BL-8040 CXCR4 antagonist is safe and demonstrates antileukemic activity in combination with cytarabine for the treatment of relapsed/refractory acute myelogenous leukemia: An open-label safety and efficacy phase 2a study.

BACKGROUND: CXCR4 mediates the retention and survival of acute myelogenous leukemia blasts in bone marrow and contributes to their resistance to chemotherapy. The authors evaluated a combination of the high-affinity CXCR4 antagonist BL-8040 with high-dose cytarabine (HiDAC) chemotherapy in a phase 2a study of patients with relapsed and refractory AML. METHODS: Forty-two patients received treatment with BL-8040 monotherapy for 2 days followed by a combination of BL-8040 with HiDAC for 5 days. Six escalating BL-8040 dose levels were investigated (0.5, 0.75, 1.0, 1.25, 1.5, and 2.0 mg/kg), and 1.5 mg/kg was selected as the dose for the expansion phase (n = 23). RESULTS: BL-8040 in combination with HiDAC was safe and well tolerated at all dose levels. Clinical response was observed with BL-8040 doses ≥1.0 mg/kg. The composite response rate (complete remissions plus complete remissions with incomplete hematologic recovery of platelets or neutrophils) was 29% (12 of 42) in all patients and 39% (9 of 23) in the 1.5-mg/kg phase. The median overall survival was 8.4 months for all patients, 10.8 months in the 1.5-mg/kg phase, and 21.8 months for responding patients in the 1.5-mg/kg cohort. Two days of BL-8040 monotherapy triggered the mobilization of blasts into peripheral blood, with significantly higher mean fold-changes in responders versus nonresponders. This was accompanied by a decrease in bone marrow blasts. CONCLUSIONS: The current results demonstrate the efficacy of CXCR4 targeting with BL-8040 and support continued clinical development in acute myelogenous leukemia.

BL-8040, a CXCR4 antagonist, in combination with pembrolizumab and chemotherapy for pancreatic cancer: the COMBAT trial.

Programmed cell death 1 (PD-1) inhibitors have limited effect in pancreatic ductal adenocarcinoma (PDAC), underscoring the need to co-target alternative pathways. CXC chemokine receptor 4 (CXCR4) blockade promotes T cell tumor infiltration and is synergistic with anti-PD-1 therapy in PDAC mouse models. We conducted a phase IIa, open-label, two-cohort study to assess the safety, efficacy and immunobiological effects of the CXCR4 antagonist BL-8040 (motixafortide) with pembrolizumab and chemotherapy in metastatic PDAC (NCT02826486). The primary outcome was objective response rate (ORR). Secondary outcomes were overall survival (OS), disease control rate (DCR) and safety. In cohort 1, 37 patients with chemotherapy-resistant disease received BL-8040 and pembrolizumab. The DCR was 34.5% in the evaluable population (modified intention to treat, mITT; N = 29), including nine patients (31%) with stable disease and one patient (3.4%) with partial response. Median OS (mOS) was 3.3 months in the ITT population. Notably, in patients receiving study drugs as second-line therapy, the mOS was 7.5 months. BL-8040 increased CD8+ effector T cell tumor infiltration, decreased myeloid-derived suppressor cells (MDSCs) and further decreased circulating regulatory T cells. In cohort 2, 22 patients received BL-8040 and pembrolizumab with chemotherapy, with an ORR, DCR and median duration of response of 32%, 77% and 7.8 months, respectively. These data suggest that combined CXCR4 and PD-1 blockade may expand the benefit of chemotherapy in PDAC and warrants confirmation in subsequent randomized trials.

Long-Term Efficacy of GMP Grade Xeno-Free hESC-Derived RPE Cells Following Transplantation.

