCL-2) inhibitor, in patients with relapsed or refractory chronic lymphocytic leukemia or small lymphocytic lymphoma (R/R CLL/SLL) and other hematologic malignancies (HMs). Patients and Methods: Maximum tolerated dose (MTD) and recommended phase II dose were evaluated. Outcome measures were safety and tolerability (primary) and pharmacokinetic variables and antitumor effects (secondary). Pharmacodynamics in patient tumor cells were explored. Results: Among 52 patients receiving lisaftoclax, MTD was not reached. Treatment-emergent adverse events (TEAEs) included diarrhea (48.1%), fatigue (34.6%), nausea (30.8%), anemia and thrombocytopenia (28.8% each), neutropenia (26.9%), constipation (25.0%), vomiting (23.1%), headache (21.2%), peripheral edema and hypokalemia (17.3% each), and arthralgia (15.4%). Grade ≥ 3 hematologic TEAEs included neutropenia (21.2%), thrombocytopenia (13.5%), and anemia (9.6%), none resulting in treatment discontinuation. Clinical pharmacokinetic and pharmacodynamic results demonstrated that lisaftoclax had a limited plasma residence and systemic exposure and elicited rapid clearance of malignant cells. With a median treatment of 15 (range, 6–43) cycles, 14 of 22 efficacy-evaluable patients with R/R CLL/SLL experienced partial responses, for an objective response rate of 63.6% and median time to response of 2 (range, 2–8) cycles. Conclusions: Lisaftoclax was well tolerated, with no evidence of tumor lysis syndrome. Dose-limiting toxicity was not reached at the highest dose level. Lisaftoclax has a unique pharmacokinetic profile compatible with a potentially more convenient daily (vs. weekly) dose ramp-up schedule and induced rapid clinical responses in patients with CLL/SLL, warranting continued clinical investigation.

Daratumumab is an anti-CD38 mAb, used frequently in combination with lenalidomide and pomalidomide. No studies compared daratumumab plus lenalidomide and dexamethasone (DRd) to daratumumab plus pomalidomide and dexamethasone (DPd) in lenalidomide-exposed multiple myeloma. We identified 504 consecutive multiple myeloma patients who received daratumumab at Mayo Clinic between January 2015 and April 2019. We excluded patients who received daratumumab in the first line, received more than four lines of therapy prior to daratumumab use, did not receive lenalidomide prior to daratumumab, or had an unknown status of lenalidomide exposure, and patients who received daratumumab combinations other than DRd or DPd. We examined the impact of using DRd compared to DPd on progression-free survival (PFS) and overall survival (OS) in patients with relapsed/refractory multiple myeloma. Out of 504 patients, 162 received DRd or DPd and were included; 67 were lenalidomide-exposed and 95 were lenalidomide-refractory. DRd was used in 76 (47%) and DPd in 86 (53%) patients. In lenalidomide-exposed multiple myeloma, there was no difference in median PFS; 34.2 months [95% confidence interval (CI), 22.8–44.6] for DRd compared to 25.2 months (95% CI, 4.9–35.3) for DPd, P = 0.2. In lenalidomide-refractory multiple myeloma, there was no difference in median PFS; 18.6 months (95% CI, 13–32) for DRd compared to 9 months (95% CI, 5.2–14.6) for DPd, P = 0.09. No difference in median OS was observed in DRd compared to DPd. Our study shows combining daratumumab with lenalidomide in patients with prior lenalidomide use is a viable and effective treatment option.

Bcl-2 and Mcl-1 proteins play a role in multiple myeloma (MM) cell survival, for which targeted inhibitors are being developed. AT-101 is an oral drug, which disrupts Bcl-2 and Mcl-1 function, impedes mitochondrial bioenergetic processes and induces apoptosis in MM cells. When combined with lenalidomide and dexamethasone (Rd), AT-101 significantly reduced tumor burden in an in vivo xenograft model of MM. These data provided rationale for a phase I/II study to establish the effective dose of AT-101 in combination with Rd (ARd regimen) in relapsed/refractory MM. A total of 10 patients were enrolled, most with high-risk cytogenetics (80%) and prior stem cell transplant (70%). Three patients were lenalidomide-refractory, 2 were bortezomib-refractory and 3 were daratumumab-refractory. The ARd combination was well tolerated with most common grade 3/4 adverse events being cytopenia’s. The overall response rate was 40% and clinical benefit rate was 90%. The median progression free survival was 14.9 months (95% CI 7.1-NE). Patients responsive to ARd showed a decrease in Bcl-2:Bim or Mcl-1:Noxa protein complexes, increased CD8+ T and NK cells and depletion of T and B-regulatory cells. The ARd regimen demonstrated an acceptable safety profile and promising efficacy in patients with relapsed/refractory MM prompting further investigation in additional patients.

