A 17-gene stemness score for rapid determination of risk in acute leukaemia.

Refractoriness to induction chemotherapy and relapse after achievement of remission are the main obstacles to cure in acute myeloid leukaemia (AML). After standard induction chemotherapy, patients are assigned to different post-remission strategies on the basis of cytogenetic and molecular abnormalities that broadly define adverse, intermediate and favourable risk categories. However, some patients do not respond to induction therapy and another subset will eventually relapse despite the lack of adverse risk factors. There is an urgent need for better biomarkers to identify these high-risk patients before starting induction chemotherapy, to enable testing of alternative induction strategies in clinical trials. The high rate of relapse in AML has been attributed to the persistence of leukaemia stem cells (LSCs), which possess a number of stem cell properties, including quiescence, that are linked to therapy resistance. Here, to develop predictive and/or prognostic biomarkers related to stemness, we generated a list of genes that are differentially expressed between 138 LSC+ and 89 LSC- cell fractions from 78 AML patients validated by xenotransplantation. To extract the core transcriptional components of stemness relevant to clinical outcomes, we performed sparse regression analysis of LSC gene expression against survival in a large training cohort, generating a 17-gene LSC score (LSC17). The LSC17 score was highly prognostic in five independent cohorts comprising patients of diverse AML subtypes (n = 908) and contributed greatly to accurate prediction of initial therapy resistance. Patients with high LSC17 scores had poor outcomes with current treatments including allogeneic stem cell transplantation. The LSC17 score provides clinicians with a rapid and powerful tool to identify AML patients who do not benefit from standard therapy and who should be enrolled in trials evaluating novel upfront or post-remission strategies.

In vivo targeting of B-cell lymphoma with glycan ligands of CD22.

Antibody-mediated cell depletion therapy has proven to provide significant clinical benefit in treatment of lymphomas and leukemias, driving the development of improved therapies with novel mechanisms of cell killing. A current clinical target for B-cell lymphoma is CD22, a B-cell-specific member of the sialic acid binding Ig-like lectin (siglec) family that recognizes alpha2-6-linked sialylated glycans as ligands. Here, we describe a novel approach for targeting B lymphoma cells with doxorubicin-loaded liposomal nanoparticles displaying high-affinity glycan ligands of CD22. The targeted liposomes are actively bound and endocytosed by CD22 on B cells, and significantly extend life in a xenograft model of human B-cell lymphoma. Moreover, they bind and kill malignant B cells from peripheral blood samples obtained from patients with hairy cell leukemia, marginal zone lymphoma, and chronic lymphocytic leukemia. The results demonstrate the potential for using a carbohydrate recognition-based approach for efficiently targeting B cells in vivo that can offer improved treatment options for patients with B-cell malignancies.

CellCelector™ as a platform in isolating primary B cells for antibody discovery.

The most robust strategy in antibody discovery is the use of immunized animals and the ability to isolate and immortalize immune B-cells to hybridoma for further interrogation. However, capturing the full repertoire of an immunized animal is labor intensive, time consuming and limited in throughput. Therefore, techniques to directly mine the antibody repertoire of primary B-cells are of great importance in antibody discovery. In the current study, we present a method to isolate individual antigen-specific primary B-cells using the CellCellector™ single-cell isolation platform from XenoMouse® (XM) immunized with a recombinant therapeutic protein, EGFR. We screened a subset of CD138+ B-cells and identified 238 potential EGFR-specific B-cells from 1189 antibody-secreting cells (ASCs) and isolated 94 by CellCellector. We identified a diverse set of heavy chain complementarity-determining region sequences and cloned and expressed 20 into a standard human immunoglobulin G1 antibody format. We further characterized and identified 13 recombinant antibodies that engage soluble and native forms of EGFR. By extrapolating the method to all 400 000 CD138+ B-cells extracted from one EGFR immunized XM, a potential 1196 unique EGFR-specific antibodies could be discovered. CellCelector allows for interrogating the B-cell pool directly and isolating B-cells specific to the therapeutic target of interest. Furthermore, antibody sequences recovered from isolated B-cells engage the native and recombinant target, demonstrating the CellCellector can serve as a platform in antibody discovery.

Bryostatin-I: a dendritic cell stimulator for chemokines induction and a promising adjuvant for a peptide based cancer vaccine.