PURPOSE: Retinal pigment epithelium (RPE) dysfunction underlies the retinal degenerative process in age-related macular degeneration (AMD), and thus RPE cell replacement provides an optimal treatment target. We characterized longitudinally the efficacy of RPE cells derived under xeno-free conditions from clinical and xeno-free grade human embryonic stem cells (OpRegen) following transplantation into the subretinal space of Royal College of Surgeons (RCS) rats. METHODS: Postnatal (P) day 20 to 25 RCS rats (n = 242) received a single subretinal injection of 25,000 (low)-, 100,000 (mid)-, or 200,000 (high)-dose xeno-free RPE cells. BSS+ (balanced salt solution) (vehicle) and unoperated eyes served as controls. Optomotor tracking (OKT) behavior was used to quantify functional efficacy. Histology and immunohistochemistry were used to evaluate photoreceptor rescue and transplanted cell survival at 60, 100, 150, and 200 days of age. RESULTS: OKT was rescued in a dose-dependent manner. Outer nuclear layer (ONL) was significantly thicker in cell-treated eyes than controls up to P150. Transplanted RPE cells were identified in both the subretinal space and integrated into the host RPE monolayer in animals of all age groups, and often contained internalized photoreceptor outer segments. No pathology was observed. CONCLUSIONS: OpRegen RPE cells survived, rescued visual function, preserved rod and cone photoreceptors long-term in the RCS rat. Thus, these data support the use of OpRegen RPE cells for the treatment of human RPE cell disorders including AMD. TRANSLATIONAL RELEVANCE: Our novel xeno-free RPE cells minimize concerns of animal derived contaminants while providing a promising prospective therapy to the diseased retina.

1-(sulfonyl)-5-(arylsulfonyl)indoline as activators of the tumor cell specific M2 isoform of pyruvate kinase.

Cancer cells preferentially use glycolysis rather than oxidative phosphorylation for their rapid growth. They consume large amount of glucose to produce lactate even when oxygen is abundant, a phenomenon known as the Warburg effect. This metabolic change originates from a shift in the expression of alternative spliced isoforms of the glycolytic enzyme pyruvate kinase (PK), from PKM1 to PKM2. While PKM1 is constitutively active, PKM2 is switched from an inactive dimer form to an active tetramer form by small molecule activators. The prevalence of PKM2 in cancer cells relative to the prevalence of PKM1 in many normal cells, suggests a therapeutic strategy whereby activation of PKM2 may counter the abnormal cellular metabolism in cancer cells, and consequently decreased cellular proliferation. Herein we describe the discovery and optimization of a series of PKM2 activators derived from the 2-((2,3-dihydrobenzo[b][1,4] dioxin-6-yl)thio)-1-(2-methyl-1-(methylsulfonyl)indolin-5-yl) ethanone scaffold. The synthesis, SAR analysis, enzyme active site docking, enzymatic reaction kinetics, selectivity and pharmaceutical properties are discussed.

Selective reduction of graft-versus-host disease-mediating human T cells by ex vivo treatment with soluble Fas ligand.

Previous work done in our laboratory, using mouse models, showed that soluble Fas ligand (sFasL) can efficiently delete donor anti-host T cells during their activation against irradiated host cells in MLCs. In the mouse models, this ex vivo sFasL treatment abrogated graft-vs-host disease (GVHD) while sparing donor T cells with antitumor reactivity. The present work was performed with human cells, to extend our work toward reduction of clinical GVHD. PBMC responders from a given individual (first party) were stimulated in vitro with irradiated PBMC stimulators from a second person (second party), in the presence of sFasL. In control MLCs without sFasL, alloreacting T cells began to up-regulate Fas (CD95) detectably and became sensitive to Fas-mediated apoptosis by as early as day 1-2. In MLCs containing sFasL, there were greatly reduced numbers of alloreacting CD3(+)CFSE(lo) cells, activation Ag-expressing CD4(hi) and CD8(hi) cells, IFN-gamma-producing CD4(+) and CD8(+) cells, and CD8(+)CD107a(+) CTLs. Furthermore, mice transplanted with the ex vivo sFasL/MLR-treated cells had prolonged time to fatal GVHD in an in vivo xenogeneic GVHD model. Responder cells harvested from primary MLCs containing sFasL had reduced proliferation in response to second party cells, but proliferated in response to CMV Ags, PHA, and third party cells. In addition, sFasL/MLR-treated cell populations contained influenza-specific T cells, CD4(+)FOXP3(+) T cells, and CD4(+)CD25(+) T cells. These data indicate that this ex vivo sFasL/MLR depletion of alloreacting human donor anti-host T cells was efficient and selective.

Ex vivo soluble fas ligand treatment of donor cells to selectively reduce murine acute graft versus host disease.

