Manna, Alak

The immunosuppressive tumor microenvironment (TME) remains a major barrier to effective cancer immunotherapy. Among the central regulators of immune suppression, CD38, a multifunctional ectoenzyme and surface glycoprotein, has emerged as a pivotal orchestrator. CD38 is abundantly expressed on regulatory T cells (Tregs), regulatory B cells (Bregs), myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), and tumor-associated neutrophils (TANs), where it enhances survival, metabolic fitness, and suppressive activity. Invariant natural killer T (iNKT) cells, which can either promote or suppress antitumor immunity, also express CD38 upon activation, suggesting a role for CD38 in directing their context-dependent fate within the TME. Mechanistically, CD38 regulates immune suppression through NAD + hydrolysis, calcium signaling, and promotion of fatty acid oxidation (FAO) while impairing effector T-cell glycolysis and mitochondrial fitness under chronic hypoxia—conditions that favor exhaustion rather than enhanced cytotoxicity. By depleting extracellular NAD + , CD38 diminishes glycolysis and mitochondrial oxidative phosphorylation in effector T cells, while sustaining regulatory cell persistence through FAO. Its enzymatic products, cyclic ADP-ribose (cADPR) and NAADP, further mobilize calcium fluxes that reinforce suppressive function. CD38 also integrates with hypoxia-driven pathways; in CD38 + Bregs, stabilization of HIF-1α and induction of FAO-related genes such as CPT1A and PPARα/γ promote angiogenesis, immune-evasion, and therapeutic resistance. Therapeutically, targeting CD38 with monoclonal-antibodies, small-molecule inhibitors, or combinations with checkpoint blockade and macrophage-reprogramming agents has shown promise. Such interventions reverse immune suppression, restore effector T cell activity, and enhance tumor responsiveness to immunotherapy. In summary, CD38 functions as both a metabolic regulator and an immunologic checkpoint, coordinating suppressive networks and shaping iNKT cell fate. These multifaceted roles position CD38 as a transformative target for next-generation immunotherapies.

e19501 Background: Relapsed and/or refractory (R/R) disease is observed in patients who receive CD19 targeting chimeric antigen receptor (CAR)-T cells to treat B-cell lymphoid leukemias and lymphomas. We generated a novel CAR, MC10029 that targets B-cell activating factor receptor (BAFF-R) to address this unmet medical need. Methods: We engineered a second-generation BAFF-R CAR (MC10029 CAR). The construct includes the single chain variable fragment (scFv) of our newly developed BAFF-R antibody with CD28 costimulatory and CD3ζ signaling domains. We confirmed MC10029 CAR-T cell antigen-specific activation with in vitro degranulation assays and a direct cytolytic assay. We used BAFF-R positive leukemia and lymphoma cell lines in both in vitro and in vivo models to further characterize MC10029 CAR-T cells, and finally, we enriched tumor primary cells from chronic lymphocytic leukemia (CLL) patients as a target for MC10029 CAR-T cells. In preparation for our Phase I clinical trial, we have manufactured three batches of CAR-T cells using a MC10029-encoding lentiviral vector produced under GMP conditions. The production batches of MC10029 CAR-T cells were evaluated for product quality with Viability, Identity and Potency as well as Safety from adventitious viral agents. Antigen specific cytotoxicity of the MC10029 CAR-T cells was confirmed using our standard degranulation assay. Results: MC10029 CAR-T cells elicited in vitro antigen-specific cytotoxicity against Nalm-6 cells, Z138 cells, MEC-1 cells, and a CD19 knock out Nalm-6 model designed to mimic CD19-negative R/R disease. We demonstrated in vivo antitumor effects of MC10029 CAR-T cells in NSG mice challenged with these same cell lines. We showed cytotoxicity of MC10029 CAR-T cells against CLL patients’ tumors. We also showed our quality control results to highlight our clinical trial trajectory. Conclusions: Novel MC10029 CAR-T cells showed broad utility against BAFF-R positive B-cell lymphoid malignancies in a series of in vitro, in vivo, and ex vivo models. With these robust data, we have moved forward to generate clinical grade MC10029 CAR-T cells using cGMP manufacturing and quality protocols in preparation of a MC10029 CAR-T cell Phase I clinical trial.

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 Purpose: Development of B-cell lymphoma 2 (BCL-2)–specific inhibitors poses unique challenges in drug design because of BCL-2 homology domain 3 (BH3) shared homology between BCL-2 family members and the shallow surface of their protein–protein interactions. We report herein discovery and extensive preclinical investigation of lisaftoclax (APG-2575). Experimental Design: Computational modeling was used to design “lead” compounds. Biochemical binding, mitochondrial BH3 profiling, and cell-based viability or apoptosis assays were used to determine the selectivity and potency of BCL-2 inhibitor lisaftoclax. The antitumor effects of lisaftoclax were also evaluated in several xenograft models. Results: Lisaftoclax selectively binds BCL-2 (Ki < 0.1 nmol/L), disrupts BCL-2:BIM complexes, and compromises mitochondrial outer membrane potential, culminating in BAX/BAK-dependent, caspase-mediated apoptosis. Lisaftoclax exerted strong antitumor activity in hematologic cancer cell lines and tumor cells from patients with chronic lymphocytic leukemia, multiple myeloma, or Waldenström macroglobulinemia. After lisaftoclax treatment, prodeath proteins BCL-2‒like protein 11 (BIM) and Noxa increased, and BIM translocated from cytosol to mitochondria. Consistent with these apoptotic activities, lisaftoclax entered malignant cells rapidly, reached plateau in 2 hours, and significantly downregulated mitochondrial respiratory function and ATP production. Furthermore, lisaftoclax inhibited tumor growth in xenograft models, correlating with caspase activation, poly (ADP-ribose) polymerase 1 cleavage, and pharmacokinetics of the compound. Lisaftoclax combined with rituximab or bendamustine/rituximab enhanced antitumor activity in vivo. Conclusions: These findings demonstrate that lisaftoclax is a novel, orally bioavailable BH3 mimetic BCL-2–selective inhibitor with considerable potential for the treatment of certain hematologic malignancies.