Bryostatin-1 (Bryo-1), a PKC modulator, was previously shown to activate monocytes and lymphocytes to produce cytokines. In this report, we investigated the adjuvanticity of Bryo-1 both in vitro and in vivo. First, Bryo-1 was found to induce the release of CCL2 and CCL3 from mouse bone marrow-derived dendritic cells (BMDC) in a dose-dependent manner. As little as 0.1nM Bryo-I induced release of chemokines from BMDC and the maximal induction could be achieved at 5-10nM. Both PKC and ERK inhibitors attenuated the release of CCL2 and CCL3. Consistently, Western blot indicated that Bryo-I activated ERK in a dose- and time-dependent manner. Experiments with the NF-κB inhibitor, MG-132, demonstrated that NF-κB was involved in the induction of CCL2 but not CCL3. Because chemokines have been demonstrated to have profound effects on immune reactions by regulating the trafficking of DC and other lymphocytes into lymphoid organs, Bryo-I was tested as an adjuvant in an E7 peptide (MHC class I-restricted peptide epitope derived from human papillomavirus (HPV) 16 E7 protein)-based cancer vaccine. Mice immunized by s.c. injection with Bryo-I/E7 had enlarged draining lymph nodes and showed an antigen specific T-cell response demonstrated by the release of IFN-γ from isolated splenocytes and in vivo CTL activity. Finally, immunization with Bryo-I/E7 totally prevented the E7-expressing TC-1 tumor growth in mice. In conclusion, for the first time, we demonstrated that Bryo-I induced chemokine release from dendritic cell and was an effective adjuvant for peptide cancer vaccine.

Strategies for Generating Diverse Antibody Repertoires Using Transgenic Animals Expressing Human Antibodies.

Therapeutic molecules derived from antibodies have become a dominant class of drugs used to treat human disease. Increasingly, therapeutic antibodies are discovered using transgenic animal systems that have been engineered to express human antibodies. While the engineering details differ, these platforms share the ability to raise an immune response that is comprised of antibodies with fully human idiotypes. Although the predominant transgenic host species has been mouse, the genomes of rats, rabbits, chickens, and cows have also been modified to express human antibodies. The creation of transgenic animal platforms expressing human antibody repertoires has revolutionized therapeutic antibody drug discovery. The observation that the immune systems of these animals are able to recognize and respond to a wide range of therapeutically relevant human targets has led to a surge in antibody-derived drugs in current development. While the clinical success of fully human monoclonal antibodies derived from transgenic animals is well established, recent trends have seen increasingly stringent functional design goals and a shift in difficulty as the industry attempts to tackle the next generation of disease-associated targets. These challenges have been met with a number of novel approaches focused on the generation of large, high-quality, and diverse antibody repertoires. In this perspective, we describe some of the strategies and considerations we use for manipulating the immune systems of transgenic animal platforms (such as XenoMouse®) with a focus on maximizing the diversity of the primary response and steering the ensuing antibody repertoire toward a desired outcome.

Allogeneic Human Double Negative T Cells as a Novel Immunotherapy for Acute Myeloid Leukemia and Its Underlying Mechanisms.

Purpose: To explore the potential of ex vivo expanded healthy donor-derived allogeneic CD4 and CD8 double-negative cells (DNT) as a novel cellular immunotherapy for leukemia patients.Experimental Design: Clinical-grade DNTs from peripheral blood of healthy donors were expanded and their antileukemic activity and safety were examined using flow cytometry-based in vitro killing assays and xenograft models against AML patient blasts and healthy donor-derived hematopoietic cells. Mechanism of action was investigated using antibody-mediated blocking assays and recombinant protein treatment assays.Results: Expanded DNTs from healthy donors target a majority (36/46) of primary AML cells, including 9 chemotherapy-resistant patient samples in vitro, and significantly reduce the leukemia load in patient-derived xenograft models in a DNT donor-unrestricted manner. Importantly, allogeneic DNTs do not attack normal hematopoietic cells or affect hematopoietic stem/progenitor cell engraftment and differentiation, or cause xenogeneic GVHD in recipients. Mechanistically, DNTs express high levels of NKG2D and DNAM-1 that bind to cognate ligands preferentially expressed on AML cells. Upon recognition of AML cells, DNTs rapidly release IFNγ, which further increases NKG2D and DNAM-1 ligands' expression on AML cells. IFNγ pretreatment enhances the susceptibility of AML cells to DNT-mediated cytotoxicity, including primary AML samples that are otherwise resistant to DNTs, and the effect of IFNγ treatment is abrogated by NKG2D and DNAM-1-blocking antibodies.Conclusions: This study supports healthy donor-derived allogeneic DNTs as a therapy to treat patients with chemotherapy-resistant AML and also reveals interrelated roles of NKG2D, DNAM-1, and IFNγ in selective targeting of AML by DNTs. Clin Cancer Res; 24(2); 370-82. ©2017 AACR.