Abstract Glioblastoma (GBM) is an aggressive primary malignant brain tumor with poor prognosis. Surgical resection followed by chemoradiotherapy (temozolomide [TMZ] + radiation) results in a median survival of only 12-15 months. Recently, immunotherapy with targeted antibodies, therapeutic vaccination and adoptive cell therapy offers a promising option for treating GBM. Herein we observed that CD38, a catalytic receptor and regulator of NAD homeostasis, is expressed on GBM cells and their microenvironment in primary patient-derived GBM tissues. Blacher et al have shown that blocking CD38 enzymatic functions results in reduced GBM cell growth. This evidence prompted us to investigate the antitumor and immune engaging effects of a clinical grade anti CD38 therapeutic monoclonal antibody (Daratumumab/DARA) in preclinical models of GBM. We demonstrated that DARA significantly enhanced the direct apoptotic effect of TMZ and induced ADCC/CDC of GBM cells, irrespective of the presence of microglia. In an in vitro 3D simulated GBM + T cell coculture system, DARA induced activation (CD69/GITR upregulation/decreased PD1) and enhanced proliferation of CD8+ T cells/cTL activity against GBM cells. DARA also resulted in increased IFNγ and decreased IL-10; suggesting an improved antitumor cytokine milieu and ensued in downregulated inflammatory cytokine associated JAK2/STAT3 signaling pathway. Finally, we tested TMZ+DARA in a human GBM xenograft in vivo model and noted significantly prolonged survival of mice [TMZ+DARA vs. vehicle: 98.13±1.88 vs. 63.63±3.77 days, p<0.001]. Altogether, these observations led us to investigate the clinical activity of DARA+ TMZ in GBM patients in a phase I/II clinical trial.

Abstract Chronic Lymphocytic Leukemia (CLL) is considered a disease of antigen-naive CD5+CD23+CD19+κ/λ+ B-lymphocytes, in which the tumor microenvironment is highly immunosuppressive. Among several human B cell subsets, B10 [CD24hiCD27+CD38hi]/B regulatory cells (Bregs) are functionally discernable in patients with aggressive CLL as they support and regulate immunological tolerance via production of IL-10 and transforming growth factor β (TGF-β). In an ex vivo setting, we noted CD38hi CLL Bregs to promote transformation and expansion of CD4+CD25+FoxP3+Tregs; an effect that was abrogated in the presence of IL10/TGF-β neutralizing antibodies. In patients with CLL, Bregs also prohibit the expansion of cytotoxic T cells (CD8+T cells; Tc), natural killer (NK) cells and other pro-inflammatory lymphocytes (CD4+ helper T cells; Th1 and Th17). We noted that patients with CLL had a significantly higher % of Tregs (11.8±2.0) compared to healthy donors (1.9±0.3) and similar to Bregs, a substantial proportion of CLL Tregs had high CD38 expression (MFI=616.8±36.2). We demonstrated that an anti-CD38 therapeutic antibody was able to eliminate CD38+ Bregs and Tregs from CLL patients by immune effector mechanisms (ADCC, CDC, ADCP) as well as direct mitochondrial/FcγR-mediated apoptosis. Furthermore, in a PDX model of CLL, mice treated with anti-CD38 therapy had decreased % Bregs and Tregs but increased Th17 and CD8+ T-cells (vs. vehicle; p<0.05). These observations carry implications beyond CLL for any Breg/Treg-dependent cancer or disease (i.e. Systemic Lupus Erythematosus, Rheumatoid arthritis, Sjögren’s syndrome), wherein CD38-targeted therapy could potentially rescue/improve an immune tolerant microenvironment by eradication of Bregs.

Abstract Hodgkin’s lymphoma (HL) is characterized by malignant CD30+ Reed-Sternberg (HRS) B-cells surrounded by an ineffective infiltrate of inflammatory immune cells. T-helper and T-regulatory cells presumably provide survival signals for HRS cells; rescuing them from immune-surveillance by CD8+ cytotoxic T lymphocytes and natural killer (NK) cells, which are also functionally anergic. Clinically, ≈10–30% of HL patients resulted in relapse after front line therapy; highlighting the need for development of new treatments. We investigated a novel bispecific CD16axCD30 antibody, AFM13 (Affimed Therapeutics, AG), which engages NK cells and recruits them towards CD30+ tumor cells. Safety of single-agent AFM13 has been demonstrated in a phase I clinical study, however, disease remission was observed in only 23% of patients. We hypothesized that combining AFM13 with another agent whose safety profile is already been established in HL and also activates NK cells (lenalidomide) could potentially increase anti-tumor efficacy of AFM13. We observed increased expression of CD107a, CD69, CD25 and IFNγ (activation markers) in NK cells isolated from HL patients (HL-NK), that were stimulated ex vivo with IL- 15 + lenalidomide. Pre-treatment with IL-2/IL-15 antibody has been shown to activate and restore T/NK cell cytolytic function. In line with this, IL-15+lenalidomide-stimulated HL-NK cells showed functional restoration by inducing specific lysis of human CD30+ lymphoma cells (30.55±1.49%) in a co-culture assay. While, AFM13 is being investigated in combination with anti-PD1 antibody therapy (NCT02665650), our preclinical data provides the rationale for an AFM13 + Len combination regimen for HL and potentially other CD30+ malignancies.