BACKGROUND: Allogeneic bone marrow transplantation (BMT) and donor lymphocyte infusion (DLI) provide valuable treatments for a range of diseases. However, the therapeutic utility of BMT and DLI is reduced by the high incidence of graft-versus-host disease (GvHD) mediated by activated donor T lymphocytes directed against recipient alloantigens. METHODS: Using mouse models, we developed and evaluated a strategy to selectively enhance activation-induced cell death (AICD) of anti-recipient T cells within transplant donor cell populations, with the goal of reducing GvHD. Responder T lymphocytes were incubated ex vivo with irradiated allogenic stimulator cells in a mixed lymphocyte reaction (MLR) in the presence of soluble Fas ligand (sFasL) to induce AICD in alloreactive cells. RESULTS: This ex vivo sFasL treatment reduced proliferation to the allogeneic stimulator cells in vitro and abrogated acute GvHD capacity in vivo. In contrast, the secondary immune responsiveness of the ex vivo sFasL-treated responder T cells to an unrelated model antigen was preserved. Furthermore, upon adoptive transfer in a DLI model, ex vivo sFasL-treated T cells were able to reject a model tumor. Finally, ex vivo sFasL treatment of bone marrow cells did not reduce their hematopoietic engraftment capacity. CONCLUSIONS: Thus, ex vivo treatment with sFasL appears to have potential for translation to clinical cell processing of BMT allografts and DLI infusions.

Extracellular phosphorylation of C9 by protein kinase CK2 regulates complement-mediated lysis.

Ecto-protein kinases (ecto-PK) are expressed on many cell types, both normal and malignant, yet their functions are largely unknown. An ecto-PK capable of phosphorylating the C9 component of the complement system is described. This C9 ecto-PK could be inhibited by TBB, Emodin and DRB, selective inhibitors of protein kinase CK2. Treatment of Raji human B lymphoma cells with these CK2 inhibitors augmented cell killing by Rituximab (anti-CD20 antibodies) and human complement. Analysis of C5b-7-bearing Raji cells showed that extracellular inhibition of the ecto-CK2 enhanced cell lysis by C8 and C9. Blocking of the membrane complement regulator CD59 with monoclonal antibodies further enhanced the effect of the CK2 inhibitors on Raji cell death by complement. C9 ecto-CK2 activity was increased on cancer cells relative to normal fibroblasts and blood cells. Therefore, ecto-CK2 appears to be an additional factor protecting cells from complement-mediated lysis, probably by phosphorylation/inhibition of complement C9.

Fas ligand as a tool for immunosuppression and generation of immune tolerance.

The role of Fas ligand (FasL) in physiologically limiting immune responses and maintaining immune-privileged sites has led to a body of research aiming to confer protection to allogeneic grafts by expressing FasL on the allogeneic tissue or by administrating FasL-transduced donor dendritic cells. In addition, several studies have used FasL to abrogate autoimmune responses. This review presents the results of these studies and discusses the problems associated with FasL usage.

Cell signals transduced by complement.

The complement system is composed of soluble blood plasma proteins and cell membrane proteins. A major function of the soluble complement proteins is to bind to and destroy invading pathogens. The membrane proteins of the complement system are divided into complement receptors and complement regulatory proteins. Complement receptors on phagocytic cells promote binding and engulfment of pathogens coated with complement opsonins, whereas complement regulatory proteins protect healthy tissues from accidental damage by the soluble complement proteins. Upon binding of complement proteins or protein fragments that are generated during complement activation, these receptors and regulatory proteins transduce various signals into cells bearing them. The complement membrane attack complex C5b-9 binds to cell membranes, independent of any receptor, and also activates multiple signaling pathways. The receptor-dependent and -independent signals transduced by complement components are of great consequence to health and disease. Complement plays an important role in immunoregulation by activating B and T lymphocytes. It may also exert pro- or anti-apoptotic effects on various cell types. At sublytic doses, the complement membrane attack complex has wide-range effects on many cell types leading to cellular responses, such as secretion, adherence, aggregation, chemotaxis and even cell division. Sublytic complement also induces increased cell resistance to lytic doses of complement. Finally, certain pathogens take advantage of complement membrane proteins to gain entry into cells. The emerging data on these complement-related signaling pathways is hereby described.