An Integrated Analysis of Heterogeneous Drug Responses in Acute Myeloid Leukemia That Enables the Discovery of Predictive Biomarkers.

Many promising new cancer drugs proceed through preclinical testing and early-phase trials only to fail in late-stage clinical testing. Thus, improved models that better predict survival outcomes and enable the development of biomarkers are needed to identify patients most likely to respond to and benefit from therapy. Here, we describe a comprehensive approach in which we incorporated biobanking, xenografting, and multiplexed phospho-flow (PF) cytometric profiling to study drug response and identify predictive biomarkers in acute myeloid leukemia (AML) patients. To test the efficacy of our approach, we evaluated the investigational JAK2 inhibitor fedratinib (FED) in 64 patient samples. FED robustly reduced leukemia in mouse xenograft models in 59% of cases and was also effective in limiting the protumorigenic activity of leukemia stem cells as shown by serial transplantation assays. In parallel, PF profiling identified FED-mediated reduction in phospho-STAT5 (pSTAT5) levels as a predictive biomarker of in vivo drug response with high specificity (92%) and strong positive predictive value (93%). Unexpectedly, another JAK inhibitor, ruxolitinib (RUX), was ineffective in 8 of 10 FED-responsive samples. Notably, this outcome could be predicted by the status of pSTAT5 signaling, which was unaffected by RUX treatment. Consistent with this observed discrepancy, PF analysis revealed that FED exerted its effects through multiple JAK2-independent mechanisms. Collectively, this work establishes an integrated approach for testing novel anticancer agents that captures the inherent variability of response caused by disease heterogeneity and in parallel, facilitates the identification of predictive biomarkers that can help stratify patients into appropriate clinical trials.

Targeting B lymphoma with nanoparticles bearing glycan ligands of CD22.

CD22 is a member of the siglec (sialic acid-binding immunoglobulin-like lectin) family expressed on B cells that recognizes glycans of glycoproteins as ligands. Because siglecs exhibit restricted expression on one or a few leukocyte cell types, they have gained attention as attractive targets for cell-directed therapies. Several antibody-based therapies targeting CD22 (Siglec-2) are currently in clinical trials for the treatment of hairy cell leukemia and other B cell lymphomas. As an alternative to antibodies we have developed liposomal nanoparticles decorated with glycan ligands of CD22 that selectively target B cells. Because CD22 is an endocytic receptor, ligand-decorated liposomes are bound by CD22 and rapidly internalized by the cell. When loaded with a toxic cargo such as doxorubicin, they are efficacious in prolonging life in a Daudi B cell lymphoma model. These B cell targeted nanoparticles have been demonstrated to bind and kill malignant B cells from patients with hairy cell leukemia, marginal zone lymphoma and chronic lymphocytic leukemia. The results demonstrate the potential of using CD22 ligand-targeted liposomal nanoparticles as an alternative approach for the treatment of B cell malignancies.

Antigen delivery to macrophages using liposomal nanoparticles targeting sialoadhesin/CD169.

Sialoadhesin (Sn, Siglec-1, CD169) is a member of the sialic acid binding Ig-like lectin (siglec) family expressed on macrophages. Its macrophage specific expression makes it an attractive target for delivering antigens to tissue macrophages via Sn-mediated endocytosis. Here we describe a novel approach for delivering antigens to macrophages using liposomal nanoparticles displaying high affinity glycan ligands of Sn. The Sn-targeted liposomes selectively bind to and are internalized by Sn-expressing cells, and accumulate intracellularly over time. Our results show that ligand decorated liposomes are specific for Sn, since they are taken up by bone marrow derived macrophages that are derived from wild type but not Sn(-/-) mice. Importantly, the Sn-targeted liposomes dramatically enhance the delivery of antigens to macrophages for presentation to and proliferation of antigen-specific T cells. Together, these data provide insights into the potential of cell-specific targeting and delivery of antigens to intracellular organelles of macrophages using Sn-ligand decorated liposomal nanoparticles.