Abstract Increased CD38 expression on chronic lymphocytic leukemia (CLL) cells correlates with a negative prognosis, however, its utility as a therapeutic target in this malignancy remains unknown. Our investigations show that the anti-CD38 therapeutic mAb, daratumumab (Dara) induces direct apoptosis of CLL cells and promotes their immune-mediated clearance (through ADCC). Notably, CD38 expression is quite high on a subset of CLL-cells (CD19+/CD38hi/CD24+/IL-10+; B-regulatory-like phenotype), which promote Treg expansion while prohibiting CD8+ cytotoxic T-cells (cTLs). Indeed, immunosuppression is a hallmark of CLL and patients often present with increased number of Tregs and Th2 cells but decreased effector T cells and cTLs. It has recently been reported that CD38hi Tregs are significantly immunosuppressive and when examining this population in peripheral blood mononuclear cells (PBMCs) from CLL patients (n=13), we noted that >50% of CD4+/CD25+/FoxP3+ cells were CD38hi (median MFI ~600). We hypothesized that Dara would kill CD38hi CLL cells à decreased CD38hi Treg formation/direct killing à expansion of cTLs. Indeed, ex-vivo treatment of CLL-patient derived PBMCs showed that Dara was highly lethal to both CD38hi "Breg-like" CLL cells and Tregs; associated with decrease in IL-10, increase in IFN-γ, effector T cells, Th17 subsets and proliferation of cTLs. Altogether this pointed towards recovery of anti-tumor immunity which was further evidenced by enhanced tumor-specific cytolytic activity of cTLs. Our pre-clinical data demonstrates that targeting CD38 in CLL has an anti-tumor effect via 1.) lethal effect on CLL-Bregs and Tregs and 2.) tumor-specific expansion of cTLs, which we will test clinically in an upcoming trial.

Abstract We have previously shown (AAI2009) that dendritic cells (DC) contribute to the survival of long lived plasma cells in the bone marrow, which are important to maintain persistent antibody titres. What is not clear are the molecular interactions that are involved. Both normal and malignant plasma cells(like myeloma cells) express the T-cell co-stimulatory molecule CD28, and both interact with DCs in the bone marrow microenvironment. CD28 activation on MM triggers similar molecular partners downstream as observed in T-cells. Gene expression analysis of normal and malignant plasma cells shows that CD28 expression correlates with poor prognostic subgroups that are associated with specific genetic lesions. In our data, while activation of CD28 with antibodies or DC-CD80/CD86 (B7) protects myeloma cells against cell death, the ligation of B7 on DCs induces DC production of plasma cell survival factor IL-6, and immunosuppressive factor indoleamine 2,3-dioxygenase (IDO). Use of Lenalidomide (a common myeloma drug) to inhibit DC-B7 expression or anti-CD28(Fab)2 fragments to block CD28-B7 interactions reduced DC ability to protect MM cells. Experiments with αCD28(Fab)2 or inhibitors of notch pathway suggest a cross talk that regulates IL-6 production. IDO activity in DC-MM cocultures, suppressed T-cell proliferation in MLRs while an IDO inhibitor alleviated this. Our data suggests a role for CD28-B7 in plasma cell survival by modulating the bone marrow microenvironment.

Abstract Purpose: IgM monoclonal gammopathy of undetermined significance (MGUS) and Waldenström macroglobulinemia (WM) represent a disease spectrum with highly varied therapeutic management, ranging from observation to chemoimmunotherapy. The current classification relies solely on clinical features and does not explain the heterogeneity that exists within each of these conditions. Further investigation is warranted to shed light on the biology that may account for the clinical differences. Experimental Design: We used bone marrow (BM) clonal CD19+ and/or CD138+ sorted cells, matched BM supernatant, and peripheral blood serum from 32 patients (7 MGUS, 25 WM) to perform the first multi-omics approach including whole-exome sequencing, RNA sequencing, proteomics, metabolomics, and mass cytometry. Results: We identified three clusters with distinct pathway activation, immune content, metabolomic, and clinical features. Cluster 1 included only patients with WM and was characterized by transcriptional silencing of genes involved in cell cycle and immune response, enrichment of mitochondrial metabolism, infiltration of senescent T effector memory cells, and aggressive clinical behavior. Genetic/structural alterations of TNFAIP3 were distinct events of this cluster. Cluster 2 comprised both MGUS and WM patients with upregulation of inflammatory response, senescence and glycolysis signatures, increased activated T follicular helper and T regulatory cells, and indolent clinical behavior. Cluster 3 also included both MGUS and WM patients and exhibited intermediate features, including proliferative and inflammatory signaling, as well as glycolysis and mitochondrial metabolism. Conclusions: We have identified three distinct molecular clusters, suggesting a potential biologic classification that may have therapeutic implications.